Session 20: Plenary Session A3 1 2 Genome-wide screen for Hirschsprung disease susceptibility loci in a Correction of the high-SCE phenotype in Bloom's syndrome (BS) cells Mennonite kindred segregating an endothelin-B receptor mutation. by transfection of the wild-type gene. J. German. M. Proytcheva. T.-Z. Ye. E.G. Puffenberger. A. Lynn, C. Kashuk, and A. Chakravarti. Dept. of Genetics, Case M. Sanz. N. Neff. N. A. Ellis. New York Blood Center, New York, NY. Weserin Reserve Univ., Cleveland, OH. BS is a rare autosomal recessive disorder characterized bv short stature and Genome-wide screens using polymorphic markers in segregating multiplex families are predisposition to developing cancer. An excessive rate of sister-chromatid exchanges now routine for localizing disease genes. In principle, susceptibility loci may even be (SCEs) in proliferating BS cells is a unique and diagnostic feature of the syndrome. mapped in simplex families by searching for allelic associations between genetic Recently, the BS gene, BLM, was isolated and shown to encode a 1417 amino acid markers and disease phenotype. This exercise is most efficient in isolated populations protein product homologous to RecQ helicases (Ellis et aL, 1995). We have introduced where large genomic segments will be shared by affected individuals on the basis of the wild-type BLM cDNA into BS cells in order to test its ability to correct the high- common ancestry. We demonstrate the identification of multiple genomic regions that SCE phenotype of BS cells. harbor putative susceptibility loci for Hirschsprung disease (HSCR) in a large, inbred A full-length BLM cDNA, R 12, was transferred into pOPRSVI-CAT, a mammalian Mennonite kindred by using genome-wide association mapping. We previously expression vector that contains the RSV promoter and dominant selectable marker neor. demonstrated the segregation of a missense mutation (W276C) in the endothelin-B The recipient cell line was the SV40-transformed fibroblast cell line GM08505B, which receptor gene (EDNRB) on chromosome 13q22 in this kindred. This mutation is derived from an Ashkenazi Jewish person with BS who is homozygous for a 6 bp demonstrated a sex and dosage dependent penetrance consistent with the complex deletionl7 bp insertion that results in termination at amino acid residue 740. Levels of pattern of inheritance of HSCR in the Mennonites. BLM mRNA in GM08505B are reduced and protein is undetectable by Western blot We analyzed the genotypes of 527 autosomal and X-linked microsatellite markers in analysis (Neff et al., this meeting). The SCE rate in GM08505B is high (mean 55.0 33 simplex HSCR cases and their parents. For each locus, associations between allele SCEs/46 chromosomes and range 15.9-88.4 SCEs/46 chromosomes of 20 cells). frequencies on the transmitted (T) and untransmitted (U) chromosomes were evaluated Ten micrograms of control cDNA, pOPRSVI-CAT, and of BLM cDNA, pOPRSVI- by chi-square statistics and multipoint association lod scores across each chromosome. R12, each were introduced by lipofection into GM08505B. Forty-eight hours after These analyses were repeated for chromosomes transmitted to affected W276C transfection, Geneticin selection was applied, clones were isolated and expanded, and heterozygotes (Ti) only. the SCE test was carried out. The SCE rate in cells transfected with the control cDNA The genome-wide screen for linkage disequilibrium identified several statistically sig- (I clone examined) was unchanged (mean 64.8; range 40.4-95.6; 20 cells) compared to nificant genomic regions in T and TI patient samples. As expected, a large region on that in untransfected GM08S05B cells, whereas the SCE rate in cells transfected with chromosome 13q22 (EDNRB) and a smaller region on chromosome 10qil (RET) were the BLM cDNA (2 clones examined) was corrected towards normal (mean 17.7; range detected in both T and Ti samples. No other statistically significant regions were identi- 8.5-41.0; 20 cells; and mean 18.0; range 10.3-25.4; 17 cells). fied in the total sample (T). However, when affected W276C heterozygotes were analyzed This phenotypic correction of GM08505B proves that the BLM cDNA encodes a independently, two other chromosomal segments were identified, viz. chromosome 6p and functional protein capable of complementing the high-SCE phenotype, and presumably 8q. The 8q locus barely exceeded the multipoint lod score threshold, while the 6p lo- the BS defect. This transfection system serves as a starting point for characterization of cus yielded results second in magnitude to EDNRB. Both of these loci are being studied the effects of mutations that we will introduce into the BLM cDNA in vitro and is the with additional markers in order to determine their role in the development of Mennonite first step toward therapy of the BS defect using the wild-type BLM gene. HSCR.

3 4 Coverage of new genetic technologies: What matters to insurers? M. Schoonmaker, B. Toward a third generation genetic map of the human genome Bernhardt and N.A. Holtzman. Johns Hopkins Medical Institutions. Baltimore, MD. based on bi-allelic To examine the role of third party payers in the diffusion of new genetic technologies, we polymorphisms. surveyed 751 insurance companies. 166 (22%) responded to a questionnaire using scenarios D. Wangl, R. Sapolsky2,1, Spencer.1, T. Riouxl, L. KruglyAkl, L to assess how coverage decisions would be made for cystic fibrosis carrier screening (CFCS), Hubbe[I2, G. Ghandg2, T.; Hawkjls1, T. Hudson1, R. Lipshut2 and BRCAl testing, and gene therapy for CF (GTCF). 76% have never made a coverage decision E. Lander1. 1Whitehead Institute/MIT Center for Genome for CFCS. 39% of insurers would cover the baseline scenario of offering CFCS to all pregnant Research, Cambridge, MA 02139 and Inc., Santa Clara, CA women at a cost of $40 per test and a sensitivity of 85%. Changing the scenario to only 2Affymetrix, 77% of 95051; pregnant women with a positive family history would result in coverage by companies. and studies a decreased endorsement ACOG and a demand would Large-scale genetic mapping epidemiology require Increased test sensitivity, cost, by high and robust for at hundreds of each also significantly increase coverage. For BRCA1 testing, 85% have never made a rapid technology assessing genotypes polymorphic loci in parallel. We have begun the construction of a coverage decision. 26% would cover a baseline scenario of testing high risk women using a of bi- $250 test detecting 80% of BRCA1 mutations. Changes significantly increasing the proportion third generation genetic map of the human genome consisting covering include an improved detection rate, reduced cost, endorsement by a national consensus allelic single nucleotide polymorphisms (SNPs) and the development conference and high consumer demand. For GTCF, 27% would cover the baseline scenario of a high-density DNA probe array for human genotyping. Bi-allelic of paying the medical costs of a trial of presymptomatic children. Coverage would be SNPs offer the advantages of ease of isolation (because of their great significantly more likely if therapy was offered only to severely affected children or adults, if abundance), highly parallel detection (using DNA probe arrays), and its effectiveness was proven, or if administered in a regional hospital or outpatient setting. automated scoring (because of their plus-minus nature). There were no differences in coverage of any technology by type of insurer (HMO, PPO, To identify SNPs, we have developed a semi-automated system for indemnity or self-insured payers). 36% thought patient demand for CFCS would be high, comparative sequencing of random PCR products from multiple versus 66% for BRCA1 testing and 91% for GTCF. Only 27% agreed that there is great unrelated individuals. Based on our results to date, SNPs with the potential for future cost savings in covering BRCA1 testing, versus 42% for CFCS and 58% minor allele a > 30% to occur at a rate of for GTCF. Fewer than 20% that for of these would having frequency appear agreed offering coverage any technologies 1/1000 To date, we have identified and more than 400 make their benefits policy more competitive. bp. mapped We conclude that few insurers have for coverage of selected new SNPs. developed policies genetic a of 1000-2000 well- technologies. Improvement in the sensitivity of testing, limiting tests to populations at highest Analysis shows that genetic map consisting risk, increased public demand, endorsement of technologies by professional societies, and spaced SNPs should suffice for comprehensive coverage in genetic reduced cost could lead to a greater likelihood of coverage. mapping studies.

5 6 Exoni of the Huntington's disease gene carrying highly expanded CAG Uptake and impact of BRCA1 testing in members of hereditary breast-ovarian repeats is sufficient to generate a progressive neurological phenotype in cancer (HBOC) families. C. Lerman, Georgetown University Medical Center. transgenic mice. G. Batesl. L. Mlangiarinil. K. Sathasivaml, \1. Seller', A. Harper2, Washington. DC; S. Narod, Women's College Hospital, Toronto, Ontario, Canada; and Y. Trottier3, J.-L. Mandel3 and S. Davies4. 1. Guys Hospital, UMIDS, London, UK. 2. H. Lynch, Creighton University, Omaha, Nebraska. St. Thomas's Hospital, UNIDS, London, UK. 3. IGBMIC, Strasbourg, France. 4. and University College, London, UK. We report the results ofthe first large-scale prospective cohort study ofthe uptake Huntington's disease (HD) is one of an increasing number of inherited neurodegenerative short-term impact of BRCAI testing on quality of life and medical decision-making. diseases caused by a CAG/Glutamine repeat expansion. The repeat is located in the first BRCAI test results were offered to 279 adult male and female members of BRCAI- exon of a ubiquitously expressed gene of unknown function and normal and expanded linked HBOC families after education and counseling sessions in a research setting. repeat ranges of 6-34 and 35-121 respectively have been reported. There is an inverse Forty-three percent of all HBOC family members and 60% of those who completed a correlation between the repeat size and the age of onset of the disorder, the larger interview BRCAI test results. was and associated with the form of the disease. Whilst features baseline (n=192) requested Uptake significantly repeats being juvenile many a number of of the genetics and molecular biology of this class of neurodegenerative diseases are positively related to: having health insurance; having greater first-degree comparable, they differ in the patterns of neuronal cell death. Transgenic models will be relatives affected with breast cancer; higher levels of knowledge about BRCAI testing; essential to unravel the molecular events underlying the neuropathology of these diseases. and perceiving the benefits oftesting as important. At one-month follow-up, individuals As part of our work directed toward the construction of a mouse model of HD, we have identified as noncarriers ofa BRCAI mutation showed statistically significant reductions introduced the 5' end of the mutated human HD gene into the mouse germ line. We have in depressive symptoms and functional impairment compared to BRCA1 mutation derived three transgenic lines with CAG expansions ranging from 115-150 repeat units Mutation carriers and who which develop a progressive neurological phenotype. The age of onset and severity of carriers and to individuals who declined testing. persons the disease is related to the size of the repeat expansion and the number of integration declined testing did not show any changes in these quality of life outcomes. However. events. The phenotype includes a constant tremor, epileptic seizures and involuntary analysis of subgroup differences in responses to a positive BRCAI test provided movements. The transgene is ubiquitously expressed in these three lines. A fourth line. evidence for possible adverse effects in unmarried individuals. Finally, 18% ofcarriers carrying a CAG expansion of 142 repeat units has shown no symptoms after twelve reported that they intended to have prophylactic mastectomies and 33% intended to have months, and in this line the transgene is not expressed. A comprehensive molecular and We conclude that of BRCAI will be far neuropathological characterisation of these mice is in progress. One of these lines has prophylactic oophorectomies. uptake testing now been bred to the fifth generation and repeat instability has been observed. from universal, even among very high risk individuals. For high risk individuals who receive BRCA1 testing in aresearch setting with extensive counseling, there are potential psychological benefits. Additional research is needed to assess the generalizability of these results, to explore individual differences in responses to testing, and to evaluate the long-term consequences. A4 Session 20: Pleinary Session (cont.) 7 8 Structure and function of the human chromosome 15 imprinting center. Mutation analysis of PXAAA1, a new peroxisome assembly gene responsible B. Horsthemke'. Barbel Dittrich', Bernd KornM. Sarah Rickard3. Jessica Buxton. Shini for a subgroup of the peroxisome biogenesis disorders N. Braverman, T. Yahraus, A. Moser, D. Gordon, S.J. Gould, D. Valle. Howard Hughes Med. Saitoh . Robert D. Nicholls4. Annemarie Poustka . Andreas Winterpacht:. Bernhard Inst., Kennedy-Krieger Institute and Johns Hopkins Univ. School of Medicine, Baltimore, Zabel' and Karin Buiting'. 'Institut fur Humangenetik, Universittit Essen. Germranv. MD 2Deutsches Krebsforschungszentrum. Heidelberg, Germany. Institute of Child Health. The peroxisome biogenesis disorders (PBD) are a heterogeneous group of lethal, auto- London, UK, 'Department of Genetics, Case Western Reserve University and Center for somal recessive diseases that are characterized by defective import of peroxisomal matrix Human Genetics, Cleveland, Ohio, sKinderklinik, Universitat Mainz, Germany. (Intro by proteins. Zellweger syndrome is the most severe phenotype; neonatal adrenoleukodystro- E. Passarge) phy and infantile Refsum disease are milder. We are currently identifying human genes Imprinting of a 2 Mb chromosomal domain within proximal 15q is regulated by an that cause PBD based on homology to yeast peroxisome assembly genes. We recently imprinting center, which has been mapped to a 100 kb region including exon I of the identified a new PBD gene, PXAAA1 , predicted to encode a member of the AAA family SNRPN gene. From this region we have identified novel transcripts, which represent of putative ATPases and responsible for complementation group (CG) 4 (Yahraus et al. alternative transcripts of the SNRPN gene. The novel exons (BDIB, BDIB', BD1B*. EMBO, in press) CG 4 is the second largest PBD CG, with all three classical pheno- types represented. We have now undertaken mutational analysis of the PXAAA1 gene BDIA, BD2, BD3) lack protein coding potential and are expressed from the paternal to determine genotype/phenotype correlations and provide information on gene structure chromosome only. Five Angelman syndrome patients who have a paternal imprint on the and function. We have identified 13 different mutant alleles from 11 CG4 probands by maternal chromosome because of a maternal imprint switch failure, have intragenic SSCP and direct sequencing of RT/PCR products. Interestingly, a genotype/phenotype deletions affecting one. two or three of the BD exons. One AS patient has a point relationship is emerging: the milder PBD phenotypes have normal levels of PXAAA1 mutation in the splice donor site of exon BD2. In another family, a 6 kb deletion mRNA and all result from missense mutations located predominantly in the 3' end of immediately distal to exon BD3 may interfere with the expression or splicing of the BD the ORF. The more severe PBD phenotypes result from frameshifts, deletions, splice site transcript. These results suggest that the BD transcript is causally involved in the changes and 2 missense changes. They are associated with decreased levels of PYAAA1 paternal->maternal imprint switch. We propose a model in which the BD transcript is the mRNA and the mutations are distributed throughout the gene. Surprisingly, normal peroxisome morphology is restored in fibroblasts from the single patient in CG 6 by imprintor. It acts in cis on an imprint switch site close to SNRPN exon 1, which is transfection with PXAAA1 . These results, taken together with the complementation deleted in Prader-Willi syndrome patients who have a maternal imprint on the paternal data, are consistent with either extragenic suppression or intragenic complementation chromosome because of a paternal imprint switch failure. In the XX germline, a trauis between CGs 4 and 6 patient cell lines. acting factor specific for the female gernline is involved in completing the switch and/or initiating the bidirectional spreading of the maternal imprint on the paternally inherited chromosome. In the absence of the trans acting factor (XY germline), the maternal imprint on the maternally inherited chromosome is lost starting from the switch initiation site (spreading of the paternal imprint). (Supported by the DFG, HFSP, NIH and MRC)

9 Complex genome rearrangements to create a mouse model (3 transgenes and 2 knock-out alleles): insights from the total replacement of murine hemoglobin with human sickle hemoglobin. C. Piszty1, C. NI. Brioni, E. Witkowska2, N. Mvlohandas5 and E. NI. Rubini. 'Human Genome Center, Lawrence Berkeley Laboratory, University of California, Berkeley. 2Childrens Hospital Oakland Research Institute, Oakland, CA. i 2Northern California Sickle Cell Center, San Francisco. Sickle cell disease (SCD) has served as a paradigm in the field of human genetics with numerous firsts occurring during the deciphering of it's molecular basis and pathophysiology. In spite of this wealth of information treatment options for SCD have remained limited thus prompting a now decade-old effort to develop a mouse model for SCD. Although a number of transgenic mice expressing sickle hemoglobin (HbS) have been described they lack many of the genetic and clinical hallmarks of SCD due to the fact that murine globin chains are still being produced in these animals. To circumvent these limitations we have undertaken an extensive series of transgenic and gene-targeting based manipulations of the mouse genome focused on the inactivation of the murine globin genes coupled with expression of human globin transgenes. We have generated a line of mice expressing human o-globin, -yglobin and 0'-globin. Through successive rounds of breeding of these transgenic mice with murine a-globin and 0-globin knock-out mice we have successfully created mice that no longer produce murine adult globin chains but express exclusively human sickle hemoglobin (HbS, ha2hoP2). These mice contain sickled red cells and die several hours after birth. In parallel we have also generated mice expressing exclusively normal human hemoglobin (HbA, hCa2h)2). These HbA mice reach adulthood and appear normal thus demonstrating that normal human hemoglobin can functionally replace mouse hemoglobin and, moreover, that mice producing exclusively HbS die postnatally as a result of red cell sickling. We are currently exploring approaches to extend the survival of these HbS mice. The creation of mice expressing exclusively HbS provides a useful new substrate for the development of improved therapies for SCD and also serves as a reminder of the challenge of moving from knowledge of the genetic basis of a disease to the development of an authentic mouse model. Slide Session 21: Cancer Genetics 1: Familial Predisposition 10 11 An ATINl antisense vector confers an ataxia-telangiectasia phenotype on normal Reconstitution of the G2 checkpoint by a human Forkhead homolog. cells. N. Uhrhammger, E. Fritz, L. Boyden, and M. S. Meyn. Departments of S.E. Plon2. C. KeIlerLandD. ajl.Texas Children's Cancer Center, Departments Genetics and Pediatrics, Yale University School of Medicine, New Haven CT. of Pediatrics and Molecular and Human Genefics2, Baylor College of Medicine. Ataxia-telangiectasia (A-T) is an autosomal recessive disease marked by Many aspects of the response to DNA damage are conserved among neurodeaeneration, immunodeficiency and cancer. A-T cells exhibit spontaneous eukaryotes. In particular, cells arrest in multiple phases of the cell cycle after DNA genetic instability and respond abnormally to ionizing radiation. The gene mutated in damage caused by radiation or chemicals. Some of the genes responsible for the A-T patients, ATM, may mediate multiple cellular responses to DNA damage. DNA damage inducible checkpoints have been identified in eukaryotes. In at least although its exact function is unclear. To facilitate studies of ATM, we have one instance the ATM/WEI1/MEC1/TEL1/ESR1 genes share sequence homology developed an ATM antisense vector that confers an A-T phenotype on normal cells. across multiple organisms including S. cerevisiae, D. melanogaster, and H. sapiens. We cloned a 1.3 kb segment of the ATM cDNA 5' to the P13 kinase domain into the Mutation in the ATM gene is responsible for the recessive disorder ataxia pRepS episomal expression vector in antisense (p46a) and sense (p46s) orientations. In contrast other genes such as mammalian p53 which plays a The p46a and p46s plasmids were tested by transfection into normal (GM639) and A- telangiectasia. T (AT5BIVA) human fibroblasts. Four independent p46a-expressing clones of crucial role in the GI checkpoint do not appear to be conserved in lower eukaryotes. GM639 were studied. All four were radiosensitive. Colony survival after 3 Gy X- We developed a screen for the isolation of new human checkpoint cDNAs by irradiation averaged 3.2% for the GM639-p46a clones and 2.6% for A-T cells, demanding high copy suppression of the yeast checkpoint mutations rad9 and compared to 35% for the GM639 parent and three GM639-p46s clones. All four mecl. In this way, human cDNA libraries can be effectively screened to obtain both GM639-p46a clones exhibited the low threshold for radiation-induced apoptosis seen homologous cDNAs and cDNAs which may function further downstream in the in A-T cells: an average of 10.8% of GM639-p46a cells and 11.2% of A-T cells were checkpoint pathway,. This screen yielded one cDNA, CCC1, which can suppress apoptotic 72 hours after 5 Gy, compared to 4.4% of GM639 and GM639-p46s cells. the sensitivity of a mecl strain to multiple forms of DNA damage including UV, p46a transfection did not affect colony survival or apoptosis of irradiated A-T cells. ionizing radiation and MMS exposure. CCC1 also suppresses rad9, dunf and Cell cycle analysis after irradiation showed similar cell cycle checkpoint defects in rad53 strains for UV sensitivity. In the absence of damage the expression of CCC1 both GM639-p46a and A-T cells. Like A-T cells, GM639-p46a fibroblasts failed to to GM639 cells. does not alter cell cycle kinetics but did result in nearly wild-type reconstitution of a increase p53 protein levels after irradiation, in contrast parental a Measurements of genetic instability in GM639-p46a clones are underway. G2 delay after UV irradiation in both mecl and rad9 mutant strain background. Our results are the first demonstration that interference with ATM gene expression This data suggests that CCC1 may function late in the pathway of G2 arrest or results in the A-T phenotype and provide functional evidence linking the ATM gene to activate a parallel pathway for G2 delay in yeast. The CCC1 cDNA encodes a novel cellular DNA damage responses. The p46a plasmid should prove an effective tool for member of the Forkhead/Winged Helix family of transcription factors. This family studying loss of ATM function in cell types not readily available from A-T has not previously been implicated in the response to DNA damage and the homozygotes, e.g., normal and malignant breast epithelium, and neural-derived cells. restoration of the G2 delay does not require new protein synthesis. Current studies Cells defective in other genes involved in radiosensitivity, cell cycle checkpoints, and are underway to determine the role of CCC1 in the response of mammalian cells to apoptosis can be transfected with p46a in order to create double mutant phenocopies DNA damage. that may facilitate delineation of ATM-dependent DNA damage responses. Slide Session 21: Cancer Genetics 1: Familial Predisposition (cont.) A5 12 13 Microsatellite mutation rates in mismatch-repair-deficient and BRCA1 mutations in women with a first degree family history of breast repair-proficient cultured cells. R.A. Farber, M.G. Hanford, J.C. Boyer, cancer identified from a population-based case-control study. IEL B.C. Rushton, and L.C. Gowen. University of North Carolina at Chapel Hill Malonel, I.E. Thompso2 Laingjl, AA. Ostrander. Public Health Science and Microsatellite sequences are unstable in tumors from patients with hereditary nonpoly- 2Clinical Research Divisions, Fred Hutchinson Cancer Research Center, Seattle, WA. posis colorectal cancer (HNPCC) and in significant numbers of sporadic tumors of 98104. types that are common in this syndrome, including colorectal, endometrial, gastric, Inherited mutations in the BRCA1 gene are associated with a high risk of breast and ovarian cancers. In most HNPCC patients this instability results from mutations in ovarian cancer in some highly selected families. Little is known, however, about the of several genes coding for enzymes involved in DNA mismatch repair, most commonly contribution of BRCAI mutations to breast cancer in the general population. We have hMsh2 or hMlhl. Some estimates of the rates of mutation in cells with mismatch-repair utilized DNA samples from two population-based case-control studies of breast cancer defects have been made by assays of subclones for the presence of new alleles at vari- in women under age 45 to directly assess the spectrum and frequency of germline ous endogenous microsatellite loci; however, it has been difficult to measure these BRCA I mutations in young women who have a first-degree family history of breast accurately using this approach, particularly in repair-proficient lines, in which mutation cancer. The first study included 847 Caucasian women born after 1944 who were rates are not expected to be very high. We have used a selectable system to determine diagnosed with a first invasive or in situ breast cancer from January, 1983 through mutation rates within a 17-repeat CA-dinucleotide tract in human cancer cell lines April, 1990 and were residents of a three county area in Washington State. The second normal embryonic fibroblasts. This sequence has been inserted near the 5' end of a bacte- study involved 641 women of all races diagnosed before age 45 from May, 1990 rial neomycin-resistance gene in a plasmid vector, such that the reading frame of the through Dec. 1992. Both studies included age-matched randomly selected controls. gene is disrupted. The plasmid is introduced into cells by electroporation and becomes Blood samples are available on 190 of the 259 cases and 66 of the 101 controls with

integrated in the cellular genome. Clones with insertions or deletions of CA-repeats that a first-degree family history of breast cancer. To date. we have examined 121 cases restore the normal reading frame of the neo gene are selected in G418, and mutation and 35 controls with a first-degree family history. An additional 100 randomly rates are determined by fluctuation analysis. The rates of reversion in the colorectal selected general population controls, who lack a first-degree family history, are also cer lines HCT116, which is deficient for hMlhl, and LoVo, which is deficient for hMsh2, currently under analysis. Genomic DNA was analyzed for BRCA1 mutations using are on the order of one in a thousand per generation, which is approximately two both single-strand conformation polymorphism analysis (SSCP) and allele-specific ders of magnitude higher than in the repair-proficient HT1080 human fibrosarcoma assays (ASO). Specific alterations were defined by DNA sequencing. 1 line. Nearly all of the revertants of the repair-deficient lines have deletions of a single Germline BRCA mutations causing either disruption in the putative RING finger or premature stop and no CA-dinucleotide from the microsatellite sequence, whereas repair-proficient lines have domain codons have been identified in nine cases (7.5%) broader spectrum of mutations. We have not observed any microsatellite mutations controls. BRCA1 positive women displayed a wide range of family histories. Some women had a normal human embryonic fibroblasts; the upper limit on the mutation rates in these cells had multiple affected first- and second-degree relatives and others is about one in a million per generation. minimal family history with only one affected first-degree relative. In some cases that relative was diagnosed late in life. Ovarian cancer was noted in the family histories of 4 of the 9 women. but in only one case was it seen in a mother or sister. Overall these data suggest that BRCA1 status is not easily or directly correlated with family history profile in women under 45..

14 15 Analysis of and Homozygous deletions in Wilm's tumors localize the WT2 gene. plI6N1.K4A, pl15NK4B pI9IYV4AJ mRNA expression in 1'3D.J.Mute 21. Pelle1ier Morgen aser,3E. lic, 4D. Prawitt 2L.L. CQu 3L.Zuo.1J. melanoma cell lines by semi-competitive RT-PCR. N.A. Gruis"3, Nowak, P.A. Van der Veldens, M. D.E. Prins', XV. tR Weiss. Davis a. Griffioen2, Bergman3 and R.R. Frantsi. Landers. N. M.J. Dzuzzii S.5ai6C. iS. 2isgs 4R. Lgkbbertl 1T. slion. 3A. Buckler, D. tathppl~e, 7E.Maher. 4A. Winterpacht. 4B.U. NIGC-Dept. Human Genetics, Leiden University, 2Dept. Clinical Oncology, Leiden Housman. University Hospital, 3Dept. Dermatology, Zabel, 6G.Evans. 5T.B. Shows and lD.E. Center for Cancer Research, MIT, Leiden University Hospital, The Netherlands. Potential tumor suppressor function Cambridge, MA; 2McGill University, Montreal, Quebec; 3Sequana Therapeutics, San has been attributed to the cyclin dependent kinase 4University of Mainz, Mainz, Germany; inhibitors 1NK4A and In Diego, CA; 5Roswell Park Cancer Center, Buffalo, p16 pl51INI4B, that play a major role in cell cycle regulation. 1I the mouse, growth arresting potentials NY; 6Clromosome Genome Center, Dallas TX 7Cambridge University, Cambridge, were also induced by aproteinp19lNK4A0 which is generated from an alternative reading frame of the p16' K4A gene. Homozygous dele- tions of and Strong evidence supports the view that a second Wilm's tumor locus (WT2) is p6lI1NK4A p151IN4B, more than point mutations or frame shift mutations seem to be a favorite located on chromosome 11, band p15.5. Sporadic Wilm's tumors (WT) exhibit a high mechanism by which these genes are inactivated. Due to tandem frequency of loss of heterozygosity (LOH) for markers at lipl5.5. Similar LOH is also localization ofp6lINK4A, pl15IK4 andpl9//NKt4A4 genes on chromosome 9 and reuti- observed in a number of other tumor types including breast cancer, rhabdomyosarcoma, lization of coding sequences between pl6"l K4A and pl91IN4AO, a conventional method, adrenal cortical carcinoma, and heptocarcinoma, suggesting the presence of like loss of heterozygosity, cannot address the target gene of inactivation. Therefore, we suppressor with a broad tissue distribution. These data are further supported by cell- have applied quantitative RT-PCR to study 17 human melanoma cell lines for their rel- mediated chromosome transfer experiments which demonstrate that DNAs containing ative levels ofp16INK4A, p151NK4B andp19INK4A0a mRNA. Shared and unique primers 11pl5.5 sequences are capable of suppressing in vivo tumorigenesis in Wilm's tumor, for the individual genes were used in a semi-competitive RT-PCR. In 9 out of 17 cell rhabdomyosarcoma, and breast cancer model systems. lines no extensive deletions svere present and enabled to quantify expression. Relative We have carried out a positional cloning effort aimed at identifying this tumor expression levels ofpl5INK4B and pl9gNK4A# differed marginally, whereas p16INK4A ex- suppressor gene(s). In brief, we have assembled a complete cosmid/BAC contig (>300kbp) pression varied widely. Seven cell lines showed reduced to nearly undetectable p16 iVK4A which spans the smallest region of overlap (SRO) for LOH in Wilm's tumor expression. Two cell lines had comparable levels of the three transcripts, but were shown suppression of tumnorigenesis by cell-mediated chromosome transfer on11p15.5. The entire to be mutant in exon 2 ofp16INK4A andp9INK4AB One of the latter cell lines is de- cosmid/BAC contig has been sequenced and subjected to'exon amplification' resulting rived from a Dutch melanoma patient and has the 19 basepair deletion within exon 2 of generation of an ordered, high resolution, integrated physical and transcription map across p16 NK4A. This deletion might explain the functional redundancy of carriers since the this region. Probes from across this interval have allowed us to define an area p91NK4A0 transcript encodes, due to a frame shift, the third and fourth ankyrin domain homozygous deletionin WT. The boundary and extent of homozygous deletion in four ofp16 INK4A. The results provide further evidence thatpl61NK4A is a main target in has been established. The SRO for this interval is less than100kbp and encodes at melanoma. -four overlapping transcripts. Homozygous deletions have been detected in 12% hemizygous deletions in 30% of over 100 WT examined thus far. Candidate transcripts currently being analyzed for mutations.

16 17 Somatic confined uniparental disomy of the NFl gene region in myeloid The neurofibromatosis 2 gene product schwannomin interacts with cells of an NFl patient mimics a loss of heterozygosity due to deletion. PII-spectrin. D. R. Scoles'. D. P. Huvnh'. E. R. Coulsell'. N.G. G. Robinson. F. Stehens. M. Weaver'KI. L.E. Side2. K. Shannon2. K Maruama Leppis' Tamanoi:. and S. -M. Pulst'. 'Neurogenctics Laboratory and Division of Neurology, Burns and University ofWashington', Seattle, WA and University of California , San Francisco, Allen Research Institute, Cedars-Sinai Medical Center, Universitv of California at Los Angeles. CA. School of Medicine, 8700 Beverly Boulevard, Los Angeles, CA 90048.. Department of We identified a 10 mo old male with sporadic neurofibromatosis I (NFI) and Microbiology and MolecularGenetics, University of California at Los Angeles, Los unusual myeloproliferative disorder whose apparent loss of heterozygosity (LOH) Angeles, CA 90024. myeloid cells resulted not from a deletion, but rather from somatic uniparental disomy Mutations in the neurofibromatosis 2 (NF2) gene are the most common cause of inherited confined to an interstitial segment of chromosome17q. Genotypic analyses tumors of Schwann cell, meningeal or ependymal origin. Although germline mutations in the performed on the parents and blood, unfractionated marrow, Epstein Barr vinrs NF2 gene are relatively rare. somatic mutations are the most common cause ofsporadic transformed lymphoblastoid cellline, and normal tissue (lung) of the patient. schwannomas, meningiomas, and ependymomas. In addition to tumors, causes non-tumor child's blood, marrow, and EBV cell line showed complete absence of maternal NF2 features suchas retinal hamartomas and cataracts.The NF2 gene is for lociUT172,NFID7S806, D17S787, D17S789, andGH. The region product, schwannomin, a suppressor of function. GH tumor unknown Amino acid homologiesto loss extended from a location proximal ofNFI(I 7q1 1.2) to the distal side merlin, ezrin. andradixin suggest that may interact with (17q22-24). Flanking loci D17S33 and D17S928 were heterozygous, schwannomin the cell membrane or the cytoskeleton. To elucidate thefunction of schwannomin we used the yeast two that allelic loss was confined to an interstitial segment of the q arm of the hybrid GAL4-based system for the identification of binding proteins. A total of 6.6 x chromosome 17. However, 2 methods demonstrated that the region was not 106 human brain cDNA clones were screened using a plasmid encoding isoform 2 of schwannomin but intact in both the maternal and paternal chromosome 17: FISH of the EBV fused to theGAL4 binding domain. Six positive clones resulted, one of which encoded a with a P1 clone of the NFlI gene and a PCR-based gene dossge assay of the patient's portion of the central domain ofP31-spectrin. The interaction was blood, unfractionated marrow, EBV line, and blood from both parents. Lung spectrin-schwannomin confirmed by retransformingpurified plasmid and retesting by the method, in by retained both parental NFI alleles, while the marrow and EBV line carried only two-hybrid and vwvo inmunocytochemical colocalization. The mediated tranaduction cascade mutant allele with a premature truncation codon R1276X consistent with spectrin signal has been well studied in lymphocytes, platelets and epithilial cells. Identification of spectrin as a evidence that NFl is a tumor suppressor in myeloid cells. Together, these data binding partner for schwannomin suggests that tais signal transduction pathway be that the new NF mutation occurred on the paternal allele and that a gene should investigated in Schvann and arachnoidal cells. The with may also explain event in a malignant clone, which gave rise to both B and myeloid cell lines, interaction spectrn non-tumor features of NF2. Dissasemblage of the the normal maternal chromosome 17 segment with paternally-derived sequences. spectrin membrane skeleton is known to he a cause of cataract formation. Similarly, spectrin plays important role in anchoring is the first reported case of somatic uniparental disomy that is confined to an plasma membrane proteins such an the Na',K-ATPase in chromosomal segment. This may be a previously unrecognized mechanism retinal pigment epithelium (RPE). Loss of function of these proteins in the RPE due to expression of tumor suppressor and/or imprinted genes that contribute to inactivation of schwannomin may explain the RPE hamartomas seen in NF2 patients. which has remained undetected because it mimics LOH due to chromosomal A6 Slide Session 21: Cancer Genetics 1: Familial Predisposition (cont.) 18 19 Mapping of the susceptibility gene for Cowden disease to 10q22-23 and Identification of a novel gene family responsible for hereditary multiple genetic analysis in related tumours. C. Engl,2. M.R. Neled.D.-Marshl. exostoses (EXT). Dominique Stickens, David Burbee, Purita Ramos, A.Y. Lin4, V. Coulon5. AM odti4. 1.1. MulvihWl6. M.A. Tucker4 J and Glen A. Evans. McDermott Center for Human Growth and Development, Zedeniu7. B.A.J. Ponder2. H. mer3. M. Longy5 G.W. Padber3. International University of Texas Southwestern Medical Center at Dallas, Dallas, Texas. Cowden Consortium. IDana-Farber Cancer Institute, Harvard Medical School, Multiple exostoses is a genetically heterogeneous disease characterized by the develop- of Hospital ment of bony protuberances at the ends of all the long bones, causing serious skeletal Boston, MA, 2University Cambridge, UK, 3University Nijmegen, deformities, short stature and limb length inequalities. Exostoses frequenly undergo ma- Netherlands, 4National Cancer Institute, MD, 5lnstitut Bergoni6, Bordeaux, France, lignant degenaration to chondrosarcomas or osteosarcomas. We recently isolated the 6University of Pittsburgh, PA, 7Karolinska Institute. Stockholm, Sweden. EXT2 gene on chromosome tp11I and defined the EXT2 gene product as a 82 kD pro- Cowden disease (CD) or multiple hamartoma syndrome is an autosomal dominant tein of unknown function. The gene product is ubiquitously expressed and preleminary inherited cancer syndrome with a high risk of breast and thyroid cancers. Expression is studies using EXT2 antibodies indicate a nuclear localization. Interestingly, the EXT2 variable and penetrance is high, probably virtually complete by the age of 20. The main gene defines a new family of putative suppressor genes including the EXTI gene, lo- characteristics of CD include hamartomas of the skin, breast, thyroid and oral mucosa. cated on chromosome 8q24 (Wells, et al, 1995) and two additional ESTs identified in Because of the protean manifestations of CD which affect derivatives of all three germ the Expressed Sequence Tag database of the Genbank. The EXT1, EXT2 and EST a cell layers, it is believed that the putative susceptibility gene might play an important and b proteins share significant sequence similarity of up to 70% and extremely high role in normal development and in neoplastic transformation. In 12 families comprising evolutionary conservation with the mouse EXT1 and EXT2 genes. A physical map of 40 affected individuals, a maximum lod score of 8.92 (theta=0.02) was obtained with 2.5 mb surrounding the human EXT2 gene region was constructed and the 200 kb EXT2 the marker DlOS573 on 10q22-23. Haplotype analysis revealed the suceptibility gene and to be localised to a 5-cM critical interval between D1OS215 and D1OS564. There was gene sequenced in entirety, defining the intron-exon organization promoter region. no genetic heterogeneity among these 12 families originating from the USA, UK, Although the function of the EXT2 gene (as well as EXT1) is unknown, we recently France and the Netherlands. Subsequently, typing of a 13th family with the markers isolated and characterized the mouse EXT2 gene which allow more detailed functional D1OS580, D0OS219, DOS573, DOS215, DOS564, DOS583 and DlOS 198 studies including "knock-out" models. Analysis of the EXTI, EXT2 and EXT3 genes on suggested genetic heterogeneity. However, further analysis with markers between chromosome 8q24, 11pil and 19q, as well as the mouse homologs, will allow a rational D1OS215 and D1OS564 confirmed that CD in this family segregated with a single approach to dissecting mechanisms of bone development and malignant transformation. haplotype within the established critical interval and revealed that the initial discordant haplotype resulted from a double recombinant. Thus, in large informative families, predictive testing is possible with linkage analysis using markers within the critical interval. Loss of heterozygosity (LOH) analysis was performed on a breast cancer from a Cowden patient belonging to a lOq-linked family and 40 sporadic thyroid tumours, important component tumours of CD. Preliminary results revealed no LOH. However, additional benign and malignant tumours from Cowden patients should be examined to determine whether there is evidence for the Cowden gene being a tumour suppressor.

20 Virtually 100% of melanoma cell lines harbor alterations within the pI6/p15 genes or one of their downstream targets. G.J. WValker, F.E Flores, JAI. Glendening, E. You, I.D.C. larkl, LD.C. Markl, and J.W. Fountain. Univ. of So. California, Inst. for Genet. Med., L.A., CA. We have recently characterized equal numbers of melanoma cell line and tumor D.N.As for mutations/deletions within the p16 gene. While homozygous deletions and mutations of this gene have been identified both in cell lines and tumors, the frequency of these alterations is much higher in the cell lines. Specifically, homozygous deletions of p16 have been observed in 55% (21/38) of the cell lines versus only 22.5% (9/40) of the uncultured melanomas. Similarly, intragenic mutations have been detected in 24% (9/38) of the cell lines versus only 12.5% (5/40) of the melanoma tumors. On first glance, this would suggest that inactivation of p)6 may confer a selective advantage in vitro or that mutations/deletions of this gene are missed in half of the uncultured melanomas. We chose to investigate the first possibility by determining the frequency with which the downstream targets of p16 and p15, including CDK4, cyclin DI and pRb are altered in the melanoma cell lines that do not harbor obvious homozygous deletions or mutations within either of these two genes. Surprisingly, at the DNA level alone, 95% (36/38) of our melanoma cell lines are either (i) deleted/mutated for p16/p15 (N=31), (ii) mutated or amplified for CDK4 (N=3); or (iii) methylated at the p16 locus (N=2). In all likelihood, the remaining two cell lines also harbor alterations that either affect the transcription or translation of one of these gene products. This implies that either a melanoma cell line can only survive in vitro if this pathway is disrupted or all melanomas in vivo also carry mutations within one of the members of this pathway. In the latter regard, we have only detected such alterations in 65% (26/40) of our uncultured melanomas, including (i) homozygous deletion of p16 (N=9); (ii) hemizygous loss and intragenic mutation of p16 (N=2); (iii) hemizygous loss and hypermethylation of p16 (N=1); (iv) hemizygous loss of p16 (and p15) only (N=10); (iv) intragenic mutation ofp16 only (N=3); and homozygous deletion of pRb (N=1). The possibility, therefore, exists that 35% of melanomas evolve through the disruption of another growth control pathway.

Slide Session 22: Genetic Counseling and Genetic Education 21 22 The Washington (WA) State Genetic Education Pr'ect (GEP) for Primary An educational program in genetics for the primary care provider. J.G. Davis and Care Providers (PCPs). RLM. Fineman. N.J. Wit K.L uL K.S. Stemn Cornell University Medical College, New York, N.Y. aylte~,CSni. J. Bentvelzen-Smithk WA State DOH', March of Dimes2, Despite the explosion of genetic knowledge and increased ability to diagnose University of WA3, Seattle, USA. individuals with specific genetic diseases, little attention has been paid to how genetic The goal of this GEP (aka: Genedcs & Your Pracdce) is to provide genetic information will be incorporated into modem medical practice. We report on the health care (GHC) education /training to 50% of WA's PCPs (about 3,000 results of a SPRANS project* designed to educate 300 primary care providers (PCPs) individuals) by October 1997, as one way of helping to ensure that WA's about genetic concepts and information relevant to their practice. The major goal of residents: (1) gain new knowledge regarding GHC issues and services statewide, the project was to develop an innovative approach to educate PCPs about genetics so and (2) have improved access to appropriate, regionalized, high-quality GHC that they can and will incorporate genetic information into their practice. The target services throughout the continuum of life. Utilizing a train-the-trainers model, audience consisted of 300 obstetricians, pediatricians, nurse midwives, nurses and this project is attempting to effectively integrate and expand trainings allied health personnel who work in medically underserved and economically deprived coordinated by other existing educational programs in order to help build a neighborhood clinics in NYC. coordinated, comprehensive, statewide clinical genetics education program. Following a needs assessment a flexible, interactive curriculum was developed, The project began with a PCP needs assessment and utilizes a benefits-based consisting of a series of modules each with a central theme. Case studies were used to clinical marketing approach to ensure high rates of PCP participation and satisfaction. to highlight both genetic concepts as well as illustrate issues related A practice. Written materials including copies of all slides and information presented three module curriculum (preconception/prenatal; infancy/childhood; and each module. Taken adulthood) was written for the trainings and it is being taught by genetic in the interactive teaching sessions were developed to accompany counselors and medical who have received additional training in adult collectively these modules serve as chapters in a book entitled A Handbook in geneticists In order to evaluate the we education. Evaluation of this GEP includes the use of standardized, PCP pre- Genetics for the Primarv Care Provider. project's impact, to assess their fund of and and data collected the WA administered pre- and post-tests to all participants knowledge post-training questionnaires, through CORN in were identified. Pretest MCH Block PRAMS (Pregnancy Risk Assessment Monitoring and to measure information gained. Gaps knowledge MDS, Grant, scores revealed that most have limited of Post test and a statewide Genetic Resource Line. Thus more than participants knowledge genetics. System), 1-800# far, scores showed an overall in Dam obtained from chart 500 PCPs have been trained with a 95% satisfaction rate. In this gain participants' knowledge. addition, is reviews and focus groups revealed major changes in clinical practice parameters as a the first time, to the best of our knowledge, that PRAMS data have been used to result of this project. evaluate any aspect of a statewide genetics program. Lastly, this GEP can be * This work was supported in part by Project #MCJ 361020-02, Maternal and Child used as a model education program for other states or geographic locations. Health Bureau (Title V, Social Security Act), Health Resources and Services (Supported in part by grant MCJ-531004, Genetic Disease Branch, MCHB, Administration, Department of Health and Human Serivces. HRSA. DHHS.) Slide Session 22: Genetic Counseling and Genetic Education (cont.) A7 23 24 Diving into the Gene Pool: The public's point of view-A non-traditional Knowledge and Attitudes about BRCA1 Testing Among Community model for active learning about ethical and social aspects of the Human Oncology Professionals. LE. Stopfero K.A. CalzoneT.R. Rebbeck, ILL Genome Project J. L. M. K. and C. C. Carlsn2. Weber!. I)Hospital of the Univ.of PA, Philadelphia, PA 2)Univ.of PA, Phila, PA. (HGP). BenkendorfI, Mller2 Health care 1Georgetown University, Washington, D.C. and 2The Exploratorium, San Francisco, professionals will be increasingly called upon to provide care for CA. individuals considering predisposition genetic testing. The Cancer Risk Evaluation The HGP is raising social, ethical and medical issues that will affect the health-care Program (CREP) at the University of Pennsylvania Cancer Center offers BRCAI decisions of an increasing range of individuals. The challenge remains for the genetics predisposition testing for selected individuals who receive extensive genetic counseling community and science educators to bring these technological advances to the public prior to and after testing. Professional education outreach efforts within CREP have clearly, responsibly and with proper attention to their human ramifications. focused on 24 community based oncology programs. Knowledge and attitudes about Between April and September, 1995, the Exploratorium, a science museum in San predisposition genetic testing for BRCA1 were surveyed before and after attendance at Francisco, mounted a major exhibition on basic genetics entitled Diving into the Gene a one day educational program designed to educate community oncology providers Pool. The exhibition included a separate area, called Points of View (POV), for about risk evaluation, genetic testing and cancer risk management. museum visitors to explore ethical and social issues arising from the HGP. The POV The one day educational program was attended by 58 oncology professionals who section housed stations at which museum visitors could read and write responses to expressed interest in offering predisposition genetic testing locally. Prior ethical questions posed by three case scenarios-a case of non-paternity to the a disease program, 42% could correctly identify the risk of passing an altered BRCA1 gene serendipitously found upon prenatal diagnosis for CF, Huntington's testing from parent to child. all case involving the right to know or not know ones genetic status, and the propriety Afterwards, correctly identified the risk as being 50%. and boundaries for germilne gene therapy. Responses were posted to encourage Respondents' estimates of the lifetime breast cancer risk associated with a BRCAI visitor interaction. 66% of the over 6000 written responses collected contained alteration prior to the program ranged from 5-100%. Only 42% responded in the narratives reflecting significant time and thought spent at a single exhibit station. While correct range prior to the program, while 92% responded correctly after the program detailed analysis of the content of the responses is not yet completed, preliminary (Fisher's Exact Test p<.001). Genetic testing for BRCAI was thought to be demographics for age and sex demonstrate that the POV scenarios engaged a broad appropriate for all women, regardless of family history by 35% of participants before, audience. and 16% of participants after the program (p =.08). This suggests that some POV demonstrated that a science museum, with technical support from the genetics professionals may offer testing to women who are not appropriate candidates. There community, can be a two-way classroom for genetics education. Beyond grasping social issues was a significant change, from 80% before to 96% after (p=.035) in the number of basic scientific principles, museum visitors are interested in ethical and participants who believed that testing should only be offered to women who have and proved capable of applying them to case scenarios about which even the received prior genetic scientific community has not reached consensus. For many visitors, POV may have counseling. The opinion that testing should be offered to been their first thoughtful contemplation of these issues. POV has established the individuals less than 18 years changed from 14% responding that this was appropriate science museum as an important laboratory for geneticists, bioethicists and science before the program, to 5% after (p=.015). Prenatal testing for BRCA1 was thought to educators to learn of public perceptions, misperceptions, choices and values about a be appropriate by 8% of respondents before and 6% of respondents after the program. major international scientific endeavor that will affect their lives and medical care. An While knowledge significantly improved in many areas after the program, further overview of POV, selected viewer responses, and preliminary interpretations will be innovative strategies will need to be implemented to ensure that health care presented. professionals offering risk assessment and predisposition genetic testing for cancer susceptibility are adequately prepared.

25 26 Attitudes toward BRCA1 testing among physicians and medical students. C.A. James, Parental attitudes to genetic counselling and predictive testing for childhood cancer. D. Malkin, K. B.A. Bernhardt. T. Doksum. N.A. Holtzman. and G. Geller. Johns Hopkins Medical Australie. C. Shuman- M. Barrera- R. Weksberg. The Hospital for Sick Children. U. of Toronto. Institutions, Baltimore, MD. Toronto, Ontario. Canada. As part of an ongoing multi-disciplinari study of the of Genetic testing, especially for common diseases, will be offered increasingly by non- psychological impact predictive genetic testing for cancer in parents ofaffected children, we set out to determine of geneticist physicians. To assess physician attitudes towards BRCA1 and whether parents' perceptions genetic testing, risk for cancer. and their attitudes toward the availability of genetic counselling and predictive testing. vary we 632 in five attitudes by stage of medical training, surveyed Maryland physicians Parents of all newly diagnosed childhood cancer patients at The Hospital for Sick Children (regardless specialties internal medicine (IM), medical oncology (ONC), obstetrics/gynecology of family history), of children with Beckwith-Wiedenann syndrome (BWS), and of children with Li- (OBIGYN), family practice (FP), and general surgery (GS) (48% response rate) and 514 lst Fraumeni syndrome (LFS) or its variants are eligible for entry into the study. A set of parents whose and 4th year medical students at two Maryland medical schools (71% response rate). Support child is diagnosed with a non-genetic/non-cancer disease constitute a 'control' group. Parent interviews for widespread BRCA1 test offering decreases with medical training, with 45% of tst year conducted by a genetic counsellor address issues of cancer genetics, population screening, and genetic students but only 23% of 4th year students and 25% of physicians (41% OB/GYN, 32% GS, risk applicable to their particular situation. The interviews are conducted within 3 mths of diagnosis. 27% IM, 16% ONC, 14% FP) believing BRCA1 testing should be offered to all with follow-ups 6 and 12 months later. To date, 103 patients (Imparents) have been enrolled (75 cancer premenopausal women. Students in favor of widespread offering are more willing to accept - non-specific, 12 BWS, 4 LFS, 12 'controls'). Initially in the childhood cancer population, 370/ of parents believe the risk for cancer in the a test with low sensitivity and more likely to value patient knowledge. Among physicians, general population to be >50%. This perception does not over interviews. in addition to specialty differences, those supporting widespread offering are older, less change subsequent Although fully 60% of parents believe that genes play a role in cancer development in 30% felt the a role in their directive, and more likely to believe trust use of new general, only genes played child's cancer. patients physicians' technologies. Interestingly, in the parents of BWS and LFS 93% and 43% Regarding prenatal testing, 76% of tst year students, 61% of 4th year students and 64% of patients, respectively, considered genes to play a role in their child's cancer development. of and limitations to ONC) would favor a Perceptions implications physicians (78% IM, 62% OB/GYN, 61% GS, 58% FP, 57% telling predictive genetic teats for cancer varied widely; although >80% of parents in all groups felt that genetic of a test. pregnant woman with a familial BRCA1 mutation about the availability prenatal testing should be made available to all cancer patients, and would have such testing performed on their Follow-up recommendations given to a woman with a familial BRCA1 mutation also vary by affected child, only 47% ofLFS parents would have their affected child tested. 82% of parents (100% of stage of medical training, with 11 % of 1st year students, 23% of 4th year students, and 34% BWS parents) would have their children tested if the test wmas 99%/6 predictive, yet only 37% (93% of of physicians likely to recommend prophylactic mastectomy. Two-thirds of both lst and 4th BWS parents) would permit the test if it was <50%/ predictive. Preliminary evaluation of the 'control' year medical students, and 56% of physicians believe it is important to obtain informed set of parents suggest that they are more likely to believe that genes play a more prominent role in consent from women prior to BRCA1 testing, consistent with current recommendations. We childhood cancer than in adult-onset cancer. Even though none of their children had documented risk factors. 75% of the would have found that non-directive physicians and ONCs and OB/GYNs (specialists expected to be parents their child tested if predictive genetic testing were available. If highly involved in BRCA1 testing) are most likely to think obtaining consent is such testing ere 99%/ predictive, all parents interviewed to date would avail themselves of it. Our important. observations indicate that At successive stages of training, providers appear to become less willing to offer genetic although genetic counselling appears to reduce anxiety related to perceived genetic risk at the time of it is not effective in of must testing for a common disease, more to recommend a treatment of diagnosis, improving knowledge genetic risk, and but willing option unimown therefore be repeated in timed sessions. efficacy. appropriately follow-up

27 28 After cancer risk counseling: Consultands' satisfaction and subsequent behavior. M.P. A long term (-5 years) prospective assessment of psychological Stadler and J.J. Mulvihill. Cancer Genetics Program, University of Pittsburgh Medical Center consequences of predictive testing for Huntington disease (HD). S. and Magee-Womens Hospital, Pittsburgh, Pa. Wigoins. T. Green. S. Adam. and M. R. Hayden. for the Canadian Collaborative Study In 1995, we formally established a multifaceted cancer genetics program of genetic of Predictive Testing counseling, research, and education. In the first year, 58 families received genetic Methods: A total of 209 individuals were followed at reaular intervals over a fti e year consultations, mostly by physician referral. The consultands' primary reasons for seeking period in each of 4 study groups: increased risk (n=49). decreased risk (n=97). consultation were to explorethe possibility of presymptomatic DNA testing and to understand uninforrnative. (n=33). and those who chose not to be tested (n=30). Predictive testing risk for cancer in themselves or relatives given their family history. To determine level of was performed using DNA analyses of genetic markers linked to HD. Psycholooical satisfaction with services and track behavior, 51 of the 58 consultands were successfully data were obtained through self-report questionnaires completed before and after testing. surveyed by telephone by staff not involved in their consultation, 3 to 12 months after Results: At the time of entry into the study, the four study groups were homogeneous counseling. All 51 were Caucasian, ages from 17 to 72 years; all but 1 was female. Final with respect to their psychological well being (as measured by standard depression. diagnoses were 16 likely for hereditary breast-ovarian cancer syndrome, 3 hereditary distress. life satisfaction and general well beino indices), education, occupation. marital nonpolypotic colorectal cancer IHNPCC), 1 familial pancreatic cancer, 25 otherfamilial cancer status. gender and family size. The decreased risk group had a higher mean age than the (breast, colon, or ovarian), and 6 sporadic cancer cases. According to the respondents, the other groups (39.2 years vs. 35 years; p=0.02). Over the five years of follow up there risk of cancer quoted during counseling was lower than they expected for 47%. higher for were improvements in the psychological well being of both the increased and decreased 8%,and what they expected for 31 %; 14% had no opinion. Those few patients who recalled risk groups. There was a linear decline in the levels of psychological distress reported the numerical risk for cancer quoted usually had it wrong. 82% patients recallet4discussion by the increased and decreased risk groups (p=.005 and p=.001 respectively): levels of of cancer screening and 57% had a physician's examination since their consultation. Of 44 depression decreased over time for the increased and decreased risk groups (p=.009 and consultandsat increased risk forbreastcancer, 28163%l had a subsequent mammogram, and p=.043 respectively). Additionally the levels of general well being reported by both 75% report monthly breast self-examinations. Of 18 patients at increased risk for ovarian groups increased over time (p=.009 and p=.0005 respectively). Comparisons of follow to cancer, 5 had CA-1 25 testing and 2 of the latter also had ovarian sonography. Only one of up pretest scores indicated that, for the increased risk group, distress and depression three patients at risk for HNPCC had follow-up colonoscopy. None of our patients at an declined significantly at the 12 month follow up (p=.02 and p=.03) while general %vell increased risk for breast cancer had prophylactic mastectomies, although 8 out of the 27 being increased significantly at the 12 month and five year follow ups (p-002 and eligible patients stated that they had considered it. Four of the 27 reported having p=.04). For the decreased risk group. general well being scores increased significantly prophylactic oophorectomies. 84% of the 51 consultands indicated that the consultation met at the 10 day follow up (p=.O001). No significant changes were observed over time for their expectations. 88% discussed the consultation with family members, and 78% shared the uninformative or untested group on any of the psychological measures assessed. their summary letter among the family. 75% would recommend the service to their relatives, However the uninformative group reported consistently higher distress and depression and 90% to friends. 90% foundthe consultation to be worth their time and money. 42% scores and lower well being and life satisfaction scores than the other study groups. stated that anxiety level related to their family history of cancer had decreased following the Conclusion: These analyses suggest that significant psychological benefits are derived counseling session, while 56% said there was no change. Overall, the service was positively from the clarification of risk status for participants requesting predictive testing. This received by the majority of patients. benefit accrues regardless of the direction of risk modification. In this study. we have not frequently seen the serious psychological disturbances that many feared might occur when predictive testing for HD was first introduced. A8 Slide Session 22: Genetic Couinseling and Genetic Education (cont.) 29 30 Inconsistencies in the provision of genetic counseling services for consanguineous "Utility" of Preference Measures: Research and Clinical Applications in unions: The need for developing practice guidelines. RL Bennett, L Hudgins, AG Genetic Testing. A. Youn', D. Feeny2, M.Cappelli' and L.C. Surhl. 'Children's MotulsI. Div. ofMedical Genetics, University ofWashington, Seattle. Hospital of Eastern Ontario, Ottawa; 2Mc.6aster University, Hamilton, Ontario, The pregnancies and offspring ofconsanguineous unions may be evaluated in clinical Canada. genetics centers, and couples who are cousins may seek pre-conceptual genetic Preference measurement in genetic testing provides more insight into decision making counseling. Baird and McGillvray (JPediatr 1982), and Hall (Am JDis Child than typical attitude/knowledge surveys and may be used as a research tool to evaluate 1978) of life as a recommended comprehensive pre-adoptive assessment ofchildren from quality and clinical tool to aid client decision making. Using Cystic Fibrosis incestuous (CF) carrier as a different outcomes unions including: serial physical examinations and testing model, resulting from testing were explored developmental assessments, meta- including "information" (1 partner is a carrier vs both are carriers) and 'reproductive" bolic studies, vision and hearing screening, and skeletal survey. No definitive guide- outcomes (birth of a baby with CF vs abortion of a fetus with CF vs not having chil- lines to assess the health of the offspring offirst cousin unions have been established. dren) Preferences for these outcomes were evaluated to assess how individuals might use To determine current practices ofgenetic counseling for consanguinity, we sent a their test results, while assisting clients with carrier testing and reproductive decsions. questionnaire to board certified counselors and clinical Preferences or "utilities" were elicited using the Standard Gamble (SG) method from genetic geneticists in the U.S.. risk 3 There was no consensus regarding the actual risks to offspring for congenital defects high groups (family hx of CF) differing in knowledge/experience with CF: Obligate Carriers: 49 parents of children with CF; Tested: 59 previously tested high risk relatives; and mental retardation. For incest between 1st degree relatives, disease risks quoted and Recruited: 98 not tested from previously high risk relatives. SG has been widely used in ranged 6% to 50%; and for first cousin unions, risks given were "not increased quality of life research and cost-utility analyses. Its unique incorporation of uncertainty above background" to as high as 10%. Recommended screening practices for and risk, makes it an ideal decision aid for those considering genetic testing. SG assesses consanguineous couples pre-conceptually, during pregnancy, and following birth preferences by asking clients to gamble with varying chances of clinically relevant testing differed. Most respondents recommended genetic screening based on ethnicity yet outcomes, deriving utility scores ranging from 1.0 (perfect health) to 0.0 (death).Results. No differences were disagreed as to which diseases to include. The issue of pre-adoptive evaluation of found between groups on information and reproductive outcomes, children from incestuous parentage was although mean utility scores across the groups showed that having a child with CF contentious. (X=0.63, S.D.=0.30) and having no children (X=0.67, S.D.=0.29) were significantly Given the availability ofDNA and biochemical testing for carrier and disease status, more desirable than aborting a fetus with CF (X=0.47, S.D.=0.35). In addition, utilities newborn screening, and prenatal diagnosis, guidelines to assess the health ofthe for CF carrier test results where 1 partner is a carrier (X=0.85, S.D.=0.18) vs both offspring from consanguineous unions need to be developed. Consideration should be partners are carriers (X=0.71, S.D.=0.26) were significantly higher than all reproduc- given to screening based on ethnicity, particularly in population groups where consan- tive outcomes. Conclusion: The use of utility assessment in DNA predictive testing as guinity is common (e.g. Middle Eastern, Amish). The actual disease risks for various a non-directive genetic counseling aid is novel and may provide important information regarding the effective provision of genetic services, while facilitating patient decision degrees ofconsanguinity need better definition. making.

31 The natural history of 5p monosomy (Cri-du-Chat syndrome) in 1996. M.E. Crlin, John Peter Smith Hospital & Cook's Children's Medical Center, Fort Worth, TX. Questionnaires were mailed to nearly 400 families registered with the 5p- Society. Although phenotypes varied some among the 1 15 respondents depending on cytogenetics (90% terminal deletions; 8% translocations) and perinatal factors (33% preterm; 20% SGA; 18% required C-section), > 90% demonstrated the characteristic cry and microccphaly, 85% showed early hypotonia and 60% reported epicanthal folds, micrognathia, low-set ears and simian creases. Almost all showed some postnatal growth delay and > 95% had poor suck/feeding problems and multiple ear infections. Constipation affects 85% and 50% report GE reflux; 70% have had some surgical procedure, with PE ventilation tubes and gastrostomy/fundoplication topping the list. Developmental milestones are delayed in most and still emerging in many since >50% are <6yrs. Over 60% of those >18 mos. are walking; hypotonia and orthopedic deformities delay ambulation. Less than 1/3 have toileting skills; <20% can dress independently. Some signs are used by 80%, with 75% of those beginning before age 3 yrs.; 15% speak a few words and 30% sing songs. Although most have received some PT/OT, 90% still have a wide-based awkward gait and poor coordination. More than 80% experience chewing-swallowing dysfunction. Adaptive equipment is/has been required by 60% and 20% use glasses and/or hearing aids. More than 90% demonstrate some self-stimulating activities; aggressive behaviors and ADHD increase with age. Medications are frequently not helpful. Other problems seen with aging include: bruxism, poliosis, hypertonicity, periodontitis and scoliosis. Roughly 2/3 of those >18 yrs. are in some vocational setting, but skills are limited and inappropriate behaviors interfere with performance; <5% are reimbursed. Of the 60% who noted residence, 85% lived at home even as adults. Some health insurance is available for 50%; 30% have dental insurance and 15% have life insurance. Distraction, time-out, immediate rewards and stem reinforcement of rules are the most successful behavioral modifiers; play, music and equestrian therapy have also proved beneficial. Initial responses to the same questionnaire from cohorts in Great Britain, Germany, Belgium and Holland have been similar.

Slide Session 23: Molecular Genetics of Disease I 32 33 A novel X-linked gene, DDP, shows mutations in families with deafness Unraveling the genetic and biological bases of ocular albinism type I (DFN-1), dystonia, mental deficiency and blindness. H. Jinl, M.MVay2, M. V. Schiaffino l, M. T. Bassi 5, C.Baschirt I, G. Pellegrini2,3, S. Mona i5 £ L. Tranebjaerg3, E. Kendall', T. Freeman4, G. Fontan5, J. Jackson6, TacchettiS, M. De Luca2,3 and A. Ballbiol. 'Telethon Institute of Genetics and S. H. Subramony7, F. Arenaa, H. Lubs3,ks, S. Smiith6, R. Stevenson2, Medicine (TIGEM), San Raffaele Biomedical Science Park, Milan, 31DI, Istituto C. Schwartz2 and D. Vetriel. Div. of Medical and Molecular Genetics, UMDS, Guy's Dermopatico dell'Immacolata, Rome, 41stitute of Anatomy, University of Genoa, Genoa, and St. Thomas's Hospitals, London, U.K.', Greenwood Genetic Center, Greenwood, and 5Departement of Molecular Biology, University of Siena, Italy. S.C.2, Dept. of Medical Genetics, University Hospital, Tromso. Norway.3, Sanger Ocular Albinism type I (OAl) is an inherited disorder characterized by severe Centre, Hinxton, U.K.4, Immunology Unit, Hospital La Paz, Madrid, Spain5, Div. of reduction of visual acuity, photophobia, and retinal hypopigmentation. Ultrastructural Medical Genetics, Univ. of Mississippi, Jackson, MS6, Dept. of Neurology, Univ. of examination melanocytes reveals the presence of macromelanosomes. suggesting a defect Mississippi, Jackson, MS', Dept. of Pediatrics, Univ. of Miami, Miami, Florida8. in melanosome biogenesis. Our group has recently identified the human and mouse OA I The unique combination of sensorineural deafness (DFN-1 type), dystonia, mental de- gene, which is exclusively expressed in pigment cells and encodes a putative membrane ficiency and blindness (Mohr-Tranebjaerg syndrome/DFN-1/MTS) is a rare, progressive protein sharing no similarities with previously identified molecules. Several functionally disorder which maps to the Xq2l.3-Xq22 region of the X chromosome. Using positional significant mutations have been identified by SSCP analysis within the OAI gene in only information from a patient with a 21 kb deletion in Xq22 and sensorineural deafness one-third of over 60 OAIpatients analyzed. We are presently concentrating our efforts on along wiht dystonia, we characterised a novel transcript lying within the deletion as a unraveling two main questions that remain unanswered: firstly, what is the main candidate for this complex syndrome. We now report small intragenic deletions in this mutational mechanism underlying OAI? and secondly, which is the actual OA I protein candidate gene in the original Norwegian DFN-1/MTS family, and in a family with deaf- function? Based on the genetic homogeneity shown by the disease by linkage analysis, ness, dystonia, and mental deficiency but not blindness. This gene, named DDP, shows we have hypothesized the presence of a common mutational mechanism, or mutation hot highest levels of expression in fetal brain. The DDP protein demonstrates striking sim- spot, accounting for the 60% "lacking" mutations. At present, we are testing this ilarity to a predicted Schizosaccharomyces pombe protein of no known function. Thus, hypothesis intensively by several strategies including long range genomic sequencing, it is likely that the DDP gene encodes an evolutionarily-conserved novel polypeptide analysis of the OAI RNA and protein in patients' cultured melanocytes, and searching for necessary for normal human neurological development. In order to help elucidate the genomic rearrangements. Preliminary results suggest the presence of a marner function of this novel gene, we have also characterised the murine homologue of DDP transposon-like element located within the gene, which could enhance the frequency of (murDDP) and have used RT-PCR and in situ hybridisation to look at the expression DNA rearrangements in the nearby genomic region. The role of the OAI protein in patterns of murDDP in a variety of tissues. melanocyte biology has been studied by biochemical and immunochemical techniques. Using polyclonal antibodies we have identified the endogenous OAI protein in pigment cells as a 60 kDa glycoprotein, and a 48-45 kDa unglycosylated doublet. Upon subcellular fractionation and phase separation with nonionic detergents, the OAI protein segregated into the melanosome-rich fraction and behaved as an authentic integral membrane protein. Immunofluorescence and immunogold analyses on normal human melanocytes confirmed the melanosomal membrane localization of the endogenous OAI protein, consistent with its possible involvement in melanosome biogenesis. Slide Session 23: Molecular Gienetics of Disease I (cont.) A9 34 35 and of a novel human homeobox Cloning characterization gene Identification of a for Involved in the Rieger syndrome and its mouse homolog. EL.S gene Leber's congenital amaurosis (LCA1). Semina. J.C. Murray. B. Reiter. N. Leysens. N. Datson. G. Mondt. W.L.S. J. Kaplan'. I. Perrault'. J. M. Rozet'. P. Calvas'. A. Camuzatr. S. Alward. P. Bitoun. D. Bierke-Nelson. K. Small. B. Zabel. J. Carey. The Gerber'. H. University of Iowa, Departments of Pediatrics, Biological Sciences, and DollfUs2. S. Chatelin'. E. Soueid'. I. Ghazi2. J. L. Dufier?. Ophthalmology, Iowa City, IA; Leiden University, Netherlands; SIDVA, S. Pittler3 and A. Munnich'. 'Unite INSERM 393, H6pital Necker, France; University of Florida, Department of Ophthalmology, Gainesville, FL; Paris, France. 2Service d'Ophtalmologie, H6pital Necker, Paris, University of Utah Medical center, Salt Lake City, UT; University of Mainz, France. of Department of Pediatrics, Germany. 3Departments Biochemistry & Molecular Biology and The Rieger Syndrome is a rare, autosomal-dominant human disorder Ophthalmology, Mobile, Alabama, USA. characterized by variable expressivity and including dental hypoplasia, Leber's congenital amaurosis (LCA) is an autosomal recessive glaucoma, mild craniofacial dysmorphism, and a protuberant or stalk-like disease responsible for congenital blindness. It is umbilicus. The presence of large cytogenetic deletions suggested a locus for the earliest and the this disorder might be present on the long arm of chromosome 4, and most severe form of inherited retinopathy. The existence of non allelic subsequent linkage analysis confirmed it to the region of the epidermal growth heterogeneity in LCA has long been suspected. Recently, we have factor gene at 4q26. We report here a positional cloning strategy relying on a gene for LCA to balanced translocations in two patients with Rieger syndrome as well as linked mapped responsible chromosome 17pI3 (LCA1) by families to identify a novel homeobox gene causing this disorder. We describe homozygosity mapping in seven, consanguinous families of North cDNA sequence and genomic structure for the human gene and the identification African origin. Therefore, we have constructed an overlapping YAC of 6 etiologic mutations in unrelated individuals segregating for this disorder, the LCA1 cDNA sequence for the mouse homolog and its in situ expression pattern on contig encompassing gene from the DNA markers flanking whole mounts mouse embryos and sections. The gene itself is a novel the LCA1 genetic interval. One candidate gene mapping to this contig homeobox gene named Solurshin, consisting of four exons surrounding a 183- harbored an homozygous one bp deletion in exon 1, leading to a base-pair homeobox element. Mutations have been identified within the codon homeobox and in splicejunctions. Interesting features of the gene include premature stop in two affected sibs. The identification ofthe first apparent alternative splicing in both human and the mouse and a strongly LCA gene will be presented. conserved element located in the 3' untranslated region which has 97% nucleotide homology between human and mouse over a 270-base-pair region.

36 37 Mutation in PAX 2 in a patient with coloboma-ureteral-renal syndrome. Prevalence of large NF1 gene deletions in neurofibromatosis type 1 (NFI). SA LA, Schimmentil. K.A. Byely-. S.R. Braddocki'. M.R. Ecciesl.Y.H. Zhanei. Rasmussen', SD Colman', CR Abernathy', CE Schwartz2, PH Am3, MR Wallace' H.H. ShimL. University of California. Los Angeles, CA; 2 University of Missouri at Columbia. Columbia. MO; 3University of Otago, Dunedin. New Zealand. 'Pediatric Genetics, University ofFlorida, Gainesville, FL; 'Greenwood Genetics Center, Coloboma-ureteral-renal syndrome is an autosomal dominant disorder consisting of Greenwood, SC; 3Nemours Children's Clinic, Jacksonville, FL. eye colobomas. vesicoureteral reflux, and abnormal kidneys that may lead to renal Mutation detection in NFl is a challenge, with no NF1 gene mutations recurring failure. We demonstrated that mutations in the PAX 2 gene are responsible for the frequently. Most identified mutations result in premature protein truncation and disorder in two families. PAX 2. a transcription factor expressed in the developing presumably loss of protein function. Since large gene deletions would also kidney, eye. central nervous system sequences result in and otic vesicle, binds target through a absence of functional and can be paired domain. We describe a patient with optic nerve colobomas, hypoplastic protein missed by conventional mutation detection kidneys. we proteinuria, end stage renal disease, abnormal ears and hearing loss in whom techniques, sought to identify the prevalence oflarge constitutional gene deletions in we NF1. We demonstrated a mutation in PAX 2. This 20 year old woman was referred to genetics to have screened a set oftypical NFl patients for large NFI gene deletions by rule out CHARGE syndrome. The patient developed 3+ proteinuria at age 3. At age IO, comparing patient and parent genotypes using up to 10 intragenic polymorphisms (RFLPs a renal biopsy revealed focal segmental glomeruloscierosis and renal ultrasound showed and microsatellites). We have studied 69 patients and their parents (49 new mutation small kidneys. She progressed to end stage renal disease and underwent transplantation. patients and 20 affected parent-child pairs) at eight or more intragenic markers. Five Ophthalmologic examinationatage 6 revealed bilateral optic nerve colobomas. Hearing evaluation patients (7.2%) demonstrated loss of heterozygosity (LOH), indicative ofNF1 gene revealed moderate unilateral hearing loss at 6000-8000 Hz. She had abnormal deletion, which that pinnae with under-folded superior helices, a square nose with a thick tip, up-slanting suggests large AFT deletions constitute a significant proportion of palpebral fissures and hyperextensible elbows, hands and feet. Intelligence was normal. germline mutations in typical NFl cases. All five deletions were de novo and maternally- Karyotype was 46, XX. Both parents were examined by an ophthalmologist and do not derived, in contrast to linkage reports that the majority ofNFl mutations are paternally- have colobomas. We performed single stranded conformational polymorphism analysis derived. These five patients do not have severe NFl manifestations, dysmorphic features, of the PAX 2 gene on DNA from the patient, her parents and normal controls and found or mental retardation, in contrast with patients previously reported with large NFI an abnormally migrating band in exon 2 from the patient. Automated sequencing of exon deletions. Two 2 suggested a patients demonstrated partial LOK, consistent with somatic mosaicism for frame shift mutation. PCR products from the patient's exon 2 were cloned the deletion, that and sequenced. Sequencing of 6 clones revealed an inserted G into a normal sequence of suggesting mosaicism may be more frequent in NFl than previously 7 G's in 3 clones, leading to a stop codon 27 amino acids downstream and deleting the recognized. paired domain of PAX 2. Sequencing of the parent's DNA did not reveal a mutation. We recently extended LOH studies to plexiform neurofibromas. Preliminary studies Dot blots performed with labeled oligonucleotides, confirmed that the patient was demonstrated LOH, indicative of somatic NFI gene deletion, in 2 of 6 plexiform heterozygous for the mutation parents were while the and normal controls homozygous neurofibromas studied thus far. Our studies suggest that large NF) deletions are an for the normal allele. Thus this mutation appears to have developed de novo in this important mechanism as constitutional mutations in typical NFI patients, as well as patient. Clinical evaluation combined with understanding of the molecular basis of this somatic mutations disorder further delineates the coloboma-ureteral-renal syndrome. in plexiform neurofibromas.

