patients were significantly different across pop mosomal level, but not generally at both levels. Abstracts of Lectures ulations with frequency maximum of the com In this view, the aneuploid karyotype is the read mon mutations in EastEurope (W151X, V326L), out of an underlying chromosomal instability NorthWestEurope (IVS81G>C), and SouthEu (CIN). In a small proportion of cancers display 1. Symposia rope (T93M). ing CIN the loss of this checkpoint is associated Carrier frequency analysis of the IVS81G>C, with the mutational inactivation of a human ho W151X, T93M, and V326L mutations in 2250 mologue of the yeast BUB1 gene. BUB1 controls S1 healthy individuals from different European pop mitotic checkpoints and chromosome segrega ulations revealed much higher frequencies for tion in yeast. The Human SHOX Mutation Database these common mutations (e.g. 1:50 for the IVS8 Because the MIN and CIN forms of instability are Beate Niesler, Christine Fischer and Gudrun A. 1G>C in Austria, and 1:84 for the W151X in rarely found to coexist in tumours, it would seem Rappold Poland) than expected from the reported preva that one form of instability is sufficient to drive Institute of Human Genetics, University of lence of the SLOS. Based on these frequencies tumorigenesis. Heidelberg, Im Neuenheimer Feld 328, the expected incidence of SLOS patients with Genetic instability appears early in tumorigene 69120 Heidelberg, Germany null mutations ranges from 1:2000 to 1:16.000. sis and is believed to play a critical role in the The human SHOX gene (Short Stature Home This discrepancy might be due to underdiagno malignant process. Cells with CIN are found to obox gene on the XChromosome) has been iso sis of both severe and mild cases, but also to activate or increase the number of copies of lated by positional cloning and resides in the embryonic/fetal loss of homozygotes for fre oncogenes and lose tumorsuppressor genes, pseudoautosomal region 1 of the sex chromo quent null alleles (W151X, IVS81G>C). whereas cells with MIN accomplish the same somes (Rao et al. 1997, Elison et al. 1997). Het Using eight cSNPs in the DHCR7 gene haplo through mutations in repetitive DNA sequences. erozygousous SHOX mutations have been types were constructed for 52 SLOS chromo This relationship is well documented, and the ef shown to cause idiopathic short stature (ISS), somes. All chromosomes carrying the most fects of genetic instability in cancer develop short stature phenotype in Turner Syndrome (TS) common European mutation IVS81G>C shared ment are straightforward in terms of Darwinian (Rao et al. 1997) and LèriWeill Dyschondrosteo the same haplotype suggesting a founder effect. evolution: Genetic instability provides a reper sis (LWD) (Belin et al. 1998, Shears et al. 1998). Mutations T93M and R404C which involve CpG toire of mutants from which the environment se

The homozygous loss of SHOX has been corre islands were found on 4 and 3 different haplo lects favorable variants. Abstracts GfH ÖGH SGMG Tagungsband lated with the Langer type of mesomelic dyspla types respectively suggesting that they are re Recent experiments have shown that CIN and sia (Belin et al. 1998). current. The W151X mutation was present on MIN instabilities reflect resistance to different The Human SHOX Database has recently been different related haplotypes and is probably old carcinogens and that genomic instability in can established to provide clinicians and scientists er than the SNPs used for haplotype construc cers may mirror the mutagenic environments in access to a central source of information about tion indicating that it may be the oldest common which they evolve. It is therefore interesting to all known SHOX mutations associated with short SLOS causing mutation in Europeans. The data speculate that both MIN and CIN develop stature phenotypes. So far, the database con suggest an intriguing high frequency and hetero through selection pressures that can readily be tains 29 unique intragenic mutations of the geneity of the ages and origins of common understood in Darwinian terms rather than aris SHOX gene. These mutations were detected in DHCR7 mutations in ing in a random and mysterious manner. a total of 39 patients from different families. Fourteen of these mutations have been found Functional dissection of signal transduction from the SHOX research group at the Institute of pathways in mammalian cells using RNA Human Genetics in Heidelberg, Germany; 25 S2 interference mutations are from data reported in the litera Anja Schramme, Christine Sers, Oleg ture. Not included in this database are complete Tchernitsa, Katrin Barth and Reinhold Schäfer Genetic instability and darwinian selection SHOX gene deletions which represent the ma Laboratory of Molecular Tumour Pathology, in tumours jority of all detectable SHOX mutations ( Rap Charité, Schumannstr. 20/21, D10117 Berlin Alberto Bardelli pold et al., 2002). The RASRAFMEKERK pathway is the major The Johns Hopkins University, The The database is accessible via the web site player in the regulation of oncogenic signalling Oncology Center, VogelsteinKinzler http://www.shox.unihd.de. It contains general in various types of cells. Most cells express dif Laboratory, Room 520, 1650 Orleans, information about the SHOX gene, allows remote ferent isoforms of the effector kinases down Baltimore MD 21231, USA users to search the data and to submit new mu stream of RAS, however, the contribution of in Whether and how human tumours are genetical tations into the database. The gene structure is dividual kinases is poorly understood. To eluci ly unstable has been debated for decades. There incorporated in order to facilitate data submis date the function of kinase isoforms in control is now evidence that most cancers may indeed sion. ling cell growth and transformation, we have in be genetically unstable, and that the instability References troduced siRNAs (small interfering RNA douplex exists at two distinct levels. 1. Belin et al., 1998. Nat Genet 19:679. es) specific for RAS, RAF, MEK and ERKiso In a small subset of tumours, the instability is 2. Ellison et al. 1997. Hum Mol Genet 8:13417. forms into HRAStransformed rat (FE8) and observed at the nucleotide level and results in 3. Rao E, et al.1997. Nat Genet 16:5463. mouse (NIHpEJ) fibroblasts and into human EJ base substitutions, deletions or insertions of a 4. Rappold etal. JCEM 87: 13841388. bladder carcinoma cells harbouring endogenous few nucleotides. This instability is most easily 5. Shears et al. 1998. Nat Genet 19:703. activated HRAS. HRASspecific siRNA strongly observed at short sequences of DNA repeats inhibited growth in all cells and morphological scattered throughout the genome, called mi transformation in FE8 and NIHpEJ cells. crosatellites, thus generating the characteristic KRASspecific siRNA had no effect. Targeting ki FREQUENCIES AND ORIGINS OF COMMON microsatellite instability (MIN) seen in these tu nases downstream of RAS in mouse NIHpEJ by DHCR7 MUTATIONS CAUSING THE SMITH mours. MIN tumours have nucleotide mutation RNA interference revealed a differential contri LEMLIOPITZ SYNDROME rates two to three orders of magnitude higher bution of MEK1 and MEK2 to growth control. Martina WitschBaumgartner, J. Löffler, M. than normal cells or mismatchrepairproficient Whereas the MEK1specific siRNA inhibited cell Gruber, I. Braun G. Utermann cancers of the same cell type. This form of insta growth only weakly (20%), interference with Institute of Medical Biology and Human bility arises from inactivation of DNA mismatch MEK2 expression resulted in 50% growth sup Genetics, Innsbruck, Austria repair (MMR) genes such as MSH2 or MLH1. pression. Interference with either MEK isoform SmithLemliOpitz syndrome (SLOS [MIM Strikingly, unlike the vast majority of solid tu had almost no effect on cell morphology. How 270400]) is an autosomal recessive multiple con mours, MIN cancers have a normal complement ever, the combination of both siRNAs fully re genital anomaly/mental retardation snydrome of chromosomes. These cancers retain a diploid verted the transformed phenotype of the cells caused by mutations in the D7sterol reductase karyotype ¯ one pair of each of the chromo and inhibited cell growth completely. This indi (DHCR7, E.C.1.3.1.21) gene. Based on case fre somes. cates a strong synergism between MEK1 and quency surveys the incidence of the SLOS has In most other cancers, the instability is observed MEK2. In a similar way, full morphological rever been estimated to range from approximately at the chromosome level, resulting in losses and sion was achieved upon cotransfer of ERK1 and 1:20.000 to 60.000 in populations of European gains of whole chromosomes or large portions ERK2specific siRNAs. Cell growth however, was origin. thereof. NonMIN tumours have a wide variation inhibited up to 50% by either combining ERK1 We are analysing the frequencies, origins, and in chromosome number their karyotypes are and ERK2 siRNAs or after transfer of individual ages of DHCR7 mutations in Europe (Italy, aneuploid. Such observations have led to the siRNAs. Knockdown of kinase isoform expres Spain, Poland, Austria, Germany, and Great suggestion that cancers develop instability ei sion was confirmed by Western blot analysis. In Britain). Mutational spectra analysed in 81 SLOS ther at the sequence level (MIN) or at the chro

medgen 14 (2002) 1 hibition of ERK1 resulted in increased phospho S4 cation at the right time. Genome Biol 2, RE rylation of ERK2 and vice versa. This suggests a VIEWS1024. crosstalk of the activated, phosphorylated Fulllenght cDNA and gene annotation forms of ERK1 and ERK2 kinases compensating Sumio Sugano inhibitory effects. Our data show that RNA inter Human Genome Center, The Institute of Molecular identification of genes and ference is a powerful genetic tool for functional Medical Sciennce, The pathways involved in skeletogenesis by EST ly dissecting the complex network of signalling University of Tokyo, 461, Shirokanedai, sequence analysis and microarray pathways in mammalian cells. Minatoku, 1088639, Tokyo, Japan expression profiling of human mesenchymal Although a huge data of draft and finished se stem cell differentiation quences of the human genome are now avail Zabel Bernhard (1), Schlaubitz, S. (1), Stelzer, High prevalence of BRCA2 mutations in able, it is still not a trivial task to identify genes C. (1), Luft, F. (2), Schmidt, E.R. (2), Hankeln, T. pancreas cancer families and further from genome sequences. Thus, a fulllength (2), Hermanns, P. (3), Lee, B. (3), Jakob, F. (4), evidence for a CDKN2A mutation associated complementary DNA (cDNA), which is a com Noeth, U. (4), Mohrmann, G. (5), Tagariello, A. pancreas cancer melanoma syndrome plete DNA copy of mRNA, is required for the (5), Winterpacht, A. (5) Rieder, Harald (1), Hahn, S.A. (2), Lang, S. (3), identification of a gene and the determination of (1) Children’ s Hospital, University of Mainz, Frey, M. (1), Korte, B. (2), Wild, A. (3), Gerdes, its structure. We are now participating FLJ cDNA Germany, (2) GENterprise GmbH, Mainz, B. (3), Kress, R. (4), Ziegler, A. (5), Rehder, H. project to collect and sequence fullleng th cD Germany, (3) Baylor College of Medicine, (1), Rothmund, M. (3), Schmiegel, W. (2), NAA clones under support of Houston, Tx, USA; (4) Orthopedic Clinic, Bartsch, D.K. (3,6) ministry of economy, industry and international University of Würzburg, Germany, (5) (1) Inst. f. Klin. Genetik, Marburg; (2) Klinik f. trade. In this project, we extensively used cDNA Institute of Human Genetics, University of Innere Med., Knappschaftskrankhaus, libraries made by Oligocapping method, a cap ErlangenNuernberg, Germany Bochum; (3) Klinik f. Visz., Thor. und targeted selection procedure for fulllength cD The differentiation of mesenchymal stem cells Gefäßchirurgie, Marburg; (4) Inst. f. Med. NAs. So far, we have accumulated more than into the chondrogenic or osteogenic lineage is Biometrie und Epidem., Marburg; (5) Inst. f. 800,000 5’ end sequences through random se an integral part of multistep processes important Med. Biometrie und Statistik, Lübeck; quencing of cDNA clones from about 100 kinds in pattern formation, ossification, postnatal GfH ÖGH SGMG Tagungsband Abstracts GfH ÖGH SGMG Tagungsband Germany. (6) für die FaPaCa Studiengruppe of human cDNA libraries. Among them, 12340 growth and development of the skeleton. Our Familial pancreatic cancer (FPC) is a rare dis clones have been fully sequenced. The accura project aims at the identification of genes and ease for which no major causative gene has cy of the sequences was more than 99.95%. The pathways involved in these complex processes. been reported. Members of hereditary average length of cDNAs wwhose sequence Candidate genes are expected to be of value for breast/ovarian cancer (HBOC) families with were determined was about 2200bp, which dis diagnosis and treatment of monogenic and BRCA2 germline mutations, and of families with tributed from 1kb to 5kb. Furthermore we could multigenic heritable disorders of the skeleton. familial atypical multiple molemelanoma (FAM cluster 5’ end of many mRNAs (more than 5000 So far, 5´end sequencing of randomly selected MM) syndrome and with CDKN2A mutations genes), which could be mapped to onto the hu clones from an unique human fetal cartilage have an increased pancreatic cancer (PC) risk. man draft genomic sequence. The detail of this cDNA library (provided to us exclusively by Dr. Provisionally, the latter have already been sum project and its results will be presented. B. Lee, BCM, Houston) has lead to the genera marized under the pancreas cancermelanoma tion of 5.000 ESTs. 4.000 of these sequences syndrome (PCMS). The German National Case have been run through ESTsweep, an EST iden Collection for Familial Pancreas Cancer (Fa tification task which we initiated in close collab PaCa) has been established for the clinical and oration with the HUSAR bioinformatics team (Dr. genetic analysis of familial pancreas cancer Highly parallel subcellular localization and HotzWagenblatt, Dr. Glatting, DKFZ/Heidel (FPC) and of families suggestive of PCMS. Con functional analysis of proteins encoded by berg). Most of the ESTs (69,6%) show significant stitutional DNA of 14 index patients of FPC fam novel fulllength cDNAs similarity to known genes of the human RefSeq ilies was screened for sequence variants of the Wiemann, Stefan; Poustka, Annemarie; collection (NM_#), and another 4,8% correspond BRCA2 gene. Three different frame shift muta Pepperkok, Rainer to human model RNAs (XM_#). About 23% do tions were identified in three cases (21%) of Deutsches Krebsforschungszentrum Im not show any similarity to defined mRNAs or which two have been described as oncogenic Neuenheimer Feld 280 proteins, but to genomic clones or anonymous mutations and one as an unclassified variant in 69120 Heidelberg Europäisches ESTs. A number of these genes of unknown HBOC families. The latter was accompanied by Molekularbiologisches Laboratorium function are currently under detailed functional an unclassified missense variant on the same al Meyerhofstr. 169117 Heidelberg investigation. Preliminary data revealed putative lele. At the time of recruitment, none of the fam cloning / localization: 200 open reading frames transcription factors as well as genes probably ilies with BRCA2 mutations presented with a tu have been subcloned into expression vectors involved in RNA transcription/processing. Al mor pattern suggestive of a HBOC family. Index within the reported period, totaling over 300 ready in the present stage the data provide an patients of 18 FPC families and of 5 families with ORFs. All ORFs have been cloned into YFP and interesting insight into the gene expression pat potential PCMS were screened for CDKN2A se CFP fusion vectors, the encoded proteins have tern of the developing skeleton. All EST se quence variants. In none of the FPC but in 2 of been localized.Database: the clone/ORF track quences will be stored in a dedicated database, the PCMS insinuating families CDKN2A frame ing database has been established. This data and will be used to generate cDNA microarrays shift mutations were identified. Thus, CDKN2A base is currently extended to be accessible for the expression profiling of human mesenchy mutations seem to contribute to the familial oc through a webinterface. This will allow the sub mal stem cells (hMSC) which can be differenti currence of PC and MM in 2/5 of the cases. This mission of queries, but also the uploading of ated into osteoblasts/osteocytes and chondrob provides further evidence for PCMS to represent data by the EMBL group to optimize the time be lasts/chondrocytes. This enables us to select a distinct hereditary cancer predisposition syn tween data generation and database integra candidate genes involved in bone/cartilage de drome. Screening for BRCA2 and CDKN2A mu tion.Assay development: Assays for the identifi velopment for further experimental analysis, and tations in index patients of FPC and PCMS fam cation of proteins that are involved in the regu to characterize the pattern of gene expression ilies may be feasible to allow a individualized PC lation of the regulatory pathway have been es during stem cell differentiation. risk assessment of family members by predictive tablished and are routinely applied to all proteins genetic testing. However, penetrance data of the localizing to the respective compartments (e.g. respective gene mutations and procedures sen ER, Golgi, TGN, but also microtubules). Current Comparative methylation analysis of CpG sitive enough for an early detection of PC are ly, assays to monitor protein involvement in cell islands in human Xq28 and mouse still lacking. The continuation and extension of proliferation and/or apoptosis are being devel Galgoczy, P; Platzer, M. FaPaCa may provide the logistic infrastructure oped. Automated 96well microscope: the high Dept. Genome Analysis, Institute of for the prospective acqua in mutation carriers content screening microscope has been set up. Molecular Biotechnology, Jena and for the evaluation of newly developed sur Hard and Software are operational and used for Xq28, the distal end of the long arm of the hu veillance programs for early PC detection. assays that are carried out in the project. man Xchromosome, is one of the most gene Supported by the Deutsche Krebshilfe, grants Simpson, J. C., Wellenreuther, R., Poustka, A., dense regions of the human genome. To date 702362Ba2 and 702828Ba3 Pepperkok, R. and Wiemann, S. (2000). System more than 30 human disease genes have been atic subcellular localization of novel proteins mapped to this region. We use comparative identified by large scale cDNA sequencing. mapping and sequencing of 6 Mb syntenic re EMBO Rep 1, 287292.Pepperkok, R., Simpson, gion between Ids and Dmd of the mouse X chro J. and Wiemann, S. (2001). Being in the right lo mosome to investigate the organization/evolu

2 medgen 14 (2002) tion of Xq28 and as a tool for detection of novel RT1 sequencing effort and will present a prelim Clinical Variability of Opitz BBB/G regulatory elements. inary comparative analysis with the human and syndrome: a challange for clinicians and Methylation of CpG islands in higher eukaryotic the currently available mouse MHC sequence. biochemists insulates sequences from the interaction with Acknowledgement of funding: RT1 sequencing Susann Schweiger 1, Jennifer Winter1, Sybille DNA binding proteins. Only unmethylated pro is supported by the German National Genome Krauß1, Vanessa Suckow1, Zofia Kijas1, Peter moter regions are accessible targets for binding Research Network (NGFN). Lunt2, Koen Devriendt3, Helen Firth4, Beate and interaction. Albrecht5, Marieke Baars6, Claude Moraine7, We have analyzed more than 600 kb syntenic J.J. van der Smagt8, Albert Schinzel9, Frank sequences in mouse and man for the occur Majewski10, Eric Sistermans11, Vera rences of CpG islands: In the regions ZFP92 to Kalscheuer1, HansHilger Ropers1, Rainer S5 CDM (375 kb) and FLN to NEMO (240 kb) we de Schneider12 tected 28 CpG islands in man, 23 of them were (1) MaxPlanck Institut für Molekulare also present in mouse. Most of the CpG islands Fetal Determinants of Adult Health Genetik, Berlin (2) Bristol (3) Leuven (4) (19 human and 18 murin) are associated with the Judith G. Hall, OC, MD Addenbrooks (5) Essen (6) Amsterdam (7) 5’ end of known genes. Four CpG islands are lo University of British Columbia, BC Tours (8) Leiden (9) Zürich (10) Düsseldorf cated within genes in both species. The addi Children’s Hospital, 4480 Oak St., Room (11) Nijmegen (12) Innsbruck tional CpG islands (5 human and 1 murin) are 2D19,Vancouver, BC V6H 3V4 Opitz BBB/G is a malformation syndrome of the not close to any known gene. We try to eluci After organogenesis has been established and is ventral midline, which is characterized by a large date, whether the se CpG islands represent ele proceeding, a number of important developmen spectrum of symptoms including hypertelorism ments of alternative promoters or are clues for tal processes occur in the human fetus. Recog and hypospadias, cleft lip and palate, laryngo yet unknown genes. nition of these processes and their importance tracheoesophageal fistulas, congenital heart de Therefore we have examined the methylation for adult health will undoubtedly impact research fects, developmental delay and imperforate status of 13 selected CpG islands conserved in related to complex genes. Fetal maternal mi anus. Autosomal dominant (chromosome 22) as both species using the bisufite genome se crochimerism, thrombophilias, fetal movement, well as Xlinked recessiv inheritance have been quencing technology. Two of the analyzed inter cytokines and steroids, maternal nutrition and shown in families with Opitz BBB/G syndrome.

nal CpG islands were unmethylated and repre intrauterine growth retardation all interact with While a gene has been identified on the Xchro Abstracts GfH ÖGH SGMG Tagungsband sent elements of alternative promoters. Four of each other and have individual longterm affects. mosome (called MID1 gene), the underlying ge the human CpG islands that are not associated Fetal maternal microchimerism plays an impor netic defect on chromosome 22 remains un with known genes are unmethylated. Here we tant role in autoimmune and connective tissue known. There is an ongoing discussion among detected at least two new genes and a new, yet disorders; fetal movement and use are essential clinicians wheather or not particular symptoms unidentified 5’UTR of the PMCA3 gene. to the development of most organs; cytokines can be defined eather for the Xlinked or for the Furthermore, we present detailed comparison of and steroids play a role in the maturation and autosomal form of OS. We have now summer methylated versus unmethylated CpG islands to growth of various fetal structures; maternal nu ized the phenotypes of all patients in which we improve the prediction of functional CpG islands trition is important to intrauterine growth and have identified mutations in the MID1 gene and in man and mouse. may also have transgenerational affects; and in compared them to patients with the autosomal trauterine growth retardation is being recognized form. Interestingly, the whole spectrum of OS as having long term effects producing diabetes, features can be found in both groups. Moreover, Genomic sequencing of the rat major hypertension and heart disease in adulthood. the variability of the phenotypes even within one histocompatibility complex (RT1) region family is enormous. The MID1 protein function, Peter Hurt1, Lutz Walter2, Eberhard Günther2, e.g. the regulation of phosphatase 2A, a central Hidetoshi Inoko3, Takashi Shiina3, Hans Phenotypic Spectrum of Noonan Syndrome serin / threonin phosphatase, pave the way for Lehrach1, Ralf Sudbrak1, Richard Reinhardt1, Patients with Mutations in the PTPN11 Gene speculations regarding the molecular basis of Heinz Himmelbauer1 Hoeltzenbein, Maria (1), Kehl HG (2), Musante L the observed variability. 1MaxPlanckInstitute of Molecular (1), Majewski F (3)†, Meinecke P (4), Gillessen Genetics, Berlin, Germany, 2Division of Kaesbach G (5), Schweiger S (1), Göldner B (6), Immunogenetics, University of Göttingen, Tinschert S (7), Hinkel GK (8), Ropers HH (1), Göttingen, Germany, 3Tokai University, Kalscheuer VM (1) Isehara, Japan MaxPlanckInstitut für Molekulare Genetik, S6 The human major histocompatibility complex Berlin(1); Kinderkardiologie, Univ. (MHC) plays a major role in human disease, Münster(2) and Charité, Berlin(6); Inst. für Predictive Testing for LateOnset most of them being of autoimmune or infectious Humangenetik, Univ. Düsseldorf(3) and Univ. Neurodegenerative Diseases nature, for instance insulin dependent diabetes Essen(5); Abt. Med. Genetik, Hamburg(4); Olaf Riess mellitus (IDDM), rheumathoid arthritis, and mul Inst. für Med. Genetik, Charité, Humboldt Department of Medical Genetics, University tiple sclerosis. Situated on human chromosome Univ. Berlin(7) and Univ. Dresden(8) Tübingen, Calwerstrasse 7, 72076 Tübingen 6 and occupying 3.6 Mb of genomic DNA, the Noonan syndrome (NS, MIM 163950) is a well The recent identification of numerous gene de MHC is one of the most genedense regions de known autosomal dominant disorder character fects causing neurodegeneration in humans scribed so far in the human genome. Of the 224 ized by short stature, facial dysmorphism and promises widespread genetic predictive testing identified human genes, an estimated 40% have cardiac defects (i.e. pulmonary stenosis and hy also for late manifesting disorders. Due to its functions associated with immune response and pertrophic cardiomyopathy). Recently mutations current limitations (lack of treatment, disease participate in diverse pathways, e.g. antigen pro in the PTPN11 gene have been described by progression, and for some diseases inadequate cessing, antigen presentation and Tcell interac Tartaglia et al. (Nat. Genet. 29: 465468, 2001). test sensitivity and specificity), however, genet tion. To shed further light onto evolutionary, bi We have screened the PTPN11 gene for muta ic testing is not generally recommended. There ological and biomedical aspects of the MHC, we tions in 92 familial and sporadic NS cases as fore, it is anticipated that predictive analysis of have set out to sequence the rat MHC (RT1 well as 5 patients with cardiofaciocutaneous late manifesting diseases should be accompa complex) on rat chromosome 20. Towards this syndrome (CFC), that has been suggested to be nied by testing programs. The issue of genetic end, we have generated a BAC/PAC contig allelic to NS. Mutations were identified in 32 pa testing will become even more complex as ge across the RT1 complex region and have estab tients with NS (5 families and 18 unrelated indi netic susceptibility factors for socalled sporadic lished a minimal tiling path that consisted of 39 viduals). 86% of patients with mutations had a diseases (e.g. Alzheimer’s disease, Parkinson’s clones. As of May 2002, HTGS data have been cardiac defect, with pulmonary stenosis (76%) disease) are being defined in an increasing num submitted to Genbank for 24 clones (For up and/or septal defects (14%) being the most ber. The benefits, limitations, and risks are de dates see http://seq.molgen.mpg.de/hgs/chrom_ common. Catheter intervention or surgery were scribed. First results of genetic testing programs rat.html). The entire region is actively being necessary in nearly half of them. Mild mental re are being discussed in the context of ethical, le worked on, by completing draft sequence and tardation or developmental delay were observed gal, and social issues. by annotation of finished clones. Annotation is in 42% of patients. 75% of boys had malde carried out using the program Genotator, up scended testes. Apart from the absence of hy graded by us by implementing additional se pertrophic cardiomyopathy, patients with muta Predictive Testing for Breast and Ovarian quence analysis tools for efficient comparisons tions could not be distinguished clinically from Cancer between mRNA and genomic sequence and be patients without mutations. We did not find any R.K. Schmutzler tween genomic sequences from different organ mutation in patients with CFC, suggesting ge University of Bonn isms. We will describe the present status of our netic heterogeneity.

medgen 14 (2002) 3 Rund 5% der Brust und Eierstockkreb gation analyses and heterologuos expression of arabidopsis, Drosophila, C. elegans, yeast, mul serkrankungen sind erblich bedingt und folgen mutant alleles followed by classical patch clamp tiple bacterial species etc.) as well as stimulat einem autosomaldominanten Erbgang. Der techniques. Furthermore we identified a com ing the growth of our technology base. An im erbliche Brust und Eierstockkrebs tritt familiär mon polymorphism which is associated with IGE portant consequence of this effort was the ap gehäuft auf und ist durch ein frühes as shown by the results of a populationbased plication of high throughput process technolo Erkrankungsalter gekennzeichnet. Mutationen in as well as familybased association study com gies to discovery phase of research, specifical den Genen BRCA1 und BRCA2 sind für rund prising two independent samples. Thus, our re ly the introduction of automation and informat 50% der Erkrankungsfälle verantwortlich und sults highlight CLCN2 as the first gene of which ics into the biology work place. The project has können mit molekulargenetischen Methoden rare mutations can cause the whole spectrum of also stimulated a paradigm shift in research as identifiziert werden. Das lebenslange Risiko ein common IGE subtypes as well as a common se the gathering and presentation of information er Mutationsträgerin beträgt 8090% für quence variant conferring susceptibility to IGE has become an end itself, resulting in the disso Brustkrebs und 3060% für Eierstockkrebs. Das probably in a significant number of patients. ciation of data acquisition from classic hypoth Risiko für ein Zweitkarzinom der Brust oder der esis based research. Eierstöcke liegt bei 50%. Auf Grund des hohen The goal of this talk will be to review the utility Erkrankungsrisikos der Mutationsträgerinnen ist of available genetic systems for target discovery Genome scan and families with myocardial ein standardisiertes Früherkennungsprogramm and target validation. Special emphasis will be infarction (sekundäre Prävention) zu empfehlen, welches placed on invertebrate model systems as they Heribert Schunkert, Jeanette Erdmann die speziellen Charakteristika des erblichen offer the opportunity for systematic genetic Clinic and Poliklinik if Internal Medicine II, Brust und Eierstockkrebses berücksichtigt. Für screening in the context of well established un Cardiology, University of Regensburg, die Verhütung (primäre Prävention) des erblichen derstanding of organismal biology, the availabil Germany Brust und Eierstockkrebses gibt es operative ity of high quality genomic/genetic information Myocardial infarction is a trait with a strong fa und medikamentöse Optionen. Die Inanspruch and tools as well as advanced technology for milial component. Especially, manifestation at an nahme einer Genanalyse sowie primär präven germline modification. I will also discuss the age of 60 years or less points to a genetic back tiver Maßnahmen hängen von der individuellen translation of fundamental approaches, first pi ground. However, generally speaking, the patho Situation ab und verlangen eine interdisziplinäre oneered in simpler genetic systems, into tools physiology of myocardial infarction is rather

GfH ÖGH SGMG Tagungsband Abstracts GfH ÖGH SGMG Tagungsband Beratung und Betreuung. for genetic dissection of vertebrate models. complex and involves several environmentally Eine erste Evaluation in unserem Zentrum an and genetically determined risk factors. More über 800 Frauen mit einer familiären Belastung over, haemodynamic, metabolic, inflammatory, SLC12A7 encoding KCl cotransporter ergab eine hohe Akzeptanz für das Früherken coagulatory, and apoptotic mechanisms are ac KCC4: another gene associated with nungsprogramm (>85%), während sich nur tivated to a variable extent. With such complex deafness and renal tubular acidosis wenige Frauen für eine prophylaktische Opera ity the prediction of candidate genes that may Hübner, Christian A (1), Boettger, T (2), Singh, tion entschieden (5%). Die Effizienz der explain the familial background of myocardial in A (2), Maier, H (3), Rust, MB (4), Beck, FX (5), Früherkennung wurde an 429 Frauen, die seit farction is extremely difficult. In fact, prioritizing Jentsch, TJ (6) mindestens einem Jahr an dem Früherkennung of candidates into more or less important genes (1),(2),(4),(6) Zentrum für Molekulare sprogramm teilnehmen, untersucht. Die should be considered to be rather speculative. Neurobiologie, Universität Hamburg, prospektive erhobenen Inzidenzraten für das We thus preformed random genome screens in Germany; (3) HNO Klinik, Universität familiäre Mammakarzinom lagen 40fach höher pairs of siblings with premature myocardial in Hamburg, Germany; (5) Physiologisches als in der Allgemeinbevölkerung. Die bisherigen farction (n = 1406 individuals) and large families Institut, LudwigMaximiliansUniversität Daten belegen, dass die Detektion früher Karzi with up to 20 affected members with coronary München, Germany; (1) Institut für nome durch eine multimodale und engmaschige artery disease. We identified a chromosomal lo Humangenetik, Universität Hamburg, Früherkennung möglich ist. cus (14 q 32) with a significant LOD score for Germany myocardial infarction (Nat. Genet. 2002; 30:210 Hearing depends on a high K+ concentration of 4). In addition, the family studies suggest that the endolymph bathing the apical membranes of further loci may contribute to the heritability of sensory hair cells. According to the K+ recycling S7 myocardial infarction. Of note, we also identified model K+ that has entered hair cells through api chromosomal loci for genetically determined cal mechanosensitive channels is transported to cardiovascular risk factors. However, there was the endolymph producing epithelium (stria vas Molecular basis of common forms of human no overlap with the MI locus on chromosome 14. cularis) for resecretion into the scala media. K+ idiopathic epilepsies This suggests that the genetic risk of myocardial probably exits outer hair cells by basolateral Armin Heils infarction is partially independent from tradition KCNQ4 K+ channels, and is then transported via University of Bonn, Institute of Human al risk factors. This finding is not surprising giv a gap junction system connecting Deiters’ cells, Genetics en that epidemilogical surveys already demon which support outer hair cells, and fibrocytes Epilepsy affects about 23 % of the world´s pop strated the independent nature of the familial back to the stria vascularis. We show here that ulation. Idiopathic epilepsies account for 40% of contribution irrespective of potential covariates. mice lacking the KCl cotransporter KCC4 (en all cases. During the last decade several genes However, without knowledge on potential mech coded by SLC12A7) are deaf because their hair causing monogenic forms of epilepsy have been anisms the functional implications of the gene cells degenerate rapidly after the beginning of identified, however, the molecular basis of com variant related to myocardial infarction in our hearing. In the mature organ of Corti, KCC4 is mon subtypes of idiopathic generalized epilep families will be difficult to predict. We therefore restricted to supporting cells of outer and inner sy (IGE) remains elusive. Common forms of IGE currently engage in further phenotypical charac hair cells. Our data suggest that KCC4 is impor including idiopathic absence epilepsy (IAE), ju terization of our sample including analysis of tant for K+ recycling by siphoning K+ ions after venile myoclonic epilepsy (JME), and epilepsy coronary angiographies. their exit from outer hair cells into Deiters’ cells, with grandmal seizures on awakening (EGMA) where K+ enters the gap junction pathway. Like are characterized by unprovoked seizures in the in some human genetic syndromes, deafness in absence of any detectable brain lesion. Family Kcc4/ mice is associated with renal tubular studies and segregation analyses have clearly acidosis. It probably results from an impairment shown, that common forms of IGE are inherited S8 of Cl recycling across the basolateral mem as genetically complex traits. Results of a recent brane of acidsecreting aintercalated cells of genome search for IGE susceptibility loci provid Genetics in a PostGenomics Era: Lessons the distal nephron. Screening of patients suffer ed significant evidence for the existence of a from Model Systems ing of deafness and renal tubular acidosis so far novel IGE locus on chromosome 3q26 (Sander Geoffrey Duyk, M.D., Ph.D. did not reveal any mutations in SLC12A7. et al., 2000). This chromosomal region harbors The human genome project has reached its mid several potential candidate genes including point. Its initial goals were to complete genetic CLCN2, encoding the voltagegated chloride Mouse model for Xlinked mental and physical maps of the human genome in an channel ClC2. Since ClC2 is essential for an retardation by targeting the Arhgef6 gene ticipation of completing the primary nucleotide inhibitory response towards GABAergic trans Kutsche, Kerstin (1), Kiemann, K. (1), sequence. The project was initially aimed at pro mission in the brain, we followed a positional Rosenberger, G. (1), Bösl, M.R. (2), Gal, A. (1) viding the infrastructure necessary for the iden candidate gene approach searching for muta (1) Institut für Humangenetik, tification of the genetic basis of common dis tions and common sequence variants in 46 IGE Universitätsklinikum HamburgEppendorf, ease. This effort spawned „sister projects“ fo index cases. We identified three diseasecaus Germany, (2) Transgenic technologies cused on model systems (mouse, rat, zebrafish, ing mutations as shown by the results of segre

4 medgen 14 (2002) group, Zentrum für Molekulare and stable mouse lines established. In order to nine models several clinical phase I studies on Neurobiologie, Hamburg, Germany verify the function of these mouse lines we haemophilia A and B patients have been initiat Mutations in ARHGEF6, the gene encoding a crossbred the ataxin3 responder lines with a ed of which one preliminarily was reported to be guanine nucleotide exchange factor for Rho GT wellcharacterized prion protein promoter mouse successful. Pases, have been reported to cause Xlinked line (kindly provided by Dr. S. Prusiner). Double non specific mental retardation. We have char transgenic mice of two disease model mouse Molecular therapeutic approaches for acterized the orthologous mouse gene. Arhgef6 lines express the ataxin3 protein with an ex muscular dystrophies is expressed in various mouse tissues suggest panded polyglutamine repeat. Studies are ongo Hanns Lochmüller ing an ubiquitous expression pattern, as found ing to characterize these mouse lines and to de Genzentrum, FriedrichBaurInstitut, and for ARHGEF6. The mouse gene product shows fine their suitability as a model of SCA 3. These Dep. of Neurology; LudwigMaximilians high homology to the human protein with all models will allow us to turn off or on ataxin3 ex University; Munich; Germany structural motifs highly conserved. A genomic pression at different developmental stages and We will give a review on preclinical experimen cosmid clone containing Arhgef6 exons 1 and 2 will demonstrate whether or not the disease tation that will hopefully result in safe and effec has been isolated and used to map the gene to phenotype will be reversible. tive therapy of muscular dystrophies. At pres the mouse X chromosome. Subsequently, to es ent,different approaches such as gene replace tablish a Arhgef6 knockout mouse, we replaced Transgenic rat model of Huntington’s ment therapy, utrophin upregulation and muscle exons 1 and 2 by the taulacZ marker gene and disease progenitor cells are investigated. Adenoviral a neo cassette. The start codon of the lacZ O. Riess1, I. Schmitt2, H.P. Nguyen3, C. transfer of therapeutic genes such as dystrophin marker was placed exactly over the start codon Holzmann4, Th. Schmidt1, Th. Walther5, D. is hampered by low transduction efficiency of of the endogeneous Arhgef6 gene. Detection of Stiller6, A. Kask7, J. B. Schulz8, U. Grasshoff1, adult skeletal muscle. This is largely due to the homologous recombination events in ES cell I. Bauer4, B. Landwehrmeyer9, A. Bauer10, X. lack of appropriate virus attachment receptors clones was done by PCR and Southern blotting. J. Li11, and St. von Hörsten3 on the myofiber surface. Two independent recombinant ES cell clones Univ. Departm. of 1Med. Genet. Tübingen, Recent studies in transgenic mice revealed that were used to generate chimeric mice and germ 2Neurol. Bonn, 3Funct. and Appl. Anatomy upregulation of CAR (Coxsackie and Aden line transmission was achieved. By PCR analy Hannover, 4Med. Genet. Rostock, 5Cardiol. ovirus Receptor) improves gene transfer efficien

sis on tail DNA, F1 females were found to be Abstracts GfH ÖGH SGMG Tagungsband Berlin, 6Neurobiol., Magdeburg, Germany, cy by approximately 10fold. Conversely, the heterozygous for the Arhgef6 mutation whereas 7Pharmacol. Tartu, Estonia, 8Neurol. vector load that needed to be administered to all F1 males carried only the wildtype allele. Tübingen, 9Neurol. Ulm, 10Res. Center achieve sufficient gene transfer could be low Heterozygous F1 females were mated to C57bl/6 Jülich, Germany and 11Hum. Genet., Emory ered significantly. Reduced viral vector loads males. Among the offspring, males and females Univ. Atlanta, USA may help to control virally mediated toxicity and were almost equally represented. Males of the Huntington’s disease is a neurodegenerative immunogenicity. To date, there are no drugs or F2 generation carrying the targeted Arhgef6 CAG trinucleotide repeat disorder in humans. We methods known to increase CAR expression in gene showed no gross morphological abnormal report a rat model transgenic for Huntington’s skeletal muscle that would be easily applicable ities. By RTPCR, we confirmed the absence of disease, which carries a truncated Huntington in humans. However, alternative strategies such exon 1 and 2 in the Arhgef6 transcript indicating gene fragment including 51 CAG repeats under as vector retargeting are currently being investi that the full length Arhgef6 cDNA is not present control of the native rat Huntington promoter. gated that may allow for an increase in binding in these mice. The Arhgef6 deficient male mice These rats exhibit an adult onset neurological of adenoviral vectors to skeletal muscle. There will be tested for the presence of behavioural phenotype with reduced anxiety, cognitive im fore, vector retargeting may be achieved by di and cognitive deficits as well as possible struc pairments, and slowly progressive motor dys rected genetic alteration of adenoviral capsid tural abnormalities in the brain. function as well as typical histopathological al proteins. terations in the brain. As in Huntington’s disease Inducible mice model of Spinocerebellar patients, in vivo imaging demonstrates striatal Genetic modifications of hematopoietic Ataxia Type 3 shrinkage in magnetic resonance images and a stem cells as treatment strategy for Boy, Jana (1); Schmidt, Th. (1); Holzmann, C. reduced brain glucose metabolism in highreso disorders of the blood and immune system (2); Ibrahim, S. (3); Grasshoff, U. (1); Schmitt, I. lution fluordeoxyglucose positron emission to Hanenberg, Helmut(1), Schindler, D.(2), (4); Zimmermann, F. (5), Prusiner, S. (6) and mography studies. This is the first transgenic rat Rethwilm, A.(3), Leurs, C.(1) Riess, O. (1) model of a neurodegenerative disorder that ex (1) Department of Pediatric Hematology and (1) Department of Medical Genetics, presses an intermediate number of CAG repeats. Oncology, Heinrich Heine University, University of Tübingen, (2) Departments of This model allows longitudinal in vivo imaging Duesseldorf, Germany; (2) Institute of Medical Genetics and (3) studies and is therefore ideally suited for the Human Genetics, University of Wuerzburg, Immunology, University of Rostock, (4) evaluation of novel therapeutic approaches in Germany; (3) Institute of Virology, University Department of Neurology, University of Huntington’s disease. of Dresden, Germany Bonn, 5) ZMBH, University of Heidelberg, (6) Ex vivo genetic modification of autologous Department of Neurology, University of hematopoietic stem cells (HSCs) and their sub California, San Francisco sequent transplantation is a promising therapeu Spinocerebellar Ataxia Type 3 (SCA3) or Macha S9 tic strategy for a variety of inherited hematopoi doJosephDisease (MJD) is an autosomal dom etic and metabolic disorders. Retroviral vectors inantly inherited neurodegenerative disorder have been the most widely used vectors for caused by the expansion of a CAG stretch in the Gene Therapy of Haemophilia gene transfer because the vector genome inte MJD1 gene encoding a polyglutamine repeat in Rainer Schwaab grates into the target cell chromosomes result the respective ataxin3protein. In order to study Institut für Experimentelle Hämatologie und ing in stable expression of the transgene(s). In the course of the disease we generated an in Transfusionsmedizin; Universitätsklinikum the past years, several improvements for the ducible transgenic mouse model using the „Tet Bonn; SigmundFreudStr. 25; 53105 Bonn transduction of HSC with type C oncovirus OffSystem“ developed by Dr. Bujard (Heidel Hemophilia A and B are Xlinked bleeding disor based vectors were implemented in clinical pro berg). This system is based on two constructs: ders caused by mutations within the factor VIII tocols leading to successful treatment of two The promoter construct controls the expression and factor IXgene, respectively. Although both types of inherited immunodeficiencies by stem of the so called tTA (Tetracycline transactivator) disorders can be excellent treated by substitu cell gene therapy. In addition, there are other gene product. The binding of this protein to a tion of factor VIII and factor IXconcentrate, con hematopoietic disorders such as Fanconi ane Tetracycline responsive element (TRE) in the re siderable efforts are undertaken to develop a mia where in vivo and in vitro survival advan sponder construct induces the transcription of gene therapy for haemophilia in order to improve tages of corrected cells suggest that these dis the gene of interest. The expression can be patients’ life quality and also to reduce high eases will be amendable to stem cell gene ther blocked by the addition of Tetracycline which al costs of therapy. The principle of gene therapy apy. However, the major problem with currently losterically inhibits the tTA protein. is the introduction of an intact copy of the fac used recombinant replicationincompetent on For the ataxin3responder mouse lines two dif tor VIII factor/factor IX gene in somatic cells coretroviruses are that efficient transduction with ferent full length constructs containing 15 re compensating the defect cell gene. For this pur these vectors requires cytokine prestimulation of peats (control lines) and an expanded repeat pose vectors systems based on e.g. retroviral, the target cells for breakdown of the nuclear with 77 glutamines (disease model) were used. adenoviral and adenoassociated virus are used. membrane during cell cycle and extended in vit So far three founders for the control line and six Encouraged by the results of many animal ex ro manipulation periods with multiple infection founders for the disease model were generated periments comprising preliminarily mice and ca cycles. Since these protocols interferes with the

medgen 14 (2002) 5 engraftment capacity of HSCs, the initial two genes. These studies demonstrate that it is now ic region. Using a complementation assay we successes in longterm genetic correction of the possible, contingent on appropriate funding, to show that Dsh and Shh/ are allelic. The het defective lymphoid lineage within the determine the expression pattern of all genes of erozygous mutant exhibits foreshortened digits hematopoietic system were only possible due to the mouse genome. caused by missing/fused second phalanges of a strong selective advantage for the corrected all digits and missing interphalangeal joints. Us progency of few transduced HSCs. An alterna ing in situ hybridization for Dsh/+ embryos of tive solution to this obstacle is the development stages 10.5 to 14.5 we show that Shh expres Sox9deficient mouse models for mild and of gene transfer systems that efficiently trans sion is reduced in limbs of Dsh/+ mice at stage severe forms of campomelic dysplasia duce quiescent cells. Lentivirusbased vectors E11.5 compared to wt. However Ptc, Gli1, 2,and Kist, Ralf (1,2,3); Dohrmann, U. (1); Imai, K. (2); allow efficient transduction of nondividing cells 3 Bmp2, 4, 7 and Fgf8 expression were not al Schrewe, H. (4); Balling, R. (2); Scherer, G. (1) including hematopoietic stem cells. However, the tered at this stage. At E12.5 expression of Ihh, (1)Institute of Human Genetics and toxicity associated with the lentiviral protease re Ptc, Gli1, 2, 3 and Noggin and Gdf5 were sub Anthropology, University of Freiburg, quires the use of complex inducible systems for sequently altered or reduced, reflecting the al Freiburg, Germany; (2)Institute of production of replication defective vectors in terations in chondrogenesis and joint develop Mammalian Genetics, GSF, Neuherberg, stable packaging cell lines. These difficulties so ment. Our data indicate that the underlying de Germany; (3)Current address: The Insitute far prevented the clinical exploration of lentiviral fect is expected to be a regulatory mutation of Human Genetics, Newcastle upon Tyne, vectors in humans, e severe nature of diseases leading to disruption of a cis or transenhanc UK; (4)Max Planck Institute of associated with lentiviruses. Finally, the use of ing element affecting Shh expression. Dsh/+ Immunobiology, Freiburg, Germany recombinant vectors based on wildtype viruses provides a model to analyze human Brachy Campomelic dysplasia (CD) a semilethal human that are absent in humans and are not accociat dactyly A1. skeletal malformation syndrome with XY sex re ed with any disease in their natural animal hosts versal, is caused by heterozygous mutations in or in accidentially infected humans would add an the SRYrelated transcription factor gene SOX9. additional safety level for human somatic gene The SOX9 gene is expressed during chondroge therapy approaches. These criteria are fulfilled nesis and male gonadogenesis and acts as a S11 by foamyviruses (FV), a family of complex retro key regulator of cartilage differentiation and

GfH ÖGH SGMG Tagungsband Abstracts GfH ÖGH SGMG Tagungsband viruses whose members are widely spread testis development. SOX9 is also expressed in a among mammalians and are apathogeneic in all Genetic testing and insurance no interest variety of other embryonic tissues, suggesting hosts. Findings on foamyvirusbased gene in see through patients further functions during organogenesis. Indeed, transfer into hematopoietic stem cells in surro Dr. Achim Regenauer CD patients show malformations in nonskeletal gate assays for human hematopoietic stem cells Muenchener Rückversicherungs organs like brain, heart and kidneys. will be discussed. Gesellschaft, Koeniginstr. 107, 80802 To provide tools for the analysis of Sox9 func Muenchen, Germany tions in skeletal development and the formation Contrary to the spirited debate that is currently of other organs, we have generated a condition being conducted about genetic testing in insur S10 al Sox9 mutant allele in mice using the Cre/loxP ance, these tests to date play virtually no role in system. Crossing of Sox9 +/flox mice with Cre risk assessment in life and private health insur deleter mice results in Sox9 +/ mice that die Visualizing Genomewide Gene Expression: ance. Nevertheless, insurers are taking the pop shortly after birth and that recapitulate defects From Highthroughput Tools (GenePaint) to ulation’s concerns seriously and have therefore observed in CD patients such as respiratory fail the Internet (GenePaint.org) decided to ease the pressure that is being exert ure, malformed scapulae and bending of long Yaylaoglu, MB, Visel, A, Ahdidan, J, Carson, ed by the general public on legislators in con bones. J+, Chiu, W+, Thaller, C+ and Eichele, G nection with this issue by drawing up a declara We have also established two hypomorphic MaxPlanck Institute of Experimental tion of commitment, which regulates the use of Sox9neo lines that, in the heterozygous state, Endocrinology, Dept. of Molecular predictive genetic tests. show a 30% reduction of wildtype Sox9 tran Embryology, Hannover, Germany, + Baylor This extensive declaration of commitment pres script resulting from aberrant splicing into the College of Medicine, Dept. of Biochemistry ents an opportunity to discuss the issue at neo cassette in intron 1 of the gene. These mice and Molecular Biology, Houston, Texas, USA length in society and to include in this discus have a milder phenotype with little or no bowing The human and mouse genomes are now se sion the trend and results of future medical de of the long bones, reminiscent of the acampom quenced to an extent that had not been expect velopments in the field of genetic engineering. lic form of CD; most mice develop kypholordo ed until the middle of this decade. As a result of The declaration of commitment issued by the sis as seen in some surviving CD tranlocation this remarkable progress, the need to decipher German Insurance Association and the Associ patients. In the homozygous state, normal Sox9 gene function has become even more pressing. ation of Private Insurers essentially comprises transcript is reduced by 70% and the severity of To advance understanding of gene function it is the following points: the phenotype is correspondingly increased, necessary to study gene expression at tissue 1. Predictive genetic testing shall not be a pre with marked dwarfism, drastic shortening of the and cellular levels. We have deveoped a unique requisite to the conclusion of an insurance long bones, and perinatal lethality, resembling and powerful tool (GenePaint) to study and doc policy. the most severe forms of CD. ument gene expression on tissue sections. 2. Prospective clients shall not be required to Specifically, we have submit to the insurer the results of voluntary constructed instrumentation for highthrough The Mouse Mutant „Short Digits“ (Dsh) as a predictive genetic tests, except when very put, automated in situ hybridization on tissue Model for the Role of Sonic Hedgehog high sums insured are concerned (in excess sections that yield data with cellular resolution, during Limb Development of Euro 250,000 in life insurance and annual assembled an automated highthroughput slide Schwabe, Georg (1,2), Niedermaier, M. (1), payments above Euro 30,000 in occupational scanning microscope that digitalizes tissue Fees, S. (3), Jaeschke, B. (3), Mundlos, S. (1,2) disability, general disability and longterm sections and their gene expression patterns, (1) Max Planck Institute for Molecular care annuity insurance). developed programs for automated annotation Genetics, Berlin, (2) Institute for Medical 3. In addition, special data protection regulations and quantitative analysis of digitized gene ex Genetics, Charité, Berlin, (3) Children’s shall apply. Premium rebates based on genet pression patterns, Hospital University of Mainz, Germany ic tests shall not be allowed. Where policies established a database (GenePaint.org) that Sonic hedgehog (Shh) plays a central role in ver involve high sums insured, only physicians makes accessible annotated gene expression tebrate patterning and is a key signal during limb may take the findings of genetic tests into patterns. development. The mouse mutant „short digits“ consideration. (Dsh) is a semidominant, lethal in the homozy The declaration of commitment will be valid for We will discuss the individual components of gote mutant with a limb reduction phenotype in an initial period of five years. All life and private GenePaint hardware and chemistry, compare its the heterozygote. The homozygote is character health insurers in Germany have signed the dec performance with conventional lowthroughput ized by multiple internal and skeletal malforma laration in the meantime. procedures, discuss our techniques for annotat tions and strongly resembles the Shh k.o. The medium and longterm future of molecular ing gene expression patterns and discuss the mouse. We have mapped Dsh to a 16 centimor medicine cannot be predicted at present, as the salient aspects of the Genepaint.org database. gan (cM) region comprising the Shh gene. North extensive findings of the Human Genome Pro We shall illustrate the power of the integrated ern Blot analysis revealed that Shh expression is ject have yet to be implemented. One thing al system by describing the results of expression absent in the homozygote and reduced in the ready appears to be certain, however: it is un analysis of regulatory genes and of all known heterozygote. However, no mutation was detect likely that a general genetic test providing clues mouse orthologues of human chromosome 21 ed within the Shh coding or surrounding genom as to a person’s life expectancy will exist.

6 medgen 14 (2002) Frequently, the uneasiness about providing in decades before. Phaenotypical realisation of ge Characterization of possible signal surers with access to the results of genetic tests netic disposition has to be recognized during transduction pathways leading to cardiac that have already been conducted springs from every examination as well as all that is told structural changes in renal failure the fear that insurers might also be able to draw about family anamnesis. Biochemical analysis of Christian Maercker (1), Christiane Rutenberg conclusions as to a person’s identity. Every hu genproducts is helpful to identify hidden risks (1), Eberhard Ritz (2, Gerhard Mall (3), Kerstin man being has a distinctive genotype that that will find their manifestation far in the future, Amann (4) makes him or her unique. Today, the judicial sys for example deficiency of Alpha1Antitrypsine. (1) Resource Center for Genome Research, tems of several countries are already taking ad But: The employee is not yet ill and he will not Heidelberg, (2) Dept. of Internal Medicine, vantage of this knowledge (e.g. DNA fingerprint get ill immediately if he would work for example University of Heidelberg, (3) Dept. of ing in the USA), which raises the question as a fire fighter. But doing his job with heavy Pathology, Darmstadt, (4) Dept. of whether this information might also be of inter breathprotection will double his risk to get a Pathology, University of Erlangen est to insurance companies. However, insurers lung emphysema. We have to give him a knowl Death from cardiac causes is the most common are sure not to use or evaluate this data because edge of that and a warning, of course or not? fatality in uremic patients. In particular, left ven it is completely irrelevant to a policy. Insurance And what about recent methods of genome tricular hypertrophy (LVH) is frequent. The car companies will not look into the genotype of a analysis? They will give us far more answers to diac alterations develop very early in renal insuf new client, let alone that of an existing one, just our questions and we will be able to protect ficiency. However, the pathomechanisms in because they are curious or want to be on the people against much more health risks in work volved are currently not fully understood. Thus, safe side. Not only would this overstrain their ca life than today. Do we want that? Do they want additional information about the expression lev pacity but it would also not make any sense. Af that? This will be subject to many and long dis el of the genes in the affected tissue could be ter all, the actual purpose of insurance is to cov cussions. Staff manager will ask us if they can extremely helpful in identifying the pathogenesis er unforeseeable and uncertain risks. Neverthe employ a certain man without risk of severe ill of these lesions. Here, SpragueDawley rats less, when a person applies for insurance, the ness in 20 years. They know that we could know were subjected to subtotal nephrectomy (SNX) insurer has to request information on all of the about that, if we would. Should we, would we or sham operation (SHAM), serving as a model factors relevant to the risk to be insured, includ answer? system for a gene expression profiling analysis. ing diseases, provided of course that these fac The animals were followed for 2 and 12 weeks,

tors are known to the applicant. This socalled The Association of German Occupational Health respectively. Blood pressure was monitored at Abstracts GfH ÖGH SGMG Tagungsband disclosure requirement that is imposed on the Physicians has developed a distinct position to regular intervals and the experiment was termi applicant is regulated in Art. 16 of the German these questions, that is presented here. nated by retrograde perfusion fixation with ice Insurance Contract Act. If the applicant fails to cold NaCl. Poly(A)+ RNA was isolated from the disclose any information, even in part, for in hearts of SNX and SHAM rats, 33P labeled, and stance by holding back relevant data or because hybridized with RZPD Rat Unigene1 cDNA ny of a legal restriction, this is likely to lead to anti lon arrays containing about 27.000 gene and S12 selection, with adverse consequences for the in EST sequences (Bento Soares clone collection, sured community as a whole. Univ. of Iowa). Phosphorimaging of the radioac A genetic test that produces conspicuous find Molecular mechanisms of blood vessel tive signals and extensive interpretation of the ings is practically always equated in public dis growth in health and disease data with array analysis software and database cussions with a potential declinature of life and J. Schoch, R. Mailhammer, M. Hautmann, E. tools revealed substantial changes in gene ex health insurance. There are even fears that a so Knapik & A. Hatzopoulos pression in SNX compared to SHAM animals. In cial group will be formed that is no longer GSFNational Research Center for terestingly, some extracellular matrix genes, mo deemed insurable by life and health insurers be Environment and Health, Institute of Clinical tor protein genes genes as well as growth and cause ist genetic test results are positive. What Molecular Biology and Tumor Genetics, differentiation markers were upregulated in SNX is often overlooked, however, is that, even now, Marchioninistr. 25, D81377, Munich, rats. Activation of some of these genes could be without the use of genetic testing in risk assess Germany involved in expansion of the nonvascular inter ment, not all applicants are deemed insurable. Endothelial cells maintain oxygen and nutrient stitial tissue, reduced capillary supply and in The concern that the currently low declinature supply, manage waste removal, play a critical creased wall thickness of intramyocardial arter rates (less than 2% of new clients) might rise in role in hemostasis, and control cell trafficking ies in uraemic animals. Initiated by the activation future as a result of genetic testing is under across the vessel wall. Blood vessel growth is of the renin angiotensin system (RAS), at least standable but given the rapid progress made in essential for normal development, wound heal two pathways seem to be involved in ECM acti medicine and in view of developments in the ing and plays a crucial role in many diseases. vation, one going directly via Gproteins, second 20th century, the insurance industry is in fact ex We have developed an in vitro system of en messengers, MAP kinase, proteoglycans, cad pecting the opposite to occur. dothelial progenitor cells to address the molec herins and catenins (short term signaling), the ular basis of endothelial cell differentiation and other via Gproteins, second messengers, mo activation. We have found a large number of tor proteins, actins, focal adhesions, and inte Actual and future discrimination genes expressed in endothelial cells that take grin (long term signaling). Some of the gene ex Christiane Lohkamp part in angiogenesis, thrombosis or inflamma pression profiling results, s and integrin, could The increasing knowledge in genetics and fur tion. We have identified Raf/Mek/Erk as a criti be confirmed by in situ hybridization and im ther discoveries of genetic dispositions for dis cal signaling knot that connects these genetic munohistochemistry. These results are an impor eases and handicaps open new perspectives of pathways and thus regulates the dynamic bal tant prerequisite for the ongoing work with a possible discrimination in the fields of reproduc ance of endothelial functions. We currently focus newly designed subarray for the investigation tion, social context, employers interests, juris on a genome wide functional analysis of the of special cell types, time points, and treat diction, creation of legal precedence, legislation identified genes associated with this critical sig ments, respectively, but also for more special and insurance matters, not only in refusing life nal transduction pathway, in order to validate in ized experiments on candidate genes involved and disability insurance but also in refusing vivo individual components in animal models of in cardiac structural changes in chronic renal medical treatment. The necessity of awareness myocardial infarction, stroke and tumorigenesis. failure. by all groups involved like selfsupport groups, The aim of this work is to identify a „new gener counsellors, and public awareness in general ation“ of drug targets that can be used to design can help to define actual and future menacing therapeutic approaches to safeguard and specif discrimination. Selfsupport organisations play ically adjust the diverse functions of endothelial the keyrole in detecting and monitoring discrim cells. ination and in defending and protecting individ Improved DNAMicroarrays for SNP uals against it. Detection and Transcriptional Profiling based on Nanoparticle Monolayers GENETIC DIAGNOSIS IN OCCUPATIONAL N. Hauser, K. Zeller, A. Weber, B. Steitz, T. MEDICINE DO WE WANT IT? Schiestel, H. Brunner, G. Tovar, S. Rupp Dietrich, M. Fraunhofer Institute for Interfacial Verband Deutscher Betriebs und Engineering and Biotechnology & University Werksärzte e.V., Wilhelmshavener of Stuttgart, Nobelstr. 12, D70569 Stuttgart, Heerstraße 79, 26125 Oldenburg (Germany) Germany Genetic diagnosis is quite usual in modern oc We have developed a new type of microarray cupational medicine and it has been many that can be used for SNP detection based on

medgen 14 (2002) 7 APEX or LDR methodology, as well as for tran 2. Workshops truncating mutations versus others) or localiza scriptional profiling. The array is based on tion of the mutation within the JAG1 gene at nanobeads that are fixed to a solid support, e.g. 20p12. A novel significant correlation was found, microscopic slides, using the LayerbyLayer W1 however, for kidney anomalies and cerebrovas (LbL) method. With this technique, polyelec cular disease. Kidney anomalies have been re trolytes are deposited as nanoscopic layers, ComputerAssisted Recognition of ported only for patients with truncating muta forming the binding support for the stable immo Syndromic Faces tions (n=24) with 25% of them being affected, bilization of nanoparticles. The surface of the Dagmar Wieczorek1, Hartmut S. Loos2, e.g. by hypoplastic kidney or even Wilms tumor. particles is designed for improved DNAimmobi Christoph von der Malsburg2, Bernhard On the other hand, about 22% of all patients lization. This approach allows for tailormade Horsthemke1 with missense or splice site mutations (n=18) de surface chemistry and enhances the active sur 1Institut für Humangenetik, veloped cerebrovascular diseases (e.g. Moy face area by its specific morphology. Universitätsklinikum Essen, 2Institut für amoya disease), features having not been re Initial experiments showed that signal intensities Neuroinformatik, RuhrUniversität Bochum ported, however, for patients with truncating mu for detection of SNP´s using the APEX method Syndromes are often defined by a specific facial tations. Our data provide also evidence that is significantly improved if compared to conven appearance. Experienced geneticists usually atypical symptoms like deafness or mental retar tional polylysin slides. The probe can be fixed on make a diagnosis through immediate pattern dation tend to be associated with respective the nanobeadmodified surface by spotting the recognition. We have investigated how well a „private mutation“ within a given pedigree. respective oligonucleotides using contact or computer can do this. In view of the fact that pa noncontact spotters or by fixing the DNA sep tients with a syndrome look more similar than Subtelomeric screening in clinical practice arately to the nanobeads and spotting of the unrelated individuals do, we have chosen a pat Spranger, Stephanie (1); Mehl, Burkhard (2); functionalized nanobeads, thus enabling a more tern recognition program that was developed to Wagner, Michael (3); Hagendorff, Angela (4); flexible design of the arrays. These nanobeads identify a person by matching his face to faces Huppertz, HansIko (4); Lauber, Peter (2); Koch, are currently also tested to design protein ar stored in a database. It is not based on anthro Hartmut (5); Kazmierczak, Bernd (1) rays. pometric measurements, but uses digital photo (1) Praxis für Humangenetik, Bremen, (2) graphs of 256x256 pixels which are subjected to Kinderzentrum, Bremen; (3) Kinderzentrum, GfH ÖGH SGMG Tagungsband Abstracts GfH ÖGH SGMG Tagungsband a Gabor Wavelet Transformation to create a vec Oldenburg, (4) Prof. Hess Kinderklinik, ZKH Autosomal dominant inherited obesity by tor with 40 complex coefficients (jet) for every St. JürgenStr., Bremen, (5) Kinderklinik, mutations in the Melanocortin4 Receptor pixel. For the purpose of this study, each face Vechta Gene was automatically labeled with 48 nodes. The Mental retardation is one of the main reasons for Raab, Maximilian(1), Grassl, W.(1), Fontenla jets attached to each node of a face were then parents to present their child in a Department of Horro, F.(1), Agricola, E.(1), Geller, F.(2), compared to the jets of all nodes at the same Clinical Genetics. In up to 10 % submicroscop Schäfer, H.(2),Remschmidt, H.(1), Hinney, A.(1), fiducial points of every face in the data base ic subtelomeric chromosome defects have been Hebebrand, J.(1) (bunch graph). Classification was based on a found as a cause of mental retardation. Recent (1) Clinical Research Group of Child and majority decision of all analysed nodes of a face ly, fluorescence in situ hybridisation (FISH) has Adolescent Psychiatry, University of (jet voting). Analysis of 32 innerfacial nodes from become available to investigate these subtelom Marburg, (2) Institute of Medical Biometry 55 frontal view photographs of patients with mu eric rearrangements. Because of the technical und Epidemiology, University of Marburg copolysaccharidosis type III (n=6), Cornelia de complexities and cost of screening, a clinical Introduction: Mutations in the melanocortin4 Lange (n=12), fragile X (n=12), PraderWilli preselection including an indication checklist rezeptor gene (MC4R) are associated with early (n=12), and WilliamsBeuren syndrome (n=13) with a minimal cutoff score of three points was onset obesity. 41 muations are known so far, revealed correct syndrome recognition in 42/55 suggested (de Vries et al., 2001). however the effects on receptor function of sev (76%) of the patients. In another four patients We present data of 120 FISH assays we per eral mutations remain undetermined. Excluding (7%), a correct and an incorrect diagnosis formed during the last six months. We found two polymorphisms, almost all mutations were scored equally well. Our results indicate that it seven positive cases (6%); whose average found in severely obese indexpatients. Family may be feasible to develop a program which checklist score was 3.7 (range 27). Two cases studies revealed rare cases of mutations in lean may aid the clinical diagnosis of genetic syn only had two points. The family history was pos subjects.Methods: We screended 887 severely dromes and the study of genetic variation of fa itive only in one case. Besides mental retarda obese children for mutations in MC4R by single cial patterns. tion facial dysmorphies, especially apparent hy strand conformation polymorphism analysis. The pertelorism, were always present. Mental retar following phenotypical data were collected: Genotypephenotype correlation in Alagille dation ranged from severe (2/7) to mild (5/7). Weight history, eating behavior, body mass in syndrome resulting from JAG1 mutations Postnatal growth retardation was more frequent dex (BMI), waist hip ratio, bodycomposition by Kujat, Annegret; Röpke, A ; Giannakudis, J ; than prenatal growth retardation. Chromosomal bioelectrical impedance analysis, serum leptine Hansmann, I abnormalities comprised single deletions (4/7) levels.Results: We identified 17 different muta Institut für Humangenetik und Medizinische and complex rearrangements (3/7). tions in 27 heterocygous mutation carriers. Two Biologie, MLU HalleWittenberg; Halle Conclusion: Since the suggested clinical prese subjects werde compound heterocygous, no ho (Saale), FRG lection may lead to underdetection of subtelom mocygous mutation carriers were detected. In 9 The Alagille syndrome (AGS, MIM 118450) is an eric rearrangements, the FISH assay should be patients we found a nonsense mutation at codon autosomal dominant disorder with reduced pen performed in patients with mental retardation in 35 (Y35stop), in two cases we discovered a etrance and significant variable expression. It is combination with facial dysmorphies. A positive frameshift mutation (4bp deletion at codon characterized by 5 major symptoms: chronic result in subtelomeric screening in the early 211).We recontacted the families and investigat cholestasis, typical face and variable heart, stage of the diagnostic procedure could avoid ed 22 pedigrees, including 189 subjects (82 mu sceletal and ocular anomalies. Minor symptoms cost intensive further diagnostics. tation carriers, 107 wildtype subjects). 71 of the and various other features have been reported 82 mutation carriers had a current BMI exceed as well. So far, no explanation can be given for Molecular analysis of TP53: Identification of ing the 90th percentile (39 exceeded the 99th the variable expression and the occurence of familial and sporadic cases of german origin percentile), whereas only 57 of the 107 wildtype symptoms not considered to belong to the spec Ines Bendig, Nicole Mohr, Bernhard H.F. Weber subjects exceeded the 90th percentile. 61% of trum of AGS. In approx. 70% of classical AGS Institute of Human Genetics, Biocenter, the female haploinsufficiency mutation carriers patients diseasecausing mutations within the University of Würzburg, Germany had a BMI above the 99th percentile. We could JAG1 gene are being found. For analysing a LiFraumeni syndrome (LFS) is an autosomal not find additional common abnormalities in genotypephenotype correlation we identified 51 dominant disorder with high occurrence of ma phenotype in the mutation carrier s apart from different JAG1 mutations in our sample of non lignant neoplasms caused by mutations in the obesity.Conclusion: Our family studies demon related patients with a mutation and correlate TP53 tumorsuppressor gene. Inherited germline strate a major gene effect of MC4R in early on the mutation spectrum with clinical features. TP53 mutations predispose to an increased risk set obesity. Based on a standardized questionnaire we eval for cancer at an early age and for developing uated the occurence of the 5 major symptoms multiple primary cancers such as sarcomas, as well as additional features like kidney dis brain tumors and breast cancer. The 12 exons of ease, vascular anomalies, pancreatic insufficien TP53 encode a phosphoprotein which is impli cy, deafness, short stature etc. For the 5 major cated in cell cycle arrest, DNA repair and apop symptoms we did not observe any correlation tosis. The majority of mutations are clustered between phenotype and genotype, i.e. type (e.g. between exon 5 and 8 preferentially affecting

8 medgen 14 (2002) codons 248, 273, and 245. More than 80% of ptosis, microcephaly, partial agenesis of corpus binucleated lymphocytes and lymphoblast cells detected alterations are missense mutations, callosum, short stature, and mental retardation. (LCLs). The study included 1. heterozygote whereas deletions (~ 9%) and nonsense muta The clinical presentation of our patients expands NBS1 women who show a significantly reduced tions (~ 6%) are less common. De novo germline the spectrum of symptoms seen in the mildest rate of spontaneous abortions which might be TP53 mutations are rarely reported in the litera expression of HPE. Our results stress the impor due to a particularly low rate of meiotic nondis ture. tance of 7q36 microdeletion studies in patients junction and 2. a woman (A.B.) from a consan We have analyzed three index cases with con with even minimal symptoms of HPE. All pa guineous Arab family with three trisomy 21 chil spicuous anamnesis of cancer for germline mu tients with terminal 7q deletions should be dren suggestive for an increased rate of meiotic tations in TP53. In family A, a 12 month old boy screened for symptoms of Currarino syndrome. nondisjunction. No evidence was found for go was affected with rhabdomyosarcoma, while no nadal mosaicism or premature centromere divi other relative developed cancer. In this patient a sion. FISH analysis was performed with pericen nonsense mutation in codon 196 was identified, tric probes for chromosomes 18, 21, 22 and X, ZimmermannLaband syndrome associated while neither his parents nor his sister carry this and 1000 binucleated cells per individual were with a balanced reciprocal translocation change. Paternity was confirmed with several scored. The frequency of nondisjunction for t(3;8) (p21.2;q24.3): delineation of the highly polymorphic DNA markers suggesting a chromosomes 21 and 22 was in the normal breakpoint regions by FISH de novo mutation in the patient. Alternatively, a range in lymphoblast cells of NBS1 heterozy Fuchs, Sigrid (1), Stefanova, M. (2), Atanassov, germ cell mosaic could be present in one of the gotes and homozygotes as compared to normal D. (3), Gal, A. (1), Kutsche, K. (1) parents. In family B, two relatives are affected control subjects. However, in the lymphocytes (1) Institut für Humangenetik, with brain tumors at the ages of 12 and 34 years and the lymphoblast cell lines of the woman with Universitätsklinikum HamburgEppendorf, and three siblings under 30 years with breast the three trisomy 21 children, the frequency of Germany, (2) Department of Medical cancer. Testing of BRCA1 and BRCA2 did not re nondisjunction of chromosome 21, but not of Genetics, Medical University, Plovdiv, veal diseaseassociated alterations. Subse 18, 22 or X, was significantly increased com Bulgaria, (3) Department of Oral Surgery, quently, a novel germline mutation in TP53 was pared to all other individuals studied so far. In Medical University, Plovdiv, Bulgaria detected in codon 105 leading to an amino acid contrast, it was in the normal range in her moth ZimmermannLaband syndrome (ZLS) is a rare change from glycine to cysteine. In family C, al er, grandmother and, surprisingly, also in her disorder characterized by coarse facial ap

though fullfilling the classic criteria of LFS, most three trisomic children who carry the same chro Abstracts GfH ÖGH SGMG Tagungsband pearence including bulbous soft nose, thickened family members developed cancer at a relative mosomes 21. Thus, the high rate of maternal lips, thick and floppy ears, gingival hypertrophy, ly old age (ranging from 12 to 70). We found a meiotic nondisjunction is paralled by an in aplasia or dysplasia of hand and toenails, vari deletion of eleven base pairs encompassing the crease of mitotic nondisjunction, however, con ous skeletal anomalies including hypoplastic splice acceptor sequences in IVS 5. The patho fined to chromosome 21. Experiments are in changes in the terminal phalanges, hyperexten logic consequences of this intronic deletion on progress to study the basis of this new phenom sibility of joints, and, in some cases, he the splicing of the gene is currently being exam enon by treating these cells with various agents patosplenomegaly, hypertrichosis, and mental ined in vitro. Our findings in the three cases rep that affect cell cycle progression and chromoso retardation. The genetic basis of ZLS is unknown resent the first report of germline TP53 muta mal nondisjunction and measuring the rate of whereas autosomal dominant inheritance has tions in patients of German origin. segregation of chromosomes 21 and 22. been suggested. Here we report on an apparent ly balanced chromosomal aberration, 46,XX, Three patients with minimal expression of t(3;8) (p13p21.1;q24.1q24.3), in an affected Partial deletion of the common 1,5 Mb the holoprosencephaly spectrum and mother and daughter. Neither the healthy son critical region in an infant with classical cytogenetic rearrangements involving the nor one of the healthy parents carries the WilliamsBeuren syndrome loss of the Sonic Hedgehog gene at 7q36 translocation. In order to define the cytogenet Winterpacht, Andreas(1), Rauch, A. (1), Endele, Horn, Denise (1), Neitzel, H. (1), Tönnies, H. (1), ic breakpoints more precisely, we performed S. (1), Schröder, B. (2), Steglich, C. (2), Kunze, J (1), Hinkel, G.K. (2), Bartsch, O. (2) FISH experiments with BAC clones widely Lüttgen, S. (2), Heller, R. (2,3) (1) Institute of Human Genetics, Charité, spaced across the entire chromosome bands. (1) Institute of Human Genetics, Erlangen, Humboldt University Berlin, Germany,(2) By using 10 BACs for chromosome 3p13p21.1 Germany, (2) Institute of Human Genetics, Institute of Clinical Genetics, Medical and 6 BAC clones for the region 8q24.1q24.3, Hamburg, Germany; (3) Dept. of Clinical Faculty, Technical University of Dresden, we successfully refined the breakpoints to Genetics, The Churchill Hospital, Oxford, UK Germany 3p21.2 and 8q24.3 and, thereby, narrowed down WilliamsBeuren syndrome (WBS; OMIM Among the heterogeneous causes of holopros both breakpoint regions to approximately 1.5 194050) is a contiguous gene deletion disorder encephaly (HPE), mutations and deletions affect Mb. In conclusion, our data suggest that the ZLS with a variable clinical phenotype that is caused ing the Sonic Hedgehog (SHH) gene at 7q36 gene maps to one of the above given break in most cases by a heterozygous microdeletion have been identified. The phenotypic anomalies points and that the trait follows autosomal dom in 7q11.23. Due to two highly homologous flank of HPE are extremely variable, with symptoms inant inheritance. Further FISH experiments will ing ~300 kb duplicons, the microdeletion is usu ranging from the most severe form, alobar HPE identify BAC clones overlapping both breakpoint ally of similar size in almost all cases and en to microsigns such as single maxillary incisor. regions. Subsequent database searches should compasses a common ~1,5 Mb interval that We report on clinical, cytogenetic, and molecu reveal whether a known or a putative gene is contains at least 17 genes mostly of uncertain lar cytogenetic studies of three patients with disrupted by one or both breakpoints. Finally, pathogenetic relevance. Phenotypegenotype subtle or submicroscopic 7q36 deletions show mutation analysis of any candidate gene(s) iden correlation studies for WBS are hampered by the ing only microsigns of the HPE spectrum. tified in the breakpoint regions in five sporadic uniform size of the microdeletion. Here we report The absence of a single copy of the SHH gene ZLS patients with normal karyotype will be per the case of a 1yearold boy with a full WBS in the first patient with a de novo 7q36.1qter formed. phenotype that is caused by a partial deletion of deletion did not have gross consequences for the common ~1.5 Mb interval. Initial analysis structural brain development except for micro with two sets of commercially available FISH cephaly. Further anomalies of this 37 month old probes (Appligene/Oncor and Vysis) yielded con W2 patient included bilateral ptosis and sacral age flicting results. We therefore carried out deletion nesis. The loss of the homeobox gene HLXB9 mapping with microsatellite markers and an ar which is located telomeric to SHH may cause Analysis of mitotic nondisjunction in ray of targeted FISH probes. We mapped the this bony sacral anomaly as a minimal form of binucleated cells: evidence for a genetic proximal and distal breakpoint of the deletion on Currarino syndrome. disposition two BAC clones. The deletion spans a region The second patient, a 13 year old female with a (1) Ramel, Christian, (2) Al Gazali, L., (3) Krebs, between the elastin gene (ELN) and the distal 7q36 microdeletion, showed microcephaly, bilat A., (4) Seemanova, E., (5) Neitzel, H., (6) duplicon and therefore excludes STX1A and eral iris coloboma, absence of maxillary and Sperling, K. FZD9 from the WBS minimal critical region of mandibular incisors, short stature, mental retar (1, 3, 5, 6) Institute of Human Genetics, deletion. Fine mapping and sequencing of the dation, and normal brain findings. The 7q36 mi Humboldt University Berlin, Germany, (2) breakpoints is in progress. Evidence from two crodeletion in this case was found by FISH Dept. of Pediatrics, United Arab Emirates similar cases in the literature also suggests that analysis and is the unbalanced product of a University, AlAin, UAE, (4) Dept. of Clinical the centromeric portion of the 1,5 Mb interval is t(7q;10q)mat. Genetics, Charles University Prague, Czech not always deleted in patients with full WBS In the third patient, a 13 year old male, the de Republic phenotype, defining a WBS critical region esti novo 7q36 microdeletion was detected by FISH. The rate of mitotic nondisjuncton was studied mated to be less than 1 Mb. The conclusions He had with a single maxillary central incisor, on interphase nuclei of cytochalasin Bblocked from our work concern diagnosis and molecular

medgen 14 (2002) 9 etiology of WBS: 1. Quantitative differences in GC. Recently, we have shown that this isochore Assignment of normal diploid metaphases signal intensity after FISH analysis with commer boundary marks exactly the transition from low to the leukemic cell population by cial probes for a WBS microdeletion should be to high recombination frequency. To study the chromosome banding analysis of followed up carefully if partial deletions are not correlation between sequence composition and immunomagnetically selected cells in acute to be missed. 2. Even in patients with full WBS replication timing in detail a 300 kb DNAstretch lymphoblastic leukemia phenotype the underlying molecular defect may containing the isochore transition was analysed Fritz, B (1); Bachmann, I. (1); Busch, S. (1), be more variable than previously thought. by performing interphase FISH for a number of Schwartz, S. (2); Thiel, E. (2), Hoelzer, D. (3, 4); cosmid clones from the respective region. The Rieder, H. (1) technique applied results in single fluorescent (1) Institut für Klinische Genetik, Universität Characterization of human small marker signals, if the target sequence is still unreplicat Marburg; (2) Klinik für Hämatologie und chromosomes by centromerespecific ed and in doublets in the case of replicated se Onkologie, Universitätsklinikum Benjamin multicolorFISH (cenMFISH) and high quences. The results clearly demonstrate that Franklin, Berlin; (3) Klinik für Hämatologie resolution multicolor banding (MCB) GCrich sequences are replicated early during und Onkologie, Universitätsklinikum Liehr, Thomas (1), Nietzel, A. (1), Oliver Bonet, the SPhase, whereas neighbouring GCpoor se Frankfurt, Frankfurt; Germany. (4) für die M. (1), Starke, H. (1), Heller, A. (1), Weise, A. quences are replicated late. The boundary be GMALLStudiengruppe (1), von Eggeling, F. (1), Claussen, U. (1) tween early and late replicating sequences is In about 40% of the patients with acute lym Institute of Human Genetics and sharp and precisely coincides with the boundary phoblastic leukemia (ALL) no clonal chromo Anthropology, Jena, Germany between the GCrich and the GCpoor iso some aberrations are detectable using chromo The origin of markerchromosomes is nearly im chores. Moreover, fiberFISH experiments with a some banding analysis (CBA). With molecular possible to establish by banding cytogenetics cosmid contig spanning the isochore boundary cytogenetic methods masqued and submicro alone, while FISH methods are highly suited for revealed, apart from linear structures, a number scopic aberrations may be identified. However, that purpose. 24colorFISH approaches using of Yshaped structures, directly demonstrating after chromosome preparation an assignment of wholechromosomepaintingprobes can only be that the replication fork is arrested for a longer normaldiploid cells to the leukemic cell popula used successfully for the determination of the time period within the isochore transition region. tion is not possible anymore. For a well directed markers chromosomal origin if it is larger than use of molecular cytogenetic methods for the 17p. Thus, smaller supernumerary markerchro

GfH ÖGH SGMG Tagungsband Abstracts GfH ÖGH SGMG Tagungsband detection of cryptic chromosome rearrange mosomes (SMC) found in clinical cytogenetics in Experience with subtelomeric screening in ments, the information would be usefull, if the 0.010.05% often cannot be characterized by over 150 prospective patients with normal diploid metaphases do represent the those FISH analyses. For rapid characterization developmental delay leukemic cell population. Therefore, immuno of small SMC recently a technique was estab A Rauch (1), U Trautmann (1), M Zenker (1), M. magnetic cell selection using antiCD19 and lished which allows a onestep identification of Beese (2), D. Wenzel (2), W. Kreß (3), U. antiCD7 coated immunomagnetic particels pri all human centromeric regions, excluding Hüffmeier (1), A. Reis (1) or to CBA was chosen to try to assign the re #13/21, by their individual coloring (centromere (1) Institute of Human Genetics, Friedrich spective cytogenetic finding to the leukemic cell specific multicolorFISH = cenMFISH [Nietzel et Alexander University of Erlangen population. To adjust the separation procedure al., 2001, Hum Genet 109:199204]). To clarify if Nuremberg (2) Pediatric Hospital, FAU the purity of the target cells in the positively se euchromatic material is present on the SMC the ErlangenNuremberg, (3) Institute of Human lected cell fraction was determined in titration recently developed multicolor banding (MCB) Genetics, University of Würzburg experiments with CD7+ and CD19+ cell lines us technique has been shown to be suited. The re Since the group of Jonathan Flint and others ing different beads concentrations and beads: liability of cenMFISH for the characterization of have shown that subtle subtelomeric rearrange target cell ratios. The proportion of the target small SMC has been proven in 50 prenatal, post ments may account for more than 7 % of pa cells was evaluated by interphase FISH and natal or tumorytogenetic cases. Thus, cenM tients with unexplained moderate to severe men chromosome banding analysis. A purity of the FISH fills a gap in multicolor karyotyping. As tal retardation, we started a prospective investi separation procedure of up to 100% was clinical findings associated with SMC may also gation to evaluate the incidence and significance achieved. The procedure was tested on 20 bone be related to uniparental disomy (UPD), UPD an of subtelomeric aberrations in children with un marrow/and or blood samples including 11 B alyzes have been done in about 10 cases with explained developmental delay. The study in cell precursor, 5 preT/T, and 1 mature BALL SMC. UPD has been detected in 2 of the test cluded all consecutive patients referred for de as well as 3 AML. Chromosomes of the positive ed cases (partial UPD4 and UPD12). The char velopmental delay, in whom after clinical evalu selected fraction, of the supernatant, and of acterization of small SMC can help to explain ation and karyotyping by GTG banding at app. control cultures were prepared. Metaphase cells clinical symptoms of a patient, or, if there are 500 bands resolution the reason for develop were present in 6 CD7+ and in 7 CD19+ select none, provide important information about ge mental delay was still unknown. So far we have ed fractions. In no case metaphase cells were netically inactive regions in the human genome. analysed 139 patients seen personally by the found in CD19+ as well as in CD7+ fractions. Moreover, in case of identification and charac first author in our genetic clinic and 13 patients Clonal chromosome aberrations were present in terization a small SMC by GTG banding and referred from other centres. Subtelomeric all CD19+ fractions. Most excitingly, only normal cenMFISH and the exclusion of partial trisomy screening was performed with two colour FISH diploid metaphases were found in 3/6 CD7+ by MCB, a test for UPD of the two normal sister for each chromosome separately. Initially we fractions. Thus, the normal diploid cells in these chromosomes by molecular genetic methods is used a set of BAC/PAC probes described by CD7+ fractions most likely represented leukemic reasonable [e.g. Liehr et al., 2001, Clin Genet Knight et al. (AJHG 2000). With this set we found cells. In all cases with metaphases detectable in 60:8385]. Supported by the Wilhelm Sander a 2q deletion in 5 of the first 65 patients (7.7%). positively selected cell fractions, flow cytomet Stiftung (99.105.1) and the EU (ICA2CT2000 Further studies showed that all of these were ric analysis revealed expression of the respec 10012). simple polymorphisms. After delineation of the tive antigen on the lehromosome analysis after size of the deletion polymorphism against the immunomagnetic cell selection (MCIC) may be deletion size in true „2q“ patients we subse An isochore transition in the NF1 gene used for combined immunologic and cytogenet quently used BAC RP1111B2 for the 2q sub region coincides precisely with a switch in ic analyses in ALL. This method allows the aim telomeric region. In the highly selected 13 cas replication timing. directed use of additional cytogenetic tech es from other centers we found 3 significant Schmegner Claudia, Hameister H., Gläser B., niques in the analyses of normal diploid cells aberrations (23 %), while in our consecutive pa Assum G. with respect to the detection of cryptic re tients only in 2 % kryptic aberrations were de Abteilung Humangenetik, Universität Ulm, arrangements. In addition, it facilitates the inter tected. All in all we found 6 pathological aberra D89081 Ulm, Germamy pretation of normal diploid chromosome findings tions (3,9 %): two deletions, three unbalanced The mammalian genome is a mosaic of long with respect to their representativeness of the translocations, and one de novo balanced stretches of alternate DNA sequence composi leukemic cell. translocation. Further investigation of genotype tion. This socalled isochore structure is corre Supported by the Deutsche José Carreras phenotype correlations revealed a 3 Mbp 5q lated, on one hand, with the visible banding pat LeukämieStiftung, grant R24 deletion adjacent to the Sotos syndrome dele tern of chromosomes Gbands being com tion region and allowed the delineation of a nov posed of GCpoor and Rbands of GCrich iso el, clinically recognizable subtelomeric 5q mi chores and, on the other hand, with a number crodeletion syndrome. Breakpoint mapping of of functional features like gene density, recom the balanced translocation is in progress. bination frequency and replication timing. With in the human NF1 gene region a 350 kb stretch with an average of 39% GC is immediately fol lowed by a stretch of several 100 kb with 51%

10 medgen 14 (2002) W3 regulatory sequences within the 3´UTRs. Per drome and the factors that initiate their expres forming electrophoretic mobility shift assays we sion in tissues such as the aorta will represent identified five protein regions (NF1PBR15), two an important step toward the development of Large Scale Genotyping in Haemophilia A of them (PBR1 and 2) carry AUrich elements new therapeutic strategies for this disorder. An update on 1350 patients (AREs). On the one hand AREs were shown to We and others have shown that fibrillin1 muta Oldenburg J(1,2,3,), Schröder J(2), Graw J(4), be cisacting elements which target specific mR tions can increase the susceptibility of fibrillin Pavlova A(1), Brackmann HH(3), Schramm NAs for rapid degradation. A family of four pro fragments to proteolysis. We speculate that W(5), Seifried E(1), MüllerReibleC(2), teins, the neuronspecific proteins HuD, HuC and these proteolytic degradation products may pos Schwaab R(3) HelN1 and the ubiquitously expressed HuR sess signaling properties, and are able to alter (1)Institute of Transfusion Medicine, were shown to be involved in mRNA destabilisa the expression of genes by cells such as medi Frankfurt, (2)Institute of Human Genetics, tion via binding to AREs. On the other hand the al smooth muscle cells of the aorta involved in Würzburg, (3)Institute of Transfusion binding of HuD to specific AUrich sequences in turnover of extracellular matrix components, Medicine, Bonn, (4)GSF, Institute of 3´UTRs can also prolong the mRNA lifetime. Us such as matrix metalloproteinases. To test this Mammalian Genetics, Neuherberg, ing electrophoretic mobility shift assays we were hypothesis, we have generated a series of re (5)Medical Clinic, Munich able to demonstrate the specific interaction be combinant fibrillin fragments. Fibroblast and oth The phenotype of haemophilia A is due to the tween HuD (a kind gift from Dr. Furneaux) and er cell cultures have been incubated with the deficiency or absence of coagulation factor VIII NF1PBR1 but not with PBR5, one of the other fragments, following which protease activity was (FVIII) caused by a great number of heteroge RNA fragments with proteinbinding capacity, monitored by zymography and the mRNA ex nous mutations within the large FVIII gene. The not containing an ARE. To determine the physi pression of selected proteases by quantitative gene consists of 26 exons comprising a cDNA of ological role of the observed PBR1/HuD interac RTPCR (TaqMan). Initial results have suggest 7.1 kb that encodes a mature FVIII protein of tion we performed northernblot experiments af ed several alterations in activity following incu 2332 amino. Our consortium is supported by the ter cotransfection of HeLa cells with HuD cDNA bation. Current results will be presented. German Human Genome Project and aims to and reportergeneconstructs carrying various determine the genotype in a substantial propor parts of the NF1 3´UTR. tion of the about 6000 German haemophiliacs as The abnormal spermatozoon head shape a basis for further studies on phenotype/clinical (azh) mutation in the mouse is caused by a course genotype correlations. Mutated human neural cell adhesion deletion in the Hook1 gene Abstracts GfH ÖGH SGMG Tagungsband So far more than 1350 patients from 900 fami molecule L1 affects neurite outgrowth in a Juergen Neesen, Peter Burfeind, Wolfgang lies with severe and nonsevere haemophilia A cell culturebased model of L1 disease Engel and Irene MendozaLujambio have been analysed. 35.5% of the patients Michelson, Piret (1), Hartwig, C. (1), Schachner, Institute of Human Genetics, University of showed an intron 22 inversion, 0.9% an intron 1 M. (2), Gal, A. (1), Veske, A. (1), Finckh, U. (1) Goettingen, 37073 Goettingen, Germany inversion, 47.2% a point mutation (38.0% mis (1) Institute of Human Genetics and (2) In mice carrying the autosomal recessive muta sense, 9.1% nonsense), 10.2% a small inser Zentrum für Molekulare Neurobiologie, tion „abnormal spermatozoon head shape“ (azh) tion/deletion, 3% a large deletion and 2.4% a University Hospital HamburgEppendorf, all spermatozoa display a highly abnormal head splice site mutation. Patients in whom no muta University of Hamburg, Germany morphology that differs drastically from the com tion could be initially identified by the screening Mutations in L1CAM, the gene encoding the pact and hookshaped head of the normal methods were diagnosed by the GSF (Munich; multifunctional neuronal adhesion molecule L1, murine sperm. Moreover, the azh mutation caus Uen, Graw et al., for details see separate ab are associated with neurodevelopmental disor es tail abnormalities often resulting in coiled stract). The causative mutations could not be ders including Xlinked hydrocephalus and men sperm tails or in the decapitation of the sperm identified in about 2% of the patients, even by tal retardation. It is largely unknown how these head from the flagellum. We have isolated and sequencing the complete cDNA, which may be mutations result in neurodevelopmental distur characterized the murine Hook1 cDNA and ana due to mutations that are located outside the F8 bances and whether the effects of mutations on lyzed the corresponding genomic structure. Fur cDNA (either allelic or nonallelic). Remarkably, neurodevelopment can be modeled in vitro. We thermore, the Hook1 gene was mapped to the about one third of all point mutations have not stably expressed fulllength human wild type L1 same region on chromosome 4, where the azh been described before, thus underlining the and known pathogenic missense mutations locus was previously linked. The Hook1 gene is great variety of the mutations in the F8 gene. I179S, R184W, Y194C, and C264Y in NIH3T3 predominantly expressed in haploid male germ Three mutation hot spots could be verified: i) the cells and used them as substrate for primary cells and immunohistochemical analysis re prevalent intron 22 inversion, ii) CpG sites and murine cerebellar neurons. Two of the four mu vealed that Hook1 is responsible for the linkage iii) two series of adenines in exon 14 that ac tations affected posttranslational processing and of the microtulular manchette and the flagellum counted for 25% of all small deletions /inser surface localization of L1, whereas all four mu to cellular structures. Here, we report that the tions. A total of 56% of point mutations resulted tations showed reduced stimulation of neurite azh mutation is due to a deletion of exons 10 from either C>T or G>A nucleotide exchanges. outgrowth. Measurement of neurite outgrowth of and 11 in the murine Hook1 gene leading to a Consequently, arginine that is encoded by CpG neurons grown on transfected substrate cells nonfunctional protein. Our results indicate that containing codons was affected in 37.4% of all may be a suitable model for studying neurode loss of Hook1 function results in ectopic posi point mutations. Missense mutations were high velopmental disturbances associated with vari tioning of microtubular structures within the ly underrepresented in exon 14 that forms the ous mutations affecting extracellular domains of spermatid and causes the azh phenotype. middle third of the FVIII gene. L1. Supported by DFG, SFB444 C3. Therefore, the human HOOK1 gene could serve In conclusion our high throughput mutation as a candidate gene for male infertility due to screening approach allowed successful charac teratozoospermia or decapitation defects. Expression of Proteases in Cell Culture terisation of the underlying genotype in 900 Influenced by Incubation with Fibrillin1 haemophilic A families representing 1350 pa Fragments The tumor suppressor gene Patched and tients. This database will represents an impor Patrick Booms (1), Pregla, R (2), Pletschacher, rhabdomyosarcomagenesis tant tool for future large scale genotype phe A (1), and Robinson, PN (1) Roland Kappler, Julia CalzadaWack, Udo notype correlation studies in haemophilia A. (1) Institute of Medical Genetics, Charité Schnitzbauer, Heidi Hahn University Hospital, Berlin, and (2) Institute of Human Genetics, University of Specific binding of HuD to the NF1 3´UTR; Deutsches Herzzentrum Berlin, Germany Goettingen, 37073 Goettingen, Federal analysis of RNA stabilizing effects The Marfan syndrome (MFS) is an autosomal Republic of Germany Striebel, Andrea, Bader, T., Haeusler, J., dominant heritable disorder of connective tissue, Inherited mutations of Patched (PTCH) in the Assum, G. caused by mutations in the gene for fibrillin1. nevoid basal cell carcinoma syndrome (NBCCS) Department of Human Genetics, University Fibrillin1 is a large (320kDa) multidomain glyco lead to several developmental defects and con of Ulm, Germany protein that is a main component of a class of 10 tribute to tumor formation in a variety of tissues. The 8.5 kb coding sequence of the neurofibro to 12nm extracellular microfibrils. The microfib PTCH mutations have been also identified in matosis type1 (NF1) gene is followed by an un rils are thought to be important for elastogene sporadic tumors associated with NBCCS includ usually long 3´untranslated region (3.5 kb) sis, elasticity, and homeostasis of elastic fibres. ing basal cell carcinoma and edulloblastoma. which is highly conserved between mouse and The pathogenesis of MFS is still unclear, but Mice heterozygous for Ptch recapitulate the typ human. 3´UTRs have been found to influence the some evidence has suggested that a progressive ical developmental symptoms of NBCCS and fate of mRNAs in several ways, including intra loss of microfibrils may be an initiating factor in develop rhabdomyosarcoma (RMS) and medul cellular localisation, control of its stability and the development of aortic dilatation and dissec loblastoma. These mice are therefore a useful regulation of translation efficiency. These func tion. Identification of the proteases likely to be model to study Ptch function in disease and de tions are often mediated by proteins, binding to involved in the pathogenesis of the Marfan syn velopment.

medgen 14 (2002) 11 PTCH is widely assumed to be a tumor suppres to separate active inflammation from chronic fi quence, 14 transcripts without exonintron sor gene. Tumor suppressor genes normally ex brosis. boundary and 40 EST clusters without signifi ert a negative control on cell growth and the par cant homology to known sequences. For all 295 adigm is that inactivation of both alleles is re EST clusters, reverse Northern bloting was per FUNCTIONAL ANALYSIS OF MAMMALIAN quired for tumor formation. formed to identify abundantly expressed genes GENES BY A LARGE SCALE GENE TRAP To determine if deletion of both copies of Ptch is in the RPE and to prioritize subsequent analy APPROACH IN MOUSE EMBRYONIC STEM a prerequisite in RMS development in heterozy ses. Representative clones were spotted onto a CELLS gous Ptch+/ mice, we examined whether the nylon membrane and hybridized with probes Floss, T.*, Ruiz, P.+, Vauti, F.#, Füchtbauer, E. normal Ptch allele was deleted or inactivated by from cDNA of driver (heart and liver) and tester M.§,Van Sloun, P.$, Arnold, H.H.#, Lehrach, a mutation in these tumors. Furthermore, we (RPE) used in the cDNA library construction. H.+, von Melchner, H.$, Hansen, J.* and tried to elucidate if both alleles contributed to Subsequently, 107 normalized EST clusters were Wurst,W.* the high Ptch mRNA expression previously subjected to Northern blot hybridizations. These * GSF Nat. Research Center, Inst. of found in RMS of these mice. Our data show that analyses resulted in the identification of 6 RPE Developmental Genetics, Neuherberg; + the wildtype Ptch allele is retained in tumor tis specific, 3 retinaspecific, 8 RPE/retinaspecif MaxPlanckInstitute (MPI) for Molecular sue. Interestingly, the high levels of Ptch mRNA ic, and 8 tissue restricted transcripts, while 33 Genetics, Berlin; §MPI of Immunobiology, in these tumors result from overexpression of the EST clusters were ubiquitously expressed, and Freiburg; # Inst. of Biochemistry a. mutant Ptch transcript. Our results suggest that evaluation was not possible for another 49 EST Biotechnology the wildtype Ptch allele might be selectively si clusters. The genes showing specificity or tissue We have established a research centre in the lenced in RMS tissue or, alternatively, that hap restriction are currently being analysed further. framework of the German HUGO project to loinsufficiency of Ptch is sufficient to promote This include the characterization of their full perform a large scale functional analysis of RMS formation in mice. length and the generation of genederived sin mammalian genes taking advantage of the gle nucleotide polymorphism (SNP) maps to en gene trap technology. The gene trap able gene association studies in AMD patients technology is based on insertional and controls. mutagenesis in ES cells and provides an W4 important tool for the identification and GfH ÖGH SGMG Tagungsband Abstracts GfH ÖGH SGMG Tagungsband characterisation of the function of mammalian genes in vitro and subsequently in vivo. The Protein detection and purification using GENE EXPRESSION FINGERPINTS IN mutated genes are identified using RACEPCR motifspecfic monoclonal antibodies HUMAN TUBULOINTERSTITIAL allowing to establish an archive of mutated Blazek E., Kremmer E., Meisterernst M. INFLAMMATION AND FIBROSIS genes generated in ES cells. At present, we Institute of Molecular Immunology, Dept. for Anna Henger*, Matthias Kretzler*, Peter have established 16 000 mutant ES cell clones. Gene Expression, GSF, Marchioninistr. 25, Doran+, Stephen Madden+, Elisabeth F. For 7332 clones, the site of gene trap vector D81377 München, Germany Gröne#, Detlef Schlöndorff*, Peter J. Nelson* integration has been determined using RACE Here we present a novel approach to character and HermannJosef Gröne# PCR and direct sequencing of these RACE ize and purify subsets of functionally or struc + Genomics and Bioinformatics Research products resulting in 4530 high quality turally relevant proteins, that greatly reduces Unit, University College of Dublin; # sequences. From these 4530 sequences, 2463 complexity of cell extracts or other protein sam Department of Cellular and Molecular (54 %) show homology to nonredundant ples for further analysis of signaldependent Pathology, German Cancer Center, genes, 794 (18 %) to expressed sequence changes. Heidelberg; *Medical Poliklinik, LMU, tag’s (EST’s) and 1273 (28 %) show no First, we have optimized the existing methods Munich homology to sequences present in the NCB1 for cell fractionation by both physical and bio Gene expression profiles of human kidneys will GenBank database. For about 75 of those lines chemical criteria enabling us to primarily focus provide insight into the molecular basis of dis we have established germline transmission and on nuclear and chromatinassociated proteins. ease, and may also detect novel diagnostic pa the we are presently studying the mutant In order to identify proteins of interest we have rameters. To identify molecular markers of renal phenotypes. Some represent animal models for developed a number of antibodies that bind pro inflammation and scarring, gene expression human disease e.g. nephrodic syndrom. The teins according to their catalytic domains or screening was performed on human kidney sam individual data for each clone, i.e. sequence, structural features. Functional domains of pro ples. expression pattern and eventually mutant teins frequently contain a set of wellconserved Total RNA was isolated from 9 hydronephrotic phenotype, are stored in a public database amino acids accompanied by residues, which and 4 control kidneys (tumor free, unaffected re which is accessible to the scientific community are more variable among proteins of the same gions of tumor nephrectomies). Random primed (http://genetrap.gsf.de). These data will type. Monoclonal antibodies raised against a radioactively labeled cDNA probes were hy contribute to unravel gene function genome specimen of a functional domain may or may not bridized to Panorama Human Cytokine Gene Ar wide. discriminate between these aminoacid substi rays with more than 350 genes representing cy tutions. By selecting for antibody clones of low tokines, chemokines, their receptors, cellcell er specificity a considerable number of proteins contact proteins and matrix turnover molecules. Identification and characterization of genes containing the domain can be detected in im In parallel, a pathologist unaware of the gene ex from the retinal pigment epithelium (RPE) as munoblots of cellular extracts. So far we are pression data determined the degree of tubulo candidates for age related macular able to demonstrate this principle for the ATP interstitial inflammation, fibrosis and tubular at degeneration (AMD) binding domains of Kinases and Helicases and rophy assigning a score value to each kidney. Faisal M. Rahman(1), Faisal M. Moula(1), other motifs of lower complexity. The respective Differential gene expression profiles were Andrea Gehrig(1), Claudia Keilhauer(2), monoclonal antibodies we have developed bind processed by self organizing maps and cluster Bernhard H. F. Weber(1) several proteins in a Tcell nuclear extract. Some ing analysis. The dendrogram reflecting the re (1)Institute of Human Genetics, Biocenter, of these proteins are apparently modified or dif lationship of molecular similarity among the Am Hubland, D97074 Würzburg; ferentially expressed upon Tcell activation, samples generated three distinct groups: (I) 5 (2)University Eye Clinic, Würzburg which we use as a model system for signal in kidneys with low inflammation and high fibrosis AMD is the leading cause of visual impairment in duction. Our approach could be very helpful to scores, (II) 4 kidneys with high inflammation and the elderly and a major cause of blindness in the purify novel enzymatic activities and also to low fibrosis score and (III) the control group. Dif developed countries. To date, the molecular identify diseaserelated proteins by their differ ferential regulation of 7 out of 10 cDNAs could mechanisms of the disease are not well under ential regulation. be confirmed by realtime RTPCR. stood although in recent years a primary involve 28 genes yielding stringent separation between ment of the retinal pigment epithelium (RPE) has inflammation and fibrosis were selected as po become evident. tential predictive diagnostic markers, incl. i.e. The aim of our study is to systematically identi CXCR2, CXCR5, IL11, HGF, TIMP2 and CD40. fy and characterize novel genes active in the Results indicate the feasibility of gene expres RPE and to clarify their role in the pathogenesis sion based disease categorization in a defined of AMD. Towards this goal we have generated set of kidney samples. Analysis in a comprehen 1002 ESTs from an inhouse constructed RPE sive collection of tubulointerstitial samples from suppression subtracted cDNA library. This has the European renal cDNA bank will help to de identified 188 known human genes, one partial termine the diagnostic utility of this marker set transcript with exonintron boundary, 52 predict ed proteins in the human genome draft se

12 medgen 14 (2002) W5 potonia and severe mental retardation, with the for mice, and other currently available transgenic 3p syndrome. By FISH and LOH analysis, we mice models are not viable for long periods of demonstrated that this gene resides on chromo time. Goal of this study is to develop a cell cul Molecular characterisation of two novel some 3p25 and is deleted in 3p patients. ture system in which the SMN gene expression genes disrupted in a mentally retarded MEGAP mRNA is predominantly and highly ex can be varied using catalytic nucleic acids and patient with a 46,X,t(X;8)(p11.2;p22.3) pressed in fetal and adult brain and specifically double stranded RNA interference. Catalytic nu balanced translocation in the neurons of the hippocampus and cortex, cleic acids are short sequences of RNA (ri Hagens, Olivier (1); Barbi, G. (2); Menzel, C. (1); structures known to play a pivotal role in higher bozymes) or DNA (DNAzymes) capable of se Chelly, J. (3); Fryns, J.P. (3); Moraine, C. (3); cognitive function, learning and memory. We de quence specific cleavage of a target mRNA, thus Hamel, B. (3); Tommerup, N. (4); Ropers, H.H. scribe several MEGAP transcript isoforms and downregulating gene expression. RNA interfer (1); Kalscheuer, V.M. (1) show that MEGAPa and b represent functional ence is the phenomenon by which double (1) Max Planck Institute for Molecular GAP proteins by an in vitro GAPassay. We pro stranded RNA complementary to a specific Genetics, Berlin, Germany; (2) Department pose that haploinsufficiency of MEGAP leads to mRNA sequence can strongly inhibit gene ex of Human Genetics, University of Ulm, abnormal development of neuronal structures pression. We designed three ribozymes and Germany; (3) European XLMR consortium, that are important for normal cognitive function. three DNAzymes targeted against the murine Berlin, Paris, Leuven, Tours, Nijmegen; (4) Smn RNA sequence. All ribozymes and DNA Wilhelm Johannsen Centre for Functional zymes effectively cleaved the full length Smn Genome Research, Copenhagen, Denmark Identification of a novel polymorphism that RNA in a sequence specific manner, while inac Mild mental retardation (MR) is likely to be a affects an exonic splicing enhancer in exon tive versions of the molecules had no effect. multifactorial condition, whereas severe cogni 3 of the SMN1 gene and causes mild SMA in Cleavage of target RNA was observed at mag tive disorders are believed to predominantly in heterozygous individuals nesium concentrations as low as 2 mM, which volve single gene defects. It has been realised Verena, Schwarzer, Hofmann, Y. Sun, Y. corresponds to the intracellular Magnesium con for a long time that mental retardation has a Helmken, C. Raschke, H. Wirth, B. centration of mammalian cells. Cleavage in higher occurrence in males than in females. The Institute of Human Genetics, University creased in a time and concentration dependent observation of a skewed sex ratio implies an in Bonn manner. Preliminary RNA interference results volvement of genes located on the Xchromo Proximal spinal muscular atrophy (SMA) is a

show a reduction in SMN expression in NIH 3T3 Abstracts GfH ÖGH SGMG Tagungsband some in at least part of the cases with MR. neuromuscular disorder caused by homozygous cells when treated with double stranded RNA By combining the arguments of ‘single gene’ in deletions/mutations within the survival motor constructs. These results indicate that catalytic volvement and Xlinked MR (XLMR), investiga neuron gene 1 (SMN1). All SMA patients retain nucleic acids and double stranded RNA have the tion of mentally retarded patients with balanced at least one of the SMN2 copy gene which pro potential to effectively cleave Smn target RNA in X;autosome rearrangements is a promising start duces mainly alternatively spliced transcripts the cell and could be valuable tools to study ing point in the search for genes important in lacking exon 7. This is the result of a silent nu SMN function in different cell types. brain function and/or development. cleotide exchange in exon 7 that disrupts an ex Here we report the study of a balanced onic splicing enhancer. 46,X,t(X;8)(p11.2;p22.3) translocation in a men Here we describe an additional silent nucleotide Establishing reliable diagnosis of PROMM/ tally retarded girl suffering from epileptic exchange (G to A transition) in exon 3 responsi MD2 experience with the first 44 cases seizures. ble for alternative splicing of exon 3. Minigenes Jakubiczka, Sibylle (1), Vielhaber, S. (2), Kreß, Using FISH we recovered a breakpoint spanning containing genomic DNA from exon 2 to exon 4 W. (3), Reuner, U. (4), Kunath, B. (4), Wieacker, BAC clone on the Xchromosome. Southern with either the G or the A variant were construct P. (1) blotting with a Xspecific probe narrowed down ed and the splicing pattern analysed by RTPCR (1) Institute of Human Genetics, (2) Clinic of the breakpoint region sufficiently to enable chro after transient transfections in HEK293 cells. Al Neurology, University of Magdeburg, mosome walking from X to 8. On the Xchromo though both minigenes show alternative splicing Germany; (3) Institute of Human Genetics, some the translocation disrupts a novel gene of exon 3, the amount of variant A is ~10x high University of Würzburg, Germany; (4) Clinic with a predicted PDZ and Leuzipper domain er. The GtoA transition lies within a GArich ex of Neurology, University of Dresden, and on chromosome 8 a novel gene with a pre onic splicing enhancer that is Htra2ß1 depend Germany dicted Fbx domain is disrupted. Genomic organ ent as shown by RNAprotein interaction stud Myotonic dystrophy is a multisystem disorder isation of these two genes was established and ies. and the most common form of muscular dystro expression analysis was performed. For both We identified 5 type III SMA patients who are phy in adults. In contrast to myotonic dystrophy genes splice variants were recovered. heterozygous for SMN1 and carry the A variant type 1, proximal myotonic myopathy/myotonic Mutation screening of the Xchromosomal gene as the only mutation within the SMN1 coding re dystrophy type 2 (PROMM/DM2) is charac in the >300 unrelated patients of the European gion. All show abundant alternatively spliced terised by a proximal rather than a distal muscle XLMR consortium is in progress. This screen is SMN transcripts lacking exon 3 as compared weakness with sparing of the facial muscles, of utmost importance as it is a powerful ap with control individuals with the G variant. Also muscle pain, and absence of congenital cases proach to show the involvement of this gene in the SMN protein level is significantly reduced. and mental deterioration. The underlying gene XLMR. Exon 3 encodes the essential Tudor domain of defect could recently be identified as an expan the SMN protein that interacts with the Sm pro sion of a (TG)n(TCTG)n(CCTG)n repeat tract in teins, important components of the splicing ma intron 1 of the zinc finger protein 9 (ZNF9) gene The novel RhoGTPase MEGAP has a chinery. The amount of fulllength SMN1 protein localised on chromosome 3q21. The patholog putative role in severe mental retardation is significantly diminished through the A variant ic expansions show a great variation from 75 to Volker Endris (1), Birgit Wogatzky (1), Uwe in exon 3 and leads even in the presence of one more than 11,000 CCTG repeats. Intraindividual Leimer (2), Dusan Bartsch (2), Malgorzata SMN1 copy to a mild SMA phenotype. variation can be explained by extensive somat Zatyka (3), Farida Latif (3), Eamonn R. Maher The exon 3 polymorphism will be analysed and ic mosaicism and instability. Therefore, expand (3), Gholamali Tariverdian (1), Stefan Kirsch (1), discussed as an additional SMAmodifier, as sig ed alleles can often only be visualised as faint Dieter Karch (4) & Gudrun A. Rappold (1) nificant genetic implications are expected. smears that could easily escape detection in (1) Institut für Humangenetik, Southern blot hybridisation experiments. In our Universitätsklinikum Heidelberg, INF 328, laboratory, we achieve the most unequivocal re 69120 Heidelberg, (2) ZI für Seelische Towards a cell culture model of Spinal sults by a combination of pulsed field gel elec Gesundheit, J5, 68159 Mannheim; (3) Muscular Atrophy trophoresis and quantitative Southern blot analy Section of Medical and Molecular Genetics, Trülzsch, Barbara (1), Davies, K.(2), Wood, sis. Additional information can be obtained in University of Birmingham, B15 2TT, UK MJA(3) the majority of cases by using the linkage dise In the last few years, several genes involved in Dept of Human Anatomy and Genetics, quilibrium reported. Four flanking markers were Xspecific mental retardation have been identi South Parks Road, Oxford, OX13QX tested in DNA samples of all our PROMM/DM2 fied using genetic analysis. Although it is likely, Spinal Muscular Atrophy (SMA) is an autosomal patients (n = 44) and in samples from 100 unaf that additional genes responsible for idiopathic recessive disease caused by loss of functional fected controls. For the most informative mark mental retardation are also localized on the au survival of motor neuron gene (SMN) product. er CL3N59, we observed an allele that was not tosomes, cloning and characterisation of such SMA ultimately leads to progressive loss of mo seen on any of the control chromosomes in genes have been elusive so far. Here we report tor neuron function and muscular atrophy. Al about 85 % of our patients, confirming a strong the isolation of a novel gene, MEGAP, which is though the SMN gene is ubiquitously expressed, linkage disequilibrium. Interestingly, in one of our disrupted and functionally inactivated by a the cause for selective motor neuron loss is un patients with a severe manifestation no normal translocation breakpoint in a patient, who shares known. Study of the disease has been hampered fragment could be seen in Southern blot hybridi some characteristic clinical features, such as hy by the fact that the condition is embryonal lethal

medgen 14 (2002) 13 sation experiments. Furthermore, amplification coding RNAs (ncRNAs). We isolated the very which may account for their more circumscribed using a repeat specific marker failed repeatedly large BCMS gene, which consists of at least 50 growth and better prognosis. in this patient. Since he is heterozygous for all exons and spans more than 560kbp. In addition, four flanking markers a large deletion could be it is excessively spliced, giving rise to more than ruled out. Further Investigations are in progress. 20 different splice variants. While tissuespecif Microarray Technology; Applications in ic expression of RNA variants was observed, Cancer Research there was no evidence for the expression of a Jörg D. Hoheisel variant specific for BCLL. Identification and functional Division of Functional Genome Analysis, DNA sequence analyses have failed to detect characterization of an R621C mutation in Deutsches Krebsforschungszentrum small mutations in one of these genes, suggest the synphilin1 gene in Parkinson’s disease (DKFZ), Im Neuenheimer Feld 280, D69120 ing a different pathomechanism, most likely hap F.P. Marx (1), C. Holzmann (2), K.M. Strauss (1), Heidelberg, Germany loinsufficiency. We therefore tested BCLL sam L. Li (2), M. Cookson (3), M.R. Farrer (3), J.B. The Division of Functional Genome Analysis at ples for epigenetic aberrations by measuring ex Schulz (1), O. Riess (4), R. Krüger (1) the DKFZ is involved in the development of tech pression of the majority of genes localized be (1) Neurodegeneration Laboratory, nologies for the analysis of large genomic areas tween the Retinoblastoma gene (RB1) and the Department of Neurology, University of or the entire genome with respect to encoded chromosomal marker D13S25 located 3MBp dis Tübingen, Tübingen, Germany; (2) functions and their regulation. Functional as tal to the RB1 gene, including the critical region. Department of Medical Genetics, University pects are being studied by means of DNA, pro With two exceptions, all genes were significant of Rostock, Rostock, Germany; (3) tein and peptidemicroarrays. Beside other ap ly downregulated in BCLLpatients, with RFP2 Neurogenetics Laboratory, Mayo Clinic, plications, analyses on diseaserelevant poly showing the most pronounced loss of expres Jacksonville, USA; (4) Department of morphisms, comparative studies on transcrip sion. To test whether this loss of gene expres Medical Genetics, University of Tübingen tion levels, the actual protein expression and sion is associated with methylation of CpGis The search for interacting proteins of alpha epigenetic variation are performed. Also, sys lands in the respective promotorregions, we synuclein, a protein involved in the pathogene tems are being developed toward early diagno performed methylationsensitive quantitative sis of Parkinson’s disease (PD), yielded in the sis, prognosis and evaluation of the success of PCRanalyses and bisulfite sequencing on DNA identification of synphilin1, which is also a com disease treatment.

