Posters: Cancer Genetics A59 305 306 Monosomy X as a Recurring Sole Cytogenetic Abnormality Associated with Representational Difference Analysis of tumors for detection of DNA Myelodysplastic Syndrome. F. Abruszzese. N. Rao. J. Cruz. B. Powell, M. PettenatL losses. H. Aburatani, Y. Hippou and T. Kodama. Third Dept of Internal Med., Bowman Gray School ofMedicine of Wake Forest University, Winston-Salem, NC Univ. of Tokyo. Tokyo, Japan. Intro. by: David J. Munroe. Representational Difference Analysis(RDA) has been applied to detect the genetic Single autosomal trisomies as an acquired, primary clonal abnormality are lesions occuring in tumor cells. To define the tumor suppressor loci. wve performed rare and clinical associations of many remain uncertain. Loss of sex chromosomes RDA with using tumor DNA as driver on 15 individual cases of various tumors: 6 as the sole cytogenetic abnormality in leukemia has not been firmly established. cervical. 2 renal. 2 hepatocellular, 1 gastric, I hepatoblastoma and 3 neuroblastoma. Monosomy Y has been suggested to be age-related but is also encountered in RDA failed in 3 cases, 2 cases of cervical dysplasia, presumably with little genetic AML and MDS. In contrast, X chromosome monosomy is not recognized as a abberatioris and a case with significant contamination of normal tissues into tumor recurrent abnormality in acute leukemia or as an age-related phenomenon. specimen. In all others, difference products were produced and were verified to We identified 5 patients with loss of X as the sole cytogenetic abnormality be lost in tumor D.NA in homo- or hemizygous fashion by Southern blot. From to in the bone marrow. Four of the patients ages ranged from 60 79. One patient, the cervical carcinoma cell line CC6 eleven homozygously deleted fragments were been to a coal tar All 5 cases with various age 23, had exposed therapy. presented amplified and all were localized in 3pl2-13. CC6 deletion is overlapped with the of anemia and bone marrow 3 had 1 had ANLL-M6 with degrees dysplasia: MDS; homozygous deletion in the SCLC cell line U2020. suggesting the locus as a possible an 1 with a marrow. All 5 were evolving MDS; slightly dysplastic patients married, tumor suppressor. In the analysis of neuroblastoma, a polymorphic marker 12-2 had children and were 46,XX constitutionally. A FISH study on bone marrow showing LOH in 33% of cases has been mapped on 3p and further genomic analysis blood smears demonstrated the involvement of monosomy X in both myeloid and is underway. Thus, RDA is a valuable tool to produce DNA probes in the search lymphoid lineage suggesting a stem cell abnormality consistent with MDS. of tumor suppressor genes. The idic(X)(ql3) and other structural abnormalities of Xql3 region have been associated with MPS and appear to be inactivated. Inactivation of Xql3-> pter and loss of Xql3-> qter would be comparable to the monosomy X state. Loss of genes from the X chromosome may allow cells to become neoplastic. Clinical course, presentations, cytogenetics and FISH analysis will be presented along with a review of monosomy X in acute leukemia (14 cases) and leukemia in Turner syndrome (10 cases). Based on this small series and limited literature cases, monosomy X as a sole non-constitutional cytogenetic abnormality in bone marrow appears to be associated with myelodysplastic diseases.

307 308 Mutations of the tuberous sclerosis complex 2 (TSC2) gene in an isolated Chromosomal mapping in childhood acute lymphoblastic leukemia of a anglomyollpoma. K-S. Au.' E S. Roach2. H. Northrup. 'Dept. of Texas Medical School- candidate tumor suppressor gene in chromosome 12p12.3. A. Baccichet Houston; 2Dept of Neurology, University of Texas Southwestem Medical School, Dallas. J.-M. Leclerc and D. Sinnett. Charles Bruneau Cancer Center, Research Tuberous sclerosis complex (TSC) is a dominantly inherited disorder of hamartoma Center, H6pital Ste-Justine and Universit6 de Montraal, Canada. and hamartias affecting 1 in 10,000 In the population. Both hamartomata and hamartia Acute lymphoblastic leukemia (ALL) is the most common of the pediatric representdevelopmental anomaliescharacterized byabnormal cellortissue arrangement cancers. Cancer can be considered a genetic disease which develops through and defective function. The hamartomas seen in TSC have been reported to occur in a series of mutational events affecting proto-oncogenes and/or tumor virtually every organ system but are most commonly seen In the brain, kidneys and suppressor genes (TSG). However, studies of TSGs have failed to identify heart. Common tumors found in TSC patients are: cortical tubers and astrocytomas genes whose inactivation could explain a significant number of ALL. Using (brain), anglomyollpomas (kidney), and rhabdomyomas (heart). TSC exhibits genetic cytogenetics techniques, chromosomal rearrangements (deletions and heterogeneity with one gene mapping to chromosome 9 (TSC1) and another to translocations) involving the short arm of the chromosome 12 are frequently chromosome 16 (TSC2). The TSC2 gene has been cioned. Loss of heterozygosity for observed in hematopoetic malignancies including ALL. The involvement of a DNA markers on chromosome 16 has been observed in tumors from TSC patIents. We tumor suppressor gene has been postulated. In order to identity candidate have tested 14 tumors from TSC patients as well as an isolated angiomyolipoma. Two TSGs we used a strategy based on the detection of specific chromosomal of the tumors (angiomyolipomas) from TSC patients showed LOH with chromosome 16 deletions (deletion mapping) since frequent loss of heterozygosity at markers surroundingthe TSC2 gene. DNAfromthe isolated angiomyolipoma wastested chromosomal loci in tumor genomes may indicate the presence of a such with the TSC2 cDNA and found to have a deletion of at least three kb at the 5' end of the genes. We have screened normal and tumoral genomic DNAs isolated from gene. The patient from whom the angiomyolipoma was obtained was evaluated and no 17 ALL patients (13 pre-B and 4 T-cell) for allelic losses using 12 signs of TSC ware detected. DNA extracted from lymphocytes was obtained from the microsatellites markers covering the chromosomal interval 12pll-pl3.2. patient and family members. The large deletion observed in the tumor was not observed Deletions in chromosome 12pl2.3 were found in nine patients (7 pre-B and 2 In DNA from lymphocytes from the patient or family members. Using RT-PCR and direct T-cell) constituting the most frequent type of alterations found in childhood sequencing, mRNA from the tumor and the lymphocyte DNA of the patient were ALL. The shortest region of deletion overlap was delimited by the distal compared. An additional change was detected in the tumor that was not observed in locus D12S77 and the proximal locus D12S320, indicating the localization of the patient's lymphocyte DNA. At nucleotlde #2555 a change from T to A which a candidate tumor suppressor gene within this region. So far we have replaces a non-ionic aromatic Phe with an aromatic Tyr at codon 846 was observed. identified a contig of 6 yeast artificial chromosome clones that contain the This change makes the corresponding peptide sequence (E-F -Lto E-Yee-L) of tubenn entire region of interest. The characterization of these clones and the search simiar to a recognition motif of the EGF receptor kinase (E/D-Y-L/A/V). This finding for candidate genes are in progress. (This work is sponsored by the suggeststhat the TSC2 gene, like other genes involved in phakomatoses (NF2and VHL), Leukemia Research Fund of Canada). Is Important in formation of isolated tumors in individuals not affected with the dise. Additional isolated tumors are currently under study to confirm this speculation.

309 310 Mapping of SEN6, a gene involved In immortalization of human Detailed molecular characterization of APC gene alterations in K. esophageal cancer: Evidence for additional sechanisas in tuaorigenesis. fibroblasts, to chromosomal region 6q26-27. S. Banga, S. Kim, Bayat. L. Xi. Eskandarian. A. Hitri. B. Reid. M. Redston. A.G. Hubbard, K. Jha, P. Kraemerl and H. Ozer. Dept. of Micro. and Mol. Genes., Casson. Mount Sinai Hospital and the University of Toronto, Ontario, UMD-NJMS, Newark, NJ. lCell. and MoL Bio. Group, LANL, Los Alamos, NM Canada. Normal human diploid fibroblasts (HF) have a limited life span in contrast to cancer The molecular genetic events underlying the development and cells which often continue to divide indefinitely. At the end of their proliferative life progression of human esophageal cancer (EC) are unknown. Frequent loss of heterozygosity (LOH) of chromosome Sq, which harbors the APC tumor in cease to - a span culture, normal cells display morphological changes and divide suppressor gene, has been reported for EC. APC mutations are implicated process called cellular senescence. Expression of SV40 large T antigen in HF extends in the pathogenesis of many human solid tumors, and the majority of their finite life span and ultimately rare immortal clone(s) arises due to mutation(s) in thsse result in truncation of the APC protein. However, the regulation the cellular genome. One approach to identifying gene(s) whose inactivation leads to of APC transcript expression has not been investigated in EC, to date. immortalization is to characterize a genetically matched set of preimmortal and We conducted a detailed investigation of APC gene alterations at the transcriptional and post-transcriptional levels in a prospective series immortal SV40 transformed cell lines. Cytogenetic and Southern blot analyses of of ECs. such a matched set of cell lines have revealed genetic alterations involving the long Twenty-five patients (22 males, 3 females) who underwent total arm of chromosome 6. In addition, introduction of a normal chromosome 6 or 6q into esophagectomy were studied. Tumor histology was primary esophageal SV40 immortalized cell lines by mnicocell fusion results in senescent-like morphology adenocarcinoma (ADC), 20 patients; and squamous cell carcinoma (SCC), and growth suppression. These data indicate that the long arm of chromosome 6 5 patients. For each tumor, histologically normal esophageal mucosa from the distant resection margin was available. APC mutations were analysed encodes a gene, SEN6, whose loss of function leads to immortalization. To further by an 'in vitro synthesized protein' (IVSP) truncation assay. Genomic map the position of this gene, preimmortal and immortal cell lines were charactrized DNA was analysed for LOH for Rsal polymorphism in exon 11. Differential for the presence or absence of both polymorphic and non-polymorphic markers by allelic expression of APC was quantitated by RT-PCR and RFLP analysis polymerase chain reaction, Southern blotting and fluorescence in situ hybridization. of the same locus, and assessed by densitometry. Allelic expression These experiments revealed that (1) One of the immortal cell line (ARS) has retained levels with a difference of three-fold or more were considered significant. Results were confirmed in triplicate, somatic truncating only one copy of chromosome 6. (2) Another closely-related immortal cell line APC mutations were not identified by IVSP analysis. 13 tumors (9 ADC, (Halneo) has retained a single but different copy of chromosome 6 in addition to a 4 SCC) were heterozygous and informative for APC-Rsal polymorphism. LOH small chromosomal fragment containing a part of region 6q26-27. Moreover, the of the APC locus was observed in 2 tumors (1 ADC, 1 SCC). More chromosome 6 present in Halneo has undergone mitotic recombination with the result importantly, unequal expression of APC alleles was seen in 2 other tumors (1 ADC, 1 SCC), in comparison to allelic expression patterns of that a set of markers from region 6q26-27 shows the same alleles in single copy that matched histologically normal esophageal mucosa. APC alterations did are characteristic of AR5 cells. This observation suggests that a mutated copy of the not correlate with clinico-pathologic findings or outcome. These studies SEN6 gene was transferred to chromosome 6 present in Halneo. (3) Characterzation further implicate the APC tumor suppressor gene in esophageal of independent sets of preimmortal and immortal cell lines also exhibited loss of tumorigenesis, and suggest that the transcriptional regulation of APC in the Taken these results gene expression is yet another molecular mechanism involved in the heterozygosity region 6q26-27. together, strongly suggest of that the SEP16 gene is located in the chromosomal region 6q26-27. pathogenesis esophageal malignancy. A60 Posters: Cancer Genetics (continued) 311 312 Co-amplification of two non-syntenic regions within the same Analysis of the FACC and XPAC genes as candidate genes for the basl cell double minute chromosomes in a gastric carcinoma cell line. nevus syndrome. John W. Bare, Jingwu Xie. Anthony G. Quinn. Ervin H. L Bar-Aml, Yarinj2, S. Lavil and L. Avivi3 Epstein Jr. Department of Dermatology, San Francisco General Hospital, 1 Cell Research and Immunology Dept., George S. Wise Faculty of University of California, San Francisco. Life Science, Tel Aviv University, Israel. 2 Spectral Diagnostics Ltd., The mapping of the Basal Cell Nevus Syndrome (BCNS) to chromosome 9q22-31 Migdal Haemek, Israel. 3 Human Genet. Dept., Sackler School of and the high frequency of allelic deletion of chromosome 9q in both familial and Medicine, Tel Aviv University, Israel sporadic BCCs indicate that inactivation ofa gene in this region is a critical event in Multicolor FISH was used to follow the mechanism by which extra- the development ofthese tumors. Linkage studies and haplotype analyses have chromosomal amplified regions (double minute chromosomes; localized the gene to a region flanked by D9S196 and D9S287. Two genetic DMs) are being formed and stabilized in transformed cells. We took disorders, Fanconi anemia group C (FACC) and xeroderma pigmentosum advantage of a gastric carcinoma cell line harboring co-amplified complementation group A (XPAC), which somewhat resemble BCNS in that sequences of two non-syntenic chromosomal regions corresponding affected individuals are susceptible to the mutagenic effects of ionizing and to the Cmyc and Ksam oncogenes. Following hybridization with the ultraviolet radiation, both map to the same chromosome region as the BCNS gene. corresponding probes, the fluorescent signals were detected using a We therefore have examined these genes as possible candidate genes for the BCNS. novel spectral-imaging system, SpectraCube, that enables the SSCP analysis of the 14 inown FACC exons and the six XPAC exons and the simultaneous recording of a large number of dyes in a single XPAC promoter region failed to detect aberrant bands in DNA from leukoytes measurement, thus preventing the need for complex filter sets or from 16 unrelated BCNS patients or DNA extracted from 23 sporadic BCCs. filters' wheels. Both amplified regions appeared in almost every cell We also assayed these genes for gross chromosomal rearrangements by Southern of the tumorogenic line. The amplified regions in every cell were blot hybridization of digested DNA resolved by pulse field or continuous get organized in a large number (over 50) of DMs varying in size. electrophoresis. We found no aberrant bands when DNA from 30 unrelated BCNS Moreover, most DMs carried both amplicons while the others patients were probed with either the FACC cDNA or with the promoter region of exhibited exclusively only one. We therefore assume that the two XPAC. Hence, it is unlikely that mutations in either gene are the cause of the amplicons were formed independently and were capable of BCNS. independent segregation, but have subsequently undergone extensive fusion and recombination events to form DMs with a heterogeneous complement of both amplicons.

313 314 Mutational analysis of mismatch repair genes in Hereditary Different patterns and frequencies of p53 mutation in primary breast cancers in Non-Polyposis Colon Cancer. A. Bellacosa. M. Genuardi. F. cohorts ftom two low-risk populations: northern and southern Japan. H. Leonardi. S. Cavallo. A. Ventura. M. Anti*. M. Ponz de Leon'. and G. Bk~lazk. A. Hartmann. y. _TaMUral3. S. Saitoh".3. j.M. Cunningham'. R.M. Neri. Institutes of Medical Genetics and *Internal Medicine, Catholic McGovern'. J.J. Schroeder'. D.J. Schaid.' A. Hirata. A. Okazakd3. K. 1i3. J.S. University Medical School, Rome; and 'Departrnent of Internal Medicine, Kovach2. and S.S. Sommer'. 'Mayo Foundation, Rochester, Minnesota; 2City of University of Modena, Italy. Hope, Duarte, California; 'Collaborating Institutions in Japan. Molecular basis of hereditary non-polyposis colon cancer (HNPCC, The pattern of acquired mutations in the p53 tumor suppressor gene is potentially Lynch syndrome) relies on a defect of the post-replicative DNA mismatch useful in determining factors contributing to carcinogenesis in populations differing repair, due to germline mutations of the genes in incidence and/or morbidity from the disease. We previously reported differences hMSH2, hMLHI and, to a in mutational patterns of the p53 gene in primary breast cancers (BC) of Caucasians lesser degree, hPMSI and hPMS2. Clinical diagnosis of HNPCC is based from rural upper U.S. Midwest, African-Americans from Detroit, and women from on the so-called Amsterdam criteria. However, some families which Austria. To determine the pattern of mutation in women at low risk ofbreast cancer, segregate colorectal cancer (CRC) and other neoplasms of the HNPCC exons 2-11 and adjacent intronic sequences were analyzed in tumors of women from spectrum, although not formally fitting those criteria, do warrant a northern (Sapporo) and southern (Tokushima) Japan. 19 independent mutations were clinical suspicion of HNPCC. Furthermore, a fraction of the apparently found among 21 primary BC (81 %) from Sapporo (two tumors had a double missense sporadic CRC cases originating in young adults (<40-45 years), could be mutation), and 13 mutations among 46 BC (28.3%) from patients from Tokushima. due to de novo germline mutations of the mismatch repair genes. The frequency of p53 gene mutation in the Sapporo group is the highest reported in We have conducted a mutational analysis of hMSH2 and hMLHI on breast cancer so far. Thirteen of the 19 mutations (68.4%) in the Sapporo cohort CRC patients belonging to HNPCC families fitting the Amsterdam criteria were beterozygous, an unusually high frequency for p53 mutations in any tumor type. (n=12), "suspect" HNPCC families (n=6) and on 3 young CRC patients There were three missense mutations at codon 175, a known hotspot for alterations without family history. Screening for mutations was conducted on in the p53 gene and three missense mutations (H>R) at codon 179. These peripheral blood lymphocytes by RT-PCR followed by SSCP and differences in type and frequency of acquired mutations, including an abundance of sequencing. SSCP analysis of hMSH2 was also conducted on DNA.We base substitutions at a site seldom altered in other cohorts (codon 179) and the rare have identified a new germline mutation in exon 12 of hMSH2 in a patient occurrence of double mutations in two tumors, which are rare in breast and other cancers raise the possibility of biological differences among BC based on frequency from an HNPCC kindred. During the analysis, we have also identified two of altered p53 function and support the hypothesis that different factors contribute to apparently normal splicing variants of hMLHI. breast cancer mutagenesis in different populations.

315 316 Towards new genes and imprinted regions involved in Wiedemann-Beckwith Mutation screening and further characterisation of the NF2 tumour syndrome and associated tumors. C. Boulvin. * V. Chehensse. C. Jeanpierre. suppressor gene. D. BournI, S. Masonl, S. TekesI, L. A. Trueman1, S. A. S. Luce, C. Junien and I. Henry. INSERM U383, Paris. France. Carterl, D. G. R. Evans2, T. Strachanl. IDepartment of Human Genetics, The Wiedemann-Beckwith syndrome (WBS) is a malformation syndrome associated Molecular Genetics Unit, Ridley Building, University of Newcastle upon with predisposition to different types of tumors (WT. ADCC). The WBS locus maps to Tyne, 11p15.5 and genomic imprinting is implicated in the expression of the syndrome. 25% Newcastle upon Tyne, NEI 7RU, United Kingdom. 2Department of Medical of the sporadic WBS studied displayed uniparental disomy (UPD) corresponding to a Genetics, St. Mary's Hospital, Manchester M13 9JH, United Kingdom. paternal isodisomy for region lIp15. Partial isodisomy for lip and somatic mosaicism for Type 2 neurofibromatosis (NF2) is a dominantly inherited disorder UPD were observed in patients presenting isodisomy, explaining possibly the incomplete characterised by a predisposition to multiple tumours of the central nervous forms of WBS and the association of hemihypertrophy. Moreover, the risk of developing system. We have screened for germline mutations in the NF2 tumour a tumor seems higher for patients with paternal lp UPD than for WBS patients in suppressor gene in a range of clinical general. Two genomic libraries specific for region 11pl5.5 were used to isolate the gene(s) patients with phenotypes, using mainly responsible for WBS and/or tumor progression: 1) 25 microdissection clones with 11 SSCP and heteroduplex analysis of PCR-amplified genomic DNA. DNA clones evolutionarily conserved: 2) 4 clones for a genomic EMBL3 library established sequence analysis of cloned amplification products has revealed mutations in with hybrid J1.1 (only region 11pi5) containing HTF islands or evolutionarily conserved over thirty unrelated individuals. In addition, Southern blot analysis has been sequences. The screening of a cosmid library with the whole microclone library or with used to detect large deletions in several families. The results of linkage analysis the evolutionarily conserved microclones allowed us to isolate 141 and 39 cosmid clones in families with atypical presentation have generally been consistent with the respectively, some of which are elements of already defined contigs. About 60% of these cosmids map to region 1 lp 15. The 39 cosmids are used to isolate expressed sequences with NF2 gene also being involved in these cases. magnetic beads technique. Characterization and localization of the 21 cDNAs isolated Mutation screening has improved the prospects for presymptomatic diagnosis are in progress. Five CA repeats genetically mapped in 11pil were used to isolate YACs in affected families where the germline mutation has been detected, and has (CEPH). These CA repeats are now physically mapped with a panel of 11p15-specific allowed tentative conclusions to be drawn about genotype/phenotype hybrids, and the contigs of YACs mapping in the regions of interest will be used to isolate correlation. However, no mutation has been found in the majority of the coding sequences. In order to study the imprinting of the I pil region, several approaches are used: 1) Hybridization of anti-methylcytosine antibodies on metaphases from normal families studied, suggesting that a large proportion of pathological NF2 and disomic WBS lymphocytes to detect possible differences of methylation between the mutations are not detected by screening the coding sequence of the NF2 gene by paternal and maternal 11piS regions; 2) Study of molecular asynchronous replication of PCR-based methods. We are currently extending characterisation of the NF2 different 11pl5 loci: 3) Direct genomic sequencing after Frommer's technique to study all gene to include analysis of the 5' and 3' flanking regions. 5-methylcytosine residues in 11p15. Posters: Cancer Genetics (continued) A61 317 318 Analysis of mutations in brain, thymus and testis from lact containing transgenic Terminal deletion and multiple breakpoint cluster in the chromosome mice: do the spontaneous mutation frequencies and patterns change with age? 3p region of sporadic and hereditary renal cell carcinomas. P. Bugert. V. L. Buettoer'. H. Nishino'1. J. Haavikl'. and S. S. Sonneri Mayo Clinic/Mayo MI. Wilhelm,. C.Kenck, G. Staehler and G. Kovacs. Clinical Research Group, Foundation, Departments of Biochemistry and Molecular Biology' and Neurology', Molecular Oncology, Dept. Urology, Ruprecht-Karls-Univ., Heidelberg, Germany. Rochester, MN. The development of nonpapillary renal cell carcinomas (npRCC) is associated Recently developed transgenic mouse detection systems offer a powerful tool for with the loss of 3p sequences. The most distal breakpoints have been found at the analysis of spontaneous mutations in vivo. Big Blue' mice carry chromosomally boundary between the chromosomal bands 3pl4.1 and p13. Thus. the RCC gene integrated lambda bacteriophage shuttle vectors (LIZ) containing the E.coli lacl gene is thought to be located in the 3p region. Recently, the VHL gene has been cloned as a Lambda shuttle vectors rescued target for mutagenesis. by in vitro packaging from the chromosome 3p25-26 region. The gene is considered to be the RCC can be screened for mutations in the ci gene lacI gene by using complementation and X-gal but only about 50 % of analyzed tumors with the deletion of one allel showed a as a substrate. Blue' mice were used to examine the rate and Herein, Big pattern mutation of the other VHL allel So. another tumor is involved of spontaneous mutation in vivo with to each were supressor gene respect age. Four mice sacrificed in and should be located more at 3 and 10 months. Three tissues were examined: and testis. Nine npRCC development proximal to the VHL locus, brain, thymus to The million plaques were screened from all three tissues and a total of 287 blue presumably, proximal 3pl4.2. microsatellite analysis was used to determine (mutant) the RCC locus in and plaques were harvested and replated. The mutant frequencies in the 3 and 10-month- gene sporadic hereditary tumors including those of which old mice were not statistically different (2.9xlO-5 and 2.8xl05 in the brain, 2.8x10-5 are associated with constitutional balanced translocations 3;6 and 3;8. All tumors and 4.1xlO5 in the thymus, and 3.9x15 and 3.6x10-5 in the testis). The entire lacd analyzed showed a terminal deletion distal of the D3S1300 locus at 3pl4.2 and no gene (1.2 kb) was sequenced from 69 blue plaques harvested from brain. A single interstitial deletion was found. In about 30% of the tumors a common breakpoint mutation was found in the lacd gene from 68 of the plaques. When recurrent was found between the D3S1251 and the D3S1101 locus with about 1 cM genetic mutations from the same mouse were excluded, 63 independent mutations were found distance at 3pll.2. Other breakpoints clustered between the D3S1663 locus (p11.2) in total. There were no statistically significant differences in the mutational spectra and the D3S13f0 (p14.2) locus including the constitutional breakpoints in the 3;6 in the brain between 3 and 10 month old mice (p=0.4; Fisher's Exact Test). This and 3:8 translocation families with renal cell carcinomas. suggests that a 3-fold increase in age does not affect the mutational frequency or pattern in the brain. Analysis of the age-dependent pattern of mutation in thymus and testis is in progress.

319 320 Frequency and spectrum of inherited mutations in BRCA1 from Increased risk of stomach cancer In BRCA2 carriers. L.A. Cannon- a population-based study. L.M. Butler 1. B. Neewmnan 2, L.S. Friedman 1, Albriaht'. S. Neuhausen'. M. Higbee'. M. Skolnick'2. 'University of Utah E.A. Ostermeyer 1. J.E. Morrow 1. P. Dowd 1. P.G. Moorman 2, R. Millikan 2, School of Medicine, 2 Myriad Genetics, Inc., Salt Lake City. M-C. King 1. 1 Univ. California, Berkeley: 2 Univ. North Carolina. Chapel Hlill. Increased risks for ovarian cancer as well as colon and prostate cancers Heretofore. highly penetrant alleles of BRCA1 have been identified in families selected for high risk of breast or ovarian cancer. But are there BRCA1 mutations are recognized for BRCA1 mutation carriers. It is not yet clear whether that confer lower risk than those found in high-risk families? What is the frequency BRCA2 increases risks for cancers other than breast; however, in a kindred of inherited BRCA1 mutations among breast cancer patients from the general pop- with evidence of BRCA2 linkage, an excess risk of stomach cancer has been ulation? Does the frequency or spectrum of inherited BRCA1 mutations vary by noted. Kindred K4328 descends to 310 individuals in six generations. The age at diagnosis or by race? Do environmental factors influence penetrance of some kindred was ascertained for an excess of prostate cancer, 8 cases were BRCA1 mutations? What is the role of genes such as the androgen receptor (AR). observed and 2.42 were expected (p<0.01). Examination of the descendants at which polymorphism may have a moderate influence on breast cancer risk? To revealed an excess of breast cancer (6 obs., 2.43 exp., p=0.03), an excess address these questions, a population-based series of 900 breast cancer patients was of stomach cancer (6 obs., 0.30 exp., and a selected from 24 counties of central and eastern North Carolina. The sample was p <.001), single early onset designed to include equal numbers of African-American and Caucasian patients, ovarian case (0.40 exp., p <0.3). Because 4 of the breast cancer cases and equal numbers of women diagnosed younger than 50 years and at age 50 or were diagnosed before age 50 the family was typed for BRCA1 and BRCA2 older. Family histories and information on environmental risk factors was obtained linkage. No linkage to BRCA1 was observed for all cancers, nor for the by interview. Age and race-matched controls were also sampled Based on prelimi- breast cancer cases alone. The four breast cancer cases which could be nary data from 5% of BRCA1 sequences. three distinct mutations have been found typed share an 11 marker haplotype on chromosome 13q from D13S290 to in four patients. These early data suggest that: (1) the BRCA1 mutation spectrum D13S267. In addition, the 4 stomach cancers which could be typed also will vary considerably across populations. with founder effects important within share this 11 marker haplotype. Two stomach any one group: (2) mutations leading to splicing errors may be relatively more fre- cancers at age 47 and 62 quent in the general population of patients; (3) distinguishing rare disease-related were untyped. Other cancers observed in BRCA2 susceptibility haplotype mutations vs rare polymorphisms requires a large and appropriately matched se- carriers include pancreas at 71, prostate at 86, lung at 49, and cervical ries of controls; (4) BRCA1 mutations occur among probands with both younger cancer at 25. Allele loss in gastric and lung tumors at 13q12-22 has been and older ages at onset and among both races. Penetrance of these mutations will reported. Only one of the 8 prostate cancer cases carries the BRCA2 be evaluated by testing both affected and unaffected relatives of probands. The susceptibility haplotype. Although this family differs from the typical high polymorphic polyglutamine repeat in the N-terminal domain (CAG repeat in exon penetrance BRCA2 kindreds, it may represent a lower penetrant variant 1) of the androgen receptor (AR) will be evaluated in the same series to deter- which could be responsible for a portion of breast cancer and stomach mine whether inherited variation in this functionally significant region contributes to breast cancer risk in the general population cancer which is less commonly recognized as familial.

321 322 Loss of heterozygosity at TSC1 and TSC2 loci In TSC-associated and Molecular Characterization of der(1)t(1;2)(p36;q31) in non-Hodgkin sporadic hamartomas. C Carbonara(1) L.Longa(l) E-Grosso(1 I lymphomas. K. Carlson, G-Mazzucco. C.Borronae M-L.GarrA.MBriio. A.Giannotfi. P.Falzonoi S. Bajalica and M. Nordenskjold. Department of Clini- G.Monga, G.Garini M.Gabrielli PRene C.Danasino, M.Ruggieri cal Genetics, Karolinska Hospital. Stockholm. Sweden. G.Magro. G.Fiippi. A.Scabar. F.Bonetti. M.Pea- G.Martignoni. N.Migonef 1). Chromosomal rearrangements are frequently associated with neo- (1 )CNR Centro Immunogenetica ed Oncologia Sperimentale and plasias.Translocations involving lp36 are found in 17% of all NHL while 80-90% Dipartimento di Genetica, Biologia e Chimica Medica, Universith di Torino; of all folicullar non-Hodgkin lymphomas (NHL) are found to contain a 14;18 TS Italian Collaborative Group, Italy. translocation. Using fluorescence in situ hybridization (FISH) with biotin labeled Loss of heterozygosity (LOH) at TSC1 and TSC2 loci, as well as at seven chromosome-specific libraries (chromosome painting) on metaphases from short- tumor suppressor containing regions (p53, NF1, NF2, BRCA1, APC, VHL term cultures from follicular non-Hodgkin lymphomas, we identified three cases and MLM) has been investigated in 20 TSC-associated hamartomas and In 17 patients carrying sporadic angiomyolipomas (15 renal and 2 hepatic). The with der(1)t(1:2)(p36;q31). Furthermore, two normal chromosome 2 homologues 20OTSC-associated hamaulomas included 8 renal angiomyolipomas, 8 giant were found in addition to the der(I)t(1;2) indicating a partial trisomy of the dis- cell astrocytomas, 1 cortical tuber and 3 rabdomyomnas, derived from a total tal part of chromosome 2 in the three cases. In one of the cases, the der(l)t(l,2) of 18 patients (14 sporadic and 4 familial). In the TSC-associated lesions, appeared as the sole aberration. By PCR analysis we were able to show that TSC2 LOH was observed in 5 angiomyolipomas and in 4 nstrocytomas; none of these tumors had the 14;18 translocation despite their follicular growth TSCI LOH was found in only one astrocytomna from a TSC1-linked family pattern. We performed subtraction with (Carbonara C. et al. Hum. Mol. Genet. 3:1829, 1994). Overall a significant hybridization both chromosome I and preponderance of TSC2 LOH was documented. A series of possible chromosome 2 specific libraries and a genomic phage library derived from one explanations were considered: a) a TSC2 gone higher mutation rate; b) the of the der(l)t(l,2) patients. A genomic clone containing an open reading frame likelihood that the TSC1 defects are mostly microdeletions or point which maps to chromosome 2 was identifed. Continued analysis of this and other mutations, undetectable by the LOH method; c) hamartomas bearing TSC2 clones from our subtraction screen may give us more insight into the genetic mutations might more frequently necessitate surgical excision. No loss of ma- heterozygosity has been observed at the others tumor suppressor containing terial involved in lymphoma-associated translocations. In addition, two genes, the regions. leukemia-associated phosphoprotein, p18 and a cAMP-dependent protein kinase LOH spanning different regions has been observed in two samples are known to map to lp36. These will be regarded as potential genes involved in (astrocytomna and renal angiomyolipoma) from the same patient. Thus the development of follicular lymphomas which do not involve the t(14;18). supporting the muitifocal origin of the second mutation. Among the sporadic angiomyolipomas, TSC2 loss of heterozygosity has been observed in 2/11 informative renal angiomnyiolipomnas and in 1/2 hepatic angiomyolipomas. This observation is consistent with a role of TSC2 in the development of sporadic hamartornas. A62 Posters: Cancerr Genetics (continued) 323 324 Detection of Pas mutations by degenerate mutagenically separated polymerase Functional evidence for a tumour suppressor gene on chain reaction (DMS-PCR). LQ. ha& JLM yuan T.Y. Ymang H.J. Liu. chromosome 8 by monochromosome transfer into colorectal Department ofMolecular Medicine, Taipei Municipal Jen-Ai Hospital, Taipei, Taiwan. cancer cell lines. G. Chenevix-Trench. C. E. Gustafson, L. Annab- Ras oncogene plays a very important role in the tumorigenesis. Recently, the and C. Barrett*. Queensland Institute of Medical Research, Brisbane, success of several drugs to inhibit the abnormal function of mutant ras protein in Australia and tLaboratory of Molecular Carcinogenesis, NIEHS, several kinds oftumors augments the importance ofdetection of mutant ras gene. The Research Triangle, North Carolina analysis of ras gene in tumors has shown that the mutations are mainly located at the We have examined a series of 136 human colorectal adenomas and codon 12, 13, or 61. In this study, we develop a very rapid and simple PCR-based carcinomas, and 20 colorectal cancer cell lines, for loss of heterozygosity method called DMS-PCR to detect the mutations of ras gene. We used four or five (LOH) or hemizygosity on chromosome 8p. The relative rales of LOH in primers in each reaction tube to perform DMS-PCR. There is a longer primer (40mer) carcinomas with metastases (71%) and without (38%; P = 0.035) which is specific for normal allele to recognize the normal sequence ofcodon 12, 13, or suggest that there is a gene involved in the progression towards late stages of colorectal carcinoma, perhaps including metastasis, at 8p22. 61, respectively, and 2 or 3 shorter primers (20mer) for mutant alleles. The first shorter Data obtained from analysis of 10 microsatellite markers spanning the mutant allele-specific primer has a degenerate base and a mutagenic base at the 3' end. entire short arm of chromosome 8 indicate that the shortest region of This degenerate base in combination with the mutagenic base will make the primers overlap is at 8p22, within 21cM of the LPL locus. In order to provide amplify all the mutant alleles, which have different base at the first base ofthe mutant functional evidence for this putative tumour suppressor gene, microcell- codon. The second shorter degenerate mutant-specific primer has the same function, mediated chromosome transfer of normal human chromosome 8 was but they recognize all the second base of the mutant codon. The third degenerate carried out. Three colorectal cancer cell lines, SW480, SW620 (the shorter primer is for the third base of the mutant codon. In combination with other metastatic counterpart of SW480) and HT29, which are all hemizygous polar primers, these 4 or 5 primers will recognize all the changes of the normal or the at 8p, were used as recipients. Hybrid clones generated from the fusion mutant codons in PCR. The 20-bp difference of PCR products from normal and of chromosome 8 microcells to the recipient cell lines, when compared to mutant alleles can be easily differentiated under UV light after electrophoresis in the parental lines, displayed altered cell morphology, decreased growth agarose gel. More than 50 cases of different cancers have been tested by this method rate in vitro, decreased cloning efficiency in soft agar, and significantly with good results. delayed tumour formation in athymic mice. These hybrid cell lines are This approach can also be applied to the detection of hotspot p53 being compared to the parental lines by differential display of mRNA mutations or other genes with hotspot mutations. species to isolate genes involved in the suppression of tumorigenicity.

325 326 Identification and characterization of a novel Kruppel type zinc-finger DI Cytoge -cand FISH Rult h a26 YearO e With Ear TCeALL locus on human chromosome 9q22-31 in the Nevoid Basal Cell D. HE W & A. Rsafoi. R. Blancbd andA. NmamkL Bodon Unbriy Carcinoma Syndrome (NBCCS) region. A hiamara , B. Gerrard 1, Schoolof Medlckewnn Bosdon Clly Hosital, MA. A. Bale2, A. M. Goldstei3, C. Stewartl and M. Dean4. 1BCDP, SAIC A 26 yer-d manb we seen ba ora rgMtmndr kVand Frederick, NCI-FCRDC, 4LVC, NCI-FCRDC, Frederick,MD, 2Dept. of nodules i hisform. He had a WBC o 33,000,a plt coa d 125,00Dard a noml hm.xalln Apaeal bbod smnr wd atypical moncdand mo cswth Genetics, Yale University School of Medicine,New Haven, CT, 3Genetic 10-16%bles. Furlhrv tion realed a 3 x 4cm r wlbmuurhord lymph node, Epidemiology Section, NO, Bethesda, MD. maddland asily and kauhW Vmph nodes, pa sp tinpl, we numeo sndals. The Nevoid Basal Cell Carcinoma Syndrome (NBCCS) has been usnomriodu vrbolhforarmb Bone marw showed nor meWpimcy -w red mapped to human chromosome 9q22.1-q31 by linkage analysis. Consti- cal as, b* a m rlad iree inlhci as wih d80% blts. Irmwaphantping tutional deletions of this region in NBCCS patients and loss of hetero- shwed 95%CD5d 9%CD7 o aW h ely T-l ALL:6% C033s hdodof zygosity of polymorphic marker flanked arbid ad pse. A kmbwrpan showed CNS hkeomw ithlymphobless CTscan loci by D9S197 and D9S180 have ofthedomx a been in as reseld 34cmararo mdlasina s. The palt wustreatd *wi c- observed familial well as sporadic tumors. We have utilized a twat lanai, predisonm and mudtbsple sof imhscl ARA C. Aflbr2 da sthe pted s positional cloning approach involving the construction of YAC contigs blood smears norm. Bonemarowwsrhypeacleruth %bleatsnds*m decrae spanning this region and Alu-PCR probes derived from these YACs to hi ha 1poatDoloeameus Cyogsbtus swed a noal kwype hi antaphas. select chromosome 9 specific cosmids for more detailed molecular H er, bluoresce hiask on (FL" showd 80% ofd phaeswkhonlyon1sgn analysis of this genomic region. One of the cosmid clones contained a usig te outnom probe, D7Z1 (Onoor), spedaic for dmomom 7. A repe bone marrow (CA)n repeat region. of the sequences of this 10dayasryrsldad fewanaab mtaphswsnbil gm amarruithi ilphs b FISH. Analysis flanking repeat Sit-w m hs mfromthetsm bormarrrowsamples wetbod by FISH. Fortyonewe region demonstrated considerable homology to the 'A' box of the frm allshybriddwh heosromri prbe: 19 nmtapham hsdtIogn perdpod Kruppel-like family of zinc-finger genes which have been shown to act as comlement (5 oell wereterpkd; 21 had one sign; 1 had no sia Anode 21 maS- transcription regulators. PCR analysis using primers designed to the con- phases, aflrhybdkkatowha-p ngprobe (Vyss)specfordcmomwe7, yielded 12 served linker region of the zinc-finger repeats amplified multiples of the witwoomplt no. Ta; 3 wih one no.7; 4 wih one ompide no.7 & one prtl pbId 100 bp repeat in an overlapping cosmid. Sequence analysis of a 4 Kb EcoRI dchrmosoe, "idiathig st wms cal were mig a rml no.7 due tobunsction sh subdone containing the repeats revealed the presence of a unique conti- ahhrdcahooom. The ddcrpat resl bewen G-besnin and FISH shis we remarbible. nuous open frame that contained 13 of Abnrmal matsp asse studied byFISHwere _b*ctaun asyz byG4byG.bn , roug astbong reading repeats the C2H2 type. lhical bisforanisof noralcelb byroutne ndho. Wbelew hi may nrt This sequence has been assigned the gene symbol ZNF169 and its presence bean unusu phemenonh cen dorders divngcromse 7. in the NBCCS region is intriguing since several tumor suppressor genes contain zinc-finger regions with transcription regulatory properties.

327 328 Poster Symposium-Session 30 Use of a microsatellite marker in Rbl gene in molecular analysis of Identification and molecular cloning of a 20q13.2 amplicon is breast carcinoma. hereditary retinoblastoma. L Chiu2 PS Lai.' JSH Tay.' Y Ling.2 PYY Colin CollinsI, JeffFroulal, Minna Tanner3 Jan-Fang ChengI, Tony Godfrey2, David Kowbel'I Cheong.2 PK Wong3 and A Lim.2 Department of Paediatrics, National Joanne Cochran', Andrea Shaerl, Chris Martinl, Michael Palazzolol, Mary Hintz2, Ulli Weierl, University of Singapore, 2Singapore National Eye Centre, Department of Wen-Lin Kuo2, Olli Kallioniemi3, Jeff Gingrich4, Johanna Rommens5, Dan Pinkell,2 and Joe Ophthalmology, University Hospital, Kuala Lumpur, Malaysia. Gray12. kLawrence Berkeley Laboratory, Berkeley, California; 2University of California, San The Rbl-20 polymorphism is a microsatellite marker consisting of Francisco. California; 3Tampere University Hospital, Tampere, Finland; 4Lawrence Livermore (C'IT(T)). (n-14-26) repeats. It occurs at 54 bp from the 3' end of exon 20 National Laboratory, Livermore, California; 5Hospital for Sick Children, Toronto, Ontario. in the Rbl gene and can be used in molecular analysis of patients and kindreds Increased copy number involving chromosome 20ql3 has been reported previously in 1S- affected with hereditary retinoblastoma. Sequencing of these repeats using -y 20% of primary breast carcinomas using Comparative Genomic Hybridization. The region of "2P-ATP end-labelled primer enabled identification of different genotypes highest amplification was defined to be at 20ql3.2 using FISH with 33 locus specific cosmid and P1 among 45 unrelated Chinese individuals (18 males and 27 females) analysed. probes distributed along chromosome 20. The frequency of tbe 20q13.2amplification has now been These repeats were found to be very polymorphic assessed in 132 pnimary breast carcinomas and 11 metastases by FISH using probe RMC20C001, in our population, and a total localized to the amplicon core. The frequency of RMC20CO01 amplification (>1.5-fold) was 29% of 7 different alleles (344 - 380 bp) was observed. 63% of the females studied (38/132) with 6.8% (9/132) showing high level (>3-fold) amplification. 20q13 amplification was were heterozygotes for this polymorphism. Eleven affected families identified associated with a high S-phase fraction (p=0.0055) and high level amplification with short disease- from local eye clinics were studied, and nine were informative (82%) for this free survival (p=0.04), especially innode-negative patients (p=0.002). These observations sugest microsatellite marker. This marker was found to be useful in the that the amplifiedgene(s) confers a more aggressive phenotype on the tumor. The ampliconhas beencloned as a 12 member S Mb YAC contig and used to construct an presymptomatic prediction of retinoblastoma for identification of gene carriers, -2 Mb PI contig across the amplicon. Free chromatin FISH was employed to verify the contig and to exclude individuals who do not carry the mutant gene, especially in structure and to precisely measure gap distances. Small gaps are being closed by screening BAC families where occurs. library DNA pools by the PCR. The only gene known to map to the amplicon is vitamin D 24- incomplete penetrance hydroxylase. However, this gene does not appear to be expressed, in breast cancer celllines, using RT-PCR and therefore is not alikely candidate oncogene in breast cancer. Thus, a novel oncogene appears to be driving tHe copy number increase. To identify candidate oncogenes, we have performed exon trapping and cDNA direct selection on a set of P1 and BAC clones comprsing the minimal tiling path and have initiated the directed genomic sequencing of the amplicon core with the Lawrence Berkeley Human Genome Center. We will report on the molecular characterization of cDNAs and exons identified using these complimentary approaches. Itis expected that the combination of these technologies will culminate in the isolation of the hypothesized oncogene(s) and contribute to the development of novel molecular diagnostic and therapeutic approaches for the management ofbreast cancer. This work was supported by grants from US DOE contract 0376SF00t95, USPHS grant CA52807 and Vysis. Posters: Cancer Genetics (continued) A63 329 330 Cardiac myzomas are not associated with activating mutations of the Gs Chromosomal gains and losses, DNA-ploidy and p53- alpha gene. L. De Marco'. W.L. Boson'. L.M. Andrade' V. Foneca. and E. expression during the genesis of colorectal tumors Fneodman'. Departments of Pharnacology' and Pathologye, Universidade Federal Qldu R. Kmc Auer'zne g.B #. de Mines Gerais, 31270 - Belo Horizonte; Brasil, and DNA Diagnostics #Diagnostic Development Branch, NCHGR/NIH, Bethesda,MD. *Karolinska Chaim Sheba Medical Center, Tel-Hashomer - 52621, Israel. Laboratory', Institute, Stockholm, Sweden; llnstitute of Germany The molecular basis for the development of cardiac atrial myxomas is unclear. To document the chromosomal aberrations thatPathology,Flensbur$,occur during the initiation and Pathological myocardial function and myocardial hypertrophy have been associated progression of colon tumors we used a molecular cytogenetic approach, termed with alterations in several copo of the signal transduction cascade that comparative genomic hybridization to screen the DNA from rnicrodissected, regulatees cAMP generation, in particular the heterotrimeric GTP binding proteins histologically defined stages of colon tumorigenesis, i.e., normal mucosa (n=14), (G protew). The postulated proto oncogenic character of the gene encoding the low grade adenoma (n=17), high grade adenoma (n=l1) and invasive carcinomas (n=19). This material was investigated immunohistochemically for p53, WAFI alpha subunit of the stimulatory GTP-binding protein Gsa (gap) in pituitary and expression and for proliferative activity. DNA-ploidy was measured on the tissue thyroid tumors, and the recent finding of identical somatic gsp mutations in hearts sections. of patients with the McCune-Albright syndromc, prompted us to investigate the The transition of normal mucosa to low grade adenoma is associated with a occurrence ofthes miasense mutations in the Gsa gene in seven atrial myxomas. tetraploidization, an increased proliferative activity and the acquisition of few chromosomal imbalances. The most frequent changes are overrepresentation of The polymerase chain reaction (PCR) was used to ampli* the appropriate chromosome 7 (or 7p), 20 and underrepresentation of 18. p53- and WAF1- regions ofthe Gsa gene. Then, these DNA fragments were subjected to denaturing expression are coordinatedly elevated, indicating functionally active p53. The gradient gel electrophoresis (DGGE) and direct DNA sequencing of PCR transition from high grade adenomas to carcinomas is accompanied by an important fragments, methods that can readily detect mutations. No atypical migration increase of chromosomal aberrations. Interestingly, p53 protein is non-functional in a patterns wore demonstrated by DGGE and this was confirmed by direct sequencing large fraction of the carcinomas. Recurrent gains (including high level copy number ofPCR products, indicating that the DNA do not contain point mutations increases) are mapped on chromosome 20q (60%), 13q (57%), 8q (42 %) and 7p analyzed (36%). Recurrent losses are observed on chromosome 18 (consensus region 18q21, or sequence alterations. Thus, Gsa activating mutations are not associated with the location of the DCC-gene), 8p, 4 and 17p. The CGH-data are highly concordant to development of atrial myxomas, and the significance of these mutations in studies using karyotyping (Bardi et al) or LOH for the chromosome 17p and 18q. A symptomatic patients with McCune-Albright syndrome remains to be determined. discrepancy exists when CGH-data are compared to established genetic models for This work was supported by PRPqlUFMG, and FAPEMIG, Brazil. colorectal carcinogenesis, i.e. on Sq. This indicates that the APC-gene is inactivated by other mechanism than gross deletions (same remark for K-RAS). Our results suggest that the gain of 7p and 20q are early events during colorectal carcinogenesis. The gain of 13q occurs after the progression to carcinomas.

331 332 Detection of polymorphisms and miaense mutations in BRCA1 gene. RadT on of He, K-, and Raoneogenepointmualns byatomAa pec F DU , D.2 M sk23, D.E. g3and L analyis: pleationto humn camFA EgaEld Q auMn2A , B . SamAgl. 1 Laboratory of Molecularndocrinology, CHUL Reearch Center & Laval 1Peft live Foster and University, Quebec City, GlV 4G2, Canada, 2 Myriad Genetics Inc Salt Lake City, EwCrrion, Apied By Ciy, Calforia, Memail Utah; 3 Department of Medical Informatics, University ofUtah, Salt Lae City, Utah. SloaKleulng Cancer Center, NewYork, New York. The structuof BRCA1, agene predipoing to famlialealy-oeseastad Thehumanhs genebi* ccsi orieecloselyreltedgenes, Hs K-ms, and N-rns, ovarian cancer, was recently cided Mutations in BRCA1 are responsbl far - whichencode eled21 kDapo n ned Inignaltansduclion. Ra genes acquire 45% of inherited breast cancer, mor than 80% of inherimed ovarian small oncogenicpcenalasa sultofpoirn emulions in codons IZ 13, or61 ofhe gene, and proprion of sporadic ovarian tumors. The BRCA1 gene contains 5592 bp ofcoding munaed ins gnsasanonge mxtonwwenongeelc ories huracanc aoccng sequence encompasaed within 22 exons sprad over 100 kb encodn a pote of in aout0 fdpanreall cwcdnon 50%ocoloncanners, and 20k3096ofacebukemias 1863 ino acids. isoation of the BRA1 offerd possiility forgenesd Dkirtbnore s showspecidt donWgs geeacvion;forexwwr, aclied H-asis testing of asymptotic women with family histuy and for providing bed tundmostobn baddercancei K-insm ga eni and lungbnrs, and N-tsIn on allelid heterogeneity. Frameshift, nonsens, sple or regulatory m o, which result in a truncated BRCA1 protin accountedfor 85% of mutaons, while the remaining are due to substituon of a nle amino acid. Until a functional test is AivHd ead andauuband alpl_ E*a gy has been Doped tb defec and available, population frequency analysis of sequence variants in dth BRCA1 coding chma **acgpt irtnmulonsinasgenes inhunanmnor PCRargIfcabonofexn I rion is essential to support the putative role of missen mutations when (codons12 and 13) adaeon2(odon 61) of huanrasgenes isfoIoed by a higIy specific cosegregation analysis with predisposing BRCA1 alleles cannot be established. dgion ldepmo ndnam triondeeioL Nomnal datmutatgedypesae Moreoe, analysis ofpolymorphisms is essendal to detect regulatoy mutations and is e o useful to detemine the common origin of frequent mutations. We determine de anayzedb probet Mtultion-speilcPobes are frquency of twenty sequence variants in BRCA1 gene, which were first detected in sLe rpossibnfa e lase, nondet MnO incodons 12 13, and 61. Nomal DNA samples om baovanan cancer patients, in a normal population by direct andmAtalecpobes hyd inpftjoion1ob acnon, domwne pxrbe onlypobes sequencig and/or ASO hybridization. Among the eight frequent polymorphisms fAp iedsmtch *h DNAsequenwlgL C peen b arelbeldwith analyzed, the following five yield to a change of amino acid: Gin 356 + Arg (12%), Iuouhones anddec*pbs each hawd derentrengrt Geope s d igon products Pro 8714- Leu(53%), Glu 1038 Gly (29%), Lys 1183 Arg (54%) and Ser 1613 d uishd bybol ho scen osizealerpheis onantmedm DNA a-Gly(51%)(* o beterozygosity). Ths uent polymorphisms were in linage disequilibium. Thee rae we also 197 -+ polymorphisms analyzedi Cys Cys (4%), anb Asp 693 Asn (8%) and Ser 1040 -* Asn (7%). Three silent mutations were not DNAisdtedfbmhun rspewngw astmlyzhnsdtrb iangisum muonusing found in our population. Finally, the variants Met 1008 -+ lle, which was found in ts novel molecuargenedic assay. Mutlonsdeecled by his assay ve beencorirmed by three unrelated patients, Glu 1219 -+ Asp, Arg 1347 Gly, 1hr 1561 Ile, Met cloning and sequencing. The assaycand rsmuWonstn umorelsmading up Iess 1628 Val and Thr 1852 Ser were not found in more than 78 chromosomes, thus 5%ofthedcrisanille. Idenmn ofn mutonmWashdha veI seddlinic suggesting hat they correspond to eal missense mutations. Iknuas eC1rlihSibt D wUao *s aed*v oped.

333 334 Mutations in the BRCA1 Gene in Japanese Breast Family history of cancer Is a risk faetor for squamous cardnoma ofthe head Cancer Patients. ((M.Eaiil.2.3, T.Katagiri2, I.Ito2, and neck: a case-control study from Brazil. W.D. Foulkes'. J-S. Brunet'. L.P. K.Kobayashil, F.KasuMi2, Y.Miki2,4, M.H.Skolnick4 and Y.Nakamiura",2.)) 1Inst.Med.Sci., Univ.of Tokyo, Tokyo; Kowalski'. E.L. Franco' and S.A. Narod' . Division of Medical Genetics, Dept. 2Cancer Inst., Tokyo; 3Inst.Gerontol., Nippon Med.Sch., Medicine, McGill University, Montreal General Hospital, QC, Canada'; Ludwig Kawasaki, Japan; 4Univ. of Utah Ned. Cntr, Utah. Institute for Cancer Research, Sao Paulo, Brazil'; Division of Epidemiology, Dept. Oncology, McGill University, Montreal, QC, Canada'. Predisposing mutations in a ERCA1 gene have been Squamous cell carcinoma of the head and neck (SCHN) can occur in some recently identified in germ-line of 17q-linked breast hereditary cancer syndromes. In addition, mutagen-induced chromosome damage and ovarian cancer families. we examined primary breast is seen in SCHN patients with a family history of cancer. To determine the role cancer patients consisting of 46 early-onset cases (<35 of age), 12 cases from multiply-affected families, and of familial factors in SCHN we analysed data from a hospital-based case-control 59 cases with bilateral cancer for mutations in entire study of SCHN in Brazil. There were 754 cases of SCHN and 1507 age- and coding exons of ERCA1 gene using single strand gender-matched hospital-based controls with non-malignant diseases. Subjects conformation polymorphism (SSCP) analysis. Four mutaions provided information on the occurrence ofcancer in first degree relatives, as well were detected in this panel of 103 patients; a flame- as information about other risk factors, including tobacco and alcohol shift due to 2-bp deletion at codon 797, a nonsense consumption. Relative risks (RRs) were estimated for developing mouth, pharynx mutation at two one codon 1214, missense mutations, at cancer cancers in were observed. Relative risks were codon 271 leading to Val->Met substitution, and the and larynx when relatives other at codon 1150 leading to Pro->Ser substitution. adjusted for age, sex, alcohol and tobacco consumption. All of them were germ-line mutations; no somatic The RR for developing SCHN was 3.65 (95% CI: 1.97-6.76) i, the first degree mutation were found in these tumors, a finding that relative had SCHN. Specifically. significantly elevated risks for developing SCHN support a rather confined role of BRCA1 in breast were associated with siblings with SCHN (RR=8.57, 95% Cl: 2.72-27.04) and carcinogenesis. The aman age of onset in these Japanese to a lesser extent with fathers with SCHN (RR= 2.49, 95% Cl: 1.04-5.95). The patients was 49, in contrast to the mean age of 35 RR for if first relative had cancer at was observed among the ERCA1 mutation carriers in a U.S. SCHN any degree any site 1.97 (95% study. Among the three selection groups, all mutations CI: 1.49-2.61) These data show that familial, possibly genetic, factors are were found in patients with bilateral tumors. important in the etiology of SCHN. A64 Posters: Cancer Genetics (continued) 335 336 Patterns of allelic loss on chromosome 17q in sporadic carcinomas of Viability of homozygotes for a germline deletion of 19 bp in the coding ovary and breast. T. S. Frank, R. F. Caduff. and S. M. Svoboda. The Univer- region of the CDKN2/pl6 gene in Dutch melanoma families. LEraaL!Rd. sity of Michigan Hospitals, Ann Arbor, Michigan. P.A. Van der Velden'. L.A. Sandkuil1. D.E. Prins'-2 W. Berghnan and N A It has been speculated that an area of deletion on chromosome 17q distal Gruis-. iMGC-Dept. Human Genetics, Leiden University, Dept. to BRCA1. particularly involving 17q22-23, may be involved in the development Dermatology, Leiden University Hospital, Leiden. The Netherlands. of sporadic ovarian carcinoma. We analyzed tumor and normal DNA extracted The CDKN2 (p16) gene is frequently deleted and mutated in several types from 86 paraffin-embedded sporadic ovarian carcinomas for allelic deletion at poly- ofhumancancer, implicating a general tumor suppressor function. CDKN2 has morphic markers on chromosome 17q. Two of the markers tested. D17S855 and D17S1323, are intragenic to BRCA1 and a third at EDH17B is closely associ- been shown to be mutated in some melanoma-prone kindreds, suggesting it to ated with BRCA1. Two other markers, GH and D17S1330 ("CTT16") have been be the 9p21-linked gene for melanoma susceptibility (MLM). Analysis of localized to 17q23 and 17q25. respectively. 53 of 86 (61%) ovarian carcinomas CDKN2 coding sequences by single strand conformation polymorphism (SSCP) demonstrated loss of at least one marker on 17q, and 13 (15%) demonstrated LOH analysis in Dutch familial atypical multiple mole-melanoma (FAMMM) of all informative 17q markers tested. Of the 74 ovarian carcinomas informative for pedigrees identified a 19 basepair germline deletion in 13 of these 15 families. at least one of the three 17q21 markers, 42 (57%) demonstrated LOH of this locus. The germline deletion causes a reading frame shift, predicted to result in a When more distal markers were analyzed. LOH was identified in 29/57 (51%) ovar- severely truncated protein. Two family members were identified as ian carcinomas informative for GH on 17q23 and 11/64 (17%) cases informative homozygotes for the deletion, indicating that homozygosity for this CDKN2 for D17S1330 on 17q25. 27 ovarian carcinomas with LOH of 17q21 were infor- mutation is viable. Moreover, the expression of other malignancies in gene mative for GH. eight (30%6) of which showed no LOH at this locus. Conversely. carriers strongly support the view that CDKN2 is a tumor suppressor gene and five of 29 (17%) ovarian carcinomas demonstrated loss of GH but not 17q21. ln suggest the presence of functionally redundant genes that can fulfill the role of order to determine whether a difference in the prevalence of LOT! on 17q could p16 in its absence. The occurrence of apparent clinical FAMMM cases with be identified between sporadic carcinomas of ovary and breast, normal and tumor melanoma but without the 19 basepair deletion, asks for caution in definitely DNA was extracted from archival breast cancers and analyzed. Loss of informative markers in 17q21 was identified in only 14 of 40 (35%) of sporadic carcinomas of considering CDKN2 to be the MLM gene. the breast, a significantly different prevalence than that observed in carcinomas of the ovary (X2. P = .0266). We conclude that allelic loss on chromosome l7q in sporadic carcinoma of the ovarv more commonly involves markers associated with BRCA1 than does sporadic carcinoma of the breast. A minority of ovarian carcino- mas demonstrating loss of markers on 17q 23 but not 17q21 support the possibility that other tumor suppressor genes on 17q may be involved. albeit infrequently. in ovarian carcinogenesis.

337 338 Spectrum ofRET protooncogene mutations in 52 MEN 2A and FMTC families. The AF4/FEL gene: differential expression and the first six exons. LL C Franz12, D. Chi13, S Dou 1, K.Js a4, K. Carlson1, S. A. Wells , a. Frestedt and JL . Kerse. University ofMinnesota, Minneapolis, Minnesota. oiLs-Kell'.1,5 tDiv. of Human Molecular Genetics, 3Div. General Surgery, Dept. The AF4/FEL gene has been described (genbank L13773) and several subtle sequence discrepancies have been published which alter the open reading frame. of Surgery; 5Depts. of Psychiatry and Genetics, Washington Univ. School of The AF4 gene is involved in the 4;11 translocation which has been associated with Medicine, St. Louis, MO, USA, 2Dept. Surgery, Univ. Clinic, Hamburg, Germany, infant leukemia and a very poor prognosis. We cloned and sequenced 3294 bp of 4Second Dept. of Surgery, Nippon Medical School, Tokyo, Japan. the AF4 open reading frame from a placental cDNA library. Our sequence was We and others have found that germline mutations in the RET protooncogene identical to L13773 from base 493 to base 3783 with the exception of an additional are responsible for multiple endocrine neoplasia type 2A (MEN 2A) and familial triplet AAG inserted after base 3710 ofthe genbank sequence. These three bases medullary thyroid carcinoma (FMTC). RET mutations have also been found in tumor would likely result in the insertion ofan arginine residue as defined by the sequence DNA from sporadic cases of medullary thyroid carcinoma, suggesting a significant in this area ofthe gene. We have also identified the boundaries ofthe first six role for RET in sporadic as well as inherited forms of MTC. We have completed exons ofthis gene using a novel intron/exon trapping technique and a review ofthe analysis of 52 unrelated families (47 MEN 2A and 5 FMTC) and have identified 17 literature. Introns were identified at bases 438, 559, 1459, 1504, and 1591 ofthe different point mutations in a cluster of 5 cysteine codons (609, 611, 618, 620, 634) L13773 sequence. located in the extracellular domain close to the putative transmembrane segment of We also used our 3.3kb clone (named PL12) to probe several Multiple Tissue RET. The most commonly mutated codon was Cys634 (48%; 25 of 52 families), and Northen Blots and found that placental tissue showed a higher level of AF4 mRNA 12 of these families revealed a Cys634->Arg634 substitution. The remaining 27 expression than lung, liver, heart, brain, skeletal muscle, kidney, and pancreas families revealed mutations at Cys618, to Arg, Ser, Tyr, Phe, or Gly (27%; 14/52 (using Beta-actin to control for the amount and stability ofthe mRNA loaded). An families), Cys 620, to Arg, Tyr, Phe, or Ser (15%; 8/52 families), Cys6O9->Tyr (8%; alternate transcript of 5kb was found in placental tissue along with the expected 4/52 families), and Cys61 I->Trp (2%; 1/52 families). FMTC mutations were found 10.5 and 12kb transcripts. This smaller 5kb transcript was not seen in any ofthe for codons 609, 618, and 620 and were therefore no different than those found to be other tissues or cell lines that we examined. These findings suggest that the AF4 responsible for MEN 2A. The molecular basis of the difference between FMTC and gene is upregulated in placental tissue and may have specifc altemately spliced MEN 2A families that harbor the same RET mutation yet differ in phenotype remains products in the placenta. to be elucidated. However, it is possible that phenotype classification may explain the apparent disparity, at least in some instances. For example, one of our FMTC families has recently been reclassified as MEN 2A due to the appearance of hyperparathyroidism in one affected family member. Using a combination of restriction enzyme analysis, SSCP, and DNA sequencing we have been able to identify mutations in all MEN 2A families that we studied. For 80% of families, a simple restriction enzyme analysis is sufficient to detect RET mutations. For 2 FMTC families RET mutations have not been found despite a relatively thorough search of germline DNA. Additional studies are underway to reevaluate these families clinically and to further investigate the role of RET in the development of MTC.

