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Published Abstracts: Cancer A361

2109 2110 Clinical correlation of complex chromsomes rearangements (CCR) in Telomerase activity in skeletal sarcomas. G Aue, M Bethi, HS Schwartz, hematological malignancies. M. N. Ahmed and M. B. Oumsiyeh. Duke MG Butler. Vanderbilt University Medical Center University Medical Center, Durham, NC. Telomerase is a ribonucleoprotein which adds TTAGGG nucleotide repeats There are few reports on clinical correlation of complex chromosome onto the ends of eukaryotic chromosomes to maintain telomere integrity. Somatic rearrangements (CCR) in hematological malignancies. The origin and cells do not express telomerase and will stop dividing when the chromosomal prognostic significance of CCR is still debated. We report ten cases of CCR ends are shortened critically after many cell divisions. Immortal cell lines and in disorders: CML cases: cancer cells apparently have telomerase activity that contributes to an unlimited hematologic 1) t(1;9;22) (p21;q34;q I1), t(2;9;22) number of cell cycles. The purpose of our study was to investigate whether p21;q34;qll), t(6;9;22) (p12; q34;qll). 2) AML cases: t(9;11;15) telomerase activity is expressed in malignant tumors of the skeletal system and to Vp21;q23;q21); t(22;16;12) (p10;p11;p12), t(15;16;17) (q22;p13;q12), compare with adjacent normal tissue. Fresh tumor and normal tissue were t(8;13;21) (q22;q13;q22). 3) idiopathic myelofibrosis syndrome t(8;11:11) collected from 14 patients (10 males, 4 females, age range of 8 to 76 years) and (p12;p24;q13). 4) ALL: t(1;1;11) (p22;q22;q23). 5) diffuse histocytic protein extraction performed. The tumors included: 10 osteosarcomas (3 before : t(2;6;9) (p21;q15;q23). Fluorescent in Situ Hybdrization (FISH) and after chemotherapy), 2 chondrosarcomas, 2 spindle cell tumors, 1 using painting probes augmented conventional cytogenetic interpretation. hemangiopericytoma 1 chordoma and 1 adamantinoma. Telomerase activity was Eight cases out of ten were of myeloid origin. It was proposed that genomic analyzed by using a highly sensitive polymerase chain reaction (PCR) based recombination leading to myeloid originates from non-specific assay (telomere repeat amplification protocol-TRAP). Protein extraction samples DNA breakage whereas that leading to lymphoid neoplasm originates were examined and telomerase activity compared with telomerase positive control during B and gene rearrangements. This would explain both the cells (HeLa). Among surgically resected tumor samples, telomerase activity was higher number of CCR cases of myeloid origin and the relatively poorer found in 8 of 14 (57%) patients using the TRAP assay. There was also a variable in complex than simple translocations. number of telomeric repeats generated by telomerase. Compared to HeLa cell extract, telomerase activity was reduced in the tumor specimens and ranged between 0 (in osteosarcoma) to 12% (in hemangiopericytoma). Seven of the 10 osteosarcomas demonstrated no telomerase activity. Histopathological analysis was performed and the number of tumor and normal cells recorded. A positive correlation was noted with the number of tumor cells and telomerase activity. However, no correlation was seen between the mitotic rate, presence of necrosis or extracellular material and telomerase activity. Telomerase activity may be an oncogenic sustaining event required to maintain the transformed phenotype seen in malignant tumors of the bone. Telomerase may be useful as a new target for therapeutic drugs as well as a diagnostic marker.

2111 2112 THE DETECTION OF Ph-POSITIVE CELLS BY CYTOGENETIC AND FISH Two of nine tumors in p53-deficient mice have an elevated ANALYSIS IN INTERFERON-TREATED PATIENTS WITH CML. C. L. frequency. VL Buettner, KA Hill, A Halangoda, M Kunishige, S Moore, W Borovikl, E. A. A. Bittencourtd, N. S. Loghin-Grosso', J. C. Guerra2 and N. Li, and SS Sommer. City of Hope, Dept of Molecular Genetics, Duarte, CA. Bacaf 3. tHereditas Sao Paulo; 2Centro de Hematologia Sao Paulo; 3Hospital p53 is a tumor suppressor gene which is mutated in a wide variety of cancers. Israelita Albert Einstein, Sao Paulo, Brazil. Mice which are nullizygous for the p53 gene are developmentally normal, but tumors Chronic myelogenous leukemia (CML) is characterized in 90% to 95% of occur by six months of age. In heterozygous mice, tumors occur by 20 months of cases by the Philadelphia (Ph) translocation, t(9;22)(q34;ql 1). According to age. p53/Big Blue double transgenic mice provide a model for comparing the Hochhaus et al. interferon-a induces remission in relationship between spontaneous in vivo somatic mutation and tumorigenesis. Big (1996), (IFN-a) hematologic Blue mice carry a chromosomally integrated lambda bacteriophage shuttle vector about 60% to 80% of CML patients in chronic phase and patients on IFN-a containing the E. coli lacl gene as a target for mutagenesis. The lambda shuttle who obtain major chromosomal responses survive significantly longer than vector can be rescued by in vitro packaging and screened for in the lad those who do not. However, since cytogenetic analysis of bone marrow (BM) gene by using a - complementation and X-gal as a substrate. Previously, we and aspirates is limited by the requirement for dividing cells, fluorescence in situ others found that mutation frequency and pattem were unchanged in normal tissues hybridization (FISH) is a very helpful tool as it can be used to detect and from p53 (-/-)/Big Blue mice. Herein, seven thymic and two quantify the BCR-ABL rearrangement in interphase cells (Bentz et al, 1994; hemangiosarcomas arising in p53 (-/-) or p53 (+/-)/Big Blue double transgenic mice Zhao et al, 1993). were examined and the mutation frequencies and pattem were determined. DNA In this report we present the results obtained from 96 patients referred for was extracted from the tumors as well as normal tissue. Ten million plaques evaluation due to suspected or clinically diagnosed CML, IFN treatment were screened and a total of 985 blue mutant plaques were harvested. The entire response or bone marrow transplant (BMT). A total of 122 bone marrow (BM) laclgene was sequenced from a representative number of the blue plaques. Jackpot were standard Of the mutations (i.e. clonal expansions early in embryogenesis or tumorigenesis) were samples processed using techniques. 76 patients excluded referred for confirmation of CML diagnosis, 12 were found to have a normal and the mutation frequency was determined. Normal thymus (p53 -1-) had a karyotype and 3 had chromosome abnormalities different from t(9;22); two mutation frequency of 5.5 x 10-5. Thymic tumors 100.86 (p53 +/-) and 100.175 (p53 -/ patients had two in addition to -) had a 2.5-fold and 2.1-fold increase, respectively, in mutation frequency relative to complex rearrangements; patients had, t(9;22), normal respectively, a second Ph chromosome and a 17q isochromosome. thymus tissue in p53 (-/-) mice (p=0.001 and 0.018, respectively). Tumor 100.86, but not tumor 100.175, had an altered mutation pattem (based on 11 34 of the BM samples came from 17 patients who had undergone IFN-a treatment. In 7 categories of mutation) relative to normal thymus (p=0.01 and 0.7, respectively). samples in which a cytogenetic response was present, FISH Further analysis revealed that deletions and insertions were underrepresented in was made with a / probe (Vysis, Inc). The cytogenetic findings tumor 100.86 (p=0.008). In conclusion, most of the nine tumors have a normal correlated well with the results of FISH analysis. mutation frequency. When the mutation frequency is elevated, the pattern of Our data represent further evidence that FISH analysis may be used to mutation may be unchanged. This is unexpected, since the pattem of spontaneous monitor patient response to IFN-a. mutation results from the aggregate of multiple mutational mechanisms.

2113 2114 Molecular histopathological analysis of signet ring cell carcinoma of the 7 in different Dukes' stages of colorectal cancers. S.-D. Cheng sigmoid colon. P. Chaudari, M.D. J. A. Varkey, M.S.1.2, M. Gupta, M.D. 1, S. and C. -M. Chen. Department of Anatomy, Chang Gung College of Medicine Luke, D.Sc.1 2, and C.T. Ladoulis, M.D.'. Maimonides Medical Center', Adelphi and Technology, Kweishan, Taiwan, R. 0. C. University 2. It is known that the trisomy 7 has been a chromosomal abnormality Molecular studies specifically fluorescence in situ hybirdization (FISH), flow identified in various tissues including bladder carcinoma, brain tumor, cytometry and were conducted on a rare case of lung immunohistochemistry signet cancer, renal tumor, and colorectal cancer. Thus, trisomy 7 may be on ring cell carcinoma (SRCC) of the sigmoid colon. SRCC is a rare variant of the mucinous adenocarcinoma and accounts for less developing routes of some tumors. In this study, we identified the trisomy 7 than 2.5% of all primary in the colorectal carcinomas in adults, and is very rarely seen in children. However, we cells from colorectal cancer tissues of different Dukes' stages. The report a case of SRCC of the sigmoid colon in a 11-year-old Middle Eastem boy, cells were obtained from short-term primary culture of colorectal cancer who died 6 months after initial diagnosis. tissues, and their interphase nuclei were detected with biotin-labeled alpha FISH, flow cytometry and immunohistochemistry where performed on satellite DNA probe D7Z1 (Oncor) with fluorescence in situ hybridization malignant tissue sections which were paraffin embedded and formalin fixed. (FISH). Our results indicated that the percentage of the cells with trisomy 7 Using FISH technology, chromosomes 1, 3, 4, 7, 8, 9, 11, 12, 16, 17 and 18 as increased with the development of the tumor according to the Dukes's well as the c-Erb B2 and RB genes were analyzed in 100 interphase cells. staging system. The average percentages of the trisomy 7 cells raised from Chromosomes 1, 4, 11, and 17 exhibited polysomies in over 10% of the cells. stages B1 (3.08%, n=7), 82 (3.62%, n=31), 83 (3.88%, n=6), C2 (4.11%, Over 20% of the cells indicated for chromosome 3, 8 and 9. While, n=32), to C3 (5.28%, n=5), and dropped slightly in stage D (4.7%, n=5). We chromosomes 7, 12, 16 and 18 exhibited a heterogeneous distribution, from concluded that the percentage of trisomy 7 cells may be related to the monosomy to . Amplification of c-Erb B2 was observed in 50% of the development of the colorectal cancer. tumor cells while of one copy of the RB gene was observed in 66% of the interphase cells studied. The apoptotic index was calculated as 1.3% using an in situ localization technique, which labeled the fragmented nucleosomes of malignant cells. Flow cytomety indicated an aneupolid population with a DNA index of 1.66 and a S-phase fraction of 7.73%. Immunohistochemistry indicated positivity for proliferation cell nuclear antigen (PCNA). The biological aggresiveness of the tumor which was indicated by the clinical the history patient is substantiated by 1) FISH and flow cytometry, both of which indicated that there were extreme chromosomal and gene abnormalities, 2) rate of high proliferation cells as indicated by the S-phase fraction and PCNA the low 3) apoptotic index suggests that the viability of malignant cells population high. (Supported by Maimonides Research and Development Foundation Grant). A362 Published Abstracts: Cancer Genetics (cont.)

2115 2116 Complex, variant translocations involving chromosomes 11 and 22 in PCR ANALYSIS OF CHROMOSOMAL ABNORMALITIES IN REHO-1, A PRE- two Ewing sarcomas: identification by cytogenetics and FISH. A. M. B LEUKEMIC CELL LINE. Jeannette C. Engle', Hope H. Punnetf and Sheldon Cherry, D. Spak and C. D. Bangs. Stanford Health Services, Stanford, N. Finvert.Department of Microbiology, Immunology and Molecular Genetics, California. Marshall University School of Medicine, Huntington, West Virginia1, St. for The ability to distinguish Ewing sarcoma (ES) and primitive neuroectodermal Christophers Hospital Children, Philadelphia, Pennsylvania2. tumors (PNET) in a group of small round cell tumors of childhood that includes ES, Chromosomal abnormalities in leukemic cells have proven to be a rich source PNET, neuroblastoma, rhabdomyosarcoma and lymphoma, is a task that is a for identifying cellular oncogenes and tumor suppressors which regulate growth diagnostic challenge based on histo athology alone. In 1984, a recurrent and development. The cytogenetics of Reho-1, a cell line established from a t( 1;22)(q24;q12) was first described in ES. A cytogenetically identical translocation male patient with pre- leukemia, revealed several chromosomal was also observed in PNET and Askin tumor. Since that time, the t( 1 ;22), or a aberrations: a der(19)t(119)(q23;pl 3) previously characterized, an variant, has been observed in approximatel 90% of ES and PNET. The add(9p)(p13), as well as undefined complex abnormalities of chromosomes 2 demonstration of the t(11;22), or more sp ically the der(22), has become a and 12. Based on karyotypic analysis suggesting that part of the short arm of 9 confirmation of the diagnosis of ES or PNET. Recently, we studied two ES tumors has been replaced by 1q23 ->qter, as well as previous studies showing frequent that had complex, four-way translocations involving chromosomes 11 and 22. Both loss of material in carcinoma and experiments evaluating gene 1 9p lymphoma, had the characteristic der(22) that is commonly associated with ES/PNET. Case of chromosome 9 markers were conducted to localize the 9p was from a boy with multiple bone lesions. His tumor had a karyotype of dosage 46,XY,?dup(1 )(q42q21 ),t(1 1;22;13;18)(q24;q12;q32;q21.1), del(16)(q 12)[18/ translocation breakpoint as well as detect any deletions. Using oligonucleotide 46,idem,-?dup(1),+1,der(16)t(1;16)(q21;q12)[2]. Chromosomal fluorescence in situ primers for a series of 16 polymorphic markers (11 tetra-, 1 tri- and 4 hybridization (FISH) using a probe cocktail containing D22S75 and D22S39 (Oncor) dinucleotide) covering chromosome 9p from telomere to centromere, Reho-1 showed that D22S39 was translocated to , and whole chromosome genomic DNA was amplified by polymerase chain reaction (PCR) and the paints (Oncor) for chromosomes 13, 18 and 11, showed that chromosome 13 resulting products were visualized following electrophoresis. Interpretation of material was translocated to , that chromosome 18 was translocated gene dosage data suggested the following: 1) An area of hemizygosity between to and that chromosome 11 was translocated to . markers D9S917 (telomeric) and D9S304 (band 21.12), reflecting a large The t(1;16) has been described in the literature as a secondary change in interstitial deletion of chromosome 9p, leaving the telomere intact, and 2) a approximately 20% of ES. Case 2 was a biopsy from a thigh mass of a giri. Her second, smaller interstitial deletion occuring within band 9p13. Evidence thus tumor had a karyotype of 47,XX,+8,t(10;14;11;22)(q22;q32;q24;q12). Chromosomal far, demonstrating loss of heterozygosity of tested markers, is inconsistent with FISH using a chromosome 14 specific telomeric probe, D14S308 (Oncor), revealed a simple translocation event in the etiology of the add(9p)(p13). In an additional that the chromosome 14 telomere had been translocated to one chromosome 11 series of experiments involving reverse transcription PCR, Reho-1 has been long arm. The seen here has also been described in the literature as a shown to harbor a cryptic translocation t(12;21), reflecting the possible secondary change in approximately 40% of ES. Further molecular analyses using RT- production of a chimeric tel/aml fusion product. Future studies should determine PCR are currently underway to see if the EWS/FLI1 fusion transcript is present in the relevance of these chromosomal aberrations to the development of B-cell these two cases. leukemia.

2117 2118 Genetic polymorphism in the p21WAF1/CIP1 cyclin-dependent kinase Non-random chromosomal aberrations in two Mixed Mulerian inhibitor gene and susceptibility to cancer. E. A. Facher and J. C. Law. Sarcomas of the endometrium. SA Farjqi, RB Deger and JS Noumoff. Department of Human Genetics, University of Pittsburgh, Pittsburgh, PA. Crozer-Chester Medical Center, Upland, PA. Cellular replication is a complex process requiring the proper balance of Mixed Mullerian tumors of the uterus are rare neoplasms that have both both positive and negative regulators of cell growth. Positive regulators carcinomatous and sarcomatous elements. When the sarcomatous commit the cell to replicate by inducing it to pass the Gl restriction point. components demonstrate heterologous elements, the prognosis in general Negative regulators stop the cell at specified checkpoints giving a is adversely affected. To date, few studies describing chromosomal replicating cell time to repair damaged DNA, preventing its incorporation aberrations in such tumors have been reported. We present two cases of into the genome. The p21WAF1/ClP1 gene is a cyclin-dependent kinase uterine mesodermal mixed tumors, both with heterologous elements; with inhibitor which primarily acts as a negative regulator of cell proliferation at the sarcomatous element including rhabdomyosarcoma. The first case, with the Gi cell cycle checkpoint. As this gene plays a role in halting the cell in tumor limited to a uterine polyp had two diploid subclones and few polyploid response to DNA damage, alterations in it may lead to unchecked cellular cells. Chromosome 12 trisomy was present in all cell populations. The proliferation or cancer. Two polymorphisms have previously been second case, presenting with diffusely metastatic demonstrated characterized in the p21 gene, the occurrence of both together having been tumor cells with chromosome number ranging from 47-60 with trisomy of shown to occur with an increased frequency in certain types of cancer. In present in all cells. of chromosomes 2 and 3 were the present study we report that a C to G base substitution 16 nucleotides present in 87% and 74% of the cells respectively. Similar reports are rare, downstream of exon 2 (A2) did not occur with an increased frequency in fifty and they describe complicated genomic constitution with numerical and prostate adenocarcinoma samples [Al/Al (0.46), A1/A2 (0.44), A2/A2 structural anomalies. Aberrations included in one study are structural (0.10)] or fifty non-small cell lung carcinoma samples [Al/Al (0.45), A1/A2 abnormalities of chromosomes 3 and 5; with another study reporting (0.44), A2/A2 (0.11)] when compared with fifty healthy, unrelated controls deletion of 1 1q22. These studies substantiate the current report's findings of [Al/Al (0.42), A1/A2 (0.46), A2/A2 (0.12)]. This polymorphism, which leads abnormalities of chromosomes 5 and 12. Additional studies including to the loss of an ALW441 enzyme recognition site, apparently plays no role molecular-genetic analysis directed to these abnormalities may provide in increasing a person's susceptibility for either of these two forms of cancer. insight to the etiology of these tumors.

2119 2120 Genetic diagnostic testing identifies VHL mutations in 62 of 65 Androgen receptor genotypes and risk of human prostate cancer. K. kindred: associated family phenotypes show one or more von Hippel- Kaartinent, J. Schleutkerl, M. Matikainenl, T. Tammelal, T. Visakorpil, O- Lindau disease neoplasms of , brain, spine, pancreas, adrenal P. KallioniemP. 'Lab Cancer Genetics, Inst. Medical Technology, Univ. glands, retina, and inner ear. G. M. Glenn1, M. Walthert, P. Choyke2, J. Tampere and TAUH, Tampere, Finland, 2Lab Cancer Genetics, NHGRI, Humphrey1, K. Hurley', A. Manolatos2, C. A. Stolle3, B. Zbart, and W. M. NIH, Bethesda, MD 20852. Linehan'. I National Cancer Institute, 2Warren G. Magnuson Clinical Center, Androgen receptor (AR) is a critical mediator of mitogenic effects of Bethesda, Maryland; 3University of Pennsylvania School of Medicine, androgens in target tissues and represents a candidate gene for conferring Intro. Dr. susceptibility to human prostate cancer (PCA). Exon 1 of the AR gene Philadelphia. by: Dilys Parry. trinucleotide CAG and GGC, encoding at contains two polymorphic repeats, Germline mutations in the VHL tumor suppressor gene 3p25-26 tracts. Low number of CAG leads to tumors and are polyglutamine and polyglycine repeats predispose to early onset of specific benign and malignant and has been individuals an AR protein with increased transactivational activity, now detectable in about 95% of kindred, identifying requiring to be associated with cancer. To test this hypothesis in include or suggested prostate clinical screening. VHL gene mutations rearrangements, single Finnish we collected peripheral and the genetically homogeneous population, nucleotide deletions, insertions, nonsense with 200 PCA multiple substitutions, blood specimens from A) 400 unselected patients PCA, B) A kindred's VHL demonstrated in a with missense mutations. genotype clinically with (2-5 affecteds), C) 90 for in patients positive family history patients affected family member may be used genetic diagnosis biologic and 400 blood donors. Simultaneous At-risk relatives are advised to benign prostate hyperplasia, D) relatives, following . genotyping of CAG and GGC repeats, and four flanking genetic markers baseline clinical and ABI undergo comprehensive screening yearly monitoring DXS1194, DXS1213, DXS1216 and DXS1275 was performed with an for VHL tumors. More precise phenotype-genotype information is needed to of tests of 310 fluorescent sequencer. reduce the economic and psychological burden screening statistical showed that the mean CAG repeat We and others Preliminary analyses families and individuals during their lifetime. in affected length was 22.2±2.8 in A, in B, and 22.1*3.2 group VHL clinical group 21.9*3.3 group previously described 3 subtypes of (family phenotypes) by between the distributions was substantial, a fraction of mutations. This D. Although the overlap criteria, and later found examples correlating germline cancer from A and B had very short confirmed mutations from of the prostate patients (5.6%) groups current Further analysis employs independently germline CAG repeats that were not detected among any of the controls. a CLIA that are correlated with the results of approved laboratory studies of these and other AR variants as well as the flanking markers may comprehensive clinical screening by CT and ultrasound of kidney, pancreas for and adrenal glands, 24 hr urinary catecholamines, ophthalmoscopy, MRI of allow improved assesment of susceptibility gene prostate brain, spine, and internal auditory canal, audiology, and ultrasound of cancer, including identification of high-risk haplotypes. epididymis in males. Current subtypes are being correlated with additional germline mutations, and additional subtypes may be found. Published Abstracts: Cancer Genetics (cont.) A363

2121 2122 The role of BLM for microsatellite instability and p53 mutation. H. t(2;13)(q35;q14) and other chromosomal changes helped establishing Kaneko, T. Fukao, R. Inoue, E. Matsui, S. Hayakawa, K. Kasahara and N. the diagnosis of alveolar rhabdomyosarcoma. J.H. Lin, H. Qiu, R. Kondo. Gifu University School of Medicine, Gifu, Japan. Hammami, 1. Mirza, S. Kosuri, D. Sabatino, M. Chester, H.O. Shah. Nassau Bloom syndrome (BS) is a rare autosomal recessive genetic disorder County Medical Center and SUNY Stony Brook, NY, USA. characterized by lupus-like erythematous telangiectasias of the face, This 13-year old black boy was admitted to hospital with complaints of sunsensitivity, stunted growth and . In addition BS skin rash, petechiae/ecchymoses over his right thigh and abdominal pain. patients are highly predisposed to cancers. In 1995 the causative gene of Physical examination revealed masses in the right abdomen. The CT scan BS (BLM) was identified as a DNA helicase homologue. disclosed the right pelvic mass, right hydronephrosis and hydroureter and We identified two siblings with a nonsense mutation in BLM. Both mild . The blood count showed RBC of 4.58x10(6)/ul, Hb siblings revealed homozygous 3-bp deletion of BLM causing stop codon at 11.6gm/dI, Hct 34.3%, platelets of less than 10x10(3)/ul and WBC of 186 amino acid residues. They developed B-cell lymphoma in their 6.5x10(3)/ul with 6%immature cells mimicking primitive myeloblasts. Right twenties. In the originally developed tumor p53 mutation was not detected. groin mass biopsy studied with light and electron microscopy and However, after treatment by ionizing radiation metastatic B-cell lymphoma immunocytochemistry narrowed down the diagnosis to between occured and it showed a 9-bp deletion in exon7 of p53. intrabdominal desmoplastic small round cell tumor versus alveolar The length of telomeric repeats and telomerase activity in peripheral rhabdomyosarcoma (AR). Bone marrow aspirate exhibited abundant blood lymphocytes of two siblings were comparable to normal controls. In immature cells in singles and in clusters. The marrow cytogenetic study lymphoma originating from two siblings, strong telomerase activity was revealed 46,XY[15y43-53,XY,t(2;13)(q35;q14), t(5;15)(q35;q22),+der(11),- detected. 18,-21,+der(21),-22,1-5marqCP5], thus AR was established. This case is a Microsatellite instability was detected by the reduced length of good example in which the chromosomal study specifically delineated not microsatellite DNA markers (HUMARA and DXS1 113) in both original and only the diagnosis of AR, but also the distinction of AR from its kindred, metastatic lymphoma. These results suggest that BLM might be involved in embryonal rhabdomyosarcoma, for AR poses a more grave prognosis and DNA mismatch repair and that the accumulation of replication error by the may require different chemotherapeutic agents. Most ARs exhibited a loss of BLM function might predispose BS patients to lymphoma or other characteristic chromosomal rearrangement t(2;13)(q35;q14). This change forms of neoplasia. was almost exclusively found in ARs and it had also been occasionally associated with other complex structural and numerical abnormalities, such as unusual additions demonstrated in this case. The patient has been improving on chemotherapy for the last 8 months.

2123 2124 Alpha satellite and whole chromosome "paint" studies of c- Germ-line NF2 mutations and tumor growth in young 1 amplification in ovarian cancer Interphase cells. W.Liu Z. Wang S. T.Smith neurofibromatosis 2 patients. V. F. Mautner, M. E. Baser, L. Kluwe, 2 C. S.R. Young. Department of Obstetrics and Gynecology, University of South Funsterer, 3 M. Sami 'Allgemeines Krankenhaus Ochsenzoll, Hamburg, Carolina School of Medicine, Columbia, South Carolina 29203 2University Hospital Eppendorf, Hamburg, 3Hamburg-Othmarschen MRI DNA amplification is a process whereby a limited part of genome is Institute, Hamburg, 4Nordstadt Hospital, Hannover increased in copy number with various consequences for the cell. It is Frameshift and nonsense germ-line neurofibromatosis 2 (NF2) mutations frequently observed in cancer cells. It has been common to use multicolor cause severe disease, and splice-site mutations cause variable FISH in the study of DNA amplification. Usually DNA probes of the sequence phenotypes. To further study genotype- phenotype correlations in patients centromeric region are labeled in different under study and its correspondring with severe disease, we examined 19 young NF2 patients from 18 families. some colors and analyzed in interphase cells. In studies it has been noted that The ages of onset of symptoms or diagnosis were 1-16 yrs and the patients genes can be arranged in either a "cluster" or "dispersed" pattern amplified had other nervous system tumors in addition to vestibular schwannomas. and that the centromeric region can sometimes appear displaced. It appears Head and full spine GE-MRIs were performed biannually from 1989-1996, that chromosomal rearrangement and the location and pattern of oncogene could be clinically important. To investigate the relationship and temperature gradient gel electrophoresis and direct sequencing were amplification used to identify mutations. The germ-line NF2 mutations that were identified the DNA sequence in question, the appropriate centromeric region between in 8 of 18 unrelated patients (44.4%) included four splice-site and the whole chromosome involved, we decided to also study the mutations, chromosome specific *paint". Therefore we would have the unique sequence, three nonsense mutations, and a frameshift mutation. The mutations the centromeric region and "paint' all in the same interphase cell. We report occurred throughout the NF2 coding region, from exons 2-15. The average here some results of our study of c-myc and . In 43 Ovarian annual growth rates of vestibular schwannomas ranged from 0.0-2.1 cm/yr; cancer cases, we found 16 (37 per cent) to have c-myc amplification, In four of intracranial meningiomas, from 0.0-2.6 cm/yr; and of spinal tumors, from cases we found disomy 8, c-myc amplification to be "cluster" and the "paint" 0.0-0.4 cm/yr. These results are consistent with previously published NF2 appeared intact. In seven cases there was disomy 8, "diffuse" amplification genotype- phenotype correlation studies, and the neuroradiological data and the "paint" was intact, In the remaining five cases there was disomy/ indicate that tumor growth rates are highly variable in young patients with polysome of 8, "diffuse" amplification and the "paint" appeared in rearranged severe disease. However, growth rates should be interpreted with caution pattern. With this small sample and short follow up period it is difficult to make due to the space-filling nature of the lesions and posible self-limiting growth. a clinical correlation but there is a trend toward "clustered" c-myc signifying pooprognosis. The complex and powerful molecular cytogenetic characterization of tumors will hopefully lead to a letter understanding of the malignant process and be useful in early diagnosis, prognosis and treatment of cancer.

2125 2126 Identification of Three Distinct Regions in Chromosome 5 Involved in Aberrant with no apparent involvement of Non-Small Cell Lung Carcinoma. P. Mendes-da-Silva1, A. Moreira 1,2 j. in acute promyelocytic leukemia with fulminant Duro-da-Costa 3, M. J. Monteiro 1, D. Matias 3, J. Rueff1 and C. Monteiro 1 clinical outcome. I. Mirza, J.H. Lin, H. Qiu, R. Abbas, E. Bayani, M. Faculty of Medical Sciences, Department of Genetics, Lisboa, Portugal; 2 Chester, D. S. Kosuri, D. Sabatino, H.O. Shah. Nassau County Medical Department of Medical Oncology and 3 Unit of Pneumology, Oncological Center, SUNY at Stony Brook, NY, USA. Hospital Dr. Francisco Gentil, Lisboa, Portugal. This 17-year old black male was admitted to hospital with complaints of Lung cancer is one of the most prevalent in the western world. disorientation, back pain and coffee ground vomiting for 2 days. The blood In attempt to determine the location of putative tumour suppressor gene(s) count showed RBCs of 2.52x10(6)/ul, Hb 8.Ogm/dI, Hct 22.6%, platelets involved in lung tumorigenesis, we performed loss of heterozygosity (LOH) less than 10xlO(3)/ul, The 13.4x10(3)u/l WBCs disclosed 8% myeloblasts studies using a panel of 14 short tandem repeat (STRs) markers spanning the and promyelocytes, 6% bands, 3% neutrophils and 10% lymphocytes. The entire chromosome 5. A total of 25 lung squamous cell carcinoma and 8 lung PT was 25 sec [reference range (rr) 11.0-12.8 sec], aPTT was 25 sec (rr adenocarcinoma samples and matched control constitutional DNAs were 21.1-34.7 sec), and fibrinogen 103mg/dl (rr 165-450 mg/dl). The brain CT using semi-automated fluorochrome- allelotyped for microsatellite alterations scan revealed right ventricular hemorrhage. Bone marrow aspirate and based methodology. A 373A DNA sequencer with GeneScan 672 software spinal tap confirmed the diagnosis of acute promyelocytic leukemia scoring and quantification. (PE-ABD) was used for electrophoresis, (APL)(AML FAB M3v). The marrow chromosomal study showed loci was detected in 60% of the squamous LOH for one or more informative 46,XY,der(17)t(?;17)(?;q23). The patient was managed for the acute clinical cell carcinoma samples and in 50% of the adenocarcinoma samples. problems and ultimately expired 4 days later. Marrow leukemic infiltrate, The study lead us to the identification of three distinct regions of loss in hypofibrogenemia and bleeding diathesis constituted the triad uniquely chromosome 5: a 46 cM region in 5pi 1 -q21.2, a 9 cM region in 5q21.3-q22.3. present in APL. Most APL showed t(15;17)with a new PMURARA hybrid and a third one of 53 cM in 5q35.3-qter. Although there was previous gene a remarkable moleculocytogenetic module -leading to APL patients of large regions of loss in chromosome 5 our results clearly identify evidence initially responding to the treatment with retinoic acid. It remains unclear three specific regions which may contain putative tumour suppressor genes. what role does a RARA gene alteration in chromosome 17 without instability was observed in any of the studied cases. No microsatellite chromosome 15 involvement play in the pathophysiology of APL. Therefore, the RER+ phenotype does not seem to be a common event in NSCLC. Acknowledgments: PRODEP, CIENCIA/PRAXIS XXI, Liga Portuguesa Contra o Cancro-Nucleo Sul, Perkin-Elmer (ABD) and CMDT/IHMT. A364 Published Abstracts: Cancer Genetics (cont.)

2127 2128 The potential relationship of Muir-Torre Syndrome (MTS) to Hereditary FISH characterization of variant Ph-producing translocations in two Non-Polyposis Colon Cancer (HNPCC). J. A. Peters 1,2, /. Kirsch 3, E. newly diagnosed CML patients. E.Rajcan-Separovic1, P. Wells2, I.Bence- Dimond L. Grogan 3, B.B. Biesecker1, D.W. Hadley'. 1 National Human Bruckle,3, HS Wang'. 'Children's Hospital of EastemOntario, 2Civic Genome Research Institute, Bethesda, MD; 2 Johns Hopkins University Hospital, 3Ottawa General Hospital, Ottawa, Ontario, Canada School of Medicine, Baltimore, MD; 3 National Cancer Institute/National We have applied GTG banding and FISH analysis to define complex Naval Medical Center, Bethesda, MD. variant translocations from two newly diagnosed cases of Ph positive CML. Through our multi-disciplinary HNPCC research protocol, we recently In one case GTG banding sunested a balanced t(9;22;9;l 1) provided psychosocial evaluation, genetic education and counseling to a 64 (q34;q11;p22;q23). FISH using the MBCR year old man regarding his personal and family history consistent with the ABL fusion gene probe and the painting probes for chromosomes 9,11 Amsterdam criteria for HNPCC. He has had 3 separate primary and 22 confirmed this finding. So far rearrangements involving bands 9p22 malignancies of the cecum, duodenum, and skin as well as a tubular villus and 1 1q23 in addition to the t(9;22) were described only in the blastic phase adenoma of the colon and keratoacanthoma on his nose. His colon tumor of CML and AML-M5a. and polyp are positive for replication errors (RER+). After thorough informed In the second CML patient an apparently balanced t(19;22) was found in consent, the proband elected to undergo genetic susceptibility testing. all cells analyzed using the GTG banding. FISH with the MBCR/ABL probe Molecular analysis of a blood specimen revealed a mutation in codon 621, detected the fusion gene on the indicating the exon 12, of the MSH2 gene, changing an arginine (CGA) to a stop codon involvement of chromosome 9. Further FISH analysis with selected painting (TGA). MSH2 is one of the mismatch repair genes responsible for HNPCC. probes suggested that the t(19;22) was a result of an unbalanced complex There is reported clinical and molecular overlap between HNPCC and MTS, translocation involving chromosomes 9, 19, 21 and 22. It could be a dermatological syndrome involving charactenstic skin tumors along with at speculated that some of the simple t(19;22) reported in CML may be in fact least one internal malignancy. There have now been several reports of of complex nature. MSH2 as well as MLH1 mutations in MTS patients. This presentation will explore the potential relationship between MTS and HNPCC as well as the implications for medical management and genetic counseling.

2129 2130 Complex pseudotriplold karyotype in a gastrointestinal stromal Diagnostic implications of a BCR polymorphism in a BMT donor: a sarcoma. B. Roland and WS.Hwang. Departments of Pathology and case report. LS Rosenblum-Vos and CA Griffin. Dept. of Pathology, Johns , University of Calgary, Alberta, Canada Hopkins Univ. School of Medicine, Baltimore, MD. A 39 year old female had previous surgery for a gastointestinal stromal Chronic myelogenous leukemia (CML) patients typically harbor a reciprocal sarcoma. Subsequently, the tumor recurred, and an abdominal and pelvic translocation of the abl proto-oncogene on chromosome 9 to the breakpoint cluster mass measuring 38 cm in greatest diameter was resected. The light and region (BCR) of chromosome 22. This gene rearrangement is detectable semi- electron microscopic features of the recurrent mass were those of a quantitatively by genomic Southem hybridization (GSH) for a non-germline BCR gastrointestinal stromal sarcoma. A portion of the tumor was cultured for 8 pattern or with high sensitivity by RT-PCR for the presence of a BCR/abl fusion and the chromosomes with the found transcript. We report a novel case: a CML patient exhibiting a false positive BCR days, harvested, analyzed, karyotype gene rearrangement following bone marrow transplant (BMT). The patient, a 27-year- to be 69,XXX,der(3)t(3;7)(ql1 .2;ql1 .2),+5,-10,+1 1, add(12)(q24.1),-13,-14,- old male with a diagnostic karyotype of 46,XY,t(9;22)(q34;ql 1), received an 17,-17,-17,+18,-19,+20,+20,-22,-22,-22,+2mar [cp5]. This is the first allogeneic BMT from his sister; at ENT no BCR rearrangement was seen. After BMT reported karyotype of a gastrointestinal stromal sarcoma. the patient *as followed by molecular diagnostics for engraftment and/or BCR gene Although some sarcomas have a simple abnormal karyotype, others rearrangement; initially the patient had fluctuating levels of mixed chimerism, with 45- have multiple chromosome aberrations. Indeed, a complex karyotype, or 50% host pattern at 90 days post-BMT. At 120 days, RT-PCR showed no evidence by flow cytometry, is for some mesenchymal tumors a hallmark of a BCR/abl fusion transcript, but GSH detected a BCR gene rearrangement with of malignancy. A very complex karyotype is also a feature of some one of three restriction enzymes, at a level corresponding to 40-45% of the DNA (80- stromal sarcoma is 90% of the cells) in the sample. Additional testing showed this rearrangement to be a advanced, metastatic sarcomas. This gastrointestinal in the donor DNA, at the 50% level associated with allelic endometrium in polymorphism similar to other stromal sarcomas of breast and having polymorphism. There was insufficient material to assay for chimerism in the 120 day significant aneuploidy. Several specific abnormalities in this tumor are sample. However, DNA from a 150 day BM sample showed only the donors notable. The loss of all chromosomes 17 and 22 may be significant in that hybridization pattem, with no evidence of mixed chimerism. By extrapolation, the 120 both of these chromosomes include tumor suppressor loci. Structural day sample may have been approximately 80-90Yo donor, consistent with the 40- abnormalities of are relatively common in sarcomas, but 45% relative level of DNA present in the apparently rearranged BCR allele derived abnormalities of 3 and 12 are less frequent. The abnormalities of from the donor in the 120 day post-BMT sample. Compilation of the BCR tumor in the marker results and mixed chimerism data provided an over-all picture negative for a chromosomes 3, 7, 12, 17, and 22 may be important pathogenesis in fact due to a of stromal sarcomas. of additional BCR gene rearrangement in the patient. The aberrant pattern was and progression gastrointestinal Analysis rare constitutional polymorphism at the BCR in the donor, complicated by cases of this rare tumor will help to clarify the significance of these mixed chimerism. This novel case illustrates difficulties in interpreting a tumor marker observations. rearrangement in a post-BMT sample and emphasizes the need to modify interpretative criteria to address differences between constitutional and cancer genetic diagnostics.

2131 2132 HER-2lneu oncogene amplification by fluorescence in-situ hybridization The Philadelphia chromosome as a secondary change in leukemia: (FISH) in ovarian neoplasms. J.S. Ross, F. Yang, R.A. Ambros, B. V.S. three case reports and an overview of the literature. Z Chen, R.Morgan, Kallakury, C.E. Sheehan, J.H. Malfetano, P.J. Muraca. Albany Medical M.Notohamiprodjo, A.Meloni-Ehrig, RT.Schustert, JS.Bennett, JD.Cohern, College, Albany, NY and Oncor, Inc., Gaithersburg, MD JF.Stone, AA.Sandberg. Genzyme Genetics, Santa Fe, NM; 'Paoli Background: The HER-2/neu (C-erbB-2) oncogene encodes for a tyrosine Memorial Hospital, Paoli, PA; Pediatric Hematology-Oncology Associates, kinase growth factor receptor with extensive homology to the epidermal growth P.C., Phoenix, AZ. and factor receptor. HER-2/neu gene amplification (AMP) protein of acute leukemia with a in and We report three cases nonlymphocytic overexpression have been associated with prognosis breast, lung The Ph was studied in ovarian carcinoma. chromosome as a secondary abnormality. late-appearing prostate cancers, but have not been extensively in the other two formalin fixed tissue sections from one patient and occurred as an additional anomaly ptients. Methods: Five micron paraffin-embedded first identified the of a minor(m)-BCR/ 74 cases ovarian were selected from the FISH studies of the patient presence of epithelial neoplasia randomly ABL The of these cases together with those surgical pathology files of the Medical Center Tumors were rearrangement. findings Albany Hospital. in the literature indicate that(1) some leukemia specific graded, staged and evaluated for amplification of the HER-2/neu gene using reported chromosome rearrangements, sch as the Ph, inv(3)(q21q26), the Oncor chromosome in-situ hybridization system and unique sequence HER-2/neu probe (Oncor, Inc., Gaithersburg, MD). Hybridization was t(15;17)(q22;q11-21), and inv(16)(p13q22) which are to be considered in the genesis of leukemia, could also appear as performed on the Ventana Gen II automated in-situ hybridization instrument primary changes involved (Ventana Medical Systems, Tucson, AZ). Results: HER-2/neu AMP was secondary anomalies involved in the progression of the disease; (2) several present in 3 (23%) of 13 serous, mucinous, and endometrioid tumors of low chromosomal anomalies seem to be closely associated with the malignant potential (LMP) whereas AMP was found in 40 (66%) of 61 epithelial appearance of a secondary Ph, such as 9p-, t(7;11)(p15;p15), and -7/7q-; carcinomas (p=0.005). In the carcinoma group AMP did not correlate with and (3) the p190 variety of BCR/ABL rearrangements may occur more often stage, grade or tumor type. With a mean follow-up of 31 months, 1 (8%) of the than the p210 variety in association with a Ph related disease progression. borderline tumor patients died of disease whereas 32 (53%) of the carcinomas More data are needed to clarify whether different expression varieties of died from disease. On univariate and multivariate analysis stage of disease BCR/ABL rearrangements are associated with different clinical courses of correlated with survival (p=0.0001) but not with HER-2lneu AMP. Conclusions: the diseases with a Ph as an additional anomaly. Thus, the Ph may play a HER-2/neu AMP by FISH can be performed on tissue sections of ovarian role not only in leukemogenesis but also in disease progression. neoplasms; HER-2/neu AMP is uncommon in LMPs, but is present in 66% of ovarian epithelial carcinomas. HER-2/neu AMP does not predict outcome in ovarian epithelial neoplasia but may play a significant role in tumor development. Published Abstracts: Cancer Genetics (cont.) A365

2133 2134 Chromosome Catastrophe and Cancer: Wilm's Tumour. J.Satish, Microsatellite instability in malignant melanoma of soft parts. H. S. Ph.D., B.S.Bhagavan, M.D., R.Luddy, M.D., M.Dadash-Zadeh, M.D. Schwartz, G. Aue, and M. G. Butler. Vanderbilt University Medical Center Molecular Pathology-Genetics and Pediatric Oncology Sinai Hospital of Malignant melanoma of soft parts, also termed clear cell sarcoma (CCS) Baltimore, Baltimore, Maryland, USA is a distinct clinical pathological entity separate from other soft tissue Increasingly, cytogenetic diagnosis of solid tumours is finding meaningful sarcomas. This rare malignancy is of origin and demonstrates applications in clinical oncology case management. A variety of karyotypic changes histological, immunohistochemical, ultra-structural and clinical features in a variety of tumours have been catalogued in comprehensive surveys. It is crystal which overlap with sporadic cutaneous malignant melanoma. Recent clear from these studies that chromosome changes in cancer are not random. cytogenetic investigations of CCS have demonstrated a distinctively Control of accuracy of non-disjunction and loss of control of chromosome number important 12;22 translocation not seen in malignant melanoma. Yet, there appear to be critical events. However, a major problem in such studies is to are similar chromosomal anomalies seen in CCS and malignant melanoma distinguish between the primary changes essential in establishing the tumour and the which include: 1q, 6q and extra copies of chromosomes 7 and 8. The fusion secondary changes which mark tumour progression from the cytogenetic noise which gene chimeric from the CCS translocation represents the background level of non-consequential aberrations. Deciphering, the protein produced 12;22 consists clinical relevance of chromosome catastrophe in cancer still remains a challenge to of the N-terminal domain of the Ewing's sarcoma gene. The purpose of this both the cancer cytogeneticist and the clinical oncologist. A series of clinical cases investigation is to further differentiate CCS from malignant melanoma using presenting with Wilm's tumour was studied cytogenetically. The relationship between molecular biological techniques. Repetitive DNA sequences termed the clinical, cytogenetic and pathological profiles were examined. Wilm's microsatellites are widely distributed throughout the human genome. Tumour(WT) or nephroblastoma is a malignant embryonal neoplasm of the kidney. Microsatellite instability (MIN) is a marker for defective DNA mismatch WT accounts for nearly 6% of pediatric tumours with an incidence of 1:10,000 live repair and manifests as somatic alterations in repeat lengths of births. Both sporadic and hereditary forms are observed. Some rare kidney microsatellites. MIN has been described in colon cancer and other neoplasms such as malignant rhabdoid tumour and leiomyosarcoma of the kidney carcinomas including 25% of malignant melanomas. Paraffin embedded can mimic Wilm's tumour histologically. Cytogenetic diagnosis, is critical in the of this class of renal neoplasms. The objective of these clinical neoplastic and non-neoplastic control cells were obtained from 11 case studies was to identify specific and recurrent chromosome changes that serve individuals with CCS (5 M; 6 F; mean age 37.4 years; age range from 7 to as diagnostic and prognostic markers of disease and survival. WT case studies 60 years). Isolated DNA from normal and tumor cells was PCR amplified at showed specific chromosome changes of 1q,11p and ploidy status to be useful 17 separate microsatellite regions using radio-labeled primers. Tumor tissue diagnostic and prognostic indicators.Abnormalities of 1q were associated with was compared to normal tissue in each instance. No MIN was detected. favourable histopathology and clinical prognosis. A summary review of current However, a single instance of loss of heterozygosity was detected at the literature on the genetics of WT is provided. Genetics of WT is complex and locus in a 48-year-old patient. Lack of MIN in CCS make it distinct from demands a re-examination of the simple notions of causality relating genotype to melanoma when using MIN for classification. phenotype.

2135 2136 Detection of a BRCA1 germllne mutation In a 45 year old male with breast Construction of Genomic Contig of the Tumor Suppressor Locus at cancer. R.J. Shnigley', P. Silverman2, S.E. McCandless', G.L. Wiesner' .2 1 1q23.3. Tanigami, A., Gomyo, H., Arai, Y., Hosoda, T., Arai, K, and Ohki, Department of Genetics and Center for Human Genetics', Department of Medicine M.. Radiobiology Division, National Cancer Center Research Institute, and Ireland Cancer Center2, Case Westem Reserve University School of Medicine Tokyo, Japan and University Hospitals of Cleveland, Cleveland, OH 44106. Chromosome band 1 1q23.2-23.3 harbors the tumor suppresser gene(s) Breast cancer in American men is an uncommon tumor with a lifetime risk of causing breast cancers, lung cancers and neuroblastomas on the basis of appoximately 0.02%. This risk is markedly increased for men who are germline carriers of a breast cancer susceptibility allele. The lifetime risk for male BRCA2 loss of heterozygosity study. We have made genomic contig, using P1 and carriers is estimated to be between 5-7%. However, the risk for breast cancer in PAC (P1-derived artificial chromosomes) clones to isolate the causative male BRCA1 carriers is not clear, mainly due to the small number of cases in known gene(s). We screened 2.5-genome equivalent DuPont's P1 library, but BRCA1 families. could not find any clones to fill three gaps in the contig. We constructed We report a small family that was identified by the occurrence of a male with PAC sublibrary from YAC which covers the gap and got some PAC clones breast cancer. The index case was a 45 year old previously healthy man who was filling the gap. However we have to screen other libraries such as whole- diagnosed with unilateral invasive ductal carcinoma at the age of 42. His family genome or chromosome-specific PACs, BACs or Fasmids library because history revealed three cases of female breast cancer. His only sister had died of we still have two gaps. Our contig spans more than 1.5-Mb region. breast cancer at the age of 30, his mother died of breast cancer at age 40, and his containing three Notl recognition sites. The chromosome band 1 1q23 maternal grandmother died of breast cancer at an unknown age. There were no cases of ovarian, prostate, or other types of cancers reported. The cancer diagnoses belongs to G-band, where it is reported that LINE sequences are rich, SINE in the index case, his sister and mother were verified by medical record review. is poor, and that gene is big and the number is a little. We developed -60 Molecular diagnostics was performed by a commercial laboratory on a sample of STSs and found five LINE sequences and only seven SINE sequences. DNA from the index case after giving his informed consent. Mutational analysis for Also shot-gun sequences of two clones show very poor SINE sequences both BRCA1 and BRCA2 revealed a mutation in exon 11 of the BRCA1 gene, (38 out of 671). We are trying to isolate cDNAs from these contigs, using causing a stop codon at position 563 of the BRCA1 protein. No mutation was conventional cDNA library screening, exon trapping, CpG island rescue and identified in BRCA2. This mutation has been described previously in other families genome sequencing. Unfortunately, it is refractory to isolate cDNA although with early-onset female breast cancer, but has not been reported in families with our efforts are still going on. cases of male breast cancer. This case illustrates the importance of counseling and testing for both BRCA1 and BRCA2 in families with male breast cancer, since the occurence of male breast cancer has previously been thought to indicate a mutation in BRCA2. Predicting the underlying genetic mutation in families with an autosmal dominant form of breast cancer is clinically difficult and should be supported by genetic counseling and laboratory analysis whenever possible.

2137 2138 Potential demand for genetic evaluation services for colorectal cancer Cytological, cytogenetic and FISH studies of a recurrent predisposition in an inpatient surgical service at an academic hospital. astroblastoma. M. Tyrkus, J. Wyatt-Ashmead, K. Zilch, Y. Takaoka, M. B.L. Tumer1, S.M. Jones2, W.F. CohnW, S. Miesfeldt' . 'Division of Hematology/ McCorquodale, J. Diaz-Nazario, and D.J. McCorquodale. MetroHealth Oncology, 2Cancer Center, 3Department of Health Evaluation Sciences, Medical Center, Cleveland, OH, Rockford Memorial Hospital, Rockford, IL, University of Virginia Health Sciences Center, Charlottesville, Virginia. and Columbia Michael Reese Hospital, Chicago, IL. Demand for cancer genetics service is potentially great. Identification of risk Astroblastomas are rare neoplasms of glial cell origin that occur primarily in older or the for, presence of, a heritable cancer predisposition syndrome (HCSD) may children and young adults. We here report a primary (PT) and recurrent (RT) affect medical and surgical management of patients. To examine the needs for astroblastoma in a 25 year-old pregnant woman. Initially, at 24 weeks gestation, she inpatient cancer genetics services, we reviewed the charts of all patients (N=32) complained of left frontal headaches. Radiologically, she had an extra-axial, focally admitted to a university hospital in 1995 for surgical management of newly cystic tumor (7.5x6.0x6.0cm). The tumor had a broad base along the dural surface diagnosed, resectable, pathologically-confirmed colorectal cancer. Complete and did not extend to the ventricle. At 28 weeks gestation, the PT was resected. The medical records were available for 30 patients. Family histories were recorded in PT was focally attached to, but did not infiltrate the dura. At 40 weeks gestation (3 28 charts. Of these, 22 showed a positive family history of cancer, including 14 months after resection) her baby was bom vaginally. Although no residual tumor was suggestive of an HCSD. Eight were suggestive of HNPCC, 1 of attenuated FAP, seen radiologically for the first 4 months after resection, the tumor (5.1x4.5x4.0cm) and 2 of the breast and ovarian cancer syndrome; an additional 3 patients had recurred within 8 months. The RT was resected along with a rim of surrounding family histories that did not conform to any described HCSD, but were parenchyma. The PT and RT had similar macroscopic and microscopic findings. suspicious (because of the numbers and types of cancers recorded) for an Microscopically, the tumor glial cells radiated around myalinized vessels. A portion of HCSD. In 5 cases where a family history was gathered, little evidence was seen the RT was submitted for cytogenetic studies. Cells were harvested after 7 days in suggesting the presence of an HCSD. Three charts contained family histories culture and examined by GTG banding. A karyotype of too incomplete for interpretation. Thirteen of the 30 patients with complete 47,XX,+2,t(5;22)(q31 ;ql1 .2),del(11 ?)(p14?)[18]46,XX[2] was observed. We then medical records had a personal history (multifocal tumors, >1 primary cancer, evaluated both tumors with Fluorescent In Situ Hybridization (FISH) to determine if and/or young age of onset) suggestive of an HCSD. Of these, 11 also had a the trisomy 2 was present in the initial tumor. Paraffin embedded tissue sections of of cancer. tumor and adjacent tissue wre studied using a centromeric probe (D2Z) for family history A total of 19/30 patients (63%) had either or both a chromosome 2 and/or medical an (Oncor). Three signals (indicating trisomy 2) were observed in a personal family history suggestive of HCSD. Of these, 2 of the cells from the but 2 were seen in have been referred to the cancer majority RT, only signals the PT, suggesting (-11%) university genetics clinic. We conclude that the second tumor had either evolved cytogenetically or was a new primary. The that there will be a significant need for cancer genetics consultation for patients literature suggests that numeric anomalies of chromosomes 10, and 22 may be admitted for surgical resection of a newly diagnosed colon cancer. The results of frequent findings in these tumors while structural changes of chromosome 2 and this pilot study reveal the need for: 1) inpatient cancer genetics services; 2) others are less often observed. To our knowledge, the present report is the first case education of physicians regarding the role of family and or personal medical documenting trisomy 2 and t(5;22) in an astroblastoma. histories in identification of heritable cancer predisposition. A366 Published Abstracts: Cancer Genetics (cont.)

2139 2140 Characterization of clonal chromosome rearrangements using FISH In a cell Clinical diagnostic laboratory experience with BRCA1 and BRCA2 line derived from ovarian epithelial cancer (EOC). J. C. Wang', N. Lagousakos', utilizing DNA sequencing technology. BE Ward, S Manley, M McClure, M. M. Bielanska1, A. M. Mes-Masson2, P. Tonin1, D. Provenchet2, S. Demczuk1, P. M Staebell, T Scholl, K Gumpper, B Lingenfelter, A Oliphant, J Haskell, TS Eydoux' 3. 1McGill University and 2Universite de Montreal, Montreal, Quebec, Frank. Myriad Genetic Laboratories, Salt Lake City, UT. Canada; 3Hopital Robert Debre, Paris, France. Deleterious mutations of BRCA1 and BRCA2 in our clinical laboratory We characterized clonal abnormalities in an EOC cell line (OV90) using classical are identified by comprehensive DNA sequencing. Over 6,400 DNA base and molecular cytogenetic techniques. Clonal cytogenetic abnormalities included a pairs are sequenced in both the forward and reverse direction for diagnostic homogeneously stained region (HSR) replacing chromosome 3p, a complex analysis in BRCA1 and over 11,100 base pairs for BRCA2. Frameshift translocation identified with classical cytogenetics involving chromosome 9 and 17, mutations are with a loss of the derivative (9p;17p)(p10;p10), and an add(13)(p1l). Using mutations, nonsense mutations and other reported causal chromosome specific centromeric, telomeric and painting probes, we were able to defined as deleterious. We here present the referral patterns and results of further characterize these conal abnormalities. The karyotype as established with the first 200 clinical specimens analyzed for deleterious mutations in FISH was: BRCA1 and/or BRCA2 by comprehensive sequencing or analysis of three 46,XX,der(1)t(1;1 0)(p36;pl 5),hsr(3)(pI1 ),der(9;1 7)(qlO;ql0),der(10)t(10;1 7)(pl5;p1 specific mutations in individuals Qf Ashkenazi descent. Greater than 80% of 2p13),der(13)t(13;13)(p11;q14). The HSR originated from chromosome 22, but did patient specimens were referred to our laboratory from institutions with not involve loci D22S75 in region 22q11.2 and D22S39 in region 22q13.3. To our comprehensive genetics and/or cancer units. Documentation of informed knowledge, this is the first example of amplification originating from chromosome consent was obtained in all cases. The presence of two or more family 22 in EOC. The translocation (9;17) was identified with FISH as a complex members affected with breast or ovarian cancer was noted on 86% of rearrangement involving chromosomes 1, 9, 10 and 17. Breakpoints were located requisitions which reported family history (118 of 137 requisitions). For at chromosome bands 1p36, 10p15, 17p or q10 and 9p or q10. Resulting and had a chromosome imbalances were monosomy 3p and 9p, partial monosomy 17p, and patients who were both affected with breast or ovarian cancer partial trisomy for the distal end of chromosome 13q. Loss of heterozygosity (LOH) positive family history the following results were obtained; deleterious at some of these chromosomal regions has been described previously in ovarian mutations were diagnosed in 24% of patients referred for combined BRCA1 cancer, including within chromosome 3p, 9p and 17p. To our knowledge, no plus BRCA2 sequence analysis and in 31% of Ashkenazi Jewish patients recurrent translocation breakpoints and associated cancer genes have been referred for analysis of three specific mutations. Within the combined identified at chromosome bands 1p36 and 10p15. Conversely, chromosomal population of all 200 specimens, including currently unaffected individuals, breakpoints at 17p12-13 have been seen in neoplasms of numerous organs. The 13.5% had a deleterious mutation. Laboratory analysis of these specimens chromosomal changes observed in this cell line was consistent despite of long term was completed in an average of 21.4 days. These data indicate that clinical cell culture and repeated freezing and thawing; they may be related to testing for BRCA1 and BRCA2 demonstrates an appropriate referral pattern tumorigenesis, tumor evolution or even in vitro cell transformation. Cytogenetic and molecular cytogenetic studies aid in the identification of critical sites involved in as well as a clinically significant proportion of patients with deleterious cancer induction or progression, and can be used to orient further molecular studies. mutations.

2141 2142 Frequency of loss of heterozygosity at the ATM and the BRCA1 loci in Renal cell carcinoma with X;1 translocation in a child with Klinefelter women with Stage III and IV breast cancer. M. J. Worsham, S. R. Wolman, U. syndrome. A. Yenamandra, X. T. Zhou, S. Sastry, L. Trinchitella and L. Raju, N. Bamabas, S. D. Nathanson, G. Pals . Henry Ford Hospital, Detroit, Mehta. North Shore University Hospital - N.Y.U. School of Medicine, Uniformed Services University of the Health Sciences, Bethesda, MD, and Vrije Manhasset, NY. Universiteit, Amsterdam, The Netherlands (KS)is a sex chromosome abnormaity occurring in 1 Five to ten percent of all breast cancers are attributable to hereditary in 1000 males. An association with leukemia, germ-cell tumors and male predisposing genes. Mutations in the BRCA1 and LOH from chromosome 17q breast cancer has been suggested in KS. Such information is important for (BRCA1 locus) in both familial and sporadic tumors support the hypothesis that professionals caring for KS patients as the condition is frequently not BRCA1 is a tumor suppressor gene. LOH in this region is observed in 30-70% of clinically recognizable until after puberty. We report on a 10 year old boy sporadic breast cancers examined as well as in sporadic ovarian cancers. The with KS, the fourth child of a now 51 year old mother. He developed ATM (ataxia telangiectasia, mutated) gene may contribute to or account for risk in intermittent hematuria at age 10 years and was diagnosed with a right 8% of all breast cancers, with cancer predisposition in heterozygotes estimated to kidney mass which on pathology was identified as renal cell carcinoma. In be three to fourfold that of the general population. In the heterozygote, the mutant delays. ATM may also play a tumor suppressor role in the development of breast cancer. addition, he was known to have learning and language ll flanking and Peripheral blood chromosome analysis had revealed a 47,XXY karyotype. We evaluated 31 women with stage and IV breast cancer using involving translocation intragenic markers in the ATM gene and intragenic markers for BRCA1. Archival Analysis of tumor cells showed clonal abnormalities specimens from 31 women were the source of DNA obtained from normal breast between chromosomes X and 1, designated (a) 46,Y,t(X;1)(p11 ;q21)[47%] and nodal tissue, and primary tumor tissue microdissected from 4 micron paraffin (b)47,Y,t(X;1),+X [40%], (c) 47,Y,t(X;1),+r(Xp) [13%]. Renal cell carcinoma sections. The mean age at diagnosis was 61. Tumor stage, type and grade is rare in childhood and is not previously reported in KS. The Xp11 included: 21 stage 3, 9 stage 4, and 1 undefined; 23 invasive ductal carinomas, 3 breakpoint, a region with highly repetitive sequences, has been reported to invasive lobular and 5 undefined; and 17 grade 3, 8 grade 2, 1 grade 1, and 4 be involved in translocations with chromosomes 1 and 17 in adults with undefined. LOH for BRCA1 employed intragenic markers )D17S855 and sporadic renal cell carcinoma. The significance of the association of these D17S1323, and for the ATM gene, markers D11S1818, D11S1819, and unusual events in our patient is not clear. It is possible that the D1 1 S2719. Using these markers both LOH and microsatellite instability (MI) could constitutional aneuploidy increases the risk of genomic be determined simultaneously. LOH/MI for any one of the two markers for BRCA1 instability. and for any one the three markers for the ATM gene was scored as an LOH/MI. The frequency of LOH/MI instability for BRCA1 was 45% (14/31) and for the ATM gene 32% (10/31). The similarity of results support an interpretation that both these genes function as tumor suppressors. Correlation with tumor stage, grade, hormonal status, survival, and other parameters will be reported.

2143 2144 Molecular Changes in Adenomas, Adenocarcinomas and Metastatic Assessment of cytogenetic results of 1339 cancer bone marrow Tumors within the Same Individual Presenting with Colorectal chromosome studies. S. M. Znelmer, J. B. Ravnan, L. S. Jenkins. Kaiser Permanente, of San Jose, CA. P. M. Saint Barnabas Department Genetics, Disease. N. P. Zauber, S. Marotta, Sabbath-Solitaire. Cytogenetic analysis for the diagnosis of hematologic malignancies has become standard Medical Center Livingston, NJ 07039 among an array of feats for oncologists. Currently, there are many non-random chromosome Mutations of the Adenomatous Polyposis Coli (APC) gene located on abnormalities associated with specific hematologic malignancies which play a major role in the in colorectal diagnosis and prognosis of these disorders. What remains unclear is the overall frequency of chromosome 5q21 is recognized as an early change cytogenetic abnormalities found with different types of and lymphomas, especially tumorigenesis. Mutations in the Ki-ras oncogene on chromosome 12p is an from initial indication versus final clinical outcome. This study is a review of three years of additional, later change present in many sporadic colorectal adenomas and cytogenetic results of cases with an indication of a known or suspected hematologic adenocarcinomas. We evaluated samples of colon carcinomas, adenomas malignancy. These results were obtained from a laboratory performing 600-800 bone marrow of the APC tumor specimens per year for at least 100 providers in a non-specialty cancer center. Results are and distal metastases for Loss of Heterozygosity (LOH) categorized based on the initial indication for study, documented by the referring physician, suppressor gene and for mutations of Ki-ras oncogene from 14 patients and are shown below. classified as having sporadic colorectal carcinoma. Ten of these patients with adenocarcinoma were also found to have an adenoma. All 14 patients iC1dw" IotL11AWLUO % N o .13l displayed metastatic disease. DNA was extracted from previously selected MDS 481 36 22 3.3 were in a Polymerase Chain CML 230 17 71 6.5 paraffin-embedded tissue and all amplified 6.4 using sets. The LOH for the APC gene was AML 189 14 23 Reaction (PCR) specific primer ALL 137 1() 40 11.7 detected by amplification of a polymorphic dinucleotide repeat, and Lcukemia 11)2 X 41 1(.8 mutations of codon 12/13 of Ki-ras oncogene was assessed using the Lymphimna 92 7 14 5.4 normal muscularis mucosa was used as the CLL 70 5 26 2.9 SSCP method. Autologous 31 20 7.9 negative control for each case. In our evaluation of individual patients MPD 3 of presenting with adenomas, carcinomas and metastatic disease, 50% Results show a clear predominance with the indication of myelodysplasia (and associated them presented with a molecular progression between their adenomas and anemias), and the frequency of abnormal cytogenetic results is very low (22%). CML, the a progression from their second volume test indication, shows a 71% abnormal result rate, which is lower carcinomas. Fifty percent also showed molecular highest by for low abnormal carcinoma to their metastatic tumor. Specifically, no progressive than the widely reported 85-95% level. Possible explanations frequencies primary based on inital indications include the physician's criteria for ordering studies and different change in Ki-ras mutation was observed between the primary carcinoma patient populations seeking care at a general facility rather than a specialty cancer center. and the metastasis for most of our patients (13 out of 14). Data regarding Published reports, when specified, often correlate final clinical outcomes with abnormal specific types of Ki-ras mutations will be presented. Also, those patients cytogenetic results, and in addition there is little consensus of abnormal frequencies for each their primary tumor to their disease. Laboratories such as ours that are independent from clincial oncology groups do not showing a molecular progression from access to final clinical outcomes. For assurance purposes, it may be best to LOH for APC gene. have easy quality metastatic tumor all presented with correlate abnormal frequencies based on initial indications. Published Abstracts: Clinical Genetics, Malformations and Dysmorphology A367

2145 2146 Melnick-Needles syndrome: report of the first Brazilian case with FISHing for a new syndrome or chasing a mirage. G.M. Azar, R.A. mother-daughter transmission and literature review. L.M.J.Albano, Conte, S.M. Kleyman, A.Z. Logush, R.S. Verma. Institute of Molecular Biology C.A.Kim, S.M.M.Sugayama, C. Y.Utagawa, C.E.F.Andrade, M.F.Barba, V.K. and Genetics at InterScience, Brooklyn, The Stanley S. Lamm Institute for Lee, L.Monteiro, C.H.Gonzalez. Department of Pediatrics. Instituto da Child Neurology and Developmental Medicine & Long Island College Hospital- Cnanga - HCFMUSP, S. Paulo, S.P, Brazil. (Intro. by M.Zatz) SUNY Health Science Center at Brooklyn, N.Y. This syndrome - first described in 1966 by Melnick and Needles - is The dysmorphic features associated with a variety of chromosomal characterized by a typical facial appearance, that usually provides the abnormalities have been a fundamental basis of clinical genetics. For the past diagnosis, with exophtalmus, micrognathia, full cheeks, malalignment of three decades, exhaustive attempts have been made to zero-in-on an teeth and by a severe generalized bone dysplasia with: sclerosis of skull abnormal morphology which is apparently associated with gene(s) rather than base and mastoids, metaphyseal flaring, shortening and bowing, S-shaped with a chromosomal segment. Consequently, many syndromes which were appearance of tibia, cortical of clavicles, tubular bones and nibs thought to have chromosomal basis are due to a single gene. This approach irregularities has resulted in describing a new syndrome with obscure chromosomal (ribbon-like), vertebrae with increase in height and anterior concavity and abnormalities. For example, a 7-year-old boy was referred for cytogenetic abnormalities in other bones, as the flat ones and sternal. The mode of evaluation because of psychomotor delay and behavioral problems. The inheritance has been described as X-linked dominant, lethal in males with a physical examination revealed: coarse facial features, frontal bossing, bushy few surviving male cases, probably due to new mutations. Up to now, there eyebrows, prominent ears, a small uptumed nose and a history of repaired are about 48 well-documented cases reported, mainly single cases, having inguinal hernia. Routine karyotype showed an additional material on only 10 familial reports, involving 11 kindreds (6 mother/daughter and 5 chromosome 4q, which could easily be matched with bands 18q21.2, 2p24- mother/son) and 4 surviving male cases. p25, 16p21-p23, 10p12-p14, 20q12-q13.2, 15q25-q26.2, 8p23-p24.2 and A case of mother-daughter transmission of this so rare syndrome and 6p22.3-p24. Parents were unavailable for cytogenetic evaluation in a search also being the first Brazilian case reported up to now, led us to report a for a balanced translocation. Therefore, using FISH technique, a battery of female, aged 14 years, propositus, product of a full-term gestation with probes had to be used to determine the origin of the extra material, which prominent forehead, exophtalmus, full cheeks, micrognathia and turned out to be 3q26.3-qter. Consequently, the child had a partial trisomy 3q malalignement of teeth and with the typical radiological abnormalities of this syndrome, which was not identified clinically. However, should FISH technique syndrome. We have also studied her mother whose features and have not been used, the patient would have been diagnosed as trisomic for radiological findings were compatible with Melnick-Needles syndrome. any one of the aforementioned segments and a new syndrome would have emerged. Obviously, previous cases of a so-called Atrisomy 3q syndrome@ need to be re-evaluated using current approach. Nevertheless, this suggestion is neither practical nor feasible in present health care environment!

