Published Abstracts: C ancer Genetics A301 1743 1744 lluorescence in situ hybridisation (FISH) studies on nuclei released Identification of BCR/ABL gene in Chronic Myeloid Leukemia cases using from paraffin-embedded tissue (1ST) in low stage ebryonal carcinoma Fluorescence In Situ Hybridization (FISH) in Indian S.Bose, of the testis. RI. Blouch. PAlbers. TA. Smolarek. R. ster. and population. N.A...earma, Indiana University Medical Center, Indianapolis, K.Kucheria, V.P.ChoudharX, H.K.Kumbnani*. All India Institute of Mialrl- Indiana, USA. Sciences, Delhi University-, Delhi, India. cytogenetic abnormalities in testicular germ cell tumors (TGCTs) Identification of bcr/abl fusion gene in Chronic Myeloid Leukemia frequently include the presence of i(l2p), as well as other (CML) is diagnostically significant and crucial for monitoring therapy. abnormalities of 12p. we have developed p and q -specific painting 95% of all cases show probes for chromosome 12 for use in FISH, and with them a protocol for Approximately reported the Philadelphia(Ph') the study of distribution of 12p and 12q sequences within interphase translocation. Remaining Ph'-negative cases are either bcr-positive nuclei released from formalin-fixed, paraffin-embedded tissues (PET). or bcr-negative. Since the latter group comprise of heterogenous In an effort to look for a cytogenetic feature which could discriminate myeloid disorders it is important to evaluate cytogenetically uninformative pathologic stage I GCTs from pathologic stage II tumors, we have or Ph'-negative cases further by molecular or Fluorescence Insitu applied this technique in a retrospective study of low stage nonseminomatous germ cell tumors. Approximately 30t of patients who Hybridization (FISH) analysis. FISH is also a sensitive and efficient present with clinical stage I nonseminomatous testis cancer are in fact tool for monitoring Ph'-negativity after interferon (IFN) therapy or bone stage II. Previous studies at Indiana University have showed an marrow transplantation (BMT). increasing volume of embryonal carcinoma in the orchiectomy specimen We analysed 48 treated and untreated CML cases by conventional to be associated with a higher likelihood of pathologic stage II cytogenetic method. O 22 untreated cases, 20 were positive for Ph' diagnosis. However, not all patients with pure embryonal carcinoma in the primary were pathologic stage II. Whole nuclei were released from chromosomes, 1 was negative and 1 was mosaic. Of 26 treated cases, archival material (PET) and studied using bicolor FISH with chromosome 23 were positive, 2 were negative and 1 was mosaic. Sequential cyto- 12 p and q arm-specific painting probes developed through the use of genetic follow up study was done in 15 cases receiving Interferon-alpha chromosome microdissection. FISH was performed and the results /chemotherapy. Post therapy 2 became negative and 2 mosaic. Six analysed using blinded specimens. In all cases, distinct regions of cases were analysed using FISH for bcr/abl gene which confirmed the 12p and 12q probe hybridization could be simultaneously observed, results. Cases allowing identification of probable normal chromosme 12s, as well as cytogenetic showing negativity/mosaicism, post therapy regions of amplification of 12p sequences, including identification of are being further confirmed using FISH to assess the minimal residual possible i(12p)s. In 5/14 specimens, a distinct and peculiar pattern malignant cells. Clinically CML, Ph'-ve cases at diagnosis are also of 12p hybridization was observed, and termed multi-focal 12p, or 12p being evaluated further by FISH. 'disarray'. Of the five specimens demonstrating 12p disarray, 4 were Cytogenetic results show similar percentage of Ph-positive CML pathologic stage B, while 1 was pathologic stage A. Whether the trend patients toward 12p disarray portends metastatic potential or activity in NSGCTs In Indian population as reported elsewhere. Our results support the will need to be assessed using a larger series of patients. importance of using FISH technique to assess the presence of malignant cells in evaluating the cytogenetically negative cases at diagnosis and mosaic or negative cases post therapy.

1745 1746 Familial brain gliomas: s report of two families. CaL. Dal B. AzzaIi2. E. Unusa Cytegenet Finga hI A Choroid Plexus Cacnona. FoxI. and G. H. Vancel. Department ofMedical and Molecular Geneticsl, ((You-S. Li'. Ross F. Armstrong'. Yao-Shan Fan Department of Department ofPathology2, Indians University, School ofMedicine, Indianapolis, IN, Pathology, Victoria Hospital, London, Ontario, Canada. 801 St. Mary's Dr., Evansville, IN 477143. Choroid plexus tumours are rare, and most occurrence frequently occur in early Familial ofbrain gliomas is a rare finding. In some families the diagnosis childhood. Only a few choroid plexus turnours have been karyotyped. ofbrain gliomas in one or more family members is associated with other malignant In a in the proband and family members. These other tumors can involve patient with choroid plexus carcinoma, cytogenetic studies tumors tissues revealed a such as breast, skin, muscle and bone marrow. The pattern of cancer may suggest that hyperhaploid stemline, 32, XY, + 1, + 7, + 9, + 12, + 13, the family has a familial cancer syndrome or preneoplastic syndrome, such as Li- + 14, + 19, + 20. Endoreduplication and doubling of the stemline up Fraumeni syndrome, familial polyposis, tuberous sclerosis or neurofibromatosis. In to 200-400 chromosomes per cell and variation in numerical changes some families, brain tumors are the sole neoplasia identified suggesting tamilial site- were also noted. Telomeric association was present in most cells. specific cancer. We report on two families with familial brain gliomas. Within the first The 1 2p and 20q were by far the most frequently involved family, Family A, a total offour individuals spanning two generations have been chromosome . It is believed that telomeric association triggered diagnosed with glioblastoma multiforme. Additionally, breast, ovarian, and colon further structural changes in this case since the 12p and 20q were cancers have been identified. A tentative diagnosis of Li-Fraumeni syndrome, a rare always involved in the few structural abnormalities identified. A inherited cancer syndrome, was given. Testing for p53 mutations is in progress. A review of the literature suggests that choroid plexus carcinoma seems karyotype performed on the probind demonstrates normal peripheral blood to be characterized by hyperhaploidy and that choroid plexus chromosomes. The second family, Family B, has two sisters one diagnosed with papilloma by hyperdiploidy. glioblastoma multiforme and the other with astrocytoma at the respective ages of 31 and 28 years. This family fits the familial site-specific cancer model. Neither family demonstrates cutaneous abnormalities or reports exposure to environmental carcinogens. In addition to describing these two families, we will discuss the inheritance pattern for site-specific brain cancer and explore genetic counseling issues for familial cancer syndromes and site-specific brain cancer including the availability of genetic testing and screening protocols.

1747 1748 The use of Non-Isotopic RNase Cleavage Assay for p53 mutant Chromosome analysis of distal common bile duct carcinomas. screening in prostate cancer. C.J.Godec_ .J.Macera, N.Sharma and R.S.Verma. Long Island College Hospital/SUNY C. A. Griffin. P. P. Long. L A. Morsherr T. Eilingham. R. H. Hrub=n Health Science Center at Brooklyn, N.Y. CJ.L.. Johns Hopkins School of Medicine, Depts. of Oncology, The most common form of cancer in men today, prostate car- Pathology and Surgery, Baltimore, MD. cinoma (244,000 new cases in 1995), is still poorly under- stood at the genetic level. p53, a known tumor supressor Bile duct carcinoma is a rare malignancy which may share some genetic gene has been shown to be involved in lung, colorectal, abnormalities with other GI tract breast and other tumors. Involvement neoplasms.We analyzed eight primary of p53 in early pros- adenocarcinomas of the distal common tate cancers is uncommon but studies indicate a 20-25% in- infiltrating bile duct for volvement in advanced stages. The majority of mutations chromosome abnormalities as afirst approach to describing genomic found in p53 tend to be point mutations clustered in exons changes in this tumor. All eight of the carcinomas were obtained fresh 5 through 8. The most common methods of detecting these from surgical pancreaticooduodenectomy specimens. Six of the carcinomas mutations is either through direct sequencing or single were differentiated and two strand conformational moderately were poorly differentiated. Clonal polymorphisms (SSCP) of polymerase chromosome abnormalities were found in seven of the neoplasms. The chain reaction (PCR) products. Direct sequencing is very modal number cumbersome and SSCP is limited to fragment sizes of 200-300 was near triploid in two cases, near tetraploid in one case bps. We used the non-isotopic RNase cleavage assay (NIRCA- and near diploid for the rest. Four cases had complex chromosome Ambion) where transcripts are made in vitro to PCR products rearrangements in addition to whole chromosome losses and gains. The that contain opposable T7 and Sp6 promoters. Sense and most frequent losses were chromosomes 9 (4 cases), 10 (3 cases) and 18 (3 anti-sense strands are hybridized to wild type controls and cases). Chromosome 7 was gained in 3 cases (1 of these had a small the reuslting duplex is digested with RNase. Mismatch mu- interstitial deletion in Five cional tants are identified ethidjum 7p). deletions or rearrangements of byagarose gel electrophoresis, chromosome 6 were seen in total of bromide staining and UV illumination. This method not only a three cases. Chromosome fragments examines both sense and anti-sense strands but allows the or di's were seen in two cases, and in one cell of another case. Similar to p53 exons 5- 9 region to be analysed in only two fragments. our findings in adenocarcinoma of the pancreas (Genes Chrom Cancer Primary cultures of tissue from 9 radical prostatectomies 9:93-100, 1994; Griffin et al, Cancer Research in press) were the were established and DNA extracted. Five of the tumors were involvement of 6q, losses of 18, and apparent dms. A small metacentric stage T1 and T2 and four were stage T3. No mutations in - marker was observed in three Characterization of the marker exons 5 9 were detected in any of the samples. The use neoplasms. of the NIRCA method should allow the detection of additional by FISH is underway to determine whether a site specific chromosome mutations within the p53 gene providing a better under- abnormality characterizes this group of neoplasms. standing of the genetic mechanism of cancer. A302 Published Abstracts: C.ancer Genetics (continued) 1749 1750 Epstein-Barr virus is an early event in gastric carcinogenesis and Is Molecular cytogmetic characterization of the thyroid cancer cell Use PTC1113A. Independent of bcl2 expression and p53 accumulation. M. 1.. QGlULy ME. Herrnann'-'~ G.B. JTAloe2 N. Abumrad N. Blin' E. Meese P.A. D. R. Pulitzer, P. A. Eagan C G. Cho. B. G. Schneider Department of Pathology, Lalloevi University of Texas Health Science Center at San Antonio and the Audie L. Murphy SUNY Department of Surgery, Stony Brook', NY, Henry Ford Hospital', Center for Memorial Veteran's Hospital, San Antonio, TX. Moleluclar Biology3, Wayne State University, Detroit MI, Departments of Genetics, Epstein-Barr virus (EBV) was detected in 11 of 95 adenocarcinomas of the University Tabingen and Homburg/Saar, Germany, National Technical Institute for stomach by EBERI in situ hybridization. When present, the virus was localized to the Deaf, Rochester Institute Technology, Rochester NY'. malignant epithelial cells and to dysplastic gastric epithelium, but was not seen in The recently established thyroid cancer cell line PTC-11 13A was characterized by normal appearing gastric epithelium or in intestinal metaplasia. The EBV DNA was molecular cytogenetic means, to delineate chromosomal areas for altered candidate monoclonal in all three cases tested by Southern blot analysis of the EBV terminal repeat fragment. These findings suggest that the virus was present before malignant genes. Tumors were grown in an enriched RPMI 1640 medium. Cytogenetic studies transformation. The presence of EBV was correlated with increased numbers of were followed by fluorescent in situ hybridization (FISH) of two passages (P16, P17) tumor-infiltrating T-lymphocytes, however this cellular immune response did not using the alphoid sequence lql.77 and whole chromosome libraries as probes. PTC- appear to portend improved overall survival among the cohort of patients with EBV- 1113A continued growing as a monolayer with doubling times sharply increasing related carcinomas. Neither p53 nor bcl2 were consistently detected in the EBV- after passage 3. Clonal cytogenetic aberrations were detected with double minutes in associated tumors. Five of ten EBV-positive carcinomas had accumulation of p53 a proportion of cells (-5%) and marker chromosomes: a short and medium sized protein by immunohistochemical analysis, which was similar to the prevalence ofp53 metacentric marker and a rearranged chromosome lp36+. FISH evaluation revealed accumulation in EBV-negative cases and suggests that EBV infection does not substitute for p53 mutation during tumorigenesis. The bcl2 oncoprotein was chromosome 1 rearrangements with gain of Iq as prominent change. The repetitive expressed in half of the carcinoma specimens tested, but bc12 expression did not sequence lql.77 showed different dosage for appearantly normal chromosome 1 correlate with the presence of EBV or with expression of EBV latent membrane compared to rearranged chromosome 1, and hybridized in some metaphases to lp- protein 1. However, bc12 may play a role in physiologic renewal of gastric mucosa telomer. In additon. we found iso-lq and of breakages at lpl-2. Chromosomes 1, 3, based on the observation ofbcl2 protein in basal cells of normal gastric epithelium. 4, 6, 11, 12, 15, 16 and 17 are involved in marker formation and/or translocations. Chromosome 11 contributes to the formation of the small metacentric marker. The chromosome 4 library hybridized to extrachromosomal genetic material ins meta- phase containing double minutes. We did not find gross chromosome 10 rearrange- ments, which could be expected due to the rearranged ret gene detected in this cell line (P19 and 21). Further definition of molecular genetic changes applying microfish may facilitate the use of this cell line to study amplification and possible loss of suppressor gene function. (Supported in part by HFH funding to GBT).

1751 1752 Exression of alternatvely spliced RET trcripts In the developing Amplification of c-myc correlates with tumer ty In SV40 T-mmortallxed human kidney and Wimn tumor. S. Ivanchuk. *C. Eng. S. Myers and human prostate cancer sublnes: A new system for analysis of tumor progres- LM- Mulligan, Department of Pathology, Queen's University, Kingston, sion. C.K. Jackson-Cook'. V. L. Bae2. and J.L Ware. Departments of Human Ontario K7L 3N6 and ACRC Human Cancer Genetics Research Group, Genetics' & Pathology2, MCV/Virginia Cmmonwealth University, Ricbmond Department of Pathology, University of Cambridge, Cambridge C82 1OP, Previously we developed and carcterd a new model of prostate cner U.K progression using sequential in vwvq4n vitro culture of sublines derived from SV40T- The RET proto-oncogene encodes a receptor tyrosine kinase thought to immortalized human prostate epithelial cells (P69SV40T). Following injections into play a role In development and kidney induction. In the munne model in athymic nude mice, P69SV40T induced tumors in 2/18 mice, designated M2182 and situ hybridization has shown RET expression at early developmental M2205. These new sublines, and the daughter sublines of M2182 (named M15 and stages while RET -/- mice display kidney dysgenesis or agenesis. To M12) have increased tumorigenicity and reduced latency intervals as compared to examine the involvement of RET in human kidney development, we have P69SV40T. Cytogenetic studies, using GTG-mdig, FISH (with multple probes), used RT-PCR and Southern blot analyses to Identify RET exresion in and CGH revealed that each line was near diploid and had several consstent fetal kidney at gestational ages 57 days through 24 weeks as well as in abnormalities. Two structural anomalies were seen in all lines: a der(7)t(5;20;7) and adult kidney. Similarly, RET expression has been determined in a panel a der(5)t(5;9). Aberrations that were acquired and retained in sublines M2182, M1S, of Wims tumors (WT), an embryonal renal neoplasm. We have and M12 following in vivq/n vitro selection included a der(3)t(3;14)Xpl;qll.2), demonstrated expression of RET in all stages of developing kidney der(20)t(7;20)(p22;q12), der(X ;II)(p22.I;q21), and a der(1)t(1;8)(q42;q24.I). examined and in Wr. Recently, multiple mRNA isoforms of RET The latter finding is of particular interest since it resulted in 3 copies of c-myc in generated by alternate splicing at its 5' and 3' ends have been identified. each of these lines, Additional anomalies seen in the icreasingly tumoigenic RNase protection analyses are currently belng used to compare relative (M15,M12) and metastatic (M12) daughter lines were a t(I6;19)(q24;qI3.3) and -Y. levels of expression of each transcript type in both fetal kidney and Wr. The breakpoint on 16 is consistent with the location of the c-myc transciption factor. Loss of this gene could lead to increased transcription of cnmyc. In the M2205 line unique -jumping" rlocatons involving co somes 8 and 13 were seen. These resulted in multiple copies of c-myc (> 6 copies/cell), with its locus being juxtaposed to that of active ribosomal genes (having double NORs). Based on these findings, as well as reports of other groups, the role of c-myc and other genes whose products regulate c-myc, warrant further study within the context of human prostate cancer tumorigenesis and progression. (Supported by NCI-ROCA58126).

1753 1754 eoumnic Instability - A predisposing factor for cancer of cervix. The use of YAC. for mapping tumour suppresor genr on human Nalini Jaiawal, G.Anuradha, N.Rajeswari and Y.R.AhuJa. Mahavir Hospital chromoo 1. LA. James.Y. He. B. Brntnell. D. Jone. E. M Itchel and 10-1-1, tshavir targ, Hyderabad - 500 004, India. J.MV.Ma L CRC Department of Cancer Genetics, Paterson Institute for Cancer of the cervix uteri is the most common aaligeant tumor in Cancer Research, Manchester, UK. the Indian wamen. It develops through distinct preneoplastic epithe- Introduced byJ. Hewitt. lial changes called dysplastic lesions. in this cancer although We have identified recently two regions of human chromosome 1 which involvement of certain chromosomes have been reported there is no potentially harbor genes involved In breast cancer. Areas showing high loss convincing information known as yet to correlate any of the chromo- of consational hetarozygolsity (LOH) were seen at both 1p31.1 and 1p13. some aberrations with disregulation of specific onogene sequence. In order to cone target genes in these regions we have undertaken to Therefore, it would be desirable to pursue an ideal biomarker which construct YAC contigs using clones from both the CEPH and Zeneca YAC will help in identifying the individual at risk. libraries. We have used 5 markers from each region which show high LOH Almost all cancers are associated with genomic instability. In the to screen the YAC libraries by PCR. We have On used singie and multi- present study it was intended to quantitate DNA damage in cervix their Those epithelial calls of patients with precancerous and cancerous lesions colour FISH to order and localise YACs and assss integrity. of the cervix. The cervical epithelial scrapping was collected from YACs which did not appear chimeric by FISH were used to constuct 126 female subjects comprising 26 normal controls, 45 patients with Vectorette libraries to obtin the insert terminal regions for walking. dysplasLa and 52 patients with carcinoma of the cervix. The cells Contig construction In the 1pl3 region was considerably more difficult were processed by comet assay. DNA damage of eplthelial cells was compared with 1p31.1. On average, each marker from 1p31.1 identffed a quantitated with and without the treatment of mutagen (MNNG). Repair larger number of YACs from both the Zenece and CEPH libraries than efficiency was also studied after 2hrs of incubation at 37'C after markersfrom Ipi3. Of theYACsfrom 1 p31.1, 29% of Zenea and 66% of treatment. The cells showing DNA damage appeared as comets under the CEPH were chimeric by FISH. However at la two YACs for each marker microscope. 50 cells per treatment were scored for the length of the were not chimeric and were therefore suitable for walking. In contrast, comet tail. For various comparisons, Maim Whitney U-test was pfozd. although the percentage of 1p13 YACs identfed as chimeric was similar A significant increase in DNA damage and repair deficiency was seen (27%, of Zeneca and 50% of CEPH) far fewer YACs then remained to be from controls through precancerous stages to carcinoma in both untrea- used for walking. In addition, only 36% of insert terminal regions could be ted and treated epithelial cells. Inter and Intra-Lndividual hatero- from Vectorette libraries of the 1 YACs whereas 78% of the as The mean obtained p13 geniety was observed In DNA damage as well repair. DMA termini of 1p31.1 YACs were obtained and used to generate STS markers damage and the number of unrepaired cells in each individual gave an for to obtain further YACs. indication of her genomic instability. The individuals with high PCR screening to to Cloarly, although YACs are a very useful resource for contig generation genoeic instability were considered be highly predisposed still remain of the are cervical cancer. (Supported by the Indian Council of Medical and gene cloning, dffiulties whore regions genome Research No.74/7/90/pers/EMS). under-represented in the lIbraries available at present Published Abstracts: Cancer Genetics (continued) A303 1755 1756 Molecular characterization of a variant 46,XX,t(9;17;22) Comparative Genomic Hybridisation in relation to (q34;ql2;qll) CML by RT-PCR and FISH analysis. M.J.Macera, L.J.Smith, A.Braverman, S.Gogineni and R.S.Verma. Long Is- molecular and cytogenetic analysis Elisabeth Nacheva. land College Hospital/SUNY Health Science Center, Bklyn, NY. Michael Bittnerl. David Ledbettcrl. Robert Jenkins2. Steve Seeli&3 and Approximately, 5-10% of the reported cases of chronic Colin Grace4 Department of Haematology, Addenbrooke's NHS Trust myelogenous leukemia contain what is referred to as variant Hospital, Cambridge, UK; 1-NIH, National Centre for Human Genome translocations; those where at least one additional chromo- Research, Washington DC,USA; 2-Mayo Clinic, Rochester, Mn, USA; sone is included in the translocation with chromosomes 9 & 22. 3-VysisNaperville, Ma, USA;4- Digital Scientific Ltd., Cambridge, UK Clinically, no difference is observed between these cases and those patients with the more traditional translocation. Comparative Genomic Hybridisation (CGH) is a technique by which In the few variant translocations analysed, the BCR/ABL genomic imbalancies are detected by in situ hybridisation of whole translocation has occurred. We were referred a case of CML genomic DNA and image analyis. with a 46,XX,t(9;17;22)(q34;ql2;qll) karyotype in all of Here we compare the results of CGH analysis with other traditional the bone marrow cells. Hybridization with the dual color methods used routinely for the detection of genetic abnormalities. Several BCR/ABL probe (Oncor) identified the BCR/ABL chimeric gene types of specimen were used as models for this study - amniotic fluids, located on the Ph chromosome. RT-PCR was done on RNA ex- LBL with a single chromosome band aberrations (deletions and tracted from bone marrow cells with Transprimers (Oncogene duplications), malignant cell lines with complex genomic alterations and Science) and a PCR product was identified at approximately biopsy material from solid tumnours. 579 bps. A fragment this size indicates the presence of The CGH results were obtained using direct labelled fluorochromes BCR exon 3, a breakpoint-within the intron between exons 3 (Spectrum Orange and Spectrum Green) following published and 4 and having a b3-a2 fusion junction. The majority of hybridisation protocols and a dedicated image analysis software. traditional CML's have either a B3-a2 or b2-a2 junction with the later type missing exon 3. We then hybridized the The CGH data were compared with the results of G-banding, Her-2neu (ERBB2) loci probe located at 17qll.2-12 and the chromosome painting and molecular genetic analysis. RARA loci probe located at 17q21.1 (Oncor) to chromosomes from this patient. In all bone marrow cells, signals from the Her-2neu probe were always located on chromosome 17 while one signal of the RARA loci was always seen on 17q while the other one was always found translocated to the der(9). Thus establishing that the breakpoint on the 17q is located between the two marker loci,at 17q12. It is hoped that studies of this nature will increase our under- standing of this deadly disease.

1757 1758 Differential expression ofgenes in human malignant glioma cells following treatment A case of Infantile Fibrosarcoma with Translocation(12;13). with BCNU. Norman, S.A., Shapiro, J.R, and Scheck, A.C. Neuro-Oncology S.B.Reddy! A.K.Royland Subha Reddv? 1St.Joseph Medical Research, Barrow Neurological Institute of SJHMC, Phoenix, AZ 85013. Center, Detroit, Michigan and2DMC Huron Valley Hospital, Despite surgical resection, followed by irradiation and chemotherapy with the Wayne State University, Novi, Michigan. alkylating agent 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU), human malignant A 7-month-old boy presented with a large intrathoracil gliomas usually recur. Although it can be demonstrated that approximately 90% of Fibrosarcoma. Computerized tomography revealed a homogene- the cells in the primary tumor are killed by BCNU, the recurrent tumor is often ous, dense shading of the right thoracic cavity with no detected within 34 months following treatment. Our data suggests that there are evidence of metastatic lesions. The histopathologic exami- nation demonstrated a tumor more genes involved in resistance to BCNU than have been previously described. We spindle cell composed of inter- lacing fascicles in a "herring-bone" pattern with abnormal are using the differential mRNA display technique to identify genes whose expression mitoses. Cytogenetic analysis of this tumor showed the is induced in a human glioblastoma multiforme cell line following treatment with presence of a translocation(12;13) and numerical changes of 5pg/ml BCNU. Seventeen cDNA fragments have been isolated and sequenced, 3 chromosomes 17 and 20. A total of 15 metaphase spreads were having high nucleotide sequence similarity to heat shock proteins 90 and 86, analyzed. All tumor cells have a rearranged chromosome 13 transfonring growth factor P1 binding protein, and a ribosomal protein. The in common, likely derived from a reciprocal translocation t(12;13) and chromosomes 17 and remaining cDNAs had less than 60%/o nucleotide sequence similarity to any known (q24;ql3-14) supernumerical or 20. Eight cells showed a t(12;13) (q24;q13-14). Five of and novel sequences in the GenBank and EMBL databases may represent genes them had trisomy 17 and trisomy 20, two cells had only involved in the growth ofBCNU resistant cells. trisomy 17 and one only- trisomy 20. Seven cells displayed the same translocation but with an additional derivative chromo- some 13. Four of these cells were trisomic for 17 and 20, two were trisomic for 20 and one was trisomy 17. Thus the karyo- type of this tumor can be described as 47-48,XY,t(12;13) (24;q13-14), +17,+20[cp8]/48-49,XY,t(12;13) (q24;q13-14), +der(13)t(12;13) (q24;q13-14),+17,+20[cp7].

1759 1760

Imprint analysis of small, potentially neoplastic breast lesions by FISH. j, An unusual t(2;11)(p1L2;q23) Eba with Intact MLL eae In as adult Sanford'. R. A. Giraldez'. K. J. Flom'. H. D. Feiner2. S. R. Wolman'. Oncor, Patient with Phladphia postie alnte lymphoblastic leukeia. S. Inc', N.Y.U. Medical Center LeVin.5 LC Copt&2 j. LoCker.2 ME. Sherer.1 N. Wad& and s.M Gollin-1 Deps Definition of genetic aberrations by Fluorescence in situ Hybridization (FISH) of Human Genetics,' Pathology,2 and Environmental and Occupational Health, may aid in the diagnostic/prognostic assessment of small breast biopsies, Univ. of Pittsburgh and the Univ. of Pittsburgh Cancer Institute, Pittsburgh, PA. particularly in lesions whose role in neoplastic evolution is uncertain. Utilizing Cels in approximately half of adult acute lymphoblastic leukemias (ALL) have FISH, we have evaluated touch preparations (imprints) from freshly excised clonal structural chromosome abnormalities, of which the t(9;22)(q34;qll) breast biopsies for copy number of chromosomes 1, 16, 17, 18, X and the translocation appears to be the most frequent, specifically in ALL with immature HER-2/neu (ERBB2) gene 1H2). Probe selection was based on prior experience B-cell differentiation. This finding suggests a dismal prognosis with a short (Micale et al. Human Path 25;29, 1994) and published reports of genetic leukemia-free interval and a high relapse rate. Chromosome Segment 11q23 is alterations associated with breast cancer. At least 100 cells were evaluated involved in translocations with a variety of other chromosomes in a small for each probe study. All analyses were performed without prior knowledge percentage of adult ALL The splitting of the MLL gene (also called HRX, of pathological findings or patient outcome. We used cut-offs of 20% + to ALL-1) at 11q23 and the concomitant fusion with a gene on a partner chromosome indicate significant loss and 10% for gain. Borderline gains (<15%) of results in production of chimeric transcription factors, deregulation of the chromosome 1 were observed in 2 non-malignant lesions, possibly related to subordinate genes and worsening of the clinical outcome. We present a 25-year- man split signals causing apparent low levels of trisomy. In the first 10 cases old with a newly diagnosed B-cell lineage ALL Chromosome analysis of 19 analyzed, we identified 8 cases with significant loss or gain. One case was trypsin-Giemma banded metaphase cells from a 24-hr harvest of unstimulated bone marrow aspirate cell cultures revealed what appeared to be a excluded because of high % of cells with 0 signals, and the remaining 7 were 46,XYt(2;11) karyotype. Metaphase dual-color malignant. Extra H2 cosmid signals are informative of proliferation, reflecting (pll.2;q23),t(9-22)(q34;qll)[16]/46,XY[3] fluorescence iunS hybridization with a 2p telomere DNA probe (D2S445) the G2M fraction; they were elevated in 4/5 ductal malignancies. One case (FISH) and an a-satellite chromosome 11 DNA probe (DllZl) confirmed the presence was near-triploid for all chromosomes except 18 and the increased H2 copies of the translocation between 2p and 11q. However, the rearrangement corroborated trisomy 17 and increased cell division. Extensive, non-uniform expected of the MLL gene was not observed by Southern blot analysis. The clinical aberrations distinguished malignant from benign lesions. (Support attributed significance of this unusual translocation and the possibility that other genes than to NIH CA6217). MLL at band 11q23 may be involved in leukemogenesis remains to be elucidated. A304 Published Abstracts: Cancer Genetics (continued) 1761 1762 Cytogenetic characteristics of a malignant histiocytic tumor (? ilterdigitating AMulti-Center Clinical Validation ofthe Reproducibility ofSpectrumCEP 8 DNA reticulum cell sarcoma). M. Tharumvelu. M. E. Greene, F.G. Crusi. Probe Kit. K Theil. P.N. Rao2. MJ.Pettenati2. N. Heerema. S. Seelig P. Hsu4. L. yscwich. M. KleUe and P. M Chou. Departments ofObstetrics and 'Ohio State University, Columbus OH; 'Bowman Gray School of Medicine of Gynecology and Pathology, NorthwestenUniversity, Chicago, Illinois. Wake Forest University, Winston-Salem NC; 31ndiana University Medical Center, Aggressive tumors composed predominantly ofhistiocytes are uncommon in Indianapolis IN; 4VYSIS, Inc. Downers Grove IL children. Those onginating as primary bone tumors fall within a complex group of A multi-center study was conducted to evaluate site-to-site, day-to-day, lot- lesions, the reficulohistiocytosis, whose diagnosis is compounded by the ubiquity of to-lot, assay-to-assay, and observer-to-observer reproducibility of the Spectrum the mononuclear phagocyte system. Many tumors previously diagnosed as true hist- Orangecm CEP 8 DNA probe kit's ability to provide a rapid and reliable method iocytic lymphomasareactually B-cell, T-cell, Ki- l positive non-T, non-B cell. The for the detection and quantitation of chromosome 8 in interphase nuclei. The kit t(2;5X)p23;q35), observed in some Ki-l positive lymphomas have also been reported contained SpectrumOrangeTm fluorescent labelled DNA probe specific for in malignant histiocytosis. We describe one example in an 8month old infant with chromosome 8 centromere, reagents and instructions for the assay. Routine multiple lytic lesons in the left tibia, left ulna and right radius, which we believe are of cytogenetic slide preparations from lymphoblast suspensions and bone marrow histiocyte lineage and possible related to the interdigitating reticulum cell by immun- were prepared at +8 concentrations of 0%, 5%, 6% and 10% by the sponsor. ohistochemical panels and EM studies. Histologic, immunoldatochemicaL electron Each site was blinded to the status of the samples. Two technologists at each site microscopic and flow studies are often insufficient in delineating the full spectrum followed strict guidelines in the use of the kit and in signal enumeration. Assays of dendritic cell diferentiation. We studied the chmmosomal constitution ofthe were performed at each site during 8 days using 4 separate lots of the Spectrum cultured tumor cells. Metaphase cells were observed in cultures stimulated with CEP8 kits. 500 interphase nuclei and 20 metaphases were analyzed per specimen. GM-CSF, but not in the culture without GM-CSF. All metaphase cells observed There was no deviation from the prescribed assay, cell enumeration procedures, were cytogenetically abnormal. The karyotype was 46,XY,der(l)t(l;lXpl3;q21). or randomization schedules. A total of 180 assays were performed without failure. add(6)p2l),+g,-10[16J/NCA:47,XY,idem,+marf I]. GM-CSF appears to be an Intra-assay reproducibility was statistically demonstrated for all levels of + 8 effective mitogen in stimulating abnormal cells in some cases of histiocytosis. both among the 3 sites and between the 2 observers indicating high observer- The presence oftrisomy 8 is an interesting finding since this abnormality is to-observer reproducibility. There was no significant difference among the 4 probe observed in malignant tumors of myeloid origin. Further studies are required to lots (p-029), between the 2 observers at each investigation site (p=0.07) or determine the effectiveness of GM-CSF in culture to obtain metaphase among the 4 study days (p=0.93) demonstrating high inter-assay reproducibility. for chromosomal analysis of tumors. The chromosomal constitution The kit was found to be of high quality, easy to use, highly reproducible, cells histiocytic and capable of providing reliable quantitative information for trisomy 8 in the ofthese tumors may be useful in determining their lineage as well as subtyping. samples studied. Demonstration of equivalency between observers indicated that only 1 trained and experienced observer is needed for signal enumeration.

1763 1764 Linkage and clinical analysis in the identification of high risk family members Screening ior pi: educations in coloreccal cancer. h. P. M.P. Triantafillou, M.J.Macera, L.J.Smith, L.Newman, D.Earle, I. with Multiple Endocrine Neoplasia 1 (Meni). Thirukkumaran, 1, Sawicki, Grosman and R.S.Verma. Long Island College Hospital/SUNY 2 KLJ. Gratton! and C.j. johnsacL Department of Medical Genetics, University of Cal- gary, Alberta. Canada1 and Department of Surgery, Wadsworth VA Medical Centre, Health Science Center at Brooklyn, NY. UCLA, Los Angeles. CA.2. Mutation and allelic loss of the tumor supressor gene p53, dom- highly conserved exons 5 through 8, have been reported inj Multiple Endocrine Neoplasia Type I (.MENi) is characterized by an autosomal of such inant predisposition to develop various endocrine tumors (classically parathyroid, pan- variety of cancers. Although a large proportion family mutations are point mutations, larger deletions have been creatic, pituitary). The MEN1 gene has been localized to chromosome 11q13 by for these linkage and tumor loss of heterozygosity studies. The clinical features associated with observed. The most common techniques detecting A mutations are DNA sequencing and single strand conforma- MEN1 are often vague and appear unrelated until a history has been established. of PCR A third method, lack of familiarity with the syndrome has led to poor ascertainement and misdiagnosis tional polymorphism (SSCP) products. the the non-isotopic RNase cleavage assay (NIRCA, Ambion) at- in most families. The development of highly informative molecular markers spanning and to PCR con- region provides the opportunity to trace haplotypes through MENI families and taches opposable T7 SP6 promoters products MEN1 taining multiple exons. RNA transcripts are hybridized to assess carrier status for the mutant MEN-1 gene. Thirty-five families with clinical features are di- lines wild type transcripts and the resulting duplexes suggestive of MEN1 were clinically characterized and DNA samples, lymphoblastoid gested with RNase. Mismatch mutants are detected by aga- and tumors collected. Inclusion criteria included a positive family history of a wide range and illumu- histories rose gel electrophoresis, ethidium bromide UV of endocrine tumors or a family history of parathyroid adenomatosis. Clinical niation. A patient with adenocarcinoma of the colon that were obtained on the presumed affected individuals in each family and autopsy/pathology had metastasized to the liver and another patient with information was carefully confirmed. DNA analysis proceeded in those families with con- were resected both-and 4 colon cancer and retroperitoneal sarcoma firmed pathology and an inheritance pattern. The 11q13 region was haplotyped using DNA was extracted from all samples. No p53 mutations in polymorphic markers spanning a 10 cM region contalning the MEN1 gene. Interpretable in the colon and liver con- exons 5-9 were detected using NIRCA haplotype data was obtained on all but one presumed recombinant. The analysis mets. PCR of the DNA from the colon tumor in the other firmed the association of the classic MEN1 phenotype with the 11q13 region and provided no mutation was found. However, nc ade patient was present and carrier inclusion and exclusion in 25 families. Eight families segregating parathyroid PCR products for fragment l(exons 5&6) or fragment 2(exons nomatosis did not demonstrate linkage to the MENI region. The use ofMEN1 haplotype DNA from the sarcoma. A tumor 7,8&9) were observed in PCR pro- analysis provides accurate carrier information and focuses the clinical efforts of duct for exons 8 and 9 combined was present and exons 5,6 surveillance on those family members carrying the gene. The future cloning and charac- in both The two accurate and 7 were individually present samples. terization of the MEN1 gene will provide phenotype/genotype correlation but different 3' primers for exon 6 are separated by 55 bps haplotype analysis will focus cloning efforts and provide an efficient method of determin- while the two primers for 5' exon 7 are seperated by 25 bps. ing carrier status. It is apparent that in this patient, a 500-600 bps deletiot in intron 6, located between the primer sites, has occurre( in only the sarcoma, but ns. iD theg wis cmancne-r

1765 1766 and mplfscatle ia breast cancer: detectian fluorescent i sit. Bi-phenotypic leukemiacase showing both acute lymphold HER-l/2me acute myolold leukemia karyotypic changes. S. M. Zneimer. H. hybrlIlad nu. A.K. Yenasnandra' J. Yelaick3 JDrmd M. Schwalb'and ofPathology2, Edrissi. J. Nebbia. and L. Soon. Department of Genetics, Eeinaaitl, Center forHuman and Molecular Genetics', Dept. CytogeneticsLaboratory, KaiserPerrnanente Medical Center, 280 W UMDNJ,Newark,NJ,07103. 94611. The is amplified in 25% ofbreast cancers. MacArthur Blvd., Oakland, CA. oncogene HER-2/neu approximately a case of a 2 old who with The as a copy in normal cells andhas boee We present year girl presented HER-2/neu gene is present wengi and an excess of blast cells in her to e 17 in the reon 17ql 1.2-12. Three bret cancer cell bruisability, enlarged spleen mapped com elevated white blood count. Bone marrow (BM) was sent for lines (obtined from ATCC) and formbfin fixed, paffn -embedded tissue out leukemia and showed the of cytogenetic analysis on day 1 to rule sections (41am )from a femle breast tumor were examined for presence normal cells and 2 abnormal cell lines: 50,XX,+6,+8,+8,+21 and HER-2/neu (c-bB-2) We ni o by fluoresent in situ hybridizat 46,XX,del(3)(q21),add(6)(q12). The hyperdiploid cell line is most (FISH) uaing HER-2/eou cosmid( Oncor Inc. G ath eMD) We have acute leukemia observed commonly seen in childhood lymphoblastic (ALL); typical cion profile(h en disin of cels with >4 however, structural abnormalities involving chromosome 3q21 are siYaa~in onecell line and in al the tumor ells ofthe tissue sections. There was generally associated with acute nonlymphocytic leukemia, type M7 noc-ted variation in the hybridization sinl. The higb sensitivityand or megakaryocytic leukemia. Based on the patient's pathology iy ofthiste auiquend its use in a tissue- based system will allow the results, which showed thepresence of lymphoid blasts, she was put study of a range ofposs precursor lesions ofbreat cancerfbr evidence ofc- but after one week, did not improve in status. p ion on an ALL treatment, erbB-2 amplification ad itsinvolvement inthe d t and/or A second BM was sentfor cytogenetic analysis on day 7 and results ofthe breast cancr. showed only the 46,XX,del(3)(q21),add(6)(q12) cell line. We conclude that this patient's lymphoid cell line died off due to the ALL treatment, leaving the myeloid cells to proliferate, causing further disease. This case Is a good example of bi-phenotypic genetic expression, whose cytogenetic results affected both this patient's diagnosis and prognosis of disease. Published Abstracts: Clinical Genetics, Malformations and Dysmorphology A305 1767 1768 Pena-Shokeir Phenotype with Normal Intelligence. M.A Al- PROGRESSIVE FAMILIAL INTRAHEPATIC CHOLESTASIS Ghamdi.1 Robert PolomenoA David Chitavat.' Michael Azouz.3 Ahmad S. Teebi.2 The F. Clarke Fraser Clinical Genetics Mohammed M. Al-Ghanim, Hesham H. Kandil. Mohammed A. Abu-Taleb. Unit, Division of Medical Genetics,' Department of Ophthalmology,2 Department of Radiology,3 The Montreal Talast I. Farag. Children's Hospital and McGill University, Montreal, Department of Pediatrics, Al-Jabra Hospital and Medical Genetics Center. Quebec, Canada. Maternity Hospital, Kuwait. The majority of cases with Pena-Shokeir Syndrome or Two Bedouin siblings suffering from progressive intrahepatic cholestasis have Fetal Akinesia/Hypokinesia sequence are stillborn or die been ascertained and followed up in the Jahra Regional Liaison Community - of the complications of pulmonary hypoplasia in early Genetics life. The survivors often have severe delays. Programme. The proband is 7 years old boy presestwed with persistent jaundice since early infancy with episodic exacerbations every 1 - 2 months We report on a 6 year-old French-Canadian boy born with accompanied by severe prunitus, associated findings were steatoiThoea, clay clinical manifestations consistent with Pena-Shokeir coloured stools. stunted growth. rickets, clubbing and hepatosplenomegaly syndrome. These included characteristic dysmorphic facial Investigations revealed biphasic hyperbiliribinemia, normal aminotransferases, features, cleft palate, multiplex v - transferase and elevated alkaline TC 99 HIDA congenita, pulmonary hypoplasia and unusual glutamvl serum phosphatase ophthalmological findings including bilateral SCAN showed normal hepatocyte tracer extraction and delayed excretion ophthalmoplegia and optic disc hypoplasia. There was no Repeated liver biopsy in two years interval demonestrated paucity ofintrahepatic intrauterine growth retardation or decreased fetal bile ducts with slowly progressive portal inflammation Lipovitamins were movements. There was no history of oligohydramnios 'or supplemented Cholestyramine and phenobarbital were unsuccessful in polyhydramnios. Despite the poor expected prognosis the controling prutitus Now ursodeoxycholic acid and rifampin are used A patient presented with normal mental capability on follow- younger sister rims a similar but more agressive course Clinical up. The differences between Pena-Shokeir syndrome and and laboratory phenotype are discussed and the possibility that this findings are consistent with the autosomal recessive Byler disease. child represents a distinct entity is entertained.

1769 1770 The mlcrocephaly-mesobrachyphalangy-tracheoesophageal fistula (MMT) An acardiag twin with a microscopic heqrt: a case report. syndrome in a Chaldean male. D. J. Aughton. J. A. Berger. and S. M. LaCroix. P.1. Bader1 and David J. Huddleston2. 'Parkview Memorial Hospital, Ft. William Beaumont Hospital, Royal Oak Michigan. Wayne, In., 'Ball Menurial Hospital, Muncie, In. The MMT syndrome is an autosomal dominant disorder (McK 164280) We describe an acardiac female twin who was delivered by cesarean characterized by microcephaly, normal intelligence, tracheoesophageal fistula section after 33 weeks gestation. Karyotypic analysis of fibroblast (TEF), and digital anomalies, most typically fifth-finger . To date, cultures fraL several tissues including skin showed 46,XX. A single there have been 4 reports describing 23 affected individuals distributed among placenta with diamnionic-snnochorionic dividing membrane was noted. 5 families. We present the case of a 5-year-old Chaldean (Christian Iraqi) male Both cords had two vessels and were connected by one large and many with microcephaly, TEF, digital anomalies, and normal intelligence. smaller vessels. External features of this twin included a non-rec- J. A. was born at term to a 24-year-old woman following a pregnancy ognizable head with proboscis, fused eye slits, and a primordial mauth. complicated by gestational diabetes mellitus. His birth weight was 2.96 kg (-1 The trunk revealed an canhalocele and female external genitalia. SD); length, 48. cm (-1 SD); and head circumference (HC), 34 cm (slightly >-I Anus were absent although a single digit with a nail was on the right. SD). F with esophageal atresia was recognized and subsequently repaired. The legs were well-formed with only four toes on each foot. The other Multiple minor anomalieswere noted, including downslanting palpebral fissures, twin did not appear dysmorphic and had no ancaralies. Multiple internal small-normal mandible, mildly increased space between the first and second anomalies in the acardius were bilateral cystic hygrcnas, alctar holo- toes, and unusual thumbs, with decreased range of motion at the interphalangeal prosencephaly, hypoplasia of the lungs, esophageal atresia, intestinal atresia joint. Skeletal survey and renal sonography were normal. Cardiologic (type IIIa), asplenia, a single fused adrenal, and gross ab- evaluation showed only closing patent ductus arteriosus. Subsequent sence of the heart. The twin was categorized as acardius anceps (holo- ophthalmologic examination showed only minimal refractive error. On follow- cardius) until microscopic examination of the tissues revealed cardiac muscle and a rare up at age 5-5/12 years, J. A. had normal intelligence. His height, weight, and fibers Anitschokow cell. The microscopic presence of cardiac tissue proves that HG were 116.5 cm (p75), 21.2 kg (p75), and 48.5 cm (

1771 1772 A combined cytogenetic and molecular approach for diagnosing delayed deformity is a diagnostic clue In . G. puberty. J. K. Bayleran. D. B. Seifer. C. H. Mevers-Seifer and H. F. L. Mark. Brown University School of Medicine, Providence, RI and Eastern Maine Medical Pinelli. F.M. S6n6s. L. Franchi. A. Palmieri. A. Bertola. C. Bellini, E. Center, Orono, ME. Bonioli. Divisione Ortopedia I, Istituto G. Gaslini - Istituto di Clinica The patient, a phenotypic female aged 15-4/12 years, was referred because of Pediatrica and Istituto di Puericultura e Medicina Neonatale, State delayed puberty and short stature. A chromosomal analysis of 60 peripheral blood University, Genova, Italy. leukocytes revealed two subpopulations of cells. The modal cell line, consisting of 95% The minimal diagnostic criteria of Larsen syndrome (Ls) usually of the total cell population, was hypodiploid containing 45 chromosomes with a missing X chromosome. The other cell line, consisting of 5% of the total cell population, include dislocation of multiple major joints, especially and , contained 46 chromosomes, with one X chromosome and a G-sized chromosome that flat face with depressed nasal bridge and prominent forehead, and resembled a Y chromosome in morphology. Fluorescent in situ hybridization (FISH) cylindrical fingers with broad, spatulate thumbs; both autosomal studies on peripheral blood slides yielded results consistent with the above conventional recessive (MIM 245600) and dominant (MIM 150250) patterns of cytogenetic studies. Using an a-satellite, commercial probe of the Y chromosome, inheritance have been reported. We diagnosed Ls in a weak hybridization signals could be observed in a small percentage of cells. Therefore, female child, it was hypothesized that the G-sized marker, present in a low percentage of cells, might aged 20 days, with congenital bilateral dislocation of both knees and be of Y chromosome in origin. To provide unequivocal evidence of the Y-chromosome hips, a typical flattened (dish) face, spatulate thumbs and material, molecular analyses using the polymerase chain reaction (PCR) and various calcaneovalgus . The parents were not consanguineous. primers were carried out. Amplification of the DNA with six sets of primer pairs In the proband's mother, who had no joint identified an intact short arm and centromere of the Y chromosome and structurally dislocation, Ls was diagnosed altered long arms. The presence of-he Y chromosome material raised the possibility of only after the birth of the proband, on the basis of a typical dish facies neoplasic transformation in the gonads. In view of this risk, a laparoscopic bilateral and bilateral congenital talipes equinovarus. The foot deformity, while gonadectomy was performed. A cytogenetic analysis of the gonadal tissue revealed described in every patient in the original report of Larsen et al., is 85% of the cells containing 45,X and 15% of the cells containing 46,X,?del(Y)(ql2). actually listed among the additional, i.e. minor, features of Ls, like short The present report illustrates the usefulness of a combined approach to study and undermineralization and diagnose delayed puberty: clinical evaluations, conventional cytogenetics (GTG- stature, overtubulation of the diaphysis of banding), molecular cytogenetics (FISH) and molecular (DNA) analysis. This approach bones, carpal , precocious development of multiple carpal can provide a work-up of the patient that is more complete than that derived from any ossification points, thoracolumbar , vertebral segmentation single methodology. abnormalities and duplication of the ossification center of the calcaneous. In the present pedigree Ls could be traced back to the proband's mother on the basis of a typical facies and clubfoot, which may be regarded as a major diagnostic clue in this family in this family. A306 Published Abstracts: Clinical Genetics, Malformations and Dysmorphology (cont'd) 1773 1774 A Rossible new variant of nonsyndromic lissencephaly, associated Beemer Lethal Malformation Syndrome: A fourth case and clarification with myoclontc epilepsy of thne syndrome. Nanette S. N. Gilpin. Christie Clinic Association .3 and University of Illinois at Champaign-Urbana. S.Farah,_2IM.Rudwan.2 S.Khafagi.2 A.Khuraibet,1 B.Qasrawi.3 This paper describes a fourth patient with the Beemer Lethal heart W.Al-Buaijji 3 M.Hassann3 H.Hasseeb.3 S.A.EI-Gamal.3 It A.Sby Malformation Syndrome. He lacks hydrocephalus and congenital T.1. Farag. disease; however, he has the profound hypotonia and facial 1 ~~~~~~~~~2 characteristics that are consistent with this syndrome. He is still Neurology Department & Neuroradiology Department, Ibn Sina alive at 7 weeks of age. The purpose of this paper is to further hospital; Medical Rehabilitation Centre, Ministry of Social Affairs, clarify the syndrome. Inheritance pattern is discussed. Medical Genetics Centre. - Kuwait.

We report an Arab kindred with two sibs and a distant cousin showing the clinical, electrophysiological and radiological features of nonsyndromic lissencephaly. They presented with profound mental retardation, spastic paraparesis, myoclonic epilepsy and a brain imaging of lissencephaly. No underlying cause for the myoclonic epilepsy was identified. Although the presence of seizures represents a recognised feature of lissencephaly, the association with myoclonic epilepsy has not been described before. The clinical syndrome is suggestive of an autosomal recessive variant of the lissencephaly sequence.

1775 1776 Clinical follow up of an infant with mosalcism for trisomy 18q and Prenatal diagnosis of a unilateral pelvic multicystic dysplastic tetrasomy 18p. J. Habecker-Green, C. Kanaan, L. Bayer-Zwirello J. P. kidney in a fetus with a 47,XXY karyotype and congenital megacolon. O'Grad , R. Naeem, G. Cohn. Baystate Medical Center, Springfield, C. Kanaan, J. Habecker-Green, G. Cohn. Baystate Medical Center, Massacusetts. Springfield, Massachusetts. Clinical follow up to age 6 months of a patient with prenatally A 31-year-old female presented for genetic counseling at 20.5 weeks diagnosed anomalies of chromosome 18 is reported. The fetus appeared gestation following a positive maternal serum screen and amniocentesis on ultrasound to have multiple anomalies including clubbed feet, at another center. The fetal karyotype was shown to be 47,XXY. The abnormal hand positioning, edema of the scalp, cleft palate. and family history was significant for the patient's sister's four children polyhydramnios. The karyotype on amniocytes was 47,XY,+iso 18p. reported to have hydronephrosis. Prenatal ultrasound findings were Postnatally, the peripheral blood karyotype was 46.XY,+iso 18q, while significant for a unilateral, pelvic, multicystic dysplastic kidney the skin fibroblast karytoype was 47,XY,+iso 18p. Uniparental disomy that was first noted at our center at 31 weeks gestation. This is suspected. The infant had many features consistent with those diagnosis was confirmed at birth. During the neonatal period the previously described in cases of tetrasomy 18p, and some consistent infant was additionally noted to have a scalp defect that upon biopsy with trisomy 18q. He also showed some features found in association proved to be an ectodermal dysplastic lesion. At 5 months of age the with connective tissue disorders. baby was readmitted with a history of no spontaneous bowel movements The patient's physical exam, growth, and development are significant since birth. A rectal biopsy showed aganglionosis of the anal verge. for a birthweight of 2460 gm. (10th to 25th centile) with severe post- Upon review of the prenatal ultrasound tapes evidence of minimal bowel natal growth retardation. Feeding difficulties have been present from distension was noted. The rectosigmoid colon was obscured prenatally birth. The patient is alert and laughs and smiles appropriately. by the cystic dysplastic pelvic kidney. There is a question of developmental regression. The thin, hypertonic Anomalies associated with a 47,XXY karyotype have been previously infant has microcephaly; MRI studies of the brain showed hypogenesis reported, including one case of unilateral renal agenesis. To our of the corpus collosum and slightly dilated ventricles. Anomalies of knowledge, this is the first case report of an individual with this the GI system were noted. Studies of the heart and kidneys were unre- karyotype and congenital megacolon or ectodermal dysplastic lesions. markable. The patient's sclerae are blue, and he has translucent skin. One previous study has suggested that there is increased risk for A skeletal survey noted several congenital skeletal anomalies and a genitourinary malformations in individuals with a 47,XXX karyotype. resolving fracture. A skin biopsy for collagen studies is pending. Together with previous reports, the findings in this case could provide the basis for a re-evaluation of the counseling dogma with respect to the incidence of additional anomalies associated with a 47,XXY karyotype.

1777 1778 Computed tomography (CT) bone density measurements A possible new Cranio Facio Fronto Digital dysplasia? in a family with osteogenesis imperfecta (OI)- type 1. B.M. Qasrawi. S.A. Elaamal.T.I. Faraa S.A. AI-Awadi M.E. Miller and T. N. Haneartner. State University School of Medical Rehabilitation Center, Ministry of Wright Aluendil M.A. Sabry2. 2 Medicine, Dayton, Ohio. Social Affairs, Medical Genetics Center Kuwait. We have studied CT bone density of the radius in 2 children and 4 We report an institutionalized 25 year old Kuwaiti male with adults with 01-type 1 from a large family with classical features of proportionate short stature, , dysmorphic features, O-type 1. The affected adults in this family noted a decreasing hand and feet abnormalities, acanthosis nigricans, moderate mental fracture frequency as they got older. CT bone density measurement retardation and generalized tonic-clonic epilepsy. He shows broad is the most accurate and precise of any method for bone density and forehead, frontal bossing, hyperplasia of the supra-orbital ridges, can determine both the trabecular bone density (TBD) and the , depressed nasal bridge, broad nose, short philtrum, cortical bone density (CBD). The results are shown below: flat cheek, macrognathia, prognathia, and teeth malocclusion. He Subject Age(yr) TBD (s.d.)* CBD (s.d.)* shows different abnormalities of the hand and feet including 1. 6.3 -1.5 -4.0 camptodactyly of the 3rd, 4th and 5th fingers in both hands, 2. 7.5 -3.0 -2.5 abnormal palmar dermatoglyphics, , fleshy +2.0 , and hypoplasiarof the terminal phalanges of both hands 3. 14.8 -3.0 and feet. 4. 26.5 -2.5 +3.0 X-Ray spine showed thick skull bones and increased height of 5. 34.4 -2.5 +2.0 the lumbar vertebrae. CT skull showed normal ventricular system. 6. 39.4 -3.0 +2.0 Karyotyping peripheral blood with banding techniques showed *(s. d.= # of standard deviations from mean of 800 normal adults) normal 46, XY constitution. His non-consanguineous healthy The results indicate that both children and adults in this family parents have no similar conditions. have abnormal bone density, and that there is an evolution with This patient seems to have a new Cranio Facio Fronto Digital age from a bone of low TBD and low CBD in childhood to a bone of Syndrome. With the absence of similar conditions in his family it low TBD and high CBD in adulthood. This change reflects an increase seems to be due to a de nova autosomal dominant mutation. in bone strength with age, and may explain, in part, why affected individuals in this family experience less fractures as they get older. Published Abstracts: Clinical Genetics, Malformations and Dysmorphology (cont'd) A307 1779 1780 X-Chromosomal abnormality in Crohn disease. Q.H. Qazi, K. A girl with branchio-oto-renal syndrome (BOR) and severe mental H. Ramesh and R.S. Verma. Long Island College Hospital- SUNY Health Science Center at Brooklyn, NY 11201. retardation. F. J. Ramosl2, M. J. L6mezl, 1. Buenol,2, J. L. Olivaresl, J. L6pez- Crohn disease is an autosomal recessive disord&r and M. Buenol. Genetics, of Pediatrics, of the of the familiar pattern does not reflect a simple mendelian EPisg3, 2Dept. Hospital University inheritance. Its etiology is not known at present. The Zaragoza Medical School; 3Dept. Neurology, Hosp. "Miguel Servet", Zaragoza, Spain. relationship between chromosomal abnormalities and Crohn Branchio-oto-renal syndrome (BOR) is a high penetrant autosomal dominant disease has not been established. The patient is a 16 disorder with variable clinical expression of branchial anomalies, ear defects, hearing year old afro-American male, born to normal parents and and renal having several normal siblings whose ages range from 11-31 impairment, maldevelopment. To date, virtually all the patients with BOR years. Physical examination revealed unslanted eyesa ears have been considered to have normal intelligence, with only a few cases reported as 7 cm in length with a long philtrum, a highly arched having borderline or mild psychomotor delay attributed to their deafness. We present a palate and maloccluded teeth. There was no facial, 21/2-year-old girl with BOR and severe mental retardation. auxiliary or pubic hair. The penis was small (1.5 cm) The proband, the second child of non consanguineous parents, was the product with hypoplastic scrotum and no palpable gonads either of a 36-week uneventful pregnancy. Apgar score 9/10. Birth weight 3,100 g (75%), in the scrotum or in the inguinal canal. The CT scan also length 49 cm (80%) and head circumference 34 cm (80%). She had bilateral showed the absence of right kidney and enlargement of the preauricular pits and bilateral cervical fistulae. Renal ultrasound showed a right kidney left kidney (compensatory hypertrophy). Testes were not 4 mm smaller than the left. Subsequent IVP and renal gammagraphy (R 43%, L 57%) visualized. Thickened mucosal folds with nodules in the were consistent with the presence of a mildly hypoplastic right kidney. Renal function jejunum suggester pseudopolyp formation. Histological was not At was examination of biopsied material showed chronic impaired. age one, she severely delayed: she did not sit and had no inflammatory changes and noncaseating granuloma. GTG speech. Hearing was reportedly normal. Head MRI, BAER and high resolution were banded metaphases revealed a normal 46,XY karyotype. chromosomes normal. Family history showed a maternal great-great mother with However, FISH-technique revealed that the euchromatic bilateral preauricular pits; the proband's mother had no external features of BOR, material of Y-chromosome (pll.l-pll.2) has been directly although no further studies could be done on her. At age 21/2, the proband does not inserted into the long arm of the X(q21.2). The walk and her speech is limited to a few bisyllables. cytogenetic finding was modified to 46,XY,dir ins (X;Y) The association of BOR and severe mental retardation has not been previously (q21.2;pll.lpll.2). It is assumed that the function of reported. One can speculate either that this is just an sporadic coincidence or that this SRY-gene has be-n disrupted due to the insertion, patient represents a new example of a nonrandom association, i.e. a contiguous gene causing loss of testicular development. syndrome. Molecular analysis of the recently discovered BOR gene locus at 8q13 in this patient will be helpful. If the association is confirmed, it may have implications in the genetic counselling of BOR families.