339 340 Cosmid contigs and transcripts in the region of the Gorlin Genetics and multiple cancers. F. Gilbert. I. Arzimanoolou. Syndrome Gene. A.-M. Fdschauf1l F. Obermayr1, B. Chadwick 1, D. R. Tiwari. T-C. Hsu. S. Pathak. M. Osborne. and D. Miller. 2 Cornell Medical College and Strang Cancer Prevention Center, Moms 2, R. Mont 1, J. Williamson 1, D. Sheer1, H. Lehrach 1, A. Reis New York, NY; M.D. Anderson Cancer Center, Houston, TX. 1 Imperial Cancer Research Fund, London; 2 Inst. Human Genetics, A proportion of cancer survivors will develop additional Virchow-Klinikum, Berlin. unrelated primary cancers subsequently. Most will have a The gene for nevoid basal cell carcinoma syndrome (NBCCS or Gorlin family history consistent with a cancer family syndrome; some syndrome) is located on chromosome 9q22.3-q31. We are in the will not. All presumably carry cancer predisposition gene process of establishing a genomic clone and transcript map of the changes, though all such genes have yet to be defined. How candidate region. We hybridised inserts from 14 previously described best to identify patients at highest risk for multiple YAC clones spanning the region and interrepeat PCR products of DNA cancers in their lifetimes is the question posed. from 7 irradiation hybrids to high density arrays of the chromosome 9 We studied 16 patients (15 families) each of whom has had specific cosmid library LL09NC01 (obtained from Biomedical Sciences 3 or more primary (non-metastatic) cancers. 7/16 fit a Division, Lawrence Livermore National Laboratory). On the basis of defined family cancer syndrome; 5/16 have pedigrees positive for cancer but do not fit known cancer family syndromes; 4/16 moderate to strong hybridisation we selected a subset of 1400 cosmids do not have a first degree relative with cancer. We measured for contig building. The clones were reordered in microtiter plates and DNA repair capacity (DRC), mutagen sensitivity (MS), and spotted onto 7 by 11 cm nylon filters. To order the cosmids into contigs chromosome rearrangements (CR) in white cells of patients and by a sampling without replacement strategy 151 individual cosmid age/sex matched controls. CR were increased in 10/12 patients probes were hybridised to the subset and identified 566 cosmid clones who had received chemo- and/or radiotherapy, and in only 1/4 (including themselves). 22 contigs contain at least 2 (of 151) probes patients who received neither. MS (chromatid breaks post- and the location of 9 of those contigs on 9q22.3-q3.1 has been treatment with bleomycin or 4-NQO) was increased above confirmed by FISH or marker content. 2 larger contigs are located at control levels in 11/16 cases. %DRC were reduced in 15/16 9q34 pointing to cross hybridising sequences there. Cosmid DNA from cases; the exception is a patient with familial medullary 7 contigs representing approximately 700 kb of genomic DNA were thyroid carcinoma, associated with inherited mutations in the used in a cDNA selection and 14 of RET oncogene (unlikely to involve DNA repair). experiment overlapping groups In this series, measurement of DNA repair capacity, along cDNAs including 2 corresponding to the XPAC and FACC genes with combine to who known to be in this were family history data, predict presenting region identified. Unless several of the groups with a first cancer is most likely to develop additional of cDNAs correspond to different parts of longer transcripts this would cancers in his/her lifetime. Studies of individual genes indicate that the Gorlin gene lies in a gene-rich region. contributing to DNA repair in these patients are in progress. Posters: Cancer Genettics (continued) A65 341 342 Expression of the neurofibromatosis 1 and 2 (NF1 and NF2) genes in P53 and BRCAI mutation detection using the Enzyme Mismatch adult glial cell malignancies. MJ. Giordano, D.S. SilbergeldL. A. uha2.and Cleavage (EMC) method. C. Giunjai, R. Youill, M. Knightl, D. Venter2, D.H.Luimann'. lWashington University School of Medicine, St. Louis, MO and C.W. Chow2, G. Sorers2, B. Kemper3 and R.G.l. Cottolni. IThe Murdoch In- 2University of Toronto, Toronto, Ontario, Canada. stitute, 2The Royal Children's Hospital, Department of Anatomical Pathology, Individuals with neurofibromatosis 1 (NFl) and neurofibromatosis 2 (NF2) develop both benign and malignant tumors at an increased frequency relative to the general Melbourne. Australia and 3Institute for Genetics, University of Cologne, Germany. population. Reduced or absent NFI expression has been reported for Schwann cell The Enzyme Mismatch Cleavage (EMC) method has been shown to have at tumors and pheochromocytomas in individuals with NFl while reduced or absent NF2 least a 98% mutation detection rate (1,2,3). To extend our study with this new expression has been observed in schwannomas and meningiomas in individuals with system, we scanned exons 5--9 of p53 from 77 CNS tumours for mutations. We NF2. In addition, reduced or absent NFI and NF2 expression has been reported in found a total of 11 mutations and 9 had a known polymorphism. Three of the tumors in individuals without NFl or NF2, suggesting a more general role for these genes in the development of malignancy. mutations have not been previously described (GC- A in codon 159, del A in codon Individuals with NFl and NF2 both develop glial cell tumors (astrocytomas) with 235, and one sample had C--T and G--A mutations present in codons 175 and 273 increased frequency. To determine the contribution of NFl and NF2 to the respectively). We have extended the application of EMC to the diagnosis of a child development of astrocyte tumors, the expression of these two tumor suppressor genes who had developed multiple tumours and identified a p53 germline mutation (GC-A was examined in thirty sporadic adult glial cell tumors. Expression of NFl and NF2 at codon was detected in murine cultured astrocytes, normal human white matter, and cultured 14690nt in 248). We have also scanned the p53 gene from osteosarcoma human astrocytes by reverse-transcribed RNA PCR (RT-PCR) and Western tumour DNA from two brothers and found a GC-A mutation at 14780nt in codon immunoblotting. No loss of NF1 expression was demonstrated in these thirty glioma 245 in their respective turnours suggesting a possible germline p53 mutation. The tumors as determined by RT-PCR and Western immunoblotting. In contrast, reduced EMC method is also currently being used to scan the BRCAI gene for inherent or absent NF2 expression was detected in seven of 21 high-grade gliomas. One of mutations in probands that are likely candidates for BRCAI mutations. 1. Youil. three anaplastic astrocytomas (WHO grade I) and six of eighteen glioblastoma R., Kemper. B. and R.G.H. Cotton. (1993) The American Journal of Human multiforme tumors (WHO grade IV) had absent or reduced NF2 expression. This loss of NF2 expression was confirmed on the protein level in selected glioblastoma Genetics. Vol 53. No. 3 Supplement. Abstract No. 1257. 2. Youil, R., Kemper, B. multiforme tumors. None of nine low-grade gliomas (WHO grade A) had reduced or and R.G.H. Cotton (1994) The American Journal of Human Genetics. Vol 55, No. absent NF2 expression. These low grade tumors included two oligodendrogliomas, 3 Supplement, Abstract No. 1468. 3. Youil. R.. Kemper, B. and R.G.H. Cotton one giant cell astrocytoma and six grade II astrocytomas. The findings in this report (1995) Proc. Nati. Acad. Sci. USA. 92:87-91. suggest that NF2 may function as a tumor suppressor gene in the development or progression of human glial cell malignancies.

343 344 lOq26: Consisnt crooso alb kpont In two cardiac nyonas fo a Genetic alterations in diffuse large cell lymphomas in patients with AIDS. patient WitM Cae sdrome S. M. Gollin.Y R. B. Jenldns.4 W. P. Swanev1 LG Go= M Sumin J-D Broome. mid P-R-K Koduns North Shore University J. J. MulvihilL' D. Bartelt. Jr.! and N. A. Nwokoro.', 'Dept. of Human Genetics Hospital Manhasset, NY. and 2Univ. ofPittsburgh Cancer Institute, 3Cleft Palate-Craniofacial Center, Univ. of Pittsburgh, PA and 4Dept. of Pathology, Mayo Clinic, Rochester, MN. With increased use ofanti-viral drugs longevity ofpatients with AIDS virus Cardiac myxomas are benign tumors which can be an isolated finding or part infction is increasing. On the other hand, the prolonged state ofimmune suppression of a syndrome. Carney syndrome [OMIM 160980] is a rare multi-system in these patients is increasing the risk for the development ofdiseases such as Kaposi's preneoplastic syndrome characterized by cardiac and/or cutaneous myxoma, sarcoma and non-Hodgi's lymphoma (NHL). It is predicted that AIDS-NHL will be myxoid mammary fibroadenoma, psammomatous melanotic schwannoma, spotty the leading cause ofdeath in these patients in the near future. Unlike the NIL utaneouspigmentation,pigmentednodularadrenocorticaldiseasetesticular in immune competent individuals, the genetic etiology ofAIDS-NHL is poorly tumor, and pituitary adenoma; at least two must be present for a diagnosis. We studied. Histologically these tumors are classified as diffuse large cell lymphoma karyotyped independently two atrial myxomas from a 36-year-old female with (DLCL) and small non-cleaved cell lymphoma (SNCCL) types. Recent studies on Carney syndrome who had additional findings of myxoid mesenchymal neoplasm AIDS-SNCCL showed the accumulation ofmultiple genetic changes affecting cMYC, of the uterus, recurrent invasive follicular carcinoma of the thyroid, Barrett p53, RAS and Rb genes in these tumors. In contrast to this, cytogenetic and metaplasia, preneoplastic colonic polyps, and growth hormone elevation. molecular genetic studies on AIDS-DLCL are obscure. We have investigated the Cytogenetic analysis of metaphase cells from an 8 day (passage 1) tissue culture spectrum ofEB virus infection, cytogenetic changes and molecular alterations in of tumor A revealed a 46,XXadd(10Xq26)[cp12]/46,XX karyotype. Metaphase dominantly acting cellular genes cMYC, BCL1, BCL2, BCL3, BCL6, RAS, and cells from 6 and 7 day primary tissue cultures of the synchronous but distinct recessively acting genes p15, p16, p18 and p53 in nine AIDS-DLCL. EBV infection tumor B had a 46,XX,der(1O)t(10,12Xq26;qll)[cp8]/46,XX[3] chromosome was identified in six tumors (671%). Cytogenetic study performed on six tumors pattern. To our knowledge, eight other cardiac myxomas have been karyotyped. showed conal chromosome abnormalities in each. Three ofthe nine tumors (33%) had Of these, four expressed telomeric associations, two had clonal loss of the Y- t(8;14Xq24;q32) (two by cytogenetic analysis, one by Southern blot analysis). One chromosome, one had a sizeable tetraploid subpopulation, and one had a complex tumor had t(2;I4)(pl2;q32). No changes were identified in BCL1, BCL2, BCL3, p15, karyotype with clonal structural abnormalities including a der(10)t(10;?Xq22;?). p16 and p18 genes. Polymerase chain reaction - single strand conformation The two tumors from our patient express different rearrangements of band 10q26. polymorphism analysis and nucleotide sequence analysis identified a mutation in exon These tumors, along with the prior reported cardiac myxoma with a 8 ofp53 in one tumor which also had t(8;14). However, in contrast to DLCL arising rearrangement involving band 10q22, suggest that key gene(s) involved in in immune competent individuals in whom changes in BCL6 gene occur in 40%/6 of mesenchymal cell proliferation may be localized in this region. cases, no changes in this gene were found in the present series. These data may indicate a distinct pathogenic mechanism in AEDS-DLCL.

345 Poster Symposium-Session 29 346 Microdlssection-mediated cDNA capture: a strategy for Narrowing a Region of Prostate Tumour Suppression at the Chromosomal Location the rapid Isolation of cDNAs amplified in human cancer. 1 Oq23-q25. I.C. Grayl, S.M.A. Philles2, Sj, Lee3, J.P. Neopgolemos2 and fiL E. Grads. A Elkahloun I M. Trnt and P. S Meltzer. National Ssurrs.1 1 ICRF Clare Hall Laboratories, Herts, UK. 2Dept. of Surgery, Queen Center for Human Genome Research, NIH, Bethesda, MD. Elizabeth Hospital, Birmingham, UK. 3Dept. of Pathology, University of We have recently shown that homogeneously staining regions Manchester, Manchester, UK. (hsr) in human cancers may be complex structures carrying large cancer in derived from Prostate adenocarcinoma is the most commonly diagnosed amplified DNA domains multiple chromosomes (Guan men and is the second commonest cause of cancer death et al. Nature Genetics 8:155, 1995). It is therefore important to European and American after lung cancer. Despite this, only a limited number of studies have addressed the devise strategies for the rapid isolation of cDNAs expressed from tumour and these structures. We now report the recovery of hsr specific genetic processes underlying prostate development progression. Cytogenetic and allele loss studies have pointed to a number of chromosomal regions eDNAs using a strategy based on chromosome microdissection. of potential involvement, most notably 8p, 16q and 1 Oq. More specifically, a Nine cDNAs encoded by a complex hsr in the sarcoma cell line OsA- number of tumours exhibit precise loss of the region lOq23-q25. We have CL were isolated. Linkered cDNA prepared from OsA-CL was assessed 37 prostate tumours of various histopathological grades and stages for hybridized to OsA-CL metaphase chromosomes under conditions allele loss at 24 highly informative dinucleotide repeat markers spanning 10q23- which preserved chromosome morphology and allowed G-banding q25. A total of 23 tumours (62%) showed allele loss at one or more markers. A after stringency wash. The hsr was microdissected, and hybridized number of samples of benign hyperplastic tissue, thought to be a precursor to cDNA was recovered by PCR and cloned. Twenty four clones from were also but no loss was observed, the recovered PCR product were analyzed. Of these 15/24 tumour development, assayed, allele suggesting (63%) that loss of 1 Oq occurs during tumour progression rather than genesis. Combining recognized amplified restriction fragments on Southern blot the loss of heterozygosity studies with genetic and physical map data reveals a analysis of OsA-CL DNA. On sequence analysis, these 15 clones fall minimal of deletion at the 1 to the into 9 groups, 2 genes previously mapped to the hsr, and 7 common region Oq23-q24 boundary, proximal CYP2C gene cluster. This region spans approximately 9cM. previously unmapped genes. On the basis of these results, been that the at 1 which encodes a selection holds as a It has recently proposed Mxil gene Oq25, microdissection-mediated hybrid promise negative regulator of the Myc oncoproteln, is a tumour suppressor gene; mutations means of gaining rapid access to cDNAs encoded by amplified number of have been described. However, in genes. of Mxii in a-small prostate tumours our study specific loss of Mxii, as opposed to loss of other 1 Oq23-q25 regions or of the entire region, was observed in only 1/23 tumours, and was accompanied by loss of markers at the 1 Oq23-q24 boundary. Furthermore, we failed to detect any mutations in Mxil in those tumours showing Mxii-associated marker loss by either single-strand conformation polymorphism (SSCP) analysis or direct DNA sequencing. It is therefore likely that there is a tumour suppressor gene (or genes) at 10q23-q24 of greater significance than Mxil in prostate tumour progression. A66 Posters: Cancer Genetics (continued) 347 348 An dofpoflpbablsn omnlRNApm clnghnpdm yWU=sbmo. K.B. Gunning, V. Huf. Expression of the RB gene in an RB-negative bladder carcinoma cell line University ofTexas M.D. Anderson Cancer Center, Houston. prevents apoptosis. T.Hashimotol), T.Nakaiimal), J`.Furmgl), T.Tamao2), RNAprocessing errors for WT1 have been suggested to play a role in the development ofWilms and R.Takahashi3). O)Dept. Genet., Hyogo Coll. Med., Nishinomiya, 663, Japan, 2) nr(WT). Incell mdeed ogrfsofprnimaryWimsbmors, exon 2 has been observed 4N1, Canada, 3)Dept. to be aberrnlyspliced out and the cell lines which maintalined their turnorgenicity also maintained Dept. Med. Biochem., Univ. Calgary, Calgary, Alberta T2N aberrantexon2splcing. RNAedlnginexon6ofteratWrl holog, involvingaTtoCtransiton Pathol., Kyoto Univ., Kyoto, 606, Japan. atcodon 280 (human codon 281), has been dIonslied to occur in both rat kidney and testis. Both We have established an RB-positive cell line, H-CL2, from an RB-negative of these RNAprocesslng events resultina ctional change in the transcribed WT1 protein and are human bladder carcinoma cell line, HTB9, by transfecting with an RB expression potential mechanisms by which WrI alterations could occur in the absence of DNA mutations. the plate within 24 hours after reaching To assess the possible involvementof these Wr1 RNA processing events in the developmentof vector. HTB9 cells detached from WT, RT-PCR was perlbiredonobtal RNA isolated from 15 pnmary tumors. Loss ofrestiction sites confluency. H-CL2 cells, on the other hand, showed contact inhibition for several in the PCR productwas utiizedt lookfor both RNA processing events, the lossofa Mnl site atcodon days. The detached HTB9 cells were trypan blue-dye exclusive and showed 281 beingspeciic toexon 6 editing andthe oss oftwo MM I sites and a BamH site being specilic fragmentation of nuclei. In addition, DNA extracted from these cells showed ladder 1otheaberrantspicingoutofexon2. Almrekictonsiteioeswouldresultinaneaslydetcted mobity formation characteristic of apoptotic cells. In contrast, H-CL2 did not show the alionobservedallergesia ny reed CR productwhihdisptayed an aled mobity paternwas sequencedI diarninethe exactatation. To detnTinethe potential presence apoptotic features following confluency. ofthe alteration athe genonic level, DNA from the corresponding tumor was also sequenced. Bd-2 was not expressed in confluent HTB9 cells, whereas it was expressed Therewas no evidenceofexon 6 edting at codon 281. All RT-PCR products fromthe 15 tumors, weakly in confluent H-CL2. c-Fos was expressed in confluent HTB9 cells but at a aswell as onenonld kidney sample, displayed a Mnl restriction pattem predicted by the unedited lower level than H-CL2 cells. c-MWc was expressed in HTB9 but not in H-CL2. T at codon 281. Serum stimulation of HTB9 at confluency resulted in increased c-myc expression Only one Wmoroutof 15 primary tumors displayed aberrant splicing out of axon 2. In this tumor, -50%ofthe Wr mRNA lacked exon Z which was confirmed by sequencing ofte abnormal RT-PCR followed by DNA synthesis, whereas no increase in the c-myc or DNA level was product SequencingofithetWmor DNA revealed no alfterations within.a region flanking exon 2 (from observed in H-CL2 cells. Both cell lines expressed equally high levels ofFas antigen IVS1-30 thru IVS2+38). Southern analysis ofthe wr DNA using seven diteent restriction enzymes detectable with an anti-Fas monoclonal antibody; however, only HTB9 cells revealed no decable genonic deletions, supporting the interpretation thatexon 2 was absentfrom showed apoptotic features 24 hours after treatment with an anti-Fas antibody. the W11 mRNA due to aberrantsplicing ratherthan a heterozygous DNA deleton. This tumor was also the only one to display the relatively rare histology ofanaplasia. These results suggest that RB expression causes the difference in the expression ofc- These resuitssuggestthatneiterexon 6editing noraberrantexon 2 spicing arefrquentmechanisms myc, c-fcss, and bcl-2, and in the sensitivity to an anti-Fas antibody and that RB by which Wr1 function is altered during t ge. These observations also suggestthat aberrant expression prevents HTB9 cells from undergoing apoptosis. exon 2 spicing may be & ited othe small subsetof Wdimstumors displaying an anaplastic histoiogy.

349 350 Evidence for an effect of UV in mutation of the CDKN2 gene in Identifying Philadelphia chromosomes In DNA and RNA isolated melanoma cell lines. N.K. Hayward. P. Pollock. F. Yu. L. Qiu. and P. Parsons. rapidly from wsie same sample using non-toxic reagents. E. M. Queensland Institute of Medical Research, P.O. Royal Brisbane Hospital. Herston. Heath. R. M. ShUman. S J. Larson and C. R. Seemann. Gentra Systems, Inc. 4029. Australia. Minneapolis, MN The CDKN2 gene. encoding the cyclin dependent kinase inhibitor pI6, is a tu- Laboratories use RT-PCR routinely to identify the presence of the mour suppressor gene involved in melanoma and maps tooma cases and somatically Philadelphia chromosome in RNA isolated from peripheral blood leukocytes. in melanoma cell lines. Previous mutation analyses of melanoma cell lines have in- This is often followed by Southern blot analysis of DNA isolated from an dicated a high frequidine sites. Including three melanoma cell lines carrying tandem additional blood sample to confirm genomic rearrangements. We have CC to TT mutations, the ion in the mutagenesis of this gene in some tumours. To developed a protocol based on a combination of Purescriptf" RNA Isolation further examine this hypothesis we have characterised mutations of theDKN\2 gene reagents and Puregene® DNA Isolation reagents to isolate DNA and RNA in 30 melanoma cell lines. Nineteen lines carried complete or partial homozygous efficiently and safely from the same sample. This new protocol is not only deletions of the gene. Of the remaining cell lines. 8 were shown by direct sequencing rapid, but is useful when the amount of sample material available for analysis of PCR products from exon 1 and exon 2 to carry a total of 9 different mut U.V light is limited. To test the protocol, we examined whole blood and cultured cell samples known to be positive by independent laboratory testing for the in the genesis of melanoma, with one target being the CDKN2 tumour suppressor Philadelphia chromosome. For each sample the amount of material used for gene. RNA and DNA isolation was either .3 ml whole blood or 2 x 106 cultured K562 cells. In the protocol, total RNA is isolated initially from the whole blood or cultured cell sample using a modified salt precipitation procedure while DNA and protein are collected as byproducts. DNA is then isolated, also using a modified salt precipitation procedure. The entire procedure requires under 1 hour. In this study RNA yields averaged 1 9g per .3 ml whole blood and 8 pg per 2 x 106 cultured cells while DNA yields averaged 10 1lg per .3 ml whole blood or 2 x 106 cultured cells. RT-PCR analysis of isolated RNA using primers that detect the bcr-abl fusion genes was positive for all known positive samples tested. Southern blot analysis of DNA isolated from the same sample also was positive when BgI and Pvu II restriction fragments were hybridized with a bcr probe. This study demonstrates that Purescript and Puregene reagents may be used successfully to isolate RNA and DNA from the same sample for Philadelphia chromosome analysis.

351 352 A minimal interval for del(5q) in acute myeloid leukemia and myelodys- SomaticmutationoftheW lmstumor (WT) suppressor gene, WTI, In a WTpatient plasia. S. K. Horrigan', C. W. Westbrook' A. H. Kim' WV. Stock2, and with a constitutional 2;15 unbalanced transocation: evidence for the Involvement R. A. Larson' University of Chicago Medical School.Chicago,IL1. and Loyola Uni- ofmultiple genes in the etiology of a Wilms tumor. V. Hff', N.R. Schne I LJ. SUng', C.F. immns2, G. Tomlinson2. 'Univ. TX M.D. Anderson Cancer Center, versity Medical School. Chicago-IL2. Houston, 2Univ. TX Southwestern Medical Center, Dallas. Interstitial loss of the long arm of chromosome 3, or complete loss of the entire In rare cases, Wilmns tumor (WT), a childhood renal tumor, is associated with chromosome is a well-recognized entity in malignant myeloid disorders. including Constional karyotypic abnormalities. Germline nmtations at the lIpl3 WT gene, WTI, acute myeloid leukemia (AMIL) and myelodysplasia (MDS). We proposed the ex- have ben observed in children with bilateral tumors and/or an early age ofonset, implying istence of a tumor suppressor gene which is important to hematologic transforma- that WTI mutations predispose to WT. However, the observationofWT associated with tion. We previously specified the IL9-D5S166 interval at 3q31 as being commonly genetically defued syndromes or with non-lIp chromosomal anomalies suggests that other genetic loci play a critical role in tumorigenesis, perhaps independently of WT1 involved. To further localize the gene, we investigated loss of heterozygosity (LOH) mutations. We report here the occurrence of a somatic WT1 mutation in a tumor from using short tandem repeat (STR) markers within and flanking 3q31. We report a a WT patient with a germline unbalanced 2;15 translocation. YAC contig spanning this interval, and the order of STR markers used to detect The child was constitutionally monosomic for distal 2q and trisomic for distal 15q LOH in this study. The order of markers is cen-D5S806-D5S393- D5S816-IL9- as a result of an unbalanced translocation, der(2)t(2;15)(q37;q22), inherited from her D35S399-D5S479-afm330ybl-D5S476-D3S414-D5S500-D5S805-tel. LOH was de- unaffected father who was a balanced translocation carrier. She had multiple congenital tected with these STR's by comparing AML or MDS cells to normal tissue de- abnormalities, including a horseshoe kidney and bilateral ovarian dysgenesis. A unilateral rived from lymphoblasts or buccal mucosa. Thirty specimens were analyzed. in- Wilms tumor was detected at 25 months of age. This early age of diagnosis associated a possiblc a 22 AML and 8 LOH was found in 7 cases. Six cases had exten- with developmental anomalies suggested genetic predisposition involving cluding MDS: gene on 2q or 15q which acted independently ofmutations atthe WT1 locus. Mutational sive. cytogenetically-visible deletions, while one had a small deletion including analysis of WTI by PCR amplification of exons and SSCP analysis of PCR products afm330ybl- D5S476, with retention of IL9 and D5S414. This case thus defines revealed a heterozygous, tumor-specific, two base-pair deletion in exon 2 at codon 162 a minimal deletion interval for the the tumor suppressor gene which is estimated ofthe gene. This mutation is predicted to result in a truncated protein lacking the normal to be 1000 kb in extent. amino-terminal 287 amino acids, including those of the zinc finger domains required for normal protein function. To date, no WT1 alteration has been detected on the other allele, although mutational analysis is incomplete. These data suggest that, if the 2;15 translocationisplaying a role intmorigenesis, itisdoing so inconjunction with mutation of the WT1 gene, perhaps by altering normal kddney development so as to increase the number of cells at risk for sustaining WT1 mutations and/or to lengthen the critical time period during which abrogation of WTI function will result in tumorigenesis. Posters: Cancer Genetics (continued) A67 353 354 Localization of XFl-related sequences on chromosome 22 BRCA1 mutation testing: resufts from 68 ealy-onset bilateral T.J.M. Hulsebos. Institute of Human Genetics, Academic breast cancer Medical Center, Amsterdam, The Netherlands. families. S.A. Inales'. A. Die2'. S.-Y. Xue'. D. Shattuck-Eidens3. R. Sparkes2. Neurofibromatosis type 1 (NF1) is characterized by a R. Haile'. 'University of Southern California, 2University of Califomia at Los very high mutation rate (1 x 10-'). As a consequence, about Angeles, and Myriad Genetics, 3Salt Lake City, Utah. 50% of the cases of NF1 are the result of new mutations. A BRCA1 mutation detection strategy is described which aims to minimize To explain this high mutation rate, Marchuk et al. (Geno- brute force sequencing while maintaining high sensitivity. DNA samples from mics 13: 672-680, 1992) have proposed that NFl-related all cases in a family are first screened by ASO or RFLP for ten known mutations, sequences (pseudogenes) are reservoirs of mutations that which (according to preliminary results) may account for more han 40% can be crossed into the functional NF1 gene on 17q by of interchromosomal gene conversion. To test this hypothesis, BRCA1 mutations. In those families which screen negative for all 10 mutations, we are in the process of identifying all NFl-related each exon is screened for mutations using a fluorescent RNAse mismatch sequences in the human genome. Previous work of Marchuk et assay, followed by sequencing of any exons which screen positive. To al. suggested that such sequences say be located on determine the sensitivity of this strategy, all exons are being sequenced for chromosome 22. We isolated cosmids from a chromosome 22- cases from each familiy. enriched library (LL22NC03) that cross-hybridized with The mutation detection method is applied to 68 families from an probe AC5. The latter originated from the functional NF1 ongoing gene on 17q. The RFl-related sequences were localized on population-based study of early onset bilateral breast cancer. Cases of bilateral chromosome 22 by probing an extended chromosome 22 mapping breast cancer with onset before age 50 were identified from the Los Angeles panel with AC5 and by FISH analysis, using the cosmids. County Tumor Registry (1970-1989). the Connecticut Tumor Registry (1935- Three types of cosmids were found: Type 1 cosmids exclusi- 1989), and from hospitals which identify 95% of all cases in southern Quebec vely hybridized to the centromeric part of 22q. Type 2 Province (1975-1989). Sixty-eight families with mutliple cases of breast cancer cosmids hybridized to the same position on 22q and, in were identified. addition, to the centromeric part of 14q and to the central part of 2q. Type 3 cosmids exclusively hybridized Thus far, only one mutation has been freqently identified. The previously to the telomeric part of 22q. In additional FISH experi- reported AG deletion in exon 2 (185 -AG) (Shattuck-Eidens at al., 195) was ments, the type 1 and 2 cossids revealed positive signals found in 5 of the 68 families (7.4%/). The relatively common insertion mutation on the chromosome 22 part of the 17/22 translocation, in exon 20 (5382 +C) was found in only one family (1.5%). The distribution of contained within cell line NF13.2- This cell line harbours mutations across exons will be presented. a small part of 22q very close to the centromere. We conclude that NFl-related sequences are present in the most centromeric and telomeric regions of 22q.

355 356 Loss of heterozygosity on the short arm of chromosomes 3 and 9 in oral cancer. Genetic analyses of borderline ovarian tumor using comparative C. S. Ishwad'. R. E. Ferrell'. K. M. Rossie2. B. N. Appel2. J. T. Johnson'. E. N. genomic hybridization: Hiroshi IWABUCHI1, Hotaka SAKUNAGA3, Mvers'. J. C. Law'. S. Srivastava4. S. M. Gollin'. Departments of 1 Human Genetics, 2 Oral Medicine and Pathology, 3 Otolaryngology, Univ. Pittsburgh, Masaru SAKAMOTO3, Teresa L. YANG-FENG2, Dan PINKEL1, Joe W. Pittsburgh, Pennsylvania, National Institutes of Health, Bethesda, Maryland. GRAY1 (I Dept. of Lab. Med, Univ. of Calif. San Francosco., 2Dept. of Loss of heterozygosity (LOH) on chromosomes 3p and 9p has been Genetics, Yale Univ., 3 Sasaki inst, Kyoundo Hosp.) documented in a variety of malignancies, which suggests the presence of tumor We have performed comparative genomic hybridization (CGH) using DNA suppressor genes on these chromosomes. We have studied 85 oral carcinomas for obtained from 71 epithelial ovarian tumors (benign 12, borderline 15, low grade LOH using 16 microsatellite markers distributed over 5 human chromosomes. 18, high grade 26) with the goal of identifying grade specific genetic abnormalities Fifty-five (65%) of these tumors showed LOH at one or more marker locus. A in epithelial ovarian tumors. We report here on the analyses of the histologically significant proportion of LOH in the informative tumors was observed on borderline cases, having previously described CGH analyses of the benign and chromosomes 3p and 9p, 53% and 43%, respectively. A majority of the tumors malignant tumors. Borderline tumors fell into two classes: those that showed no showed loss at on or or multiple loci chromosomes 3p 9p both. A smaller chromosome-level DNA sequence copy number abnormalities (CNAs, 11/15) and proportion of the tumors (6-11%) showed allelic loss at markers located on chromosomes 6, 7 and 11. However, these changes occurred primarily in the those that showed multiple CNAs (4/15). Three of the 4 borderline tumors with presence of LOH on 3p and 9p. These results suggest that tumor suppressor CNAs were serous cystoadenomas. The average number of CNAs per tumor was genes located on the short arms of chromosome 3 and 9 may be involved in the high for the four tumors showing CNAs. Furthermore, the spectrum of aberrations pathogenesis of oral carcinoma. Our data on chromosome 9 is also indicative of was similar to that for advanced cancers (e.g. increased copy number for the existence of two tumor suppressor genes on this chromosome. The deleted chromosomes lp and Iq, 2, 3q, 12 and decreased copy for chromosomes 16, 17 regions observed in oral cancers overlap those reported in other neoplasms. We and 19. These data on borderline tumors suggest that a subset of borderline tumors did not find any evidence of these changes in matched adjacent oral mucosa with having multiple CNAs may be genetically unstable and thus may be expected to be early pathological changes. more aggressive clinically. Verification of this hypothesis will require additional follow-up. This work supported by USDOE contract 0376SF00098 and Vysis.

357 358 Mutation Analysis of the vHL Gene in Individuals with Sporadic and Loss of Heterozygosity (LOH) of chromosome 11 in Carcinoid Familial Pheochromocytoma. S..yengar l, G.Tallini2, G. Sirugo A.E. Bale I tumors. Orit Jakobovitz. Devorah Nash, Yuri Kopolovitch. Luiz DeMarco. and KSL Kiddl. Depts. of 1: Genetics and 2: Pathology, Yale University School of Gideon Rechavi & Eitan Friedman. The Departments of Hematology and Medicine, New Haven, CT 06520. Pathology, Tel-Hashomer, Israel; Pharmacology and Pathology UFMG, Pheochromocytomas are tumors of chromaffin cells of adrenals. About 80-90 % of Belo-Horizonte, Brazil. pheochromocytomas occur as sporadic tumors without any associated family history. Little is known about the molecular mechanisms of tumorigenesis of When this tumor occurs in families, it is often associated with genetic cancer syndromes carcinoid tumors and their clonal origin. Carcinoid tumors are rare involving neuroectodermal tissues like Multiple Endocrine Neoplasia types 2A and 2B neuroendocrine tumors that secrete Serotonin that may be encountered either (MEN2A/2B), Neurofibromatosis (NF), von Hippel-Lindau syndrome (vHL), Sturge sporadically or as part of the familial tumor complex most notably Multiple Weber syndrome and Tuberous Sclerosis. There are a few families described in the Endocrine Neoplasia type I (MEN1). The MEN] gene localizes to the long literature in which pheochromocytomas are expressed in several individuals without any arm of chromosome II (II q13) and presumably functions as a tumor other associated tumors. Several investigators have speculated that familial suppressor gene, as inferred from the frequent LOH observed in MENI- pheochromocytomas, with no other associated tumors, are allelic variants of known associated tumors. To establish whether carcinoid tumors are truly part of the syndromes. Sporadic pheochromocytomas may also involve mutations in these candidate MFEN1 syndrome complex and develop via a similar LOH mechanism of genes. Recently, investigators have found that 10-20 % of apparently sporadic chromosome 11, and indirectly determine the clonality of these tumors, we pheochromocytomas have mutations in the RET proto-oncogene. performed LOH analysis of 32 sporadic carcinoid tumors using seven Using both SSCP analysis and sequencing we analyzed DNA extracted from 17 chromosome 11 microsattelite markers. Twenty two were lung carcinoids, 8 sporadic pheochromocytomas and a family with pheochromocytoma only for mutations were appediceal/ colonic with one intrathymic and one gastric tumors. All in the vHL gene. We were able to identify the same C to T transition at nucleotide 712 patients were heterozygous for at least one marker. Twenty three (70%) of the vHL gene in two sporadic pheochromocytomas. This mutation occurs in a CpG displayed allelic loss of more then onc marker, 4 displayed LOH of an entire island which is a mutational hotspot in vHL families with pheochromocytoma and renal allele and the rest exhibited a discontinuous pattern of allelic loss. 10/20 cell carcinoma. This implies that the nt 712 mutation is important in pathogenesis of informative patients had LOH encompassing develop via an inactivation of a pheochromocytoma in both familial and sporadic cases. We also performed linkage tumor suppressor gene on chromosome 11 (presumably the the WTI gene analysis for markers at or near candidate gene regions (lOql 1.2, 3p25, 17q1 1.2, 22q12 region on Ilpl3 and 11/21 informative cases displayed LOH of the PYGM and 1 1q13) in the pheochromocytoma only family. Markers tested to date at or near marker at the MEN] gene locus. We are currently evaluating more markers 3p25, 17q1 1.2 and 1 1q13 have not given definitive results; but no co-segregation was from within the narrowed down MEN 1 region, to define the area of minimal seen with either the RET proto-oncogene (lOqll.2) or the NF-2 gene (22ql2). Lod deletion. No tumor displayed LOH when chromosome 9 markers were used. These data support the notion that carcinoid tumors MENI gene but the WTI scores of -1.78 (0 = 0.0) were obtained with two markers sTcL2 (RET) and gene is also a candidate) that the majority of them are monoclonal and that GGAT3A1 1 (NF2). (Supported in part by NIH grant CA32066) they may be genetically related to the MIENI tumor complex. A68 Posters: Cancer CAenetics (continued) 359 360 Diagnostic evaluation of patients at risk for MEN2A by direct sequencing and spread of X inactivation resulting in retinoblastoma in a boy with an ASO analysis of the RET gene. D.R. Johnson, M.W. Glynn, C.L. Pressman, A.E. unbalanced X;13 translocation. C.L, Jones, D. Rita, C. Booth, Department of Genetics, Lutheran General Hospital, Park Ridge, Bale. Yale University School of Medicine, New Haven, CT, Illinois. Mutations of axons 10, 11, and 13 of the RET proto-oncogene have been found in greater than 97% of MEN2A and 86% of FMTC kindreds (Mulligan et al, 1994; When more then 1 copy of the X chromosome is present, one of the Eng et al., 1995). The frequency of germline RET mutations in patients with X chromosomes is inactivated. This occurs as a random event in females and males with Klinefelter syndrome. However, in X-autoscaal medullary thyroid carcinoma and no family history (SMTC) is unknown. For translocations, the normal X appears to be preferentially inactivated. screening patients on a clinical diagnostic basis, we developed allele-specific This is believed to occur to prevent spread of inactivation from the X oligonuelotides for all of the common and virtually all rare mutations in exons 10 to the autosome. A male infant was born by C-section because of oligohydramnios and 11 (40 ASOs total). The one reported mutation in exon 13 could be detected at 37 weeks gestation. He was noted in the nursery to have multiple by restriction digestion because it alters an Alul site. congenital anomalies including micrognathia, hypertelorism, bilateral Seventy patients from 38 families were tested for mutations by direct sequence hip dislocations, imperforate anus, and a VSD. chromosomal analysis analysis or diagnostic restriction enzyme digestion. The mutational spectrum in showed an unbalanced translocation between Xq and 13q[46,XY,-13ider(l3)t(Xql3q) . Because chromosome 13 was involved, probands was as follows: an ophthalmologist was asked to look for the presence of Codon containing mutation retinoblastoma. A unilateral, apparently unifocal, retinoblastoma was found. Fluorescent in situ hybridization (FISH) studies showed the Disorder 609 618 634 No mutation found be fused in the derivative - centromeres of both 13 and X to apparently SMTC 1 - 14 chromosome. FISH studies also showed the XIST locus to be present on FMTC 1 2 - 1 both the normal and translocated X and the RS locus on both the normal MEN2A 2 3 12 2. 13 and the translocated 13 was also present. In late replication In studies, most of the Xq in the derivative translocated chromosome was the 21 kindreds for which a mutation was identified, 10 of 28 1st degree late replicating in all cells visualized. Spreading inactivation onto relatives of probands were found to be gene carriers. the autosome was also present. Inactivation of one RB locus by the For ASO analysis, pools of up to 6 mutation-specific 1 5mers were hybridized spread of X-inactivation from the X to 13 is postulated as the first to dot blotted PCR products. Analysis of 60 individuals for exon 10 and 11 hit in the development of the retinoblastoma. mutations yielded 1 false positive and zero false negatives. The reason for the false positive result is being investigated. ASO analysis is a rapid, accurate method of mutation screening in the RET gene. The finding of a germline mutation in one patient with apparently sporadic MTC provides support for mutational screening in this group of patients.

361 362 Fine tumor deletion mapping localizes a putative 17q25 gene involved Diagnosis of Multiple Endocrine Neoplasia [MENI 2A, 2B & Familial Medullary in breast carcinogenesis. ICalikn' , ' TS rap' DPtsAuff. SM Thyroid Cancer LFMTCI by multiplex PCR and heteroduplex analyses of RET proto. Svoboda'ADJ Law'..and EM Petty. Depts. of 'Int. Med., 2Path., and 'Hum. Genet., oncogene mutations. M. Kambouris. C.E. Jackson & G.L. Feldman. Medical Univ. ofMichigan Med. School, Ann Arbor, MI. 48109 Genetics and Birth Defects Center, Henry Ford Hospital, Detroit, Michigan, USA. Breast cancer is one of the leading causes of morbidity and mortality for women in endocrine 2 [MEN 21 is an autosomal dominant cancer the United States. Although significant progress has been made in elucidating several Multiple neoplasia type molecula genetic mechanisms involved in breast carcinogenesis, it is clear that there syndrome with two subtypes, 2A and 2B. MEN 2A and medullary thyroid cancer are additional genes that may play pivotal roles in the development and/or progression [MTC] are caused by over 25 different point mutations in exons 10, 1 1 and 13 of of these neoplasms as suggested by genome wide amplification and loss of the RET proto-oncogene, while MEN 2B is caused by a single exon 16 point heterozygosity (LOH) studies. Like other solid tumors, it is hypothesized that mutation. Various molecular methods have been used to identify the different alterations in multiple growth regulatory genes and interactions between these genes mutations, including DNA sequencing, restriction enzymatic analyses, Single may be critical to tumor development. Characterization ofall involved genes will be Stranded Conformational Polymorphism ISSCPI and Denaturing Gradient Gel UsCial to u rding the pathogenesis ofand facilitating the medical management of Electrophoresis [DGGEI. These techniques, while useful and accurate, are labor breast cancer. Our efforts confinn and further localize a putative gene involved in intensive and some involve the use of radioactivity. We have developed a breast tumorigenesis which maps to chromosome 17q25 (Saito H et al. Cancer Res multiplex PCR assay to simultaneously amplify exons 10, 11 and 13 of the RET 1993; Slovak ML et al Cancer Res 1992). proto-oncogene. The multiplex PCR product is then analyzed on a modified DNA exaced from 39 matched normal and punmary sporadic breast tumor paraffin- Mutation Detection Enhancement [MDE1 matrix for identification and embedded microdissected tissue samples are being analyzed for (LOlH) with short heteroduplex tandem repeat polymorphic markers (STRPs) from loci which span 17q25. LOH visualized with ethidium bromide. Distinct heteroduplexes were detected for each studies to date demonstrate significant rates of loss: 24%, 33%, 45%, 30%, 23%, and RET proto-oncogene mutation available in our laboratory (eight in exon 10, five in 25% for STRPILocus GATAlOF10 1D17S968, [UT40, UT968], AFM07ye3/ axon 11, one in axon 13, and the single exon 16 mutation). We are currently D17S789, AFMI63yglID17S937, and UT962/D17S914, respectively (listed using this method for diagnostic purposes and we are analyzing the remaining proximal to distal except [ ] markers where the order is not known). The smallest known RET mutations utilizing this system. Pre-symptomatic DNA diagnosis of distinctregion of tumordeletion overlap is limited to the region of 17q25 between MEN 2 is essential since pentagastrin-stimulated calcitonin studies can produce D17S968 and D17S914, although the boundaries remain tentative given the small false positive results and lead to unnecessary thyroidectomies. Prophylactic number oftumors studied to date. Analysis of tumors with localized 17q25 loss for thyroidectomy is recommended by age five or six once a mutation is identified in involvement ofBRCAI and TP53 is underway. Continued LOH analysis within the a patient, since penetrance is essentially 100%. MDE heteroduplex detection smallest region ofoverlap with more tumors and markers, including those developed a efficient and method of for RET from YACs and Pls we have isolated in this region, will facilitate gene isolation. provides quick, inexpensive screening mutations in MTC patients with unknown mutations, or for presymptomatic diagnosis in individuals at risk for inheriting a known RET mutation.

363 364 Loss of heterozygosity in acute myeloid leukemia and myelodysplastic Deletion ofCDK4 inhibitor genes P15 and P16 in nom-Hodgla's lymphoma syndrome at 7q. S. Kiuru-Kuhlefelt, S. Knuutila and J. Kere. Department of PRK_ K =durand I D _Brnnme North Shore University Hospital, Manhasset, NY. Medical Genetics, University of Helsinki, Finland. Deletion of a part of chromosome 7q (7q-) and monosomy 7 (-7) are amongst A simple definition for cancer is uestrained cell growth. Conceivably, any the most frequent aberrations in patients with acute myeloid leukemias (AML) and change in cellular mechanism that regulate cell growth (division) may have implication myelodysplastic syndromes (MDS). To study the variation of the size and location in tumongenesis. In non-Hodgkin's lymphoma (NHL), activation ofoncogenes (such of the deletions we collected blood samples from 12 AML or MDS patients with 7q- as cMYC, BCL1, BCL2 and BCL6) has been implicated in the histogenesis of or -7 exclusively or among other aberrations. In order to compare the normal and diferent histologic groups. In addition, inactivation ofP53 gene, a cell cycle affected cell lineages, mononuclear cells (representing the normal cell lineage) and regulator, has been implicated in progression ofthe disease to an advanced histologic granulocytes (representing the affected lineage) were separated by Ficoll gradient grade. Recently several genes such as those ofPI5, P16, P18, P21 which inhibit the shown to centrifugation and DNA was extracted. Loss of heterozygosity (LOH) was studied cell cycle have been characterized, and two ofthese, P15 ad P16 have been by PCR amplifications of 50 microsatellite markers on 7q. In a parallell approach, be frequentlydeleted in some types ofprimay tumorsbut not in others. We have comparative genomic hybridization (CGH) was used to confirm the presence of imnestigated the status ofthe four genes in a series of155 B-cell NHL comprising three histologic groups - small lymphocytic lymphoma (42 tumors), follicular small deletion in the granulocyte and not lymphocyte DNA. LOH was detected at four du to nine informative loci on 7q in four patients, in agreement with the CGH results. cleaved cell lymphoma (36 tumors) and large cell lymphoma (77 tumors) using Southern blot and chain reaction - strand The proximal breakpoints varied considerably as did the size of the deleted area. anlsis poymerase single conformation were no nucleotide All deletions in these four were interstitial. The common deleted polymorphism analysis. There acquired changes (point mutations) patients segment in the ofthe in ofthese we found was delineated by the microsatellite markers D7S515 and D7S685. coding regions genes any tumors. However, deletion ofboth P15 and but not ofPI8 or in 10 tumors of Our results indicate that deletions at in AML and are more homozygous P16, P21, the 7q MDS patients but not in the other two In these 10 there variable in both size and location than suggested earlier. The recurrent area of diffuse histology histologic groups. tuMnors was no observable of the siteto which these genes have LOH is located in an area where the presence of a tumor suppressor gene in other karyotypic abnormality 9p21, been mapped. These data suggest that loss ofP16 and / or P15 may contribute to the than myeloid malignancies has been suspected. tumorigenesis ofdiffuse NHL. Posters: Cancer Genetics (continued) A69 365 366 ANDROGEN RECEPTOR GENE AMPLIFICATION IN RECURRENT Gene-environment interaction in an African-Amerian child with nevoid basal cell PROSTATE CARCINOMAS FROM PATIENTS TREATED WITH carcinoma syndrome. U. Korczak' JJ. DiGiovanna'. J. Brahimn. R.G. Kase5 and A.M. Institute Research, ANDROGEN DEPRIVATION. P. Koivisto T' viakrp E Hyynen. M Tane Goldstein'. 'National Cancer Institute and National of Dental Bethesda, MD,3Westat, Inc., Rockville, MD. C_Palmher. T- Tamiea. bola and -P Lab. of Cancer Genetics, basal cell carcinoma syndrome (NBCC) is an dominant disorder FIN-33521 Finlandi Nevoid autosomal Tampere University Hospital, Tampere, with variable expressivity. The major clinical findings include basal cell carcinomas Recurrence ofprostate cancer during androgen deprivation therapy is a major (BCCs), jaw cysts, palmar/plantar pits, skeletal anomalies, and ectopic calcification. The problen The molecular mechanisms ofendocrine treatment are clhical faiudre poorly frequency of BCCs differs in whites with NBCC (90%) vs. African-Americans with kow. Studi by comparative genomic hybridization have indicated that specimens NBCC (30%), suggesting ultraviolet radiation protection from increased skin from prostate cancers from patients treated by conventional androgen recurrent pigmentation in African-Americans. Reports of NBCC in African-Americans are rare. 1 deprivation (surgical or chemical castration) contain amplification of the Xql-q12 Medulloblastomas occur in 2-5% of affected individuals, with an average age at regio. Sincehuman androgen receptor (AR) gene maps to Xql 1, we studied whether diagnosis of 2 years. ARisthe targ gene oftheamplfcation.FSH-anabsis with an AR-specific P probe We report the case ofa 6-year-old African-American male with NBCC. At 2 years showed high-level amplifcation (3-40 AR copies per cell) in twelve (27%) of 44 of age, he had surgery, radiation and chemotherapy for a desmoplastic medulloblastoma. reounust, hormone-refactosytumors. None ofthe primary tumor specimens obtained At 6 years of age, he developed a swollen and misshapen jaw and was diagnosed with from the same patients before therapy showed AR amplification. AR gene was also an odontogenic keratocyst. Physical examination revealed a large head circumference, highlyewressed basd onimmnohistochemical studies. frontal bossing, high-arched palate, and extensive pitting of the palms and soles. In the These results suggest a novel molecular mechanism by which resistance to addition, the patient had numerous BCCs (2 biopsy proven) that were restricted to of the androgen deprivation therapy may develop. Gene amplification apparently leads to zone of previous radiation therapy for his medulloblastoma. Examination mother revealed she also had NBCC. She had calcification of nResedARexpressaon, enabling tumor cells to grow and avoid apoptotic cell death patient's that extensive the falx cerebri and dura mater, multiple jaw cysts, impacted and supernumerary teeth, in an androgen-eficient environment. Geneamplification has often been implicated in a large head circumference and high-arched palate, but no BCCs. drug resistance in vitro. ARamplification is the first example of such a mechanism as This case is noteworthy in several regards. First, to our knowledge this is the first a cause oftherapy resistance ina common solid tumor in vivo. documented case of medulloblastoma in an African-American NBCC patient who developed BCCs subsequent to radiation therapy. Second, it illustrates the complex interaction between a genetic predisposition to BCC development and an environmental stimulus, i.e., radiation therapy. Finally, one should consider NBCC in the differential diagnosis of a patient who presents with medulloblastoma and to develop treatment protocols that consider the hypersensitivity of patients with NBCC to ionizing radiation.

367 368 Identification and characterization of genomic loci preferentially COMPARISON OF GENETIC CHANGES IN PRIMARY BREAST affected by a mutator phenotype in cancer using an Alu-PCR approach. CARCINOMAS AND THEIR DISTANT METASTASES BY CGIL T. Kasiu, M. J. Lab of M.Krinovic. C. Richer. D. Labuda and D. Sinnett. Charles Bruneau Cancer KmkasRvi, IL Mi Kfihkdnen, Tanner,O-P. Kallioniemi, Isola. Center, Research Center,H8pital Ste-Justine and Univ. de Montrdal, Canada. Cancer Genetics, Institute of Medical Genetics, Tampere University Hospital and A mutator phenotype (genomic instability), usually revealed as University of Tampere, P.O. Box 607, FIN-33101 Tampere, Finland expssion/contraction in microsatellites, has been observed in several types While metastasis is perhaps the most important step ofbreast cancer progression, of cancer (both hereditary and sporadic). In order to study the pattern of the molecular mechanisms underlying metastasis as well as the degree of clonal these instabilities in different cancer genomes we propose a PCR-based DNA relatnshipbeeen the metastatic cells and the cells ofthe priary tumor are unknown. genomic has now enabled analysis ofthe degree of fingerprinting approach. The 3' A-rich tail ofAlu elements and sequences Comparative hybridization (CGH) downstream often display instabilities in cancers making thus a good target genetic similarty between genetically complex tumor specimens obtained from archival to of clonal relationship (CR) for a genome wide screening for a mutator phenotype. Using Alu-specific tissue. We have used this CGH approach study the degree 19 their asynchronous distant primers we amplified by PCR the genomic segments between inversely between primary breast carcinomas and metastases oriented Alu elements. The comparison of Alu-PCR fingerprints (>50 appearing 3-168 months post-operatively in patients who received no systemic therapy. of the 19 pairs (47%) showed either a distinct or probable CR bands/gel) from tumoral and normal genomic DNA of the same individual Nine specimen and metastasis. Common clonal was defined as the allows the detection of tumor-associated genetic alterations (gain, loss or shift between the prinmay tumor its origin of bands). In particular, the analysis of DNA of 8 patients affected with presence of three or more identical genetic changes and breakpoints. In contrast, six specimen pairs (32%) showed no evidence of CR with virtually all changes and hereditary colon cancer (HNPCC) revealed a number of instabilities in each being different. The four remaining cases could not be reliably evaluated for Alu-PCR fingerprint. Some loci were seen unstable in only one patient while breakpoints CR due to the absence or ofinformative genetic changes and breakpoints. others were altered in most patients tested suggesting that certain genomic snallmimber Themean time to metstasis washigher in specimen pairs that showed no CR (67.5 regions are preferentially affected by the mutator phenotype observed in mo., range 15-168) as compared with those showing CR (30.4 mo, range 3-108). cancer cells. cloning and sequencing we determined the nature of the By five (59%) ofthe nine specimens with CR showed a higher number of sequence instability in these genomic loci which were then assigned to a Surpsisingly, genetic changes in the primary tumor than in the metastasis. In these cases, the chromosome. This approach was also used to detect and localize to metastnasi may have originated from an ancestral clonal variant that has departed very chromosome 9q an Alu-PCR fragment lost in tumor tissue of one sporadic early on from the primary tumor. In four cases (41%), the metastases contained more patient. In this work we have demonstrated the usefulness of colon cancer genetic changes than the corresponding primary tumors. Gain of Xq appeared to be fingerprinting to identify and isolate genomic loci associated with Alu-PCR selected for the metastases suggesting that this chromosomal region may contain of and genomic instabilities. (Supported by MRC of in losses heterozygosity genes with important role in the metastatic process. Canada). an

369 370 Detection of gene rearrangement by interphase cytogenetics Specificity of p53 gene mutations, in oral cancer and the role of tobacco smoke in acute promyelocytic leukemia in early clinical remis- exposure. J.C. Law'. J.T. Johnson2. R. E. Ferrell'. D. Kerestan'. A. Deka'. and S.G. sion. J.LaSala M.J.Macera. J.Godwin T-Szatrowski and R.S. Gollin'. Departments of Human Genetics' and Otolaryngology2, University of Vprma. New York Hospital-Cornell Medical Center, New York, Pittsburgh and the Pittsburgh Cancer Institute. N.Y. and Long Island College Hospital/SUNY Health Science Oral cancer is one of the most common cancers worldwide. Mutations of the p53 Center at Brooklyn, N.Y. tumor suppressor gene are the most common genetic alterations associated with In acute promyelocytic leukemia (APL), the most common human cancer. Mutations of p53 have been shown to occur frequently in various translocation is between chromosomes 15 and 17. The re- head and neck cancers including those originating at oral and oropharyngeal sites. tinoic acid receptor alpha gene on 17q is disrupted by this Tobacco smoke consists of potentially mutagenic substances and is a significant risk to an abnormal translocation and leads accumulation of factor for oral cancer. Environmental agents which induce specific mutational events Treatment with all-trans retinoic acid has promyelocytes. may exert a general field cancerization effect on the exposed tissues and may in inducing differentiation of the leukemic been effective produce mutational profiles which are characteristic of the mutation- cells resulting in clinical remission. At the time of tissue-specific inducing agents. The mutational frequency and specificity of the p53 gene was complete remission, cytogenetic analysis of the marrow is typically normal. determined for 70 oral tumors. An overall p53 mutational frequency of approximately 27% was observed. All mutations were single base substitutions with the sample from a patient with APL at the time (19/70) A bone marrow mutations of first remission, was apparently normal (46,XY) by QFQ- majority (59%) being transition mutations. Eighty percent of the transition banding in all 30 metaphases counted. Cells from the same were G:C-'A:T, all of which occurred at CpG dinucleotide sites. This type of mutation is associated with processes, primarily methylation of cytosine to 5- sample were then hybridized with a dual color APL specific endogenous probe (Oncor). Evaluation by FISH revealed 10 metaphases methylcytosine and subsequent spontaneous deamination to thymine. Smokers which were negative for the translocation. Forty inter- comprised the majority of the total study population (76%) and represented 82% of phase nuclei were also evaluated; six of these revealed the the patients identified with p53 mutations. Surprisingly, the type of p53 mutation APL translocation identifying a leukemic lineage. commonly identified in lung cancer, G:C-T:A, was present in only one of the oral Since retinoic acid may have a role in the treatment of tumors identified with p53 mutations. Polycyclic aromatic hydrocarbons (PAHs) in de novo and relapsed APL, an accurate assessment of leu- tobacco smoke (ie., benzo--pyrene) are known to cause G:C-T:A nucleotide has been kemic cells is imperative. Cytogenetic analysis substitutions. Our results suggest that while PAH-induced mutations may play a for clinical stage verification. This type of helpful significant role in lung cancer their role in oral cancer is less certain. analysis is limited, however, to cells actively undergoing cell division. Through the use of interphase cytogenetics, it is now possible to identify the persistence of clonal gene rearrangements in bone marrow samples. This techniqua may, therefore, be useful in guiding therapeutic decisions. A70 Posters: Cancer Genetics (continued) 371 372 Molecular and cytogenetic alterations In bepatoblastoma.1E-Lee 11q13 a.p.leto ad celn DI rp o oralcam oma. 1N&L&Schneider.sLiTonl.2E&.gJDaulaA 3 uFinegd G E. Tomlinson. . Rossie.2 Michalides4 N. M tlUniv. Texas Southwestern Medical Center at Dallas, 2St. Christopher's Depts. of Human Genetics, 'Oral Medicine and Pathology, andOolrnlg, Children's Hospital, Philadelphia, PA, 3Texas Children's Hospital, Houston. Univ. of Pittsburgh and the Univ. of Pittsburgh Cancer Institute, Pithbrgh, PA Hepatoblastoma is A rare liver tumor of early childhood. Its pathogenesis and 4Netherlands Cancer Institute, Amsterdam, The Netherlands may involve a combination of both constitutional and acquired genetic The 11q13 region is amplified in 30"6Wo of head and neck squamous cell factors. Hepatoblutoma has been observed to occur in conjunction with carcinomas. In it reside oncogenes and cell cycle genes including IN12M, HSM1, several cancer predisposition syndromes including Beckwith-Wiedemann in this a Syndrome (BWS), familial adenomatous polyposis coli (APC) and rarely in CCND1, and EMS1. Speculation surrounds which gene region plays key Li-Fraumeni syndrome. role in tumor development and/or progression. To examine alterations in the To better understand the genetic basis of hepatoblastoma, cytogenetic and 11q13 amplicon in oral carcinogenesis, we are analyzing the INTD andCCND IoeL. molecular genetic studies of a series of hepatoblastomas were undertaken. Using dual color FISH with an a-satellite probe for chromosome 11 ad an INM Loss of heterozygosity (LOH) profiles were determined at the loci of known cosmid (Oncor, Gaithersburg, MD), we examined DNT2 amplification i 26 oral cancer predisposition genes. Seven of twelve informative cases squamous cell carcinoma (OSCC) cell lines established in our laboratory, 13 demonstrated LOH at the BWS locus at Ilpl5. Retention of heterozygosity and 5 fresh tumor cell harvests for was observed at the APC loci in 11 informative sporadic cases, however LOH corresponding fresh tumor cell harvests, which was observed in one patient from a polyposis kindred. At the p53 locus, cell lines were not developed. 9/13 (69%) paired OSCC cell lines and fresh amor etention of heterozygosity was observed in 5 of 5 informative cases and no cell harvests, 8/13 (62%) cell lines for which fresh tumor cell harvests we not mutations of the p53 gene were observed by SSCP analysis. available, and 1/5 (20%o) fresh tumor cell harvests without cell lines epessed Karyotype studies revealed abnormal karyotypes in 14 of 24 cases of 1NT2 amplification. To study the expression of cyclin DI in these OSCC hepatoblastoma; however, no cytogenetic abnormalities were observed specimens, immunocytochemistry with a polyclonal antibody to the cylin DI involving the two predisposition loci most commonly implicated in Res 55:975-978 was carried out on formalin-fixed, hepatoblastoma, APC or BWS. One tumor demonstrated a del(17)(pl2), protein [Cancer (1995)] paraffin however immunostaining of this tumor for p53 was negative and SSCP embedded OSCC and matched adjacent mucosa (when available). 16/30 (53%) analysis revealed no mutation in the retained copy of the p53 gene. OSCC and 5/24 (21%/6) adjacent mucosa overexpressed the cyclin DI proteinL These findings suggest that, although both constitutional and acquired Eight cases showed discordant results between INT2 amplification and cyclin DI genetic changes at the BWS locus may play a role in the development of overexpression. Fie were positive for INT2 amplificaton, but negative for cydin kepatoblastoma. other predisposition genes APC and p53 may play less of a DI overexpression. Three were negative for INT2 amplification, but positive for role in the sporadic forms of this tumor. demonstrate correlation between This work performed in part in conjunction with the Pediatric Oncology cyclin DI overexpression. These results strong Group Hepatoblastoma Biology Study. INTD amplification and cyclin Dl overexpression and also suggest thatmeanis other than 11q13 amplification are responsible for cyclin Dl overe on

373 374 Dicentric, Tetracentric, and Octocentric Ring Chromosome 15 in AML-M6 A novel hMSH2 mutation In an extended colorectal cancer kindred J G Levine M R Rossi G Gadi and P R Paphausen Dept. of Cytogenetics, with *xtracolonic cancers. C. M. Lewis. J. Swensen. F. J. Black, D. Lab Corp ofAmerica, RTP, NC. Daley. L. A. Cannon-Albright. M. H. Skolnick. University of Utah School of Erythroleukemias (AML-M6) frequently exhibit a variety ofclonal cytogenetic Medicine, Salt Lake City. anomalies. We report complex clonal anomalies including an unusual large dicentric The mismatch repair gene hMSH2 is responsible for approximately 30% r(15), also present in tetracentric and octocentric versions, with no nonnal 15 of cases of hereditary non-polyposis colorectal cancer (HNPCC). We homologues. Cytogenetic analysis of90 GTW-banded metaphases from 24-hr screened 14 young cases from 11 colorectal cancer kindreds for gemiline unstimulated bone marrow cultures from a 68 year old male with AML-M6 revealed mutations in hMSH2. Eight kindreds had previously been tested for complex polyploid clonal anomalies consistent with refractile AML. Ofthe 90 linkage to hMSH2 using D2S119, D2S391 and D2S123. Two kindreds metaphases, 9 were hypodiploid: 44, XY, -4, der(5)t(5;17Xql 1;ql 1), add (11)(q23), - (K2064, K2166) had high lod scores suggesting linkage to hMSH2. The +marl, +mar2; 42 were hypotetraploid; were seven unlinked kindreds were tested to eliminate the possibility that a 12, dicr(l5), idic(15),-16,-17, 38 subset of the cases in each kindred is due to hMSH2, but would be hypooctaploid and 1 was normal (46, XY). undetectable through linkage analysis. Three kindreds were too small or The dicentric r(15)'s and idic (15Xql3)'s were positive by FISH using chromosome had too few DNA samples to test for linkage. 15 paint (ONCOR) and GABRB3 probe (ONCOR), respectively. This suggested that Genomic DNA was PCR amplified and screened by sequencing. the distal PWS/AS-associated breaksite may have been involved. No monocentric Only one germline mutation was detected. This novel mutation in K2064 Is r(15)'s or normal chromosome 15's were present. Hypodiploid metaphases contained I a frame shift due to a single base-pair deletion in exon 16 (2730 del C dicentric r(15); tetraploid and octaploid metaphases had 2 and 4 dicentric r(15)'s and ->tAer 891). The hMSH2 mutation carmers in the five generational kindred idic(15Xql3)'s, respectively. Some metaphases had highly unusual tetracentric or include 10 confirmed cases of colorectal cancer, four cases of endometrial octocentric r(15)'s or chain-like forms. Because the rings contained symmetrically cancer (including two colorectal and endometrial double primary sites), and arranged head-to-tail portions ofchromosome 15, they most likely arose by a one each of thyroid, ovarian, ureter and prostate cancer, The carrier status mechanism involving sister chromatid exchange following initial ring formation of three kidney cancers and one parotid cancer remain to be determined. between the translocated 15q13 -e q ter and the other 15. Subsequent duplication of the Site-specific and age-dependent penetrances will be presented. K2064 was 1Sp -+ q3 to form the idic (15) is proposed. the only kindred sampled which met the Arnsterdam criteria for HNPCC. To our knowledge, this is the first report ofdicentric, tetracentric and octocentric One kindred (K2053) had a rare hMSH2 splice site polymorphism r(15)'s in human leukemia and the third literature report of any human octocentric ring (exon 7, -10 T->C) which was present in 2/83 random individuals (frequency chromosome. 1.2%). This polymorphism gives a lod score of 1.5 in K2053, suggesting linkage to hMSH2, although no germline mutation was detected.

