Published Abstracts: Cancer Genetics A341 1984 1985 Detection of amplified 7q22 DNA sequences In chorlocarcinoma by Loss of heterozygosity for 10q24-q26 loci in human gliomas comparative genomic hybridization. K. A. Alexander. B. R. Haddad. J. Traweek and M. B.Oumsiveh, Departments ofOBIGYN and Pathology, Duke R.Almida M.Mafta , A. Safaia , M.E. Marques da Silva , JRueff University Medical Center, Durham, NC and National Institute of Health, ) Bethesda, MD. and C.(Monteiro( Women with complete hydatidiform moles are at a 5% risk for developing (1) Dep Genetics, Fac Mcd Sciences UNL, R. Junqucira 96, P-1300 Lisboa, Portugal, persistent trophoblastic disease or choriocarcinoma. Genetic changes underlying the (2) Dep. Pathology. Hospital S. Jos6. P-t000 Lisboa. Portugal development and progression of complete mole to choriocarcinoma are poorly characterized. We have used comparative genomic hybridization (CGH) and Gliomas are tumours of the glial cells and constitute the majority of fluorescence in situ hybridization on archived tissues in order to identify regions turnours of the Central Nervous System Previous cytogenetic and molecular that may contain genes important to the development and progression of genetic analysis showed that at least three regions of the chromosome 10maybe choriocarcinoma. CGH is a powerful method to detect gross genomic copy-number involved in the development of this kind of tumour, namely one telomerical to map on from changes and these changes normal chromosomes. Tissue samples region on lOp and both proximal and distal regions on 10q therefore making them complete mole, invasive mole, and choriocarcinoma were aquired from the Division of Gynecologic Oncology, Duke University Medical Center. High molecular weight putative sites for location of turnour supressor genes. In the present study we DNA was extracted from very small amounts of archived tissues and resulted in have screened 41 glial tumors at different histopathological grades and stages for adequate concentrations for CGH analysis. CGH was perfomed on these samples loss of heterozygosity (LOH) at 4 highly informative dinucleotide repeat markers and the results analyzed using a digital image analysis system. CGH analysis spanning the q24-q26 region of chromosome 10. highlighted a region ol DNA amplification in one of the six specimens examined on Microsatellite allele sizes and LOH assignment were performed in a 373A chromosome 7 at band 7q22. This case was further investigated with FISH probes DNA Sequencer with GENESCAN 672 software (PE-ABD). representing centromeric regions of chromosomes X, Y, and 7 and the case was found tobe diploid with XX complement and two copies of chromosome 7. This A total of 27 (66%) tumors showed allele loss at one or more markers indicated atrue amplification with no no triploidy or polysomy. A review of the which strongly predicts the involvement of this region in glioma tumorigenesis. literature showed that a previous study of chromosome breaks in choriocarcinoma From the results obtained so far, we are able to guess the critical locus had reported excess breaks in three bands: 2q21, 5q24, and 7q22. Two potential centromeric to DIOS190. oncogenes are known to be l(oated in this region, the niet gene and a gene for a cAMP dependent protein kinase. FISH studies using probes for these candidate genes are in progress and will be followed by mutational analysis. The data implicate 7q22 as a genomic area which contains candidate genes associated with the development and progression of choriocarcinoma.

1986 1987

Genetic analysis of the DCC gene and investigation of LOH on 18q in Presence of 14 p+ with ClassIc Chromosomal Change -inv(16)(p13q22) - colorectal cancer. P.Baharuddin, R.Sud and J.D.A.Delhanty. Human Genetics In Acute Myelogenous Leukemia, FAB M4e. E.Bayani,.LHLin', Group, The Galton Laboratory, University College London, Wolfson House, 4 G.DelaFuenlg, S.Khan, M.Chester, L.O.Sha. Nassau Cty. Med. Ctr. E. Stephenson Way, London NW1 2H1E, U.K. Meadow, N.Y., Health Sci. Ctr. State U. N.Y. at Stony Brook', N.Y. We are investigating the status of the tumour suppressor gene, DCC (Deleted in This 31-year old Caucasian man was referred to our clinic Colorectal Cancer) in several colonic cell lines and colorectal cancers by cytogenetic because of high and molecular approaches. Cytogenetic analysis in the cell lines, AA/CI/SH/IOC leucocyte count with myeloblasts in peripheral blood on a routine (transformed adenoma), JW/33 & LIM 1215 (hereditary carcinomas) and tIM 1899 examination. At the clinic, the spleen was enlarged to 3 cm below the left (sporadic carcinoma) showed 2 copies of chromosome 18 in the carcinoma lines but coastal margin. There was no hepatomegaly or lymphadenopathy. The chromosome rearrangement and loss involving chromosome 18 was seen in the white blood cell count was 49 thousand per cubic mm with 30% AA/CI/Stl/IOC line and confirmed by FISHI with chromosome paints and myeloblasts and monoblasts. The marrow showed 100% cellularity with probes. FISH with 7 YAC contig probes from the DCC locus (18q21) alpha-satellite over 30% myeloblasts and monoblasts, 20% promonocytes and monocytes, showed that 2 copies of the DCC gene were present in the cell lines but evidence for 18.5% eosinophils and others. DNA rearrangement was also seen. Using the PCR-SSCI' technique with 3 The marrow microsatellite markers on 18q (D18S61), including the DCC gene (DCC1 & DCC2), chromosomal studies showed 46,XY,14p', inv(16)(p13q22). intragenic DCC dleletion was found in the AA/Cl/SIB/1OC cell line. He was treated with cytosine arabinoside and Idarubin and went into .We have extended our observations to resected sporadic colorectal carcinomas in 15 remission. Five months later, he exhibited anemia with severe leucopenia patients. Interphase FISHI on fixed normal and tuimour smears with DCC YAC probes and thrombocytopenia and he terminated in a gram-negative septicemia. detected less LOH on 18q compared to the PCR based procedures. The D18S61 marker About two-thirds of all AML patients disclosed karyotypic abnormalities. was able to detect LOll of 18q in 2/15 cases (13.3%) and replication error phenotype in an inv(16)(pl3q22) typically occurred in M4e which was associated with high additional 2/15 cases (13.3%) by the PCR-SSCP method. Using another marker, D18S8- M2 which detects an MSpI polymorphic site within the DCC gene, LOll of 18q was found complete remission and long remission duration. The significance of in a further single case 1/15 (6%) by the RFLP analysis. These molecular changes may presence of additional 14p+ in this case was unclear, apparently this be involved in the progression of adenoma to carcinoma. Correlation between these additional change had never been reported. The patient was treated with changes with the pathological stage of the tumours will provide information on the use traditional ways according to the clinical manifestations; however the of chromosome 18q loss as a prognostic marker in patients with non-metastatic colorectal response was apparently poorer than those expected in the general practice. cancer. Whether or not presence of extra 14p+ affected the shorter outcome for this patient was inderminate.

1988 1989 An adult biphenotypic acute leukemia with the FISH evaluation of chronic myelocytic leukemia (CML). Z. Chen, M. Notohamiprodiao P.D. Richards. FB8 Lane, R. Morgan, J.F. Stone, A-A. Sandberg. Genzyme t( I; 14)(p34;q II) and del(I1)(q I4q23). J. Caldwell. B.J. N. Bone. J.R. Mendiola J. McClure. R.C. Genetics. Scottsdale. AZ: Oncology and Hematology Associates of Southwest Streifel. J. Ahmad. Virginia. Inc.. Roanoke. VA: Hematology and Oncology Consultants. Tampa. FL McGlennen*. M. Arnesen. R.R. Higgins. Allina Health System and From 1994 to 1996 cosmid probes specific for the major bcr/abl rearrangement University of Minnesota*, Minneapolis, MN. (Oncor or Vysis) were employed by us to evaluate 134 patients with the clinical The rare translocation t(1;14)(p34;ql 1) has been described in diagnosis of CML. The following points represent several main observations: 1) No significant difference regarding a false positive fusion signal was approximately 20 acute lymphocytic leukemia (ALL) cases. seen in the distribution pattern between the Ph negative(-) cases (110) and the Apparently all but one of those cases had a T-cell normal controls (3.23±1.97 vs 2.8i1.92). immunophenotype, and usually Li morphology. Bernard et al. 2) The mean percentage of cells with a positive fusion signal in the Ph (1993) reported the B-lineage ALL case with the t(1;14). We positive(+) cases at presentation was 70*12.3 in bone marrow samples (12 cases) and 45*12.4 in blood samples (4 cases). describe a biphenotypic ALL case of a 71 year old male. Flow 3) A significant discrepancy was seen between the interphase FISH and cytometry detected expression of CD7 antigen and TdT cytogenetics data (see Table) in the monitoring of 4 out of 8 patients post- characteristic of T-ALL and CD13, CD33, and CD34 characteristic therapy. The clinical significance of this observation needs to be clarified. of AML in a biphenotypic pattern. His bone marrow cytogenetic SampleFISH Cytogenetics marrow 271 Ph+ analysis had 9 normal cells and two abnormal cell-lines: 10 cells [20] marrow 30% Ph+ [20] I) karyotype and 2 cells with a with a 46,XY,t(1;14)(p34;ql marrow 22% Ph+ [19]/Ph- [ 1] 46,XY,del(l I)(ql4q23) karyotype. An Iq- is a common marrow 221 Ph+ 91/Ph- [11] aberration found in myeloid disorders. especially MDS, but is also 4) Three Ph(t-I patients with significant teutocytosis nao closely adjacent but not fused signals (20%. 16%. and 20% of cells, respectively. vs the control: common in acute biphenotypic leukemias. FISH using particularly 41). This may indicate a temporal and spatial association between chromosomes 9 wcpl4, D14ZI, D22ZI, DIZ2 confirmed the t(l;14). FISH using a and 22 prior to the bcr/abl rearrangement (Ph chromosome formation). MLL probe for the I 1q23 region was not informative. PCR showed no rearrangement of the MLL gene. Additional molecular study of the t(1;14) is presently in progress and, will be compared to the results of Bernard et al. A342 Published Abstracts:: Cancer Genetics (cont.) 1990 1991 Hematogeous dissemination of prostate cancer cells as the result of Loss of Heterozygosity (LOH) at the SRDSA2 Locus on needle biopsy: detection of circulating prostate cells by a quantitative Chromosome Arm 2p During Prostate Cancer Progression nested multiplex reverse-transcription PCR assay. D. R. Clontz. C. M. Donald A. Elmaiian. Gary Lieskovsky. Donald G. Skinner. Juergen K.V. Reichardt. USC School of Medicine, Los Angeles, CA. Teigland W. Woodard. J. Daniels. and D.K. Price. Carolinas Medical Center, Prostate cancer is the most common cancer in American men. Charlotte, NC. Molecular markers for progression and outcome, however, are rare. The incidence of adenocarcinoma of the prostate is escalating with 317,000 Cytogenetic data suggest that a gene on the short arm of chromosome new cases estimated for 1996 resulting in approximately 41,000 deaths. 2 (2p) is involved in prostate cancer progression. We have initiated Patients are still monitored clinically by a serum prostate-specific antigen (PSA) molecular investigations into a candidate gene, SRDSA2 encoding level, however clinical staging to determine the extent of metastasis is now steroid Sa-reductase type 11 located on 2p23, during prostate cancer progression. being approached from the molecular level. Many groups have recently used We have collected 200 constitutional ("germline") DNA samples RT-PCR in order to detect PSA-expressing cells or prostate-specific membrane from patients who underwent radical prostatectomy as treatment for antigen (PSM) expressing cells in the circulation of prostate cancer patients. prostate cancer. These samples were genotyped for a polymorphic Due to the controversy over which of these markers more accurately provides dinucleotide repeat marker in the SRD5A2 gene. 35 patients who were information as to the progression of disease, we developed a multiplex RT-PCR heterozygous for the repeat marker were selected for further analysis that simultaneously detects mRNA from both PSA and PSM. Because of their tunor DNA. We have completed the genotypic analysis of the assay first 8 pairs of matched constitutional and tumor DNAs in this series. this assay also detects a ubiquitously expressed internal control, we have One of our eight samples showed LOH (loss of heterozygosity) in the incorporated a method to quantitate our RT-PCR results. We feel that our SRD5A2 gene. assay not only will help determine the ability to stage prostate cancer by These data indicate that the SRDSA2 gene can be involved in molecular analysis, but also allow for the study of the pathophysiology of genomic alterations during progression of prostate cancer. Our LOH metastatic prostate cancer, thus providing anwsers to some relevant clinical result suggests that the SRD5A2 gene on 2p23 is a good candidate questions. In order to answer the question: are prostate cancer cells released gene for a prostate cancer progression gene on 2p which was into the blood by needle "punch" biopsy?, and therefore actually promoting hypothesized previously by others. metastasis, we are conducting a study using the nested multiplex RT-PCR assay to compare the level of expression of PSA and PSM before and after needle biopsy. Blood from patients suspected of having prostate cancer is being drawn prior to biopsy, 30 minutes after biopsy, and at follow up visits to determine if biopsy causes the dissemination of prostate cells, and how quickly, if ever, these cells are cleared from the bloodstream.

1992 1993 Tumor and normal cell fusion and origin of polyploidy in ovarian cancer Molecular diagnosis of von Hippel-Lindau disease in familial of low malignant potential. S. A. Faruqi. R. B. Deger and J. S. Noumoff. pheochromocytoma. A. Garcia. X. Matias-Guiu. R. Cabezas. A. Chico. Department of OB-GYN, Oncology Division, Crozer-Chester Medical Center, A. de Leiva. J. Prat. M. Baiget. Departments of Pathology, Genetics and Upland, PA. Endocrinology. Hospital de la Santa Creu i Sant Pau. Universitat Monosomy of chromosome 12 was seen as the only abnormality in Autbnoma de Barcelona, Spain. diploid cells of a mucinous ovarian tumor of low malignant potential Objectives: To study the presence of germline mutations in the von associated with "pseudomyxoma peritonei." Presence of 91,XXXX,-12 and Hippel Lindau gene (vhl) in a kindred with familial pheochromocytoma in 182,XXXXXXXX,-12,-12 could he explained only through fusion of a tumor order to confirm the diagnosis of Von Hippel Lindau disease (VHLD) as cell with a cell of normal genome and then its subsequent doubling. Somatic well as to identify asymptomatic members. mammalian cell fusion is known since 1960 when Barski et aljl1 provided the Design: DNA extracted from peripheral blood was amplified by the evidence of a hybrid cell in a cell mixture. Since then, somatic cell fusion was Polymerase Chain Reaction using oligonucleotide primers corresponding described between tumorogenic human and hamster or mouse host cells and to exon 3 of the vhl gene. Specific mutations in codon 238 were screened also between tumor cells of the same species. Recently Petkovic[21 using for by restriction endonuclease digestion of PCR products with Msp I. The chromosomal analysis showed spontaneous cell fusion and origin of polyploidy results were confirmed by DNA sequence analysis. in embryonal rhabdomyosarcoma. We report evidence of cell fusion and Patients: Two generations of a family consisting of 15 individuals origin of polyploidy for the first time in an ovarian tumor. were studied. 1. Barsky et al., (1960) Compt Rend 251:1825-1827 Results: A germline missense point mutation at codon 238 of the vhl 2. Petkovic et al., (1994) Cancer Genet Cytogenet 76:125-128 gene (CGG-->TGG; Arg-->Trp) was detected in all patients with Pheochromocytoma and only one of the asymptomatic family members. Conclusion: Mutational analysis ofvhl gene in patients with familial pheochromocytoma may allow for the specific diagnosis of VHLD, and it is a good method to identify asymptomatic individuals at risk for VHLD.

1994 1995 Analyais of genomia instability in a patient with Rothaund- Cytogenetic analysis of pancreatic cell lines and their primary tumors. C. Thomson syndrome. S. G. Grant' S. L. wengerl. J. J. Griffin. L. Morsberger. T. Ellingham. A. Hawkins. P. Long. E. Jaffee. Johns Lati~er'. D. Thu112. L. W. Burke,-University of Pittsburgh Hopkins School of Medicine, Depts. of Pathology and Oncology, Baltimore, MD. and Allegheny General Hospital, Pittsburgh, PA. Adenocarcinoma of the pancreas is the fifth leading cause of cancer death in the Rothmund-Thoason (RT) syndrome is an autosomal recessive U.S. with a dismal survival rate. There are very few established cell lines. We disorder characterized by poikiloderaa, skeletal abnormali- have karyotyped 4 previously unreported lines and compared them to the ties, hypogonadism, juvenile cataracts and a high incidence karyotypes of the primary tumors. All 4 cell lines have complex karyotypes, which of malignancy. It has been postulated that a primary defect are almost identical to those of the primaries. Within each cell line, there is in DNA repair is responsible for the premature aging and remarkable consistency in the karyotypes from cell to cell and, unlike the cancer susceptibility associated with this syndrome, although primaries, no normal karyotypes are found. The first cell line is 75-79, XX, no definitive test results have been reported. Our patient der(X)t(X;l)(p?22.l;q?25), +1, +1, der(l;3)(qlIO;qlO)x2, del(l)(q23), +2, +3, +5, presented at birth with bilateral radial reduction defects, der(5;14)(qlt);qlfl)x2, -6, add(8)(q24.3)x2, del(8)(p21)x2, -9, +11, add(l 1) an umbilical hernia and IUGR. She developed severe anemia (p15)x2, +12, -13, +14, +15,+16, +17, add(17)(pl 1)x2, -18, +19, by 9 months. She developed pigmentary changes in her skin add(19)(ql3.4)x2, +20, -22, +marl, +mar2, +mar3, 1-2dmin[cp5J. The primary by 4 months, with blistering after sun exposure and atrophic tumor is also hypertriploid. The second cell line is 78-85c4n>, XXX, -X, scarring upon healing. At 34 months she had a skin biopsy i(l)(qlO)x2, +add(3)(pl 1), -4, i(5)(qlO)x2, -6, add(6)(p22), del(6)(q22)x2, consistent with poikiloderma congenitale. Her karyotype was del(8)(p21), der(9;13)(qlI);qIO)x2, -10, -10, 14, add(I5)(pl 1)x2, del(16)(p12)x2, 46,XX. One out of 50 banded cells was endoreduplicated, del(17)(p I1.2)x2, -18, -21, -21, -22, -22, +2r, +marl, +mar2, l-4dmin[cp7J. In otherwise there was no evidence of trisomy mosaicism or contrast, the primary is hypodiploid. The third cell line is 81, XX, -X, -X, -6, -6, chromosomal rearrangements. Several established assays of der(8;14)(qlO;qlO)x2, der(15;20)(qlO;qlO)x2. -17, -17, -18, -18, -22, -22, DNA repair capacity and genomic instability were performed +martx2[2J. The primary has both hypodiploid and hypoteiraploid stem lines. on blood samples from the patient and her mother. There was The fourth cel line is 39-4(0, X, -Y, der(l;14)(qlO;ql)), add(3)(ptl), no evidence of increased frequencies of chromosome breakage der(4;10)(plO;qlO), del(6)(ql5), i(8)(qlO), -13, -14, -17, -18, -21, -22, -22, +r, in untreated or DEB-treated cultures or of SCE in untreated +marl, +mar2[cp6J.rev ish der(l0)r(10;4:l). Microdissection and FISH cultures. Similar levels of breakage and SCE were induced successfully identified the origin of a ring and a marker chromosome. The with mitomycin C in patient and control cultures. primary's karyotype is 46, XY. probably representing only normal cells. The Unscheduled DNA synthesis following UV2 exposure was not abnormalities observed in these cell lines are consistent with our previous elevated in the patient or her mother. GPA analysis of observations regarding consistent involvement of chromosomes 1, 3, 6, 11, 17, 18, somatic mutation showed normal frequencies in the patient, 19 in pancreas cancer. Analysis of additional lines is in progress. but evidence of a clonal expansion of a mitant clone in the mother. Discrepant results in the literature may reflect true heterogeneity in the disease or the fact that a consistent set of tests have not been applied to RT patients. Published Abstracts: Cancer Genetics (cont.) A343 1996 1997

Characterization of prostate tumor suppressor region of human Acute lineage leukemias: three cases with unique chromosomal alterations. C chromosome 8p22. X. GuO, R. Lebovitz2, C. Chinault2, and 1). E. Wells',. 'Dept. H. Hashimoto. C. F. Stephenson, and P. Woodbhdge. Oncogenetics, Phoenix, of Biology and Institute for Molecular Biology, University of Houston, Houston TX. Arizona. and 'Baylor College of Medicine, Houston TX. In recent years, genetic testing in hematology has exploded providing a wealth of Polymorphic alleles in chromosome 8p22 are frequently lost in prostate cancer patients suggesting the existence of a tumor suppressor gene for prostate cancer in this region. information. This in conjunction with the comprehensive genetic testing concept We have developed a detailed physical map of the region and confirmed previous LOll where immunologic, cytogenetic, and molecular genetic test results are correlated, findings in this region. Our data suggest there are 2 distinct regions of LOll in 8p22 that has aided in the evaluation of patients with challenging hematologic disorders. The may be contributing to tumor formation. We are currently isolating genes from these significance of this comprehensive approach involving the interpretation and regions to develop a transcription map in order to provide candidate tumor suppressor correlation of results is illustrated in the evaluation of acute mixed lineage leukemias genes. To achieve this goal, we have made use of a modified hybrid selection protocol. (AMLL) which can be difficult to characterize. This poster presents 3 such cases: Using YACs spanning 8p22 as probes, we have isolated binned sets of cosmids. This was accomplished by hybridizing inter-Alu PCR products from the YACs to arrayed, Case #1: T.G., elderly male with an acute leukemia showing bilineage expression chromosome 8 specific cosmid filters. In total, seven YACs, spanning about 8 megabases of T-cell (CD5, CD7) and myeloid (CD33, CD34) antigens by flow cytometry. were used and over 400 cosmids were isolated. Cosmid DNA was biotin labeled and Cytogenetic analysis revealed 2 abnormal cell lines: 46,XY,del(7)(q32q36), to of hybridized inserts from selected cDNA libraries following blocking repetitive DNA add( 12)(pl 2), and 46,XY,t(2; I 6)(q3 I ;q22). elements. cDNA/cosmid hybrids were purified using avitin coated magnetic beads and Case #2: G.L., middle aged male with acute leukemia demonstrating mutilineage cDNAs were elated. Following a second round of selection, cDNAs were recloned. The selected cDNAs are subsequently characterized to determine if they indeed map back to expression including T-cell (CD7), B-cell (CD19), and myeloid (CD13, CD33) the cosmids and YACs in this region. Sequence analysis is currently being performed on antigens by flow cytometry. Cytogenetic analysis: 46,XYt(2;3)(p21 ;p25). these clones. Most appear to be novel, as yet uncharacterized, cDNAs, however some Case #3: D.S., elderly male with leukopenia and thrombocytopenia. No evidence clones which are known to map to this region, such as Lpl, have also been identified. of an aberrant population by flow cytometry. Cytogenetics: 46,XY,del(9)(p21). T- cell and B-cell gene rearrangements are unusual, demonstrating lineage crossover which is highly associated with precursor lymphoid neoplasms of B and T lineage. D.S. was finally: diagnosed with a bilineage preleukemic state. These cases demonstrate how the combination of the tests performed and the information obtained provided 1) a diagnosis which if tlhe tests were performed independently would not have been as informative and 2) critical information relevant to the clinical management ofthe patient.

1998 1999 Alterations of p53 are an uncommon event in pheochromocytomas and A Philadelphia (Ph) chromosome as a secondary abnormality in a patient medullary thyroid cancers. KL K-F Herfarthl. M. R. Wick2. H. N. Marshall'. with anemia and myelofibrosis progessing to AML R. Honchel, G.l. Vance, E. OartnefI S. Lumt and J. F. MolCX1.3. Dept. of Surgeryl and Dept. of K. Pennington, N.A. leerenia. Department of Medical and Molecular Genetics, Pathology2, Washington University School of Medicine, St. Louis, MO, V.A. Indiana University Medical Center, Indianapolis, IN. Medical Centcr3, St. Louis, MO. A Ph chromosome as a secondary cytogenetic abnormality is a rare phenomenon, The role of the TP53 gene in the development of inheritcd and sporadic with approximately 20 documented occurrences in the literature. A 77 year-old male pheochromocytomas and medullary thyroid cancers (MTC) has not been clarified. diagnosed 17 months earlier with anemia, throinbocytopenia, and myelofibrosis demon- This is due to conflicting reports and to limitations in the assays previously used to strated an increased WIBC (23.600 vs. <11,000 x 103 /ml on all previous counts). detect mutations. We exhaustively screened a large number of pheochromocytomas Cytogenetic analysis of a bone marrow aspirate revealed 46,XY,t(5;20)(ql3;qll.2) and MTCs in an effort to determine the frequency of p53 alterations. [14J/46,XY[l]. Cytogenetic analysis of an sunstimulated peripheral blood sample also Twenty-two pheochromocytomas and 29 MTCs were screened for loss of showed 46,XY,t(5;20)(qt3;qll.2) [41/46,XY [1t. The role of this apparently recipro- heterozygosity (LOll) on 17p using four different markers. Single-strand- cal (5;20) translocation in any of the patients symptoms is unknown. Sixteen weeks conformation-variant (SSCV) analysis of exons 4 through 9 of the TP53 gene was later, the patient presented with increased fatigue, marked splenomegaly, and a WBC performed in the 22 pheochromocytomas and in 22 of the MTCs. The expression of of 198,000 x 103 /ml with 92% blasts. Cytogenetic analysis of an unstimulated blood p53 was determined by immunohistochemistry in 19 pheochromocytomas and in 17 sample demonstrated 46,XY,t(5;20)(q13;ql1.2) [2J/46,idem,t(9;22) (q34;qll)[28]. Blast MTCs, using two different antibodies (DOI and D07) on both frozen and paraffin- immunophenotyping results were consistent with the diagnosis of AML. FISH analysis embedded tissues. was performed using Oncor BCR/ABL probes. For the Ph positive blood sample, only 7 Four of 22 pheochromocytomas and none of the MTCs showed LOH on 17p. No of 200 were positive nuclei (within normal limits) using the major BCR/ABL breakpoint mutations were detected in any of the tumors screened using SSCV analysis. probes. However, 175 of 200 nuclei were positive lusing the minor BCR/ABL breakpoint Immunohistochemical staining of frozen and paraffin-embedded tumor sections did probes. Additionally, 25 of 200 nuclei from the earlier Ph negative bone marrow sample not show p53 overexpression in any of the examined tumors. were positive using the minor breakpoint probes. To the best of our knowledge, sec- Our findings, combined with previous reports, indicate that mutations in the TP53 ondary development of a Ph chromosome after initial diagnosis of myelofibrosis has not gene are an uncommon event in the tumorigenesis of pheochromocytomas and been previously reported. Conclusions: 1) This patient developed a secondary Ph chro. medullary thyroid cancers. mosome (minor breakpoint) possibly associated with progession to AML; 2) The patient was likely in the early stages of transformation when the first bone marrow aspirate was received.

2000 2001 The Sheffield Cancer Genetic Service: Experience of a National Fluorescent in situ hybridization assessment of chromosome copy Health Service(NHS) Regional Clinical Genetics Unit in the United number in gestational trophoblastic disease. C. Lathrop. K. Santoro. A. Kingdom Kuaar, Dhavendra 111; Radstone, David [21; Quarrell Oliver and H. F. L. Mark. Rhode [1). [1] Centre for Human Genetics, Sheffield Childrens Hospital, Afify Island Hospital and Brown University 121 Department of Clinical Oncology, Weston Park Hospital School of Medicine, Providence, RI. ,Sheffield, England, United Kingdom (UK). Fluorescent in sitit hybridization (FISH) is an investigational In view of rapid increase in public awareness about hereditary technique that is used as an adjunct to conventional cytogenetics both in risks in common cancers ( breast, ovary, and colorectum), a cancer clinical practice and in research. Although it has multiple applications, genetic service (CGS) is being offered by the North Trent Regional the most unique and powerful is the FISH analysis of archival cancer Genetics Service in UK, serving a population of approximately specimens. Mark, Ahearn and Lathrop have previously reported on the 2,500000. The aim is to offer a comprehensive rationalised and integrated multidisciplinary service to families and relatives findings of constitutional trisomy 8 mosaicism and gestational anxious to have information on risks and advise on developing common trophoblastic disease (GTD) using FISH and archival materials that were cancers. CGS include a monthly Cancer Family Clinic , and weekly nearly 20 years old. To confirm and extend the above study and to General Genetic Clinics. The main functions in medical care of the determine the frequency of trisomy 8 in GTD, we analyzed patient cancer genetic clinics lie in providing proper information on risk specimens of various age using FISH and formalin-fixed paraffin- assessments, advising on various options, preparing for predicitive embedded material. Although only a limited number of specimens have genetic testing for having a cancer predisposing gene (e.g. BRCA1), and initiating and monitoring appropriate periodical cancer been studied thus far, we have demonstrated that FISH is feasible for the screening programmes. A total of 388 individuals from 253 families study of interphase cells in this cancer. Preliminary analysis indicated with a history of cancer have so far been counselled 1991 to 1995; that out of a total of 6 cases studied, 3 (50%) were trisomic for 224 family members were visited at home prior to formal clinic chromosome 8, if a cut-off point of 6% trisomic cells is adopted. consultation to collect preli.inary information and preclinic Additional data from this ongoing study will become available to confirm and the clinic visit to reinforce the appraisal post genetic or refute the hypothesis that a subset of gestational trophoblastic disease counselling. Experience in dealing with few illustrative families exists that is characterized chromosome 8 as was with breast/ ovarian and colorectal cancers is presented. Referral by trisomy, recently guidelines to the CGS are given for comments and suggestions to make found in breast, prostate and other cancers. these more practical and cost effective. Protocol for predictive gene testing for breast cancer susceptibility gepes (BRCAI, BRCA2) is provided. Attention is drawn on the ethical, legal, and socio- economic, and psychological implications of CGS, particulalry the predicitive gene testing for cancer susceptibility. The current problems and future scope of the CGS in the UK are highlighted. A344 Published Abstracts;: Cancer Genetics (cont.) 2002 2003 Genetic polymorphisms in the CYPlAl gene and susceptibility to oral Complex rearrangement induced by human papillomavirus DNA cancer. J.C. Law, S.M. Gollin, A. Deka, and E.A. Facher. Department of Human Genetics, University of Pittsburgh, Pittsburgh, PA. integrated within the 12q15 Multiple Aberration Region gene in a The CYPIAI gene encodes the cytochrome P450 enzyme P4501AI which is responsible cervical carcinoma for the metabolic activation of polycyclic aromatic hydrocarbons to reactive carcinogenic Maria 1. Gallego and Pedro A. Lazo Unidadde Geiefica Molectilar (Consejo intermediates. A noncoding Mspl restriction fragment length polymorphism (RFLP) and Superior de hInvesigacionues (Oientificas), (Centro Nacional de Riol/gia ('elular, Inshtildo an exon 7 single base change (A to G substitution at codon 462) have been previously de Salad Carlks 111, 28220Majadahonda, Spain. Email: plazozbi~isciii.es identified in the CYPlAI gene and have been associated with an increased risk of lung In human cervical carcinomas papillomavirus DNA is integrated in the cell genome We have cancer in Japan. An analysis of these polymorphisms was conducted in oral cancer since with cloned the integration site of HPV-I DNA in human chromosome region 12q1 present in the SW756 cancer of the oral cavity shares tobacco exposure as a common major risk factor cervical carcinoma cell line. Viral DNA is broken from nt 2643 to 3418 resulting in a deletion of 775 lung cancer. Comparison of Mspl allele frequencies between 70 U.S. patients with oral viral nt. Cloning and sequence analysis of the rearranged and gernline alleles shows that there is no cancer and 114 healthy controls demonstrated an excess of Mspl rare alleles in the patient homology betowen the target cellular and viral DNA. suggesting it is a non homologous recombination. population. The rare allele, defined by the presence of the Mspl site in the 3' flanking The target cellular region is called ppillommvxrus associaled locus 2 (PAL2). The 5' and 3' flanking region of the CYPlAl gene, was present at a frequency of 15% in the patient population probes derived from the hybrid viral-cellular clone detect completely different genaline restriction as compared to 9% in the control population. Additional analysis of the CYPlAI exon fragments. There is no overlap betiseen the restriction maps of the target germline clones obtained isth 7 single base substitution failed to demonstrate any difference between the frequency of 5' and 3 flanking probes. Probes from these germline clones beyond the breakpoint position do not the CYPIAI exon 7 alleles in the control and patient populations. detect any DNA rearrangement in SW756 cells DNA. These data prove that there is a deletion of cellular DNA as consequence of the integration, with an estimated minimum size or 14 kilobases. The rearrangement induced bh HPV18 DNA is complex and also includes an inversion of cellular DNA Both flanking probes arc outside the amplicon of this chromosome region identified in the OSA and RMSI3 sarcoma cell lines. comprising SA4.S-CIIOP-(DK2-AfI)A12 genes. We determined its precise map location by screening an arrayed cosmid human library specific for human chromosome 12 (LLI2NCt)I). P.112 is located approximately It kb from STS marker RM33. which is the most proximal to the 3. flank ofviral DNA. The integration site is present in human 433Cl and 227E8 clones from CEPH-I1 YAC librarv. This location is within the Multiple Aberration Region (MAR) which is affected in scvral types of tumors swith a mesenchymal origin. The integrated tiral DNA is located 50 kb from a CpG island and 1.5) kb from the HIIAIG-fC gene towards the centromer. HA1(1-C' gene and integrated HPV-18 DNA have opposite transcriptional orientations. There is no ovcrexprcssion or altered message from the 1A0 (;1- gcne in three cervical carcinoma cell lines. The integration at 12ql3- IS might have been sclected be its contribution to the tumor phenotype.