1781 1782 VACTERL with hydrocephalus and Hemifacial Microsomia. R. M. Ruzicka, P. A possible new syndrmsic situs inversus totalis in an institutiona- J. Baygot. V. A. Pallante. J. W. Seeds. and T. C. Markello. Medical College of lised patient. Virginia, Richmond, Virginia. Intro. by: Arti Pandya M.A.Sabry, M.A.Mubashir, N.Hasseeb, S.A.Al-Awadi. S.A.Fara,3 Hemifacial microsomia (HFM) has been reported in 33 cases ofVACTERL associa- B.Qasrawi, 2 R.M.AI-Dabbous, 1 W.Al-Busairi, 21 T. I .Farag. tion, but rarely in those cases that include anal atresia. HFM has not been reported I in children with "VACTERL with hydrocephalus" syndrome, a distinct X-linked or Kuwait Medical Genetics Centre, Medical Rehabilitation Centre, of Social autosomal recessive syndrome which often occurs in consanguineous We Ministry Affairs,2 Neurology Department, Ibn Sina matings. hospital 3 - Kuwait. report a child with the complete VACTERL association, hydrocephalus, and HFM who is the product of a nonconsanguineous mating. 'Ihere are no other known affect- ed family Dextrocardia/situs inversus has been associated with many mono- members. Prenatal ultrasonography revealed: ventriculomegaly (lateral and genic syndromes e.g. BOR (MIM 113650), Holt-Oram (MIM 142900), third), hypoplastic cerebellar hemispheres, microtia, micrognathia, abnormal posturing SLE (MIM 152700), Marfan (MIM 154700), Poland (MIM 173800) of fetal hands, clubbed feet, and polyhydramnios. Fetal echocardiogram demonstrated Springel (MIM 184400), VATER/VACTERL (MIM 192350), agnathia/ transposition of great vessels and ventriculo-septal defect. The fetal karyotype was holoprosencephaly/situs inversus (MIM202650), Ivemark (MlM208530), 46,XY. Hemifacial microsomia (L>R), frontal bossing, right microtia, left Fraser (MIM 219000), dextrocardia/abnormal facies/microphthalmia (MIM preauricular ear tags, short neck, tracheoesophageal fistula, asymmetric nipples (right 221950), Ellis Van Crevald (MIM 225500), Kartagener (MlIM 244400), Bardet Biedle (MIM 209900), Laurence-Moon (MIM 245800), superior to left), absent right first digit, digitalized left thumb, cryptorchidism, and Microphthalmia/mental deficiency (MIM 251500), a galactocialidosis anal atresia were noted at birth. Radiological findings included multiple thoracic variant (MIM 256540), TAR (MIM 274000), as well as at least one hemivertebrae, fused cervical vertebrae, 8 missing , a bifid rib, and right radial X-linked syndrome (MIM 306955). aplasia. Renal ultrasound revealed a left cortical cyst. The infant was initially stabilized by gastrostomy and colostomy. MRJ, EEG, and neurologic examinations Herein, we report on an instutionalised mentally retarded 19-year- old female with situs inversus revealed major CNS deficits, and further aggressive support was declined by the totalis, associated with proportionate short stature, facial dysmorphism, contracture of ankle joints, parents. Smith's Recognizable Pattern of Human Malformations states that infants bilateral talipus equinovarus, brachydactyly of fingers and toes. with VACTERL "merit vigorous attempts toward rehabilitation...". This infant and brisk reflexes. Head CT scan showed a deformed fourth ven- represents a salient exception to the conventional counseling that patients with tricle and ambient cisterns with effacement of sulci. Thyroid VACTERL association will have a good neurologic prognosis. function tests, lysosomal enzymes, amino acid chromatography and karyotype, were all normal. The profile described above has not been reported before.

1783 1784 Heterogeneity of Trichothiodystrophy-Neurocutaneous syndrome. Hyoerekplexia: Familial startle disease, a diaanostic puzzle. R. Sasi,1 B. Rosenfeld.2 Patrice Eydoux.3 Ahmad S. Teebi.1 H A Tbro. San Juan City Hospital, Rio Piedras Medical Center, The F. Clarke Fraser Clinical Genetics Unit,1 Division of San Juan, Puerto Rico. Medical Genetics, Department of Dermatology,2 Cytogenetics Diseases that mimic other entities are a problem in Medicine. Laboratory,3 The Montreal Children's Hospital and McGill Such a disease is hyoerekplexia. Early diagnosis will facilitate University, Montreal, Quebec, Canada. planning strategies of intervention and genetic counseling. Trichiothiodystrophy (TTD), sulfur deficient hair, is a A 5 yrs old male was referred for evaluation. Physical examina- clinical marker for several autosomal recessive tion and careful review of his perinatal history gave us a neurocutaneous syndromes. This rare congenital hair retrospective diagnosis. He was born rigid, hypertonic and dysplasia is seen in Tay syndrome (IBIDS). In addition to hymoactive. Developed multiple omplications as a neonate, becninng trichiothiodystrophy and nonbulous ichthyosiform a diagnostic puzzle to Neonatoloqy, Neurology, Radiology and erythroderma, it may be also associated with peculiar facies, Orthsoedics. Was described as having "atypical seizures" for he microcephaly, cataract, poor motor coordination, short would maintain a fixed position of the upoer and lower extremities stature, impaired intelligence, decreased fertility and with a sodden increase in muscle tone. During the episodes, easily increased susceptibility to infections. When elicited by a snap or touch, a staccato cry was audfible and he was photosensitivity is also present the condition is called also considered dvsmorphic. His face was broad with saqgy cheeks, (PIBIDS), found to have mutations present similar to that a small south and small lips. A umbilical hernia had been corrected known in xeroderma pigmentosum complementation groups. at age 4 months. Here we report on a 2½ year old child with relatively mild He received Phenobarbital until at 3 yrs the parents discontinued manifestations to demonstrate the heterogeneity of TTD. the the medication on their own accord because they thought the infant's patient was the first child born to his normal, hypoactivity was due to it. He then continued doing well, without nonconsanguineous Tamil parents. He showed the hair the"seizures". At 5 yrs his soasticity was still present and at manifestations characteristic of TTD. In addition to that he school the teacher crplained that if other kids touched him, he has congenital nonbulous icthyosiform erythroderma (improved) would extend the ants and "hit back". Upon tapping his glabella he and hyperactivity. There was no microcephaly, cataracts or would jerk his head backwards and sametimes fall. when sleeping recurrent infections and no apparent photosensitivity, he would exhibit myoclonic jerks. pigmentation or freckling in the regions exposed to sunlight. All studies done including Psychometric tests, Cr scan, 12 hour High resolution chromosomal analysis was normal. There was EEI's, metabolic screening were within normal limits. no increased gaps or breaks and sister chromatid exchanges A family history disclosed a maternal male cousin who had the same were normal. This case suggests a possible mild mutation or hypertonicity and who is notorious in his mall town for he is locus of heterogeneity of TTD neurocutaneous syndrome. described as "jurpinq all the time". A308 Published Abstracts: Clinical Genetics, Malformations and Dysmorphology (cont'd) 1785 1786 umphajoce.e and Radial Aplasia: Report of Two Patients Mlltiple Bilateral Meningoceles in a Boy with Hypotonia. Ligmentous F.R.Vargas; D.G.Ilorovitz; J.C.Llerena Jr.; A.Cunha; J.C. Laxity, Ptosis, , and Mental Retardation.R.Wallerstein, C.I. Cabral de Almeida. CGM-IFF-FIOCRUZ; RSVP; HUGO, Rio de Scott Jr., L.Nicholson. A.I. Dupont Institut.,Wilmington, Delaware. Janeiro, Brazil. We report a 9 year old boy who presented with congenital hypotonia, We report on two sporadic male patients with the associa- kyphosis, ptosis, and mental retardation. An MRI done to investigate tion of omphalocele and radial defects. Case 1: omphaloce widened interpedicular vertebral spaces revealed dramatic multiple le, left radial and thumb agenesis, mild thenar hypoplasia symmetric bilateral meningoceles in the thoracic and upper lumbar spins. on the right hand, single umbilical artery, bilateral ingui His other features include prominent occiput, ridged metopic ribs, left renal agenesis. Normal suture, flat supraorbital ridges, malar hypoplasia, and thin nasal nal hernia, 13 pairs of narrow with dental ane bridge. He has bilateral ptosis, a high palate chromosomes on GTG banding and DEB-protocol for Fanconi crowding. His ears have decreased cartilage resistance. He has dorsal mia negative. Case 2: omphalocele, agenesis of the right ra kyphosis and . His ligamentous laxity is exaggerated dius and thumb, proximal cutaneous sindactyly 2-3 on the bilaterally with cubitus recurvatum, , subluxable pat- left hand, scoliosis with fused lumbar vertebral bodies. He ellae and acromio-clavicular joints, pes planus, and dramatically hours due to pulmonary hypoplasia. Cultu- hypermobile fingers and wrists. He has had normal chromosomes, plasma died in the first ceruloplasmin. showed 46,XY karyotype. Our 2 patients re- amino acids, collagen I and III, copper, and red fibroblasts We believe that this constellation of findings represents a semble the 5 cases reported by Robinson et al (Proc Greenwo unique syndrome as his findings do not fit any previously described od Genetic Center 6:130,1987) and Hall (Amer J Hun Genet syndrome associated with multiple meningoceles. He presents a challenge Suppl:A1215,1990) as OURA association (Omphalocele and Uni- in management as surgery is proposed for the management of his lateral Radial Aplasia). Our case 2 is the only one with a kyphosis, but the risks of surgical repair in the setting of multiple right-sided radial defect. Both cases had mild contralateral meningoceles is unknown. hand anomalies and case 1 had also ipsilateral renal agene- sis, not previously described. The sporadic male patient re ported by Gershoni-Baruch et al (J Med Genet 27:403-404 ... 1990) may also be another example. Our 2 cases may contribu te to the phenotypic delineation of this unusual associatiom All cases roported have been males and sporadics. An X-link ed or autosomal recessive mutation however canot be excluded.

Published Abstracts: Cytogenetics 1787 1788 Rvviw and Hypotheses :-i Structure and Distribution of Are all phenotypically normal Turner syndrome fetuses Telomeric Sequences : A Unified Model of Chromatin mosaics? A. Amiel. D. Kidron. I. Kedar. R. Tepuer. Function. S. S. A. Adekunle, c/o. Dept. of Biology, L. Gaber and M. Feicin. The Genetics Institute, Neir Boston University, Boston, MA. General Hospital, Sapir Medical Center, Kfar-Saba, The telomeric sequence is highly conserved through Israel. evolution and is functionally inter-changeable among Fluorescent in situ hybridization technique was many organisms. It is capable of forming non-Watson- applied to tissue samples obtained from various organs Crick (Hoogsten) chemical bonds and alternate Z-DNA (placenta, lung, kidney, muscle, brain and fetal blood) conformation. Telomeres and telomere related sequences taken from: three "normal appearing" fetuses diagnosed were once thought to reside only at chromosome ends as Turner syndrome (45,XO) by amniocentesis, but but have now been shown to be dispersed at interstitial without external or internal malformations: one 45,XO sites in the human genome and the genome of other fetus with characteristic malformations: a large cystic species. Also telomeric sequences were once thought hygroma, subcutaneous edema, congenital heart defect to be responsible for only the replication of chromosome and others: one known mosaic 33% 45,XO/46/XX; one ends but are now known to be involved in a wide range 46,XX normal fetus which was aborted because of of diverse chromosome activities including DNA annionitis. All 3 phenotypically normal Turner syndrome amplification, sister chromatid exchange, fragile sites, fetuses, as well as the mosaic fetus, were found to be targeted chromosome breakage, DNA transposition and mosaic 45,XO/46,XX in-the various tissues. All tissue integration of viral and other exogenous DNA into the samples from the malformed Turner fetus were 45,XO. genome e.t.c. Here, I present a review of all the Conclusion: A fetus diagnosed as Turner syndrome but chromosome activities where the presence of the telomeric lacks the characteristic malformations, is likely to be sequence has been demonstrated and propose hypotheses mosaic 45,XO/46,XX. on how the interactions of the sequence with physiological and pharmacological substrates in the body can elicit these activities.

1789 1790 Unique interstitial deletion of chromosome 7q. M. Artiga Mecurreece of trisomy 21 Down Syndrom. ez Su-h0OTMQ11gr. Tj Diamndl~. Y Ess*rim-ahl .R.Bz*L. D.G.Mayin. R. JGarcia-awta. - L.NalfnL. F.Eocha. C.Hernhndes. Instituto 'Wie des be a female infant with pre- and bostnataj Mexico growth retardation, single umbilical artery facial Nacional de Perinatologia. city. toe abnormalities, hypotonia and deveiopmental Free trisomy 21 in two or more siblings of dysmorphy, interstitial d2 lay Cytogenetic anal sis showed an fenotypically normal young parents has two deletion of 7q, pt-r>q3j::q33>qter mosaicism or Fluorescence in situ 46,XXiy ,j.(7)ridlzat on e*ploying whQli explanations: a low percentage the derivative We 14 chromosome painting confirmed that predisposition to nondiajuntion. studied chromosome was comprised entirely of chromosome 7 with one or more children with trisoWy of the couples material. DNA anajivis allowked furt er defrinition 21 in a of one year, with banded chrom- molecular breakpoints and confirme the unique nature of period and the deletion. Parental karyotypes confirmed th de novo some preparations from cultured lmphocytes nature of the chromosome rearrangement. Literature skin fibroblasts and one case from ovarian reyiew allowed comparison of the clincal findings of stroma cells using standard techniques. we this case with other cases of 7q de leton and one case with showing cytoqenetic overlap. analised 100 metaphases from each tissue The most frequent clinical manifestations in deletion GTG banding, satellite association and chromoso- 7a? include pro and postnatal growth retardation, mal hateromorphisms. m crocephaly ai abnordmalears. These features were also a woman with a in seen in our Single umbilical artery has not We found sos 46,11/47,11+21 patient. ovarian cells and a man -of a different couple- inithe can be correlated in blood cells. Two PThrev11dkfes presenjthe c Amtitntsim larity to other with a mos 46,XY/47,-IY+21 with 7q31-33. However, iicat 21 in a cell in "deletion 7a" patients and the monspecific rature of most cases with trisomy single of the find ngs do not support a distinctive phenotype blood; and a man with 78% satellite association e to cl alone. TB5 which is ameniab Nical Oiagnosis the between acrocentric chromosomes. We concluded nature the cytogemetic abnormality in unique or future the mosaicim and present patient makes it impossible to say whether that beyond parental predispo- cases with a simnlar deletion will have identical sition to nondisjuntion, other numerical and clinical manifestations. structural anomalies may interfere the process of meiotic disjuntion. Published Abstracts: Cytoigenetics (continued) A309 1791 1792 The detection of a cryptic chromosomal rearrangement Partial trisomy 2q as the result of a de novo unbalanced involving t(6;16)(q27;q23). S.D. Batish and R.S. Verma. translocation. B. Braddock, R. Gasoarini, M. Claydon, W.A. Division of Genetics, Long Island College Hospital-SUNY Miller. Prenatal Diagnostic Center, Lexington MA. Health Science Center at Brooklyn, NY 11201. 2q3 trisomy has been previously reported in the literature Genetic abnormalities play an important role in as being associated with a distinct abnormal phenotype. To spontaneous abortions. We were referred a couple for date, all reported cases of 2q trisomy have been the result cytogenetic evaluation because they had experienced fetal of abnormal chromosome segregation from a parental loss. The mother was cytogenetically normal while in the rearrangement. We report a case of de novo 2q3 trisomy father, one of the chromosome 16's revealed an apparent ascertained following amniocentesis. The patient was deletion of the last band of the q arm. The deleted referred to our center at 17 weeks gestation by LMP for segment could not be detected elsewhere in the genoma- genetic counseling, targeted ultrasound, and possible using GTG banding. Therefore, we applied whole chromosome amniocentesis because a previous ultrasound examination 16 painting [WCPt probe and the missing part of chromosome revealed oligohydramnios. Targeted ultrasound revealed a 16 was found to be translocated to the distal end of the fetus small for gestational age, mild oligohydramnios, a long arm of one chromosome 6. Nevertheless, when WCP probable ventricular septal defect and a grade II echogenic probe for chromosome 3 was used, no hybridization signals bowel. Based on these findings, an amniocentisis was were detected on chromosome 16, suggesting a very minute performed and revealed a 46,XX,16p+ karyotype. Based on its DNA segment was involved in the balanced translocation. G-banding pattern, the additional material on 16p was Furthermore, a telomeric probe specific for chromosome suggestive of 2q31-qter. Parental chromosome analyses were 16 also lit up the 6q suggesting: 46,XY,t(6;16)(q27;q23). normal. After follow-up genetic counseling the couple Of course, sufficient copies of DNA must be present to elected to terminate the pregnancy. FISH studies utilizing identify the chromosome abnorsailittesh-by'.ISH.ete-chntqfue. a library probe for chromosome 2 (ONCOR) confirmed the Presently, at least 10 Kb of genomic sequences are additional material on 16p was from chromosome 2. Partial required for FISH analysis. Nevertheless, aberrent trisomy for 2q has been reported as arising from familial chromosome 6 could not be identified by GTG-techniques. rearrangements involving various chromosomes. Non-paternity These findings are highly significant for couples who are is denied and paternity testing is currently being pursued still in reproductive ages and have very minute cryptic for confirmation. To our knowledge, this may represent the rearrangements. first reported case of de novo 2q3 trisomy.

1793 1794 Novel Unbalanced Translocatlon: 46,XX,der(11)t( 1:12q24- p11.2). Abnormal segegation of two parental reciprocal trasslocations resulting in M. Decker-Phillios. A. McConkie-Rosell, M.B. Qumsiveh. A.K. lafolla. partial trisomy 16q23-.qter. S.H.Gagos. M.R.Delgado2. J.Mills2. L.G.Shaffer' Duke University Medical Center, Durham, NC. and S.K.Shapiral 'Baylor College of Medicine, Houston, TX 'Texas Scottish Rite We report an infant with a novel unbalanced translocation of 1 1q and 12p. Hospital for Children, Dallas, TX Features included: intrauterine growth retardation, oligohydramnios, Unbalanced segregation of parental reciprocal translocations can lead to viable prematurity, hypotonia, , bushy eyebrows, hypertelorism, offspring with compound phenotypes representing a mixture of the characteristics depressed nasal bridge, broad nasal root, low set, small, posteriorly rotated of both the monosomic and the trisomic chromosomal regions. We present two ears, cleft soft and hard palate, broad flat mouth with downturned corners, children with partial trisomy 16q23-qter and diverse phenotypes. The first patient and long smooth philtrum. Additional features included: , was referred for cytogenetic evaluation to exclude Prader-Willi syndrome. He is an accessory nipple, imperforate anus and sacral spinal defect, bilateral single obese mentally retarded 11 year old boy showing an unbalanced 46,XY, -13, palmar creases, high plantar arches, and bilateral talus equinovalgus. +der(13) t(13; 16)(q34;q23)pat karyotype. This karyotype is consistent with partial Radiologic evaluation revealed a frontal lobe cyst, posterolateral trisomy 16q and a small monosomy for 13q34-sqter. The second patient is a 6 diaphragmatic hernia and intestinal malrotation. Additional clinical features month old female infant who was evaluated at birth for possible trisomy 18. She has included: severe feeding intolerance, gastrointestinal reflux, failure to thrive, multiple structural heart defects and a 46,XX, -11, +der(l 1) seizures, hypothyroidism, partial diabetes insipidus, thrombocytopenia, t(I 1; I6)(q22.3;q23.2)mat karyotype resulting in partial trisomy 16q and partial profound developmental delay and bilateral hearing loss. monosomy l1q22.3-qter. This second patient had dysmorphic features including Family history revealed multiple fetal losses by the maternal grandmother, wide saggital suture, short palpebral fissures, low set and dysplastic ears, small and first cousin with multiple congenital anomalies including atrioventricular mouth, and telecanthus. Additionally, she had complex congenital heart defects canal defect, hypoplastic aortic arch, oomphalocele, pulmonary hypoplasia, involving dysplasia of the septum, endocardial cushion, aortic and mitral valves, widely spaced nipples, right duplicated ureter with ureteroplevic junction and branch pulmonary arteries. The first patient confirms earlier reports that partial obstruction and hydronephrosis. Cytogenetic evaluation of this family showed trisomy 16q23-qter can result in a mild phenotype of characteristic facial the proband to have a chromosomal complement of 46, XX, der(11) t(11;12) dysmorphisms, mental retardation and hypogonadism, since he had only a minor (q24;pl 1.2) mat, and both the mother and maternal grandmother to have the monosomy in addition to his partial 16q trisomy. In contrast, the second patient's chromosomal complement 46, XX, t(11;12) (q24;p11.2)., confirmed by multiple heart defects and more severe presentation are most likely associated with fluoresence in situ hybridization. the partial monosomy for distal 1 q.

1795 1796 Heteromorphisms of centromeric alphoid DNA sequences of Using FISH to further describe two patients with the chromosome 21. S.K.Gogineni, .D. Batish, S. Kleyman and R.S. Verma. Division of Genetics, The Long Island College cytogenetic aberrations dup(17)(pl1.2->pl2) and Hospital- SUNY Health Science Center at Brooklyn,N.Y.11201 del(4)(p16.3). RIR- Hgns M. A- MoreA A. Fertitta S Rh Heteromorphisms of the short arms of human acrocentric IMcCord*. M F Pirot* L- Buratens Brekken. 1I Ahmad- chromosomes are well recognized and have been classified into Abbott Northwestern Hospital, AUnity Medical Center, "University of size, intensity and color polymorphisms. Recently, Minnesota, MN; Children's the monomeric repeat units of 171 bp of alphoid sequences Minneapolis, AAEmeritus, Mercy Hospital, result in polymorphic centromeres which in some cases the Kansas City, MO; *Children's Health Care, St. Paul, MN. number of targeted sequences may be far below the We describe two patients where FISH was used to confirm and detection level. In the present investigationwe used further elucidate their abnormalities. Patient one is a 9 15 normal and 9 cases with 21 to cytogenetic trisomy investigate the month old male referred for to centromeric alphoid sequence heteromorphism of chromosome chromosomal analysis due apnea 21. FISH-technique was performed as per standard protocol episodes, and later noted to have hypotonia, motor delay and and a commercially available biotin labeled alpha seizures. Traditional cytogenetics diagnosed a de novo duplication of satelliteDNA probe specific for loci D21ZL and D13Z1 of the negative G-band 17pl1.2. FISH defined the duplication to chromosome 21 and 13 was used (Oncor, Gaithersturg, MD). Size of the pericentromeric region has been arbitrarily include two copies of the probe D17S29*, a probe which may be classified into five classes by comparison with the length deleted in patients diagnosed with the Smith-Magenis syndrome. of the short acm (p) of chromosome 18 as suggested earlier The patient's phenotype is compared to the phenotype of full blown (Babu et al., Chromosoma 1987;95:163-166). They are: Smith-Magenis syndrome. Patient two is a 15 year old moderately negative (1), small (2), medium (3), large (4) and very large (5) with incidence of 3.5%, 24.6%, 56.1%, 15.8% and retarded female having static encephalopathy with a history of 0.0% respectively. Obviously,3.5% of the chromosomes seizures and global neurologic impairment involving both motor and lacked suff'cient copies of alphoid sequences. If cognitive abilities. Traditional cytogenetics diagnosed a deletion of interphase cytogenetics by FISH technique is performed, the G-band FISH defined the deletion to be these individuals will lack and negative 4p16.3. distal signals escape the the a which be deleted in detection of onempioidy that can have dire consequences probe D4S96*, probe may patients during prenatal diagnosis. These incidences should assist diagnosed with the Wolf-Hirschhorn syndrome. FISH further cytogeneticists to predict the sensitivity of the defined the deletion by showing it includes the telomere probe procedure. D4F26*. The patient's phenotype is compared to the phenotype of full blown Wolf-Hirschhorn syndrome. [*ONCOR.l A310 Published Abstracts: Cytogenetics (continued) 1797 1798 Cytogenstic and fluorescence in situ hybridizatIon studies In the Cystic hygroma sad massive hydrops i a 45,X fetes with obstructive uropathy. cherctrican of a narker chromosoe. C Kant. Johnson. J-P E.S.Ki>.AigdwcvlMensse-Par.RMaznand A.L.SalskA. Departments Temple 0 on University of Wisconsin-Madison, Waisman Center, ofPediatrics and Pathology, Albert Einstein College ofModicine,Montefiore Medical Madison, Wisconsin. Center,Bronx,N.Y. We report an an eight year old female with developmental delays, It has been estimated that fewer than 1% ofthe number of45,X conceptuses present particularly in speech, socialization, hypotonia, and a 46, XX/47, XX, + mar at week five ofgestation survive pregnancy. It is not clear why 45,X embryos and karyotype. The marker chromosome was investigated with Ag-NOR, fetuses have such a high mortality. Though the Turner phenotype is associated with DANDAPI techniques. Human centromere-specific alphoid probe, renal anomalies in over 60%/. and with cardiovacular defects in about 20%/o of acrocentric beta satellite-specific probe, chromosome 15 PW region cases,the relationship between specific anomalies and mortality is not clear. Many A(D15S1 1), region B (GABRB3) and SIO cosmid probes were also used to believe that lymphatic hypoplasia and lymphedema contribute to decreased viability. characterize the marker by FISH technique. The cosmid probes showed We have recently examined a 25 week stillborn with evidence ofmassive edema due two sites at opposite positions on the marker chromosome. The beta to both lymphatic malformations and obstructive uropathy. satellite probe showed short arms on both sides of the marker Case report: A 990 gm phenotype female fetus was born by assisted breech delivery chromosome and alphoid satellite probe showed the dicentric nature of the to a 20 year old primip.The post-mortem exam revealed a massively edematous fetus. marker. The extent of the duplicated region and mode of formation of the A large pale placenta contained immature tissue with villous edem and a single marker will be presented. umbilical artery. A large cystic hygroma and an exreme degree ofswelling ofall the subcutaneous tissues was noted. The swelling was so severe as to result in spontaneous disruption ofthe skin ofthe neck expoing the underlying structures. The lungs were hypoplastic and there were no anatomic defects ofthe heart. Visceral exam revealed an omphalocele,2 accessory spleens, thymic and adrenal hypoplsiabilateral hydronephrosis and dilated pelvices and proximal ureters. The distal ureters were stenotic and the uterus,vagina and ovaries were present. A fibroblast culture revealed an abnormal female karyotype in each of20 cells analyzed. We conclude -at klcal death in our case was due to a unique combination oflymphatic malformations and obstructive uropathy which resulted in a dramatic degree ofhydrops. There is a great need for fMrther examination ofthe 45,X phenotype in embryos and fetuses in order to better understand the lack ofprenatal viability.

1799 1800 Molecular characterization of a de novo t(Y;9)(qll.2;q22) Tandm duplication of beta satellite, satellite III and by FISH technique. V. Klein, R.A. Conte, M.G. Bialer, S. rDNA resulting in an extreme variant of cbromsome 22p. S. Klesyman and R.S. Verma. North Shore University Hospital, Kleyman, R.A.Conte and R.S. Verma. The Long Island College Manhasset, NY and Long island College Hospital-SUNY Health Hospital-SUNY Health Science Center at Brooklyn, N.Y.11201. Science Center at Brooklyn, NY. Even prior to the invention of various staining A phenotypically normal male was found to have a de novo procedures, the variable nature of the short-arms of human translocation between the long arms of chromosomes Y and acrocentric chromosomes was well recognized. The 9, i.e. [46,X,t(Y;9)(qll.2;q22)] revealed by GTG-technique. cytological variations (heteromorphisms) have been This unreported aberration was first ascertained through classified into size, intensity and color polymorphisms amniocentesis with the GTG technique and later confirmed whose causes and consequences remain obscure. Recently, in the proband using dual color whole chromosome painting it has been shown that these regions consist of distinctly probes (Vysis, Gaithersburg, MD). Due to the location of different DNA families that are organized in a specific the breakpoints on the Y chromosome the proposita's array or pattern and usually the arrangement of these DNA fecundity may be in question. Vital gene(s) on the Yq families is: alpha-satellite, satellite IlI, beta- called the azoospermic factor (AZF) which apparently satellite, ribosomal DNA, beta-satellite, cytological control spermaLogenesis may have been deleteriously satellite and telomeric DNA. The order of these DNA affected by a microdeletion due to the translocation. families, which for the most part are tandemly arranged Most de novo cases involving Y; autosome translocations sequences and can be duplicated in a unintertrpaed~otder manifest a multitude of testicular afflictions including for hundreds to thousands of copies resulting in extreme a variety of male gamete deformities, azoospermio and variations. We encounted an individual with a chromosome sterility. Fertility testing of the proband at a post- 22 that has an unusually long p arm. Employing a series pubescence age will determine this status. It is believed of molecular and routine cytogenetc techniques it was that the sex determining region (SRY) on the Yp, which determined that a tandem duplication of rDNA, satellite has been Considered allelic to the testis determining III and beta satellite in the pll.2+pl3 regions have factor (TDF) are intact due to the presence of what apparently occurred resulting in an enlarged short arm of appears to be normal developing testis. Due to the nature chromsome 22. An apparent duplication of bands pll.2+pl3 of the Y chromosome and the limitation of the GTG was found to consist of DNA families in the following technique FISH permitted the delineation of the order: alpha-satellite, satellite III, beta-satellite, translocation with a higher level of certainty. rDNA, beta-satellite, satellite III, beta-satellite, rDNA, beta-satellite, cytological satellite and telomeric region. The duplication of such regions can occur by a variety of mechanisms restlting in extreme variants whose clinical signi icance remains obscure.

1801 1802 Doubk aneupleidy: trisemy 21 and cn do chat. PAL~xev AL. Shainrksndi Itiller-Dieker and Norman-Roberts syndromes. FISH investigat- LShmdmW Department of Peds.,Albert Einstein College ofMedicineMontefiore ion in two distinct entities with lissencephaly tipe I. J.C. Medical CenterBromcNY' and Corwagmetpath Clinical LabsTeterboroNJ2. Llerena Jr., F.Vargas & J.C.Cabral de Almeida. Centro de Ge- Double aneuploidy is an extremely rare event. Down syndrome is seen occasionally netica 1e~dica (IFF/FIOCRUZ) & Unidade de Citogenitica Humana in association with other aneuploidies. It may wise from either a double mutation in (IBCCFQ/UFRJ), Rio de Janeiro, Brazil. the chromosomes ofone gamete or even more unlikely from separate events in We report FISH studies on two patients with lissencepha- in both or from a event in one gamete ly type I; one female infant with Miller-Dieker syndrome gametogenesis parents non-dijunctional with Nor- followed by a post-zygotic mutation. We recently had the opportunity to identify a 2 (MDS) and one boy from non-consanguineous parents with the salient features ofboth the Down and cri du chat syndromes man-Roberts syndrome (NRS) whose younger brother,sintlarly year old child NRS who represeon to the best ofour knowledge the first concurrence ofthese disorders. affected,died with one year and a half. Both MDS and Case report: G.F. was the 5# 13oz female product ofa term uneventful pregnancy patients were studied by FISH using probes for lIDS region, old gr 3 2002 mother. The is unremarkable D17Z1 and all human teloreres (Oncor, Inc.). In the MDS meta delivered to a 23 year parn family history deletion has a normal She had many admissions for pneumonia and phases FISH demonstrated a "de novo" interstitial and the mother karyotype. where in failure prior to surgical repair ofa VSD. Her development has been delayed and her (cryptic parental translocation excluded by FSIH) mother describes a cry "like a a" during a . Her physical examination at 2 years the NRS metaphases no 17p deletion was detected confirming and 3 months revealed a stigmatized miocephic youngster with bilateral epicanthal different etiology. folds,upward obliquity ofthe palpebral flsreunilateral single palmar crese and a tongue-thrust habit. She had genelized hypotonia and a well-healed sternotomy scar. A peripheral leucocyte culture revealed an abnormal female karyotype: 47,XX,+21/47, XX, del(SXplS.lpIS.3),+21. All cells were trisomic for chromosome 21 and 35% ofthe cells showed a terminal deletion ofthe short arm ofchromosome 5. Trisomy 21 has been reported in association with a number ofother autosomal and sex chromosome aeuploidies. Petitet alreported in 1968 complex chromosomal anomalies including the "cri du chat comosome" in a leukemic patient with trisomy 21. Our patient represents the first reported cae ofthe concurrence ofthese two constitutional abnormalities. Published Abstracts: Cytogenetics (continued) A311 1803 1804 Microdeletion Studies in Pediatric Cases - Molecular Cytogenetic Complete deletion of short arm of Chromosome 21. T. Mathews, R.A. Conte, S. Kleyman and R.S. Verma. Division Clarification of Clinical Cases. EMak-TaM. E.apua. and P.R.Wvt. of Genetics, Long Island College Hospital-SUNY Health Department ofGenetics, North York General Hospital, North York, Science Center at Brooklyn, N.Y. 11201. Ontario, Canada. Over three decades ago, Gunz and his associates (1962) A noted a G-group chromosome which had lost all or a major retrospective recall of past patients, and prospective portion of its short arm and was termed Christchurch (Ch) institution for new patients, of in situ hybridization methods for clinical chromosome, which was then considered an abnormality. care has improved the diagnostic services to patients with microdeletion Later, it was found in a Down syndrome and was designated chromosome 21 or G1. The short arm of all human disorders. acrocentric chromosomes have attracted much attention due Over a twelve month period we recalled all patients previously seen for to variability in size, intensity and color heteromor- the suspected diagnoses of: Prader-Willi, phisins. Angelman, Williams, Fortuitously, within a one month period we were referred DiGeorge, and Smith-Magenis. In addition, all new referrals were two individuals for routine genetic amniocentesis where reviewed for these diagnosis. A total of66 cases were studied using the one chromosome 21 of each patient had lost all of its short arm and a major portion of its centromeric alphoid available commercial probes (ONCOR) for these conditions. sequences in their amniocytes. By FISH-technique, it was 24 ofthe cases had demonstrable molecular cytogenetic deletions using revealed that breakage had occurred within alphoid FISH probes which clarified the clinical diagnosis. sequences resulting in an extreme variant. The 21p- chromosome identified was similar in both cases, though The use ofmolecular cytogenetics has now become a standard and they are unrelated. The incident of such a rare variant essential tool for cytogenetic laboratories involved in the care of patients in the general population is unknown. On a cautionary with possible microdeletions. note, if an individual with an additional chromosome 21 lacks signals at interphase by FISH it may result in a rapid but wrong diagnosis. Obviously, this is a rare variant whose molecular structure has presently been characterized, which does not manifest clinical consequences and its origin was maternal in both cases.

1805 1806 Terminal d.116 in a fetus with and CNS anomalies. Familial Hypogonadotrophic Hypogonadism In four sibs with 46,XYq-. E McPherson, D Huff. M Clemens. E Smith. A Niklaus. S Kochmar. and U I.Mohapatra, K.Kucheria, K.Saravanan, A.C.Ammini. All India Institute 2urti, Magee-Womens Hospital and University of Pittsburgh. of Medical Sciences, New Delhi, India. A 20 Y chromosome is the most variable chromosome in normal population. week fetus with multiple anomalies had the karyotype 46XY -16 Most of the variations appear to be in the Yq chromosome arm acnd + derl6 t(2; 16) (q37. 1;q12. 1)pat resulting in monosomy for 1 6q12. 1- considered to be unassociated with phenotypic expression. Reports qter. Externally the fetus had macrocephaly, frontonasal dysplasia are also available showing deletion of Y chromosome long arm associated (extreme hypertelorism, broad nasal bridge, bilateral cleft lip/palate), severe with short stature, azoospermia, mental retardation, etc. We present micrognathia, and blunt fingertips. Autopsy revealed hydrocephalus, a family of four hypogonadotrophic hypogonadism sibs, phenotypically normal with a large deletion of Yq. Physical examination showed lack Dandy-Walker malformation, agenesis of the corpus callosum, arhin- of secondary sexual development, small size phallus and hypogonadism. encephaly, and optic nerve hypoplasia. There was Leydig cell hypoplasia, Ultrasound of abdominal region did not show any evidence of mullerian suggesting hypogonadism, but no major internal anomalies. duct structures. Leucocyte culture showed 46 chromosomes with a Terminal deletion of 1 6q has been reported in only 3 previous cases. small deleted Y chromosome 46,XYq-. PCR was carried out Of these, one was later shown to have an apparently balanced t(4; 16), one analysis using a series of Y long arm specific primers amplifying single DNA loci Yql1 at interval 6 sYl'47, sY152, was mosaic and unbanded, and the third dates from 1977 when banding sY153, sY157, sYS58, sYl60. All were positive including sYl60 which methods were not adequate to delineate breakpoints. Interstitial deletions is the last in interval 6. AZF is a genetic factor located in Yql 1 known of 16q have been reported in 18 cases. Monosomy of 16q12-13 (9 cases) to control human spermatogenesis. Our results showed no Interestitial or 16q22 (8 cases) causes a recognizable phenotype with wide fontanelles or terminal deletion in this region. Normal phenotypes and height in and sibs without breakpoint at Yqll suggests no loss or disruption of Yq sutures, hypertelorism, micrognathia, brachydactyly, cryptorchidism, euchromatin. and developmental delay with occasional internal malformations. The virtual Presence of Yq euchromatin and the deletion of heterochromatin absence of reports of del 16q24 (one case q23-24.1 and 2 unconfirmed beginning distal to sY160 indicates the presence of the AZF gene. terminal dell 6q) suggests that del 16q24 is rarely compatible with livebirth. Deletion of this region may cause partial infertility. Since the father Our proband had facial anomalies similar to but significantly more is mosaic with XYq- cell line, it further confirms that Yq deletion does severe than those reported in other del 16q patients. The larger size of the not involve deletion of azoospermia gene(s). the The height of four sibs is of normal average Indian height again deletion, involvement of 1 6q24, and the associated trisomy for a small confirms the presence of Yql 1 .23 as confirmed cytogenetically. The segment of 2q (which has been reported to cause hypertelorism, minor deletion of only heterochromatic region of Yq after sY160 of interval facial anomalies and developmental delay) are probably contributors to the 6 may be related to hypogonadotrophic hypogonadism. This Is the first greater severity of the facial and CNS anomalies in our patient. report of familial deletion of Yq In four Indian male sibs.

1807 1808 An unusual Interstitial duplication of the long arm of 4p trisomy syndrome due to der (17) t(4;17). H.O. Shah, K. chromosome l0-a problem in karyotype-phanotype H. Raesh, J. Sherman, J.H. Lin and R.S. Verma. Long correlation. .i.S. Sakhon. S. Kirkparc S. Morriscn-DeLaP. Island College Hospital-SUNY Health Science Center at University of WVisconsin-Madison, Waisman Center, Madison Brooklyn, NY and Nassau County Medical Center, East Meadoti Wisconsin. NY. A unique tandern invers on duplication resulting in partial There are several syndromes which cannot be recognized prior to trisomy for the region I Oq22.1-->10q23.4 is reported in a boy cytogenetic evaluation. One such clinical entity is 4p-trisomy whose mother is mosaic for the same chromosomal aberration. syndrome. We rcport a case with derivative chromosome 17 which could Dysmo.-phic features of the boy arecompared to those not be characterized by routine G-banding, since the additional resulting from partial tresomy cf region 10q22.1-->10q23.4, and material on 17q overlapped with chromosme bands 2q35 qter, 3p25 pter, contrasted to h's 4pl5 pter, 5q33 qter, 7p15 pter, 12pll.2 pter, 14q24 qter and 16q22 clinically nornal mother. qter. Therefore, whole chromosome paint and region specific probes were used to identify the abnormality by FISH-technique which revealed a 46,XY, der(17) t(4;17)(pl5.2;q25) karyotype. The proband was partially trisomic for 4pl5.2 pter and monosomic for the distal 17q25 qter regions. The major clinical features included: anti- morgoloid slanted palpebrae, colobouas of zigh. iris, depressed nasal bridge, high arched palate, protruding tongue, micrognathia and small penis. The MRI of the brain revealed mildly hypoplastic cerebellar vermis, and a normal septum- pellucdum. I. is interesting that some clinical features are comon to both trisomy 4p and monosomy 4p (Wolf-Hirschhorn) syndromes. In our case, one cannot undermine the importance of the FISH- technique in aiding precise cytogenetic evaluation, for its resolving powers superccded conventional methods. However, application of the FISH-technique, although essential in arriving at an accurate diagnosis, is sometimes not cost-effective as in the present case. Not only was it labor intensive, but at the same time a large number of WCP probes had to be used to identify the chromosomal abnormality. A312 Published Abstracts: Cytogenetics (continued) 1809 1810 An unusual euchromatic band within the secondary constri- Fragile sites in children with congenital mental retardation. X.Y. ction (qh) region of human chromosome 1. R.S.Verma, Wan2 , P. Li2, F. Liu1, and W.Y. Gaol. 1 Institute of Pediatric Research, S.Kleyman and R.A. Conte. Division of Genletics, Long Tianjin Children's Hospital, P.R.China. of Medical Genetics, island college Hospital-SUNY Health Science Center at 2Laboratory Brooklyn, N.Y. 11201. University of Alabama at Birmingham. Fragile sites (FS) are the nonrandomly distributed gaps and breaks on The morphological variation of the secondary human chromosomes. The classification of different fragile sites have been constriction region (qh) of chromosome 1 is due to established based on the frequency (i.e. common or rare, CFS or RFS) or heterochromatic DNA. Structural abnormalities involving the mode of induction. In the present investigation, the frequencies of or in association within the heterochromatic blocks have expressed fragile sites were compared in children with congenital mental been difficult to characterize and therefore have gone undetected by routine banding techniques. The presence retardation (MR group, N=65) and normal children (NC group, N=60). Blood of an "euchromatic" and "G-positive bands" within the was cultured according to standard cytogenetic protocol with addition of 10 9qh region have been considered unusual variant without pg/ml MTX (methotrexate). The results showed the frequencies of clinical significance. Fortuitously, we were referred expressed CFS were significantly different between the MR group and NC two cases where an euchromatic band within the secondary group, especially for CFS in A and C group of constriction region of chromosome 1 was embedded. Whole chromosome karyotype chromosome #1 specific paint, did hybridize to the band (P<0.01). Among the highly expressed CFS, 3p14 was the most frequent but it was digested by restriction enzyme Alul and Tacd one with a considerably higher frequency in MR group. Other significantly suggesting a GC rich region and originated from expressed CFS were 1p31, 1p36, 2p13, and 7q22. Four RFS (2q11, 5q35, chromosome 1. Both patients have minor neurological 8q22, and 17q21) and two new FS (4p14 and 6p21) were also observed. To problems. Unfortunately, the parents were not available analyze the chromosomal variations in different loci, FS at 1p31, 1p36, to confirm the inheritance. We are tempted to hypothesize 3p14, 3q25, 4q27, 7p13, 9p27, 15q22, and 16q23 were that this is an unusual variant where a G-negative band 6p23, scanned apparently became genetically inert, when it was using microspectroscopy to compare the gap distances, absorption sandwiched between two blocks of heterochromatin which spectrum, and relative DNA contents. Three FS (1p36, 6p23, and 15q22) raises a perplexing enigma regarding gene expression. We were found to have increased relative DNA contents. Our results suggested wish to shed some light on its biological and that some FS could probably predispose to phenotypic abnormalities. evolutionary significance and suggest that this new However, an alleviation of symptoms from autosomal heterozygosity could variant has previously escaped our eyes. have obscured the clinical association. Molecular characterization of FS is required in order to elucidate this point.

Published Abstracts: Gene Structure and Function 1811 1812 Identification of an eabryonic cDNa for a five- DNA methylation at CpG dinucleotides within the coding region of the hu- tranamebra juztanuclear protein preferentially man neurofibromatosis (NFl) gene. J.D. Andrews, D.N. Mancini, S.M. Singh and expressed in adult hematopoietic cells. D.I. Rodenhiser. Child Health Research Institute. Children's Hospital of Western On- Chaker N. Adra and Bing Lia. Division of Departments of Paediatrics and Hematology/Oncology, Department of Medicine, Beth tario, Zoology, University of Western Ontario. London, Israel Hospital/Harvard Medical School, Boston, MA Ontario, Canada. Intro by: S.M. Singh. 02215. CpG dinucleotides have been shown to be hotspots for mutation in a number of genes, with transitions at CpGs estimated to show a 24-fold increase over transitions at other We describe the cloning of a human cDNA for a sites (Koberl et al 1990. AJHG 47:202). The most likely hypothesis for this phenomenon gene that is also expressed in nurine esbryonal is that cytosine residues within CpG dinucleotides are subject to DNA methylation and stem cells. During embryonic development the gene subsequent spontaneous deamination of 5-methvlcytosine residues. A number of patients is expressed in both henatopoistic and non- afflicted with the common genetic disease neurofibromatosis type 1 hematopoietic tissues. The presence of the (NF1) have been transcript in yolk sac cells suggest that the gene identified to have sporadic mutations occurring at CpG motifs in the NF1 coding region. is also active in primitive hematopoistic These sites include a recurrent mutation site within exon 31 of this gene. We have precursors. In cell lines the gene is expressed previously shown that DNA methylation occurs at several sites flanking the NF1 gene as specifically in henatopoietic lineages and in well as at CpGs within the gene (Rodenhiser et al 1993, Hum Mol Genet 2:439). However, normal adult tissues, the *RNA is preferentially relatively few CpGs were evaluated, due to the limitations inherent in the isoschizomeric detected at high levels in lymphoid and myeloid restriction enzyme-based protocol. Our objective in the present study was to develop tissues. The predicted 262 amino acid protein is a a methylation map within the coding region of the NF1 gene, especially within those pentaspanner with no homology to known genes and is exons where mutated CpGs have been reported. We have used a sensitive highly conserved across evolution. A specific bisulphite antibody against the protein detected a 29 kD genomic sequencing method (Clark et al 1994: Nucl Acids Res 22: 2290) to generate protein of juxtanuclear distribution around the a strand-specific positive signal for cytosine methylation. The results confirmed our golgi, co-localizing best with lysosomes. The previous findings and extended the range of DNA methylation within the NFl coding overall pattern of expression of the gene, region by mapping strand-specific cytosine methylation patterns over several exons in designated HOTm5, indicates that the protein is DNA from a variety of human tissues. We have found that DNA methylation in this gene essential during embryogenesis and in adult animals occurs only at CpG-associated cytosines and operates in a strand-specific manner. Our performs a function of importance in hesatopoietic results strengthen the hypothesis that methylatable CpGs in the NF1 gene contribute to cells. the high frequency of spontaneous mutations associated with this gene. (Funded by the Child Health Research Institute) 1813 1814 Chsreterlaton of BTFp44, a traascrptionfactor lxolized to the SMA gem Physical analysis of the homologous sequences to the imprinted region. 'TA Carter. _'C_ Ibici.IAnBonean.Eg'£hcaa'A.nma mouse gene U2liabprs in the human genome. V. Chapman. IS. Pearsall, a.._%Ross. !LKunkeL YTC Gilliam. 'Columbia University, New York NY. 'A. Brozowska. 2P.J. de Jong, 2C. Plans. '1. Kalcheva, 'S. Sait. IT. Shows. 2Children's Hospital, Boston MA. 2H. Shibata3 and Y. Hayashizaki3. Molecular and Cellular The Spinal Muscular Atrophy gene region lies on chromosome 5ql3. Physical. Biology Department. genetic and linkage disequilibrium mapping have previously been used to localize the Roswell Park Cancer Institute, Buffalo. New York.' Human Genetics Department, gene to a relatively small region characterized by repetitive loci. These repetitive Roswell Park Cancer Institute, Buffalo. New York.2 and Genome Science Labora- sequences include not only microsatellite markers, pseudogenes, and STS's, but gene tory, RIKEN- Tsukuba Life Science Center. Tsukuba Ibaraki, Japan.3 sequences, as well. SSCP analysis of the last two exons of the SMN gene identifies a The imprinted mouse gene Ub2afbprs maps to mouse chromosome 11 and is ex- single copy of the gene which is deleted in the majority of all SMA patients, although pressed ubiquitously and exclusively from the paternal allele. We have undertaken no phenotypic correlation has been described. The BTP2p44 protein has been a comparative analysis of this gene in the human genome to determine if the im- previously described and a single copy has been mapped to the region. We report the printed regulation is conserved. The initial analyses identified a single human locus genonucstructure of the gene and a more refined mapping of certain of its multiple (U2AFBPL) on 5q23-31. We have initiated the physical analysis of the homol- loci adjacent to genes previously described. The multi-copy nature of the gene, as ogous human sequences. Seven with insert sizes well as its proximity to SMN, may indicate a possible role for BTF2p44 in human genomic clones. ranging phenotypic severity of the disease. Data indicate that the copy number of BTF2p44 from 110-150 kbp, were isolated from a PAC library using a 600 bp mouse cDNA varies in individuals, We will report further work in selectively examining single loci probe. Two seperate groups of overlapping clones were recovered that identified of the gene copies and possible functional roles of these copies, as well as more different fragment sizes with several restriction enzymes when hybridized with the general studies in the copy number of this gene relative to adjacent genes. mouse U2afbprs probe. Each set of clones had a single hybridization band that corresponded to one of the fragment sizes observed in Southern analysis of human genomic DNA. These data indicate that the homologous sequences of the mouse UIaffbprs gene are duplicated in the human genome. and that only a single copy is present in each of the PAC clones. FISH and somatic cell analvsis localized one copy to human chromosome 19p and the other copy to human chromosome 5q23, the previously identified region that shows conserved homology to mouse chromo- some 11. Sequence analysis of the copy on 19p (U2AFBPL2) indicates that there is a 300 bp Alu element in the coding region of this copy suggesting that this may be a pseudogene. Published Abstracts: Gene Structure and Function (continued) A313 1815 1816 Functional Conservation of Amino Acid residues in Factor IX: Evidence from Epiphycan (Pg-Lb), a small dermatan sulfate proteoglycan expressed in Missense Mutations in Hemophilia B Patients and from comparison with eight embryonic cartilage, is conserved between species. M. Deerel, L mammalian species. S-H Chen, M. Zhang. J. M. Schoof C. Yu. A. R. Thompson and Tohnson2, S. Garza2, Y. Yamada3, M. Hook2, and T. T. Hecht1. C. R.Scott. Univ. of Washington, Children's Hospital and Medical Center, and Puget 1University of Texas Health Science Center, Houston, 2Texas A&M Sound Blood Center, Seattle, Washington Missense mutations disease University Institute of Biosciences and Technology, Houston, 3National causing are good indicators offunctional and structural Institute of Dental Research, of the amino acid residues of In addition, variation Bethesda, MD. requirements proteins. interspecies Epiphycan (Pg-Lb) is a small dermatan sulfate proteoglycan that has of a gene sequence can provide important insight into the necessary structure of a We have identified mutations in the factor IX in 124 families with previously been characterized in chicken. Epiphycan contains leucine protein. gene rich B, and found 91 missense mutations 50 amino acid sites. repeats present in decorin and biglycan, two small dermatan sulfate hemophilia involving but has a our data with the 1994 data F et al. NAR 22: of proteoglycans, higher homology within the leucine repeats to Combining base(Giannell 3534) human hemophilia B, 177 of 461 amino acid residues (40%/6) have had missense mutations. osteoglycin. Chick epiphycan is encoded by a 1701 nucleotide cDNA that codes for a 316 amino acid protein with a molecular Mutations were identified in 2 of the 3 triad 9 of 12 weight catalytic residues, gamma-carboxyl- of 35,854 daltons. Epiphycan may participate in osteogenic processes, glutamates of the Gla domain, and 22 of 24 residues. All of the above are Cys since it to be in the zone considered structurally conserved residues. It is estimated that the total number of appears exclusively expressed of flattened conserved amino acid residues offactor IX is 210 (45%). We have determined the total chondrocytes (ossifying area) of the developing chick limb cartilage. There is only one other proteoglycan, aggrecan, that is known to be fictor IX in five nammalian human, coding sequences species: chimpanzee, macaque, solely expressed in cartilage, and aggrecan is a major cartilage matrix rat and rabbit. Sequence analyses between these species and three other mammals in the constituent. It is therefore important to characterize epiphycan in literature show that 68% ofthe amino acid residues are conserved in all the species. By humans to determine the of in human the differences with the B we find importance epiphycan limb comparing interspecies hemophilia mutations, that development. We have sequenced epiphycan from a human only II residues with missense mutations demonstrate interspecies variation. Of these chondrocyte expression library and a control human chondrocyte RNA 11 residues, 7 are "benign" substitutions (such as Arg-Gn, Ble-Val-Leu and Asp- A domain, in the variation. There are one and three amino acid differences sample. potential calcium-binding four cysteines that may Asn) interspecies form disulfide-bonds, and a between human / and human / There is potential glycosaminoglycan attachment site chimpanzee macaque sequences, respectively are conserved. The human and chicken amino an of 30% residue variation between man and the other mammals and acid sequences for average between epiphycan are > 70% conserved. these mammals. The higher degree of sequence homology between man and the primates for factor IX is another example oftheir genetic similarity and slower rate of evolutionary change.