2147 2148 Cardlomyopathy in Kabuki Syndrome. C.A. Bay 1.4 V. TsueP3, A. Mank- Bilateral cleft lip and palate, agensis of corpus callosum, multiple Seymour, G. Matika1, and S. Webber2'4. 1 Division of Medical Genetics, Children's congenital anomalies: A possible new syndrome. S. Beiraghi, M. Hospital of Pittsburgh 2 Division of Ped. Cardiology, Children's Hospital of Pittsburgh MacDonald, G.B. Schaefer andA. Olney. University of Nebraska Medical 3 University of Pittsburgh School of Medicine 4 Dept of Pediatrics, University of Center Pittsburgh Agenesis of the corpus callosum is often associated with craniofacial, skeletal We recently evaluated a 19 month old male with facial features consistent with and other developmental defect of cerebrum. We report on an 8 month old white Kabuki syndrome, who required cardiac transplantation at 5 months of age due to male with severe severe congenital hypertrophic cardiomyopathy and progressive left sided ventricular bilateral cleft lip and palate, agenesis of the corpus callosum, outflow obstruction. Although cardiac structural abnormalities are common in Kabuki associated with lobar holoprosencephaly, CNS lipoma, diabetes insipidus, syndrome, we believe this is the first example of cardiac hypertrophic hydronephrosis and secondary . Physical examination showed cardiomyopathy in this disorder. facial dysmorphism, with prominent nose, high nasal bridge, dysmorphic ears and Growth parameters were between 5-10 centiles. Features included a typical facial tapered fingers, with 5th finger clinodactyly, as well as partial syndactyly of 4th appearance of Kabuki syndrome due to long palpebral fissures with eversion of the and 5th toes bilaterally, , slight on left and moderate on right side. lateral portion of the lower eyelids, epicanthal folds, ptosis, blue sclerae, and a short Pregnancy was complicated by hypertension and subsequent delivery at 33 nose. He had low set, posteriorly rotated ears with uplifted lower lobes, overfolded weeks gestation by C-section. Apgars were 8 and 8 at one and five minutes superior helices, a webbed appearance to his neck, a low posterior hairline, respectively. He was on apnea monitor for a period of 3 months due to the hyperconvex nails, and pectus excavatum. He did not have prominent finger fat prematurity; chromosomal studies were normal male 46,XY. Family history is pads. Developmental delays were apparent. There were no other affected individuals significant in that mother has Klippel-Feil anomaly, Sprenges deformity, a broad in the family. Echocardiography prior to transplantation revealed progressive severe short and webbed neck, submucous cleft palate, and subluxation of the left hypertrophic cardiomyopathy, with left ventricular outflow obstruction. He had marked humeral head. Her father has sloping shoulders and maternal two cousins have global hypertrophy of the entire left ventricle. Outflow obstruction worsened, at at 5 cleft palate. She has had one spontaneous at 6 weeks of gestation. months he underwent successful cardiac transplantation. He was initially considered Her first son was bom with bilateral cleft lip and palate, hypoplastic left heart, to have Noonan syndrome due to the typical cardiac findings, webbed neck and vertebral anomalies and, dysmorphic features, which included , pectus excavatum. Routine chromosomes and renal ultrasound were normal. short neck with low posterior hairline, narrow shoulders, and mild webbing of the In a recent series Hughes noted 55% of individuals with Kabuki Syndrome to neck. The baby died after repair of his hypoplastic left heart. Chromosomal have congenital heart disease. The differential diagnosis considered in this patient studies for FISH .22q was normal for all three of them. Clinical findings in this included both Kabuki and Noonan syndromes, since he had features of both family clearly points toward a syndrome. However, our search of literature did not disorders. However, his facial apperance, so typical of Kabuki syndrome, and the reveal any between clinical manifestation our blue sclerae the true similarity of patients and reported suggest diagnosis is Kabuki syndrome. Altematively, he could syndromes. It therefore seems reasonable that this case might represent a new represent an overlap between the two syndromes. This autosomal dominant disorder with bilateral cleft lip and palate with agenesis of the case suggests that Kabuki syndrome should be considered in the differential corpus callosum and other major multiple congenital anomalies. diagnosis of patients presenting with hypertrophic cardiomyopathy.

2149 2150 Partial agenesis of the corpus callosum in LEOPARD syndrome. C. CLINICAL, HISTOPATHOLOGICAL AND MOLECULAR ANALYSIS OF Be//ini1, A. Di Stefano, V. Per, S. Costabel, M. Mazzarello, E. Bonioli. THANATOPHORIC DYSPLASIA IN A 19 WEEK FETUS. C. Brown' and M. Istituto di Clinica Pediatrica e 11stituto di Puericultura e Medicina Neonatale Levin. *Department of Molecular and Human Genetics, Baylor College of dell'Universitadi Genova - Italy. Medicine, Houston, Texas, USA and Department of Pediatrics, University of The multiple lentigines (LEOPARD) syndrome is an autosomal dominant entity Nevada School of Medicine, LasVegas, Nevada, USA. with high and variable expressivity. It consists of a generalized, We describe a male fetus, stillbom at 19 weeks gestation following induced symmetric distribution of Lentigines associated with Electrocardiographic vaginal delivery. A skeletal dysplasia was diagnosed by sonogram. Prenatal abnormalities, Ocular hypertelorism, Pulmonary stenosis, Abnormal genitalia, findings included severe micromelia of all long bones, short ribs, curved femurs and growth Retardation, and sensorineural Deafness. Minimal diagnostic criteria a small chest. The skeleton appeared appropriately mineralized. No fractures were include a combination of most or all of the above findings. We diagnosed Leopard identified. The family history was negative for consanguinity, skeletal dysplasias and birth defects. The stillbom fetus had a length of 18 cm (<5th percentile) and an syndrome in a 4 old month child. His was 5.420 5th weight Kg (below percentile), arm span of 14.5 cm. The upper to lower segment ratio was 13.5cm:4.5 cm. The length was 59 cm (1 0 percentile), and head circumference was 40 cm (5t face was non-dysmorphic. The chest was narrow with a bell-shaped appearance. percentile). Hypertelorism, epicanthus, strabismus, downslanting palpebral The abdomen was protuberant. Both upper and lower extremities had mesomelic fissures, large and low-set ears, enlarged nasal tip, and mandibular hypoplasia and rhizomelic shortening with abnormal curvature. The genitalia were normal. were evident. Multiple, generalized and symmetrically distributed lentigines were Radiographs showed normal bone density with shortening and campomelia of all observed, ranging in size between 0.2 and 0.5 cm. Neurologic examination was long bones; flattened, hypoplastic thoracic and lumbar vertebrae; a hypoplastic normal and the motor milestones were within the normal range. pelvis; and moderately shortened ribs. A post mortem examination revealed a Echocardiography demonstrated partial anomalous pulmonary venous return, malformed foramen magnum, flattened vertebrae, and pulmonary hypoplasia. Bone with the left pulmonary vein draining into the left innnominata vein. Brain stem histology showed normal resting cartilage with a severe disturbance in auditory evoked potentials were compatible with right periferal hypoacusia and endochondral ossification, lack of columnar alignment, and ingrowth of vascular defective truncal conduction. Brain echography and MRI demonstrated partial mesenchymal tissue interrupting the growth plate. The radiographic findings were posterior agenesis of the corpus callosum. most consistent with an atypical form of thanatophoric dysplasia; platyspondylic of is lethal Agenesis corpus callosum considered to be consecutive to an insult to the neonatal dwarfism (SanDiego type). The histologic findings were less clear, commissural not plate during early embriogenesis and is frequently associated with though inconsistent with this diagnosis. This case illustrates the difficulty that anomalies due to can be encountered cell migration defect. However, as an isolated when trying to distinguish the variants of thanatophoric it be in fetuses of this A phenomenon may compatible with normal cerebral function. Although about dysplasia age. clinical description including autopsy results, 30% of with LEOPARD of the bone patients syndrome present mental retardation, to our photographs fetus, histology, radiographsand the results of mutational at the FGFR3 locus will be knowledge partial agenesis of corpus callosum has never been reported in the analysis presented. Although specific genetic mutations literature. evaluation would be in the variant Neuroimaging should be part of the diagnostic work up of helpful distinguishing among forms, recent evidence that LEOPARD syndrome. suggests the same 'common' mutations at the FGFR3 locus may be responsible for both common and variant forms of thanatophoric dysplasia. N368 Published Abstracts: Clinical Genetics, Malformations and Dysmorphology (cont.)

2151 2152 3ycnodysostosis in Eniwetok: coincidence or founder effect? J. Microtla and short stature: Report of a new syndrome. V.D'Almeida;, 3rumblay1l4, B. R. Powel1',4, K ReinkeW2, G. W Henry2, T. GallagheP3, Y. F.T.Lima;, A.B.A.Perez;, D.Brunoni. Centro de Genetica MWdica - Escola . Hsia"'4. Kapiolani Medical Center for Women and Children1, Shriners Paulista de Medicina - UNIFESP - SAo Paulo - SP - Brazil. iospital2, Tripler Army Medical Center3, University of Hawaii4, Honolulu, A 12-year old boy, born of nonconsanguineous parents, a japonese lawaii. father and a black Brazilian mother, is described. He has short stature, birth weight and poor weight This rare disorder (McKusick 265800) has classical clinical and microtia, , mental retardation, low adiographic features. Toulouse-Lautrec had this phenotype. Many affected gain, characteristic facial features, skeletal anomalies and delayed bone groups have consanguinous parents. age. All these characteristics are seldom seen together, and they are likely :hildren from several ethnic with Marshallese boy was short with acromelia, to be a new syndrome. This patient shares many features in common CASE 1: An eight year-old two sibs seen by COHEN et al. (J Med Genet 28: 786, 1991); two unrelated arge, square, bathrocephalic head; huge fontanelle, widened sutures; and a boy Protuberant wide-set eyes, small nose, mouth, chin and ears; broad chest, boys reported by HURST et al. (Am J Med Genet 29: 107, 1988); vide-set nipples; short stubby telescoping fingers, short deformed legs; and reported by GORLIN et al. (BDOAS 11: 39, 1975). These similarities might i deep voice. Cranial sutures were extremely widened, with multiple expand the knowledge of a new syndrome. flormian bones; remarkably absent mandibular angle; extensive frontal, )eriorbital and axial osteosclerosis. His paternal grandmother was said to )e similar and several others in Eniwetok were thought to have similar )hysical features. CASE 2: A 20 month-old, apparently unrelated Marshallese boy had a )rominent forehead, box-shaped skull, wide anterior fontanelle; prominent ayes, midface hypoplasia; lax joints, wide rounded hands and feet. X-rays ;howed brachycephalic skull, large fontanelles, hypoplastic mandible; iormal pelvis, hands and feet. A paternal cousin and two maternal second cousins were "midgets" with big heads and short hands. The older boy has the classical phenotype, and the younger boy has tarly signs of pycnodysostosis; Hadju-Cheney syndrome seemed unlikely. rhe family history was tantalizing. Eniwetok in the Marshall Islands, must iave a very high inbreeding coefficient. A high prevalence of )ycnodysostosis is likely to have arisen from founder effect in an isolated Population of three to four thousand.

2153 2154 a further delineation of the Yunls-Varon syndrome. 4eurocutaneous melanosis sequence- unusual manifestations in A new case report and ' iewborn. G de Jong and HF Jordaan. Tygerberg Hospital and University of M.Descartes13, A. Lalisan2, L. Baldwin1, A. Carroll1 and W. Carlo23. Laboratory 3tellenbosch, Tygerberg, South Africa of Medical Genetics, 2Division of , and iDepartment of Pediatrics, at Birmingham, Birmingham, Alabama, USA. Neurocutaneous melanosis(NM) is a sporadic condition with melanosis Univ. of Alabama is often involved when the The main characteristics of the Yunis-Varon syndrome are craniofacial )f the skin and pia-arachnoid, of which the latter disproportion, micrognathia, bilateral absence/hypoplasia of the thumbs, first ievi involve the scalp region. Patient: Newbom female of mixed ancestry, metatarsals and distal phalanges. The inheritance is autosomal recessive. The Nith healthy, non-consanguineous parents and normal perinatal history. cardiovascular, respiratory, skeletal, ectodermal, genitournary, and central nervous She had right-sided microphthalmia, darkly pigmented nevi over the systems have been reported to be variably affected in this syndrome. We report shoulders, occiput and mandible, as well as scattered nevi on the trunk. another case which adds cleft palate and malrotation of the gut to this syndrome. kddisionally there were: a high thoracic kyphosis and a round, keratotic Our patient, a newborn male, was the first child born to 25 year old white female nass over the midline, lumbar area, with no palpable underlying bony at 34 weeks of gestation. No complications were reported during this pregnancy. Jefect. Systemic examination was normal, but hypotonia was present at 2 He developed respiratory distress and was diagnosed with Biopsy of lumbar lesion was (TOF). The birth weight was 2.4 kg (50th), length was 48 cm (75th), and FOC was months of age. Special examinations: were sparse, and the eyebrows Histologically compatible with a dermal dendrocytic hamartoma. CT scan of 31.5 cm (50th). The scalp hair and eyelashes enhancement. absent. The face was dysmorphic with hypoplastic facial bones, small eyes, :rain; calcification of right eye globe and localized meningeal epicanthal folds, a short upper lip, and dysplastic ears. He had a cleft soft palate. conclusion: Microphthalmia has not been described before with NM, but The thumbs and great toes were hypoplastic, the fingers and toes were short and Ocular pathology in neurocutaneous disorders is well known. The presence pointed, and the nails hypoplastic. Radiographical examination showed hypoplastic Df the unusual dermal dendrytic hamartoma has not been described with first metacarpals. one ossified phalanx on each thumb, and no ossified phalanx on NM. These manifestations are most likely expansions of the phenotype. the great toes. The clavicles were normal. In addition there was functional closure of the cranial sutures, and bilateral cervical ribs. The karyotype was normal. The patient's features and radiographic findings were consistent with the diagnosis of Yunis-Varon syndrome. The patient died of severe brain injury and sepsis at age 40 days. Internal examination of the patient showed a hypoplastic cranial anterior fossa, and a midline ascending colon. The brain showed severe anoxic damages, thinning of the lateral hemispheric cortex, and no pituitary tissue was identified. The family history was negative for congenital heart defects. Consanguinity was denied. Our patient is the second case of Yunis-Varon syndrome with TOF. This report adds soft cleft palate, and malrotation of the gut to the list of malformations associated to this syndrome.

2155 2156 DNA Analysis for the DIfferentIal Diagnosis of 46, XX Males versus True CONGENITAL BILATERAL PERISYLVIAN SYNDROME (CBPS) IN A CHILD Hermaphrodites. P. Goonewardenal, E. Louie2 and H. N. Bass3. Genetics WITH 47 XYY KARYOTYPE. AL Gropman 4, C Samango-Sprouse2, LG Vezina3, Department, Kaiser Permanente of Northern California" 2 and Southern CJ rfft4. Departments of Neurology', Genetics2, Radiology3, Children's National California3. Medical Center, Washington,DC and the National Institutes of Health4, Bethesda, A 16-year-old phenotypic male was referred to Genetics because of a history MD of penile hypospadias with chordee and small testicular size. The patient was The Congenital Bilateral Perisylvian Syndrome (CBPS) is a following an uneventful pregnancy. There neurodevelopmental anomaly characterized by developmental and cognitive delay, born to a non-consanguineous couple restricted tongue movement: is no history of hypospadias or other urogenital abnormalities on either side of the pseudobulbar symptoms: dysphasia, dysarthria, with family. The hypospadias was repaired in multiple stages with excellent results. congenital anomalies and epilepsy. MRI findings consist of open opercula normal intelligence. He bilateral perisylvian polymicrogyria due to a neuronal migration disorder occurring Now at age 17, he weighs 86 kg, is 164 cm tall and is of with anomalies, has slight a phallus of normal size, gonads measuring 3x4 cm in a during the second trimester. A 30% association congenital gynecomastia, IV- especially arthrogryposis, has been found in several series. Sex chromosomal somewhat bifid scrotum, and secondary sexual development at Tanner stage in twins and V. Low normal testosterone and elevated FSH levels were noted. Pelvic abnormality has not been reported. CBPS has been reported siblings, suggesting a genetic basis. We report a seven year old male with a 47, XYY ultrasound revealed no evidence of mullerian structures. A serum estradiol of CBPS deficits, measurement and sperm count are pending. karyotype and clinical and MRI features (neurocognitive DNA oralmotor dysfunction, distal arthrogryposis) and symmetric perisylvian Banded chromosome analysis revealed a 46, XX karyotype. Genomic was felt to be obtained from peripheral blood lymphocytes was analyzed by PCR for the polymicrogyna on MRI. The proband's neurocognitive profile atypical specific DNA. The SRY, Y-alphoid, Y- for the XYY karyotype. 47,XYY has not previously been reported in association presence of Y-chromosome with arthrogryposis. He had oral motor dysfunction in the newborn period with heterochromatin and X-alphoid regions were tested. The results showed absence disorder. Neuromotor of Y material in the patient's DNA. Prior to undergoing molecular analysis for the development of oral motor apraxia and a primary language of material dysfunction was apparent with muscle atrophy, right sided weakness and delayed presence of Y-chromosomal material, histologic analysis gonadal skills. The child had borderline intelligence (IQ=76, s.d.=10) with obtained at the time of a varicocelectomy at age 16 identified only testicular ambulation he with Y-negative, reexamination preserved area of development in spatial cognition. At age six presented tissue. Following discovery that the patient's DNA was headaches, dizziness and jerking movements. EEG was normal and neuroimaging of gonadal material revealed the presence of both immature testicular tissue and that of the oocytes. There was no evidence of was subsequently obtained. We suggest many patient's neurocognitive ovarian stroma containing immature and clinical features that were atypical for 47,XYY karotype are attributable to the malignancy. Serum HCG and AFP levels were normal. Thus, the gonads are of CBPS. These difficulties correlate with the functional a true hermaphrodite. Additional research is concurrent diagnosis ovotestes and the patient is anatomy of the operculum as a center for oromotor functions including speech and necessary to elucidate the mechanism responsible for our patient's male swallowing. In the presence of oral motor apraxia, especially in the child with phenotype. This case represents another example where sex determination is delay and arthrogryposis, MRI for evaluation of CBPS should be controlled by genes other than the SRY or additional loci on the . developmental disorders who with findings on and/or considered. Known chromosomal present atypical Similar cases would be of value to help identify other genes should further MRI evaluation for CBPS. the X chromosome that may be involved in sex determination. undergo Published Abstracts: Clinical Genetics, Malformations and Dysmorphology (cont.) A369

2157 2158 Secondary amenorrhea due to an apparent Xp(pl 1 .2-pi 1.3) deletion. Six week survival of a 69, XXX triploid infant. An unusual phenotype B. Kohn, G.M. Azar, V. Mizhiritskaya, R.A. Conte, R.S. Verma. Division of revisited. G.S. Kupchik, S.K.Barrett, R.Mehta and E. Lieber. Maimonides Endocrinology and Metabolism, Long Island College Hospital-SUNY Health Medical Center, S.U.N.Y @ Brooklyn, New York, USA Science Center at Brooklyn, Institute of Molecular Biology and Genetics at A 2015 g term female born by NSVD to a 24 year old mother (G 2 P InterScience, Brooklyn, N.Y. 1001) had multiple dysmorphic features including: prominent occiput, Ovarian dysfunction due to an X is usually microphtalmia, simple ears, retro-micrognathia, short neck, hypoplastic thought to be associated to the long arm of the X chromosome, specifically nipples, bilateral single palmar transversal creases, bilateral syndactily of to region Xq13-26, whereas Xp deletion appears to result in short stature 3rd and 4th fingers, irregular size toes and hypoplastic toenails. Apgar and the other stigmata of the Ulrich-Tumer syndrome. However, we report a scores were 7/7. Baby required intubation, ventilatory support and transfer case of a 16 year-old girl with secondary amenorrhea and high FSH level to the NICU. An echocardiogram revealed a large ASD, large PDA, (74.6 MIU/IML, in whom an Xp deletion was found. The patient had her moderate TR with dilated RV and RA. Brain U/S revealed dilated atria, menarche at 14 years, menstruated for one week and had only two absent corpus callosum and lobular choroid plexus. Pregnancy hx was menstrual of 4 at 28 interval. On significant for polyhydramnios and U/S at 20 weeks of gestation showing a subsequent periods days days physical large placenta previa and multiple luteal cysts. The karyotype was 69, XXX. examination, she was 152cm tall and weighed 45kg. Breast development The survived 6 weeks. The of the extra chromosome was at Tanner IV and the pubic hair at Tanner II. She had no webbed neck baby origin or other abnormalities. FISH technique was performed to determine the complement could not be determined due to lack of parental cooperation. deleted region on Xp, using the Kallman (Xp22.3), the Duchenne muscular Placental pathology, initially reported as unremarkable, upon revision dystrophy (Xp2l), the X alpha centromere and the X inactivation region confirmed a large placenta with hydatidiform changes. A recent review of (Xq13) probes. All these regions hybridized with probe. Consequently, the placental pathology and triploidy by McFadden and Pantzar, associated deleted bands were ascertained to be Xpl 1.2-pi 1.3, which may be hydatidiform chanqes with diandry. They also conclude that contrary to apparently associated with this abnormal function. This case, along with previous reports, digyny is the most common cause of triploidy. some previous reports of gonadal dysfunction associated with Xp abnormalities, suggests that the Acritical regionO responsible for ovarian development on the X chromosome has to be reevaluated.

2159 2160 Deformations In infants born to mothers with uterine malformations: Branchio-oculo-facial syndrome. F. T.Lima; A.B.A.Perez; M.A.P.Ramos; report of 4 new cases. A.F Lewanda. Center for Medical Genetics, D.Brunoni. Centro de Gendtica Medica - Escola Paulista de Medicina - Rockville, MD; Johns Hopkins University School of Medicine, Baltimore, MD. UNIFESP - Sdo Paulo - SP - Brazil. Women with uterine malformations including unicomuate, bicomuate, The branchio-oculo-facial syndrome is an autosomal dominant condition didelphus, and septate uteri, have decreased reproductive success. These of bilateral branchial cleft sinuses, abnormal overlying skin, nasolacrimal abnormalities are associated with an increased risk of infertility, pregnancy duct obstruction and ocular anomalies. Distinctive craniofacial, auricular, loss (25-50%), preterm birth (20%), malpresentation (20-45%), and and oral anomalies may be present. Kidney anomalies are unusual. Forty- cesarean delivery (60-95%). three patients were already described (Am J Med Genet 56: 42-59, 1995). Four infants with congenital anomalies are reported. A genetics We report five additional patients with this syndrome, with variable consultation was requested for each child without the pediatrician's prior expression. Among them, one is an unrelated isolated case - a 17-year old knowledge of maternal uterine malformations. Two infants (one with boy, and there are two other families, a 2-year old boy and his mother and a unilateral ectrodactyly, one with a porencephalic cyst) were bom to women 13-month old girl and her mother. All the children have neuropsychomotor with bicomuate uteri, and two (one with cleft lip and palate, one with delay in variable degrees, and all the adults were mentally retarded and micrognathia, hypoplastic right leg and right club foot) to women with showed premature graying of the hair. None of the patients have the typical septate uteri. Such anomalies may be due to fetal constraint or to vascular branchial sinuses or hemangiomatous cervical skin lesions, nor they have irregularities when implantation occurs on or near the poorly vascularized hearing impairment. No renal malformations in the ultrassonografic studies uterine septum. In both patients whose mothers had bicomuate uteri, were found. This syndrome was first delineated by HALL et. al. (Am J Med prenatal sonograms documented implantation to be on or very close to the Genet 14: 135-8, 1983). The phenotype may show a wide range of septum. Implantation in the patient with oral clefting was on the right lateral expression and there is an overlap of manifestations with the branchio-oto- wall, and the implantation position of the fourth patient is unknown. renal syndrome (external ears abnormalities, deafness, nasolacrimal duct These cases suggest that an apparent congenital malformation may stenosis, branchial arch remnants and renal defects) that may represent an actually be deformational in nature, with significant impact on recurrence exemple of allelic heterogeneity. There is an underlying disorder of tissue risk for both parent and child. Maternal uterine malformations should be differentiation and a defect in closure of embryological structures of the considered in the evaluation of an infant with congenital anomalies, and anterior embryo, that may be caused by mutations of a developmental gene fetal deformation should be included among the risks of pregnancy in (Am J Med Genet 49: 414-21, 1994). The lack of any molecular linkage plus women with uterine abnormalities. the marked degree of variability of expression arise the need of a clinical documentation of a large number of pacientes that should be refered to molecular studies in the uture.

2161 2162 Presentation of Duchenne muscular dystrophy during the first year of Terminal transverse limb defect in a patient with velo-cardio-facial life. IT. Lott, M.Simon, M.Smith. Dept. of Pediatrics, University of syndrome (VCFS) and the 22q11.2 deletion. A.R. Mank-Seymourl, J.H. Califomia, Irvine. Cummins1, S.L. WengeW2, and C.A. Bay1'3. 1Children's Hospital of We report the case of a male infant who presented at the age of 11 Pittsburgh, Pittsburgh, PA 2West Virginia University, Morgantown, WV months because he did not adopt a normal crawl position and because he 3University of Pittsburgh, Pittsburgh, PA had difficulty raising himself to a standing position. Cognitive development was normal. Velo-cardio-facial syndrome is associated with hemizygous deletions of Deep tendon reflexes were reduced in upper and lower 22q1 1.2 which are detectable by fluorescence in situ hybridization (FISH) extremities. The calf muscles appeared hypertrophic. The creatine using the DiGeorge syndrome critical region probe (D22S75) in 75% of phosphokinase level was markedly elevated (17,420 units, normal 0-200 cases. VCFS is known to be extremely variable in its presentation and has units). Muscle biopsy revealed atrophic, hypertrophic and hypercontracted features which commonly include conotruncal heart defects, cleft palate, muscle fibers with myofiber necrosis and interstitial fibrosis. No positive subtle facial membrane was dysmorphic features, and developmental delays. We have staining observed with a series of to Dystrophin evaluated a case of a protein. Analysis of genomic DNA (carried out at Baylor DNA diagnostic unique VCFS with confirmed 22q1 1.2 deletion by revealed FISH. The patient, a five year-old Korean female, presented with laboratories) a deletion of exons 46 to 51 in the dystrophin gene. hypertelorism, a prominent nose, cleft of the soft palate with hypernasal This mutation was not detected in maternal genomic DNA. The dystrophin speech, ASDNSD status post repair, hemimelia of the left forearm, and deletion in this patient occurred within the deletion hotspot located within mild the central of the This case developmental delays. region gene. illustrates the importance of CPK Limb or in infants who with anomalies have been described in patients with DiGeorge screening present muscle weakness or delay in CHARGE syndromes and the 22ql 1.2 deletion such as preaxial achieving gross motor skills. polydactyly, 'club hands", bifid thumb, and hypoplastic metacarpals(C. Prasad et al. 1997]. We report the first case of a terminal transverse limb defect associated with a confirmed case of VCFS and the 22q1 1.2 deletion. We propose that the variable clinical picture of VCFS may also include limb anomalies, and that this inclusion may aid in recognizing previously undiagnosed cases. A370 Published Abstracts: Clinical Genetics, Malformations and Dysmorphology (cont.)

2163 2164 A multimodal approach to prenatal assessment In a family with a new Holoprosencephaly, a previously unreported finding, In a prenatally syndrome of cleft lip/palate, congenital heart defects, tooth and digital diagnosed case of distal 6q deletion. C. McKennal, K. Rawlinson2, P. K. anomalies and short femurs. K.D. McGowan' and H.F.L. MarA2. Greystone/ Berryand M. G. Bialer' I North Shore University Hospital, Manhasset, Radiology Associates', Rhode Island Hospital2, and Brown University2, NY, Great Neck Women's Medical Care, Great Neck, NY, 3Genzyme Providence, RI. Genetics, Yonkers, NY. A 26yoG5P3013 was referred at 18 wks. gestation due to 2 daughters from a et al. J prior union with unilateral cleft lip/palate (CUP). The maternal serum screen was Deletion 6q is an uncommon cytogenetic diagnosis. Hopkins (Am received genetic counseling for CUP Med Genet 70:377-86;1997) recently summarized 60 cases and delineated 3 negative. The patient had previously phenotypes corresponding to proximal (6q1 1-q16), middle (6q15-q25) and following the delivery of each daughter. After the delivery of her 2nd affected cases with daughter, and in view of multiple relatives with apparently isolated CP, the patient distal (6q25-qter) deletions. The latter group included 26 typical had reportedly been given a recurrence risk for CUP of 25-50% for any offspring findings including mental retardation (100%), ear anomalies (88%), hypotonia from the prior union. Targeted (Level II) ultrasound examinations in the current (86%), limb anomalies (71%), cardiac anomalies (52%), microcephaly (82%), pregnancy revealed a male fetus with progressive marked shortening of the growth failure (61%), eye anomalies (50%), genital anomalies (48%) and cleft femurs, relative shortening of the tibiae/fibulae, positional foot deformities, lack of palate (19%). Brain anomalies were common and included hydrocephalus, stomach filling, and CUP. Review of the family history revealed a brother with cerebral atrophy and agenesis of the corpus callosum. profound mental retardation; congenital heart defects in 1 affected daughter and 2 A 27 year old woman presented at 22 weeks 5 days gestation for a nieces; several relatives with digital and tooth anomalies, and 2 with early death. comprehensive ultrasound which showed a 16 day growth lag, abnormal 4- High resolution banding analyses yielded normal results in the patient's peripheral chamber view of the heart, thickened posterior nuchal fold (7.4 mm), rocker blood and amniotic fluid, as well as in peripheral blood from her mother and bottom feet and single umbilical artery. The brain had a monoventricle with no brother. Fluorescent in situ hybridization (FISH) using various chromosome interhemispheric fissure, hypotelorism and a midline facial cyst probably painting and alpha-satellite probes, as well as the DiGeorge Chromosome Region representing a proboscis. Amniocentesis was performed and revealed a (DGCR) probe (D22S75) all yielded apparently normal results. Newer FISH- 6q25>qter deletion which was de novo. The pregnancy was terminated by a D based techniques, such as spectral karyotyping (SKY) will be needed to detect & E procedure and post-mortem examination was not possible. The growth other possible chromosomal rearrangements, such as cryptic translocations. failure, limb anomaly (rocker bottom feet) and presumed congenital heart Fragile X studies are in progress. At 30 weeks, the fetus remains viable. disease are typical of 6q25>qter deletions. Single umbilical artery (2 cases) The constellation of findings in this family may represent a new syndrome, with has occasionally been seen, but this appears to be the first case of deletion anticipation suggested by the progression in severity over subsequent 6q25>qter with alobar holoprosencephaly. Genes for holoprosencephaly have generations. The differential diagnosis includes a subtype of oro-facial-digital been mapped to 21q22.3 (HPE1), 2p21 (HPE2), 7q36 (HPE3/SHH) and syndrome, although autosomal dominant inheritance and cardiac defects make 18p11.3 (HPE4), but none has thus far been mapped to this location. this less likely. Popliteal pterygium syndrome is not usually accompanied by mental retardation and cardiac defects.

2165 2166 Misdiagnosed acute leukemia in a patient mosaic for chromosome 8 Simpson-Golabi-Behmel syndrome in a family with an apparently X- trisomy. J. A. Miller and H. F L. Mark. Laboratory of Cytogenetics, FISH linked dominant mode of inheritance. D. Montanad, E. Rua, B. Minoli, M. and Genotoxicology, Rhode Island Hospital and Brown University School of Rittler. Medical Genetics Unit, Hospital Materno Infantil Ramon Sarda, Medicine, Providence, RI and Division of Medical Genetics, University of Buenos Aires, Argentina. Iowa, Iowa City, IA Simpson Golabi Behmel syndrome (SGBS) is a multiple malformation Trisomy 8 mosaicism is a well-known clinical and cytogenetic entity usually syndrome whose main features are overgrowth of prenatal onset, coarse associated with features such as mild to severe mental deficiency, prominent facies, cleft palate, polydactyly and a high perinatal lethality. It was originally forehead, deep-set eyes, prominent ears and deep palmar creases. Mark described in 1974 by Simpson et al. and its mode of inheritance was (1994) suggested that the phenotypic variability of trisomy 8 may be due to the considered to be X-linked recessive. In 1994 the gene of SGBS was presence of different proportions of trisomy 8 and disomic 8 cells in different mapped through linkage analysis to Xq25-27. We report on a male newborn tissues of the body. Depending on which tissue contains the trisomy 8 with macrosomia, cleft soft palate, broad mouth, skin redundancy, creases karyotype, different specific phenotypes may be observed. Furthermore, the on both earlobes, postaxial polydactyly and exomphalos. A brother, hematologic picture for individuals with trisomy 8 mosaicism may be confusing. misdiagnosed as Beckwith-Wiedemann syndrome, and a sister were Here we report an astonishing case of how an 8-month-old boy, now 29 similarly affected, including the exomphalos, and both died in the neonatal months old, was misdiagnosed with acute leukemia at a major midwest period. The mother had a coarse face with a broad nose and mouth and teaching hospital. The misdiagnosis was based partly on the finding of trisomy short distal phalanges, features already reported by other authors as minor 8 mosaicism (60%) in the patfent's bone marrow. As a result, the patient anomalies in heterozygote female carriers. On the basis of three sibs of received 6 weeks of intensive chemotherapy, including intrathecal Ara-C, both sexes severely affected and minor anomalies in the mother, we intrathecal methotrexate, vincristine, daunomycin, L-asparaginase and suggest an X-linked dominant mode of inheritance for the SGBS and prednisone. This oncological treatment occurred despite the fact that the although the heterozygote females usually seem to show a mild clinical patient had classical features of mosaic trisomy 8 syndrome, including mild picture, the possibility of severe manifestations cannot be excluded. hypotonia, small VSD, hypospadius, short stature, chest wall abnormality and deep creases in his palms and soles. This case underscores the importance of ruling out constitutional trisomy 8 mosaicism before diagnosing a malignancy involving trisomy 8. It is conceivable that the hematologic picture is atypical in such in dividuals, but this has not been systematically studied. It would be of interest to perform H & E staining of the bone marrow to compare the morphology of the bone marrow of individuals mosaic for trisomy 8 and that of normal diploid individuals. The invasive nature of the bone marrow procedure, however, may pose a challenge for such future studies.

2167 2168 Male siblings of congenital unclassified dyserythropoietic anemia (CDA) with Association studies of cytochrome P45011D6 gene polymorphisms microcephalous, hydrops and intrauterine growth retardation. K Okajima', M with familial and sporadic Parkinson's disease. A. Parsian, B. Racette, Nagahama2, M EguchP3, and Y Wadal. Nagoya City Univ. Nagoya', Aichi Pref Z.H. Zhang, M. Rundle and J.S. Perimutter. Departments of Neurology and Colony Kasugai2, Dokkyo Univ. Tochigi3 Japan , Washington University School of Medicine, St. Louis, MO 63110. Case 1. G1 P1 to healthy non consanguineous Japanese 24 years old parents. In order to determine the role of debrisoquine hydroxylase in the Intrauterine growth retardation was detected at 25weeks of gestation. development of Parkinson's disease, we genotyped patients with idiopathic Microcephalous was found prenatally with US and MRI. Delivery was uneventful at Parkinson's disease (IPD) with (n=61) and without (n=93) a family history, 37weeks 0 day, with birthweight of 1846g. Multiple joint ankyrosis, scoliosis and matched normal controls (n=57), and epidemiological normal controls mild hydrops were seen. RBC count was 4.99x106/ Hgb 13.9g/dl, Hct 44.7% at (n=88) with two mutations and a microsatellite marker in the cytochrome birth. He developed jaundice at 2 days and phototherapy was done for 5 days. P45011D6 gene. Since there was no significant differences between lPD EEG was flat. Optic nerves were atrophic. He developed convulsion which was for the of the mutations and alleles of microsatellite refractory to any drug therapy, progressive anemia, chronic diarrhea, mild liver samples frequencies the two normal and recurrent and died at 227days. RBC was anisocytotic and marker, these two samples were combined. Similarly, dysfunction were combined. There were differences in genotype serum iron (SI) was high and UIBC was low. Bone marrow sampling was not done. groups significant for G Hepatosplenomegaly was not seen. Autopsy was not granted. Karyotype was frequencies between the total IPD sample and the total control sample 46XY. Case 2. Younger brother of patient 1. Intrauterine growth retardation and to A transition (X2=7.74, p=0.02). However, the difference between the two small bitemporal diameter were detected at 24weeks of gestation. Delivery was samples for the mismatch in the gene was not statistically uneventful at 35weeks 6days, with birthweight of 1782g. He had microcephalous, signficant (X2=0.45, p=0.59). Analysis of the microsatellite marker, multiple joint ankyrosis and hydroptic. He developed jaundice at 1 day, and RBC comparing all alleles together, revealed statistically significant difference count 5.37x106/ Hgb 17.0g/dl, Hct 52.1%, anisocytosis was severe, siderocytes between total lPD and total control samples (X2=18.37, p=0.03). These were seen in peripheral blood. Bone marrow smear showed ringed sideroblasts results show that the mutations in the cytochrome P45011D6 gene may play and multilobulated erythroblasts which resembled those seen in congenital a role in susceptibility to idiopathic Parkinson's disease but the gene effect dyserythropoietic anemia (CDA) type 111, although they are not macrocytic. No is very small. evidence of hemolysis, no abnormal hemoglobin pattern were seen. There were no hematological, histological, serological and biochemical data compatible to CDA and 11. Si was slightly high and UIBC was low. Hepatosplenomegaly was not seen. EEG was flat. Optic nerves were atrophic. Mild liver dysfunction was seen. Anemia was mild. Karyotype was 46XY. Due to chronic pulmonary failure, he died at 553days. Autopsy findings: Bone marrow was hypercellular with erythroid cells. Many histiocytes were seen in . Brain tissues were atrophic. Ganglioneuroblastoma was seen on adrenal gland. So far no normal delivery has been seen. No exposure to any drugs and chemicals were seen. Published Abstracts: Clinical Genetics, Malformations and Dysmorphology (cont.) A371

2169 2170 A rare recessive skeletal dysplasia and infantile obesity: Partial trisomy 16p due to tandem duplication of 16p13. R. Perrone, A. (UPD) ruled out. RJ Peoples1 and U Franckel2. Stanford University Medical Yenamandra, X. T. Zhou and M. G. Bialer. North Shore University Hospital- School Dept. of Genetics and HHMI2, Stanford, CA. NYU School of Medicine, Manhasset, NY. Uniparental isodisomy can be the cause of rare autosomal recessive Partial trisomy 16p is an uncommon cytogenetic anomaly. Most cases have disorders. If the chromosome involved carries an imprinted gene, UPD may also been the result of an unbalanced translocation, in which case the concomitant lead to distinct syndromes involving abnormal growth pattems or neurological deletion may have contributed to the phenotype. The phenotype consists of dysfunction. We have studied a female who first presented at 9 months with prenatal and postnatal growth retardation, mental retardation, cleft palate, craniosynostoses, telecanthus and marked obesity. Parents are and features family history is negative. Birth parameters congenital heart disease, hypertonia dysmorphic (ocular nonconsanguineous and the were hypertelorism, sparse hair, low-set ears, micrognathia, short wide neck, thumb normal. Progressive obesity was noted between 2 and 4 months of age. Weight at anomalies, long We have described a child evaluation was 13.6 kg, 6 standard deviations (SDs) above the mean and at the tapering fingers). previously mosaic for a small chromosome 16p marker [Am J Hum Genet 50th percentile for 34 months. Height was at the mean for age. Multiple caloric 55(Suppl):1881;1994] which helped define the features of proximal 16p intake evaluations were normal suggesting an altered metabolic rate. 1 Endocrinologic evaluation, head MRI and high-resolution karyotype were normal. trisomy. We now describe a 7 year old with distal 6p trisomy. During the second year of life, her weight gain fell off dramatically but linear The patient was the 4 lb 4 oz product of a 35 wk gestation pregnancy growth slowed as well. At 29 months her height was 2.8 SDs below the mean. A complicated by pre-term labor. At birth she was noted to have Pierre-Robin skeletal survey showed multiple abnormal findings including wedge-shaped anomaly, finger and wrist contractures, hypertonia, deep pilonidal dimple and a epiphyses of the knees diagnostic of metaphyseal acroscyphodysplasia. This left iris coloboma. A chromosome study showed 16p+. She had numerous disorder has been reported in 4 previous cases, including a pair of siblings, but in episodes of otitis media. She walked at 18 mo. Presently she speaks only 3-4 neither case was it associated with obesity or craniosynostosis. words, but signs more than 100. She is only now beginning to feed by mouth. Because of the precedent of infantile or childhood overgrowth and transient She developed premature adrenarche at 6 yr. At 7 yr her height is slightly metabolic derangements associated with abnormalities at imprinted loci (Beckwith- <5%, weight is 75% but head circumference is <<5%. Previous tindings were Wiedemann syndrome, Prader-Willi syndrome and transient infantile confirmed. Dysmorphic features include synophrys, broad nasal bridge and tip, hyperinsulinism) and the potential for unmasking of recessive loci by UPD, we hypoplastic philtrum, thin upper lip, downturned mouth, round face, wide neck investigated the hypothesis that this patient had UPD. and cupped right ear with hypoplastic anthelix. Fluorescence in-situ The patient and parents were typed for 69 highly informative microsatellite hybridization (FISH) with a whole chromosome #16 painting probe revealed markers chosen from chromosomes 1 - 6, 8 - 12 and 17 - 20 as the UPD that the extra material on 16p is derived from chromosome #16. FISH with phenotypes for these chromosomes have not been established. We were able to inv(16) cosmid probe revealed a duplication of 16p13. The patient has many demonstrate biparental inheritance at one or more loci for each chromosome features characteristic of trisomy 16p but lacks the severe growth retardation, tested ruling out uniparental iso- and heterodisomy as the cause of the complex congenital heart disease and sparse hair. Further cases of distal 16p trisomy phenotype observed. will help define the phenotypic features.

2171 2172 Mutations in Fibroblast Growth Factor Receptor Genes: Chinese Data. Hadju-Cheney syndrome: a case report of the first Brazilian patient. F. J. Tsail, J. Y. Wt?, C. T. Peng', C. H. Tsail. 'Department of Pediatrics, C.Y. Utagawa, C.A.Kim, S.M.M.Sugayama, M.FBarba, C.E.F.Andrade, 2Department of Medical Research, China Medical College Hospital, L.M.J.Albano, C.H.Gonzalez. Department of Pediatrics. Instituto da Cranca Taichung, Taiwan, R.O.C. - HCFMUSP, S. Paulo, S.P., Brazil. [Intro. by M. Zatz] Mutations in human fibroblast growth factor receptor (FGFR) gene families Hadju-Cheney syndrome, a rare autosomal dominant disorder with have been found to be associated with skeletal dysplasia and craniosynostotic variable expressivity, is characterized by a distinctive facies, bizarrely syndromes. One mutation of FGFR3 (Gly38OArg) was reported worldwide shaped skull, premature loss of teeth, osteolysis of the distal phalanges and suggesting the homogeneity of this specific mutation among different ethnic short stature. Basilar skull invagination is a serious which can populations. This study was performed to determine whether mutations result in hydrocephaly, progressive neurologic deterioration with associated with either skeletal dysplasia or craniosynostotic syndrome in involvement of cranial nerves and cerebellar dysfunction. About 40 cases Chinese patients were similar to those reported in other ethnic populations. were already reported in the literature. Unrelated cases of Apert syndrome (n=7), Crouzon syndrome (n=6), Pfeiffer We studied the first Brazilian with Hadju-Cheney syndrome, a 10-year- syndrome (n=2), achondroplasia (n=18) and hypochondroplasia (n=5) were old boy, who was the third child of healthy, unrelated parents. The collected. Utilizing techniques of PCR-based restriction analysis and direct pregnancy was normal with delivery by a cesarean section. The birth weight the above-mentioned syndromes were sequencing, mutation analyses of was and the length was 51cm. At the age of 2 years he had a a unique mutation 3,800g performed. In achondroplasia, G380R in FGFR3 was premature loss of teeth. At the age of 8 years his weight was detected in all of our patients studied. Of 7 patients with Apert syndrome, 6 22,600g, height= 122,5cm, OFC= 53,5cm and he showed a characteristic facies, had S252W mutation and 1 had the P253R mutation in FGFR2. As for patients synophrys, generalized hirsutism, hypodontia, small mouth, receded chin, with Crouzon syndrome, mutations were found in 3 out of 6 cases. One had N289P mutation in FGFR2, another had C342Y mutation in FGFR2 and the short neck, cardiac murmur, joint laxity, skin hyperextensibility, and short other had P250R mutation in FGFR3. In hypochondroplasia, N540K mutation fingers with nail dysplasia. Radiological findings were: multiple wormians had been identified in two cases: one with of C1659A and the bones in skull, protuberant squamous portion of the occipital bone other with C1659G, respectively. In summary, mutation of G380R in FGFR3 (bathrocephaly), widening sutures, basilar impressions, osteolysis of was homogeneous in Chinese patients with achodroplasia. Ratio of Chinese several distal phalanges of fingers and toes, and generalized osteoporosis. patients with Apert syndrome of 2 different mutations (S252W and P253R) is Echocardiography study revealed an atrial septal defect. Cervical spine C.T. also similar to published report. On the other hand, mutations in other scan showed basilar inva ination but cranial scan was normal without syndromes with skeletal dysplasia or craniosynosis were heterogeneous like cerebellar compression. The patient is assymptomatic with normal other ethnic populations. More effort will be needed to characterize the neurological evaluation. remaining unidentified mutations in Chinese patients affected with syndromes of skeletal dysplasia or craniosynosis.

Published Abstracts: Cytogenetics

2173 2174 De Novo duplication and dynamic mosaicism of chromosome Saggital/coronal craniosynostosis associated with balanced 22(ql1>q12), and structural rearrangement (translocation 5, 7, 14) in reciprocal tranlocation [t(2;8)(2pl6;8p22)] in an Arab girl. NA Al-Torkil, the father: A case report. S.J. Abul Hasan*' D.S. Krishna Murthy, M.A. S J Abulhassan', M A Sabiy', M A Abdel Rasoold, / AI-Jameh2, S A Al- Rasool, KK Naguib, S.A. Al-Awadi. Medical Genetics Center, P.O. Box Awadil. 1Kuwait Medical Genetics Centre, Matrenity Hospital;2Pediatrics 31121, Sulaibikat, 80901, KUWAIT Department, Sabah Hospital; Kuwait The characteristic phenotype in (CES) is due to the Sagital/coronal craniosynostosis associated with balanced reciprocal presence of a supernumerary bisatellited 22 (inv dup translocation [t(2;8)(2p16;8p22)] in an Arab girl. N A Al-Torki1, S J 22pter->22q11.2), resulting in 4 copies of this region. However, in some Abulhassan', M A Sabry', M A Abdel Rasool', Al-Jameh2, S A Al-Awadil. atypical, de novo cases unusual structural anomalies (22)] [deletion/ring 'Medical Genetics Centre, Maternity Hospital; Department, Sabah and mosaicism have been reported. 2Pediatrics Hospital; Kuwait. We report an Arab girl who presented with saggital and right A newborn male with multiple anomalies, down congenital including, coronal synostosis, microcephaly, blephrophemosis, strabismus, bilateral slanting palpabral fissures, absence of the lower eyelids, abnormal ears and central blindness with poor visual fixation, depressed nasal bridge, short nose, pre-auricular tags was investigated. Chromosome analysis showed, an long philtrum, low-set cup-shaped ears, and micrognathia. The child was extra small marker metacentric chromosome in of the cells. The majority delivered by CS following uterine rupture with complicating birth asphyxia and marker was confirmed to be derivative bisatellited in some chromosome 22, respiratory distress. There was postnatal growth delayed metaphases. Multiple cell line was retardation, mosaicism, including ring(22) observed developmental milestones, abnormal EEG, an spasticity. for the der (22) chromosome, which is characteristic of the cat eye Chromosomal study showed the presence of a balanced reciprocal The marker chromosome was confirmed to syndrome. be derivative by translocation [t(2;8)(2p16;8p22)] that was identified in the popositus and two of whole chromosome painting using digoxiginin labelled DNA probe for her phenotypically normal siblings. The detection of normal paternal karyotype chromosome 22 (Oncor). Karyotype of the mother was normal. However, in suggested the possible inheritance of the chromosomal abnormality from the the father balanced translocation involving chromosomes, 5, 7, and 14 and deceased mother. On the other hand, the presence of parental gonadal aneuploidy was observed. This is the first case of mcat eye syndrome' mosaicism stands as an alternative possibility. This report tends to suggest the among 10,600 cases karyotyped in this population. The signficance of presence of new potential candidate ene(s) for craniosynostosis at the break mosaicism and parental chromosomal anomalies in cat eye syndrome is points of the described homosomal rearrangement. discussed. A372 Published Abstracts: Cytogenetics (cont.)

2175 2176 Analysis of BTF2p44 and SMN gene copy number in the SMA region Identification of the origin of a supernumerary chromosome 6 using using interphase FISH on lymphoblast and chromosome 5 mono- spectral karyotyping. J.M.Cowan1, T.L.Stewart1, B.Haddad2, G.Gupta , chromosomal human-hamster hybrid cell lines. T. A. Carter, K. Das, M.B.Irons1. 1Tufts-New England Medical Center,Boston,MA,2Georgetown and T. C. Gilliam. Departments of Psychiatry and Genetics \& Development; University, Washington,D.C. Columbia University, New York, NY. We report a 15 month infant with early onset scoliosis, developmental The spinal muscular atrophy (SMA) locus in humans is characterized by delay and microcephaly. She was the product of a full term pregnancy. Birth a chromosomal duplication which includes at least three genes; SMN weight was 2.4 kg. The family history is significant for recurrent NAIP, and BTF2p44. Both the Survival Motor Neuron (SMN) and basal in the patient's mother and grandmother. The proband's medical history is at nine months of and transcription factor, p44 subunit (BTF2p44), genes exist as highly significant for severe thoracic scoliosis (diagnosed age) On exam she was with homologous centromeric and telomeric copies. Although homozygous strabismus. physical microcephalic dysmorphic a fissures, SMNT loss appears to be sufficient and necessary to cause SMA, SMNC features including broad, slanting forehead, upslanting palpebral bilateral a broad and a thin She was delayed. copy number may be correlated with the severity of illness. Alternatively, ptosis, nose, upper lip. globally studies identified a small, satellite-like supernumerary increased copy numbers of SMNC in mildly affected patients might reflect Cytogenetic chromosome in all cells; in 9/20 cells there was an additional, SMNT/SMNC hybrids created through gene conversion. To evaluate the larger chromosome. identified the larger marker correlation between gene copy number and disease severity we combined supernumerary Spectral karotyping molecular analysis of mono-chromosomal hybrids with interphase FISH as derived from chromosome 6, which was confirmed by chromosome painting. The smaller, more frequent marker did not show a consistent signal analysis. The use of human-hamster hybrid cell lines containing a single on and is to be derived from a satellite of human chromosome 5 from SMA families has allowed the direct analysis of spectral karyotyping, thought region an acrocentric chromosome. Maternal chromosomes were normal, and individual chromosomes in severe and mild SMA patients. Two color FISH chromosomes are SMN paternal pending. experiments have been used to directly compare the copy number of While small marker chromosomes have been found in in supernumerary and BTF2p44 from the individual chromosomes of patients differing 0.01-0.05% of liveborn infants (Buckton et al. 1980), markers derived from disease severity. chromosome 6 are extrememely rare. Comparison of our case with two previous reports, and with descriptions of partial trisomles of 6p and 6q (resuiting from unbalanced translocations), lead us to conclude that the marker in this patient is derived from the short arm of chromosome 6. This case illustrates the utility of spectral karyotyping in identification of marker chromosomes. While the precise band of origin is not identified, It narrows significantly the regions to be considered in phenotype comparisons.

2177 2178 Mosaic partial trisomy 2p: Completion of chromosomal characterization 17 Identification of a marker chromosome without detectable alpha years after initial visit. J.H. Cummins1, U. Surt '3, P. Mowery-Rushton1 3, C.A. satellite sequences by spectral karyotyping. B. Huanq1, Y. NMng3, C. Bay1'3. Children's Hospital of Pittsburgh, 2Magee Womens Hospital, Sandlin1, A. Lamb2, M.Jamehdor4, T. RiecP, J. Bartley1. Genzyme 3University of Pittsburgh. Genetics, Long Beach, CA; 2Genzyme Genetics, Santa Fe, NM; We have re-evaluated a dysmorphic, moderately delayed white male that 3NHGRI,NIH, Bethesda, MD; 4Southern Califomia Permanente Medical up 15 cells from peripheral was originally worked in 1980. Originally metaphase Los CA blood lymphocytes were analyzed with an apparently normal 46,XY karyotype. Group, Angeles, is a tool for Eight years later another chromosome study was done based on the clinical Spectral karyotyping (SKy) powerful new characterizing suspicion of atypical trisomy 8 mosaicism. A mosaic 22p+ cell line was found in chromosome rearrangements. We demonstrate the use of SKy in identifying twenty-two of fifty cells, karyotype was 46,XY,22p+[22y46,XY[28]. Parental an unusual marker chromosome. karyotypes done at that time were normal. The origin of the extra material was A newborn giri with normal birth weight was ascertained due to multiple not able to be elucidated. Recent reappraisal identified the extra material as congenital anomalies including severe hypotonia, pardiovascular defects, chromosome 2 by whole chromosome painting using a 2 Coatasome hearing loss, CNS anomalies and dysmorphic features. The infant died at probe(ONCOR, Inc.). Further characterization using the N-myc(2p23-p24) and 12 days of age. Cytogenetic analysis revealed a de novo supernumerary 2p telomeric(?p25-pter) probes(ONCOR, Inc.) demonstrated the extra material marker chromosome in all the cells examined. The marker appeared to be to be 2p23-2pter in origin. Our child's final revised karyotype is metacentric with a primary constriction, but was negative for CBG banding. 46,XY,der(22)f(2;22)(p23;p1 1.1 )/46,XY. Fluorescence in situ hybridization(FISH) with a combination of chromosome in to Close to 50 cases of nonmosaic partial trisomy 2p have been described specific alpha satellite probes and all human centromere probes failed the literature and a characteristic phenotype has been described by other show to the marker, indicating that the marker chromosome 1995]. hybridization authors[Lurie et al. lacked detectable satellite sequences. SKy was performed and is more affected compared alpha Our child although dysmorphic and delayed mildly revealed that the marker was chromosome 15 in origin. This was confirmed to other cases. His features include: short stature, downward slanting palpebral FISH with a subtelomeric which showed fissures, left exotropia, simple ears, high arched palate with narrow oral cavity by 15q specific probe, hybridization and mouth, horseshoe kidney, mild hypospadias, scoliosis, moderate mental to both ends of the marker chomosome. Combining this information with the retardation with autistic features, expressive language delays, hypotonia and G-banding pattem, the marker was determined to be an inverted duplication seizures. In the only other report describing trisomy 2p mosaicism, Dahl et al., of 15q23-q26. Since the marker chromosome is mitotically stable, it most We that the DNA (1988) described a newborn mosaic for a complete 2p trisomy cell line and a likely has a functional centromere. hypothesize sequence triploid cell line. We believe that our case is the first describing a child with at the breakpoint can function similarly to alpha satellite sequences and was mosaicism between an apparently normal diploid cell line and a partial 2p activated through the marker formation. trisomic cell line which may explain his milder phenotype.

2179 2180 Prenatal diagnosis of mosaic trisomy 5 with poor outcome and failure ANALYSIS OF NUCLEAR GENOME DNA POLYMORPHISM IN to confirm postnatally. T.Jewett1, J.C. Veille1, K.K.Nicol1, S.L.Jackson1, POPULATIONS OF THE VOLGA-URAL REGION. E.Khusnutdinova, R.A.Silve?2, S.b. Wellman2, P.N.Rao1 and M.J.Pettenati1 . 1 Bowman Gray T.Victorova, I.Khidatova, R.Fatkhlislamova, A.Galeyeva, S.Limborskaya. School of Medicine, Winston-Salem, NC and 2Frye Regional Medical Dept. Biochem. and Cytochem., Ufa Sci.Centre Acad. Sci., Ufa and Center, Hickory, NC. lnst.Mol.Genet., Acad.Med.Sci., Moscow, Prenatal detection of trisomy 5 mosaicism is rare. To date, there have The genetic structure and genetic relationship in populations of the been 5 cases reported: 3 with normal liveboms and 2 with abnormal Volga-Ural region (the Bashkirs, Komi, Tatars, Mari, Udmurts, and outcomes. In those cases with normal outcomes, trisomy 5 was not Mordvinians) have been studied. As genetic markers in this investigation: confirmed in either blood or tissues. Trisomy 5 was confirmed only in tissue diallelic loci MET and D7S23, linked to the cystic fibrosis gene; samples of those cases with abnormal outcomes. hypervariable apolipoprotein B gene and hypervariable sequences DNA, We report a case of trisomy 5 mosaicism detected in amniotic fluid detected by the help of the hybridization DNA probe bactenophage M13, (47,XY,+5[4y46,XY[14]). The mother was referred during pregnancy due to have been used. Reliable interpopulational differencies were determined by fetal growth retardation. The pregnancy was continued: no further prenatal distribution of allele and genotype frequencies of the studied cytogenetic studies were performed per patient request. The child was genetic distances for the studied loci were calculated by using the alleles delivered at 34 wks by C-section and was small for gestational age with and hybridization fragments frequencies, and on the basis of multiple dysmorphic features. The child died at 4 months of age due to corresponding dendrogrammes were built. Among the Volga-Urals region complications following heart surgery. Confirmational studies on peripheral populations the Bashkirs and Tatars turned out to be the most closed, blood were normal as were 2 tissue samples. FISH with VYSIS probe the Komi was the most distant from the other studied populations. D5S23 did not show trisomy 5 in tissue. whole, the data, we have received, reflected the assumed degree This is the first case of prenatally diagnosed mosaic trisomy 5 with an populations genetic affinity quite enough and demonstrated the possibility abnormal outcome and without postnatal confirmation. A review of all for using in population- genetic analysis of the DNA polymorphism. prenatally diagnosed mosaic trisomy 5 cases shows that all abnormal cases had features in common with our case. Trisomy5 mosaicism has not been confirmed in any case using prenatal or postnatal blood. Therefore, fetal blood sampling may not provide useful information. A review of prenatally diagnosed trisomy 5 cases will be presented. Published Abstracts: Cytogenetics (cont.) A373

2181 2182 Association of reproductive abnormalities with pericentric inversion of Pericentric inversion of and 9 [46,XY,inv(1)(p12;q13), chromosome 9. J.M. Kim, H.M. Ryul, Y.M. Kim, S.Y.Park, S.K. Choi, I.S. inv(9)(p11 ;q12), 16qh+] in a male with reproductive failure. D.S. Krishna Kang'. Genetic Research Laboratory, Department of Obstetrics and Murthy, T.LFarag', and S.A. Al-Awadi. Medical Genetics Center, Gynecology', Samsung Cheil Hospital & Women's Healthcare Center, Maternity Hospital, KUWAIT'Dal Housie University, Halifax, NS, Canada Samsung Medical Center, Seoul, Korea. Pericentric inversion (pi) is a relatively common structural rearrangement in Pericentric inversion of the chromosome 9, inv(9)(p1l;q13), is occurs man considered to be a normal heteromorphism in the general population. commonly with an incidence of 1% to 1.65% and some cytogeneticists However, it has been reported to be associated with reproductive failure/ would consider it as a normal variant. This entity is categorised as a minor repeated fetal loss. A 36-year old normal, healthy male (Indian) was chromosomal rearrangement with normal phenotypes. However, many investigated for primary infertility during 4 years of marriage. His wife (32 reports in the literature suggested that it may be associated with subfertility, years) had undergone complete gynecological investigations, including D and recurrent abortions or other chromosomal abnormalities as a result C on two occasions, and chromosome analysis, with no abnormality detected. arising General examination of the was unremarkable, for of habing inv(9). Herein, we analyzed the incidence and clinical significance physical proband except of those who had abnormalities. blood the varicocoel and recurrent urinary tract . There was no history of inv(9) among reproductive Peripheral reproductive failure or fetal loss in the family. Semen analysis showed, karyotypes (2070 cases) performed due to recurrent spontaneous abortions hypospermia (ejaculate volume 1 ml), Sperm count 150 million/ml; Motility 30% or infertility in Samsung Cheil Hospital over the last six-years were selected. active; Weak 10% and Dead 60%, WBC +ve. Hormone analysis and Cases for prenatal genetic diagnosis were excluded from this study. Thirty- histopathological studies of the gonadal biopsy could not be carried out as the one cases from 30 couples had inv(9)(pl 1;q13) which gave an incidence of patient did not come for follow up. Metaphase chromosomes were prepared 1.5%. One case of inv(9)(p12;q12) was found and excluded from the from peripheral blood culture and analyzed by standard G- Q- and C-banding statistics. There were two cases with 47,XXY,inv(9). Among 30 cases with techniques. His karyotype showed 46,XYinv(1 )(p12;q13), inv(9), 22 couples(73.3%) had history of more than two spontaneous inv(9)(pll I;q12),16qh+. Parents were not available for cytogenetic analysis. abortions. Five families(16.7%) had the problem of infertility. Three From the family history it is very unlikely that either of the parents is a carrier couples(10%) previously had babies wity major congenital anomaly. for double inversions (father died). The occurrence of double pericentric Interestingly, one case had homologous inversion 9 but she was inversions in human is extremely rare. The possible sterilizing effect of phenotypically normal. Although there may be a selective bias because pericentric inversion in heterozygous males and the very few cases reported relatively older subfertile couples were included retrospectively, these data emphasizes the importance of new cases in understanding the clinical suggest that inv(9) may often cause clinical problems such as recurrent significance of pericentric inversions and reproductive fitness in males. The spontaneous abortions, subfertility or chromosomal abnormality in the unusual clustering of polymorphisms and infertility in our patient is noteworthy. offspring of the carriers. *143, Ranchview Place, NW, CALGARY, T3G 1 H7, AB., CANADA* E-mail : smurthy acs.ucalgary.ca

2183 2184 Decreasing trend of the detection frequency of in A rapid combined cytochemistry and fluorescent in situ hybridization Korea:impact of prenatal screening for Down syndrome. E. H. Lee and for the identification of fetal chromosome aneuploidy in uncultured H. R. Moon. Green Cross Reference Laboratory, Seoul, South Korea. amniotic fluid: A pilot study of 20 cases. YK Lee1, WB Kim1, SH Lee2, Trisomy 21 is the most common chromosomal abnormality. A study was DWLee1. 'Department of Clinical pathology, College of Medicine, conducted to assess the efficiency of prenatal screening using free beta- Soonchunhyang University; 2Human Genetics Lab, Infertility Medical hCG in combination with AFP, uE3 and matemal age in Korea. Center, College of Medicine, Pochon CHA University, Seoul Korea We reviewed our 3,112 karyotype results in children whose ages were under 3 old the last four Even though traditional cytogenetic study has been the mainstay of years during years. Blood samples for chromosome a were obtained from children with prenatal diagnosis, it requires cultured cells necessitating significant analysis congenital anomalies from or 1994 to 1997. delay. To overcome its limitation, the advent of molecular cytogenetics January May fluorescent in situ hybridization(FISH) has been added as an adjunctive Among 3,112 results of chromosome analysis in children with congenital 308 tool. However, the presence of maternal cells in uncultured amniotic fluid anomalies, cases(9.9%)were trisomy 21, of these, 194 may result in errors in the interpretation of aneuploidy detection by FISH. cases(63%)were male and the remaining 114 cases(37%)were female.Of The reason is that maternal and fetal cells cannot be distinguished by all Down SD cases, 94.5% were a homogeneous trisomy 21, 3.6% were morphologic characteristics alone. cases due to translocation and 1.9% were caused by mosaicism with We tried to fetal cells normal Most the second chromosome involved in the identify by simple and cheap cytochemical methods karyotype. commonly using a Periodic acid Schiff's reaction with diastase treatment and/or a translocation was . However, that chromosome is alkaline phosphatase reaction with levamisole treatment to detect fetal cells chromosome 14 in western countries.The detection frequency of Down SD originated to was as follows.: 100 in 104 from gastrointestinal tract. A rapid and simple-to-use interphase according years cases(14.7%) 1994, FISH using X- or Y-chromosome probes(Oncor) doing on the same slide cases(12.5%) in 1995, 86 cases(7.6%) in 1996 and 29 cases(5.9%) in Since 1994 we have been subsequently. Data from our initial pilot study of 20 cases show that the 1997(Jan.-May). Aug. performed prenatal simultaneous use of a cytochemistry and interphase FISH is feasible as screening using AFP, free beta-hCG, uE3 and maternal age in Korea and potential future screening tool for the prenatal detection of chromosome as above mentioned the detection frequency of Down SD have been aneuploidy. gradually decreasing. These results suggest that this decrease resulted from prenatal maternal serum screening for Down SD. This benefit of the decreasing trend of Down SD should encourage us to perform maternal serum screening program for Down SD as routine prenatal test in Korea.

2185 2186 Prenatal diagnosis of a fetus with two balanced rearrangements: Familial sex chromosome mosaicism: Report of two sisters with 46,XX, t(2;14)(q31;q24)pat,inv(8)(p21;q22)mat. A. L. Medearis, and M. R. mosaic and an abnormal phenotype. M.A. Micale, Rahman. Prenatal Diagnosis Center, Loma Linda University, Loma Linda T. W Kurczynski and C. Gaba. Medical College of Ohio Toledo, OH Ca, Clinical Genetics Center, La Mirada, Ca. We report two cases of mosaic Turner Syndrome ascertained in two sisters A 36 year old, G2, P0, SAB1 woman was referred for genetic who both presented with mental retardation, seizures and limb spasticity. amniocentesis because of advanced maternal age. This is the first Peripheral blood chromosome studies revealed the karyotype 45,X/46,XX in pregnancy of the couple, however, the family history revealed that the M.H. (age 45). Her sister, G.H. (age 49) presented the karyotype 45,X/46,XX/ woman has one sister, three brothers and her mother lost a twin pregnancy 47,XXX. GTG-banding studies revealed the 45,X karyotype in 7/30 cells (23%) (SAB) around twelve weeks of gestation. Her first pregnancy with another in M.H. and in 3/50 cells (6%) in G.H., with 2/50 cells (4 /o) in G.H. presenting a person was an early miscarriage (SAB). Her husband belongs to a family of 47,XXX karyotype. No structural chromosome anomalies were noted in either 6 sibs. Two brothers and two sisters are living well and one sister was a sister at the 500-band level of resolution. Flourescence in-situ hybridization stillbom (SB). His previous two marriages ended up in four early utilizing a chromosome X alpha-satellite DNA probe revealed similar spontaneous abortions. The patient consented for amniocentesis which was percentages of abnormal cells in both sisters, with M.H. demonstrating one performed at seventeen weeks of gestation. Fetal ultrasound was signal (suggesting monosomy X) in 10/111 cells (9%) and G.H. demonstrating apparently normal. Chromosome analysis from amniocytes revealed a fetal one signal in 8/110 cells (7%). karyotype indicating: 46,XX,t(2;14)(q31;q24),inv(8)(p21;q22). Parental Familial mosaic Turner Syndrome has been rarely reported. The finding of blood mental retardation, seizures, and limb spasticity in these patients would not be penpheral karyotype showed: 46,XY,t(2;14)(q31;q24) for the father expected with these karyotypic findings. In addition, neither sister presented and 46, XX,inv(8)(p21 ;q22) for the mother. Repeat ultrasound at twenty two with weeks of revealed normal any genital anomalies, nor were they found to have any other Turner gestation apparently fetal structure. Any single X DNA were translocation or stigmata. Fragile analyses performed on both sisters and found to rearrangement (balanced inversion) inherited from a single be negative. Because of the observed phenotype in these patients, and a parent in most instances gives rise to a normal fetus. Rarely, the fetus may of be family history mental retardation in the mother, maternal aunt, and maternal abnormal most probably due to parental imprinting, resulting from grandmother, it is likely that an as yet undiagnosed Mendelian disorder is reorientation of interphase nuclear architecture following meiosis. The risk segregating in this family. Chromosome studies of other family members will of abnormality in the fetus that inherits two balanced rearrangements, one be performed in an attempt to document further sex chromosome mosaicism. from each parent, may be apparently normal. However the effect of non- It is interesting to speculate on the possibility that a dominant gene mechanism mendelian genetic factors, such as, Imprinting and mitochondrial influence (either autosomal or X-Iinked) responsible for X chromosome instability may be ruled out. be in operation in this family. In addition, it may be possible that this gene is linked to the putatively aberrant gene or gene region responsible for the genetic disorder in these sisters. A374 Published Abstracts: Cytogenetics (cont.)