375 376 Theray-Rated Chromosomi Chune in Human Gloma. ((You.S. Identification of a novel amplified gene in human breast cancer Li'. David A. Ramsay'. David R. Macdonald'. Rolando F. Del Maestro'. containing a polymorphic trinucleotide repeat. T. Lin. X-Y. Guan. Yao-Shan Fan'.)) Department of Pathology', and Division of S.L Anzick. J. Xu. M. H. Polymeropoulos. J.M. Trent and P. S. Meltzer. and Brain Tumour Research Laboratory2, Victoria National Center for Human Genome Research, NIH, Bethesda, MD. Neurosurgery Breast cancer is frequently characterized by amplification of cellular Hospital, London, Ontario, Canada; and Regional Cancer Centre3, genes. In an effort to isolate novel amplified genes, we have used a London, Ontario, Canada. chromosome micodissection based strategy to isolate candidate genes The karyotypes of 20 primary and 9 recurrent gliomas from 26 encoded by a homogeneously staining region from the human breast cancer patients are described based on observations using standard cell line MCF-7. This approach has isolated several cDNAs including one cytogenetic techniques. A comparative study between the primary (designated Group 1) which hybridizes to an 11 kb mRNA. Sequence and recurrent gliomas revealed therapy-related chromosome changes, analysis of the Group I cDNA demonstrated a CAG trinucleotide repeat. including (1) deletions of the long arm of chromosome 7 with Because trinucleotide repeats are frequently polymorphic and their a role in a number of dominant breakpoint at q22, possibly induced by alkylating agents, and (2) expansion__1---- plays--- - autosomal neurodegenerative disorders, we investigated the group 1 gene for numerous single cell abnormalities or unrelated clones of structural polymorphism in this region. PCR analysis with specific primers Identified 5 abnormalities without a prevailing clone, presumably induced by alleles in CEPH families on polyacrylamide gels of which three were radiotherapy. All these abnormalities occurred exclusively in recurrent sequenced. Sequencing established a variable portion of the trinucleotide gliomas. The radiation-induced abnormalities were non-random with repeat consisting of (CAG)M^ CAA (CAG)8.g followed by a conserved two recurrent breakpoints, 1 p36 and 1q42, which may harbour sequence of (CM CAG)4 CAG CAA (CAG)2 CAA. The Group 1 gene was tumour suppressor genes. It is postulated that these mapped to human chromosome 20ql 1 by fluorescent in situ hybridization radiochemotherapy induced genomic changes may have contributed and was localized by linkage analysis to the genetic interval from D20S119 to the growth of recurrent gliomas. to D20S75. The multiple interruptions in the trinucleotide repeat pattem suggests that this gene is unlikely to undergo trinucleotide repeat expansion. Sequence analysis is in progress to evaluate the function of this gene and its possible role in mammary neoplasia. Posters: Cancer Gene1tics (continued) A71 377 378 Asnchrony In replication timing of allelic DNA sequences in cells Aberrant hypermethylation of the Major breakpoint cluster region (M- derived from ovarian cancer patients. T. Litmanovitchl, A. DotanI, bcr) In Philadelphia chromosome (Ph) positive chronic myeloid leukemia M. Altaras2. (CML). C. E. Litz K. T. Stieglbauer. M. N Hanson. C. M. Cphaviw Dept of T. Leibovici3 and L. Avivil. lHuman Genet. Dept., Lab. Med. and Pathology, Univ. of Minn. Medical School, Minneapolis, MN Sackler School of Medicine, Tel Aviv University, Israel. The t(9;22), Ph, is the biologic hallmark of CML. The Ph breakpoints on 2Gynecological Oncology Unit, Sapir Medicine Center, Kfar Saba, chromosome 22 are usually confined to the 6Kb M-bcr in the BCR gene. An isolated Israel. 3Pathology Dept., Sackler School of Medicine, Tel Aviv 600bp island of hypomethylation has been described in the M-bcr in normal University, Israel. he atopoiic cells and coincides with the region where most CML breakpoints occur. The pattern of replication timing of a-satellite allelic DNA Inl CML, this island is frequently aberrantly hypermethylated (Litz et al, Cancer Res. 50:4984). The pme t study determined whetherthe M-bcr brakpoint lation in CML sequences of chromosomes 10, 11, 17 and X was studied by FISH in crrelaes with M-bcr hypermethylation. The M-bcr methylation status of marrow normal female cells (lymphocytes), in malignant cells (ovarian specimens from 49 CML patients in chronic phase was examined by a Southern bot tumors), and in non-malignant cells (lymphocytes) of ovarian cancer y employing B ll genomic digests probed with the 5' Bgl M-bcr patients. Normal cells showed a high level of synchronization in f nt.The M-bcrtrakpint in each specimen was restriction mapped to one of six replication timing of the two allelic sequences of each pair of M-bcr subregions by Souten blot. 17 cases (34%) had abantly hypermethylated autosomes, and a pattern of early and late replication timing of the rearranged M-bcrs when compared to the unrearranged M-bcrs of normal marrow control specimens (n +3 S.D.). All 17 cases had M-bcr breakpoints located 5' of two sequences of the X-chromosomes. Evidently, the a-satellite the M-bcr ScaI site (p<0.000l). The 32 remaining cases had M-bcr breakpoints located sequences on the X-chromosome do not escape X-chromosome 3' of the M-bcr BspHI site. Sequence breakpoint determination of the normally and inactivation. However, both in malignant and in non-malignant hypermethylated cases cells of these patients, the allelic sequences of the autosomes in each wiXth M-bcar eBsWR PATAL RESTRICTIONMAP in of the three loci exhibited replication asynchrony. Since replication breakpoints the w MAIWe l timing is known to be correlated with DNA transcriptional activity, ZO0bp M-bcr~Bogg, Exdo apiHmd * we assume that the observed in the was BspHUScaIfragment asynchrony patients' cells ieeld no comanton| _ II associated with epigenetic phenomena affecting DNA-function. of methylation status ' ' T. BJH lIMdao Whether the asynchrony in non-malignant cells of ovarian cancer and breakpoint 1Kb NaP"Sa patients is a consequence of the disease, an early sign of malignancy oction within thisfragment. It is concluded that the aberrantly hypermetylated M-bcr or a marker of genetic predisposition is currently under study. occurs in approximately one third of cases of CML and is associated with M-bcr breakpoints 5' to the Scal site. The aberrancy does not appear to be the result of the translocation of 3' M-bcr DNA sequences. The role of this hypermethylation in the t(9;22) is beingexamined.

379 380 TeDIB: a database re for telomeres. C. Helms1,L.il, Proximal 3p allele loss in VHL-associated and sporadic pancreatic islet leiU,- jH Donis-Kellerl2 1Division of Human Molecular Geneties, Dept. cell tumors suggest a multistep model for VHL tumor development. SurgerY, 2Depts. of Genetics and Psychiatry, Washington Univ. School ofMedicine, S. T. Lott,L S. A. Curley. M. L. Frazier and A. M. Killary. M. D. Anderson Can- St. Louis, MO, USA. cer Ctr., H6usthonTeixas. The study of telomeres and proterminal regions has intensified during the past Von Hippel-Lindau (VHL) disease is an autosomal dominant disorder character- few yew and continues to expand. For example, more than 200 telomcrc papers were ized by the development of retinal and central nervous system hemangioblastomas, published between 1992 and 1994. A number of model organisms have been utilized renal cell carcinomas (RCC)., pheochromocytomas and pancreatic islet cell tumors and a variety of experimental approaches have been applied including physical (PICT). We report a multigeneration VHL kindred in which three affected female mappin genetic analysis, cytology, and enzymological and biochemical methods. siblings developed PICT at an early age. Analysis of the three coding exons of the In order to unify these diverse elements and to provide a convenient source of VHL gene in these affected individuals has revealed a single, missense mutation in information for the scientific community, we have begun construction of a telomere codon 238. This mutation has been reported to be the most frequent mutation in database em (TelDB), and have a working prototype that is accessible over the pheochromocytomas in individuals affected with VHL (62%). Our pedigree, how- intrnet. TeIDB provides an electronic bulletin board for discussions of telomere ever, fails to identify any individuals with pheochromocytomas with the exception research issue, and includes meeting announcements, data submission forms, and of the mother of the three affected siblings. Thus, the VHL codon 238 mutation en-line help. Telomere information is categorized by organism and topic and and may predispose to pheochromocytoma in this family; however, it does not explain includes raphical representations of maps and associated data (e.g. in situ the preponderence of PICT in the third generation. These data suggest that a hybridization images). Telomere data has also been assembled in several specialized strict genotype/phenotype correlation may not be sufficient to explain the progres- databases within TeIDB that can be queried, and reports of searches can be printed by sion to malignancy in VHL associated neoplasms. We have hypothesized that the the user or stored as electronic files. Forexample, the human polymorphism telomere VHL gene may be required for benign hyperplastic growth; and in fact, most of database includes genetic markers placed within the terminal band of each the tumors associated with VHL are benign. However, since the two tumor types chromosome. The citation database will be comprehensive, including publications fom 1936 to the present (within the and can be searched that most frequently show malignant conversion in VHL are RCC and PICT, we previous month) using therefore explored the possibility that loss of additional tumor suppressor loci were such as, author, year of publication, reviews, organism, and a variety of required for malignant conversion in the siblings with We other key terms such as human cancer. In addition, a telomere tutorial aimed at the PICT. have examined undergraduate teaching level will be available for newcomers to telomere research, these affected individuals for loss of heterozygosity (LOH) in the proximal region and a 'kids page' will provide a learning resource for junior scientists (K-12). of chromsome 3p. which has been implicated by high frequency LOH in both the Curton of TelDB will be the responsibility of staff and volunteer editors. TelDB is familial and sporadic forms of RCC. Our results have shown losses in the affected Pa of GenLink: a resource for human genetics, and is available over the internet siblings involving the entire short arm of chromosome 3. In addition, LOH stud- (1stp:/Awww.gelink.wust.edu/teldb/). ies conducted on sporadic PlCT have shown high frequency losses at 3p21. These data suggest the involvement of proximal genetic loci on chromosome 3p in the development of PICT, and suggest a stepwise mutation mechanism as a potential explanation for the development of VHL-associated tumors.

381 382 p16 (CDKN2) is commonly mutated in melanoma predisposed patients, dlc(9;20) In four patients with childhood ALL KQ. Maben1, J. Be rn, but rarely In melanoma tumours. M. Lynche, A. Ruizl, S. Eui&L_ P. lBreifeld2, P-S. Nelman3, G.H. Vencel, arnd KAt Heermal. 11360 of L&u LZL=.am2jX- stiyilAd-LZ l,Cancer Research Institute and 2,Hospital Clinic, Barcelona, Spain Medical and Molecular Genetics, 2Dept of Peditcs, 3Dept of Pathology, Cutaneous Malignant Melanoma (CMM) is a fatal form of skin cancer whose Indiana UnIv School of Medicine, Indianapolis, IN. incidence is rising steadily. Hereditary malignant melanoma constitutes 10% of all cases A new recurring chros abnorality, dic(9;20)(p1l;q1), has of CMM and occurs in association with other malignancies, predominantly in families recently been described in three adult patients with acute Vynhobasc with atypical multiple mole melanoma syndrome (FAMMM sd.). The melanoma locus leukemia (ALL) (Reider et al., Gerns Chromosom Cancer 13:54, 1995). This (MLM) has been defined at 9p21. The characterisation of genes in the 9p2l region led rnlocdan pr a dicentric chomosom containing O to the detection of mutations in a gene that encodes an inhibitor of cyclin dependent kinase 4, (p161NK4 ) in melanoma cell lines and melanoma predisposed families. sequences from both cro 9 and 20. These patients all had CD10+ However, since no mutations have been described in many families with linkage to 9p Pre-C1ll ALL. and few mutations have been described in primary tumours, the role of p)6 in CMM We report four childhood ALL patients with a dic(9;20). The translocatlon auigeness has still to be defind To elucidate the role ofp16 mutations in CMM we was verified by using dual-color FISH with centromeric probes for have studied 49 tumours (25 primary and 24 metastatic) from 42 patients, 7 patients hromosomes 9 and 20 (Oncor) on interphase nuclei. Three of thm four with more than one primary CMM, 3 patients with melanoma in addition to another patients had multiple chromosomal abnormalities In addition to the pe of cancer, and 6 FAMMM ad. families. SSCA was performed for each of the 3 exons of p16 and deletions were studied with a p16 intragenic and several flanking tranlocation. One patienf karyotype was hypodiploid, ore was mierosaellite markers. Forty percent of tumours had deletions that involved the p16 pseudodiploid, and two were hyperdlplold (one with 50 cho and one locus, but four tumours (8%) had deletions that did not involve the p16 orpl5 genes. with 48 chromosomes) in the primary done. Of the 49 tumour samples analysed, 3 metastases from the same patient carried a AlI four patients were diagnosed with pre-B-cell ALL, and the three for missense mutation (D145N), one of the tumours was hemizygous for this mutation. A previously described polymorphism (Al401) was identified in 3 other metastases from whom infornation was available were CD10+, consistent with th patients 3 unrelated patients. Of the patients with more than one primary CMM, 23% had reported by Reider at al. The age of diagnosis of our patients ranged from 23 abnormal bands in SSCA; one of these patients had a single base pair deletion months to 12 yeare. AlI patients achive remission. Two ae in continuous (334delG) that changed the reading frame causing a premature stop codon. One of the remission for 2 yr 6moand 3 yr. One patent relapsed, dying 3 yr2 moaftet patients with another cancer in addition to CMM had 2 different abnormal bands. diagnosis. The fourth patient was lost to follow-up. Abnormal SSCA bands that are under characterisation have also been identified in two studies show that the is not limited to adult FAMMM ad. families. We conclude that while p16 is a CMM predisposition gene, it is Our dic(9;20) patients with ALL, not involved in the early development of most sporadic melanoma tumours. but is also present in childhood ALL cases. Due to te subtle nature of ts translocation, FISH is very useful in confirming the chromosomal abnormality. A72 Posters: Cancer Genetics (continued) 383 Poster Symposium-Session 29 384 Statistical method for testing genetic gain or loss in CGH profiles. Integration of HPV 16 sequences into human chromosome 13 in cervical G.G. Magrane, D.H. Moore II, J.E. Cronin, L.C. Yu, D. Pinkel, J.W. Gray. cancer cells demonstrated by FISH. H F. L Mark W Seart K. S. Santor. DMC/MCB 230, Box 0808, University of California, San Francisco, CA 94103-0808. E. Hann. R. Mikumo. S. Lauchlan and L. Braun. Brown University School of Comparative Genomic Hybridization (CGH) is performed by simultaneously Medicine, Providence, RI. hybridizing differentially labeled test and normal DNA samples to metaphase We previously reported the establishment of three cell lines from keratinizing and chromosomes. Variations in the relative intensity of signals from the hybridized test nonkeratinizing cervical carcinomas. These cells lines were analyzed for growth and normal samples along the chromosomes reflect variation in relative DNA copy properties in vitro and in vivo and cytogenetically characterized using GTG-banding and number between the two samples. We present a statistical method of interpreting these fluorescent in situ hybridization (FISH). Molecular data suggested that TCI40, derived variations. from a keratinizing cervical tumor, may contain HPV 16 in the episomal state, while Using a set of 11 separate hybridizations. we determined the mean and standard TC146, derived from a nonkeratinizing large-cell cervical carcinoma, may contain HPV deviation for the ratios along the entire autosomic genome when two copies of each 16 in the integrated state. We therefore undertook a FISH study using biotinylated HPV chromosome were present. These formed a control data set to which measurements on 16 DNA as a probe in order to confirm and to corroborate the original molecular study, test samples were compared using a s-test. The t-statistic for a point in the genome was as FISH is the most direct approach for mapping cellular and viral sequences in calculated as the differences of the means of the control and test data divided by an mammalian chromosomes. The results from a previous abstract demonstrated the expression containing the variances of these measurements. Variance estimates were presence of positive hybridization signals on the long arms of the apparent homologs of based on a model that takes into account two sources of variation in the ratio a human D-group chromosome in cell line TC146. The results of recently completed measurement: one due to variability among different hybridizations and another due to experiments clearly indicated that while the predominant state of viral existence in the differences between metaphases within a hybridization. A region of a test chromosome TCl40 cell line was apparently episomal, consistent viral integration can be found in the was considered to have a gain (or loss) if there were 3 or more contiguous points with TC146 cell line. Furthermore, where viral sequences of HPV 16 integration was t-statistics above (or below) a selected threshold. The threshold was selected so that the observed in cells of TC146, integration was apparently nonrandom, thereby confinning number of false positive aneusomies in this study was zero. the results of our previous experiments. FISH using various chromosorne-specific, a- CGH was performed on test samples of cell lines established from clinical cases of satellite and HPV 16 probes, that hybridized simultaneously to the slides of the TC146 trisomies 13, 18, 21, as well as other known segmental aneusomies. Using the cells, excluded chromosome 15 as the site of viral integration. Experiments with statistical method described, all whole chromosome and segmental aneusomies were chromosome 14 using both probes above further revealed the presence of viral detected. However, the estimates of the extent of the abnormality was inaccurate in integration signals on a D-group chromosome other than 14. By the process of some cases. This t-statistic analysis provided an unbiased method of classifying elimination, we hypothesized that viral integration occurred nonrandomly on regions of the genome having increased or decreased DNA copy number relative to the chromosome 13. To unequivocally demonstrate the integration of viral sequences on average for the cell population. This work was supported by NIH grants HD 17665 chromosome 13, we utilized a chromosome 13-specific, a-satellite probe and the HPV and CA58207. 16 probe simultaneously in a FISH experiment. With the aid of chromosome morphometry, the viral integration site was localized to 13q14-13q21.

385 386

Fuorescent in sila hybridization (FISH) evaluation of N-ayc copy aumber Involvement of the APC/MCC region in non-small cell lung cancer in seuroblastona J.W. Moore. J. Labanow S.J. OuImn. and G.D. Wener. A- More M .Mnefl 1 R o .Mta.J e Dept. ofLaboratory Medicine, Children's Hospital, Columbus, OH. and C MonteiroQP, The N-myc oncogene is presumed to have a significant regulatory role in ("Faculty of Medical Sciences, Departnent of Genetics, P-1300 Lisboa, Portugal; neuroectodernal development and differentiation. In the pediatric tumor ODepartment of Medical Oncology and 'Unit of Pneunology, Oncological Hospital Dr neuroblastoma (NB), amplificaton ofN-myc is associated with advanced stage of Francisco Gentil, Lisboa, Portugal. disease, rapid progression, and poor prognosis Genetic indicators N-myc Lung cancer is one of the main causes of death in western countries which makes the oncogene amplification, tumor ceil ploidy, and deletion or loss ofheterozygosity study of its tumourigenesis process of great interest. Several tumour suppressor genes (LOH) for a putative tumor suppressor on chromosome I- have clinical utility in are considered to be involved in the aetiology of lung cancer. Loss of beterozygosity guiding choice of therapy for NB. We have deternined N-myc copy number by (LOH) allows to screen for the presence of tumour suppressor genes in a certain both Southern hybridization and FISH for 28 patient samples with a diagnosis of genomic region. Several LOH studies in the 5q21 region have shown the possible NB. A biotinylated N-myc probe (Oncor) was used for Southern blotting; copy involvement of the APC/MCC gene cluster in this process. Our aim is to investigate the number was quantified by laer desitomery by comparison to simultaneous involvement of the 5q21 minimal region in the aetiology of lung cancer, for which we control samples. FISH studies were perfrmed using biotin- or digoxygenin- have genotyped STRs loci such as D5S299 and D5S346 (flanldng the APC gene) and labeled N-myc probes (Oncor) on touch preparations or cytospins of tumor nuclei another within the MCC gene. The electrophoresis, scoring and quantitadon were isolated from paraffin blocks. Results ofFISH are in agreement with Southern blot performed in an automated DNA sequencer (373A, with GENESCAN 672 software, analysis in all cases. In 9/9 tumors for which a pattern of increased fluorescence ABI)). In summary, our results are: (i) LOH is present in 36% of the squamous cell was determined by FISH, amplification was confirmed by Southern hybridization;, carcinomas analysed (n-25 informative samples) and none of the 8 adenocarinme gene copy number was detennined to be 6 to 100 times above that ofcontrol. In screened show LOH in the region under study-, (ii) there is a narked increase in the addition, FISH distinguished double minutes (dmin) vs. homogeneously staining LOH frequency towards the MCC locus; (iii) 2 cases were found with LOH for both regions (her); correlation ofFISH with cytogenetc results was found in 4/4 cases D5S346 and MCC but retaining the allele for the centromeric locus D5S299; (iv) in the in which cytogenetic analysis was performed. Advantages ofFISH include the samples analysed we did not find any case with LOH for D5S299 and/or D5S346 and abilities to assess morphology ofcells with increased signa, to determin the simultaneously retaining the allele for the MCC locus; (v) in one case we could find extent of cellular heterogeneity, and to assess the form of plific (bsr vs LOH for all the loci under study in normal distal bronquial mucosa which is not in dmin); the requirement for a snmple touch prep as opposed to 100 mg oftumor agreement with the idea that the 5q21 region is involved in a late event in squamous cell tissue, and reduced turnaround time. These studies demonstrate the utility and lung cancer. Our results support the hypothesis that at least one tumour suppressor gene efficiency ofFISH for detection ofN-myc amplification in NB in the MCC region is involved in the genesis of squamous lung cell carcinoma. Acknowtedgrnu - CIDC5A ProgamOM/CT; SC it DODG XI[-G -3-O0032 LPCC, CMDT.

387 388 Comparative genomic hybridizations with direct-labeled Inadertet Off-Protocel Testing for Colon Cancer-Predlsposlng Gene hMH2. reference DNA prepared In large-scale reactions. LL.E Mo &is. J. J. Mulvihill.`2 C. E. Ellis.' M. P. Stadler.i2 and M. C. LAce.4 Departments of DeVriS2. and F. Waldman2. Vysis, Inc., Downers Grove, IL1, University of 'Human Genetics and 'Medicine, University of Pittsburgh; 2Mage-Womens California at San Francisco, San Francisco, CA2. Hospital, Pittsburgh, PA; and 4Roche Biomedical Laboratories, Research Triangle Comparative Genomic Hybridization (CGH) has proved valuable in Park, NC. identifying regions of amplification and deletion In tumor specimens and in Germline mutations of WMSH2 have, to date, been associated only with the shortening the path to the discovery of genes associated genetic clinical phenotype of the Lynch cancer family syndrome or hereditary nonpolypodc abnormalities. Production of all the required reagents for CGH can be time colorectal cancer (HNPCC), as defined by the Amsterdam criteria. Professional consuming however, since DNA from the abnormal cells as well as normal cells must be isolated and labeled with two spectrally distinct fluorophores. consensus advises testing in research protocols only. A 34-year-old woman sought To simplify matters and reduce problems with preparation-to-preparation the 'colon cancer gene test' when her 64-year-old mother developed ssgmoid cancer variations, we have prepared the labeled normal reference DNA's in large- without polyps. After the consultand refused formal pretest counseling, a hospital scale direct-labeling reactions such that many CGH experiments can be house officer facilitated testing. Molecular genetic analysis showed that the conducted with the identical reference DNA. Direct-labeling also reduces consultand and her mother were heterozygous for a constitutional frame-Shifting the number of post-hybridization steps and reagents required to complete deletion of codons 668 to 736 in exon 13 of WSHW, resulting in asop codon 22 the assay. Large-scale preparations of human placental DNA (mixed sex), nucleotides downstream from the exon-intron splice site. Formal counseling then normal adult male DNA, and normal adult female DNA, have been prepared ensued with cancer screening for the consultand and her mother and with pretest and tested in competitive hybridizations with each other, with directly counseling for the sole at-risk sibling, whose biggest concern was privacy. At first, conjugated positive control lines, and with nick-translated tumor DNA. When the consultand was unsure about the history of cancer in her maternal grandmother, the reference DNA's of different fluorescent colors were hybridized against but we documented her right colonic carcinoma diagnosed at age 86 years. No one another, or against tumor DNA nick translated with the opposite color, other relative in the small extended family had cancer, and there was no evidenee of bright hybridizations and smooth ratio profiles were obtained. Large-scale Muir-Torre or Cowden This experience illustrates that the clinical production of direct-labeled reference DNA, therefore, may improve the syndromes. accuracy of CGH analyses and provide greater reproducibility. phenotype of lMSH2 mutations need not be the full Lynch cancer family syndrome or HNPCC, but may include even apparently sporadic colon cancer. Further, the breaches of consensus practice guidelines that will inevitably occur may suggest feasible alternative strategies for conducting predisposing gene testing, as long as adequate genetic counseling is included. Posters: Cancer Genetics (continued) A73 389 390 Characterization of RET proto-oncogene 3' splicing variants and Characterization of Ring Chromosome in Dermatofibrosarcoma Protubens polyadenylation sites: A novel C-terminal for RET. S. M. Myers', Using Cytogenetic, FISH and CGH Approaches. R. Naeeml, M. Lux2, & C. Eng23, B. A. J. Ponder2 and L. M. Mulligan1 1 Departments of Pathology and Huang2 S. Naberl, J. Corson2 and j.Flejthe2. Baystate Medical Center Paediatrics, Queen's University. Kingston. ON. K7L 3N6, Canada, 2. CRC Human and Tufts Medical School1, The Brigham and Women's Hospital, Harvard Cancer Genetics Research Group, University of Cambridge. Level 3 Laboratories Medical School2, Springfield and Boston, Massachusetts. Block, Box 238, Addenbrooke's Hospital. Hills Road, Cambridge. CB2 2QQ. 1K Dermatofibrosarcoma protuberans (DFSP) is a potentially progressive and and 3. Department of Medicine. Dana-Farber Cancer Institute, Harvard Medical recurrent dermal tumor of uncertain histogenesis. In atypical cases, School, Boston, MA 02115-6084, USA. differential diagnosis from fibrohistiocytic neoplasms, benign neural tumors The RET proto-oncogene, which encodes a receptor tyrosine kinase. displays and myxoid liposarcoma is difficult. We have characterized a series of 5 alternative variants. of sequences 3' of exon 19 to gen- DFSP using a combined approach of karyotyping, chromosome painting and multiple splicing Splicing comparative genomic hybridization (CGH). Three of these cases had a ring erate several coding and UTR sequences has been previously reported. We have chromosome and each of these ring chromosomes were composed of sequenced the full length RET coding sequence and the 3' UTRs and character- discontinuous, interwoven, sequences from chromosomes 17 and 22. CGH ized the transcripts generated by alternative splicing of the RET 3 region. These revealed amplification of 17q21-qter and 22q sequences in each ring analyses were performed using both RET cDNA cloned from a pheochromocytoma chromosome. In addition, amplification of chromosome 17 and 22 sequences library and reverse transcriptase PCR products generated from RNA from a neu- was demonstrated by CGH in one of two cytogenetically unremarkable DFSP. roblastoma cell line (LA-N-2). Three different carboxyl termini were identified. In These studies revealed an identical chromosomal derivation in each DFSP addition to the 9 and 31 terminal amino acid forms already known, we identified ring chromosome. This mechanism appears to be novel and potentially a third isoform with 43 terminal amino acids predicted to encode a novel RET unique to DFSP as ring chromosomes derived from either chromosome 17 or protein isoform. A total of 3620 base pairs of DNA 35 of exon 19, which span the 22 have not been described in any other type of neoplasia. Our findings indicate that increased copy number, rearrangement, and/or aberrant alternatively spliced exons and RET UTRs. were sequenced. Four polyadenylation regulation of novel oncogenes on 17q and 22q contribute to neoplastic sites were identified. The observed combinations of polyadenylation sites and 3' initiation or progression in DFSP. coding sequence suggest that RET transcripts with up to 10 different 3' sequences and up to 40 different full length RET transcripts may exist.

391 392 Cytoeretic and FISH studies of heterozygous twins with Juvenile chronic Tumorsuppression and the p21.3regionofchromosome 3. SL- Nalor' C.H HeL. myelogsyous leukemia (JCML). V. Nalfeld. A. VlachM W. Burnett. L D- an. R. Parker and A. 6fline. Tumor Cytogenetic Lab., Divisions of R.H Xiang'. M.C D&'- Garcia'- C.H.CM Buye2 H. Drabkin3' H. Carlson'- K.Kok'- Hematology and Pediatric Hematology/Onoology, Departments of Medicine and and K. KerhacherL 'The University of Texas Health Science Center at San Antonio, Pediatrics, Mount Sinai School of Med., NY, and Children's Med. Ctr. Stoney 2University of Groningen, The Netherlands, and University of Colorado Medical Brook NY. Center, JCML is a Philadelphia chromosome negative, clonal myeloproliferative Denver, CO. disorder of early childhood which arises in a multipotent hematopoietic Human chromosome 3 has been implicated in a number of human cancers. One locus has progenitor cell, and has persistence of fetal erythropoiesis. The prevalence of oases in infancy may imply the existence of an unidentified predisposition been defined at 3p2l.3 which shows homozygous deletion in small cell lung cancer to myelodysplasia. JCML in twins has not been reported. LP presented at age (SCLC). A fragment of human DNA overlapping the deleted region can suppress tumor 7 months with JCML and had 45,XY -7/48,XY,-7, +mar karyotype in May 1993. formation by mouse A9 cells. The region in common spans approximately 400 Kb. Using While his siblings were HLA typed for allogeneic bone marrow transplantation In 1/95, the clinical findings of his twin brother, ZP, were consistent with AIuPCR fragments from the somatic cell hybrid containing the fragnent of human DNA JCML. The bone marrow cytogenetic analysis of ZP showed 46,XY,-7,+mar on the mouse background, we have designed primers for the inter Alu sequences in the karyotype in 42 metaphases. Using chromosome 7 oentromere-specific probe (Vysis) FISH study revealed: 9.3% of interphase cells with monosomy 7 and region. The markers D3S 1573 and D3S1235 as well as the GNAI2 gene are encompassed 87.7% with disomy 7 (control: 0.3% with -7). Discordant results were in the common region. Using the end sequences from our entry clones a P1 contig was Interpreted that the only proliferating cells were those 9.3% with moneomny constructed. Each of the PI clones were cotransfected with ptkhyg plasmid into A9 cells 7. LP underwent allogeneic BMT from the non-twin HLA identical sibling and on day #28 post BMT, 12% of metaphases had 46, XY,-7,+mar karyotype and and resistant clones were screened for chromosome 3 markers. Each transfectant was then FISH results showed 24/1,025 (2.3%) of interphase cells to have -7 (control tested for their growth in nude mice. One of the PI clones (P1 294) suppressed tumor 0.3%). The bone marrow cytogenetios on day #70 poet BMT showed 5.8% of cells with -7, +ar karyotype while FISH test showed ii1868 (1.2%) (control 0.9%) formation in A9 cells. This PI clone which has 74 Kb of human DNA was used to isolate of Interph calls with -7 (control 0.9%). To understand discordant results cDNA clones using exon trapping, hybridization selection, and identification of CpG and to identify the mar chromosome the G-banded metaphases of ZP were destained and the results of metaphase FISH studies with centromeric and islands. This region of the genome appears to be quite gene dense. One gene was found whole chromosome 7 probes indicated that the mar chromosome contained two which is homologous to the semaphorin gene family and is 50% homologous to chicken copies of DNA sequences from the centromere of chromosome 7. Similarly, collapsin. As ioss ofheterozygosity is 100% in this region in small cell lung cancer, metaphases with the mar chromosome after BMT of LP showed the nearly hybridization with both centromeric and whole chromosome 7 probes we expect to find mnutations in the SCLC tumor samples and cell lines when we isolate the Indicating that two copies of centromeric chromosome DNA 7 sequences were bona fide tumor suppressor gene. However, mutations were not found in the semaphorin inserted in the mar. The combined studies of these patients provide evidence that monosomy 7 as well as the mar chromosome may have embryonic origin, homolog in lung cancer samples. The a transducin (GNAT1) gene is also located on this which in turn suggests predisposition toJCML in boh twins. Interphase FISH PI clone; however, GNATI is retinal specific and not expressed in lung. We have found with enumeration probes should not be utilized in clinical diagnostics unless the origin of mar chromosome Is fully Identified. at least 4 other genes on PI 294, three of which are expressed in normal lung. These transcripts do not have homology to known genes in GenBank. We are screening lung cancer samples for mutations in these genes. (Supported by CA56626).

393 394 Chromosome aberrations in four hepatoblastomas. J. A. Neidich. C. D. Bansa. C.-L A physical map of the BRCA2 region constructed In Pis, BACs, Hsieh, T. A. Donlon, and G. Dahl. Stanford Univ. School of Medicine, Stanford, CA. and PACs and assessment of loss of heterozygosity (LOH) In is a rare Hepatoblastoma pediatric tumor, however, it is the most common pediatric familial BRCA2 tumors S.L. Nauhausenl, CA. Marshall , J. Swensfn1, liver neoplasm. Cytogenetic analysis of hepatoblastomas have identified partial or full Y Miok1i, L Canon A1ght', J Weaver-Feldhaus2, M.StMngfellowgg2EjL trisomy of chromosomes 2 and 20 as common events, but only 20 cases have been ki2: DEG lM,M A. Kam andS2T V. Ttijg 2, published. Previously, the smallest duplication of chromosome 2 has been q23-q35. 'UnIversity of Utah and 2Myriad Genetics, Salt Lake City, Utah. Findings in hepatoblastoma are similar to reported karyotype aberrations seen in A second familial breast cancer gene, BRCA2, has been localized to embryonal rhabdomyosarcoma. Hepatoblastoma has been seen in Beckwith Wiedemann 13q12-13 by linkage analysis (Wooster et al., 1994, Science 265: 2088- syndrome characterized by loss of heterozygosity of chromosome lp. 2090). Using published markers in the region, we constructed a YAC contig We present data from four consecutive hepatoblastomas. In one tumor, there was a spanning the interval from D13S289 to D13S267. STSs generated from translocation of chromosomes 3p and 15q and what was interpreted as a translocation YAC ends and the published markers were used to isolate initial PIs, PACs, of 21q and 22q as the only anomalies (46,XY,t(3;15)(p25;q21),?t(21;22)(q22;qll.2)). and BACs In the region. Chromosomal walks from the ends of those clones The other 3 tumors had at least one clone with trisomy 2. Of these, one tumor had allowed us to complete a Pi/PAC/BAC contig spanning our geneticaly trisomy 2 and trisomy 20 in addition to a derivative 22 resulting from a tranlocaton defined region of - 2 Mb. Polymorphic short tandem repeat markers between 17q and 22q as the primary clone and a secondary clone with trisomy 8 in (STRs), isolated from the genomic clones, were used to refine the BRCA2 addition. Fsve tetraploid cells representing doubling of both clones were observed. In region. the second tumor with trisomy 2, a small unidentfied ring chromosome was seen in 24 LOH was examined in breast and ovanan tumors from both carriers and of 25 cells, one cell appearing normal. Additional aberrations included a derivative I sporadic cases of five BRCA2-linked kindreds using eight BRCA24lnked with unidentified material on lp as one clone and a secondary clone with trisomy 2 as polymorphic STRs and one STR near retinoblastoma (RB). Two of the three well as the der(l). The possibility that the ring chromosome represented a constitutional sporadic cases examined did not show LOH, whereas one exhibited LOH of anomaly remained. The last tumor had as a primary clone trisomy 2 with a duplication the entire region including RB. Of the seven tumors from carriers, two of 2q31-q33 in two of the three chromosome 2 homologs and clonal po ion as exhibited no LOH, two had LOH of the entire region including RB, and three demonstrated by an isochromosome lq in the secondary clone. had interstitial loss. In all cases where LOH was observed, the wild type Our cases increase to 24 the number of cytogenetic reports of hepaoblasoma. a re allele was lost. These results confirm the findings of Collins et al. (1995, childhood tumor. Fullor partialtrisomy 2wasseen in3 out of 4 of our caes, but we Oncogene 10: 1673-1675), and provide additional evidence that BRCA2 is found trisomy 2 as a secondary aberration in one tumor. In addition, in one tumor the a tumor suppressor gene. duplicated portion of chromosome 2 was q31-33, the smallest reported 2q duplication in hepatoblastoma. Finally, trisomy 20 was seen in only one of 4 tumors. A74 Posters: Cancerr Genetics (continued) 395 396 Risk of prostate cancer, breast cancer and ovarian cancer in parents and Loss of Heterozygosity on chromosome 14 inversely correlated with metastatlc siblings ofprostate cancer patients. D Nguyenl M. WaChC2.aH.yamil. potential. P. O'Connell, V. Pekkel. S. Fuqua. C.K. Osborne. D.C. Allred. Veterans Affairs Medical Center. University of Texas Health Science Center, San Antonio, Texas, 78284. Oregon Health Sciences University and 2Portland We are examining evolutionary stages of breast cancer to assess the impact of It is not known if cancers of prostate, breast and ovarian have a common cumulative genetic changes in the development of this disease. We carried out susceptibility gene. To examine familial aggregation of these cancers, we studied Loss-of-heterozygosity (LOH, or allelic imbalance) studies on DNA recovered their incidence in 579 parents and siblings of 112 Caucasian prostate cancer patients from the normal and tumor components of microdissected paraffin-embedded and 467 parents and siblings of 90 Caucasian spouse controls. Cases were chosen breast cancers and metastases. LOH studies identify genetic events participating in sequentially from Portland VAMC registry, regardless of family history and age at the clonal evolution of breast cancers. Genetic alterations at twelve loci showing We a form for the proband and phone high rates of LOH in invasive breast cancers were tested in either node negative onset. used self-administered family history breast cancers or node positive breast cancers. Marker D14S62 showed 23% LOH interviews with other relatives to verify the data, Kaplan-Meier survival analysis to (13/52) in this study of all breast cancers, suggesting this locus marks the location estimate incidence rates, and logistic regression to estimate age-adjusted odds ratios. of a breast cancer related tumor suppressor gene. When LOH at D14S62 was re- In relatives of patients (289 men, 290 women) 36 had prostate cancer, 23 had analyzed by stage, 42% (11/26) LOH was observed for the node negative breast breast cancer and 3 had ovarian cancer. In relatives of controls (244 men, 223 cancers while only 8% (2/26) LOH was observed for node positive breast cancers. women) 8 had prostate cancer, 14 had breast cancer and 5 had ovarian cancer. The This apparently contradictory result was confirmed with nearby marker D14S68, life-time (by age 100) cumulative incidence rates in parents and siblings of cases and and suggests that in addition to the putative tumor suppressor gene detected by the LOH, there is another gene (e.g. a protease, angiogenesis factor or cell adhesion controls were .45 ± .09 and .10 ± .04 (P=.0004) for prostate cancer, .15 ± .05 and .1 molecule) that is important for the process of metastasis. Loss of this tumor ± .03 (P=.5) for breast cancer, and .01 ± .01 and .03 ± .01 (P=.3) for ovarian cancer. suppressor locus also may also delete the metastasis-related gene, reducing the Age-adjusted odds ratios were 4.1 (P=.0004) for prostate cancer, 1.3 (P=.4) for breast metastatic potential of the invasive cancer. These results, if confirmed in other cancer, and .50 (P=.4) for ovarian cancer. Given that common cancers are often tumor banks, can be used to assess the metastatic potential of primary breast heterogeneous and the genetic subtypes are associated with earlier onset, we analyzed cancers. (Supported by NC! P50 CA58 183) relatives of early onset cases (age at diagnosis < 65) separately. The odds ratio rose to 5.3 (P=.03) for prostate cancer, but remained low and insignificant for breast (1 1. P=.9) and ovarian (.26, P=.23) cancers. These data confirm familial aggregation of prostate cancer and provide no evidence for clustering of breast or ovarian cancer with prostate cancer. If there is a common gene for these cancers, it is probably not involved in the majority of cases.

397 398 Accuracy of SpectrumCEP X/Y Dual Color DNA Probe Kit for Detection ofXX WT1 and GATAl expression patterns in human leukemia. 122masiziril-fr and XY Cells in Opposite-Sex Bone Marrow Transplant Patients: A FPmiza. IT-T iantaijiakn_ AR_ T)twnt~h_ aA G_ R Sauntr. Univ. of Texas M.D. Multicenter Clinical Validation Study S. PatilI E. BOantI2. , Theil3. £ Selid Anderson Cancer Center, Houston, Texas. P. Hsu4. 'Univ. ofIowa, Iowa City, IA, 2Fred Hutchinson Cancer Res. Ctr., Seattle, Hematopoietic specific regulation of the Wilms' tumor gene (WT1) expression WA, 3Ohio State Univ., Columbus, OH, 4Vysis, Inc., Downers Grove, IL requires the regulatory elments found in many hematpoietic specific genes. One of these elements, the GATA binding site, is also fownd within the WTI promoter and Fluorescence in situ hybridization (FISH) technique using the X and Y chromosome enhancer regions. GATA-I trasactivates both the WTI promoter and 3' enhancer. probe(s) has been employed to detect the sex chromosome complement in interphase However, he co-a n of hese tansc o factors and its implication in human nuclei. However, there remains a need to validate the sensitivity and specificity of leukemia is not well understood. WTI expression in AML and ALL has been commercially available probes. described peviously but the co-expesion ofWT1 and other transcription factors in Three clinical cytogenetic laboratories participated in this study to validate the human leukemia has not been reported. Using RT-PCR, we examined WTl and ability ofthe SpectrumCEP X/Y DNA probe kit (VYSIS) to detect the presence of GATA transcript levds in blood and/or bone marrow samples from 62 patients with XX or XY complement (signals) in marrow cells ofbone marrow transplant (BMT) acute leukemia and myelodysplastic syndrome. WT1 and GATAl were quantitated patients. All technologists were identically trained in techniques and signal by normalizing to the P-actin transcript level under optimized PCR conditions. High enumeration. Slides were made from archived fixed cell suspensions. Probe signals WT1 expression was found in CD34-positive normal hematopoietic stem cells from were scored in 500 nuclei and 20 metaphases per slide. bone marrow, suggesting that the WTI gene could be activated in early hematopoietic The hybridization efficiency was determined in 120 normal bone marrow samples development and inactivated during differentiation. Normal blood samples express from both male and female donors with a cut offpoint for presence ofdonor or very low or undetectable levels of WTI mRNA while GATAI transcript levels are recipient cells to be 0.4%. To date, we have studied 158 archived specimens from slightly higher. High WT1 transcript levels were observed in most of the AML and patients who received BMT. The sensitivity for detecting donor cells was estimated ALL patients; granulocytic lineages showed higher levels of WTI mRNA than the to be 100% based on 72 opposite-sex specimens. Similarly, the specificity for monocytic and lymphocytic lineages of leukemia. Interestingly, 12 of 13 AML cases detecting donor cells in the absence ofchimerism was estimated to be 100%!. based on with chromosome 16 (q22) abnormalities expressed high levels of WTI mRNA and 86 specimens from same-sex specimens. This accuracy is consistent among very low levels of GATAl mRNA (lower than normal). Chromosomal breakpoints specimens from patients with AML, CML, MDS and MPD. Even detection ofa at 16q22 disrupt the CBFP trnsription factor gene. It is possible that the interaction small percentage ofhost or donor cells was evident, which had been missed with of WT1 with various transcription factors, i.e., GATAl, CBFP, RB, p53, plays a role conventional cytogenetics. Details ofour study design and results will be presented in normal h , and dgulaton may contribute to human leukemogenesis.

399 400 High resolution mapping of a common region of deletion Allelic mapping of a tumor suppressing region on chromosome 10q in in 10q25.1 in endometrial cancers. S. Peifferl,1. Mutch2, L human glioblastomas. M. A. Pershouse. H. Lin. A. C. Cavender. W. K. A. Herzog2, K. Call3, P.J. Goodfellow 1. iDepartments of Surgery and Yung. and P. A. Stick. Univ. of Texas M. D. Anderson Cancer Center, 2Obstetrics and Gynecology, Washington University, St. Louis, MO; Houston, TX. 3Collaborative Research Inc., Waltham, MA. Attempts at mapping a tumor suppressor gene on chromosome 10 by loss- Endometrial cancer is the most common gynecological of-heterozygosity (LOH) studies have been hindered, in part, by a low malignancy in the United States, with an estimated 31,000 new number of tumors with partial chromosomal losses. The majority of tumors cases diagnosed annually. Endometrial carcinomas are thought to show the loss of an entire copy of chromosome 10, making localization difficult. We have used an alternative approach to map the suppressive arise and progress as the result of an accumulation of mutations regions of chromosome 10 by microcell-mediated chromosomal transfer. This in both proto-oncogenes and tumor suppressor genes. Loss of functional mapping method allowed us to propose the existence of a tumor heterozygosity (LOH) of polymorphic markers in the regions of suppressor gene on 10q24-q26 in human glioblastoma, as well as a suppressor tumor suppressor genes is frequently observed in cancers. gene on lOp. The localization of tumor suppressor genes to this region was Our initial allelotype study identified 10q as a region performed by analysis of various retained regions of inserted chromosome 10 frequently deleted in endometrial cancers (Peiffer et al. Ca Res that had fragmented during or shortly after transfer. Further analysis of the 55:1922, 1995). To further define the common region of loss we retained fragments has lead to three candidate regions on lOq; from D1OS583 investigated additional endometrial adenocarcinomas with and to CYP2C, a region of at least 3 centiMorgans (cM); from D1OS566 to D1OS575, without squamous differentiation. Of the 70 adenocarcinomas a region of approximately 30 cM; and from DlOS169 to qter, a region of at examined, 18 (26%) had interstitial deletions of the q24.3-q26.3 least 6 cM. To prioritize these three regions for a more focused search for the region with a smallest region of overlap in 10q25.1. Genetic and tumor suppressor gene, we have attempted to find regions of overlap physical maps suggest that the smallest common region of loss is between homozygous deletions in 21 human glioma cell lines and any of approximately 3 cM and 2 Mb respectively. these functionally suppressive regions. Homozygous deletions on 10q were Additional tumor and normal DNA pairs are being evaluated studied using microsatellite analysis of twelve loci covering the region from were from for LOH. We have begun to map CpG islands in a YAC contig 10q24 to 10q26. Homozygous deletions confined to the region the of one cell spanning the smallest region of deletion. Mapping of CpG islands D10S566 to D1OS575 (4 of 5 multiple loci losses)with exception had two non one in the will of the tumor suppressor line which contiguous losses, D1OS566-D10S575 hopefully facilitate the identification and one in the gene in this region. region, D1OS583-CYP2C region. Posters: Cancer Geneltics (continued) A75 401 402 A Multi-Center Clinical Validation of the Accuracy of the SpectrumCEP 8 DNA Genomic structure of human mammnary-derived growth inhibitor Probe Kit for Detection of Trisomy 8 in Bone Marrow Specimen from Patients and mutation analysis in sporadic human breast tumor C. M. with Myelold Disorders. M". Pettenati. P.N. Rao! N. Heerema2. K Theil. S Phelanl,2, S. Baird3. C. Larssonl, P. A. Futreal4. R. G. Korneluk N &eflkt P. Hsu4 'Bowman Gray School of Medicine of Wake Forest University pollak6 and S. A. Narod2. NC University Medical Center, Indianapolis IN, 3Ohio State I.Department of Clinical Genetics, Karolinska Hospital, Stockholm, Sweden, Winston-Salem 2Indiana 2.Division of Medical Genetics, Departments of Human Genetics and Medicine, University Medical Center, Columbus OH, VISIS, Inc. Downers Grove IL Montreal General Hospital, Montreal, Quebec, Canada, 3.Molecular Genetics Trisomy 8 in leukemia has diagnostic and prognostic value and can be used Laboratory, Childrens Hospital of Eastern Ontario, Ottawa, Ontario, Canada. to monitor disease progression and treatment response. Detection of + 8 is usually 4.Department of Surgery and Genetics, Duke University Medical Centre, accomplished through standard cytogenetic analysis. However, application of FISH Durham, North Carolina, USA, 5.Lady Davis Research Institute, Montreal, to detect +8 offers distinct advantages. With the increase use of FISH to detect Quebec, 6.Department of Oncology, McGill University, Montreal, Quebec. Transfection of the human mammary-derived growth inhibitor (MDGI) gene these abnormalities, there is the need to validate the sensitivity, specificity and into human breast cancer cell lines reduces their tumourigenicity and suggests reference ranges of commercially available probes. that this gene has tumour suppressor function. These findings combined with A multi-center study evaluated the ability of the SpectrumCEP8 DNA the evidence for loss of expression in breast tumour cells compared to adjacent probe kit (VYSIS) to detect + 8 in archived bone marrow specimens from patients normal tissue and the localization of MDGI to human chromosome Ip32-35, a with acute nonlymphocytic leukemia, myelodysplastic syndromes and common region of deletion in sporadic breast tumours, support a role for this myeloproliferative disorders with and without + 8 by cytogenetic analysis. The kits gene in human breast tumourigenesis. We have determined the genomic structure of the human MDGI gene, which covers approximately 1Okb of DNA contained SpectrumOrangeTm direct labelled DNA probe specific for chromosome and have sequenced the constitutional and tumour DNA of 30 sporadic breast 8 centromere, reagents and instructions for the assay. Two technologists at each tumours (8 of which showed LOH in the Ip32-35 region), for mutations in the site followed strict guidelines in the use of the kit and in signal enumeration. 500 coding regions and splice sites. No mutations were found in either in tumours interphase nuclei and 20 metaphases were analyzed per specimen. showino loss, or those retaining the lp32-35 region. A sequence variant was The hybridization efficiency was determined in 60 normal bone marrow found in the constitutional DNA of one case which changed a lysine to an samples with a threshold for + 8 determined to be > 2.2%. The study population, arginine at codon 53. This variant was also present in the constitutional DNA of 2 of50 unrelated controls. Despite experimental evidence that the MDGI plays a to date, has classified 119/272 cases as positive for trisomy 8. Compared to role in breast cancer development, our data suggest that mutations in the coding standard cytogenetic analysis, the specificity was estimated to be 99.4% based on region are uncommon mechanism in human breast carcinogenesis. 153 specimens without + 8 while the sensitivity was estimated to be 99.2% based on 119 specimens with +8. The SpectrumCEP 8 DNA Probe kit was highly accurate in the detection of trisomy 8 when compared to standard cytogenetics. Detailed analysis of the complete data will be presented.

403 404 Follovup of two hyperparathyroidism-jaw tumor syndrome Isolation of candidate sequences from the Gorlin syndrome region families reported in 1971 and 1981 reveals that they are under-expressed in related neoplasms. C. L. Pressmanl. M. R. Gailanil. related and that parathyroid cancer is a part of the D. J. Leffeill. R. Yavaril. M. Dean2. B. Wainwright3. A. E. Balel. Yale Univ. syndrome. G. N. Pid 1nyv, J. Szabo2, M. Hobbs2, H. Heath New NCI. MD2. Ccntre for Molecular and C. E. Jackson . iTranscona Clinic, Winnipeg, School of Medicine, Haven, CT1. Frederick, Manitoba, Canada 2University of Utah School of Medicine, and Cell Biology, University of Queensland, Brisbane. Australia3. Salt Lake City, iHenry Ford Hospital, Detroit, Michigan. Gorlin syndrome is an autosomal dominant cancer predisposition syndrome char- An autosomal dominant syndrome of hyperparathyroidism acterized by basal cell carcinomas, medulloblastomas, and ovarian fibromas. The and fibro-osseous jaw tumors (HPT-JT) has been reported in gene maps to 9q22.3 and is probably a tumor suppressor based on allelic loss studies 10 apparently separate families and the responsible gene (HRPT2) mapped to chromosome lq21-q31 (Szabo et al, Am J in related neoplasms. By analogy to retinoblastoma, it is likely that some tumors Hum Genet 56:944, 1995) in 5 families. In that report, will underexpress the gene. To isolate candidate sequences, 800 gridded cosmids markers flanking the HRPT2 locus were widely separated at from this region were screened with cDNA from basal cell carcinomas and cor- about 60 cM of genetic distance. Use of additional responding normal tissue. Approximately a% of clones gave strong to moderate markers has narrowed the distance to about 33 cM, but cosmid gave a strong hybridization signal with normal tissue but a de- additional family studies are needed to find more signals. One recombinants to disclose closer flanking markers. creased hybridization signal with one basal cell carcinoma. A restriction fragment Recently we were able to obtain clinical data and DNA from that hybridized to cDNA identified a 1.2 kb transcript on Northern blot. When a Canadian family that had been reported by Kennett & it was used to screen a cerebellar cDNA library (Stratagene, La Jolla, California), Pollick in 1971. Inquiry revealed that the proband had Three positive clones were obtained. One clone had an open reading frame with no subsequently expired of parathyroid carcinoma, that the we are to that the po- family was a branch of a kindred reported by Rosen & known homology. Currently, designing PCR primers verify Palmer in 1981 and that a member of this second branch had tential coding seqquence maps to 9q22. A gene that maps to the correct region and also died of parathyroid cancer. This newly united is underexpressed in tumors related to Gorlin syndrome will be a strong candidate kindred and 3 as yet unreported kindreds from Australia, for further testing. Sweden and Portugal offer substantial numbers of meioses that will allow further refinement of the map location for HRPT2. Moreover the combined data establish that parathyroid cancer is a part of the HPT-JT syndrome occurring in 42% of the 12 known families.

405 406 Detection of circulating prostate cancer cells by a multiplex reverse Heterogmenty of breast cancer risk in the fmle first degree transcriptase polymerase chain reaction assay: a new molecular method relatives of breast cancer patient - Evaluated by 'comet assay'. to evaluate prostate cancer. D. K. Price. W. Woodard. H. Godwin. N.Rajeswari, Malini Jaiswal, G.Anuradha and Y.R.Ahuja. Genetics Unit, Bhagwan Mahavir Medical Research Centre, 10-1-1, Mahavir and C. M. Teigland. Carolinas Medical Center, Charlotte, NC. Marg, Hyderabad-500 004, India. Adenocarcinoma of the prostate is the most common internal cancer in males Breast cancer is the second most common cancer affecting women with an estimated 244,000 new cases per year and 38,000 deaths. Patients are in India. Estimation of genetic instability by direct quantitation followed clinically by monitoring a serum prostate-specific antigen (PSA) level, of DNA damage and repair is an important aspect in biomonitoring an however clinical staging to determine the extent of metastasis is often difficult individual's risk to cancer. Single cell gel electrophoresis with current modalities. Approximately 40% of patients with clinically diagnosed (SOGE) assay or comet assay was carried out for the first time to cancer female first seminal vesicle biomonitor individual risk of in the degree organ-confined disease actually have capsular penetration, relatives of breast cancer patients. The assay was carried out on invasion, and lymph node metastasis at the time of a radical retropubic the peripheral blood leucocytes of 58 breast cancer patients, 128 prostatectomy; thus, there exists a great need for a more sensitive staging tool. first degree female relatives of the breast cancer patient and 75 Recently several groups have used reverse transcriptase PCR in order to controls for evaluating the basal DNA damage, individual's sucepti- detect PSA-expressing cells or prostate-specific membrane antigen (PSM) bility towards a mutagen (MNNG, an alkylating agent) and repair expressing cells in the circulation of prostate cancer patients (Katz et al., 1994; efficiency. 50 cells per treatment were scored for comet tail which an estimate of strand DNA damage. The Israeli et al., 1994). However, controversy has arisen over which marker more length gives single mean DNA damage in untreated, treated and repaired leucocytes accurately provides information as to the extent of metastases. We have chosen increased significantly from controls to cancer patients. First to examine both PSA and PSM expression in circulating cells by developing a degree relatives of breast cancer patients showed a significantly novel multiplex RT-PCR assay that allows the simultaneous detection of mRNA higher mean DNA damage than the control group but lesser damage from both of these genes, as well as a ubiquitously expressed internal control all when compared to cancer patients. Among these relatives, however, within the same reaction. At present, the sensitivity of our assay allows the there was a considerable inter and intra-individual variability. Since cancer is in the unaffected breast detection of one cell within a of monoclonal origin, among prostatic population 1,000,000 lymphocytes. cancer family members, the females showing a relatively high mean Our study includes patients with bone metastasis, hormone refractory DNA damage in untreated, treated and repaired leucocytes, as well patients, and negative control groups consisting of women, and men under the as having highly damaged individual cells would be considered as age of 40. At present, we are actively collecting patient samples, and we have carrying the predisposing factors for cancer development. had no false positives among our negative control samples using this assay. (Supported by Council of Scientific and Industrial Research With the use of our multiplex RT-PCR assay we hope to provide a sensitive 9/132(463)94-EMR-I). method of molecular staging of adenocarcinoma of the prostate. A76 Posters: Cancer Genetics (continued) 407 408 Hereditary nenpolypeosl colorectal cancer (ONPCC) which Is due to a mutation Genetk and environmental factors in lung cancer suseptIbIlity. L RiWm in bL1 affects predominanty males in a large South African kindred. P! Yang. AG Schwartz. A Adedovn. M Rornkes. R Landreneau. K Mauro. R 'RUS I t 2MYMaddenan C AWork. 'MRC Research Unit for Medical Branch. University of Pittsburgh Medical Center, Pittsburgh, PA 15261 Genetics, University of Cape Town Medical School, %Groote Schwr Hospitl, Cape Individual susceptibility to lung cancer has been linked to genetic Town, South Africa and 'Section of Hesnatology/Oncology, University of Chicago polymorphisms in drug/xenobiotic metabolizing enzyme activities. It is Medical Cetr, Chicago, USA hypothesized that a balance of the activity of the enzymes (both CYP and phase II) In South Africa the inadence of colorectal cancer is second only to lung cancer. involved in either formation of proximate carcinogens or production of non-toxic Our inveigaions have been aimed at deteriningi the genetic defect in larg HNPCC metabolites will determine cancer risk and that family members of lung cancer failies in this country. After initial linkage ofthe disorder in a large family NPCI to patients will have a higher incidence of cancer, given the heritable nature of chromosome 3, we used infbrmation on intron/exon boundaries for the mismach r differencesin enzyme activity. Genotyping analysis of CYP2D6, GSTM1 and NA72* genc, hMLHI, to screen for disease-associated mutations. The genetic defect in and phenotyping studies using debrisoquine (CYP2D6) and dapsone (N-acetyl- NPCI was shown to be a C ->T Urnsveion within exon 13 ofh}LH1, which resulted transferase) were completed in 43 primary lung cancer patients ascertained through in the conversion of the CAG codon to a TAG (amber) stop codon. The disorder in the University of Pittsburgh Medical Center. Patients were interviewed for NPC1 is noteworthy since it seems to manifest predomina i males. Interestingly, information regarding family history of malignancy and occupational exposures. Of a 70 year old female canier of the mutation has not manifested with HNPCC (or 359 first degree relatives, 42 were reported to have cancer with known sites. This reed cacers ofthe Lywh II spectrum), while her mutation-canying son developed is 3.6 times the expected number of 11.6 after adjusting forage, sex and cancer site the disorder by age 35. In a second notably lage HNCC kindred, NPC5, there was (SEER, 1987). The greates disparity between observed and expected incidence of esidence of disease association with chromosome 2 makers in the vicinity of the cancer occurred in the s 49 year age group (9 vs. 0.3). The distribution of hMSH2 gene. No sex bias was apparent amongst affected individuals and exminton genotype or phenotype of patients with or without a family history of cancer was of the hMSH2 gene revealed a (0 insertion within exon 9, resulting in a premature not statistically different. However, lung cancer patients did display variability in teation codon being introduced downstream of the mutation. The informaton acetylation activity, within NA72*genotype, with occupational exposures to asbestos from our investigations is currently being used to provide an accurate pm atic or chemicals. Patients with asbestos exposure and who were homozygous wild type indication for colonoscopic screening of farmly members. We are curenty (NA72*4/*4) had a mean acetylation rate twice as high as those without asbestos investigating possible mechanim for the skewed gender distribudon ofthe disorder in exposure (1.11±0.06 vs. 0.50±0.12). The mean acetylation rate was also higher in patients exposed to chemicals versus those with no chemical exposure in the NPC1. NAT2*4/*4 genotype group (0.77±0.03 vs. 0.32±0.08). Therefore interactions between environment and genotype in the expression of metabolizing enzyme activities should be considered in order to identify families at high risk of cancer.