1817 1818 A DNA fragment with homology to the 1,25-dihydroxyvitamin D receptor localizes CLONING OF THE HUMAN TYPE XVII COLLAGEN GENE to the heterochromatic region of chromosome 9. S.C. Fossey', N. Raw2 MJ. (COL17A1): A CANDIDATE GENE FOR JUNCTIONAL EPIDERMOLYSIS Pttenatim G-M Kiehzak' R.A Meer. Jr.'. and C.A. Brown'. 'Carolinas Medical BULLOSA. BiAn Gatalica. Kehua Li. John A. Mc Grath. M. Chriatiano and Center, Charlotte, NC and Bowman-Gray School of Medicine, Winston-Salem, NC. Angela The human D has been cloned and .ifn o. Thomas Jefferson University, Philadelphia, Pennsylvania. 1,25-dihydroxyvitamin receptor (VDR) gene maps Type XVII collagen is a 180-kDa transmembrane protein localized to hemidesmosomes to chromosome 12qI2-q14. While mutations in this gene can result in vitamin D- in the cutaneous basement membrane zone. It serves as an autoantigen in patients with resistant rickets, specific VDR gene polymorphisms have also been reported to correlate bullous pemphigoid, and was previously designated as 180 kDa bullous pemphigoid with bone mineral density. One of these reported polymorphisms, detected by the antigen, BPAG2. Furthermore, mutations in COLl7AI have been shown to underlie restriction endonuclease TaqI, is found in exon 9 and is detectable by Southern blot some cases with junctional epidermolysis bullosa, a blistering skin disease. In this study, analysis using a 2.1 kb cDNA probe encoding the full length VDR. In order to facilitate we have elucidated the complete genomic organization of the human type XVII collagen the detection of the PCR primers were from the VDR gene. Screening of a genomic A-phage DNA library with a 1.0 kb cDNA yielded six TaqI polymorphism, designed clones a cDNA to the the site from DNA. overlapping spanning 21 kb. Furthermore, 45 kb cosmid clone was isolated, and sequence amplify region surrounding TaqI genomic Southern analysis demonstrated that this clone contained most of the gene. The remaining Amplification with these primers produced two different size amplimers with individuals 5' and 3' ends of the gene were completed by direct PCR amplification from genomic exhibiting a 1600 base pair and/or 300 base pair PCR product. These PCR products DNA, using oligonucleotides corresponding to the cDNA sequences, and then subjected were sequenced directly using a Taq Dyedeoxy Terminator sequencing kit (Perkin to direct cycle sequencing. The entire gene comprises 55 exons which vary between 27 Elmer) and reactions run on an automated sequencer (ABI 373A). DNA sequence and 390 bp, while the introns are highly variable in size.The entire gene spans 48 kb. The revealed that the 300 was to the 3' codon are always split at the second nucleotide at the splice site, and all intron-exon analysis bp product completely homologous portion the consensus of the VDR cDNA while 60 at the 5' end and 280 at the 3' end of junctions depicted splice sequence, ag-EXON-gt. A common non- sequence only bp bp pathogenetic missense polymorphism was identified at the nucleotide position 735 and the larger fragment matched the VDR cDNA sequence. The TaqI restriction analysis of assessed in 100 normal control alleles: minor allele C (ACG, threonine; frequency 0.37); the 1600 bp PCR fragment generated the same TaqI genotype as that identified by major allele T (ATG, methionine; frequency 0.63). This sequence variant alters a BsrDI Southern blot analysis in all individuals tested. Further analysis indicated that both the restriction site, and the PIC value for this polymorphism is 0.348. Elucidation of the 300 and 1600 bp DNA fragments were transcribed and that the segments of non-VDR intron-exon organization and identification of polymorphisms in COLl7Al will facilitate sequence analyzed-to-date were found to be identical among individuals. Fluorescence identification of further pathogenic mutations in this gene. in-situ hybridization (FISH) studies using the 1600 bp pair fragment as a probe showed a strong hybridization signal only to chromosome 9qI 1. In conclusion, we have identified a fragment of DNA homologous to the 1,25-dihydroxyvitamin D receptor gene which maps within the heterochromatic region on chromosome 9.

1819 1820 Isolation and characterization of a sterol regulatory element (SRE) in Isolation of cDNAs from differentially expressed genes by arbitrary RT-PCR: the human mevalonate kinase (MKase) gene. K.M. Gibsonl. C. Martin Optimization and analysis of specificity, sensitivity and reproducibility. I W Tn and R.W. BishoR. IBaylor Res. Inst., Dallas, TX; 2Amis I. Hernandez and B.P. Sokolov.Department of Psychiatry and Seaver Center for Pharmaceuticals, S. San Francisco, CA; 3Geron Corp., Menlo Park, CA. Autism Research, Mount Sinai Medical School, One Gustave L Levy Place, New In cholesterol biosynthesis, stero-dependent regulation of transcription is controlled York, NY 10029. Intro. Dr. an SRE in the of the HMG-CoA by: D.Greenberg. by eight bp Y-flanking regions synthase and we described a reductase, and the.LDL receptor, genes. We identified an SRE element in the 5!- Recently simple and rapid arbitrary RT-PCR based method to untranslaed region of the human MKase gene. MKase is the next enzyme in the isolate cDNA fragments from differentially expressed genes (Sokolov and cholesterol pathway afterHMG-CoA reductase. The MKase 5-flanking region was Procklop, 1994) Here we examine the sensitivity ofthe method and parameters isolated from a bacteriophage Xgtl 1 genomic library, and a single clone was sequenced that affect its specificity and reproducibility.We found that the method is sensitive which contained a 7 of 8 bp match to previously reported SREs. To determine enough to detect quantitative differences (as low as 2-fold) in levels of expression functionality, the region immediately upstream of the transcription initiation site was and can detect 'rare' mRNAs with a copy number ofabout I per cell. The method inserted into the reporter for of plasmid pSV2cu analysis transcription regulation is highly reproducible. However, variations in RNA concentration over 4-fold may (pMKWT We created three additional plasmid). plasmids by PCR mutagenesis, one effect the of cDNAs Evidence has been (pMKCon) the consensus SRE in other sterol-responsive genes and the others significantly patterns amplified obtained (pMKmut A & B) previously shown to produce the largest decrease in sterol- that competition between cDNA species plays a crucial role in selection ofcDNA responsiveness in other genes. The four plasmids were transiently expressed in COS- fragments amplified in arbitrary PCR. Even abundant mRNA species with I 10% 7 cells and transcriptional activity assessed using a chloramphenicol acetyl transfease homology to arbitrary primers may not be amplified by some sets ofprimers due (CAT) assay (Table; mean ± IlSD, n=6-10 assays/plasmid; parenthetic values are % to the competition with other cDNAs. We found that reverse transcription oftotal fetal calf serum in WT and (FCS) post-transfection medium). Con plasmids showed RNA is efficient even without significant transcriptional induction (p < 0.004) in 0.5% FCS (low cholesterol), while highly adding any primers ('background' priming) mus A & B plasmids showed no induction. The Con Dlasmid had higher induction in Because of the 'background' priming, oligo(dT) containing primers do not provide

medium to < efficient selection of mRNA both compared WT(p 0.03). The data suggest that the SRE in the 5'- in reverse transcription Nevertheless, DNA patterns flanting region of the MKase gene confers sterol-responsive transcriptional control. obtained in arbitrary PCR from total RNA and mRNA were very similar. This work (This supported by a grant-in-aid from the American Heart Association) suggests that in arbitrary PCR selection of mRNA sequences from more abundant s ribosomal and transport RNAs is provided by the very high sequence complexity (Uvnoiug roitein/0min) SRE (5'->3') - of we have pMKWT ±1activity mRNA. Finally, further optimized the method and demonstrated that 0.70 0.30 (10); 1.10i± 0.30 (0.5) CACCCCAG PCR pMKCon 1.00 ± 0.40 (10) 2.90 ± 0.60 (0.5) CACCCCA-C highly reproducible patterns can be achieved with total RNA from autopsy brain tissue 5 pMKnutA 0.02 ± 0.06 (10);0.07 ± 0.06 (0.5) CAACCCAG with postmortem interval ofup to days that had been stored for pMKmut B 0.00 ± 0.00 (10);0.06 ± 0.06 (0.5) CACQACAG several years. A314 Published Abstracts: Gene 'Structure and Function (continued) 1821 1822 Towards an animal model for Canavan disease: Isolation and The yis-yang regulation of human thromboxane synthase gene expression. characterization of mous aspa gene. R. Kaul. K. Balamurugan. M. C. K. D. Lee, S. J. Back and R.-F. Shen. Div. of Human Genetics, Univ. of Maryland Algya and R. Matalon. Miami Children's Hospital, Miami, FL. School ofMedicine and the Medical Biotech. Center, Univ. ofMaryland Biotech. Inst. Canavan disease (CD) is an autosomal recessive leukodystrophy caused Baltimore, MD 21201 by deficiency of aspartoacylase (ASPA); that leads to megalencephaly, Thromboxane synthase (TS) catalyzes the conversion of prostaglandin H2 into TxA2, hypotonia and mental retardation. The disease is characterized by N- a potent inducer of vasoconstriction and platelet aggregation. The biological function acetylaspartic aciduria and accumulation of N-acetylaspartic acid in brain. of TxA2 is mediated by the TxA2 receptors. A deficiency of platelet TS activity or defects in the TxA2 receptor gene have been associated with dominant inherited Recent Isolation and characterization of human ASPA cDNA and gene have bleeding disorder, while over-production of TxA2 is implicated in cardiovascular resulted in identification of seventeen different mutations In patients with disease. We previously mapped the human TS gene to chromosome 7q33-q34 and CD. Carrier rate for CD among Ashkenazi Jews has been determined to be recently cloned its entire genomic sequence. The human TS gene is extraordinarily about 1:36. In order to create a knock out mouse model of CD, we have large, containing 13 exons which span more than 150 kb. To better understand its now isolated and characterized mouse aspa cDNA and gene. The mouse regulation, we characterized the TS gene promoter. RNase A/TI mapping and 5'- aspa cDNA codes for 312 residue protein, compared to 313 amino acids RACE indicated that the gene is transcribed by multiple transcriptional initiation sites, predicted by the human ASPA cDNA. The mouse and human ASPA are 88% independent of a canonical TATA box. Sequencing analysis revealed many identical. The aspa is coded by six exons and spans about 25 kb of mouse transcription factor recognition sites including PU.1, PEA3, AP-1, etc. In addition, genome. Genomic organization of mouse aspa is similar to the human a 1.2 kb retrotransposon was localized between -900 and -2100 bp in the promoter. ASPA and their exonrintron boundary junctions are conserved. The aspa This sequence, a deleted prototype of the LINE sequence, shares - 80% homology was localized to mouse chromosome 11 by florescent in situ hybridization; with the Li element found in the intergenic sequence of the globin genes and exerts using mouse tk gene as a marker for this chromosome. The region of 20% repression on TS gene transcription. No homolog could be found in the mouse chromosome 11 is syntenic to human chromosome 17p, where the corresponding position in the murine TS gene promoter, suggesting that the transposition is a relatively recent event. Transient reporter gene expression analyses human homolog of aspa is localized. The isolated clones will be used to indicated that the TS gene is controlled primarily by a proximal positive regulatory create a knock out mouse model of CD. If such a model has CD like sequence (PPR) and distal repressive sequences, including a silencer. The PPR is features It will be useful for: studying the mechanism of pathophysiology in enhancer-like, since its stimulation ofTS transcription is independent oforientation but CD; understand the role of N-acetylaspartic acid in maintenance of normal dependent on position. The PPR was more efficiently utilized by TS-expressing cells white matter in CNS; and explore enzyme and gene therapy of CD. than non-TS expressing cells. (supported by the American Heart Association)

1823 1824 Light microscopic anaiysis of the distribution of the Huntington disease Methylation mapping of cytosines in the mouse neurofibromatosis (NFI) protein in murine brain.C MOalla homaakiWondk _Ln gene region following sodium bisulphite modification of genomic DNA. D.N. Mancini, J.D Andrews. S.M. Singh. and D.I. Rodenhiser Child Health Re- PS H&arper, ERLowenntuin2. Queensland Clinical Genetics Service, Royal search Institute, Children's Hospital of Western Ontario, Departments of Paedi- Children's Hospital, Herston, Brisbane, Qld 4029 Australia1' Institute of atrics and Zoology. University of Western Ontario. London, Ontario, Canada. Intro Medical Genetics, University of Wales College of Medicinel, School of by: S.M. Singh. Differential methylation of specific transcription factor binding motifs within CpG Molecular and Medical Biosciences, University ofWales, Cardiff; Wales, UK2. islands of 5 promoter regions is a recognized component of the complex regula- The Hunington disease gene (IT15) encodes a -350kd protein of unknown tory mechanism governing eukaryotic gene expression. Errors in DNA methylation finton which contains an expanded polyglutamine tact in the disease state. patterns could alter gene expression causing aberrant developmental programs or We have used an affinity purified polycional rabbit antibody raised against a in somatic tissues. is to 2110-2121 (Hunt3) of the human inappropriate expression The neurofibromatosis (NFl) gene synthetic peptide corresponding residues expressed constitutively in most cell types, although at different levels, and is an huntingtin sequence, which are conserved in the mouse sequence, to map the of cell and of a important regulator growth differentiation. The promoter regions distribution of huntingtin in the murine brain. This antibody detects -35OkDa both the human and mouse a of tran- band on Western blot analysis ofwhole brain homogenate and bands of 71, 87 NF1 homologues possess variety putative and 135kDa corresponding to bacterially expressed huntingtin fusion proteins scription factor binding motifs including several shown to be methylation-sensitive containing the Hunt3 sequence. when localized within the promoters of other tumour suppressor genes. Our ob- Murine huntingtin homologue (Hdh)-like immunoreactivity is distributed jectives in this study were to define a methylation map of cytosine residues in the widely in the brain and is seen in a cytoplasmic distribution in cell bodies in the mouse NF1 promoter, to specifically assay methylation within putative regulatory Purkinje cell layer ofthe cerebellum, layers II and V ofthe cerebral cortex, the elements and to evaluate these methylation patterns in sodium bisulphite method lateral amygdaloid nucleus and in focal collections ofcells in the brainstem. It is for modification of genomic DNA and DNA sequencing (Clark et al 1994; Nucl distributed in a diffuse punctate fashion in striatun, thalamus, layer IV of the Acids Res 22., 2290-2297) to obtain a strand-specific positive signal for methylated cerebral cortex and in specific layers in the hippocampus. Preimmune serum, cytosines. We have developed a site-specific methylation profile of a 300 bp region serum preabsorbed with a 500 fold excess of Hunt3 peptide and control of the NF1 promoter which includes the transcription start site and a number of (normal mouse) serum show no staining ofany cells/structures. putative transcription elements. Such a map will be invaluable in understanding the tissue-specific and temporal regulation of NF1 gene expression in normal tissues as well as determining how inappropriate methylation of such regulatory sequences mav lead to disease manifestation. (funded by the Child Health Research Institute)

1825 1826 e Sequencing of a human PAX-9 cDNA clone and discovery of a Structural da of the 5' reon of the myosin-VUagene implated in Usher polymorphic PCR product homologous to PAX-9 in primer sequence syndrome type lb. D. J. Orten. P. M. Kelley and W. J. Kimbeing. Boys Town National NLEiishimn , N.J. Lesens, dB ila L J.C. Murray. Research Hospital, Omaha, NE. 'Department of Pediatrics, University of Iowa, Iowa City IA v~epartment of Usher syndronetype 1 causes profound conial deafness, retinitis pigmnstosa and vestibular Physiology, University of Iowa, Iowa City IA 3Washington University, St. Louis areiexia. Mutations at the USHlb locus at 1 lql3.5 am responsible for about 70% of all cases of the more severe ty 1 Ushe syndrome. A myosin-Vila protein Sg is a strong candt for the Paired box containing genes, or PAX genes, have been found to play important roles USHlb gene and signific msto in beth the hunMn gon ad the nurine hontlogaue have in development. Two human disorders, Waardenburg's syndrome and aniridia, have in a of To been identified. However, specific ntaos have been identified only smnll perentage been mapped to mutations in the human genes PAX-3 and PAX-6 respectively. Usher lb We c c n te ' of the gene to gain insigiss into its generate candidate gene information for eraniofacial disorders, we are sequencing a patients. arcurrently PAX-9 cDNA clone to extend the limited sequence data currently available. The rgulaton, and to ientify aditial mutations. The myosinn-Va gene is expressed in km&ghdIery, cDNA clone was isolated from a human embryonic craniofacial cDNA library using a retia, cochlea and testis (Well et al, Nature 374:60.61, 1995; Glbsac et a., Nature 374:62-63. degenerate oligonucleotide directed to the PAX domain ofother paired genes. 1995). Very low levels of expression are observed in skeletal muscle. heart brain and spleaL Roughly 2,200 base pairs of sequence have been generated from this clone, of which Nonnasscle myosin mRNAs can be expressed in a variety of tissues, but a single isofom nay be 492 bases have been previously published. The full length clone has been estimated at domen in each specific tissue However, regulation of the expression of nonsele myosins is 2,600 bases by PCR analysis using T3 and 17 primers. Oligonucleotide primers have not well understood been generated within the paired box sequence to find a SSCP for genetic mapping. We have isolated genoeic clones from a cosmidcontaining sequen upstamfromprevio One pair of primes in addition to amplifying the desired PAX-9 product, has shown characterized 5' exons, wich we expect to encode the promoee and enhancer regions of the gene coamplification of alarger PCR fragment. The fragments have been isolated from a Restriction have constructed, partial sequc have been following silver stained polyacsylamide gel, reamplified, and sequenced to confirm the identity of maps been and dameined The subcionng of aproria resiction fragns into pBhlescript. Transcritonal initiatin sie(s) each fragment. The larger fragnt is only homologous in the primer sequences. wiin the clones extension and RNase assays, using a of the will bemapped genonic by primer protection larger fragment displays SSCP which has been mapped to the distal region of the start site(s) will be short arm of chromosome 2, a region not known to contain PAX-like genes. hunun testis RNA (Ciontech). Featues dte sequence surromding Sequence analysis is underway in attempts to identify this product. The completed conpaed withthose of other nonrnscle myosins. The sequence upstream of the start site(s) will PAX-9 cDNA sequence will facilitate studies of its role in developmental pathways be analysd for consensus transcription factor binding sites which will be candidate sites for that may result in human disorders and assist efforts to find new PAX-like genes. disease-related natations. Future analysis of 5' and 3' deletion nutalt cou cts will determine elenmets essential for core pronrer activity and cell type-specific activation of the nyosin-Vlla g (Supported bybggrant from NIH #R01R DC00677.) Published Abstracts: Gene Structuire and Function (continued) A315 1827 1828 A paragenetic model of non-Mendelian diseases. A. Petronis. Clarke Institute of Characterization of Neurofibromatosisl -homologous locl.S.M.Purandare'. Psychiatry, Toronto, Canada. (Intro. by Dr. J.L. Kennedy) H.Breidenbach2. Y.W2 X.L.Zhu2. S. Sawada'. S.Neil'.A.Brothman'. R.White3. Families with multiple cases of breast cancer, diabetes mellitus and major R.Cawthon2. and D.Viskochil'.'Dept of Pediatrics,'Dept of Human Genetics, psychosis have demonstrated recombination supression at the loci of linkage. The 'Oncological Sciences and Huntsman Cancer Institute, Univ of Utah, Salt Lake origin of this aberrant recombination is unknown but it may be due to abnormal DNA City, Utah 84112, 'Dept of Dermatology, Jikei Univ, School of Medicine, modification, or paragenetic defect. It is well known that DNA modification plays an Tokyo, Japan. important role in genetic recombination, DNA repair and mutagenesis. Characterization of sequences homologous to the NF1 gene on chromosome 1 7q1 1.2 was carried out by direct sequencing, fluorescence in situ hybridization The paragenetic model has the potential to explain a number of unclear issues in (FISH) and PCR of somatic cell hybrids. We have identified seven non-Mendelian diseases. A paramutation occurs when genomic imprinting amplification changes NF1 homologous loci, two separate loci on chromosome 2, NF1-HL(2q21) and its pattern to sex the The same allele in according the of embryo. different gametes NF1-HL(2q33-34), and one locus each on five other chromosomes NF1- of an individual can be affected to varying degrees, and therefore lead to high HL(14q11.2), NF1-HL(15q11.2), NF1-HL(18pl 1.2), NF1-HL(21q1 1.2-q21l and variation in the clinical disease phenotype. After conception, tissue-specific and NF1 -HL(22q1 1.2). Application of PCR using NF1 primer pairs and genomic DNA age-dependent DNA modifications interact with a paramutation. Asymmetry in these from monochromosomal somatic cell hybrids identified an additional locus on post-conceptional processes in MZ twins may lead to their phenotypic discordance. chromosome 12, NFl -HLI1-2). Sequences homologous to NF1 exons Two opposite trends in paramutation dynamics occur across generations. In the 8,9,13,15,16,17,18,19a,19b,21,22,23-1,24,27a & 27b were detected by majority of cases, paramutation reverts back to the normal paragenotype, and the monochromosomal somatic cell hybrid analysis. Sequenced PCR products disease is not transmitted to the-offspring. If the reversion does not occur and the representing segments corresponding to NF1 exons from homologous loci disease becomes familial, paramutation progresses in the subsequent generations to demonstrated greater than 95% homology with the NF1 locus. Most of these produce an earlier age of onset and more severe course. This phenomenon is known segments had small deletions/insertions in addition to missense and silent as genetic anticipation, and has been so far ascribed to the unstable DNA diseases. mutations in intronic as well as exonic sequences, which suggests that they are The relationship between paramutation and DNA mutation is of major interest. It non-processed pseudogenes. The existence of homologous loci complicates NF1 mutation detection from DNA in the 5' of the is possible that paramutation predisposes to structural DNA mutation. Structural DNA genomic portion gene. Using sequence differences between bona fide NF1 and loci, we have mutation a NFl-homologous is necessary condition for most Mendelian diseases, while in non- strategically designed primer sets for 28 of 35 NF1 exons within the 5' portion Mendelian disorders paramutation plays a major role, with germline or somatic DNA of the gene to specifically amplify from the NF1 locus, which should facilitate mutation possibly also relevant. mutation analysis. The model can be falsified by recombination analysis in sporadic cases of non-Mendelian disorders and in cases of de novo mutations in Mendelian diseases.

1829 1830 Nearest-neighbour sequence effects associated with the high frequency of Enhancers, intergenic spacer promoters, and p53 involvement mutations in the neurofibromatosis type 1 (NFl) gene. D.I. Rodenhiser, in the transcription of human ribosomal RNA genes. J.E. D.N. Mancini, J.D. Andrews and S.M. Singh. Child Health Research Institute. Children's Sylvester and W.-M. Li. Medical College of Pennsylvania Hospital of Western Ontario, Departments of Paediatrics and Zoology. University of West- and Hahnemann University, Philadelphia, PA. ern Ontario. London. Ontario, Canada. Introby: S.M.Singh. As opposed to the transcribed region of the human The neurofibromatosis type 1 (NFl) locus has a high frequency of spontaneous mu- ribosomal gene (rDNA), the intergenic spacer (IGS) is much tations, with up to i07c of patients representing new mutations in this gene. Many less characterized. The IGS of many other species, such as laboratories have been involved in a collaborative effort to identify NF1 mutations for frog, mouse and rat, contain enhancers, spacer promoters diagnostic purposes and to derive an overall picture of the mutational basis of NF1. At- and gene promoter-proximal terminator which regulate the tempts to address the molecular mechanism(s) responsible for the high mutation rate of transcription from the gene promoter. Using an in vitro this gene can now be undertaken. given the relatively large number of NF1 mutations re- run-off transcription assay, we have found that a 4.5 Kb ported to date. We have analyzed DNA sequences flanking the sites of 90 NFI mutations fragment (CBE) upstream of the human rDNA gene promoter has reported in the literature and by the NF1 mutation analysis consortium. Our objective cis-stimulation and trans-competition activity for the gene was to determine whether common sequence elements or motifs could be identified in promoter function, which suggests that CBE fragment has association with NF1 mutations. For this analysis, mutations were grouped as substitu- enhancer function for the gene promoter. Furthermore, tions or small insertions (and deletions) involving 1-2 bases, 3-20 bases or over 20 bases. deletion analysis has demonstrated that a 0.3Kb subfragment Sixty-five percent of single base substitutions leading to stop codons occurred at CpG or within CBE, located -3.4kb upstream of the transcription CpNpG motifs, with only one of the 14 base changes (7%) that lead to amino acid sub- initiation site (TIS), has appreciable enhancer function stitutions being associated with such motifs. Of the 22 non-CpG substitutions, 17 (77%) especially when protein extract is limiting. p53 were associated with purine dimers (predominantly GA or AA) as nearest-neighbours. involvement is supported by the fact that this fragment Interestingly, purine dimers or multiple purine residues were also associated with 60% of contains a p53 consensus binding site and that antibody to the small deletions and insertions, as compared to only 22% involving pyrimidine dimers. p53 eliminates the cis-stimulatory and trans-competition Although a role for methylatable CpG and CpNpG motifs in mutagenesis is well rec- activity of the subfragment. other experiments provide ognized. the effect of purine dimers as nearest neighbours of mutational events has not evidence for a spacer promoter located -800 bp upstream of been investigated. It could be hypothesized that such dimers may undergo dimerization TIS by in vitro run-off transcription assay, although its followed by repair defects in this tumour suppressor gene. If future evaluations establish functional role has not, as yet, been determined for the purine dimers as a preferred site for mutation, one may argue that replication and/or human system. All of these results indicates that the human repair errors are major contributors to the relatively high rate of mutation in the NFl ribosomal gene contains enhancer(s) and spacer promoter(s) gene. (funded by the Child Health Research Institute) as do other species and suggests a conservation of function even though the nucleotide sequence has drastically diverged. A testable model for p53 action is proposed.

Published Abstracts: Genetic Counseling and Genetic Education 1831 1832 Influence of education on knowledge and attitudes to Biotechnology and Genetic counselling in Alzheimer disease: Practical Information for the Genetic Engineering. C. D. Laughlan and S. S. Mastana. Human Department, Clinician. A. D. Sadovniick. ' H. Karlinsky. 2 . Burgess2. University of British Loughbrough University Loughborough. Columbia,' and Riverview Hospital. A questionnaire survey using two opportunity samples from two university Depart- Given increasing public awareness and demand, genetic counselling and testing ments was conducted to determine the influence of an education in human genetics for Alzheimer disease (AD) is often requested. especially for individuals with early- on knowledge and attitudes towards biotechnology and genetic engineering. The onset (age<60) familial AD where the pedigree is either consistent with autosomal dominant inheritance (FAD-Aut.Dom.-Subtype) or there is at least one other family results show that students with an educational background in biology and genet- member with early-onset AS (FAD). The following is a practical guide for genetic ics are more knowledgeable regarding biotechnology and genetic engineering., gene testing and counselling for symptomatic individuals: therapy and genetic screening and are more poitive in their attitudes towards new genetic technologies. Conversley a lack of human genetics education was associated with less knowledge of biotechnology and genetic engineering and less positive, less Early-Onset FAD- Early-Onset Aut .Dom.-Subtype FAD ploarised, and frequently more negative attitudes towards these technologies. _- _- _- _- _- _- _-_- _- _- _- _- _- _- _- _- _- _- -_ SYMPTOMATIC INDIVIDUAL Apriori-risk 50% empiric and pedigree analyses Test for APP Mutation Yes Yes Disclose APP Results, with Caveats Yes Yes IF NO APP MUTATION: Chromosome 14 Linkage(i) Yes Disclose CHR14 results, with Caveats Yes ALL SUBGROUPS EARLY-ONSET AD Longitudinal Follow-up Yes Yes Recommend Neuropathology Yes Yes Recommend DNA Banking (future gene(s), e.g. Chrl4; anonymous ApoE screening, etc. Yes Yes (1) Linkage depends availability of informative family members A316 Published Abstracts: Genetic Counseling and Genetic Education (continued) 1833 On the importance of genetics teaching in secondary school and college courses for all students. L.A. SUSCA. New York Medical College, Valhalla. 'tre have reported for at least ten years on the inadequacy of knowledee of genetics as it is tau.-ht to students in american schnoos. ever 500 students interviewed during past years as well as numerous talks with obstetricians emphasize that cenatics knowledge is poor in most of those populations. Recently we have interviewed close to 100 teachers in the field and they agree that students' knowledae. in the area is lack.na. While laboratory research in genetics is excellent,. it is ultimately tha consumer who will benefit from that research. Yet, the consumer knows little about the field except for that which appears periodically in headlines. We must instruct the american population at all levels if it is to understand the implications of genetics to its well being. Such implications include academic, religious, social and psyc-olovic considerations. Leaders of education at. all levels including law) should investigate curriculums in order to try to bring knowledge of the subject in adequate fashion ,o the a-erican people.

Published Abstracts: Genetic Epidemiology and Population Genetics 1834 1835 The epidemiology of Down syndrome in California, 1989-1991: Use of a clinical trials population in gentic researh P. Chan' C.W. prevalence, Impact of prenatal diagnosis, and sex ratios within a Shults2, C.M. Tanner', , J.S. Fink3, M. Kurth4, K.L. Marek3, L.W. Elmer' and racially diverse population. LK jishopl, C.AJ Hujj rl, C 2, and Parkinson Study Group. 'The Parkinson's Inst., Sunnyvale, CA;2Univ. Cal. San F.L&reX3. 1. University of Cincinnati, Cincinnati, OH. 2. California Birth Diego, La Jolla, CA; 'Mass. Gen. Hosp., Boston, MA; 'Barrow Neurol. Inst., Defects Monitoring Program, Emeryville, CA. 3. Genetic Disease Branch of New 'Univ. Ann Arbor, MI. California, Berkeley, CA. Phoenix, AZ; 5Yale Univ., Haven, CT; Mich., The study population in California consisted of counties which were monitored Genetics may contribute to the cause of the common neurodegenerative by the California Birth Defects Monitoring Program during 1989-1991. disorder, Parkinson's disease (PD). To investigate genetic risk factors or Objectives of this study were 1) to calculate prevalence and quinquennial maternal susceptibility genes for PD, DNA samples from a large, well-characterized age risk rates; 2) to estimate the impact of prenatal diagnosis services and cross-sectional population will be critical. Assembling such a group is costly, subsequentelective termination of affected fetuses on the observed prevalence; time consuming and difficult to fund. Patients participating in a clinical trial of and 3) to examine sex ratios of affected livebirths and fetuses within the resource for such and therapeutic agents represent a unique and efficient establishing population and racial subpopulations ofCalifornia. The racial diversity large this The Parkinson size of the population allowed the data to be stratified into five race categories-- a study population. We report an example of approach. Hispanics, Whites, Asians, Blacks, and Others. For 1989-1991, the modified Study Group, a consortium of 34 centers across the United States and Canada, total prevalence, which took into account the termination of affected fetuses used DNA samples from over 450 persons with early PD and enrolled in the following prenatal diagnosis, was 1.53 (per 1000 births). Among races, variation DATATOP (Deprenyl and Tocopherol Antioxidative Therapy ofParkinsonism) of modified total prevalence rates was primarily due to differences in maternal age clinical trial in 1987-88 (Arch. Neurol. 1989; 46:1052-1060) to establish a DNA structure. Hispanics and Whitesvwere the only races which displayed repository, housed at the Parkinson's Institute in Sunnyvale, CA. Regular significantly different quinquennial maternal age risk rates of Down syndrome, 1995. with Hispanics exhibiting higher rates in maternal ages <40 years. Overall evaluations of neurological function were performed through Serum, reduction of livebirths with Down syndrome in 1989-1991 was 26%, with 49% cerebrospinal fluid and urine samples are also available and a brain bank has reduction observed in maternal ages > 35 years. The impact of prenatal diagnosis been established. To our knowledge, this represents the largest collection ofwell varied greatly among races, indicating differential utilization of prenatal diagnosis characterized, prospectively followed PD patients from whom DNA has been services. In 1990-1991, Hispanics had the lowest overall reduction of 10%, collected. DNA from this repository is likely to be useful in investigations of while Whites had the highest reduction of 46% (71% for maternal ages .35 genes that may be associated with PD. Investigators interested in use of this were lower than those of livebirths years). Sex ratios of all livebirths significantly should contact Dr. C.W.Shults (Fax: 619-552-7513, e-mail: with Down syndrome, and race was found to have a significant effect upon sex repository ratios within both of these categories. cwshults vapop.ucsd.edu).

1836 1837 Heritability of risk factors for-coronary heart disease: the Framingham Is there a maternal effect in Multiple Sclerosis?. study. LA. Cuooles' R.H. Myers2JM rdovas3 pW.F. Wilson'4E.J. Schaefe_. D. Dvment'. A. D. Sadoynick!N. R. Rischr, G C. Ebers" and the Canadian Col- Boston Univ. Schools of Public Health' and of Medicine, USDA HNRC, Tufts laborative Study Group. University of British Columbia,' Stanford University,2 Univ. School of Medicine3, Framingham Heart Study4, Boston, Massachusetts. We examined the heritability of a variety of risk factors for coronary heart and University of Western Ontario.3 has been disease in the Framingham Heart Study. The data consisted of a cohort of 5209 The female preponderance in multiple sclerosis (MS) long recognized. men and women who entered the study in 1948 and their offspring (n=5124) who The female:male ratio in population-based studies approaches 2-1 and is even higher entered the study in 1972. We calculated the following correlations as well as in childhood MS. Family studies clearly show that offspring are at a higher risk if avuncular and cousin correlations using the FCOR procedure in S.A.G.E., there is a mother rather than a father with MS. for aender. aae. smokina. alcohol and estroaen theraov. edstina consumption As part of the Canada-wide Canadian Collarobative Study on Genetic Sus- Parent-Offspring Sibling Spouse ceptibility to MS, data were collected on 882 maternal half-sibs and 697 paternal Variable Correlation Correlation Correlation half-sibs of MS index cases. The age adjusted risk for MS did not differ significantly HDL Cholesterol 0.26 0.25 0.09 according to the gender of the common parent. LDL Cholesterol 0.21 0.24 0.02 These data therefore do not support any maternal effect on MS susceptibility. log Triglyceride 0.23 0.23 0.06 109 Lp(a) 0.29 0.39 0.08 Apolipoprotein Al (apo Al) 0.20 0.15 0.01 Apolipoprotein B 0.17 0.21 0.12 Systolic Blood Pressure (SBP) 0.17 0.21 0.02 Body Mass Index (BMI) 0.23 0.20 0.13 The biologically related individuals have higher correlations than do the spouses suggesting that there are significant heritable components in all these measures. All correlations for 2nd and 3rd degree relatives are similar to those of spouses except for Lp(a) for which the correlations remain similar to 1st degree relatives. The magnitude of these correlations suggest that a simple genetic model is not sufficient to explain the heritability of these traits. Rather, complex genetic models including the possibility of several genes and potential environmental interaction are implicated. Published Abstracts: Genetic Epidemiology and Population Genetics (continued) A317 1838 1839 Fragile X premutation Frequency in Northern Thailand Genetic correlations with human ageing. P.M. Frossard and G.G. Lestringant. Faculty of Medicine and Health Sciences, Al Ain, United Arab Emirates. A. Eigel, M. Zygulska, T. Dolscheid, N. Bogdanova, B. Dworniczak, F. Steger', T. Several investigators have demonstrated that the field of molecular genetics Sanguansermsri', G. Flatz, J. Horst provides invaluable tools to understand the biological basis of human survival Institut flir Humangenetik, Vesaliusweg 12-14, D-48129 Monster; *prof em. of and longevity. Human Genetics, Medizinische Hochschule, Konstanty-Gutschow-Str. 8, 30625 Han- We carried out an association study aimed at identifying correlations between nover, Germany, ' Chiang Mai University, Human Genetics Unit, Faculty ofMedicine, genetic markers and human ageing. To that end, we isolated DNA samples from Chiang Mai 50002, Thailand 667 unrelated subjects (352 men, 315 women) who had been referred to the U.C.S.F. Medical Center (San Francisco, CA) for lipid check-up; their ages FraX syndrome, the most common heritable cause of ranged from 3 to 84. We determined the frequencies of 45 restriction fragment neurodevelopmental disability, is (RFLPs) at 22 loci (atrial natriuretic peptides, ANP caused by inactivation ofthe FMRI gene; it occurs in around I in 1.200 male and in I length polymorphisms gene in 2.000 receptor, apolipoprotein Al-CIII-AIV, All, B. Cl and CII, alpha and beta-ATPase, female births. Inactivation is caused by expansion of a CGG repeat in the 5' FGF, beta-globin, insulin, LDL receptor, non-pancreatic lipase, phospholipase A2, untranslated portion of the FMRI transcript. In regard to the CGG unstable triplets at P53, renin, tyrosine hydroxylase and vitamin D receptor genes) in the subjects the FMRI locus, individuals can be divided into three groups according to the number pooled into four age groups: 3-29, 30-49, 50-69, 70-84. We also studied the of repeats: (i) about 6 to 50 are found in the general population, (ii) - 52 to 200 associated frequencies of apolipoprotein E alleles s2, E3 and E4. (-premutation) predispose female carriers to bearing children with fraX syndrome, Our data shows that 8 of the 45 RFLPs (18%) display statistically significant (iii) sizes over 200 (full mutation) exceeding 1.000 repeats in affected individuals. altered distributions in the different age groups (p<0.07); 3 RFLPs (BamHl at the Prevalence figures for the FMRI mutation in different populations are only rarely beta-globin locus, BgAI and Hphl at the renin gene locus) showed frequency available if at all. A few studies suggest frequencies for the premutation to be about I changes after age 70, 2 RFLPs (Sstl in APOC3, EcoRI in ANPR) after age 50, 1 in 500 to 1.000 X chromosomes. In the present investigation 1.075 X chromosomes RFLP (HindlIl at the alpha-ATPase gene locus) showed a constant frequency from 644 genetically unrelated individuals from Thailand (431 females and 213 males) increase with age, and 2 varied without coherent patterns. Sixteen other from families unselected for mental retardation or fragile X were analyzed by comparable trends were observed but did not reach statistical significance. Southern blot analysis for the presence of FMRl mutations. In addition, the exact While Q3 frequencies remained constant across the four age groups, E2 number of CGG-repeats was determined. Earlier findings have to be corrected. None frequency increased and e4 frequency decreased significantly after age 70. of the premutation size alleles could be confirmed; only three FMR-I alleles of bor- Although these results have to be interpreted with great care, they indicate derline size (52/54/54) were found, out of the 1075 chromosomes analyzed. that genetic components do contribute to the determination of human ageing and longevity. Supported by the Volkswagen-Stiftung.

1840 1841 Direct measurement of nucleotide sequence variation at a conserved Genetic Referrals of Middle Eastern origin in a western and a polymorphic exon in two different human populations. K. A. Hill Citys Inbreeding and Disaase Profile. Elizabeth Hoodfar. and S. M. Singh, Molecular Genetics Unit, Department of Zoology and Ahmad S. Teebi. The F. Clarke Fraser Clinical Genetics Unit, Division of Medical Genetics, The University of Western Ontario, London Division of Medical Genetics, The Montreal Children's Ontario, Canada N6A 5B7. (Intro. by: S. Sommer). Hospital and McGill University, Montreal, Quebec, Canada. It is now realistic to characterize the DNA sequence variation for defined regions of any genome and address many issues of population Inbreeding or consanguineous marriage is a common the traditional practice of the Middle Eastern countries with genetics using direct sequencing of PCR products. The total extent of Muslim majority. Studies from various countries and genetic variation is measured, which is ideal for the description of the rate and communities of the region showed that the frequencies range pattern of neutral nucleotide substitutions. The nucleotide sequences of the from 20% in Lebanon to greater than 70% among the Bedouins third exon (147 nucleotides) of the conserved Adh2 locus and the second in Kuwait. Inbreeding is known to have adverse effects on exon (197 nucleotides) of the polymorphic HLA-DQ81 gene were determined morbidity and mortality, in particular with respect to the for a random sample of individuals from two different human populations. The autosomal recessive disorders. This study examined 200 survey included 32 subjects from Southwestem Ontario (SWO) and 38 Dogrib couples representing all referrals of Middle Eastern origin individuals from the Northwest Territories (NWT) of Canada. The SWO seen at a large Clinical Genetics Unit in Montreal. They population has mixed ancestry, but is predominantly European, while, NWT were compared with a similar size group of different individuals have a common ancestry. All NWT individuals were homozygous cultural backgrounds from among the same referrals. The for the allele of the Adh2 as were two rate of inter-cultural marriages and inbreeding was found 131 locus, all but of the SWO individuals, to be 24% and 23.5% respectively among the Middle Eastern one of whom was heterozygous, possessing the B 1 and 132 alleles and the group while they are 22.5% and 5% among the comparison other, who was homozygous for the B2 allele. NWT and SWO populations group. Excluding the referrals for consanguinity only, the contain eight and twelve different HLA-DQ81 alleles, respectively. The two rate of inbreeding among the study group was 16.4%. Within populations differ in the type and frequency of HLA-DQ81 alleles. The alleles the Middle Eastern group, autosomal recessive disorders are at each exon in NWT and SWO populations are consistent with the different more than twice as common among the inbred than the non- -volutionary histories and structures of the two populations and the different inbred families, the pattern of which is consistent with selective forces affecting the Adh2 and HLA-DQ81 gene products. Relative previous observations. stability of the DNA sequences of highly conserved and highly polymorphic protein coding regions of the human genome is indicated by the lack of novel (i.e., intra-allelic) nucleotide sequence variation.

1842 1843 Allele and genotype frequencies of two hypervariable repeat sequence in Effect of Passive Smoking on GYP1A2 Activity in Twins. B.E. Llewelly. factor VIM gene among Singapore population. PS Lai.' I Fong.' TCOuah.' . Kalow2 B. K Tangs. A. Pandval. R.M. Schieken. J .1. Meyer. W.E G Duraisamy2 and JSH Tay.' 'Department of Paediatrics, National University Nance'. 'Medical College of Virginia/Virginia Commonwealth University, of Singapore, 2Pusat Perkhidmatan Darah, Hospital Besar, Kuala Lumpur, Richmond, Virginia; 2University of Toronto, Toronto, Canada. Malaysia. Individual variation in the inducibility of p450 enzymes such as CYPlA2 by polycyclic hydrocarbons is thought to be related to the risk Two types of hypervariable repeat sequences have been reported in factor of developing smoking related cancers. Formal genetic analysis of these Vm gene, a dinucleotide repeat sequence of the form (CA), in intron 13 and enzymes has long been impeded by the lack of reliable in vivo assay of the form (CA). (TC). in intron 22. -y nP-dATP end-labelled primer were systems. However, Kalow and Tang have shown how the activity of several used in amplification reactions to examine the variability of these repeats in p450 enzymes, including CYPlA2, XO, NAT2, and CYP2El, can be estimated genomic DNA from 327 unrelated individuals comprising 50 males and 59 from analysis of the observed pattern of urinary caffeine metabolites. females from each racial groups in Singapore, namely Chinese, Malay and To compare the causes for variation in p450 enzyme activity among A passive smokers and non-smokers, we measured the urinary caffeine Indian. total of six alleles with CA repeats (n) ranging from 19 to 25 was metabolites excreted by two groups of adolescent twins. One group found in the intron 13 dinucleotide polymorphism. Allele frequencies ranged included 20 pairs with one or more smoking parent; the parents of the from 0.002 to 0.557. In the intron 22 hypervariable region, five alleles of the remaining 20 were non-smokers. Each twin drank a Coke-Cola at bedtime order n=2 to 7 were observed with frequencies ranging from 0.006 to 0.774. and the relative amounts of 10 caffeine metabolites were measured in There was no significant difference in allele frequencies between the three their first morning urine by HPLC. The 10 x 10 metabolic covariance matrices of the passive different ethnic Chinese were to smokers and non-smokers showed a highly groups. females found be more heterozygoes _ for the significant differences (X2 127, 45df, p10 in CAMBODIAN 23% 2.6% KOREAN O.AS% .008% fold difference in 20-24 year olds. Closer racial rates the <20 group may be deceiving: Gastroschisis increases with CHINESE 0.4%.. .308% LAOTIAN 10.2% 1.4% lower age by year, so lower average black maternal ages in Numbers FIUPINO 0.5% .008% MIDDLE EAST 0.11% 0.00% merged data would bias aggregate rate comparisons. are small, but there may indeed be greater differences by HMONG 1.7% .12% THAI 10.3% 0.86% year in <20 age adjusted racial rates than aggregate data of . i VIETNAMESE 2.6% 0.86% suggests. Our findings of decreased rates gastroschisis in blacks are unexpected. Most factors associated with decreased maternal age should increase risks for blacks in All cases of HbE/Beta Thalassemia occurred in Southeast Asian groups, with the every age group. Still, factors that shift risks towards majority being Laoian. Beta Thalassemia Major (Cooleys) was distributed fairly evenly lower ages would be consistent with our data. Alternatively, among the groups, with the highest prevalence in Asian Indians (1 case per every 2600 genetic population differences may decrease susceptibilities births)and no cases detected among Japanese or Cambodians. The distribution of in blacks to events that potentially cause gastroschisis. homozygous EE among the groups does not conform with Hardy-Weinberg expectations

1846 1847 Molecular Genetic Diversity in North Indian Populations: Analysis of Recent trends in the prevalence of Down syndrome. P. Genetics Hu- Medeiros, Y. Alembik. B. Dott. M.P. Roth, C. Stoll. AMP-FLPs (DISSO and D17S30). S. S.lMastana. Human Lab., Institut de Pudriculture, Centre Hospitalo-Universitaire, man Sciences Department. Loughborough University. Loughborough. LEl1 3TU. 67000 Strasbourg, France. UK. Examination of data from our regional registry of Allele and genotype frequencies for two highly polymorphic amplifiable VNTRs. congenital anomalies indicated that 217 children with Down were determined in five Northern Indian syndrome (DS) were registered (liveborn, stillborn or ter- MCT118(DlS80) and YNZ22(D17S30) mination of pregnancy) to mothers living in the Strasbourg populations(Brahmins, Khatris, Jat Sikhs, Lobanas and Rajputs) by amplified- area from 1980 to 1992 inclusive. This represents a period fragment-length polymorphism(AMP-FLP) technique. The Lobanas untill recently prevalence of 1.25 per 1000 total births. From 1980 to lived as gypsies and had a highly conserved genetic constitution. Other populations 1987, 93 children with DS were registered (prevalence of are also endogamou groups of varying social and economic status. For MCT118, in 0.79 per 1000) whereas from 1988 to 1992, 124 trisomic 21 from to 780bp in size and from 0.005 to were identified (prevalence of 1.81 per 1000). In our all populations, 23 alleles ranging 340bp country since 1980, prenatal diagnosis is offered, free of 0.3 in frequency were detected in 488 unrelated individuals of these caste groups. charge, to all women being 38 years old or more. The The expected heterozygosity varied from 72% (Lobanas) to 78% (Jat Sikhs). The percentage of women in whom prenatal diagnosis was per- discrimination power of this locus showed interesting variation which ranges from formed was "en plateau" since 1986, around 60 per cent. No 0.88(Brahmins), 0.90(Jat Sikhs and Rajputs) and 0.93 (Khatris) screening program based on detection of makers in maternal 0.86(Lobanas), in our region. For YNZ22, a total of 14 alleles of sizes from 168 bp to 1078bp were detected in serum (AFP, hCG, U3) is available found A changing pattern of risk in relation to maternal age a sample of 470 unrelated individuals. The small sized alleles (1 to 5) were was identified. In the general population, women under 35 to be more frequent than larger alleles. The significant differences were observed years gave births to 91.7 per cent of all children in 1986 when the present populations were compared with previous studies on Caucasions and to 81.1 per cent of all children in 1990. From 1980 to of affinities of showed that molec- 1987 34.7 per cent of the children with DS were registered and Japanese. Overall data analysis populations 1988 to 1992 similar results as observed with conventional poly- in mothers older than 35 years of age, from ular polymorphisms provide this percentage was 46.3. From 1982 to 1987, 33 DS were morphisms. These findings demonstrate the potential utility of highly informative born alive from mothers over 35 years whereas from 1988 to hypervariable loci in population genetic research as well as in forenisc and paternity 1992, 25 livebirths DS were born from mothers over 35 determination. years although interruption of DS pregnancies was more frequent in women older than 35 years of age. The total number of liveborn DS born during these two periods of time was 74 and 84, respectively.

1848 1849 in elderly women. II an tribal from India. Familial aggregation of bone mineral density Physiogmy and life-style of Isolated population Nguyen J R Center.P N Smbrook.N AMonPJK Hy-JAEssan.Bone and B. Muralidhar. P.J.M. Rao and J.D. Goud. Laboratory of Human Genetics, Mineral Research Division, Garvan Institute of Medical Research, St Vincent's Hospital, Department of Zoology, Osmania University, Hyderabad-500 007, India. (Intro. Sydney 2010, Australia. Intro. by: Dr. G. Chenevix-Trench by: M. Bethi). Osteoporosis is a metabolic disease associated with loss of bone mineral density The genetic structure of Gutta Koya, a tribal group from Andhra Pradesh, (BMD), which ultimately results in an increased susceptibility to fractures. It has been twin studies that as much as 80% ofvariance of BMD could be attributable India, was studied by analyzing protein (blood) polymorphisms. The gene estimated from of to measure gene diversity along with various other to genetic factors. In this study, we aimed at detning the familial resemblance frequency data was subjected for environmental factors in elderly women. tribal groups of Andhra Pradesh, under hierarchical model, and the results have BMD while controlling specific of In this The Dubbo Osteoporosis Epidemiology Study is a longitudinal, community study been reported elsewhere (Am J Phys Anthropol 90:169-183, 1993). factors that contribute to osteoporotic fractures, involved more than 2000 men and women presentation, we like to show slides that vividly depict interesting physical features aged 60 or above, who lived in Dubbo, a city of 400 kan north-west of Sydney of this tribe and their simple mode of life-style. Majority of the men and women (Australia). BMD was measured as g/cm2 at the lumbar spine and femoral neck, by dual are slender built with typical proto-Australoid physical features. Most of the men energy x-ray absorptiometry with precision of measurement of around 1.5%. and some women wear loin-cloth just to cover their nakedness. This tribe is under We examined data from three groups of relatives: 48 pairs of sisters, 14 pairs of isolation for centuries and thus retained their unique physiognomy along with mothers-daughters, 17 pairs of brothers-sisters, who had complete medical history data cultural integrity. They live in thatched-roof homes in thick forest on steep high and baseline BMD, whose average age was 69 years (range: 60-86). After regression but adjustment for the variability explained by age, sex, weight, height and lifestyle factors by hills, where no other human population inhabits. They are settled agriculturists, in each of relatives has a mean of zero are at hunting and make baskets with multiple regression analysis so that.BMD group mainly depend on forest produce. They good and unit variance, we found that at the femoral neck BMD site, intraclass and its standard bamboo. They tic two large size baskets one on each side of a long bamboo stick was 0.37 ± 0.11 (r + SE; brother-sister pairs: error0.1(SE)+for.0.sister-sisterTeegrssonpairsoeficent(S) o dugher p)0.01),vesu mothers-wAsU.± and carry it on their for transporting the forst products to sell in the 0.12 ± 0.09. The regression coefficient (SE) of daughters versus mothers was 0.45 ± nearby plains. In conclusion, though the tribe is exposed to neo-culture and 0.19 (p

1852 1853 Genetic Structure of Miane Dora: A Tribal Population of Age at OnsetofAlzheimerDisease Is Influenced by multlpl g notic and non-genetic Andhra Pradesh, India. G.V. Ramana*, J.M. Naidu** and factorn: The MIRAGE Study V.S.Rao. L.A.CuDDs. E.J.Folev. and L.A.Farrer for the J.S. Murty.* Department of Genetics, Osmania University, Hyderabad, India ** Department of MIRAGE Study GrouD Boston University School of Medicine, Boston, MA Anthropology, Andhra University, Visakhapatnam, India. Molecular genetic studies of Alzheimer disease (AD) suggest that age at onset is Manne Dora, a small tribe inhabiting the north coastal determined in part by genotype. Epidemiological studies have implicated numerous risk districts of Andhra Pradesh, South India, has been studied for 24 genetic markers comprising blood groups, factors for AD, but the influence of these factors on age at onset is unknown. We plasma proteins and red cell enzymes. Genetic distance evaluated the effects of several genetic and non-geneticfactors In 831 consecutively cer- analysis was made to test whether this tribe was part tained and rigorously diagnosed AD cases in a muitl-center study called MIRAGE. The of another tribe Konda Dora and to understand its likelihood of carrying a major gone for AD was calculated for each subject using a divergence from other tribal populations of Andhra which Pradesh. The dendrogram and principal component Bayesian approach incorporates information on aggregation of the disease, ages at analysis reveal clustering pattern of the tribal onset and censoring ages among first degree relatives. Unear regressIon techniques were populations in close agreement with geographical employed to evaluate complex statistical interactions for onset adjusting for confounding distance. The study indicates that Manne Dora may have among risk factors. Subjects witht a likely genetic form of AD tended to have younger been genetically part of Konda Dora and seperated only recently. onset ages. While earlier onset was asociated with higher education, smoking and a prior history of depression, subjects with a positive history of arthritis and high blood pressure had older onset ages. Among heavy smokers who had a low probability of major gene for AD, younger onset ages were noted for greater alcohol consumption. Also, our resuits indicate that patients within the United States (both population- and clinic-based) had older onset ages than the non-American patients suggesting possible dfferene In the referral patterns and access to health care. Mechanisms leading to onset of symptoms may differ among individuals with and without a genetic predisposition to AD.

1854 1855 Lack ofan apoecatien between the 4 allele ofapolipoprotein E and Alzheimer Genetics and epidemiology of lib reduction defects in 6 disease in elderly Nigerians A. Sahota' . 0.Os.ntoku2 M Yan' S. L. Hui European countries. C. Stoll. E. Calzolari. M. Cornel S. F. W. UnMet4 K. S. Hall4 and H C. 'Departments ofMedical and Garcia-Minaur. E. Garme. N. Nevin. European Working Group Molecular Genetics, and on limb anomalies. Institut de Putriculture, Centre 'Medicine, LPyciaty, Indiana University School of Hospitalo-Universitaire, 67000 Strasbourg, France. Medici, s and ofMedicne, University ofIbada, Nigena. We have proposed a new classification for the As part of a comanty-based study of Alm diseas (AD) in an elderly identification and description of congenital limb populaio l~group fom Thdan we hav detemined ap ip E OE) reduction defects (LRD). This study was undertaken to genotypes on 56 sbects (39 subjects who were adjudged normal on cdnical evaluate the use of this new documentation for the study assessment and 17 subjects with dementia, includig 12 with AD). Dried blood spots of LRD in 6 European countries (France, Denmark, Northern we mailed from lbadan to Indianapolis and DNA was euted an Ireland, Italy, The Netherlands and Spain). using improved The total number of births including livebirths, procedure. This procedure (the dry method) involves incubating a 5mm dried blood stillbirths and terminations surveyed during 7 years by spot in 80 ul water in a 1.5 nl tubeoverght at 60-Cwith the cap open. Fift ul the 6 registries of congenital anomalies participating to water is then added and the tube incubated for 10 min at SOC. Half of the eluted this study was 611,150. The total prevalence of LRD was DNA is used for APOE genotyping by PCM The e4 alele of APOE was not 7.06 per 10,000 births including the following categories assoated with dementia or AD in the Nigerian population sample. The e4 alle prevalence terminal transverse, 3.97, longitudinal, fiequencywas 17.6% in demented 1.75, proximal-intercalary, 0.10, split had/foot, 0.54 patients and 16.7% in AD patients, compared with and multiple types, 0.70. The prevalence of LRD was 20.5% in control subjects. These findings are in marked contrast to the strong statistically higher in Basque Country, Odense, asocation between APOE e4 and AD in several Caucasian population groups and Strasbourg and Groningen than in Belfast and Emilia our previously reported study with Aflican Ameicans. We have also noted Romagna ; 6% of the cases were chromosomal anomalies, previously that the prvalence rate for AD for age 65 and over is lower in the including 15 trisomies 18 out of 26 cases, 49.5% (201 Nigerian population tand in the African American population (1.41% versus 6.26%). cases) were isolated LRD and 50.5% (206 cases) had other Diffee in major non LRD associated malformations. The recurrence expression of APOE or its receptors may account for the lack of risk was 1 in 33 and the precurrence risk 1 in 37. association between e4 and AD intheNigerian population Factors other thanAPOE This study demonstrated on one hand the usefulness of genotypes may be involved in the etiology ofAD in the Nigerian population. the use of the new classification for the study of LRD and on the other hand, a geographical difference in the prevalence of LRD in Europe. A320 Published Abstracts: Genetic Epidemiology and Population Genetics (continued) 1856 1857 and Malay popula- epidemiology of BandSynome in Cafonia. T-cell receptor B-chain RFLP in the Chinese, Indian Descriptive Amiodc 'T tions in Singapore. J. A. M. A. Tan, J. S. H. Tay and N. Saha. Department of 1,2M. M. Tolarovi 'J A. Harris and F. Bateson. iCalifngBirth Dect Paediatrics, National University of Singapore, Lower Kent Ridge Road, Singapore Monitoring Program, Emeryville, California; 2Center for Craniofacial Anomalies, 0511. University ofCalifornia San Francisco, San Francisco, California. Restriction fragment length polymorphism (RFLP) of the gene encoding the B- The amniotic membrane participates directly in the production of anoralities chain of the human T-cell receptor was studied in three ethnic groups in Singapore through several mechanisms: (I) strips of amnion separated partially or completely by Southern Blotting analysis. Polymorphism in the B-chain genes was identified from the amniotic membrane and suspended in the amniotic fluid may encircle digits or in Bg! II digested DNA samples using a 770 bp TcR cDNA clone containing the entire limbs compromising blood supply and thus disrupt growth and development of joining (J) and constant (C) region segments. The TcRB/Bgl II polymorphism limbs; (2) when bands of amnion still attached to the membrane are swallowed, the fetus becomes tethered and through continued swallowing effort, causes cutting of of 9.2 kb and 10 kb fragments were studied in 136 Chinese. 93 Indians and 88 crcum Malays. The frequency of the rare allele (TcRB'10 kb) in all the ethnic groups was orofacial and cranial structures; (3) in other the amnion may rupture, (0.46). Indians collapsing about the embryo or fetus and causing amniotic rupture sequence. significantly lower (0.15-0.29) (P<0.01) than that in the Caucasians characteristics in birth had a significantly lower frequency of the rare allele (TcRB' 10 kb) (0.13) compared The purpose of this study was to analyze epidemiological defects related to the pathology of the amniotic membrane as described above. The with the Chinese (0.29, P<0.05) and Malays (0.26). The genotype distribution of cases ethnic most common term ofAmniotic Band Syndrome (ABS) was used to classiy the the TcRB-chain RFLP was at Hardy Weinberg equilibrium in the three Birth II in the Chinese, Indians asid in our study Using population-based data compiled by the California Defects groups. Our study of TcRB/Bgl polymorphism with ABS were identified births be useful in the study of population genetics. Monitoring Program, 330 children from 2,221,755 Malays indicates that it may from 1983 to 1992 (prevalence"0.15 per 1,000 birts, i.e., 1 in 6,733 births). The majority of these children (88.18%) were liveborn. However, the prevalence among stillborns (2.66 per 1000) was more than 20 times higher compared to liveborn. In our study, ABS incidence was similar in males and females (overall sex ratio-0.94). However, a sex ratio of 1.67 was found in babies of mothers older than 30 years. Teen-age mothers of all races were at greatest risk of having a baby with ABS (rate=0.22, confidence interval (CI)'0.16-0.28) and at highest risk were White teen- age mothers (rate= 0 27, C00. 17-0.42). In Whites, the risk decreased with increasing maternal age In contrast, in Blacks and Hispanics there was a U-shaped relationship between ABS and maternal age.