2187 2188 Direct duplication of the long arm of [bands q12q21.3]. Prenatal supernumerary ring 16 chromosome characterized by V. Mizhirtskaya, G.M. Azar, R.A. Conte, J.Pitter, and R.S.Verna. Institute multiprobe FISH, with normal pregnancy outcome. A.Paoloni-Giacobino, of Molecular Biology and Genetics at InterScience,Brooklyn, The Stanley S. M.A. Morris, S. Dahoun. Div. Medical Genetics, Geneva University Hospital, Lamm Institute for Child Neurology asnd Developmental Medicine & The Switzerland Long Island College Hospital- SUNY Health Science Center at Brooklyn, De novo marker chromosomes identified prenatally always pose a N.Y. dilemma, since the fetal phenotype cannot be reliably predicted. A 41 year Duplication of long arm of chromosome 4 [4q] resulting in partial trisomy old woman, gravida 3, had an amniocentesis for advanced maternal age. has been reported in approximately forty cases. Nevertheless, the Approximately 50% of the cells from 4 different cultures showed a small duplicated segment varied from 4q12 to 4q34 resulting in different clinical supernumerary ring-like marker with one centromere (one darkly staining C- masnifestation in those patients. The distal segment is usually more band region), non satellited (Ag-NORs negative). The marker was identified frequently involved than the proximal one. We present an unusual case with as a der(16) by multiprobe FISH (Cytocell). Further investigations confirmed duplication of 4ql2q21.3 that to the best of our knowledge has not been the karyotype as 46,XY/47,XY,+m.ish r(16)(wcp16+,D16Z3+). previously described. Our patient, an 8 year-old girl was referred for Chromosomal analysis of both parents was normal. The pregnancy was cytogenetic evaluation because of mental retardation, hearing loss, bilateral continued after genetic counselling where the parents were informed of the ptosis of the eyelids and asthma. Routine cytogenetic technique revealed results, despite the lack of information for this specific abnormality. At term, inserted material on chromosome 4q, which was later identified as being a healthy 3560g boy, Apgar 9/9/10, was delivered. Neither dysmorphic part of 4q by FISH-technique. Although hearing loss has not been described features nor malformations were noticed. At 10 months, clinical examination previously, clinical findings are variable and nonspecific. The most common was normal. The chromosomal findings were confirmed from blood and phenotypic features include: growth and psychomoror retardation, low set cord fibroblasts; approximately 75% of the cells had the r(16). This is the ears, narrow palpebral fissures, hypotonia, epicanthic fold, hypertelorism, first case to our knowledge, of a prenatally diagnosed marker derived from ptosis of the upper lids, microcephaly, congenital heart defects, carp mouth an extra 16. However the absence of phenotypic abnormalities suggest that and antimongoloid slant. In summary, the clinical findings do not seem to the extra genes present are not responsible for the high pathogenicity correlate to delineate a distinct syndrome. observed in full trisomies 16.

2189 2190 Identification of a balanced chromosome translocations and prenatal An HRC G-banding Investigation in a family with t(11;17). Y. Qi and C.L. cytogenetic diagnosis In carriers. S.Y. Park, Y.M. Kim, J.M. Kim, H.M. Zhang. Dept. of Clinical Genetics, Qingdao Sencond People's Hospital, Ryu', J. Y. Jun', SK. ChoL. Genetic Research Laboratory, Obstetrics & Qingdao, Shandong, 266033, P.R. China. Gynecology1, Samsung Cheil Hospital & Women's Healthcare Center, The balanced reciprocal translocation is a common chromosomal Samsung Medical Center, Seoul, Korea. abnormality in clinical cytogenetics. But this type of decease has wide During the years 1988-1996, cytogenetic analysis was performed on variations. 1462 women and 1321 men with history of adverse reproductive outcome. This poster is to report a family with t(1 1;17). The subject is a 29-year-old 104 balanced chromosome translocation carriers were detected. 70 cases Chinese woman. She requested genetic counselling because of three first were reciprocal translocation carriers and 34 had Robertsonian trimester abortions. She has a weak constitution but no particular facies. translocations. Chromosome aberrations were more frequent in women (73 She had been attacked by tracheitis and rheumatism but otherwise her cases) than in men (31 cases). No phenotypical abnormalities were found in health is generally good. Clinical physical examination did not reveal any all carriers, but it is shown that they experienced repetitive spontaneous abnormality. Her intelligence has been normal. She does not smoke or abortions, anomalous offsprings or infertility problems. The use of high drink. She is married and her husband is healthy. Her parents are resolution banding technique and fluorescence in situ hybridization in the unrelated. Her father had suffered from hyperaldosteronism and had been chromosome analysis has improved the correct detection of balanced cured by surgical operation. Her brother and sister are also healthy. Her reciprocal translocations. Prenatal diagnosis was carried out on 35 sister has been married with a healthy two-year-old daughter. subsequent pregnancies of balanced translocation carriers. The fetal Cytogenetic analysis was used for this study, with samples of High karyotypes showed that twelve cases were normal, twenty-one were Resolution Chromosome (HRC) by G, C, R and NOR banding. The balanced translocations, and two were unbalanced translocations. Most of pripheral blood lymphocytes karyotyping confirmed that the subject and her the fetal samples showed normal karyotypes and had balanced father has the same chromosomal aberration with 46, t(11;17) (11pter- translocations. The incidence of fetal chromosomal imbalance was relatively >q2$::17q21.3->qter; 17pter->q21.3::1 1q25->qter). This abnomality is low in prenatal diagnosis. And high resolution banding technique and clearly the reason for her previous abortions. fluorescence in situ hybridization were useful tools in the precise evaluation This case is an unusual translocation. This particular chromosomal of chromosome aberrations. anomaly has not been previously reported. This finding suggests that prenatal diagnosis will be potentially useful for patients with t(11;17) to avoid repeated abortions.

2191 2192 Chromosome 4 long (q) arm tr somy: longest autosomal trisomy Translocation 2;19 in a patient with probable relapsed AML. P. Rintels, of and compatible with life - pro- and postnatal observation. M. R. Rahman, X. Y. Gray, R. Griffith and H. F L. Mark. Departments Pathology Leduc, A. L. Medearis and F. H. Anderson. Clinical Genetics Center, 14241 Medicine, Rhode Island Hospital and Brown University School of Medicine, E. Imperial Hwy #C, La Mirada CA., Prenatal Diagnosis Center, Loma Linda Providence, Rhode Island University, Loma Linda, CA. We report the cytogenetic and hematopathologic results from a second Sex chromosome trisomies, whether complete, partial or mosaic are specimen derived from a patient diagnosed with acute myelogenous compatible with life with minimum dysmorphic features and in most instances it leukemia. Although the initial specimen revealed an apparently normal male is the gonads that are affected. Among the autosomal trisomies, #21, #18 and karyotype, a translocation, t(2;19)(q21;p13), was detected in the second #13 are the most frequent and are mild to severely phenotypically affected. specimen. In an extensive search of the recent medical literature database Other autosomal trisomies are non-viable and mostly contribute to fetal (Medline 1966-present, CancerLit 1983-present, MDX Health Digest 1988- wastage. Partial autosomal trisomies are viable depending on the size of the present, HealthSTAR 1975-present and CINAHL 1982-present), we found region that is triplicated. We report here a fetus and the family with, no previous report of this specific translocation. This case is of interest der(8)t(4;8) chromosomal abnormality, which is the longest trisomy for the only because of its cytogenetic rarity and its unique clinical features, but chromosome 4 long arm to our knowledge. The mother of the fetus is a carrier also because of its possible relevance to genotoxic exposure. Specifically, of balanced translocation and her karyotype is 46,XX,t(4;8)(q21;p23). The this patient worked in construction management, performing off-shore effect of the size of the trisomic region of chromosome 4 long (q)arm has been drilling in oil fields for several years and also worked with plastics and studied with respect to the extent of the morphological abnormality and polymer film for about 4 years. In addition, it is also of interest to point out viability. Midtrimester fetal ultrasound revealed hydramnios, small frontal area that one of the translocation breakpoints, 19p13, is apparently identical to of the brain and probable tetralogy of the fallot. Postnatal findings showed that found in the 1; 19 translocation associated with pre-B-ALL. smaller size fetus than gestational age, probable hypoplastic aortic arch, microcephaly, minimal hypotonia, syndactyly of 2nd and 3rd toe and excessive skin over the neck and back. The 18 month old patient and the 5 year old sib with, der(8)t(4;8), chromosome abnormality is trisomic for 4.04% and monosomic for 0.25% of the total haploid autosomal length (HAL) for the chromosome 4 Iong arm and the chromosome 8 short (p) arm, respectively. Partial trisomy and monosomy did not show significant additive imbalance affect which is contrary to the prevailing HAL effect view of viability. The effect may be chromosome specific and depend on the type and nature of the genes involved. This study supports the general view that addition has a milder lethal effect than deletion and is more tolerated in nature. Published Abstracts: Cytogenetics (cont.) A375

2193 2194 Identification of chromosome 15q11-12 haplo-Insuffiency in the FISHING for origin of satellite on the long arm of chromosome 4. H.O. Autistic Disorder. MA Sabiyt, A Al Shubaii', MA MaghrabP, S Farah2, S Shah, R.S. Verma, R.A. Conte, M.Chester, T.V. Shklovskaya, S.M. J Abulhassan1, MA Abde Rasooll, S A Al Awadi'. 'Kuwait Medical Kleyman, V. Diaz-Barrios, B. Feldman, J.H. Lin and J. Sherman. Nassau Genetics Centre, Maternity Hospital; 2Neurology Department, IbnSina County Medical Center, East Meadow, Institute of Molecular Biology and Hospital; 3Psychological Hospital; Kuwait Genetics at InterScience, Brooklyn, and Long Island College Hospital- We describe a 7 year old Egyptian boy who underwent formal neuro- SUNY Health Science Center at Brooklyn, N.Y. psychological assessment in Kuwait and the USA because of specific The origin of satellited material remained illusive by routine cytogenetic concerns regarding his delayed speech, steriotypic behaviours, short techniques since all acrocentric chromosomes are cytologically identical. attention span, and lack of social interaction. The child did not have any The recent availability of probes specific for the short arms of acrocentric communicative intent, comprehension of language, or speech patterns. He chromosomes prompted us to characterize a familial [matemal] satellited was indifferent to positive reinforcement, did not take pleasure in success, long arm of chromosome 4 in amniocytes from a 35 year old mother at 16 and exhibited little problem-solving skills. His resulting score in various week gestation. Molecular cytogenetics using a variety of probes by FISH clinical and psychological testing was consistent with the diagnosis of technique revealed the following impressions: Whole chromosome paint Autistic Disorder (DSM-IV 299.00) with a secondary diagnosis of Moderate [WCP] probe specific for chromosome 4 suggested that satellited 4q is Mental Retardation (DSM-IV 318.0). Fluorence In Situ Hybridisatio (FISH) intact. A beta satellite DNA probe specific for all human acrocentric technique was undertaken in Kuwait on high resolution metaphase spreads chromosomes hybridized to the 4q terminal satellited region. The Ag-NOR of his peripheral blood lymphocyte chromosomes, using three different region was positive by silver staining confirming the presence of active NOR PWS/AS probes: GABRB3, D15S11, and SNRPN (Oncor). A hemizygous on 4q. We have clearly demonstrated that the alpha satellite DNA material 15q1 1-12 microdeletion was detected with probe SNRPN. The same on 4q originated from either chromosome 14 or 22. Unfortunately, at present microdeletion was not identified in another Egyptian child diagnosed with there is no available probe specific for either of these two chromosomes Autism. This report emphasises a subset of the Autistic disorder as a since their alphoid DNA sequences are closely related. Nevertheless, the member of 15q1 1-12 haplo-insuffiency cluster and highlights the origin of satellited material on 4q has been narrowed down to these two heterogenous nature of the disorder. chromosomes. The mother is clinically normal suggesting it may have originated from a translocation between 4q and either 14p or 22p without an apparent loss of genetic material from 4q. The birth defects noted in children with a 4q satellited chromosome in earlier cases apparently are not related to this anomaly as our case was clinically normal.

2195 2196 FISH characterization of unusual familial variants involving mobile Repository of Human Chromosomal Anomalies in China. Dong-sheng acrocentric elements. V Sulcova and KS. Reddy. Quest Diagnostics, San Tang, Jia-hui Xia, Lu-yun Li, He-ping Dai, Su-lan Jiang, Chun-yu Liu, Zhi- Juan Capistrano, CA gao Long. National Laboratory of Medical Genetics of China, Hunan A maternally inherited chromosome 21 with additional material on the short arm Medical University, Changsha, Hunan 410078, P. R. China was found in a 4-month-old boy with trisomy 21 and Down syndrome features. Repository of Human Chromosomal Anomalies in China contains a Fluorescence in situ hybridization (FISH) studies revealed a tricentric chromosome computer database of abnormal karyotypes and several editions of with a 21 centromere followed by two 15 alpha, classical and beta satellite of Human Abnormal sequences, interspersed by three AgNOR+ve regions and a terminal beta satellite irregularly published Repositories Karyotypes edited in sequence. There was a single telomere at each end of the chromosome. The English and Chinese. National Laboratory of Medical Genetics of China breakpoint was shown by FISH to be proximal to SNRPN on chromosome 15. His began to collect and verify the abnormal karyotypes among Chinese, and karyotype is 47,XY,+add(21)(p1 1).ish psu trc(15;15;21)(D15Z+,SNRPN- registered the abnormal karyotypes reported firstly in the world in 1985. ;D15Z+,SNRPN-;D13Z1/D21Z1+)mat There are 1210 kinds of firstly reported karyotype which were found by An 18-month-old boy with developmental delay and slight dysmorphic facial 658 cytogenetists from 257 laboratories of China in the Repository of features carried an insertion on the short arm of chromosome 7 inherited from his Human Chromosomal Anomalies. Every entry in the repository include the normal mother.The insertion was C-band positive and AgNOR negative. Coatasome fields of karyotype, abnormal chromosomal atlas, clinical data, reporter, 7 probe revealed a gap in the painting in the proximal short arm region of institute and reference etc. The chromosome 7. The all centromeric did not to the insertion on address, computer database of abnormal probe hybrdize karyotypes can be inquired by chromosomal band, karyotype, reporter and chromosome 7, but the acrocentric beta satellite probe did. The karyotype is institute. The in 46,XY,ins(7)(p13;?).ish ins(7)(p13; 0-s)(wcp 7-,a-s -, acrocentric 0-s+)mat entries the published repositories are sorted by A satellited chromosome 2 long arm was found in two normal and two cases chromosomal band, karyotype and indexed by reporter. suspected to have . The satellited 2 had a small C-band followed by a AgNOR positive region terminating in a larger C-band. Fluorescence in situ hybridization (FISH) was negative for individual 1-22, X and Y centromeric probes but gave a small positive signal for the all centromeric probe. The acrocentric beta satellite probe gave signals that mimicked the C-banding pattem. All telomeric probe hybridized to the two ends of 2qs, therefore the terminal tip of the long arm of chromosome 2 was lost. The satellited 2 is mostly repetitive alpha and acrocentric beta satellite DNA. The karyotype in the 4 patients is 46,XX/XY,psu dic(2;?)(q37.3;?). ish psu dic(2;?)(q37.3;?)(a-s+, acrocentric p-s x 2, tel+). FISH proved to be a powerful tool to explore chromosome variants. It not only provided the molecular information but also gave the spatial organization of the elements involved. Thereby establishing not only the constitution of the variant but also the mechanism by which they arose.

2197 2198 Partial trisomy 10p and partial monosomy 17p due to a paternal The natural outcome of mosaic and nonmosaic trisomy 21 zygotes. G. translocation. M.K. Thong, Y.M.Chin1. Department of Pediatrics, Faculty of Viot and M. Vekemans. Department of Genetics H6pital Necker-Enfants Medicine, University of Malaya, 50603 Kuala Lumpur, Malaysia. 1: Division Malades Paris - France of Hematology, Institute for Medical Research, 50588 Kuala Lumpur, Using the data published in the literature, one can estimate in a cohort of Malaysia. 100 000 recognized pregnancies, the outcome of mosaic and nonmosaic An 11-month old Malaysian boy presented with global developmental trisomy 21 zygotes. The incidence of this mostly derived trisomy is delay, multiple dysmorphic features and recurrent seizures.The patient equivalent to 320 cases (312 non-mosaic and 8 mosaic) in 100 000 showed a karyotype of 46,XY,-17,+der(17)t(10;17) that had originated from recognized pregnancies. It is likely that most of the mosaic cases derive by adjacent-i-segregation of paternal balanced reciprocal translocation reduction to disomy from trisomy 21 zygotes. About 66% of the non-mosaic 46,XY,t(10;17)(p11.2;p13.3). Our patient had no features of Miller-Dieker cases are lost prenatally whereas the remaining cases are reaching term. syndrome and no lissencephaly on imaging studies. Fluorescent in-situ Among the 8 mosaic cases, 3 will reach term whereas the remaining is hybridisation (FISH) studies using a 17p telomere probe showed positive either detected as parental mosaicism or undetected because of prenatal hybridisation to normal chromosome 17 homolog only while 2 cosmid loss, cryptic mosaicism or uniparental dsomy. These figures are very similar probes corresponding to the lissencephaly region on 17p13.3 had positive to the estimates made on the natural outcome of (Wolstenholme signals to both normal and derivative 17 homolog, indicating a terminal J. Prenat. Diagn. 15: 109-122, 1995). In particular, both studies show that deletion of der(17) chromosome with a familial translocation point distal to chromosome mosaicism and aneuploid rescue might be an important the lissencephaly region. To the best of our knowledge this karyotype is modifier of the natural outcome of trisomy 21 zygotes. One need to better hitherto not reported in the literature and highlights the importance of FISH understand the mechanisms involved in generating chromosome mosaicism study to exclude Miller-Dieker syndrome despite the presence of monosomy as it might provide a basis for correction of aneuploid conditions. 17p13.3. The implication for genetic counselling is discussed. The authors would like to thank Dr D.H. Ledbetter, University of Chicago Genetic Services, Chicago, Illinois for his service and kind assistance in using the above FISH probes for the studies. A376 Published Abstracts: Cytogenetics (cont.)

2199 2200 Familial mosaicism for small supemumary chromosomes. Prenatal detection of traomy (9p) J. Xul, J. Chemosl,2, and B. HE.Wyandt1, X-L.Huang1, J.Milunsky1, A.Milunsky1 1Center for Human Rolanf2'3. Cytogenetics Laboratory, Alberta Children's Hospital1, and Genetics and Department of Pediatrics,Boston University School of Department of Pathology2 and Department of Medical Genetics3, University Medicine, Boston, MA. of Calgary, Calgary, Alberta, Canada Two cases of small familial supemumary chromosomes were Isochromosome (9p), reported in 22 cases to date, has tissue-limited ascertained for different reasons. Case 1, from a 35 yo normal male was mosaicism as a feature, with the chromosome present preferentially in referred because his wife had two first-trimester miscarriages. Chromosome peripheral blood cells, and either present, to a less extent, or absent from skin study showed an extra small marker in 11/23 (47.8%)lymphocytes. FISH fibroblast cells. We present here the first report on prenatal detection of idic identified the marker as derived from chromosome 22 and to include the (9p) using amniocentesis. DiGeorgeNCFS region and the telomere. Silver staining showed a NOR on Amniocentesis was performed for late maternal age on a 35-year-old one arm. Chromosome studies of the man's parents revealed his 58 yo woman at 14 weeks gestation. G-banding of in situ cultured cells showed that mother to have the same marker in 29/78 (37.2%) lymphocytes. Both 15 mitosis, from 13 colonies plus 2 solitary cells, all had an abnormal male mother and son are normal with no apparent features of cat-eye syndrome karyotype:47,XY,+ldlc(9)(q12). The fetus was electively aborted at 17 weeks or other clinical abnormalities. gestation and the products of conception were karyotyped using cultured fetal Case 2, a 37 yo female, had an amniocentesis for advanced maternal cells. G-banding of fetal metaphase cells showed 61.9%(13/21) of the cells age. study revealed an extra small marker in 58/59 with 46,XY, and 38.1%(8/21) with 47,XY,+idkc(9p). Interphase FISH using a Prenatal chromosome whole chromosome paint for chromosome 9 showed 28.6%(30/105) of the celils (98.3%) cells. Chromosome study of the mother revealed she had the same with 2 hybridization domains, presumably representing normal diplold marker in 10/100 (10%) lymphocytes. FISH identified the marker as a small cells, and 71.4%(75/105) with 3 domains, representing the cells with the extra ring derived from chromosome 9, consisting of centromere and proximal idic(9p). The karyotype of the mother is normal. The karyotype of the father is short arm, but lacking heterochromatin from the long arm and telomeres. unknown. The infant was delivered without complications. A chromosome study Because the fetus was not Intact, the pathology was difficult to evaluate. revealed the r(9) in 35% of lymphocytes. Neither mother nor child appear to There was, however, no evidence of congenital heart disease, or of hand or have phenotypic abnormalities. foot anomalies. Clinical outcomes of patents with (9p)vary from prenatal death to mild anomalies, depending on size of the isochromosome and the degree of mosaicism in blood cells and, particulary, in skin fibroblast cells. The findings in this case illustrate that even with a high proportion of abnormal cells in amniocentesis and fetal cells, there may be no detectable ultrasound abnormalities. The present case also demonstrates the possibility of prenatal detection of tetrasomy (9p) even though tissue-limited mosaicism is the rule.

2201 Constitutional supernumerary marker chromosome 15 In a girl with Down syndrome. KK Yelavarthi, L Brannon and J Zunich. Northwest Center for Medical Education, Indiana University, Gary, IN 46408. Constitutional supernumerary marker chromosomes are not uncommonly seen in the course of cytogenetic studies. Most common chromosome markers is the inv dup(15) which may rep resent as many as 50% of small supernumerary chromosomes detected during routine karyotyping. We report a case of a neonate who was referred for karyotyping because of suspected Down syndrome. Chromosome analysis on peripheral blood revealed a female chromosome complement with trisomy 21 and an additional small metacentric marker chromosome in each of the 20 cells analyzed. Further characterization of the marker chromosome indicated that it was C-band and distamycin/DAPI positive. FISH using Oncor's chromosome 15 related probes are as follows: WCP 15 detected possibly no signal, D15Z4(alpha satellite 15 probe for the p1 1-q 1 region) showed a single focus of hybridization and D15Z1(classical satellite 15 probe for the Pencentromerc heterochromatin of the 1 5pI 1.2 region) detected two hybridization signals on either side of the heterochromatin present on the marker chromosome. in light of these results, the marker chromosome is identified as pseudodicentnc #15 with localized breakpoints at q1 1. Parental studies demonstrated that the marker was inherited from the father. Since the father is pheonotypically normal, this marker chromosome with extra material from pter-ql 1;q1 -pter may not pose any additional burden on the clinical symptoms of the proband with Down syndrome. The proband's karyotype was 48,XX,+dic(15;15),+21.ish psu dic(15;15)(q11;ql1)(WCP- ,D1 5Z1++,D1 5Z4+)(pat).

Published Abstracts: Development and Prenatal Genetics

2202 2203 Pulmonary atresla In a fetus with molecularly confirmed Apert Prenatal counseling for anatomic variants" detected by sonography. syndrome. IJ Anderson. Developmental and Genetic Center, University of P.M. Blakley, R.M. Hoar and L.B. Holmes. Genetics and Teratology Unit, Tennessee Medical Center, Knoxville. Massachusetts General Hospital, Boston, MA. Apert syndrome is an autosomal dominant craniosynostosis syndrome Prenatal screening by sonography often identifies minor anatomic characterized by craniosynostosis, midfacial malformations and symmetric findings, such as mlld hydronephrosis, mildly dilated lateral cerebral syndactyly of the hands and feet. A variety of other associated ventricles and cysts of the choroid plexus. These findings cause anxiety in abnormalities have been reported at lesser frequencies. These include cleft parents because of the concern that they may reflect a significant degree of palate, cervical vertebral anomalies, genitouninary, gastrointestinal and abnormal development. We have learned of two aspects of such anatomic respiratory system anomalies. Cardiovascular anomalies have been findings or 'variants which help when counseling these pregnant women. reported at a frequency of approximately 9-10%. Recently, specific First, some of these features have also been identified during the development missense mutations involving adjacent amino acids Ser252Trp or of other species, such as ,physWlogic or Umild" hydronephrosis In rats Pro253Arg in the linker between the second and third extracellular (Teratology 1972; 6:191-196) and Omild* hydrocephalus in mice (Neuropath Appl Neurobiol 1988; 14.263-274) and rabbits (J Am Col Tox 1985; 4:218) immunoglobulin domains of the Fibroblast Growth Factor Receptor 2 gene birth and maturation of the have been shown as causative. Atrial and ventriculoseptal defects, which generally return to normal* following species foramen in question. Many other anatomic variants have also been identified in coarctation of the aorta, dextrocardia, tricuspid atresia, patent experimental animals, such as wavy ribs (which disappear after birth), extra ovale and mitral valve prolapse have been reported in individuals with ribs, delayed ossification and others, which could also be surprise findings molecularly confirmed Apert syndrome. during prenatal sonography. Full documentation of the frequency of these Reported here is a fetus with clinical features of Apert syndrome and a variants in animals has been published (Cong Anom 1987; 27:147-206 and molecularly confirmed FGFR2 mutation with atresia of the pulmonary 1997; 37:47-138. Second, some anatomic findings, such as chorold plexus outflow tract and single ventricle. cysts, have been identified commonly in all age groups among humans. These This suggests that pulmonary atresia and single ventricle be added to two findings do not mean that anatomic *variants" are not potential markers of the list of cardiac anomalies seen in Apert syndrome and that Apert significant disorders. However, noting that they are most often part of the syndrome be considered in the differential diagnosis of prenatally detected spectrum of normal anatomic development could avoid conveying the initial severe congenital heart disease. impression that the fetus is definitely abnormal. Published Abstracts: Development and Prenatal Genetics (cont.) A377

2204 2205 Hydranencephaly associated with absence of the Internal carotids: a new Clinical applications of preimplantation genetic diagnosis. M. condition with X-iinked or autosomal recessive Inheritance. C. Boerkoel, 1 D. Bonduelle, K. Sermon, K. Staessen, H. Joris, W, Lissens, E. Van Assche, Chitayat, 1 J.B.M. Mullen, 2 and S. Nag.2. 'Prenatal Diagnosis Program and P.Nagy, M. Vandervorst, P. Devroey, A. Van Steirteghem and 1. Liebaers. Departments of 'Medical Genetics and 2Pathology, University of Toronto, Toronto, Dutch-speaking Brussels Free University, Brussels, Belgium. Ontario, Canada. Clinical applications of preimplantation genetic diagnosis. M. Bonduelle, Hydranencephaly (HE) is frequently a disruptive disorder caused by prenatal K. Sermon, K. Staessen, H. Joris, W, Lissens, E. Van Assche, M. vascular insult to the brain. In most cases this is a sporadic disorder with a low P.Nagy, recurrence risk; however, we now report two male siblings who were prenatally Vandervorst, P. Devroey, A. Van Steirteghem and 1. Liebaers. Dutch- diagnosed with HE and postnatally found to have bilateral absence of the internal speaking Brussels Free University, Brussels, Belgium. Preimplantation carotids. genetic diagnosis (PGD) is a novel procedure which can be offered to Case 1: The mother (G1P0) and father are unrelated and of East-Indian descent. couples in high risk situations for genetic disease as an alternative for They were 24 and 26 years, respectively. The pregnancy was uncomplicated until a prenatal diagnosis. Using polymerase chain reaction (PCR) or fetal ultrasound at 17 weeks gestation revealed a clubbed left foot and HE. The Fluorescence in Situ Hybridisation (FISH) the genotype or chromosomal pregnancy was terminated, and fetal autopsy confirmed the ultrasound findings. content of biopsied cleavage stage embryos obtained after in vitro TORCH analysis and alloimmune antiplatelet studies on the mother were fertilisation can be determined and selected embryos transferred to the negative. The fetal karyotype was 46,XY. uterus. Prenatal is then later in to confirm Case 2: Conceived 18 months after case 1, the second pregnancy of this couple diagnosis performed pregnancy was uncomplicated until a fetal ultrasound at 17 weeks gestation showed HE. The the PGD, since a small risk for misdiagnosis still exists especially in case of pregnancy was terminated, and fetal autopsy confirmed the HE and revealed absent PCR based diagnosis. Intracytoplasmic sperm injection combined with in internal carotid arteries. The fetal karyotype was 46,XY. vitro fertilisation (IVF) is the method of choice to reduce contamination risk Familial hypoplasia of the internal carotids has been reported as a rare, late by residual sperm DNA for embryos analysed with PCR technique, whereas onset, autosomal recessive disorder (OMIM 243100) presenting with symptoms IVF could be used if FISH is applied. In our series 53 PGD were performed attributable to cerebral ischemia [Austin et al., Arch. Neurol. 24: 1, 1971]. However, for monogenic disease through PCR and 8 PGD were carried out with FISH in contrast to hypoplasia, absence of the internal carotids has only been reported in for X-linked disease. Of 61 PGD cycles for 29 couples at risk in a period of isolated cases. In infants, bilateral absence of the internal carotids has been reported 4 years, the ongoing was twice but only in association with congenital cardiac abnormalities and without HE. pregnancy rate per cycle 15%, per transfer 19% To the best of our knowledge, this is the first report of siblings with HE caused by and per patient 31%. Of the 10 pregnancies obtained 1 ended in an early bilateral absence of the carotids. It illustrates the near inability of the fetus to develop miscarriage, 4 are ongoing and 5 led to 6 live born children of whom 1 (part collateral cerebral circulation and thus the vulnerability of the fetal brain to vascular of a monozygotic twin) died in the early neonatal period. Children born after disruption. Furthermore, this demonstrates that not all cases of HE have a low PGD will be prospectively followed for morphological abnormality, growth recurrence risk and emphasizes the need to examine the carotids when HE is parameters and development at 2 months, 1 year and two years. Until now detected prenatally. six morphologically normal children were bom.

2206 2207 Prenatal Detection of a Deletion in the 15 (ql1.1q13) Prader Wiili Syndrome/ Polymorphisms of (CA) dinucleotide repeat in intron 13 and 22 of Region. P.B. Buchanan1, C. H. Laundon', Y. Ning2, and factor ViII gene in Korea and its application to prenatal diagnosis of T. Medical Donlon3. IGeneCare Genetics Center, Chapel Hill, NC; 2NHGRI, hemophilia A. 1 YH Cho, 1SH Shim, SR Chung. Department of Medical Bethesda, MD, 3Kapiolani Medical Center, Honolulu, HI. Genetics, 2Department of Obstetrics & Gynecology, College of Medicine, Individuals with abnormalities in the proximal long arm region (q11.1q13) of Hanyang University, Seoul 133-791, Korea. chromosome 15 usually have either Prader Willi Syndrome (PWS) or Angelman Syndrome (AS) depending upon the parental origin. When the genetic material in CA dinucleotide tandem repeats or microsatellite polymorphisms are useful this region is altered from a paternal 15 chromosome, PWS can occur. When the genetic markers especially in prenatal diagnosis of genetic disorders by same region is altered from a maternal 15 chromosome, AS can occur. PWS is linkage analysis. Two CA dinucleotide repeat polymorphisms within intron 13 characterized by hypotonia and failure to thrive in early infancy, short stature, and 22 of factor VIII gene were analyzed with 144 healthy unrelated persons hypogonadism, hypopigmentation, mental retardation, small hands and feet and (94males and 50 females). One of the PCR primers were end-labeled with 33- obesity due to hyperphagia beginning in early childhood. Characteristics of AS P and the amplified products were separated on denaturing sequencing gel, include mental retardation of a greater severity, poor to absent speech, and autoradiographed. For the intron 13 (CA)n dinucleotide repeat paroxysms of laughter, ataxia, microcephaly and disorders. These polymorphism 7 alleles, (CA)24, (CA)25, (CA)26, (CA)27, (CA)28, (CA)29 and differences in phenotype between PWS and AS depending on which parental chromosome 15 is altered is due to . Approximately 70% of (CA)3, were identified with the frequencies of 0.0211, 0.6421, 0.2211, 0.0211, PWS and AS are due to deletions in the 15(ql 1.1 q13) region. These patients are 0.0632, 0.0111 and 0.0211, respectively, and the expected rate of usually diagnosed in the early childhood period. We report here the prenatal heterozygosity for females were 0.5336. For the intron 22 (CA) n(TC)n detection of a 15(q11.1q13) deletion [del(15)] in amniotic fluids cells from an dinucleotide repeat polymorphism 4 alleles, 81bp, 83bp, 85bp and 87 bp amniocentesis performed at 18wks gestation for increased maternal age. GTG alleles, were detected with the frequencies of 0.0109, 0.2880, 0.6304 and banding at the 550+ band level showed a suspected deletion in one of the 15 0.0707, respectively, with the expected heterozygosity of 0.5146. With these chromosomes. FISH using two probes for the detection of PWS and AS (SNRPN two markers, we could expect the female heterozygosity of 0.6756, which was andD1 5S1 0-Vysis) confirmed the deletion of the PWS/AS genetic material in one somewhat lower than in white Europeans. But these polymorphisms are still but not the other 15 chromosome. Analysis of both parental bloods showed no very useful in many cases, especially when the other polymorphic loci were detectable cytogenetic deletion by GTG banding and no deletion of either the noninformative. In a three generation family study, one female was confirmed SNRPN or D15S10 probes. QM variant analysis of the parental and fetal as a carrier with St14.1 VNTR, intron 18/Bcll and intron 22/Xbal chromosomes shows a unique polymorphism which suggests maternal origin of But she was the fetal del chromosome and that the polymorphisms. homozygous for all polymorphic loci analyzed, so (15) fetus would most likely be affected was we with AS. and microsatellite studies are to prenatal diagnosis impossible. When applied intron 13 CA dinucleotide Methylation being performed determine repeat polymorphism, fortunately she was heterozygote and the parental origin of the del (15). (CA)25/(CA)26 prenatal diagnosis was made successfully. Afterwards she delivered healthy boy.

2208 2209 Biochemical screening for Down's syndrome: experience and impact of A proven case of materno-fetal transfusion determined by cytogenetic four years screening in the west of Scotland. JA Crossley, DA Aitken, E and DNA analysis. M. H. Dutoit1, M. A. Morris1, M. A. Brundle(', S. Berry, JM Connor. Institute of Medical Genetics, Yorkhill, Glasgow, G3 8 SJ, Dahoun1. 1 Div. Medical Genetics, Geneva University Hospital, Switzerland UK. 2 Dept. Pathology, Geneva University Hospital, Switzerland From September 1991 biochemical screening based on hCG/AFP/age The first pregnancy of a 31 year old woman was apparently normal until analysis at 15-20 weeks gestation was offered to pregnant women of all ages fetal death in the 35th week. A stillborn male of 3090g without any in the west of Scotland. In the first four years, 110,047 women from a total dysmorphic signs or malformations was delivered. The death could be pregnant population of 142,000 chose to have a screening test. Using a mid- explained by fetal anoxia: a spiral torsion of the umbilical cord with 4 twists trimester threshold risk of 1:220, 5.2% of women were assigned to the high risk was observed as well as a recent thrombosis of the umbilical artery. The group. Using multiple sources of ascertainment, a total of 212 Down's was anemic and syndrome pregnancies were identified in the whole of placenta the heart and lungs had multiple sites of Tardieu's pregnant population, As of routine utero, a which 146 were in women who had been screened. Of the Down's syndrome ecchymosis. part analysis for death in 46,XX pregnancies in the screened population, 98 (67%) were assigned to the high karyotype (13 cells analysed) was found in PHA-stimulated blood culture risk group and 83 of these identified by prenatal diagnosis. In addition, 2 from an intracardiac puncture. Analysis of the microsatellite polymorphism Down's syndrome pregnancies were prenatally diagnosed in women over 35 D12S79 revealed that approximately 1/3 of PHA-stimulated, fixed cells from years of age who had been assigned to the low risk group, but opted for the fetal cardiac puncture were of maternal origin, whereas pleural cells prenatal diagnosis. A further 20 Down's syndrome pregnancies were were all of fetal origin. As maternal cells are frequently found in murine prenatally diagnosed in women who did not have a screening test (10 first fetuses during normal pregnancies, this phenomenon could be simply trimester CVS, 5 amniocentesis, 4 cystic hygroma, 1 fetal blood sample). In physiological or explained by traumatism of the placenta. However in the total 105 (49%) of the total Downs syndrome pregnancies in the whole case described here, it is probable that the fetal death (or its cause) pregnant population were identified prenatally, resulting in a marked reduction contributed to the transmission of maternal blood to the fetal circulation. In in the birth incidence of Down's syndrome in the region. Since 1991 the similar situations, it is important to be aware that karyotypes of fetal blood demographics of the pregnant population have been changing, with a fall in may be misrepresentative due to preferential or exclusive culture of the birth rate of around 3% per year and an increase in the median age of the maternally-derived cells and lead to misdiagnosis of the fetal pathology. pregnant population at the time of screening from 26.4 years in 1991 to 27.8 years in 1997. This has resulted in a 60% increase in the proportion of pregnant women aged 35 years and over, from 6.8% in 1991 to 11.1% in 1997. This has consequences for both the rate of Down's syndrome pregnancies expected per 1000 births and the follow-up rate from prenatal screening. A378 Published Abstracts: Development and Prenatal Genetics (cont.)

2210 2211 Female pseudohormaphroditism with prune belly syndrome and A large deletion of 21q In a pregnancy with multiple congenital multiple caudal defects. J.S. Geer, Jr., W R. Blackbum and N.R. Cooley, anomalies. C.A. Gibbons1, A.M. Summers', E. Mak-Tamr, M.M. Silvet2, V. Jr. Greenwood Genetic Center, Greenwood, SC Jay2, E. Capual. 1Dept. of Genetics, North York General Hospital, North Prune Belly Syndrome (PBS) or the urethral obstruction sequence is a York, and Dept. of Pathology, 2Hospital for Sick Children, Toronto, Ontario, well described malformation complex consisting of deficient abdominal Canada. musculature and urogenital anomalies. Cryptorchid males represent the Partial or full monosomy remains a rare cytogenetic finding. We report of reported cases. majority (90%) here a 22 year old G2P1 woman, referred because of abnormal findings on Recently, we have evaluated at autopsy three PBS females with clitoral her 18 week ultrasound. Her amniotic fluid karyotype showed 46,XX,- hypertrophy, labio-scrotal fusion, agenesis of Mullerian derivatives and 21,+mar. At 19 weeks, the pregnancy was induced because of fetal demise. ectopic or imperforate anus. Megacystis, urethral outflow obstruction, and The marker was further characterized with fluorescence in situ renal hydronephrosis/dysplasia were present in each fetus. Ovaries were hybridization (FISH). Hybridization with whole chromosome paint 21 present in all cases. showed that, as expected, the marker originated from chromosome 21. Previous investigators have proposed mechanical urinary obstruction as Break points appear to be del (21)(ql 1-qter). A partial centromere was seen the primary etiology of PBS whereas others have suggested a umesodermal by C-banding. It was expected to be functional as the marker was found in field defect' with secondary urethral obstruction. every cell. In PBS females, pseudohermaphroditism is a consistent feature. Some of the ultrasound and autopsy findings were consistent with Previous studies have demonstrated the absence of the sex determining previously reported cases of partial monosomy 21. These included ("SRY") region in several of these cases. The possible role of hypertelorism, epicanthic folds, wide mouth, club foot, and hypoplastic- developmental genes (e.g. PAX2 and HOX4E) has been proposed to dyplastic kidneys. Demineralized bands at the end of the long bones were explain the incomplete differentiation and masculinization in female PBS. observed on the post mortem radiograph. This fetus also had semilobar Other evidence suggests transforming growth factors beta and their role in holoprosencephaly, hypertrophy of both cardiac ventricles with hydrops and vasculogenesis as playing an important part in PBS. We present further changes suggestive of retinal dysplasia. The three-vessel cord was so studies of the relationships between vascular dysgenesis and urogenital twisted that on ultrasound there appeared to be more than three- vessels. septal fibrosis as these processes relate to the urinary obstructive lesions in Cases with varying deletions of chromosome 21 are needed in order to PBS. gain a better understanding of the full range of phenotype for partial and full monosomy 21. This case presents a number of features commonly associated with deletions of 21q but also findings which are more atypical.

2212 2213 SGAP: The skeletal genome anatomy project. L. Jial D. J. Wilkin1, M. Bittner1 P. Intrauterine growth restriction (IUGR) associated with confined Robe9, M. Young2 Y. YamadaW D. KrizmarP, L. Liotta, R. Bonner4, G. SchuleA, M. placental mosalclsm of ring choromosome 15. E.S.Kim, H.M.Ryu, BoguskP,J. Powel, G. Lennon, D. Roodman8, R. Hotchkiss9, P. Meltzer1, J. J.H.Yang, M.Y.Kim, S.Y.Parki, S.K.Choi1, S.R.Hong2, H.WHan. Division of Trent1, and C. A. Francomano1. 1NHGRI, 2NIDR, 3NCI, 4NCRR, 5NCBI, 6DCRT, NIH, Maternal Fetal Medicine, Department of Obstetrics and Bethesda, MD, 7Lawrence Livermore National Laboratory, Berkeley, CA, 8University Gynecology, Genetic Research Laboratory', Department of Cheil for Surgery, New Pathology7, Samsung of Texas Health Science Center, San Antonio, TX, 9Hospital Special Hospital, Sung Kyun Kwan York, NY. University, Seoul, Korea. Genetic known as The skeletal genome anatomy project, SGAP, is a genotypic and phenotypic inconsistency between embryo/fetus and placenta, characterization of bone and cartilage. SGAP is intended to be a resource for confined placental mosaicism, is an extremely rare clinical condition. The scientists interested in both normal and abnormal skeletal development and growth. present report describes a case that showed a normal fetal karyotype in an SGAP will include a catalogue of genes expressed in bone and cartilage, as well as a antenatal genetic study but an abnormal placental karyotype of 46,XX,r(15) tissue bank of normal and abnormal bone and cartilage, tendon, ligament and on postnatal examination. The pregnancy was complicated by fetal nuchal synovium. translucency in the first trimesters. A 1780 gm female baby was born after Genes included in SGAP are those which are necessary for general bone 40 weeks of gestation, but and on the related died of respiratory distress sepsis development and growth and those which result in the skeletal dysplasias and 10th day of life. Our case monogenic and complex disorders of skeletal growth and development. The initial was unique in that the placental chromosomal aberration was a aberration stage of this project will be to characterize expression patterns of catalogued structural abnormality instead of a numerical that is seen expressed sequence tags (EST's) in cartilage and bone. This will be done using in most reported cases of confined placental mosaicism. 15,000 sequenced human ESTs from the dbEST database that have been PC amplified and robotically arrayed on glass. Hybridization of fluorescent probes, derived from mRNA of various cell types of bone and cartilage, to the microarray will determine the EST expression pattern in these cells. EST's representing previously uncharacterized genes, shown to be expressed in skeletal tissue, will be further studied. Concurrently, cDNA libraries from a variety of skeletal tissues will be assembled. All sequence data will be available to the public on the internet. These clones, which are likely to include novel expressed sequences, will be used to create a skeletal-enhanced microarray, which will then be used to analyze gene expression using probes from specific skeletal tissues in normal and disease states. We have sequenced greater than 65 clones from a human bone marrow stroma fibroblast cDNA library, with at least 6% novel sequences. It is anticipated that SGAP will greatly accelerate the process of gene discovery related to skeletal growth and development, as well as our understanding of the pathophysiology of skeletal disorders.

2214 2215 Prenatal Diagnosis of Monozygotic Twins Concordant for Non-mosaic Outcome of unexplained high amniotic fluid alpha-fetoprotein. A. Pai, 45,X Turner Syndrome. E. Lieberl, MT. Yt?, R. Shiffman', G. Lynch', J. S. Conacher and D. Chitayat. The Prenatal Diagnosis Program, The VuletinW, D. Menos1 and L. Y.F. Hsui. N.Y. Methodist Hospital, Brooklyn, Toronto Hospital-General Division, University of Toronto, Ontario, Canada. N.Y.1 &Prenatal Diagnosis Laboratoy of NYC/Medical & Health Research Elevated level of amniotic fluid alpha-fetoprotein (AF-AFP) (>2.5 MOM) Association of NYC, New York, N.Y. I is commonly associated with a variety of fetal abnormalities including open neural tube defects, abdominal wall intestinal Monozygotic (MZ) twins with Tumer syndrome (TS) are rare. Most MZ openings, cystic hygromas, twins with TS show phenotypic and karyotypic discordance and/or atresias or congenital nephrosis. Not much is known about the prognosis of cases with elevated with mosaicism. We report a prenatal diagnosis of non-mosaic 45,X in MZ twins. AF-AFP associated negative acetylcholinesterase and normal fetal karyotype. A 16 year old black female GI PO (who was herself one of triplets) was We reviewed all cases with AF-AFP detected during found on ultrasound to be carrying twins, both with cystic hygromas and one unexplained high the last 3 years at our June 1994 and 1997, 7076 being much larger than the other. Amniocentesis at 16 weeks of gestation program. Between May amniotic fluid samples were and 31 had high AF-AFP yielded clear fluid from one sac with a normal amniotic fluid alpha- analyzed samples levels (>2.5 MOM) and Of these 9/31 had fetoprotein (AFAFP) (from Twin A) and brown colored fluid with a highly negative acetylcholinesterase. elevated AFAFP) (18 SD above the norm) and positive acetylcholinesterase normal fetal ultrasound(unexplained AF-AFP). The AF-AFP levels among the eight cases from -11.9 All 9 cases were carried to (from Twin B). Analysis of amniocytes from both Twin A B showed a non- ranged 2.65 MoM. term and no abnormalities. mosaic karyotype in 30 metaphase spreads. Because of the massive postnatal examination and follow-up showed 45,X The above results cases with AF-AFP level, cystic hygroma of both twins and fetal demise of Twin B, the patient opted suggest that most high negative acetylcholinesterase and normal detailed fetal ultrasound have for termination of the pregnancy at 19 weeks. Except for the cystic good outcome. hygromas, the twins, both phenotypically female, showed no gross abnormalities. Twin B was macerated and much smaller. The placenta displayed two sacs with fusion and three vessel cords. Chromosome analysis of fetal tissues (fibroplasts) of Twin A confirmed the diagnosis of non-mosaic 45,X TS; Twin B did not grow in culture. 0-band comparison of chromosome polymorphisms from Twins A B showed concordance of all visible markers, indicating monozygotic orign. Although both twins were non- mosaic 45,X, twin B was only 12% of the weight of Twin A, and suffered intra-uterine demise. This difference in severity and viability of TS twins shows that MZ twins are not necessarily indentical. Published Abstracts: Development and Prenatal Genetics (cont.) A379

2216 2217 Functional analysis of two genes in the velo-cardio-facial syndrome Acrofacial dysostosis type Rodrig uez: prenatal diagnosis and autopsy commonly deleted region. A. Puechl, H. Sirotkin2, B. Saint-Jorel, R. findings. N. Quercia2, D. Chitayat1 , A. Toi1, M. Silver, M. SermerA. The Kucherlapati2, A. L. Skoultchil. 'Dept of Cell Biology, 2 Dept of Molecular Prenatal Diagnosis Program, The Toronto Hospital-General Division1 and Genetics, Albert Einstein College of Medicine, Bronx, NY. Intro. by: Bernice the Divisions of Clinical Genetics2 and Pathology3, Hospital for Sick Morrow Children, University of Toronto, Toronto, Ontario, Canada. Velo-cardio-facial syndrome (VCFS) is an early developmental disorder The acrofacial dysostoses (AFDs) are an heterogenous group of disorders characterized by heart defects, facial dysmorphology, cleft palate and with mandibulofacial dysostosis and limb abnormalities. Three types have been learning disabilities. A large deletion of the 22q1 1 chromosomal region has delineated: Nager type with preaxial limb defects, Genee-Wiedemann type (Miller been detected in most patients with VCFS and the closely related DiGeorge syndrome) with postaxial acrofacial dysostosis (POADS), and the most recently syndrome (DGS). Molecular analysis showed that the smallest commonly delineated Rodnguez type with phocomelia. We report a prenatally diagnosed deleted in VCFS +/- 1 Mb. Rare male fetus with AFD, Rodriguez type. region patients encompasses patients The mother was 28-years-old (G1P0) and the father was 25-years-old. The restrict the VCFS/DGS critical region to +/- 400 kb and several genes and couple was of Italian descent and non-consanguineous. A fetal ultrasound done ESTs that map to the critical region have been identified. We initiated an at 24 weeks gestation showed severe micrognathia, bilateral short humeri, absent effort to ascertain the role of the genes in the critical region by 1) generating radii and ulnae, and four digits on each hand. The left calf had only one bone and mice in which individual genes are inactivated, 2) generating a mouse which a rocker-bottom foot and the right leg showed metatarsus varus. The couple carries a deletion encompassing several genes that are hemizygous in decided to terminate the pregnancy. A male fetus was bom with dysmorphic facial VCFS patients. We have chosen to inactivate the IDD and ARVCF genes features including a short and slopped forehead, flat occiput, hypoplasia of the which flank the critical region and to delete the DNA between the two supraorbital ridges, flat nasal bridge, broad nasal root, severe micrognathia, and genes. The strategy for generating the deletion involves the incorporation of dysplastic auricles with absent external auditory meati. Both upper limbs and left LoxP sites into each of the Idd and Arvcf genes, bringing the two mutations lower limbs were abnormal. Neuro- and ophthalmo-pathological investigations together by transfection or animal breeding and inducing the deletion by were normal. Radiographs showed short humeri, absent radius and ulna Cre-mediated recombination. We have allready generated mice containing bilaterally, postaxial oligodactyly, short left tibia and absent fibula, 11 rib pairs and Idd and Arvcf knock-outs. Mice which are heterozygous for mutations in Idd coronal clefts in vertebra C2 through C5. Chromosome analysis was normal (46, and Arvcf are viable and are being bred together to assess the effect of a XY). null AFD is an etiologically heterogenous disorder with most cases being sporadic. mutation on mouse development. Rodriguez et al. [Am J Med Genet 35:484-89, 1990] reported three sibs with AFD and post-axial limb anomalies. Our case resembles the previously reported cases with AFD Rodriguez type. The lack of neurological and ophthalmological abnormalities on autopsy is surprising in view of the assumption that the defect occurred early in blastogenesis.

2218 2219 Controversies in prenatal diagnostic techniques and counseling. R. M. Prenatal diagnosis of unbalanced structural chromosomal abnormalities. H.M.Ryu, Roberts. Genetics and Prenatal Diagnostic Center M.Y.Kim, E.S.Kim, S.Y.Park', S.KChoi1, Y.H.Lee2, S.J.Yoo2, H.W Han. Division of The American College of Obstetrics and Gynecology quotes the risk from a maternal Fetal Medicine, Department of Obstetrics and Gynecology, Genetic Research conventional amniocentesis as 1 in 200. However, that figure is from a time when Laboratory', Department of Radiology2, Samsung Cheil Hospital, Sung Kyun Kwan experienced ultrasonic guidance was not always employed. Appropriate University, Seoul, Korea. counseling should provide that risk, but also an operator-specific risk when Unbalanced structural chromosomal abnormalities are rare. The purpose of our study possible, in order to allow the patient a fully-informed consent. In many prenatal were to calculate its incidence in fetal life and to evaluate its fetal sonographic findings. there is no The study population consists of 1,685 cases who underwent fetal karyotyping in recent diagnostic centers, including my own, statistically increased risk of two years. When there was structural chromosomal abnormality, we performed a FISH miscarriage over that of women who do not have the procedure. One argument to for detailed karyotyping. There were 10 cases of unbalanced structural abnormality, and provide risk analysis from triple screening to women over age 34 is dependent on its incidence was 0.59%. The of the data is as the assertion that many normal fetuses will be miscarried as a result of the summary patient's follows; procedure. Eary amniocentesis is looked upon with suspicion by some used to Case-Karyotype------Fetal ultrasound finding performing C.V.S. However, in my experience, the risk of causing a miscarriage is 1---46,XY,del(4)(p15.1)------Diaphragmatic hernia, IUGR likely less than 1 in 200, utilizing free-hand technique, and the volume removed 2---46,XY,der(5)------Holoprosencephaly safely is much higher than the usual 1 ml per week rule. Risk counseling would be 3---46,XX,add(5)(p15.3)------Mild ventriculomegaly altered if a procedure were available to seal ruptured membranes. This has 4---46,XX,der(1 1)------HLHS, Hydrops accomplished in the single attempt published in the medical literature, successful 5---46,XX,add(13)(q32?)------IUGR, Oligohydramnios at 21 weeks gestation. present one first trimester case of iatrogenic premature 6---46,XYdel 13)(q21.2)------Duodenal atresia, Hydramnios rupture of membranes in which a repair was first attempted with a 10 ml maternal 7---46,XXdel (17)(p13)------Lissencephaly blood patch, which lasted 24 hours, and then again attempted injecting one unit of 8---46,XYdel(20)(q13)------Hydronephrosis and hydroureter cryoprecipitate, and the platelets derived from one unit of the mothers blood, into 9---47,XX,+der(22)t(11 ;22)mat--Dandy Walker malformation the amniotic space. The amniotic fluid volume was restored, but the fetus 10--46,XY,t(9;20)/47,XY,t(9;20),+20--IUGR, Oligohydramnios inexplicably died the 2nd day. With regard to PUBS, the risk quoted of fetal IUGR;lntrauterine growth restriction HLHS;Hypoplastic left heart syndrome demise due to unexplained post-procedure cardiac arrest is between 1-2%. The The maternal age ranged from 22 to 36 years. The most common indication for fetal standard of practice is to console the parents should cardiac arrest occur. karyotyping was abnormality at fetal sonography(80%). The sonographic findings were present a case of resuscitation utilizing CPR (CardioPlacental Resuscitation) one or more of the findings known to occur frequently in association with chromosomal demonstrating effective placental and chest compression, as well as another case abnormalities. However, the findings were variable. The chromosomal abnormality offering an alternative procedure to PUBS--choriocentesis. Aspiration of a occurred de novo in all cases except one who had an abnormality of maternal origin. In cavernous chorioangioma in a male fetus at 22 weeks resulted in a mixed sample conclusion, although the unbalanced structural chromosomal abnormalities are rare, but of 46,XX and 46,XY karyotype. 4 subsequent attempts in 4 male fetuses at risk of as the abnormalities are associated with fetal anatomical abnormalities in most cases, Down syndrome have all yielded maternal cells only. fetal sonographic screening is important.

2220 2221 Sirenomelia and prune belly syndrome in siblings. KR.Schmidt1, Growth, differentiation and function of human airway epithelial cells C.L.Fligne&2, and L.Shields3. 1Division of Congenital Defects, Department of cultured on a tubular support. M. Tolar, W.E.Finkbeiner. Department of Pediatrics, 2Department of Pathology, 3Department of Obstetrics and Pathology, University of Califomia in San Francisco. Gynecology, University of Washington, Seattle. When cultured on a filter support, epithelial cells can acquire a We report a case of sirenomelia and prune belly syndrome occurring in considerable degree of differentiation. They get polarized into mucosal and siblings. This is the second family reported in which this combination of serosal surfaces, they perform and control a secretory function. However, congenital disorders has occurred in sibs. an exact measurement of a rate of liquid transport and its monitoring for The first patient was a female infant with sirenomelia, renal agenesis, a extended time periods has not been possible. We developed a model single ovary, lumbar meningomyelocele and a two vessel umbilical cord. system, in which epithelial cells are grown inside a hollow fiber and nutrition Five years later, a sister was delivered with megalocystis, urethral atresia, is supplied from an outside space. In series with it, a transparent tubing is vaginal atresia, imperforate anus, incomplete rotation of the bowel with connected, in which changes of liquid volume can be visually monitored. colonic atresia, cervical and thoracic hemivertebrae, and a three vessel Cultures of cadaveric human airway surface epithelial cells were maintained umbilical cord. The external genitalia were absent in both siblings. for several weeks; a rate of secretion or resorption was measured and Karyotypes of both were apparently normal 46,XX. The mother of these intraluminal liquid samples were collected every other day and their patients is a 22 y/o G4P, Hispanic woman with a previous first trimester composition analyzed. Detailed results will be presented. Such a functional spontaneous abortion and a living 2 1/2 y/o daughter with no abnormalities. model system of human secretory cells has a great potential to be used for There were no known teratogen exposures. pathogenetical analysis of human genetical diseases affecting a cellular Each sibling in this case had a typical presentation of either sirenomelia secretory and/or resorptive function (e.g. cystic fibrosis), as a monitoring or prune belly syndrome, except prune belly syndrome is more common in system for testing efficiency of transfections mediated by adenovirus- males. There were no findings which might have indicated an increased associated virus carriers that are intended for use in genetic therapy of such recurrence risk for urogenital abnormalities. Our familial case supports a human genetic diseases, and for a wide range of toxicological studies. lower mesodermal sequence etiology for some cases of sirenomelia and prune belly syndrome, and expands the phenotype of hereditary urogenital adysplasia. This has important implications for genetic counseling and patient management. A380 Published Abstracts: Development and Prenatal Genetics (cont.)

2222 Maternal uniparental disomy of chromosome 14 In an Infant with mild dysmorphology and confined placental mosaicism. D.D. Walgenbacht, S.P.Yang1, C.McCaskilP, L.G.Shaffei2, andD.R.Townera. 1UC Davis Health System, Sacramento, CA. 2Baylor College of Medicine, Houston, TX. Previously described cases of uniparental (UPD) for chromosome 14 mostly involved translocations. Matemaldisomr and paternal disomy 14 have both been described with distinct phenotypes. We report a liveborn female infant with maternal UPD for chromosome 14 with a normal karyotype and confined placental mosaicism. The 41 year old mother had an amniocentesis at 17 weeks because an Expanded AFP screening test showed an increased risk for Trisomy 21. All amniocytes were 46,XX. At delivery the infant was noted to have mild dysmorphic findings, IUGR, mild hypotonia, and poor suck-swallow coordination. Cytogenetic analysis of peripheral blood showed a normal 46,XX karyotype whereas placental tissue revealed 47,XX,+14 in every colony studied. Molecular analysis of peripheral blood was performed using four polymorphic loci on chromosome 14. Maternal disomy was found with loss of the paternal chromosome 14. All markers studied showed isodisomy for chromosome 14. The placental DNA showed maternal and paternal alleles, consistent with a trisomy rescue mechanism for the UPD. Molecular studies of polymorphic loci from chromosomes 17 and 22 were consistent with biparental inheritance and correct paternity. Additional studies using DNA polymorphic markers for chromosome 14 are underway to further distinguish isodisomy from heterodisomy along the length of the chromosome and to investigate the mechanism of the confined placental mosaicism. The clinical relevance of this case highlights the importance of studying placental tissue in pregnancies with unexplained IUGR in the absence of a familial structural chromosome abnormality.

Published Abstracts: Gene Structure and Function

2223 2224 The SMN gene is conserved during evolution. S. Bertrandy, 0. Exclusion of FMFc7, a putative C2H2 type zinc finger protein as a candidate Clermont, P. Burlet, C. Cruaud1, C. Fondraf, D. Thierry-Mieg3, A. Munnich gene for Familial Mediterranean fever (FMF). X. Chen', M. Hamon', M. and S. Lefebvre. INSERM U-393, Institut Necker, IFREM, Paris; G&n6thon, Centola2, J.I. Rotter N.Fischel-Ghodsian1 and The FMF International Consortium. Evry 1; Citi2, Paris2 and CNRS, Montpellier3, France. [Department of Pediatrics and Medical Genetics, Cedars-Sinai Medical Center, Spinal muscular atrophy (SMA) is a common fatal autosomal recessive Los Angeles; 2Arthritis and Rheumatism Branch, National Institute of Arthritis and neuromuscular disorder characterized by degeneration of spinal Musculoskeletal and Skin Diseases, National Institute of Health, Bethesda, motorneurons leading to muscular paralysis. We have described the Maryland 20892. Survival Motor Neuron (SMN) gene as the SMA-causing gene. The SMN In an effort to clone the gene responsible for Familial Mediterranean fever gene encodes a 294 amino acid protein of unknown function. Studying (FMF), an autosomal recessive disease characterized by periodic bouts of fever homologies between the human gene and other species during evolution and serositis, a cDNA fragment of 670 bp was recovered by direct cDNA selection can provide insights into functional and structural aspects of the SMN using a cosmid mapped within the FMF candidate region defined by intrafamilial recombinants. Northern analysis using this cONA fragment as probe revealed a protein. Southern blot analyses of genomic DNA have shown that the SMN ubiquitously expressed gene, named FMFc7, with two transcripts of 3.0 kb and 3.2 gene is conserved among vertebrates. The specific cDNA of various kb. A consensus sequence of 2603 bp, with an open reading frame of 831 bp species were searched by PCR from poly A+ RNA, using degenerated encoding 277 amino acids, was established by 5' RACE and sequence analysis of primers from the human sequence. In addition, a genomic DNA sequence the RAE products. BLAST search of the nonredundant protein database from C. elegans was detected in datapase by a computer search using the identified bight C2H2 type zinc finger motifs within the predicted protein sequence. human SMVN protein sequence. The C. elegans cDNA orthologue was The 2603 bp cDNA sequence was interrupted by two intronic sequences of cloned and sequenced to confirm its homology with the human gene. aproximately 262 bp and 1240 bp. Typical splicing signal Sequences were revealed Southern blot analyses demonstrated that the gene is encoded by a single at the intron-exon boundaries. Except a Tto C transition in its 3' untranslated copy gene. A genomic clone encompassing the entire coding sequence was region, with the C allele in strong linkage disequilibrium with FMF, no sequence isolated by long range PCR and allowsboth the analysis of the expression changes in its coding region were found between normal individuals, carriers and pattern and the identification of mutant phenotypes by gene disruption in C. patients. Ancestral haplotype analysis using the T to C transition as a marker revealed a historical recombination event in an affected individual. Additional elegans. These approaches should contribute to the understanding of the historical recombinants with markers D1683275 and' D16S3373, both of which SMIN gene function. map several kb 5' to FMFc7, have been identified during the course of this strudy. Taken together, these data strongly argue against this gene causing FMF.

2225 2226 Neuronal expression of human alpha-tocopheral transfer protein In Structural characterization of the murine lysosomal acid lipase gene. transgenic mice. H-X Deng1, JZu1, M Kim1, W-Y Hung1, J.G. SutcliffeR, T M Duanmu.H Du. Human Genetics, Chidren's Hosp.and Univ.of Cincinnati Siddique'. 'Northwestern University Medical School Neurology Dept, Lysosomal acid lipase (LAL) is crucial for the hydrolysis of intracellular lipids. Chicago, IL. ?Department of Molecular Biology, Research Institute of Two inborn error of lipid metabolisms, Wolman disease (WD) and cholesteryl Scripps Clinic, La Jolla, CA. ester storage disease (CESD), result from mutations in this gene. Reduced enzyme level causes massive cholesteryl ester and triglycerides accumulation. Alpha-tocopheral is the most biologically active form of vitamin E. It plays Premature atherosclerosis is noted in CESD. To study the structural similarity a primary role in anti-lipid peroxidation in cells. This anti-oxidant function is between human LAL (hLAL) and mouse LAL (mLAL), and to provide further mediated and enhanced by alpha-tocopheral transfer protein (a-TTP). insight into the regulation of LAL gene expression and its function in Mutations in a-TTP gene have been identified in ataxia associated with atherosclerotic lesion, we have cloned both hIAL and mLAL cDNAs. Deduced vitamin E deficiency, a neurodegenerative disease, suggesting the amino acid sequences from hLAL and mLAL have 95% similarity and 75% importance of a-tocopheral in anti-oxidant defense in neurons. Oxidative identity, with conservation of two active center motifs (G-X-S-X-G) and five N- stress in neurons is thought to be important in aging and neurodegenerative glycosylation consensus sequences. The expression of mLAL mRNA and protein diseases. To test the hypothesis that enhancing anti-oxidant defense may is tissue and cell type specific. High level expression was detected in adult liver, slow down the aging processes and prevent neurodegenerative diseases, spleen, adrenal cortex, pancreas, renal tubular epithelium, and choroid plexus of we have created a transgenic mouse model overexpressing the a-UTP in the developmental embryonic brain. The expression pattern is well correlated to the nervous system. the pathological finding in human WD. Structural analysis of the mouse gene is fundamentally important for studying the promoter elements, which are essential As a-TTP is only expressed in liver, we used neuro-specific enolase in tissue and cell type specific expression. We have isolated 4 mLAL genomic promotor to drive a-UTP expression in brains of transgenic mice. a-TTP clones from'X-FIX 11 library. Analysis of these clones indicated that they cover 2/3 expression was detected in brain and spinal cord, but not in muscle of these of the coding region. Two clones from mouse genomic P1 library were also animals. These mice are at present five months old with normal phenotype. isolated and analyzed. These 2 clones cover the promoter and entire gene with Further phenotype and biochemical characterization of this model is overlapping. A detailed restriction map of the mLAL gene was established by underway and may facilitate understanding mechanisms of the aging subdloning, restriction enzyme digestion and Southern blot. The genomic processes, the pathogenesis of some neurodegenerative diseases, and organization of mLAL, consists of 10 exons expanding around 48 kb, issimilar to may serve as a neuroprotective model for stroke and head injury. that of hLAL. The noncoding exon 1, partly retained in the cDNA clone, was identified by 5'RACE-PCR and S1 nuclease mapping, then confirmed in gsnomic clones. Intron/Exon junctions of mLAL were analyzed by PCR with primers designed according to hLAL gene. Sequencing of subcloned PCR products of intron/exon boundary revealed the exact match from exon 2 to 9 between mLAL and hLAL. These studies provide the structural basis for further investigation of LAL gene regulation and its role in pathogenesis of the WD and CESD. Published Abstracts: Gene Structure and Function (cont.) A381

2227 2228 Stable Myelin Protein Zero expression in S2 Insect cells using a new Calcium channel beta 4 (CACHLB4): Gene structure and chromosomal constructed all-in-one plasmid. A. B. Ekici, B. W. Rautenstrauss. Institute of location of the human ortholog of the mouse epilepsy mutant, Human Genetics, Friedrich-Alexander-University of Erlangen-Nomberg, Germany. lethargic. Miriam H. Meisler, Andrew Escayg, Julie M. Jones, and Daniel L. Myelin protein zero (MPZ, P0) is located in the myelin sheath of peripheral Burgess. Department of Human Genetics, University of Michigan, Ann Arbor nerves. The wt protein is able to form homotetramers and is essential for the The mouse mutant lethargic exhibits two types of neurological compactness of the myelin sheath. Recently point mutations within the P0 gene abnormalities. The absence seizures are brief periods of were described as cause of Charcot- Mane-Tooth (CMT) disease type 1 B, immobility Dejedne-Sottas-Syndrome (DSS) and congenital hypomyelination (CH). These accompanied by generalized cortical spike-wave discharges, with a duration peripheral neuropathies are characterized by reduced nerve conduction velocity of 1.5 sec and frequency of 100/hr, and resemble the absence seizures of (NCV<38m/s) and onion bulb formation of the myelin. Although the three human petit mal epilepsy in their electrophysiological and pharmacological dimensional structure of the extracellular domain is cleared up the in vivo effects characteristics. Episodes of uncontrolled limb activity of subcortical origin of mutations in the P0 gene remain to be determined. Therefore expression occur once or twice daily. We recently demonstrated that the lethargic studies of the P0 gene have to be carried out in cell culture or animal systems. phenotype is the result of a mutation in the beta 4 subunit of the voltage We developped an adhesion test system based on cloned P0 cDNA in gated calcium channel (Burgess, Jones, Meisler and Noebels, Cell 88:385- Schneider2 (S2) insect cells. Several P0 mutations causing DSS and CMT1B 392, 1997). The cytoplasmic beta subunit is required for the channel activity were introduced by PCR-directed in vitro mutagenesis and cloned 3' to the of the membrane spanning alpha subunit. The lethargic mutation is a 4 bp Drosophila metallothionein promotor. insertion into a splice site that results in exon skipping and synthesis of a Up to now we used two different plasmids, an expression plasmid and a truncated beta 4 protein. The truncated protein lacks more than 60% of the selection plasmid, which is able to integrate in the insect genome, for transient C-terminus, including the interaction domain for binding to the expression. To prevent complications of this two plasmid system we designed alpha and constructed an integrating, combined expression and selection plasmid for subunit. To evaluate the role of the lethargic ortholog, CACNLB4, in human S2 cells. This new plasmid carries 3 properties: 1. a heavy metal ion inducible disease we have determined the chromosomal location of the human gene expression modul which consists of the metallothionein promotor, a polylinker and using the Genebridge 4 radiation hybrid mapping panel. The human cDNA polyadenylation signal, 2. the ability to integrate in the genome for stable has been isolated by RT-PCR. The open reading frame of 1560 nucleotides inheritance 3. the bacterial neomycin gene as a dominant marker which allows encodes a 520 amino acid protein with 98% amino acid sequence identity to the selection of stable tranfected cells with the aminoglycoside G418, a derivative the rat beta 4 protein. The exon/intron borders of the 13 exons of the human of gentamycin. gene have been determined by inter-exon PCR. We are screening human This plasmid construction makes sure that all selected cells carry the P0 gene families with inherited ataxia or epilepsy to detect linkage to CACNLB4, and should be positive for protein expression. Furthermore, a transient using closely linked microsatellite markers. coexpression of mutated P0 cDNA's by transfecting the POwt S2 cell line allow to determine the dominant or recessive nature of mutations in the P0 gene.