409 410 Ce se of d sad Geomoic Sober. Hlybridiatio for detecte of bc-2 resrraagenent Does the human c-MYC variant Asnll-+Ser predispose to neoplasia? Pa. ia boe marr traasplawt patens. L S losenblum-Vas R. Yonsc and C. A. Griffin. Johns Rothberg'- Y. M.Otto1. D. W. Baker! and Y-F. Wonj. lIThe Childrens Mercy Hopkins University School ofMedicine, Baltimore, MD. Hospital, Molecular Genetics Laboratory, and the School of Medicine, Univ. of The t(i4;15Xq32;q21) chromosomal translocation, involvingjuxtaposetion of the bcd-2 oncogene into Missouri-Kansas City, Kansas City, Missouri, and 2Chinese University of Hong thejoining region ofthe IgH goe, occurs in up to 85% offollicular small cleavd cell and 30-35% of dius lr oell non-odglcin's lyunhonan (NHL) and ha important prognic implications in Kong, Shatin, New Territories, Hong Kong. (Intro. by: David J. Harris) patients undergoing high dome d _ apytlowed y bone marrow transplant (BMI). Speimes Mutations in the transcriptional activation domain of the c-myc proto-oncogene with csal malignant cells bering this'alteration can be identified by either Genomic Southern have been implicated in its oncogenic activation. We found a G for A substitution in HIbbidization (GSH) or PCP, but repor conflict as to which approach is optimally employed in exon 2 that results in a serine at codon II instead of the usual asparagine. This is a clinical dioeis. To tesolve thise, we directly conpd OSH and PCR to dect tbe bl-2 ma fairly common variant of the human c-myc gene. This alteration, which causes an breakpoint region (MBR) and minor clus aequence (MCS) rearrngeent to evalue DNA prenrd is outside of the cluster of mutations frequently seen in fron 75 born mrow (3M), peripheral blood (PB), and lymph node (1N) samples. Of 68 PB or BM altered SSCP pattern, DNA preparation all wer negative by GSH using resriction entomicleses BanHI, EcoRL and Burkitt's Lymphoma. The Asn I I codon is invariant in all other vertebrates whose c- Hindll, and hybridization probes pFL-I (MBR) and pFL-2 (MCS). Hower. 16'68 (23%) of the myc gene has been sequenced, including chimpanzees, gibbons, marmosets, eamplee were poive by PCR with neared primer soes and queo-oqpecific hybridizon. The woodchucks, mice, rats, cats, chickens, rainbow trout, zebra fish, common carp and distribon of identifiedrearanemen wan unusual: MBR n-6 (38%), MCS n-9 (56%). both n-l both c-myc genes in xenopus. It is also conserved in N-, S-, and B-myc. A serine for (6%), and may be explsied by the emall number of semples teetef The diacrepency between GSH asparagine substitution is a conservative mutation with respect to charge, andFCR results reflects the low snsitivity of GSH (detection liSit5% of cells) in identifying minimal hydrophilicity/hydrophobicity, and contribution to the formation of secondary ria disea in paen eir just prior to or imnedistely following BMT. Such pients will require sequentia saseplings to determine if the PCR-deeectsbWl lynsphomn cells ar prognostic of structures. Its impact on the biochemical function of Myc is unknown. We reliu S b.... ispo. to corrate PCR inding withdi lburn a menrd by flow developed a rapid assay to detect the Asnl I -*Ser variant. We are using this assay to cyt sy/cel uace marker dta nd cell morpholop. Additionally, two f sven DNA sampls evaluate the frequency of the variant in cancer patients and in the general population. iolatedfroi lymph nodes with known cytogenetic content mw negative by all three analysis methods. By comparison we will see if the frequency of the variant is significantly different in Five LNs with the t(14;18) were MDR-positive by GSH but only four had positive findings by PCR; The conclusions are that this variant is associated with a bsed on the GSH pstea-, we believe the GHS+, PCR- patient haibored a variant an ment not cancer patients. possible detectable byou PCRaesay. Our dam suggest that while lymph node samples front patients with NHL neoplasia of some type, is negatively associated with carcinogenesis, or has no can be analye by GSH, bone marrow and penpbawl blond specimens requir the sensitivity of PCR impact on the incidence of cancer. for detection ofthe bl-2 r .

411 412 Identfication of nonrandom chromosome breakpoints at Xq26 and 2q33 Subeelular localization of nenrofibromstosal type 2 protein merl in human which characterize cemento-osalfyIng fibromas of the orbit. fibroblasts. B. Sdmsker' and Ma Kressel2. 1 Iatitute of Hmn Genetics, J.R SAWM1.3, A.F. Trykal, J. M. Bell', F. A. Booo2. University of Erlangen, 2 Institute of Anatomy, University of Erlangen, Germany Departments of Pathology' and Neurosurgery2 University of Arkansas for (Intro. by: K H Grzeschik). Medical Sciences and Cytogenetics Laboratory3, Arkansas Children's The recently cloned gene for nerofibromatosis type 2 (NF2) encodes for a 595 Hospital Little Rock, AR. amino acid protein, named merlin, due to its striking homology to the ERM fmily, (moesm-sim-nadixin-lke protein). The ERM family members are localized at cell- Cytogenetic reports of histologically benign fibro-osseous lesions are rare, cell and call-substrate adherens junctions and thought to play a role i anchoring of with only nine previous cases being reported. None of these previous actin filaments to the plastm membrane. Based on its homology, merlin has beoe studies have revealed consistent numerical or structural chromosome to its in aberrations, and to our knowledge no recurrent karyotype abnormalities In supposed to be linked the plasma membrane, but hitracellslar localizto fibromas of the orbit have been reported. Short term in-situ cultures and cellsphysiologically expressing NF2 protein is still unknown. Giesma-band chromosome methods were used to analyze three cemento- In order to studythe cellular localization ofmerlin, we used a monoclonal anibody ossifying fibromas of the orbit, one from a 1 3-year old Black male, one from a directed agint the N-terminal amino acids I - 168 (Transduction Laboratories). h 14-year old Hispanic male, and one from a 17-year old White male. Wester blotting experiments, the NF2 protein was highly expessed in human Cytogenetic findings in these three cases revealed the presence of fibroblasts and detected by a single band at about 75kD. Fibroblasts were cultivated nonrandom chromosome breakpoints In all the three tumors at bands Xq26 in RPMI and used in the 4th to 7th passages. mmunofluorescent labelling and and 2q33. Two of the tumors showed identical t(X;2)(q26;q33) reciprocal e nat in by conIcal lser camg microscopy revealed a maked tig o the translocations as the sole abnormality, while the third tumor revealed an central pas or the entire cell body, in areas of rding membranes and in fopodia. interstitial insertion of bands 2q24.2-33 into Xq26 as the sole abnormality. The In addition, ints labeling was also found near the cell membrane ofspontaneously finding of identical breakpoints associated with two different types of spoptotic cells, as revealed by double labelling with ant-NF2 and in situ 3'ead structural chromosome aberrations strongly suggests these breakpoints are labelling ofDNA double strand breaks. After preabsorption ofthe antibody with a N- diagnostic for this tumor subtype. The localization of breakpoints in cemento- terminal protein firgment (residues 1-265) of the NF2 protein no immnmoreactivity ossifying fibroma of the orbit to Xq26 and 2q33 provides a new example of was found. Selective solubilization of the fibroblast membrane fraction using first nonrandom chromosome aberrations in cancer that correlate with tumor Lubrol WX and then NP40 extraction, reveal a single band at 75 kD exclusively in specific location and tumor specific histopathology. the Lubrol WX fraction. In contrast, no band could be detected in the NP40 fraction. In concluion, merlin is concentrated in or linked to the plasma membane and the localizion in the spreading membranes may indicate a cell-substrate adhesion fimction. Posters: Cancer Genetics (continued) A77 413 414 Report of the first recurring chromosome translocation In Sequence of genetic events In the carcinogenesis of tumors of hepatoblastoma: der(4)t(1;4)(q12;q34). N. R. SchnaiderlJL. D. the uterine cervix Evelin Schrbck1, Kerstin Heselmeyer2, Stanislas du Manoir1, Keerti Shah3, Rtdiger Qoole2 M. J. Fi d3.E. G. E. Tomlinson1. 1Univ. of National Center Southwestem Medical Center at Dallas, 2Univ. of Texas Medical Steinbeck4, GertAuer2, Thomas Ried1, tDiagnostic Development Branch, Texas for Human Genome Research/NlIH, Bethesda, Maryland, Department of Pathology, School at Houston, 3Baylor College of Medicine, Houston, TX, 4St. 2Karominska Institute and Hospital, Stockholm, Sweden, 3Johns Hopkins University School of PA. Christopher's Hospital for Children, Philadelphia, Hygiene and Public Health, 4TumorarchivFlensburg, Flensburg, Germany The most common recurring cytogenetic abnormalities in hepatoblastoma, We have studied normal epithelium, mild dysplasias, moderate dysplasias, a rare malignant liver tumor of very young children, are trisomies of chromo- severe dysplasias/CIS and invasive carcinomas of the uterine cervix to somes #2 and #20. The only reported recurring structural chromosome identify chromosomal aberrations that occur during carcinogenesis. Defined abnormalities, dup(2q) and i(8)(q10), involve only a single chromosome. We regions of formalin fixed and HE-stained tissue sections were report 4 cases with a derivative chromosome #4 from an unbalanced microdissected and investigated using comparative genomic hybridization, translocation between the long arms of chromosomes #1 and #4. The DNA-ploidy measurements, a proliferation marker and HPV-genotyping. breakpoints in three cases appear to be der(4)t(1 ;4)(q12;q34), whereas the Early stages (mild and moderate dysplasia, n=7) revealed tetraploidization fourth case has breakpoints (q25;q32). All patients are males, 17-22 months and increased proliferative activity. No recurrent chromosomal aberrations old, with stage 3 or 4 tumors of unremarkable histology. All had hyperdiploid or HPV infections were observed. Severe dysplasias and carcinomas were the to tumor karyotypes; however, in the case with (q25;q32) breakpoints, characterized by pronounced genetic instability (scattered DNA values up in the risk class der(4) was the only abnormality in the stemline. C-banding, performed 8c) and proliferative activity. Infection with HPV of high one case, confirmed the 1q12 breakpoint by showing a small C-band in the (HPV16, 31, 33, 45, and 58) was frequently observed. A gain of middle of the long arm of the derivative #4 chromosome. The der(4) in these chromosome 3q was detected once in severe dysplasias (n=13). In hepatoblastomas results in partial trisomy of lq. Trisomy for 1q involving carcinomas (n=1 0), however, the overrepresentation of chromosome 3q was and detectable other chromosomes has been reported in three other hepatoblastomas the most consistent chromosomal aberration (90%), and was also is a well recognized secondary karyotypic change in solid tumors. Therefore, as a high level copy number increase (amplification). We conclude, that a in the we speculate that the oncogenetic event in our cases may be the loss of increases in proliferative activity and tetraploidization are early events gene or genes on distal 4q or their inactivation by juxtaposition to 1q12 genesis of cervical carcinomas. In addition to high risk HPV-infection, has heterochromatin. As yet, no known oncogene or tumor-suppressor gene aneuploidy, and high proliferative activity, the specific gain of chromosome seen been mapped to this region of 4q and neither of these 4q breakpoints is 3q sequences is the most consistent chromosomal aberration, that occurs nonrandomly in other tumors. when severely dysplastic cells progress to invasive carcinomas. Two of these cases were studied in conjuction with the Pediatric Oncology Group's Hepatoblastoma Biology Study.

415 416 Complex chromosome mechanisms leading to the loss of wild-type allele Chionsosen 3 q in ANNL break points clarified by chromosomepainting of APRT gene In hetlroplold cells. C Shao P K Gupta Y SunA. Sahota. P Sinclair. V.Broadbendt. P Harper.ARGreen* and E Nacheva JL A] Tkafiald. Indiana Univ. School of Medicine, Indianapolis. Dcpt. Haematology, NHS Trust Addenbrooke's Hospital *Dept. Haematology, University Cambridge Loss of the wild-type allele of a tumor suppressor gene, or loss of heterozygosity (LOH), is one of the most important mechanisms of On the basis of a review of the literature and our own observations of 3 ANLL cases carcinogenesis. We have used the adenine phosphoribosyitransferase (APRT) with 3q abnormalities clinicopathological sub types based on chromosome break gene as a model to study mechanisms of LOH. A previous study with an point involvement are suggested. normal or HT1080-derived human fibrosarcoma cell line showed that recessive 1) 3q21. Translocations involving 3q21 and lp36 are associated with, raised platelet counts, dismegakaryopoiesis and diserythropoiesis at the APRT locus was related and phenotype expression heterozygous predominantly 2) 3q26. Translocations between 3q26 and 21q22 while invariably therapy caused by the loss of the wild-type allele. Here we report the further frequently displaying total / partial loss of chromosomes 7 & 5 show neither raised characterization of the pathways leading to LOH. A series of CA microsatellite platelet counts or dismegakaryopoiesis. A wide range of translocations partners for repeat DNA markers along chromosome 16 were employed to determine the the distal third of 3q have been reported , the most frequent of these being and intervals of LOH in different APRT - clones. We also karyotype 2pAssigned break points on chromosome 3q have varied between q26 q29 maybe performed reflecting the limited resolution of banding analysis in leukemia cells. Following and FISH with an we have the analysis, chromosome 16 specific painting, APRT-containing dual colour chromosome painting on our own case of t(2;3) assigned cosmid on four clones and their heteroploid parental cell line. Our findings break point to 3q26. We propose the existence of a sub type of ANLL with suggest that (i) LOH occurred in tetraploid cells as well as in diploid cells, and t(2;3)(p22;q26) which shares the clinicopatrhological characteristics of the diploid cells with LOH may undergo endoreduplication to attain tetraploid. (ii) in t(3;21)(q26;q22) sub-type. or are well addition to LOH caused a event such as mitotic recombination and 3) Both 3q21 and 3q26. Inversions of 3q21/q36 t(3;3)(q21;q26) by single documented and known to be associated with clinicopathological features of both of different deletion, there was LOH caused by combination sequential events, subgroups described above. such as mitotic recombination or translocation followed by chromosome loss. 4) 3q25.1. Translocation between chromosomes 3 and 5 are non randomly associated The instability of the genomes of the heterozygous parental cells, which has with ANMUJ M6. They show trilineage dyspalsia but no raised platelet counts and do been demonstrated to contain both activated N-ras and inactivated p53 genes, not seem to be therapy related. As with t(2;3) break point assignations have varied that these cases share of for evolution. Similar though evidence from high resolution G banding suggests may may have provided a greater diversity options genetic chromosome we common break points at 3q25.1 and 5q34. Using FISH painting of in thus further karyotypic evolution may occur at late stage carcinogenesis have assigned the break points in our case to 3q 25 and 5q34 providing vivo.(Supported by NIH grant DK38185) evidence for a t (3;5) sub-type with a break point on chromosome 3 distinct from other chromosome 3 ANLL sub-types. Molecular evidence relating to the existence of these different sub-types involving chromosome 3 in ANLL is discussed.

417 Poster Symposium-Session 30 418 IS INHERITANCE LINKED TO BRCA1 A FACTOR OF BAD PROGNOSIS IN Constitutional chromosome inversion 7 [inv(7)(ql1.2q22j and acute leukemia. W.S.Stanley1.S.Burkectt.B.See2.A.O BREAST CANCER? B. George2 T. Lobet2 and N. Shah2. lGenetics & IVF Institute, Fairfax, H Sobol, J Jacquemier, F Eisinger, C Julien, D Bimbaum, VA, 2Geisinger Medical Center, Danville, PA. Deparunent of Oncogenetics, INSERM U 19,. INSERM U379 Institut Psoli- Acquired aberrations of chromosome 7 are common in the Calmettes, 13973 Marseille Cedex 9 France. myelodysplastic syndromes (MDS) and acute leukemias, especially acute myeloid leukemia. These abnormalities include loss of the entire Whether hereditary breast cancer (HBC) may harbour a specific morphological patten chromosome, deletions on the long arm, and unbalanced and-prognosis has not yet been solved. To address this question, we compared HBC translocations. It has been suggested that band q22 represents a with 4414 cases from the "Bouches-du-Rh6ne' breast cancer registry (BDR registry), critical region. This same chromosome band has also been described as for the histological subtypes and grades, according to age. HBC,women with breast a critical region in uterine leiomyomas. cancer, were selected froim families with a positive lod score using chromosome 17q We report here two cases of acute leukemia where the sole markers and/or a germ-line mutation of thc BRCA1 gene, and the availability of cytogenetic abnormality was a paracentric inversion of chromosome paraffin blocks. 7[inv(7)(qll.2q22)J. In both cases this inversion was constitutional. The histology of 22 breast cancers, from 9 families (5 with at least one ovarian The first patient was a 7 year-old male diagnosed with acute cancer), was reviewed. Mean age at diagnosis in HBC was 45 years, 36% (8/22) of lymphoblastic leukemia. The second patient, unrelated to the first case, with acute leukemia. This also which were below 40, vs 59 years and 7% (308/4414) below age 40 in the BDR was a 70 year old female myeloid patient registry (p<0.001 and p<0.0001, respectively). Below the age of 40, 100% of HBC has three first degree relatives who have been diagnosed with were of infiltrating ductal carcinoma subtype, vs 63% in the BDR registry, (p=0.026, hematological disorders. These are a mother, now deceased, who had Fisher's exact test) and a grade 3 was found in 100% of HBC vs 31.5% in the BDR leukemia, a sister with MDS, and a son with idiopathic registry (p=O.00013). thrombocytopenia who has been cytogenetically examined and carries In our study, BRCA I-associated breast cancer is significantly associated with a grade the same inv(7). Orye and Van Bever (J. Med. Genet., 20, 231-232,1983) 3 when considered both as a whole and below age 50 at onset. A grade 3 is a factor of have also reported a case of acute lymphoblastic leukemia in an 11 female with the same constitutional bad prognosis, so it implies that BRCAl-associated breast cancer is of worse year-old inv(7)(qll.2q22). that this either prognosis than sporadic breast cancer. This finding is quite unexpected, in the sense These observations raise the possibility inversion, by that HBC and sporadic breast cancer are usually considered of equivalent prognosis. gene loss, mutation or position effect, is a predisposing event for the Were a reduction of survival observed in HBC, it would have important implications in development of acute leukemia. Molecular studies of the breakpoint terms of care management and preventive behaviour. It implies to set up a cooperative junctions may help identify the gene(s) within the 7q22 critical region propective study including additional families with regard to the other breast cancer associated with these leukemias. genes. A78 Posters: Cancerr Genetics (continued) 419 Poster Symposium-Session 30 420 Genetic scoing for ERCA1 mutatlow in breast and/or ovarian cancer famillie. Interphase analysis of aneuploidy in hematological malignancies: How well R Tarft1lini,k M Rd7inmh 1 Andrann sand sF . .2und. Univ. Texas M.D. do the results correlate with cytogenetics? S. A. Tharapel and L. Balazs. VA Anderson Cancer Center, Houston, Texas. Medical Center and The University of Tennessee, Memphis, Tennessee. In 1994, a gene for hereditary breast and ovarian cancer, designated BRCA1, was In situ hybridization (ISH) using non-isotopic probes for chromosomes cloned. This gene maps to chromosome 17ql2-21 and contains 22 coding exons. and chromosome regions has provided the ability to detect chromosome BRCA1 encodes a protein of 1,863 amino acids which contains a RING finger domain abnormalities that are beyond the resolution of cytogenetics. This near its amino terminus. To date, 38 distinct germline mutations in the BRCA1 gene technique is particularly attractive in the area of cancer cytogenetics to have been reported. We are analyzing a total of 19 breast-ovaian and 26 site-specific evaluate and monitor aneuploidies on interphase nuclei. To compare the breast cancer families identified through patients at M.D. Anderson Cancer Center. sensitivity and accuracy of nuclear in situ hybridization (nuc ish) with We initially concentrated on screening 19 families that had not been excluded by metaphase cytogenetic analysis, we retrospectively performed nuc ish on 50 linkage to the BRCA1 locus. We selected one or two affected women from each samples of bone marrow/peripheral blood from hematologic malignancies, family and analyzed their PCR amplified genomic DNA for germline mutations in the following routine cytogenetic analysis. These included 21 cases of coding region of BRCA1 using heteroduplex analysis and direct sequencing. Up to Myelodysplastic Syndrome (MS) or leukemia with trisomy 8, eight cases of now we have identified mutations co-segregating with breast and ovarian cancer in 4 MDS/leukemia with monosomy 7 and 21 cases of Chronic Lymphocytic of 19 (21%) families: three of these are frameshifts (two mutations in exon 2 and one Leukemia (CLL). The CLL cases were tested for trisomy 12 irrespective of in exon 11) and one is a nonsense mutation (this mutation, not previously observed, their karyotype. Alpha satellite probes for chromosomes 7, 8 and 12 were is a C toT transition in exon 15). In addition, we observed several other variants in commercially obtained (Oncor Inc.). Slides were freshly prepared from different families that have not 5 stored cell suspensions, aged in 2XSSC and hybridized with appropriate been sequenced yet and polymorphisms that appear probe for 2 hours. A minimum of 300 nuclei were scored for each case. Of to form a haplotype in some families. We also have in progress linkage studies to the 21 CLL cases, nuc ish identified six cases of trisomy 12, including the BRCA2 (on chromosome 13q 12-13) and other susceptibility loci. Some of these four identified on cytogenetics. In MDS/leukemia cases with trisomy 8 and families contain male breast cancer in addition to a high incidence of female breast monosomy 7, most showed concordance with cytogenetics. In three cases of cancer. We finally propose to study tumor tissue of patients with primary breast cytogenetic trisomy 8 and one case of monosomy 7, nuc ish either failed to cance (tunor and normal tissue specimens embedded in paraffin blocks and on slides identify the aneuploid cells or the proportion of aneuploid cells was lower are also available for some of the affected) to detect loss of constitutional than what was seen on cytogenetics. Further, 17 of these 50 cases had heterozygosity and somatic mutations. In fact it is likely that BRCA1 may be revealed additional abnormalities, unrelated to the respective aneuploidy. responsible not only for hereditary breast-ovarian cancer, but also for the more This study suggests that interphase analysis could be most useful and common *sporadic' form of the same tumor type. informative when performed in conjunction with cytogenetics.

421 422 Detection of minimal residual disease in breast cancer patients by RT. Frequent Loss of Heterozygosity of Chromosomes 8 and 9 in Follicular PCR and ICC. M.DIT stman B Murphy I G Sharp A Kessinaer CA Thyroid Cancer. K TungD.ITribune,k D Sheylin. S. Wells,A J Kuszynk. S Pirruccello S Tarantolo. E Zuvanich and E Reed University of Jr Nebraska Medical Center, Omaha NE. Goodfellow. Washington University School of Medicine, St. Louis, A sensitive and specific detection method that would identify occult tumor cells in Missouri. the bone marrow and peripheral blood of breast cancer patients would provide a more Follicular thyroid cancers (FTC) constitute a small proportion of effective tool to evaluate therapy and prognosis. The aim of this study was to evaluate differentiated thyroid cancers and appear to be genetically and biologically the use of two new techniques, reverse transcriptase polymerase reaction (RT-PCR) distinct from papillary thyroid cancers. Our current understanding of the and immunocytochemistry (ICC), for the presence of minimal residual disease (MD). genetic mechanisms behind the development of these cancers remains The RT-PCR technique used nested primers that would detect the presence of an limited. The identification of markers that characterize FTC mRNA transcript from the cytokeratin 19(K19) gene. The ICC technique used a genetic may monoclonal antibody, CAM5.2, that would detect human cytokeratins 8 and 18. The prove clinically useful for diagnosis by fine needle aspiration and for the 53 samples obtained from 26 patients were analyzed by RT-PCR and included 19 bone determination of prognosis. We have undertaken an allelotype analysis marrow aspirates, 29 peripheral stem cell harvests and S bone marrow harvest 12-week of 32 FTC specimens prepared by microdissection from archival, paraffin- cultures obtained from patients at UNMC prior to transplant. RT-PCR identified embedded tissues. Thus far 22 chromosomal arms have been assessed for positive K19 transcripts in 4/19 (21%) bone marrow aspirates, 5/29 (17%) peripheral loss of heterozygosity (LOH) using 27 highly polymorphic microsatellite stem cell harvests and 2/5 (40%) bone marrow harvest 12-week cultures. Of 26 markers. The percentage of LOH at various from 0% 6 patients, 11 (42%) were positive by RT-PCR. Preliminary ICC results available only regions ranges (at on the RT-PCR samples identified one positive peripheral stem cell harvest of the five chromosomal arms) to 41% (at 9q). The chromosomal arms characterized identified by RT-PCR. Two bone marrow harvest cell cultures originally positive by by the highest frequency of loss are 9p (9/27), 9q (11/27), 3p (7/25), 3q (6/22), RT-PCR were negative by ICC. Follow-up cultures on these patients using samples 8p (6/22), 8q (6/18), and 2p (4/12). For chromosomes 8 and 3, loss of the collected at a later time point and analyzed by ICC alone proved to be positive. These entire chromosome seems to be a feature of FTC. These data confirm a results suggest that RT-PCR is a more sensitive detection technique for monitoring previous report of loss chromosome 3 LOH in FTC. We have identified MRD in breast cancer patients that will help to determine (1) the effectiveness of LOH chromosome 8 and 9 in the of FTC adjuvant therapy, (2) the potential of mobilization of tumor cells into the circulation frequent of sequences panel with adjuvant chemotherapy and/or cytokine therapy and (3) the disease-free and specimens examined. Further refinement of the regions of allelic loss will overall survival times following stem cell transplantation. serve to localize tumor suppressor genes important in the development of FTC. (Supported in part by LB595 Nebraska Department of Health and NCI Grant P30CA36727.)

423 424 The gene for Bazex-Dupr6-Chrlstol syndrome maps to A major role for the 3p21 region and lack of involvement of the t(3;8) chromosome Xq P Vabredl D. Lacombe. LG. Ranowl=A0.Aubrt breakpoint region in the development of renal cell carcinoma sug- C-E AndEreOnA Ta-ieb2 .J-L.Banaf4A- Munnichi W-C_ Hort-eCavial I gested by loss of heterozygosity analysis. A. van den Berg, M.M.F. Hulsbeek, Unit8 INSERM U 393, H6pltal Necker Enfants-Malades, 75743 Paris Cedex D. de K. Kok, 15, France. 2 Department of Medical Genetics and Department of Pediatric Jong. P.M.J.E. Veldhuis, JRobsel. C.H.C.M. Buys. Dept. of Med- Dermatology, Hopital Pellegrin-Enfants, Bordeaux, France. 3 Departments of ical Genetics, University of Groningen, The Netherlands. 'Div. of Medical Oncol- Pediatrics and Dermatology, Medical College of Wisconsin, MACC Fund ogy. University of Colorado Health Sciences Center, Denver, USA. Research Center, Milwaukee, WI, USA. 4 Department of Dermatology and In a loss of heterozygosity analysis of 3p we examined 44 sporadic cases of renal Dermatological Research Laboratory, Rangueil Hospital, Toulouse, France. 5 cell carcinoma (RCC) and matched normal tissue with 27 microsatellite markers. Division of Medical Genetics, Jefferson Medical College, Philadelphia, PA, The majority of these markers clustered in three regions that have been suggested USA. to be involved in the development of RCC, namely the p25 region, where the Von Bazex-Duprb-Christol syndrome (OMIM 301845) is an inherited condition Hippel Lindau (VHL) gene is located; the p21 region, which has been identified with skin cancer predisposition characterized by follicular atrophoderma, hypotrichosis and early onset of muitiple basal cell carcinomas. Previous as the common region of overlap of heterozygous deletions: and the p14 region, reports suggested an X-linked mode of inheritance. We therefore performed which is the location of the constitutional t(3;8) breakpoint occurring in an RCC linkage analysis with DNA microsatellite markers of the X chromosome In three family. Our analysis shows that when deletions were detected the 3p21 region families. We obtained evidence for linkage of this syndrome to Xq24-q27 was always included. The t(3;8) breakpoint region showed the lowest percentage (maximal lod score - 5.26 wit a recombinaton fraction of 0% at the DXS1192 of loss of heterozygosity. Moreover, in three cases the t(3:8) breakpoint retained iocus). Although the entire family set was small due to the rarity of this disorder, heterozygosity, whereas a region more proximal to the breakpoint showed allelic linkage analysis performed in each family yielded maximal lod scores over 2.0 In two families. The hereditary conditions described by Parrish et al. (OMIM losses. This supports exclusion of the t(3:8) region from a role in the development 146530) and Oley et a/. (109390) may represent phenotypical variations of of sporadic RCC. In a number of tumors two or three 3p deletions were present Bazex syndrome, because of clinical similarities (hypotrichosis, milia, separated by a region of retention of heterozygosity. In these tumors deletions associated or not with basal cell carcinomas) and putative X-linked at 3p2l occurred in combination with deletions of either the VHL region, or the Inheritance. Thus linkage to Xq24-q7 markers should be tested in these region proximal to the t(3;8), or both, suggestive of multiple gene involvement in families. This represents a first step towards the identification of a gene the development of sporadic RCC with a primary role of the 3p21 region. Involved in hair follicle development and skin tumour formation. Posters: Cancer Genetics (continued) A79 425 Poster Symposium-Session 33 426 A novel multi-probe FISH procedure for the simultaneous detection of CDKN2 (P16) MUTATIONS IN AUSTRALIAN MELANOMA chromosome aberrations, disomy and diploidy In sperm of Hodgkin patients KINDREDS. G.J. Walkerl,2, J.F.Flores-lCJ. Hussusiaa, LM, receiving chemotherapy. P. Van Hummeleal X Lowel -M.L Meistrch2. AJ. Glendeningl, N.C. Draci4, N.K.Haywagd2,and J.WFouni I. IUniv. of WvXbkl. IBio. Biotech. Res. Prog., Lawrence Livermore National Laboratory, So. California, Inst. for Genet. Med., Dept. Biochem. and Mol. Biol., L.A., CA; Livermore, CA; 2MD Anderson Cancer Center, Dept. Exptl. Radiother., Houston. TX. 2Queensland Inst. Med. Res., Brisbane, QId. Australia; 3Dept. Surgery, Chromosomal aberrations and aneuploidy in sperm represent important categories Washington Univ. School of Medicine, St. Louis, MO; 4Sequana Therapeutics, of paternally transmitted genetic damage. Chemotherapy for Hodgkin's disease Inc., La Jolla, CA. includes mutagens that induce both types of chromosomal damage and persistence in The cyclin dependent kinase inhibitor gene 2 (CDKN2), mapping to chromosome germ cells would have important implications on the reproductive health of survivors. 9p21, encodes a protein termed pl6INK4 (Serrano et al., 1994) that is involved in Since no practical assay exists for monitoring chromosome aberrations in sperm, a control of the GI-S phase of the cell cycle. Inactivation of this gene presumably new multi-probe FISH method was developed. The labeling strategy was designed to results in the loss of the "brake" at GI, with concomitant increase in cell detect sperm carrying terminal duplication or deletion of the short arm of proliferation. p16 controls the transition by inhibiting the of chromosome 1 and aneuploidy involving chromosomes 1 or 8. Base levels were GI-S activity protein established for 4 healthy donors; reproducibility between hybridizations and multiple complexes consisting of cyclin dependent kinases (CDK4 or CDK6) and cyclins samples from the same donor was demonstrated. The frequencies of telomeric (Dl, D2 or D3) and it has recently been shown (Ranade et al., 1995) that missense mutations within the to duplications and deletions of chromosome I were similar and ranged from 3 to 9 and p16 protein impair its ability inhibit these complexes. from 0 to 19 per 10,000 respectively. Disomy for chromosomes 1 or 8 ranged from 1 CDKN2 is potentially involved in the genesis of many tumour types, and has been to 13 and diploidy from 7 to 14 per 10,000. under intense investigation as a melanoma susceptibility gene. Semen from 3 patients undergoing NOVP chemotherapy and from 4 patients 3-25 years after MOPP chemotherapy were also analyzed to determine the induction and In a study just completed with 18 Australian melanoma kindreds, we analyzed all 3 persistence of defective sperm. Patients undergoing chemotherapy had significant 5- exons of the CDKN2 gene by SSCP and sequencing. Overall, we detected 6 fold higher levels of telomeric duplications (16 to 40110,000) and significantly different mutations that segregated with melanoma in 7 families, including the elevated frequencies of disomic and diploid sperm (15-30/10,000 and 80-130/10,000 largest 6 families. Of these seven families, four had been previously shown to be resp.). For semen samples provided many years after chemotherapy, significantly linked to markers on chromosome 9p2l, while the other three have now been higher levels of diploid and disomic sperm were observed for one donor and a higher determined to contain one to three sporadic cases of melanoma which originally frequency of telomeric loss of chromosome 1 was found for another. confounded previous linkage analysis. Similar analyses with US kindreds by These findings indicate that this FISH assay can detect structural and numerical Hussussian et al. (1994) and Kamb et al. (1994) have yielded somewhat conflicting chromosome abnormalities in sperm and demonstrate that chemotherapy can induce results, with 9/18 and 2/13 families respectively, harbouring mutations. We now both types of genetic damage in sperm which may persist in some patients. report analysis of another 31 Australian melanoma kindreds. SSCP assays have initially revealed what appear to be novel variant bands in 5 ofthese families. These IWork was performed under the auspices of the US DOE by the Lawrence Livermore Nations. Laboratory under W-7405-ENG-48; P.V.H. was sponsored by NATO-Belgiuml. mutations are at present being characterized by sequencing, with functional studies contact also being carried out on all mutations detected in our 49 melanoma kindreds.

427 428 Amplification of c-myc as the origin of the homogeneous RT-PCR for detection of t(11;22) in pediatric seft tissue saromas. .D staining region in ovarian carcinoma detected by micro-FISH Wener, M.K. Roth S.J- Qlan. K Theil, and J.W Moore. Dept. of Laboratosy approach. N. Wang. T. Tyan. J. Xu. and E. Cedrone. Department of Medicine, Childrei's Hospital, Columbus, OH. Pediatrics, University of Rochester School of Medicine, Rochester, Several pediatric tumors, including Ewings sarcoma (EWS), primitive NY. neuroectodernal tumor (PNET), hm neuroblastoma (NB), and Homogeneous staining region (hsr), a cytogenetic indicator of gene lymphoma, appear cr i as uniform, ma, round ceob without evidence of amplification, has been frequently found in ovarian carcinoma (ovc). d ention. Differential dinsis icasl important as specific scessful To identify the origin of the hsr, chromosome microdissection therapies are developed for each tumor type. Detectio of charceristic tumor- combined with polymerase chain reaction (PCR) and fluorescence in specific chromosomal trarelocations can fiditate diagnosis; however, cytogenic studies are not succsum . Molecular assays for dntification of situ hybridization (FISH) was applied to a ovc cell line GR. A biotin always specific translocations and genetic indicators associated with each tumor type can provide labelled DNA probe generated from the hsr hybridized only to the rapid, specific diagnstc information. RT-PCR analysis for the t(l 1;22Xq24;ql2) chromosomal region of 8q24 which coincides with the chromosomal trns ion chac ofEWS and PNET is described for a series oftumors, location of the c-myc oncogene. To verify the involvement of the c- incuding two interesting cases. Cell lines and 13 prmar tumors have boen tested to myc in hsr formation, in situ hybridization with the probe specific for date. Of6 tumor smpls tested with a diagosis ofEwings sarcoma orPNET, five the c-myc oncogene was conducted and confirmed the amplification of were positive for the t(l1;22)tranlocati, including that ofa patient who presented the c-myc as the origin of the hsr. To determine the origin of the with tumor in an exremity diagnosed as EWS. e klaeretroperitoneala entire hsr bearing chromosome, micro-FISH approach was then surrnltumorwasfound;inthistumorsank evidenceofN-myc performed on the whole hsr bearing chromosome. FISH with the ampfication was found by FISH; however, RT-PCR did ident a 350 bp fiion probe generated from the whole hsr bearing chromosome hybridized scipt allowin definitive diagnosis as metastatic EWS a oppoed to to 5q(qter-pl 1) in addition to 8q on the metaphase spread of GR. The NB. Sev other small, round cell tumors were nepgive, incuding one, tentel whole hsr bearing chromosome can then be designated as identified aS PNET, which had the baryotype 45,Xt(X;5;15Xpl1.2;ql3;qll.2), der(5)t(5;8)(pll;q24)hsr(8q24). FISH with the probe generated from t(7;12Xql I .2;p13),t(l 1;22Xq24;ql2),t(13;14)qlO;qlO),-14; the paiens peripheral the hsr of the cell line GR to two other hsr containing ovc cell lines blood katyotype was 46,XX,t(13;14). The t(X;5;18) is a variant ofthe typical paints the entire hsr of the cell line MLSP. This indicates that the t(X,18) associated with synovial sarcoma, combined cytogenetic and RT-PCR rests suggst a dgnosis ofsynovial scoma This method of amplification of 8q24/c-myc as the origin of hsr may be a nonrandom detecting fusiontanscripts can form the basis for specific, nsite diagnotic of genomic alteration in ovc. (supported by NIH grant CA52761). rapid, testing selected tumors.

429 430 Allelotype analysis of medulloblastoma. E.C. White. S. Sih. L. Daneshvar. M.S. Genetic and physical mapping of the gone for nevold basal von Haken. N. Abrikosova. E. Choi, M. Bumnett. J. D. McDonald.and P.H. Cosen. cell carcinoma syndrome. C. Wickdng. S. 1il11es. K. Neou. l.Smvth. Univ. of Chicago, Chicago, Ill. J. Berkman. S. Shanlov*. G. Chenevix-Trench'. and B. Wainwright. Brain tumors remain the second most common cause of cancer death in children, Centre for Molecular and Cellular Biology, University of Queensland and yet there is little known about their molecular genetic etiology. Using a panel of 22 'Queensland Institute of Medical Research, Brisbane, Australia chromosome arm 17p probes, we have previously determined that LOH with markers Nevoid basal cell carcinoma syndrome (NBCCS, or Gorlin syndrome) a located on l7pl3 is seen in 13 of 37 (35%) specimens of medulloblastoma, one of is an autosomal dominant disorder characterized primarily by to basal carcinomas of the skin. The the most common childhood brain tumors. Detection of 17p deletion was associated predisposition multiple cell (BCCs) syndrome is highly penetrant but variable in expression, giving rise to a with prognosis negative. Investigation of the known p53 tumor suppressor gene in vast array of additional features, many of them indicative of abnormal these same tumor specimens using a denaturing gradient gel electrophoresis (DGGE) embryonic development. The gene responsible for NBCCS has been technique showed only two mutations. These results suggest that an additional gene mapped to human chromosome 9q22.3-q31, and loss of heterozygosity or genes located on 17p is involved in the etiology of medulloblastoma. To extend of markers in this region in sporadic and familial BCCs suggests that It is these observations and determine the role of LOH at sites other than 17p, we a tumour suppressor gene. Based on combined multipoint and analyzed 35 of these same tumor specimens using restriction fragment length hapiotype analyses in an extensive collection of Australasian pedigrees polymorphism (RFLP) and microsatellite marker techniques. A total of 47 loci on we have refined the iocalization of the gene to between the markers 21 autosomes including 17q were studied. The markers that we selected included D9S196 and D9S180, an interval of approximately 2cM. Los of those located at or near sites of both putative and previously identified tumor heterozygosity studies have yielded several BCCs which would suggest suppressors and oncogenes. LOH was detected in the informative tumor specimens a more likely location for the NBBCS gene In the more distal portion of as follows: 6p (1/13), 7q (1/14), 8q (2/3), 9q (1/29), 10q (2/21), 13q (2/22), and 16q this interval. YACs spanning the candidate region have been used to (2/10). Analysis of one tumor revealed multiple sites of LOH at 9q and 13q. This screen a chromosome 9 only cosmid library, and resultant cosmids are being ordered into contigs around key marker loci. In an attempt to tumor and 3 others showing autosomal loss also had 17p deletions. Nine of the 10 further narrow the region known to contain the NBCCS gen, Southern patients with autosomal LOH showed tumor recurrence and died despite aggressive blots of pulsed-field gels containing DNA from over forty probands are therapy. The results of this study show that although extra-chromosome 17p LOH being analysed for the presence of germline deletions. Simultaneously, is rare in medulloblastoma, it is associated with a poor treatment outcome. we are searching the region for expressed sequences using the Allelotype analysis using a more intensive marker approach may reveal additional techniques of exon trapping and Island Rescue PCR. sites of LOH in these specimens as well predicting patient outcome and guiding therapy. A80 Posters: Cancer Cjenetics (continued) 431 Poster Symposium-Session 30 432 CHROMOSOMAL SUBLOCALIZATION AND CLINICAL CON- Origins of heterogeneous ovarian carcinomas: insights from a genetic SEQUENCES OF LOSS OF HETEROZYGOSITY FOR CHROMOSOME 11 analysis of histologically benign, low malignant potential, and IN HUMAN BREAST TUMORS. R. Winqvistl.2,3. G. Hampton', A. Mannermaa2. carcinomatous components. N.G. Wolf. F. Abdul-Karim. and S. Schwartz. Case V. Launonen2. G. M. Alavaikko4. H. Kiviniemis. P. J. Taskinen3. G. Western Reserve University and University Hospitals of Cleveland, OH. Blanco3. Evans6, Ovarian epithelial neoplasms are classified into 3 subtypes: benign, low maligant F. A. Wright78, I. Newsham89 and V.Caveneel910. Ludwig Institute for Cancer potential (LMP; proliferative) and malignant (carcinoma; invasive). It is unclear whether Researchl. Departments of Medicine9 and Family & Preventive Medicine7, Cancer these are separate entities (each arising de novo), or whether they represent a continuum Center8, Center for Molecular Genetics"5. University of California at San Diego, La of tumor progression. The histologically benign- and LMP-appearing components Jolla, CA 92093-0660, Departments of Medical Genetics2. Oncology and Radiotherapy3. found in heterogeneous carcinomas have been interpreted in 3 different ways: Pathology'. and Surgery5, University of Oulu, FIN\-90220 Oulu, Finland, McDermott (1) evidence of tumor progression (remnants of pre-existing tumors that underwent Center6, University of Texas Southwestern Medical Center at Dallas, Dallas, TX 73235- malignant transformation), (2) areas of maturation within the malignant tumors, and 8591. (3) evidence of de novo origins (coexisting lesions which arose independently). Loss of heterozygosity (LOH) for various chromosomal regions is a typical feature of To gain new insights into the clonal relationships of benign-appearing, LMP, and cancer. Such events encompassing chromosome 11pl5 and 11q23 are frequently seen in malignant components in ovarian carcinomas, we utilized fluorescence in situ human breast tumors. Here, we have analyzed genetic and clinical characteristics of a hybridization (FISH) and confocal microscopy to study each tissue subtype series of primary breast tumors in order to determine. (a) a more finely mapped estimate independently. Aneuploidy for chromosomes 8, 12, and 17 was examined in intact of the involved regions regions, (b) whether there is a relationship in the presentation of paraffin sections of 10 heterogeneous carcinomas and 5 normnal (control) ovaries. Six LOH between the two regions, and, (c) whether a correlation exists between such LOH of the tumors were abnormal: two exhibited only trisomy 8; one had only trisomy 12; and any of the clinical parameters pertained to each patient. LOH for 1ipl5.5 and 11q23 two were trisomic for chromosomes 8 and 12; and one tumor, with trisomy 8, 12, & occurred in 35% and 46% of the 86 carcinomas, respectively, but in none of the 10 benign 17, may be triploid. Further analysis of five tumors revealed that two were aneuploid in tumors examined. The minimal region of LOH for 11piS was found in an approximately all components, two lacked the abnormality in their benign-appearing components, and 2 M1b region between loci TH and D11S988. The majority of the studied tumors showed one lacked aneuploidy in LMP- and benign-appearing components. No histologically LOH simultaneously at both 11pl5.5 and 11q23. but unique LOH for either of these benign areas contained abnormalities that were not also present in the other chromosomal sites was also seen. among these genetic groups, most clinical features components. No aneuploidy was detected in stromal tissue of the tumors, nor in were not markedly different. However, LOH of 1iq23 (either alone or in conjunction with epithelial or stromal cells of the controls. These results have far-reaching implications LOH of I1pi5) in the primary tumor was found to be highly predictive of aggressive post- for ovarian carcinogenesis. They support the theory of tumor progression, and suggest metastatic diseasecourse with substantially reduced survival (P-0.0004, logrank test). we that some benign tumors which become cancerous have genetic aberrations also observed a trend towards a more rapid development of metastatic lesions, without predisposing them to malignant transformation. We also conclude that the benign- site-specificity, in primary tumors showing LOH for chromosome 11 in the pathogenesis of appearing components in a subset of heterogeneous ovarian carcinomas do not human breast cancer, suggesting that its effects are late in the progression of the disease. represent maturation of malignant tissue.

433 434 Fluorescent In Situ Hybridization Using Centromeric Probes to Assess Defining lethal mutations of the HRASI minisatellite. N. Wyborn. J. King. Aneuploidy for Chromosomes 7 and 8 in Myelodysplastic Syndrome and Other C. Wang. B. Heideman-Joosten. and T. Krontiris. Tufts-New England Medical Hematological Disorders. H.E. Wyandt, S. Ioannidou, D. Chinnappan, M. Center, Boston, MA. Salama, C. O'Hara and A. Milunsky. Boston University School of Medicine and Mallory Institute of Pathology, Boston, MA. Mutations of the HRASI minisatellite are, as a class, strongly associated with The detection of aneuploidy in interphase using chromosome-specific several common adult cancers, including carcinomas of the breast, colon, and probes has been widely applied to many types of tissues including those bladder, and acute leukemia. We have shown that several members of the relINF-gB from cancers and leukemias. Many studies have been done without the in- family of transcriptional regulatory factors bind the minisatellite and lead to allele- clusion of controls. We present data on 57 samples of bone marrow: 29 specific activation of transcriptional reporters. To obtain further evidence that the from patients with myselodysplastic syndrome (MDS) and 28 from patients genetic risk associated with certain HRASI VNTR alleles lies solely within the with hematological disorders which include leucocytosis, pancytopenia, minisatellite, itself, we have sought evidence that the HRAS1 minisatellite locus may MPD, ANLL and CML. Collectively, 20 of these cases show structural and/ or numerical chromosome abnormalities by routine cytogeneties. Samples demonstrate transmission distortion. The heterozygosity rate of )RASI is for fluorescent in situ hybridization (FISH) were selected on the basis approximately 65 % which, under one model of neutral mutation, is consistent with of whether aneuploidy for 7 or 8 was found. These were matched with a mutation rate near 104/gamete. Typing nuclear families for HRASI and VTR1.1, samples from about the same time which showed no evidence of chromosome a chromosome 1 minisatellite with a 95% het rate, revealed one new mutation for abnormalities. Slides were prepared from fixed cells which had been each locus in 200 gametes tested, or a mutation rate of 0.005. We then began typing stored at -200C for as long as 4 years. Probes for centromere sequences, D7Zl and D8Z2 (Oncor and Vysis) were applied to the right and left halves single genome equivalents of sperm DNA and, in two individuals examined thus far, of each slide. Hybridization was done in batches of 4 or 6 and included have demonstrated mosaicism at the 1% level. The mutation rate may be as much matched normal and abnormal cases. Slides were scored blind for the di- as ten-fold higher. Finally, mosaicism involves alleles much smaller than any we stribution of 0-4 signals in 100 interphases per each half. FISH results have found in the adult population for which we have established allele and genotype were compared with the results of chromosome analysis. The results for frequencies. Together, these results suggest that HRASJ minisatellite mutation is not D8Z2 show good correlation with the percent aneuploidy detected cyto- neutral and that certain very small mutations may actually represent developmental genetically. For D7Z1, however, this correlation is less perfect. Of particular interest are several cases (two with EDS) which showed 301 or lethals. more aneuploidy (monosomy) for chromosome 7 by FISH, but which were chro- mosomally normal by cytogenetic analysis. The questions of why aneu- ploidies in some cases are detected cytogenetically, why others are not and the possible prognostic relevance of FISH are discussed.

435 436 ocg lenoteeemdsf Fe7qbrege ionguinuleriebksmyaom . YP Co-amplification of int-2 (FGF3) and hst-1 (FGF4) genes on gi,2, S.W. Scherer3,4 Lj-C. Tsi34 and G.C. Morton'12. 1 Dept of Pathology, Brigham homogeneous staining regions (HSRs) in small cell lung and Women's Hospital, 2Harvard Medical School, Boston, MA 02115; 3Dept. of Molecular and cancer Identified by chromosome microdissection and FISH J. Xu. E. Cedrone. and N. Wang. Department of Pediatrics, University Medical Genetics, University of Toronto, and 4Dept. of Genetics, Research Institute, The of Rochester, Rochester, NY. Hospital for Sick Children, Toronto, Ontario. A HSR was found in the pleural effusion cells (BHII) of a patient with Uterine leiomyomata(UL) are the most common tumors in women and a major public SCLC after failure to multidrug treatment. Using chromosome health problem for women of reproductive age. Cytogenetic studies of UL have revealed microdissection and reverse chromosome painting (MICRO-FISH), non-random, tumor-specfic chromosome aberrations in approximately 25% of specimens. In 1 lql3.3 amplification was found to be the origin of the HSR. FISH with the Micro-FISH probe generated from the HSR in BHUI to cell line an analysis of 86 UL in our laboratory, aberrations of 7q are the most frequently observed BHI, established from the lymph node metastasis of the same patient cytogenetic subgroup and comprise about 35% of karyotypically abnormal tumors. The 7q prior to chemotherapy, revealed the preexistance of 1 1ql3.3 aberrations are of special interest as they have not been found consistently in any other solid amplification. This ruled out the possibility of therapeutic induction of tumors. Seven tumors in our series appeared at the cytogenetic level to have identical gene amplification. However, the 1 Iql3.3 amplicons in BHI and BHII deletions of 7q22-q32 while one tumor had a smaller deletion, 7q22-q31. Three UL were found involve in different chromosomal rearrangements. To identify the genes involved in the amplicons, FISH and Dot-blot hybridization with to have apparently balanced reciprocal translocations involving band 7q22 indicating that the probes specific for int-2, hst-l, CCND1 and ZNF6 were performed to pathogenetic region in the del(7)(q22.q32) subgroup is likely in band q22. Our efforts are BHI and BHLI. The results revealed that the amplicons on the HSRs in focused on doning the breakpoint region at 7q22 in UL to understand genetic contributions in BHI and BHII include the int-2 and hst-I genes. Previous studies have the origin, growth and natural history of these tumors. Fourteen YACs and 23 cosmids of reported that the int-2 and hst-I genes are frequently co-amplified in known order spanning the region of 7q21 q31 have been mapped by fluorescence in situ various human cancers including breast cancer and squamous cell tumors. Our result shows that int-2 and hst-1 co-amplification also hybridization to tumor metaphase chromosomes containing del(7)(q22-q31). Two adjacent occurs in SCLC and may play a role in the tumor progression in this cosmids about 1 Mb apart were found to flank the breakpoint within a small region of 7q22 in patient. one tumor with del(7)(q22-q32); this finding will be valuable in efforts directed toward identification of genes important in the biology of uterine lelomyomata. Posters: Cancer Geneltics (continued) A81 437 438 A Philadelphia chromosome positive chronic mydogenous leukemia patient with Stdies of HER2IM Nd c-Myc auadia a cam raft dea cwr a unique translocation studied via GTG-banding and fluorescent in situ fohreuee lo hfbybidltsa (M . SR YgUns W-H Liu. JK Bro AM Tute. hybridization. C. T. Young. I. Di Benedetto. L. Glasser . H. F. L. Mark. Dept. of an ST Smith. Department ofObstetrics and Gynecology, Universi of South Pathology, R. L. Hospital and Brown Univ. Sch. of Medicine, Providence, RI. Carolina, Columbia. The Philadelphia (Ph') chromosome was the first consistently occurring Ovarian cancer is the fifth leading caui of death in the United States and is often chromosome abnormality associated with a single cancer type, chronic myelogenous not diagnosed until an advanced tg ofdisease. It is ofgreat importance to botter leukemia (CML). The present report describes a case of Ph' chromosome positive s CML with a unique complex translocation. understand the biology dictating the eidology. progression and meae ofovarian A 68-year-old female presented with an elevated leukocyte count on a pre- cancer to ficilitate earlier diagnosis and batter treatment. Recently tnere have boe operative evaluation for cataract surgery. On physical examination there was a reports ofonoene plif associated with the maligna process in several palpable spleen, 3 cm below the left costal margin. A complete blood count showed a tissues. We report our studies ofthe aooge HER/2nad c-myc and the alpha hemoglobin of 13.6 g/dl, platelet count of 713,000/p1 and a leukocyte count of satellite sequences for c o #17 and #8 conducted on4 ovarian cancer 205,000/1l with segmented neutrophils 36%, bands 21%, metamyelocytes 14%, samle. Surgical biopsies were enzymaticaly disaggregated and d studied ng myclocytes 9%, promyelocytes 4%, blasts 2%, eosinophils 3%, basophils 3%, commercially available DNA probes (Vysis, Naperville, Dl). Two hundred cells were monocytes 5% and lymphocytes 3%. The bone marrow was hypercellular with scored for centromere copy nunbe and distribution ofoncogene probe sias. Eight numerous megakaryocytes. A differential count showed blasts 1.6%, promyelocytes samples studied with the HER2/neu probe showed moderate aplification (8-12 5.2%, cosinophils 12.8% and basophils 1.6%. The myeloid/erythroid ratio was 16:1. and 5 showed t (10-30 oopies/coll). Te These findings were diagnostic of CML, chronic phase. The cytogenetic studies were copiescell) samples high done prior to treatment. remaining 31 samples had the normal 24 copies ofHER2/neu present in each cell. GTG-banding revealed abnormalities in chromosomes 9, 13, 17 and 22. The amplification is very apparent and can sometimes be so in "clusters", ofte near Fluorescent in situ hybridization (FISH) studies were performed as an adjunct to a chromosome #17 costrme. Twenty-five ofthe 44 samples showed some conventional cytogenetic analyses. Using FISH with the Oncor bcr/abl probe, the Ph' amplification of c-myc and 4 samples showed high as lio. Tea samples were translocation previously hypothesized was confirmed. Applying FISH with paired shown to be amplified or highly amfplified for both HER2/neu and myc. N isese painting probes in various combinations, a complex translocation involving samples showed c-myc amplifia but did not show HER2/neu amplificetion; 3 chromosomes 9, 13, 17 and 22 was observed. The results of the GTG-banding and samples showed HER2/neu amplification but had normal c-myc. Only 12 ofde 44 FISH studies were compared and correlated with those of the hematological findings. samples were not amplified for one or the other oncogene. Thee is som tendency for In a search of medical literature databases (Medline, Health, Cancerlit, and the HER2/n amplified samples to also be mononomic for chromosome #17. In CINAHL) spanning nearly three decades (1966-1995), we found no previous report of this specific translocation. Therefore, to the best of our knowledge, this is a unique addition there appears to be a tendency for c-myc amplified smples to have polysomy translocation associated with Ph' chromosome positive CML. ofchromosome #8. Attempts to correlate these result with clinical state ar currently in progress. We are hopfl dtht s ofthis typo will lad to the identification of molecular markers usefil in early diagnosis, prognosis, or treatment ofovarian cancer.

439 Clinical molecular genetic screening in MEN2A/FMTC. B.A. Zehnbauer, J. Shields, C. Franz. H. Donis-Ke.Ig. J. Milbrandt. J. Moley, M. DeBenedetti, J. Ivanovich, B. Dowton, S. Wells. Jr. Molecular Diagnostic Lab. Depts. of Pe- diatrics. Surgery, and Pathology, Washington University School of Medicine, St. Louis. MO. MEN2A is an inherited disease characterized by medullary thyroid carcinoma, pheochromocytoma, and parathyroid adenoma. We describe our experience with direct mutation screening of the RET proto-oncogene in clinical specimens from 741 individuals of 48 MEN cancer families. We examined exons 10 and 11 for the 5 most commonly mutated Cys codons. Each specimen was tested by PCR amplifi- cation with two different primer pairs followed by either RFLP or direct sequencing analysis of the PCR product. Sequencing was also performed in duplicate with two different primer pairs on both strands. Genotyping assays confirmed the diagno- sis in 81 cases with previous surgical treatment and positive pathology findings. 125 of 623 presymptomatic, at-risk individuals were genetically positive for RET gene mutations. Results were consistent with the specific mutation identified in an affected family member and position in the kindred. 27 genotype-positive individ- uals (7 < 15 yo.) had negative results for plasma calcitonin stimulation. Seven people demonstrated positive stimulation in the absence of RET mutation: none of these had positive pathology results consistent with malignancy. Multiple spec- imens from 90 individuals were evaluated in independent assays. In every case the repeat DNA assay confirmed the findings of the initial molecular test. The different alleles identified included C609Y, C61IW, C618S, C618G, C618R, C618Y, C618F, C620Y. C620R. C620F, C620S, C634Y, C634R, C634G, C634W. and C634F. This study establishes the routine accuracy of clinical molecular RET screening in a large population of individuals at risk for MEN2A/FMTC.