2004 2005 Centronmeric satellite DNA probes for the enumiueratlon of trisomy 8 In Emergence ofAberrant Chromosomes in ChronicMyelogenous Leukemia myeloid disorders. C.C. Lin, W. Etches, B Roland*, and C. Lee. Department of in Blastlc Crisis. J.H.Lin', E.Bayni, F.Wasty, V.J.Shah, LCarmosial, Laboratory Medicine and Pathology, Univcrsity of Alberta, Edmonton, Alberta, and M.Chesfar, HkOLSah. Nassau Cty. Med. Ctr., E. Meadow, N.Y., Health the 5Department of Pathology, Univcrsity Or Calgary, Calgary, Alberta, Canada. Sci. Ctr. State U. of N.Y. at Stony Brook', N.Y. Fluorescent in sitlt hybridization (FISH) studies tising chromosome 8 specific This 65 year old man was referred to our hematologic clinic 2 years ago ccrntromeric satcelitc DNA probes for the enumeration of trisomy 8 was performed on because of high white blood cell count of 107 thousands per cubic mm 75 cases of patients with mycloid disorders including 23 cases of AML, 23 cases of without any other abnormal laboratory tests or constitutional symptoms CML, 13 cases of MDS, and 16 cases of MPD. Twenty five control cases (without except moderate hepatosplenomegaly. Peripheral blood and marrow mycloid disorders) were also used in this study. Routine cytogenetic study results revealed presence of Ph' chromosome. He was managed with Hydroxyurea were available on all these cases. Approximately 4()) interphase nuclei and 40 he of metaphase chromosome spreads were examined by FISH with each of an alpha 8 and and Interferon with a good result. Recently complained malaise, bony a gamma 8 satellite DNA probe. In total, eight cases had 5 % - 50 % of the interphase pains and fever for 34 weeks. Blood tests revealed mild anemia, marked nsuclei exhibiting three or more hvbridi7ation signals and 7 % - 68 % of the metaphase thrombocytopenia and WBC of 58 thousands per cubic mm with 19% spreads showed three or more cluromosomes exhibiling positive hybridization signals. myeloblasts and others. No bone marrow smear was obtained; however, the Routine cytogenctic analyses confirmed 5 of those 8 cases with trisomy 8 cells, I case marrow core exhibited 100% cellularity and there were clusters of up to 300- with a hyperdiploid cell population, and 1 case with cells that acquired an additional cell immature granulocytes in paratrabecular and non-paratrabecular regions. chromosome 8 which was involved in a structural rearrangement. However, one case were at those of chronic leukemia (CML) in with cells having an extra chromosome 8 material was detected by FISH studies but The pictures least myelogenous not by routine cytogcnetic analyses. As well, tetrasomy for chromosome 8 was accelerated phase and easily in the stage of blastic crisis. Marrow identified by FISH in the case with a hyperdiploid cells. In this study, there appeared chromosomal study revealed 48,XY,+8,t(9;22)(q34;qlI), +der(22)t(9;22), to be no significant differences in the use of either alpha or gamma satellite DNA - 21,+mar. Two weeks later, the peripheral blood showed 35% myeloblasts. probes in the enttmeration of additional chromosome 8s. Furthermore, in some The family and patient opted to the conservative and supportive approach complex chromosome rearrangements and certain numerical aberrations involving following a detailed discussion. He expired in a month. chromosome 8, FISH studies may have an advantage in deciphering the presence of is in CML. When disease progresses, extra chromosome 8 material. Generally, t(9;22) present typical over 2/3 of cases develop additional chromosomal aberrations, most common ones being chromosomes 8,17,22. Combined 8, 22 was seen in less than 10% of cases with CML in blastic crisis. Presence of marker was very rare and its significance remains unclear.

2006 2007 Possible inherited predisposition to myelodysplastic syndrome, an Multicolor spectral karyotyping of primary and immortalized mammary uncommonly familial myeloid malignancy. S Mandla' S Goobie'. D.L. epithelial cells. McCormack SJ, Liyanage M, Goldstein D, Lippman ME, Trock B, Guernsey'. T. Kumar'- 0. Hayne2 and W.L. Greer'. 'Div. of Molecular Pathology Ried T and Dickson RB. Lombardi Cancer Center, Washington DC., NCGR/NIII, and Molecular Genetics and 2Dept. of Medicine, Dalhousie UJniv., Halifax, NS, Bethesda, MD. Canada, and 'Commissioner's Medical Assoc., London, ON, Canada. We have begun to optimize a novel system for early detection of breast cancer, namely syndrome is a hematological malignancy, occurring the use of nipple aspirate fluid (NAF) to detect cytogenetic changes associated with the Myelodysplastic (MDS) early stages of breast cancer progression. The ultimate goal is to use this system for primarily in the elderly as an acquired, sporadic disease. Familial cases of MDS early detection and genetic studies in women with high risk of familial breast cancer. are rare. We have identified a family with three affected individuals, with early Currently, the methods used to identify genetic alterations in epithelial cells have not age ofonset, suggesting a possible inherited predisposing abnormality. Using been applied to cells shed into NAF. To begin this study, we have developed and tested linkage analysis, we examined whether chromosomal regions commonly protocols for the culture and immortalization of epithelial cells derived from NAF. Be- associated with sporadic MDS are involved in familial expression of this cause NAF samples may yeild relatively small number of epithelial cells amidst other disorder in our Monosomy 5 (-5) or interstitial deletions involving cellular contamination, the ability to conduct pilot diagnostic cytogenetic studies can pedigree. be greatly enhanced by culturing epithelial cells derived from NAF and immortalizing band 5q3 are among the most frequent karyotypic abnormalities in sporadic them with human papillomavirus oncogenes E6 and E7. This increases the number of MDS. Although -5 was observed in bone marrow (BM) from one affected family available cells and provides a renewable resource for cell biological and genetic studies member, polymorphic DNA marker analysis showed that the critical region, requiring large cell populations. To assess any changes induced by the E6/E7 mediated 5q3 1, did not cosegregate with MDS. However, we found cosegregation of MDS immortalization of cells, we have immortalized nilk-derived mammary epithelial cells with markers from four other chromosomal regions associated with and compared them to parental populations by cytologic tests. We hypothesize that 21 3q26, and I q2 1. Cytogenetic early genetic changes in mammary epithelial cells of women with high risk of familial hematological malignancy: q22, 7q22. breast cancer may be subtle and are best studied by multicolor spectral karyotyping. In abnonnalities involving one or more of these chromosomes were detected in preliminary applications of this technique with human breast cancer cells, very detalied affected family members. Candidate genes (AMI, I, PMS2, MDSI and MCI,I, study of chromosomal rearrangements was feasible (Schrock et al, in press). respectively) have been identified in these regions and are presently being screened for possible tnutations. Characterization ofthe gene defect responsible would prove valuable by providing a basis for early diagnosis and treatment, through identification and monitoring ofindividuals at risk for MDS, as well as a better understanding of the molecular mechanisms underlying leukemogenesis. Published Abstracts: Cancer Genetics (cont.) A345 2008 2009 Family history of cancer among cases ofnon-Hodgkin's Lymphoma (NHL), Hodgkin's Breast and ovarian cancer syndrome in an identical trvin pair. S. Miesfeldt. Disease (HD), Multiple Myeloma (MM) and population-based controls. H. I. McDuflie M. A. Lovell. S. M. Jones. B. L. Turner. University of Virginia Health Sciences and the Cross Canada Pesticides and Health Study Group. Centre for Agricultural Center, Charlottesville, Virginia. Medicine, Saskatoon, Saskatchewan, Canada. BRCAI accounts for approximately 45% of autosomal dominant breast cancer Several rare tumour types: NHL, HD and MM share common putative risk and the majority of cases of heritable breast and ovarian cancer. Up to 10-15% of factors including a positive family history of site specific tumours and cigarette smoking. BRCAI mutation carriers do not develop cancer. The factors governing the Occupational exposure to certain pesticides have previously been implicated. Our penetrance and expression of this gene are poorly understood. Breast cancer risk objective was to simultaneously evaluate and compare the relative importance of several and ovarian cancer risk, in general, are known to be associated with non-Mendelian risk factors including a family history of cancer in first degree relatives of cases of NHL, factors including age, menstrual and reproductive history, hormone use and HD and MM compared to population-based controls. environmental exposure(s). However, the ways in which these various factors We conducted a Canadian (six province) case/control population-based study of interact with inherited predisposition in the etiology ofdisease are not known. Here 517 cases of NHL (ICD-9 200, 202), 316 cases of HD (ICD-9 201), 347 cases of MM we report the first documented case of the breast and ovarian cancer syndrome in (ICD-9 203) and 1506 age and province of residence frequency matched controls. All an identical twin pair. individuals were males and the age range was 19-84 years. We used structured Both twins developed histologically similar ovarian cancer in their mid-fifties. questionnaires to collect demographics, lifetime occupational history and expostires, One twin was diagnosed with stage I disease but ultimately developed an medical and smoking history and details of family history of cancer in first degree unresectable recurrence. She has been treated with multiple cytotoxic relatives. are as age Results reported and province of residence adjusted conditional chemotherapy regimens and remains clinically stable. The other twin was logistic regression analyses derived odds ratios and 95% confidence intervals (CI). diagnosed with stage Ill disease and died of refractory metastatic disease in April Family history cancer were of data adjusted for family size where applicable. 1996, following several chemotherapeutic trials. Neither twin developed breast or While a strong minority of individuals (33% to a 48%) reported positive history colon cancer. Their family history is significant for multiple cases of early-onset of cancer in first degree relatives, the adjusted odds ratios were all less than 2 for "any breast and ovarian cancer. The twins have both similarities and differences in termns cancer", cancer in parents, siblings or offspring and in models which adjusted for ofnon-genctic cancer-related risk factors. Zygosity testing revealed monozygosity. additional suspected risk factors. Statistical at was significance .01 achieved for many Results of BRCA I analysis are presented. This report has important implications of the adjusted odds ratios. Siblings experienced higher risks than either or parents concerning thie role of genotype in the ultimate penetrance and expression of offspring. We conclude that a positive family history of cancer is one factor contributing disease among BRCA I mutation carriers. to the risk of these neoplasms. A history of cigarette smoking or occupational exposure to pesticides 2 1O hours per year is less important. (Supported by Health Canada).

2010 2011 Analysis of RET and RET ligand (GDNF) in small cell lung carcinoma. LOH studies in humanbrain tumors with markers derived from the Cowden Lois M. Mulligan', Stacey M. Ivanchuk', Barbara G. Campling2, Vasi Sundaresan3, critical region M.R. Nelen', A. von Deimling2, D. Boermanl, E. Mariman3, C. Eng4, Pamela H. Rabbitts3, Charis Eng4. Departments of Pathology1-2 and Oncology2, international Cowden consortium, H. Kremer3, G.W. Padberg'. I Dept. Of Neurology, Queen's University, Kingston ON, Canada K7L 3N16; MRC Centre, University of University Hospital Nijmegen, 2 Inst.for Neuropathology, University Clinics Bonn, 3 Cambridge, Cambridge UK3; Dana Farber Cancer Institute, Harvard Medical School, Human Genetics, University Hospital Nijmegen, 4 Dana-Farber Cancer Inst., Boston. Boston MA4. Cowden's disease (Cl)) (MIM 158350), or multiple hamartoma syndrome, is a rare auto- The RET proto-oncogene is expressed in cells and tumoiirs derived from neural crest somal dominant familial cancer syndrome. Patients with CO have a high risk to develop precursors. In one published study, RET mutations of codon 664 in exon 11 have been breast and thyroid cancer. Charateristic of the disease is the hamartomatous feature of found in 2 of 6 small cell lung carcinoma (SCLC). To investigate the occurrence of the skin, breast, thyroid, oral mucosa, and the intestinal epithelium. Central nervous these arid other nutations, we examined 38 SCLC cell lines for RET mutations in system manifestations include niegalencephaly, epilepsy and dysplastic gangliocytomas exons 10, 11, 13-16. These represent the exons where RET mutations have been found (Lhermitte-Duclos disease). Expression of the disease is variable and virtually complete in SCILC or other neuroendocrine tumours and in the inherited cancer syndrome at the age of twenty. Only recently we localized the gene for CD to chromosome sub-band multiple endocrine neoplasia type 2. No RET mutations were detected in any of our lOq22-23. Tumor suppressor genes seem to play an important role in other phakomatoses panel of SCLC lines in these exons. To investigate the possibility that activating RET such as neurofibroinatosis, Von HIippel-Lindau disease and tuberous sclerosis. Since CD mutations are found elsewhere in RET in SCLC, RT-PCR analyses were used to has clinical features analogous to the other phakomatoses the involvement of a tumor examine the expression of RET in 21 SCLC cell lines. We detected RET expression in suppressor gene seems likely. Furthermore, LOHI studies in various human brain tu- 15/21 (71%) of these lines. In four of these cases, the sequence of all 21 known RET mors have implicated chromosome lOq. To test the hypothesis that the Cowden gene exons was examined for mutations by sequence analysis of RT-PCR products. No acts as a tumor suppressor gene and is involved in different types of brain tumors we mutations were identified. Further, no clear correlation was detected between the performed LOH studies in 45 brain tumors including: 2 dysplastic gangliocytomas, 3 as- presence or level of RETexpression and other features of neuroendocrine trocytomas, 1 ependymona, 6 glioblastoma, 3 gangliogliomas, 2 hemangiopericytomas, differentiation in the cell lines. WAe next examined expression of glial cell line-derived 5 meningiomas, 3 medulloblastomas, 4 metastatic carcinomas, 2 schwannomas, 2 oligo- neurotrophic factor (GDNF), the putative RET ligand, in the same set of turmour cell dendrogliomas, 7 oligoastrocytomas, 2 pilocytic astrocytomas, I pituitary adenoma, 2 lines. GDNF was expressed in 7/21 (33%) SCLC cell lines. There was no apparent pleomorphic xantho astrocytomas. The markers used were flanking or derived from the correlation between RET and GDNF expression. As yet, no mutations in the GDNF Cowden critical region. Allelic loss was seen in one dysplastic gangliocytoma (mark- coding sequence have been detected in our panel of SCLC lines. We conclude that the ers: D1OS573, DIOS215) and possibly in one metastatic brain tumor (primary tumor simple misseiise RET mutations found in other neural crest-derived tumour types are unknown). Thus, an important role for the Cowden gene in the development of human not common in SCLC. Whether mutations of RET or its ligand GDNF play an brain tumors seems unlikely. Ilowever, its function in the pathogenesis of the dysplastic important role in tumorigenesis in SCLC requires further investigation. gangliocytomas might be important, marked by LOll in a region which partially overlaps the Cowden Critical region.

2012 2013 Pesticide Exposure and Sister Chromatid Exchange Frequency. The Pesticides and Allelic deletions of the von Hippel-Lindau (VHL) disease gene detected in Genetic Instability Study Group. Centre for Agricultural Medicine, Saskatoon, islet cell tumors in VHL patients. S. Packi, I. Lubenskyl, S. Amadal, Saskatchewan, Canada. P. Queramil, J. Gnarra2, M. Walther2, M. EmMert-Buck, W.iLinehan2, L. Liotta, Pesticide is a generic term which for agricultural purposes includes herbicides, ,. Zhuangl I Laboratory of Pathology arid 2Surgery Branch, National Cancer National Institutes of MD insecticides, fimmigants, fuingicides and rodenticides. A wide variety of types of Institute, Health, Bethesda, chemicals The VIil, disease gene has been localized to chromosome band 3p25.5 and recently utilizing different metabolic pathways to control pests is implicit in the term pesticides. cloned. Patients with ViIL disease develop multiple neoplasms in the central nervous Numerous epidemiological studies have reported an increased risk of certain rare system, kidneys, pancreas and adrenal medulla. VIIL gene alterations have been reported neoplastic diseases among farmers and others occupationally exposed to pesticides. in renal cell carcinoma and pheochromocytoma of VIII, patients. Although 17% of VII L Several molecular studies have found post-pesticide exposure increases in frequency of patients develop pancreatic islet cell tumors (ICT), it is currently unknown whether the genetic markers. alteration of the VIIL gene is responsible for ICT development. We studied islet cell We recruited volunteer farmers, commercial pesticide applicators and urban tumors from 4 VIIL patients for allelic deletions of theVIlL gene using a FISH probe to theVIlL gene and primers for the polymorphism (104/105) upstream of the coding region dwellers who did not have occupational exposure to pesticides. The unexposed control of the VIIL gene. Allelic loss of the VIIL gene was detected in all 4 VHL-associated ICTs. subjects were matched to the exposed ones by age ± 5 years, sex and cigarette smoking The results show, for the first time, that the Vil. gene is involved in the tumorigenesis history. All volunteers completed a screening questionnaire which allowed us to exclude of ICT in VIIL patients, and also suggest that ICT is a part of the VHL syndrome. potential subjects with medical conditions or exposures known to contribute to genetic instability. Each individual will be tested at least three times: baseline in winter, immediately following the herbicide spraying season (June) and in the autumn. Some individuals are exposed all year and we have received additional samples from them. We used standard methodology for the sister chromatid exchange analysis and counted 20 metaphases per sample. Among the first 125 samples, the intra-individual variation in frequency of sister chromatid exchange on any one day was large (the widest range for one person was 4- 27). The immediate post-exposure mean values were not significantly higher than baseline. We conclude that sister chromatid exchange is not a sensitive indicator of pesticide exposure broadly defined as occupational versus environmental. Supported by Saskatchewan I ealth Services and Research Commission. A346 Published Abstracts: Cancer Genetics (cont.) 2014 2015

Differentially expressed genes in Ovarian Cancer D. K. Price. Z. Bahrani- Risk assesent in first degree female relatives of rpay usal Mostafavi. J.R. Ball. R.V. Higgins. J.B. Hall. M.C. Marroum. and M. Harris- and postnenopausal breast cancer patients. N.R ari, Owens. Cannon Research Center, Carolinas Medical Center, Charlotte, NC. M.Jaiswal, G. Anuradha, -S. , sN.Jain and YR. Ahuia. Genetics Unit, Bhagwan Mahavir Medical Research Centre, H erabad - Only 5-10% of all cases of breast and ovarian cancer are attributed to 500 004, India and *IJ Cancer Hospital, Hyderabad-500 004, India. autosomal dominant susceptibility genes. Ovarian cancer alone is responsible The first degree female relatives (FDFRs) of a breast cancer for approximately 14,000 deaths per year in the United States, and it is patient have been reported to have 2-3 fold increase in breast hypothesized that the lack of significant symptomatology in the early stages of cancer risk compared to the control population. Human breast ovarian cancer accounts for this high mortality rate. Seventy percent of women cancer is a multi factorial disease and there is substantial heterogeneity of risk for breast cancer among relatives of with ovarian cancer present with extensive intraabdominal disease, which is breast cancer patients. However, the risk to the FDFRs depend upon the difficult to surgically eradicate, and many patients have poor responses to family history of the disease, age at which diagnosis was made and chemotherapy. Currently, early diagnosis of non-inherited ovarian cancer is whether the disease was unilateral or bilateral. Several dependent on imaging techniques and serum levels of the tumor marker CA125. oncogenes and antioncogenes have been found to play a role in the These diagnostic tools are severely lacking, however, due to the non-specific causation of breast cancer. But no bicaaker is available in nature of the CA125 assay (reported to vary up to 15% day to day), and the identifying individual risk. inability of imaging to distinguish between malignant and benign ovarian tumors. Carcinogenic process in general is associated with gencanic instability. In our search for biomarker for individual risk, Due to the lack of specificity of CA125 and the absence of a good marker for ccnet assay was carried out for the first time to study DNA damage diagnosing ovarian cancer, we have begun a study focusing on the isolation of and repair in first degree relatives of breast cancer patients. ovarian specifically expressed genes. We are studying ovarian cancer by direct Individual mean cwmet tail length was based on 50 cells per comparison of mRNAs from tumor and normal ovaries utilizing the mRNA treatment. Cancer being monoclonal, consideration of inter-indi- differential display technique. Different tumor types including endometrioid vidual and inter-cellular DNA damage and repair was considered reasonable. Although unilaterality or adenocarcinoma, serous carcinoma, mucinous cystadenocarcinoma and clear bilaterality did not show significant association for breast cancer risk, first degree are cell adenocarcinoma all being used in this analysis. We have currently relatives of premenopausal cancer patients showed low DNA repair isolated several interesting fragments that are differentially expressed in either efficiency (ie, high cancer risk) as caspared to the first degree normal or tumor tissue. These fragments are being analyzed by DNA relatives of postemenopausal cancer patients. Long term follow up sequencing, northern blot analysis, computer database analysis, and are also studies are required to confirm this conclusion. being used as probes for cDNA library screening. Each differentially (Financial support frcm CSIR is acknowledged 9/132(463)/94 expressed product is a potential tumor suppressor gene, oncogene, or tumor -EMR-I-'). marker.

2016 2017 Prostate Cancer Genetics and Clinical Outcome Sadasivan, R., VanVeldhuizen, Unusual Complex Cbromosomal Aberration P.J., Dwyer, T. and Tranin, A. University of Kansas Medical Center, Department of [46,XX,del(9)(q22),del(13)(q21)Jfoundin AgnogenicMyeloidMetaplasla. Medicine, Kansas City, Kansas Trinity Lutheran Hospital, Kansas City, Missouri H.O.Shah, I.Mia, M.Siddigui, A.Alidin , M.Garison, M.Ches , Background. Multiple genetic alterations including the activation of oncogenes and J.H.Lin'. Nassau Med. E. Health inactivation of tumor suppressor genes are important in the pathogenesis of many malig- Cty. Ctr., Meadow, N.Y., Sci. Ctr. State nancies. Prior chromosomal analysis of human prostate carcinoma has revealed frequent U. of N.Y. at Stony Brook', N.Y. loss of portions of chromosomes 8p and 10q but the clinical significance of this is un- This 64-year old black woman developed an agnogenic myeloid metaplasia known. We have examined allelic deletions in human prostate cancer to evaluate the (AMM) five years prior to this first visit to our clinic. She complained of frequency anti pattern of loss ofheterozygosity (LOll), and to note any correlations with worsening of abdominal discomfort, easy satiety and lower extremity edema. other biologic and clinical including prognostic factors such as stage and variables grade She had a massive splenomegaly down to the iliac crest, 100-150 thousand of tumor. Using cDNA probes for and 8p loci, Southern blot analyses were Methods. l0q white count per cubic mm with lencoerythroblastic pictures. The spleen performed on prostate tumor samples and peripheral blood controls. A set of restriction was treated with low dose radiation for relieving endonucleases were used to reveal the polymorphism. Results. A subset of patients associated abdominal were identified with deletions at these loci. Of the patients analyzed, 7/20 (35%) had symptoms. The treatment was followed by improvement of WBC count to allelic deletions involving both alleles at 8p (PLAT locus: 8pl2). 1/20 (5%) had loss of 9-18 thousand, platelet counts of 170-450 thousand per cubic mm and Hct one allele at 8p. On chromosome 10q, 9/20 (45%) had loss of both 10q alleles (at the of around 30%. She had been continued on Erythropoietin for over one PLAU locus, 10q24-qter). All patients who had loss at 8p also had loss of 10q alleles. year. Currently chromosomal study revealed unusual complex anomaly Interestingly, of the 9 samples that showed allelic losses at 8p and/or 10q, all were from [46XX,del(9)(q22),del(13)(q21)]. patients with early stage (pathologic stages A and B) disease. Retention of these alleles Chromosomal studies in patients with AMM are limited. Non-random were noted in all 11 patients with advanced stage (pathologic stages C and D) disease. changes were reported in chromosomes Conclusions. These results suggest that retention of genes on 8p and 10q may be neces- 1,5,7,8,9,13 and 20. Structural sary for the development of metastasis. Conversely, allelic loss at these loci may inhibit changes were mostly seen in lq,5q, 13q and 20q. 13q as a sole anomaly is the development of metastasis. present in about 10% of patients with AMM having chromosomal alteration. Characteristic alteration in chromosome 9 was commonly numerical. These combined anomalies were apparently very rare. Since antioncogenic activity of retinoblastoma gene (RBI) resides in 13ql4, a possibility of untoward outcome might be expected in this case.

2018 2019 Exon scanning for BRCA1 mutation in a set of 170 families with members Non-ralndom association betwseen trisomy 4 and double minutes in referred to a breast/ovarian cancer family clinic. D Stoppa-Lyonnet, G Ithier, patients with acute non lymphocytic leukemia.M.Varela' F.Tsienl. S.Ca[stjto2. S Pages, P Laurent-Puig, G Thomas. Institut Curie, Paris, France. Intro. by: A 'llutian Genetic lProgramn Hayward Gettetic Center, Tulane School of Medicine,' Touro M-unn-ich. Infirmary New Orleans, Ia. Genetic epidemiology indicates that about one in 200 women is carrier of an altered rhe association between trisomy 4 and double minutes has been reported in only gene associated with a high risk of breast carcinoma. BRCA1 has been implicated in 6 previous patients. Doutble minutes are a cylogenefic manifestation of gene amplification 45% and 70% of individuals with a genetic predisposition to breast and ovarian and disease progression. 'Il[ty are found more comomonly in solid tumtors bst are seen carcinoma respectively. In May 1996, about 80 different deleterious mutations had been occasionally in leukemia. According to the FAB classification these abnormalities were reported to the BRCA1 Information Core data base. The majority of these mutations seen in ANLL of the M2 or M4 type. are small sized and scattered over the 22 coding exons or intron-exon junctions, and Our pa.tiet an 84 year old female presented with thrombocytopenia and anetia. lead to putative truncated proteins. A marrow aspirate revealed increased cellularity. the myeloid cells showed marked shift The genetic bases of breast and/or ovarian cancer susceptibilities was investigated in to the left and itcreased tnumber of blasts. Maty of (lte early tmyeloid cells show irregular 170 families with at least one member referred to our cancer family clinics. Three folded nuclei (moosocytoid). Trhe erythroid precursors were sparse and the negakaryocytes families included 2 cases of ovarian carcinoma, 60 families with at least one case of diminished. The differential shossed 18% of blasts, 14%, myelocytes, 47% metamyelocytes, breast and one case of ovarian carcinoma and 107 families with at least 3 cases of band forms and segmented neutrophils 28%, erythroid precursor 5% and lymphocytes 4%. breast carcinoma. In each family, the cancer patients were all first or second-degree Iltese results are consistent ssith a myelodysplastic syndrome. (Refractory anensia with relatives from the same lineage. After informed consent, a blood sample was obtained Excess Blasts) itt transfortoation to ANILt.. Chronmosonte sttdies its the unstimutlated from the family member with the highest probability of being gene carrier. Genomic marrow revealed trisoniv 4 and between 6-10 double minutes in all of the cells analyzed DNA was scanned for BRCAI mutations by DGGE on all coding exons and (20 cells). With G battding double minutes appear light but they stain darker with plain intron/exon junctions. Geimsa stain. Thirty-nine mutations leading to truncated proteins were identified (34 frameshift, 4 Trisomtty 4 is an uncotnmott finding in letuke,,sia but has been reported itt nonsense, 1 splice alteration): 3 mutations in the ovarian-only cancer families, 21 approximately 36 palietts. Hoswever. lthe association between trisomy 4 and double minutes mutations in the breast/ovarian cancer families, 15 in the breast-only cancer families. appears to he tot-randotit and has only been described in six previous patients. The only In addition 9 mutations (7 missenses and 2 mutations in intron/exon junctions) with common features atttong Iltese patients and ottrs seets to be the advanced age oflthe paliettts no proven deleterious consequences were detected. and the type of Icukeitia There is also a predominance of females (6: 1). Anitplificatiott Using MLINK with the segregation model, parameters and taking into account the of ftie c-tttyc oncogette was foultd it one of the patients published before with two copies ofthe on of the double genetic heterogenity of these cancer susceptibilities (Easton,1993), the expected number oncogeene each minutes. A probe specific for the c-myc oncogene was used in our patient but to of BRCA1 deleterious mutations in this set of individuals was evaluated to 86. The 50% hybridization the double minutes was not observed. difference between the observed and expected number of mutations may be due to the lack of sensitivity of the mutation screening and/or to inaccuracies of the parameters derived from genetic epidemiology. Published Abstracts: Cancer Genetics (cont.) A347 2020 Altered expression of p2l(Wafl/Cipl) during differentiation of hepatoma cells. H.Yamamotol), T.Hlashimoto2), W.Changl), T.Uckil), J. Fuiimotol), K.Sasaki3), H.Tachibana3), M.Sugita3), T.Nakajima2), N.Yamanakal), EOk-amotot), T.Tamaoki4), and J.Furuvama2). 1) Ist Dept. Surgery, 2) Dept.Genetics, and 3) Vth Dcpt. Intern. Med., Hyogo Coll. Med., Nishinomiya, 663, Japan, 4)Dept. Med. Biochem., Univ. Calgary, Calgary, Alberta T2N4N 1, Canada. p21(Wafl) is one of the factors that inhibit the binding between cyclins and cdks, leading to arrest of cell cycle progression. The expression of the wild-type p53 but not mutant p53 proteins induces expression of p21. Recently, p21 has been shown io be induced during cell differentiation of neuronal, myogenic, p53-deficient hematopoietic and hepatoma cell lines. Mutation of p53 has been frequently found in hepatoccllular carcinomas (HCC). The expression of p21 in human primary HCC tissues is highly variable. We found little correlation between expression levels of p21 and tumor size, stage or p53 mutation in these samples. In HCC cell lines, lower levels of p21 were found in cells expressing mutant p53 than those expressing the wild-typc p53. In HB cell lines which express the wild-type p53, p21 expression was higher than all HCC cell lines. With all HCC and HB cell lines, treatment with sodium butylate (I mM) to induce cell differentiation resulted in growth inhibition. In all HCC cell lines, marked induction (4 to 50 times) of p21 mRNA was observed within 4 hours after treatment. In HB cell lines, on the other hand, no change in the p21 level was found. In human and mouse embryonal carcinoma cell lines, which expressed p21 at high levels, the expression of p21 was also stable after treatment with retinoic acid to induce neuronal differentiation. These results suggest that the basal levels of expression of p21 is high in primitive, immature cells and low in mature cells and that induction levels of p21 by the treatment of cells with differentiation-inducing agents may vary with the developmental stage of cells.