2229 2230 Characterization of transgenic mice overexpressing the neuronal Identification of a new member of the myotonic dystrophy protein apoptosis Inhibitory protein. J. F. Ingram1, D. Xu2, Z. Yaraghi1, F. Jirik3, kinase gone family. D. W Johnson and D. A. Marchuk. Department of A. BorowskP, G. Robertson2, A. E. MacKenzie1. 'Solange Gauthier Karsh Genetics, Duke University Medical Center, Durham, NC. Laboratory, Children's Hospital of Eastern Ontario, Ottawa, Canada, In an effort to discover novel serine-threonine kinases involved in 2Departement of Pharmacology, University Of Ottawa, Ontario, Canada, vasculogenesis and angiogenesis, we used endothelial cell RNA in reverse 3Biomedical Research Center, University of British Columbia, Canada. transcriptase polymerase chain reaction (PCR) with. degenerate Proximal Spinal Muscular Atrophy (SMA) is a common autosomal oligonucleotide primers. The primers were designed to a number of distinct recessive neuromuscular disorder characterized by degeneration of anterior conserved regions of kinase domains. A segment amplified between one hom cells in the spinal cord leading to the atrophy of voluntary muscles. set of primers was used to extend the sequence in both directions using the Acute SMA is associated with deletions of the SMN-te/ (Survivor Motor rapid amplification of cDNA ends (RACE) technique. A large single open Neuron) gene and,frequently, with deletions of a gene that encodes the reading frame (ORF) was found to contain a unique putative kinase domain. neuronal apoptosis inhibitory protein (NAIP). in vitro and in vivo All invariant (and most of the conserved) residues are present in the relative of NAIP cells from cell death triggered locations expected for a serine-threonine kinase domain. The 264 amino overexpression protects programmed acids of this domain were found to exhibit a high degree of identity to other by a number of apoptotic stressors. In an effort to (i) clarify the role of NAIP in normal murine development and (ii) to generate resources with which to kinases in the myotonic dystrophy protein kinase (MDK) family. For test NAIP's effectiveness in treating neurodegenerative diseases, example: human Rho-associated kinase, 57%; mouse MDK, 57%; human transgenic mice expressing NAIP under the control of the chick actin and MDK, 56%; human MDK-like, 52%; human pk428, 56%. CMV promoters, were generated. Western analysis of various tissues revealed NAIP transgene expression in the heart, smooth muscle and brain. Preliminary immunohistochemical studies of brain sections revealed expression of the NAIP transgene in cortical and hippocampal neurons. No distinct differences in phenotype, longevity or fertility have been detected to date. Further characterization of this transgenic mouse lineage, including detailed CNS expression will be presented.

2231 2232 The physical structures of GDNFR-a and NDNR-a. S.M. Myers1, C. Eng2, Identification of a novel noncoding nuclear RNA from the DiGeorge C. Hession3, R. Cate3, M.D. Kogon' and L.M. Mulligan' . (1 ) Department of syndrome critical region at 22q11. G. Novelli1, A. Pizzutf, F. AmatiP A. Pathology, Queen's University, Kingston, Canada, (2) Division of Cancer Ratti, A. Mari1, L. Fogh2, E. Conti1, M. Bengala1, R. BordonP, E. Bellone3, Epidemiology and Control, Dana-Farber Cancer Institute, Boston, USA. and P. MandichP, A. Colosimo1l, F Pandolf/, B. Dallapiccola1. Tor Vergata (3) Department of Molecular Biology, Biogen Inc., Cambridge, USA. University and CSS. Mendel Institute, Rome1; Neurology Department, The RET receptor tyrosine kinase may be activated by interacting with University of Milan2; IBIG, University of Genoa3; Catholic University of either glial cell line derived neurotrophic factor receptor alpha (GDNFR-a, with its putative ligand GDNF, or neurturin neurotrophic factor receptor alpha Rome4, Italy. (NDNR-a), with its ligand NDN. Activating mutations in the RET proto- We describe the cloning and characterization of a novel transcript oncogene have been shown to cause multiple endocrine neoplasia type 2 (22K48), identified by cDNA selection on YACs mapping in the DiGeorge (MEN2) whereas inactivating mutations result in the congenital abnormality syndrome region at chromosome 22q1 1. This transcript is polyadenylated Hirschsprung disease (HSCR), characterized by lack of innervation of the but not spliced and has a predominant nuclear expression in different cell hindgut. Mutations in RET are responsible for the majority of MEN2 cases types. The cDNA sequence of 22K48 is contiguous with its genomic DNA while only 50% of the inherited HSCR cases appear due to RET mutations. sequence and contains two polymorphic STRs at both ends. This cDNA has We have determined the exon/intron boundaries of both GDNFR-a and NDNR- not any significant homology to known sequences. No ORF is present in the a to facilitate the study of the possible role of these molecules in cancer and in 3 Kb cDNA, suggesting it is not translated. 22K48 is expressed in all fetal early development and more specifically in the HSCR phenotype. We found 6 and adult tissues tested, including and B- and T- cells clones. of the 8 exon/intron boundaries of GDNFR-a by long distance PCR across High levels of expression have been observed in heart and skeletal muscle. introns. The remaining 2 boundaries flanking intron 4 and intron 7 could not be Southern blots of genomic DNA from human and other species probed with determined using PCR, suggesting these introns might be very large. These 22K48 demonstrates a single band only in human and monkey DNA lanes. boundaries were determined by subcloning PACs from this region using Hind The two STRs at this locus show 7 (PlC 0.58) and 4 (PIC 0.42) alleles Ill. Subclones containing the exons of interest were isolated and the intron/ respectively in the range of 71 to 142 bp. These' markers detect boundaries sequenced using exonic primers. A GDNFR-a containing hemizygosity in all examined DGS patients. These results may have PAC was used to FISH map the gene to 10q26.NDNR-a belongs to the same implications for the etiology of DGS and related disorders. In fact, the gene family as GDNFR-a and shares 48% homology. As families of genes discovery of a novel noncoding RNA from the 22q11 critical region raises have conserved exon/intron boundaries, primers were designed for the possibility that complex mechanism of gene regulation could be NDNR-a based on the corresponding positions of boundaries in GDNFR-a. implicated in the pathogenesis of DGS. Work supported by Telethon grants PCR performed on genomic DNA, as well as cDNA made from cell lines (E 3.9 and D27) and MURST. expressing NDNR-a, confirmed that exon/intron boundaries were conserved. A382 Published Abstracts: Gene Structure and Function (cont.)

2233 2234 Gene Organisation of Human Cystathionine P-Synthase. J. Comparative sequence analysis Identifies highly conserved, Oliverusova1'2, E. Kraus1, J. Sokolova2, C. Vlcek3, R. de Franchis1, G. functionally Important regions of the ATRX gone. DJ Picketts, RJ Bukovskat, M. Janosik2, D. Patterson4, V. Paces3, V. Kozich2, and J. P. Gibbons, and DR Higgs. (Intro. by: RM Harding). Institute of Molecular Kraus1. 1 Departments of Pediatrics and Cellular and Structural Biology, Medicine, Oxford, UK University of Colorado School of Medicine, Denver, CO 80262, 2Center for Mutations in the human ATRX gene give rise to the ATR-X syndrome Inherited Metabolic Diseases, Charles University, Prague, Czech Republic, which is associated with severe psychomotor retardation, characteristic 3institute of Molecular Genetics, Academy of Sciences of the Czech facies, genital abnormalities and an unusual form of a-thalassaemia. The 44Eleanor Roosevelt Institute, Denver, ATRX gene encodes a widely expressed member of a novel subgroup of Republic, Prague, Czech Republic, the SNF2-like family of DNA dependent ATPases and putative helicases. CO 80206. Mutational analysis has confirmed the importance of the ATPase/helicase Cystathionine 0-synthase (CBS) catalyzes the condensation of serine domain and suggested a role for the carboxyl terminus of the protein in and homocysteine to form cystathionine and it is the first enzyme in the genital development. To complement our mutational analysis of this large transsulfuration pathway of eukaryotes. CBS deficiency is the most gene we sought to identify other domains of potential functional importance common cause of homocystinuria and may be a significant risk factor for phylogenetic comparison. Here we report the cDNA sequence (7673 nts) cardiovascular disease. The CBS gene is located on chromosome 21, at of the murine ATRX gene including the entire coding sequence and a q22.3. It is about 30kbp long and contains 23 exons. The 5'UTR contains comparative analysis with its human counterpart. The predicted ATRX five alternatively used exons, -1a to -1e, and an invariable exon 0. The protein is comprised of two highly conserved domains (>90% identity) coding region consists of sixteen exons. Exon 15 is alternatives spliced and separated by a region of relatively poor homology (<70% identity). The is found only in a minor mRNA species. The 3'UTR is located in exons 16 highly conserved regions include the ATPase/helicase domain, the region and 17. In order to determine the complete structure of the CBS gene, we suggested to be important for genital development and the amino terminal have isolated two genomic clones, one from a P1 and one from a cosmid 300 amino acids in which we have identified a PHD-like domain. library. Restriction fragment maps were established and fragment libraries of both clones constructed. We determined the exon/intron junctions of the entire gene. DNA sequencing of the entire gene is about 80% completed. We are establishing conditions for PCR amplification of the individual exons from within the introns. The CBS gene sequence will be helpful in the studies of normal and mutant CBS and in unambiguous detection of carriers.

2235 2236 Characterization of the 5' Upstream Region of the Wilson Disease Characterization of Human Prosaposin Promoter. Y. Sun, P. Jin, and Gene and Identification of a Putative Promoter Region. AB Shah1, BM G.A. Grabowski. Children's Hospital Medical Center, Cincinnati, Ohio. Ross2, and TC Gilliam1 2. Departments of 1Genetics and Development and Prosaposin is a multifunctional protein that encodes four glycoproteins, 2Psychiatry, Columbia University, New York, NY. named saposin A, B, C and D. They participate in the catabolism of recessive disorder characterized glycosphingolipids in lysosomes. When secreted, intact prosaposin may Wilson disease (WD) is an autosomal We have demonstrated that copper in the liver, and subsequently in the brain function as a neurotrophic factor. murine by toxic accumulation of displays highly regulated temporal and spatial expression. To and other organs, leading to liver disease and/or extrapyrimidal neurological prosaposin transcriptional regulation control of this locus, we have syndrome in the first or second decade of life. Based upon sequence understand the of mouse and human prosaposin genes. Here we homology to known genes, the WD gene (ATP7B), located on Chromosome characterized 5 region the and analysis of human 5 region of gene 13, appears to be a copper-transporting P-type ATPase. The gene is report subcloning prosaposin but most predominantly in the liver. from PAC clones. The subclone H9 is 9.5Kb in length with 3.5Kb 5. to the expressed in the placenta and kidney, and 6Kb in the first intron. The first intron in date, approximately 91 mutations have been identified within or flanking translation initiation site ATG To is more than 15 Kb. The murine promoter coding regions of the WD gene. and functional human prosaposin gene proven the Genotype/phenotype and the putative human promoter lack TATA box motifs; i.e., they studies suggest a high degree of complexity in the mechanisms of copper region belong to TATA-less housekeeping gene family. The transcriptional transport. human is located about 90 bp 5 to ATG compared to 88 bp We have sought to identify the WD promoter region in an effort to better initiation site in mouse. By sequence motif analysis, human and mouse share some understand WD gene regulation through the identification of tissue-specific in the transcriptional factor binding sites within proximal promoter region. They transcription factors binding sites or other components necessary for The It is interesting that at least two studies of WD patients both have GC box for Spl binding and ROR alphal binding elements. transcription activity. SpI sites and U region that were present in the mouse were of Northem European or Mediterranean decent indicate the presence of 5. overlapping sequence. The functional analysis of these elements in mutations among their WD patient sample. not found in human regulatory ongoing. Although was well characterized, the human Although the WD transcript is estimated to be 7.5 kb, only 6.6 kb of human gene is gene displays similar temporal and spatial expression patterns sequence have been identified. We have used a combination of techniques, prosaposin including direct genomic sequencing, primer extension, promoter capture, as the mouse. Thus, the tissue specific modulators of prosaposin to extend the 5' untranslated region of expression are probably similar in both species and those responsible for Northern hybridization, and RT-PCR, CNS expression probably reside within 400 bp 5 to the ATG. This study will the WD gene and to identify a putative promoter region for the gene. provide insight into the mechanism of the regulation of this locus.

2237 2238 Molecular analysis of human age-related genomic stability: a High Conservation of Dopamine Transporter Sequences Among longitudinal study. S. Thomas and A.B. Mukheree. Department of Human Individuals. D.J. Vandenberghl, M. Thoompson2, E. Cook3, J. Biological Sciences, Fordham University, Bronx, NY. Schaeffer4, S. George2, E. Bendahhoul, J.T. You1, M. Hazama1 ' D. The possibility of human age-related changes in DNA fragment Comings3, B. O'Dowf, and G.R. UhI16A 1Mol. Neurob. Branch, NIDA-IRP, rearrangement, loss and/or amplification was examined using Southern blot NIH; 2Dep. Pharm.; U. Toronto, Canada; 3Dept. Psych., U. Chicago, 4Dep. for RFLP analysis and by AP-PCR for genomic DNA fingerprinting. Skin Chem., Cal. St. U., Northridge; 5Dep. Med. Genet, City of Hope Med fibroblasts derived from 8 individuals, each at younger and at older ages, Center, and 1'6Depts. Neurol. & Neurosci., JHUSM, Bait., MD 21224 were used in this study. Genomic DNA was extracted from each cell sample Altered dopamine transporter (DAT) sequences could contribute to and 7 highly polymorphic DNA probes (derived from 6 different interindividual differences in vulnerability to neuropsychiatric disorders. We chromosomes) and 10 arbitrary primers (10-20 nucleotides long) were used scanning each of the 15 DAT gene exons results indicated that sought DAT polymorphisms by for RFLP and AP-PCR analyses, respectively. The from more than 200 unrelated individuals by gels detecting single strand there were no obvious age-related changes in the 7 highly polymorhic DNA direct sequencing. No common each PCR reaction conformation polymorphism and/or regions in various individuals. In our procedure, polymorphisms were found that change DAT amino acids or DNA produced 150-200 discrete and reproducible DNA bands on a No disease-associated in sequences for RNA splice donor/acceptor sites. polyacrylamide sequencing type of gel. In 2 individuals, some differences polymorphisms were found in individuals with attention deficit hyperactivity the banding pattem was observed with specific primers, presumably syndrome comorbidity by results indicated that disorder with (n=35) or without (n=24) Tourette's indicating age-related genomic alterations. These City of Hope criteria, or drug abuse ( n=16). Rare conservative amino acid genomic alterations has been taken place in certain individuals during aging substitution variants in exons 2 and 8 (both Val/Ala) were identified but not and the pattem of alteration is individual - specific. disease-associated. Genetic associations between DAT polymorphisms and neuropsychiatric disorders are thus more likely to arise from DAT promotor variants that alter levels of expression, possibly including those in linkage disequilibrium with the exon 15 variable number tandem repeat marker that has been associated with attention deficit and variant psychostimulant responses. Supported by NIDA, & NIMH, NIH; Med. Res. Coun., Canada.; & Shaw Foundation Published Abstracts: Gene Structure and Function (cont.) A383

2239 2240 Functional characterisation of the TSC1 gene. Maron van Slegtenhorst1, Comparative mapping of the WSCR and MLL loci in higher primates. Mark Nellistit, Ronald de Hoogt', Remco Bakker1, Ans van den Ouweland', R.S.Verma, R.A.Conte and R. V.Samonte. Institute of Molecular Biology and Peter van der Sluijs2, and Dicky Halley'. 'Department of Clinical Genetics, Genetics at InterScience,Brooklyn, The Long Island College Hospital-SUNY Erasmus University and University Hospital, Rotterdam, The Health Science Center at Brooklyn and Natural Science Research Institute, Netherlands;2Department of Cell Biology, Utrecht University School of University of the Philipines, Diliman, Quezon City Medicine, Utrecht, The Netherlands Comparative mapping of the WSCR and MLL loci in higher primates. R.S.Verma, R.A.Conte and R.V.Samonte,lnstitute of Molecular Biology and Tuberous sclerosis complex (TSC) occurs when either one of the TSC1 Genetics at InterScience,Brooklyn,The Long Island College Hospital-Suny or TSC2 tumor suppressor genes is inactivated. The disease is Health Science Center at Brooklyn and Natural Science Reseach Institute, characterized by the appearance of hamartomatous lesions in many University of the Philippines Diliman, Quezon City. Chromosome banding different tissues, and a broad phenotypic spectrum which may often include patterns of higher primates suggested strong genomic homologies at the DNA disturbed mental function, renal problems and dermatological abnormalities. level. The evolutionary aspect of chromosome evolution is currently being There is no evidence for clinical differences between the two genetic types. investigated by various approaches. We used loci specific probes, specific for We have recently employed a conventional positional cloning approach to William syndrome chromosome region [WSCR] and the MLL gene locus and identify the TSC1 gene (TSC1 consortium, Science in press). Subsequently hybridized to the metaphase chromosomes of human [HSA],chimpanzee using SSCP analysis we have identified 5 mutations, all of which lead to [PTR],gorilla [GGO] and orangutan [PPY] to determine the phylogenetics premature truncation of the predicted 1 30kD TSC1 protein product, homologies and their locations in higher primates. Fibroplast cell lines of PTR, hamartin. There is a coiled coil domain at the C-terminus of hamartin and GGO and PPY were obtaned from Coriel Institute for Medical Research, BLAST searches show weak but significant homology to several coiled coil Cadmen,NY. The DNA probes were obtained from ONCOR, Gaithersburg, proteins thought to be associated with early endosomes. The TSC2 gene MD. A deletion in the WSCR at 7q1 1.23 causes while MLL was isolated in 1993 and encodes a large (200 kDa) protein of unknown gene locus at 1 1q23 has been associated with human myeloid-lymphoma or fuction, although there is evidence that it has a role in regulating the rate of mixed lineage leukemia. The WSCR signals were observed on the human endocytosis (J Biol Chem. 272, 6097-100). We have tested directly for chromosome 7q1 1.23 and at equivalent position at 6q1 1 in PTR and GGo interactions between TSC1, TSC2 and endosomal proteins using the yeast while at 10pI1 in PPY which was diverged. As expected, the MML probe two-hybrid system and transfection analysis of mammalian cells. hybridized human chromosome 11 q23 and equivalent location at 9q23 for PTR and GGO but at 8q23 for PPY. The diverged position may support the views that orangutan's phylogenetics position is the furthest from human than chimpanzee and gorilla. The loci specific probes like these should be used as phylogenetic markers to further delineate pattern at greater resolution.

2241 Generation of a mouse model for Smith-Magenis syndrome disease. Q. Zhao, K-S. Chen, L. Potocki, J.R. Lupski. Department of Molecular and Huamn Genetics, Baylor College of Medicine, Anderson Room 609 E, One Baylor Plaza, Houston, TX 77030. Smith-Magenis syndrome (SMS) [del(17)pl1.21 is a contiguous gene apparently caused in the majority of cases (>90%) by homologous recombination of a flanking repeat gene cluster at human chromosome 17p1 1.2 (K-S., Chen et al.). This mechanism leads to a common deletion. Clinical features include mental retardation, short stature, brachycephaly, , and behavioral problems. It is hypothesized that the haploinsufficency of one or several genes within the deleted region is responsible to this complex phenotype. To understand the molecular mechanisms underlying this disease, we are developing a mouse model by using gene knock-outs and chromosome engineering techniques. Initial experiments have focused on the physical characterization of the mouse chromosome 11 region syntenic to human 17p1 1.2. We have constructed a mouse YAC contig spanning the SMS syntenic locus in the mouse chromosome 11 by a combination of approaches including: using existing physical mapping data base (Whitehead MIT); identifying mouse EST homologs to the mapped human genes; screening mouse cDNA library with SMS intemal human genes and STS content mapping. A number of reported human genes from SMS critical region have been mapped on this contig, including Aldh3, Fli, Mgl and Znfl79. Our future plan is to make knock-outs with LoxP sites and to make contiguous genes deletions with Cre-LoxP system, then phenotypically characterize mice deficient for specific genes or/and large genomic regions.

Published Abstracts: Genetic Counseling and Genetic Education

2242 2243 Folate prophylaxis and anti-convulsant medications: a case report Development and assessment of a series of cancer genetics teaching cases with counseling implications. K.Filkins and D.Berry2. I Department of designed to educate physicians and other health care professionals about

Obstetrics and Gynecology, UCLA School of Medicine, Los Angeles, CA, inherited cancer predisposition. S. Miesfeldt1, S.M. PowelfP, J.M. Jackson , 2Westem Pennsylvania Hospital, Pittsburgh, PA. W.F Cohn4, B.L. Tumer1, S.M. Jones5. 'Division of Hematology/Oncology, Preconceptual folate supplementation has been associated with a 2Division of Gastroenterology, 3Office of Medical Education, tDepartment of decrease in the occurrence of neural tube efects. However, protective Health Evaluation Sciences, and 5Cancer Center, University of Virginia effects have not been specifically documented in patients taking anti- Health Sciences Center, Charlottesville, Virginia. convulsant medications. We report here a 22 year old, gravida 1, para 0, SA Many physicians and other health care professionals have not been trained in 0 referred for prenatal testing due to her exposure to Depakote and the new and burgeoning area of clinical cancer genetics. To meet a demand for Tegretol. The patient had taken folate supplementation prior to and during education regarding inherited cancer predisposition, staff of the Cancer Genetics the first trimester of pregnancy, and believed that this would provide her Program at the University of Virginia Health Sciences Center developed a series with complete protection against neural tube defects. Ultrasound evaluation of teaching cases designed to instruct physicians and other health care at 16 weeks gestation revealed hydrocephaly and spina bifida. The patient professionals in this area. The series consists of: an introduction to heritable elected termination of the pregnancy and post mortem examination cancer predisposition; an explanation of autosomal dominant inheritance; a list of confirmed these abnormalities as well as additional findings consistent with characteristics of personal and family medical histories that raise suspicion of an fetal Valproate syndrome. This case highlights a critical issue in inherited cancer predisposition disorder; a glossary of basic genetic terms; and a preconceptual counseling. While folic acid supplementation appears series of self-directed teaching modules composed of individual cases, with beneficial, the effect is not 100 percent. Avoidance of known teratogenic questions and answers. The cases were selected to instruct the leamer about three cancer and ovarian cancer agents should be the primary approach whenever possible, and a realistic breast predisposition disorders (breast cancer and Li-Fraumeni and two discussion of versus benefits should be provided. syndrome, breast syndrome, syndrome) risks colorectal cancer predisposition disorders (hereditary nonpolyposis colon cancer and familial adenomatous polyposis). A general list, as well as five case-specific lists, of teaching objectives were developed and included: recognition of inherited cancer predisposition risk; genetic counseling; DNA testing; medical and surgical management strategies; and psychological issues. We will describe our experience evaluating this case series among primary care physicians. A384 Published Abstracts: Genetic Counseling and Genetic Education (cont.)

2244 2245 Patient Indications for mutation testing after referral for genetic Group Supervision: A better way to train Medical Geneticists. John. G. counseling for breast/ovarian cancer. M. Nunes, M.H. Fries, KM. Rogers, Karen Dunn. VCGS, Murdoch Institute, Royal Children's Hospital, Murphy, J. Flanagan, M.W. McClellan, D.Bartholomew. 81 st Medical Melboume, Australia. Group, Keesler AFB, MS Genetic counseling demands psychological insight and sophistication to Autosomal dominant inheritance may account for 5-10% of breast cancer convey information and to deal with the issues. Traditionally this has been cases in the U.S., with a high lifetime risk for developing cancer in carriers of provided on a one to one basis which is time consuming and expensive. We mutations in BRCA 1 and BRCA 2. A program of genetic counseling and have adapted group supervision, a method used in the training of accelerated follow-up was established at Keesler AFB, MS to assess the outcome psychiatrists, psychologists and psychotherapists, for the training of medical of intensive education, genetic counseling and monitoring of such patients on geneticists. quality of life and survival. After counseling, testing for mutations in BRCA 1 and Group supervision utilises the collective wisdom and experience of the BRCA 2 was offered to women with breast or ovarian cancer who (1) had two or group, and provides a diverse range of views. The challenge to look more affected first or second degree relatives; (2) one first or second degree differently at an issue often comes from a fellow trainee. It gives a direct relative who was affected prior to age 45; (3) were younger than 45 years at experience of group dynamics which operate in any interview of a diagnosis; (4) had multiple primary cancers or bilateral disease, or (5) to family, any and allows an opportunity for modelling and affected male. Testing was also offered to unaffected individuals with family role play. The range of topics is broader as each is exposed to members with known BRCA1/BRCA2 mutations. Of the 23 women and 3 men trainee topics brought by another. As the who were counseled in the first 2 months of the protocol, 18 (69%) wanted group progresses, bonding and inter-relationship allows for increasing self- information to help their families; 7 (26%) wanted to ensure their providers disclosure and reflection within the group. understood their risk or to be followed in the accelerated follow-up clinic; and 5 We will present information about the themes that emerged during the (19%) wished to participate in a research project. Only 11 of the 26 patients were sessions which included the handling of difficult families, issues of guilt, eligible for the protocol; 13 patients were ineligible because they did not have shame, bad news, self-awareness, time constraints, responsibility, interview cancer and had no affected relatives with known mutations; two patients had no dynamics, personal issues affecting the process of counseling and ethical breast or ovarian cancer in themselves or their families. Three eligible patients and legal issues. who initially desired testing later declined, citing family objections, fear of insurance/job discrimination for children, and reluctance to discuss breast cancer with the family. The remaining eligible patients who wished testing confirmed motivations to provide information for their children; two also felt that testing would help guide further career and/or surgery options; one felt research participation was a significant motivator. Patients who are not eligible for the protocol appear to have more personal motivations than those who are eligible, confirming the necessity for careful genetic counseling in this area.

Published Abstracts: Genetic Epidemiology and Population Genetics

2246 2247 Age of Onset and Age of Death In African Americans with Epidemiological clinical studies of GSTM1 gene polymorphism. Three Huntington's Disease. R.K Abramson, H.H. Wright, E. Frank, D. years experience. H.V.Baranova, M. Canis, E.Albuisson, J.Perriot, Zimmerman, and A.D. Vafini. W.S. Hall Psychiatric Inst. and Univ. of South M.Bruhat, P. Malet. Hospital University Center, Medical Faculty, Clermont- Carolina School of Medicine. Columbia, SC. Ferrand, France. The population prevalence of Huntington's Disease (HD) differs in White and Glutathione S-transferase MI gene (GSTM1) belongs to the detoxication African Americans. Studies from Europe, Africa, and the United States indicate a system genes family (phase 11) and codes the 4STM1 conjugation enzyme, lower prevalence in African Americans. The exception is the Maryland study which is highly specific for certain electrophilic chemicals, tranc-stiblene (Folstein et al, 1987) for which the population prevalence for African Americans is oxide and carcinogenic metabolites of benzopyrene. It also functions as an 6.37 and Whites, 4.94. The South Carolina prevalence for African Americans is intracellular drug- and hormone-binding protein. 2.11 (Wright et al, 1981). Folstein also found that the age of onset in African GSTMI gene appears in 2 active alleles :GSTM1-A and GSTM1-B and yrs, the Americans with HD was significantly lower (36.20 SD=12.6, n=61) and one null allele: GSTM1-0. The combination of two null alleles number of juvenile cases were higher than expected, mean age of onset =16.00, corresponds to GSTM10/0 which does not have mRNA due to SD=3.8 years. If the HD gene is the same for Whites and African Americans, this genotype, any product in 40-50% in might indicate that there are racial differences in genes which affect age of onset extended deletion. This deletion is presented general and/or paternal anticipation occurs more frequently. This study examines 12 Caucasian population and affects a lot the detoxification process. families with 52 diagnosed HD patients taken from the South Carolina HD GSTM1IO/ genotype has been detected by direct PCR in patients with Registry with information on age of onset (AO) or age of death (AD) on 41 different types of pathology: 1) environmentally provoked diseases (EPD, indivdiuals. For 20 individuals with reliable AO data, the mean AO was 39.2 like chronic bronchitis and endometriosis, 2) infectious diseases (ID), yrs.,SD=13.08, not significantly different from the 41.8 years, SD=4.88, n=8, including tuberculosis and hepatites B and C, 3) cystic fibrosis (CF) (more reported previously for South Carolina. AD was 58.56 years, SD=14.75, n=32, then 800 cases altogether). The percentage of GSTM1-deficient subjects within the 20 years generally reported from AO to AD. Two juvenile cases, AO 18, was significanty higher in EPD group - up to 86.0% (p=0.0001); ID - 63.3% SD=2.0, were identified, which is higher than expected. Paternal anticipation was (p=0.0O1) and was elevated in the group of CF (55.1%). responsible for the majority of cases with age of onset below 30. One case of We suggest that detection of GSTM10/0 deletion can be proposed as a anticipation was documented. AO differences for and maternal African Americans convenient prognostic test for development of EPD and might be helpful in Whites may reflect small sample sizes and ascertainment versus racial the choice of therapy for ID and CF. differences. A larger collaborative study is needed to address this issue.

2248 2249 Power and Selection in MultivariateQTL Mapping. Dorret1. Boomsma, Distribution of Factor VilI haplotypes in major racial groups in Conor V. Dolan. Dept of Psychology, Free Univ, Amsterdam, The Singapore. L.L. Chiul, P.S. Lail, P.S. Low1,/. Fong2 and N. Saha3. Netherlands. 1Department of Paediatfics, National University of Singapore, 2lnstitute of The power to detect quantitative trait loci (QTLs) can be substantially Molecular Agrotechnology, National University of Singapore, 3Division of increased by considering multiple indicators of the phenotypic trait of Human Genetics, University of Pittsburgh, USA. a marker interest followed by fitting multivariate model simultaneously to the As DNA polymorphisms of Factor VilI gene have been known to be and phenotypic data. Simulation studies show that the gain in power influenced by ethnic variations, a study on the distribution of haplotypes of depends on the multivariate genetic model that underlies the data, these polymorphisms was carried out on the 3 major racial groups in especially on whether there are correlated genetic background or Singapore to determine their usefulness for genetic counselling for environmental correlations among measures in addition to a pleiotropic Hemophilia A. Altogether 462 chromosomes comprising 169 Malays, 156 QTL. However, under all circumstances power to detect a QTL in studies Chinese and 137 Indians were analysed for four intragenic bi-allelic (one that employIBD (identity-by-descent) mapping in sibling pairs is higher in sequence and three RFLP) polymorphisms, namely intron 7 (G/A), intron 18/ multivanate than in univanate designs. Multivariate selection of extreme BAil, intron 19/Hindill and intron 22IXbal, and two hypervariable VNTR scoring sibling pairs can be carried out on a weighted combination of polymorphisms (introns 13 and 22). In all the three racial groups, the intron observed phenotypes or on latent genetic factors underlying the 7 sequence (G/A) polymorphism was not found to be very polymorphic on extremes may lead to incorrect observations. Selection phenotypic (frequency of rare haplotype was 0.02) or heterozygous (2.4%) as parameter estimates in the selected sample and to correlations between compared to Caucasian white populations. Boil and Hindcll RFLPs were genetic and environmental influences in the selected groups, although G found to be at complete allelic association. The frequency of the BcA- and E are uncorrelated in the parent population. Selection based on latent (Hindll+) haplotype was significantly lower in Chinese (0.14) than in Malays factors leads to correct parameter estimates. We show that individual (0.33) and Indians (0.38)(p<0.01). In contrast Xbal+ frequency was on may in estimates latent genetic factors reliably be obtained and used significantly higher in Chinese (0.44) than in Malays (0.28) and Indians QTL analysis. (0.29). The results for these haplotypes differ from other population groups such as Caucasians, Japanese, Thais and Brazilian Blacks. The average heteroxygosity rates based on these RFLP haplotypes were 37% Tor Malays, 23% for Chinese and 50% for Indians. For the VNTR polymorphisms, highest heteroxygosity rates were in the Malays and lowest in Chinese. A combination of Bell, Xbal, and introns 13 and 22 VNTR polymorphisms was found to increase the informativity to 82.0% for Malays, 75.9% for Chinese and 68.9% for Indians. Published Abstracts: Genetic Epidemiology and Population Genetics (cont.) A385

2250 2251 The H.Sema IV,D3S1442 and D20S210 loci specific probes revealed Evidence of linkage with a specific HLA Class II alleles and Alopecia positional divergence in the great apes. R.A.Conte, R. V. Samonte and Areata. M. de Andrade, C. Jackow, KA. Johnston, J.D. Reveille, M. Duvic. R.S.Verma . The Long Island College Hospital, Brooklyn, Natural Sciences UT M.D. Anderson Cancer Center, Houston, Texas, USA Research Institute, University of the Philippines, Diliman, Quezon City, Alopecia Areata (AA) has been associated with other autoimmune Institute of Molecular Biology and Genetics at InterScience-SUNY Health diseases, and it is characterized by an inflammatory T-cell infiltrate with Science Center at Brooklyn, NY production of cytokines around the affected hair follicles. There is a wide Comparative studies of karyotype and mapping data using loci specific spectrum of disease activity ranging from patchy of alopecia with total probes are being currently used to explore the evolutionary aspects of the regrowth to persistent loss of all scalp and body hair. Previous studies have genome. Chromosomal alterations due to pericentric inversions account for suggested that there is an association of AA with genes on chromosome 6 the majority of chromosomal alterations. Loci specific probes can provide a in the major hismcompatibility locus (HLA). In this study, 38 families with clue for chromosomal rearrangements. In this investigation we used a cases of AA (<75% hair loss) and/or AT/AU (> 75% hair loss for Alopecia 3p21.3 specific probe which has been associated with small cell lung Totalis (AT) to total scalp and partial body hair loss for Alopecia Universalis carcinoma corresponding to the locus for the H.Sema IV gene; a 3p26-pter (AU)) signed consents for HLA typing of peripheral blood lymphocytes. specific probe which detects the D3SI442 locus and a 20pl3-pter specific Families were ascertained from dermatology clinic populations. HLA-DR probe which detects the D20S210 locus. Human chromosomes were from a and DO alleles were ascertained by PCR amplification and dot potting with normal male. Chimpanzee [PTR], gorilla [GGOJ and orangutan [PPY] allele-specific oligos according to the Eleventh Workshop. The association chromosomes were derived from fibroblasts cell lines [CIMR;Camden, NJ]. between AA and HLA Class II alleles were included as a part of the analysis FISH-technique was employed as per standard protocol.The hybridization by assuming that the carrier probabilities varied as a function of the HLA signals corresponding to the D3SI442 loci were observed at conserved Class II alleles in each founding parent. This approach for linkage analysis locations at the terminal region of the short arm in PTR and GGO, but at a improves the power to detect genetic linkage. For the analysis we assumed diverged position in PPY at 2ql6. The H.Sema IV loci signals were seen at that the penetrance was 16% and the population prevalence was .1%. conserved regions at 2p21 for PTR and GGO, but at a diverged location in Results of this analysis support genetic linkage between AA and Class II PPY at 2q21. Signals from the D20S210 probe were conserved at the short loci with a maximal LOD score of 2.16 to HLA-DQB1 at 5% recombination arm termini in PTR and GGO, but of were at a diverged location in PPY at and a maximal LOD score of 1.24 to HLA-DR at 0% recombination. 21 qll-12. Present data suggests that the evolutionary mechanism(s) of chromosomal evolution is highly complex and the classification and nomenclature need to be re-evaluated.

2252 2253 Apolpoprotein E allele frequencies in3patients with the Sudden Infant HLA genotype does not affect ferritin or MSAFP levels in pregnant Death Syndrome (SIDS). TA Donlon1'2 HR Zielke4, A Eberly1, C women. R.Fisher, V.Leykam, D.Peterson, J.Beggs, R.Paneth-Pollak, and Kuslichl, and D Crowelk"5. 1 Molecular & Cytogenetics Laboratory, K.Friderici. Michigan State University, E.Lansing, Ml 48824 Kapiolani Medical Center, Honolulu, Hi 96826-2150 3 Kapiolani Health We previously reported that in Caucasian women, high maternal serum Research Institute, Honolulu, Hi 4 Department of Pediatrics, University of ferritin (MSF) in the presence of high maternal serum alpha-fetoprotein Maryland, Baltimore, MD 21201-1559 5 Department of Pediatrics, (MSAFP) was predictive of increased risk of preterm delivery over the risk University of Hawaii, Manoa, HI associated with high MSAFP alone. In African American women, increased risk was associated with low MSF. We question whether these differences are Apolipoprotein Epsilon (Apo E) plays a major role in maintaining plasma attributable to ethnic cholestrerol homeostasis differences in gene frequencies for Hereditary and in the mobilization and redistribution of lipids during Hemochromatosis Increased serum ferritin levels can occur in HH normal development of the nervous system (HH). and in the regeneration of peripheral individuals homozygous for a C282Y mutation or for nerves after There is substantial support for an association compound heterozygotes injury. between C282Y and H63D mutations in the HLA-H gene. C282Y carriers have also cholinergic function and Alzheimer Disease AD. Similarly, substantial support been reported to have higher ferritin levels in some circumstances. This work demonstrates that Apo E4 is a risk factor for AD development. AD patients with the between MSAFP and MSF the Apo E4 allele have a more severe cholinergic deficit than AD patients without investigates relationship levels and HLA-H C282Y and H63D allele were it. Kinney et al (1995) presented evidence for a decreased Muscarinic Receptor genotype. frequencies determined in pregnant Binding in the Arcuate Nucleus in SIDS patients. The cholinergic system is (1) the women undergoing routine MSAFP screening. Initially we examined 109 most continuous and extensive neuronal network in the mammalian central Caucasians, 296 African Americans and 51 Hispanics. DNA was extracted nervous system; (2) commands, regulates, and moderates almost all muscle. from blood clots. Genotyping was done using PCR, digestion and organ gland, and neural regions; and (3) contributes to all physiological and electrophoresis. Assuming three allelic haplotypes: C282-H63 (a, Y282-H63 behavioral activities. Apo E genotypes were determined using the polymerase (b) and C282-D63 (c), we expected six possible genotypes aa,ab,ac,bb,bc,cc. chain reaction and restriction enzyme digestion from DNA isolated from frozen Genotypes bb and bc are at risk to develop H4H. Based on our observed brain sections. We genotyped 81 SIDS patients (41 African American, 40 frquencies, we expected the ab genotype to occur in 1/12 Caucasians, 1/52 Caucasian) with regards to ApoE to determine if there was an increased African Americans and 1/29 Hispanics. Compound heterozygotes were frequency of the ApoE4 allele. The ApoE frequencies for the African American expected in 1/71, 1/2,500 and 1/227 respectively. Haplotype frequencies were SIDS patients were as follow: 2/2=0, 2/3=0.195, 2/4=0.025, 3/3=0.463, 3/4=0.268, compared in Caucasians having MSAFP > 1.50 and < 1.49 MoM and were and 4/4=0.049, none of which were significantly different from frequencies found to be identical in the two groups. We compared ferritin levels in published for control African American individuals. The ApoE frequencies for the Caucasians with aa and ab genotypes. After gestational age corrections, we Caucasian SIDS patients were as follow: 2/2=0, 2/3=0.125, 2/4=0.025, 3/3=0.625, found that C282Y carriers had ferritin levels similar to non carriers. We 3/4=0.175, and 4/4=0.005, none of which were significantly different from conclude that MSAFP and MSF levels in pregnant women are not usually frequencies published for control Caucasian individuals. related to HLA-H genotype and that differences in HLA-H haplotype frequencies are not related to differences in the MSAFP/MSF prematurity risk.

2254 2255 Children with cleft lip show seasonal variation in birth date, males Familial Hypercholesterolemia (FH) diagnostic criteria: Application to the more than females. F.C. FraserandA. Gwyn. Departments of Human 1988-94 National Health and Nutritional Examination Survey (NHANES) III Genetics and Biology, McGill University, Montreal, Quebec, Canada. data. GB Gardiner', M Khoury2, RR Williams3, CL Johnson1, MD Carroll1. Liability to neural tube defects is increased by maternal dietary 'National Center for Health Statistics, Centers for Disease Control and Prevention deficiency, and children with neural tube defects show seasonal variation in (CDC), Hyattsville, MD,2National Center for Environmental Health, CDC, Atlanta, date of birth. Since maternal dietary insufficiency may also increase liability GA,3Cardiovascular Genetics, Univ. of Utah School of Medicine, Salt Lake City, UT. to cleft lip, we looked for seasonal variation in date of birth of a sample of Familial hypercholesterolemia (FH) is an autosomal dominant condition which children with cleft lip. The multifactorial threshold model predicts that any can lead to early death for men (40's) and women (50's). Upid profiles (serum effect would be more apparent in males than females, since cleft lip is more cholesterol [SC], triglyceride [TG], high and low density lipoproteins [HDL and frequent in males. LDL], and very low density lipoprotein [VLDLJ) along with positive family history can Month of birth was obtained from records of 598 children with cleft lip make a dinical diagnosis. This is more accurate than molecular diagnoses for with or without cleft palate, seen at The Montreal Children's Hospitai populations, as lipid profiles and family history confirm phenotype. Early detection, between 1950 and 1996. Children with syndromes or associated diet and pharmacologic intervention can avert premature deaths. malformations were excluded. There was a significant tendency CDC's National Center for Health Statistics has conducted cross sectional for children NHANESs since 1960 to monitor the nation's health. NHANES lIl included with cleft lip to be born more often in the summer and autumn than in winter >40,000 participants matching US census composition by ethnicity, age, gender and and spring. The difference was greater in males than in females. The socioeconomic status. Of those, seasonal fluctuation in month of birth of children 24,424 (ages four to >90) participated in blood with cleft lip is consistent studies including lipid profiles. Cardiovascular family history was also taken. with the presence an environmental of teratogen with a maximal effect in Selection of SC>300 mg/dl and TG<300 mg/dl yielded 312 participants December-January. This may be related, at least in part, to a seasonal (1.28%). This is within the expected combined prevalence of the three genetic- fluctuation in maternal diet. Analyzing the data for the sexes separately related conditions that result in these levels of SC and TG: FH, Familial combined amplifies the effects of variation in liability for multifactorial threshold traits hypercholesterolemia, and Polygenic hypercholesterolemia. Additional anaylsis that have different frequencies in females and males. This approach could using NHANES lIl population means and percentiles for lipid profiles and positive be useful in the study of other gene-environment interactions. family histories provided an opportunity to make strongly suggestive clinical diagnoses for these conditions. Interpretation of NHANES Ill data to identify a fatal (if not treated) genetic condition may suggest ethical, legal and policy issues. Public use tapes provide a resource (>7,000 variables/participant) for population genetics and epidemiologic study (e.g. MCV values offer thalassemia carrier information). NHANES's capabinty for prevalence analysis utilizing standard FH diagnostic criteria may set precedent for other databases. A386 Published Abstracts: Genetic Epidemiology and Population Genetics (cont.)

2256 2257 Trends in Fetal Alcohol Syndrome: Albert Einstein College of Medicine Molecular analysis of the oxytocin receptor gone: a novel missense Affiliated Hospitals, 1985-1996. J. D. Hoffnan and A. L. Shanske. Center mutation and Its prevalence In pregnant women. W X. Liao, A. C. Roy, for Congenital Disorders, Montefiore Medical Center, Albert Einstein S. Chua, S. Arulkumaran, S. C. Ng and S. S. Ratnam. Department of College of Medicine, Bronx, NY. Obstetrics and Gynaecology, National University of Singapore, National Although a six-fold increase in the incidence of Fetal Alcohol Syndrome University Hospital, Lower Kent Ridge Road, Singapore 119074 (Intro. by: (FAS) in the period 1979-1993 was reported by the Birth Defects Monitoring Chew Kiat Heng). Program (BDMP) of the U.S. Department of Health and Human Services, Oxytocin, a neurohypophysical nonapeptide hormone, is involved in a one of the above authors noted a significant decrease in the incidence of variety of physiological responses such as uterine contraction, milk ejection, FAS in the hospitals affiliated with the Albert Einstein College of Medicine penile erection, and yawning. Clinically, oxytocin has been extensively used (AECOM), Bronx, NY. The purpose of this study was to test the author's in obstetrics for decades to increase the frequency and force of myometrial observation. contractility. The physiological effect of oxytocin could be perturbed by Data including Intemational Classification of Diseases discharge code mutations occurring in the sequence of the oxytocin receptor (OXTR) gene. 760.71 ("noxious influences affecting fetus via placenta or breast milk, Molecular characterization of mutations in OXTR gene would lead to better specifically alcohol; includes fetal alcohol syndrome") were gathered from understanding of the structure and function of OXTR. We used polymerase five of the AECOM hospitals for the years 1985-1996. The total number of chain reaction (PCR)/single-stranded conformational polymorphism (SSCP) reports of code 760.71 per live births per year was then calculated. and direct sequencing to study the coding regions of OXTR gene in In contrast to the six-fold increase in the incidence of FAS seen nation- pregnant women, and identified a novel missense mutation and a silent wide by the BDMP in the period 1979-1993, a sixteen-fold decrease in polymorphism in the exon 3 of the gene. The missense mutation resulted in combined incidence of FAS was found in the population of the AECOM amino acid replacement from Ala218 to Thr218, located in the fifth affiliated hospitals in the period 1985-1996. The incidence of FAS was transmembrane domain of OXTR. We used restriction fragment length found to be only 0.07/1000 in 1996 in this study as compared to 0.67/1000, polymorphism (RFLP) to screen this mutation and found out that it occurred the incidence of FAS reported by the BDMP for 1993. in 25% of the pregnant women studied. Two women who are homozygote Factors such as young maternal age, socioeconomic status, minority had normal delivery at term and normal milk ejection. This result indicated race, high rate of poly-drug use, high rate of abortion, high level of physician that this mutation may be a polymorphism. Therefore, the Ala218-Thr218 awareness of drug and alcohol abuse, and data reporting may have substitution does not seem to change the function of OXTR, though this contributed to the low and decreasing incidence of FAS seen in the Bronx substitution might have altered the hydropathy profile of OXTR, and as compared to the nation. possibly the conformation of OXTR. This study also demonstrated for the first time the molecular heterogenicity of OXTR.

2258 2259 DNA DIVERSITY IN EAST EUROPEAN POPULATIONS. S.A.Limborska, Associations of Argl6Gly and Gln27Glu polymorphisms of 02- P.A.Slominsky, M.I.Shadnna, S.N.Popova, O.V.Belayeva, E.V.Shabrova, adrenoceptor gene with asthma in Chinese. Z. Liu', Z. Wang1, B. T. V.Pogoda, M. V.Khorte, E.K.Khusnutdinova, V.A. Spitsyn, A.l. Mikulich. Wang', T. Niu1, Z. Fang2, X. Xu'. 'Program for Population Genetics, Institute of Molecular Genetics, Russian Academy of Sciences, 123182, Harvard School of Public Health, Boston, MA, 2Anqing Public Health Moscow, Russia; Medical Genetic Scientific Centre, 107012, Moscow, Bureau, Anqing, China. Russia; Institute of Ethnography, 220015, Minsk, Belorussia Bashkir Asthma is a disease characterized by reversible airway obstruction, Science Center of Ural Branch of Russian Academy of Sciences, Ufa, airway inflammation, and airway hyperresponsiveness. 02-adrenoceptor Russia (02AR), a G-protein receptor localized to a number of cell types in the lung, tone and be involved in In Russia there is a vast space inhabited by a lot of mixed populations participates in establishing bronchomotor may with different degree of Caucasoid and Mongoloid anthropological types. asthma. Previous studies suggest that the Arg16Gly and Gln27Glu The comparison with nearest neighbours and distant populations let to polymorphisms of W2AR may have functional relevance in the regulation of components and adenylyl cyclase coupling, and may contribute to the pathogenesis of evaluate the role of Caucasoid and Mongoloid in origin the roles of and of population studied. We have analyzed the normal asthma. In an attempt to examine Arg16Gly Gln27Glu ethnoqenesis in we conducted an association variability of minisatellite and microsatellite markers in some East European polymorphisms of P2AR asthma, study among 143 86 from China. populations. There are Slavonic (Russians, two urbanic samples, and individuals (57 cases, controls) Anqing, With respect to the for Belorussians, one urbanic and three rural samples), Finnic (Udmurts, Arg16Gly, genotype frequencies Arg/Arg, Arg/Gly, urbanic and rural samples), Romanian (Moldavians, urbanic sample), and and Gly/Gly were 26.2%, 51.2%, and 22.6%, respectively, for nonasthmatic mixed Turkic with Finic component (Bashkirs, four rural samples). Data subjects, and 27.2%, 47.3%, and 25.5%, respectively, for asthmatic patients. With to the for obtained was analysed by use of Philip 3.5 and GDA computer programs respect Gln27Glu, genotype frequencies Gln/Gln, for the analysis of variance. Maximum likelihood estimates of the Gln/Glu, and Glu/Glu were 83.4%, 15.3%, and 1.3%, respectively, among allelic nonasthmatic and and was constructed. Most divergent positions was obtained for subjects, 83.6%, 14.6%, 1.8%, respectively, among phylogeny We found no association between asthma and the Bashkirs and Udmurts. The principal components analysis was also used asthmatic patients. for Udmurt position estimation and the Udmurts were shown to occupy an genotypes of either Arg16Gly (X2=0.23 df=2, p=0.89) or Gln27Glu (X2=0.1 1 isolated position among the populations representing the Northern branch df=2, p=0.95). In conclusion, we could not find any evidence suggesting that of Europeoids. these polymorphisms of J2AR influence susceptibility to asthma in our population.

2260 2261 Occupational pesticide exposure and genetic Instability. HH McDuffiel, R The analysis of polymorphism of the angiotenzinogen, ACE and Nordaf, A kirsch3, K Nakahara3, G Cortopassi, P Pabello A Uedas A Gibson'. glukokinase genes In NIDDM patients from Russia. O. V.Miloserdova, MN; IUniv of Saskatchewan, Saskatoon,SK, Canada; 2United Hospital, St. Paul, M.l. Shadrina, P.A.Slominsky, MN. Balabolkin, S.A.Limborska . Institute of 3NCI Navy Med Oncology, Bethesda, MD;4Univ of Southem Califomia, Davis, CA; Molecular Genetics, Russian Academy of Sciences, Moscow 5Kagoshima University, Kagoshima, Japan. The analysis of CA dinucleotid polymorphism in the angiotensinogen and Herbicides, insecticides and fungicides utilize a variety of metabolic pathways to glukokinase genes and insertion/deletion in the ACE in control pests. Epidemiological studies report an increased nsk of rare neoplastic polymorphism gene a a of with NIDDM from the diseases among pesticides exposed workers. Molecular studies report increased random subsample and sample patients frequency of genetic markers assayed in peripheral lymphocytes (or DNA) of Moscow population were spent. We not found any differences in ACE gene pesticide exposed workers compared to controls. To evaluate biological markers as polymorphism in our two groups - but microsatellite allele frequency was measures of exposure to pesticides, we chose (a) a chromosome 7 inversion, which quite different. In the angiotensionogen gene alleles with the number results in a transient increased frequency of a hybrid antigen receptor gene; (b) G- repeatsu 16 were higher in the NIDDM patients compared with a control Banding emphasizing the 14:18 translocation found in non-Hodgkin's lymphoma group and S - alleles group with the smaller number of repeats (NHL);*c),1-2 frequency which is increased in NHL, in smokers and with increasing (10,1 1,12,13,14,15,16) in a random subsample was 0,48 and in the NIDDM age; d) sister chromatid exchange, effect indicators of mutagens and carcinogens. patients was 0,69. The observed differences of these two allelic groups The chromosome 7 inversion results in a hybrid antigen receptor gene. The inversion frequency of this hybridantigen receptor gene is determined by polymerase between a random sample and NIDDM patients were significant (X2=9,8, chain reaction (PCR) amplification of the target sequence in sequential dilutions of <0,001). The L group (alleles with n.>16) frequence were higher in a DNA from peripheral blood lymphocytes. The highest dilution in which the frequency random subsample of the Moscow population. In the case of glukokinase of the target sequence can still be detected determines the frequency of the hybrid gene some differences between NIDDM patients and control group was gene per microgram of DNA. An administered questionnaire obtained demographic, found - for example, major allele frequency was 0.59 in NIDDM patients occupational, medical and smoking history. Pesticide use diaries were utilized. The group and 0.71 in a random These differences are not statistically study population consists of Saskatchewan workers (n=77) exposed at work to sample. - of in herbicides, insecticides and fungicides and unexposed workers (n=46); men, women, significant but comparision glukokinase gene genotype frequencies to RxC method revealed nonrandom smokers. nonsmokers. Subjects were tested more than once. Of the 161 samples the same groups according analyzed to data, 9(5.6%, 8 exposed men, and 1 exposed woman) were positive at a association of glukokinase gene with NIDDM dilution to 62ng or 125ng. Eight percent of samples from exposed men were positive compared to 4.2% of nonexposed men. Among women, 4.3% of exposed women and no exposed women were positive. The instability noted may be a manifestation of the biologically effective dose of a pesticide for the tissue (lymphocytes) which has been shown to be the site of increased disease risk (lymphoma) (HSURC). Published Abstracts: Genetic Epidemiology and Population Genetics (cont.) A387

2262 2263 Mutation analysis of the HLA-H gene associated with hereditary A NORMAL VARIABILITY OF CTG REPEAT IN THE MYOTONIN hemochromatosis In African-Americans. Kristin G. Monaghan1, Ben Rybickf, PROTEIN KINASE GENE IN SOME EAST EUROPEAN POPULATIONS. Muhammad Shurafa3, and Gerald L. Feldman1. Henry Ford Hospital, Detroit, Ml. Popova S.N., Shadrina M.I., Slominsky P.A., Spitsin V.A., Mikulich V.A., 'Department of Medical Genetics; 2Division of Biostatistics and Research E.K.Khusflutdinova, Limborska S.A. Institute of Molecular Genetics, Epidemiology; 3Department of Hematology/Oncology. Russian Academy of Sciences, 123182, Moscow, Russia; All-Russian Hereditary hemochromatosis (HH) is an autosomal recessive disorder of iron Medical Genetic Centre, 107012, Moscow, Russia; Institute of metabolism resulting in accumulation of excess iron in tissues and organs. It is one Ethnography, of the most common inherited diseases among individuals of northern European 220015, Minsk, Belorussia; Bashkir Science Center of Ural Branch of descent, affecting between 1 in 200 and 1 in 400 individuals; however the frequency Russian Academy of Sciences, Ufa, Russia. among African-Americans is unknown. Two point mutations (Cys282Tyr and We have analyzed the normal variability of CTG repeat number in the 3' His63Asp) in a gene on the short arm of chromosome 6, designated HLA-H, were UTR identified in studies of patients with HH. These studies focused exclusively on of the myotonin protein kinase gene in some East European Caucasian populations, where the carrier frequency for the Cys282Tyr mutation was populations. We have studied a random samples from Bashkiria (a reported as 6.4 percent in one study and 15 percent in another. The carrier population with a high myotonic dystrophy frequency, 2.9610-), Russia frequency for the His63Asp mutation, which is in complete linkage disequilibrium (two samples - from Moscow and from Reazan's region; it is a populations with and to have a much lower was 17 for Cys282Tyr appears penetrance, percent low - Moldavia, both the control and hemochromatosis patient populations. We performed a PCR- with myotonic dystrophy frequency 0.05610-5), Belorussia based assay followed by RFLP analysis for these two specific mutations on a control (four populations from different parts of Belorussia), Udmurtia. Overall, we population of 172 African-Americans and four African-American patients with found 17 different alleles in population studied. No alleles with >27 CTG hemochromatosis to determine the frequency of these two mutations in this group. repeats were observed in all populations tested and frequency of 20. A similar distribution of the mutations causing hemochromatosis in Caucasians are significantly less CTG repeat number was found early in some population from Asia (Japan, prevalent in both the African-American control and hemochromatosis patient Tibet) and South America (native americans). A high frequency of CTG20-27 population, suggesting that hemochromatosis in African-Americans may be due to a alleles in Bashkir population with a high of myotonic dystrophy frequency mutation in a gene other than HLA-H or to other mutations in the HLA-H gene. support a suggestion of Novelli et al (1994) about North Asian origin of myotonic dystrophy mutation.

2264 2265 Genotype analysis of unaffected sibs in families with familial A RARE HAPLOTYPE OF [S GENE IN THURPU KAPU AND THE Mediterranean fever does not reveal evidence for heterozygote CHARACTERISTIC ARAB-INDIAN HAPLOTYPE IN AN INBRED advantage. E. Pras1 ,. Aksentijevich3, J.E. Balow Jr.3, M. Pras4-5, DL. ISOLATE OF RELLIS OF ANDHRA PRADESH, INDIA. Ramana, G. V., Kastner . IInstitute of Human Genetics, Departments of Medicine 2C and Chandak, G.R., Singh, L. Centre for DNA Fingerprinting and Diagnostics, 4F, 5Heller Institute of Medical Research, Sheba Medical Center, Israel; CCMB Campus, Uppal Road, Hyderabad 500 007, India 3Arthritis & Rheumatism Branch, NIAMS, NIH, Bethesda. In India, sickle cell gene is widespread in tribal populations inhabiting Familial Mediterranean fever (FMF) is an autosomal recessive disease malarial endemic areas. Several mendelian isolates (caste populations) characterized by recurrent attacks of fever accompanied by peritonitis, pleuritis also possess high frequency of sickle cell gene.We have examined an array and/or arthritis. The gene causing this disease has been mapped chromosome of nine restriction site polymorphisms, by PCR analysis, in the gene 16p, but has not yet been cloned. FMF segregates in specific geographical cluster of 10 sickle cell homozygotes,and 36 sickle cell heterozygotes in areas, mainly in the Middle East and around the Mediterranean basin. An Reltis, a high frequency population for sickle cell gene. The restriction especially high frequency of the disease has been noted in North African and enzymes used were Hinc II site 3E gene, Xmn I site 3'Gy gene, Hind IlIl site Iraqi Jews, -and among Armenians. Surprisingly, haplotype analysis suggest in Gy and Ay genes,Hinc II site in pseudo and 3'pseudo IS, Dde site, Ava that several different mutations segregate within each of these population 11 site in globin gene and Hinf site 3 P gene. In Rellis, the sickle cell gene groups. These findings could be explained by a selective advantage for is associated with the characteristic Arab-Indian haplotype. We have also heterozygotes. Previous studies among obligatory FMF heterozygotes have found 3 sickle cell homozygotes from Thurpu Kapu, yet another isolate failed to detect any genetic, immune, or environmental advantage. A having high frequency of sickle cell gene. For the first time, in this group, we heterozygous advantage might be manifested by an increase in the carrier to have encountered a rare 'atypical'(+------).In all sickle cell homozygotes, non carrier ratio among the unaffected sibs of the FMF families. We examined the clinical behaviour of the disease is benign with elevated fetal this ratio in 35 of our FMF families, in which both parents were obligatory hemoglobin levels (3.9%-21.0%). The importation of sickle cell gene into the heterozygotes. Genotype was determined with 3 tightly linked polymorphic Indian populations is presumably through Arab traders and its high markers from a 1 cM interval containing the FMF gene. Of the 67 unaffected frequency in Indian populations is due to its selective advantage in malarial sibs included in this analysis, 43 were carriers and 23 were none carriers. environment. the of sickle cell in caste we did not observe a distortion of the 2:1 carrier to non carrier However, presence gene populations Thus, expected is due to flow as shown et al our ratio, among the unaffected sibs. No apparent advantage for heterozygotes possibly gene by Murty (1995). Thus, study could be demonstrated in this study. Several explanations could account for demonstrates high frequency of sickle cell gene these results: 1) The selective advantage of the heterozygotes is too small to Arab-Indian haplotype in nontribal caste populations of Andhra Pradesh, be detected by this study. 2) Selective pressures that were significant in the India. past are no longer operating today. 3) Factors other than heterozygote advantage are responsible for the high prevalence of FMF in these regions.

2266 2267 Molecular heterogenicity of luteinizing hormone in Singapore Biochemical and mutational profiles, heterogeneity and haplotype association population. L. N. Ramanujam, W. X. Liao, A. C. Roy, S. Arulkumaran, S. of G6PD deficiency In Portugal. M.O. Rodrigues', C. Monteiro2, G. Gaspar', J. C. Ng and S. S. Ratnam. Department of Obstetrics and Gynaecology, Pereira1, and M.C. Martins1. 11NSA; Av Padre Cruz; P-1699 Lisboa Codex; T: 3511 National University of Singapore, National University Hospital, Lower Kent 751 9200, Fax: 3511 759 0441, email: carolino@ mail.telepac.pt. 2 Fac Cidncias Road, 119074 Chew Kiat Medicasd UNL, R Junqueira 96, P-1300 Lisboa, Portugal. Ridge Singapore (Intro. by: Heng). The glucose-6-phosphate dehydrogenase (G6PD) deficiency in erythrocites have Luteinizing hormone (LH) is important in the stimulation of follicle growth, in the caucasoid population of Portugal a prevalence of 0.51% 0.11 (confidence steroidogenesis and maturation of the oocyte. Molecular variants of LH limit of 95%; individuals under study: 13785 males). The frequency for deficiency- have recently been reported. These are due to some missense mutations in confering genes is 0.39. We performed the biochemical characterization of the G6PD LH beta-subunit gene. Two point mutations were reported in healthy and in erythrocytes in 22 of the deficient individuals through the study of its enzymatic infertile women. They caused two single amino acid substitutions of Arg8 for activity, Km for G6P and NADP, electrophoretic mobility and the efficiency with Trp8 and Thr15 for Ilel15 in codon 8 and 15 respectively, which change the 2dG6P and GAL6P. From our work 31.4% (22/70) of the studied deficient individuals of LH. The purpose of this was to are included in class 2 according to WHO. The remaining are included in class 3. immunogenicity study determine the Six RFLPs exon of these two variants of LH and new intragenic were studied: 5, nt 376 A®G, Fok I; intron 5, nt 611 presence identify mutations in C®G, Pvull; intron 8, nt 163 COT, BspHI; exon 10, nt 116 GOA, Pstl; exon 11, nt Singapore population. We identified a novel missense mutation, in the 1311 COT, Bcl1; and intron 11, nt 93 T®C, Nlall. New haplotypes were constructed codon 102 (G1502 to A1502), which resulted in a replacement of Gly102 by and only 5 out of the 64 possible were found showing a marked linkage disequilibrium Ser102 using Polymerase Chain Reaction (PCR)/ Single Stranded for several RFLPs and also for mutations and specific haplotypes. The control Conformation Polymorphism (SSCP). We used PCR, Restriction Fragment population (n=168 males) presented the G6PD B variant and were asociated to Length Polymorphism (RFLP) and DNA sequencing to screen the above haplotypes ( - - + + -- ).a(--+ + - -+). and Vlla(-+ + + + ), in 91.8%, 2.3% and mutations in Singapore population. A total of 202 subjects consisting of 95 5.9%, respectively. Chinese, 56 Indian and 51 Malay participated in this study. Four Chinese The sequencing analysis showed: 48.6% (16/33) of individuals with the G6PD A- subjects were detected heterozygotes of G1502 to A1502 mutation. This mutation, associated to haplotype Via (+ + - + - +); 9% (3/33) with the Betica mutation was not found in Indian and mutation, 18% (6/33) with the Santamaria mutation, both of them associated to Malay subjects. For the two mutations haplotype lVa ( + - - + - + ); 6.1% (2/33) with the Mediterranian mutation associated to at codon 8 and 15, nine Chinese, four Indian, and seven Malay subjects haplotype VIla; 12.3% (4/33) with the Seatle mutation, 3.0% (1/33) with Gahoe were heterozygotes, and one Chinese was homozygote. In conclusion, the mutation and a new mutation, 3.0% (1/33), which we designated by G6PD Flores, all prevalence rate of variant LH due to G1502 to A1502 mutation occurs in of them associated to haplotype 1. 0% and A 4%, 0% in Singapore Chinese, Indian and Malay population global idea of the mutated gene flow with a haplotype association was not workable since such genetic studies for the world are not available. respectively. The known immunological LH variant occurs in 10% of population yet it is not a full of Chinese, 4% of Indians and 7% of Therefore, yet possible understanding the genetic epidemiology of the Malays, which is lower than both the UK deficiency in G6PD within the portuguese population. (15%) and the Finnish (28%) populations. A388 Published Abstracts: Genetic Epidemiology and Population Genetics (cont.)