38 39 PID1 gene involvement and renal cystic disease in LIMK: a second gene that is deleted in patients with Williams tuberous . ?clerosis. J. Sampson. M. Maheshwar. R. syndrome. Aaijnwall P. Thompson. J. Cheadle. D. Ravine. S. A.P.Readi, M.Tassabehji', K.Metcalfe', X.Mao2, C.Proschel3, N.J.Gutowski3, Royf. E. HaanJs J. Bernstein". Harris'. Institute of W.D.Fergusson', M.J.A.Carettel, J.K.Do e, D.Donnai' and D.Sheer2. 'Department of Medical Genetics, CardiffI UK. Institute of Molecular Medical Genetics, St Mary's Hospital, Manchester M13 OJH, UK. 2Imperial Cancer Medicine, Oxford, UK. Institute of Child Health, Research Fund, London WC2A 3PX, UK. 3 Ludwig Institute for Cancer Research, London, UK, 'Centre for Medical Genetics, Adelaide, London WIP 8BT, UK. Australia, 4William Beaumont Hospital, Michigan, USA. Williams syndrome (WS) is believed to be a contiguous gene disorder caused by a Renal cystic disease of variable severity (RCD) is a submicroscopic chromosomal deletion at 7qll.23. The elastin gene (ELN) is deleted in well recognised complication of tuberous sclerosis most WS patients, and hemizygosity for ELN is thought to cause the vascular and (TSC) . The TSC2 gene lies at 16p13.3, immediately connective tissue abnormalities seen. Features such as mental retardation, growth adjacent to the autosomal dominant polycystic kidney deficiency and hypercalcaemia, which are not seen in patients with mutations confined disease (ADPKD) associated gene, PKD1. A second TSC to 28 the ELN gene, are likely caused by deletion or transcriptional silencing of other gene, TSC1, maps to 9q34. We studied patients with genes flanking the elastin gene. One such gene is the LIMK protein kinase gene, TSC and carefully documented RCD of different degrees located within 30kb of the 3' end of the elastin gene. of severity. In 26 cases we identified deletion LIM kinase (italicize LIMK is a novel protein kinase containing two LIM domains, mutations involving TSC2 and PKD1. In 18 cases with each composed of a double zinc finger motif. Human and mouse homologues of this severe disease c~haracterised by total cystic gene have been isolated and been shown to replacement of both kidneys have map to 7q1123 and 5G1 respectively. and early decline in We have isolated a cosmid containing the entire LIMK gene. The 36 kb cosmid also glomerular filtration rate, the PKD1 gene was largely contained the 3' part of the ELN gene. We used the or completely deleted. 10 cases had less severe LIMK cosmid as a fluorescent in RCD and situ hybridisation (FISH) probe on a panel of WS patients. LIMK was deleted in all preserved renal function. Of these, 6 appeared to be patients with the classical WS who also mosaics for similar deletions, 2 had smaller deletions phenotype had deletions of the elastin gene (N=20). The deleted chromosome 7 from a WS patient was isolated in a somatic cell involving TSC2 and only the most 3' part of PKD1 and 2 PCR had large deletions hybrid. analysis showed that the entire LIMK gene was deleted. of TSC2 which did not involve the The mouse is PKD1 gene. Our data indicate that, despite histological homologue, limk , expressed in the developing central nervous system. Thus LIMK is a candidate gene for the neurological features of Williams differences between renal cysts in TSC and ADPKD, RCD its role will syndrome. in TSC is usually associated with involvement of Establishing require evidence that haploinsufficiency for LIMK produces the some or all of the mental characteristics of Williams PKD1 gene and that RCD severity reflects the nature of syndrome. the underlying mutation. A10 Slide Session 23: Molecullar Genetics of Disease I (cont.) 40 41 Duplication of the proteolipid protein gene (PLP) is a frequent cause of A novel mechanism of aberrant splicing. J.D. Cogan. . E.M.S. McCarthy. M.A. Pelizaeus Merzbacher disease. E.A. Sistermans', I.J. de Wijs', Prince. S. Lekhakula. A. Futrakul. S. Bundev. and J.A. Phillips III. Vanderbilt I.F.M. de Cool and B.A. van Oost2. 1: Dept. of Human Genetics, University Hospital University School of Medicine, Nashville, TN; Mahidol University, Bangkok, Nijmegen, Nijmegen, The Netherlands, 2: Current address: Dept. of Clinical Sciences and of UK. of Companion Animals, University of Utrecht, Utrecht, The Netherlands. Thailand; University Birmingham, Birmingham, Pelizaeus Merzbacher disease (PMD) is an X-linked recessive neurological disorder Eukaryotic mRNA splicing is regulated by consensus sequences at the intron characterized by dysmyelination of the central nervous system. General features of PMD boundaries and branch site. Recently, Sirand-Pugnet et al reported the importance ofan patients are rotatory nystagmus, optic atrophy, spasticity, dementia, microcephaly and intronic (A/U)GGG repeat in chicken P-tropomyosin which is a binding site for a retarded growth. In a number of patients different pathological mutations in the PLP protein required for spliceosome assembly. we have gene located on Xq22 have been found. However, these mutations are found only in 25% Interestingly, detected mutations of the families that show linkage with Xq22. Recently it was discovered that duplication in IVS3 ofhuman growth hormone (GH) that affect a putative, homologous consensus of the PLP gene also causes PMD. We therefore combined direct genomic sequencing sequence and which also perturb splicing. In a series of dominant-negative GH with the determination of the copy number of the PLP gene for the molecular analysis mutations that cause exon skipping we found two mutations that do not occur within of patients from 11 independent families in which the disease cosegregated with Xq22 the donor, acceptor, or branch consensus sites. The first mutation deletes 18bp markers. Due to this combined effort we were able to find a mutation in 10 out of 11 families (91%). In 5 of the families a missense mutation was found, in 5 other families a (del+28-45) within and the second is a G-+A transition of the 28th base (+28G-+A) duplication of the PLP gene had occurred. In a separate analysis of 19 sporadic patients of GH IVS3. Both mutations are located 28bp downstream from the donor splice site suspected of PMD 1 missense mutation and 5 duplications were found (32%). From these in a G-rich region of IVS3. These mutations segregated with autosomal dominant GH results it can be concluded that duplication of the PLP gene is an important patholog- deficiency in both kindreds and no other allelic changes were detected. RT-PCR ical mutation mechanism in familial as well as in apparently sporadic PMD. Molecular diagnosis of PMD patients should therefore always include duplication analysis. amplification of transcripts from expression vectors containing the del+28-+45 or +28G-*A alleles yielded products showing a >10 fold preferred use of alternative splicing similar to findings previously reported by us for IVS3 donor site mutations. Examination of the del+28-+45 and +28G0-A mutations revealed they perturb an intronic XGGG repeat similar to the repeat found to regulate mRNA splicing in chicken P-tropomyosin. Our data demonstrate XGGG sequences shown to regulate alternative splicing in chicken 3-tropomyosin may also regulate this process in the human GH gene resulting in alternative splicing in the latter and suggest that mutations ofhomologous intron sequences may cause other human diseases.

42 Complete and specific antisense inhibition of gene expression using U I snRNA as a vehicle for the delivery of targeting sequences. RL.A. Montgomery and H.C. Dietz, Johns Hopkins Univ. School of Med., Baltimore, MD. In the pursuit of targeted inhibition of gene function, the in situ expression of antisense RNAs has many theoretical and practical advantages over antisense techniques based upon the administration of synthetic DNA oligomers. When considered in the context of therapeutic application for inherited disorders with dominant-negative or gain- of-function pathogenetic mechanisms, foremost among these is the potential for enduring activity. Disadvantages have included low expression or limited stability of antisense RNAs, their non-specific targeting, or low efficiency of inhibition of gene expression. Many naturally occurring antisense RNAs have been described in prokaryotes, all possessing stable hairpin loops that encompass or flank the targeting sequence and that confer resistance to nucleases. This structure is highly similar to that for the snRNAs, essential components of the spliceosome complex that are abundant and stable in the nuclei of all mammalian cells. A sequence exactly complementary to the first 30 coding nucleotides of FBN1, interrupted in its center by an autocatalytic hammerhead ribozyme loop, was substituted for the Sm protein binding site between the hairpin loops of U 1 snRNA within a transcriptional unit that is under the control of the potent and constitutively active Ul promoter. An osteosarcoma (MG63) cell line that overexpresses fibrillin-l, the dominant-negative gene product in Marfan syndrome encoded by FBN 1, was stably transfected with either an insertless vector or the chimeric construct (pUl/FIB). Fibrillin-I was not detectable by immunohistochemical analysis of clonal colonies harboring pUl/FIB, while mock transfected cells were identical to untransfected controls. Fibronectin synthesis and deposition was unaltered by expression of pUl/FIB. Northern analysis showed an undetectable level of FBN1 transcripts but normal abundance of 0-actin message in cells expressing the chimeric antisense RNA. Only minimal inhibition of fibrillin-l expression was seen in the absence of U I structural elements within the antisense RNA. The biological properties of Ul snRNA may provide an ideal vehicle for the in vivo delivery of antisense targeting sequences directed against transcripts expressedby inherited mutant alleles or a variety of infectious agents. Slide Session 24: Prenatal and Perinatal Genetics I 43 44 Fetal gamma globin: a highly specific marker for identification and isolation of fetal Fetal cell quantitation in maternal blood samples from normal and aneuploid nucleated erythrocytes from maternal blood. Y.L. Zheng'. D.K. Zhen'. K Wolcott'. pregnancies. D.W. Bianchi', J.M. Williams2. C. Pelletier2. K.W. Kinger'. A.P. Shuber3. D.W. Bianchi'2 Depts. ofPediatrics' and Ob/Gyn2, New England Medical Ctr. 'Depts. ofPeds and Ob/Gyn. Tufts Univ. School ofMedicine, Boston, MA; 2Genzyme Hospital/Tufts Univ. School ofMedicine, Boston, MA. Corp, Cambridge, MA, 'Integrated Genetics, Framingham, MA. Monoclonal antibody to the y chain offetal hemoglobin (HbF) has been proposed as a Noninvasive prenatal diagnosis by the isolation and analysis offetal cells in maternal fetal-specific reagent. Persistence ofHbF cells in normal adults and pregnant women blood is now possible. To date, results suggest that low numbers offetal cells are present raises the question ofwhether HbF is a uniquely fetal marker. We determined the in 20 ml samples. This study was performed to determine ifintrinsically low numbers of specificity ofthe HbF by immunocytochemical staining on pure fetal blood and adult fetal cells exist in maternal whole blood samples or ifthere are initially higher numbers that mononuclear cells, and by flow and magnetic sorting ofartificial mixtures and maternal are progressively lost during fetal cell separation techniques. samples. Peripheral venous samples were collected from 219 pregnant women and subjected to a Patient material studied included blood from first trimester fetuses (n=7), pregnant whole blood lysis DNA extraction. PCR amplification ofa Y chromosome sequence (49a) women (n-6) and non-pregnant (n=3) women. Immunocytochemical studies assayed the was used as an indication ofthe number offetal cells originating in male fetuses. Reaction percentage ofHbF' and HbF cells present. We scored 500 nucleated fetal cells and products were separated by electrophoresis, phosphorimaged, and compared to results of l0xl06 adult nucleated cells per sample. Artificial mixtures (n=7) were made ofmale cord amplification using known amounts ofmale DNA. Values were normalized to represent blood into female adult blood and maternal samples (n=7) were depleted ofCD45' cells the equivalent number offetal cells in two 8 ml vacutainers. using MACS, stained with anti-HbF, and analyzed by FISH using X and Y probes. Results # ofMale Cells Detected The mean percentages ofHbF' cells were 90% (fetal blood), 0.0003% (pregnant Fetal Kasyotype N (Mean) (Range) (S.D.) (Median) women), 0.0002% (non-pregnant women). The mean number ofHbF cells obtained after 46,XY 83 18 0-91 21 10 MACS depletion ofartificial mixtures was 70 (range 7-150) and ofthese, 91% were male 47,XY, + 21 18 115 1-650 160 57 by FISH. In the maternal samples, HbF' cells were detected in 6/7 samples. The mean 47,XY, + abnl 14 56 1-420 107 26 number ofHbF cells per sample detected was 4 (range 0-11). An aggregate total of22 46,XX 104 2 0-24 3.6 0.3 HbF' cells were seen from 4 pregnancies with male fetuses. Ofthese, 21/22 were male by These results indicate significant differences exist in the number offetal cells detected in FISH. 46,XY vs. 46,XX (p<0.0001), 46,XY vs. 47,XY,+21 (p<0.001), or 46,XY vs. other male This data demonstrates that HbF is highly expressed in early fetal blood cells and that the abnormal karyotypes (p<0.05). Male cells detected in some 46,XX pregnancies are number ofHbF' cells is extremely low in pregnant and non-pregnant women. FISH possibly due to prior male pregnancies or transfusions. Low, although analyzable, analysis on artificial mixtures and maternal samples with male fetuses demonstrates the numbers ofmale fetal cells are present when the fetus is cytogenetically normal, but high specificity (91-95%) ofHbF as a fetal cell marker. significantly more cells are detectable when the fetus is aneuploid. This implies that routine noninvasive detection offetal aneuploidy is feasible. Slide Session 24: Prenatal and Perinatal Genetics I (cont.) All 45 46 Five-color combinatorial FISH for simultaneous detection of X, Y, 13, 18 and 21 in Prenatal diagnosis of single gene disorders by analysis of fetal cells in maternal blood: fetal cells isolated from maternal blood. F.Z. Riachffo . D.D. Neun1. S. Murrell53 Successful application in sickle cell anemia and P-thalassemia. MC. CheunLgJ.D. ,W. Schober3 T. Scott, 1. Boi off, I.L. Simpsonl,. D.E. Jst3 and S. Eliasjl2.4. MS. Golbus and Kan. Howard Hughes Medical Depts. OB/GYN I, Molecular/Human Genetics2 and Microbiology/Immunology, GoldberL Y.W. Institute and Department Baylor College of Medicine Houston, TX; U. of Tennessee4, Memphis, TN. of Obstetrics & Gynecology, Unversity of Califomia, San Francisco, CA. Cytogenetic analysis of fetal cells isolated from maternal blood by fluorescence in Currently, amniocentesis, chorionic villus sampling (CVS), and fetal blood sampling are situ hybridization (FISH) would be a major advance, potentially obviating the need used to obtain fetal cells forprenatal diagnosis. are invasive for the fetus and Because detection of all 24 chromosomes in a single Theseprocedures for invasive procedures. posea low, but not completelynegligiblerisk Recoveringfetal cells from maternal circulation interphase nucleus is not currently feasible, a strategy to concurrently detect cells, rather chromosomes X, Y, 13, 18 and 21 is required. However, multi-color FISH analysis for diagnosis will be noninvasive and safe for the fetus. Fetal nucleated red than on interphase nuclei is limited by 1) limited numbers of fluorochromes, 2) fetal lymphocytes, have been chosen because the latter could circulate in the maternal blood limitations in excitation/emission filters for fluorescence microscopy, 3) availability for many years. The eniiched fetal nucleated red cells have been utilized for in situ of chromosome-specific probes, and 4) vicissitudes in spatial relationship among hybridization or polymerase chain reaction (PCR) for chromosome analysis and sex various hybridization signals. Slunl Dsign We have developed a FISH strategy in in the prenatal diagnosis of single gene which combinatorial mixing of fluorophore-labeled probes was used for determination. Our laboratory has been interested simultaneous-color identification of the five target chromosomes. Using three disorders. To achieve fetal diagnosis of these disorders, pure fetal cells are necessary. We centromere and two locus-specific probes (VYSIS, Inc.), FISH analysis was performed chose aprocedure which includes the following steps: density gradient separation ofnucleated on flow sorted (CD71+, CD45-) maternal blood specimens. Results (Hybridization cells, magnetic bead separation using anti-transferrin receptor antibodies, staining with anti- Efficiencyl A mean of I,100 cells per case were scored. fetal or anti-embryonic hemoglobin antibodies, identification and isolation of nucleated red lybndization Etficiency M% cells with all exected signals) Cases (N) cells by microdissection on light microscopy, and nonradioactive PCR analysis. In 10 100% 2 we 97% to 99% l pregnancies, at the time ofchorionic villus biopsy from the10th to 12th week of gestation, 91% to 96% 4 were able to ident 7-22 fetal nucleated red cells starting from 16 to 20ml of maternal blood 85% to 96% 1 1 drawn before the CVS. In two ofthese pregnancies, fetal cell isolation was done in patients e intensity of each probe signal was 10 to 20 told greater than the background who requested prenatal diagnosis for sickle cell anemia and forP-thalassemia We identified fluorescence as determined by PowerGene probe system (PSI, Inc.). Analysis of fetal nucleated red cells from their blood, PCR and reverse signal distribution for each of the probes further revealed no trisomic or and isolated18 and8 performed monosomic cells. Failure to detect two FISH signals corresponding to the locus- dotblot analysis. Bothfetuses were ofthe AA genotype and did not carry the sickle cell or P- specific probes was on average 5 to 10 fold higher (0.25% to 0.5%) as compared to thalassemia mutations. The diagnoses agreed with those determined by direct DNA analysis the centromere-specific alpha satellite probes. These observed differences mav be on CVS. We conclude that fetal diagnosis of single gene disorders using fetal blood from unavoidable given innate characteristics of the probes and their target NA when the genotypes ofthe are This procedure sequences. Overall, fetal sex was correctly identified in 30/40 (75%) cases with 0% maternal circulation is possible parents known. false positive detection. Conclusion Five-color FISH analysis is a reliable method to canbe applied to other genetic disorders such as cystic fibrosis or Gaucher's Disease, where detect fetal chromosomal aneuploidy in fetal cells isolated from maternal blood. the mutations have been defined. Supported by NICHD #NOIHD43203.

47 48 Identification of trophoblast cells circulating in maternal blood during 24-Color FISH using spectral karyotyping: applications for prenatal I.J. van WiMk. J.M.G. diagnosis and potentials for preimplantatlon diagnosis. B. R. Haddad'.2, E. early pregnancy for non-invasive prenatal diagnosis Shrock1, S. du Manoiri, B. Bourddlat-Parksl, T. Veldmani, J. Meck2, M. R. Hughes1.2, and van Vugtl, M.A.M. Mulders. A.A.M. Konst and C.B.M. Oudeians. T. Ried' INCHGR/NIH, Bethesda, MD, and 2Georgetown Univ., Washington, DC Departments of Clin.Chem. and lObstet./Gynecol., Free University Hospital, Despite major advances in molecular cytogenetics during the past decade and the Amsterdam, The Netherlands. important diagnostic role that FISH plays in the characterization of chromosomal cells circulating in maternal blood during pregnancy are useful abnormalities, the usefulness of this technique remains limited by the number of spectrally Trophoblast distinguishable fluorochromes or fluorochrome combinations. Overcoming this major for non-invasive prenatal diagnosis. These trophoblast cells are good limitation would allow the use of FISH to screen the whole human genome for candidates as they (1) circulate in maternal blood very early during chromosomal aberrations which was, until recently, only possible through conventional pregnancy, (2) are unlikely to persist, (3) can be cultured in vitro, (4) are not karyotyping. The applications of such an approach are obvious in clinical cytogenetics both present in normal adult blood and (5) these cells could be informative for pre- and post-natally and in cancer cytogenetics. 24-Color FISH using spectral karyotyping is a recently described molecular cytogenetics technology (Schrdck et al. Science, in pregnancy associated diseases (e.g. pre-eclampsia). For enrichment of chromosomes in 24 different was press) that allows the simultaneous visualization of all human mononuclear trophoblast cells a density gradient centrifugation colors. This technique is based measurement of individual spectra of each chromosome combined with depletion of maternal white blood cells using the miniMACS specific probe followed by computer classification of the different chromosomes. Most X/Y PCR as a model system, we previously showed in a chromosomal aberrations detected during prenatal diagnosis can be resolved using procedure. Using dual color FISH. group of 36 patients, that fetal cells can be enriched and isolated as early as routine cytogenetic techniques alone or in combination with single or of the cases. However, some cases remain unresolved, in particular de novo supernumerary marker week 8 of pregnancy. Fetal sex was correctly predicted in 92% chromosomes and de novo structural rearrangements, causing major concems. We have Direct evidence that fetal cells are trophoblast cells was provided by RNA in used the 24-color FISH technique to detect different chromosomal aberrations that situ hybridization. As a new trophoblast specific marker we used HASH2. present important prenatal diagnostic dilemmas. In particular, we identified chromosome of the mouse basic helix markers of various sizes and origins as well as small unbalanced rearrangements. Diagnosis HASH2 is the human (achaete scute) homologue can potentially be loop helix protein encoding gene Mash2. Mice deficient for Mash2 die at was confirmed in all cases using specific FISH probes. This technique of applicable to preimplantation genetic diagnosis. Preliminary experiments to apply this midgestation because of abnormal placental development due to a lack technology using either whole chromosome probes to detect aberrations in polar body spongiotrophoblast formation. We have recently cloned and partially chromosomes or using centromeric probes to detect numerical aberrations in interphase sequenced HASH2. RNA in situ hybridization on human placental tissue cells confirmed the feasibility of this approach. shows high expression in extravillous trophoblasts. Using this detection of Spectral karyotyping has considerable diagnostic applications in the prenatal diagnosis ml field because of its reliability, speed and ability to visualize all human chromosomes in HASH2 mRNA, we are able to identify trophoblast cells enriched from 20 different colors, in a single FISH experiment. peripheral maternal blood during first trimester pregnancies.

49 50 Preifplantation diagnosis of age-related aneuploidies by Single cell analysis of Survival Motor Neuron gene deletions using first and second polar body FISH analysis. Y. Verlinskv. allele-specific single nucleotide primer extension. T. 1wata'. M.R. J. Cieslak. V. Ivakhnenko. M. White. A. Lifchez. B. Kaclan. Abbaszadegan'. R.E. Gore-Lan ton' and M.R. Hughes'3. NCHGR, NIH, Bethesda J. Moise. J. Valle. N. Ginsberg. C. Strom. and A. Kuliev. MD, and Dept. 'Ped./POb. Gyn., Georgetown Univ. Med. Ctr., Washington, D.C. Reproductive Genetics Institute, Chicago, IL Spinal Musc ular Atrophy (SMA) is a fatal autosomal recessive disorder characterized Age-related aneuploidies originating from non- by degeneration of lower motor neurons, leading to progressive paralysis with muscular disjunction in the first or second meiotic divisions atrophy. SMA affects about I in 6000 births and is the second most common contribute considerably to early pregnancy loss. We autosomal recessive disorder after cystic fibrosis. Development of a specific and introduced testing of oocytes by sampling and fluorescent sensitive test which can be used for preimplantation diagnosis and parental screening in situ hybridization (FISH) analysis of the first (IPB) will be a tremendous benefit for affected families. Deletions of the region containing the and second polar body (IIPB), to avoid the age-related risk exons 7 and 8 of Survival Motor Neuron (SMN) gene have been observed in the in patients of advanced maternal age. IPBs and IIPBs were majority of cases with the disease. The 20 kb gene, encoding a 294 amino acid protein removed following their extrusion from the oocytes and with yet unknown functions, is localized in the telomeric portion of a 500 kb inverted studied by FISH, using probes specific for chromosomes X, duplication in chromosome region 5ql3. The existence of an almost identical copy in 18, and 13/21. From a total of 261 couples of advanced the centromeric repeat has complicated PCR-based DNA tests for clinical diagnosis of maternal age (34 to 46 years) 1694 oocytes were biopsied the disease: PCR products amplified from telomeric SMN gene must be distinguished and subjected to FISH analysis, with FISH results available from products from centromeric SMN gene copy. The two genes differ by only a in 1365 oocytes (80.6%). Of these oocytes 852 (62.4%) were single nucleotide in each of exon 7 and 87 Previously, these sites in the copy gene were predicted to be normal and 513 (37.6%)-aneuploid. The cleaved by restriction enzymes. enabling distinction of the genes by fragment sizes. embryos resulting from the latter oocytes were followed up However, nested PCR on a single diploid cell or following whole genome amplification by FISH analysis to confirm the PB diagnosis, while 638 by primer extension preamplification (PEP) are required in preimplantation diagnosis. embryos from oocytes with normal FISH results were PCR products from control single lymphoblasts with a homozygous deletion of the transferred back to patients. A significantly higher critical region did not give complete digestions, which may lead to misdiagnosis. We pregnancy rate was observed in the group of patients have now successfully applied the single nucleotide primer extension (SNuPE) to studied by both IPB and IIPB (27.9%) compared to that in identify the absence of exons 7 and 8 in the telomeric SMN gene. SNuPE primers were patients of advanced maternal age without PB diagnosis designed to be adjacent to the sites of single nucleotide differences. Primer extension (16.2%). Overall, 25 healthy children were born and 15 reactions were performed with radiolabeled nucleotides, followed by denaturing- pregnancies are ongoing following confirmation of the PB polyacrylamide electrophoresis and a short autoradiography. Results of multiple diagnosis by CVS or amniocentesis. The study suggests a experiments indicate that single-cell SNuPE assay can detect affected single cells from practical value of PB diagnosis for avoiding the age- unaffected cells without any ambiguity. This method is straightforward, specific, and related risk for common aneuploidies. rapid, making it suitable for preimplantation diagnosis of SMA. A12 Slide Session 24: Prenatai and Perinatal Genetics I (cont.) 51 52 Preimplantation diagnosis of single gene disorders by two- Evidence for interchromosomal effects on pairing in female meiosis. step oocyte genetic analysis. S. Rechitsky. C. Strom. S.M Gartler E-Y Cheno Bonnet K Storck University ofWashington, J. Cieslak. V. Ivakhnenko. M. White. A.Kuliev and G. Seattle, Y. Verlinsky. Reproductive Genetics Institute, Chicago, IL. Washington. A high crossover frequency presents problems in Observations of associations between parental chromosomal rearrangements or predicting the denotype of embryos resulting from oocytes aneuploidy and chormosomally abnormal conceptions involving a different whose first polar body (IPB) was found to be heterozygous. chromosome have been reported. These associations, including parental sex To overcome this limitation of IPB preimplantation genetic chromosome aneuploidy and trisomy 21, parental DID translocation and aneuploidy, diagnosis (PGD) we introduced a two-step DNA analysis of and parental supernumery marker chromosome and trisomy 21 suggest that a oocytes using both IPB and second polar body (IIPB) to identify mutation free gametes following second meiotic primary chromosome abnormality may affect the meiotic behavior ofother division. The purpose of this work is to investigate the chromosomes. In a study ofthe meiotic segregation pattern ofsperm from 2 men reliability of the two-step genetic analysis for predicting with a supernumery marker, Martin et al. (1986) found that one man had the the genotype of the embryos resulting from heterozygous expected aneuploidy rate of 5 % found in the general population while the other man oocytes. Thirty four oocytes whose IPBs were found to be had an aneuploidy rate of 16%. Using single, dual color, and triple color FISH, we heterozygous for different alleles, including cystic studied the rate of asynapsis in an unrelated chromosome in at pachytene fibrosis (CF) Delta F-508 (9 oocytes), beta-globin (3) and oocytes linked polymorphic markers (22) (4-bp repeat (GATT) at the from a 19 week fetus with an unbalanced 8; I translocation and a 23 week fetus with 3' end of intron 6 in CF gene (2); short tandem repeat trisomy 18. The asynapsis rate was compared to that of previously studied euploid (STR) for Von Willibrands Disease (VWF) located on material at 15 to 25 weeks gestation. chromosome 12 (14); STR located on chromosome 21 (3)], have Sumnmary of Asynapsis of an Unrelated Chromosome in Pachytene been followed by DNA analysis of the IIPB and embryos t(8;1 1) (%) (%) Euploid (%) resulting from corresponding oocytes. In all 34 oocytes the Tri-1S genotype of the embryos originating from heterozygous Chromosome oocytes was reliably predicted by DNA analysis of their 7 NA 25/210 (11.9) 1/411 (0.2) corresponding IIPBs based on the presence of the alleles 13 16/329 (4.8) NA 3/512 (0.6) other than those in the resulting embryos. The data 16 31/509(6.1) 14/186(7.5) 2/236(0.8) demonstrate the accuracy and reliability of the two-step 18 6/257 (2.3) NA 0/755 oocyte analysis by IPB and IIPB for PGD of single gene X 2/79 (2.5) NA 0/832 disorders. The increased rate of asynapsis observed in the chromosomally unbalanced specimens suggests that the presence of a chromosome abnormality may alter the pairing ofother chromosomes in human female meiosis.

53 The Integrated BAC Resource: A Powerful New Molecular Tool For Prenatal Genetics X.-N. Chen', S. Mitchell', Z.-G. Sun', D. Nova', S. Ma', G.S. Sekhon2, K. 17psW2 W.-T. Hsu3, P. Wong3, N. Warm', R. Schreck', R. Falk', and J. R. Korenberq' 'Division of Medical Genetics, Bums and Allen Research Institute, Cedars-Sinai Medical Center, UCLA, Los Angeles, CA; 2Waisman Center, Madison, WI; 3Rush Presbyterian St. Luke's Medical Center, Chicago, IL; 4University of Rochester, Rochester, NY An ulfimate dream of human geneticists is push-button access to perfect cones from each region d the human genome forthe analysis and diagnosis ofdisease. These clones would be stable, easily manipulated, sequenceable, integrated with the genetic and physical maps, and ideal for molecular cytogenetics, the step beyond chromosome banding. We now present our progress toward creating The Integrated BAC Resource and describe its application ID prenatal genetics. An array of BACs and PACs (bacterial artificial- and P1 arfificial- chromosomes), has been defined either at random or by library screens using consensus alpha or TTAGGGn olgonudeotides representing 1he centrmeres and teblmeres respectively. Each clone has been mapped by high resokution fluorescence in situ hybridization (FISH) to regions of2.6 Mb and/or integrated with the genetic map by multi-dimensional PCR screening. The Resource now includes 4500 clones representing about 24% of the entire human genome, 22 telomeres, 24 centromeres, and 630 of which uniquely contain a polyCA marker from the Genethon set. The BAC Resource has been applied as the critical diagnostic device to provide rapid prenatal diagnosis using multiplex muticotor FISH of complex or subtle cases. Duplication: Using 8 BACs, the 19p 13.1-13.2 direct duplication and breakpoints were defined in 48 hours in a fetus with a diaphragmatic hemia. Deletion: Using 6 BACs, a subtle deletion of 9p24.3 was defined in a 6 month old dysmorphic infant with delay. Translocation: Using 5 BACs, a telomeric 21;22 translocation was established as balanced. Inversions: 1. Using 16 unique or repeat sequence BACs, subtle different inversion/deletion of 8p23.1-.2 was shown in a child with multipleanomalies and in the normal mother. 2. Using 3 BACs to define a subt&e pencentromeric inversion of 20 also carried in the mother, the fetus was not terminated. The era of pos-banding cytogenetics has arrived and in each of these cases, the results provide the tools for understanding the genes responsible forhuman development and disease. Slide Session 25: Cytogenetics I 54 55 Examination of the underlying pattern of chromosomal exchange in meioses S Chromosomal abnormalities in sperm from cancer patients before and after leading to trisomy 21: Evidence for initiation of all maternal meiotic errors at p chemotherapy. meiosis I. Neil E. Lamb' Eleanor Feingold'. Terry J. HassoId2. and Stephanie L. Sherman'" . 'Emory University, Atlanta, Georgia, 2Case Western Reserve University, R.H. Martin'. S. Ernst2. A. Rademaker4. L. Barclav3. E. Ko3. N. Summers. 'Dept. of Cleveland, Ohio. Medical Genetics, University of Calgary; 'Tom Baker Cancer Centre, Alberta Our recent studies of trisomy 21 have shown that altered levels of recombination Children's Hospital, Calgary, Alberta, Canada and 4Cancer Center, Norwestern are associated with maternal nondisjunction (NDJ) occurring at both meiosis I (MT) University Medical School, Chicago, IL. and meiosis II (MU). For MI NDJ, recombination is significantly reduced whereas Sperm chromosome abnormalities were assessed in testicular cancer patients before recombination is increased for MIt NDJ. We examined the underlying pattern of and after treatment with BEP (bleomycin, etopiside, cisplatin). The frequencies of chromosomal exchange to determine if specific meiotic configurations lead to a disomy for chromosomes 1, 12, X, Y and XY were assessed along with diploid higher risk of NDJ than others. To do this, we used the pattern of recombination frequencies and sex ratios by multicolour fluorescence in situ hybridization (FISH). For observed among four relatively equally spaced regions (about 20 cM each) along 21q each cancer patient, a minimum of 10,000 sperm was assessed for each chromosome to predict the underlying exchange pattern occurring at the four-strand stage during probe pre- and post-chemotherapy (CT). Data was analyzed "blindly" by coding the meiotic prophase. This was done for three types of data: 1) normal female meiotic slides. A total of 161,097 sperm were analyzed: 80,445 before and 80,642 after events (n=212), 2) meiotic events leading to MI NDJ (n=253), and 3) those leading treatment. The mean disomy frequencies were 0.11% pre-CT vs 0.06% post-CT for to MU NDJ (n=91). Using this approach, we found that nearly one-half of MI errors chromosome 1, 0.18% vs 0.15% for chromosome 12, 0.10% vs 0.9% for the X were predicted to have no exchanges (achiasmate), a 3-fold increase when compared chromosome, 0.13% vs 0.10% for the Y chromosome and 0.25% vs 0.20% for XY with normal events. Among the remaining chiasmate bivalents, almost one-half were sperm. There was no significant difference in the frequency of disomy pre-CT vs post- predicted to have a single distal exchange, whereas only 10% were predicted for CT for any chromosome except that chromosome 1 demonstrated a significant decrease normal rneiotic events. In contrast, chromosomal exchange was increased along 21q after CT. The "sex ratios" and frequency of diploid sperm were also not significantly among MU cases, particularly in the proximal region of the chromosome: over 27% different in pre- and post-CT samples with 50.2% X-bearing sperm pre-CT and 50.5% were predicted to occur in the most proximal region compared with 6% predicted for X-bearing sperm post-CT and 0.14% diploid sperm pre-CT vs 0.15% diploid sperm normal events. These data show that homologous exchange, a process which occurs post-CT. There was no significant donor heterogeneity among the cancer patients. during meiosis I, appears to be disrupted and/or altered for both meiosis I and None ofthe values in the cancer patients differed significantly from 10 normal control meiosis UI NDJ. This surprising result challenges the widely-held concept that events donors. Thus our study suggests that BEP chemotherapy does not increase the risk of occurring at meiosis I are largely independent of events occurring at meiosis II and numerical chromosomal abnormalities in human sperm. suggests that all nondisjunction events may be initiated during prophase of meiosis I. Slide Session 25: Cytogenetics I (cont.) A13 56 57 Mechanism of chromosome malsegregation in male mammalian meiosis: Effects of heterozygosity for Robertsonian translocations on meiotic lessons from chemically-induced disturbances and stage-specific analysis of segregation and fertilization competence of male germ cells. F. Marchetti, spermatocytes. J. Lahdetie and M. KalliW, University of Turku, Turku, Finland. X. Lowe and A.J. Wyrobek. Biology and Biotechnology Research Program. Lawrence (Intro. by: Pertti Aula) Livermore National Laboratory, Livermore, We have studied mechanisms operating during male mammalian meiosis in order to CA. understand how in is induced. Robertsonian (Rb) translocations are common in mammals. In man about 5% of aneuploidy sperm The male mammalian meiotic divisions individuals with Down syndrome have a Rb translocation. Also, individuals Rb have unique features that differ from mitotic divisions especially due to the presence of heterozygous have elevated bivalents and resolution of chiasmata at the reductional (fsrst) division. frequencies of unbalanced gametes, and therefore are at We used the stage-specific model of rodent spermatogenesis to characterize normal high risk for spontaneous abortions and chromosomally imbalanced offspring. To study divisions both meiotic segregation and fertilization competence of sperm produced by Rb meiotic and disturbances induced by chemicals. In the stereomicroscope, under translocation carriers, transmitted light, seminiferous tubules show differences in light absorption in a wave-like we performed FISH painting analysis of mouse metaphase II fashion, due to the organization of germ cells in the epithelium. Spermatogenic cell (MII) spermatocytes and first-cleavage (1-Cl) zygotes in male mice doubly populations at defined stages of their development can be identified. Isolation of a 1 mm heterozygous for the Rb(6.16) and Rb(16.17) translocations. These mice are used to segment of the tubule by microdissection yields a highly enriched population of generate trisomy 16, which is a mouse model for Down syndrome. Fifteen Rb mice meiotically dividing cells for cytological and cytogenetic analyses. were mated with normal B6C3F1 females. 1-Cl zygotes were collected from mated Inhibitors of tubulin polymerization such as colchicine and vinblastine disturb the females 18 h after fertilization and prepared for metaphase analysis. Testes were then formation and dynamics of the meiotic spindles and lead to arrest and cell death at isolated from four randomly chosen mice and testicular preparations for MII divisions. These chemicals are weak inducers of in spermatocyte analyses were prepared. Hybridization was performed with a cocktail surprisingly micronuclei spermatids - mixture containing three biotin-labeled probes signs of lagging of chromosomes and chromosome loss at meiotic divisions - while they specific for chromosomes 8, 16 and 17 produce high frequencies of micronuclei in mitotic cells of e.g. the bone marrow. This plus a digoxigenin-labeled probe specific for chromosome Y. The frequencies of suggests that chromosome alignment at metaphase and stabilization of kinetochore- aneuploid male-derived complements were very similar before and after fertilization. microtubule connections is controlled more efficiently at meiotic divisions than at mitosis. Balanced male complements were 60.6% (57/94) in MII spermatocytes and 57.8% This may be a result of a previously undetected mechanism of assembly of meiotic (63/109) in 1-Cl zygotes. Disomy or nullisomy for 16 represented 20.2% and 18.1% in spindles; confocal analysis showed that the functional spindle elongates from a small MII spermatocytes and 23.9% and 18.3% in 1-Cl zygotes, respectively. Disomy for 17 bipolar spindle in the middle of the chromosome mass after nuclear envelope breakdown was 1.1% in MII spermatocytes and 0.9% in 1-Cl zygotes. No aneuploidy involving (NEB), while in mitosis, spindle pole separation and migration to the opposite sides of chromosome 8 was found, as expected, while a possible segmental aneuploidy involving the nucleus are known to preceed NEB. the Y was observed in a 1-Cl zygote. These results show that Rb translocation carriers Two DNA topoisomerase II directed compounds (VP-16 and merbarone) induced are at high risk of producing gametes unbalanced for those chromosomes involved in aneuploidy, polyploidy, micronucleus formation, cell death and centromere breakage. the translocation and suggest that aneuploid sperm are not selected against during Reports of similar effects of VP-16 in mammalian oocytes suggest that basically the same fertilization and the zygotic cell cycle. mechanisms operate in male and female meiosis and indirectly point to the fundamental Work performed under the auspices of the U.S. DOE by the Lawrence Livermore role of topoisomerase II function in chromosome segregation at meiosis. National Laboratory under contract W-7405-ENG-48 with support from NIEHS Y01-ES-10203-00.

58 59 De novo rob(13ql4q) and rob(14q21q) Robertsonian translocations form Evidence for the presence of chromosome rearrangement hotspot in through common breakpoint regions during female meiosis. iLfage, £ 15q13: towards understanding the mechanism for chromosome McCaskill, P.H. Gunaratne, L.G. Shaffer. Baylor College of Medicine, Houston, TX. rearrangements. B Huang', SL Christian', T Kubota', IL Lesser', Peter Robertsonian translocations (ROB) are the most common chromosomal rearrangements in with Papenhausen-, DH Ledbetter 'Diagnostic Development Branch, National Center for humans, rob(13ql4q) and rob(14q21q) most frequently observed. Previous work Human Genome Research, National Institutes of Health, Bethesda, MD; 2Lab Corp in our laboratory showed a consistent location of breakpoints to specific short arm regions in 95% of of America, Research Triangle Park, North Carolina. rob(13ql4q) and 100% of rob(14q21q), supporting the hypothesis that these Chromosome rearrangements involving band lSq 13 such as inv dup(l5) and 15q translocations occur through recombination between homologous DNA sequences present triplication are relatively common in humans. To the in the short arms investigate molecular proximal of chromosomes 13, 14, and 21. Parental origin studies of mechanisms for the frequent rearrangements in lSq, we analyzed the parental origin some balanced de novo homologous ROBs have shown biparental inheritance, and breakpoints of a) large inv dup(15) chromosomes (containing the Prader- indicating formation during mitosis early in development. Molecular studies have not been Willi/Angelman Syndrome critical region) and b) interstitial 15q triplication patients. possible for de novo nonhomologous ROBs because one cannot distinguish between the The parental origin was determined by microsatellite analysis and/or DNA alleles corresponding to the translocation and the free-lying homologues. To further methylation studies. A maternal origin was found for all of the 19 individuals delineate the mechanism(s) of ROB formation, we investigated whether the translocations analyzed [17 with inv dup(15) and 2 with and no was form meiosis or In we triplication] uniparental disomy during mitosis. this study, unequivocally determined the parental found in these cases. In order to characterize these breakpoints, a YAC contig and origins of the chromosomes involved in 16 de novo nonhomologous ROBs by STR-PCR physical map were constructed for 15q distal to the PWS/AS critical analysis of somatic cell hybrids which segregated the translocations from their region including The translocations D15S165 and S144. FISH analysis using P1 and YAC clones in this region homologues. analyzed included 8 rob(13ql4q), 3 rob(14q21q), 2 demonstrated that the breakpoints clustered in a single YAC clone, y8Ofl , in rob(15q21q), one rob(14ql5q), one rob(14q22q), and one rob(15q22q). In the majority of the ROBs the 13/13 inv dup(l5)s and both of the 2 triplication patients studied. Interestingly, the (14/16), chromosomes involved were both of maternal origin, while the same breakpoint is also involved in the pericentric inversion event in chimpanzee remaining two were derived from both paternal chromosomes. These findings are chromosome different from those 16, which is homologous to human chromosome 15. This significant significantly expected for random formation in mitosis, where about clustering of human constitutional breakpoints as well as an evolutionary breakpoint half of the ROBs would form between chromosomes of opposite parental origins strongly suggests that hotspot for chromosome rearrangement exist in this region (p

60 61 Identification of a small (100-200kb) region which contains the Chromatin structure: evidence that the RB1gene is spread on two loops. breakpoint in all large (Class III) inverted duplications of chromosome M. Boutouil, R. Fetni, C.L. Richer and N. Lemieux. Dept Pathol, Universitede 1S. A _EWandstrat and S Schwartz Case Western Reserve University and of Montreal, Montreal, Quebec, Canada. University Hospitals Cleveland, OH. The retinoblastoma gene (RB1)of 200 kb contains 27 exons distributed in three clus- The favored theory for the mechanism of formation of inverted duplications of ters the DNA. To chromosome 15 has been along genomic study the organization of the RBI gene, different [inv dup(15)] illegitimate recombination between cDNA probes were hybridized simultaneously and differentially visualized by electron homologous chromosomes involving a U-type exchange followed by nondisjunction and centromere inactivation. Low DNA microscopy (EM). We have used three cDNA probes complementary to exons in each of copy repeat sequences along the clusters I (exons 1-2), II (exons 3-17), and III (exons 18-27) to individually illuminate chromosome, several of which have been identified in proximal 15, are believed to each cluster two distinct sizes facilitate such aberrant To test using of gold particles. This study shows, using EM in situ recombination. this hypothesis, chromosomes from 20 hybridization on banded patients were used to delineate the breakpoints of inv dup(15) which include the (EMISH) high-resolution chromosomes, that as many as three critical spots per chromatid are observed when total cDNA of RBlis hybridized resulting from Prader-Willi/Angelman syndrome region (15ql -q13). YAC and cosmid DNA or absence of fusion located in were used for FISH in incomplete between the three hybridization networks of the three probes 15qll-q14 analysis order to detect the separate exon clusters. of distance between the presence or absence ofmaterial on the inv dup(15). Analysis three spots shows that one spot In addition to small inv dup(15), which break in 15ql1 (Class I), and medium inv corresponding to cluster III is clearly separated and the other two spots corresponding which break between and D1SS24 in we have to clusters I and It are always either fused or distinguishable but immediately adjacent. dup(1S), D15Sl2 15q13 (Class II), To further these we examined in detail 13 Class III cases that break just proximal to D15S144 in 15ql3- identify spots used double-labeling and double-detection of differ- q14. FISH was used to study four YACs that map to the D15S144 locus. Only one ent RB1segments corresponding to the three exon clusters. Simultaneous hybridization of these YACs, 810fl 1, was found to span the breakpoint in the Class Imcases. Alu of cluster II and cluster III confirmed the variation of distance between the two spots and restriction element mapping four YACs in the on nuclei and chromosomes. This variation was found to be independent of chromatin fingerprinting comparing breakpoint condensation. no region, including 810fl1, revealed that a small, discrete area (100-200kb) accounts for Also significant distance variation between cluster I and cluster IIwas all of the breakpoints in the Class III cases. Such a clustering of breakpoints suggests observed. A housekeeping gene such RB1,is expected to be localized on an R-band. a hotspot for crossover breakage. Studies are underway to further refinethe map of According to our finding, the proximal part of RBI containing exon clusters I and II the breakpoint region in order to identify any low copy repeat sequences that may be is on the early replicating R-band 13ql4.3, whereas the distal part containing the exon responsible for such exchange. Parent-of-origin studies demonstrated that in all of the cluster III might be located on the late replicating G-band 13q21.1.We demonstrate that cases analyzed, including all of the Class III cases, the inv dup(15) was maternal -in the number of spots corresponds to the number of exon clusters differentially identified origin. We conclude that the homogenous nature of the breakpoints in Class mcases using the appropriate probe and gold particles of two sizes. The variation of distance may indicate that there is a single repeat sequence which allows aberrant recombination between exon clusters suggests that the RB1 gene is located on two separate chromatin and that these inv dup(15) are probably the result of a U-type exchange followed by loops which are separated by an active SAR (scaffold attatchement region). The future maternal nondisjunction. purpose of this project is to localize and show in vivo the presence of this putative SAR. A14 Slide Session 25 Cytogenetics I (cont.) 62 63 Several mechanisms at the origin of ring chromosomes E.Rossil23, Y.Ning4, Studies of "acentric" and "dicentric" marker chromosomes: Implications for the S.Giglio3, R.Carrozzo2, O.Zuffardi' 3, D.Ledbetter4. lLaboratorio di Citogenetica and definition of the functional centromere. S Schwartz, TW Deoinct. Center for Human 2Servizio di Genetica Medica, Ospedale San Raffaele, Milan, 3Biologia Generale e Genetics, Department of Genetics, Case Western Reserve University and University Genetica Mtedica, Pavia, Italy, 'National Center for Human Genome Research, N.I. H., Hospitals of Cleveland. Ohio. Bethesda, Md. To better understand the mitotic behavior of abnormal chromosomes with either no We analyzed 27 cases of apparently entire ring chromosomes by FISH with simple detectable (thus. "acentric") or two copies ("dicentric") of the centromeric region, the telomeric sequences (TTAGGG)n and with chromosome-specific telomeric probes for role of centromere proteins and the process of centromere activation/inactivation, we both arms. We found that in 7 cases the (TTAGGG)n sequences were preserved beside undertook a study of six supernumerary markers which had no detectable alpha-satellite the chromosome-specific telomeric sequences ofeither both arms (2 cases) or a single arm (5 cases). Among the latter, 3 were mosaic with a seemingly normal cell line. This was DNA and 16 accessory dicentric marker chromosomes. Previous studies of non- in fact normal in one case, while in the remaining two the chromosome corresponding to homologous dicentric Robertsonian translocations have demonstrated the importance of the ring had chromosome-specific signals at one end and (TTAGGG)n signals at both centromere protein C (CENP-C) and E (CENP-E) in the functionally active centromere. extremities. Among the 20 cases in which the (TTAGGG)n were lacking, 18 also lost In this study. two contrasting groups of marker chromosomes ("acentric" and both the chromosome specific telomeric sequences, 1 lost only that of one arm, and I had "dicentric") were studied with antibodies to CENP-B, CENP-C and CENP-E to further both the telomeric sequences in half of the mitoses and only one in the remaining half. evaluate centromere activity. Fourteen of these cases were mosaic with a normal cell line. These data demonstrate These studies not only confirm the importance of CENP-C and CENP-E, but also that some rings might originate after a single breakpoint on one arm which sticks to the (TTAGGG)n sequences of the opposite arm, while others originate as a result of two illustrate other important factors in understanding the activity of centromeres. breakpoints followed by a joining of the opposite arms. The presence of a normal cell line Examination of these data indicate: (1) Even though "acentric" markers lacked confirmed the postzygotic origin of the ring in a single case in which uniparental disomy detectable alpha-satellite DNA and CENP-B, all ofthese markers exhibited both CENP- was ruled out. Moreover phenotypic alterations associated with apparently entire ring C and CENP-E. (2) While 15 cases of chromosome 15 dicentric chromosomes chromosomes can be due not only to ring chromosome instability but primarily to the demonstrated two regions which hybridized with chromosome 15 alpha satellite DNA loss of chromosome material. and showed the presence of CENP-B, the majority of these markers (10115) showed only one active centromere and the presence of CENP-C and CENP-E at only one centromeric region. (3) Only the smallest group of markers (with the least amount of material between the two centromeres) demonstrated two active centromeres with CENP-C and CENP-E at both centromeric regions (detected in 5 of the 6 cases studied). and (4) Four of these five cases were mosaic suggesting a correlation between the presence of two active regions and mitotic instability.

64 Functional status of centromeres in dicentric X chromosomes: evidence for the distance-dependence of centromere/ldnetochore assembly and correlation with malsegregation in anaphase. BA Sullivan and HF Willard Case Western Reserve University, Cleveland, OH. The stability of dicentric human chromosomes has generally been attributed to inactivation of one of the two centromems; such chromosomes, therefore, while structurally dicentric, are functionally monocentric. To examine the structure and behavior ofactive and inactive centromeres, we studied 8 dicentric X isochromosomes (isoX) using indirect immunofluorescence with antibodies against centromere proteins (CENPs) previously implicated in centromere function. In 3 of7 i(Xq)s and 1 idic(Xp) studied, CENP-C and -E were detected at a single centromere only, consistent with the centromere inactivation hypothesis. In contrast, CENP-C and -E were detected at both centromeres in 4 isoXs, suggesting that such isoXs are functionally dicentric. Notably, the functional status ofthe two centromeres correlated with the distance between them. Thus, the 4 largest isoXs, with -1680 Mb between the centromeres, were functionally monocentric and appeared stable mitotically. However, 4 smaller i(Xq)s, with 8-12 Mb between the centromeres, were functionally dicentric with two CENP-C and -E signals; further, these cases were associated with the greatest degree of mosaicism. To determine directly if functional dicentrics show an increased frequency of abnormal segregation, we developed an assay to enrich for anaphase/telophase cells after treatment with nocodazole and dihydrocytochalasin B to prevent cytokinesis. Thus, the daughter sets of chromosomes can be visualized directly on the mitotic spindle and segregation of individual chromosomes scored after in situ hybridization. Using this assay, segregation ofthe normal X, the i(Xq), and a control autosome was scored in >100 anaphase/telo- phase cells foreach cell line. While 3 functionally monocentric i(Xq)s (as well as control chromosomes) segregated normally in >99% ofcells, a functionally dicentric i(Xq) showed abnormal segregation (chromosome lag/loss) in -25% ofanaphases scored. These data (i) demonstrate directly that chromosomes with two functional centromeres encounter segregation difficulties in anaphase, (ii) suggest that mosaicism associated with many functionally monocentc i(Xq)s may reflect loss early in development rather than constitutive mitotic instability, and (uli) provide evidence for a distance-dependence of dicentric chromosome structure and centromere/ idnetochore assembly and behavior.

Slide Session 26: Inborn Errors of Metabolism and Biochemical Defects 65 66 Desmosterolosis: A New Disorder of Cholesterol Biosynthesis Association of the fragile X mental retardation protein with David R FitzPatrick', Jean W K ing Kevin Mills3, Peter Clayton3. Department of somatodendritic polyribosomes in brain. Y. Feng, C-A. Gutekunst S Clinical Genetics, Molecuila Medicin Centre, WGH, Edinburgh EH4 2XU, M. Hersch. S. T. Warren. Emory Univ., Atlanta, GA. 2Department of Paediatric Pathology, RHSC, Edinburgh EH9 ILF, 'Biochemical Unit, Fragile X syndrome is a frequent form of inherited mental retardation. Institute of Child Health. London WC1N' 1EH The absence of FMRP, a selective RNA binding protein encoded by the We describe a child with multiple malformations including macrocephaly, hypoplastic FMR1 gene, has been demonstrated as the sole determinant for the clinical nasal bridge, thickened alveolar ridge, gingival nodules, cleft palate, total anomalous pul- manifestations. FMRP associates with translating ribosomes and potentially monary venous drainage, ambiguous genitalia, short limbs and generalised osteosclerosis. shuttles between nucleus and that FMRP be Gas chromatography and mass spectroscopy of the neutral sterol profile in this infant cytoplasm, suggesting may revealed an accumulation of the cholesterol precursor, desmosterol (cholesta-5,24-dien- involved in RNA nuclear transport and/or translation. Here, we show that 3B8-ol), and relative deficiency of cholesterol in every tissue examined. In the brain this FMRP predominantly cofractionates with rough endoplasmic reticulum and accounted for 49% (normal <15%) of neutral sterol content, in the liver 15% (normal membrane-free polyribosomes in neuronal and non-neuronal tissue. To <2%)and the kidney 24% (normal <2%). There was moderate elevation of plasma levels visualize subcellular localization of FMRP at high resolution, of desmosterol in both parents (mother 12.27 micromoles/l, father 39.62 micromoles/l, immunocytochemical and immunogold studies with anti-FMRP antibody normal range 0.87-6.33 micromoles/l). The biochemical phenotype in this infant is highly were carried out in rat brain. FMRP expression was restricted exclusively in suggestive of a novel inborn error of cholesterol biosynthesis with an autosomal reces- neurons with no FMRP detected in the non-neuronal cells. Intense neuronal sive deficiency of the enzyme delta-24 reductase. Triparanol, a specific inhibitor of this labeling of FMRP was found throughout the brain, particularly of the enzyme is highly teratogenic in rodents producing frontonasal dysplasia, anophthalmia perikaryon and proximal dendrites. Nuclear labeling of FMRP by and renal dysplasia, thus strengthening the suggestion that desmosterol accumulation immunogold electron microscopy was also evident. Immunogold particles has an adverse effect on specific developmental processes. The suspected enzyme defect within the nuclear pores were visualized, supporting the nucleocytoplasmic in this child is in the final step in one of two different pathways mediating the conversion shuttling of FMRP in vivo. Immunogold particles primarily localized to of lanosterol to cholesterol in humans. The final enzymatic step in the other pathway neuronal polyribosomes and rough endoplasmic reticulum in the neuronal is 7-dehydrocholesterol reductase which is deficient in patients with Smith-Lemli-Opitz cell body, and to dendritic spine regions where ribosomes commonly occur, syndrome. such as spine apparatus, dendritic branch points, and the origins of dendritic spine. This is supported by the cofractionation of FMRP with synaptosomal polysomes. These results suggest the possibility that FMRP may play a role in localization and/or translation of mRNAs to the postsynaptic sites, and the fragile X syndrome may be a result of abnormal postsynaptic protein synthesis. Slide Session 26: Inborn Errors of Metabolism and Biochemical Defects (cont.) A15

67 68 Biochemical Analysis of the 3460, 11778, and 14484 Leber's Hereditary L - [1"CJ-phenylalanine oxidation inhyperphenylalaninemia (HPA); analysis of Optic Neuropathy (LHON)mtDNA Mutations in Lymphoblasts and Trans- genotype/phenotype correlations. C.R. Scriver'. J.J. Delente2. G. Elkas'. K. Carter'. mitochondrial Cybrids. M.D.Brown.I.Trounce.B.Jun. A.Panov. and D.C. M. Lambert'. P. Waters'. E.P. Treacy'. 'Dept. of Human Genetics, McGill Univ.- Wallace. Department of Genetics and Molecular Medicine, Emory University School of Montreal Children's Hospital, Canada,2Martek Biosciences Corp., Columbia, MD and Medicine, Atlanta, GA. of Canada. To investigate the pathophysiological mechanism of LHON, we studied respiration and 'Dept. Genetics,Hop. Ste-Justine, Montreal, mitochondrial enzyme activities in lymphoblasts and lymphoblast-derived trans- Hyperphenylalaninemia (HPA) results from deficiency of the enzyme phenylalanine mitochondrial cybrids for the "primary"3 LHON mtDNA mutations at nucleotide hydroxylase, with 302 known mutations at the PAH locus (McKusick 261600; positions 3460, 11778, and 14484. Each of these mutations alters a polypeptide http://www.mcgill.ca/pahdb), including 33 that have been expressed in viro. constituent of Complex I (NADH:ubiquinone oxidoreductase). Hypothsis The invivo metabolic counterpart (HPA) is an emergent property and may For respiration studies in lymphoblasts, statem rates using NADH-linked substrates not correlate consistently with the in vitro unit-protein (enzymic) phenotype. Methods: were reduced for all three mutations, although most markedly in 11778 mitochondria, in i) We developed a novel, rapid (60 min), non-invasive breath test to estimate whole- which the statem rate was 30% lower than controls. Uncoupled rates were generally body phenylalanine oxidation from cumulative "CO2 recovered at 60 mins. (isotopic reduced for all three LHON genotypes, but ADP/O ratios did not differ significantly from PKU control ratios. Similar results were observed in cybrid cell lines, but with less marked plateau) in: 7 classic PKU (phe tolerance <500 mg); 6 variant (phe tolerance differences between patients and controls. For enzymological analysis in lymphoblasts, >500 mg/day); 4 non-PKU HPA subjects; 17 obligate heterozygotes; and 8 controls. Complex I activity was reduced for all mutations (3460 =70% reduction relative to We correlated in vivo data with genotype (PAH mutations analyzed by DGGE and DNA cybrid controls, 11778 = 30% reduction, 14484 =20% reduction). Citrate synthase sequencing) and In vitro expression data (where known). We also compared the density (CS) normalized Complex I activities exhibited the same degree of dysfunction. There function for noon-time plasma phenylalanine level and the phenylalanine/tyrosine ratio were essentially no differences between patients and controls for Complex [1+11. m, or with ICO2 data in obligate heterozygotes.Results: (i) Oxidation data (%"C oxidized), CS Complex IV was elevated in 3460 and 11778 assays, although activity mitochondria. differed between groups (mean ± SD): all HPA cases, 0.28±0.58; seen which exhibited a heterozygotes In cybrids, an enzymatic defect was only for the 3460 mutation, 4.77±1.88; controls, 7.95±1.43; p <0.001; ii) differences existed between the 73% reduction in absolute and normalized Complex I activities. subclasses of and 'mild' PAH mutations This study represents the first time that a direct comparison of the biochemical HPA; iii) heterozygotes harbouring 'severe' phenotypes of the important LHON mutations can be made. Using a single tissue and could be differentiated; iv) discrepancies were noted between observed(in vivo) oxidation with uniform assays, we have shown that: (1) Complex I defects were found for all three rates and predicted effect (in vitro expression) of alleles. Conclusion: The "IC mutations, with the most marked respiration defect found in 11778 mitochondria and the phenylalanine breath test is a reproducible non-invasive rapid measure of in vivo most marked enzymological defect found in 3460 mitochondria, (2) the 14484 mutation phenylalanine oxidation. It can discriminate between mutant, homo- and heteroallelic has the most benign biochemical phenotype, and (3) cybrid transfer tended to reduce phenotypes of differing severity; and between the heterozygous and wildtype. HPA has differences between patient and controls values relative to the lymphoblast data, thus both Mendelian (major locus) and emergent (complex trait) properties. demonstrating the influence of the nuclear genetic background. We are currently investigating the kinetic parameters of mutant Complex I in an attempt to identify a common pathophysiological mechanism for LHON.

69 70 Possibility of gene therapy for globoid cell leukodystrophy (GLD, Krabbe CORRECTION OF ORNITHINE ACCUMULATION PREVENTS RETINAL DEGENERATION IN A MOUSE MODEL OF disease) is indicated by successful transfer of GALC activity from GYRATEH overexpressing cells to deficient cells. M.A. Rafil, J. Fugarol, T. Victoria' ATROPHY OF THE CHOROID AND RETINA Tao Wang, Ann Mil, and David Valle. Howard Hughes Medical Institute andthe epartmentof Pediatrics, Beggs2. H.Q. Tong2 and D.A. Wenger'. 'Jefferson Medical The Johns Hopkins University School of Medicine, Baltimore; Department of College, Philadelphia, PA and2Children's Hospital, Boston, MA. Ophthalmology, University of Washington School of Medicine, Seattle. Krabbe disease or GLD is an autosomal recessive disorder caused by the deficiency of Deficiency of ornithine-b-aminotransferase in humans results in galactocerebrosidase (GALC) activity. This enzyme catalyzes the lysosomal hydrolysis (OAT) gyrate atrophy an autosomal recessive disorder characterized by and a of galactosylceramide, an important component of CNS and PNS myelin. Two animal (GA), hyperornithinemia slowly progressive chorioretinal degeneration. To a mouse model for of the models, twitcher mice and terrier dogs, are well characterized and available for gene develop study patho- genesis and treatment of GA, we an OAT-deficient mouse by gene trials. GALC is extremely hydrophobic, existing in very low endogenous levels produced targeting. therapy OAT-/- mice similar to human GA as a high molecular weight complex (>700 kD) composed of its 30 and 50 kD subunits. Post-weaning, develop hyperornithinemia patients ornithine mean ± OAT-/- 1172 ± normal 95 ± and Human GALC cDNA with a modified start site was placed in the MFG retroviral (plasma SD; mice, 251; mice, 21) vector, a retinal Mean is and used to transfect cells. The MFG-GALC from the packaging cell line suc- slowly progressive degeneration. electroretinogram (ERG) amplitude 0-crip normal at 2 months but reduced to 40% 12 months. At 2 months the retinas of cessfully transduced many cell types including fibroblasts from patients and dogs with by OAT-/- mice are normal but 7 months the RPE cells are swollen with GLD, human CD34+ hematopoietic cells, rat brain astrocytes, mouse Schwann histologically by cells, loss of basal and microvilli and the outer are and myoblasts from twitcher mice. Deficient cells reached GALC levels 20,000 times infoldings apical photoreceptor segments highly disorganized and reduced in length. Retinal amino acids show ornithine accu- pre- infection levels (about 100 times normal) with no ill effects. These cells secreted mulation 15 x control. To determine the effect of chronic reduction of ornithine on the GALC into the media that was efficiently taken up by other deficient cells. Normal val- retinal we OAT-/- mice on an diet at 2 months of ues were obtained in recepient cells in 2-3 days. This compares to the very low transfer phenotype, placed arginine-restricted Plasma ornithine was reduced to near control levels and of GALC activity by untransduced normal cells. The high uptake of GALC from media age. (mean 167, range 74-249) normal growth was maintained.. At 12 months, ERG and retinal was inhibited 50% by mannose-6-P indicating that significant uptake was via a non- amplitudes histology of the treated OAT-/_ mice were normal. We conclude that the OAT-/- mice are an mechanism, probably pinocytosis. Astrocytes and Schwann cells were receptor-mediated excellent biochemical and retinal model of that accumulation is central to also transduced to levels 10-20 times baseline by this vector, and these GA; ornithine successfully cells the pathophysiology; and, that systemic correction of ornithine accumulation were able to donate GALC to deficient fibroblasts with high efficiency. As bone mar- chronic, prevents the retinal degeneration. row transplantation of affected patients using a normal donor has been demonstrated to have a positive, but not curative, effect on the clinical course, it is postulated that transplantation of cells overexpressing GALC would provide significantly more enzyme for delivery to neighboring cells.

71 72 disease: enzyme replacement therapy in o-galactosidase A deficient Fabry Amelioration of the skeletal disease in hypophosphatasia by bone marrow mice. Y.A. loannoul, K.M.Zeidnerl, B. Friedman2 and R.J. Desnick'. 1 Department transplantation using the alkaline phosphatase-knockout mouse model. of Human Genetics, Mount Sinai School of Medicine, NY 2 Genzyme Corporation, Feddeml L Blair'- F. Terzicl HC Anderso6n S.Narisawa3 J.L.Ma3,MyP WhAte'-4. Washington Framingham, MA Univer ofMed, St. Univer Fabry disease is an X-linked lysosomal storage disorder that results from the deficient Schl Louis, MO'; ofKansasMed Cente2,TheBurnham Institute, progressive La CA3; and Shriners for Crippled Children, St. Louis, MO4. activity of the lysosomal hydrolase a-galactosidase A (a-Gal A). The accu- Jolla, Hospital mulation of its major glycolipid substrate, globotriaosylceramide (GL-3), particularly in Hypophosphaasnia (HPP) is an untreatable autosomal recessive skeletal disease vascular lysosomes, results in premature death due to vascular disease of the kidney, (ricketslosteomalacia) resulting from mutations in the tissue-nonspecific alkaline phosphatase heart or brain. The recent availability of mice with n-Gal A deficiency, generated bv (TNSALP) gene and deficient TNsALP activity. Infantile HPP typically manifests with the evaluation of the potential therapeutic effectiveness of a-Gal gene targeting, permits progressive skeletal disease developing during the first 6 months-of-life with lethality A replacement. Studies were conducted in the enzyme deficient mice to determine the reported in 50% ofpatients. trials by intravenous infission of ALP appear to be plasma clearance and biodistribution of four different a-Gal A glycoforms which varied Therapeutic we a (null allele) mouse in their levels of sialylation. The plasma clearance half-lives for all four recombinant unsuccessful. Accordingly, investigated lNSALP gene-knockout a model for HPP and tested glycoforms were about 3 to 5 min, with the most highly sialvlated glycoform having the the therapeutic efficacy ofbone marrow transplantation. mouse longest half-life. All four glycoforms were primarily taken up by the liver, with a small The HPP (-/-) has absent TNSALP mRNA and TNsALP activity (all isoforms; bone, amount present in kidney and spleen 1 hr after intravenous administration. The in vivo liver, kidney). F.adiographsof lower limbs show skeletal disease in -/- mice by 10 days-of- was two of the a-Gal A glycoforms; one that was sialylated stability evaluated for and age with rachltic changes and failure of appearance of 20 ossification centers. Noted one that was non-sialylated. Ninety-six hours after administration, the amount of a-Gal absence ofperitrabecular fibrosis, osteoclastosis, or excessive plump osteoblasts is evidence remaining in the liver of each of these two glycoforms was 10% and 5% respectively. against underlying 2' hyperparathyroidism. Plasma and urine calcium levels are nonnal in relatively long stability is consistent with the being localized in lysosomes. --- - enzyme -i- +/- ants mice, also arguing against secondary causes for the rachitic disease-. effect of enzyme replacement on the accumulated substrate, an initial series Bone marrow cells (from +/+V's) were injected cells) intravenously into newborn intraveneous injections of 5X105units (nmol/hr) of the sialylated glycoform of c>- (I.Sx10' HPP mice after irradiation. Transplanted HPP mice were compared to administered at 48 hr intervals into each of four mice. Compared to untreated PBS-injected,

the GL-3 levels in littermates, plasma, liver, and heart were markedly decreased, while irradiated control and 1PP mice. Semi-quantitative ALPhistochemistsy of blood smears decrease was observed in only slight the kidney. However, a series of eight injections from transplanted HPP mice indicated the presence ofdonor leukocytes; we estimated -35% same dose at 48 hr intervals decreased the Ultra- levels of renal GL-3 by 30-50%. engrafiment. From 1toI 44 day-of-age, radiographic studies showed amelioration of the structural examination of liver revealed empty vacuoles indicating that the administered skeletal disease in all 13 transplanted HPP mice. enzyme gained access to and hydrolyzed the accumulated substrate. These precdinical We find that the TNSALPgene-knockout mouse appears to be a good model for infantile provide the rationale for enzyme replacement in patients with Fabry disease. HPP. Using the HPP mouse model,these preliminary observations provide evidence for the efficacy ofbone marrow transplantation for inbomnerrors of osteoblast function. A16 Slide Session 26: Inborn Errors of Mletabolism and Biochemical Defects (cont.) 73 74 Adenoviral and Retroviral Gene Therapy for Hereditary Tyrosinemia Type Human apolipoprotein E2, E3 and E4 transgenic mice: A model for Alzheimer's I Ken Overturf Muhsen Al-Dhalimy Milton Finegold Markus Grompe. Texas disease and other neurodegenerative disorders. -T. D. ] Children's Hospital, Houston, Texas Oregon Health Science University, Portland, Xu1, Schmehael', Oregon Rothrock', H.-L. Qi N. M1asda2e A-M. Saunders', A..BRoses' and LJR. Gilbert. 1. Hereditary Tyrosinemia Type I (HTI) is an autosomal recessive disease caused by de- Duke University Medical Center, Durham, North Carolina, 2. University ofNorth ficiency of fumarylacetoacetate hydrolase (FAH). Patients suffer from progressive liver Carolina at Chapel Hill. dysfunction, renal Fanconi syndrome and hepatocellular carcinomas. FAH deficient mice Apolipoprotein E4 (APOE4) (APOE4 gene; apoE protein) allele is associated with treated with the drug NTBC provide a suitable model for the study of HTI. In humans increased risk and earlier onset oflate-onset Alzheimers disease (AD) than the and mice transplanted wild-type hepatocytes are positively selected in FAH deficient APOE3 or APOE2 allele. To gain insight into the isoformn specific effects ofapoE on liver. We therefore used a first generation El deleted adenoviral vector to deliver the hu- brain pathogenesis, late-onset AD and other neurodegenerative disorders, we have man FAH gene into the livers of mutant animals. 5x109 infectious virions were injected into the tail vein or the portal vein of mutant animals. After 8 months, 12 surviving generated human apolipoprotein E2, E3 and E4 allele specific transgenic mice whose animals were harvested and examined. Two animals displayed normal appearing healthy endogenous APOE gene was inactivated. Three APOE2, five APOE3 and four livers while the remaining ten animals had varying degrees of hepatocellular cancer. FAH APOE4 transgenic lines were established which contain multiple copies (1-8) of staining of the livers from these animals revealed the presence of the enzyme in all ani- human APOE genomic DNA fragments with their associated regulatory regions. The mals with some livers displaying almost 80% FAH positivity. Liver functions tests were human APOE transgenes were expressed and transcribed appropriately in the brain, close to normal in some animals, but were elevated in mice with cancer. The pattern of liver, kidney, heart, spleen, and other tissues. Human apoE expressed in the FAH immunostaining was consistent with clonal expansion of single adenovirus trans- duced cells and suggests the possibility of stable integration of the vector in some cells. transgenic mice results in normalization of serum cholesterol in APOE "knockout" We conclude that adenoviral gene transfer can yield high level long-term expression in mice. Mouse brain sections fluorescein-stained with anti-apoE (Calbiochem) showed FAH- mice. However, tumors arising from remaining FAH deficient cells present a ma- apoE immuno-reactive neurons in cerebral cortex and hippocampus. Double-staining jor obstacle to adenoviral liver gene therapy. We have previously reported successful results showed that the apoE-stained neurons were co-stained by rhodamine-labeled retroviral gene transfer in this same mouse model. 5 corrected mice were followed > 10 Neurotag (Boehringer Mannheim), anti-NFL and anti-MAP2. ApoE immuno- months after gene therapy. All 5 retained normal liver function tests and renal function. reactivity has been detected in neuronal cytoplasma, dentrites and axon. In the Partial hepatectomy at 9 months demonstrated FAH negative tumors in 2/5 mice. At least 90% of all hepatocytes in other areas were FAH positive indicating sustained ex- transgenic hippocampal and cortical cultures, the apoE immuno-reactivity was also pression from the viral LTR. We conclude that hepatic malignancies are not completely detected in neuronal bodies, dentrites and axons. When the sections were stained prevented by retroviral gene transfer and that successful gene therapy will require the with anti-Alzheimer Precursor Protein A4 (APP) and anti-Alz 90 (Boehringer selective elimination of FAH negative hepatocytes. Mannheim), differences in APP immuno-staining patterns were found in APOE4 and E2 transgenic lines. These animals may serve as valuable models for the study ofthe effects ofapoE on AD and other neurodegenerative disorders.

75 Mucopolysaccharldosis and gangliosidosis In mice lacking both subunits of lysosomal 0-hexosaminidase. K. Sangol, M. L. Macki, CA nMfi6M,M McDonaul, J. N. Crawgy2, E Sko CMSar3, A. Hoffmann4&, Sandhoff4. K. Suzuki5 and R.oial. IGBB, NIDDK: 2ETB, NIMH; NIH, Bethesda, MD 20892; 3Glyko Inc., Novato, CA 94949; 41nstitut ffir Organische Chernie und Biochemie der Universitlit. Bonn. Germany; 5Dept. of Pathol., Univ. of North Carolina, Chapel Hill, NC 27599-7525 and'Dept. of Med. Genetics, Children's Natl. Med. Center, Washington, DC 20010. The GM2 gangliosidoses, Tay-Sachs and Sandhoff diseases. are caused by mutations in the HEXA (a-subunit) and HEXB (P-subunit) genes, respectively. Each gene encodes a subunit for the heterodimeric lysosomal enzyme, 3-hexosaminidase (P- hex) A, as well as for the homodimers 0B-hex B and S. Thus in Tay-Sachs disease, a deficiency of the a-subunit results in an absence of F-hex A (ap) and S (aa) and the presence of only B (Np). In Sandhoff disease, the lack of the p-subunit leads to a absence of P-hex A (ad) and B(PP) leaving only the residual activity of S (aa). Since GM2 ganglioside can be degraded only by the heterodimer P-hex A, the absence of either the a-subunit or the p-subunit results in a massive accumulation of GM2 ganglioside and related glycolipids. The lysosomal storage of these substrates in neurons ultimately causes severe dysfunction of the nervous system. Other organ systems do not appear to be seriously affected. We have now produced mice with both 5-hex genes disrupted that are totally deficient in all three forms of lysosomal P-hex (A, B and S). In addition to gangliosidosis, the totally F-hex deficient mice showed pathologic and biochemical features of a second class of lysosomal storage disease, mucopolysaccharidosis. The mice displayed facial and physical dysmorphia. Analysis of skeletons revealed dysostosis multiplex. The mice stored glycosaminoglycans in their cells and tissues and excreted large amounts of glycosaminoglycans in their urine. They exhibited severe movement difficulties, and had a shorter life span (1-4 months) than mice with a single disrupted 3-hex gene. The novel phenotype of the totally 3-hex deficient mice shows that glycosaminoglycans like gangliosides are critical substrates for n-hex and that their lack of storage in Tay-Sachs and Sandhoff diseases is due to functional redundancy in the P-hex system. Slide Session 27: Linkage Mapping and Polymorphisms 1: Mapping Mendelian Traits 76 77 Physical map of the Xq27 candidate region for X-linked Human Tenascin-X deficiency causes an Ehlers-Danlos-like phenotype. recessive in two kindreds Grant H. Burch. Yan Gong.Cynthia Currv. Walter L. Miller and James D Bristow, hypoparathyroidism merged by Universitv of California. San Francisco; San Francisco. CA. Intro. by: Charles Epstein mitochondrial DNA analysis. 1.2S, Mumm.2M.,p Why= 3.V Tenascin-X (TNX) belongs to the tenascin family of extracellular matrix proteins, and Thakker. and ID.Schlessinger. I Department of Molecular Microbiology, is encoded by a gene overlapping the opposite strand of the 2 1-hydroxylase B gene Washington University School of Medicine, St. Louis, MO USA; 2Metabolic (c2IB). A duplicate c2IA gene and partial duplicate TNX gene (XA) are tightly linked in Research Unit, Shriners Hospital for Crippled Children, St. Louis, MO USA; the class III region of the HLA locus. TNX and tenascin-C (TNC, cytotactin) are 3MRC Molecular Endocrinology Group, Hammersmith Hospital, London, UK. expressed in connective tissues of muscle, tendon, and skin. A 25 yo male with A rare form of hypoparathyroidism (X-linked recessive H.PT) has been reported congenital adrenal hyperplasia due to c2lB deficiency, and Ehlers-Danlos-like only in two Missouri families. We have used mitochondrial D-loop sequence phenotype (consisting of hyperextensible skin and joints, patellar chondromalacia and analysis to show identity in the maternal lineage from both kindreds, supporting a easy bruising) was referred for evaluation of a possible contiguous gene syndrome single founder. Based on new linkage mapping in the merged pedigrees, the involving c2lB and TNX. To investigate this hypothesis we obtained skin fibroblasts interval containing the gene has been narrowed between F9 and DXS 1205. We and DNA from the proband, his parents and seven normal controls. Western blotting of have constructed a YAC contig across the interval, which spans 2.5 Mb of Xq27 heparin-Sepharose concentrated fibroblast conditioned medium with anti-human TNX DNA. The contig is formatted with 22 STSs. We find that this region is relatively antiserum showed no TNX in samples from the proband (passage 3-12) and reduced gene-poor, according to its content of CpG islands, detected by rare-cutter amounts of TNX in conditioned medium from both parents compared to controls. restriction enzyme mapping, and its overall content of GC. Nevertheless, three of Blotting for TNC showed normal parental TNC production and increased TNC by the the STSs appear to represent transcribed sequences, and one EST and three known proband's fibroblasts. Southern blots showed reduced intensity of Taql and BglII genes (MCF.2, SOX3, and CDRI) fall within this region. It is notable that this genomic fragments representing the 3' end of TNX when compared to XA in the region has been reported to contain the genes for X-linked albinism-deafness and proband and his father. 30 kb deletions due to unequal crossover between duplicate c2 thoracoabdominal syndrome and is included in the current candidate region for at genes frequently occur in this locus. To evaluate whether a similar deletion occured in least two other syndromes. To infer additional potential candidate genes for these this family, we performed PCR experiments using an XB-specific sense primer paired diseases, bacterial artificial chromosome clones containing two of the small number either with an XB-specific antisense primer or an XA-specific primer normally located of CpG islands in the region have been subcloned for total sequencing. Finding 32 kb away from the sense primer. A normal XB product was found in all family the gene responsible for X-linked recessive HPT will help elucidate the members and controls, while a 2kb XA/XB product, indicating a 30 kb deletion, was embryogenesis of the parathyroid glands. found only in the proband and his father. This results in loss of c2IB and creates an TNX/XA hybrid causing early termination of TNX translation. The nature of the molecular lesion of the proband's 2nd TNX and c2IB alleles is unknown. HLA typing revealed crossover events between the class I and m regions on both of the proband's chromosomes 6, suggesting a de novo mutation. We conclude that the patient's Ehlers- Danlos phenotype is due to loss ofTNX and represents the first tenascin related disease. Slide Session 27: Linkage Mapping and Polymorphisms 1: Mapping Mendelian Traits (cont.) A17 78 79 A third Stickler syndrome locus is linked to COL1lAl, the gene encoding Triple A the al subunit of collagen XI. D. A. Sirko-Osadsal, J. Zlotogora2. G. E. syndrome maps to markers on human chromosome 12q13 near Tiller3, the type 11 keratin gene cluster. A. Weber'.T. R. G. Knowlton", and M4. L. Warman'. Department of Genetics, Case Western Reserve F. Wienker2. M. Jung2. University School of Medicine, Cleveland, OH1, Department of Human Genetics. The D. Easton3. H. J. Dean4. C. Heinrichs'. A. Reis2e and A. J. L. Clark7.'Children's Hebrew University, Jerusalem, Israel2, Department of Pediatrics, Vanderbilt University Hospital, Technical University Dresden, Germany, 2Mikrosatellitenzentrum, School of Medicine, Nashville, TN3, Department of Biochemistry and Molecular Max Delbruck Center Berlin, Germany, Department of Community Medicine, Biology, Thomas Jefferson University, Philadelphia, PA4. University of Cambridge, UK, 4Department of Pediatrics, University of Stickler syndrome, hereditary arthro-opthalmopathy, is an autosomal dominant disor- Manitoba, Winnipeg, Canada, 5Children's Hospital Queen Fabiola, Free der with a broad range of phenotypic manifestations both within, and among, families. University of Brussels, Belgium, 'Institute of Human Genetics, Humboldt Clinical features can include vitreoretinal changes, sensorineural deafness, arthropathy, University Berlin, Germany, Department of Chemical Endocrinology, St cleft palate, micrognathia and mid-face hypoplasia. Stickler syndrome exhibits locus het- Bartholomew's Hospital, London, UK. erogeneity; mutations in COL2A1 and COLIlA2, the genes encoding two of the three The triple A or subunits of collagen XI, have been identified in affected families. We have identified and Allgrove's syndrome (MIM*231550) is a rare autosomal used intragenic and tightly linked markers to COLliA1, the gene encoding the third recessive disorder characterized by the triad of adrenocorticotropic hormone subunit of collagen XI, to show that this locus is also linked to the Stickler syndrome (ACTH) resistant adrenal insufficiency, achalasia and alacrima.The syndrome phenotype. Three CA-repeat polymorphisms (7B1, DlS495 and D1S248) were used to is frequently associated with progressive neurological degeneration presenting obtain a lod score of 3.6 at zero recombination in a large family for whom linkage to as a mixed pattern of upper and lower motor neuropathy, sensory impairment, COLllA2 and COL2A1 were excluded. Data from this family, coupled with data from autonomic dysfunction and mental retardation resulting in a severely disabling several smaller families, suggest that COLliA1 is a third Stickler syndrome locus. The disease. Additional features include hyperkeratosis of palms and soles, optic polypeptide subunits of collagen XI also participate in the formation of other collagen nerve atrophy and nerve deafness. After excluding the genes for ACTH molecules. In addition, the genes encoding collagen XI are subject to tissue dependent receptor, vasoactive intestinal alternative mRNA splicing. Comparison of similarities and differences among families peptide (VIP), VIP-1 receptor, pituitary linked to each Stickler syndrome locus can now be used to explore the bases of interfa- adenylate cyclase activating peptide (PACAP) and neurotrophin-3 as milial variability as well as pleiotropy. candidates for the triple A syndrome we have performed a systematic linkage scan in 8 families with this condition including the large highly inbred kindred described by Moore et al 1991. We obtained conclusive evidence for linkage of the triple A syndrome locus to markers on chromosome 12q13 (D12S368, 8=0, Z,,.=10.81). Multipoint and haplotype analyses revealed obligatory recombination events allowing us to refine the interval of the triple A gene to a genetic segment of 4 cM between D12S368 and D12S1586. This region harbors the type 11 keratin gene cluster, and potential candidate genes include Scn8a and HOXC5.