Published Abstracts: Clinical Genetics, Malformations and Dysmorphology 2021 2022 Geroderma Osteodysplastica in a Bedouin sibship: Clinical analysis of Plagiocephaly in 15 patients and their parents. further delineation of the syndrome. NA AI-Torki. SA Al- CJ Arroyo-Kuribrefial, VF Morin-Barrosol, S Chavira-Stephanl, MID Saavedra-Ontiveros'. 'Research Subdirector. Hospital General "Dr Manuel Gea Awadi, L Cindro-Heberiel, MA Sabry. Medical Genetics Centre, Gonzilez" Maternity Hospital & Pediatric Neurology Unit, Sabah Hospital1, Cranyosinostosis is one of the most frequently found abnormalities in the genetic prac- Kuwait. tice, they include plagiocephaly, which is a malformation caused by the early closure of We report on the first Bedouin a cranial sutures with the corresponding compensatory perpendicular growth; explained family with Geroderma by the Virchow law. The patients manifest a cranio-facial asymmetry characterized by Osteodysplastica, in which two affected female siblings are a deviation of the mid facial line directed toward the affected side; with a convexity in described. They have delayed motor milestones, nomnal inteligence, a the malformed side of the face and a concavity on the contralateral hemiface, associ- prematurely aged face with loose and wrinkled skin, high and ated to orbitary strabism and crossed bite. The inheritance is an autosomic dominant prominant forehead, maler hypoplasia, mandibular prognathism, joint trait with a variability in its expression and penetrance. The genetic counseling in this laxityldislocation, and osteoporosis. The disease profile includes some cases is a complicated one, because of the minimal expression of this entity in one of the unusual traits that have not been previously reported in association parents, making it necesary to confirm this diagnosis in the ancestors. In this paper we with report the characteristics of 15 patients with plagiocephaly, their cephalometric analysis, Geroderma osteodysplastica, e.g. ear anomalies and abnormal classification according to their severity and the parents analysis in order to detect the EEG records. Phenotypic heterogeneity has been observed between minimal expression of this abnomality in the parents of this Mexican population from the two sibs in terms of the more pronnuniced phenotype of the the General Hospital "Dr Manuel Gea Gonzalez" in Mexico city. disrease in the younger sib with the presence of delayed milestones, abnormal EEG record and brachycephaly, compared with the milder profile in her elder sib with normal milestones and EEG records and the absence of brachrycephaly. This is the first time that Geroderma Osteodysplasica is diagnosed in an Arab family. The previous detection of the syndrome in a Jewish family from Morocco suggests that tihe disease might not be unfamilier in the Arab/Middle East area and supports the notion that the syndrome is probably under-diagnosed in this part of the world.

2023 2024 Normal plasma cholesterol and increased 7-dehydrocholesterol in a girl with Isolation and DNA sequence of a t(X;11) breakpoint. Sam Atallah, a few manifestations of Smith-Lemli-Opitz syndrome. M. Artioas. R. Sutohen. Stephen Siqing Wang, Michelle Gilbert, Kenneth Kupfer, Dana Jones, Sylvia Thomas B.G. Kousseff. S. BenfordA and G.C. Ness*. Division of Genetics and David Burbee and Glen A. Evans. McDermott Center for Human GrowthainT Development, University of Texas Southwestern Medical Center at Dallas, Dallas, Department of Biochemistry and Molecular Biology*, University of South Texas. Florida, and Genzyme geneticsA. Tampa. A balanced t(X;It)(q26;q23) was originally described in a 26-year-old female patient Several reports have demonstrated a correlation between severity of Smith- with a complex phenotype including gonadal dysgenesis and imniortalized in transformed Lemli-Opitz (SLO) phenotype and biochemical derangement. We report a 5 cell line GM-3552. In the course of detailed physical mapping of human chromosome year 10 month-old Caucasian girl with hypotonia, developmental delay, failure 11, we constructed a STS/YAC/PAC contig including 2 Mb of region surrounding the to thrive, short stature, hypoplastic labia minora, congenital talipes valgus, translocation breakpoint linking 10 STSs and other markers into a continuous map. symphalangism at the mid-distal phalanges of the left second finger and YAC clone yMY9ttf2 was found to span the t(X;tt) breakpoint. A 160 kb contig of bilateral 2-3 toe chromosome-specific cosmids was isolated by screening a chromosome 11-specific cosmid syndactyly. Facial features suggestive of Smith-Lemli-Opitz library with PCR products of the YAC clone. A single cosmid was isolated spanning the (SLO) syndrome included bitemporal narrowing with prominent metopic breakpoint and the sequence of 34.5 kb determined by M13 shotgun sequencing. Open suture, ptosis, depressed nasal bridge with broad nasal root, and low set and reading frames (ORFs) were determined within the sequenced region and compared posteriorly rotated ears. She also showed short columella, short philtrum, to sequences deposited in GenBank using BLAST. This region was found to contain maxillary hypoplasia and high palate. Pregnancy was complicated by genomic sequence from a recently described t(l1;14)(q23;q32) occurring in a patient gestational diabetes. Fetal activity was normal. Delivery was by breech with non-Htodgkin's Lymphoma, the LPC/PC8 proprotein convertase gene, and an open presentation at 42 weeks by dates. Birth weight was 2270 g. Ambiguous reading frame encoding a novel 267 amino acid protein. The precise sequence of the t(X;tt) breakpoint will be determined to identify the relationship between the site of genitalia was noted. Evaluation of heart murmur showed ASD, left pulmonic the abnormality, the genes identified at the breakpoint, and the components of the stenosis and vascular ring due to right aortic arch and aberrant left subclavia. phenotype. Further analysis of this and other chromosome translocations relative to Poor feeding led to gastrostomy tube placement. A karyotype was normal genomic sequence should allow future correlations of genotype and phenotype. 46,XX. Due to clinical evolution over time SLO syndrome was suspected. Plasma cholesterol was 1 16 mg/dl (normal for age) and 7-dehydrocholesterol was 2.01 mg/dl (100 times normal). Among patients with documented 7DHC elevations, this case may represent the mildest SLO phenotype. Her findings suggest that 7DHC assays should be considered even with mininal clinical suspicion of the diagnosis. A348 Published Abstracts: Clinical Genetics, Malformations and Dysmorphology (cont.) 2025 2026 4p- Wolf Hirschhorn syndrome with Multiple Odonigenic Keratocyst. A SE !E ORMQF FAMILIAL MICROCEPHALY WITH NEUROLOGIC DEFICITS ANQ INFANTILESPASMS. S. Beiraghi- M. Safari and G. B. Schaefer, University of Nebraska Medical P.J. Benke*,P. Jayakar*. M. Bauer* and R.Tuchman*. Center, Growth and Development and Pediatrics, Omaha, Nebraska. *Miami Childrens Hospital and '*University of Miami,School of Medicine. Wolf Hirschhorn syndrome (WHS) is associated with a deletion of distal Autosomal recessive microcephaly is generally characterized by mild neurologic deficits and chromosome 4p. The size ofthe deletion and complication associated with the seizures. We report 2 siblings (1 male, 1 female) aged 4 1/2 years and 15 mths, who had an syndrome has impact on survival rates of children born with Wolf-Hirschhom unusual presentation of microcephaly. Parents were non-consanguinous, of Italian decent and syndrome. The common anomalies in liveborn WEHS are; Intrauterine growth had no family history of microcephaly or birth defects. Both sibs presented with severe Retardation, Ilypotonia, Seizures, microcephaly, craniofacial asymmetry, psychomotor retardation and infantile spasms at ages 4 and 7 mths respectively. Physical examination revealed severe microcephaly, high sloping forehead, bitemporal narrowing, large congenital heart defect,( atrial septal defects), and other multiple congenital ears, high arched palate, and no craniosynostosis. Neurologically, both were extremely anomalies ( Estabrooks et al, 1995) Dental findings in this syndrome is limited restless with poor eye contact, visual agnosia, generalised hypertonia and brisk reflexes with and reported literatures only cites fused and widely spaced teeth We report a frequent dystonic posturing. There was no strabismus and examination of the fundus was fourteen years old white male with WHS. Medical history includes distal deletion normal. of chromosome 4p1 6, mental retardation, controlled seizures, typical WHS EEGs in both sibs showed hypsarrythmia. Neuroimaging studies were normal as were chromosome studies, TORCH titres and metabolic profile including amino acids, organic acids, craniofacial manifestation. Oral and dental findings were; micrognathia, and studies for glucose transport defect, GABA metabolites and neurotransmitters. microstomia, high arched palate, crowding of lower anterior, missing fourteen Microcephaly presenting with infantile spasms and hypsarrhythmia in non- permanent teeth, multiple impacted primary teeth and most significant bilateral consanguineous siblings has not been previously reported. Two studies have described odontogenic keratocyst in posterior maxillary region and one cyst in posterior microcephaly with severe neurologic deficits and perinatal seizure onset affecting 5/7(Gross- mandibular region. Most of Odontogenic keratocysts will cause bone expansion Tsur et al, 1995) and 2/2 (Silengo et al, 1992) siblings, respectively. The high risk of sibling and aside from high recurrence rate, neoplastic changes are possible within the involvement may be an important consideration in genetic counseling of this rare but severe form of familial microcephaly. epithelial lining. Therefore, it is critical to screen every patient with WTIS for Jaw cysts and other possible pathology in oral cavity, and treat them accordingly.

2027 2028 Description a unique choreoathetoid movement disorder in two Hutterite A new autosomal dominant trigonocephaly syndrome with dysmorphic fades & siblings. F.P. Bernier', O.Suchowerskyl, C. Adams2, D.R. McLeod' Departments of congenital heart defects. A. Bogdanow. J. Goodrich F.. Hantman. A. Shanske. R. Medical Genetics' and Pediatrics2, University of Calgary, Calgary, Alberta, Canada. Marion. Montefiore Medical Ctr/Albert Einstein College of Medicine., Bronx, NY. The Hutterites are an endogamous religious isolate of German descent. We describe Trigonocephaly is a major feature in several craniosynostosis syndromes: Opitz two Hlutterite male siblings who have a unique movement disorder with choreoathetosis, gait ataxia and developmental delay. syndrome, Frydmnan syndrome, Say-Meyer syndrome, and some syndromes with Our proband was delivered at term following an uncomplicated pregnancy. His motor chromosomal etiologies. We describe a family with an apparently unique multiple milestones were delayed and his mother noted he was quite unsteady at one year of malformation syndrome that includes trigonocephaly as an important component. age. He did not take unassisted steps until the age of three years and at that time he The family consists of a mother and her 2 daughters, all affected with similar cranio- had choreic movements of the limbs and trunk with dystonic posturing of the hands. facial features: trigonocephaly with hypotelorism, ptosis, epicanthal folds, prominent At seven years of age, he still falls frequently and he cannot tandem walk. lie has eyes, hypoplastic supraorbital ridges, raised nasal bridge, lowset ears. thin philtrum, and speech delay and problems with hyperactivity and attention deficit. His height and high arched palate. All have normal stature without limb anomalies. Both daughters head circumference are at the 5th centile while his weight is at the 60th centile. his vertical smooth pursuit eye movements are saccadic with the remainder of his cranial have a glabellar capillary hemangiomna and a congenital heart defect. The older child nerve examination normal. his tone, muscle strength and reflexes are normal. His has mild pulmonic stenosis and the younger child had an atrial septal defect that choreoathetotic movements persist and his gait is ataxic. Neurological investigations required surgical closure. Both underwent cranial reconstruction for metopic including EMG, nerve conduction studies, ABRs, EEG and cranial MRI are normal. synostosis, and both have mild developmental delay. Chromosome analysis and CNS The only persistent laboratory abnormality has been an elevated CK (300-800 IU/I). imaging are normal. The mother has no heart defect, and never underwent cranial Normal investigations include CBC, lactate, ammonia, AFP, vitamin E, plasma amino reconstruction. She has borderline intelligence and required special education acids, copper, ceruloplasmin, immunoglobulin profiles and urine organic acids. This patient was treated with Tetrabenazine with significant functional improvement. His (although her unaffected parents and brother have above average intelligence). The younger brother is not walking at 4.5 years of age, has the same movement disorder, is children's father is unaffected and consanguinity was denied. The mother's father was developmentally delayed and has the same facial appearance. There are no individuals 41 at the time of her conception, which suggests the possibility of a new mutation in in the extended family with a similar movement disorder. the mother. Blood from the mother and both children was sent to Dr. Max Muencke's These siblings appear to have a unique choreoathetoid movement disorder not previously lab at UPenn for mutational analysis of FGFRI and FGFR2. but thus far, no mutation described in the lHutterite population. Dysequilibrium syndrome is another neurological those in has been identified. condition prevalent in this population but the features differ significantly from what to be a our family. These conditions may be amenable to mapping using molecular techniques The nmother and her 2 daughters are affected with appears unique for identifying recessive genes by autozygosity.The application' of these techniques will trigonocephaly syndrome. Autosomal dominant inheritance is suggested, but the first require careful clinical characterization of the disorders. pattern is also consistent with X-linked dominance. Further work is necessary to define this entity.

2029 2030 Typification of the Neural Tube Defects in Aborted Fetus according to the Congenital absence of the right lung in Down syndrome. S. Cheney'. J. Bodurthal. C. Multi-Site theory. S Chavira-Stephan', VF Morin-Barrosol, CJ Arroyo-Kuribrefial, Jackson-Cook'. G. Gutcher2. 'Department ofHuman Genetics, 2Department ofPediatrics, MD Saavedra-Ontiveros'. Research Subdirector. Hospital General "Dr Manuel Gea Medical College ofVirginia/ Virginia Commonwealth University, Richmond, VA. Gonzalez" Agenesis of the lung is a developmental abnormality arising from the absence of lung The Neural Tube Defects (NTD) are frequently found entities, but their worldwide has distribution is variable depending on the country we refer to, suggesting a multifactorial tissue, bronchi, and pulmonary circulatory structures. Unilateral pulmonary agenesis pattern. The reported incidence varies from 1:1000 live born, up to 4:1000, as reported an incidence estimated at 1:10,000 to 1:30,000. More than halfof the reported cases have by Mutchinick in Mexico. Recently Torriello proposed a new model to explain the NTD other associated congenital malformations but chromosomal abnormalities are not physiopathology, based on the multi-site closure theory. The present study reports the generally seen. The mortality rate is high due to increased susceptibility to infections, NTD found in the abortion fetuses obtained from a Mexican population from the General although survival to adulthood is possible. Hospital "Dr Manuel Gea Gonzalez", in Mexico city; their analysis, correlation to the a old female at 38 weeks who severity and anatomical situation in anatomical situation in relation to the multiple-site We present a case of an infant born of 30 year gestation closure model. died at 3 weeks of age. The patient presented with probable right unilateral pulmonary agenesis, microphthalmia, small nose with anteverted nares, and short fingers with simple creases. Ileight. weight, and head circumference were all in the 10th to the 25th percentile. Antenatally, the right lung was "undetectable" from 34 weeks and polyhydramnios developed at 37 weeks. No lung tissue was visualized on ultrasound postnatally and no right bronchus was detected by bronchogram and bronchoscopy. Cytogenetic studies revealed a 47, XX, +21 karyotype by GTG banding. FISH studies were performed because of the unusual phenotype. Whole chromosome paints of chromosome 21 confirmed the GTG banding results whereas the locus specific probes (D212S259, D212S341, D212S342) used for the enumeration of the chromosomes 21 detected the presence of mosaicism. Approximately 78% of the cells showed 3 signals (trisomy 21) and the remaining 22% of the cells showed 2 or fewer signals. To our knowledge, this is the first reported case of pulmonary agenesis occurring concomitantly with Down syndrome, although there have been reports ofalveolar hypoplasia in Down syndrome. Published Abstracts: Clinical Genetics, MalftDrmations and Dysmorphology (cont.) A349 2031 2032 The del (9p) syndrome: Some peculiar cognitive and linguistic aspects. A, A third case of microcephaly, microphthalmia. ectrodactyly and prognathism (MMEP): Battagiia, D. Brizzolara, A. Chilosi, P. Cipriani. Stella Maris Scientific Research Expansion of the phenotype to Include scoliosis and deafness. A.E. Chudley. B. Bvbel and M.H. Reed. Children's Hospital and Faculty of Medicine, the University of Institute- Institute of Child Neurology and Psychiatry - University of Pisa- Manitoba, Winnipeg, MB, Canada. Calambrone. Pisa. Italy. Two previous reports delineated an apparently previously unrecognized MCA Interest towards the description of behavioral phenotypes has greatly increased disorder consisting of severe mental retardation, Microcephaly, Microphthalmia, during the past few years. We wish to present three patients (all white females, ages gctrodactyly of the feet and Prognathism designated by the acronym MMEP (Viljoen respectively: 9 yrs. 6 mon., 8 yrs. 10 mon., and 9 yrs. 6 mon.) admitted to our DL and Smart R: Clin Dysmorph 2:274,1993; Suthers G and Morris L: Clin Dysmorph Institute because of developmental delay/mental retardation, seizures and learning 5:77,1996). The 1st case was a female with the above features plus a cleft lip and disabilities. Chromosome analysis showed a deletion of the short arm of palate and an apparently balanced karyotype 46,XX t(6; 1 3)(q21 ;q1 2). The 2nd case chromosome 9 (9p-) in all these patients. They all had a peculiar cognitive and was a male with a normal karyotype. The etiology and inheritance is unknown. linguistic profile in the context of mental retardation of variable degree. The We assessed a 35 year old male with severe mental retardation, scoliosis, deafness cognitive profile was characterized by a significant discrepancy between verbal and and other features of MMEP. He was the 3rd of 5 children born to healthy non- performance IQ, due to a marked deficit in the visuo-praxic and visuo-spatial skills, consanguineous parents following an uncomplicated pregnancy. Examination associated with a memory disturbance. The analysis of linguistic abilities showed revealed short stature 0131 cm), with a severe thoracic scoliosis, microcephaly (50 that, although on the whole adequate to mental age, there were some internal cm), small eyes, upslanted palpebral fissures, facial asymmetry, hypoplastic alae dissociations leading to an atypical profile. The overall style of language use in a nasae, a short philtrum, abnormal teeth, small mouth, marked prognathism, low-set simple dysplastic ears, and a short neck. testes were He conversational context had a somewhat cocktail party-like trait, because of inadequate Both undescended. had in of flexion contractures of the elbows, fingers and knees. There was ulnar deviation of semantic-pragmatic competencies, spite good enough morpho-syntactic abilities the 3rd digit of the right hand and both 5th digits which were hypoplastic. There (lack of integration between language form, content and use). Within the lexical were 4 toes on both feet with the 5th toe being part of a bifid 4th toe. Radiographs domain it did, as well, appear a dissociation between the quantitative-qualitative showed a severe thoracic scoliosis convex to the right with no obvious segmentation aspects of spontaneous versus elicited vocabulary. In fact the performance in a defects; anterior dislocation of the radial heads; 4 metacarpals with the 4th being confrontation naming task was very atypical with a high proportion of semantic bifid distally and articulating with 2 fingers; the 5th digits had 2 phalanges; several paraphasias and bizarre answers. In conclusion the behavioral profile of our patients intertarsal bone fusion of both feet; fusion of the 4th and 5th metatarsals with 4 toes showed some suggestive analogies with the 'typical" pattern of cognitive and in which the 4th toes were bifid distally with 2 phalanges; the 2nd toes were shorter linguistic functioning described in the Williams syndrome. than the 3rd toes. Chromosome analysis was normal 46,XY. Metabolic investigations were normal. We believe our patient represents the 3rd case of MMEP. Our patient has additional features of deafness and severe scoliosis. To date, all cases have been isolated cases with an unknown pattern of inheritance.

2033 2034 Autoimmune disease in three patients with Cohen syndrome. Saethre-Chotzen syndrome in a family cosegregates with a pericentric J. Clayton-Smith and D. Donnai. Regional Genetic Service, St Mary's Hospital, inversion of chromosome 7. 1D.L. Daentl, 2'4M.L. Cunningham, 3G. Ehrlich, 4& Manchester, UK. Beck, 5J.D. Stephens, 6M.Q..Zapala. and 7EW..Jabs. 'Shriners Hospital-San li 1973 Cohelr et at. reported three patients with hypotonia, truncal obesity, mild Francisco, San Francisco, CA; Univ. Washington School of Medicine, Seattle, WA; mental handicap, chorioretinal dystrophy and dysinorphic facial features. Most 3Univ. Pittsburgh, Pittsburgh, PA; 4Children's Hospital & Medical Center, Seattle, notably, these patients had a short philtrum and an open-mouthed appearance with WA; 5San Jose, CA; 6Children's Medical Center, Dayton, OH; 7Johns Hopkins Univ. prominent central incisors. Around 100 cases of Cohen syndrome have since been reported, but there is still much debate about how distinctive the clinical features of School of Med., Baltimore, MD. Saethre-Chotzen is an autosomal that this condition really are. Cohen syndrome has an autosomal recessive pattern of syndrome (SCS) dominant disorder has inheritance and has been mapped to chromosome 8q22-23 in Finnish families. been mapped to distal chromosome 7p and is variably expressed with a phenotype We report three further patients with Cohen syndrome to aid clinical delineation of the of craniosynostosis, low frontal hairline, ptosis, deviated nasal septum, condition. One patient, diagnosed at the age of 54 years is one of the oldest reported brachydactyly and partial cutaneous syndactyly. We have evaluated an interesting Cohen syndrome patients. All three have the characteristic facial features, truncal obe- family in which a pericentric inversion, inv(7)(p21.2;q34), with the same breakpoints sity with slender arms and legs and pigmentary retinopathy. They also behaved in a in all affected individuals, cosegregates with the SCS phenotype. The mother is 'cocktail party' manner, a feature which has been described in previous patients. One very mildly affected with low frontal hairline, deviated nasal septum, and high patient had diabetes mellitus, one autoimmune thyrotoxicosis and the third an autoim- cutaneous webbing between the 3rd and 4th fingers. Her 10 y.o. son was bom with mune haemolytic anaemia. We therefore suggest that predisposition to autoimmune bilateral coronal sutural synostosis, high forehead, mild ptosis, low frontal hairline, disease may be an additional feature of this syndrome. small pinnae, and partial cutaneous syndactyly between the 2nd and 3rd fingers and 2nd and 3rd toes. An 8 y.o. son has more severe congenital coronal synostosis with a high forehead, bilateral ptosis, small pinnae, and more severe partial cutaneous syndactyly between the same fingers and toes. A phenotypically normal daughter does not have the pericentric inversion of 7. This case, together with the more than 20 cases reported with chromosome rearrangements (deletions, translocations) involving 7p in which there is cosegregation with SCS is further confirmation that haploinsufficiency is the most likely mechanism of the phenotype. The inversion breakpoint maps within the region previously implicated by molecular studies.

2035 2036 Intrafamilial variability in velocardiofacial syndrome (VCFS)/DiGeorge Creatine kinase brain isoenzyme (BB-CK) presence in serum distinguishes syndrome (DGS)/deletion 22ql 1.2 (del22q): Implications for recurrence risk osteopetroses among the sclerosing bone disorders. M.C. Eddy', A. Chines'2. counseling. MB Dinulos. RA Pagon. VP Sybert. L Hudgins. Univ. of Washington D.P. Silva. Jr.2. Y. Landt2. J.H. Ladenson'. M.P. Whyte". Shriners Hosp for School of Medicine and Children's Hospital and Medical Center, Seattle, WA. Crippled Children' and Wash U School Med2; St.Louis, Mo. The broad phenotype observed in patients with VCFS/DGS and del22q has made Creatine kinase (CK) isoenzyme BB-CK is predominantly found in brain and is genetic counseling difficult for individuals at 50% risk. To determine the extent of not normally detected in the blood. A few recent reports, however, have described intrafamilial variability in this group of disorders and develop data for recurrence risk BB-CK in serum from several patients with osteopetrosis (OP). counseling, we reviewed the findings in I I familial cases from our (5 families) To evaluate the presence and specificity of BB-CK in serum in the osteopetroses institution and 57 cases (23 families) fromn the literature. among disorders that increase skeletal mass, we total CK and We conducted a retrospective review of 66 patients from our genetics program who quantitated activity carried the diagnosis of VCFS [palatal abnormalities, +/- conotruncal heart defects CK isoenzymes in 15 patients representing the five major clinical forms of OP [two (CHD), characteristic facies, learning disabilities (LD)J; DGS [2 of 3 findings: CHD, infantile, three intermediate, seven adult (two type I, five type II) and three hypocalcernia or T cell deficiency]; and del22q as determined by fluorescence in situ carbonic anhydrase It (CA II) deficiency cases] and in 22 patients representing 14 hybridization using the N25 probe. We identified 5 families (3 VCFS/DGS with other types of sclerosing bone disease. deletion; I VCFS/DGS without deletion; 1 del22q without VCFS/DGS). In all All OP patients (except the two adult, type I subjects) had BB-CK readily detect- families the proband was a child. ed in their serum. Conversely, only one of the 22 patients with other sclerosing Two of 5 and 3 of 5 families were concordant for the presence or absence of palatal bone disorders had detectable BB-CK in sertim [one of three patients with fibro- abnornalities and CHD, respectively. There was discordance for the severity of the dysplasia (myositis) ossificans progressiva who had barely measurable activity]. In malformation. For example, one parent had a ventriculoseptal defect and her child had three OP patients (one of two with the infantile form and two of five with adult, hypoplastic left ventricle. Four of 5 parents had LD; developmental assessment was type 1I disease), BB-CK values were sufficiently high that serum total CK activity not possible in the infant probands. Three of 6 probands had hypocalcemia or T cell was elevated. In a newborn with malignant OP, both cord blood and peripheral deficiency; parental data was not available. One family had postaxial polydactyly. blood serum had substantial amounts of BB-CK. In three subjects (with adult, type 11 Of 23 families reported in the literature, 13 (-60%) and 7 (30%) were concordant for OP), who were re-studied 2-6 years later, BB-CK was still elevated in their blood. the presence or absence of palatal abnormalities and CHD, respectively. The most BB-CK in serum appears to distinguish the osteopetroses among the sclerosing intrafamilial occurred in a woman with a striking variability deletion who had normal bone disorders. Absence of serum BB-CK in I disease that this intelligence and subtle facial anomalies, whose 3 affected children had severe adult, type suggests not be a form of OP. of BB-CK in fetal blood could manifestations (Wilson etla., 1991). condition may genuine Assay be assessed as a means for of OP. the osteoclast As illustrated in our families and supported by the literature, it is not possible to prenatal diagnosis malignant Why accurately predict the severity of VCFS/DGS/del22q in offspring of affected failure that characterizes all true forms of OP is associated with BB-CK in the individuals. circulation is a new question for skeletal biology. A350 Published Abstracts: Clinical Genetics, Malformations and Dysmorphology (cont.) 2037 2038 Autosomal recessive inheritance of primary microcephaly with Hypercholesterolernia in patients with del(17)(pll.2) (Smith-Magenis normal intelligence. H. Farao. B.G. Kousseff. R. Sutghen. syndrome). B. Finucanel, A. C. . Smith2, S. i1. Elsea3, F. Greenberg2, Division of Medical Genetics, University of South Florida, and P. 1. l'atel3. 'Elwyn, Inc., Elwyn, PA, 2NCHGR-NIH, Bethesda, MD, and 'Baylor Tampa. College of Medicine, Houston, TX. We report on two male sibs with primary microcephaly Smith-Magenis syndrome (SMS) is a clinically recognizable condition associated with an normal psychomotor development, no dysmorphy and no clinical interstitial deletion at 17ptl.2. Reported clinical findings include physical dysmorphia, evidence of immunodeficiency. Pregnancy and delivery mental retardation, and behavioral and sensory impairments. Fasting cholesterol profiles histories were unremarkable. Smoking, alcohol and drug use were obtained on 13 patients with SMS, ages 6 to 55 years. Eleven of 13 patients showed were denied. The older brother presented at the age of 18 significantly elevated total serum cholesterol, requiring dietary intervention. Mean IIDL months with an occipito-frontal circumference (OFC) of 41.8 and triglyceride levels were normal, while mean LDL was patients. cm (5 standard deviations below the mean). His OFC at birth elevated in SMS The was 30.5 cm (4 SD below the mean). His height and weight recent mapping of the gene for sterol regulatory element binding protein 1 (SREBFI), were at the 10th centile at birth but dropped gradually known to be involved in LDL metabolism, to 17p1l.2 suggests a possible underlying below the 5th. He had a large hyperpigmented macule on his mechanism for the clinical observation of elevated cholesterol in SMS. Correlation of left calf. Diagnostic testing included a skull X-ray, brain hemizygosity for the SREBFI locus in SMS patients with hypercholesterolemia is being CT and MRI, ophthalmologic evaluation, chromosomal analysis, investigated. We conclude that moderate hypercholesterolemia, requiring dietary inter- fragile X study, TORCH titers, serum amino acids, chemistry vention, is a common associated finding in both children and adults with SMS. Con- profile and thyroid hormone studies; all were normal. The versely, the study of inherited hypercholesterolemia in patients with SMS may provide younger sib's OFC was 30 cm at birth (4 SD below the mean). insights into lipid disorders in the general population. Height and weight are at the 10th centile. Immunologic and breakage studies to rule out Nijmegen syndrome (MIM *251260) are in progress. Neither child has the bird -like facies or recurrent infections described in this syndrome. The OFC of the mother and father were at the 25th and 90th percentile respectively. Autosomal dominant inheritance is unlikely. X- linked recessive inheritance cannot be ruled out. This suggests a new variant of autosomal recessive microcephaly with normal intelligence.