2268 2269 Polymorphism patterns in the BRCA1 gene in the Native American Molecular basis of human erythrocyte Miltenberger glycophorins In population of the Midwest. F. V, Schaefer, C. Shults 4nd L. Whetsell. HA Chinese. Mu-Ching Shih, Li-Huey Yang, Jan-Gowth Chang (1). Department Chapman Institute of Medical Genetics, 5300 E Skelly Dr, Tulsa OK 74135 of Clinical Pathology, Changhua Christian Hospital, Changhua, Taiwan. The Native Americans have been shown to have arrived in the United Department of Molecular Medicine, Taipei Municipal Jen-Ai Hospital, Taipei, States by entirely different migration patterns than the Caucasian population. Taiwan (1) In order to examine the genetic diversity between these populations with Molecular basis of human erythrocyte Miltenberger glycophorins in respect disease association, especially breast cancer, we examined the Chinese. Mu-Ching Shih, Li-Huey Yang, Jan-Gowth Chang (1). Department polymorphism pattern of exon 11 of the BRCA1 gene. Our sequencing of the of Clinical Pathology, Changhua Christian Hospital, Changhua, Taiwan. BRCA1 gene has found two common polymorphism patterns. The most Department of Molecular Medicine, common pattern is represented by the sequence published in for Taipei Municipal Jen-Ai Hospital, Taipei, Genebar*_ Taiwan (1) Human erythrocyte Miltenberger (Mi) glycophorins series are the the BRCA1 gene (Pattern A). However, a C2201T, T2430C, C2732T A3232G varants of and an A3667G pattern of polymorphisms (Pattern B) is found commonly in the MNSs blood group.system which are due to the rearrangements association as a second sequence pattern. In our control Caucasian between glycopho'hn A (GPA) and glycophorin B (GPB) population 17 of 58 alleles (30%) display the latter polymorphism pattern. genes, and the hotspot recombination regions are located between exon 2 We were able to examine 6 100% Native Americans (4 Cherokee and 2 and exon 5 of GPA and GPB genes. The MiV ahd MiXI are both encoded Choctaw) for their polymorphic sates. The sequencing found that (1) 10 of the by A-1B hybrid glycophorin whose unequal crossing-over points reside in Native American allele patterns were the same as the Genebank sequence different sites of intron 3. The Mill, MiVI and MiX are encoded by B-A-B (Pattern A) and two were the same as the less common polymorphism pattern hybrid genes. The Mil, Mill, MiVII, MiViII and MilX are encoded by A-B-A (Pattern B) for an incidence of 16% and (2) no additional polymorphism genes. We take advantage of the differences between GPA and GPB genes patterns were observed. Although it might be speculated that the lower to develop a PCR-based rhethod to detect all the Mi 9lycophorinvariants. incidence of the second pattern (Pattern B) may be interest, the low Native We use GPA or GPB specific primers or combination with mismatch primers American sample size does not allow the difference to be yet significant. Of to amplify the GPA or GPB or hybrid gene specifically, and then the PCR more interest is that the same two sets of polymorphism pattems exist in the products are digested with restriction enzymes to recognize the different Native Americans and they lack of a third, unique polymorphism pattern. hybrid genes. Totally 250 cases of Chinese from general population of Therefore, the genetic constraints on variability in the BRCA1 gene appears to Taiwan were studied, 13 (5.2%) of them were Mill, 15 (6.0%) of them were be very strong. However, two distinct populations of alleles have arisen during MiIV, 8 (3.2%) of them were Sta, 2 (0.8%) of them were MiVII, and one evolution and they suggest a common ancestor between the two ethnic (0.4%) of them was MiV. Our method is the first PCR-based method to populations. Furthermore, subtle variations in the polymorphism patterns may detect all the Mi variants and the first genetics data of Mi glycophorins in be informative in tracing subpopulations within larger ethnic groups. Chinese. Similar approach can also be used to detect other MNSs variants.

2270 2271 Alcohol dehydrogenase (ADH2) genotypes In Infants with fetal alcohol Genetic alterations in homocysteine metabollzin9 genes and coronary effects. Stoler JM and Holmes LB. Genetics and Teratology Unit, heart disease. L. Wan 9, B. Upson2, M.R. MalinoW , P. . Due/F, D.L. Massachusetts General Hospital Hess2, R.A. Gluckmarn P.C. Block2, C.R. Holzgang2, P.H. Andersorn, D. Theoretically some alcohol-exposed fetuses have increased SeltzeP3 and W Kruger1. 1Fox Chase Cancer Center, Phila. 20regon Health susceptibility to damage due to genetic factors. We have recruited 448 Sciences University, OR 3Carle Foundation Hospital, Urbana IL. women during pregnancy to identify by self-report and maternal blood Total plasma homocysteine (tHcy) is a significant independent risk factor markers those women who have consumed significant amounts of alcohol. for coronary heart disease (CHD). We examined a population of 142 CHD The infants have been examined at birth for the facial features and growth patients and 110 controls from the Portland Oregon area for several retardation attributed to alcohol by a study physician who was unaware of polymorphisms and mutations in genes directly involved in homocysteine exposure status. 71 infants have been identified with fetal alcohol effects. metabolism. Using PCR/RFLP methodology we typed this group for the To date ADH2 genotypes have been determined for 75 infant-mother pairs C677T (Al 14V) polymorphism in the methylene tetrahydrofolate reductase (45 black, 30 white). The DNA was amplified using using primers designed gene (MTHFR), the G919A (G307S), T833C (1278T) and 68bp duplication to introduce an artificial Alul restriction site at the base pair difference mutations/polymorphisms in the cystathionine beta synthase (CBS) gene, between the ADH2-1 and ADH2-3 alleles. The alleles were scored after and the A2766G (D919G) polymorphism in the methionine synthase (MS) digestion by Alul and are designated as 1-1 or 1-3. Similarly the ADH2-2 ene. We analyzed the gene frequencies for a relationships with CHD and allele was determined by a Maelil restriction digest. Only one mother and fasting tHcy levels. The only statistically significant difference occurred in no infants had the 1-2 genotype. 50% of the 14 affected black infants had a the frequency of the C677T MTHFR polymorphism (P<.008). In addition we 1-3 genotype compared to 33% of the 31 unaffected black infants (p=.04). found that T677 homozygotes had average fasting homocysteine levels By comparison, 59% of the black mothers of affected infants had the 1-3 20% higher than T677 heterozygotes or C677 homozygotes. Our results genotype compared to 48% of the black mothers of unaffected infants. 50% suggest that the C677T polymorphism in MTHFR is a risk factor for CHD, of the white mothers of affected infants were 1-3 compared compared to while the polymorphisms in CBS and MS are not. 29% of the white mothers of unaffected infants. Not enough white affected babies have been analyzed to date. These findings suggest that the fetal 1- 3 ADH2 genotype is associated with the teratogenicity of alcohol in black infants. Previous work has shown conflicting results (McCarver-May DG et al, Alcohol Clin Exp Res 20(2):122A, 1996). Additional studies need to be done to further evaluate the role of ADH2. Supported by the William Randolph Hearst Foundation, NIH# 5R03M09049-02, March of Dimes (MA Chapter), John W. Alden Trust and the Christopher D. Smithers Foundation.

2272 2273 Associations of molecular variants of ALDH2 and EPHX1 genes with Association of breast cancer with vitamin D receptor gene spontaneous abortion. M. Wang', T. Niu1, Z. Wang1, A. Kuo', B. Wang1, polymorphism. Zentaro YAMAGATA, Yingning ZHANG, Akio ASAKA, JJ Rogus', Z. Fang2, X. XuW 1Program for Population Genetics, Harvard Masao KANAMORI, Takashi FUKUTOMI. Dept. of Health Sciences, School of Public Health, Boston, MA, 2Anqing Public Health Bureau, Yamanashi Medical University Anqing, China. The previous reports indicated that breast cancer related with BRCA1 or Reproductive health is a major public health concern in both developed and BRCA2 is rare in Japanese women. Recently, it was reported that Vitamin developing countries. One important reproductive outcome is spontaneous D derivatives have been shown both to inhibit the proliferation of cultured abortion, which is defined as a loss of embryonic viability prior to 20 weeks of breast cancer cells and to cause regression of experimental mammary gestation. While partially influenced by genes, the genetic basis of spontaneous tumours in vivo. Furthermore, James et al indicated that vitamin D abortion remains poorly understood, though previous investigations suggest that derivatives may play a role in regulating the expression of genes and polymorphisms in genes responsible for the metabolism of xenobiotics may protein products implicated in apoptosis. We hypothesized that vitamin D an spontaneous To explore the of the an confer increased risk for abortion. roles may be an important determinant of breast cancer. We analyzed Mboll polymorphism of the human aldehyde dehydrogenase 2 gene (ALDH2) and association between the vitamin D receptor (VDR) gene polymorphism and gene the HisI 39Arg polymorphism of human microsomal epoxide hydrolase breast cancer using case-control study 3f 60 Japanese patients with breast (EPHX,) in spontaneous abortion, we conducted a case-control study in Anqing, cancer and 120 age-matched controls. VDR Bsml genotype was China. 155-women (Mean age: 46.5 years, SD=15.0 years) who experienced determined by PCR-RFLP described by Morrison et al. The frequency of bb spontaneous abortions and 143 (Mean age: 48.4 years, SD=14.1 years) who had in cases was more than controls. The odds ratios for breast cancer was healthy pregnancies were enrolled in the study. 3.90 confidence interval, 1.63-9.30) Women who are homozygous Regarding ALDH2, the genotype frequencies of / / (95% ALDH2-1 ALDH2-1, ALDH2-1 risk of for the presence of Bsml site appear to have 3.90 times of the ALDH2.2, and ALDH2.2 / ALDH2-2 were 61.7%, 31.6%, and 6.7%, respectively, developing breast cancer as women who are heterozygous or homozygous among the cases, and 59.7%, 32.4% and 7.9%, respectively, among the controls. of the absence for the Bsml site. Though there have no study on We found no statistical evidence of association (X2=0.21 df=2, p=0.90). With association between breast cancer and VDR gene polymorphism, Taylor et respect to the His139Arg polymorphism of EPHX1, the genotype frequencies of al reported the significant association of prostate cancer with VDR gene His/His, His/Arg, and Arg/Arg were 86.7%, 11.8%, and 1.5%, respectively, among polymorphism. They indicated allele T of Tagl polymorphism of V DR cases, and 75.6%, 23.6%, and 0.8%, respectively, among controls. In this case, a increased the risk of prostate cancer. The allele T links allele b, so we are significant association was found (X2=6.14, df=2, p<0.05). In summary, our study interested in this result. suggests that the His1 39Arg polymorphism of EPHX1 may play a role in susceptibility to spontaneous abortion in our study population. Published Abstracts: Genetic Epidemiology and Population Genetics (cont.) A389

2274 2275 Mutation detection methods and preferential amplification of wild type Distribution of Apolipoprotein E genotypes in the Fragile X syndrome, allele of the human P2-adrenergic receptor gene. C. N. Yandaval, D. M. Batten disease, and Down syndrome populations. L-L. Ye, W. Ju, W-M. Cooper', A. Pillari1, S. B. Liggett, and J. M. Drazent. 'Department of Xu, N. Schupf, E. C. Jenkins, W. T. Brown, N. Zhong. New York State Institute for Medicine, Harvard Medical School and Pulmonary Division and Crtical Basic Research in Developmental Disabilities, Staten Island, NY 10314. Care and Women's MA Apolipoprotein E (ApoE) is a polymorphic protein with E2, E3, and E4 being the Medicine, Brigham Hospital, Boston, 02115, three common alleles. The E4 allele has been implicated in Alzheimers disease (AD) 2Department of Medicine, University of Cincinnati Medical Center, and associated with dementia in neurodegenerative disorders, while E2 appears to Cincinnati, Ohio be a protective allele. We employed a PCR based assay to genotype the ApoE Sequence variations at nucleotides 146 and 179 of the human b-adrenergic alleles in three syndromes associated with mental retardation including Fragile X receptor gene result in Arg ("wild type') or Gly at amino acid 16 and GIn ("wild (FX), Batten Disease (BD), and Down Syndrome (DS). A total number of 325 type") or Glu at 27 of the encoded protein. Polymorphisms at these loci may be genomic DNA samples, 98 FX, 37 BD, 91 DS, and 99 unrelated normal controls associated with asthmatic subsets and/or the to (NL), were PCR amplified followed by digestion with restriction enzyme Hha I. This response b-adrenergic agonists. generated two fragments (91 and 83 bp) for the E2 allele; three fragments (91, 48 In order to detect these polymorphisms, we have developed an oligo-specific and 35 bp) for the E3 allele, and four fragments (72, 48, 35, and 19 bp) for the E4 polymerase chain reaction (PCR) and studied over 200 asthmatic patients. As allele. Combinations of these three alleles could result in six genotypes: 2/2, 2/3, 2/4, oligo-specific PCR involves two PCR reactions for each polymorphism, we tested 3/3, 3/4, and 4/4. Our results were consistent with published studies in normal and the feasibility of sequencing directly by dye primer and dye terminator automated DS populations. We noticed that ApoE2 is more frequent in FX (11.5%) and BD sequencing of a PCR product that comprises this variable region. When we tested (9.5%) compared with DS (6.6%) and NL (7.4%). The genotype 2/3 was 18.4% in the 8 patients, we found discrepancies in genotyping assignment between oligo- FX and 13.5% in the BD compared to 7.4% in the NL. Further study may be needed specific PCR and sequencing. Individuals who were identified as heterozygous by to determine whether the E2 allele is correlated in population-based samples. oligo-specific PCR method typed as wild type homozygous individuals by Distribution (%) of ApoE in FX, BD, and DS sequencing. DNA from individuals heterozygous by oligo-specific PCR was amplified by 3 separate sets of primers and cloned into TA vectors. In this small populations |jenotypes Alleles scale approach, all the colonies sequenced were found to be wild type with one of 2/2 213 2/4 3/3 3/4 4/4 E2 E.L b4 the primer sets. In order to rule out the possibility of allele specific preference, NL 2.0 10.1 1.0 72.7 14.1 0 7.6 84.8 7.6 about 100 colonies from one heterozygous individual were transferred onto (99) duplicate nylon membranes and were grown. Each filter was probed separately FX (98) 1.0 17.3 3.1 57.1 20.4 1.0 11.2 76.0 12.7 either with a wild or mutant type probe Tor polymorphism at 27. Of the 41 positive BD (37) 2.7 13.5 0 64.9 16.2 2.7 9.5 79.7 10.8 colonies 33 were positive with wild type probe, whereas only 8 colonies were DS (91) 1.1 8.8 2.2 65.9 18.7 3.3 6.6 83.0 10.4 mutant type. The specificity of some of the clones was checked by sequencing. The deviation in the observed number of mutant colonies clearly indicates a This study was supported in part by NYS OMRDD, by Children's Research preferential amplification of wild type allele and the unsuitability of direct PCR Foundation, and by NIH 1RO1HD2947-01, AG090400, and PO1AG11531. product sequencing method for mutation detection at this locus.

2276 2277 Identification of an African American community In Colombia with a A mutant CCR5 alleles is absent in Chinese Han population. Y.P. low prevalence of hypertension: An potential founder effect conferring Zhang*, L. Nie*, J. Qi*, C.L. Zhu*, S.K Gou , A.H. Liu , S. Y. Lint and X. protection against hypertension. /. Zarante, R. Mendoza-Londono, JE. Zhou#. *Laboratory of Cellular and Molecular Evolution, Kunming Institute Bernal. Human Genetics Institute, Javeriana University, Bogota of Zoology, Chinese Academy of Sciences, Kunming 650223, P.R. China; Expedicion Humana visited 36 Amerindian, African American (AA) and #Kunming Blood Center, Kunming, P.R. China. isolated communities in Colombia. 861 adults from five AA communities were HIV-1 and related viruses require co-receptors to infect target cells. The interviewed and evaluated by physicians and dietitians. Data included age, chemokine receptor CCR-5 is a co-receptor for macrophage-tropic HIV-1 gender, diet, nutritional status and blood pressure. The 5 AA communities were strains. A CCR-5 mutant with 32-base-pair deletion within the coding region compared to each other, and with other ethnic groups. Average systolic pressures was found recently. Homozygotes of this allele was demonstrated to be were 122 mm Hg in AA, 110 mm Hg in Amerindian and 112 mm Hg in Hispanics (p<<0.001). Diastolic and mean arterial pressures also showed significant highly resistant to infection by M-tropic HIV-1 viruses. This allele was found differences between groups (p<<0.001). The prevalence of hypertension (HBP) in to be common in the Caucasian population with a frequency of 0.081 to AA groups was 27.3%, with 10.3% having values in the normal high range. Of 0.092, but was not found in African or Asian populations (Samson et al., those with HBP 54.5% were in stage 1, 28.5% in stage 2, 12.3% in stage 3 and 1996; Huang et al., 1996). Huang et al. (1996) found this allele was absent 4.7% in stage 4. The only risk factor that was significantly associated was obesity in 91 Chinese. Unfortunately, they did not have information on the locality (p<<0.001), determined by body mass index. In contrast, we identified a and nationality of those Chinese. There are more than fifty nationalities in prevalence of HBP of 7%, all stage 1, in an isolated community in rural Colombia. China, among which Han is the majority. We have investigated 622 Han This settlement has been geographically and culturally isolated since it was individuals from Yunnan, Sichuan, Guizhou, Fujian, Jiangsu, Xinjiang, founded in the 18th century. Henan, Shanxi, Guangxi and Qinghai Provinces of China. The mutant allele Essential hypertension results from influences of environmental and polygenic has not be found in this population. Our results suggest that the mutant factors. A number of genes have been proposed to contribute to the polygenic allele is absent in Han population. Further studies on the minority effect of HBP. The angiotensinogen gene shows allelic variants that show linkage nationalities are to determine if to certain forms of hypertension in African-Caribbean populations. RFLP studies required this allele is absent in the whole of the renin loci show characteristic distributions among hypertensive individuals. Chinese population. Other interethnic differences have been proposed for the higher prevalence of HBP in AA; these include differences in the rate of sodium excretion, alterations in the sodium-lithium counter transport, and an increase in cytosolic calcium. This is the first report of an isolated AA community with a low prevalence of HBP. We hypothesize that members of this community share a protective gene or lack alleles of genes that predispose to HBP. Such a genetic effect would be amenable to gene localization studies, which are underway.

Published Abstracts: Genetics Services and Tests, Genetic Screening and Public Policy

2278 2279 screening by using Mass spectrometry. S.S.Al Arrayed, E.AI Complete mutation scanning of the acid beta-glucosidase gene by Jishi, A.Abbasi, M.S.Rashed, P.T.Ozand. Genetic department,Salmaniya Medical fluorescence-assisted mismatch analysis (FAMA) using universal center,Bahrain. King Faisal Specialist Hospital and research centerSaudi Arabia. primers. D. Germain, J.P. Puech, C. Caillaud, A. Kahn, L. Poenaru. Department of Introduction:Several newborn screening programs exist on wide scale in different Genetics and INSERM U129, CHU Cochin, Paris, France. countries.These programs vary widelydetecting disorders before frank symptoms of Among the useful technologies for the detection of sequence variation, not one is ideal disease exist.Screening programs are also helpful to determine the real frequency of the in all situations. Despite constant improvements, direct automated DNA sequencing is population. costly and not always sufficiently reliable for definitive identification or exclusion of Material and Method: We applied newborn screening on 1000 Bahraini newborns heterozygous mutations in a diagnostic setting. Comparisons of different DNA scanning rendom for two years.The blood samples were collected from infants heelon methods applied to different genes should provide indications as to the best choice(s) in a to fifth day after birthon special card and send to the laboratory in King Faisal diagnostic context. We use the FAMA procedure to search for mutations responsible for specialist hospital in Saudi Arabia,where Electrospray Tandem mass spectrometry(ESI- Gaucher disease. The underlying pathogenic mechanism of this autosomal recessive MS/MS)MS/MSwas applied. It analysis blood samples for thirty metabolic diseases such disease is the deficient activity of the lysosomal acid beta-glucosidase. The beta- amino acids,organic acids and carnitine esters ets. Abnormal results were received glucosidase gene is scanned on a set of 6 amplicons, which comprise the eleven exons in 48 hours to start treatment. and all exon/intron boundaries. For each region a first round of PCR is performed using Results: We analysed 1000 Bahraini newborn,out of it 21 infant were found to have unlabeled specific primers that do not match the highly homologous pseudogene. These results. Ten infants were definitely sick. In five case the follow up samples primers are extended by 'universal" arms at their 5' ends. An aliquot of the first showed normal resultsand those infants are normal until now. Six cases were lost for amplification is submitted to a second round of PCR, using a pair of primers which are up. The preliminary results showed ten abnormal cases out of 1000 newborn, complementary to the universal extensions and labeled with different fluorophores at their which give the incidence of 1%.We expect to have 100 sick infants with metabolic 5' end. The conditions of fluorescent PCR are rendered uniform by using such universal diseases out of the 10,000 newborn annually. Diseases such as maple syrup urine primers. Heteroduplexes are formed from the bichrome PCR products and mismatched disease, medium chain acyle co A dehydrogenase deficiency, primary carnitine bases are modified using hydroxylamine or osmium tetroxide. The modified bases are deficiency, methyl malonoc acidemiamethylene tetrahydrofolate reductase deficiency cleaved with piperidine and the samples are separated on a polyacrylamide denaturing were found to be common. These results are not comparable with other studies as we gel. Data are collected and analyzed using an automated DNA sequencer and appropriate arestudying large number of diseases at neonatal period, each of these diseases have software. Both Gaucher disease mutant alleles were identified in all 12 patients studied, frequency rate. and 5 new mutations were also characterized. FAMA, in the 'universal" primers Conclusion: Prevalence rate of metabolic diseases is high among our newborn, and configuration used in this study, is a reliable and sensitive mutation scanning method. a metabolic screening program is essential to provide better and cost effective medical Upon labeling PCR products with strand-specific fluorescent dyes, it also provides care for patients.The clinical picture of the diseased infants will be presented. information on the localization and nature of the nucleotide change prior to sequencing, and thus distinguishes new mutations from those that are recurrent or Dreviouslv identified. A390 Published Abstracts: Genetic Services and Tests, Genetic Screening, and Public Policy (cont.)

2280 2281 Incidence of phenylketonurla In Institutionalized mentally retarded Individuals The role of primary care providers in the delivery of genetics services. S. In Iran. N. M. Ghiasvand'3, S. Seyed', F. Seyedin Boroojenit and Saeb2. IGenetic J. Hayflick, K. Silvey and M. P. Eif. Oregon Health Sciences University, Center, Sch. of Med., Shahid Beheshti Univ. of Med. Sci., Tehran, Iran. 2Behzisti Portland. Theran, Tehran, Iran. 3Div. Hum. Mol. Genet., Dept. Surg., Washington Univ. Sch. Though genetics knowledge is advancing rapidly, application of this Med., St. Louis, MO 631 10.(lntro. by: Helen Donis-Keller). information has not increased at the same pace. With substantial resources In countries where the frequency of PKU in the newborns is relatively high, PKU directed to the Human Genome Project, a commensurate effort must be screening of the newborns and implementation of early diet therapy can be a cost placed on educating all health care providers about the application of genetics effective national health program. However, in many countries, where PKU knowledge to clinical care. In order to develop educational programs that teach frequency is very low or cost effectiveness of PKU screening has not been relevant genetics to a diverse group of providers of primary health care, the demonstrated yet, this preventive practice is not included in the national health role of these providers in delivering genetics services must be delineated. After priorities. To predict whether inclusion of PKU screening in a countrys health we program, is cost effective, the frequency of this disease needs to be determined. obtaining input from the genetics and primary care communities, propose From our direct experience and observations it appears that PKU frequency in that the role of primary care providers is to incorporate primary level genetics Iranian newborns is relatively high. presumably due, at least in part, to a relatively services into routine health care. We further propose that primary level high rate of consanguinity. To test our hypothesis we needed to study the frequency genetics services be defined as the following: of PKU in Iranian newborns. However, since funds were not available to support Identify individuals who may benefit from genetics services, including those such a project, we sought to determine the incidence of PKU among the with a genetic disorder and those at increased risk for having or transmitting a institutionalized mentally retarded individuals, which could, to some degree, reflect genetic disorder the frequency of this disease in the whole population. Recognize historical and physical features of common genetic conditions In a retrospective study we utilized the Guthrie test on the dried blood samples and features that suggest a genetic disorder from 4512 mentally retarded individuals institutionalized in sixty governmentally Monitor the health of Individuals with a genetic disorder, in conjunction with administered centers across the nation. Among these, 87 had blood phenylalanine genetics health professionals concentration of 15 mg/dl or higher. We considered these individuals as Provide basic genetics information to patents and families in a culturally phenylketonurics, and calculated the incidence of PKU among the institutionalized competent way using non-directive genetic counseling skills to facilitate mentally retarded population of Iran, at 1.92%. comprehension and informed decision-making These results show that the incldence of PKU in the mentally retarded population Coordinate care for individuals with complex genetics service needs of Iran is relatively high and provide indirect evidence for a high frequency of PKU in Recognize the special psychosocial issues for a family in which one or the Iranian population. A prospective study to estimate the frequency of PKU in the more members is affected with a genetic disorder newborn population of Iran, would furnish definitive results that could be utilized in Know the full range of genetics services in the community and in estimating cost effectiveness of a national-wide PKU screening program. neighboring and regional communities from which patients might benefit ppropriately refer patients with additional genetics services needs Facilitate utilization of local and regional genetics services

2282 2283 The effects of base change, fragment length and base content on the Re-contact to advise of testing available to detect high risk for detection of point mutations by heteroduplex analysis and SSCP. Q. carcinoma: response to letters sent to persons initially ascertained for Jin', J. M. O'Conno2, V. D. Burlancf, W. R. Bauboni?, F. P. Curtis2, M. M. other genetic reasons. B. A. Swiglo*, R. R. Lebel. Genetics Services, The Garnet2, and W. E. Highsmith, Jr.1. 'Dept. of Pathology, University of Helix Building, Glen Ellyn, IL. Maryland Medical School, Baltimore 2FMC BioProducts, Rockland ME. A computerized database of over 5000 persons, whose four-generation Mutation scanning (searching for unknown base changes over a pedigrees were entered over a period of nine years, was employed to relatively long DNA fragment) is most often performed using gel ascertain prospectively families with apparent elevation of risk for electrophoretic methods which are sensitive to DNA conformation, i.e. carcinoma of the breast, ovary, colon and prostate. The individuals Heteroduplex (Htx) analysis and Single Stranded Conformational presented for genetics consultation for reasons other than familial Polymorphism (SSCP). The sensitivity and specificity of either assay carcinoma risk, most commonly for prenatal indicated by depends, among other things, on the base change, the size of the fragment maternal age or abnormal results of prenatal screening. being studied, the location of the base change within the fragment, and the Persons who had two or more relatives, within three degroes of overall base composition. It remains difficu t to predict how any of these relationship, affected by one of the tumors of interest were offered (by letter) parameters will affect the electrophoretic resolution of hetero vs. consultation to learn more of the possibilities for predisposition testing in homoduplex molecules and single strands in SSCP. To address these regard to risk for such disease (BRCA-1, BRCA-2, etc.) questions we have constructed a set of molecules of varying GC content Those who responded to the letter with interest were scheduled for (40, 50, and 60% GC) having all possible base changes at a particular genetics consultation to explore the psychological, ethical, economic and location in order to characterize the dependence of separation for both Htx other issues associated with testing. Those who chose to pursue the testing analysis and SSCP. Confirmatory sequencing demonstrated that there were were referred for neuropsychological testing designed to uncover occult no other base changes in any of the clones. Using this set of "mutation depression or risk of adverse response to disturbing test results, and were toolkit' clones as PCR templates, amplicons of various lengths with the asked to identify both a personal support individual to be present with them same base mutated to all other bases were generated and their behavior in at clinic visits and also a professional with crisis intervention skills who manual and automated heteroduplex analysis and SSCP was analyzed. would be ready to work with them in the event of adverse outcomes. Test Separation in SSCP is dramatically affected by GC content, whereas it is results obtained from reference laboratories were provided at face-to-face not in Htx analysis. For both methods, the base substitution can significantly visits, followed by appropriate follow-up contacts. alter the electrophoretic pattern. These results, as well as length and position effects, will be presented.

2284 The evaluation of the rubella vaccination program to women in reproductive age in Taiwan C. F Yuan, M. L. yang, H. T. Ng (Intro. by: Bing-Wen Soong). Veterans General Hospital-Taipei, Taipei, Taiwan, R.O.C. A Series of mass immunization programs against rubella infection has been implemented in Taiwan as a means of preventive strategy to reduce the occurrence of congenital rubella syndrome (CRS) since 1986. In 1991, females of age 20 to 35 were included in the vaccinated target population. It is anticipated that 1.4 million doses would be injected to child-bearing age women during the period of 1991 to 1996. Serum samples of 2768 pregnant women were collected and tested for rubella IgG. The overall susceptibility rate was 14.7%, which is lower than the previous rate from the 1986 survey (14.75 vs 19.4%, P=0.000). However, this rate did not meet our expectation, as it remained higher than results reported by studies from other countries. A total of 2562 questionnaires were sent to those subjects, inquiring them of their previous vaccination history to rubella and their willingness to be vaccinated. Of those only 815 (32%) responded. The percentage of women at child-bearing age receiving rubella vaccination during five years was relatively low, only 28%. In the returned surveys, 598 of them had immunity against rubella, and 51% (305) of them have received vaccination in the past. However, 80% of those in survey who were not immune from rubella showed strong interest in receiving vaccination. Our results also demonstrated that women who possessed immunity against rubella had a better understanding about the disease than those without immunity. Furthermore, the study indicated a correlation between knowledge of rubella and their education, profession, religion and residential environment. These information can be referenced when enforcing rubella vaccination programs. Our data indicated that a more vigorous program has to be implemented, enacted and enforced in Taiwan. Published Abstracts: Genomics A391

2285 2286 SSCP ANALYSIS OF THE FMR1 GENE. K. Brondum-Nielsen, K. Structure and chromosomal localization of novel Hmob33 sequence Gronskov, A. Hallberg, A. Sand, H. Hjalgrim John F Kennedy Institute, from human brain cDNA library. L. V. Dergunova, I. P. Vladychenskaya, Glostrup, Denmark N. M. Raevskaya, L. G. Polukarova, G. P. Lelikova, S. A. Limborska . Rare point mutations and deletions in the FMR1 gene have been Institute of Molecular Genetics, Russian Academy of Sciences, 123182, reported to be a cause of fragile X syndrome. To screen for pointmutations Moscow, Russia in FMR1 we have set up SSCP analysis. 130 mentally retarded males that The novel cDNA clone Hmob33 was isolated from cDNA library, were referred to us for fragile X syndrome, but who did not show an constructed from human medulla oblongata poly(A)+ RNA. Hmob33 expanded CGG repeat in the 5 UTR of the FMR1 gene were selected for sequence expressed in different structures of human brain, but not in mutation screening. 35 of the subjects were selected by more strict criteria kidney, skeletal and uterine muscle. Northem blot analysis showed that and were only included if they displayed one or more of the following brain mRNA contains two transcripts more than 5 kb. Hmob33 sequence features: hyperactivity, familial predisposition, suspected clinical fragile X includes 1567 nucleotides and has polyadenilation signal AATAAA at 3'- syndrome, macroorchidism, or autistic behavior. end. The comparison of Hmob33 with EMBL / GenBank databases revealed We found 2 patients with a silent mutation in exoni (13979G-T), and one homology with some expressed sequence tags (ESTs) which do not patient with a 2 bp deletion in intron 1 (14100delCT). Exoni was correspond to any known genes. These ESTs and Hmob33 sequence have investigated in a total of 121 patients and 38 controls. Neither of the two overlapping regions of high identity. They represent new tentative human changes were found in controls. 20 of 117 patients had a bp change in consensus sequence which consists of 1800 nucleotides. Hmob33 intron 1 at 23603, changing A to G. sequence was mapped on the chromosome 10 by a mapping panel of Furthermore, we found a polymorphism in exon5 in 6 of 114 patients human-rodent hybrid cells and by hybridization in situ. (G30584A), this polymorphism was also found in 5 of 58 controls. In 4 patients we also performed Westem blot analysis using an anti- FMR1 antibody to cell extracts from lymphoblastoid cells. All 4 patients displayed macroorchidism and mental retardation, and all showed normal expression of FMRP. From these data we conclude that pointmutations in FMR1 are a rare cause of mental retardation and that a fragile X-like phenotype may be caused by other as yet unknown genes.

2287 2288 Analysis of genetic instabilities of RS447 megasatellite DNA region in human Comparative physical and genetic mapping studies of the murine and 4p15. Y. Gondo12, T. Okada', S. Hadano2, Y. Saitohl, J. GoO3, I. Kanazawa? and human genomes encompassing the Adrenoleukodystrophy gene. S. L. J.-E. Ikedal 2. 'The Inst. of Med. Sci., Tokai Univ., Isehara, Japan, 2JST/Canada J. Hawkes1, S. A. Rowland', C. Morris2, M. A. Kennedy2, D. R. Love1. Int'l Res. Exchange Program 'Neurogenes, 3Sch. of Med., Univ. of Tokyo, Japan. 1School of Biological Sciences, University of Auckland, Private Bag 92109, A novel tandem repetitive sequence, RS447, was identified and cloned from the Auckland, New Zealand; 2Cytogenetics and Molecular Oncology Unit, human 4p15. We designated RS447 as 'megasatellite DNA' based upon its similarities to microsatellite DNA including its head-to-tail tandemly repeated Christchurch School of Medicine, Christchurch, New Zealand. structure and polymorphic nature but the unit size of 4.7 kb (Gondo et al., ASGH The human Adrenoleukodystrophy (ALD) gene is localised to the meeting 1996). The repetitive unit sequence of RS447 megasatellite DNA is 4746 disease gene-rich region of Xq28. Pulsed field gel mapping studies have bp. The unit sequence is almost identical from one copy to the other according to placed the ALD gene less that 200kb proximal to L1CAM and approximately the restriction analysis of human genomic DNA with various restriction enzymes. 600kb distal to DXS15 (Sarde et al., 1994). Extensive comparative physical Within the unit sequence, a large ORF sequence was identified and corresponding mapping of the human Xq28 region and band B of the mouse X cDNA clones are also isolated (Saitoh et al., ASGH meeing 1997). These findings chromosome has demonstrated a high degree of synteny and sequence strongly indicate some biological function(s) of RS447. It was also supported by conservation. the conservation of RS447 sequence in various mammalian genomes by zoo blot' Southern hybridization as well as PCR analysis. In spite of very little divergence of We have concentrated our efforts on the physical mapping of the murine the RS447 unit sequence among the repetitive arrays, the copy numbers of the Adrenoleukodystrophy gene (Aldgh) homologue. Fluorescence in situ tandem repetition are highly polymorphic ranging from 12 to 90 depending each hybridisation studies using an 800kb YAC containing the Aldgh gene has allele so far examined. When the transmission pattern of RS447 alleles was shown it to be non-chimaeric with positive hybridisation to band B of the examined in 6 independent pedigrees, all but 3 cases were stabIly inherited to the mouse X chromosome. Restriction enzyme mapping of this YAC and offspring. In such three cases, the copy number changed only one larger or smaller subsequent hybridisation analysis has localised the Aldgh gene within the than the parental alleles. These occasional copy number changes seem to be the 800kb cloned genomic DNA. PCR amplification of murine loci known to map cause of the polymorphism but the molecular mechanisms remain to be elucidated to band B of the mouse X chromosome, together with mouse backcross because neither slippage nor unequal crossing over would explain only one copy mapping have been undertaken to the more of such tandem A studies, map Aldgh gene change large repeats. linkage analysis between RS447 (4p15.3) precisely on the genetic and physical map of the mouse X chromosome. and 4pter including the HD locus (4p16.1) also indicated higher recombination frequency around this region than the rate expected from the physical These studies have shown that the Aldgh gene is localised close to chromosomal length. Above all, genetic instabilities of the tandem repetitive DXMit12O rather than the more distal location, flanked by Gabra3 and numbers of RS447 megasatellite DNA are inferred and some recombination G6pd, that would be predicted based on synteny with human Xq28. hotspot(s) are also implied at the RS447 chromosomal region.

2289 2290 Two regions of human chromosome 21p share extensive sequence similarity Revealing of the unstable region in human ribosomal intergenic with Y centromere low copy number sequences. C. Holmes1, B. Casey', V. spacer. N.S. Kupriyanova. K.KNetchvolodov, A.P.Ryskov. Institute of Bauman', S. Y. Wang2, C. Van Broeckhovenf, M. Cummings3 and J. Doering'. Gene Biology, Russian Academy of Sciences, Moscow 'Dept. of Biology, Loyola University Chicago, 2Laboratory of Neurogenetics, Several promoter, enhancer and other control elements have been University of Antwerp (UIA), Belgium, 3Dept. of Biological Sciences, University of shown to reside approximately 2kb upstream of the rRNA transcription Illinois at Chicago. initiation sites (tis) in different organisms. In humans, only upstream control The DNA sequences required for human centromere function have not yet been element and spacer terminator have been mapped in this region of the clearly defined. While the tandemly repetitious alphoid sequences may contribute ribosomal intergenic spacer (rIGS). Recently, in the course of to centromere function, a number of studies have indicated comparative that other as yet study of rDNA containing cosmid clones of the human we uncharacterized sequences must also be involved. The human Y centromere chromosome 13 have shown that 2kb upstream region of rRNA genes displays a contains at least 8 low copy number repeat sequences (LCNR) in addition to significant alphoid and satellite IlIl sequences (Cooper et al., 1993). We have mapped these 8 intrachromosomal polymorphism owing to nucleotide substitutions and sequences on chromosome 21 using a panel of somatic cell hybrids that divides microsatellite (mcs) variations. DNA stretch of this region formed by 90bp the short arm/centromere into at least 9 regions. Five of the eight LCNR are found repeats, flanked by the two Alu elements includes a set of different mcs in the region of 21p distal to the ribosomal RNA genes (21p13). All eight of the motifs (Alu-mcs-Alu region). We have shown that discrete mcs motif from LCNR are also found in the centromere region distal to D21Z1. None of these this cluster reveals specificity to the human rIGS and can be used for the sequences are found on the long arm of chromosome 21. The LCNR in the human rDNA polymorphism study. With the use of this specific mcs probe centromere region were further mapped to a previously-constructed 21p YAC we have shown the presence of the 5-methyl cytosine islands in the contig using probe hybridization to EcoRI digests of the YACs. All eight of the LCNR to this and genomic Alu-mcs-Alu region. Using PCR technique and hybridization with mapped contig, while these additional markers confirmed the specific oligonucleotide probes we have shown also that some rDNA original YAC order, the extent of was reduced in some cases. From overlap their monomers in human individuals are avoided of a major part of placement on the contig the LCNR in the chromosome 21 centromere appear to Alu-mcs-Alu have a linear order very similar to that in the Y centromere. However, some of the region suggesting enhanced instability of this part of the rIGS LCNR that are present at single loci in the Y centromere are present at multiple loci in the chromosome 21 centromere. The LCNR sequence similarity between the Y centromere and two regions on chromosome 21 could account for the interaction between these two chromosomes that is apparently involved in some trisomy 21 paternal origin (Griffin et al., 1996). Sequences similar to the LCNR have found the centromeres of acentric marker chromosomes. This, together with the results on chromosome 21, suggest that the LCNR are candidates for sequences involved in centromere function. (Supported by the Down's Syndrome Research Fund). A392 Published Abstracts: Genomics (cont.)

2291 2292 A HUMAN MITOCHONDRIAL DNA STANDARD REFERENCE MATERIAL FOR A model for the origin of a complex repetitive sequence family on QUALITY CONTROL IN MUTATION DETECTION, , AND human acrocentric p-arms. S.M. McCutcheon', M.M. Mullen1, J. FORENSIC IDENTIFICATION. B.C. Levin1, H. Cheng2, and D.J. Reeder1 (Intro. by Doering2, and M.R. Cummings'. 1Department of Biological Rosalie K. Elespuru) 1National Institute of Standards and Technology (NIST), Sciences, University of Illinois at Chicago, Gaithersburg, MD, and 2GEO-CENTERS, Inc., Newton Centre, MA. Chicago, Illinois, 2Department of Biology, Every human cell can have up to several thousand mitochondria, each containing Loyola University, Chicago, Illinois. mitochondrial DNA (mtDNA). The sequence of human mtDNA (16,569 bp)is used by The Acro repeat is a subfamily member of a repetitive sequence family the forensic community for human identification and by the medical community for which includes members on HC13,5,10, and the FSHD-associated D4Z4 diagnoses of diseases associated with specific mutations and deletions. Examination repeats on HC4. Our characterization localized this Acro repeat subfamily to of the mutagenic effects of chemical and physical agents is also being explored. A the p-arm of all acrocentrics as a 2.8-kb repeat interspersed within P- mtDNA standard reference material (SRM)is being prepared by NIST to provide satellite domains. This genomic organization contrasts with the tandemly- quality control to investigators who sequence human The mtDNA SRM will mtDNA. arranged, 3.3-kb D4Z4 repeats on our of include both extracted and cloned DNA and all the information necessary to perform HC4. From sequence comparison the polymerase chain reaction (PCR) amplification process, cycle sequencing steps, the Acro and D4Z4 repeats, we hypothesize that the Acro repeats were gel separation and data analysis to determine the DNA sequence of the two derived from the D4Z4 repeats by a minimum of two recombinational templates (CHR and 9947A) that are provided in this SRM. The DNA from 9947A events. In this model, one event involves an intramolecular recombination of consists of total genomic DNA, whereas that from CHR was extracted by a method to D4Z4 sequences that produces a full-length LSau domain at the 3' end. The enhance the concentration of mtDNA. The SRM also includes cloned DNA from the other recombinational event involves the insertion of this modified D4Z4 HV1 region of the CHR cell line which includes the difficult to sequence C-stretch. repeat into an acrocentric 3-satellite cluster. The insertion event is facilitated The sequences of fitty-eight sets of unique primers are supplied with the SRM to by the sequence present at the distal junction of the D4Z4 allow any area or all of the mtDNA to be amplified and cycle sequenced. to P-satellite Compared repeats on HC4. The insertion event results a the Anderson sequence, the mtDNA from the tissue culture cell lines, CHR and in truncation at the 5' end of 9947a, have 46 and 32 differences, respectively. None of these differences the modified D4Z4 repeat producing an Acro repeat of 2.8-kb with a full- correspond to the published mtDNA mutations that have been associated with length LSau domain at the 3' end. We also investigated the genomic specific disease states. An interlaboratory evaluation of the amplification, sequencing, organization of this repeat family on HC1,3, and 5 and found a genomic and analysis of the data from the CHR template was conducted by four laboratories organization different from HC4 or the acrocentrics. We examined the including NIST to determine any difficulties others might experience. Investigators will genomic organization in several species of higher primates. In all higher be able to purchase this SRM from NIST and use it as a control when they amplify primates studied, both an HC4 and an acrocentric genomic organization and sequence their test samples. Attainment of the correct results with the SRM will were present. Therefore, the recombination events that produced Acro provide quality assurance that the experimental mtDNA is being sequenced correctly, repeats may have occurred before the radiation of higher primates. the forensic identifications are right, and the medical diagnoses are accurate. (Supported by the Down's Syndrome Research Fund).

2293 2294 Searching for novel X-linked genes involved in retinal disease. KE Cloning and localization of novel gene mapped on human McTaggart1, N.J. Nesslingerl, P. W. Wong2, M.A. Walter1, and I.M. chromosome 1q25. S.-H. Park, C.-M. Kim, Y.-J. Chang, l.-J. Rhyu, Y.-H. MacDonald1. Departments of Ophthalmology1 and Biological Sciences2, Chun, H. Kim. Institute of Human Genetics and Department of Anatomy, University of Alberta, Edmonton, Canada. College of Medicine, Korea Univ., Seoul 136-705, Korea. A group of human genomic clones containing human X chromosome Chromosome microdissection technique has become a very powerful sequence expressed in chorioretinal tissue have previously been isolated method to generate chromosome band-specific library and isolate the using a lateral screening approach (Wong et al., 1989 Gene 85: 59-65). In genes localized in a specific chromosome band. We made Uni-amp cDNA this approach, a genomic library constructed from a humra-niX chromosome/ pool from 18 weeks old human fetal brain assuming it expresses many hamster hybrid was cross screened with a human chorioretinal cDNA library genes related with brain development. In situ hybridization was performed to isolate genomic clones from the X chromosome containing genes with these Uni-amp cDNAs on human chromosomes, and chromosome expressed in the choroid and/or retina. Such genes could have a role in X- 1q25 was microdissected. The cDNA fragments bound on this band were linked retinal disease. Expression pattern and chromosome sublocalization amplified by PCR. Unknown cDNA clones were isolated by sequence have previously been determined for one of the clones (R8) (Wong et al., analysis and developmental expression patterns of these new clones were 1993 Genomics 15: 467-471). Exon trapping of a second clone (R43) has investigated with in situ hybridization histochemistry on the various tissues resulted in tne isolation of an exon (R43-8) with homology to exon 20 of the including brain of developing and adult rats. The expression of one novel human P gene, located on human chromosome 15. Mutations in the P gene clone was found only in the early postnatal period and restricted to the may result in type 11 occulocutaneous albinism (OCA2). Preliminary results brain, especially in the entorhinal cortex. From this result, it may be suggest that R43 is located in Xq. Interestingly, X-linked albinism-deafness suggested that this novel gene located on 1q25 may play an important role syndrome (ADFN) has been localized through linkage analysis to Xq26.3- in development of entorhinal cortex. For the further, we isolated human q27.1 (Shiloh et al., 1990 American Joumal of Human Genetics 47: 20-27). genomic DNA encoding this novel clone and confirmed the location on the As a result, clone R43 could contain a canaiaate gene for AuFN. Work is human chromosome 1q25 by fluorescent in situ hybridization. underway with exon R43-8 to determine the precise expression profile as well as precise sublocalization on the X chromosome. The isolation of this gene fragment demonstrates the feasibility of the original lateral screening approach in the isolation of genes with potential roles in retinal disease.

2295 2296 Physical mapping and distance refinement of a marker (D13S232) in A human olfactory receptor gene, recently mutated in pseudogene, complete linkage disequilbrium with Limb Girdle Muscular Dystrophy defines a new olfactory receptor gene family in mammals. Sylvie type 2C (LGMD2C). J.M. Rochelle1, K. Ben-Othmane1, F. Hentatfi, Rouquier, Cecile Delettre, Dominique Giorgi. CRMB, ERS 155 CNRS J.Barker", M.A.Pericak-Vance1, E.M. McNally3 L. Kunkel3 and The olfactory system has the remarkable capacity to discriminate a wide J.M. Vance1. 1 Duke University, Durham, NC Instituteof range of odor molecules. The identification of a large multigene family NeurologyTunisTunisia 3Childrens Hospital, Boston, Mass. encoding odorant receptors that belong to the G-protein coupled receptors gene superfamily (seven-transmembrane-domain has LGMD2C maps to chromosome 1 3q12 and is associated with a molecules) provided new insights in the of the olfaction mechanism. This OR disruption of the gene coding for the dystrophin-associated-glycoprotein understanding gene family contains hundred members the human gamma-sarcoglycan (SG). The complete linkage disequilbrium found dispersed throughout genome. In the course of new members of the OR between a rare allele(122bp) of the marker D13S232 and the 6521-T characterizing gene family, we isolated a OR consensus mutation in exon six helped to as PCR product, generated by degenerate lying lead the identification of SG the domains LGMD2C defect. Therefore, it is of interest to know the distance primers chosen in the coding region between the conserved 2 and 7, that was very from all other known OR This relationships between the 6521 -T mutation and D1 3S232, as linkage divergent sequences. sequence was on chromosome 11 a monochromosomal disequilibrium is a significant tool in fine mapping. We have physically mapped using somatic cell hybrid and used to screen a 5.8 chromosome mapped the eight exons of the 1.4 kb SG transcript over a 100 kb region panel coverage 11-specific cosmid library A single clone was isolated and the complete using a PAC and YAC contig. Using this physical map in the region and sequence was established. This OR all characteristic long PCR, we placed D13S232 in intron 2, lying between exon two and receptor presents three. This places the D13S232 locus 42-60 kb from the 6521-T mutation. features of an OR gene but defines a new receptor gene family in mammals. Surprisingly, the sequence reveals an in-frame codon in On the telomeric side of 6521-T, we identified a new SSCP polymorphism: stop position NH2-ter due to a single non-sens to 537.CAG. This marker showed an ancestral cross-over in 1/11 LGMD2C mutation, probably GAA TAA (Glul stop). Genomic DNA seven unrelated Caucasian individuals families tested and maps in intron seven, 12 kb or less from the mutation. from were sequenced and confirmed that this mutation was not a PCR or sequence-induced error or a polymorphism. Analysis of non-caucasian individuals is on the way. Since this gene did not accumulate more than one ponctual mutation, it is likely that it has been recently mutated. Therefore, given its highly divergent sequence, this new OR sequence constitutes an excellent model for expression studies and ligand analysis. Published Abstracts: Genomics (cont.) A393

2297 2298 Genomic organization and expression of the gene encoding human Detailed physical mapping of the centromere region of human Ral guanine nucleotide dissociation stimulator which maps at the chromosome 21. J. So1, C. Stevens1, 1. Wiese', C. Holmes1, M. centromeric end of the TSC1 candidate region on chromosome 9q34. Cummings2 and J. Doenng'. 1 Dept. of Biology, Loyola University Chicago, M. Smith, K.A. Handa. Dept. of Pediatrics, University of California, Irvine. 2Dept. of Biological Sciences, University of Illinois at Chicago. RaIGDS controls the cycling of Ras and Rap1b related GTPases We are preparing a detailed physical map of the sequences in the centromere between a GTP bound active state and a GDP bound inactive state of chromosome 21 using the hybrid cell line 153E7BX. Our previous mapping (Peterson et al. J.Biol. Chem. 271:29903-29908 1996). We determined that studies indicated that this cell line appears to contain the elements of the human Ral GDS sequence (Genbank HSU14417) is distributed in 29 exons functional centromere, while lacking essentially the entire short arm. DNA from in the chromosome 9q34 cosmid AC00395 (Genbank). In addition the cell line was restricted with a number of enzymes that create large fragments to in the 5' of mouse in the centromere region, subjected to pulsed field electrophoresis and probed. sequence highly homologous sequence region Ralgds Physical linkage between two sequences was established if probes for them cDNA (Genbank MUSG NDSA) is present in cosmids AC00395 and hybridize to the same pulsed field gel fragments made by at least two different AC001643 and is distributed over 6 exons. We probed Northem blots of enzymes. This centromere map is linked to the long arm physical map using the polyA mRNA from adult and fetal tissues with a probe corresponding to the D21S190 marker. The major centromeric alphoid cluster, D21Z1, is no more than central portion of Ralgds cDNA and determined that there are tissue 1.0 Mb long and contains organizational heterogeneity within its q arm end. A specific and temporal specific differences in Ral GDS expression. 3.6kb satellite cluster less than 0.4 Mb long is found within 0.2 Mb of the q arm end of transcripts were identified in a number of tissues. Transcripts of 12 and D21Z1. Still uncharacterized repetitive sequences apparently extend from both 6.0kb were identified in adult heart and brain. In fetal kidney the most ends of D21Z1 for distances of at least 1.0 Mb. A second cluster of alphoid abundant transcript was 2.4kb. The presence of different forms of Ralgds sequences, a21-Il, is located at least 1.6 Mb from the p arm end of D21Z1. This mRNA in different tissues may have implications for the function of this cluster, which is at least 1.0 Mb long, has a complex internal organization. molecule in different tissues. Interspersed within its alphoid sequences are a satellite IlIl cluster and several low copy number sequences (LCNR) also found in the Y centromere. In other studies we have identified these LCNR as candidates for sequences involved in centromere function. Thus, the a21-ll region may be a critical part of the centromere. The length of the overall physical map for the chromosome 21 centromere is nearly 5.0 Mb, which correlates well with estimates of centromere size for other human chromosomes. This pulsed field map will serve as the basis for checking the accuracy of YAC and BAC contig maps being constructed for portions of the centromere region. (Supported by the Down's Syndrome Research Fund).

2299 2300 Myosin VI (MY06): A candidate gene for non-syndromic sensorineural Molecular study of AZF locus by STS-PCR in 126 patients with deafness. T. Sobel, R. T. Taggart', D. A. Vasquez', N. Ahituv1, and K. B. idiopathic infertility. L. Stuppial, V. Gattal, G. Calabresel, P. Guanciali Avraham1. 'Dept. Human Genetics, Sackler School of Medicine, Tel Aviv Franchi1, F Pompetti', E. Morizio1, R. Mingarell2, G. Palka1. Dipartimento University, Tel Aviv, Israel. 2Genetics Division, Dept. of Pediatrics, SUNY di Scienze Biomediche, Sezione di Genetica Medica, Universita "G. Buffalo, Children's Hospital, Buffalo, NY. D'Annunzio", Chieti, Italy'; Ospedale CSS, S. Giovanni Rotondo, Italy2. Deafness is the most common sensory impairment in humans and may In recent years, a number of studies evidenced the presence of Y result from mutations in as many as 100 genes. The extensive genetic chromosome microdeletions detectable by STS-PCR in patients with heterogeneity and small size of deafness families has made identification of idiopathic infertility. We previously described microdeletions of the Y causative genes difficult. The similarity of human and mouse auditory systems chromosome in 6 out of 33 patients with oligo-azoospermia of unknown provides an alternative means to identify candidate deafness genes by origin. In the present study we report data concerning 126 infertile patients circumventing the need for conventional linkage analysis. We previously studied by cytogenetic analysis and STS-PCR of 27 loci mapped within demonstrated that the mouse recessive deafness mutation, Snell's waltzer, interval 6 of the Y chromosome (AZF locus). In 17 patients (16 azoospermic contains a deletion of the unconventional myosin VI gene (Myo6; Avraham et and 1 oligozoospermic) we detected a chromosome abnormality. The al., 1995). The extensive homology between human and mouse genes, and remaining 109 patients with normal karyotype were investigated by STS- the existence of additional unidentified human deafness loci suggest that PCR, and microdeletions within interval 6 of the Y chromosome were myosin VI may also be involved in human deafness. Human myosin VI detected in 15 cases (14%). Microdeletions in is in the human detected azoospermic (MY06) expressed fetal cochlea and maps to chromosome were one et We patients scattered along the entire interval 6, and involved in half 6q13 (Avraham al., 1997). recently isolated the homologous human of the cases the DAZ gene. On the other hand, microdeletions in cDNA clone and now report isolation of the entire MY06 gene in six partially PAC clones. The exon-intron structure of the is oligozoospermic patients were clustered within subinterval E, outside the overlapping MY06 gene being DAZ region, suggesting the presence of a specific locus involved in determined both by long-range PCR and sequencing of PAC subclones. To examine the involvement of MY06 in human deafness, we are performing oligozoospermia. Overall, we detected a genetic cause of the disease in mutation analysis of the conserved functional domains of myosin VI by SSCP, 25% of our patients. These results suggest the usefulness of molecular ddF, REF and a newly developed single strand DNA conformation fragment analysis of AZF locus by STS-PCR in patients with idiopathic infertility length polymorphism (CFLP) method. We are screening deaf individuals for showing diploid karyotype. MY06 mutations in multiplex families and in families from geographical isolates by analysis of RT-PCR products and exon scanning. Identification of MY06 mutations in the human deaf population will aid in understanding the biology of sensorineural hearing loss and the function of the inner ear.

2301 2302 Localization of CAVI, a candidate gene for neural tube defects, to The identification of three additional genes contiguous to the 7q31. K.D. Viles, J.Gilbert, W.Strittmatter, T.M.George, M.C.Speer, and the glucocerebrosidase locus: implications for Gaucher disease. S. L. NTD Collaborative Group. Duke University Medical Center, Durham, NC. Winfield, N. Tayebi, B. M. Martin, E. I. Ginns, and E. Sidransky. Clinical Neural tube defects (NTDs) are among the most common birth defects. Neuroscience Branch, IRP, NIMH, NIH, Bethesda, MD. The relationship between folic acid supplementation and the risk for NTD The human glucocerebrosidase gene locus (GBA) on chromosome 1q21 recurrence has been well established. Recent studies have shown that is complex, with a duplication which gave rise to pseudogenes for both GBA folate receptors, needed for folate internalization, are clustered in caveolae and a divergently transcribed gene, metaxin (MTX). Other genes have also in the plasma membrane of cells (Smart et al 1996, Ritter et al 1995, and been identified downstream to glucocerebrosidase, including Ying et al 1992). Caveolin-1 (CAV1) is a 22kD integral membrane protein thrombospondin 3 (Thbs3) and mucin 1 (MUC1). We now report the and has been identified as the major component of these caveolae sequence and organization of three new genes within the 32 kb upstream to membranes. Thus, caveolin-1 is a candidate gene for NTDs. The published GBA, all of which are transcribed in the same direction as GBA and are cDNA sequence of CAVI contains 838bp with 537bp of this being coding expressed in all tissues studied. Of these three genes, that most distal to sequence. To establish chromosomal localization and begin candidate gene GBA is a previously described protein kinase (clk2). The other two, propini mutation analysis, primers were designed from this cDNA sequence and and cotel, are novel and their function is not yet known. Propini shares are as follows: 5 (coding region) and 5 (noncoding region). Using the some homology with a gene encoding a rat Secretory CArrier Membrane Stanford G3 Radiation Hybrid panel, CAV1 was localized to 7q31, 28.6cR Protein (SCAMP37) and a 1.5 kb cDNA forTropini has beeiTisolated. from D7s2877 with a LOD of 8.2. The two flanking markers are D7s2502 Cotel lies most proximal to GBA and extends 6-14 kb upstream to GBA. A and D7s1764. Further characterization and analysis of the gene is in 1.8 kb partial cDNA has been isolated for cotel, while the full length cDNA process. is predicted to be 3.15 kb. Thus this region on chromosome 1q21 is This work is supported by grants from the NICHD (HD33400), the NINDS particularly gene rich and contains seven functional genes and two (NS26630), and the March of Dimes. pseudogenes. The possible contributions of these closely arrayed genes to the more atypical presentations of Gaucher disease are now under investigation. A394 Published Abstracts: Genomics (cont.)

2303 2304 Construction and Application of Chromosome Band-specific Probe Mapping of human MAD2 gene to chromosome 5q23.3. L Xul, H-X Pools and pUCI9 Libraries. Jia-huiXia, Oing-guo Ruan, LeiXu, Dong- Dengl,2, Y Yang2, JH Xial, WY Hung2 and T Siddique. 1 Nationai sheng Tang, Chun-yu Liu, Han-xiang Deng. National Laboratory of Medical Laboratory of Medical Genetics, Hunan Medical University, Changsha, Genetics of China, Hunan Medical University, Changsha, Hunan 410078, P. China 2Department of Neurology, Northwestem University Medical School, R. China Chicago. IL 32 human chromosome band-specific probe pools have been Human mitotic arrest defective protein 2 (hsMAD2) is required for the constructed by microdissection and PCR: 1q11, 1q12, 1q44, 2p23-pter, execution of spindle assembly checkpoint during mitosis of cells, which 2q13-q22, 2q32-qter, 3p21, 3p21-p23, 3q27-q29, 4p12-p16, 4q35, 6q25- guarantees the proper chromosome segregation under normal growth q27, 7p15, 7p14-pter, 7q32-q33, 7q34-qter, 8q24.1, 11p12, 11p15, 12q12, conditions. Dysfunction of hsMAD2 may lead to malignancy or degeneration of 12q23-q24.1, 12q24.1-qter, 13q12, 14q11.1, 14q24.3, 16p11.2-pter, cells. We are omterested om this gene because of its possible functional 16p11.1-q12.1, 16q11, 19q11.1, Xp11.2, Xp21.1-p21.2, Xpl1.3-pl1.4. importance in degenerative diseases or cancer due or cancer due to the role 27 human chromosome band-specific pUC19 libraries have been that it plays in maintaining the normal function of cell. The hsMAD2 gene has constructed using the chromosome band-specific probe pools: 1 p32, been cloned, but the localization on chromosome remains unknown. To map lp36.3, 1q12, 1q32, 1q42, 2q33, 2q32-qter,3p21-p23,3q27-q29, 4q35, this gene, we synthesized a pair of DNA primiers based on its cDNA sequence 5q12, 5q13, 6p21, 6q25-q27, 7q32-q33, 8q24.1, 11p12, 11q13, 11q23-qter, to amplify specific DNA frahment from human genomic DNA. Two genomic 12q23-q24.1, 13q12, 14q24.3, 15q12, 16p11.2-pter, 16p21-pter,17p13, DNA clones ranging from 16 to 18kb in size were isolated from bacteriophage Xpl1.2. human genomic DNA library using the amplified DNA fragment as a probe. Using the chromosome band-specific probe pools and pUC19 libraries, These two bacteriophage clones have been verified to contain hsMAD2 gene we have constructed chromosome cDNA isolated by DNA sequencing. DNA from one of the bacteriophage clones was used as band-specific libraries, probe for fluorescence in situ hybridization (FISH) on human metaphase chromosome band-specific DNA markers including single copy DNA and chromosomes. The result indicates that hsMAD2 gene is on chromosome segments (CA)n microsatellites DNA, identified the origin of marker It has been verified in vitro that some tumor are in chromosome. 5q23.3. cells defective the mitotic checkpoint. Dysfunction of hsMAD2 may lead to malignancy or degeneration of cells. Interestingly, chromosome 5q23-31 has been defined as a 2critical re9ion2 which is deleted in all nonlymphocytic leukemia and myelodysplastic syndrome patients. HsMAd2, required for mitotice arrest, is now assigned to this important region, suggests that hsMAD2 may be a candidate gene for malignant diseases. Further work has to be carried out for the structure, expression and function of hsMAD2 in tumor cells.

Published Abstracts: Inborn Errors of Metabolism and Biochemical Genetics

2305 2306 Improvement of biochemical testing for carriers of Tay-Sachs disease f(-Glucuronidase deficiency presenting antenatally as a unilateral multicystic- by molecular testing of inconclusive specimens. Y. Ben-Yoseph and dysplastic kidney. D.Day-Salvatore', T.Cimarofi1, C.Benito2, D. Kamali, E. Guzman2, D.A. Mitchell. Wayne State Univer~sity, Detroit, Michigan. A. Vintzileos. Divisions of Clinical Genetics' & Matemal-Fetal Medicine2, UMDNJ- Robert Wood Johnson Medical School & St. Peter's Medical New NJ. Classical Tay-Sachs disease or GM2 gangliosidosis type B is caused by Center, Brunswick, P-Glucuronidase deficiency (MPS Vii) has previously been reported in association with mutations in the gene encoding the a subunit of hexosaminidase A. 24 cases of hydrops fetalis or fetal edema/ascites. We report on a 34-year-old German/ Biochemical testing for carriers of Tay-Sachs disease is routinely performed in Dutch/American Indian/Polish gravida 2 para 1 who presented at 17 weeks gestational the Ashkenazi Jewish population in which the carrier frequency is about 1 in 30. age by dates with an elevated M SAFP of 4.2 MoM. The patient denied consanguinity with The biochemical test is based on the determination of the labile her 32-year-old Polish husband. An amniocentesis had been performed prior to the hexosaminidase A activity that is destroyed by heat inactivation. Some overlap patient's referral which revealed a 46,XX fetal karyotype, normal AFAFP, and negative exists between carrier and non-carrier values and this range is defined as the acetylcholinesterase. A level 11 ultrasound demonstrated an average gestational age of inconclusive range. Since 3 mutations account for more than 97% of the Tay- 19.5 weeks by fetal measurements, a unilateral right multicystic-dysplastic kidney, and normal amniotic fluid index (AFI). A follow-up scan 3 weeks later revealed appropriate Sachs mutations in the Ashkenazi Jewish population, we elected to test interval fetal growth, normal left kidney, and no additional sonographic anomalies. At 24.1 biochemically inconclusive specimens by molecular techniques. We used PCR weeks GA the total AFI measured 33.1cm, indicative of polyhydramnios. Three weeks amplification for identification of the exon 11 insertion mutation (1278 insTATC) later, the following abnormalities were noted on ultrasound in addition to the cystic- by formation of heteroduplex. PCR amplification followed by restriction dysplastic kidney: macroglossia, hepatomegaly (liver span 6.9 cm [normal<4.2 cm]), endonuclease digestion was used for detection of the intron 12 splice mutation moderate ascites, and significant polyhydramnios (AFI=48.9cm). An autopsy performed (1421+1 G-+C), the exon 7 glycine to serine adult GM2 gangliosidosis mutation after an intrauterine fetal demise at 28 weeks gestation confirmed the sonographic findings and was additionally notable for esophageal stenosis at the level of the carina. (G269S; 805 G-+A), and two exon 7 pseudo-deficiency mutations (R247W; Radiographic findings were unremarkable. A lysosomal enzyme panel was performed on 739C-+T and R249W; 745C-+T). About 1.3% of 5,540 specimens tested amniocytes and revealed a marked 0-glucuronidase deficiency; the remainder of the panel previously were found to be in the inconclusive range (73 specimens). Nearly was normal. To our knowledge, this is the first report of MPS VIll presenting antenatally 50% of these specimens (36) were identified as Tay-Sachs carriers by with a unilateral cystic-dysplastic kidney and the first report of esophageal stenosis as an molecular testing (26 insertion, 8 splice and 2 adult mutations) and one was associated finding. Cystic-dysplastic kindeys have been previously described with other identified as pseudo-deficiency. The remaining specimens were either metabolic disorders (e.g. Zellweger, glutaric acidemia Il)and have also been associated homozygous normal or heterozygous for mutations other than those examined. with non-renal malformations including TE fistula. While these findings are suggestive of This that molecular of inconclusive can an expanded phenotype for MPS VII, the possibility of an intercurrent condition can not be study indicate testing specimens excluded. markedly narrow the inconclusive range of the biochemical testing.