GfH ÖGH SGMG Tagungsband Abstracts GfH ÖGH SGMG Tagungsband from BCLL samples. No difference in the ponent of Lewy bodies. (http://www.dkfzheidelberg.de/funct_genome) methylation patterns could be detected in any In addition synphilin1 was identified as a sub CpGisland of the minimally deleted region. strate for parkin, an ubiquitin ligase, mutated in Downregulation of genes within chromosomal Caveolin1 gene silencing by methylation at an autosomal recessive form of PD. Since syn band 13q14.3 in BCLL is in line with the con four CpG sites in the promoter region philin1 forms a direct link between these pro cept of haploinsufficiency, but this tumor specif Haeusler Juergen, Reutter P., Bochum S. teins involved in PD we analyzed the synphilin ic phenomenon is not associated with DNA Haeussler J., Vogel W 1 gene in 416 familial and sporadic PD patients methylation. Dept. Human Genetics, University of Ulm, of German origin for mutations. In two patients Germany we identified an amino acid substitution from The CAV1 promoter contains seven CpG dinu arginine to cysteine in position 621 (R621C) of Genomic and Expression Profiling of cleotides four of which are methylated in two hu the peptide sequence, that was not found in 350 Pleomorphic Xanthoastrocytomas man breast cancer cell lines. Both cell lines do healthy German controls. Because synphilin1 Weber, Ruthild G. (1,2), Ehrler, M. (1,2), not express CAV1 mRNA, thereby suggesting produces cytoplasmic inclusions in transfected Kaulich, K. (3), Blaschke, B. (3), Jauch, A. (1), CAV1 gene regulation by CpG methylation. cells, we tested the inclusionforming capacity Weber, S. (1), Wiestler, O.D. (4), Reifenberger, Here we report on CAV1 expression in both, hu of wild type (R621) and mutant (C621) synphilin G. (3) man prostate cancer and normal prostate cell 1 in dopaminergic SHSY5Y cells. Cells express (1) Dept. of Human Genetics, University of lines. The methylation status of the promoter ing C621 synphilin1 displayed a significantly re Heidelberg; (2) Dept. of Human Genetics, was found to vary at the first four CpG sites and duced number of aggregates compared with University of Magdeburg; (3) Dept. of may correlate with differing CAV1 activity in cells expressing wt synphilin1, when subjected Neuropathology, University of Düsseldorf; these cell lines. CAV1 transcription was com to proteasomal inhibition. In viability assays (4) Dept. of Neuropathology, University of pletely absent from the human prostate cancer C621 synphilin1transfected cells were more Bonn, Germany cell line LNCaP. In these cells, the distal part of susceptible to staurosporininduced cell death Pleomorphic xanthoastrocytomas (PXAs) are as the CAV1 promoter was methylated at the first than cells expressing wt synphilin1. Thus the trocytic neoplasms that mainly affect children CpG site suggesting this CpG to be part of a C621 mutation in the synphilin1 gene may be and young adults. PXAs usually show a circum main regulatory element. Methylationdepend causative for PD due to a deterioration of cell vi scribed growth and favorable prognosis despite ent transcription compatible with the repression ability possibly mediated by the accumulation exhibiting a high degree of cellular pleomor of CAV1 in LNCaP cells was also revealed by not aggregation of toxic intermediates. phism. We report on the first comprehensive reporter gene assays. Demethylation by 5aza analysis of PXAs for aberrations at the chromo 2’deoxycytidine restores CAV1 transcription some, gene and mRNA levels. Comparative ge demonstrating the presence of an intact gene nomic hybridization of 50 PXAs revealed a dis copy in this cell line and confirming a possible tinct pattern of chromosomal imbalances. The W6 role of methylation in gene silencing. EMSAs de hallmark alteration detected in 48% of PXAs was tected a nucleoprotein interacting with the loss on chromosome 9. Less common recurrent methylated CAV1 promoter and this protein is The tumor suppressor mechanism localized losses were on chromosome 17 (10%); 8, 18 and different from MeCP2. In conclusion, silencing of in chromosome band 13q14.3 involves 22 (4% each). Recurrent gains were identified on the Caveolin1 gene in LNCaP cells is associat downregulation of genes in Bcell chronic chromosome X (14%); 7, 9q, 20 (8% each); and ed with CpG methylation in the promoter region, lymphocytic leukemia 19 (4%). Amplifications were found in 2 tumors suggesting this epigenetic event playing a po Daniel Mertens*, Stephan Wolf*, Petra and mapped to 2p23p25, 4p15, 12q13, 12q21, tential role in prostate cancer progression. Schroeter, Hartmut Döhner, Stephan 21q21 and 21q22. Molecular genetic analysis of Stilgenbauer, Peter Lichter selected candidate genes revealed TP53 muta Deutsches Krebsforschungszentrum, INF tions in only 3 of 62 (5%) PXAs analyzed. The Epigenetic inactivation of the RASSF1A 280, 69120 Heidelberg CDKN2A, CDKN2B and p14ARF genes on 9p21 gene in human cancers A critical region distal to the retinoblastoma lo did not show homozygous deletion, mutation, Dammann, Reinhard (1), Schagdarsurengin, U. cus is frequently deleted in a variety of tumor promoter hypermethylation or complete loss of (1), Otto, N. (1), Pfeifer, G.P. (2), HoangVu, C. entities. In Bcell chronic lymphocytic leukemia, mRNA expression. None of the tumors showed (3) more than 50% of patients have lost genomic amplification of the EGFR, CDK4 or MDM2 (1) Institute of Human Genetics, University material in this critical region. Three candidate genes. Expression profiling of 1188 cancer relat of HalleWittenberg, Germany, (2) tumor suppressor genes are localized in the ed genes using cDNA array analysis revealed a Department of Biology, City of Hope Cancer 400kbp large region: Bcell neoplasia associat distinct set of genes that were differentially ex Center, USA, (3) Universitäts und Poliklinik ed gene with multiple splicing (BCMS), BCMS pressed in PXAs versus diffuse astrocytomas für Allgemein, Viszeral und Gefäßchirugie, upstream neighbor (BCMSUN) and Ret finger and/or nonneoplastic brain tissue. Taken to University of HalleWittenberg, Germany protein 2 (RFP2). Whereas the RFP2 gene has an gether, our study indicates that PXA are associ Loss of genetic material from chromosome open reading frame (ORF), the BCMS and BCM ated with genetic and transcriptional aberrations 3p21.3 is one of the most common and earliest SUN genes probably exert their function as non that differ from those in diffuse astrocytomas events in the pathogenesis of lung cancer and

14 medgen 14 (2002) many other solid tumors. The chromosomal area ma has not been described as yet. Thus, our is the last of the seven amino acids which rep 3p21.3 is thought to harbor at least one impor three families may represent a new hereditary tu resent the predicted NLS2. tant tumor suppressor gene, which despite many mor predisposition syndrome. To analyze the functional consequences of these years of investigation, has remained elusive. In Supported by the Deutsche Krebshilfe, grants mutations, we constructed expression plasmids our previous studies, we have identified and 702362Ba2 and 702828Ba3 of wildtype and mutant TRPS1GFP fusion pro cloned a gene from the common homozygous teins. As expected, the wildtype TRPS1GFP deletion area at 3p21.3. The gene, named fusion protein was only detectable in the nucle RASSF1A (Ras Association domain Family 1A), us. Interestingly, the deltaexon6TRPS1GFP has homology to a mammalian Ras effector. The construct was still able to enter the nucleus, W7 RASSF1A gene is epigenetically inactivated in a whereas the R952HTRPS1GFP construct was large percentage of human lung cancers, in par exclusively located in the cytoplasm. ticular small cell carcinomas. A high frequency The LeriWeill and TurnerSyndrome Our data indicate that the predicted NLS1 is not of methylation of RASSF1A is found also in homeobox gene SHOX encodes a celltype a nuclear localization signal, at all, and that the breast cancers, renal cell carcinomas and ovar specific transcriptional activator predicted NLS2 is the only true NLS in TRPS1. ian cancer. Recently, we have analyzed the epi Rüdiger J. Blaschke*& Ercole Rao*, Antonio Furthermore, it shows that the deltaexon6 genetic inactivation of RASSF1A in thyroid car Marchini, Beate Niesler, Michael Burnett, and TRPS1 behaves like a functional nullallele like cinomas. In 9 thyroid cancer cell lines and in Gudrun A. Rappold** alleles with premature truncating mutations. This 71% (27/38) of primary thyroid carcinomas the Institute of Human Genetics, University of is in contrast to the TRPS1 proteins with mis RASSF1A CpG island was hypermethylated. Heidelberg, Im Neuenheimer Feld 328, sense mutations in the GATAtype zincfinger Methylation frequency was higher in the aggres 69120 Heidelberg, Germany which lead to the more severe TRPS type III sive forms of thyroid carcinoma. Furthermore, Functional impairment of the human homeobox phenotype. we have investigated the methylation status of gene SHOX causes short stature and Madelung RASSF1A in pancreas carcinoma. In 64% deformity in LeriWeill syndrome and has recent Imprinting defects in PraderWilli syndrome (29/45) of pancreas carcinoma RASSF1A was si ly been implicated in additional skeletal malfor and Angelman syndrome: a molecular study lenced. Interestingly, RASSF1A methylation was mations frequently observed in Turner syndrome. of 133 patients

detected in 41% (7/18) of pancreatitis samples. To enhance our understanding of the underlying Abstracts GfH ÖGH SGMG Tagungsband Karin Buiting, Christina Lich, Stefanie Gross, Thus, RASSF1A inactivation may play a crucial mechanism of action, we have established a cell Bernhard Horsthemke role in the malignancy of human carcinoma. This culture model consisting of four stably transfect Institute of Human Genetics, Clinic of work was supported by the BMBF (NBL3 FKZ ed cell lines and analysed the functional proper University Essen, Germany 01ZZ0104) and in part by Land SachsenAnhalt. ties of the SHOX protein on a molecular level. The PraderWilli syndrome (PWS) and Angelman Results show that the SHOX encoded protein is syndrome (AS) are neurogenetic disorders which located exclusively within the nucleus of a vari A possibly new hereditary tumor are caused by the loss of function of imprinted ety of cell lines, including U2Os, HEK293, COS7 predisposition syndrome characterized by genes in 15q11q13. The most frequent lesions and NIH3T3 cells. In contrast to this celltype in pancreatic cancer and basaloma are a deletion of the entire region or uniparental dependent nuclear translocation, the transacti SinaFrey,Mercedes (1), Bartsch,D.K. (2), disomy. In a small group of patients, the disease vating potential of the SHOX protein on different Ziegler, A. (3), Hahn,S. A. (4), Przypadlo, is due to aberrant imprinting and gene silencing. luciferase reporter constructs was observed only E.(2,3), Kress, R.(3), Gerdes, B.(2), Rieder, H. Incorrect imprints can occur de novo without in the osteogenic cell line U2Os. Since Ctermi (1) any mutation in the DNA sequence or as the re nally truncated forms of SHOX lead to LeriWeill (1) Inst. f. Klin. Genetik, Marburg; (2) Klinik f. sult of a microdeletion affecting the imprinting syndrome and idiopathic short stature, we have Visz., Thor. und Gefäßchirurgie, Marburg; centre (IC), which controls the whole imprinted compared the activity of wildtype and truncat (3) Inst. f. Med. Biometrie und domain. The IC maps to the SNURFSNRPN lo ed SHOX proteins. Interestingly, Cterminally Epidemiologie, Marburg; (4) Klinik f. Innere cus and appears to consist of two elements. truncated SHOX proteins are inactive with re Med., Bochum; Germany One element is required for the maintenance of gards to target gene activation. These results for Pancreas cancer (PC) is the fifth leading cause the paternal imprint during early embryogenesis. the first time provide an explanation of SHOX re of cancerrelated mortality with a very poor The second element is required for maternal im lated phenotypes on a molecular level and sug prognosis. Its etiology is still largely elusive. The printing in the female germline. gest the existence of qualitative trait loci modu only consistent environmental risk factor is cig Here we describe the molecular analysis in a se lating SHOX activity in a celltype specific man arette smoking. A family history of PC provides ries of 133 patients with an imprinting defect. ner. a 18 and 53fold risk among first degree rela Fifteen (12 %) of these patients were found to tives of PC patients with two and with three or have an IC deletion. Sequence analysis in 62 AS more affected family members, respectively. It is Report of a novel TRPS1 missense mutation nonIC deletion patients and 27 PWS nonIC estimated that 35% of all PC cases are caused and functional analysis of the two predicted deletion patients did not reveal any point muta by a genetic predisposition. In July 1999 the nuclear localization signals of the TRPS1 tion in the critical IC elements. German National Case Collection for Familial PC transcription factor The presence of a faint methylated band in 25% (FaPaCa) was setup to collect families with PC Kaiser, F., Horsthemke, B., Lüdecke, H.J. of AS nonIC deletion patients suggests that (FPC) and with pancreas cancermelanoma syn Institut für Humangenetik, these patients are mosaic for an imprinting de drome (PCMS) for the evaluation of the clinical Universitätsklinikum Essen, Germany fect that occured postzygotically. This is in and genetic characteristics of familial PC. The The TRPS1 gene on human chromosome 8q24.1 agreement with the finding that in AS patients prevalence of PC as well as of other tumors and encodes a nuclear zincfinger transcription fac the imprinting defect occured on the chromo diseases was studied in families with at least 2 tor. It is the only known zincfinger protein with some that was inherited from either the mater first degree relatives with histologically con an unusual combination of nine zincfinger mo nal grandfather or grandmother. In contrast, in firmed PC or with at least two first degree rela tifs of four different types (Momeni et al., 2000). all informative PWS nonIC deletion patients the tives with PC or MM. At total of 32 families has One of them is a GATAtype DNAbinding zinc imprinting defect always occured on the chro been collected so far. A combination of PC and finger. It is flanked by basic amino acids stretch mosome inherited from the paternal grandmoth breast cancer, which may indicate a predispos es predicted as nuclear localization signals er. These data suggest that the parental identity ing BRCA2 germline mutation, was found in 4 (NLS1: LRRRRG and NLS2: RRRTRKR). of the two homologues is or can be maintained (12.5%) families. PC and melanoma, which may In our ongoing attempts to augment the spec through spermatogenesis. point to a CDKN2A germline mutation, was pres trum of TRPS1 mutations, we identified two mu ent in 6 (18.8%) families. Most surprisingly, in tations which affect the predicted NLSs in pa The ICSNURFSNRPN transcript serves as three families an accumulation of PC and basa tients with TRPS type I. One is a mutation of the a host for multiple small nucleolar RNA loma was identified. The median age at diagno splicedonor of intron 6, IVS6+1G>T, causing an species and as an antisense RNA for UBE3A sis of PC in these families was 67 years (range inframe skipping of exon 6. The resulting trun Maren Runte (1), Alexander Hüttenhofer (2), 4378 years). The median age at diagnosis of cated TRPS1 protein lacks the entire GATAtype Stephanie Gross (1), Bernhard Horsthemke (1), basaloma was 63 years (range 56 74 years). All zincfinger as well as the last four amino acids Karin Buiting (1) basaloma patients were first degree relatives of (RRRG) of the predicted NLS1. Fusion of exons (1) Institut für Humangenetik, PC patients. Most interestingly, one patient de 5 and 7 does not create a novel NLS. The sec Universitätsklinikum Essen, Essen, veloped basaloma at first and PC 5 years later. ond is a transition G>A at position 2885 in exon Germany; (2) Institut für Experimentelle No other tumors had occurred in all three fami 7, found in two unrelated patients. It leads to the Pathologie / Molekulare Neurobiologie, lies. A familial accumulation of PC and basalo single amino acid change R952H. Arginine 952 ZMBE, Muenster

medgen 14 (2002) 15 The imprinted domain on human chromosome A 1.4 Mb deletion within the 5´ control tides are maintained throughout these updates 15 consists of two oppositely imprinted gene region of SOX9 causes acampomelic where possible. The EnsEMBL website displays clusters, which are under the coordinated con campomelic dysplasia and XY sex reversal genomic data for multiple species. All genomes trol of an imprinting centre at the 5’ end of the Pop, Ramona (1); Pfeifer, D. (2); Conz, C. (2); are presented with the same user interface. Hu SNURFSNRPN gene. One gene cluster spans Briault, S. (3); Blesson, S. (3); Zimmer, J. (1); man and mouse displays are thoroughly inter the centromeric part of this domain and contains Scherer, G. (1) linked, providing easy species comparison infor several genes that are transcribed from the pa (1)Institute of Human Genetics and mation by mouseclick. Third party data can eas ternal chromosome only (MKRN3, MAGEL2, Anthropology, University of Freiburg, ily be added using the DAS protocol. EnsEMBL NDN, SNURFSNRPN, HBII13, HBII85, HBII Freiburg, Germany; (2)Genescan Europe AG, is an open source software engineering project 52, HBII436, HBII437, HBII438A and HBII Freiburg, Germany; (3)Service de Génétique, and all code and data is made available for 438B). Apart from the HBII small nucleolar RNA Hopital Bretonneau, Tours, France download with a nonrestrictive license. The de (snoRNA) genes, each of these genes have a 5’ Campomelic dysplasia (CD; MIM 114290), an velopment process is open to everyone and in differentially methylated region (DMR), and pa autosomal dominant skeletal malformation syn volves a number of international collaborations. ternalonly expression appears to be regulated drome with XY sex reversal, is caused by het The whole system is fully portable. The web in by methylation of the maternal allele. The sec erozygous mutations in and around the SOX9 terface contains advanced visualisation and ond gene cluster maps to the telomeric part of gene on 17q. SOX9 has an extended control re search tools. the imprinted domain and contains two genes, gion as indicated by translocation breakpoints UBE3A and ATP10C. For ATP10C preferential that scatter over 1 Mb 5´ to the gene. We have The GenomeMatrix Information Retrieval maternal expression has been demonstrated in screened a sample of 13 classical and unclassi System fibroblasts, cultured lymphoblasts and brain, fied campomelic cases with no SOX9 coding re Hewelt, Andreas(1), Ben Kahla, A(2), Hennig, whereas maternalonly expression of UBE3A is gion mutation or translocation for deletions in S(2), Nagel, A(1), Himmelbauer, H(2), Zehetner, restricted to brain. As there is no evidence for a the SOX9 5´flanking region by comparative ge G(2), Haas, S(2), Vingron, M(2), Yaspo, ML(2), DMR at the UBE3A locus, maternalonly expres nomic hybridization (CGH) on a DNA microarray. Lehrach, H(2) sion of UBE3A may be regulated indirectly Using largeinsert clones covering a 2.5 Mb re (1)RZPD German Resource Center for through a paternally expressed antisense tran gion around SOX9, a deletion was thus detect Genome Research, Berlin, Germany (2)Max

GfH ÖGH SGMG Tagungsband Abstracts GfH ÖGH SGMG Tagungsband script. We report here that a processed anti ed in an XY female patient with features of CD PlanckInstitute for Molecular Genetics, sense transcript of UBE3A starts at the imprint such as Robin sequence, tracheobronchiomala Berlin, Germany ing centre. The paternally expressed SNURF cia and cervical kyphosis but who lacks bending A number of databases hold information on SNRPN sense/UBE3A antisense transcription of the long bones (acampomelic CD). Size and genomes, genes, gene products and their func unit spans more than 460 kb, contains at least location of the deletion were refined by STS con tion. Most of these are either restricted to one 154 exons and serves as the host for all snoR tent mapping on DNA from a somatic cell hybrid organism or to one type of information. Inter NAs, which are encoded within introns of this clone that carries the deleted chromosome 17 in faces allow access to information on one transcript. To find out if and how the antisense the absence of the normal chromosome 17, gene/gene product at a time. Few databases al transcript might regulate imprinted expression of placing the deletion breakpoints at 380 +/ 5 kb low an overview of many genes at a time. The UBE3A we have compared expression levels of and at 1830 +/ 10 kb 5´ to SOX9. The deletion functional analysis of a specific gene requires in both transcripts in human blood and brain. In does not remove the five evolutionary conserved formation on the function of orthologs of the blood, where UBE3A is biallelically expressed, sequence elements previously identified by us gene in other species. To simplify multigene, we found very low expression of the antisense that are up to 290 kb 5´ to SOX9. This indicates cross species analyses, GenomeMatrix (www. transcript compared to UBE3A. Interestingly, in that one or more additional cisregulatory ele genomematrix.org), a new database/interface brain, where UBE3A expression is imprinted, an ments must exist within the deletion interval that system, able to integrate a wide range of infor equal expression level of both transcripts could are essential for correct SOX9 expression during mation resources, was established. Information be detected. This suggests that upregulation of chondrogenesis and gonad development. The on genes and the different types of information the antisense expression may lead to silencing milder phenotype and the acampomelia seen in is displayed as a matrix of colored boxes, where of paternal UBE3A expression. the patient, who is now over 4 years of age, may columns represent the different genes, and rows be attributable to residual SOX9 expression from the different information types linked to the the deletion chromosome. Gene expression changes in brains of genes. Depending on the type of information the MECP2 knockout mice color of a box encodes either its presence or by Nuber, Ulrike A. (1), Guy J. (2), Selfridge J. (2), a spectrum of colors within a type gives insight Barr H. (2), Hendrich B. (2), Kriaucionis S. (2), into the underlying information. Diverse types of Steinhoff C. (1), Ropers H.H. (1), Bird A. (2) W8 information are linked to genes, e.g. related dis (1) MaxPlanck Institut für Molekulare eases, protein interactions, mouse gene traps Genetik, Ihnestrasse 73, 14195 Berlin, and biological material available at the RZPD EnsEMBL, a genome information system Germany; (2) The Wellcome Trust Centre for (www.rzpd.de) to facilitate the work of the scien (1)(2) The EnsEMBL Team (1) Birney, E. (2) Cell Biology, Institute of Cell and Molecular tific community. To improve the list of biological Clamp, M. (2) Hubbard, T. Biology, University of Edinburgh, The King’s materials a genespecific product portfolio is de (1) EMBLEBI, Hinxton, Cambridge, UK (2) Buildings, Edinburgh EH9 3JR veloped. Whereas some parts of the product The Sanger Institute, Hinxton, Cambridge, Rett syndrome is caused by mutations in portfolio are already available, others are under UK MECP2, a gene that encodes methylCpGbind development. Genome matrices are currently EnsEMBL (www.ensembl.org) is a joint project ing protein 2. MeCP2 has been shown to act as available for man, mouse, worm and fly. Orthol between the Sanger Institute and EBI funded by a transcriptional repressor, and the Rett syn ogy relationships between genes of different or the Wellcome Trust to provide a bioinformatic drome phenotype is assumed to result from ganisms are used to combine information on framework for handling genomic information. It changes in the expression pattern of MECP2 tar gene function across species. currently contains annotation for human, mouse, get genes. We have used cDNA microarrays to mosquito and zebrafish. All data is publicly avail search for gene expression differences in brains able through a website interface and flat files via The human endogenous retrovirus family from Mecp2 /y knockout mice and normal age an ftp site. EnsEMBL offers a variety of gene HERVK(HML3) and its genomic impact matched controls. Several differentially ex prediction methods which utilize different de during primate evolution pressed mRNA species were identified in this grees of similarity to existing peptide and dna Jens Mayer, Eckart Meese way, and the expression changes were found to sequences. One method places known genes Human Genetics, Building 60, University of coincide with the onset of symptoms. Possible using a fast, exact dna/protein matcher (pmatch) Saar, Medical Faculty, 66421 Homburg, implications of these findings for the pathogen to match known human proteins. This is fol Germany esis of Rett syndrome will be discussed. lowed by a refining of the genestructure with ge A substantial amount of the human genome newise. Another method uses aligned non hu comprises human endogenous retroviruses man proteins to get novel gene predictions. (HERVs). Manifold HERV families have been Alignment supported ab initio predictions (gen identified and are represented in Repbase, scan) and est based genes complete the set of among them several HERVK families. While the the current prediction methods. Sequencing HERVK(HML2) family has been studied in de projects like human and mouse go through fre tail by us and others in recent years, other quent iterative improvements of the sequence HERVK families are much less characterized. data. Identifiers for genes, transcripts and pep The availability of the human genome draft se

16 medgen 14 (2002) quence now provides an excellent source of in genitor cell during development, and switching W9 formation to study HERV families at the nu from the insulinproducing ßcell to insulin sig cleotide level regarding structure and specific naling will then provide the necessary informa Screening for Down’s syndrome a evolution. We here describe the HERVK(HML3) tion to understand the meaning of this cell type bioethical point of view family in more detail. We analysed proviral loci for the regulation of energy metabolism on a cel Dagmar Schmitz regarding structure and variations. We estimate lular and systemic level. Moreover, PheGe will Institut für Ethik und Geschichte in der about 140 proviral loci or remains of such per guide through the peripheral regulation of ener Medizin, Universität Tübingen haploid genome. Most loci are severely mutat gy homeostasis, the central control of food in Prenatal screening strategies for Down’s syn ed. Proviruses displaying larger deletions in gag take and the supply of nutrition to the organism. drome have become an established part of ob and pol are common. A multiple alignment of 73 stetric practice and a frequent subject of genet HERVK(HML3) sequences displays several po A Simplified Computer readable Cytogenetic ic counselling services. They are offered with the tentially important differences compared to Notation (SCCN) for the automated analysis aim of minimising the number of „affected“ preg HERVK9I, the HERVK(HML3) corresponding of tumor cytogenetic findings nancies that are missed, reducing the number of consensus sequence found in Repbase. We Bradtke, Jutta (1); Balz, H. (1); Döhner,H. (2); miscarriages due to amniocentesis or chorionic generated a consensus sequence from the mul Heinze, B. (3);Jauch, A. (4); Mohr, B. (5); villus sampling and reducing costs. Today, ante tiple alignment displaying open reading frames Schoch, C. (6) ; Rieder, H. (1) natal care providers have several strategies in for all retroviral genes. As reported for HERV (1) Institut für Klinische Genetik, Marburg; addition to screening based on maternal age K(HML2) and (HML6), for instance, dUTPase (2) Klinik für Hämatologie und Onkologie, (e.g. foetal nuchal translucency measurement motifs indicating a formerly active dUTPase en Ulm; (3) Institut für Klinische Genetik, Ulm; combined with maternal serum tests) at their zyme were observed within the protease ORF. (4) Institut für Humangenetik, Heidelberg; disposal. Variants of the envelope gene displaying differ (5) Klinik für Hämatologie und Onkologie, A comparative analysis of screening strategies ent coding capacities were observed. Phyloge Dresden; (6) Klinik für Hämatologie und regarding their consistency with principles of netic analysis shows nearmonophyly with dis Onkologie, München Großhadern medical ethics is performed. Implications and tinction of two closely related subgroups. From The aim of the project „Zentrale Zytogenetik“ of consequences relating to the availability and util LTRLTR comparisons it is concluded that the Competence Net „Akute und chronische isation of screening for Down’s syndrome are

HERVK(HML3) homologous proviruses formed Abstracts GfH ÖGH SGMG Tagungsband Leukämien“ is to identify new rare chromosome elucidated. Implications concern physicianpa about 36 million years ago, immdiately after the aberrations, which are relevant for leukemogen tient interactions as well as interactions between split of Old World from New World primates. esis, and to evaluate them with respect to their parents and children or between health care pro However, and in contrast to the HERVK(HML2) clinical significance. Additional aberrations and fessionals and people with Down’s syndrome. It family, for instance, no continuous activity hot spots of chromosomal rearrangements, is stressed that routine screening for Down’s throughout primate evolution is indicated in gains and losses should be found. Therefore, syndrome apart from screening based on ma terms of generation of new proviruses. Our flexible definitions of aberration keys, which de ternal age is particularly conflicting with regard analysis contributes to the understanding of scribe distinct chromosomal aberrations and to the principle of respect for autonomy. Be repetitive elements, specifically human endoge combinations of it, are neccessary to make cy sides, it might lead to a remarkable shift in nous retroviruses, as important factors for the togenetic information available for the correla physicianpatient interaction in suggesting that evolution of the host genome. tion with results of therapy studies. The analysis selection of children with Down’s syndrome and of distinct leukemia entities presenting with termination of affected pregnancies is medical Approaching virtual organism by bridging of identical chromosome aberrations should con ly justified or authorized. cellular actions tribute to the clarification of the relation between In conclusion, it is pointed out that several Klaus Seidl genotype and phenotype in leukemias. Type and screening strategies for Down’s syndrome are German Research Centre for Biotechnology frequency of therapyrelated chromosome ab potentially inconsistent with the principles and (GBF), Braunschweig, Germany, email: normalities should be evaluated in different standards of medical ethics if offered as part of [email protected] leukemias in order to clarify the mutagenic ef routine antenatal care. They should therefore be With the steadily emerging number of interaction fects of the drugs in different leukemias and to embedded in a process of shared decisionmak data gained by genomewide methods such as give clues to the genetic mechanisms of ac ing between the health care professional and the the chip technology, mass spectrometric analy quired resistance against different cytostatic patient. Standards of disclosure and informed sis and twohybrid technique, interaction data drugs. A large amount of cytogenetic findings is consent prior to antenatal screening procedures bases become more and more important for the needed for these studies which will be generat need to be developed to encourage au classification of the individual components into ed by the compilation of the data from cytoge tonomous, competent choices and prevent pa the network of regulatory systems. Several com netic centers involved in therapy trials of ternalistic physicianpatient interactions. putational approaches have been presented ad leukemias. This includes data (>10000 patients) dressing the problem of signal interaction and from chromosome banding analysis and molec Genetic counseling following prenatal signal transduction. The main focus of all these ular cytogenetic methods. The manual analysis diagnosis of chromosomal abnormality a interaction databases is to provide knowledge of such a large amounts of cytogenetic data is support in deciding a conflict situation? about interaction profiles on molecular levels. time consuming. Automated procedures are not Dr. med. Britta Maria Schmitz, Prof. Dr. med. Seizing the idea of systemlevel thinking we available as yet. The ISCNkaryotype of unbal Ursel Theile have to take into account that interactions take anced aberrations includes more information Institut für Humangenetik am Klinikum der place within distinct biological compartments than written in signs. Therefore, we developed a Johannes GutenbergUniversität, and that organism function is managed by the Simplified Computer readable Cytogenetic No Langenbeckstr. 1, 55131 Mainz, Germany concerted action of a multitude of different cell tation (SCCN) to allow an automated analysis of The empiric study focuses on the special coun types. Therefore, to attack the problem of virtu cytogenetic findings. The information of the seling situation that follows after prenatal diag al network analysis on a systemic level it is cru ISCN karyotype is splitted into the qualitative nosis of chromosomal abnormality and the con cial to link the individual interactions to their re and quantitative contents. Using predefined cri flict for those who seek for advice. About 200 spective cell systems and to express signal teria the ISCN karyotyp is manually converted case histories were retrospectively surveyed transduction events as cellspecific phenomena. into this two types of data strings which are now since 1993. The common basis of all cases was In order to approach automated network analy ready for computer analysis with two special chromosomal abnormality and the fact that there sis on organism level the relational platform software modules programmed in VBA (Visual was no genetic counseling prior to prenatal di PheGe was generated. PheGe is a pilot scheme Basic for Applications). After successful analy agnosis. It was shown that factors like age of for virtual biology on a systemic (multicellular sis, results and error prone findings are report parents, number of common children, stage of organism) level. It represents a prototype of ed in tables or graphs. Every step of the analy pregnancy at time of diagnosis, indication for knowledge base platforms that organize data sis can be retraced by the operator. In future, the and method of prenatal diagnosis and especial driven interactions in a way that attack the prob manual conversion of ISCN into SCCN will be ly the results of the chromosome analysis have lem of digital recording and simulation of cell replaced by automated procedures. an enormous influence on the parents´ decision overlapping signal cascades and cellular differ Supported by BMBF Grant No: 01GI9974 wether or not to continue the pregnancy. Effects entiation programs. By means of directed graph of the decision are mostly mourning and feelings technology one can follow up signaling stepwise of guilt. In consequence of this study we recom by an automated datadriven organization of in mand that all parents should be offered a quali teraction profiles. As an example, someone can fied genetic counseling before prenatal diagno pursue the fate of a pluripotent pancreatic pro sis. During the coping process they should also

medgen 14 (2002) 17 be accompanied by an interdisciplinary team Universitätsklinikum Charité, Humboldt families the RYR1 was genetically screened (geneticists, obstetricians, psychologists) to Universität zu Berlin, Schumannstr. 20 identifying a mutation in 46. This indicates a high come up to medical, psychological, ethical and Nausea and vomiting still belong to the feared success rate of the mutation screening (46,6%) social aspects of the individual decision. side effects of cancer chemotherapy. 5Hydrox as compared to other studies. 18 different ytryptamine (5HT3) receptor antagonists like RYR1point mutations were found, out of these ondansetron or tropisetron play the major role in four have not yet been published. 38 RYR1 mu An unbalanced translocation the current antiemetic treatment of acute eme tation carriers were classified as MH positive t(X;15)(q22;q12) in one fetus of a dizygotic sis. However, about 2030% of the patients do without the invasive muscle test. twin pregnancy diagnosed after pathologic not respond satisfactorily. These individual dif Conclusions: Under exact and critical evaluation ultrasound findings: a case report ferences in drug response may be explained by each of its potential, one of the three pillars of Kuechler, Alma (1/2), Hesse, M. (3), Hagemann, variation in drug biotransformation by genetical MH diagnosis the clinical phenotype, the IVCT M. (4), Thiele, H. (4), Hansmann, I. (4), Liehr, T. ly polymorphic enzymes, such as the cy or the DNA analysis can preferentially be used (1), Claussen, U. (1) tochrome P450 2D6 enzyme (CYP2D6) and by to obtain certain information. However, these (1) Institute of Human Genetics and genetic variations in the 5HT3 receptor gene. methods should not be regarded as competing Anthropology, Jena, Germany, (2) Therefore we analyzed the antiemetic outcome with each other but should be complementary Department of Radiation Oncology, Jena, in 270 cancer patients. Nausea and vomiting used to increase sensitivity and specificity of MH Germany, (3) Sudharzkrankenhaus, was documented by using standardized inter diagnosis. In MH families in which a MH related Nordhausen, Germany, (4) Institute of views before and in two observation periods fol mutation is detectable the molecular genetics Human Genetics, Halle, Germany lowing the chemotherapy. We genotyped all in may represent a less invasive and specific diag An ultrasound scan performed on a 39 year old dividuals for CYP2D6 and measured the tro nostic tool for determining the MH disposition. woman pregnant with twins at 11+6 weeks re pisetron serum concentrations in 42 patients. We vealed a pathological nuchal translucency in one sequenced the whole 5HT3B and 5HT3A re of the twins. Therefore, diagnostic amniocente Prospects and Limits of Pharmacogenetics: ceptor gene of all patients. About 30% of all pa sis was performed at 16+3 weeks of gestation. The TPMT Experience tients suffered from emesis. Genetically defined GTGbanding showed a cytogenetically normal Jan van Aken, Mechtild Schmedders, Günter poor metabolizers of CYP2D6 had higher serum male karyotype in one twin, whereas the twin Feuerstein & Regine Kollek

GfH ÖGH SGMG Tagungsband Abstracts GfH ÖGH SGMG Tagungsband concentrations of tropisetron than all other pa with the suspicious ultrasound findings showed Forschungsschwerpunkt Biotechnik, tients (p<0.03). Ultrarapid metabolizers of a female karyotype with additional material on Gesellschaft und Umwelt, Universität CYP2D6 had higher scores of vomiting in both the long arm of one Xchromosome [46,X, Hamburg, Germany observation periods than all other patients add(X)]. An increasing number of pharmacogenetic stud (p<0.001, p<0.03). Sequencing of the 5HT3B Characterization of the origin of th is chromoso ies is rapidly elucidating the genetic influence on receptor gene revealed 13 polymorphisms, two mal material using MFISH and centromeric drug safety and efficacy and may help to im amino acid exchanges (Tyr129Ser, Ala223Thr) probes revealed a translocation der(X)t(X;15). prove future drug therapy. Thiopurine drug me and two deletion variants. Patients homozygous Multicolor banding (MCB) performed according tabolism is a quintessential case of pharmaco for the 100AAG deletion variant showed signif to Liehr et al. (Int J Mol Med 2002, 4:335339) al genetics and has repeatedly been brought for icantly more vomiting in both observation peri lowed the specification of breakpoints: der(X) ward as a perfect example for the promises of ods than all other patients (p<0.001, p<0.04). In t(X;15)(q22;q12). Thus, the fetus had beside a pharmacogenetic diagnostics. A wealth of ex conclusion, ultrarapid metabolizers of CYP2D6 partial monosomy Xq (deletion of the region perimental and clinical data on polymorphisms and individuals homozygous for the 100AAG Xq22—>Xqter) a nearly complete trisomy 15q in the thiopurine metabolizing enzyme thiopurine deletion variant of the 5HT3B receptor gene (15q12—>15qter). The parents’ karyotypes methyl transferase (TPMT) has been generated had the highest severity of nausea and vomiting. showed that the aberration occurred de novo. in the past decade. Pharmacogenetic testing pri These patients need a different antiemetic ap FMR 1 repeat analysis revealed the paternal ori or to thiopurine treatment is already being prac proach. gin of the Xchromosome involved in the translo ticed to some extent in the clinical context, and cation. it is likely that it will be amongst the first phar Although there were nearly no information about Molecular genetics as an alternative macogenetic tests applied on a regular basis. the expected clinical outcome of this specific diagnostic tool for maligant hyperthermia Here, we analysed the published TPMT data to aberration, the pregnancy was continued. After H.Rüffert (1), D. Olthoff (1), C. Deutrich (1), UG. identify some lessons to be learned for the fu premature birth at 31+4 weeks because of se Froster (2) ture implementation of pharmacogenetics for vere maternal heart insufficiency, the affected 1) Dept. of Anaesthesiology and Intensive thiopurines as well as in other fields. While there twin showed apart from mild growth retardation Care Medicine, Univ. Leipzig, 2) Institute of are undoubtedly clear benefits of pharmacoge and developmental delay no facial dysmorphic Human Genetics, Univ. Leipzig netic TPMTtesting, we identified some prob features and no further external or internal ab Malignant hyperthermia (MH) is a pharmacoge lems and pitfalls which should be taken into ac normalities. To the best of our knowledge this is netic and potential lifethreatening disorder trig count in future studies. These include the first case of an almost complete nonmosa gered by inhalation anaesthetics and depolariz the appreciation of the limits of pharmacoge ic translocation trisomy 15. According to t he lit ing muscle relaxants. The disposition to MH is netics, which may be able to avoid some, but erature, patients with unbalanced Xautosomal inherited in an autosomaldominant manner. The by far not all adverse side effects of drug ther translocations can have attenuated phenotypes objective of the presentation is to give a survey apy; clinical studies with thiopurine drugs because of the complex mechanism of inactiva of experiences and results obtained within a pe showed that an average of 78% of side effects tion of autosomal material attached to the X riod of 15 years of diagnosis of malignant hyper in heterozygous patients were not associated chromosome. This leads to varying effects on thermia in the MHcentre in Leipzig. The new with TPMT polymorphisms; the patients’ phenotype (Abuelo et al. Am J Med branch of MHdiagnosis the molecular genet the need for comprehensive and unbiased data Genet 2000, 94: 392399). ics and its general diagnostic potential shall be on allele frequencies relevant to all populations presented more in detail. worldwide; the vast majority of TPMT studies Methods: The halothane/caffeine in vitro con published so far are restricted to the most fre tracture test of a skeletal muscle specimen rep quent alleles from one or two populations, resents the standard method for determining the which entails the risk of generating ethnically W10 MH disposition. In 1999, the MH diagnosis in our biased data; centre was supplemented by molecular genetic a careful approach towards dose increases for Pharmacogenetics of Antiemetic Drug examinations of the skeletal ryanodine receptor patients with high enzyme activity, which may Treatment gene RYR1 (direct sequencing of the three hot increase adverse drug reactions due to an in Kaiser, Rolf (1,2), Papies, A.(2), Tremblay, PB spot regions). This fast calcium releasing recep crease of toxic byproducts. (2), Orhan, S. (3), Bauer, S. (2), Rösler, N. (2), tor (RYR1) located in the membrane of the sar the necessity to address the dual information Schelenz, C. (2), Possinger, K. (3), Roots, I. (2), coplasmic reticulum appears to play the most problem and thoroughly study possible disease Brockmöller, J. (1,2) important role in the pathogenesis of MH. More susceptibiliy information provided by a phar (1)Abteilung für Klinische Pharmakologie, than 20 RYR1mutations were found to be asso macogenetic test, which may entail serious Universitätsklinikum, GeorgAugust ciated with the disorder. ethical, social and legal issues; Universität Göttingen, RobertKochStr. 40, Results: A total of 1456 muscle tests (carried out The TPMTexperience provides some valuable 37075 Göttingen, (2)Institut für Klinische from 19862001) in patients having a potential insights into the power and the limits of pharma Pharmakologie und (3)Abteilung für MH disposition provided 376 MHS, 121 MHE cogenetics. A careful analysis of this experience Hämatologie/Onkologie, and 921 MHN results. In 99 independent MH

18 medgen 14 (2002) may help to avoid some of the shortcomings and ation probe panel“ [Ref. 2] and by using the ULS A new subcentromeric probe set for the pitfalls in the future. / Universal Linkage System ® labelling tech characterization of centromerenear *Projekt „Individualisierung medikamentöser nique, high labelling specificities and hybridis rearrangements Therapie? Implikationen der Pharmakogenetik ation efficiencies could be reached. This allows Starke, Heike (1), Heller, A. (1), Weise, A. (1), für Patienten und Gesundheitswesen. BMBF the unequivocal analysis of 21 respectively 20 Nietzel, A. (1), Claussen, U. (1), Liehr, T. (1) Förderkennzeichen 01KV9906 probes per hybridisation with two to four (1) Institute of Human Genetics and metaphases each. In contrast to other subtelom Anthropology, Jena, Germany eric multicolour techniques, the high multiplic A variety of FISH approaches have been devel ities of Subtelomere COBRA FISH make it pos oped in the last decade, covering the entire hu sible to differentiate e. g. long arm and short arm man genome in multiple ways. Nonetheless, W11 subtelomeric regions of a given chromosome there is still a chromosomal region which is not thus permitting the diagnosis of cryptic pericen well investigated with the presently available Dynamics of DNA Topoisomerases I, IIalpha tric inversions in addition to translocations and probe sets: the pericentric region. This region is and beta in Living Human Cells deletions. The technique and its validation on of special interest when small supernumerary Morten O. Christensen, HansUllrich known aberrations as well as on normal controls marker chromosomes (sSMC) shall be charac Barthelmes, Fritz Boege, and Christian Mielke will be presented. References: [1] Genome Res. terized. Neither whole nor partial chromosome Department of Clinical Chemistry, (2000) 10:861865, [2] Am J Hum Genet (2000) painting (wcp and pcp) probes are informative if Medizinische Poliklinik, University of 67:320332 centromerenear euchromatic material is pres Würzburg, Klinikstrasse 68, D97070 ent on a sSMC. This question can be answered Würzburg, Germany best when hybridizing centromerenear probes Screening for subtle subtelomeric Constitutive expression of active human DNA like BACs. At present, we are working on 24 rearrangements in 75 mentally retarded and topoisomerases as fusion proteins with green probe sets, each for one human chromosome, dysmorphic children fluorescent protein enabled us to monitor these which consists of a centromere specific satellite Martin, Thomas; Christmann, A., Reichardt, S., enzymes in living cells going through mitotic cell probe, one centromerenear BAC in q and p (ex Brand, M., ÖhlJaschkowitz, B., Zankl, M., cycles. Using photobleachimg techniques we cluding the acrocentric chromosomes) and arm Zang, K. D.

determined enzyme mobilities and resident specific pcp probes. Finished probe sets are Abstracts GfH ÖGH SGMG Tagungsband Institute of Human Genetics, Saarland times at various nuclear locations. Our key find used after characterizing the sSMC by cenM University, 66424 Homburg, Germany ing is that each topoisomerase species consti FISH (Nietzel et al., 2001, Hum Genet, 108, 199 Subtle chromosomal rearrangements have been tutes a single diffusible population in interphase 204). Up to now two cases with cat eye syn found to be an important cause of human genet and mitosis. The heterogeneous subnuclear and drome (CES) chromosomes [inv dup(22)(q11.2)] ic diseases. Especially among moderate to se chromosomal distribution is determined by dif and four sSMC derived from #2, #16, #19 and vere mentally retarded children without any ex ferences in residence times, resulting for in #22, respectively, have been characterized by planation for their handicap a frequency of 7.4% stance in upconcentration in nucleolar substruc centromerenear BAC probes. As expected, cen chromosomal rearrangements involving the ends tures. tromerenear euchromatic material was present of chromosomes has been described (1). In this During mitosis, almost the whole complement of in the CESchromosomes. For the derivative #2 study we analysed 75 children with mental retar topoisomerases I and IIa localizes to chromo qarm specific centromerenear material could dation and dysmorphic features with the Chro somes throughout the cycle, whereas topoiso be detected, while the derivatives #16 and #22 moprobe MultiprobeT system (Cytocell). It is a merase IIb can not be discriminated at chromo had no detectable euchromatin. Additionally, a FISH based approach, which has a predefined somes before anaphase. Such an appearance clinical case with a normal karyotype apart from hybridisation area for the p and q telomeric implies that only topoisomerases I and IIa func the very small pericentric inversion could be de probe of every single chromosome from 1 to 22, tion in chromosome condensation, segregation tected using the chromosome 2 specific peri and X and Y. We found 4 subtelomeric re and decondensation. Moreover, the entire pop centromere probe set. Recently, a case with a arrangements according 5.3%. Case 1 was the ulation of topoisomerase IIa was found to be centromeric duplication including the cen unbalanced offspring of a mother carrying a mobile between individual chromosomes and tromerenear region of 5p could be identified, as cryptic balanced translocation 4p;20q. Case 2 nuclear substructures. Thus the concept of well. In summary, we present a probe set use was a subtle de novo deletion 8p. Case 3 was a topoisomerase II participating in the building of ful to characterize centromerenear rearrange partial duplication 7p and a partial deletion 7q a fixed chromosome scaffold or nuclear matrix ments. Supported by Herbert Quandt Stiftung resulting from a very large pericentric inversion must be reconsidered. der VARTA AG and the EU (QLRT199931590). 7 of the father. Case 4 was a de novo unbal anced translocation 1;22 leading to partial tri Subtelomere COBRA FISH: A novel somy 22q and partial monosomy 1p. None of the The establishment of highresolution multicolour FISH method for the efficient chromosomal aberrations could be seen by high phenotypegenotype maps detection of subtelomeric rearrangements resolution banding. Only two of the cases (cas Jürgen Kraus a,b, Christine Fauth a,b, Monika Engels, Hartmut (1, 2), Zahn S. (1), Ehrbrecht es 1 and 4) could have been detected also by Cohen c, Heide Seidel d, Imma Rost d, Michael A. (1), Bosse K. (1), Vrolijk J. (2), Propping P. other molecular cytogenetic techniques like SKY R Speicher a,b (1), Schwanitz G. (1), Raap A.K. (2), Wiegant J. or MFISH. In case 1 only the derivative chromo a Institute of Human Genetics, Technical (2), Tanke H.J. (2) some 4 was recognisable with painting probes University, Munich, b Institute of Human (1) Institute of Human Genetics, University while the mother’s derivative chromosome 20 Genetics, GSF National Research Center of Bonn, Germany, (2) Laboratory for appeared inconspicuously. While handling and for Environment and Health, Neuherberg, c Cytochemistry and Cytometry, MCB4, hybridisation efficiency of the FISHchip proved Kinderzentrum München, d Institute of Leiden University Medical Center, The to be sufficient for routine diagnostics there is Medical Genetics, LMU München, all Netherlands one major pitfall: The probe for the subtelomer Germany Cryptic subtelomeric chromosomal rearrange ic region of 2q (marker D2S2986) does not hy In the „postgenomic“ era one of the most im ments represent a significant cause of unex bridise with a polymorphism known to occur in portant challenges is to link genomic to function plained mental retardation and are also being about 5% of the population (2). In 3 patients a al data. Patients with chromosomal aberrations described increasingly in neoplasia. FISH using „deletion“ was indicated that had to be ruled out can facilitate this task if the chromosomal defect subtelomeric probes is the most widely used by a second hybridisation with another probe for is corroborated with the corresponding clinical technique to detect such rearrangements. Since 2q (VYSIS TelVysion Probe; D2S447). In all three data. However, in order to establish highresolu for a complete investigation of all euchromatic cases the polymorphism could be shown to be tion phenotypegenotype maps Gbanding may human subtelomeres 41 different FISH probes familiar and without clinical relevance. By PCR be too imprecise. The use of publicly accessible must be analysed, multicolour techniques have analysis of D2S2986 it was furthermore exclud BACclone resources should allow the finemap been developed for subtelomeric probes to fa ed that the polymorphism is based on a mi ping of chromosomal aberrations to the kbres cilitate a more efficient analysis. COBRA (COm crodelet Ref. olution range. As a consequence, strategies are bined Binary RAtio) labelling combines ratio and 1. Subtle chromosomal rearrangements in chil needed to utilize both appropriate technologies combinatorial labelling to attain especially high dren with unexplained mental retardation. and probe sets to work up cases in an efficient multiplicities [Ref. 1]. With „Subtelomere COBRA Knight et al. Lancet. 1999; 354: 167681. way. We propose a hierarchical approach. In a FISH“, the specific detection of all FISH probes 2. he promise and pitfalls of telomere region first step a screening method (e.g. MFISH) is is possible in only two hybridisations using four specific probes. Ballif et al. Am. J. Hum. used to identify the involved chromosomes. spectrally separated fluorochromes. By strict Genet. 2000; 67:13561359. Subsequent steps are aimed to fine map break probe selection mostly from the „second gener points. (e.g. application of BACclones, mi

medgen 14 (2002) 19 crodissection of chromosomes, etc.). To exem for the biological process of interest, much like Haar; Depts. of Psychiatry, (4) Univ. plify our point we demonstrate two examples. a mutant in classic forward genetics. We have Würzburg, (5) Univ. Dresden, (6) Univ. Mainz, The first is a familial terminal deletion on the used chemical genomics to study chromosome (7) Hadassah Univ. Jerusalem, Israel; (8) long arm on one chromosome 6 which segregat segregation during mitosis in human cells. I will Inst. Medical Biometry, Informatics, and ed through at least two generations in several use this project as an example to illustrate the Epidemiology, Univ. Bonn; (9) Dept. Of family members. Contrary to the common be technical challenges and the potential of the Psychiatry, Univ. Bonn; (10) Central Institute lieve that subtelomeric imbalances result usual chemical genomics approach. of Mental Health, Mannheim. ly in severe malformations affected family mem Bipolar affective disorder (BPAD) is a severe bers had no dysmorphic features and were only psychiatric disorder that has a lifetime preva mildly mentally retarded. The BAC tools allowed lence of about 1% in all human populations. In to map the deletion to a 2.8Mb region which har order to identify chromosomal regions contain W13 bors 15 genes. The second example exemplifies ing BPADrelated genes we have conducted a that chromosomal aberrations may be much complete genome screen at an average marker more complex than initially thought. The initial Molecular genetic findings in obesity spacing of 9.3 cM in a sample of 75 BPAD fam diagnosis of an inv ins(5;7)(p13;qterq31) turned J Hebebrand (1), A Hinney (1), A Hamann (2), F ilies. Pedigrees were of German, Israeli, and Ital out after detailed characterization with a Geller (3), K Saar (4), K Reichwald (5), M ian origin, respectively. The highest twopoint YAC/BACclone cocktail to involve not 3 but at Platzer (5) LOD score was obtained on 8q24 (D8S514; LOD least 6 breakpoints including a deletion in the (1) Clinical Research Group, Department of score = 3.62), in a region that has not attracted Mbrange. The deletion would had been com Child and Adolescent Psychiatry, Philipps much attention in previous linkage studies of pletely missed by standard cytogenetic meth University of Marburg, Germany, (2) BPAD. The second best finding was seen on ods. These fine mapping efforts will provide if Department of Internal Medicine, University 10q25q26 (D10S217; LOD score = 2.86) and done on a large scale a wealth of information of Heidelberg, (3) Institute of Medical has been reported in independent studies of and should improve our understanding of geno Biometry and Epidemiology, University of BPAD. Other regions showing „suggestive“ evi type/phenotype relationships. Marburg (4) Max Delbrück Centre, Berlin, (5) dence for linkage localized to 1p33p36, 2q21 Institute of Molecular Biotechnology, Jena q33, 3p14, 3q26q27, 6q21q22, 8p21, 13q11,

GfH ÖGH SGMG Tagungsband Abstracts GfH ÖGH SGMG Tagungsband Obesity in adulthood is defined via a body mass and 14q12q13. In addition, we aimed at detect Chromosomes are chromosomes index (BMI; kg/m²) ³ 30 kg/m². Approximately ing possible susceptibility loci underlying ge throughout the whole cell cycle 50% and 20% of the German population is over nomic imprinting by analyzing the autosomal Claussen, Uwe (1), Lemke, J. (1), Claussen, J. weight (BMI ³ 25 kg/m²) and obese, respective genotype data with the recently developed ex (1,2), Chudoba, I. (3), Liehr, T. (1), Muehlig, P. ly. Obesity is associated with an increased risk tension of the GENEHUNTER program, GENE (4), Sperling, K. (2) for development of type 2 diabetes mellitus, car HUNTERIMPRINTING. Putative paternally im (1) Institute of Human Genetics and diovascular disorders, specific types of cancer printed loci were identified in chromosomal re Anthropology, Jena, (2) Institute of Human and other disorders. Both environmental and ge gions 2p24p21 and 2q31q32. Maternally im Genetics, Berlin (3) MetaSystems GmbH, netic factors predispose to overweight. Heritabil printed susceptibility genes may be located on Altlussheim (4) Institute of Molecular ity estimates for the quantitative phenotype BMI 14q32 and 16q21q23. Biotechnology, Jena, Germany typically range from 5080%; nonshared envi Interphase chromosomes analysed with current ronment is considerably more important than ly available techniques do not present any rec The QTc interval duration is correlated to shared environment. Genetic research into hu ognizable structures such as bands, cen KCNQ1 polymorphisms in a population man obesity has greatly profitted from the simi tromeres, telomeres, or specific shapes. Microir based MONICA survey larity of regulatory pathways in mammals. Both radiation experiments and molecular cytogenet J Erdmann, C Herrmann, C Hengstenberg, H spontaneous mutations in mice and transgenic ic investigations with whole chromosome paints Löwel, GAJ Riegger, S Holmer, H Schunkert, A models have led to the identification of single and region specific microdissection probes have Jeron monogenic forms of human obesity, which are confirmed a territorial organization of chromo Clinic and policlinic Internal Medicine II, all inherited autosomal recessively and associ somes in interphase nuclei. Until now, however, Cardiology, University of Regensburg, ated with diverse endocrinological abnormalities. their structure is not well understood. Using Prolongation of the QTc interval is known to be Mutations in the melanocortin4 receptor gene laser scanning microscopic examination and the a risk factor for ventricular arrhythmias. The in have been detected in 24% of obese probands highresolution DNAbased multicolour banding fluence of polymorphisms in genes encoding for in different European populations. Current evi (MCB) technique, we have generated a banding ion channels on QTc duration in the regular pop dence suggests that these mutations underly an pattern and have determined the length of hu ulation without structural heart disease or inher autosomal dominant form of obesity, which is man chromosome 5 in lymphocyte interphase ited long QT syndrome is not known. not associated with other symptoms; these nuclei, and in nuclei of HeLa cells arrested at dif Digitized ECG’s of 1516 probands from the same mutations have only rarely been detected ferent phases of the cell cycle. The shape and MONICAAugsburg survey were used to calcu in nonobese individuals. However, because MCB pattern of chromosome 5 in interphase nu late QTc interval duration. Furthermore, the re nonobese mutation carriers have been identi clei is similar to that of metaphase chromosome sults were correlated with echocardiographic fied among family members of index patients the 5 at all stages of the cell cycle. The length of the and electrocardiographic parameters as well as associated relative risk for development of obe chromosome axis is comparable to that of a with age, gender, and medication. The coding sity are is unclear. Several genome scans have metaphase chromosome at a 600band resolu region of the KCNQ1 gene was analyzed by sin been performed in different populations. tion. Therefore, the concept of chromosome gle strand conformation polymorphism (SSCP) Chromosomal regions of interest include 1p, 3q, condensation during mitosis has to be re in all probands with a QTc above (group 2, n=44) 4q, 10p, 11q and 20q. Finemapping of these re assessed. Interphase chromosome banding can and below (group 1, n=38) the second standard gions and identification of candidate genes is be used to identify chromosome aberrations and deviation as well as in a sample of probands ongoing. opens new fields in cytogenetic analysis. with a median QTc interval (group 3, n=55). Our molecular genetic studies are being sup The table summarizes the significant differences ported by the Bundesministerium für Bildung between the three groups, while ejection frac und Forschung (DHGP and NGFN). tion, left ventricular diameters and blood pres sure were not different. W12 A GENOME SCAN IN FAMILIES WITH BIPOLAR AFFECTIVE DISORDER 123p Analyzing Human Cell Division by Chemical Cichon S(1),Schumacher J(2),Müller n 384455 Genomics DJ(2),Hürter M(2),Albus M(3),Borrmann Age 42+14 60+13 54+12 0.01 JanMichael Peters Hassenbach M(3),Franzek E(4),Fritze Heart rate 57+8 76+18 67+9 0.01 Research Institute of Molecular Pathology, J(4),Kreiner R(5),Weigelt B(5),Minges J(6),Lerer QT interval 367+25 397+43 377+26 0.01 Vienna, Austria B(7),Kanias K(7),WindemuthKieselbach QTc interval 345+9 444+17 397+1 0.001 The field of chemical genomics proposes to use C(8),Wienker TF(8),Baur MP(8),Lichtermann small molecule inhibitors to study gene and pro D(9),Maier W(9),Rietschel M(9),Propping Next to 9 known polymorphisms we identified tein function. Chemical compounds that interfere P(2),Nöthen MM(1) four new intronic polymorphisms in KCNQ1 (IVS with a biological process can be obtained in (1)Dept. Med. Genet.,Univ. Antwerp, 10 +46A>T; IVS 11 +14T>C; IVS 12 +36G>A; highthroughput screens. These compounds can Belgium; (2)Inst. Human Genet.,Univ. IVS 14 +32G>T). Three different haplotypes, dif then be used to identify the enzymes required Bonn,Germany; (3)Mental State Hospital fering in polymorphisms in exon 10 (+46A>T)

20 medgen 14 (2002) and exon 13 (+43T>C), were identified. The re are studies especially in animal models which Establishing an aneuploidy screening for sulting haplotype frequency results in a signifi might rise the theory, that ICSI itself is responsi single cells using CGH cant correlation with QTc and heart rate (0.58 ble for these problems, other studies may sup Gross, Claudia (1), Suess, F. (1), Paulmann, B. and 0.57, p<0.01). port the idea, that these risks are not related to (1), KunzSchughart, L. (2), Hofstaedter, F. (2), The QTc interval duration as well as resting heart the techniques used, but to parental background Hehr, U. (1), Seifert, B. (1), Hehr, A. (1) rate are modified by polymorphisms in the factors. Data from surrogate motherhood also (1) Center of Gynecological Endocrinology, KCNQ1 gene in the general population. These demonstrate, that here the risks are lower as Reproductive Medicine and Human results provide evidence for a genetic influenced compared to pregnancies from IVF mothers, who Genetics; Regensburg, Germany, QTc duration independent of gender and age. carry there own child to birth. Therefore, there andreas.hehr@humangenetik are more infertility related problems than tech regensburg.de, (2) Institute of Pathology, nique related ones. Finally, however, a risk relat University of Regensburg, Germany Common haplotypes in five genes influence ed to the techniques itself cannot be excluded Several methods are currently used for the char genetic variance of LDL and HDL totally by the actual available data. acterization of aneuploidy in single cells. FISH cholesterol in the general population and multiplex PCR with polymorphic markers Hans Knoblauch (1,3), Anja Bauerfeind (2), enable the analysis of small cell or DNA Christine Krähenbühl (3), Aurelie Daury (3), Morphological anomalies in spermatozoa amounts, however only a limited number of Klaus Rohde (2), Stéphane Bejanin (3), Laurent reflect chromosomal aneuploidies chromosomes can be simultaneously analyzed. Essioux (3), Herbert Schuster (1,3), Friedrich C. Ditzel, Nicole (1,2), Gläser, B. (1), Jelinkova, L. In contrast, comparative genome hybridization Luft (1,2), Jens Georg Reich (2) (2), Vogel, W. (1), Sterzik, K. (2) (CGH) is a powerful method to detect gain or (1) Franz Volhard Clinic, HELIOS Kliniken, (1) Department of Human Genetics, loss of chromosomal regions or entire chromo Berlin, Germany; (2) Max Delbrück Center University of Ulm, (2) ChristianLauritzen somes ideally covering the whole set of chromo for Molecular Medicine, Medical Faculty of Institute, Ulm somes. Limitations of the current CGH protocols the Charité, Humboldt University of Berlin, Recent studies have shown that reproductive include the necessary amount of probe DNA as Germany; (3) Infogen, Köpenickerstr. 48/49, difficulties can be associated with cytogenetic well as the time consuming hybridization steps. Berlin, Germany abnormalities like chromosomal aneuploidies. Here we report an optimized CGH protocol We studied the association between common Therefore intracytoplasmic sperm injection (ICSI)

based on whole genome amplification enabling Abstracts GfH ÖGH SGMG Tagungsband haplotypes in six relevant lipid metabolism as an treatment for severe male infertility may us to analyze small cell amounts of up to one genes with plasma lipid levels. We selected lead to an increased incidence of chromosomal cell for aneuploidy. Initially, single mononuclear SNPs in the cholesteryl ester transfer protein abnormalities in the resulting offspring. So far, blood cells were isolated by FACS, lysed and (CETP), lipoprotein lipase(LPL), hepatic triglyc Fluorescence in situ hybridisation (FISH) studies subsequently whole genome amplification was eride lipase (HL), low density lipoprotein choles with probes specific for chromosomes 1, 7, 18, performed by ligation mediated PCR. After bi terol receptor (LDLR), apolipoprotein E (ApoE), X and Y were performed to analyse the aneu otin and digoxigeninlabeling of the resulting and lecithin:cholesterol acyltransferase (LCAT) ploidy rate in morphologic al aberrant sperma PCR products of probes and controls respec genes and studied 732 individuals from 184 Ger tozoa of infertile men. Other studies focused on tively, CGH was carried out on metaphase man families. Total cholesterol (TC), lowdensity the correlation between sperm motility and non spreads of male control individuals. Following lipoprotein cholesterol (LDL), and highdensity disjunction. The aim of our study was the com hybridization for up to 3 days, washing and im lipoprotein cholesterol (HDL) were similar to parison of aneuploidy rates in spermatozoa of munodetection slides have been evaluated us those reported in other European and American infertile men prior and after selection according ing the Metasystems ISIS/CGH module. Results populations. Haplotypes derived from SNP com to morphology and motility, used in practise for of the analysis of cells with different gonosomal binations resulted in more significance and of a ICSI. Specific probes for chromosomes 13, 21 constitutions will be presented, demonstrating higher degree than did single SNPs in the geno and 16 served for FISH as disomy 13 and 21 the potential of this assay to detect aneuploidies typephenotype association analysis. Reduction could lead to trisomic severe effected livebirths in single cells. In addition to mononuclear blood of the polygenic variance attributable to haplo and trisomy 16 ist one of the frequently found cells this optimized single cell CGH has been types was estimated using variance components cytogenetic aneuploidie in abortions. Four infer successfully applied to first polar bodies from analysis. Under the biometrical genetic model, tile patients were included in this preliminary unfertilized oocytes. Potential applications might allelic association of haplotypes was highly sig study. The inclusion criteria were: oligoas include the evaluation of the frequency of aneu nificant for HDL, LDL, and the LDL/HDL ratio. thenoteratozoospermia with sperm concentra ploidies in polar bodies of women with an in The residual kinship correlation was reduced tion lower than 10x106/ml, sperm motility <30% creased risk for unbalanced chromosomal aber accordingly. The ApoE gene had a strong effect and normal sperm morphology <10%. Sperm rations in their offspring as well as after further on trait variation; however, the other genes also selection was performed under 400xmagnifica optimization of the protocol as an alternative contributed substantially. An epistatic interaction tion, using Hoffman modulation contrast. FISH procedure to detect aneuploidies prior to fertil could not be demonstrated in this sample. The procedure was made by standard protocol, us ization of the oocyte during assisted reproduc data are consistent with the notion that common ing probes for chromosome 13 and 21 (locus tion. genetic variants influence common traits. specific probes) and the probe for chromsome 16 (centromeric satellite probe). For each pa tient, we analysed 100150 spermatozoa prior and after selection, respectively. The significanty Intracytoplasmic sperm injection (ICSI) may lower aneuploidy frequencies were observed in increase the risk for imprinting defects W14 the selected group in comparison to native Bürger Joachim (1,2), Cox GF (3,4), Lip V (3), sperm population (34% vs. 431%). Incidence Mau UA (5), Sperling K (1), Horsthemke B (6), ICSI and congenital malformations: of disomy in native sperm ranged from 2% to Wu BL (3) infertility or techniquerelated risks? 7%, 2% to 10% and 1% to 4% for chromsome 1 Institut für Humangenetik, Humboldt Michael Ludwig 13,16 and 21, respectively. In the selected Universität, Berlin; 2 Munich Re, Life Division of Reproductive Medicine, group, the rates of disomy ranged from 0,7% Sciences Centre of Competence, München; Department of Gynecology and Obstetrics, to2%, 0% to 0,7% and 0,7% to 1% for chromo 3 Division of Genetics, Harvard University, University Clinic, Lübeck some 13, 16 and 21, respectively. The frequen Boston, USA; 4 Genzyme, Cambridge, USA; The course of pregnancies and health of children cy of diploidy was 15% in ected in the selected 5 Institut für Humangenetik, Universität born after assisted reproductive technologies group of sperm. Our results support the hypote Tübingen; 6 Institut für Humangenetik, are two of the most important outcome param sis of positive correlation between abnormal Universität Essen eters of the quality of the techniques. There is an sperm morphology and motility and their cyto In germ cells and the early embryo, the mam ongoing discussion, whether these parameters genetic anomalies. Spermselection prior ICSI us malian genome undergoes widespread epigenet may show worse results as compared to spon ing 400 x magnification significantly reduces the ic reprogramming. Animal studies suggest that taneous conception. Recent studies have shown incidence of aneuploidy in gametes of infertile this process is vulnerable to external factors. increased risks for the pregnancy course follow men. However, this method cannot exclude ane Abnormal DNA methylation and gene expression ing conventional IVF (e.g. premature birth, low uploid offspring of paternal origin. were found in cloned animals. In addition, birthweight), and a higher rate of major malfor cloned sheep preembryos showed hypomethy mations after conventional IVF as well as after lation of the imprinting control element of the ICSI. Of course, especially in the situation of maternal Igf2r allele, thus demonstrating that ICSI there have to be taken several genetic risks maternal imprinting in the preembryo is vulner of the father into account. However, even if there able to external factors.

medgen 14 (2002) 21 We report two children who were conceived by Genetic testing to be judged a worthwhile enter cases come from three diagnostic conditions in intracytoplasmic sperm injection (ICSI) and prise has to provide results, which from the re which increases in diagnostic complexity and found to have a chromosome 15 imprinting de cipients’ point of view have to increase their predictive power translate into individual per fect (ID) causing Angelman syndrome (AS). Mu ability to enrich their lives. sons’ practical problems of valid decisionmak tations of the imprinting centre were excluded. Parallel to medical issues with questionable ben ing: Counseling concerning i) familyrelated can Observing 2 IDs in ICSI children by chance ap efit for the individual patient a whole class of cers, ii) anomalous findings in prenatal diagno pears unlikely. One would expect 2 ICSI AS ID public policy questions emerge: genetic informa sis and iii) infertility disorders. To date we coun individuals in a sample of 40 ICSI AS patients. tion has the potential to be used out of medical seled 61 families with suspected HBOC and 27 However, we do not know of any AS ICSI patient context in ways that are contrary to the interest families with suspected CRC. Analysis in the first with a 15q11q13 deletion, a uniparental disomy of patients. In general DNA profiles can provide 30 families (2x20 clients) counseled because of or a UBE3A mutation. insights into many intimate aspects of a person suspected HBOC revealed that clients receiving The ID occurred on the maternal allele, thus pro and their family as possession of and suscepti the enriched format had a significantly better viding two explanations: (i) The failure occurred bility to certain medical conditions. It becomes knowledge and comprehension of relevant facts in the oocyte (imprint switch failure), i.e. the ma increasingly necessary to decide whether and to than clients receiving the standard format ternal chromosome carried a paternal imprint what extend third parties as employers, health (KruskalWallis OneWay Analysis of Variance: that is maintained in the zygote. (ii) The failure care and insurance companies etc. may get ac p< 0,001). The preliminary results in the sub occurred in the zygote, i.e. the oocyte carried a cess to an individual’s genetic information thus group of clients counseled because of suspect normal maternal imprint, that was not main providing them with an opportunity for genetic ed HBOC indicate that i) forclient letters can im tained in the zygote. discrimination. prove client´s knowledge and comprehension of In summary there are some indications that ICSI Steps to be taken to prohibit and to eliminate relevant facts and ii) form and content of the let might interfere with the establishment of the ma genetic discrimination include action of law ter influence this effect. ternal imprint in the oocyte or preembryo and in givers in developing legislation to protect indi Supported by BMBF, FKZ 01KU9904 crease the risk for imprinting defects. We sug viduals from it an action of medical profession gest to address this question in a longterm fol als to teach general public about it. lowup study of a large cohort of children. One We contacted ministries of health and law in

GfH ÖGH SGMG Tagungsband Abstracts GfH ÖGH SGMG Tagungsband may also consider to perform a prenatal methy several relevant countries to collect information W16 lation test in all foetuses conceived by ICSI. about awareness of the problem, legislation proj ects and laws prohibiting genetic discrimination. Over the past years the Internet has become an Patent on method of diagnostic, the BRCA1 effective medium for reaching large numbers of gene the real problem health consumer and the general public. Health Jacques Warcoin, Paris W15 related Web sites are among those pages of the In vitro diagnostic tests are patentable subject Internet with the highest rate of interest. An matter. Example of such type of patent is Privacy Protection and Human Genome analysis of Web sites with genetic discrimination BRCA1 diagnostic tests. But what is the situa Testing as topic was performed using predefined crite tion visàvis of the patent dealing with mutated Dr. HansJ. Menzel ria for the evaluation of the content. genes per se. Is it an infringement to use the Der Hamburgische Datenschutzbeauftragte, According to our preliminary analysis neither ac knowledge of the mutated gene itself or of the Baumwall 7, 20459 Hamburg, Germany tion of lawgivers nor information of general pub normal gene itself ? Is there any remedy if no so Personal data, gained by genome testings, are lic are satisfying and appropriate for the magni lution is found with the owner of the patent ? different: They reveal possible or probable future tude of the problem. French government has proposed a solution for incidents more than actual facts. Nevertheless patented inventions dealing with human health, they can determine life – not only the life of the are these solutions ForClient Letters in Genetic Counseling for tested person, but also that of untested rela satisfactory or not ? and what happen if these Hereditary Breast/Ovarian (HBOC) and tives. This has consequences for the right to ig tests are used for research purpose only ?“ Colorectal Cancer (CRC): How can their norance as well as to the requirement of techni Communicative Value be Enhanced? cal data protection. Schäfer, Dieter (1), Stein, C. (1), Raedle, J. (2), Predictive genome testings for medical purpos Kettner, M. (3) es need a medical indication, profound informa (1) Institut für Humangenetik, tions about how and how long the institution will Universitätsklinik Frankfurt/M., (2) deal with the sample and with the data, and an Medizinische Klinik II, Universitätsklinikum explicit consent. Frankfurt/M., (3) Kulturwissenschaftliches Genome testings for research purposes raise dif 3. Quality Workshops Institut Essen ferent questions: Is it allowed to use samples The knowledge about hereditary cancers is obtained for medical reasons for basic genetic growing rapidly. However, the social, ethical and QW3 research without the consent of the person af legal consequences of this knowledge are hard fected? What does anonymity really mean in re to assess. The concept of informed consent and gard to genome data? Which procedure is safe QUALITY CONTROL IN TUMOR the intention to help clients by genetic counsel enough to prevent the deciphering of a pseudo CYTOGENETICS – CAN IT BE PERFORMED ing to arrive at valid decisions obviously come nym? AT ALL? under pressure. Hence ensuring the quality of In October 2001 the conference of the data pro Christa Fonatsch counseling is of great importance. Our project is tection commissioners in Germany again de Institut für Medizinische Biologie, concerned with forclient letters, serving as a manded clear binding rules in order to protect Universität Wien, Währingerstr.10, A1090 medium for recording the actual state of knowl privacy and selfdetermination in human genome Wien edge as well as its individualized interpretation, testing. It published a detailed proposal of legal External quality control assessment in the cyto facilitating clients’ deliberation beyond the tem regulations, which now is discussed within the genetic analysis of constitutional chromosome poral limitations and other constraints of the ac political parties and the responsible administra aberrations was implemented in Germany more tual counseling session. It studies the effective tion. than ten years ago. A comparable enterprise in ness, the scope and the ethical significance of tumor cytogenetics was started last year, when enhancing the flow of communication by for participants of the tumor cytogenetics workshop Genetic discrimination a challenge for client letters. An established standard format for have decided that quality control in tumor cyto medical education of general public and such letters is varied and enriched systematical genetics is an essential prerequisite of this dis lawgivers action ly. One half of the clients receive a standard let cipline.In the meantime, Harald Rieder and Rein Tuengler, Victoria, Pelz, J. ter, the other an enriched letter (randomized, er Siebert, who have been elected as the lead Reformstudiengang Medizin, Humboldt double blinded). For this purpose information ers of the quality control group, have dispatched Universität zu Berlin, Charité Campus Mitte, gained by taperecording the whole counseling a questionnaire to all laboratories in Germany Germany session is used. Comprehensibility and useval and Austria that are concerned with tumor cyto Genetic tests are becoming a routine tool for ue of the standard format and the enriched for genetics. This is the first step in our quality as medical diagnosis. Information revealed allows mat are empirically compared, both by quantita sessment strategy and corresponds to the individuals and their families to understand and tive (interview and questionaire with a 20 point phase I of the quality assessment in cytogenet sometimes to control their inherited health risks. score respectively) and qualitative methods. The ics of constitutional chromosome abnormalities.

22 medgen 14 (2002) The problems and limitations of the standardiza Questions about interphase molecular cytoge with respect to response to therapy and survival tion of quality in cytogenetics in general are due netic analyses included detailed information in acute myeloid leukemia (AML). During the last to the used different protocols for cytogenetic about the types of samples, types of probes, years knowledge about karyotype abnormalities analysis, and limited tissue availability. In tumor and of the analytical procedures. Over 75% of and treatment outcome has increased and led to cytogenetics,however, the spectrum of methods the institutions answered the questionnaires. treatment decisions based on the karyotype of for cell cultivation and harvesting, as well as of Personal and institutional identifying information AML blasts. Therefore, a high quality of culture metaphase staining, is much broader than in was removed from the forms prior to evaluation. techniques, metaphase preparation, chromo „constitutional „ cytogenetics, and the amount The results of the survey will be presented and some banding and karyotype interpretation is of material available for cytogenetic analysis is, discussed in detail. necessary in order to provide a correct cytoge in most cases of leukaemias, lymphomas and This work was supported in part by the Bun netic result as a reliable basis to guide treatment solid tumors, very restricted. Thus a standardi desministerium für Bildung und Forschung, com decisions. Since October 1998 bone marrow zation of techniques in tumor cytogenetics is petence net ?Akute und chronische Leukämien?, and/or blood samples of 1753 AML at diagnosis very difficult to perform. project ?Zentrale Zytogenetik?, grant 01GI9974 were sent to our laboratory for chromosome One of the intentions of this session is to banding analysis. More than 85% of samples demonstrate that for different tumor types differ were sent overnight. Cells were cultured in RPMI ent procedures have proved most successful.To 1640 medium with 20% fetal calf serum and the THE PROBLEM OF SMALL CLONES – HOW prepare and stain chromosomes, but also to addition of antibiotics and antimycotics. Five MANY METAPHASES NEED TO BE evaluate the karyotype, a number of different cultures were set up in parallel for each patient: ANALYZED FOR AN ACCURATE methods have to be used even within a group of one culture without further supplements, one CYTOGENETIC DIAGNOSIS IN MDS? related diseases, such as myelodysplastic syn with the addition of thymidine and three cultures D. Haase, R. Steffens and C. Steidl dromes and acute myeloid leukaemias. with the addition of a cytokine cocktail (CC) con Department of Hematology and Oncology, Another important aspect is related to the insti taining erythropoietin, GCSF, GMCSF, SCF and Hematological Cytogenetics, GeorgAugust tutions where tumor cytogenetics is performed. IL3. One plain culture (R24) and 3 stimulated University, RobertKochStr. 40, 37075 Therefore representatives of institutes of human cultures were cultivated for 24h, colcemid was Göttingen genetics, of hematology and oncology clinics added for 2h followed by standard slide prepa Hematological diseases like MDS, typically show

and of private laboratories will present their spe ration. One culture was cultivated for 24h with Abstracts GfH ÖGH SGMG Tagungsband mosaic karyotypes at the timepoint of first diag cific points of view of tumor cytogenetics. Our out colcemid and then another 24h after the ad nosis correlating with a minor infiltration of the primary aim is to provide clinicians with correct dition of colcemid followed by standard slide bone marrow by the malignant clone. cytogenetic diagnoses that may be of great im preparation. Metaphases were analyzed for G The karyotype in MDS is one of the most signif pact on prognosis and therapy planning. Sec bands using a modified GAGbanding technique. icant prognostic markers and has a strong im ondly, the cytogenetic findings constitute the In cases showing an aberrant karyotype 20 pact on the differential diagnosis, especially if base for the isolation and characterization of af metaphases were karyotyped, in cases with no nonclonal reactive bone marrow dysfunctions fected genes, and thirdly, specific chromosome chromosome abnormalities 25 metaphases were have to be discriminated from clonal myelodys anomalies may give hints to substances and karyotyped. 947 cases (54%) showed clonal plastic syndromes. The therapeutic spectrum is mechanisms associated with chromosome aber chromosome abnormalities. In 21 of 1753 cases extremely broad, ranging from supportive care rations. In order to achieve these goals, quality (1.2%) no analyzable metaphases could be ob to intensive „AMLlike“ regimens or myeloabla assessment in tumor cytogenetics is of pivotal tained. In another 26 cases (1.5%) less than 10 tive radiochchemotherapies with retransfusion importance. normal metaphases could be analyzed and were of autologous or allogeneic stem cells. There scored as no result. A series of 273 cases with fore, it is of major interest, how much metaphas AML M0, M1 or M2 with normal karyotype were es have to be analyzed to detect even smaller investigated using FISH with a variety of differ A survey about tumor cytogenetic cell clones with an acceptable expenditure. ent probes. In 9 cases (3%) karyotype abnormal diagnostics concerning hematological In 1986 Heim and Mitelman calculated that if ities were detected. Furthermore 25 AML with malignancies in Germany and Austria 25 metaphases are completely analysed the normal karyotype were analyzed using 24color Rieder, Harald (1); Bradtke, J. (1), Siebert, R. (2) risk to miss an existing cell clone is 10% related FISH, in one patient a t(17;21) was identified. In (1) AG Tumorgenetik, Institut fuer Klinische to a clone size of 9% and 1% related to a clone conclusion, in 97.3% of AML at diagnosis a valid Genetik, Marburg; (2) Institut für size of 17%. result from chromosome analysis can be ob Humangenetik, Kiel; Germany In a pilot study we examined 77 pts. with sus tained. With high quality chromosome banding The assessment of the genetic changes using pected or verified MDS who displayed mosaic analysis a screening with FISH is not necessary metaphase chromosome and interphase nuclei karyotypes in their bone marrows. We retrospec in every case. In cases with less than 10 analyz analyses has become a mandatory part of the tively determined the analytical effort for the ver able metaphases or poor chromosome quality a diagnostic requirements in hematological malig ification of an abnormal cell clone. In the mean, screening with FISH probes covering the most nancies. Because the techniques as well as the 2.8 metaphases were needed to detect the first frequent abnormalities in AML is necessary in or procedures may vary depending on the disease (range: 1 to 25) and 5.8 (range 2 to 26) der to provide the clinician with the best possi in question, the type and the spectrum of the metaphases were needed to detect the second ble information. analyses and also the operating expenses may abnormal cell. Our analyses showed an increase Acknowledgement: supported by the Compe differ between the cytogenetic institutions. of sensitivity depending on the amount of tence net „Acute and chronic leukemias“, Pro Therefore, we started a survey among the tumor metaphases analyzed. 5 metaphases: 81% of ject „Central Cytogenetics“, Grant No. 01GI9974 cytogenetic institutions in Germany and Austria abnormal clones were detected, 10 metaphas to collect information about the diagnostics con es: 84% detection, 15 metaphases: 94% detec cerning hematological neoplasias. Investigations tion, 15 metaphases: 94% detection, 20 Quality control in solid tumor cytogenetics of metaphase and interphase chromosomes metaphases: 97% detection, 25 metaphases: Henn, Wolfram; Zang, K. D. were addressed and the persons responsable for 100% detection. Institute of Human Genetics, Saarland the respective methods were asked to fill in a Summarizing, at least concerning MDS, it has to University, Homburg/Saar, Germany mailed questionnaire. Data about the spectrum be discussed critically, to which extent the ana Solid tumors display a variety of recurrent cyto of the ages of the patients, of the diseases and lytical effort can be reduced without endanger genetic markers, some of which are of prognos of the samples used for the analyses were col ing the accuracy of cytogenetic analysis and tic and therapeutic significance. Unlike for lected. Detailed information was requested con possibly missing a clonal disease which might hematological malignancies, however, clinically cerning cultivation procedures for metaphase need intensive treatment. sound cytogenetic classification schemes are chromosome preparation including precultiva scarce for solid tumors. Major reasons for this tion treatment of the samples, cultivation media, are difficulties in cell culturing that make some Necessity for high quality in cytogenetic cultivation time, and the use of cytokines. The tumor types virtually inaccessible to chromo results in acute myeloid leukemia spectrum of the chromosome banding tech some analysis, as well as the sometimes ex Claudia Schoch, Mirjam Klaus niques, the avarage time spent on the analysis, tremely complex patterns of aberrations in solid Labor für LeukämieDiagnostik, and the type of the documentation of the find tumors that hamper the establishment of clini Medizinische Klinik III, Klinikum ings were asked. The extend of the use of mo cally valuable diagnostic schemes. This is why Großhadern, LudwigMaximilians lecular cytogenetic methods in metaphase chro cytogenetic laboratories working on solid tumors Universität München, Marchioninistr. 15, mosome analysis was addressed, e.g. the types are mostly specialized on few and sometimes 81377 München of probes, the analytical procedure, and the doc rare tumor types. Consequently, quality manage The karyotype of the leukemic blasts is the most umentation and interpretation of the findings. important, independent prognostic parameter

medgen 14 (2002) 23 ment strategies for solid tumor cytogenetics Postersession 1 a corepressor associated with mammalian tran face some specific challenges: Sept. 30th 2002 scriptional regulators, the tumor suppressor Multicenter evaluation studies for a given tu gene and trigger of terminal muscle differentia mor entity are not feasible, Foyer tion cadherin15, as well as numerous other The prognostic relevance of findings often re musclespecific genes. We observed downreg mains elusive due to limited published data, ulation of the glutathioneStransferase M1 and P103 01 (GSTM1) associated with drug metabolism. Results may be biased by specific biological The 11 EWS showed upregulation of genes en and technical problems of solid tissue cytoge coding the inhibitor of DNA binding 2 (Id2H), netics, like intratumoral heterogeneity and Frameshift mutations of RIZ, but no point probably an inhibitor of tissuespecific gene ex clonal selection in vitro, that sometimes lead to mutations in RIZ1 exons in malignant pression, as well as cyclin D, a cell cycle regu conflicting results between different tech melanomas with deletions in 1p36 lator and oncogene. Beyond it, we observed niques. Nonetheless, these shortcomings must Micaela Poetsch (1), Thomas Dittberner (2), downregulation of genes encoding the bridging not give rise to nihilism in quality management, Christian Woenckhaus (3) integrator binding protein 1 (BIN1), a tumor sup because there are in fact rules of good prac (1) Institute of Forensic Medicine, (2) pressor gene and inducer of apoptosis. tice that must be obeyed and can, in part, be Department of Dermatology, (3) Institute of In summary, two clusters totaling 33 differential externally assessed even in this field: Pathology, University of Greifswald, lyexpressed genes are able to differentiate RMS Laboratory procedures using uptodate tech Germany from EWS. These genes are likely to be instru niques aiming at the best possible number and Recently, the retinoblastoma protein interacting mental in maintaining tumorspecific character resolution of analyzed cells, zinc finger gene RIZ has been proposed as a istics and, therefore, participate in key signaling Correct documentation and interpretation of candidate for the tumor suppressor locus on processes. RMS associated genes may inhibit data with particular caution on the background 1p36, because of the common loss of RIZ1 RNA terminal muscle tissue differentiation, whereas of clinically valid experience on the given tu in human tumors. In addition, frameshift muta EWS associated genes may have profound im mor entity, tions of this gene have been demonstrated in a plications on cellcycle regulation. Internal evaluation of results by clinical follow variety of tumors with microsatellite instability. Since alterations in this region have been de

GfH ÖGH SGMG Tagungsband Abstracts GfH ÖGH SGMG Tagungsband up within multidisciplinary quality circles in cluding clinicians and pathologists, and scribed in malignant melanomas, we investigat P103 03 Continuous education in tumor cytogenetic ed DNA of paraffinembedded sections from 16 methodology. typical naevi, 19 atypical naevi, 33 primary melanoma lesions and 25 metastases by PCR InterphaseFISH using YACderivated and direct sequencing analysis of RIZ. probes reveals losses on 10p1415 in Frameshift mutations in the RIZ gene were found cervical cancer Standardisierte tumorzytogenetische in 17% of melanoma samples and 8.6% of nae Meyer, Birgit(1), KühneHeid, R.(4), Bartsch, Analysen im Routinelabor vi, but we could not demonstrate any missense O.(2), Kalscheuer, V.(3), Köllner, S.(4), Rudolph, Elisabeth Gödde, Datteln mutations in the exons of RIZ1. No LOH of the B.(1), Schneider, A.(1), Dürst, M.(1), Backsch, RIZ gene nor any microsatellite instability in six C.(1) Tumorzytogenetische Analysen sind inzwischen dinucleotide markers or in the mononucleotide (1) Gynäkologische Molekularbiologie, Teil der Differentialdiagnostik bei der Abklärung repeats IGFIIR, hMSH3, and hMSH6 could be Abteilung Frauenheilkunde, Frauenklinik der unklarer hämatologischer Befunde in onkolo demonstrated in the samples with RIZ frameshift FSU Jena, Germany, (2) Institut für klinische gischhämatologischen Schwerpunktpraxen mutations. Although our results do not explain Genetik, TU Dresden, Germany, (3) Max sowie der Kontrolle ambulanter Therapien chro the high rate of deletions in 1p36 found in this PlanckInstitut, Berlin, Germany, (4) Institut nischer Leukämien. Dementsprechend sollten tumor, they assign RIZ a potential role in the für Pathologie der FSU Jena, Germany tumorzytogenetische Befunde qualifiziert, zügig multistep tumor forming process of malignant In a previous study we had demonstrated that und kostengünstig in die kassenärztliche Ver melanoma of the skin. putative senescence genes are located on the sorgung integriert sein. short arm of chromosome 10 (10p1415). The Bei den Proben zur tumorzytogenetischen high frequency (38,7%) of LOH in this region in Analyse handelt es sich in der Regel um Blut cervical cancers (including early stage tumors) oder Knochenmarkproben, die auf der Basis der suggests that a loss of gene function at this par P103 02 „Leitlinien zur tumorzytogenetischen Labordiag ticular locus may be significant for the develop nostik“ (Berufsverband Medizinische Genetik ment of cancer. e.V.) bearbeitet werden. Ausgehend von der (Ver Expression profiling of pediatric In this study we used 10 different YACderivat dachts)diagnose werden Laborprotokolle rhabdomyosarcoma versus ewing sarcoma ed probes for interphase FISH of paraffin sec gewählt, die so in die Routine integriert sind, using oligonukleotid microarrays tions in order to detect or exclude allelic losses dass systematischen Fehlern vorgebeugt und Bär Claudia (1), Wai DH (2), Schaefer KL (2), within 10p1415 in a large number of cervical tu gleichzeitig die Qualität der Arbeitsprozesse Breit S (1), Selle B(1), Kulozik A (1) and mors and cervical high grade lesions (CIN3). nicht beeinträchtigt wird. Hierzu gibt es zum Poremba C (2) So far we could confirm frequent loss of 10p in einen des Basishandbuch zu den Labor (1) Dept. Of Pediatric Oncology, 20 cervical cancers. The analysis of 20 CIN3 le prozessen und zum anderen Entscheidungsdi Hematology, Pulmonology and Immunology, sions is under way. Those YAC clones which de agramme zur Festlegung der Kultur, Färbe und University Cildren´s Hospital Heidelberg, tect the most frequently deleted region are suit Analyseschritte für den Einzelfall, die auf der Ba Germany, (2) GerhardDomagkInstitute of able for further functional analysis (complemen sis aktueller wissenschaftlicher Erkenntnis jew Pathology, University of Muenster, Germany tation of gene defects by spheroblasts/cell fu eils nach den anamnestischen Angaben abgear The aim of this study was to identify patterns of sion). None of the genes so far mapped to chro beitet werden. differentiallyexpressed genes in pediatric rhab mosome 10p1415 represent obvious candi Die Standards eines genetischen Labors im Rah domyosarcoma (RMS) in comparison with pedi dates for tumor suppressor genes. men einer humangenetischen Kassenpraxis, das atric ewing sarcoma (EWS). We used Affymetrix auch tumorzytogenetische Analysen durchführt, Human Genome U95A oligonucleotide microar werden vorgestellt. rays carrying approximately 12,000 genes in or P103 04 der to investigate the expression profile of 23 pediatric primary tumors [12 RMS (3eRMS, 9 aRMS), 11 EWS]. Subsequent hierarchical clus Evaluation of the potential significance of a tering identified 22 of 23 tumor entities and re senescence gene locus on chromosome 4 vealed a group of 33 gene probe sets exhibiting (q31>4qtel) involved in cervical tumortype specific upregulation and clearly dif carcinogenesis ferentiating the two tumor entities. Verification of Rudolph, Bettina (1), Meyer, B.(1), KühneHeid, the mRNA levels of genes in the microarray ex R. (2), Bartsch, O. (3), Kalscheuer, V. (4), Beer, periments was carried out by duplicate hybri za K. (1), Jansen, L.(1), Köllner, S. (2), Schneider, tions and quantitative realtime polymerase A.(1), Dürst, M.(1), Backsch, C.(1) chainreaction (PCR). (1) Gynäkologische Molekularbiologie, The 12 RMS exhibited upregulation of genes Abteilung Frauenheilkunde, Frauenklinik der encoding the Cterminal binding protein (CtBP2), FSU Jena, Germany, (2) Institut für

24 medgen 14 (2002) Pathologie, FSU Jena, Germany, (3) Institut P103 06 A predominance of de novo mutations in the pa für klinische Genetik, TU Dresden; Germany, ternal germline has been reported for several (4) MaxPlanckInstitut, Berlin, Germany disorders, but parental origin of APC mutations Various LOH events on chromosome 11 lead Putative senescence genes can be mapped to in familial adenomatous polyposis (FAP) has to oncogenesis of paragangliomas specific chromosomes by the introduction of been systematically examined scarcely so far. Kathrin Riemann1,2, Simone Braun1,2, Markus chromosomes obtained from normal cells into FAP is an autosomaldominant inherited precan Pfister2, Karl Sotlar3, HansPeter Zenner2, immortal or tumour cells via microcell fusion. For cerous condition characterized by the occurence Nikolaus Blin1, Susan Kupka1,2 HPVinduced cervical cancer cells we had of hundreds to thousends of colorectal adeno 1. Department of Anthropology and Human demonstrated via microcell fusion that putative mas and extracolonic manifestations. Germline Genetics, University of Tübingen, Germany, senescence genes may be located on the long mutations in the tumor suppressor gene APC are 2. Department of Otolaryngology, University arm of chromosome 4 (4q31>4tel). In the pres responsible for FAP. We examined 916 unrelated of Tübingen, Germany, 3. Department of ent study we are evaluating the potential signif FAP families for germline mutations. Family his Pathology, University of Tübingen, Germany icance of loss of gene function in this region by tory was known sufficiently in 552 families; in Paragangliomas of the head and neck region are microsatellitePCR and interphase FISH of 8,5% out of them both parents of the index pa usually benign tumors developing from paraffin sections using YACderivated probes. tient had not developed colorectal adenomas chemoreceptors of paraganglionic origin in the Paraffinembedded tissue sections from cervical suggesting a de novo mutation. A germline mu majority of patients. These receptors play an im cancer (25 cases) were selected. Specific FISH tation was detected in 45 patients with suspect portant role in sensing and regulation of the probes from 4q were generated from YAC ed de novo mutation, in 38 of them the mutation blood CO2level. Genetic alterations in the mi clones (MPI Berlin). After slide preparation fluo was excluded in both parents. The 5 bp deletion tochondrial enzyme complex II (SDH), which is rescence in situ hybridization (FISH) was per at codon 1309 was overrepresented in the group involved in respiratory chain and citric acid cy formed. For microsatellite PCR only histological of patients with proven de novo mutation (17/38 cle reactions, have been shown to lead to spo ly verified microdissected areas consisting of = 45%), when compared with the group as a radic as well as familiar cases of these tumors. 103 104 dysplastic or tumour cells were used. whole (58/916 = 6%), thus the high frequency of Therefore we analyzed our collective containing Cases with smaller amounts of material were this mutation is not due to a founder effect but sporadic cases of patients with paragangliomas amplified by DOPPCR first. rather due to de novo mutation events. Parental for genetic changes in the SDHgenes SDHD,

Thus far interphase FISH analyses revealed al origin of de novo mutations could be traced in Abstracts GfH ÖGH SGMG Tagungsband SDHC and SDHB. We detected several new lelic losses in the 4q terminal region in about 13 families, including 3 families with large, cyto DNA mutations in samples derived from tumor 12% of cervical cancer cases. Data from LOH genetically detectable deletions. 6 mutations patients. Furthermore we demonstrated loss of analysis is still being generated. were of maternal and 7 mutations of paternal heterozygosity (LOH) usually connected with origin. Interestingly, the large deletions were of oncogenesis of various tumors. Elucidation of paternal origin only. Mutations in the paternal the genetic regions involved in tumor develop germline included two base exchanges (R564X; P103 05 ment is a basis for understanding their contribu S1201X), a 5 bp deletion at codon 1061 and a 2 tion to normal and pathogenic cell physiology. bp deletion at codon 1465. The 5 bp deletion at Breast cancer in young women: Loss of codon 1309 was found three times, but only in chromosome 8p23 reveals strong the maternal germline. In addition in the mater association with metastatic phenotype and P103 07 nal germline a 2 bp deletion at codon 1186, a disease free survival single bp deletion at codon 1548 and a splice Susanne Weber (1), HansPeter Sinn (2), site mutation at codon 653 were detected. In Chronic myeloid leukemia with a variant Susanne Popp (1), Martin Bentz (3) Claus R. conclusion, in our sample de novo APC germline Philadelphia translocation: Bartram (1), Anna Jauch (1) mutations show almost the same frequency in t(9;22;11)(q34;q11.2;q13) (1) Institute of Human Genetics, University the paternal and maternal germline, but we ob Emberger, Werner(1), Sodia, S.(1), Seewann, of Heidelberg, Germany, (2) Institute of served a difference in parental origin regarding H.L.(2), Zierler, H.(1), Petek, E. (1), Kroisel, Pathology, University of Heidelberg, the 1309 mutation and large del P.M.(1), Wagner, K.(1) Germany, (3) Department of Internal (1)Institute of Medical Biology and Human Medicine, University of Ulm, Germany Genetics, University of Graz, Harrachgasse Breast tumors occuring in young women are 21/8, A 8010 Graz, Austria (2) Department P103 09 generally more aggressive and have a higher of Hematology, Landeskrankenhaus metastatic potential than tumors of older Fürstenfeld, Krankenhausgasse 1, 8280 women. So far, the genetic alterations that may Juvenile Polyposis: Massive Gastric Fürstenfeld, Austria be responsible for these differencies are largely Polyposis is More Common in MADH4 We report a 57yearold female chronic myeloid unknown. Therefore, we applied comparative Mutation Carriers than in BMPR1A Mutation leukemia patient, first diagnosed in March 2000, genomic hybridization (CGH) to analyze DNA Carriers who was admitted to the Department of Hema copy number changes in breast tumors of 88 Friedl, Waltraut (1), Uhlhaas, S. (1), Schulmann, tology in June 2001 for further evaluation and young women (2135 years at time of diagnosis). K. (2), Stolte, M. (3), Loff, S. (4), Back, W. (5), therapy adaptation. Cytogenetic analysis re The most frequent gains of chromosomal mate Mangold, E. (1), Stern, M. (6), Knaebel, H.P. vealed the following karyotype: 46,XX t(9;22;11) rial were found on 1q (64.8%), 8q (61.4%), 17q ( (7), Sutter, C. (7), Weber, R.G. (8, 9), Pistorius, (q34;q11.2;q13). Detailed molecular cytogenet 50%), 20q (33%) and 3q (20.5%). Losses of ge S. (10), Burger, B. (1), Propping, P. (1) ic studies showed that the BCR/ABL rearrange netic material affected chromosomes 8p (1) Institute of Human Genetics Bonn, (2) ment is present and BCL 1 (CCND1) is com (19.3%), 11q (11.4%), 16q (11.4%) and 17p Dep. Medicine Bochum, (3) Dep. Pathology pletely translocated to the derivative chromo (11.4%). Gaines occured significantly more of Bayreuth, (4) Dep. Pediatric Surgery and (5) some 9. The prognostic implications as well ten than losses compared to CGH findings in the Pathology, Mannheim, (6) Dep. Pediatrics as the relevance of the involvement of the addi cohort of older patients. Loss of 8p23 and gain Tübingen, (7) Dep. Surgery Heidelberg, (8,9) tional breakpoint region 11q13 and the BCL 1 of 14q were strongly associated with positive Institute of Human Genetics, Heidelberg gene are discussed. lymph node metastasis, suggesting that these and Magdeburg, (10) Dep. Surgery Dresden genetic alterations are involved in the metastat Juvenile polyposis syndrome (JPS) is an autoso ic process of breast cancer in young women. maldominant predisposition to multiple juvenile Univariate analysis of our patients revealed a P103 08 polyps in the gastrointestinal tract. Germline mu higher risk of relapse for tumors with loss of tations in the MADH4 or BMPR1A genes have 8p23, gains of 18q and positive lymph node been found to be causative of the disease in a Frequency and Parental Origin of de novo metastasis. Using a multivariate analysis loss of subset of JPS patients. So far, no genotypephe APC Mutations in Familial Adenomatous 8p23 emerged as significant predictive variable notype correlation has been reported. We exam Polyposis (FAP) (p<0.05). These findings indicate that 8p23 plays ined 29 patients with clinical diagnosis of JPS Aretz, S. (1), Uhlhaas, S. (1), Caspari, R. (2), an important role in breast cancer of young for germline mutations in the MADH4 or Mangold, E. (1), Pagenstecher, C. (1), women and may harbor a novel tumorsuppres BMPR1A genes and identified MADH4 mutations Propping, P. (1), Friedl, W. (1) sor gene. in seven (24%) and BMPR1A mutations in five (1)Institute of Human Genetics, University of patients (17%). A remarkable prevalence of mas Bonn, Germany, (2) Department of Medicine, sive gastric polyposis was observed in patients University of Bonn, Germany with MADH4 mutations when compared to pa tients with BMPR1A mutations or without iden

medgen 14 (2002) 25 tified mutations. This is the first genotypephe P103 11 ment of a tumour suppressor gene. The DICE1 notype correlation observed in JPS. (deleted in cancer1) gene is a tumour suppres Acknowledgments. The study was supported by sor gene deleted in various sporadic cancers, in TP53 mutation and LOH analysis as a non the Deutsche Krebshilfe (Project No. 702371 cluding carcinomas of the head and neck, invasive diagnostic tool in serum and urine Pr5). breast, ovary, prostate, and others and is lo samples of bladder cancer patients calised in 13q14.3. DICE1 maps app. 4 Mb dis Bettina Schimmel(1), Kerstin Junker(2), Michael tal from the retinoblastoma gene (RB1). CCA and Utting(2), Wolfram Werner(2), Uwe Claussen(1), FISH analyses were performed on bone marrow P103 10 Regine Dahse(1) cells and unstimulated lymphocytes from 14 B (1) Institute of Human Genetics and CLL patients. Locusspecific FISH probes were Anthropology, University of Jena, Laser capture microdissection in 2D used for ATM (Ataxia teleangiectasia mutated Kollegiengasse 10, D07740 Jena, Germany coculture models as a novel tool to study gene) in 11q23, RB1 and DICE1 in 13q14.3 and (2) Department of Urology, University of tumorstromainteractions p53 in 17p13.1. Three patients showed a dele Jena, Lessingstr. 2, D07740 Jena, Germany Regine Dahse (1), Alexander Berndt (2), Peter tion of ATM, two a deletion of p53 and five pa Molecular markers are needed for better distin Hyckel (3), Frank. D. Böhmer (4), Uwe Claussen tients a deletion of RB1 and DICE1. The same guishing noninvasive papillary (pTa) and mini (1), Hartwig Kosmehl (2) percentage of RB1 and DICE1 deletion was de mally invasive (pT1) bladder carcinomas and for (1) Institute of Human Genetics and tected in 3 of them. Two of them showed a high identifying individual tumors with a high risk of Antropology, (2) Institute of Pathology, (3) er percentage of RB1 deletion than DICE1 dele recurrence or disease progression. Clinic of tion. This study indicates, that the DICE1 gene First aim of our study was to evaluate TP53 mi Cellular adhesion, migration and invasion are es also may be involved in BCLL patients. As the crosatellite and mutation analysis as an effective sential processes in tumor progression including rule, patients with 13q deletions have a concept for the characterization of superficial actions by malignant cells as well as by the stro favourable outcome. For delineation of the prog bladder tumors with different biological aggres mal microenvironment. In vitro, tumor cell stro nostic factor of DICE1 deletions in BCLL pa siveness. Mutation screening in the TP53 hot mal fibroblast interaction can be studied by 2D tients further patients have to be examined. spot region was performed in 55 microdissect and 3D coculture models. In conventional co ed superficial bladder tumor samples by direct

GfH ÖGH SGMG Tagungsband Abstracts GfH ÖGH SGMG Tagungsband culture systems, the different cell types are genomic sequencing. PCR based LOH analysis grown together in glassslide based chambers was done with two markers at 17p13. P103 13 with medium. Immunohistochemical detection at Second, there is considerable interest in the de the protein level can be easily performed, how velopment of noninvasive techniques that ever, for downstream protein and gene expres Constitutional Hemimethylation of the would detect recurrent bladder neoplasia. In or sion analysis the cell compartments have to be STK11/LKB1 Gene Promoter Predisposing der to evaluate TP53 alterations as a potential dissociated and sorted, for instance by fluores to PeutzJeghers Syndrome a Single marker for a noninvasive diagnosis of recur cence activated cell sorting or magnetic cell Case? rences or residuals and to determine whether tu separation. These procedures are labor intensive Ballhausen, Wolfgang (1), M. Werner (2), W. morspecific DNA exhibiting LOH or sequences and could possibly result in stressinduced al Fiedler (1), W. Friedl (3), U. Trautmann (4), A. harbouring a mutation can be detected in body terations in RNA expression. Abed (1) fluids, mutation screening was performed in Here we present a novel technique for cocultur (1) Department of Internal Medicine I urine, plasma and serum of patients with a mu ing and separating fibroblasts and carcinoma Section Molecular GI Oncology, University tated primary tumor. LOH analysis with two cells. It is based on cell cocultivation on a HalleWittenberg; (2) Department of markers at 17p was done in the corresponding PALM LCM membrane followed by rapid im Pathology, University Freiburg, (3) Inst. of urine and blood samples of 31 primary tumors. munostaining and laser capture microdissection Human Genetics, University Bonn, (4) Inst. As seen from our results, TP 53 inactivation by of the cell compartments. The 1.35 µm thin of Human Genetics, University Erlangen mutation seems to characterize higher malignant membrane has been originally used for the Nuremberg, Germany superficial bladder tumors which tend to recur mounting of histological tissue sections on reg We report the first case of PeutzJeghers syn and in which the probability is higher that the ular glass slides for the PALM LCM System drome (PJS)due to constitutional hemimethyla rezidives progress to muscle invasive growth (P.A.L.M. GmbH, Bernried, Germany). For iden tion of the serine/threonine protein kinase 11 pattern. tifying the tumor cell compartment, immunola (STK11/LKB1) gene promoter. The severely af Only in 2/8 cases, the TP 53 mutation from the beling for the laminin gamma 2 chain was per fected young female exhibited characteristic primary tumor could be redetected in patients formed, a marker that is expressed only in ep symptoms of PJS including pigmentation abnor urine and blood. 17 p microsatellite changes ithelial tumor cells. malities and multiple intestinal lesions. Typical with at least one marker were found in 30/31 Laser capture microdissected tumor and stroma mutations inactivating the STK11/LKB1 gene body fluids of the tumor patients (97%). Corre cells from the presented membrane based co were excluded at the RNA and DNA level (Abed lating the 17 p status found in body fluids to the culture model can be used for gene expression et al., 2001). However, hemimethylation of the status of the primary tumor, the concordance profiling and DNA based analysis. Isolation of STK11 gene was identified in mononucleated was only about 52%. amplificable DNA from the microdissected cells peripheral blood cells, and also detected in ex We conclude that TP 53 genotyping as a nonin can be performed with standard procedures. foliated oral squamous cells, and in normal gas vasive diagnostic tool in outpatient samples is Analysis of RNA molecules has been more trou tric and duodenal mucosa. Interestingly, mucosa of limited value for clinical practice. blesome and can be a major limiting step for of two polyps resected from the antrum and the downstream applications because of loss of duodenum demonstrated complete biallelic RNA quality and quantity during RNA sample methylation of the STK11/LKB1 promoter. How preparation from a limited number of immunos P103 12 ever, examination of microdissected stromal and tained cells.The RNA quality from our microdis epithelial cells of one harmartomatous polyp re sected cocultured cells was successfully vealed complete methylation restricted to the Chromosomal aberrations in patients with proved by RTPCR for a housekeeping gene epithelial fraction, while stroma cells showed BCLL the role of DICE1 transcript and for the laminin gamma 2 chain hemimethylation in accordance with the consti Pelz, AntjeFriederike; Weilepp, G; Röpke, A; gene transcript used in immunostaining the tu tutional situation. Hence, functional loss of Wieacker, P mor cell compartment The laminin cDNA was STK11/LKB1 due to two consecutive epigenetic Institute of Human Genetics, University of amplificable only in tumor cells and not in the mutations was demonstrated as a very early Magdeburg cocultivated fibroblasts indicating no cellcross event in the initiation of the hamartomaadeno B cell chronic lymphocytic leukemia (BCLL) is contamination during microdissection. macarcinoma sequence in PJS tumors. Since the most common type of leukemia in adults in constitutional hemimethylation was not found in Western countries. Clonal chromosomal aberra any of 10 PJS patients, who did not harbour a tions are found in about 50% of cases using typical germline alteration in STK11/LKB1, it conventional chromosome analysis (CCA) and in could be of significance that our sporadic PJS more than 75% using molecular cytogenetic patient simultaneously exhibited a de novo Xq+ methods, such as fluorescence in situ hybridis chromosomal aberration resulting from a X/Y ation (FISH). The most frequent changes are translocation. deletions in 13q14, 11q22~23, trisomy 12q, (Funded by BMBFNBL3 WilhelmRouxProgram deletions 17p13 and 6q21. Deletions in chromo 990038) somal band 13q14.3 may indicate the involve

26 medgen 14 (2002) P103 14 Methods: The investigation was carried out by The candidate tumor suppressor gene DICE1 questionnaires. We received 67 questionnaires (DDX26; OMIM *604331) is located within a pre completed by RP and 30 questionnaires com viously reported LOH critical region telomeric to Hereditary Nonpolyposis Colorectal Cancer: pleted by P. the RB1 gene on human chromosome 13q14.3. Frequent Occurrence of Large deletions in Results: Nearly 90% of persons in both groups Expression of DICE1 is downregulated in lung MSH2 and MLH1 genes accept predictive diagnosis. Only less than a and prostate tumors. To elucidate the reduced Mangold, Elisabeth (1), Wang, Y. (1,2), fifth see problems in taking it. Those were psy DICE1 expression in tumor cells the putative Lamberti, C. (3), Jungck, M. (3), Mathiak, M. chological problems and problems with insur promoter sequence upstream of the DICE1 tran (4), Pagenstecher, C. (1), Propping P. (1), Friedl, ance companies. Coping strategies are mainly scription start site was analyzed. This sequence W. (1) active and related to the disease: 2.4 for RP and shows a high GC content and is rich in CpG (1) Institute of Human Genetics, University 2.1 for P on a five degree rating scale. Other sites. Several fragments of the putative promot of Bonn, Germany, (2) Jiangsu Institute of coping strategies are distraction and self esteem er sequence were analyzed for promoter activi Cancer Research, Nanjing, PR China, (3) upgrading: 2.8 for RP, 2.9 for P. The profile of ty by transfecting chimeric ßgalactosidase re Department of Internal Medicine, University mood states (POMS) show less dejected (1.7 for porter constructs into COS7 cells. Promoter ac of Bonn, (4) Institute of Pathology, RP, 1.5 for P) and disgruntled mood (1.9 for RP, tivity was identified within three overlapping University of Bonn 1.8 for P), but more thirst for action (3.5 for RP, fragments. When reporter constructs exhibiting Hereditary nonpolyposis colorectal cancer (HN 3.2 for P). promoter activity were methylated by SssI PCC) is due to a deficiency in DNA mismatch re Conclusions: Compared with coping strategies methylase decreased promoter activity was ob pair. In a considerable proportion of HNPCC pa of persons at risk for late onset neurodegenera served in COS7 cells. Band shift assays tients no germline mutation in DNA mismatch re tive disorders, persons at risk for hereditary can demonstrated that the predicated DICE1 pro pair genes can be uncovered. However, large cer diseases show a more positive thinking, di moter binds proteins from HeLa nuclear extract genomic deletions cannot be detected by the version and active coping. For them genetic enriched for transcriptional factors. By analysis methods currently used. In this study we used a counselling plays a more important role than of of lung and prostate tumor cells with methylation semiquantitative multiplex PCR to detect the fered psychological care. They know that in case sensitive enzymes, a methylation pattern of the proportion of large genomic deletions in patients of positive predictive diagnosis specific prophy CpG sites in the upstream sequence of the suspected of HNPCC. Of 368 unrelated colorec

lactic and treatment programmes are available. DICE1 gene corresponding to the expression Abstracts GfH ÖGH SGMG Tagungsband tal cancer patients fulfilling the Bethesda crite level was found. Prostate tumor cell lines LNCaP ria for HNPCC, 180 exhibited microsatellite in and Du145 as well as lung tumor cell line Calu stability in their tumor tissue. Among these pa 3 and SKMes1, which show low DICE1 expres tients 68 diseasecausing point mutations (38%) P103 16 sion, are hypermethylated at all investigated re had previously been detected in the MSH2 and striction sites. In contrast, the higher DICE1 ex MLH1 genes by SSCP, heteroduplex analysis or INTERPHASE CYTOGENETICS IN CHRONIC pressing lung tumor cell line SW900 is hy DHPLC followed by direct sequencing. The re LYMPHOCYTIC LEUKEMIA USING SEVEN pomethylated. From these results we conclude maining 112 patients were examined for large DIFFERENT DNA PROBES that 1) the DICE1 promoter is rich in CpG sites genomic deletions. We identified deletions in 19 Krömer Elisabeth (1), Agis H. (2), Jäger U. (2), and shows promoter activity; 2) DICE1 is down patients (10.6%). 11/19 (58%) deletions were lo Lechner K. (2), Fonatsch C. (1) regulated in lung and prostate tumor cell lines by cated in MSH2, and 8/19 (42%) in MLH1, re (1) Institut für Medizinische Biologie der hypermethylation of the DICE1 promoter and spectively. The size of deletions ranged from one Universität Wien, (2) Universitätsklinik für may associate with tumor progression. exon to a deletion of a whole gene. Five break Innere Medizin 1 This work was supported by the Dr. Mildred points of deletions were sequenced. Alurepet Bcell chronic lymphocytic leukemia (CLL), the Scheel Stiftung. itive elements were involved in all of them. In pa most frequently diagnosed leukemia in adults, is tients meeting the Amsterdam criteria the pro characterized by a low mitotic activity of the portion of genomic deletions was 12.6%. A sim leukemic cells. Therefore by means of classical ilar proportion of deletions was found in the P103 18 cytogenetics clonal chromosome aberrations group of patients with a positive family history can be revealed in only 4050% of cases. It has for colorectal cancer and MSIH tumors, not been demonstrated that fluorescence in situ hy Detection of altered CpG methylation by meeting the Amsterdam criteria. The results of bridization (FISH) on interphase nuclei allows the denaturing highperformance liquid this study suggest that genomic deletions in detection of genomic anomalies in up to 80% of chromatography (DHPLC) both MSH2 and MLH1 genes play a consider patients. Chromosome aberrations in CLL have Betz, Beate (1), Florl, A. R. (2), Seifert, H.H. able role in the pathogenesis of HNPCC and proven to be important predictors of disease (2), Schulz, W. A. (2), Dall, P. (1) and should be part of the routine HNPCC mutation progression and survival time (Döhner et al., Niederacher, D. (1) detection protocols. 2000). Thus for riskadapted clinical manage (1) Dept. Obstetrics & Gynecology, (2) Dept. Acknowledgement: The study was supported by ment it is of great importance to characterize the Urology, HeinrichHeineUniversity, the Deutsche Krebshilfe (Grant 702371Pr 5). leukemic Bcells by FISH analysis at diagnosis. Düsseldorf, Germany In our study peripheral blood mononuclear cells Background: Promoter methylation at CpG din of 50 patients with CLL were analyzed by FISH ucleotides is an established mechanism of tumor P103 15 for deletions in chromosome bands 11q22~23 supressor genes (TSGs) downregulation in can (ATM locus), 13q14 (Rb1) and 17p13 (p53), for cer development and progression. DHPLC is a trisomy of band 12q13 and for translocations in reliable, highly sensitive technique primarily de Attitudes towards molecular genetic testing volving band 14q32 (IgH gene). With this DNA veloped for mutation detection. In this study, the and coping strategies of persons at risk for probe panel we could detect at least one chro use of DHPLC as a prescreening method for de hereditary cancer diseases and those of mosome aberration in more than 70% of the tection of CpG methylation was investigated. their partners blood samples, two changes were observed in Methods: The method was established by ana Aehnelt, Michael, Kreuz, F. R. about 25% of the aberrant cases. Consistent lyzing the methylation pattern of the CDKN2A Institute of Clinical Genetics, Medical with the data from a larger study, the most fre p16 promoter using DNA samples with different, Faculty „C G Carus“, Technical University quent anomaly was a deletion in 13q14 followed well characterized methylation patterns, either Dresden by the loss of one 11q22~23 signal and trisomy from tumor specimens or generated artificially Introduction: Gene analysis has been possible of 12q13. by treatment of unmethylated blood DNA with for several hereditary cancer diseases: HNPCC, DNA methylases. Bisulfite treatment of genomic HBOC, FAP and MEN. As there is a dominant DNA was used to deaminate unmethylated cy cancer predisposition, for persons at risk predic tosines converting them to uraciles. A 392 bp tive diagnosis can help to decide to take part in P103 17 fragment of the p16 promoter region containing early diagnosis. However, the social and psycho 35 CpGs, was PCRamplified with primers suit logical problems may grow with this new knowl DICE1 gene expression is regulated by CpG able for unmethylated as well as methylated al edge. methylation in the promoter region leles. PCR products were denatured and rena The aim of our study was to investigate the atti Röpke, Albrecht, Meyer, J., John, K., Wieacker, tured permitting the formation of heteroduplex tudes towards molecular genetic testing and P.F., Wieland, I. DNA detectable by DHPLC. To verify DHPLC coping strategies of persons at risk (RP) for Institute of Human Genetics, Ottovon positive results, PCR products were subcloned hereditary cancer diseases and those of their GuerickeUniversity Magdeburg, Germany and sequenced. partners (P).

medgen 14 (2002) 27 Results: After establishing the method, DNA Wittenberg, Halle, Germany. (2) Institut f. matography). Several missense mutations could samples from sporadic breast and ovarian can Pathologie, Universität Leipzig, Germany be determined and characterized as so far un cers were analyzed, revealing hypermethylation Intrahepatic cholangiocarcinoma (ICC) is the known polymorphisms. No functional mutations of the p16 promoter in 10% of cases. While second most common intrahepatic neoplasm were found. To investigate altered promoter complete methylation results in a peak shifted in accounting for 1030% of primary liver cancers. methylation as an alternative mechanism for ob retention time compared to the unmethylated Since the carcinogenic mechanisms leading to served TSG silencing a methylation screening blood DNA, partial methylation can be distin the development of ICC are poorly understood analysis by DHPLC has been established. To guished by appearance of additional signals rep we examined whether alterations of the fragile characterize activation of putative oncogenes, resenting diverse heteroduplex structures. histidine triad (FHIT) gene, a putative tumor sup gene expression was analysed by hybridization Conclusions: DHPLC is suitable as a prescreen pressor gene on chromosome 3p14.2, contribute of CPAs or RTPCR. One presumable oncogene ing technique to detect promoter methylation of to the carcinogenesis of ICCs. Additionally, (bt11) was shown to be frequently overex presumable tumor suppressor genes. The oncogenic mutations within exon 3 of bcatenin pressed especially in OC but gene amplification method is now used for a methylation screen which essentially lead to a constitut ively acti determined by quantitative differential PCR analysis of several putative TSGs associated vated Wnt signaling pathway and microsatellite could not be shown in BC and OC samples. The with sporadic breast cancer. instability (MSI) in the tumors as a consequence pathohistological evaluation of TSG bn39 and (funded by Deutsche Krebshilfe, Bonn) of mismatch repair deficiency were examined. oncogene bt11 in breast and ovarian tumors and LOH at the FHIT/FRA3B locus was detected in the functional characterization of both genes in two of 10 (20%) informative cases using the BC and OC cell lines will further elucidate the marker D3S1300 and in one of seven (14%) in role of these genes in development and progres P103 19 formative cases by marker D3S1234. Further sion of breast and ovarian tumors. (This project more, an altered expression pattern character is part of the DHGP „Gynecological Cancer Con Variations of DNA double strand break ized by an unusual intracellular distribution of sortium“, coordinated by Edgar Dahl, metaGen repair genes in 120 patients with bilateral FHIT protein was detected by immunohisto Pharmaceuticals GmbH) breast cancer chemistry analysis in these cases with LOH. In Gerriets, Katrin (1), Bremer, M. (2), Hector, contrast, oncogenic mutations were excluded in

GfH ÖGH SGMG Tagungsband Abstracts GfH ÖGH SGMG Tagungsband A.(1), Steinmann, D. (1,2), Karstens, J.H. (2), exon 3 of the bcatenin gene and MSI could not P103 22 Dörk, T.(1) be detected in the tumor specimens tested by (1) Clinics of Obstetrics and Gynecology and appropriate markers. In two of nine (22%) in (2) Department of Radiation Oncology, formative cases loss of heterozygosity (LOH) A physical map for the rat genome Medical School Hannover, Hannover, was displayed by marker D5S346, which is lo C. Gösele, H. Zimdahl, T. Kreitler, S. Blachut, Germany calized nearby the adenomatous polyposis C. Bräuer, A. Feldner, H. Himmelbauer, M. Defective DNA double strand break repair has (APC) gene. Thus, the Wnt pathway may be af Knoblauch, D. Ganten, H. Lehrach, N. Hübner been implicated in the etiogenesis of breast can fected in ICCs rather by the APC gene than by MaxDelbrückCenter for Molecular cer. We are investigating a hospitalbased series mutations within exon 3 of the bcatenin gene. Medicine, RobertRössleStr. 10, 13092 of 120 consecutive patients with bilateral breast MSI seems not to contribute to the development Berlin; MaxPlanckInstitute for Molecular cancer for the prevalence of inherited mutations of ICCs in our cohor t of tumors analyzed. Our Genetics, Ihnestr. 73, 14195 Berlin and variants of genes involved in chromosome results of parallel investigations of several genet One of the key goals of the German Human break repair. Truncating mutations of the BRCA1 ic alterations in ICCs provide further insights in Genome Project is the identification of genes and BRCA2 genes were identified in eight unre the carcinogenesis of ICCs. The detection of al that cause or contribute to multifactorial dis lated patients (4 BRCA1, 4 BRCA2). After a me lelic losses of genomic DNA in intronic intervals eases and the characterization of diseaseasso dian followup of 72 months (first cancer) and 30 of the FHIT gene is the first demonstration of ciated allele variants. The rat provides the most months (second cancer), local recurrence was FHIT alterations occuring in the carcinogenesis thoroughly studied experimental models for observed in 3 out of 8 identified BRCA1 and of ICCs. Therefore, the FHIT gene could be iden common human complex genetic diseases. A BRCA2 mutation carriers (38%) whereas this tified as a new player in the multistep carcino large number of quantitative trait loci (QTL) have was the case in 8 out of 103 nonBRCA1 and genesis of ICCs. been identified in the rat, which lead to the sub BRCA2 carriers (8 %). Two further patients car sequent generation of numerous congenic and ried truncating ATM gene mutations. We also subcongenic rat strains in QTL regions Tanta identified several patients with common mis mount to the identification of the underlying dis P103 21 sense substitutions of the p53, XRCC2 and ease genes is the establishment of a powerful XRCC3 proteins, or with the promoter variant set of genomic tools and reagents for this 135g>c in the RAD51 gene. Some patients har Molecular analysis of differentially species. boured alterations in more than one gene. One expressed candidate genes in breast and We have built a physical map covering the rat particularly young patient who had her first ovarian tumors genome by integrating markercontent informa breast cancer by the age of 29 and her second Czystowska, Malgorzata (1), Betz, Beate (1), tion with the constructed radiation hybrid map. by the age of 30, was found to be heterozygous SadrNabavi, Ariane (2), Meindl, Alfons (2), The integrated map shows the location of ~5500 for BRCA1 mutation 4184del4 as well as for the Dall, Peter (1) and Niederacher, Dieter BAC and PAC loci, providing BAC and PAC land RAD51 gene variant 135g>c and for a XRCC3 (1) Dept. Obstetrics & Gynecology, Heinrich marks with an average spacing of approximate missense substitution. Homozygosity for a HeineUniversity, Düsseldorf, Germany, (2) ly 600 kb across the rat genome. YAC coverage XRCC2 missense substitution was found in the Dept. Ped. Genetics, Childrens Hospital, extends well beyond 90% of the rat genome only affected patient from a family segregating LMU, München, Germany represented by some 30,000 clones. The cloned the BRCA1 mutation 5382insC. Our data sug Putative tumor suppressor genes (TSGs) and proportion of the rat genome in BAC and PAC gest a genetic defect in DNA double strand oncogenes were identified as differentially ex clones affords to 27%. break repair in at least 10% of cases with bilat pressed by „insilico“ database searches. In or We will discuss the integration of the physical eral breast cancer and a potential interaction of der to characterize the TSGs a strategy includ map with large scale genomic sequencing efforts different DNA repair gene variants in these pa ing expression analysis, LOH (loss of heterozy in this species. We propose to utilize the estab tients. gosity) analysis and mutation screening were ap lished physical map for the generation of region plied. To confirm the electronic Northern data specific BAC/PAC contigs providing a minimal TSG candidates were investigated by hybridiza tiling path for the fine mapping, sequencing, and tion of cancer profiling arrays (CPA; Clontech) cloning of disease genes in rat models. P103 20 with genespecific probes. Two TSGs (bn39, on78) were found to be differentially expressed ALTERATION OF THE FRAGILE HISTIDINE in up to 80% of breast (BC) or ovarian cancer TRIAD (FHIT) GENE IN INTRAHEPATIC (OC) samples, respectively. In a set of 200 BC CHOLANGIOCARCINOMAS and 70 OC samples gene specific LOH analysis Fiedler, Wolfgang (1), Koch, E. (1), Tannapfel, was performed to find at least 20 tumors with al A. (2), Fleig, W.E. (1), Ballhausen, W.G. (1) lelic loss of the corresponding TSG. LOH was (1) Klinik und Poliklinik f. Innere Medizin I, found in 20% to 47% of informative cases. For Sektion Molekulare Gastroenterologische two TSGs (bn39, on98) LOH positive tumor sam Onkologie, MartinLutherUniversität Halle ples were screened for mutations by DHPLC (denaturing high performance liquide chro

28 medgen 14 (2002) P103 23 and 17qgain on the one hand as well as 3p from either normal or tumour tissue data bases and 11qaberrations on the other were highly as (metaGen Pharmaceuticals). sociated with each other (p<0.001). Contrary, Aim: To validate differential gene expression of Detection of loss of heterozygosity in MYCNamplification and 11qalterations tend to a subset of 15 tumour suppressor gene candi primary bladder carcinoma show an inverse correlation (p=0.07). The out dates and 5 oncogene candidates by realtime Kommerau, Markus (1), Kuschel, C. (1,2), come of patients with alterations in 1p, MYCN RTPCR and reverse Northern analysis. Huland, H. (2), Friedrich, M. (2), Finckh, U. (1) amplification and 17qgain was inferior com Results: For this purpose total RNA was extract (1) Institute of Human Genetics and (2) pared to those without aberrations (p=0.01 and ed from histologically verified ductal breast can Department of Urology, University Hospital p<0.001). Chromosomal changes in 3p and 11q cer tissue and corresponding normal tissue. HamburgEppendorf, Hamburg, Germany were determined as markers of poor prognosis RNA from the two tissue types was then subject In bladder carcinoma, loss of heterozygosity in localized and 4s NB (p<0.01). ed to realtime RTPCR. RNA from the same tis (LOH) is a common phenomenon in chromoso Conclusion: Investigation of 5 chromosomal re sues was also used as hybridisation probe for mal loci harboring tumor supressor or related gions allows to define high and low risk sub reverse Northern analysis (cDNA arrays derived genes. In a pilot study we analyzed 20 mi groups in NB to forecast disease progression from IMAGE clones). Twenty one different tu crosatellite markers from various chromosomal and to optimize therapy. mour samples were analysed. Differences in regions in primary bladder carcinoma specimens gene expression in relation to a pool of normal of 11 patients. Microsatellites were analyzed us ductal tissue (n=5) were scored as ing PCR and fluorescencebased detection sys 1) no difference, tem ABI 310. All aberrations detected in a pri P103 25 2) up or downregulated by a factor of 24, and mary screen were checked twice by repeating 3) up or downregulated by a factor of > 4. the complete assay. This eliminated several sus Interphase FISH in myelodysplastic Six of 20 genes showed to be of particular inter pected positive results, particularly additional or syndrome with normal conventional est because a high percentage of cancers were aberrant peaks previously reported to be asso cytogenetic result consistently either up or downregulated by a ciated with bladder carcinoma. LOH or allelic im Trost, Detlef(1), Hildebrandt, B.(1), Germing, factor > 2. These are bn22, bn39, pct56, ot59 balance (AI) was found with one or more of 10 U.(2), RoyerPokora, B.(1) and ot89 (all downregulated) and bt11 (upreg markers in a total of 7 out of the 11 samples.