80 81 Paroxysma dystonic-chorcoathetowis: tight linkage to chronmosome 2q L.iSlk. X1 Autosomal dominant Pallister-Hall syndrome maps to 7p13. _ ~ ~~~~~at R. _hnTz" sikiTrein,"I~ssN-aad.TY i /B. Otteru ;j2e- S Kanga, J M Graham Jr2, M Abbott3, A Schaffer', ED-Green', -M oy _nv Mihgn An Aror pt- HumnG ts-niv:. Utah. Rosenberg', J Allen4, C Clericuzio5, T Grebe", A Haskins-Olney', La Bieseckerl 1 National Center for Human Genome Research, NIH, Paroxysmal dystonic chorcoathetosis is characterized by attacks of involuntary movements that last up to several hours and occur at rest both spontaneously and Bethesda, MD, 2 Division of Genetics, Cedars-Sinai Medical Center & following caffeine or alcohol consumption. We analyzed a Polisb-American kindred UCLA, Los Angeles, CA, 3 Department of Psychiatry, Johns Hopkins with autosomal dominant PDC. After a thorough genome wide search, genetic linkage University, Baltimore, MD, 4 NYU Medical Center, NY, NY, 5 Univ of New was established to a set of contiguous genetic markers on chromosome 2q33-35. The Mexico School of Medicine, Albuquerque, NM, 6 Univ of Arizona Medical maximum pairwise lod score obtained is this family, under the assumptions of 0.90 School, 7 Univ of Nebraska Medical Center. penetrance and an autosomal dominant ene, was 4.77 at 9=0 for the marker D2S 173. An additional three markers, D2S434, ?2S377. and D2S339 also yielded lod scores greater than 3.0. Previous physical ma ping of microsatelite markers allows us to Pallister-Hall syndrome (PHS) is a rare disorder characterized by assign the PDC locus to chromosome 2q33-35. Presently, our data localizes this polydactyly, hypothalamic hamartoma, and occasional craniofacial and region to a 15 cM interval, between the flanking markers D2S164 and D2S159. Our visceral anomalies. The original reports were all perinatal lethal sporadic results clearly establish the existence of a locus for autosomal dominant PDC on distal cases. Autosomal dominant inheritance of this chromosome 2q, The fact that 3 other paroxysmal neurologic disorders (periodic syndrome has been ataxia with myokymia, hypo- and hyperkalemic periodic paralysis) are due to ion associated with a less severe phenotype in several families. After channel gene mutations raises the possibility that PDC is also due to an ion channel conducting a genome-wide search with highly polymorphic markers in four gene mutation. It is noteworthy that a cluster of sodium channel genes is located on affected families including 32 affected and 36 unaffected persons, we found distal chr. 2q near the PDC locus. linkage initially to D7S672. Physical mapping data from this region resulted Our patients differ in two important respects from autosomal dominant paroxysmal in refinement of marker order in this choreoathetosis/spasticity (CSE recently mapped (Auburger et al. 1996) to a 2 cM region. Genotyping of additional region on chromosome lp. First, episodic dyskinesia in our patients occurs markers in this region resulted in a maximal two point LOD score between spontaneously at rest; physical exercise was a precipitating factor for CSE patients. PHS and D7S691 of 8.04 at theta=0. Multipoint analysis shows that the Second, none of ourpatients had spasticity; five of 8 CSE patients had constant phenotype maps between D7S521 and D7S678 with a peak LOD score of spastic paraplegia. We consider the disorder in ourkindred to be "pure PDC" in so far 13.6 at D7S691. Haplotype and physical analyses demonstrate a candidate as paroxysmal extrapyramidal disturbance is not accompanied by ataxia or other neurologic impairments and patients are neurologically nomsal and asymptomatc region of approximately 2 Mb. This region contains two interesting between attacks. In this circumstance, it seems appropriate to consider autosomal candidate genes, GLI3 and inhibinlA. GLI3 is the gene implicated in Grieg dominant PDC a clinically heterogeneous group ofdisorders with i identified for cephalopoly-syndactly syndrome based on three affected individuals with "pure PDC" (chromosome 2q33-35) and "PDC/spastiiy" (also known as CSE; translocation breakpoints in that gene. InhibinBA is a good candidate chromosome Ip). This clinico-genetic classification undoubtedly will be modified as because it is expressed in the hypothalamus. Mutational analysis of these loci are discovered for additional paroxysmal extrapyramidal syndromes. candidate genes is underway.

82 83 Localization of the gene for autosomal dominant The gene for Schnyder's crystalline corneal dystrophy maps to human Iridogoniodysgenesis to 6p25. A.J.Mears. F. Mir~ayans. chromosome lp34.1-p36. A.M. Shearrnanl, T.l. Hudson2, I. M. Andrsenl, X,.WU2, W.G. Pearce and M.A. Walter. Department of Ophthalmology, R.L. Sohnl, F. Haluskal3, D.E. Housmanl and IS. Weiss4'5. lCenter for Cancer Research, University of Alberta, Edmonton, Alberta, Canada. Massachusetts Institute of Technology, 2Center for Genome Research, Whitehead Autosomal dominant iridogoniodysgenesis (IGD) is Institute, Massachusetts Institute of Technology, 3Department of Hematology/ Oncology, characterized by iris hypoplasia and juvenile glaucoma. lGD is Massachusetts General Hospital, 4Department of Ophthalmology, University of the result of aberrant migration or terminal induction of the Massachusetts Medical Center, 5Kresge Eye Institute, Wayne State University School of neural crest cells involved in the formation of the anterior Medicine, Michigan. chamber of the eye. Gonioscopy of affected individuals typically Schnyder's crystalline corneal dystrophy (SCCD) is an autosomal dominant eye reveals excess tissue in the iridocorneal angle which can disease characterised by a bilateral clouding of the central cornea, arcus lipoides increase resistance to aqueous outflow, leading to glaucoma. and/or visible crystalline deposits of cholesterol in the stroma. Accumulation of After phospholipid, unesterified cholesterol and cholesterol ester in the stroma is thought eliminating candidate regions for other ocular disorders, a to occur as a result of an imbalance in the local factors affecting Upid/cholesterol genome-wide scan using linkage analysis was performed on an transport or metabolism. To determine the chromosomal location of the SCCD eight-generation lGD kindred, originating from the Maritime locus, genome-wide linkage analysis has been performed in two large kindreds from region of Canada. Approximately 95% of the genome was central Massachusetts; both kindreds are of Swede-Finn descent and trace their excluded with >300 microsatellite markers before significant ancestry to the same region of the southwest coast of Finland. Analysis of 219 linkage was demonstrated between IGD and chromosome 6 microsatellite markers excluded over 90% of the genome. We here identify the markers. From chromosomal assignment of the gene for SCCD in both families to be lp34.1-p36; haplotype analysis and identification of for one pedigree zmax=3.17 at 0=0 using DlS489, for the other pedigree recombinants, the lGD locus is mapped to an 8.3 cM region zmax=3.93 at 0=0 using D1S214. Using data from both pedigrees the maximum distal of D6S477, at 6p25. Our linkage results are consistent multipoint lod-score was 8.04 in the 7 cM interval between DlS214 and DlS503. with the ocular findings in rare cases of individuals with From haplotype analysis, the SCCD locus lies in the 16 cM interval between chromosomal anomalies involving 6p. Fine mapping of the lGD DlS214 and D1S228. Several candidate genes for SCCD have been localised to the critical region with additional families and identification of 1p34.1-p36 region. Two candidate genes, cytidine 5'-triphosphate synthetase candidate genes for lGD are (CTPS) and sterol carrier protein(SCP2) , previously mapped to this region of underway. chromosome 1, have now been excluded from the SCCD interval by radiation hybrid mapping. A18 Slide Session 27: Linkage Mapping and Polymorphisms 1: Mapping Mendelian Traits (cont.) 84 85 Homozygosity maping of the Chediak-Higashi syndrome gene to Homozygosity mapping of Hallervorden-Spatz disease to chromosome 20pl2.3- chromosome lq42-q44 in a segment of conserved synteny that includes the pl3. T. D. Taylor, P. Kramer M. Litt and S. J. Hayflick. Oregon Health Sciences mouse beige (bg) locus. J. Ohl, K. Fukail, K.J. Mloore2, H.H. Kandil3, H. Ito4, and R.A. Spritz'. 'Dept. of Medical Genetics, Univ. of Wisconsin, Madison, WI; University, Portland, Oregon. MNillennium Pharmaceuticals, Inc., Cambridge, MA, 3Pediatric Dept., Al-Jalra Hallervorden-Spatz disease (HS, OMIM #234200) is a rare, autosomal recessive Hospital, Kuwait: and 4First Dept. of Pathology, Kobe Univ. School of Medicine, disorder of brain iron accumulation. It is characterized by progressive rigidity, Japan. dystonia, retinitis pigmentosa and basal ganglia densities on MRI. The objective of (CHS) is an autosomal recessive disorder characterized by hypopigmentation or oculocu- our study was to determine the chromosomal location ofthe HS gene. taneous albinism and severe immunologic deficiency with neutropenia and lack of natural Samples were obtained from 10 families, one each of Amish, New Zealand and killer (NK) cell function. Most patients die in childhood from pyogenic infections or an Australian descent, two Spanish, and five Italian. Highly polymorphic microsatellite unusual lymphoma-like condition. A hallmark of the disorder is giant inclusion bodies seen in all granule-containing cells, including granulocytes, lymphocytes, melanocytes, markers were first run near candidate genes, and these regions were excluded. A mast cells, and neurons. Similar ultrastructural abnormalites occur in the beige (bg ) primary genome scan for the HS gene was performed using samples from the large, mouse, which has thus been suggested to be homologous to human CHS. High-resolution consanguineous, multi-generation Amish family. Two-point linkage analysis was run genetic mapping indicated that the bg gene region of mouse chromosome 13 is likely ho- using MLINK and haplotypes were constructed for all markers. mologous to the distal portion of human chromosome lq. Accordingly, we carried out Homozygosity mapping of genes in families from isolated populations is a powerful homozygosity mapping using markers derived from distal human chromosome lq in three inbred patients with CHS. Our results indicate that the human CHS gene maps to a 18.8- method for determining linkage. However, in the case ofthe large Amish family we centiMorgan interval in chromosome segment lq42-q44, and that human CHS therefore found that the lod scores dropped dramatically as we moved away from the gene is very likely homologous to mouse 6g The mouse bg gene has recently been cloned, region. Multiple inbreeding loops and untyped individuals in this family hindered and we are currently carrying out analysis of the homologous human gene to identify linkage analysis. Simulations studies determined that only three out of nine mutations in patients with CHS. inbreeding loops needed to be included to get reliable lod scores. Additional loops had no significant effect on the lod scores, but increased computation time drastically. Linkage was demonstrated between the Amish family and genetic markers on chromosome 20pl2.3-pl3. Analysis ofthe other nine fanilies supported linkage of the HS disease gene to this region with a total maximum two-point lod score of 13.75 at 0=0 for the marker AFMaO49ydl. Homozygosity in the Amish family and recombinant haplotypes in three of the other families suggest that the HS gene is located in a 4 cM interval between markers D20S906 and D20S I 16.

86 Familial Hypobetalipoproteinemia (FHBL): linkage to chromosome 2p and evidence for genetic heterogeneity. R. J. Neuman, J. Pulai, G. Schonfeld. Washington Univ. School of Medicine, St. Louis, MO. FHBL is a genetic disorder of lipid metabolism characterized by extremely low levels of apolipoprotein B (apoB). FHBL is purported to have an autosomal dominant form of inheritance and to affect less than 1/2% of the general population. Two families. family D and F, initially screened for low cholesterol levels were selected for genetic analysis be- tween the FHBL phenotype and the apoB gene using both internal and flanking markers for the apoB gene. Initially, individuals with abnormally low apoB levels (< 40 mg/dl) were considered affected with FHBL. Under the assumption of an autosomal dominant model of disease with no sporadics, positive linkage of FHBL to markers in the 5' region of the apoB gene was established in family D with a maximum lod score of 4.9 (6 = 0.0) and in the 3' region with a lod score of slightly over 4 (8 = 0.05). Lod scores were also computed using residual levels of apoB (corrected for age, sex, and BNMI) as the pheno- type. Linkage was confirmed for this quantitative phenotype with lod scores of around 4 for family D in both regions. Linkage was excluded at the VNTR site of the 3' region for family F with a lod score of almost -22 for the dichotomous phenotype and less than -2 for the quantitative phenotype. Several studies had found various truncated forms of apoB cosegregating with the FHBL phenotype; accordingly attention was focused on sequencing the coding and regulatory regions of the apoB gene for family D. However. no truncation producing defect or other mutation cosegregating with FHBL has been found. Thus, it is likely that a mutation heretofore undescribed for apoB may shed light on the regulation of synthesis of apoB. These two kindreds illustrate the genetic heterogeneity of FHBL since linkage with apoB has been ruled out for family F. This latter family offers the possibility of finding new low cholesterol specifying genes. A20 Slide Session 33: Clinical Genetics, Malformations and Dysmorphology 1: Syndrome Delineation 87 88 The expansive phenotype of velo-cardio-facial syndrome: a review of 206 cases. Velocardiofacial syndrome in the newborn and infant. R.]. Hopkin. E.K. Schorry. R.I Shprintzen. A. Shanske. R. Marion. R. Goldberg. Monteliore Medical Ctr. Bronx, NY. M. Bofinger. H.M. Saal. Division of Human Genetics, Children's Hospital Research The purpose ofthis paper is to update the phenotype of velo-cardio-facial syndrome with Foundation. Cincinnati OH. many features which have not been published, including joint dislocations, spontaneous Velocardiofacial syndrome (VCFS) is a condition of multiple anomalies associated oxygen desaturation, asymmetric crying facies, chronic constipation, Sprengel shoulder. with a microdeletion of chromosome 22q1 1.2. In spite of recent interest in the clinical thrombocytopenia, and hypertrophic arytenoids leading to laryngomalacia. Exhaustive study phenotype and molecular genetics of this condition there has been little written on the of 206 patients with VCFS at a single center has resulted in the continued delineation of the presentation and management in infancy. phenotype. VCFS has one of the most expansive and varied phenotypes of genetic We report a series of 12 infants diagnosed between the ages of birth and 9 months. disorders with over 60 delineated features involving all physical systems and behavioral Diagnosis was confirmed by FISH analysis in all cases. Seven patients had congenital functions. Many VCFS anomalies are not easily found on physical examination and therefore heart disease (CHD), I had cleft palate, 1 had recognized submucous cleft palate, 1 require specialized tests which can only be implemented if the phenotypic spectrum is patient had hypocalcemia; however, 4 patients had none of these findings. Facial known by the clinician. For example, the palatal and pharyngeal anomalies in VCFS can not dysmorphia were present in all patients. The features were distinct and recognizable, be detected without nasopharyngoscopy and magnetic resonance angiography. Using a full but differ from those in older patients. Nine patients had feeding problems requiring battery oftests in 206 consecutive patients, anomalies were found in the following physical intervention; five had apnea with cyanosis only one of whom had CHD; eight required systems: musculo-skeletal, CNS, peripheral nervous, GU, GI, cardiovascular, peripheral multiple hospitalizations. Two patients died as infants. Feeding problems and re- vascular, cerebro-vascular, respiratory, integumentary, renal, endocrine, ocular, auditory, hospitalization were equally frequent in patients with and without CHD. immune, and hematologic. Behavioral disorders were also common including speech We conclude that VCFS is recognizable at birth, but many cases will be missed if disorders, language impairment, voice/resonance disorders, feeding disorders, conductive the diagnosis is considered only with major malformations. Many patients have and sensori-neural hearing loss, learning disability, psychosis, mood disorders, temperament important medical needs not related to CHD or other major malformations. Early disorders, and ADHD. Several sequences occur secondary to the primary genetic cause of diagnosis can facilitate anticipatory guidance and management of these infants. VCFS, including Robin sequence (1 7'/s of VCFS cases) and DiGeorge sequence (9% of VCFS cases). The most common anomalies in VCFS are the behavioral manifestations and noncardiac vascular anomalies. Cardiac anomalies and cleft palate occur with frequencies of 78% and 85% respectively. Because there is phenotypic overlap between VCFS and a number of other syndromes, including Opitz, fetal alcohol, CHARGE, and Noonan syndromes, the accurate delineation of the full phenotype becomes important in making the fine distnctions between disorders which at initial observation may be confused with VCFS.

89 90 Psychiatric illness in children and adults with velo-cardio-facial syndrome The 22qI1.2 deletion in African-American patients: ? an underdiugnosed with/without 22q11 deletions. B. Morrowl, C. G. Carlson, I, R. Goldberg'5, population. D.M. McDonald-McGinn. D.A. Driscoll, B.S. Emanuel. E.H. Zackai. The Children's R. G. S. H. Hospital of Philadelphia. Philadelphia. PA. Shprintzen 2, Faedda', Veit', Lachmanl, Clinical characteristics associated with the 22qI 1.2 deletion may include the following: cardiac R. Kucherlapati' and D. Papolos'. ', Albert Einstein College of Medicine, Bronx, NY, anomalies, cleft palate/VPI. hypocalcemia, hypoplastic or absent thymus with T cell deficiency. a 25Montefiore Medical Center, Bronx, NY learning disability, and a characteristic facial appearance. Often the clinical suspicion of the diagnosis Velo-cardio-facial syndrome (VCFS) is characterized by conotruncal cardiac defects, in a patient with one or more of these findings is heightened based on the presence of the typical facies. cleft palate, learning disabilities and minor facial dysmorphology and is associated with The facial characteristics have been described as a prominent nose with a squared root and hypoplastic deletions. A clinical feature of VCFS is the of alae nasi (75%). a thin upper lip. small auricles with thick helices (60%). malar flatness (70%), vertical 22qll newly recognized presence psychi- maxillary excess (85%), and micrognathia. Our experience has been that there is a paucity of these atric illness in adult VCFS patients. Clinical evidence has begun to accumulate which findings amongst our African-American patients. This prompted us to review. in detail, the facial suggests that psychiatric disorders in VCFS patients may begin during childhood. To characteristics of our African-American patients with a 22q1 1.2 deletion. Eighteen of our 88 patients assess this possibility, we have evaluated 26 VCFS patients that range in age from 5 (20%) are African-American, 10 female and 8 male. Four of the 18 patients are adults, 3 were - 32 yrs, using a structured clinical interview (SCID; DICA-R) to establish a DSM-IV ascertained through an affected child, and I was previously diagnosed in childhood with velocardiofacial consensus diagnosis. The VCFS children and adolescents were found to share a common syndrome through the Cleft Palate Clinic. The remaining 14 patients are children; I was the offspring set of psychiatric disorders including ADHD and bipolar disorder. The adult patients of the parent with velocardiofacial syndrome. 5 came to attention in infancy because of their cardiac were more severely affected with schizoaffective-manic and bipolar I disorder, some with defects and dysmorphic features. 4 were evaluated solely because of cardiac anomalies, 3 carried the psychotic symptoms. To define the deletions in each patient and to correlate the psv- clinical diagnosis of DiGeorge anomalad. and I was referred because of a learning disability and chiatric phenotype with the extent of the deletion, we utilized 13 ordered genetic STRP hypocalcenia at age 9 years. Review of the clinical genetics evaluation performed in these patients We a between the prior to the diagnosis of a 22q1 1.2 deletion (with the exception of one cardiac patient diagnosed markers in 22qll for haplotype analysis. did not find correlation prenatally) revealed that:- 10/18 (55%) patients had none of the typical facial characteristics; and only size of the deletion and the presence of physical anomalies and/or major psychiatric 2/18 (11%) had four characteristics. each having a bulbous nasal tip. malar hypoplasia and a long face. illness in VCFS patients. Bipolar spectrum disorders occurred in 2 non-deleted VCFS with I having a prominent nasal root. and the other having small ears. The most common feature patients as determined by haplotype analysis and FISH. The remarkably high incidence amongst our African-American patients was a bulbous nasal tip in 4/18 (22%); the findings of of psychiatric disorders and the congenital anomalies of VCFS and its occurrence among prominent nasal root, a long face. malar hypoplasia, and thick helices were the next most common non-deleted VCFS patients suggests a common genetic pathway for the physical as well features each being present three times (16%) in this population. Features other than those typically as the psychiatric findings. One candidate gene present within 22q11 that may modify found in patients with a 22qll.2 deletion were reviewed. 7 patients (39%) were noted to have the susceptibility to psychiatric illness in VCFS patients is the gene for catechol O-methyl hypertelorism, 4 (22%) patients had excess nuchal skin in infancy (I without a heart defect). 3 (16%) transferase (COMT). A polymorphism in the COMT gene that leads to a variation in had protuberant ears. and 2 (11%) had ptosis. Thus, there is a paucity of typical facial characteristics in enzymatic activity was identified. We found an association between the low-activity our cohort of African American patients with a 22q1 1.2 deletion. This underscores the need for allele of COMT and the development of rapid cycling bipolar spectrum disorders. microdeletion studies in African-American patients who have findings other than those related to their facies. In fact. we propose that this may be an underdiagnosed population of patients based on the fact that only 16% of our 22q1 1.2 deletion patients are African-American as compared to the 42% of African-Americans in our hospital's patient population.

91 92 A genetic etiology for interrupted sortie arch type B. M. LewinL2.,_E CHARGE association: report of 47 cases with genotypic analysis of Linsy1aJ2lA.ldiniI. (1) Dept of Molecular and Human chromosomes 7q36 and 22qll AL.Tellier. D.Bonnet. V.Cormier. Genetics, (2) Division of Pediatric Cardiology, Baylor College of Medicine, One D.Thiophile. Baylor Plaza, Houston, Texas. V.Abadie. P.Scambler*. M.Vekemans. A. Munnich and S. Lvonnet. Department of 22q1 1 deletions are associated with DiGeorge (DGS), Velocardiofacial (VCFS) Genetics INSERM U-393, Hopital des Enfants-Malades, 75743 Paris, France; *Institute syndromes, and conotruncal heart defects; about 80% of deleted patients have cardiac anomalies. We sought to: l)Identify specific congenital heart defects (CHD) most of Child Health, London, UK. frequently associated with 22q1 1 deletion, and 2) study non-deleted patients with the The acronym CHARGE refers to a non-random clustering of congenital identified CHD to understand whether the etiology of the cardiac anomaly in these malformations. We report a series of 47 CHARGE patients studied for the frequency of patients related to the chr. 22 locus. FISH was performed on cell lines from patients with CHD. Some patients were suspected of having DGS or VCFS based upon the major malformations namely retinal coloboma (79%), heart malformation (85%), associated phenotypic abnormalities (i.e. hypocalcemia, immune disorders, or facial choanal atresia (57%), growth (75%) and/or mental retardation (100%), genital anomalies anomalies), while others were referred solely based upon the presence of a conotruncal or aortic arch defect. Of the first 50 patients studied, 4 of the 5 deleted patients had (34%), ear anomalies (91%) and/or deafness (62%). In addition, we report on prenatal Interrupted Aertic Arch Type B (IAA-B). Therefore, we tested an additional 23 patients echographic anomalies and other non-classical features in neonates and infants. These with LAA-B and found 12 to be deleted (52%). Truncus Arteriosus (TA) is also a defect often associated with deletions. Interestingly, the first 10 TA patients recruited had no were: facial dysmorphy, brainstem dysfunction with cranial nerve palsies, and labyrinth deletion, this is clearly a small patient group, but if confirmed, the finding suggests that malformations. We propose the following criteria for poor life prognosis (p<0.05) i) the etiology of TA is more heterogeneous than that of IAA-B. We analyzed the 11 IAA- B non-deleted patients by FISH (using probes throughout the commonly deleted male patients ii) CNS and/or oesophageal malformations iii) bilateral choanal atresia and region) and Southern blot using probes representative of the genomic region containing iv) conotruncal heart defect. However, no predictive factor regarding developmental two transcipts disrupted in a balanced translocation patient (ADU) with severe coarctation of the aorta and DGS/VCFS phenotype. To date, no deletions or prognosis could be identified. The family history was not informative but we found a rearrangements have been detected by FISH (5 loci tested), or Southern analysis (3 significant increase in the mean paternal age at the time of conception suggesting the probes tested). Interestingly, we incidentally found that the genomic region tested by role of a de novo dominant mutation or a undetectable chromosomal Southern is normally duplicated within 22ql 1.2. The significance of this finding is possible presently being investigated. In conclusion, this is the largest case study to evaluate deletions or abnormality in the CHARGE association. Cytogenetic analyses were negative in all otherrearrangements at the DGS/VCFS lous in IAA-B. Ourresults show that 1) LAA- patients tested, including FISH with 22ql 1 and 7q36 cosmids. Finally, the combination B is the single clinical sign which most predictably correlates with a 22ql 1 deletion; 2) IAA-B is the CHD most frequently associated with a specific conosomal mutation. of these malformations strongly supports a polytopic developmental field defect involving neural crest derived cells and favors the view that the CHARGE association could be regarded asu syndrome. Slide Session 33: Clinical Genetics, Malformations and Dysmorphology 1: Syndrome Delineation A21 93 94 Lessons from the Lissencephaly Research Project: five new malformation Lissencephaly syndromes: Does the face reflect the brain? syndromes. W.B. Dobyns. Departments of Neurology and Pediatrics, University of .LF.Allansonn, D1 .Ledbettereand-W-B.Dobyn Children's Hospital of Eastern Minnesota, Minneapolis, MN. Ontario and University of Ottawa, Ottawa, Ontario, Canada; Center for From its inception in 1982, the lissencephaly research project has ascertained over Medical Genetics, University of Chicago, Chicago, IL; University of Minnesota, 600 children with lissencephaly or related malformations of cortical development such Minneapolis, MN. as polymicrogyria and diffuse or focal heterotopia. Previous studies delineating a sin- gle 'lissencephaly syndrome" have progressed to recognition of over 30 such syndromes. Lissencephaly is a brain malformation characterised by a smooth cerebral The best known of these are Miller-Dieker svndrome and isolated lissencephaly sequence surface, the spectrum of which includes agyria, mixed agyria/pachygyria, and (Allanson et al., Chong et al., Follin et al., ASHG96), X-linked lissencephaly and sub- pachygyria. Type lissencephaly, which results from arrest of neuronal cortical band heterotopia (Srivastava et al., ASHG96), bilateral periventricular nodular migration at about 10 to 14 weeks gestation, is found in Miller-Dieker heterotopia, Walker-Warburg syndrome (muscle-eye-brain disease), and Fukuvama con- syndrome IMOSI and isolated lissencephaly sequence ILS), both of which are genital muscular dystrophy. From this large data set, I report five new and apparently rare MCA/MR malformation syndromes, three of which were first delineated by this associated with deletions and mutations in the gene on 17p. In project. (1) The cobblestone lissencephaly only syndrome consists of the same brain lissencephaly is associated with a particular facialLISaappearance, consistingMoS,of malformation observed in Walker-Warburg syndrome, but without abnormalities of the a tall, prominent forehead, which may show vertical furrowing; bitemporal eyes or muscle. Inheritance is autosomal recessive, but it remains unmapped. (2) The narrowing; wide-spaced eyes; markedly short nose with low bridge, BPNH/MR syndrome consists of bilateral periventricular nodular heterotopia, cortical anteverted nares and flared, thin alae; long and thick upper lip with rounded dysplasia, cerebellar hypoplasia, severe mental retardation, epilepsy and syndactyly. In- philtral pillars and thin, inverted vermillion border; flat midface; small chin; and heritance is X-linked, and linkage to Xq28 appears likely (Fink et al., ASHG96). (3) ' ' Ramer syndrome consists of lissencephaly and striking facial dysmorphism including flattened ear helices. In contrast, no typical gestalt hhas been defined in ILS. trigonocephaly, shallow orbits, ptosis and colobomas. It may be causally heterogenous We have evaluated 5 individuals with MDS (3 months to 6 years) and 25 as the brain malformation has been variable. (4) Winter-Tsukuhara syndrome consists with ILS (1 to 15 years) by clinical examination and craniofacial measurement. of lissencephaly with intermediate increased cortical thickness, facial dysmorphism espe- Z-score pattern profile analysis reveals craniofacial similarities between these cially hypertelorism, heart malformations, distal joint contractures, and early death. (5) two conditions which have not been appreciated previously. Microcephaly is The lissencephaly and ambiguous genitalia syndrome consists of lissencephaly with inter- mediate increased cortical thickness, ambiguous genitalia in genotypic males, neonatal c common in both, with brachycephaly in MDS. Craniofacial widths are close onset seizures and temperature instability. While presumed to be genetic, the basis of to, or may exceed, normal, while depths and heights are reduced. Nasal and the last three syndromes is not known. mouth widths and inner canthal distances are above average, particularly in MDS. Maxillary dimensions are closer to normal than their mandibular counterparts. In MDS, nasal height is markedly reduced. Additional molecular data may explain the craniofacial and cerebral similarities and differences.

95 96 Differences in age at of with Johanson-Blizzard syndrome maps to chromosome 17p13.3 based on diagnosis patients Prader-Willi syndrome due to observation of a child with der(17) and both Johansson-Blizzard and uniparental disomy 15 and deletion 15q. Gunay-Aygun M.. Hccneer S.. Schwartz S. and Miller-Dieker syndromes. A.C.M. Smithi, D.H. Ledbetter2. M. Mlacha2. Cassidy S.B. Center for Human Genetics. Department of Gcnetics. Case Wcstcrn Reserve J.C. Mvlurray3, W.B. Dobyns4. Medical Genetics Branch, NCHGR, NIH, Bethesda, University School of Medicine and University Hospitals of Cleveland. OH MD; 2Center for Medical Genetics. Univ. of Chicago. Chicago, IL: 3Dept. of Pediatrics. Prader-Willi Syndrome (PWS) results from absence ofnormally active patcrnally inherited Univ. of Iowa, Iowa City, IA; and of Univ. of Minneapolis, MIN. 4Dept. Neurology, Minnesota, genes on proximal l5q, due to del( l5)(q lq13)pat or by maternal uniparental disomv 15 (UPD). Minor phenotypic differences between deletioni DEL) and UPD patients have been Johansson-Blizzard syndrome (JBS) is a multiple congenital anomaly syndrome mani- fested by severe growth deficiency. ectodermal dysplasia with midline scalp defects, char- reported including lower birth weight in the DEL group, shorter birth length in males with acteristic facial appearance including hypoplasia of the nasal UPD and shorter course of gavage feeding and later onset of hyperphagia in females with severe alae, sensorineural hearing loss, pancreatic insufficiency, and other abnormalities. It is inherited as an au- UPD. Cassidy et al. found that those with UPD had less "typicar' facial appearance, and tosomal recessive trait based on reports of eight families with multiple affected children they less often had skin picking, skill with puzzles and high pain threshold. Those with UPD of both sexes and normal parents, and five families in which the parents were related were older than those with DEL on average, suggesting delay in diagnosis of this group. (Gershoni-Baruch et al., 1990). The gene responsible for JBS has not been mapped. We We sought to investigate this possibility. report a child who has a constellation of complex congenital anomalies consistent with Charts of both JBS and Miller-Dieker syndrome (MDS). Chromosome analysis and fluorescence in all patients followed in our PWS clinics were reviewed. Sixty of 158 patients situ hybridization (FISH) demonstrated a deletion of which included had complete molecular testing (41 DEL., 19 UPD). Differences were as follows: L132 and 17p13.3 probes 71, (D17S379) 8-1from the lissencephaly and MDS critical regions (Chong etal., DEL UPQ .p ASHG96). Her mother was found to be a carrier of a balanced 16p;17p translocation. Mean age at diagnosis(yr) 3.76 9.29 0.043 The proband inherited the der(17) not the from her mother, leaving her with Mean age at diagnosis(yr) (males) 1.69-p=0.016 8.64 ns 0.076 an unbalanced but der(16) karyotype designated 46,XX,-17,+der(17), t(16;17)(p13.3;p13.3)mat. The (females) 6.40/ 10.017 ns most likely mechanism of co-occurrence of these two syndromes is inheritance of a mu- Birth weight (by) 40.39 22.89 0.025 tation of her paternal JBS gene which was uncovered by deletion of the maternal JBS gene. This mechanism requires that the JBS gene be located within the relatively large Birth length (bl) 68.20 37.73 0.006 deleted segment of chromosome 17p13.3 or near the site of the translocation breakpoint Delay in the diagnosis of patients with UPD might be explained by lower frequency of on chromosome 16pl3.3. Because of its much greater size and gene content, the former typical features. Males with DEL were found to be the earliest diagnosed, suggesting that is more likely. This mechanism has been observed in other autosomal recessive disorders males with DEL may have more hypoplastic genitalia than other groups. The such as metachromatic possible leukodystrophy (Coulter-Mackie etal., 1995). reasons for these phenotypic differences include incompleteness of imprinting effects and absence ofnon-imprinted genes contributing to the phenotype in deletion patients. Delay in diagnosis may influence the natural history of the disorder, and may be prevented by lowering the threshold ofclinical suspicion ofPWS.

97 African-Americans with Prader-Willi syndrome are phenotypically different. S.B.Cassidv. J.S.Geer. L.Hudzins. Case Western Reservc UniversitvLnivcrsitv Hospitals of Cleveland, OH. Greenwood Genetics Center, Greenwood, SC. and Children's Hospital Medical Center/University of Washington, Seattle. Prader-Willi syndrome (PWS) includes characteristic dysmorphic appearance. hypotonia, hypogonadism, development deficiency, behavioral and growth abnormalities. Diagnostic criteria have been developed, and recently a single diagnostic marker, methylation analysis at SNRPN or PW71, has been developed that can detect all cases to date. However, diagnostic suspicion still must rest with the clinician. In the course of seeing patients in genetics clinics, few African-American patients have been referred for this diagnostic possibility. Among those referred, clinical differences often delay diagnosis. We here report on 10 African-Americans (5 males. 5 females) with confirmed PWS and delineate and differentiate their clinical phenotype. The most striking measurable differences among these patients compared with Caucasians related to their larger hand length (> height percentile in 90%) and foot length (>height percentile in 88%). In addition, facial phenotype was not typical in the majority of patients; specifically, the mouth did not show thin upper lip and downturned corners, and the eyes appeared relatively large and were not almond- shaped in most patients. These phenotypic differences between African-Americans and Caucasians with PWS may lead to delayed or missed diagnosis and lack of fulfillment of diagnostic criteria. The threshold for suspicion of PWS in these patients should be lower. A22 Slide Session 34: Cytogenetics II: Clinical Cytogenetics 98 99 A new partial trisomy of murine chromosome 16 (TslO8Cje) as an Correlation of X inactivation pattern with phenotype in females with an animal model for Down syndrome. H. Sago. T.T. Huang, E.J. Carlson, and abnormal X chromosome. Di Wol L Carrel CL Kelly HF Willard S C.J.nEstein. University of California, San Francisco, CA Schwartz. Case Wessen Reserve Univ School of MedicinDUniversity Hospitals of Mice trisomic for mouse chromosome 16 (MMU146) have been used as an animal Cleveland, Ohio. model for Down syndrome (DS). Although trisomy 16 (Tsl6) mice have several X inactivation is the process by which mammalian females achieve dosage phenotypic features suggestive of DS, their value as a model for DS has been compensation by transcriptionally silencing one of two X chromosomes. In somewhat limited because they live beyond the end of gestation and because chromosomally normal females, this process is random. However, females with one MMU16 contains many genes located on human chromosomes other than 21. abnormal X chromosome demonstrate skewing of X inactivation presumably as the Recently, Davisson et al. developed a viable partial Tsl6 mouse (Ts65Dn) with a result of cell selection. This non-random X inactivation pattern has been inferred translocation producing segmental trisomy of only the region of MMU16 from App indirectly from the qualitative study of late replication banding. To study this to Mx that exhibits male sterility, neuronal deficits, and behavioral abnormalities. phenomenon on a more quantitative basis and to correlate X inactivation pattern with We now report the development of a new partial Tsl6 mouse (TslO8Cje) with an phenotype, we utilized molecular techniques (methylation analysis of androgen even smaller region of duplication than Ts65Dn. During the construction of a Sod) receptor and/or FMR1, and expression studies of an XIST polymorphism) to assess knock-out mouse by homologous recombination, we obtained what we believed to X inactivation pattern of females with abnormal X chromosomes including 16 be heterozygous mutant animals by virtue of a 50% reduction in CuZnSOD activity, balanced X;autosome translocations, 2 deleted Xs, 2 duplicated Xs, 3 r(X)s, and 3 the presence of the neo resistance marker, and the expression of mRNA of a size i(Xq)s. In 20 cases with complete (>97%) skewing of X inactivation, the phenotype expected from the mutant gene. However, when bred to wild type animals, aberrant was either normal or that of classical Turner syndrome. In contrast, five cases did not segregation was noted in the progeny of these mice. About 30% of the time, have complete skewing and were phenotypically abnormal. Carriers of two balanced offspring were obtained that carried the neo marker and, in addition, also expressed translocations [46,X,t(X;9) and 46,X,t(X;17)], one duplicated X the normal allel present in the heterozygous parent and had 100% of wild type [46,X,dup(X)(q21.2q22.l)1, one deleted X [46,X, del(X)(q26.3-q27.3)], and two CuZnSOD activity. By FISH analysis it was found that, in addition to the two 45,X/46,r(X) presented with complex phenotypes including multiple congenital normal MMU16, a segment of MMU16 that contains both Sod) and Ets2, but not abnormalities and mental retardation. Analysis of X inactivation pattenm of App, is translocated to the distal end of MMU12. The cytogenetic data suggest, lymphocyte or fibroblast samples revealed that the translocated or abnormal X, was therefore, that the proximal end of the translocated segment lies between App and active in a significant percentage of cells (up to 50% of the cells). The abnormal Sod]. Furthermore, the presence of 100% rather than 150% of wild type CuZnSOD phenotype is hypothesized to be due to functional nullisomy or disomy of X-linked activity indicates that the Sodl gene in the translocated segment is disrupted, genes in these cases. Based on these results, we propose that X inactivation studies presumably by the original homologous recombination event. The same 12;16 should be performed on all female clinical cases with abnormal X chromosomesa translocation chromosome, along with a cytogenedcally normal but presumably This should aid in the understanding of abnormal phenotypes in livebom individuals mutated MMU16, has also been identified in the original Sod) knock-out ES clone with abnormal X chromosomes and may help to predict phenotypes for prenatally used to derive the animals being described. The new partial Ts16 mouse (TsI08Cje) detected cases in the future. should be valuable for assessing the contribution of the region from App to Sod] to the phenotype of Ts65Dn.

100 101 Noncontiguous spreading of X inactivation Into autosomal material in a Prospective prenatal diagnosis studies in supernumerary marker 15 aormal t(X;4) carrier. WM WhiteIHWrnamd DL Yan Dyke' DI Wolff. Case cases: uniparental disomy risk and molecular mechanism of marker Western Reserve/University Hospitals Cleveland, OH, Henry Ford Hosp, Detroit, ML formation. S. L. Chrisil, B. Hu 2, J L. Lesser2, M. S. Verp3, and lD. H. The process of X inactivation involves initiation sped and maintenance of the X inactivation signal with the spreading phenomenon being the east well understood. LSedetterlICenter for Medical Genetics, The University of Chicago, Chicago, IL, Unbalanced Xautosome transcations exhibit a range of phenotypes, hypothesized to be 2National Center for Human Genome Research, NIH, Bethesda, MD, 3Department correlated with the extent ofX inactivation sped into the adjacent autosome, and as such of Obstetrics and Gynecology, The University of Chicago. Chicago, IL. provide an excellent model to study the process of spading. To examine the molecular for spreading we have assayed the transcrptonal activity of loci spanning an Supernumerary marker chromosomes involving chromosome 15 occur with a inactivated t(X;4) present in a phenotypically normal 31 yo f with a karyotype of frequency of -0.1-0.4 per 1000 live births. These psu dic(15;15) chromosomes are 46,Xder(X),t(X;4Xq22;q26). Since pal trisomy of 4q usually manifests with subdivided into two general groups based on the size of the marker chromosomes. dysmorphic features, and severe growth and mental rtardaton, normal phenotype of Small markers can be either de novo or familial and are usually associated with a our pauent suggests Go g of inacaton throughout the autosomal material. normal phenotype. Large markers, containing euchromatin from the Prader- RT-PCR analysisof a somatic cell hybrid containing only the inactive t(X;4) for 8 X- Willi/Angelman region on 15qI l-q13, are associated with an abnormal phenotype linked genes revealed results consistent with normal inactive X chromosomes. We next including mental retardation. Additionally, both Prader-Willi and Angelman examined expression of 6 transcribed sequences (2 known genes and 4 ESTs) spanning syndromes have been observed in patients with marker 15 chromosomes in which 4q26-qer. In contrast to hybrids containing the normal 4, hybrids containing the inactive uniparental disomy for the normal 15 homologs accounted for the abnormal t(X,4) did not express 3 of the ESTs, consistent with spring of inactivation into the phenotype. In this study, 17 prenatal cases presenting with either small (N=12) or autosomal material. However, I EST and 2 genes (IL 15 in 4q31 and FRGI in 4q35) large marker 15 chromosomes (N=5) were analyzed prospectively for the presence of were expressed from the t(X;4) and thus "escaped" spreading. Based on the map location uniparental disomy. All de novo cases (N=12) were associated with advanced of the 6 transcribed sequenes tested, spread of inactivation into chromosome 4 material maternal age (34-43 years), and DNA polymorphism analysis in all informative cases appears tobe noncontiguous, as loci escaping or subject to inactivation were intespersed. (N=7) showed a maternal origin for the marker chromosome. Analysis of distal This direct assay ofexpression demonstrates that although a proportion of the autosomal markers demonstrated biparental inheritance of the normal 15 homologs in all 17 cases material is transcrptionally silentdue to the spreading of inactivation from the adjoining X, ruling out uniparental disomy. Analysis of placental tissue from one case with a large the spreading is incomplete. These findings are broadly consistent with the existence of marker chromosome showed the presence of an additional weak maternal band using genes known to escape inactivati on nonnal inactive Xs; however, the fact that 3 of 6 distal markers demonstrating the presence of mosaic trisomy 15. The presence of chromosome 4 loci tested escape inactivation may indicate that there are gene promoter or trisomy in the placenta provides evidence of a trisomic conception which was domain element differences in autosomes compared to the X chromosome. Future analyses "rescued" by formation of the marker chromosome. These results indicate that of the trnription status of additional loci in ths particular t(X;4), as well as investigation supernumerary marker 15 chromosomes show a low risk of uniparental disomy and of other translocations in same manner between this will further elucidate not only the link demonstrates correction of a trisomy 15 conception as one mechanism of marker X inactivation spreading and phenotype, but also the mechanism by which normal X formation. inactivation spreading occurs.

102 103 A gene Involved in XY sex reversal maps to chromosome 9p. W.L. Cytogenetic and molecular analysis of subtle structural abnormalities. I FleIter. J.L. GorsjC and M. Llovd 1. lDepartment of Pediatrics, University o1 Utah Experience with 12 cases. CL Kelly. K Tsuchiva. LA Becker, JM Conrov, TW and -Departments of Human Genetics, and Pediatrics and Communicable Diseases, Deninet. D Wolff and S Schwartz. Case Western Reserve University and University University of Michigan. Hospitals of Cleveland, OH. The genetic mechanisms involved in sex differentiation are poorly understood and Even with the advent and the increasing utilization of molecular techniques, high in the involved has been slow. The fortuitous of progress identifying genes finding resolution chromosome remains an for the detection chromosomal rearrangements in association with a sex-reversed has led to analysis important methodology phenotype of subtle chromosome To and determine a to better the isolatiot, of two genes, SRY and SOX9, both shown to be involved in the sex abnonnalities. investigate way determining pathway. Mutations in SOX9 have been observed in XY females with quantify, confirm and detect subtle cytogenetic abnormalities, we have utilized both campomelic dysplasia, and mutations in SRY have been identified in a relatively small molecular (PCR) and molecular cytogenetic (FISH with YACs and cosmids) analysis number (15%) of XY females. These findings suggest that sex reversal in the majority to study 12 different subtle chromosome abnormalities which were either detected or of cases involves alterations of other genetic loci. Reports of XY females with partial suggested by high resolution chromosome analysis. These abnormalities include 3 -duplications of Xp, and alterations of chromosomes 9 and 10, suggest that these tandem 2 interstitial and 2 terminal deletions, and 2 denovo and 3 chromosomes also harbor involved in sex determination. Recently, we duplications, may genes familial unbalanced These studies indicate that resolution have obtained cell lines from four XY females, each of whom exhibit rearrangements. high gonadal 625-750 band can detect a of 2-5 cM. dysgenesis and other variable malformations, and who have terminal deletions of distal analysis, at approximately level, genetic change chromosome 9p resulting from unbalanced autosomal translocations. PCR Based on the data from these cases, we can conclude that: (1) interstitial tandem amplification and DNA sequence analysis of SRY revealed no mutations in the highly duplications are detectable with as little as 5-7 cM of DNA duplicated; (2) both conserved binding domain (HMG box) in any of the four cell lines. Cytogenetic and terminal and interstitial deletions can be seen with less than 5 cM of DNA missing; FISH analyses of metaphase chromosomes from these patients suggest that the (3) derived chromosomes, whether denovo or familial, can be detected with as little smallest of (SRO) of deletions involves an 0.5- .0 Mb region overlap approximate as an overall of 3 cM of DNA; and. (4) the detection of these alterations region of band 9p24. To more clearly define the SRO, cells from the shown to exchange patient are facilitated their location in and the telomeric resolution of the carrythe smallest deletion were analyzed for LOH using 13 microsatellite markers and by the genome by FISH with eight cosmid and PI clones previously mapped to distal 9p. The data preparations. This approach was also utilized to detect a 3-5 cM deletion in a denovo show that the SRO lies distal to marker D9S143 and three clones have been identified "balanced" translocation. This data supports the utility of high resolution banding, in that lie within the region of interest. Further studies using YAC clones and additional conjunction with both molecular and molecular cytogenetic techniques, in confirming microsatellite markers are currently in progress, and clones that map within the deleted and more precisely defining subtle cytogenetic abnormalities. This will allow for a region are being used to screen a chromosome 9-specific cosmid library to identify more definite correlation between genotype and phenotype. additional mapping resources. Those clones will be used to construct a complete contig of distal 9p and will serve as starting materials to ultimately identify a gene on distal chromosome 9p that is involved in the sex determining pathway. Slide Session 34: Cytogenetics II: Clinical Cytogenetics (cont.) A23 104 105 Eight patients with deletion of chromosome lp36: clinical, cytogenetic, A second locus for DiGeorge syndrome and velocardiofacial syndrome in and molecular investigations. S.K Shapiral. C. McCaskill'. H. Northrup2_ 10p13. S. Daw, M. Kraman. C. Taylor. K. Call'. J. Mao'. T. Lipson2. J. F.F.B. Elder2. F. GreenbergI.3. and L.G. Shafferl. lBaylor College of Medicine, Goodshio3, P. Scambler. Institute of Child Health, London, UK, 'Genome Houston, TX, 2University of Texas Medical School, Houston, TX, and 3National Therapeutics Corporation, Waltham, MA, 2(deceased) Royal Alexandra Center for Human Genome Research, NIH, Bethesda, MD. Hospital, Parramatta, Australia, Less than twenty cases of deletion of the tenminal short arm of chromosome 1 (lp36) 3University of Newcastle-upon-Tyne, have been previously reported. Many patients have had similar phenotypic features, Newcastle, UK. including severe developmental delay and mental retardation, somatic growth delay, DiGeorge and velocardiofacial syndromes (DGS and VCFS) may result in microcephaly, and craniofacial dysmorphic features. Some patients have seizures, brain several abnormalities including cardiac defects, hypoparathyroidism, T-cell anomalies, eye abnormalities, hearing deficits, minor limb anomalies, and/or skeletal immunodeficiency and facial dysmorphism. They are frequently associated deformities. there are common features the Though phenotypic among reported patients, with deletions within 22q1 1.2, but a few single case reports have the variable features appear to be unrelated to the presumed size of the deleted region, DGS or VCFS based on cytogenetic analyses. However, molecular studies to define the extent of each suggested may also occur in some individuals with deletions patient's deletion have not yetbeen reported. of 1 Op. By investigating the extent of deletion in 5 such patients (four with Here we describe eight patients with isolated partial monosomy for chromosome Ip terminal deletions and one with an interstitial deletion) using FISH and that have various deletion breakpoints within 1p36. This deletion can be difficult to quantitative Southern analysis we have defined the SRO for this second detect, but by fluorescence in situ hybridization using probes p1-79 or p58, which map locus (DGSII). A YAC contig at the 1 3/lOp14 to distal Ip36, all eight patients were as having a deletion. The smallest Op boundary spanning the confirmed SRO has been assembled, and we estimate its size to be approximately deletion among the patients is most likely interstitial since probe p58 is deleted, but the 2Mb. As with deletions more distal probe, pl-79, is retained. Though the phenotypes of these patients vary, as of 22q 1 1, phenotypes vary considerably between a group they share many of the reported features of this condition. To determine if a affected patients. These results strongly support the hypothesis that phenotype-genotype correlation exists among the patients, fifteen polymorphic (CA),, haploinsufficiency of a gene or genes within 1 Op can cause the DGS/VCFS microsatellite markers were examined in the patients and available parents. Marker spectrum of malformation. Markers from the DGSII region are currently D1S243 was informative and hemizygous in six of eight patients, and marker D1S468 being used in screens for submicroscopic deletion. was informative and hemizygous in another patient, indicating that the deletion in six patients was mammal in origin and in one patient was paternal in origin. The natural deletion panel generated from our patients has facilitated the map ordering of markers in distal Ip36. Two patients with smaller deletions, based on biparental inheritance of marker D1S468, had less severe developmental delay, and lacked the nasal hypoplasia that is present in patients with larger molecular deletions. Phenotypic features in lp36 deletion patients seem to be unrelated to the parental origin of the deletion, but differences appear to exist based on the extent of the deleted region in each patient.

106 107 Point mutations and an intragenic deletion in three ILS patients confirm Molecular Overlap in Wolf-Hirschhorn and LISi as the lissencephaly causative gene in isolated lissencephaly Michael R. Pitt-Rogers-Danks sequence and Miller-Dieker syndrome. Syndromes. Altherr'2. Karen Denison'. Michele Clemens.. Olive S.S. Chonq,'2 C. Lo Niqro."4 A.V. Quarrel4 and Tracy J. Wright'. 'Genomics, LANL, Los Alamos, NM. 87545, Roschke,'A.Taniqami,'S.D.Pack,'A.C.M.Smith."R.Carrozzo,'W.B. Dobyns, Biol. Chem., Irvine, CA 2Dept. andD.H.Ledbetter. 'NCHGR/NIH, Bethesda, 'Georgetown UCI. 92717, 3Dept. of Genetics. Magee-Womens Hospital, MD. Univ. Med. Ctr., Pittsburgh, PA 15213, 4North Trent Clinical Genetics Service, Centre for Washington, DC, 4TIGEM and 4Ospedale San Raffaele, Milano, ITALY, 5NCCRI, Genetics, Sheffield, UK. Human Tokyo, JAPAN, and 'Univ. Minnesota Med. Sch., Minneapolis, SlO 5DN, MN. Clinical dysmorphology strives to categorize patients based on phenotype and often Miller-Dieker syndrome (MDS) is a multiple malformation syndrome characterized represents the first in the molecular by classical lissencephaly and characteristic facies. It is associated with visible step characterization of a genetic abnormality. The or two abnormalities considered here have been described as distinct clinical submicroscopic deletions within chr. 17p1 3.3. Classical lissencephaly without facial Wolf-Hirschhorn entities. The dysmorphism has also been observed syndrome (WHS) is a multiple anomaly dysmorphic malady and is referred to as isolated lissencephaly characterized by mental and developmental defects including prominent sequence (ILS) (see Allanson et al., this meeting). (A second lissencephaly gene and glabella, has been mapped to chr. Xq22.3-q23; Srivastava this hypertelorism, convulsions, hypospadias heart defects. Pitt-Rogers-Danks (PRD) et al., meeting). syndrome is a rare, often less severe, condition which is characterized by a number of Apparently partial and nonoverlapping deletions of the 5' or 3' end of a candidate similar features gene LISt in one ILS and one patient had suggested that dysmorphic including hypertelorism. microcephaly, maxillary MDS MDS was a single hypoplasia and short stature. Both of these conditions have been associated with gene disorder and that LISt spans in excess of 400 kb. However, the originally chromosome 4 from the loss of presumed 5' end of LISi was found to belong to the 14-3-3E gene residing more hemizygousity resulting genetic material from the terminus of the short arm. In this study we describe two patients, one diagnosed as distally on 17p1 3.3. We have isolated the true 5' end of LISt, constructed a -500 PRD and one as WHS. We have kb genomic contig encompassing and determined analyzed both individuals with a series of landmark LISt, its gene extent to be cosmids from 4p 16.3. These have an 4 approximately 100 kb. FISH analysis of an ILS with a patients approximately Mb terminal deletion patient 17p;19q balanced and both breakpoints are localized with a 70 kb segment. We have identified a number translocation, as well as several other key MDS and ILS deletion patients localizes of genes common to these deletions. the lissencephaly critical region within the LISt gene. Characterization of We hypothesize that there are several genes the common to these clinically distinct syndromes, that the dose of one or more of these structure of LISt indicates the presence of 11 exons. SSCP analysis of individual genes is critical at some in exons was performed on 18 ILS patients who showed deletions point development and that the phenotypic differences no detectable by between these syndromes is due to allelic variation. This work was FISH. Mutations were identified in three patients: (1) a dA-4dG transition in exon 6 DOE Contract W-7403-ENG-36. supported by resulting in a H149R missense mutation, (2) a dC-4dT transition in exon 8 causing a R273X nonsense mutation, and (3) a 22 bp deletion at the exon 9-intron 9 junction predicted to result in a splicing error. Therefore, these data confirm mutations of LISt as the cause of the lissencephaly phenotype in ILS and MDS. Together with the deletion analysis results for other ILS and MDS patients, these data are also consistent with our hypothesis that additional genes distal to LISi are responsible for the facial dysmorphism and other anomalies in MDS patients.

108 A discriminant function using Mitomycin C stress test to Fanconi anemia. D. Kuffel. A. R. N. Lindor. M. identify Rochester, MN. Zinsmeister, Litzow and G. Dewald. Mayo Clinic, Fanconi anemia (FA) is difficult to diagnose because the phenotype is highly variable. FA is associated with aplastic anemia (AA), but is treated differently than the acquired AA and requires a marrow and modified pre-bone transplant regiment to lessen toxicity post-transplant mortality. FA involves an inability to repair mutations b y DNA cross-linking agents such as Mitomycin C (MMC) and in particular, chromosome radials form. This is the first Diepoxybutane (DEB); the study to use cytogenetic data from MMC stress test to establish a discriminant function based on radials in normal individuals and patients with FA. We studied 20 from Feb patients with clinically confirmed FA '82 to Nov '94, and 84 normal controls from Mar '82 to Oct '95. For each individual, we used a 72-hour blood culture (PHA) with 50 MMC for 48 hrs. scored 20 non-banded metaphases for radials. Total ng/ml and of cells with radials in 20 cells and percentage radials for normal individuals and patients with FA were calculated. An upper bound (95% confidence interval for for [Cl]) normal individuals and a lower bound (95% Cl) patients with FA were estimated (Table). The Cl for total radials was based on the Poisson distribution, and distribution. These percentage of cells with radials, the binomial distributions did not account for all the variation in the data, and so alternatively a linear discriminant function combining both each subject was used to cytogenetic measurements for discriminant classify normal individuals and patients with FA. A function consistent with FA is defined as the % cells with radials + 1.6 x total radials > 40. This function data set. The provided 100%sensitivity and 100% specificity in this discriminant function applied to 16 additional normal parents and of patients with FA yielded siblings 100% specificity; this finding 95/ Cl Bound 95% Cl Bound is consistent with investigators who suggest GrOUs Total # of Radials % of Cells with 21 Radial the MMC stress test will not Normals identify carriers of a gene for (upper) 4 74 21.8 FA. Further studies are Abnormals needed to validate this (lower) 4830 72.5 discriminant function in dinical practice. A24 Slide Session 35: Molecular Genetics of Disease 11 109 110 Endothelin signaling pathway mutations in non syndromic Tuberin associates with a 115 kD protein that interacts with a rab Hirschsprung disease. C.Biaud, l Amiel, A.&elet. P. Edecy T. Atti6 Q, GTPase. Xiao GH. Shoainelad F. Jin F. Yeung RS. Fox Chase Cancer Valdenaire', A. Munnich, S. Lyonnet. Department of genetics and Unite Center, Philadelphia. PA INSERM U-393, H6pital Necker-Enfants-Malades, 75743 Paris, France; Tuberous sclerosis (TSC) is an autosomal dominant disorder of *Hoffman-Laroche, Suisse. hamartomatosis linked to two genetic loci. The TSC2 gene on 16pl3 encodes a Hirschsprung disease (HSCR) is a frequent congenital malformation -180 kD membrane-bound protein, tuberin, with in vitro GAP activity towards related to an abnormal migration of neural crest cells towards the hindgut. A rapla. Its biologic function is consistent with that of a tumor suppressor gene as significant proportion of sporadic (15-20%) and familial (50%) HSCR cases demonstrated by in vitro and in vivo assays in the Eker rat model of hereditary has been ascribed to mutations of the RET proto-oncogene. However, the cancer. To elucidate the mechanism of TSC2 function, proteins that associate involvement of other susceptibility genes is supported by several lines of with tuberin were identified using the yeast two-hybrid system. One such clone evidence. These include : i) segregation analyses, ii) chromosomal anomalies that was isolated using the C-terminal fragment of pTSC2 as 'bait' represents a (trisomy 21, 13q deletions), iii) RET exclusion in some HSCR families and iv) 115 kD protein with predicted coiled-coil structure. In vivo binding assays using the existence of several mendelian models for megacolon in mice. Recently, exogenously expressed and endogenous proteins confirmed the physical homozygous mutations of the endothelin receptor B (EDNRB) and endothelin 3 interaction between tuberin and p1 15. Data from an independent study showed were identified in the that the latter also interact with a member of the rab family ofGTPase. Based on (EDN3) genes Shah-Waardenburg syndrome. Here, we domain mapping analysis, the pI15-tuberin occurred on mutations of the EDN3 and the enzyme endothelin 1 binding between the N- report converting of and a (ECE1) genes in isolated HSCR patients. We have screened a series of 173 terminal region p115 C-terminal region of tuberin, distal to the GAP HSCR families, 114 SSCP domain. In contrast, the p1 15-rab association lied near the C-terminus of p1 15. probands (59 sporadic cases), by using analysis of This led us to test if tuberin PCR amplified exons. We have identified missense mutations of possesses GAP activity for this rab protein. heterozygous Immunoprecipitated native tuberin as well as recombinant tuberin GAP-domain the EDN3, EDNRB and ECEI genes in a total of 6 sporadic HSCR cases. demonstrated substantial and EDNRB mutations seem to fragment GTPase activating activity in vitro. Our Thus, while homozygous EDN3 predispose to model is consistent with the recruitment of the p115 protein to membrane surface features of the Waardenburg syndrome in addition to HSCR, heterozygous by activated rab GTPase followed by the targeting of the vesicle to tuberin-bound EDN3, EDNRB and ECE1 mutations were found in isolated HSCR patients as organelle where docking and fusion take place. Upon GTP hydrolysis by well.These data suggest that the endothelin-signaling pathway mutations are tuberin, p1 15 and rab recycle to the cytosol. Functional analysis linked tuberin dosage sensitive and support its role in the development of neural crest- with the endocytosis pathway and suggested a novel association between a tumor derived enteric neurons. suppressor gene and the vesicular transport system.

111 112 COL2A1 mutation analysis demonstrates that Kniest dysplasia Mutations in the COL4A3, COL4A4 and COL4A5 genes in Alport predominantly results from small deletions in the type II collagen triple syndrome (AS) L. Forestier, C. Breillat, C. Arrondel, B. Knebelmann, MI.C. Gubler, helix. D. J. Wilkin', C. Yoo2, D. L. Rimoin2, and D. H. Cohn2. 'Medical Genetics C. Antignac. INSERNI U423, Necker Hospital. Paris, France Branch, NCHGR, NIH, Bethesda, MD, 2Cedars-Sinai Medical Center and UCLA AS is a mainly X-linked hereditary disease of basement membranes (BM) characterized School of Medicine, Los Angeles, CA. by progressive renal failure, deafness and ocular lesions. It is associated with mutations Kniest Dysplasia is a moderately severe type II collagen disorder characterized by short of the COL4A5 gene located at Xq22 and encoding for the a5 (IV) collagen chain. The limbs with prominent joints, a short trunk with kyphoscoliosis. myopia, cleft palate, and COL4A3 and COL4A4 genes, encoding for the o3 and a4lV) chains and located head- distinctive radiographic features. A unique cartilage morphology, termed "Swiss-cheese" to-head at 2q35-q37, have been shown to be involved in the less frequent autosomal cartilage, also distinguishes this phenotype from the other type II collagenopathies. By recessive form. We have screened 48 out of the 51 exons of the COL4A5 gene by SSCP heteroduplex analysis of PCR amplified genomic DNA fragments followed by DNA se- analysis and identified 62 mutations (in 61 patients) and 10 sequence variants among 131 quence analysis, we have identified novel COL2Al mutations in three Kniest dysplasia unrelated AS patients with either a probable X-linked inheritance or a sporadic disease. patients. One patient was heterozygous for an 18 bp deletion within exon 19. The other This represents a mutation detection rate of 49%. There was no hot spot of mutation and two patients were heterozygous for 5 bp and 4 bp deletions that removed the consensus no recurrent mutation in our population. All types of mutations were found (nonsense, splice donors of introns 14 or 20 respectively, predicting skipping of the corresponding frameshift, splice-site and missense mutations, 40% being glycine substitutions in the exons during splicing. All three mutations are therefore expected to result in small collagenous domain). Two of these occured on the same allele in one patient and segre- deletions within the type II collagen molecule. The pathologic mutation has now been gated with the disease in the family. We showed that most of the glycine substitutions identified in 13 patients with the Kniest phenotype. Eleven of the mutations result in are associated with the lack of immunological expression of the o3-5(WV) collagen chains small deletions, all located between residues 91 and 378 (exons 12-24) in the collagen in the glomerular BM. De novo mutations occured in 13% of cases, which is consistent triple helix. The remaining two mutations result in aspartate for glycine substitutions, with the previously estimated frequency from pedigree analyses. We have also screened also within this region. Thus COL2A1 mutations of distinct type and location, primar- 6 and 4 exons of the COL4A3 and COL4A4 3' ends respectively, in AS patients with a ily resulting in shortened type II collagen chains, are consistently found in patients with likely autosomal recessive inheritance or a sporadic disease. Six mutations (1 nonsense this phenotype. These data support the hypothesis that Kniest dysplasia results from mutation, 2 frameshift deletions, 2 conserved substitutions and one substitution in an disruption of a functional domain within the type II collagen triple helix important in intron creating a novel acceptor splice site) in 7 unrelated patients and one sequence vari- the organization and structure of the cartilage extracellular matrix. ant have been detected. Three of these were homozygous mutations in offsprings from consanguineous unions. All these patients have severe juvenile type AS, whereas the parents who are heterozygous for the mutation are either asymptomatic or have micro- scopic hematuria. Thus, heterozygous COL4A3 and COL4A4 mutations could account for a number of familial benign hematuria cases.

113 114

Fibroblast growth receptor 2 mutations in a craniosynostotic and A fibroblast growth factor receptor 3 (FGFR3) mutation causes Pfeiffer demiatologic condition, BeareStevenson cutis gyrata syndromesrK-A. Przyltu, syndrome and isolated craniosynostosis. G.A. BellusL, K. Gaudenz2 ' 'W. Paznekas,2M. Golabi, 'W.Bias, 3M. J.Bamsran J. C.Carey, aB.D. Hall,'I LH. Zackai., L.A. Clark', J. Szabo', C..A. Francomanol& M. Muenke2. Medical Genetics Branch, MD & Center for Stevenson, "S. J. Oow,"MIIM.. Cohen Jr., 'E. W. Jabs. 'Johns Hopkins Univ., NCHGR, NIH, Bethesda, Medical Genetics, Johns Baltimore, 2UCSF, Francisco,CA; 3Univ. Utah,Salt Lake City, Utah; 4Univ. Hopkins University School of Medicine, Baltimore, MD 2Children's Hospital of

KY; Genetic Div. of Human Genetics and Molecular Kentucky,Lexington, sGreenwood Center, Greenwood, SC; 6NYU, NY, NY; Philadelphia, Biology, Depts. of Pediatrics and "Dalhousie Univ., Halifax, Canada. Genetics, University of Pennsylvania School of Medicine, Philadelphia, 3Department of Medical British Columbia Institute of cutis Genetics, Child and Vancouver, gyrata syndrome (BSCG) (MIM123790) is a rare, autosomal Family Health, British Columbia, Canada. characterized by cutis gyrata or furrowed skin with a corrugated Pfeiffer syndrome (PS; MIM is an autosomal dominant syn- appearance (especially on the face, axilla, and and 101600) craniosynostosis perianal/genital areas) acanthosis drome with characteristic craniofacial anomalies and broad thumbs and toes. Ge- nigricans (verrucous hyperplasia with great hyperpigmentation). Other consistent findings netic heterogeneity is recognized in PS and mutations in FGFR1 (chromosome 8) and craniosynostosis, downslanting palpebral fissures, ocular hypertelorism, and FGFR2 (chromosome 10) have been identified. However, linkage to chromosomes 8 and proptosis, digital anomalies, umbilical and anogenital abnormalities, and early death; 10 have been excluded in several large families with PS phenotypes. Here we report a features include natal teeth,pyloric stenosis, and a coccygeal tail in some recurrent single point mutation in the FGFR3 gene, located on chromosome 4p, in 10 patients. craniofacial features are reminiscent of other craniosynostotic conditions unrelated families with craniosynostosis syndromes (including several sporadic cases). Crouzon, Apert,and Pfeiffer syndromes with mutations in the fibroblast growth This mutation (C749G) results in a Pro 250 Arg substitution in the highly conserved receptor (FGFR2) extracellular domain. In addition,Crouzon syndrome patients linker region between the second and third immunoglobulin-like domainsof the FGFR3 acanthosis nigricans have a recurrent mutation (Ala391Glu) in the FGFR3 trans- protein. What is most striking is that this common mutation occurs precisely at the membrane domain. We searched within the FGFR2 and FGFR3 as candidate genes for BSCG. In analogous position FGFR3 protein as the mutations in FGFR1 (Pro 252 three cases, novel missense and FGFR2 253 described in Pfeiffer sporadic mutations were found in FGFR2 causing an Arg) (Pro Arg) previously and Apert syndromes to be a respectively. The of individuals with this mutation varied acid replaced by cysteine; two had the identical Tyr375Cys mutation in phenotypic spectrum from PS with broad thumbs and transmembrane domain and typical toes to isolated with normal the third had a Ser372Cys mutation in the carboxyl- great craniosynostosis hands and feet. This was even terminal the linker appearing phenotypic variability seen within the same region between the immunoglobulin and transmem- III-like family. These results indicate that FGFR3 mutations are for some isolated It is likely that the introduction of a responsible domains. cysteine causes an alteration in the cases of craniosynostosis and highlight the functional significance of this proline residue disulfides of two cysteine residues in the immunoglobulin domain, in FGFRs. interactions with the two cysteines in the transmembrane domain, or covalent dimerization between two mutant receptors. Interestingly, these mutations are in the same position as analogous amino acids in FGFR3 which are mutated (Gly37OCys and Tyr373Cys) in thanatophoric dysplasia, a lethal dwarfing condition. In two BSCG patients, neither of these mutations were found, suggesting genetic heterogeneity. Slide Session 35: Molecular Genetics of Disease 11 (cont.) A25 115 116 A new skeletal dysplasia with severe tibial bowing, profound developmental The level of normal CFTR transcripts required for normal pulmonary function. delay and acanthosis nigricans is caused by a Lys 650 Met mutation in B. Kerem'N. Rave-Harel'. 0. Chiba-Falek'. E. Kerem2. A. Augarten3 T. Shoshani'. fibroblast growth factor receptor 3 (FGFR3). C.A. Francomano"2, A. Tal'. Y. Yahav. M. Aviram'. L. BenturS. A. Szeinberg!. Dept. of Genetics. G.A. Bellus"a, J.Szabo' 2, 1. McIntosh"2, J. Dorst3, R. Lee , 0. Hurko2A, Hebrew University, Jerusalem', CF clinic Shaare Zedek Medical Center, Jerusalem2, A.E. Fraley' & M.J. Bamshad'. 'Mledical Genetics Branch, NCHGR. NIH., Bethesda, CF clinic, Sheba Medical Center, Ramat-Gan3 CF Clinic, Soroka Medical Center. MD, 2Center for Medical Genetics 3Dept. of Radiology and 4Dept of Neurology, Johns Beer-Sheva4, CF clinic Rambam Medical Center, Haifa5. Israel. Hopkins University School of Medicine, Baltimore, MD, 5Dept. of Pediatrics, The severity of lung disease varies considerably among patients carrying the same University of Utah Health Sciences Center, Salt Lake City. CFTR mutations. One molecular mechanism associated with this variability is Recurrent mutations in the FGFR3 gene have been associated with several different hu- alternative splicing in which 2 possible transcripts are generated: one normal and the man skeletal dysplasias including achondroplasia, hypochondroplasia and thanatophoric other aberrantly spliced. We have analyzed the relative level of the normal and dysplasia types I and II (TD1 & TD2). FGFR3 mutations have also been reported in aberrantly spliced RNA transcribed in respiratory epithelial cells from 12 patients with craniosynostosis syndromes not affecting long bone growth including a variant of Crouzon the 3849+10kb C-> T mutation and 11 patients with the ST allele. We have tested, the syndrome associated with acanthosis nigricans. We report here the discovery of a novel relative of the FGFR3 mutation (A1949T: K650M)in two unrelated patients with a previously unde- stability aberrantly spliced transcripts and found that aberrantly spliced scribed skeletal dysplasia characterized by extreme short stature, severe tibial bowing, transcripts from the ST allele were as stable as normal transcripts. However, aberrantly profound developmental delay and acanthosis nigricans. This mutation occurs at the spliced transcripts from the 3849+10kb C-> T allele, which carried a stop codon, were nucleotide adjacent to the TD2 mutation (A1948G: K650E) and results in a different three 3 less stable than normal or AF508 transcripts. In addition, we employed amino acid substitution at the same codon located within the distal tyrosine kinase do- differential and non-differential RT-PCR on the same individual and found no main. Both individuals with K650M mutations (proband 1, age 5 years & proband 2, difference for the 3849+10kb C- >T mutation. The level ofaberrantly (shorter) spliced age 29 years) have skeletal findings that are distinct from TD1 and TD2. These in- RNA transcribed from the 5T allele was higher in the differential than in the non- clude absence of craniosynostosis or cloverleaf skull anomaly and moderate bowing of differential RT-PCR (factor of difference of 2.5). Thus, we have analyzed all patients the femurs with reverse bowing of the tibia and fibula. Proband 2 has bilateral tibial using quantitative non-differential RT-PCR and revealed considerable variability in the pseudoarthroses. Other clinical and physical features common to both patients include: level of aberrantly spliced RNA transcribed from the 3849+10kb C- >T (0-48%) and survival past infancy; periods of respiratory compromise during infancy but without the from the 5T allele (1444%). The actual levels of normal RNA transcribed from the need for prolonged mechanical ventilation; development of acanthosis nigricans in the 3849+10kb C-> T mutation were considered in the calculation. A significant non linear cervical and flexural areas; seizures and hydrocephalus during infancy with severe limi- correlation (r=0.82 p=0.002) was found between the level of normal transcripts and tation of motor and intellectual development. Proband 1 has structural anomalies of the pulmonary function of the entire group. Patients with normal pulmonary function (FEV, brain including a hypoplastic corpus callosum and abnormal development of the cere- 83-92 %predicted) had >25% normal transcripts and patients with severe lung disease bellum. These patients represent a new and unique phenotype resulting from a specific (FEV, 18-44% predicted) had 2-8%. Thus, in this class of mutations > 25 % of normal missense mutation in the FGFR3 gene. transcripts are required to maintain normal lung function. These data are important for the design of gene therapy aiming to restore normal lung function.