Posters: Clinical Genetics, Malformations and Dysmorphology 440 441 Somatic mosamicism in a case ofsporadic neurolibromatosis. TheiUtlity of Intraocular Presu as a Quanttative Truk for Ienti age Analysis Pil.Ain2w R. C Shuan. tMedical Biochem., Hospital, R.R. allingharn1, .ERuMnifl,s a 1, L P 1,hd Wej&=3, Victoria Van . Duke Un sy WedS Center, Du m, NC; and 2Dept. Genetics, Children's Hospital of Western Ontarlo, London, Ontario, Hospial, Boston, MA. Canada. 3Dept. Genetics, Hospital for Sick Children, Toronto, Ontario, Canada Neurofibromatosis type I (NFl), affecting prmarily the growth of neural crest- Purpos: Fluid pressure within the eye, inracua pressure (lOP), whe an Y derived tissues, is one of the most commo aut dominant human genetic elevated is a major hxft associated with the deveopmnt of primary open ang glaucomn (POAG), a complex dir which Is a leIng ause of IIndM se wvodldes. disorder with an incidence of 1/3000. A ificat feature of NFl is its high Understanding gen(s) which control IOP may help elucd t the diology of P0A4. spontaneous mutaton rate, by some estimates as much as 1x0Igamete/ Although lOP Is flund by many factors Including p, tim Of dey. ad eseansinin generation1. It has been hypthesized that an NFl patient with an apparent new technique, It has a significent get component with an setimded h _dsLiy of 0.4.0.6 mutation is often a sotc mosaic for the mutation2. We would like to report a case (Annay. 1867; Leve, 170). IOPK pproidmase a nomnl distbon Inth ge tora of spoRdic NFI in which we confirmed the presence ofa deletion involving the NFl population (16 +V 3mm Hg), en In P0.4 Is usually 3- 21 mm Hg. We wuetook a plot gene, and which, consistent with the above hypothesis, appears to be a somatic study to determne t utility of lOP sea quantive 0taor11'h2g analsis. Meds: mosaic. Two examiners pefon appWann (Pefkin) toMihy oan 14mmnl comrle (4M, A bank of laucyte DNA samples derived from a panel of NFl probands was 10F, men a 27 y). OP was me at se s me da on two _ocss 2 reviewed with a view to selecting individuals who showed no h oity at a wek apar In a contld, masked fashlon. seriesof DNA pol morphic sites, found in exon 5 and inos 27, 38and 41ofthe R_elt: Mn IOP +- Sd (mm per for Each Visit NP1Senem One iubind-ividual, who was known to have sporadically occurring NFl, IVIsit I Visit 2 MMRnq (be vislts diagnewd by the presence of multiple CAL's, nr ib and bilateral Lisch 15.5 Or2.1 115.7 +1.6 12-20.5 nodubs, and who had no family history of this disease, was initially determined to Exmlner2 14.3+1.9 14.1+1.3 8.17 have henuzygosity involving the NFl gene locus. This appeared to be the result of a The diffeenc between examiners wes stetoslly agi Namedt ( * 4.30, Pi 0.0001). de novo ddedor event, extending at least from inuns27 to41, which occured on Th mean dif in lOPbewn vbist wee 0.10.5-2.00mm Hg for exa d the maternally-derived chronosome. However closer o f the hapte 0.186+.1.56mm Hg for exmlr2 wh werenot statistiollygt-lsui dat revealed that a trace of the matnally-derived NFI haplotp was detecble i Conclusions: it maybe possibletoperform genetic lnlmg snalyasusiglOPas all of the informative PCR-aplified intragenic markers. he mosicism at the NFl quantative treal provided facto affecting IOP ar contolled for and one m rw Is locus in this affected individual's leuocyte DNA was confirmed by Souten used or difrencas bethwen examiners are cored. Meppin the ges for IOP may analysis coupled with densitometry. Furdter studies on the frequency of somatic be a usef approach for identying genes Involved In POAG. mnicism and the parental origin ofofNFI pon o mutations, will be required to b understand the associated clinical p p and genetic mechanism(s) contribing to the high spontaneous mutation rate for NFl. Rdferenem:t 1. Huson et aL, (1989)1. Med. Genet 26:704-711 2. Zlotog (1993) Am. J. Med. Genet. 46: 182-184 A82 Posters: Clinical Genetics, Malforrmations and Dysmorphology (continued) 442 443 The Neurofibromatosis-Noonan syndrome: continued An unusual form of autosomal recessive spastic controversy.MoM. r.M. Articas-Loez. R. Sutphen J.D. paraplegia, developmental retardation, Rael nd g.cG K0314serf. mlcrocephaly, and progressive deterioration in a Pakistani family. F Ande maEu.E Dlylsion or Genetics. Deartment of Pediatrics. Anderann2 D Mac Gre*or'. M-P Du_. and G RouA'. Department of Neurology' and University of South Florida, Tampa. Typ I Npurofibromatosis (NF-1) is a commpon Neurogenetics Unite, Montreal Neurological Institute and Hospital, McGill University, with an incidence of 1 in 3000 live geneticgirths. Montreal, Quebec, Canada; Hospital for Sick Children', Toronto, Ontario, Canada; and condigionInheritange is autosomal dominant with 50% of cases representing new mutations. The gene has been mapped to Centre for Research in Neuroscience', Montreal General Hospital, Montreal, Quebec, the proximal lonig arm of chromosome 17. Penetrance is Canada. high, approaching 100%. However there is a wide We wish to describe a Pakistani with fourteen variability of the phenotype even within families. family individuals affected in seven sibships Noonan syndrome has an incidence between 1:1000 and with familial spastic paraplegia, progressive loss of ability to walk and talk between 20-30 1:2$00 live births. The inheritance is also autosomal years of age, and death in the fourth and fifth decades. with variable expression of the gene. Candidate dominantgene id in the long asm of 12. The proband, a four-year-old girl, was the product of a normal pregnancy and delivery. atients have been chromoqomedescribed with clinical Developmental milestones were delayed. At 18 months, she was noted to have a spastic gait, manifestations of both NF-1 and Noopan syndrome (NF-1- and for NS). This has led to a designation of NF-1-Noonan referred investigation at the Hospital for Sick Children. Height and weight were at syndrome. It is not clear whether the syndrome is a 25th percentile and head circumference at 3rd percentile. Reflexes and tone were increased monogenic separate entity or its phenotype is a result of in the clonus was present, and there was abnormal and lumbar lordosis. At that pleiotropy of one of the genes. Since bdth conditions are legs, gait fairly common, cosegregation of two genes also has to be time, somatosensory evoked potentials showed poorly delineated central cortical responses considered. with low amplitude, and visual evoked potentials were abnormal. Brain stem auditory evoked Recently Carey et al reviewed 21 patients with NF-1- Noonan syndrome phenotypes. Only six of them showed responses, EMG, neve conduction studies, CT, MRI of head and spine, as well as amino enough of the Voonan syn rome clinical manifestations to acids, organic acids, and a metabolic screen were normal. confirm the diagnosis. This prompted the review of 14 atients with NF-1-NS evaluated through the USF Regional At four years of age, muscle biopsy revealed atrophy of histochemical type II-B fibers, enetics Program between 1/2J85 and 12/30/94. They were most likely related to upper motor neuron involvement. Nerve and skin biopsies were part of 23000 families evaluated during this period. normal. Among them there were 494 NF-l families and 145 Noonan syndrome families. 14 patients were diagnosed as Probable The parents are first cousins, and there is multiple consanguinity in this 6-generation NF--.NS. The review of them showed that 6 had enough Seven of the inical manifestations of Noonan syndrome for the pedigree. fourteen affected individuals are deceased, but no autopsy is dcla nosis of NF-l-NS. available. This appears to be a new autosomal recessive syndrome with probable founder Thus NF-1 is infrequent yet real, requires strict affect in this Pakistani family, which originates from Lahore. Blood for DNA has been diagnostic criteria and may represent a separate entity. obtained on 47 family members, and linkage studies with the known markers for familial spastic paraplegia are in progress.

444 445 Effect of-a a sl tation on dopamneand deopme outcome in Coielation between high frequency of ap olipoti E allele at and Down Syndome. G.L Arnold' E. G. Schul. V. Riher. . Kroh and G.L. dereasing maternal age in mothers ofchildrenwith Down syndrome. leG=2. 1)University of Rochester Medical Center, Rochester, NY, 2)University of D. Avramopoulos'. M. Mikkelsen2. D. Vassiop oO. M. Grisoriadoul' and Arkansas for Medical Sciences and Arkansas Children's Hospital, Little Rock, AR. M. B. Petersen'. of Child Health Tone and developmental milestones were improved in one infant with Down Syndrome 'Inst Athens, Greece; The J.F. Kennedy during amantadin prophylaxis for influenza. Further, preliminary studies suggestpossible mist, Glostrup, Denmark; 3Dept of Neurology, Univ. of Athens, Greece. nEurotranamitter deficits in this population. We therefore studied six infants ages 1-2 years An increased risk of Alzheimer disease (AD) has recently been reported in a double blind crossover study which evaluated cognitive, motor and speech-language in young modters ofDown syndrome (DS) probands. Allelic variation in the skills at baseline and after three months of amantadine (5 mg/kg/day in two divided doses) apolipoprotein E (apoE) gene is related to plasma cholesterol level, and or placebo administration followed by crossover into the other group. A twenty four hour allele e4 has been identified as a risk factor for early and late onset AD. We urine collection and two serum caolaine smples were collected prior to and post therefore decided to examinete distribution of apoE alleles in families with amantadine therapy. Two subjects withdrew, one because of parentl complaint that the DS children amantadine caused irritability. Amantadine administration did not affect the expected age The material consisted of 161 Danish liveborn cases of dependent decline in MDI (mental) or language milestones. PDI (motor) quotient increased non-mosaic, free in two subjects after amantadine therapy, stabilized in one and was indeterminate in the trisomy 21 of maternal origin detmined by DNA polymorphism analysis other. Urine and plasma epinephrine and norepinephrine were at the lower limits of and their parents from a population-based study of DS. We compared the normal. Baseline urine dopamine excretion was low in one subject, and urine dopamine frequency of the apoE allelea4 in the parents of children with DS and in a excretion and plasma dopamine increased after amantadine therapy where measured. Danish control sample (466 men at 41 years of age). The frequency of allele Changes in PDI did not correlate with changes in dopamine levels or excretion. Additional e4 in mothers was 18.6% as compared to 17.4% in controls and 12.2% in studies will be required to determine if the stabilization in motor function is a consistent fathers. A significant correlation between decreasing maternal age and finding and if this finding can be related to reticular activation or other dopaminergic in the of was found in the effects. incease frequency apoE e4 mothers of DS children (r - -0.85, p<0.05). The correlation was particularly strong when only cases of 2nd meiotic division errors were studied (r- -0.99, p<0.01 ). In addition the frequency of allele e4 in the fadters was found significantly lower than that in controls (p<0.05). The results mightsupport the finding of an increased risk of AD in young mothers of DS children and might also support the hypothesis of accelerated ageing in young mothers of children with D6.

446 447 SelfMtuilastiom Ia the SmIth LemU OpItz ayndrome: The effect of abnormal Unusual presntation of two-generation velo-cardio-facial syndrome: cholesterol metabolism oe the brain. (R. Babull,lD) Chitalat,M- Silverl,. hydronephrosis diagnosed on prenatal ultrasound. J.Q. Bgrs",IA.Alegk1, A. Ganclave11masi2 ) The Hospital forSick Chienl, Univasity of Toronto, Toronto, Einbeo2. and T.A. Grebe2. 1University of Arizona, Phoenix, AZ and ZPhoenix Ontario; SaultSt. Marie General Hoisptal2, Sault St. Marie, Ontario, Canads. Pennatal Associates, Phoenix, AZ. Tem Smith LemliOpitzsyndrome (SLO) [Smith et al, 19641 is aninborn esrrof Velo-cardio-facial syndrome (VCF) is an autosomal dominant condition cholestero metabolism Mnt etA., 1994]assoncated with prefoond mental mtardation,sver faluhe to thrive. craniofailand urogenital anomaliesanda high mortality ras. Since 7.dehydrocbhlesterol characterized by clefting of the secondary palate, congenital heart defects, learning Is highand plasma cholesemol is low in SLOpatients, the meubolic defect is lilely a deficiency of disabilities, and characteristic physical features. Recent findings have shown that 3"bydroy-Wqduacve individuals with VCF have a deletion in the DiGeorge region of chromosome 22q1 1, .We reporttwo adUltpaten who wereclinicaly disposed with the SL syndromes which can be detected by fluorescent In-situ hybridization (FISH). The ability to infants. Of te peviously undescribed with this both patients have ahistory of self-mutilation. ureand syndme, detect this deletion has led to an increased number of confirmed diagnoses of VCF. We report a patient who presented in-utero via ultrasound with bilateral Patient I ssan18 yerold male who ws noted at bith to havedynarphicfad] feaurs a small penis, udescends left astisand bilateal syndactylyof the 2nd.3rd toes. Healsohad feeding hydronephrosis and hydroureters. At birth, no anatomic site of obstruction was difficulties As 12 yass, hednonstrated ggressive behavkorad hadepisodes ofsef-mutilation found, but rather a neurogenic bladder without a spinal cord lesion. She also which involved bead bangi and hand biting. At 18yeas,his weight could notbe assessed his exhibited the Pierre Robin sequence, microcephaly, hypoplastic nasal alas with height was 124 cm (<brachydactyly. whorl and IQ of 70 were noted at 6 years. CASE 3 is a 2 year old In reviewing the literature, our patient seems unique in that short stature is her only born to a woman who received 100 mg of mthx biweekly to induce finding. This case of a chromosome abnormality in a child who presented with short abortion from 11 through 17 weeks and 200 ng biweekly from 19 through stature in the absence of any dysmorphia or medical problems illustrates the importance 25 weeks after a failed elective abortion at 9 weeks. He has short of cytogenetic analysis in the work-up for unexplained short stature in males and stature, bitemporal receding frontal hair line, low set ears and females. normal psychomotor development. Cases 2 end 3 suggest fetal exposure to mthx at 11 weeks post conception and onwards in large doses can lead to growth retardation and congenital malformations.

450 451 Long survival of two brothers affected by spondilothoracic Familial thoracic aortic aneurysms: a case-control study. dysostosis (Jarcho-Levin syndrome). C. Bellini. M. Scarsi. M. A dingerLM.R kln' Cl lli2M M ''Department of Internal Mazzarello, A. Bertola. E. Bonioli. Istituto di Puericultura e Medicina Medicine, University of Texas Houston Medical School, Houston, TX; 2Baylor Houston, TX. Neonatale dell'Universita - Divisione Ortopedia II, Istituto G. Gaslini - College of Medicine, 6560 Fannin, Suite 1.144, Istituto di Clinica Pediatrica dell'UniversitA - Genova - Italy. The Jarcho-Levin syndrome (JLs) is a condition characterized by Aortic aneurysms can be classified based on the anatomical origin. Familial aggregation aortic aneurysms (aortic aneurysms occurring below the malformations of vertebral bodies and ribs. Two subtypes of JLs have of abdominal been proposed: patients with malformations of vertebral bodies and renal arteries) has been clearly documented but no comparable studies have been done of thoracic aortic aneurysms (aortic aneurysms originating in the thoracic flaring of the ribs in a '"an-like" or "crab-like" pattem, without intrinsic cavity). rib have been as dysostosis defects, classified spondylothoracic This study contrasted the aggregation of thoracic aortic aneurysms and sudden (STD), while patients with vertebral malformations without 'fan-like" death in 892 first-degree relatives of patients referred for thoracic aneurysm or "crab-like" ribcage deformity, but with intrinsic rib defects, have surgery (probands) and their spouses' first-degree relatives (585 controls). been classified as spondylocostal dysostosis (SCD). The two Proband first-degree relatives were 5.6 times more likely to develop thoracic subtypes differ in survival rates, associated malformations, and aneurysms and 2.0 times more likely to experience sudden death than controls. inheritance patterns. STD, most commonly described in children of Proband fathers and brothers were particularly at risk for thoracic aneurysm Puerto Rican origin, has an autosomal recessive pattern of development (relative risks=6.2 and 20.0, respectively). There were no inheritance, and is characterised by early death likely occurring as a differences in the prevalence of abdominal aortic aneurysms, cerebral aneurysms result of progressive thoracic restriction during growth. We diagnosed or myocardial infarction between the two groups. A single pattern of aneurysm STD with severe thoracic deformity in two Italian brothers, who inheritance could not be determined from visual inspection of the pedigrees. In nevertheless are in fair conditions at the age of 7 and 10 years. The conclusion, proband first-degree relatives are at greater risk for developing reason of their longer survival compared with the other STD patients thoracic aneurysms or experiencing sudden death as compared to controls. is unclear. This report emphasises the nosologic and prognostic Formal segregation analysis may elucidate the mode(s) of thoracic aneurysm difficulties associated with this disease, and underlines the need of inheritance and provide a basis for genetic counseling and the development of cost caution in predicting the course of a severe structural chest deformity. effective non-invasive screening programs.

452 453 Familial unilateral acoustic neuroma: a new hereditary disease or an Isolated congenital eyelid absence with 25-year follow-up. J.jN Incomplete form of neurofibromatosls type 2? N. A. Bikhazil. W. Rodurtha KE. Teague and K. McNeer. Medical College of Virginia, SItt A. K Lawanii. R. K. Jackleri. D. E. BrackmAnn'. 'Department of Virginia Commonwealth University, Richmond, Virginia. Otolaryngology-Head and Neck Surgery, University of California San Francisco. Our 24 yo patient was born with absent upper and lower eyelids 2House Ear Institute, Los Angeles, California. bilaterally, a hydrocele, and no other birth defects. He was the fullterm product of a pregnancy complicated only by nausea for which Acoustic neuromas (ANs) or vestibular schwannomas present clinically in one of Bendectin was taken. His 23-yo parents were healthy without two forms: unilateral sporadic variety or bilateral hereditary tumors associated with consanguinity, birth defects, skin or eye problems. He had a maternal intracranial and spinal tumors as part of neurofibromatosis type 2 (NF2). In NF2 or male first cousin with mild ichthyosis without eyelid problems and a bilateral acoustic neurofibromatosis, acoustic neuromas are usually bilateral; maternal aunt and her son with benign familial tremors. Our patient's occasionally, the AN may be unilateral but is associated with other stigmata of NF2. globes were normal at birth without unusual membranes, cataracts, Familial unilateral acoustic neuroma in the absence of associated intracranial or coloboma, glaucoma, or retinal abnormalities. Lashes and tear ducts spinal lesions has not been previously described. were absent. He had one febrile seizure but was otherwise healthy In this report, we describe seven families in which two members in each family throughout childhood with normal intelligence. Mild hand tremors began had surgically confirmed unilateral ANs but do not have evidence of NF2. There in adolescence. He had normal tooth development, nail growth, wound was no evidence of presenile lens opacities in 10 patients who have been examined. healing, overall growth, and development. Immediately at birth Additionally, these patients do not have a first degree relative with NF2. In four of therapies to prevent drying of the eyes were begun. He has had many and the seven families the affected were parent-offspring, while in the other three the extensive surgeries with skin grafting from multiple body sites to affected were sibling-sibling, cousin-cousin, and aunt-nephew. There was no create lids. There has been mild keloid formation and no ichthyosis. gender predilection among members (7 men and 7 women). There was no evidence The differential diagnosis of ablepharon includes Neu-Laxova syndrome, of genomic imprinting. Presenting symptoms were typical of sporadic AN, and Fraser syndrome, and ablepharon-macrostomia syndrome. These were not included hearing loss, tinnitus, imbalance, and headache. Nine of fourteen apparent in our patient. The lgHl open-eyelids-at-birth mutation in members have had follow- up MRI scans (range up to 25 years after surgery) with mice usually includes blindness which was not present in our patient. no evidence of contralatl tumor or other intracranial neoplasms. These defects may be hormone-responsive. We consider the possibilities A tumor suppresser gene transcribing the merlin protein has been found to be of a germinal mutation homologous to that in the mouse vs. a somatic responsible for NF2. Familial unilateral AN may simply be a variable expression of craniofacial mutation in counseling our patient about potential risk to mutation within the NF2 gene or may be due to a mutation in a novel gene for AN. offspring. Alternatively, the finding of affected relatives with AN may be coincidental. Further study of these families will elucidate the underlying molecular mechanisms involved in the clinical expression of acoustic neuroma and ascertain whether this represents a distinct new gene or partial expression of NF2. A84 Posters: Clinical Genetics, Malfornnations and Dysmorphology (continued) 454 455 Assodatim between Russe-SilverSadroe (RSS) and Attention Decit Disorder Childhood alopecia areata (AA) and atopy: a significant disease (ADD)/Atteatioa Defcit Disorder with Hyperactivt (ADHD) and/or Leaing association. J. Bork, M. Dallaire. S. Lewis. C. Boeck. and M. Hordinsky. University ofMinnesota Health System, Minneapolis, MN, USA. . Lesse P. Disabilities (LD): Report of 16 ases. dL AA is postulated to be an immunologically mediated disease with an inherited Lma and.R-arion Moteflore Medical Center/Albert Einstein Coleg ofMedicin susceptibility based on review of family history information and analysis ofHLA Bronx, New York class II alleles. Results of several epidemiologic studies primarily involving adult First described by Silver et al. in 1953 and Russe in 1954, RSS consists ofgrowth AA patients, suggest a less favorable prognosis if a history of atopy is present. defiiency (uually ofprenatal onset), keletal asymmetry (a variable feature), fifth digit Atopy is defined as a type ofhypersensitivity reaction associated with circulatin and a facies. Other abnormalities include immature antibody resulting in the clinical features of asthma, allergic rhinitis or atopic clinobrchydactyly small, triangular dermatitis. Hereditary factors have also been implicated in the pathogenesis of osseousdevedopmnt, ddelayed grossor fine motor development, bluish scirae in infancy, atopy. In this study, we wanted to (1) validate the diagnosis of atopy in a pediatric eacssive sweang, hypoglycemia, and urogenital anomalies. The etiology is uncetain. population presenting to the Children's AA Clinic, (2) classify patients based on ntelligence isusuwly normal; alt hsom caesodevel nal delay or even mental presence or absence of atopy and (3) initiate studies comparing AA patients with retadation hv boen reported, very little has been published rgtrding specific cognitive their siblings in relationship to atopy. An allergy questionnaire was administered problems. to 40 children with AA, 19F/21M3 to 16 years of age. Serum inmmunoglobulin and/or (IgB) levels, prick skin testing (ST), and IgE antibodies (EAST/CAP) to common WerepM on 16 patientswho hve been diagnod with RSS and ADD/ADHD Midwest aeroallergens were assessed in all patients. Over 50% of the patients had LD. Tweve patients itially refred for developmental evaluation because of a postive family history for atopy. Based on the allergy evaluation, patients were hyperativty, attention or learning problems were ultimately diagnosed with RSS based subdivided into 4 groups: A) elevated IgE levels and atopy (I1); B) elevated IgE onthe prene ofthe pyic feaurdescribed above. Four patients with known RSS levels and no atopy (2); C) nonnal IgE levels and atopy (8); D) normal IgE levels were rebved fbr de nta evaluation and were found to have ADD/ADHD and/or and no atopy (19). Group A patients had more severe allergic symptoms and were LD. Ofthse 16 patients, 8/16 (50%/.) had both ADD/ADHD and LD; 6/16 (37.5".)had reactive to multiple allergens. In all 4 groups, children with extensive disease did not respond well to standard therapies. We found 48% of the children (7M,12P) LD (usually language based); and 2/16 (12.5%/) had ADD or ADHD alone. to be atopic as compared to the estimated 20% in the general population which is These data suggest that children with RSS may be at increased risk ofADD/ADHD significantly different - 95% CI (32%,63%). It remains to be established whether and/or LD despitenormnl intelligene. Ihrefore, early delopm l evaluation should or not the association of atopy with childhood AA is related to disease extent or be considered in all children with RSS. Further research is necessary to confirm that treatment outcome. However, there is a significant association between these two ADD/ADHD and/orLD is, in fact, avariable feature ofRSS. diseases. This information will be useful to those asked to counsel families with children who have AA.

456 457 Aaere a nb Ph24Whe. Cbala pb.etypn,cmdeltl.aad p als, IsP SuccesfuIl Pregnancy in a Woman with Diabetic Emboyapath. *' C-M Rltare.. Ji " k K 1.saadL.IN! 'Dhriv of .1-3Lyme- Baylor College of Medicine, Houston, Texas. MleOmosDepLofCHd h Div ofa wHi,y 'Diviimf -oarwy,DqW. of Congenital malformations are increased in the infants of diabetic mothers two to Sargery, Univesi of M mi-Combia, Combia, MO, 'Dept. of _Aadeiogy', GerIm four fold over that of the general population. Although not pathognoonc, anomaies Uniqs, Wapoa, D.C., 'St MatedePee Hoa M a hl i of the caudalenbryo form a unique class of defects that are less caomon in non- Eaeephaoel. are dseasiaosated abmemaly as wla a hainra of avral multiple diabetics. We report a woman born to an insulin-dependent diabetic who presented in mafrahwmiMmy&emm l00as do o xly-rqt-eawep Mumdiefawbaaltabeeloure. Thusem the newbom period with imperforate anus, abnormal genitalla and severe ureteral aesrt my of thni9oo~o sinsdoth oasiial portio. of the .irI ambe, raeslting ai thm PNra1 of lmatiate: eio, parieta md ooeipilal _WkSpb00110L A p araests i thi oecipil reflux. She had a total reconstruction by pull-through procedure at eight hours em oafe e _ignifieaad am*rcee ti a n Uato d Stamm Wmntes age, and later requIred a unilateral nephrectomy with removal of an ectopic ureter. EBoqtadadimmbb arme-fteqady asmainTaihll the- l ibppino, [m1 At age 14, with a history of progressive severe dymnorrhea, she underwent Malaysia, wdpar ofoa ThermaDI forthi dorly eismcer. TeperpoedoftaisMfdyut exploratory laparotomy for an abdominal mass. A septuat uterus with a non- beatr olusfy ior m nte Philippim by doemetiog phnoeypic varisiew ian th com municatig hor and masive hematometrolpos was found. A vanal septun ialeshdiqupwhotbmmy be aoaalreatkteshi leediegtrtmiorraodioideeeof was Inised and a neocervix was made at the same time. airiaremeptaalocole inWhier ofthewedd At age 25, she became pregnant after 5 years of infertility. Her pregnancy was Tesm-dcdeldemwen utsied for aoew Waooes Th ywereevalaeda Male 4 complicated by chronic uriay tract infection, symptomatic uterine prolapse due to potat recotnetinveoramihaial aor1y. Thalmied 6 fema d Al o r d e5 of normal structures, continence and intrauterine monthz 12 yea.7mont.(m : 6yen3 ment). Reurintmime swe.-reporteda20% d lack pelvic support difficulties oslarsimentoftoevadlesioi 0% alhoegb ady twohId asraqetredemu icehitem.& growth retardation. Screening for gestational diabetes was negative. At 37 weeks Derel~aldehywn dimadlyda&0b 6 N steiedh ladd etow_rin- g taio the patient delivered by cesarean section a healthy 2340g female infant (3110,yaga(VI0), bifid nvm(2/10), mropbaip, meofthsoeapecalmuA ArsiChiei with apgsrs 9/9 at 1 and 5 minutes Her postpartum course was unconpllcated. naseta, aboenaalhkpa pe sk- - alwolrndge, aba1sol post There Is a paucity of literature available regarding the counsli of women with aolxarpil, etiuw)el Faunlyhireimwame _ ibiamy. multiple genitourinary and mcllerian anomalies regarding reproductive risks and 1JaI.Phlipa rme cthite. are relntvey ad _lyietod en. Oay options for obstetrical management. The recurrence risk In our patient should be effic dnWA _rehe f *p k . 8S very low, especially given the probable causal relationship with diabetes in her maiergamofmvsieowa ofdisop be rao prveitobe emoiasedwiwhapkt mother. Continence is a major concern in patients who have had surgery for pejbolm idmo Nteidahmslmmudevolpm_. A gooeae eleloewasufd, th Although there is no data to that elective cesarean would be bleitainPropamofomoheleeleisle Phtppimaeoolaridedwtthtdkot inothereemfrioeaithe imperforate au. suggest relw Futhrrgudie. "E.wrudterdnineboehw pipradiopositam aaorev mI dft protective, an abnormally short perineal body, lack of rectal sphincter and abnormal weovolvedintpethogmeeioofdiemalfoeu*a plvtic muscular support structures place her at very high risk for serious sequels from obstetric laceration during a vaginal birth. More information is needed about the natural history of these disorders.

458 459 Pallister-illian syndrome (PKS) with diap ra atice hernia (DH1) and Dandy What factors contribute to the increased incidence of oculo-auriculo- Waker nalfonation (DWM). N S Ca_ H n _N. Harwma vertebral dysplasia (OAV) In Ecuador? M E. Lk T. L. Doinnnbr2 LL M. Per and I uars Indiana Universt and Methodist Hospital of QBiesne 3 andL&SMlJ lJohn Peter Smith Hospital, Fort Worth, Tea, Integraed Indianapolis, Indiana. Miami Florida, and 3PorCristo, Boston, MA. A woman presented for evaluation of fetal hydramnios, short limbs and Genetics, sacral appe ge at 32 w gestation. In addition to the above findings, OAV designates any combination of defects arising from abnormalities in the growth DWM wa observed on targelsd ultrasound, and t heart and stomach and development of derivatives from the first and second branchial arches, including were difficult to visualize. hemifacial microsomia and Goldenhar syndrome. Microtia and mandibular hypopasa ae Amnocentesis was peromed. KAryotype showed 46, XY/47, XY, el(12p), the most common findings, but asymmetric malar bones/orbits, epibulbar dermoids, idified by G-banded metaphases and FISH anaysis with a d o preauricular tags/pits, vertebral anomalies, cleft lip/palate, malocclusion and hearing loss of are often noted as well. More than 2/3 of cases are unilateral, with right sided 12 painting probe (ONCORE). The tetrsomy of 12p was observed in 18 involvement and/or severity predominating. Males outnumber females 3:2. OAV occurs 20 colonies. Terasomy 12p is consistent with PIKS. The extra crmosome in every racial group, but reported frequencies vary from 1/3500 - 1/6000. In contrast, is oftn not fomd in poriphorlIymphocyte and may be lost in cultured call figures for S. America range from 1/500- 1/3000, with the highest incidence reported in lines. I cordocntsis had been used to obtain tissue for CygNsltics In this Quito, Ecuador. We recently evaluated 40 patients (21 males; 19 females) from ca , as it often is in th third trImester t extra ch som may not have Guayaquil, Ecuador (Por Cristo). The involvement was right sided in 25(62.5%), left ban found. sided in 10(25%) and bilateral in 5 (12.5%). Other findings included: cleft palae in 5, On repast uson DH was observed. A review of the literature hearinaloss in 18, MR in 3 and other anomalies in 17. Seven patieats adparents with indicated ta all PSfetuses with DH died shortly afer birth. Hydramnios, measurable facial asymmetry; 4 other parents showed malar flaening and7 pedigree short limbs and DH ae common in PKS and sacral has been contained another reative with some OAV anomaly, usually microtia. but Most OAV cases are sporadic, but documented cases with chromosomalahnormalitles, reported. idvkdais with PICS and hpydrocsphaus haw been repoed, as well as AR and AD inheritance exist. Multifactorial inheritance best explains most this aears to be th first seoatd with DWM. This baby deli dar pedigrees. Vascular disruption and/or damage to cephalic neural crest cells ar spontaneos onset Of labor at 35 weeks. The family chose only comfort documented causes of OAV. Many unaffected Ecuadorians with a strong Indian measures and he died shortly awtbirth. background show a flattened malar area, retrognathia and posteriorly rotated c. Our The clinical features of PK8 resemble Fryns syndrome, an auosoma perinatal histories also note many potential risk factors such as heavy coffee intake, first reesive condition, which may have bee In the absence ofan trimester infections, seizures, hypertension, placental abnormalit, large babies, and accurate karyotype. Tnsu or born marrow rate ta peripheal or cord monozygotic twinning. These findings suggest that an interplay between a possible blood, is optimal for analysis when PKS is p d. Once a corrt genetic predisposition and a heterogeneous group ofenvironmental factors may partially dgnosis is made, proper pr s and r risk counseling can be explain the significandy elevated incidence ofOAV in this population. Posters: Clinical Genetics, Malformation:s and Dysmorphology (continued) A85 460 461 Females with midline malformations and complete reversal of left-right Segregation analysis of primary congenital glaucoma. K.C. Chen', . asymmetry in a family with apparent X-linked heterotaxy. B. Caseyl, aateman2, A.. Iach, R.A.Martin, P.L. FlodmanM, MSlncc I'Division of GehbialM. Devot02, D. Nelsonl, and A. AylswortP3. lBaylor College of Medicine, Human Genetics, Dept. of Pediatrics, University of California, Irvine, 2The Glaucoma Houston, Texas; 2Columbia University, New York, New York; 3University of North Research and Education Group, San Francisco, California. 3Dept. of Ophthalmology. Carolina, Chapel Hill, North Carolina. University of Colorado. Heterotaxy arises from inappropriate lateralization of thoracic and abdominal organs This study investigated the segregation of primary congenital glaucoma (PCG) in the during embryonic development. Typical manifestations, described together as situs United States. PCG is defined as congenital glaucoma due to malformation of the ambiguus, include complex heart malformations, gastrointestinal malrotation, and trabecular meshwork unassociated with other ocular anomalies or multisystem splenic abnormalities. Autosomal and X-linked inheritance have been established from malformations. The onset of symptoms occurs before age one year. In this study. clinical studies, which also suggest that complete reversal of normal left-right PCG was further defined as the anatomical defect of isolated trabeculodysgenesis. asymmetry (situs inversus) is extremely rare in familial heterotaxy. Recently we Probands were ascertained at the office of the Shaffer Associates Medical Group in San localized a gene for X-linked heterotaxy, H7XI, to Xq25-q26 in a single, large family. Francisco, California. Chart review was performed to collect data regarding associated All eight obligate carriers in that kindred are anatomically normnal, and some of the findings suggestive of multisystem syndromes, other ocular conditions, and secondary eleven affected males had other midline malformations in addition to classic laterality forms of glaucoma. After excluding patients with non-isolated PCG, questionnaires defects. A second family with apparent X-linked heterotaxy has now been studied. were mailed to 188 subjects to confirm diagnoses and elicit family history. A total of Two maternally-related male first cousins died shortly after birth with autopsy-proven 49 probands met the inclusion criteria. Segregation analysis was performed using features of heterotaxy, including severe heart defects and splenic abnormalities. One of direct calculations and SEGRAN. The recurrence in siblings in this data was consistent these affected males, who also had imperforate anus, has a sister and two maternally- with the recurrence observed by Demenais and colleagues (1979) in France and by related nieces with complete, asymptomatic situs inversus. The sister also has a double Gencik (1989) in the Slovakian non-Gypsy population. The sex ratio and laterality in urinary collecting system, and the two nieces had imperforate anus. Fifteen the current data were also consistent with the French data (Demenais et al., 1979). polymorphic markers in and around the HTlXI critical region have been tested in fifteen Using likelihood ratio criteria, the model of autosomal recessive inheritance with no members of this family. Nine markers show no recombination with the disease gene. sporadic cases was rejected for the data. The remaining models described 1) a DXS984 is fully informative and yields a maximum two-point lod score of 1.73 at segregation ratio for PCG of 0.01 for all families or 2) a small proportion of cases e=0.0. The recombinant markers DXS425 and DXS1215 define the centromeric and segregating PCG in an autosomal recessive manner with 95% of cases sporadic. telomeric border of the disease gene, respectively, and encompass the previously- Neither model was significantly better than the other. defined critical region for H7X1. Clinical evaluation and linkage data suggest that this family harbors a mutation in HTX1. We suggest further that cases of apparent sporadic situs inversus in females, particularly in combination with midline malformations. may represent new mutations in HTXI with subsequent risk of transmission to future offspring.

462 463 A new case of the Zunich neuroectodermal dysplasia (CHIME) syndrome. K A new syndrome of bydrocephalus due to foramen of ilonro obstruction 1. C and congenital bilateral neurosensory deafness in siblings. &L5, .b A.Buck Reidz RR Schnurl. 1. Children's Hospital of Philadelphia Chudlev and DW. McCullouah. Children's Hospital and Univ. of PA, 2. Cooper Hospital/University Medical Center, Camden, New Jersey. Manitoba, Winnipeg, Canada. The Zunich neuroectodermal dysplasia syndrome (MIM #280000) is a rare disorde A non-dysmorphic male infant was noted at birth to have previously reported in only four patients (including two sibs), and characterized by macrocephaly. He was the first born to healthy 26 year old mother and £olobomas of the eye-Ileart defects (3/4 previous reports)-Lchthyosiform dermatosis- 28 year old father who were second cousins of Mennonite ancestry. The pregnancy, labor and delivery were unremarkable. CT scan of the Mental retardation, Ear defects (acronym from N. Esterly). Cleft palate is a variable brain showed marked asymmetrical dilatation of the lateral feature. We evaluated a girl with multiple features of this syndrome. Major problems ventricles, particularly of the temporal and occipital horns and included a migratory ichthyosiform dermatosis resistant to Accutane, topical emollients normal third and fourth ventricles. A ventriculoperitoneal shunt was and exfoliants, bilateral retinal colobomas, developmental delay, seizures, macrosomia, placed at 1 week of age. Chromosomes were normal, 46,XY. At 2 years facial dysmorphia, and a duplicated renal collecting system. Family history was of age speech was delayed and severe bilateral neurosensory deafness (BNSD) was diagnosed. He was fitted with hearing aids. His vision negative for similar features and the parents were nonconsanguineous. At 3 yrs, 8 was normal. At 10 years of age he was in good health. He attends a months, height was at the 80th%ile, weight > 97th%ile, and head circumference was regular grade 4 class, has acceptable speech, and average 60th%ile for age. She was just beginning to speak and exhibited hyperactivity. Her intelligence. skin demonstrated diffuse scaling and an underlying erythroderma with scattered A second pregnancy resulted in a phenotypically normal male with "islands of sparing". She had brachycephaly, a mild upslant to her eyes, strikingly pale no evidence of deafness or hydrocephalus. A third pregnancy resulted in a spontaneous abortion. The fourth and last pregnancy resulted in blue irides, and hypertelorism. The midface was flat, the upper lip thin, and there were a female infant who was confirmed at 6 weeks of age to have BNSD. excessive creases around the mouth. Ears were low-set, rotated, and helices were thick She was fitted with hearing aids at 4 months. On assessment at 5 and over-folded. The nasal tip was underdeveloped and the nares upturned. The mouth months of age she was developmentally normal. She exhibited was very wide and broad in shape, and the teeth were widely spaced and squared. She macrocephaly, hypertelorism and a prominent forehead. The remainder had pectus excavaturn and small, low set nipples. She also had a supernumerary nipple, of the examination was normal. A CT scan of the brain showed massive dilatation of the right lateral ventricle and the right temporal a ventral hernia, flat feet, hyperextensibility of the digits, deep plantar creases, and horn, moderate dilatation of the left ventricle and normal third and clinodactyly of the 2nd and 4th toes. Her gait was wide-based. Diagnostic evaluations fourth ventricles. This suggested an obstruction of the foramen of included a 46,XX karyotype, normal echocardiogram (performed because of the Monro. At 15 months she underwent a craniectomy and neuroendoscopic suspected diagnosis), normal head MRI (at age 5 months), normal urine/plasma amino opening of the foremen of Monro and fenestration of the urine chain acids & acid. interventricular septum. At 31 months she was developmentally and acids, organic acids, very long fatty phytanic Multiple neurologically normal. sulfatase deficiency & neutral lipid storage disease were also excluded. Skin biopsy Congenital BNSD and obstruction of the foremen of Monro resulting revealed features of an epidermal nevus (deep rete pegs, hyperkeratosis, and a markedly in hydrocephalus has not been previously reported, based on a review increased granular layer). The etiology of the CHIME syndrome is unknown, but of POSSUM(1994) and Gorlin, Toriello and Cohen's book Hereditary recognition is easy because of the striking phenotype, and important because of Hearing Loss and Its Syndromes (1995). On the basis of associated congenital heart disease, significant neurologic compromise and probable consanguinity, affected sibs of both sexes, and in the absence of adverse pro- and perinatal events, this is likely an autosomal autosomal recessive inheritance. recessive disorder.

464 465 4p In a child with Pitt-Ro~gers-Danks syndrome. M. Macrocephaly In Neurofibromatosis Type 1. M.S. Cohenl3. C.A. Sarango- microjleleton ~~~~~~~~. Clemens' E.W. McPherspn"2. U. Surti"_ 'Department of Genetics, Sprousel. H.J. Steml,2. K.N. Rosenbaum', and C.J. Tifft',4. Depts. of Medical Magee-Womens Hospital, Department of Human Genetics, University Geneticsl & Lab. Medicine2, Children's Natl. Medical Centr, Washington, DC., Albert of Pittsburgh Graduate School of Public Health, Department of Einstein College of Medicine3, Bronx, NY, & GBB/NIDDK/NIH4, Bethesda, MD. Pathology, University of Pittsburgh, Pittsburgh, PA. We have evaluated the frontooccipital circumference (FOC) in patients with neurofibromatosis type 1 (NFl). Previous investigators have reported that patients with Pitt-Rogers-Danks syndrome (PRDS) is a rare, presumably autosomal NF1 more frequently have larger head sizes than expected for a normal population. In recessive, syndrome with pre- and postnatal growth retardation, this study we compared the FOC in patients with NFl to the head circumference growth microcephaly, characteristic facial appearance, seizures, unusual palmar curves for normal individuals developed by Roche and Nellhaus. Head circumference creases and developmental delay. Although first described in 1984, growth curves specific for children with NFl arc presented. only sIx cases have been reported. Our patient had typical features of Children diagnosed with NFl by the NIH consensus criteria in the Department PRDS Including IUGR and subsequent slow growth, hypotonia, of Medical Genetics at Children's National Medical Center were considered for the microcephaly, and developmental delay. Her facial appearance is study. All children had intracranial imaging either by computerized tomography or distinctive due to a short facial height, wide mouth, short upper lip, magnetic resonance imaging. No children with hydrocephalus or intracranial tumors, maxillary hypoplasia, vertically short mandible, and slightly beaked nose. other than T2 weighted high intensity foci (UBO's) or asymptomatic optic nerve Her ears are borderline low set and mildly antiverted. Her eyes are gliomas were included in the study. Multiple head circumference measurements for 114 prominent, giving a wide spaced appearance but lacking true boys and 113 girls recorded during regular office visits were used. hypertelorism. She had a grad mal seizure at 6 months of age and has When comparing this data (444 measurements for boys and 355 measurements since had a seizure disorder. Her extremities are normally proportioned for girls) to that of Roche, 80% of the boys and 78%o of the girls had positive z-scores, and she Is mildly hyperextensible. She has bridged palmar creases and with 34% of the and 23% of the z-scores than 2. One extra creases Her mental boys girls having greater sample bilaterally. development has been slow; t-test comparisons of the z-scores were made for the following age groups: <3 months, walking at 28 months and beginning to babble at 4-5 years of age. Her 3-5 months, 6-8 months, 9-1 1 months and yearly thereafter. After 9 months of age the photographs and medical history were reviewed and the diagnosis of boys had significantly larger FOC at each age group (p<.01). The girls also had PRDS was confirmed by Dr. John G. Rogers. FOC at each for the 8 old Traditional chromosomal and metabolic studies were normal. Due significantly larger age group (p<.01) except year group. results were obtained when the data were that of Nellhaus. There to some between and the associated with a Similar compared to similarity PRDS syndrome were no differences in z-scores for or based on race or the of NFl deletion of 4p we performed FISH analysis using the D4S96 girls boys presence chromosome in other family members. 4p16.3 specific probe (Wolff-Hirschhorn region). 20/20 are more to be than children in the were for the Children with NFl likely macrocephalic metaphase cells positive microdeletion. the increase in head size a The use of to contact other PRDS and FISH normal population and begins at very early age. Attempts patients perform analysis specific head circumference growth curves may allow for more accurate and is ongoing. conservative management of children with NFl. A86 Posters: Clinical Genetics, Malforrrnations and Dysmorphology (continued) 466 467 Clinical heteoeeit and T lymphocyte studies in patients with latae abdominal aits lumvesus associatd wiei an mblulsed 1:16 microdeletions of 22q11 and conotruncal cardiac anomalies. K. Coleman'-', L trsloecao L.N Cos . A H.ge S.J Koch.ar U. SuE Mg XSQUA, L. M alidhunad, J. Doer, F. Em e', K. May, L.J. Elsas2. Egleston N. Lazebnik and L.M. Hill, Mageo-Womens Hospital, Pittsburgh, PA: University of Children's Hosp', Divs Med Genetics2, ImmunolP, Cardiol', Dept Peds, Emory Univ Pittsburgh, Pittsburgh, PA. Sch Med, Atlanta, GA. The of 22q11 deletions is estimated to be as high as 29% in isolated Abdominal situs inversus is extremely rare and most reported cases ore frequency associated with additional congenital malformations. We report a 26 year old conotruncal cardiac defects. This group of infants would require evaluation ofthymic female who was seen for a dating ultrasound at 18 weeks gestation, which function and heritability. This prospective study describes 51 consecutive conotruncal revealed the apparent presence of isolated situs invereus of the fatal stomach end anomalies which were evaluated for a microdeletion of 22ql1 by routine chromsome a BPD/FL ratio of 1.72 (normal range < 1.70). The patient was counseled on the banding and the FISH probe N25 (Oncor). Two patients were eliminated because extremely rare occurrence of isolated abdominal situs inversus and the increased of 45,X Turner syndrome or no growth of cells. Of the remaining 49 patients, S risk of Down syndrome that has been reported with an Increased BPD/FL ratio. (10%) were positive for the microdeletion. Of these, 3 were female and 2 male. The Genetic amniocentesis was offered and declined. Multiple marker screening cardiac diagnoses of the 5 with 22q11 was within normal limits with an adjusted risk for Down syndrome of 1/19000. deletions included three with tetralogy of Repeat ultrasound at 21 weeks confirmed the earlier finding of abdominal situs Fallot (TOF), one with a ventricular septal defect (VSD) with aortic override (a inversus but BPD/FL ratio was at 1.56. Fetal echocardiography was normel. Fetal variant of TOF), and one with a muscular VSD. One patient with a deletion of growth was monitored through serial ultrasound and remained consistent. 22qll died at 3 months of age from central apnea. Central apnea like conotruncal The patient had an uncomplicated delivery at 38 weeks of an otherwise cardiac defects is pathogenetically related to problems with neural crest tissue during healthy male. Clinical examination confirmed the abdominal situs inversus and embryogenesis. The mother of one deletion positive patient also had the 22q I1 borderline microcephaly at the 10th percentile was additionally noted. The deletion, velocardiofacial syndrome (VCFS), mild to moderate mental retardation but remaining examination was unremarkable. Cytonetic studies on cord blood no known heart defects. Her daughter had a more severe phenotype of VCFS revealed a karyotype of 45, XY, -1,-15, + der(l)t(l 15)(p36.3;q1 1 1), indicati partial monosomy for 1 p and I 5p. Parental bloods were normal. including marked developmental delay, VSD with mild aortic override and bilateral Evidence from Robertsonian translocations suqgest that deletion of the 15p talipes equinovarus. The percent of CD3* and CD4* cells were similar among region should have little phenotypic effect. There is limited information available patients with and without microdeletion: CD3* 72+1 vs 70±16; CD4* 52+9 vs on the phenotypic effects of monosomy for the 1 p36 region. Two reported cases 50+15. However, of 18 patients studied by flow cytometry absolute numbers of T suggest some common features not apparent in this child, Including congenital lymphocytes were lower in 3 patients with microdeletions compared to 15 without heart disease, wide fontanelles, low set ears, flat nasal bridge, bulbous nasl tip 22qll microdeletions: CD3+ 918+650 vs 1941+1115 and CD4+ 648±455 vs and cleft and thin lipa. This third case implies that a full syndrome of effects, 1403±842 p <0.05. This preliminary data indicates that screening for microdeletions including abdominal situs inversus and microcephaly, may be associated with a of 22q11 should be considered in infants with conotruncal cardiac defects and that deletion in the 1 pter region. This case also suggests that cytogenetic analysis be a proportion of these patients have lower than normal T lymphocytes and a heritable considered upon ultrasound visualization of abdominal situs inversus. microdeletion.

468 469 Trisomy 12 mosaicism in three tissues In a live born infant. J. M. Coan DNA fluoreecnt probe use to detect microdeletlons In paents wfth one and KR.liba New England Medical Center Hospitals, Boston, Massachusetts. The rarity of trisomy 12 in spontaneous abortion material suggests that additional or more featues of CATCH 22. P. CrifasiI. V. Michels', Drscoll2. S Jolse copies ofchromosome 12 are incompatible with fetal development. We report a case of and G Dewald Mayo Clinic Departments of Medical Genetics', Carddoogy_, trisomy 12 mosaicism in a live born infant, with several tissues showing both and Laboratory Genetics populations of cells. Velo-cardio-facial (VCFS), DiGeorge (DGS), and related CATCH 22 Baby Girl T. was born at 31 weeks gestation to a 26 y.o. healthy primagravida. (cardiac, abnormal facies, thymic hypoplasia/aplasia, cleft palate, Prenatal course was marked by a decreased maenal serum alphafetoprotein. hypocalcemia) syndromes are often associated with microdeletion of Amniocentesis was declined. At birth she was noted to have dysmorphic features and a 22q11.22-q11.23. This study was done to assess the usefulness of DNA cardiac murmur. Peripheral blood and placenta were sent for cytogenetic analysis. fluorescent to Cardiac evaluation identified transposition of the great vessels, ventricular septal defect probes detect these microdeletlons in patients with 1, 2, or 3 and patent ductus arteriosus. She had periods of apnea and brachycardia. Following a features of VCFS, DGS, and CATCH 22. The probe D22S75 was used with a grade m intraventricular hemorrhage she developed post-hemorrhagic hydrocephalus. control probe for 22qter. The probe efficiency was 99.9% in 10 normal Death occurred at 312 months. individuals. Of 42 patients referred for these disorders, 9 had deletion by Cytogenetic analyses oftissues from this patient revealed the following: FISH. Only 1 of these patients had a microscopically visible deletion and 2 Placenta: 46, XX (16]/47, XX, +12 [13] were questionable by high resolution chromosome analysis. Patients were Peripheral blood: 46, XX [50] examined and scored for presence of conotruncal heart defects, cleft palate Skin: 46, XX [47]/ 47, XX, +12 [3] or velopharyngeal incompetence, and typical facies. Four of 4 patients with Initially the placental trisomy 12 was thought to be confined tissue mosaicism or a all 3 CATCH 22 features had the deletion and 5 of 13 others who had 2 tissue culture artifact because all the abnormal cells were seen in a single culture, but the features were also deleted. None of our 25 patients with only a single observation of a small number oftrisomy 12 cells in the fibroblast cultures confirmed feature had the deletion. We conclude that the use of DNA fluorescent probe this as a rare case of true trisomy 12 mosaicism. Fluorescent in situ hybridization is useful for detection of microdeletions of 22q1 1.22-11.23, especially when (FISH) with the probe for alpha satellite sequences of chromosome 12 (Oncor), two or more features of or CATCH 22 are identified a small population oftrisomic cells in the peripheral blood preparation VCFS, DGS, present. (30/1,000) and confirmed the presence of the small population in fibroblasts (42/1,000). Parental karyotypes were normal by GTG banding analysis and FISH. This case illustrates that the examination ofmultiple tissues from a dysmorphic child may yield results in cases with < 5% mosaicism, when traditional karyotype analysis may fail to identify abnormal clones. FISH should be considered also, because ofthe ease with which a large number ofcells can be examined.

470 471 1-LINN XCMTOUIJa A 1133 I L CU IN COMPlR12aN WITI Concordance of clinical findings in siblings with the Smith-Lemll. ICN!3T0815 VULGUIS^A IN NIXICAN POPULATION.1i~nJ%-Aa. Opitz syndrome. CbM Cunniff.1 DN Abuelo.2PLArn? MLBamhad' SuaxaLaroj s.A. uMana H. Koft l-Alflro. J of NA j ulyhl l.5 and 4 ",QW-0 g2 Kira Ea.A~sther Orozco Orozco Caw=4 NWokor.5 EH Zackai-6 RI K& 7 IThe Univ. of a an C-- D1&Z-Z&UQXA,- (Intro by: Alicia Cervantes). Arizona, Tucson; 2Rhode Island Hospital, Providence; 3Nemours Children's Clinic, 8Bervicio de GCn6tica, Hospital General de MHxico and Jacksonville, FL; 4Univ. Utah Medical Center, Salt Lake City; 5Univ. of Pittsburgh, 2Dopartamento do Bioquluica, Facultad do MNdicina, UNAN. Pittsburgh; 6Chldren's Hospital ofPhiladelphia, Philadelphia; and 7Kennedy-Krleger 3CINVESTAV, IPN. tEXICO D.F., MEXICO. Institute, Baltimore. Rocossive X-linked ichthyosis (XLI) and ichthyosis In order to determine the inter-and intfamilial variability ofthe Smith-Lemli.Opitz vulgaris (IV) are the most frequent types of ichtyosis. The (SLO) syndrome,9 sibshipsin which at least 2 children were affected with SLO wae frequenco of IV is considered 1:300 and the frequence of identified through a laboratory providing biochemical testing for this disorder. At XLI is 1:2000. Although clinical diagnosis is hard to be least one of the siblings had diagnostic confirmation of the diagnosis by elevated 7- done, enzymatic assay of steroid sulfatase allows the clear DHC and decreased cholesterol levels or by an abnormal cholesterol to 7-DHCratio. classification of both types of ichthyosis since this There were 2 affected children in eight sibships and 3 affected children in the ninth. enzyme is deficient in XLI. The present study analyzed 25 For single malformations, cleft palate showed the greatest inafamilialvaiton,wit cases referred by ichthyosis to the General Hospital of 4 of 9 showing discordance. Three of 9 sibships were discordant for polydactyly,2 Mexico and Dermatological Center of Pascua during 1993- each for cardiac defects and Hirschsprung's disease, and 1 each for renal anomalies, 1994. From those cases, we found that 21 were classified as seizures, pyloric stenosis and cholestatic liver disease. Sibling survival was XLI through steroid sulfatase assay. This means that XLI particularly variable, with 5 of 8 families having one living and one deceased child. had, by far, a higher prevalence in the sample analyzed There was general concordance of severity scores within sibships. with an average than reported for other populations. On the other hand, 80% sibling difference of 5.1 points of a total 23 possible points. The source of most of of the sporadic cases corresponded to inherited cases, as the variation in severity scores was accounted for by differences in survival. In only their mothers were clasified as carriers by the enzymatic one sibling pair were the malformations sufficientlydifferent to suggestthat one child assay. This is very important for genetic counseling. In could be classified as "classical SLO" and the other as "type II SLO." addition, 14 patients were analyzed using PCR for the STS It appears that most siblings with SLOwill have general concordance ofseverity gone and none of them presented amplification of the and clinical findings. However, the range of phenotypic effects may be great, and product, indicating a complete deletion of the gene as overall survival rates appear to be particularly variable. The degree ofconcordance of reported for other populations. In conclusion, this sample these findings has implications for counseling about the range ofphenotypic effects shows a higher prevalence of XLI in comparison with IV in which might be expected in subsequent offspring offamilies with one affected child. contrast with that reported for other populations. Study of the source of variability seen among sib pairs may provide clues to Nevertheless, complete deletion of the gene seems to interventions that will improve the outcome in affected individuals. correspond to a common feature for all populations analyzed. Posters: Clinical Genetics, Malformationis and Dysmorphology (continued) A87 472 473 Bilateral periventricular nodular heterotopia, cerebellar hypoplasia, mental Ana*yss ofpedgrees affected by juvenile onset myopia: Evidence for antosomal retardation and syndactyly: a malformation syndrome mapping to chromosome dominat inheritae. E. A DelBoolo J. E. Gwiada. J. UL.H . and J. L. Xq28. D.K. Czapansky-Beilman. B. Hirsch. M.E.M. Piermont. C.L. Truwit. W.B. Wigg'. 'Dqeatnt ofOphtlhalmogy, New England Medical Center;' tmnent ofBrain and Cognitive Sciences, Massachusetts Institute ofTecoology; and 2QgXM. University of Minnesota, Minneapolis, MN. 3Departnent ofMolecular Neurogenetics, Mschusetts General Hospital, Boston, Bilateral periventricular nodular heterotopia (BPNH) are nodular masses of gray Massachusetts. matter which line the walls of the lateral ventricles. Most probands are female, Myopia (nearsightedness) is a risk factor for several important eye disorders, and have normal intelligence, epilepsy and mild cerebellar hypoplasia. Several including glaucoma, retinal detachment and abnormalities ofthe optic nerve. Myopia multiplex families have been reported in which only females were affected. The Otat develops during childhood is particularly severe and is the form ofthe disorder most likely to contribute to additional ocular pathology. Several lines ofevidence gene was recently mapped to chromosome Xq28. We report two unrelated boys have suggested that genetic factors may influence susceptibility to the development of with BPNH and severe mental retardation, one of whom had a subtle structural myopia. This includes an increased incidence ofmyopia between identical twins, and abnormality of chromosome Xq28. an increased incidence ofmyopia in first degree relatives ofmyopic individuals. Both boys had a malformation syndrome comprised of BPNH, cerebellar hypo- However, many studies have suggested that environmental factors may also play an plasia, severe mental retardation, epilepsy, and partial syndactyly of the hands important role in the development ofthe process, especially studies that have correlated and feet. One of them had cortical dysplasia of both central regions, and normal the accommodative effort exercised by the affected individual with the degree of realized myopia. Because ofthis interesting relationship between environmental and chromosome analysis. The other had a subtle structural abnormality of Xq28 genetic factors, myopia is an ideal system to study the influence ofgenetic susceptibility inherited from his mother, who had normal intelligence and a normal MRI scan. in the face ofvariable environmental cues. In an attempt to begin to locate gene(s) Familial BPNH has been reported in four multigeneration families in which all which may contribute to the development ofmyopia, we have identified 52 two and affected individuals were female (DiMario et al., 1993; Huttenlocher et al., 1994; three generation pedigrees with two or more individuals affected byjuvenile onset Kamuro et al., 1993; Oda et al., 1993). The rate of pregnancy loss was myopia Individuals were determined to be affected ifthey had developed 0.75 D of ratio of livebom sons to daughters was decreased, which myopia by the age of 15, without any clinical findings ofother ocular or systemic increased, and the disorders. Among this collection ofpedigrees, affected individuals are found in every suggest that most of the miscarriages were males. Multipoint linkage analysis generation, equal numbers of men and women are affected, and approximately 50% of gave a maximum lod score of 3.65 for markers in distal Xq28 (Walsh et al., individuals at risk in each generation are affected. These findings are consistent with an 1995). Based on these two independant observations, we hypothesize that a autosomal dominant model ofinheritance. An autosomal recessive model would be gene involved in neuronal migration is located in Xq28, and that mutations of this possible ifa high incidence ofheterozygous carriers ofa mutant allele is present in the BPNH gene cause BPNH in mosaic to either studied population. Linkage analysis assuming each model independently is planned. putative typical individuals (due in NIH EY09847 (JLW) and EY10886 (JLW)). Lyonization in females or somatic mosaicism in rare males), and increased loss (Supported part by grat of males embryos or severe, syndromi BPNH in surviving males.

474 475 r Ia aemaly-esgaIta heart. defeat plus aiereoaphaly. IL Autosomal dominant inheritance of Barber syndrome: Distinctive facial ssp~rta.. Xemlpr. A. J. Carroll and W. H. Finlay. features with ocular hypertelorlsm, ectropion, macrostomia, cleft palate, Laboratory of Medical Genetics, University of Alabama at Birminghaa, iainghaa, Alabama. hirsutsm and redundant akin. MB Dinulos and RA PaEon. University of Loss of function of the Paz6 gone (11p3) underlies many cases of Washington, Seattle, WA. familal end sporadic anLiridia. Ivdeno. shows that PAS6 g9ne may have A motwer and son with unusual facial features (ocular hypertelorism, ecropion, a extenive role in causing malformations in the anterior chamber ef the ey, including Peters anomaly. The PAX6 gone a member of the bulbous nasal tip with hypoplastic alse nasi, macrostomia with thin upper lip, paird box-containing PAx family of developmental genes with major micrognathia), small cup-shaped ears, mild conductive hearing loss, hirsutism and sites of PASS expression including the neural tube, brain and the redundant skin appear to have the same disorder as the 3 year old girl described devlopiag Peter's anomaly has bee described in association with abnormalities, Kivlia syndroms, and Gillespie syndrome. by Barber et al. (Syndrome Identification Vil (1):8-9, 1982). The presence of this lebohi et al. in 1955, and Chsn and D'Souaa in 1990 described two constellation of findings in mother and son suggests that this is a dominantly eass with Peter a anomaly and oongenital heart defoats te.g. ASD, VaDn, disorder. tetralogy of Pallet, dSxtrocardia and conotruncal defeats). The inherited sondition knoAwn as Peter's anomaly-congenital heart defeat. The mother was the 2920 gram product of a 39 week gestation born to a 23 year VryologLcally, thes 2 olies my result from abnormal developeant old mother and unrelated 28 year old father. In 1968, at 4 1/2 years of age when of the neural crest. evaluated by the late David W. Smith, she had the above findings and midline cleft report a famale infant who was born at 39 weeks of gestation to palate, a 'plucked chicken skin' appearance of her neck, normal intelligence and a a 31 y/o mother 0P2, by spontaneous vaginal delivery. The pregnancy Wa oomplioated with gestationel diabetes. At birth she had microce- normal unbanded karyotype. Her siblings and parents were healthy and noncon- paly 131 am) and bilateral corneal opacities. Bar birth weight was sanguineous. Her son was the 5050 gram product of a 42 week gestation born 2.9 kg 125th percenLtle) and her length was 48 eon 10th percentile). when she was 21 years old. The father was unrelated. When evaluated atS days ead CT scan shod punctuate calcification. in the inferior basal panglia bilaterally and iA the medial left temporal lobe. of age, her son had a low anterior hairline with unusually broad eyebrows with very ephthalmologioal examination showed bilateral dense corneal opacities long hairs, primary hypospadias with a shawl scrotum and no cleft palate. Banded ad Peter's anomaly. Cardiovasoular evaluation revealed tricuspid karyotype was normal. Development was normal at 16 months of age. atresia, dextrooardia, and transposition of the great veseels. Karyotyps revealed 46,X5. Tha molecular cytogenstic analysis for Although there are similarities between the disorder present in our patients and Seoqrge syndrome was negative. An infectious etiology (Cv, rubella) Sexleis syndrome (bulbous nasal tip with small abe nasi, hirsutism, low frontal was ruled out. The family was negative for We problems or hairline), the differences are significant (lack of temporal cuts aplasia, distichlasis, consaaguinity. in our Our patients significant microcephaly in addition to the Peter's thick lips, and developmental delay patients); furthermore, Setieis syndrome enomaly-congenital heart defeat supports the hypothesis that PAX6 may is inherited in an autosomal recessive manner. be involved in this condition. Molecular detection of a R26G iStation Despite the differences between our patients and the patient of Barber et al. who asing sequenoe analysis in the PaX6 g9e product as reported by Manson had borderline intellect (IQ of 72), 'atrophic skin' and growth retardation, there are et al. currently being performed in our patient. The molecular characterization of thes conditions will help in identifying other enough similarities for us to consider that she and our two patents have the sJam loci involved in eye development. rare genetic disorder.