1858 1859 Performanoe and allele frequeneies of the YNS22 and DiSC0 Populaton genetic charactenstics of human short tandem repeat vTyS in paternity testing. B. Whitfield1. M. J. DAsouki2, polymorphIsms and 3' UTR variants In a human diversity panel. IL and G Niblackl Gene Proof Technologies' and Vanderbilt WiI1 ,J juE IKH.tw2 VA-shffiidl. lThe University University of Iowa, Iowa City, IA. 2Fox Chase Cancer Center, Philadelphia PA. of short tandem elements has Variable number tandem repeats (VNTRs) are a powerful The generation markers based on repeat class of polymorphic genetic markers that are used in increased to thousands of markers since 1989. Since they are highly identity testing. Analysis of the allele frequency distri- polymorphic and widely distributed throughout the human genome, they btion at the VNTR loci detected by probes YNZ22(D17S5) and have been instrumental in creating high-density linkage maps. The NCT118(DlS80) in two populations (Caucasian and African majority of the loci that have ben well characterized have heterozygosity American) was completed using the polymerase chain but few studies have attempted reaction(PCR). A total of 15 segregating alleles were levels in Caucasians greater than 70%, detected at the D17S5 locus in a population of 2892 to define the population genetic properties of these markers. A pilot individuals, consisting of 1777 Caucasians and 1170 African study was designed utilizing markers generated by the Cooperative Americans. A total of 28 segregating alleles were detected Human Linkage Center (CHLC) and indivuduals representing a diverse at the MCT118 locus in a population of 3038 individuals, population base. Four classes of repeats were investigated in sample consisting of 1868 Caucasians and 1170 African Americans. obtained from the Coriell 4 was the most common allele individuals comprising ten human populations For the YNZ22 locus, allele were followed by allele 2 in Caucasians. Allele 3 was the most Cell Repository. In addition, a panel of nine non-human primates common in African Americans, followed by allele 4. At the investigated using primers designed from human sequence. STRP NCT1l8 locus, allele 24 was the most frequently observed markers thought to be contained within genes, and those in the 3' allele in both populations. Heterozygosity for the YNZ22 included in this We have studied and 87.56% in untranslated region, were also study. locus was calculated at 80% in Caucasians 30 GATA, 3 GGAAT and 14 3' UTRs to date for a total of 47. 22 of 27 African Americans. At the MCT118 locus, heterozygosity was and calculated 79% in Caucasians and 87% in African Americans. markers also amplified in one or rsore of chimpanzees, gorillas The mean power of exclusion for YNZ22 was 60% and 76% for orangutans. Results showed rno deviation from heterozygosity values -bbth populations respectively, and for MCT118 was 58% and found in Caucasians. These results suggest markers found in Caucasians 73%. are likely to be equally useful as genetic markers in most other populations and many primate species as well.

1860 A improved metod for duting DNA from dried blood spots and its applcaton to apollpoproteia E genotyplag in Albhelmer disease. 1 yal H C. Heie' K. S. Hall2 B. 0. Osuntbkun' and A. Sahotal. 'Departments of Medical and Molecular Gentics and 2Psychiaty, Indians Universi School of Medicine, i and r t ofMedicie, University ofIbadan, Nigeria We are coctng a longitudinal std on the prevalence and incidence of Aizbeimer disease in two elderly populaion groups of African origin, one from Indiaapos and the other from Ihadan. A major part of this study is the demInationofapoipoproteinlE (APOE) genotypes in these groups Wehaveused liquid blood samples frm the Indinapis subjects for th purpose, but it has not been posi to obtain liquid samples in good conditon fom the badn subjects because of logscal prblems. To overcome these problems, we have developed a simple procedure (the dry mehod) for eluting DNA from dried blood spots (DBS). A 5mm disc from aDBS was cut into four pieces and placed in a l. ml tube. Eighty ul water was added and the tubes incubated with the tops open in a 60C heating blockuntil thewater had evaporated (usuayyfourhourstooveniglt). Another 50 ul waterwas added and the tubes incubated at 80C for 10 mi. Halfofthe duted DNA sohton was used for APOE genoypg by PCR The dry method was mnch more effective in eluting DNA for PCR amplification fiom DBS compared with the previously descibd boiling and direct methods. Ninety-five perct of the 236 samples tested to date have been successflly typed for APOE by the dry method, wheas only nine samples out of20 eluted by the boiling method gave unambiguous genotypes. Identical genotypes were obtained from DNA eluted by the dry method and that obined from liquid blood smples. The dry method could also have applicatons in other screeng or diagnostic situations. Published Abstracts: Inborn Errors of Metabolism and Biochemical Genetics A321 1861 1862 Newborn Screening for Homocystinuria. H.K. Berry. Children's Hospital Medi- Isolation and characterization of mouse cDNA cal Center, Cincinnati, Ohio. fusion-proteins that Interact with human In 1981 the frequency of homocystinuria identified through newborn screening was estimated to be I in 200,000. Subsequently, rate of detection of homocys- beta-amyloid precursor protein. S.L. BRESSLER. tinuria in countries that had screened at least 200,000 infants was reported to be M.D. GRAY. B.L. SOPHER, M.G. H-EARN. 0. HU. K.-I. I in 332,000. FUKUCHI AND G.M. MARTIN. Alzheimer's Disease A survey to provide current information on affected patients was carried out Research of Box by contacting 20 states screening for homocystinuria and approximately 300 in- Center, Department Pathology, dividuals in metabolic centers. Four of 9 states responding reported screening a 357470, University of Washington School of Medicine, combined total of 923,200 newborns without having found an affected infant. Seattle, WA 98195-7470. Five states found 20 affected infants among approximately 21,000,000 infants The of Fields and who were tested. yeast two-hybrid system Song Data were received on 17 subjects: 10 identified between 2 and 5 days and 7 (Nature 1989, 340:245-246) as modified by between 2 and 6 weeks. Two of these had normal tests on first screening Hollenberg and co-workers (Cell 1993, 74:205-214) specimens and were identified following a mandatory second test. Low- was used to screen a mouse embryo cDNA library for methionine diet, usually accompanied by vitamin B6 was initiated at ages ranging proteins that interact with the C-terminal 106 amino from 10 days to 4 months. None were responsive to vitamin B6 alone. Symptoms in subjects identified through newborn screening are descried and acids of the human beta-amyloid precursor protein compared with those reported for 13 subject identified because of symptoms. (bPP). 2-3 million clones were screened and eight It seems likely that most infants are tested before the rise in plasma positive clones were identified. Presently, we are methionine is sufficient to permit detection of homocystinuria. If so, and if the incidence is as great as I in 200,000 to 300,000 mahy infants with homocys- isolating the genes that code for these proteins in tinuria may be missed by newborn screening to be recognized later on the basis both mouse and humans. These bPP-interacting of symptoms which led to the first description of the disease: detached lens, proteins are being further characterized according to skeletal abnormalities, and thromboembolic episodes. a variety of genetic and biochemical criteria in order to evaluate their relative importance in the pathogenesis of Alzheimer's disease as well as to further investigate their potential roles in the complex biology of beta-amyloid.

1863 1864 Fibrillin immunofluorescence in "rule-out" Marfan syndrome. M. Godfrey1, Medium Chain Acyl-CoA Dehydrogenase Deficiency: Autopsy J. Cisler'. S. De Bie5. L.J. Kuglerl, K.J. Andersonl. and A. De Paepe2. I Univ. Ne- Findings In 26 Children AXKafolla. J. Butts, C.R. Roe. Duke University braska Med. Ctr., Omaha; 2Univ Hospital, Gent, Belgium. Medical Center, Durham, NC. Chief Medical Examiners Office, State of Since the first reports of abnormal fibrillin in the Marfan syndrome (MFS). there has North NC been a Carolia, Chapel Hill, great deal of interest in the possibility that fibrillin immunofluorescence (IF) may Medium chain acyl-CoA dehydrogenase deficiency (MCADD) is an be a useful diagnostic tool. Individuals were referred for fibrillin IF for "rule out" MFS autosomal recessively inherited disorder of beta oxidation of 6-12 carbon between January 1992 to May 1995. None of the patients had a clear clinical diagno- characterized recurrent and sudden death. The sis of WFS. Patients with MFS who were referred for or fatty acids, by hypoglycemia linkage mutation analyses and of this was to define and the in the patients with other known heritable connective tissue disorders were not included. A purpose study emphasize variability of in an to total of 200 patients, 107 males and 93 females ranging from neonates to 70 years of age autopsy presentation MCADD attempt improve its post-mortem were studied. Patients were referred by 75 physicians from 55 centers in six countries. recognition, preventing the unnecessary death of subsequent siblings. Direct fibrillin immunohistochemistry was performed on 14 micron skin sections from Thirty five patients with a postmortem diagnosis of MCADD made 155 patients. Fibrillin IF in hyperconfluent fibroblast cultures was performed on 190 between January, 1988, and December, 1994, were identified by review of patients. Both studies were performed or are in progress on 141 patients. The clinical lab files. Autopsy reports were available for review on 26 patients, ranging parameters tested were: Skeletal -dolichostenomelia, pectus change, scoliosis, arachn- in age at death from: 3 days -5 years (mean = 15 mos). Post mortem odactyly, high arched palate, joint laxity, joint dislocation. pes planus; Ocular - ectopia analysis was requested because of: newly diagnosed sibling (10), lentis; Cardiovascular - aortic dissection, dilatation. aneurysm. regurgitation, or insuf- parental request (7), SIDS protocol (6), history of hypoglycemia (1) and ficiency. Preliminary statistical correlations were performed using Pearson Chi-Square fatty liver (2). All deaths occurred prior to diagnosis of MCADD. Twenty five analyses. We determined the association between abnormal fibrillin IF, skin or fibroblast patients (96%) had fatty infiltration of the liver, eleven (42%) had fatty cultures, and major clinical features of MFS. The latter being: 1) dislocated lenses: 2) infiltration of the kidneys and 9 (35%) had fatty infiltration of the heart. aortic abnormalities (dissection, dilatation, and/or severe regurgitation); 3) skeletal in- Cerebral edema was found in 18 patients (69%). Only one child did not volvement. Skeletal manifestations were considered present if at least three of the above have gross or microscopic evidence of fatty infiltration of the liver. Fatty noted findings were present. Slight positive correlations were found between abnormal liver appears to be the most reliable marker for MCADD. Biochemical fibrillin IF. skin or clutured fibroblasts. and dislocated lenses and aortic abnormalities. A testing for fat oxidation disorders should be performed in all cases of slight positive association between abnormal fibrillin IF in fibroblast cultures and skeletal sudden unexplained death in childhood accompanied by fatty changes in abnormalities. These studies may show the utility of fibrillin IF analysis in some cases of internal "rule out" MFS. organs.

1865 Biotin Kmestimation in intact holocarboxylase synthetase (HCS)- deficient fibroblasts. C.E. MizclM i.J.enjetL. T.P. Le2. B.A. BarshoAk W.L. Nvhan2 O.N. lagan I. Depts. of Peds., 1Southwestern Med. Ctr., Dallas, TX and 2Univ. of Calif., San Diego, La Jolla, CA; 3Metabolic Screening Lab, Shaare Zedek Med. Ctr., Jerusalem, Israel; 4Baylor Res. Inst., Dallas, TX. HCS deficiency (< 20 reported patients) results in defective biotin linkage to inactive apocarboxylases. HCS deficiency is detected by 1) direct assay monitoring biotin incorporation into various substrates (not routinely available) or 2) normalization of carboxylase activities in cells subcultured in high biotin (which does not estimate biotin Km). We tested an intact-cell, biotin-titration method to identify HCS deficiency and estimate biotinKm. Control and HCS-deficientfibroblasts (n=2 each) were cultured in low biotin (2.7 nM) medium, harvested and washed, and equally seeded into duplicate flasks containing biodn-depleted (BD) medium (generated by twice chromatographing low biotin medium over excess avidin-agarose). BD medium was supplemented with0-410 nM biotin, cells grown to confluency and harvested for standard carboxylase assays. Control fibroblasts revealed stationary carboxylase activities at all biotin levels(see figure for typical results), while patient fibroblasts showed with intact-cell value of 25 ± 7.7 nM ± biotin-saturable carboxylases Km (mean SD, range 15-38, n=6 for 2 3 carboxylases, 2 cell lines) : compared to9nM (mean; con- > < M trol 5 nM) for the same cell 3 IL _ lines using direct HCS assay. If biotin uptake and/or trans- 1 port is not rate-limiting, then ; I our method provides another -0- PAMENT useful approach to study HCS- 5 A CONTROL deficiency. The mild biotin Km d elevation may be consistent 0 with the late age-of-onset of 150 200 300 4;0 500 our patients (20 & 21 months). KOTN(in) A322 Published Abstracts: Linkage Mapping and Polymorphisms 1866 1867 Linkage mapping of zeta crystallin on human chromosome ip: application to Cefnti ofc toa ofVan Der Woude gene to lq32-41 rega S PlAgbiL hereditary cataracts. L , ' 5J 2'j1 .is,'Lfr j, A. Qgahger D_ Hoover' A- Dobi' T. Eowler S- Tinle K. Br~ennn W K berting! 3mand LE. AIhe.4D epm t ofOphthalmology', University ofColorado, Healt Univei ofNebrakaMedical Center,Pediatric Dentiatty,Meyer Reb oiln Institute, Sciences Center, Denver, CO 80262; Jules Stein Eye Institue?, Department of Omaha, NE and Boys Town National Research Hospital, Omaha, NE. Ophthalmology', UCLA, Los Angeles, CA 90095; Department ofPediatrics, University Van der Woude syndrome is an autosomal dominant craniofiil disorder with high ofCalifornia, Irvine, CA 92613; Division ofGenetics,' Vanderbilt University Medical penetrance (98%) and variable expression. The hallmark ofclinical findin are: cleft lip, Center, Nashville, TN 37232. deft paramedian lower lip pits, and hypodontia. This syndrome accounts for 2°/.of Zeta (Q crystalis is a minor component ofthe human lens, but is the predominant all cleft and palae (Schinzel, 1986). crystallin in the guinea pig lens. A mutation in the 4-crystallin gene (CRYZ) has been The gme hasbeen localized to region Iq32-41 (Murray et al, 1990). The purpose ofthis shown to caue autosomal dominant congenital cataracts (ADCC) in guiea pigs. Hence study was to verify previous findings in our fiuly and fiather refine the localization. CRYZ is aviable candidate gene for human ADCC, which is genetically heterogeneous Our study population consisted of60 individuals with 18 affecteds in five generations (a Using RFLPs, we previously reportedpotentiallikagebetween ADCC and CRYZ in two single kindred). Thirty ml. venous blood from antecubital vein was collected under IRB unrelated families. approval and signed consent by the subjects. Lacldng more infomnative CRYZ polymorphisms, we constructed a multipoint linkage DNA was extracted by an automated 340 nucleic acid extractor, and DNA gopes map ofhuman chromosome lpin the region surrounding CRYZ in order to facilitate the were determined by PCR amplification according to the method ofWeber, et al. (1989). study of CRYZ in cataractogenesis. We amplified segments of CRYZ by PCR and Mikrosaelliterepeat markers covering more than 43 eM of diromsune Iq were used. Two screened for single-strand coor on polymorphis (SSCPs). We identified a two point linkageanalysis resulted in positive LOD scoresfor 3 markers, DIS205 (Zmax = 3.79, allele SSCP in intron 1 and genotypes CEPH families in order to construct a mutipoint theta = 0), DIS414 (Zmax = 2.05, theta = 0), and DIS245 (Zmax = 3.62, theta = 0). Marker D1S414 is 1 cM distal to DIS205 and DIS245 is 2cM proximal ofDS205. Our linkage map. CRYZ is most tightly linked to MCAD ("-0.02 at z=12.99) and lies within on a 4 cM interval between DlSS00/D1S465 and D1S464/DlS481. We analyzed the two results confirm the previous findings that the gene is closer to DIS205. Racombina his potentially CRYZ-1inked ADCC fiamilies with the flanking microsatellite markers Usig been deted with othermarkers. We are expandingthese results to refine thelocalization multipoint analyses, CRYZ could be excluded as the disease-causing gene in both ofthe gene with newly ascuined families and new markers. families. These CRYZ-inked markers permitted the exclusion oflinkage between the disease loci and CRYZ in three additional unrelated ADCC families The localization of CRYZrelative to other markers on human chromosome lp augments the human linkage map and enhances our ability to examine the role ofCRYZ in cataractogenesis as well as in other inherited disorders mapping to this region.

1868 1869 Homozygosity mapping of a new form of HMSN: autosomal Halley-Hley ie Subloe: lizatlon ofthe gene onchromosme 3q and recessive Hypermyelinating Neuropathy. A. Bolinol. V. idesitlfiatsof one Idndred with a 3q deletion. M . Jr Brancolini2- A. Quattrone3. A. Qambardella3. G. RomeoI. and M . Peluso k Ike"a Zhilan Hu Ervn Department of Devotol.2. 1Istituto G. Gaslini, Genova, Italy; 2Columbia University, New Dermatology, San Francisco General Hospital, University of California, San York, N.Y.; 3Facolti di Medicina e Chirurgia, Catanzaro, Italy. Francisco. We described a large Italian pedigree with Hypermyelinating We have reported previously that tew gene for Hailey-Hailey (HE Disease is Neuropathy showing an autosomal recessive inheritance. The affected located on chromosome 3 in the approximately 14 cM interval between D3S1589 members have an early onset motor and sensory neuropathy with 4 rather severe clinical course, in which progressive distal and (cen) and D3S1316 (tel). The maximum combined two point lod sore in proximal weakntess. foot deformities and scoliosis as well as severe limb families was 14.60 (8=0) at D3S1290. Further analysis of an additional 11 deformities are observed. The six patients belonging to the seventh familes identified recombinations with D3S1541 in an unaffected 33 year old generation are related to each other through multiple consanguineous individual. These recombinations localize the HE gene so a region less than S cM marriages starting from an original couple of founders. To assess and D3S1541 whether the mutation segregating in our pedigree is allelic to any of in size between D3S1589 (cen) (tel). the other HMSN loci, we performed a linkage study using markers In one of the 15 families non-Mendelian inheritanc was observed with the from the following four candidate regions: 17p11.2 (CMTIA), Iq22-q23 microsatelite markers D3S1541, D3S1587 and D3S1292. Affected individuals in (CMTIB), 1p35 (CMT2A), and 8ql3-q23 (CMT4A). Standard two point this family appear to be homozygous and to inherit allels at these loci only from linkage analysis yielded significant negative lod scores. A search for the unaffected parent and never from the affected parent, whereas unaffected mutations in PMP22 (the CMTIA gene) and PO (the CMTIB gene) members show Mendelian inheritance. Alleles at the flanking loc D3S1589 (cen) produced negative results. Taking full advantage from the particular from (Mendlian). Thes data are structure of this pedigree, only five patients have been typed with 362 and D3S1290 (tel) are inherited both parents Gdndthon's markers, distributed throughout the genome. In this way, constent with the affected chromosome having a deletion so that patients transmit we have identified only two markers on chromosomes 4 and 10 at null alleles, and this interpretion is confirmed by FISH data indicating which all five patients are homozygous for the same allele and three heterozygous loss of D3S1292. markers on chromosomes 12, 14, and 18 at which the five patients If the production of an altered prtn determines the autosomal inheritance share nine out of 10 possible haplotypes. These five candidate regions (i.e. a dominant negative mutation), it is likely that the Hailey-Hailey gene will be further screened using additional markers and including the pattern remaining family members. crosses the breakpoint ofthis deletion.

1870 1871 S. An investigation into Genetic and Behavioral Aspects of Dyslexia. Genetic heterogeneity of atrial septal defects ((D Roonn .Lynnec. m Contrino A. Young de et INSERM U-393, DM. Bradway (1.31. J.S. Ramforth (2). A. (1) R. Sid I.Kach A. Mmlich)) Dipartment Pediatric Unite (3). BE. Ginsburg (3.4) and M. Sarfarazi (1) (1)-Surgical Research H0pital des Enfants Malades, Pars, Frane. Center, Department of Surgery, University of Connecticut Health Atrial septal defects (ASD) of ostium secundum type with or without Center, Farmington, CT; (2)-Edmonton Genetics Clinic, University of atrioventricular conduction defects are alinost always sporadic. However, autosomnal Alberta Hospitals, Alberta, Canada; (3) -Departments of Psychology and dominant inheritance is possible in both types of ASD (MIM 108800 & 108900 (4) Biobehavioral Sciences, University of Connecticut, Storrs, CT. resctively). Recently, we and others reported on the mapping of a gene for Holt-Oram Developmental dyslexia affects between 3-15% of all school aged children. of this condition is complicated as result of syndrome (HOS) to the distal long arm of chromosome 12 (12q23-q24). Since ASD is Diagnosis of of extensive variability that exists in patient populations and lack the most frequent cardiac defect in this rare cardio-skeletal disorder and several lines are aiming to clarify heart standardized classification and definition. We evidences support the co-localization of syndromic and isolated congenital the definition and/or diagnosis of this condition through the study diseases (Williams syndrome and supravalvular aoric stenosis, DiGeorge association of perceptual/motor difficulties, by positional mapping and cloning and conotruncal cardiac anomalies), we investigated HOS and familial ASD for allelism of the defective gene(s). We have so far ascertained 12 Canadian (131 subjects; 62 affected) and 3 American families (141 subjects; 50 at the HOS locus. Patients were classified as dyslexic individuals and relatives to two non affected) with this condition. Fifteen affected 38 belonging consanguineous if their ability was 2 years below the level as In one only reading multiplex families were tested for linkage to chromosome 12q23-q24. family, predicted by their age and IQ. Due to the reported genetic linkage ASD was isolated and in the other. ASD was associated with progressive atrio- of this condition with DNA markers from Ip34-p36 and an apparent ventricular block. No radial ray defect was observed in either families excluding the association of a balanced translocation of lp22;2q31 with dyslexia are in this condition. we genotyped our families with 12 diagnosis of HOS since skeletal deformities fully penetrant and delayed speech development, of markers to this STRP markers from lp region. Similarly, due to an earlier report Linkage studies using microsatellites DNA mapping region gave disorders in dyslexics and lodscores in the 22cM interval an increased incidence of autoimmune significantly negative pairwise (<-2) genetic reported linkage of this condition with DNA markers from 6p21 region encompassing the HOS locus. Haplotypes study supplemented multipoint analysis by (i.e., site of the HLLA system), we genotyped our families with 11 providing direct evidence ofrecombinant events and confirmed that the gene responsible markers that span the 6p21-pI2 region. As yet, we are unable to of these markers for ASD in these families is not linked to the HOS locus. conf irm or refute the reported linkage relationship the of heart septal defects. with the dyslexia locus. This may be attributed to small family These results underline genetic heterogeneity meioses, heterogeneity the view that at least two a crucial role in atrial size, lack of sufficient informative genetic Moreover, they support genes play are currently our diagnostic and/or misdiagnosis. We refining with septation during embryogenesis. criteria by developing a perceptual/motor test that correlates the dyslexia phenotype. This is in accordance with previous reports of coordination difficulties in dyslexic patients. Published Abstracts: Linkage Mapping and Polymorphisms (continued) A323 1872 1873 Genetic susceptibility to multiple sclerosis: the role of MAG, MBP, Familial Hemiplegic Migraine: identification of a second locus. Anne MOG, OMGP and PLP. R. ue'. D. Ph Dinh2 0. Boespflur- Dlucros1, Anne Joutel1, Katvoun Vahedi1/3, Antonio Ereima2, Evxlynn M. Lathropt. J. J.L. J. Oksenberg. Tanuy5 D. .E. Goodkin4. Rimmlcer. Haings7. Dsrnad2, Michaelle Cecillon', Alain Verrier2, Marie-Germaine Bousser3 and C. Fizamesi. R. Lincoln'. M.A. Pericak-Vance6. J. F. Gusella!. A. D. Roscs . J. Elisabeth Tournier-Lasservel. 1: INSERM U25, Facultd de Midecine Necker, Weissenbach' .A. Dautign. S.L. Hauser'. E. Seboun'. 1) Gindthon, 1 rue de Paris, France; 2: Centre Hogitalier, Lens, France; 3: Service de Neurologie, lInternationale, 91002 Evry Cedex, France; 2) URA 1488 CNRS, 9 Quai Saint- Hopital Saint-Antoine, Paris, France. Intro. by A. Munnich. Bernard, 75005 Paris, France; 3) INSERM, U384, Faculte de M6decine de Clermont-Ferrand, France, 4) UCSF, San Francisco, CA; 5) H6pital Saint-Louis, 75010 Paris, France; 6) Duke University, Durham, NC; 7) Molecular Neurogenetics unit, Mass. General Hospital, Charlestown, MA. We previously mapped the first gene responsible for Familial Hemiplegic Multiple sclerosis (MS) is a polyfactorial disease characterized by scattered areas Migraine (FHM) on the short aim of chromosome 19 and demonstrated the genetic of demyelination in the central nervous system. Because myelin destruction is heterogeneity of this condition, 50% of the families being linked to chromosome thought to be due to an aberrant immune response in genetically susceptible 19. We hypothesized that the other FHM gene(s) would belong to a common gene individuals, we evaluated the role of MAG, MBP, MOG, OMGP and PLP/DM20 family or would be implicated in a common metabolic pathway. Therefore genetic genes in a panel of 52 MS multiplex families. MAG is located on chromosome 19 mapping of other FHM genes and comparative analysis of cloned genes within and two microsatellites markers flanking the gene are respectively 100 kb and 400 these distinct regions may suggest strong candidate genes for this condition. kb away from it. MBP is located on chromosome 18; a 4 bp tandem repeat is In order to identify a second locus for FHM, we selected a large family present between the promoter region and exon 1. MOG is located on chromosome unlinked to chromosome 19 (28 living members including 12 affected), and 6, close to HLA-F. Two polymorphic microsatellite sequences are located within conducted a linkage analysis by a sequential search covering the whole genome. the gene. OMGP is embedded within the NF1 gene located on chromosome 17. A total of 207 markers have been analysed so far. We have been able to Two microsatellites are located 2 and 20 kb upstream from exon 1. PLP is located exclude several chromosomes, including chromosomes 7, 19, 20, 21 and 22, and on chromosome X and contains a microsatellite sequence. Physical mapping data build an exclusion map covering 62% of the total genome. allowed to locate MAG on the Gtnethon genetic map. All the other genes were Several positive lodscores were obtained with 6 markers spanning a 30 cM mapped on the G6ndthon genetic map by linkage. For each gene, at least one marker region and 2 recombinants were identified with 2 markers bracketing a 2 cM located within the gene and two flanking markers located 1cM away from the gene interval.There is no additional marker within this interval and in order to furteher were analyzed. Genetic analysis was based on the sibpair method. Preliminary confirm linkage to this locus, we are currently analyzing five additional unrelated results suggest that MAG, MBP, OMGP and PLP do not play a major role in MS FHM families unlinked to chromosome 19. Results will be presented at the susceptibility; however, the role of MOG could not be excluded. meeting.

1874 1875 Microsatellite allele frequencies and heterozygosities in a large set Demonstration of founder effect in carbonic anhydraseIl deficient Arabic leads to the identification of a hot of Caucasian, non-Hispanics in the United States. II. J. Edenberg.' patients spot recombination site in chromosome 8 q 21. D.M. Fathallah M Bejaoui. & K T. A. Goate 2 and J. Indiana University School of Medicine. Indi- Foroud.' Rice2. Uelagi. Institut Pasteur & Charles Nicolle Hospital, Tunis, Tunisia anapolis IN' 1 and Washington University School of Medicine, St. Louis, MO.2 Microsatellite markers are increasingly important in genetic studies. Better data Six Tunisian families with a history of osteopetrosis, renal tubular acidosis, on the allele distributions and observed heterozygosities of these markers in different cerebral calcification, mental retardation and carbonic anhydrase II deficiency, populations are therefore valuable. previously characterized by mutational analysis were studied to test the hypothesis that the unique mutation ( A splice junction at the CA II gene exon 2-intron 2 As part of the Collaborative Study on the Genetics of Alcoholism, a large, boundary) shown to underly the CAU deficiency in all the families arose in a markers are on a set of one family-based study. microsatellite being typed nearly common ancestor originating in the Arabic Peninsula. The segregation of a Taq I thousand individuals in 106 families across the UiS. From this growing dataset bialleic restriction marker upstream of the CARl gene with the mutation was studied we have identified about 180 unrelated. Caucasian, non-Hispanic founders in 82 by STS analysis in all the families members and showed a linkage desequilibrium families. of the Taq I (-) allele with thc mutation in 4 famillies out of 6 indicating a founder effect within this An cthno-historical to trace all the We report the allele distribution and observed for more population. study permitted frequency heterozygosity families to a common arabic tribe that setteled in the Maghreb in the 10 th century than 20 markers and compare them to published values Observed heterozygosi- thus confirming the common ethnic origin of these families. The genetic analysis ties are within 10 Although there are fewer African-American families in the of the two remaining famillies showed that a recombination occured between the dataset. we will also present the allele frequency distributions and test for racial Taq I marker and the CAll gene mutated site. Parentage assessment in all the heterogeneity. families was performed by DNA fingerprinting using VNTR probes. Given the small size of this population sample, the observed high recombination These data are for the establishment of for genomic important screening panels frequency suggest the presence of a hot spot recombination site within this region surveys. We find unreported alleles, typically at the ends of the size distribution. of chromosome 8 (8q2 1). for most markers. These can affect the choice of markers for multiplexing

1876 1877 Two large South African families of diverse origins with Parametric linkage analyses of diabetes mellitus in Pima ADIP genes on 17p and 17q. R.G. Goliathl. S. Bardienl. Indians. RL Hanson. S Kobes. DB Thompson. RC Janssen. M S. Bhattacharya2. R. Ramesarl and J. Greenbercil. 1 Univ. Prochazka. WC Knowler. NIDDK, Phoenix, AZ. of Cape Town, South Africa, 2 Department of Molecular Genetics, Institute of ophthalmology, London. Segregation analysis suggests that a major gene may Retinitis pigmentosa (RP) is a retinal degenerative influence the risk of diabetes mellitus in Pima Indians. disorder which can be transmitted as an autosomal Sib-pair analyses have revealed evidence for linkage dominant (AD), autosomal recessive or X-linked trait. (p = 0.01) of diabetes to chromosome 7 (D7S479) and of The AD form has to date been shown to be linked to 2 linkage with metabolic precursors of diabetes to chromosomes genes and 6 anonymous loci. The two most recent ADRP 1 (D1S198), 3 (GLUT2) and 4 (FABP2). These observations genes, on 17p and 17q, have been localized in two large were extended with parametric linkage analyses of diabetes. South African (SA) families, from different origins. The analyses included 140 Pima Indian pedigrees with Four retinal specific cDNAs have been localized to the genotype information in a maximum of 1114 individuals. 17p region. The recoverin gene has been excluded by Parameters for inheritance of diabetes with variable age of linkage analysis using an intragenic CA-repeat onset were defined on the basis of previous segregation microsatellite marker. The pigment epithelium-derived analysis. The LINKAGE program was used to calculate the factor (PEDF) gene, could not be excluded from linkage maximum lod score (ZMAx) and to estimate the recombination due to the uninformativity of an intragenic RsaI RFLP. fraction (0). PEDF as well as the guanylate cyclase (GC) and Lod scores were low for linkage of diabetes with D1S198 phosphatidylinositol transfer protein gene cDNAs (PITPN) = 0.0, 0 0.5), FABP2 = 0.0, 0 - and are currently being screened. (ZMAX - (ZMAX 0.5) GLUT2 (ZMAX - 0.1, 0 0.3). Weakly positive lod scores were Candidate cDNAs at the 17q locus include the gamma observed with D7S479 - 0.9, 0 = 0.2) and with a marker- phosphodiesterase gene which has been excluded based on (ZMAX 5 cM telomeric, D7S662 = 0.9, = 0.1). By the HOMC'G an intragenic detected SSCP (ZMAX polymorphism using analysis. program (A-test), there was no evidence for heterogeneity The tissue inhibitor metalloproteinase 2 and the protein (proportion unlinked families = 0.0). kinase C are screened. To alpha genes presently being These that of a date, no disease mutations have been analyses suggest tight linkage major causing yet disease with GLUT2 or identified in of these retinal candidate susceptibility gene D1S198, FABP2, any specific these markers linked with diabetic genes. YAC-based of the critical despite being precursors, physical mapping is to the observed of diabetes regions to refine localization of the two SA RP loci are unlikely explain segregation in Pima Indians. The lod scores for the on being undertaken. markers chromosome 7 are consistent with the the sib-pair analyses, and this area deserves further investigation. A324 Published Abstracts: Linkage Maipping and Polymorphisms (continued) 1878 1879 GenTink: a database resource for human genetics. C. ms 1, S. Steinbreck 1, L Occlusion Mapping of Autosomal Recessive Primary Congenital Glaucoma Lii 1, Q. ggggly2, H. Deonis-Kller. 1,3 Division ofHuman Molecular (tuphthalmos). A. Hossain. AN. Akarsu. ME. Turacli (1). SG. Aktan Genetics, Dept. Surgery, 3Depts. of Genetics and Psychiatry, Washington Univ. (1). M. Barsoum-Homas (2). L. Chevrette (2). BS. Sayli (3) and M. School of Medicine, 2Dept. ofChemistry, Washington Univ., St. Louis, MO, USA. Sarfarazi Surgical Research Center, Department of Surgery, A working prototype version of GenLink has been available over the internet University of Connecticut Health Center, Farmington, CT; (1)- since May 1995, and new developments are underway. GenLink provides linkage Departments of Ophthalmology, (3)-Medical Biology and Genetics, mapping information and software that will facilitate map integration and positional University of Ankara Faculty of Medicine, Ankara, Turkey and (2)- cloning projects. GenLink is comprised of a set of databases, including a genotypes Department of Ophthalmology, University of Montreal, Quebec, Canada. database, where public genotype information is stored. Users wishing to submit Primary Congenital Glaucoma (PCG) is a pediatric eye disorder genotype data and to perform linkage mapping experiments will find a user interface that is inherited as an autosomal recessive trait in the majority of designed to provide easy access to the data. GenLink information will include families studied. Association of congenital glaucoma with various detailed meiotic map graphics with links to marker information. Index maps of all the abnormalities of chromosomes 1, 2, 3, 4, 6, 7, 8, 9, 10, 11, 12, 13, human chromosomes made available through GenLink will be periodically updated. 14, 16, 17, 18 and 21 has already been reported in the literature. In addition, GenLink will include published map information, and enable the Most of these patients presented with multi-system abnormalities that comparison of maps in a meaningful way, including the ability to query a database of include congenital glaucoma. Recently, we have excluded major map discrepancies. The database will also include genotypes verification candidate regions on lq21-q31, 6p21, 6p25, 9p24-pter, lip, and 11g as information. Another associated database, TelDB (see Helms et al. abstract from this likely sites for the PCG locus. In our continued effort to map the meeting), provides a comprehensive telomere resource, including published PCG locus, we studied a series of STRP markers from certain region of information for all species. Another key resource in GenLink will be a set of meiotic chromosomes 2, 3, 5, 7, 8, 12, 14, 16, and 20. A group of 19 mapping panels for the human genome that will be constructed using the BINS pedigrees consisting of multiply affected children (99 meiosis: 44 program package developed by Dr. Pam Fain and colleagues. We will develop an affected and 55 unaffected) and 1st-cousin consanguinity in most of interface to BINS which will assist users in constructing meiotic mapping panels. the parental generation were selected as our initial screening panel. Other services GenLink provides includes an FTP site for the distribution of Polymerase chain reaction (PCR), denaturing polyacrylamide gels and information and publicly available software packages, and an electronic bulletin Silver staining were used to genotype our families for linkage board for discussion of mapping issues. The GenLink database will reside in evaluation. Lod scores were calculated under the assumption of GemStone, an object-oriented database management system. The database will have autosomal recessive inheritance and full penetrance. we have so far links to other related databases including Genome Topographer (GT). Genome genotyped our PCG panel with a total of 117 PCRable DNA markers and Database (GDB), and the Cooperative Human Linkage Center (CHLC). Another excluded those chromosomal regions that have been reported to be series of interfaces to the data will be built using the object-oriented Smalltalk associated with the PCG phenotype, including regions of 6p, 9p, llp languagc, enabling researchers to access additional data and share data from and l1q. Therefore, the apparent reported association between PCG collaborative projects using the GenLink server. The prototype version of GenLink is and candidate regions of these chromosomes are either coincidental or available on the WWW server over the internet due to the genetic heterogeneity between different populations. (http://www.genlink.wustl.edu). Supported in part by AHAF-NGR, FPS-NSPB and Knights Templar Eye Fnd.

1880 1881 Linkage analysis of Palliator-Hall Syndrome. S. Kang'. J. M. A gene for X-linked infantile spinal muscular atrophy (a severe lethal Graham2. Jr., M. Abbo 3. and L. G. Biesecker1. 1. Laboratory of Genetic form of X-linked arthrogryposis) maps to Xpll.3-qll.2. H. Kobayashir, Disease Research, NCHGR, NIH, Bethesda, MD, 2. Division of Genetics, L-BaumbachU, T.C. Matise#, A Schiai. F. GreenbergS, E.P. Hoffman*. - U'ni Cedars-Sinai Hospital, Los Angeles, CA, 3. Dept. Psychiatry, Johns versity of Pittsburgh School of Medicine, a University of Miami School of Medicine, Hopkins University, Batimore, MD. # Columbia University. $ National Institutes of Health. We have initiated efforts to dissect the phenotypes, pathophysiology, X-linked infantile muscular et al. 1988) is a rare dis- and embryology of malformations of the brain and limbs. One member of spinal atrophy (Greenberg this family of disorders, Pallister-Hall syndrome (PHS), is characterized by order showing hypotonia, areflexia, and multiple congenital contractures (arthro- , hypothalamic hamartoma, imperforate anus, and other visceral gryposis) associated with loss of anterior horn cells and death in infancy. Elec- anomalies. Although originally described in sporadic cases, we and other trornyography and muscle biopsy findings are consistent with autosomal recessive have found it to be inherited in an autosomal dominant pattem. We are spinal muscular atrophy (Werdnig-Hoffman disease). We have ascertained four X- analyzing 4 autosomal dominant pedigrees that contain 13, 22, 2, and 4 linked families (Three N. American, one European), and have used one of these affected persons, respectively. The family with 22 affecteds manifests for linkage analysis. Seventeen members of an extended kindred (Greenberg et al. variable polydactyly and hypothalamic hamartoma. All obligate 1988) were analyzed using CA repeat markers spaced approximately 10cM on the heterozygotes manifest polydactyly. The hypothalamic hamartoma is X chromosome. A meiotic breakpoint analysis (concordance analysis) based on present in 5/6 individuals that have been evaluated. We have performed shared of the founder X chromosome was successful in localizing the X- on these regions genetic linkage studies to candidate chromosome segments linked infantile muscular gene to In this region, the families based on a previous report that showed a 3p;7q translocation in a spinal atrophy Xpll.3-qll.2. patient who has malformations similar to PHS. This child had premaxillary highest two-point lod score was found with DXS991 (Zmax = 2.63. theta = 0.00). agenesis with microphthalmia, cleft lip and palate, agenesis of nasal In multipoint linkage analysis covering the entire X chromosome, only the region septum. He did not have polydactyly. PHS families were analyzed by PCR- defined by MAOB and DXS991 showed positive lod scores, and all other regions based microsatellite genotyping with the polymorphic markers D7S486, showed negative lod scores. These data establish the first gene mapping assignment D7S483, D7S550, D7S559, D7S594, D3S1307, D3S1270, D3S1211, and of an X-linked lethal form of human lower motor neuron disease. D3S1300. Our results do not support linkage of PHS to these regions. We conclude that the translocation patient is etiologically distinct from familial PHS. This result is consistent with reports of hypothalamic hamartomas in a number of other disorders, demonstrating that they are not specific to PHS. We will present our results of the linkage analysis and the clinical evaluation of the largest pedigree.

1882 1883 Linkage analysis of the MNS and esterase D chromeosmal regions with alcohol Homozygosity mapping of an autosomal recessive Osteogenesis dependence. D. L. Keller. T. Foroud. P. M. Comeall. T. Reed R J. Edenber Imperfecta. Founder effect In the Atikamekw Indians. M.Labuda and ad T.-KI Li Departments of Medical and Molecular Genetics, Bioclemity and F. H.lorieux. Genetics Unit, Shriners Hospital, McGill University, Montrdal, Molecular Biology, and Medicine, Indiana University School of Medicine, Qudbec, Canada. Osteogenesis imperfecta (01) is a congenital disease that affects connective tissues, Indianapolis, IN. with the bone brittleness as a hallmark. This condition is phenotypically and geno- 223 pedigrees containing 2645 individuals ascertained via the Consortium on the typically heterogeneous with a variety of subtypes, and is usually inherited as a dominant Genetics of Alcoholism study were analyzed for linkage to the regions of the MNS trait. We have recognized seven patients with autosomal recessive 01 who belong to a blood group (4q25-31) and erythrocyte esterase D (13ql4) loci for which suggestive complex pedigree of the Atikamekw Indians ofQuebec. This disease is presumably due evidence has been reported. 1803 persons were genotyped for MNS, and 1912 for to a founder effect. Identifying a cause underlying this disorder is important for etcase D. All available family members were evaluated independently for alcohol understanding 01 forms other than the typical dominant ones. Itwill give us an insight dependence by the DSMHR and Feiglner criteria. 917, 925, and 1131 individuals into the mechanism of skeletal development and might eventually contribute to the were classified as affected using the criteria ofDSMl alcohol dependence, Feiglner treatnent of the bone impairment The candidate i COLlAl, COLlA2 and COLSAl, COLSA2 (encoding the major and minor bone collagens, respectively) as well as that of definite alcoholism, and Feighner probable alcoholim, respectively. Nonparamtic with linked markers. We anya was u the affected sibling pair and affected ostonectin were excluded by segregation analysis undhtook a lige performed (ASP) tri- or tetra-nucleotide on the pooled DNA of or either genome-wide screening by typing repeats pedigre member (APM) methods. No evidence for linekge using DSMIIR affected individuals to look for loci ofreduced heterozygosity, since the region adjacent Feighaer diagnostic criteria was found with MNS or esterase D. The overall mean to the disease is expected to be identical by descent in patients resulting from the same proportion of ailes shared identical by descent (IBD) for sibling pairs affected under founder. Only loci that express reduced heterozygosity among affected individuals were each of the thre diagnostic criteria was <0.50 (corresponding p-values >0.5). tested in all family members. Out ofthe 116 markers typed on DNA pool ofpatients, 20 Anals using the APM method did not suggest linkage under any of the diagnostic were further examined but no evidence of linkage to the disease locus was obtained. criteria with either of these markers. 937 individuals in 105 of these pedigrees were Among 116 markers tested in the pedigree with the affected individuals being offspring for higy microaateilite markers in the chromosomal regions of 9 parents related as first, second or third relatives, most of the loci were represented genotyped polymorphic we that around MNS and esterase D. Markers D4S1625 and D4S1629 4 cM by 3 or more alleles. Because of the high degree of allelic diversity conclude (approximately to in this It is cost and 15 cM from the MNS locus, respectively) and Dl3S765 and D13S318 (each homozygosity mapping is an appropriate approach pursue study. very effective and may be applied to rae diseases where the probability of allelic approximately 15 cM from esterase D) were used These 4 markers show no evidence is low. Two hundred more markers in order to criteria ASP and APM heterogeneity are being typed presently for linkage with the three diagnostic using analysis. cover the whole genome. We acknowledge Research Genetics Inc. for DNA typing. Published Abstracts: Linkage Mapping and Polymorphisms (continued) A325 1884 1885 for Usher I Linge and haplotype analyses of bipolar affective disorder in the Old Evidence for a fourth locus responsible syndrome type (USID). Q S D T V Der Kaousfan3. Order Amish. M. C. LaBuda K. Otten2. J. A. Eldand3. C. Allen3, LaMRg-Pig, ,rbe1, 1,M Mathllu2, D. L. Pauls4. and D. S. Gerhard', 'Dept. of Psychiatry and Behavioral Sciences, A Munnichl and * n de GOntique et Unltd de Recherches sur les Handicaps Gdndtiques de l'Enfant, INSERM U. 393, Paris France. 2 CHRU Johns Hopkins University Medical Institutions, Baltimore, MD, drnAmiens, Paris, France. 3 Mac Gill University, Montrdal, Quebec. 'Depts. of Genetics and Psychiatry, Washington University School of Medicine, Purpose: Thie aim of this study was to emphasize the existence of a fourth locus St. Louis, MO, 3Dept. of Psychatry, University of Miami, Miami, FL, rsponsible for Usher syndrome type I in addition of the three previously reported 4Child Study Center, Yale University School of Medicine, New Haven, CT. loci: USIA on chromosome 14q32.2, USIB on chromosome 11q13.5, USIC on chromosome 11p15.1. Methods: Six affected individuals and eight healthy relatives belonging to two Geec linkage analyses have been completed on 281 loci (data from 225 loci consanguinous families were ascertained, one of Maroccan origin and the other of ar unpublished) as part of a comprehensive genome search for genetic Turkish origin. For each of these 14 individuals, a 20 ml EDTA blood sample was polymorphisms linked with bipolar affective disorder in the Old Order Amish collected. The minimal criteria for diagnosis of US1 have been described elsewhere and the six affected probands fulfilled these criteria. For genotyping these families, (OOA). Standard pedigree lod-score analyses were conducted using 162 members we used the same DNA markers as those previously reported to analyse the three of the OOA of which 125 had DNA available for genotyping. Three diagnostic loci already known to account for USI. schemes were used and analyses were conducted assuming an autosomal dominant Results: Consistently negative lod-score values were obtained for aI markers mode of inheritance with an age-ependent penetrance (maximum penetrance exploring the three chrom regions. Multipoint linkage analyses showeta large exclusion of around 40 cM. 45 cM and 30 cM for USIA, USIB, USiC regions = 0.5). No significant evidence for linkage was obtained however there were respectively and haplotype analyses confirmed the unambiguous exclusion of the several markers throughout the genome showing small positive lod scores. Given three already reported tod for Usher syndrome type 1. the power of linkage disequilibrium mapping in genetic isolates, extended Conclusion : Here, we report genetic exclusion of the three bl previously reported for USI and suggest the existence of at least afourth locus (USID) in two haploqypes of 5 distantly related and clinically homogeneous individuals were Turkish examined. Two chromosomal regions (9q34 and 9ql3-q22.3) contained several families of Maroccan and ancestry. loci resulting in small positive lod scores in the pedigree analyses and were therefore the site for initial linkage disequilibrium analyses in the OOA. Preliminary analyses indicate no significant haplotype sharing among the affected individuals however there exist large untyped regions 25 cM distal and 21 cM proximal to the marker producing the most positive lod score. Additional marker typings in this region are currently underway and other chromosomal regions ae being examined.

1886 1887 Natural clinic and genetic history of rd ultiple exotoses. L Lgaj- A new protocol to confidently screen for mutations using SSCP and 1-P. nn , P MarotawxA u M La Merrrl. 1 Service de keteroduplex analysis. T. Lian. N. Udar. S. Dandekar. S. Sheikhavandi. GUndtiqueMan jtMddlcae et Unite de Re su; es Handches G&Atue de R.A. Gatti. Dept. of Pathology, UCLA, Los Angeles, Califonida. rEnfant INSERM U. 393, Paris, France. 2 Unitb de Recherches drEpidrnimlogie Single stranded conformational polymorphism (SSCP) and heteroduplex G6ndtque INSERM U. 155, Paris, France. fomnation are widely used methods to quickly screen for mutations and Hereditary multiple exostoss Is a autosomal dominant affection polymorphisms. Efforts to screen for mutations in new and unknown regions characterized by pnce of multiple cartilage capped exostoses with shortness using these methods are often frustraing because all too often the bands and raely sarcomatous den n. The prevaln of the disease is communly visualized are unvaryng in their migration pattern through the gel. The lack estimated at 1 in 100 000 but some authors argue for an incomplete penetrance of of difference between these bands may be due to no mutations being present in the dise especially in females. Three localizatons were described 6q24.1 the DNA sequence or to sub-optimal running conditions of a gel that fails to (EXTI), 11 can (EXT2) and 19p (EXT3). In a pel of 29 pedigrees, the a prn resolve mutated strands from the normal strands. A simple and inexpensive Dbability to belong to either of the localizations was estimated by linkage study. approach was developed to resolve this problem. Positive controls are created Twenty-eight per cent of the families were linked to chromosome 19, % to by incorporating site-directed mutations into primers for amplifying a region chromnosome 8 and 28 % to chromosome 11. A possibility of a fourth localization tat was designed for was excluded. For each family, an a probabiity of linkage with EXTI, must be analyzed for mutations. A specific mutation EXT2, EXT3 was esmated. In 19 famls, thl probability to be Inked to one of the only one primer from a set of PCR priners. The PCR product for the was more 70 % (9 families were linked to EXTI, 5 for EXT2 and 4 for EXT3). astificial mutations is used as a standard along with the PCR fragments being However in 11 familbs, the values were too low to conclude. analyzed. Thus, if subtle differences can be observed in the mutated controls, From a clinic study of 177 affected proposants, our estimation of the the gel conditions are considered satisfactory for detecting unknown mutations. peneance and the frequency ot the disea was calculated. These data together This protocol gives the researcher a higher degree of confidence in with the linkage sudy should aliow us to perform a correlation btween genotype interpreting results. and phenotype with a likely variation of penetrance for each locallzadon.

1888 1889 Asigment of Pmp22 and PO, candidate genes for Charcot-Marie-Tooth (CMT) Generation of a swine chromosome 6 specific library by microisolatlon. 1LX dises type IA and 16, to rat cir. 10q22 and 13q24-26 by FISH MendiolgS. Amba'X2 L u n A.A. Paszekl. F. A. Ponce de Le6f2. and C.F. T. Lishr. A Ekici. R. Hillenbrand'. M. Schacner'. B. Rautenstua, Institute of Lguils. 'Univ. of Minnesota, St. Paul and 2Univ. of Massachusetts at Amherst. Human Getics, Schwabachanlag 10, D- Similarities in gastrointestinal and cardiovascular physiology make swine an Fiedrich-Akexnder-University, including obesity. Our goal is to and 'Institute of Neurobiology, ETH H , CH- excellent genetic model for human disease processes 91054 Erlangen, F.R.G. identify and localize swine genes which regulate traits such as fat deposition and 8093 Zuerich, Switzerland growth rates. This is facilitated by the availability of over 600 swine genetic markers, Hereditary motor and sensory neuropathies (HMSN), synonymous to CMT the majority of which are highly polymorphic microsatellite markers. Swine disease, are a clinically and genetically heterogenous group of slowly chromosome 6 has been suggested to carry genes important for fat deposition and progredient disorders of peripheral nervous system. CMT type 1A is linked growth due to the localization of the gene causing Porcine Stress Syndrome (PSS) on to chr. 17p11.2 comprising te PMP22 gere, type 16 is linked to chr. 1q22 chromosome 6. PSS is the range of symptoms shown by certain breeds of pigs which including the myelin protein zero PO. To carry out the chromosomal assignment exhibit an increased likelihood of sudden death in response to stressors such as by FISH of the corresponding rat genes we used cDNA probes. Rat Pmp22 transport, restraint, fighting or exposure to certain anesthetics. In addition, PSS animals are well known for their low fat content and high degree of muscling. The clone CD25 (kindly provided by H. Mueller, University Duesseldof, F.R.G.) with molecular defect responsible for PSS in pigs, and malignant hyperthermia in some an insertion of 1.8 kb and PO cDNA (kindly provided by G. Lenke, Salk Institute, human families, has been shown to be a point mutation in the skeletal muscle to M. Schachner, Neurobiology, ETH Zuerich, Switzerland ) with only 0.75 kb sarcoplasmic reticulum calcium release channel (ryrl) which is located on swine probe size were used. The chromosomes were derived from rat call line Ratl. chromosome 6. While the mutation in the ryrl gene has been demonstrated to cause About 50 metaphases have been analyzed each. As our results indicate Pmp22 PSS, the associated low fat, high muscling phenotype is thought to result from allelic maps to rat chr. 10q22 and PO to rat chr. These results are in good variation at distinct, but linked, loci on chromosome 6. Additionally, another loci has 13q24-26. been identified on chromosome 6 which regulates growth rate and muscle concordance with investigations indicating homology between the development. To more precisely map these loci which regulate fat deposition, corresponding rat and human chromosomal regions. By means of FISH it is muscling and growth, additional genetic markers are needed on swine chromosome 6. possible to map even small cDNA probes quite exactly. This method should be We havc. therefore, used chromosome microisolation techniques to generate a used more intensively to complete especially the poorly inveigated genetic chromosome 6-specific population of DNA inserts from which microsatellite markers card of the rat can be derived. 10 copies of swine chromosome 6 were scraped from metaphase Acknolaedgement: We thank H. Mueller and G. Lemke for the cDNA probes, spreads and amplified by adaptor-PCR. The purity of the microisolated population T.L holds a Herbert Quandt fellowship, M.S. is supported by the Swiss national was verified by fluorescence in situ hybridization. The adaptor-PCR product was ligated into lambda-ZAP vector and has been screened for (GT)n microsatellite science fund, B.R. holds a grant of the DFG. markers. To date, 10 microsatellite markers have been derived from the microisolated library and 4 have been genotyped on a reference family. All 4 markers map to sw ;e chromosome 6. This work is supported by USDA NRI grant 58-1265-3-073. A326 Published Abstracts: Linkage Maipping and Polymorphisms (continued) 1890 1891 Mapping the mouse gene for primary carnitine deficiency. K. Okita, J., The assignement of the locus for Meckel syndrome with multiple con- T. Tokino,' H.Nishimnori.1 H..Nikaido.2 J.Hayakawa.2 A.Ohno, M.Kuwajima,3 genital malformations. P. Paavola, R. Salonen, 2 J. Weissenbach and Y.Matsuzawa,3 and Y.Nakamural. Lab. Molecular Medicine. I.M.S. Univ. Tokyo, L. Peltonen'. National Public Health Institute, helsinki, Finland,' Helsinki Uni- Tokyo, Japan,' Institute of Experimental Animals,School of Medicine.Kanazawa versity Hospital, Helsinki, Finland2 and Genethon, Evry, France.3 University,Kanazawa,Japan.2 Second Department of Internal Medicine.Osaka Uni- Meckel syndrome (MS) is an autosomal recessive malformation syndrome leading versity Medical School,Osaka,Japan3. to death of the fetus in utero or shortly after birth. The hallmarks of the syndrome Carnitine is an essential cofactor for mitochondrial fatty acid oxidation by trans- are occipital meningo-encephalocele, very large kidneys with multicystic dysplasia, porting long-chain fatty acids as acylcarnitine esters across the mitochondrial inner cystic-fibrotic changes of the liver and polydactyly. Although a rare disease, MS membrane. Carnitine deficiency results in failure of energy production and accu- has been reported in most parts of the world and it represents a significant part mulation of triglyceride in tissues including skeletal and cardiac muscles, and leads of the genetic congenital malformation syndromes with severe neural tube defect. to recurrent episodes of coma and hypoglycemia, chronic skeletal muscle weakness In Finland MS is relatively frequent with an incidence of 1-9000. As in most of and cardiomyopathy. Juvenile visceral steatosis (JVS) mouse is an animal model of the malformation disorders the molecular pathogenesis of MS is still completely systemic carnitine deficiency, and jvs is inherited in an autosomnal recessive mode. unknown. Here we report the assignement of the gene for MS to a defined 13-cM For a positional cloning of a gene responsible for JVS, we have performed linkage chromosomal region by random genome screen. analysis on 190 back cross progeny, using microsatellite markers. We were able to map the jvs gene to a region of approximately 1.6cM on mouse chromosome 11. Furthermore. we have covered this region with three YAC clones.