117 118 Variable phenotypes for nine CFTR mutations associated with pancreatic Correlation between Waardenburg Syndrome phenotype and genotype in a population of sufficiency in cystic fibrosis (CF) patients. A. Hamoshl and M. Corey for the CF Individuals with identified PAX3 mutations. A. L. DeStefano for the NIDCD Waardenburg Genotype-Phenotype Consortium, 1Johns Hopkins Hospital, Baltimore, MD; Syndrome Consortium 2Hospital for Sick Children, Toronto, Canada. Most (85-90%) CF patients have pancreatic insufficiency (PI). Compound Waardenburg Syndrome (WS) type I is an autosomal dominant disorder characterized by heterozygosity for a subset of CFTR mutations appears to dominantly confer a sensorineural hearing loss, pigmentary abnormalities of the iris, hair, and skin, and dystopia phenotype characterized by pancreatic sufficiency (PS). The hypothesis is that the canthorum. The phenotype is variable and affected individuals may exhibit only one or a vast majority of patients carrying these alleles will be PS, however controversy exists combination of several of the over the effect upon pulmonary disease. Participating centers identified all patients who associated features. A mutation in PAX3, a paired domain protein were compound heterozygotes for one of 9 CFTR mutations previously associated that binds DNA and regulates transcription, has been identified in each of the 48 families (271 WS with PS and a second identified mutation known to be associated with PI. For each individuals) included in this study. Forty five of the 48 mutations are unique. To assess the case, a control patient matched for sex and age and homozygous for AF508 was relationship between phenotype and gene defect, mutations were grouped in five mutations selected from the same center. W1282X was accepted for controls in one center where that mutation is predominant. Data recorded included age at diagnosis, sweat chloride. categories; amino acid (M) substitution in the paired domain, AA substitution in the height and weight percentiles, % predicted FVC and FEV1, pancreatic status, homeodomain, deletion of the Ser-Thr-Pro-rich region, deletion of the homeodomain and the Ser- Pseudomonas colonization, and history of meconium ileus or pancreatitis. Results: Thr-Pro-rich region, and deletion of the entire gene. These mutation classes are based on the 211 pairs of subjects from 14 centers were documented. The number of cases and % structure of the PAX3 gene and were chosen to group mutations predicted to have similar defects with reported PS. for each mutation group were: R1 17H (38, 84%), R334W (24, 87%), R347H (8, 86%), R352Q (6, 83%), 3849+10kbC->T (53, 77%), 2789+5G-A (23, in the gene product. The associations between mutation class.and the presence of hearing loss, 68%), A455E (16, 69%), R347P (25, 44%), and G85E (28, 33%). Among the control eye pigment abnormality, skin hypopigmentation, or white forelock were evaluated using patients, 5 (2%) were reported as PS. Overall the patients with PS mutations had generalized estimating equations (GEE) to estimate logistic regression coefficients. Use of GEE significantly better values for every variable except for pancreatitis, which was seen in 13 (8%) cases and no controls (p<0.001). Surprisingly, two missense mutations, allowed for incorporation of a correlation structure that accounts for potential similarity among R347P and G85E, were associated with PS in less than half of patients. The group of members of the same family. Significant associations were detected for the traits involving mutations (2789+5G->A, 3849+10kbC->T, and A455E) which result in reduced pigmentary disturbances. Odds for the presence of eye pigment abnormality, white forelock, and synthesis of normal CFTR had intermediate PS frequencies. Only the 4 missense were and 5 times mutations (Ri 17H, R334W, R347H, R3520) with >80% PS had significantly better skinhypopigmentation 3, 7, greater, respectively, for individuals with deletions pulmonary function than matched controls (mean case-control difference in FEV1 was of the homeodomain and the Ser-Thr-Pro-rich region compared to individuals with an AA 18% predicted, p=0.001). Multiple regression analysis showed that the patterns of substitution in the homeodomain. Differences in the odds for these traits may indicate that the FEV1 differences in the 3 subgroups were not explained by the differences in reported gene products resulting from different classes of mutations act differently in the expression ofWS. PS status. It appears that mutant genotype is not the sole determinant of pancreatic or No associations were detected between loss and mutation which pulmonary phenotype suggesting a prominent but variable role for epistatic, significant hearing class, stochastic, and/or environmental modifiers depending upon genotype. indicates that allelic heterogeneity within PAX3 does not influence this phenotype.

119 Association of the dopamine transporter gene (DAT1) and Attention Deficit Hyperactivity Disorder in children. LD. Waldmann, D.C.RoweJ I .X. Abramowitz", S. Kozel'. J. Mohr', S.L. Sherman3, HiH. Cleveiand4, M.L. anders', C. Stever4. 'Departments of Psychology, 2Psychiatry, and 3Genetics and Molecular Medicine, Emory University, Atlanta, GA; 'FCR. University of Arizona, Tucson. AZ. Attention Deficit Hyperactivity Disorder (ADHD) affects approximately 3-5% of chil- dren in the United States, with more boys than girls being diagnosed. In the current psychiatric nomenclature, ADHD comprises three subtypes - Inattentive, Hyperactive- Impulsive, and Combined - based on extreme scores on inattentive and/or hyperactive- impulsive problem dimensions. Family, adoption, and twin studies have suggested that ADHD is familial and that genetic influences contribute substantially to its etiology. Dopaminergic activity also has been hypothesized to be important in the etiology of ADHD, consistent with the fact that many medications used to treat ADHD inhibit dopamine transporter. In a recent study, inactivation of the dopamnine transporter gene (DAT1) resulted in extreme hyperactivity in mice. In this study, we examined the asso- ciation of the DAT1 gene and ADHD. Our sample included 101 children referred to psy- chiatric clinics for behavioral and learning problems including but not limited to ADHD. Children were further selected based on having moderately or severely extreme scores on the inattentive and/or hyperactive-impulsive dimensions. DNA samples for children and their parents were gathered from cheek epithelial cells via a mouthwash procedure. Within-family association analyses using the Transmission Disequilibrium Test (TDT) suggested that ADHD was associated with the DATI gene more strongly at severe levels of ADHD symptoms (TDT x2 = 4.17, df = 1, p<.05) than at moderate levels (TDT x2 = 3.27, df = 1, .10>p>.05). In additionthe DATI gene was more strongly associated with the Combined (TDT x2 = 5.76, df = 1, p<.025) and Hyperactive-Impulsive (TDT X= 7.35, df = 1, p<.01) subtypes than with the Inattentive subtype (TDT x2 = 3.00 df = 1, .10>p>.05). We will conduct further analyses on a larger sample to examine sex and age differences in, and the effects of related psychiatric disorders on, the association of DAT1 and ADHD. A26 Slide Session 36: Genomics I 120 121 Whole genome radiation hybrid maps with 500kb average resolution. AN STS-BASED MAP OF THE HUMAN GENOME E.A. Stewart, A. A arwal. E. Bajorek, A. Chu. N. Fang, D. Hadley, M. Harris, T. Hudsonl, L,. Steinl, L. Huil, J._Mal, A.Castlel, J. Silval, D. Sloniml, X S. Hehert. HuIsinA. S. Perkin .Piercv, F. Qin, T. Reif. Baptista1, L C. S. Rozen1, R. Nah1, X. Wu, C. Sanders, X. She, W-L. Sun, P. Tabar, S. Voytick Kruglyak1, Rosenberg', Vestereaardl, L.Horton1, M.Andersonl1, A.CollymoreIW. Ye IR. Kouyu ia1, J-B. Fan, S. Cowles, J. Quackenbush, D.1lzit, R.M. Myers, D.R, Cox. Department 1 of Genetics and the Stanford Human Genome Center, Stanford University School of L. Tam1, I. Zemtseva R. Devine1, D. Courtney1, S. GerholdI.D.-Grav1, S. Foote1, Medicine, Stanford, California Q. Gyapay5, .ib5,J. Morissette5, J. Sikela3, J. Korenhu.-2, X. Chen2, E3aYam2, The Stanford Human Genome Center (SHGC) has built medium-resolution whole G. Shulg4, M. Bocuski4, J. Weissenbach5, D. Page1 B. Birrenl, and E. Lander1. genome radiation hybrid maps. These maps, constructed from markers assayed against lCenter for Genome Research, Whitehead Institute/ MIT, 2Cedars Sinai Medical Center, the G3 RH panel (available from Research Genetics), have an average resolution of 3University of Colorado Health Sciences Center, 4National Center for Biotechnology 530kb/interval. Markers placed in these maps represent 5049 unique positions, with 1766 Information and 5Genethon. high confidence bins. There are an additional 3015 markers which are either duplicates We report the construction of an STS-based physical map of the human genome to the unique positions (945 markers) or positioned within the best possible bin (2070 containing 20,186 STSs, with an average of 148 kb. The involved markers). These maps include 1999 Genethon markers, 1367 genes or spacing project cDNAs, ESTs, of a radiation map of the loci and and 1205 random genomic clones. The maps and data are available assembly hybrid humarWenome containing 11,357 ancillary publicly incorporated a genetic linkage map of the human genome loci. This on our web site (http://www-shgc.stanford.edu). An of the markers within the containing 5,264 analysis information was combined with the results of STS-content screening of 11,400 loci map shows that the markers are distributed randomly. With the current map coverage, against a YAC library to produce an map, anchored the radiation and 75% of random new markers can be positioned with a lod 6 or greater link with at least integrated by hybrid genetic maps. The map provides radiation hybrid coverage of 99% and physical coverage one mapped marker. the the markers is also an If chromosomal assignment of known, of 94% of the human genome. additional 15% can be positioned with a lod score between 3 and 6. The final 10% of Current mapping efforts focus on map verification, transcript mapping, and new markers will not have a link greater than lod 3 due to errors in the scoring data, cytogenetic integration. New STSs are mostly derived from non-redundant ESTs which low retention of the marker or gaps in the map. SHGC is to markers on continuing type are part of the UniGene and UniEST set prepared at NCBI. To date, 8402 STSs the G3 panel, with the goal of reaching a total of 10,000 mapped markers. In addition, designed from 3end of ESTs have been mapped to the GeneBridge 4 radiation we are beginning to construct our next set of whole genome maps using the new, hybrid high scale PCR automation. BACs identified over 800 STSs are resolution TNG3 hybrid set, which is also publicly available. The first stage of this panel using large by being FISH mapped to serve as integration points between the STS maps and the cytogenetic map construction is to type markers from the G3 maps on the new panel. With this map. approach we are improving the G3 map by integrating G3 and TNG data while at the STS-content data generated at the Center is made accessible by ftp to same time constructing a framework for the additional 20,000 markers needed for the genome.wi.mit.edu, directory '/distribution/ human_STSreleases' or via our web site at high resolution maps. http://www-genome.wi.mit.edu.

122 123 Large-scale human genomic sequencing as a tool for gene discovery. YAC-based map of the X chromosome at 85 Kb J. Quackenbush. J.-B. Fan, Hussain, She, inter-STS S. A. Mlaratukulam, X. M.Y. Tsai, resolution reveals extremes of recombination and A.x Urband, L Fine, C. Mader, K.B. McKusick, S. Cowles, E.A. Stewart. D.R. Cox, activity GC R.M. Myers, and D. Vollrath. Stanford Human Genome Center, Stanford University, content. R. Nagaraia. S. MacMillan,J. Kere. S.Cox. C. Jones, M. Schmatz,J. School of Medicine, Palo Alto, California. Terrell. M. Shoemakr Jerak. C. Hott M. Masisi. S. $umm. A Srivaslava G. Pilia.T. B. B. The Stanford Human Genome Center has recently embarked upon a large-scale Featherstone. S..Kesterson. McCauley Radey F. BuEough. B. se- Brownstein. V. D. States. M. and quencing project, the goal of which is the determination of a minimum of 17.5 NMb of high Nowotny. D'Urso D.Schlessinger. Center for Genetics in Medicine, Wash. Univ. Sch. St. 10 quality, unique human genomic DNA sequence over a three period. This sequence Med., Louis, MO 631 USA year A YAC/STS of the X chromosome has is determined using bacterial 6 transposons to establish the priming sites for the map reached 85 Kb inter-STS 'y resolution. The and resolution of the map majority of sequencing reactions, allowing the use of a directed approach with formatting physical is provided largely universal markers sites" primers. Our initial targets are contained within two areas of human chromosome 21q: by unique-sequence ("sequence-tagged STSs; ) scorable by PCR. The current map 1900 STSs a current a 409 kb region between D21S334 and D21S267 that is involved in Down Syndrome and incorporates (of total of 2037) in 5,144 YACs and 569 cosmids across the 160 Mb of the a 1.0 Mb region around D21S25 containing genes responsible for progressive myoclonus overlapping chromosome. The order of markers was inferred the SEGMAP epilepsy (EPM1) and autoimmune polyglandular disease type I (APECED). The com- using program (Magness and Green, 1995), and contigs were aligned and oriented unequivocally a combination of bination of existing gene prediction programs with EST and other database homology by marker deletion and a searches has proven to be an extremely powerful tool for the elucidation of the FISH, published orders, panels, linkage maps derived from genomic number of sources. The STSs include 947 YAC-ends; 190 dinucleotide and 21 structure of a number of previously mapped genes, including SIM2 and PFKL, as well tri- and tetranucleotide repeat markers; 190 of the known genes; 100 ESTs; and as to identify novel candidate genes. Upon completion of these regions, our goal is 438 other unique sequences. to focus on the sequencing of human chromosome 4. We are currently constructing a The WEB representation at http://genome.wustl.edu/cgm/cgm.html shows the sequence-ready, large-insert bacterial clone map spanning a 10 Mb region of 4q25 that ordered STSs along an ideogram of the X, with approximate cytogenetic band will serve as our next sequencing target. This map is being constructed by STS-content positions and an index scale in Mb. Lines join the corresponding position of using a high-resolution radiation hybrid map as a framework. This approach is rapid dinucleotide repeat markers on the physical and the CEPH/Genethon 198.1 cM and robust and is being expanded to the construction of a sequence-ready map of the en- genetic linkage map (Weissenbach et al., 1996). tirety of human chromosome 4. Finished sequence data are available through GenBank The STS density is sufficient to detect deletions or gaps in contigs; to provide and both finished and preliminary data are available thourgh our World Wide Web site, YACs or recover other large-insert clones as sequencing substrates; and to . localize regions of high or low recombination and GC/gene level -- indices of interest to sequencers and gene-hunters. For example, five regions of relatively highGC concentration are detected, with the most gene-rich in Xq28; and a20 Mb region with very low recombination falls between the Xql3.3 XIST and Xq2 1.3 XY homology loci.

124 125 Features of human chromosome organization revealed with the completed Towards a complete gene map of human chromosome 12. Beatrice Renault', physical map of human chromosome 16. N.A.Doggetl, RLaD. Sutherland'. M. Ivonne \Iarondel'. KateMontgomery', Sung-Joo Yoon'. JoanChang', Alilerr . D.C.Torneyl, E.H. KnillI, L.L. Deave 1, D.F. Callen. and R.K. Moyzis'. Stephanie Lau'. Amy Banks2, PatriciaBray--Ward2. Prakash .Nadarnil, KeiChung2, 'Center for Human Genome Alamos National Laboratory, Los Studies. Los Alamos, llyaChumakov3, Lincoln Stein4, NM 87545,'Women's & Children's Hospital, Adelaide, Australia Jean Weissenbach5,Daniel Cohen3, TomHudson4, EricLander4, Perry Miller2, David Ward2, RajuKucherlapatil. 'Albert Einstein We have previously reported on the construction of an integrated physical map of human chromosome 16 (Doggett et al., Nature 377:Suppl:335-365, 1995). The College of Medicine, Bronx, NY, 2Yale University, New Haven, CT, 3Jean Dausset physical map consists of a low resolution YAC contig map and a high resolution Foundation. Paris, France, 4Whitehead Institute/MI1T. Cambridge, MA, 5Ginithon, cosmid contig map, both of which are aligned to a somatic cell hybrid breakpoint map Paris, France. comprised of 90 intervals (1.1 Mb average size). The low resolution YAC contig More than fifteen genetic disorders have been linked to genes on chromosome 12 by map currently consists of 900 CEPH megaYACs, and 250 flow-sorted 16-specific linkage analysis and/or molecular studies (eg, Noonan syndrome, Maturity Onset YACs that are localized to and ordered within the breakpoint intervals with 800 STSs. Diabetes of the Young type III, MODY3). In addition, an increasing number of The YAC/STS map provides practically complete coverage of the euchromatic arms breakpoints and rearrangements associated with a wide range of tumors have been of the chromosome and contains STS markers on average every 125 kb. The high identified (eg., acute lymphoblastic leukemia in 12p13, lipomas in 12q13-15 and germ resolution "sequence ready" cosmid contig map consists of 4000 fingerprinted cell tumors in 12q22). To facilitate the identification of genes involved in these diseases, cosmids assembled into contigs covering 60% of the chromosome and is anchored to by positional cloning or candidate gene approaches, the availability of an integrated the YAC and cytogenetic breakpoint maps via STSs developed from cosmid contigs physical map of the entire chromosome is essential We constructed such a map and by hybridizations between YACs and cosmids. Integrated with the physical maps consisting of overlapping sets of yeast artificial chromosomes (YACs) from the CEPH II are a gene map of 336 genes, expressed sequence tags (ESTs), and exon trapped mega YAC library. This map contains 1100 markers covering nearly 110 megabases of sequences (XTSs) and a genetic marker map of 387 polymorphic markers (60 DNA, thus providing an average resolution of 105 kb. In constructing the map, we markers in the CEPH consortium framework linkage man). placed special emphasis on the integration of different types of markers generated by We have used this integrated map to investigate various features of chromosome different groups. The map contains 376 highly polymorphic markers which provide a organization and have found among other things 1) highly uneven rates of resolution of 1 marker every 0.5 centiMorgans. In addition, more than 300 unique recombination along the chromosome--ranging from <0.04 cM/Mb across the genes or EST clusters have been ordered relative to the remaining 800 markers on the centromere to >20 cM/Mb near the telomeres, 2) highly non-uniform distribution of map. Several individual YACs that are part of the YAC contig were used for FISH genes--with light bands having 7x greater density of genes than gray bands and 22x analysis enabling us to anchor the physical map to the cytogenetic map. We are now greater density than dark bands, 3) a greater frequency of Eco RI and Hind III converting the YAC contigs to sequence-ready maps in different regions of interest of restriction sites in cosmids mapping to dark bands than light bands, and 4) a higher the chromosome, such as 12q12 (lipoma breakpoints), 12q13 (lymphoma breakpoints) G+C content for STS sequences mapping to light bands (0.51) than dark bands (0.44). and 12q24 (Darier's disease and SCA2). As a step towards our goal of obtaining the Implications of these results for the structure of human chromosomes will be complete nucleotide sequence of human chromosome 12, the 12p13-p12 ETV6 gene presented. As a result of 2) we suggest that large scale genome sequencing should locus known to be involved in leukemias is currently being sequenced. initially focus on light bands to achieve a greater initial return in gene discovery. Slide Session 36: Genomics I (cont.) A27 126 127

Distances between 9q34 cosmid contigs determined by FISH on molecular XREFdb: Cross-referencing genes, genetics and mammalian genomes combed genomic DNA. R.Ekongl, S.Rousseauxl, . NIichalet2, C.Schurra. D.E. Bassett Jr1.2, M.S. Boguski2. F. Spencerl. R. Kim', T. WVeaverl, R. Reevest, N.Hornigold3, J.Nahmiasl, M.van Slegtenhorst4. J.VWolfe3 S.Povey' and A.Bensimon2. and P. Ilieterl. 'Johns Hopkins Medical School. Baltimore, MD, 2National Center for Biotechnology Information, NIH. (Intro. by: David Valle) IMRC Human Biochemical Genetics Unit and 3The Galton Laboratory, University We have initiated a project that is systematically transferring College London, UK. 2Departement des Biotechnologies, Institut Pasteur, Paris, information about genes in model organisms onto the mouse and human France. 4Department of Clinical Genetics, Erasmus University, The Netherlands. genetic maps. Novel ESTs exhibiting significant sequence similarity with model organism Our approach to identifying the TSC1 gene in human 9q34 has been to construct proteins are being systematically mapped in the mouse and human genomes by cosmid contig that spans the interval to which TSC1 maps (D9S149 to D9S11l). By the XREF group. These EST mapping data. supplemented with data from the conventional physical methods, we have produced a deep cosmid contig (with one International RH Mapping spanning the TSC1 interval (Hornigold et. al., abstract at this meeting). Consortium, cross-reference the genomic position of mammalian cDNAs with associated with genes in model attempts to assist in contig construction by measuring distances on extended functional information organisms. This information should facilitate the identification of genes mutated in human chromatin by previously described methods (chromatin released from fixed disease states. In addition, the identification of mammalian homologs and Fidlerova et. al. 1994, Cytogenetics & Cell Genetics 65:203-5; fibre-FISH using family members of model organism proteins allows the experimental approaches available in from PFGE blocks, Heiskanen et. al. 1995, Genomics 30:31-6) have not, in our hands, multiple organisms given quantitative and reproducible results. to be employed synergistically in the study of the those genes. par XREFdb, a relational database, has been designed as a Molecular combing of DNA was previously described for lambda DNA (Bensimon free, public resource to manage, and disseminate similarity al. 1994, Science 265:2096-7). To determine the size of the remaining gap in our cross-reference, search, mapping, and mammalian phenotype data and accelerate the establishment of contig, we applied molecular combing to total genomic DNA and used LL09 cosmids valuable cross-species connections. can an account and query proteins and mouse the contigs as probes for FISH experiments on combed DNA molecules. Control Users establish and human map regions of interest. Protein queries are automatically searched experiments on combed genomic DNA confirmed a 40kb distance between cosmids 117F9 against both dbEST and the and 165A9 previously measured by EcoRl restriction mapping. The distance non-redundant protein database on a monthly basis. Positional cloners can take advantage of the map query option, which returns ESTs 109C8 and 18OF1 has been determined on combed DNA to be about 76kb. This mapped to regions of interest, to similar genes in model organisms with associated has recently been covered by two PACs of yet unknown lengths. The length of the linked functional information. Users are automatically informed of similarity search remaining gap in our contig was determined using cosmids (50D9 and 180F1) near results, EST mapping data, and on related to their protein ends of their respective contigs and close to the gap. Using molecular combing and information human phenotypes and map queries each month via an e-mail summary, and can access detailed results via the distance between 50D9 and 180F1 is about 50kb. These data, together with the the project World Wide for each of the contig data, make the TSC1 interval (D9S149 to D9S114) approximately Web site. XREFdb tracks results accountholder, flagging new results for close in context find molecular combing of DNA to be highly reproducible and the most reliable inspection each month, placing them the of results viewed previously. par To access XREFdb or view project information, so far employed for determining distances on extended genomic DNA. visit the project World Wide Web site (http://www.ncbi.nlm.nih.gov/XREFdb/).

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Merged Tandem (M/T)-nested PCR: low and single copy genome amplification Analyzing human mitochondrial DNA variation by hybridization to high- with de radable dUTP outer primers. M.B. Grace'. G. S. Buzrd2 E. Gore- density oligonucleotide arrays. M. S. Chee. R. Yang. E. Hubbell and A. Langton and M.R. Hugies'3. 'NCHGR-NIH, Bethesda MD, -SAIC Frederick, NCI- Berno. Affymetrix, 3380 Central Expressway, Santa Clara, CA 95051. FCRDC, Frederick, MD, Georgetown University Medical Center, Washington, D.C. Arrays containing probes complementary to the 16,569 bp human Amplification from low or single copy genomes is an increasingly important mitochondrial DNA (mtDNA) have been synthesized. Each position in diagnostic tool in such diverse fields as preimplantation familial genetic screening, themtDNA is represented by a set of 4 probes. The cancer diagnosis and microbiological detection. To obtain sufficient specificity and probes are varied at a sensitivity of specific target or allele target detection, two sequential amplification single position which contains either A, C, G, or T. The array also contains reactions with nested primer pairs are often required. Typically aliquots of product additional sets of haplotype-specific probes. are diluted into the secondary 'inner' reaction from the primary 'outer' reaction highly The entire mitochondrial sequence was hybridized to the 1.64cm2 array has Tms for specificity. Nevertheless, the presence of carry-over that elevated primer in a single experiment. After hybridization, the biotinylated target 'outer' primers can result in undesired primer activity and background bands. While sequences were labelled with a phycoerythrin-streptavidin conjugate. A several 'clean up' techniques will eliminate 'outer' primers from PCR products, quantitative fluorescence image of the array was generated in a separation methods risk loss of irreplaceable genetic material in rare or single genomic single specimens. As an alternative, we have adapted uracil-N-glycosylase (UNG)-mediated detection scan requiring only a few minutes. degradation of dU-containing amplified DNA for prevention of first round primer Each target sequence had a reproducible and characteristic pattern of carry-over. Targeting the most common mutation in exon of the human hybridization intensities. Differences between samples were found by hexosaminidase A gene, we have merged single-tube amplification with intronic comparing normalized intensities. Physical differences were rapidly and flanking 'outer' dU primers and a tandem-nested amplification of only the 'inner' dT accurately detected, without any need for base calling. primers (hence, merged tandem (MIT)-nested PCR). Primary amplification When a readout of actual sequence was required, sequences were interrupted for the addition of UNG and dT 'inner' primers to the mixture by comparing the intensity patterns incubated to degrade free dU primers and primer-extended 5' product-ends. In serially determined of each set of 4 probes to diluted wild-type (wt) genomic DNAs (100 ng/ml to I pg/ml), amplifications the pattern from a reference sample of known sequence. Most successful with as few as 5 'outer'/20 'inner' cycles for 30,000 diploid genomes polymorphisms were read with high accuracy by automated base calling 12'outer'/26'inner' cycles for a single genome. Only the specific 128-bp product Some polymorphisms could II 2 software. clustered be detected but not read detected with SYBR Green in all samples with 300 diploid genomes and -90% because of multiple mismatches to the probe. These regions were samples with 30 to I diploid genome without carry-over contamination automatically flagged, and read by conventional sequencing. (n=20/dilution). Single cells from lymphoblastoid lines were lysed, neutralized, MfT-Nest amplified for exon wt and1277insTATC genotypes. Heteroduplex We have demonstrated the hybridization analysis of a 16.6 kb sequence at analysis showed enhanced PCR specificity in an abridged protocol. single nucleotide resolution. We estimate the rate of sequence analysis to be 50 to 100 fold greater than for conventional gel-based methods.

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Identification offive novel human Li elements capable of retrotransposition. D. M. Sassaman. B. A. Dombroski. J. V. Moran. H. KiazZia.L IL Dept. of Genetics, Univ. of Penn. Sch. Med., Philadelphia, PA. LINE-1 or LI elements are repetitive DNA sequences. asmall minority of which the ability to retrotranspose. Two of these "active" elements, LREI and LRE2, have isolated previously by us; however, the total number of active Ll elements in the genome is unknown. To identify additional LI elements capable of retrotransposition, assayed a number of full-length elements for their ability to encode functional transcriptase in in vitro and in vivo yeast assays and to retrotranspose in HeLa culture. First, we tested two LI elements (Ll.3 and LI.4) which are members of the LRE1 subfamily. Both of these elements encode functional reverse transcriptase interestingly, are able to retrotranspose in human cultured cells at 5-10 fold greater frequencies than LREL. L.3 and Ll.4 differ from LREI by only 2 and 4 amino residues, respectively, out of the 1619 total amino acids encoded by these elements. Second, we tested thirteen LI elements belonging to the Ta subset. Ta subset comprise 20% of transcribed Lis and account for 6 of the 7 known recent insertions the human genome. Of these thirteen elements, seven encoded detectable transcriptase activity and three of these were able to retrotranspose in cultured Based on the estimated number of full-length Ta subset elements in the human genome (about 60) and our observed frequency of Ta subset elements capable of retrotransposition (3 of 13), we estimate that the average human genome contains about twenty active I elements. In total, we have now isolated seven of these active elements. A28 Slide Session 37: Genetic Epidemiology and Population Genetics 1: Mapping Complex Traits 131 132 Evaluation of susceptibility in disease risk assessment genes T.R. Rebbeck'j M. Evaluation of mutational spectra using a hierarchical model: application Viana2. HA. Jordan3. B. L. Weber'. A. Rooatko3. 'U. of Pennsylvania, Philadelphia; 'U. to of Illinois, Chicago; 3Fox Chase Cancer Center, Philadelphia, PA. BRCAI. J.S. Witte and R.C. Elston. Case Western Reserve University Once the role of a susceptibility gene in disease etiology is established, additional Department ofEpidemiology and Biostatitics, Cleveland, Ohio. Information may be required to determine when it is appropriate to use this gene in Hierarchical modeling-incorporating a prior model into a conventional disease risk assessment. We propose a Bayesian statistical method that uses epide- analysis-can give estimates that are more plausible and stable than miological study data to evaluate the usefulness of genotypes in disease risk assess- conventional estimates, while offering a solution to problems of multiple ment. This method is dependent on observed genotypes, sensitivity and specificity of inference. We use this method to contrast the age-specific breast-cancer rates the genetic test, the disease odds ratio (OR), and the background cancer rate (BCR) in corresponding to different BRCAI mutations. A conventional (first-stage) the target population. We compute a statistic (H) that measures the degree to which approach to estimating the incidence rate corresponding to each BRCA1 the genetic test distinguishes affected from unaffected individuals (higher values of H mutation might entail using, for example, a proportional-hazards model. Such imply better risk prediction ability). We also estimate a range of BCR values for which an approach invariably leads to unstable estimates of mutation-specific it may be appropriate to use these genes in disease risk assessment. relative risks because most of the distinct BRCAI mutations occur infrequently: the GENE OR H BCR ,% We apply this method to evaluate the use eight most common BRCA1 mutations reportedly make up only 45% of all BRCAI 72.8 4.3 >1 of a) BRCA1 genotypes in breast cancer (BC) mutations CYP1A1 2.9 1.1 >28 risk assessment, and b) CYP1A1 and glu- observed to date, and many of the remaining fifty-plus detected CYPIAUxGSTMI 5.4 1.7 >22 tathione-S-transferase p (GSTM1) genotypes mutations have been observed only once or a few times. To stabilize the in lung cancer (LC) risk assessment. We find mutation-specific estimates, we use a second-stage (prior) model in which that BRCA 1 is a good predictor of BC risk (i.e., large H) in populations with background effects of the first-stage covariates depend linearly on second-stage covariates BC rates of >1% (see Table). This result confirms knowledge that BRCA1 provides that describe the BRCAI mutations. These second-stage covariates useful information about BC risk. Based on published data of Kawajiri et al. (1993), distinguish: 1) single base-pair substitutions versus frame shift mutations; 2) CYPIAI alone and the interaction CYP1A1xGSTM1 are relatively poor predictors of mutations in different exons; 3) splice site mutations versus coding region LC risk (i.e., small H), and should only be applied when the background LC rate is mutations; 4) 3' versus 5' mutations; and 5) assays of protein expression or _22% (see Table). Since most individuals do not belong to populations with such high function. In addition, when looking jointly at more than one type of cancer LC rates, it may not be generally appropriate to apply CYPIA1 or CYP1A1xGSTM1 in (e.g., breast and ovarian), we recommend extending this hierarchical approach LC risk assessment. Even if genes such as CYP1AI or GSTM1 have an established to model factors specific to each cancer site in the second stage. role in cancer etiology, our results suggest they may be less useful predictors of can- cer risk than genes such as BRCA1. The proposed method can be used to objectively evaluate the conditions under which susceptibility genotypes may be applied in dis- ease risk assessment.

133 134 Fifty percent of Jewish high-risk breast and ovarian cancer families are not Interactions between genetic explained by the three known BRCA1 or BRCA2 founder mutations while a 0.7% and epidemiological risk factors in cutaneous malignant combined BRCA1 founder mutation frequency Is reported in a Jewish cohort. LM melanoma in France. F. Demenais'. A. Chompret2. M. Guilloud-Bataille'. N. Feineold3. 1 . F. Grange2 and M.F. Avrinl. 'INSERM U358, Paris, France, 2Institut Gustave Roussy, E.Schubert.C. Oddoux2.J.Morrowl. E.eich2..L.Hull H.Ostrer M-C. Villejuif. France, 3INSERM U155. Paris. France. iflUl, Univ of Washington, Seattle l; New York University, New York City2. In a series of 175 families at high risk of breast and/or ovarian cancer ascertained by our The role of genetic and epidemiological risk factors in the etiology of cutaneous laboratory, defined by 4+ cases of breast cancer or 3+ cases of breast and ovarian cancer, malignant melanoma (CMIM) was investigated in a sample of 295 families, ascertained 16 families are of Ashkenazi Jewish ancestry. This subset of Ashkenazi Jewish families through 295 melanoma probands followed at Institut Gustave Roussy (France), between was screened for the BRCAI founder mutations 185delAG and 5382insC as well as 1986 and 1989. Information on melanoma history and melanoma risk factors (phototype, BRCA2 founder mutation 6174delT by SSCA, ASO hybridization and genomic number of nevi, degree of sun exposure, ability to suntan. propensity to sunburn) was sequencing. Six families from this subset were shown to transmit BRCAI.185delAG. obtained from all probands and 90% of first-degree relatives. All melanoma reported in Ages at diagnosis of breast cancer in female heterozygotes ranges from 34-77. Other relatives were confirmed by pathological reports. Prior to segregation analysis, the probands cancers in heterozygotes include ovarian (2), thyroid (1) and urethra (1). One family from were compared to types of controls, unrelated spouses and unaffected sibs, with respect to the Ashkenazi Jewish subset was shown to transmit BRCAI.5382insC. Ages of onset of the measured risk factors. Propensity to sunburn, high degree of sun exposure and presence breast cancer in this family are 48 and 49 and one case of ovarian cancer at age 48. One of multiple nevi (2 50 nevi) increase family from the Ashkenazi Jewish subset was shown to transmit BRCA2.6174delT. Ages significantly the risk of melanoma with the spouses as of onset in women with breast cancer range 34-63. Other cancers in heterozygotes include controls whereas only the two latter factors are significant using the blood-related controls. ovarian (1), thyroid (1) and pancreatic (1). The eight remaining families from this subset Segregation analysis was conducted on 295 nuclear families using the class D regressive are wild-type for the three founder mutations. The pattern of cancer in these unexplained model incorporating survival analysis concepts to account for a variable age of onset of families is consistent with that in the mutation positive families, suggesting the presence disease (Abel and Bonney, Genet Epidemiol, 1990, 7:391-407) and implemented in the of a predisposing mutation. Two of the eight unexplained high-risk families share alleles computer program REGRESS (Demenais and Lathrop, Genet Epidemiol. 1994, 11:291). at markers on chromosome 17 across 150 kb inclusive of BRCAI suggesting the presence The probability for each person to be affected is expressed as a function of a major gene of another ancient mutation in this population. A cohort of 623 young adults of Jewish effect, residual family dependence (due to other genes and/or shared environment) and ancestry from New York City were also screened for the two BRCA1 founder mutations. measured risk factors. Our results show evidence for the segregation of a rare dominant The frequencies of heterozygotes for the two BRCA1 mutations in this cohort were major gene (gene frequency = 0.0002) with a lifetime risk in gene carriers of 0.50 in males 0.0048 (3/620) forl8SdelAG and 0.0018 (1/558) for S382insC, for a combined frequency and 0.72 in females and a high proportion of sporadic cases (98%). Inclusion of risk factors of 0.0066 with standard error of 6.87x1o-S. The frequency in this cohort is substantially (propensity to sunburn, high degree of sun and lower than that reported previously in an American and Israeli series (Nature Genetics exposure presence of 2 50 nevi) 11:198, 1995). With the knowledge of yet to be defined founder mutations and the significantly improve the fit of all models. The segregation of a rare dominant gene is still possibility of other predisposing loci to account for the high incidence of breast cancer in highly significant (p<0.001), fitting Mendelian transmission. There is indication of the Ashkenazi Jewish population, mutation screening and genetic counseling efforts significant interactions between this gene and two risk factors, propensity to sunburn and should observe that wild-type sequence at these three sites does not exclude inherited presence of 2 50 nevi. These results may have important implication for further linkage breast cancer susceptibility among Jewish women. studies and risk assessment. Supported by NIH ROICA27632, DOD BCRP 2340, and the Kornen Foundation.

135 136 HLA class II DR-DQ amino acids and IDDM. G. Thomson. A. NM. Valdes Assesment of Cytokine, Collectin and Vitamin D receptor gene variants with S. McWeenev. H. Salamon. Department of Integrative Biology, University of California, Berkeley, CA. altered susceptibility to tuberculosis in Africans. C.Ruwvende, R. Bellamy, T. P. The HLA component to insulin dependent diabetes mellitus (IDDM) involves Corrah*, W. McGuire%, K.P.J. McAdam*, D. Kwiatkowski+, H.C. Whittle*, A.V.S. multiple loci, multiple alleles, genetic heterogeneity and interaction effects. Current Hill. The Wellcome Trust Centre for Human Genetics, University ofOxford, Oxford, molecular models of IDDM in the HLA region, namely: Asp at DQB 1 57 protective UK, The Medical Research Council Laboratories, Fajara, The Gambia; +The Institute and Arg at DQA 1 52 predisposing, do not explain the known associations, ethnic of Molecular Medicine, John Radcliffe Hospital, Oxford, UK differences, and genetic heterogeneity of IDDM. We have developed a number of independent methods to identify the amino acids in the HLA region involved in Epidemiological observations and various twin studies implicate a role for host IDDM. (1) The "unique combinations" method employs an efficient computer genetic factors in determining the clinical outcome ofMtuberculosis infection. algorithm to detect all amino acid combinations (singles, pairs, triplets, etc.) which Though several associations with HLA alleles have been reported, the only consistent distinguish a set of sequences from another set of sequences, e.g., haplotypes or result is an association of HLA-DR2 with increased susceptibility to tuberculosis in genotypes Which are common in IDDM patients versus those that are virtually absent Asian populations. TNF is important in host defense against but there is from patients yet common in controls. (2) The "haplotype" method is a test of pathogens whether all relevant amino acids involved in disease have been identified. We evidence that in excess amounts this cytokine is responsible for the immune- mediated consider the haplotypic amino acid combinations at putative predisposing sites. For tissue damage. We find that no TNF promoter variant is associated with increased each of these haplotypes we expect the relative frequencies of amino acid variants at susceptibility to tuberculosis (P > 0.05). Mannose binding protein (MBP) is polymorphic sites not involved in disease to be the same in patients and controls, if all a member ofthe collagenous lectins (collectins) that a role in innate sites plays immunity. predisposing have been identified. The ratio for non-predisposing polymorphic By attaching to non-host carbohydrate moieties on the surfaces ofpathogens, MBP sites in patients and controls will differ if not all predisposing sites are included in the analysis. Statistical features of tests using this method, including the complication of can activate the classical complement pathway or opsonise organisms for phagocytosis high correlation between amino acid sites within the HLA genes, have been Low serum MBP levels lead tan opsonic defect characterised by recurrent childhood considered. When multiple sites are involved, the test statistic gives a closer fit to the infections and a life long risk of severe infections. In our study, homozygotes for null expectation when some, compared to none, of the true predisposing factors are structural variants associated with very low or absent serum MBPlevels are not at an included. This method can unequivocally determine ifall sites in a region involved in increased risk of tuberculosis (P > 0.05). There is some evidence that vitamin disease have not been identified. - Our results prove that the combination DQAI 52 have an to DQB1 57 does not include all the IDDM elements. Given the direct D may important role play in anti-tuberculosis immunity and we find that predisposing (3) for a RFLP role of HLA DR-DQ genes in IDDM, we have considered the ratio of patient to homozygotes Taq vitamin D receptor gene variant that increases expression control haplotypes and genotypes, to give relative estimates of their penetrance of the receptor, is associated with resistance against tuberculosis (0.=0.31, P<10.2). No values. Combining the results from all three methods, specific amino acids in HLA associations were found with any HLA-DR allele nor the NRAMPI gene implicated in DRBI, DQA1 and DQB 1 have been identified which correlate well with IDDM resistance to mycobacterial and other intracellular infections in mice. These data identi- incidence, and and effects in a number of predisposition protection ethnic groups. fy non-HLA genes that influence the risk of disease caused by Mtuberculosis infection. Slide Session 37: Genetic Epidemiology and Population Genetics 1: Mapping Complex Traits (cont.) A29 137 138 Quantitative variation in IGFBP-3 serum concentration is linked to Localizing genes with pleiotropic effects on platelet-derived growth factor and chromosome 6 in Mexican Americans. J. Blangero. L. Almasv. A.G apolipoprotein E in the San Antonio Family Heart Study. M. C. Mahaney. Comuzzie. M.C. Mahanev. P.B. Samollow. J.W. MacCluer. J. E. Hixson. A.G. Comuzzie. D.L. Rainwater, L. Almasy. P.8. Samollow. J.E. Hixson, J.W. Southwest Foundation for Biomedical Research, San Antonio, TX. MacCluer, and J. Blangero Southwest Foundation for Biomedical Research, San Insulin-like growth factor binding protein 3 (IGFBP-3) is the Antonio, TX. major binding protein for insulin-like growth factor 1 (IGF-I) and In vitro, very low density lipoproteins (VLDL) potentiate platelet-stored platelet- its serum levels are growth hormone-dependent. The main functions of IGFBP-3 are to modulate access of IGF-I to the IGF-I receptor and to derived growth factor (PDGF) stimulation of arterial smooth muscle cell proliferation potentiate IGF-I action. Little is known about the genetic basis of and migration. Apolipoprotein E (apoE) is a major protein constituent of VLDL normal quantitative variation in IGFBP-3 serum levels. To examine the particles. Previously, we reported moderate heritability for variation in PDGF levels genetic determinants of IGFBP-3, we assayed serum levels in 416 and pleiotropy (shared additive genetic effects) between PDGF and plasma apoE Mexican Americans. These individuals were members of 10 large levels in Mexican American families. Using a maximum-likelihood based variance pedigrees that were randomly ascertained in the San Antonio Family components linkage analysis that utilizes full pedigree data, we searched the genome Heart Study. Quantitative genetic analyses determined that IGFBP-3 to localize these pleiotropic genes in 484 individuals from 10 extended pedigrees for levels were significantly heritable. To localize genes influencing whom we have genotyped STR marker loci at 20 cM intervals. This method IGFBP-3 variation, we performed a 20 cM genomic screen using STR simultaneously estimates the proportion of phenotypic variance due to a marker locus, markers. In order to systematically search for quantitative trait residual additive genes, and random environment, plus mean effects of selected loci (QTLs), we employed a new multipoint variance component method covariates. Phenotypes included PDGF levels, apoE levels, and the proportion of that is appropriate for pedigrees of arbitrary size and complexity. A apoE on VLDL particles. We observed possible co-incident linkage, i.e., linkage of the major benefit of this method is that it provides unbiased estimates with of both QTL location and genetic effect size without having to same locus both PDGF and the VLDL-apoE proportion, in three genomic regions. specify a penetrance function. Two-point quantitative trait linkage Of these, anonymous loci in the 17p12 and the 8q23-24.2 regions display the analysis provided a maximum lod score of 3.61 with the anonymous strongest evidence of co-incident linkage. The former accounts for 22% and 29% of marker D6S1031. Multipoint analysis utilizing all available marker the variance in PDGF (p = 0.001, lod = 2.2) and VLDL-apoE (p = 0.002, lod = 2.0), information improved the lod score to 4.57 and localized the putative respectively; and the latter accounts for 20% and 25% of the variance in PDGF QTL to a region approximately 74 ± 6 cM from the pter end of (p = 0.008, lod = 1.5) and VLDL-apoE (p = 0.04916, lod = 0.84), respectively. The chromosome 6. This QTL was estimated to account for 38% ± 7% of the third marker, located in the chromosome 12 IGF-1 locus, accounts for 19% of the phenotypic variance in IGFBP-3 serum concentration. This finding variance in PDGF (p = 0.024, lod = 1.1) and 28% in VLDL-apoE (p = 0.118, demonstrates that QTLs influencing normal physiological variation are lod = 0.6). Therefore, we conclude that these three chromosomal regions likely resolvable using randomly ascertained extended pedigrees. contain one or more of the genes contributing to the previously detected pleiotropic Supported by NIH Grants HL45522, GM31575, and DK44297. relationship between PDGF and apoE. This study was supported in part by NIH grant HL45522.

139 140 A search for functional mutations in the lipoprotein lipase (LPL) gene region that Identification of haplotypes of the apolipoprotein (Apo) B gene region influence quantitative intermediate risk factors for coronary artery disease hypothesized to carry functional DNA variations using a cladistic analysis. MB.l (CAD). S.L. Reilly Kardia'. M.B. Haviland'. RE. Ferrell'. C.F. Sing'. 'University Haviland'. A.R. Templeton2. R.E. Ferrell3. C.F. Sing'. 'Univ. of Michigan. Ann Arbor. of Michigan, Ann Arbor, MI, 'University of Pittsburgh, Pittsburgh, PA. MI, 2Washington Univ.. St. Louis, MO, 3Univ. ofPittsburgh, Pittsburgh. PA. One ofthe most complex and challenging problems in human genetics is Meaningful inference about the relationships between genetic and phenotypic identifying the DNA sequence variations that influence individual variation in variation in the population at large requires an understanding of the evolutionary quantitative risk factors for complex diseases having a multifactorial etiology. It is processes that produced the current distribution ofgenetic variation. We used a becoming widely accepted that each susceptibility gene will have many DNA sequence cladistic approach to address the question: Which haplotypes ofthe Apo B gene carry variations, yet only a fraction ofthose variations will contribute singly or in some functional DNA variations? Unambiguous haplotypes were determined for 472 combination to a significant phenotypic effect on levels of intermediate risk factors. unrelated non-Hispanic White participants from the parental generation ofthe We applied cladistic analysis to five RFLP markers (5'- (Asp9-Asn) - Pvull- HindIII - Rochester Family Heart Study using five markers in Apo B (5' Ins/Del--XbaI--MspI-- (Ser447-Ter) - BSTNI-3') in the LPL gene region to identify haplotypes that may carry EcoRl--VNTR 3'). The distribution ofthe 12 observed VNTR alleles was bimodal and one or more functional DNA sequence variations with significant effects on CAD risk recent evidence suggests that there are multiple sequence differences between the high factor levels. Five RFLPs and eleven intermediate risk factors (systolic BP, diastolic and low modes. The VNTR alleles were thus dichotomized. Hypotheses about BP, and fasting plasma levels of cholesterol, triglycerides. HDL-C, and Apo Al, All, recombination and homoplasy were tested and the Ins/Del marker was omitted based B, CII, CIII, and E) were measured in 191 unrelated individuals in the parental on the results. A maximum parsimony cladogram was estimated and coalescent theory generation and 228 unrelated individuals in the grandparental generation ofpedigrees and information about the gain/loss of RFLP sites was used to resolve cladogram relatedness was ambiguity. The resulting cladogram still contained one loop and this ambiguity was collected as part ofthe Rochester Family Heart Study. Evolutionary incorporated into the nested analysis ofthe association between haplotype variation used to estimate a parsimonious cladogram ofthe 12 observed haplotypes. The and variation in adjusted plasma levels of total cholesterol, HDL-C, triglycerides and cladogram was used to define a nested analysis which incorporated the evolutionary apo Al, All. B. CII, CIII, and E in females and males separately. The results suggest relationships among haplotypes. We identified seven classes ofhaplotypes that were that the 12 haplotypes observed in this sample can be grouped into 7 functional associated with significant effects (P<0. 1) on levels oftriglycerides, Apo CII, Apo CIII. haplotype classes. One ofthe haplotypes was associated with significantly (p<0.05) Apo E as well as cholesterol, Apo B, Apo Al, Apo All, systolic BP, and/or diastolic increased levels ofapo B in both males and females while three other haplotypes BP. In particular cases, the associations were dependent on gender and generation. (grouped because they were not significantly different) were associated with increased These results suggest that there are multiple mutations in the LPL gene region, rather apoAI, apoAII and HDL-C levels in females only. In conclusion, evolutionary than a single mutation, with significant effects on levels of established quantitative information empowers one to infer that Apo B contains multiple functional DNA CAD risk factor traits. Individuals that carry these haplotypes are candidates for further variations, carried by multiple haplotypes. some ofwhose effects are pleiotropic and characterization ofthe responsible mutations. gender dependent.

141 Heritability of Bone Mineral Density (BMD) at Four Sites and Evidence for Linkage Disequilibrium for the Vitamin D Receptor and low BMD in Younger but not Older Persons. R.H. Myers.' X.F. Kong. X.H. Zhu.' J. Ordovas,2 D.T. Felson.' J.A. Eisman. P. Sambrnok,3 T.V. Neuven? M.F. Holick,' D.P. Kiel,' 'Dept. of Neurology, Arthritis Center and Bone Res. Lab, Boston U. Sch. of Med., 2Tufts U., 'Garvan Inst. of Med. Res. and St. Vincent's Hosp, Sydney Australia 'Hebrew Rehah. Center for Aged, Harvard Med Sch., Boston. Previous studies of familial aggregation of BMD have been small and have focused on mother-daughter similarity. We examined the familial resemblance of BMD for Ward's area, femoral neck, trochanter, and one third radius in 160 spouse pairs and 109 sibling pairs (mean age=76.4+5.1) participating in the Framingham Study. BMD was adjusted for the effects of age, weight, smoking, alcohol use and for women, estrogen replacement therapy. All analyses were performed on covariate adjusted standardized residuals of BMD computed for each sex separately, using the Statistical Analysis for Genetic Epidemiology program controlling for comparisons within nuclear families. Significant sibling correlations were found for all sites studied. The only significant spouse correlation was for the radius, suggesting that this site may be more susceptible to environmental influences. Sib correlations varied from 0.22 for Ward's area and radius to 0.21 for trochanter and 0.19 for femoral neck. Inconsistent results on the role of the vitamin D receptor (VDR) in BMD have recently been reported. We compared BMD for VDR linked BsmI genotypes (BB, Bb, bb) among 94 younger (mean age=47+12) persons. We also assessed the BsmI site for 328 Framingham participants, sampled equally from each quintile of femoral neck BMD. No association was seen between BsmI genotype and BMD in the Framingham sample, even after stratifying for dietary calcium and serum vitamin D level. However, the BB genotype was more frequent among persons with low BMD in the younger age sample (p<05). This analysis suggests that the role of the vitamin D receptor in osteoporosis may be confined to a young onset phenotype. A30 Slide Session 38: Genetic Epidemiology and Population Genetics II: Molecular Evolution 142 143 The PAR locus and population genetic variation: The Quebec example. S. Byck. The PAH Locus and Rare Alleles causing Phenylketonuria (PKU) in a Neo. K. Morgan. L. Blanc and C.R. Scriver. Dept. of Human Genetics, McGill University, K. Carter. S. B. Montreal, Quebec, CANADA. European Population (Quebec). Byck. Richards. P.J. Waters. R. Rozen and C.R. Scriver. Montreal Children's Hospital Research Institute and Dept. Origins and affinities of populations can be analyzed using two types of DNA Human Genetics, McGill Univ., Montreal, Quebec, CANADA. variation: 1) rare disease-producing alleles, 2) polymorphic neutral alleles. The PAH locus (McKusick OMIM 261600; GenBank U49897; http://www.mcgill.ca/pahdb) has Using denaturing gradient gel electrophoresis (DGGE) and sequencing, mutation analysis is now 93% complete on 141 independent chromosomes in the PKU patients > 300 rare alleles, widely ascertained by newborn screening for phenylketonuria, and resident in 11 polymorphic sequences (7 biallelic RFLPs, 2SNPs (Q232Q and and Quebec. They harbour 38 different mutations on kmown polymorphic V245V) 2 core and multiallelic sites (STR (bp 226-254) and VNTR (3-13 copies)). The polymorphisms haplotypes [H] show clear geographic and demographic stratification in possess several different rates of mutation and provide informative core haplotypes. contemporary Quebec populations. Nine disease-producing alleles (MIV, K421 French Canadians are one of the best documented populations in the world for history, 165T, R157N, E280K[H2], A309D, 1054/55 del G, D338Y, R408W[Hl]), and one genealogies and genetic differentiation and earlier, by use of rare PKU alleles, we found silent allele (IVS6 nt-55), were first discovered in Quebec. Three mutations (K421, evidence of regional genetic differentiation in Quebec. Here we report on the R157N, D338Y) are so far unique to French Canadians and six mutations were polymorphic PAH alleles. We analysed 100 normal chromosomes from families long found on novel polymorphic haplotype backgrounds. When further analyzed, two resident in the Saguenay-Lac-St-Jean region (North east Quebec (NEQ)) and 65 (E280K [Hl,H2], R408W [Hl,H2]) were shown to be recurrent (Byck et al. 1994, chromosomes from South eastern Quebec (SEQ) on the opposite side of the St. 1996). Three mutations sharing dual haplotypes (S67P[H1,H4],G218V[Hl,H2], Lawrence River and compared them with families from 3 regions in France: Ile de R6 V245A[H3,H7]), analyzed for relative codon mutability with MUTPRED software (n=39), Mortagne-Perche (n=22) and Lyonnais (n=36). PAH haplotype distributions (D. Cooper and M. Krawczak), involved non-hypermutable codons. They are each were different within Quebec and between Quebec and France. Percent of haplotypes compatible with a single homologous recombination event between two different shared by individuals in NEQ and SEQ was 48 and 25 respectively, vs 13, 18, and 22 haplotypes. The sixth novel/haplotype association, an intronic mutation (IVS12 in France (IdR, M-P and L respectively). NEQ and SEQ regions had two different ntl[H44/45]), also not involving a hypermutable site, is apparently the result of a predominant haplotypes: 1.Q-.V-.238.8 and 4.Q+,V+.234.3 respectively. Analysis rare double recombination event within the 100 kb locus. Whereas mutation types site-by-site shows that NEQ and SEQ differ and in turn differ from France. The (missense 64%, nonsense 6%, splice 9%, frameshifts 6%, silent 15%), resemble findings indicate a founder effect for NEQ and different descents for NEQ and SEQ those for the world population (see PAH Mutation Analysis Consortium Database, French Canadian populations. Since PAH polymorphic haplotypes can disclose subtle http://www.mcgill.calpahdb; Nucl Acids Res 24:127-131, 1996) the Quebec allelic population genetic variation, in conjunction with the disease-producing allelic variation, profile differs from that of any European population. The Quebec spectrum reflects historical and genealogical information, this locus could be a very informative range expansion, founder effects, genetic drift, and assimilation. contributor to the Human Genetic Diversity Project. (We thank Gerard Bouchard and Bernard Brais for Quebec DNA samples).

144 145 HLA.E amd natural selection. C.Grimsley-and C. Obe The University of Chicago, The origin and history of modern humans: Insights from 100 mi- Chicago, Illinois. crosatellite loci. L. Jin. P. Underhill. A. Lin. E. Minch. B. Sun. C.T. Lam The non-classical HLA genes (lb), HLA-E, -F and -G, are thought to have either P. Srinivasan. and L.L. Cavalli-Sforza. Department of Genetics, Stanford different or more specialized functions than the classical (la) HLA genes because they University, Stanford, California. have limited tissue distributions and less allelic variation. These genes may therefore be Microsatellites, which are abundant in human genome, are highly under the influence of a than poly- different form of selection that acting on other HLA genes, morphic due to allelic variation in the number of 2-5 base pair repeat units. or they may be evolving neutrally. We previously reported (Grimsley and OberAm JHum The Genet 57 utility of such genetic markers in human evolutionary studies are ex- (supp):A163, 1995) that the nearly equal frequencies of the two HLA-E alleles plored systematically in our lab. Recently ewe have finished genotyping in African American, Hispanic, and Caucasian populations are suggestive ofsome form a of collection of worldwide populations at more than 100 highly informative mi- Stabilizing selection acting to maintain these alleles in diverse populations. In this study, crosatellite loci in 14 worldwide we tested this hypothesis by comparing the populations. The genotyping effort has also observed allele frequencies to the exiect been extended to 30 distribution ofallele frequencies under 1 additional worldwide populations (mostly from Asia and a neutral model using the Watterson's statistic. at 30 to 60 selected Homozygosity values (F) were 0.513 for African Africa) markers which seem to be more informative than Americans, 0.508 for Hispanics, 0.506 the others. Ten individuals were for outbred C sians, 0.512 for the Hutterites, and 0.635 for Chinese. These values are Chimpanzee typed as an outgroup popu- significantly lower than that lation. This study has been conducted to address the following questions expected for a neutrally evolving gene in Caucasians both and (p=0.047) and the Hutterites (p=0.020) and marginally significant in African empirically theoretically: (1) the reliability of the reconstruction of Americans in terms of the (p=0.063) and Hispanics (p=0.053). The low homozygosity values for HLA-E are similar population phylogeny selection of genetic distance measures to other HLA loci but different from several as well as the choice of tree-making methods; (2) the utility of microsatellites non-HLA, MHC encoded genes. Thus it is in the unlikely that the lower than expected homozygosity values are due to hitchhiking or other studies of distantly related populations as well as closely related pop- effects of selection acting on neighboring class la genes Rather, the low homozygosity ulations where migration is not negligible: (3) the robustness of phylogeny of index is consistent with some form of stabilizing selection, such as balancing selection, individuals; (4) the criteria and procedures that allow us to select a subset acting to maintain these alleles. Consistent with this hypothesis is the conservation of the of genetic markers that can be recommended to worldwide genotyping effort exons encoding the extracellular domain compared to intronic sequences. Whereas only a in the future; and (5) the reliable estimation of the time of divergence be- Single base pair substitution was found in exons 2, 3 and 4 in 67 individuals, four tween major racial groups using genetic distance measures that have a linear substitutions have already been identified in intron 3 in the first six individuals examined. relationship with the time of divergence. Collectively, these data suggest that selection is influencing the evolution of this gene and that the HLA-E protein is functional Supported by SBR-9423509.

146 147 Human genomic diversity created by the insertion of Alu retroposons. Amassing a wealth of Y chromosome polymorphisms for evolutionary MU.A\.Batzer123, S.S. Arcot4, M. DeAngelis',3, B. Kanagyt 2. A. W. Adamson4, studies. P. A. Underhillt. L.Jint. L. L. Cavalli-Sforzat- R. Davist and P. J. J. E. V.Carrano4,M.Stonekingsand P. L. Deininger2. 'Depts. of Oefnert. Departments of iGenetics and -Biochemistry, Stanford University, Pathology, 2Biochemistry and Molecular Biology, and 3Biometry and Genetics, Stanley Stanford, California. S. Scott Cancer Center, Louisiana State University Medical Center, New Orleans, LA. The non-recombining portion of the Y chromosome preserves haplotype 4Human Genome Center, Lawrence Livermore National Laboratory, Livermore, CA. information over time scales spanning modem human history. In addition, most 5Dept. of Anthropology, Pennsylvania State University, University Park, PA. nucleotide substitutions, as well as small insertion/deletion polymorphisms on Over 500.000 Alu repeats are dispersed throughout primate genomes in a semi-random the Y display geographical localization making them particularly valuable manner. Alu elements may be divided into subfamilies of different genetic ages. Three indices of gene flow. Nearly 100 kb of primary Y chromosome sequence has subfamilies (Ya5, Ya8 and Yb8) appear to be of very recent evolutionary origin, having been generated from cosmids, creating an abundant reservoir of new Y-specific largely expanded within the human genome sometime after the divergence of humans STS's. Our ongoing screening effort for polymorphisms, which is based upon a from African apes. We have screened a randomly sheared total human genomic library sensitive and rapid heteroduplex detection method, is accomplished using for members of the Ya5 and Ya8 Alu subfamilies. Over 60 individual loci were subjected denaturing high performance liquid chromatography (DHPLC) to comparatively to DNA sequence analysis, and oligonucleotide primers complementary to the 5' and 3' sequence males (n 2 50) from globally diverse populations. Each automated flanking unique DNA sequences of each element were used in polyrnerase chain reaction analysis compares two genomes as a mixture of denatured and reannealed (PCR) based assays to determine the chromosomal location, phylogenetic amplicons, in only minutes for - 20 ¢ per assay. We describe a collection of distribution, markers which are defined both in terms of geographic affiliation and frequency and human genetic variability of each Alu repeat. All of the young Alu elements were re- within stricted to the human genome, and appear to have integrated randomly within the human indigenous populations. Approximately 50% of the markers display a genome. The polymorphic Alu fossil relics displayed frequency > 15 %. The majority of polymorphisms discovered to date have a significant amount of variation in accumulated in African and some Oceanian populations, reflecting the antiquity presence/absence allele frequencies within human population groups from different geo- of those populations. Our estimate The combination of over updated of nucleotide diversity for the Y is graphic origins. twenty identical by descent polymorphic Alu not much smaller then that observed in autosomes. This insertions provides an unprecedented set of nuclear markers for human reinforces our earlier lation resolving popu- observation that selection is not a significant factor in explaining the reduction relationships. Work at Lawrence Livermore National Laboratory was performed of variation observed on the Y chromosome. under the auspices of the U.S. Department of Energy contract No. W-7405-ENG-48. Slide Session 38: Genetic Epidemiology and Population Genetics II: Molecular Evolution (cont.)A31 148 149 Tracking male-mediated migrations in Asia: the origin of the Jomonese. MS Dispersion of human Y chromosome haplotypes based on five microsatellites In Hamme 1. T. Karafetl. S. Harihara2. S. Horai3. and M. S'&kUA IDivision of global populations. R. Dekal. L Jin2. M.D. Shriver'. LM. Yul. N. Sahal. R. Biotechnology, University of Arizona, Tucson, AZ; 2Dept. Biological Sciences. Barrantes . R. Chakraborty' and R.E. Ferrell' l Department of Human Genetics, The University of Tokyo, Tokyo, Japan; 3Dept. Human Genetics, National Institute University of Pittsburgh, Pittsburgh, Pennsylvania 15261, USA, 2 Department of of Genetics, Mishima, Japan; 4Dept. Anthropology, Pennsylvania State University, University Park, PA. Genetics, Stanford University School of Medicine, Stanford, California 94305, We have used markers on the Y chromosome to test hypotheses on the origin of USA,3 Istituto de Investigaciones en Salud, Universidad de Costa Rica, San Jose, the ancient Jomon people who are thought to be the first inhabitants of the Japanese Costa Rica, Human Genetics Center, University ofTexas Health Science Center, archipelago. One hypothesis, based mainly on evidence from tooth morphology, Houston, Texas 77225, USA states that the Jomon had a southeast Asian origin and arrived in Japan around We have analyzed five microsatellite loci from the non-recombining portion of 12,000 years ago via Okinawa and the Sunda shelf'. An alternative hypothesis the human Y chromosome in 15 diverse human populations to evaluate their posits that modern humans first entered into Japan about 30,000 years ago from usefulness in the reconstruction of human evolution and early male migrations. northeast Asia 2. We suggested previously that Y chromosomes bearing the Y Alu The results show that there exists a set of alleles that are, in general, most polymorphic (YAP) element migrated to Japan with the Jomon people and that a frequent across all populations. A reconstruction of hypothetical ancestral large infusion of YAP chromosomes entered Japan with the Yayoi migration haplotypes, based on these alleles and their close derivatives are shared by starting -2,300 years ago3. Upon examination of more than 1000 Y chromosomes multiple populations across racial and geographical boundaries. Phylogenetic trees from 20 Southeast Asian and Oceanian populations we did not identify a single did not depict true genetic relationships of Y haplotypes. A network of the YAP+ chromosome. In contrast, in a survey of more than 700 males from 13 North observed haplotypes also characterized the lack of clustering of haplotypes from Asian populations, we found YAP+ chromosomes at low frequencies in the same population or geographically proximal populations. In spite of this, few southwestern Siberian Altai (3.2%) and Mongolian (2.6%) populations, and at high frequencies in Tibetans (53.3%). In combination with archeological data and distinct clusters of closely related populations emerged in the network, which are results from other Y-specific polymorphisms our data support the hypothesis that associated with population-specific alleles. A tree based on allele frequencies also the ancient Jomon people had a northeast Asian origin and migrated to the Japanese shows similar results. Lack of haplotypic structure associated with the presumed archipelago perhaps as long ago as 30.000 year bp. ancestral haplotypes consisting of individuals from almost all populations indicate extensive male migration during human evolutionary history. The convergent References: 1 Turner, C.G. Science 193, 911-913 (1976). 2 Nei, M. (1995) in The nature of microsatellite mutation confounds population relationships. Original Past ofModern Humans as Viewedfrom DNA. p. 71-91. 3 Hammer, M.F. Optimum & Horai, S. Am. J. Hum. Genet. 56.951-962 (1995). resolution of Y chromosome evolution will require the use of additional microsatellite loci, and diallelic genetic markers, with lower mutation rates.

150 151 Amerindian Y chromosomes: molecular characterization by the use of six polymorphic Use of mitochondrial D-loop polymorphisms to determine genetic markers. N.O. Bianchi'. G. Bailliet'. C.MU. Bravi'. R.F Carnese. F. Rothhammer2. V.L. relationships between ancient remains and a contemporary population over Martdnez-Marignac'. and S.D.J. Pens'. IMBICE, La Plata, Argentina'; Inst. Cs. a four hundred year period. E.Pereral, A. Schiavi', J.DaV2, C. Moraess, Antropol6gicas, Fac. Filosoffa y Letras, Univ. Buenos Aires, Argentina2; Dep. Biologia D.WVallace3, and L. Baumbach'. 'University of Miami Sch of Med, Miami, F1, Celular y Gendtica, Fac. Medicina, Univ. Chile, Santiago, Chile'; Dep. Bioquimica e 2Research Atlantica, Coral Springs, Fl, and 3Emory University School of Med, Atlanta, Imunologia, Univ. Federal de Minas Gerais, Belo Horizonte, Brazil'. GA. We analysed the frequency of six Y-specific polymorphisms in 105 Amerindian males To prove an ancestral relationship between seven 17th century human skeletons from from seven different populations, in 42 Caucasian males. and in a small number of males a Bahamian archaeological site and the contemporary inhabitants of a neighboring of African, Chinese and Melanesian origin. The combination of three of the six genetic isolate with Laron syndrome (LS), we have implemented a screen for polymorphisms produced four different Y-haplogroups. The haplogroup A (non-variant) was mitochondrial DNA (mtDNA) polymorphisms in the D-Loop hypervariable regions the most frequent one. Eighty five percent of Amerindians showing haplogroup A have the (HVR-1 and HVR-2) in contemporary and ancient DNA (aDNA) specimens with the alphoid l (alpha hlI) and the DYS19A Y-specific markers; an association that is found only aim of detecting lineage-specific polymorphisms. We describe here the completion of in 10% of Caucasians and that has not been detected in Mongolians or Africans. these studies in an adult female skeleton and nine contemporary island inhabitants. Haplogroups C (YAP+) and D (YAP+ plus an A-GG transition in the locus DYS271) are Detailed DNA sequence analysis of HVR-1 and HVR-2 has identified specific and of African origin. Four percent of Amerindians and -12% of Caucasians showed the different polymorphic changes in each of the contemporary subjects, identifying at least haplogroup C; - 1% of Amerindians and -2% of Caucasians had the haplogroup D. The four matriarchal lineage. This information is consistent with historical and genealogical haplogroup B is characterized by a C-T transition in nucleotide position 373 of the SRY information which suggests that all LS patients are derived from six island founding gene domain; this haplogroup is found in Caucasians (-12%) and Amerindians (-4%). surname lineage. All individuals studied share significant D-Loop homology to the None of the Amerindians exhibiting the haplogroups B, C or D show the haplotype published consensus sequence, indicating they are of British ancestry. alpha Similar analyses of the aDNA detected a mtDNA T16304C polymorphism which was hII/DYS 19A. By haplotyping the Alu insert and the DNA region surrounding the insert in shared with one of the LS lineage, however additional polymorphisms in HVR-2 were YAP+ individuals, we could demonstrate that Amerindian Y chromosomes bearing African detected only in the aDNA sample, thereby excluding a directancestral relationship be- markers (haplogroups C and D) are due to recent genetic admixture. Most non-alpha tween the skeleton and this contemporary lineage. Further definition of the mtDNA hap- hIIIDYS19A Amerindian Y-chromosomes in haplogroup A and most cases in haplogroup lotype of the island subjects by restriction enzyme analysis indicated that they belong to B are also due to miscegenation. We show that haplotype alpha hill/ DYS19A is in linkage the same haplogroup, defined by an Alu I site loss at nt 7025, however the aDNA sample disequilibrium with a C-T transition in the locus DYS199. Our results confirm that most does not share this Alu I polymorphism. Delineation of the aDNA mtDNA haplotype or all Amerindian Y-chromosomes derive from a single paternal lineage characterized by is ongoing to determine the genetic background of the T16304C polymorphism which the alpha hII/DYS19A/DYS199T Amerindian-specific haplotype. was determined to be heteroplasmic in the aDNA specimen using two independent DNA isolations and three different detection methods. The results of these combined stud- ies demonstrate the usefulness of mtDNA polymorphism analysis in addressing ancestry investigations in genetically defined populations over a several hundred year period.

152 A Moment Measure of Linkage Disequilibrium for Microsatellite Loci M. Kimmell, V. S. Pankratz'. and R. Chakrabortvy. 'Rice Univ. 2tniv. of Texas-Houston School of PublicefalTh Linkage disequilibrium has been successfully used for gene mapping in recent years. This method requires that the measure of linkage disequilibrium be related with the recombination fraction between loci. This presentation develops one such measure of linkage disequilibrium between microsatellite loci. at each of which mutations may lead to either contraction or expansion of allele sizes. Using the Fisher-Wright model applicable to two linked loci, we showthat under the mutation-drift-recombination equilibrium. the expected value of the covariance of the squared differences of allele sizes at two loci normalized by the product of their variances (s'), is equal to the recombination fraction subtracted from 2. In contrast, at an early generation of evolution of such loci, the expectation of aris equal to one, independent of the recombination fraction. Application of the theoryto data on four intronic tetranucleotide loci at the DMID gene shows that the polymorphisms at these loci are too recent to make them useful for predicting their map distances using this index. (Research supported by grants NIH-GM58545, GM41399, NSF-DMS9409909, and by Keck Center for Computational Biology). A32 Slide Session 39: Gene Structure and Function I 153 154 Cloning and characterization of a human regulator of nonsense transcript Determination of Ll functional domains using a high-frequency stability. H.A. Perlick. S.M. Medghalchi. F.A. Spencer. and H.C. Dietz. Johns retrotransposition assay in HeLa cells. J.V. Morani* S.E. HoIMCI,.. Hopkins University School of Medicine. 2 2 Baltimore, Maryland. Naq X Deeris 1. Q. Feng I.D. Boeke and H.H. inn I. iU. Penn. All eukaryotes that have been studied to date possess the ability to detect and degrade Sch. Med., Philadelphia, PA and 2 Johns Hopkins Sch. Med., Baltimore, MD. transcripts that contain a premature termination codon. Nonsense mediated RNA decay Human Lls are high copy number repetitive elements. The vast majority of (NMRD) likely protects the organism from deleterious peptides that could be expressed these elements are 5' truncated; however, a small minority are full-length and can from nonsense alleles if the corresponding transcripts were stabilized. NMRD has been retrotranspose. We previously isolated two active LI elements that gave rise to disease- most comprehensively studied in yeast where at least three trans-acting factors (Upf1-3) producing insertions into the factor VIII (LREI) and dystrophin genes (LRE2). Both are required. Despite the observation that mammalian NMRD occurs in elements contain two open reading frames (ORFI and ORF2), end in a poly A tail and predominantly are a the nucleus while the process to be we to determine flanked by target site duplication. ORFI encodes a 4OkD protein of unknown yeast appears cytoplasmic, sought function, and ORF2 encodes a reverse whether the two pathways utilize functionally related proteins. We have identified a transcriptase and a putative metal binding motif. We tagged LREI and LRE2 with a neo selectable marker and developed an episomal human cDNA encoding a protein with strong homology to Upfl, termed rentI (ggulator vector system to detect LI retrotransposition in HeLa cells. We find that LREI nonsense was seen at the extreme N- and C- and of transcripts). Although divergence LRE2 autonomously retrotranspose at remarkably high frequencies (I in 10-2-10-3 termini, the large central regions of the two proteins show 58% residue identity and 80% cells). We characterized four retrotransposition insertions and found that: 1) they insert conservation. Rentl is the first identified mammalian protein that contains all of the at different chromosomal locations. 2) they are significantly 5' truncated, 3) they end in putative functional elements found in Upf including zinc finger-like nucleotide binding a poly A tail ranging from 37-74 bp, and 4) they have variable target site duplications domains and all of the motifs common to members of helicase superfamily L. Our ranging from 0-215 bp. Site-directed point mutations in conserved domains of ORFI, cloning of the mouse gene, and subsequent alignment of the sequences for all related or in either the RT active site or metal binding domain of ORF2 drastically reduce the helicases, demonstrates that rentl, Upfl, MovlO, and SenI define a distinct subset ability of LREI to retrotranspose (1 in 10-6-10-7 cells). These results demonstrate that within the superfamily. Expression of a chimeric protein, containing the central distinct domains of both ORFI and ORF2 proteins are essential for retrotransposition. region Biochemical and of rentI flanked by the extreme N- and C-termini of Upfl, complements the Upf 1- mutational analyses further revealed that the N-terminus of ORF2 deficient growth phenotype in yeast. These data provide significant evidence that rentl encodes an endonuclease activity that is also critical for retrotransposition in HeLa cells. We propose that the endonuclease nicks chromosomal DNA exposing a 3'-OH is a mammalian orthologue of Upf 1. As expected for an essential component of the that apparently ubiquitous NMRD pathway, rentl is expressed in all tissues tested. serves as a primer for reverse transcription of LI RNA. Interestingly, this LI endonuclease domain shares extensive homology with a family of Although tumorigenic or accelerated be to result from evolutionarily- aging phenotypes might predicted conserved DNA repair endonucleases including E. coli exonuclease HI. In summary, the somatic accumulation of nonsense or frameshift mutations on an NMRD-deficient we have developed the first system to analyze a mammalian retrotransposon. Using no to background, apparently relevant phenotypes have been linked the map positions this system we show that distinct functional domains of the LI proteins are required for for the murine or human genes on syntenic regions of chromosomes 8 and 19pl3.2- activity and propose a model for LI retrotransposition. *Supported by the Cancer Research p13.11, respectively. Ongoing work includes functional studies in mammalian cells as Fund of the Damon Runyon-Walter Winchell Foundation Fellowship, DRG1332. well as targeted disruption of the murine gene by homologous recombination.