(1)Institute of Human Genetics and ulated). According to the in silico analysis of Abstracts GfH ÖGH SGMG Tagungsband The 10 markers included one, four, and five on Anthropology, HeinrichHeine University ot59 and ot89 we would have expected to ob chromosomes 2, 8, and 9, repectively. In a sec Düsseldorf, Germany, (2)Department of serve an upregulation of RNA in tumour tissues. ond series of tumor specimens from 10 addition Haematology, Oncology and Clinical Ongoing research: For further analysis, the full al patients we used the 10 markers with positive Immunology, HeinrichHeine University length cDNAs from the six candidate genes were findings in the pilot study. Seven out of the 10 Düsseldorf, Germany amplified by RTPCR and were cloned into pET samples showed LOH or AI in one or more of the Acquired loss of a great portion from the long 29a (Novagen) and pBK (Stratagene) for expres markers also verified by replication. None of the arms of chromosome 5 or 7, loss of whole chro sion of their protein in bacteria (generation of an 10 markers showed signs of LOH or AI in surgi mosomes 5 or 7 and trisomy 8 are frequent cy tisera for immunohistochemical analysis) and eu cal bladder tissue specimens of 21 control pa togenetic aberrations associated with myelodys karyotic cells (functional tests) respectively. Fu tients without bladder carcinoma. In summary, plastic syndromes (MDS) or myeloid leukemias ture functional tests will focus on the putative using the 10 markers selected allowed the de (AML). In this study we performed fluorescence senescence or immortalization properties of the tection of LOH or AI in 14 of 21 tumor speci in situ hybridisation (FISH) analysis on inter genes under investigation. mens (67%) but in none of 21 control specimens phase nuclei of 32 patients with AML secondary (twotailed Fisher exact test p < 0.0005). Detec to MDS and a normal karyotype in conventional tion of LOH/AI in tumor tissue may be helpful for analysis prior to and after progression of dis confirmation of histopathologically suspected P103 27 ease. BAC probes mapping to two critical inter carcinoma. vals on 5q and 7q as well as centromeric FISH probes for chromosomes 7 and 8 were analysed Tissuespecific microdissection coupled in order to search for hidden chromosomal aber with protein chip array technology to P103 24 rations of these chromosomes. In one patient a analyse proteomic differences between hidden terminal deletion of the long arm of chro normal and highly proliferative noninvasive mosome 5 was present. Monosomy of chromo squamous epithelium of the hypopharynx Alterations in 5 chromosomes define high some 7 was found in 4/32 cases. All these clon and invasive hypharyngeal carcinomas risk groups in neuroblastoma al aberrations occurred after progression to Ernst, Guenther (1), Melle, C. (1), Schimmel, B. Spitz, R, Hero, B, Ernestus, K, Berthold, F AML. Deletions of 7q31 and trisomy 8 were not (1), Bleul, A. (1), Koscielny, S. (2), von Eggeling, University Children’s Hospital, Pediatric detectable. The presented ?7 and 5q cases F. (1) Oncology, Cologne, Germany could only be detected by interphase FISH and (1) Institute of Human Genetics and Introduction: Amplification of the MYCNonco were not assessed in conventional metaphase Anthropology, Jena, Germany, (2) ENT gene and deletions in 1p36 in a subgroup of analysis. Such hidden anomalies may contribute Department, Jena, Germany neuroblastoma (NB) are well known factors of to the poor outcome in cases of myeloid disor For the molecular pathological analysis of ep poor prognosis. The prognostic impact of other ders with progression of disease and a normal ithelial hyperproliferation and invasive carcino nonrandom aberrations (17qgain, deletions in cytogenetic result in dividing cells. ma of the hypopharynx 20 deepfrozen (80 C) 3p and 11q) as determined by LOHanalyses or tissue samples from normal epithelium and hy CGHdata is not yet clear. popharyngeal cancer as well as one sample of a Methods: Using interphaseFISH on large series noninvasive but highly proliferative epithelium of primary NB the frequency and prognostic po P103 26 (HPE) were microdissected from unstained his tential of aberrations in the following chromoso tological sections with Laser Capture Micro mal regions were investigated: 1p36 (n=191), Validation and functional analysis of scope (Palm). The crude protein extracts were 2p23p24(nmyc) (n=202), 3p26 (n=188), 11q23 candidate tumour suppressor and analyzed on a strong anionic exchanger (SAX2) (n=202) and 17q21.3q23 (n=141). Deletion oncogenes in sporadic breast cancer ProteinChip Array in a ProteinChip Reader (SEL events were defined as: deletion (=monosomy of Größl,Susanne(1),Jansen,L.(1),Kühne DI PBS II; Ciphergen Biosystems). In HPE promi a specific region) and imbalance (at least two in Heid,R.(2),Gelling,S.(3),Himmelfarb,M.(3),Dahl, nent new protein peaks not detectable in the tact and additional truncated copies of the in E.(3),Schneider,A.(1),Dürst,M.(1) normal epithelium appear at 3533 Da, 5207 Da, vestigated chromosome). (1)Gynäkologische Molekularbiologie, Abt. 9955 Da, 10462 Da and 13606 Da. A peak at Results: Of al1 analyzed NB, aberrations were Frauenheilkunde, FSU Jena, (2)Institut für 10171 Da is in comparison to the normal epithe found in: 1p36: 19% deletions, 9% imbalances; Pathologie, FSU Jena, (3) metaGen lium considerable increased. In the invasive tu MYCNamplification: 19%; 3p26: 15% deletions, Pharmaceuticals, Oudenarder Str. 16, 13347 mor tissue the protein of 3533 Da is in compar 4% imbalances; 11q23: 19% deletions, 8% im Berlin ison to HPE massively overexpressed. The ex balances; 17qgain: 62%. Aberrations were Background: Over 600 putative tumour suppres pression of the 5207 Da, 10464 Da and 13606 highly associated with unfavorable clinical stage sor and oncogenes were identified as being dif Da proteins however is downregulated. The pro 4 and occurred rarely in favorable localized ferentially expressed in gynecological tumours teins of 9955 Da and 10171 Da stayed quantita stages 13 and 4s. The age of patients showing by an „in silico“ approach. This was based on tively unchanged. The results indicate that pro chromosomal alterations was significantly in the analysis of more than 4 million ESTs derived tein pattern correlate with epithelial hyperpro creased. MYCN amplification, 1p36alteration liferation and malignant growth in the hypophar

medgen 14 (2002) 29 ynx, allowing the identification of proteins asso mosome der(9)t(9;22) subdivide CML patients in carcinoma and one adenoma. This group of tu ciated with hyperproliferation on the one hand two distinct clinical subgroups: Patients with a mors showed gains of chromosomes 1, 3, 5, 6, and invasion on the other. Further analyses to deletion have median survival of about 38 7, 9, 12, and 15. Chromosome losses were identify these proteins are in progress. Moreover months, while the median survival of patients found less frequently and affected chromosome the results demonstrates the suitability of the without a deletion is about 88 months (Sinclair 2, 3, 4, 11q, 16 and 17. All patients in this study combination of tissue microdissection and pro et al, 1999). Virtually all adult patients with a are recorded in the interdisciplinary and multi tein chip array technology as a new powerful deletion in der(9) lack the ABL1BCRtranscript center trial study GPOHMET 97 (German Soci tool in cancer research. and the respective gene. Inversely, only 50% of ety of Pediatric Oncology and Hematology Ma the ABL1BCRtranscriptional negative patients lignant Endocrine Tumors). Evaluation of the exhibit a deletion in der(9) by FISH (Loncarevic clinical data and correlation to specific geno et al, 2002). We examined the incidence of types are part of our ongoing work. P103 28 ABL1BCR gene deletion and gene transcription in 54 children with CML. Lack of an ABL1BCR MEN1 gene mutations in 11 MEN1 families transcript was found in 18/54 (33%) individuals. P103 31 and their associated tumors Among the 18, 10 (55%) exhibited a deletion in Peters, Hartmut(1), Völker, D.(1) Jamrath, T. (1), der(9) by FISH. This data show for the firsttime Tuschy, U. (3), Gerl, H.(2), Ventz, M.(2) a deletion in der(9)t(9;22) in childhood CML and Frequency of the Heterozygous Germline (1)Institut für Medizinische Genetik und demonstrate that this deletion occurs at similar NBS1 Mutation 657del5 in Cancer Patients (2)Medizinische Klinik mit Schwerpunkt frequency (~33% (18/54)) as in adults. None of from Poland Gastroenterologie, Hepatologie und the children showed variants of standard t(9;22). Varon, Raymonda 1, Thomas, M 1, Maurer, M Endokrinologie des Universitatsklinikums This stays in contrast to the data we obtained 1, Stumm, M 2, Nowakowska, D 3, Charite, Berlin (3)Klinik für Innere Medizin, with adult ABLBCR RNA negative patients. 1 Institute of Human Genetics, Charité, Klinikum Erfurt, Germany Therefore, variants of standard t(9;22) do not HumboldtUniversity, Berlin, Germany MEN1 is an autosomal dominant disorder char contribute significantly to ABL1BCRRNA neg Blood relatives of patients with Nijmegen break acterized by the combined occurrence of tumors ativity in childhood CML. Moreover, we found age syndrome were reported to have an in

GfH ÖGH SGMG Tagungsband Abstracts GfH ÖGH SGMG Tagungsband of the parathyroid glands, the endocrine pan only one variant t(9;22) among 137 children with creased risk of malignancy, implying that het creas, the duodenum, and the anterior pituitary CML after GTGbased karyotype analysis. The erozygous carriers of the 657del5 mutation of gland. With the cloning of the MEN1 gene, we clinical significance of the deletion in der(9) the NBS1 gene should be more frequent among are able to identify the carrier status of individ could not be evaluated in a long time follow up, cancer patients than in the general population. uals at risk. The MEN1 gene contains 10 exons as 75 % of the children where subjected to stem Following molecular screening of blood samples and encodes a ubiquitously expressed 2,8kilo cell transplantation within a period of 1.5 years from 1683 nonselected patients with malignant base transcript. The predicted 610amino acid after diagnosis. Our data indicate a common tumors, mostly from Central Poland, we found protein product, termed menin, exhibits no ap mechanism that acts in both children and adults 16 carriers of the 657del5 mutation vs. approxi parent similarities to any previous known pro generating the deletion in der(9). The deletion af mately 9 expected. Germline 657del5 mutation teins. fecting all of the ABL1BCR gene in our cohort was found in 5/145 patients with melanoma, Eleven unrelated German MEN1 families and of patients occurs most likely simultaneously 4/232 patients with breast cancer, 3/246 patients their associated tumors were characterized us with the t(9;22) in the double strand break repair with colorectal cancer, 2/49 patients with non ing PCRSinglestrand conformation polymor process. (Sinclair PB et al (2000) Blood 95:738 Hodgkin’s lymphoma and in 1/183 patients with phism (SSCP), DNAsequencing and LOH stud 743 / Loncarevic IF et al (2002) Genes Chromo soft tissue sarcoma. No carriers were found ies on chromosome 11q13. All probands were some and Cancer 34, 2, 193200) among 164 patients with malignant tumors of clinically evaluated at university hospitals. Famil the testis and only one carrier was identified ial MEN1 was defined as endocrine tumor in two among 388 patients with various gynecologic of the three principal MEN1related tissues plus malignancies. The average age of carriers did P103 30 at least one first degree relative with an MEN1 not differ from the mean age of noncarriers with related endocrinopathy. All individuals were also the same tumors. Of 11 carriers interviewed, 9 screened biochemically, with measurements of Characteristic genetic changes in childhood reported 14 cancers in 1st and/or 2nd degree intact parathormone, gastrin, insulin, cpeptide, adrenocortical carcinoma and relatives. One of the probands with melanoma and prolactin. LOH studies revealed deletions at pheochromocytoma detected by had a first degree relative with melanoma, two 11q13 in tumor samples in 8 cases. comparative genomic hybridization probands reported lymphoma in a relative and We identified 7 novel mutation (2 deletions, 3 Swoboda Antje.(1), Loncarevic I.(1),Michel one proband with breast cancer reported two nonsense, 1 spliceside, 1 missence) in exons 2, S.(1), Ernst G.(1), Claussen U.(1),Kloetzer cases of prostate cancer in the family. In addi 8, 10 and intron 3. MEN1 mutations are distrib Ch.(2), Parlowsky T.(3), Bucsky P.(3), tion 6 patients (3 colorectal tumors, 1 melanoma, uted throughout the open reading frame with no Loncarevic IF.(1) 1 nonHodgkin’s lymphoma and 1 with sarcoma) apparent hotspots for mutation. Mutation (1) Institut für Humangenetik und were carriers of the R215W aminoacid ex screening can be offered to affected families. As Anthropologie, FSU Jena, Germany, (2) change, which is believed to be a polymorphism. there were no clinical signs of MEN1 in members Klinik für Urologie, FSU Jena, Germany, (3) These results imply that heterozygous carriers of at low risk in many of the families studied, it (3) Klinik für Kinder Und Jugendmedizin, the 657del5 mutation may have indeed an en should be possible to identify persons at risk Medizinische Universität Lübeck, Germany hanced risk to develop tumors, in particular with a mutant allele for regular clinical and bio Pheochromocytoma and adrenocortical carcino melanoma and breast cancer, and perhaps also chemical examinations. ma in children are very rare. Pheochromocytoma colorectal cancers and lymphomas. To asses the occur sporadically or within the scope of famil potential tumor supressor function of the NBS1 iar disease like VHL, MEN 2a/b or NF1. Differen gene and hence the risk factor of the 657del5 tiation of benign and malignant lesions is often mutation for the Slav population, tumor materi P103 29 difficult by standard criteria. In order to detect al of the heterozygous patients found here was characteristic chromosome imbalances that may further analysed for loss of heterozygosity. Incidence and significance of a deletion in serve as a clinical parameter 12 pheochromocy der(9)t(9;22) in children with CML toma and 11 adrenocortical carcinoma were an Schell, B.(1), Harbott, J(2), Haas, O.A.(3), alyzed by comparative genomic hybridization P103 32 Claussen, U.(1), Suttorp, M.(4), Loncarevic, (CGH). In Pheochromocytoma loss of chromo I.F.(1) some 3/3p and 11/11p was found always to (1)Institut für Humangenetik und gether and in 10 of the 12 patients analyzed. Genomic heterogeneity in advanced head Anthropologie, FSU Jena, Germany, (2) Gain of chromatin was found less frequently and and neck cancer detected by comparative Onkogenetisches Labor, Kinderklinik JLU affected chromosome 6,7,12,15,17. The data genomic hybridization Gießen, Germany, (3) St. Anna Kinderspital, differ from those obtained with adult patients, Susanne C. Tremmel (1), Karl Götte (2), CCRI Wien, Austria,(4) Bereich who show losses of 1p and 3q as the most fre Susanne Weber (1), Susanne Popp (1), Karl Hämatologie/Onkologie, Klinik und Poliklinik quent anomalies (Edström et al 2000). One tu Hörmann (2), Claus R. Bartram (1), Anna Jauch für Kinder und Jugendmedizin, mor showed an near tetraploid karyotype and a (1) Universitätskl. CGC, Dresden, Germany relatively underrepresentation of chromosome 3 (1) Institute of Human Genetics, University Data obtained with FISH probes targeting ABL1 and 11 as determined by interphase FISH. In ad of Heidelberg, Germany, (2) Department of and distal to BCR located sequences on chro dition we examined 11 childhood adrenocortical

30 medgen 14 (2002) Otolaryngology, Head and Neck Surgery, sträger in den beiden Familien haben entweder cused on APC gene and involved PCRHD, University Hospital Mannheim, Germany in dem einen oder anderen Gen die Verän PCRSSCP, PTT and DDF methods. Genetic Although the knowledge of genetic changes dur derung. Im Gegensatz zu den bisher publizierten testing involved APC gene fragments encom ing carcinogenesis of head and neck squamous Fällen zeigen in diesen Familien die Doppelmu passing exons 5, 8, 11, and 3’end of exon 15. cell carcinomas (HNSSC) has enlarged during tationsträgerinnen einen früheren Erkrankungs The study allowed us to establish distribution of the last decade, very little is known about the zeitpunkt und einen schwereren Krankheitsver mutation of APC gene in Polish population and extent of intratumoral genomic heterogeneity. lauf als die alleinigen BRCA2Mutationsträgerin identify mutations in 38% of Polish FAP families. Comparative genomic hybridization (CGH) pro nen. Insbesondere kann in der ersten Familie vides an excellent tool to gain more detailed in gezeigt werden, dass die Mutationen in den sight in genomic differences between multiple einzelnen Anlageträgern zu unterschiedlicher Ex P103 35 biopsies from advanced HNSSC. Therefore, 79 pression führen und die BRCA2 Mutationen mit HNSSC samples stage III and IV from 35 pa einem späteren Erkrankungsalter einher gehen. tients were investigated for DNA copy number Die Daten zeigen, dass BRCA1 und BRCA2 Complex karyotypes in two cases of changes. In 32 cases, the biopsies were taken Mutationen in nicht AshkenasiFamilien häufiger gastrointestinal stromal tumors from the primary tumor and a corresponding ip als bisher angenommen auftreten können und Gerresheim F(1), KolinGerresheim I(1), Boltze silateral lymph node metastasis. In 6 cases, ad deshalb eine gründliche Stammbaumerhebung C(2), Lasota J(3), Epplen JT(1), Miettinen M(3), ditional biopsies came from the primary tumor unabdingbar ist. Sollte der Stammbaum Hin Roessner A(2), SchneiderStock R(2) and in 6 cases, contralateral lymph nodes were weise ergeben, dass in beiden Linien mit BRCA (1) Department of Molecular Genetics, Ruhr analysed. In both primaries and metastases the Veränderungen assoziierte Tumore auftraten, University Bochum, (2) Department of most frequent gains of chromosome material sollte nach Detektion einer BRCA1Mutation die Pathology, OttovonGuericke University were 3q, 11q, X, 19, 5p, 8q, 1q, 7q, and 17. The Analyse des BRCA2Gens ebenfalls durchge Magdeburg, Germany (3) Department of Soft most frequent losses affected chromosomes 3p, führt werden. Die unterschiedliche Expressivität Tissue Pathology, Armed Forces 9p, Y, 18q, and 8p. There was a slightly higher der einzelnen BRCAMutationen in diesen und Washington, U.S.A. mean total number of aberrations per tumor in weiteren Familien aus unserem Beratungskollek Gastrointestinal stromal tumors (GIST) constitute primaries (11.6) compared to metastases (10.2). tiv stützt die Beobachtung, dass sich, im Gegen the most common mesenchymal group of tu

Losses were 1.5 times more frequently detected satz zu einer BRCA1Mutation, die Mammakarzi mors of the gastrointestinal tract. Cytogenetic Abstracts GfH ÖGH SGMG Tagungsband in primary tumors than in metastases. Between nome, die auf eine BRCA2Mutation zurück studies of this entity are rare, and only two mo primaries and metastases a more extensive het zuführen sind kaum von den sporadischen Karzi lecular cytogenetic studies using different chro erogeneity (average discordance 32.8%) was nomen unterscheiden. mosomespecific probes have been conducted. found compared to multiple synchronous metas We performed detailed karyotypic analysis of tases (26.5%) or within primaries (24.3%) two malignant GISTs (case 1: f/80; 30mi toses/50HPF; 22x16x8cm, liver metastases; P103 34 case 2: m/60; 170 mitoses/50HPF; 30x45 x15cm). Both tumors were CD117positive im P103 33 DNA bank of familial adenomatous munohistochemically. Mutational analysis of c polyposis and mutation spectrum of APC kit gene detected a 6bp insertion in exon 9 in Keimbahnveränderungen in beiden BRCA gene in Polish population the first case, whereas the second GIST had a Genen bei Ratsuchenden aus Plawski, Andrzej (1), Banasiewicz, T. (2), duplication of codons 574586 in exon 11. Hochrisikofamilien mit Mamma und Paszkowski, J. (2), Lubinski, J. (3), Kruszyna, T. Both GISTs revealed losses of chromosomes 14, Ovarialkarzinomen (4), Strembalska, A. (5), Slomski, R. (1) 15, and 22, alterations, which are commonly re Arnold Norbert(1), Crohns C.(1), Andreas S.(1), (1) Institute of Human Genetics, Polish ported in GISTs. Formation of a dicentric chro Fischer B.(2), Siebert R.(2), Albacht B.(3), Academy of Sciences, Poznan, Poland, (2) mosome involving chromosome 1 was another Gerber D.(3), Grote W.(2), Jonat W.(1) Department of Surgery, Medical School, feature in common to case 1 and 2. In case 1 a (1)Klinik für Gynäkologie und Geburtshilfe; Poznan, (3) Department of Genetics and stable dicentric chromosome (1;15) was charac Universitätsklinikum Kiel (2)Institut für Pathomorphology, Medical School, teristic. In case 2 the rearrangement of chromo Humangenetik; Universitätsklinikum Kiel Szczecin, (4) Department of some 1 involved a different acrocentric, presum (3)Institut für Medizinische Psychologie; Gastroenterology, CMJU, Cracow, (5) ably chromosome 14. In addition, a second di Universitätsklinikum Kiel Department of Pathophysiology and centric chromosome dic(6;11) was remark Weltweit wurden nur wenige Familien publiziert, Genetics able due to its high variability within the fusion in denen in beiden BRCAGenen Mutationen Familial adenomatous polyposis (FAP) is inherit region. Supernumary small variable rings and an auftreten und darunter nur wenige Mitglieder, die ed as autosomal dominant predisposition to ini obligatory marker chromosome indicate a more Träger dieser beiden Mutationen sind. Die meis tiate numerous polyps in colon that lead to de complex and heterogeneous karyotype than in ten Familien gehören der Gruppe der Ashkenasi velopment of colorectal carcinoma. FAP is case 1. By use of whole chromosome painting Juden an, in denen die drei Gründermutationen caused by heredited or germ line mutations in and centromer probes the composition of the 185delAG und 5382insC in BRCA1 und 6174 APC gene. Early recognition of mutation carriers marker chromosome was shown to contain parts delT in BRCA2 relativ häufig vorkommen. Eine is very important for medical treatment of per of chromosomes 15 and 22 and a piece of the Möglichkeit für die geringe Detektionsrate in sons from highrisk group. DNA bank for Polish rearranged chromosome 6. Exclusive partial nicht AshkenasiFamilien kann darin bestehen, FAP patients at the Institute of Human Genetics monosomy of chromosomes 15 and 22 repre dass die Stammbäume nicht gründlich genug er in Poznan was established in 1996. Central reg sents a novel observation in GISTs. hoben und nach Entdeckung einer BRCA1Mu istry of genetic data prevents from double analy These results indicate that chromosomal re tation die Analyse nicht fortgeführt wurde. Im sis of mutations for the same families living in arrangements in malignant GISTs are much more Rahmen unserer Analyse bei Mitgliedern aus different regions of country. Performing genetic complex than assumed to date. Increased com Hochrisikofamilien für hereditäres Mamma und tests in single laboratory covering large part of plexity of the karyotype in case 1 may reflect an Ovarialkarzinom fanden sich nun zwei Familien, Poland may lead to significant reduction of the extraordinarily high tumor growth. in denen Mutationen in beiden Genen auftreten. analysis costs due to lower investment in labo In einer Familie wurde die 185delAG in BRCA1 ratory equipment and analytical tools. DNA sam und die 5950delCT Mutation in BRCA2 und in ples from 233 persons affected with FAP and P103 36 der anderen Familie die 1081G>A Trp321Stop in 141 their family members belonging to 145 FAP BRCA1 und die 2459C>G Ser744Stop in BRCA2 families were collected. In these group 212 cas nachgewiesen. Bei der ersten Familie trat die es with typical FAP, 11 cases of attenuated FAP The breakpoints of the benign thyroid BRCA2Mutation in der väterlichen (nur Mam and 10 cases of Gardner syndrome were collect tumors with 2p21 aberrations cluster to a makarzinome) und die BRCA1Mutation in der ed and evaluated. All available DNA samples region of less than 450 kb mütterlichen (Mamma und Ovarialkarzinome) from FAP patients and their family members Belge, Gazanfer, Rippe, V., Meiboom, M., Bol, Linie auf. In der zweiten Familie traten alle were isolated from peripheral blood. FAP pa S., Drieschner, N., Bullerdiek, J. berichteten Mammakarzinomfälle nur in der müt tients and families come from NorthWest, Cen Center for Human Genetics, University of terlichen Linie auf. In beiden Familien gibt es tral, SouthWest and SouthEast regions of Bremen, Bremen, Germany eine Patientin, die Mutationen in beiden BRCA Poland. Institute of Human Genetics cooperates Cytogenetic studies of about 450 hyperplasias Genen trägt und in einer Familie noch einen with clinics of medical schools in Gdansk, Cra and adenomas have been reported in the liter männlichen nicht erkrankten verwandten mit Mu cow, Poznan, Szczecin and Wroclaw. Molecular ature. Cytogenetic aberrations occurred in about tationen in beiden Genen. Die anderen Mutation diagnostics of collected DNA samples was fo 20% of these lesions (Belge et al., 1998) and

medgen 14 (2002) 31 were divided in different subgroups. One of P103 38 Our female sample consisted of 11 sporadic these subgroups is characterized by structural breast cancer patients and 10 controls (mean rearrangements involving the chromosomal band age 45,6, range 2481) and the sample of male Evaluation of automated MNT by computer 2p21. To narrow down the breakpoints of these patients included 9 sporadic prostate cancers based image analysis aberrations, we established two cell lines from and 10 controls (mean age 54, range 2774). Dominic Varga, Brenda Patino Garcia, Isabell benign thyroid tumors showing translocations in The breast cancer patients could be identified by Michel, Karina Eiwen, Walther Vogel, Silke volving 2p21. These two cell lines and one addi higher micronucleus counts compared to con Jainta tional primary tumor were used for FISHstudies trols in nonirradiated and irradiated cells (logis University of Ulm, Department of Human with 18 BACclones derived from band 2p21. tic regresion OR:10.6, p<0.02). The prostate Gentics, AlbertEinsteinAllee 11, D 89081 The results show that the breakpoints of these cancer patients did not differ from the controls Ulm, Germany tumors map within a segment of about 450 kb in the MN counts of irradiated cells but were al The micronucleus assay (MNT) in human lym flanked by the two BACclones 339H12 and most completely separated when looking for the phocytes is a frequently used tool to assess 1069E24. As to known genes in the breakpoint spontaneous MNfrecuency (contingency table chromosomal damage or instability. It has been region, a database search allowed us to identify chi2Test:13.38, p>0.0003; due to the separation suggested to discriminate between cancer pa the zinc finger gene ZFP36L2, a member of the of the groups no OR could be calculated). tients and controls. In order to eliminate interob TIS11 early response family, within BAC clone Our results are too preliminar to draw a conclu server differences from MN scoring we started 339H12. This makes it a welllikely candidate sion out of the conunted slides, but demonstrate to establish an automated scoring procedure for gene for the 2p21 aberrations seen in benign that it may be promising to evaluate further the the cytokinesis blocked MNT based on comput thyroid tumors. G0 Micronucleus Test and to see whether or not erized image analysis (Metafer 4 vers 2.12 by it is suitable for predictive testing. Metasystems). The evaluation was based on slides of blood P103 37 samples after irradiation with gammarays with 2 Gy. The slides were stained with DAPI and P103 40 counted automatically. Our sample consisted of An infrequent haplotype of the PCTA1 gene, 26 (13 patients with sporadic breast cancer and

GfH ÖGH SGMG Tagungsband Abstracts GfH ÖGH SGMG Tagungsband located in the susceptibility region 1q42.2 Characterization of chromosomal 13 age matched controls) whom we compared 43 (PCaP), indicates linkage and association aberrations in 23 diffuse astrocytoma using to 328 counts by human observers in order to to prostate cancer cytogenetic and molecularcytogenetic obtain information on the origin of interobserver Maier, Christiane (1), Pietsch, B (1), Paiss, T (2), methods variability.For automated analysis two classifiers Roesch, K (1), Herkommer, K (2), Cancel Holland, Heidrun (1), Krupp, Wolfgang (2), with different symmetry criteria were used to Tassin, G (3), Vogel, W (1), Assum, G (1) Meixensberger, Jürgen (2), Froster, Ursula G (1) identify binucleated cells.The mean values of the (1)Abteilung Humangenetik, (1) Institute of Human Genetics, University human observers (range:284320) and the two Universitätsklinikum Ulm, Germany, (2) of Leipzig, Germany, (2) Hospital of classifiers (102106) had a large variation (coef Abteilung Urologie, Universitätsklinikum Neurosurgery, University of Leipzig, ficient variation: 0.3)and they varied concerning Ulm, Germany, (3)Centre de Recherche pour Germany their „discriminative“ power. Classifier 1 showed les Pathologies Prostatiques, Evry, France Informations on combined cyto and molecular a clearcut differentiation between carcinomas Prostate cancer (PCa) is a complex disease with cytogenetic analyses of astrocytomas are rare. and controls (logistic regression: OR:13.16,p various genetic factors involved in its etiology. We analyzed 337 metaphases of 23 primary cell value:0.0029), whereas classifier 2 did not. We While familial aggregation of PCa, observed in cultures (5 astrocytomas WHO grade II, 2 astro therefore assume that automated scoring has about 10% of all affecteds, could be best ex cytomas WHO grade III, 16 glioblastomas) using the advantage of invariant standards which al plained by the cosegregation of rare high risk classical karyotyping (CK), additionally spectral low reproducible results whereas human ob mutations, common gene variants may effect karyotyping (SKY) was performed in two, multi servers may vary in their scoring criteria and as sporadic disease risk with a low penetrance color FISH (mFISH) in three and Comparative a result end up with different numbers in MN contribution. Within the susceptibility region Genomic Hybridization (CGH) in eight of the cell scoring. The variation between human observers PCaP (1q42.243), expected to harbour a high cultures. which impairs any use of the MNT seems rather risk gene predominant in the population of We found 155 structural aberrations with high due to the identification of binucleated cells than SouthWest Europe, we characterized the can est frequency on regions 2p21>p22 and 7q32 on the definition of micronuclei. didate gene locus of the prostate carcinoma tu (CK). mor antigen1 (PCTA1) by SNP genotyping and The following translocations were identified us identified five gene haplotypes coding for four ing CK and SKY: t(1;10)(p32;q32); t(2;13)(p21; different PCTA1 proteins. The rarest of these P103 39 q22), t(2;17)(?;?), t(2;17)(p23;p11), t(3;5)(q29; variants, which we termed haplotype C2, was q33), t(7;8)(q10;p10), t(8;9)(q11;q12), t(8;12)(q11; associated with both sporadic and familial PCa. p12), t(12;20)(p13;q13), t(15;21)(p10;q10). The First results on the use of the G0 In a population based association study involv majority of these translocations are reciprocal Micronucleus Test to detect cancer ing 265 controls and 263 cases, heterozygous and the t(3;5)(q29;q33) occured in some meta patients. carriers of the C2 haplotype carried a relative phases as a balanced change. Some small intra Brenda PatinoGarcia, Dominic Varga, Isabel risk of 2,9 (CI: 1,396,13) in sporadic cases and chromosomal aberrations could only be speci Michel, Karina Eiwen, Silke Jainta, Walther 3,6 (CI: 1,309,83) in familial index patients. In fied by CK. Vogel order to elicit transmission disequilibrium of hap Conclusions: The broad spectrum of aberrations University of Ulm, Deparment of Human lotype C2 in high risk prostate cancer, familial encountered in astrocytomas requires a com Genetics, AlbertEinsteinAllee 11, D 89081 based association tests were in performed 57 bined approach. We were able to identify a suf Ulm, Germany prostate cancer pedigrees, revealing an extent ficient number of high quality metaphases in There is a considerable number of studies of cosegregation exerted by haplotype C2. Para spite of in several studies described difficulties. demonstrating a difference in micronucleus in metric linkage analyses indicated no linkage to To our knowledge most of the identified translo duction and other mutagenicity tests between the PCaP region in the total set of families, but cations and the aberrations on 2p21>p22 are controls and a variety of cancers. However, the a LOD score of 1,6 was obtained from the sev described for the first time in astrocytomas. results of these studies are frequently conflict en pedigrees with a C2haplotype carrier as in ing (Berwick and Vineis). Since the G0 Micronu dex patient. These positive association and link cleus Test (MNT) has been shown to detect age results concerning haplotype C2 may be BRCA 1 and 2 mutation carriers with high effi P103 41 due to the conspicuous allele itself, because it ciency we decided to look whether this test can codes for a unique PCTA1 protein, or could re be used for sporadic breast cancer and prostate flect linkage disequilibrium to a neighbouring Protein Profiling of Head and Neck Cancer cancer as well. gene predisposing for prostate cancer. using protein chip arrays and SELDI The blood samples where either used directly or technology irradiated with 2 Gray at the beginning of the Melle, Christian (1), Bleul, A. (1), Ernst, G. (1), culture and the MNT was performed using Schimmel, B. (1), Claussen, U. (1), Wötzel, D. cythocalasin B. The slides were stained with (2), von Eggeling, F. (1) Giemsa and counted in the microscope by dif (1) Institut für Humangenetik und ferent observers. Anthropologie, Klinikum der FSU Jena,

32 medgen 14 (2002) Jena, Germany, (2) BioControl Jena GmbH, about 20% of the cells due to a monosomy 8. P103 44 Germany The Hodgkin`s lymphoma showed aditionally a The analysis of the proteome is a possibility to homozygous deletion of the NBS1 gene in about A regulator of the Wnt signaling pathway is monitor specific changes in physiological differ 45% of the cells. Three tumors (1 colorectal tu strongly downregulated on RNA and ent tissues. The surfaceenhanced laser desorp mor, 1 melanoma, 1 neuroblastoma metastases) protein level in breast cancer tion/ionisation – mass spectroscopy technology demonstrated an amplification of the NBS1 gene Klopocki, E. 1, CastanosVelez, E. 1, Klaman, I. (SELDIMS) is a new tool to investigate particu due to a trisomy to pentasomy of chromosome 1, Kristiansen, G. 2, Leibiger, H. 1, Essers, L. 1, larly these differences by using protein chip 8. These results suggest a „second hit“ causing Pilarsky, C. 1, Weber, B. 1, Dahl, E. 1 and the technology. In this respect we compared protein a dominant negative effect of the mutant NBS1 gynecological cancer consortium profiles of head and neck cancer (HNC) to nor allele or loss of the NBS1 wildtype allele, as 1 metaGen Pharmaceuticals GmbH, Berlin; 2 mal tissue. In the present study we examined fif possible mechanisms for tumorigenesis in NBS Institut für Pathologie, Charité, Berlin teen normal and seventeen HNC tissues, re heterozygotes. CGH analyses and moleular Candidates for novel tumor suppressor genes spectively. The protein samples of both tissue analyses are in progress to get additional infor have been identified as differentially expressed species were generated and separated using mation about the mechanisms gtes. in gynecological carcinomas by analyzing four laser capture microscopy (LCM) and studied ac million ESTs with software tools developed at cordingly concerning signal constitution and in metaGen Pharmaceuticals. The selected genes tensity. Rough data were subjected to a evalua P103 43 are validated on the RNA and protein level to tion by the Ciphergen Biomarker Wizard soft confirm the in silico expression data. ware. Differences in signal intensity could be de One of the candidate genes, secreted Frizzled tected. Namely, sample deduced from HNC tis Expression analysis of mt101, a new tumor related protein 1 (SFRP1), encodes a regulator sue showed peaks that were overexpressed, suppressor candidate gene, in sporadic of the Wnt pathway. The dysregulation of the compared to the same signal in normal tissue. breast cancer Wnt signaling pathway is known to play a role in These peaks could be mainly found in the range Himmelfarb, M., Gelling, S., Hinzmann, B., cancer development. Among the target genes of of 3 to 10 kDa. A proper identification of these Klaman I., Dahl, E. and the GCC (Gynecological this pathway are the known oncogenes cmyc absorbing peaks as well as a further analysis of Cancer Consortium) and cyclinD1. SFRP1 is abundantly expressed

the data with bioinformatic tools is in progress. metaGen Pharmaceuticals GmbH, Abstracts GfH ÖGH SGMG Tagungsband on the RNA level in many human tissues, the The here described technology represents a Oudenarderstr. 16, Berlin strongest expression is observed in heart, kid powerful feasibility for the discovery of specific A candidate for a novel tumor suppressor gene, ney and mammary gland. The chromosomal lo signals in unregulated proliferating cells as well called mt101, has been identified as differential calization of SFRP1 on chromosome 8p12 is a as in cancer. ly expressed in breast carcinomas by analyzing site of frequent alterations in various tumors in four million ESTs with software tools developed cluding sporadic breast carcinoma. The SFRP1 at metaGen Pharmaceuticals. The candidate transcript is specifically downregulated in gene, mt101, is located on chromosome 5q34. P103 42 breast tumors. Hybridization of RNA from mi This region is often deleted in patients with in crodissected normal and tumor tissues to a cus vasive breast carcinomas. (Richard et al., 2000). tom designed Affymetrix chip revealed a two FISHanalysis detects amplification and LoH (loss of heterozygosity) analysis of 32 ma fold downregulation of SFRP1 in 72% of the ex deletion of the NBS1gene in tumor samples lignant ovarian germ cell tumors revealed a dele amined breast carcinomas. To investigate the from NBSheterozygotes tion in 5q34 region in 46% of the tumors protein expression pattern of SFRP1 in breast Stumm Markus (1), Varon R (2), Tönnies H (2), (Faulkner, 2000). Here we present expression cancer, we characterized an SFRP1specific an SchneiderStock R (3), Bolze K (3), Seemanova analysis of mt101 on RNA level. tibody. A downregulation on the protein level E (4), Nowakowska D (5), Steffen J (5), Sperling Mt101 encodes a transmembrane protein that is was observed in breast tumor cell lines. Protein K (2) and Pelz AF (1) expressed in the hippocampus and in several expression of SFRP1 was analyzed by immuno (1) Institut für Humangenetik, Magdeburg, nonneuronal tissues including the lung, stom histochemistry on paraffin sections of 83 breast (2) Institut für Humangenetik, Charité ach, uterus, ovaries and in the epithelial cells of tumors. Strong immunostaining is detected in Berlin, (3) Institut für Pathologie, mammary gland. The mRNA is 3,3kb in size and epithelial, stromal and endothelial cells of nor Magdeburg, (4) Institute of Biology and is specifically downregulated in breast tumors. mal mammary tissue. We observed a loss or re Medical Genetics, Charles University Northern blot hybridization on cancer profiling duction of SFRP1 expression in 73% of the ex Prague, (5) MSC Memorial Cancer Center array (Clontech) showed in 75% of mammary amined breast carcinomas. Future experiments and Institute of Oncology Warsaw carcinomas a more than two fold downregula will include functional studies to evaluate the There is still the open question, whether the tion of mt101. Real Time PCR analysis on 6 antiproliferative potential of SFRP1 after forced NBS1gene has a tumorsupressor gene (TSG) matching patient pairs (normal and tumor tissue) expression in established human tumor cell function. Recently, we could demonstrate that and 10 tumor samples with invasive mammary lines. NBS1 gene deletions are not a major cause or carcinoma confirmed the significant downreg This study is a research project within the gyne primary event in tumorigenesis of human B and ulation in breast tumors. cological cancer consortium. Tcell nonHodgkin lymphomas (NHL) (Stumm et We further examined the expression and local al., 2001). These results were in line with the ization of mt101 transcript by RNA in situ hy findings of Stanulla et al. (2000), who did not bridization. A strong expression was observed in found NBS1 mutations in NHL of childhood and normal epithelial breast cells and in benign pa P103 45 aldolescence. In contrast, Varon et al. (Cancer pilloma epithelial cells but no signal could be de Res 2001) detected missensemutations in the tected in invasive ductal carcinoma. Rare MSH2 and MLH1 missense variants are NBS1gene in childhood acute lymphoblastic Future experiments will include antibody design a frequent finding in HNPCC patients, but leukemia (ALL) patients. These latest results led and analysis of the protein expression pattern in should be treated with reserve in predictive us to perform further studies wether NBS acts breast tumors. The antiproliferative potential of testing as TSG. For this purpose we have investigated mt101 will be evaluated after forced expression Pagenstecher, Constanze (1), Wang, Y. (2), by FISH analysis 9 tumor samples (5 colorectal in established human tumor cell lines. Addition Mathiak, M. (3); Lamberti, C. (4), Leister, M. (1), tumors, 2 melanomas, 1 Hodgkin`s lymphoma, 1 al functional studies of mt101 using cell culture Gloeckner, C. (1), Steinhoff, M. (1), Ohlendorf, neuroblastoma metastases) of NBSheterozy assays will be performed. M. (1), Aretz, S. (1), Friedl, W. (1), Propping, P. gotes from Poland and Czech Republic (see also The presented study is a research project with (1), Mangold, E. (1) abstract from R. Varon). Interphase FISH was in the gynecological cancer consortium (GCC). (1) Institute of Human Genetics, University performed on isolated nuclei from parafin em of Bonn, Germany, (2) Jiangsu Institute of bedded tumor samples, by means of a Cy3la Cancer Research, Nanjing, PR China, (3) beled BACprobe (BAC159I23), containing the Department of Pathology, University of whole NBS1 gene region. A FITClabeled BAC Bonn, (4) Department of Medicine, probe flanking the NBS1region and a chromo University of Bonn some 8 centromeric probe were hybridised as Identification of germline mutations in HNPCC controls. Three samples failed, because they did patients is of major importance for predictive not contain tumor cells or there were not enough testing in family members at risk. From a sam nuclei for FISH analysis. Three tumor samples (1 ple of 569 unrelated index patients 169 meet the colorectal tumor, 1 melanoma, 1 Hodgkin`s lym Amsterdam criteria for HNPCC (AC+), 400 meet phoma) showed a deletion of the NBS1 gene in

medgen 14 (2002) 33 other Bethesda criteria for HNPCC. Tumour tis were explored by cluster analysis. Differentially likelihood ratio test implemented in GENE sue was available from 115 AC+ patients and expressed proteins were found by comparing HUNTERPLUS v1.2 in order to calculate the from 277 AC negative patients. High microsatel the serum proteins from controls and patients nonparametric Zlr and the associated LOD lite instability (MSIH) was found in 92 (80%) of with RCC. Further examinations will comprise scores. the tumours from AC+ patients and in 125 (45%) screening of a valid number of cases including We applied the aggressiveness of prostate can of tumours from AC negative patients, 10 tumour the comparison of metastatic and nonmetasta cer given by the pathological tumor grade of had low microsatellite instability (MSIL). Screen tic RCC. It can be concluded that applying this each individual and the mean age of onset of a ing for germline mutations in the MSH2 and fast and powerful ProteinChip array technology family as covariates and constructed two MLH1 gene was performed in 227 patients with it becomes possible to find tumor specific mark weighted models. The first (weight01 model) MSIH or MSIL tumours and 21 AC+ patients, ers in serum. puts weights on families with at least two cases of whom no tumour tissue was available. We of GIII prostate cancer. The second (weight 02 found 101 definitely pathogenic germline muta model) also adds weights to families with early tions (61 mutations in MSH2, 40 in MLH1). The and late onset cancer respectively. The un P103 47 mutation MSH2,c.942+3A>T was found in 9 un weighted analysis gave no evidence of linkage related index patients and represents a hot spot. to chromosome 7q. The Zlr scores increased 38 rare missense variants (18 in MSH2 and 20 in BCR/ABL DFISH should be a mandatory when including the covariates, to 2.60 (p=0.005) MLH1) and 8 intronic variants of unknown rele examination for primary diagnosis of CML using the weight01 and to 3.02 (p=0.001) using vance were detected. In some families segrega HansChristoph Duba (1), Andreas Petzer (2), the weight 02 model for late onset prostate tion of a variant with HNPCC tumours could be Adriane Mehringer (1), Martin Erdel (1), Thomas cancer. The associated LOD scores were re demonstrated, and in most of these cases no Kühr (3), Gerd Utermann (1) and Josef Thaler spectively 1.47 (p=0.009) and 1.98 (p=0.002). other mutation could be identified; immunohis (3) The markers that gave most evidence for linkage tochemistry data point towards a mutation in the (1) Inst. f. Med. Biologie und Humangenetik were exactly in the range of the published gene harbouring the variant. On the other hand, der Universität Innsbruck, Schöpfstrasse 41, prostate cancer aggressiveness region. Our re the MLH1 variant T682I was identified in a pa A6020 Innsbruck; (2) Uniklinik Innsbruck, sults support a widespread relevance of this lo tient, who was later found to carry a MLH1 non Klin. Abt. f. Hämatologie und Onkologie, cus and suggest that aggressive and late onset

GfH ÖGH SGMG Tagungsband Abstracts GfH ÖGH SGMG Tagungsband sense mutation as well. The MLH1 variant Anichstr. 35, A6020 Innsbruck;(3) IV. Int. prostate cancer is linked to chromosome 7q31 Q701H apparently segregates with the HNPCC Abteilung, A.ö. KH Wels, Grieskirchner Str. 33 in the German population. phenotype in two generations, but not in the 42, A4600 Wels, Austria youngest generation. These observations Imatinib and Interferonalpha alone or in combi demonstrate, that before offering genetic testing nation with cytostatic drugs can induce major P103 49 for persons at risk to families with unclear vari and durable cytogenetic responses in chronic ants, very careful evaluation of these variants myelogenous leukaemia (CML) patients. Since has to be undertaken. these patients have a significant survival bene Mutation and expression analysis of TRAIL Acknowledgement: This study was supported by fit, frequent follow up investigations have be receptors in human breast cancer the Deutsche Krebshilfe come clinically important. It has been shown by Seitz,Waßmuth,Fischer,Nothnagel,Jandrig, Duba et al. [Int J Oncol 17; 12451249; 2000] Schlag and Scherneck that fluorescence in situ hybridisation (FISH) MDC für Molekulare Medizin, Robert strategies reveal results comparable with con RössleKlinik, Berlin, Germany P103 46 ventional cytogenetics. In a recent report Hunt The chromosome region 8p12p22 shows fre ly et al. [Blood, 98, 17321738, 2001] have quent allelic loss in a variety of human malignan Comparative analysis of serum proteins demonstrated that a highly sensitive BCR/ABL cies, including breast cancer (BC). The tumor from patients with Renal Cell Carcinoma DFISH probe detects deletions of ABL and BCR necrosis factorrelated apoptosisinducing lig (RCC) and normal controls using protein sequences on the derivative chromosome 9. and (TRAIL) receptors TRAILR1, R2, R3 and chip arrays and SELDI technology These deletions provide a powerful and inde R4 are located on 8p21p22 and might be can Gneist, Jana (1), Melle, C. (1), Junker, K. (2), pendent prognostic indicator in CML. We have didate tumor suppressor genes in this region. Schubert, J. (2), Claussen, U. (1), von Eggeling, therefore performed FISH with the highly sensi We studied the mRNA expression of the four re F. (1) tive LSI® BCR/ABL Dual Color, Dual Fusion ceptors in a group of BC cell lines and tumors. (1) Institute of Human Genetics and Translocation Probe probe from Vysis in CML In most of the cell lines the expression of the Anthropology, University of Jena, Germany, patients treated with Interferonalpha and YNK1 TRAIL receptors was reduced. In cancer tissue (2) Clinic of Urology, University of Jena, (n=147), Imatinib (n=22) and on archived cell a 1.7, 1.2, 3.6, and 3.5fold reduction of Germany suspensions from CML patients at first diagno TRAILR1, R2, R3, R4 mRNA levels was ob The common aim in molecular analysis of malig sis (n=22). We have detected deletions in 12% served when all cancer tissues were compared nant tumors is to find specific markers which al (22/191) of patients. A BCR/ABL sensitive D with the corresponding normal samples. low an early diagnosis, the monitoring of pa FISH for detection of the Philadelphia chromo To understand the tumorspecific downregula tients after primary therapy and the optimization some and deletions of the derivative chromo tion of TRAIL receptors we have analyzed the of individual therapy regimes. In renal cell carci some 9 should therefore be incorporated into fu entire coding region of TRAILR2 and the death noma (RCC), 30 % of patients had already de ture diagnostic strategies as well as manage domain regions of TRAILR1 and R4 for the de veloped metastases at the time of primary diag ment decisions of CML. tection of mutations in a series of breast tumors nosis. Since 40 to 50 % of patients develop and BC cell lines, lymph node metastases and metastases in course of disease, a precise pre patients with a family history of BC. Although diction of the metastatic potential of primary tu functional studies have not yet performed we as P103 48 mors by reliable markers may help selecting pa sume that most of the alterations found do not tients for adjuvant therapies. In this context we alter the function of TRAILreceptors and, con tried to establish a serum cancer assay to detect Linkage of aggressive prostate cancer to sequently, do not play a significant role in BC. protein markers in serum specific to RCC. As an chromosome 7q3133 in German prostate Taken together, our mutation studies indicate alternative to 2DE in this preliminary study a cancer families that death domain receptor mutations occur at new technique was used to generate protein ex Wörner, Sonja (1), Kurtz, F (1), Haeussler, J (1), a low frequency and are not the primary cause pression patterns from serum of patients with Hautmann, R (2), Gschwend, J (2), Herkommer for the altered mRNA expression of the TRAIL RCC and controls. Surfaceenhanced laser des K (2) Vogel, W (1), Paiss, T (2) receptors in BC cell lines and breast tumors. orption and ionization (SELDI) allows the reten (1) Abteilung Humangenetik, Universität tion of proteins on a solidphase chromato Ulm, Germany, (2) Abteilung Urologie, graphic surface (ProteinChip Array) with direct Universität Ulm, Germany detection of retained proteins by timeofflight Chromosome 7q32 has been suggested to con mass spectrometry (TOFMS). Up to now we an tain genes that influence the progression of alyzed blood sera from ten patients with RCC prostate cancer from latent to invasive disease. and ten controls. The serum mixed with a spe In an attempt to confirm linkage to prostate can cial binding buffer was directly applied to differ cer aggressiveness 108 German prostate cancer ent chip surfaces (anion/cation exchanger, hy families were genotyped using a panel of 8 poly drophobic) and analyzed by the SELDI system. morphic markers on chromosome 7q. We used Differences between controls and RCC patients a multipoint allele sharing method based upon a

34 medgen 14 (2002) P103 50 comparative PCR for quantitative DNA analysis generate protein expression patterns from urine to search for STK15 and PTPN1 amplification in of patients with TCC and controls. Surfaceen gliomas previously characterized by CGH. Five hanced laser desorption and ionization (SELDI) Quantitative expression of p16INK4a and out of 16 tumours (31%) of different grade (1x II, allows the retention of proteins on a solidphase p19ARF in blood leukocytes of patients with 1x III, 3x IV) showed lowlevel DNA amplification chromatographic surface (ProteinChip Array) chronic myelogenous leukaemia (CML) of STK15 whereas we could not detect amplifi with direct detection of retained proteins by Franke, Dirk, Loncarevic, IF., Hoppe, C., von cation of PTPN1. We hypothesize that amplifica timeofflight mass spectrometry (TOFMS). Up Eggeling, F. tion of the STK15 gene may be a nonrandom ge to now we analyzed free protein in urine from Institute of Human Genetics and netic alteration in human gliomas playing a role eight patients with TCC and eight controls. For Anthropology, Friedrich Schiller University in the genetic pathways of tumourigenesis. that purpose the urine was centrifugated to re Jena, Germany move cells, supernatant was mixed with binding Recent studies revealed the CDKI p16INK4a buffer, then directly applied to strong anion ex and p19ARF, potent blockers of p53inactivation, changer chip surfaces and analyzed by the SEL as important inhibitors of cell cycle progression. P103 52 DI system. Differences between controls and The p16 gene encoding both proteins is located TCC patients were explored by cluster analysis. on chromosome 9p21. In many solid tumours Interaction of chromosome 20p11.212 and Four differentially expressed proteins were found both cell cycle inhibitors are inactivated. The pri Xq2728 in German prostate cancer families by comparing the urine proteins from controls mary aim of this work is to find a correlation be Kurtz, Florian (1), Schedel, M (1), Wörner, S (1), and patients with RCC. Especially one cluster tween p16INK4a/p19ARF expression pattern Herkommer, K (2), Häussler, J (1), Vogel, W (1), with about 17 kDa displayed a high significance. and disease progression in CML as it could be Paiss, T (2) Further examinations will comprise screening of shown in many other tumours. Valid quantifica (1) Department of Human Genetics, a valid number of cases and the identification of tion results are obtainable using real time PCR University of Ulm, GERMANY, (2) differentially expressed peaks. It can be con for amplification of p16mRNA. For our experi Department of Urology, University of Ulm, cluded that due to the application of this fast ments we used the LightCycler instrument with Germany and powerful ProteinChip array technology tu the SYBRgreen detection system and examined Chromosome 20q13 has been suggested to har mor specific markers could be also detected in blood cell derived cDNA of about 60 patients in

bour a prostate cancer (PC) susceptibility locus urine. Abstracts GfH ÖGH SGMG Tagungsband different leukaemic stages. 13 samples derived in families that were characterized by a low from chronic phases with tendency of progres number of affected familiy members, late onset sion, 26 showed no progression, 9 were cytoge disease and no maletomale transmission. As netically in remission and 10 were staged as P103 54 this epidemiologic profile is characteristic for blast crisis. Because specimen spanned a wide many German PC families we performed linkage range of mRNA concentration we decided to use analysis using 9 markers on chromosme 20. Different amplification pattern of the STK15 crossing points (CroP; calculated cycle number, There was no evidence for linkage in the whole gene in cancer cells at which a critical fluorescence level is reached) sample of 108 PC families and in the subsets Klein, Alexandra; Reichardt, W.; Wemmert, S.; as an indirect value of concentration. p16 ex with mean age of onset < or > 66 or the number Jung, V.;Zang, K.D.; Urbschat, S. pression level was determined as a ratio of of affecteds/family < or > 3 as criteria for strati Institute of Human Genetics, Saarland crossing point of sample to crossing point of fication. In a previous study we identified a sub University, Saarland University Hospital, house keeping gen. We found different levels of set of families that were linked to chromosome Building 60, D66421 Homburg/Saar, p16INK4a and p19 ARF expression in various Xq2728 (NPL > 0) but at the same time had an Germany phases of CML. To correlate distinct expression affected individual in the paternal line of their Gene amplification is found in many tumor cell values further data analyses with different CML pedigree. In this subset with the conflicting char types and is thought to be associated with tu stages or clinical outcome are in progress. The acteristics of maletomale transmission and X mor progression. Amplification of STK15, a cen median survival of CML patients varies signifi chromosomal allele sharing, two point paramet trosomal serine/threonine kinase located at cantly between individuals. Expression analysis ric linkage analysis (Genhunter 1.3) showed a 20q13, is particularly interesting because this of p16 and p19 may provide a new clinically rel maximum LOD score of 3.47 at D20S112. We aberration is commonly detected in breast can evant parameter for individual prognosis. correlated the nonparametric NPL Z scores of cer and correlates with poor prognosis. the two loci using the Kong and Cox allele shar In previous investigations we detected an ampli ing model (ASM) implemented in Genehunter con at 20q in gliomas by comparative genomic P103 51 plus v1.2. We observed an interaction of chro hybridization. The aim of our study was to detect mosomes 20p11.212 and Xq2728 in pedigrees STK15 amplified cells, especially their frequen whith maletomaletransmission and early on cy and the delineation of the amplification. Amplification of the Serine/Threonine kinase set disease (p<0.001). We examined the breast tumor cell line MCF7 STK15 gene in low grade and high grade and three primary cell cultures of gliomas gliomas (T5135, T3868, TX3868) by using simultaneous Reichardt, Wilfried (1); Brunner, C. (2); fluorescence in situ hybridization (FISH) with the Wemmert, S. (1); Jung, V. (1); Romeike, BFM P103 53 centromeric probe for chromosome 20 and a (3); Zang, KD (1); Urbschat, S (1) STK15 gene specific probe. (1) Institute of Human Genetics, Saarland Comparative analysis of proteins in urine We found in 50% of the MCF7 cells increased University, Homburg/ Saar, Germany; (2) from patients with bladder carcinoma and copy numbers of the STK15 gene in comparison Department of Otolaryngology Head and normal controls using protein chip arrays to the signal numbers of the centromeric probe. Neck Surgery, Saarland; University, and SELDI technology Surprisingly in 29% of the T5135 glioma cells we Homburg/ Saar, Germany; (3) Department of von Eggeling, Ferdinand (1), Gneist, J. (1), observed a cytogenetic visible high level ampli Neuropathology, Saarland University, Melle, C. (1), Schubert, J. (2), Claussen, U. (1), fication of the STK15 gene. T3868 and TX3868 Homburg/ Saar, Germany Junker, K. (2) cells did not show an increase of STK15 signals. Searching for amplifications in low grade and (1) Institut für Humangenetik und Interestingly we found two complete different high grade gliomas we observed an interesting Anthropologie, FriedrichSchillerUniversität pattern of the copy number gain of the STK15 correlation between the recurrence and progres Jena, Germany, (2) Klinik für Urologie, gene. In the MCF7 cells we could detect addi sion of astrocytic low grade gliomas and the am FriedrichSchillerUniversität Jena, tional distinct single signals, in contrast to the plification of the STK15 gene located in the Germany glioma cells, where we saw an expanded ampli chromosomal region 20q13. Chromosome copy The development of noninvasive methods for fication signal. These different pattern of STK15 gains in this region have been reported in astro the diagnosis of transitional cell carcinoma (TCC) gene copy gain may implicate different cytic gliomas and glioma cell lines before as well of the bladder is required, because 70 % of the pathways of tumor cell development, which have as in many cancer types including breast, col cases show relapse in the first two years. There to be proved in further investigations. orectal and ovarian cancers. The serine/threo fore, the aim is to find specific markers, which nine kinase STK15 has been reported to be am allow an early diagnosis, the monitoring of pa plified and overexpressed in breast cancer cell tients and the optimization of individual thera lines and colorectal cancer. Another candidate py regimes. Consequently, we tried to establish gene located in this region is PTPN1, a protein an assay to detect protein markers in urine spe tyrosine phosphatase nonreceptor type 1 that cific to TCC. As an alternative to 2DE a new might play a role in cell cyclus control. We used technique was used in this preliminary study to

medgen 14 (2002) 35 P103 55 P103 56 somies 3, 7, 12, 18, and translocations or aber rations including the MLT, IgH, BCL6, BCL10, p53, MYC locus by FISH on isolated nuclei Germline mutations in both BRCA genes Denaturing High Performance Liquid preparations. Using centromere probes only tri found in members of two high risk families Chromatography (DHPLC) in Screening for somies 3 and 18 (but not 7 or 12) were found in with hereditary breast and ovarian cancer Mutations in the APC (Adenomatous 29% of the cases. The trisomies were most of Arnold Norbert(1), Crohns C.(1), Andreas S.(1), Polyposis Coli) Gene ten associated with tumour stage EII, however, Fischer B.(2), Siebert R.(2), Albacht B.(3), Heinritz, Wolfram; Froster, U.G. without prognostic value. Using a new 18q21 Gerber D.(3), Grote W.(2), Jonat W.(1) Insitute of Human Genetics, University of breakpoint flanking probe set for the MLT locus (1)Department of Gynecology and Leipzig; PhilippRosentalstr.55, 04103 we were able to reliably detect the translocation Obstetrics; University Hospital Kiel; Leipzig, Germany t(11,18) on paraffin embedded material. The Germany (2)Institute of Human Genetics; FAP (OMIM: *175100, McKusick 1986) is a rare t(11;18) was found as the sole aberration in one University Hospital Kiel; Germany form of hereditary colorectal cancer. Germline lowgrade MALT only. Interestingly a higher rate (3)Institute of Medical Psychology; mutations of the APC gene were reported in pa of trisomic patterns was found with this probe University Hospital Kiel; Germany tients with Familial Adenomatous Polyposis set as compared to the centromere 18 probe. To date, only a few families have been reported (FAP). Inactivation of the APC tumor suppressor Similar results were also obtained with a BCL6 with two mutations in the BRCA genes and also gene plays a significant role in the development probe set for 3q27. Additionally we observed an individuals with two mutations. Most of the re of early onset colon cancer based on a polypo unexpected high rate of a pathological probe ported families belonged to the Ashkenazi Jew sis of the colorectum. The location of germline patterns at the IgH locus on 14q23 that does not ish population were three founder mutations, mutations in the APC gene appears to correlate seem to result from a MALT specific t(1;14). The 185delAG and 5382insC in BRCA1 and with the clinical phenotype (number of colorec results from the ongoing study will allow to eval 6174delT in BRCA2 occur relative frequently. tal adenomas, concomitants like occurence of uate the prognostic value of the detected aber One reason of the low detection rate in non further adenomas in other digestive organs, rations. Ashkenasi families could be a not extensive desmoid tumors, osteomas, thyroid cancer and evaluated pedigree and a termination of analy Congenital Hypertrophy of the Retinal Pigmen sis after the detection of a deleterious mutation tal Epithel [CHRPE]). The APC gene encodes for GfH ÖGH SGMG Tagungsband Abstracts GfH ÖGH SGMG Tagungsband in the BRCA1 gene. During the mutation analy 2843 amino acids and more than 800 mutations P103 58 sis on members of hereditary breast and ovari are described. To provide a fast mutation an cancer families we found two families with screening we analyzed the APC gene by using VHL2C Phenotype in a German von Hippel the presence of two mutations, one in BRCA1 DHPLC (Denaturing High Performance Liquid Lindau Family with Concurrent VHL and the second in BRCA2. The alterations in the Chromatography) mutation analysis followed by Germline Mutations P81S and L188V first family were 185delAG in BRCA1 and automated sequencing of suspicious fragments. Weirich Gregor(1), Klein B (2), Wöhl T (3), 5950delCT in BRCA2 and in the second family We investigated the genomic DNA of 11 patients Engelhardt D (4), and Brauch H (2) 1081G>A W321X in BRCA1 and 12459C>G with a clinical diagnosis of FAP. 16 different se (1)Institute of Pathology, Technische S744X in BRCA2. The BRCA2 mutation in the quence variations could be identified: 8 muta Universität München, (2)Dr. Margarete first family was detectable only in the paternal tions and 8 polymorphisms. All mutations lead FischerBosch Institute of Clinical branch (only breast cancer cases) whereas the to a truncation of the predicted protein and have Pharmacology, Stuttgart, (3) Wilex BRCA1 mutation segregated from the maternal disease causing character. According to our re Biotechnology GmbH, München, branch (breast and ovarian cancer cases). In sults DHPLC is an efficient and fast screening Medizinische Klinik II, Klinikum Großhadern both families double heterozygote affected fe method to identify mutations in the APC gene Universität München males and one unaffected double heterozygote which can be applied to optimize further diag VHL germline mutations predispose to von Hip male were observed. Other affected and unaf nostic and therapeutic strategies in families with pelLindau disease (VHL), a tumor syndrome in fected family members harbored the mutation ei hereditary colon cancer. volving multiple sites including eyes, cerebellum, ther of BRCA1 or BRCA2. In contrast to the up spinal cord, kidney, adrenal gland, pancreas and to now reported cases the affected double het epididymis. VHL is distinguished into type 1 dis erozygote patients displayed an earlier onset P103 57 ease without pheochromocytoma, and type 2 and severe progression of disease than the disease with pheochromocytoma. The VHL pro members with the solely BRCA2 mutation. In tein (pVHL) is a key player of a regulatory path particular in the first family further analysis re MolecularCytogenetic Analysis of 38 way of controlled protein degradation. Some vealed differential expression of the BRCA2 mu Gastrointestinal MALT Lymphoma VHL germline mutations show a preponderance tation especially a later onset of breast cancer. Erdel Martin (1), Dirnhofer S (2), Tzankov A (3), for adrenal gland tumors. They are located at nu Thus previous reports and the present study em Duba HC (1), Utermann G (1), Siebert R (4), cleotides affecting amino acid changes in the phasize the fact that the presence of two inde Krugmann J (3) pVHL beta or alpha domain critical for protein pendent mutations sepsiegating in the same (1)Institut für Medizinische Biologie und protein interaction. Phenotypically these muta family may not be infrequent outside the Ashke Humangenetik, Universität Innsbruck, tions differ with respect to a low or high risk for nazi Jewish community. It is important to obtain Austria, (2) Institut für Pathologie, additional renal cell carcinoma. Here we de an accurate family history in the context of ge Universität Basel, Switzerland, (3) Institut scribe a familiy with two concurrent VHL netic testing. As to the implications of double für Pathologische Anatomie, Universität germline mutations affecting both the pVHL beta heterozygosity on genetic counselling we recom Innsbruck, Austria, (4)Institut für and alpha domain resulting in a pheochromo mend complete screening of both genes BRCA1 Humangenetik, Universitätsklinikum Kiel, cytoma only phenotype. Among 16 members of and BRCA2 in families suspicious for heredity of Germany a VHL family 9 members were previously diag breast and/or ovarian cancer in both paternal We are studying archival paraffin embedded tis nosed for pheochromocytoma and identified to and maternal branch. Without the knowledge of sues from 38 well documented cases of surgi carry a nucleotide 775 C>G (L188V) germline the second mutation in these families, many as cally resected primary gastrointestinal lym mutation (Glavac et al. Hum Genet 98:271280, symptomatic individuals would have been given phomas of the mucosa associated lymphoid tis 1996). Refined VHL mutation analysis including a negative test result and be falsely reassured. sue (MALT) for the occurrence of different report new exon 1 primers 13F and 13R, SSCP, DH The variable expression of the different BRCA ed cytogenetic abnormalities. The aim is to cor PLC and sequencing analysis detected a 454 mutations in the reported and other families of relate cytogenetic aberrations with the tumour (C>T; P81S) mutation that cosegregated with our cohort support the evidence that in contrast grading, staging and the clinical outcome, and the 775 (C>G; L188V) mutation in all patients. to breast cancers due to BRCA1 alterations tu to establish the prognostic value of the aberra The concurrent P81S may influence the pheno mors attributable to BRCA2 mutations are bare tions detected. The tumour samples included 15 typic consequences of VHL disease resulting in ly distinguishable from sporadic cases. classical marginal zone Bcell lymphomas (low the rare pheochromocytoma only phenotype grade MALT), 6 lymphomas with low and high (VHL type 2C) of this family. grade component (mixed type), and 17 diffuse large Bcell lymphomas (high grade MALT). The lymphomas were staged according to the mod ified Ann Arbor system by Musshoff: 17 patients presented with stage EI, 19 with EII, and 2 with EIV. So far we tested for some of the most fre quently reported genetic abnormalities, i.e. tri

36 medgen 14 (2002) P103 59 H2 respectively. Regarding the Amsterdam I and predict whether or not a BRCA1/2mutation is II families only, the percentage of mutation pos present. itive families is 13/33. Additional 12 truncating Supported by Deutsche Krebshilfe Evidence of hidden monosomy 13 in a mutations were found in families or patients ful cohort of 32 multiple myeloma patients filling the Bethesda criteriaIncluding these fam Wieland, Claudia (1), Schneider,P. (2), ilies, the percentage of mutation positive fami Hildebrandt,B. (1), Redmann,A. (1) and Royer P103 62 lies in the cohort of families with hereditary col Pokora,B. (1) orecatal cancer is 25/45. All tumors with truncat (1) Institute of Human Genetics and ing mutations were MSIH whereas none of the Familial occurrence of lowgrade Anthropology, Heinrich Heine University of tumors without mutation in H1 or H2 was MSI astrocytomas Duesseldorf, Germany, (2) Department of H. IH however gave a protein expression for H1 Müsebeck, Jörg (1), Lohner, R. (1), Haematology, Oncology and Clinical in more than 10% of the tumor cells for two tu Reifenberger, G. (2), Weber, R.G. (3) Immunology, HeinrichHeine University of mors caused by mutations in H1, one tumor (1) Zentrum für Humangenetik, Universität Duesseldorf, Germany without mutation showed a loss of expression Bremen, (2) Institut für Neuropathologie, We analysed 32 multiple myeloma (MM) patients for H1. The tumor with mutations in H1 or H2 HeinrichHeineUniversität, Düsseldorf, (3) in part with complex cytogenetically visible aber were found in the whole colon, whereas tumors Institut für Humangenetik, Ottovon rations not involving chromosome 13, for the of the mutation negative group clustered in the GuerickeUniversität, Magdeburg presence of deletions in the chromosomal region rectum and sigma. H1 and H2 negative families Brain tumours like astrocytomas are most often 13q14 or monosomy 13. For the FISH analyses show the highest relative percentage of colorec sporadic diseases and show two age peaks in we used BACs from the critical region shown to tal tumors (79%), compared to families with mu frequency, namely in childhood and in the sixth be deleted in CLL covering the markers tations in H1(68%) and families with mutations to seventh decade, respectively. Mortality due to D13S663E, D13S1168, D13S1220, D13S319 and in H2 (58%). Families with mutations in H2 show brain tumours accounts for about three per cent D13S272; parts of this region have also shown the most associated tumor diseases. The mean of all tumours in men. In most cases, a multifac to be deleted in multiple myeloma patients. age of onset for colorectal cancer in the first torial genesis is thought to be causative. Less Close to this region, overlapping with D13S272, group was 39 years versus 48 years in the sec often, brain tumours occur because of an in the BCMS gene was identified, which is dis

ond. born, genetic susceptibility to tumours of differ Abstracts GfH ÖGH SGMG Tagungsband cussed as a candidate tumor suppressor gene Of the families with a hereditary predisposition ent tissues (e. g. LiFraumeni syndrome, Turcot for BCLL. The following 13q14 BACs were for colorectal cancer, only one third carry muta syndrome) or as part of a syndrome with fea used: RPCI11 236M15, RPCI11 346I19, RPCI tions in the DNA mismatch repair genes. The tures in addition to tumour predisposition (e. g. 11 34F20 and RPCI11 480P3. We performed residual families most likely carry mutations in the neurofibromatoses, tuberous sclerosis, and FISH on interphase nuclei of methanol/acetic genes not involving the DNA repair system as Cowden syndrome). We report on a family with acid fixed bone marrow samples using one the tumors are MSS. These results show that four affected males diagnosed with lowgrade 13q14 BAC indirectly labelled with digoxigenin there are at least two entities of hereditary col astrocytomas but without any other symptoms together with a 13q telomeric probe indirectly la orectal cancer and that these entities show dif suggesting a syndromic disorder. An astrocy belled with biotin to prove the presence of ferent clinical features. toma was first observed in a 12yearold boy, monosomy 13. Hybridization of the probes was later in his brother at age 19 and his son at the detected with antiDIGFITC and avidinCy3 re age of 8 years. The second, today 56yearold spectively. Only bone marrow samples of the brother of the index patient has not suffered first aspiration were analysed, containing >20% P103 61 from any tumour, whereas his son recently de plasma cells. We scored 300 nuclei for each veloped an astrocytoma at age 22. The tumour probe set. Cut off values for all probes were de A comparison of methods to determine the of the latter patient can be analysed for chromo termined on 4 control samples. We found mono risk to develop breast cancer and to predict somal imbalances by comparative genomic hy somy 13 in 46,8% (15) of the patients. 7 of the the BRCA1/2 mutation status bridisation (CGH). All patients were operated on patients had a poor prognosis and 6 of them The German Hereditary Breast and Ovarian successfully and are alive. Maletomale trans showed monosomy 13. A deletion was found Cancer Consortium, Goecke, T.(1) mission and the healthy 56yearold male sug only in one patient, where the 4 BACs used, (1) Institute of Human Genetics, University gest autosomal dominant inheritance with in were absent. Further analysis of the extension of of Düsseldorf, Germany complete penetrance. To our knowledge, famil the deletion is in work. The results indicate a The Deutsche Krebshilfe has granted a multicen ial occurrence of dominantly inherited lowgrade correlation of monosomy 13 with poor progno ter project to 12 German centres. The centres astrocytomas is exceedingly rare. We are cur sis in MM. Statistical computation has to be offer persons from breast and/or ovarian cancer rently investigating the TP53 tumour suppressor done. families qualified genetic and medical coun gene and would continue to perform CGH analy selling, participation in an intensified early can sis mentioned above to reveal the genetic aber cer detection program, and provision of psycho ration underlying the disease in this family. P103 60 logical support. Families who satisfy the inclu sion criteria are offered genetic testing. At pres ent more than 1000 families are under study; in Hereditary colorectal cancer: two different P103 63 about 1 out of 3 families a mutation in BRCA1 or entities in regard of genetics and clinic BRCA2 has been detected (German Consortium MuellerKoch,Yvonne1, Vogelsang H5, Keller for Hereditary Breast and Ovarian Cancer, Int. J. Detection of Complex Germline G7, Kopp R2, Lohse P3, Gross M4, Baretton Cancer 97: 472480, 2002). Rearrangements of the BRCA1 Gene Using G5, Aust D5, Kerker B1, Henke G1, Daum J1, One aim of this project is to develop criteria that Semiquantitative Multiplex PCR Method Heydrich S5, Plaschke S7, Schiemann U4, better identify BRCA1 or BRCA2 positive fami Wera Hofmann (1), Heike Görgens (2), Denise Muders M5, HolinskiFeder E1 lies. A better identification of those families Horn (1), Christine Hüttner (3), Hans K. Dept. Med. Genetics1, Surgery2, Clinical could allow a more selective referral for genetic Schackert (2), Siegfried Scherneck (1) Chemistry3, Gastroentrology4, Pathology5, counselling, intensified surveillance, and genet (1) Department of Tumor Genetics, Max University of Munich, Germany; Dept. of ic testing. Delbrück Center for Molecular Medicine, Surgery6 and Pathologyc, Technical As yet data of 1001 persons with BRCA1 and Germany (2) Department of Surgical University of Munich, Germany 731 persons with BRCA2 testing completed Research, University of Technology The families described here are taken from a have been reported to the central register. The Dresden, Germany (3) Department of large cohort (254) of suspected HNPCC families. risk of breast and ovarian cancer was evaluated Gynecology and Obstetrics, Charite Virchow Here we report about the microsatellite analysis, in the reported families using various instru University Hospital, Germany the immunohistochemistry (IH), age of onset, tu ments (Claus tables, MLINK as incorporated in Most described germline BRCA1 mutations are morspectrum and tumorlocalisation of two dif Cyrillic 2.1, BRCAPro, Gail model). We also test single base pair substitutions or small sequence ferent entities: First, 25 families with truncating ed the accuracy to predict the mutation status deletions and insertions causing frameshift and mutations in hMLH1(H1) or hMSH2 (H2) and mi using standard methods (MLINK, Couch, Shat truncation of the BRCA1 protein. However, at crosatellite instability in the corresponding tu tuckEidens, Frank, BRCAPro). present only twothirds of expected mutations mors and second, 20 Amsterdam positive fami The preliminary results demonstrate that the var are detected in high risk breast and ovarian can lies without mutations in H1 or H2 and without ious methods produce quite different probabili cer families. One reason for this is the existence MSI in the tumors. The 25 mutations described, ties of becoming affected by breast or ovarian of complex germline rearrangements in the include 14 different new mutations for H1 and cancer. Also neither method could accurately BRCA1 gene that are not detectable by standard

medgen 14 (2002) 37 diagnostic techniques like direct sequencing. To Dept. of Obstetrics and Gynaecology at the strategies, major genes involved in tumorgene detect such large deletions or duplications en University,81377 München sis and metastasis may be identified. compassing one or more exons of the BRCA1 The German Consortium for Hereditary Breast Using virtual northern, the metaGen company gene we developed a semiquantitative Multiplex and Ovarian Cancer (granted by the Deutsche has identified over 600 genes differentially ex PCR method. Short exon fragments correspon Krebshilfe) has recently published BRCA1 and pressed in gynecological tumors and 50 of them, ding to the 22 proteincoding exons and one BRCA2 mutation profiles and frequencies for either putative tumor suppressor genes or onco fragment containing sequence of the 5?untrans breast and ovarian cancer families (Int. J. Can genes, could be localized to regions of LOH or lated region of the BRCA1 gene were PCRam cer 97: 472480). This comprehensive study (to amplification. Initial experiments with 25 of these plified from genomic DNA using fluorescent la date more than 1700 distinct families have been genes indicate that one third of them could be beled primers and a limited number of cycles. investigated) revealed that further predisposing confirmed after characterization by hybridization The method was tested with the study of eight genes have to be identified in the German pop on array filters or by real time PCR. In a similar complex germline BRCA1 rearrangements pre ulation and that rare sequence variants (unclas approach, we plan to construct SAGE libraries viously reported. In order to assess the possibil sified variants = UVs) with unknown relevance from microdissected material to identify differ ity that German families with a strong history of for tumorgenesis have to be validated further (60 entially expressed genes in gynecological tu breast and/or ovarian cancer negatively tested from about 100 families). mors. The resultant list of candidate genes will for codingregion mutations in BRCA1/2 by di Recently, a novel predisposing gene for breast be limited to genes localized to regions of inter rect sequencing could carry complex germline cancer has been published (The CHEK2Breast est. BRCA1 rearrangements, we screened them us Cancer Consortium, Nat. Genet. 31, 5559, This work will be done in conjunction with the ing Multiplex PCR method. Data as to the fre 2002). The CHEK2 gene encodes for a cellcy group in Bonn who is currently investigating dif quency of germline BRCA1 rearrangements in cle checkpoint kinase that participates in DNA ferentially expressed genes located in an LOH German breast and/or ovarian cancer families repair processes and a single frameshift muta interval on chromosome 15. Eight prognosis will be presented and discussed with respect to tion (1100delC) was found in about 5% of genes for metastasis, defined by array tech their relevance in BRCA1 diagnostic. BRCA1/2 negative families tested. Our group niques in recent publications, and 20 candidate and members of the GCHBOC have started to genes, selected due to their involvement in tu screen over 600 families where BRCA1/2 muta mor associated pathways, are being tested by

GfH ÖGH SGMG Tagungsband Abstracts GfH ÖGH SGMG Tagungsband tions were excluded. Preliminary analysis of 80 real time PCR on microdissected material. The P103 64 families, mainly from Southern Germany, has not results of these ongoing experiments will be pre revealed a 1100delC mutations so far. sented. Mutation analysis of BRCA1 and BRCA2 To further characterize UVs found in the genes in Iranian breast and ovarian cancer BRCA1/BRCA2 proteins from families with families breast/ovarian cancer, we have collected the P103 67 Pietschmann, Andrea (1), Hofmann, W.(2), corresonding microdissected tumor samples. Mehdipour, P.(3),Scherneck, S.(2), Peters. H.(1) The UVs identified either in the BRCA1 (T1685A; (1)Institute of Medical Genetics, Charité, I1385A) or the BRCA2 (R2502H; K1286del; The Role of Trisomy 7 in Thyroid Neoplasms Humboldt University Berlin, (2) Department S1221Y) protein, respectively, are rare variants Korabecná, M. 1, Elgrová L.1, Ludvíková, M. 2, of Tumour Genetics, Max Delbrück Center which to date have been twice, once or not list Reischig J. 1 for Molecular Medicine, Germany, (3)Tehran ed in the International BIC database. Currently, 1 Department of Biology, 2 Department of University of Medical Sciences, School of the tumors are being tested for LOH in the Pathological Anatomy, Charles University Public Health & Inst. of Public Health BRCA1 region in 17q21 or BRCA2 region in Medical faculty, Plzen, Czech Republic Research, Department of Human Genetics, 13q13. After demonstration of LOH in the ac AIM: Trisomy 7 is often found in short term cul Iran cording tumor, the remaining allele will be se tures of different epithelial neoplasms, including Germline mutations in either BRCA1 or BRCA2 quenced for the presence or absence of the mis malignant tumors of the thyroid, and also in be genes are responsible for the majority of hered sense mutation. nign lesions such are thyroid hyperplasias and itary breast and ovarian cancers. Most disease Conclusions: A low prevalence (<5%) of the adenomas. This trisomy has also been reported causing mutations spread throughout the genes 1100delC variant in the CHEK2 gene in the Ger in normal cells from different tissues. As the role and generate stop codons. However, specific man population is very likely and the relevance of trisomy 7 in the malignant process from hy mutations are found to be common within par of UVs for tumor initiation can be addressed by perplasia to adenoma and finally to folicular car ticular populations, resulting from genetic the characterization of microdissected tumor cinoma of thyroid or from hyperplasia to papil founder effects. So far, germline mutations in the samples. lary carcinoma of thyroid is still discussed, we BRCA1 and BRCA2 genes in families with a present this study. strong history of breast and/or ovarian have not METHOD: We performed fluorescence in situ hy been identified within the Iranian population. To bridization (FISH) using probes for chromosome P103 66 estimate the frequency and spectrum of 7 in paraffinembedded samples of 2 hyperplas BRCA1/2 mutations in Iranian families we col tic thyroids, 6 papillary carcinomas and 4 folic lected a large number of breast and ovarian can Differentially expressed genes from ular adenomas. Four samples of the normal thy cer families in cooperation with Tehran Univer gynecological tumors: approaches and roid were used as controls. Evaluation of results sity of Medical Sciences, Department of Human perspectives was done by counting at least 200 nuclei per Genetics. Ten families with more than five breast Dufault, Michael R. (1), Kreutzfeld, R (2), Sadr slide according to the description in recent liter cancer cases have been chosen for BRCA1/2 Nabavi, A. (1), Gelling, S. (3), Schmutzler, R. K. ature. mutation screening by direct sequencing of the (2), Dahl, E. (3), Ramser, J. (1), Meindl, A. (1) RESULTS: We found no trisomy 7 in the normal protein coding regions including all exons and (1) Abt. Päd. Genetik der Kinderpoliklinik der and hyperplastic samples, but tumors with and exon flanking regions of the introns as well as LMU, München; (2) Onkogenetisches Labor without this trisomy were found in the both re the 5’ and 3’ untranslated regions of both genes. der Universitätsfrauenklinik, Bonn; (3) maining groups. It suggests that trisomy 7 does Results as to novel sequence alterations and metaGen, Gesellschaft für Genomforschung not specify the pathway of malignant develop common BRCA1/2 polymorphisms defining hap mbH, Berlin ment, it may be included either in the process lotypes will be presented and discussed. Differentially expressed genes in tumors can be leading to the formation of papillary carcinoma identified by several approaches that include mi or to the folicular carcinoma. The findings of croarray, SAGE or „in silico“ techniques. Much cases without trisomy 7 demonstrate that it does of the information available in literature and pub not imply the development of tumor cells in the P103 65 lic databases is varying based on the source thyroid. material and bioinformatic programs. Indeed, so Relevance of BRCA UVs for familial breast far only a few of the candidate genes defined by cancer and CHK2 analysis in BRCA1/2 these approaches are overlapping. In order to in negative families crease the reliability of the data, laser capture Schmidt,Dorothee E.,Dufault M.,Straub microdissected material should be used. In ad J.(*),Wilsch B.,v.Lindern C.,Untch M.(*),Meindl dition, to increase the likelihood of identifying in A. teresting candidates from the complex data, Dept. of Medical genetics at the Ludwig genes can be restricted to known regions of Maximilian University,80336 München,(*) LOH or amplification. By implementing these

38 medgen 14 (2002) P103 68 delayed dentition, syndactyly of the toes 23 and cific clinical features. However, there remain retarded psychomotor development. The clinical UPDs such as maternal UPD7 in SilverRussell examinations during the first weeks of life syndrome in which both imprinting and chromo Deletions of the long arm of chromosome 10 showed neither abnormalities of the cardiovas somal aberrations appear to influence the phe have no prognostic impact in t(14;18) cular nor cerebrovascular system. Chromosomal notype. positive lymphomas analysis of lymphocytes at the age of 10 weeks Gesk, Stefan (1), Plötz, S. (1), GarcíaGranero, showed a normal female karyotype. The pano M. (2), Harder, L. (1), MartínSubero, J.I. (1), ramic radiograph at the age of 2 3/12 years de Schlegelberger, B. (1), Grote, W. (1), Calasanz, P104 03 picted an oligodontia. OFCDsyndrome was M.J. (3), Siebert, R. (1) suggested because of the girl’s eye anomalies, (1) Institute of Human Genetics, University dental abnormalities, her syndactyly of toes 23 Clinical and molecular cytogenetic analyses Hospital Kiel, Germany, (2) Asesoría and her facial appearance. Oculofaciocardio of three patients with interstitial deletion of Bioestadística, Pamplona, Spain, (3) dental (OFCD)syndrome is a very rare probably chromosome 6q Department of Genetics, University of Xlinked dominant inherited disorder. Because of Fritz Barbara (1), Dietze I (1), Mandel T (2), Navarra, Pamplona, Spain the small number of all patients with OFCDsyn Rehder H (1), Friedrich U (1,3) The t(14;18)(q32;q21) translocation initiates lym drome described in the literature and the report (1) Institute of Clinical Genetics, Phillips phomagenesis via deregulation of the BCL2 of a female patient with the typical findings of Universität Marburg, Germany, (2) FA für oncogene. Nevertheless, the sole presence of a OFCDsyndrome and consanguinous parents Kinder und Jugendmedizin, Marburg, t(14;18) is not sufficient to render cells onco autosomal recessive inheritance could not be Germany, (3) Institute of Human Genetics, genic and secondary chromosomal alterations excluded. The term OFCDsyndrome summaris Microdissection Department, University of are necessary for malignant transformation. es the combination of congenital cataract, den Aarhus, Denmark Deletion of 10q is observed in around 1015 % tal abnormalities like root gigantism and delayed Interstitial deletions involving chromosome 6q of the t(14;18)positive lymphomas. The com dentition, characteristic facial findings and car are rare. Only 11 patients with deletions of the monly deleted region has been narrowed down diac abnormalities. Despite the absence of car segment 6q136q21 have been reported. Char to 10q24 by cytogenetics and FISH. To study the diac abnormalities our patient fullfills the other acteristic features correlating with the specific clinical impact of 10q24 deletions in t(14;18)

criteria for OFCDsyndrome. region are: low birth weight, mental retardation, Abstracts GfH ÖGH SGMG Tagungsband positive lymphomas, we correlated the 10q24 hypotonia, microcephaly, mild facial dysmor deletion status as detected by cytogenetics and phisms with prominent forehead, hypertelorism, FISH with the most relevant clinical parameters low set and malformed ears, depressed nasal by uni and multivariate statistical analyses in a P104 02 bridge. Respiratory distress is described in near retrospective multicenter survey of t(14;18) ly half of these patients. PraderWillilike pheno positive follicular and diffuse large Bcell lym UPD and pregnancy complications: The type (feeding problems in infancy, polyphagia, phomas. resulat of a parentoffspring conflict or adipositas) is reported in 5 patients with intersti The survival analyses showed well known prog simply of a chromosomal disturbance? tial deletion of 6q16 or q21. We present the mol nostic factors like histology or clinical stage to Katja Eggermann (1), Susanne Mergenthaler ecularcytogenetic investigation of three patients clearly differentiate groups with different prog (1), Hartmut Wollmann (2), Thomas Eggermann with interstitial deletion of the q15q21 region of nosis in t(14;18)positive lymphomas (p<0.01). (1) the long arm of chromosome 6. The karyotypes Different treatment regimens did not significant (1) Institute of Human Genetics, RWTH were analysed at the 500 band level. The break ly influence overall survival of the population un Aachen, (2) Section of Pediatric points have been determined accurately by us der study. Regarding 10q24 deletions, the medi Endocrinology, Tübingen ing MicroFISH. Patient 1 is a 3yearold girl with an survival in patients with and without 10q24 Uniparental Disomy (UPD) has been observed an interstitial deletion, del (6)(q15q16.3), devel deletion was comparable (p>0.5). After adjust for almost all chromosomes. In some instances, opmental delay, low birth weight, short stature, ing the 10q24 deletion status for important clin it gives rise to distinct phenotypes (UPDs 15, 11, mental retardation and minor craniofacial anom ical parameters by multivariate analysis, its im 7), UPD of others is not associated with a clini alies. Patient 2 is a 2 5/12yearold boy with fa pact on survival was not significant either. Re cal phenotype (UPDs 13, 21, 22). In most syn cial dysmorphisms, high arched palate and hy stricting the analysis to the follicular lymphoma dromes associated with UPD, pregnancy com potonia. Reverse chromosome painting identi patients, the major prognostic parameters were plications include intrauterine growth retardation fied an interstitial deletion del(6)(q15q21). In the retained and 10q24 deletions remained not as (IUGR). This indicates that genomic imprinting third case of a 5yearold girl with developmen sociated with prognosis. Our analyses show that plays a fundamental part in regulating antenatal tal delay, VSD, respiratory problems, and dis deletions in 10q24 are not related with the out growth. However, the influence of imprinting is creet facial stigmas an obviously balanced com come or other clinical parameters in t(14;18) not obligate considering that trisomic rescue is plex structural aberration with insertion of chro positive lymphomas. Therefore we speculate the most frequent UPD formation mechanism: mosome 22 material into a chromosome 6 and that 10q24 deletions are involved in the early on trisomic rescue might lead to either placental or simultaneous inversion of chromosome 6 was set of t(14;18)positive lymphomas or represent fetal tissue mosaicism. This mosaicism may re prediagnosed. Reverse painting with a microdis a very late event common to different prognos main undetected and renders the delineation of section library probe, which was generated out tic groups. a phenotype more difficult. of the derivative chromosome 6, however, This work was supported by the Deutsche Kreb We tested a large cohort of IUGR patients with showed that in the area of the distal breakpoint shilfe. We gratefully acknowledge all our collab and without further clinical features for UPD of in 6q21 genetic material is deleted, so that par orators for providing cytogenetic material and chromosomes (namely 2, 6, 9, 14, 16, and 20) tial monosomy exists. Interstitial deletion of the clinical data. the imprinting situation of which is ambiguous. precise segment (q21) deleted in our patient has The heterogeneous clinical pictures of UPDs 2, not been reported previously. The investigations 9 and 16 and the frequently detected additional showed that through chromosome microdissec P104 01 chromosomal mosaicisms suggest that placen tion in combination with the usual cytogenetic tal mosaicism is the underlying mechanism examination, as well as FISH analyses with lo causing the phenotype. Paternal UPD6 is re cus spefication of chromosome structural aber A new case of OFCDSyndrome stricted to IUGR in association with transient rations can be achieved. Such investigations will Kleier, Saskia(1), Lüttgen, S.(2), Meinecke, P.(3), neonatal Diabetes mellitus. In case of UPD14, a allow a direct comparison of reported cases and MarschnerSchäfer, H.(1) maternal UPD14 syndrome could be delineated enable a more accurate diagnosis as well as (1)Praxisgemeinschaft für Pränatale which includes IUGR. An imprinting effect prognosis in patients with 6q deletions. Diagnostik und Humangenetik, Hamburg, should also cause the clinical features of UPD20 Germany, (2) Institute of Human Genetics, since prenatally detected mosaic trisomy 20 University of Hamburg, Germany, (3) does usually not affect the pregnancy outcome. Department of Medical Genetics, Children In conclusion, most of the known UPDs with Hospital Altona, Hamburg, Germany clinical abnormalities can be divided in two We report on a 2 5/12 yearold girl, first child of groups: the ones belonging to imprinting syn healthy nonconsanguinous parents, with bilat dromes and the ones caused by placental dys eral microphthalmia, bilateral congenital function as the result of a chromosomal aberra cataract, coloboma of the left iris, submucous tion. cleft palate, small median cleft palate, bifid uvu Both groups include growth abnormalities and, la, gothic palate, broad nasal tip, long philtrum, in case of putative imprinting syndromes, spe

medgen 14 (2002) 39 P104 04 somy 20q13.1qter and 2p24.1pter and the first quenced. Here, we describe the results for a set report on the meiotic segregation pattern of a of 54 patients. We tested our patients for the in t(2;20) (p24.1;q13.1) in a male carrier. tron1 inversion, 5 (#3, 14, 33, 76, 79) patients Multicolor chromosomal bar coding are characterized as carriers of this inversion. In characterizes a de novo interstitial deletion 11 patients mutations have been identified, (5)(q33.3q35) in a child with multiple which have been described previously. Addition congenital malformations P104 06 ally, 13 novel mutations have been characterized Schiffer Christiane (1), Popp S. (1,2), Moshir S. during this study in 14 patients. The mutations (1), Rupprath G. (3), Düngfelder E. (3), Hager Deletion of 16q23: A recognizable facial are mainly base pair substitutions. The second H.D. (1), Tariverdian G. (1), Jauch A. (1) phenotype group are deletions or insertions of 1 bp; in two (1) Inst. of Human Genetics, Univ. of Claudia Walter (1), Dagmar Wieczorek (1), Anja cases, 4 nucleotides have been inserted. The Heidelberg, Germany (2) Deutsches Weise (2), Thomas Liehr (2), Gabriele Gillessen mutations are predicted to cause amino acid ex Krebsforschungszentrum, Division of Kaesbach (1) changes or frameshifts leading to premature Genetics of Skin Carcinogenesis, (1) Institute of Human Genetics, Hospital of stop codons close to the mutation points. Heidelberg, Germany (3) Childrens Hosp., University Essen, Germany; (2) Institute of During the systematic sequencing of the 54 pa WestpfalzKlinikum Kaiserslautern, Human Genetics and Anthropology, tients, three known and 1 novel polymorphic Germany FriedrichSchillerUniversity Jena, Germany sites were identified: the first one at codon 1241 Phenotype correlations of partial chromosome We report on a 9 6/12yearold boy with short (C to G; Arg to Glu) occurs five times in this set 5q deletions are yet poorly defined. stature, normal head circumference and devel of patients. The silent polymorphic site in exon We describe a boy with multiple congenital opmental delay. He shows specific facial fea 14 at codon 1269 (A to C, Ser to Ser) was ob anomalies including a severe complex heart de tures such as a long, narrow face, high forehead, served in four patients and does not have any fect, club feet, adducted thumbs and facial dys small palpebral fissures and low set, posteriorly consequence for the amino acid composition. morphic features. He died at the age of two rotated ears. In addition, the examination of the Moreover, the known polymorphic site in the 3’ months following cardiac surgery. Gbanding re limbs revealed short and broad thumbs and first UTR of exon 26 (position 8728 A to G) was con vealed an abnormal chromosome 5q interpreted toes as well as a decreased length of the distal firmed in 8 patients. The novel polymorphic as an interstitial deletion (5)(q23.2q31.3). Break

GfH ÖGH SGMG Tagungsband Abstracts GfH ÖGH SGMG Tagungsband phalanges. Retardation of speech and delayed site T to A is located in intron 19 at position points of the deleted segment were adjusted to motor milestones were present. Due to his be 86.nt and occurs two times (patients 68, 69). del (5)(q33.3q35) by multicolor fluorescence in havioural problems, especially aggressiveness, This study was supported by DHGP01kw situ hybridization (FISH) using two sets of com he was accommodated in an institution for 9905/9. binatorially labeled band specific YAC clones. handicapped children. Our findings underscore the importance of exact Cytogenetic banding analysis and molecular cy breakpoint analysis for genotypephenotype cor togenetics using the multicolor banding tech relations in patients with uncertain chromosomal P104 08 nique (MCB) and region specific BAC probes re aberrations such as interstitial deletions and vealed a distal interstitial deletion of segment 23 show the importance for genetic counseling. on the long arm of chromosome 16 [karyotype: Clinical, cytogenetic and molecular 46,XY,del(16q23)]. Werner et al. (1997) previous investigation in a fetus with WolfHirschhorn ly described a case of a 5yearold boy with syndrome and loss of paternal 4p16 alleles P104 05 deletion 16q23.1 >q24.2. The propositus Dietze Ilona (1), Huhle D (2), Simoens W (3), showed striking facial similarities to our patient. Fritz B (1), Rehder H (1) In contrast to previous reports we speculate that (1) Institute of Clinical Genetics, Philipps Partial monosomy 20q13.1qter and 2p24.1 a deletion of 16q23 leads to a clinically recog University Marburg, Germany, (2)Laboratory pter is associated with multiple abortions: nizable facial phenotype. for Clinical Genetics, Wetzlar, Germany, (3) molecular characterization and segregation Clinic for Gynaecology and Obstetrics, analysis in a family bearing a t(2;20) Kassel, Germany translocation WolfHirschorn (4p) syndrome (WHS) has been Trappe, Ralf (1), Kohlhase J. (1), Böhm D. (1), P104 07 extensively described in children and young Weise A. (2), Liehr T. (2), Essers G. (1), Meins adults. Knowledge on fetuses with WHS is still M., (1), Zoll B (1), Bartels I. (1), Burfeind P. (1) 13 novel point mutations in the Factor VIII limited due to the small number of published (1) Institute of Human Genetics, Georg encoding gene lead to severe or moderate cases. We report on a fetus with club feet defor August University Göttingen, Germany; (2) Hemophilia A mity and intrauterine growth retardation on pre Institute of Human Genetics and Uen, C., Oldenburg, J.(1,2), Klopp, N. (4);, natal ultrasound. Chromosome analysis after Anthropology,FriedrichSchillerUniversity Brackmann, H.H. (2), Schramm, W. (3), amnioncentesis at 23. gestational weeks dis Jena, Germany Schwaab, R.(2), Graw, J. played a de novo deletion of chromosome 4p; Fetal chromosome aberrations account for about (1) Institute of Transfusion Medicine and 46,XX,del(4)(p16.1>pter). The terminal deletion 50% of firsttrimester pregnancy losses. Numer Immune Haematology of the DRK Blood was confirmed by FISH with a WHSCR probe ical abnormalities represent the bigger part Donor Service, D60528 Frankfurt, (4p16.1) (Vysis), a subtelomere probe (4p16.3) (86%) while a low percentage of abortions is Germany;(2) HemophiliaCenter and (Vysis) and a D4S96 chromosome 4p16.3 spe caused by structural chromosomal rearrange Institute of Transfusion Medicine, University cific probe (Oncor). By use of highly polymorphic ments (6%). About half of the structural abnor of Bonn, D53105 Bonn, Germany; (3) DNA microsatellite markers we determined the malities are inherited from a parent carrying a Medical Clinic, LudwigMaximilians extent of the deletion. The markers D4S2366 balanced chromosomal translocation or an in University of Munich, D80333 München, (4p15.316.2) and D4S3023 (4p16.2) showed version. We analysed a novel reciprocal translo Germany; (4) GSFNational Research Center lack of paternal alleles. Correct paternity was cation t (2;20)(p24.1;q13.1) and its segregation for Environment and Health, Institute of demonstrated by use of D4S2623 markers lying in a three generation family. The rate of miscar Epidemiology, D85764 Neuherberg, in 4q2526. Parents decided for termination of riages in pregnancies from male translocation Germany pregnancy. Post mortem examination confirmed carriers of 5 out of 10 could clearly be explained Hemophilia A is caused by heterogeneous mu a WHS phaenotype, the female fetus showing by adjacent 1 misbalanced translocation bear tations in the gene coding for factor VIII (gene characteristic features like „greek warrior hel ing spermatozoa found with a frequency of symbol: FVIII); it maps to Xq28 and consists of metfacies“, carpshaped mouth, micrognathia 48.2% in the entire sperm population of one of 26 exons. In Germany, approximately 6000 pa and low set simple ears. Our findings in the fe the t (2;20)(p24.1;q13.1) carriers. While the tients are suffering from Hemophilia A. In a sys tus are in agreement with the observation of loss translocation is clearly associated with an in tematic largescale analysis we will identify the of mainly paternal 4p16 alleles in female WHS creased risk of early abortions (7/12) in male and genotype of all severe cases (approximately patients, while male WHS patients show loss of female carriers, no malformed livebirths were 3000 patients). A first screening for mutations maternal 4p16 alleles. observed. This suggests complete embryonic causing Hemophilia A analyzes the exons from lethality of 2, der(20) and der(2), 20 imbalanced genomic DNA by methods like DGGE, SSCP or offspring. With respect to known cases of par dHPLC. Since this approach covers only approx tial trisomy 2p and 20q we consider that their imately 97% of the mutations, the FVIIIgene of corresponding monosomies provoce fetal the remaining patients have to be sequenced in wastage. This is the first study describing multi total. Therefore, all exons including their flank ple abortions associated with a partial mono ing regions were amplified and subsequently se

40 medgen 14 (2002) P104 09 ni dysplasia of the cochleas. Cochlea implanta in combination with region specific YAC and tion was performed on the left side. Recently, in BAC probes, the breakpoint could be corrected two families with SHFM and hearing loss the un and refined to 2p12p11.2. The missing chromo PARTIAL TRISOMY OF CHROMOSOME 22 derlying gene has been mapped in 7q21(Tack somal material could not be detected in the DUE TO INTERSTITIAL DUPLICATION OF elsHorne et al., 2001) and a deletion in 7q21 karyotype of any of the three studied probands 22q11.2 IN A CHILD WITH TYPICAL CAT EYE has been detected in a child with SHFM and using these FISH techniques. The reported dele SYNDROME Mondini dysplasia (Haberlandt et al., 2001). In tion is located more proximal than a known case Meins, Moritz (1), Trappe, R. (1), Motsch, S. (2), our case, no deletion could be detected by con of a familial deletion without phenotypic penal Schulz, R. (3), Langer, S. (4), Speicher, M. (4), ventional chromosome analysis. Therefore, mo ties [Lambert et al., 1991, J Med Gent Vol. 28, Mühlendyck, H. (2), Bartels, I. (1), Burfeind, P. lecular cytogenetic studies are in progress in or p62: del(2)(p13p12.2)]. As the region 2p12p11.2 (1), Zoll, B. (1) der to detect a possible microdeletion. is known to be gene rich it is surprising that no (1) Institute of Human Genetics, (2) Eye phenotypic consequences can be observed. Clinic, and (3) Paediatric Cardiology, Thus, a new region within the human genome is University of Göttingen, (4) Institute of identified, which seams to have no clinical con Human Genetics, Technical University P104 11 sequences when present in one copy, only. Sup München and GSF, Neuherberg ported by the DFG (PO284/61) and the EU Background: Cat Eye syndrome (CES) presents Autistic disorder and chromosomal (ICA2CT200010012 and QLRT199931590). as a genomic disorder with a variable combina mosaicism 46, XY [120] / 46, XY, tion of congenital features including ocular del(20)(pter>p12.2) [10] coloboma, anal atresia, defects of heart and kid Sauter, Simone; von Beust, G; Lenz, E; Zoll, B ney, craniofacial anomalies, and mild to moder P104 13 Institute of Human Genetics, GeorgAugust ate mental retardation. CES is usually character University Göttingen, HeinrichDükerWeg ized by the cytogenetic finding of a bisatellited, 12, 37073 Göttingen The role of the BamHIpolymorphism of dicentric marker chromosome containing mate Autism is characterized by impairments in social heparan sulphate proteoglycan 2 (HSPG2) in rial of the CES critical region on proximal 22q interaction and communication, stereotyped pat the development of CAD (inv dup(22)), resulting in a partial tetrasomy. We

terns of interests and activities as well as devel S. Schulz1, P. Greiser1, U. Schagdarsurengin1, Abstracts GfH ÖGH SGMG Tagungsband report here a patient with a rare interstitial dupli opmental abnormalities. It is estimated to affect U. MüllerWerdan2, K. Werdan2, C. Gläser1 cation of 22q11.2 and typical features of Cat Eye approximately 5 per 10000 individuals with a 1Inst. of Human Genetics, 2Dep. of Internal syndrome. Clinical findings: The patient was male to female ratio of 4:1. Mental retardation Med., Univ. Halle, Germany born after uneventful pregnancy, parents are occurs in approximately 75 % and seizures in HSPG2 is one of the three major classes of he healthy and unrelated. The boy presented at the about 1530 % of autistic patients. In most cas parin sulphate proteoglycans acting within the age of 3 weeks with a combined heart defect, es of infantile autism the behaviour of affected cardiovascular system and controlling various kidney anomalies, and anal atresia. Characteris children is abnormal from early infancy. We re aspects of vascular development. It’s involved in tic craniofacial features included preauricular port on a 3yearold boy with a moderate to se the lipid metabolism by binding lipoprotein li pits, colobomata of iris and uvea, downslanting vere mental retardation, autistic behaviour pat pase and apolipoprotein B and may therefore be palpebral fissures and ptosis, short nose with terns and myoclonic epilepsy of early childhood. related to vascular disease. Methods: In a case flattened nasal bridge, hypertelorism, long Facial dysmorphic signs are a flat nasal bridge controlstudy we examined the interactions of philtrum, high arched palate, and retrognathia. and prominent ears. Magnetic resonance imag the BamHIpolymorphism (PM) of HSPG2, lipid Reexamination at the age of 8.5 months ing of the brain, electrophysiological examina metabolism (LDL, HDL, triglycerides, total cho showed moderate delay of psychomotor devel tions as well as metabolic sreening and molec lesterol) and coronary atherosclerosis (CAD). We opment. Cytogenetics: Standard GTL banding ular testing for Angelman syndrome and FraX investigated 353 patients with angiographically techniques revealed a structural anomaly of one syndrome showed normal results. The cytoge confirmed coronary diagnosis depending on chromosome 22 (46,XY,add(22)(q11.2)). FISH on netic analysis of blood lymphocytes revealed a their angiographically confirmed coronary and metaphase spreads with probes for the DiGe deletion of chromosome 20p12.2 occuring as lipid state, 126 patients without any coronary orge critical region (D22S553, D22S942) showed mosaicism in 8 % of the analysed metaphases: symptoms having an abnormal lipid metabolism only one signal on each chromosome 22. How 46, XY [120] / 46, XY, del(20)(pter>p12.2) [10] by (mean age: 51.4y) and 227 CAD patients having ever, FISH with YAC probes for the paracen conventional banding analysis and fluorescence a distinct pathological lipid profile (mean age: tromeric region of 22q11.2 showed hybridization in situ hybridization. Several studies lead to the 50.6y). As controls we examined 300 long patterns suggestive for an inverted interstitial conclusion that many cases of autism are standing healthy blood donors with normal lipid duplication of proximal band 22q11.2. Conclu caused by chromosomal disorders involving metabolism (mean age: 43y). Results: In order to sion: To our knowledge this is the first case of chromosome 15 and the sex chromosomes. To evaluate the interrelation of the HSPG2 BamHI CES with the complete pattern of anomalies in date it is not clear whether genes on other chro PM, lipid metabolism and CAD the genotype fre cluding coloboma, preauricular anomalies, heart mosomes like chromosome 5, 8, 13, 17 and 18 quencies were examined according to a domi defect and anal atresia caused by interstitial du or on chromosome 20 as in our case play a role nant, codominant and recessive genetic model plication of the CES critical region on 22q11.2, in the development of this disease. within in three proband groups. The analysis of resulting in partial trisomy 22q11.2. the codominant genetic model showed signifi cantly decreased frequencies of the homozy gous mutationcarriers TT among CAD patients P104 12 P104 10 (0.02) and patients without coronary afflictions (0.04) compared to healthy blood donors with Deletion 2p122p11.2 without abnormal normal lipid profile (0.07, p<0.032). Based on a Split hand/split food malformation with clinical findings detected over three Trecessive genetic model a comparison of CAD Mondini dysplasia generations patients showing the most pathological lipid pro Muschke, Petra (1); Berkholz, J (2); Wieacker, P Weise, Anja (1), Belitz, B. (2), Pfeiffer, L. (2), file and healthy blood donors revealed a 4.2 (1) Claussen, U. (1), Liehr, T. (1) times higher risk for TTcarriers of developing an (1) Institute of Human Genetics, (2) (1) Institute of Human Genetics and unbalanced lipid metabolism connected with se Department of Ear, Nose and Throat, Anthropology, Jena, Germany, (2) vere coronary atherosclerosis (p>0.006; 95% CI: University of Magdeburg, Germany Laboratory for Medical Genetics, Berlin, 1.4212.4). Conclusions: Depending on the Split hand/split foot malformation (SHFM) occurs Germany pathological lipid profile the TTgenotype fre either as isolated birth defect or as feature of dif We report on a family with a deletion in chromo quency of the BahmHIPM of HSPG2 decreased ferent malformation syndromes. Most cases are some 2 del(2)(p12p11.2) without apparent significantly. Beside the pathological lipid profile inherited as an autosomaldominant trait, but X phenotype. The deletion of the short arm of the angiographically confirmed coronary state linked and autosomalrecessive inheritance also chromosome 2 was initially detected in the fetus seems to be only a more minor fact in this asso have been described. SHFM loci have been of a 38 year old woman referred for amniocente mapped in 7q21 (SHFM1), Xq26 (SHFM2), 10q sis. Karyotypic analysis of the mother and the (SHFM3); as well as 3q27 (p63 gene), 6q and 2q grandfather showed a morphologically altered for EEC syndrome. We report on a 3 yearsold chromosome 2, as well. According to GTG boy with split hand/split foot malformation as banding the breakpoint was thought to be in well as syndactyly of the 3rd and 4th digits of 2p12. Using high resolution multi color banding the right hand and deafness because of Mondi (MCB) applying the probe set for chromosome 2

medgen 14 (2002) 41 P104 14 At the age of twenty months regression and loss P104 17 of already acquired skills was obvious. At the age of 30 months the girl has lost purposeful Genetic analysis of patients with hereditary DNADiagnostik for NoonanSyndrome: hand function, had intermittent episodes of hy hemorrhagic telangiectasia (HTT) MutationScreening in the PTPN11Gene perventilation and was gritting with her teeth. Pasche, Bastian (1); Folz, Benedikt (2); Zoll, Schlüter, Gregor; Rossius, Malte; Zoll, Barbara; Chromosome analysis, molecular analysis for Barbara (1); Engel, Wolfgang (1) Engel, Wolfgang Fragile X as well as for CATCH 22 showed nor (1) Institut für Humangenetik, Universität Institute of Human Genetics, University of mal results. By sequence analysis of the MECP2 Göttingen, HeinrichDükerWeg 12, 37073 Göttingen, Heinrich Düker Weg 12, 37075 gene the 808 C?T mutation, a mutation which Göttingen (2) UniversitätsHNOKlinik, Göttingen leads to a stop codon, was detected. The diag Universität Marburg, Deutschhausstr. 3, The Noonan Syndrom is a complex syndrome nosis Rett syndrome was therefore stated. 35037 Marburg with a highly variable phenotype, typically com Conclusion: Even if the symptoms of a patient Hereditary hemorrhagic telangiectasia (also prising hypertelorism, low set ears, pulmonary have already been adjoined to a known syn known as OslerWeberRendu Syndrome or stenosis, sternum anomalies. Though phenotyp drome it has to be kept in mind that the syn Morbus Osler) is a rare autosomal dominant dis ically similar to the 45,X TurnerSyndrom it af drome may be superimposed by symptomes of order that affects 1 in 10000 individuals. It is het fects males and females and chromosomes are a further disease. erogeneous both between and within families in normal. Recently, mutations in the SHP2 Gene age of onset and severity of clinical manifesta have been reported to account for approximate tions. The first symptom is generally epistaxis, ly 50% of all NoonanCases. in most cases followed by arteriovenous malfor P104 16 We have performed mutation screening in the mations (AVM) of different inner organs (lung, liv SHP2 gene in patients referred to our depart er, brain, gastrointestinal tract). ment with the clinical diagnosis of NoonanSyn Pure trisomy 12pter12p11.21 in a girl with The heterogeneity can be explained in part by drom. Here, we report on the mutation spectrum Xautosomal translocation: minor mutations in at least three different genes, two and genotypephenotype correlation in our pa congenital anomalies and moderate of them are known. Mutations in the Endoglin tient population. All mutations were missense developmental delay gene on chromosome 9 are associated with HHT mutations. Few recurrent and mostly private mu

GfH ÖGH SGMG Tagungsband Abstracts GfH ÖGH SGMG Tagungsband Dufke, Andreas (1), Stötter, M.(2), Starke, H.(3), type 1, which is a more severe form with a high tations were identified. Our data indicate a high Liehr, T.(3) er incidence of pulmonary AVMs. The milder er rate of cardiac hypertrophy in those patients (1) Abteilung Medizinische Genetik, form of HHT (type 2) is associated with muta were mutations could be identified. Universität Tübingen, Germany, (2) tions in the ALK1 gene, which is located on Kinderklinik, Universität Tübingen, chromosome 12. Both gene products are trans Germany, (3) Institut für Humangenetik und membrane proteins, they are expressed in en Anthropologie, Universität Jena, Germany P104 18 dothelial cells and are connected to the TGF? Trisomy 12p has a distinct chromosomal pheno signalling pathway. type including heavy birth weight, macrocephaly, We screened six single patients and two families A disease associated balanced turribrachycephaly with high and prominent with six members for mutations in both genes. chromosome rearrangement (DBCR) in a girl forehead, midface hypoplasia, broad nasal We amplified the 10 (ALK1) or 14 (Endoglin) ex with multiple congenital anomalies (anal bridge, upturned tip of the nose, long philtrum, ons by PCR followed by direct sequencing. Be atresia, colon aplasia) and normal mental everted prominent lower lip, short broad hands side several polymorphisms we observed mis development and fingers, early hypotonia, severe mental re sense and nonsense mutations as well as dele Spiegel, Miriam (1), Kalscheuer, V. (2), Werner tardation and seizures. Over 35 cases have been tions. All mutations are located in the extracel W. (1), Bartsch O. (1) reported, including 10 observations of complete lular domain, but no hot spot region could be (1) Institute for Clinical Genetics, Medical pure trisomy 12p and 4 cases of trisomy 12p in identified. Faculty Carl Gustav Carus, TU Dresden, volving mosaicism. We now report the first pa Germany (2) MaxPlanckInstitute for tient with trisomy 12p in conjunction with an X Molecular Genetics, Berlin, Germany chromosomal translocation. Disease associated balanced chromosome re P104 15 Our patient was born at 42 weeks of gestation arrangements (DBCRs) are a powerful tool for (birth weight 4.800g, length 53cm, OFC 37cm). the identification and isolation of disease genes She had unilateral postaxial hexadactyly as sole Fetal alcohol syndrome in association with and for our understanding of their physiological malformation. Dysmorphisms included hyper Rett syndrome and pathogenetic role as well. telorism with inner epicanthus, thin upper lip, Zoll, Barbara (1), Huppke P. (2), Schulz R.(3), We report a 12yearold girl with multiple con drooping lower lip, sparse eyebrows, hypopig Bartels I.(1), Laccone F.(1) genital anomalies and a balanced translocation mented skin areas, bilateral inverse nipples and (1) Institute for Human Genetics,(2) Dept. of 46,XX,t(12;14)(q24.1;q22.1) de novo. The prominent great toes. Mental retardation was Neuropediatrics,(3) Dept. of Pediatrical proposita was born at 42 weeks of gestation as obvious at the age of 3 years. Motor develop Cardiology GeorgAugustUniversity, the second child to healthy unrelated parents. ment was in the normal range. Seizures have not Goettingen, Germany She had mildly dysmorphic facial features with been observed. At the age of 4 3/12 years Fetal alcohol syndrome (FAS) is a common syn slight asymmetry, a prominent left zygomatic (height 105cm, weight 18,7kg, OFC 49,5cm) she drome which affects about 1/1000 livebirths. It bone, strabismus divergens, high nasal root, had a vocabulary of 200?300 words. is recognizable by characteristic craniofacial ab short philtrum, and a thin vermilion border. She Cytogenetic analyses revealed an Xautosomal normalities, malformations, growth retardation also had anomaly of the iris, hearing loss of the translocation which was characterised as and deficits in intellectual functioning. Severe right ear, horseshoe kidney with nephrocalci der(X)(Xpter>Xq28::12p11.21>12pter) by vari FAS is mostly diagnosed in the first days of life. nosis, colon aplasia, anal atresia, right hammer ous molecularcytogenetic techniques. A sub On the contrary, patients with Rett syndrome de toe and streaklike hypopigmented areas on telomeric probe Xq (cosmid CY29) hybridised to velop normally until the age of 6 to 18 months. both arms and legs. Her mental development the derivative Xchromosome. Xinactivation Thereafter, a stagnation of development occurs was normal. studies showed that the normal Xchromosome followed by regression of mental and motor abil Detailed FISH analysis was performed. For chro was inactive in 41/57 analysed lymphocytes. ities and autistic features. Rett syndrome is less mosome 12, we used YAC probes 855e2, 979a5, The patient gives us the opportunity to show the frequent than FAS but with an incidence of 753g1, 654b2 (order, cen—>qter) and found the role of Xinactivation in mitigating the effect of 1/10000 in girls it belongs to the more common breakpoint between probes 979a5 (locus data chromosomal imbalance. Different Xinactivation genetic diseases. not available) and 753g1 (D12S354). For chro ratios in various tissues could explain noncon We report on a girl who was born in the 36th mosome 14, we used YAC and BAC probes 959 cordance of the phenotypic expression with the week of gestation with hypotrophy, micro b11, RP112G1, RP1158E21, RP11286 O18, inactivation status in peripheral blood cells. The cephaly, ASD and VSD and typical features of al and 772d1 (order, cen—>qter). BAC RP11286 clinical picture of the patient will be demonstrat cohol embryopathy. The mother conceded a dai O18 (AL133485) showed signals on the normal ed in comparision to cases with pure trisomy ly consumption of about 150 to 200 g pure alco chromosome 14 and both derivative chromo 12p and cell line mosaicism. hol. During the first months of life the child pro somes and is therefore breakpointspanning. gressed in her motor and psychomotoric devel We suggest that her phenotype resulted from the opment but was at the very beginning of her life inactivation of a gene(s) at one or both break severly retarded. points, either by direct disruption or possibly by

42 medgen 14 (2002) an undetected minimal unbalanced rearrange odactyly can occur and the big toe is often short P104 22 ment at the molecular level. and broad. Other fingers or toes are usually not affected. Brachydactyly A2 follows an autosomal A comprehensive molecular cytogenetic dominant mode of inheritance; the causal gene analysis of a familial complex chromosome is unknown. Only a few families with brachy P104 19 rearrangement involving four chromosomes dactyly A2 have been published in the literature and at least 8 breakpoints (Mohr and Wried, 1919; FreireMaia et al.,1980). C. Fauth (1, 2), S. M. Gribble (3), J. Leifheit (4), Dup(13)(q14.2q21.1) yet another differential We present a large German kindred with brachy S. Uhrig (1), H. Fiegler (3), N. P. Carter (3), M. diagnostic aspect for short stature like dactyly A2 (MohrWriedt) and typical clinical R. Speicher (1, 2) phenotype findings. The manifestations show a high vari (1) Inst. für Humangenetik, TU München, Schreyer, Isolde (1), Beensen, V. (1), Eichhorn, ability among the affected individuals. The most Germany (2) GSF, Forschungszentrum für K.H. (2), Heller, A. (1), Liehr, T. (1), Claussen, U. severely affected person is characterised by a Umwelt und Gesundheit, Neuherberg, (1) missing middle phalanx of the index finger. We Germany (3) The Wellcome Trust Sanger (1) Institute of Human Genetics and have performed a genome scan using a set of Institute, Wellcome Trust Genome Campus, Anthropology, Jena, Germany, (2) 395 polymorphic microsatellite markers to map Hinxton, Cambridge, UK (4) Kinderklinik der Ambulance of Gynecology, Weimar, this type of brachydactyly. Two point linkage TU München, Germany Germany analysis (maximum LOD score [Zmax] 5.2 at re We report a complex chromosome rearrange We report on a case of a healthy pregnant combination fraction [theta] 0.00) located ment (CCR) involving 4 chromosomes and at woman with hyposomia. She was 23 years old brachydactyly A2 in this family to chromosome least 8 breakpoints which was observed in both and it was her first pregnancy. The short stature 4q. Haplotype analysis of the pedigree confined a mother and her daughter. This is to the best of was conspicuous (length only 146 cm). No oth the locus within an interval of 21.6 cM to chro our knowledge the first case in which such a er clinical symptoms and no menstrual irregular mosome 4q22q26 on the cytogenetic map. complex rearrangement was transmitted through ities were registered. 15 years ago she has been Candidate genes are currently being investigat two generations. suspected having a Turner syndrome and chro ed in this region. An extensive molecular cytogenetic workup of mosomal analysis revealed a very mild mosaic this CCR was appropriate for two reasons: First