1892 1893 Linkage analysis of autosomal recessive Juvenile parkinsonisen A cDNA clone sharing homology with ceruloplasmin maps to the X to GTP cyclohydroase 1 gene locus on chromosome 14 chromosome: A candidate for occipital horn syndrome?. R. Sakthivel, M Saito', Hailme Tan', Atsushl Ishikawa', Sholi TWuAi' C. Kunsch*. J. Harris. C. Venditti and M. Chorney. The Penn State College of 1 Department of Neurology, Brain Reserch Institite, Nilgata University, Medicine. Hershey, PA and *Human Genome Sciences. Inc., Rockville, MD. Niigata, Japan 2 Department of Neurology, Nishi-ojiys Byoin National During the course of our studies designed to clone the hemochromatosis (HFE) Sanatorium, Niigata, Japan gene, we have adopted a positional candidate approach as one of our avenues of investigation. Based on this approach, we chose to initially characterize a par- Juvenile pakionsonism (JP) is defined as pakinsn manifestations beginning tial cDNA clone which demonstrated a marked homology to human ceruloplasmin before the age of 40. In contrast to the situation in western countries, approximately (Cp). This decision stemmed from the fact that mutations and deletions in the 10% of patien with idiopathic parkinsonism fall under the category of JP sn Japan. Cp gene, which cause aceruloplasminemia in some humans, result in a general- On the basis of mode of inheritance and clinical features includin# diurnal fluctuation ofsymptoms and sleep benefit, we have proposed that juvenile parkinsonism ized hemosiderosis with iron accumulation occuring in a variety of tissues including with autosomal recessive inheritance (AR-JP) is a distinct entity among those classifical brain. As opposed to HFE's map position at 6p2l.3, the Cp gene has been shown as P. to reside on chromosome 3 while a processed Cp pseudogene has been shown to On the hereditary progressive with fluctuation otherhand, dystonia mared diurnal a the clone on the (HPD) shows similar clinical symptoms mainly consisting of foot dystonia, diurnal reside on chromosome 8. Analysis of hybrid panel places Cp-like fluctuation of symptoms and similar clnicopharmscological responce to L-dopa. X chromosome. Based on Southern analysis, homologous sequences were found to It has been suposed that common abnormalities in pathophysiology can exist between be present in both mouse and hamster genomes. Overall, the novel Cp-like clone JP and HPD. possessed slightly less than 50%c amino acid homology to Cp; a stretch of 10 amino Recently we have discussed that GTP I is the causative gene for cyclohydrolase was to conserved in both This motif was reiterated three HPD. To investigate if AR-JP is allelic to HPD, we performed linkage analysis in 12 acids found be sequences. families with AR-JP. These families are characterized by: autosomal ressesive times in the Cp cDNA sequence while a less conserved stretch was also observed to inheritance, mean age of onset of 27.8 ±9, foot dystonia. marked effect of L-dopa, be reiterated in the factor VIII gene. Sequence homology to Cp as well as expression diurnal fluctuation of symptoms and alleviation after sleep. studies suggest that the novel gene may a role in copper metabolism; Linkage analysts were perfonned with 9 microsateilite markers (D14S69,75, 79, Cp-like play status as a 47,52,66,63,77,43) flanking GTP cyclohydrolase 1 gene. Summed lod scores for 12 further consideration of the clone's candidacy gene potentially responsi- families excluded linkage to the gen. It is considered AR-JP locus is not closely linked ble for milder forms of X-linked copper overload, such as that observed in occipital to chromosome 14q, and AR-1P seems to be another clinical entity. horn syndrome, are discussed.

1894 1895 Hunm M-ICR gene is located at chromosome 23 band qll in Genetic Linkage Study of Adult-Onset Primary Open Angle Glaucoma. the great ape. R.V. Samonte K.H. Ramesh and R.S. Verma. Diliana Stoilova. Anne Child (1). Ivavio Stoilov. Rez& R. (2). and Hansoor Sarfarazi. Long Island College Hospita-MY health Science Center at Seohatoleslami. Pitts Crick Surgical Brooklyn, New York 11201. Research Center, Department of Surgery, University of Connecticut Svolutionary divergence associated with point mutations has been Health Center, Farmington, CT; (1)-Department of Cardiological United and (2)- an important part of the speciation process and views concerning Sciences, St. George's Hospital, London, Kingdom human descent remain elusive. The chromosomal basis and most recent International Glaucoma Association, King's College Hospital, London, findings based on highly polymorphic repeat DNA sequences have United Kingdom. created conflicting views concerning evolution. We wish to narrow Glaucoma (POAG) is a common ocular condition the gap by deciphering the phylogenies of the hominoid species at a Primary Open Angle of the nerve and molecular level by using a single gene M-BCR probe, present on presenting with a characteristic degeneration optic with an elevated chromosome 22qll in humans to establish whether convergence or visual field loss that is often associated to the chromosomal location divergence of this gene has occurred in the great apes. intraocular pressure. In order identify we 15 with mid to late of Ape chromosomes were obtained from fibroblast cell lines (Coriell of Adult-onset POAG selected pedigrees age consisted of cell repositories, Camden, NJ) of chimpanzee (AGO 5253, Pan onset in 2-4 generations. Our initial screening panel 78 There troglodytes, PTR), gorilla (AGO 5251, Gorilla gorilla, GGO) and 131 individuals (72 affected) providing a total of meioses. in of the studied in this orangutan (AGO 6217, Pongo pygmaeu6, PPY) using the standard are no skipped generations any pedigrees a we have used di-, tri-, procedure. Human chromosomes (Homo sapiens, HSA) were prepared from panel. Using candidate gene strategy, blood lymphocytes from normal tetra- and penta-STRP markers from numerous regions of the genome. phytohemagglutinin-stimulated Silver staining individuals. The FISH-technique was employed with minor PCR amplification, denaturing gel electrophoresis and the POAG families. modification. For localizing the M-BCR region in the great apes, of the DNA fragments were utilized to genotype out with the KLINK the digoxigenin-labeled human M-BCR DNA probe (Oncor, Gaithersburg, Two-point linkage analysis was carried program for an autosomal dominant condition with full MD) was used. (FASTLINK version) lod has been obtained for The human M-BCR DNA probe hybridized to human chromosome 22 band penetrance. A significantly negative score more than 25 candidate genes and many other loci that harbor qll while in the chimpanzee, gorilla and orangutan the hybridization analogous genes. Amongst the candidate genes excluded are a- signals were detected on chromosome 23 band qll; the equivalent of Collagen 2A, 3A, chromosome 22 band ql. The conservation of the human M-BCR Spectrin, Fibronectin, Desmin, a-Crystallin, (1A, human Fibrillin, slastin and we have (22qll) at the corresponding locus (23qll) in the great apes has 4A, SA, 6A), 6-Tubulin, s-Spectrin. of these families to 1q21-31 region, indicated that the gene is conserved. While our earlier results also excluded linkage Adult-POAG the reported site of Juvenile-POAG. Genetic linkage study of [Verma and Luke: Mol Gen Genet 1994;243,369-3731 suggest that the and other STRP markers is currently in position of the human ABL protooncogenc was at a non-corresponding additional candidate genes chromosome locus, thus revealing the evolutionary divergence of progress NIH and Glaucoma Association. human chromosome 9. Supported by (EY-09947) International Published Abstracts: Linkage Mapping and Polymorphisms (continued) A327 1896 1897 SSCP polymorphisms in the human tumor necrosis factor receptor type I Comparison of two sampling methods for linkage study of Alzheimer's disease using affected sibpair methods. EW Talor. I Xu. DAMey. gene (TNFR1) on chromosome 12pl3. C. Tasasra. F. J. Rosenberg. and J. M. The Johns Hopkin Medical Institutions, Baltimore, Maryland. Puck. Laboratory for Gene Transfer, National Center for Human Genome Affected sibpair analyses are often used in genome searches for susceptibility Research, NIH, Bethesda, MD 20892. loci in complex disorders. Computer simulations were used to determine the increase The recent discovery that mutations in the Fas gene, a mediator of in power added by genotyping unaffected sibs in such studies. In diseases with a late lymphocyte apoptosis, are associated with human autoimmune disease age of onset such as Alzheimer's disease, clinically unaffected sibs may still be gene prompted investigation of other members of the tumor necrosis factor carriers and data on the parental generation is rarely available. Two different pedigree structures were simulated and then analyzed using two different methods of sibpair receptor (TNFR) family. Single strand conformation polymorphism (SSCP) analysis. The first structure (SI) was two affected sibs with no parents available (200 was used to screen the 10 exons of the TNFR1 gene, on human chromo- of these pedigrees), and the second (S2) was two affected and two unaffected sibs some 12p13. Primers pairs were chosen to flank each exon and surrounding with no parents available (100 pedigrees). In both samples, 400 individuals would be splice sequences to amplify individual PCR products of around 200 bp. In genotyped. Data for a major gene with dominant inheritance were simulated (gene screening DNA from six individuals, SSCP mobility shifts were noted in frequency = 0.02). The marker data were simulated assuming 8 alleles, the first allele multiple exons, including exon 8. An expanded panel of samples from with a frequency of 0.50 and the remaining alleles have equal frequency (heterozygosity = 0.72). Unequal allele frequencies such as these are often observed unrelated individuals revealed 3 distinct migration patterns for the 195 bp for the highly polymorphic markers used to perform genome screening. For analysis segment between the forward (5'-TCCAGGGTGCCAGGGCTGAGAGA) and of the simulated data, allele frequencies were calculated from the data sets by randomly reverse (5'-GCGAGCCCCCGGAAAGTGAAGGATGATT) primers. Allele selecting one person per pedigree. When 50% of the families are linked, for the S I frequencies in 105 chromosomes were: allele 1(fastest migration), 0.22; pedigrees, p values less than 0.01 were obtained 83% of the time using SAGE and allele 2, 0.741; allele 3 (slowest migration), 0.04. Sequence analysis of cloned 78% using ERPA. For the S2 pedigrees, p values less than 0.01 were obtained 74% of alleles showed a T to C point mutation 9 bp 5' to the beginning of exon 8 in the time with SAGE and 72% of the time using ERPA. There was little difference in 1, consensus sequence in allele and a deletion of the A residue 42 the expected power between the two pedigree structures when 80% of the families are allele 2, linked, for each structure every replicate was significant at the p=0.01 level. The type I bp 3' to the end of the exon in allele 3. Of 51 unrelated individuals (20 from error rate for the two pedigree structures was also calculated with both analysis CEPH pedigrees) 43% were heterozygous for allele 2 with either alleles 1 or programs. For the SI sample, the type I error rate with p=0.01 is 0.0138 for SAGE 3. Mendelian inheritance for the polymorphisms was confirmed by and 0.0322 for ERPA, while for the S2 samples, the type I error rate with p=0.01 is genotyping a multigeneration pedigree. This polymorphism is a useful 0.0078 for SAGE and 0.018 for ERPA. These results suggest that in the presence of linkage marker for chromosome 12p and can now be used to determine significant heterogeneity, a larger sample of affected sib pairs is more informative than whether TNFR1 is linked to an increased risk for autoimmune disease. a smaller number of families which include unaffected sibs.

1898 1899 Clouston syndrome (bldrotic ectodermal dysplasia) is not linked to T-CELL RECEPTOR GERM LINE POLYMORPHISMS AND MULTIPLE keratin gene dusters on chromosomes 12 and 17 or keratin-related genes SCLEROSIS SUSCEPTIBILITY. E.C. Twist,. E. Mowat RB. Bell; Department on chromosome 11. T.D. a l, LZonA' W. McKinnon2, M. Littl, and S. of Clinical Neurosciences, University of Calgary, Calgary, Alberta, Canada T2N Hayffickl. 'Oregon Health Sciences University, Portland. 2University of Vermont, 4N1 Burlington. Clouseon syndrome is an autosomal dominant ectodermal dysplasia characterized by Multiple Sclerosis is a complex neurological disease ofpresumed autoim'une the triad of nail dystrophy, alopecia, and palmoplantar hyperkeratosis. Teeth, facial etiology where an interaction between environmental and genetics factors is appearance and sweating are narmal. Though the pathog s ofClouston syndrome is responsible for the development of disease. Epidemiologic data strongly support the unknown, a defect in keratin has been hypothesized and is supported by depleted hair role of multiple genes in conferring susceptibility. Considerable controversy matrix proteins in affected individuals, accompanied by failure to form disulfide bonds surrounds the role of T cell receptor germ line polymorphisms in conferring disease in the remaining keratin molecules and by disorganization of fibrils with loss of risk. A putative disease associated gene has been suggested to reside in a region of cuticular cortex. Based on these findings, candidate genes for Clouston syndrome include the keratin gene family and genes encoding keratin-associated matrix proteins. approximately 110 kb in length located within the TCRB locus. Keratin genes are kcnown to map to chromosomes 12, 17, and 11. The objective of our In identifying genes for complex diseases, both affected sib pair analysis and tudy was to investgate co-segregation of Clouston syndrome with highly polymorphic case control association studies have significant limitations. The extensive number madrkes on these chrmnosoms of mapped markers now available however allows for genetic association studies DNA was isolated from three families, two of French-Canadian descent and one based upon linkage disequilibrium (LD) between a disease locus and particular Scottish. Linkage was tested to 69 markers spanning chromosomes 12 and 17, marker alleles or haplotypes. LD methods are based upon the presence of a low rate including those flanking the keratin gene clusters, and those adjacent to keratin-related genes on chromosome 11. Two point linkage analysis was run using MLINK. of recombination between tightly linked marker and disease loci and therefore under Complete penetrance and equal allele frequencies were assumed. Haplotypes for each conditions of LD, associations are preserved between alleles at these loci. The LD family were construct. approach utilizes the population as the focus of investigation rather than families as Linkage was excluded to the keratin gene clusters on chromosomes 12 and 17 and to are used in standard linkage methodologies. By evaluating LD through the keratn-related genes on chromosome 11 in the three families. As in all genetic identification of conserved haplotypes in patients, this methodology has proven to disorders, the possibility of non-allelic genetic heterogeneity must be considered in be beneficial in positional cloning of disease genes. We report here a linkage Clouston syndrome. disequilibrium study using twelve highly polymorphic markers evenly distributed This work was supported by OHSU Foundation grant MRF9319 (SJH) and NEH grant across the genetic region encoding the T cell receptor variable beta chain genes. The ROI-DEIl1311-5 (JZ). sample population includes 100 nuclear families with at least one affected child. Genotype and haplotype relative risks were assessed. The results and interpretation of this data will be discussed.

1900 1901 Delimitation of the region for the long QT syndrome locus (LQT1) Towards the identification of a third gene involved in familial type iMa on 11p. A. H. van der Hout,1' E. Verlind.' M. B. Tan-Sindhunata.' hypercholesterolemia by exclusion mapping in two families. M. Varret J. W. Viersma,2 H. Scheffer.' and C.H.C.M. Buys'. Department of Medi- 1,J.P. labes 1-, B. Saint-Jor= J.C. Marinoni', M. Krempf, D.Erlc, cal Genetics, State University.1 and Department of Cardiology. University C. Bonj5,C. Junien1 . C. Boileau"2. ' INSERM UR383. Paris; 2 CHU Am- Hospital,2Groningen, The Netherlands. broise Pare. Boulogne; CHU Hotel-Dieu. Nantes; 4 CHU. Saint-Louis, Paris; Long QT syndrome (LQT) is an autosomal dominantly inherited cardiac disorder. INSERM UR331, Villejuif - FRANCE. It is characterized by a prolonged QT interval in the electrocardiogram (ECG). LQT Familial type IIa hypercholesterolemia (FH) is the most common monogenic causes syncopes and sudden death from ventricular tachyarrhythmias. It appears inherited disease. Genetic heterogeneity in this metabolic disorder has been proven to be a genetically heterogeneous disorder as linkage has been established with by the occurence of FH-related molecular defects in the low density lipoprotein markers in 3p2l-p24. 4q25-q27, 7q35-q36, and llplSS, whereas several families are receptor (LDLR) and apolipoprotein B (APOB) genes. We have demonstrated the known to be unlinked to any of these regions. In the 3p and 7q regions mutations existance of a third major locus (called 'non LDLR/non APOB") that accounts for have been found in LQT patients in a cardiac sodium channel gene (SCN5A) and about 25% of FH. This third locus is responsible for FH in two families for which a putative potassium channel gene (HERG), respectively. In a large pedigree with involvement of LDLR and APOB genes has been excluded by using both genetic LQT (4 generations, 12 affected individuals) we found linkage with markers in and biochemical approaches. We undertook the identification of this third locus 1ip15i3. Based on a published recombination event LQT1 has been placed proximal using linkage analysis. In a first step. a candidate region approach was used: 18 of HRAS. We found a crossover between the markers D11S1318 and DlS1338. The candidate genes encoding proteins involved in cholesterol metabolism and mapping individual involved has not shown any signs of the syndrome. at age 24. Dependent to 14 different genomic regions were selected. Thirty-three microsatellite markers on the result of a 24 h ECG to be made soon, LQT1 lies proximal to D11S1338 in that widely overlap these regions were tested. Linkage was excluded between these case our individual would be affected. or is located between HRAS and Dl1S1338, markers and FH. To map the 'non LDLR/non APOB" locus we then undertook with a genetic distance of less than 12 cM, when unaffected according to the ECG. an exclusion mapping approach, 103 new microsatellite markers located througout the genome were tested. To date, about 70% of the genome has been excluded and no linkage identified. A328 Published Abstracts: Linkage Mapaping and Polymorphisms (continued) 1902 1903 Coastruction of highly enriched markee libraies of rat gemooic DNA Flrnbelng methods fr hybridization probe and PCR. J. M- Worls 1. C. M. for the rapid dev et of high qualt p r irkers. R.XYJWaWL ]2QO'P EA 1IH nE S. a . 'Molecular Dynamics, Sunnyvale, CA 94086. AiL Mark Mt A miad V Univ. ofIowa, Iowa City, IA. 'Amersham Ufe Science Inc., Arlington Heights, IL 60005. Despite the recent development ofa linkage map ofthe rat genome, genetic studies Southern blotting and are two of the most common molecular genetic in the rat are still limited by a paucity of genetic markers. The constrution of small PCR amplification insert DNA libraries, which are highly enriched for clones containing short tandem re- analysis technques. The development of non-radioactive probes and chemiluminescent peat (STR) elements, by a method known as marker selection, has allowed the Coop- detection methods for blots hasdecreeed the use of radoactivity, butthe dynamic range and erative Human Linkage Center based at Iowa to develop over 2500 high quality short linearity of signal is til limited by the use of film. PCR product analysis usually employs ethidium tandem repeat polymorphic markers (STRPs) in 24 yews. In order to facilitate the bromide staining and UV detection, which is hazardous and damaging to DNA. We have rapid and efficient development of large numbers of STRPs for the laboratory rat, we evaluated the use of fluorescence labeling and detection methods for both blots and gels on the have constructed marker-selected libraries. Rat genomic DNA was sheared by sonica- Fluorlmagertm System (Molecular Dynamics, Sunnyvale CA). tion to achieve a random distribution of genomic inserts, without the site preference observed with restriction enzyme digestion, size selected (300-900 bp), ligated to We validated two fluorescent labeling methods for DNA hybridization probes: random prime Bs:XI linkers and inserted into the BsMM site ofthe phagemid vector, pJCPI. The pri- being of DNA fragments with fluorescein-1 1 -dUTP, and 3-end labeling (also with fluorescein- mary libraries are maintained in the E. colh strain JMGI (F+ duitung) and have an av- dUTP) of oligo probes using Vistra reagent kits (Amersham Lie Science, Arlington Heights, IL). erage sized insert of 530 bp and consist of 8 X 106 CFU, corepondg to The fluorescent signal is amplified using an anti-fluorescein antibody conjugated to alkaline approximately 1.3-fold coverage ofthe rat genome. Single sanded DNA was purified phosphatace and a fluorogenicsubstrate. We achieved limits of detection of 0.25 amol of target from the primry libries and extended with several STR primers. The marker- in human genomc Southerns using random prine labed probes and less than 10 amol of target seleced diueleotide repeat libraries generated with p(CA)1s were highly enriched for the dinucleotide repeat sequence. By hybridization analysis, approximately 45% ofthe in dot blot hybridizations usinga 3-end abeled oligo probe. clones were positive. DNA sequence analysis of a random sampling of362 CA repeat We also tested two fluorescent labeling methods for analyzing PCR products in gels. We contining clones revealed that the minimum STR length in these clones is 10 repeat compared the sensitivity of ethidium bromide detection on a UV transilluminator with SYBRTm units with an average length of 18 repeat units. Primers were selected from a random Green staining (Molecular Probes, Eugene OR) and scanning in the Fluorimager System. The sample ofthe CA repeat containing sequences. Approximately 75% ofthe STRs wer labter method, which utilizes laser scanning, is approximately 100 fold more sensitive than polymorphic when analyzed in a panel of 8 rat strains. Marker-selected libraries gen- tradtional UV detection of ethidium bromide. SYBR Green is suitable for agarose and acrylamide erated with tri- and tetranucleotide repeats are being caacterized. The library gener- including denaturing gels. We also 5-end labeled unmodified olgos using 5iodo- ated with p(GATA)IO was found to be highly enriched for these STR sequences. By gels, hybridization, approximately 19%/ of these clones were positive and DNA sequence acetamidofluorescein for use in gel shift assays and PCR. We have detected 2 fmol of product analyses ofa random sampling of 50 positive clones revealed that the selected repeat made with prelabeled primers, and less than 10 pg of DNA in gels with SYBR Green staining. sequence was present and at least 8 repeat units long. Our marker-selected libraries will allow the rapid development ofa large number ofpolymorphic markers across the rat genome.

1904 1905 Mapping of 10q microsatellite repeats using a de novo inverted tandem azolusiem mapping of chromosomal regions whioh oreas- P. hybridise to isuia associated markers in rSZlI. duplication 10q25-q26.2. M.J. Worsham'. DL. Van]Dyke' E.W. Jabs2. aka. .c. p J. StaJih. K. w Czarecki'. G.L. Feldman'. Henry Ford Hospital' Detroit, Ml, and The Johns Oiu. A. Kuar J. vane. eC Stewart. N. *nea A. n Hopkins University School ofMedicine2, Baltimore, MD. s I. A. Per 1-Vane and J. R- Gilbert- Division of We evaluated a patient with dysmorphic features, including upslanting Neurology, Duke University Medical Center, Durham, NC. Facioscapulohumeral muscular dystrophy (FSID) is a palpebral fissures, bilateral simian creases, posteriorly rotated ears, bitemporal genetically heterogeneous, autosomal dominant primary narrowing, frontal bossing, camptodactyly, and head circumference and weight disease of muscle. The dominant form of FSHD, FMIDlA, has less than the 5th percentile who was found to have a de novo inverted tandem been localized to the 4q34 region of human chromosome 4. of IOq25-q26.2. Our patient has many ofthe skeletal and The disease locus (loci) for the remaining FSHD families, duplication FSHDlI, has not yet been identified. craniofacial manifestations ofother patients with duplication in this region The D4Fl04S1 marker detects copies of a 3.2kb tandem (minor malformations ofthe hands and feet, and major malformations ofthe repeat (D4Z4). These repeats contain several types of heart, skeleton and kidneys). PCR-based amplification ofdinucleotide repeat repetitive sequences including Hox gene like elements and and have been shown to be closely linked to the chromosome 4 markers were used to compare DNA dosage, both quantitatively FSD disease locus The loss of an integral number of the qualitatively. Patient and parental DNA were studied using several such marks 3.2kb repeats has been associated with FSHD1A. When to (1) confirm duplication of lOq25-q26.2; (2) determine the parental origin of hybridized to chromosomal spreads these sequences cross- the duplicated l0q; and (3) to refine the physical map ofgenetic markers on lOq. hybridise with heterochromatin on acrocentric chromosomes and D1OS216 and specific areas on human chromosomes lq, 3p, lOq and Six markers, D1OS254, D1OS464, DlOSIO8, D10S468, D1OS587, loen. These regions have been suggested as potential were informative for parental origin and mapped within the region ofthe sites for the FSHD1B locus. To examine this possibility duplication. For these markers, densitometric measures ofthe paternal allele we have carried out linkage studies in our largest F8SHDlB were twice the intensity ofthe maternal allele, identifying the duplication as family, Duke 689 (Total-89, Affecteds-32), using three polymorphic markers for each of the four candidate paternal in origin. Markers DlOS608 and D1OS4211 at 1Oq23.3and 10ql1.2-qter chromosomal areas. The majority of two point lod scores respectively, were not duplicated and thus, mapped outside the lOq25.-q26.2 were negative for all members tested for both age region PCR-based amplification ofdinucleotide repeats was useful in mapping adjusted and affecteds only analysis. In addition, each & to the of of the four candidate regions was excluded by haplotype two previously unmapped lOq markers (D1OS254, D1OS464) region analysis in affected family members. There was no the duplication and in refining the location offour more markers (DlOSIO8, evidence for significant linkage to any of the four D1OS468, D1OS587, & DIOS216) within lOq25-q26.2. chromosomal regions.

1906 MitoDat - a mitochondrial protein information resource. S. Zullo, M.A. Chipperfield2, P. Lemkin3, C.R. Merrill. 'Laboratory of Biochemical Genetics, NIHM Neuroscience Center at St. Elizabeths, Washington, DC. 2Biomedical Information Sciences, Johns Hopkins University, 2024 E. Monument St., Baltimore, MD. 3Image Processing Section, Laboratory of Mathematical Biology, NCl/FCRDC, Building 469, Rm 150, Frederick, MD. The mitochondrion was once a free-living organism which has been utilized by more complex organisms during the course of their evolution. The host organism benefited from the increased energy production through electron transport systems provided by the mitochondrion, while the mitochondrion adapted itself to import proteins from the surrounding cytoplasm, thus reducing the coding capacity of its genome. Given this central role of the mitochondrion in the life of the eukaryotic cell, we have created a resource of genetic information pertaining to the mitochondrion. This database (MitoDat) is a repository of genome mapping and compartmental localization information, supplemented by reference data. MitoDat consolidates information from several publicly-available online resources, such as the GDB, SwissPro and OMIM. Currently, MitoDat focuses on mammalian mitochondrial data, but it will soon be expanded to include mapping information from other species such as plant, yeast and Drosophila. In addition to its use as a resource for mitochondrial protein data, MitoDat may also be a valuable tool for drawing new conclusions about synteny among the nuclear genomes of disparate species. The database design is generic, so as to lay the foundation for a more general comparative mapping database. The MitoDat search interface is available through the World Wide Web (WWW), at: http://www-lmmb.ncifcrf.gov/m-toDat/. Published Abstracts: Molecullar Etiology of Disease A329 1907 1908 Use of patient buccal swabs for molecular genetic studies of families with limb- Continued efforts in cloning the hemochromatosis disease gene. girdle muscular dystrophy. J.A Bedell, M.C. Krane. C.B. Wagner-McPherson and M. Chorney, J. Harris, C. Venditti. C. Kunsch7. J. Knoll+. K. Chorney and JLI. McPherson. University of California, Irvine, CA. R. Sakthivel. The Penn State College of Medicine. Hershey, PA, *Human Genome We have undertaken an effort to rapidly collect LGMD patient and family DNA Sciences. Inc.. Rockville, MD and Children's Hospital, Boston. MA. samples to identify families suitable for linkage analysis. The common hereditary disease, hemochromatosis (HFE), resides distal to the Buccal cell collection with sterile cytological brushes has proven to be a simple, I.82 polymorphic marker located 100 kb centromeric of the HLA-A locus within rapid and effective method of obtaining patient material. The non-invasive nature and the MHC. HFE resides in the state of linkage disequilibrium with the polymor- relative convenience of the protocol have facilitated the collection of 228 samples out phisms falling within a region demarcated by 1.82 and the marker D6S105. situated of249 brushes sent out (91.5%). A much greater compliance in the use of the buccal over 3 Mb from HLA-A. Large insertion/deletion polymorphisms within the MHC swab collection method performed by the patients and their families in their own class I region have been suggested by our group to be responsible for a general homes, versus scheduled appointments for blood-drawing, has enhanced the level of suppression of recombination and the cemented MHC-D6S105 haplotypes segregat- representation within families partaking in our studies. The brush collection method ing in HFE pedigrees. Moreover, based on extant HFE haplotype data obtained has proven to be cost effective, as well. from colleagues in Brittany exchanges between chromosomes appear nonrandom Molecular genetic analysis was done on crude preparations of the cytological brush in nature: in toto. these characteristics may suggest that the telomeric end of the samples. The brush tips were cut off into a lysis buffer solution (1OmM Tris HCl class I region has genomic properties similar to the T complex of the mouse. Al- (pH8.3), 50mM KC1, 2 5mM MgC12, 0. lmg/ml gelatin, 0.45% Tween20, 0.4% lele 8 of the VNDR marker D6S1O5. which is found with a 60% frequency among TritonX-100, 60ug/ml proteinase K), incubated at 500C overnight, then I/100th of Brittany hemochromatotics, was present at only a 40% frequency among our own the sample was analyzed by PCR PCR with polymorphic markers was completed in U.S. patients. The less rigid retention of HFE haplotypes comprised of HLA-A3 order to type individuals within families suitable for linkage analysis. The specimens and D6SO5 allele 8 reported in Furope and Australia, along with the advent of have been stable for 10 months at -700C allowing for long term screening by PCR additional polymorphisms within this interval. will allow us to further refine HFE s and subsequent sequencing of these PCR products. map position via an ancestral haplotype mapping strategy. We are also exploiting a positional candidate approach and have isolated cDNA clones possessing homology to ceruloplasmin and a variety of yeast metal regulatory sequences such as FET3. CTR1 and others. The testing of these candidates on our YAC contig spanning the MHC to D6S105 region, as well as the continued application of cDNA selec- tion technology which has yielded a variety of additional candidate sequences. may facilitate the identification of the HFE disease gene

1909 1910 Presymptomatic Molecular Diagnosis of Adult Dominant Polycystic Kidney Disease Identification of the mutation R329X in exon 7 of the low-density using PKDI and PKD2-Linked Markers. C. D. Constantinou-Deltas and A. Pierides. lipoprotein receptor gene in a French Canadian family with familial The Cyprus Institute of Neurology and Genetics and Nicosia General Hospital, Cyprus hypercholesterolemia. P. Couturel, S. Mooljani2, M.C. Yohl2, C. Gagne2, E. The Adult Dominant Polycystic Kidney Disease (ADPKD), is a heterogeneous disorder, Labriel, P.T. Lupien2 and I. Simardl. lLaboratory of Molecular Endocrinology primarily characterized by the formation of epithelium line cysts in the kidneys. In the and 2Lipid Research Center, CHUL Research Center and Laval University, Caucasian population, the estimated incidence of the disease is 1:1000 people. The age Quebec, Canada. (Intro. by V. Raymond). of onset is usually after the fourth or fifth decade of life and it is manifested as end-stage Familial hypercholesterolemia (FH) is one of the most common renal disease. Three genes are implicated in causing ADPKD. One on chromosome 16 autosomal dominant diseases. FH is caused by mutations in the low-density accounts for 85-90% of cases and the rest is accounted by the PKD2 gene on chromosome lipoprotein (LDL) receptor gene and is characterized by raised plasma LDL- 4. A very rare third locus is of unknown location. We used PKDI and PKD-2 linked cholesterol (C) and premature coronary heart disease. FH has higher highly polymorphic markers to perform pre-symptomatic diagnosis in affected families. frequency among French Canadians (FC) in northeastern Quebec than in We showed that in young members of families where clinical diagnosis cannot be most other populations, 1:154 vs 1:500. In our FC cohort, six mutations definitely established, haplotype and linkage analysis can provide unambiguous results. account for 85% of heterozygous PH, thus leaving 15% of patients still In one such family a young individual of 18-yo had one cyst on one kidney by ultrasound uncharacterized at the molecular level. To identify potential novel examination. However, molecular analysis showed clearly that he inherited from his mutation(s) in these patients, single-strand conformation polymorphism affected father the normal haplotype. In another family, in one part of the pedigree there (SSCP) analysis of the 18 exons of LDL receptor gene was carried out. An was co-inheritance of the disease with a PKDIl-linked haplotype which originated to a abnormal SSCP pattern was observed in exon 7. Direct sequencing of non-affected 78-yo father, and not from the reportedly affected deceased mother. Analysis asymmetric PCR fragments revealed the presence of a nonsense mutation with PKD2-linked markers excluded the PKD2 locus. Upon the request of the parents, in heterozygous form. This mutation results from C to T transition at young children below the age of 15 were ultrasonographically tested and found to be nucleotide 1048, converting codon 329 (CGA) encoding Arg into TGA stop- affected as expected by the inherited PKD1 haplotype. In this particular family, we codon (R329X). The mutation R329X occurs at a CpG dinucleotide and has suggest that the reportedly affected deceased individual actually had renal disease of a already been reported in a Norwegian family with FH. Sixty additional different aetiology, not ADPKD. Consequently, her affected daughter was the first person unrelated FC patients with suspected FH were analyzed by SSCP and 5 of to have the disease, most probably because of a new sporadic mutation that occured in them showed abnormal SSCP pattern in two different exons. The relative one of her normal father's chromosomes. In conclusion, the combined use of markers frequency of the R329X mutation in our cohort of 343 children is < 1%. High around the PKDI and the PKD2 locus provides more definitive answers in cases wher- frequency of FH among FC is attributed to a founder effect due to a high pre-symptomatic diagnosis is requested by concerned families. prevalence of few mutations. It is thus likely that the low prevalence of R329X mutation may be due to a more recent entry in FC population.

1911 1912 Unusual genetics of schizophrenia explained by the retroviral hypothesis. A homozygous mutation detected in exon 4 of the Cu,Zn P. Deb, R. L. O'Reilly and S. M. Singh. Molecular Genetics Unit. Departments superoxide dismutase gene associated with familial of Zoology and Psychiatry. The University of Western Ontario. London, Ontario, amyotrophic lateral sclerosis. G Dengl, H-X Dengl, S. Alston2 Canada. W. CamMbell3, I ~lL.&IZu,JI Caliedl, W-Y Hungl. AL The genetic predisposition for schizophrenia, a psychiatric disorder with an inci- Siddiguejl.i Department of Neurology, Northwestern University dence of 1% in most populations, is well recognized. The role of non-genetic factors Medical School, Chicago, IL. 2Department of Pathology, University complicates investigations on its causation. The offspring of schizophrenic parents of Virginia Health Sciences Center, Charlottesville, VA. have a higher risk of developing the disorder (13%c) than the parents of schizophrenic 3Department of Neurology, Veterans Affairs Medical Center, probands (6%c). The risk in dizygotic twins (17%c) is higher than in full siblings Richmond, VA. (9%c) and the concordance rate in monozygolic twins approximates 44%. The phe- Familial ALS (FALS) is a motor neuron disease inherited in an nomenon of anticipation, an increase in severity of symptoms across successive autosomal dominant pattern. Twenty percent of FALS cases have a generations. has been reported recently. Such results are compatible with Crow's detectable mutation in the CuZn superoxide dismutase (SODI) gene. retroviral hypothesis (1984) where exogenous/endogenous retroviral elements may There are currently over thirty mutations within the SODI gene serve as mutagenic/pathogenic elements. The possible insertional mutagenesis by known to be associated with FALS. All of the mutations have been the putative retroviriis accomodates the higher incidence of schizophrenia in chil- found to be heterozygous. However, we have reported a dren of probands, reduced concordance among identical twins and anticipation. Al- homozygous polymorphism found in Sweden in exon 4 of the SODI though the hypothesis is attractive, attempts to link retroviruses with schizophrenia gene resulting in an aspartic acid to alanine mutation in codon position 90. This polymorphism has an estimated carrier have not been successful. We present Representational Difference Analysis as an frequency of 2.5% in Northern Sweden. experimental strategy, which is expected to be highly sensitive iii detecting any We have recently detected this homozygous Asp9OAla mutation in unknown sequence of retroviral origin, unique to schizophrenic DNA. PCR is used the United States in FALS patient born of consanguineous to amplify all retroviral-related sequences from the genomic DNA of monozygotic unaffected parents. This mutation has been verified by twinrs with primers complementary to a conserved region of the RT polymerase sequencing to be homozygous. This is the first report of a gene. Linker ligated amplicons from the schizophrenic patient (tester) are annealed homozygous CuZn SODI mutation in the United States found to be in the presence of excess unaffected (driver) amplicons towards the identification of associated with FALS. We also have autopsy confirmation of ALS on unique tester sequences. The site specificity of insertion (representing the disrupted his affected brother which will be analyzed to confirm the gene) can then be established by means of DNA based homozygous Asp90Ala mutation. Marklund et al. have previously long-range representational reported this mutation to be associated with FALS in Sweden. difference analysis. Conversely, negative results will rule out the role of retroviruses We have also compared the Cu,Zn SOD protein levels between this and focus research in other important areas. patient and normal individuals measured by ELISA. There was no significant different in the protein level. A330 Published Abstracts: Molecular Etiology of Disease (continued) 1913 1914 TheClRRmutation16I+t16kbIAGfoundinSpanishcysticfibrosisduosomesis A single tube, nom-radioactive PCR -y for the detectioe of the IWI spectrum of FMR1 CGG repeats seam in the lorma, carer and fragie X syadpme presentinNorthAmencanfHfl panics. P n a I Kaiser Medical Cater, San Jose, C. C. dePagter and LA. Penny. Division of Medical Genetics, University of California, iodividualCalifosia, USA. San Diego, La Jolla, CA 92093. Fragile X syndrome is the most common inheited form ofmental retardation, nex A mutation in the CFrR gene, 11811+1.6kbA-G, has recently been described and is present to Down's syndome. The underlying mutation, with a few exceptions, is due to the Because a expansion ofthe CGG triplet repeat in the 5' u nslated region ofthe FMRl gene in 2% of the cystic fibrosis chromnosomes found in Spanish individuals. at Xq27.3. CGG repeat nunbers upto 50areconsidered normal. Repeats significant proportion of Hispaiiic individuals presenting in our laboratory for cystic betweenssted 51 and 200 are stions and >200 are fill mutations. Individuals who fibrosis mutation testing are liktsely to be of Spanish ancestry, we screened this carry the pemtation arc no expressing carriers. Clinical expression of the population for the 1811 mutaticon. Of the 60 individuals that have been retrospectively syndrome is observed when the expansion ofthe COG repea reaches a threshold of This 200 repeats. Accurate determintion ofthe CGG repeats is helpil in screened thus far, one individusal has been shown to have this mutation. the corrcet identification ofthe diffent classes ofmutations which is important for individual originally presented before the discovery of 1811, at 30 weeks of gestation the appropriate genetic counseling. PCR methods acrrently available to identify the with an echogenic bowel and didated bowel loops. Prenatal diagnosis detected only the allele sizes in the normal, p tion and fill mutation situations require the use of mutation. A was born at32 weeks gestation with meconium ileus. Two more than one reaction and dierent st ofPCR primers. G542X male We have developed a singletube PCR say that detecs the whole spectrum of iced boarderline results. Both parents daim Mexican (O~ leeszsse ntedfeetmtto~Tepiesue r rmr sweat chloride tests have prods (CGG)n aide mm eseen 11kthe dimwiet nat aTe prier wed are primr I ancestry for at least three generaations. Work is in progress to determine the frequency and 3 as reported byBrown et al., (JAMA 270:1569,1993). The PCRproducts ar of this mutation in Hispanics in nthe Sain Diego area. Because this mutation is Irun on a daturing polyc e gel, blotted on to a nylon membrane and American Hispanics than many of the other hybidzed to a (CGG)n probe. Hybridiztion products ar detected by potentially more common in Ncirth chemiluminescnce. The entire analysis is simple, accurate and f&at, requiring only mutations currently assayed forr in the mutation panels used in North America, testing four days to complete. We have completed the analysis on more than 500 X for this mutation should be offered to Hispanic individuals presenting for CF mutation chromosomes and the results are compared to the Southern blot analysis. Control p ion, and screening in North America. studies done on previously identified normal, Sill mutation methylation mosaic cases also will be presented.

1915 1916 and loci involved in autosomal recessive retinitis sh s loss of syoitn th region of the Analysis of genes pig- Isolated corcal dysplaia in 47 families. " M. A. 1 AlG Vei mentosa Spanish D. Grinberg, Bayes.' Martinez, Tuberous Sclerois, gem I R. MA Earradl. 'DepL of Clcl Genetics, Addenbroke's NMS Trust, D. Valverde. A del Rio, 2 C. Ayuso,3 Ll. Vilageliu. Gonzalez-Dusrte, t Univ. de de U 2Dept. ofPathlg, Univerniy of bd ,UK p M. Baiget2 and S. Balcellsl. Dept. de Genetica. Barcelona.i Hosp. of and Laboroy Medine, UCLA Scool of Medicin, Sant Pau. Barcelona2 and Fundacion Jimenez Diaz. Madrid.3 Spain. USA. 'Dept of Neuropathology, Beaumont Hospital, Dublin, Several genes and loci have been reported to be involved in autosomal recessive retinitis pigmentosa (ARRP)- the genes coding for two subunits of the rod cGMP- of resembles that foumd in channel. The _ cortical dysplasia dlosely phosphodiesterase (PDEA and PDEB), rhodopsin. the rod cGMP-gated acortical cases two u sclosis (1SC). Hm m ,including tber, from peripherin/RDS and ROM1 (the two latter in a digenic form of RP), and of TSC show loss of heterozygosity (LOH) for DNA markers in the region loci on 6p and lq respectively. However, they explain only a minority of ARRP or the on of the TSC2 gene on chromosome 16p13.3 TSC1 gene cases. The purpose of this work was to determine whether some of these genes both mome 9q34. These dat are onstent withte hypot s and loci are responsible for the disease in Spanish pedigrees. The genes for PDEB, adtas grwthsuppressorS. On sbass, the eaos seen in TSC may ROM1 and the loci at 6p and lq were analysed. Linkage of one rhodopsin. peripherin. ccu asan Idadfinding due totwo somac inactig mutaos and homozygosity studies were carried out on 47 Spanish ARRP families. SSCP in of a TSC gene mutation. In or other TSC ge, fteabsence germline was to search for mutations in the genes cosegregating with the rise to analysis performed some caes, the mutation may be enough to give disease in Only three homozygous mutations in the PDEB LOH. particular pedigrees. detectabbe were found in the affected members of three consanguineous families. Although was extracted from brain and cortical gene DNA parafflwe~ddednormal several were detected in the analysed, no other disease causing have polymorphisms genes dysplstic brin frm 3 patients with cortical dyspaia, who did mutation neither significant evidence for the involvement of the loci at 6p and lq TSC. DNA was used as tempaate for PCR anysisof were found. In view of the present data, it is unlikely that these genes and loci DNA marker in the region of the TSCI and TSC2 genes. One of the cause of the disease in most ARRP families. mutations in TSCl No could be the However. ortcal dysplasia tisses sowed LOH in the regon of the gene. PDEB seem to account for about of all reported cases. LOHwas found in any of theisues for markes flanking the TSC2 gene. 69c Wehavethusshownp ntary evidence tdatcortical dysplasia mayaria as a result of ina tin of both somatic allels ofa TSC gene, analogous to a somtc localisedo form of tuberous rosi.

1918 1917 Swiss CF of A new mutation, 3905insT, is prominent among Gkitsthlone S-Transferase Ml Genotype and Age-Related Cataracts: Lck Association In an toilan Popubltion. J. F. Heitmanclk G. AlbertF. 0ounf. M. chromosomes. eg be J alakrishnl. T. Betteck=2x C. S. VWklsna M. Kalser-Kuof, and Podoork. R.D. SpordutoIR S. Tomar- Grassf: .Bz ,C. Brfig% L4 R. Burge4 I. Ei schenkl. M. Morris of OOGCS, Q- hfmkrains.3nltuto of Ophthalmology, Un~vrslty Panna, Parmna, Rely; Schorderet6. F. F., Institut National Eye Inti, National Institutee of Hesith, Bethesda, Maryland, USA; ThonnevP. Ht. ar~nd N. M 71 fiir LMDB, Universitit Zurich, Switzerland; fur National Eye Insttute, National Isitu of Healt, Bethesd, Maryland. USA; Medizinische Genetik, 2Abteilung USA Switzerland; National Eye InSti, Naional situts of Health, Bthees, MaWland, Medizinische Genetik, Universithts-Kinderklinik, Bern, Studleawer carded outto Investate possise ebetween the gene 3Hopital Debrousse, Lyon, France; 4Universitats-Kinderklinik, occurrence number and allefc faumn of Glutathione 5-Transfese Ml (GSTMI) and the Switzerland; 5Division de m6dicale, Switzerland; of nucear and coilical age-related cataracts. Indiduals with cortical cataracts (102), Genetique Geneve, cataract autonome de m6dicale, CHUV, Lausanne, nuclear cataracts 29). mbxed nuclear and cortlcal cataracts (71), and wihout 6Division Gdndtique (98) were syeymeticallyselected from Indduma evaluated In the fitaln-Arnerican Switzerland; 7 Abteilung fir Medizinische Genetik, St* of the Natural History of Age-Related Cataract The patientswere typed forthe A, Universititskinderklinik, Basel, (Intro. by: Roland Spiegel) mutation Switzerland. B, and null allels of GSTMlusfn a variation of am on refractory The DNA from members of 650 families of Swiss descent affected 45% mainly system (ARMS). Forty nin percentof patients (50/102) wIth cortical cateraeb, was for 8 mutations in the fibrosis cataracts, by cystic fibrosis (CF) analyzed cystic (1329) with nuclear cataracts, 51% (36171) wth Med nuclear and cortical conductance regulator (CFTR) gene. 90% of all mutations and 50% of controls (49/98) were homozygous forthenull GSTMI allele. Twet fie transmembrane An insertion cortical 24% (729) with nuclear cataracts, have been recognized in this heterogeneous population. peroent of patients (28/102) with cataracts, was discovered. This 31% with nbxed nuclear and cortical cataracts, and 27% of cont (268) displayed mutation in exon 20 of the CFTR gene, 3905insT, of with cortical was in 5% of the mutations in onlythe A alll forGSTM1. Twenty four percent patients (24/102) mutation found approximately CFTR gene cortical of cataract, 24% (7/29) with nuclear cataracts, 14% (10/71) with mixed nuclear and Switzerland, and has since been identified in other populations the B for GSTMI Two percent of three cataracts, nd 18% of controls showed only allel Swiss descendant. Haplotype analysis with intragenic 4% wM potential patients 2lZ) with cortical cataracts. 7% 229) W nuclear cataracts. (3/71) microsatellites in the CFTR gene showed that the mutation is associated A and B mixed nuclear and cortical cataracts, and 5% of controls (5/6 showed both with a haplotype rarely identified on other CFTR alleles, and therefore the allels for GSTMI. No assocIations between the GSTM1 alleles, includIng the null allle, of the mutation in Switzerland is explained by a founder effect were detected in this study. frequency and cataract after a mutation event in historical times. The same haplotype was found on chromosomes of two French families canying the mutation 3905insT, which are therefore likely to descend from the same mutation event. This mutation has been identified in a high percentage of CF patients of the Amish and the Cajun populations. Interestingly, these two populations both descend to some extent from Swiss immigrants. Published Abstracts: Molecular Etiology of Disease (continued) A331 1919 1920 Mutations In the PCCB gene encoding the P subunit of propionyl-CoA Lack of expansion of triplet repeats in the FMR1, FRAXE, and FRAXF carboxylase In Spanish patients with proplonic acidemla. L Hoenicka. C. genes in multiplex male families with autism and pervasive developmental Pdrct-Cerdd P.Rodrfguez-Pombo and M Uggte. Centro de Biologia Molecular disorders. 1,2J.UA. Holden. 2M. Chalifoux. IC.Julien-Inalsing. IC. Schutz. 'P "Severo Ochoa" Universidad Aut6noma de Madrid. Madrid. Spain. Robinson. JB.N.White, JS Bryson, 3W. Mahoney. 3G 5aolu .M.lB. Propionic academia is an autosomal recessive metabolic disease resulting from Jones,. P. Szatmari. Queen's Univ. and 'Ongwanada, Kingston, Ontario, a deficiency of propionyl-Coa-carboxylase (PCC) activity. Most patients cell Canada;7McMaster Univ., Hamilton, Ontario, Canada; 4York Univ., North lines are heteroallelic for two different mutations and, at the moment, there are York, Ontario, Canada; tHershey Medical Center, Hershey, PA. twelve disease causing-mutations reported ocurringin Caucasian and Japanese Autism is a developmental disability characterized by impairments in recipro- patients with P-subunit defects. Since the most common mutations involved the cal social interaction, and verbal and non-verbal communication, as well as a MspI restriction site, we have previously studied this region in sixteen patients pattern of repetitive, stereotypic activities, representing the most extreme form of assigned to be P-PCC deficient. We found five patients having the a spectrum of conditions termed the pervasive developmental disorders (PDD) insertion/deletion mutation. In order to test for other mutations in this region we that share these clinical features. Sibling, twin and family studies have shown performed analysis on PCR products derived from reverse-transcribed fibroblast that a genetic etiology exists for many cases of autism, with a portion of autism RNA (RT-PCR). In two unrelated patients we found the band of expected size associated with a fragile X chromosome. Three folate-sensitive fragile sites are plus an additional band approximately 100 bp shorter. Sequencing analysis located within the Xq27- > Xq28 region. With the cloning of all of these fragile showed a 101 bp deletion between nucleotides 1199 and 1299 corresponding sites and the identification of polymorphic trinucleotide repeats in the respective precisely to the exon of the PCCB gene involved in the insertion/deletion genes, which are amplified and methylated in individuals who are positive for the mutation. This deletion resulted in a frameshift and a stop codon in the new different fragile sites, it is possible to test whether repeats in any of these genes frame. The same effect has been described in a Japanese patient having a deletion (FMR-1, FRAXE, and FRAXF) are amplified in autistic individuals. VW have of 8 nucleotides after the second base of the intron in the 5' splicing signal. We tested affected boys and their mothers from 20 families with two or more showed by sequencing and restriction analysis in our patients that the molecular autistic/PDD boys for amplification of the triplet repeats and concordance of lesion in the genomic DNA is different. We have identified another candidate inheritance of alleles by affected brothers. In all cases, the triplet repeat numbers disease causing-mutation by single strand conformation polymorphism and were within the normal range, with no individuals having expanded or premuta- sequencing analysis in the region located between 1311 and 1616 bp in the 3'end tion-size alleles. For each locus, there was no evidence for an increased frequen- of the cDNA. We have found in this fragment the C--to--T transition leading te a cy of concordance, indicating that mutations within these genes are unlikely to be substitution of Ala497Val. The point mutation eliminated a BsoFI restriction site responsible for the autistic/PDD phenotypes in the affected boys. Based on our in the cDNA and this enzyme can be used for patients screening. We are findings, we believe it is important to re-test those autistic individuals who were currently searching for other mutations that could account for the rest of our cytogenetically positive for a fragile X chromosome using the more accurate mutant alleles. DNA-based testing procedures. This study was supported by research grants from the Ontario Mental Health Foundation and the Medical Research Council of Canada.

1921 1922 Allelic size ladders for the accurate sizing of trinucleotide repeats in Huntington Carrier detection of spinal muscular atrophy. Y.J. Jong'. J.M.Huang2. C.P.Chang2. disease and fragile X syndrome. D. B. Johnson*. F. J.A. Buchannan** an Quan*. S. P. Lin' Y. J Chen'. and J. Cha Depar of Pediatrics, Neurological Section, B.W. PopovichV. DNA Diagnostic Laboratory, Oregon Health Sciences University, Kaohsiung Medical College, Kaohsiung, Taiwan. 2Department of Molecular Medicine, Portland*, and North York Hospital, North York, Ontario, Canada**. Taipei Municipal Jen-Ai Hospital, Taipei, Taiwan. 3Department of Pediatrics, Genetic Trinucleotide repeat expansions are now known to be responsible for a wide array of genetic diseases. A CGG tnnucleotide expansion is responsible for the fragile X Section, Mackay Memorial Hospital, Taipei, Taiwan. Proximal muscular is one of the most syndrome (FXS), a CTG repeat expansion for myotonic dystrophy, and CAG repeat spinal atrophy (SMA) common autosomal expansions are responsible for spinobulbar muscular atrophy, Huntington disease recessive disease, which is characterized by degeneration of the anterior horn cells (HD), spinocerebellar ataxia type 1, Machado-Joseph disease, and dentatorubral- resulting in progressive symmetric muscle weakness. SMA has been linked to the pallidoluysian atrophy These repeats are polymorphic in the normal population and chromosome 5q1 1.2-13.3 and two candidate genes for SMA have been cloned recently become prone to expansion only after a certain size threshold is exceeded. In many One of the candidate gene of SMA was termed survival motor neuron (SMN) gene. There cases there is no clear boundary between the upper limit ofthe normal size range and are two highly homologous SMN genes, centromesic and telmenric SMN genes, and the the lower limit of the premutation or affected range. For example, in HD the normal telomeric gene is the critical determining gene of SMA. In Lefebvre's series, the telomeric CAG size range spans 11-36 repeats and the affected range is239 repeats. Alleles gene was either lacking or interrupted in 226 of 229 patients (98.6%). We take advantage with 36-38 repeats fall into a "gray zone" between the normal and affected size ofthe polymorphism of exon 7 of telomeric SMN (codon 280 and centromeric SMN ranges. The molecular diagnostics of trinucleotide diseases is thus critically TTC) (codon 280 TT) genes in view that more than 95% of SMA patients losing the SMN dependent on the accurate determination of repeat size. Repeats are typically sized exon 7. A mutagenic primer which has a mutagenic base 'T' at the last second base of 3'- using nucleotide sequencing ladders. G In order to provide a rapid, more convenient, and accurate method for end changing the sense strand base to A is designed. In combination with the trinucleotide repeat sizing, we have developed allelic size ladders for the molecular polymorphism Tat the exon 7 of centromeric SMN gene, a Dra I site is created. The PCR diagnosis of FXS and HD. In each case the allelic ladder is generated by the co- products are digested with Dra I, electrophoresed, and then further analyzed by the Bio- amplification of a mixture of genomic DNAs from individuals with repeat numbers Profile image analysis system (Vilber Lourmat, Mare la Vallee, France) to calculate the spanning the normal and premutation or affected ranges. The fragile X allelic ladder digested part and undigested part ratio. In our study, 48 cases of SMA from 42 families, was assembled using DNAs from 10 individuals with repeat sizes ranging from 5 to the parents of SMA patients, and 100 cases of over 65 years old physically normal person 56 CGG repeats. "Stutter" bands, generated during amplification (and detected using were studied We found the carriers can be differentiated from normal individuals after a Southern based acrylamide detection method) result in a continuous series of bands comparison of their respective digested/undigested PCR products ratio. We believe the differing by one repeat in the 15-56 repeat range. For HD, the ladder consists of ability to identify the carrier status of SMA from general population is valuable in both DNAs from 6 individuals with 13 to 39 CAG repeats, with a continuous series of prenatal diagnosis and carrier detection, and this simple, rapid and economic method can bands differing by one repeat through the entire range. Using these allelic ladders, repeat sizes can be determined rapidly, accurately, and most importantly reproducibly also be used in mass screening (both within and between labs). Allelic ladders are currently being developed for all diseases caused by trinucleotide repeat expansions.