476 477 Cobblestone lissencephaly only (CLO) syndrome: exclusion from chromosome FRAXE In two unrelated males: molecular, behavioral and 9q31-32. W.B. Dobyns. M.A. Patton. R.F. Stratton. I.M. Mastrobattista. S.H. neuroanatomical assessment, =.F. Doheny.2M.T. Abas. SL KnhL Blanton. H. Northrup. Univ. of Minnesota, Minneapolis, MN; St. George's Freund' and ZA.L. Reiss. 3Johns Hopkins School of Medicine and ?Kennedy Krieger Hospital Medical School, London, U.K.; South Texas Genetics Center, San Institute, Baltimore, MD, 3University of Oxford, England. Antonio, TX; Univ. of Texas Medical School, Houston, TX; Univ of Virginia, Detailed cognitive, behavioral and neuroanatomical evaluations were performed on Charlottesville, VA. two unrelated males with the FRAXE CGG triplet expansion to describe the possible phenotype associated with this mutation. Both subjects were originally evaluated for Cobblestone lissencephaly is the characteristic brain malformation observed in non-specific developmental delay and diagnosed with fragile X syndrome based on the muscleeye-brain group of syndromes which includes Fukuyama congenital cytogenetic results. Southern analysis ruled-out expansions at FMR1 (FRAXA) but musculardystrophy (FCMD), Finnish muscle-eye-brain disease (MEB), and Walker- revealed fully methylated expansions (2.3 and 2.8kb) at the FRAXE locus. Warburg syndrome (WWS). We report three children from two families with Subject A received cognitive evaluations at the ages of 8 (Stanford Bindt 4th ed.) and cobblestone lissencephaly but normal eyes and muscle (CLO syndrome), and 12 years (WISC-R) and achieved standard scores (SS) of 88 and 100 respectively. exclude linkage from the region of the FCMD gene on chromosome 9q31-32. Relative strengths in quantitative/visual spatial performance and weaknesses in criteria include cobblestone lissen- verbal/short term memory skills were observed. Standardized parental interviews were The diagnostic for FCMD, MEB and WWS used to assess adaptive functioning and to probe for symptoms of autism at the age of 8 cephaly and congenital muscular dystrophy (CMD), while the criteria for MEB years. Significant deficits were observed in all of the Vineland Adaptive Behavior and WWS also require retinal abnormalities. Our three patients came from two Scales (Composite SS=57). Additionally, Subject A met DSM-Ell-R criteria for the inbred Middle Eastern families. All had moderate to severe mental retardation, diagnosis of autistic disorder. Quantitative brain MRI evaluation indicated a reduction in nystagmus, esotropia, and epilepsy. CT or MRI scans confirmed cobblestone cerebellar vermis area and putamen volumes compared to normal and developmentally lissencephaly of moderate severity. Eye exams and serum creatine kinase were delayed controls. Unlike comparison subjects with FRAXA, this FRAXE subject did not caudates or lateral ventricles. normal in all while EMG and muscle were in one. have enlarged three, ERG, biopsy normal Subject B received cognitive evaluations at the ages of 17 months (Bayley Scales of FCMD has been mapped to a 5 cM region between markers D9S306 and D9S58 Infant Development) and 4 years (Stanford-Binet) and achieved SSs of 63 and 88 (Toda et al., 1994). We tested our CLO families using markers D9S58, D9S59, respectively. Global deficits were observed across subtests of the Stanford-Binet with D9S127 and D9S306 which lie within or close to the FCMD critical region. the lowest score being the Abstract Visual (SS=77). Low average scores were observed Linkage between CLO and each of the tested markers was excluded for distances in all areas of the Vineland at both ages. with a particular weakness in Motor Skills at ranging from 2 to 4.5 cM. We conclude that CLO is a distinct cobblestone age 4 years (SS=68). Subject B did not meet DSM-Ill-R criteria for the diagnosis of which differs from MEB and WWS because of the lack autistic disorder or pervasive developmental delay at either age. Quantitative MRI lissencephaly syndrome evaluation of Subject B indicated a neuroanatomical profile similar to controls with of CMD and retinal abnormalities, although we hypothesize that it is allelic to idiopathic developmental delay, and distinct from that of subjects with FRAXA. both MEB and WWS. It is not allelic to FCMD based on our linkage results. A88 Posters: Clinical Genetics, Malformations and Dysmorphology (continued) 478 479 Hemimazllofacial dysplasia: A report of 2 new cases and further delineation of skeletal and cardiac salforuations with throebecyto pe iat of A now syndrome? Alison M. Ell ottl.2. RosemarieRD~2 E. the disorder. S.Eisig R.Marion.R.Shonntzenand A.L.Shanske. Departments Michel Dentistry,Plastic Surgery and PediatricsAlbert Einstein College ofMedicine, Azouz3, Mark L. Bernstein". Patrice Wvou an IKaolanl.2 and Vazken M. Der Kaloustian1 2. The F. Clarke Montefiore Medical CenterBronxNew York. Fraser Clinical Genetics Unit', Division of Medical In 1987,Mlleset al reported a new craniofacial disorderhemimaxillofacial Genetics2, Division of Radiology3 and Department of dysplasia,consisting offacial asymmetryhypertrichosis ofthe facial skinunilateral Hematology4, The Montreal Children's Hospital and McGill enlargement of the maxilla and hypoplastic teeth. There have beento our University, Montreal, Quebec. knowledge,no additional cases reported since the original 2 unrelated patients. We We describe an 8 year old female patient with to young men disorder. multiple have had the opportunity examine 2 with this anomalies suggestive of a new syndrome. She was born at 35 Case 1. S.R. is a 22 year old with a history of developmental language problems and weeks of gestation after a generally uneventful pregnancy. chronic depression. He was referred because ofright-sided facial asymmetry, Birth length and weight were at the 50th centile and OFC hyperplasia of the maxillary alveolar bone and delayed dental eruption and hypoplasia. was <3rd centile. The parents are nonconsanguineous and of A biopsy of of a radio-opaque lesion in the right maxillary alveolus revealed dense Italian descent. Her mother was 28 years old and her father was 32 at the time of include bone. The physical examination revealed right facial prominence and a pigmented delivery. Physical findings microcephaly, facial asymmetry, hypertelorism, prominent nevus with hypertrichosis ofthe right cheek and forehead. nose, prognathism, high-arched palate, VSD and ASD, Case 2. L.C. is a 7 year old who was the product of a term uneventful pregnancy. His conductive hearing loss and mild developmental delay. Her growth and development have been normal. Physical examination revealed minimal radiological findings consist of bilateral carpal asymmetry with a downward displacement ofthe left maxilla and bony hyperplasia of coalition, tapering distal phalanges, bilateral coxa valga and bone expansion of the pubic and ischial with a the right hembnaxilla. A number ofright-sided maxillary teeth were missingthe right regions abortive wide puboischial synchondrosis. She has chronic upper canine was surrounded by a firm bony growth and there was a minimal thrombocytopenia secondary due to production failure. The cleft ofthe right lip. A patch offacial hair was present beneath the right lower lid and findings of bilateral carpal fusion, VSD, ASD and on the right cheek. These 2 sporadic cases meet the clinical criteria described by thrombocytopenia may be present in the Keutel Syndrome, Miles. They lack the hypertrophic changes associated with segemental IVIC Syndrome as well as the 10qter deletion syndrome. However, the full hemihypertrophy and the ear abnormalities always seen with hemifacial microsomia. none of these syndromes can explain spectrum of anomalies seen in our patient. Her may The presence offacial hypertrichosis may distinguish these patients from the closely findings represent a single gene defect, a dysplasia. contiguous gene syndrome related disordersegmental odontomaxillary or a multifactorial condition.

480 481 A clinical study of a large inbred kindred with autosomal The role of HLA class II polymorphisms in multiple sclerosis suscepti- recessive familial spastic paraplegia. H. El-Shanti. A.S. bility in Italians. M. Eoli. M. Pandolfo, A. Salmaggi. .Mlanise, M.2axiani. Daoud. Jordan University of Science & Technology, Irbid, Jordan. Istituto Nazionale Neurologico 'C. Besta". Milano, Italy. Spastic paraplegia, an uncommon neurodegenerative disorder Scveral epidemiologic observations suggest that both genetic and enviromental with phenotypic and genotypic heterogeneity, is mainly factors may influence susceptibility to Multiple Sclerosis (MS). Although associa- characterized by progressive weakness and spasticity of the lower tion between MS and HLA-DR2. DQW6 has been well documented in populations limbs. There Is degeneration of the corticospinal (pyramidal) tracts of Northern European ancestry. no clearcut associations have been identified in and in some instances the dorsal column as well. There are pure and the Italians. except for Sardinians. Furthermore, clearcut genetic linkage between HLA In order to as to complicated forms with the pure forms being autosomal dominant, and MS has not been established from family studies. verify whether multiallelic to II are associated autosomal recessive or rarely X-linked. We here present a large polymorphisms belonging HLA-class genes with or linked to MS in Italy, we 28 MS families for HLA-clasa highly inbred family with autosomal recessive pure familial genotyped multiplex II markers. Allelic characterization was carried out of restriction frag- spastic paraplegia. The clinical picture in this family has some by analysis ment length polymorphisms and oligonucleotide be- unique features. This ten generation family is composed of about typing. Linkage disequilibrium tween MS susceptibility and the haplotype DRBI*1501- DQAI*0102-DQB1*0602 200 members with about 70 affected individuals distributed over was demonstrated by using the transmission linkage disequilibrium test. Results five consecutive generations. Although autosomal recessive pattern suggesting linkage were provided by using two non parametric tests, i.e. affected is clearly demonstrated, the disease shows a of inheritance sibpair analysis and affected member analysis. LOD score analysis suggested that pseudodominant pattern probably secondary to extensive in our families MS susceptibility could be conferred by HLA-class II genes according age at onset seems to with successive inbreeding. The decrease to a low-penetrance. autosomal recessive mode of inheritance. generations, either due to a true anticipatory phenomenon or to increased awareness. All symptomatic patients were clinically affected (by physical examination) and all clinically affected patients reported symptoms. The disease is purely motor, confined to the lower limbs without sphincter dysfunction or obvious sensory involvement. However, we are currently studying the most advanced patients with somatosensory evoked potentials. Affected patients show progressive pseudohypertrophy of the calf muscles.

482 483 A sibship with Allrwe syndrome and normal high-resolution karyotypic analysis. Congenital presentation of Neurofibromatosas, Type I, in four D. N. Finegold2. W. S. Rubinstein'. M. B. Gorin"g. C. M. Lese'. and S. M. Gollin'. unrelated cases supports a role for neuroflbromln during early Depts. ofHuman Genetics', Pediatrics2, and Ophthalmologj, University ofPittsburgh, PA. morphogenesis. BD FTriedman1 CM CunniffISJ Hassed,2 and HE HoXme.1 An unusual family with Allgrove syndrome was identified because a 4 year old girl tThe University of Arizona, Tucson; 2The University of Arkansas, Little Rock. We report on four unrelated with I who presented with muscle weakness and excess pigmentation. Evaluation showed elevated patients Neurofibromatosis, Type (NFI), have congenital onset features ofthe disorder. These cases support recent findings ACTH (3910 pg/ml; normal 10.60 pg/mi) and normal electrolytes. Further history on suggesting that neurofibromin plays an important role in of embryonic development. the patient and a 11/2 year old male sibling revealed achalasia and alacrima. Evaluation Patient1 had a history of a seizure disorder, a double right ureter, congenital right the younger sibling showed undetectable cortisol and elevated ACTH (202 pg/ml). ptosis, cafe au hit macules, bilateral axillary freckling, right hemimicrencephaly, right Extensive analysis for autoimmune or metabolic causes of adrenal failure was negative. sided pachygyria and schizencephaly. High resolution karyotype was 46XX. Both children had normal intelligence and development. Microcephaly, short stature, Patient 2 was described previously by one of us in a published report in 1987. An extensive oral mass detected prenatally was confirmed at birth to be a and dysmorphic features were absent. The patient had a visual acuity of 20/200 and no gingival granular cell tumor. Follow up showed multiple cafe au lait spots and axillary an pupillary nystagmus, indicating acquired defect. She had markedly abnormal freckling. Patient 3 presented with a congenital right upper extremity nerve palsy. responses but no evidence of an afferent pupillary defect. Progressive weakness and atrophy of the right upper extremity led to the diagnosis of To rule out a visible deletion, high-resolution chromosome analysis was carried out plexiform neurofibromas of the right brachial plexus. Ophthalmologic examination and revealed a 46,XX karyotype. This excluded a visible chromosomal deletion as the revealed Lisch nodules. Patient 4 had enlarged labia and clitoral hood, multiple cafe au a and mild basis for this disorder in this fimily (although it does not eliminate the possibility of a lait macules including one in bathing suit distribution, right leg hemihypertrophy. Surgical exploration of lobulated masses to the submicroscopic deletion causing a contiguous gene syndrome (CGS)). An alternative adjacent spinal canal from L5 to thesacral region revealed a neurofibroma. the plexiform hypothesis to a CGS is a mutation in a developmental gene involved in formation of lTe cases herein described lend credence to recent findings suggesting that aberrant myenteric plexus, adrenal cortex, lacrimal gland, autonomic nervous system, and morphogenesis in the embryo may be a consequence of NFI mutations. possibly central nervous system. Another hypothesis, which assumes normal initial Neurofibromin is suspected to be involved in cell proliferation, differentiation, and function and subsequent deterioration of the components of Allgrove syndrome, is a tumor suppression. Studies revealing high levels of neurofibromin in the brain suggest a possible role for the NFl gene in neuronal Of mutation in a pathway ofneurodetoxification, analogous to that proposed for superoxide development. particular importance in this regard is the finding in Patient1 of a neuronal migration defect that gene lateral This is supported dismutase-l mutations and familial amyotrophic sclerosis. is more severe than the heterotopias more commonly seen with NFl. Clinicians by commonality of autonomic innervation to the adrenal cortex, esophagus, and lacri caring for neonates should be aware of the congenital effects of NFl, including cafe gland. In addition, shared response ofthe adrenal cortex and lacrimal gland to neuropeptides au lait macules, neurologic impairment, and tumors, since early diagnosis and and basic fibroblast growth factor (FGF-2) may underlie a common susceptibility to a uniuue treatment may decrease morbidity and allow for appropriate prognostic and recurrence risk counseling of this biochemical defect. Further studies in this funily may be able to test the above hypotheses. disorder. Posters: Clinical Genetics, Malformationms and Dysmorphology (continued) A89 484 485 Analysis of clinical sub-groups in NFl. J.M. Friedman, M. SeXwerd, P. Premature coronary artery disease results in a markedly higher prevalence of Birch. Dept. of Medical Genetics, Univ. of British Columbia, Vancouver hypertriglyceridemia among offspring. C. A. Friedrich', J. Coresh', T. H. Beaty' Canada. and P. 0. Kwiterovich'. Johns Hopkins University Schools of Medicine and Determining the sources of clinical variability in neurofibromatosis type 1 (NFl) is essential to understanding its pathogenesis. We have 'Hygiene and Public Health, Baltimore, MD. used numerical taxonomy to analyze 23 phenotypic variables in 600 A family history of premature coronary artery disease (CAD) is a known risk probands with NFl taken from the National Neurofibromatosis Foundation factor for CAD. However, the prevalence of different dyslipidemias among Internation Database. On the basis of this analysis, we postulate the offspring of patients with premature CAD is less well-characterized. The existence of 5 distinct clinical groups of NFl patients. lipoprotein phenotypes of adult offspring of 61 probands (males < 50 years Cafe au lait macules and scoliosis occur with similar frequencies in all of the groups, but the frequencies of many other features differ. old; females < 60 years old) who underwent elective coronary angiography The table lists some features that were commonly (but not always) found were determined. Thirty-two probands (15 males; 17 females) had CAD; 29 (9 in each group, along with the group's mean age and relative size (as a males; 20 females) did not. Plasma lipid, lipoprotein and apolipoprotein (apo percentage of the total). A-I and apo B) levels were determined in 93 adult children of CAD + probands and 95 adult children of CAD- probands. The distribution of lipoprotein Group 2 Age Characteristic Features phenotypes was: A 8 No other frequent features B 18% 9 Intertriginous freckling (IF) c 15% 22 IF; peripheral neurofibromas (NFs) Normal TTG D 10% 19 IF; peripheral NFs, plexiform NFs (in all patients) Normal Ila HypBNTG Hypoalpha Ilb IV HvpBHTG E 50% 18 IF; peripheral, internal & plexiform NFs; seizures; CAD+ 49 8 18 1 3 8 6 hydrocephalus; hypertension; optic glioma; CAD- 66 9 17 0 0 0 3 malignant tumours; pseudoarthrosis *0.005 < p < 0.010 for 3 high TG phenotypes combined Groups A and B contain predominantly children. As these patients age HypB - > 130 mg/dL apo B with normal (N) or high (H) triglycerides (>90th they will probably move into the other groups, and we are currently percentile) trying to develop a method of predicting which children will become members of which of the older groups. Hypertriglyceridemia (Type llb, IV and HypBHTG) was present in 18% of the offspring of CAD+ parents compared to 3% of the offspring of CAD- parents. (Supported in part by the Texas NF Foundation.) These data emphasize the importance of hypertriglyceridemia as a genetic factor in the etiology of premature CAD.

486 487 Orofacial clefts and twimming B. H. Garabedian and F. C. Fraser. Dystrophin gene deletion with Becker's phenotype but without creatine Department of Human Genetics, McGill University, Montreal, Quebec, kinase elevation. J. Y. Garbern* and J. A. Kant. University of Pennsylvania Canada. School of Medicine, Philadelphia, Pennsylvania. To investigate a possible familial association between orofacial We describe an individual found at one yearofage to have a myopathy. The child clefts and twinning, twin pairs were counted in the sibs, parents, at that time was unable to support his weight and also had proximal arm muscle aunts and uncles of 368 non-syndromic cleft lip (CL) and 154 cleft weakness. At 16 months of age, the patient underwent a muscle biopsy which revealed palate (CP) probands ascertained from hospital genetics records. a mild myopathy characterized by fiber size variation, an increase in sacolemmal nuclei Twinning rates (calculated by dividing twin pairs by total livebirth and a mild increase of interfiber connective tissue. pregnancies) were significantly high (1.6% and 2.0%, respectively) The patient did not walk until age 3, and then had a waddling gait, lumbar lordosis when compared to local population rates (1.1%) and control families and pseudohypertrophic calf muscles. Although the patient had been given a (1.0% - families of probands with Mendelian disorders). The excess presumptive diagnosis of Duchenne muscular dystrophy, at age 10, serum enzyme in the CP group was like-sexed, pointing to a monozygotic twin excess. levels for creatine kinase, aspartate aminotransferase, and lactate dehydrogenase were These findings, along with the previously reported familial normal, at 33 U/L, 19 U/L and 320 U/L, respectively. Therefore, it was felt unlikely association of twinning, neural tube defects (another schisis-type that the patient had a Duchenne - like muscular dystrophy. melformation) and non-righthandedness support the hypothesis that At age 26 his examination was notable for calf pseudohypertophy, significant there are genetic factors for developmental instability linking upper and lower extremity proximal weakness, a waddling gait and a Gower's sign. At these malformations early in development. (Supported by the Medical this time his creatine kinase level was 145 U/L (normal range 20 - 315), while that of Research Council of Canada.) his clinically normal mother was 96 U/L (normal range 20 - 150). Family history was noncontributory. Dystrophin multiplex PCR analysis with the Chamberlain primers was normal, however, by Southern blot analysis the 6.5 Kbp Bgl II fragment and of the 7.5 Kbp Hind m fragments were missing when hybridized with a cDA probe panning exons 1 through 11. Because the PCR testing reveals that exon 8 is at least partially intact, we deduce that the deletion primarily spans exon 9 of the dystrophin gene. An isolated exon 9 deletion would be predicted to result in an out of frame deleton. More specific testing is underway to confirm our findings. Our patient, whose clinical picture is consistent with a dystrophin abnormality, is the first documented with a dystrophin gene mutation and whose serum muscle enzyme levels have been normal. Therefore, the absence of muscle enzyme elevation cannot be used to exclude the presence of a dystrophin abnormality.

488 489 Identification of h ygoity for hyperhomocsnemla biochemical and A patient with the velocardiofacial syndrome and an unbalanced 1;22 genetic approaches. U. C. Garg. N. 0. Hanson. N. S. Key. K. A. chromosomal translocation. J. Garza, C.jT. Fong, E. Clark. C. Buzzard and Swchtb. '. W. Boutain and M. Y. Tsai University of Minnesota N. Wang. University of Rochester School of Medicine and Dentistry. Medical School, Minneapolis, MN. The velocardiofacial syndrome (VCFS or Shprintzen syndrome) has frequently In recent years it has become dear that hyperhomocyst(e)inemia is an important risk factor for premature vascular injury, atherosclerosis and been associated with a deletion of the 22q11.21 - 22q11.23 region using the flu- thrombosis. The prevalence of hyperhomocysteinemia, generally thought orescent in-situ hybridization technique (FISH). The syndrome is believed to be to be due to deficiency of cystathionine beta-synthase (CBS) is about 1/70 to one manifestation of a microdeletion syndrome named CATCH-22. We have diag- 1/225. As mild hyperhomocysteinemia is easily treatable, it is important to nosed a 4 month old patient with VCFS based on typical craniofacial features and identify heterozygotes who are at risk of developing premature vascular tetralogy of Fallot. On cytogenetic examination, the patient was found to have a disease. Approaches that have been used to identify heterozygotes include karyotype of 45,XY.-1.-22,+der(1)t(1;22)(p36:qll). FISH study using a CATCH- measurement of homocysteine levels before and after methionine loading, 22 region probe showed monosomy of the 22q11 region. Chromosome analyses of direct asay of cystathionine beta-synthase in cultured fibroblasts and, more the parents were normal. This case represents a rare instance of VCFS where the recently, identiflcation of mutations in the CBS gene. In this work, we is the result of studied each approach in obligate heterozygotes and controls and compared 22q11 monosomy an apparently unbalanced, de novo chromosomal the feasibility and reliability of these methods. The first two approaches are translocation. cumbersome, and the results between controls and patients show a significant overlap. To explore molecular approaches we used amplification refractory mutation system (ARMS), a modified PCR method, to screen 7 homozygotes and 12 heterozygotes for mutations at nucleotides 341, 374, 434, 833 and 919. Using ARMS we found that mutation T833C is the most common (11 out of 38 alleles had T833C mutation). Two out of 38 alleles had G919A mutation, and no mutations were found at nucleotides 341, 374 or 434. Further studies which will define the prevalence of known mutations in a large population are needed. Once this is accomplished, identification of heterozygosity should begin with molecular diagnosis for prevalent mutations. Methionine loading and CBS assays should be used only if further work up becomes necessary. A90 Posters: Clinical Genetics, Malfornnations and Dysmorphology (continued) 490 491 Non imnune hydrops feta l i s due to beta-g l ucu ron i dase def i c I ency. J. S. Unifonn genetic diagnoss code registuatiom. Geer, W.R. Blackburn, R.E. Stevenson, P.A. Riedel. Greenwood Genetic Geraedts. J.P.M. (1): Bilsma. I.B. (2): Hennekam. F.A.M. (2): Center, Greenwood, S.C. A number of lysosomal storage diseases have been associated with Lindhout. D. (3); Beemer. F.A. (2): Bakker. E. (4): Kleiier, hydrops fetalls. Fetus N. (GGC-51687) was found to have generalized W.J. (3): de France. H.F. (2): de Die-Smulders. C.E.M. (1): hydrops at 5 months gestation. At birth, the infant had diffuse Duran. M. (2): van GenniR. A.H. (5): van Mens. J.T. (6): edema with pleural and peritoneal effusions. The hematocrit was 70%, Mantel. G. (4): Verhage. R.E. (3): Zwamborn-Hanssen. A.M.N. total serum protein 3.0 gm/dl, albumin 1.4 gm/dl, ALT 110 IU/l, and uJh AST 304 IU/I. Leukocytes and fibroblasts showed less than 10t of (I) Clinical Genetics Centre Limburg, Maastricht; (2) Utrecht; (3) normal beta-glucuronidase activity. The infant died at 18 days of Rotterdam; (4) Leiden; (5) Amsterdam; (6) SIG Zorginformatie, age. Microscopic analysis showed extensive vacuolated granular Utrecht, The Netherlands. inclusions in the cells of all major organ systems including the placenta. Several systems are used to classify and code genetic disorders for the The pathogenesis of hydrops in lysosomal storage disease has been purpose of registration. Dr. McKusick developed the MIM'code for unclear. Anemia, although present in Gaucher and Wolman disease, has Mendelian disorders. The World Health Organization designed the not been found in the other lysosomal storage disorders. We suggest lCD-code which is based on organ systems. Neither system fulfils the that edema In these disorders may result from low colloid osmotic required standards for all genetic disorders. In The Netherlands, a pressure secondary to liver involvement and increased capillary uniform genetic diagnosis code is used that links a new, unique code to permeability. the arore mentioned coding systems. In this way it is possible to cover all disorders which otherwise are not clearly distinguished. New codes are assigned centrally and distributed periodically to all seven genetic centres in The Netherlands. At present a fully automated system is being built to process requests and to assign and distribute new codes. The architecture of this coding system is suitable for international use, which will make easv and reliable comparison of data possible. The disease code provides a unique, uniform and unifying method for the registration of diagnoses. Its main advantage is the user-friendliness. Searches for a diagnosis-code can be performed alphabetically on ICD/BPA-code or on McKusick-code.

492 493 Ectrodactyly-Ectoderaal Dysplasia-Clefting (EEC) Syndrome Phenotypic analysis of 18q- patients. P. D. Ghidoni. J. D. Cody, D. Galbreath R. end Hypothalamic-Pituitary Insufficiency. R Gershoni- Plaetke- D. Hale, T. Sloan C. T. Gay. R. Rauch, R. J. Leach and C-I. Kay=. The Baruch, D Goldscher and Z. Hochberg. Depts of Human of Texas Health Science San Texas. Genetics and Pediatrics, Rambam Medical Center, and the University Center, Antonio, Bruce Rappoport Faculty of Medicine, Technion-Israel 18q- syndrome is the most common human deletional aneusomy, with an estimated Institute of Technology, Haifa, Israel. frequency of 1/40,000. We have evaluated 36 subjects with this disorder, and compared The EEC syndrome (ectrodactyly, ectodermal dysplasia, phenotypic features with 66 individuals reported in the literature. Medical records were and cleft lip and palate) is a complex, pleiotropic, rare, reviewed on all subjects for identification of major congenital malformations (e.g. autosomal dominant syndrome. The most common clinical congenital heart disease, renal anomalies). Each subject was examined by the authors to manifestations are ectodermal dysplasia (anomalies of hair, determine dysmorphic neurologic findings, and status. teeth, nails, nasolacrinal ducts, and sweat glands) features, growth Thirty-three ectrodactyly (split hand and foot anomalies), cleft subjects were tested for electro- physiologic parameters (brainstem auditory evoked lip/palate and genito-urinary anomalies. Other clinical response (BAER), and cortical auditory evoked response (CAER)]. Twenty-eight subjects signs include genito-urinary anomalies, heart defects, also underwent brain magnetic resonance imaging for identification of central nervous deafness and mental retardation. The syndrome is notable system malformations. Frequencies of congenital heart disease, cleft palate and both for its reduced penetrance and variable expressivity. microcephaly in the study population did not differ from subjects described in the We report on two brothers with EEC syndrome and literature. The study subjects had a significantly greater likelihood ofbeing affected with hypothalamic-pituitary insufficiency. Both had a renal tract anomaly than patients described in the literature. Intention tremor was hypogonadotropic hypogonadism. One brother had partial growth hormone deficiency and the other had tertiary identified in 16/36 subjects examined, and 3/36 children were ataxic. Frequencies of these hypothyroidism. The association of hypogonadotropic neurologic findings in 18q- syndrome have not been reported previously. Only one subject hypogonadism with EEC was reported on one occasion had a height equal to or greater than the mean for age. Atretic/stenotic ear canals occurred previously (Van Maldergem et al. 1992). Knudtzon and in 20/34 study subjects compared with 26/66 subjects described in the literature (p=0.05); Aarskog (1987) have described two patients with EEC and 31 of 33 (94%) subjects had hearing impairment as determined by BAER, compared with isolated growth hormone deficiency. Hypothalamic-pituitary 26% of in the literature. These data indicate that renal dysfunction should be considered as yet another subjects reported tremor/ataxia, manifestation of the EEC syndrome. anomalies and hearing impairment may be more common in 18q-syndromethan Considering our cases and the cases reported in the previously reported. Further studies will explore genotype/phenotype conelations. This litterature we suggest that hypothalamic-pituitary study was funded in part by the MacDonald family, Microsoft Corp. and Genentech, Inc. insufficiency should be accepted as yet another manifestation of EEC syndrome. Since our patients were born to unrelated and unaffected parents, we reconfirm that EEC syndrome is variable, non-penetrant and pleiomorphic.

494 495 ClInical criterla for fragile X Southern blot DNA diagnostic testing. CA Increased rate of congenital heart defects among the Inuit of Baffin Is- ianarec' 2. M.W. Steelel.2. C. E. Aston2. J.H. Cummins'. S.L. Wenger' 2. land. C.A. Gilpinl, L. Arbourl, G. Pekelesl.M. Paquct2. P. Eydouxl. 1 Montreal 'Children's Hospital and 2University of Pittsburgh, PA. Children s Hospital/McGill University. Departments of Pediatrics. Human Genet- In our laboratory, 1.5-2% of index cases referred for DNA fragile X [fra(X)] ics and Pathology. Montreal. Quebec. Canada and 2 Department of Pediatrics. testing are positive. With managed care pressures, cost effectiveness of University of Sherbrooke. Sherbrooke, Quebec. Canada. laboratory testing is an acute concern for health care providers. We The Baffin Island region, Northwest Territories. Canada, has been inhabited by analyzed, retrospectively, clinical characteristics of 273 male and 62 female the Inuit for over 1000 years. The population of this area is approximately 11,300 probands (average age 5.5 years) referred for fra(X) testing from Children's with 3,800 people residing in the town of Iqaluit, and the rest clustered in 12 isolated Hospital of Pittsburgh clinics during 1992 and 1993. Twelve cases were communities spread over an area larger than 1 million km2. Although there have fra(X) positive, one being a female. Characteristics tabulated from patient been reports suggesting a higher rate of congenital heart defects (CHD), this has records included mental retardation (MR), family history of MR, large or not be previously confirmed. A chart review of consecutive livebirths over a period prominent ears, elongated face, attention deficit disorder, and autistic-like of 5 years, from April 1st 1989 to March 31st 1994, was undertaken. The rate for These were as 2 if present, 1 if 0 behavior. scored borderline present and CHD was compared to the figures of the Alberta Congenital Anomaly Surveillance if absent. Summing these gives a 'total score' with a maximum of 12. System which reports a 0.83% rate of CHD in livebirths. We found a Discriminant analysis between fra(X) positive and negative cases suggested (ACASS), the diagnostic usefulness of, primarily, the total score and, secondarily, of significantly higher rate of CHD in this population (2.09+/-0.77, 95% confidence large ears. Most referrals for fra(X) testing were on the basis of interval). These malformations include 17 ventricular septal defects, 6 atrial septal 5 developmental delay. This is a primary trait but by itself does not defects, patent ductus arteriosus, 2 pulmonic stenoses (5 combined defects) and discriminate between the fra(X) positive and negative groups. All 12 patients 3 complex heart malformations. The reason for this increase of CMID in the Inuit in our fra(X) positive pediatric age group were mentally retarded (1 of the Baffin region is not clearly understood. Further studies may elucidate the borderline), 10 had large ears, 8 had a family history of MR (1 borderline), respective contribution of genetic and environmental factors. and seven had autistic-like behavior. Using our checklist of 6 characteristics, requiring a threshold total score of 5 or more for referral would have eliminated about 60% of the negative fra(X) testing in our patients without missing any positive cases. The percentage of positive index cases would have increased to 9.5% consequently improving the cost effectiveness of fra(X) testing. Posters: Clinical Genetics, Malformationms and Dysmorphology (continued) A91 496 497 Tnw *bory 2 moslclam In alart and newbom Nver assocded wIth multpl Dominant inheritance of hearing impairment in patients with Long QT 1 3 SYStem M.no111" Gl H Sawo MM2E. 3j, ConL syndrome. G. E. Green', M. H. Lehman2 and D. G. Drescherl. 'Department of and RN. Lebo 1 UC.SF Medical Center. San Francisco: 1 Chidren's Hospital Oakland, Otolaryngologv. Wayne State University. Detroit, MI. and 2Section of Cardiology. CA,2 Prenatal Diagnostics, Inc., Mountain View, CA.3 Sinai Hospital. Detroit. MI. Long QT syndrome (LQTS) is characterized by abnormal prolongation of the Among the 58.000 amniocenteses completed, our loboratories found only QT interval (the time required to repolarize the cardiac ventricle) and is associated one case of true trisomy 2 mosaicsm. We report this case with multiple system with arrhythmia-induced syncopal episodes and sudden death. Traditionally. fa- abnormalities and low level hepatic mosaic trisomy 2. Two other mosaic trisomy 2 milial LQTS has been divided into Jervel and Lange-Nielsen syndrome (autosomal liveborns have been reported: one with Pfeiffer syndrome-Ike features and the other recessive LQTS with hearing loss) and Romano-Ward syndrome (autosomal domi- with growth failure, hypothyroidism and RDS. The 19 year old mother of our mosaic nant LQTS without hearing loss). Further heterogeneity is shown in the recently- trisomy 2 patient hod elevated MSAFP at 16 weeks gestation and amniocentesis at 19 identified ion channel genes for Romano-Ward syndrome: HERG and SCN5A. weeks when oligohydramnios was noted. The mother elected to continue the However. the distinction between Jervel and Lange-Nielsen syndrome and Romano- pregnancy even through mosaic trisomy 2 was reported In 8/25 and 7/31 omniocytes absolute. Ion channels are central to function of analyzed In each of two cultures. At 26 weeks the fetus had IUGR, hydronephrosis, a Ward syndrome does not appear double outlet right ventricle, ASD, and VSD. At 30 weeks the fetus was delivered by both the heart and the cochlea. and ion channels expressed in both tissues have cesorean section because of decelerations with a birth size at 10 percentile. The been identified. Case reports have identified children with LQTS and deafness born newborn had a large anterior fontonel, prominent occiput, prominent lower canthal to a parent with isolated LQTS. We now report two unrelated. non-consanguineous folds, beaked and prominent nose, flat molar area, prominent antihelix and flat outer families with six individuals in which bilateral. symmetric sensorineural hearing ear heix, thin lips, pointed chin, heart murmur, pectus excovatum. inguinol hernis, loss (SNHL) and LQTS are concurrently inherited in a dominant fashion. Family rocker bottom feet, and prominent heels. Head MRI at 6 months showed delayed A: The propositus is a 27 yo female with LQTS and moderate SNHL. Her 17 yo myelination, abnormal paucity of white matter, thin corpus callosum, hippocampal sister with LQTS also has below normal hearing. Her 54 yo father has SNHL (up dysplasia, and an abnormal deep left frontal lobe sulcus. Although chromosomes to 65 dB) and LQTS. Her mother has normal hearing and does not have LQTS. were normal in blood, skin fibroblasts, and ascites fluid cells, 4 of 100 analyzed Family B: The propositus is a 42 vo female with LQTS and SNHL which was first hepatic biopsy fibroblasts were 47XY,+2. Uniporental disomy was excluded in identified at age 6. With one husband she had two children - both with LQTS cultured newborn skin and lIver by Informative D2S1 19 and Mtdl28 polymorphic VNTR and one with SNHL. With a second husband she had two additional children - analysi. Although this was our only case of mosaic trisomy 2 at amniocentesis, we again, both with LQTS and one with SNHL. These inheritance patterns suggest diagnosed 11 of 10500 fetuses tested by CVS with true mosaic trisomy 2. Of these, 5 of 5 tested by amriocentesis had normal omnlocyte karyotypes and 11 of 11 fetuses that, in some cases, variable expressivity may explain the concurrence of hearing were phenotypically normal at birth. We conclude that true trisomy mosaicism at loss and LQTS. This association should not be considered autosomal recessive in amniocentesis shou d be taken seriously because the trisomic celis have a greater all cases. We recommend cardiologic evaluation for the farnily of any child with probablity ofbeing fetal in ongin and not just confined to the placenta. hearing impairment and LQTS.

498 499 Arnold-Chiari salforuation in twin brothers and father with autosomal Ocular Albinism-Lentigines-Deafness (OALD) syndrome in a female infant. dominant spondylo-epiphyseal dysplasia tarda. K.W. Gripp and C.I. Scott. M. J. Hajianpour and H. Fialkoff. Clinical Genetics Center, The Genetics Institute Alfred I. duPont Institute, Wilmington, Delaware. and Health Care Partners Medical Group, Pasadena, California. We describe twin brothers and their father with autosomal dominant We report a 22-month-old Caucasian female, who was born to a 22-year-old, G5, spondylo-epiphyseal dysplasia (SED) tarda. The proband presented at 17 delivery. Multiple years with intermittent esotropia due to abducens nerve involvement and P1, TAbI, SAb2 mother, after a full-term pregnancy, by vaginal was found to have a severe Chiari malformation involving caudal dis- hyperpigmented spots were noticed after birth. The number and sizes of these placement of cerebellar tonsils and vermis as well as an elongated and pigmentary skin lesions have continuously increased. Due to speech delay, a dilated fourth ventricle. Due to the severity of these changes and the brainstem auditory evoked response was performed at 15 months, which revealed inconsistent terminology in the literature this malformation may be profound neurosensory hearing loss. The patient was referred to our center with a regarded as an intermediate form of Chiari deformity, showing some tentative diagnosis of neurofibromatosis-l versus LEOPARD syndrome. However, findings characteristic of Chiari II (Arnold-Chiari), but not conditions. The MRI of the associated with either neural tube defect nor other hindbrain the patient did not fulfill the diagnostic criteria for these abnormalities . brain and vestibulocochlear complex, echocardiogram, renal ultrasound, and Studies performed subsequently in his twin brother and father chromosome analysis were all normal. Slit lamp examination indicated minimal revealed milder, asymptomatic Chiari I malformations. Although these peripheral iris translumination, with no visible pigment present in the iris. No Lisch malformations to our knowledge have not been described in patients with nodule was identified. Fundus examination showed complete absence of SED, they are relatively common in patients with bony abnormalities, pigmentation except for the small area of the inferonasal rim of the right disc. particularly those involving the cranio-cervical junction. The con- cordance of the finding of Chiari abnormalities in all three family Macula and fovea were normal. members affected with SED suggests that this association is not On physical examination at 22 months, the weight, height, and head circumference coincidental. Further the occurrence of Chiari I and a more severe were at 15th, 7th, and 25th centile, respectively. The head was normocephalic. She form resembling Chiari II in one family may support the theory that had overfolded upper helices, depressed nasal bridge, upturned nose, mild joint the definition of these two groups is based on historically evolved laxity, wide-based unstable gait, and numerous lentigines lesions ranging from few criteria, but that there is some overlap and the groups represent the millimeters to several centimeters in diameter. The family history was negative for extremes of a continuum rather than distinct entities. any similar clinical features. We think that OALD' is the most likely diagnosis for this patient. The OALD syndrome was first reported in a family with 7 affected males and 5 affected females in three generations (Lewis, 1978). To our knowledge, we are now reporting another case with "QALD' diagnosis in literature.

500 501 Diaphragmatic hernia and diabetic embryopathy. a.D. Hall. University of Recombinant 8 syndrome breakpoint analysis. D. Patterson'. E. Sujansky2. I. Hart'. Kentucky, Lexington, Kentucky. J. Bleskan'. K. Walton'. J. Giang'. I. Shechter'. 'Eleanor Roosevelt Inst, Denver, CO; Diaphragmatic hernia in association with diabetic embryopathy has been reported in 'Univ of Colo Health Sciences Ctr. Denver, CO. approximately 9 instances. Kucera (J Reprod Med 7:61, 71) reviewed the world's Recombinant 8 Syndrome (Rec8 Syndrome) is one of the few identified literature and placed diaphragmatic hernia 23rd on the rclative scale of single chromosomal disorders that impacts a specific ethnographic population, the Hispanic anomalies associated with maternal diabetes. He concluded its degree of specificity population of the San Luis Valley of southern Colorado and Northern New Mexico. doubtful relative to the adverse fetal effects ofmaternal diabetes. Chung and The Rec8 Syndrometrec(8), (8qter-8q22.1::8p23.1--8qter)J is due to a parental to Myrianthopoulos (BD:OAS 11(10):23, 75) also noted the rare potential association as inversion (8)(8pter-8p23. 1::8q22. 1-8p23. 1 ::8q22. 1-8qter) which has been found is characterized only 1 of 149 malformed infants ofdiabetic mothers (IDM) had a diaphragmatic originate primarily in this Hispanic population. Rec(8) Syndrome by hernia. However, Greene et a]. (Teratology 39:225, 89) and Miodovnik et al. dysmorphic features, major congenital malformations including congenital heart moderate to severe mental retardation. While the literature some (Diabetes Care 11:713, 88) noted a combined occuirencc of2 of 34 malformed IDM defects, and provides the of Syndrome, the factors contributing to the with diaphragmtic hernia. description of phenotype Rec(8) phenotypic variations seen in these individuals are unknown. As a start toward I present an additional 1DM who had a diaphragmatic hcrnia. He was the 36 week understanding the etiology of Rec8 Syndrome we have isolated a yeast artificial a and fetal gestation product of pregnancy complicated by polyhydramnios, chromosome of 280 kb and a PI clone of 66 kb which span the 8p23.1 chromosomal evidence of a truncus arteriosus. Amniocentesis had revealed a echocardiography breakpoint associated with the inversion 8 chromosome found in at least one parent of normal AFP and karyotype (including DiGeorge probe). The mother was a 17-year- all Rec(8) Syndrome individuals. These clones both contain at least the 5' coding 1 who was an 7 years age. Birth old gravida insulin dependent diabetic since of region of the gene encoding squalene synthase, the first step of the cholesterol were normal. He was weight (2845g), length (45cm), and head circumference (32cm) biosynthetic pathway specifically committed to cholesterol and steroid hormone in severe cardiorespiratory distress secondary to a right diaphragmatic hernia. He had metabolism. Squalene synthase is the most highly transcriptionally regulated step of no external defects. Radiographs showed sacral agenesis and multiple cholesterol synthesis in human cells found thus far. We are now engaged in dyssegmented/fused lumbar and thoracic vcrtebrae. He died at 12 hours and no determining whether this gene is disrupted by the chromosomal inversion and isolating autopsy was allowed. and analyzing the chromosomal inversion breakpoints associated with Rec(8) The above additional case ofdiaphragmatic hernia and IDM should serve to keep Syndrome. These studies will be relevant for molecular definition of the chromosomal the issue of an association alive. regions aneuploid in this syndrome and for analysis of the mechanisms of chromosome inversion. A92 Posters: Clinical Genetics, Malfornnations and Dysmorphology (continued) 502 503 Shpritzenh-Gldbeq syndrome wiaut fractrs and childhood onset of Mevacor - a new teratoge? A.Hes. A. Gilbert. G. Loe. W. A. hrocepalus. U. Haassd. K. Shewmake. C. To., C. Cunniff. M.A. Curtis Miller. Prenatal Diagnostic Center, Lexington, MA. University of Arkansas for Medical Scienes and the Arkansas Children's Hospital. MEVACOR5 (Lovastatin) is a widely-prescribed cholesterol-lowering Shprintzen-Goldberg syndrome was initially described as a condition with agent. Its mechanism of action is trugh inhibition of HMG-CoA craniosynostosis, proptosis, abdominal hernias, marfanoid features, and intellectual reductase, an essential step in cholesterol biosynthesis. MEVACOR* has deficits. A review of the literature indicated common features also include been shown to be highly effective in reducing total and LDL cholesterol in hypeseloism, downslantng palpebral fissures, ptosis, micrognathia, high arched palate, heterozygous familial and non-familial forms of primary low-set anomalous ca, aachnodaly, camptodactyly, talipes equinovarus, scoliosis, hypercholesterolemia and hyperlipidemia. With increasing awareness of and obstructive apnea. cholesterol-relatedmorbidity and emphasis on earlier risk-factor intervention We report a child with Shprintzen-Goldberg syndrome who presented in the neonatal for coronary heart disease, there is greater likelihood that pregnant women period with plagiocephialy, downaslanting palpebral fissures, low-set cupped ears, might inadvertently be exposed to cholesterol-lowering agents. We report bulbous nose, micr hnodactyly, camptodactyly, femoral fractures, clubfect, a 24-year-old primigravida in whom a first trimester exposre to and poor suckc Th differential at that time included congenital Marfan syndrome and MEVACOR9 was associated with an extensive neural tube defect in the Beals conural chnod ly. Echocardiogram, head MRI, chromosome analysis, fetus. Although a definite cause-effect relationship cannot be established, and TORCH titers were all normal. During the neonatal period the patient developed it is possible, based on animal and other experimental data, that hypoadrenalism; VLCFA were obtained and were normal. Obstructive apnea required MEVACOR* may have acted as a teratogenic agent in this fetus. tracheostomy. Gastrostomy tube was placed for GE reflux, poor feeding, and failure MEVACOR° is one of a group of phannacological cholesterol-lowering to thrive. Repair of bilateral inguinal hernias was performed at that time. At age agents known as vastatins which, by HMG-CoA reductase inhibition, affect the and one-half years the tracheostomy was closed, the G-tube removed, and the production of nievalonic acid (MA). MA is vital for diverse cellular adrea medications discontinued. A developmental evaluation at 4 2/12 years fuction ranging from cholesterol synthesis to cell growth control and indicated delays of about 12 months. Following an accident, he was evaluated for proliferation. If the mevalonic acid pathway is interrupted during critical head pain via head Clr which revealed hydrocephalus that required shunting. His periods ofcellular proliferation during embryogenesis, there is the potential progress was rapid following closure of tracheostomy site and VP shunt placement. for pleiotropic teratogenic effects. Caution should be exercised in the Spine rdiogrphs at 4 6/12 years revealed mild scoliosis. prescription of the vastatin cholesterol-lowering agents to women in their In patients with Sprintseni -oldberg syndrome repeat brain imaging studies may reproductive years. identify progressive hydrocephalus. If this finding is typical, it could help in diagnosis of Shprintzen-Goldberg syndrome and perhaps help to explain the cause of the reported intellectual deficits in these patients.

504 505 'flu aqtict1 of 4atel ad triso 18. ((M.U. Rieferl, J.H [er*u2o j.C. The changing phenotype of Pelizaeus-Merzbacher disease EHi ca~r',)) St. Jue's dlae's _ ital ds, n u it of Stabh R. Qlouby. Indiana University School of Medicine, Indianapolis, IN. Sdnl of £ nlci ,FAd , of ah of Mflcirn, salt IA aCty,Utih. It was 104 years between the description of Pelizaeus-Merzbacher di (PMD) and the delineation of the first mutations in the proteolipid protein gene lIsy 18 is a lathal dwmse &ramality with a prreme of 1 in 6,00D. (PLP). There are now some 38 changes, ranging from point mutations to aXivie bycnd infacy is orac, ad death in okier dideu .^anly is dce to deletions or duplications of the entire gene, that are known to cause PMD. eAcd1qfcrmy icatis. Mllns bter han bern diwread in 9 patiets with Some of the mutations occur in that portion of the mRNA that is triscni 18, ejIINizirg an iatiua bebn tin bo, ad potential for tie snmiry spliced out beig a cams of dinty. when DM-20, which seems to be important in oligodendrocyte maturation, is Ik ;rmwrt a 2-6/12 yew bilk femde with trsy 18 ad t t. formed. Although one of these mutations in DM-20 is associated with the mild Kajeypeon licdaltsin did rt zim is. nd a VSD, ht aer murine mutation rumpshaker, other mutations are associated with severe forms stabs e0 cipewtmd. An *&dami nts WE dicoered after a fwr w history of of PMD. l Isuc midre ad Welt 1b. Me dlsgcsi bsed cn ahintirr l irrong ard PMD appears in relatively mild ('classical") to severe ("connatal') forms. S da NDb tamht-t kW psad ad da epired for neins later. ogr c, wean Ran of 6 instama of s in 18, Moreover, mutations in PLP, including that in rumpshaker, have been found in lreldirrg >tc with melcias Mu prserca ofFLe ad nturDisyin X-linked spastic paraplegia (SPG). This is a condition that overlaps PMD in trlnq 18 1ircatas an ireesed risk for drl sic inis f many if not most aspects. Our attempts to correlate the phenotypes of PMD aatplodAy. Vt thn iriderse of l_-uc MINa?, ldIM t=, cld s and SPG with the genotypes have not been fruitful. This may be in part to b t possl ty edits nttind nit ocorter frsaaftly if thte because the structure of the wm = rttem saiv. proteolipid protein, PLP, has not been elucidated Alti* aBessie int nw rot be irdicated afte recfftes of a fully and in part because most patients with clinically defined PMD (ca. 70%) do nnliymcy, amiss of ti pntial fr ti= dsmte in t=is 18 Inly ip to not have mutations in the coding region of PLP. Yet, linkage studies show that inti~iha dis mrme rptly vhnt dte is a daWe In a petlait's clinical PMD in many if not all of these cases is linked genetically to the proteolipld stabm seaa to csdiplnw pirtams. Periodic &dndal ueec~~ protein locus. tefe, ny be Nstifial in Of trimy 18 szivrg brd lnfey, to assist in unitg tin nest aprerlate rat decisinsn for tim patients. It is becoming clearer that PMD and SPG constitute a spectrum of disorders that ranges from mild to very severe. Variation in expression is sen in individual families and some have individuals witb PMD, whereas other individuals seem to have SPG. This spectrum of conditions associated with mutations in PLP is expanding. Overlaps are frequent and there are both male and female patients with dementia and peripheral nerve involvement.

506 507 Polyniorogyria in two brothers: evidence for an inhejited X chromosome inctiv on inonozygotic female twins discordant for neuronal Tigration disorder. B. Wittwey , D.G. Palm Schuierer , J. Horst . Institut fur Humangenetik systemic lupus erythematosus (SLE). Q. Huang N Manolios_A Parfi, D Kinderklinik, Institut fur klinische Radiologie der MorrisJ D. Grnuan. Department of Rheumatology, Royal North Shore Hospital, Universitat Munster, Germany. St. Leonards, NSW, Australia 2065.( Intro-by: RZ.A.Trent) Over 20 syndromes with lissencephaly or other types of Genetic susceptibility to SLE has been suggested by familial aggregation neuronal migration disorders have been described. Among and higher concordance for SLE in monozygotic (MZ) twins than Otat in dizygotic them are syndromes with very different patterns of twins. One recent inheritance including chromosomal mutations and unknown large twin study showed that concordance for SLE in MZ twins etiology. The neuronal migration disorders are subdivided was 24%, suggesting that genetic susceptibility played a minor role, whilst into several different pathological types. In polymicro- environmental factors may have major contributions to the disease. Studies have gyria, normal cortex is replaced by abnormal, crowded suggested that SLE is a polygenic disease and a gene (or genes) on the X narrow gyri. chromosome may have an additive effect on clinical expression. We report on two brothers of healthy unrelated parents Epigenetic with developmental retardation, microcephaly (OFC below phenomena, such as X chromosome inactivation and genetic imprinting, are well- 3rd centile), and hemispasticity in the first and te- known factors that can cause different phenotypes in MZ twins. This study traspasticity in the second boy; until now no epileptic examined the hypothesis that the clinical discordance in MZ twins for SLE could seizures. The MRI revealed a severe asymmetric polymicro- be accounted for by different X chromosome inactivation. Three pairs of female gyria in the first and symmetric diffuse polymicrogyria Z twis (1, 2 and 3) discordant for SLE, zygosity confmned by DNA in the second child. Both pregnancies were uncomplicated except minimal in the second month. fingepriting, were studied for X chromosome inactivation. DNA was extracted bleeding In both from blood and X chromosome cases intrauterine growth retardation was observed during inactivation was assessed by methylation status in the last months. The OFC at birth in both patients were trinucleotide repeats in exon 1 ofthe Androgen receptor gene. Results showed that in the 3rd centile. the X inactivation patterns between affected and non-affected twins were the same Polymicrogyria may be evidence for a sporadic rather than in all three pairs, with nonrandom patterns in the twin pairs land 2, and random genetic etiology. The findings of our presented cases pattern in twin pair3. X chromosome inactivation was also examined suggest a genetic cause. We assume that inheritance here in DNA is autosomal-recessive although X-chromosomal recessive from buccal cells of twin]. The patterns from buccal cells were concordant inheritance remains possible. between both twins and same as the patterns in DNA from blood. In this study, these three pairs of MZ twins could not substantiate the hypothesis that the low concordance rate in SLE MZ twins may be accounted for by the differences in X chromosome inactivation. Posters: Clinical Genetics, Malformations and Dysmorphology (continued) A93 508 509 A New Skeletal Dysplasia Syndrome with Progressive Bone Changes, Camptodactyly, MABN I LSFALSY Dysmorphic Facial Features, and Feeding Difficulties. A.H.M.M. Hug. F. Greenberg. Pau Jaakar Mano Rv and Donald A l Miami Childrens C. A. Bacino. and M.L. Levin. Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, Texas. Hospital,Miami.Florida. We describe male and female siblings, who are the products of normal appearing first A wide range ofbehavioral and cognitive disturbances have been cousins once removed, that display a previously undescribed constellation of physical reported in patients with Velo Cardio Facial syndrome (VCFS), yet findings including a unique skeletal dysplasia, camptodactyly, characteristic facies, and their anatomic basis remains unknown. We report the MRI findings inability to feed normally in the newborn period. Gross motor and social development in 4 patients (2 M,2 F) aged 4 months to 11 years with VCFS. All of the older child appears normal thus far, although speech articulation problems are had clinical features suggestive of VCFS including neonatal suspected. hypocalcemia, congenital heart disease, developmental delay and Both children presented to our center with aspiration pneumonia in the neonatal characteristic facial features. Cytogenetic analysis revealed period and were found to be unable to suck. Both ultimately required gastrostomies for microdeletion ofchromosome 22qI 1 in 2 patients. MRI scans in feeding. Physical features include camptodactyly of the 3rd, 4th, and 5th fingers, in the white matter hypertelorism, downslanting palpebral fissures, hypoplastic and low appearing all 4 cases showed signal abnormalities nasal nasal tip, anteverted indicative ofmultiple areas ofgliosis and demyelination. Additional supraorbital ridge, rectangular shaped bridge, prominent spaces (n=2), nares, relatively small mouth with limitation of opening, velopharyngeal incompetence, MRI findings included dilatation ofVirchow-Robin and retrognathia. The children are intermittently hypertonic as newborns. Weaver prominence of temporal tips oflateral ventricles and hippocampal syndrome was initially considered as the diagnosis in the older child but there was no hypoplasia (n=2), hypoplasia ofthe splenium and rostrum ofthe advanced bone age, and a series of bone changes became apparent by age two months. corpus callosum (ntl) and Arnold Chiari malformation (n=1). The These changes include bowing of the tibula and fibula, metaphyseal splaying affecting severity of Central Nervous System (CNS) involvement did not the long bones and the anterior ribs, with low density of the bone matrix and increased correlate with the presence ofa demonstrable chromosomal density in the periosteal regions. Pathologic fractures have occurred. Similar skeletal deletion. changes have been recognized at age one week in the younger sibling. Our findings confirm that multifocal white matter abnormalities Chromosome analysis, including FISH for VCF, extensive metabolic studies, first reported by Mitnick et al.,(1994) are a consistent structural skeletal surveys, CNS imaging, muscle biopsy, nerve conduction and abnormality in patients with VCFS and may be causally related to the electromyographic studies and collagen studies have not been helpful in making a retardation and psychosis seen in these patients. Since the characteristic definitive diagnosis. Literature and database searches have revealed no cases similar to chromosomal deletion is not always present, MRI lesions need to be these children. in defining the spectrum ofCNS involvement in VCFS. Based on the clinical data and history of consanguinity we conclude that these cases considered represent a new autosomally recessive inherited condition that can be added to the lists of syndromes presenting with camptodactyly and/or skeletal dysplasia.

510 511 Clinical profile of patients with chromosome 22q1 1 microdeletion at the DiGeorge locus. AUTOSOfMAL RECESSIVE COMMON VARIABLE IMWAUNODEFICIENCY Salinas. G.S. Medical University of South M.Joseph. E.S. Cantu, J.M. Jastrazab. C.F. Pai. REPORTS OF SEVEN RELATED CHILDREN IN A BEDOUIN KINDRED. Carolna, Charleston, S.C. studied a total of 32 patients ranging in age from newborn to 38 years for the specific We Hesham H Kandil, Akhter S Ghulam, Fatma S Thal lab, Mohammed M locus on chromosome 22ql 1 by FISH using the molecular probe deletion at the DiGeorge AI-Ghanim, Taloat Farag. Patients with at least two of the following five anomalies were selected for the D22S75(Oncor). Department of Paediatrics, Al-Jahra Hospital and Medical Genetics irrespective of their overall clinical diagnosis: conotruncal cardiac anomaly, facial study Center, Maternity Hospital, Kuwait. dysmorphism, thymus aplasia/T ceil anomalies, cleft palate/ velopharyngeal Insufficiency and sustained hypocaicemia. Aim of the study was to determine the frequency of 22q1 1 deletion in In an Arab Kindred with Bedouin Ancestors, seven related infants of manifestations of DiGeorge syndrome, Velocardiofacial syndrome and other entitles with partial both with early onset common variable immunodeficiency syndrome overlapping manifestations. sex were ascertained and followed up in the Jahra Regional Liaison Clinical diagnosis was DGS in 11, VCFS in 8, CHARGE association in 3 and Diiantin Comnunity Genetics Programme. As early as six months of age all embryopathy In one. The remaining 9 did not have a syndrome diagnosis. 22q1 1 deletion at the patients developed recurrent ptogenic infections In the form of was detected in 13/32(41%). All the 13 deleted patients were syndromic, 9 with DGS DGS locus otitis media, epiglottitis, conjunctivitis, bronchopneumonia, 4 with VCFS. Conotruncal anomalies were present in 11/13(85%), facial dysmorphism in and cellulitis, perianal and skin abscesses. Associated disorders 13/13(100%), thymic hypoplasia/T cell anomalies in 10/11(91%), cleft palate/ velopharyngeai included: failure to thrive, autolmmune cholangitis, enteropathy, insufficiency in 4/13(31%) and hypocalcemia in 5/12(42%). Although a wide variety of intermittent neutropenia and thrombocytopenla. Immunological anomalies were present in the studied patients, the deleted group had either conotruncal evaluation revealed persistent hypogammaglobinaemia with normal Interrupted aortic arch(6/11), tetralogy of Fallot (3/11) or truncus arteriosus(2/11). Facial number of B cells and normally functioning T cells. Inspite of dysmorphism was highly variable from nonspecific to typical. Thymus anomales ranged from triweekly intravenous immunoglobuIin replacement therapy and aggre- total aplasia to subnormal numbers of T cell population. ssive use of antibiotics, five patients died within the first two Interesting associated anomalies were Wilms tumor in 2 patients with VCFS one of whom had years of life due to overwhelming sepsis. Expanded family study deletion and esophageal atresia/ TE fistula in 4, one in a DGS with deletion and 3 in seven generations demonstrated an autosomal recessive nonsyndromic patients. of common variable immunodeficiency variant with an early onset, differing Thus in our population with at least 2 of the 5 selected anomalies, 82% of the clinically the classical late-onset disease (MIM 240500 McKusick 1944). diagnosed DGS and 50% of the VCFS patients had detectable deletions by FISH. No deletion from was detected in other syndromes although they had overlapping spectrum of anomalies. Thymic/ T cell anomalies and conotruncal anomalies(interrupted aortic arch, tetralogy of Fallot and truncus arteriosus) were best correlated with the deletion in our study population.