155 156 FUNCTIONAL CHARACTERIZATION OF LISPA, THE FIRST KNOWN ACTIVE PHOG, a candidate gene for Involvement In the short stature of Turner MOUSE Ll ELEMENT. T. P. 1. V. S.F. Naas*I Moranl. Kingsmore. M.F. eldin3 syndrome. J.W. Ellison, Z Wardak, M. Webster, and W. Chiong. University and H H Kaaan. Jr.1, tUniv. of Penn. Sch.of Med., Philadelphia, USA; 2Univ. of of Califomia, San Francisco, CA. Florida. Gainesville, USA; 3Duke University, Durham, USA. The pathogenesis of Tumer syndrome is presumably the of LINE- I (LI) is a mammalian family of highly repeated DNA sequences and some result Lls haploinsufficiency of certain genes on the X can retrotranspose. Recently, it has become evident that insertions of LI elements can chromosome. Gene dosage lead to genetic disorders in humans and mice. Kingsmore et al. (Nature Genetics, considerations lead to the prediction that the culpable genes escape X 7,1994) and Muelhardt et aL (Neuron, 12., 1994) reported that the recessive mouse inactivation and have functional Y homologs. Among the genes possessing disorder spastic (spa), a prototype of inherited myoclonus in humans, results from an these characteristics are those residing in the pseudoautosomal region (PAR). L1 insertion into intron 5 of the glycine receptor beta subunit gene (Glrb). We The distal 700 kb of the short arm PAR has been implicated as containing a previously reported (ASHG 95 abstract 206) that this insertion (Llspa) is a full length gene or genes involved in linear growth: patients with only a single copy of this LI element with two intact open reading frames (ORFI and ORF2). The element ends critical region exhibit short stature. with a 64-bp poly-A tail and is flanked by a 16-bp target site duplication, both of which It seems likely that this same dosage- are characteristics of a retrotransposition event. Surprisingly, sequence analysis of the 5' sensitive locus contributes to the short stature observed in Tumer syndrome. end revealed that this L belongs to a class of elements (F-type) previously thought to be We report here the isolation of a gene from the critical region of the PAR. ancient and inactive. The gene product is likely a transcription factor, since the predicted protein Using an in vivo assay in S. cerevisiae, we found that Llspa ORF2 encodes reverse contains a homeodomain that is similar to certain homeodomains found in a transcriptase (RT) activity, whereas an allele containing a point mutation in the RT- number of diverse organisms. active-site lacked activity. We next showed Expression ofthe gene appears to be restricted that Llspa is capable of high frequency to osteogenic cells: it Is expressed in trabecular retrotransposition from an episomal vector into the genome in human cells (HeLa) and bone cells and bone marrow mouse cells (Ltk-). Efficient retrotransposition occurs even in the absence of the 5' and stromal fibroblasts, while we have failed to detect expression in a variety of 3 untranslated regions of Llspa as long as transcription is initiated from a heterologous other adult and fetal tissues. We have named this gene PHOG, for promoter. These data indicate that Llspa, a recently retrotransposed F-type element, is aseudoautosomalhomeoboxcontaining2steogenicZeno. Its location in the the autonomously mouse. first active LI element isolated from We have shown that LI pseudoautosomal region, the nature of the predicted protein, and its restricted elements of one species can retrotranspose in the cells of another species, indicating that expression in bone make this gene an attractive candidate any putative requirements are we for involvement in host conserved. In addition, have provided the first the short stature phenotype of Turner direct evidence that some retrotransposed elements are capable of subsequent syndrome. retrotransposition. (* is supported by a longterm Fellowship of the European Molecular Biology Organization).

157 158 The choroideremla escapes some gene X inactivation in females, but aot in An evolutionarily conserved gene associated with the common others: heterogeneity in control of gene expression from the inactive deletion X breakpoint regions in the Prader-Will/Angelman syndromes. Y. Ji chromosome. LCarel and HYF Willard. Case Western Reserve Univ., Cleveland, OH L Buitingi, M:J. Walkowicz2, . K. J. M. L While it is well accepted that some X-linked genes escape X inactivation and are Johnson2, Amos-Landgraf, Stubs 2, B. R. D. Nicholls. Case Western Reserve Univ., expressed from both the active X (Xa) and inactive X (Xi) chromosomes, most models for Horstherkel, Cleveland, OH. the chromosomal control of X-linked gene expression are based on the assumption that the Univ. Essen, Essen, Germany; 2Oak Ridge National Lab., Oak Ridge, TN. status of a given gene with respect to X inactivation is constant among cells within an The majority of Prader-Willi/(PWS) and Angelman(AS) syndrome patients have a individual and among females within a population. To testthis assumption, we have similarly sized - 4 Mb deletion within chromosome 15q l-q 13, with apparently examined relative levels of expression of the X-linked choroideremia gene (CHM) from the clustered breakpoints. In studying the deletion mechanism, we have found a low copy Xa and Xi in bothm somatic cell hybrids and in diploid human fibroblasts. repeat spanning the proximal and distal breakpoint regions. Here we report the CHM expression wastested by quantitative RT-PCR analysis in a series of 10independent characterization of a novel evolutionarily conserved gene family (ERY-1,-2, MN7) hybridscarrying different inactive Xchromosomes. CHM was well expressedfro 7 Xi associated with the chromosome 15 specific low copy repeats. hybrids at levels -30-100% of Xa levels. However, in 3 additional hybrids, there was no Using different parts of the ERY-1 eDNA as a probe, three ubiquitous transcrips detectable expression of CHM (<1% of Xa levels), suggesting that the control of CHM (6.7, 10, 12 kb) are detected on adult and fetal human northens. Cloning and expression varies on different Xi. sequencing identified several genes related to the 6.7 kb transcipt (MN7, homology To evaluate this finding more directly, we analyzed expression of CHM in diploid human about 90%). We have cloned and sequenced more than 7 kb of the 12 kb putative ERY- fibroblasts, using atranscribed polymorphism to distinguish Xa from Xi transcription in 8 I cDNA, the 5' and 3' sequence of which shows 90% homology to MN7 while the cell lines from females with non-random X inactivation due to the presence of a structurally middle part is unique. ERY-) has an ORF of at least 2,200 amino acids with a 500 abnormal X. In each case, complete non-random inactivation wasestablished by amino acid segment encoding RCC-I repeats, whereas MN7 has a premature stop monoaslelic expression of a transcribed XIST polymorphism and/or by methylation analysis codon and lacks RCC-I repeats. The mouse has a single Ery-l/Mn7 locus, witha 12 kb at the AR or FMR1 loci. Biallelic CHM expression was detected in 6 heterozygous cell transcript highly expressed in testis, brain, skeletal muscle and other tissues. A 2 kb lines, establishing that CHM escapes inactivation, while monoallelic CHM expression was partial mouse cDNA has 95% amino acid identity to human ERY-). Recombinant detected in 2 othercell lines, demonstrating that the gene is subjectto inactivation in those inbred strain and deletion analysis maps the mouse Ery-l locus to the p6H deletion cases.Thus CHM escapes inactivation in some females, but not in others. Further, the region, proximal to the p gene. The neurological and spermatogenic abnormalities of level of Xi CHM expression varied among the celllines examined: expression of the Xi the p6H mouse (runtiness, nervous jerky gait and male sterility, female semisterility) are allele was -40% oftheleveloftheXaalleleinonecase,-10%ofXalevelsin 5cases,but due to dysfunction of a single gene,jdf-2 (juvenile development and fertility), and we <1% of Xa levels in the cases with monoallelic CHM expression. are currently testing Ery-I as a candidate forj#-2. We conclude that the inactivationstatus ofat least some X-linked genes vanes among PAC and phage genomic clone contigs for the human gene family at either end of different fenales, either at the cellular and/or population Whether or not level. such 15qI 1-13 are under construction to further characterize the ERY- I related gene heterogeneity is familial, these data suggest that gene expression from the Xi is under a duplications. This gene family is highly expressed in ovary and testis, and we postulate previouslyunsusc level of genetic or epigenetic control, likely involving local or that active transcription in the germline may stimulate genetic recombination, to play a regional changesin ch roatin organization that determine whether a given gene, like CHM, role in the PWS/AS common deletion process. escapes or is subject to X inactivation. Slide Session 39: Gene Structuire and Function I (cont.) A33 159 160 Isolation and characterization of a gene from the DiGeorge chromosomal Features of VCFS in a patient with a balanced translocation region homologous to the mouse Tbxl gene. C. Chicafgl, N. Garvey2, L interrupting a transcript within the minimal DGS/VCFS critical region. B&i3. L,,,. Silvers2, and M.L. 1udarfl. iThe Children's Hospital of Philadelphia, S.E. HolmesI, Sin=l, W. Gongl, J. CollinsI, BRQR2, H.E. MDermid3, Phila., PA, 2Dept. Molec. Biol., Princeton Univ., Princeton, NJ, 3University of M.A. Ria3, E.H. Zackail, B.S. Eanuell and M.L. Budarfl. IThe Children's Oklahoma, Norman, OK Hospital of Phila., Phila. PA, 2Univ. of Oklahoma, Norman, OK, 3Univ. of A number of genetic disorders map to chromosomal region 22ql 1. Amongst Alberta, Edmonton, Canada. them, DiGeorge syndrome, velocardiofacial syndrome, conotruncal anomaly face, and VCFS is an autosomal dominant disorder with variable phenotype. Findings isolated forms of conotruncal cardiac defects have been found to have 22q 1I include congenital heart defects, cleft palate, characteristic facial features, and microdeletions. These syndromes are considered to result from failure of proper neural learning difficulties. The DGS/VCFS minimal critical region (MIGCR) within 22q11 crest cell migration in embryogenesis. Further, the autosomal dominant form of Opitz is -300 kb, and is the region most consistently deleted in the patients we have syndrome has been linked to chromosomal region 22q and shown to be associated studied. A transcription map of the MDGCR has been generated. A de novo, with 22q 1I deletions in some cases. This region of 22q 1I is called the DiGeorge balanced translocation within the MDGCR has been identified in a 25 year old male. Chromosomal Region (DGCR) and spans approximately 1.2Mb. Using cosmids from He has some features associated with VCFS, including facial dysmorphia, mental the DGCR and exon amplification as a method for gene identification, we isolated an retardation, long slender digits, and genital anomalies. We have cloned the exon with homology to the mouse Tbxl (Mn-Tbxl) gene. This exon was used as a breakpoint of this patient's translocation and show that it interrupts a transcript within probe to screen a cDNA library. A partial transcript was obtained. Using 5' and 3' race the MDGCR. The translocation partner is the short arm of another acrocentric a nearly full length transcript has been identified. The transcript covers approximately chromosome. Comparison of the sequence at the breakpoints with the sequence from 24 kb of genomic DNA and 813 bp of cDNA sequence. It contains at least 9 exons. the normal homologues indicates that no nucleotides were lost; rather there is one The genomic organization is similar to Mn-Tbxl. Northern blot analysis identifies a extra 'A' not derived from either of the involved partner chromosomes. Analysis of 2.0 kb transcript in human adult skeletal muscle and testis. The deduced amino acid the sequence in the immediate vicinity of each breakpoint does not reveal any repeated sequence shows 98% identity over 220 amino acids with Mn-Tbxl. Human TBX1 is sequences potentially contributing to the mechanism of the translocation. The a member of a phylogenetically conserved family of genes that share a common DNA breakpoint on the acrocentric short arm is telomeric to the rDNA cluster and falls binding domain, the T-box. The mouse brachyury gene was the first member of this within a sequence which we have found to be repeated on all of the acrocentrics. The gene family to be discovered. Brachyury previously has been shown to play a role in patient's phenotype is unlikely to be caused by this disruption, as interruptions of mesoderm development. Several mouse T-box containing genes have been identified acrocentric short arms have not been associated with an abnormal phenotype. We and recent studies suggest that these genes may function where inductive interactions postulate that the patient's phenotype is due to the interruption of the chromosome 22 occur. Mn-Tbxl expression is localized to the lateral plate mesodern, the branchial transcript in which the 3' end of the ORF is separated from its 5' end by the arches, the otic vesicle and the optic cup. Peak expression was seen in day 9.5 rearrangement. Characterization of this breakpoint strongly suggests a role for this embryos, the same time at which neural crest cell migration is occurring. Studies to transcript in the VCFS features found in our patient. The gene's structure and determine whether Human TBX1 can be implicated in the etiology of syndromes additional characterization are well under way and will be discussed. Finally, the involving the 22q1 1 chromosomal region are in progress. partial VCFS phenotype of our patient suggests the possibility that haploinsufficiency for more than one gene in the MDGCR is etiologic for VCFS/DGS.

161 162 Expression of the DMAHP gene is suppressed in cis by the myotonic Characterization of proteins which interact with the Fmrl gene product dystrophy (MTD) CTG repeat expansion. C. A. Thornton, J. P. Wymer. by two hybrid analysis. K. A. Lugenbeel and D. L. Nelson. Department of and R. T. Moxley. University of Rochester, Rochester, NY Molecular and Human Genetics, Baylor College of Medicine, Houston, TX. A putative homeodomain gene (DMAHP) is positioned within 2 kb centromeric to the Fragile X syndrome is characterized by absence of the protein product of the FMRI MTD (CTG)n expansion (Boucher et al, 1995). We RT-PCR amplified, cloned, and se. gene (FMRP). While the majority of fragile X patients demonstrate an expansion quenced partial DMAHP cDNAs to identify a coding sequence polymorphism (C to T at and methylation of the CGG repeat located in the first exon of the gene, patients have nt 4143, GenBank X84813) that eliminates a Cac8l restriction site. The (CTG)n expan- been identified with intragenic mutations that demonstrate FMR1 is the primary gene sion in MTD was in complete linkage disequilibrium with the type 1 (Cac8l restricted, involved in fragile X syndrome. We have used the yeast two hybrid system to 56% of normal chromosomes) allele. To test for an effect of the (CTG)n expansion on identify proteins which interact with Fmrp. The full length murine Fmrl cDNA was DMAHP expression, myoblasts or myotubes were cultured from subjects who were het- the "bait" construct used to screen a day 9.5 and a day 10.5 mouse embryonic "prey" erozygous for the exon polymorphism. RNA was isolated from these cultures, and RFLP libraries. Two clones, KL5 from the day 10.5 library and KL5 1 from the day 9.5 analysis was done on RT-PCR products. DMAHP mRNA synthesized from the mutant library, were true positives. Sequence analysis indicates that KL51 is a fragment of chromosome was reduced, and the magnitude of this effect depended on the extent of Fxrl (Fragile X related-I). Expression analysis of Fxrl indicates low level (CTG)n expansion. The ratio of type 1/type 2 transcripts was 94 +/- 10% in normals expression of two transcripts in all adult mouse tissues tested, except in testis where (n=3), 71 +/- 15% in mild MTD ((CTG)n < 100, n=3), and 19 +/- 8% in moderate only the smaller transcript is present at a much higher level than observed in other or severe MTD ((CTG)n > 100, n=4). In RNA isolated from postmortem tissue of 1 tissues. Sequence analysis of KL5 indicated significant similarity with FMR1 and patient, the ratio of type 1/type 2 transcripts was 33% in muscle, 38% in heart, and 41% FXR1; because of this significant similarity, the clone was named Fxr2 (Fragile X in cerebral cortex. These results are the first experimental support for a position effect related-2). Northern analysis of Fxr2 detected a transcript of 3.4 kb in all adult of the (CTG)n expansion on the expression of neighboring genes. Reduced expression mouse tissues examined. Northern analysis of FXR2 in adult human appears to be of DMAHP, which has significant homology to the muscle-specific transcription factor restricted to heart and skeletal muscle, while expression was seen in all human fetal AREC3 (Kawakami et al, 1996), may contribute to the myotonic dystrophy phenotype. tissues tested. KL5 was then used to screen mouse brain and mouse heart cDNA libraries for Fxr2. Positive clones have been identified and sequence has been obtained. Sequence comparison has indicated 90.4% identity at the nucleotide level and 98.5% identity at the peptide level over approximately 2.2 kb with human FXR2. Sequence of the 5' portion of the gene is currently underway. Work has also begun to examine the effect of a missense mutation (De Boulle et al., Nat. Genet., 1993) on the interactions of Fnrl with Fxrl and Fxr2. Preliminary evidence using a yeast mating type assay suggests that this mutation disrupts the ability of Fmrp to interact with Fxrl and Fxr2. These types of protein studies should give evidence for a pathway in which FMRP interacts, which should further our understanding of the pathogenesis of the disease.

163 Construction of a synthetic single-chain antibody phage display library and its application to genetic disease. G.R. Thomas', A. Mackenzie., D.A. Cox3, J.R. Forbes3 and S.A. Narang'. 'National Research Council of Canada. Ottawa. Ontario, Canada. 2Children's Hospital of Eastern Ontario. Ottawa. Ontario, Canada. 3The Hospital for Sick Children. Toronto. Ontario, Canada. The wide spread success of the positional cloning approach has resulted in the identi- fication of an increasing number of gene products which when mutated or absent result in inherited human disease. The isolation of antibodies with high affinity and specificity for such proteins is clearly a priority. This is currently accomplished through animal systems which are both expensive and time-consuming. The selection of single-chain variable fragment antibodies displayed on the surface of 1113 phage would circumvent the problems inherent in animal derived reagents. Single-chain antibodies consist of the variable regions of both heavy and light immunoglobulin molecules, linked as a single protein. Phage which carry high affinity single-chains can then be selected by several rounds of panning and amplification against antigen immobilized in solid phase. Clones identified in this manner are essentially monoclonal antibodies that can be produced and isolated from E. coli culture. We have constructed a synthetic single-chain library by recursive PCR for use in selecting antibodies against the spinal muscular atrophy associated Neuronal Apoptotic Inhibitor Protein (NAIP) and the Wilson disease protein (ATP7B). The use of a synthetic single-chain phage display library permits control over cloning sites and the level of diversity. Moreover, clones with moderate affinity can be subjected to error-prone PCR followed by further rounds of panning to select higher affinity variants. The library has been designed such that the third complementary de- termining region (CDR) of the heavy chain gene mimics the high level of variation seen at this location in vivo. The length and amino acid content of CDR3H have been varied to produce a potential variation of l01' different clones. A34 Slide Session 40: Cancer Genetics II: Breast Cancer 164 165 BRCA 1 Mutation Detection Using High Density Oligonucleotide Rapid automated mutation scanning of farge multi-exon genes: Application to BRCA2 Arrays. Joseph G. Hacial, Lawrence Brody', MarLk Chee2, Steve Fodor2, gene. T.Ganguly. RDhulipala. K.D.Leahy, L Godmilow and A. Ganguly. Genetic Francis S. Collinsi. National Institutes of Health, National Center for Human Diagnostic Laboratory, Department of Genetics, University of Pennsylvania, Genome Research, Laboratory of Gene Transfer, Bethesda, MD, 2Affymetrix, Philadelphia, PA 19104. Inc., Santa Clara, CA. Mutation analysis of cancer susceptibility genes has opened up a new era in clinical Allelic heterogeneity of BRCAJ mutations represents a considerable technical genetics. However, clinically feasible testing is technologically challenging as the challenge for developing a diagnostic test capable of identifying quickly, efficiently, genes, such as BRCA1 and BRCA2, are usually large and multi-exon with multiple and inexpensively all possible mutations in this large gene. We have been mutations. With a view to offering rapid and reliable mutation analysis results to exploring the use of "DNA chips" for BRCAI mutation detection. In this patients with a positive family history of breast and ovarian cancer syndrome, we have approach, oligonucleotides 20-nt in length and of known sequence are synthesized automated our conformation sensitive gel electrophoresis (CSGE) mutation scanning directly on a solid support in a gridded array. Over 45,000 oligonucleotides are assay. The BRCA2 gene is amplified in 54 separate fragments covering all 27 coding present on a 1.28 x 1.28 cm chip representing the entire 3.5 kb exon 11 sequence exons and the exon-intron boundaries from genomic DNA using PCR primers, of as well as all possible nucleotide substitutions at each position. Probes representing which one is fluorescently labelled with one of the three dyes (FAM-blue, HEX-yellow all possible single base pair insertions, one through five base pair deletions, and and TET-green). After PCR, PCR products of three different color are analyzed on a other known mutations are also contained in the array. PCR amplification and single lane of the ABI 377 machine allowing analysis of 108 PCR products in a 2.5 fluorescent labelling of BRCA1 exon 11 from a patient sample, followed by chip hour run. This translates into preliminary screening of the entire coding sequence of the hybridization and data collection using scanning confocal microscopy examines BRCA2 gene for 4 patients, or the BRCAI gene for 7 patients, in two days when the whether a mutation is present. The method is particularly powerful if two-color analysis time is taken into consideration. The gel image is analyzed using the fragment analysis is used where a normal control labelled in one color and patient sample analysis software, Gene Scan (ABI, CA) and directly exported to specific database for labelled in another color are competitively cohybridized to the array. Localized each individual. The advantages of this technology are the unparalleled reproducubility differences in color ratio measurements highlight regions of sequence change. The and resolving power of the scanning method. Multiplexing of different PCR products is exact sequence of these changes can be inferred from hybridization to allele specific now been attempted. Only the aberrantly-migrating heteroduplex bands are sequenced. oligonucleotides on the chip and confirmed by dideoxysequencing. This approach To date, we have completed mutation analysis of the BRCA2 gene of 26 individuals shows great promise for the rapid detection of BRCA1 mutations and should be affected with breast and or ovarian cancer and a positive family history, but negative for generalizable to detection of mutations in any gene. BRCA1 mutations. Numerous polymorphisms have been detected. Five mutations have been confirmed in the BRCA2 coding sequence including two 6174 delT mutations in exon 11 of BRCA2. None of these families had any other male member affected with breast cancer nor was there any distinctive cancer in any of these families to suggest which BRCA gene should be analyzed. (Supported by a grant from NIH, IR21CA66179 and use of an ABI 377 machine from PE ABD, CA)

166 167 Automated detection of BRCA1 185delAG mutation in Jewish population The Carrier Frequency of the BRCA2 6174delT Mutation Among Ashkenazi using fluorogenic PCR. M.R. Abhaszadegan'. T. Iwata'. B.C. Lanpher' JP Jewish Individuals is Approximately 1%. C. Oddoux J. P. Struewing2.C.M, Struewing'. L.C. Brody'. and M.R. Hughes . National Center for Human Genome Claon' L C-Broly S_Neuhausenl M KaaCk' D.Goidga' H_Ostrer'-K Research, NIH, Bethesda, MD and 2Georgetown Univ., Washington D.C. Offit". 'New York University Medical Center, New York NY, National Institutes of The Breast and ovarian cancer susceptibility gene (BRCAl) is associated with high Health, Bethesda, MD, University ofUtah, Salt Lake City, UT, 4University incidence of breast and ovarian cancer in some families. Mutations in BRCAI gene of were detected in 45% of families with high incidence of breast cancer and 80% of California, San Diego, CA, 'International Agency for Research on Cancer, Lyon, families with increased incidence of both early-onset breast cancer and ovarian cancer. France, Memorial Sloan-Kettering Cancer Center, New York, NY In high risk pedigrees, female carriers of BRCAI mutations have an 80-90% lifetime Certain germline mutations in either the BRCAI or the BRCA2 gene confer a risk of breast cancer, and a 40-50% risk of ovarian cancer. Recently, we reported that lifetime risk ofdeveloping breast cancer that may approach 90°/.. The 185delAG approximately I in 100 of Ashkanazi Jewish individuals carry the BRCAI 185delAG BRCAI mutation was found in 20%/ and the 6174delT BRCA2 mutation in 8% of frameshift mutation. In contrast, the BRCAI carrier frequency for all mutations AshkenaziJewish women with early-onset breast cancer. The 185delAG combining in the general population is estimated to be about I in 800. Large studies are mutation needed to obtain more precise estimates for the BRCAI carrier frequency and predictive was observed in 0.9h/ of858 Ashkenazi Jews unselected for a personal or family value of the 185delAG mutation in the Ashkanazi Jewish population. We developed a history ofcancer. Assuming comparable age-specific penetrances, a frequency of rapid, reliable and a high through-put PCR-based screening system using the Applied 0.3% wasestimated for the 6l74delT BRCA2 mutation. To test this hypothesis, we Biosystem PCR technology. This system uses fluorogenic probes to quantitatively performed a population survey of 1255 Jewish individuals. In two independent detect specific nucleic acid sequence. These probes consist of an oligonucleotide with groups, ascertained from heterozygote detection programs for other conditions, a both a reporter and quencher dye attached. AmpliTaq DNA polymerase 5'-3' prevalence of 1% 0.6 - was observed of6174delT. endonucleolytic activity cleaves the probe during PCR when it hybridizes to the approximately (CI 1.5) From these template, resulting in an increase of reporter fluorescence signal that can be detected on findings, the relative risk ofdeveloping breast cancer by age 42 was determined to be a fluorometer. PCR products are quantitated by changes in fluorescence emission of 9.3 (C.I. 2.5 - 22.5) for 6174delT, compared to 31 (C.I. 11 - 77) for 185delAG. both reporter dye and quencher dye, before and after cleavage of the fluorogenic probe Although these findings may be explained by chance variation, the more likely that occurs during PCR. The presence of the wild-type BRCA1 sequence is explanation is a difference in age-specific penetrance for the two mutations. These distinguished from the mutant sequence by differential fluorescence emission of two results indicate that one in 50 Ashkenazi Jewish individuals harbor specific germline different reporter dyes (FAM for wild-type probe and TET for mutant probe). A set of mutations in BRCAIor BRCA2 and that recombinant wild-type and mutant BRCA1 target sequence was constructed to calibrate genetic counseling for these individuals must the LS-50B Luminescence Spectrometer. This method allows 96 patient samples to be be tailored to reflect the different risks associated with the two mutations. analyzed at this BRCA I mutation in about 20 minutes without the need for radioactivity, gel electrophoresis or membrane blotting/hybridization.

168 169 Ashkenazic Jewish population frequencies of common BRCA1 and BRCA1 Mutations in African-American Women L F.ArenaI, S. Snmithl, BRCA2 breast cancer gene mutations. B.B. Roa, A. A. C. S. of Molecular and Boyd. Richards. M1. Plewinska'. L. Gayol', E.Pereral, P.Murphy2, and H. Lubs1 .'Universityof Dept. Human Genetics, Baylor College of Medicine, Houston, Texas. Miami School of Medicine, Miami Florida. The gene on chromosome and the BRCA2 on 'Oncor!vled Inc., Gaithersburg, Maryland. BRCAI 17q21 gene 13q12-13 are the Breast cancer in African-Americans associated a two penetrant autosomal dominant that account is with poorer prognosis than in Cau- highly genes for the majority of the 5% to casians. African-American women tend to an 10% of inherited breast cancer cases in the Mutations in present at earlier age with larger tumors population. the BRCAJ gene and a more advanced stage disease. Studies to detect a confer inherited susceptibility to breast and ovarian cancer, and mutations in are designed possible molecular basis BRCA2 for this difference have not been reported. As part of a primarily associated with breast cancer, including male breast cancer. Although the total pilot project investigating the frequency of all known BRCA1and BRCA2 mutations is relatively low in the general presence of BRCA1mutations in the South Florida population, 3 African- American mutations were identified patients with a strong family history of breast cancer were investigated. Mutations were population, specific recurrently in Ashkenazic Jewish patients. found in all three This study will analyze the frequency of multiple recurrent mutations in the Ashkenazic cases and all were previously unreported mutations in exon 11(943 Jewish the ins 10bp, 3888 del GA and 4160 del AG). Because of these findings, we investigated population, including 185delAG, 188del1I, and 5382insC mutations in forty-two additional African-American BRCAI, as well as the 6174delT mutation in the BRCA2 gene. Five thousand patients with early onset breast cancer for the anonymous DNA samples from Ashkenazic Jewish and 1000 from presence of mutations in exon 11 of BRCAL.Eight patients (19%) had a positive family individuals, samples history for breast/ovarian cancer with at one more a reference mixed-ethnic population, all of whom were unselected for breast cancer, will least affected relative. All others cases be screened for a number of recurrent BRCAJ and BRCA2 mutations. Preliminary (81%) were sporadic. Our search, using four sets of PCR primers, was directed to the studies have been completed on approximately 3000 Ashkenazic Jewish DNA samples regions in exon 11 surrounding these mutations. Only one mutation was found, a second that were screened for a panel of five BRCAJ mutations. A total of 34 out of 3108 943 ins 10bp mutation in an unrelated family. However, we found a previously unde- samples (1.09%) were identified as heterozygotes for the BRCAJ 185delAG mutation, scribed polymorphism in exon l1(A3557G), which waspresent in 4 of 100 chromosomes which is consistent with the published carrier frequency of approximately 1% in the from African-American controls and in none of 46 chromosomes of White controls. We Jewish population. Four out of 3116 samples (0.13%) tested positive for the 5382insC conclude that, at least in our series, African-Americans with early onset breast cancer mutation, which appears to be about 8-fold less frequent than the 185delAG mutation. and strongly positive family histories may carry BRCA1 mutations different from other Zero carriers were found for the 188de11 1, C6IG, and the 4184deITCAA mutations. ethnic groups and that further studies are urgently needed to elucidate the possible role Preliminary screening for the BRCA2 6174delT mutation identified 8 heterozygotes out of genetics in producing a worse breast cancer prognosis in this ethnic group. of 652 samples tested (1.2%). Thus, the 185delAG mutation in BRCAI and the 6174delT mutation in BRCA2 appear to constitute the two most significant breast cancer susceptibility gene mutations in the Ashkenazic Jewish population. The accurate population-based determination of BRCAI and BRCA2 mutation frequencies from these studies can clarify the genetic breast cancer risk among Ashkenazic Jewish individuals. Furthermore, these results may have wide-ranging implications for the consideration of potential carrier screening for this population, should other fundamental issues regarding breast cancer carrier screening be resolved in the future. Slide Session 40: Cancer Genetics II: Breast Cancer (cont.) A35 170 171 Complete genomic sequence and analysis of 117 kb of human DNA Characterization of BRCA2 mutations and cumulative risk of breast and containing the BRCA1 gene. TNM Smith', MK Lee2, Cl Szabo2 N Jeromet. ovarian cancer associated with BRCA2 M McEuenl, M Taylor'. L flood', and M-C King2. Depts. of Mlolecular E.L.Schubertl J. Morrow! R. Argonzal S. Grimmond2 M. Lee! J.Hullj Biotechnology', Medicine2 and Genetics2, U. Washington. Seattle. 'Presenting author N. Havward, M-C King Over 100 distinct disease associated mutations have been identified in the breast-ovarian 1University of Washington, Seattle and 2Queensland Institute of cancer susceptibility gene, BRCA1. Loss of the wild type allele in > 90% of tumors from Medical Research, Brisbane, Australia patients with inherited BRCA1 mutations indicates tumor suppressive function. The Cancer is linked to BRCA2 in seven families from our series with 4+ low incidence of somatic mutations suggests that BRCAl inactivation in sporadic tu- cases of breast and/or ovarian cancer. Families were not selected mors occurs by alternative mechanisms, such as interstitial chromosomal deletion, or for occurrence of male breast cancer, but it appeared in five of the reduced transcription. The genomic sequence of BRCA1 identifies possible features con- seven large kindreds. The seven BRCA2-linked kindreds include 56 tributing to chromosomal instability and potential transcriptional regulatory elements. cases of breast cancer in women, 10 cases of breast cancer in men, and 6 cases of ovarian cancer. BRCA2 exons 2-27 and the A 117143 bp DNA sequence encompassing BRCA1 was obtained by random sequencing splice junctions were screened using SSCA in genomic DNA from linked affected of four cosmids identified from a human chromosome 17 specific library. Sequence chro- mutations in four of the seven kindreds. matograms were analyzed using the phred base-calling program (P. Green. B. Ewing) individuals, revealing Mutations were confirmed sequence All four mutations and the phrap assembly algorithm (P. Green). The 23 exons of BRCA1 span an 81 kb by analysis. cause frameshifts: 1533 del 4 (family 16), 2034 ins A (family 9), exon region which has an unusually high density of A.u repetitive DNA (46%). The richness 11 ins 5 (family 99) and 6174 del T (family 11B). In the linked of repetitive sequences may render this region highly recombinogenic. Miscellaneous families without identified mutations, other detection methods will be repeats constitute 5.9% of BRCA1 intronic sequences. In contrast, the BRCA2 region contains only 18% Alu repeats but has 20.2% miscellaneous repeats. Intron lengths for used to detect splice junction and nonsense mutations missed by genomic SSCA. Among women with identified BRCA2 mutations, cumulative both genes are comparable, ranging from 1-500 bp to > 8 Kb. BRCA1 intragenic mi- risk of breast cancer is 0.48 by age 50 and 0.72 by age 65 and crosatellite markers D17S1323, D17S1322, and D17S855 localize to introns 12, 19 and cumulative risk of ovarian cancer is 0.26 by age 60. Among men with 20, respectively. In addition to BRCA1, the contig contains two complete genes: Rho7, identified BRCA2 mutations, cumulative risk of breast cancer is 0.55 a member of the rho family of GTP binding proteins (X95456), and VatI, a choliner- by age 70. Thyroid and esophageal cancer each occurred once among gic synaptic vesicle membrane protein (U18009). CpG islands precede the three genes. women with BRCA2 mutations, at ages 37 and 78, respectively. No Partial sequences of 1A1-3B (U37574), which encodes CA125, and Ifp35, an interferon within the An L21 ribosomal cancers other than breast developed by age 70 aaong men with induced leucine zipper protein, also reside contig. protein identified BRCA2 mutations. is intron The order of on the chromosome pseudogene embedded in BRCA1 13. genes Supported by NIH R01 CA27632, DOD BCRP 2340, and the Komen Foundation. is: centromere-Ifp35-VatI-Rho7-BRCA1-lA1.3B-telomere. Complete structures for the genes were determined by a combination of similarity searching and analysis with the gene prediction algorithms, genefinder and grail 1.3.

172 173 BRCA1 and BRCA2 mRNAs are coordinately elevated in breast cancer High frequency of germline BRCA1 and BRCA2 mutations in familial cells by estrogen. A..M. Bowcock and M.A. Spillman. University of Texas ovarian cancer. S.A. Gayther 1. P. Russell 1, P. Harrington 1. P. Pharoah 1, 2 Southwestern Medical Center, Dallas, Texas. K. Foster 1, R. DiCioccio .1, D. Easton 2, B.A.J. Ponder 1, Estrogen modulates growth and differentiation of human breast epithelium: however. The UKCCCR Familial Ovarian Cancer Study Group. 1. CRC Human Cancer the exact pathway by which it exerts its effects has not been elucidated. Inherited predis- Genetics Research Group, Cambridge University, Cambridge. UK; 2. Department of position to breast cancer affects 3-10% of women, and the recent isolation of the breast Community Medicine, University Forvie Site, Cambridge. UK; 3. The Roswell Park cancer susceptibility genes BRCA1 and BRCA2 has permitted the examination of their Cancer Institute, Department Gynecologic Oncology Research, Buffalo, USA. role in breast tumorigenesis. The steady state levels of BRCA1 and BRCA2 mRNAs were We have analvsed 125 ovarian cancer families ascertained in the UK for germline shown to be coordinately elevated by the steroid hormone estrogen but not progesterone mutations of the BRCA1 and BRCA2 genes. Families were selected using a minimum in the human breast cancer cell lines BT-483 and MCF-7. Two different classes of antie- criteria of two or more epithelial ovarian cancer cases amongst first degree relatives. Forty strogens, trans 4'-hydroxytamoxifen and ICI 182,780 blocked the elevation of BRCA1 five of these families contained three or more cases of ovarian cancer while 24 families and BRCA2 mRNA levels, confirming that the effect of estrogen was mediated through contained two or more cases of breast cancer under the age of 60 years in addition to the estrogen receptor. In BT-483 cells, BRCA1 and BRCA2 mRNA levels were both ovarian cancer. A combination of the protein truncation test and SSCA/heteroduplex elevated 18 to 24 hours after estrogen stimulation suggesting that the effect of estrogen analysis were used to screen the entire coding sequence of both genes. Mutations were was indirect. Cycloheximide blocked the estrogen effect implying that estrogen induces detected in 58 families of which 47 (37%) were BRCA1 and 11 BRCA2 (9%). Almost all of synthesis of an unidentified estrogen-responsive protein(s) that is then responsible for these mutations are predicted to result in premature truncation as a result of frameshift, the coordinate elevation of BRCA1 and BRCA2 mRNAs. Failure to induce the postu- nonsense or splice site alterations. BRCA1 or BRCA2 mutations were identified in 24% lated estrogen responsive protein or alterations in the regulatory pathway involving the of families with two cases of ovarian cancer only, 61% of families with three or more elevation of BRCA1 and BRCA2 mRNAs could result in the malignant transformation cases and 75% of families with ovarian cancer and two or more cases of breast cancer of some cells through the loss of BRCA1 or BRCA2 transcripts. under the age of 60. Although a proportion of families selected for this analysis are likelv to result from chance clustering of ovarian cancer, the absence of either a BRCA1 or BRCA2 mutation in approximately half of all families suggests that other ovarian cancer susceptibility genes exist.

174

Hereditary breast and ovarian cancer syndrome: A significant fraction of familial cases is not caused by mutations in BRCAI or BRCA2 genes. A.Ganguly. K.D.Leahy. R.Dhulipala. L Godmilow and T.Ganguly. Genetic Diagnostic Laboratory, Department of Genetics, University of Pennsvlvania, Philadelphia. PA 19104. Mutations in two breast cancer genes, BRCAI and BRCA2. are alleged to account for 85 to 90% of familial disease. However, we have obtained different results from mutation analysis in women with a positive family history of breast and or ovarian cancer syndrome but no linkage information. We have screened genomic DNA from 96 affected individuals for all coding exons as well as the intron-exon boundaries of the BRCA1 gene by conformation sensitive gel electrophoresis followed by sequencing of the aberrant heteroduplex bands. This panel included 18 individuals with premenopausal breast cancer but with only one affected first degree relative. Five of these women had mutations in the BRCA1 gene. The remaining 78 individuals had variable age of onset with at least 2 first degree affected relatives and or multiple second degree affected relatives. Only 17 (22%) significant mutations were detected in these individuals including two novel mutations in exon 12 and one in exon 11. Twenty seven Jewish women were part of this screening panel. Four patients carried the delAG185 mutation in exon 2(15%) while 3 carried the InsC5382( 11%) in exon 20 of the BRCA1 coding sequence. This result has special relevance to the Jewish population as at least one commercial enterprise at present is offering screening for delAG185 mutation in BRCAI without simultaneously offering screening for the InsC5382 mutation. When the first 26 BRCAI-negative patients were scanned for mutations in the entire coding sequence of the BRCA2 gene by similar strategies as those used for BRCA1, only 5 (18%) mutations were detected. The above results can be summarized as follows: About 40% of familial patients have germline mutations in the BRCAI(22%) or the BRCA2(18%) gene. Although a major class of mutations involving the non-coding regions of the BRCA genes. such as promoter or intron mutations has not been ruled out, our data suggest the existence of other as yet unidentified genetic loci causing disease susceptibility. (Supported in part by a grant from NIH, IR21CA66179 ) A36 Slide Session 41: Clinical Genetic.s, Malformations and Dysmorphology 11 175 176 A Better Than Expected Developmental Outcome in Pfeiffer Syndrome Type 2 Clinical and molecular comparisons between severe forms of the Marfan and 3. NH Robin', JA Scott'. JE Arnold2, JA Goldstein3, BB Shilling4. R Marion5, syndrome and congenital contractural arachnodactyly (CCA) M. Godfreyl, M Muenke', and MM Cohen. Jr.' 'Depts Genetics, 2Otolaryngology, and 'Plastic M. Wang', S. Bellehl, K. Mathews', M. Wahl2, S.D. Cederbaum2, J.M. Graham2, Surg, Case Western Reserve Univ School of Med, Univ Hosp Clev; 4Dept Plastic andCiL. eliricuzio'. 'University of Nebraska Medical Center, Omaha: 2UCLA School Surg, Univ Mimn; sAlbert Einstein College Med; 6 Children's Hosp Phil; 'Dept Oral of Medicine and Cedars-Sinai Medical Center, Los Angeles, California; 'University of Maxillofacial Sciences, Dalbousie Univ. New Mexico, School of Medicine, Albuquerque. Pfeiffer syndrome (PS) is a clinically and genetically heterogeneous autosomal At the most severe end of the MFS spectrum is neonatal Marfan syndrome (nMFS). dominant disorder consisting of craniosynostosis, a flattened midface with a beaked Individuals with nMFS share some features of classic MFS including arachnodactyly, nasal tip, and broad and medially deviated thumbs and great toes. Because ofthe clinical pectus deformity, aortic root dilatation, and mitral valve prolapse. They also have pul- I monary and tricuspid valve prolapse, which are rarely seen in classic MFS. Many nMFS variability, it has been divided into three subtypes. Type represented the cases with patients share some of the previously noted features of CCA, including crumpled ears more mild findings and a good developmental prognosis. Types 2 and 3 were and joint contractures. A spectrum of severity has also been noted in CCA. Some cases associated with a more severe phenotype, often with other associated findings, of severe/lethal CCA have been reported. In addition to the clinical characteristics that mental deficiency, and a greater risk for early demise. We have reviewed a series of 7 define CCA, the infants with severe/lethal CCA often have atrial and/or ventricular sep- patients, two with PS Type 2, and five with PS Type 3. All were sporadic cases, and tal defects, patent ductus arteriosus, interrupted aortic arch, and esophageal or duodenal range in age from 6 months to 5 years. Severe ocular proptosis necessitated atresia. Here we report the clinical features of20 nMFS patients and 6 severe/lethal CCA patients who have been referred for molecular studies of their FBN1 and FBN2 genes, tarsotomy in 3. Additional findings include hydrocephalus (I case), sensorineural (I) respectively. We have identified eight FBN1 mutations in nMFS, including two outside and conductive (2) deafness, and partial or complete ankylosis ofthe elbows (6). All the 'neonatal region" (between exons 24 and 34) and two FBN2 mutations in patients seven required tracheostomy because of severe respiratory distress due to congenital with severe/lethal CCA. Molecular analyses can help to confirm the clinical distinction upper airway abnormalities. Notably, development has been essentially normal for 3; between nMFS and severe lethal CCA, which is suggested by differing patterns of cardio- mild delay has been noted in 2, and moderate delay in 1. One child, who was vascular and other visceral anomalies. Further, the observation of atrial and ventricular profoundly delayed, died from an overwhelming infection at 15 months. This report septal defects and gut atresias in individuals with FBN2 mutations suggests a possible suggests a greater potential for long-term survival with near-normal intellectual role for fibrillin-2 in some idiopathic cardiac septal defects and gut abnormalities. development in PS type 2 and 3 than what was appreciated from earlier literature reports. However, these children do require an aggressive multisystem medical approach, with particular attention to ailrway management. Prolonged hypoxemia may account for a significant part of the neurologic morbitity associated with PS types 2 and 3. Therefore early recognition and intervention of upper airway problems would be crucial ifany hope of a favorable outcome is to be achieved.

177 178 Mdmsmu*EnIciaIom hsi nalhesi heFM1 puss ot andfinutatio.Rl FRAXE. PB Jackyl. RW DL JA wJR OhsmuWE OClthm'sl-oitaL 8urman2._BW2=2Xich2. Nel=3. ReisslM HIM= RidkdA S&bty AKTa4. UvfCo adoHealth Mageisa2. and JA Pearsonl, I Kaiser Permanente NW, and 20regon Health Sciences SCnterd ,C . a University, Portland, OR; 3Baylor College of Medicine, Houston, TX. kItsweldf wydwcan afruu wihdiFMRl uiSn mhruesnmpphysicaldbdiiol FRAXE and the FMR2 gene have in several instances explained the occurrence of ds,$ offie X* imTher iskiedvAe= dit = ,rphical 1ie ad roidne individuals who historically have been cytogenetically fragile site positive, given the pnblmnuybealbosemxsf rnlsvvkht inhepreaxlyamuissu& swltzthmeryha edbt diagnosis of Fragile X Syndrome, but more recently failed to show repeat expansion on mmin&mllswith thprnainard rsalwithtefl nudncxxi utocartmk lmbeeoad FRAXA FMR1 molecular evaluation (Brown WT, Am J Hum Genet 58:903-905, u smat d bdv ftm60 l1SO.Arneficalarblvbal 1996). FRAXE appears to be much less common than FRAXA, and clinically a milder thapagmfl18-58 wbhQs Siy condition both in physical stigmata and intellectual and behavioral lismy, apl o:e irxd 1&letdpy~i aswelasxarso nicwi anetsaxinQ impairment. A We describe two brothers for whom the diagnosis of Fragile X Syndrome was made in tetwrepa ei St wfdW anymvedonenddatatokbkfrolibrt sind = 1983, based on physical appearance, mental impairment with significant speech deficit, and Xq27.3-q28 fragile site frequencies of 22% and 14% respectively. There 1in lmvightidm dme and ficdy m cdtiiet toaheadvapISa emmmutn was an otherwise negative family history of mental retardation. Polymerase chain inanutlrfarmdiEarmnmo fivan dhesdB tlum aw asforqd reaction based FMR1 testing in 1995 demonstrated normal CGG repeat values of 30 ai ard i h&Ac for both boys. FRAXE PCR testing demonstrated no amplification of the CGG repeat at dirical&hidntI- emssb geas, gh pabfk,lorE& dt ard dul the FMR2 locus. This result would either ,oidu4 sm mm,i a oat~q- Ien be explained by repeat expansion beyond pdoaees, ndthedvrdo tsd m and amplification limits with PCR, gene deletion, or poor primer annealing at the locus. be vhaIN y ir~d nxx pmbl mn y~wkhthefi minadonth in rosin secS iaito FRAXE Southern analysis with a pEB.475 probe, demonstrated CGG repeat values of md forred aWt gra rml ,a iesin eyesmc,andharddqfg Hwv, rr 438 and 667 for the two boys. There was no evidence of significant mosaicism. No withtheful mum dwadIeznd WVandovainqy dmcm&Frilswk thep uai FMR2 protein could be demonstrated in fibroblasts tested from one of the boys. a xrec the the As near-adult males, these boys are generally higher functioning than FRAXA dIMFleIsf~*icn degmefatwearp Ixa pre ofpr affected rat1w, the d PI 9cFwb nothive male individuals and do not exhibit the degree of language deficit or ess, h jghpaixdpabite ad Wesvtthpedaid mannerisms frequently apparent with FRAXA. They have a striking FRAXA facial AMorpbkmdcn alnhepdgthemdcad miobelmy. AmeWwdiniaarb appearance, but no macroorchidism. This is our first case of FRAXE mental retardation was ndoultforthndar d~miShsayf 1 whichyidoda lO~eabeHstm lvc(M in approximately 150 FRAXE chromosomes examined, and emphasizes the importance sco e wilchoondy I i P52,81/ oftd hel lba lmaio(AR-t epropord dfhe of molecular studies for individuals previously diagnosed with Fragile X Syndrome rmarlFMR Weonth daw6eXdymso) inth fre wIt te su rruttio caue *t based solely on cytogenetic evaluation. It also points to the dilemma created in the p:foIiaeQ t0.04)ardca dosetso ~m withS le.IQ(p -.07) lntriotwkhv IQ current circumstance where cytogenetic fragile site studies are no longer considered 0P-.1 lbeARahol wbdl hdieIH.008)butratthePI(0211 How~whencorafx in the workup of potential FRAXA/FRAXE patients. SESwasmrade, dm r dan werenokWrsocrt lwese 9ast dutnotMyfina wit lemtanmbttalbobrg swithhe wi lluswo ~~e~sfanfFmhe~,lImut

179 180 1 Natural History Experience with 11 Cases of Wolf-Hirschhorn Syndrome. A. Discriminant function analysis of the Turner syndrome neurocognitive Battaglia, J.C. Careyo, P. Cederholmo, D. Viskochiloand A. Brothmano. IRCCS phenotype. J.L. Ross', H. Kushner2, andA.R, Zinn'. 'Thomas Jefferson Universitv Stella Maris,Institute of Child Neurology and Psychiatry, University of Pisa, Italy; and 2Biomedical Computer Research Institute, Philadelphia, PA and 'UT oDivision of Medical Genetics, Department of Pediatrics, of Salt Southwestern Medical School, Dallas, TX. Lake City, Utah, USA. University Utah, Turner syndrome (TS) is the complex disorder associated with the absence of all or We have evaluated 11 part of one sex chromosome. Physical features include short stature, ovarian failure, patients (9 females, 2 males) in 2 centers with the Wolf- and anatomic abnormalities. Although the phenotype varies, girls with TS typically syndrome. The combined cases of these 2 centers show selective neurocognitive deficits in nonverbal as considerableHirschhorn 4p- represent domains such visual-spatial abil- experience inthis important deletion syndrome. Fourofthe cases, three ity. In order to measure the neurocognitive phenotype of individual subjects, we per- from Italy, one from Utah, have had greater than 5 years follow-up, including the formed a discriminant function (DF) analysis using data from 83 girls with 45,X TS and Utah case being followed for 15 years. Nine cases were detected by standard 165 healthy control girls matched for age, verbal IQ, race.and socioeconomic status. cytogenetics,either regular G-banding (6) or high resolution banding (3), whereas Neurocognitive tests administered evaluated the following domains: general cognition, the remaining two cases (Italian), required FISH. On the other hand, 6 of the Utah achievement, memory, language, visual-spatial ability, visual-motor ability, attention cases, the 2 were confirmed and impulsivity, and affect recognition. Over 80 variables were examined, and a subset except oldest, using FISH probes in the critical Wolf- that differed significantly between TS was Our with girls and controls identified using univariate Hirschhorn region. experience natural history expands literature reports: analysis. We then applied a stepwise DF analysis. Variables selected by the DF were 18% (2/11) have heart defects (VSD, PDA and ASD), 55% (6/11) have oral-facial as follows: WISC-R Freedom from Distractibility (FFD), WISC-R Picture Completion, clefts, 73% (8/11) have a seizure disorder, and 100% have significant, profound Test of Facial Recognition, Rey Osterreith Figure Copy, Motor Free Visual Perception developmental retardation. One Italian case has sensorineural deafness and one Utah Test, Beery Test of Visual Motor Integration, and the Wide Range Achievement Test- case has a right split hand defect. Of note is that two of the Italian cases were able Reading. We developed a formula based on these variables by converting standardized to walk unassisted at coefficients to integers, revising the scale of the function, and minimum and ages 4 years and 5 years 9 months respectively. This is maximum imposing different from the literature where it is limits of ±2 standard deviations for any particular test result. The sum of conventionally stated that ambulation is not the seven weighted z-scores yielded a of DF scores of -16.0 to seen in this All the range possible +16.0. The syndrome. patients have the characteristic facial phenotype, mean scores for the TS and control populations differed significantly (-7.7+4.4 versus which, while changing over time, remains easy to recognize into adolescence. The -1.1±4.5, p<0.001). We chose a cutoff score of <-9.0 to define the Turner syndrome seizures are somewhat difficult to control, with prolonged status epilepticus occurring neurocognitive phenotype. Using this cutoff, the DF identified 37/83 45,X subjects and in two cases. EEGs have shown multifocal spikes, polispikes/wave, and sharp 5/165 female controls as having the TS neurocognitive profile, yielding a sensitivity of element-spike/wave complexes, either diffuse or prominent posteriorly, activated by 45% and a specificity of 97%. We are using this DF to explore relationships between eye closure and by slow wave sleep. The4 older cases represent considerable karyotype, neurocognitive phenotype, and physical features in subsets of TS subjects. experience with older individuals with as there are a few reports available of older children.Wolf-Hirschhornsyndrome, only Slide Session 41: Clinical Genetics, Malfornnations and Dysmorphology 11 (cont.) A37 181 182 Neural tube defects in chromosomal sbnormalities: further evidence for non-random Fluconazole teratogenesis: A multiple malformation embryopathy distribution of abnormal closure sites LA, Evans. KM. MacDonald and B.N. resembling Antley-Bixler syndrome. KA. Aleck and D.L Chodirker. University of Manitoba and Health Sciences Centre, Winnipeg, Canada. Bartley. Inongoingepidemiologicalinvestigations ofneural tube defects (NTD) inManitoba, University of Arizona, Tucson and Maricopa Medical Center, Phoenix, we are studying patterns of associated anomalies and the sites of the closure defect. Arizona. To date, we have identified 16 fetuses or infants with NTD and chromosomal Fluconazole,a bis-triazole antibiotic used against a variety of fungal anomalies: 8 with trisomy 18, S with trisomy 13 and 3 with triploidy. These represent pathogens, is currently being promoted for single dose oral treatment of 15% of trisomy 18 cases examined through our program, 36% of trisomy 13 and 60% vaginal candidiasis. In 1992, Lee reported an infant exposed to oral of triploid infants or fetuses (30% of all triploid pregnancies reviewed). In all trisomy fluconazole in utero who exhibited a pattern of malformation consistent with 13 and triploid cases, the defect occurred in the sacrum (site S or canalization defects). In Antley-Bixler syndrome. Two subsequent patients with in utero fluconazole the 7 trisomy 18 cases with confirmed sites, one had a sacral meningocele, four had exposure exhibited similar findings. All three pregnancies were treated long lumbar or one had anencephaly a lumbosacral defects (site 5), and lumbosacral defect term and with relatively high doses for fungal meningitis. We report here an (sites 2,4 and 5), and one East Indian fetus had iniencephaly and rachischisis (sites 24,1). additional patient with fluconazole exposure and similar malformations lending further credence to the existence of a fluconazole The restriction of NTD to lumbosacral sites in trisomy 13 and triploidy was noted embryopathy. by Seller (Clin Dysmorph 4:202-7, 1995) who suggested that this generally held true BR was the 31 week product of a pregnancy complicated by Coccidioides for trisomy 18 and that the high incidence of cranial defects recorded in this groupwas immitis meningitis treated for the first 9 and the last 18 weeks of pregnancy due to biased reporting. As our provinical program includes a birth defects registry, a with high dose oral fluconazole. At birth the child was of normal size but single ytogenetica laboratory and centralized medical genetics, prenatal diagnosis and exhibited multiple craniofacial and skeletal abnormalities. The calvarium was screening programs, the ascertainment of our cases is likely unbiased. Thus trisomy 18 soft with widely split sutures and a prominent forehead. The eyes exhibited would appear to have cranial and upper spine defects as well as lumbosacral ones. exorbitism. The nose was large and pear shaped with a hemangioma at the The cause ofNTD in chromosomal anomalies is unclear and their frequency varies tip. The ears were small and posteriorly rotated with overfolded helices. The between reports presumably due to numbers and background small differing rates. upper extremities exhibited radiohumeral synostosis. The hands and feet While effects of specific gene products may be important in trisomy 13 where dosage were slender with hypoplastic nails. The hips were dysplastic and the the closure site is unique, trisomy 18 may cause a general imbalance unmasking an feet showed a rocker No underlying predisposition to various NTD types. If so, NTD in trisomy 18 should be bottom configuration. femoral bowing was noted. more common in cases at increased risk and in areas with a high NTD frequency. Follow-up one year shows an infant with the prominent metopic suture in Other multifactorial defects should show similar patterns. Among 52 trisomy 18 cases, addition to the newborn findings. Developmentally, he is mildly delayed. we found 7 with cleft liptpalate including one with a sib with isolated cleft lip. This patient, and the previously reported patients, present a pattem of skeletal and craniofacial changes that appear to be fluconazole induced.

183 184 Congenital horizontal gaze palsy, deafness, central hypoventilation, and Anticipation in Autosomal Dominant Cyclic Hematopoiesis. S. E. Palmer2, developmental impairment: A brain stem syndrome prevalent in the Navajo K. Stephens1, and D. C. DaleI. Dept. of Medicine, Univ. of Washingtont, and population. B. D. Friedman.' T. J. Tarbv.2 S. Holve.' D. Hu.' P. O'Connor?3 S. Dept. of Pediatrics, Univ. of Texas Health Science Center at San Antonio2. Johnstone.2 F. C. L. S. Richter C. H. E. Gonzalez.' Clericuzio.4 Cox.' Cunniff.' Autosomal dominant cyclic hematopoiesis (ADCH) is a rare familial form Hovme Tucson and ''Deparunent of Pediatrics, The University of Arizona, Phoenix; of the disorder also known as cyclic neutropenia. It is characterized by neutro- 2Children's Rehabilitative Services, St. Joseph's Hospital and Medical Center. Phoenix; penia recurring in approximately 21-day cycles. We previously characterized 'Department of Pediatrics, Tuba City Indian Medical Center, Tuba City, AZ; 4The 9 ADCH families in which variable expressivity within families was common, University ofNew Mexico. Albuquerque. but found no clinical evidence to suggest non-penetrance or heterogeneity. Several Native American tribes. including the Navajo, are of Athabaskan descent. However, the pattern of expressivity suggested anticipation: no families ap- Many genetic disorders are seen with increased frequencyin this population. We peared to display milder phenotypes in successive generations, and the most report on seven Athabaskan children with congenital horizontal gaze palsy, deafness, severe cases seemed to occur only in children in the youngest generations. and central hypoventilation. All of the children have global developmental delay and This pattern was independently noted by several families. To assess the three have seizures. Convergence and vertical eye movements remain intact. Brain possiblity of anticipation, phenotypes of 42 affected individuals were ranked stem auditory evoked response testing in six and cold caloric testing in five of the by assigning severity categories ranging from asymptomatic to very severe. children show no response, consistent with lack of cranial nerve VIII function. Facial These were based on frequency of symptoms and hospitalization as ascer- movements are sparse, but cranial nerve VII function is intact. Three of the children tained by questionnaire, interview, and record review. The spectrum of sever- have congenital cardiac defects, two of whom have outflow tract anomalies. ity included death from necrotizing enterocolitis, a complication of neutro- These cases represent a unique syndrome of unknown cause with distinctive penia. Death occurred in 2 three-generation families, but only in children in abnormalities ofbrain stem function. Cardiac anomalies are a variable feature. The the youngest generation. Another case of life-threatening enterocolitis occured distinct ophthalmologic findings, lack of cranial nerve VII palsy, and central in a sibling. Two boys died in a third family; their mother was unaware of her hypoventilation distinguish this disorder from the Moebius syndrome. The high disease at that time. No other cases of life-threatening illness could be identi- representation ofNavajo cases suggests a genetic etiology with a founder effect. A fied. Two families consisted of only parent-child pairs with similar pheno- candidate genetic region is chromosome 22q, since mutations at this locus have been types. One family showed a narrow range of phenotype severity when 3 gen- associated with sensorineural deafness and cardiac outflow tract anomalies. erations were compared. However, the remaining 6 families displayed Recognition of any one of the findings of this brain stem disorder should prompt increasingly severe phenotypes across several ranked categories in successive evaluation for the other features. generations. Patterns of increasing clinical severity were comparable to those seen in diseases with anticipation due to trinudeotide repeat expansions.

185 Facial dysmorphism in disorders of oxidative phosphorYlation. V. Cormier-Daire. A. Rotig. P. Rustin. NI. Le Mutrcr A..unnich. Dept of Genetics and INSERM U-393, HOpital des Enfants Malades, Paris, France. In the last ten years, mitochondrial disorders have been largely regarded as neuromuscular diseases. In fact. oxidative phosphorylation is present in all tissues and genetic defects of mitochondrial energy supply can give rise to any symptom in any organ or tissue and possibly express during fetal development. Recently, we observed 4 unrelated patients with mitochondrial respiratory chain defect and facial dysmorphism including round face. high forehead, short nose, flat philtrum. low set and abnormally shaped ears and short neck. Pre- and postnatal growth retardation with microcephaly was present in all cases. Additional malformations affecting the skeleton (hypoplasia of distal and middle phalanges). external genitalia (cryptorchidism) and the gastrointestinal tract were frequently noted (duodenal atresia). Progressive multivisceral involvement and metabolic acidosis led to consider the diagnosis of mitochondrial disease. which was further confirmed by enzymological and molecular studies. The combination of facial anomalies, prenatal short stature and malformations in our patients is highly suggestive of the antenatal expression of respiratory chain deficiency affecting growth and development. A38 Slide Session 42: Molecular Genetics of Disease Ill 186 187 The gene for anhidrotic (hypohidrotic) ectodermal dysplasia encodes a Two novel mutations in KI codon 479 cause a unique form of ichthyosis. J. S. small transmembrane protein. J. P. PL L. T. Kerel,0.U Montonenl, Bayhavan2, Francis' 2. Smith'. V. P. Sybert' 2. K. Stephens. L. D. Corden'. and W.H. 1. Chen2, S. EzerI, 1. ZonandN.Thomas4, Saarialho-Kere 15,A. de la Chapelle1, McLean'. iUniv ofWashington, SeattleWA, USA; 2Children's Hospital & Medical D. Schlessinger6, and A.K. Srivastava6'7. [Department of Medical Genetics, Haartman Center, Seattle, WA. 3CRC Cell Structure Research Group, Univ ofDundee, UK. Institute, University of Helsinki, Finland; -Advanced Center for Genetic Technology, We report four individuals from two families with a unique inherited skin disorder. Applied Biosystems Division, Foster City, CA; 3Department of Molecular and Medical Manifesting erythema and superficial erosions at birth, which improved in the first few Genetics, Oregon Health Sciences University, Portland, OR: 4lnstitute of Medical months, affected individuals went on to develop palmoplantar hyperkeratosis with patchy Genetics. University of Wales College of Medicine, Cardiff, U.K.; 5Department of erythema and scale elsewhere on the body. Three ofthe four exhibit dramatic episodic Dermatology, Helsinki University Central Hospital, Helsinki, Finland; 6Department of flares ofannular, polycyclic erythematous plaques with scale that coalesce to involve most Molecular Microbiology, Washington University School of Medicine, St. Louis, MO; ofthe body surface. The flares last weeks to months and in between the skin Self Research Institute of Human Genetics, Greenwood Medical may be 7J.C. Center, normal except for palmoplantar hyperkeratosis. Due to the unusual clinical phenotype and Greenwood, SC the Ectodermal dysplasias comprise a large group of syndromes involving defective presence ofclumped keratin filaments limited to spinous cells (epidermolytic development of hair follicles, sweat glands, teeth, and nails. Although more than 150 hyperkeratosis) and not present in granular cells, we postulated that the causative clinically distinct ectodermal dysplasias exist, the molecular pathogenesis has remained mutation might reside in KI, so that expression ofthe late differentiation type II keratin, unknown in all types. X-linked anhidrotic, or hypohidrotic, ectodermal dysplasia (EDA) K2e, could compensate for the abnormal KI. For the first patient, mRNA was prepared one or absent is of the common types, and is characterized by sparse hair, abnormal from snap frozen biopsy material and the entire KS coding sequence was amplified by RT- teeth, and inability to sweat due to lack of sweat glands. We have positionally cloned the PCR. Direct cycle sequencing ofthe fragment revealed a heterozygous mutation T->C at gene in which defects cause EDA, based on the study of CpG islands and their relative base location with respect to X-chromosomal translocation breakpoints in two female patients 4220, changing isoleucine to threonine at codon 479 in the highly conserved helix with X;autosome translocations and EDA. Cloning and sequencing of a CpG island termination peptide motif The mutation created a new BsmAI restriction endonuclease adjacent to one such translocation breakpoint revealed a putative exon in the expected site which was used to confirm the mutation in patient cDNA and genondic DNA and also orientation. A cDNA was recovered from a sweat gland library and includes two exons exclude it from the unaffected parents and 50 normal individuals. To determine ifthe are an in DNA. The cDNA encodes a that separated by intron of 200 kb genomic second family carried a similar defect, we amplified the K1 gene from DNA predicted 135 residue protein with a single putative transmembrane domain. The EDA genomic and found a A->T gene was found to be disrupted or mutated in at least 13 patients in large clinical series heterozygous mutation at base 4219, changing isoleucine to phenylalanine from Cardiff (U.K.) and Portland, OR. The gene is expressed in several fetal and adult in the same codon 479. This base change was confirmed in all three affected family tissues by northern analysis. In situ hybridization revealed that the expression in adult members. These amino acid substitutions within the KI gene have not been previously skin is localized to keratinocytes, hair follicles, and sebaceous and eccrine sweat glands. reported and may explain the unique phenotype. We speculate that K27e expression may The predicted protein may belong to a novel functional class with a role in epithelial- modulate and partially compensate for the abnormal KI, but the cause of the episodic mesenchymal signaling and in the development of ectodermal appendages. flares remains unknown.

188 189 Genomic organization of ITGB4 encoding the f34 integrin Epidermolysis bullosa simplex with muscular dystrophy: cloning of the subunit, and demonstration of a homozygous splice-site human plectin gene and pathogenic mutations in four families. XV. H. mutation In junctional epidermolysis bullosa with pyloric 1. McLean', L. Pulkkinen2, F. J. D. mith', H. Shimizu3, R. A. J. Eadv4. atresia. Leena Pulkkinen, Kenneth Kurtz, Yili Xu, Leena Bruckner- I. M. Leigh'. E. B. Lanei, and J. Uitto2. lUniversity of Dundee. Scotland, UK: Tuderman, and Jouni Uitto, Thomas Jefferson University, Philadelphia, 2Jefferson Medical College, Philadelphia, PA: 3Keio University, Tokyo, Japan; 4St PA, University of MOnster, Germany. Thomas's Hospital, London, UK; 5Royal London Hospital, London, UK. The a6J34 integrin is expressed primarily within epithelial basement Epidermolysis bullosa simplex with muscular dystrophy (MD-EBS), is an autsomal recessive disorder characterized by hereditary membranes, and participates in the formation of hemidesomes skin blistering in combination with a pro. gressive late onset muscular dystrophy. Ultrastructural examination of patients skin necessary for stable association of epidermis to the basement showed an abnormality in the connection of keratin intermediate filaments within the membrane. A variant of junctional epidermolysis bullosa associated with basal epidermal keratinocytes to the hemidesmosome, a transmembrane protein complex pyloric atresia (JEB-PA) has been shown in one case to be deficient for connecting cytoskeleton to basal lamina. Immunohistochemical analysis with antibodies the f4 integrin expression. To facilitate identification of further to hemidesmosomal antigens revealed that plectin, a widely expressed high molecular pathogenic mutations in the gene, we have weight cytoskeleton-associated protein, was absent from hemidesmosomes and other cy. 34 integrin ITGB4, locations in determined the intron-exon organization of the entire gene. We have toplasmic MD-EBS epidermis. Similarly, plectin was found to be absent from MD-EBS muscle, where it is normally associated with the sarcolemma and Z-bands. also developed a strategy for mutation detection, based on PCR The complete 14.8 kb human plectin cDNA and the corresponding gene consisting of 33 ex- amplification on of genomic DNA with primers placed intronic sequences ons spanning >26 kb of genomic DNA were characterized and the gene was mapped by flanking the exons, followed by heteroduplex analysis and direct FISH to 8q24. The predicted 518 kDa protein was found to consist of an N-terminal actin automated nucleotide sequencing. The entire gene was shown to binding domain, a central coiled-coil rod domain and a putative C-terminal intermediate consist of 40 exons spanning -36 kb of the genomic DNA on filament binding region. Homozygous insertion and deletion mutations leading to pre- chromosome 7q11-qter. The intron-exon borders demonstrated the mature termination codons (5135del8; 5907ins8; 5866delC) were found in three MD-EBS families where plectin immunoreactivity was completely absent. In each case, mu- consensus splice sites DNA from a the ag-EXON-gt. consanguineous family tation was excluded from normal controls. A homozygous in-frame deletion (2179ded9) with an individual with JEB-PA, who had died at age 1 was the of month, was detected in a fourth MD-EBS kindred, where plectin was found to be present in the subjected to mutation analysis. Both parents showed a G-to-A epidermis by immunofluorescence staining but was greatly attenuated. This mutation was substitution in the first nucleotide of intron 30 predicting aberrant splicing excluded from 135 normal individuals. These results demonstrate that plectin ex- of exon 30. Since,B4 integrin is expressed not only in the skin but also in pression is essential for cytoskeleton-basement membrane connection in the epidermis. the epithelial lining of the stomach, reduced expression of this integrin A similar role for plectin in muscle would explain the muscle pathology in MD-EBS. would explain the blistering tendency and development of pyloric atresia in this patient.

190 191 Delta-Sarcoglycan: A Novel Component of the Dystrophin Glycoprotein- MyoD induced myo-differentiation: application in Duchenne Muscular Complex Is Involved in Autosomal Recessive Umb-Girdle Muscular Dystrophy diagnosis, mutation detection and research. P.A.M. Roest, Dystrophy Unked to Chromosome 5q33-q34. F. Duclos. D. Jung'. V. J.J. Heus, G.J.B. Van Ommen, J.T. Den Dunnen. MIGC-Dept. Human Genetics, Straub'. B. Aoostol'. R. Shiana2..V. Allamand'. J. Lee'. C.- Leveille'. C. Leiden University, The Netherlands Moomaw3. C.Slauahter3.T. 0. Crawford4. J. McPherson5 and K. P. Diagnosis of muscular dystrophies is often hampered by the absence of muscle tissue Cambell. from the 1: HHMI and Dept of Physiology and Biophysics, Univ. of Iowa, City. 2: patient. We have tested in vitro myo-differentiation as an alternative to study Iowa muscle-specific proteins in non-muscle cells. Differentiated Dept of Biological Chemistry, Medical SciencesI, Univ. of Califomia, Irvine. 3: cells, including fibroblasts and chorionic villi cells, are forced into myogenesis transformation with a HHMI, Biopolymer Facility, Univ. of Texas, Dallas. 4: Univ. of Johns by retroviral vector Hopkins, containing the MyoD differentiation gene. Transfected cells are selected with Dept of Pediatric Neurology, Baltimore. 5: Dept of Genetics/GSC, Washington neomycin, grown to confluency and myogenesis is induced by a transfer to low serum medium. Univ. School of Med., St. Louis. Differentiation is first observed by the appearance, within a few days, of early myogenic The dystrophin-glycoprotein complex (DGC) acts as a structural link between proteins like desmin. Later, cells change shape, fuse and form multi-nuclear aggregates. the actin cytoskeleton and the extracellular matrix in skeletal muscle. Defects in During these later stages, a time-dependent range of muscle-specific proteins become several components of the DGC are involved in muscular dystrophies, including detectable, including myosin (embryonic and adult), titin, nebulin, M- and C-protein Duchenne muscular dystrophy, limb-girdle muscular dystrophies (LGMDs) and and dystrophin. Titin and dystrophin appear simultaneously after 4-7 days. After 7 days, the titin pattern shows a clear cross-striation and all cells also congenital muscular dystrophy. We have cloned 8-sarcoglycan, a new titin-positive have dystrophin. These observations new for component of the DGC and of the sarcoglycan subcomplex. Several opened possibilities prenatal diagnosis, 8- carrier detection and mutation detection and gave a new tool for DMD-research. In sarcoglycan transcripts are detected at high levels in skeletal and cardiac cells derived from DMD-patients, dystrophin is absent while those from carriers become muscle. 8-sarcoglycan encodes for a 35kDa transmembrane glycoprotein, with titin/dystrophin double positive in only a portion of the cells. The increased expression a large C-terminal extracellular domain and shares high homology with y- of the dystrophin gene were exploited in RNA-based mutation detection, enabling us sarcoglycan. We have mapped the human 8-sarcoglycan gene to to identify several new point mutations in DMD/BMD-patients. DMD-research was chromosome facilitated 5q33-q34 in a region overlapping the recently linked autosomal recessive by myoD-induced myo-differentiation of different mammalian cell lines which were transfected with a YAC-derived copy LGMD2F locus (Passos-Bueno et of the complete dystrophin gene (see Heus et al, Hum. Mol. Genet, 1996) and have al.) identified a patent with mutations in this gene. Immunohistochemical analysis of this patients skeletal muscle biopsy revealed that a-, 3-, y- and 8-sarcoglycan are greatly reduced at the sarcolemma. Thus, mutations in 8-sarcoglycan are responsible for the disruption of the sarcoglycan subcomplex and lead to LGMD2F. Slide Session 42: Molecular Genetics of Disease Ill (cont.) A39 192 193 Ruled-Out Congenital Myotonic Dystrophy - Think Prader-Willi G.'Mettler, Minimal definition and function of the imprinting center for Prader-Willi M.McGill, S.Leblond, D.Lahev, L.C.Surh Department of Genetics, Children's Hospital of Eastern Ontario, Ottawa, Canada syndrome. T. Ohtal, P.K. Rogan2, S. Saitohl, G. Deng3, K. Buaiin,4, K As a molecular diagnostic laboratory providing publicly supported and regionalized Horsthei , D.J. Driscofl3, R. Weksbrg5,T. Ishikawa6, M.G. Butler7. patient services to an entire province, we have virtually a complete catchment of DNA GabrielJl, R.D. Nichollsl. tCase Western Reserve Univ., Cleveland. OH; testing for myotonic dystrophy samples from a population of 10 million. From January 1 2Pennsylvania State Univ., Hershey, PA; 3Unv. Florida, Gainesville, FL; 4Univ. 1992 to May 31 1996, 1468 samples were received for mutation testing of the trinucleotide Essen, Essen. Germany; 5Hospital for Sick Children, Toronto, Canada; 6Nagoya City CTG expansion in myotonic dystrophy (DM). Of these, congenital myotonic dystrophy Univ., Nagoya, Japan; 7Vanderbilt Univ., Nashville, TN. (cDM) was the primary referral reason for 34 samples which also had the following A subset of patients with disorders involving imprinted genes, such as Prader-Willi features:(a) samples were from infants of 12 months or less and (b) there was no family (PWS) and Angelman (AS) syndromes, have been found to have a familial mutation in history of DM. Only 3/34 (3%) of these infant samples revealed a DM mutation, which the imprinting mechanism. Previously, we identified a bipartite imprinting center (IC) were also in the largest expansion size range commonly observed in cDM. Given that within chromosome 15qI 1-q13, with adjacent microdeletions found in the majority of differential diagnosis in the floppy infant for genetic disorders includes spinal muscular PWS and AS imprinting mutation patients, and proposed that IC mutations block dystrophy (SMA) and Prader Willi Syndrome (PWS) as well as the recent availability resetting of the imprint in the germiline. This fixes on that chromosome the parental of 'DNA diagnosis" for these disorders, infant samples negative for the DM expansion imprint (epigenotype) on which the mutation arose. The PWS microdeletions spanned were further investigated. Deletion of exons 7 and 8 from the SMN locus is observed in 45-200 kb and overlapped a 30 kb region including SNRPN exon 1. We have analysed about 85-90% of patients with SMA. Only 1/34 (3%) samples referred for cDM revealed six additional PWS families with an imprinting mutation in order to further define the this defect. Of note, both cDM and SMA testing was requested, suggesting enough IC element involved in PWS. clinical suspicion to consider SMA. In contrast, molecular diagnosis of Prader Willi In the PWS-T family, the affected sibs and father have a 450 kb microdeletion Syndrome (PWS), through methylation defects with PW71 and SNRPN along with 15q between D15JS3 and IPW. In the PWS-J family, the affected sibs and father have an microsatellites from the PWS critical region, revealed abnormalities suggestive of PWS -50 kb deletion spanning SNRPN and upstream. In the PWS- 14 family, the patient has in 5/34 (15%) of the samples sent for rule out cDM. For comparison, over the same 5 year a de novo 7-9 kb microdeletion in the SNRPN exon 1 region and SNRPN, IPW, time period, 87 samples were received for PWS molecular diagnosis. Of these samples, 4 ZNF127, PAR-i and PAR-5 transcripts are repressed. Combined, our data show that were from infants of 12 months or less of whom 2 revealed molecular defects suggestive of the IC element for PWS is in a 3 kb region of the SNRPN 5' promoter and exon 1, and PWS. Thus in the differential diagnosis of the floppy infant for genetic disorders, clinical does not overlap the IC element for AS (termed the imprinter: see Horsthemke et al., suspicion and signs may not be obvious enough to consider PWS in the infant. Our this meeting). This minimal element (termed the switch initiation site) controls the findings suggest that PWS is more likely than cDM or SMA in the isolated case of a maternal -> paternal imprint switch in the male germiine throughout 2 Mb of 15q II- floppy infant who has no specific family history. q13. The sequence and function of the PWS-IC element in humans, including non- deletion patients (PWS-B, PWS 103), and mouse is currently under study to determine the molecular basis of imprint switching by the IC.