1923 1924 Mutation and Haplotype analysis of a Druze pedigree with McArdle's Sensitivity of direct testing for SMN gene deletions in autosomal spinal Disease. H. 2, Kalinsky 1, S. Iyengar S. Weiss 1, M. Sadeh3, B. Bonne-Tarir and muscular atrophy. LA. Kant. H. Rennert. Joshi. and R.B. Wilson. University of K. K. Kidd2. 1. Dept. of Human Genetics, Sackler Faculty of Medicine, Ramat- Aviv Pennsylvania Medical Center, Philadelphia, PA. 69978, Israel, 2. Dept. of Genetics, Yale University School of Medicine, New Haven, Spinal muscular atrophy (SMA) is a common autosomal recessive disorder classified CT 06520, 3. Dept of Neurology, Sheba Medical Center, Ramat-Gan 52621, Israel into types I, HIand IIIdepending on clinical severity. All3 types map to chromosome McArdle's disease is a rare hereditary disorder resulting from the absence of functional 3ql3,and linkage analysis has been a mainstay of prenatal diagnosis. Several genes muscle glycogen myophosphorylase. Patients with the disease are characterized by a have recently been identified in this region as candidates for SMA. The survival motor lifelong history of exercise intolerance, muscle pain, stiffness on exertion and recurrent neuron (SMN) gene, like others in the critical region of 3q13, is known to be duplicated. myoglobinuria. We examined a large consanguineous Druze family with McArdle's Lefebvre et al found that the telomeric copy of SMN was homozygously deleted or disease for mutations in the glycogen myophosphorylase gene. This family is the first interrupted in 226 of 229 SMA-affected samples and mutated in the remaining 3. Exons reported case of McArdle's disease in Israel. 7 and 8 of the telomeric and centromeric copies of SMN differ by one nucleotide in each All affected individuals were homozygous for a single Gto A transition in the first exon, and these differences can be exploited by SSCP or RFLP analysis to assess the nucleotide of intron 14 of the glycogen myophosphorylase gene. This splice-site presence or absence ofcentromeric and telomeric copies of SMN in clinical samples. mutation results in activation of an upstream cryptic splice site in exon 14. Thus, 67 Using these techniques, we analyzed 38 unrelated patient samples submitted with a base pairs of the 3' end of exon 14 are deleted when the cryptic splice site in exon 14 is presumptive clinical diagnosis of SMA. Most were from families where linkage analysis used. The G to A transition is a rare mutation, previously having been reported in had previously been performed. These included SMA types 1, HIand IIIin 33, 1 and 4 combination with another, more common, mutation at codon 49 in a Caucasian patients, respectively. 32 samples (84%) demonstrated loss of exon 7 from the individual. We have observed a marked variability in the clinical findings among family telomeric SMN gene; all but2 also exhibited loss of exon 8. Direct analysis was members with the same mutation. performed retrospectively in 11 prenatal samples. Five fetuses predicted as affected by To determine the ancestral haplotype on which this mutation occurred we typed nine linkage showed loss of exons 7 and 8.while six fetuses predicted as carriers showed microsatcllites at or near the PYGM locus; Dl 1S1368, Dl IS1392, Dl 1S1783, PYGM retention of these two exons. Six (16%) of the 38 patient clinical samples showed (AT), PYGM (CA), INT2, DIIS916, DIIS787 and DIIS33. All affected retention of exons 7 and 8; five came from patients with SMA, type I,and the other from individuals shared a core haplotype between markers D 1S1783 and IiNT2. We were, a patient with SMA, type IILWe did not observe evidence of other nucleotide changes however, able to detect recombinants both proximal and distal to this region in a number by SSCP analysis in exons 7 or 8 of these six patients. of affected individuals. Most recombination events were new,but we also detected an Our data support results indicating deletion of SMN is strongly associated with ancestral recombination event that created two different haplotypes carrying the same SMA. Our assay sensitivity of -85% is less than reported by others with standardized mutation in an affected family member (A). The inferred haplotype of A's father was patient groups and mostlikely reflects variability in diagnostic criteria among submitting consistent with the hypothesis that the ancestral recombination event occurred in a prior clinicians. A higher percentage of SMN point mutations than previously reported seems generation. (Supported by a grant from Tel Aviv University) unlikely, as we saw noevidence of this by SSCP analysis. Recent experience with cases lacking exons 7 and 8 butfelt NOT to be SMA by clinical or histologic criteria suggests an opportunity to revisit clinicopathologic correlations in SMA. A332 Published Abstracts: Molecular Etiology of Disease (continued) 1925 1926 Molecular analysis of Mucopolysaccaridosis IV A : mutation screening of Mutations involved in Tay-Sachs disease among Moroccan Jew the Colombian patients with non-RI SSCP-PCR. Z. Kato,', S. Tomatiu,1 M. Kaufinm**5 M. Karplati L. Pelai. B- Goldman#np E. Akateins R- Navon#5 H. Vega,' S. Fukuda, N. Yamada, A. Yamagishi,L A. Basrea,' K. Sukegawa. #Department ofHuman Genetics,Sackler School of medicine, Tel Aviv University, Tel N. Kondol and T. Orii.' Dept. of Pediatr., Gifu Univ. Sch. of Med., Gifu, JAPAN,1 Aviv and Molecular Genetics laboratory Sapir Medical Center, Kfar Sava, Israel. im and CIBI, Universidad de los Andes, Bogota. COL1OMBIA.2 Institute of Human Genetics, Sheba Medical Center, Ramat Gan, Israel. -sMinistry of Mucopolysaccharidosis IV A (MNPS IV A) is an autosomal recessive disorder caused Health, Jerusalem, Israel. by a deficiency in N-acetylgalactosamine-6-sulfate sulfatase (GALNS). Patients are Moroccan Jewry (N>500,000) is the only non-Ashkenazi Jewish cosnmunity in characterized by specific spondyloepiphysial dysplasia. short trunk dwarfism, coxa which Tay-Sachs disease (TSD) is not extremely rare. Previous studies among 6 valga, odontoid hypoplasia, corneal opacities, preservation of intelligence, and ex- unrelated Moroccan Jewish TSD families identified 3 Hex A mutations: AF305; cessive urinary excretion of keratan sulfate and chondoroitin 6-sulfate. Studies on Tryl8O-sestop; Argl70-+Gln. The first two were not described before and all three the molecular basis of MPS IV A have been facilitated by isolation of the cDNA and were different from the known 'Ashkenazi" mutations. Extension of the molecular genomic DNA. To date we reported large deletions, small mutations and several investigation to Moroccan TSD carriers who were identified enzymatically, revealed 4 polymorphisms of GALNS gene in Japanese and Caucasian patients. In this study additional mutations. One of them is a novel, IVS5-2 a-*g substitution resulting in we screened promoter region, 14 exons and exon-intron boundaries of GALNS gene exon skipping, while the other three, Gt"4-+A; 1278+TATC; IVS12+I g-+e; have of 9 Colombian patients using a non-RI SSCP-PCR method with silver staining. We been previously described in other populations. The distribution of the 7 mutations amplified the abnormal fragment from the SSCP gel by PCR and directly sequenced [AF304/305; Argl70-Gln; IVS5-2 a-+g; Tryl0S-stop; G1444-A; 1278+TATC; the products. The results showed the presence of 4 kinds of missense mutations and IVS12+1 g-c] among 53 unrelated Moroccan Jewish TSD carriers were one silent mutation. 901C/T. Three of missense mutations were novel mutations 24:19:6:1:1:1:1. Other g carriers are unidentified at the DNA level. However the (F69V, S162F and G301S) and the other was A393S. the same mutation previously enzymatic assay using 4-MUG gives also false positive results, we assume that some of identified in the Caucasian and Japanese populations. A393S was thought to be the 8 individuals are not TSD carriers. benign polymorphism as demonstrated from the results of the expression study and This relatively rich repertoire of mutations, probably reflects both the antiquity of the prevalence in the normal population. F69V and G301S were found in unrelated this community, established more than 2000 years ago, and its genetic hete ty, one patient and sibling patients. respectively. The mutation S162F was found in that resulted mainly ftom influx ofJews expelled from Iberia in late 15th century CE. both alleles of two sibling patients who were clinically severe and in one allele of another unrelated patient. also severe type. This mutation. S162F, may correlate the severe phenotype of the disease. These new findings will help us for better understanding the genotype-phenotype correlation of MPS IV A

1927 1928 Quantitative screening of family members with heteroplasmic mutant Mutational spectrum of 21-hydroxylase gene in Korean patients mtDNA: A noninvasive method using halr follicles and blood. C. W. with congenital adrenal hyperplaala Jjin-Su"gI=', UgY2n2, Lams. D. IL Sen2 B. Wane. J. , andLj.C.Wong.3IDept. of HoaSmg_..Km3, 1k-Hi Kial. 1- Dept. of Pediatrics, 2. Dept. of Pathology, Princess MgaretHospital, Hog Kong, 27he MolecularDiagnostics Microbiology, Yonsei University College of Medicine, 3. Dept. of Pediatrics, l~by,1he ical Genetics Divison, C nslHospital ofILsAngeles, Los Ewha Women's University Medical College. Seoul, KOREA Congenital adrenal hyperplasia (CAH) is a common genetic disorder Thelincalydfledmltsysemcmilochondrial syndrone,ntchdra caused by defects of an enzyme in steroidogenesis and inherited as an ncephalomyopasathy, lactic acids, and sAoke li episodes (MELS) isa autosomal recessive trait. Steroid 21-hydroxylase deficiency is the most maenallyineritedmDNA disorder. An A toG change at nt3243 is responsible common cause of the disease. The gene for this enzyme is located on formoe than 80% of this disease Th phenotypic expression of the tplasmic chromosome 6p2l.3 in the midst of the major histocompatibility complex. a e mutation of mtDNA isdewmined by dierelative pipion of the There are two 21-OH genes, one active gene and one pseudo gene, in normal mutant DNA to normal DNA. Such tehold effect vanes from se to tissue individuals. We performed mutation analysis of the 21-OH genein order to dep ing on the a bility of the pasicuartssue todamage in therespiatmy investigate mutational spectrum in 28 Korean patients with CAH. Genomic chain. For MEAS,it been repoed ht thepeentageof mutantDNA is DNA of the active gene was selectively amplified into two fragments using signicanily higherin affected tissues such as muscle. It also ccxelases with the known mutations within the iq of symptomns For epurpose ofgentic ounseling, it is imprtant to active gene specific primers. Frequent gene deterine dhedepse of h y in an ambes in dte affected were screened and the rest of exons were directly sequenced using PCR- it be to the amplified genomic DNA fragments as sequencing templates. There was no family. Due sodtesaismofhe plc i , would best test was in affected ts Howeritisusuallyidifflculttperformmnvasivesamplingon homozygous deletion, and heterozygous deletion detected 10.7% aWY t car We havercdy made diag on two MEIAS families (6/56) of tested alleles. The frequency of each mutation was as follows; fromlHong Kong. An uan tscreetingwas performed on a total splicing mutation in intron 2 was 23.2% (13/56), Ilel73Asn was 14.3% (8/56) Of 15 st ic, mildly cted, d ampraic family members. Hair Gln319stop was 3.6% (2/56) and Arg357Trp was 3.6%. A new mutation, follies re used as an alternative tisc. The amount of mutant DNA in both His245Tyr, was identified in the present study. A silent mutation, blood samples and hairfollkles was detemined. Ourresilts indicate that the Argl03Lys, appeared to be frequent (28.6%) (15/56) in Korean patients. The degreeofA32430 eteplamn inthesetissues a ab . Although it may relationship between genotypes and phenotypes was not different from the no e the true amuntofmutantDNAin de affecteddsu it does pride results of Western populations. In total 57.2% (33/56) of the disease causing valuableinform an the degree of etplasmy. Tbus, the useofhr follicles, alleles were found to carry known mutations and further study is in inaddiiontoblood forquntiaveanalyiofmu mtDNA, should be progress to identify rest of the mutations in the gene. In conclusion, the reccmmendedtoasymaticindlvldulsofaffected fily. ur ermore, the underlying causes of 21-OH deficiency in Korean patients with CAhseems cvenie ofuf n hairfolliclesin specimen sampling, shipment and storage can to be similar to that of Western populations. On the other hand, biological not be over ha effect of the new mutation have to be studied in order to investigate the phenotypic effects of this mutation.

1929 1930 Nnlecular basis of spinal muscular atrophy in Chinese. S.P. Lin', Y.J. Potential for a false positive diagnosis of myotonic dystrophy (DM) due to a Jong2, Y.J. Chen 1, W.S. Wang2, J.M. Huang3, C.P. Chang', J.G. Chang-. Bg1l polymorphism. D AKMgr K.A. Lppig. L p 'Department of Pediatrics, Mackay Memorial Hospital, Taipei, of Washington. Medical University Washington, Seattle, 2Department of Pediatrics, Kaohsiung College, Kaohslung, Molecular diagnosis for DM was performed for an at risk Ahican American 'Department of Molecular Medicine, Taipei Municipal Jen-Ai Hospital, DM Taipei, Taiwan. infant. Testing included PCRwith primers flanking the CTO repeat ofthe gene The gene for autosomal recessive proximal spinal muscular atrophy (19q13.3) and Southern analysis using the probe MlOM6 with BglI digested DNA. (SMA) had been mapped to chromosome 5q11.2-13.3. Two possible Prior analysis offamily members demonstrated that the mother had an expanded candidate genes - survival motor neuron (SMN) gene and neuronal DM allele of-2.5 kb (>800 repeats) and two maternal halfsiblings had expaded apoptosis inhibitory protein (NAIP) gene - were identified recently by alleles of-2.9 kb (>900 repeats) and -3.1 kb (>1000 repeats). two different groups (Lefebvre et al, Cell 80:155; Roy et al, Cell 14 the By PCR analysis, the infant showed two normal sized alleles with 11 and 80:167, 1995), respectively. In this study, we characterized a 3.4 kb molecular defects of these 2 genes in 48 Chinese SMA patients by using repeats. However, Southern analysis with BgII showed normal fragment a sensitive mini-gel non-isotope SSCP method and the multiplexed PCR and a -3.9 kb fiagment, indicating an expansion of-0.5 kb. Identical results were technique. We found that all the cases had intact exon 5 and exon 6 of obtained from a second blood sample. To confirm the presence ofa CTG the NAIP gene. All 48 cases lost exon 7 of telomeric SMN gene. Among expansion, Souther analysis was repeated with BamI and PstL enzymes which, them, 38 cases lost exon 8 of telomeric SMN gene, and the remaining 10 have restriction sites the CTG Only normal sized 8. The of non-deletion form of exon 8 like Bgll, Banking repeats. cases had intact exon percentage with either BaniHI or The existence of an was much higher in our series. There was no relationship between the fragmnts were observed PstA. expanded disease severity and the types of gene defects. Our results are DM allele was not confirmed. somewhat different from that obtained by other groups. It may be We concluded that the infant had two normal sized DM alles of 11 and 14 attributed to racial variation in molecular defects. repeats. The apparendy panded allele observed only in the BgHI digest is most likely the result of a BglI polymorphism rather than a CTG repeat e on. This BglI polymorphism is presumably the same as one ofunreported size and frequency observed in Bantu speaking South Africans with the probe pSBl.4 (J. Med. Gen. 31:37-40). Although the frequency ofthe DM BglI polymorphism in ethnic populations remains to be defined, it is imperative that expansions of-0.5 kb detected by Southern blot analysis with BglI be confirmed with BamI or PstI. Routine confirmation ofthese cases will prevent a false positive molecular diagnosis ofDM. Published Abstracts: Molecular Etiology of Disease (continued) A333 1931 1932 Malattia Levaatiaese: exclusion of known disease causing genes associated Linkage analysis in early onset, recurrent unipolar depression with macular dystrophies. F. L.Munier"2. B. Piguet' E. Hdonl2" G. Pescia' V. (EORUD) families of genes mediating neuroendocrine and serotonin L.Sheffield3 D. F. Schorderet' and E. M. Stone3. 'H6pital Jules Gonin, functions. K. Neiswnnoer. G. S. Zubenko. J. L Lewis. N. Zezza. G. Departement d'Ophtalmologie, Universit6 de Lausanne, 'Division deGdndtique Wiorecht. H. B. Huhes Ill, R. A. Galus. E. Frank. D. J. Kuofor. and B. B. M6dicale and Unitede GidtiqueMoleculaire, CHUV, Lausanne, Switzerland, Kaolan. Department of Psychiatry, University of Pittsburgh, School of 3The Uiversit ofIowa College ofMedicne, Iowa City, USA. Medicine, Pittsburgh, Pennsylvania. hBygmued:Age related macular d on(ARMD) remains the leading cause We have conducted a linkage analysis of 16 families ascertained of blindness in the Western counties in adults over 65 years of age. Malattia through probands with EORUD. Genotyping was completed on 126 Levantinese is an autosomal dominant macular dystrophy mimicking the natural individuals, using 37 STRPs from chromosomal regions containing 12 course of ARMD except for an earlier onset around the first or second decade of genes involved in neuroendocrine or serotonin functions. Pairwise li. In a first attempt to identifiy the underlying genetic defect of Malattia linkage analysis was carried out with the software package FASTLINK Levantinese a mutational analysis ofknown macular genes was performed. The affected phenotype was defined four ways: recurrent unipolar Methods: We have asceraied three large Swiss pedigrees with history ofMalsotia depression, any major affective disorder, any major or minor affective Levantinese in a least 4 generations, all originating from the Levantine valley, disorder, and depression spectrum disorder. Both dominant and Ticino. After informed consent, a comprehensive eye examination was performed recessive modes of inheritance were analyzed. Sex- and age-specific on individuals at risk of carying the disease gene. Blood was drawn for DNA penetrances were incorporated, and recombination was assumed to be extraction. A mutational analysis of known macular genes (RDS, exon 5 of equal between sexes. Regardless of the affection status employed, no TMM) was performed using a combined SSCP/DGGE approach. significant evidence for linkage was found with any of the markers. The Rets: Of a total of 58 individuals at risk, 28 were documented to be affected most positive lod score was +1.71 at E) = 0.1 for any major affective with Malattia Levantinese. No mutation has been identified in the RDS gene or in disorder and D1S214, a marker near the serotonin receptor 1Da. Taken exn5 ofthe TIMP3 gene. together, these results suggest that mutations in the genes for growth Conclusions: Our results suggest that Malattia Levantinese is caused by a distinct hormone, growth hormone releasing factor, somatostatin, corticotropin gene which remains to be identified. Identification of this genetic defect will be releasing hormone, corticotropin receptor, glucocorticoid receptor, critical to a better understanding ofthe pathogenesis ofARMD. serotonin receptors 1A, IDa, 1DO, and 2A, serotonin transporter, and tryptophan hydroxylase do not play a major role in disease susceptibility in a large majority of EORUD families.

1933 1934 P-Thalassemia mutations found in Koreans. Park'. Y. J. Lee'. K. Y Linkage analysis in a large Brazilian family with Knobloch syndrome. yionL S. I. To_'. H. I. Cho' Y. Hattri2. and Y. Ohha2 Dept. of Clinical Pathology, MIR.P A.Jtil Murra S.K.Marhe Seoul Nat'l Univ. College of Medicine', Seoul, Korea; Dept. of Clinical Laboratory M yaz. 'Dept. Biologia, IB and 2FMUSP, Universidade de SdoPaulo, SP, Science, Yamaguchi Univ. School of Medicine2, Ube, Japan. Brazil; 3University ofIowa, USA. 0-Thalassemia is particularly common throughout the Mediterranean region, parts We have recently reported a large inbred Brazilian family with 12 of the Middle East and Southeast Asia. However in Korea 0-thalassernia was known affected patients with the diagnosis ofKnobloch syndrome (Passos-Bueno et to be absent or very rare until 1988 when the first case of [-thalassemna was al., 1995: Am.J.Med..Genet.52:170-173). This syndrome is characterized by discovered. Since then, we have diagnosed twenty five cases of 0-thalassemia and high myopia, vitreoretinal degeneration with retinal detachment and occiptal identified eight types of 0-thalassemic mutations using PCR-SSCP and direct cephalocele. These alterations may occur early in embryogenesis suggesting sequencing (Table 1). Among these mutation types initiation mutation ATG-AGG is that a developmental gene or a gene that interferes with the migration ofthe most prevalent type 5/15) in Koreans and is thought to be highly specific to neuronal cells may be involved in the pathogenesis of this disease. Because Koreans. Codon 17 A-T and codons 41/42 -TCTT are common types in China Knobloch syndrome is a rare disease (to date there are only 3 families and Southeast Asia, but the other mutations are very rare types worldwide. in the and the present genealogy is highly inbred from an Table 1. Identified 0-thaasseeia autations in 15 Koreans with -thalasaeaia reported literature) isolated area in Brazil, it is most likely that probands are homozygous by Site Nltation Etiology Koreans Other Ethnic groups descent at the disease locus and at nearby DNA markers. Therefore, we have undertaken a linkage study of this genealogy using "homozygosity Initiation CD ATG-ASG Initiation 5 families Korean, Japanese, Chinese mapping". Until the present moment, we have analysed 121 microsatelites CD 17 AAG-TAG Nonsense 4 Chinese markers spaced at 20 cM intervals across 9 chromosomes. Althouhg one CD 121 GM-TM, Nonsense 1 Polish, French, Japanese positive region was observed on chromosome 2p (Zmax=2.43 at 0-0.10), CD 41/42 TTCITT - IT Frazeahift 1 Southeast Asian, Chinese this region was excluded with additional markers. Therefore, we are IVS-I-130 G-C Splicing 1 Turlcish, Japanese the to IVS-I-130 U--A Splicing 1 Egyptian extending study additional markers and regions. (FAPESP, PADCT, IVS-II-I G-IA Splicing 1 Mediterranean CNP,. CD 33/34 -GGT` Thalassenic Hb 1 [Hb Korea] Dominant types of 0-thalassenia

1935 1936 UNSTABLE DNA AND HUMAN DISEASE The Identification of fibrillin mutations in Marfan syndrome using heteroduplex analysis and nudeotide sequencing. F. san1,2, L. ak2 and B.W. Popovich1l2. Sally An Price, Munir A. Kadhimt, Eric 0. Wglstt and Duncan J. Shaw lOregon Health Sciences University and 2Shriners Hospital for Crippled Children, Depament of Molecular and Cell Biology, University of Aberdeen, Scotland, UK. Portland. tMRC Radiobiology Uni,Oxford, Enland. Marfan syndrome (MFS) is an autosomal dominant disorder characterized by involvement of the skeletal, ocular, and cardiovascular systems. Patients may Simplbsequence repats, consistn of repating u of name to five base pairs, manifest with dolichostenomelia, ectopia lentis, dural ectasia, and aortic dilatation are naturally ubiquitous, abundant and highly throuhoutt and dissection. MFS is caused by mutations in the fibrillin (FBNI) gene, a dispersed eukaryotic of the extracellular matrix. The FBN1 is in genomes. The underlying cause of a nunber ofhunan heritable disorders is an unstable component gene organized 65 exons and trinucleotide rpat, whose disease repertoire is growing rapidly. spans approximately 165 kb of genomic DNA. Mutation detection methods based on SSCP analysis of RT-PCR products have detected mutations in only 10-20% of One reason for sudc instability could be environmental. a-particl irradiation patients. A higher yield has been reported using heteroduplex analysis of PCR of a roducts from DNA et al. Amer. J. cellinduces gandine instability nvitro. Our preliminary results indicate tha a- amplified genomic (Nijbroek [19941 Hum. Genet., and X-ray irradiation may a demonstrated 55, A233). Fibrillin exons are amplified using an intron primer set and heteroduplex particles mnagnify previously repeat is Mutations are instability, at a specific disease locus. Subject to corfirmation, this leads us to suggest analysis performed using Hydrolink-MDE gels (AT Biochem). that identified by nucleotide sequencing. We have used this approach to search for FBNI ionizng radiation could be detrimental to the replication mechanism of unstable mutations 4 DNA and in patients and have identified mutations in 2. Both mutations were found trinucleotide repeat, expansion transmission. in exons which code for calcium binding EGF-like motifs. Patient K3 carries a C to Trinuclenode sequences also exhibit a high degree of T transition in codon 565 (exon 13) which results in the conversion of an arginine polymorphism. Using to a triplet repeatprobes in we have shown that codon premature TGA stop codon. Patient F3 carries a C to T transition in codon Southenhybridisation experimnots, 627 which results in individual-specific, multi-locus DNA fingerprints can be generated. These show (exon 15) the subsitution of Cys for Arg. This mutation has mendelian ihertance and represen a simplified alternative to other DNA previously been reported in an unrelated family (Hayward et. [19941 Hum. Mol. Genet., 3, 373-375). A detailed clinical evaluation of F3 is currently underway. A figepriiigm etods. comparison of the clinical findings in the 2 families will allow an investigation of genotype/phenotyecorrelations and clinical variability in MFS. Further studies are being carried out on the 2 patients in which mutations have not as yet been detected. In addition, mutation detection using the heteroduplex approach is being applied to additional MFS patients in order to better evaluate the utility of this approach for direct mutation detection in MFS. A334 Published Abstracts: Molecular Etiology of Disease (continued) 1937 1938 Screening for large NF1 gene deletions using Cornelia deLange Syndrome: Toward the positional cloning of a locus at loss-of-helerozygosily P.Rizzu. L.G. Jackson*. A. aldini. of intranic markers. S.A. Rasmussen1. S.D. Colman . V.T. Ho. 3q27. - OvraMuser*. Baylor College of C.A. Will]am1 0.J. Driscoll P.H. A'.C.R. Abernathy. M.R. Medicine, Houston, TIX Thomas Jefferson Univ. Philadelphia,PA. Walc' 'Division of Genetics, Department of Pediatrics, Cornelia deLange syndrome (CDLS) presents with charteristc cofacial Vniversity of Florida College of Medicine, Gainesville, Florida, anomalies, mental and growh retardation,hearing loss, heart defects and limb defects. Nemours Children's Clinic, Jacksonville, Florida Complications of gastroesophageal refluxand heart defects are responsible for about Neurofibromatosis type I (NFl) is an autosomal dominant 50% of the deaths. The phenotype is variable in severity and only recently has the condition, characterized by neurofibromas, cafe-au-lait macules, existence of mildly affected cases been recognized. There are no cytogenetic, and Lisch nodules of the iris. The NFl gene spans 350 kb and biochemical or genetic markers that can be used to confirm the diagnosis. CDLS is contains 59 exons. Direct mutation detection in NF1 is a mostly sporadic but a good number of familial cases have now been identified. Thanks challenge, with the same mutation found in unrelated individuals to the collaboration of the Comelia de Lange Foundation, we have so far identified 12 in only a few reported cases. Conventional mutation detection families with multiple CDLS cases and cell lines are being established for mostof these methods often miss large gene deletions, which may represent a families. It has long been recognized that the so called dup(3q) syndrome has numerous significant proportion of NFl mutations. clinical features similar to CDLS. We have applied a straightforward method to screen NF1 patients Dup(3q) syndrome is associated with triploidy of a segment of the long arm of for large gene deletions by comparing patient genotypes to those chromosome 3. We have defined the minimal region of duplication overlap critical of their parents at multiple intragenic polymorphic sites region within the chromosomal band 3q27. We have constructeda YAC contig that (restriction fragment length polymorphisms and microsatellites). spans the region and estimated a minimum size of 10-12 Mb. Recently, a patient has Thus far, we have screened 24 patients and their unaffected parents been referred to us with a CDLS phenotype and with an y paracentnc using six intragenic markers, and have identified two patients with inversion of the long arm of chromosome 3. We have mappedthe distal breakpoint in loss-of-heterozygosity of the maternally inherited allele, this patient within our YAC contig at 3q27 and clones spanning the breakpointhave been suggestive of deletion of the NFl gene. One of these patients, identified and will shortly be used for expressed sequence search. No duplication of the mildly affected, demonstrates somatic mosaicism for the deletion, 3q27 region was observed in this patient by FISH and uniparental disomy was excluded and is the first reported case of somatic mosalcism in NFl. as a possible cause of the phenotype. The chromosomal reaIrangementin this patient This method is a useful tool for identification of NFl gene was inherited from the apparently normal mother. However, because 1) the patient deletions and may contribute to genotype/phenotype correlation. phenotype is very suggestive of CDLS, 2) another CDLS patient with a balanced Our studies will be expanded to incorporate further intragenic and translocation in this region has been reported and 3) this region is implicated in the flanking markers and to include additional patients (new-mutation dup(3q) phenotype which is very similar to the CDLS phenotype, we believe that itis sets and affected parent-child pairs). Deletions will be further very likely that the rearrangement affects a gene responsible for the CDLS phenotype. characterized using other methods, such as fluorescence in situ Polymorphism analysis of familial CDLS should provide supporting data for the hybridization and pulsed field gel electrophoresis. mapping of at least one CDLS locus within 3q27.

1939 1940 Sequence analysis of the PRNP gene in hereditay Inclusion Body Genetic heterogeneity in autosomal dominant pattern dystrophy of Myopathy. L. Cervenakova. K. Sivakumar. J. Nagle. S. Isaacson. M. C. the retinal pigment epithelium (PDRPE). R. Spiegel, M. Weigell-Weber, L G. Goldfarb, National Institutes of Health, Bethesda, MD. Ch. Krvenbuehl and E. R. BuehI. Institute of Medical Genetics, University of Hereditary inclusion-body myopathy (IBM) is a progressive muscle Zurich, Switzerland, Dept. of Ophthalmology, Cantonal Hospital Luzern, Switzer- disease characterized clinically by selective limb weakness and land and University Eye Hospital Basel, Switzerland. histopathologically by rimmed vacuoles with basophilic granular lining Pattern dystrophy of the retinal pigment epithelium (PDRPE) is a rare autoso- and cytoplasmic or nuclear filaments in the muscle biopsy specimens. The mal dominant eye disorder. Common clinical characteristics of PDRPE are onset in disease is inherited in an autosomal recessive or autosomal dominant midlife with a good to fair vision prognosis and different age-dependent alterations fashion. Previously studied IBM families were too small for genetic of the retinal pigment epithelium. Reticular dystophy, macroreticular dystrophy linkage analysis, and the genes associated with familial IBM have not been butterfly-shaped pigment dystrophy and fundus pulverulentus are assumed to

studies have identified the retinal identified. Careful immunocytochemical be different expressions of PDRPE. Recently. mutations in the degenera-

in the vacuolated muscle fibers of with IBM as found in with deposits patients hereditary tion slow (RDS)/peripherin gene on chromosome 6p were patients of the Alzheimer's or admixture of AS and AS amyloid type amyloid PDRPE. as well as in families with retinitis pigmentosa (RP6). macular dystro- as seen in some of the human prion protein, spongiform In order to characterize the defect in a encephalopathies. phy (MD) and other retinopathies. genetic large Swiss PDRPE family we first performed linkage analysis with intragenic and We studied two families with hereditary IBM: a four-generation markers from the RDS region. We could exclude linkage to RDS. autosomal-dominant American pedigree that included 8 patients; and a flanking gene and in a more extended disease association to the rhodopsin gene and other family of Iranian Jewish origin with two affected individuals in a sibship study. candidate loci involved in RP and MD in this family. Furthermore, in an ongoing of 7 and phenotypically normal parents suggesting an autosomal recessive inheritance. The clinical and pathological features in all studied patients mapping attempt we have already screened approximately 50 percent of the auto- for IBM. somal genome. Exclusion of the RDS gene as the responsible locus in this clinically satisfied diagnostic requirements hereditary Complete sequences in of the PRNP coding region in two affected individuals, one from each well characterized family, however, provides evidence for genetic heterogeneity family, did not contain any abnormalities in either of the two studied retinal pattern dystrophy. patients suggesting that hereditary IBM has a disease mechanism substantially different from the pathogenesis of the known familial spongiform encephalopathies always carrying mutations in the PRNP coding region.

1941 1942 Identification of the number of the fragile X syndrome Analysis of dystrophin illegitimate transcripts : detection point CGG silver stain. M.-S. Tsail T.-Y. Chieni1 mutations and evidence for structure rearrangements Tuffery. repeat using *RG Roberts. I. Demaile and M. Claustres Laboratoire de Blochimie H and S.-M. Hwang2 Cathay General CNRS Institut de Biologie, Bd Henri Montpellier, Taiwan. 2Food Research and Development gdndtique UPR9008 Hospital, Taipei, Industry medical France. *Paediatric Research Unit, Division of Taiwan. Institute, Hsinchu, London SE19RT, UK. form of inherited genetics, Guy's Hospital, X is the most common a Fragile Syndrome Duchenne muscular dystrophy (DMD) is lethal X-linked the for X mental retardation. FMR-1, gene responsible fragile disorder, which results in approximately 65% of frameshifting and located in The The affected syndrome, has been isolated Xq27.3. gross rearrangements in the dystrophin gene. remaining a subtle number of repetitive sequence (CGG)n in FMR-1 showed individuals (35%) are supposed to carry more progressive increment in the premutation (50-200 repeats) to a cause premature translational termination. To search full mutation (>200 repeats). We report on a method to point mutations, we applied the strategy of analysis determine the number of CGG triplet repeats for nonradioisotopic the Protein Truncation Test (PIT) which specifically of chain reaction termination mutations by means of in vitro transcription/translation. detection of FMR-1. The conditions polymerase RT-PCR kb) can be screened through only (PCR) were optimized by including the nucleotid analog 7'- coding sequence (11 deaza-2'-deoxyguanosine-5'-triphosphate (c7-dGTP) due to the reactions. extraordinary high GC content. The PCR products were seperated The analysis of the illegitimate dystrophin transcripts fromn blood lymphocytes) of the non deleted patients by nonreducing 6% polyacrylamide gel electrophoresis and peripheral stained with 0.2% AgNO3,followed by formaldehyde development. frameshift mutations. Furthermore, we describe splicing indivduals We tested the silver staining to analyze 150 mental retarded resulting in exon skipping or intronic sequence supposed and revealed that it promised to provide a rapid and physiological rearrangements as it could be expected Mb-gene the radioactive-free method of direct FMR-l analysis. In addition, we have comprising 79 exons. This study contributes understanding developed the nonradioisotopic method of Southern blot analysis dystrophin mRNA structure, the gene regulation by digoxigenin-labeled StB12.3 probe. functional domains of the protein. Published Abstracts: Molecular Etiology of Disease (continued) A335 1943 1944 Repeat expanion detection In manic depression and sch reniaB. Vincent. Characterization of point mutations and deletion mutations in a series of A. Petronis. T. Kiempan. I. Gottesman. E.F. Torrey and J.L. Kennedy. Clarke null alleles at the triosephosphate isomerase locus in humans and mice. Institute of Psychiatry, University of Toronto, Ontario, Canada. B. C. Zingg*l. M. Watanabe', M. P. Thelen' and H. W. Mohrenweiser'. 'Lawrence Much attention has been given to the role of triplet repeat expansions in Livermore National Laboratory. Livermore CA and 'NIHS, Tokyo. Japan. neuropsychiaric disorders More recently attention has been focussed on the major Null alleles have been identified at the triosephosphate isomerase (TPI) locus shown to demonstrate similar psychoses which have been patterns of anticipation during the screening for activity of 10 erythrocyte enzymes in 2000 newborn chil- (inreased severity of symptoms, decreased age of onset) as observed in triplet repeat dren (Pediatr. Res. 16:960-963. 1982; Hum. Genet. 77:241-245, 1987) and in disorders, thus implying a possible role for triplet repeat expansion in psychotic mouse germinal experiments (W. Pretsch., pers. comm.). In the human population, disorders. the frequency of TPI null alleles was approximately 10 fold greater in the African We have used the ligase chain reaction (LCR) for triplet repeat expansion detection American population (0.02) than in the Caucasian population (0.0024). In the case amongst a population of 63 unrelated manic depressive individuals (RDC Bipolarl), of point mutations. genomic PCR products were sequenced. For the analysis of 27 pairs of monozygotic twins either concordant or discordant for schizophrenia, and heterozygous gene PCR products were quantitated by Perfusion a population of 58 unrelated, unaffected controls of mixed ethnic origin with ages deletions., HPLC. report the of mouse mutants. above the average ages of onset for schizophrenia and manic depression. Thus far we We characterization heterozygous TPI deletions in 3 have searched for expansions of the repeated triplets CAG/CTG and AGG/CCT. Among the human population, we describe the molecular characterization of the null alleles from 7 African American and 3 Caucasian individuals. Whereas 3 Expansies have only been observed for the CAG/CTG triplet, however no differ- ent were a common gnificant difference is observed between the experimental and control groups. In the amino acid substitutions identified in the 3 Caucasians, variant bipolar sample, for the CAG/CTG repeats 30 subjects had expansions in the 153 bp allele consisting of two nucleotide changes immediately 5 of the transcription ini- range, 9 had repeats in the 204 bp range and 6 had evidence of expansions in the 255 tiation site was found in the 7 unrelated African Americans. Two of the 3 amino bp range. The control group was not significantly different in CAG/CTG repeats, acid substitutions had not been described previously and occurred at residues not having 21, 13, 1, 1 and 1 counts of 153, 204, 255, 306 and 357bp expansions directly involved in the active site but highly conserved through evolutionary time. respectively. In the MZ twins similar distribution of CAG/CTG repeats were This suggests important roles for these residues in maintenance of subunit structure observed but no discordance was evident. and conformation. The combined results from human and mouse studies provide We conclude from the data produced thus far that expansions of these triplet insight into the mechanism of mutations and serve as models for structure-function repeats are not involved in the etiology of manic depression or schizophrenia. studies. Work performed under auspices of the US DOE by Lawrence Livermore National Laboratory; contract W-7405-ENG-48.

Published Abstracts: Molecular Pathogenesis and Treatment 1945 1946 The largest Intra-gene deletion of the dystrophin gene Identified In a A cell culture model of spinal and bulbar muscular atrophy. IL DMD case with full clinical spectrum of dystrophin deficiency. P. Brooksl K L. Paulnh E. F. Sali . D. E.MglT A. O. H. Alimsardjono. S.Y. Patria. H. Nishio. K.Honkc and M.Matsuo. Bgrinkaj3. M. J. McPhaul4. M. Oonzi-Sc tl. and K. HL Fischbeck.1 ICMR, Kobe Univ. School of Med and Iou Hospital, Japan IUniversity of Pennsylvania, Philadelphia, PA. 2University of Chicago, 3 The dystrophin gene is the largest gene in human and 3.000 kb long (Nishio Chicago, IL. Erasmus Univeristy, Rotterdam, The Netherlands. et al., J. Gin. Invest. 94:1037-1042) At least eight promoters have been localized in 4Southwestern Medical Center, Dallas, IX. this huge gene and each of promoter is considered to be expressed in tissue- or Spinal-and-bulbar muscular atrophy (SBMA) is an inherited, pessive development-specific fashion. Although many kinds of mutations of the dystrophin lower motor neuronopathy caused by the expansion of a CAG tr-nucleotid gene have been reported in Duchenne / Becker muscular dystrophy (DMD/BMD), repeat in the codingregion of the androgenreceptorge We have stabl we can not draw the full clinical spectrum of dystrophin deficiency. Bccausc, somc transfected normal- and e d-repea arn receptor genes AR-2 deletions arc so big that other genes are also deleted to deteriorate clinical phenotype. and AR-66, respectively) into a clonal mouse moor neu -neuroblastoma In other cases deletion is not big enough to delete all dystrophin promoters, and hybrid cell line. These cells high Kl ofcholine acetyle some of dystrophin isoforms arc still expressed. Here we report the largest intra- (QuAT) have a severalfold increase in ChAT activity when cocultured with gene deletion of the dystrophin gene, which spans from a brain promoter region to muscle, express no detectable endogenous androgen receptor, and can be exon 78. differentiated into a neuronal phenotype with retinoic acid and mitotic A Japanese boy was born to healthy parents. He started to wallk at the 18 inhibitors. Expression of AR-24 and AR-66 was confirmed by Western months old. Since then his developmental milestones werc delayed. At the age of blot, [3H]-R1881 binding, and immunoflourescence. Levels of AR-24 and five he was diagnosed as mentally retarded. DMD was a clinical diagnosis at 7 y.o. AR-66 appear stable upon differentiation and can be augmented by Myopia was pointed out at 13 y.o. At 14 y.o. ECG abnormalities were disclosed and treatment with sodium butyrate. CAG repeat length in AR-24 and AR-66 ultrasonographic examination disclosed mitral regurgitation and low left ventricular clones appears to be stable across at least fifteen passages. There is no ejection fraction. And he died at the agc of 17 because of cardiac failure. immediately discemable phenotypic difference between AR-24-exprtessng, The dystrophin gene was analyzed by PCR amplification method. L- AR-66-expressing, and parent line cells. We are currently studying these promoter, located 500 kb upstream of brain promoter, was present. However, all cells in hopes ofelucidating the biochemical basis of SBMA and the effects other S' region promoter such as B-, M-, P- promoters were deleted. Furthermore of androgens on motor neurons. To our knowledge, this is the first stably- exon 78 could not be amplified. And the last part of 3' end of exon 79 could be transfected neuronal cell culture model of a trinucleotde repeat disease. amplified. These results showed that the case lacked almost all region of the dystrophin gene. And all his clinical symptoms, muscle weakness, myopia, mental retardation and cardiac failure, arc attributed solely to the absence of dystrophin. It is clearly concluded that this is the most severe phenotype of dystrophin deficiency.

1947 Paternal transmission of a full mutation in the FMR1 gene: Identification of paternal CGG repeat sizes in multiple tissues. C.A Brown, C K rasington and F.L Grass. Carolinas Medical Center, Charlotte, NC. The predominant mutation in the fragile X syndrome is an amplification of a CGG trinucleotide repeat in the 5' untranslated region ofthe FMRI gene at Xq27.3. Normal alleles have 4-52 repeats, premutation alleles have 52-200 repeats and full mutation alleles contain >200 repeats. Expansions in the full mutation range are often accompanied by methylation at an adjacent CpG island which downregulates expression of the FMRI gene resulting in the fragile X phenotype. Full mutations reportedly always derive from maternal premutations. We have identified a kindred where the aunt of an affected individual carries an unmethylazed full mutation composed of a broad smear with an average size of 330 repeats in DNA isolated from lymphocytes. She was reported to have some learning difficulties in school, especially with mathematics. Analysis of the lymphocyte DNA from her parents showed that the fragile X allele was inherited from her father, who carried a premutation allele containing approximately 125 CGG repeats. He did not appear to have any phenotypic expression of the fragile X syndrome, but was reported to lack socialization skills. The father died of acute heart failure at the age of 74, and multiple organs were procured for genetic studies. DNA was isolated from heart, liver, kidney, testes, and various regions of the brain including cerebellum, cerebral cortex, hypothalamus, amygdala, and the frontal lobe. Premutation size alleles were found in all tissues examined, with the brain tissues showing alleles of slightly larger sizes. Additionally, Southern blot analysis of the DNA isolated from the cerebral cortex revealed a mosaic pattern suggesting the presence of both pre- and full mutation size alleles. In conclusion, we present a novel case of a paternal transmission of a full mutation FMRI allele. A336 Published Abstracts: Molecular FDathogenesis and Treatment (continued) 1948 1949 A mutatlon ofthe COL3A Ieae presenting with Miehers elastona. The contribution of LHON mitochondrial DNA mutations to multiple NP Burrows', AJ Richards, P Narcisit, AC Nicholls', HK Shamy3, D sclerosis. F. Carrara, MH. Eoli. L. La Manfia and M. Zeviani. Istituto Nazionale McGibbon4, FM Pope' '21MRC Connective Tissue Genetics Group, Neurologico XC. Besta", Milano, Italy. Strangeways Research Laboratory, Cambride; Department of Clinical Mutations of mtDNA commonly associated with Leber's Hereditary Optic Neu- Genetics, Addenbrooke's Hospital, Cambridge; York District Hospital, York; ropathy (LHON), have occasionally been reported in female patients from the U.K. 4St John's Dermatological Centre, St Thomas's Hospital, London, U.K. with a Multiple Sclerosis (MS)-like phenotype, suggesting that mitocondrial gene Mieschers elastoma is a common presenting feature of vascular Ehlers- abnormalities may contribute to MS susceptibility. To verify this hypotesis in the Danlos Syndrome type IV. Here we describe an example presenting with a Italian MS population, we searched the LHONI mutations at nt 11778, 3460 and cutaneous rash at adolescence which persisted into adulthood. She then 15257 of mtDNA, in 53 unrelated MS patients (18 males and 35 females; median developed severe venous varicosities and the clinical features ofvascular EDS age 38.4 SD 12). All patients were characterized by early, severe optic nerve in- IV were first noted. RT PCR of COL3A1 mRNA prepared from cultured skin volvement. We found one male patient with optic neuritis and other symptoms fibroblasts shows two products within the gene fragment which codes for of MS that carried a virtually homoplasmic LHON mutation at nt 11778. In this cyanogen bromide peptide al(IIl)CB9. Protein biochemistry is consistent with patient, serial MRI examinations showed regression and reappearance of demyeli- this observation and shows poor collagen secretion and intracellular retention. nating areas, as typically found in MS. Family history was completely negative Some cDNA clones show an infiame insertion. Further studies using genomic for MS or LHON, although the nt 11778 mutation was present in both the pa- DNA are in progress to confirm this observation. tient's mother, sister and brother, albeit in lower amounts than in the proband. In none of several hundred control individuals was the nt 11778 mutation ever found. Three additional patients, a male and two females, carried a mtDNA mutation at nt 15257. Although our sample is relatively small, the frequency of this mutation in our MS population is higher (6%) than that found in our controls (1/50= 2%). No nt 3460 mutation was found in either the MS or the control populations. Our data support the idea that LHON-associated mtDNA mutations may indeed contribute to conferring genetic susceptibility to MS in selected individuals.

1950 1951 Asseiation of dopumiue receptor genotype wit rsponse to Ckeapie ia a pr Autosomal recessive familial intestinal polyatrsaia po dis F Craf J Ferrro J Hoeae A Osborne. P Dim M Mh syndrome (71ZP) is not caused by common mutations Reskmp Laboratories, lastitute for Research in Psychiatry, Umivriy ofSouth Florida, in the CFMM gene. M.J. Dasouki and C. L. Vnencak-Jones. Ta, orida 33613. Vanderbilt University. Nashville, TN. The dopene been in because syatain has implicated the eology ofsciohr of Familial intestinal polyatresia (FIPA) is a rare autosom- deaiityof aipschticdrugsto blockdcamie receptors. Molear enetic adhiqum al recessive developmental defect of the gastrointestinal have prei ly boe applied in association studies between the disse and van in a tract that causes neonatal intestinal obstruction due to nubr ofthe ges frter dopamne receptors. The DRD2 ad DRD4 genes are onthose multiple atresias. The genetic defect in this syndrome is which contain variable sequences which have boe used fbr .nolecula genetic yet to be found. Meconium plug syndrome secondary to cystic have investigated possible association betweenpolytnorhisms in dese Ms and zophrenia fibrosis also causes intestinal obstruction in the neonate. in a cohort ofpatients. In both genes the pymoisms tuied a found in thethird The CFTR gene and a locus linked to the autosomal recessive cytoplasmic loop ofbherceor, a reion kwnto b criil fr-prtin ad sO dhe severe combined immunodeficiency (SCID) syndrome gene are variations nwybeathoting signal transduction byalering the G-prote aci4. We suspected as candidate loci. In this consanguineous ailedto deect s a disortion of lec between shi nic d non- Palestinian family, three infants (2 boys and one girl) amy frequcies presented during the neonatal period with signs and shizophrenicase. In addition we observedthe patient's responseto C a an atypical symptoms of intestinal obstruction. Multiple intestinal cwhich is effacio inte treaunent of schizophreniap S Capi atresias were found intra-operatively and all three diaa a hir affinity (lOx) for the DRD4 receptorthan for DRD2 or DRD3, and di children died within the first 3 months of life. Both doespim bindn characeristics betwe al;ic recepor variants ofDRD4 have bee noted. parents as well as an unaffected 5-year old daughter were We investigatd the efc ofthe oombined DRD2/DRD4 gnotype in reation to the pates available for evaluation. To rule out CF, the parent's response to dcozapin. Prei ary resules u thattr is an associsuo between response genomic DNA was isolated and analyzed by PCR for the F508 to cloespine medicatio and DRD2/DRD4 genotp. and W1282X mutations. These represent the most common Rebreaces: 1. O'Dowd, B. (1993). JournlofNeurochendstry, 60;3, 804- mutations in the CFTR gene in this population. Neither n mutation was detected. These findings support autosomal 816. 2. Arinari TTIokwaM,K EMNg etal (1994). LanCet 343, recessive inheritance of this disorder and suggest that 703-704. 3. Licker J, Barr C, Kae dy J, et al (1993). Hum. Mol. Genet. 2, FIPA is a distinct disorder from cystic fibrosis. 6,767-773. 4. 0 Dowd B, Hnatowich M & Lefowitz R. (1991). In Encyclopaeda ofhuman bfology 1:81-92. eds Dulbeoco, Academic Press, San Diego. S. Meltzer, H. Y. (1991). SchizophreniaBullein 17:2, 263-287.

1952 1953 Gen*c coa of gm epresn in Autoamel Desndiat Pelycyc Ki Disese. Differences between aosaicism in blood cells and skin fibroblasts suggest A. Dobin' P. A. Gabow2. A. M. Johnsonand W. J. Kimberfing'. 'Boys that kin may better predict brain function. C. S. Dobltdn'. S. L. Nolin'. 1. Town Nadtonal Reearch Hospital, Omaha, NE and 2Dep nt of Medicine, University of Cohen'. V. Sudhalt&. M. G. Bialere. X. H. Ding'. E. C. Jenkins'. N. Zhong' and Colorado Health Science Center, Denver, CO. W. T. Brown'. 'NYS Institute for Basic Research in Developmental Disabilities, Clinical expresion of Autosomal Dominant Polycystic Kidney Disease (ADPKD) vanes Staten Island, 2North Shore University Hospital, Manhasset, New York. not only between families, but even among patients within the same family. One obvious An estimated 12 to 41% of affected fragile X males are mosaics who source of such variation is due to "normal" allelic differences at either or both of the two carry both a nfull mutation" FMR1 allele which has no gene expression, and a maor Ioci involved in ADPKD. We propose to use the genedc marker data to determine "premutation" allele which usually has normal expression. We compared the whether two affected sibs that share the same "normal" PKD1 marker(s), transmitted from the mosaic X males to see the DNA in blood cells and skin fibroblasts from four fragile u d parent, are more similar in clinical symptoms than pairs whose "normal" PKDI if there was a difference in the relative amounts of premutation and full mutation homosomes differ. alleles within the tissues of individuals. Two of these males showed nine sib from 17 families were classified into two to these Forty pairs PKD1 groups according differences in the ratio of to full mutation in different tissues their identy by descent for the markers on their normal chromosome 16 (identical, group 1, strildng premutation and non-identical, group 2) and the difference between sibs were calculated for each of the sib while the other two showed only slight differences. Interestingly, behavioral pairs for several clinical parameters, inchding scam creatinine (SC), creatinine clearace evaluation suggested that the proportion of premutation in skin correlates with (CC), urinary protein excretion (UPE), kidney voume (KV). For each parameter the group I higher functioning. These observations support the hypothesis that the fragile X sib pairs, sharing the same normal alele, had less variability between siblings, than group 2, CGG expansion occurs somatically during early embryogenesis and that the having dffe normal alleles. Furtmoe.sib pair difference in group 2 were greater tha mosaicism in brain and skin, which are boh ectodermal in origin, should be in group 1. The men values with their appropriate standard errors for all sib pairs are: similar to each other but potentially different from the mosaicism in blood which 0.37±0.087 vs. 0.55±0.136 for SC, 21.39±3.25 vs. 24.50±4.24 for CC, 132.56±45.50 vs. is not ectodermal. 171.90±63.23 for UPE, 245.65±32.39 vs. 317.14±60.58 for KV for groups 1 and 2 A study of the rate of adaptive skills development in 51 affected males resptively. While the differences do not attain the statistical significance, the tend is indicated that the rate of development in non-autistic males was significantly towards the expected under the hypotesis that the normal allele influences the expression of greater in mosaic cases compared with full mutation cases. Taken together these the mutant gene. The lack of statistical significance is believed to be due to the limited sample results suggest that the presence of mosaicism is associated with a higher level of in this pilot study. Thus, our preliminary results are consistent with the hypothesis that the functioning and that mosaicism in skin fibroblasts may be a better predictor of severity of the disease influeced not only by the culprit gene, but also by its normal allele or brain function than the mosaicism in blood cells. by their inteaction. Published Abstracts: Molecular Pathogemnesis and Treatment (continued) A337 1954 1955 Quentitation of HEXA mRNA in fibroblasts of Tay-Sachs Diseases pa- Association between mutation size and cognitive/behavioral deficits in fra(X) tients and in a late-onset GM2 gangliosidosis patient with a novel mu- males: A prospective multicenter study. Gene S. Fisch. Nancy Car ter. Patricia tation. M Fernand=s, B. Boulay, P. lHechtman, F. Kaplan. McGill Univ. N. Howard-Peebles. Anne Maddalena. Richard Simensen. Jack Tarleton. Con Julien- Mutant alleles at the HEXA locus cause Tay-Sachs Disease. Mutations com- Inalsingh. Maryse Chalifaux. Jeanette J.A. Holden. SUNYIHSCB and Kings County patible with low expression of the Hex A gene product cause less severe or later- Hospital, NY; Chapman Institute, OK; Genetics & IVF Institute, VA; Greenwood onset neurological phenotypes. Quantitation of gene expression is, therefore. an Genetics Center, SC; Ongwanada Resource Centre, Kingston, Ontario, Canada. important component in understanding genotype-phenotype correlations. We have Previously, researchers reported molecular-neurobehavioral or identified a novel mutation in the HEXA gene (IVS7. -7 G->A) which is the cause molecular-behavioral associations in individuals with the fra(X) mutation. As a result, we examined of a chronic form of GM2 gangliosidosis associated with muscle wasting but not prospectively 31 fully mutated fra(X) males from 4 testing sites, ages 4.5 - 14 years, with intellectual deficit. This mutation causes both a profound reduction in the to determine if there was a relationship between mutation size and extent of either amount of HEXA mRNA and also the appearancte of a second, abnormally spliced, cognitive or adaptive behavior deficit. To measure the extent of cognitive deficit, all exon 8 is deleted. A new 3' site is by this muta- mRINA in which splice generated individuals were administered the Stanford-Binet (4th Edition) IQ test. To evaluate the new site is not used. Fibroblast mRNAs were tions, however the splice quantitated degree of adaptive behavior (DQ) deficit, all individuals were assessed with the by competitive PCR using synthetic HEXA mRNA standards containing a 322bp Vineland Adaptive Behavior Scales. To determine fra(X) status, DNA from all insert derived from pUC 19 allowing distinction between RTPCR products ampli- individuals was obtained using identical molecular protocols; gels were run at a single fied from mixtures of standard and fibroblast mRNA. Normal control fibroblast site. Members from each site, blind to the subject's identity, estimated the mutation HEXA mRNA was 17.3 pg/ug total cellular RNA (29% poly A+). In a fibroblast. size in each lane of the autoradiographs. The median value of the 4 mutation size cell line heterozygous for the most frequent classical infantile TSD mutation (ins estimates for each subject was used in the analysis. Correlation coefficients between 1277) the steady-state level of HEXA mRNAs were 8.4 pg/ug normal RNA and median mutation size and IQ score reveal a non-significant, near-zero association 0.33 pg/ug for the mRNA species carrying the insertion mutation. By contrast, (r=0.07; p>.72). Correlation between mutation size and DQ also shows a non- a fibroblast cell line obtained from a French-Canadian TSD patient homozygous significant, near-zero association (r=0.02; p>.92). We conclude that, while the for the 7.6kb deletion, a mutation incompatible with mRNA expression. showed no fra(X) mutation produces cognitive and behavioral deficits in individuals who inherit HEXA mRNA (<0.052 pg/ug cellular RNA). Cultured fibroblasts from the chronic the defective gene, there is no relationship between mutation size and degree of deficit. GM2 gangliolsidosis patient, who is a compound heterozygote for the ins 1277 mu- tation, produced 0.46 pg/ug of the ins-1277-HEXA mRNA species and 1.04 pg/ug each of normal and exon 8-deleted mRNA from the IVS7,-7 G- > A allele.