512 513 Evidence that the Setleis and Brauer syndromes of focal dermal Ultrasound characteristics of the development of the hips in infants with hypoplasia are the same entity. P. Kaplan. I. Krantz. M. Mascarenhas. W. Down syndrome. E. E. Karaikovic. J. H. Miles. E. B. Otto. R. W. Gaines, Tunnessen. S. Schulman Children's Hospital of Philadelphia, University of University of Missouri, Columbia, Missouri. Pennsylvania School of Medicine, Philadelphia, Pennsylvania. progressive Setleis syndrome is characterized by bitemporal scar-like lesions, "leonine" facies Children with Down S often suffer from a hip dislocation due to (periorbital fullness, sharply angled medial eyebrows with lateral deficiency. laxity of the surrounding soft tissues. The dislocation causes gait problems which distichiasis of upper lids, astichiasis of lower lids, flat nasal bridge, bulbous nose tip, often must be treated operatively. Knowledge about the natural history of the long columella, thick lips), alopecia/thin scalp hair, low frontal hair line, development of the hip in infants with Down S is insufficient. The ultrasound cleft chin, hypo/hyperpigmentation, imperforate downslanting palpebral fissures, modality gives a unique opportunity for a better look "inside' the child's hip. The anus, cryptorchidism, duplication of vagina & uterus, hydronephrosis, and aim of the study was to assess the maturation of the Down's hip compared with megaureters. Aulosomal recessive inheritance was suggested (two sets of affected study we followed up sibs with apparently unaffected parents and an affected child with consanguineous a normal child. In this prospective ultrasonographically parents). bony development of the hips of 13 Down S children (26 hips) of the Down Brauer syndrome (focal facial dermal hypoplasia) is characterized by bitemporal Syndrome Clinic of the Department of Child Health, Division of Medical of chin & infrequently mental "scars", vertical depressions of forehead, clefting Genetics of the University of Missouri-Columbia. Nine of them had US done in retardation. Inheritance has been postulated as autosomal dominant (AD) (affected the first three months of life, 8 between 6 and 9 months of age, and 9 from 12-15 and child) or explained as a new mutation or gonadal mosaicism (parent parent 12-15 unaffected). months of age. Six children had continuous follow up from birth until Our proband has bitemporal "scars", sparse lateral eyebrows, bulbous nose tip, months of age. Clinical examinations by a pediatrician and an orthopaedic anus & rectocutaneous fistula, thick lips, micrognathia, chin crease, imperforate surgeon were performed prior to every ultrasound exam. The standard ultrasound megaureters, hypotonia, growth and developmental delays & non- bilateral technique was used (Acuson 128 with 5.0 and 7.5 MHZ linear transducers). consanguineous parents. The patient's father has only bitemporal "scarring", bulbous Coronal and transverse views were obtained. Based on ultrasound findings, we nose tip & hypertension. modeling, shape of the bony promontory, Our proband has "Setleis syndrome" but has inherited the condition as an AD were able to describe bony trait. This case and a review of published reports suggests that Setleis and Brauer characteristics of the labrum, stability of the hip and appearance of an ossification an autosomal dominant fashion with syndromes are the same entity inherited in center of the femoral head, as well as to measure hip angles and classify the hips variable expressivity. in these patients according to Graaf. A94 Posters: Clinical Genetics, Malfortnations and Dysmorphology (continued) 514 515 total anonyohia congenita and microcephaly with normal Phenotypic spectrum of Kabuki make-up syndrome In 15 North American intelligenoc in 2 aibahips of consanguineous parents: a atients. H. KawaMe. L. Hudgins. R.A. Pagon. Children's Hospital and Medical now autosomal reoesive syndrome? Pardeei Kaurah. Amad S. enter and University of Washington, Seattle, WA. Zkab.. The F. Clarke Fraser Clinical Genetics Unit, To evaluate the Division of Medical Genetics, The Montreal Children's phenotypic spectrum of Kabuki make-up syndrome, we Hospital and McGill University, Montreal, Quebec, Canada. reviewed the findings in 15 patients with the characteristic facial appearance and developmental delay who were ascertained through medical genetics programs in Anonychia Congenita or congenital absence of nail is a Washington and Arizona. Al patients had hypotonla in infancy and seven had rare anomaly that is encountered as an isolated finding feeding difficulties; two required gastrostomy. Although developmental involving some or all nails, or in association with other quotients/1Qa ranged from 15 to 90, motor and cognitive ability in all but two was anomalies that are mostly confined to the limbs. Here we 60% or less. Eight had seizures with persistentiy abnormal EEGs; one had report on a brother and sister with a previously infantile spasms. Postnatal growth retardation occurred in 12. Eight patients had unrecognized constellation of total anonychia congenita ocular anomalies including ptosis (5), late onset cataracts (2), strabismus (2), and microcephaly with normal intelligence. The facies and coloboma (1), and mild optic nerve hypoplasia (1). Recurrent otits media was head shape are normal. Other anomalies included bilateral common, as was hearing loss, but no patients required hearing aids. Four patients fifth finger clinodactyly and bilateral single transverse had congenital cardiac two palmer creases. Skeletal survey was normal in both. The defects; required surgery for coarctation of the sorta. children and their first cousin Iranian parents have Five had renal anomalies; two required surgery (1 UPJ obstruction; I VU reflux). widely spaced teeth. A maternal female first cousin has Other features included joint hypermobility (5), patellar dislocation (2), congenital apparently similar constellation of manifestations without hypothyroidism (1), intestinal malrotation (1), and panhypoimmunoglobullnemla (1). the teeth anomalies. The presence of 2 independent Family histories were negative for Kabuki make-up syndrome. All patients had autosomal recessive trait versus a new syndrome is normal chromosomes. discussed. Given that 15 patients were identified from two genetics programs, it appears that this syndrome is more common in non-Japanese patients than previously appreciated. Patients with Kabuki make-up syndrome have a broad spectrum of neuromuscular dysfunction and mental ability, and are at risk for other severe, but less well-recognized, problems such as coarctation of the aorta, patellar dislocation, renal malformation, and immunodeficlency.

516 517 A ae of Duchenne muular dystrophy with noncontLguous parti Genetics ofvery-late-onset dementia: A prospective study. J.KaLrH. Ginmslid. deetion and partial duplcation mutations In dystroi gene. . MurdocLL D. Howieson. R. Ca ioll. A. Dame. M. Litt. and H. Payami. Oregon awanmura1LsT ij 1, IT. JaroI and &aig2. National Health Sciences University, Portland. Higashisaltama Hospitall and Department of Microbiology, Keio University, Little is known about the cause of dementia in the very old, a prevalent disorder in the fastest growing segment of the population. The aim of this study was to School of Medicine2, Japan determine if the major risk factors for Alzheimer disease (AD), i.e.; age, Apolipoprotein E genotype, and family history of dementia, continue to be risk Duchenne muscular dystrophy (DMD) is a severe X-linked lethal myopathy factors for cognitive decline in the very old. We studied 109 subjects ages 75-105, with an incidence of approximately 1 in 3500 male births. The primary who were optimally healthy at entry to the longitudinal Oregon Brain Aging Study. biohemical defect in DMD is dystrophin deficiency. The dystrophin gene, Within 2.8 ± 1.7 yrs of follow-up, 25 showed cognitive decline, 11 progressed to which spans aprox ely 2.4Mb of Xp, has a high mutation rate and meet the NINCDS-ADRDA criteria for dementia of AD type. The cumulative intagenc recombination frequency. About fifty percent of DMD patients have incidence rate rose sharply by age; by age 105, it reached 1.0 for cognitive decline, a deletion mutation and 10 percent has duplication mutation. Most mutations and .64 ± .2 for dementia of AD type. The frequency of ApoE-e4 allele, a well- ae clustered in two hotspot regions; one in the 5' portion of the gene, and the established risk factor for AD, was not elevated in the affected elderly (. I vs. .13 in ote in the 3', more distal halfof the gene. However, the occurrence of 2 healthy elderly, .15 in the population and .43 in AD). The frequency of£2 was nonontiguous mutations in the same dystrophin gene is quite rare. Here we higher in the healthy elderly (.11 vs. .04 in affected elderly, .04 in AD and .08 in the repot a patient with DMD who has noncontiguous deletion and duplication population), supporting the notion that £2 may be protective. The average age at nitltions in gene. The was found dystrophin patient to have gait disturbance onset was 85.3 ± 6.4 for 90.6 ± 6.4 for£3 and markedly elevated serum creatin kinase level at age 6 years, and became £4-positive subjects, homozygotes, and wheelchair-bound at age 12 years. He was admitted to our hospital because of 99.9 ± 7.4 for -2-positive subjects. Only 17% of the affected elderly had a positive congestive heart failure. There was no family history. Quantitative Southern family history of dementia as compared to 14% of the healthy elderly and 43% of AD patients. However, those with a family history had earlier age at onset than those blot analysis using cDNA probes revealed partial deletion mutation of exons 3-7 without aad partial duplication mutation of exons 49-52 on dystrophin gene. There have a family history (86.7 ± 3.4 vs. 91.6 ± 7.1, P < .02). These preliminary data bean two reports of a single gene suggest that (a) age is the major risk factor for cognitive decline, (b) the lifetime dystrophin carrying 2 distinct deletions, but of dementia is not our patient is a novel case with noncontiguous deletion mutation and duplication probability influenced by family history of dementia or the mutation on dystrophin gene. Our finding testifies high mutation rate for both presence of£4, although these factors can influence the age at onset, and (c) £2 is delon and duplication of the dystrophin gene. associated with delayed onset and may be protective against dementia throughoutlift

518 519 Segregation of expanded CAG alleles in the dentatorubral-pallidoluysian Two cases of multiple severe congenital anomalies associated with atrophy(DRPLA) pedigrees. I.Kondo, F.Takakubo, A.Kuwano, Y.Morimota, prenatal cocaine exposure; expanding the phenotype of cocaine related MTakhashi. T.Yoshizsa and L Kanazaxa.JUniv. of Ehime, Ehime, Univ. of dysmorphism._. Krantz. N. H. Robin.E. i. P. Kaplan. and E. H. Kochi Med Univ.. Kochi, Univ. of Tsukuba, Ibaraki, and Univ. of Tokyo, Tokyo, ZackaiL The Children's Hospital of Philadelphia, University of Japan. Pennsylvania School of Medicine, Philadelphia, Pennsylvania. DRPLA is an autosomal dominant disorder characterized by ataxia, epilepsy, Cocaine has been shown to be a potent teratogen, causing its effects mental impairment and involuntary movement (chorea and myoclonus). The trin- through vascular disruption (vasoconstriction->hypoxic damage--> ucleotide, CAG repeat number in the CTG-B37 gene is expanded in patients with hemorrhage). We report two patients with severe congenital defects not DRPLA. Genetic anticipation is definitely observed in the pedigrees and time of associated with a known syndrome, after prolonged intrauterine onset and clinical severity of the symptoms depend on the CAG repeat number. exposure to cocaine. This report expands the phenotypes attributed to We have examined the CAG repeat length in the CTG-B37 gene and performed this teratogen. segregation analysis of expanded alleles in 39 siblings from 13 pedigrees. Ages The first patient was born at 32 weeks after a severe placental at onset of the disease in probands were between 28 and 30 years. Twenty six abruption. Features included hydrocephalus, schizencephaly, bilateral siblings were clinically affected and had expanded alleles of the CTG-B37 gene. corneal clouding, rudimentary low-set ears, microstomia, severe Then, segregation ratio of the expanded allele was significantly higher than the ex- micrognathia, deft palate, small nose, anteverted nares, widely spaced pected based on a dominant form of inheritance in siblings (c2=4.33. df=l, ptetrasomy Y. A. Kyriakakos. C. Sansaricq. M. A. Perle. C. Bamabe. Departments of dysmorphism and mental retardation. L.Larizza. P.Riva. P.Castorina. Pediatrics & Pathology, NYU Medical Center, Bellevue Hospital, NY, NY. L.DalprA. L.Doneda. S.Manoukian Dept. Biology and Genetics, Medical The relationship between behavioral neurodevelopmental abnormalities and the Faculty, University of Milan, Italy. presence ofadditional Y chromosomes has been previously reported. The relationship Eight NF1 patients presenting with dysmorphism and learning between skeletal andjoint abnormalities and Y chromosome aneuploidy has been much disabilities/mental retardation were selected for study as putative carriers less focused upon. We report a case of a 9 year old Hispanic boy with mild Mental of contiguous gene syndromes. Retardation (MR) and impulsive behavior who also has joint laxity and progressive High resolution banding allowed to detect a microdeleted 17ql 1.2 band scoliosis due to leg length discrepancy. in patient 1, who also displayed severe skeletal malformations and a Family history on the mother's side was negative for MR, but included six early 'borderline' microdeletion in the same band in patient 2, who also showed miscarriages, one elective abortion and one healthy daughter. The father who was not a thalamic hamartoma. involved in any of these pregnancies was a known user of cocaine and other drugs. Fluorescence In Situ Hybridization by using 3 NF1 region-specific YACs Premature labor started on the 20th week, treated with Ritodrine, and the delivery took (y12, y23, yK57) evidenced the lack of the hybridization signal on one place in the 39th week by Caesarian Section. chromosome 17 in patient 1, while fluorescent signals were visible on both Other findings included mild facial dysmorphism, short stature, bilateral simian homologues in patient 2. creases, normal male genitalia and hypotonia. X-rays revealed spina bifida occulta, In order to approximate the limits of the deletion in patient 1 and prove scoliosis, and an irregular calcification pattern in the proximal femoral epiphysis. Bone the deletion in patient 2 segregation studies were performed by means of age was normal. Endocrine and metabolic studies were negative exccpt for low 8 DNA polymorphisms, RFLPs and microsatellites, mapping both inside testosterone reading, for age. Full-scale IQ was 55. the NF1 gene and at close proximity. Analysis of informative markers Karyotype studies showed two cell populations in lymphocytes. 45X (2/31 cells) and allowed to prove that both patients carried a NF1 gene deletion spanning, 47X+2 identical large marker chromosomes (29/31 cells). Fluorescent QFQ banding at least, from intron 27 to intron 38 Patient 1 was heterozygous at locus revealed a large block ofheterochromatic material in the middle consistent with the long pHHH202 which is closely linked to the 5' end of the NF1 gene and is arm of chromosome Y. These two identical isodicentric Y chromosomes were thus centromeric to the deletion. The distal boundary of the deletion is, at composed ofduplication ofboth the Y short arms and most ofthe long arm. The point present, the extragenic locus D17S932. The closest markers for which offusion ofthe two Y's appears to be very close to the end ofthe heterochromatic region patient 2 was heterozygous were the Rsal RFLP in exon 5 of the NF1 gene (confirmed by FISH with various Y-specific DNA probes). Skin biopsy tissue and the extragenic D1 7S933 locus. Polymorphic loci flanking the 3' of the confirmed the findings with tetrasomy Y present 100% in one flask, 99% in the other. NF1 gene are under test aiming at establishing the extent of the deletion in To our knowledge, no such karyotype has been reported. both patients.

522 523 Risk of dementia among relatives of Aizheimer disease patients: what Is In store for the Phenotypic variability associated with del(22)(qll.2): A report of five familial .oldest old' NT. Lautenschlaner. LA Cupnles VS. Rao. and LA. Farrer for the MIRAGE cases. J. Leana-Cox. S. Panakanon, K. R. SIpvitz. M. S. Curtin, and E. A. Study Groun. Boston Univ., MA. Wulfsberk. Division of Human Genetics, University ofMaryland School of Medicine, Baltimore, Maryland. Despite recent advances in the molecular genetics of Alzheimer disease (AD), several The DiGeorge (DG) syndrome, the velocardiofacial (VCF) syndrome, and the fundamental questions concerning risk of illness are unresolved, namely, do Mendelian factors conotruncal anomaly-face (CTAF) syndrome have been described as distinct disorders, account for the incidence of the disease and is AD an inevitable consequence of the aging although overlapping phenotypes have been noted. That all three syndromes may process. To address these issues and other aspects of familial aggregation of the disorder, result from apparently identical or overlapping chromosome 22q11.2 deletions we evaluated life time risk and age at onset of AD in families of 1,516 patients ascertained in questions the distinctness and nosology of these disorders. the MIRAGE Study. The risk of AD by the age of 96 in 11,216 first-degree relatives was Of 6 del(22)(qll.2) cases identified in our laboratory using FISH (DG cosmid 37.0%t2.2%. By the age of 90 this risk was more than twice the corresponding risk to probe, Oncor, Inc.), 5 were familial. Two families were ascertained through a spouses. Although genetic factors likely have a major role in AD, the risk of 37% is not proband with a DG phenotype, 1 with a VCF phenotype, and 2 with a CTAF consistent with autosomal dominant inhentance. The observations of an apparent decline in phenotype. Four individuals (2 mothers and 2 daughters) had very mild phenotypes age-specific risk of AD after age 90 and 56 apparently unaffected persons surviving to age 96 (learning disabilities and mild facial dysmorphism), while 4 children had fatal cardiac without becoming demented suggests that few centagenarians will develop AD. These results lesions. These 5 families (a total of 12 individuals) were compared with 22 support the hypothesis that AD is not an inevitable concomitant of the aging process, a del(22)(ql 1.2) families (57 individuals) described in the literature. Common features conclusion which has profound implications for basic and applied AD research. We also found included mental impairment (95%), a dysmorphic face (92%), cardiac malformations that the risk of developing AD was higher in female than male relatives at all ages indicating (69%), and cleft palate or velopharyngeal insufficiency (46%). that women are innately more susceptible than men. The risk by age 85 among children of Familial del(22)(qll.2) manifests significant clinical variation encompassing DG, affected fathers was 1.7 times the corresponding risk to children of affected mothers. The nsk VCF, and CTAF phenotypes, with both mild and severe phenotypes occurring in the of AD among children of affected mothers was not significantly different from the risk among same family. These results suggest that the same gene(s) may be involved in DG, children of two cognitively normal parents. One explanation for the gender-related effects on VCF and CTAF. We propose use of the compound term "DiGeorge/velocardiofacial risk and transmission of AD is that the proportion of hereditary cases is higher in men than (DG/VCF) syndrome" in referring to this condition, as it calls attention to the women. Risk assessment in earty-onset and late-onset families yielded conflicting results phenotypic spectrum using historically familiar names. In addition, the high frequency suggesting that such distinction has little meaning in the absence of a biological marker. The of familial deletions in our series suggests that parents of affected children warrant age- and sex-specific lifetime risks derived from this study are sufficiently robust to be a molecular cytogenetic evaluation, since they may present with mild phenotypes. reliable source of information for counseling relatives of AD patients.

524 525 Ocular manifestations of Cornella de Lange syndrome. Trisomy 18 with Transient Thrombocytopenia and Anomalies of the Radius and Thumb: E. M. B. Interfaith Medical Center A. V Levin and J. H. Shin. University of Toronto, Ontario, Canada. Lieber, Dincsoy Olijade- CdLS is a syndrome of growth retardation, developmental delay Brooklyn Jewish Hospital limb abnormalities, characteristic facial features, and Since Trisomy 18 was first described in 1960 (Edwards et al.) There gastrointestinal refiux. have been several well documented reports on the various clinical Purpose: To expand the previous single large study of the manifestations of the syndrome. Over the years the spectrum has ocular manifestations of CdLS. widened to include less common clinical (skeletal-pre axial) features. The rare findings include upper limb skeletal involvement; abnormal Methods: The CdLS Foundation holds an annual family radii dysplastic and/or absent thumbs and Hematological conference that the authors attended from 1990-5 to conduct abnormalities - Table 1. We report here a Trisomy 18, with absent ophthalmic examinations on 73 affected children. 22 patients left radius and thumb and decreased platelets (table 1, case 4 previously reported by Levin (JPOS 1990;27:94-102) are comparing our case with other reports.) Because of the skeletal and included along with patients examined at The Hospital for Sick hematologic findings a chromosome breakage study was performed which Children. Examinations included oculofacial measurements, was normal. Because of these anomalies it has been suggested that embryologically there may be a common antecedent in the developmental visual acuity testing, hand held slit lamp biomicroscopy, pattern.** cycloplegic refraction, and dilated indirect fundoscopy. TABLE 1 ** Results: The patients manifested 100% hypertrichosis with long Case 1* Case 2* Case 3* mase 4 Case 5-12 arcuate eyelashes, 98% synorphyrs, 46% ptosis, 35% blepharitis, Sex F M F F 15% nasolacrimal duct obstruction, 16% strabismus, and 5% Weight 1600 gm 2600 gm 2300 gm 1880 gm nystagmus. New findings include 91% peripapillary pigment, Thumbs Lft.HVOP1 Abs. Bilat. ypopl.Lft bs.Lft. 2 cases 23% microcornea, and 8% macular hypoplasia. Myopia was the prevailing refractive error. Oculofacial measurements allowed for Radii Hypopl. Rt Hypopl.Bilat .ypop.Lft kbs.Lft 2 cases the creation of normative age matched curves. Cardiac + + + Conclusion: Ocular manifestations are prevalent in CdLS and Hemat M. NL.? Platel. platel._ may have diagnostic significance. All children should be 60,000 48,000 PolyHydr. + + - + assessed. The recognition of blepharitis may prevent nasolacrimal duct for similar unnecessary probing symptoms. *Rabinowitz et.al Radiol/89, f88,1967 J.L. Ramirez- CastroE. Bersu, AMJMG 2, 285:1978 A96 Posters: Clinical Genetics, Malformations and Dysmorphology (continued) 526 527 BOSMA sequence (Hypoplasia of the nose, eye coloboma, anos- Histopathology of an eye from a female carrier of choroideremia. LiL mia and hypogonadotropic hypogonadism). T.A.Lima, J.C.Llere- MacDonald' N.N e M Chen' D Addison'. 'Department of R.R.Martins, J.C.Cabral de Almeida. Ophmolo andSurgical Medical Research Institute, University of Alberta, na, F.R.Vargas, 1I.Jung, Edmonton, Canada; 3Department of Ophthalmology, Universit of Ottawa, Canada. IEDE/RJ, CGM (IFF/FTOCRUZ), UCH (IBCCF2/UFRJ), Rio de Janei- A donor eye from a 76-year-old female carrier of choroideremia (CHM) was ro Brazil. subjected to histopathological emion The eye was recovered approximately 4 We report in a young man with anosmia, hypogonadotropic hours after death from natural causes. Separate PCR-SSCP analysis in the family of hypogonadism, unilateral coloboma of the iris, lens and the patient confirmed mutation creating a stop condon in exon 9 of Rab escort protein- to a severe nasal hypoplasia. Cerebral 1 (REP-1) of geranyl geranyl transferase. Light microscopic examination revealed choroid associated thinning, depigmentation and subtle changes at the level of the retinal pigment and facial TC scans with tridimensional reconstruction revel epithelium (RPE). Transmission EM findings paralleled those of light microscopy. ed no brain abnormality but severe hypoplastic nasal composi The most striking feature seen was that from scanning EM of the RPE. Cells were te. Abdominal and pelvic ultrasound examinations were normal pleomorphic with loss of usual polygonal su and villae. The RPE is High resolution karyotype analysis with special attention to significantly affected by the CHM gene in the female carrier. This represents the first Xq22.3 region was normal. A similar disorder has been descri report of scanning electron microscopy of the RPE in a female carier with bed by Bosma et al (J.Craniofacial Genet 1981, 1: 153-184) choroideremia. in two unrelated males. An embryological insult to the -ol- phactory placode region (presenting normal migration of the LH-RH producing cells) and its neighbourhood around the 7th- 11th day of development, when olphactory placode, lens inva- gination, lens detachment and nasal swelling are formed, may have been the cause of this unusual combination of nasal,eye and gonadotropic abnormalities which bears considerations about the clinical heterogeneity of Kallmman's syndrome.

528 529 Retinal dystrophy in 18q- (de Grouchy) Uausual features in a neonate with 3C (craiuo-cerebelkoerdiac) syndrome. syndrome. S. Mahant and A. Levi. University of D P Marz and J J Muihl. Magee-Womens Hospital and University of Toronto, Ontuio, Canada. Pittsburgh, Pennsylvania. The 18q-de Grouchy syndrome is characterized 3C syndrome isthe designation for a syndrome that involves the cranium, cerebellum, by growth and mental deficiency, facial and heart (cor). The cranumis notable for a high and prominent forehead, with dysmorphism, extremity and genitourinary macrocephaly common. The cardiac defects typically include a septal defect and are malformations and ocular abnormalities. Four prior variable. ITe cerebeilar vernis is hypoplastic with a posterior fossa cyst, and have been hydrocephalus is usually present. cases with retinal dystrophy reported A female see on the first day of life had features consistent with the 3C syndrome: suggesting a locus at 18q21.1 for retinal high forehead and borderline narocephaly (95-99th percentile), a posterior fbosacyst, involvement. cerebellar hypopasia, absent cerbeIlarvermis, moderate hydocephah, and tetralogy Purpose: To use deletion mapping of phenotype- ofFallot. Herparen wereno conanuineow their only previous child was a healthy genotype correlations to further refine the locus for male. Her other features previously described in 3C yndrome, included hypoplaia of retinal dystrophy. the distal phalanges, hypoplasia of the sternum, short neck, wide open anterior and Methods: We report a child with del 18q21.3- posterior fontanelles, a prominent occiput, bitemporal narrowing, small low set ears, qter and typical features of de Grouchy syndrome. and a short upturned nose. She presented inthe neonatal period with weak cry and Retinal phenotype and electroretinographic (ISCEV floppy tone, although distal grip was preserved. She had early-onset seiue and standards) changes were consistent with a cone-rod plament ofaventriculo-peritoneal sabnt, but died at 8g days oflife. Autopsy dystrophy. revaled adysmorphic gyal pattern. At birth, the placental weht was in the 90th Conclusion: A review of all reported cases of percentile. Rmarble features not previously reported were 1) multiple cutaneous cytogenetically examined cases of 18q- strongly capillary malfbrmations, 2) extensive lobulaed nodules on the back ofthe tongue and upper pharym, and 3) cutis aplasia over the hypoplastic stenmm. These new features suggests the presence of a critical locus for cone-rod cases. dystrophy at 18q21.3 in contrast to prior reports exend the phenotypic spectrum 3C syndrome. Confirmation awaits future suggesting 18q21.1.

530 531 Different pattern of cardiac defects in patients with isolated and An Intermediate allele for Huntington's Diase Inherited through the syndromic tetralogy of Fallot. B.Marino, M.C.Digilio, S.Grazioli, unafected parent D. S. Markel and E. et. University of Michigan, Ann Arbor, R.Formigari, R.Mingarelli, A.Giannotti, B.Dallapiccola. Pediatric Michigan. A person at-risk for Huntington's Disease(HD) due to a family history of an Cardiology and Genetics, Bambino GesO Hospital, Rome and Public affected father presented to our clinic for entry into our presymptomatic testing Health and Cell Biology, Tor Vergata University, Rome, Italy. protocol for persons at 50% risk for HD. Family history information was obtained Tetralogy of Fallot (TF) is a common congenital heart defect fre- to confirm the history of the disorder in this family and a blood sample was quently associated with genetic syndromes related to an abnormality obtained from the affected father for genetic analysis. The affected father's result of neural crest cells migration, including DiGeorge (DGS), velocar- confirmed his clinical diagnosis of HD with his CAG alleles reported as 41/14. diofacial (VCFS) and oculoauriculovertebral syndromes. A microdele- With this information our at-risk client pursued genetic testing and her result was found to be 34/14. With this result two explanations were entertained: 1) that the tion of chromosome k2qll (del22) has been documented in the majority client's mother also had a 14 allele and that the 34 allele that was inherited was a of DGS and VCFS patients. The present study was designed to detect contraction of her father's 41 allele, or 2) that the client inherited the 34 allele from possible anatomical differences between TF occurring in isolated and her unaffected mother and that the 14 allele was inherited from her father. syndromic patients. The additional cardiac defects of 150 consecuti- Information about the lower frequency of the 14 allele in the general population ve patients with isolated (102) and syndromic (48) TF have been eva- made the possibility of the 34 allele having come from the mother even more TF presented a significant plausible. The family considered these possibilities with us and the unaffected luated by revision of clinical records. mother agreed to have her blood sample analyzed. A family history was obtained association with additional cardiac malformations in patients with from the mother for her side ofthe family and did not show any evidence of HD. branchial arch (11/21-pC0.01) and Down (l0/20-p<0.0001) syndromes. The mother's result was reported as 34/16. With this information we concluded that The subarterial ventricular septal defect with deficiency of infun- the intermediate allele in our patient at-risk for HD due to a genetically confirmed dibular septum (4/21-p<0.01) and the right aortic arch (6/21-p<0.05) affected father, was instead inherited from her unaffected and not at-risk mother. prevailed in patients with branchial arch syndromes, while atrioven- The interpretation of her risk ofdeveloping HD due to the presence of the 34 allele associated was given as very low since her mother at age 70 was neurologically normal. This tricular canal (10/20-p.92 and for 26 test- malformations. Survivors have a distinctive phenotype: normal weight or retest interviews K for BPI =.82, BPII=.72 and RUP=.72. Subjects with RUP had macrosomia, short nose, high and thin philtrum, low set posteriorly rotated a mean of 6 RDC depression symptoms and 5 episodes while manic patients had a ears, full cheeks, large downturned mouth, redundant skin, mental mean 6 RDC manic symptoms and 6 episodes. The results of anticipation retardation, sometimes dysostosis and pigmentation dysplasia.lf suspected, analyses were strikingly similar to those previously reported. diagnosis must be confirmed by chromosome banding after CVS, or skin fibroblast karyotyping with FISH using specific probes.

534 535 Pleiotropy in Hereditary Pancreatitis: insights from a kindred reascertained 25 Further delineation of acromesomelic dysplasia (AMD): Report of 2 caaes. L years later. E. Melvin and T. Markello. Department of Human Genetics, Mcdical Menasse-Palmer. A. Bogdanow. E. Hantman H. Pritzker and R. Marion. Montefiore College of Virginia, Richmond VA. (Intro. by: Arti Pandya) Medical Center/Albert Einstein College ofMedicine, Bronx, NewYork. Hereditary Pancreatitis (HP) is an autosomal dominantly inherited single gene Characterized by marked short stature with shortening ofthe middle and distal segments disorder of unknown etiology that results in chronic pancreatic inflammation with epi- ofthe limbs, AMD is a rare autosomal recessively inherited bone dysplasia. Since its initial sodic acute exacerbations. HP is often underdiagnosed and misdiagnosed. A known delineation in 1971, approximately 30 patients have been described. We report on 2 HP kindred (Kattwinkel et al., Pediatr.51:55,1973) was reascertained upon referral of patients recently diagnosed with this condition and review the previous literature. a 23-year-old proband with pancreatic stones. The family was initially reported to CASE 1: This 4 year old girl was initially seen at 5 months because ofdisproportionate have 209 family members in 5 generations, including 27 individuals known or short stature. The 51/2 lb product ofa fill term pregnancy to a consanguineous couple, she suspected to have HP. There are currently 595 members in 7 generations, and at least was noted early in life to have distal shortening ofher limbs, with normal facial features 43 individuals with HP. Five family members, who were originally ascertained to be and head circumference (HC). Radiographs at that time (repeated at 1, 2 and 3 yrs) failed nonpenetrant for the LIP gene, havc since developed pancreatitis. Some individuals to confirm a specific diagnosis. At age 4, height was 333/ in., weight was 25/2 lb (both are only now known to be carriers because their adult grandchildren have developed «<50/le, 505/oile for a 22 month old), and HC was 48/2 cm. (200/oile). There was frontal pancreatitis. These cryptic carriers were diagnosed with other gastrointestinal prominence without dysmorphic features and mild scoliosis was noted. Repeat radiographs problems, including ulcers, gallbladder disease, diverticulitis/diverticulosis, appendici- revealed characteristic features ofAMD. tis, mesenteric adenitis, and irritable bowel disease. These symptoms occur more fre- CASE 2: This 38 year old man was seen because of short stature. Early in life, a quently in carriers compared to that of the general population, or compared to that diagnosis of achondroplasia was made. Recently, he developed pain in his lower of siblings whose offspring are normal. Previous authors have suggested that these extremities; evaluation revealed spinal stenosis. The recipient ofa Ph.D., the patient is an patients were misdiagnosed, and that they actually have pancreatitis. Some Assistant Professor ofAnatomy. On physical exam: height was 118 cm, weight was 20 kg individuals in our kindred have been misdiagnosed whereas others are symptomatic (both <5%ile, 50'/oile for 6/2 year old), and HC 53 cm (5%ile). There was striking distal carriers with no radiologic findings of chronic pancreatitis on long-term follow up. shortening of the limbs and mild lumbar kyphoscoliosis. Radiographs revealed Reevaluating a large kindred after 25 years overcomes misclassification and nonpenet- characteristic features ofAMD. rance errors that would have hindered successful mapping of the HP gene by linkage These patients add the following to the established phenotype of AMD: (I) the X-ray analysis. In addition to providing new information about the structure and function changes ofAMD are not apparent in the newborn period and may require significant bone of the pancreas and pancreatic ducts, identifying the HP gene may lead to new maturation before the diagnosis can be made, (2) AMD is associated with normal (possibly insights into etiologies of other gastrointestinal diseases. above average) intelligence; (3) spinal stenosis may be a late consequence ofthe disorder.

536 537 Deletion of the elastin gene in an atypical case ofWilliams syndrome. W.S. Asymptomatic dystrophinopathy: detection by elevated AST levels, and Meschino and J.K. Finegan. North York General Hospital and Hospital for Sick identification of an intragenic double recombination event. A. Morrone"* E. Children, Toronto, Ontario, Canada. Zammarchi*. R.C.Hoopo M.A Donati S. SeryideiA G. Galluzzi*. E_P We describe a 6 year old boy with hypercalcemia, nephrocalcinosis, hypersensitivity Hoffmnan'. 'University ofPittsburgh, School ofMedicine-Pittsburgh. *Dept of to sound, inguinal hernia and moderate intellectual disability, all features consistent Pediatrics-Florence. AInst. ofNeurology,Catholic University-Rome. **CNR with Williams syndrome. In addition, however. he has several atypical features Institute ofCell Biology-Rome including unusual scalp hair patterning, agenesis ofthe corpus callosum, congenital Duchenne muscular dystrophy (DMD) and Becker muscular dystrophy (BMD) Horner syndrome, partial diabetes insipidus. congenital contractures, cryptorchidism are X-linked recessive diseases caused by mutations in the dystrophin gene. Both and micropenis. His facies arc dysmorphic but not typical for Williams syndrome. DMD/BMD patients show high circulating muscle creatine kinase (CK) levels, Specifically, he lacks stellate patterning of his irides, and has downslanting palpebral although transaminases (AST/ALT) often show elevations above the normal fissures and down-turned corners of his mouth. Comprehensive psychological testing range. Here we report the case of a 4-year-old girl that was referred for of this patient also reveals atypical features. His overall level ofintelligence is in the evaluation for a mild but persistent serum aspartate aminotransferase (AST) moderate of retardation with an score one standard elevation detected incidentally Serum CK activity was also found to be increased range mental IQ deviation below Immunohistochemical for in her muscle showed results the mean for our The for word-list is study dystrophin biopsy school-age sample. patient's memory learning consistent with a carrier state for muscular Molecular well into the and above the mean for our other dystrophy. work-up average range sample, although showed the proband to be a carrier of a deletion mutation of exon 48 of the memory abilities are poorly developed. In some respects his behaviour is like other dystrophin Four ale relatives also had the deletion mutation, yet showed children with Williams and gene. syndrome (e.g., greeting strangers warmly remembering no clinical of muscular 8 - 58 he also has echolalia and behaviour but not autism. symptoms dystrophy (age range yrs). Linkage names), although stereotypic analysis of the dystrophin gene in the extended family showed an intragenic Fluorescent in situ hybridization confirmed a deletion of the elastin gene at 7q 1.23 double recombination event: such intragenic double recombination has not been in this patient. Recent reports suggest that Williams syndrome may involve mutations previously reported although it is expected to occur in 1% of meioses. Based on ofcontiguous genes including the elastin gene. While hemizygosity at the elastin molecular finding, this family would be given a diagnosis of Becker muscular locus likely predisposes to vascular disease and other connective tissue effects, dystrophy. This diagnosis implies the development of clinical symptoms, even mutations ofthis gene alone are unlikely to produce mental retardation or though this family is clearly asymptomatic. This study underscores the caution hypercalcemia. We postulate that a microdeletion ofan additional gene(s) not deleted which must be exerasized when giving presymptomatic diagnoses based on in classical Williams syndrome may account for the additional features seen in this molecular studies. child. Molecular studies of this patient will be important in helping to delineate the critical region involved in various aspects of this intriguing condition. A98 Posters: Clinical Genetics, Malforrmations and Dysmorphology (continued) 538 539 Coffin-Siris Syndrome: Long-term Follow-up, Case Report & Review. Wiskott-Aldrich syndrome: an autosomal dominant variant. C M. Munson, and A A. Perszyk MD. Neri. A. Bellacosa. B. Rocca*. R. De Cristofaro*. M. Della Ventura*. N. Nemours Children's Clinic, Jacksonville, Florida, USA. Maggiano'. and R. Landolfi*. Institutes of Medical Genetics, *Internal The Coffin-Siris Syndrome (CSS) is a rare, recessive condition with mental Medicine, and 'Pathology, Catholic University Medical School, Rome, retardation, distinctive features, nail hypoplasia, and severe growth failure. Italy. The majority of clinical reports deals with the first 3 years of life. Infections Wiskott-Aldrich syndrome (WAS) is an X-linked recessive disorder have been a major concern, and few older children have been reported. characterized by eczema, thrombocytopenia and recurrent infections. The main WAS biochemical defect is the impaired expression of the This is a report of a 4 year old white female diagnosed with classical features membrane glycoprotein CD43 on most blood cells. CD43 appears to be a of the CSS. She has had hypotonia and significant delay in motor areas. regulator of cell maturation, survival and/or activation, and its gene is Pyloric stenosis required surgery in the first month of life. Despite this, she failed to gain weight. She was noted to have severe growth failure without located on chromosome 16q1 1.2. A gene called WASP located on Xpl1 endocrine etiology. Microcephaly was present without CNS abnormality. has been recently isolated; it encodes a putative transcription factor which Sparse hair, thick eyebrows, and nail hypoplasia were noted in the first is mutated in WAS patients and likely controls CD43 expression. months of life. Several geneticists have concurred on her diagnosis of CSS. We describe a family, studied over three generations, with several members presenting clinical and laboratory findings of WAS, including Developmental dysplasia with dislocation of both hips occurred by 2 years. decreased CD43 expression on T lymphocytes. However, at variance with Respiratory infection and bronchospasm necessitated frequent hospitalization classic X-linked WAS, this disorder shows a segregation compatible with in the first 3 years of life. A harness was used to control the hip dislocation autosomal dominant transmission. Due to the autosomal inheritance and to problems, but eventually surgery was performed. At the time of her surgery the impaired CD43 expression, we tried to determine whether the there were no reports of individuals affected with Coffin-Siris syndrome ever primary defect was associated in this family to CD43 alterations. Southern learning to walk. Our patient is now walking! She continues to be non-verbal, blot analysis failed to detect gross abnormalities of the CD43 gene. but does use some signs. Infections have lessened, but growth is continuing Moreover, genotype analysis revealed that the affected family members below expectations. do not share a common CD43 allele. Our dilemma of incomplete natural history information on CSS points out the The genetic data and the striking similarities between classic X-linked need for on-going and collaborative collection of data on rare syndromes. WAS and the autosomal disease here described, suggest that the gene products of these two disorders are likely to be functionally related.

540 541 The Holt-Orax Syndrome: Evidence for Genetic Mosaic Trisomy 7 in a male with Hypomelanosis of Ito and Heterogeneity. R.A. Newbury-Ecob. J.A. Terrett. multiple congenital anomalies. A.C. Newlin. R. Leanage*, J.D. Brook. I.D. Young. J.A. Raeburn, Centre M.M. McCorguodale. M.M. Miller and B.K. Burton. Univ. for Medical Genetics, City Hospital, Nottingham, UK, of Illinois Eye and Ear Infirmary. University of *Paediatric Cardiology, Groby Road Hospital, Leicester, Illinois college of Medicine. Chicago. Illinois. UK. A 19-month-old male presented with persistent open The Holt-Oram Syndrome (HOS) is a dominantly inherited anterior fontanel, short palpebral fissures, bilateral disorder characterised by congenital abnormalities of the preauricular pits, sparse scalp hair, micrognathia, left heart and upper limb. A genetic study of HOS in the UK ptosis, hypomelanosis of Ito and generalized hypotonia. identified 70 affected individuals from 27 families. He was the product of an uncomplicated pregnancy with a Clinical data shows that 100% had skeletal and 95% vaginal birth at 37 weeks gestation. A muscular VSD and cardiac abnormalities. These were bilateral, bicuspid AV without stenosis and mild tricuspid asymmetrical (L>R), and affected primarily the radial regurgitation were identified by echocardiogram in the ray. The gene is fully penetrant but wide variation in newborn period. An MRI of the brain revealed a Dandy expression is characteristic. 7 isolated cases had Walker variant. Peripheral blood karyotype in the atypical abnormalities (Tetralogy of Fallot, ulnar newborn period was normal: 46, XY. On examination, aplasia, unilateral radial aplasia) which were not found height and weight were below the third percentile while in familial cases suggesting that other heart-hand head to the fifteenth associations may exist which can be distinguished circumference plotted percentile clinically from HOS by the pattern of malformation. for age. Linkage analysis using polymorphic microsatellite Developmental milestones were delayed with the patient markers was carried out on 7 multiplex families with sitting independently at 15 months and standing with evidence of linkage to 12q in 5 families (D12S79 support at 19 months. He had no language development Zmar-7.76). 2 phenotypically indistinguishable families despite a normal BAER. A skin biopsy was obtained from did not show linkage to this locus. We conclude that the an area of hypopigmentation and was submitted for Holt-Oram Syndrome may be caused by mutations in more cytogenetic analysis. Results revealed mosaicism for than one gene. trisomy 7 in three independently established cultures. 28 out of 35 cells studies were trisomic for chromosome 7. There was no evidence of any structural abnormalities or other chromosomal aneuploidy. This represents the second reported case of mosaicism for trisomy 7 associated with Hypomelanosis of Ito.

542 543 Caruncle abnormalities in Oculo-auriculo-vertebral Tetramelle milror-image like polydactyly and de novo balanced autosomal spectrum. N. 1. Siegel-Bartelt2. A. V. Levin2. trasleoateon [46,XYt(2;14)(p23.2;q13)l. H. Ohashi 1, Y. KinW, M. lwasakit, NL1hawanI, T. Ohnol, M. Satol, S. Imaizumil, T. Aiharal, A. TamuishiT , Y. University of Torontol and the Hospital for Sick Children2, Fukushima2. 1Saitama Children's Medical Center, lwatsuki, Shinshu Univ. Sch. Toronto, Ontario, Canada- Med., Matsumoto, Japan. Introduction: The oculo-auriculo-vertebral spectrum (OAV) is Mirror polydactyly is a rare type polydactyly, in which the ulnar side are believed to be caused by defective development of the 1st and symmetrically duplicated and a thumb is absent. The cause is still unknown 2nd brachial arches and the 1st brachial clefts during weeks 4- although several homeobox genes have been implicated in pattern formation of the 8 of embryologic development and includes Goldenhar limbs in embryogenesis. We report here on a male infant with tetramelic mirror- image like polydactyly associated with de novo balanced translocation between 2p syndrome. There are only 3 previous reports of abnormal and 14q. carundes in OAV. The patient, a male infant, was the second product of normal parents. There was Methods: We have reviewed our experience with a no family history of malformation. At birth, he was noted to have left inguinal consecutive series of seven OAV patients with caruncular hernia, and tetramelic polydactyly with six fingers on both hands and 8 digits on malformations and report dysplastic and/or bilobed both feet, forming a mirror-image like configuration especially in feet. Radiological carundes(2) and ectopic carunces (2 bilateral and 3 unilateral). examination revealed that all fingers had three phalanges except the first finger This series had an unusually high incidence of nasal with two phalanges on the right hand, and that 8 metatarsals on the right foot and 7 on the left. There were no abnormality in long bones of forearms and lower legs. involvement (5/7). Chromosome analysis showed autosomal balanced translocation Conclusion. Our experience suggests that the incidence of [t(2;14)(p23.3;ql3)j. Chromosomes of parents were normal. caruncular malformations in OAV is higher than previously This case might suggest that one of the genes responsible for mirror-image reported. Linking abnormalities in months 1-2 of gestation polydactyly was located at 2p23.2 or 14ql3 involved in the translocation of the causing OAV with malformations of the carundes, which patient. These two chromosome sites have not yet been known to bear any normally develop in month 3, may offer clues to the homeobox genes previously identified. pathogenesis of OAV. Posters: Clinical Genetics, Malformationis and Dysmorphology (continued) A99 544 545 Genetic aspects of Facioscapulohumeral Muscular Dystrophy. Variable age of onset and non-penetrance in a large pedigree with autosomal G.W. Padberg'. J.C.T. van Deutekom2. E. Bakker2. R.R. Frants2. dominant hemolytic uremic syndrome. S. E. Palmer1, B. S. Kaplan2, and I. 'Department of Neurology, University Hospital St Radboud, Nijmegen, Elshihabil. Dept. of Pediatrics, Univ. of Texas Health Science Center at San The Netherlands, 2MGC-Department of Human Genetics, Leiden Antonio1, and Div. of Nephrology, The Children's Hospital of Philadelphia2. University, Leiden, The Netherlands. Familial hemolytic uremic syndrome (HUS) is a rare group of disorders In order to assess the prevalence of FSHD and its various genetic with atypical presentation of HUS (without bloody diarrhea or E. coli 0157:H7 aspects an attempt was made at complete ascertainment in infection). Few families with autosomal dominant HUS have been reported; the Nether- most have onset in adults with poor renal prognosis. Autosomal recessive fam- lands. ilies present with recurrent episodes of HUS in infants or children. We recently Extrapolation of a previous study in one province suggests that the studied an unusual family with both adult and childhood presentations follow- present roster of 145 probands and families with FSHD contains nearly ing an autosomal dominant inheritance pattern, and apparent non-penetrance all expected cases. Coupled to molecular genetic studies it allows to in some individuals. A male (age 17 months) presented with acute onset of estimate the prevalence of 1 in 20.000, a mutation frequency of 10%, HUS in a manner indistinguishable from sporadic atypical HUS. He is manag- a small sex-difference (earlier onset, more probands, less asymptomatic ed with chronic peritoneal dialysis. Family history is significant for HUS af- fecting a paternal uncle (onset age 41) and aunt (onset age 19); one receives di- gen-carriers, more sporadic cases and an increased frequency of mental alysis and one was transplanted. The paternal grandmother developed renal retardation in male patients), and a frequency of 13% for non-4q35 failure with clinical and histological features of atypical HUS following child- linkage. Chromosome 4q35 linked families showed a correlation be- birth (age 35); onset of HUS during or following pregnancy has been reported tween earlier onset and a larger EcoR!-deletion detected with p1 3E- 1. in other families. The paternal great-grandfather died of renal failure at age Comparing clinical and genetic data we found FSHD clinically homo- 68; the diagnosis of HUS is not certain. His nephews son died from HUS at geneous although genetically heterogeneous. age 6. Intervening non-penetrant individuals who are obligate carriers of the gene with no known renal or blood disease include the living fathers (ages 37 and 70s) of the affected children, a grandfather and a great-great grandparent (deceased). This pattern of presentation suggests anticipation with earlier onset in successive generations. As many individuals were affected prior to the recognition of HUS and its familial pattern, this kindred illustrates the need for a thorough family history in apparent sporadic cases. As yet there are nv known genetic markers or presymptomatic diagnosis for this disease.

546 547 Sperm aneuploidy frequencies in men with Congenital Absence of the Clinical and molecular analysis in Joubert syndrome. Jl.Es elleia= Vas Deferens (CAVD) with or without identified mutations in the Cystic D.B. Flannery2, E.H. Zackail, Mh. Muenkel, P.F. Chancel. IThe Children's Fibrosis gene. P. 1. P. Van Hummelen2 E. Panica2. X. Lowe2 and AJ. Hospital of Philadelphia, University of Pennsylvania School of Medicine, Wyrowk2. 1Div. Reprod. Endocrinol., University of CA, Irvine; 2Bio. Biotech. Res. Philadelphia, PA, 2Medical College of Georgia, Augusta, GA Prog.. Lawrence Livermore National Laboratory, Livermore, CA. Total or partial absence of the inferior vermis is the most common cerebellar CAVD is a reproductive disorder that causes sterility in men which may be malformation, and may be associated with the Joubert syndrome. According to overcome by in vitro fertilization with aspirated epididymal sperm. Approximately criteria of Saraiva and Baraitser (1992), Joubert syndrome is defined by cerebellar 60% of patients with CAVD carry one or more mutations in the cystic fibrosis (CF) vermis hypoplasia, hypotonia, developmental delay and either abnormalities of gene while the carrier frequency in the general population is only 4%. CAVD patients breathing pattern, or eye movements. We reviewed clinical information on 41 and especially patients carrying a mutation in the CF gene also had lower fertility rates patients meeting these criteria. Cases of other cerebellar hypoplasia syndromes with in vitro compared with healthy men. Our objective was to determine whether CAVD overlapping features such as OFD type VI. Dekaban and Arima syndromes were patients with or without identified mutations in the CF gene have higher frequencies of excluded. There were 10 sib pairs (1 with consanguinity) and 23 single cases (2 aneuploidy or diploid sperm compared with healthy men. with consanguinity). All patients had cerebellar hypoplasia, hypotonia, and Epididymal sperm was analyzed from 4 CAVD patients and semen from two healthy developmental delay; 73% had episodic hyperpnea and 73% had abnormal eye controls. One CAVD patient was homozygous for DF508 and showed mild movements. There was clinical variability with polydactyly in 7% and colobomas in preliminary CF disease, one was compound heterozygous for WI 282/5T and two 5%. were negative on a CF screening. Ten thousand sperm were evaluated per donor using A subset of these patients were evaluated by molecular analysis. The swaying multi color FISH with alpha satellite DNA-probes for chromosomes X, Y and 8. All (sw) locus in the mouse is a recessive mutation leading to absence of the cerebellar CAVD patients showed frequencies of aneuploid and diploid sperm within the range of vermis and ataxia. The sw phenotype is due to a mutation in the wnt-1 gene, a values for healthy men. developmental gene highly expressed in the CNS. Due to the phenotypic similarity Although the number of men evaluated in this study was small, our findings suggest between the sw phenotype and Joubert syndrome, we studied the human WNT1 that epididymal sperm of the CAVD patients have frequencies of aneuploidy and gene, mapping to 12ql3, as a candidate for this disorder. Six familial cases (1 diploidy similar to ejaculated sperm of healthy men. These preliminary findings also consanguineous) and 10 isolated cases (1 consanguineous) were screened by DNA suggest that double mutations of the CF gene in CAVD patients do not have major heteroduplex analysis. However, no mutations were detected in the four exons or effects on sperm aneuploidy or diploidy. Further studies are needed to determine promotor region of the WNT1 gene. Other candidate genes should be investigated whether mutations in other specific regions of the CF gene have effects on sperm in this disorder. ancuploidy or diploidy in affected patients. [Work was performed under the auspices of the U.S. DOE by the Lawrence Livermore National Laboratory under contract W-7405-ENG-48; P.V.H. was sponsored by NATO-Beigium].

548 549 Primordial Dwarfing Condition with Cleft Palate, Neurosensory Deafness, and Evidence against genetIc antcpatIon in familial colorectal cancer. Y. Tsai. G.M. Digital Anomalies: Case Report and Critical Review. Petersen. S.V. Booker. J.A. Bacon. and F.M. Giardiello. School of Hygiene and A. A. Perszyk MD. Public Health, and School of Medicine, Johns Hopkins University, Baltimore, MD Nemours Children's Clinic, Jacksonville, Florida, USA. Background: Anecdotal reports of hereditary colorectal cancer suggest that genetic anticipation of age of onset occurs. However, problems of ascertainment bias and birth A 17 year old white female with primordial short stature, Pierre-Robin cohort effects confound the study of this phenomenon. Methods: We examined data sequence, deafness, and digital anomalies is described. She was born in the Hereditary Colorectal Cancer Registry at The Johns Hopkdns Hospital with premature and SGA She was noted to have multiple congenital anomalies methods that correct for such biases. Family history questionnaires were completed by immediately at birth, and poor weight gain was seen. Craniofacial features persons from across the U.S. who may or may not have had colorectal cancer; familial include microcephaly, frontal bossing, short pre-maxilla, with a prominent adenomatous polyposis pedigrees were excluded. Of 588 pedigrees, 326 had at least beak-like nose. Profound neurosensory deafness and high-grade myopia are one affected parent-offspring pair. Excluding 28 pairs in which only one of the present. Both upper and lower extremities show progressive, distal members had completed the questionnaire, 548 pairs were left for study. Results: We hypoplasia. The hands and feet are extremely small. The digits are observed a birth cohort effect among offspring, in that the mean ages of onset of those angulated at multiple joints due to accessory triangular phalanges. Nails on born before 1921, between 1921 and 1930, and after 1930 were 64±12 (SD), 58±9, all digits appear small and were hypoplastic at birth. The hair over the scalp and 44+9, respectively, while their parents' mean ages were 65±13, 66±14, and is sparse, fine and slow growing. 61+15, respectively. We observed no effect of parental gender on age of onset in offspring of either gender. Because of variation in observation periods, pairs in which She has a normal blood karyotype. Skeletal survey did not suggest a the parent had an older onset age than the present age of the offspring were excluded, dysplasia. Endocrine evaluation found no hormonal basis for her shortness. leaving 254 pairs for analysis. The mean age ofdiagnosis ofparents was 60.1 +12, and She has been treated with growth hormone, and yet her height is that of a 9 that of the offspring was 59.9+13; pairwise comparisons were not significant (t-.33, year old. Microcephaly is profound, yet a brain scan shows no gross p> .05). Life table analysis also showed there is no difference in onset age between abnormality. She still has no functional speech. She has moderate range two generations (Gehan Wilcoxon statistic=.034, p> .05). Parent-offspring correlation cognitive deficits. of onset age was .41. A subgroup analysis was also done on 93 pairs from families Our patient does not fit easily into any known syndrome. Early facial features classified as hereditary nonpolyposis colorectal cancer. No significant differences were have suggested Hallermann-Streiff syndrome. Individuals with similar hand observed (mean onset age was 54.1+11 for parents, 54.9±15 for offspring, parent- correlation = ConclusIon: There is no evidence that findings have rarely been seen. Our patient's features suggest a contiguous offspring .35.). genetic be anticipation or genomic imprinting of onset age occurs in this sample of colorectal gene syndrome may responsible for her uniqueness. cancer families. A100 Posters: Clinical Genetics, Malformations and Dysmorphology (continued) 550 551 Apple Peel Syndrome: Another Gastrointestinal Manifestation of Cystic Fibrosis. Systemic lupus erythematosus in a man with Noonan syndrome: Is R. Petrella1, L. Gonzalez2, and autoimmune disease a rare feature of this syndrome? EM Pety and CF L.R. Shapirol. Divisions of Medical Geneticsl and Int. Med., Univ. of Med. Ann Neonatology4, Department of Pediatrics, New York Medical Gencyuz. Michigan Center, Arbor, MI. 48109. College and Westchester We describe a 20 yearold Caucasian male who we diagnosed with Noonan County Medical Center, Valhalla, syndrome based on clinical diagnostic criteria (Duncan WJ et al. AJMG 1981). He New York. had short stature (< a broad neck with low The Apple Peel Syndrome is a specific form of jejunal 3%); posterior hairline; craniofacial atresia which is inherited in an autosomal dysmorphism characterized by ptosis, malar hypoplasia, wide-set eyes, a prominent recessive and arched upper lip, and low-set ears; strabismus; a chest wall deformity (pectus fashion and is characterized by intestinal atresia, carinatum and abnormal mesenteric vessels and the resultant twisting of superiorly, excavatum inferiorly) widely spaced nipples; the intestine which suggests an apple peel or Christmas hypogonadism; cafe-au-lait spots; learning problems; and a normal karyotype (46, tree XY). He was in good health, without known cardiac disease, until 17 years of age appearance. Ordinary jejunal atresia, intestinal when he a diffuse obstruction and meconium ileus are known to be associated developed polyarthritis, low-grade fevers, pericarditis, oral ulcers, with generalized alopecia, anemia ofchronic disease, and mixed mitral valve disease. He Cystid Fibrosis. Apple Peel Syndrome can be was subsequently diagnosed with serologically positive systemic lupus erythematosus associated with additional gastrointestinal and non- (SLE). Recent echocardiograms have documented mixed mitral valve disease with gastrointestinal manifestations; however, we have noted severe valve only one other case in the French literature linking this regurgitation (necessitating replacement at 20 years of age) but no other specific of structural cardiac defects. No first degree relatives have Noonan syndrome or SLE. form jejunal atresia to Cystic Fibrosis. The Since the first of with a patient was delivered at approximately 36½ weeks gestation report patients distinct syndrome characterized by short by C-section because of prenatally diagnosed small bowel stature, characteristic facies, and valvularpulmonic stenosis in 1963 (Noonan JA and obstruction. Ehmke DA, J Pediatr), several subsequent reports have been published further Surgical findings revealed a volvulus, delineating the cardinal features of Noonan syndrome and broadening the spectrum of multiple jejunal atresia of the apple peel type, and less common inspissated stool impacting the distal colon. Sweat manifestations. The majority of individuals have cardiac defects, most chloride testing was positive for Cystic Fibrosis. often pulmonic stenosis. Significant involvement of the mitral valve is rare and our DNA patient's mitral valve disease is most likely due to his SLE. SLE has not been reported mutation analysis for confirmation and delineation of the in individuals specific mutations responsible is presently underway. with Noonan syndrome and it may be a coincidental finding in our It is likely that the Apple Peel Syndrome is patient. Other forms of autoirnmune dysfunction, however, have been infrequently a described including case reports of hypothyroidism, vasculitis, vitiligo, and anterior gastrointestinal manifestation of Cystic Fibrosis which uveitis may account for the autosomal recessive mode of inherit- (rev. in Sharland M et al. Arch Dis Child. 1992). Given this, further studies of ance, and routine Cystic Fibrosis testing should be done autoimmune disease in adults with Noonan syndrome is warranted to determine if in newborns affected with this condition. autoimmune dysfunction is a rare feature of this disorder.

552 553 A paracentric inversion of 16q in a patient with anus, hand, and ear Brachydactyly type Al with bilateral discoid menisci and scoliosis in anomalies: further evidence for a Townes-Brocks syndrome gene at three generations. M.L Raff.K.A. Leppiig. J.C. Rutledge. R.A. Pagon. 16q12.1. C. M. Powell. P.J. Reitnauer. K.A. Kaiser-Ropers, K.W. Rao. The Childrens Hospital and Medical Center and University ofWashington School of University of North Carolina at Chapel Hill. Medicine, Seattle, WA. Townes-Brocks syndrome (TBS) is an autosomal dominant syndrome of We report a three generation family with brachydactyly type Al accompanied by variable scoliosis and degenerative arthritis of the knee as a complication ofdiscoid anus, thumb, and external ear anomalies, and sensorineural deafness. meniscL The proband, a seven year old boy with height at the 65th centile and normal Intelligence is usually normal. There have been three patients reported with development, had hand findings of severe brachydactyly type Al with short middle typical TBS and chromosome abnormalities: a father and daughter with a phalanges and metacarpals of both hands and absent middle phalanges and short pericentric inversion of chromosome 16 [inv(16)(pl 1.2q12.1)] and an infant proximal phalanges ofboth feet. He had chronic knee pain with bilateral discoid with a reciprocal translocation between chromosomes 5 and 16 menisci evident on MRI scan. There was no scoliosis by examination. His 42 year Based old father was atthe 50th centile for height and had similar hand and foot involvement [t(5;16)(p15.3;q12.1)]. on these cases, a TBS gene has been Scoliosis was diagnosed at 18 months of age and required osteotomy at 13 years and tentatively localized to 16q12.1. internal fixation at 15 years. He had left lateral meniscectomy at 8 years of age for We have seen a 16 year old male with severe mental retardation and recurrent inflammation of a cyst over the lateral aspect of the knee. Histology of the sensorineural hearing loss, an imperforate anus, cupped ears with tissue removed with the meniscus demonstrated fibrous tissue with cystic preauricular tags, and thumb anomalies. Chromosome analysis revealed a de degeneration. novo paracentric inversion of 16q: 46,XY,inv(16)(ql 1.2q12.1). Of the 15 individuals that comprise the two generations preceding the proband, nine have brachydactyly. Of these, three have scoliosis, and one has radiologically This case is the first paracentric inversion found in association with all of the confirmed discoid menisci. Three others (including the father) have degenerative knee TBS features. The breakpoint at 16q12.1 may disrupt a critical gene for arthritis but have not been assessed for discoid menisci. Males and females seem to be normal anus, hand and ear morphogenesis. The severe mental retardation equally affected in number and severity. and additional dysmorphic features may be caused by a submicroscopic Autosomal dominant inheritance of brachydactyly Al and discoid menisci have been deletion with loss of several genes resulting in a contiguous gene syndrome. reported separately. However, cosegregation of these features in one family has not previously been described and seems to comprise a unique autosomal dominant Alternatively, there may be a position effect with several genes malfunctioning condition. The combination of brachydactyly, scoliosis and unusual meniscal due to movement from a euchromatic to a heterochromatic region. The parent configuration suggests that this disorder represents a new osteochondrodysplasia of origin may determine the phenotype in patients with chromosome syndrome. Based on these observations, patients with brachydactyly Al should be abnormalities in this region. There have been three published cases of evaluated for the presence of scoliosis and knee problems. matemal uniparental disomy for chromosome 16 with imperforate anus, one case also had an ear abnormality, suggesting the possibility of an imprinted gene. Further molecular and cytogenetic studies of our patient are planned.