46,XX/45,X (72/2). Now the woman requested Abstracts GfH ÖGH SGMG Tagungsband ly, while the mother is phenotypically normal the counseling for a potential recurrence risk for the P104 21 daughter suffers from mild mental retardation. unborn child having a Turner syndrome. This suggests that a difference may exist be In gestational week 16+1 she was referred to tween the chromosome complements of mother amniocentesis. Prenatal cytogenetic diagnostics Three particular cases with p63 mutations and daughter. Secondly, there is no single showed a fetal karyotype of 46,XX,dup(13) Katarina Lehmann, S. Schweiger, G. Leschik, method which on its own can elucidate such (q14.2q21.1) ish.13q14(RBx3) In this context fa S. Mundlos, S. Tinschert complex rearrangements completely. milial chromosome analysis have been carried Institut für Medizinische Genetik Standard chromosome CGH showed a balanced out. The mother had the same karyotype as the (HumboldtUniversität), Campus Virchow profile for all chromosomes. Investigation with fetus caused by a de novo aberration. Addi Klinikum, D 13353 Berlin MFISH confirmed the rearrangement of the tionally a very mild Turnermosaic was found Mutations in the p63 gene have been estab aberrant chromosomes. YAC clones were then conforming with the earlier analysis. The father lished as the molecular basis in a number of hu used to determine the orientation of the chromo had a normal karyotype. The parents decided to man syndromes (EEC syndrome, AEC syndrome, some fragments. This higher resolution analysis continue the pregnancy. Growth arrest and poly Limbmammary syndrome, ADULT syndrome) also identified small insertions which had not hydramnion were detected by ultrasound. At 35 and non syndromic split hand and foot malfor been detected previously. A study using ordered weeks of gestation the female child was born by mation. Genotypephenotype correlations have BAC clones was then initiated to identify break cesarean section with apgar scores 6/8/9, 2750g been described for EEC (Ectrodactyly, Ectoder point spanning clones. As this is a time consum weight and only 44cm length. Postnatal exami mal dysplasia, Clefting) syndrome and AEC ing process, we employed a rapid method utilis nations revealed a healthy girl with normal de (Ankyloblepharon, Ectodermal dysplasia, Cleft ing DNA microarrays which has been developed velopment apart from the short stature and very lip and palate) syndrome. Heterozygous mis to facilitate breakpoint mapping. The transloca mild dysmorphic signs. Mainly phenotypic cor sense mutations in the SAM (Sterile Alpha Mo tion chromosomes were isolated by flow sorting relation of the presented unbalanced partial tri tif) domain are the cause for AECsyndrome; and hybridised to a 1 Mb BAC array (Fiegler et. somy 13 is a short stature. The reported chro missense mutations in the DNAbinding domain al. in press). Only two hybridisations of the flow mosomal aberration was not described previ of p63 have been found in the vast majority of sorted derivative chromosomes onto the BAC ously. The case report presents detailed pheno individuals with EECsyndrome. arrays were required to provide localization of all type descriptions and advice for a differential di We present one familiar and two sporadic cases breakpoints to within a resolution of 1 Mb. This agnosis and genetic counseling in rare chromo with unusual mutations in the p63 gene and dis clearly demonstrates the power of this approach somal aberrations associated with short stature. cuss the genotypephenotype relations. to map breakpoints rapidly. Currently we would Supported by the Wilhelm SanderStiftung (1) Father and son presenting ectrodactyly, describe the four translocation chromosomes as (99.105.1) and the EU (ICA2CT200010012). oligodontia and lacrimal duct stenosis which are der(6)(6pter>6q21::11p13>11p13::20p13>20 features of EEC syndrome. A deletion of one nu pter), der(9)(11pter>11p14.1::11p11.2>11p13: cleotide was detected in exon 14 (1913delA) re :6q23.2>6q22.1::11p14>11p14::9p21.3 sulting in a frameshift. This kind of mutation is P104 20 >9qter), der(11)(9pter>9p21.3::6q22.1>6q22.1: not typical for EEC syndrome. :11p11.2>11qter), der(20)(6qter>6q23.2: (2) A 38yearold man with features of AEC syn :11p14>11p14::20p13>20qter). Mapping of Brachydactyly A2 to drome such as chronic scalp erosions in child All breakpoints will be further refined to within Chromosome 4q2226 hood, partial hair loss, dystrophic nails, teeth ab breakpointspanning clones as our mapping ef Katarina Lehmann (1), B. Meyer (2), D. Müller normalities, partial anhidrosis, lacrimal duct atre forts continue. (3), S. Tinschert (1), S. Mundlos (1,4), P. sia, hypoplastic uvula and a mild form of brachy This extraordinary case sets a striking example Nürnberg (1,2) dactyly. Interestingly, we detected a frameshift for the effectiveness of different molecular cyto (1) Institut für Medizinische Genetik, mutation in exon 13 (16151616delCAinsG) genetic tools and how, if applied in concert, an HumboldtUniversität, Berlin, (2) which stands in contrast to previously described extremely complex rearrangement can be re Genkartierungszentrum, MDC, Berlin missense mutations (SAM domain) in patients solved efficiently. Buch,(3) Institut für Medizinische Genetik, with Hay Wells syndrome. Klinikum Chemnitz, (4) Max Planck Institut (3) A 13yearold girl with typical AEC syndrome. für Molekulare Genetik, Berlin We have identified a novel missense mutation in Brachydactyly is due to abnormal development exon 14 (I558N) that appears to be characteris of the phalanges and/or metacarpals. As an iso tic for the class of mutations found in persons lated feature, the different types of brachydacty with AEC syndrome. ly were classified according to the anatomical lo cation by Bell in 1951. Type A2 brachydactyly is a rare hand malformation characterized by brachymesophalangy and lateral deviation of the index finger and the second toe. Brachymesophalangy of the 5th finger with clin

medgen 14 (2002) 43 P104 23 explained by the Lyons hypothesis. The full clin ical picture of DMD has been described in chil dren with Turner syndrome. A 12yearold girl DiGeorge/velocardiofacial syndrome: FISH P104 26 was born to parents with no history of disease. studies of chromosomes 22q11 and 10p14, There was history of repeated falls, difficulty in and clinical reports of the proximal 22q11 walking and getting up and normal performance Severe, neonatal onset OTC deficiency in deletion in the school. On clinical examination she was twin sisters with a X;5 translocation Bartsch, Oliver (1), Nemecková, M. (2), mentally normal, had muscle pseudohypertro Zenker, Martin (1,3), Wermuth, B. (2), Kocárek, E. (2), Wagner, A. (1), Puchmajerová, phy and was Gower’s sign positive. Her clinical Trautmann, U. (1), Knerr, I. (3), Kraus, C. (1), A. (2), Poppe, M. (3), Goetz, P. (2) symptoms were less severe than typical for boys Rauch, A. (1), Reis, A. (1) (1) Institut für Klinische Genetik, Technische of the same age. Her creatinine kinase level was (1) Institute of Human Genetics, University Universität Dresden, Germany, (2) Institute elevated, Xray chest and ECG were normal, and of Erlangen, Germany, (2) University of Biology and Medical Genetics, Charles myogenic EMG. Her brother had normal creati Hospital Bern, Switzerland, (3) Children’s University, Prague, Czech Republic, (3) nine kinase level and no signs of disease. Mole Hospital, University of Erlangen, Germany Kinderklinik, Technische Universität cular analysis revealed double recombination OTC deficiency is the most common inborn urea Dresden, Germany event in patient’s Xchromosome. cycle defect inherited as a Xlinked trait. Female The DiGeorge/velocardiofacial syndrome carriers may manifest mild to moderate metabol (DGS/VCFS) may occur with different deletion in ic insufficiency but the severe neonatal form of tervals on 22q11 or 10p14. The clinical outcome the disease is normally restricted to hemizygous with the common 22q11 deletion (CDEL; size 3 P104 25 males having less than 5% residual enzyme ac Mb, some 30 genes) is well known, but the out tivity. Case report: We report on monozygotic fe come with the other deletion types is less well IDENTIFICATION OF RARE FORMS OF male twins born to healthy unrelated parents documented. We hypothesized that there could AUTOSOMAL DOMINANT HERITABILITY OF with an unremarkable family history. Both infants be differences with respect to intelligence and/or MYOCARDIAL INFARCTION developed hyperammonemic coma on the 5th behaviour disorder/psychosis (polygenic traits). Mayer, Björn (1), Fischer, M. (1), Erdmann. J. day of life. The metabolic profile suggested OTC From 1997 to 2001, we studied a series of 295

GfH ÖGH SGMG Tagungsband Abstracts GfH ÖGH SGMG Tagungsband (1), Holmer, S. (1), Lieb, W. (1), Hubauer, U. (1), deficiency confirmed in one of the patients by patients with suspected DGS/VCFS. Klein, G. (2), Nürnberg, G. (3), Saar, K. (3), liver biopsy (residual OTC activity less than 5% No 10p deletion was observed, adding evidence Nürnberg, P. (3), Löwel, H. (4), Reis, A. (5), of normal). Because of the unusual manifesta to that the 10p deletion is extremely rare. We Hengstenberg, C. (1), Schunkert, H. (1) tion of a Xlinked disorder in females, genetic found the CDEL in 52 patients and the proximal Klinik für Innere Medizin II, Universität anaylsis was started with karyotyping. Chromo deletion (PDEL; size 1.5 Mb, some 15 genes) in Regensburg (1), Klinik Höhenried, Bernried some analysis revealed a de novo, balanced X;5 5 patients. Clinical data of 4 patients with PDEL (2), Gene Mapping Center am MDC, Berlin translocation (karyotype: 46,X,t(X;5) (p21.1; were available. Their malformations represented (3), GSF, München (4), Institut für q11.2)) in both patients. No mutation could be the DGS/VCFS spectrum, as could be expected Humangenetik, Universität Erlangen (5) detected by subsequent direct sequencing of from previous studies, but two patients (age, 6 Myocardial infarction (MI) is considered to be a the entire OTC gene. No abnormality was either and 25 years) had a normal mental development. multifactorial disease that results from an inte found by Southern blot using the OTC cDNA. Normal mental outcome may also occur with the grated vascular response to common cardiovas Discussion: Manifestation of Xlinked recessive CDEL, but not frequently. This study may pro cular risk factors. Interestingly, some families disorders in females is very rare. When it occurs vide a first evidence for a better intellectual show a high incidence of MI that cannot be ex it is mostly associated with cytogenetical abnor and/or behavioural outcome with the proximal plained by these traditional risk factors. Thus, malities of the Xchromosome or abnormal Xin vs. the common 22q11 deletion. the aim of this study was to investigate whether activation pattern. In the present case the al MI can also be inherited in a Mendelian pattern. most complete absence of OTC activity might be Methods: In cooperation with 17 inhospital car explained by two effects due to the chromoso P104 24 diac rehabilitation centers, the Regensburg MI mal rearrangement: The first is presumably a family study group screened over 200.000 med disruption of the OTCgene by the chromosomal ical reports of patients with coronary artery dis break occurring exactly in the subband of the X Female child with Duchenne muscular ease in order to find extended MI families. chromosome where the OTC gene has been dystrophy and double recombination in X Results: We identified 124 MI families with at mapped (Xp21.1) although Southern blot did chromosome least 3 or more siblings who survived a MI. In 13 not suggest a breakpoint within the OTC gene. Kala, Marta (1), WigowskaSowinska, J. (2), of these families without bilinearity, a parent, an Secondly, the translocation of a large autosomal GalasZgorzalewicz, B. (2), Slomski R. (1,3) aunt or an uncle, as well as a minimum of 2 fragment to the Xchromosome results in (1) Institute of Human Genetics, Polish cousins had the history of MI. Within these fam skewed Xinactivation in favor of those cells in Academy of Sciences, Poznan, Poland, (2) ilies, 85 living MI patients and 365 unaffected which the derivative X is the active one and the Clinics of Develovemental Neurology, family members were recruited. Moreover, 49 normal X is inactivated. Medical School, Poznan, (3) Department of patients had died from MI in these families. The Biochemistry and Biotechnology, largest family had 22 affected individuals in five Agricultural University, Poznan generations. In the age group from 50 to 70 Over the past decade geneticists have searched P104 27 years, 47.7% of the family members were affect for rapid and efficient means of identification of ed. Traditional cardiovascular risk factors, such genetic diseases. Our laboratory is involved in as smoking, hypercholesterolemia, hypertension Homozygoter LDLRezeptormangel bei mutational analysis, carrier detection, and pre and diabetes mellitus, had the same frequencies eineiigen Zwillingen natal diagnosis in DMD/BMD families for the last as a populationbased sample of sporadic MI J. Zschocke, M. Soufi, G. Klaus, J. Thiery, G. F. 11 years. Till now, we have analyzed over 200 (MONICA MIRegistryAugsburg, Germany, Hoffmann, J.R. Schaefer DMD patients and 15 BMD patients. Studied n=609, p>0.1 for all risk factors). However, the Institut für Humangenetik (JZ) und Univ. group involved 15 familial cases. Out of these, predominance of male gender in this age group Kinderklinik (JZ, GFH), Universität 51% were found to have intragenic deletions as usually seen in MI registries, was less pro Heidelberg; Abt. für Innere Medizin / and 49% were nondeletional cases. 70% of nounced in MI families (69.4% vs. 87.4 % in the Kardiologie (MS, JRS) und Univ.Kinderklinik deletions were located in central deletion prone population based KORA MI registry, p<0.001). (GK), PhilippsUniversität, Marburg; Institut region, with 35% deletion originating in intron Furthermore, the patterns of inheritance argued für Laboratoriumsmedizin, Klinische Chemie 44. In approximately 10% of nondeletional cas against Xchromosomal disorder. In the MI fam und Molekulare Diagnostik, Universität es point mutations were detected. Molecular di ilies, power simulation using the SLINK software Leipzig (JT) agnostic studies of carrier status were per package and applying several potential models Wir berichten von zwei identischen Zwillingen formed for 39 DMD/BMD families. In all studied of inheritance consistently suggested that an au türkischer Herkunft, bei denen im Alter von zwei families at least one marker was informative. Ad tosomal dominant trait explained the heritability Jahren ausgeprägte Xanthome erkannt wurden. ditionally, a few cases of intragenic recombina in these families with the highest probability. Lipidanalysen zeigten eine massive Hypercho tion event were detected. Analysis of (CA)n re Conclusion: A minority of MI patients shows an lesterolämie (Gesamt/LDLCholesterol >1100/ peats located in the deletion prone region of the autosomal dominant pattern of heritability which 1000 mg/dL, Lp(a) 130 mg/dL). Beide Eltern DMD gene detects de novo mutations and pro cannot be explained by traditional cardiovascu zeigten mäßig erhöhte Cholesterinspiegel, die vides direct information about carrier status. Oc lar risk factors. The data suggest the exsistence auf medikamentöse Behandlung gut currence of symptomatic disease in girls can be of yet unknown monogenic forms of MI. ansprachen. Die Familienanamnese war

44 medgen 14 (2002) bezüglich kardiovaskulärer Erkrankungen unauf patients are known to have milder phenotype P104 30 fällig. Molekulargenetische Analysen im LDLR with only one major symptom (often hy Gen ergaben bei beiden Zwillingen Homozygotie poparathyroidism) associated with Y85C muta Congenital disorder of glycosylation Type Ie für die bislang nicht beschriebenen Mutation tion causing just mildly abnormal protein AIRE. (CDGIe) 4 patients have been reported so W556R (c.1729T>C). Beide Eltern sind heterozy Other genetic and/or environmental factors must far. The 5th case in the literature? got für diese Mutation. W556R betrifft eine von influence the phenotype and disease progres K. Bosse1, J.C. v. KleistRetzow2, T. fünf hochkonservierten YWTDMotiven des sion of individual APS1 patient. Marquardt3 LDLRProteins und beeinträchtigt vermutlich den 1Institute of Human Genetics; University of Transport vom ER zur Zelloberfläche (Mutation Bonn, Germany; 2Department of sklasse 2A). Funktionelle Studien in Fibroblasten P104 29 Paediatrics; University of Cologne, bestätigten eine fehlende Bindung und Internal Germany; 3Department of Paediatrics; isierung von LDL. Eine konservative Behandlung University of Muenster, Germany der Zwillinge brachte keine wesentliche Senkung Mutation 314del14 in GJB2 is the second Congenital Disorders of Glycosylation (CDG), a der Cholesterinspiegel. Im Alter von drei Jahren mutation in patients with nonsyndromic recently described group of multisystem disor waren die Xanthome deutlich vergrößert, und hearing loss in Poland ders, characteristically are associated with ma eine Behandlung mit Lipidapharese wurde be Waligóra, Jaroslaw (1,2,3,) Ploski, R. (4), jor nervous system impairment. The underlying gonnen. Dadurch wurde eine akzeptable MuellerMalesinska, M. (1), Pollak, A. (1), pathomechanism consists of an abnormal glyco Senkung der Cholesterinspiegel errreicht; die Korniszewski, L. (1,5), Skórka, A. (1,5), sylation of glycoproteins and glycolipids. CDG Kinder sind jetzt 4 1/2 Jahre alt und al Skarynski, H. (1) comprises two major subentities: Type I (CDG tersentsprechend gesund ohne Zeichen einer (1) Institute of Physiology and Pathology of Iae) concerns the synthesis and transfer of the kardiovaskulären Schädigung. Der molekulare Hearing, Poland, (2) II Department of carbohydrate chain. In type II CDG the process Befund erklärt sowohl die massive Hypercholes Paediatrics Neonatal Clinic Medical ing of the carbohydrate chains is altered. CDG terolämie bei den Kindern als auch den milden University in Warsaw, Poland (3) Ie is characterized by mutations the dolichol Phänotyp und die gute Therapieantwort bei den Postgraduate School of Molecular Medicine, phosphatemannose synthase1 gene leading to Eltern. Das Auftreten symmetrischer Hautläsio Poland (4) Human Molecular Genetics a deficiency of DMP1.

nen bei beiden Kindern war ein wichtiger Faktor Labolatory of II Department of Paediatrics Abstracts GfH ÖGH SGMG Tagungsband Here we report on the first child born to healthy für die frühe Diagnosestellung manchmal kann (5) II Department of Paedi parents of Sicilian origin. After an uneventful es unerwartete Vorteile haben, ein Zwilling zu Hearing loss is the most frequent disease of hu pregnancy the boy was delivered by cesarean sein. man senses. At least 60% of all cases are attrib section because of shoulder dystocia. He pre uted to genetic factors in developed countries. sented with the clinical picture of macrosomia During last few years several genes causing (4700g) and arthrogryposis multiplex with hearing loss have been discovered and beyond P104 28 clasped thumbs. In his first weeks of life he de any doubt the most important one is GJB2 gene veloped seizures. Several EEGs demonstrated coding connexin 26. Connexin 26 is a compo the pathologic pattern of burst suppression. An A Novel Mutation in AIRE Gene in a Patient nent of gap junctions in the organ of Corti. MRI of the brain showed a bilaterally reduced with Atypical Presentation of Autoimmune At first in our study we have tested 262 patients volume of the cerebral hemispheres. Screening Polyglandular Syndrome Type 1 with different levels of nonsyndromic hearing for common metabolic diseases showed normal Trebusak, Katarina, Battelino, T., Bratanic, N., loss. In order to detect 35delG mutation, which results. Cytogenetic studies carried out on pe Krzisnik, C. is the most frequent in Caucasian populations ripheral blood lymphocytes proved a normal University Children’s Hospital, Vrazov trg 1, we have used a method based on PCR mediat male karyotype. His motor and mental develop Ljubljana, Slovenia ed site mutagenesis. Among tested patients 137 ment was profoundly retarded. At examination Autoimmune polyglandular syndrome type 1 (52,3%) probands did not display 35delG, 92 with 4 years he only turned from his back onto (APS1) is a rare autosomal recessive disorder, (35,1%) were homozygotes for 35delG and 33 his stomach lacking any speech development. characterised by presence of two of the three (12,6%) were hetorozygotes for this mutation. His body measurements were below the third major clinical symptoms: hypoparathyroidism, The next step consisted in sequencing of the centile. Hypertelorism, beaked nose, high arched Addison’s disease and mucoutaneous candidia coding region of GJB2 gene in heterozygous pa palate and small teeth were observed. Addition sis. Several other endocrine or nonendocrine tients. In 13 cases mutation was found in the al investigations included a serum transferrin disorders may be present. The disease usually second allele. In 8 cases it was 314del14 and in isoelectric focussing (IEF), which serves as a occurs in childhood, but new tissuespecific two cases 333334delAA. Having regard to the convenient marker of glycosylation abnormali symptoms may appear throughout life. APS1 frequency of mutation obtained, a new method ties. Results showed a significant amount of cases are reported worldwide, with higher inci was elaborated permitting to display the three tetrasialo and disialotransferrin but hardly any dence in some genetically isolated populations most common mutations at the same time. This asialotransferrin. Clinical features and serum IEF (Finland 1/25000, Iranian Jews 1/9000, Sardinia method consists in simultaneous amplification pattern strongly suggest CDG Type Ie as the un 1/14400). The aetiology of APS1 is associated of the regions 1272 and 306464 of the coding derlying cause. Currently, further biochemical in with mutations in AIRE gene encoding a protein region of GJB2 with the use of labelled primers. vestigations on cultivated skin fibroblasts and predicted to be a transcriptional regulator. Products obtained are next subject to size molecular genetic analysis of the dolicholphos The patient was born in 1976. He presented with analysis with the application of ABI 377. 857 pa phatemannosesynthase1 gene are under way. alopecia at the age of 9 years, folowed by Addi tients were examined with this new method. son’s disease at the age of 11.5 years and IDDM Both methods were used on 1119 patients. and vitiligo at the age of 21 years. 314del14mutation was found in 21 compound Genomic DNA was extracted from peripheral heterozygotes for 314del14 and 35delG, and in P104 31 blood lymphocytes by salting out procedure. All one case homozygote for 314del14. 256 patients exons and the exon/intron boundaries of the were homozygotes for the 35delG mutation and Artificial neural networks in malformation AIRE gene were individually PCR amplified and 132 heteorozygotes for this mutation. Therefore syndrome diagnosis don’t believe and take directly sequenced. Nucleotide sequences were 314del14 mutation is second most frequent mu for granted, but weigh and consider compered to the sequence of the normal gene tation in the coding region of GJB2 in patients Graetschel, Gabriele, Pelz, J (GeneBank Access No. AB006684). with hearing loss in Poland and this fact should Reformstudiengang Charité, Humboldt A novel deletion of 1 G in exon 5 and common be taken into account when setting diagnostic Universität Berlin, Germany Finnish mutation R257X were identified. The methods in patients with hearing loss also in In this study, we developed an artificial intelli novel 540delG ( counting from AUG codon, Acc. neighbouring countries. gent diagnostic system based on an artificial No. AB006682) is a frameshift mutation, allow neural network (ANN) to support the diagnosis ing the synthesis of 180 amino acids, followed of multiple congenital malformation syndromes by 196 unrelated amino acids and then prema caused by chromosomal structural mutations. ture stop codon in exon 10. R257X is the most As a software tool a commercially available shell common mutation in Slovenian population. was used, which was suitable for development Although the patient is a compound heterozy of feedforeward ANNs trained using backpropa gote for two serious mutations resulting in short gation of errors. Several ANNs were built up us er and probably unfunctional protein AIRE, he ing standard training procedures with different only developed one out of three major clinical sets of training data. For the analysis of the re characteristics. On the contrary Iranian Jewish

medgen 14 (2002) 45 sulting diagnostic tools predefined criteria were Surgery, (3)Department of Phoniatrics, P104 35 used for the evaluation. Decisions for the correct Hannover Medical University, Germany syndrome with a high probability were favoured, A new approach for the detection of chromo MeCP2 related disorders in males while undecisiveness of the network and a dif some deletion 22q11.2 in interphase nuclei from Eric EJ Smeets 1, Ute Moog 1, Sam ferential diagnosis without the correct diagnosis buccal mucosa cells obtained by a noninvasive Schoenmakers2, Anneke MJ Schoonbrood led to a devaluation. Using different orders of procedure is decribed. FISH analysis has been Lenssen3, Kees EP van Roozendaal1, Jos cases within a training set we succeeded in the performed on samples from a group of 101 pa Herbergs1,4, Connie TRM Schrander optimisation process of different diagnostic tients that presented consecutively for speech Stumpel1,4 tools. The numbers of symptoms/combinations therapy and/or surgical correction of cleft palate. 1Department of Clinical Genetics, University for the description of cases were limited to 28, 92 patients had overt cleft palate (hard/soft), in Hospital Maastricht, Maastricht, The 55 and 78 in different series of tests. the most 9 patients, the cleft was submucous. At the time Netherlands; 2University Maastricht, efficient ANN yielded a diagnosis in more than of the investigation, most of the patients had al Maastricht, The Netherlands; 3Center for 95% of all tests the correct diagnosis was pro ready received surgical correction. Ages ranged People with Intellectual Disability,Pepijn’, duced in 74% of the analyses. A test of the final from three months to 20 years, mean age was Echt, The Netherlands; 4Research Institute ANN with more than 70 different clinical cases 7.1 years. 37 patients were female, 64 were Growth & Development (GROW), Maastricht yielded no correct answer in two cases and se male. A normal result has been obtained in 98 University, riously wrong diagnoses (the correct diagnosis patients; a deletion 22q11.2 was present in three Mutations in the MeCP2 (methylCpGbinding not within the differential diagnosis and high patients (2.8%), one with no other major clinical protein 2) gene are the cause of Rett syndrome probability value for a wrong decision) in addi finding than cleft palate. In one case, the dele which is a wellknown and clinically defined neu tional two cases. The significance of an ANN is tion turned out to be familial. With the investiga rodevelopmental disorder. Rett syndrome occurs to a great extent influenced by the experience of tion of interphase nuclei from buccal mucosa almost exclusively in females and for a long time the developer. cells, screening for 22q11.2 deletion also in was thought to be an Xlinked dominant condi groups with minor prior risk becomes feasible. tion with lethality in hemizygous males. Since The present investigation shows that in about the discovery of the MeCP2 gene as the cause 1% of patients with isolated cleft palate a dele P104 32 of Rett syndrome in 1999, a series of publica

GfH ÖGH SGMG Tagungsband Abstracts GfH ÖGH SGMG Tagungsband tion 22q11.2 occurs which may have conse tions reported the occurrence of MeCP2 muta quences to the families due to its heritability. tions also in males. These males phenotypical Clinical and moleculargenetic findings in 6 ly have Rett syndrome when the mutation arises families with cherubism as somatic mosaicism or when they have an ex Peters, Hartmut(1), Dette, B.(1), Mehdipour, P104 34 tra X chromosome. In all other cases, males with P.(2), Motamedi, K. M. H.(3), Stiller, M.(4), MeCP2 mutations show diverse phenotypes Petschler, M.(4) which are different from Rett syndrome and cov (1)Institute of Medical Genetics, Charité, Hereditary motor and sensory neuropathy er a spectrum from severe congenital en Humboldt Univ. Berlin, (2)Tehran University (HMSN) IA, developmental delay and cephalopathy via mental retardation with differ of Med. Sciences,Dep. of Human Genetics, autisme related disorder in a boy with ent neurological symptoms up to mild retarda Iran; (3)Clinic of Oral & Maxillofacial duplication (17)(p11.2p12) tion only. In this group, no de novo mutation has Surgery, Baqiyatallah Medical Center, Iran. Ute Moog1, J.J.M.Engelen1,2, J.W. Weber3, J. been reported up to now. (4)Dep. of Dental, Oral & Maxillofacial Steyaert4,6, F. Baas5, J.P. Fryns1,6 We present a 22 year old male with severe men Surgery, Free Univ. Berlin 1Department of Clinical Genetics, tal retardation, spastic tetraplegia, dystonia, Cherubism (MIM 118400) is rare autosomal 3Neurology and 4Paediatric Psychiatry, apraxia and neurogenic scoliosis. A history of dominant inherited syndrome characterised by University Hospital Maastricht, Maastricht, early hypotonia emerging into severe spastici excessive bone degradation of the upper and The Netherlands; 2Research Institute ty, slowing of headgrowth, breathing irregulari lower jaws followed by development of fibrous Growth & Development (GROW), Maastricht ties and a good visual interactive behaviour were tissue masses, which causes a characteristic fa University, Maastricht, The Netherlands; reminiscent of Rett syndrome. He had a de novo cial swelling. The bone lesion may cause prema 5Department of Neurology, Academic missense mutation in exon 3 of the MeCP2 gene ture loss of deciduous teeth and displacement Medical Center, Amsterdam, The (P225L). of the permanent dentition. Netherlands All affected family members were examined clin We present a 6yearold boy with moderate de ically and radiographically. We detected 5 muta velopmental delay, gait disturbance, autisme re tions in exon 9 of the gene coding the cAbl lated disorder and mild dysmorphic features with P104 36 binding protein SH3BP2 by DNAsequencing in a triangular face, hypertelorism and a preauric five of six families investigated. ular pit. He was seen for evaluation of his retar Photoanthropometric Investigation of Facial The destruction and expansion of the jaws is dation since the age of 2 years. At first sight, a Structures most active in patients between the age of 3 and cytogenetic analysis showed a normal 46,XY Farhud D.D.1; Mahmoudi M. 2; 7 years. The spectrum of severity may range karyotype. Neurologic examination at the age of DerakhshandehPeykar.P. 1, StengelRutkowski from extreme bilateral expansion to subclinical 5.5 years revealed a motor and sensory polyneu S. 3 unilateral involvement. In one family we ob ropathy. FISH with probe PMP22 and VAW409 1. WHO Ethical Committee, Geneva, served craniosynostosis and clubbing of the fin specific for CharcotMarieTooth type 1 (CMT1) Switzerland/2. Dep. of Human Genetics, gers in two patients. There was no difference in disclosed a duplication which confirmed the di School of Public Health, Terhran Univ. of frequency of affected males and females among agnosis HMSN IA. Subsequently, GTG banded Medical Sciences, TehranIran /2. . Dept. of 52 probands, but the onset of the disorder oc metaphases were reevaluated and a small du Anatomy, School of Medicine, Univ. of curred in females at an older age. In contrast to plication 17p was seen on retrospect. Addition Babol, Iran/3.. Genetic Diagnosis and previous reports we observed incomplete pene al FISH with probe LSISMS (Vysis) specific for Counselling Department in the Munich Child trance also in males. the SmithMagenis region at 17p11.2 again Center, Institute showed a duplication. Cytogenetic investigation The photoanthropometric method suggested including FISH for CMT1 was normal in both first by StengelRutkowski et al. (1984) was used parents. The boy had a de novo 46,XY,dup (17) P104 33 to study the facial features in 136 Iranian chil (p11.2p12) karyotype. dren with Down syndrome, aged 4 to 14 years. Up to now, 4 patients have been reported with Nineteen parameters were investigated and Search for Deletion 22q11.2 in Interphase the same duplication. The present case confirms compared to an age related control group of 100 Nuclei of Buccal Mucosa of Patients with previous findings of mild to moderate delay, neu normal Iranian children. The obtained measure Apparently Isolated Cleft Palate: A New robehavioural features and minor craniofacial ments were related to reference values in the Diagnostic Approach anomalies as the major phenotypic features. same faces. The normal range was defined by Shouman, Nadima (1), Pabst, B. (1), Arslan Furthermore, it illustrates the need of a thorough age related index values between the 20th and Kirchner, M. (1), Eckardt, A. (2), Schönweiler, R. cyto and molecular genetic workup on clinical 80th percentile in the collective of normal Iran (3), Ptok, M. (3), Mehraein, Y. (1), Schmidtke, J. grounds. ian children. Five parameters were considered (1), Miller, K. (1) as characteristic facial traits of Iranian children (1) Department of Human Genetics, (2) with Down syndrome by index values outside Department of Oral and Maxillofacial these percentiles in ¡Ý50% of the studied collec

46 medgen 14 (2002) tive: low midface; narrow, upslanted palpebral P104 38 this ratio changes is informative too.We demon fissures and short, anteriorly rotated ears. strated the increased level of chromosomal Twelve parameters were considered as addition aberrations due to exceeding of chromatide Overexpression of Xq28 causing retardation al facial traits by index values outside these per aberration type in children evacuated 910 years in a boy with der(Y)t(X;Y) centiles in ¡Ý30% <50% of the studied collec ago from contaminated Gomel area to Minsk Caliebe, Almuth (1), Tüshaus, L. (2), Siebert, R. tive: broad inner canthal distance; prominent city. Taking into account, that irradiation produce (1), Partsch, C.J. (2), Grote, W. (1) nose root; short nose back; everted nasal base; the chromosome aberration type mainly, we sup (1) Institut für Humangenetik und (2) Klinik long nasolabial distance; forwards inclined in pose, that chronic low dose radiation exposure für Allgemeine Pädiatrie, tegumental upper lip; narrow mouth fissure; can modify the genetic repair response of cells Universitätsklinikum Kiel, Germany high, prominent chin; highset, narrow ears and after another genotoxic influence. As the result, Deletions of the long arm of the Ychromosome narrow conchae. These results contribute to an environmental chemical mutagens stimulate the (Yq) cause a wide phenotypic spectrum. Loss objective definition of facial traits in children with higher frequency of chromatide aberration.Aber of the heterochromatic region is usually not as Down syndrome in a homogeneous ethnic pop rations level in newborns concerned as back sociated with abnormalities. Deletions of the eu ulation. ground pattern reflecting the basic tendencies of chromatic region are found in individuals ascer mutagenesis in a cohort. Dicentric and ring chro tained for short stature and azoospermia, but mosomes frequency as well as amount of poli also several patients with dysmorphism, micro ploid and aneuploid cells are essentially in P104 37 cephaly, short stature, and retardation have been creased in newborn of contaminated area in described. comparison with the marched group. That spec We report on a boy with microcephaly, failure to CAMURATIENGELMANN DIAPHYSEAL ifies the biologically effective influence of radia thrive, and retardation. MR imaging of the brain DYSPLASIA familial occurrence with tion exposure on the hereditary device of blood showed atrophy, retarded myelinisation, and a anticipation lymphocytes.Studies of different age/territory hypoplastic corpus callosum. The face was re Seemanová Eva (1), Leschik, G. (2), Mundlos S. groups of Belarus children population showed garded as „funny looking“ with no discernible (2), Tinschert, S. (2) that children who were born in 198688 have un dysmorphism. At the age of 15 months he was (1) Dept. of Clinical Genetics, Charles dergone most effective influence of ionizing ra not able to sit and required a gastrostomy tube

University Prague (2) Institut für diation on the somatic cells chromosomes. Abstracts GfH ÖGH SGMG Tagungsband for sufficient feeding. Chromosomal analysis on Medizinische Genetik, Charité, Humboldt Thereof the specified cohort should be referred peripheral blood lymphocytes of the patient Universität zu Berlin to group of the increased genetic risk. showed a male karyotype with a deleted Ychro We report on 3 sibs affected by muscular hy mosome described as del(Y)(q11). Multiplex poplasia and weakness, broadbased waddling PCR for the AZFregions showed complete loss. gait, hyperlordosis, leg pain, and reduction of The paternal Ychromosome was normal. P107 02 subcutaneous fat, who had been diagnosed with In 1994 Lahn et al. described three microcephal autosomal recessive myopathy. Family History – ic and retarded boys with a der(Y)t(X;Y)(q28; The parents are young and nonconsan Down Syndrome Registry fields for q11.1). Molecular studies showed a paternal and guineous. The mother and oldest brother are application in Belarus a maternal allele for markers localised in Xq. All healthy. The father has unilateral deafness and Rumyantseva, Natalia; Polityko A., Naumchik I., patients had an elevated glucose 6phosphate received surgery for coxa valga and genua vara, Asadchuk T, Zatsepin I. dehydrogenase (G6PD)activity. Overexpression cortical and endosteal hyperostosis were Institute for Hereditary Diseases, Minsk, of genes localised in Xq28 was regarded as demonstrated radiographically. His mother was Belarus causative for the phenotype. The syndrome was asymptomatic, but had severe hyperostosis on A computerannotated database Down Syn named XYXqsyndrome. As our patient showed radiographs. Clinical picture – The onset of the drome Registry (DSR) is presented. It includes a phenotype comparable to that syndrome, we disorder was reported in the sibs at the age of cytogenetics, phenotypes, genetic data of 953 performed FISH with several clones mapping to 22.5 years during different febrile illnesses, af patients detected by GTGbanding method Xq28. ter which muscular hypotrophy and subcuta (19832001 years). Unusual karyotypes are col The probes for the genes FVIII, G6PD, TMG3, neous lipoatrophy were noted. All show muscle lected in CytoVision. Cytogenetics: DSR con MTM1 hybridised not only to the X but also with weakness, joint hyperexcursibility, lumbar hyper tains 840 cases full trisomy 21, 79 Down syn the terminal portion of the derivative Ychromo lordosis and waddling gait, muscular pain in the drome (DS) cases due to de novo and inherited some. The duplicated fragment was determined legs, and have frequent headaches. 2 of 3 sib Robertsonian translocations and other structur to have a size of about 4 Mb. Functional studies lings suffer from microcystic anemia. Mental de al abnormalities, 13 complex rearrangements for the G6PDactivity showed a two to threefold velopment is quite normal in all 3 children. EMG and 21 mosaic cases. Rare aberrations were ob elevation in the patient. findings are normal, serum CK, LD, and alkaline served: numerical aberrations: 48,XXY, +21; To exclude the XYXqsyndrome FISH with clones phosphatase are in normal ranges. Radiograph structural unbalance: 46,XX,psudic(21;21)(21pter of Xq should be offered to all patients with a Yq ic findings – There is striking thickening of the >21q22::21q22>pter); 46,XY,der(9)t(9;21) karyotype. cortices of the long bones, particularly involving (p24.3;q11.2); 46,XX,dup(21)(q22q11.2); complex Lahn et al., Nat Genet 8, 243250 (1994) the diaphyses with irregular bone contours in all rearrangements: 48,XY,+21,+mar; 47,XX, t(11;21) affected persons. All show sclerosis of skull (q21;q22)mat,+21; 47,XY,t(12;22)(p11.2;q13) pat, base. Analysis of the phenotype, family history +21; 47,XX,t(9;14)(q13;q22)+21; 47,XY, inv(7) and radiological findings leads to the diagnosis P107 01 (p12q21.1)mat,+21; 47,XX, inv(9)(p11q13), +21. AUTOSOMAL DOMINANT CAMURATIENGEL Mosaic forms are presented as numerical abnor MANN DYSPLASIA (CED). DNA analysis con malities 47,XY,+21/48,XY+21;+21; 47,XY, +21/ Cytogenetic monitoring database: the firmed CED by detection of the most common 46,XY, as structural 46,XX, der (21;21) (q10; assessment of the tendencies and dynamics R218C mutation in TGFB1 gene. Conclusion – q10),+21/46,XX and complex rearrangements of mutagenesis in child Variable expression is presented in the family 48,XY,+21,+mar/46,XY. Phenotype’s data con Polityko, Anna D., Egorova T.M., Pisarik I.V., with manifestation of CED in 5 members of 3 tains clinical, ultrasonography, post mortem find Khurs O.M. generations. The age of onset and severity of ings. DSR includes different proband & parents Institute For Hereditary Diseases, Minsk, manifestation in successive generations which characteristics like sex, age, origin of mutation Belarus show an anticipation had led to the mistaken (de novo, inherited), consanguinity, ethnic origin, Several of mutagenesis tendencies were discov clinical impression of autosomal recessive mus graphic pedigree. Fields for application: DSR is ered during 19862002 cytogenetic monitoring cular dystrophy in the youngest generation. utilized in the scientific researches, genetic studies of Belarus children population. When counseling and prenatal diagnostics service, chronic internal and external exposure of popu congenital malformations monitoring system, in lation by low doses of ionizing radiation take different Teaching Genetics courses for medical place, it is productively to analyze the basic geneticists, cytogeneticists, other physicians characteristics of cytogenetic status, i.e. fre and students. DSR is an inprogress database. quency of: Our next step for update DSR is to built a new chromosome aberrations, aberrant metaphases, „prenatal DS cases“ subgroup. Rare cytogenet chromatide and chromosome aberrations, cyto ical and clinical data of our patients with DS will genetic markers of irradiation, as well as the pa be discussed. Teaching Genetics courses organ rameters of a ratio of frequency of chromo ization in Belarus will be presented. some/chromatide aberrations. The dynamics of

medgen 14 (2002) 47 P107 03 ly in amniotic fluid cells (38%) and fetal lympho P107 06 cytes (78%) and was confirmed postnatally in the umbilical cord (52%), placental biopsy A Phenotypically Normal Liveborn Male after Analphoid de novo marker chromosome (40%), lymphocytes (between 55% and 70%) Prenatal Diagnosis of Trisomy 20 Mosaicism invdup(3)(q28qter) with neocentromere in a and buccal mucosa cells (between 30% and von Beust, Gabriela, Bartels, I., Zoll, B. dysmorphic and dvelopmentally retarded 42%), demonstrating the overall prevalence of GeorgAugust University, Goettingen, girl the trisomy 8 cell line in this patient was quiet Germany Barbi, Gotthold (1), Spaich, Ch.(2), Adolph, S. high. We found no evidence of an appreciable Trisomy 20 is a common chromosomal mo (2), KehrerSawatzki, H. (1) increase or decrease in the frequency of the tri saicism diagnosed in amniotic cells, constituting (1) Department of Human Genetics, somic cell line over a period of eleven years. 16 % of all such mosaicism, and it is also one of University of Ulm, Germany; (2) Institute of Molecular genetic investigation demonstrated the most common pseudomosaic trisomies. Tri Clinical Genetics, Olgahospital, Stuttgart, the maternal origin of the additional chromo somy 20 mosaicism remains a serious dilemma Germany some 8. It is known from the literature that tri in prenatal diagnosis and genetic counseling. In We describe a marker chromosome from distal somy 8 mosaicism in liveborns can almost al prenatal chromosome analysis a true mosaic is 3q which is smaller than those reported earlier. ways be traced to a postzygotic mitotic error. present if two different karyotypes are found in The marker was ascertained during cytogenetic at least two different clones or cultures. We re diagnostics of a 5yearold girl with develop port on a case of a prenatally detected trisomy mental delay and mild facial dysmorphism. The 20 mosaicism and the pregnancy outcome. Cy P107 05 girl showed slight hirsutism and a bilateral togenetic analysis was performed on amniocytes ichthyosiform hyperkeratosis of hands and feet. (the karyotypes identified in three independent At the age of 5 years, she showed slight muscu Subtelomeric screening: new means to cell cultures were 46, XY[15]/47,XY+20[4]; lar hypotonia and hyporeflexia. Perceptive skills diagnose chromosomal changes in children 46,XY[12]/47,XY+20[6] and 46,XY[20]/47,XY and visiomotoric coordination were retarded cor with malformations of the heart +20[3], respectively)and postnatally on lympho responding to a developmental age of 33.5 Schellberg, Ruth (1); Wiebe, W. (2); Schwanitz, cytes and extraembryonic tissues.For analysing years. At the age of 8.5 years the girl attends a G.(1); Raff, R.(1) uroepithelial cells we established a new proto special school for mentally handicapped chil

GfH ÖGH SGMG Tagungsband Abstracts GfH ÖGH SGMG Tagungsband (1) Institute of Human Genetics, University col. We performed cell nuclei preparation by dren. She cannot speak properly, seeking for of Bonn, Germany; (2) Deutsches treating with potassium chloride and trypsin so words, can recognize only few letters, and is still Kinderherz Zentrum, Sankt Augustin, lution and after fixation procedure we performed unable to write. Chromosome analysis detected Germany a pepsin digestion, so that we revealed good nu a very small supernumerary de novo marker Congenital heart defects are heterogeneous. Be clei preparation of urine sediment for FISH chromosome, which was Cband negative but sides being exclusively exogenous, there exist analysis. After birth of a healthy male baby con appeared mitotically stable in cultured lympho those caused by genetic factors on the one ventional cytogenetic analysis and FISH were cytes. FISH with an alpha satellite probe detect hand and on the other hand multi factorial ones performed on umbilical cord blood lymphocytes, ing all human centromeres gave hybridization adding an exogenous stimulus to an existing ge on extraembryonic tissues (three different sites signals on all chromosomes of the proband, ex netic disposition. of the placenta) and on an urine sample. The cept the marker chromosome. The chromosomal The latter include the majority of congenital mal postnatally analysed cells showed disomy 20 by origin of the marker was clarified by microdis formations of the heart. The frequency of heart conventional and FISH analysis. The phenotyp section and reverse painting: After microdissec defects among newborns amounts to 0,6% in ically normal male baby is developing normal. tion and amplification DNA from the marker hy Central Europe. Already prenatally, a high per We compare our data to previously described bridized exclusively on the marker and on distal centage of severe malformations of the heart cases in the literature. The significance of a pre 3q. In order to characterize the extent of the can be diagnosed by ultrasound. natally analysed trisomy 20 mosaicism is still in marker chromosome along 3q, FISH with Parents of a child with congenital heart defects doubt. Review of the literature shows that it is mapped YAC or BAC clones was performed. All carry a risk of 24%, statistically, to have anoth not possible to correlate the presence of amni clones tested gave either no FISH signal on the er child with congenital heart defects. This em otic fluid trisomy 20 mosaicism with any specif marker chromosome, or a double signal. Thus pirical figure includes couples with a very high ic phenotypic abnormalities. the marker belongs to the expanding category risk (50%) and those with a very low one (less of analphoid marker chromosomes originated by than 1%), though. Furthermore, of importance is (presumably inverse) duplication of a distal chro the indication of additional malformations and/ mosome arm, which must have gained cen P107 04 or dysmorphic features like morphological tromere function by neocentromere formation. changes without illnesses. This requires classi According to the ENSEMBL database the most fication of the syndromes. Followup in a Patient with Trisomy 8 proximal clone present on the marker, RP11 About 10% of the patients with congenital mal Mosaicism over a Period of Eleven Years 634l24, maps to chromosome band 3q28, and formations of the heart show the presence of a Raff, Ruth; KalzFüller, B.; Schwanitz, G. the extent of the duplicated segment is approx chromosomal aberration detectable by conven Institute of Human Genetics, University of imately 7 Mb. tional cytogenetic analysis. Prenatally performed Bonn, Germany ultrasound at specialised clinics can even lead Trisomy 8 estimated to occur in about 0,1% of to a percentage of 40% of malformations of the all clinically recognised pregnancies. In the live heart caused by chromosomal aberrations thus P107 07 born population, trisomy 8 is almost always as justifying a supplementary chromosomal analy sociated with mosaicism, and more than 100 sis in all cases of heart defects. cases have been reported so far. Individuals with Recurrent bone fractures and overgrowth in Up to now, there was no way to unequivocally trisomy 8 mosaicism may present with a variety a retarded boy due to unbalanced exclude small structural aberrations by means of of features, including moderate mental retarda t(2;15)(q37;q26)de novo translocation conventional analyses. The development of flu tion, multiple skeletal and joint anomalies, uro O. Rittinger (1,2), G. Kronberger (1), E. Ploechl orescence in situ hybridisation (FISH) which en genital malformations, congenital heart defects, (1) and A. Rauch (3) ables the investigation of critical regions with deep palmar and plantar furrows, distinct and (1) Klinische Genetik an der (2) Landesklinik small DNAprobes offered further results con agenesis of the corpus callosum. There is a für Kinderund Jugendheilkunde, Salzburg, cerning submicroscopic microdeletions. great phenotypical variability, and several pa Austria (3) Institut für Humangenetik, Our study presented deals with the exclusion of tients displaying normal intelligence and devel Friedrich Alexander Universität Erlangen subtelomeric deletions leading to congenital opment and normal or nearnormal phenotype Nürnberg, Germany malformations of the heart. Preliminary results have also been described. Growth retardation is considered a common fea are being presented. To date, relatively few longterm studies of ture in children affected with developmental de Supported by the RichardWinterStiftung probands with trisomy 8 mosaicism have been lay caused by an unbalanced chromosomal re published. We report on the cytogenetic and arrangement. Prenatal onset of tall stature, by moleculargenetic investigations of a child with contrast, is observed mainly as a result of ma mosaic trisomy 8, analysed over a period of ternal influence eg gestational diabetes, so eleven years. The female patient showed clinical called overgrowth syndromes or distinct genet features and facial dysmorphisms characteristic ic causes like fragile X syndrome. We report on of the syndrome as well as mentally impairment. a now 2 year old boy who showed in the new The mosaic trisomy 8 was diagnosed prenatal born period macrosomia, muscular hypotonia,

48 medgen 14 (2002) long and thin hands and feet with hyperextensi trachromosomal localization of elements of the P107 10 ble fingers, somewhat unusual facial ap cytoskeleton (e.g. tubuline and tauprotein), and pearence, and transient hypocalcemia. Later on permits analyses of the locationspecific inter A Phenotypically Normal Liveborn Male after overgrowth persisted and psychomotor and action between some (e.g., by multicolor FISH). Prenatal Diagnosis of Trisomy 20 Mosaicism speech delay was noticed. In the age of 1 year Fluoreszenz analysis of interphase nuclei and von Beust, Gabriela, Bartels, I., Zoll, B. two femoral fractures occurred without an ap metaphase chromosomes after Feulgen staining Institute of Human Genetics, GeorgAugust propriate trauma. Lymphocyte karyotyping re (DNAanalysis at 63 C; excitation laser wave University, Göttingen, Germany vealed a small additional fragment on 2q. Fur length 364nm, 488 nm, 543 nm and 633 nm) al Trisomy 20 is a common chromosomal mo ther investigation by means of subtelomeric lows measurements of the DNA content in the saicism diagnosed in amniotic cells, constituting probes revealed a translocation of the subtelom different phases of the cell cycle. The integrated 16% of all such mosaicisms, and it is also one eric 15q FISHsignal to 2q, in addition to 2 nor density profiles shows in some of the nuclei of the most common pseudomosaic trisomies. mal signals on both chromosomes 15. Using a chromosomes in a compartment like structure Trisomy 20 mosaicism remains a serious dilem panel of 9 FISH probes mapping to the telomer („chromosome territories“), probably in the end ma in prenatal diagnosis and genetic coun ic 2q region, evidence was given for a very small of G2 phase. In addition to this 3D analyses we selling. In prenatal chromosome analysis a true 2qter deletion in addition to the partial trisomy made image stacks (e.g. of FISH stained mosaic is present if two different karyotypes are 15qter. The parent’s karyotype proved to be nor metaphase plates) to generate threedimension found in at least two different clones or cultures. mal. To our knowledge, our patient represents al image information, such as the creation of We report on a case of a prenatally detected tri the first observation of this unusual clinical pic stereo images and orthogonal or oblique image somy 20 mosaicism and the pregnancy out ture associated with this particular rearrange sections. come. Cytogenetic analysis was performed in ment. More detailed analysis of the breakpoints amniocytes (the karyotypes identified in three in on 15q and the respective genes might elucidate dependent cell cultures were 46,XY[15]/47,XY pathogenesis of the phenotype and possible P107 09 +20[4]; 46,XY[12]/47,XY+20[6] and 46,XY[20]/ therapeutic approach. 47,XY+20[3], respectively), and postnatally in lymphocytes and extraembryonic tissues. For A third case of proven mosaicism for analysing uroepithelial cells we established a

del(17)(p11.2p11.2) in a SmithMagenis Abstracts GfH ÖGH SGMG Tagungsband P107 08 new protocol. We performed cell nuclei prepara syndrome (SMS)patient tion by treating with potassium chloride and Schüler, Herdit M., Zerres, K. trypsin solution and after fixation procedure we Confocal laser scanning microscopy on the Institute of Human Genetics, University of performed a pepsin digestion, so that we re cytoskeleton and human chromosomes Technology, Aachen vealed good nuclei preparations of urine sedi after immunostaining, multicolor FISH and SmithMagenissyndrome (SMS) is associated ment for FISH analysis. After the birth of a Feulgen reaction with an interstitial deletion involving band healthy male baby conventional cytogenetic Volkmar Beensen (1), Thomas Liehr (1), Anita 17p11.2. Since its first description in 1982, over analysis and FISH were performed on umbilical Heller (1), Heike Starke (1), 100 patients with SMS have been reported. The cord blood lymphocytes, on extraembryonic tis Institute of Human Genetics and incidence is estimated to be 1:25.000 livebirth, sues (three different sites of the placenta) and on Anthropology, FriedrichSchillerUniversity but it is believed that this condition is easily an urine sample. The postnatally analysed cells of Jena, Germany (1), Institute of Molecular overlooked clinically and also be missed cytoge showed disomy 20 by conventional and FISH Biotechnology e.V., Jena, Germany (2), netically. analysis. The phenotypically normal male baby Institute SMS is characterised by a welldocumented but is developing normal. We compare our data to At present confocal laser scanning microscopy varying clinical phenotype including brachy previously described cases in the literature. The allows an insight into exceedingly complex or cephaly, prominent forehead, epicanthal fold, significance of a prenatally diagnosed trisomy 20 ganization of chromosomes. New results are broad nasal bridge, ear anomalies, prognathism, mosaicism is still doubtful. Review of the litera now possible by analysis of 3dimensional ultra short stature, brachydactyly, sleep disturbance, ture shows that it is not possible to correlate the structural pictures. Especially the simultaneous selfinjurious behaviour, signs of peripheral neu presence of amniotic fluid trisomy 20 mosaicism presentation of fluorochromed chromosomes in ropathy, delayed speech development, and vari with any specific phenotypic abnormalities. combination with fluorescenceimmunohisto able degrees of mental retardation. chemical presentations of the cytoskeleton pro We report a third proven mosaic case for the vides an innovative methodical approach to deletion characterising SMS. To our knowledge, study the open questions. For the simultaneous proven mosaicism for del(17)(p11.2p11.2) has P107 11 presentation of the cytoskeleton proteins with been reported before only twice. Our patient is primary and secondary antibodies (antibodies la the second who is mosaic and displays charac Partial trisomy 10p and partial monosomy beled with FITC and Rhodamine) and metaphase teristic phenotypic anomalies of SMS. The first 6p resulting from a paternal translocation chromosomes (amniocyte preparation: in situ observed mosaicism was a phenotypically nor case report and review of the literature technique; 4,6diamidino2phenylindole DAPI mal mother mosaic for del(17)(p11.2) discovered SiebersRenelt, Ulrike; Exeler,J.R.; staining), the common methanolacetic acid only because she had had a child with SMS. Kennerknecht,I.; Horst,J. fixation (3:1) was replaced with a fixing pretreat The detection of mosaic cases not only for SMS Institute of Human Genetics, University of ment of the cells with a mixture of triton X100 but also for other structural rearrangements em Muenster, Germany (2%), formaldehyde (2%) and glutaraldehyde phasises the value and usefulness of FISH We report on a 2 year old boy who is the first (0.5%). This kind of specimenpreserving pre analyses. Furthermore a generous use of FISH is child of a treatment does not cause any visible spreading recommended even though only a part of exam healthy not consanguinous couple. At delivery of chromosomes in the form of metaphase ined metaphases are suspected to bear a subtle the mother was 34 years old and already had plates Fluorochroming of individual chromo aberration. two healthy children from a previous marriage. somes or chromosome regions („multicolor It is quite possible that mosaicism for During pregnancy no adverse events were not FISH“) was performed after a method described del(17)(p11.2p11.2) and further structural re ed. Delivery was induced because of CTGab by RUBTSOV et al. (Hum. Genet. 97: 705709, arrangements may be a significantly underdiag normalities in the 38th week. Birth weight, length 1996). An efficient analysis of specimens pre nosed cause of contiguous gene syndromes, and head circumference were in the normal pared by the new developed objectpreserving particular in patients with mild expression of the range. Several craniofacial abnormalities are method described is made possible especially phenotype. present: triangular face shape, unilateral flat oc by the capability for simultaneous excitation of The frequency of mosaicism for structural chro ciput, frontal bossing, low set ears with preau all fluorochromic dyes used (including DAPI, mosomal abnormalities is unknown but this type ricular tags, deep hair insertion, long lashes. FITC, Rhodamine) and the acquisition and analy of aberration may be much more common than Moreover, the boy shows anisocoria with normal sis of data in three dimensions. For instance in was, is, or ever could be, recognised. reagibility of the pupils. In addition he developed the metaphase stage compared with an inter microcephaly (<3rd percentile). The statomotoric phase nucleus a quantitative increase in the development is severly retarded. Development tubuline and tauprotein concentration can be of speech is also severly disturbed although correlated to the increase of chromosomal DNA hearing tests were normal. Structural brain ab concentration. In the case of simultaneous test normalities or organ malformations are not pres ed ßactin such a quantitative increase was not ent. On karyotyping we found an elongation of found. The obtained data allow conclusions to the short arm of chromosome 6 derived from a be drawn on the intracellular, intranuclear and in

medgen 14 (2002) 49 previously unknown translocation of the father. cific aspects of the 21q syndrome. Here we re P107 15 The karyotype of the child is 46,XY, der (6) t port on the clinical, cytogenetic and molecular (6;10) (p25;p13) pat. Compared with cases re characterization of a pure de novo partial mono Dysmorphic stigmata and psychomotor ported in literature our propositus shares some somy 21 with a deletion of 21q22.2qter. FISH retardation in a boy with a rare interstitial features with other individuals with trisomy 10p mapping of cytogenetically and genetically an deletion del(9)(q32q34) (microcephaly, hypotonia and severe develop chored YAC and BAC clones result ed in the Volleth Marianne (1), Muschke P (1), Liehr T (2), mental delay). Cleft lip / palate is also reported identification of a BAC clone clearly spanning Starke H (2), Gedschold J (3), Brett B (3), although in association with the region p11.2 to the deletion breakpoint. Moreover, we have am Stumm M (1,4), Wieacker P (1) p12.2. Partial deletion of the distal part of 6p plified different single copy DNA probes within (1) Institut für Humangenetik, Leipzigerstr. correlates with abnormalities of the anterior eye exonintron sequences of the ETS2 (avian ery 44, 39120 Magdeburg, (2) Institut für and hearing loss. The anisocoria of our patient throblastosis virus E26 oncogene homolog 2) Humangenetik, Kollegiengasse 19, 07743 is an interesting finding in this context. In sum gene for FISH analyses. Interestingly, our results Jena, (3) Sozialpädiatrisches Zentrum, mary, the phenotype of the unbalanced translo revealed that the deletion breakpoint must lie in AdolfJentzenStr. 2, 39116 Magdeburg, (4) cation in our case seems to be influenced both a fragment of 5,2 kb, between exon 5 and exon Praxis, Kurfürstendamm 199, 10719 Berlin by partial trisomy 10p but also by haploinsuffi 8 of ETS2. Molecular studies using polymorphic We report on a 2 year old boy with a very rare ciency for genes located on the distal part of 6p. markers supported these findings and showed deletion 9(q32q34). The 30 year old father and that the derivative chromosome 21 was of pater the 29 year old mother are healthy and not con nal origin. The patient who showed mild mental sanguinous. The pregnancy and delivery were retardation, marfanoid habitus and minor dys P107 12 uneventful, the birth weight was 2080g (< 3 cen morphic features, is lacking most of the typical tile), length 48 cm (3 10 centile) and head cir features seen in the 21q phenotype and thus is cumference 32 cm (< 2 centile) after 40 weeks Cytogenetic and molecular cytogenetic quite unique. Our findings support the sugges gestation. The patient presents at the age of 2 study of an infant with MIDAS syndrome: tion that the loss of the region at 21q22.2qter is years dysmorphic stigmata including downslant First case of MIDAS syndrome in a 46,XY critical for only some minor aspects of the 21q ing palpebral fissures, prominent forehead, ret male syndrome. Genotypephenotype correlations of rognathia, deep set ears, microcephaly, broad

GfH ÖGH SGMG Tagungsband Abstracts GfH ÖGH SGMG Tagungsband Walter Werner (1), O. Bartsch (1), K. Kutsche our case and other reported cases will be dis nasal bridge, simian crease, and arachnodacty (2), P. Meinecke (3) cussed. ly. GTGbanding analysis of cultured lympho (1) Institute of Clinical Genetics, Medical cytes revealed an aberrant karyotype 46,XY Faculty, Technical University Dresden; (2) ,del(9)(q32q34). The interstitial break points were Institute of Human Genetics, University of P107 14 confirmed by highresolution multicolorbanding Hamburg; (3) Department of Medical (MCB) with a chromosome 9 probe mix and Genetics, Children’s Hospital Altona FISHanalyses with a ABL gene specific probe The MIDAS syndrome (microphthalmia, dermal An Unusual Intrachromosomal [VYSIS] and three different YACprobes (957k08, aplasia, sclerocornea) alias MLS syndrome (mi Rearrangement in Chromosom 16 in an 933c05, 750c6). To the best of our knowledge, crophthalmia, linear skin defects) is a rare X Azoospermic Man there are only 3 further patients with del linked multisystemic disorder occurring exclu Hickmann, G.(1); Chudoba, J.(2); Jauch, A.(3); (9)(q32q34) reported in the literature (Farrell et sively in XX individuals. To date, 32 cases of MI Exeler, R.(4); Schubert, R.(5); Heinrichs, S.(1); al., 1991). Currently, the small number of cases DAS syndrome have been published. In most Kozlowski, P.(1) does not allow the delineation of a characteris cases, the MIDAS syndrome was associated (1)PraenatalMedizin und Genetik, tic chromosomal syndrome. with Xpter>Xp22.2 monosomy. These observa Duesseldorf, Germany; (2) MetaSystems, tions suggest that deletions within the critical Altlussheim, Germany; Institut für Xp22.2 area are lethal in males, possibly due to Humangenetik, Universität Heidelberg, nullisomy for the causative gene. Germany; Institut für Humangenetik, P107 16 The patient, a male infant, showed intrauterine Universitätsklinikum Muenster, Germany; growth failure (week 37, birthweight 1640 g)lin Institut für Humangenetik, Universität Bonn, A molecular cytogenetic study of female ear skin defects parallel to the nose, and corpus Germany patients with various Xq deletions callosum agenesis. Findings strongly suggest Cytogenetic investigation in a couple with fertil M. Linné, O. Bartsch, G. K. Hinkel, W. Werner MIDAS syndrome. Additionally he suffered from ization failure revealed a normal karyotype for Institut für Klinische Genetik, Medizinische severe congenital lactic acidosis, and cardiomy the woman while the partner showed an aber Fakultät C. G. Carus, TU Dresden opathy. High resolution RBGbanding revealed a rant chromosome 16. The banding pattern after Efforts of more precise karyotypephenotype subtle inversion in Xp22 [inv(X)(p22.33/22. GTG, CBG and RBGbanding was not compat correlations have been made in 10 cases of var 32p22.13)] in 15 % of lymphocytes. ible with an inversion and more than two break ious Xq deletions. First, the presence of an ad The finding was confirmed by FISH and DNA points were suspected in one chromosome 16. ditional 45,X cell line was evaluated. Second, the analyses. Biochemical studies confirmed a defi After FISH using a WCP 16 translocation involv breakpoint localization as well as the X inactiva ciency of the pyruvate dehydrogenase complex. ing another chromosome was excluded. Hybridi tion status were studied by highresolution RBG To our knowledge, this is the first case of MIDAS sation using a locus specific probe for 16p13.3 banding. Third, the precise breakpoint localiza syndrome in a 46,XY male. (D16Z71) showed a normal signal localisation on tion was verified by FISH using a specific set of the normal #16 while on the der(16) the signal Xq probes: XIST (q13.2), pcos80II7 (q13.2 was observed proximal to the centromere on the q21/Yp11), and tel Xq/Yq (subtelomeric Xq28). parm. However, only by multicolor banding P107 13 Further, the presence of a minor Xq/Xp+ or Xq (mBAND) it was possible to identify the chromo autosome rearrangement was excluded by FISH somal structure and 5 breakpoints were diag using cosmid probes 2.1/2.B (in PAR1) and Clinical, cytogenetic and molecular analyses nosed. Additional information was obtained by wcpX probe. of partial 21q monosomy in a girl with hybridisation using the subtelomeric probeset The results were: 1. Nine out of ten cases of our mental retardation, marfanoid habitus and for chromosome 16 which leads to the diagno Xq deletions had a breakpoint in the proximal re minor dysmorphic features sis of altogether 6 breakpoints. The derivative gion Xq13 to Xq24; 2. One case was an unbal Ehling, Daniela (1)(2), Lemcke, B. (3), chromosome 16 was described as follows: anced Xq/12q translocation with a distal Xq Kennerknecht, I. (3), Exeler, R. (3), Schmitt breakpoint; 3. An additional 45,X cell line was John, T. (1)(2), Wirth, J. (1)(2) der(16)(pter::p12 > present in four cases; 4. In all cases the deleted (1) Praenadia GmbH, Muenster, Germany (2) p11.2::q11.2>p11.2::p12>p13.3::q12.1 Xq chromosome was always late replicating; 5. Developmental Biology and Molecular >q24::q11.2>q12.2::qter) Interestingly, all Xq deletions tested had the dis Pathology, University of Bielefeld, Germany The couple wished a intracytoplasmatic sperm tal breakpoint in the 300 kb subtelomeric region. (3) Westfaelische WilhelmsUniversitaet injection (ICSI) because of desperate desire for Finally, we tried to correlate the clinical findings Muenster, Institut fuer Humangenetik, children. The family situation and the results of of the female patients with the extent and local Germany the cytogenetic and molecular cytogenetic in ization of the Xq deletions. Special attention has Phenotypic and molecular analysis of patients vestigations will be reported in detail as well as been paid to nonmosaic Xq deletions. The re with partial monosomy 21 resulting from translo the outcome of the pregnancy. sults support the notion that the phenotype in cations, ring chromosomes or pure partial mono Xq patients is inexplicable by the size of Xq somy 21 allows to define which regions of chro deletion and a mosaic constitution alone. mosome 21 contribute to the generation of spe

50 medgen 14 (2002) made use of the fiberFISHtechnique as a com P107 20 plementary mapping strategy. This technique al lows the direct visualization of cloned DNA se P107 17 From rod to rings: Chromosome 18 replaced quences along chromatin „fibers“ released from by two ring chromosomes of chromosome interphase nuclei. In the present study we phys 18 origin Two healthy boys with normal karyotype ically mapped the gene families CDY, DAZ, Miller, Konstantin (1), Pabst, B. (1), Nürnberg P. from a mother with isochromosome t(21;21) RBMY and TSPY on released chromatin. We (2), Siebert R. (3), Schmidtke, J. (1) Arslan Neumann, Thomas E., Exeler, R.J., Herrmann, used the Y heterochromatin and the Y cen Kirchner M. (1) M., Horst, J., Kennerknecht, I. tromerespecific alphoid sequences, as well as (1) Institut für Humangenetik, Medizinische Institut für Humangenetik, Westf. Wilhelms the already mapped XKRY genes (Lahn and Hochschule Hannover, Germany, (2) Gene Universität Münster, Germany Page 1997) as markers along the fibers. Our Mapping Center, Max Delbrück Center, Robertsonian translocations are among the most mapping results received by fiberFISH allowed BerlinBuch, Germany, (3) Institut für common balanced structural chromosome re the construction of an overlapping fiberFISH Humangenetik, Universität Kiel, Kiel, arrangements in the general population with an contig spanning the nonrecombining region of Germany estimated incidence of 1 in 1000. They can be the human Y. Indeed, the results of our fiber In a patient with a single chromosome 18 and transmitted through many generations. Carriers FISH contig presented here may help to close two additional ring chromosomes, FISH analysis of a balanced Robertsonian translocation are the gaps in the Tilfordmap. Furthermore, it may with a whole chromosome paint specific for phenotypically normal. The majority of balanced help to get a more complete picture with respect chromosome 18 revealed the chromosome 18 Robertsonian translocations involve nonhomol to position and arrangement of the multicopy origin of the ring chromsomes. FISH analysis ogous chromosomes. Homologous transloca gene families investigated along the human NRY. with a chromosome 18 specific alphasatellite tions are rare and always seen as a de novo probe demonstrated a chromosome 18 specific event. Most likely homologous Robertsonian centromere on both ring chromosomes. The sig translocations are postzygotic and of maternal P107 19 nal on the normal chromosome 18 always pre and paternal origine. Fusions occur in one of the sented stronger than the signals on both individ earliest cell divisions. In contrast, most of the ual ring chromosomes, respectively. With sub

t(21;21) chromosomes are isochromosomes Computer assisted diagnosis of Abstracts GfH ÖGH SGMG Tagungsband telomeric probes specific for the short and long i(21q) arising from one homolog, either maternal chromosomal aberrations using a Bayesian arm, the normal chromsome showed the respec or paternal. Practically all conceptions of homol and a counting approach with the help of tive signals, while the ring chromosomes ogous translocation carriers result in either tri the database SYNDROC showed no signal. A chromosome painting somy or monosomy. Monosomy 21 results in Tautenhahn, Ute (1), Kunze, J. (2), Pelz, J. (1) probe specific for the short arm of chromosome abortion, as does trisomy 14, 15, 22 and most of (1) Reformstudiengang Medizin, Charité 18 revealed a positive signal on the small ring trisomy 13. Trisomy 21 results in Down syn Campus Mitte, Germany, (2) Institut für chromosome. Molecular studies showed the ex drome. Only one case of a son with ring 13 from Humangenetik, Charité Campus Virchow clusive presence of paternal alleles distal to a 13;13 translocation mother and one case of a The possibility to diagnose chromosomal aber short arm locus D18S843 and distal to long arm son with ring 15 from a 15;15 translocation rations using a computerized database was test locus D18S474. This demonstrates 1. the mater mother are known. Here we report a healthy ed using 101 patients with an established chro nal origin of the ring chromosomes, and 2. the mother with homologous translocation t(21;21). mosomal aberration using the database SYN loss of the respective short and long arm regions Chromosome analyses in all 60 metaphases DROC. This system provides the user with two distal to these loci thus indicating the break from peripheral blood lymphocytes showed a different algorithms for the calculation of a diag points in ring formation. The dysmorphisms ob 45,XX,t(21;21)(q10;q10) karyotype. She had five nosis: served in the patient also indicate deletions of miscarriages, but gave unexpected birth to two a descriptive algorithm which proposes a diag both chromosome arms, as clinical findings phenotypically normal boys with 46,XY kary nosis counting a set of phenotypic markers all partly overlap with observations in 18p and otypes. This observation confirms the theory of having the same weight. 18q syndrome and are similar to some cases of mitotic origine of the t(21;21), formed after con a Bayesianalgorithm which, evaluating calcu ring chromosome 18. This case is the first exam ception. lates probabilities for competing diagnoses by ple of the replacement of an autosome by two Most likely the mother carries a somatic mosaic analyzing phenotypic anomalies. ring chromosomes originating from the missing 46,XX/45,XX,t(21;21). To our knowledge this is Three levels of precision were used assessing chromosome. Centromere misdivision is sug the first report of a homologous Robertsonian the diagnoses: suggestion of the correct (1) gested as one mechanism involved in the forma translocation carrier with healthy children. chromosome number, (2) chromosome arm, (3) tion of the ring chromosomes. aberration type and rough location. The combination of both algorithms yielded 51 consensus diagnoses for the level of the correct P107 18 chromosome, 24 for the chromosome arm, and P107 21 15 for the aberration type. Additional diagnoses Mapping the nonrecombining region of the solely with the descriptive algorithm were yield FISH assessment of sperm aneuploidy human Y chromosome (NRY) by „fiberFISH“ ed for 43, 52 and 47 cases and using the frequencies in ICSI patients with severe Röttger, Susanne (1), Yen, PH (2), Schempp, W Bayesianalgorithm for 1, 4 and 1 diagnoses re oligoasthenoteratozoospermia (OAT) (1) spectively. Since with the Bayesianalgorithm, Ditzel, N. (a,b), Gläser, B. (b), Jelinkova, L. (a), (1) Institute of Human genetics, Freiburg, when evaluating an uncertain diagnosis using a Strehler, E. (a), Reeka, N. (a), Speit, G. (b), Germany, (2) HarbourUCLA Medical Center, combination of symptoms, one does so by cal Sterzik, K. (a) Torrance, USA culating the probability of the claim in the light a) ChristianLauritzenInstitut, Ulm, The nonrecombining region of the human Y of given information. This seems to be a much Germany, b) University of Ulm, Department chromosome (NRY), which comprises 95% of more promising for a correct diagnosis than the of Human Genetics, Ulm, Germany the chromosome, does not undergo sexual re pure counting of numbers of matches. The The aim of this study was to examine chromo combination. Thus a genetic linkage map cannot Bayesian coefficients were in the range between somes in sperm by fluorescenceinsituhybridi be constructed, and the localization of genes 0.57 and 0.05; the 0.5 margin as a trustworthy sation (FISH) with considerable differences in di and gene families within this differential region one announced by the authors of SYNDROC somy frequency for the chromosomes 13, 16 on the Y chromosome depends on physical was reached by only one of the correctly recog and 21. mapping. Complete physical mapping, however, nized cases. The prior probabilities for the cal 23 patients with oligoasthenoteratozoospermia has been found difficult in regions rich in repet culations of the Bayesianformula do not seem were involved in our study. FISH procedure was itive sequences. Recently, a first detailed map of to use serviceable weights. made by standard protocol, using probes for the human NRY has been published (Tilford et al. chromosome 13 and 21 (locusspecific probes) 2001), and nearly one half of the euchromatic and a probe for chromosome 16 (centromeric portion of the NRY has been sequenced through satellite probe). For each patient 100 2000 the publicly funded Human Genome Project. sperms were analysed. In the OATpatients, the However, there are still four gaps one in Yp and average incidence of disomy for chromosome three in Yq in this map of overlapping BACs 13, 16 and 21 is 2.12%, 1.57% and 1.87%, re and ordered STSs spanning the human NRY. To spectively. In the normospermia patients the av overcome the problems of physical gene map erage disomy incidence of chromosome 13, 16 ping occurring when building up a Y contig, we

medgen 14 (2002) 51 and 21 is 0.67%, 0.44% and 1.1%, respective cation of proteinprotein interactions an auto P110 04 ly. The incidence of aneuploidy was significant mated yeast twohybrid system has been devel ly higher in OATpatients (median: 5% vs. 2.8%; oped in our laboratory. This method is based on SELDI protein chip analysis of laser Wilcoxontest: p = 0.028). interaction mating in 384well microtiter plates. microdissected tissue optimisation and Our results support the hypothesis of positive Currently, we are subcloning ~2000 human standardisation correlation between OAT and higher aneuploidy cDNAfragments into DNAbinding and activa Bleul, Annett, Melle, C., Dahse, R., Ernst, G., rates. However, this correlation is not linear and tion domain vectors. These plasmids are then Schimmel, B., Claussen, U., von Eggeling, F. absolute, among patients with severe OAT we transformed into yeast MATa and MAT alpha Institute of Human Genetics and can observe also high proportion of men with strains for interaction mating. Using the auto Anthropology, University of Jena, Germany normal range of diploidy. mated yeast twohybrid system we will generate SELDIMS (surface enhanced laser desorption According to our results and also results from lit a proteinprotein interaction matrix of 2000 hu /ionisation mass spectrometry) allows analysing erature, we recommend a more intensive prena man proteins. To verify the proteinprotein inter protein extracts from small amounts of samples. tal control of pregnancies, originating from the actions in vitro binding experiments, coim Protein profiles of cell lysates, urine and serum, ICSI method. munoprecipitations and colocalisation studies can be generated. To gain small welldefined not are performed. The results of the twohybrid contaminated samples, especially for the analy screens and of the functional assays will be ses of tumours, microdissection is inevitable. stored in the primary database at the RZPD. P110 01 Therefore our aim was to combine both tech niques to an optimised procedure for protein profiling in cancer. The LCM (laser capture mi Expression profiling in murine P110 03 crodissection) technology offers the possibility medulloblastomas by means of cDNA to perform a tissue selection at the microscope microarrays level and to cut normal and tumour cell areas Kappler, Roland (1), Heß, I. (1), CalzadaWack, Decreased decorin transcript levels in accurately by laser. Currently, however, there are J. (2), Schlegel, J. (3), Hahn, H. (1) fibroblasts of patients with EhlersDanlos no references to what extend the SELDI gener (1) Institute of Human Genetics, Univ. of type I/II syndrome with and without COL V ated protein spectra gets affected by LCM han

GfH ÖGH SGMG Tagungsband Abstracts GfH ÖGH SGMG Tagungsband Goettingen, D37073 Goettingen, (2) mutations dling. Consequently we tested potential proce Institute of Pathology, GSFNational Just, Walter (1), Walter, S. (1), GrondGinsbach, dures for handling tissue with LCM to obtain Research Center for Environment and C. (2), Trautmann, T. (1), Hausser, I. (3) samples that can be analysed by SELDI. The Health, D85764 Neuherberg, (3) Institute of (1) Dept. of Human Genetics, University of performed experiments involved the creation of Pathology, Technical University Munich, D Ulm, 89070 Ulm; (2) Department of tissue slices, different stains, different LCM pro 81675 München Neurology and (3) Department of cedures, transfer of excised tissues by laser or Medulloblastoma is a highly malignant, invasive Dermatology University of Heidelberg, 69120 needle into a test tube and the quantifying of the embryonal tumor of the cerebellum with a pref Heidelberg minimum of required tissue. Best results were erential manifestation in children. Although the EhlersDanlos syndrome (EDS) is characterized detected by using nonstained cryostat tissue majority of medulloblastomas occur sporadical by loosejointedness and fragile, bruisable skin. slices (8 µm thickness) on microscope slides ly, this tumor is also associated with familial can EDS I is the severe form of classic EDS, and coated with a 1.35 µm membrane. Different test cer syndromes including the nevoid basal cell EDS II is the milder one. There is evidence that ed stains affected the results adversely. The carcinoma (Gorlin) syndrome. Mutations in the EDS types I and II are allelic, and that mutations membrane between section and slide did not tumor suppressor gene PATCHED have been de in COL5A1/A2 cause the syndrome in about show influence on the protein profile and thus it scribed in both familial and sporadic cases and 40% of the reported investigated cases. Anoth is advantageous, because of the better handling inactivation of one Patched allele in mice pro er molecule of the extracellular matrix is decorin. of the lasercut section. To keep the impact of motes development of medulloblastoma. In or Decorin (DCN) is a small leucinerich proteogly the laser as slight as possible it appeared der to determine candidate genes involved in tu can. Dcn null mice show an increased fragility of favourable to transfer the laserexcised areas of morigenesis of medulloblastoma we have skin and altered shape and interfibrillar distance tissue slices into testtube manually by a lancet screened tumors of heterozygous patched mice of collagen fibrils. Therefore, DCN might be a needle. By analysing a tissue lysate dilution se for differentially expressed genes by means of candidate gene for idiopathic fragile skin disor ries we defined the minimal required quantity of cDNA microarray technology. Hybridization of ders. We present data about studies in the DCN tissue that still generates satisfying results by fluorescently labeled cDNA probes isolated ei gene of unrelated patients and familial cases SELDIanalysis as about 700 cells. Using this ther from normal cerebellum or medulloblastoma with EDS types I and II, diagnosed according to procedure we are in the position to obtain opti to an array of 1000 genes revealed cell cycle clinical symptoms and ultrastructural collagen mised and standardised results in SELDI analy genes, cell adhesion genes and signal transduc fibril aberrations. We have sequenced all exons sis. tion genes to be altered in the tumor. Our data with their splice junctions of DCN on genomic suggest that formation of medulloblastoma in DNA of 16 EDS I/II individuals. Not a single mu Patched mutants is associated with an aberrant tation nor a polymorphism was found. In addi activity of mitogenic signaling pathways. tion we measured transcript levels in skin fibrob P110 05 lasts of these patients. There is a significant de crease in transcript levels to an average of about The search for proteins interacting with 60 % independent of an intact COL5A1/A2 al P110 02 bestrophin lele. We suggest that a factor upstream of Vladimir Milenkovic, Franziska Krämer, Andrea decorin contributes to the downregulation of Gehrig, Bernhard H.F.Weber Identification of ProteinProtein Interactions decorin on the mRNA level. The diminished Institute of Human Genetics, Biocentre, Am by Systematic Interaction Mating amounts of processed decorin proteoglycan Hubland, University of Wuerzburg Goehler, Heike (1), Lalowski, M. (1), Stelzl, U. molecules may be a consequence of other dis Best vitelliform macular dystrophy (BMD) is an (1), Stroedicke, M. (3), Worm, U. (3), Abraham, easecausing mutations and coinfluence the autosomal dominant disease with a juvenile age C. (1), Goedde, A. (2), Korn, B. (2), Wanker, E. precise assembly of collagen fibers, leading to of onset that causes loss of visual acuity. BMD (1) fragile skin and other connective tissue disor is diagnosed specifically by a depressed elec (1) MaxDelbrück Zentrum für Molekulare ders. trooculogram. The gene responsible for Best Medizin (MDC), RobertRössleStr.10, 13125 This work was supported by the Fritz Thyssen disease (VMD2) has been identified by position BerlinBuch; (2) RZPD Ressourcenzentrum Foundation. Grant: 1999 1071 al cloning and encodes a 585 amino acids trans für Genomforschung, INF 506, 69120 membrane protein named bestrophin. Be Heidelberg, Germany; (3) MaxPlanck strophin is preferentialy expressed in the retinal Institut für Molekulare Genetik (MPIMG), pigment epithelium (RPE) and is localized to its Ihnestr.73, 14195 Berlin basolateral plasma membranes. Based on amino The sequencing of the human genome has re acid sequence predictions bestrophin contains sulted in the identification of a large number of at least four transmembrane domains (TMDs), novel proteins, whose functions have to be de with the N and the long Cterminal portions fac termined. In order to determinate the function of ing the cytosol. Almost all diseaseassociated human proteins we will analyse proteinprotein alterations reported to date are missense muta interactions on a large scale using the yeast twohybrid system. For the systematic identifi

52 medgen 14 (2002) tions that cluster in 4 regions near the predicted scribe the pathways involved in the pathophys P110 08 TMDs. iology of DCM and heart failure. Finally conven Our goal is to identify interacting partners of be ient customarrays for routine profiling may be Characterization of Novel Proteins by high strophin and to elucidate the molecular function developed on this basis, and provide a useful di Throughput Analysis of Human Protein of the protein. Toward this goal a GAL4based agnostic resource. Networks (Automated TwoHybrid yeast two hybrid (Y2H) system was used. An oli Screening) go(dT)/random primed bovine RPE cDNA library Gödde, Astrid (1), Czerny, K. (1), Schultz, E. (1), was constructed and cloned as a fusion of the P110 07 Schatten, R. (1), Henze, S. (1), Schick, M. (1), activation domain vector pGADT7 and the Ebert, L. (1), Göhler, H.(2), Wanker, E. (2), Korn, bovine RPE cDNAs. In addition, we generated a B. (1) series of bestrophinderived baits. Thus far, Identification of differentially expressed (1) RZPD Ressourcenzentrum für screenings with Nterminal and loop fragments genes in colon carcinoma by pairwise Genomforschung, INF 506, 69120 have been completed by analysing 6 x 105 and hybridization of normal and tumor tissue on Heidelberg, Germany; (2) MaxDelbrueck 8 x 105 transformants, respectively. This result global human cDNA arrays Zentrum für molekulare Medizin (MDC), ed in the isolation of 37 (Nterminus) and 6 (loop A.Schroth1, W.Huber1, B.Sipos2, D.Güssow3, RobertRössleStr.10, 13125 BerlinBuch fragment) putative positive clones. Further char G.Klöppel2, B.Kremer4, M.Grell5, A.Poustka1, As the human genome becomes fully se acterization and verification will include reintro H.Kalthoff4, S.Matzku5, B.Korn6 quenced, our attention turns to proteomics, the duction of plasmids from positive clones into the 1German Cancer Research Center: largescale identification and characterization of yeast strain used for screening either (i) alone, Heidelberg,Germany; 2Institute for proteins. Our project is focused on the charac (ii) with the bait vector, (iii) with the bait used in Pathology, University of Kiel, Germany; terization of unknown human proteins by yeast the initial search, or (iv) with a plasmid encoding 3Merck KGaA Preclinical Research, GBT: twohybrid screening in order to assign them to a totally unrelated protein as a DNAbinding do Darmstadt, Germany; 4Clinic General particular pathways or multimeric structures. main hybrid. Subsequently, plasmids only posi Surgery Kiel, Germany; 5Merck KGaA We defined the genes of interest in the human tive with the target hybrid will be subjected to Preclinical Research, Oncology: Darmstadt, genome that lack annotated function, or that GSTfusion assays and coimmunoprecipitation Germany; 6RZPD: Heidelberg, Germany have purely predicted function attached. In total

studies. Additionaly we generated a set of hu Colorectal cancer is the second leading cause of Abstracts GfH ÖGH SGMG Tagungsband we found 5.200 of these genes, and cloned man VMD2 Nterminal mutants by site directed death from cancer in the United States. Devel 3.120 full length ORFs up to now. Most of them mutagenesis in order to asses the effect of the opment and progression of cancer is accompa have been completely sequence verified and an diseaseassociated mutations on the strength of nied by complex changes in patterns of gene ex notated, to characterize differences to the al the proteinprotein interactions. pression. The development of colorectal tumors ready published sequence data in respect to bp is a multistep process that is known to depend exchanges and alternative splice forms. Only on the deregulation or mutation of certain criti clones showing stop codon mutations and frame cal genes. Therefore colorectal tumors provide P110 06 shifts were discarded. The information on the an excellent system for the research of different cloned material has been passed on RZPD. gene expression patterns because clinical and As the initial cloning was done in a recombina Monitoring tissue remodelling in human histopathological data suggest, that most malig tion shuttle vector system, we are moving the dilated cardiomyopathy (DCM) by gene nant colorectal tumors(carcinomas) arise from ORFs in a second step to bait and prey vectors expression profilling preexisting benign tumors (adenomas). (1.150 and 96 ORFs respectively). These plas R. Grzeskowiak (1), M. Drungowski (1), R. Complementary DNA arrays provide a powerful mids will be used to perform an automated two Thermann (1), H. Witt (1), A. Perrot (2), K.J. tool for studying these complex phenomena. To hybrid interaction screening (see joint abstract Osterziel (2), H. Lehrach (1), P. Ruiz (1) measure variations in gene expression between by Goehler et al.). (1) MaxPlanck Institute for Molecular different stages of colon carcinomas, metasta We intend to characterize and verify the protein Genetics, Ihnestr. 73, D 14195 Berlin, sis and normal colon tissue we used cDNA ar protein interactions that result from novel pro Germany (2) FranzVolhardKlinik / Klinische rays carrying a global human cDNA set of teins by in vitro methods. Therefore, we have es und Molekulare Kardiologie, Wiltbergstr. 50 72.000 nonredundant sequences. Poly (A)+ tablished Baculovirus and E.coli (in vitro and in 13125 Berlin, Germany RNA from tumor, metastasis and normal colon vivo) expression systems. These systems will al In response to various pathophysiological con tissue was reversed transcribed into radioactive low us to gain access to the respective proteins ditions heart undergoes complex remodelling labelled first strand cDNA and hybridized to two in question for in vitro binding experiments. The processes, which primarily compensatory, ulti filters each. results of these studies will be deposited in the mately lead to ventricular dilation and heart fail Array expression data for colon or metastasis Primary Database at RZPD. ure. Cytoskeletal, apoptotic and calciumrelated versus normal colon tissue confirmed overex signalling pathways play a pivotal role in this re pression of several genes known to be upregu modelling gene programme. To monitor gene ex lated in carcinomas e.g. IGF2R (insulin growth pression during progression of human dilated factor receptor), several kinases and transcrip P110 09 cardiomyopathy (DCM), we collected biopsies tion factors and downregulated e.g. carbonic an from 10 patients by routine heart catheterization. hydrases and genes involved in apoptosis. Oth Heterologous expression of human An „in silico“ normalised human UniGene library er known genes with interesting functions, that TIGR/MYOC as well as selected mutations containing 33,689 cDNA clones was PCR ampli have not previously been descriped to be impli in insect cells results in insoluble protein fied and robotically arrayed on nylon mem cated in colon cancer were found upor down aggregates branes. The resulting filters were hybridised with regulated in the tumors and the metastasis. In Oezbey, Sevinc (1); MichelsRautenstrauss, K 33P labelled cDNAs derived from RNA isolated addition, many ESTs without annotation and (1); Rautenstrauss, B (1) from 4 healthy and 10 dilated cardiomyopathy functions have been found up and downregu (1) Institute of Human Genetics, Erlangen, biopsies. We identified ~650 differentially ex lated. Knowledge of gene expression patterns Germany pressed transcripts, from which ~400 were se typical for certain types and stages of tumors Several mutations in the TIGR/MYOC gene, also quence verified, and several (23) confirmed by will give insight into tumorgenetics. Molecular known as myocilin (MYOC), were identified in ju real timePCR. Global trends in gene expression changes involved in tumor development and venile open angle glaucoma (JOAG) patients, patterns were revealed by hierarchical clustering progression will provide molecular markers for and more rarely in primary open angle glaucoma and functional categorisation. tumor diagnosis, and clinical prognosis. Finally (POAG). The biological function of MYOC is un The largest number of differentially regulated differently expressed gene products offer a known. Based on HighFive insect cells, we have genes was found to be involved in energy me promising strategy for testing new targets in investigated the determination of a putative ad tabolism, sarcomere architecture and cell sig colon cancer therapy. hesion capability of normal and mutant forms of nalling. Moreover, several transcripts emerge to MYOC as well as the localization of the respec be involved in the cytoskeletonrelated sig tive proteins in transfected cells. By means of fu nalling, myocyte survival/apoptosis and calcium sion proteins carrying green fluorescent protein homeostasis. These may be essential for dilative (GFP) as a reporter the intracellular localisation remodelling and heart failure. In conclusion, this was visualized. First, selected mutations (Pro study represents the first report on gene expres 370Leu, Gln368X, Lys423Glu) have been intro sion profiling of >33,000 unique cDNAs using duced into a human MYOC cDNA by PCR di human cardiac biopsies from clinically stable pa rected invitro mutagenesis. Subsequently these tients. The resulting profiles might enable to de

medgen 14 (2002) 53 mutations have been cloned into pure expres P110 11 sion/selection plasmids (pEXIV), as well as into expression/selection plasmids in frame with the Expression of the Mtmr1 gene in the Mtm1 P110 13 GFPcoding region derived from pQI25 (Quan knock out mouse and its differential tum). To transfect these expression plasmids in alternative splicing HighFive cells we used insect cell specific lipo Variance stabilization and robust Rausch, Anne (1), Bujbello, A.(2), Halliger somes, namely insectin (Invitrogen). normalization for microarray gene Keller, B.(1), Kress, W.(1) Conclusions: The results indicated, that a par expression data (1) Institute of Human Genetics, University ticle number analysis of the cells, transfected ei v.Heydebreck, Anja (1), Huber, W. (2), of Wurzburg, Germany, (2) IGBMC, ther with wt or mutant forms of MYOC, gave no Sueltmann, H. (2), Poustka, A. (2), Vingron, University of Strasbourg, France significant alteration compared to nontransfect M.(1) Xchromosomal recessive Myotubular Myopathy ed or only GFP transfected controls. Using fluo (1): MaxPlanckInstitute for Molecular (MTM1) is a severe congenital myopathy in man. rescence microscopy, no insertion of the Genetics, Berlin, Germany, (2): German Affected boys present with general hypotonia MYOC/GFPfusion proteins into the cell mem Cancer Research Center, Heidelberg, and muscle weakness after birth (floppy babies). brane was observed. In all cases, GFPfluores Germany A histological examination of muscle biopsies cence was clearly visible in cytosolic vesicles, Due to variations in the experimental conditions, shows myotubelike fibres with centrally located but not in the nuclei. GFPalone transfected cells measurements from different gene expression nuclei. Private mutations distributed all over the show fluorescence also in the nuclei as well as array experiments are generally on different nu myotubularin gene, coding for a phosphatidyl in the whole cell. With these experiments we merical scales and need to be normalized before inositol3phosphate specific phosphatase, are have excluded its putative role as adhesion mol further analysis. Furthermore, the analysis of causative. Bujbello et al. (unpublished) estab ecule, and furthermore no secretion of MYOC replicate experiments shows that the variance of lished a knock out mouse model for MTM1 with from this cells was observed. MYOCwt protein the measured expression intensities depends on a similar phenotype and histology. We examined as well as mutant expression results in aggluti their mean value. Thus the statistical signifi by quantitative RTPCR the expression of the nates and MYOC accumulates insoluble in cy cance of a measured fold change depends on myotubularin gene (Mtm1) and its neighbouring tosolic vesicles followed by a preliminary cell the intensity level at which it was observed. Our homologue myotubularin related protein 1

GfH ÖGH SGMG Tagungsband Abstracts GfH ÖGH SGMG Tagungsband death. The MYOC agglutination and insolubilisa approach for dealing with these problems is (Mtmr1), both originating from a common ances tion after overexpression or mutation represents based on a statistical model of measurement er tor gene, in various tissues of Mtm1knock out possibly one of the pathomechanisms. ror for gene expression data. From this model, mice and wild type mice. Myotubularin mRNA using a mathematical technique, we derive a was present in the knock out mice in its full family of variancestabilizing transformations of length (without exon 4, which is knocked out), the measured intensities which are of the form P110 10 and there was no compensatory upregulation of y=arsinh(ax+b). Differences on this new intensi the Mtmr1 gene. Also, no mutation was found in ty scale have nearly uniform statistical signifi MTMR1 till now in any patient with Myotubular The Berlin Protein Structure Factory cance. For large intensities x, our transformation Myopathy. Consequently, MTMR1 does not play Konrad Büssow coincides with the usual logarithmic transforma a role in the pathogenesis of the myopathy. We MPI Molekulare Genetik, tion, such that differences can still be interpret observed three new coding exons in intron 2 of Proteinstrukturfabrik, Heubnerweg 6, 14059 ed in terms of fold changes. For small values of the mouse Mtmr1 gene leading to differential al Berlin, Germany x, the transformation diminishes the fluctuation ternative splicing in different tissues. Each organ The Protein Structure Factory (PSF, http:// www. of the intensities that is usually visible in log has its own individual pattern of transcripts. This proteinstrukturfabrik.de) is a joined initiative of transformed data. For the normalization of data observation supports the idea of a special func research institutes and companies in the Berlin from different mRNA samples, we use a robust tion of Mtmr1 isoforms in many tissues. area covering the fields of protein structure statistical estimation technique. This is applica analysis, biophysics, genomics, protein expres ble as long as between the samples under con sion and bioinformatics. Is was established to sideration, the majority of genes have roughly characterise the structures of proteins encoded P110 12 unchanged expression levels, while possibly dif by the large number of human genes being dis ferent numbers of genes may be up or down covered, using automation technology to accel regulated. Finally, we evaluate the performance Gene Expression Changes in Stem Cell erate the process from the protein sequence to of our approach on real data sets, comparing it Differentiation studied with DNA the structure. with standard methods. We find that our method Microarrays Proteins are selected for structure analysis, leads to higher power of statistical tests to iden Georg Wieczorek, Ulf Gurok, Constanze which have an unknown structure, do not con tify differentially expressed genes. Scharff, Christine Steinhoff, H.Hilger Ropers tain nonglobular domains and for which full and Ulrike A. Nuber length cDNA clones are available. In addition to MaxPlanckInstitut für Molekulare Genetik, whole proteins, there is a focus on predicted sin Department Human Molecular Genetics, P110 14 gle domains. Ihnestr. 73, 14195 Berlin In the E. coli expression project of the Protein Research on adult stem cells has gained in Structure Factory, human cDNA sequences are OLIGONUCLEOTIDE VERSUS cDNABASED creased attention since these cells have been subcloned into expression vectors for overex MICROARRAY SYSTEMS: A COMPARISON found to show a high plasticity and differentiate pression in E. coli. A parallel project for the ex USING NORMAL HUMAN COLONIC into many different cell types. pression in yeasts has been established for pro MUCOSA Mesenchymal stem cells from bone marrow, so teins that are difficult to express using bacterial Mah, Nancy (1), Thelin, A. (2), Costello, C. (1), called bone marrow stromal cells (BMSC) differ systems. Subcloning is performed by standard Lu, T. (1), Gurbuz, Y. (3), Eickhoff, H. (4), entiate into cells of mesenchymal, but also non techniques, which are adapted to the microtitre Nikolaus, S. (1), Lehrach, H. (4), Mellgård, B. mesenchymal origin, including cells with a neu plate format to increase the throughput. PCR (2), Schreiber, S. (1) ronal phenotype. Another type of adult stem cell products are purified and bacterial colonies are (1) 1st Medical Clinic, ChristianAlbrechts that can differentiate into neurons and glial cells picked by robotic systems. University Kiel, Kiel, Germany, (2) exists in the mammalian brain and proliferates in Human cDNA clones as templates for sub AstraZeneca R&D Mölndal, Mölndal, vitro forming so called neurospheres. BMSC cloning are obtained either from the Berlin Sweden, (3) Department of Pathology, which are easily accessible in humans, but also Ressource Center of the German Human ChristianAlbrechtsUniversity Kiel, Kiel, neuronal stem cells from the brain represent a Genome Project (RZPD, http://www.rzpd.de) or Germany, (4) MaxPlanck Institute for source for cell replacement / transplantation from expression libraries developed by the E. Molecular Genetics, Berlin, Germany therapy. coli expression project. These libraries are ar The human genome project has identified We are analyzing the differentiation pathways of rayed in microtitre plates and onto highdensity ~32,000 genes, many of which are predicted these two types of adult stem cells using the colony filters using robot technology. Screening genes supported by expressed sequence tags. DNA microarray technology. Investigating ex of highdensity protein arrays, protein expres Functional characterisation of unknown genes pression changes in the course of differentiation sion and purification in microtitre plates and can be assisted through the use of high in mouse models and BMSC from patients with mass spectrometry identify protein expression throughput microarray expression screening. In central nervous system disorders will help us to clones with the desired properties. this study, we compare the two main types of understand underlying pathomechanisms of the expression screening platforms—an oligonu diseases. cleotidebased and a cDNAbased platform. Ex

54 medgen 14 (2002) pression profiles of 5 normal controls from sig models have become an indispensable and ver identification of candidates for mutated genes in moid colon mucosa were investigated using a satile tool to study development, genetics, be critical regions. Data generated in this project cDNA filter microarray (Human UniGene Set havior, and disease. To interpret experimental are a major tool and have successfully been RZPD1) and an oligonucleotide array (Affymetrix data, the extend and diversity of gene expres used to identify new mouse models for human HGU95Av2). Each platform performed in a sim sion for the system under study should be well genetic diseases based on the differential ex ilar manner with regard to variability in probe de characterized. Up to now the vast majority of pression of known marker genes. In addition, tection (coefficient of variation= 0.26±0.19 and these experiments have focused on specific bi new and functionally not annotated genes have 0.20±0.16 for the Affymetrix and clonebased ological processes. Unfortunately little is known been associated with specific mouse mutant systems, respectively). Quantitatively, there was about the normal variance of mouse gene ex models. One of the largest European resources no statistically significant rank order correlation pression in vivo. To study the normal variance of mouse models (over 400 mutant lines) from between the mean expression data generated by we analyzed the expression profile of normal the Munich ENU mutagenesis screen is directly the two platforms (rs=0.140). Replication rates male C57Bl6, C3H, Balb/c, DBA/2, and FVB accessible. The MouseExpress Consortium of gene detection on both platforms were relat mice, respectively. In total, we profiled gene ex combines the power of mouse genetics, expres ed to BLAST scores between cDNA clones and pression of 75 mouse samples of 15 different in sion profile technology, and bioinformatics. Such probe sequences on oligonucleotide arrays, in dividuals. We used a U74A mouse Genearray comprehensive transcriptome analyses will lead dicating that observed differences are influenced from Affymetrix to quantitate transcript levels in to the identification of new gene functions and by probe design. Overall, probes detected tran brain, liver, heart, spleen, and kidney in the five coregulated synexpression groups of genes, scripts for genes known to be linked to the func genetic backgrounds. which are the basis for the description of regu tion and structure of the colon, such as genes The expression levels of several genes varied latory networks. involved in immunity, signal transduction, and significantly in more than one tissue. These vari transport. This study demonstrates the comple ances in gene expression might be one of the mentarity and overlap between two different reason for observing different phenotypes of P110 18 techniques for expression profiling. transgenic mice based on a different genetic background. These studies will help to define the baseline level variability in mouse gene ex Eliminating CrossHybridisation: High

pression and accentuate the importance of repli Quality Expression Profiling Data For The Abstracts GfH ÖGH SGMG Tagungsband P110 15 cated microarray experiments. Furthermore, this Molecular Phenotypic Analysis Of Mouse dataset will be used as a data base for normal Mutants Towards the analysis of the mouse expression profiles, to reveal insights into the Alexei Drobyshev1, Marion Horsch1, Christine transcriptome MouseExpress mechanisms of transcriptional regulation, and to Machka1, Michael Mader2, Sabine Tornow2, Frohme M(1), Korica T(1), Seltmann M(2), study disease etiology in future transgenic mod Marcus Frohme3, Tamara Korica3, Martin Drobyshev A(2), Vingron M(3), Mewes W(2), els. Finally, we are developing a free and public Vingron4, Werner Mewes2, Jörg Hoheisel3, Hrabé de Angelis M(2)*, Beckers J(2)* and ity database to allow the scientific community to Martin Hrabé de Angelis1#, and Johannes Hoheisel J(1) use this resource. Beckers1# (1) Deutsches Krebsforschungszentrum, #coordinators of the MouseExpress Heidelberg Germany (2) GSF consortium (DHGP), 1GSF, Institute of Forschungszentrum für Umwelt und Experimental Genetics, Munich, Germany. P110 17 Gesundheit, Munich Germany (3) MPI für 2GSF, Inst. of Bioinformatics, Munich, Molekulare Genetik, Berlin Germany (*) Germany, 3DKFZ, Division of Functional coordinators of MouseExpress (DHGP) MouseExpress: In Silico Analysis Of Genomics, Heidelberg, Germany, 4MPI for RNA from various mousesources, derived from Expression Patterns In Mouse Mutants Mol. Genetics, Dept. of Computational Mol. the ENU mutagenesis screen (GSF, Munich) are Matthias Seltman*1, Marion Horsch*1, Alexei Biology, Berlin, Germany subjected to analyses on DNAchips. The aim of Drobyshev1, Michael Mader2, Sabine Tornow2, The cDNAchip technology is a powerful tool for the collaborative initiative is to get new insights Marcus Frohme3, Tamara Korica3, Martin the comprehensive analysis of gene expression into the interrelations of gene function, expres Vingron4, Werner Mewes2, Jorg Hoheisel3, at the transcript level. The biological significance sion and networking on the one hand and the Martin Hrabe de Angelis#1, and Johannes of such expression profiling analyses critically (mutagenized) phenotype of an animal on the Beckers#1 depends on the quality and specificity of hybridi other. For a global transcriptome analysis more *equal contribution, #coordinators of the sation data. Based on experimental data, we de than 60.000 PCRproducts from two independ MouseExpress consortium (DHGP), 1GSF, scribe methods to >discriminate between gene ent nonredundant clone libraries are used (one Inst. of Experimental Genetics, Munich, Ger, specific signals and signals resulting from exten being the MouseExpress RZPD library, the oth 2GSF, Inst. of Bioinformatics, Munich, Ger, sive crosshybridisation. We identify criteria that er originating from LION Bioscience). 3DKFZ, Division of Functional Genomics, can be used for each individual probe on com The system was established with a lesscomplex Heidelberg, Ger, 4MPI for Mol. Genetics, prehensive DNAchips to correct expression chip of some hundred PCRproducts, which are Dept. of Computational Mol. Biol., Berlin, data to achieve high quality results. For this, we important in embryonic development of the Ger apply in situ fractionation of hybridised targets mouse. We have implemented a platform for the func by means of contiguous washes with increasing Additionally a set of external controls for normal tional analysis of mouse mutant lines using RNA stringency. In the course of such washing steps, ization has been developed, used for spiking ss expression profiling technology on a large scale distinct fractions of hybridised target are washed DNA made of phage lambda instead of RNA. and high throughput basis. The routine analysis out at different stringency. The fluorescent inten Furthermore, we approached the question of dif of RNA expression patterns from organs sup sity data at each step and for each probe of a ferential gene expression in mutant mice by rep ports the understanding of the underlying mo microarray comprise the fractionation curve. resentational difference analysis (RDA) of cDNA. lecular biology of such mouse mutants and pro Based on this information, unreliable data can Using a Delta(minus) mutant, various modifica vides new insights in mammalian gene function. be filtered and gene specific probes relevant for tions of RDA have been tested for improved sen To achieve this goal standardised protocols high quality expression data can be identified. In sitivity and selectivity. (SOPs) from mouse husbandry and tissue sam the MouseExpress project we apply this technol pling to the molecular techniques of DNAchip ogy for a systematic and comprehensive analy hybridisations were developed and implement sis of expression profiles from a set of organs in ed. We generate comprehensive high quality a compendium of mouse mutant lines (more P110 16 DNA glass chips with 20.000 and 48.000 than 400 mutant lines) derived from the Munich 3?UTRs and ESTs, respectively. Expression pro ENU mutagenesis screen. Such comprehensive DNAMicroarray technology defining the files that have been obtained in a first high transcriptome analyses will lead to the identifi normal variance in mouse gene expression throughput series were used to establish a com cation of new gene functions and coregulated Michael Bonin, Sven Poths, Dany Bhugon, Olaf prehensive database system for gene annotation synexpression groups of genes, which are the Riess and functional data mining. Genes differentially basis for the description of regulatory networks. Institut fuer Humangenetik, Abt. expressed in mouse mutant lines have been Medizinische Genetik, Universitaetsklinikum identified and were confirmed by alternative Tuebingen, Calwer Str. 7, 72076 Tuebingen methods. The detection of affected pathways Highthroughput gene expression has become has become an integral part of the molecular an important tool to investigate transcriptional phenotypic analysis of mouse mutant resources activity in a variety of biological samples. Mouse and, together with mapping data, supports the

medgen 14 (2002) 55 P110 19 sessed essential sequences for directing expres beyond their mere sequence. Protein domains sion exclusively to the mammary gland. In fluo can sometimes be identified based on sequence rescence in situ hybridization (FISH) experiments similarities with other known proteins, but there Transcriptional analyses of a Lupusnephritis transgene was mapped to telomeric region on are currently no reliable methods available to mouse model long arm of chromosome 7. Purification of hu predict the spatiotemporal expression pattern of Frohme M(1), Klopp J(1), Beißbarth T(1,3), man growth hormone from milk and releasing its a gene in a living organism, it can only be deter Hoheisel J(1), Schwarz M(2), Radeke H(2) active form was performed by affinity chro mined experimentally. We have therefore devel (1) Funktionelle Genomanalyse, Deutsches matography followed by thrombin cleavage. Bi oped machinery for high throughput gene ex Krebsforschungszentrum, Heidelberg ological activity of growth hormone was meas pression analysis on tissue sections by in situ Germany (2) Institut für Pharmakologie und ured by immunoreactivity and adding to culture hybridization. Automation of various steps of the Toxikologie, Klinikum der Goethe Universität of hormone dependent NB211 cell line. The re procedures minimizes human handson time, Frankfurt Germany (3) MPI für Molekulare sults indicate that transgenic rabbits are suitable rendering investigations at a genomewide scale Genetik, Berlin Germany for the production of recombinant proteins in the conceivable. The results of our studies are made Systemic lupus erythematosus is a frequent milk of lactating females. available for the scientific community via a data chronic automimmune disease. One of the ma base at www.genepaint.org. Here we describe jor and most severe symptoms is an inflamma the different possibilities for data retrieval using tion of the renal glomeruli, called Lupusnephritis the webbased public interface that can be used (LN). It is one of the most common reasons for P110 21 by all members of the scientific community to renal failure resulting in dialysis and transplan access our data. Searches of the Genepaint tation. LN may be well understood with the help Cloning and expression studies of the database can be conducted based on gene of the MRLFaslprMouse model, presenting a chicken homolog for the human ARVCF, a names, descriptions, and accession numbers. In genetically dependent glomerulonephritis. gene from the CATCH22 deletion region addition, systematic annotation to a treelike hi In order to get an insight into the progression of Jung, Anita, Arnemann, J. erarchical list of anatomical structures permits the disease and the involved genes, RNA from Institute of Human Genetics, Johann searches for and comparison between expres mouse kidneys at characteristic time points dur Wolfgang GoetheUniversity Hospital, sion patterns of genes. The digital images of ing the manifestation of LN are analysed using

GfH ÖGH SGMG Tagungsband Abstracts GfH ÖGH SGMG Tagungsband Frankfurt/Main, Germany sections at singlecell resolution are very large, DNA microarrays. These arrays are constructed The human 22q11.2 chromosomal deletion pro so special attention was paid to providing a con from PCRproducts of about 1000 ESTclones duces haploinsufficiency for a number of devel venient way of viewing these images: Along with representing more than 600 genes that are char opmentally regulated genes which cause main the annotations of expression patterns, a virtual acteristic for different celltypes in the nephron ly disturbances of the migration and differenti microscope feature is available that can be used and for the various cells of the immune system. ation of neural crest cells. This leads to syn to view and zoom into images down to the orig The aim the project is to profile the inflammation dromes of the DiGeorge or CATCH22 spectrum. inal cellular resolution while keeping download processes on the transcriptional level. A focus is Still not fully understood are the contributions of times reasonable. on the characterization of the immunogenic in each deleted gene towards a pathological phe vasion of the renal cortex, which will be notype. In our study we use the chicken embryo adressed based on the analysis of chemokine as a model to study the embryonic expression related and cell type specific housekeeping P110 23 profiles of the corresponding genes. Our actual genes.The respective second kidneys of the work focusses on ARVCF (armadillo repeat gene mice used in our experiments may serve for par deleted in velocardiofacial syndrome) a mem Characterization of an alternative TSPY allel histological examination. ber of the catenin family, characterised by ar transcript (TSPYL) in LNCaP prostate madillo repeats and with a predicted function in carcinoma cells signal transduction and cell differentiation. We Krick,Roswitha(1), Eishold,M.(2), P110 20 cloned the chicken homologous gene which is Jakubiczka,S.(3), Jonas,D.(2), Arnemann,J.(1) highly conserved to human, mouse and xeno (1) pus. By means of wholemount ISH a ubiqui (1)Institute of Human Genetics, University Expression of human growth hormone in tous, but variable expression can be shown in Hospital, Frankfurt/M., Germany; (2)Dpt. of milk of transgenic rabbits with transgene different organs and stages of the chick embryo. Urology and Pediatric Urology, University mapped to telomeric region of chromosome Partially it colocalizes with the expression of N Hospital, Frankfurt/M., Germany; (3) 7q cadherin, a potential binding partner of ARVCF. Institute of Human Genetics, University Slomski, Ryszard (1,2), Lipinski, D. (1,2), Kalak, However when using RTPCR we were able to Magdeburg, Germany (2) R. (1,2), Plawski, A. (2), Jura, J. (2), Szalata, M. show alternative splicing patterns which are Recent studies revealed an aberrant expression (1,2), Kala, M. (2), Jarmuz, M. (2) Nuc, K. (1), specific for different embryonic tissues. For the of TSPY in prostate carcinoma leaving the ques Slomska, K. (1), Jura, J. (3), Korcz, A. (4), 5‘region altogether 5 variants (ae) were ob tion of its role in malignancy. We analysed the Smorag, Z. (3), Pienkowski, M. (4) served mainly due to alternative start sites, while TSPY expression profile in detail for LNCaP cells (1) Department of Biochemistry and for the 3‘ region of the gene only 2 variants (1,2) and revealed evidence for an alternative spliced Biotechnology, Agricultural University, were found. Heart and liver e.g. present only the transcript, termed TSPYL, which was cloned Poznan, Poland, (2) Institute of Human d2 isoform, while other organs like eye, kidney, and analysed in detail. This variant is transcribed Genetics, Polish Academy of Sciences, intestine or brain show various combinations. at of least 4 fold lower abundance than TSPYS, Poznan, Poland, (3) Department of Animal Any functional differences of the different splice the common form. We were able to show that a Reproduction, National Research Institute variants are still not known, however the ar presumed alternative splicing takes place in in of Animal Production, Balice, Poland, (4) madillorepeats, and thus the binding site for tron 4 using a splice acceptor site just 11 bp up PienGen Biomedical Corp cadherin molecules, are not affected by alterna stream from that of TSPYS. Due to a frameshift Targeting the expression of sequences encoding tive splicing. this leads to an altered amino acid sequence nonmilk proteins to the mammary gland of and thus to a shortened Cterminus with postu transgenic farm animals was proven as effective lated different cellular functions. There is a pre way for producing pharmacologically active pro dicted difference in molecular weight from al teins on a large scale. We report here expression P110 22 most 2 kD between TSPYS (35 kD) and TSPY pattern of human growth hormone (hGH) in L (33 kD). To test whether TSPYL is functional transgenic rabbits carrying hGH genomic se Genepaint.org A Database of Gene and not a pseudogene we raised an antipep quences driven by the rat whey acidic protein Expression Maps tideantiserum in rabbit using a peptide of 16 aa (WAP) promoter. Transgenic founder animal was Visel, Axel (1), Ahdidan, J. (1), AlvarezBolado, from the TSPYL Cterminus for immunization. generated by microinjection of genetic con G. (1), Kruse, S. (2), Eichele, G. (1) The specificity of this antipeptide antiserum (a structs to male pronuclei of fertilized rabbit (1) Max Planck Institute of Experimental 1026) was tested in immunoblot experiments oocyte and transferred to foster mother. Founder Endocrinology, D30625 Hannover, Germany, against GSTfusion proteins of as well TSPYL animal transmitted the transgene to the next (2) Orgarat GmbH, D45130 Essen, Germany (clone pRo11) as TSPYS (clone pRo18). These generations in stable form. Approximately 30% The genomes of humans and an increasing num clones cover the complete coding region of each of F1 offspring demonstrated presence of trans ber of model organisms, including the mouse, variant. As a positive control for the detection of gene. Ectopic expression of transgene was not have now been sequenced and tens of thou both GSTfusionproteins TSPY antibody a839 observed in brain, heart, kidney, liver and sali sands of mammalian genes have been predict (2) was applied which was raised against a N vary gland indicating that WAP promoter pos ed. However, for many of them little is known terminal fusionprotein common to both variants.