1956 1957 Yeast artificial chromosomes as vectors for gene transfer into human Dystrophin transcripts analysis from muscle and lymphocytes in asymptomatic neuroblastoma cells. C. T. Fong. S. Pollock, H. Dostman and B. French. Uni- BMD deleted patients. G Galluzzi*O. R De Leo". A Morrone*. E Vigneti*. S. versity of Rochester School of Medicine and Dentistry. Rochester. New York. ServideiA and L Felicettie *Institute of Cell Biology, CNR, Rome ° ULDM-Rome Due to their large cloning capacity, yeast artificial chromosomes (YACs) are Section 'Institute ofNeurology, Catholic University, Rome. potential vectors for the simultaneous transfer of multiple genes into mammalian Deletions in the distrophin gene are the most common mutations (65%) causing and Becker muscular The deletions result cells. We have retrofitted a YAC (yl H7) containing 130 kb of DNA from the Duchenne (DMD) (BMD) dystrophies in an absent (DMD) or an abnormal (BMD) protein product in the skeletal human N-myc gene region with the neomycin-resistance and beta-galactosidase muscle, depending on whether the deletion breakpoints disrupt (DMD) or genes by homologous recombination in yeast. Yeast cells containing the retrofitted maintain (BMD) the open reading frame of nucleotide triplets. The clinical range YAC (yllH7-neo-gal) were fused with the human neuroblastoma cell line NGP varies from the most severe DMD form to the mildest BMD, which includes also by spheroplast fusion. Selection with G418 was applied. After 3 weeks of selec- asymptomatic phenotypes. In addition to typical DMD/BMD patients, we studied a tion, G418-resistant colonies were obtained from the yllH7-neo-gal/NGP fusion at group of Italian families, in which several male subjects, aging from 7 to 60 years, approximately 40 colonies per million NGP cells. No G418-resistant colony was showed increased CK levels, mild calf hypertrophy, typical BMD obtained from the yllH7/NGP fusion (control). We are currently studying the immunocytochemtical pattern and deletion in the central rod domain of the stability and level of expression of the introduced genes. as well as the physical dystrophin gene. All ofthem were asymptomatic: strength was normal and they did configuration of the transferred YAC. not complain any cramp or muscle fatigue With the aim of investigating a possible genotype-phenotype correlation we analyzed by RT-PCR and cDNA direct sequencing, the dystrophin transcripts derived from muscle and/or lymphocytes in a sample of these subjects with the following genomic in frame deletions: exons 48-49 (4 patients), exon 48 (3 patients), exons 45-51 (2 patients), exons 45-55 (2 patients). In all ofthese patients the transcripts analysis confirmed at mRNA level the genomic rearrangements the sequence of the cDNA fragments encompassing each specific deletion showed that the mRNA deletions corresponded to the DNA deletions. No alternative products were found Our results suggest that the occurrence of such asymptomatic phenotypes associated with these particular deletions, is consistent with the rule of the in frame deletions, but further non transcriptional regulation mechanisms should be involved.

1958 1959 Analysis of a poly-T variant In Intron 8 of the CFTR gene In Italban patients Unusual ataxic phenotype in a patient with abnormal (CAG)n with ConnItal Bilateral Absence of the Vas Deferens (CBAVD). S. expansion at the HD locus. C Gellera. M. B. Zappacosta. F. Girotti. C. Garnrmn *, C. Ardulno A, D. Fontana **, L. Rolle a, M. Manenti A, C. Meoni B C&Atellotti. F Taroni. and S Di Donato. Istituto Nazionale Neurologico Bombieui &, M.G. Benetazzo &, P.F. Pignatti &, A.O. Carbonars *. W. '"Carlo Besta", Milano, Italy. Genews, Biology and Modsal Cheistry, CNR C/OS, UnWere of Tunn, Italy, Abnormal expansion of (CAG)n repeats in the coding sequence of IT15 gene is associated with ^ Medical Genedcs Service S. Giovanni Hospital, Tuin, Div. of Huntington's disease (HD). HD is a late onset neurodegenerative Uroogy, S. Luig Hospitl, Oibassano; Sero of Androgy ,S. Anna autosomal dominant disorder of the central nervous system, characterized by choreo- Hospital Turn; & Inst ofBiology and Genetics, University of Verona, Italy athetotic movements, intellectual decline, and mood and behavioral changes. Autosomal dominant neurological disorders caused by abnormal expansion of 70 % of with translated (CAG)n repeats in different genes include two cerebellar ataxias (SCAI and patients Congenital Bilateral Aplasia of the Vasa Deferentia SCA3) and dentatorubral-pauidoluysian atrophy (DRPLA). We present a family with CBAVD) have mutations on the Cystic Fibrosis gene (CFTR). The most an autosomal dominant disease characterized by a combination of ataxia, frequent mutations found in CBAVD patients are AF508 and RI 17H. parkinsonism and chores. Involuntary movements, bradikinesia, gait disturbances, 11 patients CBAVD have been invstigated for the presence of the mutations and cognitive difficulties were present along three generations, suggesting familial AF508 and RI 17H and for the other mutations more frequent in the Caucasian Parkinson's Disease (PD). In the index patient, gait ataxia, dysarthria, and occasional population. All patients and their relatives have also been typed for the variation involuntary movements were noticed at age 52. The patient was deemed to have PD. in ienght of the polypyrimidine tract in the spke-acceptor site in intron 8. It is At age 57, HD was suspected because of the ensuing of choreic movements in both known that one variant, the 5 thymidine (5T), is associated with high lavels of hands. Clinical examination at age 59 disclosed marked ataxia of the gait, dysarthria CFTR transcript missing a critical region of the gene (exon 9) which encodes a with rhythm speech motor abnormalities, rare involuntary movements of the fingers. non functional protein. and irregularities in slow tracking eye movements. The patient did not show Comparing the distribution of the alielic frequencies of the ST, 7T and 9T myoclonus, dystonia, and tremor. A complex task of cognitive tests showed only vanants in the CBAVD patients with that of a normal control population it results mild cognitive deterioration. CT scan and MRI showed cerebral and cerebellar that the frequency of the ST variant in CBAVD patients is significantly increased atrophy, and bilateral hypotrophy of the caudates and putamina. Genetic studies were 6/22 alieles (27%) in CBAVD patients vs 41128 (3%) in controls ( pa 0.0022). focused on mutations involved in SCAl and SCA3, DRPLA, and HD. Analysis of These results suggest that the ST alele could be one of the genefic factors the patient's DNA showed that the (CAG)n repeats in the SCA1, SCA3, and DRPLA contributing to clinical phenotype. An extsive study of CFTR haplotypes with genes were within the normal range. Interestingly, a pathological expansion was at the HD locus intra and extraknic markers in CBAVD patients and on patients affected by found (44 CAG repeats; 10-36 repeats, normal range). Thesedata Chronic demonstrate the existence of overlap syndromes among different movement disorders Obstructive Pulmonary Disease (COPD) could verify the possible in those caused mutations". influence of different chromosomal background to the clinical phenotype of humans, including by "dynamic diseases associated to mutations on the CFTR gene. A338 Published Abstracts: Molecular Pathogenesis and Treatment (continued) 1960 1961 Another look at a family with t(8;12) and hereditary spherocytosis. Proximal trisomy 15 aesooated with Angelman phenotype In V. J. Harris, L. M. Pasztor and R.A. White. Section of Genetics. The Children's monezygooutwinsD. f ronlj.-P.HainU&rgol I. Henrv2J S. Lego 1. M. Mercy Hospital and the University of Mlissouri-Kansas City. Prudent . A. Munnch3. and J.-P BonnefonO. 1:Servic de Pdliatrie The family was reascertained when the new proband was one dayold because of a Gdndtique et de Cytogdndtique, H6pital Pl6-Salpdtrlbre. 2: UnitA INSERM U 383 3: Sorvices de et de Biochimie, H6pital Necker ventricular septal defect, coarctation of the aorta. toe webbing, and a dilated renal et, Gdndtique Enfants Malades, Paes, France. on included frontal and pelvis prenatal ultrasound. Other findings bossing microg- Angelman syndrome (AS) is a cause of severe mental retardation nathia. The fact that the mother of this infant was part of the kindred reported (1/20,000 births), usually arising from a maternal deletion or from a by Kimberling, et al (Am J Hum Genet 27:586, 1975) was not disclosed until the patemal uniparental disomy for chromosome 15qi 1-13. presence of an unbalanced translocation was suspected on the preliminary micro- We report on a coupleof monozygous twin-boys with an AS phenotype scopic analysis of lymphocyte chromosomes. The study was undertaken to search associating severe mental retardation, absent speech, ataxic gait, for del(22). Analysis of maternal blood confirmed that the proband's karvotype is inappropriate laughter and characteristic EEG pattern at four yearsof age. 46,XY,- 12+der(12)t(8;12)(pll.2;pl3.2)mat. This infant does not feed well. and Microsatelite analysis of the 15q1 -q13 region revealed the presence of has had recurrent apnea. A head ultrasound is normal. The family seemed un- one paternal allele and two maternal alleles (maternal heterodisomy) in aware of the reproductive risks of the translocation carrier state, but were well both siblings. These molecular data have been confirmed by high- informed about hereditary spherocytosis. As the ankyrin gene maps to 8p11 2. resolution G-banding chromosome analysis showing a supernumerary chromosome. we have started to investigate the mother who has the balanced translocation and 15pl3-q14 These data indicate that either double or absence of maternal spherocytosis, as the the latter may be due to disruption of the coding sequence. dosage chromosome 15 can result in an AS SDS-PAGE of red cell ghosts stained with Coomassie blue demonstrated that both q11-q13 phenotype. spectrin and ankyrin were present. There were three additional bands that were also demonstrated on Western blotting with anti-ankyrin antibody. A Southern blot with pAnk58 was normal, while a single band was deleted when probed with pAnkib. The protein analysis suggests alternative spliceforms whose origins are unknown. Previously studied families have shown a deficiency of ankyrin. It is likely that the altered proteins are mechanically unstable or have aberrant binding properties to the other proteins of the membrane cytoskeleton.

1962 1963 A Cytochrome P4. genotype and pesticide exposure as predictors of Parkinson's Widespread tissue distribution and high percentage of an to G dimsea dementia. LL Hubble. SL. Glatt1. MC. Kurth GD. 5chllnbler R..S. transition at nt3243 of mitochondrial tRNALeu(UUR) gene associated Hassaein'A. Lieberan2 W.C. KoLer'. JR Kut. 'Univ. Kansas Med. Ctr., with clinico-pathologically defined Leigh's disease. a final common Kansas City, KS; 2Barrow Neurol. Inst., Phoenix, AZ; 3Univ. Washington, Seattle, WA. outcome for severe defects in OXPHOS genes. Y KoRa. T. Matsuishi. M. Parkinson's disease (PD) is a neurodegenerative disease of the elderly with probable Yoshino and H. Kato. Department of Pediatrics and Child Health, Kurume University M. Eand genetic risk factors. PD is a motor disorder, but clinical School of Medicine, Kurume, Japan. (Intro. by Nakao) dementia plays a significant role in some patients. The dementia of Parkinson's disease Subacute necrotiziog encephalopathy or Leigh's disease is a clinico-pathologically (PD+D) may be associated with risk factors distinct from PD without dementia (D-D) defined syndrome, characterized by early onset developmental deterioration, bilaeral necrosis and/or of the ccntral nervous associated with various defects in or the dementia of Alzheimer's disease (AD). We have examined enviro el, gliosis system, sociodemographic, and clinical variables as predictors for PD+D and found that lower oxidative phosphorylation (OXPHOS). There are three mitochondrial point mutations educational attainmnt, advanced age of onset, and greater motor imnpairment reported to cause this disease including T8993G and T8993C in mitochondrial ATPase 6, and A8344G in We analyzed a three year-old floppy infant who was differentiated PD+D and PD-D populations. We hypothesize that exogenous factor(s) tRNALys. as Leigh's disease and died of 10 failure. He interact with genetic element(s) to increase the risk ofPD+D among all PD patients. In diagnosed at age years by cardio-intestinal had recurrent lactic acidosis and intractable seizure as Lennox-Gastaut syndrome, but this sudy, we re-anabyed our previously described poopulations of 43 PD4-D and 51 PD- had no stroke-like Pathological findings of tissues at autopsy revealed D subjects by ain assessi environmental, sociodemographic, and clinical variables in episodes. showing bilateral necrosis and of the co~nction with 3 gene polymorphisms previously shown to be associated with an characteristic features of Leigh's disease gliosis mid brain. Muscle biopsy at 3 years of age showed ragged-red fibers (5% of total muscle nred risk ofPD or AD - cytochrome P4so poor debrisoquine metabolizer genotype fibers), scattered COX negative fibers and SSVs, pathologically, and complex 1 (15% of (CYP2D6 29B+), monoamine oxidase B (MAOB-1), and apolipoprotein E4 (ApoE4). the control) plus IV (10% of the control) deficiency, biochemically. Mitochondrial Initialy, variables were entered singly into a multivariate model. Education, age of onset, analysis in various tissues including skeletal muscle, liver, heart, kidney and and motor impairment were again predictors of PD+D. All other variables, including genomes brain obtained at autopsy showed high percentage (>90%) of an A3243G mutation in the genotype at the loci described above, were not predictive of PD+D as single risk factors. mitochondrial tRNALeu(UUR) gene in all tissues examined. RNA analysis showed Environmental and genetic varisbles were then entered intandemto assess potential consistent increase in the steady-state levels of an RNA 19, which is a transcript variable interactions. This analysis revealed that subjects with at least 1 CYP2D6 29B+ corresponding to 16S rRNA + tRNALeu(UUR) + NDl. Single fiber PCR analysis of and pesticide exposure had an 83% predicted probability ofPD+D (stepwise logistic COX-positive and COX-negative fibers using muscle specimen showed high point regrion model: This the that genetic elements in p-0.0491). study supports hypothesis mutation (>90%) in both groups. Above findings indicate that the Leigh's disease tandem with specific environmental factors distinguish PD patient populations with phenotype is a final common outcome for severe defects in OXPHOS genes. dementia from those without dementia.

1964 1965 Genetic evidence of differences in Parkiason's disease sIusceptibfity by CYP2D6 hytetical GeEnvironmnt lnteacioanod for Bipolar Dsorders.EM MC Cavallini. C Namy". Q Cadisl L Franchini- M C Mairin. A and MAOB genotypes MC LK Mirth'. J.P. Sime, B. Mmciardi. E Smeraldi Istituto H. San Raffaele, Deptment of AN. Liebm ' W.C. Koller2. 'BarrowNeurological Insituta; Phoenix, Arizona; Serretti. Sciendfico 2University of Kansas Med. Center, Kansas City, Kansas. Neuroscience, University of Milano School of Medicine, Milano, Italy. The cause ofParkdnson's disease is but likely involves environmental (PD) uncertain, are and genetic risk fictors. Several studies have shown that an allele of the cytochrome P4e0 Both the presence of a genetic component and environmental factors nes of mood disorders. We have CYP2D6 gene (29B+; associated with a poor debrisoquine metabolizer phenotype), and somewhat required to explain the eiopa a theoretial model of gen-environment interacin accounting for an allele ofthe moamine oxidase B gene (MAOB1) are associated with PD. Inheritance hypothesized susceibility to Disorders. In our the genetic component is alleles independent PD Bipolar hypothesis ofthese may signal a 2-fold increased risk for PD. An population in by candidate' genes coding for receptors or enzymes involved from Kansas (KS) was studied to determine if these alleles are associated with PD, as had represented monminergic DRD4 receptor ges and Tyosine previously beem demonstrated with PD cohorts from Txas (TX) and Arizona (AZ). KS systems (DRD2, DMD3, gene), while the environmental component Is represented by PD patients were Cauci had at least two of the three cardinal signs of PD, and Hydroxylase variables related to a "subjective" enviment the individual social sustined response to levodopa therapy. DNA from 81 male and 32 fenale patients psycho-social the dege of Self Esteem and the personality structure, with peOcal allowed alysis of 224 chromosomes for 29B+ and 133 chromosomes for MAOB1. adjustment, attention to the presence of Personality Disorders. The modld describes the following Control population consistd of 78 age and geographically matched individuals without inclusion sgns noted above. The PD MAOB1 allelic frequency was 0.504, not relationships: - Two genetic and partially overppin components directly increase susceptibility significantly different from control frequency of 0.474 (p=0.67). MAOB1 frequencies both to and to Personality Structure. were identical for the KS and TX control populations. MAOBI fiequencies for the PD Bipolar DIwrder A Structure leads to individual low social adjustment were different. The Kansas PD 29B+ allelic was maladaptative Pasonality tie populations significantly frequency and self estem, which then increase the liability to Bipolar disorders. 0.201, not significantly different from control frequency of 0.209 (p-0.92). We compiled We collected a sample of 100 Bipolar patents typed for the candidate genes allic data from our studies and the literature for additional firquency population 7.16 statistical and measured for the psycho-social variables. Using the LISREL comparisons different locations. Frequencies of between populations from geographic model 83.62 packae, we found a good agreement of our data to the theoretical (*2- 29B+ in the control were nearly identical, with a variance of 0.002. In populations with 24 d.f, GFI- 0.867, AGFI- 0.750, RMSR- 0.111). These preliminary contra, variance of29B+ the PD was 0.012, which is frequencies among populations results suggest that a specific genetic structure could interact with non genetics factors greater than for controls We that genetically different significantly (p=O.03). hypothesize to of the disease in a complex model, providing a possible rationale the susceptibility environmental stressors which with location where PD populations are selected by vary suitable for and maldng the model further extensions. the controls remain similar, supporting the notion that individuals are affected with PL as a result ofenvironmental factors and genetic predisposition. Published Abstracts: Molecular Pathogeanesis and Treatment (continued) A339 1966 1967 Fibrillin (FBN1) mutations in the "neonatal region": Toward geno- Semi-quantitative analysis of cytokines in inflammatory type/phenotype correlations in neonatal Marfan syndrome. K. Mathews, demyelinative lesions in X-linked adrenoleukodystrophy and M. Wang, C. K. Corbit, and M. Godfrey. Unix. Nebraska Med. Ctr.. Omaha. multiple sclerosis. 1,2M. C. McGuinness, 2D. E. Griffin. 3J. M. Powers, Fibrillin (FBNl) mutations are now well described in the Marfan syndrome 1,2H. W. Moser, and 1,2K. D. Smith. 1Kennedy Krieger Institute and 2Johns (MFS). MFS is an autosomal dominant heritable disorder of connective tissue man- Hopkins University School of Medicine, Baltimore, MD, and 3University of ifested by variable and pleiotropic defects in the skeletal, ocular, and cardiovascular Rochester, Rochester, NY. An and more severe that includes The childhood cerebral form of X-linked adrenoleukodystrophy (X-ALD) systems. early presentation joint contractures. with cerebral inflammation is the most common form X-ALD while abnormal multi-valvular disease. and of the adult facies, early death. usually due to congestive form, adrenomyeloneuropathy, with little or no cerebral involvement is the heart failure, has been termed neonatal Marfan syndrome (nMFS). These individ- second most common form. The reason for the variable phenotypic uals too, have FBN1 mutations, but most appear to cluster in a relatively small expression in X-ALD is unknown but segregation analysis suggests that an region of the gene, between exons 24 and 32, with most occuring between exons autosomal modifying gene is involved. Such a modifying gene might be 24 and 27. Using intron specific primers and MDE gel heteroduplex analysis we involved in initiating/ promoting the inflammatory response. The inflammatory have identified FBINl mutations in three additional MFS patients in exons 24 and nature of the demyelination seen in the cerebral form of X-ALD distinguishes 26. A G3037A transition resulting ini a G1013R substitution was found in exon 24, it from the other inheritable leukodystrophies and aligns it more with multiple a transforming growth factor-binding protein (TGF-bp) like repeat. Two patients sclerosis (MS). In X-ALD inflammation tends to be found behind the active had mutations in exon 26 a calcium binding epidermal growth factor (EGF) like demyelinating edge of the lesion and in subsequent secondary tract domain. One, had a T3258G transversion in a C1086W amino acid sub- degeneration, while in MS the inflammatory infiltrates are located at the resulting active demyelinating lesion edge. In an effort to better understand the stitution. This change was not found in either parent. An additional child had two inflammatory demyelinating process in X-ALD and MS we compare cytokine changes in the same allele. She had a T3212G transversion resulting in a 11071S expression in inflammatory demyelinating lesions taken post mortem from amino acid substitution and an A3219T transversion resulting in a E1073D amino patients with both diseases. Ribonucleic acid was isolated from histologically acid substitution. The possibility that one is a polymorphism in the black pop- defined regions of X-ALD and MS brain tissue and mRNA levels for ulation remains. Parental studies are pending. These findings provide additional interleukin-lp (IL-1a), interleukin-2 (IL-2), intedeukin-4 (IL-4), interleukin-6 (IL- evidence for the biologic significance of fibrillin in general and the "neonatal region" 6), interferon-y (IFN-y), tumor necrosis factor-a (TNF-a) and TNF receptors in particular. and 11 (TNFRI and TNFRII) were quantitated using standard procedures. IL-1i, IL-6 and TNF receptor and 11 mRNA levels were increased in MS lesions but not in ALD lesions. There was no correlation between TNF-a, IL-2, IL-4 or IFN-y mRNA levels and phenotype. These results suggest that inflammatory responses leading to demyelination in X-ALD and MS are distinct.

1968 1969 DNA hairpins:a cause for expansion and human disease. McMurray CT, Fanconi anemia fibroblasts from complementation groups A and D Gacv, A, Goellner, G, Juranic, N, Macura. S. Mayco Foundation. have normal sensitivity to MMS and hydrogen peroxide. R. E. Moses, L. We show that repeating units from all reported expansion disease genes are ca- S. Merkens. M.T. Piper. Molecular and Medical Genetics, Oregon Health Sciences of of common structure and University, Portland, OR. pable forming hairpins stability. The threshold energy Fanconi anemia (FA) is an autosomal recessive disorder. Although the basic for expansion to become probabale is roughly -50kcal/hairpin. and the threshold molecular defect is unknown, cells from FA patients show increased sensitivity to energy is influenced by the flanking sequence of the gene. Hairpin stability has two DNA-crosslinking agents such as mitomycin C (MMC) and diepoxybutane (DEB). components. sequence and length; only DNA of correct sequence and the correct This disease may be the result of a defect in DNA repair. We have measured the effect length have the ability to from hairpins of threshold energy. There is a correlation of two agents, methyl methanesulfonate (MMS) and hydrogen peroxide (H202). the to form of threshold the MMS causes bulky adducts and producers apurinic sites requiring base excision among ability hairpins energy. sequence selectivity of repair. H202 damages DNA via free radical formation. One FA primary fibroblast expansion, and the length dependence of expansion. Additionally, hairpin forma- cell line (PD20) and three different FA immortalized fibroblast cell lines (GM6914, tion provides a potentail structural basis for the constancy of the CCG region in PD224 and PD20) were used. GM6914 and PD224 are cell lines from Huntington's disease gene and the stabilizing effect of AGG interruptions in FMR1 complementation group A and PD20 is from complementation group D. Two alleles. Hairpin stability may determine the threshold and potential for expansion different control cell lines were used: GM639 and a fused cell line GM6914xPD20. and human disease Both showed normal sensitivity to MMC and DEB. Five days after treatment of fibroblasts with MMS (50mM to 400mM) or H202 (50mM to 300mM) the number of live cells was measured with a MTT (3-(4,5-dimethylthiazol-2-yl)2,5diphenyl- tetrazolium bromide) colormetric assay. There were no differences in survival rates among any of the FA cell lines or control cell lines for either MMS or H202. Attempts to show the adaptive response with MMS were not successful. We conclude the FA molecular defect does not lead to defective rapair of DNA damage caused by MMS or H202.

1970 1971 Relevant Issues In the Identfleatlon of Intermediate alleles forthe CTG expansion Mutations underlying heat lability of P-hexosaminidase (Hex) B. G. Narkis*#_ L. In Huntington disease. O.T. Mueller'2. T.M. Diamond2 and F.V. Schaefer3. 'All Jaberi# and R. Navon*#.*Molecular Genetics Children's Hospital, St. Petersburg, Florida, 'University of South Florida, Tampa, laboratory, Sapir Medical Center, Kfar Florida, and 3Children's Medical Oklahoma. Sava, Israel and the Department ofHuman Genetics, #Sackler School ofmedicine, Tel- Center, Tulsa, Aviv Israel. .The An unstable CTG expansion in the IT15 gene encoding the protein huntingtin University, Tel-Aviv, Taibe Bridge to Peace Pediatric Center and identifies the vast majority of mutations responsible for Huntington disease. There is Comunity unit Shneider Children Hospital, Petah-Tikva, Israel. variation in the number of CTG repeats in normal individuals (5 to 34) whereas the Different mutations in the HEXB gene cause heat lability of the lysosomal enzyme repeat number In affected subjects is usually greater than 37. Individuals with alleles Hex B with extremely variable consequences. We report here on identification of two between 30 and 40 (intermediate alleles) have an uncertain clinical prognosis. such mutations in homnozygotes, the one being lethal while the other is totally harmless. Out of 98 families with HD, we have encountered two subjects with intermediate 1. Momoi et al (Pediat Res 12:77, 1978) reported on two Japanese siblings, one sized alleles. Case 1: a subject with HD onset at the age of 39 was identified with who died of disease with heat-labile Hex B and the other CTG alleles of "Tay-Sachs" with the same repeat 45 and 17. Both parents are in their late 70's. One has Hex deficiency who was aborted. We this fetus' DNA and revealed progressive mild dementia without and has CTG alleles of analyzed chorea 33 and 20. The homozygosity for a A619iS G mutation in HEXB 5), first reported by Banjeree other parent had no family history and has alleles of 17 and 18. Two other sibs are (exon at risk and to be tested et al (BBRC 181:108, 1991).Incidentally we found the same mutation in heterozgously agreed anonymously without disclosure of results. Case 2: a Ashkenazi Jews. 61 year old subject with progressive chorea and dementia of HD was found to typical 2. Navon et al concluded have CTG allele sizes of 38 and 41. There is family history through only one parent (AJHG 37:138, 1985) that two related Israeli Arab (both deceased). There are no sibs but there are five offspring between the ages of perfectly healthy children, with no Hex A activity by 4-MUG were homozygotes for a 18 and 29 who have declined testing. The smaller intermediate-sized allele places the heat labile Hex B mutation. We can now confirm it and report on a novel mutation offspring at 50% to 100% risk. The results were disclosed only as positive for HD; responsible for this phenotype: it is a G1627-+A substitution at exon 14 of HEXB allele sizes were not identified. resulting in Ala543-+Thr changerWe also found the same mutation in 9 heterozygous Counselling family members with alleles between 30 and 40 poses a number unrelated Israeli Jews of who most of them originated from various middle eastern challenges:1) inconclusive prognostic information for family members; 2) the need countries some east for accurate of and from Europe. sizing alleles which would allow compilation of case Information A third mutation Hex B between laboratories; further due to in allele causing heat lability is G1514-+A substitution in exon 13 of 3) uncertainty integenerational changes HEXB. Bolhuis et al found it in sizes which can significantly aiter the disease coursd in offspring. These cases (BBA 1182:142, 1993) compound heterozygosity with illustrate the need for a concerted effort to compile HDsubjects with intermediate a lethal HEXB mutation, in a patient suffering from adult type ofSandhoffdisease. alleles in order to identify any modifying factors, whether genetic or environmental, and It is obvious that proper evaluation offinding ofheat labile Hex B must be based on to accurately predict risks to offspring. molecular characterization ofthe underlying mutation. A340 Published Abstracts: Molecular Patthogenesis and Treatment (continued) 1972 1973 HLA-DQ association determines organ-specificity for IDDM but not for The AMOE locus influences the age ofonset ofFamilial Alheimer's Disease synergistic autoimmunity in IDDM and Graves' disease. Masami Nemoto, in a pedigree unlInked to any known camative loc A Osborne. D Falib. P K. Mizobuchi, Takashi Sasaki and N. Tajima. Department of Internal Medicine, Dia. F Crawford. M Mullam Roskamp Laboratories, Institute for Research Jikei University School of Medicine, Tokyo, Japan. in Psychiatry, University ofSouth Florida, Tampa, Florida 33613. Insulin-dependent diabetes mellitus (IDDM) is an autoimmune disease with Familial Alzheimees disease is etiologically heterogeneous. Mutations in which patients are more tend to accompany with autoimmune thyroid diseases the B-amyloid precursor protein (B-APP) gene on chromosome 21, and in an as yet specifically Graves' disease (GD) than in normal healthy populations. Genetic unidentified gene on chromosome 14 are known to be responsible for themaority susceptibility for IDDM is well characterized by association with HLA dass II ofearly onset cases. It has been suggested that the APOE locus on chromosome gene,which isgenerallythoughttoberesponsible forautoimmunepathogenesis 19 may alter the pre-clinical rate ofprogression in individuals that are otherwise in these endocrine diseases. The specific mechanism, however, for the disease predisposed to the disease]. Individuals that possess two copies ofthe ApoE-s4 aggregation and determination of organ specificity have not been identified. allele have a markedly earlier onset than individuals with no copies ofthis alle2. We, therefore, intended to know whether this susceptible gene at the HLA-DQ In this study we have observed a similar age ofonset effect in a large, region for IDDM would also determine the susceptibility for GD, or would multiplex American family. Individuals homozygous for the s4 allele have an age determine only organ specificity in IDDM but not in GD. We compared ofonset 10 years earlier than those heterozygous for e4, and 12 years earlier than association in forty IDDMs, ten IDDMs accompanied by GD, twelve GDs those with no copies ofe4. Meanxum likelihood linkage analysis forthe APOE without diabetes, and fifty normal healthy Japanese who have no family locus resulted in a lod score of 1.18 at 0-0.00, reflecting the weak evidence for history of diabetes and GD. DQA1 genotypes of individuals in each group APOE as a causative locus. The age ofonset ranges from 54 to 71 years (mean were determined by PCR-RFLP method. Titer for anti glutamic acid 62.3), excluding the possibility ofchromosome 14 mutations3. None ofthe known decarboxylase antibody, specific autoantibody for IDDM, in IDDMs B-APP mutations were detected and linkage analysis with a repeat marker forB- accompanied with GD appeared higher (m=3872U/ ml) thaninIDDMs without APP gave no evidence for linkage. Therefore, given the age ofonset, the minimal GD(m=455U/ ml),suggestingthatsusceptibility forboth autoimmuneresponse lod score at APOE, and the results ofscreening procedures, it is postulated that are synergistic. Significant association to DQA1*0301 were detected in IDDMs this may be affected by unknown additional fictors which bring forward (allelic frequency; AF=81.2%, RR=10.1, p=0.0001), and IDDMs with GD family (AF-85%, RR-13.2, p0.0001), while GD without diabetes showed no onset age. These References: I Bennett et al, (1995) Newropsychiatric Genetics, 60:1-6. 2 Corder association to any genotype at this locus (AF of DQA1*0301=10%). 921. 3 et al, results suggest that DQ region has an organ specific determinant for IDDM et al, (1993) Science 261, Mullan (1993) Neuropsychiatric Genetics, but not for GD, and that autoimmunity which shows synergistic effect in 48:129-130. these diseases does not link to DQ region.

1974 1975 Ssrch for evidence of dgenic Inheritance of limbgirdle muscular dystrophy Myelin associated glycoprotein immunoreactivity and Charoot-Mane-Tooth (LGMD2A) in the Northern Inians and Pennsylvana Amish popultion. (CMT) disease type 1A: association of CMT and Multiple Sclerosis A GMt M. Sostarko2. A. Era, !.RichardL , J.S. BeckmennZ, C.E. Jackson', and GL.Eidan. Henry B. Rautenstrauss. T. Liehr. H. Grehl'. KD. Eke. Ford Hospital, Detroit Ml and G6n6thon, Evry France. Ekici. 0.ParkT.Dumn R. Hauman.R.Hlohli MSd h BA Autosomal recessive limb-girdle muscular dystrophy is due to defects in at least FPfeiffe. B. Neund&W Institute of Human Genetics, 'Department of Neurology five different genes. A gene for LGMD2A, proteolytic enzyme calpain 3 (CANP3t and Institute of Forensic Medicin, University Erlangen, F.R.G., 2erpatmet of was previously mapped to lSql5.1-q21.1. Recently, mutations in this gene were Neurology University Zagreb, Croatia, Institute of Neurobiology ETH ZUrich, identified, and at least 6 different mutations are present in the small genetic Switzerland isolate of Isle de La Rdunion, suggesting a possible multifounder effect [Richard et EcoRIl/Southem hybridization of genomic DNA with probe pNEA102 (provided al., Cell 81:27-40, 19951. However, because this population is presumed to by J. R. Lupski) and MspI/Southem hybridization with probe pVAW409R3a derive from a single ancestor, they proposed that a second permissive gene (provided by C. Van B hovn) as well as FISH analysis on 125 interphase (nuclear or mitochondrial) might be necessary to allow complete penetrance of the nuclei with probes pVAW409RI (provided by L.J.Velentijn) and cRCNEU CANP3 mutations. To test this hypothesis, we are investigating another genetic (internal standard, provided by R. White) revealed a typical tandem duplication isolate with a high incidence of LGMD2A caused by a mutation in CANP3. the within chromosome 17p11.2 indicating a CMT type IA disease. To support the Amish of Adams Co. IN and Lancaster Co. PA. A single point mutation in CANP3, clinically determined symptoms of multiple sclerosis sera of both patients were called R769Q (CPQG-CAG), accounts for all affected patients in this population. analysed by ELISA for the presence of antibodies against myolin associated The proof of the hypothesis will depend on identifying unaffected individuals who glycoprotein (MAG), GMI and the L2/HNK1 carbohydrate epitope by CD57 are homozygous for R769Q. Previous linkage studies have not identified any such antibodies. Both were positive for MAG and GMI. Preblock with CD57 did not individuals among siblings of affected patients. Using allele specific alter this result indicating a MAG specific reaction. For one patioet a oligonucleotides and a non-radioactive detection system, we have initiated a lymphoblast cell line was established. RNA extracted from this cell line wes county-wide survey of the Amish to identify such individuals. To date, we have analysed by a MAG-specific RT-PCR producing a MAG cDNA Electrophoreti¢ examined DNA from 20 spouses of siblings of affected individuals for the R769Q analysis revealed two MAG cDNAs, one of expected length (1.8 kb) and one mutation, but have not yet identified any couples in whom both individuals are shorter of ap. 1.2 kb. This may indicate a deletion in one MAG-allele. An heterozygous. We are currently collecting blood from siblings (and their spouses) instable, defect MAG protein encoded by the shorter allele could be responsible of known carriers. Absence of a non-manifesting R7690 homozygote would be ELISA. the for the autoimmune reaction indicated by evidence against a modifying gene within this population. However, T. holds a Herbert Quandt O.P. holds a Korean for a or more Acknowledgement. L. fellowship, identification of such an individual would provide evidence digenic and are funded the DFG, M.S. is by issues as but fellowship, H.G., KDD.B. B.R. by supported complex inheritance pattern and complicate such genetic counseling, the National Science we thank Prof. E. Gebhart for his support. would reflect a new general mechanism of inheritance. Swiss Fund,

1976 1977 in sura eves of with hereditay y Absence ofCOL2A1 defect in the Marshal syndrome (MIM No.154780). Eypressin of PMP-22 patients A hansleand B Einstein of to (HNPP). A. Schenone'. L. Nobbio'. A.J. Lee2. Dept.ofPeds.,Albert College Medicine, with liability pressure palsies Montefiore Medical CenterBronxNY' and Depts.ofPediatrics and Molecular Windebw k, P.Mad!i', E. Bellone'. M. Abbruzzese' and G.L. Mancadi'. ofMedicineHoustonTexas.2 n of and Human Genetics,Baylor College 'Departmet of Neurological Sciences, University of Genova, Italy. 2De In 1958,Marshall described 7 family members with ocular abnormalities including Neurology, Mayo Clinic and Mayo Foundation, Rochester, MN, USA. 'Institute of cataracts and myopiasensorineural deafixe cddle nose deformity and evidence of Biology and Genetics, University of Genoa, Italy. anhidrotic ectodermal dysplasia. Since thenthere have been to our knowledge only 3 HNPP is asciaed with a deblion on chronosome 17pl 1.2 including the gene for additional families recorded with this dominantly inherited disorder. One ofus has peripheral myelm protein 22 (PMP-22). We used sec tve RT-PCR and already reported one ofthese families which consists ofS individuals in 3 generations to study PMP-22 expression in aural nerves from 9 patients with this disorder. Phenotypic overlap has suggested to some investigators that the the 1.2 and from S normal controls. In Stickler and Marshall syndromes are alleic mutations. The identification of a defect in affeced by HNPP, all showing 17pl deletion, these 2 defects are allefic. s nitatve RT-PCR glycealdehyde 3-phosphate dehydrogenase (G3PDH) was type II collagen would help resolve the question ofwhether DNA from members of our was along with normal whole used as an internal standard to determine relative levels of PMP-22 mRNA; band Genomic family prepared blood and xsjected to a series ofsingle and double digestions with restriction intensy was measured on ethidium bromide sained agarose gel by a Fooanalyst 11 endonucleases. Southern blot hybridization analysi using type II collagen cDNA G3PDH absolute intens did nOt differ bWeen HNPP imagng system.Whereas probes was carried out. The filters were probed first with a COL2A1 3 cDNA pa d controls (! 832 ±516 vs 1103 ±528 p=n.s.), PMP-22 was ificantly spanning axons 21-52 (HC22) and then with aCOL2A1 5 cDNA spanning exons I- lower in HNPP cases than in normal subjects (I 2368 ±1644 vs 4422 ±1144 21 (HC21). Autoradiographs ofthe filters revealed identical patterns in patients and p= <0.03). However, the differece in PMP-22/G3PDH ratio between HNPP normal controls. patients and normal controls was only margially significant (1 2.84 ±1.9 vs 4.45 These studies exclude any gross type II rearnent as the basic abnormality in ±1.2 p=0.058). PMP-22 immunostaining of sural nerves was on average weaker in this family. Identification ofmore subtle mutations would necessitate PCR DNA or HNPP than in normal controls, but several myelinted fibers showed a amplificaion and analysis ofindividual exons form genomic COL2A1 patients We the to demonstrate a also in HNPP We conclude that u ession of cDNA's (from patient cartilage). feel,howeverthat inability normal PMP-22 expression cases. in this and in another Marshall (REF.Strattonet the molecular p c manism in HNPP, but the Type II abnormality syndrome family PMP-22 may be underlying al,Am.J. ofMedical Genetics41:35-38 ,1991)is a reflectionofthe fact that thebasic variable expression of the protein and its mRNA in genetically homogeneous patients defect in this disorder involves ectodermal derivatives rather than a primary dizorder warrant furher studies to correlate the clinical and neopathological features with the ofcollagen synthesis. amount of PMP-22 expression. Purally supported by Teledton, gant # 533 and 580. Published Abstracts: Molecular Pathogenesis and Treatment (continued) A341 1978 The effect of apolipoprotein E genotype on expression of an autosomal dominant schizophreniform disorder with progressive dementia and neurofibrillary tangles. D.W. Tsuang. J.B. Leverenz. E.R. Peskind. T.D. Bird. G. Schellenbera. and M.A. Raskind. Seattle/American Lake VAMC and University ofWashington, Seattle WA, USA.

Individuals carrving the apolipoprotein E (APOE) c4allele are at increased risk for the expression of Alzheimer's disease (AD). In several subtypes of familial AD, presence of the £4 allele is associated with earlier disease onset In contrast, those carrying the s2 allele may be at decreased risk for AD. It has been hypothesized that these genetic risks are mediated by differential effects of the three APOE alleles on neurofibrillary cytoskeletal degeneration. To address this hypothesis, we examined APOE genotype frequency and the possibility of differential effects of APOE genotype on age of disease onset in a family with autosomal dominant schizophreniform psychosis and progressive dementia. This disorder is manifested neuropathologically by AD-like neurofibrillary tangles in the absence of neuritic plaques or other evidence of extracellular beta-amyloid deposition. APOE e4 allele frequency appeared under- represented in this family. The one affected e4 heterozygote did not demonstrate an earlier disease onset, but the affected e2 hetero7ygote had an age of onset of 56,> 1 SD above the family mean (48.7). The two other E2 heterozygotes remain at risk at an age much greater than the mean age of onset for the disease. These results are consistent with a protective effect for the e2 allele in this family with neurofibrillary tangle dementia.

Published Abstracts: Physical Mapping 1979 1980 CAG repeats on chromosome 18, candidates for cipolar disorder. Breschel A genetic and physical characterization of the D14q24.3 familial Alzheimer's TS, Li SH. Margolis R. Ross CA.Mclnnis MG. Johns Hopkins University disease locus. K.P. Clancy'. M. Cox'. J. Dutchik'. K. Walton'. I. Hart'. J. Bleskan' School of Medicine, Baltimore MD, 21287-7463. and D. Patterson': L.L.C. Bonnycastle2. V. Sharma2 and G. Schellenberg2. 'Eleanor Roosevelt Institute, Denver CO; 'University of Washington, Seattle WA. It has been established that an inherited form of Alzheimer's Disease, described as The clinical phenomenon of anticipation in bipolar disorder has been well AD3, is tightly linked to the chromosome 14 specific dinucleotide-repeat marker A yeast documented and with the recent reports of linkage to chromosome 18 we D14S43. artificial chromosome (YAC) contig for the 14q24.3 region between the have set out to identify CAG repeats on chromosome 18. A genomic markers D14S289 and D14S74 has been constructed, a distance of -6.6 centiMorgans. Eight MegaYacs cover human chromosome genomic library (ATCC 57742) was screened using the region, with gaps between the markers WI- 4706 and D14S71; and and (CAG)15 oligonucleotide radiolabelled with 32P. Approximately 80,000 D14S43 D14S76; D14S61 and D14S53. Thirty four Olsen/St. Louis library YACs have been isolated for the markers clones were screened giving a 5x coverage of the library. IThirty positive D14S42, D14S43, D14S48, D14S53, D14S55, D14S57, D14S59, D14S61, D14S63, D14S71, clones were identified and ofthose 10 appear to be unique based on the D14S77, D14S251, D14S273, D14S277, D14S284 and > size of the insert. The subcloning from the library vector (phage D14S289, covering 60% of the D14q24.3 region. Charon2lA) was done using PCR amplification from the EcoRl cloning Single chromosome 14 somatic cell hybrids have been generated for the patient cell site and subcloned into PCRII TA cloning vector (Invitrogen). lines SNW603, SNW646, L809 and V1020. These will be used to relate pre-existing Sequencing has been completed for 3 clones containing a CAG repeat and the novel markers to the genetic and contig maps of the D14q24.3 region. varying from 16 to 24. Clone 10.2B is polymorhpic (4 alleles). Clone Mapped genes within the AD3 locus include the transforming growth factor b3 gene, 7.6A contains a 24CAG repeat followed by a 13 CTC repeat, it is highly methyleneteerahydrofolate dehydrogenase - methylenetetrahydrofolate cyclohydrolase polymorphic with an observed heterozygosity of 84%. It's closest formyl tetrahydrofolate synthetase gene, cFOS oncogene, the heat shock protein gene marker is Dl 8S41, a region ofinterest for bipolar disorder. Expansion HSPA2, farnesyl transferase P-subunit gene and dihydrolipoamide succinyltransferase analysis of 28 bipolar probands did not reveal any expansions. gene. Novel constitutively expressed genes within the D14q24.3 region have been identified by use of a cDNA selection hybridization procedure. Selection has been carried out on some -2 Mbp of DNA between markers D14S77 and D14S42 using a normalized human infant brain cDNA library, fetal and adult libraries. Novel hybridizing cDNAs have been positioned upon the D14q24.3 contig map. Full length cDNAs will be generated from size selected cDNA libraries from human brain regions. 1981 1982 Genetic and physical mapping of the early-onset Alzheimer's disease candidate Genomic analysis of the human kappa opioid receptor: no evi- region AD3 at chromosome 14q24.3. M. Cruts. H. Backhovens. J. Theuns. G. Van dence for multiple genes. DK Grandy'. K Gunter', S Hagen2, Z-W Zhu', Gassen. S.Y. Wang. A. Wehnert. J.J. Martin and C. Van Broeckhoven. Laboratory for S Toth-Fejel 2, Q-Y Zhou3, S Olson' and RE Magenis2. 'Vollum Institute and Neurogenetics, Born-Bunge Foundation, University of Antwerp (UIA), Antwerpen, Dept. Cell Biology and Anatomy.OHSU,Portland, OR2Dept. Molecular and Medi- Belgium and Laboratory for Neuropathology, Bom-Bunge Foundation, University of cal Genetics,OHSU3Howard Hughes Medical Institute. Univ. ";ashingtonS, Seattle. Antwerp (UIA), Antwerpen, Belgium. WA. A major form of familial early-onset Alzheimer's disease (EOAD) has been localised A fragment of the human kappa (k)-opioid receptor gene was cloned by homology to chromosome 14q24.3 in a 22.7 cM region between D14S52 and D14S53 (AD3). using a rat cDNA probe. A portion of this human gene (OPRK1, opioid recep- We performed linkage analysis in two large pedigrees AD/A and Belgian EOAD tor kappa) was sequenced and the deduced polypeptide corresponded to putative AD/B using additional STRs inside the AD3 candidate region. Identification of new transmembrane (TM) domains Ill and IV of the rat k-opioid receptor. Overall the to a substantial reduction the to 6.4 cM between recombinants led of AD3 region deduced amino acid sequence of the human k-opioid receptor was 98.6% identical to markers D14S289 and telomeric. Using the STRs flanking centromeric D14S61 both the mouse and the rat k-opioid receptors. This significant conservation rapidly linked to EOAD we isolated 72 yeast artificial chromosome (YAC) clones from the dropped off after the seventh residue of the extracellular loop between putative TM CEPH YAC and megaYAC libraries and constructed a contiguous YAC contig map domains IV and V. Inspection of the DNA sequence revealed the presence of an of the region between D14S258 and D14S53 with an estimated physical size between excellent exon /intron splice junction at the point of divergence. Interestingly, the 4 and 8 Mb. The AD3 candidate region is completely contained in the YAC contig position of this intron is perfectly conserved in the mouse gene. The two human and is covered by 6 CEPH megaYACs. The size of the AD3 candidate region was genomic phage clones, whose inserts contained the k-opioid receptor gene fragment estimated between 2 and 6 Mb. The cellular oncogene c-fos (FOS), the dihydrolipoyl and overlapped by approximately 7kb, were fluorescently labeled and hybridized succinyltransferase gene (DLST), the transforming growth factor f3 gene (TGFB3), in situ to denatured human metaphase chromosomes. Metaphase chromatids from and the ESTs and were localised to the YAC contig map. FOS, D14S102E DI4SlOlE forty cells were counted and with sequential R-. Q. or G-banding OPRK1 was as- DLST and were inside the AD3 candidate region. We previously excluded TGFB3 signed to 8ql1.2-12. There was no evidence for any secondary site of hybridization. FOS as the AD3 gene since sequence analyses in AD/A and AD/B did not identify Therefore, we conclude that there is only one kappa opioid receptor gene in the pathogenic mutations. We analysed DLST in one patient and one escapee of both human genome. families AD/A and AD/B by SSCP analysis and direct sequence analysis of the coding regions and the 5' regulatory domain. No pathogenic mutations were detected. Therefore, we exclude DLST as the gene causing EOAD in families AD/A and AD/B. A342 Published Abstracts: Physical Mapping (continued) 1983 1984 Resignment ofthe muscle pbospbofructokinse (PFK gene to chromosome 12q. Assignment of Gephyrin gene to human chromosome 15q22. T. D. Howard'. D. T. Moir2 and D. W. Bowden'. 'Bowman Gray School ofMedicine W-Y. HungiH. Betz2. X-X Hel.M-HL. ngl, H-X PLngjand T. of Wake Forest University, Winston-Salem, NC 27157. 2Genome Therapeutics, Inc., Waltham, MA 02154. 1) Department of Neurology, Northwestern University Medical Phosphofiuctolinase (PFK), a key rate-limiting enzyme in glycolysis, represents a major School, Chicago, IL. 2) Max-planck-institut Fur Hirnforschung, control point in the metabolism of glucose, and may indirectly play a role in insulin Frankfurt, Germany. secretion. There are at least three known isoforms ofPFK in humans, referred to as the Gephyrin, a 93 kD receptor-associated peripheral membrane muscle, platelet, and liver forms, each of which are expressed to varying degrees in protein, is associated with cytoplasmic domains of the glycine different tissues. The genes for each isoform have previously been reported to map to receptor core and may interact with elements of the human chromosomes 1, 10, and 21, respectively. A possible additional isoform, PFKX, submembranous cytoskeleton. We have used purified rat gephyrin has been mapped to chromosome 12. In an effort to create a correlated physical and cDNA as probe to screen a total human genomic phage library. Four genetic map encompassing the PFKM region, we have found that this gene does not map out of ten selected positive clones from second screening were used to chromosome 1, but instead maps to chromosome 12. PCR analysis with a somatic ceO for analysis. After labeled separately with biotinylated dUT? using hybrid mapping panel using prumers derwed from intron 6 and exon 18 ofthe PFKM gene random primed labeling kit, fluorescence in situ hybridization to consistently amplify cell lines containing chromosome 12 only (concordance =100%, human metaphase chromosome spread indicates that gephyrin gene discordance-0%). These PCR products were specific for the PFKM gene based on is located at chromosome 15q22. Gephyrin gene is homologous to Southern blot and/or sequencing analysis. To confirm this chromosome 12 assignment, two genes of E. colt which are known to be implicated in two YAC's were isolated using these primer sets from two human genomic libraries. Both molybdopterin synthesis (prior et. al., 1992). A molybdopterin YAC's were used as probes in fluorescence in situ hybridization (FISH), and both deficiency gene has also been cloned from Drosophila. The deduced kinase gene (DAGK), gene product, name cinnamon and gephyrin show extensive hybridized to chromosome 12q13, centromeric to the diacylglycerol As which maps to 12q13.3. These YAC's are also significantly centromeric to a YAC from homology over their entire sequences. molybdenum deficiencies reported to be linked to a third gene for maturity are known to cause neurological symptoms in man, the the D12S76 locus, which was recently human onset diabetes ofthe young (MODY; Vadllaire et al. Nat Gen 9:418, 1995). The physical consequences of localizing gephyrin gene on this region of distance between PFKM and D12S76 suggests that PFKM is not the gene responsible for chromosome 15 may be quite interesting. this form ofMODY.

1985 1986 Chromosomal localization of an epithilial sodium channel gene to 12pl3.3 Identification of expressed sequences from the CADASIL region on M. A. Walter2. 19p. A. Joutell, A.Ducro I S. Alamrowitchl, K. Vabedi,12 H.Chabriat2, V by fluorescence in situ hybridization. C. Leel, Domenga', Y. Roni J. MaCiazek', M. pneur3 H. MohrenWi=er4 MG B. Kamosinskaj, S. Parimi3, P. Lawczynski3, M. Duszyk3, S.F. P. Man3, gounr2 and EI m L z. 1. INSERM U25 Facult6 de mfdecine C.C. IjinL Departments of Laboratory Medicine & Pathologyt, Opthamology2. Necker- Paris- Frnce. Intro by A. Munn2ch. H6pital St Antoine- Paris. 3. and Phvsiology3., University of Alberta. Edmonton, Alberta, Canada. H6pital Necker-Paris. 4. Lawrence Livermoore National Laboratory- of sodium channel is to sodium ion con- Livermoore-CA-USA. The function epithelial genes regulate ductance in the airway. This serves to control water and electrolyte conductance in mucous which is a vital component of muco ciliary clearance. A sodium channel gene was cloned from a cDNA library and found to be homologous to the alpha CADASIL is an autosomal dominant cerebral arteriopathy subunits of previously isolated human sodium channel genes. Two cDNA clones. characterized by the recurrence of subcortical infarcts leading to dementia and pNAC2, of 1577 bp and 900 bp in size respectively. were isolated We recently mapped the CADASIL locus on chromosome 19p13 pNACI with biotin-16- (D19S221-D19S215). Linkage analysis of 18 additional families established from this library. Both DNA clones were non isotopically labelled genetic homogeneity and allowed us to reduce the size of the interval to 1 dUTP and used as probes in fluorescence in situ hybridization experiments. Twenty cM. Three overlapping YACS spanning the whole interval were identified. metaphases were examined for each probe and 22 chromosomes were found to have A long range restriction map of the interval was constructed using DNA hybridization signals at the terminal short arm of chromosome 12 at band 12pI3.3. fragments subcloned from ch.19 cosmids from Lawrence Livermoore been localized to band in the National Laboratory and the size of the critical region was estimated to be Several other ion channel genes have regionally 12p13 around 1 Mb. Sixty unrelated patients are ctgently screened for physical human genome. These include potassium voltage-gated channel genes: KCNAL, rearrangement by conventional Southern blot analysis as well as PFGE. KCNA5. KCNA6 and the calcium L-type alpha-1 channel gene CACNL1A1. The Several ongoing approaches including 1) the characterization of significance of the clustering of these channel genes to this chromosome region is interspecies conserved as well as known EST sequences mapping within still unknown. this region 2) the selection of cDNAs mapping to the YACs contig, already led to the identification of two expressed sequences which implication is currently tested in CADASIL patients.

1987 1988 Construction of a tscriptIon map in the rqgon of the R echls's Mapping of the gene for the a3 chain of type IX collagen, COL9A3, dee gene. _. Van De Vosse'. Va Der Bent'. N.A. Dso'. EJ.M. to human chromosome 20q13.3. G.E. Tiller.' M.L. Warman.2 Y. Van Ommen' and J.T. Den Dunne'. and R.G. SghuurIfin A.A.B. Bergen2 G.-L&B Gong.2 J.H.M. Knoll.3 N.A. MEkpjunath,4 . Hansmann.4 'MGC-Department of Human Genetics, Leiden University, Leiden, Ihe BrCQ._5 'Vanderbilt University School of Medicine, Nashville, TN, Netherlands; 2The Nethands Ophthalmic Research Institute, Amsterdam, The 2Case Western Reserve University School of Medicine, Cleveland, OH, Netherlands. MA, 4University of Gottingen, We have constructed a 4 Mb YAC contig in the Xp22.I-p22.2 area 3Harvard Medical School, Boston, several disease Germany, and 5University of Alabama, Birmingham, AL. the markers DXS414 to DXS451. The region encompasses with genes, including RS, HYP, CIS and KFSD. Currently, we are focussing on Type IX collagen is a minor collagen which forms mixed fibrils a in the region around RS. Retinochisis (RS, MbI 312700) is sue heredity types II and XI collagen. To investigate the role of type IX collagen vitoreinal degeneration. The physical map and linkage analysis with new vertebrate development and pathology, we have cloned the human gene marker in the region allowed a refinenent of the RS candidate encodes a mapped De for the t3 chain of type IX collagen, COL9A3. The gene to a 0.6 Mb region between DXS418 and DXS999/AFM291wf5 (Van 684 amino acid protein with alternating collagenous and non-collagenous Vosse et al. 1995). in and vitreous humor. We are constnacting a transcription map of the RS candidate region. ITe domains, which is expressed hyaline cartilage direct of Fluorescence in situ hybridization (FISH) on metaphase chromosomes techniques that are applied to identify transcripts include screening cDNA libraries, cDNA selection followed by cDNA from a control male using a phage clone of COL9A3 localized the gene hybridization-based is a screening and exon trping using several vectors. One of these, sCOGH2, to human chromosome 20q13.3. Utilizing an SSCP in the COLl the simultaneous isolation of all exons and determined the most recently developed vector facilitating domain, we genotyped the CEPH families in the inert (see abstract of Den Dunnen et al.). We have constricted 1 present cosmida ae likely locusorder tobecen-D20S l5-D20S73-COL9A3-D20S 9-D20S24 a library in sCOGH2 from a YAC spanning the RS region. These tel. FISH studies of three patients with chromosome 20 aneuploidies used to construct a contig of the RS region, to isolate new CA repeat markers revealed trisomy for this region in one infant whose phenotype included and to identify genes by exon uapping. cleft lip and palate. These data may facilitate studies of patients with deletions of chromosome and provide the means to assess E. Van De Vosse, et al. A 4 Mb Xp22.1-p22.2 YAC contig encompassing terminal 20q the disease loci for RS, KFSD, CLS, and HYP; refined localization of RS. COL9A3 as a candidate gene in human chondrodysplasias. 1995. Submitted. Published Abstracts: Prenatal and Perinatal Genetics A343 1989 1990 Prenatal detection of mosaic deletion of 16p, del(16)(p12). coincident with Abnormal serum triple screen and cri-du-chat syndrome. B.M. Berlin. B.A. the expression of fragile site. fra(16)(pl2), in amniotic fluid cells. A. Babu. Shenhard, E.R. Elias. E.C. Lazar. D.W. Bianchi. Tufts Univ. Sch. Med., and New S. Pooescu. V. Bogosian and V.S. Penchaszadeh. Beth Israel Medical England Medical Center, Boston, MA Center, Albert Einstein College of Medicine, New York, NY. An amniotic fluid specimen from a 32 year old female was received for The differential diagnosis of an abnormal serum triple screen includes incorrect prenatal diagnosis because of parental anxiety. Family history was gestational age and chromosome abnormalities such as trisomy 21. We recently cared unremarkable except for a child with dwarfism from a previous union the 35 for a 34 year old gravida 2 para 0 woman who had an abnormal AFp3. Her infant was year old father. Chromosome analysis of 40 amniocytes revealed a mosaic later diagnosed with mosaic cri-du-chat syndrome. Her obstetrical history is notable chromosome complement with 46,XY in 33 cells and 46,XY,delll 6)(P1 2) with a terminal deletion in the short arm of chromosome 16 in 7 cells. The for uterus didelphys, 9 years of infertility, and one first trimester spontaneous abortion. abnormal cells were present in two cultures examined, two out of 20, and She also has a congenitally absent kidney, bilateral , and micrognathia for three out of 20, respectively. In a follow-up repeat amniocentesis, three which she underwent surgical jaw advancement. Her family history is significant for cells out 40 were abnormal with delO 6)(pl2). In addition, two cells, micrognathia and foot deformities in several family members. This pregnancy was including 1 from each of the two cultures, showed a chromatid gap (1) and complicated by an abnormal AFP3: AFP=0.86 MOM, hCG=1.44 MOM, uE3=0.51 a chromatid break (1) at 1 6pl 2, the break-point involved in the deletion. MOM. This elevated her Down syndrome risk to 1:128 from an age-related risk of Concurrent studies on parental blood revealed normal karyotypes with no 1:309. A detailed ultrasound examination was negative for anatomic abnormalities, indication of fragile site at 1 6p1 2 under routine culture conditions. The and the parents declined amniocentesis. At birth at 36 weeks' gestation, her female couple decided to interrupt the pregnancy and no fetal morphological infant had dysmorphic features and limb anomalies. The infant's physical examination abnormalities were found at termination. Post termination chromosome included mild microcephaly, hypertelorism, shallow orbital ridges, one low set ear, analysis of fetal blood (100 cells), fetal skin (100 cells), and placenta and bilaterally dislocated hips, club feet, and transverse palmar creases. Her infrequent cry amnion (100 cells) could confirm the presence of the 1 6p deletion in a single was abnormally high-pitched. There were no other skeletal or cell in placenta/amnion. However, 5 cells (3 from skin and 2 from genitourinary abnormalities placenta/amnion) showed either a chromatid gap or a break at 1 6p1 2. It is on radiographic or ultrasound examinations. The baby's karyotype was interesting that the breakpoint involved in the deletion is coincident with a 46,XX(4 cells)/46,XX, del(5) (pI3.3) (46 cells), revealing a terminal deletion in 92% rare fragile site at 1 6p1 2 that is expressed in response to folate deficiency. of cells consistent with cri-du-chat syndrome. Parental karyotypes were normal. We The prenatal finding of mosaic deletions, and the relationship between rare believe that this is the first report of a case of mosaic cri-du-chat syndrome following fragile sites and the breakpoints involved in de novo deletions present a pregnancy complicated by an abnormal serum triple screen. challenging counseling dilemmas in view of their present uncertain pathological significance.