2307 2308 Genetic analysis of 6 patients with no detectable Butyrylcholinesterase Autoimmune/lymphoproliferative disease (ALD):is it due to a dominant activity- three novel silent genes and heterogeneities of mutations in BCHE negative effect? I.Dianzanil, M.Bragardo2, D.Di Franco', S.Bonissonp, gene in Japan. DC. Deyi, T. Kanno1, K. Sudo2, M. Maekawa3. 'Department of C.Gambarutol, U.Ramenghil, U.DianzanP. tDept.of Pediatrics and Laboratory Medicine, Hamamatsu University School of Medicine, Hamamatsu, Japan Genetics, Univ.of Torino, Italy; 2Dept.of Medical Sciences-Novara, Univ-of 2Department of Laboratory Medicine, Jikei University School of Medicine, Komae, Japan 3Clinical Laboratory, National Cancer Center Hospital, Tokyo, Japan. Torino, Italy. Several genetic variants of human butyrylcholinesterase (EC 3.1.1.8; Fas (CD95) is a surface molecule that induces programmed cell death (PCD) pseudocholinesterase; BChE) have been shown to cause prolonged apnea to of lymphocytes and plays a role in immune response control. We detected a muscle relaxant drug succinylcholine. On the other hand, the BChE activity has been functional defect of Fas in pediatric patients with autoimmune chronic and and/or (ALD) (U.Dianzani used as a marker of liver dysfunction in Japan. Thus genetic variants of the enzyme splenomegaly were sometimes misdiagnosed as a patient of liver dysfunction. We analyzed some et al. Blood 89, 2871, 1997). Prednisone-induced PCD was normal. cDNA silent variants and also summarize the distribution of BCHE allele in Japan. sequencing of the Fas gene did not identify any causal mutation. This Phenotyping was done by dibucaine and fluoride inhibition and DN, FN was distinguishes ALD from the human autoimmune lymphoproliferative syndrome calculated. Genetic analysis was performed by PCR-SSCP analysis followed by (ALPS), due to Fas gene mutations. Moreover, ALD patients do not show the RFLP and DNA sequencing. peripheral expansion of CD4/CD8 double negative T cells present in the ALPS Among the 6 atients, patients 1 and 2 were homozygous for BCHE'365R (GGA; phenotype. We found that also ceramide-induced PCD was defective in ALD Gly to CGA; Ar mutation. Patient 3 was compound heterozygous for BCHE*365R patients, which suggests a common pathway used by Fas and ceramide. The and BCHE25OP (ACT; Thr to CCT; Pro) mutations. Patient 4 was a compound pediatric onset, the consanguineous parents of one patient and the family history heterozygote for two mutations. One mutation was BCHE*435R (GGA; Gly to AGA; with autoimmunity favour a genetic component of ALD. In the present study we Arg) and another was a point mutation BCHE*365R. Patient 5 was a compound report that both parents of alTthe ALD patients show a decreased Fas function in heterozygote for two mutations. One was BCHE*446V (TTT; Phe to GTT; Val) and vitro, though they have not clinical signs of ALD. ALD may be due to another was a point mutation BCHE*365R. The remaining patient was also hyperexpression of a negative regulator of Fas signaling or to mutated molecules compound heterozygous for two different mutations. One was BCHE*451X (GM; Glu exerting a dominant negative effect on Fas-mediated PC D. To test this hypothesis to TM; STOP) and another was a common point mutation BCHE'3301 (TTA; Leu to we T cell CD4+ T cell lines from two ALD patients are and produced hybridomas by fusing ATA; Ile). BCHE alleles BCHE*435R, BCHE446V and BCHE'451X novel with a continuous T cell line sensitive to Fas-mediated PCD. Hybrids were responsible for silent phenotype of BCHE gene. In case of silent phenotype enzyme cultured in the of ceramide and anti-Fas antibodies to select Fas- zero or almost zero. with and presence activity is Linkage K-polymorphism compound resistant fused cells. were obtained from both patients, whereas no was found for the variants. We also summarize the various Hybridomas heterozygous nature resistant cell line was obtained in using four normal donors. alleles of BCHE those found until now and their distribution in Japan. Heterogeneity of parallel experiments the is a common mutation and These studies suggest: 1) ALD lymphocytes carry a protective factor inhibiting BCHE was observed among subjects. BCHE365R effect in since it found all over Japan followed by BCHE3301. In contrary, some mutations like Fas function; 2) this factor exerts a dominant negative vitro, BCHE^435R, BCHE'446V, BCHE*451X and BCHE465X are very rare, found only in inhibits the normal Fas pathway. one instance until now. Published Abstracts: Inborn Errors of Metabolism and Biochemical Genetics (cont.) A395

2309 2310 Clinical and genetic heterogeneity of succinate dehydrogenase Presenting features in the mucopolysaccharidoses (MPS): A review of deficiency in 5 Japanese patients. Y. Goto', I. Nonaka1, K. Kita2. 12 consecutive cases. L. Kramer, P. Levy, A. Bogdanow, E. Hantman, R 'Department of Ultrastructural Research, National Institute of Neuroscience, Marion. Montefiore Medical Center/Albert Einstein College of Medicine, NCNP, Kodaira, Tokyo, Japan; 2Department of parasitology, The Institute of Bronx, New York. Medical Science, The University of Tokyo, Tokyo, Japan. Although clinically disparate, common features of the MPSs include short Succinate dehydrogenase (SDH) is a key activity of succinate-coenzyme stature, hepatomegaly, coarse facies, cardiac valve dysfunction, deafness Q reductase complex between citric acid cycle and respertory chain and joint contractures. In an attempt to identify the specific reason for enzymes. The complex is composed of flavoprotein (Fp), Iron-sulfur protein referral for genetic evaluation, we reviewed the records of all patients (pts) (Ip) and two membrane anchoring subunits. We experienced 5 Japanese found to have an MPS at our center between January, 1994 and December, patients, in whom two were siblings, with SDH deficiency in muscle 1996. determined by histochemical and biochemical methods. Onset of the During this period, 12 pts were diagnosed. Ten had MPS II, 1 had MPS disease was under 2 years of age in 5 male patients and was 37 years of VI, and 1 had MPS IIIB; all pts were male and ranged in age from 20 mos to age in one female patient. All 6 patients had muscle weakness. Three of 8.5 yrs. On exam, the most common findings were coarse facies (11/12), them had central nervous system symptoms (mental retardation, joint contractures (10/12), hepatomegaly (10/12), macrocephaly (7/12), and nystagmus) and one was died of cardiac failure at 9 months of age. SDH short stature (6/12). Otolaryngologic (ENT) complaints were noted in all 12 activity was histochemically preserved in intramuscular blood vessels in 3 pts and included chronic rhinorrhea (9/12), noisy breathing and/or snoring patients in contrast with muscle cells. Cytochrome c oxidase activity was (8/12), and chronic otitis media with hearing loss (7/12). Developmental also decreased mildly in 1 and markedly in 2 patients. These findings delay was documented in 10 of the 12 pts. strongly suggest genetic heterogeneity of the disease. Major mitochondrial Interestingly, only 2 pts were referred by their primary care providers. Of DNA mutations were not found. Molecular analysis of Fp and lp subunits is the remaining 10, 7 were referred by ENTs (in none of these cases was the under way. reason for referral listed as 'rule out MPS"), 2 by developmental pediatricians, and 1 by a radiologist (due to the serendipitous finding of dysostosis multiplex on a chest X-ray). Although this series is small, these data suggest that efforts to educate ENTs about the symptoms and signs of MPS is an important key to early diagnosis.

2311 2312 A novel frameshift mutation in glucose-6-phosphatase gene causing Muscle weakness and peripheral neuropathy in a pre-pubescent girl Von Gierke disease in Chinese. C.W.Lamt, WM. But2, K.W Choy', C.C. with glycogen storage disease type Ill. P. A. Levy. Department of Shek3, C.P. Pang', N.M. Hjelm'. 'Department of Chemical Pathology, The Pediatrics, Montefiore Medical Center, the University Hospital for the Albert Chinese University of Hong Kong, Prince of Wales Hospital, Hong Kong; Einstein College of Medicine, Bronx, New York. 2Department of Pediatrics, Queen Elizabeth Hospital, Hong Traditionally, patients with GSD Ill have mild lactic acidosis, mild Kong;3Department of Pathology, Queen Elizabeth Hospital, Hong Kong. , and marked hepatosplenomegaly during childhood, which Glycogen storage disease type 1a (GSD1a) is an autosomal recessive disease improves after puberty. Generally, the hypoglycemia is much less severe caused by deficiency of glucose-6-phosphatase (G6Pase). It is characterised by than glycogen storage disease type I and requires less rigid dietary hepatomegaly, hypoglycemia, hyperlipidemia and lactic acidosis. The gene has management. A subgroup of GSD Ill have muscle weakness which is been cloned and sequenced and various mutations have been found. We report a reported as being minimal in childhood but more severe by the third or patient with most of the manifestations of GSD1a. The male patient was the fourth decades. second child of healthy unrelated Chinese parents. He was seen at about two We present an 11 year old girl diagnosed with glycogen storage disease years of age for hepatomegaly. He had been asymptomatic, except that the type Ill (GSD ll) at 14 months of age, who presents with sudden onset of parents noted that he had progressive abdominal distention since birth. He had a stocking glove sensory loss, and increased muscle weakness. Our patient doll's face and his liver was enlarged to 7 cm below the right costal margin. has been followed at multiple centers. She has been poorly compliant with Further evaluation showed fasting hypoglycemia, lactic acidosis, hyperlipidemia dietary intervention. Attempts at using uncooked cornstarch at a previous and hyperuricemia. GSD1a was suspected, but the parents refused a liver biopsy to confirm the clinical in this DNA-based center failed because the patient found the starch unpalatable. Surprisingly, diagnosis. Therefore, situation, her growth has been good. At 11 years of age, her was 146 cm techniques were the only tools available for a definitive diagnosis of this condition. height (50- We have PCR-amplified all the 5 exons and the flanking sequences of the 75th percentile), and weight was 35.5 kg (25-50th percentile). G6Pase gene. The PCR products were directly sequenced by the Hepatosplenomegaly was pronounced, with liver palpable 8 cm below the dideoxynucleotide termination method. The sequencing result showed that the right costal margin and spleen palpable 6 cm below the left costal margin. patient possessed of 2 G6Pase mutations. One mutation was the G727T She had mild elevations of her liver transaminases, as well as CPK. Our mutation, which has previously been reported in Japanese patients. The second patient had strking muscle weakness of her legs which permitted her to is a novel mutation involving a deletion of a single base, guanine (G) at position walk only with assistance. Nerve conduction studies showed slowed 339. The mutation alters the reading frame and creates a stop codon, TM, 15 conduction. A sural nerve biopsy showed marked storage of glycogen. codons downstream, resulting in a truncated, possibly inactive, protein product. The father of the subject was found to be heterozygous for the G727T mutation, and the mother was confirmed to be heterozygous for the 339delG mutation. To our knowledge, the G727T mutation has not been reported outside Japan, and the frameshift mutation has not been reported in the literature previously.

2313 2314 The significance of abnormal glycolipids and myelin proteins in the Identification of a myophosphorylase gene mutation in a family with four pathology of X-linked adrenoleukodystrophy. M. C. McGuinnessl"2, S. Itonorp, generations of McArdle's disease. B. D. Papendick', E. C. F. Villacal, R. E. R. L. Schnaar3"4, K. D. Smith"5. 'Kennedy Krieger Institute and Departments of Hillman2, L. E. Spollen3, S. H. Horowitz', and C. H. Wang'14. Departments of 2Neurology, 3Pharmacology, 4Neuroscience and 5Pediatrics, Johns Hopkins Psychiatry and Neurology1, Child Health2, Pathology3, and Biochemistry4, University School of Medicine, Baltimore, MD. University of Missouri, Columbia, M.O. X-linked adrenoleukodystrophy (XALD) is associated with elevated very long McArdles disease is an autosomal recessive metabolic myopathy characterized chain fatty acid (VLCFA) levels and manifests with two major clinical phenotypes, a by exercise intolerance, muscle cramps, and myoglobinuria. The biochemical severe childhood cerebral form (CCER) and a milder adult form, defects of this disease is a deficiency of myophosphorylase, a muscle specific adrenomyeloneuropathy, (AMN). CCER and AMN differ in (1) the age of disease glycogen phosphorylase. The myophosphorylase gene is located on chromosome onset, (2) the duration from age of onset to death and (3) the presence of a 1 1q13 with a genomic span of about 14 Kb and a coding region of 2.5 Kb. Several cerebral white matter inflammatory demyelinating reaction in CCER which is mild homozygous and compound heterozygous mutations have been reported or absent in AMN. We have been investigating immunologic parameters that could suggesting a recessive form of genetic transmission. We have identified a family in contribute to the cerebral inflammatory reaction in CCER. which four generations of females exhibit similar clinical symptoms of McArdle's Elevated levels of VLCFA have been reported in glycolipids in CCER brain disease. The patients in the second and the third generations have muscle biopsy tissue, and glycolipids are thought to play a pivotal role in lymphocyte apoptosis, proven glycogen storage and myophosphorylase deficiency. Mutation screens on cytoxicity and immunosuppression. We analyzed glycolipids in normal appearing three of the patients (the second through the fourth generations) identified a white matter from CCER patients and controls, and antibodies to these moieties in previously reported nonsense mutation (R49X) at codon 49 in exon 1 of serum from CCER and AMN patients and controls, to determine whether or not myophosphorylase gene. This nonsense mutation converts an arginine (CGA) to a these molecules be might involved in triggering the CCER inflammatory reaction. stop codon (TGA). The C to T transition results in a new Nla Ill restriction site Glycolipid extractions followed by HPTLC analyses indicate a decrease in which allows rapid identification of the mutant allele by combing PCR-restriction expression of neutral glycolipids (primarily galactocerebroside) and sulfatides and enzyme digest and gel-electrophoresis. The three affected individuals are all an increase in expression of gangliosides in normal appearing white matter heterozygous for the R49X mutation. We also screened for two other less common from samples CCER brain. mutations, G204S on exon 5 and 77A to C substitution on exon 1, but found no demyelinating diseases cell-mediated immune-reactions against myelin mutant allele in these loci. Mutation screening of the entire myophosphorylase anti' ns are thought to contribute to the disease process. Therefore, we used SDS- gene coding region using RT-PCR and direct sequencing is currently in progress. PAGE analysis of normal appearing white matter from CCER patients and controls, Families affected with McArdle's disease in multiple generations are rare and were blot analysis using serum from CCER and AMN patients and controls, thought to be the examples of manifesting heterozygotes or compound study potential myelin antigens and antibodies to these moieties in CCER. heterozygous mutations of the myophosphorylase gene. However, neither Identification of immunologic differences between CCER and AMN is important explanation is plausible in the current family. It is possible that a dominantly determining the initial steps in inflammatory demyelination in XALD and in transmitted genetic defect other than the myophosphorylase deficiency could co- providing insight for development of possible treatment for the more severe CCER segregate with the R49X mutation in this family. Alternatively, a dominantly phenotype. transmitted myophosphorylase gene defect should be considered. A396 Published Abstracts: Inborn Errors of Metabolism and Biochemical Genetics (cont.)

2315 2316 Niemann-Plck Disease Type C: Phenotypic variability often leads to delay in A CRITICAL EVALUATION OF COPPER METABOLISM IN INDIAN diagnosis. C. Prasad1, C. Pushpanathanr, R. Morris3, A. J. Davis3 and F. E. WILSON'S CHILDREN WITH SPECIAL REFERENCE TO THEIR Dougherty. Division of Genetics1, Pathology2 and Pediatrics3, Janeway Child Health PHENOTYPES AND RELATIVES. R. PRASAD, G.KAUR AND B.N.S. WALIA. Center, St. John's, Newfoundland, Canada, and the Division of Genetics and DEPARTMENT OF BIOCHEMISTRY AND PAEDIATRICS* PGIMER, Metabolism4 Children's Hospital, Boston, MA. CHANDIGARH, INDIA Niemann-Pick Disease Type C (NP-C) a lipidosis, is caused by a unique biochemical defect in cholesterol esterification. The protean manifestations of this WILSON'S DISEASE IS AN AUTOSOMAL RECESSIVE DISORDER OF condition often cause diagnostic confusion in the early stages. We present 3 cases COPPER ACCUMULATION LEADING TO LIVER, KIDNEY AND/OR BRAIN highlighting such phenotypic variations, and distinctive pathological findings of this DAMAGE. SERUM COPPER AND CERULOPLASMIN IN CONTROL disorder. SUBJECTS (141 CASES OF DIFFERENT TYPES OF LIVER CIRRHOSIS ) A 2 year and 9 month old boy presented with neonatal hepatitis, WERE SIGNIFICANTLY HIGHER AS COMPARED TO WILSON'S DISEASE hepatosplenomegaly~AnJ: and developmental delay. Initial investigations failed to (51) AND THEIR RELATIVES (58) WHILE MARKED HYPERCUPRIURIA establish a cause. A repeat study of the bone marrow showed foamy histiocytes, (145+/-7 ug/24Hrs.)WAS OBSERVED IN WILSON'S CHILDREN ONLY. providing a diagnostic clue. THERE WAS A GOOD CORRELATION (R=0.92) OBSERVED BETWEEN Case 2: A 14 year old boy presented with chronic megaloblastic anemia, COPPER NOT BOUND TO CERULOPLASMIN AND URINARY COPPER hepatosplenomegaly and short stature. There were no neurological symptoms. EXCRETION IN WILSON'S PATIENTS. INTERESTINGLY, 24 HOUR Electron microscopic examination of muscle tissue showed complex lipid storage and URINARY EXCRETION OF COPPER AND C-AMP WERE SIGNIFICANTLY cholesterol crystals in cytosol. IN WILSON'S CHILDREN ASSOCIATED WITH RENAL 3aL.a: A female infant born at 38 weeks gestation developed neonatal (P<0.01) ELEVATED hep)atni. At 4 months of age she developed respiratory failure requiring aggressive TABULAR ACIDOSIS AS COMPARED TO PATIENTS WITH ventilation unili 2 year and 3 months. She had developmental delay, generalized HEPATOLOGICAL AND/OR NEUROLOGICAL MANIFESTATIONS. 20% hypotonia and weakness. A muscle, skin and nerve biopsy showed lamellar CASES OF WILSON'S DISEASE AND 25% OF RELATIVES OF WILSON'S inclusions suggestive of NP-C. DISEASE HAD SERUM CERULOPLASMIN BETWEEN 14-20 mg./100ml. In each of the three cases the definitive diagnosis was established by THEREFORE, 13 CASES OF WILSON'S WERE CONFIRMED BY demonstration of impaired cholesterol esterification in skin fibroblasts. In conclusion, MEASURING HEPATIC COPPER (90+/-8ug/g WET TISSUE: MEAN +/- SD). these cases illustrate the diverse, but common presentations of a rare disorder. DURING THE FAMILY SCREENING BY SERUM COPPER, Pulmonary manifestations (as in case 3) are rarely described in classical NP-C, but CERULOPLASMIN AND URINARY COPPER AND HEPATIC COPPER, 10 have been reported in NP-C 2. Unless specifically sought for, a delay in diagnosis is SIBLINGS WERE DIAGNOSED TO HAVE PRESYMPTOMATIC WILSON'S not uncommon. Skin biopsy is an effective screening tool, while demonstration of DISEASE. THESE SUBJECTS WERE THEN STARTED THE D- defective cholesterol esterification remains the gold standard for diagnosis. With PENICILLAMINE THERAPY, BECAUSE PRESYMPTOMATIC TREATMENT limited treatment options, establishing an early diagnosis is invaluable. PREVENTS PROGRESSION OF THE DISEASE AND ITS COMPLICATIONS.

2317 2318 Neonatal Hemochromatosis. G. Serra, W Bonacci, C. Bellini. Servizio di Identification of two novel polymorphisms in the glucocerebrosidase Patologia Neonatale, Universita' di Genova, Italia. gene region. E. Sidransky1, E. K. Lau1, S. Winfield1, B. K. Stubblefield1, A. The female infant was the product of an uneventful 36-week pregnancy. Zimrar, N. Tayebi1, and E. 1. Ginns'. 1Clinical Neuroscience Branch, IRP, Parents were non-consanguineous and healthy. At birth the child was NIMH, NIH, Bethesda, MD; 2Shaare Zedek Medical Center, Jerusalem, jaundiced and had hepatomegaly with ascites. Laboratory studies revealed Israel. the following: total bilirubin 14.5 mgldl (direct 0.5), albumin 1.7 mg/dl, Gaucher disease, an inherited glycolipid storage disorder, is caused by a prothrombin time 30', Factor 11 16%, V 22%, Vii 16%, X 22%, fibrinogen deficiency of the catabolic enzyme glucocerebrosidase. The gene for 114 mg/dl; ammoniemia was normal. Serum ferritin concentration was glucocerebrosidase is located on chromosome 1q21 and has a highly 1,915 mg/mi. Urinary succinylacetone was absent. Alphal-antitrypsin homologous pseudogene situated 16kb downstream. We now report two deficiency was excluded. All viral and serologic studies and cultures were novel polymorphisms in the glucocerebrosidase gene region: the first one negative. The patient's condition progressively deteriorated and despite consists of a tetranucleotide (AAAT) repeat upstream to the intensive management the child died on day 21 of life of diffuse uncontrolled glucocerebrosidase gene, and the second is a series of a dinucleotide (CT) cutaneous and mucous bleeding. Post mortem evaluaton revealed repeat in the intergenic region between the glucocerebrosidase gene and its significant iron deposits in the liver as well as in other main organs; pseudogene. These two polymorphisms, along with the previously reported extensive loss of parenchyma was evident; residual hepatocytes showed Pvu II polymorphism in intron 6 of the glucocerebrosidase gene, were iron overload; giant cell transformation was also found. The pathologic analyzed in Gaucher patients (n=106) and two control populations picture was compatible with the diagnosis of neonatal hemochromatosis (Askenazi n=72 and non-Jewish n=46). Strong linkage disequilibrium was (NH). NH (OMIM 231100) is an uncommon polyvisceral iron storage found between the common N370S mutation and particular haplotypes, but disorder of prenatal onset. It is a phenotypically defined disease and it is no significant linkage disequilibrium was found in patients carrying the believed that various insults during fetal life may result in the NH phenotype. L444P or 84GG mutations. We also found exceptions to previous reports NH is determined on the basis of a specific pathological diagnosis. Its thatthe N370S/84GO genotype is always associated with a Pvl.1-/Pvl.1+ genetic or environmental bases are still unknown. NH is usually considered genotype. Several unusual cases of patients with unexpected haplotypes an autosomal recessive disorder. Parents and sibs of patients with NH are led to the recognition of novel complex alleles, and contribute to our not necessarily at increased risk of iron storage disease. NH is not understanding of the origin of glucocerebrosidase mutations. The study of genetically related to hereditary hemochromatosis. these markers may reveal possible ancestral chromosomes which led to affected alleles, and that may be diagnostically useful in Gaucher patients when the specific mutations have not been identified.

2319 2320 Maternal hyperhomocysteinemia and occurrence of orofacial clefts in Spectrum of Mutations in 21-hydroxylase deficient form of Congenital offspring. R.P.M.Steegers-Theunissenl 2, W.Y. Wongl, A.Kuijpers- adrenal hyperplasia in Singapore. Agnes TaqA, Kah-Yin Loke2, Larry Jagtman , P.H.M.Spauwen4, B.C.J.HameI6, H.J.Blom6, C.M.G.Thomas7, Poh2. 1Institute of Molecular and Cell Biology, Dept of Paediatrics,National T.K.A.B.Eskes1. Department of Obstetrics/Gynaecology1, Epidemiology2, University of Singapore Orthodontics and Dentistry3, Plastic and Reconstructive Surgery4, Clinical Congenital adrenal hyperplasia is due to a deficiency in cytochrome Genetics5, Laboratory of Pediatrics and Neurology6, Laboratory of P450 enzymes, the most common of which is 21 -hydroxylase. This enzyme St Radboud, is encoded by the CYP21 gene on chromosome 6p. Our aim was primarily Endocrinology and Reproduction7, University Hospital to determine the spectrum of genetic abnormalites responsible for this Nijmegen, The Netherlands.(Intro.by M.M.Tolarova). disease; such analysis has not been previously reported in South-East Asia. Orofacial cleft (OFC), i.e. cleft lip with or without cleft palate, is a In addition, we hope to develop rapid screening assays for mutations classical example of a multifactorial disorder. Evidence has been common in our local population. accumulated over the past decade showing that majority of OFC results Fourteen unrelated patients from the Endocrine outpatient clinic were from an interaction between environmental factors, including nutritional studied with a view to characterising the specific mutations. DNA was deficiency or toxicity, and genetic factors. Results from case-control and extracted from peripheral leukocytes. The CYP21 gene amplified from intervention studies suggest that periconceptional vitamin supplementation, genomic DNA using the polymerase chain reaction and the products of including folic acid, reduces the recurrence risk of OFC. However, the amplification sequenced. Sequencing of six exons and one intron where fundamental biological processes that underly the preventive action of folic mutations have previously been described revealed mutations in 6 out of acid supplementation are as yet unknown. Folate and the vitamin B12 and the 14 individuals. These included: intron 2 splice site mutation (3 patients), B6 are involved in the metabolism of homocysteine. 8-bp deletion in exon 3 (1 patient), 1172N missense mutation in exon 4 (1 In order to invesigate the folate-dependent homocysteine metabolism, a patient), and Q318X nonsense mutation in exon 8 (1 patient). For the intron standardized methionine loading test was carried out in 29 mothers of a 2 mutation, allele-specific oligonucleotide hybridization proved to be a child with OFC and 56 control women. reliable and rapid screening technique. Surprisingly, in 8 mothers of a OFC child and 2 controls - in the absence Sequencing of the remaining exons is ongoing and we hope to infer of liver and kidney dysfunction - hyperhomocysteinemia was established. In genotype-phenotype correlations when we have catalogued the mutations general, the folate, vitamin B12 and B6 levels were within the normal ranges. in all the affected patients. Therefore, this preliminary finding suggests a disorder in the enzymes involved in remethylation of homocysteine or in the metabolism of folate and/ or vitamin B12. Published Abstracts: Inborn Errors of Metabolism and Biochemical Genetics (cont.) A397

2321 2322 Classical galactosemia in premature monozygotic twins. C Theda, E Identification of mutations in Korean patients with atypical PKU Garcia Soto, L McHargue, G Alpan. Johns Hopkins Bayview Medical Center and caused by 6-pyruvoyl-tetrahydropterin synthase deficiency. H-W. Yoo, Johns Hopkins University, School of Medicine, Baltimore, Maryland. G-H. Kim. Department of Pediatrics, Asan Medical Center, Ulsan University Classic galactosemia is an autosomal recessive disorder caused by a defect in galactose-1-phosphate uridyltransferase (GALT). Clinical features include failure to College of Medicine thrive, hyperbilirubinemia and liver disease. Despite treatment long term outcome is Atypical PKU is caused by a deficiency of tetrahydrobiopterin (BH4), the poor in regards to mental development. obligatory cofactor for phenylalanine hydroxylase. The most common form We present the cases of a set of monozygotic twins with intrauterine growth of this cofactor deficiency is due to lack of 6-pyruvoyl-tetrahydropterin retardation who were delivered prematurely at 33 weeks gestational age. Both twins synthase (PTPS) activity, the second enzyme in the biosynthetic pathway were clinically well at birth with growth parameters at the 10th percentile. Feedings for BH4 metabolism. This study was undertaken to identify molecular with a premature formula containing lactose were started on the first day of life. defects in four unrelated Korean patients with PTPS deficiency who were Despite the lack of hemolytic disorder significant hyperbilirubinemia had developed in diagnosed on the basis of hype rphenylalaninemia concomitant with lowered both twins at less than 48 hours. One twin deteriorated on the fifth day of life when she developed bloody stools and gastric aspirates. Both twins consequently level of urinary biopterin and PTPS activity in red blood cells. Mutation developed severe coagulopathy that was found to be due to liver dysfunction. analysis was performed on the patients' cultured primary skin fibroblasts. Laboratory evaluation showed multiple abnormalities such as elevated Direct cDNA sequence analysis using reverse transcriptase-PCR transaminases, decreased levels of coagulation factors 7 and 9, and increase of technology revealed four different missense mutations; these nucleotide plasma tyrosine. Reducing substances in urine were 0.25 percent. Succinylacetone alterations were an A to G transition at nt.155 replacing an asparagine by a negative. The neonatal screening test revealed elevated levels of galactose and serine in codon 52 of the PTPS gene (N52S), a C to T transition at nt.259 absent activity of GALT. The parents are of Caucasian background, deny substituting a serine for a proline in codon 87 (P87S), a G to A transition at consanguinity and were found to have heterozygote levels of GALT activity. The molecular defect of the GALT has not been characterized yet. Despite the early and nt.286 substituting an asparagine for an aspartic acid (D96N), and a C to T severe manifestation of galactosemia neither of the twins developed cataracts. At 15 transition at nt.317 replacing a threonine by a methionine in codon 106 months both patients show evidence of developmental delay. (T106M). Three out of four patients were compound heterozygotes for these We report this case of classical galactosemia in a set of monozygotic premature mutations except a homozygote for the mutation T106M. The allele twins who presented with strikingly similar clinical manifestations. It also appears of frequency of these mutations in 4 unrelated Korean patients (8 alleles) were importance to the clinical geneticist to recognize that several advances in determined to be 3/8 for T106M, 2/8 for N52S & D96N, and 1/8 for P87S. neonatology have restlted in changes of neonatal practice (such as earlier enteral The results indicate that molecular defects of Korean patients with PTPS feedings) that can have direct impelpt on the time of manifestation of inborn errors of deficient PKU are genetically heterogeneous even though the incidence of metabolism. The overlap of the clinical features between metabolic disorders and this disease is very rare. other conditions frequently associated with prematurity may further complicate diagnosis.

Published Abstracts: Linkage Mapping and Polymorphisms

2323 2324 Autosomal recessive hypodontla: Exclusion of candidate genes MSX1, MSX2 Analysis for linkage to Bipolar Affective Disorder In select chromosomal and EGF. W. Ahmadl, H.M.Lam1, M. Faiyaz ul Haque3, M. Ahmad3, M. Haider2, A. regions: 4; 18; and Xq24-28. VM Aita 1, JA Knowles 2.3, J Terwilliger2 3, Z Wang Ahmad Maimon3, S. ul Haque3, and A.M. Chnstiano 1. 1 Dept. of Dermatology, 2,, JLiu 2J3IEndicott 2, JE Loth 2, B Lerer4, JR Alexander4, A Grunn2, G Columbia Univ., New York, NY; 2Dept. of Biological Sciences, Quaid-i-Azam Univ., Penchaszadeh 2,3, M Baron 2.3, and TC Gilliam 1, 2,3. 1Departments of Genetics Islamabad, Pakistan; 3Karachi Medical and Dental College and Abbasi Shaheed and Development, 2Psychiatry, and 3Columbia Genome Center; College of Hospital, Nazimabad, Karachi, Pakistan Physicians and Surgeons at Columbia University and New York State Psychiatric Hypodontia involves congenital absence of one or more teeth, and may involve Institute, NYC, NY, Hadassah-Hebrew University Medical Center, Jerusalem, Israel. deciduous and/or permanent dentition. We studied an inbred kindred from Pakistan Family, twin and adoption studies support a strong role for genetic factors in the with hypodontia, along with various associted dental anomalies, being transmitted as etiology of bipolar affective disorder. Relative risk to a first degree relative of an an autosomal recessive trait in five generations with 13 males and 7 females affected proband is estimated between 17-20, and heritability is estimated between showing hypodontia. Dental examination of the affected members revealed the 50-60%. Like most complex genetic traits, the disorder is frequent (0.5-1.0% lifetime presence hypodontia associated with various dental anomalies such as enamel prevalence) with a likely multifactorial etiology. Genetic analysis is complex since hypoplasia and hypocalcification, malformation, malpositioning, failure of eruption of contributing loci are likely to be heterogeneous, and to have small individual effect teeth, early carious lesions and attrition of erupted teeth and dentinogenesis sizes which may be further complicated by epistatic interactions. Limited by such imperfecta, leading prematurely to the edentulous state. We have begun linkage complexities, it has been hard to discriminate false positive findings from evidence of analysis using a modified homozygosity mapping approach and several candidate true linkage. This study incorporates one of the largest and best characterized genes shown to have critical roles in tooth development. MSX1 and MSX2 are bipolar disorder samples described to date for evaluation of previous bipolar linkage closely related homeobox-containing transcnption factors thought to be vital for tooth findings. We have collected 57 large families (12-71 relatives) from the US and development based on their expression paitems in tooth morphogenesis. We used Israel, each with a high density of bipolar disorder. Cell lines are established from microsatellite markers within the MSX1 and MSX2 genes located at 4p16 and 5q34- over 1300 individuals, 490 of whom (including 401 sib pairs) meet criteria for a 35 to demonstrate linkage exclusion in this family. Further, epidermal growth factor conservative disease definition. Using semi-automated flourescent genotyping we has been thought to play a role in tooth development in mice, and the EGF gene are testing candidate regions for linkage to bipolar disorder. Chromosomes 4p, 18 located at 4q25-27 was also excluded from linkage to hypodontia in this family using and Xq24-28 have been reported to co-segregate with bipolar disorder in an intragenic tetranucleotide repeat polymorphism. A genome-wide search for independent studies. In the initial analysis, we are analyzing the 384 most linkage at 10cM intervals is now underway in this family, to be followed by positional informative individuals in our series with a of pedigree dense array of polymorphic cloning the causative gene. Considenng the extent and severity of dental markers spanning each of these regions. The data is being analyzed by a variety of anomalies in this family, and autosomal recessive nature of the trait, it appears to be statistical methods. a distinctive case of its type.

2325 2326 An XLMR syndrome with short stature, small hands and feet, seizures, No linkage between systolic blood pressure and chromosome 8 in cleft palate, and glaucoma is linked to Xq28. K.B. Armfieldl, CE. Mexican Americans. LD Atwood1, CM Kammere,2, JE Hixsorn. 1 University of Schwartz2, H.A. Lubs3, W.L. Krause1, R. Nelson2, B. HIane 2, R.J. Schroe2, Minnesota, Minneapolis, 2Southwest Foundation for Biomedical Research, San F Arena3, RE. Stevensor?. 'Dept of Genetics, Scottish Rite Children's Antonio. Medical Center, Atlanta, GA 2JC Self Research Institute, Greenwood A recent study of diabetic Taiwanese families showed linkage of systolic Genetics Center, Greenwood, SC 3Div of Genetics, of Miami blood pressure (SBP) to a region on chromosome 8 near the lipoprotein lipase University locus. The locus School of FL. (LPL) putative accounted for a large (52-73%) proportion of Medicine, Miami, the inter-individual variation in SBP. Usipg data on 46 extended Mexican Six males in 3 generations were identified with severe mental American families in the San Antonio Family Heart Study we tested for SBP retardation, short stature, small hands and feet, relative macrocephaly (3/6), linkage to twelve chromosome 8 markers, two of which are at the LPL locus cleft palate (2/6), adult onset glaucoma (3/6), and seizures. Affected males itself. The analysis was performed with SIBPAL (version 2.9). experienced global developmental delays and ultimately had severe mental blood measures were for and retardation. All had the Systolic pressure adjusted sex, age, age2, onset of generalized seizures in infancy. Carrier body mass index using multiple regression. Individuals on high blood pressure females have no abnormalities. The entity maps to Xq28 with a lod score of medication and women taking oral contraceptives or hormones were excluded. 2.11 (0=0.00) for the fully informative markers p39, DXS8061 and Outliers (beyond three standard deviations) were also removed. DXS8103. This localization adds to the large number (over 100) of mental Results for each marker, with heterozygosity (H), effective degrees of retardation syndromes mapped to the X chromosome. Although there freedom from SIBPAL (DF), and p-value shown in parenthesis, were: Beta 3 appears to be a cluster of these disorders mapping to the gene-rich Xq28 adrenergic receptor (H=.336, DF=423, p=.756), D8S1100 (H=.636, DF=175, region, none shows the clinical findings noted in this family. However, since p=.352), D8S1110 (H=.806, DF=180, p=.869), D8S1119 (H=.771, DF=181, most of the genes in this region that cause XLMR have not been isolated, it p=.802), D8Sl128 (H=.828, DF=150, p=.180), D8S1179 (H=.862, DF=179, remains possible that some of these XLMR disorders may prove to be p=.575), D8S136 (H=.815, DF=170, p=.236), D8S1990 (H=.564, DF=175, alleic. p=.425), D8S592 (H=.746, DF=186, p=.663), FGFR1 (H=.646, DF=243, p=.243), LPL 3' marker (H=.793, DF=276, p=.671), and LPL Hind3 polymorphism (H=.351, DF=289, p=.670). To more closely replicate the Taiwanese study we repeated the analysis on non-diabetic offspring of diabetic parents. Again, no significant linkage was found. Thus, we find no evidence for linkage between systolic blood pressure and chromosome 8 in Mexican Americans. Supported by NIH HL54707 and HL45522. A398 Published Abstracts: Linkage Mapping and Polymorphisms (cont.)

2327 2328 Hereditary Multiple Exostoses (HME)in a Dutch pedigree not linked to guopioid receptor (OPRM1) variants: lack of association with alcohol the loci EXTI, EXT2, EXT3: shows sugesstive linkage for the EXT4 and drug dependence. AW Bergen1, J Kokoszka1, R Peterson', JC Long1, gene on chromosome 17q. E. Bakker M. Vilenus1, A.J. van der Linder?, M Linnoila2 and D Goldman1. 1Laboratory of Neurogenetics and I A. Cabrali, W v HuP, A v Haeringen1 Dept of Human Genetics and Dept 2Laboratory of Clinical Studies, National Institute on Alcohol Abuse and of Clinical Genetics, Leiden University Medical Centre, 2Dept of Alcoholism, National Institutes of Health, Bethesda, MD 20892 Orthopedics, University of Maastricht, The Netherlands, 3Dept of Medical The 1t opioid receptor is implicated in the reward, tolerance and withdrawal Genetics, University van Antwerp, Belgium. effects of alcohol and other drugs of abuse. This hypothesis is supported by the of and naitrexone on alcohol Hereditary multiple exostoses (HME) is a skeletal disorder which effects of alcohol on P-endorphin release, morphine primarily affects enchondral bone during growth. More than 80% of the consumption, and by the activation of the dopaminergic reward system by both alcohol and In has the murine g patients are diagnosed in the first decade of life. The diagnosis can be opiates. addition, linkage analysis implicated of opioid receptor locus, Opnn, as the major quantitative trait locus affecting the established at birth when a specific search is made. The mean number different levels of morphine consumption between two inbred mouse strains that exostoses in a patient is 15-18 but may rise to 80. So far three loci involved also exhibit differences in alcohol and cocaine consumption. in HME are reported, EXT1, EXT2 and EXT3 respectively located on We directly sequenced the human opioid receptor locus, OPRM1, to detect 8q24. 1, 11 p13 and 19q. Exostoses may degenerate in a malignant form natural variation that might affect g opioid receptor function and/or be associated (chondrosarcomas). DNA studies performed on tumormatenial shows loss of with psychiatric phenotypes related to opioid function. Four DNA sequence heterozygosity for each of these three loci, indicating that the EXT-genes variants were found: three non-synonymous substitutions (Ala6Val [rare], are tumorsuppressor genes. In a large Dutch pedigree, with 13 affected Asn40Asp [0.09-0.17], Ser147Cys [rare] and one intronic variant, IVS2 C691G patients, linkage studies shows exclusion for the three EXT gene regions. [0.45-0.62]. OPRM1 genotype and haplotype frequencies from three Because testing other candidate loci with protein homology to the EXT psychiatrically characterized population samples (Southwestem American Indian = genes also tumed out negative, a genome search was started. A positive (SAI, n = 385), US Caucasian (USC, n = 100) and Finnish Caucasian (FC, n with lod score of 2.5 over a 5-10 cM interval was found with this pedigree which 324)), were used to perform association and sib-pair linkage analyses No association of is highly indicative for the presence of an EXT4 gene located at alcohol and drug dependence diagnoses. significant genotype chromosome 17q. Further refinement and characterisation of this region is to phenotype was observed. Using these data sets, a threshold model provides nearly complete power to detect a genetic effect on susceptibility to alcohol in progress and will be discussed. dependence, while other models may provide less power to detect genetic effects. Though these data do not support a role of the la opioid receptor on susceptibility to alcohol dependence, the potential relationship between OPRM1 variation and response to opiate pharmacotherapy, e.g., methadone and naltrexone, should be investigated.

2329 2330 Case influence measures in genetic linkage analysis. S. B. Bull and D. Lack of evidence for linkage to a nuclear locus in a pedigree with non- Pinnaduwage. Lunenfeld Research Institute, Univ of Toronto, Ontario, syndromic deafness due to a homoplasmic mitochondrial mutation. Y. Canada. Bykhovskaya1, K. Ehrenman1, D. Johnson1, M. Hamon1, R. Cantor1, K. The informativeness of linkage studies to detect genes for complex Taylor', X. Bu1, L. Jabet2, J. L. Rotter1, M. Shoha?, and N. Fischel- disease is dependent on a number of factors, including specification of an Ghodsian1. 1Ahmanson Department of Pediatrics, Steven Spielberg appropriate model, the presence of genetic heterogeneity and/or sporadic Pediatric Research Center, Medical Genetics Birth Defects Center and cases, accurate diagnosis and genotyping, as well as the number of UCLA School of Medicine, Los Angeles, Califomia 2Department of pedigrees and their structure and size. Using jackknife methods, we Pediatrics and Medical Genetics, Basil and Gerald Felsenstein Medical formulate influence measures for a pedigree, and for an individual within a pedigree, for inference concerning the recombination fraction and genetic Research Center, Tel Aviv University Medical School, Petah Tikva, Israel. linkage. We propose two approaches to separate the information in the The relationship between mitochondrial genotype and clinical phenotype nature of phenotype data from that provided by the marker typing data - one for is complicated in most instances by the heteroplasmic pathogenic pedigree influence and one for individual influence. mitochondrial mutations. We have previously shown that maternally We also develop several graphical displays that allow different aspects inherited hearing loss in a large Arab-Israeli kindred is due to a of the data to emerge more clearly, particularly for multiple markers. These homoplasmic mutation in the mitochondrial 12S ribosomal RNA gene include plots of case influence measures ordered by pedigree (Prezant et al, 1993), and that there is genetic and biochemical evidence for characteristics, such as diagnostic subclass, ethnicity, or pedigree size, for nuclear gene involvement in this family (Bu et al, 1993; Guan et al., 1996). each of several markers. Simultaneous plots for several pedigrees ordered In order to identify a nuclear locus responsible for the expression of by marker location emphasize marker similarities and differences. The deafness in this family, two candidate genes were excluded through linkage methods are illustrated in several linkage datasets that include multiple analysis and sequencing, and a genome wide linkage search in family markers and a variety of pedigree structures. members who all have the mitochondrial mutation, but differ in their hearing Case influence measures are data-driven and hence exploratory in status, was performed. In two stages a total of 547 polymorphic genetic nature, and may be suited best to sensitivity analyses. They can facilitate markers were genotyped, and the data were analyzed under model- the detection of unusual podigrees and individuals for further examination, dependent and model-free assumptions. No chromosomal region was and can indicate heterogeneity among pedigrees and covariate-defined identified as a major contributor to the phenotypic expression of the groups of pedigrees. mitochondrial mutation, and thus effects of multiple genes, environmental Supported by the Natural Sciences and Engineering Research Council of factors, and/or stochastic events must be implicated for the individual- Canada. specific and tissue-specific expression of this mitochondrial mutation.(This work was supported by NIH/NIDCD grant DC01402.)

2331 2332 A Novel Polymorphism in Intron 12 of the Dystrophin Gene and Its A Genetic Mapping Study of an Autosomal Dominant Inclusion Body Implication for Deletion Detection by Multiplex PCR. B.Chen, P.E. Myositis Pedigree. K.P. Clancy', D. Patterson1, L.L. BaumbaclA, C. North, and D.M. Parham. Molecular Diagnosis Laboratory, Department of Garcia3, S. Ringef, H. Neville4. 1Eleanor Roosevelt Institute, 1899 Gaylord Pathology, Arkansas Children's Hospital; and Department of Pathology, St., Denver, Colorado, USA; 2Department of Pediatrics, University of Miami University of Arkansas for Medical Sciences, Little Rock, AR 72205 School of Medicine, Miami, Florida, USA; 3Departments of Neurology and The multiplex polymerase chain reaction (PCR) is a reliable and efficient Pathology,Louisiana State University Medical Center, New Orleans, method for detecting dystrophin gene deletions, which are present in about Louisiana, USA; 4Neuromuscular Unit, Department of Neurology, University sixty-five percent of patients with Duchenne or Becker muscular dystrophy of Colorado Health Sciences Center, Denver, Colorado, USA. D/BMD). The 9-plex PCR assay, which enables simultaneous skeletal (DM Inclusion Body Myositis (IBM) is an inflammatory disease of exons 3, 6, 47, 50, disease amplification of the muscle-specific promoter and 13, 43, muscle, which is unresponsive to anti-inflammatory treatments. The 52, and 60, is an extremely useful diagnostic test for DMD/BMD deletions. occurs in both inherited and sporadic forms. Similarities between some this study, we describe a unrecognized A to G base variation lead to In previously pathological aspects of this disease and Alzheimer's disease has in intron 12 (nt -110 from exon 13) the dystrophin gene. This variant is of the speculation that the two diseases share similar pathological located within the annealing site of the exon 13 forward primer, and may in this mechanisms. As a first step towards identifying the gene involved cause of exon 13 in the 9-plex PCR assay. Present in on an deficient amplification disease, we have carried out a genome-wide mapping study 25 of 45 (56 percent) of normal Caucasian males and 3 of 13 (23 percent) autosomal dominant IBM pedigree of Volga German ongin. Only one of normal black males, it is likely to be encountered frequently in diagnostic branch of this five transmits this disease, with a that offer dystrophin deletion analysis by multiplex PCR, generation pedigree laboratories and maximal estimated Lod score = 2.3. A panel of 371 sequence tagged site may create difficulties in interpretation of the test results. markers from the Weber version 6 CHLC marker panel were used to genotype eight members from this portion of the pedigree. By this analysis it was possible to eliminate many genetic loci for inherited Alzheimers disease for involvement in autosomal dominant IBM. After LINKAGE analysis, five chromosomal regions were found to give Lod scores greater than 1.0. A maximal Lod score = 1.86 at a recombination fraction was found upon the q-arm of chromosome 8. Further characterization of these five regions by genetic linkage analysis will allow us to identify chromosomal locus of the autosomal dominant IBM gene in this pedigree. Published Abstracts: Linkage Mapping and Polymorphisms (cont.) A399

2333 2334 Linkage of D6S305 in Alzheimer Disease families containing APOE E4/ Genome wide search for genes causing familial orthostatic intolerance E4 genotypes - The NIHM Genetics Initiative AD Study Group. J. S. syndrome. AL DeStefano, H Gavras, M Burzstyn, I Gavras, D Handy, 0 Collins1 R T Perry', B. Watson', C. J. Vanichanan1, D. BlackeP3, D. A. Joost, M Nicolaou, D Streeten, LA Farrer, and CTBaldwin. Boston Meyers2, M. S. Albern, R. TanzP, S. S. Bassett, L. Rodes2, and R. C. P. University School of Medicine, Boston, MA. Go'. 1University of Alabama at Birmingham; 2Johns Hopkins University, Orthostatic Intolerance Syndrome is a dominantly transmitted disorder of Baltimore, MD; and 3Massachusetts General Hospital, Boston. unknown etiology, characterized by upright lightheadedness worsening to 267 families with late onset Alzheimer Disease (AD) affected siblings syncope, palpitations, and blue-purple ankle discoloration, accompanied by were collected as part of the NIMH Genetics Initiative AD Study. a marked decrease in systolic blood pressure, mild increase in diastolic Chromosome 6 was scanned in these families by genotyping 21 pressure and marked tachycardia which resolve when supine. DNA and microsatellite markers at approximately 10 cM intervals. D6S305 was found clinical information were obtained from members of 3 previously reported, to be linked to AD in 85 families that contain individuals with APOE E4/E4 unrelated families with this syndrome (McKusick # 143850). Simulation genotypes. Non-parametric linkage analyses were performed by the studies indicate that in aggregate these families are of sufficient size to programs GENEHUNTER and SIBPAL, and the LINKAGE program was establish linkage. To identify the genetic basis of disease we conducted a genome wide scan in 9 affected and 22 unaffected individuals from our two used for parametric linkage analyses. The NPL score from GENEHUNTER Two unaffected carriers with both an was 1.87 (p-value = 0.03) for D6S305. Analysis with SIBPAL produced a largest families. clinically obligate affected or and affected were observed mean IBD of 0.55 (p-value = 0.02) and the LINKAGE program showed a parent sibling offspring indicating incomplete penetrance. Our initial screen comprised 200 polymorphic LOD score of 0.83 at theta = 0.10. Additional markers will be typed in this highly region to follow up these findings. D6S305 has also been linked to markers evenly distributed throughout the genome. Multipoint lod score autosomal recessive juvenile parkinsonism in another study. The SOD2 analyses were carried out using the GENEHUNTER program assuming an (superoxide dismutase-2) gene, which is a mitochondrial free radical autosomal dominant mode of inheritance with reduced penetrance (80%). scavenging enzyme, maps very close to D6S305. As free radicals have Five regions showed suggestive evidence of linkage (Z>1.5) in at least one been hypothesized to contribute to AD, this could be a possible candidate of the two families. for further We are currently saturating these chromosomal regions in all three gene study. families with additional markers to establish or exclude linkage to these locations. This study represents the first step in the identification of genetic defects that cause Orthostatic Intolerance Syndrome in these families and will contribute to the understanding of factors that control blood pressure regulation.

2335 2336 The Schizophrenic Problem. J H Edwards. Oxford University Exclusion of candidate spinocerebellar ataxia (SCA) loci provides After many publications on linkage in there is still no locus with evidence for a new SCA locus. K.Friend1, T. Duddingc, M Edwards2, G R predisposing alleles sufficiently strong and common to have been defined with Sutherland1, J C Mulleyl and R I Richards 1. Women's and Children's confidence. In manic depression the position is similar. Most studies have been Hospital, Adelaide, Australia1 Hunter Genetics, NSW, Australia2. based on large families. In insulin dependent diabetes there is firm evidence of susceptibility alleles on The spinocerebellar ataxias (SCAs) are a clinically and genetically about a dozen chromosomes. The most informative studies have been on the heterogeneous group of disorders. To date eight loci have been localised and / small family unit of an affected sib pair with parents, conveniently termed a or characterised. SCA1 (6p22-23), SCA2 (12q23-24.1), SCA3/ MJD (Machado foursome. Joseph disease) (14q24.3-32)), and SCA6 (19p13) have all been charaterised The psychoses and autoimmune disorders are both examples of inappropriate and the mutations in these disorders shown to be due to expansions of an responses by educable systems whose organs impose similar demands on space (AGC) repeat. These (AGC) repeats are normall polymorphic but in affected and energy and may have comparable rates of information flow. The human brain individuals these dymanic mutations are larger than the normal range. SCA7 differs sustantially from most other mammals, with major changes in the last few has been shown by antibody detection with 1C2 to also be due to an million years, while the has had a more leisured evolution of its expansion of an (AGC) repeat. Genetic localisations have also been made for structure. Both psychotic and autoimmune disorders show a high familiarity but, SCA4 (16q24-qter) and SCA5(11q13). We present a preliminary study on a as Penrose showed in 1953 (Acta. Genet. 4. 257-65), this is formally irrelevant to family which clinically has autosomal dominant ataxia and mild developmental the prospects of detection on samples of finite size. delay. All affected members of the pedigree have cerebellar degeneration. The failures in psychoses and the successes in diabetes must be related to Several symptomatic individuals also display dysarthria, a common feature of differences in the distribution of the strength and frequency of the alleles SCA. Affected individuals in this pedigree developed symptoms at an earlier necessarily involved, or to different strategies in collecting data, or to methods of age in each successive generation. Older affected members also have milder analysis which either miss what is there or find what is not. symptoms. Thus, this pedigree appears to exhibit anticipation, a hallmark of These possibilities will be considered and the essential difference between the dynamic mutations. Analysis of known SCA expansion loci and other SCA genetic landscapes in mendelian and other disorders discussed. The strategies localisations was therefore undertaken in this family. There was no evidence of and analytical methods for surveying these landscapes efficiently differ. The relative of families of expansion of the SCA1, SCA2, SCA3/ MJD or SCA6 (AGC) trinucleotide advantages one (population studies), two (parent and child, repeat loci. Also MLINK analysis at these loci indicated no evidence of linkage either affected) and foursomes will be compared. In multifactorial disorders, as to mendelian disorders, decline with to these loci. Microsatellite markers at / or flanking the localisations for SCA4, opposed efficiency may increasing size of and SCA7 loci were family while families selected for more than one affected individual are biased SCA5 amplified by PCR in the family. MLINK analysis of against the detection of resistance alleles. These are likely to balance these microsatellite markers again show no evidence of linkage. Thus, this susceptibility alleles in strength and frequency, and to be more important in family with spinocerebellar ataxia and mild mental retardation does not map to relation to any of the known SCA loci. Therefore it is most likely that there is another as prevention or therapy. yet unmapped genetic locus for spinocerebellar ataxia.

2337 2338 Affected sib pair analysis of families with absolute pitch (AP); Comparison of Tailing Techniques Used for Promoting Nontemplated exclusion of the Williams locus. P.K Gregersen', E. Kowalsky', M. de Nucleotide Addition to Microsatellite PCR Products. J.D.Hauser, Andrade.2 and D. Jawaheer1 . North Shore University Hospital/NYU H.S.Lee, P.Dong, A.M. Wheaton, K.Tynan, A.L.Lowe. PE Applied School of Medicine, Manhasset, NY and 2 Univ. of Texas/ MD Anderson Biosystems 850 Lincoln Centre Drive Foster City, CA 94404. Cancer Center, Houston, TX. The tendency of AmpliTaq DNA polymerase to catalyze the addition of a Absolute pitch (AP), also known as perfect pitch, refers to the rare ability nontemplated nucleotide to the 3' end of double stranded PCR products of some individuals to instantaneously identify the pitch of musical notes or can result in complex patterns of products that create difficulties in ambient sounds without utilizing a reference pitch. This cognitive ability automated allele identification, especially for dinucletide repeat markers. usually arises without specific training in early childhood, and we have This activity is affected by several factors including the marker sequence, previously estimated the relative risk to sibs (Xs) at approximately 20. We salt concentration and thermal cycling conditions. When this nontemplated have now obtained information on 400 probands with AP and their families. addition goes to completion (or does not occur at all) the PCR products 117 (29%) of these families have more than one member with AP, including generated can be unambiguously allele called by Genotyper software. 81 families with one or more sib pairs with AP. Also included in these Problems arise when the addition occurs to approximately 50% of PCR families are three sets of MZ twins all of whom are concordant for the AP products. phenotype, and one DZ twin pair who are discordant for AP. Two methods have been described to promote nontemplated nucleotide It has recently been appreciated that many individuals with Williams addition by the manipulation of the 5' end of the reverse primer (to promote Syndrome have musical ability. At least some appear to possess AP, addition on the 3' end of the forward strand). Brownstein, et al. although it is unclear whether the prevalence of AP is actually elevated in (BioTechniques 20: 1004-1009 (1996)) utilized a 5-7 base "PlGtail", while the Williams population. Therefore, we have evaluated the Williams Magnuson et al. (BioTechniques 21:700-709 (1996)) determined that the Syndrome locus at 7q11.23 as a potential candidate region in sibling pairs addition can be enhanced by modifying the 5' end of the reverse primer with with AP. 24 AP families with one or more affected sib pairs were evaluated a single 'G". with markers D7S1870 and D7S2204. An evaluation of 40 fully informative We tested both methods to determine their relative efficiencies in sibling pairs did not reveal significant allele sharing IBD at the D7S1870 promoting the nontemplated nucleotide addition to dinucletide repeat locus (10:21:9 distribution of 2,1 or 0 alleles shared IBD). The D7S2204 markers from the ABI Prism Linkage Mapping Set in both single-plex and marker also showed no significant sharing among 23 fully informative pairs. multi-plex PCR amplifications. Our results show that while both methods These data indicate that the Williams region is unlikely to contain a major generally improve allele pattems, the "PIGtail" method is more efficient at susceptibility gene for AP in the normal population. We are currently promoting nontemplated nucleotide addition for a subset of the markers studied. Issues related to the of pursuing a genome wide screen of a larger set of affected sib pairs in order implementation this technology will be to identify candidate regions for the AP phenotype. discussed. A400 Published Abstracts: Linkage Mapping and Polymorphisms (cont.)

2339 2340 GenLink and TeIDB: WWW resources for human genetics and Mapping of Testis Determining Genes by Linkage Analysis in Families telomere research. C. Helms, L. Liu, M. Gonzalez, P. Giles, S. Cole, and H. with Sex Reversal. D. JawaheerA 5, K McElreavey 2, G. Berkovitz 3, A. Donis-Keller. Div. Hum. Mol. Genet. Washington Univ. Sch. Med., St. Louis, MO. Braun 4,P. K. Gregersen 1,5, and H. Ostrer5. tNorth Shore University Now in its third year of development, GenLink, an NIH sponsored genetics Hospital, Manhasset, NY; 2 Institut Pasteur, Paris, France; 3Johns Hopkins resource, is designed to facilitate human map integration and gene identification University School of Medicine, Baltimore, MD ;4UniversitAt Munchen, projects. Through a WWW interface [http://www.genlink.wustl.edu], GenLink provides users with graphics of a wide variety of linkage maps, the underlying data Munich, Germany; 5 New York University School of Medicine, New York, NY (e.g. genotypes used to produce the maps, the position of crossovers detected in Sex determination in humans is genetically controlled. Genetic analysis reference chromosomes), and crosslinks to other databases containing marker of individuals with sex reversal (genotypic sex of one type, phenotypic sex information and maps (e.g, GenBank, GDB,WICGR). Updates over the past year of the opposite type) has been useful for identifying testis determining to GenLink's WWW interface include: 1) a user-directed program, DCOP, that genes in humans. Individuals with 46,XX maleness or true hermaphroditism crossover contributing to generates crossover panels; DCOP finds chromosomes have been identified with translocations of the SRY gene onto the X map order for any given map segment, then sorts and returns a color-coded chromosome. Individuals with 46,XY have been found the crossover 2) CEPH database genotypes; 3) a graphic of chromosomes; with mutations in the WT1 and searchable and graphical interface to Genethon's 1996 microsatellite maps. (1 Ip15) SOX9 (17q25) genes, duplications of the DSS region on the X chromosome (Xp2l), and deletions of 9p and TeIDB, the WWW telomere database [http://www.genlinkwusti.eduteldbIJ, another component of GenLink, contains a wide vanety of information about 10q. Sex reversal may affect several individuals within a family. Pedigree telomere research, mainly in the form of a citations database. TeIDB now has analysis of such families suggests sex-limited, autosomal dominant literature references for telomere-related articles from more than 175 journals and inheritance. We have identified 6 families with co-existing 46,XX maleness over 75 different specles. The references in TeIDB are annotated with additional and/or true hermaphroditism, 2 large families with 46,XY mixed gonadal information given in each article (e.g., organism(s), chromosome ends, genes, and/ dysgenesis, and 4 families with 46,XY pure gonadal dysgenesis. To test or subtelomeric repeats studied, as well as many other keywords). Review articles whether known genes were involved in these familial cases of sex reversal, are annotated in the database as such. TeIDB users may design their own query of linkage was microsatellite DNA dates) analysis performed using polymorphic the database using any of these criteria (also author(s) and/or publication markers spaced at approximately 10 cM intervals from 9p, 10q, 11p15, through a WWW interface. A report is returned listing the citations forms-based and the X chromosome. LOD score values several different database. 17q25, testing found in the annotated modes of inheritance were lower than those GenLink's data repository for journal articles includes data that are of great significantly predicted by value but often cannot be included in journal publications. Researchers can submit simulation. These findings suggest that mutations in genes other than those in cases of sex reversal and maps and/or data to the GenLink data repository through a 'no forms' submission. already identified are involved these familial Text files may be sent by email to genlink~hdklab.wustl.edu. Submissions should that identifcation of these genes will provide additional insight into the include literature references, contact information, an abstract, the data file(s) used, genetic pathway of testis determination. and other information such as map order with distances.

2341 2342 Linkage of cytokine genes to (RA). Evidence of A dopamine D3 receptor gene polymorphism is not associated with the genetic heterogeneity. S John, A Myerscough, A Marlow, A Hajeer, AJ Polycystic Ovary Syndrome. M.D. Kahsar-Miller', L.R. Boots2 and R. Azziz2'3. Silman, WE Oilier, J Worthington. ARC Epidemiology Research Unit, 1Laboratory of Medical Genetics, and Depts. of 2Obstetrics/Gynecology and University of Manchester, UK. 3Medicine, The University of Alabama at Birmingham, Birmingham, AL. The is a common endocrine Background: RA is an oligogenic disease. Whole genome screens have, as yet, Polycystic Ovary Syndrome (PCOS) reproductive 2-10% of characterized ovulatory failed to detect susceptibilty loci other than HLA presumably due to the size of effect disorder affecting women, by: i) dysfunction, clinical hirsutism, acne, and/or frontal balding) and/ of the genes and distance of markers from non-HLA loci. A candidate gene approach and ii) hyperandrogenism (i.e. levels. PCOS is and has could be more revealing. or elevated serum androgen clinically heterogeneous a causative has not been Alms: To use microsatellite markers which map close to cytokine genes to significant familial tendency, although gene yet investigate linkage to RA in 200 affected sibling pair families. identified. It has been proposed that dysregulation of dopamine action may Methods: Microsatellite markers (heterozygosity > 0.65) mapping within or less contribute to the development of PCOS. Investigators have previously identified a than 3cM from: INFa, INFO, INFy 11a, 1L1-, IL1R, IL2, IL6, ILSR, IL8R were dopamine D3 receptor gene (D3) polymorphism. This A to G polymorphism, compiled in a set for fluorescence based automated genotyping. DNA from 200 RA termed the D3-2 allele, creates an additional Msd restriction site. In U.S. Hispanic affected sibling pair families from the ARC National Repository was genotyped for females, homozygosity for the D3-2 allele (i.e. 2/2) was found to be associated the above markers. Excess allele sharing by affected siblings was tested by a with elevated serum testosterone levels and ovulatory dysfunction (Legro et al., maximum liklihood-identity by descent method (MLS-IBD) using SPLINK version Fertil Steril 63:779, 1995). Based on this data we hypothesized that the D3-2 1.05. Markers showing some deviation from random inheritance were analysed in allele was also associated with PCOS in other ethnic groups, and studied 120 subsets of the families stratified to take account of; age at onset, sex, disease consecutive non-Hispanic Caucasian PCOS patients. As controls, 78 healthy, non- severity, and HLA haplotype sharing. hirsute Caucasian women with regular menstrual cycles were studied. Genomic Results: Non-significant increased sharing of IBD alleles was observed with DNA was isolated from the blood of all subjects. A 204 bp fragment of D3 was markers for IL2, IL5R and IFNy Markers for these genes were re-tested following amplified by PCR, then subjected to Mscl restriction enzyme digestion and sized stratification of the data. Significant evidence of linkage was seen for IL5R in female on a 4% sieving agarose gel. Subjects were classified according to the presence sibling pairs (LOD 0.91 p0.03) and for INFG in sibling pairs with an affected male of the D3 polymorphism into three potential genotypes: 1/1, 1/2, and 2/2. In this (LOD 0.96 p0.03). The most significant evidence for linkage was seen for IL2 in population, no difference in the distribution of either the 1/1, 1/2 or 2/2 genotypes & sibling pairs in which one or both were persistently seronegative (LOD 1.05 p0.02). between PCOS patients and controls was observed (51, 38 & 11% vs. 44, 45 marker genotypes for this subgroup were analysed in the Transmission IL2 11%, resp., p = We can conclude that in our population of non-Hispanic an association. 0.597). Disequilibrium Test (TDT) which detects linkage in the presence of Caucasian women from the Southeast U.S. an association between the reported were more expected. Two alleles (141 and 143bp) inherited often than D3 and PCOS is not observed. Further studies in other populations Conclusion: Evidence of linkage of RA to IL5R, INFy and IL2 has been detected polymorphism may demonstrate an association between polymorphisms of D3 and PCOS, in clinical subsets of sibling pairs suggesting that RA is a genetically heterogeneous yet it is that such a disorder is associated with a disease. The data in seronegative RA provides preliminary evidence that the IL2 although unlikely heterogeneous IL2 defect. microsatellite is in linkage disequilibrium with a functional polymorphism in the gene. single gene

2343 2344 Genomic screen for linkage in a family with autosomal dominant Refined mapping of the locus for palmoplantar keratoderma type of chordoma. J. F. Korczak, M. J. Kelley, K. A. Allikian, A. A. Shah, A. M. Bothnia. L. K. Lind, A. LundstromI and G. Holmgren. Departments Goldstein, and D. M. Parry. National Cancer Institute, Bethesda, MD. Clinical Genetics/Cell and Molecular Biology and 1Dermatology, Ume(A) Chordoma is a rare, low-grade, malignant bone tumor derived from University, Ume{A), Sweden. remnants of the notochord, a structure that gives rise to the embryonic axial Hereditary palmoplantar keratodermas constitute a heterogenous group skeleton and almost always disappears by birth. Chordoma typically is a of human skin disorders characterized by thickening of the epidermis sporadic tumor; only three multiplex families, each with 2-3 affected relatives, palms and soles. Palmoplantar keratoderma type Bothnia is a distinct have been reported since 1958. variant of the disease, prevalent in northern Sweden. The disorder a a nine in three generations We evaluated family with affected individuals purely hyperkeratotic form of diffuse palmoplantar keratoderma, frequently that included two father-son pairs, a pattern consistent with autosomal the hands and feet, the complicated by dermatophyte infections. Except for was on or a mass no associated dominant transmission. Diagnosis based histopathology skin of affected individuals is normal and there are typical of chordoma on MR scans of the skull base and spine. Two additional malformations or family members had unusual MR findings that might indicate variable malignancies. We have keratoderma type expression of the disease trait. previously mapped palmoplantar chromosome 12q11-q13, a region known to contain the keratin type 11 gene To localize a chordoma gene, we conducted linkage analysis. Two families cluster. However, based on recombinational events detected definitions of affection were used: (i) the nine individuals diagnosed with the PPK Bothnia locus maps to an approximately were considered affected and (ii) these nine persons plus the two studied, type chordoma the centromeric to the 11 keratin cluster. In addition, haplotypes relatives with unusual MR findings were coded affected. Prior to screening the type gene for the family members were identical over some power detect constructed affected genome, we used the program SIMLINK to estimate the to 2 cM all of the linked markers microsatellite markers in a region) linkage. At a recombination fraction of 0.05 (corresponding to a 10 cM map), (10 could a common ancestral mutation for the disease in the families. the mean and maximum lod scores over 200 replicates were about 1.5 and imply We have constructed a YAC covering the region of 3.1, respectively. To date, we have performed two-point lod score (using contig found the order of the DNA markers in this contig to tne LINKAGE) and affected sib-pair and Haseman- Elston regression (both using dier somewnat orn genetic map. In addition, our YAC contig also contigs of SIBPAL) analyses for 208 of 369 autosomal short tandem repeat markers clones chromosome 12q, for instance do some of the 8 spaced an average of 10 cM apart. Preliminary results detect (CHLC version panel) were markers when tested in different laboratories. We suggest possible linkage of the disease locus to a region on chromosome 1, YAC clones that contained two of the microsatellite or on scores and nonparametric any 17, 19, based both parametric (lod of 1.0-1.5) harboring type (P significant at the 0.01 level) results at each of two adjacent type Bothnia. It thus appears as if the chromosomal region values nominally vectors. marker loci. Upon completion of the initial 10 cM screen, we will type additional Bothnia is not ideal for cloning in YAC markers in each region of interest and conduct both two-point and multipoint linkage analyses to localize the chordoma gene. Published Abstracts: Linkage Mapping and Polymorphisms (cont.) A401

2345 2346 Mapping of susceptibility loci for Systemic Lupus Erythematosus. A-K LindqvisO, Linkage analysis of a Greek-American kindred with autosomal M.E. Alarcdn-Riquelmel, K Steinsson2, L. Klareskog3, 1. Lundberg3, E. Svennungsson3, dominant, levodopa-responsive parkinsonism and anticipation. K. G. Sturfelt4, D. Alarcdn-Segovia5, J. Alcocer-Varela D.Commenges6 and UB. Markopoulou', B. A. Chase', and Zbigniew Wzsolek2. 'University of Gyllensten'. 'Dept of Medical Genetics, Uppsala University, Sweden, 2Dept of Nebraska at Omaha and of Nebraska Medical Center, Omaha, Rheumatology, Landspitalinn, Reykjavik, Iceland, 3Clinic of Rheumatology, Karolinska 2University Hospital, Stockholm, Sweden,4Dept of Rheumatology, University of Lund, Sweden, NE. de Mexico 6INSERM We previously described a Greek-American kindred with a phenotype of 5Depto Inmunologia y Ruematologia, INNSZ, City, Mexico, U330, and Universitd de Bordeaux II, Bordeaux, France. levodopa-responsive, autosomal dominant parkinsonism anticipation. Systemic Lupus Erythematosus (SLE) is an with variable We present initial findings from a linkage analysis performed with markers course and clinical heterogeneity. It is regarded as a prototypefor systemic autoimmune from regions previously associated with familial parkinsonism: 17q21-23, diseases. The principal clinical manifestations include skin rashes, arthritis and 4q21-23, and 6q25.2-27. Two-point lod scores with markers D17S958 and glomerulonephritis, but hemolytic anemia, thrombocytopenia as well as central nervous Hox2B (17q21-23) were -1.6 and -7.9 respectively, making exclusion of system involvement are also common. The most common autoantibodies found in SLE linkage to region 17q21-23 likely. Two-point lod scores with markers patients are antinuclear antibodies (ANA). between 0.8 and 1.59. SLE mainly affects women between 20-50 year of age with a incidence of 2.5-7.8/ D4S1578, D4S2380 and D4S1647 (4q21-23) ranged 100 000. The incidence for men is approximately eight times lower. Scores of flanking markers D4S2361 and D4S421 were -1.6 and 1.1 The aetiology of SLE is not known. Genetic components to SLE are indicated by respectively. Thus, linkage of the kindred phenotype with region 4q21-23 family studies and increased concordance rates of monozygotic twins (25-70%) cannot be excluded. Given these findings and those of Polymeropoulos et compared dizygotic twins (1-3%). SLE is speculated to be a multifactorial disorder with a al. associating mutations in the a-synuclein gene of chromosome 4 with complex mode of inheritance. several families autosomal dominant we are A collaborative network has been established to study families with multiple cases of having PD, currently evaluating SLE from well defined populations in Europe and Mexico. The patients are being studied affected kindred members for such mutations. Region 6q25.2-27 marker clinically for SLE according to the criteria of the American College of Rheumatology and testing is in progress. as agreed upon among the collaborators. Presently a full genomic screen is being performed on extended families from Iceland with multiple affected generations and 3-8 affecteds per family. Genotyping is done in a semi-automated fashion with 360 fluorescent-labeled microsatellite markers (Weber set 6.0, CHLC) ordered in chromosome-specific panels and detected on ABI 377 sequencer. A multi-analytical strategy is applied. Two-point linkage analysis is being performed using the MLINK program (FASTLINK version 3.0 with two different mode of inheritance, dominant and recessive. In parallel non-parametric analysis is performed using Weighted Pairwise Correlation statistics (WPC).