194 195 Hypopigmentation in the Prader-Willi syndrome correlates with P gene A somatic cell hybrid model to study genomic imprinting. J.M. Gabriel, deletion but not with haplotype of the hemizygous P allele. R.D. Nicholls', T. Bailin2, J.M. Amos-Landeraf, L.C. Guida, H.F. Willard, M.J. Higgins', T.B. Showsi, S M.J. Mascari3, M.G. Butler4, and R.A. Spritzz. 'Case Western Reserve Univ., Saitoh, R.D. Nicholls. Case Western Reserve Univ., Cleveland, OH; 'Roswell Park Cleveland, OH; 2Univ. of Wisconsin, Madison, WI; 3Pennsylvania State Univ., Cancer Institute, Buffalo, NY Hershey, PA; and 4Vanderbilt Univ., Nashville, TN. Somatic cell hybrids have previously been shown to maintain the correct chromatin The Prader-Willi syndrome (PWS) usually results from a paternal deletion of 15qll- states to study developmental globin gene expression, as well as gene expression on the q13 or maternal uniparental disomy for chromosome 15. Reduced pigmentation of the active and inactive X chromosomes. This suggests the potential use of somatic cell skin, hair, and eyes is a frequent feature of PWS and has been previously suggested to hybrids containing either a maternal or a paternal human chromosome as a model be associated with the 15q11-q13 deletion. The P gene, which is located in this same system to study known imprinted genes and to identify as yet unknown imprinted chromosomal region, is associated with OCA2, an autosomal recessive disorder that is genes. While cell lines are available from Prader-Willi (PWS) and Angelman (AS) the most frequent form of tyrosinase-positive oculocutaneous albinism. We carried out syndrome patients with specific parental origin 15q1I-q13 deletions or uniparental fine mapping of 32 individuals with PWS and found that hemizygosity for the P gene disomy (UPD) for chromosome 15, specific parent-of-origin deletions are not known was very significantly correlated with the occurrence of hypopigmentation among pa- for other chromosomes. UPD has been found for some other human chromosomes but tients with PWS (p<0.00004). However, we found no apparent relationship between the is rare, not usually of both parental origins, and cell lines are not always available. occurrence of hypopigmentation and the polymorphism haplotype of the intact P allele Thus, there is an important need for a general model system to study imprinted genes or with the genotype at a functionally significant polymorphism of tyrosinase (R402Q), and chromosome regions for all human chromosomes. the key enzyme in pigment biosynthesis. These results indicate that hypopigmentation Testing gene expression using RT-PCR, we have shown that imprints are maintained in PWS (and perhaps also Angelman syndrome; AS) most likely results from deletion of at SNRPN, IPW and PAR-5 in eleven somatic cell hybrids containing a single human the P gene per se. However, patients heterozygous for OCA2 mutations do not appear chromosome 15, and at IGF2 and H19 in 5 hybrids containing a single human hypopigmented, suggesting that deletion of the P gene may only result in hypopigmen- chromosome 11. Microsattelite markers were used to confirm the parental origin in 2 tation in the context of PWS or AS, in which the expression of the intact allele may be cases. Subsequently, a novel candidate imprinted gene from chromosome 11 has been reduced as the result of complex gene regulation throughout this region of chromosome identified using this system, and other genes are currently being assayed. Interestingly, 15q. DNA methylation is strictly maintained at SNRPN exon 1 in all hybrids tested, but not at H19, D15S63 (PW71) or ZNF127 Nevertheless, ZNF127 and H19 functional imprinting was maintained, in preliminary studies. It thus appears that a somatic cell hybrid model is valid for studying gene expression of some imprinted loci. Our model suggests that the factors involved in maintainance of imprinted gene expression are conserved between mouse and human as, in this system, the human chromosome has been remodeled with mouse proteins.

196 Relaxation of imprinting in patients with Prader-Willi syndrome. P.K. Rogan'". J.R. Seip J.H.M. Knoll3. L.M. White'. S. L. Wcnger4 M.W. Steele4. M.A. Sperling4. R. Menon4. 'Dept Peds, Penn State Univ Coll of Mcd, Hershev; -Dept Hum Genet, Allegheny-Singer Res Inst, Pittsburgh, PA; 3Div Genet, Children's Hospital and Dept Path, Beth Israel Hosp, Boston MA; 4Dept Peds, Children's Hosp, Pittsburgh, PA. Relaxed expression of imprinted alleles has been associated with Wilm's tumor and other neoplastic conditions, however loss of the germline imprint has not been noted in uniparentally expressed, congenital human disorders. We describe two unrelated children (BW and ZB) with Prader-Willi syndrome (PWS) and maternal uniparental disomy of chromosome 15 (UPD) whose cells express imprinted genes and display asynchronous replication timing that is characteristic of individuals with biparental inheritance. Both patients were diagnosed with PWS during infancy (poor feeding, hypotonia, developmental delay, crvptorchidism, small hands/feet), but later presented atypical findings. BW, at 5.3 and 8.3 years, was >2 standard deviations below the mean for both weight and height and was not hyperphagic. ZB, at 3.3 years, was also not hyperphagic. His height was at the 30th percentile and weight was >95th percentile. Genomic methylation (at SNRPN, D15S63, and D15S9), RT-PCR (at SNRPN, ZNF127, PARS and IPW), and DNA replication analyses (at SNRPN, D15S10, and GABRB3) have been performed. In contrast with other patients with PWS and UPD, BW expressed PAR5 and ZNFI27, and ZB expressed a single allele at IPW. Asynchronous DNA replication, which is seen in individuals with biparental inheritance, was found at D15SlO and GABRB3 in BW. Otherwise, BW and ZB displayed methylation patterns that are typical in PWS. SNRPN was not expressed in either patient, ZNF127 and PAR5 were not expressed in ZB, and IPW was not expressed in BW; DNA replication at SNRPN was synchronous in BW. In summary, imprinted genes that are usually not expressed in PWS are transcribed in a subset of patients with UPD. This pattern of gene expression and allele-specific DNA methylation appears to be coordinated with asynchronous DNA replication in BW and is being investigated in ZB. Relaxation of imprinting could be due to the failure to reset the imprint in the maternal germline or to postzygotic gene expression. Relaxed imprinting may involve different loci in 15ql lq13 and may result in milder phenotypes in PWS patients with UPD. A40 Slide Session 43: Prenatal and Perinatal Genetics 11 197 198 Impact of prenatal diagnosis on Down syndrome: trends over a 10-year period Prenatal genetic testing: What do obstetrical providers discuss with patients? (1986 through 1995) in a New England HMO population. M. D. MacMillin and B.A. Bernhardt. G. Geller. S. Larson. D. Roter. T. Doksum and N.A. Holtzman. S. P. Pauker. Harvard Pilgrim Health Care. Boston. MA. Johns Hopkins Medical Institutions, Baltimore, MD. Down syndrome (DS) cases were ascertained over the 10-year period from 1986 To study communication about prenatal genetic testing between obstetrical providers and through 1995 among obstetric and pediatric patients of a 300,000 member HMO, the patients, we audiotaped the first prenatal visits of 177 patients with 41 providers [22 Health Centers Division of Harvard Community Health Plan. Cases were identified obstetricians (OB) and 19 certified nurse midwives (CNM)]. Tape segments mentioning family through prenatal diagnosis results (CVS and amnio), the Genetics department's cross- history (FH) or genetic testing (maternal serum marker screening (MSMS), CVS. index file of DS pregnancies and newborns referred for consultation, and a computer- amniocentesis, ultrasound or carrier screening) were analyzed for comprehensiveness and based medical record search of all pediatric patients less than I year. Of the 145 DS proficiency. FH was reviewed in 65% of visits, but asking about a FH of birth defects or cases identified, 56 (38.6%) were live-bom (I died shortly after birth), 0 were stillbom. mental retardation was done in only 29% and 26% of visits respectively. Only one provider 3 (2.1%) resulted in fetal demise, and 86 (59.3%) were selectively aborted following asked about consanguinity. In 59% of all visits, MSMS was mentioned. In thbes visits, spina prenatal diagnosis. During the study period, DS screening using maternal serum bifida was mentioned in 52% and Down syndrome in 61%. A possible false positive result was markers was offered to all obstetric patients with a 78% acceptance rate. AFP alone mentioned in 30%, practical details (timing, blood test) in 95%, and abortion in only 14%. The provider indicated that MSMS is in 64%, made a recommendation in 11%, did both was available from 1986 through January 1992, AFP with HCG from February 1992 voluntary in 11% and neither in 14 %. In 33% of all visits, performing a mid-trimester ultrasound to through 1994, and uE3 was added in January 1995. Trends were analyzed to determine the of DS serum markers and and the screen for fetal anomalies was discussed. Amniocentesis or CVS was mentioned in 98% of impact screening by ultrasound, impact of visits of women 35 or older, and in 56% of visits of women 33-34. When mentioned, in only prenatal diagnosis. The percentage of DS pregnancies per year diagnosed prenatally 38% was a referral for genetic counseling offered. Down syndrome was mentioned in 60%, and the percentage of DS pregnancies per year aborted increased over the study period but further described in only 3%, and a numeric risk provided in 32%. Practical details of (33.3% in 1986 to 75% in 1995, 33.3% in 1986 to 71.4% in 1995; respectively) and amniocentesis or CVS (timing, risk, cost) were mentioned in 85%, abortion as an option after coincided with an increase in the percentage per year of DS mothers 35 or older at abnormal results in 43%, and continuing the pregnancy in 28%. The provider indicated that EDC (33% in 1986 to 82.1% in 1995.) For the years 1990-1995 when delivery data testing is voluntary in 65% of visits, made a recommendation in 7%, did both in 8% and was available, the incidence of live-bom and aborted DS cases was 3.21 per 1000 neither in 20%. The provider gave inaccurate information in 4% of all visits, but in only one second-trimester pregnancies. An increase in the incidence of DS pregnancies was also was the implication serious (patient told that her matermal FH of hemophilia was of no observed (2.1 per 1000 in 1990 to 5.8 per 1000 in 1995.) These trends suggest that the consequence). OB's were significantly less likely than CNM's to discuss MSMS, say that average maternal age of our obstetric population is also increasing. Trends in decision- testing is voluntary, or refer to a genetic counselor, and more likely to make recommendations making after prenatal diagnosis showed a recent increase in the number of women about testing. These results demonstrate that, although OBs and CNMs mention genetic testing electing to continue DS pregnancies identified at amnio; 60% of live-born DS cases at the first prenatal visit with most patients, the discussion is not comprehensive, especially with (3 out of 5) were detected prenatally in 1994 compared to 0-15% in previous years. women of advanced maternal age. Furthermore, the differences in communication style between OBs and CNMs are likely to influence genetic testing decisions.

199 200 Hyperechogenic fetal bowel: A novel quantitative measurement, clinical significance and fetal Are fetuses with cystic fibrosis at increased risk for jejunoileal atresia? a population study outcome.T.C. Falik-Zaccai'. H. Laufer'. MBen Dov'. Z Borochowitz'. S. Deganil, I.Shapira'. J. H.F. Roberts" J.D5 Crasan'.J. Cono'. MJ Khoury'. M.R, Weatherlv. C.. Moorc' 'Division of tBirth Bronstcin2.I. GaWsatcin A. Oslander3. R Dukeman3. Z.Lcibovitz'. Bnai Zion Mcd Ccnt., Detects and Developmental Disabilities, Centers for Disease Control and Prevention and 'Division of Rambam Mcd. Cent. Carmel Med. Ccnt. 1 Rappaport facultv of medicine. Haifa. Israel. Medical Genetics, 'Divisions of Cystic Fibrosis and Pediatric Pulmonary Medicine, Emory Universitv An association between hypercchogcnic fetal bowel (HEB) on prenatal sonograms and adverse School ofMedicine, Atlanta, GA outcome of the pregnancyhas been previously documented. The major disorders related to this Several case series in the surgical literature have reported an association betweenjejunoileal atresia (IIA) finding were aneuploidy, cystic fibrosis (CF), itsra uterine infections, intra uterine growth retardation and cystic fibrosis (CF). The postulated mechanisms of JIA in patients uithCF relate tothe consequences of meconium ileus. However, no population-based data arc available on the magnitude for (IUGR), perinatal death and prematurity. All studies reported in the literature were based on a ofrisk hA among infants with CF. Our purpose in this study was to quantitate the magnitude ofthe morphological and a qualitative description of the HEB, and were in association mostly performed small between JIA and CF in Atlanta using population-based data from 1968-1995, groups which had an aprion high risk for aneuploidy and cvstic fibrosis. We have developed a novel Cases included all infants with isolated JtA born from 1968-1995 to mothers residing in the five county computerized quantitative method for the evaluation of of fetal bowel a cchogenicity by using metropolitan Atlanta areaatthe time ofbirth. To ascertain cases, we reviewed records ofthe computerizedimage processing of ultrasonographic B-scan of the fetal bowel and liver. A Metropolitan pictures Atlanta Congenital Defects Program (MACDP), a population-based registry of all serious birth defects relative echogenicity coefficient (REC) was calculated which a of provided quantitative evaluation diagnosed during a child's firstyear oflife. We ascertained a total of94 isolated NA cases during the the bowel echogenicity relative to the liver echogenicity. We have examined the REC in 137 normal study period (jejunal- 67, ileal- 27), for an overall prevalence of 1.8/10,000 livebirths. Among the cases, fetuses between the 12th and 28th weeks and gestation determined mean values and 95% confidence 38 were white, 52 wvere black, and 4 avere asian or hispanic. Four ofthe 38 white JIA cases (1I%) also had interval the along pregnancy. We have then conducted a prospective study on 79 women who were CF. The expected number ofJA cases with CF is 0.019 (using even the relatively high incidenceof referred to us for the evaluation of HEB. These women did not relate to CF any high risk population. 1/2000 for CF). Giventhat there were 526,000 white livebirthsduring the study period and 1/2000 CF carrier status, fetal kazYotpig, and maternal blood IgM and IgG for Toxoplasma, Rubella, CMV and incidence, we estimated that there were 263 white infants with CF inthis population. The estimated risk Herpes infections were performed along with bowel to liver REC measurments at least twice along of JIA among white infants with CF is 1.5% (4/263). Thus, white infants with CF are atmore than 210 the pregnancy. We have followed up the pregnancies and examined the newbors between atee to six times the risk of having JIA compared with white non-CF infants (P < 0.0001 using Poisson distribution). months age. An abnormal outcome was documented in 24 out Of65 (37%) pregnancies. Fourteen Most cases of ISA have been shown experimentally to be the result of a late intrauterine mesenteric pregnancies are still ongoing. We have found high incidence of aneuploidy (5.4%), CF (2.5%), intra vascular accident. The pathogenesis ofAA in patients with CF include several possible mechanisms that uterine infections (3.5%), IUGR (8%), perinatal death (4.8%), anatomic anomaly of the are consequences ofmeconium ileus. These mechanisms include I) a localized area ofischemiathat may gastrointestinal tract (4.7%), and prematurty (9.4%). Bowel to liver REC of 1.68+0.18 was found heal with stenosis or atresias, 2) a volvulus that occurs when hvperperistalsis leads to rotation ofthe among the fetuses with hyperechogenic bowel compared to 1.35 ± 0.19 in normal fetuses matched for meconium-filled, obstructed segment resulting in ischemia at the base of the volvulus, and 3) a pseudocyst gmtacional age (P<0.001, student t-test). Bowel to liver REC higher then 1.55 identified 90% of the that results from necrosis and liquefaction of the entire volvulus. The affected bowel may reabsorb leaving pathologic cases whereas only 6.6% of the normal fetuses had REC higher then this cutoff vahie. A a long, atreticsegment and calcified remnents. Mostofthe duodenum and both thejejunum and the ileum novel quantitative method has been developed for an objective evaluation of echogemicity. The are supplied by the superior mesenteric artery. However, most of the duodenum lies retroperitoneally and method has been utilized in the evaluation of fetal HEB and was proven to be reliable in the detection becomes fixed to the posterior abdominal wall, unlike the jejunum and the ileum, which predisposesthem of most of the fbtuses who presented with a pathological condition. Fetal HEB was proven to be a to volvulus. significant cause for an adverse outcome of pregnancy in women with no apnori high risk. Careful The results of this study have implications for both the management ofinfants bornwith JIA and genetic follow-up, directed work-up and approprate genetic counseling are indicated when idenuuied. counseling for fauilies with affected infants.

201 202 Abnormal genital development in XY neonates associated with severe THE SIGNIFICANCE OF RARE TRISONIY NIOSAICISM DIAGNOSED IN AMNIOCYTES placental insufficiency and chronic intrauterine growth restriction. T. H. Hsu L.Yu M1,Neu R,VanDyke D.Benn P.Bradshaw C,Shaffer t.Higxins RKhodr Nesbitt. C. L Bodine. S. G. Kahler. and M. Decker-Phillips. Duke University Medical GItorton (,'Wang H.Brothman A.Chadwick D.Disteche (,.Jenkins L.galousek Center, Durham, North Carolina. DPantzar T & Wvatt P. Prenat. Diagn. Lab. NYC/.HRA and 16 other labs. Defective virilization of external genitalia has been demonstrated in the male offsping o1 The clinical implication of trisomy (T) mosaicism in amniocytes. several animal species exposed to intrauterine hypoxic stress. This association has not involving an autosome other than * 21. 18. 13 & 20. is related to the been described in humans. proportion of trisomic to normal cells, the role of placental mosaic- We have observed strikingly similar genital dysmorphology in four male infants with ism, & the impeinting effect of uniparental disomy (UPD). To determine early (e28 weeks) severe intrauterine growth retardation (<5th percentile) and placental the significance of such diagnoses, 151 prenatally diagnosed cases vascular pathology. All four infants were delivered prematurely because of non- were collected. These are cases of true mosaicism (mos) with known reassuring fetal status and/or maternal preeclampsia(3/4). Allhad evidence of severe pregnancy outcome and without evidence of ascertainment bias. The data placental vascular disease and abnormal doppler waveformswith a reversal of diastolic showed very high risk for abnormal outcome (>60%) for mos of T2, 16, flow. 22: high risk (40-59%.) for mos of T5, 9. 14, 15: The infants had normal XY genotype. All four boys had micropenis with tethering to a moderately high risk (20-39%) for mos T12; mode- Mos. Abn. Outcome perineal hypospadius. Internal genitalia were normal without cryptochordism.There was rate risk (up to 19SM) for mos T7, S; low risk for Type /Total % no other evidence of other congenital abnormalies or dysmorphology. There were no T17 and undetermined risk, due to small number or known teratogenic exposures and the mothers were of diverse geographical, ethnic and lack of cases for the remaining rare Ts. Abnorma- T2 10/ti (91%) socioeconomic backgrounds. Family histories were unremarkable for other congenital lities noted in prenatally diagnosed cases were T3 1/2 abnormalities. quite similar to those seen in postnatally iden- T4 1/2 We stress from tified Ts. Since most propose early hypoxic abnormal placentation may cause interference reported congenital abnor- TS 2/5 (40%) normal in malities are detectable placental steroidogenesis, resulting deficient virilization of external male prenatally with ultra- T6 0/3 to abnormal should genitalia. may secondary regulation and expression of cytochrome sound, sonography be performed in all pre- T7 1/8 (12%) products and/or Ad4BP transcription. At 20-21 weeks of gestation, P450 SCC natal cases. Cytogenetic confirmation of T mos T8 1/14 ( 7%) mRNA most abundant in the adrenals,followed by the testis, placenta, and ovary. showed a much higher confirmation rate in cases T9 14/25 (56%) is most abundant at 14-16 weeks is with abnormal outcome than with normal and very diminished by 26 weeks outcome T1I 0/2 gestation. This impliesimportant timing sequences in the expression of these gene (81% vs 55% for fibroblasts. 88% vs 46% for pla- T12 6/23 (26%) products relation to placental steroidogenesis. cental cells, 22% vs 10% for blood cells). When T14 2/5 (40%) both fibroblasts and placental tissues were T1S 6/11 (55%) studied. trisomic cells were found in one or the T16 15/21 (71%) other or both in 85%. Except for mos T8. 9, & 14, T17 0/7 PUBS is of limited use for diagnosis. DNA study T19 0/1 for UPD is suggested for chromosomes with known/ T22 7/11 (63%) likely imprinting effects (T2, 7,11.14.15 & 16). Slide Session 43: Prenatal and Pierinatal Genetics 11 (cont.) A41 203 204 A meiotic origin of the trisomy in pregnancies with confined placental Analysis of parent-of-origin specific DNA methylation at SNRPN and PW71 in tissues: mosaicism Is correlated with increased risk of fetal IUGR, UPD, and implications for prenatal diagnosis. S. high levels of trisomy in placental trophoblast W. P. Robinson'. 1. Barrett' T. Kubota', AradhyaL, M. Machab, AC.M. A. Telenius', R. D. Wilson' P.N. Howard-Peebles'. S. Langlois'. D. K. Kalousek-' Smith', L.C. Surh J. Satish , M.S. Verp!. A. Johnson, D.H. Ledbetter' ('Univ. of British Columbia, Vancouver Canada; and 'Genetics & IVF Institute, 'Diagnostic Development Branch, National Center for Human Genome Research, NIH, Fasirfx and Mcdical College of Virginia, Richmond, VA) Bethesda, MD, 2Children's Hospital of Eastern Ontario, Ottawa, Canada, 3Sinai Hospital of Molecular studies have been completed in 84 pregnancies with confined placental Baltimore, MD, 4University of Chicago, IL; 'Prenatal Diagnostic Center, Lexington, MA. mosaicism (CPM) involving an autosomal trisomy. In 44 cases, the origin of the Prader-Willi syndrome (PWS) and Angelman syndrome (AS) are distinct developmental placental trisomic cell line could be determined: a somatic (post-meiotic) origin was disorders caused by absence of paternal or maternal contributions of the chromosome observed in oniv one of 25 cases involving chromosomes 16 and 22, but in the region 15q11-q13, resulting from deletions, uniparental disomy (UPD), or rare imprinting majority (14 of 19 cases) involving chromosomes 2, 7, 8, 10 and 12. In many of the mutations. Molecular cytogenetic diagnosis is currently performed using a combination of remaining cases, the percentage of trisomic cells was too low to determine origin, and fluorescence in situ hybridization (FISH), DNA polymorphism analysis, and DNA it is thus likely that the true frequency of somatic cases is higher than we have methylation analysis. Only methylation analysis will detect all three categories of PWS observed. Fetal uniparental disomy (UPD) was found in 17 of 81 informative cases abnormalities, but its reliability in tissues other than peripheral blood has not been including maternal UPD for chromosome 2 (1 case), 7 (1 case), 16 (13 cases), and 22 examined extensively. Therefore, we examined the methylation status at the CpG island (2 cases). UPD was only detected in pregnancies where the placental trisomy was of of the small nuclear ribonucleoprotein-associated polypeptide N and at meiotic origin. (SNRPN) gene the A mciotic origin was only weakly correlated with high levels (>50%) of trisomy in PW71 locus using normal and abnormal lymphoblast (LB) cell lines (n=48), amniotic fluid cultured chorionic villi samples (CVS) (p=.05, Fisher's exact test) and cultured term (AF) cell cultures (n=25), cultured chorionic villus samples (CVS, n=17), and fetal tissues placental chorion (p=.04) but was strongly associated with high levels of trisomy in (n=18) by Southern blot analysis with methylation sensitive enzymes. Of these samples, term placental trophoblast (pj 0.0004) and presence of fetal UPD (p=.OOO3). 20 LB cell lines, 3 AF cultures, 1 CVS, and 15 fetal tissues had been previously Intrauterine growth restriction (IUGR) was also strongly associated with meiotic diagnosed as having deletions or UPD by other molecular methods. Methylation status at origin of trisomy (p=.0005) as well as presence of fetal UPD (p=.00003). The SNRPN showed consistent results when compared with FISH or DNA polymorphism association was strongest when both meiotic origin and presence of fetal UPD were analysis using all cell types tested. However, the methylation pattern for PW71 was analyzed together (p<0.00001). IUGR showed little to no correlation with high level inconsistent when compared with other tests and should therefore not be used on tissues of trisomy in cultured CVS (p= .37) or term chorion (p=.03), but was highly other than peripheral blood. We conclude that SNRPN, but not PW71, methylation correlated with presence of >50% trisomic cells in trophoblast (p= .0008). Larger analysis may be useful for diagnosis of PWS/AS on LB cell lines, cultured amniotic fluid, numbers of pregnancies need to be studied to evaluate chromosome specific effects and to determine to what degree the association of pregnancy outcome with origin of or chorionic villus samples and will allow, for the first time, prenatal diagnosis for families trisomy is attributable to each of the correlated effects of UPD and of level of trisomy known to carry imprinting center defects in which the specific mutation has not been in placental trophoblast. identified.

205 206 Methylation studies of FMR1 in paired fetal and extraembryonic tissue: Implication for First trimester screening for Down syndrome using biochemical and ultrasound prenatal diagnosis of fragile X syndrome using CVS. M Dccker Phillips-. H Kay1l LR ParksT markers. J.E. Haddow. G.E. Palomaki. G.J. Knight. Foundation for Blood I Lonashore. E Crawford; I Tarleton1 A McConkie-Rosell' 'Duke Univ. Medical Center, Research, Scarborough, Maine. Intro. by: L.A. Bradley. Durham . NC and Greenwood Genetic Ctr. Greenwood SC. As the number of CGG repeats and Since the 1986 report by Brambati et al. that maternal serum AFP was reduced in the methylation status of the CpG island of FMR1 influences the phenotype in fragile X 8 Down syndrome cases at 8 to 11 weeks gestation, many investigators have syndrome, accurate determination of these two parameters is crucial to ensure correct examined maternal serum analytes to determine the feasibility of first trimester interpretation of prenatal DNA testing and appropriate counseling to at risk couples. Down syndrome screening. Routine biochemical screening is not yet a reality, Methylation in FMR1 is associated with both normal (X-inactivation) and aberrant processes however, due in part to inconsistent findings and in part to a lack of a (expansion of the CGG trinucleotide repeat segment). The purpose of this study was to comprehensive dataset. Ultrasound measurement of nuchal translucency thickness determine the amount and correlation ofmethylation ofthe FMR1 region in paired female fetal (NTT) has also shown promise, but its use is not widespread. We are in the final and extraembryonic tissues samples in order to assess the appropriateness of prenatal diagnosis of phase of an NIH-sponsored (HD-31183) non-intervention study to evaluate maternal fragile X syndrome by CVS. 192 fetal and extraembiyonic tissue samples were collected from serum biochemical markers and ultrasound measurement of NTT as a way to elective terminations following an approved IRB protocol. Gestational age was determined by identify Down syndrome between 10 and 14 weeks. The study involves 20 prenatal transabdominal ultrasound assessmentofcrons-nrump length. SRY detection was used to confirm centers from throughout the US; the centers enroll women about to undergo either fetal sex. 27 paired extraembryonic tissue and female fetus samples ranging in weekly intervals CVS or early amniocentesis. A serum sample is obtained from each woman along from 9 to 13 weeks were available for analysis. Methylation status of the FMR1 region on the with ultrasound measurements and relevant demographic and pregnancy-related inactive Xwas determined by Southern blot analysis using EcoRI/Eagl digestion with the information. Sera are shipped unfrozen via express mail to the study center for StB12.3. The relative amount of methylation was determined by densitometry. Fetal tissue biochemical testing. Karyotype results are subsequently reported. In the first 3,285 demonstrated a consistently mature methylation pattern earlier than the extraembryonic tissue. pregnancies, 40 Down syndrome cases are confirmed. For those cases, median The amount ofunmethylated signal in the extraembryonic tissue was: 94-100°/o at 9 wks, 89-99% MoM levels are: AFP=0.85, uE3=0.94, hCG=1.64, free fl-subunit=2.13 and at 10 wks, 87-99% at 11 wks, 72-96% at 12 wks, and 90-93% at 13 wkas. Only a slight trend PAPP-A=0.52. At a 5% false positive rate, univariate detection rates (%) are: towards a mature methylation pattern by 12 weeks was identified. These results suggests that AFP=13, uE3=10, hCG=23, free f3-subunit=25 and PAPP-A=28. PAPP-A extreme caution should be used with prenatal diagnosis ofFMR1 utilizing CVS. CVS should not performance differs 46% detection below 12 weeks be performed prior to 12 wks as the transition in by gestation; and 12% at or methylation is highly variable prior to that time. above 12 weeks. At the same 5% false However even at 13 weeks some samples did not positive rate, NTT detects 28% of the 32 appear to have a mature pattem and follow up cases where measurements could be obtained. amniocentesis may still be required to resolve the methylation pattern in large premutations and An updated analysis of this study will small full mutations. be presented, including multivariate screening performance.

207 Urinary Markers: A new approach to screening for Down syndrome in the second trimester. L.H_ Kellner' I A_ Canick 2- G Palomaki' D.N Sailer4' G.M LaIrmbert- Mesgerlian2. M- Neuer'- R Osashsnondh' A T_ Bombard' 'Monteflore Med Ctr/Albert Einstein Col Med, Bronx, N.Y.; 'Womens & Infants Hosp/Brown Univ Sch Med, Providence, RI; 'Foundation for Blood Research, Scarborough, ME;4 Strong Mem Hosp, Univ Rochester Sch Med, NY; 'Brigham & Women's Hosp., Harvard Med Sch, MA. Screening for fetal Down syndrome (DS) in the second trimester using multiple maternal serum markers is both well established and considered routine. Matemal urine has recentlv been identified as a source for analytes in DS screening and shows great promise. Measurement of urinay P-core fragment (UPCF), total estrogen, and total estriol (tE3) have been reported in pregnancies affected with DS (Cuckle. et al., Canick, et al.. Kellner, et al.). In our studies, there was little correlation between UPCF and tE3 levels in both cases and controls (R=0.07 and 0.05, respectively); therefore, these independent markers may be combined. Moreover, neither marker was associated with maternal age. 32 DS (cases) and 206 unaffected (controls) pregnancies were collected between 14.9 and 21.6 weeks gestation. Case samples were obtained prior to pregnancy termination and control samples were obtained at time of routine prenatal care, amniocentesis, or ultrasound examination. Reasons for prenatal diagnosis were advanced maternal age in 25 cases, ultrasound findings in 2 cases, and positive serum screening results in 5 cases. Day-specific medians were calculated by appropriate weighted regressions using results from control pregnancies, and values were expressed as Multiples ofthe Median (MoM). UPCF values in cases were significantly increased (median=5.42 MoM); in contrast, tE3 values in cases were significantly reduced (cases median=0.64 MoM). The log standard deviations for UPICF and tE3 MoM were 0.306/0.354 and 0.221/0.237 for cases & controls, respectively. Both UPCF and tE3 MoM values fit log Gaussian distributions. Screening performance was estimated using our distribution parameters, a Gaussian model, and the U.S. matemal age distribution in 1992. At a 5% false positive rate, the UPCF and tE3 detection rates were 72% and 22%, respectively Combining maternal age and UPCF increased detection to 74%; adding tE3 increased the detection rate to 80%. Ifhowever, the DS detection rate was fixed at 60% the false positive rate using second trimester urinary markers and maternal age was only 1.3%, a marked reduction from the -5% expected using multiple serum markers. These data support the finding of Cuckle et al. that urinary multiple marker screening for DS in the second trimester may be effective. A42 Slide Session 44: Molecular Genetics of Disease IV 208 209 Four Half ABC Transporters May Heterodimerize In The Peroxisome Genotype/phenotype correlation in VLCAD deficiency. Membrane N. Shani, G. Steel, M. Dean*, D. Valle. Kennedy Krieger Inst. Dept of Pediatr and The Howard Hughes Medical Inst, Johns Hopkins Univ School of Medicine. BS Andresen'. P Bross'. H Lund'. LD Schroeder'. C Vianey-Saban2. P Divry'. CR Baltimore, MD and*Frederick Cancer Research & Development Ctr, Frederick, MD Roe;. MA Nada5. RJA Wanders4. L list'. B Poorthuis5. M Duran'. HR Scholte. Three ATP-binding cassette (ABC) transporters have been identified in the human JBC deKlerk5. TJ Kristensen'. L Bolund'. Ml Corydon'. N Gregersen'. peroxisome membrane: ALDR and PMP70. All three are half ABC transporters ALDP, Center for Medical Molecular Biology, Aarhus University Hospital and Faculty of Health and are presumed to dimerize to form functional transporters. ALDP and PMP70 have Denmark'. been in X-linked and Science. Aarhus. Hdpital Debrousse. Lyon, France-. Baylor University Medical implicated adrenoleukodystrophy Zellweger syndrome, respectively. Center. Dallas, Texas3. Academic Medical Acadenic By homology probing of the EST database, we cloned a human gene (P7 OR) that encodes Center, Amsterdam', Ziekenhuis, Wilhelmina Children's Erasmus a fourth peroxisomal ABC transporter. The P70R gene is expressed in all human tissues Leiden5, Hospital. Utrecht'. University, Rotterdam', The examined. The P70R protein (30% amino acid identity to PMP70) migrates as a band Netherlandes. Institute of Human Genetics, University of Aarhus, Aarhus, Denmark9. of approximately 69 kD and immunocytofluorescence studies localize it to peroxisomes. Very-long-chain acyl-CoA dehydrogenase (VLCAD) deficiency is a clinically The presence of four half ABC transporters in the human peroxisomal membrane raises heterogenous, potentially fatal disease in the mitochondrial fatty acid oxidation. The the possibility that these proteins heterodimerize. To investigate this possibility we disease usually present in early childhood. Phenotypically disease manifestation can used a homologous S. cerevisiae system [Shani et al P.NAS 92:6012, 1995]. PXA1 be divided into two forms. both with hypoketotic hypoglycemia and dicarboxylic and PXA2 are S. cerevisiae genes that encode homologs of the human peroxisomal aciduria. either without (1) or with cardiac involvement (2). We have shown that both are involved in ABC transporters. peroxisomal /3-oxidation We have the and characterization of the of fatty acids. Here we show that Pxa2p, like Pxalp, is associated with peroxisomes previously reported cloning human but not required for their assembly. Yeast strains carrying gene disruptions of PXA1, VLCAD cDNA sequence, together with the molecular basis of VLCAD deficiency

PXA2 , or both have similar and, in the case of the latter, non-additive phenotypes. [Hum Mol Genet 1996; 5:4614721. We have now established a method for PCR/direct- We also find that the stability of Pxalp, but not Pxa2p, is markedly reduced in the sequencing of all 20 exons of the VLCAD gene, and used it to investigate the absence of the other. Finally, we find that Pxalp and Pxa2p co-immunoprecipitate with mutational spectrum in 32 unrelated patients with verified VLCAD deficiency. an These either anti-Pxalp or anti-HA in cells expressing epitope tagged Pxa2p. genetic We show that the mutational spectrum is large. and that it was possible to data indicates that the and heterodimerize to form a and physical yeast Pxalp Pxa2p correlate the of the mutations to the complete peroxisomal ABC transporter involved in fatty acid /3-oxidation. This result, severity identified severity of the disease in the of plus the occurrence of 4 half transporters, predicts the presence of similar heterodimeric majority patients. Generally, patients which presented with the milder form (I) ABC transporters in the mammalian peroxisome membrane. had VLCAD alleles with missense mutations, and patients which presented with the more severe form (2) had mutant alleles encoding truncated proteins or proteins lacking amino acids. The fact that it is possible to correlate mutation type with disease phenotype may have important implications for future treatment of this potentially severe disease.

210 211 Phenotypic variation in patients with severe Hunter Syndrome. Aspartylglucosaminuria: structure, function and assembly of aspartyl. Kirsten M. Timms'. Marie-Louise Bondeson2 M. li Ansari-Lari', glucosaminidase with a novel type of catalytic mechanism. R. Tikkanen. A. Kristina Lagerstedt2, Shannon P. Dugan-Rochal, Donna M. luzny'. Ulf Pettersson2 Riikonen. C. Oinonen2. A. Jalanko, J. Rouvinen2 and L. Peltonen. National Public Health of Human Molecular and Richard A. Gibbs'. 1: Dept. of Molecular and Human Genetics, Baylor College of Institute, Department Genetics, Helsinki, Finland and 'University Medicine, Houston, Texas. 2: Dept. of Medical Genetics, Uppsala University, Uppsala. of Joensuu, Department of Chemistry, Joensuu, Finland. Sweden. Aspartylglucosaminidase (AGA) is a lysosomal hydrolase that participates in the of the residues in Iduronate sulfatase (IDS) deficiency results in Hunter Syndrome. a fatal X-linked lyso- degradation glycoproteins by cleaving asparagine glycoasparagines. Lack ofAGA results in a accumulation disease somal storage disorder. Patients with complete deletion of the IDS gene have been activity lysosomal aspartylglucosaminuria (AGU) that is characterized severe retardation. Several described with additional symptoms not commonly associated with Hunter Syndrome, by mental mutations resulting in AGU have been described in the gene encoding for the AGA enzyme. including the occurrence of seizures. In about 13% of patients there has been a recom- AGA is synthesized as an enzymatically inactive precursor polypeptide that is processed bination event between the IDS gene and an unexpressed IDS pseudogene-like structure into two subunits in the ER. This proteolytic processing results in the activation of the located 20kb distal of the IDS locus. The recombination results in a disruption of the IDS enzyme. The active enzyme complex which is a heterotetramer of two a and two gene in intron 7 and an inversion of the intervening DNA. These individuals have severe 0 subunits is transported into the lysosomes, where the a subunit is further trimmed by Hunter Syndrome with no variant symptoms. Sequence totaling more than 200kb in two removal of a short peptide from its C-terminus. Our recent data would imply that the contigs from the IDS region has been generated and fully characterized. Several genes activation cleavage of AGA into subunits is an autoproteolytic event that is triggered by were identified in close proximity to the IDS locus in addition to the IDS pseudogene-like the joining of two inactive precursor molecules together. structure. PCR was carried out on analysis four patients with large deletions spanning We have crystallized the human AGA enzyme purified from leukocytes and determined that a number of these are to IDS, revealing neighboring loci deleted in addition the its three-dimensional structure as well as dissected the structure-function relationship. IDS locus. .411 of these individuals have atypical symptoms in addition to severe Hunter Our data indicates that AGA has an exceptional catalytic mechanism in which a Syndrome. RT-PCR was used to analyze IDS gene transcripts from four patients where bifunctional residue Thr206 in the N-terminus of the 0 subunit plays a key role. This there has been a recombination gene structure. between the IDS and pseudogene-like residue functions both as a nucleophile (the OH group) and a base (the free a-amino Sequence analysis of these PCR products revealed that the transcripts were combina- group) in the glycoasparagine breakdown. The functional role ofthe active site residues of tion between exons from the IDS gene and two genes identified distal to IDS. However, AGA was dissected by site-directed and studies. Based on these of normal mutagenesis expression expression transcripts from these loci does not appear to be disrupted by this results, only one known AGU mutation interferes with an active site residue. mutation. These results reveal that the deletions in Hunter Syndrome patients with vari- Our data provides evidence that AGA is a member of a recently described novel family ant involve to symptoms loci adjacent IDS, supporting the theory that these phenotypes of amidohydrolases that share the fold in the active site region and exhibit very similar could be due to loss of other loci. In addition, the lack of variant phenotypes in the catalytic mechanisms with an N-terminal nucleophile/base. This family of N-terminal recombination patients, combined with normal expression of neighboring genes, suggests nucleophile (Ntn) hydrolases contains also three prokaryotic amidohydrolases with known that the loci we have identified distal to IDS should be considered candidate genes for three-dimensional structures. All Ntn-hydrolases seem to be autocatalytically activated some of these phenotypic variants. from inactive precursors. Thus, the Ntn-hydrolase fold seems to provide the capability to bring about both autoproteolytic activation and hydrolysis of the amide bond of the substrate.

212 213

Two identified and LeuI52Met) in individuals Cloning methionine synthase and identification of mutations in newly mutations (Thr2331le partially deficient for adenosine (ADA) result in differing biochemical and metabolic patients cblG complementation group. D. Leclerc, P. Govette, deaminase and Jenkins. NYU Ctr Campeau, Christensen, C.E. Adjalla. D.S. Rosenblatt, R.Rozen phenotypes. R. Hirschhom.W. Borkowsky. D.R. Yang T. Mcd .. and R.A. Gravel. MRC Group inMedicalGenetics, McGill University, Montreal, N.Y.N.Y. & S African Inst Med Research, Johannesburg, South Africa. Quebec, Canada. Deficiency of adenosine deaminasc (ADA-) results in autosomal recessive immunodeficiency Cobalamin-dependent methionine synthase catalyzes methyl group transfer from disease of varying severity. "Partial" ADA deficiency, (absence of ADA in erythrocytes but methyltetrahydrofolate homocysteine to form tetrahydrofolate and methionine, with expression of easily detectable ADA in non-erythroid cells) has also been identified primarily by cobalamin prosthetic group serving as an intermediate methyl carrier. cDNAs en- population screening of healthy adults in Africa and newborns in New York State. Normal coding synthase have been reported for several lower organisms, but not for immune function and/or minimal elevations of toxic metabolites in childhood suggested that We of specific regions homology within the methionine synthase se- partial ADA deficiency was benign and that six missense mutations. (expressing from 8-80% quences, including portion of the cobalamin to binding site, design degenerate oligonu- of normal ADA in non erythroid cells), identified in "partially" ADA deficients were not RT-PCR. These PCR reactions, as well as RACE-PCR and inverse-PCR, pathogenic. However, one of these mutations expressing the lowest activity (Argl I ICys), has yielded specific amplification products highly homologous to the sequences of the other recently been found to result in adult onset disease when heteroalieic with a large deletion. We syntheses, covering at least 90% of the coding sequence. We have identified have now molecularly and biochemically studied two additional partially ADA deficient mutations thus far in patients of the cblG complementation group by SSCP individuals who represent opposite ends of the spectrum of partial ADA deficients as to age of sequence analysis, confirming that the cblG complementationgroup corresponds to ascertainnct and biochemical abnoomalities. Homozygosity for a newly identified Leul 52Mct primary methionine synthase. This work will allow a better understanding of mutation less activity than the pathogenic mutation was of disorders of cobalamin and folate metabolism. expressing considerably Arg2l lCys found in a currently healthy 10 year old Afghanistani child (ascertained at birth). He had the highest accumulation ofthe metabolite deoxyATP among 13 "partially" ADA deficients studied, although considerably lower than in immunodeficients. Homozygosity for a newly identified Tbr2331Ie mutation expressing ADA somewhat greater than Arg2II Cys was found in a healthy young adult Kung tribesman, associated with very low metabolite concentrations. Based on the biochemical findings and a family history suggestive of immunodeficiency in a prior offspring, it is likely that the Leul52Met mutation could result in disease in homozygous individuals challenged by severe environmental insult or in heterozygosity with a "null" mutation. The pathogenicity of the Thr2331le mutation, as well as of a previously described Ala215Thr mutation with similar low activity will only be determined by long term observation of individuals carrying these mutations. Slide Session 44: Molecular Genetics of Disease IV (cont.) A43 214 215 Mutations in the PEX gene in X-linked hypophosphatemnic rickets Identification of Wilson's Disease Mutations in Various Ethnic Populations and Preliminary (HYP). I.A.Holm, X.Huang N.M.Zacconi.L.M. Kunkel. Children'sHospital Functional Analyse AB Shah'. K Pctrukhin-. HT Zhang]. BM Ross,. E Paranot. QK and Harvard Medical School, Boston, MA. Penchaszadeh2. I Sternlieb4. IH Scheinber&4. and TC Gilliam'i2 Deparments of Genetics and X-linked hypophosphatemic rickets (HYP) is the most common form of rickets in Development and -Psychiatry, Columbia University, New York, NY; 'Pediatric Clinic, University of the United States today, and is characterized by renal phosphate-wasting, abnormal Catania, Italy; 4The National Center for the Study of Wilson's Disease and St. Luke's Roosevelt 1,25-dihydroxy vitamin D metabolism, and an X-linked dominant pattern of Hospital, New York, NY. inheritance. The pathophysiology is unclear, and the map position cloning Wilson's disease (WD) is an autosomal recessive disorder of copper metabolism characterized by approach has been taken to identify the gene defective in HYP on Xp22.1. A 1916 substantial decrease in biliary excretion of copper and defective incorporation of copper into a bp candidate cDNA for HYP has been identified from the critical region and named plasma-copper protein, ceruloplasm. Toxic accumulation of copper in the liver and brain leads to PEX (Uhosphace regulating gene with homologies to Indopeptidases, on the x liver disease and/or extrapyrimidal neurological syndrome in the first or second decade of life. The chromosome) (The HYP Consortium, atureGenetics. 11:130-6, 1995). WD gene, identified on chromosome 13, encodes a P4ype ATPase with 6 metal binding sites. We have characterized the genomic structure of the PEX gene.and have analyzed We report the identification of25 WD mutations, 18 of them novel, and their relative population our '4 HYP families for mutations. A genomic phage library was constructed from frequencies in a total of five clinical samples from America, Sicily, Sweden, Russia, and Costa Rica. YAC A0472, the YAC that spans the HYP critical region, and a contig of genomic The population frequency values for these mutations confer that the spectrum of WD mutations phage was constructed across the PEX gene. To determine the intron-exon borders consists of a small number of relatively frequent mutations with a large number of rarely occurring of the PEX gene, primers were designed to the cDNA and were used to sequence mutations. Upon comparison of these and previously identified mutations, we have noted that the genomic phage clones containing portions of the PEX gene. Based on the particular residues or regions of the gene are mutated frequently, allowing us to identify new intron sequence, intron primers were designed to amplified the exons. The PEX residues or regions crucial for protein function. Although no clear genotype/phenotype correlation is exons were screened for mutations by single-stranded conformational apparent, we have observed a high incidence of fulminant cases in our Costa Rican population, polymorphism (SSCP) analysis. In females, exons migrating aberrantly were highly enriched (61%) for the asnl270ser mutation. isolated from the dried SSCP gels and sequenced. In males, PCR-amplified exons To examine the effects of mutations more closely, we studied various mutations at transcriptional were sequenced directly. 13 exons, spanning 1370 bp of the 1916 bp cDNA, have and functional levels. RT-PCR studies on lymphoblastoid cell lines derived from patients been identified. Exon range from 70 to 145 bps in length. 11 exons have demonstrated that A to G splice acceptor substitutions at the invariant -2 position lead to a high undergone SSCP analysis, and we have identified mutations in 6 of our patients: 3 degree of exon skipping, suggesting that this mutation is responsible for the onset of Wilson's nonsense mutations (2 males, I female), a 4 bp deletion of the 5' intron donor disease. Lymphoblastoid cell lines from patients with 4 different WD missense mutations exhibited splice site (male), a large deletion spanning at least 10 exons (male), and a patient significantly decreased copper-stimulated ATPse activity as compared to normals; however, these with 2 missense mutations 4 bp apart (a female). We will describe the genomic reduced levels do not correspond with the age of onset seen in patients, again indicative of a high structure of the PEX gene, the mutations in our HYP families, and make degree of complexity in the mechanisms of copper transport. phenotype-genotype correlations.

216 217 Biochemical and molecular genetics of the syndrome of increased aromaltase activity: Mild and male substitution in the from the gene and evidence for aberrant androgen insensitivity infertility: ligand- segregation with a marker within P45Oarom domain of the that transactivation but not alternative splicing of its '-end mR.NA. C.-A. Stratakiis ;. A. 'ortero A. Brodie D.DeAtiine binding androgen receptor impairs ;,O.L . S .itsiad , Y FlA% £FA.Garnica '. C.S.Nlitsiadis G.P.ChroIsos' androgen binding. E.L. Yong, A.A.R. Abdullah. C.K. Choo. J. Lim, T.G. Tut. DEB.NIICHD.NIH. Bethesda. MD. (2) Dept. Pharmacology. Univ. Maryland. Baltimore. MID. 13) R, Lumbroso. M.A. Trifiro and L. Pinsky. Dept. of Obstetrics and Gynecology, Southview Mled. Cr., Birmiogham.AL. (4) Pediatrics. Georgetown Lniversity. Washinsgon. DC. National University of Singapore; Lady Davis Institute, Depts. of Human Two unrelated kindreds have been reported with familial gynecomastia and increased Genetics, Biology and Medicine, McGill University, Montreal, Canada. extraglandular aromatization. Although X-linked inheritance was proposed tor the syndrome of In male infertility due to impaired spermatogenesis the etiology often remains increased aromatase activity with rznecomastia" (SIAG- ONtttM 107910). we recently reported a unknown. Androgens are essential for sperm production, and a small fraction of novel kindred with clinical and biochemical evidence of SIAG and male-to-male transmission. The androgen-sufficient, sperm-deficient infertile males have been found to have mild propositus (P) presented at age 9y with n-ynecomastia. His 7 v.o. sister (S) had Tanner-Itt breast androgen insensitivity due to deficient or defective androgen binding by the development and pubic hair. their father (F) had bilateral necomastia in early puberty and the paternal grandmother (PGMI) had massive macromastia. DNA and mRNA were extracted from androgen receptor (AR). We used SSCP to screen all 8 exons of the AR gene in whole blood. Skin fibroblasts and EBV-transformed lvmphoblastoid cell lines were established from 150 infertile males with defective spermatogenesis. Two unrelated subjects have the two children. their parents and paternal grandparents. Aromatase activitv. as measured by P3H1- the same amino acid substitution (Met886Val) in the portion of the ligand-binding andronestedione (A) to [3H]-estrone (El) conversion. was markedly increased in fibroblasts [S: 85. domain (LBD) encoded by exon 8. Both have severely depressed sperm counts F: 48.1, mother (,M): 0 fmol/mg proteinihr] and lymphoblasts (P -.2 S: 13.4. F: 36.1. 1: 0. PGMI (less than 1 million per ml). One had gynecomastia; the other, sparse facial hair. 23.2 fmoLmg protein/hr) from affected individuals. Immunostaining of the boy's breast tissue The AR in genital skin fibroblasts of both subjects binds normally to testosterone, A from showed expression of the aromatase gene in epithelial cells. tetranucleotide repeat marker and the mibolerone and within this gene (chrom. 15qZ segregated with the disease in all affected members (Lod score 0.6. 5ei-dihydrotestosterone, synthetic androgens, methyl- as measured Scatehard q = B). PCR amplification and sequencing of the proximal 5' region of the PIt- and negative trienoline, by analysis (B..., KJ), nonequilibrium regulatory elements of the Pl- promoter showed no alterations. RT-PCR demonstrated that trom the dissociation kinetics, and thermostability. Likewise, no changes in the binding or known promoters of this gene. only the ovarian and fetal liver promoter -ere expressed in the cells dissociation kinetics of the first three androgens were detected when the mutant of the patients. Rapid amplification of cDNA ends (RACE) showed a novel promoter being AR was expressed in COS-7 cells. However, the mutant AR has a consistently exclusively present in the affected but not the unaffected members of the family. A BLAST search reduced (-50% of normal) capacity to transactivate either of two cotransfected revealed no homologv to other knowvn sequences. including those of the P4IOarom genomic region. androgen-inducible reporter genes, pMAM-luciferase (n=7, lab A), or pMMTV- We conclude that SIAG can be the cause of isosexual precocity and massive macromastia in (i) luciferase lab We conclude that in the AR is the cause of females and is inherited as an autosomal dominant trait. (ii) increased aromatase expression in SIAG (n=6, B). Met886Val can be shown not only in skin fibroblasts. but also in EBV-transformed lymphocytes from affected impaired spermatogenesis in these two infertile males. Our data reveal that patients. (iii) the genetic defect in our kindred cosegregated with a polymorphism of the CYP19 Met886 of the AR influences the post-androgen-binding events that are critical for gene. (iv) aberrrant alternative splicing of the -'end of the P.50mR.NA appears to be the cause of normal transactivation by the AR. This is the first evidence that a particular this disease in our kindred. residue in the LBD of the AR is dedicated primarily to transcription regulation.

218 A novel germ line mutation in SOX9 causes familial campomelic dysplasia and sex reversal. A.H. Sinclair'. FJ. Cameron'. C. Cooke- Yarborought. C. Kwok . L.L. Goodwin3 and D.O. Sillence3. l.Dept. of Pediatrics and Centre for Hormone Research, University of Melbourne, Royal Children's Hospital, Melbourne, VIC. 3052. Australia. 2.Dept. of Genetics. University of Cambridge, Cambridge, CB2 3EH, U.K. 3.Dept. of Clinical Genetics, The New Children's Hospital, Sydney, N.S.W. 2124. Australia. Mutations in the gene SOX9 result in the syndrome of campomelic dysplasia (CD) which includes sex-reversal in 75% of 46,XY affected individuals. These mutations only affect a single allele of SOX9 suggesting a dominant mode of inheritance for this syndrome. Consequently, CD and autosomal sex reversal may result from haploinsufficiency of SOX9. The SOX9 gene maps to the long arm of human chromosome 17 and translocations in this region also result in C.D. We report a family in which there were three affected patients, two of whom showed 46,XY sex-reversal. Interestingly, despite all three patients being heterozygous for a familial mutation in SOX9, (insertion of a cytosine residue at nucleotide position 1096), their gonadal phenotypes varied widely. The proband was found to have 46,XY true hermaphroditism with ambiguous genitalia. The other two sibs were 46,XY and 46,XX both of whom had bilateral ovaries with normal female genitalia. The somatic cells in both parents revealed wild-type SOX9 nucleotide sequences. However, mutational analysis of the SOX9 gene in the father's germ cells revealed they were mosaic for mutant and wild-type sequences. This family is particularly informative as it demonstrates that the same SOX9 mutation can produce very different 46,XY gonadal phenotypes. Presumably, the variable penetrance of CD between these sibs reflects differences in genetic background in which the SOX9 gene operates. These results are also the first to demonstrate that paternal germ cell mosaicism of a mutant SOX9 sequence can result in a CD phenotype amongst his offspring. A44 Slide Session 45: Linkage Mapping aniId Poymorphisms II: Mapping Complex Traits 219 220 Why genomic screens of 5-10 cM can be successful in mapping genes Linkage disequilibrium mapping of a novel peripheral neuropathy in Balkan through association and haplotype sharing analysis. Gypsies. L.aayievl A. . D.AnjgeihevalK. Gerard J. Te Meerman and Martin A. Van der Meulen Dept. Medical Genetics, in'- A.Niko University of Groningen, A. Deusinglaan 4, 9713AW Groningen, The Netherlands. F.Calafelll, '.Laboratory of Molecular Pathology, (Intro. by H.Scheffer) Medical School, Sofia 1431, Bulgaria; 2Department of Human Biology, Edith Cowan Despite reports on successful genetic mapping using genomic sharing, the theoreti- University, Joondalup Campus, Perth 6027 W.A., Australia; 3Graylands/UWA Clinical cal foundation for this method is still incomplete. Association and haplotype sharing Research Unit, Perth 6010 W.A., Australia; tepartment of Anthropology, University of are caused by slow clipping off due to recombination, surrounding alleles Identical by Barcelona, Spain; 5Neurosciences and Brain Research Foundation, Centre of Hygiene, Descent. Association between disease and genetic markers at the population level is Sofia 1431, Bulgaria possible because very few mutated alleles will drift to very high population frequencies. To quantify expected genomic sharing and the variance we used computer simulation of Centuries of nomadic travel and several large migration waves have spread genetic drift and recombination by tracing single alleles. A representative result is given Gypsies to every comer of the world. An estimated 10 million live in Europe today, of for a mutated allele that has drifted over 60 generations, with a growth factor of 5.2% whom about 70% reside in or come from Eastern Europe. Linguistic evidence has been per generation. This results in a 4% probability of survival and an expected number of used to date Gypsy exodus from India to around 1000 A.D. Their population oforigin is 440 for surviving copies. After weighing for the number of alleles IBD, we found that the unknown and it is not clear whether it is identical for all Gypsy groups. Gypsy society expected genomic sharing between two haplotypes carrying the same allele in the popu- lation is 4.7 cM, with a standard deviation of 8 cM. The distribution is very skewed due has a complex stratified structure subdivided into many groups on the basis oftraditions, to clusters of comparatively closely related relatives with large genomic sharing. Because trade, history of migrations and language etc. The question of how these classification genetic drift and geographic drift are correlated, sampling of patients should be from the criteria relate to common origins and genetic similarities has never been addressed. As a smallest possible geographic region. It thus appears that under assumptions realistic result, Gypsies have not attracted the attention ofgeneticists insearch of isolated founder for many populations, appreciable genomic sharing can be expected. Genomic sharing populations. Moreover, very little is known about hereditary disorders in Gypsies. We is so large and so variable that even a 10 cM initial screen has a reasonable, but not very high, probability of being successful by exhibiting significant allelic association of provide the first example ofmapping a disease gene in a Gypsy genetic isolate. A novel very polymorphic markers and excess haplotype sharing surrounding disease alleles. We peripheral neuropathy was found to occur in several strictly endogamous Gypsy groups therefore advocate using 10 cM screens initially in founder populations, with extension which do not intermarry and differ in terms ofidentity, tradition, language and history of to the 5 and 2.5 cM level. migrations into Bulgaria. We have mapped the gene to a narrow interval on 8q. Our data strongly suggest that in all Gypsy groups the disorder is caused by a single mutation whose origin predates the divergence of the groups. The age of the mutation is at least 800 years, i.e. around the time of exodus from India and settling in the Balkans. We show that, within the complex structure of Gypsy society, genetically related endogamous groups exist which can be traced to a common small founder population.

221 222 The use of a novel strategy for genome-wide linkage studies to identify a Linkage disequilibrium mapping in the Finnish population susceptibility locus for a complex congenital heart defect. D.Y. Nishimurat. confirms localization of a susceptibility locus for celiac M.E. Pierpont, J.S. Beck', T.L. Burns'. M.A. Berg'. E..M. Stone', R.L. Lauer', dis.as.. A. Polvil. C.C. McComb. F. Zhona3. 3. Partanien. and V.C. Sheffield'. University of Iowa, Iowa City, IA and 2University of Minnesota, M. Mki2. 3P. Collin5. R.C. Elaton . J.M. Olson . and J.P. Minneapolis. MN. Micbhnuki.. Finnish Red Cross, Helsinki', University of The identification of genetic loci involved in most forms of congenital heart disease has Tampers, Finland2, University of South Alabama?, and Case been hampered by the complex inheritance patterns of these disorders. Atrioventricular Western Reserve University'. canal defects (AVCD) are most commonly associated with Down syndrome, although Celiac disease is a severe and chronic gastrointestinal nonsyndromic cases also occur. Nonsyndromic AVCD have been attributed to multi- disorder in which immune-mediated damage to the small factorial inheritance. However, the occurrence of a few kindred's with multiple affected intestine leads to malabsorption. It is well established individuals has suggested that a major susceptibility locus contributes to the disorder in that a particular HLA-DQ heterodimer is an important some families. DNA pooling was used in combination with shared segment analysis to genetic risk factor for the disease. Segregation analyses perform a high density screen of the entire autosomal human genome in an extended kin- have shown that at least one other locus is involved, and dred with 13 affected individuals. We performed extensive genotyping on four affected is, in fact, a stronger determinant of familial aggregation individuals, as well as a pool of DNA from all 13 affected individuals. Markers for which than is HLA-DQ. A recent genomic screen of microsatellite at least three of four affected individuals shared an allele, and for which the shared allele markers in our laboratory, employing linkage analysis of was a major allele in the affected DNA pool, were considered to be positives. Regions affected sib pairs from the western counties of Ireland, of the genome containing clusters of positive markers were selected for genotyping in all identified a region on chromosome 6p, about 30cM telomeric family members. By this approach. we identified a genetic region at which all of the from the HLA region, as being significantly linked to affected individuals shared a haplotype. Linkage analysis using only affected individuals celiac disease. To confirm the significance of this demonstrates significant linkage with a lod of 4.0 (theta=O). Analysis using all available locus, we performed a linkage disequilibrium mapping study of the most relevant microsatellite marker in 49 Finnish pedigree members demonstrated linkage with lod 6.7 (theta=O) using a penetrance value celiac disease of 0.65. Our data demonstrate the existence of an AVCD susceptibility gene, inherited as patients and 46 DQ2 positive controls. There an autosomal dominant with incomplete penetrance. Furthermore, our data demonstrate was a highly significant association between genotypes of the power of using key kindred's in combination with an efficient approach for this marker and celiac disease (X2 - 58.8: p-0.0004 by performing tabulated chi squares, df-27, or p-0.0006 by Monte Carlo a high density screen of the genome to identify loci involved in complex/multifactorial One disorders, such as congenital heart defects. simulation). particular allele was especially frequent, there being 11 homozygotes among the patients compared with only two among the controls (p-0.009 by Fisher's exact test). These results replicate the finding of a specific second susceptibility locus (in addition to the known HLA factor) contributing to the risk of celiac disease.

223 224 Identification of a non-ELr susceptibility locus for celiac Mapping ofthree susceptibility genes to insulin-dependent diabetes mellitus: IDDM4, disease in the of Ireland. IDDM5 and West naL.C.C.McCmb.Z-3 IDDM8: J.X. She'.D.F. Luo'. R. Buzzetti2.JI. Rottei9. L.J. gaffel3. L. J.M. Olson2. R.C. Elston2. F.M. Stevenas5, C.F. Mc Nistico2.C. Giovannini2.P. Poz-illi.G Thomson4and Noel K. 'University and J.P. . Maclaren'. Michalski University of South Alabama , Case Western Reserve University2, University College Galway, of Florida; Gainesville; 2University of Rome, Italy; 3Cedars-SinaiMedical Research Ireland3. Institute, Los Angeles, California; 'University of California, Berkeley. Celiac disease, also known as gluten sensitive The expression of Type I diabetes (IDDM) is influenced by a number of susceptibility enteropathy, is a debilitating, lifelong gastrointestinal genes and environmental factors. The HLA class II region (IDDMI)on 6p and the illness provoked by the ingestion of certain grain proteins insulin gene region on I 1ql5 (IDDM2) are wellknown to influence susceptibility to in susceptible individuals. The highest prevalence of this IDDM. HLA and INS disorder be in the However, together only explain <50% of the total genetic may western counties of Ireland, where contributions, suggesting that other factors must 1 in 300 individuals are affected. A genetic exist. Previous genome-wide 453 linkage analysis mapping studies in our own and other laboratories have provided suggestive linkage study using highly polymorphic microsatellite markers evidence several novel of 45 affected sibpairs from the West of Ireland was for IDDM loci; however, it was not sufficient to confirm the carried out to identify chromosomal regions conferring risk existence of these genes. We analyzed 265 Caucasian families with IDDM and report of celiac disease. The results confirmed the well known the first evidence that meets the standard for confirmed linkage for three loci. The linkage of alleles in the HLA region, and highlighted maximum lod scores (MLS) were 3.9, 4.5 and 3.6 in our data set, and 5.0,4.6 and 5.0 several additional regions as showing significant or for our data combined with data from the The most non-oveflapping literature, for IDDM4 on suggestive linkage. significant of these was an chromosome 1q13, IDDM5on 6q25, and IDDM8 on 6q27, respectively. However, we area on chromosome 6p, about 30cM telomeric from the HLA could not confirm of several markers linkage for several other putative intervals including IDDM3 on 15q26 region. Analysis tightly clustered in and on valuable this area indicated a multipoint maximum lod score of 4.66 IDDM7 2q31-q33. Many lessons for mapping genes involved in and a recessive mode of inheritance. The multipoint lod common diseases can be learned from this study. First, it demonstrates that genome- score for this locus was greater than that for HLA-DQ, wide scan can be used to identify genes involved in common diseases, even though it which was 4.44. These results suggest that a second remains a very difficult task. Second, variations in gene sharing between data sets are susceptibility locus for celiac disease has been localized. very common, probably due to random variations and/or Its mode of action genetic heterogeneity. Third, appears consistent with the results of the proportion of gene sharing by affected sibpairs may vary dramatically even in sample previous segregation studies, which have the - of suggested sets of 100 fully informative sibpairs and a minimum of 200 families should be presence a non-HLA linked gene, acting in a recessive targeted for most studies. manner, that is a greater determinant of familial mapping Finally, linkage evidence may decrease very quickly aggregation than the known HLA risk factor. as the distance from the disease gene increases. Therefore, a high density map of highly polymorphic markers would be very helpful to confirm real linkage. Slide Session 45: Linkage Mapping and Poymorphisms II: Mapping Complex Traits (cont.) A45 225 226 Localisation of a Type 1 diabetes mellitus polygene (!DDM16) to 200 Setting priorities for follow-up studies of a complete genome scan for kilobases of human chromosome 18q12 using microsatellites and genetically NIDDM. N.J. G.I. P. T. Cox', Bell', Concannon', C.L. Hanis3. R.S. Spielman4. diverse populations. Ierriman, R. TIwells, M1. Mlerriman, Z. .Atta, R. Cox, of S. Jenkins, L. Pritchard, A. Wilson, F. Cucca, E. Bosit, R. Buzzetti2, P. Pozzilli2, 'University Chicago, 3Virginia Mason Research Center, Seattle, 3Universitv of E. S. Texas. 4University of Pennsylvania. G. Joner3, K. Rbnningen4, Thorsby4, D. Undlien4, Bain3, Results of a genome screen of 490 markers in 330 Mexican American sib pairs with A. Barnett5 and J. Todd. University of Oxford, UK. 'University of Milan, Italy. NIDDM suggested that a locus (VIDDMI) linked to D2S125 on 2qter contributes to 2University of Rome "La Sapienza", Italy. 3Aker Diabetic Research Centre, Norway. NIDDM susceptibility in Mexican Americans, and identified additional regions with less 4The National Hospital, Norway. sUniversity of Birmingham, UK. conclusive evidence for linkage. While some ofthese latter regions may contain additional Insulin dependent (type 1) diabetes mellitus (IDDM) is a common, complex disease NIDDM susceptibility loci, some are likely to be false positives. In order to determine caused by autoimmune destruction of the pancreatic insulin producing cells. The HLA which of the regions in addition to D2S125 should be examined further, we conducted region on chromosome 6p (IDDMI) and the insulin gene region on chromosome lip multipoint linkage analyses on 2 subsets of the data: families most (all affected sib IBS (IDDM12) have long been known to be associated with IDDM and were originally de- 2 at both D2S125 and the tightly linked marker D2S395) or least (at least 1 affected sib tected in case-control studies as candidate genes. Genome linkage scans using affected IBS/IBD 0 at D2S125 or D2S395) likely to have IVIDDMI susceptibility. We found 18 sibpair families have indicated that IDDMI is the major locus, but that several other regions in which the multipoint lod score for a subset was > 0.5 and at least 1 lod unit chromosomal regions are linked. One of these, on chromosome 18q12 (IDDM6), showed higher than in the dataset as a whole. Simulations in which the data were randomly weak evidence of linkage and of linkage disequilibrium (LD) in an initial set of 195 UK subdivided into samples the same size as those most or least likely to have NIDDMI families. By cloning several hundred kilobases of DNA containing this marker and by were used to establish empiric locus-specific p-values. D15S119 was the most significant isolating further microsatellites from the contig we have been able to define a region of of the markers identified in these studies, and was therefore used in a haplotype with maximal LD of less than 200 kb. This was done using a total of 1236 diabetic fami- flanking markers to similarly subdivide the data into family subsets most and least likely lies from six separate populations, which enabled the identification of several different to derive NIDDM susceptibility from this region of chromosome 15. These analyses founder chromosomes. These results demonstrate that systematic LD mapping can be identified 16 regions in which the multipoint lod score was > 0.5 and at least 1 lod unit used to finely position a complex disease susceptibility gene in the absence of any known higher than in the dataset as a whole; 11 of these regions were among those identified candidate genes. Polymorphic microsatellites are ideal tools to detect disequilibrium. in the analyses on the iVIDDMI subsets. Simulations were again used to determine the Large numbers of families are required and, critically, several different ethnic groups empiric p-values for regions identified in both the 2q and 15q subset analyses. Based on should be analysed to provide replications for initial observations, and to establish the our results, we consider the regions around the following loci to have priority for follow- chromosome region most associated with disease. up studies: D2S125, D15S119, D2S406, D3S1600, D4S243, D8S555, D11S929, D12S393, D14S53, DiSS111, MCAG69, D17S1308, and D21S1435.

227 228 Genetic linkage studies using cellular phenotypes in transformed Genome-wide search for susceptibility genes for nephropathy and hypertension among diabetic lymphoblasts: Mapping of a locus for platelet activating factor-evoked Pima Indians. G. Imoratore. R L. Hanson. P. H. Bennett V. C. Knowler. and the Pima Diabetes calcium response to chromosome 16, and its possible implication for Genes Grup. NIDDK, Phoenix AZ. essential hypertension. LM Brztistowiczi, JP[ ardner , L Hopp2. J Ott3, Non-insulin-dependent diabetes (NIDDM) among Pima Indians of Arizona is frequcntlv E Jeanclos2, XY Yan Z Fekete2, and A Aviv2. Center for Molecular and Behavioral accompanied by diabetic nephropathy. often leading to end-stage renal disease (ESRD). Familial Neuroscience RutgersUniversity, Newark, NJ and Department of Psychiatry, aggregation of ESRD and nephropathy occur in Pima Indians with NIDDM suggesting the existence University of Medicine and Dentistry of New Jersey, Newark, NJ, 2The Hypertension ofgenes determining susceptibility to nephropathy in addition to those which lead to NIDDM itself. Research Program of the University of Medicine and Dentistry of New Jersey, Newark, To identity genetic loci for NIDDM among the Pima Indians, a genome-widc scan using over 700 NJ, 3Departments of Psychiatry and Genetics and Development, Columbia University, anonymous DNA markers is being conducted. Siblings from 260 families are potentially informative New York, NY. for diabetic nephropathy. Only diabetic subjects were included in these analyses. Nephropathv was Epstein-Barr virus transformed lymphoblasts from patients with essential hypertension defined as the presence ofat least one of the following: urinary albumin/creatinine ratio >300 mg/g. demonstrate enhanced G protein-mediated cytosolic calcium (Cai) response to platelet- urinarv protein/creatinine ratio 2500 mg/g, presence of ESRD. or serum creatinine 22.0 mg/dl. activating factor (PAF). To map genes responsible for variation in G protein coupled Because nephropathy usually develops only late in the course of diabetes and many of these subjects signaling, we used this cellular phenotype for a linkage study of transformed cell lines have diabetes of short duration, only analyses of affected pairs are shown. Hypertension was defined from reference pedigrees. PAF-elicited Caj responses were obtained in lymphoblastic as blood pressure 2140/90 mmHg or use of antihypertensise drigs. For both traits, we estimated the cell lines from five pedigrees of the CEPH collection. The PAF-evoked change in Ca, mean proportion of genes identical by descent (lMD) among sibling pairs and tested its difference ranged from 20 to 392 mmol/L and was highly reproducible within each cell line. Us- from 0.5. In addition. the Haseman-Elston (H-E) regression test over all sibling pair types was ing PAF-elicited Ca, response as a quantitative trait, two-point sibpair linkage analy- performed for hypertension. A total of-.603 diabetic sibling pairs were analyzed among whom there ses were conduced using 5,150 markers from the Cooperative Human Linkage Center were -120 pairs with hypertension and -80 pairs with nephropathy in both members ofthe pairs. database. Nine loci, located on seven chromosomes, were suggestive of linkage, with Three chromosomal regions suggestive of linkage (p< 0.001. for affected pairs) for hypertension or p values less than 7.4x10-4. Multipoint linkage analysis produced a significant linkage nephropathy were identified: finding (p=2.1x10-5) in one family at D16S1S1, with suggestive linkage results spanning Trait Marker Affected pairs H-E a 40 cM interval of chromosome 16. Multipoint analysis produced suggestive findings n IBD p-Values _p-Values of linkage to two distinct regions of chromosome 11 in another family. These results Nephropathy D7S 1804 72 0.53 0.0004 indicate that loci involved in the control of G protein-mediated mechanisms, suggested to be involved in the pathophysiology of essential hypertension, can be identified using Hypertension D5S2500 112 0.58 0.0009 0.0011 cell lines from general pedigrees selected without any knowledge of clinical pathology. D6SI40 113 0.58 0.0006 0.0080 This strategy represents an approach to rapidly and inexpensively map loci related to Not reported common, complex disorders, using phenotypes which are stable in immortalized lym- In addition, we observed p< 0.01 in 6 and 5 regions for pairs affected with nephropathy or phoblasts together with existing reference pedigree cell lines and genotype databases. hypertension, respectively. These findings need to be extended in additional Pima Indian subjects and in other populations.

229 Assignment of a QTL influencing 3chizotypal personality to chromosome 22q. K. Phillips. M1. D. Brennan. S. W. 'toselev. A. P. Matheny. Jr.. L. Zu. University of Louisville School of Medicine. Louisville. Kentucky. The non-clinical research sample on which this report is based comprises a large group of adult twin pairs and their siblings .'ho have participated in the Louisville Twin Family Study. a longitudinal research program on child development that is now in its fourth decade. We are currently conducting a whole-genowe search for the location of QTLs influencing individual differences in behavioral develmpment. Adults in the sample complete the Minnesota Xlultiphasic Personality Inventory--2 (MMPI-2). the slightly revised version of a long-standing, empirically developed questionnaire used to assess aspects of personality and psychopathology. We have genotyped our sample at several markers on chromosome 22q, a region that other researchers have identified tentatively in relation to clinical schizophrenia. and linkage and interval mapping tests of schizotypal personality as measured by the ?E1PI-2 have been conducted. Specifically, the analyses involve sib-pair identical-by-descent status in multiple regression linkage tests and interval mapping procedures developed by Fulker and coll agues for quantitative traits. Analvses of markers on 22q yield both suggestive (t = 3.41, P = 0.00033. LOD equivalent 2.32) and significant (t = 4.11, P = 0.00002. LOD equivalent = 3.66) linkages that indicate the presence of at least one QTL influencing schizotypal personality on chromosome 22q. A46 Slide Session 46: Differentiation, Development and Morphogenesis 230 231 The genetics of ROMI: evaluation of the requirement for rom-1 in Human huntingtin derived from YAC transgenes completely compensates for photoreceptor morphogenesis. and of the digenic hypothesis of retinitis loss of murine huntingtin by rescue of the embryonic lethal phenotype. pigmentosa (RP). G.A.Clarkel, W.Kedzierski3, J.Rossant2, G.H.Travis3 iand Hodeson', D. Smith2, K. McCutcheon', N. Bissadal, J. Nasir', EM. Rubin", MR R.R.xMinnesl. lHospital for Sick Children. and 2Samuel Lunenfeld Research Institute. Hayden'. ' Department of Medical Genetics, Univ. of B.C., Vancouver, Canada. Toronto. Canada; 3University of Texas SW Medical Center, Dallas. Texas. Human Genome Center, UC Berkeley, Berkeley, CA. Rom- I is a membrane protein of the outer segment (OS) of photoreceptor disks, where Huntington's disease (ND) is an autosomal dominant disorder it exists in a tetramer composed of a rom- I homodimer and a homodimer of a related neurodegenerative caused by expansion of a CAG trinucleotide repeat in exon I the protein. rds: (rom- 1)2-(rds)2. Both proteins are assumed to have a structural function. of IT15 gene. The HD protein (huntingtin) plays a critical role in Because mice without rds (Rds-4-) fail completely to form OS's, at least the rds early embryonic component of the tetramer is essential for disk morphogenesis. Rom- I and rds may also development since a homozygous targeted disruption of the murine HD gene results be required for the integrity of the mature OS. To determine whether mice lacking rom- I in lethality at embryonic day 7.5. To rescue this phenotype by transgene based also fail to form OS's like Rds-1- mice, we used gene targeting to derive animals carrying huntingtin expression it is therefore essential to express the protein early enough in an exon I deletion. No rom-I protein was detectable in the OS's of outbred Roml -I- development in the appropriate cells. Since YAC based transgenes are known to be mice. Light and electron microscopy revealed that by 8 wks. of age, Rom/-l- mice had regulated in an appropriate temporal and tissue specific manner, we sought to rescue obvious photoreceptor OS dysplasia with loss of the parallel, membranous arrays seen in the embryonic lethality by breeding YAC transgenic mice expressing human control retinas, and with numerous abnormal gaps in the OS's. The retinal phenotype is huntingtin with mice heterozygous for the targeted disruption. Following two rounds progressive: the OS's of 16 wks. Roml-l- mice were more disorganized, had increased of breeding, we generated offspring homozygous for the disrupted murine HD gene gapping. and had begun to accumulate amorphous material. The availability of Romll- but expressing human huntingtin derived from the YAC. This clearly shows that mice with a recognizable abnormal phenotype allowed us to test the ROMI-RDS digenic YAC transgene based expression occurs at the hypothesis of RP (Kajiwara et al., Science 264,1604.1994): i.e. that patients carrying a appropriate developmental stage and ROMI null allele and the RDS substitution, L 185P, will develop RP. whereas carriers of that human huntingtin is functional in a murnne background and can compensate for either allele alone will not. We reconstructed the digenic RP genotype (Roml +/1. loss of murine HD function. Additionally, we show that human huntingtin Rds+l'- tgLI85P) in mice. The retinas of RomI+b- and of Roml+i+, Rds+l' tgLI8SP expression in YAC transgenic mice follows an identical tissue distribution and mice had only mild OS disorganization, but those of 4 wk. digenic animals were very subeellular localisation pattern as that of murine endogenous HD and that expression abnormal: the OS's were short and extremely disorganized, and the inner segments were levels 2-3 times endogenous can be achieved. Taken together, these data show that and vacuolated. We conclude that i) rom- I is an essential structural component swollen YAC based human transgene expression parallels that of mouse endogenous and has of the OS, but unlike rds, it is not critical for OS morphogenesis; ii) the RDS:ROMI important implications for the development of transgenic mouse models that are truly digenic hypothesis of RP is correct iii) if the changes in -/- mice progress to Rom] representative ofHD. retinal degeneration. mutations in human ROMI may cause late-onset recessive RP. and Romi +/- mice will be an important model for slowly progressive RP.