2039 2040 The trisomy 5 (5q3l-*qtcr) syndrome. A new case due to de novo A new case of the ectrodactyly-cleft palate syndrome (ECP syndrome). rearrangement. E GalAn-G6mez. N Beriano. FM Campo-Sampedro, A Villa- J Garza, C-T Fong. Department of Pediatrics, University of Rochester School of Milla. I Lorda and JJ Cardesa-Garcia. hospital Infanta Cristina, Univ of Mwdicine and Dentistry. Extremadura. Badajoz and ECEMC, Madrid, Spain. Opitz at al. described a kindred with ectrodactyly and cleft palate, but without cleft lip or ectodermal dysplasia in 1980. Further report of this syndrome has been sparse. We present a female newborn product ofthe first pregnancy for the 33 year old We report here a 3 year old white male with ectrodactyly of the hands and feet, who woman. Due to an AFP-screening, an amniocentesis was performed at 14 weeks does not have any evidence of clefting or ectodermal dysplasia. However, the mother of gestation. The result was 46,XX,lp+. Delivery was at 31 weeks gestation by the child also has ectrodactyly of the hands and feet. In addition, she had cleft palate, vacuum extraction. Apgar scores were I and 4 (I and 5 minutes respectively). which was noted at birth and repaired during childhood. There is no abnormality of the Physical examination showed a female newborn with generalized cyanosis, skin, hair, nails or teeth, and there is no cleft lip. The mother and child are the only dysmorphic face, short chest, respiratory distress snd a systolic heart murmur. affected individuals in this family. We propose that this family represents a new case of the auitosomal dominant ectrodactyly-cleft palate syndrome of Opitz (ECP syndrome), There was an isolated umbilical artery. The limbs were short more proximally. which is distinct from the ectodermal dysplasia/ectrodactyly/cleft lip/palate syndrome Club feet were noted. Chest X-ray showed 10 ribs on the right siede and I I ribs (EEC syndrome). on the left side. The patient deceased by respiratory failure at 23 hours. Necropsy showed hypoplastic pulmonary arteries. Routine peripheral blood karyotyping showed corroborated the result of the amniocentesis. FISH by painting whole chromosomes using the ONP 5209-Coatsome 5 probe from Oncor to the chromosome 5, showed a partial duplication of the long arm ofchromosome 5 ( 5q3 l--qter). The karyotype was 46,XX,-l,+rec(I),(Sqter-sq3 1:1 pter4lqter). The karyotypes of both parents were normal. We present a new the novo case of partial trisomy of the long arm of chromosome 5.

2041 2042 Features of Ruvalcaba-Myhre-Smith Syndrome with Oliogodactyly of the Toes and Skin dimplinf in diastrop~ic dysplasia. J.H. Hersh . K. Christensen Functional PDH Deficiency: Clinkal Heterogeneity or New Syndrome? R.S. Laclhman , I. Kaitila . University of Louisville School of Medicine, Louisville, Kentuckyl, UCLA School of Medicine, Los Angeles, G.E. Graham'. G.K. Brown. B.H. Robinson3. E.P. Treacy'. R.T. Zori4 and A.S. Teebi'. California2, Helsinki University, Helsinki, Finland3. F. Clarke Fraser Clinical Genetics Unit and DeBelle Laboratory for Biochemical Genetics, Skin dimpling is a nonspecific finding that has been described in Montreal Children's Hospital and McGill University, Montreal, Quebec, Canada'; Oxford several skeletal dysplasias, most notably in the tibial region of University, Oxford, England2; The Hospital for Sick Children and University of Toronto, patients with campomelic dysplasia. Failure in development of under- Toronto, Ontano, Canada3; University ofFlorida, Gainsville, Florida, USA4. lying subcutaneous tissue due to close approximation of fetal bone to overlying skin is the presumed mechanism for this finding. We present a 17 year old male with bilateral Previously, skin dimpling had not been regarded as a feature of macrocrania, inguinal hernias, multiple diastrophic dysplasia, an autosomal recessive skeletal dysplasia, rectal polyps, hyperpigmented penile macules, cutaneous nevi, oligodactyly of the feet and characterized by micromelic dwarf ism, hitchhiker thumbs, foot deform- moderately severe intellectual impairment with autistic behaviour. The patient is the son of ities, cystic masses of the pinnae, progressive lordosis and scoliosis, unrelated Ashkenazi Jewish parents with macrocrania (>98th percentile) and normal cleft palate, and joint contractures and dislocations. We report on intelligence. His mother has intestinal polyps with pathology reminiscent of Cowden's the presence of pretibial skin dimpling in three patients with this disease, a history of thyroid follicular adenoma and a fissured tongue. The maternal disorder. grandfather also had a thyroid tumor. Findings in our cases indicate that skin dimpling appears to In early childhood the presence of an intermittently high serum lactate prompted represent a low frequency association in diastrophic dysplasia. Its enzyme studies on the PDH complex. Fresh skin fibroblasts from repeat biopsies presence may serve as another useful clinical sign that raises demonstrated normal PDH Elc activity which declined markedly to subnormal levels with suspicion of the diagnosis in infancy, particularly if more typical serial passage. This progressive decline in Elc activity was confirmed by immunoblot manifestations are absent. The origin of skin dimpling in diastrophic studies but Elc cDNA and promoter sequences were normal. Additional metabolic dysplasia is likely to be similar to other skeletal dysplasias. investigations, including fasting plasma acyl-carnitine profile, fasting urine organic acid However, the absence of marked deformity of the lower extremities does profile, and fibroblast L-CHAD enzyme activity were unremarkable. There was no physical not preclude involvement of other pathogenetic mechanisms. or EMG evidence of myopathy and serum CK was normal. Muscle biopsy demonstrated minor interstitial inflammatory infiltrates, normal mitochondria and absence of myoliposis. Our patient has a functional abnormality of the PDH complex which is likely to reflect an unknown primary metabolic dyshomeostasis. This findipg and oligodactyly are not previously recognized components of the Ruvalcaba-Myhre-Smith/Bannayan-Riley- Ruvalcaba spectrum and may reflect further clinical heterogeneity or constitute a new syndrome. Published Abstracts: Clinical Genetics, Malformations and Dysmorphology (cont.) A351 2043 2044 Induction of mineralization in cells cultured from fibrous dysplasia Imerslund Grasbeck syndrome with intrafamilial patients. G.M. Hobson. R.P. Stanton. P.A. Moses. B.E. Montgomery. and V.L. heterogeneity. EAR Ismall. 0 Al Saleh. M Zaki. MA Eunanage. Departments of Clinical Science and Orthopaedics, Alfred 1. duPont Sabv1y- Institute, Wilmington, DE Paediatrics Department, Farwanlya Hospital & Medical Genetics Fibrous dysplasia is a progressive bone disease in which abnormal fibroblast Centre, Maternity Hospital 1, Kuwait. proliferation results in the replacement of normal cancellous bone with an immature Imerslund Grasbeck Syndrome (IGS) Is a rare autosomal recessive fibrous tissue that is poorly mineralized. The disease manifests itself in the disease with megaloblastic anaemia due to selective intestinal vitamin monostotic form in which only one bone is involved and the polyostotic form in which multiple bones at different sites arc alfected. Somatic activating mutations in B12 malabsorption, for which a locus has been recently assigned on the alpha stimulatory subunit of the G protein (Gsa) have been demonstrated in the chromosome 10. The majority of previously described patients were fibrotic lesions of patients affected with either monostotic or polyostotic fibrous either Scandinavians or North African Jews. The disease has also dysplasia. been described In 3 Saudi patients. Herein, we report on the first Affected bones of fibrous dysplasia patients are weak and subject to fracture and Kuwaiti IGS patients in two unrelated Bedouln families, 3 brothers In but recurrence is seen. deformity. Treatment requires surgical intervention, always one family and a male patient In the second famlly who also has two The of our research is to a method of long-term objective develop non-surgical anaemia treatment for fibrous dysplasia. Toward this end, a tissue culture model was siblings with macrocytic (now wider Investigations). The are cinico- established to test potential therapeutic agents for their ability to modify the fibrous parents In both families first cousin Bedouins. A dramatic dysplasia phenotype. We have demonstrated that stromal cells from the marrow of haematological response to vitamin B12 injections has been elcited In affected patients can be grown in culture. These cells mineralize in the presence of the 4 patients. In the first family, with three affected brothers, the age dexamethasone or methylprednisolone in a manner similar to normal human bone of presentation was very variable (16 months, 5 years, and 15 years). marrow cells. Two of the affected sibs In family I and the patient In family 2 had raised Hb A2 (>4.5%) while the third affected sib in family I had a raised level of Hb F (10.5% at age 16 months). In all Bedouln patients, Hb electrophoresis abinormalities were corrected alter the institution of vitamin B12 therapy.

2045 2046 Hydronephrosis and neurogenic bladder in Down syndrome H. Kawame, A new skeletal dysplasia associated with severe short stature, dislocated hips, K. Kurosawa, A. Imadachi, and N. Usui. Dep. of Pediatrics, Jikei Univ. Schl. of Med., stapes fixation, articulation abnormalities and unusual craniofacial features. YE Tokyo, Japan. Kimoni '_W.H. MFAlistee. D. Binstadt and R Paule, 'SIU School of Medicine Although numerous anomalies are associated with Down syndrome, clinically Springfield.2Mallinckrodt Institute of Radioloy Washington Universit t Loui- significant urinary system anomalies do not seem to occur with greater frequency in of children with Down syndrome than in the general population. We report a 7 year old MQ. 5St Johns Hospital- Springfield- IL 'University Wisconsin-Madisorn WI girl with Down syndrome (47, XX, +21) who presented with bilateral hydronephrosis, We report two sisters age 48 and 42 yr. who have an identical skeletal dysplasia. They hydroureter, and neurogenic bladder. are the product of non-consanguinous normal appearing parents. Both sisters were This patient had a history of difficulty voiding since 3 years of age, and had an markedly growth retarded in utero; their birth weights were 3 Ibs 14 oz and 4 lbs 3 oz abdominal mass since 6 years of age. At 7 years of age, she had a recurrent cystitis, respectively at term. Both have severe short stature with relative macrocephaly since and abdominal CT revealed bilateral hydrouretonephrosis and megabladder which were birth. Congenital dislocated hips occured in both, with severe manifestation in the older confirmed with intravenous pyelography. Cystourethrography revealed vesicoureteral sister who numerous bilateral and reconstructive reflux (VUR) (lt>rt). Urodynamic study suggested detrusor-external sphincter required hip replacement acetabular dyssnergia (DSD). She had mild renal dysfunction. Clean intermittent catheterization surgeries. She also had triple arthrodesis for forefoot varus type deformity of the feet. was immediately begun by her mother to preserve the residual renal function. This Both developed hearing difficulties due to stapes fixation. Stapes mobilization surgesy management significantly changed her and her family's daily life style. was performed in the older sibling at the age of 24 yr. They also have an unusual Obstructive uropathy in Down syndrome was described by several large studies with articulation deficit with difficulty pronouncing certain consonants. low frequency, but we are aware of only four case reports of symptomatic patients with Craniofacial features noted are malar hypoplasia, downslanting palpebral fissures, hydronephrosis and neurogenic bladder in the literature. All four were female, and all hypotelorism, bitemporal narrowing, narrow palate, micrognathia and frontal bossing. severe left-sided The cause of bladder function included presented VUR. neurogenic of these features are of first and second branchial arch DSD and bladder neck stenosis. Given the high frequency of pyeloectasis in Down Many suggestive abnormalities. There is marked limb of meso-acromelic Skeletal in the syndrome fetuses, generalized hypotonia may be associated with progression of shortening the segment. x-rays dilatation of urinary tracts. Also mental retardation might contribute developing older sibling reveals stenosis of all the long and short tubular bones, with diminished incoordination between the detrusor and sphincter during toilet training. medullary cavities and thickened cortices. The ribs and clavicles are thin and dense. This case illustrates urinary system anomalies may be more common in Down Skull x-rays show malar hypoplasia, and shallow orbits and brain MRI reveals basilar syndrome than previously thought. Early detection and intervention will avoid long impression, with the odontoid process projecting through the large foramen magnum, term complication which could lead to significant morbidity. and resting against the medulla. Spine films reveal scoliosis, and lack of flaring of the interpediculate distances. Karyotype analysis, metabolic studies, review of the literature and databases were non contributory. The constellation of unulsual features in these siblings may represent a new skeletal dysplasia, with most likely an autosomal recessive mode of inheritance.

2047 2048 Atypical CHri di chat syndrome characteriized by FIS11l-technique. E. Koenig. A. Molecular characterization of an isodicentric Yq by FISI--technique. BL. Kohn. J. Parekh. S. M. Klevman. R. A. Conite. M. Bezdickova and R. S. Venna. D)eparlment S. M. Kleyvnian. R. A. Conte. M. J. Macera. K. Glassberg and R. S. Verma. Divisions of Pediatrics atid l)ivisiomi of Genetics, 1The Long Island College Hlospital-SUNY of (ierietics, The Long Island College Hospital-SUNY Health Science Center at icalh Scicnce (enter atl Brooklyn, NY. Brooklyn, NY. 1Tie cryptic dlcctiouis associated witti a iutmibier of syndronies tiase been an arduous A newsbom infiant was referred for evaluation of ambiguous genitalia. Briefly. the task in the precise identification by routine cytogenctic techiniiqucs. O)nc such clinical features included: hypertclorisni, anti-mongoloid slant to the eyes. m11ild syndrome is CHi (hi chal syndrome (C)DC) which is associated withi deletions of widening of the neck, but no definitive webbing, shield like chest and positive cubittis chromiosoine band 5p15. 1tic highi-pitched cry siiiilar lo tlie imesing of a cat, post- valgus. Examination of genitalia revealed a hypospadiac phallus measuring .5cim in natal slow growssth. mental deticicincy and niicroceplialy liase heen insauiahle length with chordee. There was incomplete fusion of the scrotum. No gonads were hallmarks of this syvidronie. A nesw hoir seas rclirred foir cylogcnctic evaltiatioti palpable within the scrotal sac. A pelvic and renal sonogram confirmed the presence because she weigtied 2tt65 got Ill percentile) ssitl a head circurirtererice of 311.5 cin of a uterus and normal appearing kidneys. The patient was assigned a female gender (I11th perceiitile), a length (if 48 cnii (251h - 501th percentile) and soniessliat high- on the basis of the presence ora uterus, the phenotypic appearance of the genitalia arid pitchled cry. Tlhe remiiaiitder of the physical examiiination was uinremnar kable. By GIG- the malignant potential of the gonads. The cytogenetic findings with QFQ-handing banding the bhind 5p15.3 ssas deleted. The band 5p1 5.2 was present as revealed by revealed an abnornmal karyotype i.e. nmos 46,X,idic(YXpI 1.2)l771/ lISI 1-tecnitirquc using 1)5S23 probe (Onmcor). Ftirticerriore the presence ofteltnieres 45,X[29]146,Xidic(Y)(pl I?)[2j/47,XYidic(Y)(pl 1.2)[2j/ on lhe short aritr swere also revealed by the all human tcloincre probe sutggestirig the 47,Xidic(Y)(pl 1.2),idic(Y)(pl 1.2)[11/46,XY[l]. delction to he irrerstitial1Tihe abniouirsal cry has been asso(ciaied wsitih the deletion of' She presence of air abnormal isodicentric Y-chromosome was evaluated by 5.3. Otlter phieniotypic tieatures that are associated with a deletioii of band 5p1 l5.2 Iluoresceisce in-sio, hybridization IFISHJI technique to ensure a finer characteri7ation are rot present ill our case resttlitirg ill atypical leattircs ol C;i (di chr syridroriie. Ihe (if its dicentric nature. Nevertheless, owing to lack orf parental cooperation, tire risilecilar ctiarctecrizaiiori has enahled us to sted soneI light coricerniig the geriotype- molectilar ssork could niot he done on the father and proband. plienoritype relatiorislhip ot (IDC syndroriie. The geriotyp-pheniotype correlations could not be established as genetic expression of sonmatic nianifestation is highly unpredictable ossing to the presence of mosaicism. The clinical presentation (if such cases are so unusual that each case needs to he reporied lor flurther understanding. A352 Published Abstracts: Clinical Genetics, Malformations and Dysmorphology (cont.) 2049 2050 Postnatal obesity, mental retardation, contractures and body aaysmetry: A new form of chondrodysplasia punctata with specific changes of knee. a new syndrome? Deborah M. Lambert, I Gordon Watters, I Vazken H. Der Kaloustian. I McGill M. Le Merrer. P. Maroteaux. A. Munnich. Dept of Genetics and INSERM U-393, University/Montreal Children's Hospital, Montreal, Quebec, Canada. H6pital des Enfants Malades. Paris, France. This report describes a patient with obesity, mental retardation, contractures, and body asymmetry. This eight year old boy was referred Chondrodysplasia punctata is an heterogencous condition characterizetl by because of psychomotor delay, absence seizures and dysmorphism. He is multiple calcifications in epiphyses and in juxta epiphyseal regions seen on the X- the second child of non-consanguineous French Canadian parents. The delivery was spontaneous vaginal at 42 weeks of gestation after an rays of newborns. Beside the rhizomelic type, a peroxysomal disorder, the Conradi uneventful pregnancy. There were no perinatal complications. He had Hunerman type and the Happle type. Maroteaux et al. described the marked developmental delay, standing without support at 2h years and walking without support at 3 years, speaking few words at age 7. brachytelephalangeal type, which is X-linked inherited and due to an aryl sulfatase E Physical examination at the age of 8 years reveals a weight on the deficiency. Here we report three cases of chondrodysplasia pttnctata in early 98th centile, a height on the 25th centile and a head circumference on the 50th centile. He has lordosis and an ataxic gait. He has a peculiar childhood with flat face, depressed nasal bridge. limited movement of knee and facies with prominent forehead, hypertelorism and malpositioned teeth multiple calcifications on knee, tarsal region and coccyx. Distal femoral epiphyses with a high and narrow-arched palate. He has bilateral single palmar became creases with camptodactyly and clinodactyly of the fifth finger, flattened, squarred and irregular in the course of the disease, and a retarded overlapping third and fourth toes, partial syndactyly of the second and ossification of the carpal and the tarsal bones was noted. Spine abnormalities were third toes and calcaneovalgus deformity of the feet bilaterally. His moderate. right lower extremity is significantly larger than of the left. Genital The absence of skin defect, asymetric limb lesions or drug exposure examination is normal prepubertal male. during pregnancy and the presence of normal distal phalanges excluded the already An EEG reveals evidence of a generalized epileptic abnormality. A CT reported cause of chotidrodysplasia punctata. The disease two brothers scan shows atrophy of the vermis. Endocrine, immunological, and affected and ophthalmologic findings are within normal limits. Blood karyotyping another case was sporadic. We believe that we are facing an hitherto unreported showed a normal pattern: 46,XY.ishl6qll.2(SNRPNx2). condition. Three similar syndromes have been described (Camera et al.[19931, Vasquez et al.[1979], Urban et al. [1979], Pagnan and Gollop 119881) that have features in common: mental retardation, obesity, camptodactyly, clinodactyly, and a normal karyotype. Features unique to this case include: seizures, prominent forehead, single palmar creases and body asymmetry. We suggest that he represents a new syndrome.

2051 2052 Cleft lip, microphthalmia, simple helices, facial asymmetry, shield Frequency of FV Leiden mutation supports the role of geneticists chest, and large hands: a new syndrome. A.F. Lewanda 1,2, in identification and referral of at-risk patients. M.T. Lomnisl, A.I C. Van der Kolkl, J. Simpson', and E.W. Jabs'. 'The Johns Hopkins University School of Medicine, Baltimore, MD 2Children's National ThonasI. H.K.Pate12, P.K. RusL-gi3. and C.L. Vnencak-lones1. 1Vanderbilt Medical Center, Washington DC. Medical Center, Nashville, TN 2Northwest Alabama Cancer Center, Florence. Oral clefting is a common congenital malformation seen in over 300 AL 3University of Alabama at Birmingham Hospital, Birmingham, AL. genetic syndromes. Isolated cleft lip occurs approximately 1/1000 The Factor V (FV) Leiden mutation has a carrier frequency of 1/20 and Caucasian births, and is less likely to be associated with a genetic results from a G to A point mutation at codon 506 in the Factor V gene. The syndrome than is cleft palate. We describe a brother and sister with Arg to GIn stibstitution renders the protein resistant to inactivation by clefting and an unusual combination of features not previously anticoagulant activated protein C and predisposes to thronibosis. To identify reported in the literature. Both patients had cleft lip (one the clinical symptoms most often associated with FV Leiden alone or in unilateral, one bilateral), facial asymmetry, microphthalmia, simple conjunction with other known risk factors for thrombophilia, we studied helices with hypoplastic antihelices, an overbite, and large hands. medical histories of 95 symptomatic patients (mean age 38) referred for FV Hearing and intelligence were normal. The brother (who had the Leiden mutation detection by DNA analysis. Heterozygosity was identified in unilateral cleft) also had microstomia and widely spaced nipples. 16/95 (17%) patients; 14/16 (88%) with venous thromboembolism and 2/16 His karyotype was normal. There is no family history of clefting, and (12%) with arterial disease. Additional risk factors for thrombophilia In the consanguinity was denied. The patients' mother had type I insulin 16 PsV Leiden predisposed patients included: oral contraceptive use, smoking, dependent diabetes, which is associated with increased oral clefting surgery, and pregnancy. In 56% of FV Leiden positive patients a history of in offspring, but not with the other features described here. As thromboembolism was present, as compared to only 21% of FV Leiden neither patient had the more common features of diabetic embryopathy negative patients. In 45/59 (76%) patients with venous and 28/30 (93%) (cardiac, skeletal, CNS, GI or GU malformations), clefting is less patients with arterial disease, FV Leiden was not detected. 0/5 patients likely to have occurred on this basis alone. Cleft lip and presenting with seizures, spinal fracture, cervical radiculopathy, visceral abnormalities of ocular size, auricular shape, mouth width, and infarcts, or orthostatic intolerance had the mutation. Included in the study nipple placement could all be explained by a genetic defect affecting was one family with three symptomatic members and in which two the fetus at approximately 6 weeks gestation. These patients may asymptomatic FV Leiden positive members were identified enabling medical represent a new autosomal recessive syndrome caused by abnormal counseling and prophylactic measures. Our findings: 1) confirm the niost expression of a developmental control gene during this critical common presentation of FV Leiden mutation as venous thrombosis, 2) embryologic period. identify asymptomatic FV Leiden mutation carriers for medical counseling and prophylactic measures, and most importantly, 32 the frequency of this mutation supports the role of the geneticist in identification and referral of at-risk patients during routine family history collection.

2053 2054 Cutis lax. with bone dystrophy ( type 11 autosomal recessive cutis laxa ): 2 new Variable expressivity of the branchio-oto-renal (BOR) syndrome in a male patients. J. A. Maatouk , O. Albaz 2, L. Almenawi 3 and W. Hammad 2. three-generation family. C.P.Macliano. C.Bellini. F.Ginevri*. E.Zullino. Department of Genetics (1), Orthopedics (2) and Pathology (3), King Fahd Hospital F.Camoone. L.Giovannini. G.Aicardi. Istituto di Puericultura e Medicina Jeddah, Saudi Arabia. Neonatale dell'Universitb di Genova, and *Divisione di Nefrologia, Istituto Cutis laxa is a heterogeneous group of diseases that are divided into five types. G.Gaslini; Genova, Italia. Atitosomal recessive type If (cutis laxa with bone dystrophy) was first reported in an Branchio-oto-renal syndrome (BOR syndrome, MIM 113650) is an affected female and ,so far, 19 patients have been described. Frequently described autosomal dominant disorder characterized by sensorineural, conductive manifestations include prenatal and postnatal growth retardation, delayed motor or mixed hearing loss, structural defects of the outer, middle or inner ear, development, cutis laxa, delayed closure of fontanels and congenital hip dislocation. branchial fistulae, preauricular pits and renal anomalies. We observed in Facial manifestations are characteristic and include dolichocephaly, triangular face, a newborn a renal dysplasia that led to profound renal insufficiency, frontal bossing, downward slanted palpebral fissures, deep-set eyes, bitemporal associated with preauricular pits. The patient's father had only preauricular narrowing, maxillary hypoplasia, pointed chin and thin upper lip. pits without demonstrable renal anomaly and/or hearing loss. Patient's In the cases reported so far the ratio of males to females is 5: 14 This is consistent grandfather and his brother showed preauricular pits without deafness with male lethality before birth. The 2 males reported here bring the total number of and/or renal anomaly. A first-cousin of the patient's father presented both males to 7 and the total number of reported patients to 21. The number of males is kidneys mild reduced in size, without reduction of renal function. still considerably lower than female I male can not be the Hearing impairment is the most common defect in BOR syndrome (80% of cases. lowever, lethality cases). It is estimated that at least 2% of profoundly deaf children have only explanation We have noticed that thirteen females were reported early on. Under of male should be BOR syndrome. Renal anomalies are usually minor and often remain reporting patients investigated. 10% of studies of otir showed of elastic fibers with no asymptomatic; patients with BOR syndrome do have clinically Histologic patients disorganization renal anomalies and at 9 to maturation in taken from the dorsum of hands and was not significant least deaths due renal agenesis- samples feet. This the have been documented in newborn infants with case in taken dysplasia confirmed BOR samples from the trunk. Clinically, loss of elasticity was noted on the syndrome. hands and there is skin over the trunk loss of face, feet. Although ekecessive , On the basis of greatly variable expressivity of BOR syndrome, as elasticity is not usually evident. confirmed by our family, we suggest a meticulous search for renal anomalies in individuals with aural and/or branchial abnormalities. Published Abstracts: Clinical Genetics, Malformations and Dysmorphology (cont.) A353 2055 2056 Trisomy 8 phenotype in a child with partial tetrasomy 8p and partial trisomy 8q A case of de novo balanced reciprocal translocation mosalcism. J.E. Martinez. C.M. Tuck-Muller, S. Li W. Wertelecki. University of [46,X,t(X;1 6)(q28;q11.2)] associated with Pal syndrome. M. South Alabama, Mobile, Alabama. Masuno. K. Imaizumi. T. Ishii, and Y. Kuroki. Division of Medical Genetics, Trisomy 8 syndrome is a well known entity with a distinct phenotype including Kanagawa Children's Medical Center, Yokohama, Japan. (Intro. by Dr. hypotonia, large ears, prominent nasal tip, everted lower lip, rib and vertebral Hirofumi Ohashi). abnormalities, characteristic deep palmar and plantar furrows and varying degrees is a rare disorder characterized of mental retardation. Cytogenetic studies usually reveal trisomy 8 mosaicism in Pai syndrome (MIM 155145) genetic by peripheral blood lymphocytes and in cultured skin fibroblasts. median cleft of the upper lip, polypoid skin masses on the nasal septum, and a of the is not We investigated a 7 month old white male infant with developmental delay, lipoma corpus callosum. Psychomotor development At the the cause of this is hypotonia and trisomy 8 phenotype who also had a cytogenetic abnormality not impaired. present time, syndrome unknown, previously reported. Chromosome analysis of peripheral blood lymphocytes by although autosomal dominant inheritance has been suggested. GTG-banding revealed mosaicism for a derivative chromosome 8 resulting in partial Here we report a female patient with median cleft of the upper lip and tetrasomy 8p and partial trisomy 8p and q 146,XY/47,XY,+der(8)lpter-q24.1: polypoid skin masses on the nasal septum associated with a de novo :p23.1-pter)l. This interpretation was confirmed by locus specific fluorescence in reciprocal translocation [46,X,t(X;16)(q28;q 1 .2)]. In X-replication studies, situ hybridization. Parental chromosomes were normal. the derivative X chromosome was constantly late replicating in all of 41 Various duplicated segments of chromosome 8 have resulted in phenotypes cells examined. She had hypertelorism, epicanthus, upward slanting of indistinguishable from those of the Trisomy 8 syndrome except for distal 8q the palpebral fissures, three polypoid skin masses on the nasal tip and on trisomy (8qter-q23 or q24.1), which appears to be associated with different the bilateral nasal septum, broad nose, median cleft of the upper lip, high- clinical manifestations (Stengel-Rutkowski et al.,1 992). Patients with tetrasomy arched palate, large ears, left hypoplastic anthelix, ventricular septal 8p, inverted duplications of 8p, trisomy for the pericentric region of chromosome defect, and ophthalmological abnormalities including persistent pupillary 8 (Spinner et al., 1995), as well as our case, have all shown overlapping clinical membranes, left corneal leukoma, and heterochromia of the iris. manifestations. These previous reports and our patient's physical stigmata and Computed tomography of the head showed mild dilatation of the anterior unique chromosome abnormality suggest that perhaps the phenotypic horn of the lateral ventricles. At 13 years she spoke no meaningful words manifestations of Trisomy 8 syndrome are primarily the result of duplications of but expressed her wills by sign language. Her weight was 31.1 kg (-2.0 chromosome 8p. SD), height 138.3 cm (-2.8 SD), and OFC 51.0 cm (-1.7 SD). The translocation breakpoints in the present patient may be candidate regions for a gene responsible for median cleft of the upper lip and polypoid skin masses on the nasal septum including Pai syndrome.

2057 2058 Greig cephalopolysyndactyly versus acrocallosal syndrome: a mild Subcortical band heterotopia associated with limb vascular case with intermediate features. C. McKenna and M.G. Bialer. North malformation. L. Mehta, I.P. Taff, S. Sastry and J.L. Zito*. North Shore Shore University Hospital, Manhasset, New York. University Hospital and *Lakeville Imaging Center, Manhasset, New York. A newborn girl was referred at birth for multiple congenital anomalies. She A five year old girl presented with bilateral 4th cranial nerve palsy, fine was the 3395 g product of a full term gestation pregnancy delivered by motor delays, visuomotor incoordination and staring spells. She had normal Cesarean section for breech presentation and cephalo-pelvic disproportion. cognitive functions. Brain MRI revealed asymmetric ventricles and extensive Parents were not consanguineous. Fetal hydrocephalus had been diagnosed 2 subcortical band heterotopia involving the right frontal, parieto-occipital and weeks prior to delivery. Features noted at birth included bilateral soft-tissue left frontal regions. EEG revealed spike-wave abnormalities in the left 3/4/5 finger syndactyly, bilateral 2/3 toe syndactyly, macrocephaly (OFC parieto-occipital region only. Physical examination revealed mild 38.2 cm), prominent forehead, broad nasal bridge and bilateral epicanthal hypertelorism, cupped ears, midface hypoplasia, flat philtrum and high, folds. CT scan showed agenesis of the corpus callosum and enlarged occipital narrow palate. Hypertrophy of the right foot and calf with overlying port- horns of the lateral ventricles. A high resolution blood chromosome analysis wine discoloration was noted. Family and prenatal history was non was normal. The patient developed severe hydrocephalus and required a contributory. Plasma amino acids, urine organic acids, chromosome analysis ventriculo-peritoneal shunt at 5 mo of age. X-rays taken at that time and FISH analysis for the Miller Dieker region were normal. Parental MRI's revealed malsegmentation of the T4 and T5 vertebrae with bifid ribs at T4 were normal. Band heterotopia is a neuronal migration disorder presenting and T5. The patient was referred to an early intervention program because commonly with seizures, developmental delays and visual abnormalities. An of gross motor delays but was discharged at 2 years of age because she was X linked form is recognized with affected males manifesting lissencephaly and felt to be age appropriate. She also required speech therapy which has been females presenting with milder abnormalities such as band heterotopia. An discontinued. At 4 years of age she is age appropriate in all areas and is excess of female patients is well documented. Other etiologies include above average on cognitive functioning tests. metabolic abnormalities and teratogen exposure. To our knowledge, no All of the findings in this patient have been described in both acrocallosal specific association with vascular abnormalities is described. However, syndrome and Greig cephalopolysyndactyly. However almost all patients Klippel Trenaunay Weber syndrome may be associated with with Greig cephalopolysyndactyly have had polydactyly. Agenesis of the hemimegalencephaly. Neuronal heterotopias are described on pathological corpus callosum has been described in only a few cases. Individuals with examination in hemimegalencephaly, but are not extensively present as in acrocallosal syndrome also usually have polydactyly. Furthermore almost this patient. She presents with a unique combination of features with all individuals with acrocallosal syndrome have been severely mentally possible implications for a vascular basis to abnormal reuronal migration. retarded. These 2 conditions have so many similarilties that it has been suggested that they are allelic, but a recent linkage analysis indicates that this is not the case. This patient's findings could be consistent with an atypical mild form of either of these conditions.