2347 2348 Population genetic mapping studies of Tourette Syndrome. CA Identification and analysis of single nucleotide polymorphisms in the Mathews1, MMC DeMille1, LD Herrera2, VI Reus1, TL Lowe1, M Spesny2, cholecystokinin-A receptor gene, a candidate gene for multi-factorial BJM van de Wetering3, and NB Freimer1. 1 University of California at San obesity. S. P. Moffett, M. Moriarty, E. C. Lawrence and R. E. Ferrell. Francisco, 2Hospital National de Nihos, San Jose, Costa Rica, 3Dipjigzt University of Pittsburgh, Pittsburgh, PA. Hospital, Rotterdam, the Netherlands. The neuropeptide cholecystokinin (CCK) is released as a satiety factor in Tourette Syndrome (TS) is a neuropsychiatric disorder that affects 5/10,000 response to food intake. The actions of CCK are mediated by two receptors, individuals and consists of multiple motor and vocal tics. Epidemiologic studies the cholecystokinin-A receptor (CCK-AR) and the cholecystokinin-B indicate a strong genetic component: however, linkage studies of TS have receptor (CCK-BR) which are differentially expressed in the gastrointestinal been unsuccessful in identifying a susceptibility locus. Because of the difficulty tract, central nervous system and peripheral nervous system, and belong to of collecting an adequate sample size using TS families most studies have the superfamily of the G-protein coupled receptors. The CCK-A receptor is used broad diagnostic definitions, increasing the risk of phenocopies. expressed predominantly in the gastrointestinal tract where it induces In order to mimimize these difficulties, we are using a population genetic gallbladder contraction and pancreatic enzyme secretion as well as in the mapping (PGM) approach to search for gene(s) predisposing to TS. PGM is a CNS where it regulates satiety. In order to determine the importance of the powerful strategy of genetic analysis based on identifying regions of the CCK-A receptor in obesity, SSCP and sequence analysis were used to genome shared identical by descent among randomly chosen affected identify common polymorphic variations in the CCK-AR gene. Four individuals in a genetically isolated population. Using this strategy it is possible individuals out of twelve were found to be heterozygous for a C to G to limit investigation to individuals with narrowly-defined (severe) phenotypes transversion at nucleotide position 402 (Genbank accession # U23427). At such as definite TS. Two populations that are primarily descended from a nucleotide position 1260, two individuals were found to be homozygous for limited number of founder for families and thus are particularly appropriate a T to A transversion. A T to C transition was found at nucleotide position PGM studies are the of the and that of population Central Valley of Costa Rica, 1266 which caused the loss of a PstI restriction site. This RFLP was used to Ashkenazi Jews in the US. simulations with varying Computer performed screen a biethnic sample of healthy, premenopausal women participating in sample sizes using these two populations indicate a high probability of the Women's Healthy Lifestyle Project. detecting TS loci even in the presence of etiologic heterogeneity. Simulations Allele frequencies at this site differed between African-Americans = and = were done using a variety of methods of statistical analysis. The results (N 36) U. S. Caucasians (N indicate that, even with a sample size as small as 50 affecteds from either 462) with the frequency of the most common allele (T) being 0.905 and population and a phenocopy rate of 40%, the power to detect a TS 0.763, respectively. No significant differences in age adjusted weight or susceptibility locus approaches 100%. For some forms of analysis, with weight related phenotypes (BMI, waist:hip ratio) were found between sample sizes of 150 or more, the power remains near 100% even for individuals with the C allele and those without. Haplotype analysis using the phenocopy rates as high as 70%. three CCK-AR single nucleotide polymorphisms may reveal a subtle influence of this locus on weight related phenotypes.

2349 2350 Distribution of Anglotensin Converting Enzyme (ACE) I and D alleles A multivariate model using haplotype sharing: Results for bipolar frequencies In a of 155 subjects of Rio de Janeiro, Brazil. K. Miranda sample affective disorder in the old order Amish. R. A. Philibert1 P. L. St. Jear?, Si/va2, E. Rondinelli A. C. Nogueira3, C. C. Sucharov, , Campos de Carvalho1, A. M. Sahbatou3, J A. Egeland4, S. M. Paul5, and E. I. Ginns1 1Clinical L. H. S. Campos3, M. Souza e Silva3 e R. S. Moura-Neto2. Instituto de Biofisica Neuroscience Branch, IRP, NIMH, NIH, Bethesda, MD; 2Department of Carlos Cha as de e Universitarfo Clementino Filho1, Depto. Genetica2 Hospital Epidemiology and Biostatistics, Case Western Reserve University, Fraga Filho - UFRJ - Brazil. Hypertension is a determinant factor of cardiovascular ailments including cardiac Cleveland, OH; 3Foundation Jean Dausset, CEPH, Paris, France; hypertrophy, coronary disease and others. However, little is known about its primary 4Department of Psychiatry, University of Miami School of Medicine, causes. The Renin-Angiotensin System develops important functions in the control Hershey, PA;5Lilly Research Laboratories, Eli Lilly and Company, of blood pressure. The system components are well characterized and the Indianapolis, IN. correspondents genes cloned, including the angiotensin converting enzyme (ACE). The ACE gene has an insertion of 287 bp in the intron 16 in certain subjects Numerous studies suggest that there is a high degree of vulnerability generating a genetic polymorphism. The genotype DD (deleted) does not show the conveyed by genetic factors to the susceptibility of bipolar affective insertion, in contrast to the genotype 11. Patients with the genotype DD can show disorder. The inheritance of bipolar affective disorder appears to be twice the concentration of circulating ACE compared to the genotype 11. The multifactorial, rather than the result of simple Mendelian transmission. We genotype Dl has intermediate levels. However, no relationship was demonstrated have used genotypes from a dense map of informative markers to construct between the polymorphism D/l and hypertension. On the other hand, a strong haplotypes for eighteen individuals from Amish family 884 (Core 110). correlation was observed myocardial between allele D and an increase risk of Using non-parametric analysis, we analyzed the contribution to infarction and other as well as an increased risk of developing left the cardiomyopathies, phenotype of candidate loci. Our analyses demonstrate genes ventricular hypertrophy. Brazilian population is extremely heterogeneous, and the that in the determination of the and D alleles frequencies in this population is of great regions represented by the haplotypes at the chromosome 4p, 6p and 15q importance. In this work, 155 non related subjects (98 men and 57 women) were loci significantly contribute to the occurrence of bipolar affective disorder. Of analyzed for the ACE gene polymorphism. The subjects were not discriminated to the models studied, most of the variance, 60%, can be accounted for by a any pathology and are provinient of a patemity test laboratory. Determination of the two way ANOVA model using the chromosome 4p and chromosome 15q polymorphism was done by PCR technique. The frequencies of 0,659 for allele D loci. These results provide further evidence that BPAD in the Old Order and 0,341 for allele were obtained, being on Hardy-Weinberg equilibrium. These Amish is inherited as a complex behavioral illness. Further data are frequencies do not differ f ound in other studies (qui2 0,726, P = discussed. 0,687) and there is no difference on frequencies between sexes (qui2 = 1,91, P = 0,566).This study is of importance on the initial frequency characterization of these alleles on the Brazilian population and for posterior analyses of cardiomiopath patients, so a correlation between the different cardiomiophathies and the ACE polymorphism can be analyzed. A402 Published Abstracts: Linkage Mapping and Polymorphisms (cont.)

2351 2352 Linkage studies in neurofibromatosis - evidence of reduced Further suggestive evidence for involvement of the a7-nicotinic penetrance of the NF1 gene? M.Poyhonen, S.Kytdla, J.Leisti. Department cholinergic receptor gene on chromosome 15q13-qi4 in of Clinical Genetics Oulu University Hospital Oulu, Finland schizophrenia. BP Riley, M Mogudi-Carterl, TJ Jenkins2, R Williamson3, In a population based study of 197 NF1 patients, 20 families with an DA Collier and RM Murray. Department of Psychological Medicine, Institute affected parent and at least one affected child were analyzed for linkage to of Psychiatry, LondonUK, 'Department of Psychiatry, Baragwanath the NF1 gene by polymorphic microsatellite markers in order either to Hospital, Soweto, RSA, 2Department of Human Genetics, University of the comfirm the diagnosis or to exclude it. From the 87 informative meiosis 54 Witwatersrand, RSA 3The Murdoch Institute, were associated with a respective nriskN haplotype. Fifty-two of these Johannesburg, Royal individuals were affected and 2 were unaffected, among them a 1 5-year-old Children's Hospital, Melboume, Australia. girl with no clinical signs of NF1 and a pair of 8 and 11 years old sisters who Recent reports have strongly linked the a7 nicotinic cholinergic receptor had inherited different haplotypes from the affected father and did not show gene on chromosome 15q13-q14 to a sensory gating deficit common in any signs of NF. All the 33 individuals who had inherited the "nonrisk" schizophrenics, and have shown positive though non-significant results haplotype were healthy. The obtained result suggest that the two linking this receptor gene to the primary phenotype of schizophrenia in a nonaffected individuals still can be nonpenetrant mutation carriers of the sample of North American families. We therefore tested for linkage between NF1 gene. markers in this region of chromosome 1 5q and schizophrenia in a sample of In the 20 families studied for genetic linkage, one was shown to have a 16 families multiply affected with schizophrenia drawn from the Bantu- deletion encompassing markers from EVI-20 to INT-38. In addition, 7 speaking black population of South Africa. The map of markers used was familial and 39 sporadic cases in informative families were screened for D15S165-4cM-D15S1360-2cM-D15S1 44-6cM-D1 5S118-7cM-D15S641. deletions with linked markers. One deletion for the INT-27 marker was We find an affected-only LOD score maximum of 1.08 at e=o.oo for found in a sporadic case. Thus, deletions were found in two of the 66 D15S1360, a dinucleotide polymorphism found on the same YAC as the aC7 studied families. receptor gene, when analysing affected individuals only under a recessive In the familial cases in which the parental origin of the new mutation in transmission model with penetrance=0.5. Nonparametric affected-only the first affected individual could be evaluated with linked markers, 6 of the multipoint analysis yields a Z-score of 1.29, p=0.098, for D15S1360, and 7 studied cases had the mutation in the paternally derived chromosome. Z=1.45, p=0.075 for D15S118. Transmission disequilibrium testing was not The sporadic deletion occured in the maternally derived chromosome. possible with D15S1360 alone due to large numbers of parents homozygous at this marker. However, haplotype data from D15S1360 and D15S144 were analysed for transmission disequilibrium. Allele-wise testing yields a chi-square of 19.74, 12 df, p=0.073.

2353 2354 Linkage and Mutation Studies of Adult-Onset Primary Open Angle Glaucoma Evidence of a major autosomal dominant mendellan gene responsible (POAG) with TIGR and Genes on 4 other Chromosomes. M. Sarfarazi', A. for nonsyndromic cleft palate. A.L. Serti6, S. Steman, M.Zatz, M.R. Childf, D. StoilovaI, G. Brice2, R. Coakes3, R.P. Crick4 'Surgical Research Passos-Bueno. Dept. de Biologia, Universidade de S.Paulo, SP, Brazil Center, Dept. of Surgery, Univ. of Connecticut Health Center, Farmington, CT; Nonsyndromic cleft palate (CP), a common affecting 1/1500 2Dept. of Cardiological Sciences, St. George's Hospital, London, UK; 3Dept. of Caucasians, has been considered a different entity from cleft lip with or Ophthalmology and; 4lntemational Glaucoma Association, King's College Hospital, without cleft palate (CUP). The most likely inheritance for this condition is London, UK. multifactorial; however, there are some cases in which CP is clearly Familial POAG is an autosomal dominant condition with clinical presentation associated with a mendelian inheritance, such as X-linked cleft palate or in between ages of 3 to 90. The juvenile POAG on 1q24 has recently been shown to the Van der Woude syndrome. In the present report, we are describing a be caused by mutations in the TIGR gene. SSCP screening and DNA sequencing large three generation Brazilian kindred with 11 individuals with CP, 1 with of TIGR fragments in 150 probands of our chronic adult-onset POAG (COAG) CUP and 2 with CL. The 2 individuals with CL and 1 with CUP were born families identified 5 kindreds with a nonsense mutation in exon Ill. In one family, from CP We observed that 6 CP patients and 1 had from COAG side with patients. CUP patients this mutation is inherited from the unaffected spouse, but not of 13 are with 1 with CUP but there is no a total of 45 offspring, whom, affected (10 CP, a clear family history. This spouse has glaucoma secondary to Iritis of family history in his very large extended pedigree. In a second family, this mutation and 2 with CL) and 22 are normal individuals. The most likely pattem is present and passed on by an 86 year old clinically normal female. A large inheritance in this family is autosomal dominant because male to male number of phenotypically unaffected adult members also inherited the same transmission was reported in two instances. Based on genetic and clinical mutation. Since this nonsense mutation is not present in general population, the analysis of this family, we considered as candidates the 3 Stickler genes exact role of TIGR in the etiology of these families still remains to be determined. (COtAl, COLl lAl and COLl 1A2), the Van der Woude as well as TGFB3 Two loci for COAG have also been mapped to 2cen-q13 (GLC1B) and 3q21- loci. In order to test the above hypothesis, we performed linkage analysis q24 (GLC1C). For GLC1 B, our original linkage report has now been confirmed in a using the closest available markers nearby these loci. We obtained negative total of 18 families with a very broad range of intraocular pressures (low to high). lod score and therefore, we considered that mutations in anotherlocus Screening of a number of genes previously mapped to the GLC11B critical region might be responsible for the condition in this family. We are currently has not identified any mutations as yet. We have also screened over 70 COAG a wide search DNA The far have not been able to confirm conducting genome using pooling strategy. families for linkage to the GLC1C locus, but so identification of this will be for the none of our large families are mapping and gene undoubtedly important linkage to this location conclusively. While clearly of CP. In will allow us to test if CL linked to this locus, a number of smaller families may be linked, but the size of understanding of the occurrence addition, these pedigrees are not adequate to provide statistical evidence of linkage. may be have a common etiology with CP in some cases. Financial support: More recently, we have identified linkage to two new loci, one in a large FAPESP, CNPq, PRONEX, HHMI. American and several other families, and another in a 4 generations of a British family with over 40 scorable meioses. Saturation mapping and mutation screening of these two new gene loci are currently in progress.

2355 2356 Mapping additional loci for Leber Congenital Amaurosis. D W A PCR based Highly informative DNA Marker with more than 65 Stockton', R A Lewis".2, K L Anderson1, E B Abboucf, A Al-Rajhf & J R alleles. Tang, JianQing*h, Seguin, Marie-Jose*., Morse, Marie-Claude*., Lupskil. 1Baylor College of Medicine, Houston, TX., 2King Khaled Eye Melancon, Serge-B*I. *Procrea BioScience Inc. Genetic Services, aHopital Specialist Hospital, Kingdom of Saudi Arabia. Notre-Dame, Universite de Montreal. Although Leber Congenital Amaurosis (LCA) is a rare autosomal recessive PCR based STR (short tandem repeats) markers are not considered as retinal dystrophy, it is arguably the most common genetic cause of retinal informative as other marker systems due to their lower heterozygosity and blindness in infants. Clinically and genetically heterogeneous, it is characterized discrimination power. Hence their utilization has been limited in forensic by severe visual impairment from birth or early infant, infantile nystagmus, evidence case-work and as genetic profiling tools as a considerable number various ophthalmoscopic features, non-recordable ERG responses, and of STR markers need to be run simultaneously to achieve sufficient occasionally various systemic associations. One LCA locus has been identified informativeness. Few previously published PCR based complex STR and the gene for a retinal specific 9uanylate cyclase (GUC2D) cloned. Two markers have demonstrated a potentially high heterozygosity and in of missense mutations and two frameshift mutations were reported patients discrimination Here, we report additional results on one very demonstrated families power. North African origin. Genetic heterogeneity was also by STR marker described and mapped to in other informative complex previously excluding this locus. A candidate gene approach screening for mutations We have components of the guanylate cyclase pathway has been unsuccessful at chromosome 17q12-q24 (Mammalian Genome, 1995, 6/345-349). identifying other LCA genes. cloned one of the many alleles of this marker and completely sequenced it. Data from our collection of families from the Kingdom of Saudi Arabia and Using specific primers designed from the non-polymorphic part of the marker and a we were able to greatly North America also exclude this locus (Z = -14.59 at e = 0.1 for marker sequencing grade gel electrophoresis, D1 7S804). LCA1, therefore, does not appear to be a major locus for LCA in either improve both amplification efficiency and resolution power. The sequencing population. data on 26 alleles indicated that a complex STR structure was involved. Its Pedigree simulation in our LCA families has demonstrated sufficient power structure is summarized as (AAAG)xAAAAG(AAAG)0-1(AG)y(AAAGG)z. In independently in several Saudi Arabian pedigrees that do not link to the LCA 1 a preliminary population study, this marker now named Sextolet 900, has locus. These families will be useful to identifi other locl. An identity-by-descent revealed 65 alleles out of 180 chromosomes. The frequency of the most screen has been completed and several candidate regions are being investigated frequent allele was 0.0797. Each allelic fragment differs in size by 1 to 5 bp. further with additional markers and formal linkage analysis. We estimated the heterozygosity and discrimination power will further the The identification of other for LCA elucidate biological that sextolet 900 genes be above 0.97. These data suggest powerful to this disease and critical to the molecular defects leading provide reagents study STR marker described so far. its application of the human retina. will enable the accurate potential developmental biology They services is We are running identification of siblings who may be carriers and have the ultimate capacity to and genetic profiling highly promising. currently determine its allelic modify or to prevent the devastating visual consequences of this disorder in future a population study to further frequencies generations. rates. Published Abstracts: Linkage Mapping and Polymorphisms (cont.) A403

2357 2358 Evidence for locus heterogeneity in Hallervorden-Spatz syndrome. T. Genetic linkage analysis in families with Charcot-Marie-Tooth type 2 D. Taylor', H. Hattorn, S. Bunde?, A. Malandnn$, M. Villanova4, G. M. neuropathy. V. Timmerman1, P. De Jonghe', E. De Vriendt1, D. Fabrizi, A. Malone1, M. Litt1 and S. J. Hayflick'. 'Oregon Health Sciences FitzPatricke, S. Malcolm3, C. Van Broeckhoven1 1 Neurogenetics University, Portland, 2Kyoto University School of Medicine, Japan, Laboratory, Flanders Interuniversity Institute for Biotechnology (VIB), Bom- 3University of Birmingham, UK, Institute of Neurological Sciences, Bunge Foundation (BBS) University of Antwerp (UIA), Antwerpen, Belgium. University of Siena, Italy. 2Westem General Hospitals, Edinburgh, Scotland, UK. Institute of Child Hallervorden-Spatz syndrome (HSS) is an autosomal recessive Health, London, England, UK. neurodegenerative disorder of childhood. Its features include extrapyramidal Clinical, neurophysiological and histopathological criteria are used to dysfunction, onset during the first two decades of life, and progression of signs differentiate Charcot-Marie-Tooth (CMT) neuropathy into 3 different and symptoms. HSS is characterized pathologically by massive iron deposits in subtypes. CMT1 or the hypertrophic form of CMT is characterized by the basal ganglia, specifically the globus pallidus and substantia nigra pars extensive de- and CMT2 is the neuronal form of CMT reticulata. We have mapped a gene for Hallervorden-Spatz syndrome to remyelination. with chromosome 20p12.3-p13 in 20 unrelated and ethnically axonal degeneration but without extensive alterations of the myelin sheaths. diverse families, axons motor indicating a single major locus (designated NBIA1, neurodegeneration with brain In the spinal form of CMT the of the anterior horn neurons iron accumulation, type 1) for this disease. seem to be affected while the sensory neurons are spared. We selected 12 Three families with Hallervorden-Spatz syndrome are not linked to this region. families with a CMT2 phenotype for linkage studies. The CMT1A duplication All affected individuals show MRI evidence of iron accumulation in the basal on chromosome 17 11.2 and mutations in the peripheral myelin protein ganglia, with extrapyramidal dysfunction and progression of disease. Post- genes PMP22, MPt and Cx32 were excluded. We performed linkage mortem examination of affected members of two families demonstrated typical analyses to confirm or exclude candidate loci for autosomal dominant CMT2 findings, including intracellular and extracellular deposition of iron-containing located on chromosomes 1p35-p36 (CMT2A), 3q13-q22 (CMT2B) and 7p14 Pigment and axonal spheroids in the globus pallidus. The inheritance in these (CMT2D). Linkage to these loci was excluded in most of our CMT2 families. families is consistent with an autosomal recessive pattem. Though the average In one CMT2 family suggestive evidence for linkage to 3q13-q22 was found. age of onset in these families is later than in linked families, at least one affected Some patients in this family have pronounced sensory disturbances leading individual from each unlinked family had onset in the first two decades of life. to poorly healing ulcerations. for CMT unusual sensory Furthermore, rate of progression of disease seemed to be slower, with individuals These, signs, living longer than those in families linked to chromosome 20p. suggest a distinct clinical phenotype for CMT2B. Exclusion of the locus for Pigmentary sensory on retinopathy was absent in unlinked families, though one individual did present hereditary neuropathy type (HSN I) chromosome 9q22 with optic atrophy. Three clinical features may help to distinguish this group from indicates that HSN with mild motor symptoms and CMT2 with prominent those with the classical form of this disease. These include later age of onset, sensory abnormalities are not allelic. Our results further support that CMT2 slower progression of disease and absence of retinal pathology. These families is a genetically heterogeneous disorder. provide evidence for locus heterogeneity in Hallervorden-Spatz syndrome.

2359 2360 Identification of candidate genes for autosomal dominant retinitis The endemic tyrolean infantile cirrhosis locus is not linked to the WND locus on pigmentosa (RP1 0) mapping to 7q31-q35. E T Tonkin, Z Mohamed, N chromosome 13. C.Wijmenga', T.MuIlet2, T.Brunrd, H.Feichtige&3, D.Schonitzer4, Haites, D J Shaw. Dept of Molecular and Cell Biology, University of R.Houwen5, WMullei5, L.A.Sandkuiyi, P.L.Pearson'. 'Dept. Human Genetics, Aberdeen, Scotland, UK. Utrecht Univ., Utrecht, hWilhelmina Children's Hospital, Utrecht, and 7Dept. Clinical Retinitis pigmentosa (RP) describes a group of clinically heterogeneous Genetics, Erasmus Univ., Rotterdam, Netherlands. 2Dept. Pediatrics, 3Pathology and hereditary retinopathies. Genetically, the disorder is complex, with 4Blood Group Serology, Univ.lnnsbruck, and 6Dept. Pediatrics, Community Hospital autosomal dominant (AD), autosomal recessive (AR), X-linked, maternal Reutte, Austria. (mitochondrial) and digenic forms reported. ADRP has been linked to at In Western countries, liver cirrhosis is rarely observed in children. In Tyrol, Austria, 138 cases of an endemic form of infantile cirrhosis were reported. This endemic least eight different loci, one of which, on 7q (RP10) has been further Tyrolean infantile cirrhosis (ETIC) is clinically and pathologically indistinguishable localised to 7q31-q35. from Indian childhood cirrhosis (ICC) and idiopathic copper toxicosis (ICT).Since the This region, flanked by the markers D7S686 and D7S530 is three phenotypes resemble that of early forms of hepatic copper overload seen in approximately 5cM in size and contains at least 30 ESTs. We will present Wilson's disease (WND), it is thought they might represent a distinct WND phenotype. data from our study of a number of these potential candidate genes, The WND gene has been identified; it maps to chromosome region 13q14.3. including one particular EST (clone 362906) known to be expressed within Since we assume that ETIC is an autosomal recessive trait, we felt encouraged to retinal tissue. Having mapped this cDNA to YAC 813f10 (between D7S680 investigate the possible role of the WND locus in the ETIC etiology. The increased and D7S514), we are currently identifying and sequencing the full length frequency of ETIC in a rural cluster in Tyrol suggests a founder effect, i.e. the transcript and screening our RP10 family for mutations. diseased individuals are descendants of one common ancestor who introduced the two ETIC mutation into the population many generations ago. Such distantly related We have also used YACs to directly select cDNAs from non- patients should share large regions of DNA around their shared disease gene, which affected retina. The YACs chosen span a previously described smaller would result in strong association between the disease and certain marker alleles. critical region of 1cM between D7S686 and D7S514. Information on Association studies were performed between ETIC and 9 genetic markers products resulting from this screen will be given at the meeting. spanning a region of 15 cM around the WND locus. Using a likelihood-based approach an over-representation of any marker-allele on more than 60% of the disease chromosomes could be excluded for all markers except D13S133. Only marker D13S133 shows weak statistical evidence for association. Similarly, the chromosome 13q14.3 marker genotypes were arranged into haplotypes to identify a shared chromosomal segment around the WND locus in ETIC- carriers. No region larger than 1 cM could be identified that was shared between several ETIC carriers. Assuming that ETIC is due to a founder effect, the association and allele-sharing experiments reveal no evidence that the ETIC phenotype is an allelic variant of Wilson's disease.

2361 2362 Polymorphic CTLA-4 alleles are not associated with rheumatoid Anticipation and lack of association with the HLA locus in familial arthritis (RA). J Worthington, A Barton, A Myerscough, S John, WEO0lier. Meniere's disease. Y.-G. Xie, K Fung', S.F Hall1, D.P. Lilicrap, S.A.M. ARC Epidemiology Research Unit, University of Manchester, UK. Taylor. Departments of Pathology and Otolaryngology', Kingston General Background: RA is an oligogenic disease for which only one susceptibilty Hospital and Queen's University, Kingston, Ontario, Canada. locus has been identified to date. In view of the T-cell mediated nature of Meniere's disease (MD) is an idiopathic condition with the symptoms of destruction of joint tissues, genes involved in T-cell regulation are potential fluctuating sensorineural hearing loss, episodic vertigo and tinnitus. candidates. Linkage and association to cytotoxic T lymphocyte associated-4 About 10% of cases of MD are familial and exhibit an autosomal dominant (CTLA4), a negative regulator of T-cell activation, has previously been pattern described in another autoimmune oligogenic disease; insulin dependent (type of inheritance with 60% penetrance. The etiology of MD remains obscure. I) diabetes mellitus in a Spanish population. It has been postulated that CTLA- Genetic predisposition has been suggested based on limited observations 4 may be a common susceptibility factor for all autoimmune diseases. of an association with the HLA locus on chromosome 6p. Recently Aim: To investigate linkage and association of CTLA-4 in RA in Spanish anticipation was reported in one study of familial MD. Here we present a and UK subjects. clinical and molecular study of two families with a total of six affected Methods: 100 RA affected sibling pair families from the ARC UK individuals. Autosomal dominant inheritance and anticipation (early onset Repository were genotyped for the highly informative microsatellite marker age and more severe symptoms in successive generations) were observed which maps to CflA-4 using fluorescence based automated genotyping. The in both families. Linkage analysis was performed by usinglfour polymorphic maximum likelihood-identical by descent method (SPLINK version 1.05) was loci on chromosome 6p: D6S89, D6S109, D6S105 and HLA-A. No used to test for evidence of linkage. Caucasoid UK RA patients (n=64) and significant evidence in favour of linkage with the HLA locus was obtained healthy caucasoid UK controls (n=96) were typed for an A/G biallelic and was likely excluded in the one family. Furthermore, due to the presence polymorphism in exon 1 of CTLA4 using PCR-RFLP (enzyme). A further 136 of anticipation, we are exporing the possibility that a trinucleotide repeat Spanish RA patients and 144 Spanish controls were also typed. Odds ratios expansion is involved in this condition. Such repeat expansions have been for the presence of the G allele were calculated. discovered in several other inherited disorders where anticipation has been Results: There was no significant evidence of linkage to CTLA-4 in the UK observed. A whole-genome screen for a trinucleotide repeat expansion in RA families (LOD 0.4 p=0.12). The frequency of the G allele-positive the affected individuals is ongoing the DIRECT method. This is only individuals did not differ using significantly in either the UK RA group (odds ratio = the second report of anticipation in familial MD, and our results also indicate 0.66; 95% Cl = 0.32 - p = 1.38; 0.23) or the Spanish RA group ( odds ratio = that genetic heterogeneity may exist within familial MD. 0.83; 95% Cl = 0.51 - 1.36; p = 0.42 ) compared with controls. Conclusion: We have no significant evidence of linkage or association between RA and CTLA-4. Our data does not support a role for CTLA-4 as an autoimmune gene however it is worthy of further investigation, particularly in conditions in which defects in apoptosis have been reported. A404 Published Abstracts: Linkage Mapping and Polymorphisms (cont.)

2363 2364 A novel sequence-based polymorphism in the 3'-untransiated region No evidence of linkage between HSCR and PDGFB on chromosome of the thrombopoletin gene and its association with human platelet 22. L. Yin1 2, Y. Shugart', G. Romeo' .2 1. International Agency for count. J Yankowitzt, SM Zeng', JC Murray2, JA Widness2, RG Strauss3. Research on Cancer, Lyon, France. 2. G. Gaslini Institute, Genoa, Italy Departments of Obstetrcs and Gynecology', Pediatrics2, and Pathology3, Hirschsprung disease (HSCR), or congenital megacolon, is a University of Iowa neurocristopathy characterized by the absence of intramural ganglion cells Thrombopoietin (TPO) is a critical regulator of platelet production. The in the hindgut. Segregation analysis shows that the disease has a complex human TPO gene has recently been characterized, but to our knowledge no genetic etiology. he RET proto-oncogene has been identified as the major polymorphism of the TPO gene has been reported. We designed primers to causative gene, while in a minor proportion of patients mutations have been amplify exon 1 to exon 6 and the 3'-untranslated region of the TPO gene. found in the Glial cell line-derived neurotrophic factor (GDNF), endothelin Using single-strand conformation polymorphism analysis (SSCP) we found beta receptor (EDNRB) and endothelin 3 (EDN3). Since mutations in the two polymorphic alleles in the 3'-untranslated region. Sequence analysis above genes account for less than 50% of HSCR cases, additional loci like revealed that the polymorphism was caused by a G to A transition at the Dom locus which causes a congenital megacolon phenotype in the position 5753. Linkage analysis showed strong linkage of the polymorphism mouse had to be investigated. This gene maps on a region of mouse to the D3S1262 locus, mapped on chromosome 3q26 (Zmax=11.33 at chromosome 15 syntenic to human chromosome 22 (Puliti A., et. al., 0=0.0680). This is consistent with the previous mapping of TPO to 3q26-28. Mammalian Genome 6:763-768, 1995). Four markers from 22q (D22S283, The allelic frequences, 0.56 for G and 0.44 for A, are in Hardy-Weinberg IL2RB, PDGFB and D22S274) were used to genotype 17 human pedigrees equilibrium in the Iowa population (p>0.05). We examined the allelic with recurrence of HSCR in two or more individuals who were all negative for mutations in the RET proto-oncogene. Using the computer frequency in 61 subjects with high (335.35 +/- 37.82 k/mm3) or low (184.40 program +/- Gene Hunter (version 1.1), a multipoint parametric analysis was performed 11.77 3) platelet counts obtained by the blood bank at the time of under both a dominant and a recessive model. Based on the assumed donation of blood products. These subjects were selected from a large genetic models, we excluded the possibility of linkage between the group of 812 healthy Iowans. Chi-square analysis demonstrated that the candidate markers and the proposed disease gene in most of our polymorphism was not significantly related to human platelet count pedigrees. Since PDGFB is a candidate gene for the Dom phenotype, we (p>0.05). Control of day-to-day platelet count may reflect interation of report the multipoint lod scores at this locus: -7.60 under the dominant several factors or may be unlinked to the TPO polymorphism we have model and -26.94 under the recessive model. Furthermore, a non- described. parametric method yielded similar negative results. More HSCR patients, with a more homogeneous genetic background, will be needed to study linkage disequilibrium with the same chromosome 22 markers.

2365 2366 Exclusion of Loci for Marfan and Stickler Syndromes, and Congenital Familial restrictive cardiomyopathy: Molecular genetic studies in a large Juvenile Glaucoma in Pedigrees of Familial Pathologic Myopia. T. Young', kindred. J Zhang', A Kumair', VJFerrans3, FJ Fricker1, MR Wallace'. 1University S.Ronan', L.Drahozal', S.Wildenberg', WOetting', L.Atwood', D.Wilkin2, and of Florida, Gainesville, FL, 2University of Texas Medical Branch, Galveston, TX, R.King'. 1Univ.of Minnesota, Minneapolis MN; 2Medical Genetics Branch, NIH, 3National Institutes of Health, Bethesda, MD. Bethesda MD. Restrictive cardiomyopathy (RC) is characterized by decreased compliance of Pathologic myopia, defined as nearsightedness greater than -6.00 diopters, myocardlum and impaired diastolic filling of the ventricle with intact systolic exhibits autofiomal dominant inheritance in some families with presentation usually function. Features include exercise intolerance, anasarca and chronic atrial within the first decade of life. Some connective tissue disorders and juvenile fibrillation. RC is usually sporadic, although it can occur in familial form secondary glaucoma also have an early presentation of high myopia. As linkage to specific to amyloidosis or hemochromatosis in an autosomal dominant fashion. Abnormal genetic markers has been established for the Stickler and Marfan syndromes, and accumulation of desmin, an intermediate filament protein that is a major juvenile glaucoma, we sought to explore the possibility of an association between component of the cytoskeleton in myocytes, can sometimes occur in RC. We have these disorcers and the high myopia in our families. Eleven families were a large 4-generation family in which RC segregates as an autosomal dominant ascertained With at least two individuals with -6.00 diopters or higher degrees of trait. Of the 34 family members, 19 were diagnosed as having RC, and we have myopia and no clinical evidence of connective tissue abnormalities. These families collected DNA from 24. Age of onset ranges from 3 to 50 years. Amyloidosis and contained 261 individuals and DNA was available for 118 ( 66 affected). The hemochromatosis were excluded and thus the molecular basis of RC in this family average age of diagnosis of myopia was 6.8 years (range 1.5-9.5). The average is still unknown. Immunohistochemical and electron microscopic studies in one spherical equivalent refractive error for the affected individuals was -10.549 patient's heart tissue showed abnormal granular deposits of desmin throughout the diopters (range -7.417 to -21.286). Candidate gene markers flanking or intragenic cytoplasm, rather than the normal pattern of 10 nm intermediate filaments located to the identified genes for the Stickler syndromes (12q13.1-q13.3 and 6p21.3), at the level of the Z bands. To test the hypothesis that the desmin gene causes Marfan syndrome (15q21.1), and the juvenile glaucoma locus on chromosome RC, Northem blot analysis of patient heart tissue was performed, which 1q21-q31 were analyzed. Lod scores were calculated using the MLINK feature of demonstrated that desmin gene expression is normal in affected heart. This the computer program FASTLINK. The cumulative lod score for Stickler syndrome suggests that the accumulation of desmin is due to post-translational effects. In type 1 using a polymorphic' VNTR located just 3' to the COL2A1 gene locus on addition, genetic linkage analysis was performed, which showed that RC in this chromosome 12q was -10.31. Intragenic markers D6S276 and D6$299 for the family is not caused by a structural abnormality in the desmin gene. A set of COL 1 A2 gene locus for Stickler syndrome type 2 yielded a cumulative lod score cytoskeleton and extracellular matrix genes was also tested for linkage and found of -8.70 and -10.09 respectively. Intragenic markers Dl 5S117 and DI 5S648 of the negative. This is in accordance with a study ruling out the desmin locus in a familial fibrillin gene locus for the Marfan syndrome yielded a cumulative lod'score of -8.20 myopathy (not RC)(Vicart et al., 1996). We are pursuing biochemical and -12.43 respectively. Markers Dl S196, DlS215, Dl S218, Dl S433 intragenic to characterization of this family, as well as linkage analysis to other possible the juvenile glaucoma locus also produced significantly negative lod scores ( -8.36, candidate genes and via a genome-wide linkage study. The identification of the RC -7.33, -8.33, -4.18 respectively). Familial pathologic myopia without systemic gene will add to our knowledge about development and long-term function of the features has been characterized in eleven families. Linkage was excluded from the heart. loci for Stickler and Marfan syndromes, and juvenile glaucoma.

Published Abstracts: Molecular Basis of Disorders with Complex Inheritance

2367 2368 Characterisation of an FBNl gene mutation, GIOl3R,In a child with neonatal Moaran Generation of clones with varying trinucleotide repeat sizes using and I (Cl) L.C. Ades' K.J. syndrome (nMFS) mitochondrial complex deficiency. 2, intrinsicinstability in E. coil. JM Boutell, LA Krusche, PS and AL Holman', K.C. Watson', M. MurrelF, J.T.R. Clarke3 and J. Chnstodoulou2. IMartan Harper Jones. Institute of Medical Genetics, UWCM, Cardiff CF4 4 XN, UK. Research Group and 2University Department of Paediatrics and Child Health, Royal Alexandra Hospital for Children, Sydney Australia; 3Division of Clinical Genetics, Hospital Several human hereditary diseases are associated with the expansion of for Sick Children, Toronto, Canada. CAG/CTG trinucleotide repeats. Previous studies of repeat instability in E. to and We previously reported an infant with nMFS and Cl enzyme deficiency. We speculated coli have used various methods generate study repeat containing that concurrence of these rare disorders might be dueto the combination of inherited inserts. We have used PCR on colony preparations, without further mutations affecting Cl and a de novo mutation of the fibrillin-1 (FRN1) gene. We screened treatment, to enable fast and accurate CAG repeat sizing. Plasmids the FBN1 gene using genomic DNA from this boy, and identified a de novo FBN1 gene generated from patient DNA containing the 5' end of the Huntington's gene mutation (G1013R) in exon 24, within the 2neonatal2 region of the gene. showed both host strain, orientation and length dependent instability of the The G1013R mutation has been described twice before, once in a case of presumed repeat tracts. We have extended this method to generate constructs of nMFS, and the other in a girl with the typical features of nMFS. Despite the severity of her without the need for a further of samples phenotype, this girl was still alive at the age of 15 years, albeit with severe physical different repeat sizes array patient with disabilities. Similarly, our patient is alive and relatively stable at age 7 years despite his or repeated cloning steps. For instance a plasmid 51 CAG repeats early severe presentation. yielded plasmidsof up to 72 CAG repeats after one round of transformation. To our knowledge, Cl deficiency and nMFS have never been described together before, Sequencing of plasmids thus generated proved them to be unchanged and a unifying mechanism remains unclear. Chance concurrence of these two disorders is apart from repeat This work also indicates that any preparation of one possibility, with the being heterozygous for mutations in a nuclear encoded Cl length. parents a tract contain a heterogeneous subunit, and the G1O13R mutation being a de novo FBN1 defect. FBN1 is an extracellular plasmid DNA containing repeat may matrix protein, but our case raises the question that FBN1 may also be located population of repeat sizes, and expansions and deletions may occur upon intracellularly, incorporatedinto the mitochondrial membranes.If this were the case, it may transfection into other systems, thus repeat size should be redetermined at be possible that the RC defect is a secondary (nongenetic) functional perturbation. each step. We suggest that more extensive biochemical evaluation of patients presenting with severe or nMFS may be warranted, particularly when there is significant hypotonia, neuromuscular and cardiac involvement. Persistent lactic acidemia (which may pass unrecognised) should be excluded, and if present muscle, liver and cultured skin fibroblast RC enzyme analyses should be considered. We are currently undertaking studies of RC enzymes in fibroblast cell strains from nMFS patients in order to investigate a possible causal relationship between FBN1 mutations and abnormalities of RC function. Published Abstracts: Molecular Basis of Disorders with Complex Inheritance (cont.) A405

2369 2370 Mitochondrial DNA and Rett Syndrome: A Search for a Genetic Marker. F Detection of polymorphic triplet repeats In the genomes of patients Castora' 2, C. Burgess3, C. Hart , D. Lewis2, S. Naidu4, M. Anvref3, Z. Zhang3, with a bipolar affective disorder. Peter Eichhammer (2), Anja Scholer (2), and F. Xiang3. Depts. Biochemistry1 and Neurology2, Eastern Virginia Med. Sch., Albert Putzhammer (2), Annette Walz (1), Josef-M. Aigner (2), Helmfned-E. Karolinska Sweden, Institute, Baltimore, MD Institute3,Stockholm, Kennedy Krieger Klein (2) and Jurgen Schlegel (1) . Department of Neuropathology, Rett Syndrome (RS) is a progressive, neurodevelopmental disorder University of Marburg, Germany (1) Department of Psychiatry, University of characterized by acquired autistic-like behavior, dementia, seizures, microcephaly, scoliosis, respiratory irregularities, and stereotypic hand movements. A number of Regensburg (2) studies have reported enzymatic and morphologic abnormalities in mitochondria of Genetic factors contribute substantially to the etiologys of bipolar disorder. RS patients. Defects in several respiratory complexes of the oxidative Recent studies support the proposal, that anticipation occurs in bipolar phosphorylation system (OXPHOS), suggesting a failure in general mitochondrial affective disorder. Anticipation of disease severity is a feature of some protein synthesis, coupled with the matnlineal inheritance of RS, have led us to neurodegenerative disorders, too. Expansions of trinucleotide repeats has investigate mitochondrial DNA (mtDNA) as a source of a genetic marker for RS. been shown to be the common genetic basis of these disorders. For this in We have undertaken an analysis of the 22 mitochondral tRNA genes and the large reason, we established a DNA fingerprinting technique for the screening of rRNA gene from a number of RS and control samples. We have utilized single expanded repeat motifs in genomes of patients, suffering from manic- strand conformational polymorphism (SSCP) gel electrophoresis and DNA depressive illness. The triplet repeat enriched arbitrarily primed polymerase sequencing. One (sample R93) of four RS patients tested from the US was found chain reaction (TREAP-PCR) is a modified PCR-based fingerprinting to have both a T-+C mutation at np 10463 in the tRNAArg region, and a G-4T technique using one primer with a repeat motif and 5' anchored specific mutation at np 15928 in the tRNAThr gene. Thirty three RS blood samples sequence, and a second primer whose nucleotide sequence is arbitrarily collected in Sweden were screened for the 10463 T-+C mutation. Five of these chosen.. Using this approach, 40 patients with a ICD-10 diagnoses of were found to possess the 10463 mutation. Three of these were found to possess bipolar the 15928 mutation as well. The combined U.S. and Swedish data indicate that 6/ affective disorder (ICD10: F31) and 25 healthy controls were investigated. 41 or 14.6% of RS patients possess the 10463 T--C point mutation and 67% of DNA fingerprints were generated with 30 different primer combinations using 6 those also possess the 15928 G-*T mutation. This compares to 3/22 or 13.6% of different triplet primers of 3 triplet categories (CAG, CCG, AAT) and 5 different controls having the 10463 mutation and one of these (33.3%/o) having the 15928 arbitrary primers. Differences seemed to occur dependent on the type of mutation. A particular region of interest in the rRNA gene was the sequence triplet, i.e. no significant differences could be found between patients and around nucleotide position (np) 2835, recently reported to be linked to RS by a controls using primers of the CAG or AAT type. However, TREAP-PCR Japanese group. None of our RS samples possessed this mutation. However, an polymorphisms using CCG triplet primers could be more often observed in the interesting mutation at np 3212 has been found in two of six RS patients examined. genomes of patients suffering from manic-depressive illness than in the DNAs This C-OT point mutation appears to be highly conserved as a search of lbenbank of the control group. These data are in accordance with recent reports sequences using BLAST or FASTA search programs indicated that zero of 84 demonstrating repeat expansion in psychoses using the RED technique which human mtDNA samples possessed the 3212 C-*T mutation. presented evidence for elongations which were more pronounced in manic- depressive than in schizophrenic patients.

2371 2372 Inhibition of Oxidative Phosphorylation (OXPHOS) in Fetal Heart and Kidney Analysis of non-amplifying (CAG)n alleles at the Huntington Disease Mitochondria by Transplacental Exposure of Pregnant Mammals to Cisplatin (HD) locus. L. C. Williams1, M. Hegde2, R. Nagappan2, K. Snow2 and D. or 3-azido-2',3-dideoxythymidine (AZT. M. Gerschenson', S. W Erhart1, L.M. R. Love1. 1School of Biological Sciences, University of Auckland, Private Anderson2, B.A. Diwan3, and M.C. Poirier . ILCCTP, NIH, Bethesda, MD, 2LCC, Bag 92019, Auckland, New Zealand; 2Molecular Genetics Laboratory, NCI-FCRDC, Frederick, MD, 3SAIC-FCRDC, Frederick, MD. Auckland Hospital, Auckland, New Zealand; Current address: Mayo Clinic, AZT and cisplatin are transplacental carcinogens that can also cause Rochester, Minnesota, USA. cardiomyopathies and nephrotoxicity in humans. The genotoxicity of these drugs occurs either by binding covalently (cisplatin) or becoming incorporated (AZT) into Huntington disease (HD) is an autosomal dominantly inherited DNA. Mitochondrial (mt) DNA encodes for thirteen polypeptides of enzyme neurodegenerative disorder. This disorder is associated with expansion of a complexes that are necessary for OXPHOS resulting in the synthesis of ATP. Thus polyglutamine-encoding (CAG) trinucleotide repeat at the 5' end of the damage to mt DNA may result in impairment of ATP production. Since cisplatin coding region of the HD gene. HD-affected individuals typically have an and AZT are both given to pregnant women, the consequences of drug exposure allele of at least 36 (CAG) repeats. Since most (CAG) repeats in the may be even more serious for the fetus, as compared to the mother. affected range number less than 100, PCR can be used as a diagnostic Transplacental exposure was examined in two animal models to investigate the tool. A concern in using a PCR-based assay is that apparent homozygosity effects of these compounds on OXPHOS capacity: for a normal (CAG) repeat length could actually represent an inability of the 1.Erythrocebus patas monkeys were exposed transplacentally to daily AZT assay an doses of 10 mg for the second half of gestation. OXPHOS enzyme specific to detect expanded allele. We have examined (CAG) repeat activities were measured in fetal mitochondria of skeletal muscle, heart, cortex, lengths in 69 apparently unrelated pedigrees (198 individuals tested). A null cerebellum, spleen, kidney, and liver. There was an overall decrease in NADH allele, associated with the use of primer Hunt 1 B, which covers the region dehydrogenase (Complex I) specific activity in all tissues in the AZT exposed fetal between the (CAG) repeat and the downstream (CCG) repeat, was monkeys compared to controls. The spleen and heart Complex specific activities detected in seven individuals from four families (5.8% of expanded alleles). were significantly decreased by 2-fold. The cytochrome c oxidase (Complex IV) There was no evidence for null alleles associated with a normal (CAG) specific activity was inhibited 10-fold only in the cardiac mitochondria of the AZT repeat allele. The entire contiguous polymorphic region of the HD gene, exposed fetal monkeys. encompassing the (CAG), (CCG) and (CCT) repeats were PCR amplified 2. Fisher rats were exposed to one dose of 5 mg cisplatin/kg body weight on for seven individuals from two families. The products were sequenced to day 18 of gestation and OXPHOS activities measured at birth. There were no determine the reason for the non-amplification, and the results compared dierences in Complex specific activities in skeletal muscle, heart, cortex, with fluorescent PCR determination of repeat lengths. cerebellum, spleen, kidney, and liver from exposed and nonexposed animals. However, Complex IV specific activity was decreased 50a% in heart and kidney of the cisplatin exposed fetal rats as compared to controls. Therefore, transplacental exposure to both drugs causes impairment of OXPHOS capacity in fetal heart and kidney.

2373 2374 Molecular analysis of Huntington's disease and dentatorubral- Case report: Three FMR-1 alleles detected in a developmentally pallidoluysian atrophy (DRPLA) in Koreans. S-I. Joo1, S. Shin1, B. delayed female child with a 46,XX karyotype. R. Kornreich', K. Kraiza', Jeor?, S.A. Lee3, S.S. Park1, H.I. Cho'. Dept. of Clinical Pathologyt and D. Debauchr?, C. Stevens2, C. Eng3, N. Mcintosh1, S. Gersent. 1DIANON Neurology2, Seoul National Univ College of Medicine and Seoul National Systems, Inc., Stratford, CT. 2University of Tennessee College of Medicine, Univ Hospital, Seoul, Korea; Dept. of Neurology, Asan Medical Center, Chattanooga, TN. 3Mount Sinai School of Medicine, New York, NY. University of Ulsan3, Seoul, Korea. We report a 3 year old female who was referred for fragile X DNA and Several human disorders are now known to be caused by expansion of chromosome analyses because of developmental delay, failure to thrive, unstable trinucleotide repeat sequences, and the analyses of those repeats and prenatal alcohol/drug exposure. PCR amplification across the CGG proved to be useful in the diagnosis and classification of the diseases. repeat region of the FMR-1 gene of DNA isolated from leukocytes revealed Huntington's disease (HD) and dentatorubral-pallidoluysian atrophy the presence of three alleles with tInucleotide repeat numbers of 19, 35, (DRPLA) are autosomal dominant neurodegenerative disorders and 53. Southern blot analysis was consistent with the presence of a small characterized by hereditary chorea with various symptoms. To assess the expansion mutation. Results were confirmed by repeat analyses of a frequencies of HD and DRPLA in Koreans, the authors performed the subsequently submitted blood sample. Routine cytogenetic analysis of G- molecular analysis of 29 families with familial or sporadic chorea, using banded metaphase cells from PHA stimulated peripheral blood lymphocyte CAG repeat analyses in HD and DRPLA genes. We found 19 families with cultures revealed a normal female (46,XX) karyotype in 20 of 20 cells HD (41 patients; CAG repeats 41-70) and 4 families with DRPLA (8 examined. To exclude chromosomal mosaicism, cytogenetic analysis of patients; CAG repeats 60-71). The CAG repeats of HD and DRPLA genes primary skin fibroblasts derived from a skin biopsy was performed. A normal in normal Korean individuals were 15-30 (150 chromosomes) and 8-24 (136 female karyotype (46,XX) in 60 of 60 metaphases examined was obtained, chromosomes), respectively. This study suggests that DRPLA frequency excluding the presence of a mosaic cell line with a frequency of 5% or might be much higher in Koreans like in Japanese than in Caucasian, and greater at the 95% confidence level. Sixty additional metaphase cells were the molecular diagnosis of DRPLA should be included in the diagnostic analyzed from the remaining pellet of the original peripheral blood culture. process of chorea tn Koreans. All cells were observed to have a normal female karyotype excluding the presence of a mosaic cell line with a frequency of 4% or greater at the 95% confidence level. STR analysis of other X-chromosome regions are in progress to address the question whether this individual has a very low grade mosaicism of , or if her FMR-1 results are due to allele instability. Currently, preliminary analysis does not appear to support the presence of low grade mosaicism. A406 Published Abstracts: Molecular Basis of Disorders with Complex Inheritance (cont.)

2375 2376 Trinucleotide repeats and neuropsychiatric disorders. RL Margolis, JJ Modifier Genes In Waardenburg Syndrome. R. J. Morell and T. B. Kleiderlein, HA Bruce, MG McInnis, CA Ross. Div. of Neurobiology, Dept. of Friedman. National Institute on Deafness and Other Communication Psychiatry, Johns Hopkins Univ. Sch. of Med., Baltimore, MD 21287 Disorders / NIH, Bethesda, Maryland, USA. Most neuropsychiatric disorders are characterized by complex patterns Waardenburg syndrome (WS) is a clinically and genetically of inheritance, including, in some instances, genetic anticipation. We and heterogeneous disease accounting for more than 2% of the congenitally others have hypothesized that trinucleotide repeat expansion mutations deaf population. It is characterized by deafness in association with might explain some of this complexity, and serve as susceptibility factors for pigmentary anomalies and various other defects of neural crest-derived bipolar affective disorder, schizophrenia, or autism. Also, expansions of tissues. The majority of WS are associated with mutations in one of two CAG repeats encoding glutamine may cause neurodegenerative disorders transcription factors, PAX3 and MITF, which are expressed by the neural in addition to those already known to stem from this particular mutation. To crest early in development. It is not known how PAX3 or MITF mutations test these hypotheses, we have cloned cDNAs containing AAT, CCA, CAG, affect neural crest development. Moreover, there is a high degree of and CCG repeats from human brain libraries. The clones have been variation in the expressivity of the clinical features of WS, even within assigned to specific chromosomal loci, and we have determined the extent families segregating a single PAX3 or MITF mutation. Stochastic events of length polymorphism for each repeat. Our analysis suggests that the influencing the migration of the neural crest cell population through the extent of polymorphism varies by repeat type; AAT/TAA repeats are more developing embryo might account for this variability. Conversely, the polymorphic than either CAG/CTG or CCA/GGT repeats. CCG repeats genetic background might be the most significant component. Mouse appear disproportionately in 5-untranslated regions. A number of the clones models of WS support both hypotheses, depending on which trait is being correspond to genes of particular interest. For instance, we have identified a investigated. Recently, we have published evidence that deafness in WS is cDNA that encodes 40 consecutive glutamines, yet is unassociated with attributable to genetic background and not stochastic events (Morell et al., disease. We have recently found CCG repeats in the human version of the J. Med. Genet. 34: 447-452, 1997). We have also shown that ocular mouse zic2 gene, the 5' UTR of a G protein, and in a variety of previously albinism, a condition that is normally inherited in an autosomal recessive unknown genes. While characterizing these genes, we are continuing to manner, is manifested as part of WS when individuals inherit both an MITF test for an association between repeat expansions and degenerative mutation and a specific allele of TYR 0 a gene whose expression is neurological disorders, bipolar affective disorder, schizophrenia, and autism. regulated by MITF(Morell et al., Hum. Mol. Genet. 6: 659-664, 1997). We hypothesize that mutations and polymorphisms in genes regulated by PAX3 and MITF could be the modifiers of deafness in WS. Their identification could provide candidates for non-syndromic deafness loci.

2377 2378 Spinocerebellar Ataxias analysis of CAG expansions at SCAl, SCA2, Molecular analysis of four types of spinocerebellar ataxias (SCA1, SCA3 and SCA6 loci in Italian families. 'A. Nardacchione, 2E. Dragone, SCA2, SCA3/MJD, SCA6) in Koreans. S.S. Park', S-I. Joo1, S. Shin1, H.I. 3L.Orsi, 3P Mortara, 3A. Franco, 3E. Pavanelli, 2E. Grosso, 1G. Matullo, Cho1, B. Jeon2. Dept. of Clinical Pathology' and Neurology2, Seoul 1"2A. Carbonara, 4G. Restagno. 1Dipartimento di Genetica, Biologia e National Univ College of Medicine and Seoul National Univ Hospital, Seoul, Chimica Medica, University di Torino, 2Unitb Operativa di Genetica Medica, Korea. Azienda Ospedaliera San Giovanni Battista, Torino, 3Dipartimento di Spinocerebellar ataxias (SCAs) are a heterogeneous group of Neuroscienze, Universita di Torino, 4Laboratorio di Genetica Molecolare, neurodegenerative disorders of which clinical classification has been very Azienda Ospedaliera OIRM-S.Anna, Torino, Italy. difficult and unreliable. The CAG repeat expansions in the causative genes were identified as the basic causes of several types of SCAs, and began to Autosomal dominant spinocerebellar ataxias (SCAs) are a clinically and with ataxia. To genetically heterogeneous group of neurologic diseases. Almost seven use for the diagnoses and classifications of the patients SCA2 and assess the frequency of each type of SCAs, the authors performed the different loci are involved in the etiology of SCAs. Recently, molecular analysis of 49 families with familial or sporadic ataxias, using SCA6 genes have been cloned: in both, the mutation involves the We as in and SCA3/MJD genes. CAG repeats analyses in SCA1, SCA2, SCA3/MJD, and SCA6 genes. expansion of an unstable CAG repeat, SCA1 found 13 family cases with CAG repeats in the four loci : two family cases of We analized the CAG expansion of SCA1, SCA2, SCA3/MJD, and of two or more affected SCA1 (48-60 repeats), six of SCA2 (38-48 repeats), four SCA3/MJD (56- SCA6 in 22 Italian families with patients. SCA1 79 repeats), and one of SCA6 (23 repeats only). The CAG repeat ranges of mutations were found in 4 families (18%); SCA2 mutations in 7 families in normal Korean individuals (32%); SCA6 mutations in 1 (5%). SCA1 and SCA2 families show a clear SCA1, SCA2, SCA3/MJD and SCA6 genes In there is a were 16-39 (204 chromosomes), 19-24 (92 chromosomes; Twenty-two clinical heterogeneity and anticipation. SCA2 families, repeats were 88%), 14-35 (188 chromosomes; Fourteen repeats were correlation between the number of CAG repeats and age of onset (r=-0.633, 42%), and 8-17 (92 chromosomes), respectively. Up to now SCA2 is the p=0.002). We did not found SCA3/MJD mutations in any patient analyzed. most prevalent type in Korean patients with SCA, and this study sugests All the sporadic cases were investigated for the SCA1, SCA2, SCA3/ that clinical analysis and stepwise molecular tests of SCA2, SCA3/MJD and MJD and SCA6 mutations: none was positive. useful in the and classification of SCAs On the basis of these data we can: then SCA1 could be quite diagnosis 1. confirm the genetic heterogeneity in Italian families; in Koreans. 2. confirm the low frequency of SCA3/MJD in Italian population; 3. confirm the intrafamilial clinical heterogeneity; 4. correlate, for SCA2 only, the size of expansion with the age of onset.

2379 2380 Intrafamiltal polymorphisms In triplet repeat detection in three large Screening ataxia patients for trinucleotide expansions in the known families with cases of manic-depressive disorders in different genes-experience in a service laboratory. M. Skuterud, P. Florio, D. generations by using TREAP-PCR. Putzhammer A. , Eichhammer P., Kennedy, S. Mosher and D.J. Allingharn-Hawkins. North York General Hospital, Scholer A. , Rohrmeier T. , Schlegel J. *, Klein H. E. Department of Ontario, Canada University of Regensburg, Germany Department of The spinocerebellar ataxias (SCA) are a genetically heterogeneous, clinically Psychiatry, indistinguishable group of neurodegenerative disorders characterized by ataxia, Neuropathology, University of Marburg, Germany dysphagia and dysarihna with onset of symptoms usually in the third or fourth In the last years it became more and more clear that expansions of decade of life. Four genes responsible for autosomal dominant SCA (SCA1 on trinucleotide repeats are the main factor in the etiology of some inherited chromosome 6, SCA2 on chromosome 12, SCA3 or Machado-Joseph disease on neurodegenerative diseases. Recent studies have shown that triplet-repeat chromosome 14 and SCA6 on chromosome 19) had been cloned and several others expansions could also be the genetic basis of psychiatric disorders. Triplet- have been mapped. In all genes cloned to date, the causative mutation is expansion trinucleotide. Other diseases with a similar clinical include repeat expansions were for example found in manic-depressive disorders of a CAG phenotype FRDA as well as in schizophrenia by using RED techniques (repeat expansion FriedreichIs ataxia (FRDA) and dentatorubral-pallidoluysian atrophy (DRPLA). the is an autosomal recessive disorder caused by expansion of a GAA trinudeotide in detection). We used a modified PCR-based fingerprinting technique, the X25 gene on chromosome 9. DRPLA is a rare autosomal dominant disorder triplet repeat enriched arbitrarily primed polymerase chain reaction (TREAP- caused by expansion of a CAG repeat in the B37 gene on chromosome 12. PCR), to investigate the genomes of members of three large families with Because of the difficulty in distinguishing these disorders at the clinical level, manifestations of manic-depressive disorders in different generations. The samples are often referred to the service laboratory for SCA1, 2 and 3 testing as TREAP-PCR uses one 5anchored specific primer with a repeat motif and a well as FRDA and DRPLA testing in some cases. To date, SCA1 has been positive second primer with arbitrarily chosen nucleotide sequence. Using primers in 1.1% of samples, SCA2 in 15.4% and SCA3 in 10.8%. Interestingly, 3 with different triplet repeat motifs we compared the intra-familial independent (5.5%) intermediate SCA1 alleles (i.e. 37 to 40 repeats) were also polymorphisms in affected and not affected members of the same family. identified among symptomatic individuals. Among 47 samples referred for FRDA testing, 6 were also referred for SCA testing and 3 were positive for FRDA while the mutant gene was not identified for the other 3. Overall, the positive rate for FRDA testing is 57.7% for affected individuals and 61.9% for possible carriers. No cases of DRPLA have been identified among the 23 patients tested, 18 of whom were referred for SCA testing as well. The experience of this laboratory suggests that patients with non-specific ataxia should be tested sequentially for SCA2, SCA3, FRDA and SCA1 to achieve the highest rate of diagnosis with a minimum of unnecessary testing. DRPLA testing appears gratuitous among this group of patients. SCA6 testing is currently being implemented and will be added to the panel imminentiv. Published Abstracts: Molecular Basis of Disorders with Complex Inheritance (cont.) A407

2381 2382 Establishment of a differential diagnostic of patients with Mental Retardation Identical premutation size In multiple tissues of a male fragile X based on Southern-blot and PCR considering the frequency of Fragile X carrier. A. K. Taylor', F. Tassone1 .2, L. Staley-Gane2, and R. J. trinucleotide repeats from normal population in Rio de Janeiro, Brazil. C. C. Hagerman2 3. 'Kimball Genetics, Inc., Denver, CO, 2Fragile X Treatment Sucharov', F. R. Varandas2, E. Rondinellf1, G. Carakushansky3, R. S. Moura- and Research Center, Child Development Unit, The Children's Hospital, Neto2. Instituto de Biofisica Carlos Chagas Filhol, Depto. de Genetica2, Instituto Denver, CO, and 3Department of Pediatrics, UCHSC, Denver, CO. de Pediatria e Puericultura Martagao Gesteira3 - UFRJ Brazil. Studies of the FMR1 mutation in different tissues are important to further our Fragile X Syndrome is associated with a fragile site in the long arm of understanding both of CGG repeat expansion in development and of the chromosome X. The gene responsible for the disease, FMR1, contains an in expansion frequency and possible clinical significance of tissue heterogeneity fragile X of a CGG repeat in the 5' untranslated region, with the major alleles as a in falling within the 29-30 repeat size syndrome. With a few exceptions, such cryptic full mutation the epithelium in the normal population. An expansion of more an a in most cases than 200 repeats is considered a of affected male with only premutation the blood, reported full mutation and people with alleles within this no differences in mutation or size between expansion range express the syndrome. Alleles sizes ranging from 43 to 200 have shown significant category copies fall inside the pre-mutation category. It is well known that the Brazilian tissues. There have been few FMR1 studies of multiple tissues from autopsy. We population is composite from three different ethnical make-up. In order to study have previously found near identical full mutation patterns by Southern blot the numerous an We pattern of repeat size in a normal sample of this population we have analyzed 100 analysis of postmortem tissues of affected male. have also chromosomes of normal unrelated individuals and compared to a related described a high functioning fragile X male with an unmethylated full mutation in Caucasian population distribution. It is important for diagnosis, to show that the the blood and most of the brain but a fully methylated full mutation in the parietal range of allele distribution is comparable to other populations, so the same pattern lobe and all other organs tested. of expansion can be expected. A total of 21 distinct repeat sizes were identified by We report here the results from Southern blot and PCR analysis of blood and PCR amplification and our mode of distribution falls within the 30 repeats, followed 13 postmortem tissues from a male carrier of fragile X syndrome. This 85 year old by the 29 repeat size. This data was them compared to the Caucasian population male had mood lability but no learning disabilities or features of fragile X showing that the frequency of distribution is in concordance with what was syndrome and was considered to be clinically unaffected. A premutation of 94 observed in other populations. To analyze patients with mental retardation for the CGG repeats was found in all tissues studied including heart, lung, liver, kidney, Fragile X Syndrome we have subcdoned a new fragment within the FMR-1 gene skin, testis, and 7 regions of the brain. This is the first description of FMR1 that can be used as a probe for diagnostic (Pt1 probe, 1.2 Kb). We have analyzed mutation status in multiple tissues of a premutation carrier male. The presence of 24 patients by PCR and by Southern Blot using the Pt1 probe. Two of these identical premutation size in all tissues supports the concept of somatic stability in patients have shown an expansion of about 0.8 Kb at the FMR1 region by the fragile X premutation carriers. Southern Blot while the PCR showed no amplification in one case and an We were also able to observe intergenerational changes in FMR1 mutation abnormal amplification in the other. This study is of great importance as it shows size by testing the blood of two daughters of this carner male. His germline that the distribution pattern of the repeats is the same as in other populations, and premutation of 94 repeats increased to 95 repeats in one daughter and to 111 that a differential diagnosis using both the PCR and the Southern Techniques can repeats in the other. These small changes in CGG repeat number are typical for be done. the passage of a premutation from a male to the next generation.