232 233 Abnormal cerebellar development in mice lacking the murine homolog of The mouse Sry glutamine-rich domain binds specifically to three proteins in both the Drosophila proneural gene atonal. N. Ben-Arie'. M. M. Matzuk'. A. E. adult and fetal testes. J.Q. Zhang, P. Coward, M.W. Xian and Y.-F. Lau, Univ. of McCall'. ' C. Guo'. D. Armstron G. Eichele'. H. J. Bellen'2 and H. Y. Zoshbi'I. California, San CA Baylor College of Medicine and Howard Hughes Medical Institute, One Baylor Plaza, Francisco, Houston, Texas 77030. The mouse sex determining gene Sry encodes a protein of two domains: a atonal is a proneural gene, which encodes a transcription factor that belongs to the HMG box DNA-binding domain at the amino terminus and a glutamine-rich (Q-rich) basic Helix-Loop-Helix (bHLH) family of proteins, and is critical for the normal domain of unknown function at the carboxyl terminus. The Q-rich domain is encoded development of the chordotonal organs (proprioceptors) and photoreceptors in by stretches of CAG repeats and its derivatives. Previously, we had demonstrated Drosophila. We rationalized that atonal will be conserved throughout evolution and will several polymorphisms within the Q-rich domain of the Sry gene from different play a key role in vertebrate nervous system development. We isolated and characterized atonal homologs from various species and observed that only the bHLH domain of musculus and domesticus strains that can be correlated to the B6.YD" sex reversal, a atonal is highly conserved among mealybug, pufferfish, chick, mouse and man. In condition which occurs when the Y chromosome from certain domesticus strains is contrast, the similarity between the mouse atonal homolog (Mathl) and the human introduced into the inbred musculus strain, C57BL/6J, background by backerossing. homolog (HATH)) is high both within and outside the bHLH domain. We mapped We hypothesize that the CAG repeats was inserted in-frame in the ancestral Sry gene in to mouse chromosome respectively. Mathl and HATH) chromosome 6 and human 4, the mouse, resulting in a Q-rich domain that has acquired a protein-protein interactive Northern analysis revealed that Math) has a single transcript, which is expressed at function with a co-factor(s) involved in sex it to be retained embryonic days 11-17 with the highest expression at ElI. RNA in situ analysis on determination. causing chick and mouse embryos detected very similar expression patterns in the developing through evolution. Recent divergence between musculis and domesticus subspecies cerebellum and throughout the dorsal portion of the spinal cord. To study the function has resulted in the observed polymorphisms and incompatibility between the of Math) in vivo, we generated mice lacking the entire Mathl coding region using domesticus Sry protein and the B6 co-factor(s) in the sex determination process. Using for the deletion homologous recombination in embryonic stem cells. Miceheterozygous the Q-rich domain as probes in farwestern blotting studies, we detected 3 specific bands null mice are alive at E18.5 but die after are viable and fertile. The homozygous shortly at 94, 32 and 28kDa only in testis extract and a 85 kDa in the brain extract from adult birth from apparent respiratory failure. Histological analysis of brain sections from null multi-tissues. The 94, 32 and 28 as mutant mice revealed that the cerebellum is devoid of the external granular layer and is kDa testicular proteins have been designated Sry slightly reduced in size compared to that of wild type and heterozygote littermates. interactive protein I (Sip-l), 2 (Sip-2) and 3 (Sip-3) respectively. Sip-l was present in Careful characterization of cranial nerve nuclei, brain stem and spinal cord is currently the testis extracts of both musculus and domesticus strains and its expression was cause of death in progress on embryos of various developmental stages to determine the associated with the presence of germ cells at late spermatogenic stages. Significantly, of the Math) mice. These data demonstrate that the vertebrate of atonal null homologs all Sip proteins were detected in extracts from genital ridges of male embryos as early show sequence conservation throughout evolution, and demonstrate that Mathl plays a as 1 1.5 day post coitus, at the the exact nature of key role in cerebellar development. time of sex determination. Although these Sry interactive proteins has yet to be defined, their detection supports our hypothesis that the mouse Sry Q-rich domain contributes to the biological finction(s) of Sry through a protein-protein interactive role(s).

234 235 Phenotypic characterization of a mouse model of Marfan syndrome. Long-term expression of human factor IX from mouse hepatocytes after L.Y. and N.L.J..Bier', L. Pereira, T.Bunton', Sakai3. F. Ramirez4 C. Dietz'. intravenous injection of AAV vectors. D. D. Koeberl, I. E. Alexander, Johns Hopkins Univ. Sch. of Med., Baltimore, MD; Univ. San Paolo, Brazil; Portland and A. D. Miller. University ofWashington and Fred Hutchinson Cancer Research Shriners Hospital, Portland, OR; Mount Sinai Sch. of Med., New York, NY. Center, Seattle, Washington. aortic Marfan syndrome (MFS) is an autosomal dominant disorder characterized by Adeno-associated virus (AAV) vectors transduce nondividing cells, which is an advan- dilatation, long bone overgrowth and lens dislocation. The phenotype results from tage for hepatic gene therapy because hepatocytes divide slowly. We have demonstrated dominant-negative mutations in fibrillin-1, the major constituent of extracellular marked enhancement of transduction of hepatocytes in vivo followingtreatment with microfibrils. Many observations suggest that microfibrils regulate the deposition of DNA-damaging agents. Exposure of mouse livers togamma irradiation allowed up to tropoelastin molecules into maturing elastic fibers. In order to study this process, we 800-fold increased transduction of hepatocytes an AAV vector for expression of human used homologous recombination in ES cells to target disruption of the mouse Fbnl alkaline phosphatase (AP) compared to unirradiated controls. The increase in transduc- gene. The resulting in-frame deletion of exons 19-24 predicts a centrally truncated tion was proportional to the dose of gamma irradiation, and atopoisomerase inhibitor monomer. Unexpectedly, there was a dramatic reduction of mutant transcript, <5% of (etoposide) caused an increase in transduction similar to low levels of gamma irradia- tion. The transduction of hepatocytes was that expressed from wild-type alleles. Heterozygous (+/-) mice were viable and fertile, increased much more than that of other cell types following DNA damage, and and showed no obvious phenotypic alteration, consistent with the dominant negative liver-targeted delivery followed tail vein injection of the AAV vector. Up to 3% of hepatocytes were transduced, and the model. There was no fetal loss of homozygous (-/-) mice, which appeared grossly efficiency of hepa- tocyte transduction was approximately 20% of that observed for tB3 cells in vitro. normal at birth. All died suddenly between 1 and 10 weeks of age and showed The presence of wildtype AAV was required to observe a full response to gamma irradiation. multifocal aneurysmal dilation of the aorta. The focal lesions are characterized by Injection of mice with an AAV vector encoding human clotting factor IX (FIX) after disruption of elastic fibers, accumulation of amorphous matrix elements, and recruitment gamma irradiation resulted in long-term synthesis of low levels of human FIX.Intra- of inflammatory cells. the architecture of elastic fibers is preserved Interestingly, venous injection of AAV vectors following DNA damage may be a useful method for lesions and in Dissection and intravascular thrombosis were between unaffected tissues. hepaticgene therapy incertain hematologic or metabolic diseases. also common findings. Dissections within the pulmonary artery and atrial wall were occasionally observed. Skeletal findings were restricted to severe kyphosis in animals that survived until an older age. Ultrastructural analysis showed scant fibrillin-l microfibrils that failed to form clusters and arrays. Fibrillin-2 microfibrils were found in normal abundance and maintained their temporally- and spatially-restricted pattern of expression. Normal appearing elastic fibers were deposited in the absence of microfibrils. We conclude that fibrillin-I is required for the homeostasis rather than the deposition of elastin. Despite subtle differences between the molecular mechanisms underlying this model and the human condition, these mice will be a useful tool in delineating modifiers and therapeutic strategies for the vascular phenotype of MFS. Slide Session 46: Differentiation, Development and Morphogenesis (cont.) A47 236 237 Molecular and Morphological Analysis of the Limb Phenotype In Novel phenotypes in synpolydactyly families segregating with HOXD 13 Hypodactyly (Hd) Mice. L.C. Post. and J.W. Innis. Department of Human of Ann alanine tract expansions. F. Goodman. W. Reardon. R. Winter. S Mundlos', Genetics, University Michigan, Arbor, Michigan, USA. Y. B. P. Limb development is a complex process that is mediated through a careful balance of Muragaki'. Olsen'. Scambler. Institute of Child Health, London, UK, cellular proliferation, programmed cell death, and differentiation into the cartilage, 'Harvard Medical School, Boston, MA. muscle, and bone that make up the final structures. Perturbation of any of these Synpolydactyly (SPDI comprises 3/4 syndactyly in the hands and 4/5 processes or pathways may markedly alter the pattern of the limb. The genes that syndactyly in the feet, with duplication of a digit in the syndactylous web. regulate each of these processes, what controls their expression and how they interact Expansion of a polyalanine tract in the aminoterminal region of HOXD1 3 has are under active investigation. Examination of mice with heritable defects in limb recently been identified in 3 SPD families. SPD is thus the first human is a useful to and development strategy identify key regulatory signals pathways. malformation to be associated with a mutation in a HOX gene. We now Hypodactyly (Hd) is a spontaneous, semidominant mutation that is characterized by shortening or absence of the first digit and mild defects in maturation of elements of the describe 8 additional SPD families. All have expansions of the Ala tract with digital arch in heterozygotes, but severe reductions in the digital arch, in utero lethality, up to 14 extra residues. Expansion carriers from a large 7 generation and infertility in rare adult homozygous mutants. We have recently identified a 50 base pedigree are variably affected, with occasional incomplete penetrance, but no pair deletion within the coding sequence of the first exon in the Hoxal3 gene as the anticipation and no increase in expansion size contrasting with the instability molecular basis for this mutation (Mortlock et al, Nature Genetics, July 1996). mRNA of many disease-causing trinucleotide expansions. The family with the levels appear to be unaffected in mutants by Northern blot analysis; we predicted that largest expansion has a consistent and more severe phenotype including no HoxaJ3 protein would be produced from the mutant mRNA. Through western blot involvement of the first digit. The 2 families with the smallest expansions analysis using an antibody to chicken Hoxal3 (kindly provided by A. Kuroiwa), we were unable to detect any wild-type protein in mutant limb buds. Despite the contain individuals with an atypical, mild phenotype including 2/3 toe anmeshift, a protein of 300 amino acids could be generated from the deleted mRNA. syndactyly. Affected males from 2 families with large expansions had This protein would contain the first 25 amino acids of the normal protein but not the hypospadias, consistent with HOXD1 3 expression in the distal genital bud. homeodomain. To determine whether such a protein is produced in Hd/Hd animals, Increased expansion size thus appears to correlate with increased severity of we are producing antibodies to a GST fusion protein incorporating the mutant phenotype. The observed involvement of pre-axial digits suggests that the sequence. Western blot analysis will be performed to determine whether this protein is Ala tract has a role in the late phase of HOXD1 3 expression in the distal limb, in Hd animals. We are also interested in stably produced determining how loss of which unlike the extends to the first Hoxa13 leads to digital arch deficiency. We are examining the cartilage/bone early phase, anteriorly digit. Since both phenotype of embryonic mutant limbs and beginning to characterize the changes in gene the tract and the late phase are absent in zebrafish, but present in chick, expression that occur in developing limbs of Hd/Hd animals. With this data, we will mouse and man, this supports the hypothesis that the appearance and correlate altered expression patterns with phenotype and begin to determine the role of expansion of the Ala tract has contributed to the evolution of the mammalian HoxaM3 in the formation of the digital arch in vertebrates. autopod. we thank Professor F. Majewski, Dusseldorf, Dr C. McKeown, Birmingham and Professor M.L. Giovannucci-Uzielli, Florence for provision of samples.

238 239 The faciogenital dysplasia (FGD1) gene is a specific activator of the p21 Full FMR1 mutations in fetal germ cells shed light on embryonic GTPase Cdc42; the isolation of three new FGD1 homologs, candidate loci In X for developmental disorders. N.G. Pasteris', M.F. Olson'. A. Hall', repeat expansion fragile syndrome. S. T. Warren'. H. Malte1r, J, and J.L. Gorski1. University of Michigan'. Ann Arbor. and University College Chastain,', J. C. Tarleton2, R. Willemsen3, E. de Graff3l, J. Leiti4, B. A. Oostra3. London2, UK. 'Emory Univ., Atlanta, GA; 2Greenwood Genetics Ctr., Greenwood, SC; Guanine nucleotide exchange factors (GEFs), activators of the Rho family of p21 GT- 3Erasmus Univ., Rotterdam, The Netherlands; 40ulo Univ., Finland. Pases, form a growing family of proteins that play crucial roles in embryonic development It has been proposed that expansion of the FMR1 CGG repeat associated and cancer. Although most RhoGEF genes were isolated as oncogenes in transfection with fragile X syndrome occurs early in development and that primordial assays, FGD1, the gene responsible for faciogenital dysplasia (Aarskog-Scott syndrome) cells are from this event since adult full mutation was cloned and an role in germ (PGCs) protected positionally plays essential embryogenesis. Mutations in FGDl We examined somatic tissues of a 16 wk female result in a developmental disorder affecting specific skeletal structures including elements sperm carry premutations. of the face, cervical vertebrae and distal extremities. We now show that FGD1 specifi- fetus with a methylated full mutation. Southern analysis of ovarian DNA cally activates the Rho GTPase Cdc42 and induces the polymerization of actin and the showed an FMR1 band similar in size and complexity as other somatic formation of actin-rich filopodial extensions when microinjected into Swiss 3T3 cells. No tissues with no evidence of premutations (including PCR). However, stress fibers or overt lamellopodia are observed. In contrast, other RhoGEFs such as methylation studies of the ovarian DNA showed an additional band not Vav and Dbl, activate Rho, Rac, and Cdc42 and yield dramatic lamellopodial forma- present in somatic tissues, consistent in size to be the unmethylated but fully tion. FGD1 also stimulates the SAPK/JNK1 cascade that leads to the activation of allele of Band is similar to that for the oocyte- the N-terminal c-Jun kinase. Our results show that FGD1 is a RhoGEF that, along expanded oocytes. intensity with its target protein Cdc42, controls essential signals required during embryonic de- specific band observed with the X-linked P3 gene and is appropriate for the velopment. In addition, by using a degenerate primer PCR cloning strategy and a low -25% oocyte content of ovaries at 16 wks. Therefore, oocytes reflect a similar stringency hybridization approach, we have isolated three new putative members of the degree of repeat expansion as somatic tissue but are hypomethylated like RhoGEF family of genes that share a striking similarity to FGD1. We are presently other X-linked loci. An additional female fetus showed similar results. Two characterizing the human and mouse homologs of a related gene. termed FGD2, that male fetuses at 13 and 17 wks with full mutations also showed no evidence contains a RhoGEF domain that is 70 percent identical to the FGD1 RhoGEF domain. of in testes PCR -4% vs the GEF domain of the closest relative to premutations by analysis (sensitivity pre- full). By comparison, previously recognized FGD1, showed in ect2, shares 30 percent sequence identity. Preliminary expression data indicates that Immunohistochemistry FMRP expression normal testes at 13 wks; FGD2 is expressed very early (e7.5) in mouse embryonic development, and at high levels none in the 13 wk affected testes; and expression in a few PGCs at 17 wks. in human fetal tissues. Based on its putative RhoGEF encoding domain, its expression These data indicate that the germline is not protected from expansion. We pattern, and its affinity to a known disease gene, we suggest that FGD2 and the other suggest that full expansions are less stable in testes and contract to related clones may be likely candidates for human developmental or cancer disorders. premutations, eliciting FMRP expression. It is proposed that such FMRP- positive PGCs outcompete the fully expanded PGCs resulting in premutation sperm in full mutation males.

240 Down syndrome congenital heart disease: Gene isolation using BAC and PAC contigs between D21S55 and MX1. RS Hubert', Y-K Huo'. S Mitchell', X-N Chen', Z-G Sun', M VanKeuren2, H Shizuya3, C Chen4, G Lyons5, K Yamakawa'. D Nova', K Ekmekii', C Gadorskl', U-J Kim3, Y Nakamura6, D Kumit2, M Simon3, P DeJong', JR Korenberg'. 'Cedars-Sinai Medical Center, L.A., CA; 2HHMI/ U. of Michigan, Ann Arbor; 3Caltech, Pasedena, CA: 4Roswell Park Univ., Buffalo, N.Y.; 5U. of Wisconsin Medical School, Madison; 'Cancer Institute, Tokyo, Japan The 4-5Mb D21S55 to MX1/2 region of chromosome 21 is significant due to its association with Down syndrome congenital heart (DS-CHD) and gut disease. Contig Construction. To provide stable clones for gene isolation and sequencing initiatives in the D21S55 to MX1 region, we constructed contigs using Bacterial Artificial Chromosomes (BACs) and P1 Artificial Chromosomes (PACs). A BAC library was screened using several YACs spanning the region; a PAC library was screened using radiolabeled STS PCR products and whole BACs in gap filling initiatives. The FISH verified BAC and PAC clones were overlapped using clone to clone Southems and 24 new STSs, generated from the direct sequencing of BAC and PAC ends, along with 35 pre-existing STSs. The STS density over the intervals covered in BACs and PACs is 1 STS every 60 kb and 79% of the clones were positive for 2 or more STSs. Approximately 3.5Mb of the 4-5Mb D21S55 to MX1 interval is covered in 85 BACs and 25 PACs representing 4 fold coverage within the contigs. The minimal contig sizes as determined by counting only non-overlapping clones are: 1100kb, 900kb, 510kb, 380kb and 270kb. Gene Isolation. The direct cDNA selection procedure using 19 BACs and 2 PACs between ETS2 and MX1 generated a total of 145 unique cDNA fragments. Genbank and TIGR homology searches using FASTA revealed matches to ETS2, HMG14, PEP19, a Na K ATPase, Titin ESTs, MX1 region ESTs, and 14 ESTs of unknown function. A cDNA library from a trisomy 21 fetal brain at 14 weeks gestation was screened using one of these cONA fragments and the novel gene CHD1 was isolated. CHD1 has an 864 bp open reading frame coding for a 288 amino acid protein. Northern analysis using adult tissues with CHD1 showed differential expression in heart versus skeletal muscle. RT-PCR studies on multiple embryonic tissues demonstrated expression only in heart from day 53 and 55 gestation and in the 6-9 week heart. The temporal and tissue specific expression patterns of CHDI suggest this gene may be a candidate for DS-CHD. The mouse homologue of CHD1 is presently being isolated for expression studies in mouse early embryonic and fetal tissues using tissue in situ hybridization techniques. The BAC and PAC contigs spanning D21S55 and MX1 are a valuable resource for gene hunting and the contigs show promise as reagents for large scale sequencing efforts. A48 Slide Session 48: Molecular Genetics of Disease V

241 242 Contrnibution of DNA sequence and (CAG)n size to mutational frequencies of HUNTINGTON'S DISEASE: IDENTIFICATION OF PROTEINS THAT intermediate alleles (lAs) in Huntington Disease (tlD): evidence from single INTERACT WITH HUNTINGTIN. P.W. C. L.M. H. Faber, Dompe. Carlies G.T. sperm analyses. E. Almqvist. S. Chong- Telenius- L. La rra). K. Nichol, 1B Barnes. J.F. Gusella and M.E. MacDonald. Molecular Neurogenetics Unit, Bourdelat-Parks. Y.P. Goldberg. F. Richards. D. Sillene. nberg. Ives, (. Van Massachusetts General Hospital East, Bldg 149, Charlestown MA 02129. den Engh. M. H-ughes M R Hayden. Vancouver, CAN. NIH, Bethesda. US; U. Wash., Huntington's Disease (HD) is an inherited autosomal dominant neurodegenerative Seattle; Parramatta, Australia; Children's Hosp., Winnipeg; Mem. U.. St. John's. disorder characterized by uncontrolled eye movements, general motor impairment, HD is associated with expansion of a CAG tract. All new mutations (NM) arise psychiatric disorders and dementia. The underlying feature ofHD is the specific loss from lAs of29-35 CAG that expand through the paternal germline to the affected range of neurons in the basal ganglia. Genetically, HD is caused by an expanded CAG- of>35 CAG. Single sperm analysis provides unique opportunities for determining CAG repeat in 4p16.3. This CAG-repeat is translated into a polyglutamine stretch near the mutational frequencies and allows direct comparison of the role of size and sequence on amino terminus of a novel 350 kDa protein of unknown finction, termed huntingtin. intergenerational CAG changes. On affected chromosomes increasing CAG size is Both the normal and HD isoforms ofhuntingtin are widely expressed in the associated with marked elevation in the frequency of expansion. To study the influence cytoplasm of cells in many tissues. Also, its expression pattern in brain does not of CAG size and sequence variability on the instability of the lAs, we performed single closely match the primary target areas of the disease. Thus the expanded sperm analyses of 1,160 sperm on lAs which have expanded into the affected range polyglutamine stretch in huntingtin leads to a gain of function mutation whose mode and NM-34), as as derived from the general population (GP-35 and (NM-35 well lAs of action remains to be established although a dominant-negative scenario, in which GP-31). Comparison of lAs of the same size and sequence (NM-35 and GP-35) showed the mutant protein inactivates the normal isoform has been ruled out as HD knock- marked differences. NM-35 had less stability of the CAG repeat 134/209 (63.8%) out mice proved to be embryonic lethal. compared to GP-35 (152/199 or 76.4%) (p-0.008). This supports the hypothesis that To obtain more knowledge about the biochemical pathways involving both the cis acting sequence influences the likelihood of expansion. A dramatic indication of the normal andHD isoforms of huntingtin we have employed yeast two-hybrid influence of the sequence of the CAG tract compared to CAG size was apparent when technology the identify their cellular protein partners. Using matched sets of yeast comparing GP-3 1 and GP-35. GP-3 1 showed more instability despite its smaller CAG two hybrid baits from the huntingtin N-terminus with either 21or 60 glutamines so size with 43/147 (36%) expansions, compared to 25/199 sperm (12.6%; p<10-6) inGP- far at least three independent interactors have been identified from a human fetal 35. Sequence analysis revealed that GP-31 had a loss of the regular CAA and CCA brain library. Although none interacts exclusively with one bait, quantitative interruption between the CAG and CCG repeats. These results show that an differences exist. Also, two interactors share a common protein motive, defining the uninterrupted sequence is more important in destabilising the CAG tract than the CAG region of interaction. These results as well as the additional characterization of these size itself. These analyses also provide risk estimates for expansion of lAs into the interactors will be presented. affected range for counselling relatives of persons with lAs from HD families and the general population. Funding: Human Science Frontier Program. Hereditasy Disease Foundation and NIEL

243 244 Polyglutamine length modulation of Huntingtin Cleavage by Slipped strand structures in neurodegenerative disease-associated Apopain/CPP32 during Apoptosis represents a gain-of-function for the trinucleotide repeats: a role for human mismatch repair and cryptic HD protein. interruptions. C.E. Pearson I, E.E. Eichler-, D. Loren 2,S. Achay3, Kramel, S. Kramerl, D. L. Nelson', H Y. Zoghbi2,R.A. Fishel3 & R.R indenl YPGoldbergl. DWNicholson2. MBromml. Kazemi-EsfAianiil. MAKalchmanl. B I) Center for Genome Research, Texas A&M University, Houston, Texas, 77030, 2) Koidel. RK Grahal. DM Raser2. Vaillancourt2. NA Thomberry3. MR HaydenI Molecular & Human Genetics, Baylor College of Medicine. 3) DNA Repair and 'Dept of Medical Genetics, University of British Columbia, Vancouver. Canada. 2Dept Molecular Carcinogenesis Program, Thomas Jefferson University, Philadelphia, PA.

of Biochem. and Mol. Biology, Merck Frosst, Montreal, Canada. 3Dept of The mechanism of neurodegenerative disease-associated expansion of Enzymology, MRL, Rahway, U.S.A. (CTG)n(CAG)n and (CGG)no(CCG)n trinucleotide repeats is unknown but is thought Recently it has been proposed that the mode of cell death in Huntington disease (HD) to involve DNA slippage at the repeats. In order to gain insights as to the possible we is apoptotic. Apopain/CPP32 is a human protease near the apex of a cascade of mechanism(s) of triplet repeat instability investigated alternative triplet DNA proteolytic events that culminate in apoptotic cell death. We have shown that apoptotic structures, their formation, and interaction with human proteins. Using genomic clones extracts and apopain itself directly and specifically cleaves huntingtin and that this of various neurodegenerative disease genes (DM, SCA-1, FRAXA) containing disease we proteolytic cleavage is strongly inhibited by an apopain-selective tetrapeptide aldehyde relevant lengths of triplet repeats report the formation of slipped strand DNA (Ac-DEVD-CHO). Apopain cleavage of huntingtin containing different polyglutamine structures during reannealing of complementary strands, assayed by gel electrophoresis. lengths revealed that polyglutamine expansion enhances huntingtin cleavage (CAG The propensity to form slipped structures increased with increased repeat length.The tract 91>80»44>15). Analysis of variance indicated that huntingtin polyglutamine size is a presence of AGG interruptions in the FRAXA CGG hinders the ability to form significant determinant of apopain cleavage (P <0.006). Moreover, in mammalian cell slipped structures. The effect of both the length and the purity of the repeat tract on the cleavage on culture, huntlngtin occurs coincident with the onset of apoptosis as propensity of structure formation correlates with their effect genetic stability in simultaneous an demonstrated by activation of apopain and concomitant PARP cleavage. human diseases. This is the first presentation of alternative trinucleotide repeat Furthermore, induction of apoptosis in a lymphoblastoid cell line (CAG=19/21). structure whose formation correlates with the genetics of the neurodegenerative disease revealed a novel huntingtin cleavage product, whereas in an HD derived line genes, suggesting that these structures may participate in triplet repeat instability. (CAG-23/69) there was an additional aminoterminal huntingtin breakdown product Slipped (CTG)-(CAG) intermediates, expected to arise during the expansion from normal (n=30) to disease (n=50) repeat lengths were used in a band-shift assay to inderived from the expanded allele, providing evidence that cleavage of huntingtin occurs vivo. Apopain cleavage of huntingtin represents an important link between neuronal investigate the binding of the human mismatch repair protein hMSH2.hMSH2 bound to cell death and theHD protein. The rate of cleavage increases with the length of the slipped substrates where both strands are of equal length (slipped homoduplex orS- polyglutamine tract providing one possible explanation for the gain-of-function DNA) and slipped intermediates where one strand is 20 repeats longer (heteroduplex or associated with CAG expansion These data show that huntingtin is cleaved by cysteine SI-DNA).hMSH2 bound preferentially to looped-out CAG repeats, implicating strand proteases and thatHD may represent a disorder ofinppropriate apoptosis. asymmetry in the mechanism of instability. Our results suggest that mismatch repair may participate in triplet tract length polymorphisms in post-replicative repair as well as by mismatch repair which may occur in the absence of replication.

245 246

Spiaocerebellar ataxia type 2 (SCA2) is caused by expansion of a CAG trinucleotide Transcriptional suppression in Friedreich ataxia: consequences of the repeat which is stablein the normal population. A.V, Nechivoruk'. T.T. NechiDoruk'. K.P. intronic GAA triplet repeat expansion. S.I. Bidichandanil, L. Monterminil Fisueroa'. S. Sahba. J R. Korenberel P.J. DeJona. S. Pearlman'. S. Gisnert4. A. Lunkes'. G. M.D.loltol, M. Pand l fo§. Departments of Neurology5 G. Rouleau 1. Loes-Ccdes Auburecr'. S.-M. Pulst'. 'Burns and Allen Research Koenig3,P.1. Patel'52, M.P. Institute. Cedars-Sinai Medical Center. UCLA School of Medicine, Los Angeles. CA 901)48; and Molecular and Human Genetics2, Baylor College of Medicine, Houston, Texas; 3 Department of Human Genetics. Roswell Park Cancer Institute. Buffalo. New York 14263: IGBMC, Strasbourg, France; §Present address: Centre de Recherche Charles-Louis 'Department of Neurology, UCLA School of Medicine, Los Angeles. CA:'Department of Simard, Montreal, Canada. Neurology, University of Duesseldorf. Germany;sCcntre for Research in Neuroscience. Friedreich ataxia, is an autosomal recessive disease, caused in the vast majority of cases Montreal General Hospital. Montreal. Canada. by an abnormal expansion of a GAA triplet repeat polymorphism in the first intron of the The dominant spinocerebellar ataxias (SCAs) represent a phenotvpically heterogeneous gene encoding frataxin. How this expansion results in disease is not known. We show group of disorders with a prevalence of familial cases of approximately per 11)0,000. The that patients homozygous for this expansion have severely reduced levels of normally SCA2 gene has been localized to a cM region on chromosome 12q24. In order to identify spliced fratazin mRNA suggesting a transcriptional or post-transcriptional mechanism. the SCA2 gene we generated a 1.1 Mb physical map consisting of Pt artificial chromosome No evidence was found for mis-splicing of the abnormally expanded first intron. The (PAC) and bacterial artificial chromosome (BAC) clones. The generation of STSs from intronic sequence immediately flanking the site of the expansion (containing a normal clone-ends resulted in a high density STS map with an average of one STS per 37 kb. repeat length) was shown not to function as a transcriptional enhancer in a transient trinucleotide repeat was identified that was expanded in patients with SCA2. This transfection experiment using mammalian cells. Polypurine-polypyrimidine sequences not contained in any of the YAC clones mapped to 12q24.1. The repeat is expressed and are known to form intramolecular triplexes, structures capable of physically blocking contained in a novel gene that does not show any homologies with other CAG expansion the passage of the transcriptional machinery. Using varying lengths of cloned GAA disease genes. In contrast to other unstable trinucleotide repeats, the CAG repeat is not triplet repeats (kindly provided by Drs. R.D. Wells and K. Ohshima, J. Biol. Chem. highly polymorphic in normal individuals and 94% of normal chromosomes have a in press), we show that in vitro transcription is severely suppressed when the the structure (CAG)sCAA(CAG),CAA(CAG)s. A rare second normal allele with a frequency repeat length is increased from 37 to 103 GAA triplets. Interestingly, this was of 6% has the sequence (CAG)14CAA tCAG)s. In SCA2 patients, the repeat is suppression only seen when synthesis of GAA-rich transcripts was attempted. The moderately expanded to 36-52 repeats. Expansion of the repeat is inversely correlated with observed directional suppression is indicative of a transcriptionally mediated non-B DNA the age of onset in SCA2 patients (tee IT Nechiporuk et al., this meeting). The shortest structure impeding frataxin transcriptional elongation. This blockade of transcriptional disease allele bas fewer repeat units than the longest normal allele in the SCAI. SCA3/MJD elongation, as a possible underlying molecular pathogenetic mechanism, is or Huntington's disease (HD) genes arguing against a pathogenic mechanism purely without precedence in human genetic disease. length of the polyglutamine tract. The sequences flanking the repeat are extremely and contain multiple palindromic sequences predicted to form stable hairpin structures.

These findings extend the search for trinucleotide repeats associated with expansion to repeats that may not be highly polymorphic. Further investigation of this repeat and its flanking regions may provide new insights into the mechanisms underlying repeat expansion in humans. Slide Session 48: Molecular Genetics of Disease V (cont.) A49 247 248 Molecular analysis of the FMR-2 gene associated with mild mental Spatial distribution of expanded trinucleotide repeat sequences in handicap. L. Chakrabarti. S. J.L.Knight and K. E.Davies. Genetics Myotonic Dystrophy (DMI) and Fragile X Syndrome (Frai) patient cells. Laboratory, Department of Biochemistry, University of Oxford, South Parks K.L. Tanejal, B. Davis2, D. Nelson3, D. Housman2, and R.H. Singerl. UK tDepartment of Cell Biology, University of Massachusetts Medical Road, Oxford, Center, Worcester; 2Department of Biology, Cancer Center, Massachusetts The cytogenetic expression of the folate sensitive fragile site, FRAXE, is Institute of Technology, Cambridge; 3Institute for Molecular Genetics due to the expansion of a CGG repeat in proximal Xq28 of the human X and Human Genome Center, Baylor College of Medicine, Houston, TX. chromosome and is associated with a mild form of mental handicap. Using a single oligonucleotide probe labeled with fluorochromes for Normal individuals have 6-35 of the repeat whereas cytogenetically in-situ hybridization, we have shown that the DM triplet repeat copies sequences (CTG) were post-transcriptionally accumulated as a number of positive, developmentally delayed males have >200 copies and show discrete foci in the nuclei of DM patient cells, whereas the FraX methylation of the associated CpG island. Phenotypically FRAXE males triplet repeat sequences (CGG) were present as a number of patches in show no consistent dysmorphology and the main clinical features appear to the nuclei of FraX patient cells. Ho reduction in foci or patches be in and writing were seen with actinomycin D treatment, suggesting that these tran- learning difficulties, particular, speech delay, reading scripts containing expanded triplet repeat sequences were not nascent problems, there may also be behavioural deficits. Through the use of transcripts. Colocalization of transcripts with the allele containing conserved sequences adjacent to the FRAXE GCC repeat, we have isolated the repeats showed that the foci were derived from only the affected the FRAXE gene which begins just distal to the FRAXE trinucleotide allele. All but one of the sites of accumulation of transcripts were different than the site of and extends to a several hundred kb distal in transcription, confirming that the repeat repeat, region Xq28. The transcripts are post-transcriptionally accumulated. The intensity and predicted protein has amino acid motifs which share significant homologies the number of foci inreased in growth-arrested DM patient muscle with the human AF4 gene which encodes a putative transcription factor. On nuclei, showing that the transcripts containing expanded repeat northern analysis, the cDNA detects a 9.5 kb transcript in human brain, sequences were continuously transcribed. This accumulation of tran- in either disease a rate and This is at low scripts may affect limiting step in mRNA placenta lung. transcript present levels in all human processing or transport. brain tissues tested but is more abundant in the hippocampus and amygdala, thus providing possible functional insights. In-situ hybridisation studies to investigate the expression of this gene in the brain reveal highest levels of FMR-2 RNA in certain cell types of the cerebellum. Work to elucidate the developmental expression profile of this gene will be presented.

249 250 Purification of a new 20 kDa protein from HeLa nuclei that binds specifically to the Abnormal Processing and Degradation of the Familial Alzheimer unstable trinucleotide repeat (CGG) in the human FMR1 gene. HL Deissler M. Disease-Associated Mutant Presenilin Genes R.E. Tanzi. T-W. Kim, R.D. Moir, Wilml' M. Manra and W. Doerfler. Institute for Genetics, University of Cologne, D.M. Kovacs, S.Y. Guenette. W. Wasco Massachusetts General Hospital and Harvard Cologne, Germany and 'EMBL, Heidelberg, Germany. Medical School. Charlestown, NIA Expansion ofthe unstable 5-(CGG)-3' repeat in the 5'-UTR ofthe human FMR1 gene The presenilin genes (PSI on chromosome 14 and PS2 on chromosome 1) appear to be leads to the fragile X syndrome, one ofthe most frequent causes ofmental retardation in responsible for the majority of early onset familial Alzheimer disease (FAD). To date, 28 human males. In studies on the molecular cause for the ofthis and its different mutations have been described in the presenilin genes in over 50 FAD kindreds. instability repeat The roles we from nuclear extracts ofhuman cells that bind to this biological of the presenilins and how their pathogenic mutations confer FAD function, investigated proteins are unknown. We set out to examine the and with extracts processing degradation pathways of PSI trinucleotide repeat. Electrophoretic mobility shift assays nuclear from and PS2 in tetracycline-repressable, human glioma cell lines expressing wild-type and several human cell lines and from primary lymphocytes demonstrated highly sequence- mutant forms of these genes. Western blot analysis revealed PS2 as a 53 kDa band and specific binding to the double-stranded trinucleotide repeat 5'-d(CGG)17-3'. Protein a high molecular weight form. The 53 kDa PS2 species was found to be processed into binding was inhibited by complete methylation of the trinucleotide repeat. A 20 kDa two stable cellular polypeptides including a 33 kDa N-terminal and 20 kDa C-terminal protein was purified from HeLa nuclear extracts by DNA affinity chromatography and fragment which was localized to the cytoskeletal-associated cell fraction. We a'so found subjected to peptide sequencing. A cDNA-clone was identified, which encoded a 19 kDa that degradation of PS2 involves poly-ubiquitination and subsequent degradation by the protein containing the analyzed peptides. This protein carried a putative nuclear 26S proteosome complex. In cells expressing PS2 carrying the N1411 (Volga German localization and showed to The nature of the FAD) mutation. the cytoskeletal-associated, 20 kDa C-terminal PS2 fragment accumu- signal homology DNA-binding proteins. lated at concentrations than in cells identified is now under we showed that the 70 higher expressing wild-type PS2 apparently due to newly protein investigation. Furthermore, inefficient degradation by the proteosome. Full-length (47 kDa) PS1 was processed into kDa subunit ofthe human single-stranded DNA binding protein was part ofthe complex a 28 kDa N-terminal fragment and a 19 kDa C-terminal fragment associated with the formed with the single-stranded trinucleotide repeat 5'-d(CCG)17-3' and HeLa nuclear cytoskeleton. As with PS2, the proteosome inhibitor, ALLN,caused the accumulation proteins. Nuclear proteins also formed complexes with the single-stranded trinucleotide of poly-ubiquitinated PS1 indicating degradaion of both presenilins by the ubiquitin- repeat 5'-d(CGG)17-3', and these complexes appeared not to be sequence-specific. proteosome pathway. Our studies indicate that abnormal processing and inefficient (Supported by the Bundesministerium fMlr Bildung, Wissenschaft, Forschung und degradation of PS2 (and perhaps PS1) are involved in the pathogenic mechanisms of Technologie, Bonn through Zentrum fir molekularbiologische Medizin Koln, TP-13.) FAD mutations in the presenilins.

Slide Session 49: Cytogenetits Ill 251 252 Primed In situ hybridization (PRINS) - a sensitive alternate to YAC probes for the molecular FISH: Applicatlons In rapid cytogenetic diagnoses. G. V. N. Velagaleti. Region-specific cytogenetic S. A. Tharapel. M. Waterhouse, P. R. Martens, L. P. Shulman, F. Pellestor and characterization of chromosome rearrangements. T. Haaf', J. Wirth' A. T. ThacagL. The University of Tennessee, Veterans Affairs Medical Center, K. Kingslev'. N. Tommeruo2. D. C. Ward3, H. H. Rovers'. 'Max-Planck- Memphis, Tennessee, USA and CNRS, Montpellier, France. Institute of Molecular Genetics, Berlin, Germany, 2J. F. Kennedy Institute, Primed in situ (PRINS) hybridization is a rapidly evolving molecular cytogenetics methodology which could become a viable alternate to the conventional Glostrup, Denmark, 3Yale University School of Medicine, New Haven, CT. fluorescence in situ hybridization (FISH). It is based on sequence-specific Because they carry an average of 1 Mb of human genomic DNA, CEPH annealing of unlabeled nucleotide primer, followed by primer elongation with Taq YACs generate high-intensity FISH signals. The available set of polymerase using labeled nucleotide (dUTP). While the reaction is similar to cytogenetically and genetically anchored YACs, approximately one every conventional PCR, the PRINS technology uses a single strand (anti-sense strand) 5-10 cM evenly spaced over almost the entire human genome, provides primer in the reaction mix. The reaction is performed in situ on nuclei or region-specific probes for molecular cytogenetics which can be adapted with metaphase preparations and the entire procedure takes less than two hours. unlimited flexibility to specific applications. Using PRINS with primers targeting the centromere specific alphoid to sequences, we have resolved a number of clinical cytogenetic problems: We have selected YACs from all chromosome ends screen mentally (1) identification of the marker chromosomes in three cases, (2) origin of retarded patients for subtle deletions/translocations in the telomeric regions. centromere in a whole-arm translocation, (3) trisomy 21 and 13 in five cases, De novo balanced chromosome rearrangements occur in one out of 3000 (4) nuclear in situ hybridization (nuc ish) analysis on uncultured amniocytes and newborns. About 6% of these cases have an abnormal phenotype, compared CVS and (5) 'nuc ish' analysis of hematological disorders (monosomy 7 in four to 3% in random newborn populations. The cytogenetic and molecular cases, trisomy 8 in ten cases and trisomy 12 in three cases). PRINS has the analysis of balanced translocations and inversions associated with specific potential to make greater impact in prenatal diagnosis, especially with nuclear in in unrelated is a to the situ hybridization. Unlike conventional FISH with alpha satellite probes, PRINS symptoms/syndromes patients powerful approach can discern alpha satellite components of the two most commonly seen trisomies, identification of disease genes. By FISH we perform a systematic search for namely trisomy 13 and 21. Based on our experience with PRINS, we conclude that YACs overlapping chromosome breakpoints that truncate or inactivate this latest technology not only provides sensitive and reliable diagnoses, but also specific genes. These YACs are employed to isolate cosmids allowing large- offers a faster, easier and cost effective alternative to conventional FISH. scale automated sequencing and computer-assisted gene finding. Comparative FISH of human YACs on primate chromosomes facilitates the study of human chromosome phylogeny. We have identified clones spanning the telomere-telomere fusion and a pericentric inversion breakpoint on human chromosome 2. In the long term, we will establish complete homology links between the human and important mammalian genomes. A50 Slide Session 49: Cytogenetics IIl (cont.) 253 254 A complete set of human telomeric probes and their clinical application. Y. to Ninh' J. Flint2 A. Roschkel K. E.C. MC. Phelan4 H. MultiFISH analysis detect microdeletions in patients with idiopathic Kvalov3 Crawford4 Rieshman' developmental delay and mental retardation. A.H. Ligon and L.G. Shaffer. and D.H. Ledbetterl. 'INCHGR, Bethesda, MD; 21MM, Oxford, UK; 3Oxford University, Kleberg Cytogenetics Laboratory, Baylor College of Medicine, Houston TX. Oxford, UK; 4GGC, Greenwood, SC. 5The Wistar Institute, Philadelphia, PA. Developmental delay and mental retardation (DD/MR) are common clinical indications Human chromosomes terminate with specialized structures including the simple for chromosomal studies. These features can also occur as part of a contiguous gene tandem repeat (TTAGGG)n and additional complex subtelomeric repeats. Unique deletion syndrome caused by deletion of several physically contiguous, yet functionally unrelated, genes. Deletions of chromosomes 7ql 1.23 (observed in Williams sequence for each telomere is located 100-300 kb from the end of most chromosomes. syndrome), chromosome l5q12 (Prader-Willi and Angelman syndromes), 17pl1.2 The human telomeric regions represent a major diagnostic challenge in clinical (Smith-Magenis syndrome, SMS) and 22q1 1.2 (DiGeorge/Velocardiofacial syndromes, cytogenetics, because most of the terminal bands are G negative and cryptic DGS/VCFS) have been demonstrated in a majority of patients with these respective rearrangements in the telomeric regions are therefore difficult to detect by conventional disorders. While particular unique physical characteristics of some of these syndromes cytogenetic methods. In fact, several submicroscopic chromosomal abnormalities in may facilitate diagnoses of patients, some syndromes (e.g. DGS/VCFS and SMS) are patients with undiagnosed mental retardation or multiple congenital anomalies have not as easily identified by physical features alone. For most microdeletion (or been identified by DNA polymorphism analysis. Combining the approaches contiguous gene deletion) syndromes, the size of the deletion is below the level of of 1) cytogenetic resolution and can only be visualized using FISH analysis of the relevant using half-YAC vector-insert junction fragments to screen chromosome specific cosmid chromosomes when a specific disorder is already suspected. We performed a FISH libraries or total human genomic P1 or PAC libraries, 2) subcloning half-YACs into a study of 200 chromosomal samples referred to our laboratory for several indications cosmid vector, and 3) using the most distal marker on the current integrated map of a (developmental delay, mental retardation, fragile X, or specifically to evaluate any of chromosome, we have isolated and characterized a complete set of specific FISH the aforementioned microdeletion syndromes) to determine whether any of these non- probes representing each human telomere. Since most of these clones are at a known specific diagnoses could be attributed to one of the tested microdeletions. FISH analysis was performed using a cocktail of probes that are routinely used independently distance of within 100-300 kb from the end of the chromosome arm, they increase the to detect deletions at the Williams, Prader-Willi/Angelman, SMS and DGS/VCFS resolution of deletion detection by approximately 10-fold over current cytogenetic critical regions. This simultaneous MultiFISH analysis was performed and scored in a banding methods (2-3 Mb resolution). The 2q telomeric probe was used to characterize blinded fashion on 200 patients. Ten patients were known by the diagnostic 3 patients with previously documented deletions of 2q37. It confirmed that the cytogenetics laboratory to have microdeletions of specific chromosomes, and each of deletions in 2 patients are terminal rather than interstitial. However, one case was these microdeletions was also detected by the MultiFISH analysis. In addition, the discovered to have a cryptic 2q;4q reciprocal translocation, which could barely be MultiFISH cocktail identified a deletion of chromosome 22ql 1.2 corresponding to the DGS/VCFS critical region in a patient referred for DD/MR, for whom no specific detected with whole chromosome painting probes for chromosomes 2 and 4 in less diagnosis had been suggested and, therefore, no prior FISH analysis had been than10% of the cells. In contrast, the 2q and 4q telomeric probes gave 100% detection. performed. This analysis demonstrates that a multiprobe cocktail including probes These results demonstrate that telomeric probes will complement painting probes and corresponding to several contiguous gene deletion syndromes can be used successfully significantly improve cytogenetic diagnosis. to identify microdeletions which may otherwise go undetected in a proportion of patients presenting with idiopathic mental retardation or developmental delay.

255 256 Detection of inapparent chromosomal aberrations by whole genome STRP scanning: results of a pilot project. LG Biesecker', High-resolution, multi-locus karyotype analysis with 'Chromosomal Rainbows (CR)': a novel technique applied to rapid characterization of MRosenberg', Y Ning',D Vaskea,J &ebr2, and the UMCAS research rearranged chromosomes in primary cultures and cell lines from thyroid group. 1 National Center for Human Genome Research, NIH, Bethesda, 2. MD, 2 Marshfield Medical Research Foundation, Marshfield, WI. cancers. H.-U.G.Weierl, B. O'Brient T.H. Quell , M.G. Won2g,W.-L.KuL3. A.E. Siprstein2, Q-Y.Duh andO.H.Clark2 lResource for Molecular Cytogenetics, We have whole genome STRP marker scanning to detect chromo- Lawrence Berkeley National Laboratory, Berkeley, CA 94720, 2Department of Surgery somal aberrations in children with multiple anomalies and a normal and 3Div. Molecular Cytometry, Univ. California, San Francisco, CA 94103, USA. karyotype. To pilot this method, 388 STRP markers from the Marshfield Rearrangements of chromosomes I and 10 are frequently observed cytogenetic changes in papillary thyroid cancer (TC), while loss of heterozygosity for several loci screening set 6 were used to genotype an affected child and both parents. on the short arm of chromosome 3 has been reported in follicular TC. The aberrations The genotypes are used to infer non-mendelian patterns including mono- range from small interstitial deletions to intra- and interchromosomal translocations and somy, trisomy, and uniparental iso- and heterodisomy. We have completed chromosome loss. We developed a screening technique termed chromosomal rainbows a pilot study of 12 affected children (and their parents) using this approach. (CR) based on in situ hybridization to assess the integrity of selected chromosomes and The first round of genotyping included all 388 markers for a total of 13,968 rapidly map deletion and translocation breakpoints. genotypes. The abnormal genotyping results are as follows: 173 required We prepared two sets of CR probes that bind specifically to human chromosomes 3 repeating (3.7%), 8 had evidence of intensity differences between alleles and 10, respectively. Each set was comprised of 16-21 individual P1 or yeast artificial (.2%), 11 suggested deletions (0.2%), 15 had mutated alleles (.3%), and 1 chromosome (YAC) probes mapping in approximately 8-10 Mbp intervals along the each were compatible with uniparental heterodisomy and trisomy. The total target chromosome. The DNA from individual clones was labeled with reporter number of genotype results that suggested an anomaly (deletion +UPHD+ molecules such that detection with fluorochrome-labeled antibodies produced repeated trisomy) was 13. Of the 1 1 genotypes that suggested a deletion, 3 of those pattern of red-infrared(IR)-green-yellowv hybridization signals along the target were confirmed on repeat testing. Two of those three were contiguous chromosomes (rainbows). Overnight hybridization to metaphase spreads from several markers on chromosome 4 in a single patient. Repeat cytogenetic analysis of our thyroid tumor cell lines followed by image analysis allowed rapid delineation of confirmed that the patient had an interstitial deletion of chromosome 4. The interstitial deletions, characterization of marker chromosomes and mapping of 1 translocation breakpoints with a resolution of a few Mbp. Metaphase cells in primary third deleted marker was on chromosome in this patient and is being tumor cell cultures could be analyzed even when the quality of chromosome spreads confirmed by FISH. We have not yet been able to confirm the UPHD result was insufficient for conventional banding analysis. in the second subject. We conclude that whole genome scanning for Complementing chromosome banding or painting analysis, CR analysis combine deletions is possible and that it can detect aberrations that are missed by high resolution analysis with speed and less stringent requirements regarding standard cytogenetic techniques. An increase in marker density and a metaphase quality, thus enabling rapid screening for oncogene-activating decrease in genotyping costs could this a practical diagnostic test and translocations, gene amplifications or deletions. provide an unbiased estimate of the distribution of these.

257 258 Spectral karyotyping (SKY) of human chromosomes E.Schrock. T. Veldman. S. du Manoir. D.H. Ledbetter. T. Ried. Endometriosis: ChromosoMal aneuploidy detected by multi-color Diagnostic National Centerfor Human fluorescence in situ hybridization (FISH) J.-C. ShinI',H.L. Ross', D. Development Branch, Genome Mitchell3, !.L. Simpson', S. Elias'2 and F.Z. Bischotfl. Dept'Ot OB7GYNtand Research/NIH, Bethesda, MD 20892-4470. Molecular/ Human GeneticsL, Baylor College of Medicine, Houston, TX; Southern Spectral karyotyping permits the simultaneous display of all human FertilityInst., Atlanta, GA3; Dept. of OB/GYN, Catholic University Medical College, chromosomes in different colors. This novel genome scanning method Seoul, Korea4. combines Fourier spectroscopy, CCD-imaging, and optical microscopy to Endometriosis is a common gynecologic disorder associated with infertility and visualize the hybridization of 24 differentially labeled human chromosome pelvic pain. Although our own studies have shown familial tendencies (AJOG, painting probes (Schrock et al., Science). The measurement of definitive 137:327-331, 1980), cytogenetic and molecular genetic studies have demonstrated emission spectra simultaneously at all sample points no clear etiology. Some studies have demonstrated over-expression of certain allows oncogenes c-fms, k-ras, c-erb B-I and but mutations in neither or to classify all human chromosomes and to automatically identify the origin of (c-myc, 10 -2); ras chromosomal material that could not be described using banding methods p53 were evident in severely affected women (Gynecol ObstetInvest, 38:70-71, alone. 1994). Cytogenetic analysis (Cancer Genet Cytogenet, 78:172-174, 1994) Spectral karyotyping was appliedto the analysis of chromosomal aberrations in demonstratedrandom numerical changes and rearrangements; however, ability to clinical and cancer cytogenetics. In clinical cytogenetics, the analysis of subtle detect clonal chromosomal abnormalities is limited by analysis of relatively few telomerictranslocations and the identification of the origin of marker metaphases and potential outgrowth of the selectively advantaged normal cell chromosome is greatly facilitated. E.g., a reciprocal translocation of populations. An alternative approach would be usingFISH because1) more nuclei chromosome and material was unambiguously identified and confirmed can be analyzed, and 2) analysis can be performed rapidly on uncultured tissue using telomere specific cosmid clones. Of note, SKY can be performed on biopsies. Sttidv Design-Multi-color FISH was performed on endometriotictissue previously G-banded chromosomes, allowing comprehensive analysis of obtained tromtour women undergoing surgery. To- and three-color FISH using chromosomal rearrangements in the same cell. centromere-specific alpha-satellite probes for chromosomes 7,8,11,12,16,17 and The of 18 (ONCOR) were used to determine aneuploidy frequencies in uncultured normal analysis chromosomal aberrations in solid tumors is often complicated peripheral by low mitotic indices, poor banding quality and the presence of marker blood lymphocytes, normal endometrium and endometriotictissue. A chromosomes, homogeneously staining regions and double minutes. Spectral minimum of 250 cells were scored for each probe combination. Results-Overall, mean frequency of chromosomal aneuploidy in the normal samples was 3.4% karyotyping was applied to direct preparations, short term cultures and cell lines monosomy and 1.2% for trisomy. Of the four for established from breast carcinomas, astrocytic tumors, in and endometriosis samples a significantly lymphomas higher frequency of chromosome 17 monosomy was observed in one case leukemias. In all cases the karyotypes were unambiguously reconstructed, (14.8%,X24= 53.3, p< 0.0001) and chromosome 11 trisomy in another including the identification of marker chromosomes that contained chromosomal (14.8%,X24= 96.2, p_< 0.0001). Conclusion-Chromosome-specific numerical aberrations unrecognizable using chromosome banding alone. Limitations and abnormalities may be involved in eno3meiiosis, reflecting clonal expansion of further developments of SKY will be discussed. chromosomally abnormal cells. Given that endometriosis is characterized by abnormal growth or turn-over, tumor-suppresser genes and oncogenes are attractive candidate genes. That such candidte genes have been mapped to chromosomes 11 and 17 furthersuggeststhatchromosomal loss or gain plays a role in the development and progression of endometriosis. Slide Session 49: Cytoglenetics 1UT (contj A51 259 260 Why is the human cornea cytogenetically abnormal? M.J Pettenati'. P.N. Rao ' J. Targeting evolutionary breakpoints in the Hominidae family. E. Nickerson. Reynolds . P. Lantz '. R.M. Davis'. 'Bowman Gray School ofMedicine of Wake Forest D.L. Nel Department of Molecular and Human Genetics, Baylor College of Univ., Winston-Salem NC;, Shodair Hospital, Helena MT. Medicine, Houston, TX. We report for the first time that the stromal keratocytes (fibroblasts) of Similarity between the DNA sequences of the members of the family Hominidae, which the human cornea are cytogenetically abnormal at an unprecedented rate and includes humans (Homo sapiens), chimpanzees (Pan troglodytes), gorillas (Gorilla gorilla), and orangutans (Pongo pygmaeus), is estimated to be greater than 95%. High these aberrations are acquired. The abnormalities are clonal and non-clonal, and resolution G-banding studies carefully comparing the karyotypes of the Hominidae, are primarily aneuploid in nature. showed that while 18 of 23 pairs of chromosomes appear virtually identical, several Standard cytogenetic analysis was performed on normal (n=27) and inversions and a few translocations have occurred as the Hominidae evolved. We have abnormal (n=38) human cornea, both fetal/child (n=6) and adult (n=59). Our initiated a project to identify sequences at the inversion breakpoints. data showed that 1) all fetal/child corneas were cytogenetically normal; 2) nearly Rearrangement breakpoints were assigned to cytogenetic bands. The CEPH and Whitehead physical mapping databases, as well as chromosome workshop reports and 75% of the normal (primarily donor) corneas had chromosome abnormalities; and many genetics world wide web pages, were used to identify relevant MegaYAC contigs 3) all scarred/failed graft/diseased comeas were chromosomally abnormal. The predicted to span human cytogenetic bands of interest. MegaYAC DNAs are used as loss and/or gain of sex chromosomes were the most common abnormality FISH probes against human chromosomes to determine appropriate FISH conditions, to occurring in 94% of all cases. Autosomes most commonly involved in aneuploidy confirm the probe's location in the genome and to detect chimerism. Concurrently, male were chromosomes 3, 7, 15, 20, 21 and 22. Specific disease groups had different cell lines from gorilla, chimpanzee, and orangutan have been cultured and harvested for degrees of chromosome aneuploidy. Clonal evolution occurred in a some cases. metaphase chromosomes and are being hybridized by FISH. By this method, a YAC spanning a pericentric inversion breakpoint will be detected when it produces a FISH There were several instances of structural chromosome abnormalities. signal on one arm of a human chromosome but on both the long and short arms of the The cornea is unique from a structural and functional point of view. We corresponding chromosome of the relevant primate. have demonstrated cytogenetically that the keratocytes are very unusual in Initial results indicate that the approach described is effective. A comparison of human comparison to other tissues. We hypothesize that the human cornea may use the 9q22 to chimpanzee involving 7 MegaYACs spanning approximately 8060 Kb has mechanism of chromosome aneuploidy and structural alterations to regulate its uncovered a single YAC that spans the pericentric inversion in the chimpanzee equivalent structure and function. This is based on our observations that the chromosomes We have also identified a chimeric MegaYAC, isolated for a chromosome 9 locus, whose chimeric piece appears to be rearranged in orangutan. Breakpoints in chromosomes 1, 4, involved in aneuploidy and structural abnormalities harbor genes important in 12, and 17 are also being targeted with this approach. Each region involved in a regulation of collagen synthesis, the primary role of keratocytes. These unique rearrangement is being characterized to evaluate the molecular consequences of the findings raise questions and issues concerning clinical and disease associations, evolutionary change. Genes that have been deleted, interrupted, moved under the control genetic correlations, lack of corneal tumors and research conducted on cornea of an alternate chromatin environment, or otherwise altered may be significant to tissue. phenotypic differences among the great apes.

Slide Session 50: Cancer Genetics III 261 262 Identification of a trinucleotide repeat-containing gene located on chromosome FHIT/FRA3B alterations in premalignant and malignant lesions of 3p2l.3 exhibiting loss of heterozygosity in lung cancer. S.P. Shriver. M.D. Barretts esophagus. D. Michaell,24, D.G. Beer3, CAM. Wilke"2, K. Abet2 Shriver. L.M. Bloch.. D.L. Tirpak. and J.M. Siegfried. Department of A. Hoge,2. M.B. Orringer3, D.I. Smith5, underlineT.W.Glover' 2. Departments of Pharmacology, University of Pittsburgh, Pittsburgh, Pennsylvania. 'Pediatrics, 2Human Genetics and 3Surgery, University of Michigan Medical School, Chromosome 3p is consistently deleted in lung cancer, oral squamous cell Ann Arbor, MI. 4Innere Klinik/ Tumorforschung, Universititsklinikum Essen, carcinoma, and renal cell carcinoma, and is believed to contain several tumor Germany. SDepartment of Internal Medicine, Wayne State University, Detroit. suppressor genes. Each of these tumor types has been found to be heritable in some We have previously reported that the common site at extends cases and genes on chromosome 3p could be involved in susceptibility to these fragile 3p14.2 (FRA3B) cancers. We have isolated a candidate tumor suppressor gene, TDL (trinucleotide over a broad region just distal to the hereditary renal cell cancer t(3,8) breakpoint and is repeat-containing gene deleted in lung cancer), which is located at 3p2l.3, within within the 1.3 Mb YAC 850A6. Using trapped exons derived from cosmids in t his region, the smallest region of deletion overlap in lung tumors. The TDL gene is predicted we obtained a cDNA corresponding to the FHIT gene. The cloning of this gene was to encode a 17.6 kilodalton protein with a nuclear localization signal and a probable recently reported by Ohta et al. (Cell 84: 587-597, 1996) who, together with Sozzi et al. DNA binding region. The TDL sequence contains a highly polymorphic (Cell 85: 7-26, 1996) further showed that transcripts from t he FHIT gene are altered in trinucleotide repeat array which encodes a variable-length polyalanine tract. digestive tract cancers and lung cancer. To expand on these findings and determine the Heterozygosity studies for TDL show that this locus is approximately 70% extent of FHIT mRNA alterations in early stages of tumor development, we analyzed 9 heterozygous in the normal population, compared with 25% in NSCLC cell lines patients with Barrett's metapl asia, a premalignant condition, and adenocarcinoma using (p=0.04). Cell cultures derived from normal bronchial epithelium showed a 75% DNA sequence and Southern blot analyses of RT-PCR products from FHIT mRNA. level of heterozygosity, reflecting that of the normal population. When RNA from Alterations of FHIT transcripts were observed in 100% (9/9) of Barrett's metaplasia these cell lines was examined, the heterozygous bronchial cell samples expressed and in 100% (9/9) of the adenocarcinomas from the same patients. Characterization of both alleles, maintaining the 75% heterozygosity level. In contrast, all heterozygous the altered transcripts revealed that they correspond to messages lacking two or more tumors failed to express one allele (the heterozygosity level was reduced to 0%), exons of the FHIT gene. The region of deleted exons spans our map of FRA3B fragili suggesting a functional loss of heterozygosity. In addition, cell lines derived from ty and includes exons 3 through 8, with exons 5 through 8 being the most frequently squamous cell carcinoma of the head and neck (SCCHN), also demonstrated a deleted. Southern blot characterization of genomic DNA from these tumors shows that reduced level of heterozygosity (20%; p=0.008). SCCHN has the same risk factors some contain deletions in the corresponding regions of the gene. Analyses of 7 adenocar as lung cancer and is hypothesized to have a similar etiology. Although large cinomas from gastric cardia gave similar results. In contrast, no alterations have been expansions of the trinucleotide repeat have not been observed, lung cancer patients observed in the FHIT transcripts from esophageal squamous cell carcinomas. These exhibit a wider range of allele sizes than unaffected individuals. These results results extend the range of tumor types in which altered FHIT transcripts have been suggest an association of certain alleles with an elevated risk of lung cancer, either demonstrated, and show that these alterations can be seen at the premalignant stage. directly through an effect of allele size on TLD function, or indirectly by association These and related findings raise questions regarding the association of FRA3B with this through linkage disequilibrium of certain allele sizes with mutation in TLD or a region of frequent deletion in tumors and the functional s ignificance FHIT in tumor nearby gene. development.

263 264 A human Mad gene family. GJ Riggins'. S Thiagalingam'. SE Kern' 2. KW Analysis of spontaneous mutations in MSH2 'knockout' mice using a Kinzler' and B Vogelstein'". 'Johns Hopkins Oncology Center. Baltimore, MD transgenic mutation detection system. S.E. Andrew', A.H. Reitmair'*, J. Fox', 2Johns Hopkins University School ofMedicine Department of and The L. Hsiao', T. Mak#, and F.R. Jirik'. 'Biomedical Research Center, Univ. of British Pathology Columbia, Vancouver, B.C., Canada #Amgen Institute, Ontario Cancer Institute, Howard Hughes Medical Institute at The Johns Hopkins Oncology Center. Depts. of Medical Biophysics and Immunology, Univ. of Toronto, Ont., Canada. Baltimore. MD. DNA mismatch-repair (MMR) systems are responsible for processing of mispaired bases Resistance to TGF-beta growth inhibition is a common phenotype of human arising during DNA replication and preventing recombination between non-homologous cancers, yet the mechanism by which this occurs is largely unknown. Probable DNA sequences. In prokaryotes, MMR is initiated by the MutS, MutH and MutL pro- mediators ofTGF-beta like signals have been found in Drosophila (Mofad) and C. teins. Mutations occurring within eukaryotic homologues of these genes are observed cancer The murine homo- Elegans (sma-2, sma-3 and sma-4). To explore the role of Mad genes in human in individuals with hereditary non-polyposis colon (HNPCC). logue of the MutS gene, designated MSH2, has recently been 'knocked-out' via gene- cancer, we sought human Mad homologues. Starting with expressed sequence targeting. Homozygous MSH2 deficient mice show a high incidence of malignancies, tags and with subsequent cloning, we have discovered a family of human genes especially thymic lymphomas, although other tumor types have also been observed. with remarkable homology tol/ad. Two of these genes, JV4- 1 and JV5-1, have Lambda-phage transgenic mutation detection systems offer insight into the consequences over 96% amino acid identity at the carboxyl-terminus. IV18-1 shows a 62% of MSH2 deficiency. We have bred a transgenic line (BC-1) containing a retrievable identity to .1/ad over 373 of its 467 amino acids. JV1 8-1 maps to 18q21. in a lad shuttle vector with an MSH2 gene-targeted line. The transgenic system reveals an increased mutation frequency in DNA obtained from the thymus, small intestine and region that undergoes a high rate of loss of heterozygosity(LOH) in colorectal brain in MMR deficient (BC-1/MSH2-/-) mice. Heterozygous (BC-I/MSH2+/-) mice tumors. Colorectal tumors with LOH ofthis region were screened for homozygous show spontaneous mutation frequencies similar to controls. The spectrum of mutations deletions and sequenced for mutations. One homozygous deletion and one 42 bp in MSH2-/- mice shows that in addition to the repetitive sequence instability which deletion ofa conserved region were found in JV1 8-1. Additional mutations were leads to a higher frequency of frameshift mutations compared to controls, the majority found in another Mad homolog. DPC4. suggesting that Mad genes are relativelh of mutations are transitions, primarily at CpG sites. Analysis of mutation frequencies in frequent targets for somatic mutation in human colorectal cancers. tumours, as well as in tissues not normally prone to tumour development in these mice, Interestingly. may offer a correlation between the genotype and the tendencies of different tissues to three other homologues found by this approach map to chromosomal locations undergo malignant change. This animal model may be useful in shedding light on the that undergo a high percentage of LOH in multiple tumor types. Mutational mechanism of cancer susceptibility seen in HNPCC. analysis ofMad homologues will be presented for a variety of tumors. A52 Slide Session 50: CSancer Genetics Ill (cont.) 265 266 The use of mRNA differential display to isolate genes altered in breast A Transcript Map of a 20q13.2 Breast Cancer Amplicon and Putative cancer progression. M.A. Spillman, F. Du, C. Zhao, and A.M. Bowcock. University Candidate Genes S-i Hwang', D Kowbell, J Rommens2, T Godfrey3, D Polikoffl of Texas Southwestern Medical Center, Dallas, Texas. T Cloutier', J Cochran', K Myambol, M Tanner4, O-P Kallioniemi4, F WValdman3, Aggressive breast tumors follow a progression of proliferation, invasion and metastasis, C Iartin', M.e Palazzo10l, G Hutchinson' and JW. Gray' 3, and CC Collins'. accompanied by alterations in gene expression. As an in vitro model of human breast 'Lawrence Berkeley National Laboratory, CA, 2Hospital for Sick Children, Toronto, cancer we have been examining the BT-483 breast cancer cell line that was derived Canada, 4Tampere Univ., Finland, 5RabbitHutch Biotechnology, B.C. Canada, and from a 23 year old woman with breast cancer and grows very slowly in culture, at 3Univ. of California, San Francisco, CA. the approximate in situ doubling rate of a breast tumor. We identified a fast growing Chromosome 20q13.2 is amplified in primary breast tumors and breast cancer cell lines. subpopulation of BT-483 cells that arose spontaneously in culture and had a decreased High level amplification is associated with poor prognosis. This amplicon is also detected sensitivity to estrogen and an undifferentiated morphology. The transition of BT-483 in numerous other solid tumors. To identify candidate genes that may participate in the parental cells to mutant cells therefore has characteristics of in vivo tumor progression. progression of these tumors, a 1.5M1b P1 and BAC contig spanning the 20q13.2 region The identity of the mutant subline as a derivative of the parent BT-483 cell line was was cloned. The amplicon was further sublocalized to a 0.6Mb interval by interphase confirmed by analyzing 27 polymorphic loci on 10 autosomes. Differential display was FISH on primary breast tumors. Transcribed genes in this amplicon were identified used to isolate genes that were uniquely expressed in either the BT-483 parent or mutant by exon trapping, cDNA direct selection, genomic sequencing, and computational ge- subline, permitting the identification ofgenes whose expression was lost in the progression nomics. Over 200 exons were trapped and analyzed from 32 Pls and BACs subcloned of breast cancer or genes whose expression was upregulated as a result of deregulated in the pSPL3 vector. 200 clones from a BT474 cDNA were isolated by direct selection growth. Using 60 primer pairs that displayed approximately 1800 cDNAs, 69 cDNA using a set of genomic clones from across the amplicon. A 0.6 Mb genomic interval bands (46 parent and 23 mutant) were differentially expressed. Of 29 bands (17 parent spanning the minimal amplicon is being sequenced. Exon prediction and gene modeling and 12 mutant) analyzed by hybridization to total RNA northerns, 11 failed to hybridize are carried out with XGRAIL, SORFIND, and BLAST programs. Putative gene frag- (7 parent and 4 mutant) and 10 (5 parent and 5 mutant) showed no differences in ments identified by these approaches were analyzed by RT-PCR, Northern and Southern expression. Differential expression was confirmed for 4 bands, where two were only blots. Database searches revealed several significant similarities to cytoplasmic and nu- expressed in the parental line and two were only expressed in the mutant Line. The clear proteins, and ESTs. 20 unique exons and 11 unique cDNAs were identified that insulin-like growth factor binding protein 5 (IGFBP-5) was isolated by virtue of its may define 12 unique genes within the amplicon. Candidate oncogene cDNAs are being expression in the parent line and loss of expression in the mutant line. Differential cloned from several libraries and further analyzed for sequence similarities in sequence expression of 3 additional bands (2 parent and 1 mutant) is still being confirmed. Some databases, for expression, and amplification in primary breast tumors. All putative can- of the differentially expressed sequences matched sequences present in the EST database didate genes examined thus far represent novel genes. Supported by grants from US and are currently being extended and localized to human chromosomes in a panel of DOE contract DEAC0376SF00098, USPHS grants CA44768, CA45919, CA52807 and radiation hybrids. These genes are candidate markers for breast tumor progression and Vysis. S-i H. is supported by a Human Genome Distinguished Postdoctoral Fellowship are being examined in a panel of human breast tumors. from DOE/ORISE.

267 268 The role of the human homologue of Drosophila patched in sporadic basal cell Atm-deficient mice: a paradigm of ataxia-telangiectasia. C. Barlow1, carcinomas. M. Gailani', A. Unden2, D. Leffell ', C. Wicking 3, M. Stahle-Backdahl 2, S. S. Hirotsunel, R. Pavlor2, M. Liyanage', M. Eckhaus3, F. Collins', Y. Shiloh4, Shanley , A. Chidambaram S. Gillies 3, P. Zaghiropoulos 2, E. Holmberg., K. Negus J.N. Crawlev2, T. Ried±, D. Taglel and A. Wynshaw-Boris'. 'NCHGR, 2NIMH, 1. Smth3. M. Glynn', B. Gerrard 4, A. Goldstein", B. Wainwright , G. Chenevix-Trench', 3NCRR, National Institutes of Health, Bethesda, MD 20892 and 4Tel Aviv University, M. Dean 4, A. Bale R. Toflaard. Yale Univ., New Haven, CT I, Karolinska Institute, Stockholm, Sweden Centre for Molecular and Cell Biology, Univ. of Queensland, Ramat Aviv 69978, Israel Brisbane, Australia3, N.C.I., FCRDC, Frederick, MD 4and Genetic Epidemiology Branch, Ataxia-telangiectasia (A-T) is a lethal, progressive human autosomal recessive Bethesda, MD6, The Queensland Institute of Medical Research, Brisbane, Australia s. disorder with the hallmark clinical manifestations of progressive neurological Basal cell carcinoma (BCC) is the most common type of cancer in humans. The degeneration, immunodeficiency, lymphoreticular malignancies, growth retardation, majority of BCCs have allelic loss on chromosome 9q22, and the gene for Gorlin incomplete sexual maturation, oculocutaneous telangiectasias and premature aging of syndrome (NBCCS), an autosomal dominant disorder characterized by multiple BCCs, the skin and hair. Individuals are extremely sensitive to the effects of ionizing radiation. maps to the same location suggesting that NBCCS functions as a tumor suppressor. Heterozygous carriers may have a predisposition to cancer, particularly breast cancer. NBCCS has been identified recently and encodes a transmembrane protein with We have created mice, via gene targeting, with a disruption of AItm , to study the strong homology to the Drosophila segment polarity gene, patched. To evaluate the pleiotropic function of this gene in a mammalian model. Mice heterozygous for the Atm role of patched in sporadic 8CCs, we used SSCP to screen 37 tumors for mutations. disruption appear normal and remain disease free at six months of age. Homozygous mice Mutations were identified in 9/22 tumors with allelic loss and in 3/15 tumors without have neurologic dysfunction, immunologic abnormalities, growth retardation, infertility allelic loss. Direct sequencing of two tumors without allelic loss or SSCP variants secondary to complete lack of mature gametes, extreme sensitivity to ionizing radiation, revealed mutations of both alleles, indicating that SSCP is not a sensitive screening lymphoreticular malignancies, and chromosomal instability. Cells from these mice grow technique. Eleven of sixteen mutations that would be predicted to cause protein poorly, and exhibit G1/S cell cycle checkpoint abnormalities. A tumor cell line derived truncation (8 premature stop, 2 splice site mutations, one duplication) were scattered from one lymphoma contains multiple chromosomal aberrations suggesting a high degree throughout the gene. Of four of genomic instability. The coincident finding of thymocyte maturational defects, sensi- missense mutations and an in-frame deletion, two tivity to radiation and gonadal degeneration, suggests a link between the mechanism of occurred in a large extracellular loop, one in an intracellular loop, and two in the double strand break repair occuring during recombination in the immune system, DNA intracellular carboxy terminus. Nine mutations were typical UVB-induced transitions and meiotic and that is crucial for these opposite dipyrimidines. Seven mutations were not related to UVB despite their damage repair recombination, Atm processes. in This mouse recapitulates the human disease and provides an excellent model for under- occurrence tumors from sun-exposed areas. Northern blots and RNA in situ standing the role of Atm in normal cellular function as well as pathophysiology, and for showed high levels of patched in BCCs but very little expression in normal skin. Our testing potential therapeutic agents to treat the progressive, debilitating manifestations data show that inactivation of NBCCS is probably necessary for BCC formation and of A-T. that factors other than UVB are involved in mutagenesis. That tumors express patched but RNA in situ failed to detect it in normal skin suggests that the gene may be expressed at a very low level in normal cells or that expression is restricted to a limited number of BCC precursor cells.