56 medgen 14 (2002) Using antipeptide antiserum a1026 a specific ment epithelium (RPE) is involved in the degen signal of the predicted size was obtained only erative processes. for the TSPYL type, thus confirming the speci Our aim is to identify and characterize genes P110 25 ficity of the antiserum. specifically or abundantly expressed in the RPE. As several copies of TSPY with different haplo Towards this end, a bovine cDNA library en types do exist on the human Y chromosome a Identifying novel retinaspecific genes by riched for RPE was constructed inhouse using haplotype analysis was performed on cDNA in characterising a suppression subtracted a PCRbased suppression subtractive hybridiza dicating that the GG18 haplotype is absent in cDNA library highly enriched for retinal tion technique, which normalizes sequence TSPYL transcripts. This suggests that only de genes abundance and achieves high enrichment for fined TSPY copies on the Y chromosome can Jelena Stojic(1), Andrea Gehrig(1), Heidi L. differentially expressed genes. The individual se give rise to the alternative splice product TSPY Schulz(1), Matthias Wagner(2), Bernhard H. F. quences of 1002 cDNAs were analyzed and L. Weber(1) querried against sequences deposited in the (1)Institute of Human Genetics, Biocenter, GenBank and dbEST databases. A RDBMS was Am Hubland, D97074 Würzburg; (2)Lynkeus designed and developed to organize the storage BioTech, Science Park Würzburg, D97076 and retrieval of the sequencing and blast search P110 24 Würzburg ing information. RDBMS leads to more efficient The human retina is a multilayered neuronal tis use of queries, forms, and reports, increases the Designing customized arrays in the post sue specialized for the processing of visual in reliability of the extracted information, and can genome era formation. It is a highly complex system which expand as database information requirements Michael Dahms, Michael Baum, Andrea consequently may be greatly susceptible to ge grow and change. Our results demonstrate that Schlauersbach, Kahrin Eggelbusch, Markus netic defects leading to a wide range of retinal 64% (642) of the clones are derived from known Beier, Peer Stähler disease phenotypes. To identify novel retinal genes, while 36% (360) of clones showed no febit ag, Käfertaler Straße 190, 68167 genes we have generated a human retinal cDNA matches in dbEST. In silico expression analysis Mannheim library by suppression subtractive hybridisation of the known RPE genes revealed that 7.5% are Entering the postgenome era with an increas (SSH) techniques. We have sequenced 1113 RPEspecific, 4.2 % are retinaspecific, and

ing amount of sequence data available in public clones with insert sizes ranging from 150 to 850 4.2% are expressed in both retina and brain. Ex Abstracts GfH ÖGH SGMG Tagungsband databases, for genome research a complex ma bp. On the basis of BLASTN algorithm analysis, trapolation of these data to the EST clusters with trix of possibilities is opened. Up to date more the clones were classified into four categories no significant homolgy to known genes suggests than 500 sequencing projects for the most including those with i) significant homology to that the number of novel RPE/Retinaspecific prominent model organisms have been initiated, known human genes (769/1113), ii) significant genes to be around 1015. Characterization of from which already hundred have been finished. homology to partial transcripts (43/1113) and hy the full length human orthologs of the bovine The outcome of these projects represent the ba pothetical gene predictions (119/1113), iii) no RPE/Retinaspecific transcripts have been initi sis of genome research in academia, pharma homology to known mRNAs (149/1113), and iv) ated. In a second phase, we plan to determine R&D or the diagnostic industry. To address this vector sequences and clones derived from mito sequence variants in the RPEspecific genes in vast and complex arena of genomic questions, chondrial genes (33/1113). After correcting for a large, wellcharacterized AMD patient group by new flexible analysing tools are desperately redundancy, category 1 represents 236 known highthroughput exonscanning technology. needed that translate these into meaningful bio human genes and category 2 a total of 94 un logical experiments. known transcripts. Clones from category 2 were Febits Geniom technology represents the first in selected for expression analysis by RTPCR in P110 27 tegrated solution to that problem by transform 20 human tissues. Thus far, RTPCR has been ing genomic questions into a digital software file completed for 46 EST clusters and has revealed that is used as the design scheme for potential 18 transcripts specifically or abundantly tran Identification of genes preferentially ly any genomic assay in a microarray format. Mi scribed in the human retina. Another 6 EST clus expressed in the human retina using an croarray fabrication, hybridisation, detection and ters are expressed in neuronal tissue. Cloning expressed sequenced tag (EST) approach analysis takes place within one single benchtop and characterisation of five of the retinal genes Heidi L. Schulz, Heidi Stöhr, Susanne Fröhlich, instrument. This setup allows for maximum flex is currently in progress. In addition, partial cD Claudia Berger, Jelena Stojic, Bernhard H.F. ibility in design and realisation of all kind of ge NAs of two EST clusters represent novel splic Weber nomic assays. ing variants of known genes. Our study demon Institute of Human Genetics, Biocenter, Employing proprietary algorithms for calculation strates i) a high enrichment of retinal transcripts University of Würzburg, Germany of optimal oligonucleotide probes febit has start in our inhouse constructed cDNA library and ii) The retina is a highly specialized tissue com ed a broad initiative to make a wide variety or the presence of many as yet unidentified tissue posed of more than 50 cell types. Many active ganisms available for expression profiling stud specific genes, as well as unknown alternative genes, especially those with retinaspecific ex ies on the Geniom platform. So far complete ly spliced variants for known genes. Therefore, pression are required to establish the structure sets for yeast, E. coli and mayor parts of human isolation and characterisation of these novel and maintain the integrity of this tissue. Studies are available. Further projects include mouse, N. genes will further our understanding of retinal of the etiology of more than 80 singlegene crassa, A. thaliana, drosophila and analysis of physiology and will provide novel candidates for retinopathies have shown that mutations in the splice variants for human genes. Starting from retinal disease genes. genes abundantly expressed in retina are re these calculated libraries customized array de sponsible for many retinal disorders. Because signs for expression profiling experiments are the genetic causes of more than 70 retinopathies easily set up following a kind of drag & drop are as yet unknown, we aim to identify novel P110 26 scheme. New array designs and therefore new genes preferentially expressed in the retina. genomic assays maybe developed either from Moreover, a catalogue of genes preferentially ex scratch or more simply by combining parts of Construction of a Relational Database pressed in the retina would facilitate the study of existing designs stored in the database. This Management System (RDBMS) for the agerelated macular degeneration (AMD). This kind of combinatorial approach will simplify ac analysis of RPEenriched expressed multifactorial disorder, currently affecting 11 mil cessing the huge amount of data produced by sequence tags lion people worldwide, accounts for most cases the various genome projects in the nearby fu Faisal M. Moula(1), Faisal M. Rahman(1), of vision impairment in the elderly population. ture. A further advantage that results from the Andrea Gehrig(1), Claudia Keilhauer(2), In order to identify retinaspecific or retinaabun digital nature of the assay format is that the as Bernhard H. F. Weber(1) dant genes we performed an expression profil says are always backward compatible. To run an (1)Institute of Human Genetics, Biocenter, ing of the EST clusters assembled in the human assay that was set up years ago it’s only neces Am Hubland, D97074 Würzburg; UniGene database. From the 6190 UniGene sary to pull up the old array design file from the (2)University Eye Clinic, Würzburg clusters containing at least one retina EST, 2201 database and load it onto the system. Agerelated macular degeneration (AMD) is the represent known genes and were not further an Details on the procedures how these probe li most common cause of legal blindness in indus alyzed. We then selected all clusters containing braries have been generated will be presented. trialized countries and predominantly affects the only retina ESTs (673 clusters) and those that It will be shown how customized expression pro elderly population over 75 years of age. Al had at least a 30% retina EST content (568 clus filing arrays are easily selected from these li though the primary events in AMD pathogenesis ters). Exhaustive bioinformatical analyses of braries through easy to handle software tools. are not well understood, it is thought that the these clusters were carried out to further priori Furthermore, ways for data analysis of will be deterioration of the highly metabolic retinal pig tize for in vitro expression analyses. Subsequent described. RTPCR of the 248 chosen clusters led to the

medgen 14 (2002) 57 identification of 41 retinaspecific or abundant Computational Molecular Biology, Berlin, P110 31 and 41 neuronal EST clusters. Thus far, we have Germany, (3) febit AG, Mannheim, Germany completed the cloning and characterization of a The filamentous fungus Neurospora crassa has ‘Functional Genomics of Osteoarthritis’ total of 18 genes and fulllength cloning of the been used as a model organism in basic science from gene expression to function and remaining transcripts is currently in progress. In for more than 60 years. application addition, we are designing an interactive Retina Until now, only a fraction of the estimated 10 Dietz Uwe1; Brimmer, A1; Maier, P 1; Gerwin, Gene Expression Database. This information 13,000 genes of N. crassa was characterised. To N1; Obermayr, F2, Weiss, T1, Zimmer, R3, system will provide easy and integrated access overcome this limitation to the understanding of Aigner, T4 and Bartnik, E.1 to pertinent information about the genes identi the fungus’ biology, a genome initiative was es (1) DG Thrombosis & Degenerative Joint fied in our study as well as other known genes tablished. Whole genome shotgun sequencing Diseases, Aventis Pharma Deutschland, expressed in the retina and will represent an im was started in 2000 at the Genome Research Industriepark Höchst H825, 65926 Frankfurt portant resource for ophthalmogenetic research. Center of the Whitehead Institute, Massachus am Main; (2)GPC AG Genome setts, USA. In february 2002 an assembly of 821 Pharmaceuticals Corporation, contigs >2kb containing 38,044,343 bp was Fraunhoferstr. 20, 82152 made publicly available. In addition, a set of P110 28 Martinsried/München; (3) Institut für 10.082 putative genes was annotated. Informatik, LMU München, Theresienstrasse For transcriptional profiling analyses on N. cras 39, 80333 München; (4) Cartilage Research, Functional analysis of gene and protein sa, we started with a nonnormalised cDNA li Pathologisches Institut, Friedrich networks in Drosophila with microarray brary containing 4800 ESTclones (Nelson et al., AlexanderUniversität, Krankenhausstrasse based genome expression studies 1997). After amplifying the inserts of the clones 810, 91054 Erlangen Koch, B.(3), Beckmann, B.(2), Haas, S.(1), by PCR, high density microarrays were pro Osteoarthritis, the degenerative disease of artic Vingron, M.(1), Hoheisel, J.(2), Sauer, F.(3), duced by spotting the PCRproducts on polyL ular cartilage, is the leading cause of disability Paro, R.(3), and Hild, M.(3) lysinecoated glass slides. with a very substantial economic impact of di (1)MPI für Molekulare Genetik, Having the genome sequence as well as anno rect and indirect cost, but without causative Computational Molecular Biology, tated genes, expression analyses by using mi treatment. Since three years a consortium of 12

GfH ÖGH SGMG Tagungsband Abstracts GfH ÖGH SGMG Tagungsband Ihnestrasse 73, 14195 Berlin (2)DKFZ, Abt. croarrays based on oligonucleotides became industrial, academic, biotech and clinical part Funktionelle Genomanalyse, Im possible. Therefore a subset of 25 genes known ners is engaged in a functional genomics ap Neuenheimer Feld 506, 69120 Heidelberg. to be lightregulated and another 30 selected proach to identify and validate genes and pro (3)Universität Heidelberg, ZMBH, Im genes were investigated employing oligonu teins to enable the development of disease Neuenheimer Feld 282, 69120 Heidelberg cleotide chips. The oligonucleotide chips were modifying drugs. In a first step an array of 4.500 The complete genomes of several dozen bacte either fabricated by spotting presynthesized cartilage specific genes was employed to detect ria, Caenorhabditis elegans, Drosophila melano 70mers or by using in situ oligonucleotide syn genes differentially expressed in human articu gaster and man have been sequenced. The most thesis on febits GeniomÒ technology. lar cartilage. In a second step, the initial analy challenging task arising from this „digital“ se Oligonucleotide probes of 2030 bp in length sis was refined with a further microarray contain quence data will be to decipher the role and func were calculated using 3 different algorithms from ing 6.800 preselected genes and 20 human tis tion of the identified genes and their correspon 3 different groups for comparison. sue samples for each group to be analysed: ding protein products in the context of an entire Results on performance and concordance of the macroscopically intact, early and late os organism. To tackle this challenge, we combine different approaches will be presented. teoarthritic human knee cartilage. Bioinformat DNA microarray based gene expression analyses ics analysis tools included text mining tools to with in vivo protein function studies to dissect collect interaction of gene expression candi gene and protein networks in Drosophila. P110 30 dates with contexts defined by expert lists of rel We generated a new annotation of the Drosophi evant factors for OA. Furthermore, network mod la melanogaster genome that identified 21.396 els generated from databases and literature min putative ORFs. Specific primer pairs were de Studies on the gene expression profiles in ing are used as further evidence for gene candi signed to generate PCR fragments representing patients with abdominal aortic aneurysm dates based on their pathway/signalling con all ORFs. So far we could successfully produce Korcz, Aleksandra (1), Waliszewski, K. (2), texts and the expression of neighboring genes products for 20.903 ORFs (98%). The first ver Lipinski, D. (1), Gabriel M. (2), Zapalski, S. (2), in the resulting biochemical networks. As the sion of our Drosophila microarray is available Slomski, R. (1) first drug targets have entered the Aventis drug since May 8th 2002 and contains two copies of (1) Institute of Human Genetics, Polish discovery pipeline, a more refined validation is the 20.903 PCRproducts, 70 nucleotide Academy of Sciences, Poznan, Poland, (2) now the focus of the interaction of the consor oligonucleotides for smaller ORFs, and 64 differ Karol Marcinkowski Medical School, tium. ent controls (summarized, it contains 46.848 Poznan, Poland The validation process includes further expres baits). Analyses based on this microarray will al Abdominal aortic aneurysm (AAA), a localized sion profiling and in situ hybridisation of human low identification of genes whose activities are abnormal dilatation of aorta, is a lifethreatening and murine tissues, proteinprotein interaction required for the execution of complex develop condition affecting 49% of population with a analyses and the study of target genes in mod mental gene networks and signal transduction risk increasing with age. The results of the stud el organisms. pathways. In a first series of experiments, we will ies that has been done so far on the AAA indi So far comparative expression analyses during use mRNA pools from every stage of Drosophi cate as the reason for an aneurysm development skeletal development have proved to be very la development (e.g. 04 h old embryos, 48 h the loss of the arteries walls resistance caused helpful for generation of concepts to study gene old embryos, etc.) to establish a gene expres by degradation of their structural elements (e.g. function in vitro and in vivo. sion profile of Drosophila development. This ex elastin). Risk factors include: hypertension, ath pression profile will serve as a reference for all erosclerosis, cigarette smoking and also familial of our other microarray gene expression analy occurrence indicating genetic factors to be in ses and in addition may provide first clues to volved. Since 1999 in our research group the ge P110 32 wards how many of the additional genes present netic studies on the determination of the fea in our annotation are expressed during the tures favorable to intensification of degeneration Expression analysis of the dynamin related Drosophila life cycle. The results of this study processes within the aortic wall had been car protein OPA1 in rat tissues will be presented at the meeting. ried out. DNA and RNA isolated from a group of Bette, Stefanie (1), Kohler K. (2), Wissinger, B. 30 AAA patients were collected. Here, we pres (1), Pesch U.E.A. (1) ent the preliminary results of the studies on the P110 29 (1)Molecular Genetics Laboratory, University gene expression level in AAA patients using DNA of Tuebingen, Germany, (2) Division of macroarray system. Experimental Ophthalmology, University of Transcriptional profiling on Neurospora Tuebingen, Germany crassa employing spotted and in situ Autosomal dominant optic atrophy (ADOA) is the synthesized microarrays most prevalent hereditary optic neuropathy char Aign, Verena (1), Rahmann, S. (2), Dahms, M. acterized by an insidious onset of optic atrophy (3) and Hoheisel J. (1) in the early childhood with moderate to severe (1) Functional Genome Analysis, Deutsches decrease of visual acuity. Mutations in the OPA1 Krebsforschungszentrum, Heidelberg, gene are responsible for ADOA. This OPA1 gene Germany, (2) MPI for Molecular Genetics,

58 medgen 14 (2002) encodes a 1015 amino acid polypeptide (ap designed in collaboration with outstanding ex ily later according to the large capacity of the proximately 110 kDa) with similarities to specif perts in the field. They represent 1,200 genes of tree model we use. We have received positive ic GTPbinding proteins of the dynamin protein immunological and 1,600 genes of oncological user feedback on features such as an instant family. The presence of typical sequence fea relevance, respectively. Additional indication view of the pedigree while editing, graphic out tures (i.e. basic leader, MPP and MIP cleavage specific arrays are in preparation. put of the calculation results, the ability to rep sites) at the Nterminus suggests that the OPA1 resent the pedigree and do the computing on polypeptide is imported into the mitochondria. the same screen, and the unique function of IBD OPA1 may represent the human ortholog of the pattern enumeration. P111 01 S. cerevisiae MgmI and the S. pombe MspI pro teins, which are important for mitochondrial in heritance and maintenance in these organisms. A New Syndrome of Cardiac, Cerebral, P114 02 Northern Blot analyses show that OPA1 is ex Renal, Gastrointestinal, and Skeletal pressed in all tissues examined, with the highest Malformation and Intrauterine Death transcript level in the retina. Newly generated Authors: Nicolai Kohlschmidt (1,3), Tamara Haplotype Estimation and Linkage polyclonal antibodies raised against a car Ehresmann (2,3) and Wiltrud Coerdt (3) Disequilibrium for phaseunknown SNP boxyterminal peptide of OPA1 were used for Institute of Human Genetics, Mainz (1), genotypes Western Blot analyses and immunhistochemical Institute of Human Genetics, Frankfurt (2) Nothnagel, Michael, Fuerst, R., Rohde, K. studies. The OPA1 protein was detectable in all and Department of Paediatric Pathology, MaxDelbrueckCentrum, Berlin probed tissues. In liver several bands between Mainz (3), Germany An extension of the expectationmaximization 70 and 110 kDa were detected, which may rep We present three fetal sibs, who died in 24th, (EM) algorithms for the haplotype estimation for resent different processed forms of the OPA1 25th, and 14 th gestational week. phaseunknown SNP genotypes applied to sam protein. Some of these processed forms are The first fetus, male, was small for gestational ples of single individuals or of nuclear families specific for different cellular fractions. In addi age. Autopsy revealed hydrocephalus, cystic has been used to carry out pairwise and multilo tion, we performed immunhistochemical stain dysplastic kidneys, fusion of cervical and tho cus Linkage Disequilibrium (LD) estimations. ings to examine the spatial localization of OPA1 racic vertebral bodies, and mesenterium com It can be shown, that for the identification of LD

in neuronal tissues. Studies in rat retinal sections mune. A second pregnancy ended in the un blocks along the genome a series of successive Abstracts GfH ÖGH SGMG Tagungsband revealed a highly localized staining of specific eventful delivery of a healthy girl which is now pairwise linkage disequilibria sometimes may cells in the ganglion cell layer (GCL), the inner five years old. Fetus 2, female, had prosen underestimate LD blocks and should therefore plexiform layer (IPL) and the inner nuclear layer cephaly, a hypoplastic left ventricle and endo be supplemented by multilocus LD estimates or (INL). cardial fibroelastosis, and cystic dysplastic kid the whole matrix of pairwise LD. neys. Fetus 3, female, had a complex cardiac The effect of different LD measures to identify malformation, intestinal malrotation, and cystic LD blocks and the power to detect them, has dysplastic kidneys. Cerebral anomalies were been investigated by simulation studies, infor P110 33 suspected in ultrasound studies. Cytogenetic mation to support the process of finding haplo examinations were normal. Both parents are of type and/or LD blocks along the genome. RZPDToolbox for Gene Expression Profiling Turkish origin and are unaware of any consan Radelof,Uwe(1), Wagner,F.(1), Maercker,C.(2), guinity between them. We were unable to match Neubert,P.(1), Schwarz,F.(2), Heil,O.(2), the malformation pattern to a known syndrome. P114 03 Maurer,J.(1), and Korn,B.(2) We propose a new syndrome for which autoso RZPD Deutsches Ressourcenzentrum für mal recessive inheritance is likely. Genomforschung GmbH, (1) Berlin, (2) Calculating precise genetic distances for Heidelberg common markers in and around the DMD The complexity of any biological system is ulti Gen P114 01 mately determined by its genes their mere Schargus, Marc (1), MüllerMyhsok, B. (2), number and composition as well as their inter Meng, G. (3), Müller, C.R. (3), Grimm, T. (1) actions and regulation. Gene expression profil PEDMST, a New Program for IBD Pattern (1) Abteilung für medizinische Genetik im ing is the method of choice for analyzing expres Enumeration and Identity Institut für Humangenetik, (2) Universität sion levels of genes in given tissues. Detection CoefficientsCalculation Würzburg, Germany; (2) BernhardNocht of changes in gene expression provides a van Zhu Chenchen, Graham, J. Institut für Tropenmedizin, Hamburg, tage point for indepth analysis of mechanisms Zhu Chenchen, Graham J., Department of Germany; (3) Institut für Humangenetik, relevant for tissue and organ development, car Computing Science, Simon Fraser Universität Würzburg, Germany cinogenesis and its treatment.Within the last two University, Canada DMD (and BMD) are the most common Xlinked years RZPD developed a broad expertise in Currently, no specific algorithm has been built to lethal muscle dystrophies in man, affecting ~1 in gene expression profiling based on an integrat list all possible IBD patterns between the genes 3000 (~1 in 14000) live born males. Problems in ed portfolio of products and services available at a given autosomal locus for individuals in a genetic counseling occur in sporadic cases with to RZPD’s customers. RZPD is one out of only pedigree. We describe a labeled tree structure to out living index patients or in some cases with six Affymetrix Service Providers worldwide. As enumerate such patterns and develop a recur female carriers. In these families a multipoint an alternative and/or complementary approach, sive algorithm to construct this tree. When the linkage analysis may be one possibility to define RZPD also offers an Expression Profiling Service construction is done, the patterns can be easi the haplotype at risk. About 60% of the affected based on our proprietary Unigene Clone Collec ly listed using the path searching on the tree. boys have a deletion and 40% a point mutation tion for human and mouse. The Human Unigene Based on these ideas, We develop a Visual C++ (neglecting duplications). According to our for Set RZPD III comprises 40.000 and the Mouse program, PEDMST (short for Pedigree Master) mer publications the sex ratio of mutation rates Unigene Set RZPD II 35.000 sequence verified which implements the three major functionalities. is different for deletions (µdel ~ 0,2 vdel) and clones. Our clone selection procedure makes First, the function of pedigree operation allows point mutations (µpm ~ 4,6 vpm). The a priori them ideally suited for hybridization experiments the user to construct the pedigree or import the probability of a patient’s mother of being carrier on micro/macroarrays. Genes represented on pedigree from a text file used by other software will be 108/55 µ for deletions and 112/55 µ for Affymetrix GeneChipsÔ are bioinformatically such as PFIDDLER. A special graphdrawing al point mutations. When these data are used in linked with clones from RZPD’s Unigene Sets. gorithm based on the generations of the individ Bayesian risk calculations together with inform Therefore, after a first genomewide screen uals is designed and implemented for import. ative DNA markers carrier detection is much based on Unigene Sets and/or Affymetrix Second, the function of kinship and identity co more precise than using the simpler model of GeneChipsÔ indication specific subsets can be efficients calculation gives the user not only nu equal mutation rates. We have analysed the data assembled from RZPD’s Unigene Clone Collec merical results but also graphic results in terms of 455 pedigrees which were tested in our de tions applying RZPD’s proprietary Rearraying of detailed or condensed identity states. Gener partment from 1982 to 2001. After remodelling technology. Rearrayed subsets are used for the alized kinship coefficients are used to compute and selecting 21 specific markers in and around fabrication of custommade microarrays that the identity coefficients. Third, the function of the dystrophin gene we recalculated the genet provide an economic way for the analysis of IBD pattern enumeration allows the user to view ic distances in cM between markers. Our results large sample sets e.g. from patient cohorts. Cur all possible IBD patterns between the input were compared to previous linkage studies. The rently, RZPD provides two predefined „Indication genes whose maternal or paternal source is application of these new data in multipoint link Arrays“, the ImmunoArray RZPD I and the On specifiable. The maximal number of the genes age analysis will result in more precise risk as coArray RZPD I. Both microarrays have been currently is set to 10, but can be expanded eas sessments for genetic counseling.

medgen 14 (2002) 59 ic effects, pvalues of individual allele combina 4), and pseudogenes with 92% identity to RTD tions) are used to rank the importance of alleles are clustered. The genomic approach has iden and combinations thereof. Graphical represen tified an new highly cationic bdefensin (DEFB3) P114 04 tations allow for convenient evaluation of two in 8p22p21, which was experimentally charac way interactions. ACs can be ranked according terized (1, 2). SNPtyping experiments for molecular to several criteria, thereby selecting the most rel With the exception of the arrangement of the epidemiological studies on flexible, in situ evant ACs. Analysis of typing data concerning disulfides and the property mediated by posi synthesised oligonucleotide microarrays Multiple Sclerosis illustrates the relevance of the tively charged amino acids, the defensins show Alexandra Nieters, Robert Fleischer, Peer methods. little sequence conservation reflecting the vari Stähler, Markus Beier, Nikolaus Becker and ation of the antimicrobicidal potency and salt Jörg D. Hoheisel sensitivity of the individual defensins. The broad Division of Functional Genome Analysis and spectrum antimicrobial activity is mediated pre P116 01 Division of Clinical Epidemiology, Deutsches sumably by binding to and permeabilizing cell Krebsforschungszentrum, Im Neuenheimer membranes. To dissect the structurefunction Feld 280, Heidelberg, Germany, febit AG, The German cDNA Consortium relationship we use a recombinant system for Käfertaler Straße 190, Mannheim, Germany Wiemann, S., Weil, B., Wellenreuther, R., the DEFB3 expression (2 )and the isolation of The investigation of geneenvironment and Gassenhuber, J., Glassl, S., Ansorge, W., variants thereof, generated by random and site genegene interactions in complex diseases re Bocher, M., Blocker, H., Bauersachs, S., Blum, directed mutagenesis. The variants are tested for quires large, epidemiologically well charac H., Lauber, J., Dusterhoft, A., Beyer, A., Kohrer, saltdependent antimicrobial activity towards E. terised populations and flexible, highthrough K., Strack, N., Mewes, H. W., Ottenwalder, B., coli, Pseudomonas aeruginosa, Salmonella put genotyping technologies. Important for the Obermaier, B., Tampe spec., and Staphylococcus aureus. It is aim to study of contextspecific single base polymor Deutsches Krebsforschungszentrum, Im isolate DEFB3 variants with differential high phism (SNP) combinations is a flexible chip de Neuenheimer Feld 280, D69120 Heidelberg specifity against bacterial targets. sign. The most successful German cDNA Consortium, References For the analysis of SNPs associated with the ex which had been established in the first phase of 1. Peng Jia et al. 2001. Gene 263: 21118

GfH ÖGH SGMG Tagungsband Abstracts GfH ÖGH SGMG Tagungsband istence of lymphoid neoplasms, such a microar the DHGP as the second project worldwide, has 2. Harder et al., 2001. J Biol Chem 276:570713 ray has been established. Initial aim of this proj been extended to continue with the isolation of ect is the comparison of epidemiological data fulllength cDNAs of novel human genes. In the obtained from a casecontrol study of 600 pa first phase of the project, 7.87 Mb of high quali tients and 600 controls with molecular informa ty sequence were generated, equaling 3,300 cD P116 03 tion on some 100 appropriate SNPs. While a set NAs. In the reported period of the second phase, of evaluated polymorphisms (in cytokine genes, another 924 cDNAs (total ? 3,285,985 bp) have cytokine receptor genes and other genes of rel been finished. The total achieved in the second Mouse models for deafness and inner ear evance to the immune system) already exists, funding period is 14.4 Mb finished sequence. The development more are currently selected. To this end, the Ge average insert size of the cDNAs was pushed Fuchs Helmut (1), Soewarto D.(1), Kiernan niom system is used, which permits onsite pro from 2.4 kb to over 3.5 kb.The first publication of A.E.(2), Ahituv N. (3), Vreugde S.(2), Erven A. duction and use of oligonucleotide microarrays the consortium activities has been published (1), Kros C.J.(5), Marcotti W. (5), Kurima K. (6), of entirely flexible design. Controlled by a mask (Wiemann et al., 2001. Genome Res. 11, 422 Wilcox E.R. (6), Friedman T.B. (6), Griffith A.J. free, lightinduced synthesis process, up to 435). The cDNAs analysed by the consortium (6), Balling R. (4), Avraham K.B.(3), Steel K.P. 40,000 different oligonucleotides can be synthe have been basis for a number of publications, (2) and Hrabe´ de Angelis M.(1) sised on a single microarray. Also the detection both systematic functional genomic?s approach (1)GSF Center of Environment and Health unit is contained in the system, permitting qual es and projects dealing with individual cDNAs. Institute of Experimental Genetics, ity control during synthesis and, subsequently, The international importance of the consortium Neuherberg, Germany; (2)MRC Institute of quantification of signal intensities upon the hy is reflected by two notes that have been pub Hearing Research, Nottingham, UK; (3) bridisation of DNAtargets. The complete free lished in Nature and Science, respectively.Wie Department of Human Genetics and dom in the choice of oligomer sequences is ele mann, S., Weil, B., Wellenreuther, R., Gassenhu Molecular Medicine, Tel Aviv University, mentary for the evaluation and selection of in ber, J., Glassl, S., Ansorge, W., Bocher, M., Israel; (4) GSF Center of Environment and formative SNPs. Currently, 24 oligonucleotides Blocker, H., Bauersachs, S., Blum, H., Lauber, J., Health Institute of Mammalian Genetics, are being used for each SNP, 12 for either DNA Dusterhoft, A., Beyer, A., Kohrer, K., Strack, N., Neuherberg, Germany; (5) School of strand. The PCRproducts of all studied SNPs Mewes, H. W., Ottenwalder, B., Obermaier, B., Biological Sciences, University of Sussex, are analysed simultaneously in a single hybridi Tampe, J., Heubner, D., Wambutt, R., Korn, B., UK; (6) laboratory of Molecular Genetics, sation. Klein, M. & Poustka, A. (2001) Genome Res 11, NIH, USA Results from the analysis and the various steps 422435. Within the Munich ENUMouseMutagenesis leading from the identification of potentially in Screen deaf mutants and mutants showing head teresting SNPs for epidemiological studies to a tossing or head shaking behavior indicating de practical genotyping chip will be discussed. fects in the inner ear have been detected. A Eu P116 02 ropean deafness consortium was launched to analyse these valuable mutants in more detail. Antimicrobial peptides on human chr. 8p22 Two mouse lines will be presented: Beethoven P114 05 p21 (Bth) is a semidominant mouse model for pro R. A. Siddiqui, K. Reichwald, U. Möllmann1, P. gressive hearing loss. Beethoven was mapped Descriptive methods in large scale Zipfel1, J. Harder2, J. M. Schröder2, and M. to mouse chromosome 19 to a 4.4cM region. association studies involving many Platzer As a candidate gene Tmc1, the mouse ortholog candidate genes Institut für Molekulare Biotechnologie, of the known human deafness gene TMC1, was Stefan Böhringer (1,4), Cornelia Hardt (2), HansKnöllInstitut1, Beutenberstr. 11, sequenced. A T /A transversion in exon 13 was Bianca Miterski (3), Ansgar Steland (4), Jörg 07745 Jena; Klinik für Dermatologie2, detected and identified as causative for the Thomas Epplen (3) Universität Kiel, Schittenhelmstr. 7, 24105 Beethoven phenotype. Bth provides deeper in (1) Human Genetics Essen (2) Med. Kiel sight into molecular factors affecting age relat Hochschule Hannover (3) Molecular Human Defensins (DEF) are small cationic peptides con ed progressive hearing loss. Genetics Bochum (4) Mathematical tributing to innate host defense of higher organ A second mutant, headturner (Htu), is a domi Statistics Bochum isms against microorganisms. Depending on the nant mutant that displays missing posterior and In association studies for complex diseases pattern of disulfide bridges, three branches are sometimes anterior ampullae, structures that many markers are accumulated in the process of known, designated a and bdefensins (DEFA, house the sensory cristae. Headturner mutants investigating candidate genes. A systematic and DEFB), and the cyclized species, rhesustheta also demonstrate a significant reduction in the comprehensive analysis of associations of sin defensin (RTD). In the course of our genomic se numbers of outer hair cells in the organ of cor gle alleles and allele combinations (ACs) requires quence efforts to characterise diseaselinked re ti. Htu was mapped to a 6.6 cM region on mouse proper statistical tools. We present descriptive gions on human chr. 8 we have made two con Chromosome 2. A missense mutation at position and inferential statistics to meet these require tigs of 0.7 and 0.3 Mb in 8p22p21 available 1134 in the Jag1 gene (notch ligand) was identi ments. Several measures of association (odds (http://genome.imbjena.de ), where 5 a fied to cause the Htu phenotype. Although there ratio, normalized odds ratio to measure epistat (DEFA14, HE2/EP2) and 4 bdefensins (DEFB1 existed already Jag1 knock outs, only via the

60 medgen 14 (2002) headturner allele a new function in inner ear de acteristic ones are the highly polymorphic class ty (finished sequence). In addition to the clinical velopment could be assigned to the Jag1 gene. I and class II genes, whose gene products con relevance, this effort represents a contribution to References: trol specific immune responsiveness. the generation of the finished sequence of the Kiernan et al. The Notch ligand Jagged1 is re The rhesus macaque (Macaca mulatta) serves as entire human genome, which is scheduled to be quired for inner ear sensory development. Proc an animal model for several human infectious achieved in April 2003. Additional sequencing Natl Acad Sci U S A. 2001 98(7):38738 diseases, e. g. AIDS and susceptibility to viral in targets of the mouse genome comprise regions Vreugde et al. Beethoven, a mouse model for fection is mainly controlled by class I molecules. on chromosome 2 and 9. In total, we proposed dominant, progressive hearing loss DFNA36. However, knowledge of rhesus macaque class I to generate approx. 3,3 Mb of genomic se Nat Genet. 2002 30(3):2578. genes is based on cDNA sequence information quence in highest quality. and the genomic structure of the rhesus MHC is In the first 16 month (April 2002) of the project largely unknown. Thus, we have screened a cos we initiated the genomic sequencing of all pro mid library with a probe derived from the highly posed regions in human and mouse but concen P116 04 conserved exon 4 of the rhesus macaque MHC trated on human chromosomes. We generated class I gene MamuA, that should crossreact 1,9 Mb of finished and 1,2 Mb of working draft Sequence Variation and Haplotype Analysis with most, if not all rhesus class I genes. A total sequence within this period. In collaborations in Target Genes for CNS Active Therapeutics of 122 class I genecarrying clones could be iso with clinical groups we are involved in disease in a Representative Sample of European lated. So far, for 95 clones end sequences have gene identification projects for HaileyHailey dis Population been obtained. These rhesus macaque se ease (HHD), nephronophtisis (NPHP3), Malignant FreudenbergHua, Yun(1), Freudenberg, J.(1), quences are aligned to the human MHC se Hyperthermia (MHS4) and Myotonic Dystrophy Cichon, S.(2), Propping, P.(1), Nöthen, M.M.(2) quence, selected clones are/will be sequenced. (DM2). So far, we were successful in the identi (1)Institute of Human Genetics, University of Furthermore, the sequences of two cosmids fication of the HHD and the Bartter syndrome Bonn, Germany, (2)Department of Medical have been completely determined and sequenc underlying mutations. Genetics, University of Antwerp, Belgium ing of a third clone is in progress, two overlap Knowledge about DNA sequence variation ping cosmids that map to the so called extend among individuals and their respective popula ed MHC class II region and contain the SACM2L P122 01

tion frequencies is important for a wide range of and KE4 genes as well as a cosmid that con Abstracts GfH ÖGH SGMG Tagungsband biomedical applications. In our project, a gene tains the gene coding for myelin oligodendrocyte based SNP detection approach is under way in glycoprotein (MOG), the major autoantigen in Candidate gene testing for EmeryDreifuss a representative sample (n=96) of the European volved in multiple sclerosis. muscular dystrophy population. In this panel, coding regions and Wasner, Chr., Bethmann, C., and Wehnert, M. exonintron boundaries of 102 candidate genes Institute of Human Genetics, University of are being resequenced, which are potential tar Greifswald, Germany P116 06 gets for CNSactive therapeutics. Candidate Until now STA and LMNA, have been assigned genes are selected according to their known or to EmeryDreifuss muscular dystrophy (EDMD). assumed biological function. A semiautomated Disease geneoriented genomic sequence Scanning 93 patients suffering EDMD or associ analysis pipeline was established for large scale analysis in man and mouse ated phenotypes at our unit revealed that muta polymorphism detection and analysis. The re Sudbrak, Ralf (1), Ramser, J. (2), Yaspo, M.Y. tions in STA and LMNA together account only for sults obtained so far show, that 71% of SNPs (1), Gosslar, A. (3), Beck, A. (1), Lehrach, H. (1), 36 % of the patients. Thus, further genes are detected in the representative European sample and Reinhardt, R. (1) likely to be involved in EDMD. Forced by the of individuals, are still unknown in public data (1) MaxPlanckInstitut für Molekulare lack of families, suitable for a classic positional bases. In our sample, 22% of all detected SNPs Genetik, Berlin, Germany, (2) Dept. of cloning approach, we started a candidate gene are frequent (minor allele frequency >= 0.2), 22% Paediatric Genetics, LMUniversity approach. Emerin and lamin A/C are compo are infrequent (minor allele frequency between Muenchen, Germany, (3) Institute of nents of the inner nuclear membrane and the nu 0.05 and 0.2), 30% of SNPs are rare (minor al Molecular Biology, Med. Hochschule clear lamina. Thus it seems very likely, that de lele frequency < 0.05) and 26% are private (mi Hannover, Germany fects in other genes encoding functionally relat nor allele occurring only once). About 58% of Largescale genomic sequence analysis is an in ed peptides to or interacting with emerin and SNPs from public databases, annotated to the dispensable tool for modern biosciences and lamin A/C could cause EDMD. Thus we consid genes examined in the present study, could not their application. Together with complementing ered lamin B1 (LMNB1) and B2 (LMNB2), lamin be detected in our European samples. The rate technologies like cDNA and SNP analysis, ge B receptor (LBR), laminaassociated polypep of nonsynonymous substitutions is 20%. Our nomic sequence analysis will lay the ground for tides 1 (LAP1) and 2 (LAP2), nurim (NRM), inte results not only represent knowledge about vari life sciences over the next decades. In the frame gral inner nuclear membrane protein (MAN1) ations in potential targets of CNSactive drugs, of the Human Genome Project the Germam hu barrier to autointegration factor (BAF), DEAD/H but also enable computational estimation of man genome sequencing consortium is part of AspGluAlaAsp/Hisbox polypeptide 16 gene based haplotypes. This dimension of our the worldwide public „Bermuda“ initiative, which (DDX16) and proteasome activator subunit 3 project is also important for the analysis of ge will guarantee free access to the genomic se (PSME3) as candidates for EDMD. Additionally, netically complex diseases, since knowledge on quence data not only for man but also for the we considered genes, which are expressed the complete pattern of haplotype variation in a sequence of other model organisms. specifically in heart and skeletal muscle such as certain population can guide SNP selection in During the first phase of the German Human FLNC encoding filamin C and SMPX encoding a LDmapping studies. Genome project (DHGP1) 7,1 Mb of human ge small muscular protein. The candidate genes nomic sequence were generated in highest qual were scanned for DNA variations by heterodu ity (finished sequence) at the MPIMG. The main plex analysis in 54 mostly German patients who focus of our centre was the generation and were excluded to carry mutations in STA or P116 05 analysis of the finished sequence of the entire LMNA. In 126 exons of the candidate genes. 39 chromosome 21 based on gapless sequence unique aberrant heteroduplex patterns have Isolation and sequence analysis of cosmid ready maps constructed by the group of M.L. been found so far and 30 DNA variations were clones mapping to the rhesus major Yaspo at the MaxPlanckInstitute in Berlin. This validated by sequencing. The frequency of the histocompatibility complex (MHC) project was done as a collaboration of two DNAvariations has been estimated in approx. Sudbrak, Ralf (1), Flugge, P. (2), Gunther, E. (2), Japanese groups (RIKEN Sagamihara, Keio Uni 400 chromosomes of a German reference pop Kube, M. (1), Lehrach, H. (1), Reinhardt, R. (1) versity Tokyo) and the three German sequencing ulation. So, seven novel single nucleotide poly and Walter, L. (2) centres (GBF Braunschweig, IMB Jena and morphisms (SNPs) in FLNC, three in LAP2, two (1) MaxPlanckInstitute for Molecular MPIMG Berlin). Up to now, chromosome 21 is in DDX16, LMNB1 and MAN1, respectively, and Genetics, Dept. Lehrach, Berlin, Germany, one of four chromosomes declared to be fin one in NRM were identified. whereas SMPX, (2) Dept. of Immunogenetics, University of ished. Other main targets during the first phase LMNB2 and LAP1 were proven to be monomor Goettingen of the DHGP were chromosome and X, where phic. Until now, no disease causing mutations The major histocompatibility complex (MHC) we sequenced selected medically important re have been found. However, the novel SNPs pro plays a major role in graft rejection and controlls gions. vide tools to study association or linkage of the susceptibility to many diseases mostly of au Based on the results and achieved experience of respective genes with other disease phenotypes. toimmune or infectious nature. Among the 120 the first phase of DHGP we proposed to se The candiurther genes encoding components of expressed genes of the 3.8 Mb encompassing quence medically important regions of human the recently more precisely defined nuclear en human MHC, the HLA complex, the most char chromosomes 1p, 3q, and 17p in highest quali velope proteome.

medgen 14 (2002) 61 first partial epilepsy found to be segregating as P122 05 a single gene disorder. It is characterised by nocturnal motor seizures, which often occur sev P122 02 Extended phenotype of pontocerebellar eral times a night. We have now identified the hypoplasia with infantile spinal muscular fourth CHRNA4 mutation, a T265I amino acid atrophy (PCH1) Congenital myasthenic syndrome with exchange located at the extracellular end of the RudnikSchöneborn, Sabine (1), Szrtiha L. (2), episodic apnea in patients homozygous for second transmembrane domain. The mutation Houge G. (3), Seeger J.(4), Huppke M. (5), a ChAT missense mutation was found in all seven affected individuals from Zerres K. (1) Kraner, Simone (1), Sieb, Jörn P. (2), a fourgeneration German ADNFLE family, but (1) Inst. f. Hum. Genetics, Univ. of Aachen, Laufenberg, I. (1), Straßburg, H. M. (3), not in a sample of 158 control chromosomes. Germany,(2) Dept. of Pediatr., UAE Univ., Un. Steinlein, O. K. (1) Most of the affected individuals had a mild AD Ar. Emirates, (3) Center for Med. Genetics, (1) Institute of Human Genetics, University NFLE phenotype, and in several of them the Univ. Bergen, Norway, (4) Germ. Clinic f. Hospital Bonn, Germany, (2) Max Planck nocturnal seizures were misinterpreted as noc Diagn., Wiesbaden, Germany, (5) Elisabeth Institute of Psychiatry, Munich, Germany, (3) turnal motor activity or hyperactivity. The mutat Children’s Hosp. Oldenburg, Germany Department of Pediatrics, University of ed threonine is highly conserved in CHRNA4 Pontocerebellar hypoplasia (PCH) is rarely asso Würzburg, Germany subunits from different species as well as in ho ciated with anterior horn cell disease and desig Impairment of neuromuscular transmission can mologous nAChR proteins. Electrophysiological nated as PCH1. This phenotype is character be either acquired or inherited. Congenital myas experiments in Xenopus oocytes showed that ized by severe muscle weakness and hypotonia thenic syndromes (CMS) are due to gene muta the T265I amino acid exchange increases the re starting prenatally or at birth with a life span not tions in proteins located in the presynaptic, ceptors apparent affinity for ACh. The mutation exceeding a few months in most cases. Milder synaptic or postsynaptic part of the neuromus identified here further supports the role of disease courses with later onset and longer sur cular junction. The presynaptic syndrome of AChRs in idiopathic epilepsy. vival are normally not diagnosed as PCH1. We congenital myasthenia with episodic apnea herewith describe the clinical and neuroradiolog (CMSEA) presents usually in the neonatal peri ical findings in nine patients out of six sibships od with hypotonia, ptosis, dysphagia and respi P122 04 with evidence of cerebellar defects and early on

GfH ÖGH SGMG Tagungsband Abstracts GfH ÖGH SGMG Tagungsband ratory insufficiency with apnea. If the patient sur set spinal muscular atrophy (SMA), representing vives this initial phase the condition improves, a broad spectrum of clinical variability. In all pa but recurrent crises with the possibility of sud LGI1 is mutated in familialtemporal lobe tients, the diagnosis of SMA (WerdnigHoffmann den death or anoxic brain damage can occur epilepsy characterized by aphasic seizures disease) was made on the basis of electrophys due to infections, fever, or overexertion. CMS Gu, W. (1), Brodtkorb, E. (2), Wevers, A. (3), iological data and muscle biopsy, however, ge EA can be caused by mutations in the gene cod Schröder, H. (3), de Vos, R. (4), Steinlein, O.K. netic testing failed to confirm the diagnosis of in ing for the enzyme choline acetyltransferase (1) fantile SMA with a gene defect on chromosome (ChAT). ChAT catalyses the synthesis of the (1) Inst. of Human Genetics, University 5q and resulted in clinical reevaluation. Age at acetylcholine from acetyl CoA and choline in Bonn, Germany, (2) Department of onset was after a normal period in the first neuronal cells. Direct sequencing of ChAT cod Neurology, Trondheim University, Norway, months of life in three sibships and pre and ing exons identified a previously unknown mis (3) Inst. for Anatomy II, University of Köln, postnatally in the other three families. Life span sense mutation affecting a highly conserved Germany, (4) Laboratorium Pathologie Oost was 24 years in patients with later onset, and amino acid residue (I336T) in a consanguineous Nederland, Enschede, The Netherlands age at death occurred after birth or within Turkish family. The high degree of conservation Sporadic and familial temporal lobe epilepsies months in the more severe group. Two sibships in different species strongly suggests that I336 can be divided into two main categories accord showed discordant ages at death despite simi is a functionally important amino acid residue. ing to the seizure semiology, one with medial lar treatment. The absence of I336T from a large control sam temporal lobe symptoms and one with lateral In contrast to the previous definition of PCH1, ple further supports a pathogenic role in CMS symptoms. The symptoms of sporadic and fa our observations suggest the existence of milder EA. This is the second report of ChAT mutations milial lateral temporal lobe epilepsy usually con phenotypes with pontocerebellar hypoplasia or causing presynaptic CMS. Interestingly, none of sist of simple partial seizures with mainly olivopontocerebellar atrophy in combination with the CMSEA patients reported so far was ho acoustic and / or even visual hallucinations. Au anterior horn cell loss. A pontine involvement is mozygous for a nullmutation. The residual ChAT tosomal dominant lateral temporal lobe epilep not necessarily seen by neuroimaging methods. activity is probably the reason why CMSEA pa sy (ADLTE) has previously been linked to chro The genetic basis of PCH1 remains to be deter tients, despite the important function of ChAT in mosome 10q22q24, and recently mutations in mined, the gene locus for infantile SMA on chro brain, have no signs of central cholinergic dys the LGI1 gene (leucinerich, glioma inactivated mosome 5q could be excluded by linkage stud function. (Kraner et al., submitted) 1) have been found in some ADLTE families. The ies. Parental consanguinity and affected sibs LGI1 gene is a putative transmembrane protein make autosomal recessive inheritance most like characterized by an extracellular leucinerich re ly. peat (LRR) domain, which is flanked by con P122 03 served cysteine clusters. We have now identified a missense mutation C46R affecting one of the A new CHRNA4 mutation in familial frontal conserved cysteine residues in the extracellular P122 06 lobe epilepsy region of the LGI1 protein. This mutation is ab Steinlein, Ortrud K. (1), Hufnagel, A. (2), sent in 104 Norwegian and 212 German control Mutation analysis of 1433 zeta and two Leninger, T. (2), Bertrand, S. (3), Bertrand, D. chromosomes. The C46R mutation is the first homologues genes in German Parkinson’s (3), Kananura, C. (1) point mutation reported affecting a conserved disease patients (1) Institute of Human Genetics, University extracellular residue in the LGI1 protein. The as Hermann, K. (1), Holzmann, C. (1), Krüger, R. Hospital, Bonn, Germany, (2) Department of sociated ADLTE in a large Norwegian family (2), Berger, K (3) and Riess, O. (4) Neurology, University Hospital Essen, shows unusual clinical features like short lasting (1) Dept. of Medical Genetics, Univ. Rostock, Germany, (3) Department of Physiology, sensory aphasia and auditory symptoms. Im Germany, (2) Dept. of Neurology, Univ. of CMU, Geneva, Switzerland munohistological experiments demonstrated Tuebingen, Germany, (3) Inst. of The neuronal nicotinic acetylcholine receptors that LGI1immunoreactive neurons are found in Epidemiology and Social Medicine, Univ. of (nAChRs) are involved in signal transduction by all layers of the frontal and temporal cortex with Muenster, Germany, (4) Dept. of Medical fast synaptic transmission, axoaxonic transmis strongest labeling intensities in layer II/III. LGI1 Genetics, Univ. of Tuebingen, Germany sion, and in the modulation of presynaptic trans presented the first evidence that a gene not ap 1433 proteins participate in diverse biological mitter release. Presynaptic nAChRs can increase parently coding for an ion channe l can cause id processes, including neuronal development and the release of excitatory as well as of inhibitory iopathic epilepsy. We also identified three for cell growth control. 1433 is abundant in brain, transmitters, and can thereby control neuronal merly unknown LGIlike genes (LGI2, LGI3, and comprising approximately 1% of its total soluble excitability. Thus, the genes coding for the dif LGI4) and characterized their genomic localiza protein, and has been found in the neurofibrillary ferent nAChR subunits are likely candidates for tion and expression pattern. The four LGI genes tangles in patients with Alzheimer’s disease and neurological disorders. Since 1995, three belong to a new gene family and are possible in Lewy bodies of Parkinson`s disease patients. CHRNA4 (S248F, S252F, 776ins3) and two candidates for epilepsy and some malignant dis 1433 functions as a protein kinasedependent CHRNB2 (V287M, V287L) mutations were found eases . (Gu et al., Annals of Neurology, in activator of tyrosine and tryptophan hydroxy in families with autosomal dominant nocturnal press; Gu et al,. FEBS Letters, in press). lases. It therefore regulates the synthesis and frontal lobe epilepsy (ADNFLE). ADNFLE was the

62 medgen 14 (2002) excretion of bioamines as dopamine. Recently, and underline the wide range of phenotypic vari Aims: Molecular genetic confirmation of clinical it has been shown that synuclein shares physi ability of this mutation. Therefore, a priori pre diagnosis of AD and delineation of phenotypic cal and functional homology with 1433 pro diction of the mutation carrier status in dyston variability. Methods: Diagnosis of AD was based teins and binds to 1433 proteins and to its lig ic patients and genetic counseling of affected on clinical and neuroradiological criteria. DNA ands. Mutations in alphasynuclein have been families with respect to the clinical manifestation was extracted from blood lymphocytes. Frag identified in some rare families with autosomal may prove difficult. ments containing all coding exons of GFAP were dominant Parkinson’s disease (PD). We therefore PCR amplified and sequenced using the ABI dye investigated whether 1433 proteins are also in terminator method. Detected mutations were volved in the pathogenesis of PD. We searched confirmed by digest with suitable restriction en P122 08 for mutations in the 1433 zeta (YWHAZ) gene zymes. Results: We report heterozygous GFAP on human chromosome 2p25.2p25.1 and two mutations in 6 patients with clinical and neuro homologous genes on chromosome 10p12.33 Detection of repeat expansions in patients radiological features of infantile AD, including a and Xp11.4, respectively. The YWHAZ gene and with PROMM by PCR and nonradioactive pair of monozygotic twins. Novel mutations were its homologous genes consist of one coding hybridization detected affecting nucleotides 304T>C (L97P) exon and one untranslatated exon. Hellenbroich, Yorck (1), Gehlken, U. (2), and 730G>C (R239P) in two other patients. We performed a detailed mutation search of the Schwinger, E. (3), Zühlke, C. (4) None of the parents carried the mutations coding region in 303 sporadic and familial PD Institut für Humangenetik, Medizinische stressing dominant de novo mutations as the patients by SSCP screening and sequence Universität Lübeck, Ratzeburger Allee 160, cause of infantile AD. The presence of an iden analysis. 23538 Lübeck tical mutation 250G>A (R79H) in monozygotic We found no mutation in the 1433 zeta gene, Proximal myotonic myopathy (PROMM, DM2) is twins with infantile AD points to the origin of but analysis of the homologous gene on chro a recently delineated multisystem disorder in these GFAP mutations in germ cells or very ear mosome X revealed an A95G transition leading cluding dystrophic myopathy, myotonia and ly postzygotic stages. Conclusions: Our current to a Gln32Arg exchange in a single PD patient. cataract, which is genetically distinct from my data confirm the association of GFAP mutations This mutation was not found in any of 226 exam otonic dystrophy type 1 (DM1). PROMM is with AD. Molecular genetic analysis facilitates ined controls. The functional implication of the caused by a CCTG expansion in intron 1 of the the diagnosis and should be provided in sus

A95G transition is currently being investigated. zinc finger protein 9 (ZNF9) gene located on pected Alexander`s disease. Abstracts GfH ÖGH SGMG Tagungsband Furthermore we identified a singlebase pair chromosome 3q21. ZNF9 is expressed in a deletion of G98 in the 1433 homologous gene broad variety of tissues, especially in heart and P122 10 on chromosome 10 in one patient but in none of skeletal muscle. ZNF9 contains 7 zinc finger do 360 unaffected control individuals. However, an mains and is thought to be an RNAbinding pro affected sister of the index patient did not car tein. Gene targeting of Gemin2 in mice reveals a ry this mutation questioning whether the G98 The range of expanded allele sizes is large, correlation between defects in the deletion is causative for PD. spanning from 75 to ~11000 CCTG repeats with biogenesis of U snRNPs and motoneuron a mean of approximately 5000. Very large re cell death peats, like in PROMM, are difficult to detect by Jablonka, Sibylle (1), Holtmann, H. (1), Meister, standard PCR assays, therefore we performed G. (2), Bandilla, M. (2), Rossoll, W. (1), Fischer, P122 07 nested PCR analyses followed by nonradioac U. (2), Sendtner, M. (1) tive hybridization with a DIGlabeled oligonu (1) Institute of Clinical Neurobiology, Josef Frequency and phenotypic variability of the cleotide. Using this method we tested 80 sam SchneiderStr.11, D97080 Wuerzburg, (2) GAG deletion of the DYT1 gene in an ples from patients with a myotonic myopathy MPI of Biochemistry, Am Klopferspitz 18a, unselected group of dystonia patients without repeat expansion in the DM1 gene. In 9 D82152 Martinsried Grundmann Kathrin (1), LaubisHerrmann U.(1), patients we detected CCTG expansions varying Autosomal recessive spinal muscular atrophy Bauer I. (2), Dressler D. (3), VollmerHaase between hundred and a few thousand copies. 64 (SMA) is one of the most frequent monogenic J.(4), Bauer P. (2), StuhrmannSpangenberg M. samples were heterozygous for wildtype alleles. causes of death in infancy and childhood mor (6), Schulte T. (7), Schöls L. (7),Topka H. (8), The PCR based detection procedure reveals ex bidity and characterized by progressive degen Riess O. (5) treme mosaicism of the PROMM repeat expan eration of motoneurons resulting in atrophy and (1) Dept Neurol,Univ.Tuebingen,(2) Dept. sion showing a broad variation of size in affect weakness of voluntary muscles. The SMA caus Medical Genetics, Univ. Rostock. (3)Dept ed individuals. ing gene (SMN) exists in two copies termed Neurol,Univ.Rostock, (4) Dept Neurol,Univ. SMN1 and SMN2 on human chromosome 5q13. Münster, (5) Dept Medical Genetics Univ. Whereas the SMN1 gene allows expression of a P122 09 Tübingen, (6) Inst. Human Genetics Medical fulllength protein, the major product of the School, Hannover, (7) Dept Neurol,Ruhr SMN2 gene is a differentially spliced, truncated Univ.Bochum, (8)Dept MUTATION ANALYSIS OF THE GFAP GENE and nonfunctional protein. Patients with ho Neurol,Univ.MünchenBogenhausen IN GERMAN PATIENTS WITH ALEXANDER`S mozygous absence of SMN1 express only low Dystonia is a clinically and genetically heteroge DISEASE levels of a functional SMN protein and therefore neous movement disorder characterized by sus Meins, Moritz (1), Brockmann, K. (2), Sperner, develop the disease phenotype. The SMN pro tained muscle contractions affecting one or J. (3), Stephani, U. (4), Hanefeld, F. (2), Engel, tein and its interacting partner Gemin2 are ubiq more sites of the body, frequently causing twist W. (1) uitously expressed and form part of a macromol ing and repetitive movements or abnormal pos (1) Department of Human Genetics and (2) ecular complex (SMNcomplex) that mediates tures. A 3basepair (GAG) deletion in the DYT1 Department of Neuropaediatrics, University assembly of spliceosomal U snRNPs. SMN has gene is held responsible for most cases of ear of Goettingen, (3) Department of been shown to facilitate the formation of the Sm lyonset primary generalized dystonia in Ashke Paediatrics, Medical University of Luebeck, core domain of the snRNPs, most likely by reg nazi Jews as well as in nonJewish patients. To (4) Neuropaediatric Department, University ulating the proper binding of Sm proteins onto U investigate the prevalence of the GAG deletion of Kiel snRNAs. A model arising from these data sug in the DYT1 gene and the phenotypic variability Background: Alexander disease (AD) is a rare gests that patients expressing only low levels of in the general population, we tested patients disorder of cerebral white matter due to a dys SMN, exhibit defects in the biogenesis of with different subtypes of dystonia from four dif function of astrocytes. The most common infan snRNPs, and hence suffering from SMA. Using ferent movement disorders outpatient clinics in tile form presents as a sporadic, megalencephal mouse genetics we investigated the function of Germany. Six out of 256 patients did carry the ic leukodystrophy. Affected infants show devel this complex in motoneuron maintenance. Re GAGdeletion in the DYT1 gene. However, only opmental delay, macrocephaly, seizures, spas duced Smn/Gemin2 protein levels lead to dis two of the six mutation carriers present with fea ticity and rapid deterioration. Juvenile and adult turbed U snRNP assembly as indicated by re tures of earlyonset primary generalized dysto forms of AD are characterized by a more slowly duced nuclear accumulation of Sm proteins. nia. The DYT1 mutation was also detected in progressive course. Neuroradiological features This correlates with enhanced motoneuron de two patients with multifocal dystonia, one of include various white matter changes, enlarged generation in Gemin2+//Smn+/ mice. Our data them showed involvement of cranial and cervi ventricles, and basal ganglia abnormalities. Neu provide the first in vivo evidence that impaired cal muscles, and in two patients with writer´s ropathological examination reveals megalen production of U snRNPs contributes to mo cramp of both hands with only slight progres cephaly, Rosenthal fibers, astrocytosis, and de toneuron degeneration. sion. Our findings demonstrate that the mutation myelination. Recently de novo mutations in the may be associated not only with generalized but glial fibrillary acidic protein gene (GFAP) have also multifocal and segmental forms of dystonia been demonstrated to be associated with AD.

medgen 14 (2002) 63 P122 11 On clinical grounds, the 41 patients were subdi more laboratories to participate in the EQA vided into four categories: 61% (n=25) with typ schemes in the future. Lessons drawn from the ical FSHD; 14.6% (n=6) with facialsparing development of both the scheme and European Identification of modifying pathways FSHD; 12.2% (n=5) with atypical features for guidelines should help in harmonising laborato involved in the phenotypic variability of FSHD; 12.2% (n=5) without FSHD. ry standards for the genetic diagnosis of CMT. spinal muscular atrophy (SMA) Seven patients presented with typical FSHD and Helmken, Claudia (1); Hofmann, Y. (1); Raschke, >9 repeats (>35kb), 6 patients classified as atyp H. (1); RudnikSchöneborn, S. (2); Zerres, K. ical or no FSHD within the diagnostic range of (2); Wirth, B. (1) P122 14 <9 repeats (<35 kb). Five patients with facial (1) Institute of Human Genetics, University sparing FSHD exhibited repeat numbers within of Bonn, Germany; (2) Institute of Human the diagnostic range. Thus a clear diagnostic Searching a genetic predisposition of Genetics, University of Aachen, Germany threshold was missing. Complex Regional Pain Syndrome Proximal spinal muscular atrophy (SMA) is a In the borderline region of 8 to 14 repeats, we Hühne, Kathrin (1) ; Leis, S (2); Schmelz, M (3); neuromuscular disorder, caused by homozygous find a high percentage of atypical clinical pre Birklein, F (4); Rautenstrauss, B (1) mutations of the survival motor neuron gene 1 sentations. This could give rise to considerations (1) Institute of Human Genetics, Erlangen, (SMN1). SMN1 is part of an 800 kDa protein of an expanded clinical heterogeneity in FSHD. Germany, (2) Neurological Department, complex (gems) with a crucial role in snRNP bio On the other hand one may discuss diagnostic Erlangen, Germany, (3) Institute of genesis, premRNA splicing and a presumable limitations of the test in this borderline region. Physiology und Experimental function in neural transport. SMN2, a nearly Whether FSHD with facial sparing represents an Pathophysiology, Erlangen, Germany, (4) identical copy of SMN1, predominately produces own clinical entity or an abortive form of FSHD Neurological department, Mainz, Germany exon 7skipped transcripts, whereas SMN1 remains to be answered. Further data on the Several clinical symptomes of the complex re mainly produces fulllength transcripts. The SR clinical and genetic features of the patients with gional pain syndrome (CRPS) suggest „neuro like splicing factor Htra2beta1 was shown to in borderline D4Z4 repeat numbers will help in di genic“ inflammation which coincides typical in teract with SMN and to restore fulllength agnosis and genetic counselling, and might even flammation characteristics together with trophic SMN2mRNA in vivo. contribute to the understanding of the genetic changes and hyperalgesia. The controlled re In rare cases siblings with identical 5q13ho

GfH ÖGH SGMG Tagungsband Abstracts GfH ÖGH SGMG Tagungsband mechanisms in this disease. lease of neuropeptides from afferents or mole mologs and homozygous absence of SMN1 cules in their subsequent signalling pathway show variable phenotypes, suggesting that SMA seemed to be impaired in CRPS. Our recent data is modified by other, yet unknown factors. By show that substance P (SP) is more effective to analyzing seven discordant families with affect P122 13 induce edema in CRPS patients – independent ed and unaffected siblings, we demonstrate that from disease activity or affected body region in EBVtransformed cell lines homozygously Lessons from 3 years european external compared to normal controls. In a first step we deleted unaffected individuals not only display quality assessment for CharcotMarieTooth therefore screened for mutations in functional significantly higher levels of SMN protein, but disease candidate genes related to the SP signalling and also exhibit high expression levels of Gemin2, Rautenstrauss, Bernd (1); Barton, DE (2); neuropeptide degrading enzymes, as there are Gemin3, and ZPR1 as compared with their af O’Brien, O (3); Timmerman, V (4) neurokinin 1receptor NK1R, neutral endopep fected siblings. In contrast, in primary fibroblast (1) Institute of Human Genetics, Erlangen, tidase NEP and angiotensinconverting enzyme cultures no protein expression differences could Germany, (2) National Centre for Medical ACE. The patient collective consists of 6 famil be observed. Genetics, University College Dublin, Ireland, ial cases and 28 sporadic patients who suffered Additionally, Htra2beta1 was found to be regu (3) Northern Molecular Genetics Service, from CRPS once or repeatedly. For the NK1R lated similarly. Interestingly, other SMNinteract Institute of Human Genetics, Newcastle gene we could not find any deviation from the ing proteins, which are not components of gems, Upon Tyne, UK, (4) Molecular Genetics wildtyp sequence when investigating the genom such as p53, are not subject to a SMNdepend Department, VIB, University of Antwerpen, ic DNA of 10 repeatedly affected CRPS patients. ent regulation. Since we could not find any se Belgium In the ACE gene we checked for the deletion quence or transcription differences among in CharcotMarieTooth disease (CMT) type 1A is deletion D/D intronic polymorphism and we did trafamilial siblings, we postulate that the SMA usually caused by a 1.4Mb tandem duplication neither find a significantly increased frequency phenotype is modified by a factor that directly in chromosome 17p11.212 comprising the pe of the (D/D) genotype as reported in a Japanese influences the SMN complex, most probably ripheral myelin protein 22 (PMP22) gene. Pa CRPS population nor a cosegregation of the acting upstream of Htra2beta1, which for its tients show reduced nerve conduction velocities D/D genotype with CRPS in familial cases. Final part upregulates SMN, which then upregulates (NCV<38 m/s). Genetic testing for CMT1A is rou ly, for NEP a Met8Val mutation could be identi other „ gems“complex members. Furthermore tinely offered and performed using a wide vari fied for one of the familial CRPS patients and her this modulating effect is tissuespecific. ety of methods like: detection of CMT1A junc yet unaffected mother. Her father is in the group tion fragments, STR and RFLP analysis to as of repeatedly affected CRPS patients. The sess dosage or the presence of 3 alleles, FISH, Met8Val variation couldn’t be verified for the pa P122 12 quantitative PCR to detect PMP22 dosage and tient’s father. Furthermore, a dinucleotide repeat other techniques. The main benefit for the pa vaiation in intron 6 of the NEP gene was discov tient and clinician is to offer differential DNA di ered. It is homozygous elongated compared to Facioscapulohumeral Muscular Dystrophy agnosis for CMT disease. In 1999, a pilot Exter the wildtype sequence (TA)= 8 to (TA)= 13 in 13 (FSHD) – clinical presentation in patients nal Quality Assessment (EQA) scheme for CMT out of 32 CRPS patients. This variation could with borderline D4Z4 repeats was offered by the European Molecular Genet have some influence on the gene expression. Butz M (1), Koch MC (1), MüllerFelber W (2), ics Quality Network (EMQN) for the first time. Quantitative PCR experiments will show whether Schreiber H (3) Fourteen laboratories from 12 European coun CRPS is caused by altered expression levels of (1) Institut für Allgemeine Humangenetik, tries registered. Following this successful trial, NEP, ACE and NK1R. Philipps Universität Marburg, (2) Friedrich the scheme was offered to a wider audience in Baur Institut, Ludwig Maximilians the year 2000 with 20 laboratories from 11 coun Universität München, (3) Neurologische tries participating. One genotyping error leading Klinik, Universität Ulm P122 15 to a misdiagnosis was detected. Thirthy out of FSHD is associated with a deletion of an integral 31 laboratories from 15 countries reported for number of D4Z4 repeats at 4q35. The role of the the 2001 CMT EQA. Two genotyping errors oc Recurrent parkin mutations: Hot spots or deletion in this noncoding region remains un curred. However, due to the use of multiple founders? clear. In healthy controls, the repeat number methods these errors did not lead to misdiagno Hedrich, Katja (1,2), Eskelson, C. (1,2), Marder, varies from 10 to 100 repeats (fragment sizes sis of the CMT1A duplication. The obvious trend, K. (3), Vieregge, P. (2), Kann, M. (1,2), 38300 kb). In FSHD only 1 to 9 repeats (< 35 a higher number of participating laboratories re Schwinger, E.(1), Ozelius, L.J. (4), Pramstaller, kb) are left. A few cases with more than 10 re sulting in more genotyping errors, is an indica P.P. (5), Kramer, P. (6), Klein, C. (1,2) peats are reported. tor for the necessity of not only EQA schemes, (1) Human Genetics and (2) Neurology, To systematically assess the genotypepheno but also the construction of national CMT diag Medical University, Lübeck, Germany; (3) type correlation of borderline D4Z4 repeat num nostic networks to equalize the individual labo Neurology, Columbia University, New York, bers (1014) and to explore their validity for di ratory performance. The development and im NY, USA; (4) Molecular Genetics, AECOM, agnostic cutoff, we reexamined 41 of our provement of European guidelines for genetic di Bronx, NY, USA; (5) Neurology, Regional FSHD patients with 8 to 14 repeats. agnosis of CMT (www.emqn.org) may encourage

64 medgen 14 (2002) General Hospital, Bolzano, Italy; (6) no association of these two novel SNPs to Germany, (5) Atrium 7, 63589 Linsengericht, Neurology, OHSU, Portland, OR, USA EDMD. Additionally, four different unique se Germany Earlyonset parkinsonism (EOP) is a neurode quence variations have been identified in DDX16 Prosopagnosia (PA), a term introduced by Bo generative disorder and has been associated of three unrelated sporadic patients, which did damer (1947, Arch Psych Nerven 179:6ff), de with mutations in the parkin gene (PARK2). not occur in 400 chromosomes of a normal pop fines the inability to identify a person by it´s face Some mutations were found to be recurrent. ulation. However, a segregation of these se alone. The specifity of this deficiency is support Periquet and colleagues (2001) addressed the quence variations with EDMD in the patient’s ed by double dissociation between probands origin of a range of recurrent parkin mutations, families could not be validated. with impairment of e.g. object agnosia but not concluding that exon rearrangements occurred prosopagnosia and vice versa. Almost all reports independently, whereas some point mutations (#259 citations in medlineR) are single casuistics have a common founder. or collections of unrelated patients who acquired P122 17 We studied 182 EOP patients of different ethnic prosopagnosia after brain injuries, strokes, or at background and 350 healthy blood donors from rophy of at least the right occipitotemporal South Tyrol for mutations in the parkin gene and Disruption of the serine threonine kinase 9 area. There are only a few reports of congenital found 33 different mutations, 13 of which were gene (STK9) as the cause of severe Xlinked PA almost all of them sporadic cases (#5 cita detected recurrently. Using 12 DNA markers for infantile spasms tions in medlineR). The only hints of familial the PARK2 locus, we genotyped 44 mutated Kalscheuer, Vera M(1), Tao J(1), Kuebart S(1), ecurrence in 2 generations are given by Mc chromosomes with recurrent mutations. Allele Hollway G (2), Hoeltzenbein M(1), Eyre H(2), Conachie (1976, Cortex 12:76ff) and by Haan frequencies were taken from databases Tommerup N(3), Schwinger E(4), Ropers HH(1), (1999, J Clin Ex Neuropsy 21:312ff). The family (www.gdb.org) and generated by genotyping an Gecz J(2) trees were not studied in any more detail and no other 132 chromosomes from Central European (1)MaxPlanckInstitute for Molecular entries were added to OMIM. We have found 4 controls. Genetics, Berlin, Germany;(2)Dep. of distinct families of at least 5 persons each with A common founder is conceivable for four Cytogenetics and Molecular Genetics, congenital PA ascertained in 4 generations. We (Ex2del, Ex34del, Ex4del, Ex7del) of the six University of Adelaide, Australia; (3)Inst. of are also aware of several other unrelated fami studied exon deletions, two (1072delT, 256delA) Medical Biochemistry and Genetics, The lies with 2 persons showing congenital PA. This

of three small deletions, and one (924C>T) of Panum Institute, Copenhagen, Denmark; indicates a much higher prevalence than repre Abstracts GfH ÖGH SGMG Tagungsband three point mutations but not for the duplication. (4)Institute for Human Genetics Luebeck, sented by literature data. The cumulative segre Interestingly, we confirmed that one of the most Luebeck, Germany gation ratios are compatible with a simple auto common mutations (924C>T) seems to have a Xlinked infantile spasms (ISSX, MIM # 308350)), somal dominant mode of inheritance. Evaluation distant European founder, suggested by the sometimes called Westsyndrome (WS) are char of the prevalence in the general population is in presence of a rare allele (in 3 % of controls) at acterised by onset of generalized seizures be progress. Linkage analyses in all 4 families have the marker D6S305 in all mutation carriers tween 3 and 7 months of age, hypsarrhythmia been started. By whole genome scan 75% of the (10/10 published and 10/10 this study). on the electroencephalogram (EEG) and mental genome could be excluded until now. In conclusion, we demonstrate that both point retardation (MR). In our study of diseaseasso mutations and exon rearrangements may be ciated balanced chromosome rearrangements founder mutations. (DBCRs)we have acquired two patients with bal P122 19 anced translocations 46,X,t(X;6)(p22.3;q14)and 46,X,t(X;7)(p22.3;p15) and clinical features of se vere ISSX/WS. Both patients had early onset of Clinical features and neuropathology of P122 16 seizures at age 23 month, and developmental autosomal dominant spinocerebellar ataxia arrest with profound mental retardation. Cloning (SCA17) caused by CAG expansion in the Candidate gene testing for EmeryDreifuss of the Xchromosome breakpoints revealed the TATAbinding protein muscular dystrophy serine threonine kinase 9 gene (STK9) to be in Ingrid Bauer, Arnulf H. Koeppen, Arndt Rolfs, Bethmann, Cornelia, Wasner, C., Wehnert M. terrupted by these rearrangements. Mutation Sven Buhlmann, Helge Topka, Peter Bauer, Institute of Human Genetics, University of screening of the 21 exons of the STK9 gene in Ludger Schöls, Olaf Riess Greifswald, Germany three ISSX families was negative. In the mean Department of Medical Genetics, Children’s Until now two genes, STA and LMNA, have been time the ISSX/WS gene interval was refined on Hospital, University of Rostock, Germany; associated to EmeryDreifuss muscular dystro one Canadian family to ~7 Mb region in Xp21.3 Stratton V.A. Medical Center, Albany, USA; phy (EDMD). Scanning 93 patients suffering Xp22.1 (Bruyere et al. Clin Genet 55:173, 1999); Department of Neurology, University of EDMD or associated phenotypes revealed that importantly, excluding the STK9 gene. Rostock, Department of Neurology, Ruhr mutations in STA and LMNA together account More recently, mutations in the Aristalessrelat University Bochum; Department of Medical for only 36 % of the cases. Obviously, further ed homeobox gene (ARX) have been found in Genetics, University of Tübingen, Germany genes are likely to be involved in EDMD. Forced four ISSX/WS families and in families with syn Autosomal dominant inherited spinocerebellar by the lack of families suitable for a classic po dromic and nonspecific mental retardation ataxia (SCA) is a neurodegenerative disorder sitional cloning approach, we started a candi (Stromme et al., Nat Genet 4:441, 2002, Bien clinically characterized by lateonset ataxia of date gene approach. Beside others we consid venu et al., Hum Mol Genet, 11:981, 2002). gait, dysarthria, and in some patients with ex ered genes encoding factors interacting with Based on the identical phenotype of our two pa trapyramidal dysfunctions. Several forms (SCA1, emerin and/or lamins A/C candidate genes for tients with balanced Xautosome translocations SCA2, SCA3, SCA6, SCA7) are caused by un EDMD. Thus, BAF (barrier to autointegration fac we suggest, that there are at least two genes for stable CAG repeat expansions in the coding re tor), DDX16 [DEAD/H (AspGluAlaAsp/His) box ISSX on the human Xchromosome. We pro gion of the responsible genes. Recently, CAG re polypeptide 16] and PSME3 (proteasome activa pose, that lack of a functional STK9 protein is peat expansions in the TATAbinding protein tor subunit 3) were scanned for DNAvariations responsible for a severe form of Xlinked infan (TBP) have been found in a new form of SCA by using primers to amplify all exons including tile spasms. which has been assigned SCA17. We deter the exon/intron boundaries in 56 mainly sporadic mined the frequency of SCA17 in a large sample German EMD patients who were excluded to of 1318 SCA patients of German origin for whom carry mutations in STA or LMNA. Until now, we CAG repeat expansions in the SCA1, SCA2, P122 18 have analysed 33 exons excluding the 5‘ and SCA3, SCA6, SCA7, SCA8, SCA12 and Friedre 3‘UTR regions of the three genes mentioned ich’s ataxia genes had been excluded. As con above by PCR/heteroduplex analysis. By scan Congenital prosopagnosia indicative of trols we studied 150 people older than 60 years ning the patients, nine unique aberrant heterodu simple autosomal dominant inheritance with no neurological signs, 250 patients with plex patterns have been found so far in exons 1 Kennerknecht, Ingo (1), Grüter, M. (1), Meyer B. dystonia, 81 patients with genetically confirmed and 2 of BAF, exons 2 and 18 and introns 5, 11, (2), Grüter, T. (3), Sperling, K. (4), Laskowski, W. Huntingtons disease, and 198 SCA patients with 18 and 19 of DDX16 and intron 2 of PSME3. (5), Nürnberg, P. (2) known mutation. Seven DNA variations have been validated by (1) Institut fur Humangenetik, Westfälische In total, 3 autosomal dominant SCA families and sequencing so far. Six sequence variations have Wilhelms Universität, Münster, Germany, (2) two apparently sporadic patients with CAG re been tested in 400 chromosomes of a German MaxDelbruck Centrum für Molekulare peat expansions in the TBP gene ranging from normal population. Two of them occurred with Medizin, Berlin, Germany, (3) Nottulner 45 to 54 repeats were identified. In 481 control frequencies for the rare allele of 0,0053 and Landweg 33, 48161 Münster, Germany, (4) individuals the largest allele detected consisted 0,0158, respectively, thus validating them as sin Institut fur Humangenetik, Charite Berlin, of 42 alleles and in the group of SCA patients gle nucleotide polymorphisms (SNPs). There was with known mutation alleles of 43 CAG repeat

medgen 14 (2002) 65 were found defining a small nonoverlapping re view of epidemiological, family, twin, linkage, P122 23 gion between normal (43) and expanded (45) al and theoretical studies allows to conclude that leles. The clinical features of SCA17 differ from there exist risk alleles in several genomic loci in Mutationsanalyse des Pank4Gens in other SCA subtypes by manifestation with psy order to fully explain the proportion of AD attrib Parkinson Patienten chiatric detoriations in addition to neurological utable to genetic factors. However, up to now Hering, Robert (1), Riess, A. (1), Krüger, R. (2), symptoms. Based on routine histological exam none of the numerous studies involving more Schöls, L. (3), Bauer, P. (1), Riess, O. (1) ination, the neuropathology of SCA17 can be than 100 candidate genes revealed convincing (1) Medizinische Genetik, Universität classified as a „pure cerebellar“ or „cerebello evidence for any predisdposing risk alleles in Tübingen, (2) Neurologische olivary“ form of ataxia. However, intranuclear genes other than APOE. In the published litera Universitätsklinik Tübingen, (3) St. Josef neuronal inclusion bodies with immunoreactivi ture there are more than 45 loci with positive as Hospital, Neurologische Universitätsklinik ty to antiTBP and antipolyglutamine were sociation findings. Most of these findings (in Bochum much more widely distributed throughout the cluding these of our group) could not be con Ätiologie und Pathogenese der Parkinson’schen brain gray matter than in other SCAs. firmed. There are only few loci deserving further Erkrankung sind weitgehend unklar. In einigen replication studies. This presentation briefly re wenigen Familien konnten Mutationen im alpha views possible reasons for this lack of success SynucleinGen und im ParkinGen nachge and proposes criteria for a more efficient selec P122 20 wiesen werden. Mittels Kopplungsanalysen wur tion of positional and functional candidate genes den kürzlich 2 Genloci in der chromosomalen for AD. Supported by DFG, grant FI 704/13. Region 1p3536 identifiziert, die mit einer früh Mutation analysis of genes involved in brain manifestierenden Parkinson Erkrankung einher iron homeostasis in Parkinsonian patients gehen sollen. Durch den Nachweis von Mutatio Daniela Berg (1),(2), Helmine Hochstrasser (2), P122 22 nen in einem PantothenatKinaseGen (Pank2, Thomas Franck; (2), Uwe Walter (3), Jorg Genlocus 20p13) konnte kürzlich die Ätiologie Spiegel (4), Stefanie Behnke (4), Ulrike der HallervordenSpatzErkrankung geklärt wer Sommer; (5), Georg Becker (4), Olaf Riess (2) Analysis of association of Alzheimer disease den. Diese Erkrankung geht mit einer Eisenakku Departments of Neurology Universities of (1) with allelic variants of TFAM located on mulation im Globus pallidum und parkinsonähn

GfH ÖGH SGMG Tagungsband Abstracts GfH ÖGH SGMG Tagungsband Wuesrzburg, (3) Rostock, (4) Homburg, (5) chromosome 10 lichen Symptomen einher. Mittels Daten Dresden, (2) Institut of Human Genetics, Günther, Claudia (1), MüllerThomsen, T. (2), banksuche gegen das menschliche Genom find University of Tuebingen Alberici, A. (3), Binetti, G. (3), Hock, C. (4), et man eine hohe Sequenzhomologie zwischen In the pathogensis of Parkinsons disease a dis Stoppe, G. (5), Reiss, J. (6), Finckh, U. (1) Pank2 und Pank4, welches in die Region 1p36 turbance of iron metabolism at different levels (1) Depts. of Human Genetics & (2) kartiert. Wir untersuchten die DNA von 184 including iron uptake, storage, intracellular me Psychiatry, University Hospital Hamburg Parkinsonpatienten (AAO <= 50. LJ) auf das Vor tabolism and posttrancriptional control has Eppendorf, Germany (3) IRCCS Centro S. liegen von Mutationen in Pank4. Nach PCRAm been discussed for many years. Using transcra Giovanni di Dio, Brescia, Italy, (4) Division of plifikation der 19 Exons führten wir ein initiales nial ultrasound an accumulation of iron in the Psychiatry Research, University of Zürich, Mutationsscreening mittels dHPLC (Transge substantia nigra is being detected in about 90 % Switzerland, (5) Depts. of Psychiatry & (6) nomic WAVE System) durch. Die dabei auffälli of Parkinsonian patients and there is accumulat Human Genetics, University of Göttingen, gen Proben wurden anschließend sequenziert ing evidence indicating that increased iron con Germany (Beckman Coulter CEQ 2000). Mittels dHPLC tent of the brain may serve as a marker for ni TFAM encodes mitochondrial transcription fac wiesen wir in den 184 Proben insgesamt 415 grostriatal vulnerability. To investigate whether tor A that participates in mitochondrial genome (zum Teil fragliche) Shifts nach, die zu 53 unter this ultrasound feature may be associated with replication and in activation of mitochondrial schiedlichen ShiftGruppen zusammengefasst mutations in genes encoding for proteins of the transcription. Both mechanisms are essential for werden konnten. Bisher konnten 7 Gruppen se iron metabolism we performed mutation analy mitochondrial function and integrity. Mitochon quenziert werden: in einem Fall zeigte sich bei sis in 180 patients with Parkinsons disease who drial dysfunction may be involved in neurode einem fraglichen Shift eine Wildtypsequenz; 5 displayed the feature of increased substantia ni generation observed in lateonset Alzheimer dis Shifts konnten als intronische Mutationen iden gra iron levels on transcranial ultrasound in com ease (AD). TFAM locates to chromosome 10q21, tifiziert werden; bei einem Shift (6 Proben) kon parison to 180 controls without this ultrasound a region linked to AD[1] and plasma level of nte eine Mutation in Exon 13 nachgewiesen wer feature. Mutation analyses were performed of Ab42 in AD families.[2] Linkage to 10q21 was den (1640 C>T; 547 Ala>Val). Die optimierten the genes encoding for IRP2 and ceruloplasmin. most evident in APOE e4 positive samples.[1] Bedingungen des Screenings von Pank4 mittels IRP2 is one of the cytosolic iron regulatory pro Therefore, TFAM represents both a positional dHPLC und die vollständigen Ergebnisse der teins involved in the regulation of iron transport and functional candidate gene for AD. We geno Mutationsanalyse werden vorgestellt. ing and storing proteins, whereas ceruloplasmin typed a polymorphism in evolutionary noncon seems to play a major role in cellular iron import served codon 12 of TFAM (S12T) in 372 patients and export. All 22 exons of the IRP2 gene and all with AD and 295 nondemented controls. There 19 exons of the ceruloplasmin gene were ampli was a trend towards an association between ab P122 24 fied by polymerase chain reaction. Heteroduplex sence of T12 (T) and AD (p=0.062). Conditional analysis was performed using the Wavesys logistic regression analysis entering covariates QUANTITATIVE ANALYSIS OF SMN1 AND tem(R), which forms a mixture of hetero and ho age, gender, TFAM (T vs. T+), APOE (e4+ vs. SMN2 COPY NUMBER USING TAQMAN TM moduplexes with pooled probes in individuals e4), and interaction [APOE x gender x TFAM] re TECHNOLOGY with heterozygous or homozygous mutations. vealed an interaction between the three covari Anhuf, Dirk, Eggermann, T., Rudnik Exons of individuals with evidence of heterodu ates with an exp B („OR“) of 3.39 (95% CI 1.93 Schöneborn, S., Zerres, K. plex formation were amplified for sequence 5.94) in addition to significant associations with Institute for Human Genetics, University of analysis. Comparison of mutations and polymor AD for APOE (OR 1.96, 95% CI 1.342.85) and Technology, Aachen, Germany phisms in these genes in Parkinsonian patients TFAM (OR 0.74, 95% CI 0.590.92). These data Objective: Spinal muscular atrophy (SMA) is usu and controls may help to elucidate the role of suggest a possible association between ab ally caused by homozygous deletion of the iron homeostasis in Parkinsons disease. sence of TFAM allelic variant T12 and AD which SMN1 gene. The detection of heterozygotes for is influenced by gender and APOE genotype. SMN1 deletion has many clinical implications. Supported by DFG, grant FI 704/13. The number of SMN2 copies correlates statisti [1] Myers et al., Science 290:23052305, 2000 P122 21 cally with the severity of SMA. The SMN2 copy [2] ErtekinTaner et al., Science 290:23032304, number could be of interest in the view of pos 2000 sible future therapeutic trials. The quantitative False positive association findings in 45 SMN analysis, however, is difficult since the hypothetical candidate genes for Alzheimer SMN1 and SMN2 genes differ in only a few base disease ? pairs. In order to allow a simple and reliable test Finckh, Ulrich to determine the number of SMN1 and SMN2 Institute of Human Genetics, University copies, we developed a quantitative test using Hospital HamburgEppendorf, University of the well known TaqManTM technology. Hamburg, Germany Methods: Minor groove binding (MGB) probes The genetic association between APOE e4 and were used for the detection of single nucleotide late onset Alzheimer disease (AD) was discov polymorphism in order to differentiate between ered almost one decade ago. A cumulative re

66 medgen 14 (2002) SMN1 and SMN2. As a reference locus exon 3 and our understanding of the genetic control of genetic approach in cases suspected of having of the factor VIII gene was used. Genomic DNA normal and abnormal brain development. ARCMT. First, we exclude a mutation in the from 40 unrelated parents of SMA patients car PMP22, Cx32, MPZ, and EGR2 genes. The next rying one SMN1 copy and 100 normal controls step depends on the size and structure of the were analysed. The number of SMN copies was family under investigation: Linkage studies are P122 26 determined by analysis of the multicopy mark performed in extended pedigrees, homozygosi ers AG1CA and C212. The reproducibility was ty mapping in case of parental consanguinity, evaluated on the basis of 5 independent PCRs Mutations in the gangliosideinduced and direct mutation analysis of known ARCMT and revealed non overlapping results. The pro differentiationassociated protein1 (GDAP1) genes in isolated patients. In case a family is posed method allowed a clear distinction be gene in autosomal recessive CharcotMarie compatible with one of the ARCMT loci, haplo tween typical carriers (one SMN1 copy) and con Tooth neuropathy type analysis is followed by direct sequencing of trols (two SMN1 copy) with ranges from 0.60 to Senderek, Jan (1), Bergmann, C. (1), the respective gene (if available). Thus far, two 1.20 (carriers) and 1.602.20 (controls). Low Ramaekers, V. T. (2), Makowski, A. (1), truncating mutations in GDAP1 and one copy numbers of SMN2 were precisely classified Schröder, J. M. (3), RudnikSchöneborn, S. (1), frameshift mutation in PRX could be identified. whereas the differentiation of very high numbers Zerres, K. (1) Linkage has been established in two ARCMT was more difficult. (1) Institute for Human Genetics, (2) multiplex families to loci on chromosome 5q32 Conclusion: The TaqManTM based quantitative Department of Paediatrics, (3) Institute for and 11p15 respectively. Linkage analyses as well analysis of SMN1 and SMN2 can be regarded as Neuropathology, University of Technology, as haplotype sharing will help to further narrow a reliable method to determine the SMN1 and Aachen, Germany down candidate regions if the responsible gene SMN2 copy number. While the determination of Mutations in the gene for the gangliosidein is not yet known. Appropriate candidate genes the SMN1 copy number has numerous clinical duced differentiationassociated protein1 are currently under investigation. The identifica implications, the SMN2 copy number is so far (GDAP1) on 8q21 were recently reported to be tion of gene defects in ARCMT is important for mainly of scientific interest. responsible for about 25% of cases with auto genetic counselling of affected families and for somal recessive CharcotMarieTooth sensori a better understanding of the different pathome motor neuropathy (CMT). Neurophysiology and chanisms underlying peripheral nerve dysfunc

nerve pathology were heterogeneous in these tion. To further pursue these studies we are Abstracts GfH ÖGH SGMG Tagungsband P122 25 cases: a subset of GDAP1 mutations was asso highly interested in additional families with pos ciated with peripheral nerve demyelination while sible ARCMT. Clinical and molecular studies of others resulted in axonal degeneration. In the Holoprosencephaly present study on autosomal recessive CMT in Hehr, Ute (1), Gross, C. (1), Diebold, U. (2), cluding 11 families, we identified two families P122 28 Wahl, D. (3), Diepold, K. (4), Hehr, A. (1) with novel mutations disrupting the GDAP1 (1) Center of Gynecological Endocrinology, reading frame. Homozygosity for a single base Reproductive Medicine and Human pair insertion in exon 3 (c.349_350insT) was ob CharcotMarieTooth disease: A novel Genetics; Regensburg, Germany; served in two affected children from a Turkish in Tyr145Ser mutation in the Myelin Protein ute.hehr@humangenetikregensburg.de, (2) bred pedigree. The other novel allele detected in Zero (MPZ, P0) gene causes different Social Pediatric Center Hannover, Germany, a German child from unrelated parents was a phenotypes in homozygous and (3) Medical Geneticist, Augsburg, Germany, homozygous mutation of the exon 4 donor splice heterozygous carriers (4) Department of Neuropediatrics, site (c.579+1G>A). Our patients with GDAP1 mu Leal, Alejandro (1,2); Kayser, C (3); Berghoff, M University Göttingen, Germany tations displayed severe, early childhoodonset (4); Hernández, E (2); Barrantes, R (2); Reis, A Holoprosencephaly (HPE) is a common congen CMT neuropathy with prominent pes equino (1); Del Valle, G (2); Heuss, D (3); ital defect resulting from loss or incomplete for varus deformity and impairment of hand mus Rautenstrauss, B (1) mation of midline structures of the forebrain and cles. Nerve conduction velocities (NCV) were be (1) Institute of Human Genetics, Erlangen, face. The etiology of HPE is heterogeneous and tween 25 and 35 m/s and peripheral nerve Germany, (2) University of Costa Rica, includes environmental factors, nonrandom pathology showed axonal as well as demyelinat INISA, San José, Costa Rica, (3) chromosomal aberrations as well as gene muta ing changes. There was no specific morpholog Neurological Department, Erlangen, tions. Currently, mutations in SHH (7q36), SIX3 ical feature that allowed to distinguish our pa Germany, (4) Neurological Department, (2p21), ZIC2 (13q32) and TGIF (18p11.3) account tients from other types of CMT. Our findings un Wuerzburg, Germany for appr. 1620% of all HPE cases. Here we re derline the role of GDAP1 for the pathogenesis Mutations in the MPZ gene cause Charcot port the results of the analysis of HPE patients of CMT and allow further insight into the mech MarieTooth type 1B (CMT1B), a demyelinating in the German population. Molecular studies in anisms vital for axonal integrity and Schwann hereditary motor and sensory neuropathy clude direct sequencing of the coding regions of cell properties. Identification of GDAP1 as the (HMSN). Mutations in MPZ have been related SHH, SIX3 and ZIC2. DNA samples without causative gene defect allows direct mutation also with axonal neuropathy (CMT2), particular identified mutation are further analyzed for mu analysis in individual patients also from small, ly due to a T124M mutation in exon 3. Patients tations in additional HPE candidate genes in a nonconsanguineous families. A molecular ge who carry this mutation are additionally affected collaborative research study (M. Muenke, netic diagnosis is important for accurate genet with Argyll Robertsonlike pupils and frequently NIH/Bethesda) after written consent of the pa ic counselling of affected families. with deafness and dysphagia. Here we present tients or parents, respectively. Identified muta a Costa Rican family affected mainly with tions include the previously reported SHH non CMT1B; the parents presented a mild neuropa sense mutation Glu324(amber) as well as a new thy with a late age of onset (50 years), whereas P122 27 SHH missense mutation Gly27Ala, both occur the two children presented an earlier age of on ring in dominant HPE families with solitary me set (late 30 years) and a more severe neuropa dian maxillary central incisor (SMMCI) and wide Molecular genetic approach in families with thy. Clinical signs are distal sensory deficits and intrafamiliar clinical variability. A new SIX3 mis possible autosomal recessive Charcot absent ankle jerks. Pupillary abnormalities, dis sense mutation Arg218Pro was observed in the MarieTooth neuropathy (ARCMT) tal weakness and atrophies are restricted to the highly conserved homeo domain in a 3 year old Bergmann, Carsten, Senderek, J., Rudnik children. Electrophysiological studies revealed girl with semilobar HPE and profound develop Schöneborn, S., Zerres, K. axonal degeneration and demyelination. A sur mental delay. Analysis of the parents unexpect Institute for Human Genetics, University of al nerve biopsy from one of the children showed edly revealed the clinically healthy mother to be Technology, Aachen, Germany signs of axonal degeneration as well as demyeli a mosaic carrier. The low grade mosaicism in While the majority of CMT cases is autosomal nation. Both in the maternal and the paternal maternal genomic DNA prepared from peripher dominantly or Xlinked dominantly inherited, au family histories other patients with this type of al blood was only detected by RFLP analysis us tosomal recessive CMT (ARCMT) is much rarer peripheral neuropathy exist. After sequencing of ing a restriction enzyme exclusively cutting the and highly heterogeneous. The distribution of all 6 coding MPZ exons for the parents and chil normal allele. A new truncating ZIC2 mutation weakness and sensory deficits is similar to the dren, a Y145S mutation in exon 3 was found; the del A in codon 221 was identified in a 7 year old dominant forms, but the clinical course is gen parents are heterozygous for this mutation, boy with alobar HPE and relatively mild facial erally more severe with an earlier age of onset. whereas the children are homozygous carriers. features. Further analysis of the phenotype At least ten loci responsible for ARCMT and six In contrast to the T124M mutation, only the ho genotype correlation in HPE patients will im genes have been identified so far. Thus, a rou mozygous Y145S carriers are affected with prove genetic counseling of affected families tine molecular genetic analysis is not available pupillary anomalies, and the Y145S heterozy yet. We have developed a stepwise molecular gous patients are less affected than the T124M

medgen 14 (2002) 67 heterozygous. We propose that by the exchange P122 30 FRETprobes in a PCR/melting curve analysis, of similar side chains (uncharged polar) the and by conventional RFLP analysis. The study slightly altered protein conformation has only included 367 alcohol dependent individuals and Identification of a <5Mb interval on proximal low impact on the protein function; this could 335 control individuals of German origin for the Xp playing a major role in nonsyndromic X explain the mild phenotype of heterozygote car case control study, and 72 trios of Polish origin linked mental retardation NSXLMR and riers. However, a homozygous change could be for the family based study. For NMDAR1, there largescale mutation screening of all sufficient to cause a neuropathy associated with was a significant difference in the genotype dis relevant genes pupillary anomalies. tribution between alcoholic patients and con Kalscheuer, Vera (1), Hoeltzenbein M (1), trols. Furthermore, patients with the homozygote Lenzner S (1), Jensen LR (1), Roloff TC (1), mutation had a significantly less ethanol intake Chelly J (2), Moraine C (3), Fryns JP (4), than those bearing a G allele. For NMDAR2B the P122 29 Yntema H (5), Hamel B (5), Haas S (1), Vingron T allele prevalence was significantly reduced in M (1) and Ropers HH (1) patients with an early age at onset, in patients 1 MaxPlanckInstitut für Molekulare Investigations into the Molecular with vegetative withdrawal syndrome, and in Genetik, Berlin, D; 2 Institut Cochin, CHU Mechanisms of Brain Function: Disruption Cloninger type 2 alcoholics. Furthermore, pa Cochin PortRoyal, Paris, F; 3 Service de of a Serinethreonine Kinase is Associated tients carrying the Callele showed higher rates Génétique, CHU Bretonneau, Tours, F; 4 with Motor and Cognitive Deficits in a in the personality trait subscale of impulsivity Centrum voor Menselijke Erfelijkheid, Patient with a t(Y;4) Translocation compared to those lacking it. Our family based Universiteit Leuven, B; 5 Anthropogenetisch Shoichet, Sarah (1), Duprez, L. (2), Suckow, study revealed a preferred transmission of the C Instituut, Universitair Medisch Centrum, V.(1), Menzel, C.(1), Hagens, O.(1), Vamos, allele by fathers to their affected offspring. Nijmegen, NL E.(2), Schweiger, S.(1), Ropers, H.H.(1), and Xlinked gene defects may account for one third Kalscheuer, V.M.(1) of all idiopathic forms of mental retardation in P122 32 (1)Max Planck Institute for Molecular males, and most of these cases are nonsyn Genetics, Germany (2)Department of dromic. Causative mutations in 12 different Medicine, University of Brussels, Belgium Splice mutations of the LIS1 gene leading genes as well as linkage studies have docu

GfH ÖGH SGMG Tagungsband Abstracts GfH ÖGH SGMG Tagungsband Numerous genes, coding for proteins with a to severe classical lissencephaly mented that NSXLMR is highly heterogeneous, wide variety of functions, play some role in nor Stiegler Johannes (1), Uyanik G (1), Hehr U (2), and until recently it was believed that up to 100 mal cognitive development. It is estimated that Hehr A (2), Flügel D (1), Aigner L (1,3), Winkler Xlinked genes might be involved. However, as in a significant percentage of cognitive disor J (1) judged from the recent finding that up to 20 per ders, there is a direct link between genotype and (1) Department of Neurology, University of cent of all patients with this condition have mu disease phenotype, and recent advances on the Regensburg, (2) Center of Gynecological tations in the ARX gene (Stromme et al., Nat. X chromosome provide the framework within Endocrinology, Reproductive Medicine and Genet. 2002; Bienvenu et al., Hum. Mol. Genet. which molecular cascades important for cogni Human Genetics Regensburg, (3) VW 2002), the total number of genes may be much tive function can be elucidated. We have utilised Foundation Junior Group, University of lower. As a prerequisite for identifying other ma data from routine chromosome analyses in pa Regensburg jor genes in the etiology of NSXLMR, we have tients with early onset, nonsyndromic forms of Classical lissencephaly is a severe brain malfor analyzed all available linkage data from the Eu mental retardation and established cell lines mation with absent (agyria) or poor sulcation ropean XLMR Consortium and from other from patients for which balanced chromosome (pachygyria) because of an impaired neuronal sources to estimate the distribution of gene de rearrangements provide likely explanations for migration. It occurs isolated or associated with fects underlying NSXLMR along the length of the phenotype. Here we report the results of a other symptoms. The most common form is the Xchromosome. Apart from several peaks study involving a patient carrying a translocation present as MillerDieker Syndrome with its char corresponding to ARX at Xp22 and other known t(Y;4)(q11.2;q21) and presenting with a severe acteristic facial dysmorphic features. Clinically genes for NSXLMR, a <5Mb interval on proxi neurodegenerative disorder, characterised by a isolated lissencephalies (IL) are associated with mal Xp was identified which seems to harbor progressive loss of speech and other motor ac refractory epilepsy, severe mental retardation (one or several) novel important XLMR genes. An tivities that was first observed at the age of thir and poor life expectancy. Two genes are associ integrated, functionally annotated map of this in teen months. Motor degeneration was accom ated with isolated (classical) lissencephalies. Au terval was constructed, and DHPLCbased mu panied by severe mental retardation and a tosomal dominant lissencephalies are linked to tation screening of all brainexpressed genes in seizure disorder which persisted despite admin alterations of the LIS1 gene on chromosome 17 this region is in progress. istration of classical antiseizure medication. We and Xlinked lissencephalies to mutations of the have determined that, while the breakpoint on DCX gene, respectively. The brain malformations chromosome Y lies within a highly repetitive re due to LIS1 mutations present predominantly gion that likely contains no gene, the breakpoint P122 31 with occipital agyria, whereas DCX mutations on chromosome 4 disrupts a serinethreonine ki lead to frontal predominant agyria. Most com nase that plays an established role in apoptosis mon mutations are submicroscopic deletions of NMDA RECEPTOR POLYMORPHISMS IN and stress response, and based on its restrict the LIS1 gene. This microdeletions may be de ALCOHOLIC PATIENTS ed expression pattern, is likely involved in CNS tected by FISH in up to 40% of the cases. But Wernice Catrin(1), Samochowiec J(2), Schmidt development and function. Further, we have also intragenic mutations of the LIS1 gene are LG(3), Gallinat J(1), Rommelspacher H(1) confirmed that the truncated protein is ex reported and detectable in approximately 12% (1)Departments of Clinical Neurobiology and pressed in the patient, suggesting that the un of ILs. While missense mutations of the LIS1 Psychiatry, University Hospital Benjamin derlying molecular mechanism is not one of sim gene might lead to milder forms of lissencephal Franklin, Free University of Berlin, Germany; ple haploinsufficiency. Current studies aim to ic brain malformations, nonsense mutations are (2)Department of Psychiatry, Pomeranian determine the role that this mutant protein plays associated with more severe grades of Academy of Medicine, Szczecin, Poland; in the patient disorder, specifically, to under lissencephaly. Here, we present our results of (3)Clinic of Psychiatry, University of Mainz, stand how its presence may affect the normal mutation analysis of the LIS1 gene and the de Germany signaling cascade within which the wild type tection of splice mutations in two patients with Alcohol dependence is a clinically heteroge protein functions. severe form of lissencephaly. neous syndrome caused by complex interac tions of genetic and environmental factors. The estimated genetic effects from twin studies ac count for 4060% of the variance in the liability P122 33 to develop alcoholism. The ionotropic gluta matergic NmethylDaspartate receptor is Filamin 1 mutation in a female patient with known to be reduced acutely by physiologically periventricular nodular heterotopia (PNH) relevant concentrations of ethanol. After chron Özcan Bahar (1), Uyanik G (1), Schröder M (1), ic ethanol exposure tolerance develops, and Aigner L (1,2), Stefan H (3), Kraus B (3), Plötz S NMDAR function increases. In this study we (1), Winkler J (1) tested, if recently found polymorphisms in the (1) Department of Neurology, University of genes of the NMDAR1 (G2108A) and NMDAR2B Regensburg, (2) VWFoundation Junior (C2664T) subunits are associated with ethanol Group, University of Regensburg, (3) Center dependence. Genotyping was done using both

68 medgen 14 (2002) for Epilepsy Erlangen, University of that the subcortical band heterotopia is charac women, mostly married. Major findings: A. Fam Erlangen terized by high rate of somatic mosaicism and ily relations. Majorities (US 75%, Germany 76%, Periventricular nodular heterotopia (PNH) is a incomplete penetrance. In addition we detected France 88%) thought warning relatives at genet heritable neuronal migration disorder with defec a somatic mosaic mutation in the DCX gene in a ic risk takes precedence over patient confiden tive migration of neuroblasts from their origin, male patient leading to subcortical band hetero tiality. About half (US 53%, Germany 42%, the subventricular zone to the neocortex. The topia. France 55%) thought spouses had automatic brain malformation is characterized by nodules rights to genetic information, without consent; of heterotopic neurons resting along the lateral 44%, 32%, 41% favored automatic access for ventricles due to a defect in the initiation of the blood relatives. 61%, 34%, 45% would test chil P122 35 neuronal migration. PNH is mostly inherited in an dren for genetic predisposition to Alzheimer. B. Xchromosomal dominant manner and is linked Autonomy. 60%, 48%, 45% said patients had a to mutations in the Xlinked filamin 1 gene Congenital myasthenia in Brahman calves right to any service they could pay for out of (FLN1). While mutations in the Xlinked filamin 1 caused by homozygosity for a CHRNE pocket; 69%, 46%, 48% thought refusal was gene (FLN1) at Xq28 cause PNH in heterozy truncating mutation denial of rights. C. Privacy. There was universal geous females, in hemizygeous male embryos Kraner, Simone (1), Sieb, J. P. (2), Thompson, distrust of insurers and employers. Few (20%, filamin 1 mutations usually lead prenatally to P. N. (3), Steinlein, O. K. (4) 11%, 6%) would protect confidentiality of a bus death. Families with filamin mutations are char (1) Institute of Human Genetics, University driver at high genetic risk for heart attack. Most acterized by an excess of female family mem Hospital Bonn, Germany, (2) Max Planck favored DNA fingerprinting for people convicted bers and a high rate of abortions. This neuronal Institute of Psychiatry, Munich, Germany, (3) of (8397%) or charged with (7182%) serious migration disorder leads to epilepsy, vascular Department of Production Animal Studies, crimes. D. Prevention. Majorities (64%, 49%, abnormalities and less frequent to moderate Faculty of Veterinary Science, University of 70%) thought people should know their genetic mental retardation. In patients with symptomatic Pretoria, Onderstepoort, South Africa status before marriage, but fewer (31%,5%, epilepsy this disorder is diagnosed more fre Congenital myasthenic syndromes (CMS) are in 29%) thought states should require carrier tests. quently in the last years due to improved MRim herited disorders in which the safety margin of Most (8093%) thought women at high risk aging techniques. Here, we present a female pa neuromuscular transmission is compromised by should have PND, but fewer (21%,43%, 67%)

tient with bilateral PNH suffering from epilepsy presynaptic, synaptic or postsynaptic defects. thought they should abort if tests were positive. Abstracts GfH ÖGH SGMG Tagungsband with seizures refractory to antiepileptic treat Most CMS patients have a history since infancy E. Disability. Most (7890%) would respects par ment. She underwent epilepsy surgery due to an or early childhood of fatiguable weakness involv ents’ wishes to refuse a lifesaving operation on epileptic focus of the left parietal lobe. The mu ing ocular, bulbar and limb muscles. There are a handicapped newborn. 44%, 48%, 77% said tation analysis revealed a novel 2 bp insertion in some reports of inherited myasthenic weakness bringing a child with a disability into the world the 9 exon of FLN1 gene leading to a frame shift in domestic animals but so far the molecular ba was unfair to the child, if the birth could be pre and premature stop codon. This is the first de sis in these cases has remained elusive. The vented; 26%, 27%, 56% said it was socially ir scription of an FLN1insertion mutation leading analysis of such spontaneous animal models responsible. About 30% fewer in US would abort to PNH. can contribute to our understanding of the for each of 24 genetic conditions than in Ger pathophysiological mechanisms underlying CMS many. On most issues, patients’ views differed in humans, too. We were now able to identify a from geneticists in their own country. It is time homozygous 20bp deletion within exon 5 of the to examine reasons for these differences. P122 34 acetylcholine receptor esubunit (bovCHRNE) in Brahman calves affected by CMS. The 470del20 Mutation analysis of the DCX gene in mutation causes a frame shift in the predicted P201 02 Lissencephaly / Subcortical Band bovCHRNE protein after 129 codons, substitut Heterotopia Spectrum ing 342 wildtype amino acids residues by 40 Uyanik Gökhan (1), Hehr U (2), Gross C (2), aberrant amino acids followed by a stop codon. Does Knowledge influence attitudes Stiegler J (1), Schröder M (1), Martin P (3), The frame shift occurs 90 amino acids residues towards psychiatric genetics? Flügel D (1), Aigner L (1,4), Winkler J (1) Nterminal of the first transmembrane region. Illes, Franciska (1), Rietz, C. (2), Matschinger, (1) Department of Neurology, University of Thus, the bovCHRNE mutation reported here H. (3), Rudinger, G. (2), Angermeyer, M. (3), Regensburg, (2) Center of Gynecological leads to a nonfunctional allele, which is likely to Maier, W. (1), Rietschel, M. (1) Endocrinology, Reproductive Medicine and be the primary cause for myasthenia in the af (1) Department of Psychiatry, University of Human Genetics Regensburg, (3) Epilepsy fected calves. Interestingly, all four calves were Bonn, Germany, (2) Department of Center Kork, KehlKork, (4) VWFoundation severely affected at a very young age. This is in Psychology, University of Bonn, Germany, Junior Group, University of Regensburg contrast to human myasthenic patients with ho (3) Department of Psychiatry, University of Lissencephaly and subcortical band heterotopia mozygous truncating CHRNE mutations, most of Leipzig, Germany (SBH) (synonyma: Double Cortex Syndrome) are them showing a less severe course of the dis In the framework of the German Human Genome heritable neuronal migration disorders. The oc ease. ? (Kraner et al., Neurogenetics, in press) Project we conducted a study to assess the at curence of lissencephaly and SBH within the titudes towards psychiatric genetic research and same pedigree, even within the same patient un testing and to identify factors influencing them covered their relationship and lead to the term in a representative sample of 3077 persons of „Lissencephaly / Subcortikal Band Heterotopia Postersession the German general population. One factor as Spectrum“. This spectrum range hereby from sumed to influence the attitudes is knowledge complete absence of gyri (agyria) to broad gyri 2 Oct. 1st 2002 about psychiatric disorders and genetics, which with reduced sulci (pachygyria) and merges with Foyer was assessed by questions about psychiatric ill subcortical band heterotopia. Genetically these nesses and genetics. diseases are heterogeneous with different Our results show that a higher knowledge con P201 01 modes of inheritance. Xlinked lissencephaly and cerning psychiatric illnesses and genetics is cor subcortical band heterotopia are allelic disorders related with more critical but still positive at caused by mutations in the doublecortin gene Patient’s Views on Ethical Issues; Surveys in titudes towards psychiatric genetic research (r = (DCX or XLIS) at Xq22.3q23. Mutations in hem USA, Germany, France .200, p =.000), predictive testing(r = .125, p = izygeous male patients usually lead to Nippert, Irmgard (1), Wolff, G (2), Aymé, S (3), .000), prenatal testing (r = .255, p = .000) and lissencephaly with severe mental retardation, Wertz, DC (4) access to genetic information (r = .323, p = epilepsy and poor life expectation. In heterozy (1) Institut of Human Genetics, UKM, .000). Further analyzes concerning the means of geous female patients with DCX mutations, sub Muenster, Germany (2) AlbertLudwigs the investigated scales, show significant differ cortical band heterotopia is associated with nor Universitaet Freiburg, Germany (3) INSERM, ences between the different knowledge groups. mal development or moderate mental retardation Villejuif, France (4) University of For a further analyzes of the influence of knowl and epileptic seizures. Here we demonstrate a Massachusetts Medical School, Waltham, edge on attitudes in the field of psychiatric ge large series of familial and sporadic cases with MA, USA netics, knowledge about schizophrenia and eth Xlinked lissencephaly and/or Double Cortex We surveyed patients’ ethical views at 12 genet ical standards in the application of psychiatric Syndrome analized for mutations in the DCX ics clinics in USA, 2 in Germany, 5 in France. genetics was tested in 88 medical students be gene. We have identified 13 different mutations, 476 (67%) in USA, 593 (65%) in Germany, and fore and after a short teaching program. Before including 9 novel nonsense and missense mu 394 (51%) in France returned anonymous ques the intervention the knowledge about schizo tations and 1 insertion. Our analysis revealed tionnaires. Over 9/10 in USA and Germany were phrenia was alarmingly low and misbeliefs with

medgen 14 (2002) 69 respect to clinical symptoms and course of cists, various academic disciplines and the pub schizophrenia widespread. Although knowledge lic. The informational content of technical terms, could be significantly increased common misbe which have a (nearly) clear meaning in the realm P202 01 liefs showed to persist in a substantial part of of medicine in general, is liable to be garbled the students. when transferred to and uncritically used within Our findings show that knowledge influences at human genetics. One of the technical terms we Tissue rather than genespecific models titudes and furthermore indicate that specific focussed on is ‘prevention’, which as primary help to elucidate regulatory networks via training programs are warranted to fight wide prevention keeps disease from occurring at all promoter sequence analysis spread misbeliefs. by removing risk factors but in human genetics Doehr, Stefanie (1), Maier, H. (1), Werner, T. especially in prenatal diagnosis is mainly under (1,2) stood as prevention of the ill. (1) Institut fuer Experimentelle Genetik, GSF Fourteen scenarios were developed from genet Forschungszentrum fuer Umwelt und P201 03 ic counselling before conception to abortion of Gesundheit, Neuherberg, Germany, (2) a child with trisomy 21. Three groups of students Genomatix Software GmbH, Muenchen, Defining and understanding disease in the of medicine differing in their level of clinical and Germany. Contact: [email protected] genomics era theoretical experience (1st, 3rd and 6th years of The aim of our group is the description of regu Hoffman, Elisabeth, Preß, B., Pelz, J. medical education) were interviewed using these latory promoter elements for identification of Reformstudiengang Medizin, Humboldt scenarios in a structured questionnaire. Con gene regulatory networks. For this purpose, we Universität zu Berlin, Charité Campus Mitte, founders, concepts of disease and assessment collected promoter sequences of genes related Germany of human genetics in the realm of medicine were to the monogenic disorders of MODY type 1 to The term ‘genetic disease’ is used on a daily ba recorded. 6 (maturity onset diabetes of the young). The sis, but the concept of genetic disease seems to About 50% of the study group made a distinc MODY syndromes are a subtype of noninsulin be far from clear. Most human diseases have a tion between prevention of a disease and ‘pre dependent diabetes mellitus type II (NIDDM) with complex aetiology, involving genetic, and among vention’ of a (genetically)diseased human being, autosomal dominant inheritance. Our in silico others physiological, psychological and life style while on the other hand rating human genetics modeling techniques (GEMS Launcher, Geno

GfH ÖGH SGMG Tagungsband Abstracts GfH ÖGH SGMG Tagungsband factors on their causal pathway. Genetic expla highly responsible for the prevention of geneti matix) allowed us to identify conserved tran nations of a disease are highly context depend cally caused diseases. The judgement of the dif scription factor binding sites in the genes for the ent (state of knowledge, background of non ferent scenarios by the participants was done MODYassociated transcription factors HNF1al genetic factors, study population). They have without a clear concept of ‘prevention. pha (MODY 3), HNF1beta (MODY5), HNF4alpha been discussed as a reflection of increased (MODY 1), PDX1 (MODY 4), NeuroD1/BETA2 technological capacities. (MODY 6) and HNF3beta (pancreatic network Even with a complete knowledge of aetiology a key regulator) and to reconstruct part of the the P201 05 classification of diseases in terms of cause MODYassociated regulatory network. A model would not be satisfactory. Diseases have to be derived from promoters of insulin genes from hu described in terms of aetiology as well as in the Attitudes of Medical Personnel to Ethical man, mouse, rat and apes proved to be too spe pathology and pathophysiology which results. D.D. Farhud 1,2,; N. Nikzat 2 ; H. Sadighi 2 cific for identifying sufficient coregulated genes. Six groups of students of medicine differing in 1. WHO Ethical Committee, Geneva, Solving the problem of overly specific models, their level of clinical and theoretical experience Switzerland/2. Dep. of Human Genetics, we developed a general method for detecting (1st, 2nd, 3rd and 6th year of medical education, School of Public Health, Terhran Univ. of tissueassociated genes. Our tool „LILIAN“ (lit 300 from the regular curriculum, more than 200 Medical Sciences, TehranIran erature linkage analysis) was used to identify po from the reformed curriculum) were interviewed A WHO meeting of experts in genetics was con tentially pancreasassociated transcription fac by a structured questionnaire focussing on their vened in Geneva, Switzerland, in december tors. These data were used to refocus the fol concept of disease in general and genetic dis 1997, to review proposed international guide lowing in silico promoter modeling to the most ease in particular, the latter understood as a lines on ethical issues in medical genetics and important transcription factors and associated causally oriented classification on the one hand genetic services. The medical application of ge genes. Thereby, we were able to apply more and as an assignment to the specialty human netic knowledge must be carried out with due generalized promoter models for our database genetics within the realm of medicine on the oth regard to the general principles of medical searches. The new models were more tissue er. ethics; Autonomy, Beneficience, Nonmalfi than gene specific and allowed us to identify Answers of study participants reflected the pres cience, Proportionality and Justice. several more potentially coregulated genes. ent prominence of human genetics for the diag Human genetics with its advances, specially in Thus we could enlarge the existing molecular nosis of monogenetic diseases and diseases the last two decades, has created new ethical, regulatory and metabolic network related to with a well established mode of inheritance; they legal and penal issues. MODY. highly overestimated the importance of human This study was carried out with the purpose of genetics in the diagnosis of multifactorial dis obtaining points of view of 756 physicians, nurs eases and in therapy and prevention of disease es, midwives and common people, with regard P202 02 with a known genetic aetiology. A condition has to ethical principles in medical genetics. The a significant genetic or environmental compo study was performed by questionary method nent only relative to a range of genes or a range and the descriptive and analytical assesment Interaktives Patienteninformationssystem of environments. In the teaching of medicine was accomplished on the results. The results für Humangenetische Institute und Praxen more emphasis has to be placed on the devel showed that the application of these ethical Joerg Schroeder opment of a realistic estimation of human genet principles in health care, have been observed Institut für Humangenetik Biozentrum am ics for medical practice in general. with different views. Hubland Universität Würzburg According to these views, the principles were Alle bekannten Patientenverwaltungsprogramme categorized based on priority of acceptance: sind ausschließlich auf einzelne Patienten 1 Proportionality; 2 Beneficience; 3 Justice; zugeschnitten und erfüllen die im Fach Human P201 04 4 Autonomy and 5 Nonmaleficience. genetik erforderliche Verwaltung von Familien Analytical assesments suggest that a number of daten nicht adäquat. Daher wurde am Institut für Human genetics and the meaning of personal, cultural and social variables were tak Humangenetik an der Universität Würzburg eine ‘prevention’ unprecise concept causes en in to account and the relationship between Datenbank unter der Microsoft Access Entwick confusion in contemplation and application negative views and the variables were assessed. lungsumgebung entworfen. Die schon beste Preß, Björn, Pelz, J. Autonomy, with age and marriage statues, have hende Datenbankversion unter Dbase wurde hi Reformstudiengang Medizin, Humboldt shown statistical significance. More adults than erbei als Grundlage genommen, die vorhande Universität zu Berlin, Germany middle aged, and more singles than married in nen Daten aller Patienten übernommen und um Within the specialities of medicine human genet dividuals, responded negatively. For benefi die hinzugekommenen technischen und logistis ics is one of the youngest. The introduction of its cience, the variable of profession has shown chen Anforderungen erweitert. Seit Anfang 2000 concepts and its molecular methods has an in statistical significance. Nonmalficience and pro wird das gesamte Patientenaufkommen des In fluence on medical thinking and the use of basic portionality showed to be not statistically signif stituts für Humangenetik mit der neuen Anwen medical concepts. Genetic research is highly in icant by the variables. Residents of Tehran pro dung in Access verwaltet. Die Erfassung der terdisciplinary and collaborative which makes duced more negative responses to justice than Daten erfolgt in drei Schritten. Die Eingabe der necessary the communication between geneti those living in other cities. Familie mit allen relevanten Personen, die jew

70 medgen 14 (2002) eiligen Patientenstammdaten mit dem Unter P202 04 sues or stages. The high rate of misannotation suchungsfortschritt und die Ein und Ausgabe further decreases the reliability of this data. der Untersuchungsaufträge für die einzelnen La Based on our GeneNest gene index database Bioinformatics Support within the DHGP bore. Alle drei Bereiche sind auf eigens dafür we analyze the tissue distribution of ESTs relat Community entworfene Eingabemasken verteilt, sodass dem ed to the same gene. We assign a tissue to Mechthild Falkenhahn, KarlHeinz Glatting, Benutzer eine klare Struktur vorliegt, die einfach every EST semiautomatically. The statistical Agnes HotzWagenblatt, Barbara Pardon, Coral zu bedienen ist und alle erforderlichen Eingaben evaluation of this tissue information leads to the del Val and Sándor Suhai auf einen Blick erkennen lässt. Durch die grafis detection of potential tissuespecifically ex Deutsches Krebsforschungszentrum che Benutzerführung werden fehlende Informa pressed genes. (DKFZ), Abt. Mol. Biophysik, Im tionen sofort erkannt und Eingabefehler weitest Neuenheimer Feld 280, D69120 Heidelberg, gehend vermieden. Da Access netzwerkfähig ist, Germany kann von jedem an ein Netzwerk angeschlosse Bioinformatics is a major requirement for most P202 06 nen Computer die Daten Abfrage und Eingabe areas of biological and biomedical research. The erfolgen. Ein besonderer Vorteil ist der kon growing complexity of data and bioinformatics tinuierlich abrufbare Untersuchungsstand jedes Exploring Sequence Space from Genome to tools requires welltrained scientists with knowl einzelnen Patienten. Damit ist ein effizientes Proteome edge in the underlying biological concepts and Monitoring auch bei hohen Patientenzahlen Antje Krause, Stefan A. Haas, Thomas Meinel, the different computing methods. Often it is dif gewährleistet. Abfragen des Datenbestandes Martin Vingron ficult and time consuming for the researcher to können für alle Anforderungen mit geringem MPI for Molecular Genetics, Computational select the correct combination of applications Aufwand erstellt werden. Hierzu gehörten unter Molecular Biology, Ihnestr. 73, D14195 and databases. Therefore, in order to bridge the anderem Auftragslisten für die jeweiligen Berlin considerable gap between largescale datacol Laboruntersuchungen wie auch Wiedervorlage The SYSTERS protein sequence cluster set pro lection and its interpretation we have developed listen, z.B. für schwangere Patientinnen um eine vides an automatically generated classification a task system that allows the integration of ap möglichst schnelle Diagnostik zu gewährleisten. of protein sequences into disjoint protein fami plications and methods to create tailormade Diese Listen können für Laborbesprechungen ly and superfamily clusters. For the remaining analysis. This can be used in high throughput

unter Angabe der gewünschten Kriterien auf ein layer in the hierarchy, the domain level, we rely Abstracts GfH ÖGH SGMG Tagungsband analysis e.g. genome annotation, cDNA map er Bildschirmmaske mittels weniger Mausklicks on one of the currently established domain data ping, gene and protein domain function assign erstellten in Relation zueinander zu setzen und bases, namely the Pfam collection of protein do ment, gene structure prediction, protein second miteinander zu vergleichen. Das System kann mains. The underlying data set consists of all se ary structure prediction, array oligos design. mit relativ geringem Lernaufwand selbst erweit quences from the SWISSPROT, TrEMBL, and In collaboration with a DHGP research group we ert und gewartet werden. Die Abhängigkeit zu PIR databases as well as currently available pro developed a task which allows the semiauto teuren Wartungsverträgen kann entfallen. Die Er tein sets of fully sequenced organisms. The matical analysis of EST sequences supporting fassung der erbrachten Leistungen ist durch das SYSTERS cluster set is available at http://sys the search of functional annotations of novel Programm weitgehend automatisiert. ters.molgen.mpg.de/. It is possible to enter the transcript sequences. In this task, repeats, vec hierarchy at any layer, e.g., by searching for a tor parts and low quality sequences are masked keyword, choosing a certain taxonomic compo within the input DNA. A further analysis is than sition, or selecting a domain. For each family P202 03 performed into successive steps, firstly by the cluster a consensus sequence is generated. All identification of already known transcripts pres consensus sequences together build a database ent within human mRNA and genomic DNA ref Statistical Service Unit at the Genome searchable by BLAST. SYSTERS family clusters erence database. Secondly tools for the cluster Mapping Center of the MDC are linked to our GeneNest database based on ing of `anonymous` ESTs and for further data Rohde, Klaus (1), Rueschendorf, F. (2), Gunia, precomputed sequence similarities. GeneNest is base searches on the protein level are com W. (1), Lucius, H. (1), Nuernberg, G.(2), Thiele, a database of gene indices derived from ESTs prised. Finally the relevant results are presented V. (1), Wienker, T.F. (2) and mRNAs.The sequences of a GeneNest clus in a descriptive summary. (1) MaxDelbrueckCentrum, Berlin, ter are assembled and consensus sequences Our comprehensive services include hotline, Germany (2), Institute for Medical build a searchable sequence database. The regular news, introductory courses and ad Biometrics, Informatics and Epidemiology, GeneNest cluster set is available at http:// vanced workshops on specialized topics: se University of Bonn, Germany genenest.molgen.mpg.de/. The visualization pro quence comparison, multiple alignment, data It is already a fairly long time that the dissection vides further information about the sequences, base searching, profiles and HMMs, promoter of complex genetic traits is high on the agenda the represented genes and open reading frames. and gene prediction, phylogeny and protein pre in the analysis of complex genetic traits, with its The integration of SYSTERS and GeneNest into diction. still unresolved practical and theoretical prob one framework permits an overall exploration of Support and free registration: phone 06221/42 lems as genetic heterogeneity and multifactori the whole sequence space covering protein, 2349 or email genome@dkfzheidelberg.de, al etiology. An undissputed prerequisite to over mRNA and EST sequences, as well as genomic Home page: http://genome.dkfzheidelberg.de come these problems lies in recruitment and DNA. genotyping of larger samples (using microsatel lites and more and more SNP). The high number of data, the use of different P202 05 P202 07 types of genetic markers, the rapidly upcoming new genotyping methods and lab equipment as Statistical analysis of tissueannotation well as the need for compatibility to other gene MouseExpress: Repository and data mining derived from EST libraries mapping centers, requires a high amount of flex of expression patterns in mouse mutants Marc Bruning, Stefan Haas, Martin Vingron ibility in the management of the laboratory infor from the Munich ENU Mutagenesis Screen MPI for Molecular Genetics, Ihnestr. 73, mation management system in order to check, Sabine Tornow2, Michael Mader2, Matthias 14195 Berlin control and monitor the different work flows Seltman1, Marion Horsch1; Alexei Drobyshev1, The huge number of expressed sequence tags through the GMC. Marcus Frohme3, Tamara Korica3, Martin (ESTs) in public databases represents a major In the analysis of the data the focus has shifted Vingron4, Jörg Hoheisel3, Martin Hrabé de part of the entire set of human genes. Gene in from conventional family based linkage studies Angelis1, Johannes Beckers1, Werner Mewes2 dices are clusters of ESTs related to the same to population based studies with their special 1GSF, Institute of Experimental Genetics, gene. They are used to reconstruct the se problems in quality checking, HardyWeinberg Munich, Germany; 2GSF, Institute for quence of transcripts by aligning ESTs. Addition Equilibrium, Linkage Disequilibrium, haplotype Bioinformatics, Munich, Germany; 3DKFZ, al information about the expression of those estimations and subsequent association studies Division of Functional Genomics, transcripts is covered in the sequence annota of haplotypes to qualitative and quantitative Heidelberg, Germany; 4MPI for Molecular tions. traits, as well as identification of haplotype (link Genetics, Dept. of Computational Molecular Most ESTs are annotated with information about age disequilibrium) blocks along the genome. Biology, Berlin, Germany tissue, tumor, or developmental stage from We developed a repository to store, preprocess which they are derived. However, the annotation and statistically evaluate expression data of the of these features is often inconsistent, thus com mouse mutants from the ENU Mutagenesis plicating the assignment of ESTs to specific tis Screen in highthroughput.