554 555 CONGENITAL PANCREATIC HYPOPLASIA IN BEDOUIN SIBS arain anomalies, &etardation of mentality and growth, jctodermal UshaRajaram, R..Una, K.Tahseen, M.M.AI-Ghanim, T. I .Faraq, Department dysplasia, Skeletal malformations, Iiirschprung's disease, Ear deformity of Pedi;atrics, AI-Jahra Hosp tal and Medi -cri7 'fi-csenitre,Kuwa It. and deafness, eye hypoplasia, Wleft palate and Kidney dysgenesis / hypogenesis (BRESEK / BRESHECK). A new X-linked syndrome..42ij Combined exocrine and endocrine pancreatic insufficiency, due to Res1.6 2 3 3 4 congenital pancreatic hypoplasia is a rare syndrome iMIM 260370i. L obSeLtGorlin ,MafaiaHorinsky L.UEllen9-Rst ElitharaBurke andLSusan We report a consanguineous Beduoin couple with four healthy sons and A. BeMv15 Department of Pediatrics1, Oral Pathology2, Dermatology3 Laboratory two daughters, with very earIy neonataI presentation. The mother had Medicine and Pathology4, and the Institute of Human Genetics5, University of non-insulin dependent gestational diabetes durinq these last two and preanancies. Neither parent had islet-cell antibodies and their Minnesota, Minneapolis, MN, Sackler School of Medicine, Tel Aviv University6. trypsin like reactivity was not low. The clinical features In the We present two half brothers (maternally related) who had a similar daughters Included intrauterine growth retardation, persistent hyper- of malformations. These glycemia without ketoacidosis, delayed passaqe of cast like meconium, recognizable pattern congenital included hydro-microcephaly followed steatorrhic in both and dilatation of the spinal canal with thalamus fusion in one. Primordial growth by stools. The fasting and peak C-peptide delay was noted in both with severe mental retardation in the surviving brother. Both insulin levels were very low and duodenal fluid showed absent trypsin had ectodermal with hair and lipase activity. Insulin, pancreatic enzyme supplementation and dysplasia scaling, hyperkeratosis, sparse but normal sweat nutritional glands. Skeletal anomalies included hemivertebrae with abnormal segmentation in one support improved growth velocity, and normal mental deve- and scoliosis with polydactyly in the other. Ears were large and protruding in both with lopment over a 3 year follow up in case 1, and 1 year follow up In one case 2. The mixed hearing loss in the who survived. Both had dysplastic / hypoplastic kidneys neonatal, simultaneous presentation of exocrine and endo- of a variable of which crine insufficiency in the contrast with the degree severity resulted in the early post-natal death of one present sisters, only sibling. Features that were noted in only one or the other sibling included aganglionosis other report of similar disorder in two brothers where the endocrine of the rectum and and sub-mucous abnormality presented earlier and exocrine much later. Neonatal IDDM colon, microphthalmia cleft palate. The spectrum of these defects has not been previously described. We suggest that this comprises a new alone has been reported rarely with maternal diabetes. Buth the syndrome with X-linked inheritance. The BRESEK reflects the evidence of combined deficiency can be best explained on an autosomal probable acronym recessive features shared in common while BRESHECK denotes the broadest spectrum seen in mode of inheritance. one or both patients: jrain, Retardation, Ectodermal dysplasia, Skeletal deformities, liirschprung's disease, Ear / eye anomalies, Cleft palate and Kidney dysplasia i hypoplasia. Possible mechanisms for this syndrome could include a submicroscopic contiguous gene deletion or mutation of an undefined single gene expressed in the affected organs. Posters: Clinical Genetics, Malformations and Dysmorphology (continued) Al01 556 557 Temporary brittle bone disease-proof beyond a reasonable doubt. R M. Roberts. Sagittal Craniosynostosis with Hand and Foot Soft Tissue Genetics and Prenatal Diagnostic Center, East Ridge, Tennessee. Syndactyly in a Five Generation Kindred: A New Syndrome? The features ofan osteogenesis imperfecta-like syndrome have been delineated by Dr. Cohn Paterson, characterized by brittle bones primarily in the first year oflife, with N.H. Robin1, B. Segal2, G. Carpenter23, M. Muenkel. IChildren's paradoxically no bruising, even in affected individuals with abnormally easy bruising; in Hospital of Philadelphia; 2Geisinger Clinic, Danville; 3Thomas 2/3rds ofcases, one parent has unusual joint laxity; most factures occurred only in the Jefferson Univ., Philadelphia, PA, USA. first 6 months oflife, many affecting the ribs; many affected children had large fontanels and blue sclerae. Osteopenia commonly is not visible in radiographs. Many affected The Acrocephalosyndactyly (ACS) syndromes are a group of children have been removed from their parents, some ofwhom have been prosecuted and genetically related disorders that share the features of imprisoned for child abuse. Geneticists have acted as expert witnesses to help prosecute craniosynostosis and hand and foot anomalies. Here we present some ofthese cases, because the conventional wisdom in the USA among expert the clinical findings of 8 affected individuals from a 5 generation radiologists ofsyndromes holds drh ribfractures with no evidence ofosleovnia are kindred segregating a novel type of ACS. typical ofchildaabrse. In the case here presented, one local geneticist was to help the Sagittal craniosynostosis was present in 1/8. while 6/8 had prosecution ofthe patient's father because ofthis erroneous belief I present a novel head shapes consistent with premature closure of the sagittal statistical analysis which would have been used by the defense attorney to force the suture. In addition, 2 other affected family members were genetic witness for the prosecution to calculate that the odds that this child was imot reported to have had surgery to correct sagittal synostosis. All 8 affected with brittle bone disease were greater than one in 500 billion. This analysis is affected individuals examined had soft tissue syndactyly of accomplished simply by multiplying the chance ofeach unusual feature which may be digits 2-5 of the hands and associated with temporary brittle bone disease be present by chance in an abused. 1-5 of the feet. Radiographically, unaffected child. In this instance: 1/400 for a clavicular fracture at birth there was hypoplasia and sclerosis of the distal phalanges, but having (no no fusion. There shoulder dystocia) times 1/10 for multiple wormian bones, times 1/50 for an unusually boney were no other findings radiographically large fontanel, times 1/500 for physician-documented easy bruiseability with a negative or clinically, such as facial dysmorphia, hypertelorism or hematological work-up, times 1/50 for usually blue sclera, times 1/100 for a mother with midfacial hypoplasia. Intelligence was normal. no athletic training with joint hypermobility such that she could do the splits side-to-side; While this family shares a number of general features with further unusual features suggestive ofcollagen abnormalities were also present. This the classic ACS syndromes, the type of craniosynostosis, the analysis I hope will lead to a reassessment in this country of the utility oflack of absence of facial dysmorphia, and the type hand and foot radiographic evidence ofosteopenia in helping to convict parents ofchild abuse in these anomalies are distinctive, representing an apparently novel ACS cases. syndrome.

558 559 Fancoai ama (FA) and idiopath6c interstitial lung disease (ILD). )W. S.f binstW, subnormal binocular vision in the Williama . L. R. M. and J. J. Mulvihill"2. Departments of Human syndrome.LS..LSadJer Genetics', Pediatrics', and Medicine', University ofPittsburgh, PA. S.E.01itsky.J.Reyolds. SUNY at Buffalo and the Many syndromes are knownfortheir congenital or pediatric n o and the serious Children' a Hospital. occasonal adult-onset features are poorly recognized. The possible association ofRLD with Strabismus is a frequently recognized feature of the FA was raised in a 38 year old Caucasian male carrying a diagnosis ofHolt-Oram syndrome Williams syndrome (WS)(Greenberg et al.,1988; Kapp et who preanted with diabietesm ius(sDM). He had recently taken prednisons for idiopathic ILD al.,1995). We prospectively evaluated 5 males and 7 arldtlmtoin _ wh miforUsinusitis Thobc Mpnapror etoi w females with the WS in order to further delineate The patient had a dry cough, prpuIbertal voice, short stature, m ccep 1e ophthalmologic function in this disorder. Four patients ingmented , petechiae, sparse facial and pubic hair, gynecomastia, inpiratory and had a manifest esotropia (33%). Of the remaining 8 xpirstory wheezing, and dry basilar crackles. Radiologically there was absence ofthe right patients, examination of binocular vision was possible in th b right and left first eals, hypoplastic left Ftimb phalanges, delayed bone age in 6, all of whom demonstrated reduced stereoacuity. infan, an anomalous right hrd rib, crossed fued ectopy of the kidneys, and IVC Subnormal binocular vision without manifest stabismus transpositn. He underwent VSD repair at age 6 and pacemaker insertion at age 30. He is regarded as a sensory defect rather than an ocular stopped snoking at age 32 after 21 pack-years, dyspnea began in his late 20's, and chest X- motor problem. Reduced binocularity may itself predispose rays showed increased interstitial markings at age 25. At age 38, chest CT showed bilateral to the development of strabismus. Previous reports of NRI upper lobe fibrosis, lower lobe honeycombing and bronchiectasis. Pulmonary fimction tests studies of individuals with the WS have disclosed were compronsed at age 29, and by age 38 showed a moderately severe obstnuctive/ consistent structural abnormalities including a relative restrictive pattern, with FEVI/FVC ratio 0.39, FEVI 16%, FVC 33%, FRC 93%, and DLCo decrease in posterior brain width (Bellugi et al.,1990). only 38% predicted. Alpha-l antitrypsin level was 335 mg/dL and Pi phenotype was Ml. Gross and histopathologic studies of the brain of an Karyotype was 46,XY with spontaneous gaps and breaks in 5/100 cells. The diepoxybutane- adult patient with the WS showed small abnormal gyri in induced culture contained 33 gaps, breaks, trir quradials, a dicentric, acentric the region of the mesial occipital surface and scattered fragments, and interchangefigures among 100 cells (normal< 10 gaps or brea/100 cells). areas of immature neuronal organization, most notable in Marked phenotypic variation in FA is observed in age of diagnosis, dysmorphism, the visual cortex (Galaburda et al.,1994). Studies of hemaologic iolv ~ and related illnessesuch asDM and malignancy. The early onset non-human primates have linked binocular function to and degree ofpulmonary disease in this patient cannot be fily explained by ev r l or cells in the striate cortical regions 17 and 18 (Poggio known genetic causes. The International Fanconi Anemia Registry contains no example ofa et al.,1977 and 1984), those areas found to be most simar pulmonary presentation (A. Auerbach, personal communication); the uniqueness may abnormal in the case report referenced above. We suggest rlate in pat to te older age ofthe patient. A clearer understanding ofthe pulmonary disease that subnormal binocular vision, strabismus, and the sam inPtispatient must awaitfit insight into the cellular defects imposed by FA mutations. severe visuo-spatial deficits observed in patients with the WS may be related to abnormal brain morphogenesis ir the region of the occipital cortex.

560 561 Tricho-Rhino-Phalangeal Syndrome Type II (Langer-Giedion) with Two male siblings with macrocephaly, mental bilateral agenesis of tibia and first metatarsus in a boy with 8q interstitial retardation, seizures, cavum septi pellucidi and cavum deletion. C F Salinas1l.E S Cant1, G S Pail, and C Turleau2. 1Medical verga. A new hereditary syndrome?. 0. SAnchez, J.A. Nastasi, J.A, Escalona. Unidad de Genetica Medica, University of South Carolina, Charleston S C and 2H6pital Necker- Zscuela de Medicina, Universidad de Oriente, Ciudad Enfants-Malades, Paris, France. Bolivar, Venezuela. The Langer Giedion Syndrome (LGS) belongs to a group of conditions apparently caused by the deletion of a set of contiguous genes on a Cavum septi pellucidi together with persistence of chromosome (Schmickel, 1986). We present a 5 year old white male with cavum vergae is rarely present in healthy adult and with individuals. Both may normally appear in a relatively LGS, compare the clinical and chromosomal findings of a similar high percentage of premature infants, but usually have case reported by Turleau, 1982 (Hum Genet 62:183-187). Our patient was completely disappeared by the end of the first year. born with bilateral absence of the tibia and first metatarsus, hypoplastic Their presence in adults have been occasionally and posteriorly placed fibula and deficient femur. The knee was held reported associated to psychiatric conditions, flexed with popliteal web and he had severe ankle varus. The especially schizophrenia. There is only one report in abnormalities led to bilateral leg amputation. High resolution G-banded literature about the concomitant presence, in ten chromosomes were unrelated patients, of cavum vergae, duplication of interpreted as: 46,XYdel(8)(q22.3q24.11). septum pellucidum, macrocephaly and neurological In over 50 cases reported with LGS, the unusual association with skeletal alterations. malformations has been found only in the case described by Turleau and We report two brothers from non-consanguineous in the present case. Both cases also showed similar interstitial 8q deletions parents, who share a particular constellation of involving q24.1 which has been assigned as the shortest region of deletion clinical and radiographic findings including overlap (SRO) for LGS (Ludecke, 1991). The findings in these two patients macrouephaly, mental retardation and seizures as well that as fronto-parieto-temporal cerebral atrophy, cavum suggest the skeletal malformations may be due to a common genetic septi pellucidi and persistence of cavum vergae. defect in addition to the SRO found in LGS. This is consistent with the The similar characteristics of these two brothers contiguous gene model. However, it is also possible that these two cases suggest a new hereditary syndrome. The inheritance could represent the most severe expression of the LGS gene or an mechanism can not be defined. unmasked recessive deleterious gene responsible for the striking phenotype. A102 Posters: Clinical Genetics, Malformations and Dysmorphology (continued) 562 563 Congenital anomalies in individuals with mental retardation. RA Saul, Maternal transmission of Syndrome X in familial coronary artery RE Stevenson, and RJ Schroer. Greenwood Genetic Center, Greenwood, disease (CAD). M T. Scheuer. R.M. Cantor S.S. Kahn. J.S. Forrester. J.I. SC. Rotr Cedars-Sinai Research Institute, UCLA School of Medicine, Los Angeles, Evaluation of 2412 individuals with mental retardation in California. residential facilities and 4871 individuals with mental retardation Syndrome X is a common genetic tait characterized by insulin resistance, type 2 receiving services in community-based programs has provided diabetes (DM) and cardiovascular diseases. Excess maternal transmission ofDM has information about the frequency and spectrum of congenital anomalies been observed in type 2 DM pedigrees. To investigate this phenomenon in Syndrome associated with mental retardation. Twenty-one percent of both X, pedigrees from 214 probands with CAD were analyzed for patterns of disease residential (515/2412) and community (1030/4871) patients had transmission. Family history data regarding the diagnoses of CAD, hypertension congenital anomalies. (HIN), peripheral vascular disease (PVD), stroke, and DM were collected in 1,424 Diagnosis is more commonly reached in individuals with facial first degree and 2,048 second degree relatives (1,003 maternal and 1,045 paternal). dysmorphism than those without , possibly implying that the abnormal A positive family history of CAD (at least one affected first degree relative, or 2 or craniofacies assisted in establishing the diagnosis. The presence of more affected second degree maternal or paternal relatives) was observed in 70% of other anomalies (by individual category) does not increase the the pedigrees. Within the 150 familial CAD pedigrees, 56% (84) demonstrated clear likelihood of a diagnosis. parental transmission, with 64% paternally and 36% maternally derived (p=0.009). This study provides some preliminary data about the frequency of This paternal excess may be explained by an earlier age of CAD onset and death in congenital anomalies in a relatively unselected mental retardation fathers, and competing causes of death in mothers. A non-significant difference of population--individuals with mental retardation have a substantially CAD prevalence between the second degree maternal and paternal relatives (9.3% vs. higher (7-lOx) frequency of congenital anomalies than the general 8.0%) provides further support for this explanation. population. These data do not provide information about the frequency Familial Syndrome X was defined as the occurrence of DM in the case or a relative, of mental retardation associated with congenital anomalies since and DM, HTN, PVD, CAD or stroke in at least 3 relatives from one side of the mental retardation was the primary basis for ascertainment. pedigree. It was identified in 70 of the 214 pedigrees, and the majority (80%) were Congenital anomalies can be critical clues to diagnosis and medical also classified as familial CAD. Thus, Syndrome X comprises a significant subset of management in individuals with mental retardation. Our on-going studies familial CAD. Exclusive paternal or maternal transmission was seen in 79% of the 70 in this population of patients will be important for these patients and Syndrome X pedigrees. In contrast to the findings for CAD, 71% were maternally critical to developing management protocols and prevention strategies and 29% paternally derived (p=0.003). Prevalences of the associated Syndrome X for future generations. diagnoses (other than CAD) in second degree relatives also showed significant maternal excess. These results suggest maternal transmission of Syndrome X disorders and provide a genetic explanation for the clinical heterogeneity observed in CAD.

564 565 Faclo-ocular-acoustico-renal (FOAR) syndrome: A case report and Unbalanced translocation of chromosomes 4p and lOq associated a consideration of a PAX2 causative mutation. D.LB.S Iter'. L.A. with split hand/split foot malfornation suggests novelM locus. Grego&X P. Schutt'. Raj K1ur2 Fredeick Luthardt3. E. Nunes'. Schimmenti2. R.E. Kalina' and R.A. Paaon'. Universit of Washington', Department of Pediatrics' and Pathology2, University of Washington, Seattle, WA; Seattle, WA and University of California Los Angeles, Los Angeles, CA. 3Laboratory ofPathology of Seattle, Seattle, WA. We report a patient with FOAR syndrome, a rare familial disorder. Split hand/split foot (SHSF), also known as ectodactyly, is a human limb Clinical findings in the four previously reported patients include distinctive developmental malfornation cha d by absent central diital rays, a deep facial features (prominent brow, fiat nasal bridge, ocular hypertelorism, and median cleft, and syndactyly of remaining digits. The mafortn can be seen in eyes), ocular abnormalities (high myopia, iris isolated or syndromic form, and is usually inherited as an autosomal dominant. An antimongoloid slant to the autosomal locus, SHFM1 , has been assigned to 7q21.3-q22.1. Cytogenetic heterochromialtransillumination defects/coloboma, cataracts, and retinal analysis of sporadic SHSF patients and linkage studies in extended SHSF pedigrees detachment), congenital sensorineural hearing loss, renal abnormalities both suggest more than one autosomal locus. We report a stillborn twin infant with (proteinuria and variable renal structural abnormalities), and risk for split foot, polydactyly, cleft palate, and hypertelorism, found to have inherited an developmental delays/mental retardation. Our patient, an 11 year old boy unbalanced derivative from a balanced paternal translocation t(4;l0)(plS.1;q25.2), with characteristic facial features, ophthalmologic findings (typical iris suggesting a novel SHSF locus at 4p or lOq. detachment), The twin pregnancy had followed several miscarriages, and the father was coloboma, iris hypoplasia, cataract, high myopia and retinal found to carfy a balanced translocation 46,XY,t(4;10)(plS.1,q25.2). Twin A was moderate sensorineural hearing loss, proteinuria, and normal intellect, had selectively euthanized at eighteen weeks when found to carry an unbalanced less severe hearing loss and better developmental outcome than expected. derivative; twin B carried a balanced translocation. Oligohydramnios preceded We question whether a PAX2 mutation is causative. PAX2 is a member premature delivery at 24 weeks gestation. Twin B had no major anomalies. Twin A of a family of transcription factors containing a certain 'paired box! DNA had hemimelia and ectrodactyly of the right lower extremity, pre-axial polydactyly of binding which have a unique expression pattem in fetal and adult the left foot, cleft palate, and mild hypertelorism. Chromosomal analysis of twin A region, revealed 46,XX,-4,+der(4) t(4;10)(plS.1,q25.2)paL tissues and play a central role in development. Based on the localized Deletions of 4p (specifically band 4pl6.3) are characteristic of the Wolf- expression of PAX2 in early embryogenesis in the developing ear, eye and Hirschhom syndrome. Up to 15% ofWolf-Hirschhorn cases result from unbalanced kidney, we propose that a novel PAX2 mutation may be responsible for the translocations. Although cleft pala and hypertelorism are seen with 4p deletions, findings in FOAR syndrome. These studies are in progress. ectrodactyly and polydactyly have not been previously reported. Major limb malformations have not been reported in patients with trisomy for distal lOq. The recent demonstration of linkage of isolated SHSF to markers on lOq make trisomy 10q25.2-qter an appealing candidate region for genes giving rise to the limb phenotype in twin A. We suggest that the region 10q25.2-qter may contain a gene involved in limb development.

566 567 Association of a family histories of polyps and colon cancer with risk of Exclusion of Ilnitage of a familial Ectrodactyly, Ectoderma Dysplasia and Clefting (EEC) adenomatous and hyperplastic polyps. T.A. Sellers*, S.S. Rich, R.A. syndrome to the chromosomal region 7q21.3.q22.1. D.F.Shaw'. S.W.Schere_9. L.-C.Tsu?. King, G. Grandits, RiM. Bostick, P. Elmer, P. Grambsch, J.D. Potter. C L.Johnson'. L.L.Field'. 'Univ. of Calgary, Calgary, Alberta; 2Univ. ofToronto & Hospital (University of Minnesota, Minneapolis, MN, 55454) for Sick Children, Toronto, Ontario, Canada. The ectrodactyly, ectodermal dysplasia and clefting (EEC) syndrome is a human absence of the central rays of hands and feet with for colorectal cancer, and several developmental disorder characterised by Family history is an established risk factor abnormalities of hair, skin, nails and teeth, and cancer susceptibility. Far less is fusion of remaining digits (1lobster claw"), genes have recently been linked to colon clefting of the lip and/or palate. The hereditary form is rare, segregating as an autosomal known about pre-disposition to polyps other than autosomal dominant dominant trait with variable expressivity. Cytogenetic and molecular studies of both sporadic Familial Polyposis. The authors conducted a case-control study of incident, and familial cases of isolated ectrodactyly (split hand/split foot, SHSF) have demonstrated pathologically-confirmed, adenomatous polyps in cases aged 30 to 74 years. chromosomal rearrangements of 7q21.3-q22.1, have shown that the disease-associated hree control groups were identified: 1) colonoscopied patients free of cancer breakpoints are clustered in a common interval of less than 1 Mb, suggesting a SHSF locus and polyps, 2) colonoscopied patients with hyperplastic polyps, and 3) maps to this region. In addition, the chromosomal rearrangements of two EEC patients mapped population controls identified from the drivers license list, frequency matched to the same interval, suggesting a putative EEC syndrome locus at 7q21.3-q22. 1. on age and sex. Data on family history of polyps and cancer in parents and We report an extended Canadian pedigree in which the EEC phenotype segregates with high siblings were collected through mailed questionnaires on 574 cases, 707 penetrance and variable expression. Blood was collected and DNA isolated from 31 family polyp-free controls, 219 controls with hyperplastic polyps, and 549 members of which 18 have EEC. Subjects were typed with highly polymorphic microsatellite community controls. Cases with adenomatous polyps were 2.3 times as likely markers spanning 7q21. lq31 [order: cen, D7S524, D7S558, D7S492, D7S527, D7S821, (95% C.I.: 1.18 - 3.10) to report a family history of polyps than community D7S479, D7S491, AFM088ye9, D7S554, D7S518, D7S501, D7S523, D7S500, qter]. Linkage was the controls, and 2.7 times as likely (95% C.I.: 1.80 - 4.03) to report a family between the EEC locus and these markers analysed using LINKAGEprogram package. history of colon cancer. The reported frequency of family histories of polyps In addition, haplotypes were constructed from markers D7S558, D7S527, D7S821, D7S479, withthe EEC was and colon cancer among hyperplastic polyp controls was even higher than AfnMO88ye9, D7S491, D7S554 and D7S518, then segregation gene analysed. was obtained from among adenomatous polyp cases; compared to community controls, the No significant evidence for or against linkage pairwise analysis. analysis failed to reveal a haplotype or haplotype segment that segregated with the corresponding odds ratios were 2.9 for polyps (95% C.I.: 2.01 - 4.30) and 3.4 Haplotype EEC gene. Three-point linkage analysis using the LINKMAP program significantly excluded cancer 2.10 - 5.52). Additional analyses revealed that for colon (95% C.I.: the EEC syndrome locus from sequential intervals D7S524-D7S492, D7S492-D7S527, adenomatous polyp cases with a family history of polyps were 10.0 times as D7S527-D7S491, D7S491-D7S554, D7S554-D7S518, D7S518-D7S501, D7S501-D7S523 and likely to have a family history of colon cancer than those without; for the confirming results ofhaplotype analysis. These results indicate that the EEC was Additional research is D7S523-D7S500, hyperplastic polyp controls the associated OR 9.8. mutation segregating in this family is not located in the 7q21.3-q22.1 region, suggesting that is a reflection of warranted to determine if the observed clustering of polyps genetic or locus heterogeneity exists for EEC. shared genes, shared environment, or their interaction. Posters: Clinical Genetics, Malformations and Dysmorphology (continued) A103 568 569 A method to record the effects on the fetus of drugs taken during preg- Bowed long bones, unusual hands, narrow chest, dysmorphic facies, and cystic nancy using computerised prescriptions linked to the records of a birth hygroma: a new, autosomal recessive skeletal dysplasia. B. A. Shephard. B. M. defect register. L.J. Sheffield, R. P. Batagol and H. B. McNeil. The Murdoch Berlin. R. G. McCauley. M. Irons. Floating Hospital for Children and Tufts Institute. Roval Children's Hospital, Melbourne, Victoria, Australia. University School of Medicine, Boston, MA. 697 public antenatal patients were enrolled in a study where drug intake dur- We describe two siblings with congenital anomalies and a skeletal dysplasia. ing pregnancy was documented by using computerised prescription records which Their mother is a 30 year old woman with a history of cocaine, alcohol and cigarette were validated against face to face interview, hospital records of drug intake and a use during pregnancy. She has had 4 spontaneous abortions and 2 healthy children self report diary. There was a 94% agreement of recording of drug intake by com- with other partners. Case 1 was a male, diagnosed prenatally with anomalies including puterised prescriptions with direct interview. The written hospital record of the shortened, bowed long bones, cystic hygroma, small chest, one multicystic kidney, and prescription was incomplete as was the self report diary. The diary contained very possible in-utero seizures. Fetal karyotype was 46, XY. This infant died 3 hours after complete information about prescription and over the counter drugs but was only a term birth. No postnatal assessment is available. In case 2, prenatal ultrasound returned to the investigators by 237 (37%)women despite reminder interview(s) examination revealed bowing of femora and ulnae, and cystic hygroma. This female and telephone contact. It was concluded that the diary is of limited use unless the infant delivered prematurely at 25.5 weeks gestation. Her physical exam is remarkable women are well motivated. Pregnancy outcome was evaluated from the records of for a dysmorphic facies with deep-set, creased, broad nasal bridge, small, malformed a well validated Birth Defect Register by linking the perinatal records. Pregnancy ears, brachydactyly, hypoplastic nails, and bilateral index and fifth finger clinodactyly. outcome was also recorded using hospital records (written and computerised) and Her birth length and interval growth are appropriate for gestational age. Radiographic prenatal ultrasound records. The most record of findings include a narrow chest with moderate shortening of the ribs. There is complete birth defects was the anterolateral of the lateral of the radius and ulna Birth Defects Register and 20 babies were found with birth defects. This study bowing femora, bowing proximal and of the humeri and tibiae. Distal are small shows that computerised records which are relatively inexpensive can be used to bilaterally, attempted bowing phalanges and rounded. Karyotype is XX. Other than of extreme accurately record pregnancy drug intake of prescription drugs and this information 46, expected problems her course has been unusual for seizures which have resolved. There are can be readily linked to a well validated birth defect which prematurity, very register records no pertinent intracranial or ophthalmologic abnormalities. Despite an extensive search, pregnancy outcome. we have been unable to fit these patients' abnormalities to a known skeletal dysplasia syndrome. Although some features suggest Jeune's asphyxiating thoracic dystrophy or campomelic dysplasia, each of these diagnoses has features which seem to exclude our patients. We conclude that this may represent a skeletal dysplasia syndrome not previously described with an apparent autosomal recessive inheritance pattern.

570 571 Congenital Nephrotic Syndrome and Brain Dysgenesis: A New Syndrome? Clinical severity of congenital heart disease in Holt-Orasn syndrome. Meredith Silver, Malgorata Nowaczyk, David Chiayat, Venita Jay, Ants Toi, Denis Lehotey, LI. Sletten and M.E. Pierpont. University of Minnesota, Minneapolis, Minnesota. Leslie Solomon, and Micki Thomas. Holt-Oram syndrome (HOS) is an autosomal dominant disorder with variable The Toronto Hospital and The Hospital for Sick Children; The University ofToronto, Toronto, Ontario, Canad expression where limb anomalies and cardiac malformations occur together. Most genetic reference texts indicate that the expected cardiac problems include ASD, Congenital nephnoic syndrome (cNS) is characterized by neonatal or infantileproteinuria, VSD, PDA and other single malformations. We describe a family with 5 affected hypoalbuminemia, andedema In 1968, Galloway and Mowat eporeed 2 sibs with individuals. The index case is a 19 month old boy with left thumb hypoplasia. At microcephaly, hypotonia, dysmorphic facial features, histu hernia and cNS. To date 27 cases age 6 weeks, complex congenital heart disease including endocardial cushion defect with cNS and brain dysgensis have been reported as Galloway-Mowat syndrome (OMS). with primum and secundum ASDs, severe left ventricular hypoplasia, hypoplastic In 1986, Palm et al reported two siblings with periventrvcular heteotepia and cNS, one having mitral valve, apical muscular VSD and left superior vena cava connecting to the hydrocephalus due toaqueduct stenosis. We reporta similarcase with cNS, periventricularbrain coronary sinus was found on echocardiography. Operative procedures have dysgenesis, aqueduct stenosis, hydcephalus, and normal facial featuren. included pulmonary artery banding, Damus-Kaye-Stanzel procedure, central shunt The mother was a 31-year-old G4P(fA3LO woman. The couple were healthy and non- and bilateral Glenn anastomoses. Currently the patient is thriving, but will require consanguineous. Maternal senan screen at 17 wees gestation showed AFP level at 23 MobL further surgery. This patient's mother and aunt both have HOS and have undergone Fetal ultrasound showed marked dilation of the lateral and thirdcerebral ventricles with for macrocephaly. Matemal TORCH sdi, and aLoimmuneantiplatlet antibodies were negave. surgery secundum ASD. A maternal uncle and grandfather have limb anomalies Amnioce s showed AF-AFP level of770.7 mg/L (2 MoM =15 mg/L). Amniotic fluid but no heart abnormalities. viral cultur organic acid analysis, and acetyl choline esterase were negative. The fetal Because of the severe heart disease in the index case, we surveyed the world karyotype was 46,XY. The pregnancy was terminated, Fetal autopsy revealed normal literature from 1974-1995 to learn more about the expected incidence of congenital facial feaue, findings of Fmuish type cNS, and peiventricular cerebral heteotospia. Spinal heart anomalies in HOS. Of 189 patients, 125 (66.1%) had a single cardiac cord and eyes were nomal. abnormality such as ASD or VSD. Combinations of mild defects including VSD, Like the sibs reported by Palm et al, this male fetus had normal facial features. Two of the three ASD, PDA and others occurred in 31 (16.4%). Moderate combinations of cardiac cases were diagnosed prenatally by detecting elevated AF-AFP and brain abnomalities on fetal anomalies including endocardial cushion defect and tetralogy of Fallot and severe U/S. Of the two cases with hydrocephalus due to aqueduct stenosis and penventricular combinations of cardiac anomalies (such as hypoplastic left heart, total anomalous heterotopia presented as two years ofage, while the other was detected prenatally. We propose pulmonary venous return and others) occurred in 33 (17.5%). This group of that these 3 patients born to two usrelated families representa new syndrome distinct from individuals with more serious congenital heart anomalies has a mortality of at least GMS. Since all 3 cases were males, the inheritance could be either X-linked or autosomal 36%. Clinical evaluation and genetic counseling in Holt-Oram syndrome should recessive. include discussion of the variable range of severity of congenital heart anomalies, the potential for multiple cardiac operations and possible increased mortality. The frequency of moderate and severe cardiac anomalies in HOS has not previously been appreciated.

572 573 Ocular manifestations of Jacobson syndrome Elevated Lp(a) levels hi abdominal aortic aneurysmal disease. P.L.St.Jeaa:. ( 11 q-). S. Somanil1. A. Levinl,2. M. A. M.W.Webster2 D L. Steed2. RE. Ferrell', Department of Human Genetics' NowaczykO. and Surgery2, University of Pittsburgh, Pittsburgh, PA. Feigenbaum1 ,2 R. Davidson 2, T. Costa2. University of Torontol Elevated Lp(a) levels are associated with a significantly increased risk of and The Hospital for Sick Children2, Toronto, Ontario, Canada. atherosclerotic coronary disease. Abdominal aortic aneurysms (AAA) have Jacobsen syndrome is caused by a terminal deletion of historically been viewed as late manifestations of atherosclerosis. We chromosome 1lq with a common breakpoint at 11q23. This measured plasma Lp(a) levels in 153 AAA probands and compared them to syndrome Is characterized by dysmorphic facies, pre and postnatal a population sample of 457 controls. Lp(a) levels are neither age nor gender growth retardation, hematologic disturbances, cardiac and ocular dependent but are higher in African-Americans than Caucasians; both the malformations. case and controls in this study were Caucasian. Purpose: To describe the largest series of ocular examinations on Median rather than mean levels of Lp(a) were compared using the non- affected children. parametric Kruskal-Wallis test as Lp(a) distributions are highly skewed in Methods: Five consecutive affected children received complete Caucasian populations. The median Lp(a) level of 10.8 mg/dl in AAA cases ocular exams and cytogenetic studies. was significantly higher than that of 3.8 mg/dl in controls (p<0.0001). We also Results: Observed features In our patients included hypertelorism measured Lp(a) levels in siblings of patients with AAA. These siblings had (4/5), downslanting palpebral fissures (3/5), nasolacrimal system AAA phenotype confirmed by medical records and/or abdominal ultrasound. anomalies (215), microphthalmia (3/5), nystagmus (2/5) and optic The 49 affected sibs had a median Lp(a) level of 12.7 mg/dl which was nerve hypoplasia. All required surgery for strabismus, or ptosis. At significantly higher than that of controls (p<0.0001), but did not differ from surgery, one patient had anomalous extraocular muscles. Two cases (p=0.50). The 48 unaffected sibs had a median Lp(a) level of 6.4 mg/di children required nasolacrimal duct surgery one of which was which did not differ from controls (p=0.26), but was lower than that of cases necessitated by life threatening dacrocystitis. (p=0.02). Conclusion: Ocular malformations are prevalent in Jacobsen These findings could be interpreted as supporting the historic syndrome. An analysis of candidate genes in the breakpoint atherosclerosis model forAAA. Altematively, these results may support recent regions may offer etiologic clues. research which has suggested that AAA disease may have an underlying inflammatory component Elevated levels of acute phase proteins, including Lp(a), would be expected in the presence of an active inflammatory proces- A104 Posters: Clinical Genetics, Malformations and Dysmorphology (continued) 574 575 Three ya flow up of a pate wit Torlello-Cry Syndrome. LI Sulibrey nanism in two U. S. patients R. S=phen. M. Spolt and E.A. WAIhr. University of Maryland School of Medicine, Artigas-Lonez and B.G. Kousseff. Univ. of South Division of Human Genetics, Baltimore, Maryland. Florida, Tampa. Only one report from the U.S. details findings in Torielo-Carey (- syndrome is an autosomal recsive multiple malfomaon a patient with Mulibrey nanism, a rare autosomal Msdrome c ized by growth and developmental delay, mioephaly, recessive condition found primarily in Finland. We micrognahiacleftpalate, agenesis of the corpus callosum, typicl fades, cardiac report two U.S. patients with characteristic defects (ASD, PDA), and hypotonia We report a three year old boy with T-C findings: growth failure, frequent pulmonary syndrome to provide additional prognostic information on this rare condition. infections, typical facies, J-shaped sella turcica, KLS. was the 37 week product of an unomlcted gestation to a 32 year old mild enlargement of cerebral ventricles, peculiar mother. Birth was 2000 gram he was noted to a high-pitched voice, gracile long bones and GM weight (SGA) and have 2- hepatomegaly. Associated findings in one or both vesd cord, mic athia, cleft palate, microcephaly (HC - 29 cm.), and an ASD. patients include abnormal pigmentary retinal A CT scan showed partial agenesis of the corpus callosum. At 2 months of age, changes, delayed bone age, hypodontia and cutaneous he had a traeostomy bemu of severe micro and a laryngeal naevi flammei. Fibrous dysplasia, previously abnormality, followed by a pylorolasty for pyloc stenosis. His course for the reported only in the tibia, was found to have a more first two years of life was complicated by reflux, frequent p o , allergies to widespread distribution in one patient. Pericardial milk products, and roomy complicaions. At age 3 KS. as constriction, a frequent and life-threatening yean, presents abnormality in this syndrome and sometimes a small (ht - 10th %, wt < 5th %), developmentally delayed, young boy with no associated with myocardial and endocardial fibrosis, major medical c ens. His DQ was 70 with ADHD, and hypotniia. Little led to the first cardiac transplantation in a follow-up is available on children with T-C because of a high neonatal mortality patient with Mulibrey nanism. Findings in these resulting from their multiple anomalies. A second 3 y/o child (Tiello and Carey, patients broaden the clinical description of 1988) without a cardiac defect had a DQ of 60 with hypotonia suggesting that Mulibrey nanism and show that it is more common in infants who survive the first of life show modwer mental retardation. T-C the United States than previously thought. cardiac year transplantation in one patient adds new information syndrome should be conidered in any child with cleft palate and/or agapesis of regarding therapy for cardiac th corpus callosum. involvement.

576 577 Microcephaly, Microtia, Cleft Palate, Severe Mandibular Hypoplasia, Limb Hep tobasStoma and cadoopaffy: fatal outcome In a 4.5 month old girl. Anomalies and Short Stature: Possible New Autosomal Recessive Syndrome 1R . Dcpanment of Genetics Hospital for Sick Children, Resembling the Ear-Patella-Short Stature Syndrome. Ahmad S. Teebi and Toronto, Ontario Canada Alison M Elliott Division of Medical Genetics, Montreal Children's We present a female infant with hepatoblastoma, severe hypertrophic Hospital and McGill University, Montreal, Canada. cardiomyo thy and dysmorphic facial features. She was born to a 31 year old G3P2Al of English/German descent. Oltrasound at 26 weeks gestation showed We report an apparently new syndrome with marked variability in two polyhydramnios and a discrete mass in the liver. TORCH screen and fetal sisters born to phenotypically normal and nonconsanguineous parents. The echoardiogram were normaL There was premature onset of labour followed by a proband was born at term with OFC, length and weight below the 3rd spontaneous vaginal delivery at 31 weeks gestation. Birth weight was 1647g (15- centile. An amniocentesis revealed normal chromosomes. An ultrasound at ).Early respiratory distress was evident due to compression of the chest by an 32 weeks of pregnancy detected polyhydremnios and IUGR. Anomalies noted enlarged abdomen. Examination showed a ventilator dependent infant with a couse at birth included microcephaly with sloping forehead, almost closed face,rsutism, depressed nasal bridge, anteverted nares and cgnathia. She had fontanelles, short palpebral fissures, epieanthic folds, hypertelorisa, asymmetric rght ventrculr hypophy and marked hepatosplenomegaly. By 4 flat nasal bridge, cleft palate, severe aicrognathia, and severe weeks of age, progressive, symmetric biventricular hypertrophy was evident with microtia with atresia of the right external ear canal. Hands showed subpulmonary obstion. tapering and overlapping fingers, lax joints, fifth finger clinodactyly, The patient underwent a resection of the left lobe of the liver. Pathology unusual palmar creases and nail hypoplasia. There was bilateral talipes confirmed hepatoblastoma, fetal type. 2 weeks post-operatively, AFP increased to deformities and a large gap between the second and third toes. Skeletal 4184. There was progressive abdominal distention and ascites and within 3 weeks survey showed micrecrania, severe mandibular hypoplasia, multiple the AP was 33473. segmentation anomalies in the cervical vertebrae and bilateral hip This infant progressively deteriorated. and had an upper GI bleed. The infant dislocation. CT of head revealed prominent ventricles and CSF spaces. died following multiple cardiac arrests. MRI showed relative narrowing of the foremen magnum. At 9 months, (fed Anatomical diagnosis at autopsy included small for postnatal age, facial by nasogastric tube) measurements were still below the 3rd centile, she dysmorphism, and no tumor recurrence. Biventricular myocardial hypertrophy was had marked developmental delay. Her sister (13 years old) was found to seen with the heart weighingS 3X the expected amount. There was a pericardial be remarkably short with OFC, height and weight well below the 3rd effusion and mild thickening of the pulmonary valve leaflets. centile. Anomalies are milder than the sister and they include: sloping The differential diagnoses considered included the following: metabolic storage forehead, aicrotia, severe micrognathia, telecanthus, epicanthic folds disorders, Schinzel-Gideon syndrome, Simpson-Golabi Behmel syndrome and and high arched palate. She had mild developmental delay and school Beckwith-Wiedemann Syndrome. difficulties. Skeletal survey was unremarkable including normal patellae and appropriate bone age Chromosomes were normal. Comparing the anomalies noted here to those in various mandibulofacial dysostosis syndromes and the Ear-Patella-Short stature syndrome, the pattern is quite distinct.

578 579 Candidate Gene Analysis of Neural Tube Defects (NTDm) in Humans. L Genetic variations of human gender orientations. W.J. Turner. Trembathl, A. SherbondX1. D. an~ykgl, R. Finnalt2, A_ Murrayl. University of New Mexico, Albuquerque, NM. 1The University of Iowa, Iowa City, IA. 2Texas A&M University, In 1993 Hamer et al. identified a DNA sequence in Xq28 which College Station, Texas. segregated with male homosexuality. In 1995 Turner presented We are using candidate genes identified in human and non-human evidence from studies of kinships that both male and female studies to search for genetic factors involved in NTDs in humans. homosexuality is of Xq28 origin. He proposed that the mechanism Searches for polymorphisms, as well as potential etiologic mutations are productive of homosexuality lies in variations of trinucleotide conducted strand conformational repeats and their methylation. Now evidence is offered that the being using single analysis and direct same mechanisms are productive of such clinical variation as DNA sequencing of candidate genes, including PAX3, 5- asexuality, male cross-dressing, male-to-female and female-to- methyltetrahydrofolate (5-methyl THF) receptor, and human folate male transsexuality, and some instances of infertility. While receptor beta (FR-P). An NTD population consisting of 96 affected genes for sex are determined by DNA sequences in the Xp and Yp individuals from Iowa is being analyzed for linkage disequilibrium using regions, it is asserted that a gene for gender is in Xq28/Yqll. parental as controls when or Consideration of persistence of homosexuality in all human genotypes available using control populations despite very low reproductive rate of affected individuals from the same geographic and cultural background. We individuals leads to the conclusion that it is the reproductive have identified SSCP variants for FR-Y which could be used in our case- rate of their mothers which is relevant. control study. We have been unable to demonstrate an association using a CA repeat polymorphism for PAX3, despite its role in another human developmental disorder, Waardenburg syndrome, that occasionally involves neural tube defects. These efforts are designed to help further elucidate genetic factors involved in neural tube defects and provide background for genetic counseling to parents. Posters: Clinical Genetics, Malformations and Dysmorphology (continued) A105 580 581 Joubert Syndrome Among Bedouins: An under estimate ? Chronic Fibrosis of the extraocular Muscles and Polyneuropathy segregating in a large Belgian Pedigree. Usha RajaramLR.l imaL K.TahseeniLRobeen Arafat, M.M.AI-Ghanim, L. VanMalderaeml. M. Cordonniee. E. Enale3, A. Melebeck4, P. Gilsonf4 T.l.Faraa, Department of Pediatrics, Al-Jahra Hospital and Y. Gillerot lCentrede Genetique Humaine, Institut de Pathologie et de Kuwait Genetics Center, Kuwait. Genetique, Loverval, Belgium. 2.Dept of Ophtalmology, H6pital Erasme, Universit6 Libre de Bruxelles, Belgium, 3.Division of Genetics, Children'Hospital, We report from Kuwait, 8 children 14 males and 4 females) from Boston, Massachussets, U.S.A. -Services d'Orthopedie et de Neurologie, 3 unrelated consanquinous Bedouin families, with Jouberts Syndrome Clinique St Joseph, Lobbes, Belgium (MIM 243910). All were ascertained in the Al-Jahra Regional A congenital fibrosis of the extraocular muscles (CFEOM) and Hospital serving a population of 200,000 with 85 S Bedouins with progressive ataxia, together with severe lower limbs polyneuropathy large family size and high inbreeding coefficient. The clinical (HSMN) was observed in a 42 y-old Caucasian female. Her father, as features included episodic hyperapnoea, irregular respiration, well as four of her six siblings, also presented polyneuropathy. abnormal eye movements, rhythmic tongue protrusion, hypotonia, Interestingly, two sisters had normal eye movements and polyneuropathy. hypertelorism, severe motor and mental retardation. In the family The first one had two with CFEOM and whereas the with three affected two had cleft palate and one had boys speech delay siblings, a A was occipital encephalocele. Two male children had retinal colobomata. latest had boy with unilateral ptosis. diagnostic work-up Self mutilation, with resultant tears and ulcers on the lips & hand performed in the index patient. Brain MRI was normal, as well as eye was evident in two siblings of one family, hitherto unreported fundus and biochemical evaluation of the respiratory chain on fresh feature In the spectrum of this syndrome. Uric acid levels were deltoid muscle extracts (Dr. R. Van Coster, Gent) No ragged-red-fibers normal in these two cases. Brain C.T.SCANS of all the eight were observed and morphology of the muscle was normal. There was no children showed hypoplasia of cerebellar vermix and dilatation deletion of mitochondrial DNA. A sural nerve biopsy indicated a of cisterna magna. Ventriculomegaly was detected in six patients. demylinating process with some clusters of axonal regeneration. There the on With marked drop of the incidence of neural tube defects among was no duplication of CMT la locus chromosome 17. We suggest BedouIns responding to dietetic counselling programme, we expect this unusual association is not fortuitous and represent pleiotropic the surfacing of, and detection of, more autosomal recessive manifestations of a single dominant gene dysplaying variable disorders like Meckel Gruber and Joubert Syndrome among this expressivity. Linkage studies with probes in the vicinity of the CFEOM inbred population. The incidence of Joubert Syndrome among locus on chromosome 12 (Nature Genet, 1994, 7, 69 ) are underway and Bedouins in this studyislin 6000 births. will be presented.

582 583 Analysis of CAG Trinucleotide Expansion Associated with Machado-Joseph Detection of SRY and ZFY sequences in two XX males, two XX Disease (MJD): M. Watanabe'2, K. Abe', M. Aoki', M. Shoji2,Y lkeda2, S. Hirai2 and pseudohermaphroditic females and two XY gonadal dysgenesis. Y. Itoyama'. School Department of Neurology, 'Tohoku University School of Medicine, Sendai, 'Gunma X.J. Wen1, 0. Li2, and P. Li3. 1Department of Anatomy, Morehouse University School of Medicine, Maebashi, Japan. of Medicine, Atlanta. 2Department of Obstetrics & Gynecology, Guangxi Machado-Joseph disease (MJD) isan autosomal dominantneurodegenerative disorder Medical University, Nanning, P.R.China. and 3Laboratory of Medical characterized by cerebellar ataxia, pyramidal and extrapyramidal signs, amyotrophy, Genetics, University of Alabama at Birmingham. peripheral nerve palsy, external ophthalmoplegia and bulging eyes. The genetic locus Recent studies of male sexual development have demonstrated a sex- for MOD was mapped on the chromosome 14q24.3-32. 1. A recent study has identified Y located on the short arm of the Y an unstable trinucleotide CAG repeat expansion as the mutation causing MID. Twenty determining region (SRY) gene, chromosome, functions as a genetic switch in gonadal ridge initiating testis cases of MID in 13 unrelated Japanese families were genetically and clinically examined determination. Another gene located nearby, zinc-finger Y (ZFY) gene, has in comparison with 20 cases of age at onset- and duration-matched spinocerebellar nucleic acid motif and acts as a factor on ataxia type 1 (SCA 1). The CAG repeat number of expanded MID and SCA1 allele was the binding probably transcription 72.2 ± 3.1 (mean ± SD), and 47.3 ± 4.4, respectively. The repeat size was inversely the spermatogenesis. In the present investigation, two 46 XX males, two XX correlated with age at onset, and the repeat number in leukocytes increased from pseudohermaphroditic females, and two XY gonadal dysgenesis (phenotypic parents to children with acceleration of age at onset (anticipation) in both MJD and female, Swyer syndrome) were diagnosed by the physical examination of SCA1. The number of CAG repeats in sperm was reduced in MID from that of external genitalia, biopsy examination, hormonal level study, pelvic and leukocytes at 1.0 ± 0.8 (n=3) ranging from -2 to 0, but was increased in SCAI at +3 abdominal ultrasonography, exploratory laparotomy, and chromosomal and +4 repeats (mean=3.5, n-2). However, the number of peaks in the expanded allele To the or absence of SRY and ZFY in was greater in sperm than in leukocytes in both MID and SCAl (increased somatic analysis. verify presence sequences mosaicism). MID was clinically characterized by relatively high frequencies of ocular the above cases, specific primers were designed to amplify the SRY gene signs such as eyelid retraction, bulging eyes, ophthalmoparesis, and nystagmus. spasticity and the ZFY gene from extracted DNA samples. All six patients were shown in lower limbs, and sensory and urinary disturbances in contrast to the SCA 1 patients to have the SRY sequence. The two XX males and two cases of XY gonadal that showed less spasticity, sensory and urinary disturbances, and ocular signs except dysgenesis also had the ZFY sequence, but the ZFY sequence was absent in for slow eye movement. the two XX pseudohermaphroditic females. Our results indicated that: 1) the These results suggest that the expanded CAG repeat and clinical features were well of the Y-chromosome male in correlated in MID, and the clinical characteristics are different in some aspects from presence determining fragments 46,XX genome could cause the sex reversal and ambiguous genitalia, 2) the XY cases of SCA 1. gonadal dysgenesis probably resulted from mutations in the SRY gene, molecular defects on SRY gene or ZFY gene in the cases are currently under investigation.

584 585 Autosomal dominant Inheritance of malformations of the anterior A controlled study of longitudial IQ changes in females and males with fragile segment of the eye in a large Australian pedigree. SJ Withers1-2, G Golel, X syndrome. C. Wright-Talamante. A. Cheema. J.E. Riddle. D. Luckey. A. Taylor. EM Sumnmers2. 1 Roval Children's Hospital, Brisbane, Queensland, Australia; R.J. Hagerman, Child Development Unit, The Children's Hospital, UCHSC, & 2 Department of Medicine, University of Queensland, Brisbane, Australia. Kimball Genetics, Denver, CO The eye develops from the neural tube early in embryonic development. The anterior A longitudinal decline in IQ has been reported in males but not consistently so in segment of the eye is derived from surface ectoderm (comea and lens) and mesoderm females with the full mutation of fragile X syndrome. This study evaluated the IQ (iris). A number of developmental abnormalities of the anterior segment have been decline in 54 males and 21 females with fraX to 40 control who described, including aniridia, Peters anomaly (corneal opacity with or without compared patients iridolenticulocorneal adhesions) and congenital cataracts. Aniridia and Peters anomaly were comparable in the initial IQ, initial age and first and last IQ intertest intervals. have been shown to be due to mutations in the paired box gene PAX6, located on The formula by Anastasi (1982) was utilized to evaluate significant changes in IQ chromosome 11. Peters anomaly usually occurs in isolated cases although a number scores and 31% of fraX males, 33% of fraX females, 26% of male controls and 19% of small families with several affected members have been described. We present a of female controls showed a significant IQ decline. The differences between groups large Australian family (of British ancestry) with both Peters anomaly and congenital was not significant. Only when utilizing the same IQ standardized test between the cataracts. The family consists of thirty seven living members in five generations. Te first and score was a difference seen between fraX males and index case, in generation two, had bilateral cataracts removed at age 12 years. None of last significant only her four sisters and one brother or any individuals in previous generations had eye controls (p=0.02). This suggests that using the same IQ measure gives a more problems. Three of her four children had congenital cataracts, detected between ages 9 accurate assessment of IQ change during a critical period in midchildhood. However, and 20 years. The fourth daughter has Peters anomaly and congenital cataracts and has the differences between fraX males and controls disappears when one looks at a larger very little residual vision at age 50 years. Six of the nine offspring (five female, one intertest interval over the life span using different standardized IQ measures. The lack male) of these women had congenital cataracts, with lenses removed at ages 10 to 26 of differences between fraX and controls is present because of an IQ decline in a years. Three of the four offspring of these patients have Peters anomaly, detected at was and that decline is not birth. Cataracts have not yet been detected in these young children. No members of subgroup of controls which unexpected suggests IQ unique the family have evidence of aniridia. Anterior segment malformations appear to be to fragile X patients. When comparing 3 molecular subgroups of fraX males, 31% inherited in a autosomal dominant fashion in this family. There is a high incidence of of the full mutation fully methylated males, 45% of the mosaic males and 18% of the affected females (13/14 affected). One at risk female and three at risk males have full mutation partially methylated males experienced a significant IQ decline. normal eyes. In addition, the condition appears more severe in the youngest Although these differences are not significant (p=0.39) by chi square analysis, it is generation, with all affected children having Peters anomaly, which was found in only important to note that none of the partially methylated males with less than 50% one of 11 individuals in previous generations. Preliminary studies are consistent with decline. linkage to the PAX6 locus, and we arc verifying this with a range of chromosome 11 methylation experienced a significant IQ markers. Detailed study of the clinical phenotype together with analysis of the mutation involved is being used to understand the factors involved in the phenotypic variability in eye malformations in this family. A106 Posters: Clinical Genetics, Malformations and Dysmorphology (continued) 586 587 Further delineation of the 14q- syndrome: 'Double mosalcism' or chimera in a profoundly Molecular genetics guides clinical geneticist: bedside to bench-now a two way mentally retarded individual? X-L Yao. S.A. Adelman. M. Genovese. I. Wlsniewski W.T. street L 7:akail, D M mc ,onjld-Mcntil, L Ravnl, LRjligham' L Pelleginol, LiegclIL Brown. E.C. Jenkins. Institute for Basic Research in Developmental Disabilities, Staten DJ Wolff2 D. Younkinl, PL Chnel, DB. Spinansl. Thc Children's Hospital of Philadelpiua, Island, N.Y. Psiladelphia, Penssylvania, 2Case Western Reserve University, Cleveland, Ohio A 33-year-old male individual with static encephalopathy associated with mental The following series of recent cases illustrates how knowledge of and access to molecular genetics profound must be part of the clinical geneticists' "doctor's bag" in order to unravel seemingly puzzling retardation and a seizure disorder of unknown etiology was referred for evaluation. combinations of clinical findings and guide geneticcounseling. Physical examination showed dysmorphic features and macrocephaly; neurological A 6 year old male with mid-lumbar myelomingocele was referred because of severe mental retardaton, examination demonstrated spastic quadriparesis, while EEG results were compatible with dysmorphic facial features including synophrys, telecanthus, bulbous nasal tip, and saesorneural hearing Lennox Gastaut syndrome. Short-term whole blood cultures revealed two different cell loss. Chromosomes at another institution were interpreted as normal. In examining the child, the lines. Among 100 cells studied from four separate cultures, 30 exhibited the loss of a Y clinical diagnosis of Waardenburg type I with associated myelomingocele was made. Because of the chromosome with an otherwise normal while the 70 cells exhibited known PAX-3 mutation in Waardenburg I and its location on distal 2q, it was suggested to the karyotype remaining cytogeneticist that special attention be paid to the 2q region. A de novo paracentric inversion of a normal male sex chromosome complement but there was material missing from the distal 2q23q37.1 was found. portion of the long arm of chromosome 14 in every cell with the male sex chromosome A 27 month old male presented with motor development significantly more impaired than cognitive karyotype. For the 46,XY Hue, it appears that an interstitial deletion occurred that began development He was known to have monosomy 10q and dup 17p resulting frem an apparently balanced within band 14q31 and continued to the interface or beyond band 14q323, described maternal seanslocation t(10;17)(q26.3p11.2). Knowing that the CMT type IA gene region is on 46,XY,del(14)(q3105q32.3). FISH studies showed that both 14 chromosomes were 17pl1.2-12 and is associated with a tandem 1.5mb duplication led us to suggest a neurologic woscup in with a chromosome 14 DNA Parental cultures this child. The discrepancy in motor development was subsequently found to be due to a perperal completely painted specific probe. were neurpathy. normal so that the structural chromosome abnormality occurred de novo. The combination A 12 year old girl was referred because of significant retardation with no speech and seizures. Past of a 4SX line with normal 14 chromosomes together with a separate line that contains an history revealed that an amniocentesis performed for advanced maternal age revealed a paternally XY sex chromosome complement, but has a deleted chromosome 14, has not been reported. transmitted balanced 13;15 translocation in the fetus. Based on the physical exam and knowing that The present and three previously reported cases that overlap the deleted region, Angelman syndrome is due to loss of maternal 15q11-13, adiagnosis of Angelman syndromewas made. 14q31g-qer, share the following phenotypic abnormalities: psychomotor retardation, ear Studies for uniparental disomy conflemed heterodisomy for paternal 15 in the patient. arched and skeletal anomalies. The lack of and Our finding of haploinsufficiency unmasking a recessive disorder due to a mutation on the normal abnormalities, highly palate, mkrognathia chromosome homologue explained why a child with severe hypotonia also had classical albinism and the presence of macrocephaly seen in this case but not present in any of the previous cases, why a child with minor dysmorphia and a VSD also had Bemard-Soulier bocy The clinical may be related to the extent of the deletion as well as to the tack of the Y chromosome in findings of Prader-Willi syndrome at age 6 in the former and the characteristics of velocardiofacial the second cell line. At this time, the origin of the two different cell lines is unknown. syndrome in the latter led to molecular deletion studies (Lee, et al. Hum Mol Genet 3:2047-2051, 1994; Follow-up studies with DNA including RFLPs and microsatellite markers will resolve this Budarf Hum Mol Genet 4:763-766, 1995). issue. TUSnscrWASSUvouDINsAatTvYsv ONiWYOsSTAT5OMI5CEOWMNALRETAsnATNCNAYS In the past the clinical geneticist brought these cases to the bench. Now the bench knowledge is D ovzLowizNTAL DeuAsi5rs5 guiding the geneticists' clinical management. This together with the beach knowledge of gene mutat n localization helped unravel the two seemingly puzzling clinical situations.

588 589 An autosomal form of dominant familial spastic paraplegia affecting Juvenile Rheumatoid Arthritis (JRA) in two individuals with Velo- almost exclusively males. Mavana Zatz. Suelv K. Marie, M. Rita Passos- Cardio-Facial syndrome. R.T. Zori. S.A. Rasmussen. E.M. Avoub. Bueno. and Daniella Unaar. Dept. de Biologia, Inst. de BiociAncias and Dept. B.A. Gray. A. Bent-Williams. H.J. Stalker. C.A. Williams. Raymond C. Philips Research and Education Unit, Division of Genetics, de Neurologia, Fac. de Medicina, Universidade de SAo Paulo, S.Paulo, Brazil. Department of Pediatrics, University of Florida, Gainesville. Autosomal dominant (AD) uncomplicated familial spastic paraplegia We report on a 9-year-old female with Velo-cardio-facial (FSP) is a genetically heterogeneous degenerative disorder characterized by syndrome (VCFS), confirmed by FISH to have deletion of N25 a progressive spasticity of the lower limbs,. Three locus responsible for this (022S75). She had microcephaly, a congenital heart defect (VSD, condition have been identified until now: 14q, 15q and 2p. PS, PDA), and velo-pharyngeal insufficiency. At age 7 years she We are reporting a three-generation Caucasian family with 39 affected developed onset of joint pain and polyarticular JRA was diagnosed. and/or asymptomatic transmitters of the FSP gene (29 males and 10 females). Awareness of this case led to the subsequent diagnosis of VCFS in Neurological examination of affected subjects revealed spasticity and another, unrelated, 12-year-old girl (confirmed by FISH) who had facial dysmorphia, no cardiac defect, VPI, obesity and JRA. The JRA hyperreflexia in the lower limbs, weakness of hip flexion and ankle was first diagnosed at age 5 years, leading to severe joint dorsiflexion, extensor plantar responses, diminished vibratory sense and pes involvement. Her initial ANA was positive (1:10,240) and a repeat cavus. The mean age of onset ranged from 15 to 51 years old Although two years later showed a titer of 1:320, both with homogenous autosomal dominant inheritance is proven through male to male transmission pattern. Rheumatoid factor (RF) was negative. In the 9-year-old, it is extremely interesting that only males have a clinical picture typical of the initial ANA was positive (1:160,homogenous pattern) and her RF FSP. Among 19 females at-risk (older than 20) neurologically examined was negative but became positive during the next 12 months. In both (including 7 obligate carriers), 11 were asymptomatic, 7 had brisk reflexes in girls, the following tests were either normal or negative: C- the lower limbs and only one was minimally affected. On reactive protein, antibodies to dsDNA, Sm, RNP and streptococcal the other hand, 12 antigens. The 12-year-old also had normal or negative studies for of 20 males at-risk, neurologically examined, are clinically affected 2 had Scl-1, SSA/Ro, SSB/La, centromere, and Lyme disease antibodies. brisk reflexes and 6 are asymptomatic. Neither case had evidence of lymphopenia or of hypocalcemia during Linkage analysis, with the closest available markers for the candidate early childhood. regions, excluded the locus 14q ( marker D14S266; z= -3.19; 8 = 0.10) as well Arthritis has been associated with several chromosomal as 15 q ( marker z = - 2.37; 0 = 0.10).We are abnormalities but chromosome 22 has not been involved in these D15S128, currently testing previous cases. Review of VCFS literature likewise does not markers on chromosome 2p and the lod scores obtained so far are suggestive implicate JRA as a complication of the clinical syndrome. Our of linkage to this locus (marker D2S405, Z = 2.18; 8 = 0.05). Closer markers observations may represent a heretofore unrecognized association to the 2p candidate region will be tested in order to confirm linkage to this of VCFS with JRA. region. Supported by FAPESP and CNPq.

Posters: Cytogenetics 590 591 Marker chromosome evolution in a three generation family. S. G. Adhv~av. M. An infant with complete duplication of 17q. K.A. Aleckl, Decker-Phillips T Peters-Brown E Livingston and M B QumsiYrh. P.J. Nuttina2, and T.V. DeLaCruz2. 1Phoenix Genetics Departments of Pathology, OB/GYN, and Pediatrics, Duke University Medical Program, University of Arizona School of Medicine and Center, Durham, NC. 2Phoenix Perinatal Associates, Phoenix. Confirmed duplication of the entire long arm of We report the first case of marker chromosome evolution in three generations of a chromosomes 17 is previously unreported. Duplication of family. The index case was an amniotic fluid sample from a 33 year old, G4P3, distal segments of 17q is also uncommon, with only 16 diabetic woman referred for elevated MSAFP. Family history was unremarkable previously reported cases. The initial report of partial and amniotic fluid AFP was normal. A mosaic chromosome complement was duplication 17q by Berberich et al, and subsequent reports detected: 46,XY/47XY,+r(15); the ring chromosome was NOR negative and DAPI have suggested a clinical phenotype consisting of positive. Family studies were performed and the mother and one of her two brachyrhizomelia, hexadactyly, large mouth, and ocular and children had a bisatellited i(l5p) present in a non-mosaic state. The maternal central nervous system abnormalities. We report an infant grandfather also had this bisatellited 15 but in only 2% of his cells. FISH probes with complete duplication of 17q from a derivative confirmed the identity of the marker in the index case as ring 15 and in other family translocation 46,XX,-21,+der(17q21q). members as dic(15)(ql 1). The apparently normal male was born at full term and The patient was born at 28 weeks gestation. Physical cord tissue and blood confirmed the prenatal diagnosis. At 7 months of age, the examination at birth revealed widely split sutures, baby continues to have normal growth and development. It can be concluded that a frontal bossing, hypertelorism, bilateral cataracts, mutaion in a mosaic form in the grandfather was passed to non-mosaic state in the webbed neck, and low posterior hairline. The extremities daughter and subsequently to one of her sons and that in the index case, the marker showed asymmetric rhizomelic shortening and polydactyly. evolved by excising the satellites and forming a ring chromosome. Progressive Radiographs revealed short humeri and femora, 13 ribs and changes in the frequency and subsequent genomic change in the structure of this occult cervical spinal dysraphism. Pathologic evaluation marker chromosome again illustrates the unusual property of the proximal region of of the brain showed a Dandy-Walker cyst. lSq. Remarkably, karyotypic-phenotypic correlation from 16 reported cases and the present report suggest that the 17q duplication phenotype occurs with even the smallest distal duplication (17q25->qter), suggesting that greater amounts of duplication contribute to the severity, but not types of malformations seen.