2383 2384 Prader-Wilii Syndrome RT-PCR analysis using cultured cells fixed for Unstable DNA in the etiology of major psychoses and mood disorder. JB cytogenetic studies. J. H. Tepperberl1, 8. Williford1, L. N. W. Kam-Morgan1, C. G. Vincent', SV Panikh, A Petronis1, HY Meltze,3, R Krof', M Kovacs4, CL Barr5, JL Binnie1, M. Eisenbergl, P. N. Mowrey L. K. Gadi1, G. Mengden2, G. Khod 2, P. R. Kennedy. 'Neurogenetics Section, 2Mood Disorders Clinic, Clarke Institute of Papenhausen'. LabCorp 1912 Alexander Drive, RTP NC 277091, SW Genetics San Psychiatry, University of Toronto; 3Vanderbilt University, Nashville TN; 4University Antonio, TX 782292. of Pittsburgh School of Medicine, Department of Psychiatry and Western Prader-Willi syndrome (PWS) is characterized by severe hypotonia, hyperphagia, Psychiatric Institute and Clinic, Pittsburgh, PA; 5Dept. Of Psychiatry, The Hospital mild MR and obesity. A candidate gene for PWS, small nuclear ribonucleoprotein N for of (SNRPN), mapped to 15qi 1.2 appears to be functionally imprinted in these patients. Sick Children; Department Psychiatry, University of Toronto, Canada. The primary causes of PWS are: 1) a detectable paternal del(15)(ql 1q13)(approx. There has recently been much scientific interest in the possibility that unstable 70-75% of all reported PWS cases); 2) maternal uniparental disomy (UPD) (25-30% DNA may be involved in the etiology of schizophrenia and bipolar affective of PWS cases); and 3) rare paternal methylation defects or undetectable mutations. disorder (BPAD), following several reports of increased frequency of large CAG/ PWS is correlated with the lack of expression of the paternal SNRPN gene and is CTG trinucleotide repeats in affected individuals. Summarizing our own analyses consistent with one of the 3 mechanisms listed above. A recently reported diagnostic using the Repeat Expansion Detection (RED) technique, we have performed test for PWS uses RT-PCR for SNRPN gene expression from cell cultures or matched control pair studies for both disorders and have found no significant peripheral blood obtained in EDTA or sodium herparin vacutainers (Glenn et. al., differences in CAG/CTG repeat sizes for schizophrenia or for bipolar disorder 1993; Wevrick and Franke, 1996). In the present study, peripheral blood from (p=0.2102, n=99; p=0.4209, n=91, respectively). In addition no intergenerational patients previously identified with either a deletion at 15q11.2 by FISH or maternal expansions were observed in schizophrenia families. Furthermore, no differences UPD 15 was obtained in sodium heparin vacutainers, cultured, fixed in 3:1 in clinical features were observed between the bipolar affecteds with large methanol:acetic acid (Carnoy fixative) and analyzed by RT-PCR for SNRPN gene repeats and those with small repeats. However, our studies of an unstable CTG expression. Analyses of the extracted RNA from the fixed pellet showed these repeat, known as 7,6A, in a transcription factor gene (SEF1i-2) located at patients lack SNRPN gene expression. Samples referred for PWS that were not chromosome 18q21.1 suggest that 85% of bipolar and 50% of schizophrenic deleted by FISH and showed biparental chromosome 15 inheritance had normal affected individuals with large RED repeats had expanded CTG alleles at 7,6A. SNRPN expression. These observations appear to be consistent with those studies Moreover, we did not find a higher frequency of expanded 7,6A alleles for either using RNA extracted directly from blood samples collected in EDTA or sodium disorder at (p=0.4725, n=1 00; p=0.2742, n=97, respectively). Thus it appears that heparin vacutainers. large unstable CAG/CTG repeats at this locus are not involved in disease The advantages of using Camoy pellets PWS testing are: fixed for 1) it allows etiology. Possible evidence of unstable DNA has been identified in an individual routine cytogenetics, RT-PCR, FISH, and UPD (parents to be required) performed with childhood (pre-puberty) onset depression, where an actual CAG/CTG repeat from one sodium heparin vacutainer; 2) patients need only one blood draw; and 3) it expansion event has been detected from the parental generation to provides clean gel banding patterns. the affected child. The 7,6A repeat has been ruled out for this expansion, so it is possible that In conclusion, RT-PCR using fixed pellet offers a reliable, rapid and accurate initial test for ruling out PWS. Furthermore, PWS patients who lack SNRPN another rare unstable CAG/CTG repeat is present that may be pathogenic. gene Attempts are currently underway to localise and characterise this expression can be rapidly analyzed for a deletion using FISH. repeat.

2385 EXCLUSION OF CAG REPEAT EXPANSION IN THE DNA POLYMERASE y GENE AS A FREQUENT CAUSE OF MITOCHONDRIAL DNA DELETIONS. Jurgen-Christoph von KLEIST-RETZOW, Pierre RUSTIN, Arnold MUNNICH, Agnes ROTIG. INSERM U393 and Departement de Genetique, Hopital Necker, 149 rue de Sevres, 75015 Paris, France) Autosomal dominant inheritance of multiple mitochondrial DNA deletions has been occasionally observed in progressive external ophthalmoplegia and the disease causing genes have been mapped to chromosomes 10q23.3-24.3 and 3p14.1-21.2. Among the genes known to be involved in mtDNA control in human, the recently cloned mtDNA polymerase y represents an attractive candidate for mtDNA deletions, especially as this gene contains a (CAG)10 trinucleotide repeat encoding a polyglutamine tract within the first exon of the gene. We therefore investigated the possible involvement of CAG expansion of the DNA polymerase y gene in patients harboring mtDNA deletions. None of the patients carrying either single or multiple mtDNA deletions presented trinucleotide expansion of their DNA polymerase y gene. Yet, the role of the DNA polymerase y gene cannot be formally excluded as genetic heterogeneity of multiple mtDNA deletions suggests that various loci could be involved in the triggering of the deletions. A408 Published Abstracts: Molecular Basis of Mendelian Disorders

2386 2387 Familial early-onset Alzheimer disease associated with mutations in Sensitivity to oxidative stress of fibroblasts from patients with familial presenilin-1. D.N. Abuelo1, A. Walsh1, K.S. Kosik2, WK. SeltzeA3. 'Dept of and sporadic amyotrophic lateral sclerosis. T. Aguirre 1.2, L. Van Den Pediatrics, Rhode Island Hospital, Providence and Brown University School Bosch 1, G. Matthijs2, J. j. Cassiman 2 and W Robberecht 1. 1 Laboratory of Medicine, 2Brigham and Women's Hospital, Harvard Medical School, of Neurobiology and2 Center for Human Genetics. University of Leuven, Boston, Mass., 3Athena Diagnostics, Inc., Worcester, Mass. Leuven, Belgium. Alzheimer disease (AD) is the most common cause of dementia in the elderly Mutations of the SODi gene have been identified in about 15-20% of and is characterized by defects in recent memory, progressive intellectual decline familial Amyotrophic Lateral Sclerosis (FALS) cases. In the remaining famiial and personality changes. Approximately 5-10% of patients show mendelian cases or in sporadic ALS (SALS), no genetic defect has been demostrated. inheritance and many of these have onset of disease as early as the 3rd or 4th However, the clinical and pathological expressions of the disease in FALS and decades. Early-onset familial Alzheimer disease (EOFAD) has been associated SALS patents are identical, suggesting a common etiology. Fibroblasts of with mutations in 3 different genes: the amyloid precursor gene on chromosome Belgian FALS patients with SOD1 mutatIons (G93C, L38V and D9OA) and age 21, presenilin-1 (PS1) on chromosome 14, and presenilin-2 on chromosome 1. matched controls were treated with H202, the NO donor SIN-1 as well as Most of the mutations occur at various loci within the PS1 We describe a gene. serum deprivation. Cell survival was assessed using the MTS assay. Not family with autopsy-proven EOFAD and two mutations in the PS1 gene. effect of serum withdrawal was observed. On the other FALS fibroblasts Case 1: A Caucasian male with a history of severe head trauma at age 22 died hand, at age 38 after a dementing illness. Autopsy revealed extensive neuritic plaques were significantly more sensitive to SIN-1 and H202. Analysis of the different and neurofibrillary tangles typical for AD. Case 2: One of his 4 children had groups shows that this effect is evident for the L38V and G93C fibroblasts but congenital malformations of the toes, a single central incisor, microcephaly and not for the D9OA cells. In order to investigate if this increased sensitivity was mild mental retardation. She developed dementia in her early 30s. Two nucleotide also present in SALS fibroblasts, the G93C (n=5) FALS fibroblasts were substitutions were found in her presenilin-1 gene: a T to C substitution at codon compared to ae and sex matched SALS fibroblasts. The SALS fibroblasts 143 which changes an isoleucine to a threonine (reported previously in a were significant more sensitive to SIN-1 than FALS (G93C) fibroblasts but Caucasian family), and an A to G substitution at codon 439, which changes an less sensitive to H202. These results suggest that In both cases there is an isoleucine to a valine. Additional studies are in progress to determine whether the increased sensitivity to oxidative stress that seems to be specific. However, two mutations are in cis or in trans configuration and to determine whether the our findings are compatible with a crucial role for H202 in cells harboring an codon 439 base substitution has phenotypic effects or represents a benign polymorphism. SODi mutation, as has been suggested in biochemical studies.

2388 2389 Involvement of abnormal type V collagen in a family with recurrent identification and analysis of non-deletion dystrophin mutations. osteogenesis imperfecta. M.G.EM. Ausemsl, G. Pals2, FA. Beemerl, M.A.Austint2, B.R.Korfn. IBoston College, Chestnuthill, MA,2Children's A.C. Nicholls3. iClinical Genetics Center Utrecht, 2Dept. of Clinical Hospital, Boston, MA Genetics, Vrije Universiteit Amsterdam, The Netherlands, 3Strangeways We have undertaken an analysis of the dystrophin gene in males with Research Laboratory, Cambridge, UK. Duchenne or Becker muscular dystrophy in an effort to identify non-deletion Osteogenesis imperfecta (01) is a generalized connective tissue disorder mutations. Primers were made to amplify 74 of the dystrophin exons from DNA. PCR were submitted to and characterized by frequent fractures. 01 is a clinically and genetically genomic products SSCP analysis any heterogeneous condition, usually caused by dominant mutations in the variant patterns were sequenced. Among DNA samples from 90 patients, COLIAl or COLlA2 genes that result in abnormal collagen 1. Collagen V co- 10 mutations were identified for an 11% detection rate. Four were single assembles with collagen and plays an important role in collagen base pair changes that introduced stop codons. The remaining six were fibrillogenesis. Recently it has been shown that collagen V is causally involved mutations that altered splicing consensus sequences and are presumed to in Ehlers-Danlos syndrome type and 11. Up to now, no collagen V affect splicing. abnormalities have been detected in other connective tissue disorders. We Including these ten mutations, a total of 144 unique mutations have report on a family with recurrent osteogenesis imperfecta and an abnormality been reported to date in the literature and DMD Data Pages (httpJ/ of collagen V. Two male sibs, born to healthy consanguineous parents ruly7O.MedFac.LeidenUniv.nVduchenne/). In order to gain a better presented with spontaneous fractures in the first year of life. Physical understanding of the mechanisms that lead to mutation, we nave examined examination revealed hypertelorism and retrognathia. The youngest boy also the sequence environment of the mutations for common nucleotide had a large inguinal hernia on both sides and a large umbilical hernia which sequences, small repeats, CpG and CpNpG motifs, etc. Sixty-two percent were surgically corrected. Radiography revealed wormian bones of the skull, (62%) of mutations are base pair substitutions. Forty-five percent (45%) of and osteoporosis of the spine and long bones. Collagen studies in cultured these occur at CpG dinucleotides or CpNpG trinucleotides, indicating a skin fibroblasts showed normal collagen and a normal collagen I/l1l ratio. possible role of methlyation-mediated deamination. Thirty one (31%) are However, there was a slow migration of the ?1 (V) and ?2(V) chains as microdeletions and 7% are insertions. Seventy-three percent (73%) of compared to controls, indicating overmodification of the ? chains of collagen V. single base insertions or deletions involve homonucleotide repeats. We Linkage analysis showed that the children were not both homozygous at either have analyzed the sequence environment of a set of mutations in exon 44, the COL5A1 or COL5A2 loci, thereby excluding a recessive mutation in these 2 microdeletions we identified, one of one base pair and one of for including genes. Germline mosaicism in one of the parents is more likely. Screening five base pairs. This region forms a hairpin, which may be related to the a dominant mutation in the COL5A1 and COL5A2 genes is being performed. occurrence of these mutations. Our that specific for V are findings may suggest In conclusion, our family demonstrates that abnormalities collagen of are at risk of mutation, allowing the mutation in a of regions dystrophin greater also involved subtype osteogenesis imperfecta. analysis to focus first on these sites.

2390 2391 Diversity in the structural rearrangements of Red and Green color Detection of DNA Duplications In Turkish CMT1 Patients. Bissar- genes in individuals affected with Blue Cone Monochromacy. R. Tadmoun, Nisrnie (1); Y., Gulsen Parman(2); F., Deymeer (2); C., Ozdemir Ayyagari, Y. Toda, L. Kakuk, J. Szczsny, E. Bingham, P.A. Sieving. Kellogg (2); E., Battaloglu (1). (1) Bogazici University, Department of Molecular Eye Center, University of Michigan, Ann Arbor, Ml 48105. Biology and Genetics, 80815 Bebek, Istanbul, Turkey; (2) Istanbul Blue cone monochromacy(BCM) is an X-linked disease. We have University Medical School, Department of Neurology, Istanbul, Turkey. studied the structure of red and green color genes in 23 affected males from Charcot-marie-Tooth typelA (CMT1A) is an autosomal dominant disorder of 11 families. Phenotypic symptoms of BCM observed in these families were the peripheral nervous system characterized by progressive weakness and highly diverse. Visual acuities of the affected males ranged from 20/80 to 20/ atrophy of distal limb muscles. Molecular genetic analyses of CMT1A patients 400. Some affected males could identify up to four Ishihara plates. The rod revealed a 1.5 Mb tandem DNA duplication in the chromosome 17p11.2-p12 ERG was normal in 13 males but was reduced in 10 males. Eight males region to which the peripheral myelin protein 22 (PMP22) was mapped. Patients cone ERG of 8-30 microV normal witHhHMSN1A show a dosage effect when more than one allele is present. We had photopic responses (15-50/% EcoRi- amplitude). In one family two affected brothers had bilateral macular performed Southem analysis in 26 patients using one Mspl and one Hincil RFLPs from the The used were EW401 HE, and atrophy. In a different family one affected male had 20/200 acuity but duplication region. probes PTZ-G8R1, and gene dosage was assessed visually from southern blots. This normal color while an affected had nine and D15 discrimination, nephew major was complemented by analysis of the microsatellite marker RM1 1-GT D15 errors. detection of the duplication on silver-stained gels. In this case, the duplication was Structure of the color genes was analyzed using Southern blot analysis, confirmed by the presence of either three bands representing three alleles, or two PCR and sequencing. Ten out of the 11 families studied showed bands with one of them double the intensity of the other. These intensities were rearrangements in the structure of red/green color genes. In one family only compared using densitometry. The same strategy was used to detect HNPP the locus control region of red/green pigment genes was missing. In 7 deletions which are reciprocal products of the same mechanism leading to families part of the red gene was missing besides the locus control region. CMT1A duplications. patients revealed a 1.5 Mb tandem DNA duplication in the In one family both the locus control region and the entire red gene are chromosome 17pD 1.2-p12 region to which the peripheral myelin protein 22 missing. One carrier female in this family showed pathologic pigmentary (PMP22) was mapped. Patients with HMSN1A show a dosage effect changes of perimacular retinal pigment epithelium. One family had intact than one allele is present. We performed Southem analysis patients using action spectra for red and blue cones (Stiles' pi and pil) and BCM three Mspl and one EcoRl-Hincil RFLPs from the duplication region. probes phenotype, but no major rearrangements in red and green genes and was used were VAW409R3a, VAW409R1ib, EW401HE, PTZ-G8R1, gene also negative for an earlier reported P307L mutation. Overall the dosage was assessed visually from southern blots. complemented by RMI 1-GT and detection rearrangements found in color genes are highly variable. No obvious analysis of the microsatellite marker duplication were correlation was observed between the and the silver-stained gels. Band intensities compared using densitometry. phenotype genotype. HNPP deletions Foundation Blindness same strategy was used to detect reciprocal products Funding: Fighting A of the same mechanism leading to CMT1 duplications. Published Abstracts: Molecular Basis of Mendelian Disorders (cont.) A409

2392 2393 Detection of the Intron 8-5T allele in the CFTR gene in general population Cacnl1a4 gene mutations in Italian families with familial hemiplegic based cystic fibrosis genetic screening. T.P. Brown and C.G. Binnie. LabCorp, migraine (fhm). P.Carrera 1, S.Stenirri 1, M.Piatti 1, M.Ferrari 1, Research Triangle Park, NC P.G.Righetti2, D.Mauri2, L.Capelli 2, C.GeSfi 3. 1)IRCCS H S Raffaele - Intron 8 of the Cystic Fibrosis Transmembrane Regulator (CFTR) gene Milano 2)DBAI - University of Verona 3)CNR - ITBA - Milano ITALY. contains a (T) tract with 5, 7, or 9 Ts. Congenital absence of the vas thymidine FHM is a rare autosomal dominant disorder classified as a subtype of deferens (CAVD) and bronchiectasis have been associated with the presence of ictal the IVS8 ST allele in the CFTR gene. The 5T allele has been shown to cause migraine with aura, and characterized by hemiparesis and, in some skipping of exon 9 in about 50-60% of transcripts, resulting in an abnormal CFTR families, progressive cerebellar atrophy. A locus for FHM has been mapped gene product. Approximately 5% of the general population has the ST allele. We to chromosome 19p13, in about 50% of families, thus suggesting genetic have currently screened 340 patients for 16 common CFTR mutations and the 5/7/ heterogeneity. Recently, FHM linked to chromosome 19p13 and two other 9 T tract. When family history or clinical symptoms warranted, an additional 54 neurologic diseases (Episodic Ataxia type2 and SCA-6) have been mutations were analyzed. Indications for testing were: CAVD (4), other male associated to different classes of mutations within an open reading frame infertility (3), family history of CF (67), spouse with family history of CF (40), encoding for a putative protein >90% homologous to rat and rabbit brain parent of fetus with echogenic bowel (37), fetus with echogenic bowel (11), specific P/l-type Ca2+channel alfal subunit. routine screening (46), Jewish heritage screening (44), and suspected diagnosis In order to identify mutations in our cohort of patients, the entire coding of CF (62). The ST allele was found in 7.4% (25) of those tested. No ST region and exon-intron boundaries were analysed by Double Gradient- homozygotes were found. A total of 42 non-5T mutations were found. Only one DGG E. Search for mutations was performed in 6 unrelated patients for patient, a female Caucasian, was a compound heterozygote (5T/deRaF508), and whom the disease status resulted to be linked to chromosome 19p13 and in at age 21 exhibited no clinical symptoms. All of the patients with CAVD or other cases with FHM. bands were forms of male infertility were negative for ST. 13% of parents and 11% of fetuses three sporadic Heteroduplex found in 8 tested for fetal echogenic bowel had a single ST allele. 11% of those tested for a different exons, and were analysed either by restriction analysis, or PSDM suspected diagnosis had the ST allele and no other mutation. ST positive patients systems or direct sequencing. 3 were previously described polymorphisms, with symptoms included an adult, Caucasian female with chronic bronchiectasis, 5 were new variants. Within affected pedigrees, cosegregation of new a Caucasian infant with failure to thrive, two Caucasian children with chronic variants with the disease status was analysed. They were also tested in the asthma, an African-American child with rectal prolapse, an African-American child general population to evaluate if they are normal polymorphisms or new with respiratory symptoms, and an African-American adolescent with only nasal mutants. polyps. Of the 12 African-American patients tested, 4 (33%) had the ST, and 5 In FHM families, the polymorphic CAG repeat in the 3' of the gene was (10%) Jewish patients had the ST allele. Only 1 of 46(2%) of patients having analysed (normal range:4-14n) to see whether any significant contribution routine screening had the ST allele. We are continuing to collect data to further to phenotypic variability within families exists. study the population incidence and ethnic distribution of the ST allele.

2394 2395 The screening for small rearrangements and point mutations of the LDL Analyses of haplotype and coamplification of dystrophin gene and Y- receptor gene in the Korean patients with familial hypercholesterolemia. Jae specific gene using PEP-PCR In single fetal cells. S.K.Choi, J. WKim, Jin Chae Sung Han Kim2, Sung Soo Hong1 3, Young Bae Park4, Chung Choo E.H.Cho, H.M.Ryu', IS.Kang'. Genetic Research Laboratory, Obstetrics & Lee2, and Yong Namkoongj. Dept. of IMolecular Biology and 2Biology, Seoul Gynecology 1 Samsung Cheil Hospital & Wonen's Healthcare Center, National University, Seoul 151-742, Korea, 3Dept. of Anthropology, Pennsylvania Samsung Medical Center, Seoul, Korea. State University, University Park, PA 16802, USA, 4Dept. of Intemal Medicine, Seoul National University Hospital, Seoul, 110-744, Korea, 5Dept. of Biology, Duchenne and Becker muscular dystrophy are the major neuromuscular Kangnung National University, Kangnung, disorders with X-linked recessive inheritance. Preimplantation sex 210-702, Korea determination has been generally used to avoid the pregnancy with these To assess the frequency and nature of mutations of the low-density lipoprotein diseases. However, in order to determine if the embryo is normal, carrier or (LDL) receptor gene in Korean familial hypercholesterolemia (FH) patients, 80 a unrelated FH heterozygotes were screened by single strand conformation affected regardless of the sex, there is a need for combined analysis of polymorphism (SSCP) analysis. Nine RFLPs within the LDL receptor gene were specific exon on dystrophin gene as well as sex determination of embryo also analyzed to construct haplotypes in order to estimate the number of different using the same biopsied blastomere. If the exon deletion is not mutant alleles in the sample. All of the 18 exons and a promoter region were determinable, further diagnosis of carrier or patient can be performed by amplified and analyzed. Eighteen different LDL receptor defective alleles were haplotype analysis. In this study, we applied the primer extension identified, comprising eight missense mutations: Arg94 -e His'; Val1r -e lie*; preamplification (PEP) method, which amplifies the whole genome, in 40 Glu119 -e Lys; AsP200-e Asn*; Val602-e Met; Leu54 -e Phe*; Pro664 Leu; and cases of single amniocyte and 40 cases of chorionic villus cell. We Glu693 Lys*, three nonsense mutations: Cys74 Stop*; Glu207 -e Stop; and analysed haplotypes using two (CA)n dinucleotide polymorphic markers Arg329-e Stop', two splicing mutations: Cgt Cat in the first base of 5' splicing located at the end of 5' and 3' region of the dystrophin gene. Exon 46 of donor site of intron 3 and Glt-e Git* in the first base of 5' splicing donor site of dystrophin gene and DYZ1 on chromosome Y were chosen as a target intron 4, three small deletions: 36-bp', 9-bp', and 2-bp* deletions in exon 11, and sequence for coamplification-PCR. After optimizing the conditions, the two small insertions: 4-bp insertion' in exon 15 and 1-bp insertion* in exon 16. amplification rates were 91.25% (73/80) for haplotypes (92.5% in * Among them 13 mutations denoted by were novel ones, that have not been amniocyte, 90% in chorionic villus cell) and 88.75% (71/80) for reported. These eighteen mutations were found in 25 unrelated FH patients and coamplification (85% in amniocyte, 92.5% in chorionic villus cell). The this implies that the LDL receptor gene mutations account for 31.3% of the FH present study indicates that haplotypes and coamplification using PEP can causing genes in Korean population. The missense mutation, Prom4 -e Leu was be applied to prenatal and preimplantation diagnosis in Duchenne muscular detected in DNA samples from five unrelated FH probands and thus a particularly dystrophy making it possible to determine if the fetus is a carrier or an common that account for about 6% of the FH-causing genes and for about 18% of affected one. the LDL receptor gene mutation in the Koreans and is regarded as a founder- mutation, though the frequency is not high enough to that of the founder-mutations detected in other populations.

2396 2397 Data services and computational tools for Identification of genes and The Duchenne/Becker muscular dystrophy data pages. J. T. den mutations causing retinal degeneration. S.P. Daiger1, B.J.F. Rossitem2, J. Dunnen, G.J.B. van Ommen and E. Bakker. MGC-Departments of Human Greenberg3, A. Chrstoffels4, W. Hide4. 1The Univ. of Texas Houston; 2Houston, and Clinical Genetics, Leiden University Medical Center, Leiden, the Texas; 3Univ. of Cape Town, South Africa; 4Univ. of Western Cape, South Africa. Netherlands The many inherited forms of retinal degeneration include diseases that result in severe visual impairment or blindness, such as retinitis pigmentosa, macular Although the 'Duchenne/Becker muscular dystrophy data pages" point to degeneration and Usher syndrome. To date, more than 80 forms of retinal and provide information for lay man, they have primanly been set up for the degeneration have been mapped to specific chromosomal sites or otherwise scientific community to provide information on Duchenne and Duchenne- identified. Many research laboratories throughout the world are engaged in linkage like muscular dystrophies (DMD, BMD, SCARMD, LGMD). The Intemet site mapping, positional cloning, and/or mutation screening of patients and families with contains several sections covering issues like: descriptions of the diseases these diseases. To aid in the process of mapping, gene discovery and mutation and affected genes, antibodies, physical mapping data (clone access), identification, we have established an consortium to provide informal computer sequences (gene, cDNA and protein), diagnostic techniques (protocols, database services and computational tools related to inherited retinal degeneration. primers, images), point mutation databases (DMD / BMD and The first resource for research on retinal degeneration is a collection of tables LGMD), listing mapped and/or cloned genes causing retinal degeneration, rhodopsin and literature references, the latest research news and links to related Internet peripherin/RDS mutations, and Internet resources. The Internet site is RetNet', httpJ sites. The data displayed are a mixture of published data, summarized by www.sph.uth.tmc.edu/RetNeV. the Leiden authors, and an increasing set of unpublished data provided by The second resource is a set of Internet browsing routines and analysis software researchers throughout the world. The lists of mutations currently cover the for locating mapped retinal expressed sequences, which are likely candidate genes dystrophin, alpha-, beta-, gamma- and delta-sarcoglycangenes.These lists for retinal degeneration. Details on the software and resulting data tables are are planned to gradually evolve into databases containing more detailed available at the South African National Institute, Bioinformatics http:ll clinical information, facilitating e.g. genotype www.sanbi.ac.za/. / phenotype correlations, for which access will be restricted to registered users. The direct Finally, we have established a moderated list server to distribute e-mail regarding research on retinal degeneration to interested members of the scientific community. of the information displayed and the unique properties of the Intemet to Subscription to the service, 'RD-List', is available through Dr. J. Greenberg cheaply update the information instantly should stimulate researchers to visit the site and to submit all their relevant 6g~anat.uct.ac.za), or subscribe directly to RD-L~uct.ac.za. This list server is regularly (un)published data. In similar to the patient-oriented RP-List" (httpJ/www.netspace.org/cgi-bin/ Iwgate/ this way scientists help each other and they keep the knowledge on the rplist/) except that RD-Ust is oriented to the scientific community, not patients. subject as up to date as possible. Our pages can be found at http:ll Supported by grants from the Foundation Fighting Blindness, the Williams ruly7O.MedFac.LeidenUniv.nVduchenne/. Stamps Farsh Fund, the MD Anderson Foundation, the Retinal Preservation Foundation of South Africa, and the National Eye Institute National Institutes of Health. A41 0 Published Abstracts: Molecular Basis of Mendelian Disorders (cont.)

2398 2399 Exclusion of Endothelin-1 as a candidate gone in major bronchial arch Analysis of ataxia-telangiectasia at the protein and DNA levels in a anomaly syndromes in human. B. Doray, R. Salomon, A. Pelet, J. Amiel, A. Japanese population. T. Fukao1, Xiang-Oian Song', H. Kaneko', T. Munnich, S. Lyonnet. Department of Genetics and INSERM U-393, Necker- Yoshida1, H. Tashita', T. Teramotol, R. Inoue', A. Bar-Shira2, S. Gilac2, Y. 75743 Paris, France. Enfants Malades, Shiloh2, D. Watters, M.F. Lavirn, N. Kondol. 1Dept.of Pediatr.Gifu Univ. Endothelin-1 (ET-1), the EDN 1 gene product, is a 21-amino acid peptide Sch. Med., Gifu, Japan; 2Dept. of Hum. Genet. Tel Aviv Univ., Tel Aviv, that exerts its effects via heptahelical receptors coupled to the G proteins Israel; 3Queensland Cancer Fund Res.Unit, Queensland Inst. of Med. Res. namely endothelin receptor-A (EDNRA) and -B (EDNRB). ET-1 is mainly involved in vasoconstriction and cell proliferation. Surprisingly, the Edn 1-1- Brisbane, Australia. homozygous mice displays morphological abnormalities the pharyngeal Ataxia-telangiectasia (A-T) is an syndrome, arch-derived craniofacial tissues: hypoplastic mandible, absent hyoid bone, which is characterized by cerebeller ataxia and ocular telangiectasia. We thin anterior neck, hypoplastic auricles and anomalies of the middle ear. In analyzed Japanese A-T patients at the protein and DNA level.Seven typical A- addition, Edn 1-/- mice presented with cardiovascular malformations including T patients from 5 families were analyzed. Full-length open reading frame of interrupted aortic arch, tubular hypoplasia of the aorta, aberrant right ATM cDNA, 9168 bp, was amplified dividing 8 fragments. First, mutated subclavian artery and ventricular septal defects with abnormalities of the fragments were detected by restriction endonuclease fingerprinting and outflow tract. sequenced. In some patients, we amplified ATM cDNA dividing 18 fragments These features suggested that EDN 1 may be involved in branchial arch and sequenced. anomaly syndromes in human as well. We screened a series of patients with NY was a homozygote of 5319G to A transition at the last nucleotide of sporadic forms of syndromes involving mainly the Il-IV pharyngeal arches exon 37. This mutation caused the exon 37 skipping in some transcripts and namely (17 patients), CHARGE association (13), Velo- also caused 9-base insertion to cDNA by an abberant splicing with a cryptic Cardio-Facial syndrome without 22q1 1.2 deletion (13) and Goldenhar splice donor site, 9-bases downstream from the authentic site. KS and MS had syndrome (2) by using a combination of SSCP and direct DNA sequencing. a gt to ga mutation at the splice donor site of intron 33, heterozygously, This study only detected two DNA variants in the EDN 1 gene: i) a G to T causing exon 33 skipping. MS's another mutation was R1917X. YH had a transversion at nucleotide 9272 resulting in a missense (KI98N); and ii) an R2909G mutation at the most highly conserved region. TF had a 5-bases insertion at nucleotide -131 in the 5'UTR region (-131, +A). Both variants were deletion (8050TATTA). We identified several other missense substitutions, alsopresent in controls and should be regarded as polymorphisms. however, at this point, we could not regard them as causative mutations. Our findings largely exclude EDN 1 as a disease causing gene in those In immunoblot analysis, ATM protein was clearly detected in 30 microgram branchial arch anomaly syndromes in human. However, other genes protein of control EB-transformed lymphoblasts but not detected in those from participating to the endothelin-signaling pathway could be involved, such as TF, YH, and KS. Since we could not detected ATM protein in even 120 the endothelin-converting enzyme gene for example. microgram PBMCs' protein of a control subject, EB-transformed lymphoblasts are needed for diagnosis of A-T at the protein level. Fibroblasts were also an available source for immunoblot analysis.

2400 2401 A 26-kilobase deletion spanning CFTRaxons 17a to 18 and intron 19 Mutation Analysis of GDNFR-a in Hirschsprung Disease Patients. in cystic fibrosis (CF). N. Gigarel, P. de Dreuzm, L. Thuillier3, C. A.Gosling', S.M. Myers2, A. von Deimling', and L.M. Mulligac?. (1) Institute Rochette3, G. Lenoi?2, A. Munnich', J.P. Bonnefoni. 'Department of for Neuropathology, University of Bonn, Bonn, Germany and (2) Genetics, 2Department of Pediatrics, 3Laboratory of Biochemical Genetics, Department of Pathology, Queen's University, Kingston, Canada. Hopital Necker Enfants Malades, Paris, France. Hirschsprung disease (HSCR), a congenital abnormality characterized More than 600 mutations have been identified in the CFTR gene but very by the absence of enteric parasympathetic ganglia in the terminal hindgut, few large scale deletions have been hitherto reported (Morral et al. Hum. occurs in about 1 in 5000 births. Ten percent of HSCR cases are inherited Mol. Genet. 1993; 2:677). Here, we present the molecular characterization and of these 50% have mutations in the RET proto-oncogene. RET, which of a large deletion in three CF sibs (two boys and one giri) born to first encodes a transmembrane tyrosine kinase, is activated by binding of a cousin-Senegalese parents. The children are currently aged of 14 years, 12 ligand complex comprising a soluble molecule, glial cell lined derived years, and 1 month, respectively. All three had a severe form of CF with neurotrophic factor (GDNF) and a cell surface bound protein (GDNF pancreatic and lung involvement. receptor alpha). Several studies have looked for mutations in the sequence The presence of a homozygous deletion was suggested by failure of of GDNF that could contribute to the HSCR phenotype. We examined the amplifying the proband's exon 17b while exons 4, 7, 11, 13, 14a, 19, 20, 21 sequence of GDNFR-a in DNA from HSCR patients, as well as control DNA were easily amplified. Long range PCR amplification using primers located samples, for mutations that might be responsible for this disease. We in exons 16 and 19, respectively, enabled us to clone the breakpoint. The screened over 80 HSCR DNA samples using SSCP. Where conformation deletion spans exons 17a, 17b, 18, and the major part of intron 19 (26 kb). variants were detected, we screened over 50 control DNA samples to The parents were heterozygous for the deletion. To our knowledge, this is distinguish whether these changes were normal polymorphisms. PCR the first report on a homozygous large scale deletion in CF. fragments which contained SSCP variants were isolated and sequenced. We identified five nucleotide changes that did not affect the amino acid sequence; three in the 5' UTR, one in exon 4 and one in intron 5. We observed three nucleotide changes that did affect which amino acids were coded for; one in exon 2 resulting in Y85N and two in exon 7, Q350H and T361A. These nucleotide changes were present in both the HSCR DNA sequence as well as our control DNAs. We sequenced the coding region of GDNFR-a in 4 HSCR DNA samples and found no nucleotide changes other than those (described above) which were also present in the control DNA samples. Thus it appears unlikely that mutations in GDNFRIx make a significant contribution to the HSCR phenotype.

2402 2403 LATTICE CORNEAL DYSTROPHY TYPE I IN A CANADIAN KINDRED IS A novel SOD1 mutation In familial amyotrophic lateral sclerosis. W-Y. ASSOCIATED WITH THE ARG124-*CYS MUTATION IN THE KERATO- Hung', Y. Yang', J.P. Kaplan', G. Deng H-X. Deng', N. Siddique', A EPITHELIN GENE. S. K. Gupta', W. G. Hodge', K. F. DamjiI24, D. L. Eiser?, T. Siddique'. 'Northwestern University Neurology Department, Guemse9, P.E. Nuemann". 1.University of Ottawa EyeInstitute, Ottawa Chicago, IL.2Vancouver General Hospital, Vancouver, BC, Canada. General Hospital, Ottawa, Ontario, Canada 2. Division of Molecular Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease. Pathology and Molecular Genetics, Department of Anatomy and Mutations in the gene encoding for Cu,Zn superoxide dismutase (SOD1) Neurobiology, Dalhousie University, Halifax, Nova Scotia, Canada 3. account for 20% of familial ALS cases. There are 53 known SOD1 mutations in FALS them are 47 missense Department of Anatomy and Neurobiology, Dalhousie University, Halifax, identified worldwide. Among mutations, a deletion mutation, a stop codon mutation, two intronic Nova Scotia, Canada. mutations that alter the splicing sites, and two insertion mutations that result The purpose of this study was to identify the mutation responsible for in a truncated SOD1 protein product. lattice comeal dystrophy type I (CDLI) in an extended Canadian kindred. Here we report a novel point mutation in SOD1 at codon 84 (exon 4) A search for a mutation in the candidate gene kerato-epithelin, was resulting from a G to C transition causing a leucine (L) to phenylalanine (F) carried out by polymerase chain reaction amplification of coding exons and substitution. The mutation was detected by SSCP analysis and confirmed sequencing. by DNA sequencing. A C-*T mutation at position 417 was detected in exon 4 of the kerato- Clinically the patient, a Canadian female, presented at the age of 43 with epithelin gene, which is expected to cause an Arg124-+cys change. This is leg weakness. She is currently 46 and on a ventilator. Family history the same nucleotide change described previously in two Swiss families with includes the patients brother and father. Her brother died of ALS after a one CDL1. year duration at the age of 41 and her father died at the age of 43 following Although the possibility that the three families (two previously described a two year duration. Swiss families and this Canadian kindred) are related has not been Leucine 84 occurs at the end of the Zn-binding loop where it joins theP excluded, it appears that the unique phenotype of CDLI is caused by this barrel and is highly conserved in the SOD1 gene of more than11 species. particular amino acid change. Previously we have reported a L84V with a 25% reduction in SOD1 enzyme activity. We are currently assaying the enzymatic activity for this novel L84F mutation. (Supported by Muriel Heller fellowship and in part by an NIH grant). Published Abstracts: Molecular Basis of Mendelian Disorders (cont.) A411

2404 2405 Abnormalities of dystrophin, the sarcoglycans and merosin in the A multi-step sequential approach for tuberous sclerosis gene 2 (TSC2) muscular dystrophies. KJ. Jones, S.S. Kim, K.N. North. Royal Alexandra mutatln analysis. L. Longa, S. Polidoro, S. Padovan, T. Meo1, C. Hospital for Children, Sydney, N.S.W., Australia. Carbonara, A. Brusco,E. Grosso, N. Migone. CNR-CIOS & Dip. Genetica, Abnormalities of dystrophin, the sarcoglycans and merosin are Biol. e Chimica Med., Torino Univ., Italy; tUnit6 d'lmmunogenetique and responsible for a subset of the limb-girdle (LGMD) and congenital (CMD) INSERM U.276, Institut Pasteur, Pans, France. (Intro. by: A.Piazza) muscular dystrophies. In this study we aim to characterise the nature and Tuberous sclerosis complex (TSC) is an autosomal dominant frequency of abnormalities of these proteins in an Australian population, hamartomatosis due to a germline defect of one allele at either TSC1 or and to formulate an investigative algorithm to aid in approaching the TSC2 locus, followed by a somatic mutation of the second allele. At diagnosis of the muscular dystrophies. present, only TSC2 has been cloned and appears to act as a Rab5 GTPase To reduce ascertainment bias, biopsies with dystrophic (n=131) and non- activating protein in modulating endocytosis. Efficient screening methods for dystrophic myopathic (n=71) changes were studied with antibodies to mutation detection are needed, since approaches such as linkage are rarely dystrophin, a-, 0-, and P-dystroglycan and merosin, and results were applicable due to the preponderance of sporadic cases: 107/148 TSC correlated with clinical phenotype. Abnormalities of dystrophin, the patients, referred to us by the Italian TSC collaborative group, appear sarcoglycans or merosin were present in 61/131 (47%) dystrophic biopsies, sporadic. Only 10 of 41 familial cases received a locus assignment: 7 to in 0/71 myopathic biopsies, and the diagnostic yield was highest in those 9q34(TSC1) and 3 to 16p13.3 (TSC2). Here we present our strategy for with a severe Duchenne-like LGMD phenotype (94%). Merosin deficiency TSC2mutation screening based on a sequential approach of 3 different was identified in 5/131 (4%) patients; 2/5 patients presented after infancy, methods: southern blot, protein truncation test (PTT) and fluorescence demonstrating that abnormalities of merosin are not limited to the CM D assisted mismatch analysis (FAMA). The preliminary test is southern blot phenotype. (HindlIl+EcoRI double digestion and a full-length cDNA probe): 8 of 126 We conclude that immunocytochemical study of dystrophin, the non-TSC1-linked patients showed partial % sarcoglycans and merosin can be restricted to patients with dystrophic biopsies. Abnormalities of dystrophin should not be used to exclude patients from sarcoglycan analysis without confirmation of primary dystrophinopathy. Differential staining of g-sarcoglycan may provide the most useful information to distinguish between primary dystrophinopathy and primary sarcoglycanopathy, and immunocytochemical studies can be used to guide mutation analysis. On the basis of this study, we have formulated a diagnostic algorithm for the investigation of patients with muscular dystrophy.

2406 2407 Polymorphisms of GLRAI and GLRB genes in hyperekplexia. N. Milani, Nlomozygesity mapping of a consanguineous Greek family with K. Reuter, C. Mulhardt, C.-M. Becker. Institut fur Biochemie, Universitat autosomal recessive cone-rod dystrophy. M.G.Papaioannou1, A.Payne3, Eriangen-Numberg Erlangen, Germany. D.Bessant3, M. Moschos2, A.Loutradi-AnagnostouI, A.Balassopoulout, Hereditary hyperekplexia (STHE, MIM 149400) is a congenital motor disorder 5.S. Bhattachaya3. Unit of Prenatal Diagnosis, Center for Thalassemias, characterized by infantile muscular stiffness and exaggerated startle response. LAIKO GH, Athens, 2 Dpt. of Electrophysiology, University Eye Clinic, Dominant forms of STHE have been attributed to mutations in the al subunit gene of of 3 of Molecular of the inhibitory glycine receptor (GLRA1), that give rise to amino acid exchanges within University Athens, Greece, Dpt. Genetics, Institute M2 or the extracellular loop M2-M3. Recessive cases have been reported to result Ophthalmology, London, UK. from a mutation within Ml and a null allele of GLRA1 carrying a deletion We have ascertained a large inbred family with autosomal recessive encompassing exons 1-7. In a case of dominant STHE, we identified a G1180A cone-rod dystrophy, originating from the SouthEastem Greek transition in GLRA1 leading to a S267N substitution within M2. In addition, a islands.Clinical features include loss of central and colour vision, with recessive case was found to be caused by a novel homozygous C1073G mutation in central and GLRA1, leading to a S231R substitution within Ml. These amino acid exchanges atrophic changes typical bone spicule pigmentation in the mid may result in the disruption of agonist-induced ion conductance, or affect the periphery. Electrophysiological evaluation confirms both rod and cone assembly or stability of the glycine receptor protein complex. An expression study of involvement. Of the 24 members of this family, spanning three generations, the new mutant GlyRal subunits is in progress. Murine phenotypes reminiscent of six were identified as affected. Genetic analysis by means of homozygosity human hyperekplexia, encountered in the recessive mutants spd, spdft and spa are mapping excluded linkage from the previously assigned loci for autosomal caused by mutations in genes encoding both al and P subunit of the GlyR. In recessive retinitis pigmentosa on chromosomes 1p, 1q, 4p, 5q, 6p and 16p, humans, mutations resulting in hyperekplexia have only been identified for the al and from the cone-rod dystrophy loci on 17q and 18q reported in single subunit gene. By analogy, we considered the human subunit encoding gene cases only. The family did however show linkage to the recessive Stargardt (GLRB) as a candidate gene for hyperekplexia. Screening of a human hippocampal locus (STGD1) on 1p21 with markers D1S2813, D1S2804 and D1S236. library resulted in the isolation of cDNA clones encoding two P subunit variants, Recently, Allikmets et aI.(1997) reported 19 different mutations in the ABCR reflecting either altemative splicing of the 5'-UTR or usage of alternative promotors. gene in with disease. This which is an ATP- The genomic organization of GLRB revealed nine exons and a of patients Stargardt's gene, high degree cassette to the same chromosomal as homology to the structure of GLRA1. SSCP screening of the GLRB coding regions, binding transporter, maps position including exon/intron boundaries, was performed in STHE cases unrelated to GLRAI STGD1 and is expressed exclusively and at high levels in the retina. So far, mutations. Our analysis detected a frequent T1024C polymorphism in exon 8 which screening of the first twenty-one published exons of the ABCR gene has was found in 2 families affected by hyperekplexia and in 8 healthy individuals. These failed to produce any mutations in our family and further screening of the data did not indicate any other GLRB sequence change in the 26 index cases remaining exons is underway. analysed so far. However, these findinge do not exclude mutations in promoter and introns regions not yet analyzed or, aitematively, a further genetic heterogenity of STHE.

2408 2409 A GENETIC, CLINICAL AND MOLECULAR APPROACH IN BRAZILIAN DGGE analysis of the PKD1 gone In 150 unrelated patients with FAMILIES WITH MARFAN 1 SYNDROME. A B A Perez L VPereira 2. M Polycystic Kidney Disease. R. Pemchot (1, 2), B. Mercier (2), I. Quere (2), D Brunoni 1. M R 1 - Zatz2. Passos-Bueno 2. Centro de Genotica M6dica 0. Ragu6nes (2), H.E. Thebaud (3), P. Simon (4), J. C1Ides, C. F6rec (2). UNIFESP-EPM - Sao Paulo - SP - Brazil. 2 Laboratorio de Miopatias - (1) Nephrology Department, CHU La Cavale Blanche, Bd Tanguy Prigent, - - - de Biociencias USP Sao - Instituto Paulo SP Brazil 29200 BREST France, (2) ETSBO 46 rue F6lix Le Dantec, 29275 - Brest - Marfan Syndrome (MFS) is a systemic disorder of connective tissue with France, (3) CH Vannes, (4) CH Saint Brieuc AD inheritance and variable To expressivity. date, missense mutations on FBN- With a worldwide incidence of about 1 / 1 000 individuals, Autosomal 1 are the most frequent molecular defect in MFS patients. Thirty-four brazilian Dominant Polycystic Kidney Disease (ADPKD) is one of most common families 54 with the comprising patients MFS were evaluated by genetic, clinical gene disorder. It accounts for up to of cases and molecular In the 34 single 10% all of end stage approachs. families, 23 patients represented sporadic renal disease. PKD1, the major locus on chromosome 16p13.3, accounts cases (67,7%). Among them, 13 patients met the diagnostic criteria (56,5%). for 85% of In PKD1 Out ADPKD. gene, mutation detection is of the 31 patients from the I families that represented familial cases significantly hampered because it lies in a duplicated region including more than 70% of (32,4%), 18 met the diagnostic criteria (58,1%). Among the patients that did the PKD1 not meet the diagnostic criteria, the differential were made, and it is gene. The detection of mutations in PKD1 is important to improve diagnosis our possible that some of them have the so called related phenotypes of MFS. understanding of the disorder. At this time, only a few percent of Thus, the diagnosis od MFS should mot be excluded even if the patient does mutation have been characterized in this gene. Thus we have started PKD1 nor meet the diagnostic criteria, once carefully investigated. mutation screening in the 3' single copy area of the gene using Denaturing Molecular alterations in 12 probands and two frequent polymorphisms Gradient Gel Electrophoresis (DGGE) scanning. One hundred fifty detected by the SSCP analysis, showing an efficacy of 41% of mutation unrelated patients with identified polycystic kidney disease, from the detection. Alterations in 6 probands were detected with the Heteroduplex western part of France, were included in this study. Analyses of the PKD1 analysis, all previously identified by the SSCP analysis, showing and efficacy gene from exon 39 to exon 46 showed us about fifteen different nucleotidic of 20,6% for this method. The molecular alteration in all cases but one were variations including novel mutations and polymorphisms. In order to confirmed by sequencing. Eight patients had missense or nonsense understand the molecular mechanisms underlaying ADPKD, we must pathogenic mutations. All of these mutations ocurred in EGF-like motifs, four of screen for additional mutations along the entire length of the PKD1 gene. being substitution of conserved cysteine residues. Three mutations are This will help in elucidating the physiopathological roel of the gene product. apparently being described for the first time, two missense mutations in exon 44 one nonsense in exon 14. The other 4 alterations were considered polymorphisms sinse they were also found in healthy individuals from the family. Support for Research FAPESP, CNPq, PADCT. A412 Published Abstracts: Molecular Basis of Mendelian Disorders (cont.)

2410 2411 Mutational analysis of the promotor region of the low-density Identification of new TIGR Mutations in Juvenile-Onset Primary Open Angle lipoprotein receptor gene. CL Scholtz1, A V Peeters1, CF Hoogendijkt, R Glaucoma (JOAG) Families from Different Populations. Difiana Stoilova1, Thiart1, JNP de Villiers', DR van der Westhuyzen3, R StreiffA, J Lit?, MJ Anne Chikf Bran W. Fleck3, Magda Barsoum-HomsA, Nusret Ozdemir5, Glen Kotze1. 'Dept. of Human Genetics, University of Stellenbosch, Tygerberg, Bnce2, R. Pitts Crck3, and Mansoor Sarfarazi' . Surgical Research Center, Dept. South Africa. 2Research Service, VA Medical Center, Boise, ID; 'Dept. of of Surgery, Univ. of Connecticut -lealth Center, Farmington, CT; 2Dept. of Internal Medicine, University of Kentucky, KY, Lexington, USA Cardiological Sciences, St. George's Hospital, London, UK; 3Princess Alexandra Eye Numerous gene mutations underlying human lipoprotein abnormalities Pavilion, Royal Edinburgh Infirmary, Edinburgh, UK; 4Dept. of affect protein coding regions. Mutations in regulatory regions of these genes Ophthalmology, Univ. of Montreal, Canada- 5Dept. of Ophthalmology, Cukurova that alter transcriptional efficiency appear to be relatively rare. Common Univ. Faculty of Medicine, Adana, Turkey; 4lntemational Glaucoma Association, variations in regulatory gene regions may, however, account for subtle King's College Hospital, London, UK. differences in serum lipid levels and risk for cardiovascular disease in the Positional mapping of JOAG families has previously identified a locus for this general population. To date one deletion and 7 single base changes have condition (GLC1A) on 1q24. Recently, mutations in the TIGR gene (Trabecular been reported in the promoter region of the low-density lipoprotein receptor Meshwork Inducible Glucocorticoid Response protein) have been described in a (LDLR) gene. Interestingly, 4 of these mutations have been identified in number of GLC1A-linked families. Herein, we report the identification of two new mutations and two other in 4 JOAG South Africans. In vitro studies have indicated that a CCT deletion at amino alid changes in the TIGR gene typical nucleotide position -92 in repeat 1 and C-59T in repeat 2 of the LDLR families. The first mutation is in a 6 generation well documented Edinburgh kindred. An A' to "Go transition in this pedigree has changed Glutamine to promoter markedly reduce transcriptional activity, resulting in a clinical Arginine at position 337. This mutation created a new Mspl restriction site that phenotype of heterozygous familial hypercholesterolemia (FH). We has segregated perfectly with, the JOAG phenotype and, furthermore, it has not furthermore screened more than 600 white, black and coloured South been observed in 142 chromosomes from the same population (Ophthal. Genet., Africans for two 5-upstream base changes, G-174T, in a newly defined FP2 In Press). The second mutation is in a French-Canadian kindred. A 'C' to To cis-element and C-124T polymorphism flanking a FP1 element, in order to transition has changed Proline to Leucine at position 370. This change created a determine their possible physiological consequences. These Pase changes new AlwNI restriction site that has co-segregated with the affected phenotype. We were found exclusively in black and coloured individuals. Mutation G-174T, also detected one normal family member who carries this mutation. This was occured at a significantly higher frequency in black FH patients (40%, expected, as he has inherited the same affected haplotype and, therefore, he is a p<0.001) and dyslipidemics (15%, p<0.03), compared to its frequency in the proven case of a gene carrier. In two other JOAG families from Turkey and general population (5%). These findings suggest that mutation G-174T ngland, two different sequence changes have also resuited in amino acid contributes to the development of dyslipicdemia in blacks. substitutions (missense mutations), one in exon and other in exon Ill. However, as these sequence changes have not been tested in their respective general populations as yet, the possibility of them being rare polymorphisms still remains to be determined.

2412 2413 Molecular characterization of the a- and 13-genes in 22 P-thalassaemia Exclusion of the human Sonic Hedgehog coding and promoter regions as a major families in a Malayan aboriginal group (DUSUN). J.A.M.A. Tan, candidate gene for sacral agenesis, familial neural tube defects, trlphalangeal S.F Yap, K.L. Tan, L.L. Chan1, H.P. Lin1. Department of Allied Health thumb and mirror polydactyly. F. Vargas', E.Roesslerl, S. Whitehead', G.HoopeP Sciences, University of Malaya. Deapartment of Paediatrics, University of R.E.Stevenson4, I.Cordekr5, P.Correia, I.Schwa&,i S.Antonrerakis8, T.StracharP, Malaya1 R.Gereige'0, E.Belloni11, S.Scherer", L.C.Tsui"l, M.Muenke'. Children's Hospital of PhiladelphisUSA'; U of Dublin,ireland2; Prince M Rose Orthopedic The aboriginal population or SOrang Asli in Malaysia is composed of various Hospital,Edinburgh,Scotland3; Genetic groups living in both Peninsular and East Malaysia. Alpha and Greenwood Center,Greenwood,SC,USA"; P-thalassaemia Hosp genes have been detected in the Orang Asli population. However, there is a Santa Maria,LisbonPortugal5; Iff-Fiocruz,Brazil6; HCPA,Porto Alegre,Brazil7; paucity of data on the frequency and types of thalassaemia mutations in this Geneva Medical School,Switzerland8; U of Newcastle-upon-Tyne,UK9; South Florida population. College of Medicine, Tampa,Fl,USA10; Hospital for Sick Children,Toronto,Canada". Molecular characterization of the 1-gene region was carried out in 22 1- Sonic Hedgehog (SHH) is the human hornologue of the Drosophila segmentation thalassaemia major patients and their families belonging to the Dusun group. The gene hedgehog. The hedgehog family of secreted signalling proteins is responsible Amplification Refractory Mutation System (ARMS) was used to study 9 mutations for developmental patterning in a variety of systems, including the neural tube, limbs, along theE-gene: -28 TATA BOX, Codon 15, Codon 17, Codon 19, Codon 26 (Hb and somites. Within the neural tube, at the level of the spinal cord, SHH is proposed E), IVSI #1, IVSI #5, Codon 41-42 and IVSII #654. The parents of the P-major to function as a ventral patterning influence, with the capability of inducing floor plate patients were found to be neative for all 9 mutations. Amplification of DNA and motor neurons. In humans SHH is located in 7q36, the same region where extracted from the 22 13-major patients indicated a possible deletion of the entire 1- autosomal dominant sacral agenesis isolated or with a presacral mass and anorectal abnormalities (so called Currarino OMIM autosomal dominant gene as the 443 bp Codon 41-42 normal 1-gene fragment (nt. 46 - nt. 438 of the triad, #176450), the 861 bp internal control fragment (nt. 1242 - nt. 2101) was not triphalangeal thumb (OMIM #190605), and au*tqsomal'dominant tibial aplasia / mirror A-gene)and polydactyly / triphalangeal thumb (OMIM #188770, unpublished data) have been detected after PCR. Molecular analysis of the Gy, and Ay region using DNA linked. In the present study we have performed single stranded conformation amplification with primers flanking the genes showed no deletion in this region of polymorphism (SSCP) analyses in four different trip4alangeal / mirror polydactyly the 13-gene. Molecular analysis is presently being carried out along the delta-gene families, six sacral agenesis / Currarino trina farnflIs, and 17 sacral agenesis / region. sirenomelia sporadic cases. No mutations wore foind within the coding and / or Molecular analysis of the a-genes showed the presence of both a-genes in the promoter regions of SHH. We also sequenced the entre SHH promoter region (> 1 families. The Southeast Asia a-deletion was not detected in any of the patients. Kb 5' of the start site) and coding region from a representative proband in one of the Alpha-thalassaemia is absent in this population. sacral agenesis families and one of the tibiql aplasla mirror polydactyly families In conclusion, 0-thalassaemia in the 22 families of the Dusun group appears to known to be tightly linked to the SHH gene artV fourd no mutations. SSCP analysis be caused by a large deletion in the f1-gene region with or without invoivment of is also currently being undertaken for a saplpe cnsistlng of 51 probands from the delta-genes. The y-genes are intact and analysis is currently being carried out families in which a neural tube defect is sbgregatjng. Preliminary data show no to located the 5' and,3'-breakpoints of the deletion in this group. mutations in SHH.

2414 2415 Segmental and Mosaic Forms of Neurofibromatosis-1: Molecular and Environmental and genetic factors In variable expression of autosomal EmbryologicalInsights. M Wallace', M Baue92, S Rasmussen1, and L dominant reinitis pigmentosa. A.A. Ybarral, C.I. Amos2, S.H. Blanton,-, W.H. Baumbact. 'University of Florida School of Medicine, Gainesville, FL, 2Miami Murphel, R.G. Weleber4 and S.P. Daigera 5. 'Human Genetics Center and 5Dept. Children's Hospital, Miami, FL and 3University of Miami School of Medicine, of Ophthalmology, Univ. of Texas HSC, Houston; 3MD Anderson Cancer Center, Miami, FL. Univ. of Texas, Houston; 4Univ. of Virginia, Charlottesville; 4Oregon Health Sciences The neurofibromatoses (NF) represent a clinically heterogenous collection of Univ., Portland. disorders which share certain common clinical manifestations, while the underlying Autosornal dominant retinitis pigmentosa (adRP) is a set of inherited disorders molecular and pathological mechanism(s) responsible for the diverse disease characterized by progressive nightblindness and loss of visual fields, often resulting manifestations remain unclear. One of the most intriguing variant forms is in blindness. There is significant variation in expression of adRP, including variable hallmarked by non-systemic, unilateral cutaneous manifestations typical of NF-1, age of onset, progression and final outcome, even between individuals sharing the confined to a segmental or dermatomal distribution only (segmental NF; SNF). As same mutafion. A better understanding of factors wthich modify expression may well, the recent identification of somatic and germinal mosaicism for NF-1 suggest ways to improve clinical outcome. mutations set the precedent that tissue-specific mutations can occur. It is likely The aim of this study was to identify environmental and genetic influences on the that both of these mosaic forms are phenotypically, and probably embryologically, course of retinal degeneration in a single, large adRP family with the Pro23His distinct from SNF. mutation in rhodopsin. A telephone-based questionnaire was administered to 43 We report our clinical data arising from a collection of approximately twenty-five affected members of the family. All participants had been examined by a retinal SNF cases, either sporadic or with familial involvement, as well as three cases specialist. Information was collected on smoking, alcohol consumption, pregnancy, medication use, vitamin use and with isolated NF features that may represent somatic mosaicism for NF-1 the presence of common medical conditions, ight then established for each mutations. We also present results from our most recent investigations of the NF-1 exposure. Several measures of clinical severity were locus in this patient cohort using nucleic acids extracted from phenotypically- participant. Environmental factors were analyzed using the Cox proportional hazards the correlation, for all affected and unaffected areas, as well as peripheral blood. Experiments are model; genetic factors were assessed by determining and differences in clinical measures. focusing first on the detection of NF-1 protein truncating mutations using the participant-pairs, between kinship were no Protein Truncation Assay, followed by measurement of gross gene deletions Though several suggestive associations noted, statistically significant of disease or status, assaying for loss of heterozygosity. For the cases in which mutations are not effect was found on the course by gender, smoking drinking or use of medications or vitamins. An detected, exon-specific PCR, followed by point mutation scanning techniques light exposure, common medical conditions, increase in of and onset of tunnel vision was found with increasing (SSCP, heteroduplex analysis) is being performed. DNA sequencing techniques age diagnosis will be used to confirm mutations. pregnancies. Genetic factors did not have a significant effect on the course of in this subset of the factors were not These studies represent the first detailed molecular investigations of mutations disease family. Although major modifying this established useful for more in-depth in the NF-1 locus in patients with variant forms of NF-1. A comparison of tissue identified, pilot project methodology of families with variable of dominant diseases. distribution of NF-1 mutations will provide insights into the embryologic timing of investigation large expression from NEI-NIH (EY07142), Foundation Fighting Blindness, these events. This data will also prove useful in the diagnosis and counseling of Supported by grants such patients. Research to Prevent Blindness, the Farish Fund and the MD Anderson Foundation. Published Abstracts: Therapy for Genetic Disorders A413

2416 2417 FETAL HEMATOPOIETIC STEM CELL TRANSPLANTATION THERAPY: In vitro expressed dystrophin fragments do not associate with each DEVELOPMENT OF A RAPID POLYMERASE CHAIN REACTION TEST FOR other. Y.M.Chan and L.M.Kunkel. Howard Hughes Medical Institute, QUANTIFYING ENGRAFTMENT LEVELS. K.D. Carter, J.R. Leipprandt, S.A. Division of Genetics at the Childrens' Hospital, Boston, Massachusetts, USA Kraemer, and M.Z Jones. Department of Pathology, Michigan State University, The gene responsible for Duchenne Muscular Dystrophy (DCMD) and East Lansing, Ml 48824 Becker Muscular Dystrophy (BMD) has been shown to encode a 427 kD P-mannosidosis, an autosomal recessive inherited disease, is characterized by cytoskeletal protein known as dystrophin. On the basis of homology with deficiency of lysosomal P-mannosidase activity. The phenotype of the caprine other cytoskeletal proteins, such as a actinin and spectrin, dystrophin has disease includes severe neonatal neurological and physical abnormalities. The been proposed to function as dimer. However, there is no biochemical data goal of our research is the development of in utero hematopoietic stem cell (HSCs) to support the dimer hypothesis. In this study, we used an in vitro translation transplantation protocols for the treatment of caprine j-mannosidosis. To facilitate system to express dystrophin fragments that were stable and had a their detection after interperitoneal transfusion, male donor heterologous HSCs derived from fetal liver are transfused into immunotolerant female fetal conformation similar to the native full length dystrophin. The expressed recipients. were tested for their to interact with each other as well The objective of this investigation is to develop a PCR-based test to quantify fragments ability as low levels of engraftment of male HSCs in the tissues of female recipients. First a full length dystrophin by both immunoprecipitation and blot overlay assays. PCR test was developed in order to detect the presence of the Y chromosome. A Using these methods, we demonstrated the dimerization of a actinin and set of primers was designed to amplify a 129 bp segment of the caprine SRY gene, spectrin but failed to detect any interaction between dystrophin fragments. a Y-chromosome-specific sequence. This amplicon was found to be produced Although we can not unequivocally prove that dystrophin is a monomer, our exclusively from male genomic DNA (gDNA). Application of this test revealed that it result provides the strongest evidence today that dystrophin, in the absence could detect male hepatic gDNA in female hepatic gDNA at levels as low as one of other accessory proteins, does not associate with itself in vitro. In part in 10,000. A 101 bp truncated form of this amplicon, designed to act as a PCR addition, our finding could provide important information on the structure of competitor, co-amplifies equally with the SRY sequence from 15 fg of the dystrophin which will enable researchers to design better mini-dystrophin competitor and 55 ng of male genomic DNA. Tests have begun to determine the gene for gene therapy in the future. reproducibility of co-amplification products and linear amplification ranges for differing levels of male gDNA in a constant level of total gDNA. Preliminary resuhs indicate that this test will be useful for the evaluation of donor HSC engraftment in the capnne P-mannosidosis treatment model. Supported by NIH NS33911 to MZJ.

2418 2419 Use of amphotropic recombinant SL3-3 retroviruses in the stable Cloning and Sequencing of Caprine CD34 cDNA: Development of reversion of the Tay-Sachs phenotype in human brain cells. M.A. Hematopoietic Stem Cell Enrichment Protocols for in utero Gama Sosa, R. DeGasperi, N.S. Sugandhi, T.J. Lehrerfeld, S. Undevia and Transplantation in Animal Models. J. R. Leipprandt', D. S. Anson2, and E.H. Kolodny. Laboratory of Experimental Neurogenetics, Department of M. Z. Jones1. 'Department of Pathology, Michigan State University, East Neurology, New York University School of Medicine, New York, NY, USA. Lansing, Michigan, and 2Department of Chemical Pathology, Women and Tay-Sachs disease is an autosomal recessive inherited Children's Hospital, Adelaide, Australia. neurodegenerative disease caused by the deficiency of the lysosomal P- Hematopoietic stem cell (HSC) therapy for lysosomal storage disorders hexosahninidase A activity (Hex A) due to defective or absent a-subunits. A involves the transplantation and engraftment of normal HSCs into affected promising approach to the stable therapeutic treatment of Tay-Sachs and individuals, followed by secretion of the missing enzyme and its uptake into other inherited neurodegenerative diseases involves the ex vivo transfer of lysosomes of affected cells where the storage product can be catabolized. We are the missing genetic function into the affected cells. We have generated developing in utero HSC transplantation in normal immunotolerant goat fetuses at novel amphotropic recombinant retroviruses harboring the human - 60/150 days gestation for eventual treatment trials in the capnnej-mannosidosis hexosaminidase a-subunit cDNA in animal model. A limitation is the number of HSCs that can be administered to a addition to the neo gene under the fetus using our current crude HSC fractions from fetal livers. Other systems, control of the T-cell tropic SL3-3 virus LTR. Transduction of these including human, have benefitted from HSC enrichment by selecting for cells that recombinant retroviruses into SV40-transformed fetal astrocytic cells from a express the CD34 cell surface antigen using anti-CD34 antibodies. Toward that Tay-Sacls patient resulted in the isolation of neoR clones stably expressing end we have initiated a project to clone and express caprine CD34, in order to P-hexosaminidase A activity. The levels of Hex A activity in the genetically produce anti-CD34 monoclonal antibodies for HSC enrichment procedures. engineered Tay-Sachs astrocytic cell clones were 321-1628 nmoles/hr/mg A composite caprine CD34 cDNA sequence was identified from a series of protein, while the Hex A levels in the control and parental Tay-Sachs SV40- overlapping RT-PCR amplicons. The amplicons were produced from normal transformed astrocytic cell lines were 725 and 43 nmoles/hr/mg protein, caprine cDNA using primers based on the published canine, mouse, and human CD34 cDNA sequences. The 1,030 bp that have been sequenced code for the respectively. These results indicate the feasibility of stable gene transmembrane region, most of the cytoplasmic domain, and the complete replacement therapy using SL3-3 recombinant retroviruses in the treatment extracellular domain. The intracellular regions share 82-85% amino acid identity of Tay-Sachs and other inherited neurodegenerative disorders. with the published sequences whereas the less well conserved extracellular domain shares 50-58% identity. The 251 amino acid extracellular domain has the potential for extensive glycosylation, with 11 possible N-linked glycosylation sites and 60 serine and threonine residues in the first 150 amino acids for possible a linked glycosylation sites. A 754 bp amplicon that codes for only the extracellular domain has been produced and will be cloned into an expression vector for production of a CD34/lgG fusion protein. Supported by NIH NS33911 to MZJ.

2420 2421 Intragenic suppression of Human Cystathionine P-synthase Mutations. Investigation of tissue specific promoters in the correction of X. Shan and W D. Kruger. Fox Chase Cancer Center, Philadelphia, PA. ornithine transcarbamylase deficiency in mouse models. A.H. Trainer Mutations in cystathionine 0-synthase (CBS) are known to cause and R.J. Akhurst. Intro by M.EPorteus. Department of Medical Genetics homocystinuria, a recessive disorder which results in extremely elevated University of Glasgow Yorkhill Glasgow G8 3 SJ Scotland plasma homocysteine levels. Using a previously developed yeast functional Ornithine transcarbamylase (OTC) deficiency is an X-linked urea cycle assay, we have identified a second mutation in the CBS gene, which disorder with and is several high mortality morbidity. OTC normally expressed in the suppresses mutant alleles of human CBS, based on their ability to liver and small intestine. The aim of this work was to investigate the complement a CBS null yeast strain. This suppresser mutation causes feasibility of correcting the metabolic abnormality in the two OTC deficient truncation of the C-terminal 25% of the protein. Truncated CBS mutant mouse sparse fur and sparse alleles are able to models, fur ASH mice, by targeting OTC complement growth in yeast as well as wild type CBS. In expression to non-hepatic tissues by means of tissue specific promoters.As vitro enzymatic data shows that deletion of the C-terminus of CBS increases a mitochondrial enzyme, OTC will have its effect within the target organ and enzyme activity of the protein. More significantly, the deletion also hence in this success will on the of target elevates CBS in system depend ability the tissue activity several mutant alleles to near wild type levels. In to as a an act 'metabolic sink'. 3 gene expression constructs were formed addition, SAM, allosteric affector of CBS, can no longer stimulate the C- the rat OTC cDNA sequence A promoter terminal truncated CBS. The linking with 1) keratin V with its results suggest that the C-terminus of CBS endogenous last intron and polyadenylation sequence (skin-specific). 2) the interacts with the rest of the protein as a negative regulator for its enzyme These results alpha-actin gene promoter(muscle- specific)and 3) the albumin activity. suggest that drugs could potentially be designed to promoter(hepatocyte- specific). Polyadenylation and intronic sequences disrupt this interaction, offering a way to modulate plasma homocysteine levels. We believe this were added to allow efficient mRNA processing. 3 founder transgenic lines yeast assay could be developed into a high were then produced with each construct and bred onto both a sparse fur throughput screening system that will allow the identification of drug and candidates that elevate the CBS sparse furASH background. Preliminary results suggest that, although enzyme activity. trasnsgene expression was been demonstrated, no phenotypic correction of the mouse models was noted.This is in contrast to previous studies which demonstrated phenotypic correction using viral promoters. Tissue-specific OTC activity data in these transgenic mice will be discussed.