medgen 14 (2002) 71 The Oraclebased storage conforms recent stan hybridizations at different times following a stim combining this specific software program with dards (e.g. MAGE) and organizes data in distinct ulus or during a developmental time course is an experimental approach. annotation spaces. Modularized webinterfaces more complicated, and often will attempt to for upload, reannotation, quality control, and arrange genes into groups, or clusters, of genes online statistics are located at http://mips. displaying similarities in their expression pat P202 11 gsf.de/proj/mouseExpress/ME.html. The privacy terns. of datasets is controlled via an authentication Many methods are available to analyze microar module. Preprocessing routines include flag ray timecourse data including hierarchical clus Architecture for Information Retrieval and handling, various normalizations, and genomic tering, kmeans clustering, and selforganizing Analysis of Integrated Molecular Data as well as functional annotation. maps, and there is as yet no clearcut way of T. Töpel, R. Hofestädt With the help of advanced clustering and biclus determining which clustering method is best OttovonGuerickeUniversity, Department tering tools as well as sophisticated statistical suited for a given data set. of Technical and Business Information tests we are looking for significant differentially We have developed a novel adaptation of Koho Systems, Germany; University of Bielefeld, expressed genes of the mutant relative to the nen’s selforganizing map (SOM) algorithm us Technical Faculty, Bioinformatics and wild type lines and are investigating the coher ing a mutual information content (MIC) metric for Medical Informatics, Germany ent change of the expression patterns due to the the analysis of microarray data, and implement Due to the increasing number of internet data mutation. For a functional analysis of the expres ed it as a Java application. bases and information systems with information sion data we partition the data in existing anno Shannon’s definition of the entropy of a discrete concerning mechanisms and substances in tation schemes and are using supervised and random variable can be thought of as a measure volved in inborn errors of metabolism, we creat semisupervised approaches to score experi of the uncertainty of a random variable; MIC, in ed the prototyp of an integrated information sys ments (different mutations) and significant co— turn, refers to the amount of information shared tem of molecular and clinical data. It integrates expression. by two random variables. Our implementation of various databases representing the current the SOM algorithm involves transforming the knowledge of enzymes and their catalyzed bio realvalued gene expression data into discrete chemical reactions (BRENDA), biochemical path bins, and using a mutual information (coherence) ways (KEGG) and transcription factors (Trans P202 08

GfH ÖGH SGMG Tagungsband Abstracts GfH ÖGH SGMG Tagungsband metric to determine the winning SOM nodes fac), using the idea of federated database sys (most implementations of the SOM algorithm tems. Additionally, medical knowledge (OMIM) Identification of proteins containing a use a Euclidean or Pearson metric for this step). and clinical data (RAMEDIS) is integrated, too. MethylCpGbinding domain (MBD) Preliminary testing indicates that our algorithm Based on the integrated data and prepared in a TimChristoph Roloff, H.Hilger Ropers, Ulrike „learns“ to classify genes into related clusters well structured and uniform way, casebased A. Nuber such that the average coherence between indi search algorithms are performed to retrieve new MaxPlanckIstitute of Molecular Genetics, vidual genes and the node of the SOM cluster information to detect inborn errors of metabo Dept. of Human Molecular Genetics increases to over 90% of the maximum possible lism. As a second step methods for integration MeCP2 is a transcriptional repressor binding to value. Algorithmic details and results of cluster of tools, simulating gene controlled networks will single CpGs by a domain called MBD (Methyl ing on several publicly available microarray data enhance this system and lead to an integrated CpGbinding Domain). Mutations in this gene sets will be presented. server for gene controlled biochemical networks. lead to the Rett syndrome phenotype, a kind of This work is part of the project „Modelling of mental retardation with an onset at the age of 6 gene regulatory networks for linking genotype 18 month after an apparently normal period of phenotype information“ and is supported by the P202 10 growth and development. Rett syndrome clini German Ministry of Education and Research in cally presents with regression, loss of speech the German Human Genome Project. and purposeful hand use. Other features include A systematic search for unique sequences autism, ataxia, and stereotypic hand movements on the Xchromosome such as handwashing and wringing. Tauchen, Anika (1), Ehling, D. (1)(2), Choudhuri, P202 12 The disease is almost exclusively found in fe J.V. (3), Weidner, J. (2), SchmittJohn, T. (1)(2), males and with an incidence of 1 in 10000 Wirth, J. (1)(2). 15000, it is one of the most common causes of (1) Developmental Biology and Molecular Modeling the Architecture of Signal mental retardation in females. Pathology, University of Bielefeld, Germany Transduction Networks: Insulin Receptor Although MeCP2 is ubiquitously expressed, mu (2) Praenadia GmbH, Muenster, Germany (3) Signaling Pathways tations in this gene lead to an exclusive neuronal Faculty of Technology, University of Potapov, Anatolij P. and Wingender, Edgar phenotype. This might be explained by compen Bielefeld, Germany GBF German Research Centre for sation of its repressional activity in other tissues By using the REPuter program we have per Biotechnology, Mascheroder Weg 1, D by different proteins also containing a MBD. So formed a systematic search for large DNA se 38124 Braunschweig, Germany far four more proteins with a MBD (MBD1, quences without repetitive structures of different Modeling the integral activity of large regulatory MBD2, MBD3, MBD4) were known and studied regions on the Xchromosome. The program al networks is a great challenge of modern molec intensively. lows an efficient and complete search of various ular biology and bioinformatics. We focus on We performed blast and database searches and types of repeats as well as the selection of modeling the architecture of signal transduction could identify several new MBD proteins in hu unique sequences within a chromosome band or networks which represents causal connections man, mouse, rat, Xenopus laevis, C. elegans of the whole chromosome. We have investigat between network elements and is a logical genomes as well as in different plants. Further ed the genomic organization of the DMD gene skeleton of each regulatory system. To model studies on these proteins might lead to the un (Duchenne muscular dystrophy) on Xp21, the such an architecture, we have applied a special derstanding of the neuronspecific phenotype of XIST (X inactivespecific transcript) locus on formalism that treats processes of different com MeCP2mutants as well as a better insight into Xq13 and the FMR1 (fragile X mental retarda plexity as multiple conditional events and is the cause of the Rett syndrome. tion) gene on Xq27. These sequences are pub based on propositional logic. By using this for licly available in the database. As REPuter allows malism, regulatory pathways could be expressed the setting of different parameters for the com in an algebraic form suitable for storage and putation, we were able to visualize all repeated computerassisted analysis. While representing P202 09 substrings in the regions of interest according to the status of pathways, we evaluate the robust our own criteria. This led us to the observation ness of the corresponding constructions. The ro Mutual Information Mapping of „blank“ regions, i.e., without any repeated bustness is the measure of the logical redundan Peter Robinson substring. To determine the usefulness of these cy of network compositions and reflects the abil Institut für Medizinische Genetik, unique sequences, we have designed primers ity of a system to continue functioning in face of Universitätsklinikum Charité, Berlin and amplified several DNA fragments within substantial changes (mutational damage) of its Microarray technologies make it possible to these subsequences and used them in Fluores components. An important advantage of our monitor the expression of many thousands of cence in situ hybridization (FISH) experiments. modeling approach is its simplicity. Our models genes simultaneously. Analysis of experiments In addition, we have selected BAC and PAC are basically static and do not need to simulate in which a test sample is compared against a clones of these specific regions and established step by step the corresponding processes. The control generally involves ranking genes accord them as high quality FISH probes. Here we pres approach proposed enables a quantitative eval ing to their degree of up or downregulation. ent a promising strategy for the identification of uation of the architectural role of individual ele The analysis of experiments involving a series of unique sequences on the Xchromosome by ments in signal transduction pathways. This

72 medgen 14 (2002) might be particularly useful for modeling effects P202 14 Main general information, e.g.. diagnosis, eth of pathologically relevant mutations in distinct nic origin, author, abstract etc. components of signal transduction and tran Symptoms clinical symptoms in a hierarchical Cooperative Research Project: Network scription systems, as well as for protein target manner. Modeling for GenotypePhenotype finding. The approach has been applied to in Laboratory laboratory data, ordered alphabet Correlations sulin receptor signaling pathways as they are ically with SIunits and qualitative ranking Döhr, Stefanie (1), Potapov, Anatolij (2), Seidl, provided by the TRANSPATH database on signal which enables extended graphical presen Klaus (2), Ehrentreich, Frank (3), Hoffmann, transduction. Particular attention has been paid tation. Oliver (3), Töpel, Thoralf (4), Mischke, Ulrike (5), to pathways regulating the activity of transcrip Pictures possibility to include figures as in ar Scheible, Dagmar (5), Wingender, Edgar (2, tion factors. The pathways are displayed accord ticles. coordin.) ing to their hierarchical organization, e.g., as a Molecular Genetics molecular genetics using (1) GSF, Oberschleißheim, (2) GBF, combination of many paths. Each path is repre the terminology of S.E. Antonarakis. Braunschweig, (3) University of Cologne, sented as a sequence of steps, each of which, Therapy/Development general therapy and Köln, (4) University of Magdeburg, in its turn, can be decomposed into ‘reactions’ growth parameters Magdeburg, (5) Kreiskliniken Reutlingen, and ‘molecules’ of the TRANSPATH database. Diet/Drugs special dietary and drug therapy University of Tübingen, Reutlingen Within the last 2 and a half years, the project at A statistic function is integrated, to show the tempted to achieve a bioinformatics description content of RAMEDIS. P202 13 and characterization of regulatory and metabol So far (Mai 2002), 375 cases have been submit ic networks and their clinically relevant aberra ted from different centres. First studies (e.g. con tions. These descriptions of the molecular caus Construction of a Relational Database cerning extended newborn metabolic screening) es of certain diseases were to be connected Management System (RDBMS) for the have been performed. with proper representations of clinical phenotype analysis of RPEenriched expressed To use RAMEDIS in prospective studies and to descriptions as they were contributed by physi sequence tags improve the performance of the data acquisition, cians. In the course of the project, a number of Faisal M. Moula(1), Faisal M. Rahman(1), a form based input tool will be developed. databases on transcriptional regulation (TRANS

Andrea Gehrig(1), Claudia Keilhauer(2), In summary, an anonymous electronic documen Abstracts GfH ÖGH SGMG Tagungsband FAC, PathoDB), signal transduction (TRANS Bernhard H. F. Weber(1) tation system for patients with genetic metabol PATH), enzymatic reaction (BRENDA) and meta (1)Institute of Human Genetics, Biocenter, ic diseases has been developed which is acces bolic diseases (METAGENE, MDDB, RAMEDIS) Am Hubland, D97074 Würzburg; sible on the internet. were successfully adapted and integrated at dif (2)University Eye Clinic, Würzburg ferent levels, i. e. within the individual subpro Agerelated macular degeneration (AMD) is the jects, bilaterally, and in a generic system. A new P202 16 most common cause of legal blindness in indus system has been developed to bridge phenotype trialized countries and predominantly affects the and genotype information (PheGe). Enhanced elderly population over 75 years of age. Al DNA sequencebased virtual human data acquisition has been initiated by newly de though the primary events in AMD pathogenesis chromosomes veloped text mining routines. To enable compu are not well understood, it is thought that the Haas, Oskar A. Schmidt, A., Strehl, A. tation with regulatory and metabolic networks, deterioration of the highly metabolic retinal pig CCRI, St. Anna Children’s Hosp., Vienna, methods for formal descriptions of these net ment epithelium (RPE) is involved in the degen Austria ([email protected]) works were developed. Boolean functions have erative processes. The human genome can be analyzed morpho been applied to represent the architecture of Our aim is to identify and characterize genes logically with microscopic and chemically with regulatory networks and to estimate their robust specifically or abundantly expressed in the RPE. molecular genetic means. At present, it is not ness. The tools developed have been combined Towards this end, a bovine cDNA library en possible to directly compare, correlate, ex with existing ones to analyze genomic se riched for RPE was constructed inhouse using change, or jointly analyze data that are generat quences for regulatory signals and to apply this a PCRbased suppression subtractive hybridiza ed the one or the other way within a single plat information to the identification of new candi tion technique, which normalizes sequence form. One prerequisite for such an integrated date genes that may be relevant in a certain dis abundance and achieves high enrichment for approach is the transformation of the descriptive ease area. The first comprehensive application differentially expressed genes. The individual se cytogenetic and molecular cytogenetic data into example was MODY (Maturity Onset Diabetes of quences of 1002 cDNAs were analyzed and a sequence format. The availability of the human the Young) where we could identify new target querried against sequences deposited in the sequence (http://genome.ucsc.edu/) enabled genes. GenBank and dbEST databases. A RDBMS was now for the first time a direct evaluation of the designed and developed to organize the storage relationship between the sequence and the and retrieval of the sequencing and blast search chromosomal banding pattern. Our reconstruc ing information. RDBMS leads to more efficient P202 15 tion of the human chromosomes with electron use of queries, forms, and reports, increases the ic means revealed an astonishing concordance reliability of the extracted information, and can between these two banding patterns. This ob RAMEDIS Collecting clinical and molecular expand as database information requirements servation proofs that the „largescale“ chromo genetic data of inborn metabolic diseases grow and change. Our results demonstrate that somal banding solely reflects the DNA sequence via the internet 64% (642) of the clones are derived from known and that it is hardly modified by epigenetic fac Mischke, Ulrike (1), Töpel, Thoralf (2), Scheible, genes, while 36% (360) of clones showed no tors. However, virtual chromosomes will provide Dagmar (1), Hofestädt, Ralf (3), Trefz, Friedrich matches in dbEST. In silico expression analysis also a unique basis for the joint processing of (1) of the known RPE genes revealed that 7.5% are cytogenetic, FISH, and molecular genetic data (1) Klinikum am Steinenberg, Childrens RPEspecific, 4.2% are retinaspecific, and within a single DNA sequencebased framework. hospital, University of Tübingen, Reutlingen 4.2% are expressed in both retina and brain. Ex The accurate, scaleindependent, and highly re An internet portal (www.ramedis.de) for the elec trapolation of these data to the EST clusters with gionspecific banding pattern can be easily tronic publication of patients with rare metabol no significant homolgy to known genes suggests adapted to that of natural chromosomes irre ic diseases has been established. The aim of the that the number of novel RPE/Retinaspecific spective of their state of condensation. In com patient database is to provide information about genes to be around 1015. Characterization of bination with the possibility to electronically re the clinical, biochemical and molecular genetic the full length human orthologs of the bovine construct any chromosome rearrangement with data of patients with rare metabolic diseases. In RPE/Retinaspecific transcripts have been initi a hitherto unknown molecular precision, this addition, follow up of treated patients is possi ated. In a second phase, we plan to determine may provide the basis for novel cytogenetic pat ble. sequence variants in the RPEspecific genes in tern recognition systems. Moreover, we are also The home page describes the intention and a large, wellcharacterized AMD patient group by able to map and display the chromosomal posi function of RAMEDIS, which runs with version of highthrough put exonscanning technology. tion of any sequence or set of sequences, in 11th Jan 2002 on an internet server at the Insti cluding the distribution of the currently available tute of Technical and Business Information Sys approximately 14.000 genes. The superimposi tems at the OvGUniversity of Magdeburg. tion of such a graphic interface onto molecular The data can be accessed in a read only modus genetic databases will thus enable the visualiza (analysis tool) or with the input and editing tool tion of molecular events in a chromosomal fash respectively. The following data are document ion. In addition to virtual gene mapping, such a ed: tool will enable the display of micro arrayde

medgen 14 (2002) 73 rived DNA and gene expression profiles. It also corresponding to the linkage peak. We also P206 05 remains possible to integrate more fuzzydefined found two other regions showing negative asso ined with conventional cytogenetics and with a ciation with psoriasis, at D19S917 on 19p13.1 (p Periodic catatonia: confirmation of linkage variety of FISH methods. = 0.0034), and at D19S425 (p = 0.0005), com to chromosome 15 and further evidence for patible with the hypothesis of protective loci. We genetic heterogeneity explored these two novel regions in more details Ekici, Arif B. (1, 5), Seelow, Dominik (1, 3), using novel in silico identified microsatellite P206 01 Rüschendorf, Franz (1, 4), Stöber, Gerald (2), markers at an average distance of 100 kb and and Reis, André (1, 5) identified further associated microsatellites in (1) MaxDelbrückCenter, Gene Mapping The candidate genes TGFA and MTHFR in a both regions. Analysis of SNPs from these re Center, Berlin, (2) Department of Psychiatry German CL/P population fined regions will eventually allow identification and Psychotherapy, University of Würzburg, Pletsch, N. (1), Selle, A. (2), Großmann, U. (2), of the underlying susceptibility alleles. (3) Fachbereich Biologie, Chemie, Hochban, W. (2), Austermann K.H. (2), Koch In addition, we investigated SNPs in candidate Pharmazie, Free University Berlin, (4) IMBIE, M. (1) genes at two further psoriasis susceptibility loci. University of Bonn, (5) Institute of Human (1) PhilippsUniversität Marburg, While we could confirm a strong association in Genetics, University of Erlangen Bahnhofstrasse 7a, 35037 Marburg, our cohort for SNPs within the HCR gene on 6p We earlier reported on significant evidence for Germany; (2) ? (p = 0.000058), we failed to confirm association linkage on chromosome 15q15 in periodic cata Nonsyndromic cleft lip with and without cleft to the recently presented candidate SLC12A8 on tonia, a subphenotype of the schizophrenic palate (CL/P) are complex disorders involving 3q. psychoses. The disorder is characterized by several genes and environmental factors. Asso qualitative hyperkinetic and akinetic psychomo ciation studies of CL/P have implicated a num tor disturbances through acute psychotic ber of candidate genes but have proved difficult P206 04 episodes, and debilitating symptoms in the long to replicate. Here, we report the findings of two term with psychomotor weakness, grimacing fa candidate genes, MTHFR and TGFA, in a Ger cial movements, and apathy. Here, we confirm man CL/P population. Developmental genes as candidates in mapping of a major gene locus on chromosome

GfH ÖGH SGMG Tagungsband Abstracts GfH ÖGH SGMG Tagungsband Maternal folic acid supplementation during ear cases with neural tube defects (NTD) 15q15 in a second genome scan in a new set of ly pregnancy has been suggested to play a role Felder B, Schultealbert A H, Röper B, Koch MC four multiplex families. Nonparametric multi in the prevention of oral clefts. Several study Institut für Allgemeine Humangenetik, point linkage analyses identified a broad region groups tested the 677C®T polymorphism of the PhilippsUniversität Marburg with a maximum peak of Zall = 3.91 (p =0.0063) gene for 5,10methylenetetrahydrofolate reduc Little is known about the identity of genetic fac and Zlr = 3.04 at D15S1234 (p = 0.0013), satis tase (MTHFR) in their CL/P population. Some in tors involved in the complex aetiology of neural fying conventional criteria for confirmed linkage. vestigators reported about an association of the tube defects (NTD). Reasonable human NTD Parametric affectedonly analyses gave maxi 677TT genotype to CL/P, whereas others could candidate genes are the human homologues of mum HLOD score of 1.65 (D15S1234) with an not support these findings. In order to contribute developmental genes in mice for which a func estimated 47% of families linked. Analysis of in to this discussion we examined this MTHFR tional role in neural tube closure can be as dividual families showed that one large family polymorhism in a control population (n=233) and sumed. In mice, Twist, Bmp4 and its specific in showed linkage while two other could be clear in CL/P patients (n=72). The casecontrol study hibitor Noggin are expressed in tissues adjacent ly excluded, confirming genetic heterogeneity. does not reveal a significant difference in C and to the developing neural tube. Twist knockout No other locus reached suggestive levels of sig Tallele frequencies or in genotype distributions mice fail to close the neural tube in the cephal nificance. Haplotype analysis on chromosome (CochranArmitage trend tests P=0.13). There ic region, whereas in Noggin knockout mice a 15 in this and previously linked families placed fore it is questionable if this polymorphism plays failure of neural tube closure can be observed in the susceptibility region to a 11 cM interval be an important role in the susceptibility for CL/P in cranial and in lumbar regions. We therefore test tween marker D15S1042 and D15S659. Period the German population. ed the three genes as candidates that might be ic catatonia is the first subphenotype of the In contrast to this, we were able to demonstrate involved in the aetiology of human NTD. The schizophrenic psychoses with confirmed linkage in agreement with casecontrol studies from oth coding sequences were screened for mutations despite existence of considerable genetic het er countries, that there is a statistically signifi in 200 NTD patients (anencephaly, encephalo erogeneity. cant association between the D4 bp polymor cele, spina bifida aperta) using single strand phism of the transforming growth factor a (TGFA) conformational analysis (SSCA). In TWIST, no and CL/P (Cochran Armitage trend tests P = sequence alterations could be detected. In the 0.03). Therefore, the results of our casecontrol single exon of NOG we found a point mutation P206 06 study support TGFA as a modifier gene in CL/P. in one spina bifida aperta patient (SBA) that Biological support for a role of TGFA arises from leads to a change of the predicted amino acid Evaluation of two voltagegated potassium its presence at high levels in the epithelial tissue sequence at an evolutionaryly conserved posi channel subunits in idiopathic forms of of the medial edge of the palatal shelves at the tion (G92E). In BMP4 four different missense epilepsy time of shelf fusion in mice. mutations in four unrelated SBA patients were K. Haug1, K. Hallmann1, B. Rau1, J. found: three mutations were located in the Rebstock2, T. Sander3, A. Heils1,2 propeptide region of the protein (S91C, T225A, 1University Department of Human Genetics, R226W) and one in the carboxyterminal domain P206 03 Wilhelmstr. 31, 53111 Bonn, Germany. characteristic for TGFß2family (S367T). All se 2University Clinic of Epileptology, Sigmund quence alterations change amino acids which FreudStr. 25, 53105 Bonn, Germany. Further Fine Mapping Within The Psoriasis are highly conserved among mammals and thus 3Department of Neurology, University Susceptibility Region On Chromosome 19 In may influence the stability or function of the pro Hospital Charité, Virchow Clinic, German Trios tein. In all cases (BMP4 and NOG) the mutation Augustenburger Platz 1, 13353 Berlin, U. Hüffmeier (1), C. Windemuth (3), P. Hensen was inherited from one parent. Germany (2,4), T. Wienker (3), H. Traupe (4), A. Reis (1,2) The present study displays new genetic variants Idiopathic generalized epilepsy (IGE) affects (1) Institute of Human Genetics, University in patients with neural tube defects which, to about 1% of the population worldwide. Common of ErlangenNürnberg, Germany, (2) Max gether with other factors, may have contributed subtypes of IGE including childhood absence DelbrückCenter, Berlin, Gemany, (3) IMBIE, to the patients’ phenotypes in these individual epilepsy (CAE), juvenile absence epilepsy (JAE), University of Bonn, Germany, (4) cases. Further investigations are necessary to juvenile myoclonic epilepsy (JME), and epilepsy Department of Dermatology, University of support these observations. with grand mal seizures on awakening (EGMA) Münster, Germany are inherited as genetically complex traits and Previously we performed a genome wide linkage defined by recurrent seizures presenting with scan for psoriasis and identified a further sus characteristic clinical and electroencephalo ceptibility locus at the pericentromeric region of graphic features in the absence of any de chromosome 19. In a followup study using an tectable brain lesion. Results of a recent genome independent cohort of 210 FalkRubinsteintrios, wide search (Sander et al., 2000) provided sug we scanned this 40 cM region for association gestive evidence for the existence of two novel using a dense set of 45 microsatellites. We ob IGE susceptibility loci: on chromosome 14q23 tained a positive association for the markers (ZNPL = 3.28 at D14S63; P = 0.000566) and on D19S922 (p = 0.008) and D19S916 (p = 0.016)

74 medgen 14 (2002) chromosome 2q36 (ZNPL = 2.98 at D2S1371; P P206 08 presumably complex. Current concepts assume = 0.000535). In these regions we identified two interactions of one or more environmental trig potential candidate genes both coding for sub gers with an inherited susceptibility of the pa RNomics: identification and function of units of voltagegated potassium channels: tient. According to this hypothesis are genes in imprinted, brainspecific small non KCNE4 (alternate symbol: MIRP3) on chromo volved in innate immunity candidate susceptibil messenger RNA genes in the PraderWilli some 2q36 which belongs to the Iskrelated ity genes in sarcoidosis. Toll like receptors (TLR) Syndrome region subfamily of voltagegated potassium channels are a main component of innate immunity. TLR Hüttenhofer, Alexander (1), Vitali, P. (1), and KCNH5 (alternate symbol: Eag2) on chro comprise a group of transmembrane molecules Skryabin, B. (1), Buiting, K. (2), Horsthemke, B. mosome 14q23 which belongs to the subfamily with an extracellular leucinerich repeat domain (2) and Brosius, J. (1) H of eagrelated potassium channels. We sys and a cytoplasmic interleukin1 receptor like re (1) Institut für Experimentelle Pathologie, tematically searched for mutations in 32 IGE pa gion. Different TLR respond selectively to spe ZMBE, Münster, Germany. (2) Institut für tients. We identified two common polyorphisms cific bacterial components, and lead to en Humangenetik, Universitätsklinikum, Essen, (KCNH5: G2233A; KCNE4: G435T) which were hanced activity of proinflammatory cytokines, Germany further analyzed in a familybased association that are involved in the typical sarcoid immune In our quest to identify novel nonmessenger study including 120 parentchildtrios. However, reaction. So far, eight members of the TLR gene RNAs (snmRNAs) in model organisms (an ap TDT analyses did not reveal a significant devia family have been characterised in more detail. proach for which we coined the term RNomics), tion between alleles transmitted and nontrans Two of them, TLR4 and TLR9 reside at chromo we have identified three brainspecific snmR mitted, and we thus conclude that genetic vari somal regions on 9q and 3p that exhibited mod NAs, designated as HBII13, HBII52 and HBII ation in both genes does not contribute to an in erate nonparametric linkage peaks (p < 0.05) at 85 from a mouse brain cDNA library. We have creased IGE risk. a previous genomewide linkage scan in sar isolated the human orthologues of the three sn coidosis families. We have studied the TLR gene mRNAs and mapped them between the SNRPN loci using close microsatellite markers and intra and UBE3A genes on chromosome 15q11q13. genic polymorphisms in an extended panel of P206 07 Thereby, we have sequenced 180 kb encom patients and families suffering from sarcoidosis. passing the HBII52 locus. The region contain ing the three snmRNA genes has been implicat

Genetic background of the TNFa mRNA Abstracts GfH ÖGH SGMG Tagungsband ed in the etiology of the PraderWilli syndrome expression in patients with coronary (PWS), a neurogenetic disease resulting from a P206 10 atherosclerosis deficiency of paternal gene expression. Two of U. Schagdarsurengin1, S. Schulz1, T. Süß1, U. the snmRNA genes, HBII52 and HBII85, are MüllerWerdan2, K. Werdan2, C. Gläser1 No evidence for involvement of the encoded in tandemly repeated arrays of 47 or 27 1Inst. of Human Genetics, 2Dep. of Internal promoter polymorphism 866 G/A of the units, respectively. Interestingly, these RNAs Med., Univ. Halle, Germany UCP2 gene in earlyonset obesity in humans were absent from a PWS patient cortex and from The TNFa is an important cytokine in the com Nadine Schaeuble (1), Geller, F. (2), Siegfried, a PWS mouse model, demonstrating their pater plex signalling pathway involved in the develop W. (3), Goldschmidt, H. (4), Remschmidt, H. (1), nal imprinting status and pointing to their poten ment of atherosclerosis. Methods: We studied Hinney, A. (1), Hebebrand, J. (1) tial role in the etiology of PWS. By a bioinformat the interaction of the TNFa mRNA expression (1) Department of Child and Adolescent ical approach, we were able to identify three ad (competitive RTPCR), a promoter polymorphism Psychiatry, University of Marburg, Germany, ditional snmRNA genes within the same locus, (PM) of TNFa (G238A; SSCP) and their effect on (2) Institute of Medical Biometry and designated as HBII436, HBII438A and HBII coronary atherosclerosis. In this study 256 pa Epidemiology, PhilippsUniversity of 438B, which are also subject to imprinting and tients with angiographically confirmed diagnosis Marburg, Germany, (3) Obesity Treatment are predominately expressed in the brain. were involved: 128 patients with severe coronary Centre Insula, Berchtesgaden, Germany, (4) Due to conserved sequence and structure mo atherosclerosis (CAD; 81 males, average age: Spessart Klinik, Bad Orb, Germany tifs, the six snmRNAs can be assigned to the 50.3y) and 128 patients without any coronary Recently, association between obesity and the class of small nucleolar RNAs (snoRNAs). This symptoms (WAS; 83 males, average age: 50.4y) Gallele of the 866 G/A polymorphism in the class of RNA molecules has been shown to tar as controls. Results: As proposed by recent promoter region of uncoupling protein2 gene get ribosomal RNAs by a short antisense ele studies the mRNA expression of TNFa was (UCP2) was reported (Esterbauer et al., Nat. ment located within snoRNAs. By this mecha shown to be elevated in the CAD compared to Genet. 28: 17883, 2001). Both allele frequencies nism, ribosomal RNAs are targeted for modifica the WAS group (7.4 vs. 6.7ag/cell; n.s.). In order and genotype distributions for this polymor tion at the site of complementarity to snoRNAs. to prove the role of the genetic background we phism differed between obese individuals and All six brainspecific snoRNAs from the PWS re examined the dependence of the mRNA expres neverobese controls. We attempted to confirm gion lack complementarity to ribosomal RNAs. sion on the G238A PM: Whereas the mRNA ex this finding. Genotyping was performed by poly In one case, the HBII52 snoRNA, we could pression in WAS patients was only slightly in merase chain reaction with subsequent restric show a potential interaction with a brainspecif creased in dependence on the genotype tion fragment length polymorphism analysis ic mRNA, the serotonin receptor mRNA 5HT2C (AG+AA: 7.4 vs. GG: 6.6ag/cell) the mutation (PCRRFLP). We analyzed transmission disequi at a site where the mRNA is regulated by alter carriers among the CAD patients showed signif librium of the (wild type) Gallele for 200 ex native splicing and editing. This implies (a) nov icantly higher expression than wildtype carriers tremely obese children and adolescents from 93 el function(s) of this class of snmRNAs, namely (AG+AA: 12.6 vs. GG: 6.9ag/cell, p<0.012). An concordant sib pair families using the pedigree the regulation of gene expression. analysis of the genotype distribution of the G transmission disequilibrium test. Additionally, us 238A PM revealed no significant differences in ing a onesided asymptotic Pearson’s chi the frequency of AG+AAcarriers (CAD: 0.09 vs. squaretest, we tested, if the Gallele occurs WAS: 0.12). However the evaluation of the im P206 09 more frequently in 277 extremely obese children portance of this PM for the early development of and adolescents (including the 93 index patients severe CAD described by an early age of onset of the concordant sib pairs) than in 188 never Study of TollLike Receptor Genes in (<45y) revealed significant changes in genotype obese controls. The onesided asymptotic Sarcoidosis. distribution. The CAD patients with an early age Cochran Armitage trend test was used to deter Schürmann, Manfred (1), Valentonyte, R. (2), of onset were significantly more often mutation mine differences in genotype frequencies be Albrecht, M. (1,2), Hampe, J. (2), Müller carriers than CAD patients who exhibit coronary tween extremely obese and healthy underweight Quernheim, J. (3), Schwinger, E. (1), Schreiber, symptoms at higher age (<45y vs. >45y: 0.18 vs. individuals. The PDT analysis revealed no evi S. (2) 0.02; p<0.012). This result underlines the impor dence for transmission disequilibrium in obesi (1) Institute of Human Genetics, Medical tance of the mutant genotypes AG+AA of the ty. Allele and genotype frequencies did not dif University Luebeck, Luebeck/Germany, (2) TNFa G238A PM for having a more pronounced fer between the extremely obese and never Department of General Internal Medicine, mRNA expression and also for the development obese subjects. In conclusion, we cannot con ChristianAlbrechtsUniverstität, of coronary symptoms at younger age (<45y). firm the results of Esterbauer et al. in our young Kiel/Germany, (3) Department of Conclusions: Investigating the role of the mes sample. Pneumology, Medical University Hospital sage expression of TNFa in the development of Freiburg, Freiburg/Germany coronary atherosclerosis the importance of the Sarcoidosis is a multifactorial disease of gran genetic background always be taking into con ulomatous inflammation, affecting a variety of sideration. organs, primarily the lung and lymph system. The aetiology of sarcoidosis is unknown and

medgen 14 (2002) 75 P206 11 entries in databases leading to conflicting gene P206 14 models. mRNA 1 is composed of two exons, whereas mRNA 2 was deduced from genomic Polymorphism of genes involved in bone DIFFERENTIAL EXPRESSION OF ADIPOSE DNA and corresponds to exon 2 of mRNA 1 but metabolism and association with TISSUE GENES IN OBESITYASSOCIATED is elongated upstream. Comparison of allele and osteoporosis in Poland HYPERTENSION IN MAN USING cDNA genotype frequencies of the silent SNP rs133073 Kalak, Robert (1,2), HorstSikorska, W. (3), MICROARRAYS C/T located in exon 1 of MCHR1 indicated asso BaszkoBlaszyk, D. (3), Ziemnicka, K. (3), Stefan Engeli1, Sergei Baranov2, Aleksey ciation of the Callele with obesity. Transmission Nürnberg, P. (4), Toliat M.R. (4), Slomski R. Soldatov2, Hans Lehrach2, Arya M. Sharma1 disequilibrium tests (TDT) confirmed a preferen (1,2) 1Department of Nephrology and tial transmission of the Callele. As no function (1) Department of Biochemistry and Hypertension, FranzVolhardClinic at the al significance of rs133073 is evident, we se Biotechnology, Agricultural University, MaxDelbrück Center, University Clinic quenced MCHR1 and 4.6 kb of its upstream re Poznan, Poland, (2) Institute of Human Charité, and 2MaxPlanckInstitute for gion in nine obese children and adolescents ho Genetics, Polish Academy of Sciences, Molecular Genetics, Berlin, Germany mozygous for C at rs133073 and who had con Poznan, Poland, (3) Endocrinology Clinics, Obesity is the principal risk factor for the devel tributed to the positive TDT, as well as in ten Medical School, Poznan, Poland, (4) Gene opment of hypertension, but the molecular ba obese children and adolescents homozygous for Mapping Center, MaxDelbrück Center for sis for this relationship remains poorly under T. These two genotype based groups should be Molecular Medicine, Berl stood. Obesity is characterized by an enormous most divergent for functionally relevant SNPs. Osteoporosis is a common disease character growth of adipose tissue, and despite an in We identified 18 SNPs 12 of which contribute to ized by decrease in bone mineral density (BMD) creasing body of knowledge on the endocrine two ancestral haplotypes. Since the majority of and microarchitectural deterioration of the bone and paracrine role of adipocytes, the pathophys the SNPs is located upstream of the transcribed structure leading to higher susceptibility to frac iological role of adipose tissue in obesityrelat region, promoter fragments representing the two tures. Development of osteoporosis is multifac ed hypertension has not been investigated in haplotypes are functionally tested in luciferase torial process in which environmental and genet depth. In this study, we examine differential reg reporter gene assays. ic factors play an important role. Recent studies ulation of adiposetissue genes in lean and *authors contributed equally have indicated that the majority (up to 80%) of obese normotensive and hypertensive subjects

GfH ÖGH SGMG Tagungsband Abstracts GfH ÖGH SGMG Tagungsband Supported by Bundesministerium fuer Bildung the variability in bone mass and density is genet in acrosssectional study and follow those genes und Forschung ically determined. Molecular genetic basis of os during dietary weight reduction and salt restric teoporosis remains difficult to define because tion, as these interventions lower blood pressure the bone mass, a major determinant of osteo in obese subjects. porosis fracture risk, is quantitative trait, influ P206 13 All volunteers are characterized by 24 hour am enced by interaction between many genes and bulatory blood pressure and anthropometric environmental factors. Several candidate genes measurements. Blood and 24 hour urine sam Sequence diversity of KIAA0027/MLC1: are are investigated in search for association of ples were taken for further hormonal character megalencephalic leukoencephalopathy and polymorphisms or mutations with this disease. ization. Total RNA was isolated from adipose tis schizophrenia allelic disorders? We studied polymorphisms of osteoprotegerin sue obtained by subcutaneous needle biopsy. Gerald Stöber, Claudia Rubie, Peter Lichtner, gene (OPG), transforming growth factor b1 gene The complete RZPD2 Human UNIGENE Set Jutta Gärtner, Markus Siekiera, Graziella Uziel, (TGFb1), vitamin D receptor gene (VDR), estro (about 76,000 clones) was spotted on 6 nylon Bernd Kohlmann, Alfried Kohlschütter, Thomas gen receptor 1 gene (ER1), collagen type 1a1 membranes. A subset of 29 adipose tissue RNA Meitinger, Thomas Bettecken gene (COLIA1) and analysis of 4 polymorphisms samples was hybridized to the complete library Department of Psychiatry, University of of ANKH gene (craniometaphyseal dysplasia to obtain a list of differentially expressed clones. Würzburg; Institute of Human Genetics, gene) was performed. Statistical analysis of all This subset of clones (about 3,000) is currently Technical University of Munic; Department single polymorphisms and analysis of interaction spotted on a smaller membrane that will be used of Pediatrics, Heinrich Heine University of between these polymorphisms were performed. to obtain gene expression data of the whole Düsseldorf; Istituto Nazionale Neurologico, For majority of polymorphisms no statistically study population (crosssectional and interven Carlo Besta Milan; Department of significant influence on osteoporosis was ob tions). Paediatrics, University of Hamburg served. Only analysis of OPG 9G/C polymor 90 adipose tissue biopsies have been performed The aim of the study is to validate the etiologi phism showed statistically significant results. in the crosssectional study, and the weight re cal role of KIAA0027/MLC1 in childhoodonset Genotypes with allele C were overrepresented duction study is currently underway. Results on megalencephalic leukoencephalopathy with sub in patients with severe osteoporosis as com candidate gene expression (adiponectin, 11ß cortical cysts (MLC) and in schizophrenia, par pared with Polish population group (p<0.02). A HSD1 and 2) help to explain findings on low ticularly the catatonic subtype, which were re tendency for influence on development of osteo adiponectin plasma levels and increased local ported to be allelic diseases. Among a series of porosis was observed for allele G in ANKH exon formation of cortisol in obese subjects. Exam five patients with MLC, four mutant alleles were 5 51A/G polymorphism, allele A in ANKH exon ples for preliminary results from the use of cDNA detected: one case of compound heterozygosi 5 52A/G polymorphism, allele C in TGFb1 arrays suggest increased expression of the fork ty for a splice site mutation and a 6 basepair in 29T/C polymorphism and allele f in VDR FokI head transcription factor gene FOXP1 in hyper frame deletion, one patient with a homozygous polymorphism. tensive subjects irrespective of weight, and of frameshifting insertiondeletion, and a further the gene encoding fructose1,6bisphosphate case heterozygous for a A157E substitution. A aldolase B (ALDOB) in obese subjects irrespec systematic mutation screening in 140 index cas tive of blood pressure. P206 12 es with schizophrenia revealed 13 different sin gle nucleotide polymorphisms (SNPs): one SNP in the 5’UTR, seven SNPs in intronic regions, Involvement of a Melaninconcentrating two synonymous codon variants (T52, Y199), P206 15 hormone receptor 1 allele in human obesity and three coding variants. Two of them, C171F Reichwald K1*, Wermter AK2*, Geller F2, and N218K, were observed in controls at a sig Huse K1, Platzer C3, Platzer M1, Gudermann Differentially expressed genes in rat during nificant frequency, and the L309M variant that T4, Hess C4, Hinney A2, Hebebrand J2 development of cardiac hypertrophy was previously supposed to be the causative 1 Institute of Molecular Biotechnology, Hahn, Torsten; Schlicker, M.; Hansmann, I.; factor for chromosome 22qtel linkedperiodic Dept. of Genome Analysis, Jena; 2 Clinical Schlote, D. catatonia was found nonsegregating in a fur Research Group, Dept. of Child and Institute of Human Genetics, University of ther multiplex pedigree. Furthermore, a 33 bp in Adolescent Psychiatry, Philipps University, Halle (Saale), Germany sertion/deletion polymorphism in the 3’end of Marburg;3 Institute of Anatomy II, FSU, To identify candidate genes contributing to the exon 11 of MLC1 was found at equal frequency Jena; 4 Institute of Pharmacology a initiation or progression of cardiac hypertrophy, among schizophrenic patients and controls. In The orexigenic neuropeptide melaninconcen an adaptive response to chronic increased work summary, our study provides further evidence trating hormone (MCH) is involved in regulation load, we have screened differential gene expres for allelic heterogeneity in megalencephalic of food intake and energy balance. MCH is the sion in the heart of spontaneously hypertensive leukoencephalopathy, exclude MLC1 as a sus endogenous ligand of the receptor MCHR1 rats (SHR) at different stages of their develop ceptibility locus for schizophrenia, and thereby which through coupling to multiple G proteins ment in comparison to the wild type WistarKy rule out that MLC and schizophrenia are allelic mediates diverse intracellular signalling path oto rats (4, 12, 26 weeks pp; male). We estab disorders. ways. Presently, there are two MCHR1 mRNA lished a subtractive hybridization system based

76 medgen 14 (2002) on suppression PCR and cDNA selection using P206 17 subjects for both the patient and control group mRNA from these stages resulting in identifica by 06/2002, and to include a matched popula tion of 145 different cDNA clones. Screening of tionbased control group from the KORA survey Expression of HERVK on protein and RNA these clones revealed 56 cDNAs as candidates 2000 (Augsburg). So far, 530 patients with breast level both in rheumatic and in normal for differentially expressed genes. Northern blot cancer and 550 populationbased controls have synovial tissue analysis of 42 cDNAs identified 16 upregulated been recruited between 08/2000 and 0/4 2002. Ehlhardt, Sandra, Seifert, M., Zang, K.D., and 4 downregulated genes in cardiac tissue of All patients and controls are interviewed with a Mehraein, Y. SHR. In silico analysis of these cDNA sequences standard questionnaire to obtain information on Institute of Human Genetics, Saarland identified several known genes which are being potential risk factors, i.e. reproductive factors University, Homburg, Germany discussed already in the context with cardiomy and hormone intake, nutrition and life style as Rheumatoid arthritis is the most common au opathy (gsarcoglycan) or cardiac hypertrophy well as others. DNA obtained from blood mono toimmunologic disease in man. Although clini (acylcoA dehydrogenase) and in addition 8 so nuclear cells is currently analyzed at polymor cally well defined, primary pathologic mecha far unknown genes. Validating our results of phic key players potentially involved in the meta nisms are still unclear. Besides genetic, infec northern blot analysis we established quantita bolic pathways of the assessed exposures. tious, and unknown endogenous factors also the tion of gene expression through real time PCR Genotyping includes fragment analysis, Taq involvement of human endogenous retroviruses for our candidate genes. Using these method we man®, MALDITOF, and Lightcycler technolo (HERVs) has been discussed as increased levels could indicate an significant upregulation of g gies. Protein expression of selected prognostic of antiHERV antibodies have been found in sarcoglycane and an other so far unknown gene and predictive markers in breast tissues is ana rheumatic patients in several studies. While most in cardiac tissue of SHR rats during develop lyzed by immunohistochemistry. All data are cur HERVs are defective, a nearly intact member of ment to cardiac hypertrophy. Mapping of these rently collected in a MS ACCESS database for the HERVK(HML2) family was identified on novel genes with respect to rat chromosome future statistical analysis including conditional chromosome 7. Moreover mosaictrisomy 7 is segments harboring known QTLs for cardiac hy logistic regression analysis. Our study aims to frequently found in synovial tissue from rheuma pertrophy and analysis of their expression pat estimate women’s relative risks to develop toid arthritis, osteoarthritis but rarely in normal tern might be relevant to understand processes breast cancer under both genetic and environ controls. To identify a trisomy 7 related expres leading to cardiac hypertrophy in rat and man. mental influences. sion of HERVK(HML2) sequences on mRNA and protein level we performed RTPCRanaly Abstracts GfH ÖGH SGMG Tagungsband ses for HERVKcORFRNA and immunofluores P206 16 cence labeling of the cORF protein in 28 primary P206 19 synovial cell cultures from patients with autoim mune rheumatic disease (RA), osteoarthritis Association analysis of CD86 gene FAMILYBASED ASSOCIATION STUDIES OF (OA), and normal synovia. In all cases the per polymorphisms with infantile atopic TRACE AMINE RECEPTOR GENES ON centage of trisomy 7 was investigated by FISH. dermatitis and atopy 6Q23.3 IN BIPOLAR AFFECTIVE DISORDER A significant correlation between trisomy 7 rate Lee, YoungAe (1,2), Kehrt, R. (1), Tarani, L. (3), Schumacher, Johannes (1), Diaconou C (2), and HERVK expression, however, was not ob Gustafsson, D. (4), Oranje, A. P. (5), Abou Jamra R (1), Kaneva R (1), Hua Y (1), vious. Unexpectedly cORFProtein was detect Rüschendorf, F. (1,6), Nürnberg, P. (2), Reis, A. Schulze TG (3), Müller DJ (4), Gross M (5), ed in all normal synovial cultures. The same, but (2,7), Wahn, U. (1) Ohlraun S (6), Golla A (7), Rietschel M (6), on a reduced level, holds true for the majority of (1) Pediatrics, HumboldtUniversity Berlin, Cichon S (8), Nöthen MM (8), Propping P (1) RA and OA cases. By RTPCR likewise expres (2) MaxDelbrückCenter Berlin, (3) 1)Inst. Human Genet.,Univ. Bonn,Germany; sion of HERVK specific mRNA could be proven Pediatrics, Rome University, Italy, (4) 2)Inst. Virology,Univ. Bucharest,Romania; in all cases of normal synovia and OA, and in Pediatrics, Örebro Hospital, Sweden, (5) 3)Dept. Psychiatry,Univ. Chicago,USA; 90% of RA cases. Additionally several different Dermatology, Rotterdam University, The 4)Mental State Hospital Bonn; 5)Dept. HERVK specific transcripts were identified pre Netherlands, (6) Inst. of Medical Biometry, Psychiatry,Univ. Bonn; 6)Central Institute of senting a variant expression pattern between Bonn University, (7) Human Genetics, Mental Health,Mannheim,Germany;7)IMBIE, normal and rheumatic synovial cells. By se Erlangen University Univ.Bonn; 8)Dept. Medical quence analysis the expressed sequences could Atopic dermatitis (AD) is a chronic inflammatory Genet.,Univ.Antwerp,Belgium be assigned to several chromosomal loci. The skin disease that is characterized by an intense There is evidence suggesting a role of trace reduction of HERVK expression and the change ly itchy rash with a typical morphology and dis amines (TA) in the etiology of bipolar affective of the expression pattern might be a specific tribution. Infantile atopic dermatitis is common disorders (BPAD). A functional deficiency of TA genetic marker in rheumatic disease. ly the first clinical manifestation of allergic dis has been proposed as a potential etiological fac ease and the majority of affected children devel tor in depression, increased levels of TA were op asthma or allergic rhinitis later in life. We have found to be associated with manic phases of recently identified a susceptibility locus for P206 18 BPAD. The genes for two trace amine receptors, atopic dermatitis and atopy on chromosome TA1 and TA4, are both located on chromosome 3q21. CD86 is a type I membrane protein of the 6q23.2. Interestingly, this particular chromoso The GENICA Study: Assessment of Breast immunoglobin superfamily that has been map mal region has shown evidence for linkage to Cancer Risk Factors ped to this region. CD86 interacts with CD28 to BPAD in a genomewide screen (Cichon et al., Hamann Ute (1), Brauch H (2), Brüning T (3), provide costimulatory signals for T cell activa 2001). This prompted us to search for genetic Fischer HP (4), Harth V (3,5), Justenhoven C tion and has been implicated in the activation of variants in the TA1 and TA4 genes and test (2), Ko Y(5), Pesch B (3) for the GENICA Th2 cells which are thought to play a pivotal role them for association with BPAD. Systematic se Network in mediating allergic inflammation. Furthermore, quencing of the TA1 and TA4 genes in 96 con (1)Deutsches Krebsforschungszentrum, overexpression of CD86 on B cells of AD pa trol individuals identified two common SNPs in Heidelberg; (2)Dr. Margarete FischerBosch tients has been demonstrated. CD86 therefore the TA4 receptor gene (914A/G and 993A/G) Institut für Klinische Pharmakologie, represents an interesting positional and func and one in the TA1 receptor gene (1212A/G). Stuttgart, (3)Berufsgenossenschaftliches tional candidate gene for atopic dermatitis and TDT analysis of the three SNPs was performed Forschungsinstitut für Arbeitsmedizin, atopy. in 118 parentoffspring triads that were partially Bochum, (4)Institut für Pathologie und To test whether CD86 may be a susceptibility derived from the genome screen sample. No as (5)Medizinische UniversitätsPoliklinik, gene for infantile atopic dermatitis or atopy, we sociation between the SNP in the TA1 receptor Universität Bonn have screened the coding region of the CD86 gene and BPAD was found. However, we ob There are about 46,000 incident breast cancer gene and have identified five polymorphisms in served a preferential transmission of the 914G cases each year in Germany most of which are Exons 1, 2, 4, 5, and 8. The coding variants of allele of the TA4 receptor gene (p=0.014) and a unexplained with respect to genetic origin. We the CD86 gene were genotyped using minise nonsignificant trend of the 993G allele (p=0.06). foster the hypothesis that most breast cancers quencing protocols. We used the study group Replication is clearly necessary to support a may develop due to complex genegene and/or that showed strong linkage to chromosome possible role of the TA4 receptor gene in BPAD. geneenvironment interactions. To clarify this 3q21 consisting of 199 families with at least two point we are currently conducting a population children affected with atopic dermatitis. Trans based casecontrol study in an area of one mil mission disequilibrium revealed no association lion inhabitants in Germany (city of Bonn and of the mutant allele of coding variants with either neighboring districts of Siegburg and Eu atopic dermatitis or allergic sensitization. skirchen). The ultimate goal is to include 600

medgen 14 (2002) 77 P206 20 independent. In a first step, eight microsatellite six common SNPs in the CETP gene with HDL markers were tested for LD that are located in and LDLcholesterol was studied in 286 individ the highest linked region (about 5 Mb). Individ uals. A cladistic approach, allowing for grouping FAMILYBASED ASSOCIATION STUDIES OF ual markers and twomarker haplotypes were of haplotypes based on evolution, was applied. ALPHAADRENERGICRECEPTOR GENES IN tested for LD. TDT analysis revealed two regions Results: Two major groups of haplotypes could REGIONS SHOWING LINKAGE TO BIPOLAR of interest: One is characterized by preferential be distinguished in a cladogram: The putative AFFECTIVE DISORDER transmission of two particular twomarker hap „wild type“ haplotype and those haplotypes re Abou Jamra R (1), Schumacher J (1), Kaneva R lotypes (markers AC, p=0.008; markers BC, sulting from mutation and recombination events (1), Otte A (1), Golla A (2), Müller DJ (3), p=0.035). A second region is located more cen in the population („variant haplotypes“). The Schulze TG (4), Fangerau H (5), Ohlraun S (6), tromeric. A particular allele of maker D (p=0.01) haplotype frequencies did not much differ be Rietschel M (6), Cichon S (7), Propping P (1), and the twomarker haplotype DE (p=0.001) tween male and female persons. The variant Nöthen MM (7) were preferentially transmitted. To follow up this haplotype group was associated with higher 1)Inst. Human Genet.,Univ. Bonn,Germany; finding, we have isolated new polymorphic STR HDL and lower LDL levels compared to the wild 2)IMBIE,Univ. Bonn; 3)Mental State Hospital markers in the region which are currently being types. This effect was observed only in males, Bonn; 4)Dept. Psychiatry,Univ. Chicago,USA; analyzed by TDT. not in females. The association was more pro 5)Dept. Psychiatry,Univ. Bonn; 6)Central nounced in individuals with lower triglyceride Institute of Mental Health, Mannheim, concentration compared to those with higher Germany; 7)Dept. Medical Genet.,Univ. triglycerides. Discussion: These data support the Antwerpen (UIA),Antwerp,Belgium P206 22 following hypothesis: 1) When CETP is saturat Several lines of evidence suggest an involve ed by its substrates (triglycerides and choles ment of the noradrenergic neurotransmitter sys Testing of GRID2 and TACR3 as candidate terol esters), the plasma concentration of LDL tem in the pathogenesis of bipolar affective dis genes for migraine with aura and HDL cholesterol is not responsive to the ef order (BPAD). Drugs that enhance the activity of Todt, Unda (1), Heinze, A. (2), Zumbroich, V. (2), fect of genetic variants within the CETP gene. 2) norepinephrine can precipiatate maniclike Ramirez, A. (1), Stiller, A. (1), Goebel, I. (1), The results suggest an interaction between gen episodes in some individulals while precipitous Propping, P. (1), Göbel, H. (2), Kubisch, C. (1) der, genetic variation within the CETP gene and withdrawal of these drugs can be associated

GfH ÖGH SGMG Tagungsband Abstracts GfH ÖGH SGMG Tagungsband (1) Institut für Humangenetik, the plasma cholesterol concentration. 3) SNP with depressive symptoms. Three genes for al Universitätsklinikum Bonn, Germany; (2) derived haplotypes appear to be better markers phaadrenergicreceptors (ADRA) are located in Schmerzklinik Kiel und Neurologische Klinik for functional alleles, since the effects of SNPs chromosomal regions that showed evidence for der Universität Kiel, Germany were much less significant. linkage in a genome wide screen for linkage to Migraine with aura (MA) is a common neurolog BPAD in 75 families (Cichon et al., 2001): The ical disease with complex inheritance. Very re ADRA2A gene maps to chromosome 10q25, the cently a first genome wide scan for MA has been ADRA2C gene is located on chromosome 4p16, P206 24 published describing a highly significant locus and the ADRA1Agene maps to 8p21. We on chromosome 4q24. We therefore analyzed analysed a Cys492Arg variant in exon 2 of the this susceptibility region and were able to iden GenomeWide Scan for Childhood and ADRA1A gene, a 1291G/C variant in the 5´UTR tify two genes which proposed function made Adolescent Obesity Genes in German of the ADRA2A gene, and STR marker adra2c1 them attractive candidates for MA. GRID2 en Families in the 5´UTR of the ADRA2Cgene. 117 parent codes the delta2 subunit of the ionotropic glu Saar, Kathrin (1), Geller F (2)., Rüschendorf F offspring triads with BPAD partially derived from tamate receptor and is predominantly expressed (3)., Reis A. (4), Friedel S. (2), Schäuble N. (2), our genome screen sample (n=53) were geno in cerebellar Purkinje cells, where it is supposed Nürnberg P. (3), Siegfried W. (2), Goldschmidt typed and analysed using the Transmission Dis to be involved in the regulation of neuronal ex H.P. (2), Schäfer H. (2), Ziegler A. (2), equilibrium Test (TDT). No significant differences citability. TACR3 codes for a tachykinin receptor, Remschmidt H. (2), Hinney A. (2), Hebebrand J. in transmissions were observed for any of the which is thought to be involved in pain percep (2) three ADRAgenes. Thus, our data do not sup tion in the central nervous system. We elucidat (1) MaxPlanckInstitute for molecular port the hypothesis of an involvement of the ed the genomic structures of both genes and es genetics, Berlin; (2) Dept. of Child and three studied adrenergicreceptor gene variants tablished a mutation screening strategy to am Adolescent Psychiatry, PhilippsUniversity, in the development of BPAD. plify all coding exons and adjacent splice sites Marburg; (3) MaxDelbrück Centre for from genomic DNA. GRID2 is composed of 17 molecular medicine, Gene Mapping Centre, exons and TACR3 consists of 5 exons. Direct se Berlin; (4) Institut for Human Genetics, P206 21 quencing of PCR products from index patients FriedrichAlexanderUniversität, Erlangen, from 24 multiplex families with MA revealed one Germany common silent polymorphism and one intronic Objective: Up to now, several genome scans LINKAGE DISEQUILIBRIUM ANALYSIS IN A polymorphism in GRID2, whereas no alteration have been performed for adult obesity. Single SUSCEPTIBILITY REGION FOR BIPOLAR was found in TACR3. We are currently perform formal genetic studies suggest a higher heritabil AFFECTIVE DISORDER ON 8Q24 ing association studies in a casecontrol sample ity of body weight in adolescence. In addition, Schumacher, Johannes (1), Kaneva R (1), Van with these two novel SNPs. In parallel, further genes influencing body weight in adulthood Den Bogaert A (2), Schulze TG (3), Müller DJ candidate genes from this susceptibility locus might not be the same as those relevant in child (4), Gross M (5), Fangerau H (5), Becker T (6), are investigated to identify the MA susceptibili hood and adolescence. We therefore performed Richter C (1), Ohlraun S (7), Rietschel M (7), ty gene on chromosome 4q24. a whole genome scan for childhood and adult Propping P (1), Nöthen MM (2), Cichon S (2) obesity genes in german families. 1)Inst. Human Genet.,Univ. Bonn,Germany; Design: The genome scan based on 89 families 2)Dept. Medical Genetics,Univ. Antwerp P206 23 with two or more obese children (sample I). The (UIA),Belgium; 3)Dept. Psychiatry,Univ. mean age of the index patients was 13.63 ± 2.75 Chicago,USA; 4)Mental State Hospital Bonn; Genetic dissection of LDL and HDL years. 369 individuals were initially genotyped 5)Dept. Psychiatry,Univ. Bonn; 6)IMBIE,Univ. cholesterol concentrations: Role of for 437 microsatellite markers. A second sample Bonn; 7)Central Institute of Mental Cholesterol ester transfer protein (CETP) of 76 families was genotyped using microsatel Health,Mannheim,Germany Bauerfeind, Anja (1), Knoblauch, H. (2,3), lite markers that localize to regions for which A genome wide screen for linkage to bipolar af Hafez, K. (1), Luft, F.C. (1,2), and Reich, J.G. (1) maximum likelihood binominal (MLB) LOD fective disorder (BPAD) has suggested a new (1) Max Delbrück Center for Molecular scores upon use of the concordant sibpair ap susceptibility locus on 8q24 (Cichon et al., Medicine, Medical Faculty of the Charité, proach exceeded 0.7 in sample I. 2001). Marker D8S514 gave a twopoint LOD Humboldt University of Berlin, Germany; (2) Results: Regions/loci on chromosomes 1, 2, 4, score of 3.62 and a GENEHUNTERNPL score of Franz Volhard Clinic, HELIOS Kliniken, 8, 9, 10, 11, and 19 fulfilled these criteria in sam 3.56 (p=0.00029). The positive linkage region is Berlin, Germany; (3) Infogen, Köpenickerstr. ple I; MLB LOD scores on chromosomes 8p and large and extends over 30 cM. In an attempt to 48/49, Berlin, Germany 19p exceeded 1.5. In sample II MLB LOD scores narrow down the linked region, we have per Introduction: Cholesteryl ester transfer protein of 0.68 and 0.71 were observed for loci on chro formed linkage disequilibrium (LD) analysis in a (CETP) mediates the exchange of cholesteryl es mosomes 10 and 11. respectively. sample of 119 parentoffspring trios with BPAD ters and triglycerides between apoproteinB Conclusion: In conclusion, whereas our scan did using the Transmission Disequilibrium Test (TDT). (chylomicrons, VLDL, LDL) and apoproteincon not reveal MLB LOD scores ³ 2, we nevertheless Part of the triads (53) were extracted from the taining lipoproteins (HDL) in the plasma. The as consider the fact that several of the previously genome screen families, the other 66 triads were sociation of familyderived haplotypes based on

78 medgen 14 (2002) identified peaks also gave a signal in this scan morbidity and mortality. It belongs to hyperten chromosome aberrations for the occurrence of as promising. sive diseases in pregnancy (HDP), which com CID by means of a selfadministered standard plicate 57% of all life births. Due to familial ized questionnaire. Cytogenetic studies had clustering a strong genetic influence in the aeti been performed to elucidate abnormal results in ology of HDP has been established. In the prenatal diagnosis or to unravel reasons for in P206 25 pathogenesis of HDP diverse mechanisms, like fertility or recurrent miscarriages. Of the 72 abnormal placentation, immunological maladap (45%) questionnaires returned so far, 19 Heritability of left ventricular mass and tation, endothelial dysfunction and disorder of probands (27%) reported to suffer from CID related echocardiographic parameters lipoid metabolism, have been proposed. There (Crohn’s disease/ulcerative colitis: 5; psoriasis: Golla,Astrid(1), Erdmann,J.(3), Kraus,J.(3), fore, the analysis of certain candidate genes, in 5; diabetes/pancreatitis: 4; periodontitis: 5) and Fimmers,R.(1), Hengstenberg,C.(3), Hense,H. patients diagnosed with HDP, is a strategy to 6 probands (8%) described to have relatives af W.(2), Schunkert,H.(3) identify genetic risk factors. In a systematic fected with CID (Crohn’s disease/ulcerative co (1)Institut für Medizinische Biometrie, study with at least 250 families with HDP we litis: 1; psoriasis: 4; asthma: 1). Interestingly, two Informatik and Epidemiologie started among other strategies to screen for mu patients with colitis ulcerosa and psoriasis, re (IMBIE),Universität Bonn, Bonn, Germany, tations in a couple of candidate genes. One at spectively, presented alterations in regions of (2) Institut für Epidemiologie, Universität tractive candidate gene is the HADHA gene en chromosomes 10 and 16 which were suggested Münster, Münster, Germany, (3) Klinik und coding the asubunit of the trifunctional protein by recent linkage studies to contain candidate Poliklinik für Innere Medizin II, Universität (TFP) which is responsible for the longchain 3 loci for CID. For delineation of breakpoints we Regensburg, Regensburg, Germany hydroxy acylCoA dehydrogenase deficiency used a sequential FISH approach applying bac Left ventricular hypertophy (LVH), i.e. a left ven (LCHAD). Recently an association between ma terial artificial chromosome (BAC) clones with tricular mass (LV mass) exceeding clinically de ternal HELLP and LCHAD deficiency has been sequence information available. In this way, we fined threshold levels, is an important risk factor proposed. The majority (69,4%) of LCHADdefi identified clones spanning the breakpoints in for cardiovascular morbidity and mortality. Age, cient children are homozygous carriers of a chromosome 10. Additional analyses are under blood pressure, and body weight are known de G1528C mutation in the HADHAgene. way to determine the breakpoint on chromo terminants of left ventricular mass. Interestingly, Mutational analysis of the HADHA was per some 16 and to investigate whether the break

the variability of blood pressure and body mass formed in 112 mothers with HELLP syndrome 90 point regions indeed contain genes associated Abstracts GfH ÖGH SGMG Tagungsband index has substantial genetic components. In of their children, 22 fathers and 110 women with with CID. addition, as shown previously by our group, uncomplicated pregnancies the women with Supported by BMBF there is growing evidence for independent ge HELLP by SSCP and, if necessary, by direct se netic factors influencing LV mass and hypertro quencing. The G1528C M was tested in all indi phy. Specifically, in comparison matched pop viduals by using PCR and RFLP analysis. P208 01 ulation controls, siblings of LVH patients display None of the women with HELLP showed a M in an increased risk (odds ratio 1.7) for the condi the coding region resulting in an amino acid ex tion as well, even when adjusted for blood pres change. One of 103 patients showed a homozy The Overexpression of the Insl3 in Female sure and body mass index. We now estimated gote G/T M at position 1, which was not de Mice Causes Descent of the Ovaries the strength of the genetic component, i.e. the tectable in 109 controls. Whether this mutation Adham, Ibrahim (1); Steding, Gerd (2); Thamm, heritability of left ventricular mass and related causes a shifting of splice site is now investigat Tarvo (1); Büllesbach, Erika (3); Schwabe, echocardiographic parameters by analysing sib ed. An incorrect original genomic sequence in Christian (3); Paprotta, Ilona (1); Engel, ling correlation and by variance component intron 9, 11 and 15 was identified. In intron 15 Wolfgang (1) analysis (using the program SOLAR). we additionally found a polymorphism (T/G) (1) Institute of Human Genetics, University The study population consisted of 212 individu which showed almost identical allele frequency of Göttingen, D37073 Göttingen, Germany; als with LVH and their siblings. The probands in patients and controls. None of the studied in (2) Department of Embryology, University of were ascertained as a subgroup of individuals dividuals showed a G1528C M. Göttingen, D37075 Göttingen, Germany; participating in the World Health Organization The HADHA gene dos not have any relevant im (3) Department of Biochemistry and MONICA project in the city of Augsburg and two pact on the pathogenesis of HELLP syndrome. Molecular Biology, Medical University of adjacent counties in Southern Germany. We South Carolina Charleston 29425, USA found a substantial heritability of LV mass (h2 = Developmental impairment of the gubernaculum 0.36, p=0.004) and LV end systolic diameter in male mice lacking the insulinlike 3 factor P206 27 (LVESD) (h2 = 0.48, p<0.00001), which repre (Insl3) leads to disruption of testicular descent. sents a measure of cardiac contractility. Wall Results derived from in vitro experiments thickness of the posterior wall and septum Breakpoints in balanced constitutional demonstrated that Insl3 and androgens induce showed lower heritabilities of (h2 = 0.16, p=0.07 chromosome aberrations as indicators of outgrowth of the gubernacular primordia. To pro and h2 = 0.14, p=0.05). Whereas part of the her candidate gene loci associated with vide in vivo evidence that the Insl3 mediated ac itability of LV mass is due to genes influencing complex genetic diseases tivity is responsible for induction of the guber body weight and blood pressure (which deter MetzkeHeidemann, Simone 1*, MartínSubero, naculum development in an androgenindepend mine about 0.20 of the variance), LVESD seems JI 1*, Gesk, S 1, Schürmann, M 2, Caliebe, A 1, ent manner, we generated transgenic male and to be more independent from these parameters. Kautza, M 1, Hinrichs, F 2, French, L 3, female mice that overexpressed Insl3 in the pan The relatively high heritability renders a search Earthrow, M 3, Deloukas, P 3, Grote, W 1, creas during fetal and postnatal life. Functional for genes involved in the determination of left Schwinger, E 2 & Siebert, R 1 * contributed complementation with the transgene restored ventricular mass and contractility (as measured equally to this work the cryptorchid phenotype of the Insl3deficient by LVESD) promising. Knowledge of genes un 1Institut für Humangenetik, male, suggesting that the regulatory secretory derlying these traits would be of great interest, Universitätsklinikum Kiel, Schwanenweg 24, pathway present in islet ßcells efficiently since it could eventually lead to better preven 24105 Kiel 2Institut für Humangenetik der processed the Insl3 to the functional hormone. tive and therapeutical management of cardio Medizinischen Universität Lübeck, All transgenic females displayed bilateral in vascular disease. Ratzeburger Allee 160, 23538 Lübeck 3The guinal hernia. The processus vaginalis devel Wellcome Trust Sanger Institute, Wellcome oped and contained peritoneal contents with in Trust Genome Campus, Hinxton, Cambs testinal loops. The Müllerian derivatives devel CB10 1SA, UK oped into oviduct, uterus and upper vagina, and P206 26 Classically, the first step for positional cloning of Wolffian duct derivatives were absent, indicating genes associated with complex genetic dis the loss of the androgen and antiMüllerian hor Role of HADHA gene encoding the asubunit eases, e.g. chronic inflammatory diseases (CID), monemediated activities in transgenic females. of trifunctional protein as candidate gene involves linkage studies of affected families. A The ovaries descended into a position over the for HELLP syndrome less explored approach is the study of balanced bladder and attached to the abdominal wall via P. NeumaierWagner, I. Ahillen, S. Rudnik constitutional chromosomal aberrations. the well developed cranial suspensory ligament Schöneborn, T. Eggermann, S. Kuse, W. Rath, Prospective cytogenetic studies are hampered (CSL) and the gubernaculum. Administration of K. Zerres by the low incidence of chromosome aberra dihydrotestosteron during the prenatal develop Institute of Human Genetics, University of tions, which (excluding Robertsonian transloca ment suppressed the development of the CSL Technology, Aachen, Germany tions and the inv(9)polymorphism) is only and thereby the descent of the ovaries into the The HELLP syndrome (HELLP) is a multi sys ~0.5%. Therefore, we retrospectively interviewed processus vaginalis. These results suggest a temic disorder with high maternal and perinatal 161 adult carriers of balanced constitutional crucial role of the Insl3mediated activity in the

medgen 14 (2002) 79 induction of gubernaculum development and ex are expressed and if they could play a role in the enzymemodulating activities. Calgizzarin is a clude a potential role of androgen in this stem cell differentiation. member of the S100 familiy of proteins, it has process. Furthermore the transgenic females ex To answer this question we performed in situ hy two calcium binding domains and it is highly hibit reduced fertility, which is due to fetal death bridizations on sections of adult mouse stom conserved in human, mouse, rat and chicken. during midgestation. ach. Using probes for the fork head genes Calgizzarin is thought to be involved in Sertoli Foxa1, Foxa2, Foxa3 and Foxq1 we found ex cellgerm cell interaction, being expressed in pression of all four genes specifically in the Sertoli cell culture and in coculture of Sertoli pepsinogen producing zymogenic cells, but in cellspermatid,spermatocyte fractions. P208 02 different stages of differentiation. While Foxa1 However, a high mRNA level of this gene is and Foxa3 are expressed in preneck and neck maintained until day 15 of postnatal develop Simultaneous statement of two clustered cells (which will become zymogenic cells later), ment in testis (when pachytene spermatocytes ADAM familiy genes, Testase 2a and Testase Foxq1 expression starts in the prezymogenic appear) and then declines sharply, as opposed 2b, during development of mouse germ cells stage. Foxa2 is only expressed in the complete to ovary, where the expression pattern is con BolcunFilas, Ewelina (1)., Rzymski, T. (1), ly differentiated zymogenic cells. We postulate stantly high. In ovary, the calgizzarin expression Grzmil, P. (2)., Nayernia, K. (1), Engel, W. (1) that the different fork head genes are not suffi is maintained highly during all the five stages of (1) Institute of Human Genetics, University cient for the initiation of the differentiation, but the oestrus cycle. of Goettingen, D37073 Goettingen, they play an important role in the differentiation Calgizzarin is expressed in Sertoli cells (testis) Germany of zymogenic cells. and granulosa cells (ovary). In order to establish (2) Department of Genetics and Evolution, the role played by calgizzarin gene in Sertoli cell Jagiellonian University, Krakow, Poland germ cell interaction, we carried out expression Testase 2 (also known as ADAM 25) is a member studies, using prenatal and postnatal testis and P208 04 of ADAM (A Disintegrin And Metalloprotease) ovary tissues in different stages of development. family of proteins.The ADAM family presents the We also generated a knockout model, using best characterized candidates for mediating ga Multifactorial infertility in proacrosin PTK Neo as the vector back bone of the knock mete interaction and membrane fusion in mam deficient mice out construct. The process of obtaining null mu

GfH ÖGH SGMG Tagungsband Abstracts GfH ÖGH SGMG Tagungsband mals. As previously reported and confirmed in Nayernia, Karim, Adham, I.M., Shamsadin, R., tants is ongoing. Further studies are needed in this study this gene show testis specific expres Müller, Ch., Wolf, S., Sancken, U., Engel,W. order to assess the role of calgizzarin gene in sion. During experiments we found two different Institute of Human Genetics, University of this kind of testis cellcell interaction. restriction patterns of subcloned fragments of Göttingen, D37073 Göttingen the gene, suggesting presence of two tran In human, male and female partners contribute scripts. Further experiments and Celera data more or less equally to the infertility problem. In P208 06 base search revealed that these two transcripts about 20 % of infertile couples the concurrence are the products of two separate genes, which of male and female factors is suggested to be show high similarity to the published testase 2. responsible for infertility. Neither any of these Polycystin2 is required for leftright axis Testase 2a and testase 2b as we called them are factors are known nor a model system to prove determination in mice similar to published testase 2 in 87,8% and this assumption is established. We present such Pennekamp, Petra (1), Karcher, C. (2), Fischer, 95,4% respectively, and in 87,4% to each other. a model system in the mouse, in which the lack A. (2), Schweickert, A. (2), Blum, M. (2), Horst, Both genes are located on chromosome 8 (as of acrosin in the male and modifications of the J. (1), Dworniczak, B. (1) well as ADAM 3, ADAM 5, ADAM 9) in close dis zona pellucida in the female result in a signifi (1) Institut für Humangenetik, tance of 24 kb. Genomic structure of testase 2a cant reduction of fertilization rate in vitro. We Universitätsklinikum Münster, Germany, (2) and b differs from that of other ADAM family have generated mice carrying a deletion in the Institute of Toxicology and Genetics, members, like cyritestin or fertilin which contain prolinrich domain (PRR) of the proacrosin gene Forschungszentrum Karlsruhe, Germany around 20 short exons, they are composed of which results in the absence of proacrosin in the The establishment of leftright asymmetry can only two exons. First one is very short, about 85 homozygous PRR / male mouse. Modifications be divided into (1) initial breakage of symmetry, bp while second is over 2.5 kb long. Both genes of the zona pellucida of oocytes after superovu (2) establishment of asymmetrical gene expres demonstrate the same temporal and spatial ex lation were achieved by their treatment with di sion and (3) transfer of this positional informa pression pattern during testicular germ cell de methylsulfoxide (DMSO) and aroclor1254 and tion to the developing organs. Molecular events velopment with onset of expression in haploid by in vitro ageing. It is known that under these involved in the first and third step are hitherto stages. Further functional analysis of these mol conditions a timedependent hardening of the largely unknown, but much progress has been ecules will contribute to a better understanding zona pellucida occurs. The rate of fertilization in made in elucidating the establishment of asym of the molecular mechanisms underlying mam vitro of treated and aged oocytes, respectively, metrical gene expression. Embryological and ge malian spermegg fusion. using spermatozoa from PRR / mice was found netic experiments revealed a conserved asym to be significantly reduced when compared to metric signaling cascade, which during early em that rate reached with wildtype sperm. The rel bryogenesis transmits asymmetric cues from the evance of the acrosin as well as of the zona pel embryonic midline to the lateral plate mesoderm P208 03 lucida for fertilization success is further substan and the forming organs. The central players in tiated by the result that fertilization rate depends this scheme are four asymmetrically expressed Differential expression of fork head genes in on the thickness of the zona pellucida. Our re genes: nodal, Lefty1 and Lefty2, and Pitx2c. In the stomach mucous gland sults demonstrate that the lack of acrosin in mouse monocilia on ventral cells of the node are Janssen, Astrid; Pasche, Bastian; Zoll, Barbara spermatozoa in combination with modifications required upstream of the nodal cascade; in chick Institut für Humangenetik, Universität of the zona pellucida can affect fertility and can and frog gap junctions are essential prior to Göttingen, HeinrichDükerWeg 12, 37073 be a model for unexplained infertility in human node formation. It was hypothesised that differ Göttingen couples in which male and female derived fac ential activity of ion channels results in unidirec The stomach mucosa is a proliferating tissue tors are suggested to be the underlying causes. tional transfer through gap junctions, resulting in with a high potential of regeneration. But the asymmetric gene expression. high number of patients with ulcer or stomac Recently we have generated a Pkd2 knockout cancer shows that the maintenance of the func mouse. Pkd2 encodes polycystin2, an intracel P208 05 tioning tissue can be easily disturbed. There is lular Ca release channel which is expressed not much known about the genetic background abundantly in the endoplasmic reticulum mem of the stomach regeneration and differentiation Sertoli cell germ cell interaction: brane. In humans mutations in PKD2 account for of the mucosal stem cells. Therefore we are in expression studies and functional analysis 15 % of polycystic kidney disease. terested in genes which are involved in the re of the murine calgizzarin gene Surprisingly, Pkd2 knockout mouse embryos generation process. Nica G., Mannan A., Nayernia K., Adham I., displayed leftright positional defects: embryon Fork head genes are transcription factors which Engel W. ic turning, heart looping and placement of ab play important roles in biological processes and Institute of Human Genetics, Göttingen, dominal organs were randomized and the lung are involved in embryonic development, cell cy Germany showed right pulmonary isomerism. Lefty1, cle regulation, regulation of tissuespecific gene The familiy of S100 proteins is a large familiy of Lefty2 and nodal were not expressed in the left expression and cell signalling. Some fork head calcium binding proteins, with various functions lateral plate mesoderm and Pitx2 was absent genes are expressed in the stomach and we in extracellular and intracellular processes, such from heart and lung primordia and bilaterally ex were interested, in which cell types these genes as: apoptosis, cellular growth and differentiation, pressed in body wall, mid and hindgut. The em

80 medgen 14 (2002) bryonic midline, however, was present and nor testis as fertility gene and/or in the placenta as P208 10 mal levels of hnf3beta and shh were expressed. growth gene. Therefore it is expected that spe We suggest that in mouse the ion channel poly ciation genes should affect the function of cog The Ror2 Knockout Mouse as a Model for cystin2 acts upstream of the nodal cascade in nitive abilities and the function of the testis the Development of Spondylocostal leftright axis determination and Pkd2 might be and/or placenta simultaneously. During this Dysostosis in Autosomal Recessive one of the master genes of the body plan. study the expression pattern of Arhgef6, a gua Robinow Syndrome nine nucleotide exchange factor for Rho GTPas Schwabe, Georg (1,2), Trepczik, B. (1), es, was analyzed during murine development. In Mundlos, S. (1,2) the embryo proper and especially the brain of P208 07 (1) Max Planck Institute for Molecular the embryo no specific expression was ob Genetics, Berlin, (2) Institute for Medical served. Beginning from day 7.5 p.c. a highly Genetics, Charité, Berlin, Germany Screening for retinal degeneration specific signal was observed in the placenta. Robinow syndrome (RS) is a shortlimbed mutations in the mouse, Mus musculus The major function of the placenta is to enable dwarfism characterized by abnormal morpho Dalke, Claudia (1), Löster, J. (1), Blanquet, V. the fetus and the mother to interact for the pro genesis of the face and external genitalia, and (2), Fuchs, H. (2), Soewarto, D. (2), Favor, J. (3), motion of fetal growth and viability. To identify vertebral segmentation. Homozygous mutations NeuhäuserKlaus, A. (3), Pretsch, W. (3), the cells expressing Arhgef6 coybridizations in the receptor tyrosine kinase ROR2 are respon Meitinger, T. (3), Hrabé de Angelis, M. (2), were performed with the marker genes, Mest sible for autosomal recessive RS by a loss of Graw, J. (1) (mesodermis specific transcript), for endothelial function mechanism. Ror2/ mice, exhibiting the GSFNational Research Center for cells of the syncytiotrophoblast, 4311 for the typical RS features, serve as a model to analyze Environment and Health, Institutes of (1) spongiotrophoblast, and PL1 (placental lacto the development of the underlying vertebral mal Developmental Genetics, (2) Experimental gen), for the trophoblast giant cells. Arhgef6 is formations in RS. During development the struc Genetics and (3) Human Genetics, D85764 specifically expressed in the decidua basalis and ture of the vertebrate axial skeleton is based on Neuherberg, Germany in the trophoblast giant cells. In the junctional the metameric structure of the somites. The To identify genes involved in retinal degeneration zone, high levels of Arhgef6 mRNA are observed somites are formed in a dynamic segmentation we examined mice of different strains with a high in the spongiotrophoblast and in the trophoblas process that is subject to an oscillatory mecha

throughput electroretinography (ERG) method. tic cells of the labyrinth. Abstracts GfH ÖGH SGMG Tagungsband nism in the presomitic mesoderm (PSM). Using This noninvasive screening method allows a In his pioneering thesis on Xlinked mental retar alcian blue, alizarin red staining Ror2/ mice ex clear separation of affected from unaffected in dation R.G. Lehrke has already pointed out that hibit hemivertebrae and rib fusions and a short dividuals. The animals (between 3 weeks and 3 impaired intrauterine development may be a ened tail. Whole mount in situ hybridizations of months old) were darkadapted for at least 12 contributing side effect of Xlinked mental retar embryos of stages E9.5 and E10.5 with probes hours and anaesthetized. After pupil dilation, in dation genes. for Paraxis, Myf5 and Uncx 4.1 reveal that the dividual mice were fixed on a sledge (Steinbeiß pattern of somite boundaries is disturbed. Hy Transferzentrum, Tübingen, Germany) and ERGs bridization with probes for Notch1, Delta1 and were recorded in a Ganzfeld stimulator (Espion, P208 09 Delta 3 show that the PSM is strongly reduced Cambridge, UK) with two illumination intensities in size. Expression of Pax1, Pax9 and mCer1 (500 or 12,500 cd/m2). suggest that the somite pattern is disturbed in We have established the wild type baseline for Expression of mouse Tbx22 supports its role the presumptive somites or while a newly several mouse strains (101, 129/SvJ, AKR, in palatogenesis and glossogenesis formed somite separates from the PSM. Current BALB/c, C57BL/6, CBA, CD1, DBA, JF1); as a Meunier Dominique (1), Herr A. (1), Müller I. (1), ly the expression of oscillatory genes is under in positive control for hereditary retinal degenera Rump A. (2), Fundele R. (1), Ropers H.H. (1), vestigation, in order to better characterize the tion we used C3H/El mice. During these investi Nuber U.A. (1) role of Ror2 in vertebral development and RS. gations, we identified several animals with low (1) MaxPlanckInstitut für Molekulare ered bwave amplitudes in ERG among the CD1 Genetik, Berlin (2) metaGen outbred mice as well as among the 129/SvJ Pharmaceuticals GmbH, Berlin and the JF1 inbred mice. Histological analysis of We have investigated two Xchromosomal dele P208 11 some of these mice revealed a good correlation tions at Xq21 by InterAlu array CGH and PCR between the lowered electrophysiological re and found TBX22 to be deleted in one of the two The Runx Genes: Roles in Cartilage sponse and the retinal morphology. In 129/SvJ studied cases. TBX22 codes for a transcription Development and Evolutionary Implications we detected the same retroviral insertion asso factor of the Tbox gene family, mutated in pa Stricker, Sigmar (1), Seitz, V.(1), Fundele, R.(1), ciated with the Pde6b/rd1 allele that is known to tients with cleft palate and ankyloglossia (CPX), Vortkamp, A.(1), Vilcinskas, A.(2), Mundlos, be causative for the retinal degeneration in C3H and our male patient had been earlier described S.(1) mice. In contrast, molecular analysis of CD1 and with a cleft lip and palate. Although is had been MPI for Molecular Genetics, Berlin, JF1 mice excluded this retroviral insertion. Ge assumed that TBX22 is a humanspecific gene, Germany (1), Institut für Biochemie und netic confirmation of these variations is in we were successful in identifying mouse Tbx22, Biologie, Universität Potsdam, Germany (2) progress. The method is now being used to which allowed us to study its expression during The three mammalian Runx genes belong to a screen for hereditary retinal degenerations embryogenesis by RTPCR and in situ hybridiza small family of transcription factors. Runx Pro among the offspring of ENUtreated C57BL/6J tion. Tbx22 transcripts were found in distinct ar teins play pivotal roles in various developmental males. eas of the embryonic head during the time of processes such as hematopoiesis (Runx1), os Supported by the German National Genome Re palate development. Its expression pattern teoblast differentiation (Runx2) and intestinal de search Network (KBP5T0516). points to a yet unknown, primary role of the velopment (Runx3). We demonstrate Runx2 and nasal septum in secondary palate development Runx3 expression in dynamic patterns during and explains the ankyloglossia phenotype in P208 08 early and late chondrocyte development. We CPX. To learn more about the regulation of used overexpression experiments in chicken em TBX22 gene expression, we have identified po bryos and a transgenic mouse approach to as The Xlinked nonspecific mental sitionally conserved transcription factor binding sess the role of Runx genes in cartilage differen retardation gene Arhgef6 is specifically and sites by comparing genomic sequences of tiation. We show that Runx2 is a positive regula highly expressed in the fetal placenta mouse, rat and human TBX22. In this way, tor of chondrocyte condensation and differenti Matthias Kohn1, Horst Hameister1, Reinald strong upstreamacting candidates were found, ation and that Runx2 and Runx3 act coopera Fundele2, Hildegard KehrerSawatzki1 one of which has been implicated previously in tively during chondrocyte maturation. Furtermore 1 Abteilung Humangenetik, Universität Ulm, facial clefting. we demonstrate that Runx2 is, albeit essential, D89069 Ulm, Germany;2 MPI für not sufficient to induce osteoblast differentiation Molekulargenetik, Abt. Ropers, D14195 and thus bone formation. Berlin, Germany Features such as hematopoiesis and cartilage According to our working hypothesis the genes have newly emerged during vertebrate evolution. for Xlinked nonspecific mental retardation, The development of these features might have MRX, are the best candidates for human speci been promoted by the stepwise duplication of a ation genes. The specific trait developed during single founder Runx gene. We cloned the Runx human speciation is general cognitive ability. gene of the primitive chordate Branchiostoma This trait gets introduced simultaneously with re lanceolatum. We found that Branchiostoma has productive isolation which exerts its effect in the

medgen 14 (2002) 81 only one Runx gene (Brrunx) which shows the P208 13 nation at the age of 4 years showed moderate closest homology to mammalian Runx3. Brrunx motor and mental retardation, turribrachy is expressed in the intestinal tract. This is in cephaly, midface hypoplasia, ptosis, beaked Alternative splicing of the MID1 gene, good accordance to recent reports that show a nose, high palate, microstomia, lowset dysplas mutated in Opitz BBB/G syndrome, leads to function for C. elegans Runx in intestinal devel tic ears, short and broad neck, hypoplasia of the the introduction of premature termination opment and involvement of human RUNX3 in radius bones and thumbs, severe hypoplasia of codons: implications for regulatory gastric cancer. Thus it is likely that Runx3 has the penis and scrotum, cryptorchidismus. The mechanisms of MID1 gene expression conserved ist original function in intestinal de karyotype was 46,XY, 15ps+, without increased Winter, Jennifer(1), Krauß, S. (1), Lehmann, T. velopment and that the newly emerged paralogs spontaneous chromosome breakage. At the time (1), Trockenbacher A. (2), Kijas, Z. (1), Suckow Runx1 and Runx2 acquired new functions dur of examination no evidence for Fanconi anemia V. (1), Kalscheuer V. (1), Ropers H. (1), ing craniota evolution. was found. Our patient confirms the clinical vari Schneider, R. (2), Schweiger, S. (1) ability of BGS, autosoma l recessive inheritance (1)MaxPlanckInsitute for molecular is supported by parental consanguinity. Several genetics, Ihnestr. 73, 14195 Berlin, authors have questioned whether BGS is a dis P208 12 (2)Institut fuer Biochemie, PeterMayrstr. tinct entity. The wide variability of the BGS phe 1a, A6020 Innsbruck notype and its overlap with other disorders will Mutations in the ubiquitously expressed MID1 NBS1 founder mutation and Darwinian be discussed. gene cause Opitz BBB/G syndrome, a specific fitness defect of the developing ventral midline. Al (1)Seemanova, Eva, (1)Jarolim, P., (2)Varon, R., though we have acquired detailed knowledge of (2)Pelz, J., (2)Sperling, K. the protein function of the MID1 gene product P209 02 (1)Dept. of Medical Genetics, Charles during the last few years, the basic question of Univesity, Prague, Czech Republic, how mutations in the ubiquitously expressed (2)Institute of Human Genetics, Charité, De novo distal deletion (6) (q23q25) with MID1 gene can produce a specific ventral mid Humboldt University, Berlin, Germany mild phenotype in a two year old boy line phenotype, remains open. Tissue specific The NBS1 founder mutation is present in individ Welling,Brigitte (1), Fritz,B. (2), Exeler,R. (3), splicing is an attractive model to answer. NIX

GfH ÖGH SGMG Tagungsband Abstracts GfH ÖGH SGMG Tagungsband uals of eastern, western and southern slavic ori Lemcke,B. (4), Horst,J. (5) analysis of the genomic sequence of the human, gin. Thus, the original mutational event occurred (1) Institute of Human Genetics, University murine and Fugu MID1 gene predicted several while the slavic people were still together in their of Münster, Germany, (2) Institute of Human exons in addition to the published MID1 se homeland, the Pripet swamp, more than 1,500 Genetics, University of Marburg, Germany quence. By RTPCR most of them could be years ago. We found an unexpectedly high car Distal deletions of chromosome 6 are associat shown to be transcribed and spliced into the rier frequency of this mutation in newborns of ed with growth and mental retardation, micro MID1 cDNA in a tissue dependent manner. Inter five different slavic populations: Czech Repub cephaly, short neck, retinal abnormalities, skele estingly, all splice variants found, introduced lic (8/1234), Poland (18/3569 ), Ukraine (Lvov, tal, heart, anal and other variable defects. Stop codons into the MID1 coding sequence. 5/908), Bulgaria (9/1002), and the Sorbs (family We present a two year old boy with mild clinical These exons might be fundamental for two dif studies, 8/326). This frequency rate is much features. He was born after an uneventful preg ferent mechanisms regulating MID1 expression higher than the one found in Berlin (1/900). If one nancy at term (body weight 2770 g [3. per and function: Some of them, that use the polyA+ excludes that this independent increase in allel centile], body length 52 cm [25. percentile], head tail of the MID1 gene, are carrying premature ter ic frequency is simply due to chance, it must re circumference 35 cm [25.50.percentile]). mination codons and lead to nonsense mediat sult from a different reproduction rate of the Our propositus presented after birth mainly ed mRNA decay of the respective MID1 tran gene carriers. We have therefore performed a healthy, only breathing gave suspicion for an as scripts. Others, comprising own polyA+ tails cohort study on the reproductive history of per piration. The physical examination showed a might code for Cterminally truncated MID1 pro sons from 18 pedigrees from the Czech Repub slightly dystrophic male newborn with inconspic teins. This goes in line with proteins of 25kD, lic. Each individual was personally interviewed, uous genitals. Only the right testis was not pal 30kD, 35kD and 45kD that can specifically be and blood samples were analysed for the NBS1 pable. Later on some mild clinical features such detected on Western blots using an antibody founder mutation. We found that the normal ho as a tall and thin stature, lowset ears, a high against the Nterminus of MID1. We have shown mozygous female probands (n=45) suffered a to forehead, antimongoloid lidaxes, a receding previously that Cterminally truncated MID1 pro tal of 9 miscarriages, while in the heterozygous chin, deep nasolabial creases and muscular hy teins have dominant negative effects on the group (n=39) only 2 were registered. In addition, potonia led to a cytogenetic analysis because of MID1 protein function. the female carriers of the founder mutation gave suspicion for a syndrome. birth to a total of 90 children compared to 78 The cytogenetic and FISHanalysis exhibited a children for the normal homozygotes. Thus, the P209 01 distal deletion (6) (q23q25) for the propositus. total number of pregnancies per woman of 2,3 in Our case report gives an overview concerning the heterozygotes and only 1,7 in the normal ho the development of the now two year old boy llerGerolg syndrome the clinical findings mozygotes showed a clear difference. These and reviews the known literature. in a 4years old boy findings were confirmed in principle by compar Although our propositus has only a mild pheno Naumchik, Irina; Rumyantseva N.; Polityko A.; ing the number of offspring between normal ho type and now at the age of two years mainly mo Chmel R. mozygous and heterozygous sibs from the same torical and sensomotorical deficits without any Institute for Hereditary Diseases. Minsk, generation. In principle, a higher number of off known abnormalities of inner organs, especially Belarus spring from NBS heterozygous mothers could heart and brain, a mental retardation can not be BallerGerold syndrome (BGS) is a rare disorder explain the high frequency of this deleterious excluded. presumably autosomal recessive in nature (MIM founder mutation in the slavic population. Our 218600). The diagnosis of BGS is based on the preliminary data suggest that the higher fertility presence of craniosynostosis, and preaxial up might be due to a lower miscarriage rate. Since per limb reduction defects. Different skeletal, P209 03 about 50% of all spontaneous abortions are due oralfacial, cardiac, genitourinary, anal malfor to aneuploidy, this would implicate that the NBS mations, along with mental and motor retarda heterozygotes show a lower nondisjunction rate Overgrowth and Transient Mental tion have also been described in a few patients. compared to normal homozygous females. Retardation in a Family: Sotos Syndrome ? We present the clinical findings of a 4yearsold Nicolai Kohlschmidt boy affected with BGS. The patient is the third Intitute of Human Genetics, Johannes child of healthy consanguineous parents, his two GutenbergUniversity Mainz, Germany older sisters are healthy. The mother had two Intellectual impairment is common reason for a prior first trimester miscarriages. The pregnancy child to be referred to a genetic clinic. To diag was uneventful, birth weight was 2025 g, length nose a syndrome which includes mental retarda 47 cm. First examination of the newborn boy de tion more specific features are necessary. These tected brachycephaly with flat occiput and may be discrete or remain unconsidered. Over prominent forehead, ptosis, clubhands, hypopla growth narrows the diagnosis to relatively few sia of the thumbs, ambiguous genitalia, bilater possibilities. We present family, a 37 year old al clubfoot. Further investigations at the age of woman, her ten year old son and her 15 months 2 months showed atrial septal defect, mild hy old daughter with transient mental retardation, drocephaly and slight hip dislocation. Reexami extreme overgrowth, cardiac defects, and a pe

82 medgen 14 (2002) culiar face. Diabetes I is present in the mother et al. (2001). The latter found missing permanent P209 07 and may be an additional feature. teeth in three of six patients with Kabuki syn We consider Sotos Syndrome to be the likely di drome. Small microdeletion 1p 36 in a 12 year old agnosis. It is quite uncommon though, that more Here we report a 14y old boy with scoliosis, dis boy with a mild clinical phenotype but than one person in a kindred is affected by the locations of hips and typical facies of Kabuki sudden lethal course Sotos syndrome and that mental retardation set syndrome, however, without postnatal growth re Neumann, Luitgard M.(1); Polster,T.(2); Kunze, tles before puberty. tardation (the body length corresponds to the J.(1), Spantzel,T (2); Bartsch, O.(3) Other diagnoses will be discussed. 25th centile) but with ectodermal abnormalities Institute of Human Genetics and University like hypoplastic nails and oligodontia. The fol Children’s Hospital, Charité Campus lowing dental germs were missing: 13, 12, 22, VirchowKlinikum, Berlin, Germany (1); 23, 33, 32, 31, 41, 42, and 43. Yet, serration was P209 04 Kinderzentrum Krankenanstalten Gilead, anticipated in the 2nd month of life. This agree Bethel, Bielefeld, Germany (2) Institute of well with descriptions in the literature. In addi Human Genetics and University Children’s A novel mutation that causes BardetBiedl tion to mental retardation he shows behaviour Hospital, Dresden(3) syndrome 4 (BBS) disorders like unsociable behaviour, thumb Monosomy 1p36 is a recently delineated con AbdelAleem Alice (1), Elruby M (1), sucking, jactatio capitis nocturna, and fits of tiguous gene syndrome, which is now consid Hoffmeister H (2), Grzeschik K.H. (2), Oeffner laughing. The spectrum of symptoms in Kabuki ered to be one of the most common subtelom F (2) syndrome may be larger than assumed so far. eric microdeletion syndromes. The phentype (1) Department of Human Genetics, National Ectodermal abnormalities and behavioural dis usually consists of psychomotor and growth re Research Centre, Cairo, Egypt; (2) Institute orders are cardinal symptoms in Kabuki syn tardation, microcephaly, seizures and typical of Human Genetics, PhilippsUniversity, drome, too. craniofacial anomalies. The incidence is estimat Marburg, Germany ed to be about 1: 10000. BardetBiedl syndrome (BBS) is a rare, multi We report a 12 year old patient born to healthy system disorder with an autosomal recessive P209 06 German parents. He had mild dysmorphic fea mode of inheritance. The cardinal features are tures: strabismus on both sides, mild bilateral

central obesity, rodcone dystrophy, postaxial Abstracts GfH ÖGH SGMG Tagungsband ptosis, slightly deep set eyes, mild progenia; polydactyly, learning difficulties, hypogenitalism Fetal valproate embryopathy in monozygotic there was a fusion of the right canine tooth with in males and renal dysplasia. The population twins the neighboured molar tooth, alopecia areata oc prevalence ranges from 1:13 500 livebirths Lemcke, Beate, Kleier, S., Kennerknecht, I., cipital (2X2cm diameter). The body length of our among the Bedouin of Kuwait to approximately Horst, J. patient was 138 cm (P3), his weight 34 (25 per 1:160 000 in WesternEurope. BBS is genetical Institute of Human Genetics, University of centile) and head circumference 53.3 cm (2550 ly heterogeneous with at least six different chro Münster, Germany percentile). He walked at 2 ½ years. At the age mosome loci linked to the disease: BBS1 on The use of valproic acid periconceptionally and of 3 years generalized seizures occured and he 11q13, BBS2 on 16q21, BBS3, on 3p12p13, during pregnancy is associated with adverse fe developed hyperphagia. Under low calorie diet BBS4 on 15q22.3q23, BBS5 on 2q31 and BBS6 tal outcome including intrauterine growth retar obesity could be avoided. Speech development on 20p12. To date, three BBS genes ? MKKS, dation, prematurity, abnormalities of the central was delayed. At 12 years he was moderately re BBS2, BBS4 ? have been identified. The BBS4 nervous system and of the cardiovascular sys tarded. He could speek whole sentences but gene product comprises 519 amino acid tem as well as musculosceletal abnormalities. was not able to read. He knew some figures. He residues and shows strongest homology to O Other organ systems are affected less frequent showed stereotypic hand movements. One linked Nacetylglucosamine transferase (OGT) ly. The risk for stillbirth or perinatal death is in month after presentation the child became ill from several species, including humans. Here, creased in children exposed to valproic acid dur with pneumonia, he suddenly suffered an asys we report the identification of a novel c.1247 ing pregnancy. Fetal valproate syndrome is char tolia requiring resuscitation. A causal status delG mutation in exon 14 of BBS4, homozygous acterized by typical dysmorphic features such as epilepticus could be excluded. The boy devel in an Egyptian BBS patient, who presented with high and broad forehead, bitemporal narrowing, oped severe cerebral edema resulting in an apal the typical clinical features of the syndrome. The hypertelorism, short palpebral fissures, epican lic syndrome. He died within 8 weeks. deletion causes a frameshift resulting in a trun thal folds, flat and broad nasal bridge, shallow Monosomy 1p36 was considered clinically. FISH cated protein of presumably 467 amino acid philtrum, hypoplastic midface, small ears, hy analysis was performed and confirmed the sus residues. This is the first report of a homozygous poplastic nails, clinodactyly and camptodactyly picion. A microdeletion 1p36.3 could be identi single base deletion causing a truncated BBS4 and often mental retardation. We present female fied: ish del(1) (pterp36.3336.32)([TelVysion 1p] gene product. monozygotic twins who were exposed to 2.4 g ,CD2L1,HKR3+) The deletion is less than 34 of valproic acid through all over pregnancy. We Mbp. Chromosome analysis demonstrated a saw the girls first at the age of 11 months and normal karyotype 46, XY. FISH analysis of the later of two years. The twins were delivered P209 05 mother did not reveal a microdeletion 1p36.3. spontaneously in the 37th week of gestation. The father was not available for testing Body length, weight and head circumference are Hypodontia as a cardinal symptom in at the lower normal limit. The girls show almost Kabuki syndrome identical dysmorphic features (high and broad Kreuz, Friedmar R. forehead, hypertelorism, infraorbital groove, flat P209 08 Institute of Clinical Genetics, Medical and broad nasal bridge, small posteriorly rotat Faculty „C G Carus“, Technical University ed ears, shallow philtrum, thin upper lip, camp Mesoaxial syndactyly with cataract: A Dresden todactyly, broad distal phalanx of fingers). A distinct syndrome In 1988 Niikawa et al. described a new syn hearing loss is treated with hearing aids. The Bohring, Axel (1), Caliebe, A. (2), drome with mental retardation, postnatal girls have VSD and pulmonary stenosis. They Kennerknecht, I. (1), Horst, J. (1) dwarfism and a peculiar facies characterized by show developmental delay and feeding difficul (1) Institut für Humangenetik, Westfälische long palpebral fissures with eversion of the lat ties. Surprisingly identical clinical signs are pro WilhelmsUniversität, Münster, Germany, (2) eral third of the lower eylids, higharched eye duced by a drug in genetically identical individ Institut für Humangenetik, Christian brows with lateral thinning, broad and depressed uals. A high recurrence risk of valproate embry Albrechts Universität, Kiel, Germany nasal tip, large prominent earlobes, a cleft or opathy is known in sibships. Possibly the metab Syndactylies are a heterogeneous group of higharched palate, scoliosis, persistence of fin olism of valproic acid either in the mother or in anomalies of the hands and feet and occur ei gerpads, radiographic anomalies as dislocation the fetuses is responsible here. ther as an isolated congenital malformation or as of hip joints and recurrent otitis media in infan part of a syndrome. In 1993, Pavone et al. re cy. Later, other authors described ectodermal ported on a boy with mesoaxial syndactyly, abnormalities as hypoplastic nails, abnormal cataracts, brachycephaly, mild facial anomalies, tooth crown shape, developmental defect of mild generalized hirsutism, thinning of the lower enamel, premature breast development and legs, and mental retardation. breast enlargement in girls. In 1997, Lerone et al. The authors suggested that the findings in t his reported a girl with Kabuki syndrome and ecto patient were unique and differ from cases previ dermal abnormalities like hypodontia, hypoplas ously reported. Here we report on a second case tic nails and brittle hair. These findings were with mesoaxial syndactyly of hands and feet and confirmed by Courtens et al. (2000) and Matsune

medgen 14 (2002) 83 congenital cataract, however, with apparently P209 10 strated hypoplastic cerebrum and cerebellum re normal mental and motor development. The girl sulting in a largely empty intracranial space. In a is the second child born to healthy consan 28 yearsold woman dysgenetic appearance of Microdeletion del (14)(q11.2q13.1) with guineous parents of Turkish origin. She has the cerebral cortex with a midline interhemi severe phenotype brachydactyly on both hands with partial cuta spheric cerebral cyst was diagnosed. A newborn Kroisel, Peter Michael (1), Windpassinger, C (1), neous syndactyly between fingers IIIV on the girl showed agenesis of corpus callosum, hy PleckoStartinig, B (2), Wagner, K (1), Zierler, H left, and osseous syndactyly between fingers III poplasia of the cerebellar vermis and a dysge (1), Petek, E (1) IV and minimal cutaneous syndactyly between netic cerebrum with pachygyria and a medially (1) Institute of Medical Biology & Human fingers II and III on the right hand. located dorsal cyst. Genetics, University of Graz, Austria, (2) There is complete cutaneous syndactyly be The genetic heterogeneity and the phenotypic Department of Pediatrics, University of tween toes II and III and partial cutaneous syn variability of the Seckel syndrome is discussed. Graz, Austria dactyly between toes IIIV bilaterally. On exam Microdeletions of the proximal segment of the ination at age 4 month a leftsided microphthal long arm of chromosome 14 are rare. Here we mos, microcornea, and nuclear cataract was describe such a chromosomal aberration that P212 01 diagnosed which was considered to be congen occurred de novo in the second child of a ital. In addition, there are mild facial anomalies healthy unrelated Austrian couple. Already at and a low hairline on the back. Although the girl Coping strategies of pregnant women after birth (10 days after date), following an unevent seems to be less severe affected than Pavone’s socalled „TripleDiagnostic“ and those of full pregnancy, several phenotype anomalies case, this second case may support the sugges their partners were noticed. Most obviously a pronounced mi tion that this pattern of malformation represents Jahn, Susanne, Kreuz, F. R. crocephaly with an occipitofrontal cirumference an unique condition of apparently autosomal re Institute of Clinical Genetics, Medical (OFC) of 32.5 cm (< 3 centile) was noticed, cessive inheritance. The findings are very simi Faculty „C G Carus“, Technical University whereas weight of 4435 g and length of 55 cm lar to the cataractwebbed Peromyscus manic Dresden were both > 90 centile. MRT of the brain per ulatus autosomal recessive mouse mutant de Objective: To compare coping strategies of preg formed at an age of 9 month did not reveal any scribed by Huestis [1951] and Anderson and nant women after triple diagnostic, genetic obvious anomaly of cerebral structural but just

GfH ÖGH SGMG Tagungsband Abstracts GfH ÖGH SGMG Tagungsband Bruns [1979]. counselling and prenatal diagnosis and those of enlarged ventricles. At one year of age he shows their partners. an even more pronounced microcephaly (<< 3 Background: Triple diagnostic has become a centile) with weight and length at the 50 centile, prenatal screening method by combination of P209 09 a severe psychomotor retardation, a bilateral op three serum parameters (alphafetoprotein, un tic atrophy, several distinct facial dysmorphic conjugated estriol, human chorionic go features, like a long philtrum and genitourinary Microcephalychorioretinal dysplasia nadotropin) with personal parameters to define anomalies as a hypospadia III and a bilateral hy König, Rainer (1), Ebru U.(1), Zubcov, A.(3), the individual’s probability to bear a child with a dronephrosis IIIII. Kieslich, M.(2), Fuchs, S.(1) chromosomal aberration, especially trisomy 21. High resolution banding allowed to confirm the Institut für Humangenetik(1), Kinderklinik(2) The screening often takes place without suffi microdeletion 14q, which was suspected already und Klinik für Augenheilkunde(3) der J.W. cient information and so a „bad result“ is syn from standard cytogenetic preparations leading Goethe Universitaet onymous with a handicapped child. to the karyotype 46,XY,del(14)(q11.2q13.1)de The combination of microcephaly, mental retar Methods: Our investigations were carried out by novo. Subsequently performed FISH analysis us dation and chorioretinal dysplasia was first de questionnaires. Coping strategies, mood states ing region specific BAC clones allowed to further scribed by Tenconi et al. in 1981. Until now, only and attitudes of 92 women and 52 partners were define chromosomal breakpoints. Additional mi 4 further families were reported. We here de evaluated. crosatellite and BAC contig analysis narrowed scribe a mother and her daughter, supporting Results: Only 65% of the women were informed down the deleted segment to 5 Mb and the the supposed autosomal dominant inheritance about this screening method by their gynaecol number of potential candidate genes related to of the syndrome and showing the great variabil ogist. Their partners were informed to about the phenotype anomalies already mapped to ity. At the age of 2 years the index patient had a 27% by their wives and to about 23% by the gy that chromosoaml segment is currently less than weight of 6,7 kg (3kg <3P), height of 80,3 cm (1 naecologist. After having been informed of a el 20. Therefor this case could be helpful in identi cm <3P) and a head circumference of 38 cm (8 evated risk, 87% of the women and 72% of their fing one gene or several genes that are related cm < 3P). Ophthalmologic examination showed partners felt depressed and a little part felt acti to severe phenotype anomalies. This should im rightsided mild microphthalmia, and a fibrovas vated (2% and 10%). After genetic counselling, prove our understanding on the genetic factors cular membrane with a densely pigmented reti there is a trend to activated mood (women: only involved in the regular and altered formation of nal periphery. On the left eye she had a retinitis 37% feel depressed and 46% feel activated, craniofacial and genitourinary structures. pigmentosa. Psychomotor development was se partners: only 45% feel depressed and 50% feel verely retarded. activated). After genetic counselling, negative The mother had a head circumference of 50 cm coping strategies dropped down (women: 22% (2,2 cm <3P), a weight of 77 kg (2 kg >97P) and P209 11 to 3,5%; partners: 13% to 5%) in favour of ac a length of 160 cm (2550P). Fundoscopic ex tive coping. A quarter of the pregnant women re amination revealed chorioatrophic regions, hy jected offered prenatal chromosomal analysis by Seckel syndrome with central nervous perpigmented areals, retinal folds and optic at invasive methods. system anomalies rophy. She had a normal psychomotor develop Conclusions: Genetic counselling holds up an Kautza, Monika (1), Caliebe, A.(1). Lemke, H. ment. important position in development of positive (2), Gerhardt, B. (2), Grote, W. (1) Fryns et al, (1995) argued that microcephaly coping strategies. It should give not only infor (1) Institut für Humangenetik, UKK Kiel, (2) lymphedema, microcephalychorioretinal dyspla mation but also therapeutic support for pregnant Kinderabteilung, Kreiskrankenhaus sia and the cases, reported by Jamas et al. women and their partners. Eckernförde (1981) with microcephaly and retinal folds all Seckel syndrome is a rare, heterogeneous form have the same condition. Our of primordial dwarfism. The clinical delineation two cases may support this hypothesis. of this disorder has been inconsistent, using P212 02 References even Seckel´s original criteria. There are only few Tenconi R et al. Clin Genet 1981;20:347351 cases with a detailed description of the central Fryns JP et al. Clin Genet 1995;48:131133 Social, medical and pedagogic care in nervous system anomalies. patients with Klinefelter´s syndrome We report on a newborn boy with Seckel syn Bier, Andrea, Hinkel, G.K. drome showing marked intrauterine dwarfism, Institute of Clinical Genetics, Technical severe microcephaly with holoprosencephaly, fa University of Dresden, Germany cial anomalies including a receding forehead and Klinefelter´s syndrome is the most common chin, large beaked nose and bulging eyes. There gonosomal aberration in males. However, little is was no known parental consanguinity, family known on how adult patients and parents of af histories were unremarkable. fected boys cope with the disease. In order to Imaging studies of the central nervous system in evaluate coping strategies we sent out question Seckel syndrome have only been described in naires to adult patients with Klinefelter´s syn three other reports: A 2 monthold boy demon drome and parents of affected boys focussing

84 medgen 14 (2002) on familial, professional, and social aspects. In P213 02 of organisms and to describe gene action in addition, data on medical care were analyzed. terms of the „norm of reaction“. However, it 129 uestionnaires 70 from adult patients and turns out that these considerations did not re The situation and attitudes of parents with 59 from parents of affected boys under the age strict the claim of genetics to primacy but children suffering from a hereditary disease of 18 years were evaluated. The karyotype was widened it. At the same time, the inherent con Hölzel, Beate, Kreuz, F. R. 47,XXY in 102 cases (79,1%), mosaic in 7 cases ception of nature and nurture functioned as the Institute of Clinical Genetics, Medical (5,4%), 48,XXYY in 3 cases (2,3%), 48,XXXY and basis of discriminating health policy even Faculty „C G Carus“, Technical University 49,XXXXY in 2 cases (1,6%), respectively. For though the selfimage of geneticists and human Dresden the remaining cases of Klinefelter´s syndrome geneticists was mostly not political. Considering The aim of our study was to investigate the sit the karyotype was unknown. Among the group this historical experience it seems possible that uation and attitudes of parents with children suf of affected boys and juveniles the diagnosis was recent consideration of environment in genomics fering from mental retardation (like Down’s syn made prenatally in 66,1%. This high rate can be will end in a description of human beings in drome or fragile X syndrome; group A) and my explained by a low termination rate and the mild terms of genetic risk. Genetic discrimination, opathies (like Duchenne’s progressive muscular phenotype, especially in childhood. Only 15% of then, may not be the matter of ethics but the dystrophy; group B). We wanted to find out, the parents and 17% of the adult patients felt to outcome of the factual constraints of genetic re whether due to heredity there are specific fea be adequately informed on the disease and duction. tures in the process of coping. Points of interest counselled, but most of the parents (69,5%) and were effects on partnership, lifestyle, family adult patients (74,3%) were satisfied with their planning and attitudes to prenatal diagnosis. medical care. Among adult patients 91,4% com Method: The current study was carried out ques P215 01 pleted one or more professional trainings, of tionnaires. To find out the coping strategies we those 17,2% had a university degree. Altogeth used standardized questionnaires. Results: 42% er 86,8% enjoyed their profession. 30% of the Chromosomal factors of infertility and of the parents from group A and a quarter of parents and 49% of the adult patients felt to be recurrent pregnancy loss group B had to restrict their occupational life. disadvantaged by the disease. Our results may Sodia, Sigrun; Emberger, W.; Petek, E.; 44% of group A and 20% of group B felt con be helpful in genetic counselling of patients with Zierler,.H.; Kroisel,.P.; Wagner, K. fronted with more social exclusion. About 48%

Klinefelter´s syndrome and of parents faced with Institute of Medical Biology and Human Abstracts GfH ÖGH SGMG Tagungsband parents of group A and only 7% of group B had the prenatal diagnosis of Klinefelter´s syndrome. Genetics, University of Graz, Austria conflicts with family members. Quite different are Chromosomal abnormalities are one of the the effects on partnership: Roughly half of group known factors of infertiliy and recurrent pregnan A reported conflicts with their partners and in cy loss. Cytogenetic screening was done in can P213 01 19% the partnership was dissolved. In group B didate couples for assisted reproduction and ex 72% of the parents held the view that difficulties ploration of semen alteration (Infertility group) of their situation had strengthened the partner The social situation, family relations and and couples who had more than 2 spontaneous ship and only about a quarter had conflicts with medical support of families suffering from fetal losses ( Abortion group). their partners. Similar are the opinions about heredoataxias in Germany In our retrospective study (19882000) we com family planning and towards prenatal diagnosis: Teige, Robin, Kreuz, F. R. pared frequency of chromosomal abnormalities 23% of the parents planned further children. Institute of Clinical Genetics, Medical in these indication groups. In Infertility group cy About 90% wished to undergo prenatal DNA di Faculty „C G Carus“, Technical University togenetic diagnosis of 982 candidates were per agnosis. About two thirds thought that termina Dresden formed, 403 female (41%) and 579 male (59%). tion of pregnancy after positive prenatal diagno The aim of the current study is to evaluate the The chromosomal aberration rate was in total sis is justified. Conclusion: For parents with chil social situation, family relations, medical and so 6,9 % (n=68), 5,4% female (n=22) and 7,9% dren suffering from hereditary diseases the doc cial support in German families suffering from male (n=46). Structural aberrations were found tor is important not only for giving information heredoataxias. The study is carried out by ques in 2,1% (n=21), 24% female (n=5) and 76% male and treatment, but also for supporting the tionnaires and interviews. Both, atactic persons (n=16). In the Abortion Group a total of 1877 process of coping with the affected child in or and their partners, were asked. candidates was tested, 1129 female ( 60%) and der to strengthen relationships in the family. Only a minority of the patients received the in 748 male ( 40%). In 4,2% (n=79) aberrant kary formation about their disease from their doctors. otypes were found, 4,9% female (n=56) and 3% Most gathered their knowledge from relatives or male (n=23). Structural aberrations were found through own searches. Lack of information is the P213 03 in 2,6% (n=49 ), 62% female (n=30) and 38% most common problem. All patients still take male (n=19).By detailed aberration analysis in part in a wide spectrum of physiotherapy, logo both indication groups we try to define chromo The dialectics of the genotypephenotype pedics and ergotherapy. Both, patients and their somal factors of infertility. talk and genetic discrimination partners, consider these therapies suitable to An overall increased frequency of chromosomal Alexander v. Schwerin help them. However, the numbers of sessions aberrations was found, that confirms that it is Zentrum für Human und are not sufficiently as necessary. In general, the necessary to perform genetic counseling and cy Gesundheitswissenschaften der Berliner unaffected partners see more problems in their togentic investigation of both partners in assist Hochschulmedizin, Institut für Geschichte partnership than the affected persons. The main ed reproduction and exploration of recurrent der Medizin, Klingsorstr. 119, 12203 Berlin problems are the limitation of their freedom in pregnancy loss. This historical study is concerned with innova the spare time and the inability of their affected tion in genetics, the genetic conceptualisation of partner to cope with the handicap. There are organisms and a genetic concept of „Ganzheit“ more problems with sexuality than for the pa that reestablishes the primacy of nature over P215 02 tients. On the other hand, the affected partners nurture. It casts some light on actual demands are more reserved in sexual contacts but do not in genomics towards the integration of epigenet feel less attractive for their partner. Most have Cytogenetic findings in patients prior to ic and environmental conditions. The case study no problems in relation to their family members. assisted reproduction deals with a debate between geneticists, gynae However, they reported problems with friends Suess, Franziska, Kaesbauer, J., Gassner, P., cologists, and radiologist around 1930 in Ger since first atactic symptoms had been seen. Seifert, B., Hehr, U. many. It will be shown that the conflict was As a result, we found some resources to give Center of Gynecological Endocrinology, mainly one about the claim of geneticists to the certainty to the patients and their partners, es Reproductive Medicine and Human primacy of their methods and the genetic knowl pecially concerning the progress of the disorder. Genetics, Regensburg, Germany, edge: Genetic findings about mutability can be The knowledge what progress stands for in ute.hehr@humangenetikregensburg.de transferred to the usage of Xrays in medicine. everyday life is very important for the partner Approximately 10 to 15% of Caucasian couples Analogous reasoning and the validity of animal ship in order to cope in a better way. Especially experience temporary or permanent fertility models should be investigated thoroughly in the medical professions should be better in problems. The heterogeneous etiology includes epistemological and systematic terms. In this formed about heredoataxias in order to inform an increased frequency of chromosomal aberra epistemological perspective modelling has to be their patients and the relatives. tions in both males and females. In order to understood as a process of production. Histor identify individual genetic risk situations, a thor ically it has been connected to remarkable trials ough examination of the medical and family his within the „higher mendelism“ 1930 to come to tory of both partners should be performed prior a „Ganzheitsauffassung“ (TimoféeffRessovsky) to assisted reproduction. In addition, depending

medgen 14 (2002) 85 upon the obtained clinical and anamnestic data mother of the index patient carrying the CFTR P218 03 karyotyping and/or additional gene analysis 5T allele on both chromosomes. Subsequently might be indicated. Here we report the cytoge all patients with two known pathogenic muta Comparative Primate Genomics netic findings in 1401 male and female patients tions and/or clinical CF minor signs were re Ebersberger, Ingo (1), Hellmann, I. (1), Enard, after genetic counseling and prior to assisted re ferred to a CF ambulance for adults for clinical W. (1), Heissig, F. (1), Khaitovich, P. (1), Kitano, production (7/2000 to 4/2002). All cases were work up and counseling. Sufficient amounts of T. (1), Metzler, D. (2), Nickel, B. (1), Schwarz, C. analyzed by GTG banding and additional special spermatozoa were retrieved by testicular biopsy (1), Winkler, M. (1), Zöllner, S. (1), Pääbo, S. (1) staining techniques, if necessary. At least 12 in 4 of the 5 patients; 11 IVF/ICSI cycles so far (1)Max Planck Institute for Evolutionary metaphase spreads were examined per patient resulted in 2 completed pregnancies. This report Anthropology, Leipzig, Germany (2) and 20 metaphases in case of a single abnormal further underlines the importance of genetic Mathematischer Fachbereich, Johann metaphase. In patients with two or more counseling prior to ICSI which should include Wolfgang Goethe Universität Frankfurt, metaphases with identical aberrations or at least the search for atypical CF phenotypes in Germany 3 different cell lines 30 to 50 cells were tested by „healthy“ men with oligo or azoospermia. Although humans and their closest evolutionary conventional analysis and/or additional 100 relatives, the chimpanzees differ only in a minute metaphases by FISH (wcp). 673 of the analyzed amount of their nucleotide sequence, they are patients had a primary indication for ICSI. The P218 02 unlike in many morphological, behavioral and second group (728) comprised patients, where cognitive aspects. Here, we explore the under initial genetic counseling had identified other risk lying molecular principles of those differences in factors in a patient and/or their close relatives, Gainoffunction screen for a systematic an extensive chimpanzee human omparison which required exclusion of chromosomal aber functional analysis of X chromosomal genes project that addresses the question using three rations („nonICSI“). In total, 53 (3.8%) aberrant in Drosophila melanogaster different approaches. In a first approach we karyotypes were identified (ICSI group: 27/673 = Schäfer, U., Beinert, N., Werner, M., Dowe, G., compared human and chimpanzee genomic or 4.0%; nonICSI group: 26/728 = 3.6%). In the Zunker, E., and Jäckle, H. thologous sequences to get an insight into the ICSIgroup 11 autosomal and 1 gonosomal MaxPlanckInstitut für biophysikalische patterns and dependencies of differences across structural aberrations were observed; 4 patients Chemie, Abt. Molekulare the genome. 0.1 % of the chimpanzee genome