2059 2060 Acromelic frontonasal dysplasia in a 17 month old female without developmental Kabuki syndrome in seven children from the Canadian Prairies: A delay. E. Melvin'. J. Bodurtha' and R. Gray2 'Department of Human Genetics, comprehensive review of clinical, skeletal, and dental findings. A. Mhanni. Medical College of Virginia/Virginia Commonwealth University, Richmond, VA, M.H. Reed. HA. Cross. N.G. Giddins. M. Shepertvcky. A.E. Chudley . 2The University of the West Indies, Kingston, Jamaica. Childrens Hospital and Faculty of Medicine, University of Manitoba, Acromelic frontonasal dysplasia is a subtype of frontonasal dysplasia distinguished Winnipeg MB., Canada. by brain malformations and limb anomalies. Children with this subtype have been The cardinal features of the Kabuki (Niikawa-Kuroki) syndrome (KS) include described as having median facial cleft, hypertelorism, hand and feet polydactyly, characteristic facial dysmorphic features, skeletal abnormalities, mild- epibulbar dermoids, and brain malformations. Mental retardation is found in most moderate mental deficiency, and postnatal growth retardation. The specific surviving children. In contrast, nonsyndromal frontonasal dysplasia does not dysmorphic features distinguishing KS are present in 100% of the cases and typically include mental retardation. Most cases of the acromelic subtype are diagnosis can be made directly from the facial "gestalt." We identified seven reported as sporadic. patients with KS in the Winnipeg genetics clinic over the past five years; a We describe a 17 month female who has a median facial cleft (including review of their clinical, radiographic, and dental features is presented. The unilateral right cleft lip and palate), hypertelorism, craniosynostosis, posterior diagnosis in these children was made at a mean age of five years (with a fontanelle displaced to the right, right bifid hallux, fifth finger clinodactyly, minimal range of 3-9 years). Four of seven patients had growth parameters less than syndactyly, umbilical hernia, low set ears, widely-spaced nipples, rectum diverticuli, the fifth percentile, two had normal growth parameters, and one had above and decreased sulci as seen on CT scan. She was the product of a term, average growth parameters. Significant growth failure occurred in one uncomplicated pregnancy to a 39 year old G2P2 Jamaican female after an elective patient between the age of 3.5 and 6 years with a markedly delayed bone confirmed hormone C-section due to placenta previa. Apgar scores were 8 at 1 minute and 9 at 5 age. Growth hormone stimulation tests growth minutes. Frontonasal dysplasia was diagnosed at 6 weeks. There is no family deficiency and growth hormone therapy was commenced with good history of frontonasal dysplasia or limb defects. We classify the patient as having response. All patients were mildly developmentally delayed; four had a DQ the acromelic subtype of frontonasal dysplasia due to the findings of a median cleft of 70 or above. Six of seven patients had heart defects. Complex face and limb anomalies. However, unlike many other reported cases with this coarctation of the aorta occurred in two of the seven patients. Clinodactyly subtype, our patient is developing normally thus far. Hler height is at the 50th %tile and brachymesophalangy of the fifth fingers, abnormal vertebrae, delayed and weight is just under 50th %tile. She sat at 5 months, had a good finger-thumb bone age, and various other skeletal abnormalities were found. Dental grasp at 10 months, took independent steps at 11 moqths, and has a 11 word findings included frequent missing teeth, particularly lower incisors, and vocabulary at 17 months. We suggest that developmental delay is not necessarily anomalies in the tooth shape (screw driver incisors). We believe the a defining feature of frontonasal dysplasia. presence of these unique dental findings will prove useful in the diagnostic assessment of Individuals with KS. A354 Published Abstracts: Clinical Genetics, Malformations and Dysmorphology (cont.) 2061 2062 Skeletal anomalies in patients with a 22q11.2 deletion. J.E. Ming. D.M. Oral Facial Digital abnormalities, myelomeningocele, stenosis of the McDonald-McGinn, T.E. Megerian. D.A. Driscoll, B.S. Emanuel. R.I. Markowitz. aqueduct of Sylvius and heart anomalies. A new Oral-Facial-Digital ELH. Zackai. The Children's Hospital of Philadelphia, Philadelphia, PA. Syndrome or Genetic Heterogeneity and Variability. VF Mordn-Barroso', The literature contains few reports of skeletal abnomnalities in patients with a 22q t 1.2 M Valdds-Flores', R Garcia-Uavazos2, S Kofnsan-Alfarol, D Saavedra-Ontiveros3. deletion. Thus, we report the skeletal findings amongst a large series of patients known 'Genetics Department. tlospital General de Mdxico, SSa. Universidad Nacional to have a 22q 11.2 deletion. Ninety-five of our patients with a 22q 1 1.2 deletion were Aut6noma dte Mexico. 2Cenetics Department. Instituto Nacinnal de Perinatologia, examined. Five patients had abnononalities of the upper extremities (5%); 2 of tile 5 SSa. 3Research Subdirector. patients had unilateral preaxial polydactyly, I had postaxial potydactyly, I had unilateral Hospital General "D)r Manuel Gea Gonzilez" ulnar deviation of the fingers, and I had camptodactyly of the fifth finger. Upper limb There is a group of entities that include oral, facial and digital manifestations known malfomiations have been occasionally reported, including postaxial polydactyly, ulnar as "Oro-Facial-Digital" (OFD) syndromes which show a great heterogeneity. In this angulation of the distal phalanx of the thumb (Shalev et at. Am J Med Genet 62:302, syndronies the major and minor malformations include those corresponding to the three 1996), preaxial polydactyly (Saal et al. Abstract #79 ACPA Annual Meeting, 1996; mentioned areas, described as: nodulated cleft tongue, frenula, cleft palate, brachi- Cormier-Daire et al. Am J Med Genet 56:39-41, 1995), club hands with hypoplastic first dactyly, sindactyly, polydactyly and other less common abnormalities, as a result of metacarpal (Cormier-Daire et al.), lobster claw deformity (Wilson et al. J Med Genet genetic heterogeneity and variability. In 1988 Toriello suggested such heterogeneity and 30:852-856, 1993), and triphalangeal thumbs (Saal et al.). Abnormalities of die lower variability seen in those OFD syndromes and in 1992 she classified these into tX different extremities were found in 11 patients (12%): 5 of the Il patients had club foot, 2 had types, later on two more types swere described. but until now, there are still few reports severely overfolded toes, 3 had 2-3 toe syndactyly, and I had metatarsus vacus. of these syndromes that might support the individuality of each one. The purpose of this Chest films of 54 patients were examined; 9 patients had 13 ribs (17%), 7 had thoracic paper is to report a patient with OFD syndrome with typical clinical, radiological and butterfly vertebrae (13%) (three had both the rib and vertebral anomalies), I had rib pathological manifestations but nevertheless it can sot be classified in any of the OFD fusion and a hypoplastic thoracic vertebral body, I had thoracic and lumbar coronal clefts described syndromes, due to dysplastic atrin-ventricular valves; lumbar myelomeningo- of the vertebrae, I had narrow intervertebral space, I had liypoplastic scapula, and I had cele, stenosis of the aqueduct of Sylvius and hydrocephalus; in view of this facts we could hypoplastic cervical vertebrae. In total, 17 of 54 patients (32%) had abnonlial be talking about a new type, number X1I that explains the involvsnent of another gene radiographic findings on chest filsn. in this disorders (genetic heterogenety) or support the phenotipic variability of a group This study suggests that skeletal anomalies should be included in the list of findings of genes. associated with the 22ql 1.2 deletion. In addition, since butterfly vertebrae are extremely rare in the general population and are only seen in association with cardiac disease in die Alagille and Kabuki makeup syndromes, the 22ql 1.2 deletion should be added to die differential diagnosis of butterfly vertebrae

2063 2064 A patient with SRY negative XX male precise clinical and Three Feet: A Sign of Multiple System Ahnormalities and Disorganization of pathological evaluation. T. Nagai', H. Kawame , K. Kurosawa5, T. Embryogenesis. R. Petrella. M.B.Stosser. and L.R. Shapiro. Division of Medical Ocata3, T. Hasegawa3, N. Matsuo3. 'Tokyo Metoropolitan Kiyose Children's Genetics. Departnients of Pediatrics and Pathology, New York Medical College and Westchester County Medical Center, Valhalla, New York. 10595. USA Hospital, 2Jikei Medical School, and 3Keio Univ. School of Medicine. of I he finding of three feet is due to a partial duplication of the lower limb. An Tokyo, Japan. The pathogensis of SRY negative XX male is not known and association with absence of the ipsilateral kidney has been previously described in 3 there are a few descriptions about precise clinical state. We saw a female patients. Tlsis female infant has a partial duplication of the left leg and absent Japanese patient with this disorder and evaluated his clinical state left kidney but was also found to have additional congenital anonsalies not previously described. The pregnancy was complicated by gallbladder illness and an unshielded precisely. He is now a 1 5/12 y/o social male. He was born normally and abdominal x-ray dtsring the t st trimester. Routine prenatal ultrasound at 3 months visited us for hypospadias at 1 1/12 y/o. He had bitidscrotum with testis gestation and maternal serum AFP screening were reported as normal. At delivery, palpable 1.8 cm in length in right and streake gonad-like mass in left. Penile there was a nucial cord and a single umbilical artery. A multisystem evaluation length was 1.8 cm. Chromosome and DNA analyses revealed 46,XX and revealed congenital heart disease consisting of ASD, VSD, PDA, coarctation of aorta; left renal and with SRY negative. HCG test increased serum testosterone level from c0.1 to 1.7 agenesis, right hydronephrosis reflux. Chromosome analysis of peripheral blood was normal. At 8 months of age, marked hydrocephalus with ng/ml and HMG revealed no response. Echogram showed vagina at the findings suggestive ofaqueductal stenosis was noted. backward of bladder but uterus could not be identified. Urethrography Incomplete twinning or inclusion of a polar body can not account for multisystem detected vagina aroud 4.0 cm in length. The gonadectomy of left gonad involvement, and the widespread malformations suggest a disorganization of and right testicular biopsy were done. Testis was 1 7x1 0x 0 mm in size and embryogenesis. Ihe forisation of limbs. kidney and heart occurs during the middle of the 1st trimester, and it is possible that this constellation of anomalies is a consequence vas deferens connected to the vagina. Left gonad was ovary 7x5x2 mm in of abnormal function of a gene(s) that normally regulates multiple organ size with uterine tube vanishing at inguinal canal. On pathology the testis embryogenesis but results in disorganization of embryologic development when was slightly hypoplastic with increased interstitial tissue and the ovary had functioning abnormally. many primordial ovarian follicles. Additional precisde clinical evaluations could contribute to identify the pathogenesis of this disease and also functions of sex differential genes.

2065 2066 A diagnostic problem about multiple malformations: Holt-Oram or Tel Hashomer Camptodactyly Syndrome with atrial septal defect and cri-du-chat syndrome? Y. Qi.). Qingdao Second People's Hospital, Qingdao, retinal detachment: extension of the phenotype. M.L.Raff and Shandong, People's Republic of China. (Intro. by: Yaping Zhang). K.A.Leppig. Div. of Medical Genetics, University of Washington School of The purpose of this study was to identify the pathogeny of certain congenital or herita- Medicine, Seattle. ble disorders for making definite diagnosis and giving proper treatment. Results of the Tel Hashomer Camptodactyly Syndrome (THCS) is an autosomal recessive study also may be useful for finding method of parental diagnosis to determine risk of skeletal dysplasia of unknown etiology that consists of short stature, brachycephaly, their children. The subject of our study was a 3-year-old child. The methods used in this small mouth, camplodactyly, clinodactyly, clubbed feet and muscular hypoplasia. It study include interviewing, studying famihiy history, performing physical examination, has been described iu different ethnic groups in offspring of both consanguineous electrocardiogram, electroencephalogram, X-ray, ultrasound scan, cytogenetic analysis, and nonconsanguineous unions. To date, only mitral valve prolapse has been and studying dermatoglyphics. The investigations revealed that the child had multiple reported as a cardiac finding. Ocular findings have never been reported. malformations that included particular facies, low-set ears, cleft uvula, cardiac murmur, We report the case of a 35 year-old Korean male with features consistent with cor triatriatum with atrial defect, undescended testicle, atrophy of brain, and abnormal THCS and a large secundum atrial septal defect and retinal detachment. History was palm prints. Mental retardation was also present. is G banded chromosomes showed notable for bilateral congenital clubbed feet and delayed growth. Unilateral retinal that the great majority karyotypes were 46,XY, but two karyotypes were 46,XY,b(5ql3). detachment with vision loss occurred at age 29. Congestive heart failure secondary The child's father and mother were healthy and unrelated. Family history was noncon- to a large secundurm-type atrial septal defect occurred at age 35. Physical exam tributory. The clinical symptoms of this patient were compared with those of several findings included short stature (157 cm) relative to his parents, brachycephaly, symdromes reported previously, such as Holt-Oram symdrome, cri-du-chat syndrome, isigrognathia with small mouth and dental crowding, thoracic scoliosis, elongated human malformations similar to those in the mouse mutation disorganization. It is con- thoracic cage, loud systolic heart murmur, bilateral tapering brachydactyly with cluded that the prominent finger pads and 4th and 5th finger clinodactyly and camptodactyly. There diagnoses for multiple malformations are not easy to be confirmed at the was generalized reduced muscle mass. clinical level. Further study is underway to find more diagnostic features that could be The patient's cranliofacial and skeletal findings are consistent with the diagnosis of used to evaluate these types of congenital anomalies. THCS. Thus, the findings of atrial septal defect and unilateral retinal detachment add to the phenotypic spectrum that may be observed in this syndrome. Published Abstracts: Clinical Genetics, Malfoirmations and Dysmorphology (cont.) A355 2067 2068 Auriculo-digito-mandibular dysplasia. A new syndrome of short stature of Phenotype overlapping of the Hajdu-Cheney and the Serpentine Fibula- Polycystic prenatal onset. 1M.L. Ramirez Duelas. 'L.E.iuera,N.O. Davalos and 2L.C. Kidney syndromes: Are they one single entity ? F.J. Ramosl, B.S. Kaplan, R.A. Bellah, _I1. Zackai, P. Kaplan. (I) Hospital Cl'nico Universitario, Zaragoza, Spain. The Children's Hospital of Villarroel. 1Divisicn deGenmtica, Cestrode Investigacion Bwrnidica de Occidafte, y2 Departmento Philadelphia, Philadelphia, PA. dendocroogia, Hospital de Pediatria. CMNO Instituto Mexicano del Seguro Social Guadalajara The Hajdu-Cheney syndrome (HCS) is a rare autosomal dominant disorder characterized by Jalisco distinctive facies, short stature and progressive skeletal dysplasia. We have reported two unrelated affected patients, both having bilateral polycystic kidneys, intestinal malrotation and basilar Two Mexican sisters were studied because short stature. Their parents were normal, and invagination. One had serpentine fibulae. consanguinity was denied. The older (Case I) is product of the fullterin second pregnancy, The term 'serpentine fibula-polycystic kidney syndrome" (SF-PKS) was first used by Exner, who at birth weight was 2500 g and bilateral microtia was recorded. Physical examination at 5 reported a 5 year-old girl with short stature, unusual facial appearance, polycystic kidneys and skeletal dysplasia with elongated curved fibulae. To date, further 5 cases of SF-PKS have been reported. After years revealed height 93 cm, arm span 83 cm, lower segment 41 cm, U/L segments ratio an extensive review of the literature, we found striking similarities between the HCS and SF-PKS 1.27 and OFC 49 cm, all but OFC, below the 3rd percentile for Mexican standards by age phenotypes. The Table below summarizes the most prominent features shared by both syndromes. and sex, disproportionate small stature by short extremities, and apparent macrocrania is Feature HCS (n=42) SF-PKS (n=6) Clinical observed. Her pinnae were well placed, small,( 3.4 and 3.5 cm in length) and all the Short stature ++ 6/6 auricular structures were present. Her face was small, mainly by malar reduction, her chin Scaphocephaly, bathrocephaly ++ 3/6 was short and retruded, and the palate was high and long. She had short hands and feet, and Bushy, arched eyebrows ++ 6/6 Prominent wide-spaced eyes ++ 6/6 her finger tips were wide. Her sister (Case 2) was product of the fullterm third pregnancy, Fuol cheeks ++ 6/6 birthweight was 2,080 g. At 14 months of age, the physical exploration showed a ""small Dental anomalies ++ 2/6^ baby". Her height was 65 cm, arm span 59 cm lower segment 27 cm, U/L segments ratio Micrognathia ++ 6/6 Short neck ++ 6/6 1.41, OFC 43 cm, all but OFC below the third percentile. She has identical facies and body Ilirsutism + 3/6 proportion as her older sister, her auricular length were 2.8 and 2.7 cm. Radiographic Hearing impairment + 3/6 studies demonstrated: craniofacial disproportion by small face, an open mandibular Normal intelligence ++ 3/6^ angle, Radiological almost 1800, the mandible body was thin, and the chin erased. All the long bones were Acroosteolysis ++ 2/6b slender, and in some physes were crossed by bony bridges joining the epiphyses with the Vertebral anomalies ++ 3/6 metdaphyses, the pelvis and vertebrae were normal, on the hands the tubular bones Bowing of long bones + 5/6 were Elongated, serpentine fibulae + 4/6 short, and the distal part of the 3rd phalanges were broad, as the head of a drumstick. The Polycystic kidneys + 3/6 carpal bone age of Case I was 3 years and in Case 2 of 2 months at the examination date. In Intestinal malrotation + 2/6 the differential diagnosis, the 3 M bone slender nanism, Silver-Russell S., Dubowitz S. and (++) Present in >50% of the patients, (+) Present in <50% of the patients; (a) Not applicable in three toddlers; (b) At the time of report, two patients lacking acroosteolysis werk under I year of age, the Mulibrey nanism were included, but none ofthese entities had tde particular type of microtia, remaining two were 5 and 9, respectively. and the drumstick phalanges found in the present cases. Based on these data, we hypothesize that the HCS and the SF-PKS could be one single entity with vari- able phenotypical expression. Reports of further patients and, ultimately, identification of the molecular defect will be necessary to prove our hypothesis.

2069 2070 Williams syndrome with cleft palate, expanding the phenotype. J.D. Ranells. Transmission of Mild Proteus Syndrome From Mother to Daughter? M. Artigas and B.G. Kousseff. Division of Genetics, University of South M.E. Rietsch and J.J. Hoo. Genetic Counseling Program, Binghamton and Florida, Division of Genetics, Department of Pediatrics, SUNY Health Science Tampa. Center, Syracuse. We report a 3 year and 5 month-old Caucasian girl with facial features Proteus syndrome is characterized by massive and asymmetrical hamar- typical of Williams syndrome, cleft soft palate, mild developmental delay and tomabous growth of various tissues involving mainly the skin, subcutan- self abusive behavior. Pregnancy was remarkable only for somewhat eous tissue, and skeletal system. Almost all reported cases are spora- decreased fetal activity. Amniocentesis was performed for advanced maternal dic cases. It has been postulated that the syndrome is caused by a age, karyotype was 46,XX. Two sonograms during the second trimester were lethal dominant gene surviving mosaicism. However, Goodship et al. normal. Delivery was at term, vaginal vertex with (1991) and Kruger and Wiedemann (1993Y reported the possible transmis- vacuum assist. Cleft soft sion of mild Proteus syndrome from father to son and from mother to palate and heart murmur were noted at birth. Aortic stenosis was suspected son respectively. clinically but not confirmed by echocardiogram. Early developmental Our index patient is an 8 year old girl, who was first noted to have milestones were delayed. At age 18 months self abusive behavior (hitting her asymmetric feet at 5 years of age. The size difference has become head in frustration) was noted. On developmental evaluation she was found more obvious over the past 3 years. There is no macrodactly. At age to be sociable, mildly delayed, hypotonic and hyperactive, not autistic. 7 years the left side of her maxilla was noted to be smaller than the right side. She is normal mentally. Her mother does not have asym- Lactate, pyruvate, ammonia, plasma amino acids, urine organic acids, metrical body sizes, but she is remarkable for a large, about 4 inch biotinidase, serum carnitine and Fragile X study ordered in a previous wide, non-homogeneous, and non-elevated nevus starting sharp from the neurologic evaluation were normal. An MRI of the brain was also normal. midline of her back, running down and under her left breast, and ending Genetic evaluation revealed subtle dysmorphy suggestive of Williams sharp in the midline of her abdomen (Figure 5 a and b). syndrome, including periorbital fullness, epicanthal folds, broad nasal tip, long It was initially thought, that the unilateral large nevus of the philtrum, microretrognathia, widely spaced teeth, prominent ears and bilateral mother has no relationship to the daughter's hemihypertrophy. However, clinodactyly. Growth and realizing that hemihypertrophy and nevi are the two hallmarks of head circumference were normal. FISH studies Proteus syndrome, it is suggested that the above mother-daughter pair showed a microdeletion within the qi 1.23 region of chomosome 7, may be another example of transmission of mild Proteus syndrome in two consistent with Williams syndrome. While subcutaneous cleft of the palate generations. It is noteworthy that in all three families the parent has been reported on a couple of occasions, this appears to be the first is Clinically much milder affected than the offspring. patient with cleft palate; thus it expands the phenotrpe.

2071 2072 Thalidomide:teratogen and mutagen? R. Tenconi, M. Clementi. Genetica Chromosomal mosaicism with hypomelanosis of Ito and moyamoya Medica, Dept. Pediatrics, University of Padova, Italy disease. S.ungr. D.Chitayat..R.Babul. ITeshima. nd S.Blaser. Hospital for Thalidomide is the best known teratogen. Recently the observation of congenital Sick Children and Univ. of Toronto, Ontario,Canada. malformations (CM) in children of thalidomide victims, raised the question if Hypomelanosis of Ito is a cutaneous sign consisting of hypopigmentation generally thalidomide could be also a mutagen. following the lines of Blaschko. It is often seen in association with mosaicism for Daring the last year we observed two children with CM, born to thalidomide victim aneuploidy or chromosomal rearrangements. To the best of our knowledge,there has women. They present the typical pattern of limb defects. Their anamneses reveal been one other report in the literature of a patient with hypomelanosis of Ito and maternal intake of thalidomide during the first trimester of pregnancy. moyamoya disease (without chro e analysis). We report a child with an The first boy has almost complete absence of 3rd and 4th fingers with necrotic tissue abnormal karyotype (46,XX,inv(6)(p21p25)/ 46,XX,6p+) associated with attached to the tip, 2nd finger with a deep constriction ring at the proximal phalanx hypomelanosis of Ito and moyamoya disease. with hypertrophic distal segment and hypoplastic nail. Annular constriction is also At 3.5 years of age the proposita had an afebrile generalized tonic-clonic seizure. present between the Ist and 2nd phalanx of the thumb associated with the presence of Also noted at that time were a mild left hemiparesis and a whorled pattern of a fibrous hypopigmentation on her trunk. Investigations, done at that time, included a normal remnant, while the 5th finger is normal. The final diagnosis is amniotic band brain CT scan. Approximately ten months later, a brain spect scan showed a focal sequence. The second boy has a duodenal and anal atresia with annular pancreas and perfusion defect in the right frontal region. An MRI, done at nine years of age, ventricular septum defect. Both pregnancies were uneventfull. revealed occlusion of the internal carotid arteries as they entered the intracral sa These observations raise the doubt that thalidomide could be involved as a mutagen in and an associated marked collaterilization of vessels in the radiographic pattern or the determination of CM. Although the observation of these cases might only be due to moyamoya. Also seen was a left middle cerebral artery aneurysm. A subsequent chance, a causal relationship between CM in the offsprings of thalidomide victims and catheter angiogram confirmed the diagnosis of moyamoya disease in this patient. parental thalidomide exposure cannot be excluded, because the observed frequency of Chromosomal analysis revealed mosaicism for two cell lines both with CM is statistically unexpected on the basis of the available epidemiological data and of abnormalities of 6p. In 40/50 lymphocytes the karyotype was the consideration that the offsprings of thalidomide victims are few (limited number 46,XXinv(6)(p2lp25) and in 10/50 lymphocytes the karyotype was 46,XX,6p+ and presumable reduced fitness). In our Genetic Clinic these two women are the only (with the additional material being of unknown origin). This finding was later consultations about thalidomide victims in a 16 year period and over 4,000 confirmed in fibroblasts. Both parental karyotypes were nomal. consultations. We speculate that chromosomal mosaicism may be involved in the pathogenesis of Retrospective and prospective studies are recommended, also considering that thalido- moyamoya disease and should be investigated for when this rare neurological mide is still used for leprosy and biliary cirrhosis therapy. disorder is diagnosed. A356 Published Abstracts: Clinical Genetics, Malformations and Dysmorphology (cont.) 2073 2074 Albers-Schonberg disease (autosomal dominant osteopetrosis) is tightly Filippi syndrome: Report of two additional cases with expansion of the linked to the macrophage colony stimulating factor (CSF-1) gene on phenotypic features i.L. Williams1, M.S. Williams2, D.S. Wargowski3, chromosome lp2l. W. Van Ilul', E. Van Ilul', W. Wuyts', J. llerslev2, J. gram, .hA. Pletcher4, F. I)esposito'. 'La Crosse Regional Genetic Services Project, La P.J. Willems'. 'Department of Medical Genetics, University of Antwerp, Belgium; Crosse, WVI; 2 Gundersern Lutheran Medical Center, La Crosse, WI; 3 University of 2Department of Endocrinology, Rikshospital, Oslo, Norway; 'Department of Wisconsin, Madison, WI; 4 University of Medicine and Dentistry, Newark, NJ. Endocrinology, Odense University Hospital, D)ennark. Filippi syndrome is an anitosomal recessive condition characterized by variable soft- Albers-Schonberg disease, the classical form of osteopetrosis, is an autosomal tissue syndactyly of the fingers and toes, microcephaly, pre- and postnatal growth re- dominant condition with generalized increased skeletal density due to reduced bone tardatiosn, mildly dysmorphic craniofacial appearance together with mental retardation. resorption. Patients suffer from back pain, bone pain, headache, an increased fracture We report two additional unrelated individuals who exhibit characteristics of Filippi rate and comnpression of cranial nerves leading to deafness, loss of vision and facial syndrome. Individual one has microcephaly, significant growth and developmental de- nerve palsy. (characteristic radiological findings of Albers-Schbnberg disease are lay, dysmorphic craniofacial appearance (including plagioceplialy, short, downslanting generalized osteopetrosis with most typically end-plate sandwich-like thickening of the palpebral fissures, apparent telecanthus, overhanging nasal tip and mandibular reces vertebrae (rugger-jersey spine) and bone-within-bone (endobones) phenomenon. sion) Duane anomaly, soft-tissue superniumary digit postaxially on the left hand and In an extended Danish family with this disease, we found linkage with several mark- cutaneous syndactyly of toes 2 through 5 bilaterally. Initially the diagnosis of an acro- ers from chromosome lp2l. A maximum lod score of +4,09 was obtained at loci cephalopolysyndactyly syndrome was raised on the basis of syndactyly, polydactyly and DtS239/D1S248 in multipoint analysis and a candidate region of 8.5 cM was delin- plagiocephaly. CT scan showed normal cranial sutures and mild cerebral atrophy. The eated flanked by markers D1S486 and D1S2792. Possible linkage of Albers-Schbnberg plagiocephaly subsequently resolved. Indivdual two has microcephaly, downslanting disease to this chromosomal region was analyzed because the macrophage colony stimu- palpebral fissures, growth and mental retardation, extremely tapered fingers, cutaneous lating factor (CSF-1) gene is located in 1p21. CSF-1 is an haemnatopoietic growth factor syndactyly of toes 2 through 5 bilaterally and a broad great toe. Neither of these in- that plays an important role in the proliferation of macrophages and osteoclasts from dividuals have marked cutaneous syndactyly of the fingers. Polydactyly has not been haematopoietic stem cells. Op/op mice, homozygous for a single base insertion in this previously reported in Filippi syndrome. Features of Filippi syndrome will be presented gene disrupting the protein, develop osteopetrosis. To investigate the possible role of the and contrasted with these two additional cases. CSF-l gene as disease-causing gene in Albers-Schbnberg disease, SSCP and mutation analysis is currently performed in patients.

2075 Autosomal dominant hypohidrotic ectodermal dysplasia in a large family, possibly of the Jorgenson type Williams MS', Josephson KD2, Aswegan AL3, Mowbray R , Pauli RM5. 'Guindersen Lutheran Medical Center; 21aCrosse Regional Genetic Services Project, 3Gundersen Medical Foundation; 'University of Wisconsin, LaCrosse, WI; 'University of Wisconsin, Madison, WI A large family is presented with an asitosomal dominant hypolhidrotic ectodermal dys- plasia syndrome. The disorder has been recognized in 31 individuals in five generations. 25 affected indivduals in three generations are still living. Characteristic features in this fainily include: sparse, slow-growing hair (scalp and body), absent eyebrows, fewer eyelashes, and abnormally shaped or missing teeth. Nail involvement is more variable with some individuals having brittle, slow-growing nails and others having normal nails. Most individuals have some decreased sweating and/or unusual pattern of sweating. A few report significant heat intolerance. Individual IV:16 is said to sweat normally. Some individuals report normal sweating when younger, but decreased sweating when older, suggesting age-related decrease. Extensive literature review identified 40 autoso- mal dominantly inherited syndromes associated with some form of ectodermal dysplasia. Our family seems to bear a resemblance to the family reported by Jorgenson, et al.