269 270 Towards the understanding of the mitochondrial involvement in the ageing Major Mitochondrial DNA Changes with Age in a variety of Tissues of process in human skeletal muscle A. Barrientos', F. Cardellach2, J . Casademont2, Mouse detectable by Southern Blot and Long Extension PCR (LX-PCR) of A.Urbano-Mirquez2. X. Estivill', V. Nunes'. 'Departament de Genetica Molecular, the Mitochondrial Genome. S. Melov, D.A. Hinerfeld, L. Esposito. D.C. Wallace. Institut de Recerca Oncolbgica (IRO), Barcelona, Spain. 2Departament de 'Medicina Department of Genetics and Molecular Medicine, Emory University, CA. Interna, Hospital Clinic i Provincial, Barcelona, Spain. Mitochondrial DNA (mtDNA) rearrangements have been found to accumulate with Oxidative phosphorylation appears to decline with age and this impairment could age in a variety of organisms as diverse as nematodes and humans. This has suggested contribute to the general decline in physiological functions. We investigated the effect that the accumulation of mtDNA mutations is either a reliable biomarker for aging or of ageing on skeletal muscle mitochondrial function in 101 patients, with ages ranging fundamentally related to senescence itself. Caloric restriction in rodents has been shown from between 13 to 94 years, who underwent surgery for femur fractures. We found to extend life span. Hence if mtDNA rearrangements are related to longevity, then caloric oxygen consumption with different substrates decreased with age. Respiratory control restriction might be expected to inhibit some of these effects. We have examined DNA and ADP/O rates did not change. No correlation was found between any individual extracted from liver. kidney, heart, and brain from young (2-4 months) and old (32-35 enzyme activity of the electron chain complexes and age, indicating that other factors months) mice (C57BI/6) both ad libitum and caloric restricted for changes in the mtDNA are responsible for the decrease in the respiratory rates. Analysis showed that the using full length mtDNA amplification by LX-PCR. We find large scale rearrangements proportion of deleted mtDNA molecules increased with age but was never more than 1 in old post mitotic tissues relative to young tissues. We have sequenced the breakpoints per cent of the total mtDNA. Only two individuals of 68 and 71 years old showed, at a of a number of these rearrangements, and find that in some rearrangements direct repeats low proportion, the A3243G transition typical of MELAS syndrome, while their are involved, while in other cases they are not. In addition, we have developed a novel mitochondrial respiratory chain function was normal. No individual showed point PCR assay for the detection of mitochondrial DNA 'mini circles" and have discovered mutations typical of MERRF or NARP syndromes or of Leber optical atrophy. The 2-4 kb mini circles only occur in old tissues. To further characterize mtDNA from young, total mtDNA amount content increased with age. At the same time we found a old, and calorically restricted mice, we performed Southern blots with Field Inversion decrease in the transcription and translation rates associated with age. We conclude Gel Electrophoresis (FIGE) of unrestricted DNA hybridized with a total mtDNA probe. that muscle mitochondria are biochemically undamaged with age, that the mtDNA Substantial changes in the structures of mtDNA were observed to accumulate in the alterations that increase with age are insufficient to produce a functional deficit and brain with age. The exact identity of these complex conformational changes is uncertain, that the decrease in mitochondrial transcription and translation rates is partially however mtDNA analyzed from brain of calorically restricted old animals, is intermediate compensated for an increase in the replication rate. in its conformational complexity relative to young and old ad libitum fed animals. The (Supported by DGCYTPB93-0019, CICYTSAF913/93 and FIS94/1563. AB is detection of major mtDNA changes with age in the brain by Southern blot, together depositary of grant MECPF9237289410). with the variety of defined rearrangements detectable with LX-PCR argues that there are substantial global changes with age in the mtDNA, and the amelioration of some of these effects by caloric restriction argues that mtDNA rearrangements may play a role in the senescence process. Slide Session 51: Gene Structure and Function 11 A53 271 272 A gene regulatory region polymorphism alters serotonin transporter Neurobehavioural and hippocampal abnormalities in Spinocerebellar Ataxia type 1 (Scal) null mice. T. Matifla'.S BanWi. N. Lu'. D. Armstrong'. E. Burrieht'. expression and function in humans. D. Bengell,2, K.P. Lesch2, A. Heils2, DL E.D. Roberson'. J.D Sweatt'. H.T. Orr. M. Monzk' and H.Y. Zhb'. 'Baylor Ham=r3, D.LJ Muw. ILab. of Clinical Science, NIMH, NIH, Bethesda MD, College of Medicine, Houston, TX, USA, 'TIGEM, Milan Italy, University of 2Dept. of Psychiatry, Univ. of Wurzburg, 97080 Wurzburg, Germany, 3Lab. of Minnesota, Minneapolis, MN, USA. 'Howard Hughes Medical Institute. Biochemistry, NCI, NIH, Bethesda MD. Spinocerebellar ataxia type 1 (SCAl) is a neurodegenerative disorder characterized by Transporter-facilitated serotonin (5-HT) uptake plays a key role in the ataxia, progressive motor deterioration and loss of cerebellar Purkinje cells. SCAI is modulation of serotonergic neurotransmission and is the site of action of caused by an expansion of a CAG trinucleotide repeat which codes for glutamine in ataxin- widely used antidepressant and antianxiety drugs. We recently found that 1, the SCAJ gene product. Both the wild-type and mutant proteins are translated and human serotonin transporter (5-HTT) gene transcription is modulated by a detected in affected individuals. Neurons expressing ataxin-l reveal nuclear localization, common 44 bp insertion/deletion polymorphism in its upstream regulatory however in cerebellar Purkinje cells the protein is localized in both the nucleus and with transfected chorioncarcinoma cytoplasm. The Scal RNA shows the highest expression in the hippocampus and region. Initial in vitro studies placental cerebellum by in situ hybridization analysis. cells revealed considerably different transcriptional efficiency in the long and To gain insight into the function of ataxin-l, a pioneer protein with no known short variants of this 5-HTT gene linked polymorphic region (5-HTTLPR). In homologies, we inactivated the murine SCA) homologue, Scal, using homologous the present study we tested the effect of the 5-HTTLPR on 5-HTT gene recombination in embryonic stem cells. Mice with homozygous deletions of the Scal gene promoter activity in lymphoblastoid cells derived from individuals with are viable and fertile. Immunoblot analysis confirmed that homozygous mice were null for defined 5-HTT genotypes. Functional expression was studied at the level of ataxin-1. Neither heterozygous nor homozygous null mice displayed ataxia based on both 5-HTT gene transcription as well as 5-HT uptake capacity under basal clinical and Rota-Rod analyses. Mice heterozygous for the Scal deletion are and stimulated conditions. Individuals with one or two copies of the short phenotypically normal whereas homozygous mice display neurobehavioral abnormalities. allele demonstrated both decreased 5-HTT mRNA levels and 5-HT uptake Open field test analysis showed a decreased exploratory behavior in homozygous null capacity. We therefore suggest that the polymorphism has more of a mice (p<0.01), but it did not show differences in general locomotor activity (p>0.05). In codominant/recessive than a codominant, additive effect on these measures of addition, null mice showed diminished paired pulse facilitation (PPF) (p<0.01) using expression and function of the 5-HTT. These data indicating that this Schaffer collateral stimulation in area CAL of the hippocampus. Histopathological analysis polymorphism is functionally relevant are intriguing in light of our recent using TRIM staining of brain sections from null mice demonstrated the absence of two in a bands of reactivity which are normally present in the molecular layer of the dentate gyrus finding that it is linked with anxiety-related personality traits population suggesting altered input into the hippocampus. and family based study. The normal phenotype in heterozygous mice argues against the human SCAl phenotype being due to haploinsufficiency caused by CAG repeat expansion. The neurobehavioral, neurophysiologic, and neuropathologic findings in Scal null mice suggest that ataxin-I is involved in hippocampal mediated function. More detailed analyses are now in progress to identify the precise nature of the hippocampal abnormalities in these mice.

273 274 Behavioral and Pathologic Characterization of SCA) transgenic mice. A novel hypersynchronous neocortical EEG phenotype in mice deficient in the lE.N. Burrigb, 1W._YuiiaisC,C Wilcox, IL.A. Duvick 2A. Servadio, 3H.Y. Zoghbi, neuronal nicotinic acetylcholine receptor (nAChRs) a7 subunit gene. A. Off- IH.T. Orr, and IBI. Clark. lUniversity of Minnesota, Minneapolis, 2TIGEM, Urtrege?- J.L. Noebels2. F.M. Goldner', J. Patrick3 and A.L. Beaudet". 'Molecular and Milano, Italy, and 3Baylor College of Medicine, Houston, Texas. Human Genetics, Neurology, Neuroscience,Baylor College of Medicine and 'Howard Spinocerebellar ataxia type 1 (SCAI) is one of several human neurodegenerative Hughes Medical Institute, Houston Texas. diseases caused by the expansion of a CAG trinucleotide repeat within the coding region Genes neural nicotinic are of the associated gene. The shared clinical, genetic, and molecular features of these encoding acetylcholinereceptors(nAChRs) expressed disorders suggest they are caused by a common mechanism of pathogenesis. throughoutthe brain. One class ofnAChRs known to be widely distributed in the brain We have recently reported the development of an animal model for CAG repeat- binds a-bungarotoxin(BTX). The nAChR subunit a7 is an importantcomponent of the induced neurodegeneration by expressing an expanded human SCAl allele with 82 CAG receptorthat binds BTX. When expressed in the xenopus oocytes, the a7 subunit forms repeats within the Purkinje cells (PC) of the mouse cerebellum. Transgenic animals a homo-oligomericligand-gatedion channel that is activated by nicotinic agonists. The develop adult onset ataxia reminiscent of human SCAI and have PC degeneration. Here the is to and the we report a more detailed behavioral and pathologic characterization of these animals. ion channel formed by a7 homo-oligomer highly permeable calcium, Transgenic animals from line B05 displayed normal behavior as juveniles and then agonist-induced current through this channel desensitizes rapidly. Gene targeting was gradually developed progressive ataxia beginning at 12 weeks of age. To assess used to delete exons 8-10 (corresponding to transmembrane domains II - IV and the cerebellar function more quantitatively, animals were tested for the ability to maintain cytoplasmic loop) to produce a7-deficientmice to assess the normal functionofthe gene their balance on an accelerating rotating bar (Rota-Rod). Transgenic animals had and possible involvement in neural-behavioral disorders. Western blot analysis impaired Rota-Rod performance as early as 5 weeks of age (p<0.01). a absence of the a7 in brain from mutant animals. Pathologic alterations preceded the clinical onset of ataxia. Cytoplasmic vacuolization demonstrates complete protein of some PC was the first detectable change, occurring in the fourth postnatal week in Preliminary BTX binding assays show a striking loss ofBTX binding in brain sections. transgenic animals. Cytoplasmic vacuoles continued to be seen within some PC The null mice show no obvious anatomical or neurological abnormalities. However, a throughout the course of the disease. By EM, the vacuoles appeared to be cisternal novel neocorticalexcitabilityphenotype was identifiedin EEG recordings fromthe adult dilations, possibly from smooth ER. There was also dwindling of PC perikarya and mutants, characterizedby a continuous pattern ofgeneralized hypersynchronous 4-7 Hz their dendritic trees with shrinkage and gliosis of the molecular layer. Transgenic sharp wave activity. Taken together with the report that a mutant in the a4 subunit, animals had extensive PC loss by one year of age. To determine the amount of PC loss at the onset of ataxia, we performed PC count experiments on sagital sections and another member ofthis gene family, is associated with autosomal dominant nocturnal determined the linear density of PC along the PC layer. There was no significant frontal lobe epilepsy, these data suggest an important role for nAChRs in neocortical difference in linear density of PC between wildtype and transgenic animals at 12 weeks excitability. of age, indicating the PC dysfunction and ataxia precede cell loss. These findings suggest that to ameliorate the clinical features of SCAI, and possibly other neurodegenerative diseases, the emphasis should shift to understanding the patho- physiologic changes in the affected neurons rather than prevention of neuronal cell death.

275 276 Construction of a murine model of the human Charcot-Marie- Transgenic mice expressing full-length mutant Huntingtin. L,.O'Kusky. Y.P. Goldberg, P. Kazemi. K. Nicol, G. M. Kalchman. R.K. Graham. B. Tooth disease type IA, by microinjection of YACs in mouse Hodgson. Koide. F. Jirik2. M.R. of Medical Genetics, UBC, Vancouver, Canada. oocytes. M. Fontes 1, E. Passagel, A.Manson2, J.F.Pellissier3, Hayden. Dept 'Dept of Pathology, UBC, Vancouver, Canada 2Biomedical Research Centre, UBC, I . C.Huxley2. INSERM U406, Fac de Medecine, Marseille, France. 2 . Vancouver, Canada. Dpt of Biochemistry and Molecular Genetics, St Mary's Hospital, London, Huntington disease (HD) is a devastating autosomal dominant progressive UK. 3 . Laboratory of Anatomo-Pathology, Fac De Medecine, Marseille, neurodegenerative disorder. Expansion of the CAG repeat in the HD gene beyond 35 France. repeats, confers upon huntingtin a 'gain-of.function'. Our goal is to create an animal model by expressing high levels of huntingtin with an expanded polyglutamine repeat. A 560 Kb human YAC, encompassing the proximal part of the CMT1A To achieve this, we have produced HD transgenic mice containing the full-length HD duplicated region (including PMP22 and its regulatory elements) has been cDNA with 44 CAG repeats. The transgenes have integrated intact and under the injected in murine oocytes, to create a murine model of this pathology. 7 influence of the cytomegalovirus (CMV) promoter, are expressing full-length founder mice carry at least part of the YAC, including PMP22. All of huntingtin in a wide variety of tissues in a distribution similar to the known ubiquitous expression of the CMV promoter. Detection of the transgenic protein is achieved by them have had litters, and have transmitted the transgene over 5 western blotting using a human specific anti-huntingtin antibody (GHMI). generations so far. These lines have integrated from one to 7 copies of the Alternatively, using an antibody which detects both mouse and human huntingtin human CMT/PMP22 region. No preferential site of integration have been (BKPl), we are able to identify both endogenous and transgenic huntingtin observed FISH. analysis has demonstrated that the human simultaneously, since the transgenic protein contains a longer polyglutamine tract by Expression causing it to migrate slower. The latter approach allows for relative quantitation of the PMP22 gene is expressed iA the same pattern as the endogenous murine level of expression of transgenic protein compared to endogenous. Neuronal tissue gene (at a less extent, about 30%). Two levels of phenotype has been (cortex, cerebellum, brainstem) and heart showed transgenic huntingtin expression observed. One regards the conduction of nerve velocity, which begins to equivalent to murine endogenous levels. In contrast, peripheral tissues (liver, ovary, be reduced in mice with 4 The second is an apparant spleen, skin) showed only low levels of expression. At present the mice are between copies. neuropathic 3-6 months old and phenotypically normal. Development of a transgenic animal with phenotype, which is apparent in the mouse line with the greater number of neurodegenerative features of the HD phenotype will represent an important advance copies (7). Histology has confirmed that this mouse is presenting a severe and tool in the elucidation of the pathogenesis of HD. demyelinating peripheral neuropathy. Phenotypes have been transmitted over 5 generations presently. Obtention of homozygotes has confirmed that the severity of the phenotype is correlated with the level of expression. Implications of these data for the understanding of CMT1A physiopathology, and for future therapeutic approaches, will be discussed. A54 Slide Session 51: Gene Structure and Function 11 (cont.) 277 278 The X-inactivation pattern of pyramidal neurons of the hippocampus Apoptotic photoreceptor death in gene targeted mutant mice and Its shows pronounced epigenetic variation, and suggests a small number deceleration by gene therapy. S.H. Tsang 1. J. Chen2 C L Yar 3. Ml of progenitor cells. T. Singer-Sam. S. 1. Shen. and C. Gao. Beckman Simon4' P. Gouias'. D Farber3 and S.P_ Goffl. Columbia University, New Research Institute of the City of Hope, Duarte, California. York, 2USC, 3U.C.L.A., Los Angeles and, Cal. Tech, Pasadena. (Intro. by We have used the RT-PCR nucleotide extension Professor Victor A. McKusick). single primer assay, cGMP (PDE) is a key regulator of phototransduction in a quantitative assay for allele-specific expression, to analyze single phosphodiesterase the dissociated hippocampal neurons of mouse F1 hybrids. The presence vertebrate visual system. This enzyme contains a catalytic core of PDEa and PDEO of a single base polymorphism in the X-linked Rps4 gene allowed us to subunits bound to two inhibitory PDEy subunits. To examine the in vivo function of analyze the pattern of X-inactivation in individual F1 hybrid neurons. the inhibitory y subunits, we used a gene targeting approach to disrupt the PDE'ygene We also assayed individual cells for allele-specific expression of several (Pdeg) in the mouse germline. This mutation led to a rapid retinal degeneration that autosomal genes; Ncam and F3cam, for neural cell adhesion resembles various human retinal dystrophies, a group of conditions encompassing age coding related macular degeneration, diseases which are incurable and currently affecting up molecules, and the imprinted gene, Snrpn. The autosomal genes to six million Americans. showed the expected pattern of expression (bi-allelic expression for Ncam and and for but the The mutant mice revealed several novel functions of the PDEygene. First, the gene F3cam, paternal expression Snrpn), product is necessary for the integrity of the photoreceptors. Second, it has an expression pattern of the X-linked Rps4 gene was highly variable, with unanticipated role in promoting the expression of PDE catalytic activity is vivo. cells of each female pup expressing predominantly one or the other of Previous in vitro the two alleles. studies indicate that the PDEy subunit only inhibits PDE activity. These results suggest that there are only a small Surprisingly, this targeted mutation reduced rather than increased PDE activity in number of progenitor cells of these neurons at the time of X- homozygous mutant mice. These mutant mice demonstrate how in vivo studies can inactivation. In addition, the striking differences in allele-specific reveal unexpected roles for components of a signal transduction cascade. Retinal expression between hippocampi of genetically identical F1 siblings degeneration in these animals without the inhibitory PDEysubunit is apparently due to suggest a possible epigenetic basis for the incomplete penetrance high levels of cGMP. observed for some inherited neurological disorders. Morphological analyses suggest that Pdegtm)/Pdegtml mutant photoreceptors display features of apoptotic cell death. To modify the rate of this degeneration, the anti-apoptotic BCL2 was introduced as a transgene into the PdegrmlIPdegans mutant photoreceptors. This BCL2 transgene slowed the rate of retinal degeneration and partially restored retinal function. This research therefore created a model of central nervous system degeneration and demonstrated that retarding apoptosis is a new way to treat human retinal or neuronal degenerative diseases.

279 280 Prenatal diagnosis of a deleted Yq with a satellited Y-chromosome IYVf'l. R. S. Absence of a novel putative transporter causes osteopetrosis in the Verma. S. K. Gogineni. S. M. Klevman and R. A. Conte. Division ofGenetics. The osteosclerosis (oc) mouse. Long Island College Hospital-SUNY Health Science Center at Brooklyn. NY. D.R. Beier . H. Dushkin. H. Her. and K.P. Brady. Genetics Division. Brigham and Prior to the advent of banding techniques, a variety of aberrations involving the Y- Women's Hospital, Harvard Medical School, Boston, MA chromosome have been evaluated whose has been obscure. Such In an analysis of differential expression in a murine cystic kidney model we identified by Q-banding origin a novel gene that has homology to a family of 12-transmembrane domain proteins with unusual chromosomes which were thought to be abnormal have nosw been proven to transport functions. We utilize analysis of the segregation of 3' UTR-specific SSC be a so-called 'rare variants'. A non-tluorescent Y-chromosome with satellites on the polymorphisms in an interspecific cross as a means to map murine genes, and long arm has been noted but routine cytogenetic techniques could not determine the determined in this manner that this gene maps to proximal mouse chromosome 19, in a origin of the satellites. The latest addition of FISH-technique using a battery of region to which the osteosclerosis (oc) mutation has been previously assigned. The chromosome and loci specific probes may serve as an armamentarium in phenotype of oc/oc mutant mice includes osteopetrosis, and a variety of studies suggest characterizing such abnormalities. that osteoclasts in these mice are present but non-functional. The important role of A fetus was to have a -chromosome which was inherited transport functions in bone metabolism suggested the possibility that this transport prenatally diagnosed Yn's protein is a candidate for the oc gene. from the father. By the QFQ-technique, the Yqh was non-fluorescent and the satellites The transporter is expressed only in kidney and in cultured 1,25 dihydroxyvitamin were attached to the long arm. The terminal Yq arm stained DAiDAPI positive and D3-treated bone marrow. There is no detectable transporter present in kidneys from the satellites were positively stained by the NOR-technique suggesting that they were oc/oc mice when analyzed by Northern blots. Heterozygous oc/+ mice express reduced active. amounts of the transporter compared to wild-type sibs. Southern analysis of the A chromosome 15 classical satellite and a Yq telomere probe transporter locus reveals that it is intact and unrearranged in oc/oc mutants. It thus probe [DI5ZI] that the absence of the results in an in resealed the additional satellited material originated from 15p, but did not contain Y- appears likely transporter osteopetrosis phenotype chromosome telomere associated mice. Preliminary analysis suggests there is a defect in RNA splicing. Given the sequences. It is warranted that this case with evidence of abnormal osteoclast function in oc mice, and the expression of the satellited Yq does not dictate that other similar cases with satellited material originated transporter in cultured bone marrow, it is likely this gene is expressed in osteoclasts; this from 15p since all acrocentric chromosome have the propensity to be involved in is being tested using in situhybridization and analysis of osteoclast co-culture systems. translocations. Obviously, it is a rare variant whose nature is familial and may not In humans, autosomal recessive osteopetrosis usually presents during the first yea of have any clinical significance. life, and is almost always fatal. Similar to oc/oc, it is also considered to be an osteoclast defect, since they are present but non-functional. We have identified the human homolog of this novel transporter and are determining its possible role in human osteopetrosis.

Slide Session 52: Genomics 11 281 282 En masse isolation of functional sequences from the human Y chromosome using Human fetal cochlea expressed sequence tags: identification of possible 3' terminal exon trapping Y.-F. C. Lau, R.X. Ding and D. B. Krizman*. Univ. of positional candidate genes. '2C. C. Morton, 'N. G. Robertson, G. G. Lennon, California, San Francisco, CA and *NCHGR, NIH MD. and l-2A. B. Skvorak. 'Brigham and Women's Hospital, 2Harvard Medical School, Bethesda, Boston Livermore National As a first to a ofthe human MA, 3Lawrence Laboratory, Livermore CA. step generate transcription map Ychromosome, we Gene identification in human disorders of heritable hearing impairment are have applied the 3' terminal exon trapping technique to identify en masse functional complicated by genetic heterogeneity and assortive mating. Approximately 70% of sequences from this chromosome. Two approaches have been adopted for this study. inherited deafness is non-syndromic and genetic linkage analysis has been possible only First, total DNA derived from an entire Y chromosome cosmid library (with >13,000 in isolated populations; at least 19 non-syndromic hearing disorders have been mapped. cosmids) was digested with a trapping restriction enzyme and ligated to a trapping To facilitate gene discovery in these disorders and to study gene expression in the cssette derived from the vector, The DNA was then transfected human auditory system, we contributed our human fetal cochlear cDNA library to the pTAG4. Ugated IMAGE Consortium for of ESTs. To to cells and the were detected with 3' generation date, approximately 3500 individual transiently COS7 resulting transcripts RACE and cochlear clones have been 17% of the cDNAs to for the 3' exon sequenced. Approximately correspond primers specific terminal trapping system. Second, total YDNA from known genes, including mitochondrial proteins. Mutations in some genes identified are the initial cosmid library (in Lawrist 16) was transferred en masse to another cosmid pathogenic in syndromes that have hearing loss as a component e.g. COLLA2 in trapping vector, pTAG5, before the exon trapping procedure. Analysis of the resulting osteogenesis imperfecta. 3' RACE products by PCR using specific primers of 9 Y genes indicated that all were Approximately 50% of the cochlear clones match only to ESTs from other tissue specific present in the final exon products which were subsequently subcloned into pAMNI cDNA libraries, while over 800 clones, more than 25% of the cDNAs analyzed, represent and into 51 96-well micrositer dishes. indicates novel DNA sequences. To identify chromosomal locations of the cochlear ESTs for eval- plasmid arrayed Hybridization analysis uation of these as candidate genes for disorders that and 2.8% ofthese exon were transcripts positional involving hearing 8.7/o clones derived from two repeated genes, TYPY impairment, we are performing BLAST searches of the cochlear sequences for homology and RBM respectively, from this chromosome. Random sequencing of 170 individual to STSs in the GenBank database. Because STSs have been mapped, the chromosomal clones confirmed the frequencies ofboth genes. Further, over 54%ofthe clones were assignment of the matching cochlear clones can be determined. We have deduced the either homologous to sequences of genes located on this chromosome or unique position of over 40 cochlear ESTs by this method and have identified one done (H88064) sequences having characteristics of valid 3' terminal exons. Sequence analysis of 14 which may be a reasonable positional candidate for DFNA7. In addition, the Whitehead exon clones indicated that of them harbor 2-4 internal exons in addition to Institute is currently mapping over 150 of the novel cochlear cDNAs; these clones and TSPY 57%/ their will be released on their Web site the terminal exon. The results en map positions (http://www-genome.wi.mit edu). indicate that: 1) masse 3' terminal exon trapping for ESTs have been a major tool for positional cloning of disease genes. We believe these an entire chromosome is highly feasible, 2) >50%/of resulting exon clones are either cochlear ESTs, which are available to the scientific community through Research Genet- derived from known Ygenes or potential functional sequences from this chromosome, ics (Huntsville AL), will be an invaluable resource for studying the molecular biology of and 3) similar strategy should be applicable for en masse isolation of ESTs from other hearing and deafness. human chromosomes. Slide Session 52: Genomics 11 (cont.) A55 283 284 Test for presence of a modifier gene for cystic fibrosis on human A large-insert bacterial clone physical map of the imprinted chromosome 19q13. R. Rozmahel"4, J. Zielenskil, D. Markiewicz', P. Durie25 BWSCR1/tumor suppressor region in 11pi5.5 MJ Higgins', NJ Smilinichl M. Corey3-5. and L.-C. Tsuii,4. 'Depts. of Genetics, 2Gastroenterology and 3CF CD Day', LH Reid2, BE Weissman2, EJ Stanbridge3. S Sait', PR Cooper', Research. Hospital for Sick Children, Toronto, Canada. Depts. of 4nlecular and NJ Nowaki, PJ deJong', JM Gabriel4, RD Nicholls4, C Davies5, GA Evans5, Medical Genetics and 'Pediatrics, University of Toronto, Canada. Investigation of CFTR genotype and its contribution to cystic fibrosis (CF) respiratory and TB Shows'. Roswell Park Cancer Institute, Buffalo NY', Universitv of North and intestinal disease severity has demonstrated an incomplete correlation that could be Carolina, Chapel Hill2, University of California, Irvine3, Case Western Reserve attributable to environmental and secondary genetic modifiers. Since studies aimed at University, Cleveland. OH University of Texas, Southwestern Medical Center, Dallas5 uncovering genetic modifiers are not feasible in humans, CF animal models maintained in Loss of heterozygosity (LOH) analysis and chromosome fragment-mediated gene trans- controlled environments could be utilized for this investigation. Loss of CFTR function fer studies suggest the presence of one or more tumor suppressor genes in 11p15.5. in mouse models results in severe intestinal lesions including mucus accumulation leading Chromosome rearrangements in patients with Beckwith-Wiedemann syndrome (BWS), to obstructive disease. Comparable lesions consisting of mucus accumulation of diseased an overgrowth/tumor predisposition condition, disrupt the identical band of chromosome human airways and mouse intestine implicates common pathogenesis, providing support 11. This same region contains four genes (IGF2, H19, p57-KIP2, and ASCL2) that are for use of these animals in the investigations. Our previous studies in a CF mouse regulated by genomic imprinting. As part of our work to characterize human chromo- model implicated the existence of at least one genetic modifier of disease severity that some band 11pl5.5, we have constructed a contig through this region consisting of P1, was linked to markers at proximal mouse chromosome 7, a region syntenic with human PAC, and BAC clones. Established libraries were screened using STS-based PCR as chromosome 19q13. To test whether the 19q13 region contains a disease modifier in CF well as hybridization with Alu-PCR probes and whole YAC inserts. The contig extends patients, we analyzed a set of microsatellite markers in families containing two or more approximately 700 kb from D11S517 to H19 and, at present, contains one gap (estimated affected sibs with respect to concordance and discordance of respiratory and intestinal to be <100 kb) just centromeric to IGF2. The contig map is in good agreement with disease severity. Among the concordant group 22 sib pairs were found to share at least a previously-published clone map and PFGE maps of the region and also reveals sev- one marker allele and only 2 with no shared alleles, whereas among the discordant group eral potential CpG-islands. Genomic clones from the 11pl5.5 contig have been used as 13 were found to share 1 or 2 alleles while 4 shared none. Our data are also consistent probes in FISH analysis. This work confirmed the relative order of constituent clones with the presence of a genetic modifier of CF at human 19q13, however the association and mapped three BWS breakpoints and a rhabdoid tumor breakpoint in the contig. has not reached high statistical significance (p<0.05). Two P1-clones were shown to span a BWS t(11;22) translocation; two BAC clones were shown to cross a BWS inv(11); and a PAC was split by both a BWS t(11;16) and the rhabdoid tumor t(11;22). Several new transcripts have been identified by direct cDNA selection and sequencing of the large insert bacterial clones. These cDNAs are being tested for monoallelic versus biallelic expression using a newly developed somatic cell hybrid imprinting assay. CpG island clones isolated from the large insert clones are being tested for differential methylation. New transcripts isolated using this contig are candidates for BWS, tumor suppressor loci, and novel imprinted genes.

285 286 X-Linked Lissencephaly and SBH (XLIS): Mapping of a novel neuronal migration Construction of a physical map and and additional phenotyping in gene. A. K. Srivastaval, M. E. Ross2, K. M. Allen3, T. Featherstone4. J. Christopherl, G. autosomal-dominant hypertension and brachydactyly, which maps J. Gleeson2. J. H. Youne2, E. Anderman5, D. Schlessinger4. C. A. Walsh3 and W. B. to chromosome 12. S. Bahring. H. Schuster. T.F. Wienker. H. Dobvns2. IJCSRI, Greenwood Genetic Center, Greenwood SC; 2Neurology Dept., HaIller. H. Toka 0. Toka. R. Naraghi and F.C. Luft. Framz Volhard University of Minnesota, MPLS, MN; 3Neurology Dept., Beth Israel Hosp. HMS, Boston, Clinic and Max Delbruck Center, Humboldt University, Berlin, MA; 4Center for Genetics in Medicine, Washington Univ. St. Louis. MO, 5Montreal FRG. Neurological Institute, McGill Univ., Montreal, QU, Canada. We have mapped autosomal-dominant hypertension and Malformations of neuronal migration such as lissencephaly (smooth brain) are an brachydactyly to chromosome 12p. Affected persons have severe important cause of mental retardation and epilepsy. Classical lissencephaly has been hypertension (+50 mm Hg mean blood pressure) and die of stroke associated with one autosomal and one X-linked form. Mutations of the LISI gene on before age 50 years. The renin-angiotensin, and sympathetic chromosome 17pl3.3 cause isolated lissencephaly sequence or Miller-Dieker syndrome nervous systems respond normally to volume expansion and (Chong et al., this meeting). Mutations of the XLIS gene cause classical lissencephaly in contraction. Thus, the condition resembles essential hypertension. hemizygous males and a milder phenotype known as subcortical band heterotopia (SBH) We conducted magnetic resonance imaging angiography of the in females, sometimes in the same family. posterior fossa vessels in 7 affected and 6 nonaffected persons, since We have mapped the XLIS gene to chromosome Xq22.3-q23 by using a combination loops in the posterior inferior cerebellar artery are associated with of(l) linkage studies in five multiplex families, and (2) physical mapping of an X- essential hypertension. These loops may impinge on the autosomal translocation [46,XXst(X;2Xq22.3;p25.1 )de novo] in patient XLI-001, a girl venterolateral medulla and elicit a neurogenic form of hypertension. with classical lissencephaly. Linkage analysis identified an XLIS critical region between All 7 affected and no nonaffected persons had such loops. We Xq21.3 and Xq24, flanked by markers cen-DXS1002 and DXSI 001-tel with a maximum cultured fibroblasts from 15 affected and 10 nonaffected persons and two point LOD score of 3.3. Using available markers from the region, we mapped the X found by bromdesoxyuridine and formazan incorporation, as well as chromosome breakpoint to a 1-2 Mb region in Xq22.3-q23 by analysis of a somatic cell by cell counts that cells from affected subjects grow faster than from hybrid constructed from patient XLI-001 which retains the der(2). Markers DXS87, the nonaffected. This cellular phenotype will allow for phenotype DXSI 105, COL4A5 and DXS287 were placed centromeric, and DXS1072 telomeric to the cloning and gene analyses with differential display. A reported breakpoint. This was confirmed by fluorescence in situ hybridization (FISH) using deletion in chromosome 12p from a family in Japan, which features genomic probes on the metaphase chromosome from patient XL1-001. The breakpoint was the same brachydactyly, enabled us to narrow our attention to a 3 further mapped within a yeast artificial chromosome (YAC). We propose that the XLIS Mb fragment. We mapped this area to a YAC contig. We are also phenotype is caused by mutations ofa single gene located in Xq22.3-q23 and that the less examining potential candidate genes and have excluded the genes severe SBH phenotype in females is due to random X inactivation. for the L-type calcium channel as well PTHrP. Elucidation of the responsible gene may reveal a new mechanism of blood pressure elevation.

287 288 Refinement ofthe candidate region and isolation of candidate genes for Familial A transcript map of the 260kb WolfHirschhorn Syndrome critical Dysautonomia on human chromosome 9q3 1 region. Tracy J. Wrightl' Karen Denison'. Darrell Rickel.Simone Abmayri. Jason S. A. Slaugenhanlt.IA. Blum nfeld. C.B. I iebert'. J. Mull'. K. MacCorna~ck_, L CollinsI. MidiaSomer2.Theresa Yang-Feng3 and Michael R. Altherrl 4. iGenomics LeBellKO'er.X3 . Breakefield'. C. Maaya -.M. K. McCormick LS Group, Life Sciences Division,Los Alamos National Laboratory,Los Alamos.NM. Bucklr4F.B. Axelrod3. J.F. Gusel. 'Massachusetts General Hospital and Harvard 87545, 2Dept. of Medical Genetics, The Family Federation of Finland, P.O. Box 849. Medical School, Boston, MA; 2Hadassah University Hospital. Jerusalem. FIN-0Ol0l, Helsinki, Finland, 3Dept. of Genetics, Yale University, Box 3333, New Israel. Haven, CT 06510, 'Department of Biological Chemistry, University of California- 3New York University Medical School. New York, NY; Sequana, La Jolla. CA. Irvine, Irvine, CA 92717. Familial dysautonomia (FD; Riley-Day syndrome) is the best known and most frequent of Wolf-Hirschhornsyndrome (WHS) is a multiple anomaly dysmorphic malady a group ofcongenital sensory neuropathies characterized by widespread sensory and characterised by mental and developmental defects resulting from the absence of the variable autonomic dysfunction. FD is due to a recessive genetic defect with a remarkably distal segment of one chromosome 4 short arm (4p l6.3). Due to the complex and high carrier frequency in Ashkenazi Jews of I in 30. We have used genetic linkage to map variable expression of this disorder, it is thought that the WHS is a contiguous gene the defective gene, DYS, to chromosome 9q3 1 and have determined that its ethnic bias is syndrome with an undefined number of genes contributing to the phenotype. In an due to a founder effect, with 98% of FD chromosomes sharing a common haplotype. effort to identify genes that contribute to human development and whose absence The DYS candidate region originally spanned a 3 cM interval from D9S748 to D9SIO5 in results in this syndrome, we have utilised a series of landmark cosmids to characterise a chromosome 9q3 1. The identification of a new recombination event and the addition of collection of WHS patient derived cell lines. Fluorescence in situ hybridisation with these cosmids was used to refine the WHS critical region to 260 kb. This represents new markers to the haplotype has enabled us to refine the likely candidate interval to a 400 12% of the estimate for the kb the marker previously published WIHSCR. The entire genomic region surrounding D9S1677. We have constructed a detailed physical map sequence ofthis region is available and analysisof this sequence through BLAST that spans 1.1Mb and contains the mostlikely DYS interval. Using exon amplification, we detected eight independent cDNA clones in the dbEST data base. These cDNAs show have placed more than 70 exons on this physical map and have to date identified six no significant similarity to members of other DNA or protein databases. Furthermore, potential DYS candidate genes. Previously we reported on the evaluation of two of these these genes have been localised within the WHS critical region and reveal an interesting genes, which can now be excluded based on the new genetic data. Of the four DYS patternof transcriptional organisation. This work provides the starting point to how can candidates, one gene is homologous to the growing family of band 4.1 related proteins, one understand multiple genes contribute to a complex phenotype like the Wolf This was bearsamino acid similarity to a calcium channel gene, and two are unrelated to any HirschhornSyndrome. work supported by DOE Contract W-7403-ENG-36. sequences currently in the database. We are currently saturating the map with additional exonsand working to identify new cDNA clones. Details ofthe refined physical map and the analysis of potential DYS candidate genes will be presented. A56 Slide Session 52: Genomics 11 (cont.) 289 290 Transfer of the full-size DMD-gene to mammalian cells with the use of a Genotype/phenotype studies in YAC/P1 transgenic mice containing several putative mammalian artificial chromosome. J.J. Hels, A.J. Wiersma, megabases of contiguous DNA from human 21q22.2. D. J. Smith', E. de Meijer, G.J.B. van Ommen, J.T. den Dunnen. Leiden University, Dept. Human MN. E. Steyna±, N. Patil2, J.-F. Cheng' and E. M. Rubin'. 'Human Genome Center, Genetics, Leiden, The Netherlands. Lawrence Berkeley National Laboratory, Berkeley, CA 94720. 2Department of We have set out to shuttle the giant (2.4 Mb) Duchenne muscular dystrophy (DMD) Genetics, Stanford University Medical School Stanford, CA 94305. gene to mammalian cells via construction of an autonomous replicon with Nlendelian seg- Many genes on chromosome 21 contribute to the distinct phenotypic features of Down regation, i.e. a mammalian artificial chromosome (MAC). This approach would relieve syndrome and in clinical studies the 21q22.2 region has been implicated as having an most of the restrictions of current methods to study mammalian gene expression like the important role. As an approach for analyzing the genotype/phenotype relationship in limitation to cDNA and small sized constructs or the need for random integration. and a complex trait such as Down syndrome, we investigated a panel of transgenic mice would facilitate the identification of regulatory elements within the natural context of the containing YACs and P1 phage from 21q22.2. These YACs and Pls together cover ap- gene. In previous research, a 2.6 Mb YAC containing the complete DMD gene was con- proximately 2 Mb of contiguous DNA from this region. Our phenotypic analysis of the structed from a set of overlapping YACs by meiotic recombination. To obtain a putative mice has begun with behavioral traits, since defects in these processes are among the MAC we have joined this 2.6 Mb DMD-YAC to a 150 kb YAC containing pure alphoid most significant consequences of the syndrome. We utilized a battery of quantitative tests DNA (currently the best candidate for a functional centromere) by mitotic recombina- of learning and behavior for this analysis. Several of the YAC transgenic lines displayed tion on a 3 kb overlapping fragment. Human telomeric sequences and selectable markers specific but distinct patterns of abnormalities, in each case replicated by two indepen- were added, while replication origins were assumed to be present in the DMD gene. The dent lines of mice containing the same full-length YAC. Most strikingly, mice bearing two basic constructs were introduced into mouse (LA-9) and monkey cells by spheroplast YAC 152F7 displayed learning deficiencies suggestive of specific hippocampal dysfunc- fusion. Five (LA-9) clones contain the complete DMD gene with copynumbers ranging tion (memory acquistion). Analysis of mice containing fragments of 152F7, generated as from 1-7. Expression of the gene is now being studied. For all five DMD-clones, Western a consequence of the DNA manipulation, has narrowed the critical region containing the blot analysis indicates that two DMD-isoforms are expressed (Dpl4O and Dp7l). To gene(s) responsible for this behavioral abnormality from 570 kb to approximately 200 see if the muscle isoform of 427 kDa is also made from the introduced YAC DNA, as is kb. A candidate gene that maps to this region is the human homolog of the Drosophila suggested by RT-PCR data, MyoD-induced myo-differentiation is used. Some cell lines minibrain gene and it is currently being evaluated by creation of transgenic mice using a If the 150 exhibit extrachromosomal structures with variable but high copynumber. a BAC containing exclusively this gene. Mice containing another YAC, 230E8 (670 kb), kb of alphoid DNA is providing (partial) centromere function, the extrachromosomal also displayed specific learning abnormalities (defects of "perseveration") which were structures are expected to be lost at a lower rate than extrachromosomal controls with- distinct from those shown by the 152F7 transgenic mice. The gene(s) responsible for out alphoid DNA. Therefore, these lines are currently tested for mitotic stability during the YAC 230E8 defects are being finely mapped by the strategies described for YAC growth in non-selective medium. 152F7. These studies demonstrate the utility of panels of large insert transgenic mice as a substrate for linking genes to phenotype in a defined region of the genome. The approaches used here may have applicability in identifying genes contributing to other complex traits.

Slide Session 53: Services, Public Policy and Screening 291 292 Education about BRCA1 testing decreases women's interest In being tested. The use of optically scannable forms and computerized algorithms for mass family history N.A. Holtzman. B.A. Bernhardt. T. Doksum. K.A. Helzlsouer and G. Geller. cancer risk assessment. H. H4ampel, T. Kuhn. A. Markowitz, KH. Lin, S. Li, K. Brown. C. Schulz Johns Hopkins Medical Institutions, Baltimore, MD. SJ. Winawer, F. Taylor, P. Mcfiuire- A. Schiltiger B. Peshkin. K. Offit. Memorial Sloamm-Ketteriisg As a part of a study to develop a model informed consent process for BRCAI testing, Cancer Center (MSKCC), New York, NY we surveyed 587 women who had at least one Ist degree relative with early-onset breast In order to obtain hereditary cancer risk information for all patients entering a comprehensive cancer. We asked respondents to imagine they were a 35 year-old at high risk for breast cancer center, the mass-screening of family histories was initiated. An optically scannable fonm cancer who is offered testing. Before being given information about BRCA1 testing, women was developed by us and printed by National Computer Systems, Inc. (NCS). This form contains were asked to indicate their interest. Of 426 respondents (73% response rate), 82% were family history information from first, second and some third degree relatives and is called a Family initially very likely to want testing. Women were then asked how important it would be for History Questionnaire (FHIQ). The FI1Q is completed by the patient and then scanned by an NCS them to discuss each of 15 issues with their provider before deciding whether to have tle test. (TM) OPSCANB 5 optical scanner and the data is transferred and then stored through the use of The following issues were deemed most important: 1) what can be done to reduce her chance the MSKCC clinical research database (CRDB). By exporting data to the Cyrillici) pedigree of developing breast cancer (rated very important by 92%); 2) her chance of developing breast drawing software, a family tree is generated using the data gathered from the FFIQ. Standardized cancer if the test is positive (81%); 3) possible loss of insurance if she is found to carry a algorithms were then developed to identify family histories which are diagnostic of or suggestive mutation (70%); and 4) the chances that the test result would show she carries a mutation of the major cancer predisposition syndromes. In addition, published epidemiologic empiric risk (66%). Most women would prefer to be educated about testing through individual discussion estimates were used to identify individuals with a relative risk of >2.0 (based on family history with a specialist rather than with their own provider or in a group setting. The questiomuaire and/or age of onset) for developing breast, ovarian, colon, prostate, thyroid or melanoma cancers. then provided information about the risks, benefits, limitations and implications of testing. To facilitate mass-screening, these algorithms were converted into a computer program so that each after which only 48% were very likely to want testing. Factors associated with a decrease in FHQ could be quickly assessed for major cancer syndromes. The computer generates a report participate interest were greater concern about the financial cost of testing, less willingness to which accompanies the pedigree in the patient's medical record. It provides a risk assesmenit and in research on preventive drugs, and lesser likelihood of doing anything different regarding recommends genetic counseling and/or individualized screening for the canicers to which the they follow-up management if the test result were positive. Only 6% of respondents said individuals are at risk. The risk assessment program can be modified continually and can be used a 78% wouldbe likely to undergo proplsylactic mastectomy. In making decision abouttestiisg, to identify individuals eligible for research studies. To study the accuracy of the cotmmptiter of women would want their provider's reconmmendation. These data demonstrate that the algorithms, 472 FHQs were analyzed by a genetic counselor, a research assistant and a physician content of education, the likelihood that a positive test result might influence follow-up who evaluated the family histories using the same criteria as the computer. They were then behavior, and provider recommendations are likely to alter high risk women's interest in independently assessed by the computer and the results were compared. The computer made errors testing. Without education, particularly about the efficacy of follow-up options, women's on six FIiQs (1.3%), primarily due to errors in programming. The group made errors on 31 FIIQs consent for testing may not be truly or reflective of their own values. informed (6.6%). Tlme main limitations of the computer assessment involvedtie design features of the FI-IQ. Modification itt programming will address many of these limitations. The use of a computerized prograin to assist the preliminary evaluation of cancer family history data will be an important resource for both academic and managed health care environments. 293 294 AN evidence-based BRCA1 testing guideline. R.P. Bachman', H.N. Bass2, Population-based Genetic Testing for the 185delAG BRCAI Mutation in J. Bergoffen', D.L. Broome2, E.L. Harris, M. Jarnehdor2, K.M. Johnston',J.A. the Ashkenazim. C.S. Richards. P.A. Ward. B.B. Roa. A. Boyd. P. Gunaratne. G. Reiss3, D.K.C.YiM4, Kaiser Permanente, Northern California', Southern California2, Kuenzli. L.C. Friedman. S.E. Plon. Baylor College of Medicine, Houston, Texas. Northwest3, and Hawaii4. A longitudinal five year research study was initiated to assess medical and Kaiser Permanente (KP), the nation's oldest and largest not-for-profit health psychological impact of genetic testing for 185delAG in the Ashkenazim. Protocol maintenance organization, is developing an evidence-based clinical practice guideline design and initial findings will be described. Participation was open to men and women for testing of individuals who may be carriers of a BRCA I mutation. KP provides who were 21 years and older, with or without a personal or family history of breast or medical care for 6.9 million members in 12 regions throughout the U.S. This is an ovarian cancer, and at least 50% Ashkenazic Jewish ancestry. All participants who ongoing inter-regional, multidisciplinary effort that will be completed soon. The chose testing received results. Group enrollment sessions conducted at a local Jewish guideline-development process involved establishing several workgroups that reviewed community center consisted of pre- and post-education assessment of cancer genetic the literature, gathered and analyzed the various pieces of evidence and then synistesized knowledge, educational presentation, informed consent, medical and psychological the evidence following a specific model and methodology of David Eddy, MD, PhD. questionnaires, and specimen collection. Of the 333 individuals who attended a session This guideline will be released in the summer of 1996 and will identify women who 307 (92%) participated and of these 88% were female and 12% male. TheMajority should be offered genetic counseling and testing. Key elements of the guideline arethe (60%) of participants were between 40 and 59 years, and 95% were college graduates. requirements of genetic counseling and informed consent prior to testing. Participants were categorized as having negative history (67%), positive family history Probability estimates were established for the prevalence of a germliine BRCAI defined as one first degree or two second degree relatives with breast (premenopausal) or mutation, the sensitivity and specificity of the BRCA1 test,t1e penetrance of the ovarian cancer (22%), positive personal history (8%), and both positive personal and BRCAI gene and risk of death from breast and ovarian cancer for the following family history (3%). Pre-education average test score was approximately 58%, while groups: those women with no personal or family history of breast or ovarian cancer, post-education scores improved by approximately 20%. Of the 307 participants, 18 women with breast cancer developing after age 40, women in whom breast cancer individuals (6%) declined genetic testing. The most common reason for their decision occurred prior to age 40, multi-generational families with breast and/or ovarian cancer was concern about losing or obtaining health insurance. Of 2 I the 289 individuals (94%) in 3 relatives, and family members with a known BRCA mutation. We have also who opted for testing, the most common reasons for their decision included: (1) concern estimated the impact on morbidity and mortality of various interventions such as for their own risk; (2) concern for the risk of their children; and (3) desire to learn more prophylactic mastectomy, oophorectomy, and early and frequent breast and ovarian about surveillance options. Six participants were found to be lieterozygous for the cancer on women groups. not screening in each of these risk We have addressed 185delAG mutation, including two women and two men with family histories of breast chemoprophylaxis with tamoxifen in this model at this time. or ovarian cancer, one woman with a personal history of breast cancer, and one woman Women identified as most likely to benefit from genetic counseling and BRCA with a negative history. These individuals will receive genetic and psychological were those a history of breast cancer < age 40, a family history analysis2 with personal counseling as well as discussion of surveillance and therapeutic options. The 59 of 3 relatives having breast and/or ovarian cancer and those with known BRCA I mutation-negative participants with positive family histories are I being offered185deIAG mutations in their family. Asymptomatic women found to carry a BRCA mutation testing for an affected relative to clarify their risk. Follow-up of all participants will be will be offered follow-up and long-term monitoring through a multi-disciplinary conducted using questionnaires mailed at intervals over five years to assessmedical and approach. psychological impact of genetic testing. Slide Session 53: Services, Public Policy and Screening (cont.) A57 295 296 An Epidemnologlc Evaluation of Newborn Screening for Cystic Fibrosiso A Scientific Challenge Telemedicine for of clinical services- at for Public Health Action. J. Cono and M. J. Kihourv. Centers for Disease Control and Prevention, delivery genetics Experience the BlirthIt)ixtcs and (;nentic Diseases Branch, Atlanta, GA. Medical College of Georgia. DB Flannery', HB Radtke', CR Barefield2. Since 1989 there has been renewed interest in population-based newborn screening for cystic tDepartment of Itediatrics, Section of Genetics, Medical College of Georgia, Augusta. fibrosis in the United States. While the states of Wisconsin and Colorado and some countries have 2Children's Medical Services, Waycross, GA. screened neonates for CF since the mid 1980s, others have been waiting to see whether results of 'Telemedicine (TM) is a network of computers, high-resolution cameras and high- controlled clinical trials demonstrate clinical benefits from newborn screening In our evaluation, we capacity telephone lines which enables lotg-distance patient-physician audiovisual comts- applied epidemiologic principles to observational studies and to controlled clinical trials. Although muitications. TM has been shown to be an effective way to provide multiple titedical sonie observationial studies have suggested clinical benefits from early diagnoses of CF, results are specialty services to rural communities. Georgia has established one of the most exten- difticult to initerpret because of the potenitial for confounding bias (the apparent benefits miay 1: sive TM networks in the world. At the Medical College of Georgia (MCG), TM has been iclaled 1iicolacsto other than newborn screenting), selectioni bias (tle scrienes group aid the used by several specialties including pathology, psychiatry, neurology, radiology and der- utastreened group may not adequately represent their respective underlying populations), aid lead- matology. Since 1984, we have provided genetic services to Waycross, GA, a town 190 tisie bias (the screened group may appear to have a higher survival rate because earlier diagnosis miles south of MCG, in a bimonthly outreach clinic. Because of increasing demand for increases the interval between diagnosis and death). We also assessed die likelihood thae a controlled our service, the travel and the of TM in December trial can assess the benefit of detection a time, availability resources, 1995, we clinical early within reasonable time aid performed power an additional clinic via TM for consultations. calculations to determined the number of needed in sch a began monthly, half-day follow-up As with subjects study. Expected rates ofvarious all TM sessions, the consultations were and retained as of the and outcomes were derived the 1994 Fibrosis Patient videotaped part patient's mortality morbidity from Cystic Foundaiosn medical record. Based ott review of the use Antual D)ala For to show a 50% reduction in CF deaths afler 5 literature, this is the first of TMI for regularly Registry Report. example, years (fir scheduled genetics services. Attendance at TM clinics was to the attendance which the mortality rate is 2.2%), researchers using a =0.05 (two sided test) and P = 0.20 would iced compared to have CF at our regular genetics outreach clnic and to on-campus clinics. Patient compliance was 2105 inewborns with il solth slicsciectiL antiiiuscrcenoil gotip. ITo show a 5(l)/a teduction not in CF deaths alter 10 years (mortality rate 4.4%), researchers would need 1034 cluildien with Cl- ill statistically different. The attendance rate (attended/scheduled) for six TMI clinics each group. On the other hand, to show a 5(0/o Iedtuction in the rates of some of the nsorc conunon was 77% compared to 68% in the outreach clinic during the last two years, and 77% in morbidity endpoints, (for which the rate is 20%e or more depending upon the condition), iescaichcis on-campus clinics. Satisfaction surveys completed by patients seen by TM and seen at witild nee '95 to 21111 ocwtsoras with ClF il euch gitil) lii a Ityls)ilieticul regioitialstidy ofa MCG were compared. In both cases, patients were either 'very satisfied" or "satisfied" population of whites with a CF incidence rate of I in 34011 and 00,00 bahis annually, it would take with their genetics appointment. Clinical productivity was also examined. We were able more thant 50 years of citrollment to detect a 50% reduction in modiality 5 years after the childteir's to see 1.2 follow-up patients per hour via TM compared to 0.47 follow-up patients paer births. Thus unless drasmatic effects on morbidity and nsortaliiy are observed, a regional clinical trial hour for the outreach clinic when travel time is included. Physician satisfaction with may not be able to assess the clinical utility of newborn screening for CF within a ieasonsable period TM is high also. The video image quality is superb, as we can demonstrate in our pre- oftime. Ifpublic health agencies wait for the results ofsitall clinical trials, sewborn screciting liir Cl sentation. TM appears to be an efficient and effective modality for follow-up clinical may sever move forward even if the screening otitcoties show clinical benefits. he niethssloligic genetics outreach services, although larger numbers may demonstrate small statistical problems with observational epidemiologic studies and the limited saniple sizes available ill regional differences. We will next assess the utility of TM for initial patient consultations clinical trials create a dilemma for public health officials attempting to fornitilaie policy recomnimidations concerning newborn screening for cystic fibrosis.

297 298 Primary care physicians' utilization of and perceptions about genetics services. Genetic counseling for hemophilia: testing of children and adolescents. Connic ai SJ. Hayflick1. M.P. Eiffl, L. Carlen=2, and J. Steinb lrer . IOregon Health Miller and Alison N. Park-, Children's Memorial Hospital and Northwestern University Sciences University, Portland, 2University of Washington, Seattle. Medical School, Chicago, Illinois. In health care care managed plans, primary physicians (PCPs) must authorize a Carriers for the X-linked have a wide of referral in order for a patient to receive specialty services, including genetics hemophilias range factor Vil or IX levels, services. To determine utilization and perceptions about genetics services, we presumably due to X-chromosome inactivation, with a mean factor level of 50 u/dl (°'of surveyed PCPs, including family practitioners, internists, obstetricians, and normal). Since normal ranges usually end at 40-60 u/dl, close to 50% ofcarrierswill have pediatricians in the Pacific Northwest (Alaska, Idaho, Oregon, and Washington). a level below normal, revealing carrier status. 28% ofcarriers fall below the hemostatic Data was gathered on practice demographics, education and training, current referral range and may be symptomatic. Symptomatic carriers are usually mildly affected but may patterns for genetics services, knowledge of appropriate referrals for selected genetic treatment in the manner as and about services. Of 4824 PCPs require same hemophilia patients. Medical benefit may disorders, perceptions genetics surveyed, 1642 therefore accrue from measurement offactor level in children at risk. Medical (34%) responded. Demographics of respondents were representative of PCPs in the directors Northwest, where more than half of family physicians practice obstetrics. Over 90% of 132 U.S. hemophilia treatment centers were surveyed by mailed questionnaire regarding of family physicians, obstetricians and pediatricians report that genetics consultation their views on testing, with response from 60 (45%) who care for 8,035 hemophilia is available to them, whereas only 60% of internists do. One in four internists did patients. Directors, usually hematologists, were asked to review two quotes from"Points not know if genetics consultation is available to them, yet only one in ten had to Consider: Ethical, Legal, and Psychosocial Implications ofGenetic Testing illChildren additional genetics service needs, suggesting that 15% of internists surveyed know and Adolescents"(AmJHum Genet benefit to the child of no available services still no need. Consistent 78% 57:1233, 1995) regarding timely yet perceive with this, of as the for and deferral of if internists had obtained no genetics consultation in the past year, compared with 45% primary justification genetic testing testing benefits will sot of family physicians, 9% of pediatricians and 5% of obstetricians. For all accrue until adulthood. 53% agreed with these points and 32% disagreed; 15%°'ogave no practitioners, the greatest factor in prompting a genetics referral was the opinion. When asked "does knowledge of the factor VIII or IX level in a female child patient's/family's interest in the referral. The most common reason not to obtain a constitute a medical benefit which outweighs the possible negative impact on the family genetics consultation was no perceived benefit for the patient. Managed care plan if carrier status is found," 78% answered "yes," many noting that discussion of limitations were barriers in 30% of non-prenatal cases and in 17% of prenatal cases. consultation is often reproductive risks and options can be deferred to a later date in conjunction with DNA Though genetics sought for individuals with dysmorphic In recommend features or a family history of a "genetic condition", it is rarely sought for a family testing. practice, 67% carrier testing in childhood or adolescence, many history of cancer, deafness, polycystic kidney disease or congenital heart disease. noting that child-bearing age is often under 18. 32% indicated that testing would be Services provided by geneticists are perceived by PCPs to be of limited benefit performed whenever parents wished. Only 7% considered testing before age 18 to be to patients and are probably never offered to many patients who might well benefit "unnecessary." Factor level is no longer the definitive test for hemophilia carrier status, them. PCPs education about the contributions of services to from require genetics due to its high false negative rate. When factor levels are used to identify children at risk the evaluation and management of an increasing number of patients with a wide of disease. for bleeding, appropriate counseling should be provided to families on the meaning and use range ofthis information.

299 Can terathanasia explain the protective effect of periconceetual multivitamis upon neural tube and other birth defects? E. B. Hook.1' A. Czeizel3, _S.l. Samuel 1 School of Public Health, University of California, Berkeley 2Dcpt. of Pediatrics, University of California, San Francisco. 3Dept. of Human Genetics and Teratology, National Institute of Hygiene, Budapest, Hungary. Maternal dietary folic acid supplementation prior to and during early conception is associated with a lower rate of neural tube and other defects in livebirths. The risk ratio was about 0.5 in the randomized large scale Hungarian occurrence study. The mechanism for this effect is unknown. Available data from both the Hungarian occurrence study and the smaller U.K. MRC recurrence study indicate folate supplementation is, unexpectedly, also associated with a higher overall rate of spontaneous abortion. Risk ratios of embryonic and early fetal death of about 1.15 were observed in both investigations. While little attention has focused upon the latter association, it suggests that folic acid-induced selective loss of defective conceptuses (terathanasia) rather than actual inhibition of forusation of defects may account, at least in part, for the observed diminished frequency of malformation in livebirths. We reviewed and modeled available data from this perspective. We found a relatively modest increase of defects among (unexamined) spontaneous abortions (of the order of two fold) easily capable of accounting for the entire observed preventive effect upon defect in livebirths. These considerations do not detract from the likely major public health impact of folate supplementation upon malformation prevalence. But they indicate that the mechanism may well be asi unexpected and novel one worthy of serious consideration and investigation. A58 Slide Session 54: Linkage Mapping and Polymorphisms Ill: Novel Approaches 300 301 Identification of a hot-spot for microdeletions In patients with X-linked Combinations of STRPs from a New Genome Screening Set for deafness type 3 (DFN3) 900 kb proximal to the DFN3 gene POU3F4. Simultaneous Amplification and Analysis. D. A. Vaske', Y. Bo', Y.J.M. de Kok': E.R. Vossenaar1: C.W.R.J. Cremers2: N. Dahl3: J. Laworte4: C. A. Blanchette', D. David', J. Beck2, V. C. Sheffield2, and J. L. Weber'. Center for Medical Genetics, Marshfield Medical Research Foundation, Marshfield, WI. L Hu4: N. Fischel-Ghodsian5: S. Malcolmo: J.T. den Dunnen': H-H. Roners': 21)epartment of Pediatrics, Division of Medical Genetics, University of Iowa, Iowa City, F.P.M. Cremers". Depts. of 'Human Genetics and 2Otorhinolaryngo-logy, IA. University Hospital Nijmegen, Nijmegen, the Netherlands; 3Dept. of Clinical Linkage mapping of genes which influence genetically complex disorders will require Genetics and Pediatrics, University Hospital, Uppsala, Sweden; 4D6pt. de analysis of many fold more DNA samples than genes responsible for Mendelian disorders. G6n6tique Humaine, Strasbourg, France; 5Dept. of Pediatrics, Cedars-Sinai To facilitate the genotyping process for these projects, we have developed a new screen- Medical Center, Los Angeles, USA; 6institute of Child Health, London, U.K.; ing set of high quality short tandem repeat polymorphisms (STRPs) that covers all 24 7Dept. of Human Genetics, Silvius Laboratorium, Leiden, the Netherlands. chromosomes and is amenable to automation. Marshfield screening set 7 is comprised of 391 markers with an average heterozygosity of 0.76 and an average sex-equal spacing X-linked deafness type 3 (DFN3) is the most frequent cause of X- between markers of 10 cM. Tri and tetranucleotide STRPs developed by the Cooperative linked hearing impairment. Computerized tomography studies demonstrated Human Linkage Center comprise 78% of the markers within the screening set. 75% of specific structural defects in the temporal bone of DFN3 patients. Through a the markers in screening set 7 are also in screening set 6. Changes were made to replace positional candidate gene cloning approach, we recently identified a causal problematic STRPs in screening set 6 with informative tri or tetranucleotide STRPs gene, POU3F4, encoding a POU-domain containing transcription factor, and to improve spacing between adjacent markers. Multiplex PCR combinations have which contains small mutations in 7 patients with classical DFN3. been determined for screening set 7 that will allow simultaneous amplification of up to 6 Molecular analysis of two sporadic DFN3 cases yielded two novel fluorescently labeled STRPs in a single small volume reaction. Each PCR combination in the POU homeodomain of POU3F4. In one of these cases, has been put together on the basis of size: marker allele ranges were selected to avoid pointmutations overlap between adjacent markers within the PCR combination, and all markers within a mosaicism of mutated and wildtype POU3F4 sequences was observed in a given PCR combination are amplified with the same fluorescent dye attached to the EBV-immortalized B-cells and in peripheral blood cells. forward primer. All 391 markers can currently be amplified in approximately 100 PCR Four of 5 DFN3 associated deletions did not encompass the POU3F4 combinations. With gel multiplexing of differentially labeled pcr combinations, a com- gene. Two of these are situated more than 400 kb proximal to the POU3F4 plete genome scan can be completed on less than 40 gels. Screening set 7 and the PCR gene. To investigate this proximal region in more detail, we extended a combinations will be available on our Web site at http://genetics.mfldclin.edu. previously constructed 850-kb cosmid contig to 1 500-kb. Employing cosmids from the proximal part of this contig, we identified 6 novel microdeletions which overlap 900 kb proximal to POU3F4. These data suggest the presence of another DFN3 gene or of sequences involved in transcriptional regulation of POU3F4 at a 900-kb distance.

302 303 Solid-phase primer-extension technology for robust and rapid genotyping of Application of Genomic Mismatch Scanning to Mammalian genomes. cytochrome P450 polymorphisms. J.E. Reynolds', L.A. Vrolijk', J. Baisch', L McAllister, L Penland, J DeRisi, P 0 Brown. Depts Biochemistry,Cardiovascular and M.T. Boyce-Jacinol. Molecular Tool, Inc., a GeneScreen Company, Baltimore, Med,IIHMI.Stanford UStanford,CA. MD'; GeneScreen, Inc., Dallas, TX2. Genomic Mismatch Scanning(GMS), a biochesiscmaml technique for map- ping the genes Genetic polymorphisms in the human cytochrome P450 gene CYP2D6 have been im- shared by affected relative pairs, will greatly facilitate the genetic analysis of complex plicated as the etiological basis for the adverse nietabolism of greater than 30 widely traits. We show that the GMS procedure, first developed in a yeast model system, can prescribed drugs. This "poor metabolizer" phenotype is common in the Caucasian pop- be effectively applied to mammalian genomes. ulation (7-10%) and has also been suggested to be involved in increased cancer suscep- GMS employs the E.coli DNA mismatch repair system (Mut HLS) and the unique tibility. These polymorphisms are currently tested using PCR/RFLP and ASO tech- sequence polymorphism in complex genomes to isolate the regions of identity by nologies with limited success: the results sometimes being ambiguous, and the technolo- descent (IBD) shared by two related individuals. The gene(s) determining traits shared gies employed cumbersome and thus, not amenable to large scale population screening. by the two relatives will tend to be included in the regions of IBD. The trait-related We demonstrate that our solid-phase primer-extension technology, Genetic Bit Anal- loci can then be mapped by comparing the regions of IBD for several affected pairs. ysis (GBA) is ideally suited to standardize testing of these polymorphisms. In short, GMS experiments were performed on DNA from members of a simple mouse pedigree GBA involves hybridization-capture of a single-stranded PCR product to a sequence- generated by crossing two divergent homozygous strains. Using PCR to analyze specific microplate-bound primer, followed by enzyme-mediated single-base extension of polymorphic repeats segregating in the cross, enrichment of the IBD alleles through the the capture-primer across the polymorphic site. Direct determination of the base compo- GMS procedure is shown. The selection of mouse IBD sequences was quantitatively sition of the mutation is achieved through simple colorimetry and results are statistically comparable to that obtained for yeast sequences. presented. In a pilot study, we have developed a GBA typing capability for 3 common GMS was applied to a CEPH pedigree. Enrichmtnt of the IBD fraction was CYP2D6 mutations: A (Del A2637), B (G1934A), and T (Del T1795). Preliminary data demonstrated by following microsatellite repeats. To map the human GMS products a has shown a 100% genotyping success rate when compared to existing technologies with DNA micro-array comprised of genomic inter-alu fragments is under development. a substantially improved turn-around time (< 1 day) and sample capacity of 10 samples Preliminary results indicate that the inter-alu array provides a convenient and accurate per microtiter plate. Parallel testing information on 70 patient samples directly compar- mapping tool. Using GMS and the micro-array the regions of IBD shared by two ing GBA typing data with currently employed technologies for these three mutations will relatives can be mapped in a single hybridization step. be presented. Preliminary data on two additional CYP2D6 polymorphisms (G4268C and C188T) which are currently tinder development will also be presented. We will show that because of its technical simplicity, automatability, and robust biochemistry, GBA-based polymorphism testing of P450 miutations will enable larger scale clinical trials which are required to understand the therapeutic and pathological implications of polymorphic drug oxidation.

304 305 Genome sharing among relatives: theoretical considerations and application Linkage disequilibrium mapping of quantitative trait loci in isolated or inbred to the mapping of genes for complex traits. A. Lynn, C. Kashuk, populations using threshold defined cases and controls. S. Nath. P. St-Jean. B Thiel E. G. Puffenberger, A. Chakravarti. Case Western Reserve University, Cleveland, OH J. Reserve Any pair of relatives shares some proportion of their genomes identical by descent. X. Xu, A, Chakrsvarti. N. Schork. Case Westem University, Cleveland, Ohio; The size of the shared proportion depends on their genetic relationship and declines The Jackson Laboratory, Bar Harbor, Maine; Harvard University, Boston, Massachusetts. with decreasing relationship. For any specific locus, the probability that a given pair, Linkage disequilibrium (LD) mapping has been applied to many simple monogenic, or a collection, of relatives share a segment of the genome containing that locus is overtly Mendelian, human traits with great success. However, extensions and small. Ilowever, when these relatives are selected because they are carriers of a specific applications of LD mapping approaches to more complex quantitative traits have not mutation (allele) at that locus the sharing probability increases dramatically. We have of the of been straightforward or pursued in a serious way. We describe a strategy for mapping developed a theoretical framework that allows the computation distribmition trait variation in isolated or inbred populations that involves the length of a shared genomic segment surrounding a specific locus. This calculation loci influencing quantitative assumes that all mutation carriers have inherited the mutation identical-by-descent sampling individuals from opposite ends ofthe distribution ofthe trait under scrutiny. from a common ancestor. We have examined the distribution of shared genomic The purpose of this strategy is to enrich a sample with individuals likely to harbor/not segments for various relationships between affecteds, for chromosomes which have been harbor relevant chromosomal segments associated with an ancestral chromosome which sampled either randomly or because of the presence of a specific mutation. introduced a trait-influencing allele into the population from which the sample was The theoretical results have been used to derive the extent of genome sharing in a set We derive statistical models for detecting linkages and association hat of relatives and shows that genetic marker analysis can be used to detect and define obtained. simple these segments. The marker resolution required to detect shared segments depends on make use of this sampling scheme and explore these models under a variety of the population history from which the individuals are chosen; clearly, isolated conditions, such as the age of the population being studied. In addition, we discuss a populations are the most efficient for detecting genomic segment sharing. An number of issues which bear on the properties and utility of linkage disequilibrium in important consideration for complex trait mapping is the level of heterogeneity present isolated populations, such as the "multiple entry" problem (i.e., more than one founder among affected individuals. Simulation studies have been utilized to determine the a the of the size and marker characteristics, on introducing relevant allele into the population), importance evolutionary effects of heterogeneity, genetic relationship, sample that to the ability to detect shared genomic segments using multipoint disequilibrium mapping history of an ancestral mutation, and the behavior ofthe multiple loci are likely methods. Initial studies have been concentrated on young populations of 12 generations influence a complex quantitative phenotype. Models that investigate and accommodate deep, genotyped for two-allele markers at a resolution of 5 cM, for various sample sizes these phenomena are described. Analytic and simulation-based power studies, as well as and levels of heterogeneity. Our results suggest that disease loci may be mapped by the some practical examples involving real data, suggest that LD mapping ofquantitative detection of significant genomic sharing at marker loci studied throughout the genome traits is entirely possible with the proposed strategy in certain settings. in a collection of affected individuals, even in the presence of heterogeneity. Slide Session 54: Linkage Mapping and Polymorphisms Ill: Novel Approaches (cont.) A59 306 307 l he lRelative Power of Genome Screens by Linkage versus Association A mouse model for Angelman syndrome. B.M. Cattanoch'. J. Barr'. C.V. CA. Beechev'. J. Bressler2. Sutcliffe'. A.L. Bcaudet'. J. Martin JL. and Atialysis for Complex Diseases. N. Risch. Stanford Univ., Stanford, JiS. Nwebels''Department power of linkage versus association analysis to J. Jones'. 'MRC Mammalian Genetics Unit, Harwell, Oxfordshire, UK, Vo assess the relative of Molecular and Human Genetics and 'Neurology, Baylor College of Medicine, 'ialp genies for complex diseases, we employ the parameter y, the Hotiston, TX, and 'Department of Morbid Anatomy, The London Hospital Medical risk ratio for disease (AA versus Aa and Aa versus aa, where College, London. :semil!vic locus A). Using an Prader-Willi syndrome (PWS) and Angelman syndrome (AS) are distinct A is the high risk and a the low risk allele at a disease imprinting, and seen in typed parents, we calculate the sample size neurological disorders exhibiting opposite patterns of genomic atfftead sib pair design with patients with deletions and uniparental disomy in chromosome 15q 1 I-q 13. Mouse n-cessary, with 80%/6 power, to detect locus A by linkage analysis, using a genetic studies using the ls(7;X)Ct translocation have demonstrated that animals with a that is completely polymorphic (PIC=1). For maternal duplication for a central region of mouse Chr 7 syntenic to the PWS/AS region tightly linked marker (0=0) to we calculate the number of families of the same design show an imprinting effect, fail to express the imprinted Stirpn gene and are concluded consparison, represent a mouse model for PWS. A second imprinting effect observed with paternal necessary to detect locus A by association analysis using the transmission had to be of duplication for a larger region of Chr 7 studied with another translocation /disequilibrium test (TDT). For linkage, we use a lod score threshold excluded as a mouse model for AS when it appeared that the critical region was not 3, corresponding to a significance level a of 104 and a genome-wide false involved. of the translocation rate of < 5% for 500 markers. For a genome-wide association We now present evidence that the relative positions positive breakpoints and genes were incorrect. Detection of maternal-specific methylation at the study, we employ a conservative significance level a of 5x10-8, which Stirpit CpG island, similar to observations in the human, in addition to expression gives a similar false positive rate for 1,000,000 tests. For linkage analysis, studies with Stirpri, D034 and ZnfJ27 have established that all three imprinted genes as not distal as previously of families is reasonable (<1,000) only for y values well as the p locus lie proximal to the T(7;15)9H breakpoint, the required number believed. This relocates the entire interval homologous to the PWS and AS deletion of 4 or greater. By association analysis, y values as low as 1.5 are detect- interval within the cytogenetic region between the IsCt and T9H breakpoints, and able with a comparable number of families (<1,000). Thus, genes of suggests that the second imprinting effect could represent a mouse model for AS. The the paternal duplication mice exhibit a reduced postnatal growth from 7 days of age until small effect (y<4) are not detectable by linkage analysis because weaning and ire liablc to (die spontaneously. Developissent of their locomoter skills required number of families is untenable. On the other hand, the appears retarded from about 10 to 16 days, but they subsequently become hyperactive. current limitations of association analysis are not statistical but techno- At later adult stages the hyperactivity becomes less obvious, and gross obesity logical and thus potentially solvable -- the small number of genes yet develops. Brain size and cortical thickness are reduced in affected mice compared with identified and the problem of large-scale genotyping. These results may nornmal sibs. EEG recordiings revealed striking abnormalities, featuring prolonged 2-3 some recent linkage studies of lIz spike and wave discharges and giant slow waves consistent with the aberrant also explain inconsistent findings from human AS patients. complex diseases. rhythms and seizure patterns described in

308 309 Mapping genes that control Thl vs Th2 responses to fleishmania major. Results from an international collaboration to map the genes responsible for high A. M. Beebe', N. J. Schork2, S. Mauze', R. L. Coffmanl. IDNAX Research Institute blood pressure in the rat. Jacob Lab". Cowley Lab'. Dzau Lab'. Grigor Lab'. of Molecular and Cellular Biology, Palo Alto, California, 2Case Western Reserve E.M. Krieger J.E. Labi'i. Ohio. Fanestil Lab. Harrap Lab'. Harris Lab4. Krieger Lab"'. University School of Medicine, Cleveland, Printz Lab'. Provoost Lab". Roman Lab'. Sassard Lab". Schork Most strains of mice are resistant to cutaneous injection of the protozoan parasite Lander Lab'"'. a Thl response with high IFN'Y production Lab"NVincent Lab". 'MGH, 'Med. Col. Wisc., 'Stanford Univ., 'Univ. Otago, 'Univ. Leishmania major. Resistant mice develop 9MIT and healing of the infection. BALB/c mice, however, generate a predominantly Th2 Cal. San Diego, 'Univ. Melbourne, 'Heart Inst., 'Univ. SaS Paulo, "'Whitehead response to infection, characterized by high I,-4 and IgE production, no DTII, and no Inst., "Erasmus Univ., "Univ. Claude Bernard, "Case Western Reserve Univ. effective control of parasite replication. Though resistance to L. major is multigenic, Using total genome scan strategy with the rat animal model, the regions of the only one locus on chromosome 11 has been previously identified by analysis of congenic, genome containing quantitative trait loci (QTLs) responsible for more than 250 recombinant inbred, and F2 mice. Our experimental approach was designed to overcome phenotypes have been identified. We (more than 100 people from 13 institutions) have the complexity of L. major resistance by successive backerossing of resistance from the GHxBN, SHRxDRY, all re- studied seven different crosses, SHRxBN, SS/JrxBN, B10.D2 strain onto the susceptible BALB/c background. The Fl progeny were strain is the hypertensive and the are mostly dominant. Resistant progeny at each SHRxWKY, FHHxACI and LHxLN*, where the first sistant indicating that resistance genes considered to be unique since generation were backcrossed again to BALB/c mice. Twenty-five resistant n4 mice were second one is the normotensive. Each phenotype was genotyped in a full genome scan with 125 markers. Four loci were present in the resistant none of the phenotyping protocols were identical. Using a targetted genotyping mice with much higher than expected frequency, though all four loci were not required strategy, we have completed scans using 165 to 336 genetic markers in 6 of the 7 We are currently working on some of the statistical issues associated crosses. The one cross* is for full resistance. clearly remaining tinderway. with this mapping strategy, however association with L. major resistance was rhumaym n Chr o moso mes(LODM for loci on chromosomes 6, 11, 15, and 16. 113 resistant n5 progeny derived 6 significant loci Tail BP(SBP) GHxBN 2 (4.05), 2 (2.93) (4.50), 18 (4.66) from the 25 resistant n4 mice were next genotyped. Analyses confirmed candidate FHHxACI 1 (4.2) also an additional locus on chromosome Tail BP (SBP) on chromosomes 6, 11, 15, and 16, and suggested MAP SHRxWKY (4.43) 10. Based on these mapping results, we have begun inbreeding and testing congenics MAP SHRaBRY 1(4.4) for the most candidates. homozygous promising MAP SHRxBN 1 (2.6), 11 (3.16) SBP LHxLN 2 (4.4) Dubey et al., Nature Genetics April,1993 Chromosomes and 2 contain QTLs which appear to be very important for determining baseline blood pressure as well as many other blood pressure phenotypes in rats. These data illustrate the power of looking at several crosses simultaneously, and may indicate the major genes responsible for blood pressure regulation in human.