GfH ÖGH SGMG Tagungsband Abstracts GfH ÖGH SGMG Tagungsband were found to carry constitutional gonosomal Entwicklungsbiologie, 37070 Göttingen, was sequenced in a shotgun approach and was aneuploidies and additional 11 patients gonoso Germany compared to the corresponding human se mal mosaicism. Aberrant karyotypes in the non Conventional genetic screens are aimed at gen quence. The average nucleotide divergence was ICSI group included 5 autosomal and 1 gonoso erating mutations that reduce or eliminate gene determined as 1.27 %. However the substitu mal structural aberration, 1 constitutional gono functions. On the other hand, it is known from tions appear not to be distributed uniformly somal aneuploidy and 19 patients with gonoso Drosophila and other model organisms that across the chromosomes. The sex chromo mal mosaicism. Our data further confirm not knockouts do not produce any obvious lossof somes differ clearly in their nucleotide diver only couples prior to ICSI to be at higher risk for function phenotype in over 60% of the genes. gence from the autosomes, with the Xchromo chromosomal aberrations but also a subgroup of We perform, therefore, a gainoffunction screen: some howing the least substitutions and the Y couples employing other tech over or misexpression of genes might identify chromosome showing the most between the two roles for the products of otherwise phenotypical species. Further, we found evidence that the dis ly silent genes. tribution of substitutions among the autosomes The basic scheme for our gainoffunction P215 03 is not random, suggesting chromosome specif screen is adapted from P. Rorth?s (1996) modu ic influences on the mutation rates. Second, in lar system, combining random P element inser order to explore evolutionary patterns in genes, Phenotype in patients with two CFTR tional mutagenesis with GAL4regulated gene we compared transcribed sequences of the mutations identified prior to TESE expression. The P vector used is P{Mae chimpanzee to the corresponding sequences in Hehr, Andreas (1), Gassner, P. (1), Wimmer, K. UAS.6.11} (Crisp and Merriam, 1997) which car humans. For this purpose ESTs were generated (2), Schroeder, J. (3), Gross, C. (1), Bals ries at the 5? end binding sites for the yeast from chimpanzee testis and brain cDNA libraries Pratsch, M. (1), Hehr, U. (1) transcription factor GAL4 close to a basal pro for comparison with their human orthologous se (1) Center of Gynecological Endocrinology, moter. This scenario allows for the ectopic ex quences. Comparing the variation within humans Reproductive Medicine and Human pression of genomic sequences adjacent to its and between humans and chimps, it is striking Genetics, Regensburg, Germany, 5? end when a second transgene containing an that the 5’UTR within humans is relatively more ute.hehr@humangenetikregensburg.de, (2) activated GAL4 gene is introduced into the fly conserved than the between humans. Apart from Caritas Hospital St. Joseph, Department of genome. the observation of general patterns of substitu Urology, Regensburg, Germany, (3) Institute Startingpoint of the screen is the mobilization tions, we also screened for evolutionarily distinc of Pathology, University of Regensburg, of a single P element that is inserted on a dom tive genes with an excess of nonsynonymous Germany inantly marked autosome. Currently, more than over synonymous substitutions. With our ap Homozygosity or compound heterozygosity for 15,000 such crosses were performed resulting in proach we found twenty candidate genes for CFTR mutations is thought to be the major ge over 1,000 lines with an X chromosomal P{Mae positive selection out of 1266 coding region netic cause for obstructive azoospermia or se UAS.6.11} insertion. Most of these lines were al alignments screened. Third, in addition to pure vere oligozoospermia. Here we report the clini ready tested for misexpression phenotypes by sequence analysis we investigated chann the cal data of 5 patients, each with two identified introducing a ubiquitously expressed GAL4 expression of individual genes among primates. CFTR mutations and the current status of their gene, P{Act5CGAL4}. This combination results Therefore, we compared the transcriptome in fertility treatment. Only one of them had previ in detectable phenotypes, e.g. lethality, in over blood leukocytes, liver and brain of humans, ously been diagnosed as a CF patient. Mutation 30% of the cases. Up to now, the insertion sites chimpanzees and macaques using cDNA arrays. al screening for the most common 31 CFTR al for about 900 lines are molecularly determined With our approach we identified a total of 158 leles in the Caucasian population and analysis by inverse PCR and hence the overexpressed genes that differ significantly in their expression of the polythymidine tract in intron 8 (IVS8) were genes are identified. pattern between at least two of the species. Fur performed after genetic counseling. For one of The insertions lines serve as starting points for thermore, while the rates of transcriptome these patients thorough interview during genet the identification of novel gene functions in change in liver and blood were generally similar ic counseling revealed recurrent episodic salt Drosophila. Since genes as well as regulatory in chimpanzees and humans, it is almost four wastage with dehydration, at one point requiring circuitries are molecularly conserved throughout fold accelerated in the human brain. The com hospitalization for i.v. liquid substitution. After a the animal kingdom this will, eventually, help to parison of genomic sequences, EST data and positive sweat test direct sequencing of the unravel the corresponding human gene func gene expression levels between chimpanzees CFTR gene confirmed two CFTR mutations oc tions. and humans provides an insight into general curring with low frequency in the Caucasian Supported by Deutsches Humangenomprojekt evolutionary patterns of primates as and repre population: I336K and 3359delCT, which had (grant 01 KW 9917 to H. J.) sents a powerful tool to pinpoint genes of impor been missed during routine CFTR screening. A tance for either chimpanzee or human specific serious pancreatitis at the age of 4 years was re traits. ported from another patient (compound het erozygous R347P/5T). His maternal grandfather had died of pancreatic cancer after a long histo ry of recurrent unexplained serious abdominal pain attacks, which were also reported from the

86 medgen 14 (2002) P218 04 events in the presence of the MTdisrupting ciencies and autoimmune diseases. At the drugs benomyl, nocodazole and colchicine. same time, the mutant mouse lines used to iden While we failed to detect inhibition of meiosis tify these genes and pathways are supposed to How do imprinted genes correlate with specific telomere redistribution, MT disruption serve as mouse models for corresponding hu placental dysplasia in mouse (Mus) inhibited centromere redistribution and homolo man diseases. interspecies hybrids? gous chromosome pairing. In support, meiotic Out of more than 10.000 F1 animals and more Zechner, Ulrich (1), Wei, S. (2), Hemberger, M. telomere movements occurred in a haploid than 2500 G3 mice analyzed thus far we have (3), Kalscheuer, V. (2), Rüschendorf, F. (4) and meiosiscompetent yeast strain despite the dele established more than 50 mutant mouse lines Fundele, R. (2) tion of the telomere component Sir3p and the ki with dominant and more than 25 mutant mouse (1) Inst. for Human Genetics, Johannes nesinlike Kar3p motor protein that has been im lines with recessive phenotypes. Many of those GutenbergUniv. Mainz, Germany, (2) Max plicated as a meiotic telomere motor. Our results resemble known human diseases like severe PlanckInst. for Molecular Genetics, Berlin, opt for a role of cytoskeletal components other combined immune deficiency (SCID), hyper IgM, Germany, (3) Dept. of Biochemistry and than microtubules as promoters of meiotic selective IgA deficiency and different aspects of Molecular Biology, Univ. of Calgary, Canada, telomeric movements. Finally, the KAR3 disrup autoimmunity. (4) Inst. for Medical Biometry, Informatics tion rendered less cells entering meiosis, which Of these we have analyzed selected lines in and suggests that Kar3p may be important for other more detail and mapped the corresponding mu Interspecific hybridisation in mammals results in than bouquet functions possibly in the premei tation. As of today we were able to map the re several consistent hybrid dysgenesis (HD) ef otic division. sponsible mutation in four lines. The intervals for fects, most notably male sterility and abnormal the respective mutations could be narrowed growth. It can be assumed that other, less obvi down to 14cM. In one case the mutation could ous HD effects occur. Indeed, in those interspe already be identified at the molecular level. Data cific hybrids where this has been assessed to P218 06 from these studies will be presented. date, abnormal placental development was ob served. Interspecific hybrid placental dysplasia Phenotypic analysis and chromosomal (IHPD) was analysed in some detail in the two mapping of ENUinduced mouse mutants rodent genera Peromyscus (1) and Mus (2) and P218 07

with alterations of the immune system Abstracts GfH ÖGH SGMG Tagungsband strong similarities were detected between these Flaswinkel, H.1#, Rathkolb, B.2, Howaldt, M.2, groups. Reciprocal placental phenotypes, hy Faerber, C.3, Augustin, M.3, Imlau, A.3, 1) Environmental factors influencing postcoital poplasia or hyperplasia, appeared in reciprocal Servatius, A.1, Soewarto, D.4, Fuchs, H.4, 5) genetic selection. The depressed sex ratio matings; the placental tissue that was mainly af Kremmer, E.5, Sandholzer, N.1, Schubbert, following the cigarette smoke exposure in fected was the spongiotrophoblast; and placen R.6, Hrabe de Angelis, M.4, Balling, R.7, Wolf, C57BL congenic mice differing in alleles of tal dysplasia exhibited genetic linkage to the X E.2, Pfeffer, K.1 ahr gene occurs in the ahr bb progeny of chromosome. In Peromyscus only, involvement 1. Institut für Med. Mikriobiologie, ahr bb mothers sired by the younger ahr bd of the autosomal imprinted, paternally expressed Immunologie & Hygiene, Troger Str. 4a, males gene Peg3 in the generation of placental hyper 81675 München, Germany Andrzej L. Pawlak1, Ewa Strauss1, Ewa Florek2 plasia was demonstrated (3). The pronounced 2. Institute of Molecular Animal Breeding, 1 Institute of Human Genetics, Polish similarities between Peromyscus and Mus hybrid Gene Center, FeodorLynenStr. 25, 81377 Academy of Sciences, Poznan, 2 Chair of placental dysplasias suggested that Peg3, or at München, Germany Toxicology, K. Marcinkowski University of least loci linked to Peg3, should also be involved 3. Ingenium Pharmaceuticals Fraunhofer Medical Sciences, Poznan, Poland in placental hyperplasia in Mus. However, our Str. 13 82152 Martinsried, Germany The cigarette smoke (CS) induced changes in study, which combined BC analysis, allelic ex 4. Institute of Experimental Genetics, GSF the fetal resorption rates and sex ratio were as pression (lossofimprinting) analysis, and the Research Center for Environment and sessed in the series of pairings differing in ahr use of BC males heterozygous at the Peg3 lo Health, Ingolstädter Landstr. 1, 85764 alleles. The frequency of resorptions in standard cus, does not support a major role of this im Neuherberg, Germany conditions was higher in females ahr dd (5/30 = printed gene in murine IHPD. This finding sug 5. Institute of Molecular Immunology GSF 0,17), as compared to females ahr bd and ahr dd gests that different molecular mechanisms have Research Center for Environment and (7/61 = 0,11). Following the exposure to ciga been recruited in comparatively closely related Health rette smoke (CS) the number of resorptions was rodent groups to produce indiscernible HD phe 6. MediGenomix GmbH Lochamer Str. 29 found increased in the groups of ahr bb and ahr notypes. However, our BC analysis has shown 82152 Planegg/Martiensried, Germany bd females up to the frequency 0,17 (7/44), that that another imprinted chromosomal region ex 7. Gesellschaft für Biotechnologische is equal to the values in the group of unexposed hibits linkage to placental hyperplasia.(1) Forschung, Mascheroder Weg 1, 38124 ahr dd females. The CS exposition did not in Rogers, J.F. & Dawson, W.D., J. Reprod. Fertil. Braunschweig, Germany crease the number of resorptions in the group of 21, 255262 (1970).(2) Zechner, U. et al., Nature Analysis of gene function in vivo has mostly ahr dd females. This indicates that the low ac Genet. 12, 398403 (1996). (3) Vrana, P. et al., been performed by transgenic insertion, inacti tivity ahr d allele does confer resistance to the Nature Genet. 25, 120124 (2000) vation of the respective gene by homologous re cigarette smoke induced increase in fetal resorp combination in embryonic stem cells and gene tions. trapping. We have complemented this approach In the series of pairings ahr bb (female) x ahr bd P218 05 by isolation of mutant mouse lines with pheno (male), in which the increased frequency of fetal typic alterations of the immune system from resorptions was found after CS exposure during ENUmutagenized mice. the period of pairing and the early pregnancy, we Meiotic chromosome behavior in relation to In this phenotypic approach C3HeB/FeJ were have noted also the decrease in sex ratio (M/all) the microtubule cytoskeleton in yeast randomly mutagenized in two central facilities in in the ahr bb progeny as compared to the in Edgar TrellesSticken, Harry Scherthan Munich using the potent mutagen ethylni creased sex ratio in the ahr bd progeny. The MaxPlanckInstitute fur Molekulare trosourea (ENU). ENU is most potent during greater effect of CS exposure on the sex ratios Genetik, 14195 Berlin (Dahlem) spermatogenesis and consequently mutagene in progeny differing in ahr genotype was seen in Chromosome pairing is a prerequisite for homo sis was performed on male mice which were the exposed group sired by the younger males. logue segregation at meiosis I and contributes subsequently mated to female WT mice. Proge These effects may be related to the decrease in to gametogenesis and sexual reproduction. Per ny thereof were analyzed for inherited alterations sex ratio noted among children of the fathers ex inuclear telomere clustering (bouquet formation) of the immune system using a panel of immuno posed to dioxin when young, as described by occurs at leptotene/zygotene during first meiot logical parameters. Both, F1 heterozygous mice Mocarelli et.al. (Lancet, 2000). ic prophase and is thought to facilitate homo carrying one mutated allele of a given gene logue alignment and pairing. We have recently (dominant screen) as well as Generation 3 (G3) established that the bouquet motif is part of the mice harboring identical mutations in both alle meiotic pathway in the model organism Saccha les of a given gene (recessive screen) have been romyces cerevisiae. In search for factors that are used to establish mutant mouse lines with alter involved in meiotic telomere movements and ations of the immune system. since it has been observed that microtubule (MT) With this approach we aim at the discovery and poisons adversely affect chromosome move functional characterization of novel genes and ments and pairing in plant and mammalian meio pathways which are relevant for the prevention, sis, we investigated the course of chromosomal diagnosis and therapy of human immune defi

medgen 14 (2002) 87 P218 08 SMA002 is a dominant mutant mouse line devel a stable abnormal phenotype. The phenotype oped from the Munich ENUmousemutagene was transmitted to their offspring by 62 of these sisscreen (see also Mammalian Genome Vol animals, and 58 new mutant mouse lines were Doppeltransgene Tiermodelle zur Analyse ume 11, July 2000) which is bred on the genetic established. Four phenotypes got lost due to low von aSynuclein, UbcM4 und Elk1 in der background of inbred C3HeB/FeJmice. From penetrance or breeding problems. In the reces Pathogenese des Morbus Parkinson days 8 to12 pp on heterozygous mutants show sive screen 3523 G3 offspring belonging to 153 Kuhn, M. (1); Cesari, F.(2); Bonin, M. (1); growth retardation and reduced body weight pedigrees were screened and 64 variants were Nordheim, A. (2); Habers, K. (3), Kahle, P. (4); gain as well as abnormal increased cleaning and identified and tested for a mutation by confirma Rieß, O. (1) scratching behavior. These characteristics make tion crossing. Out of these 13 new recessive mu (1) Department of Medical Genetics, this mutant an interesting model for diseases as tant mouse lines were established. The urinary University of Tuebingen; (2) Institute for Cell sociated with pruritus in humans, like allergolog protein screen identified six mice with abnormal Biology, Department of Molecular Biology, ical, autoimmune or nervous disorders. Gener patterns of protein excretion in urine. Two mice University of Tuebingen; (3) HeinrichPette ally, injuries to the skin through scratching and died without having produced any offspring. The Institut, Hamburg; (4) AdolfButenandt nibbling do not occur. Histopathological exami other four cases proofed not to be inherited. The Institut, Department of Metabolic nation of the skin gave no evidence of allergic poster will present an overview of the actual Biochemistry, University of Munich reaction such as an increased number or de screening achievements, the phenotypes of the alphaSynuclein spielt eine zentrale Rolle in der granulation of mast cells. Also the total plasma lines established and the collaborations set up. Pathogenese der Parkinson’schen Erkrankung IgE level was not elevated. Blood samples were More detailed information on two of these lines (PD). So ist alphaSynuclein eine Hauptkompo collected from 3monthold mutants and wild is presented on separate posters (see Howaldt nente der intrazellulären Proteinaggregationen type littermates and were analyzed for clinical et al. and Tran et al.). bei betroffenen Patienten. Mutationen im alpha chemical, immunological and hematological pa Synucleinkodierenden Gen führen zu einer sel rameters. The mutants show significant changes tenen autosomal dominant vererbten Form der in clinicalchemical parameters, such as reduced Erkrankung. Die normale Funktion von alpha P218 11 plasma cholesterol and triglyceride levels. For Synuclein, wie auch die Ursache des Zelltods chromosomal mapping, an outcross was per dopaminerger Neurone in der Substantia nigra

GfH ÖGH SGMG Tagungsband Abstracts GfH ÖGH SGMG Tagungsband formed by mating male mutant mice to wildtype Conditional Expression of Human alpha pars compacta bei PDPatienten, sind weitge C57BL/6JIco females. From these crosses, mu Synuclein in Mice hend ungeklärt. In vitro wurden Proteinaggregate tant F1 hybrid progeny were backcrossed to S.Nuber(2), C.Holzmann(1), T.Schmidt(2), von alphaSynuclein identifiziert, die u.a. die wildtype C57BL/6JIco animals. Tail clips from I.Schmitt(3), M.Neumann(4), A.Bornemann(5),F. MAP Kinase ERK2 und dessen Substrat, den the N2 descendants were taken for isolation of Zimmermann(6), S.B. Prusiner(7), W.Kuhn Transkriptionsfaktor Elk1 enthalten. genomic DNA. Twenty DNA samples each from (8),U.Grasshoff(2) and O.Riess(2) Wir wollen nun neueste Pathogenesemodelle an carriers (mutants) and noncarriers (wildtype) Depts.of Med.Genetics hand von doppeltransgenen Tiermodellen were pooled and used for chromosome screen (1)Univ.Rostock,(2)Univ.Tuebingen;(3)Mol.Hu (Maus) in vivo überprüfen. Zum einen soll mit ing. 60 different microsatellites (polymorphic be man Genetics,Univ.Bochum;Inst.s of Hilfe von alphaSynuclein(/)/Elk1(/) doppel tween C57BL/6JIco and C3HeB/FeJ) were am (4)Neuropathology,Univ.Muenchen;(5)BrainR knockout Tieren analysiert werden, in wie weit plified from these pools by polymerase chain re esearch,Univ.Tuebingen;(6)Center of der MAPKinase Weg und nachfolgende Stof action (PCR) and analyzed by agarose gel elec Mol.Biology,Univ.Heidelberg,(8)St.Josef fwechselwege speziell des Dopaminstoffwech trophoresis. The SMA002 mutation was mapped Hospital,Bochum;Germany;(7) Inst.of sels beeinträchtigt sind. Dies könnte wichtige to chromosome 13 to the locus between the mi Neurodegenerative Hinweise auf eine physiologische Funktion bei crosatellites D13Mit20 (35cM) and D13Mit253 Diseases,Univ.California,USA der Gene im Dopaminstoffwechsel ergeben. Bei (37cM). In this region, Madh5 (Smad5) and Neu alphasynuclein, a protein enriched in presynap den jeweiligen einzelknockoutTieren ist ein rod3 are promising candidate genes which are tic nerve terminals, may have an important role Verlust dopaminerger Neurone zunächst nicht currently being analyzed. in the development of Parkinson´s disease, de offensichtlich, jedoch zeigen alphaSynuclein( mentia with Lewybodies and other neurodegen /) knockout Mäuse eine gestörte Dopamin erative diseases, also known as synucle freisetzung. alphaSynuclein und der Transkrip inopathies. To elucidate its involvement in these tionsfaktor Elk1 wiederum interagieren mit der P218 10 diseases, we have used the tetoff transactiva MAP Kinase ERK2, so dass eine Beteiligung der tor system to generate conditional transgenic durch Elk1 initiierten Genexpressionskaskade im The Munich ENUmousemutagenesis mice. The tet system allows conditional expres Dopaminstoffwechsel zu untersuchen ist. Ander project: Achievements of the clinical sion of human alphasynuclein as a function of erseits gibt es bisher keine Untersuchungen, ob chemical screen an update oral administration of tetracycline analogs. sich Elk1 wie alphaSynuclein in den Lewy Kör Rathkolb, Birgit(1), Fuchs, E.(2),. Soewarto, To control the expression of human alphasynu perchen ablagert und damit den MAPKi D.(3), Mohr, M.(1), Klempt, M.(1), Hrabé de clein in mice and to direct the expression to the naseweg bei der PD beeinträchtigt. Angelis, M.(3), Kolb,H.(2) and Wolf, E.(1) brain, we used a tetracycline controlled transac Wir haben daher alphaSynucleinüberexprim (1) Institute of Molecular Animal Breeding tivator (tTA) driven by the PrP gene control ele ierende transgene Mäuse mit Tieren mit re and Biotechnology, Gene Center, LMU ments and a tTAresponsive promotor (PhCMV* duzierter Proteindegradation (UbcM4/UbcH7(/ Munich, Germany, (2) Institute of Clinical 1) linked to the human alphasynuclein gene. )) gekreuzt. Die zu erwartende Komplexität der Chemistry, City Hospital Harlaching, To analyze the conditional expression of human beeinträchtigten Stoffwechselwege bei den dop Munich, Germany,(3) Institute of alphasynuclein in transgenic mice, we per peltransgenen Tieren wird gegenwärtig mit Hil Experimental Genetics, GSF Research formed Western blot analysis with protein ex fe der Transkriptom und Proteomanalyse (DNA Center, Neuherberg, Germany tracts from several tissues and immunostaining Microarrays und MALDI) weitestgehend charak The clinicalchemical screen detects new mouse of paraffin embedded brains. terisiert. mutants with clinically relevant phenotypes with We showed that double transgenic mice express in the offspring of ENU mutagenized founder an human alphasynuclein in a brainspecific man imals by a combination of clinical, clinicalchem ner, whereas little or no alphasynuclein protein P218 09 ical and hematological investigations. Mice are was detectet in mice carrying only the alpha weighed every other week and examined clini synuclein construct. The alphasynuclein ex cally. Routine diagnostic procedures are utilized pression in doubletransgenic mice was abro Phenotyping and linkage analysis of a new to measure hematological and 17 clinicalchem gated to basal levels upon administration of mouse line: SMA002 ical parameters, including plasma enzyme activ doxycycline for 20 days due to the binding of Howaldt, Maike(1), Krebs, O.(1), Rathkolb, ities, specific substrates and electrolytes. Addi doxycycline to tTA. We currently investigate, B.(1), Matiasek, K.(2), Hrabé de Angelis, M.(3) tionally we started to analyze spot urine samples wether doubletransgenic mice show a neu and Wolf, E.(1) for qualitative protein excretion by SDS poly ropathological or behavioral phenotype. The (1) Institute of Molecular Animal Breeding, acrylamide gel electrophoresis at the end of conditional expression of human alphasynucle Gene Center, LMU Munich, Germany, (2) 2001. A detailed description of the screening in in mice may help to gain insight to the patho Institute of Veterinary Pathology, LMU protocol was published in Mammalian Genome genesis of synucleinopathies. A future challenge Munich, Germany, (3) Institute of Vol 11, No 7, pp 543546. Until April 2002 about is to investigate, if downregulation of gene ex Experimental Genetics, GSFResearch 12000 F1 offspring from mutagenized founder pression could slow down or stop the progres Center, Neuherberg, Germany animals were screened for dominant mutations. sion of the neuropathology. This could facilitate Among these 285 mice were identified carrying the development of pharmacotherapeutics

88 medgen 14 (2002) The initial step included identifcation and char to study the causes and consequences of acterization of the orthologous murine gene. We chronic azotemia and the pathogenetic connec established the genomic organization of the tions of azotemia and anemia. A backcross was P218 12 Vmd2 gene and fully sequenced a 8.2 kb region set up to map the HST001 mutation by mi from exon 3 to the 3’UTR. The Nterminus of crosatellite linkage analysis. Investigations into the mechanism of retinal the deduced amino acid sequence is highly con degeneration in a mouse model for Xlinked served between human and mouse (83% identi juvenile retinoschisis ty), while the sequence identity deviates sub P218 15 Andrea Gehrig(1), Heinrich Schrewe(2), stantially in the Cterminal region (29% identity). Bernhard H.F.Weber(1) Via sitedirected mutagenesis, the missense mu (1)Institute of Human Genetics, Biocenter, tation Tyr227Asn was introduced into the murine Characterization of mouse models for bone Am Hubland, D97074 Würzburg; (2)School Vmd2 gene. This mutation segregates in a large and cartilage related diseases from the of Biosciences, The University of Best disease family and is located in an evolu Munich ENUMouseMutagenesis Screen Birmingham, Edgbaston, Birmingham, UK tionary highly conserved region. As selection Abe Koichiro (1), GrundnerCulemann E. (1), Deleterious mutations in RS1 encoding retino marker for the homologous recombination event, Wagner S. (1), Flaswinkel H. (2), Fuchs H. schisin are associated with Xlinked juvenile the neor gene was engineered in close proximi (1)and Hrabe de Angelis M. (1) retinoschisis (RS), a common form of macular ty to the mutation. The final Vmd2 knockin tar (1)GSF Center of Environment and Health degeneration in males. Retinoschisin is a retina geting construct is composed of a 1.8 kb 5’ Institute of Experimental Genetics, specific polypeptide of 24 kDa that is secreted fragment from exon 3 to intron 6 containing the Neuherberg, Germany; (2) Institute of as a disulfidelinked oligomeric protein complex mutation, a neor cassette and a 4.2 kb 3’frag Medical Microbiology, Immunology and from both the rod and cone photoreceptors and ment from intron 6 to intron 9. Prior to electro Hygiene, Technical University of Munich, the bipolar cells. The protein consists almost ex poration into WW6 murine embryonic stem cells, Germany clusively of a discoidinlike domain that has the construct was verified by restriction enzyme Within the Munich ENUMouseMutagenesis been implicated in cell adhesion and cellcell in digestions and linearized with XmaI. Currently Screen several mutants with defects in bone or teraction. To gain further insight into the function we are screening for ES colonies harboring the cartilage development have been detected. In

of retinoschisin and its role in the cellular pathol homologous recombination event some of these mutants, we performed an in Abstracts GfH ÖGH SGMG Tagungsband ogy of RS, we have generated a knockout (Vmd2+/Tyr227Asn). Further steps will include depth phenotypic characterization and high res mouse deficient in Rs1h, the murine ortholog of microinjection of positive clones into recipient olution genetic mapping. RS1. In histologic murine eye sections, there is mouse blastocysts, determination of chimeras Two mutant lines, ALI14 and ALI18 exhibit a marked splitting of the inner nuclear layer, over and breeding of offspring to test for germline swollen limb phenotype at adult stage. In ALI18 all disorganization of the retinal cell layers with transmission. and ALI14 heterozygotes become red initially irregular displacement of cells, and later degen then swollen around 10 weeks of age. Homozy eration of the photoreceptors which appears gous ALI18 mutants exhibit a more severe phe more pronounced in cones. The observed dis notype than the heterozygous mice. The swollen P218 14 tortion of the retinal layers could be explained by hind feet phenotype in homozygotes can be ob the loss of cellcell and/or cell matrix interac served earlier, at 48 weeks of age, and subse tions, both of which are thought to be mediated Phenotypic characterization of an ENU quently severe swelling deforms toes. by the discoidin domain. We are now interested induced mouse mutant: HST001 In ALI14 homozygotes are sterile. Different ge to investigate whether apoptosis is the final Van Tuyen, Tran (1), Rathkolb, B. (1), Wanke, R. netic backgrounds can repress the phenotype pathway of photoreceptor cell death and cur (2), Hrabé de Angelis, M. (3), Wolf, E. (1) completely. rently pursue the developmental time course of (1) Institute of Molecular Animal Breeding The recessive mutant KTA041 shows a complex the degeneration. In addition, in the Rs1h/Y and Genetics, LMU, Munich, Germany, (2) phenotype. Homozygous KTA041 mice are retinae we observed disturbed vectorial trans Institute of Veterinary Pathology, LMU, smaller than wild type mice. They have a short port of PSD95 to the outer and inner plexiform Munich, Germany(3) Institute of er tail whereas the number of vertebrae is not layers, where Rs1h is ordinarily present in high Experimental Genetics, GSFResearch changed. In most animals the tail is kinky, some amounts. PSD95 is a synapseassociated pro Center, Neuherberg, Germany times quite severe. Some rib vertebrae have ab tein and plays a crucial role in the targeting and The dominant mouse mutant HST001 was iden normal shape. Strongly affected animals have rib clustering of ligand and/or voltagegated ion tified within the clinicalchemical screen of the fusions in the corresponding ribs. channels at synaptic junctions. This has prompt Munich ENUmousemutagenesis project by el Mutants are kept on a pure C3HeB/FeJ back ed further investigations into developmental as evated plasma urea concentrations in 3month ground, mapping of dominant mutants is carried pects of the Rs1h/Y retina with specific regard old animals. Since this mutant might be an inter out via an outcross back cross breeding strate to retinal synaptogenesis. esting model for diseases associated with gy with C57BL/6Jico mice. Recessive mutants azotemia, a more detailed clinical and patholog are mapped by outcrossintercross. ALI18 was ical characterization was performed. The body mapped to mouse chromosome 4. High resolu weight gain of wildtype mice in comparison with tion mapping is in progress. KTA041 was P218 13 mutant mice is characterized by a significantly mapped to chromosome 17. steeper slope of the growth curve from day 21 Candidate gene approaches are currently under Generation of a mouse model for Best to day 180. The body weights of the wildtype investigation. The mouse mutant lines ALI14 and vitelliform macular dystrophy mice are significantly higher than those of the ALI18 could serve as models for arthritis in hu Franziska Krämer(1), Burkhard Kneitz(2), mutant mice, which show progressive emacia man. Further experiments to prove this hypoth Bernhard H.F. Weber(1) tion with increasing age. Mutant mice are char esis are underway. (1)Institute of Human Genetics and acterized by progressively increasing plasma (2)Department of Physiological Chemistry I, urea concentrations and elevated plasma total Biocentre, Am Hubland, University of protein levels, but significantly reduced plasma P218 16 Würzburg, Germany glucose and triglyceride levels. At the age of 4.5 Best vitelliform macular dystrophy (BMD) is an and 6 months the hemoglobin concentration and autosomal dominant disorder of the macula the number of erythrocytes are reduced in mu Disruption of the meiotic chromosome core characterized by an accumulation of lipofuscin tant vs. wildtype mice. Mean corpuscular he alters telomere dynamics like material within the retinal pigment epitheli moglobin (MCH), mean corpuscular volume Liebe Bodo (1), Alsheimer M. (2), Höög C. (3), um (RPE) and by a progressive loss of central vi (MCV), and hematocrit (HCT) are also decreased Benavente R. (2), Scherthan H.(1) sion. A unique electrodiagnostic feature of BMD in mutant mice. In mutant mice, the plasma urea (1) MaxPlanckInstitut für Molekulare is an abnormal electrooculogram indicating that concentrations correlate negatively with the he Genetik, 14195 Berlin; (2) Dept. Cell & Dev. the RPE is the primary site of pathogenesis. The moglobin concentrations and the numbers of Biology, Biozentrum, Univ. Würzburg, 97074 diseasecausing gene, VMD2, encodes a 585 erythrocytes. The histopathological investigation Würzburg; (3) Karolinska Inst., Stockholm, amino acid residue protein, named bestrophin, of the kidneys did not show any sign of chronic Sweden which has 4 putative membranespanning do kidney disease. Azotemia can be caused by re Meiosis is a specialized division type that medi mains. To further elucidate the molecular patho duced urea excretion or increased urea produc ates chromosome pairing, recombination and in genesis of mutant bestrophin, we directed our tion due to an elevated protein catabolism and dependent assortment of homologues vital as efforts towards generating a knockin mouse is frequently associated with anemia. Therefore pects of sexual reproduction. During meiotic model targeting the murine Vmd2 locus. the HST001 mouse line is an interesting model prophase telomeres attach to the nuclear enve

medgen 14 (2002) 89 lope (NE) via a specialized thickening of the uals or their entire genealogy. Depending on the to produce about 2000 F1 animals to further iso ends of the axial cores that run along replicated values of the measurements, screeners can post late novel dominant alleles of known and new sister chromatids. This attachment plate likely requests for animal stock breeding, additional genes. reinforces chromosome connections to the NE samples of blood/tissue or confirmation crosses Currently, more than 30.000 mice have been in during a prophase stage where telomeres move from the core facilities. vestigated for dysmorphology and blood based along the inner nuclear membrane and homo 3. Within the Sample Tracking System (STS), parameters. To date, more than 400 mutant lines logues engage in pairing. Here, we have tested sample tracking lists are produced and modified. have been isolated. Novel dominant or recessive by analysis of Scp3 knockout spermatocytes the In addition, the storage of samples (e.g. tail clips phenotypes have been identified with specific role of the axial core in telomere clustering, mor for DNA extraction, frozen sperm) is being abnormalities comprising congenital malforma phogenesis of the attachment plaque and homo recorded. For example, if a query shows no liv tions, biochemical alterations, immunological logue pairing. Using threedimensional immuno ing animal of the desired phenotype within the defects and complex traits such as behaviour or cytology and FISH we show that SCP3/ telom animal facilities, information on archived sper predispositions to allergies. eres attach to the NE and perform meiosisspe mata will be displayed instead. Mutants of clinical relevance for inherited dis cific movements that involve the formation of a MouseNet© is an essential tool for functional an eases in human have been further analysed by chromosomal bouquet. The timing of the latter notation of the data generated in the project. It backcross mapping and genomewide mi is altered in that nuclei with clustered telomeres enables us to link data in appropriate formats to crosatellite typing. Many mutant lines deriving accumulate in mutant meiosis. EM analysis dis other genomedata resources. from this ENU Screen are under detailed pheno closed an altered morphology of the attachment References: typic characterisation and have been proceed plaques with the conical thickening pointing to 1. Hrabé de Angelis, M. et al. (2000) Nature Ge ed for fine mapping and positional cloning in or wards the NE missing at Scp3/ telomeres. netics 25, 444447 der to isolate the causative mutation (Kiernan et FISH with chromosomespecific probes revealed 2. Hrabé de Angelis, M., Strivens M (2001)Brief al. 2001, Vreugde et al. 2002, Graw et al. 2001). a defect in homologue pairing. However, when Bioinform 2, 170180 Recent mapping data of new mutant phenotypes homologues were regionally paired this involved will be presented. the formation of short stretches of SC. Immuno References fluorescent staining of meiosisspecific cohesin Graw J. et al. 2001, Exp Eye Res. 73, 867876 P218 18

GfH ÖGH SGMG Tagungsband Abstracts GfH ÖGH SGMG Tagungsband axes suggests that cohesin cores are not suffi Graw J. et al. 2001, Invest. Ophtalmol. Vis. Sci. cient to generate meiosisspecific chromosome 42,29092915 cores and their termini. A systematic, phenotypedriven Graw J. et al. 2001, Invest. Ophtalmol. Vis. Sci. mutagenesis for gene function studies: 42, 15741580 Recent results of the Munich ENUmouse Kiernan A. et al. 2001. PNAS 98, 38733878 mutagenesis screen Vreugde S. et. al. 2002. Nat.Genet. 30, 257258 P218 17 Dian Soewarto (1), Wagner, S.(1), Rathkolb, This work was supported by a grant from the B.(3), Mohr, M.(3), Flaswinkel, H.(2), German Human Genome Project to R.B., E.W. Data Management and Bioinformatics of the Fuchs,H.(1), Marschall, S.(1), Schäble, K.(1), and M.H.d.A. (01KW9923) Munich ENUMouseMutagenesis Project Tiedemann, H.(1), Alessandrini, F.(5), Schäble, Karlheinz F.(1), Tiedemann H.(1), Jakob,T.(5), Fuchs, E. (6), Kolb, H. (6), Hahn, A.(2), Schneltzer, E.(1), Steinkamp, R. (1), Kremmer, E. (7), Behrendt, H. (5), Ring, J.(5), P219 01 Pargent, W.(3), Stefan Hefner, Soewarto, D.(1), Zimmer, A. (8), Pfeffer, K. (2), Balling, R. (9), Fuchs, H.(1), Hrabé de Angelis, M.(1) Eckhard Wolf, E. (3) and Hrabé de Angelis, M. (1)Institute for Experimental Genetics, GSF (1) FISH for carrier detection of large deletions Research Center for Health and (1)Institute of Experimental Genetics, GSF in the Factor VIII gene Environment, (2)Genomatix Software GmbH, Research Center for Environment and Förster, Tanja, Guttenbach, M., Oldenburg, J., Landsbergerstr. 6, 80339 Munich, Germany Health, Neuherberg, Germany Müller, C.R. (3)Ingenium Pharmaceuticals AG, (2)Institute of Medical Microbiology, Institut für Humangenetik Würzburg, Fraunhoferstr. 13, 82152 Martinsried, Immunology and Hygiene, Technical Biozentrum, 97074 Würzburg, Germany Germany University of Munich, Germany Haemophilia A is a common congenital bleeding The ENU Mouse Mutagenesis Screen has been (3)Institute of Molecular Animal Breeding, disorder caused by a deficiency of coagulation set up as a large scale mutant production, phe Gene Center, University of Munich, Germany factor VIII. The disease is inherited in a sex notyping and mapping project. It encompasses (4)MaxDelbrueckCentre, Molekulare linked recessive pattern with an estimated inci two animal breeding facilities and a number of Genetik und Mikrosatellitenzentrum, Berlin, dence of 1 per 500010000 males. The gene en screening groups located within the general area Germany coding factor VIII is large (186 kb) and is locat of Munich. (5)Division Environmental Dermatology and ed about 1 MB from the telomere in Xq28. In order to guarantee multiuser access, com Allergology, GSF/TUM, Munich, Germany Asymptomatic female carriers transmit pleteness and consistency of the data generat (6)Institute of Clinical Chemistry, Clinic haemophilia A to 50 % of their male descen ed in a such a largescale project, a profession Harlaching, Munich, Germany dants. Large deletions comprise 3 % of the mo al database management system is a necessity. (7)Institute of Immunology, GSF Research lecular defects in severe haemophilia A. While Our system is based on Sybase Adaptive Serv Center for Environment and Health, they can easily be detected in males by PCR er Enterprise 12.5. running on a HewlettPackard Neuherberg, Germany and Southern Blot it is more demanding work to cluster (2 servers) on HPUX 11.0. We have de (8)Division Molecular Neurobiology, determine the presence of a large deletion in a veloped and implemented the ENUScreen soft Polyclinic for Psychiatry, University of Bonn, female. The second intact Xchromosome com ware system which is called „MouseNet©“. It Germany plicates the results of PCR based methods and enables database access to the screeners par (9)GBF German Research Center for at a best semiquantitative results e.g. by South ticipating in the ENU project via a Graphical Biotechnology, Braunschweig, Germany ern Blotting can be obtained. In order to un User Interfa With the completion of the human genome se equivocally assign the status of a potential fe ce based on common WebBrowsers and an quence and the prospect of a complete mouse male carrier of a large deletion we have estab ApacheWebServer (using Java applet or servlet sequence within the near future, a major chal lished FISH (fluorescence in situ hybridisation) techniques). MouseNet© consists of three ma lange is the systematic determination of gene for individual FVIII exons. PCR products con jor modules: function in mammals. The growing ENU mouse taining individual exons and part of their adja 1. The first module, the Animal Management mutant resource provides a powerful entry point cent introns were amplified to generate probes System (AMS), records all animals, location of to gene function studies. of about 5 kb. After labelling with fluorescent nu their cages and other data necessary for main Here, we give an update of one of the largest cleotides the probes were hybridised to taining a large mouse colony. It enables the an ENU mutagenesis programs in Europe, the Mu metaphase chromosomes of four mothers of pa imal caretakers to record any changes immedi nich ENU Mouse Mutagenesis Project. Since tients with known deletions. An Xchromosomal ately online on the database. The database it proofofprinciple of a large scale ENU mutage probe of the centromeric region was used as an self initiates actions by automatically generating nesis program has been demonstrated during internal control. All four women turned out to be work lists. the first phase of the project by focusing on carriers of the familial deletion. With probes of 2. The Result Documentation System (RDS) dominant traits, we put our main efforts during the deleted exon only one of the two Xchromo stores the data generated by the various the last year on the recessive screen by produc somes was labelled. In contrast probes of flank screens. The screeners are able to display re ing 100 micropedigrees (with 20 G3 offspring/ ing exons showed signals on both Xchromo sults for selected groups of mice, lines, individ micropedigree) per year. In parallel, we continue somes. The extend of the deletion (as known

90 medgen 14 (2002) from PCR analysis in the patient) could be con probe set, using all human centromeric probes Savelyeva L, Claas A, Matzner I, Schlag P, Hof firmed by FISH in all cases. In conclusion the labeled in different colors, allowing the simulta mann W, Scherneck S, Weber B, Schwab M.; FISHtechnique is well suited for direct visuali neous characterization and identification of all Cancer Res 2001 Jul 1;61(13):517985 sation of a large deletion in a female carrier. chromosomes by their centromeric region (Niet Schwab M, Claas A, Savelyeva L.; Cancer Lett zel et al., 2001, Hum Genet, 108, 199204). The 2002 Jan 10;175(1):18 technique, called cenMFISH has been extend ed by the introduction of an additional probe P219 02 specific for the short arm of all human acrocen P219 05 tric chromosomes called midi54 (described in Suspension fluorescence in situ Mrasek et al., 2001, Cytogenet Cell Genet, 93, hybridization (SFISH) a versatile 242248). This acro/cenMFISH probe set re A set of high quality FISH probes for the technique for interphase analyses vealed in the present case, that the both SMC detection of human chromosome Steinhaeuser, Ulf (1), Starke, H. (1), Nietzel, A. were identical derivatives of chromosomes 15 imbalances (1), Lindenau, J. (2), Ullmann P. (2), Claussen, with two specific signals for the centromere 15 Wirth, Jutta (1)(2), Tauchen, A. (1), Weidner, J. U. (1), Liehr, T. (1) specific and the midi54 probe. Additional FISH (2), SchmittJohn, T. (1)(2), Ehling, D. (1)(2) (1) Institute of Human Genetics and experiments using the high resolution multicol (1) Developmental Biology and Molecular Anthropology, Jena, Germany, (2)Carl Zeiss or banding (MCB) technique and probes specif Pathology, University of Bielefeld, Germany Jena GmbH, Jena, Germany ic for the PraderWilli/Angelman syndrome re (2) Praenadia GmbH, Muenster, Germany FISH on human chromosomes in meta and in gion (SNRPN and D15S10), respectively, char Conventional fluorescence in situ hybridization terphase is a well established technique in clin acterized the derivative chromosomes as dicen (FISH) is a useful tool for the detection of human ical cytogenetics as well as for studies of evolu tric isochromosomes i(15)(pter>q13::q13 chromosomal abnormalities in prenatal and tional and interphase architecture. However, all >pter). To the best of our knowledge, this is the postnatal diagnostics. The quality of DNA published FISH approaches are based on the first case with partial hexasomy 15pter>15q13. probes contributes to signal detection and is im airdrying procedure of chromosome preparation. Studies to clarify the origin of the derivatives portant for a confident interpretation of every This procedure leads to well spread metaphas (UPDanalysis) are in progress. In summary, FISH result. We have developed high quality

es on the slide surface, if an adequate humidity acro/cenMFISH is a very useful approach for FISH probes suitable for the identification of ma Abstracts GfH ÖGH SGMG Tagungsband in the air is present, and to a flattening of the the one step identification of all human chromo jor trisomies (21, 13, 18) as well as common originally spherical interphase nuclei. It is well somes by their centromeres and acrocentric p structural rearrangements such as deletion of suited for nearly all FISH approaches, however, arms. Supported by the Herbert Quandt Stiftung 22q11. Most of our BAC clones were isolated by when interphase architecture shall be studied der VARTA AG, the Wilhelm SanderStiftung using the REPuter program which allows to vi the flattening of the nuclei may lead to question (99.105.1) and the EU (ICA2CT200010012 and sualize distributions of exact and degenerate re able results. Here we present a technique, where QLRT199931590). peats with a minimal length of 20 bp. Regions the whole FISHprocedure is performed on cell with high gene content and relatively low repeat suspension and the cells are brought on a con density were selected and primers were de cave polished slide as the final step of the pro signed for the identification of BAC clones. Fur P219 04 cedure, just before the evaluation. We call this thermore, for improvement of the FISH signals procedure suspensionFISH (SFISH) and show we have assembled clone contigs and used that it is possible to do 3Danalyses on totally Genomic instability of distal 9p in families these as complex probe mixtures. These BAC spherical interphase nuclei or even on threedi with BRCA2 mutation carriers clones were mapped to different regions of chro mensional metaphases. The SFISH approach Katrin Arnold (1), Larissa Savelyeva (1), mosome 21 including the centromeric region of has been tested successfully on 10 different Andreas Claas (1), Wera Hofmann (2), Siegfried 21q11, the Down syndrome critical regions on chromosomal suspensions using up to twocol Scherneck (2), Peter Schlag (3), Manfred 21q22, and subtelomeric region of 21q22.3. or FISH experiments. Evaluation was done by a Schwab (1) Several DNA probes were isolated in the region ZeissAxioplan 2 or by a Zeiss Laser Scanning (1) Deutsches Krebsforschungszentrum, (2) of 13q32, 18p/qtel, 22q11 and subtelomeric re Microscope 510 META. It could be demonstrat MaxDelbrückCentrum für Molekulare gion of 22q13. The probe set extremely facili ed (i) that it is possible to perform the complete Medizin, (3) RobertRössleKlinik tates the detection of specific chromosome im FISH procedure in suspension, (ii) that it is not About 510% of breast cancer cases are asso balances on uncultured amniotic fluid cells and necessary for the evaluation of the interphase ciated with a genetic predisposition to the dis metaphase chromosomes and is greatly useful nuclei that those are fixed (and flattened) on the ease. BRCA2 is one of the most commonly mu for the identification of partial trisomies and par slide surface, and (iii) that even metaphases can tated genes in familial breast cancer. The major tial monosomies. be analyzed in principle three dimensionally by ity of germline mutations are predicted to inac SFISH. At present we are working on the adap tivate the BRCA2 protein. Recent studies show tation of the method for five color FISH experi that the BRCA2 protein is involved in homolo P219 06 ments. Supported by the Herbert Quandt gous recombination and DNA repair. Identical Stiftung der VARTA AG, the Wilhelm Sander BRCA2 mutations can display different cancer Stiftung (99.105.1) and the EU (ICA2CT2000 phenotypes and in comparison to population Prenatal diagnosis and molecular 10012 and QLRT199931590). based studies there is a greater penetrance in cytogenetic characterisation of a de novo high risk families. Thus the individual cancer risk interstitial duplication 16q11.2~13 a case may be determined by the type of mutation and report the environmental factors but it is also modified Trimborn, Marc (1), Toennies, H (1), Sarioglu, N P219 03 by additional genetic factors. FISH analysis of (2), Wegner, RD (1), Neitzel, H (1) lymphocytes of families with BRCA2 mutation Institute of Human Genetics, Charité, Characterization of two small carriers showed constitutional chromosomal al Humboldt University Berlin, Germany, (2) supernumerary marker chromosomes by terations of 9p2324 including duplications, am Department of Pediatric Pathology, Charité, acro/cenMFISH first case with partial plifications and inversions. The 9p rearrange Humboldt University Berlin, Germany hexasomy 15pter>15q13 ments are complex in all BRCA2 mutation carri We describe the first prenatally detected case of Heller, Anita (1), Albrecht, B. (2), Nietzel, A. (1), ers of the families tested. This indicates that this a de novo interstitial duplication of chromosome Starke, H. (1), von Eggeling, F. (1), Claussen, U. chromosomal region has suffered a number of 16q. This chromosomal aberration is extremely (1), Liehr, T. (1) intrachromosomal recombinations. In the 3 fam rare. There are only five comparable postnatal (1) Institute of Human Genetics and ilies analyzed the rearrangements identify an reports of duplications involving this chromoso Anthropology, Jena, Germany, (2) Institute overlapping region of recombinations ranging mal region. These patients present with little as of Human Genetics, Essen, Germany from D9S144 to D9S269. These alterations of 9p sociated dysmorphic features but significant Cytogenetic analysis performed in a three year were not detectable in a control group of individ neurodevelopmental delay. Amniocentesis was old girl resulted in a karyotype 48,XX, +2mar uals without BRCA2 mutations. Taken together indicated by advanced maternal age. During [25/25]. She had the severe mental retardation, these results suggest an association of BRCA2 pregnancy, ultrasound examinations of the fetus microcephaly, postaxial hexadactyly at both mutations with genomic instability in 9p2324 in showed no abnormalities. Conventional and mo hands and at the right foot plus a pachygyry. at least a number of BRCA2 mutation carriers. lecular cytogenetic analyses on cultured amnio Such small SMCs often are uneasy to character References: cytes by comparative genomic hybridisation ize in standard cytogenetic or molecular cytoge (CGH) and fluorescence in situ hybridisation netic approaches. Recently, we developed a (FISH) using partial chromosome paints and a lo

medgen 14 (2002) 91 cus specific YAC clone revealed a de novo direct We report on a case of mosaicism for two struc P219 10 duplication of the chromosomal region 16q11. tural aberrations of chromosome 22 in a 1 2/12 2q12~13 leading to a partial trisomy 16q (46,XX, year old girl. The child of healthy, unrelated par Segmental duplication of the region flanking dup(16)(q11.2q12~13).ish dir dup(16) (q12)(YAC ents was referred to chromosome analysis be the proximal NF1LCR in Pongo pygmaeus 744e11++)). After genetic counseling the parents cause of mild hyperplasia of the left lower leg, Hildegard KehrerSawatzki1, Horst Hameister1, opted for the termination of pregnancy. Post mild macrocephaly and mild facial dysmor Dieter E. Jenne2 mortem examination showed slight facial dys phisms. Body weight and size were normal. At 1:Department of Human Genetics, morphic signs, minor dysgenesis of the ovaries age of 2 years the right lower leg developed the University of Ulm, Ulm; 2: Department of and an atypical origin of the arteria thyreoida same mild and slowly progressive hyperplasia as Neuroimmunology, MaxPlanckInstitute of ima. the left one. There is no retardation in mental de Neurobiology, Martinsried velopment. Sequence analysis of the human genome re GTGbanding on chromosomes of the peripher vealed an unexpected high degree of recent al blood revealed a robertsonian translocation P219 07 segmental duplications, which have accumulat t(14;21) in all 15 cells analyzed and the presence ed during primate evolution. The dynamic nature of two clones with different derivative chromo of these duplications most probably contributed Detection of chromosomal methylation somes 22. The t(14;21) was inherited from the to variations of intra and interspecies genomic patterns using bisulfite treatment and mother, while the chromosomes 22 of both par architecture. Segmental intra or interchromoso fluorescence in situ hybridisation (FISH) ents were inconspicuous. A detailed characteri mal duplications are implicated in the generation Kaiser, Antje, Nietzel, A., Heller, A., Claußen, zation of the aberration in the child was done by of new or mosaic transcripts, thus contributing U., von Eggeling, F. the multicolor banding (MCB) approach. Thus, to gene evolution. Recent analysis of the entire Institut für Humangenetik und the two derivative chromosomes 22 could be genome suggests that interchromosomal dupli Anthropologie, FSU Jena, Germany characterized as del(22)(q13.2)[26] and r(22)inv cations are enriched in pericentromeric and sub Recently, additionally to genetic DNA changes, dup(p10q13.2)[4], which has been confirmed by telomeric regions. Aberrant recombination be epigenetic alterations have gained major atten FISHanalysis using regionspecific single copy tween duplicated genomic segments termed du tion in studies of human diseases including can probes. This is the second report on mosaicism plicons or LowCopyRepeats (LCRs) is the mo

GfH ÖGH SGMG Tagungsband Abstracts GfH ÖGH SGMG Tagungsband cer. Epigenetic phenomenon such as differential for structural aberrations of chromosome 22 in lecular basis of microdeletions or duplications of replicating timing, heritable chromatin structures volving a chromosomal deletion and an inverted the genomic region between these repeats in and foremost cytosine methylation do not affect duplication combined with a ring chromosome several genetic diseases. In about 510% of NF1 the base pair sequence but nevertheless regu formation, even though the breakpoint in 22q of patients, interstitial 17q11.2 microdeletions are late the expression of a steadily growing number the presented case is more distal than that re caused by nonhomologous recombination be of genes including many, who are linked to tu ported previously (Bergman and Blenow, Europ tween 60kb duplicons separated by ~1.5Mb. morigenesis, i.e. cancer stimulating genes J Hum Genet 2000; 8: 801804). FISH analysis has shown that the duplicons (oncogenes) and inhibiting genes (p16, p53). Supported by the the Willhelm SanderStiftung flanking the NF1 gene region are already present Methylation of cytosine within CpG islands in (99.105.1) and the EU (QLRT199931590). in the genome of the great apes. In the orang gene promotors has been shown not only to be utan (Pongo pygmaeus), however, we observed jointly responsible for silencing of genes but also an additional segmental duplication of about induce mutational events in early tumor progres P219 09 40kb flanking the proximal NF1LCR. This seg sion. The appearance of altered methylation pat mental duplication contains at least two genes terns in cancer such as promotor hypermethyla and has been transposed into the short arm of tion and genome wide hypomethylation may Aimed manipulation of Breast cancer Cell the orangutan chromosome 19 (PPY19), which serve as markers in tumorigenesis and enables lines by micro cell mediated chromosome 8 is homologous to HSA17. These findings allude prediction of the clinical behavior to improve the transfer (MMCT) results in suppression of to the dynamic nature of specific segments of treatment of individual patients. Different meth tumorigeneicity the NF1 gene region during primate evolution. ods have been developed to examine the methy Weimer, Jörg (1), Seitz, S.(2), Jacobsen, A. (1), Comparative FISH analysis on great ape chro lation profile of single gene sequences or to Grams, R. (1), PagerolsRaluy, L.(1), Scherneck, mosomes with subregional probes flanking the measure the overall content of 5methylcytosine S.(2), Arnold, N.(1) paracentric inversion of PPY19 revealed that but none enables to describe the extent and (1)University Kiel, Clinic of Gynecology and PPY19 is very similar to the ancestral form of chromosomal location of altered methylation Obstetrics, (2) MaxDelbrückCentrum of this chromosome in hominoids. Therefore, the patterns of transformed tissues. In our study, we molecular medicine in BerlinBuch, Robert segmental duplication in PPY 19p might also be performed a combination of sodium bisulfite RössleClinic ancestral and has been progressively eliminated modification of genomic DNA and a subsequent Cytogenetic and molecular evidence support the during the genome evolution of the African great fluorescence in situ hybridisation. For that DNA existence of a breast tumour suppressor gene(s) apes and human. This will be investigated by isolated from human lymphocytes and a colon on human chromosome 8. To identify a function molecular characterization of this segmental du cancer cell line was treated under conditions al role for this chromosome in breast cancer, we plication and comparative FISH analysis in gib whereby unmethylated cytosine are converted to introduced a chromosome 8 into the MDAMB bons and Old World mo uracil but 5methylcytosine remains nonreactive. 231 breast cancer cell line. The stable transfer After hybridisation of the purified and nicktrans of chromosome 8 resulted in suppression of tu lation labelled probes to human lymphocyte mourigenicity in nude mice and reversion of oth metaphases a hybridisation signal of converted er neoplastic properties of MDAMB231 hy P219 11 DNA can be seen especially in the heterochro brids. The transfer and changes of chromosome matic region of chromosome 1, 9 and 16. After material were controlled by micro satellite mark The Azoospermia region AZFa: An further improvements this technique may pro ers and a wide spectra of cytogenetic tech evolutionary view vide detailed information about individual tumor niques like SKY, CGH, FISHMD, and reverse Rainer Wimmer1, S. Kirsch2, G. Rappold2, W. characteristics and may enable improved diag painting. FISH with labelled mouse DNA exclude Schempp1 nostics and treatment of cancer. the presence of mouse chromosomes in cells af 1Institute of Human Genetics and ter transfer. This procedure explains the extent Anthropology, Freiburg, Germany of transferred chromosome 8 as a complete one, 2Institute of Human Genetics, Heidelberg, and the complete cytogenetic situation before P219 08 Germany and after transfer and infection in nude mice. A The human Y chromosome is unique in its bipar reduced representation of 8pterp21 in MDA tite structure and function. At the telomeres it Mosaic del(22)/r(22): characterization of the MB231 has compensated by the transferred presents the pseudoautosomal regions (PARs), derivative chromosomes by multicolor chromosome 8. Thus, the suppression of grow that undergo meiotic recombination with the X banding (MCB) and region specific probes ing of MDAMB231 correlates with the diploid chromosome. These PARs differ from the much Hartmann, Isabell (1), Starke, H. (1), Mitulla, B. presence of 8pterp21. These results provide larger nonrecombining region on the Y (NRY). (2), Beensen, V. (1), Heller, A. (1), Claussen, U. functional evidence that a breast cancer tumour Besides the testis determining gene SRY, the (1), Liehr, T. (1) suppressor gene (s) resides on human chromo NRY contains other genes and gene families that (1) Institute of Human Genetics and some 8. play an important role in spermatogenesis. Dele Anthropology, Jena, Germany, (2) Central tions detected on the Y chromosomes of infer Clinic Südthüringen, Suhl, Germany tile men gave rise to the definition of three re

92 medgen 14 (2002) gions, AZFa, b and c, that contain azoospermia P219 13 fected healthy probands but not the high copy factors. Because of the inhibition of recombina numbers occurring in MDpatients. tion, the NRY has evolved into a patchwork of In contrast to the regular PCR protocol with Rad51 protects cells in S/G2 phase from segments that show different stages of se primers spanning the repeats, our PCR primers radiationinduced chromosome quence homology to the X chromosome, and amplify a 200 bp region located 3´ to the CTG rearrangements additional segments that are Yspecific. Bound repeatstretch. Restriction enzymes are chosen Thomas Haaf, Elke Raderschall, Isabell Grandy, aries of these XY homologous segments may to cut 5`to the repeats and 3` to the PCR region. Eberhard Fritz be defined by evolutionary breakpoints, that are Thus, both the repeat and the PCR region are on Mainz University School of Medicine and marked by differences in the XY sequence sim the same restriction fragment. In case of a re Institute of Radiation Biology, GSF, Munich ilarity (Lahn & Page 1999). peat expansion, the fragment yielded by this re In order to assess the effects of Rad51mediat Via fluorescence in situ hybridisation (FISH) we strictiondigest is of increased size as opposed ed recombinational repair on chromosome sta have analysed the AZFaregion, important for to normal alleles. Thereafter the DNA is size sep bility, both chromosometype and chromatid human spermatogenesis. Its location was de arated by HPLC and the expanded fragment is type aberrations were scored in constitutively fined by a few infertile patients with deletions in isolated from the wild type allele. The subse Rad51overexpressing rat fibroblasts and Yq11.21. The variability in the extent of these quent PCR reaction of all fractions with our parental controls following treatment with differ deletions determines its maximal length of ap 3´primers yields positive products only in the ent doses of gamma irradiation. The number of proximately 1.4 Mb. collected fraction consistent with the WT allele chromosome breaks (gaps, deletions and rings) We have mapped a human Y chromosomal PAC and in those fractions containing much higher was the same in the two populations. However, contig spanning 2.8 Mb, that includes AZFa by fragment lengths due to expanded triplet repeats the number of chromatid breaks (gaps and frag FISH analysis. The hybridisation results for the . As added control, the WT allele fragment can ments) and exchanges (triradials), which result PAC clones of the classical AZFa differ from sur be analyzed by the regular PCR reaction that from doublestrand breaks (DSBs) generated rounding clones due to a lower conservation of spans the repeats. Conclusion: by using auto during or after replication, was significantly re the X/Y homology. This allows a new, evolution mated HPLC and a laboratory robot for the duced in Rad51overexpressiong cells. ary definition of AZFa with a size of about 1Mb. pipetting steps, all Southern/Northern blotting In a conceptually related experiment, synchro Comparative mapping of the PAC contig clones applications can be replaced by fully automated nized chick embryo fibroblasts (CEF) were tran

that form this evolutionary defined human AZFa HPLC plus PCR based kits. Abstracts GfH ÖGH SGMG Tagungsband siently transfected with human Rad51 gene ac region to higher primates may result in novel in tivity or control vector. The frequency of radia formation on the conservation emphazising the tioninduced translocations between macrochro importance of AZFa for spermatogenesis. mosomes was measured by spectral karyotype P220 02 analysis. After irradiation of S or G2 phase cells with a dose of 6 Gy, the macrochromosome Preparation and charaterisation of a protein P219 12 translocation frequenceny was significantly low expression library for mouse Th1 long er in the Rad51transfected cultures, compared stimulated cells to controls. However, the transfected Rad51 had FiberFISH mapping of MHC genes in the Claudia Gutjahr (1), Derek Murphy (1), Andrea no protective effect in cells that were irradiated rhesus monkey König (1), Hans Lehrach (1), & Dolores J. Cahill during G1 phase. Collectively, our experiments Huber, Isabell (1), Wimmer, R. (1), Walter, L.(2), (1, 2) suggest an increased recombination and repair Günther, E. (2), Schempp, W. (1) (1) Max Planck Institute of Molecular function of Rad51 in S/G2 phase. Homologous 1 Institute of Human Genetics and Genetics, Ihnestr. 73, D14195 Berlin; (2) recombination plays an important role in DSB re Anthropology, Freiburg, Germany, 2 Prot@gen AG, EmilFiggeStr. 76, D44227 pair in replicating mammalian cells. Department of Immunogenetics, Göttingen Dortmund In CEF cells the microchromosomes are usually The human MHCregion that spans about 4Mb In order to make a expression library for mouse distributed throughout the entire nucleus and, on the short arm of chromosome 6 in 6p21.3, is TH1 cells, we have directionally cloned the thus, in close physical contact with the ma essential for the immune system. Its hallmark is cDNA into a pQE32NSTattB vector, which per cochromosomes, whereas in chicken DT40 cells the extraordinary polymorphic structure and mits IPTGinducible expression of His6tagged the microchromosomes are sequestered in the evolution of this region. The MHC is among the recombinant proteins. The library was then center of the nucleus. The frequency of radia most gene dense regions of the human genome transformed by electroporation into Escherichia tioninduced translocations between microchro annotated so far. A first complete gene map of coli SCS1 cells carrying the plasmid pSE111 mosomes and macrochromosomes was signifi the human MHC was published by the MHCse with the lacIQ repressor and an argU gene, cantly higher in CEF than in DT40. Evidently, quencing consortium (1999). From centromere which codes for a rare arginine tRNA. The trans higherorder nuclear organization provides the to telomere the human MHC is divided into three formed cDNA library was plated on 22x22cm structural basis for the formation of celltype regions: MHC class II (1 Mb), class III (1 Mb) and agar plates and the clones are transferred into specific chromosome rearrangements. class I (2 Mb), (Campbell and Trowsdale 1993). 384 well plates by a picking robot. To date, over The region contains more than 220 gene loci of 80,000 clones have been picked. The average which 128 are predicted to be expressed. In insert size of this cDNA library, as determined by comparison to the human MHC little is known P220 01 PCR, is 1.5kb. In order to identify clones with an about the organization of the MHC of the rhesus insert in the correct reading frame, the His6tag monkey (Macaca mulatta), the primate species expression was examined. First the clones were Alternative to Southern/Northern Blotting: most extensively used in laboratory research. As spotted by a robot onto PVDF membranes. HPLC plus PCR based technique amenable a nonhuman primate its importance especially Then, after overnight growth on LB agar, protein to full automation increased as it is a valuable model for investigat expression was induced by IPTG. The mem K.R. Huber (1), R.E. Bittner (2), C. Wolf (3), J. ing human diseases such as AIDS. In the pres branes were next screened with an antiHis an Bittner (1), W. Rossmanith (2), I. Klinghofer (1), ent study we tried to shed light on the chromo tibody. Aproximately 17% of the total clones in K. Bauer (1) somal position and genomic arrangement of the library were found to express recombinant (1) Ludwig BoltzmannInstitut f. MHC genes in the rhesus monkey. Using fluores proteins. These expression clones have been re molekulargenetische cence in situ hybridization (FISH) we could un arrayed to create a protein expression subset. Laboratoriumsdiagnostik, Donauspital; (2) equivocally show that the rhesus MHCregion We have characterised the protein expression li Institut für NeuroAnatomie d. Univ. Wien; maps to rhesus monkey chromosome 6 in 6q2.4, brary using a number of monoclonal antibodies (3) Chemische Analysentechnik, Wien, the orthologous region to human 6p21.3.Further against proteins expressed in T helper cells. The Austria more we used fiberFISH to physically map five results of the antibody screening have also been We have begun to utilize an alternative method genes of the rhesus MHC on chromatin fibers re confirmed with both Western immunoblot analy for the diagnosis of myotonic dystrophy (MD) leased from cultivated rhesus blood lympho sis of individual clones and DNA hybridisation of that is based on HPLC separation of restriction cytes. Thereby we were able to orientate the DNA arrays made from this library, and will be fragments and subsequent PCR of collected rhesus MHC and to clear up the internal order of presented (Gutjahr et al., in prep.). fractions. Mutated MD alleles show expansion of these MHC genes of the rhesus monkey. CTGtriplet repeats with up to thousands of added repeats. Normal PCR analysis for repeat expansion is not always possible for this disor der because PCR can amplify only low copy numbers CTGtriplets as it is the case in nonaf

medgen 14 (2002) 93 P220 03 In approximately 510% of SRS patients, a the possibility of other yet unidentified genes in growth hormone (GH) deficiency can be ob volved in cystinuria. In contrast to recent publi served, one third of patients response to GH cations, we could not correlate the severity of Distribution of 13 polymorphic sites in the treatment. the disease to the type of cystinuria in the pedi SLC7A9 gene indicates an Considering these different observations, we de atric patients. cided to perform an exhaustive analysis of mem involvement of silent variants in the etiology bers of the GH/insulinlike growth factor (IGF) of cystinuria axis, mutations in which should cause growth P220 06 Christa Schmidt(1), Jürgen Tomiuk (2), Elke disturbances and GH excretion abnormalities. In Botzenhart (1), Udo Vester (3), Klaus a group of more than 40 SRS patients, we Zerres (1), Thomas Eggermann (1) analysed the genes for GHRH, GHRHR, GH and The LGI1 gene involved in lateral temporal (1) Institute of Human Genetics, Technical further members of the GH cluster including lobe epilepsy belongs to a new subfamily of University of Aachen, Germany, (2) Institute CSH1, IGF2, IGFBP1, IGFBP3, IGF1R, IRS1, leucinerich repeat proteins of Anthropology and Human Genetics, GRB2 and GRB10 for genetic variants. Addition Gu, Wenli (1), Grzeschik, K.H. (2), Derst, C. (3), Tübingen, Germany, (3) Children’s Hospital, ally, in case of GHRHR, GH, IGF2, IGFBP1, IGF Steinlein O. K. (1) University of Essen, Germany BP3, IGF1R and GRB10 the chromosomal local (1) Institute of Human Genetics, University Cystinuria is a hereditary disorder of cystine and isation on chromosome 7 or other candidate re Hospital, Bonn, Germany, (2) Department of dibasic amino acid transport across the luminal gions further indicates an involvement in the eti Human Genetics, University of Marburg, membrane of renal tubule and intestine resulting ology of growth disturbances. Germany, (3) Institute of Physiology II, in recurrent nephrolithiasis. While mutations in However, apart from polymorphic sites and hem University of Freiburg, Germany the SLC3A1 gene cause type I cystinuria, pa izygosity of CSH1, we did not detect relevant Recently mutations in the LGI1 (leucinerich, tients with nontype I cystinuria carry mutations pathogenic variations in the investigated genes. glioma inactivated 1) gene have been found in in the SLC7A9 gene. Both gene products form It can therefore be concluded that mutations in human temporal lobe epilepsy. LGI1 presented the renal amino acid transporter rBAT/b0,+AT af further, so far unknown factors, are involved in the first evidence that genes not apparently cod fected in cystinuria. growth regulation and the etiology of SRS. In ing for ion channels can cause idiopathic epilep

GfH ÖGH SGMG Tagungsband Abstracts GfH ÖGH SGMG Tagungsband In the present study a total of 59 patients with vestigations on such genes are still in progress. sy. We have now identified three formerly un different ethnic background were screened for known LGIlike genes (LGI2, LGI3, and LGI4). sequence variations in SLC7A9, out of these 32 Hydropathy plots and pattern analysis showed were of German origin. For determination of al that LGI genes code for proteins with an Nter P220 05 lele frequencies of detected polymorphisms 58 minal extra and an Cterminal intracellular do healthy German controls were investigated. Mol main connected by a single transmembrane re ecular genetic analysis was performed using sin Cystinuria in children: spectrum of gion. The phylogenetic analysis demonstrated gle strand comformation polymorphism analysis, mutations in SLC3A1 and SLC7A9 that the four human LGI peptides and their restriction assays and sequencing. Allele fre Thomas Eggermann(1), Schmidt C(1), Vester murine counterparts are closely related to each quencies were analyzed statistically for the de U(2), Botzenhart E(1), Wagner C(3), Zerres K(1) other, while other leucine rich repeatcontaining tected polymorphisms. (1) Institute of Human Genetics, Aachen, (2) proteins were placed more distantly on the evo In addition to the 6 already known variants we Children´s Hospital Essen, (3) Institute of lutionary scale. The branching pattern of the identified 7 new polymorphisms. Statistical Physiology, Zürich dendrogram strongly suggested that the four LGI analyses showed a significantly different distri Cystinuria is a common inherited disorder of de proteins belong to a formerly unknown subfam bution of alleles between German patients and fective renal reabsorption of cystine, ornithine, ily of LRR proteins. The distribution patterns of German controls in case of the polymorphisms lysine and arginine leading to nephrolithiasis. LGImRNA expression in different tissues were c.147C>T (exon 2), c.386C>T (exon 3), IVS3 Two responsible genes have been identified so studied by semiquantitative PCR with cDNA +22T>G, c.584C>T (exon 4), c.610T>C (exon 4), far: Mutations in the SLC3A1 gene encoding the from different tissues. All four LGI genes are ex c.692C>T (exon 5), c.852C>A (exon 6) and heavy chain rbAT of the renal cystine transport pressed in brain as well as in several other tis c.872C>T (exon 6). These results corroborate system rbAT/b0,+AT cause cystinuria type I, sues. In silico mapping and radiation hybrid ex previously published data on different frequen while variants in SLC7A9, the gene of its light periments assigned LGI2, LGI3, and LGI4 to dif cies of SLC7A9 alleles in cystinurics and con chain b0,+AT, have been demonstrated in non ferent chromosomal regions (4p15.2, 8p21.3, trols and indicate that silent variants in the type I cystinuria. In this study, we searched for 19q13.11). Besides the involvement in epilepto SLC7A9 gene are involved in the etiology of mutations in both genes in a cohort of children genesis LGI1 is suspected to act as a tumor cystinuria. with cystinuria. suppressor gene. It was therefore interesting to We analysed 21 cystinuric children from 16 fam see if the homologous genes hLGI2hLGI4 are ilies by mutational analysis of the genes SLC3A1 located in candidate regions for epilepsy genes and the SLC7A9. The patients were classified by and/or in regions showing loss of heterozygosi P220 04 the urinary amino acid excretion profile of their ty in different malignant diseases. (Gu et al., parents. Additionally, we screened 10 unclassi FEBS Letters, in press) Analysis of components of the GH/IGF axis fied patients for genomic variants. The screen in SilverRussell syndrome patients ing techniques included single strand conforma Susanne Mergenthaler (1), Sebastian Prager tion polymorphism analysis, restriction assays P220 07 (1), Miriam Mavany (1), Esther Meyer (1), Katja and direct sequencing. Eggermann (1), Michael B. Ranke (2), Gerhard Two novel mutations were identified in SLC3A1 Binder (2), Hartmut A. Wollmann (2), Thomas and three in SLC7A9; three were missense mu Refinement of the Mapping Region and Eggermann (1) tations and two frameshift mutations. In the pe Further Delineation of the Wittwer (1) Institute of Human Genetics, Aachen, (2) diatric patients, we could demonstrate mutations Syndrome Section of Pediatric Endocrinology, in 54% of type I (SLC3A1) and in 25% of non Wieland, Ilse; Muschke; P., Wieacker, P. Tübingen type I (SLC7A9) chromosomes. For this group of Institut für Humangenetik, Ottovon SilverRussell syndrome (SRS) is a heteroge patients a total detection rate of 46,6% for mu GuerickeUniversität, Magdeburg neous clinical syndrome with evidence for a sub tations in both genes could be delineated. In the A XLMRsyndrome mapping in Xp22.3 was pre stantial role of genetic factors in its aetiology. cohort of unclassified 10 patients, 70% of mu viously described by Wittwer et al. (Am J Med Apart from other clinical key features, severe in tations could be determined. M467T and G105R Genet 64: 4249). The spectrum of clinical symp trauterine and postnatal growth retardation are were the preponderant mutations in SLC3A1 and toms includes prenatal and severe postnatal the dominant characteristics of SRS. Therefore, SLC7A9, respectively; T216M was the major mu growth retardation, blindness due to microph studies on the genetic basis of the disease tation in Turkey and Greece. talmia or optic atrophy, moderate to severe hear aimed on genes involved in growth and its reg Interestingly, the detection rate for mutations in ing loss, dysmorphic features, epilepsy, and se ulation. Another key for the identification of (a) SLC3A1 and SLC7A9 in classified cystinuric vere mental retardation with absence of speech, SRS gene(s) is the finding of disturbances of children was lower than that in 10 further pa as well as anomalies of the urogenital system, chromosome 7, i.e. structural rearrangements tients with a nonclassified cystinuria, although gut and lungs. This family was reexamined for a and uniparental disomies, in nearly 10% of pa the clinical characterisation in the first group more exact mapping of the underlying gene. To tients; a putative SRS gene on chromosome 7 was more stringent; additionally, different spec the previously found clinical symptoms progres can therefore be assumed. trums of mutations were observed. The lack of sive skeletal lesions with osteoplastic and osteo detectable mutations in many patients indicate clastic changes were identified in the proximal

94 medgen 14 (2002) femora as well as vertebras. Haplotype analysis The competing efforts to sequence the entire hu within Poland and their distinctiveness from oth using 23 microsatellite markers on Xp22 local man genome have resulted in two draft se er parts of Europe, at least in respect to their Y ized the disease locus between DXS8095 and quences generated by the Human Genome Pro STR haplotypes. DXS7108 comprising 3.9 6.1 Mb. Within this in ject (HGP) and Celera Genomics (CEL), respec terval, at least one MRX locus involved in con tively. Upon global comparison, the two se tiguous genedeletion syndromes has been sug quencebased physical maps are similar in size P220 11 gested. Because the possibility of a contiguous and quality, predict a similar number of genes, gene syndrome has to be considered in Wittwer and are in good agreement with the existing ge syndrome, PCR analysis of the known genes netic maps. In the course of a positional cloning Molecular diagnostics in transcobalamin II and ESTs in the critical interval was performed. approach, we have examined in more detail a 20 deficiency No deletion was detected, suggesting that a Mb region of chromosome 16 and found signifi Silke Pauli, J. Häberle, M. Linnebank, E. contiguous gene deletion is rather unlikely in cant discrepancies. We have compared a YAC Schmidt, P. Navratil, H.G. Koch Wittwer syndrome. based physical map covering this chromosomal UniversitätsKinderklinik Münster region with the draft sequences of HGP and Transcobalamin II (TC II) deficiency is an auto CEL, the genetic map of Marshfield and the somal recessively inherited rare disorder of computed metrical map of the Location Data cobalamin metabolism. Most patients present P220 08 base (LDB). With respect to locus order, the YAC with combined methylmalonic aciduria and ho map was in good agreement with the Marshfield mocystinuria. Here, we report on two patients of Homozygosity Mapping of a Second Gene map, whereas both sequence drafts required turkish and lebanese origin and a consan Locus for Hereditary Combined Deficiency some „inversions“ for adjustment. Many discrep guineous background. Both patients presented of Vitamin KDependent Clotting Factors ancies were found in the LDB map. Possible rea during the first weeks of life with failure to thrive, (FMFD) to Chromosome 16 sons for these disagreements are problems with vomiting, muscular hypotonia, and also pancy Fregin, Andreas (1), Rost S. (1), Wolz W. (1), assembly in the physical maps, duplications that topenia. Treatment with parenteral cobalamin re Krebsova A. (2), Muller C.R.(1), Oldenburg J. are known to occur frequently on chromosome solved all symptoms and signs. (1, 4) 16 or errors in genetic and combined maps, re Using data bank analysis (GenBank), the entire

(1)Inst. of Human Genetics, Univ. of spectively. In particular, the generation of sec structure of the TC II gene including all exons Abstracts GfH ÖGH SGMG Tagungsband Würzburg, (2)MaxDelbrückCenter for ondary maps by computational integration of with their flanking regions was elucidated. Here Molec. Med., Berlin, (3)Inst. for Human primary data appears to be errorprone. If a sim by, the diagnosis of TC II deficiency could be Genetics, FriedrichAlexanderUniversity ilar level of accuracy were to apply to other confirmed by genetic analysis of cDNA and ErlangenNuernberg, (4)Institute of chromosomal regions it will still take some time gDNA in the two patients. In the lebanese pa Transfusion Med. and Immune Haemat. of to establish „the definitive map“ of the human tient, a 2148 bp deletion was detected by gDNA the DRK (Germany) genome. sequence analysis. This mutation causes a loss Familial multiple coagulation factor deficiency of exon 7 of the TC II transcript. The turkish pa (FMFD) of factors II, VII, IX, X, protein C and pro tient showed the mutation Ivs 4175 A>T. This tein S is a very rare bleeding disorder with auto mutation results in a new 5’ splice site, leading P220 10 somal recessive inheritance. The phenotypic to an insertion of 87 bp in the TC II transcript presentation is variable with respect to the resid and premature termination. ual activities of the affected proteins, its re Homogeneity and distinctiveness of Polish Until now, only 3 patients with 5 mutations of the sponse to oral administration of vitamin K and to paternal lineages revealed by Y TC II gene were described. Since the exact gene the involvement of skeletal abnormalities. The chromosome microsatellite haplotype structure was not known, found deletions could disease may result either from a defective re analysis not be determined precisely. The complete char sorption/transport of vitamin K to the liver, or R Ploski*, M Wozniak@, R Pawlowski#, DM acterization of the entire TC II gene structure will from a mutation in one of the genes encoding g Monies&, W Branicki$,T Kupiec$, A facilitate molecular diagnostics in the future. carboxylase or other proteins of the vitamin K Kloosterman**, T Dobosz@@, E Bosch&&, M cycle. We have recently presented clinical details Nowak*, R Lessig##, MA. Jobling&&, L of a Lebanese and a German family with ten and Roewer##, and M Kayser$$ P220 12 four individuals, respectively, where we pro *Warsaw Medical Univ. @Univ. School of posed autosomal recessive inheritance of the Medical Sciences Bydgoszcz #Medical Univ. FMFD phenotype. Direct sequencing of gamma Gdañsk &Medical Univ. Lublin. @@Medical Methylation sensitivePCR improves PCR carboxylase and microsomal epoxide hydrolase Univ. Wroclaw $Inst. Forensic Research testing of myoclonus epilepsy type revealed no mutation. Biochemical investigations Krakow, POLAND Unverricht Lundborg of vitamin K components in patients’ serum Different regional populations from Poland were Weinhäusel A.1; Morris M. 2, Waldner P. 1, showed a significantly increased level of vitamin studied in order to assess the genetic hetero Antonarakis, S. E. 2, and Haas, O. A. 1 K epoxide, thus, suggesting a defect in one of geneity within Poland, investigate their genetic 1 Children’s Cancer Research Institute the subunits of the vitamin K 2,3epoxidase re relationship with other European populations (CCRI), St. Anna Children’s Hospital, Vienna, ductase (VKOR) complex. We now have per and provide a populationspecific reference Austria; 2 University Hospital Geneva, formed a genome wide linkage analysis and database for anthropological and forensic stud Division of Medical Genetics, found significant linkage of FMFD to chromo ies. Nine Ychromosomal microsatellites were Geneva,Switzerland some 16. A total maximum twopoint LOD score analysed in a total of 919 unrelated males from The UnverrichtLundborg type of progressive of 3.4 at theta = 0 was obtained in the interval of six regions of Poland as well as in 1273 male in myoclonus epilepsy (EPM1; MIM 254800) is an 3 cM. In both families patients were autozygous dividuals from nine other European populations. autosomal recessive disorder. It is caused by the for more than 20 markers in this region, suggest AMOVA revealed that all of the molecular varia functional impairment of the cystatin B gene due ing inheritance by descent(IBD). tion in the Polish dataset is due to variation with to various point mutations or an expansion of a in populations, and no variation was detected dodecamer repeat within the untranslated 5’re among populations from different regions of gion of the gene. Both expanded and mutated Poland. However, in the nonPolish European alleles can occur in a homozygous form or may P220 09 dataset 9.3% (P < 0.0001) of the total variation concur in compound heterozygotes. The status was due to differences among populations.Con of the dodecamer repeat is usually determined Comparison of the Human Genome sequently, differences in pairwise RSTvalues by Southern blot and PCR analyses, the latter of Sequence Data to Genetic and Physical between all Polish populations were notstatisti which is, however, significantly hampered by the Maps of a 20 Mb Interval on Chromosome cally significant, whereas significant differences high CG content of the respective repeat region. 16 were observed in nearly all comparisons of Pol We have developed a MSPCR strategy to as Rost Simone (1), Fregin A. (1), Wolz W. (1), ish and nonPolish European populations. Phy sess the EPM1 methylation and expansion pat Müller C.R.(1), Oldenburg J. (1, 2) logenetic analyses demonstrated tight cluster terns in the cystatin B promoter and repeat re (1) Institut für Humangenetik, Universität ing of Polish populations separated from non gions. We show that the promoter of the cystatin Würzburg, Germany, (2) Institut für Polish groups. Population clustering based on B gene is unmethylated in both normal and ex Transfusionsmedizin und YSTR haplotypes generally correlates well with panded alleles and, therefore, exclude that de Immunhämatologie des DRK the geography and history of the region. Thus, novo methylation is part of the disease process. Blutspendedienstes, Frankfurt, Germany our data are consistent with the assumption of In contrast to the FRAX syndrome, methylation homogeneity of presentday paternal lineages patterns can therefore also not be exploited for

medgen 14 (2002) 95 diagnostic procedures. However, as expected, To identify longrange regulatory elements risk of sensorineural hearing loss is shown in we found that DNA deamination significantly im around human and Fugu rubripes transcription heterozygous carriers. Functional studies are in proves the PCR conditions for the amplification factor gene TWIST we compared 2.4 Mb and 60 progress to determine the role of WOLFRAMIN of the GCrich repeat region. Thus, we were able kb of sequence around human and F. rubripes during the hearing process. The upcoming re to reliably amplify fully expanded alleles in a se TWIST, respectively. Besides TWIST, we identi sults should clarify the pathogenic effect of mu ries of homo as well as heterozygous individu fied only six and five protein coding genes in the tations in this protein. als. We therefore conclude that our MSPCR ap human and Fugu sequences, respectively. The Supported by the FritzThyssenStiftung. proach is a simple and fast alternative that can Fugu Twist showes all common features of the replace the laborious Southernblot analysis for TWIST protein family, i.e. the basichelixloop the evaluation of the EPM1associated dode helix, and WRdomain, respectively. P220 16 camer repeat expansion. TWIST and three additional genes of F. rubripes have been mapped on human chromosome 7p21.1, whereas two genes were localised on Mutations in the TYR and P gene in human chromosome 3p25 in the same direction individuals with oculocutaneous albinism P220 13 and orientation. All genes located on 7p15 Opitz, Sven (1), Kaufmann, M. (1), Schwinger, showed high conservation in their exonintron E. (1), Zühlke, C. (1), KäsmannKellner, B. (2) Random Xinactivation in sporadic and structure. Most of the coding exons are identi (1) Institut für Humangenetik, Universität zu hereditary Medullary Thyroid Carcinoma cal in size, whilst most of the introns are dramat Lübeck, Germany, (2) Augenklinik der (MTC) ically compressed in Fugu. The order of genes Universitätskliniken des Saarlandes, Petra Zeitlhofer, Andreas Weinhäusel, between human and Fugu is nearly conserved Homburg/ Saar, Germany Katharina Niederle, Petra Waldner, and Oskar with the exception of HDAC9 which is missing in Oculocutaneous albinism (OCA) is a genetically A. Haas the analysed Fugu genomic region and a mir heterogeneous disorder with autosomal reces Ludwig BoltzmannInstitute for Cytogenetic rowed position of LOC168829. By sequence sive inheritance. Caused by phenotypic variabil Diagnosis (LBICD) and Children’s Cancer comparison between human, mouse and Fugu, ity clinical diagnosis of the albinism type is diffi Research Institute (CCRI), St. Anna we identified conserved noncoding sequences cult. In contrast, genetic analysis allows precise

GfH ÖGH SGMG Tagungsband Abstracts GfH ÖGH SGMG Tagungsband Children’s Hospital, Vienna, Austria in the 3´end of human TWIST which give evi classification and may be helpful for counselling We have set up a methylation sensitive PCR as dence for cisregulatory elements. of the patients. say for determination of Xskewing using two Ch.Kosan is a fellow of the GRK767 supported Here, we report the mutation profile detected by different microsatellite markers. FMR1 CGG re by the Deutsche Forschungsgemeinschaft molecular genetic analyses of the genes respon peat and HUMARA CAG repeat which are locat sible for OCA1 and OCA2. OCA1 is caused by ed on Xq27.3 and Xq11.2q12, respectively have mutations in the tyrosinase gene (TYR) encoding been used for dignostic as well as clonality and the key enzyme in melanin biosynthesis. We in P220 15 Xinactivation studies within different approach vestigated the 5 exons of the TYR gene of 176 es using standard PCR –amplification. Based on unrelated patients by PCR and SSCP and found bisulfite deamination of DNA we have designed Identification of a new missense mutation in variations in 66 individuals (47 patients with 1 a unique quantitiative PCR assay to amplify both WFS1 in a family with low frequency variation, 17 patients with 2, and 2 in 3 patients). unmethylated and methylated alleles within sin sensorineural hearing impairment OCA2 resulting from mutations in the human ho gle reactions for both repeat markers. Quaniti Kunz, Jürgen (1), MarquezKlaka, B. (1), Uebe, mologue of the mouse pink eye gene is the most fication of MSPCR products using both mark S. (1), VolzPeters, A. (1), Berger, R. (2), common type of albinism worldwide. Analysing ers allows us to determine the ratios of Xskew Rausch, P. (2) exons 2 to 25 of the P gene by PCR and SSCP ing from almost all cases informative either for (1) Institut für Allgemeine Humangenetik, aberrant signals could be detected in 36 per one of both markers. To elucidate the effect of PhilippsUniversität Marburg, Germany, (2) sons (34x1, 1x2, 1x3 variations). In 11 patients germline mutations of the RET protooncogene, Klinik für Phoniatrie und Pädaudiologie, conformational deviations were detected by predisposing for hereditary forms of medullary PhilippsUniversität Marburg, Germany SSCP in both the TYR and the P gene. In 85 pa thyroid cancer (MEN2, FMTC), with regard to We have previously reported a three generation tients (48.3%) no mutations were found. In our clonal selection and expression of disease in pa German family with an autosomal dominant low sample of German origin about 37.5% of the pa tients we investigated the Xinactivation patterns frequency sensorineural hearing impairment (LF tients show SSCP changes in the TYR gene and in different groups of female patients suffering SNHI) linked between the genetic markers pter 20.5% in the P gene suggesting that mutations from hereditary and sporadic medullary thyroid D4S431D4S432cen (Brodwolf et al. 2001) to at the latter locus are possibly underrepresent (sMTC). In addition we investigated X skewing the nonoverlapping interval DFNA6 and ed in Caucasians. The mutation profile present in unaffected RETmutation carriers, predispos DFNA14 (Lesperance et al. 1995; Van Camp et ed here was detected by sequence analysis fol ing for hereditary disease and healthy noncar al. 1999) on chromosome 4p16. The audiograms lowing SSCP screen. riers. By our approach we could only find some show consistently a hearing threshold of 50 ± 20 females with sligthly Xskewing, without any sig db hearing loss (HL) between 250 Hz and 12 nificant difference between hereditary and spo kHz. Normal hearing level is reached between 3 P220 17 radic MTC patients as well as mutation carriers and 6 kHz in all examined children. Adult pa and noncarriers. Thus we conclude that there is tients show an additional hearing impairment (HI) no clonal selcetion with respect to the mutator in the mid and higher frequencies that seems to Three members of a new gene family of low phenotype in blood of patients. differ from presbyacusis. The HI is always bilat copy repeats in Xp22.3 expressed eral and symmetrical. Recently, missense muta exclusively in testis: implications for tions were identified in the WFS1 gene, which recombinations in this region was found to map to this chromosomal interval, MartínezGaray, Isabel (1), Sutajova, M. (1), P220 14 segregating in three families with LFSNHI (Be Steuernagel, P. (2), Gal, A. (1), Kutsche, K. (1) spalova et al., 2001, Young et al., 2001). Conse (1) Institut für Humangenetik, Comparative genomics of the TWIST region quent screening of the WFS1 gene in our family Universitätsklinikum HamburgEppendorf, in human and Fugu rubripes resulted in the identification of a new heterozy Germany, (2) Institut für Klinische Genetik Jürgen Kunz and Christian Kosan gous missense mutation 2285G>C resulting in und Zytogenetik, Oldenburg, Germany (1) Institut für Allgemeine Humangenetik, an amino acid exchange K705N. This sequence Illegitime recombinations between lowcopy PhilippsUniversität Marburg, Germany variant was not found in 100 control chromo repetitive elements (LCR) have been implicated One of the most common craniosynostosis syn somes. in the pathogenesis of various chromosomal re dromes, SaethreChotzen syndrome (SCS, MIM The gene WFS1 is known to be responsible for arrangements. In Xp22.3, two such duplicons 101400), is caused generally by intragenic mu Wolfram syndrome type 1 or DIDMOAD, an au have been reported previously, the CRIS232 el tations of TWIST. However, several cases of SCS tosomal recessive disorder characterized by di ements, involved in the generation of deletions are associated with chromosomal rearrange abetes mellitus, diabetes insipidus, optic atro in the steroidsulfatase gene, and five members ments that leave the TWIST gene intact. Further phy, and deafness (Inoue et al. 1998, Strom et of the G1.3 (DXF22S) repetitive sequence fami more, patients with SCS and significant learning al. 1998). Based on only a few data it is remark ly. By molecular characterization of an Xp22/ disability, are described to have a chromosomal ably, the hearing impairment in WFS1 patients 10q24 in a female patients translocation, we microdeletion in 7p21 of approx. 2.4 Mb includ affects especially the high frequencies (Higashi identified a duplicon of the G1.3 family in the ing the TWIST locus. et al., 1991), but it has been reported that in breakpoint region in Xp22.3. We show that G1.3 families with Wolfram syndrome an increased harbours at least three expressed genes,

96 medgen 14 (2002) FAM9A, FAM9B, and FAM9C, and three putative Biochemie, ZMBE, Münster, Germany; 3 (multiplex). MALDITOFMS based genotyping pseudogenes, all mapped to Xp22.32p22.31. HämophilieZentrum an der Raphaelsklinik, results were confirmed by conventional se The open reading frames of the three genes Münster, Germany; 4 quence analysis. Due to its accuracy and molec show about 80% pairwise identity and 4665% Universitätskinderklinik, Münster, Germany; ular resolution, this procedure can serve as an identity at the amino acid level. FAM9A, FAM9B, 5 Medizinische Hochschule, Hannover, alternative method for clinical genotyping of and FAM9C are expressed exclusively in testis. Germany HBB alleles in the first screening round. By transient transfections of EGFPFAM9 con Haemophilia A is a common Xlinked bleeding structs, we found that all proteins were located disorder caused by various types of mutations to the nucleus, whereas FAM9A to the nucleo in the factor VIII gene. The most common intron P220 21 lus, in COS7 cells. Each of the proteins encod 22inversion is responsible for about 40% of the ed by members of this novel gene family show severe haemophilia A cases while large dele about 30% identity to SYCP3, a component of tions, point mutations and small (less than 100 TFNR is a subunit of TFIIIB and interacts the synaptonemal complex located along the bp) deletions or insertions are responsible for the with a novel zink finger protein paired chromosomes during meiosis. Thus, al disease in the rest of patients. Schoenen, Frank, Kelter, A.R., Wirth, B. though the function of the proteins is not yet We report on nine novel (6 deletions, two indels Institute of Human Genetics, University known, they might play a role in meiosis. It has and one partial duplication) and five recurrent Bonn, Germany been speculated that, in addition to the high se small rearrangements identified in 15 German The transcription factorlike nuclear regulator quence homology of LCRs, other recombination patients with severe haemophilia A, negative for (TFNR) is a novel human gene that maps on promoting factors may also be present within the intron 22inversion. c.22082214delTTATTAC 5q13, distally to the duplicated region which in duplicons, e.g. genes/pseudogenes expressed / c.22072215insCTCTT and c.46654678del / cludes SMN1, the spinal muscular atrophy in germ cells. Indeed, FAM9B was found in the c.46644678insAAGGAA identified in the pres (SMA) determining gene. TFNR shows homolo Xchromosomal breakpoint region of the ent study are the first small indels described in gy to yeast B’’ protein which is required for tran Xp22/10q24 translocation. It is tempting to the factor VIII gene. Our analyses suggest that scription of both TATAless and snRNAtype speculate that active transcription of FAM9B re the prevalence of this type of mutations (pre RNA polymerase III promotors and thus is an es sults in an open chromatin structure stimulating dominantly located in exon 14) among patients sential factor of the basal RNA poymerase III

recombination in meiotic germ cells of the pa with severe phenotype and negative for the transcription machinery. Northern blot and tran Abstracts GfH ÖGH SGMG Tagungsband tient’s father that might have promoted the re common intron 22inversion, is about 30%. The scription start point analysis allowed us to de combination event resulting in a translocation correlation between these molecular defects and termine a transcript length of ~ 10 kb. The TFNR between chromosomes X and 10. formation of factor VIII inhibitors as well as the transcript is highly expressed in cerebellum, parental origin of the de novo mutations are cerebral cortex and weakly in all tissues tested. evaluated. Finally we show that denaturing TFNR encodes a 2254 amino acids (aa) protein. HPLC (DHPLC) and classic heteroduplex analy The protein contains a SANT domain, and ssD P220 18 sis (HA) are able to detect these sequence alter NA and dsDNAprotein interactions were ations on 100% and could be preferred as a shown. Xlinked mental retardation with isolated screening approach when analysing for muta The Nterminal part of TFNR reveals a strong growth hormone deficiency tions in factor VIII in severely affected patients. protein interaction to a novel zincfinger protein J Tao, S Briault, BCJ Hamel, HH Ropers and identified by yeast two hybrid screens. The zinc VM Kalscheuer finger protein interacts with the first 640 aa of MaxPlanck Institute for Molecular Genetic, TFNR. It encodes a protein that contains three P220 20 Ihnestrasse 73, Berlin 14195, Germany zinkfinger domains (Nterminus) and one The aim of the project is to identify the underly POZ/BTB domain (Cterminus). Northern blots ing gene defect(s) in two families with Xlinked Genotyping of allelic variants of the human revealed a transcript length of ~ 5,5 kb in all mental retardation (XLMR) and isolated growth hemoglobin beta gene (HBB) by MALDITOF tested human tissues. An antibody generated in hormone deficiency (IGHD). Linkage studies nar mass spectrometry. rabbit shows a stronger expression in human rowed the interval to Xq24Xq27.3 and Xq22 Humeny, Andreas (1), Vetter, B. (2), Kulozik, brain and cerebellum on western blots. In im Xq27.1 (Hamel et al., 1996, Am. J. Med. Genet. A.E. (3), Becker, C.M. (1) munofluorescence stainings a colocalisation with 64:35, Raynaud et al., 1998, Am. J. Med. Genet. (1) Inst. f. Biochemie, EmilFischerZentrum, tubulin stained structures can be seen. Coim 76:255). We first established a complete contig Uni. ErlangenNürnberg, Fahrstr. 17, D munoprecipitation and functional analysis of the of the Xq26.1Xq26.3 region by using GenBank 91054 Erlangen, (2) Klinik f. Allg. Päd., Uni. novel interactor are in progress. and Celera sequence informations. This region Berlin, Augustenburger Platz 1, D?13353 Human and mouse antibodies from the N and was chosen as a starting point because a dupli Berlin, (3) Abt. f. Päd. Onkologie, Cterminus of TFNR were established and will cation in Xq25q26 and Xq26q27 is present in Hämatologie, Immunologie, Uni. allow ontogenetic and functional analysis of two families with a similar phenotype to IGHD, Kinderklinik, Im Neuenheimer Feld 150, D TFNR. Xlinked panhypopituitary (XPH) dwarfism 69120 Heidelberg (LagerströmFermer et al., 1997, Am. J. Hum. Allelic variants of the hemoglobin beta gene Genet. 60:910; Hol et al., 2000, Genomics. (HBB) are involved in different human disorders P220 22 69:174). Subsequently, candidate genes of the and are highly prevalent especially in the Xq26.1Xq26.3 region were analysed for their Mediterranean population. While the nucleotide expression in pituitary and hypothalamus RNAs. substitution FS6 A—>T leads to sickle cell ane Multiple cases of sporadic brest and ovarian Genes expressed in these tissues were further mia, absence or reduced amounts of beta chain carcinomas in BRCApositive families analysed by mutation analysis and semiquanti due to various frame shift mutations or nu Wappenschmidt, Barbara (1), Thomas A. (1), tative RTPCR. With the latter technique, one cleotide substitutions cause bthalassemia. Due Kuhl C. (2), Wardelmann E. (3) and Schmutzler gene was found to be overexpressed in the lym to the high prevalence of sickle cell anemia and R.K. (1) phoblastoid cell line of a patient with XLMR and bthalassemia, the HBB alleles represent impor (1) Departemnt of Obstetrics and IGHD. Expression studies in other affected and tant clinical targets for high throughput screen Gynecology, (2) Radiological Center and (3) nonaffected family members are in progress. ing. In this context, matrixassisted laser des Institute of Pathology, University Hospital orption ionization timeofflight mass spectrom Bonn, Germany etry (MALDITOFMS) based genotyping offers Aim: Since 1997, 221 families with hereditary a promising technical alternative to common breats and/or ovarian cancer were testet for mu P220 19 electrophoretic and chromatographic tech tations in the BRCA1 and 2 gene in our center. niques. Following PCR amplification using ge In high risk families, each patient was tested. PREVALENCE OF SMALL nomic DNA as a template, allelic variants of the The aim of this study was to analyse, if families REARRANGEMENTS IN THE FACTOR VIII HBB gene were determined by allele specific with high penetrances also show multiple cases GENE elongation and termination of extension primers. of sporadic breast and ovarian carcinomas indi Nadja Bogdanova1, Arseni Markoff2, Hartmut Primer extension products were analyzed by cating modifying factors. Pollmann3, Ulrike NowakGöttl4, Roswith MALDITOFMS. The nucleotide substitutions, Material/Methods: DNA of patients from 221 Eisert5, Bernd Dworniczak1, Antonin Eigel1 deletions and insertions of the HBB gene were families was analysed using PCR reaction fol and Jürgen Horst1* genotyped by this approach. Using MALDITOF lowed by the DHPLC method (Denaturing High 1 Institut für Humangenetik, UKM Münster, MS, the allelic variants were unequivocally and Pressure Liquid Chromatography) or direct se Germany; 2 Institut für Medizinische reliably detectable either alone or in combination quencing. In high risk families (* 2 patients < 50y.

medgen 14 (2002) 97 or patients with breast and ovarian cancer in one P220 24 ing of the CFTR gene. Mutations were not de family), if possible all index cases were analysed. tected by screening for the 33 most common Results: In 221 tested families, 41 pathological CFTR gene mutations or by sequencing exons Molecular characterization of a mutations and 33 unclassified variants were 224 of the CFTR gene. Exon 1, however, could chromosome 1;22 translocation in a patient identified. 34/41 pathological mutations were not be amplified by the commonly used exon with Costello syndrome: implication for the found in high risk families. However, in five of flanking PCR primers. Narrowing the critical re genes of the PDGF and PDGFR families in these families, additional noncarrier patients gion with several other primer pairs revealed an the trait were identified. The age of onset was 34, 39 and unexpected longer PCR product instead of the Sutajova, Marketa (1), Neukirchen, U. (1), 42y. in three cases of breast carcinomas and 39 predicted exon 1 amplicon. Sequence analysis Czeizel, E. (2), Timar, L. (2), Gal, A. (1), and 46y. in two cases of ovarian carcinomas, re identified a substitution of 120 bp, including the Kutsche, K. (1) spectively. whole exon 1 except for the start codon, by a (1) Institut für Humangenetik, Conclusions: In 14,7 % of high risk families with sequence of 300 bp from a reverted portion of Universitätsklinikum HamburgEppendorf, mutations noncarrier patients were identified the downstream intron. This unusual mutation, Germany, (2) OKK Családtervezési Központ, with a striking low age of onset and the oc termed 135del120ins300, may have been gener Budapest, Hungary curence of ovarian carcinoma cases. ated by incorrect DNA repair. While the patient Costello syndrome is a rare congenital disorder We postulate, that in these families modifying appeared to be homozygous for this allele, characterized by postnatal growth deficiency, factors exist, which result in a higher rate of analysis of her parents confirmed this mutation mental retardation, „coarse“ face, loose skin of „sporadic“ breast and ovarian carcinomas. in the father but not in the mother. Haplotype the neck, hands, and feet, cardiomyopathy, and Therefore, these families are an excellent tool to analyses with 13 different microsatellite markers nasal papillomata. Autosomal dominant inheri identify modifying factors who may also be in throughout chromosome 7 demonstrated a pa tence of the trait with de novo mutations has volved in sporadic breast and ovarian carcino ternal uniparental isodisomy of chromosome 7 been suggested whereas the genetic basis of genesis. in the patient. Two consecutive rare events thus the syndrome is yet unknown. We are investigat explain the homozygous appearance of a prob ing a female Costello patient carrying an appar ably rare deletion/insertion mutation in the ab ently balanced translocation t(1;22)(q25;q13.1). sence of consanguinity. Cases like this may be P220 23 By FISH analysis, cosmid LL22NC0310C3 was

GfH ÖGH SGMG Tagungsband Abstracts GfH ÖGH SGMG Tagungsband taken into consideration when routine diagnos found to overlap the breakpoint on chromosome tics fails to identify CFTR gene mutations in ei 22. Sequence analysis revealed that the gene Molecular evolution of FOXP2, a gene ther a clinically confirmed CF patient or one of encoding the betasubunit of plateletderived involved in the development of speech and the parents. growth factor, PDGFB, is located on the insert of language the cosmid. By RACE and RTPCR, we identi Enard, Wolfgang (1), Przeworski, M. (1), Fisher, fied three different PDGFBfusion transcripts in S. E. (2), Lai, C. S. L. (2), Wiebe, V. (1), Kitano, the patient containing either the complete P220 26 T. (1), Monaco, T. (2), Pääbo S. (1) PDGFB coding region or only part of it suggest (1)Max Planck Institute for Evolutionary ing that the patient is a mosaic and that the Anthropology, Leipzig, Germany A novel type of mutation causes a splicing PDGFB gene is disrupted in a portion of her (2)Wellcome Trust Centre for Human defect in ATM cells. Genetics, University of Oxford, UK Pagani, Franco (1), Buratti, E. (1), Stuani, C. (1), PDGFB belongs to a family including PDGFA, Language is a uniquely human trait likely to rep BendixWaltes, R. (2), Dörk, T. (2), Baralle, FE PDGFC, and PDGFD, that have important func resent a prerequisite for the development of hu (1) tions as paracrine growth factors in develop man culture. It has become clear in recent years (1) Molecular Pathology, International ment. The various plateletderived growth factor that aspects of speech and language develop Centre for Genetic Engineering and isoforms exert their effects on target cells by ac ment are likely to be influenced by genetic fac Biotechnology, Trieste, Italy; (2) Medical tivating two structurally related protein tyrosine tors. Furthermore, the ability to develop articu School Hannover, Hannover; Germany kinase receptors, alpha and beta, encoded by late speech relies on capabilities, such as fine Diseasecausing splicing mutations described in PDGFRA and PDGFRB. Thus, PDGFB as well as motoric control of the larynx and mouth1, which the literature are mainly concerned with changes all other ligands and receptors of the PDGF fam are absent in chimpanzees and other great apes. in splice sites and, to a lesser extent, with vari ily have been considered candidate proteins/ Recently, FOXP2, the first gene of relevance for ations in exonic regulatory sequences such as genes for Costello syndrome. Mutation screen the human ability to develop language, has been the enhancer elements. In the ATM cDNA of an ing in all a.m. PDGF(R) genes in 18 Costello pa described2. A point mutation in FOXP2 coseg affected patient we have identified a 65 bp aber tients has identified so far an amino acid change regates with a disorder in a large family in which rant exonic inclusion caused by deletion of four (Val722Phe) in PDGFRB in one patient, and an half of the members have severe articulation dif internal nucleotides in intron 20. Surprisingly, other one (Val1028Ala) in PDGFRA in a second ficulties, accompanied by linguistic and gram these four bases were located 12 bp down patient. Although neither of the changes has matical impairment3. In addition, this gene is di stream and 53 bp upstream from the borders of been found in 100 controls their pathogenetic rectly disrupted by a translocation in an unrelat the cryptic exon, respectively. Analysis of the relevance remains to be determined. In conclu ed individual who has a similar disorder. Thus, splicing defect using a hybrid minigene system sion, our data suggest that the PDGF and two functional copies of FOXP2 appear to be re led to the identification of a novel Intronic Splic PDGFR genes are not the major genes involved quired for acquisition of normal spoken lan ing Processing Element (ISPE) complementary in Costello syndrome. guage. In order to investigate the evolution of to U1 snRNA. This element exerts a positive ef FOXP2, we sequenced the cDNAs that encode fect for accurate intron processing and interacts the FOXP2 protein in chimpanzee, gorilla, orang specifically with U1 snRNP particle. The deletion utan, rhesus macaque and mouse, and com P220 25 completely abolished this interaction, causing pared them to the human cDNA. In addition, we the activation of the cryptic exon. In conclusion, investigated the intraspecific variation of the a new type of intronic U1 snRNP binding site A case from a routine CF diagnostic lab: FOXP2 gene in humans. The results strongly performs an essential function for accurate in Complex deletion/insertion involving exon 1 suggest that FOXP2 has been the target of se tron removal. Deletion of this sequence is direct of the CFTR gene in a patient with paternal lection during recent human evolution. ly involved in a splicing processing defect in a uniparental isodisomy of chromosome 7 human disease. Gläser, Dieter (1), Mau, U.A. (2), Kuhn, M. (2), Dörk, T. (3) (1) Gregor Mendel Laboratories, NeuUlm, Germany; (2) Department of Medical P220 27 Genetics, University of Tübingen, Germany; (3) Clinics of Obstetrics and Gynecology, Molybdenum cofactordeficient mice Medical School Hannover, Germany resemble the phenotype of human patients Large deletions may account for some cases and a suitable therapy model with cystic fibrosis (CF) in whom the underlying Lee, HeonJin; Adham, Ibrahim; Engel, mutation of the CFTR gene cannot be identified. Wolfgang and Reiss, Jochen Here, we report on a girl severely affected by a Institute of Human Genetics, Heinrich pancreatic insufficient form of CF whose sample DuekerWeg12, D37073 Goettingen, was provided for routine molecular genetic test Germany

98 medgen 14 (2002) The molybdenum cofactor (MoCo) is essential the brain of these mutant animals. This result Mal de Meleda (MDM), or transgressive palmo for the function of the molybdoenzymes sulfite suggest that SCD1 is inactivated in the homozy plantar keratoderma of Siemens, is a hereditary oxidase, xanthine dehydrogenase, and aldehyde gous animals. To address the question if this skin disorder characterised by diffuse palmo oxidase. Therefore, mutations of genes involved gene is involved in the phenotype of the pelota plantar keratoderma (PPK) and transgressive in MoCo biosynthesis lead to the inactivation of naked mice, we are performing molecular analy keratosis. There is no involvement of other or molybdoenzymes. In humans, MoCo deficiency ses on SCD1 gene, along with anatomopatho gans but a rather broad spectrum of clinical pre is a rare and devastating autosomalrecessive logical studies, in order to detect any malforma sentations with other features is characteristic. disease for which no therapy is known. Affected tion at the tissue level (liver, pancreas, brain, MDM was first described in patients from the patients show neurological abnormalities such skin). SCD1 gene is required for the normal func isle of Mljet (Meleda) in Croatia where a founder as attenuated brain growth and neonatal tion of skin and eyelid,being also involved in the effect was supposed to be responsible for MDM. seizures. Neurological damage due to sulfite tox cholesterol homeostasis and lipid metabolism. Recently, mutations in the ARS (component B) icity, sulfate deficiency, or a combination of Therefore, any new mutation in the SCD1 gene 81/s gene on chromosome 8q24qter were iden both, is irreversible, and many patients die might contribute to our knowledge about the fat tified in patients with MDM. We have shown that shortly after birth. Because most patients harbor ty acids metabolism, and pelota naked mice a very similar phenotype of transgressive PPK is mutations in the MOCS1 gene, we generated could be a reliable animal model for this pur not linked to chromosome 8q24qter in several mice with a targeted disruption of the murine pose. families from the United Arab Emirates. Here we MOCS1 gene by homologous recombination. A present further families with transgressive PPK. targeting vector was constructed to delete exon We identified four novel mutations in ARS (com 3 which encodes a highly conserved domain ponent B)81/s in consanguineous families from P220 29 from bacteria to man. As in humans, heterozy Turkey, Palestine, and the United Arab Emirates: gous mice display no symptoms, but homozy two different mutations both resulting in the gous animals die between days 1 to 10 after Missense substitutions differentially affect amino acid change G86R, and a mutation that birth. Homozygous knockout mice appear ex the function of ATM by dominant alters the translation initiation codon. In a Ger ternally normal, but are smaller and have a hy interference in patients with breast cancer man family without known consanguinity origi perextended posture as compared to their litter Scott, Shaun (1), BendixWaltes, R. (2), Chen, nally supposed to have a dominant form of

mates. Biochemical analysis shows that they do P. (3), Clark, R. (4), Dörk, T. (2), Lavin, M.F. (1) transgressive PPK we recognized pseudodom Abstracts GfH ÖGH SGMG Tagungsband not possess any molydopterin nor active MoCo (1) Queensland Institute of Medical inant inheritance. Three children and their affect and sulfite oxidase activity is undetectable. The Research, Royal Brisbane Hospital, ed mother were homozygous for mutation W15R knowledge gained about the genetics of MoCo Brisbane, Australia; (2) Medical School while the unaffected father was heterozygous. deficiency suggests that a small amount of Hannover, Hannover, Germany; (3) St. Pseudodominance was confirmed by analysis MoCo activity is sufficient for a normal pheno George Hospital, Sydney, Australia; (4) of several neighbouring microsatellites. Our find type. The described animals therefore are suit Department of Surgery, University of ings show that the MDM type of transgressive able animal models to try biochemical and/or Queensland, Australia PPK may be caused by SLURP1 mutations in genetic therapy approaches. Ataxiatelangiectasia (AT) is characterised by patients from various origins, and allelic hetero hypersensitivity to ionizing radiation and an ele geneity was demonstrated for mutations in vated risk of malignancy. Epidemiological data SLURP1. support an increased risk for breast and other P220 28 cancers in AT heterozygotes. It has been hy pothesized that ATM missense mutations are im P220 31 Stearoyl CoA desaturase gene family: plicated in breast cancer. To investigate the candidates for a sever mouse mutation functional significance of these changes we associated with hair loss have introduced missense substitutions, identi NOVEL MUTATIONS IN LAMIN A/C GENE Nica, G. (1), Gille, M. (1), Nürnberg, P. (2), fied in either AT or breast cancer patients, into AND AUTOSOMAL DOMINANT EMERY Adham, I. (1), Engel, W. (1) ATM cDNA prior to establishing stable cell lines DREIFUSS MUSCULAR DYSTROPHY (1) Institute of Human Genetics, to determine effect on ATM function. Pathogen Albena Todorova 1,2, MarieChristine Göttingen,Germany, (2) Gene Mapping ic missense mutations and neutral missense Dabauvalle 2, Wolfram Kress 1, Clemens R. Center, Max Delbrück Center for Molecular variants were initially distinguished by their ca Müller 1 Biology, Berlin pacity to correct the radiosensitive phenotype in 1 Department of Human Genetics, Biocenter Stearoyl CoA desaturase (SCD) is a microsomal AT cells. Furthermore missense mutations abol of the University of Würzburg, Würzburg, key enzyme required for the biosynthesis of ished the radiationinduced kinase activity of Germany and 2 Department of Cell and oleate and palmitoleate, which are the major ATM in normal control cells, caused chromo Developmental Biology, Biocenter of the monounsaturated fatty acids of membrane some instability and reduced cell viability in irra University of Würzburg, Würzburg, Germany phospholipids, triglycerides and cholesterol es diated control cells, whereas polymorphic vari The LMNA gene on 1q21.2q21.3 encodes nu ters. Alteration of the ratio of saturated to mo ants failed to do so. This approach represents a clear lamina proteins lamin A and C. Mutations nounsaturated fatty acids is involved in various means of identifying genuine ATM mutations and in the lamin A/C gene are responsible for 4 dif diseases,such as: noninsulindependent dia addressing the significance of missense ferent disorders: autosomal dominant Emery betes mellitus, neurological disorders, immune changes in the ATM gene in a variety of cancers Dreifuss muscular dystrophy (EDMD); limbgir diseases, cancer and obesity. There are three including breast cancer. dle muscular dystrophy type 1B (LGMD1B); Dun isoforms of SCD in the mouse genome, which nigantype familial partial lipodistrophy (FPLD) are highly conserved in their coding regions. and dilated cardiomyopathy and conduction However, the expression pattern of these genes system disease (CMD1A). Surprisingly, muta P220 30 is tissuespecific, SCD1 gene being highly ex tions in these nuclear envelope proteins lead to pressed in liver, SCD2 genein brain, and SCD3 muscular dystrophy. genemainly in skin. SCD genes are localized as Pseudodominant inheritance of Mal de Here we report 4 different mutations in the lamin a cluster on mouse chromosome 19. Here, we Meleda (MDM), or transgressive A/C gene, associated with EDMD phenotype. report a severe spontaneous mutation, associ palmoplantar keratoderma of Siemens, with Three of them are new ones: exon 3 583A—>G ated with hair loss. Linkage analysis has re mutations in the gene for SLURP1 (Asn195Asp), exon 6 1067T—>G (Leu356Arg), vealed that the DNA marker D19Mit 119 is close Hennies, Hans Christian (1), Eckl, K.M. (1,7), exon 6 1144G—>C (Gly382Arg). The fourth mu ly linked to the SCD locus. This new mutation is Stevens, H.P. (2), Lestringant, G.G. (3), tation in exon 9 1583C—>A (Thr528Lys) was de named pelota naked (pn), as compared to ase WestenbergerTreumann, M. (4), Traupe, H. (5), tected in 3 unrelated patients. The mutations in bia (ab) or scraggly (sgl) mutations, which are Frossard, P.M. (3), Stadler, R. (4), Leigh, I.M. exon 3 and 6 fall in the helical rod domain of the exhibiting skin and hair defects, and are also as (2), Reis, A. (6), Nürnberg, P. (1) protein and according to the published data, are sociated with mutations in SCD genes. The (1) Gene Mapping Centre and Molecular expected to provoke dramatical aberrant local pelota naked mutation is inherited in an autoso Genetics, MDC Berlin, Germany, (2) ization of the lamin A/C in many cells. The mu mal dominant manner, as opposed to the asebia Cutaneous Research, St Bartholomew’s, tation in exon 9 affects the globular tail of the and scraggly mutations. Northern blot studies on London, UK, (3) Tawam Hospital, Al Ain, protein and most probably disrupts proteinpro liver tissues from the homozygous pelota naked UAE, (4) Dermatology, Klinikum Minden, tein interactions. animals have shown that SCD1 transcript is Germany, (5) Dermatology, Univ. Münster, Our present knowledge on mutations distribution lacking from this tissue, as opposed to the Germany, (6) Human Genetics, Univ. along the LMNA gene and their effect on the SCD2 transcript, which is normally expressed in Erlangen, Germany, (7) Biology, Ch protein and/or clinical phenotype support the

medgen 14 (2002) 99 speculation that the lamins A/C do not play one in humans are known to cause mental retarda P220 35 simple role in the nuclear envelope, but most tion in humans. In each gene, we sequenced probably different parts of these proteins are en about 3kb in 20 humans sampled worldwide Treacher CollinsSyndrome: Molecular gaged in structural and/or regulatory functions. and 10 chimpanzees representing two sub Spectrum, GenotypePhenotype Correlation species. Nucleotide variability within and be in 42 Patients tween species was low, suggesting a high level Özge AltugTeber1, Beate Albrecht1, Martin of constraint at these loci. Overall nucleotide di P220 32 Sprengel2, Mine ArslanKirchner3, Christof versity is about twofold lower in humans than in Hammans4, Erdmute Kunstmann4, Wolfram chimpanzees. In contrast to previous studies, we Henn5, Georg Klaus Hinkel6, Jürgen Kunze7, Mutation analysis in autosomal recessive find no correlation between diversity and recom Luitgard Neumann7, HansDieter Rott8, Anita polycystic kidney disease (ARPKD/PKHD1) bination at these loci. Strikingly, we find that a Rauch8, Wolfgang Engel9, Barbara Zoll9, Zerres, Klaus, Senderek, J., Pegiazoglou, I., summary of the allele frequency spectrum is sig Monika HagedornGreiwe10, Rainer König11, Sedlacek, B., Puglia, P., Eggermann, T., nificantly correlated in humans and chim Angelika Albert12, Heide Seidel12, Gabriele RudnikSchöneborn, S., Bergmann, C. panzees, perhaps reflecting very similar levels of GillessenKaesbach1, Dietmar Lohmann1, Institute for Human Genetics, University of constraints in the two species. A possible ex Dagmar Wieczorek1 Technology, Aachen, Germany ception is FMR2, which shows more nonsyn 1Institut für Humangenetik, Universitäts Recently two groups independently identified a onymous than synonymous substitutions on the klinikum Essen, 2Klinik für Mund, Kiefer so far unknown gene responsible for autosomal human lineage, suggesting the action of positive und Gesichtschirurgie, Universitätsklinikum recessive polycystic kidney disease (ARPKD) on selection. Kiel, 3Institut für Humangenetik, chromosome 6p. The gene which we named Medizinische Hochschule Hannover, polyductin extends over at least 470 kb and in 4Abteilung Molekulare Humangenetik, cludes a minimum of 86 exons that are assem P220 34 RuhrUniversität Bochum, 5Institut für bled into a number of alternatively spliced tran Humangenetik, Universitätskliniken des scripts. The longest continuous open reading Saarlandes, Homburg/Saar, 6Institut füt frame encodes 4,074 amino acids. The gene SALL1 mutation analysis in TownesBrocks Klinische Genetik, Medizinische Fakultät