Published Abstracts: Cytogenetics 2076 2077 Familial unbalanced t(5,17Xp15.33;q25) In two adult siblings with Comella Potential chromosomal outcomes for zygotes of a couple both of whom are de Lange-like syndrome. A. Al Saadi. M. Madercic and C. Mcya". William translocation 14121 carriers. R ASaif'. N. Sakatil. A. Al-Ashwal. N. Attia'. A. Beaumont Hospital, Royal Oak, Michigan. label'. P. T. §Mnatterlv2. and T. E. Kely. King Faisal Specialist Hospital, Riyadh, Cornelia de Lange syndrome (CDLS) is characterized by physical and Saudi Arabia', and University of Virginia Health Sciences Center, Charlottesville2. A first-cousin couple was referred for evaluation for three miscarriages. mental retardation, hirsutism, synophyrs, microcephaly, long eyelashes, 45 chromosomes small nose with anteverted nostrils, micromelia, and Karyotyping of both was performed and both were found to have characteristic lips, mouth, with a 14/21 Robertsonlan translocatlon presumably by descent from a common other variable phenotypic abnormalities. Most cases are sporadic but ancestor. They have one healthy son and plan to have more children. They autosomal dominant inheritance has been suggested, and dup(3)(q25q29) wanted to know the possible chromosomal outcomes for future pregnancies. tends to yield a CDLS-like phenotype. Variability of the phenotype and the Since both parents are 14/21 translocation carriers, more chromosome uncertainty of the etiology of the syndrome require other cases of CDLS with combinations are possible for their zygotes than if only one was a carrier. A specific genetic mutations to determine the etiology. We report here two adult Punnelt square with 6 possibilities for sperm and 6 possibilities for eggs results In siblings, a male age 34 and a female age 42, with a phenotype strongly 36 potential zygotes. Of these 36 potential zygotes, 24 are presumably lethal, 7 suggestive of CDLS including profound mental retardation, hirsutism, would be balanced translocation carriers (1 homozygous for the translocation), 4 synophyrs, seizures, spastic quadriplasia, scoliosis, inability to walk, would result in Down syndrome (2 homozygous for the translocation), and I would constipation, cryptorchidism, and cystic breast. Upon investigating a healthy have normal chromosomes. One would assume that these possibilities would not sister for inability to conceive, a balanced t(5;17)(p15.33;q25) was discovered. be equally likely. For instance, the sperm that are monosomic for chromosomes 21 or 14, or trisomic for chromosome 14, may have a lower chance of participating In Her healthy mother was a carrier of an identical translocation but the father fertilization. had a normal karyotype. The two affected siblings, who are in a community The question arose of the phenotypic effect of homozygosity by common living institution, were found to have a der(5)t(5;17) (p15.33;q25). An Oncor descent of the translocation. Our conclusion is that it would not be any different Tel 17q DNA probe was used in all five cases, confirming the high resolution from homozygosity by descent of any of the non-translocated chromosomes. In results by the presence of three signals in each of the 30 examined cells from addition to the increased risk of autosomal recessive diseases in this the affected siblings and two signals in each of the parents and the healthy consanguineous couple, the risk of Down syndrome was estimated at sister. approximately 14% for this couple (about 2% patemal and 12% matemal). The Since there is no clear etiology of CDLS, these results suggest that a gene likelihood of a balanced carrier karyotype is greater than the, likelihood of a normal or a chromosomal segment on either 5p or 17q is responsible for CDLS or karyotype. CDL-like syndrome. Additional cases with a similar translocation and cloning of a number of gene sequences from 54el5.33 and 17q25 may help delineate the etiology of CDLS. Published Abstracts: Cytogenetics (cont.) A357 2078 2079 A CASE OF TETRASOMY 9P AND REVIEW OF THE LITERATURE. X chromosome mosaicism in women with fertility A. Asamoah', K. N. Nandi', H. Chen", L. Prouty', H. disorders. Ibrahim J. Baier', R. L. Young, and S. Ursin. R.B;ez, D.G.Mayen, R.Garcia-Cavazos, M.J.Zava- leta. Genetics Department. Instituto Nacional Genetics Section, 'Neonatology Section, Department de Perinatologia. Mexico city. of Pediatrics, Louisiana State University School of Medicine, Shreveport, Louisiana. From January 1984 to december 1995 we analized We report on a Caucasian boy who presented at 2,706 peripheral blood samples for karyotype, birth with midline defects of the skull and face, 1045 samples from couples with fertility disorders: amenorrhea (11%) (primary or secon- complex skeletal and limb malformation, upper airway dary), sterility (9%) or recurrent spontaneous instability, and Hirschsprung disease. Chromosome abortion (80%). We report the karyotype-phe- analysis of peripheral blood lymphocytes and skin notype correlation of 46 women with X chromo- fibroblasts showed tetrasomy 9p involving part of some mosaicism. the heterochromatic region of the long arm of We found 16 women with mos45,X/46,XX, 9 with mos46,XX/47,XXX, 20 with mos45,X/46,XX/47,XXX chromosome 9. There was no evidence of mosaicism. and one patient with 45,X/46,XY in limphocy- Fluorescence in situ hybridization analysis tes. We performed karyotype in fibroblasts in confirmed the cytogenetic analysis. A review of the 9 patients and found them normal . Seven phenotypic features seen in 28 published cases and patients had a history of a malformed child (Trisomy congenital heart defect, VACTER our patient suggest that tetrasomy 9p is a syndrome 21, asociation) . The karyotypes, clinical corre- that can be diagnosed clinically. lation and hormonal profiles will be presen- ted. We also present the follow-up of 12 patients that became pregnant. We conclude that the women with X chromosome mosaicism have great phenotipic and endocrinological va- riations and have higher risk than other women of recurrent abortion.

2080 2081 Prenatal Diagnosis of Mosaicism for a Small Supernumerary Ring Interstitial deletion of 7q34-35: A new case report and review of the literature. Chromosome X Identified by Fluorescent in situ Itybridization M.L.Begleiter, hi Ben-Yishav. V. Pulisaal. J. Roberts. A. Pathlak. G. Reinerman R. Stoessel- B. Girz. J.C.Rogers, D.J.Hlarris and L.M.Pasztor. The Children's Mercy Hospital and the A. Shanske. ofPediatrics and Montefiore Medical University of Missouri School of Medicine, Kansas City, MO Departments Reproductive Genetics, Small supernumerary marker chromosomes identified at prenatal diagnosis present a Center. Albert Einstein College of Medicine, Bronx, New York. particularly difficult cytogenetic and counseling challenge. A 40 year old primigravida Terminal and interstitial deletions of7q have been well described. Most involve was referred to us for an amniocentesis procedure because of advanced maternal age. relatively large segments and clinical correlations have been unclear. We have had the A three generation family history revealed no individuals with genetic diseases or birth opportunity to examine a newborn with a small deletion in this region, del(7Xq34q35) defects. Twenty-one colonies from the amniotic fluid cultures were examined. In thirteen who represents only the third case with a deletion involving only these two bands. colonies there was a normal female karyotype while in eight colonies there was a small B.C. was the 2389 product ofa 38 week gestation delivered by caesarean section to a 41 extra marker chromosome which was 1/4 to 1/3 the size of a chromosome 21 and did not show evidence of satellites. In some metaphases it looked like a small ring. The year old gravida 5 para 2022 insulin dependent diabetic. He was delivered through thick following cytogenetic fluorescent probes were hybridized to slides and processed by tile meconium and received an Apgar of9 and 9. An amniocentesis done for advanced maternal in-situ technique (FISII); Y "cocktail", X alpha-satellite, X whole chromosome probe age revealed an elevated AFP (2.85 MoM), a negative AchE, and an abnormal male and XIST (Xql3.2). The hybridization signals observed indicated that the marker was karyotype, 46,XY,del(7)(q34q35). Parental karyotypes were normal. A targeted ultrasound derived at least in part from the X chromosome and that there was no evidence for study at 18 weeks showed oligohydramnios. The pregnancy was carried to term because of XIST in the ring (marker), although both standard X chromosomes showed this region. convictions. Chromosome analysis on both parents was normal: Based on the FISH findings, it would religious be expected that the marker X would not be inactivated. There is only one case report The newborn physical examination revealed a minimally dysmorphic infant. The of a child with a sinmilar karyotype who had moderate retardation, seizures and impaired anthelices and lobules were prominent and there were deep transverse furrows below the speech as well as facial dysmorphia (Clin. Genet. 47:270-273. 1995). Our patient's lower and above the upper lids. The palate was intact. lie had an interrupted right-sided pregnancy is continuing and should be delivered before these meetings where we will be proximal transverse crease. The external genitalia were abnormal with a glandular able to review the outcome. hypospadius, large right testis and left cryptorchidism. The neurologic examination was normal and the infant was discharged after an uneventful newborn period. Ultrasound imaging of the head and kidneys was normal. Our patient has only mild dysmorphic features but does have some of the abnormalities found in more than 1/3 of cases reported by Young, et al (1984, American Journal of Medical Genetics) in a large series of terminal and interstitial deletions including abnormalities ofthe ears, palmar creases and external male genitalia. Future evaluation of our patient will help to further delineate a syndrome of this deleted segment.

2082 2083 Partial trisomy 9 and Angelman syndrome in a patient with a Mapping of the X-breakpoint involved in a balanced X;12 translocation in a matermally-inherited der(9)t(9;15)(q21.2;q11.2) chromosome. M.G. female with mild mental retardation T.Bienvenu 1,1. Der Sakarian 2, Bialer. X.T. Zhou. S. Sastry. M.M. Sanz and J.H. Ray. North Shore V. Desportes 1, P. Billuart 1, M. Tissot 2, T. Bruls 1, J.P. Chabrolle 3, P. Chauveau 3, University Hospital, Manhasset, NY. M. cherry ,1,1). Cohen 2, J.C. Kaplan 1, C.tieldjord1, J. Chelly 1, D. Cherrif 2. 1: A 16 day old female presented with a right clubfoot and dysmorphic INSlEM U129, CHIU Cochin, Paris; 2: CEPH, Paris; 3: service de Cytogenetique et de features including excessive wrinkling ofskin on her forehead and above both Mdecine no-natale, Le llavre; 4:Laboratoire de Cytogenetique, Nancy, France chromosomal abnormalities such as translocations and ears, prominent nasal bridge, micrognathia, single transverse palmar creases Nonrandom balanced inversions, and 6th finger clinodactyly with sharp angulation of the DIP joints. have been identified in many genetic diseases. Cloning of the breakpoints involved has led Cytogenetic analysis revealed the karyotype to be to the identification of the affected genes. Recent reports suggest the existence of a men- 46,XX,+9,der(9)t(9;15) tal retardation locus at We report the investigation of a balanced X;12 translo- (q21.2;qll.2),-15. The child's features were consistent with trisomy 9p. XqI-t12. cation t(X;12) (qll;qlS) observed in a female patient with mild mental retardation. We Analysis of parental blood samples revealed that the mother had a balanced identified a yeast artificial chromosome (YAC) spanning the X-chromosome breakpoint translocation between chromosomes 9 and 15, also present in the maternal by using fluorescent in situ hybridization techniques. This YAC ICRFy900H0493 is grandfather. Because of the maternal inheritance of the der(9) chromosome positive for DXS159 and DXS227. Cosmids library of this YAC has been construct and in the child and the localization of the breakpoint on chromosome #15 in the the search for candidates genes is in progress. Prader-Willi syndrome (PWS)/Angelman syndrome (AS) region, an effort was made to localize the breakpoint on chromosome 15 more precisely utilizing FISH and commercially available probes for the PWS/AS region. Probes D15511 and SNRPN from the PWS region and probesD-S-1- and GABRB3 from the AS region were employed. GABRB3 was located on the der(9) chromosome of both mother and daughter whereas D15S11, SNRPN and D15S10 were located on the der(15) chromosome in the mother's cells. Thus the daughter's cells were monosomic for these 3 probes. Since D15S10 is in the critical region of AS, it was predicted that the patient would most likely exhibit features of AS as well as those of partial trisomy 9. On follow-up at 12 mo, the child had several features of AS including absent speech, frequent tongue protrusion, happy disposition and frequent outbursts of laughter. These results demonstrate the utility of combining FISH with traditional cytogenetics in the study of derivative chromosomes. A358 Published Abstriacts: Cytogenetics (cont.) 2084 2085 Characterization of a Supernumerary Marker Derived from Chromosome 17 Novel phenotypic features in a newborn with interstitial del(21 q21 .2q22.31. through MicroFISH of G-banded Chromosomes of an Adult with MR/MCA PA Bradshaw. L Linck TA Kuforiji. LD Wallen. SB Olson. RE Magenis. DM Pillera. 'Blancato, J., 2Kozma, C. WMeck, J., 'Plaia, T.. 'Jiang, Y. Oregon Child Hlth Res Cntr, Doernbecher Children's Hosp, Oregon HIth Sci U, Portland. 'Oncor, Inc., Gaithersburg, Maryland; 2Cieorgetown University Medical Center, Washington, DC; Deletion of chromosome 21q is associated with phenotypes ranging from severe 'National Center for Human Genome Research, Bethesda, Maryland psychomotor retardation with characteristic dysmorphic features to normal intelligence and appearance. We report a neonate with the karyotype 46,XX,del(21)(q21.2q22.31. Recent molecular cytogenetic techniques such as MicroFISH (rev ish) have allowed the identification FISH with Oncor Coatasome 21 tCAT.#P5221-DG.2, Lot#4H152) supports the inter- of the chromosomal origin of supernumerary marker chromosomes and the delineation of specific pretation of interstitial deletion. The patient had dysmorphic facies, including syndromes. Here we describe an 8-year-old male with profound mental retardation and a small triangular shaped head, prominent and wide nasal bridge, low set ears with redundant supernumerary marker chromosome which was identified by MicroFISH to be derived from pinnae, absent eyelashes, short palpebral fissures with slant, chromosome 17q. Past medical history indicated that the patient was born prematurely with a birth antimongoloid weight of 2.4 kgs. He had recurrent respiratory problems, mitral valve regurgitation, and surgical hypertelorism, and micrognathia. Other dysmorphic features included bridged correction of sevcre scoliosis. On physical examination, he had growth retardation, hypoplastic facial transverse palmar creases, poorly developed femoral creases, sacrococcygeal dimple, bones, small eyes, a prominent nose, abnormal ears. a barrel-shaped chest with severe kyphoscoliosis, small hands and feet, and hypoplastic nails. Other findings include abnormal extra transverse crease bilaterally, digitalization of the thumbs, long slender fingers, severe pulmonary lobation, hepatic cyst, redundant renal collecting system, and bilateral calcancovalgus deformity of the feet, and severe hypotonia. G-banding of PHA-stimulated schizencephaly. Echocardiography demonstrated a double outlet right ventricle with lymphocytes revealed a ring-like marker in greater than 80% of metaphase spreads. NOR staining for subaortic infundibulum and anteriorly displaced aorta, hypoplastic left ventricle, VSD, the identification of acrocentric markers was negative. C-banding was performed to assure that a ASD, and PDA. Born at 36 weeks EGA after an uncomplicated pregnancy, she centromere was present in the marker chromosome. FISH was performed using a number of alpha presented with respiratory distress requiring intubation. She required multiple platelet satellite DNA probes, including I, 5, 8. 9, 15,18 22, X, and Y, and the whole chromosome probe for transfusions and FFP for thrombocytopenia. Causes of consumptive coagulopathy, the 22. Clinical suspicions and morphological observations of the marker were the basis for choosing including sepsis/congenital infection, DIC, and neonatal alloimmune thrombocytopenia these probes, although none demonstrated hybridization with the marker. MicroFISH of G-banded were ruled out. At age 15 the increased apnea-bradycardia was This method entails the microdissection of unknown days, patient developed metaphase preparation performed. due to infection with virus which a rapid chromosomal material with direct PCR and fluorochrome The result is a episodes respiratory syncytial precipitated amplification incorporation. function to death. confirmed labeled probe which can be used to hybridize with a normal metaphase spread. DAPI banding allows decline in cardiopulmonary leading Autopsy the presence for chromosome identification of the normal chromosome showing the signal. In this case, a of multiple anomalies noted above, as well as a hepatic cyst and schizencephaly. cohybridization of the marker DNA labeled with Texas Red was performed with a fluorescein-laheled Many groups are attempting to delineate regions on chromosome 21 that correlate FISH probe for the myeloperoxidase locus (17q22) to confirm hybridizigion to chromosome 17. with specific phenotypic features. In monosomic regions, abnormalities may be caused either by having potential dosage effects due to hemitygosity or by unveiling mutations within genes on the undeleted homolog. Given the multiple organ systems involved in this case, the gene or genes involved in chromosomal region q21.2-q22.3 play an essential role in normal embryonic development.

2086 2087 Distal Sp duplication, Including band Sp22, has mild phenotypic effect on several X;2 Translocations in two unrelated cases: Late replication patterns and members of a family. "klower Broks'. M. Genovese. H. Gut- C. J. Duncant A implications for phenotypic variations. J.E. Chernos and S.K. Murthy. Alberta She ke Eud.LC.InkinL. 'New York State Institute for Basic research in Developmental Children's lospital and University of Calgary, Alberta, Canada. Disabilities, Staten Island, New York; 2Montifiore Medical Center, Bronx, New York. The observed phenotypes in X/A tranlocations may be attributed to the disruption of Duplication 8p has generally been considered to result in a syndrome characterized by specific genes, the breakpoints involved on the X chromosome and the pattern of severe mental retardation, mild facial dysmorphism, hand deformities, and malformations of X-inactivation attd extent of spreading into the tranlocated autosome. We report two the heart and brain. We report on a large kindred segregating a Y;8 translocation in whom unrelated cases of X;2 translocations involving nearly the same breakpoints on X and 2 several individuals have a duplication for Sp2l.3-8pter with mild phenotypic effect. The but showing significant phenotypic variations. proband presented following karyotype analysis for repeated pregnancy loss which revealed Case I was a female infant referred at 3 months of age for failure to thrive. G-banding him to be a carrier ofa balanced Y;8 translocation. Several years later, another brother was showed an unbalanced translocation 46,X,-X,+der(X)t(X;2)(q26;pl3)mat with trisomy tested and found to have the same karyotype. His wife subsequently became pregnant and of 2p13-pter. The mother was a balanced carrier of the X;2 translocation. She had no history of reproductive problems, but coincidentally had a cleft lip. Replication studies underwent amniocentesis elsewhere, which revealed a fetus with a duplication in the distal the was active in the but in the the was region of 8p. The pregnancy wvas terminated. Recently, two showed that der(X) mother, daughter, der(X) other brothers were found to inactive with incomplete spreading into the 2p region as far as 2p16. Clinical features have the same karyotype as the fetus. FISH analysis using whole chromosome painting in this patient (growth and psychomotor retardation, hypertelorism, frontal bossing, probes Y and 8 (ONCOR) confirmed our findings in both brothers with the balanced and macrocephaly) can be attributed to functional partial trisomy of chromosome 2. reciprocal translocation, as well as both with a derivative Y chromosome with extra lp Case 1I was a 17 year old female referred for primary amenorrhea but normal material are respectively: 46,XY.ish t(Y;lIXql2;p21.3)pal(wcpY+,%vcpg+;wcpY+,wcp8+) secondary sexual characteristics. Tier height was at the 5th percentile. G-banding and 46,X,der(Y).ish t(Y;8) (qI2;p21 .3)pat(wvcpY+,wcp8+). The two men with unbalanced showed an apparently balanced, de novo reciprocal tranlocation between X and 2 with karyotypes attended special education classes through high school. Each lives independently breakpoints at Xq27 and 2pl3. The normal X was late replicating and thus she is and holds a responsiblejob. One brother is married and has a two-year-old child who was functionally balanced. shown to have the same unbalanced karyotype as his father. This child has mildly delayed The occurrence of similar and rare translocations in two unrelated cases is very curious. language acquisition A recent report (Engelen el al, J Med Genet 199S;32:792-795) In addition, short stature and disruption of ovarian function in case II is unexpected describes mild mental retardation without dysmorphism in partial trisomy Rp with direct since the X breakpoint is more *listal than in the mother of case I and olstside the critical duplication of 8p22-8p23. I. This is in contrast to the severe mental retardation and region (Xqt3-q26). This siuggests that there are probably other more distal Xq genes malformations associated with inverted duplications ofSp, where the duplications are more that are respoussible for normal reproductive function. Gonadal dysfunction in case 11 could also be explained by position effect of genes in this region or effective hemizygosity proximal and overlap with band Rp22. Our family is phenotypically similar to that described of a recessive that inhibits due to skewed X inactivation. by Engelen and the duplicated segment is also comparable suggesting that duplication of gene gonadal functioning 8p22-8pter may result in a mildly affected phenotype. lwok pmp nd pw Nysoea of mmai RStdutimnd Daunpat Dabitltaakd NYS Dewbnmeal"16ut

2088 2089 Direct duplication lqll.2->16q22 in a child with cardiac and renal Sequence composition of a centromeric chromosome 19 probe. 'N. Sacchi, 2Mj anomalies. M-y Chung, J-H Lu*, B Hwang*, L-S Fang, I-T Yu and H Chiang. Darfer, 1A MadzLR. Meneveri. 1l, Maan'E. IDepartment ofBiology Departments of Pathology and Laboratory Medicine, and Pediatrics*, Veterans and Genetics. School ofMedicine, University ofMilan, Italy and 2Molecular and Cell General Hospital, Taipei, Taiwan, R.O.C. Life A twenty-five-month old girl, born full term after an uncomplicated pregnancy (G3P2) Biology R&D, Technologies Inc., Gaithersburg. MD. with low birth weight (2,400 g), was diagnosed of muliti-cystic dysplasia of the left kidney We reported a possible case ofcentromere activation in a stable marker chromosome prenatally and heart anomalies (supervalvular pulmonary stenosis, ASD and PDA) post- that contains a cryptic centromere associated with an inverted duplication ofthe natally. Her cardiac anomalies were corrected at the age of one year by surgery. Neither 14q32-+qter region (1). In an attempt to identify the cryptic centromeric sequences the limb nor digital structural abnormality was noticed. Weight and height are between the marker was flow-sorted and used to generate probes by degenerate oligonucleotide 3rd and 10th percentile. Delayed development was noticed at age of two years dite to primer polymerase chain reaction (DOP-PCR). Interestingly, reverse chromosome unable to stand and walk. Chromosome analysis of the peripheral blood lymphocyte revealed partial trisomy of the long arm of chromosome 16 due to a direct duplication painting experiments using these probes showed that the marker chromosome contains from qll.2 to q22 (karyotype 46,XX, dir dup(l6)(qll.2->q22), by G- and C-banding. sequences that are also highly represented in the centromeres ofchromosome I 8and in Previous report of duplication of a smaller region (16qll.1 to 16q13) presented moderate the pericentromeric region ofchromosome 19, respectively (2). mental retardation, severe hyperkinetic behavior, and slight dysmorphic stigmata but no Sequence analysis ofthe chromosome 19 probe has revealed the presence ofa few internal anomalies (Fryns et al., Annals de Genetique 1990;33:46-8), while the complete novel single-copy as well as repeated sequences. Most of the sequences determined so duplication of 16q always results in early deaths. l)ue to the nature of the duplication far do not match any ofthe sequences in the available data bases. We do not and the fact that this child has a healthy elder sister, this duplicated chromosome 16 genomic should be a de novo event instead of being inherited from a balanced translocation. Her know at the present time which ofthese sequences may account for the centromere parents' chromosome analyses still need to be performed. This is the first report of the function ofboth chromosome 19 and the marker chromosome. duplication of this region that is not accompanied by other chromosomal abnormality. I) Magnani I et al. Clin Genet 43: 180-185, 1993 2) Sacchi N et al. Cytogenet Cell Genet 73: 123-129,1996 Published Abstracts: Cytogenetics (cont.) A359 2090 2091

Partial aneuploidy detected by prenatal interphase FISH analysis. Direct duplication of chromosome 2q31.1q32.1 In a boy with mental LL Estab[oks', C.H. Lyl- M.J. Sapeta , J.M. andow' J.LE retardation and minimal phenotypic abnormalities. M. Fernandez. K.A. Leooia. Takacs', J.S. Hanna', R.L. Neu', J. J. Weiser', B.E. Ward', K.W. B. Vlcek. L. Hudains. University of Washington School of Medicine and A.NJ.Lanb'. 'Genzyme Genetics, Santa Fe, NM; 2UNC-CH, Children's Hospital and Medical Center, Seattle, WA. Chapel Hill, NC. Duplication of various segments of 2q are rare and are usually manifested low birth retardation; prominent forehead; Prenatal interphase FISH analysis on uncultured amniotic fluid cells by: weight; psychomotor flat nasal short beaked nose and anteverted is designed to detect non-mosaic aneuploidies involving chromosomes hypertelorism; broad, bridge; nares; long philtrum; malformed ears; and clinodactyly. Less commonly, heart 13, 18, 21, X & Y. In most cases, the detected aneuploidies are anomalies and genitourinary defects have been described. We report a 14 year straightforward trisomies or monosomies. On occasion, interphase old boy with a direct duplication of chromosome 2q31 .1q32.1 who presents FISH analysis detects partial aneuploidies due to structural with mental retardation and minimal phenotypic abnormalities. abnormalities. Out of the past year's 4,067 samples, interphase FISH This mentally retarded 14 year old boy with the diagnosis of fetal alcohol analysis has detected 5 cases with partial aneuploidy. Interphase syndrome was referred for neurological evaluation for attention deficit and FISH detected three cases of partial monosomy 18q representing two hyperactivity disorder. Chromosomal studies were ordered and initial analysis cases of ring 18 chromosomes and one terminal 1 8q23 deletion. indicated an insertion of unknown material at 2q31. Interphase FISH results were consistent with trisomy 13 in one case Genetic evaluation revealed minimal phenotypic abnormalities including a broad nasal thin lip, and wide distal phalanges. which had trisomy 13q12-qter secondary to a 1;13 translocation and bridge, synophrys, upper were normal and no abnormalities were detected. 3:1 segregation. The fifth case involved detection of Y disomy due to Growth parameters major The chromosomal rearrangement was identified by fluorescence in situ the presence of a cryptic psu idic(Y)(q12). hybridization and prometaphase preparation as a direct duplication of Although prenatal interphase FISH analysis is not designed to detect chromosome 2q31.1q32.1, thus this patient is trisomic for this region. rearrangements, this can occur if the probe hybridizes to the structural This case illustrates the utility of chromosomal analysis in patients with region involved in the partial aneuploidy. Caution must be used in mental retardation and minimal phenotypic abnormalities, even in the absence reporting the results as interphase FISH does not provide information of growth retardation and major malformations. To our knowledge, this is the regarding the specific structure of the abnormality. Chromosome first report of trisomy 2q limited to this specific segment. The minimal findings analysis is necessary in order to clarify the specific rearrangement. in our patient could be explained by the smaller size of the duplicated segment.

2092 2093 Identification of an unusual extra marker chromosome by microdissection, PCR Partial monosoay of chromosome 6q in a mentally retarded dysmorphic and FISH. I.K. Gadii, R.N. Slotnick2, A. Abuhamad2, K. Graham2. A.D. Stock3, T. patient with alopecia. Dennis3, P.N. Mowrey', P.R. Papenhausen', J.H. Tepperberg'. 'LabCorp, Center for C. Giloin.1 P. Edoux.1 J. Allanson-2 YSufrategui1 V . Der Molecular Biology and Pathology, Research Triangle Park, NC, 2LabCorp, Sierra KaloustianL Nevada Lab, Reno, NV. 3EVM Department of Ob/Gyn, Norfolk, VA. Clinical cytogeneticists are often faced with the challenge of identifying the origin 1McGill University, Montreal, Quebec, Canada; 2University of Ottawa, of extra marker chromosomes. PCR amplification and labeling of DNA from a Ontario, Canada. microdissected marker chromosome provides a marker specific DNA probe useful in We report the case of a child with multiple congenital anomalies determining the chromosomal origin of the marker. and a partial eonosomy 6q. A 37 year old women, G4P2, underwent an obstetrical sonographic evaluation at This eight-year-old girl was the second child of parents of 20 weeks gestation due to suspicion of fetal intrauterine growth retardation. Mexican and French Canadian origin, both 29 years old, with no Ultrasound evaluation was remarkable for three weeks fetal growth restriction, significant family history. Pregnancy was uneventful. Birth weight was 6 pounds. Hypotrichosis of the temporal areas was present at birth. hydrocephaly, cleft lip and a 2 vessel cord. Cytogenetic analysis of the amniotic Hicrocephaly was noted in infancy and generalized seizures fluid revealed an extra marker chromosome (47,XX,+mar) in all 20 cells analyzed. developed in early childhood. At eight years of age, physical

The patient elected to continue the pregnancy to term. PHA stimulated cord blood examination revealed a height on the 25th centile, a weight on the 50th and placental fibroblast cultures from a placental biopsy revealed mosician for the centile and a head circumference below the 3rd centile. Her hair was marker chromosome (cord blood 47,XX,+mar[15]/46,XX[5]; placenta 47,XX,+mar sparse in the nuchal, parietal and temporal regions. Fine and sparse eyelashes, mild bilateral epicanthic folds, fifth finger clinodactyly [20]/46,XX,[5J). Maternal blood contamination in the cord blood was ruled out by and hyperextensible fingers were present. The teeth were slightly isoelectric focusing. The presence of normal cells in postnatal cytogenetic analysis crowded and carious. They psychological assessment revealed mild was to apparent ring attributed mitotic instability of the marker and possible mental retardation. The high-resolution blood karyotype showed a selections against the marker. Distamycin/DAPI and fluorescent in situ partial deletion of chromosome 6q:46,XX,de(6)(q22.2q23.l). hybridization (FISH) studies with alpha satellite DNA probes for chromosomes Deletions of the long arm of chromosome 6 are rare. Only two other cases were reported in the literature with interstitial 13/21, 14/22, X, and Y chromosomes were negative. A FISH probe generated from breakpoints close to the region deleted in our patient. Two other the microdissected marker chromosome demonstrated that marker chromosome thf reports describe larger deletions encompassing the same region. was derived from the centromeric and pericentromeric regions of chromosomes 2 Considering the presence of dysmorphic findings, mental retardation and and 6. alopecia associated with a smell chromosomal deletion, the possibility of a contiguous gene syndrome is entertained.