1991 1992 Fetal lung mass associated with significant mediastinal shift: a pul- Difficulties in genetic counseling and assessing risk: couple with 2 unrelated trisamies a monary sequestration with good outcome. F.P. Bernier, 1 P. Martyn. consecutive, plus paternal 13-14 4 translocation. SM Boehmerl, RJ Hildebrandt2, MM McCorquodale3. 1The P.D. Elliott, 3 B. Simrose, 2 J. Harder, Members of the Fetal Development Clinic' Western Pennsylvania Hospital, Pittsburgh, PA, 'Center For and C.L. Johnson.. Dept of Medical Genetics,' Dept of Obstectrics and Gynecology.2 Diagnostic Ultrasound, Dayton, OH, 3 Michael Reese Hospital and of of Pediatrics.4 University of Alberta. Canada and Medical Center, Chicago, IL. Dept Radiology.3 Dept Calgary, A 38-year-old GP1SA white female was seen for genetic Foothills Hospital, Calgary, Alberta, Canada.5. counseling and amniocentesis because of maternal age. It was Fetal lung masses are rare anomalies that can be detected on antenatal ultrasonographic revealed that she had a previous spontaneous abortion which was examination. Associated include mediastinal shift. and chromosomally abnormal. Further investigation revealed the SA was findings hydrops. polyhydramnios 46,XX/47,XX,+17. Based on maternal age and past history, a rarely additional malformations. In recent years, attempts have been made to identify recurrence risk was quoted as 1%. The couple also has a daughter features associated with poor outcome. Although the presence of hydrops indicates almost who is 46,XX as determined by amniocentesis for maternal age. universal the of mediastinal shift remains controversial. Ultrasound examination of the current pregnancy -noted a large lethality, prognostic significance omphalocele, mild renal pyelectasis, absence of the middle phalanx We describe a fetal lung mass detected at 23 weeks gestation. The mother was a 30 yo of the fifth digit on the left hand, and a fused papillary muscle. G1P0 who has hypertrophic cardiomyopathy and therefore had been referred for fetal Amniocentesis confirmed 47,XY,+18 and the pregnancy was ended. In an attempt to the with an accurate risk The left sided thoracic mass was and the provide patient for echocardiography. homogeneously echogenic recurrence of a non-disjunctional trisomy in a future pregnancy, the heart was displaced into the right hemithorax. The reminder of the ultrasound was possibility of interchromosomal effect was considered. The couple normal. The mass was felt to represent either a type III cystic adenomatoid malformation elected parental blood chromosome analysis. The patient's karyotype was 46,XX and her husband's showed or a scans at 45,XY,dic(13;14)(p1l.2;pll.2). pulmonary sequestration. Repeat performed 28 and 31 weeks revealed Existing literature indicates a 2% recurrence risk for a non- improvement in the mediastinal shift with a relative decrease in the chest mass size. disjunctional event and a 50% risk for a balanced 13;14 Labor was spontaneous at 38 weeks. There was no evidence of neonatal respiratory translocation when detected at amniocentesis. CVS in the next pregnancy revealed a balanced 13;14 translocation. This pregnancy and a chest obtained was distress X-ray shortly thereafter normal. Follow-up CT scan is ongoing. The existence of interchromosomal effect, that the identified an irregular density in the posterior and medial aspects of the left lower lobe presence of a parental translocation can predispose the individual suggestive of a pulmonary sequestration. At the age of 1.5 years, our patient underwent to produce gametes aneuploid for a chromosome not involved in the translocation, is controversial. this case prove resection of an extralobar Although does not pulmonary sequestration and at the age of 2 he has normal that interchromosomal effect exists, it illustrates that the option cardiorespiratory function. Our case report adds further evidence that fetal lung masses. of parental blood studies for couples with repeated non- even when associated with impressive mediastinal shift. can have a normal outcome. disjunctional events can be of great importance in predicting risks and offering options for testing for future pregnancies,

1993 1994 Prenatal diagnosis of a mosaic, unbalanced (Y;1) translocation Long term follow-up of a prenatally diagnosed mosaic in two unrelated fetuses with multiple anomalies. J.E. Chemos' inv dup(15):clinical implications.K.L. David (1W. and K. Huvnh2. 'Alberta Children's Hospital, Calgary, Alberta, C.T. RubenstginaLli C.Puno-Cocuzza(ll.and A&.amb(2). Canada and 2HMtel-dleu de Canada. Section of Genetics, The Brooklyn Hospital Center, 121 Gasp6, Quebec, DeKalb Avenue, and Mosaicism for structural Brooklyn, NY, (2)Integrated Genetics, chromosome rearrangements are Santa Fe, N.M. extremely rare. On cytogenetic prenatal diagnosis, structural Few long term follow-ups of a prenatally detected rearrangements are often encountered in an isolated cell or colony inv dup(15) exist. We report follow-up of a 13 year old in an otherwise normal culture and are presumed to represent male diagnosed by amniocentesis to be mosaic for a d. technical artifact or pseudomosaicism without clinical significance to nova unidentified marker, 46,XY(74%)/47,XY,+mar(26%) the fetus. Here we report on two prenatally diagnosed cases of confirmed by a peripheral blood study at birth, 46,XY(43%) "true" mosaicism for an unusual structural Two /47,XY,+mar(57%). Physical exam revealed a 3884 gram term rearrangement. infant with no dysmorphic features. The child's development unrelated fetuses exhibiting ultrasound anomalies were found on was normal physically and mentally but he recently demon- amniotic fluid cell culture to have the same abnormal, mosaic strated behavioral problems in school such as disruptive cytogenetic pattem. In each case, two cell lines were present, one talking, day dreaming and inattention to homework. Parental with a normal male karyotype, and the other with an unbalanced concern prompted a revisit to the Genetics Unit at age 13. translocation: 46,X,-Y,+t(Y;1)(q12;q12) being trisomic for essentially Physical exam was unremarkable except for swirled hyper/ hypopigmentation over trunk and lateral the entire arm of chromosome 1. Parental were arms, thighs. long karyotypes Cytogenetic analysis again confirmed the mosaic karyotype completely normal. Cytogenetic analysis of fetal tissue confirmed with the marker in 64% of cells. FISH analysis enabled mosaicism in one fetus, while, skin and placenta cultured from the identification of this marker as an inversion duplication second fetus showed only a normal male karyotype. In both cases, of chromosome 15,inv dup(l5)(pter-I ql2::ql2-> pter), multiple fetal anomalies were confirmed on examination following including coding sequences from the Prader-Willi/Angelman termination. syndrome (PWS/AS) chromosome region. The patient has 4 copies of the proximal 15q. Inv dup(15) chromosomes have been reported with both a normal phenotype when the PWS/AS region is missing and with an abnormal phenotype that can include mental retardation, behavioral problems, and mild dysmorphic features when the PWS/AS region is present. This patient's very mild phenotype in spite of the presence of the PWS/AS sequences may be due to mosaicism. Caution must be exercised in counseling for such a prenatal diagnosis. A344 Published Abstracts: PrenataI and Perinatal Genetics (continued) 1995 1996 Alobar boloprosencephaly and multiple anomalies in a stillborn with ring Severe infantile cortical byperostosis (Caffey's disease) in siblings: evidence for chromosome 1, T.N. Diehn M.L. Netzloff. and PD. Storto. Michigan State autosomal recessive inhentance. B M Drinkwae J.P Crino F K Etret J University, East Lansing. Qgburn. J T ite h The University ofTexas-Houston Medical School, Houston, TX. Infantile cortical hyperostosis (ICH), Caffeys disease, is a multifocal, inflammatory A 02, P1,32 year old fmale was refeed at 34 weda gestation r furith diFretia of skeletal process with classic onset before the fifth month of life and resolution by the abnoumal ultrasound findin. Prenancy history was positive for crack cocaine use. age of3 years. A severe phenotype with early prenatal onset has also been described. Ultrasound exminatin demonstrated a markdly abnormal fetal lend with no evidence of Of the 13 prenatally diagnosed cases reported, 8 manifested the severe phenotype cerebral henispheres or a brain stem, fiued thaluni and massive hydrocephalu. characterized by hydramnios and extensive skeletal involvement, with 6 intrauterine Asymmetrical growth parmeters, a recessed nasal re , ypo bowel lop or early neonatal deaths (Tumpenny et al., 1993). Inheritance is generally accepted as dilation and bilateral hy 1phrosis were also denostrated A diagosis of alobar The autosomal dominant with variable expression and penetrance. However, two of the holprosencepbaly with rea and iestinal anomalies was nude. role of the maternal and ccme use as austve agent versus an underlying gentic defec was considered. The prenatally diagnosed cases were siblings, raising the possibility of heterogeneity w delived sllborn at 375/7 weeks after oephalentesto aow vaga deiv. the existence ofa severe autosomal recessive form. We describe a second family with Autopy findings included, is addition to fet already mntiod, efo anus and prenatally diagnosed ICH in 2 siblings. The patient was initially referred to our center peistent cloaca sacral agenesis, horse shoe kdney, absence of the right distal ureter and at 22 weeks with hydramnios. Ultrasound findings included generalized extremity thirteen ribs. Cytogenetic studies pfmed on fibroblasts showed a 46,XYinv(6) shortening with a small thoracic circumference. The fetus delivered spontaneously at (p22.2q22.1),-l8,+r kaype. Parental chromosome studies d aed the inv(6) to be 28 weeks gestation and expired on day 10 of life. Autopsy revealed skeletal findings ofmaternal orgi. To fuer dde the itiy of he ng c o a chromosome consistent with ICH. In the couple's next pregnancy, ultrasound at 14 weeks revealed 18 alpha-elle FISH probe was utiized. The probe was hybridized to p ubd unilateral bowed femur and mild lower extremity shortening. More severe sections. The maorzty of cells had two hyridization signals, indicating ta th ring was generalized extrnity shortening was apparent by 19 weeks. A closed lumbosacral most liey derived from chromosome 18. Alobar caudal regession with neural tube defect was also present The patient terminated the pregnancy. Autopsy acral agnesis, ana atresia and renal, aboin and vertebral anomales have been mot again revealed skeletal findings consistent with ICH. Histologic examination revealed reported in association with ing chmosome 18. The inv(6) is ildy unrelated to the extension ofneutrophils into medullary bone, which has not been described previously fea abnormalities. Cocaie exposure can not be excluded in the deel tsomce ofthe anomalies, however, we couclude that the majority ofthe muple anomalies wer the result in ICH. We speculate that this finding may be related to the early diagnosis This ofthe chromosomal defect. second report of siblings diagnosed with the severe prenatal form of ICH provides further evidence for heterogeneity and the existence of a severe autosomal recessive form.

1997 1998 Confined placental mosaicism (CPM): Further cases and a review. L.Ey- Prenatal diagnosis of fetal holoprosencephaly. Mehitretter. T. Peters-Brown H Ka- S AdhvNrv aN M -B -Oumive R.J.Garcia-Cavazos, S.Garza. MJ. avaletA, R. Departments ofOB/GYN, Pathology, and Pediatrics, Duke University Medical M*16ndez, E.Rendbn , R. B~ez D.G. May~n Leis Center, Durham, NC. T., Sanromfn R. Instituto Nacional de Perinato- logia. Mexico city. We report four new cases of apparent CPM which provide further evidence of the Holoprosencephaly (HPE) is a developmental impact of this problem on prenatal diagnosis: 1) cultured chorionic villi (CV) defect of the forebrain and midf ace, with an showed mosaic 47,XXj(7p)/46,XX [22:131 while amniotic fluid showed a 46,XX incidence of 1:5200 to 16000 live-born infants . karyotype in 52 cells examined with a normal pregnancy outcome, 2) Direct harvest EPE is associated with teratogenic agents and on CV 46,XY [7 cells], cultured CV showed mosaic 47,XY,+21/ genetic factors. Chromosome aberrations are 49,XY,+21,+21,+21, and amniocentesis had 47,XY,+21, 3) a spontaneous found in 50% of the cases and mendelian inheri- abortion (SA) with vili showing 46,XX (35], umbilical cord 46,XX/47,XX,+21 tance is also involved. [32:181, and anniotic fluid cells with 46,XX/47,XX,+21 [35:151, 4) SA with We studied 8 cases of HPE prenatally diagno- 45,X cells limited to the placenta. The latter case and others which have been sed by high resolution ultrasound. We performed reported suggest that CPM can be assciated with adverse pregnancy outcome even amniocentesis for cytogenetic study in all of when fetal karytype is normal. An updated database for placental mosaicism them. Two, were associated with maternal diabe- maintained in our laboratory shows that 83% of reported discrepant findings at CV tes, one have a sib with HPE and a mother with a diagnosis were false positive results. Combined with the observations from large balanced translocation 2;7. We found two semilo- surveys that CPM represents as much as 30% of all abnormal CV results, these data bar and six alobar HPE based in prenatal ultra- support the idea that confined placental mosaicism contributes to reduction in sonography and confirmed by postnatal computed specificity ofprenatal diagnosis using chorionic villi. tomography or histopathological examination. In the alobar cases we found three, with chromoso- mal aberrations, all of these include the criti- cal regions for the recently described HPE genes : 45,XX,-13,-18+der(18)t(13;18), 46,XX,t(2;7) (p23;p22), 46,XX,-14,+rob(13;14)(pll;qll), all related with the genes recently designed for HPE. The first case were comfirmed by fluorescence in situ hybridization and presents the most severe form of HPE: cyclopia and proboscis. We also found a facio-auriculo-radial syndrome.

1999 2000 A case of trisomy 5 mosaicism: Expansion of Identification and characterization of de novo bisatellited marker phenotype and example of variability with prenatally chromosomes in three prenatal cases. Diana L. Gray Robert Ball. lane diagnosed chromosome mosaicism. K Grace1. M lartel'. Bauer. Amy Crawford. Sheri Bbb. Heidi Beaver. Sua ukrbrger.and and N Xardon2. 'Virginia Baptist Hospital, Lynchburg, Chih-Lin Hsieh. Washington University School of Medicine, St. Louis, Virginia, Genetrix, Yonkers, New York. The literature describes three cases of trisomy 5 MO. mosaicism, all discovered prenatally. Two The psu dic(15;15) chromosomes are the most commonly observed pregnancies resulted in the birth of healthy marker chromosomes and have been associated with a wide spectrum of liveborns. The third had asysmetric IUGR, phenotypes dependent on the content of the chromosome. Here we report dysmorphic features, a VSD and diaphragmatic three prenatal cases with various sized psu dic(15;15). The first case was a eventration. observed in a CVS from a AB2, seen at 19 48,XXY,+psu dic(15ql3;15ql3) specimen 43-year- We present a 31 year old, G4, P1, old G3P1 woman at 11.1 weeks gestational age. The nature of the marker weeks for routine obstetrical ultrasound. Detailed 15 examination revealed an encephalocele, microcephaly chromosome was determined by FISH using chromosome paint probe. and hydrocephaly. A three chamber heart with an AV The parents chose to terminate the pregnancy based on the cytogenetic canal was suspected. Amniocentesis was performed, findings and information available in the literature. The second and third and the karyotype revealed eleven cells with 46,XY, cases were 47,XY,+psu dic(15q11;15q11) observed in amniotic fluid and nine cells, fros throe separate culture vessels, specimens from a 39-year-old G3P2 woman at 17.6 weeks gestational age each with 47 chromosomes, 47,XY,+5. Amniotic fluid 16.7 weeks The nature of AFP was normal at 0.44 multiples of the median. and a 38-year-old G3P1 woman at gestational age. these two marker chromosomes was determined by FISH using a Ieckel's diverticulum, and left, preaxial fetal hexadactyly were identified on post mortem chromosome 15 centromere-specific probe. Detail sonographic examination. evaluation in these two cases fail to reveal any structural abnormality. Clearly, the diagnosis of trisomy 5 mosaicism may Both couples chose to continue the pregnancy, and the outcomes will be be associated with a wide range of outcomes, and the presented. While very little phenotypic information on other bisatellited full spectrum must be considered when counseling the 1 and 2 have cases had normal marker chromosomes is available, type type psu dic(15;15) parents. Two previously reported been to have normal Therefore, characterization of outcome. We present the second case with proposed phenotype. significant abnormalities. This case expands the the bisatellited marker chromosomes can affect the counseling and the phenotype for trisomy 5 mosaicism and illustrates decision of parent& significantly when de novo bisatellited markers are the complexities encountered with prenatally observed. It is also intriguing that all three women are of advanced diagnosed chromosome mosaicism. maternal age. The possibility of maternal age effect will also be presented. Published Abstracts: Prenatal and Plerinatal Genetics (continued) A345 2001 2002 Cye t Fiand Frm 2015 Coanecutive First Tnmester Chorionic Vili Samples chrolosolne Ibonnautes in aall an Hispanik populton. LW.lasan' ((S. ausekarn, V.G. Dev',M. 0. Butlr, S.L Sal1y, and F. H. Boehm2.)) .D. Om__, D.M. CarrA.& Angulwn& C.P. Brown', 'eaetics Associates and 'Vandrbilt University, Nashville, TN. Medcl Center, Los Angeles, Calif. Celontioic villi samnpes were collected over the pat four years from 2015 women refured 'KiWDrew for prenatal diagosis primarily due to maternal age or previous chromosomal anomalies. 2Corning/Nichols bstitute, San Juan Capistrano, Calf. Cursenossanal analysis wanmssdertaken ssisg a dutct harvest a long-tensculture. No villi wan obtaineden 20 cases. Direct harvests were succesfully obtained on all but 72 From 1984 to 1994, 2,416 amniocenteses were successfully performed at King- sntples and log-tesincaltres were obtained on all but 23 samples. Cytgenetic Drew Medical Center in Los Angeles (KDMC). 2165 of these procedures were diaposis wan possle on 99% of the cas Of the 2015 samples. 916 were diasosed as nm al maees ad 873 were disposed an nonnal females. Te cytogenetic data we performed on women of Hispanic origin. Chromosome abnormalities were noted presented in the table. in 76(3.1%) of the cases (range: 0.8%-4.4%). A slight increase in the frequency 46,XX or 46,XY: 1937 of abnormalities was noted in the last four years. Hispanics comprised 94.7% of Abnormal 78 the abnormal cases. The indications for referral in the 76 abnormal cases included Ntsnerical Anomali;ss 34 Suctural Assmalies: 27 abnormal ultrasound child Tnismy 21: 19 Familial Rearangements: 23 advanced maternal age (86.6%), (9.2%), previous with T nisomy 13: 0 De novoR ent: 4 an abnormality (2.6%) and family history of abnormality (1.3%). The mean Tlaomy 18: 7 maternal age in the advanced matenal group was 38.9 years. The mean panty was X1YAnploidy: 8 Mosaics: 17 2.4 births and the mean gestational age was 18.4 weeks. Among the 76 cases with Triploidy: 0 Direct Only: 8 detected dcomnosome aberrations, 68 were numeric (78% autosomal and 22% sex). Culture Only: 8 of were Dibse ultue Discrpancies: 05 Both 3 Trisomy 21 was present in 57% of the cases. Eight the defects structural, Two caes merit stecil mention. On cane had a 47,)OO in the direct and a 45,X/46,XX 7 autosomal and 1 sex); 3 were de novo and 3 were hereditary structural in the culture and the prepacy wan subsequently tersinated, In the other case, the direct rearrangements. Twenty-nine of the cases with numerical aberrations (including wan 46,XY and the colture was 47,XY,+18 The fetus exhibited the clinical features of 15 with trisomy 21) and all eight cases with both de novo and hereditary structural Trisomy 18 and wan atan aborte In comparisn with the amnioctesis date(Achirind, to continue their Of those that is rearrangements elected pregnancies. pregnancies Applied Cytognetics 18:6,1992), which the frquency of abnormality wan 3.0%, the one fetus was frequency of aunematity in our CVSsudy wa 3.8%. The trisamy 21 occurrence was continued, four resulted in fetal demise or spontaneous abortion and 0.94% in CVS and ootpsrdto 0.77% is amniocentesis, The percentage of mosaicisn in stillborn. The frequencies of abnormal results at the time of amniocentesis at CVS wan 1.1% as empared to 0.26% in amniocentesis. This difference can beexplained KDMC are similar to those reported in other series. by plarental mossiism a the CVS timue. Utilizing five in-ituhybridization probes (13,18,21,XY), 37.8% of the cromosomalabrmalities in the amniocentesis data and 34.6% in our CVS survey could not have been detected.

2003 2004 AF`P and free 0-CG in prospective screening for Down syndrome in Taiwanese Prenatal diagnosis and birth outcome of a mosaic ring 18/monosomy 18 . pregnant women under 34 years of age. J. J. Hsu. T. T. Hsieh. J. D. Liou. F. J. Jaswancxl, B.A. Clark:s. G.G. Ashmeadl.. L. Ko2. D. powelI3. S.B. CassidA Hsieh.' K.S~pcer.2 Chang Gung Memorial Hospital, 'Taiwan National University and S. S1hwkI)MetroHealth Medical Center, Case Western Reserve University, Hospital, (Taiwan Down Syndrome Screening Groups), Taipei, Taiwan, ROC, 2Old- Cleveland, Ohio, 2)Department of Genetics, Case Western Reserve University, church Hospital, Romford, UK. Cleveland, Ohio, 3)Department of Pediatrics, Mt. Sinai Hospital, Cleveland, Ohio. A was conducted to assess the efficiency of prenatal screening There are a number of case reports of patients with ring 18, some of whom have prospective study mosaicism with a 45, -18 cell line believed to emerge in culture. One case of mosaic using free (3-hCG in combination with AFP and maternal age for Down syndrome in ring 18/monosomy 18 detected at amniocentesis was reported, but the pregnancy was Taiwanese pregnant women under 34 years of age. We collected 4084 women under terminated at 18 weeks gestation. We report a prenatally detected case who was 34 with singleton pregnancies screened between 14 and 22 weeks' gestation from liveborn despite severe anomalies and died at 19 days of age. A 15 year old G2 PI January 1994 to January 1995. All of these pregnant women can be followed up in presented to ultrasound at 24 weeks gestation with a hypoplastic right heart, pulmonic detail from our birth registration. Most of the study cases dates were confirmed by atresia, cleft lip and palate, and a two vessel cord. Amniocentesis revealed a karyotype of 46,XYr(18)/45XY,-18 with 25% of colonies monosomic for 18. The patient was ultrasound scan. The normative median values of AFP and free 3-hCG and maternal counseled regarding true mosaicism involving fetal and/or extra embryonic tissue; weight correction formula were established from 13000 normal pregnancies by our pseudomosaicism was considered unlikely. PUBS revealed a karyotype of 46,XY, own data. r(18) in all cells. The patient delivered prematurely at 34 weeks an 1860 gram infant In this prospective screening, 8.4% (342/4084) of screened patients were found to with Apgars of 6 at 1 minute 6 at 5 minutes after SROM. Length and weight were at be at increase risk (cut-off point is 1:270). Following genetic counseling, 4.8% the 50th centile, OFC 25th centile. Exam of the infant showed a flat midface, small palpebral fissures, absent nasal cartilage, hypotelorism, nidline cleft lip and palate, (196/4084) were accepted amniocentesis and 6 cases of Down syndrome were syndactyly and abnormal flexion and palmar creases. Cardiac ECHO identified a detected with 83.3% (5/6) detection rate. The incidence of Down syndrome is 1.47 patent ductus arteriosus, pulmsnary atresia, and a hypoplastic right ventricle. MRI per 1000 births in this study. Only one case of Down syndrome was born in term, and ultrasound could not confirm holoprosencephaly. There was a complete absence resulting in a reduction of prevalence of Down syndrome at birth from 1.16 per 1000 of serum IgA. On day 6 he developed Klebsiella sepsis. Repeat blood karyotype in previous study at Taiwan to 0.25 per 1000 in last screening year. showed 46,XYr(18)(plI.23q22.1) in 95/100 cells, double ring in 4 cells, and a We conclude that the rate with this protocol should deleted ring in one. Fluorescence in situ hybridization conftmed origin of the ring and benefit of a high detection an absence of telomeres in the chromosome. Parental chromosomes were nornal. encourage us to introduce maternal serum screening programs using AFP and free 0- After consultation, he was extubated on day 19, expired, and autopsy and biopsies hCG for Down syndrome into Taiwan. The experience of our screening programs were refused. This is the second case to demonstrate true mosaicism of a may provide e ental model for other Orientals and for further large scale r(18)/monosomy 18 at amniocentesis. This is the first reported case to survive after multicenter study. birth, living 19 days. In addition, this case demonstrates the variability of the phenotype found in ring 18/monosomy 18 mosaicism.

2005 2006 Identifying triplet pregnancies at increased risk with an Congenital Cystic Adenonatoid Mealformation of the Lune and Horseshoe atypicality index employing MSAPP and Free Beta (hCG). DA Kidneys A Case Re2ort. KA LeChien, RL Thomas; The Western Krantz'. K Svencer2.PD Buchanan'. TW Hallahan'. VR Klein4.JN Pennsylvania Hospital, Pittsburgh, PA Macril&. 1N7D Laboratories, Inc. Huntington Sta, NY, 'Oldchurch A 26 year-old Caucasian GsP3513 was medicated with phenytoin before and Hosp, Romford, Essex, UK, 'GeneCare Medical Genetics Center, during pregnancy to control a seizure disorder related to previous Chapel Hill, NC,' North Shore Univ Hosp, Manhasset, NY head trauma. At 29 weeks gestation she was transported for Use of an index in maternal serum screening of evaluation of early preeclampsia. A diagnosis of Ballantyne's or atypicality Mirror Syndrome was made with the sonographic finding of associated singleton and twin pregnancies can identify patients at fetal hydrops. Delivery was advised for maternal indications. increased risk for conditions other than open NTD and Down Labor was induced with resultant spontaneous vaginal delivery of a syndrome. We extend this evaluation to triplet pregnancies. grossly hydropic male infant. No resuscitation was attempted. At Blood was collected on 102 unaffected triplets between 14 autopsy, findings included a large congenital cystic adenomatoid and 22 weeks The MOM distribution of Free Beta malformation of the lung (CCAML), secondary hydrops fetalis, and gestation. horseshoe kidney. The resulting chromosome analysis revealed a male (hCG) and APP were as follows: karyotype with no modal or structure changes. Free Beta AFP 10th percentile 1.88 2.05 Previous medical literature regarding the effects of phenytoin 50th percentile 3.13 3.10 during pregnancy has described an association between antenatal 90th 5.52 4.87 exposures and specific anomalies - microcephaly, nail dysplasia, and percentile developmental delay. CCAML has not been associated with phenytoin Standard Deviation (LOGe) 0.4203 0.3376 exposure. Correlation LOGe(AFP) vs. LOGe(FB) r=.1889 Both AFP and free Beta distributions were tighter in triplet Additional malformations have been reported with CCAMLs. Hydrops than in singleton pregnancies. An atypicality index was fetalis is a common finding. Kidney anomalies have been described calculated Mahalanobis' distance. The formula in a few cases of CCAKL; however, the renal abnormalities previously using squared reported have been renal agenesis and multicystic kidneys. Our was as follows: abstract presents the MSD. *(Z22 ereZmxZrs+Z=) first reported case of congenital cystic 1 _-r2 adenomatoid malformation of the lung with associated finding of where, Zm number of standard deviations from the mean for horseshoe kidney. the given analyte and thus patients with extreme levels (either high or low) of AFP and/or FB would demonstrate a high MSD value. Since triplet data did not fit a multivariate log-normal distribution curve, an MSD cut-off of 11.0, resulting in 5 (4.9%) cases being classified as atypical was established. This group, considered to be at significantly increased risk, warrants further evaluation. 0ubsished Abstracts: Prenatal and Perinatal Genetics (continued) 2007 2008 Prenatal Diagnosis Of Wolf-Hirschhorn Syndrome Associated With Prenatal detection of a Y-autosome translocation Abnormal Triple Scren K. Leonard. J.LB. Byrne. S. Elias. Baylor College of [46,XX,-9,+der(9),t(Yn9)(q12; 24)]. Medicine, Houston, Texas. M.M. McCorcruod le'. B. Ranghino)2.L. Drog2. E. rmmerich2. Maternal serum analyte screening as a non-invasive method of prenatal diagnosis J. Payne' R. Ryde. and K. Kruger-Sanden . Michael has become the standard of care in modem obstetrics. Ongirnlly developed as a Reese Hospital and Medical Center, Chicago, IL, screen for women at high risk for having a child with a neural tube abnormal 2Rockford Memorial Hospital, Rockford, IL. defect, Y-autosome translocation is a rare condition. we values of alpha-fetoprotein have been associated with both structural and non- report a prenatal diagnosis of such a translocation in structural fetal abnormalities as well as chromosome abnormalities. More recently, a woman referred for amniocentesis due to advanced the development of the triple screen (aipha-fetoprotein, human chouionic maternal age. Chromosomal analysis revealed a 46,XX, gonadotropin, and unconjugated estriol) has allowed the non-invasive screening for chromosome constitution with extra material on the Down Syndrome, with a detection rate of 60-65%. This compares to a detection rate short arm of chromosome #9. Parental karyotypes were of 20% using maternal age alone. An association has been discovered between very normal, amniotic fluid AFP was normal, and 2 high low values of all three analytes and fetuses with trisomy 18, and many screening resolution ultrasounds detected no abnormalities. A programs now incorporate this into their risk profile. We describe a case of Wolf- FISH study using a WCP paint for the #9 chromosome Hirschhom Syndrome originally identified because of abnormal triple screen results. indicated that the additional material on the p-arm The patient is a 23 year old Hispanic woman referred at 16 weeks gestation for was not #9 in origin. C-banding gave a dark positive further evaluation because of an increased risk for trisomy 18 as determined by a signal on 9p and Q-banding gave a bright fluorescent triple screen. Ultrasound revealed oligohydramnios and a growth discrepancy of 2 signal. A Y-chromosome probe confirmed that the extra material consisted of Y-chromosome material. The weeks. No anomalies were seen, but visualization was sub-optimal due to lack of patient was counseled that the extra material appeared fluid. The family initially declined invasive testing, but later consented when further to be inactive, and if so should not have any clinical growth delay was documented Cytogenetic analysis from fetal blood revealed a significance. We could not, however, rule out the 46,XXdel(4)(pl 5.3) karyotype. Parental karyotypes were normal. The fetus has possibility that a small amount of 9p material may be continued to be growth retarded, now almost 6 weeks behind at 37 weeks gestation. missing, or that the break may have occurred in a Delivery is pending. critical gene. In addition, if some active Y- Although prenatal diagnosis of 4p- has been reported, these cases have usually chromosome material was present, we also could not been identified as a result of fetal anomalies seen on ultrasound, growth retardation, rule out the possibility of some abnormalities in or fetuses at risk because of familial translocation. To our knowledge, this is the sexual development and/or fertility. The patient first report of a case discovered as a result of Maternal serum screening. elected to continue the pregnancy, and she is expected to deliver in July.

2009 2010 Prent diagnos of femoral hyploplasia in a diabetic woman. AJ. PrensatalDiapasa of the Holt-Oras ayndreoe. (L- Molal, Ai'. fD_ CoaxIt. fL Meaders. P. Clark. W.A. Miller. Prenatal Diagnostic Center, Lexington, MA. TffredaL~2 LJohdmol-M 7bommslD_ shinrlM Serm~r.) 7%TaromnHotsph Maternal insulin-dependent diabetes mellitus (IDDM) increases the risk for Gom~iGaensrW Divisio,,heifiTheh3Ummt~onor~onCboloqitMme Hospital for Sick Children2, University of Tormnt. Tloroto,Ontario. a variety of congenital malformations including neural tube defects and caudal Te Holt4Oram syndrome isas msomal domninantondion compising ofupperlimb regression syndromes. The degree of risk depends to some extent on the level abnormality andcongnital eatdmieas TheMr.condtomha beta linhlod ekomaomsozaglon 12q2 of control of the diabetes in the first trimester. Hypoplastic femur-abnormal but thegone has notbeen mapped ye. Wereporsapegamw ofdan affcdmother whom fetal facies syndrome has also been reported in association with maternal insulin- ultrasounddetected anaffieadfemtatdaccuratelydetemanedtdoeaevertyofthiedefec Themotherwas a27 yewoldprhitgvids wonm with loledt-am syndroe iscitling dependen diabetes. We report on prenatal diagnosis of femoral hypoplasia in bilasmrl hypoplassic sthmnbs.conpitalhteartdani(ASDasd VSD)and bOmWabatpecewm a 28-year-old gravida 1, para 0 woman with juvenile-onset IDDM. muclk Sheunderwentaseicalcorrectmi of hterASD adbilual pollidiudonofbothl oed Glycosylated hemoglobin levels in the first trimester of pregnancy had been fingers id developedconnhallautblockdingchildhoodforwhicbap VcemarwaahTend and 12%, reflecting poor diabetic control. She was referred to us for Her prepancywas aoadand the first eutalloaddoneat 16wedoso. wn 10.4% inrepetedasnormal. A second feta ultraoondat 2S weds pstedonrevealdhypopluiaof both genetic counseling and targetedultrasound examination because ofthe diabetes. thmnbs. bilatal dubbed hands, VSDnsd unilerlcerebal alool mgaly. Ultrasound examination revealed a single fetus with a BPD corresponding to Delivery wasompaed vaginal aid the newbon wasfound tohave bilateralclubbedhads. weeks Both femurs were shortened, measuring less hypoplastic thumbs, absentpecsoalia majorand nanVSD. 15.4 gestation. markedly TheHoit-Oram syneromehasbeen lbed totdesegmo 12q2and thaeqciic pae will than one-third of the anticipated length for that gestational age. Based on this probably saon be detcted However. asia masygenctic corndo otypedxrhn oneo iadoe information, the patient opted for erminaton of pregnancy, which was is poor and fetal ultrasound will remain the most imptya st tool forestimagt aoeveriy of te accomplished by D & E. Pathology studies on the products of conception conditon is ts syndromeL ThMseportishows toHo-Oran syndromecm bedceectedprenatally confirmed bilateral femoral hypoplasia with proximal "kinking' deformation, by fetal ultrasound uidthe severityof thecondidon can bedemnined aouaaely. suggestive of a caud regression variant. Bone and cartilage histology were normal, suggesting the absence of a skeletal dysplasia. In addition, a membranous ventricular septal defect was identified. The patient was counseled that recurrence risks for this condition could probably be decreased by establishing good diabetic control prior to the onset of a subsequent pregnancy.

2011 2012 Prenatal diagnosis of an atypical case of OEIS complex. M. Murray, Prenatal diagnosis of Pentasosy X by fluorescent in J. Habecker-Green C Kanaan, S. Pfleuger, G. Cohn. Baystate Medical situ hybridisation in a fetus presenting with the Center, Springfield, Massachusetts. DandT-Walker malformation. T.D. Mvlesi. L. Bus.d2. G. A case of prenatal diagnosis in the early second trimester of t M. McCorauodale2. and D.J. McCorauodale . atypical OEIS complex is reported. The growth retarded fetus was University of Illinois at Chicago Hospital and diagnosed at 16.7 weeks gestation, and had multiple ultrasound 2Michael Reese Hospital and Medical Center, Chicago. anomalies including severe oligohydramnios, a large omphalocele Prenatal diagnosis of Pentasomy X followed the involving the liver, hydronephrosis, hydroureters, an approximately identification of a Dandy-Walker malformation in a 26 90 degree lordotic angulation of the spine, a small, funnel-shaped year-old G P1051 who was referred to us at 36 weeks chest, a multiloculated mass in the abdomen representing dilated gestation ior evaluation of a sonographically identi- ureters or bowel, and clubbed feet. The pregnancy was terminated and fied fetal anomaly. Ultrasound examination revealed the fetus was released for postnatal pathology. Findings were dilation of the 3rd and 4th ventricles, a Dandy-Walker significant for a ruptured omphalocele with adherent amnion, malformation, splaying of the cerebellum, and imperforate anus, bowel and ureter emptying into a cloaca, absent polyhydramnios. FISH analysis of uncultured amnio- appendix, positional deformities of the extremities, severe scoliosis, cytes revealed 5 X-chromosomes in 45 of 50 cells ex- ambiguous external genitalia, and single umbilical artery. The bladder amined, no Y chromosome, and no aneuploidy for chrom- was absent. The cloaca contained both colonic and transitional bladder osomes 13, 18, or 21. These results were confirmed epithelium. The fetal karyotype performed on placental tissue was by standard karyotyping in which all cells analyzed 46,XY. Based on these findings, this fetus was most likely affected were 49,XXXXX. An elective C-section was performed with OEIS complex with amnion rupture secondary to its adherence to at 39 weeks due to massive hydrocephaly. Macroceph- the omphalocele. aly was identified along with many phenotypic findings HEIS complex is a developmental field defect representing the most suggestive of Down syndrome. Reported chromosomal ab- severe of the birth defects in the exstrophy-epispadias sequence. normalities in fetuses with Dandy-Walker malformation OEIS complex itself includes omphalocele, exstrophy of the cloaca, varied from complete and partial trisomies to dele- imperforate anus, and spinal defects. Most previously reported cases tions and marker chromosomes. To the best of our include myelocystocele or other neural tube defect as the spinal knowledge, this is the first case of an association of The defect in our case was severe scoliosis. This hydrocephaly and a Dandy-Walker malformation with anomaly. spinal X. This is the second case of X case provides additional information that may be of value in determin- Pentasomy Pentasomy ing the underlying etiology of this complex. diagnosed prenatally, and the first in which the FISH technique was applied. Published Abstracts: Prenatal and Perinatal Genetics (continued) A347 2013 2014 Mild phenotype in a prenatally diagnosed case with de novo deletion for terminal Unusually low Triple Screen profile associated with major placental 18q. T Peters-Brown L Fry-Mehitretter M Decker-Phillips S. G Kahler. and infarction and prematurity. F.D Seydel and G.S Eglinton Dept. of OB/GYN, M. B. Oumsileh. Departments of OB/GYN, Pathology, and Pediatrics, Duke Georgetown University Medical Center, Washington, DC. University Medical Center, Durham, NC. We report a case of 90% placental infarction associated with an unusually low Triple Screen pattern in a 32 year old G3, P2 white woman. At 35 years of age she sought We report the mildest phenotype expression of the 1 8q- syndrome. The classical preconceptual consultation from our center. Her first pregnancy resulted in a 3410 gm. deletion 1 8q syndrome shows abnormalities such as growth deficiency, normal-term infant. The second pregnancy resulted in fetal death in utero at 18 weeks genitourinary malformations, neurologic and occular abnormalities, hypotonia, with multiple structural anomalies; cytogenetic analysis revealed a 45,X fetus. microcephaly, and mental retardation, among others. A 28 year old black female Consequently, the patient was referred for prenatal diagnosis for the third pregnancy. (G2PI) was referred for clinical evaluation because both partners were sickle-cell The Triple Screen MOM values at 16 weeks were: AFP, 0.69; hCG, 0.52; uE3, 0.7. carriers and and alcohol abuse. The patient had one normal four-year-old son from The uE3 value was 0.1 MOM unit from the threshold for high risk for trisomy 18 a different partner and no other remarkable family history. Amniocentesis was (Canick et al., 1990). Amniocentesis revealed a 46,XY fetus. The patient was performed at 15.9 week gestation and DNA testing predicted the fetus to have diagnosed at 27 weeks with hand, foot, and mouth disease. Two weeks later fetal hemoglobin AA. Cytogenetic studies were done incidentally on the cultured movement had stopped. The non-stress test was non-reactive. The umbilical cord amniocytes and revealed a terminal deletion of chromosome 18: showed absent end-diastolic velocity, but the middle cerebral artery doppler flow was 46,XX,del(18)(q22). Chromosome studies on both parents were normal. The normal. The baby was delivered that day due to fear of acute decompensation. The parents were counseled about potential outcomes and chose to continue the baby weighed 1320 grams (30th percentile); the 5 min. apgar score was 8. Placental pregnancy. Targeted ultrasound at 15.9 and 18 weeks gestation revealed no examination revealed 90% abruption. The baby was extubated at 5 days and went home abnormalities. A female baby delivered at 33 weeks gestation showed cleft palate at 6 weeks. At three years of age, the baby is currently doing well with no gross motor and no other noticeable anomalies. At 10 months, the baby has midface delay or neurological impairment. hypoplasia, mild developmental delay, possible macrocephaly (mild), and no other Both high and low MSAFP values have long been associated with adverse apparent abnormalities. It appears from this case and other reports that the severity pregnancy outcome, including low birth weight, congenital malformations, and of malformations correlates with the extent of 18q deletion. prematurity. More recently, unusually low hCG or uE3 values have also been associated with adverse outcome, including trisomy 18 and triploidy. Low uE3 values have also been associated with early fetal death at 15-20 weeks (Ventura eral., 1994). In this case, inasmuch as hCG and uE3 are placental hormones, the massive placental infarction was clearly sufficient to cause significantly low hormone values. The reduced placental circulation could also account for the low AFP value. It seems likely that some portion of the infarction had been recent, but the abnormal Triple Screen could have associated with an early portion of the infarction, or perhaps it is a marker for risk of infarction. The relationship between the viral infection and the infarction is suggestive but cannot be determined.

2015 2016 Unusual Case of 45,X146,X,del(XXp11.4) mosaiclsm In Infant of diabetic mother. Antley-Bixler syndrome: A case report and prenatal diagnosis in a subsequent L Steinberg. H. Punnett. C. Quashie. A. Schneider. Albert Einstein Medical Center pregnancy. L. M. Wolf. T. G. Kelly. J. S. Austin. P. K. Senagore. and M. L. and St. Christopher's Hospital for Children, Philadelphia, PA. Netzloff. Michigan State University, East Lansing. We report a case of 45,X/46,X,del(X)(pl 1.4) mosaicism in a female with Antley-Bixler syndrome (ABS) is a rare disorder consisting of , multiple congenital anomalies. The pregnancy was complicated by well-controlled radiohumeral synostosis, femoral bowing and breakage, choanal atresia/stenosis and maternal insulin-dependent diabetes secondary to a pancreatectomy due to a stab severe respiratory complications which often lead to death. Survivors usually have wound. An obstetrical ultrasound at 25 weeks gestation identified a single umbilical normal mental capacity. We report a ne* case with prenatal diagnosis in a artery and shortening of the long bones. A fetal echocardiogram at 21 weeks was subsequent pregnancy of the parents. unremarkable. Amniocentesis at 26 weeks gestation revealed SK was diagnosed with ABS at birth. Findings typical for ABS include frontal 45,X/46,X,del(X)(p11.4) mosaicism and a negative acetyl-cholinesterase. Peripheral blood chromosomes after birth showed only 46,X,del(X)(pl 1.4). bossing, fusion of the lambdoidal sutures, unilateral choanal stenosis, hypogenitalia At birth the baby was mildly hypotonic. Length was 46 cm (<5%), weight 2.4 with hypospadias, radiohumeral synostosis, and a history of fractures in both femurs. kg (10%), and head circumference 31.3 cm (10%). Turner syndrome-like features Chromosomes are 46,XY. Parents are unrelated. included: a short neck with low hair line and redundant skin, widely spaced nipples, During a subsequent pregnancy, ultrasound at 17 weeks demonstrated femoral marked , narrow and convex nails, and slightly puffy feet. Other bowing, shortening of the extremities, fixed elbows in a flexed position and dysmorphic features included flat nose with anteverted nares, micrognathia and . The parents opted to interrupt the pregnancy at 18 weeks. Fetal overriding maxillae, accessory left nipple, single umbilical artery and depressed left anomalies included ambiguous genitalia, fixation of the elbows, , chest wall. Of particular note were multiple vertebral anomalies from T-1 to T-8 sacral neural tube defect, ventral wall defect with bowel protrusion at the umbilical with butterfly and hemivertebrae. A rudimentary left first rib was seen with absence of insertion site and bowed femurs. Additional autopsy findings were probe-patent the next six ribs on the left side. Twelve ribs were noted on the right with partial fusion choanae, radiohumeral synostosis, sacral meningomyelocele, rhizomelia of the upper of the third and fourth ribs. Incomplete ossification of the sternal segments was also extremities, prominent clitoris, vaginal atresia and micrognathia. Chromosome noted. A kidney ultrasound identified a left pelvic kidney. There was dextrocardia studies were 46,XX from amniotic fluid obtained prior to delivery. with an ASD and VSD Head ultrasound was normal. Our experience along with previous reports of consanguinity and recurrence within Prenatal genetic counseling focussed largely on the findings that may be a sibship for ABS supports autosomal recessive inheritance. The recurrence risk of associated with this chromosome abnormality. The likelihood that the short limbs 25% and the high incidence of respiratory complications which reduce survival and single umbilical artery may be related to diabetic embryopathy was overlooked given the mother's well controlled diabetes. This case highlights the importance of suggest the usefulness of prenatal diagnosis. Our case and previous reports indicate considering all possible etiologies when counseling for prenatally detected this can be done effectively and accurately with ultrasound in the second trimester of anomalies. Well controlled diabetes does not guarantee a good outcome. pregnancy. A348 Published Abstracts: Servic,es, Public Policy and Screening 2017 2018 Advisability of testing Children and Adolescents presymtomatically for TheFmadlialCmwePre ra: A M a Appreeb toIIUin_ Bebef NFII: Families and Nurse's Perceptions Geedics k C u M (1,2), MS. =_eblel MD(104),AE. Ovace. MD Catherine Bove M.Ed., RN* Mia MacCollin M.D. ,* M. Priscilla Short M.D.* (1,2), D.W. Yaadell.5D(1,3) (1) Familial Ciaeerhopiaaf St VermostCme esCw.(2) Deborah Adams, PhD,RN+ Vrmat Rem Ginmds Cew,Dept ofPediatris, UivasfityeVeamontCAeW ef Mdisi,(3) *NF Clinic, Neurogenetics, MGH +Boston College Of Nursing Vermont Cut Cuser,Uihrity ofVermont ColldgeofMdimime; (4)Detetoeisle,Univehty Purpose:Recently molecular biological techniques have made it possible of Varnan Ceh ofMudicie. to provide presymptomatic testing in 2/3 of families with NFII. Neuro- Tie Veement Cue Ceteredb tlhs Familial Cmar Progun (FCP) s Dober, 1993. It surgery is an available treatment for both adults and children. This isa maoded prormeambaig ecsprsusefcliniciasm _d ssrobers iveiwdi eis_, raises the issue of the advisability of testing children and adolescents sology, usdmoleelar bioloa. The prorads oal is Wideiiy Smiliesulterepieswilhmeauu for a predominately adult onset disorder. The purpose of this study was of ceaeeradisepmdic_oemaeba& cura rseech--iou UNm smeing, at to 1)survey genetic nurse specialists to identify their perceptions of a pediatric age group and to *efirat 17 173 b the concerns for presymptomatic testing in Dorimg moeuhse FCPhasbebnisadftm catbmhaWee 11Irekghdo 2)correlate these perceptions with the current clinical experiences at Vermont RegieoalCOsm (VRGC). resnssab%t5oef kquirsbhs beenaelf. the NF clinic at the MGH. rssrraswihwboencerosa4bos asfaauiiyhieoryefooiem amm. Seaf~rf-deupsped wdwib pblis. Methods: A review of the literature was done to generate a preliminary masommouamtofdiediaoowais ofthe reesMSH2 ad MAlI, both isslvedin tbe develapomoef ooisq list of concerns regarding the advisability of testing children and HerditarNy Colo. Cutter (MNCC). e adolescents. This list of concerns was modified through telephone Od_ rdaralshavemu frmnavrietyofeclom. Ap i 20% md discussion with four experts who were authors in these published .idiantian ofaoaemsunugtmay balmydermronmdm reta ooumuiigfaralvied madualag investigations. Next, a written questionaire was sent to nurses who (AMA) amsnier e0dmaed from ralsf. _rIu6l20%O od~veaksefi dhaveemeerd cs d25%h afanlybtemy practice i. ge.:atics and are members of ISONG(International Society Of u sm Nurses in Genetics). To achieve the second purpose of the study, four which is tivef*llcer irms iowladoglHNPCC, Beed/OvrmCow Syndrome, families followed at the MGH NF clinic were interviewed to identify Familial Amisms Polyposis vasHippeLisdm 5yrem.,RookoblaioLio-Fui. sai Cowdensyndoec. TeU faimiedo ofit m bwa caomer syndrome adishledei s issues and concerns from the family's perception. The interview data r_1aining emtee is& wtl - from the families was analyzed for content and the resulting themes thatrage kern sunmigly lowrisktlo thomedueretmplex n qpoex with the list of concerns. risk for caner. were correlated Family hstories amereviwoduad_rurd at abi-wa.ly _amferme by a pOP Results:The list of issues and concerns generated by 1)the literature a survey of genetic tmg a uvsonag mseo lforpriaroi. Manyfunlis arweeld emtis gi *he review, 2)interviews with author/experts and 3)the oppoutumity topacstaipatisresmwer a_apimg to likkupefic _s to oaer. Is addia, a aty nurse specialist's demonstrated concerns from the following areas: lkg atpycolagcfar iszaghinkfamiies aplaud Wehleduh frmtheoedisisod legal/ethical, family&child/psychological, educational and social wil cmeboth betterctefor od expa is (insurance, employment problems). The nursing concerns of family effort famihs uedWmdsrtulisgoftaroieofgrakis relationships and decision making correlated with our experience with four different NFII affected families in the NF clinic at MGH.

2019 2020 Implementation of a comprehensive thalassemia screening program in Establishing a cancer genetics clinical program in an academic medical Massachusetts Asian And center. I. P. Stadler and J. J. Mulvihill. Department of Human conjunction with the Lowell, Southeast Birthing and infancy (SABAI) project. T. Namm', J. Cochran , L..Ql3, and A. C I. University Genetics, University of Pittsburgh Medical Center, Magee-Womens Massachusetts Department of Public Health and Lowell Hospital, Pittsburgh. PA. of Massachusetts/Lowell1, Advances in cancer genetics, media attention, and increased emphasis Community Health ContePr". on cancer detection create a demand for establishing clinical has been at the early A comprehensive thalassemia screening program fully operational cancer genetics programs, especially at academic medical institutions. Lowell Community Health Center prenatal clinic since 1993. Patients and their From 1991-4, we saw 41 consultands in a genetics clinic of a large spouses (or partners) are offered screening when one or any combination of the women's hospital. In January 1995, after gaining interinstitutional following occur (1) a family history of thalassemia, (2) a MCV value of less than 80, support from the hospital and the local cancer and genetics institutes, (3) the presence of hemoglobin E, and/or (4) elevated hemoglobin A2. SABAI health we launched a comprehensive cancer genetics clinical program with the educators, genetic counselors, and other support personnel assist patents with the classic tripartite activities of service, education, and resesrch of medical and assessment of recurrence/occurrence risk. Research comprises participation in national counseling and ethics interpretation diagnoses of a local education involves The Health Centers medical director has primary responsibility for thalassemia protocols and development protocol; and lectures in and outside the sponsoring institutions and a one-day CME screening. A physician is available to the project for staff training, chart review, Service takes in 3 4-h clinics a week, SABAI health educators accompany when prenatal course on cancer genetics. place protocol development patents for a maximum of 6 probands or families a week, in addition to monthly diagnosis is performed, and provide personal support if additional screening becomes clinics for neurofibromatosis and multiple endocrine neoplasia necessary. A Professor at UMass Lowell provides frequent training sessions for syndromes. Following initial publicity efforts, 420 inquiries were SABAI staff in Genetics and Human Anatomy & Physiology. received in the first 5 months, resulting in 61 scheduled families. The The thalassemia screening program is readily accessible to patients in greater 3 major reasons for consultation were the desire for "gene testing", Lowell. Commensurate with expansion of the referral network, infants with HbE are concern about family history of cancer, and interest in being involved from the New Newbom Screening Program (NERNSP). with research. referred England Regional intake to the consultation consisted of Screening over the two-year period has revealed a wide range of thalassemia Lengthy telephone prior trait clarification of issues, collection of family history, and retrieval of variations, including alpha-1 thalassemia (cc-1), alpha-2 thalassemia (cc-2), alpha records and tissue blocks on relatives with E disorder E trait and permissions for medical (c/A), hemoglobin H disease (HbH), homozygous (EE), (AE), cancer. To date, 30 initial consultations have been completed, with beta-thalassemia variations (>-thal). In addition, a number of iron deficiences (FeDef) clinical recogn't'on of hereditary breast and ovarian cancer in 6 have been diagnosed, as well as the E trait/iron deficiency combination (AE/FeDef). families, hereditary nonpolypotic colorectal cancer in 3, and The data shown below represent Lowell area Southeast Asian patients. Li-Fraumeni syndrome in 1. Families deemed suitable were offered linkage DATA SUMMARY: studies; DNA collection nearly always involved tissue blocks. A a-1 c-2 a/A HbH EE AE 3-thal FeDef AE/FeDef Professional costs are 0.7 MD geneticist and 1.2 genetic counselor. 168 1 6 3 5 16 30 1 20 6

2021 THIE ONTARIO MATERNAL SERUM SCREENING PILOT PROGRAMME: AN EVALUATION OF PATIENT KNOWLEDGE, SATISFACTION AND PSYCHOLOGICAL OUTCOMES A.M. Summers. V. GeeI*. S. HM geizaosl5 . YIeunsA. P. PUhA. R. GlazierA:. Dcpartmcwn of Gcnctics. North York GcnCrsi Hospital. North York: lnstitutc for Clin Evalualisc Scienecs. North York: e*Dcpt. Of Family Modicinc. Women's College Hospital, Toronto: and ADept. Of Clinical Epidmniology. Wcilcslcy Hospital, Toronto, Ontario. On July 1. 1993, a two year pilot programme looking at maternal serum (triple marker) screening was started in the Province of Ontario, funded by the Ontario Ministry of Health (MOH). Part ofthe mandate ofthe programme was to evaluate patient attitudes toward the programme. In order to assess patient knowledge and satisfaction, a standardized scale, the Maternal Serum Screening Knowledge Questionnaire (MSSKQ) was developed and validated. Anxiety and depression were measured using Spielberger's State Anxiety Inventory (STAI) and the Centre for Epidemiologic Studies Depression scale (CES-d), respectively. In addition, demographic data was obtained for each woman. The questionnaires were administered in the form oftwo booklets - Booklet I at 15-18 weeks (prior to MSS) and Booklet 2 at about 24 weeks. The booklets were administered to about 900 women registering at a centralized programme in one hospital and to a further 8SO women at seven other centres throughout Ontario. Overall, women were quite satisfied with the programme. However, the level of knowledge varied. There was a clear increase in the state anxiety score of over four points among women who received false positive results. Interpretation of this increase in score is problematic. Concern about the health ofthe newborn but not the mother's own health was significantly higher among women who screened false positive.