GfH ÖGH SGMG Tagungsband Abstracts GfH ÖGH SGMG Tagungsband products share structure homology with hepato syndrome an update Carl Gustav Carus, Dresden, 7Institut für cyte growth factor receptor and plexins which Liebers, Manuela; Kohlhase, J. Humangenetik, Charité, Berlin, 8Institut für belong to a superfamily of proteins involved in Institute for Human Genetics, University of Humangenetik, Friedrich Alexander the regulation of cell proliferation, adhesion and Goettingen, Goettingen, Germany Universität ErlangenNürnberg, 9Institut für repulsion. However, the definite gene function TownesBrocks syndrome (TBS, MIM 104780) is Humangenetik, Universität Göttingen, has still to be unravelled. a rare autosomal dominantly inherited malforma 10Institut für Humangenetik, Universität Thus far, we screened 78 patients diagnosed tion syndrome characterized by anal, renal, limb, Lübeck, 11Institut für Humangenetik, with ARPKD for polyductin mutations and iden and ear anomalies. TBS has been shown to re Universität Frankfurt, 12Abteilung für tified a total of 81 mutations making up a detec sult from mutations in SALL1, a human gene re pädiatrische Genetik, LudwigMaximilians tion rate of about 50%. We were able to disclose lated to the developmental regulator sal of Universität München two underlying mutations in 33 patients and only Drosophila melanogaster. Studies of its chick The Treacher CollinsSyndrome (TCS, Frances one mutation in 15 cases. Mutations were found homologue Csal1 suggest regulation of Sall1 ex chettiSyndrome, MIM #154500) is an autosomal in all areas of the gene but were three times as pression by Sonic hedgehog, Wnt3A/7A, and dominant disorder of craniofacial development frequent in the 5’ half of the gene compared with FGF4/8 signaling pathways. The SALL1 gene associated with mutations in TCOF1 on chromo the 3’ half. In detail the mutations were as fol product is a zinc finger protein thought to act as some 5q32q33.1. Phenotypic expression is lows: 37 missense mutations (46%), 24 non a transcription factor. It contains four highly con variable and is characterized by downward sense mutations (30%), 18 frameshift mutations served C2H2 double zinc finger domains which slanting of the palpebral fissures, lower lid (22%), and two splice site mutations (2%). Thus, are evenly distributed. A single C2H2 motif is at coloboma, hypoplasia of the zygomatic arch and about half of the changes is predicted to trun tached to the second domain, and at the amino mandible, external and middle ear anomalies, cate the protein. One recurrent missense muta terminus SALL1 contains a C2HC motif. The sensorineural hearing loss and cleft palate. tion in exon 4 most probably represents a muta protein is exclusively found in the nucleus and The TCOF1 codes for the 1411 amino acid pro tional hotspot. Two founder alleles were present localizes to pericentromeric heterochromatin, tein treacle which is a nucleolar phosphoprotein. in the Finnish population responsible for about acting as a transcriptional repressor. Here we It is composed of a 213 residue Nterminus fol 60% of mutations in this cohort. Genotype/phe present 7 yet undescribed mutations of SALL1 lowed by 10 repeated units with potential phos notype correlations could be established for the and an updated genotypephenotype correla phorylation sites and a Cterminus with multiple type of mutations rather than for the individual tion. One of the novel mutations is positioned at putative nuclear localization signals. To date, 51 mutation. the 3’ end of exon 2 and was found in a girl also distinct mutations have been reported. To ex The mutation detection rate of about 50% is mosaic for trisomy 8. Another mutation is plore the phenotypic spectrum associated with comparable to that achieved for other large mul strongly associated with eye defects in another mutations in TCOF1, we performed mutational tiexon genes showing the feasibility of direct ge family. Together, 28 out of 29 SALL1 mutations analysis in 42 patients with tentative diagnosis netic diagnosis in ARPKD. A more effective ap known to date are located in exon 2, 5’ of the of TCS. We identified heterozygous mutations in proach for mutation analysis of this huge gene third double zinc finger encoding region. These 25 of 35 patients with an unequivocal clinical di is currently worked out with the future aim of of are nonsense mutations, one of which causes agnosis of TCS, five of them being familial cas fering prenatal diagnosis based on direct geno TBS in nearly half of the sporadic cases, short es. In another seven patients with doubtful diag typing. insertions and short deletions as well as one nosis of TCS we did not identify a mutation. gross intraexonic deletion. One mutation within Of a total of 25 mutations, 23 are novel. The mu intron 2 creates an aberrant splice site. All mu tational spectrum comprises 12 frameshift, 5 tations lead to preterminal stop codons and are P220 33 nonsense, 5 splice site, one missense mutation thought to cause the phenotype via haploinsuf and one insertion of a lysin. In addition, we iden ficiency. However, Sall1 knock out mice do not tified a de novo base substitution affecting the Nucleotide diversity patterns within ten X show the TBS phenotype but homozygotes die start codon (M1I) in a patient with severe expres chromosomal genes in humans and from kidney malformations commonly seen in sion of TCS. The missense change is located in chimpanzees TBS. the first of the 10 repeated units and leads to a Kitano, Takashi, Schwarz, C., Nickel, B., substitution of a serine for an asparagine Przeworski, M., Pääbo, S. (S266N). Both amino acids have uncharged po Max Planck Institute for Evolutionary lar side chains. However, serine contains a po Anthropology, Leipzig, Germany lar hydroxyl group that may serve as a phospho Humans and chimpanzees differ in a number of rylation site. cognitive capabilities, some of which may be of We did not observe any correlation between lo fundamental importance for the differences in cation and nature of the mutation and phenotyp culture between the two species. In order to take ic expression. Moreover, mutation carriers with a first step towards studying genes that may be in families showed variable expression ranging of relevance for these differences, we have in from mild to severe phenotype. In conclusion, vestigated the pattern and extent of nucleotide diversity in 10 Xlinked genes where mutations

100 medgen 14 (2002) our data indicate that there is no obvious phe phoblastoid cell line of a patient with XLMR and nel CLCN7, demonstrating that inadequate bone notypegenotype correlation. IGHD. Expression studies in other affected and resorption by dysfunctional osteoclasts is the nonaffected family members are in progress. basis of this disorder in the majority of cases. In a small inbred family with malignant infantile os teopetrosis we could not find a mutation in ei P220 36 ther of the two known osteopetrosis genes. We P220 38 therefore started a candidate locus approach to The pseudoautosomal region is a fast elucidate the causative gene defect in this fam evolving region in primate genomes TGGE Screening in Marfan Syndrome and ily. By microsatellite analyses we excluded ho Katrin Schiebel (1), Wimmer, R. (2), Orlicz Related Fibrillinopathies: 53 FBN1 mutations mozygosity for several candidate gene loci Welcz, B. (1), Beck, S. (1), Henz, I. (1), Katzke, Stefanie (1), Booms, P (1), which cause osteopetrosis in knockout mice, Schempp, W. (2) Pletschacher, A (1), Neumann, L (2), among them cFOS on chromosome 14q24, c (1) Institut für Biochemie, EmilFischer Rosenberg, T (3), Robinson, PN (1) SRC on 20q11, OPGL on 13q14, and CSF1 on Zentrum, Universität ErlangenNürnberg, (1) Institute of Medical Genetics and (2) 1p13. In addition, we investigated a locus on Germany, (2) Institut für Humangenetik und Human Genetics Department of the Charité chromosome 6q21, that shows conserved syn Anthropologie, Universität Freiburg, University Hospital, Berlin, Germany, (3) teny to a region on mouse chromosome 10 to Germany National Eye Clinic for the Visually Impaired, which the locus for the osteopetrotic mouse The pseudoautosomal regions are located at the Hellerup, Denmark „greylethal“ has been mapped. We could tips of human X and Y chromosomes. They are The Marfan syndrome (MFS) is an autosomal demonstrate homozygosity in a region spanning identical on both chromosomes and recombine dominant heritable disorder of connective tissue approximetly 15cM between markers D6S1717 during male meiosis. Several human pseudoau with highly variable clinical manifestations, and D6S287 in the affected child. In this region, tosomal genes have paralogous genes located caused by mutations in the gene for fibrillin1 several functionally attractive candidate genes at different autosomes. Analysis of the genomic (FBN1). The cardinal features occur in the skele are located, like e. g. Fynrelatedkinase (FRK), region of the pseudoautosomal protein phos tal, ocular and cardiovascular system. BET3, and MARCKS. Currently, we are perform phatase gene PPP2R3L revealed that this ge We screened 126 individuals with MFS, other ing a systematic mutation screening of candi

nomic region is highly polymorphic in human type1 fibrillinopathies, and other potentially re date genes in this region. In summary, we pro Abstracts GfH ÖGH SGMG Tagungsband with 1 SNP in approx. lated disorders of connective tissue for FBN1 vide evidence for a novel locus for human infan 150 bp, whereas the autosomal PPP2R3 gene mutations, by developing temperaturegradient tile malignant osteopetrosis on chromosome has less than the average 1 SNP in 1 kbp. gel electrophoresis (TGGE) assays for all 65 6q21 which probably represents the human or Analysis of PPP2R3 and PPP2R3L in gorilla, FBN1 exons. We identified a total of 53 muta tholog of the murine „greylethal“ locus. chimpanzee and orangutan revealed that ex tions. Mutations were identified not only in per changes during evolution are more frequent in sons with classic Marfan syndrome but also in the pseudoautosomal than in the autosomal individuals with neonatal Marfan syndrome, P220 40 gene. As this is true for both exons and introns atypically severe Marfan syndrome, isolated we suggest that differences in selective pressure aneurysm of the ascending aorta, predominant arenot the reason for these differences. ectopia lentis, and several individuals with only DNA methylation analysis by MALDITOF Due to the high GC content of pseudoautosomal skeletal and ocular involvement. The overall mu P. Schatz, I. Schwope, J. Greese, R. genes DNA variability was expected to occur tation detection rate of 42% is typical of that re Wasserkort, M. Schuster, J. Distler predominantly within CpG ported for studies in large heterogeneous patient Epigenomics AG, Science Department, dinucleotides. Analysis revealed that differences populations. Kastanienallee 24, 10435 Berlin in C to T transitions are not sufficient to explain Together with the 53 mutations characterized in Epigenetic phenomena defined as any gene differences of variability. Further reasons may be our study, a total of 337 FBN1 mutations have regulating activity that doesn’t involve changes the genomic localization together with the ele been published to date. The mutations are to the DNA code and that can persist through vated recombination rate within the pseudoau spread over almost the entire FBN1 coding se one or more generations are manifested in tosomal region. quence, and most of the mutations have been DNA methylation, histone acetylation or methy identified in only one individual or family. The lation and formation of matrix attachment re only well established genotypephenotype cor gions. relation is the finding that almost all mutations in The connection between DNA methylation and P220 37 individuals with neonatal Marfan syndrome oc gene activity is widely recognized. Methylation cur in exons 2432, the socalled neonatal re of cytosine occurs mostly in a CG content. Aber Xlinked mental retardation with isolated gion of FBN1. We have analyzed our results to rant DNA methylation of CpG sites is among the growth hormone deficiency gether with other published results for potential earliest and most frequent alterations in cancer. J Tao, S Briault, C Moraine, BCJ Hamel, HH genotypephenotype correlations. Although the CpG methylation can be analyzed by transferring Ropers and VM Kalscheuer total number of mutations is still too low to allow the 5 base code (including 5methylcytosine MaxPlanck Institute for Molecular statistically significant conclusions to be made, 5mC) in to a 4 base code via a bisulfite treat Genetics, Ihnestrasse 73, 14195 Berlin, our analysis suggests correlations of subsets of ment. During this treatment cytosine is deami Germany mutations at the 5’ and 3’ ends of FBN1 with rel nated while 5methylcytosine stays unaffected. The aim of the project is to identify the underly atively mild clinical phenotypes lacking aortic A subsequent desulfonating step leads to an ing gene defect(s) in two families with Xlinked dissection. uracil or an unchanged 5methylcytosine, re mental retardation (XLMR) and isolated growth spectively. The information „methylated cyto hormone deficiency (IGHD). Linkage studies nar sine“ or „not methylated cytosine“ is transferred rowed the interval to Xq24Xq27.3 and Xq22 into a C/T(U) polymorphism. This polymorphism P220 39 Xq27.1 (Hamel et al., 1996, Am. J. Med. Genet. can be analyzed with standard SNP detection 64:35, Raynaud et al., 1998, Am. J. Med. Genet. assays. 76:255). We first established a complete contig Evidence for a novel locus for human Here we present a MALDITOF based methyla of the Xq26.1Xq26.3 region by using GenBank malignant infantile osteopetrosis on tion assay based on the methylation specific hy and Celera sequence informations. This region chromosome 6q21 bridisation of PNA probes to complementary tar was chosen as a starting point because a dupli Ramírez, Alfredo (1), Faupel, J. (1), Beyer, S. get sequences on standard glass slides. Glass cation in Xq25q26 and Xq26q27 is present in (1), Stöckel, C. (1), Hasan, C. (2), Bode, U. (2), slides offer the advantage that many samples two families with a similar phenotype to IGHD, Kubisch, C. (1) can be attached by standard arraying tech Xlinked panhypopituitary (XPH) dwarfism (1) Institut für Humangenetik und (2) niques and are available for simultaneous analy (LagerströmFermer et al., 1997, Am. J. Hum. Zentrum für Kinderheilkunde, sis. Oligonucleotides representing bisulfitecon Genet. 60:910; Hol et al., 2000, Genomics. Universitätsklinikum Bonn, Germany verted DNA were covalently bound to different 69:174). Subsequently, candidate genes of the Human malignant infantile osteopetrosis (OMIM activated surfaces and subsequently probed Xq26.1Xq26.3 region were analysed for their #259700) is a severe autosomal recessive dis with charge tagged PNA probes. Due to its un expression in pituitary and hypothalamus RNAs. ease characterized by osteosclerosis and pan charged backbone it is suitable for MALDI analy Genes expressed in these tissues were further cytopenia which become apparent during the sis. The modification with a tertiary ammonium analysed by mutation analysis and semiquanti first months of life. Causative mutations have re function (charge tag) enables highly sensitive tative RTPCR. With the latter technique, one cently been identified in the a3 subunit of the MALDI analysis in the positive ion mode. Charge gene was found to be overexpressed in the lym H+ATPase or the voltagegated chloride chan tag PNA libraries containing up to six different

medgen 14 (2002) 101 PNA´s were synthesized. After hybridisation of scribed linkage to chromosome 17 in PCa fami image, the sequence at the analysed SNP posi the entire library the PNA´s are bound to their lies. Germline frameshift, nonconservative mis tion is given together with additional statistics. complementary DNA sequences. Each charge sense mutations and diseaseassociated com We have applied this kind of SNPtyping scheme tag PNA has an individual mass. Therefore, the mon variants were found in this gene. However, to the genetic analysis of various variants of the methylation status of the probed DNA can be in one family the disease did not segregate green fluorescence protein (GFP). Furthermore, concluded from the mass of the cooresponding clearly with the mutation. Two common mis a broad lymphoma case control study 600 pa PNA detected by MALDITOF analysis. sense mutations S217L and A541T were identi tients and 600 controls was recently started in fied and the authors demonstrated that the collaboration with the German Cancer Research strongly linked L217/T541 variants occur more Center (DKFZ), looking at some 100 appropriate frequently in PCa cases than in agematched SNPs in parallel. P220 41 controls. This association was confirmed in sev eral studies, but no association was found in Characterization of a novel brain specific others. We have analyzed the allele frequencies P220 44 transcript on human chromosome 20q13 in 219 German PCa patients and 195 controls. Worch, Sebastian (1), Hansmann, I. (2), No association of the L217/T541 genotype with Schlote, D. (3) PCa was found in our study. In our search for a A new quantitative PCR Multiplex Assay for Institut für Humangenetik und Medizinische PCa tumor suppressor gene on chromosome 10 rapid analysis of HMSN / HNPP Biologie, MartinLutherUniversität Halle we have identified the smallest region of over Thiel, Christian; Kraus, C; Rauch, A; Ekici, AB; Wittenberg, Germany lapping deletions. This region spans 3.2 Mbp Rautenstrauss, B; Reis A The human chromosome 20q13 segment and oligonucleotides were deduced from all pre Institute of Human Genetics, University of (HSA20q13) is known to correspond to distal dicted exons. These will be arrayed onto glass ErlangenNuremberg, Germany mouse chromosome 2 (MMU2) as they show a slides and hybridized with normal/tumor RNA A 1.4Mb duplication containing the gene for pe conserved synteny of gene loci. Uniparental di pairs to identify genes that are turned off in PCa. ripheral myelin protein 22 (PMP22) on chromo somies for distal MMU2 result in different neona In addition, expression of known genes located some 17p11.212 is responsible for 70 % of the tal lethalities with opposite anomalous pheno in this region was analyzed by RTPCR using cases of the demyelinating form of Charcot

GfH ÖGH SGMG Tagungsband Abstracts GfH ÖGH SGMG Tagungsband types strongly suggesting the presence of im RNA extracted from paraffin embedded tumor MarieTooth disease (CMT1A/HMSN), whereas a printed genes in this region and therefore on the samples. For this purpose, we designed primer reciprocal deletion of the same region causes syntenic HSA20q13 as well. We have identified sets resulting in short PCR products. Some of the hereditary neuropathy with liability to pres a novel human gene within the region of interest these genes were downregulated or shut off in sure palsies (HNPP). The CMT1A duplication in whose expression appears to be restricted to tumors compared to adjacent normal tissue. creases the gene dose from two to three, the the brain as Northern analysis of 8 tissues re deletion reduces the gene dose from two to one. vealed expression of a 3.2 kb and a 3.0 kb tran Previously HMSN / HNPP patients were mainly P220 43 script in brain only. The corresponding cDNA diagnosed by analysis of polymorphic markers (AJ311122) contains a 1680 bp ORF distributed inbetween the repetitive elements flanking the on 13 exons spanning a genomic region of ap Designing & performing genotyping assays PMP22 gene and by fluorescence in situ hybridi proximately 250 kb. The homologous mouse with a flexible probing tool sation (FISH). We now developed a new real cDNA (AK005136) contains a 1680 bp ORF ei Hans Rebscher, Gilbert Karbach, Michael time qantitative PCR analysis assay for rapid de ther, and multiple tissue Northern analysis has Baum, Markus Beier, Peer Stähler termination of gene dosage. The method in revealed 3 major transcripts of 3.3 kb, 2.9 kb febit ag, Käfertaler Straße 190, 68167 volves a multiplex reaction using newly designed and 2.5 kb visible in brain only emphasizing the Mannheim FAM labelled TaqmanProbe with TAMRA human expression pattern. During detailed ex Having the sequence of the human genome fin quencher derived from PMP22 exon 3 and a VIC pression analysis using tissue from 8 different ished and more than 500 sequencing projects labelled probe with nonfluorescent quencher brain regions a complex pattern has been de ongoing, analysis of the genetic diversity repre from exon 12 of the Albumin gene as internal tected concerning the quantity of every single sents one of the next frontier of genome re reference. Reaction was carried out in a single transcript depending on analyzed tissue. A de search. With more than 60.000 estimated SNPs tube on a Taqman 7900HT. Copy number of the velopmental specific expression pattern has falling within exons the next step will be to iden PMP22 gene was determined by the compara been found during embryogenesis displaying tify those SNPs or combination of SNPs that tive threshold cycle method (DDCt). Each sam weak signals from day 10 pc and strong signals have a strong correlation to genetic disorders. ple was run in quadruplicate and analysed at from day 15 pc onwards to adult mice suggest To attack that great challenge flexible probing two different threshold levels. The level giving ing a function from late development. Character tools are needed. the smallest standard deviation was scored. We izing possible gene function, database search Employing febits Geniom technology that allows evaluated this method through the retrospective using the translated human gene product re microarray fabrication, hybridisation, detection analysis of 250 HMSN patients with known vealed homology to 3 hypothetical human pro and analysis within one single benchtop instru genotype and could confirm the previous find teins of yet unknown function sharing a striking ment, genotyping assays are easily set up. ings in all cases. Thus this method exhibits com homology of approximately 120 aa at the Cter Designing customized SNP arrays starts direct parable sensitivity to microsatellite analysis for minal end suggesting the existence of a shared ly from the gene sequences holding the SNP to duplication and FISH for deletions while it has domain. This homology seems to be conserved be investigated that are found in public databas the advantage of being a fast and uniform assay down to D. melanogaster and C. elegans. This es. Having selected of up to 250 SNPs that may for both HMSN / HNPP. work was supported by a grant of the DHGP. be screened in parallel into a spreadsheet, soft ware tools will generate an input file for the ge niom instrument, which will perform the synthe P220 45 sis of the SNParray. One of the current designs P220 42 follows a tiling scheme where each SNP is rep resented by 24 oligonucleotide probes including Spectrum of mutations found in 92 cases Association of HPC2/ELAC2 genotypes and screening for both the sense and the antisense with Noonan syndrome (NS) prostate cancer in the German population strand. Musante, Luciana(1), Kehl HG(2), Majewski and screening for other potential tumor Sample preparation starting from PCR fragments F(3)†, Meinecke P(4), Schweiger S(1), suppressor candidate genes is performed by employing terminale transferase GillessenKaesbach G(7), Hoeltzenbein M(1), Remus, R. (1), Gerasimovski, I. (1), Heller, Hilde and Dnase for labelling and fragmentation. Dur Tinschert S(5), Hinkel GK(6), Ropers HH(1), (1), Betz, B. (2), Niemann, A. (1), Niederacher, ing transferase reaction a biotin moiety is incor Kalscheuer VM(1) D. (2), Drechsler, M. (1), Wieland, C. (1), porated with in the target sequences. Fragmen (1)MaxPlanckInstitut für Molekulare Cronauer, M. (3), Scheil, H.G. (1), Royer tation of these products to appox. 150 bp in Genetik, Berlin,(2)Kinderkardiologie, Univ. Pokora, B. (1) length results in best performance during hy Münster,(3)Inst. für Humangenetik, Univ. (1) Institute for Human Genetics and bridisation. Detection is achieved through a Düsseldorf and (7)Univ. Essen, (4)Abt. Med. Anthropology, (2) Clinic for Gynaecology, (3) strepatavidinefluorophore conjugate. Genetik, Hamburg, (5)Inst. für Med. Genetik, Clinic for Urology, HeinrichHeine With the CCD based detection many fluo Charité, Humboldt Univ. Berlin and(6)Univ. Universität Düsseldorf, D40225 Düsseldorf rophores can be employed and two color detec Dresden A candidate prostate cancer (PCa) susceptibili tion permits the comparison of two samples Noonansyndrome (NS, MIM 163950) is a well ty gene on chromosome 17, HPC2/ELAC2, was within one experiment. After generation an inten recognized autosomal dominant multiple malfor described by Tavtigian et al. (2000). They de sity raw data file from the resulting fluorescence mation syndrome with an estimated incidence of

102 medgen 14 (2002) 1 in 1,000 to 2,500 live births. Affected individu cm² in normal confluent fibroblast cultures as in clinical presentation largely overlaps with the als have proportionate short stature and a char homozygous mutants. We propose that the in common Xlinked dystrophinopathies of the acteristic facial appearance with hypertelorism, creased amount of mutant TIMP3 is due to the Duchenne and Becker types, respectively. In the ptosis, downward slanting palpebral fissures, formation of protein complexes with a decreased majority of LGMD cases, the transmission mode and low set posteriorly rotated ears. In addition, turnover rate. This may lead to an unbalanced is best explained by autosomal recessive inher cardiac involvement, most commonly pulmonary activity of the metalloproteinases (MMP) and itance while autosomal dominant transmission valve stenosis, is frequently seen. NS is geneti struc has been documented in a few pedigrees only. cally heterogenenous. Linkage to a 5 cM region To date, at least 10 genes have been shown to on chromosome 12q24 has been reported pre cause some of the subforms of LGMD. These in viously and only recently, mutations in the pro clude the sarcoglycanes (a to e), a group of sar P220 47 tein tyrosine phosphatase gene PTPN11, have colemmal membrane proteins which are tightly been described. In a small sample, missense attached to dystrophin, calpain3, caveolin3, mutations were found in 50% of NS cases Activation of three different 5‘ cryptic splice dysferlin, telethonin and lamin A/C. Specific an (Tartaglia, M et al., Nat. Genet. 29: 465468, sites in exon 16 of the NF1 gene caused by tibodies to most of these proteins are available 2001). We have screened PTPN11 for mutations the same 5‘ splice site mutation in the and can be used for diagnostic screening on in 92 clinically well characterized familial and following intron. muscle tissues from biopsies. sporadic NS cases identified 16 different mis Mischung, Claudia, Tinschert, S. and Over the past 10 years we have applied an in sense mutations in 32 (35%). Six mutations, 4 in Nuernberg, P. creasing panel of these antibodies for diagnos the NSH2 domain and 2 in the PTP domain, are Institut für Medizinische Genetik, tic screening on Western blots of over 500 mus novel and have not been described before. In Universitaetsklinikum Charité, Campus cular dystrophy patients. By far the most com addition, we found 4 mutations (Asp61Gly, Virchow Klinikum, D13353 Berlin mon cause of muscular dystrophies are com Tyr63Cys, Ala72Ser, Asn308Asp) repeatedly in Detection of NF1 gene mutations is still a chal plete or partial deficiencies of dystrophin which apparently unrelated cases. Most mutations lenge, and no single technique is able to detect account for about 70 % of young patients (< 15 cluster in the SH2 domain at the Nterminus (N the entire NF1 mutational spectrum. The NF1 years at biopsy) and about 30 % of older cases SH2), which acts as a molecular switch between gene spans approximately 350kb of genomic (> 15 years at biopsy). Deficiencies of the sarco

the inactive and active protein form. No PTPN11 DNA comprising 60 exons. The open reading glycanes were rare in both age groups at 2 % Abstracts GfH ÖGH SGMG Tagungsband mutations were detected in 5 patients with car frame codes for 2867 amino acids including and 0 %, respectively. Defects in calpain3 were diofaciocutaneous syndrome (CFC), which three alternatively spliced exons. It is ubiqui observed in 9 % of the adult cases and 1 % of shares many phenotypic similarities with NS. tously expressed and encodes an 11 – 13kb the younger patients, thus constituting the most mRNA. Most mutations of the NF1 gene present frequent cause of LGMD. as base substitutions or other small lesions Upon sequencing of the calpain3 gene, we P220 46 throughout the coding sequence and the splice have identified to date 8 different mutations in 7 sites. To date, several hundred distinct NF1 mu index cases. A recurrent single base deletion Towards elucidation of the disease tations have been identified. (550delA) was observed on several alleles. mechanism underlying Sorsby fundus cDNAbased analysis is able to detect the ef dystrophy (SFD) fects of several different kinds of mutation on Galina Soboleva(1), Birgit Geis(1), Heinrich the RNA level. Missense and in rare cases silent P220 49 Schrewe(2), Bernhard H. F.Weber(1) mutations can generate cryptic splice sites (1)Institute of Human Genetics, Biocenter, which are responsible for aberrant splicing of the Am Hubland, D97074 Würzburg; (2)School transcript. In other cases mutations of the con GNAT2 is the third gene implicated in of Biosciences, The University of served intronic splice site cause activation of autosomal recessive achromatopsia Birmingham, Edgbaston, Birmingham, UK cryptic splice sites. Wissinger, Bernd (1), Baumann, B. (1), SFD is a late onset maculopathy caused by mu Exon 16 is one of the largest exons in the NF1 Rosenberg, T. (2), Kellner, U. (3), Lorenz, B. (4), tations in the tissue inhibitor of metalloproteinas gene and one exonic 5‘ cryptic splice site has al Vadala, M. (5), Jacobson, S.G. (6), Kohl, S. (1) es3 (TIMP3). In contrast to the other members ready been published to be acivated by a splice (1) Molekulargenetisches Labor, Uni of the TIMP family TIMP1, 2 and 4, TIMP3 is in site mutation (IVS 16+1 G>A). We detected the Augenklinik Tübingen, Germany, (2) National soluble and localised to the extracellular matrix same mutation in two unrelated patients and two Eye Clinic, Copenhagen, Denmark, (3) (ECM). The protein consists of 211 amino acids further mutations of exon 16 in two other pa Universitätsklinikum B. Franklin, Berlin, with 12 cystein residues forming 6 intramolecu tients (IVS 16+1 ins TT and G2709A). When Germany, (4) University Eye Hospital, lar disulfid bridges. All known mutations intro analysing the effects of the mutations on the Regensburg, Germany, (5) University Eye duce potentially unpaired cystein residues in the mRNA transcript by cDNA sequencing, we no Hospital, Palermo, Italy, (6)Scheie Eye Cterminus of the protein resulting in the forma ticed the activation of three different cryptic Institute, Philadelphia tion of higher molecular weight protein complex splice sites in exon 16. Both 5‘ splice site muta Achromatopsia is an autosomal recessively in es. We have now generated knockout as well as tions activated the known cryptic splice site at herited visual disorder that features the absence knockin mice carrying a diseaserelated cDNA position nt 2618, but in one of the patients of color discrimination from birth. We have re Ser156Cys mutation in the ortologous murine with mutation IVS 16+1 G>A two additional cently found that mutations in CNGA3 and gene. To study the nature and content of the splice sites were activated at nt 2707 and nt CNGB3 that encode the alpha and the beta higher molecular weight protein complexes and 2761 resulting in three different mutant tran subunit of the cone photoreceptor cGMPgated their possible role in the etiology of SFD we have scripts. Interestingly, one of them was the same channel can cause achromatopsia including Pin established immortalized fibroblast cell lines found in the patient with the silent mutation gelap blindness (Kohl et al. 1998, Nat Genet from the knockout and knockin mice. The G2709A (V903V). These results underline the im 19:257259; Kohl et al. 2000, Hum Mol Genet composition of the protein complexes was portance of cDNA analysis in understanding the 9:21072116). Screening for CNGA3 and CNGB3 analysed by mass spectrometry. In addition, effects of different mutations on mRNA splicing mutations in a large cohort of achromatopsia pa Western blotting was used to investigate the and their pathological consequences. tients, however, revealed further genetic hetero turnover rate of the mutant protein in the ECM. geneity in about one third of cases. We now re Further, we applied a lactic acid and other as port the identification of five independent achro says to study the metabolic changes due to the matopsia families that segregate protein trunca P220 48 mutation. Immunocytochemical techniques tion mutations in the GNAT2 gene located on were used to analyse cell morphology. To com chromosome 1p13. GNAT2 encodes the cone pare functional activities of the normal and mu Mutations in the caplain3 gene cause the photoreceptor specific alphasubunit of trans tant protein we used direct and reverse zymo most common form of limbgirdle muscular ducin, a Gprotein of the phototransduction cas grams. Taken together, our study demonstrates dystrophy cade that couples to the visual pigment(s). The a higher amount of TIMP3 in ECM from mutant Gerhard, Meng, Todorova, A., Müller, C. R., identified mutations most probably represent fibroblasts compared to normal. Moreover, ho Kress, W. functional null alleles of GNAT2, that prevent the mozygous Ser156Cys fibroblasts have a de Institute of Human Genetics, University of formation of the trimeric G protein complex or its creased mitochondrial activity. These data are Würzburg, Germany interaction with the excited photopigments. No supported by the lactic acid assay revealing 1.5 The limbgirdle muscular dystrophies (LGMD) tably, all genes now known to be involved in times less lactic acid in the conditioned medium are a genetically heterogeneous group of pro achromatopsia encode crucial components of of the mutant cells. Mutant fibroblasts are amoe gressive myopathies. In an individual male pa the cone phototransduction cascade. Due to the boid and flat. There are four times more cells per tient, the diagnosis is often difficult since the lack of cone function in these patients, it is rea

medgen 14 (2002) 103 sonable to argue that, like the conespecific Tyro 3/Axl family of receptor tyrosine kinases B) To map quantitative trait loci (QTL) in mouse cGMPgated channel, all three different types of that includes Axl, Rse, and Mer. Recent studies and rat strains it is necessary to analyse the human cone photoreceptors utilize a common demonstrated that autosomal recessive retinal genotypes of the cross progeny with a panel of transducin alphasubunit. Our results also indi dystrophy in the RCS rat is due to a loss of func markers spaced at 10 to 20 cM intervals. Allele cate that achromatopsia is a genetically very tion mutation in the gene (Mertk) encoding Mer. size data for the STR markers are provided in heterogeneous condition. Whereas mutations in Similarly, mutations in the human ortholog the public database only for a small set of inbred CNGA3, CNGB3 and GNAT2 together account (MERTK) were found in patients with autosomal strains and rarely for their different substrains. In for about 70% of the cases, the genetic etiolo recessive retinitis pigmentosa. We determined order to obtain panels of highly polymorphic gy of the remainders has still to be discovered. the exonintron structure of human GAS6 by markers applicable to different mapping projects genomewalking using oligonucleotide primers we have tested more than 300 microsatellite corresponding to the GAS6 cDNA. We per markers in 15 mouse inbred str formed mutation screening using SSCP and se ains and about 150 markers in 16 rat inbred P220 50 quence analysis of all GAS6 exons (except those strains or substrains. corresponding to nucleotides c.1389) and adja Isolated Cytochrome COxidase( COX) cent splice sites in 306 patients with autosomal Deficiency: genetic, clinical and recessive retinal dystrophy. The 2037 bp coding P220 53 neuroradiological heterogeneity region of GAS6 is divided into (at least) 15 ex P.Freisinger 1, R. Horvath1 D. Auer2, M. ons, ranging in size from 25 to 229 bp, with Jaksch 1 exonintron junctions conforming to the AG/GT A High Throughput Functional Screen For Metabolic Disease Center München consensus rule. Five sequence variants (c.862 Novel Factors That Induce Apoptosis Schwabing(1), MaxPlanck Institut for G>A, 1/712 alleles; c.1245G>A, 16/200 alleles; KönigHoffmann, Kerstin, Kazinski, M., Boche, Psychatry München(2) c.1332C>T, 48/712 alleles; c.1725C>T, 1/712 al I., Gille, H., Gnirke, A., Kesper, B., Korherr, C., COXdeficiency is the most commen cause of leles; c.1734C>T; 44/712alleles) were identified Klein, M., Liebetrau, W., Link, D., Röhrig, S., respiratory chain disorders. Combined COXde in the patient population screened. The latter Schäfer, R., Pessara, U., Hergersberg, C. ficiency is found in mutations of mitochondrial four are synonymous changes (Leu415, Cys444, Xantos Biomedicine AG, Fraunhoferstr. 22,

GfH ÖGH SGMG Tagungsband Abstracts GfH ÖGH SGMG Tagungsband (mt)DNA. In isolated COXdeficiency mutations His575, and Thr578), whereas c.862G>A effects D82152 Martinsried, Germany of mtDNAencoded structural genes and nuclear a Val284Met substitution present in the patient In the wake of the human genome effort, the encoded assembling genes ( SURF1, SCO2) are in heterozygous form. Additional sequence vari search for new pharmaceutical targets has been increasingly found. We compare molecular, clin ants identified in nearby intronic sequences are driven primarily by computational biology and ical and MRI/MRS data in 6 patients with isolat not predicted to affect splicing. Our results sug expression screening efforts. The functions of ed COXdeficiency. SURF1mutations result in gest that GAS6 may not harbour critical muta the genes identified need to be defined in sub „loss of SURFprotein“ and systemic COXdefi tions responsible for autosomal recessive retinal sequent experiments. Mammalian expression ciency. Patients present from 2 months of age to dystrophy, or that such mutations may be rare, screening approaches have thus far been limit 4 years with LeighSyndrome, encephalopathy, raising the possibility that alternative ligands ed to pool transfections resulting in limited sen developmental delay and myopathy. MRI shows may be involved in activating MERTK function sitivity. Heterologous expression screens e.g. in mainly lesions of the brainstem sometimes the necessary for phagocytosis by the retinal pig yeast have met with limited success. Single basal ganglia. ment epithelium. cDNA clone transfections offer clear advantages SCO2encodes a protein transporting Cu++ to with respect to sensitivity and hits. Also, since COX. The E140K mutation is found in all patients the functional assay is the first step in this type either compound heterozygous or homozygous. of analysis, the downstream evaluation of the P220 52 All patients develop encephalopathy, myopathy gene function is straightforward. So far, this ap and a live limiting cardiomyopathy (CM) with proach has been limited to the use of rather death during the first weeks in the heterozygous New STR marker panels for man, mouse and small selections of cDNAs due to limits in patients. Homozygous patients present later rat at the GMC throughput. We have set up a highthroughput with encephalomyopathy, peripheral neuropa Katrin Hoffmann, B. Meyer, B. Lucke, Y. Lee, K. expression screen for apoptosis where single thy and CM . MRI/MRS showed variably distrib Saar, M. Jung, G. Nürnberg, W. Gunia, T. cDNA clones are prepared on a robotics plat uted lesions in brainstemm, basal ganglia and Wienker, A. Reis, P. Nürnberg form. Subsequently, an automated transfection white matter. Genkartierungszentrum, MDC, BerlinBuch, and readout system completes the four robot A stopmutation (W58X) in COIII was found in a Germany cascade. Currently, the setup is capable of pro patient with extremely reduced COXactivity The Gene Mapping Center (GMC) in Berlin is ducing up to 150,000 complete functional as (10%) in muscle. Presenting at age 12 years with performing highthroughput genotyping for nu says and subsequent analysis for individual a nonprogressive mild myopathy his CNS is merous cooperative mapping projects in human cDNA clones per month. This assay allows for clinically and radiologically intact at 19 years and animal models to identify the underlying the unbiased screens of cDNA collections and li suggesting isolated muscle involvement. gene loci in monogenic and complex diseases. braries. The results of a first screen with exam In conclusion there is no clear correlation be We developed human, mouse, and rat short tan ples for novel apoptosis inducing genes with tween COXactivity in muscle and clinical sever dem repeat (STR) marker panels to meet the re new disease associations will be discussed. ity. In SURF1patients there is heterogeneity quirements of a broad range of project designs concerning symptoms and progression. In such as linkage analysis, homozygosity map SCO2patients E140K homozygous patients ping, QTL mapping, and haplotype sharing P220 54 show considerable differences in MRI/MRS and analysis. All marker panels are subject to regu clinical course. Surprisingly the COIIImutation lar updates. Our current marker sets are as fol causes very low COXactivity with a mild clinical lows: Molecular genetic studies in autosomal picture. A) The human marker set consists of three parts recessive hypotrichosis simplex that can be typed separately or in combination, Betz Regina C (1), Al Aboud K. (2), Al Hawsawi as needed by the project. Part I refers to our K. (2), Al Aboud D. (2), Al Githami A. (2), routine mapping set based on a modified We Dewald G. (3), Nöthen M.M. (1) P220 51 ber9Set (Marshfield Institute) and is used for (1) Department of Medical Genetics, standard genome scans (average density 10 cM, University of Antwerp, Belgium; (2) Genomic organization and mutation 411 markers, mainly tri und tetranucleotid Dermatology Section, King Faisal Hospital, screening of the GAS6 gene encoding an markers). Part II consists of 305 additional mark Saudi Arabia; Institute of Human Genetics, activating ligand for MERTK in patients with ers designed and tested at the GMC. Combina University of Bonn, Germany autosomal recessive retinitis pigmentosa tion of parts I+II results in an average density of Hypotrichosis simplex is a rare form of isolated Li, Yun (1), Thompson, D.A. (2), Gal, A. (1) 4.46 cM. For some project designs such as hap alopecia. A generalized form affecting all body (1) Institut für Humangenetik, lotype sharing analysis in isolated populations, hair is distinguished from a scalplimited form Universitätsklinikum Eppendorf, Hamburg, a higher density is needed. Therefore, 347 addi (HSS). In both forms, patients are born with nor Germany, (2) Kellogg Eye Center, University tional markers (Part III) were designed to close mal hair and show a gradual loss of hair begin of Michigan Medical School, Ann Arbor, MI, gaps in the combined set of parts I+II larger than ning at the middle of the first decade leading to USA 5.5 cM resulting in a genome wide average den a complete loss of scalp or body hair by the The protein encoded by GAS6 (growth arrest sity of 3.4 cM. third decade. All families described up to date specific gene 6) is an activating ligand for the follow an autosomal dominant mode of inheri

104 medgen 14 (2002) tance. Here, we report preliminary data from a P220 56 include missense (10/32) and nonsense (5/32) al family with an autosomal recessive form of hy terations, frameshiftcausing deletions and in potrichosis simplex, with predominant affection sertions (11/32) and splice site mutations (6/32). A Mutation in the CDMP1 Prodomain of the scalp hair. Due to the complex structure of the OPA1 gene Causes a Recessive Form of Brachydactyly The family originates from Saudi Arabia. The (31 exons), this approach is cumbersome and Type C parents are third cousins. Four out of ten chil time consuming. Moreover it may fail to detect Georg Schwabe (1,2), Türkmen, S. (1), dren are affected, six are unaffected. The affect mutations in the undefined gene promoter as Palanduz, S. (3), Stricker, S. (2), Goecke, T. (4), ed siblings presented with progressive hair loss well as larger deletions. Thus we established a Tinschert, S. (1) Majewski, F. (4), Mundlos, S. and thinning of scalp hair since early childhood. sensitive and practicable OPA1 screening pro (1) Two patients are almost bald, two show diffuse cedure by cDNA analysis. Single stranded cDNA (1) Institute for Medical Genetics, Charité, thinning of scalp hair. Furthermore, their body is synthesized from blood leukocytes by reverse Berlin, (2) Max Planck Institute for hair is sparse. No other associated abnormali transcription and four overlapping cDNA frag Molecular Genetics, Berlin, (3) Department ties are present. ments are amplified for mutational detection by of Internal Medicine, Division of Medical We have excluded linkage to the following can subsequent direct cDNA sequencing. Applying Genetics, Istanbul University, Turkey, (4) didate regions: 18p11 (hypotrichosis simplex), this technique we could detect three missense Institute for Human Genetics, Düsseldorf, 16q22.1 (CDH3), 12q13.11 (vitamin D receptor), mutations, one nonsense mutation, three dele Germany and 12q13.11 (keratin gene cluster). We are cur tions and three splice site mutations previously Hereditary brachydactylies are relatively frequent rently performing linkage analysis for chromoso found by genomic DNA analysis. Furthermore limb malformations, characterized by shortening mal regions 6p21.3 (HSS), 17q11.2 (keratin gene we were able to identify one novel deletion and of phalanges and/or metacarpals. Isolated cluster), and 8p21p22 (hairless). If no linkage three novel splice site mutations. brachydactylies are classified by Bell’s classifi can be detected with one of these loci, a total cation in seven distinct subforms. We here de genome scan will be undertaken. scribe a large consanguinous turkish family with We expect that the identification of the disease four individuals that show brachydactyly type C P220 58 causing gene will provide important insights in (BDC). They exhibit brachymesophalangy and the genetic, molecular and cellular pathways hyperphalangy of the index and middle finger

that control hair growth. Mutations of NEMO in female and male Abstracts GfH ÖGH SGMG Tagungsband with marked phenotypic variability. Some but not patients with incontinentia pigmenti type 2 all of the parents of affected individuals show Orth, Ulrike, Gal, A. minimal signs as camptodactyly or minor nail hy Institut für Humangenetik, P220 55 poplasia. Mutation analysis revealed that individ Universitätsklinikum Eppendorf, Hamburg, uals affected with BDC carry a novel homozy Germany gous missense mutation 517A>G of the CDMP1 Characterisation of the human RAB22A Incontinentia pigmenti type 2 (IP2) is a rare X gene and parents exhibit a heterozygous geno gene belonging to Rab small GTPases chromosomal dominant disorder that is usually type. The mutation is located within a highly Kussmann, Soeren, Worch, S., Hehr, A., lethal in utero in males. Affected females show conserved seven amino acid region of the Hansmann, I., Schlote, D. highly variable abnormalities of the skin, hair, prodomain of the gene and changes a methio Institut fuer Humangenetik und nails, teeth, eyes, and, in some cases, the cen nine to valine at amino acid position 173. Unlike Medizinische Biologie; Universitaet Halle, tral nervous system. The IP2 gene encodes the previously described heterozygous frameshift or Germany regulatory subunit of a multiprotein kinase com nonsense mutations this is the first report of a The mouse chromosome 2 segment (MMU2) plex required for activation of the NFkappa B homozygous CDMP1 mutation leading to BDC. corresponding to human chromosome 20 pathway (NFkappa B essential modulator, Considering the prodomain’s key function in pro (HSA20) is known to be involved in both, mater NEMO). NEMO plays a central role in immune cessing and secretion of active CDMP1, the mu nal as well as paternal noncomplementation (ge and inflammatory processes and prevents apop tation presented here is likely to lead to a re nomic imprinting). Uniparental disomies for dis tosis. Indeed, about 98% of affected females duced availability of biologically active CDMP1. tinct regions of MMU2 result in different neona show a nonrandom Xinactivation in peripheral The recessive nature of the mutation points out tal lethalities with opposite anomalous pheno blood lymphocytes due to apoptosis of the cells the importance of inspection of minimal signs in types, strongly suggesting the presence of im expressing the mutant NEMO allele. NEMO kindreds with limb malformations. printed genes in this region. These chromosomal maps in Xq28 and consists of 10 exons. It was regions show a conserved synteny of gene loci shown that a large genomic deletion (delta410) to human 20q13 segment, predicting the pres accounts for about 80% of IP2 cases, 10% car ence of imprinted genes in this syntenic human P220 57 ry point mutations or small rearrangements, chromosomal region. We have identified a nov whereas in the remaining 10% of the patients no el gene in this region of interest which is locat mutation was found. Most of the small mutations Improved screening for OPA1mutations in ed on a BAC RPCIB753L051096 proximal to result in a truncated NEMO protein. So far, no patients with ADOA by cDNA analysis GNAS1 on HSA 20q13. Cloning and sequencing clear genotypephenotype correlation has been Schimpf,Simone (1), Pesch,Ulrike E A (1), Leo the fulllength cDNA revealed a novel isoform of established. Kottler, Beate (2), Schaich, Simone (1), the human RAB22 subfamily of small GTPbind In a total of 80 apparently unrelated patients Wissinger, Bernd (1) ing proteins located in distinct intracellular com with IP, including 2 living males, we performed (1) Molecular Genetics Laboratory, partments and playing an important role in the mutation analysis. Fifty seven patients (~80%), University of Tübingen, Germany, (2) regulation of vesicular trafficking. Based on the including the 2 males, carried delta410. Both University Eye Hospital, University of EST WI12997 this new isoform was isolated males were shown to be somatic mosaic. In the Tübingen, Germany containing 2517 nucleotides and is designated remaining 23 patients, SSCP analysis of all cod Autosomal dominant optic atrophy (ADOA) is the RAB22A. Structurally, the RAB22A encodes a ing exons is being performed. To date, 5 differ most prevalent hereditary optic neuropathy char polypeptide of 194 amino acids which has 97% ent mutations have been identified in single pa acterized by an insidious onset of optic atrophy identity to the canine rab22. Northern blot analy tients, including a missense (C400Y) and a non in early childhood with moderate to severe de sis revealed two transcripts of 2.7 and 2.3 kb sense (Q290X) mutation, a point mutation in the crease of visual acuity, blueyellow dyschro adapted from two different polyA sites of the splice consensus sequence (IVS82a>g), as well matopsia, and centrocoecal scotoma of varying corresponding cDNA sequence indicating alter as two smaller gene rearrangements (deltaK290 density. ADOA occurs with an estimated disease native splicing. The genomic structure was com and c.1115delC). Cosegregation of the respec prevalence ranging between 1:10000 and pleted by database analysis and sequencing of tive mutations with the disease phenotype and 1:50000 in different populations. The predomi the isolated BAC clone RPCIB753L051096. The their absence in 50 controls suggest that the se nant locus has been mapped to chromosome gene consists of 7 exons spanning about 50 kb quence changes found are the primary genetic 3q28q29 and we have previously identified the of genomic sequence. defect responsible for the disease. Data of seg responsible gene, called OPA1, by means of po Physical and FISH mapping revealed that regation analysis in 10 families with delta410 sitional cloning. The OPA1 gene encodes a dy RAB22A is located on a BAC RPCIB753L051096 suggests that this mutation arose in 80% of the naminrelated GTPase targeted to mitochondria. proximal to GNAS1 but downstream of PCK1 on cases in the paternal meiosis. The high frequen Most of the presently known OPA1 mutations human chromosome 20q13. Supported by a cy of the delta410 deletion in the NEMO gene were found by the analysis of exons amplified grant of the DHGP. greatly facilitates diagnostic testing for IP and from genomic DNA. In our study we have identi provides a valuable tool for genetic counselling fied 32 OPA1 mutations in 97 unrelated ADOA of the families. patients (detection rate 31%). These mutations

medgen 14 (2002) 105 P220 59 (78 %) reported a positive family history with at mation in mammals. It plays a crucial role in least one affected firstgrade relative, only 8 mu chromatin structure and gene expression, and tations occurred sporadically (22 %). aberrant DNA methylation, including hypo as Hints and pitfalls of fragile X carrier testing MYBPC3 was the gene that most frequently well as hypermethylation, is often associated Gasteiger Maria., Neitzel B*, HolinskiFeder E. caused HCM in our cohort of unrelated patients. with pathogenesis, such as tumorigenesis. Al Center of Medical Genetics, Bayerstrasse Systematic mutation screening in large samples though a variety of methods are available to as 53, 80335 Munich, Germany of HCM patients leads to a genetic diagnosis in sess the methylation status in biological materi Mental retardation in fragile X syndrome is, in the about 30 % of unrelated index patients and in al, studying methylation is still limited by the low majority of cases, caused by CGG trinucleotide about 57 % of patients with a positive family his accuracy and/or the high consumption of time, amplification within the FMR1 gene. The syn tory. material, and labor of current protocols. We de drome is caused rarely by point mutations or scribe a rapid, quantitative method to assess deletions within or around the FMR1 gene. Here methylation levels at specific CpG sites using we describe a family with two boys and one girl PCR products of bisulfitetreated genomic DNA. affected by fragile X syndrome and two healthy P220 61 The new method takes advantage of the fact girls asking for carrier analysis. The two girls re that bisulfite modification of genomic DNA cre vealed two normal sized alleles in PCR and Usherin mutations in patients with Usher ates artificial single nucleotide polymorphisms Southern blot analysis. However, exact sizing of syndrome type IIA and autosomal recessive (SNPs), such as [C/T], at differentially methylat the PCR fragments brought up three different nonsydromic retinitis pigmentosa ed CpGs, which we call methylationbased (mb) FMR1 alleles (31/32 CGGs; 32/10 CGGs) for Caballero, Manuel (1,2), Ehmer, S. (1), Bolz, SNPs. We utilized the sequencingbysynthesis them, with one allel (10 CGGs) that was neither H.(1), Gal, A.(1) technique Pyrosequencing to analyse the per found for the mother or the father respectively. (1) Institut für Humangenetik, centage of methylation at a CpG supposed to be By haplotype and sequence analysis, the 10 Universitätsklinikum Eppendorf, Hamburg, hypomethylated exclusively in pilocytic astrocy CGG allel turned out to have arisen by a deletion Germany, (2) Centro Internacional de tomas (PA). Analysis of about 100 brain tumors of the mutated allel inherited from the mother. Retinosis Pigmentaria, La Habana, Cuba with the new technique, called PyroMeth, re The deletion allel did not show mosaicism for the Usher syndrome (USH) is a both clinically and vealed unspecific hypomethylation in all types of expansion and the deletion and therefore would

GfH ÖGH SGMG Tagungsband Abstracts GfH ÖGH SGMG Tagungsband genetically heterogeneous, autosomal recessive gliomas as compared to normal brain tissue. have been missed by the standard procedures disorder characterized by sensorineural hearing Methylation of the tumors ranged between 20% for molecular fragile X diagnosis, which is PCR loss and retinitis pigmentosa (RP). Based on the and 90% while the control tissues showed con and Southern blot analysis. This is the first case phenotype, the syndrome has been divided into sistent methylation between 80% and 90%. of a false negative carrier analysis due to a com three clinical types (USH13), whereas each type Data were confirmed by an independent plete deletion of the expanded repeat se consists of several genetic subtypes. Remark method. We consider quantitative mbSNP analy quences. How frequently we have missed these ably, mutations of the 4 known USH1 genes sis by Pyrosequencing a favourable alternative in affected boys and girls and weather or nor not (USH1B, C, D, and F) have been implicated not to existing highthroughput methylation assays this allele is associated with an increased risk for only in USH1 but also in cases of nonsyndromic such as Methy Light or microarraybased ap affected children is subject of further studies. It deafness. In the same way, mutations of the proaches. It combines single CpG analysis with is advisable however, to perform extended mo gene (USH2A) implicated in the most frequent accurate quantitation and is easily amenable to lecular analysis for carrier detection and for form of USH2 may also cause nonsyndromic high throughput. highly suspicious fragile X males. RP. The USH2A gene contains 21 exons and en codes a protein (usherin) of 171.5 kD with simi larity to a number of extracellular matrix proteins P220 63 P220 60 and receptors with lamininlike epidermal growth factor and fibronectin type III repeats. Using SSCP analysis, we screened USH2A for muta Detection of a new mutation (L257X) by MYBPC3 not MYH7 is the predominant gene tions in 38 and 110 German patients with USH2 complete scanning of the hereditary mutated in a Large Cohort of Unrelated and autosomal recessive nonsyndromic RP hemochromatosis gene (HFE1) using the Consecutive Patients with Hypertrophic (arRP), respectively. So far, most likely disease denaturing HPLC technology Cardiomyopathy related USH2A mutations have been identified in Mau UA(1), Deplazes J(1), Petersen I(1), Hering Dähmlow Steffen (1), Erdmann J (2), Senyuvan 10 of the 38 (26,3%) USH2 patients/families R(1), Lauer U(2), Gregor M(2), Rieß O(1) M (1), Hummel M (1), Hetzer R (1), (1), Tanis N studied, with c.2299delG in exon 13 (7,9% of (1) Department of Medical Genetics, (2) (1), Werner U (1), RegitzZagrosek V (1) disease alleles) being the most frequent change. Department of Gastroenterology, University (1) Cardiac and Thoracic Surgery Deutsches Other mutations detected include two missense of Tübingen, Calwerstr. 7. D72076 Herzzentrum Berlin, Germany (2) University mutations (C419F and L1378P) and two „pri Tuebingen Hospital Regensburg Regensburg Germany vate“ rearrangements (926insACCA (4% of dis Background: Between 9095% of German Defects in ten sarcomeric genes are known to ease alleles) and c.1416del26). In the group of hereditary hemochromatosis probands are cause hypertrophic cardiomyopathy (HCM). Mu arRP patients, two different usherin mutations C282Y or H63D homozygotes or (compound) tation types and frequencies in large cohorts of were identified; c.2276C>T (C759F) was found heterozygotes. 510% of the probands lack both consecutive and unrelated patients have not yet in 3 patients (1,36% of all disease alleles), and of these common HFE1 mutations. By the use of been determined. We therefore screened 108 c.2299delG in 2 patients (0,91% of all disease denaturing HPLC we scanned 14 DNA samples, unrelated and consecutive HCM patients for mu alleles). Our data suggest that mutations in in which either one or none of the two common tations in six sarcomeric genes: cardiac myosin USH2A are frequent (5/110, 4,5%) among pa mutations were identified. binding protein C (MYBPC3), ßMHC (MYH7), tients with arRP and are in line with findings of Methods: Analytical conditions for each coding Troponin T (TNNT2), aTropomyosin (TPM1), Tro other groups. Further studies are needed to ex exon were determined by a combination of com ponin I (TNNI3), and Troponin C (TNNC1). plore the spectrum of USH2A mutations in pa puter melting profile predictions and experimen HCM was diagnosed by echocardiography (sep tients with Usher syndrome and nonsyndromic tal melting curves. We tested samples harboring tum > 15 mm, septal/posterior wall >1.3), an RP as well as the relationship between genotype the C282Y and H63D mutation as well as sam giography or based on a state after myectomy. and phenotype. ples in which both chromosomes lack these Singlestrand conformational polymorphism common mutations. (SSCP) analysis was used for mutation screen Results: DHPLC detected all known HFE1 muta ing, followed by sequencing. Sensitivity was de tions as well as a striking heteroduplex in exon termined in comparison with denaturing high P220 62 4 in 2 unrelated patients. Sequence analysis de performance liquid chromatography (DHPLC) tected a previously undescribed stop codon mu and reached > 97 %. A total of 34 mutations in PyroMeth: quantitative analysis of CpG tation at position 257. 36 out of 108 patients were identified: 18 muta methylation with Pyrosequencing Conclusions: Denaturing HPLC can be used to tions in MYBPC3 in 20 patients, two of them be Brinckmann, Anja, Uhlmann, K., Toliat, M. R., scan for the two common mutations in the HFE1 ing founder mutations; 13 missense mutations in Ritter, H., Nürnberg, P gene in hemochromatosis probands and to iden MYH7 in 14 patients, one of them being a Gene Mapping Center at the Max Delbrück tify previously undescribed mutations. founder mutation (R870H); one amino acid ex Center for Molecular Medicine, D13092 change in TPM1, TNNT2 and TNNI3, respective BerlinBuch, Germany ly. No diseasecausing mutation was found in Methylation of CpG dinucleotides is a key ele TNNC1. Twentyeight of the 36 mutation carriers ment of the epigenetic control of genomic infor

106 medgen 14 (2002) P220 64 substantial proportion of familial and sporadic P220 67 cases. We investigated the frequency of BMPR2 mutations in a German PPH cohort. Genetic refinement of the locus for A systematic screening approach for Design: 82 patients were investigated by DHPLC Osteopathia striata with cranial sclerosis monogenic and polygenic diseases in (WAVE) and sequence analysis of aberrant exons (OSCS) to a 9cM interval on chromosome human Xp21.1Xp11.22 in the first laboratory and by direct sequencing Xp11.4 p11.22 J. Ramser (1), G. Wen (2), M. Dufault (1) I. in a second independent laboratory. The results Kraus, Cornelia (1), Koenig, R (2), MartinezGaray (3), F. Abidi (4), H. Hellebrand of both laboratories were obtained in a blinded Wurzenberger, C (1), Rauch, A (1), Rott, HD (1) (1), S. Engert (1), K. Badenhoop (5), C. setting and compared afterwards. (1) Institute of Human Genetics, Friedrich Schwartz (4), M. Platzer (2), A. Meindl (1) Results: BMPR2 Mutations were found in only 4 Alexander University of Erlangen 1) Dept. of Medical Genetics, Ludwig of the 74 sporadic patients. In addition, we Nuremberg, Germany, (2) Insitute of Human Maximilians University, Munich, Germany, found 3 unclassified variants. In the group of fa Genetics, Johann Wolfgang Goethe (2) Institute of Molecular Biotechnology, milial PPH (n=8) patients we found one mutation Univerity of Frankfurt, Germany Jena, Germany, (3) University of Valencia, and one UV. None of the groups had missed a Osteopathia striata with cranial sclerosis (OSCS) Valencia Spain, (4) Genetic Center, mutation. In only two cases we noticed se refers to the radiological appearance of longitu Greenwood, USA, (5) J.W.G.University of quencing problems which were clarified by re dinal striations of the tubular bones and fan Frankfurt, Germany sequencing or endonuclease digests. The results shaped configurations of the ilia. The skull is ab Human Xp21.1 to Xp11.22 is of high medical rel always confirmed the DHPLC data. normally dense, and the clavicles are not striat evance since several monogenic as well as poly Conclusion: We conclude that the frequency of ed. Based on various family observations OSCS genic traits have been mapped in this region. BMPR2 mutations in sporadic PPH patients is had initially been considered an autosomal dom Apart from 15 diseases for which the underlying lower than previously reported (5%10% instead inant condition with complete penetrance and genes have already been identified, 7 syndromic of 26%), at least in the German population. high clinical variability. Recently, however, we forms of Xlinked mental retardation including and others suggested Xlinked segregation due Prieto, Renpenning and XMRE and more than 20 to the observation of female preponderance and nonsyndromic MRX families including MRX18, of severely affected sons from OSCS mothers. P220 66 MRX51 or MRX56 have been completely or par Because OSCS is a mosaic carrier manifestation tially mapped to this interval. Additionally, loci Abstracts GfH ÖGH SGMG Tagungsband in females, the nonrandom Xinactivation re for diseases such as Xlinked optic atrophy, X Xinactivation and fragileXfullmutation ported by Viot et al. 2002 in OSCS women is un linked congenital nystagmus and juvenile spinal methylation in human placentas likely. Here, we studied a three generation fami muscular atrophy have also been localized there. Peter Steinbach, Sibylle Jakubiczka, Thomas ly from German origin that included a severely Diabetes mellitus type 1 and Graves’ disease are Bettecken affected male, his mother, aunt and grandmoth two polygenic disorders for which susceptibility Abt. Humangenetik, Universitätsklinikum, er with OSCS. Assessment of Xinactivation sta genes have been linked to the region. In order to Ulm, Germany (PS); Ist. für Humangenetik, tus in the three affected females with the CAG initiate a systematic mutation screening ap Universität, Magdeburg, Germany (SJ); Inst. polymorphic marker in the androgen receptor proach, we have established a detailed gene für Humangenetik, GSF Forschungs gene revealed a random Xinactivation pattern catalogue for this region. The interval, which is zentrum, Neuherberg, Germany (TB) in the grandmother. The mother and aunt unfor flanked by markers DXS1237 and DXS1204, en In female somatic cells X inactivation is associ tunately were not informative. Linkage analysis compasses approximately 18 Mb and is under ated with differential CpG methylation on the X by PCR based microsatellite marker genotyping investigation via mapping and genomic se chromosomes, e.g., the LINE1 element of the was used to identify the disease locus. Based on quencing at both the Sanger Centre (UK) and DXS255 minisatellite is extensively methylated recombination breakpoint analysis, we have de within our group. Through exploration of the ge only on the active X whereas CpGs in the andro termined that a critical interval is lovalised be nomic data, we have identified 142 genes, in genreceptor (AR) and the fragileXmentalre tween markers DXS993 and DXS1039, placing cluding 81 known genes, 65 novel genes (includ tardation gene (FMR1) are methylated only on the disease locus within a 9cM region to Xp11.4 ing transcripts with unknown function, for exam the inactive X chromosome. Full expansion of p11.22. Therefore our results confirm the X ple the KIAA series), spliced ESTs and genes the FMR1CGG repeat in male and female frag linked inheritance of OSCS on a molecular ba based on exon prediction only. In addition 35 ileX patients is usually associated with promo sis. Our observation of random Xinactivation in pseudogenes have been found. tor hypermethylation. In first trimester chorionic a OSCS female is in line with the mosaic pheno Detailed expression studies, involving in silico as villi of female placentas, Xinactivation methyla type of striated bone patterns and contradicts well as wetlab experiments, are in process. Im tion differs significantly from somatic cells. Also, the published nonrandom Xinactivation status. mobilization of the genes on membranes and differences among X inactivation and full muta Further Xinactivation and linkage studies are subsequent RNAhybridization approaches will tion methylation of FMR1 have been reported. ongoing in a second 3 generation family with 5 allow the establishment of expression profiles in Our project includes a detailed study of CpG affected patients. a wide variety of tissues. To date, mutation methylation of the FMR1 promotor and other X screening on the genomic and/or cDNA is un linked loci in chorionic villi from first trimester derway for 3 syndromic and one nonsyndromic and full term female placentas of normal and form of mental retardation, as well as for the P220 65 fragileXfullmutation individuals, in order to es polygenic trait Diabetes mellitus type I. tablish the timing of methylation on both, nor malinactive and fullmutationfragile, X chromo Frequency of BMPR2 mutations in German somes: In contrast to other Xlinked loci and to patients with Primary Pulmonary somatic tissues, the FMR1 promotor is not dif P220 68 Hypertension ferentially methylated among the active and in B Janssen1, R. Köhler1, A von Hippel1, G active X chromosomes in the trophoblast of fe MiltenbergerMiltenyi1, W Seeger3, J Winkler, Chemical mutagenesis and clone library male placentas at any stage of development. HA Katus2, E Grünig2, M Pauciulo4, W generation of mouse embryonic stem cells FMR1 methylation in the trophoblast represents Nichols4 Greber, B., Campregher, C., Lehrach, H., an abnormal feature of fragile X syndrome, is From the Institute of Human Genetics1, Himmelbauer, H. only found with full expansion of the CGG re Department of Cardiology2, University of MaxPlanckInstitute of Molecular Genetics, peat, but occurs at variable times usually be Heidelberg, Heidelberg. Germany; Ihnestr. 73, D14195 Berlin, Germany tween the 10th and 13th week. Department of Pneumonology3, University Facing a large number of genes with unknown of Gießen, Germany; Division of Human functions in model organisms, mutant collec Gemnetics, Children’s Hospital Medical tions are valuable resources for studying gene Centre Cincinnati, Ohio, USA4 function. For the mouse, ES cell technology of Study Objectives:Primary pulmonary hyperten fers the possibility to manipulate the genome sion (PPH) is a dominantly inherited disorder and select for mutations in vitro. Mutant mice characterized by remodeling and loss of paten can then be generated from clones of interest to cy of the pulmonary arteries. Recently it has study the phenotype of these animals. A major been shown that mutations in the bone morpho advantage of such genedriven approaches is genetic protein receptor 2 (BMPR2) gene, which that the lesion in the genome of a particular mu encodes a transforming growth factor ß type II tant can be identified before the animal is gen receptor on 2q33, are the cause of PPH in a erated.

medgen 14 (2002) 107 We are manipulating the genome of ES cells P220 70 In our laboratory the CFOLA kit (Cystic Fibrosis chemically using several different mutagens, one Assay) is used to screen for the most common being TMP (trimethyl psoralene). TMP is gener mutations in the Caucasian population. Surpris Determination of CTG repeats: Unstable ally known to predominantly cause „small“ dele ingly, homozygosity for the dF508 mutation was alleles in Myotonic Dystrophy locus tions in the genome of C. elegans but has not found in a healthy woman. As the homozygote Vojtiskova Marie, Froster Ursula, Enzmann been established as a mutagen in mammalian dF508 genotype is not compatible with her Gabi, Falk Martin, Silhanova Eva systems yet. We have characterized TMP as a healthy phenotype and as technical problems [1]Institute of Biophysics, Acad. sci. 61265 mutagen for mouse embryonic stem cells re have been ruled out by repeating the analysis, Brno, Czech Republic, Institut fur garding death rates, mutation rates, and muta additional studies were implemented to clarify HUmangenetik, Universitat Leipzig, 04103 tion spectrum. The majority of lesions induced the situation regarding the dF508 status. Het Leipzig, Germany, Depart. of Human at the Hprt locus were point mutations rather erozygosity for dF508 was identified by fragm Genetics, University Hospital, 70852 than deletions at the genomic level. ent lengths polymorphism analysis of exon 10 Ostrava, Czech republic Work is in progress to establish libraries of mu PCR products with subsequent verification by Human genome dynamic mutations are a new tated ES cell clones and to develop screening sequencing. In addition, the polymorphism IVS9 class of gene mutation represented by unstable procedures for the identification of mutations in 61A>G, present in heterozygous state, was de number of trinucleotide repeats and causing genes of interest. Current results of our screen tected. As the PCR primer of exon 10 spans the several human hereditary neurological and neu ing approaches will be presented at the meeting. polymorphic site, only the allele not carrying this rodegenerative diseases with extremely variable Acknowledgement of support: ES cell mutagen DNA variant will be amplified. In case of het phenotypic manifestation. esis and characterization is carried out through erozygosity for the polymorphism only one sin We present our experiences with the determina funding by the German National Genome Re gle allele will be present as PCR product. This tion of an unstable CTG repeat sequences locat search Network (NGFN). will imitate homozygosity for mutations in this ed in the 3’untranslated region of the myotonin genomic region. In consequence of this, the CF protein kinase gene MDPK [locus 19q13.3]. CTG OLA kit will produce a false positive test result. expansions up to several thousand repeats are In order to assess the relevance of this findings, P220 69 specific for a severe form of myotonic dystrophy we started rescreening of 200 CF samples. The [MD] autosomal dominant disease. The physio

GfH ÖGH SGMG Tagungsband Abstracts GfH ÖGH SGMG Tagungsband results of this study will be presented and the logical copy number rangers from 5 to 3o re Two metaphysal chondrodysplasia type data should help to evaluate the reliability of the peats that is easy determined by PCR methods Schmid cases with novel mutations CFOLA kit. using primers complementary to the CTG se Wildhardt, G. (1), Brenner, R. (2), Decker, J. (1), quence border. To reduce the time for final re Zabel B. (3) sults of DNA analysis in cases of MD patients, 1) Bioscientia, Ingelheim, 2) Department of we have introduced and modified fast and effi P220 72 Orthopaedics, University of Ulm, 3) cient methods a] triplet repeat primed fluores Children´s Hospital, University of Mainz cent PCR [TP PCR] [1] and b] XL system for long Schmid metaphyseal chondrodysplasia (SMCD) Mutation screening in the fibrinogen gamma PCR targets with partial substitution of 7deaza is an autosomal dominant skeletal disorder char chain gene in 100 patients with acute dGTP for dGTP [XLCTGPCR] [2,3] and so we acterized by short stature, waddling gait, and ischaemic stroke have suggested two steps PCR molecular diag coxa vara. The genetic cause of SMCD are mu S. Gerdes(1), H. Plendl(1), P. Zunker(2), A. nostic protocol. Thos protocol was checked up tations found in the COL10A1 gene. Collagen Allardt (2), N. Petersen(2), A. Schmied(2), G. on the group of MD positive control DNA sam type X is the most abundant extracellular matrix Deuschl(2), R. Siebert(1), W. Grote(1) ples provided us by references laboratories and component synthesized by hypertrophic chon (1) Institute of Human Genetics and (2) on the group of our MD patients. Determined drocytes of the growth plate during the transi Department of Neurology, University CTG pathological long alleles by PCR methods tion from cartilage to bone in the process of en Hospital Kiel, Kiel, Germany were confirmed with Southern blott analysis chondral ossification. Collagen type X is classi Dysfibrinogenemia is a rare defect of haemosta [SBA], too. Application of PCR protocols and de fied as a shortchain nonfibrillar collagen and the sis. Clinical symptoms of dysfibrinogenemia vary termination of unstable trinucleotide CTG repeat chains are composed of three structurally dis from mild to moderate bleeding, recurrent abor alleles we demonstrate on the example of MD tinct domains: an aminoterminal globular do tions, venous and arterial thrombosis, and renal family pedigree with severe affected girl in the main (NC2), a short triple helical region, and a amyloidosis. About half of the patients show no age of eight with normal female karyotype and carboxylterminal globular domain (NC1). The clinical symptoms. Dysfibrinogenemia is mostly clinical symptoms of myotonic syndrome. In this chains are synthesized with an Nterminal signal diagnosed by discrepant values for plasma fib MD family we have proved by the two steps peptide, which is proteolytically removed from rinogen concentration analysed by functional PCR protocol verified by SBA, the maternal ori the prea1(X) chains during biosynthesis. and antigenic tests, and a prolonged thrombin gin of CTG instability, the amplification lenght of We will present two new familial SMCD cases: time. Nevertheless, some alterations in the fib pathological allele and worse phenotype with (1) A 15yearold girl presented with short rinogen molecule detectable on the DNA level early succession to the consequent generation. limbed short stature, bowed legs and waddling may escape diagnosis by routine coagulation gait. Radiological findings consisted of coxa assays. vara and metaphyseal changes. She and her af In 100 patients with ischaemic stroke we cur fected father carried an amino acid substitution P220 71 rently screen for mutations in the three genes in position 554 (F554L). (2) The symptoms of a coding for the fibrinogen molecule by horizontal 5,5yearold boy and her equally affected moth PAGE based SSCP analysis. Here we present False positive cystic fibrosis test result due er included short stature, coxa vara and meta our results for the mutation analysis of the fib to a DNApolymorphism physeal abnormalities. In both, COL10A1 analy rinogen gamma chain gene. Trübenbach J.1, Decker J.1, Zabel B.2, sis revealed a nonsense mutation in position 653 In two unrelated patients with an ischaemic Wildhardt G1 (Q653X) and in addition a point mutation in po stroke we found a novel missense mutation in 1Bioscientia, Ingelheim, 2 Children´s sition 1729 G to C leading to an amino acid sub exon 4 (C>G at position 2525) of the gamma Hospital, University of Mainz, Germany stitution glycine to arginine (G545R) previously chain. As the family histories of the patients are Cystic fibrosis (CF), the most common metabol described as polymorphism. not indicative of further strokes or thromboem ic disorder with autosomal recessive inheritance, The two novel mutations are located in the C bolic events these findings suggest that the has an incidence in the Caucasian population of terminal nonhelical domain (NC1) of the a1(X) gamma chain A108G substitution may not act as 1:2500 and is characterized by a severe chronic chain. It is still under discussion, why with ex a major risk factor for these conditions. A newly respiratory tract disease and pancreatic dys ception of two cases all known mutations clus developed PCRRFLP assay offers an appropri function. The disease is highly variable in pres ter in this domain. The NC1 mutations were pro ate procedure for rapid screening for this muta entation and course. posed to cause reduced mRNA stability and also tion. Using this method we could not detect this Since the characterization of the gene in 1989 to affect the initial stages of the folding and mutation in a control group of 100 healthy vol more than 900 mutations were identified. Delta chain assembly of collagen X, hindering nucle unteers. F508 is the most common mutation with a fre ation of the triple helix and, therefore, impairing In a third patient we found a new mutation A>C quency of about 72% in the Caucasian popula secretion of collagen X trimers. two nucleotides after the stop codon. A recent tion. Some other specific mutations account for study in yeast [O Namy et al. EMBO Rep 2001; 215% of CF alleles, depending on the ethnic 2: 78793] showed that this location might have background of the patient studied, with the rest an important positional effect on readthrough ef of the mutations being very rare.

108 medgen 14 (2002) ficiency. Screening for this mutation in a control Biology and Genetics, Medical University of markers should be decreased considerably in group of 100 healthy volunteers is on the way. Gdansk, Poland the group of healthy blood donors because of Our results indicate that mutations in the fibrino The RomanoWardSyndrome (RWS) is the most the supposed cardiovascular susceptibility of gen gamma chain gene are a rare finding in pa common form of the so called „Hereditary Long persons carrying these genotypes. Surprisingly tients with ischaemic stroke. QTSyndromes“ (LQTS) that involve cardiac ar this expected decrease with age was not found rhythmias which can lead to sudden death. among the healthy blood donors (p=0.337). Con These syndromes are characterized by pro clusions: Our results suggest that the investigat longed QT intervals which are visible on an elec ed genetic markers are not predictors for the in P220 73 trocardiogram (ECG) and often lead to juvenile cidence of coronary atherosclerosis in general. cardiac arrhythmia. These events are based on But nevertheless these PM’s could be markers Detection of duplications of proteolipid mutations in genes encoding for cardiac ion for the progress and therefore for a practicable protein gene (PLP) in Czech families with channels leading to abnormalities in cardiac re clinical therotic disease. classical form of Pelizaeus Merzbacher polarization. To date six loci and five genes re Disease, high denovo mutation rate sponsible for LQTS were described with KVLQT1 P. Seeman, P. Krsek, M, K. Namestkova, L. being the most frequently involved in RWS (45 P221 03 Paulas, M. Proskova, M. Malikova, M. 50%). In our study, these loci were analysed by Havlovicova segregation analysis and direct sequencing in Charles University Prague, 2nd School of four RomanoWard families. Mutations in KCNE1 Vascular pathology types associated with medicine, Dept. Of Child Neurology, DANN and KCNE2 can be readily determined by se the low activity variants of MTHFR laboratory, V úvalu 84, CZ 150 06 Praha 5 quencing, the genes KVLQT1, HERG, SCN5A Ewa Strauss (1), Krzysztof Waliszewski (2), PelizaeusMerzbacher disease (PMD) is an X and the locus LQT4 were studied by segregation Marcin Gabriel (2), Stanislaw Zapalski (2), Jerzy linked disorder od central myelination. Two most analysis using different sets of fluorescentla G?uszek (3), Andrzej L. Pawlak (1) important clinical types, the more common and belled markers. Sequencing of KCNE1 and (1) Institute of Human Genetics, Polish milder clasical type and the rarer and more se KCNE2 revealed several single nucleotide ex Academy of Sciences, Poznan, Poland; (2) vere connatal type are frequently described. changes, which either were silent or polymor Department of General and Vascular

Classical type is caused in most cases by a du phisms (SNP), but no diagnostic mutation. One Surgery, Institute of Surgery, and (3) Abstracts GfH ÖGH SGMG Tagungsband plication of proteolipid protein gene (PLP). This family with only two affected persons showed Department of Arterial Hypertension, duplication is variable in its extent but also in the linkage to the LQT1 locus, but we detected no Institute of Cardiology, K. Marcinkowski location on X chromosome. Denovo PLP dupli diagnostic mutation. These results suggest that University of Medical Sciences in Poznan cations origin in most cases as an intrachromo the KVLQT1gene, which is most frequently in The low activity variants of MTHFR gene (677 somal event in paternal meiosis. Various meth volved in RWS in the US and Western Europe, C>T and 1298 A>C) are implicitly associated ods as intrephase FISH, quantitative PCR and shows different mutation frequencies in Polish with mild hiperhomocysteinemia. This last pa intragenic markers are used for detection of PLP RWSfamilies. After establishing the marker sets, rameter is considered as an important determi gene duplications. No cases with PLP duplica segregation analysis of candidate gene regions nant of vascular pathology. Both variants of tion were reported from Czech Republic yet. represents an efficient and reliable tool for geno MTHFR were originally studied in patients with We use quantitative comparative fluorescence type diagnosis of LQTS. neural tube defects, but also in vascular dis PCR (QCFPCR) in combination with an intra eases, in particular in early coronary disease. genic dinucleotide marker for detection of the In this study distribution of MTHFR gene alleles most common mutation in Pelizaeus Merzbach is compared in groups of patients displaying the P221 02 er disease. Exon 4 of the PLP gene and exon 4 specific types of vascular pathology diagnosed of the CFTR gene are simuntaneously amplified on the basis of clinical course (Tab.1). in one multiplex PCR and exon 4 of PLP gene The genetic distribution pattern of In the abdominal aortic aneurism (AAA) group together with the exon 48 of the DMD gene are polymorphisms regarded to influence the the mixed heterozygotes (MTHFR 677 T, 1298 C) amplified in a second multiplex PCR reaction. development of atherosclerosis occured at increased frequency (p<0.05) as Resulted fluorescently labled PCR products are A. Kabisch1, S. Schulz2, P. Greiser2, U. Müller compared to the control group. subsequently analyzed on the ABI310 Genetic Werdan3, K. Werdan3, C. Gläser2 The study will be extended to include the pa Analyzer using the GeneScan software. In the 1Bloodbank, 2Inst. of Human Genetics, tients with the obliteration of iliac arteries (syn presence of PLP duplication the product inten 3Dep. of Internal Med., Univ. Halle, Germany drom Leriche; 34 patients) and more patients sity ratio is shifted to the PLP peak. Intragenic Atherosclerosis is a very complex disease influ with arterial hypertension. Finally these last pa dinucleotide marked is used for detection of the enced by a variety of environmental and genet tients will be divided into groups on the basis of origin of the PLP duplication and for assesment ic factors. Among the genetic factors which are clinical symptoms and spectrum of risk factors of the carrier status in female in risk relatives. involved in different atherosclerotic processes potentially contributing to the disease. 6 PMD patients with PLP gene duplications from there are a lot of well investigated established Supported by KBN grant no.6P05A 03921 5 families were detected using this combination risk markers like e.g. polymorphisms (PM’s) in of methods. All 5 mothers of these patients were the ACE, ApoE, and Eselectingen as well as also carriers of the PLP gene duplication. In four the LeidenPM in the factor Vgen. Methods: We P221 04 of the 5 families the PLP duplication originated investigated the genotype distribution of the denovo in patients mathernal grandfather meio abovementioned PM’s in a group of 362 long sis as an intrachromosomal event. Three of the standing healthy blood donors without any coro Mutation analysis of the spastin gene patients were born in three subsequent years in nary symptoms and coronary risk factors includ (SPG4) in patients with autosomal dominant Czech Republic which shows, that the incidence ing hypertension, smoking, diabetes mellitus and hereditary spastic paraplegia in Germany of PMD must be at least 1:90 000 in our country. drug treatment (more than 3y in medical moni Sauter, Simone; Dörwald, N.; Neesen, J.; Supported by GA UK 54/2002 and by VZ toring; mean age: 41.9y; 61.3% male). All donors Engel, W. 11130003 and VZ 111300004 belong to the same Central German Caucasian Institute of Human Genetics, GeorgAugust origin. Results: For evaluation of the genotype University Göttingen, HeinrichDükerWeg distributions the DD (ACE), the 3/4 (ApoE), the 12, 37073 Göttingen Arg/Arg (Eselectin) as well as the AAgenotype Hereditary spastic paraplegias (HSP) comprise P221 01 (factor V) were considered as pathologically in a genetically and clinically heterogeneous group terms of coronary atherosclerosis in common of neurodegenerative disorders characterized by Candidate gene studies as a tool for consent. We evaluated the score of the patho progressive spasticity and hyperreflexia of the genotype diagnosis in Long QT syndromes logical genotypes taking the gender as well as lower limbs. Autosomal dominant hereditary M. Ebauer1,2, B. Haack1,2, N. Blin1, M. the age (1829 y vs. 3039 y vs. 4049 y vs. 50 spastic paraplegia 4 linked to chromosome 2p Pfister2, H.P. Zenner2, K. Ochman3, I. 70 y) into consideration. We could not detected (SPG4) is the most common form of autosomal Romanowska3, J. Limon3, S. Kupka1,2 any gender specific significant differences in the dominant hereditary spastic paraplegia. It is 1. Institute of Anthropology and Human score within the group of healthy blood donors caused by mutations in the SPG4 gene encod Genetics, Department of Molecular irrespective of age (p=0.542) even though it is ing spastin, a member of the AAA protein family Genetics, University of Tübingen, Germany, known that there is a gender specific preference of ATPases. In this study the spastin gene of 2. Department of Otolaryngology, University to the incidence of coronary afflictions. In addi HSP patients from 70 apparently unrelated fam of Tübingen, Germany, 3. Department of tion one could imagine that with increasing age ilies in Germany was analysed. We identified mu the number of these pathological genetic risk tations in 16 out of the 70 HSP families; 13 of

medgen 14 (2002) 109 these mutations have not been described before trointestinal mucosal damage. They promote cell as proteins of the podocyte foot processes and only two mutation were found in more than migration, protect and heal the mucosa and sup which are known to be essential components of one family. Among the detected mutations are 9 press tumor growth. We assume them to be reg the glomerular filtration barrier. In addition we frameshift, 3 nonsense and 2 missense muta ulated by the proinflammatory cytokines inter have found features of either dedifferentiation or tions as well as 2 mutations that affect splicing. leukin1beta (IL1 beta) and interleukin6 (IL6), arrest at an early differentiation stage which may Most of the novel mutations are located in the which trigger the transcriptional factors NFkap explain the presence of rudimentary not fully de conserved AAA cassetteencoding region of the pa B and C/EBP beta. Methods: After IL1beta veloped glomeruli. In summary it can be postu spastin gene. The relative frequency of spastin and IL6 stimulation, expression of TFF genes lated that a combination of deregulative pro gene mutations in an unselected group of Ger was analyzed by reporter gene assays using TFF cesses causes this severe disease. Which of man HSP patients is approximately 23 %. promoter constructs and by quantitative real them are caused directly by a mutated WT1 pro Frameshift mutations account for the majority of time RTPCR in gastrointestinal cell lines HT29 tein and which are secondary has to be exam SPG4 mutations in this population. The propor and KATO III. NFkappa B and C/EBP beta were ined in the future. tion of splice mutations is considerably lower transiently coexpressed. than reported elsewhere. Results: We have functionally identified tran scription factors NFkappa B and C/EBP beta to P221 08 inhibit transcription of human TFF genes. Down regulation of TFF transcription is also observed P221 05 by IL1beta and IL6, suggesting crosstalk with or Mutational analysis of the MODY genes, in response to the immune system. Downreg hepatic nuclear factor1alpha; (HNF1alpha) IDENTIFICATION OF FVIIIINHIBITOR ulation of reporter gene transcription of all three and glucokinase (GCK), in diabetic children EPITOPES USING CELLULOSE BOUND TFF genes was observed by both IL1 beta and LeeKirsch, Min Ae (1,2), Rohayem, J. (1), FVIIIPEPTIDE LIBRARIES IL6 as well as by NFkappa B and C/EBP beta. Bergert, R. (1), Winkler, U. (1), Gahr, M. (1), Albert, Thilo (1); Lange, S. (1); Oldenburg, J. IL1 beta and IL6 caused a 3 to 11fold reduction Naeke, A. (1) (2); Graw, J. (3); Schramm, W. (4); Hanfland, P. in TFF mRNA expression. displayed in realtime Klinik fuer Kinder und Jugendmedizin (1) (1); Brackmann, H.H. (1); Schwaab, R. (1) PCR. and Institut fuer Klinische Genetik (2),

GfH ÖGH SGMG Tagungsband Abstracts GfH ÖGH SGMG Tagungsband (1) Inst. f. Exp. Hämatologie u. Transf. Med., Conclusions: Downregulation of intestinal tre Technische Universitaet Dresden Bonn; (2) Inst. f. Transf. Med. u. foil factor TFF3 caused by transcriptional repres Maturityonset diabetes of the young (MODY) is Immunhämatol., Frankfurt; (3) GSF sion by IL1 beta and NFkappa B as well as by a clinically and genetically heterogeneous disor Forschungszentr. f. Umwelt u. Gesundheit, IL6 and C/EBP beta activation in vitro may re der characterized by nonketotic diabetes, onset Neuherberg; (4) Klinikum Innenstadt: flect the situation in vivo and may contribute to before the age of 35 years, and autosomal dom Medizin. Klinik, München ulceration and decreased wound healing in in inant inheritance. Owing to its mild manifestation Hemophilia A is caused by the deficiency or dys flammatory bowel disease. Downregulation of affected children are often undiagnosed. Correct function of Factor VIII (FVIII). The most serious TFF1 and TFF2 by transcriptional repression by diagnosis, however, is essential as children with complication of replacement therapy is the de the abovenamed factors may explain the mu MODY, unlike those with type 1 diabetes, often velopment of alloantibodies (inhibitors) towards cosal damage in the stomach during chronic do not require insulin and respond to oral antidi substituted FVIII in about 30% of patients with gastritis and may contribute to gastric carcino abetics. In addition, early therapy will prevent severe haemophilia A thus neutralizing FVIII ac genesis. microvascular morbidity due to chronic hyper tivity. AntiFVIII antibodies (Abs) are mainly IgG glycemia. MODY can be caused by mutations in molecules of polyclonal origin. at least 6 genes (MODY1MODY6) involved in in Until now Abbinding to epitopes is not yet sulin secretion. The aim of the study was to in P221 07 known on the amino acid level. In our project we vestigate the prevalence of mutations in the characterize antiFVIII Ab epitopes of hemophil genes encoding HNF1alpha (MODY2) and GCK ia A patients using solid phase bound FVIIIpep WT1 mutations and downstream molecular (MODY3) by sequencing analysis. tide libraries. Actually our library presents the pathomechanisms causing nephrotic 8 out of 164 diabetic children (5%) treated in our primary sequence of FVIII (2351aa) divided up in syndrome clinic had a clinical history suggestive of MODY. 13meric oligopeptides, overlapping 10aa one Schumacher Valérie(1), Pitschke G.(1), Except for one all had at least one first grade rel another. FVIII inhibitors which are specifically as Pavenstädt H.(2), RoyerPokora B. (1) ative with type 2 diabetes, gestational diabetes, sociated to oligopeptides after incubation are (1)Institute of Human Genetics and or impaired glucose tolerance. In 5 out of 8 cas detected by HRPOconjugated secondary Abs Anthropology, University of Duesseldorf, es (63%) novel mutations were detected within and chemiluminescence. Before screening the li Germany, (2) Department of Medicine, HNF1alpha (L382fsdelA) or within GCK (E399X, brary, antiFVIII Abs must be purified from hu Division of Nephrology, University of Y247X, IVS6nt1G>A). All mutations were het man blood plasma on Protein G followed by Freiburg, Germany erozygous except for the splice site mutation affinitychromatography on a FVIIImatrix. Nephrotic syndrome in the first year of life may within the GCKgene, which was found in 2 This technique allows a screen of linear anti be caused by an inherited glomerular disease mildly affected siblings. genic sites and possibly discontinuous epitopes like diffuse mesangial sclerosis. In association The molecular diagnosis of MODY can be estab on FVIII in only one assay. We have investigated with male pseudohermaphroditism and a high lished in a large proportion of carefully selected Abpreparations of different patients with FVIII risk for Wilms tumor it is known as DenysDrash patients by mutational analysis of the HNF1al inhibitors. The results showed several potential syndrome caused by constitutional heterozy pha and GCKgenes. The identification of mu epitopes including partsequences of regions geous missense mutations in the WT1 gene. tations has therapeutic as well as prognostic im described as frequently appearing epitopes in WT1 encodes a transcription factor thought to plications and may help in the identification of the literature. Many signals were observed in dif be essential in nephrogenesis and the home asymptomatic family members, thus preventing ferent patients. The pattern of patient specific ostasis of glomerular podocytes. future complications of untreated chronic hyper epitopes is actually compared with epitopes To elucidate downstream molecular pathomech glycemia. found in healthy persons. Epitope data will be anisms by which WT1 mutations lead to further correlated to genotype/phenotype data nephrotic syndrome we have compared kidney in the consortium. specimens from healthy and diseased individu P221 09 als using different techniques. RNA expression was determined after laser microdissecting glomeruli from frozen tissue samples, isolation Functional characterization of human P221 06 and amplification of the RNA and hybridization LETM1 a gene deleted in WolfHirschhorn to cDNA arrays containing 3600 different genes. syndrome Downregulation of TFF Expression in Furthermore we have compared the expression Schlickum, Stephanie (1); Endele, S. (1); Gastrointestinal Cell Lines by Interleukin1 profile of cultivated podocytes from healthy and Steglich, C. (2); Fackelmayer, F.O. (3); Dossinger, Veronika; Baus, M.; Kayademir, T.; diseased persons Protein expression of the Winterpacht, A. (1) Gött, P.; Blin N. same material was analysed by immunohisto (1) Institute of Human Genetics, Erlangen, University of Tuebingen, Wilhelmstraße 27, chemistry. The results show that the presence of Germany, (2) Institute of Human Genetics, 72074 Tuebingen, Germany a WT1 missense mutation leads to a complex Hamburg, Germany, (3) HeinrichPette Background and Aims: Proteins of the trefoil fac misregulation of genes which may be the cause Institute, Hamburg, Germany tor family (TFF1, TFF2 and TFF3) are acute of massive proteinuria. These include basal Recently, we have identified a novel gene LETM1 phase proteins upregulated in response to gas membrane and slit membrane proteins as well which is deleted in almost all WolfHirschhorn

110 medgen 14 (2002) syndrome (WHS) patients. WHS (MIM 194190) is than 15 promising candidate genes for autoso dence of approximately 1 in 2500 live birth. The caused by deletions in chromosome region mal MR and related disorders, as well as 6 can gene for CF spans approximately 250 kb and 4p16.3 and is thought to be a true contiguous didate genes for Xchromosomal MR could be contains 27 exons. CF patients have two defec gene syndrome. Rauch et al. (2001) described identified. These genes code for protein kinases tive alleles and may either be heterozygous for the first patient with a small interstitial deletion and phosphatases, synapseassociated pro different mutations, or homozygous for one of mainly restricted to a previously defined 165 kb teins, transcription factors, neuronal cell surface the mutations. WHS critical region (WHSCR) which excludes proteins and axon guidance molecules. To prove Since the identification of the gene responsible LETM1. Since the patient shows a partial WHS the identity of candidate genes for Xlinked MR, for CF, more than 900 mutations was described phenotype without seizures and severe neuro mutation screening in >350 unrelated families is in CFTR gene of patients affected by cystic fi muscular features, WHSCRflanking genes (like in progress. For autosomal MR, however, this brosis, but the prevalance of the CF shows a ge LETM1) may contribute to the complete WHS strategy cannot be used due to the enormous ographical and ethnical variations in the world. phenotype. Here, we present novel data con heterogeneity of this condition. Therefore, func The delta F508 mutation in CFTR gene accounts cerning the functional characterization of LETM1 tional approaches will be employed to substan for over 70 % all mutant CFTR alleles in the Eu and its role as a putative candidate gene con tiate the role of these genes in MR. ropeans to 20 % in the Asians. tributing to the WHS. Database sequence com The DNA samples of 24 individuals, who were parisons revealed that LETM1 is evolutionary carrier for CF, partly with their affected children conserved and shows significant homology to and their chorion villus samples (CVS) from the P221 11 proteins from different species including A. pregnant women, have been tested for five com thaliana, C. elegans and D. melanogaster. Inter mon mutations: deltaF508, G551D, G542X, estingly, all of these proteins exhibit a character Deletion Analysis and Prenatal Diagnosis in W1282X and N1303k. The study was performed istic conserved domain structure which includes Iranian SMA Patients Type, I III by using the ARMS method for mutation detec a transmembrane domain, 12 EFhand motifs, P. DerakhshandehPeykar1,2, M. Rahmani2, M. tion of CF gene. The delta F508 mutation was a specific phosphorylation site and a novel Atai2, J. Ghassemi2, D.D. Farhud 1,2 found only in one couple with first cousin mar SAF/SAP related domain. Using a „pull down as Dep. of Human Genetics, School of Public riage and three times in other partners of relat say“ we could show that in contrast to a stan Health, Terhran Univ. of Medical Sciences, ed/non related couples (28%). One of these cou

dard SAF/SAP domain the SAF/SAP related do TehranIran/2.2. Genetic Clinic, ValieAssr ples were heterozygous for two different muta Abstracts GfH ÖGH SGMG Tagungsband main possesses no conventional SAR/MAR Sq. 16, Keshavarz blvd. TehranIran tions (delta F508 and G551D). Results from this binding properties. The function of the novel do Autosomal recessive spinal muscular athrophy study revealed the following frequencies: Delta main remains unknown and will be analyzed fur (SMA) is after cystic fibrosis, the second most F508: 28%, G551D: 5%, G542X: 5%, W1282X: ther. To determine the subcellular localization of common fatal monogenic disorder and after 0%, N1303k: 0%. human LETM1 we transfected COS7 cells with Duchene muscular dystrophy is the first com cDNAfusion constructs with fluorescent protein mon severe neuromuscular disease in childhood. EGFP either on the N and Cterminal site of The disease is characterized by degeneration of P221 13 LETM1. Our results clearly demonstrate that the anterior horn cells leading to progressive paral fusion protein is located in the mitochondria, in ysis with muscular athrophy. Depending on the dicating that LETM1 may play an important (evo clinical type (WerdnigHoffmann=type I, interme Biogenesis of cellular ironsulfur proteins lutionary conserved) role in mitochondrial func diate form=type II and Kugelbergwelander=type and its implication for human disease tion. Since mitochondrial dysfunction has gained III), SMA causes early death (type I) or increaing Lill,Roland(1),Mühlenhoff,U.(1),Hörtnagel, considerable interest as a potential cause of disability in childhood (type II,III). All three types K.(2),Meitinger,T.(2) epileptic seizures and neuromuscular disorders, of autosomal recessive SMA map to chromo (1)Institut für Zytobiologie,Philipps it is tempting to speculate that LETM1 repre some region 5q13.1. Homologous deletions in Universität Marburg,Germany,(2)Institut für sents a suitable candidate for these clinical fea exon 7 and 8 of the survival motor neuron (SMN) Humangenetik,GSFForschungszentrum, tures characteristic for WHS patients with the full gene have been described in >9095% of SMA MünchenNeuherberg,Germany phenotype. patients. Ironsulfur (Fe/S) clusters are important cofac The aims of this study were to screen the dele tors of numerous proteins involved in electron tions of SMA gene (exon 7 & 8) in iranian pa transfer, metabolic and regulatory processes. In tients for prenatal diagnosis of SMA. We have eukaryotic cells, known Fe/S proteins are lo P221 10 studied 26 families with SMA types IIII, partly calised within mitochondria, the nucleus and the with their affected children and their chorion vil cytosol. Only recently, the molecular basis of the Systematic clinical, cytogenetic and lus samples (CVS). DNA deletion genotypes biogenesis of these proteins in a living cell has molecular characterization of balanced were determined by PCRRFLP analysis ampli started to become elucidated. Mitochondria per chromosome rearrangements that are fying exons 7 and 8 of SMN. Results revealed form an essential function in the biosynthesis of associated with disease the homozygous deletions of exon 7 and 8 of the cellular Fe/S proteins. The organelles harbour a Kalscheuer, Vera (1), Menzel, C. (1), Viertel, P. SMN gene in 23/26 (90%). complex „ironsulfur cluster (ISC) assembly ma (1), Kuebart, S. (1), Shoichet, S. (1), Hagens, O. The data support that homozygous absence of chinery“ consisting of some ten proteins. The (1), Klein, M. (1), Slosarek, I. (1), Madle, H. (1), SMN exon 7 & 8 is strongly associated with ISC proteins (including a cysteine desulfurase, a Tommerup, N. (2), Ropers, HH. (1) SMA. The percentage of homozygous deletions ferredoxin, and an Hsp70 chaperone) are highly (1) MaxPlanckInstitute for Molecular in the study is almost as high as that reported in conserved from bacteria to man. Their function Genetics, Berlin, Germany, (2) Wilhelm other investigations. This method is useful, fast is crucial for maturation of both mitochondrial Johannsen Centre for Functional Genome and effective for gene diagnosis and prenatal di and extramitochondrial Fe/S proteins. Recent Research, Institute of Medical Biochemistry agnosis of SMA. ly, we identified frataxin located in the matrix as and Genetics, The Panum Institute, a novel component required for cellular Fe/S Copenhagen, Denmark protein maturation. The human homologue is de Clinical and molecular characterization of dis pleted in patients of Friedreich’s ataxia. The pre P221 12 easeassociated balanced chromosome re cise role of mitochondria in the maturation of cy arrangements (DBCRs) is a very powerful ap tosolic Fe/S proteins is still unclear, but accord proach for the identification of disease genes Mutation Detection and Prenatal Diagnosis ing to a current working model, an Fe/S cluster and the elucidation of their function. To date, the of Patients With Cystic Fibrosis (CF) in Iran or a derivative thereof is assembled in the mito Mendelian Cytogenetic Network (MCN) consists P. DerakhshandehPeykar1,2, M. Rahmani2, M. chondrial matrix and exported by the „ISC ex of 305 laboratories from 51 countries worldwide Atai2, J. Ghassemi2, D.D. Farhud 1,2 port machinery“. Its first known components are and 2345 DBCRs have been submitted to the 1. Dep. of Human Genetics, School of Public the ABC transporter Atm1p of the mitochondri central database. As a prerequisite for finemap Health, Terhran Univ. of Medical Sciences, al inner membrane, the sulfhydryl oxidase Erv1p ping of breakpoints, we established a unique set TehranIran/2. Genetic Clinic, ValieAssr Sq. (human ALR) of the intermembrane space, and of >5000 FISHmapped YAC and BAC/PAC 16, Keshavarz blvd. TehranIran glutathione. Depletion of these proteins results clones which covers >65% of the human auto Cystic fibrosis (CF), the most common severe in a specific defect in the maturation of extrami somes as well as the entire Xchromosome. Dur lethal autosomal recessive disorder in whites, is tochondrial Fe/S proteins. Mutations in ABC7, ing the past 30 months, the probe set was em caused by mutation in the CF transmembrane the human orthologue of Atm1p, are causative ployed to map breakpoint regions in >70 pa conductance regulator gene (CFTR) on chromo of a form of sideroblastic anemia (XLSA/A) un tients with DBCRs, most of which were associ some 7q31. The carrier frequency among Cau derlining the importance of Fe/S protein biogen ated with mental retardation (MR). So far, more casians is approximately 1 in 25, with an inci esis for normal cell function.

medgen 14 (2002) 111 P226 01 P226 02

P221 14 CF Diagnosis using Polar Bodies Problems Preimplantation diagnosis (PGD) in Germany and pitfalls attitudes and prospective usage in genetic Tomi, Diana (1), Ludwig, M. (2), Schöpper, B. highriskcouples and matched control Genomic characterization of candidate (2), AlHasani, S. (2), Eckhold, J. (1), Diedrich, couples genes from a chromosomal region K. (2), Schwinger, E. (1) Krones Tanja (1), Koch MC(2), Hoffmann GF(3), associated with infantile benigne myoclonal (1) Institute of Human Genetics, University Huels G(4), and Richter G(1) epilepsy Hospital Luebeck, (2) Womens Hospital and (1)Center for Conflict Studies & Department Prawitt D.,Bauer K.,Philippi H.,Busch J.,Brixel Obstetrics, University Luebeck of Internal Medicine, University of Marburg; L.,Spangenberg C.,Zabel B. Introduction:Preimplantation genetic diagnosis (2)Institute of Human Genetics, University of Children’s Hospital, Univ. of Mainz (PGD) by testing one or two blastomeres for Marburg, (3)Department of General The infantile benigne myoclonal epilepsy (FBME) chromosomal or single gene disorders is an es Paediatrics, University of belongs to the idiopathic epilepsies represent tablished technique. PGD offers families at risk Heidelberg,(4)Department of General ing 40% of all epileptic illnesses in children. To to have known inherited diseases the possibility Paediatrics, University of Giessen, Germany date, only few of these conditions have been to avoid affected offspring without pregnancy In our study we assessed the attitudes, ethical linked to specific candidate genes: Mutations termination with its ethical problems and psy concerns and prospective usage of PGD in the have been described specifically in genes that chological consequences. However, PGD is not light of other possible reproductive options (re encode ion channel proteins like KCNQ2 or compatible with the German embryo protecting frain from having more children, adoption, pre SCN1B. We analysed 11 FBME patients for mu law (ESchG). The prefertilization genetic diagno natal diagnosis(PD), pregnancy without PD) in a tations in the SCN1B gene, without detecting sis by testing only polar bodies instead of blas population of 162 couples with recessive and sequence alterations. One of these patients tomeres, is not restricted by the ESchG and is in dominant genetic disorders (high risk sample) in served as starting point to persue an extended certain cases an alternative to PGD. When polar their reproductive age. These are compared to FBME candidate gene search as this case bodies for autosomal recessive disorders are responses of 149 couples, matched for age and showed a deletion of chromosomal region analysed, only the alleles inherited by the moth number of children and controlled for social sta

GfH ÖGH SGMG Tagungsband Abstracts GfH ÖGH SGMG Tagungsband 11q23.324, thus probably linking FBME to this er can be indirectly studied. Statistically, 50% of tus. As expected, the reproductive history of the genomic area. Using a hybrid cell line of the the oocytes carrying the mutation are fertilised two populations differ significantly. The high risk aberrant chromosome 11 in a CHO background, by sperms bearing the wild type allele and yield sample reports more miscarriages and abor we were able to localize three genes in this dele to unaffected children. tions. 30% of the high risk population versus tion that would meet the criteria for a FBME can Experiments: Here we present our results after 15,2% of the control population had used PD in didate gene. Two of them (KCNJ1, KCNJ5) en three cycles with polar body testing for cystic fi at least one pregnancy, 24,2% versus 10,8 % of code potassium channels and are related to pro brosis. the children were born after PD. The Odds Ratio teins that are involved in other forms of epilep Comparing the results after primer extension for the highrisk group for refraining from having sy. The third gene (GRIA4) encodes a glutamate preamplification (PEP) with the results after mul more children in reproductive age is 1,7 (CI 1,3 receptor, whose rat homologue is involved in tiplex PCR, we could show that multiplex PCR is 2,7) referred to the control group. For 17% of neurological disorders. To determine the role of more accurate and reliable than PEP for polar those high risk couples who opt for having more these genes in FBME we identified the exonin body testing. children, PGD is, although not allowed in Ger tron structure and sequenced the coding regions By testing only first polar bodies we found a rate many, the `most probable option´. The PGD in 11 patients. In three FBME DNAs we detect of heterozygosity of 54% for the deltaF508 mu group assesses the burden of having an(other) ed a CtoT transition in exon 1 of KCNJ1. We tation. In these cases no information about the affected child and their subjective genetic risk analysed 58 control DNAs and found the CtoT oocyte, whether it is a carrier of the mutation or significantly higher, has less healthy living chil change in 14 cases, thus classifying this transi not, is available. Statistically, 50% of these het dren, and their desire to have an(other) child is tion as polymorphism. Our further strategy now erozygous oocytes bear the wild type allele af stronger. An overwhelming majority of the whole includes the analysis of the remaining exons in ter the second meiotic division, but had to be sample considers the preimplantative embryo to our FBME cohort and the identification of addi discarded if only the first polar body could be be `my child´ or `quite my child´(77,1%). Only tional candidate genes. The genomic character tested. 4,4% say the embryo is `a cell cumulus´. 18% of ization of the three genes on 11q has also pro In the third cycle we were able to test four sec the whole sample think the PGD should not be vided the tools to screen for mutations in other ond polar bodies. The results indicate for anoth legalized in Germany, whereas 82 % think it patient groups with neurological defects linked er possible pitfall if only the first polar body is should be legalized in a more or less restricted to this chromosomal region. tested. In one case the second polar body was way. Our data allow a deep insight into the influ heterozygous while the first polar body had pre ences on the personal attitudes towards PGD sented with homozygosity. and other reproductive options in a population P224 01 Discussion: Prefertilisation genetic diagnosis by of high vs. average genetic risks. testing polar bodies is a possibility to perform preimplantation diagnosis. However, this alter Genotoxic effects of Oxazepam mitotic native to PGD needs a great number of oocytes. abnormalities P226 03 A large number of oocytes have to be discard Dr Slavka Ibrulj, Aida Tasevac, MSc ed, since the paternal allele can not be tested. Institut for Genetic Engineering and Oocytes with heterozygous first polar bodies Erste Erfahrungen in der Anwendung der Biotechnology, Kemalbegova 10, Sarajevo, have also to be discarded. Only by testing the Polkörperdiagnostik Bosnia Herzegovina first and second polar body the number of Buchholz Tina, ClementSengewald Annette, Genotoxic effects of Oxazepam(benzodiazepines oocytes available to be transferred can be in Thaler Christian J. anxiolitic)and its influence on the mitotic process creased. But analysis of the second polar body Frauenklinik, Klinikum Grosshadern, in human lymphocytes in vitro and in Allium can not be performed in the same cycle. There LudwigMaximiliansUniversität München roots meristem cells, are presented in this re fore, oocytes have to be cryopreserved at the Chromosomale Veränderungen in der Eizelle (EZ) port. pronuclear stage, which will result subsequent führen zu IVFVersagen und zu Fehlgeburten. Ihr It has been established that OX in concentration ly in a lower pregnancy rate as compared to Auftreten ist abhängig vom mütterlichen Alter of 0.5 50 microg/ml causes disturbances in the fresh cycles. oder der mütterlichen Prädisposition. Die Polkör kinetics, separation, structure and chromosomal perdiagnostik (PKD) stellt eine Möglichkeit dar, organization in every phase of lymphocyte divi chromosomale Fehlverteilungen bei EZ zu erken sion and in onion roots meristem cells. nen, bevor diese befruchtet werden. OX induces lagging, vagrancy, dislocation, irreg Die Entnahme der Polkörper (PK) durch Mikro ular grouping, anaphase and telophase chromo manipulation muss die Unversehrtheit der some bridges, stickness and C mitosis. Eizellen garantieren, um eine ungestörte Be The established irregularities point to the con fruchtung und Embryonalentwicklung zu clusion that OX induces spindle abberations and gewährleisten. Zusätzlich ist die Vollständigkeit aneuploidy. der PK für die nachfolgende Diagnostik mittels der Fluoreszenzin situHybridisierung (FISH) er forderlich. Der Vergleich mehrerer Methoden zur

112 medgen 14 (2002) Extraktion von PK (A: Extraktion mit spitzer (3) Prenatal Medicine and Genetics, for UPD 14 were undertaken with a pannel of 12 Pipette; B: Extraktion nach Laser Zona Drilling Duesseldorf, Germany micro satellite markers located on chromosome (LZD) mit stumpfer Pipette; C: Extraktion auss Prenatal diagnosis mainly focusses on the de 14. One of these markers, which moreover was chließlich mittels Laserlicht (Kombination aus tection of trisomie 21, which is the most com located on 14q32 a region where imprinted LZD und Extraktion mittels Laserfalle)) zeigte mon chromosomal abnormality. During the last genes have been located recently, showed only einen eindeutigen Vorteil für den Einsatz von decade a lot of ambitious efforts were undertak one maternal allele indicating segmental mater Laserlicht. en to replace the risky fetal cell sampling tech nal UPD 14. Therefore, a chromosome 22 cen Bei über 200 PK von verworfenen EZ wurden niques like amniocentesis with noninvasive pro tromer specific probe (22Z4; provided by Dr. M. mittels FISH einzelne Chromosomenabschnitte cedures like maternal blood sampling. The Rocci, Bari, Italy) was used in parallel to the mit 2 oder 5 Sonden farblich markiert. In 86% analysis of fetal cells isolated from maternal 14/22 probe. In 14 of 30 metaphases the mark ließen sich die PK nach der Präparation hybri blood is still not usefull under routine conditions. er was ascertained and to be a derivative chro disieren und in 66% auswerten. Die Aneuploi On the other hand maternal serum parameters mosome 22. As a consequence the molecular dierate ergab sich bei Verwendung von 2 Son like triple test and PAPPA in combination with result was considered as a new mutation in the den mit 26% und bei 5 Sonden mit 69%. In 44% nuchal translucency (NT) measurement and ma paternal allele resulting in a homozygous or der Fälle fanden sich mehrere Chromosomen ternal age are already used to calculate the hemizygous status in the fetus. In conclusion, for aneuploid. probability for the occurance of trisomie 21. To a confident UPD diagnosis it is necessary to find Seit Anfang diesen Jahres evaluieren wir die detect new protein markers in serum of pregnant at least two independent and informative loci in PKD in einer Studie. Bisher wurden 15 Patientin women we analysed 15 samples with the sur dicating uniparental inheritance. nen rekrutiert, davon 7 mit maternaler Transloka face enhanced laser desorption ionisation tech tion. Das Indikationsspektrum, die Besonderheit nology (SELDI). This technology is based on pro en bei der Stimulationsbehandlung und unsere tein chip arrays with solid phase chromato P227 04 Erfahrungen bei der Mikromanipulation und der graphic surfaces, which were examined by MAL FISH Untersuchung werden im Detail vorgestellt. DITOF (Matrix associated laser desorption ion isation time of flight). SELDI allows the detection Experiences of nuchal translucency of nearly all proteins present in serum. For analy screening to detect fetal aneuploidies

sis serum samples were mixed with the chip Lohner, Regina (1), Drechsler, K. (1), Bullerdiek, Abstracts GfH ÖGH SGMG Tagungsband P227 01 type appropriate binding buffer and were direct J. (1), Neumann, A. (2) ly spotted on the chip surface. After incubation (1) Center for Human Genetics, University of Influence of ethnic origin on the level of and several washing steps an energy absorbing Bremen, Germany, (2) Clinic of Gynaecology, „triplescreen“ serum parameters with matrix (EAM) was added to ensure the energy Zentralkrankenhaus Links der Weser, special consideration of a Turkish sub transfer from laser to proteins. Then the chip Bremen, Germany population in Germany was transferred to the SELDI system and OBJECTIVE: Measurement of nuchal translucen Sancken, U., Moghadam, S. analysed by an automated data collection pro cy (NT) is a widely used screening method for Institute of Human Genetics, University of tocol. The analysis of fifteen sera (12 16 weeks chromosomal abnormalities. During fetal NT Goettingen, Goettingen, Germany of gestation) from women carrying a fetus with screening, ultrasound is used to assess for a flu A database including the results of 38539 rou trisomy 21 and fifteen controls from women with id collection behind the fetal neck. Increased NT tine risk assessments for chromosomal disor an inconspicuous pregnancy resulted in at least can identify fetal aneuploidies as e. g. trisomy 21 ders and neural tube defects (socalled „Triple two significantly differentialy expressed proteins and is associated with major defects of the heart Test“) was screened for Turkish, Arabic, Persian, in the range of 5 to 15 kDa. These results indi and great arteries and a wide range of genetic and SouthEastAsian patients living in Germany. cate that beneath the already existing protein syndromes. In this study we evaluated the preg Their serum levels (AlphaFetoprotein=AFP, Hu parameters other markers can be found in ma nancy outcome after NT screening. man Choriongonadotropin=hCG, free Estriol= ternal blood to differentiate between normal and METHODS: Fetal NT thickness and crownrump uE3) were compared with those of German pa trisomy 21 pregnancies. length (CRL) were measured in 633 women with tients. singleton and 51 with multiple pregnancies. The After exclusion of all cases where the serum val risk for trisomy 21 was estimated on the basis of ues might have been influenced by other factors maternal age and fetal NT thickness for CRL. P227 03 than ethnic origin (affected pregnancies, abor The distribution of estimated risk was deter tion, gestosis, smoking, etc) 25377 cases were mined and the sensitivity of a cutoff of 1 in 300 left for a comparative analysis. 3.7% patients Pitfalls in prenatal diagnosis of a was calculated. Fetal karyotyping was per were of Turkish origin and by far the most nu supernumerary marker chromosome and formed in 89 cases (12 %). merous subgroup besides the 94.3% Germans. exclusion of uniparental disomy RESULTS: The median maternal age was 32 As the body weight of Turkish (mean: 65.0kg) Hoppe, Constance (1), Kotzot, D. (2), Langer, S. (range 1744) years, the median gestation age at and German women (mean: 68.4kg) differed sig (2), Starke, H. (1), Liehr, T. (1), Ziegler, M. (1), screening 12.7 (1114) weeks and the median fe nificantly only weight adjusted MoM values were Weise. A. (1), Ernst, G. (1), von Eggeling, F. (1) tal CRL 61.2 (3884) mm. An abnormal karyotype compared. At a 1% alpha level there were no (1) Institut für Humangenetik und was found in 21 cases (2.9 %). The estimated significant differences regarding the hCG and Anthropologie, Klinikum der FSU Jena, risk calculated by the maternal age was 1 in 300 uE3 MoM values. A highly significant difference Jena, Germany, (2) Institut für or greater in 262 (36 %) cases. The respective was stated for AFP though the AFP mean in the Humangenetik, Klinikum rechts der Isar, TU value based on maternal age and fetal NT thick Turkish subgroup (0.96 MoM) was only slighly München, München; Germany ness for CRL was ³ 1 in 300 in 85 (11,5 %) cas lower than in the German population (1.01 Clinical relevance of supernumerary marker es. These included 60 (9.2 %) of the normal fe MoM). Because of only small differences in chromosomes (SMC) found in amniotic fluid de tuses, 11 of 12 (92 %) cases with trisomy 21, 5 serum levels we conclude that adjustment of pends on the amount of euchromatic material (100 %) with trisomy 18, and 2 (100 %) with tri serum MoM values is not necessary for Turkish and might be a hint towards uniparental disomy somy 13. With a cutoff of 1 in 300, the sensitiv patients. (UPD) of the normal homologs. Problems asso ity was 95% at a falsepositive rate of 8.2 %. ciated with UPD include trisomy mosaicism, ho CONCLUSION: The combination of maternal age mozygosity of autosomal recessively inherited and fetal NT provides an effective screening mutations, and genomic imprinting. In the case method for chromosomal defects. Application of P227 02 described here amniocentesis was performed in a cutoff of 1 in 300 is efficient for the screening the 16th week of gestation due to advanced ma of fetal aneuploidies. Protein chip analysis to detect new protein ternal age. Ultrasound evaluation was unremark markers in the maternal serum specific for able. Cytogenetic analysis showed a SMC in trisomy 21 pregnancies about 48 % of metaphases. By AkroMFISH the Michel, Susanne (1), Hoppe, C. (1), Eiben, B. marker was characterized to originate from chro (2), Kozlowski, P. (3), Claussen, U. (1), Melle, C. mosome 14 or 22 and no euchromatic material (1), Gneist, J. (1), von Eggeling, F. (1) was detectable. Because of the advanced time (1) Institute of Human Genetics and and the risk of maternal or paternal UPD 14, Anthropology, University of Jena, Germany, which both are associated with growth retarda (2) Institute of Clinical Genetics and tion and dysmorphic features as well as in a pro Cytology Nordrhein, Oberhausen, Germany, portion of cases with developmental delay and skeletal abnormalities, molecular investigations

medgen 14 (2002) 113 P227 05 nation of the striking anomalies of hands, feet, which are subsequently exported from the pro the facial dysmorphism and the very hypoplas ducing cell and taken up by other cells. This tic fibulae. The mother has mild facial dysmor biodistribution increases the number of cells Prenatal diagnosis of rhizomelic phism with antimongoloid slant of palpebral fis reached by a therapeutic protein by orders of chondrodysplasia punctata sures and hypertelorism, a sign, which was re magnitude in comparison to the number of Hempel, Maja (1), DaumerHaas, C. (1), ported in other carriers of OPD syndrome type II transfected cells. Use of the native or protein Minderer, S.(1), Freisinger,P. (2), Nerlich,A (3), which is localized on Xq28. It could be demon engineered PTD from the HIVTAT protein allows Schramm,T (1), Gloning,K.P. (1) strated that the mother has a skewed Xinacti the distribution of large molecules, e.g. beta (1) PränatalMedizin München, Munich, vation pattern, further confirming the diagnosis. galactosidase, including the transport across the Germany, (2) Kinderklinik der TU München, In the next pregnancy an indirect DNA diagnos bloodbrain barrier. Munich, Germany, (3) Institut für Pathologie, tic can be offered. This approach is especially suitable for the treat Städtisches Krankenhaus Bogenhausen, ment of recessive neurological diseases, where Munich, Germany the missing gene product can be delivered to In the 19th week of the fourth pregnancy of a the brain via the blood after, e.g., production in 28year old woman a detailed sonographic in P227 07 the liver, since hepatocytes can be transfected vestigation was performed to exclude fetal mal more efficiently than many other cell types in formations. Her first 5year old son had a very Reduced amplification efficiacy of cluding neurons. Examples will be shown from short stature, extremly short humeri, contrac KIAA0027/MLC1 alleles: Implications on the murine models, which illustrate that injection of tures in distal joints, delayed myelination, bilat molecular diagnosis of MLC1 marker DNA into the liver effectively results in eral cataracts, seizures and severe developmen Thomas Bettecken, Claudia Rubie, Peter 100% of positive hepatocytes and that the tal delay. A lot of efforts have been made to es Lichtner, Markus Siekiera, Thomas Meitinger, marker protein can be detected in the choroid tablish a diagnosis for him, but without success. Gerald Stöber plexus of the brain, where the bloodcere Two further pregnancies ended in miscarriages Institute of Human Genetics, Technical brospinal barrier is crossed. Details of the vec in the first trimester. The woman and her hus University of Munic & GSF, Ingolstädter tor requirements will be explained as well as band were second degree relatives. The detailed Landstr. 1, 85764 Neuherberg; Department suitable candidate diseases, whose treatment sonographic investigation in the 19th week of

GfH ÖGH SGMG Tagungsband Abstracts GfH ÖGH SGMG Tagungsband of Psychiatry and Psychotherapy, University can be tested in murine models. pregnancy revealed no anomalies. Four weeks of Würzburg, Füchsleinstraße 15, 97080 later, in the 23st week short humeri, an abnormal Würzburg profile with hypertelorism, depressed nasal Mutations in KIAA0027/MLC1 are causative for bridge, anteverted nostrils and flexion contrac the chromosome 22qtellinked autosomal reces tures of the 3rd and 4th fingers were detected. sive childhoodonset megalencephalic leukoen We suspected that this fetus has the same auto cephalopathy (MLC). A high degree of allelic het somal rezessive syndrome as the first child. Un erogeneity has been observed with 15 disease der the hypothesis that the son and the fetus causing mutations. Exon 11 is a hot spot for mu might have a rizhimelic chondrodysplasia punc tations among patients suffering from MLC, and tata efforts were made to confirm this diagnosis in multiplex pedigrees with schizophrenia oc in the son. After these investigations the diagno curred a rare coding variant L309M of unknown sis of rhizomelic chondrodysplasia punctata was biological significance, which was observed seg nearly certain. The mother decided to terminate regating as well as nonsegregating with the dis the pregnancy. The fetus had the typical facial ease. Using DHPLCanalysis, reproduction of dysmorphism, short humeri and joint contrac earlier findings on L309M revealed homoduplex tures. The Xray showed the typical epiphyseal resolution patterns among individuals, who had stippling confirming the diagnosis of rhizomelic been described to be heterozygous for the vari chondrodysplasia punctata. Mutation analysis of ant, which was further confirmed by sequencing the PEX7 gene is in progress. In the next preg the respective PCRproducts. The preferential nancy an earlier prenatal diagnosis can be of amplification of specific alleles of exon 11 may fered with biochemical or DNA analysis. be caused by a cumulative effect of high Tm do mains, several incomplete intronic tandemre peats of 68 bp (copynumber 6.3) and of 33 bp P227 06 length (copynumber 9.5). Secondary folding structures due to tandem repeat elements, and a common polymorphism, 1165ins33bp, at the Prenatal diagnosis of otopalatodigital 5’ end of exon 11 may be causative for prefer syndrome type II ential amplification of mutant 1040A alleles. DaumerHaas, Cornelia (1), KlehrMartinelli,M. Consistent amplification was obtained only when (1), Freisinger,P. (2), Nerlich,A. (3), Orth,U. (4), we employed exonic primers directly adjacent to Gal,A. (4), Schramm,T. (1) the L309M variant. We propose a differentiated (1) PränatalMedizin München, Munich, proceeding for mutational screening of exon 11 Germany, (2) Kinderklinik der TU München, of MLC1 gene among patients with the clinical Munich, Germany, (3) Institut für Pathologie, phenotype of MLC. Städtisches Krankenhaus Bogenhausen, Munich, Germany, (4) Institut für Humangenetik, Hamburg, Germany In the 21st week of her second pregnancy a 28 P229 01 year old woman was seen for detailed sono graphic investigations. Her first pregnancy end New possibilities for the gene therapy of ed with a stillborn malformed male child in the neurological diseases 30th week. This child had shortened and curved Reiss, Jochen limbs, malformed feet, flat profile with microg Institute of Human Genetics, University nathia. A campomelic dysplasia with a low re Göttingen, Germany currence risk was the given diagnosis. However To date, gene therapy approaches can be divid no SOX9 mutation could be detected. The ed into those using viral vectors and those using anomalies detected sonographically in the 21st plasmid DNA. While the viral vector strategies week of her second pregnancy were very simi imply complex production protocols and serious lar: shortened limbs with very hypoplastic fibu risks for the patients, plasmid therapy protocols lae, malformed hands and feet and facial dys have the main drawback of a very low transfec morphism. These findings were compared with tion efficiency. The use of protein translocation the photographs and the pathology report of the domains (PTDs) in fusion proteins encoded on a first child. The Xlinked otopalatodigital syn plasmid vector allows the expression of marker drome type II was suspected due to the combi and therapeutic proteins in a transfected cell,

114 medgen 14 (2002) medgen 14(2002)

115 GfH ÖGH SGMG Tagungsband Abstracts 116 GfH ÖGH SGMG Tagungsband Abstracts medgen 14(2002)