2094 2095 A maternal balanced translocation leading to partial duplication of 4q Translocation of Satellites and Partial Centromere from Chromosome 13 to the and partial deletion of lip in a son: Cytogenetic and FISH studies Distal Long Arm of Chromosome 4 with Probable Loss of D. using band-specific painting probes generated by chromosome 4q35 ala', S. Verahese2. B. K. Burton'2. J. D. iaz-Nazario. D. J. McCorguodalel. and M. microdissection. Z Chen- T.A_ Grebe. X.Y_ Guan. M. Notohamiprodjo- M. McCorguodale'Z. Michael Reese Hospital' and University P.J. Nutting, J.F. Stone, J M. TrentAA.A. Sandberg, Genzyme Genetics of Illinoisa, Chicago, IL. and Southwest Biomedical Research Institute. Scottsdale. AZ: A 7 year old female with a history of serious University of Arizona-Phoenix. Integrated Genetics Program: Maricopa behavioural problems and alopecia was seen. Family history was significant for schizophrenia in Medical Center. Phoenix. AZ; Laboratory of Cancer Genetics. National both parents. Physical examination revealed an obese child exhibiting Center for Human Genome Research, National Institutes of Health, mild facial dysmorphism with epicanthal folds, a depressed Bethesda. MD. nose bridge, and a short neck. The scalp had bald areas A 9-month-old boy with pre- and postnatal growth retardation, and the eyelashes were sparse. The skin had a hypo- microcephaly. plagiocephaly and several minor dysmorphic features had pigmented area on the anterior chest wall. She had bilateral flat feet and an abnormal EEG. the initial karyotype: 46.XY.der(1)t(1:?)(p36.1:?). Further analysis Chromosome analysis showed an extra dark band and revealed that the der(D) was derived from an unfavorable segregation satellites on the distal long arm of one #4 chromosome and of a maternal complex chromosome rearrangement, i.e.. the absence of satellites on one chromosome 13. C-banding 46.XX.der(l)t(l:?)(p36.1:?).der(4)t(4:?)(q?:?). Whole chromosome revealed that the extra band on chromosome 4 was C- fluorescence in situ hybridization (FISH) and chromosome positive, but C-dot staining showed that this centromere was inactive. FISH analysis, using a #13 paint, showed the microdissection were used to clarify the maternal karyotype as: normal #13 chromosome with signals along its entire length 46 .XX t (1 :4) (4qter-+4q33 :: lp36 .13-+lqter;4pter-+4q33 :: lp36.13+l-pter). except for the centromere and short arm, to which this inv(4)(4pter-.4q31.3: :lp36.33-lp36.13: :4q33-n4q31.3: :lp36.33-.lpter). paint does not hybridize. The other #13 chromosome Therefore, the karyotype of the boy was subsequently revised to hybridized similarly, but had a shorter region that did not Clinical comparison of the show signals. With a #4 paint, the extra material on one 46.XY.der(D)t(1:4)(p36.13;q33). patient's of the #4 chromosomes did not give a signal, and no other clinical findings showed similarities to individuals with partial signals were seen on any other chromosomes except for the del(lp) or dup(4q). To our knowledge. thei above cytogenetic normal chromosome 4. Thus an unbalanced translocation abnormalities have not been described previously. between chromosomes 4 and 13 appears to be present such that some of band 4q35 is not present. A360 Published Abstracts: Cytogenetics (cont.) 2096 2097 Complicated structural chromosome aberration involving chromosomes 1, 4 High frequency of structural chromosome abnormalities in a population and 16 revealed by fluorescence in situ hybridization. T. Johannesson', referred for karyotyping through pediatric neurology: a retrospective study. S. Ehlers2 and J. Wahistrorn'. Departments of 'Clinical Genetics, East Hospital and Lidia Kasprzak and Patrice Eydoux. Cytogenetics and Prenatal Diagnosis, Montreal 2Child and Adolescent Psychiatry, Annedals Clinics, University of Gothenburg, Children's Itospital, McGill University, Montreal, Quebec, Canada. Gothenburg, Sweden. W%'e reviewed pediatric hospital records of 510 patients referred for chromosomal We here report on a seven-year-old normally built boy with slow psychomotoric de- analysis by four pediatric neurologists. The purpose of this project was to establish the velopment and mental retardation. According to DMS-III-R (APA 1987) the boy meets frequency of chromosomal abnormalities in a defined patient population. Patients were criteria for Attention-Deficient Hyperactivity Disorder and Oppositional Deficient Disor- selected according to the specific referring diagnosis, and classified into clinical der. G-banding analysis showed an apparently balanced reciprocal translocation, involv- subcategories, including developmental delay, mental retardation, autism, and ing the telomeric regions of chromosomes lp and 4q. Fluorescence in site hybridization hyperactivity, with or without dysmorphic features. This study included individuals (FISH) was subsequently employed to further characterize the aberration. We used assessed within a period of five years (1990-1995). Chromosome analyses were all Whole Chromosome Probes (WCP) for chromosome 4 and 16, and the midi satellite performed in one center. probe, DIZ2, for the telomere region of chromosome 1. WCP4 gave an additional signal There were significantly more males assessed in this population (69.8%). All of these to the terminal region of one of the chromosomes lp. rhe probe for the terminal region patients were subject to various diagnostic procedures, ansI karyotyping revealed an of chromosome 1, midi satellite probe DIZ2, gave unexpectedly an additional signal on underlying chromosomal anomaly as a causative factor in 8.2% of the cases. Among the telomere of one of the chromosomes 16p and WCP16 gave an additional signal on these, the most prevalent were structural chromosomal rearrangements. This study did the q-terminal of one of the chromosomes 4. Therefore, the karyotype of the patient in- not show any excess of males in the group of patients with a cytogenetic abnormality. volved an apparently balanced complex translocation 46,xy,(1;16;4)(p36.1;pl3.1;q34)de %Ve believe that a number of chromosome structural abnormalities remain undetected novo. The involvement of chromosome 16 was impossible to discover with classical cy- in patients with developmental delay, mental retardation, autism, hyperactivity with or togenetic methods only. Although the translocation appear balanced, we can not rule without dysmorphism, and we conclude that differential diagnosis in pediatric neurology out the possibility that the abnormal phenotype is caused by unbalances in the kary- should include karyotyping at a level of resolution of 400-600 bands. otype with cryptic undetected translocations or small deletions, or mutations in the translocation-breakpoints. Our data emphasize the importance of using FISH for detec- tion of small chromosomal aberrations. We also describe new critical regions in which genes of potential interest for neuropsychiatric diseases may be located.

2098 2099 A Yq- case which dissects the Yq heterochromatin region: possible Ringe hrososane 11 [46,XX,r(11)(pt5q25)] in a Bedouin child with dysmorphism and mental retardation: A case report and existence of growth control genes at Q-banding negative heterochromatin brief review. D.S.Krishna Murthy*, S.A.Al-Awadi, T.I.Farag, segment of Yq. K.Kim I. K.Matsushima l. T.Ohno 1. KWakui 2 Y.Fukushimna 2 S.A.Gouda, M.A.Abdel-Rasool. Medical Genetics Center, Maternity Hospital, KUWAIT. and H.Ohashi Saitama Children's Medical Centcr, lwatsuki, 2 Shinshu Univ. A 1-year-2-months old female child with tiny facial features, Sch. Med.,Matsumoto, Japan. marked delayed psychomotor development and dysmorphic features, The patient, a male, waLs born at 39 wceks of gestation to healthy and unrelated including - microcephaly, frontal bossing, short neck, hypertelorism, antimongoloid slant, low set ears, thin long eye parents. His birth wcight was 1,44) g (-4.4 SD) and length 4) cm (-5.3 SD). G- brows, carp-like mouth, bilateral clinodactyly and hypotonia was banded chromosome analysis revealed a 46.X,+mar karyotype. The marker investigated. Chromosome analysis using G-banding and C-banding showed a ring chromosome 11, 46,XX,r(11)(p15q255. Monosomy for chromoso)mc as submetacenritc and about 1/2 si7e of G-group chromosomes. By chromosome 11, double rings and other characteristic ring products FISH willh Y specitic ccntromcric repetitive pn)be (DYZ3), the marker wvas identified were also observed. Karyotypes of the parents were normal. The frequency of ring chromosome in human newborn and antenatal as originating from Y and wc diagnosed that the marker was Yq-. Q-banding revealed diagnosis surveys has been estimated to be 1/50,000 to 1/52,279. no Iluoresceni helerxchromatin region on the Yq, while other cytogenctic (C-banding Including the present caseonly 12 cases of r(11) have been reported. In this population this is the first case of r(11) among 10,800 and DA/DAPI staining) and FISH (DYZ3 and satellite Ill as probes) analyses detected a cases karyotyped. No precise phenotype in r(11) cases has been tin) but obvious hetrochromatin segment at its end. At 14 months his length was 71 cm delineated. The diversity of clinical manifestations in r(11) cases could be due to in vitro mosaicism and/or instability of the ring (-2.3 SD) and his development was normal. His father had a normal Y chromosome. chromosome. Other possibilities could be break points involved in The break of the Yq in the patient seems to dissect the htcrochrnmatin region of Yq the formation of ring. A wide spectrum of abnormalities has been reported in llq deletions. Molecular studies to understand the clearl) into Q-blanding positive and negative segments. Furthermore, the patient mayt genomic imprinting involving 11p15.5, where IGF2, H19 and other suggest that putatis e growth control genes exist at this Q-banding negative imprinting genes are located will be useful to establish the parental origin and to delineate the clinical manifestations more heterochromalin segment of Yq. precisely. A brief review of r(11) cases reported in the literature will be presented . *P.O.BOX 31167, Sulaibikat, 90802, KUWAIT.

2100 2101 Abnormal Phenotype in a Patient with an Apparently Pseudomosaicism for i(21) (qlO) in an Initial Amniotic Fluid Balanced 14:21 Translocation. D. J. McCorquodale'. A. Culture Proven to be True Mosaicism after Birth. M. M. Newlin. B. K. Burton'. L. Kaufman7. R. EgelV. and M. M. McCorcuodale".2, D. J. McCorguodale'. B. K. Burtonl'TJ. McCorauodalel .2 Michael Reese Hospital' and University of Santolaya. L. Cohen and Y. Bairg. Michael Reese Hospital' Illinois , Chicago, IL. and University of Illinois , Chicago, IL. An 8-month old female, born to a 22-year-old gravida 9 In our laboratory we experience about 1% pseudomosaicism para 3 mother, presented with microcephaly, hypertonia of each year. Recently, amniotic fluid chromosomal analysis the limbs, axial hypotonia, hyperreflexia, developmental of 36 cells revealed 4 cells with a Robertsonian 21,21 delay, and convergent strabismus with right horizontal and translocation in addition to a normal 21 chromosome. One rotatory nystagmus. abnormal cell was confined to 1 of 4 independently Standard karyotyping at the 550-band level revealed established in situ cultures. The other three abnormal 46 chromosomes with an XX sex chromosome complement. cells were found in a 5th culture initiated as a backup However, one #14 chromosome contained extra material, and from pooled media from the 4 initial cultures. only one normal #21 chromosome was present. A bisatellited FISH analysis of 1,267 interphase cells using a 21- marker chromosome, about the same size as a #21 specific probe was inconclusive. Whereas 8.7% of the cells chromosome, was also present. FISH studies showed that the displayed 3 signals, an average of 4-5% of interphase cells marker chromosome gave a centromeric signal with a #14 from controls showed 3 signals, and this % varies depending alpha-satellite probe, and displayed hybridization signals upon the percentage of cells undergoing DNA synthesis at in its mid-region with a #14 paint. The extra material on the time of cell harvest. one of the #14 chromosomes gave hybridization signals with The mother's chromosomes were normal, the father was a #21 paint. unavailable, and they elected to have a repeat amnio. The presence of abnormal features in this patient, Sixteen colonies were analyzed from 6 independently despite an apparently balanced de novo translocation, is established cultures. Two colonies (5 cells) from one usually explained in terms of the break points involved in culture showed the same 21,21 translocation. the translocation. If these break points interrupt Although each tap alone would be interpreted as important genes, phenotypic abnormalities can emerge. This pseudomosaicism, the two together verified true mosaicism. case emphasizes once again that breakpoints appear crucial The family elected to continue the pregnancy and the baby's in assessing the risk for abnormalities in patients with blood revealed i(21)(qlO) in 5 out of 20 cells. FISH balanced translocations. analysis revealed an i(21) (qlO) in abhut 1/4 of the patient's white blood cells. Published Abstracts: Cytogenetics (cont.) A361 2102 2103 Prenatal diagnosis of "crossover" event by routine cytogenetics T. McGuire', Parental origin of 8p deletion chromosomes. T. Nelsont, M. Schertzerl, C. Stein2, R. Artal', 0. Geifman-lloltzman'. 'Department of OB/GYN, Division of F.J. DilIl, S. Woodt, C. Danesino2 and 0. Zuffardi2. I University of British Columbia, Maternal-Fetal Medicine and Genetics;2Department of Pathology, SUNY Health Vancouver, BC, Canada and 2University of Pavia, Pavia, Italy. Science Center, Syracuse, NY. A sex-bias for the origin of chromosomal rearrangements has been shown for certain Meiosis is the reduction division resulting in haploid cells required for sexual regions of the genotne. For example, terminal deletions involving 4p, 5p, and lIq have reproduction. One of the important events during meiosis is the crossing-over or been shown to be preferentially of paternal origin. A sex-bias is not seen for most exchange of homologous segments between the paired chromosomes. If deletion, interstitial deletions. Informative inversion duplication 8p chromosomes are always of duplication or loss of genetic material occurs during this process, an abnormal or maternal origin (Floridia et al. AJIIG 58:785-795, 1996). These chromosomes have nonviable fetus would be expected. been postulated to arise from an intermediate dicentric by breakage. The reciprocal A 36-year-old, G2Pl001 underwent amniocentesis at 16 weeks' gestation due to product would give rise to an 8p terminal deletion, which necessarily would be of advanced maternal age. Karyotype analysis revealed an apparent paracentric inversion maternal origin. (PAI) of the long arm of one chromosome 9 46,XX inv(9)(q32q34.2).Peripheral blood We are studying six cases of deletion of the short arm of chromosome 8 to determine karyotypes from both parents revealed the mother to be 46,XX and the father to carry the parent of origin. Genotyping using polymorphic STS markers located on 8p was the same PAI as the fetus. Howeverthe inverted chromosome 9 in the fetus was carried out tising standard techniques. In one family the deletion is interstitial and is of structurally different, with a large heterochromatic region proximal to the paracentric paternal origin. In two families the deletion is terminal and of maternal origin. This is inversion, compared to the father's inverted chromosome 9 with a significantly smaller consistent with the hypothesis of a maternal bias for terminal deletions of 8p. proximal heterochromatic region. The father's second chromosome 9 had the standard band pattern, but a large heterochromatic region was present. The rearrangement was interpreted as a homologous recombination event in paternal meiosis I. The fetal recombinant 9 is expected to be balanced, but as in any occurrence of structural chrotnosomal variation, an unequal recombination could result in phenotypic abnormalities. The patient was counseled and level II ultrasound examinations were performed at 23 and 31 weeks, which showed no anomalies. The patient delivered a healthy female with a normal exam, except for a sacral dimple and abdominal hemangioma. In counseling the pregnant patient with a fetal karyotype which includes a structural variation resulting from ineiotic recombination, a low but unknown risk for abnormalities should be included. We recommend follow-up with ultrasound examinations and reassurance if gross anomalies are not identiied.

2104 2105 Primary Pulmonary Hypertension an Infant with monosomy of tersinal 4q and trisomy of Characterization of a de novo tandem 12p duplication by molecular the terminal segment of 16q. TIal 1. Adatia 1. Teshima. R.Babult D ChitayL The Hospital for Sick ChildrenUniversity of Toronto, Toronto, Ontario, Canada. cytogenetic methods. Primary Pulmonary hypertension (PPH) is a progressive fatal disease of an unknown etiology. Venkat R. Pulijaal'. Pulavarthi H. Rao2. Ali A. Javed'. Harold M. Nitowsky'. Some of the cases are familial with an autosomal dominant mode of inheritance with incomplete Richard Kleiaer' and Jaclyn Roberts'. 'Albert Einstein College of Medicine penetrance and possible genetic anticipation. However, the gene for PPH has not been mapped. and Montefiore Medical Center, Bronx, NY; WMemorial Sloan Kettering Tenminal deletion of 4q results in a well described syndrome with characteristic facial features, cleft palate, hand and cardiac abnormalities and developmental delay. Cases with duplication of the Cancer Center, NY and 'Community Health Plan, Poughkeepsie, NY. terminal segment of 16q have also been reported with characteristic facial features, cardiac and A variety of molecular methods can now be utilized as adjunct to gastrointestinal abnormalities. To the bestof our knowledge, PPH has never been reported with traditional cytogenetic methods for the elucidation of complex chromosome terminal deletion of 4q or with duplication of the terminal segment of 16q. aberrations. We report the case of a 3 year old boy with developmental delay We report a4-month-old male infant with PPH and sprasystemic pulmonary pressures. On physical examination he had normal weight, length and OFC. He had a 'boxy' head with a Nat and dysmorphic features, who on a G-banded study of peripheral blood occiput, high forehead and both frontal and parietal bossing giving the skull a triangular shape. He chromosomes revealed extra material on the short arm of a 12 chromosome. had a large anterior fontanelle, depressed nasal bridge, rightchoanal stenosis, bilateral epicanthic Although this finding was consistent with a 12p duplication, the possible folds, hypoplastic malar areas and small, cupped, overfolded ears. Cardiac examination showed a association with a reciprocal or other structural rearrangement could not be patent formmen ovale and trivial patent ductus arteriosus. There was mild hepaosplenomegaly and excluded. The dysmorphic features including a prominent normal male genitalia, There were dimples on both elbows with clinodactyly of the 4th and 5th forehead, fingers bilaterally. He had a normal neurological examination and mild developmental delay. hypertelorism, epicanthus, broad nasal root, long philtrum, macrognathia and Chromosome analysis showed 46,XY,der(4)t(4;l6Xq34;q22)paL brachydactyly of the 5S fingers were in accord with previously reported cases of To the best of our knowledge, there have been no previously reported patients with terminal the 12p trisomy syndrome. Parental chromosome studies showed no deletion of 4q and trisomy of the terminal segment of t6q. Moreover, PPH has not been reported abnormalities indicating a de novo origin of the rearrangement. Comparative with eitherof these chromosome abnormalities. The association of an unbalanced chromosome genomic hybridization (CGH) revealed a ratio profile indicating gain rearrangement with a single gene disorder has led in many cases to the mapping and identification of material of the responsible gene. The finding in our patient raises the possibility that a major gene(s) on 12p. This was confirmed by a painting probe of 12p indicating a tandem responsible for PPH reside in the terminal segments of 4q or t6q duplication of 12p. The child's karyotype is 46, XY dup(12)(p12p13). Further studies are in progress to delineate the boundaries of the duplication and possible gene dose effects for the genetic loci that maps to this region. This may aid in genotype-phenotype correlation for this uncommon chromosome aberration.

2106 2107 A maternally-derived recombinant inversion X chromosome in Syndrormic dextrocardia associated with a de novo partial phenotypically abnormal twin brothers. J.H. Ray. XT. Zhou, M.M. deletion of the short arm of chromosome 12 In an Arab girl. Sanz, R. Perrone, B.A. Pletcher' and M.G. Bialer. North Shore University MASab iSAA.d.M,Redjtp, KtMis ahn Wuilry.y4,$abtyJLFEtaJ Hospital, Manhasset, NY and U. of Medicine and Dentistry of NJ, Newark. KuwaltMedical Genetics Centre, Matemity Hospital, Kutai. Two mo old twin boys born at 34 wk gestation presented with mild Several reports have documented the association between dextrocardia/situs micrognathia, high-arched palate, minor ear anomalies, and symmetric Inverstt and chromosomal rearrangements, Including the association vtth growth retardation of prenatal onset and by 12 mo had developed chromosome 4 periceestric inversion, chromosome 7/X translocatlon, chromosome dolichocephaly, significant hypotonia, bilateral 5th finger clinodactyly and 1/11 translocation, chromosome 12/13 translocation, trisomy 13, partial trisomy developmental delay. Cytogenetic analysis revealed that the X chromosome 10, mosaic Turner, and tetrasomy 12p. Herein, we report an Arab girl with in both boys had extra chromatin on its short arm. Family studies revealed syndromic dextrocardia associated with a de novo partial deletion of the short arm that the der(X) was inherited from the mother and maternal grandmother. of chromosome 12 (46;XXdel(12)(p)(p12-pter). In addition to the dextroposition of the heart, the girl had generalsed hypotonia, high forehead, mild FISH using an X chromosome painting probe revealed that the der(X) hypotetorism, chromosome consisted entirely pmotmuding eyes, lw#-set, posteriorly rotated ears, dom-turned mouth, high arched of X chromatin. Probe DXS1140, localized to palate, mlcrognathia, imited ebow movements, smai hands and feet and Xp22.3, hybridized to the short arm of the der(X), indicating the breakpoint in brachydactyly of toes. Skeletal survey showed generaised melaphyseal dysplasia. Xp to be in distal Xp22.3. Probe Tel Xq/Yq, localized to Xq28-qter and Yql2- Rearrangements Invoicng the short arm of chromosome 12 have been found to qter, not only hybridized to Xqter and Yqter as expected, but also to Xpter. be non-randomiy associated witai a variety of haematological malignancies. a Thus the der(X) chromosome is recombinant X [rec(X)J chromosome with Isochromosome 12p Is considered to he a diagnostic/prognostlc marker for germ duplication of Xq that must have arisen from an X chromosome with a cell tumours. Chromosonte 12p rearrangements have alo been reported pericentric inversion [inv(X)(p22.3q27)J at least 4 generations ago. The associating several mitliple congenital anomales. rec(X) chromosome would have been selectively inactivated in the females, This is apparently lt. first report associating syndromic dextroposition of the exerting no phenotypic effect; however, when the rec(X) chromosome was heartwilt chromosome 12pdeletinn, a tlnting that wwol suggest the presence of at one gene on the arm of chromosome inherited by the twin boys, adverse effects were observed. Since neither boy least short 12 that represents part of the has ichthyosis, the Xp22.3 deletion must be distal to the steroid sulfatase heirarchy that determines biased left/right body asymmetry. gene. Individuals with deletions in this region of Xp have been described with mild mental retardation and short stature, but these boys have much more severe problems. At almost 5 yr of age they have no speech, are not walking and are severely growth retarded. This may be due to the partial disomy of Xq27-qter. Unlike Klinefelter syndrome, in which the extra X chromosome is inactivated, these boys have 2 active Xq27-qter regions with severe phenotypic consequences. A362 Published Abstracts: Cytogenetics (cont.) 2108 2109 Experience with marker chromosome identification. Z en, M. Familial cryptic translocation identified with FISH. S.L. Sell. S.L. Wenger. A.H. Urbach. Notohamiprodjo A.A. Sandberg .. F. Stone, Genzyme Genetics and and M.W. Steele. Children's Hospital of Pittsburgh, PA. Southwest Biomedical Research Institute. Scottsdale. AZ. A 17 year old white female with microcephaly, profound mental retardation, and dysmorphology had several similarly affected first and second degree relatives all related The following points are the summary of our current approaches through the proband's normal father. Peripheral blood chromosome analysis performed on the for the identification of marker chromosomes: proband both at the time of birth and again at age 17 years failed to demonstrate the suspected 1. The sequence of procedures is the same for prenatal and unbalanced chromosome translocation. Further analysis at 17 years using FISH technology postnatal cases. Both special staining/FISH and prenatal studies demonstrated a cryptic translocation: 46,XX.der(l )t(l ;3)(q43;q28). proceed simultaneously and expeditiously for prenatal cases. whereas The proband was a 4593 g product of a FTVD to a 27 year old GI white female who for postnatal cases the order of testing is determined by the nature presented neomuatally with hyperbilirubinemia, hypoglycemia, and hypotonia. At three days of of each case. life, the head circumference was 35 cm (75th percentile), weight 4.18 kg (above 95th 2. The following flow chart shows the appropriate procedures: percentile), and length 51 cm (75th percentile). Physical examination revealed a large Satellited: FISH(13/14/15/21/22) dysmorphic baby with increased subcutaneous fat. TORCH studies were normal. Markers(G/Q/C/NOR bandings)*' By 8 months, a seizure disorder had appeared. Head circumference had fallen below the 3rd percentile while other growth parameters remained within the normal range. The patient was ',Non-Satellited: FISH(1/16/18/20/X/Y) lost to follow-up until the age of 17 years at which time she was admitted for hypernatremic 3. 16 prenatal and 13 postnataT cases with a marker were dehydration and acute renal failure secondary to GJ fluid loss. Physical examination at this evaluated. The results showed that in prenatal cases the markers time revealed height, weight, and OFC below the 3rd percentile; upward slanting palpebral were chromosomes 15(5). 13 or 21(2). Y(2). X(1), 18(1) and 14 or fissures; bilateral epicanthal folds; nystagmus; anteverted nares; cupid's bow upper lip with 22(1) in origin, and in postnatal cases chromosomes X(3). 18(3). short philtrum; repaired cleft palate; micrognathia; low-set ears with fleshy, creased lobes and 20(2). 15(2). Y(1) and 13 or 21(1) in origin, indicating an a right preauricular sinus; poor tooth enamel; and hypertrophied gums. Feet were slightly apparently different distribution pattern of prenatal and postnatal rocker-bottom with polysyndactyly of left great toe, bilateral slight syndactyly of second and markers. Of note is that only 1 postnatal and 4 prenatal markers third toes, and overlapping right fourth toe. Breast development was minimal, and axillary and could not be identified this pubic hair were sparse. A deep sacral dimple was present. by approach. Cryptic translocations may account for a significant proportion of severe mental retardation. In such cases, when the family history is suggestive of a cryptic translocation, we suggest FISH analysis be utilized attempting to identify the rearrangement so appropriate genetic counseling can be offered.

2110 2111 The role of chromosomal mosaicism in women with multiple miscarriage Demonstration of subtle sex chromosome abnormalities through the use of and IVF failure. C.G.Sinclairl, A.l.ass2, I.Sku112, C.Rodeck' and J.l).A.Delhanty1. FISH. C. F. Stephenson, J. L. Wilson. C. D. Croft, D. J. Ghabrial- J. A. Rayburnm D. 4 Huluman Genetics Group, Galton Laboratory, University College London, Stephenson Sommerfeldt. D. Stegeman- C. S. Berger C. H. Hashimoto. OncoGenetics, Phoenix, Way, London NWI 211E, 2IVF Unit, HIammersmith Hospital, Du Cane Ro.ad, London and 3 Department of Obstetrics and Gynaecology, University College London, Chenies Arizona. Mews, London, U.K.. Two patients were referred for chromosome analysis. Patient A is a 34 year old The presence of small numbers of 45, X cells in lymphocyte cultures of women with phenotypic female who was referred for an initial clinical diagnosis of amenorrhea. multiple miscarriage has been regarded as important by some workers and dismissed by Patient B is a 10 year old phenotypic female who was referred for an initial clinical others. In order to answer this question an in depth study is being carried out. This diagnosis of Turner's Syndrome. Cytogenetic analysis of Patient A demonstrated that study uses a combination of G-banding and fluorescent in situ hybridisation (FIShI) 100% of blood lymphocytes appeared to have a 46, XY male chromosome with a chromosome X specific alphoid probe to screen 500 nuclei frosn miscarriage Fluorescence in-situ showed that the Y chromosome patients and age matched fertile controls. Both groups had previously been reported as complement. hybridization karyotypically normal after routine diagnostic cytogenetics. To date 12% of the consisted of both X and Y material. Cytogenetic analysis of Patient B showed that patients screened have been identified as 45, X mosaics bult none of the controls. 10% of blood lymphocytes had 45 chromosomes due to loss of either an X or Y Additionally couples with more than three failed attempts at in vitro fertilisation chromosome. The remaining 90% of cells demonstrated 46 chromosomes with a (IVF) have been karyotyped. Three women have exhibited low level mosaicism, derivative chromosome. Fluorescence in-situ hybridization demonstrated that the for chromosomes 18 and X A control of involving trisomy 11, respectively. group derivative chromosome consisted of both X and Y material. The significance of FISH women who have had previous IVF success and are under going another cycle are also being karyotyped. in elucidating structural sex chromosome abnormalities and thus enabling a more .When ever possible any embryos produced which are spare are being spread and analysed complete diagnosis is illustrated in this poster. by FISH with o-satellite probes for chromosomes 1, 9, 18, X and Y. These results will also be presented.

2112 2113 TURNER SYNDROME RELATED WITH A VERY SMALL CYTOGENETIC FINDINGS IN PLACENTAL CULTURES OF A STRUCTURAL NON-MOSAIC CHROMOSOMAL ANOMALY P Tavaresa, GESTATION WITH IUGR A. Tavares*, C Palmares, P Rendeiro, C Barbedo*-, * ** P RendeixoL, D Pignatelli2, C PalmarestL. - Centro Genetica Clinica, Porto, Portugal J Braga", P Tavares*. Centro Genitica Clinica, Porto, Portugal Servigo 2 - Endocrinol gia, Faculdade Medicina do Porto Obstetricia Hospital Santo Ant6nio, Porto, Portugal Turner syndrome has usually been described in patients with absence of an X chromo- We report a case of a pregnant woman referred for amniocentesis at 25 weeks ges- some or, in a reduced frequency, associated with various sex chromosome aberrations. tation after detection of severe intrauterine growth retardation (IUGR) with no ap- A few of the latter include microdeletions of the short or the long arm of the X chro- parent cause. The amniocytes karyotype revealed a normal chromosomal constitution: mosome, corresponding to varying degrees of phenotypic characteristics. We detected a 46,XX. A premature normal female was delivered at 32 weeks of gestation, and the very small deletion of the short arm of X del(X)(p21.3p22.1) in a 16 year old patient placenta was investigated for detection of confined placental mosaicism associated with referred with primary amenorhea and with typical Turner syndrome features, excepting abnormal intrauterine growth. Placental chromosome analysis showed three cell lines: pterigium colli and low neck implantation hairline. We discuss this case and compare 46,XX(52)/46,XX,-10,+mar(2)/47,XX,+mar(2). We agree with other previous reports with others reported in the literature. that confined placental mosaicism is strongly associated with IUGR, or may be its cause, in pregnancies with no other apparent explanation with normal chromosome fetuses. Published Abstracts: Cytogenetics (cont.) A363 2114 2115 Inverted duplication of 9p21-p23 with terminal 9p deletion. Trisomy 47,X.X, + 17 at amiiioccnlcsis, with normal 46,XX at birth 1. Wagstaff and M. Hemann. Children's Hospital and Harvard Medical School, S. Wijlaja, Nutrwansyab. S.l latlin. L. rratmnu. MadavanA. Indriastut Boston, MA. Chilren and Matcruity "Ilarapan Kila" hospital Jakarta, Indonesia We report a female patient with phenotypic characteristics of the trisomy 9p syndrome, including developmental delay, microcephaly, downslanting A 35-year-old Indonesian primigravida had amniocentesis in the 16 gestational week: 20 ml fluid was palpebral fissures, prominent nasal bridge, prominent maxdlla, small otlained tinder tihrasouind guidance and placed in two sterile tubes, centrifuged gently for 10 minutes mandible, prominent ears, and fifth finger clinodactyly, in whom and the pellets were cultsured in two separate sterile flasks with Chang medium. Alter two weeks, the chromosome analysis showed an abnormality of distal 9p. Whole- ctlttures were harvested for G-banding. From each flask. 20 cells were counted and 4 to 6 cells were chromosome painting showed that the abnormal chromosome was composed karyo-t1pcd. All ctts were 47.XX,+17. Both parents had normal 46XX, and.46AXY. Ultrasound entirely of chromosome 9 material. FISh with YACs containing loci from detected no atnalomnic abnorttiality so pregnatcy was continued, with tiltrasound every four sweks, 9p2l(D9S171) and 9p23 (D9S157 and TYRPI) showed duplication, and two- vhictt shoswed normal grossth until the baby was born spontaneous lransvaginally alter onset of color FISH showed the duplication to be inverted. FISH with YACs from distal labor in the 28th gestational sseek 9p containing D9S1858 and D9S178 showed deletion on the abnormal T1he infant looked normal. APGAR scores were 4-6, birth weight was 950 grams. Cord blood had chromosome 9. Therefore, this abnormal chromosome 9p shows inverted tortual 46,XX(no nosaicism was seen).The baby needed intubation and positive pressure vettilation duplication of loci from 9p21 and 9p23, with deletion of more distal loci. for 7 days. Enteral feeding svas wvell tolerated from the 5th day. The baby was discharged from Similar rearrangements have been reported for chromosome 8p, and hospital alter 50 days, weighing 2080 grams At 5h months, she weigled 5600 grams, had normal probably arise from recombination between repeated and inversely physical examination, was rolling over, and had passed the BERA hearing test. Skin biopsy was oriented sequences on homologous chromosomes, with formation of a requested tolook for any residual cells +-17, but the parents declined. dicentric chromosome that is broken during meiosis. The phenotype of this Trisomty 17 had never bett reported in amniocentesis, stillbirths, or livc-birttts, only in aborted tissue female patient closely resembles that of the trisomy 9p syndrome and does frott 5 to 6 week gestations. Our findings could have been dtte to mislabeling or laboratory error, bit not include dysmorphic features characteristic of the 9p- syndrome; this is sitice October 1992, onty 3 of 107 amniocentesis failed to grow, due to bacterial conlanmittafion. Tiso flasks are routinely ctllttred from each amniotic sample, from each flask 20 cellson five to six slides consistent with our previous observation of the 9p- syndrome in a patient arc counted and 4 to 6cells have G banding Aflerabnormal ultrasotind, one fetus with a with a small translocation-associated deletion flanked by D9S162 and D9S286, single umbilical artcry had trisomy 18, and a second swith proximal to the region deleted in this patient (Wagstaff and Hemann, Am. J. holopros-encephaly had trisotny 13. These siere confirmed and aborted. Normal amniotic results were alter Hum. Genet. confirmed by physical findings 56:302-309, 1995). birst. The felts mtighit Itave had mosaicism, with gradual loss of the atetiploid cell litte. That trisomy 17 occurs, is indicated by finding tiniparental disotty for 17, vith esiploidy. Six differeut reciprocal translocations were found in 35 coupIes with isto or more first rimeistcr abortions.Thtis relalively fcquicnt finditg of Iranslocations miighit du to sttail satiple size.

2116 An unusual Y chromosome variant in a male with hypospadius and lack of spermatogenesis. C-Y.Yul, A. Sobrino', A. Bryant, P. Yen2 and D. Warburton'3. Presbyterian Hospital in the City of New York 2ttarbor-UCLA Medical Center, TorranceCA 3Columbia University, New York, NY. Karyotype analysis on peripheral blood was performed on a 35 year old male be- cause of severe hypospadius, absence of one testis, and anomalies of the urinary tract. Testicular biopsy revealed tubules without spermatogenesis. The G-banded karyotype revealed a Y with apparently normal morphology, but an unusual handing pattern, and Q-banding showed absence of brilliantly fluorescing material on distal Yq. FISHI analysis using probe DYZ3 for Y alphasatellite showed two hybridization sites equidis- tant from each end of the chromosome. A primary constriction was seen at only one of these positions. X-specific painting demonstrated an apparent pseudoautosomal re- gion at each end of the chromosome. The interpretation of the karyotype was thus 46,X,idic(Y)(qi l.2).ish(DYZ3++,DYZI-,wcpX+). Cosmid probes mapping to proximal Yq were used to further define the duplicated region. Three cosmids mapping to region 51 in proximal Yq all showed two signals, as did a probe mapping to region 6C. Two very closely opposed signals were seen for a probe mapping to region 6E, indicating that the break in Yq was just distal to this region. PCR analysis is being carried out to

define the breakpoint more precisely. A 45,X karyotype was seen in 60% of cells from a gonadal biopsy and 80% of cells from a skin biopsy. Review of the lymphocyte culture revealed a 45,X line in low frequency. Thus while the deletion of material from proximal Yq may be contributing to the phenotype in this patient, the presence of 45,X mosaicism complicates this interpretation.

Published Abstracts: Differentiation, Development and Morphogenesis 2117 2118 A NEW APPROACH TO PATHOGENESIS AND ETIOLOGY OF TRISOMY 21 The human GARS-AIRS-GART gene encodes two proteins which are differentially expressed during normal human brain development and Svetlana ARBUZOVA temporally overexpressed during Down Syndrome brain development. Regional Medico-Genetic Center of Donetsk G. Brodsky, J. Bleskan, M. Cox and D. Patterson. Eleanor Roosevelt Institute, Denver, CO. Pathogenesis of trisomy 21 is still unknown. Moreover, the mechanism whereby Monoclosal and polyclonal antibodies have been generated against the glycinamide ri- the supernumerary chromosome 21 contributes to the pathology of syndrome remains bonucleotide synthetase (CARS), aminoimidazole ribonucleotide synthetase (AIRS) and elusive. glycinamide ribonutcleotide formyltransferase ((ART) enzytsatic domains of the hrtman We compared the clinical features of Down's syndrome with the effects of low trifunctiotal GARS-AIRS-GART protein. Western blot analysis of human tissue and dosage radiation damages including diagnostic and prenatal irradiation and the cultured cell protein extracts demonstrates that a 50 kDa OARS protein is expressed descendants of irradiated animals. along with the trifunctional 110 kDa GARS-AIRS-GART protein in human cells. Ex- The striking analogy in developmental malformations, mental retardation, pression studies and sequence analysis of the GARS-AIRS-GART genomic locus indicate increased to to and premature aging, susceptibility leukemias, high sensitivity infections that both proteins are encoded by the GARS-AIRS-GART gene. Western blot analysis mortality was obtained. of normal human brain tissue samples demonstrates that both the CARS and GARS- of of families with 21 the increased The results examination I5 trisorny gave AIRS-GART proteins are primarily in pre- and human brain of free enhanced in antioxidant expressed early postnatal level radicals, lipoperoxidation, disbalancing system tissue and that the relative levels of expression of the two proteins are developmentally and oxydative phosphorilation in donors of extra chromosome and in patients with of extracts from brain tissue indicates 21. regulated. Analysis protein Down Syndrome that trisomy both proteins are temporally overexpressed during early Down Syndrome brain develop- The increased level of free radicals, different grade and duration of disbalancing ment. in donors of extra chromosome explains the age-dependent frequency of trisomy 21; recurrence risk in the liveborn and abortuses; connection to other aneuploidies; degenerative, autoimmune, oncological disorders, etc. in some families and pedigrees. The effect of CuZnSOD gene in trisomy 21 with the initially increased level of superoxidic radicals promotes the formation of more active radicals and peroxides. The obtained results of dinico-biochemical investigation and the detected point mutations in mitochondrial DNA in a donor of extrachromosome 21 not described earlier illustrate the role of mitochondrial genome in etiology and pathogenesis of aneuploidies. A364 Published Abstracts: Differentiation, Development and Morphogenesis (cont.) 2119 2120 Maternal trace elements, Vitamine B12, Vitamine A, folic acid and fetal Epoxide hydrolase gene polymorphism in a family with phenytoin-induced oral malformations. B. Dott. Y. Alembik. C. Koehl. C. Stoll. Centre Hospitalo- clefting. J.K. Hartsfield. Jr.'12 and J.C. Casada'. Indiana University Schools ofDentistry' Universitaire, Strasbourg, France. and Medicine2, Indianapolis, Indiana. The demonstrated teratogenicity of a maternal zinc deficit in rats has led to a Approximately 19,000 children are born to epileptic women in the United States burgeoning interest in zinc and other trace elements as fetal teratogens in human. Levels annually. The most efficacious anticonvulsant therapy for these women is the daily use of zinc, copper, manganese, magnesium, Colic acid, vitamineB12 and vitamineA were ofphenytoin (Dilantin@), a medication that increases by four times the incidence oforal evaluated at the beginning of pregnancy in the plasma of pregnant women who clefting in humans. There is evidence of a familial predisposition to phenytoin-induced delivered later malformed newborn. Fetal chromosomal anomalies and recognizable non congenital anomalies. We tested the hypothesis that a relatively low microsomal chromosomal syndromes were excluded. The results were compared to controls women epoxide hydrolase (mEH) enzyme activity decreases the capability ofthe embryo and/or who delivered normal babies. Hundred fifty two mothers had malformed children. The mother to detoxicate the bioactive arene oxide metabolite ofphenytoin, resulting in an more frequent congenital malformations were congenital heart diseases (72 cases increased risk oforal clefting. DNA was extracted from peripheral blood samples taken including 24 VSD), musculo-skeletal malformations (21 cases), urogenital from five sisters and their parents. An enzyme assay ofmEH activity in their malformations (23 cases), neural tube defects (12 cases) and labial cleft (14 cases). The lymphocytes was performed utilizing HPLC. The mother had taken phenytoin and following results were obtained: (NS = Non Significant) phenobarbital throughout the pregnancies ofall five women. Four ofthe five sisters Mothers ofmalformed Controls were born with a cleft lip i cleft palate. There was no previous family history of Zn(pmol/l) 13.16 ± 1.61 12.99± 2.68 NS clefting, nor have there been any clefts in the children born to the sisters. DNA Cu(pmol/l) 23.32 ± 6.17 22.98 ± 5.31 NS amplification and sequencing by PCR methods were used to ascertain two candidate Mg(mmol/l) 0.81 ± 0.07 0.78 ± 0.08 NS polymorphisms, an mEH allele (YI 13-+H in exon 3) associated with approximately 40%/a Mn(nmol/l) 35.08 ± 3.92 36.70 ± 3.85 NS less mEH activity compared to the most common human allele, and an mEH allele Folate(tig/l) 3.86± 1.51 3.76± 1.19 NS (R338-+C in exon 7) associated with 60-65% less mEH activity, and increased heat Vit.BI2(psg/l) 0.41 ± 0.15 0.44 ± 0.20 NS lability ofthe enzyme activity in mice. All ofthe sisters are heterozygous for Vit.A(mg/l) 0.45±0.12 0.49 ±0.12 NS Y1 13/H1i13. The father is homozygous for Y113, while their mother, who is Thus vitamine profiles do not form a suitable means for identifying women at risk for homozygous for HI 13, has a mean mEH specific activity that is 45% less than that of having a child with neural tube defects. This endorses the hypothesis that the beneficial the father. The mEH activity ofthe sisters vary between their parent's values. Further effect of folic acid supplementation on the prevention of neural tube defects is possibly research into this and other mElI polymorphisms as genetic niarker(s) for low mEH at least partly due to the fact that it overrides a relative folic acid shortage caused by a activity associated with susceptibility to phenytoin-associated anomalies will allow metabolic disorder. identification of families and individuals at greater risk and increase our knowledge of factors that predispose to oral clefting.

2121 2122 Focal dermal hypoplasia (Goltz syndrome): a report of two unusual Development of a mouse model system for the analysis of hereditary cases. N. Lemieux, D. Marcoux. C. McCuaia. J. Milot and L. L. Olianv. multiple exostosis. X. Lin and D. E. Wells. Dept. of Biology and Institute for H6pital Sainte-Justine, Universite de Montreal, P. Quebec, Canada. Molecular Biology, University of Ilouston, Houston TX. Focal dermal hypoplasia (FDH) is characterized clinically by multiple Hereditary multiple exostosis is an autosomal dominant trait in humans. It is charac- ectomesodermal defects such as the classical numerous cutaneous atrophic terized by short stature and presence of cartilage-capped exostoses at multiple locations macules arranged in a linear fashion associated with a wide range of other throughout the skeletal system. Three distinct genetic loci can give rise to the EXT skin, nail, hair, eye, dental and skeletal abnormalities. This disorder shows phenotype. We have previously cloned one of these loci, EXTI, and have identified mu- markedly variable expressivity. It is thought to represent an X-linked tations in EXT patients. To stutdy the detail biological functions of EXTI, we plan to (Xp22.31) dominant condition lethal in non-mosaic males (no case of use mice as a model system. As the first step in this process, a mouse embryo cDNA mother-to-son transmission reported), although genetic heterogeneity is library was screened, using a human EXTI-specific probe. Positive clones were identi- hypothesized. We reviewed 9 patients with FDH seen at our institution. fled and sequenced. The mouse nucleotide sequence shows 94% overall similarity with Two patients were unusual: 1. a 35 y.o. female with FDH shows a fragile site the human EXTt cDNA. The average level of similarity holds at 94% for the large 5' at Xp22.31, with breaks in 10.6% of cells, and del(X)(p22.31pter) in 4% of untranslated region, however decreases dramatically in the 33' untranslated region to only cells. This site is known to be fragile in a small percentage of the population, 80%. This could indicate a conserved function for the 5' UTR. Like the human gene, yet it's association with FDH is puzzling; it may be merely coincidental, but the mouse gene encodes a 746 amino acid protein. It shares 99% amino acid sequence a fragile site at Xp22.31 could conceivably lead to a loss of function of that identity with its human counterpart. Northern blot analysis showed the expression of locus. 2. A karyotypically normal 2 year old boy shows the classical mouse EXTI in all tissues examined, including heart, brain, spleen, lung, liver, skeletal stigmata of FDH, including cutaneous atrophy, papillomata, and cutaneous muscle, kidney and testis. However, the expression levels in liver and kidney were much syndactyly; his parents were phenotypically normal, but his mother's first higher than other tissues. We are curently doing in situ hybridization and developmental cousin has a lobster claw deformity of her hand and a cleft palate, arguing in northern blot analysis to more closely analyze the tissue distribution and developmental favor of genetic heterogeneity and of non X-linked transmission of a onset of EXTI expression. Finally, a gene targeting vector has been constructed for syndrome phenotypically indistinguishable from FDH. establishment of EXTI mouse model. Targeted mutagenesis of mouse ES cells has been Although an abnormal collagen is suspected, the pathogenesis of FDH is performed and "knock out" EXTI mice are now under construction. unknown; it must be secondary to an abnormality that functionally impairs embryogenesis. The multiple maldevelopments affecting different organs support this thesis, as these anomalies are unlikely to solely result from a poor collagenous tensile strength. We are starting to understand the role of collagens and of the other components of the extracellular matrix in inducing cell differentiation and embryologic migration, and to grasp the complexity of this cascade of events. Genetics and molecular embryology will be discussed as they relate to the markedly variable expressivity of FDH.

2123 2124 Mutational Analysis of the SOX9 gene in South African XX True X chromosome inactivation patterns in blood and buccal Hermaphrodites. A. N. Magewu and M. Ramsay. Department of Human Genetics, mucosa in singletons and MZ twin pairs. J Montciro+. C School of Pathology, South African Institute for Medical Research and University of Derom#. R Vlietinck#. R Derom# and PK Gregersen+. +Div. Biology and the Witwatersrand, Johannesburg, South Africa. Human Genetics, North Shore Univ. Hospital, Manhasset NY/ #Center XX True hermaphrodites (XXTHs) have both ovarian and testicular tissue. of Human Gcnetics, Katholicke Universiteit Leuven, Leuven, Belgium. The South African XXTHs analyzed to date do not have Y chromosome sequences We have reported that X inactivation patterns may differ sub- nor do they have the SRY gene suggesting the involvement of autosomal or X linked stantially in blood among members of an MZ twin these dif- genes in the ofthe condition. of pair; etiology Identification causative genes and mutations ferences are seen only in dichorionic and not in mono- would provide further information about the sex determination pathway. The (DC) twins, autosomal sex reversal gene SOX9 is mutated in XY females with campomelic chorionic (MC) pairs (Trejo et al., Molec. MAd 1:62, 1994). The usual dysplasia. In these XY females, loss of function mutations have been described in explanation for this finding is that MC MZ twins share a common SOX9. Our prediction was that if SOX9 is involved in XX true hermaphroditism, the blood supply during intrauterine life. However, an alternative hypo- mutations would not be loss offunction but would result in a gain of fuznction or some thesis is that MC MZ twins may undergo embryo splitting after X inac- level of constitutive expression to give rise to testicular tissue despite the absence of tivation has occurred, and thus may have highly correlated patterns the SRY gene. To test this hypothesis, mutational analysis of SOX9 in XXTHs was of X inactivation in other tissues. Using a Hpa 11 PCR assay of the undertaken. The SOX9 gene is on chromosome 7q, has 3 exons, codes for an mRNA androgen receptor locus, a preliminary ,study of X inactivation in the of4.3 kb and a predicted protein of 509 amino acids. buccal mucosa of 10 MZ twin pairs (7DC, 3MC) revealed major The mutation E148X in exon 2, and the G -s A mutation at the intron 2 splice differences in 4 twin pairs, all of whom were dichorionic. Thus, the site, which are present in XY females have not been found in the 10 XXTH analyzed similarity of X inactivation in MC MZ twin pairs may in some cases be to date. No PCR amplification was found in XXTH individuals with all 3 overlapping explained by a twinning event which occurs later than X inactivation. primer sets spanning exon 3, although this region amplified well in the normal controls. We have also found that blood (mesoderm) and buccal mucosa This will be further investigated by Southern blot analysis. Conformational shifts have (cctoderrm) of 3/14 (21%) singleton females exhibited different been identified by SSCP analysis and the nature of the mutations causing these shifts patterns of X inactivation. Thus, similar to reports in the mouse, X will provide insight as to the role of SOX9 in the etiology of XX true hermaphroditism. inactivation may occur independently in ectodermal and mesodermal precursor cells. Examination of endodermal tlissues, in which X inactivation appears to occur later (in mice) than ectoderm and mesoderm, may be helpful for timing the twinning event in MCtwins. Published Abstracts: Differentiation, Development and Morphogenesis (cont.) A365 2125 2126 Chromosomes 22q1I microdeletion In sibs with negative FISH and discordant phenotype. R. Introduction of Yeast Artificial into embryonic stem cells via homologous recombination. R. M. Tucker and D. T. Sutphejn, 0. T Mueller B. Zeffren. D. Clayton. K Hinds-Frey. S. Komfeld and B.G. Kousseff University of South Florida and All Children's Hospital, St. Petersburg, Fl. Burke. Dept. of Human Genetics, University of Michigan, Ann Arbor, Ml Phenotypes known to be associated with microdeletions in the 22q11 region The classical 'knock-out" experiment involves construction of a vector include DiGeorge syndrome, velocardiofacial syndrome/schizophrenia and isolated containing the gene of insert with a neor cassette inserted into the coding cardiac defects. A predominance of maternally derived microdeletions has been region of the gene. The neo cassette acts as both the marker to select for reported. We report two brothers who have discordant phenotypes compatible with integration and the introduced mutation. Homology length of the introduced 22q1 1 microdeletion. DNA affects recombination efficiency: A five-fold increase in length of The proband is a six-year-old boy who was born at 32 weeks gestation due to homology demonstrates a 100-fold increase in rate of recombination. The chorioamnionitis. Tetralogy of Fallot was diagnosed in the neonatal period. Chest disadvantage of the "knock-out' technique is that it only allows for complete radiographs showed small thymus. Candidal sepsis required antibiotic therapy. disruption of the gene leading to a null allele. Attempts at generating subtle Immunologic evaluation showed T-cell abnormalities, low thymulin and zinc mutations in mammalian cells have been successful, but these are either too deficiency. Left cryptorchidism and right inguinal hernia were surgically repaired. inefficient or technically difficult to be of routine use. Frequent respiratory infections and otitis media began in infancy. TOF was Yeast Artificial Chromosomes (YACs) have been used as vehicles for surgically repaired at age 2 1/2 years. FISH analysis employing the D22S75 probe introducing entire genes and their regulatory regions into mammalian cells. in the 22q11 region revealed no deletion. The proband's brother is a 12-year-old with A number of human- and mouse-derived YACs have been transferred to a history of agitated behavior beginning at 9 years of age. Beginning at age 11 various cell lines, including mouse embryonic stem (ES) cells, and years, he has described hearing "voices" that tell him to "do bad things." He is expression of the genes located upon them has been demonstrated. Germ currently under psychiatric care and receiving psychotropic medication. The boys' line transmission of randomly integrated YAC sequences has been mother has mild psychiatric symptoms. observed in mice derived from the these ES cells. Many of the introduced Molecular analysis of the 22q11 microdeletion syndrome region employing eight genes show correct expression patterns implying that all or many of the microsatellite markers (Mueller et al., this volume) revealed that both siblings have a regulatory sequences necessary are contained in the transferred constructs. maternally derived deletion of only one microsatellite, D22S944. Data from flanking As yet, experiments involving homologous recombination of YACs microsatellites show that the siblings have apparently inherited the same matemal into ES cells have not been described. Due to their large size, YACs have alleles and different paternal alleles. Possible explanations for these data include: an advantage over small knockout constructs. The markers necessary for (1) affect of paternal alleles which differ between the two boys account for the selection of recombinants can be placed at a distance from the targeted observed phenotypic differences or that (2) there are differences in the extent of region. As a consequence, they should have little tp no effect upon the deletions between the two boys which were not apparent using the available normal regulatory elements of a given gene. The proposed experimental markers. Ongoing analysis may further define the extent of microdeletions in the strategy will allow for introduction of subtle mutations within a region and two boys and other family members. may lead to fine structure/function studies of a particular gene.

Published Abstracts: Gene Structure and Function 2127 2128 Alu Elements and Hormone Response Elements. C. Aleman, P.L. Deininger. Different gene expressions for the soluble ABH and Lewis antigens in mucinous LSU Medical Center, Stanley S. Scott Cancer Center, New Orleans, La. 70112 and serous secretions. B. D. Bosch and F. J. Grundbacher. University of Illinois Short Interspersed Repeated Elements (SLNEs) are DNA repetitive elements found in College ofMedicine, Peoria, IL. a wide variety of species ranging from fish to humans. The reason for their existence The ABH, Le' and Leb antigens have been important in blood transfusion and organ and the mechanism of amplification are still a mystery. It is hypothesized that only a few elements can undergo the proposed RNA-mediated retroposition. A goal is to transplantation because these antigens occur on red cells as well as on tissue cells. elucidate the mechanism of amplification. Alu is found in all primates and the original Mucinous secretions from secretor individuals of blood types A, B and 0 are known Alu elements are estimated to be 65 million years old. During this time, the few elements to be rich in A, B and H antigens, respectively. In contrast, concentration of the A that can amplify have mutated to generate Alu subfamilies. Alu is the only SINE found antigen has been found to be negligible in the secretions of the parotid gland, a nearly in humans at 500,000 copies per haploid genome. Alu amplification has disrupted a pure serous gland. The Le' (also called stage-specific embryonic antigen) and the Ley of genes to disease. no function has been attributed...until number leading However, antigens have recently become of interest in cancer immunology because of enhanced now. The Alu field has taken a new and exciting turn. Recently, we have shown that hormone response elements (HRE)- estrogen response elements (ERE) and retinoic acid expression of these antigens in various carcinomas. The detection of an unusual Ley response elements (RARE)- located within Alu elements in the promoter regions of antigen prompted the quantitative study ofthese soluble antigens. the human breast cancer gene (BRCA-1) and the human keratin gene (K-18) increase Saliva was membrane-filtered, concentrated and fractionated by gel filtration the transcription of reporter genes in the presence of the corresponding hormone. We chromatography. Antigen levels in these fractions were determined by an enzyme- hypothesize that the HREs enhance the transcription of Alu elements as well as adjacent linked immunosorbent assay (ELISA) utilizing monoclonal antibodies. Fractionated genes. An increase in of Alu elements may aid in its This transcription amplification. whole saliva from a group 0, Lewis-positive, secretor individual has two quantitative suggests that selection due to function aided in the amplification of Alu elements giving rise to the current subfamilies. We have transfected mouse L929 cells with vectors peaks of high-molecular weight mucins (>2,000 and 669 kDaltons) containing the H, containing an Alu element with an ERE. The level of Alu transcription was determined Lea, Leb and Le7 antigens. However, the Le' antigen alone displays a third, its highest in the presence and absence of estrogen. peak, in the protein molecular range of 29-66 kDaltons. ELISA tests of individual salivary gland secretions reveal that the levels of H, Lea and Leb are very low in parotid saliva. In contrast, the Le' levels are mtuch higher in parotid than in whole saliva, and the parotid Ley is of low molecular weight. The results reveal that, depending on genotype, the mucinous salivary glands can secrete any of these blood group substances, but in secretors only the parotid saliva contains the low molecular weight Le7 antigen in high concentration. These findings point to a different gene expression between the mucinous and serous salivary glands.

2129 2130 Isolation of human testis-specific genes by differential display RT-PCR. Molecular evolution of FMR2 gene promoter. D Curlev. N Zhong. C Dobkin. WT J.M.Cameron, J.Wolfe and K.'raylor. The Galton Laboratory, University College in London, UK. (Intro. by: S.Povey) Brown. New York State Institute for Basic Research Developmental Disabilities. We have been using differential display on a range of human male tissues (liver, lung, Staten Island, NY muscle, white blood cells, placenta, brain and testis) and have so far isolated 90 cDNAs which appear to be specifically expressed in the testis. The fragile site FRAXE, located at Xq27-8, is approximately 600 kb distal to FRAXA 25 of the cl)NAs have been sequenced and compared with Genbank, cDNA, EST and but to FRAXF. The gene for the FRAXE was cloned and SI'S databases. Of these, 2 thatch known testis-specific cDNAs: spl7, a zona pellucida proximal syndrome recently binding protein and a testis-specific cDNA clone (Genbank accession 1152864). 10 designated FMR2. A polymorphic triplet GCC repeat within FMR2 located at the 5' end match sequences expressed front other tissues not used in the differential display reaction and 13 appear to be novel sequences. is similar to the CGG repeat in the fragile X gene, FMR1. FRAXA. It is of FMR2. We are currently screening an adult testis cDNA library with the novel sequences to isolate full length cDNAs for further characterization. Northern blots containing RNA Expansion of the FRAXE GCC repeat is associated with hypermethylation ofthe FMR2 from male tissues are also being screened to confirm that the cDNAs are testis-specific. CpG island and results in a mild mental retardation syndrome. We have shown that the polymorphic GCC repeats of FRAXE vary among different populations. To understand the evolution of the gene, we analyzed the FRAXE GCC repeat region sequence in a variety of mammals. Using a pair of primers derived from human sequence we were able to amplify the FRAXE locus from 41 primate chromosomes, including 19 from Chimpanzee (repeat range 5-23, model 15); 12 from Gorilla (range of 11-21, model 18); 6 from Orangutan (range 14-24); and one from Marmoset (17 repeats). Our results showed that the primate GCC repeat region is highly conserved. Sequencing showed no imperfect interruptions were present. The conservation of this region located at 5', end ofthe FMR2 gene may indicate that it has a role for the gene regulation. A366 Published Abstracts: Gene Structure and Function (cont.) 2131 2132 Characterisation of the collagen Vial gene (COL6Al): the genomic structure of Isolation and chromosomal localization of novel the amino-terminal globular domain. 1'. Davis. D. irikka and A.M. Kessling. Academic Unit of Medical and Community Genetics (Imperial College School of Hmob3 sequence from human brain cDNA library. L.V. Medicine), Kennedy Galton Centre, Northwick Park and St Mark's NHS Trust, Harrow Dergunova, I.P. Vladychenskaya , Polukarova L.G.. IHA 3UJ, UK. (Intro. by: Prof. Peter Scambler) Lelikova G.P.. Limborska Institute of molecular The genes encoding the aI and a2 chains ofcollagen VI are located in band 21q22.3 S.A. in the telomeric region of chromosome 21. These genes are believed to have arisen genetics RAS, Moscow, Russia from a recent evolutionary duplication event. Genetic studies have indicated that they The novel cDNA clone Hmob3 was isolated from cDNA are candidate genes for the congenital heart disease phenotype associated with 45% of constructed from human medulla human trisomy 21. library, oblongata We have characterised a cosmid clone covering the amino terminal globular domain poly(A)* RNA. Hmob3 was expressed in different parts of of COL6AI plus the first two exons of the triple-helical domain. The structure of the human brain, in kidney and skeletal muscle but not in amino-terminal domain is found to be different from that in ('O.6A2 and consists of 8 other tissues. Northern blot analysis showed that muscle exons. In contrast with the triple-helical domain. the exon lengths and the intron-exon boundaries are not conserved between the two genes, with the exception of exon 6 in mRNA contains two transcripts more than 6 kb. In brain COL6AI which is conserved with exon 4 in COL6A2. Presumably these genes have only the shorter transcript was detected, in kidney - only undergone considerable divergence since the duplication event. The last cxon (21 bp in the longer one. Hmob3 sequence includes 1420 length) contains the cysteine residues which form the disulphide bridges that are required for the formation of mature collagen VI fibres. The equivalent residues in nucleotides with poly(A) tail at 3'end and polyadenilation COL6A2 are in an exon containing both globular and triple-helical domain sequences. signal AATAAA 20 bases upstream. There is no any Situated between the third and fourth exons is a microsatellite sequence which may be rather long open frame and we believe that this sequence involved in tile instability found in clones from this region and may explain the difficulty in cloning this gene. Exons I and 2 of the triple-helical domain are close to corresponds to the 3-untranslated mRNA region. Hmob3 the last exon ofthe globular domain and are conserved in length with those in COL6A2. sequence was mapped on the chromosome 10q21-q22. The elucidation of the genomic structure of COL6AI will aid in determining the role The comparison of Hmob3 with Embl/Genbank databases of the collagen VI genes in the development ofthe fetal heart. was revealed homology with some of the short (200 - 400 nucleotides) ESTs, that are not corresponded to any known genes.

2133 2134 Analysis of DNA methylation and in vivo protein-DNA interactions for the Tissue distribution and splicing patterns of axon Rl of dystrophin isoform Dp260. FMR1 gene. R. Drouinl, . Dallairel, M. Angersl, B. Lemieux3, E.W. Khandjian2, NM Duncan.' SM Rash.' VN D'Souza.2 G Daily,2 PN Ray 2 and DM Pillars'. Oregon and F. Rousseau2. Unite de Recherche en .en tique Humaine & Moleculaire, Pav. Child Health Research Center, Doernbecher Children's Hospital, Oregon Health Saint-FranSois d'Assise, CHUQ, Depts IPathology & 2Biochemistry. Universit6 Laval, Sciences University, Portland, 2Hospital for Sick Children, Toronto, ON, Canada. Quebec City, 3Dept. Pediatrics, CHIUS, Sherbrooke, (Quebec) Canada. Dystrophin is expressed in retina and is necessary for normal retinal function. An Fragile X syndrome which is the most common inherited form of mental retardation isoform of dystrophin, Dp260, has a novel exon 1 which lies within the intron results from the inactivation of the FMRI gene. Silencing of gene expression is due to 29 and 30 of full and is spliced in frame to axon the expansion of the trinucleotide CGG repeat sequence that is between exon length dystrophin abnormally methylated. 30. In mouse, this isoform is found primarily in the retina, and to a lesser extent in Regulation and inactivation mechanisms of FMR1 gene expression are poorly under- of 1 of the human of has stood. We investigated DNA methylation at the nucleotide level of resolution, the in heart and brain. Alternative splicing exon homolog Dp260 vivo protein-DNA interactions as well as chromatin structure for normal, premutated been shown. We refer to the first axon of Dp260 as axon R1 and have studied its and mutated males. LMPCR (ligation-mediated polymerase chain reaction), a power- tissue distribution and splicing pattern in mouse. Further work is in progress at the ful sensitive approach, was used as a method for high resolution genomic sequencing, protein level, including use of an antibody specific to Dp260, to clarify the methylation studies and in vivo footprint analysis. Regions of the promoter, the first importance of our findings. We isolated mRNA from C57BL/6J mouse tissues, and exon and intron of FMR1 were analyzed. In uinmethylated normal FMR1 gene from pe- performed RT-PCR with two primers sets: 1) A forward primer in the 5' untranslated ripheral blood lymphocytes, several footprints were identified and characterized. These region of axon R1 and a reverse primer in axon 31 which were used to detect the footprints were absent in peripheral blood lymphocytes and primary fibroblasts from tissue distribution and potential splicing patterns of axon R1, and 2) a forward fragile-X patients carrying a mutated and methylated FMRI gene. Also, we unexpect- primer in axon 29 and reverse primer in axon R1 were used to determine if axon R1 edly observed that EBV-immortalized normal and mutated lymphoblasts have lost these is present in any position except the first. RT- PCR with the first set of primers footprints indicating that regulation of FMR1 in normal cells from organisms might dif- showed a 385 bp product indicating R1 in the first axon position of mRNA in brain, fer from that in immortalized cultured cells. The results suggest that methylation might heart, intestine, liver, lung, muscle, spleen, stomach, testis, thymus, and to a greater play a role in the loss of protein binding to specific sequences. However, this might not extent, retina. With exception of retina, all tissues showed a larger PCR product of be the only factor causing the dissociation of the proteins from the DNA of the FMRI which included the 100 intron located between axon R1 and 30. Using gene leading to transcriptional silencing. The approach used in this project revealed 485 bp bp unsuspected footprints that were not picked up by standard transcription factor binding the second set of primers, we detected a product of 380 bp in which the majority site searches such as TFSEARCHI. of the intron between axon 29 and 30 was spliced out, with axon 29, 120 bp of intron, and exon Rl remaining. Conclusion: Using RT-PCR, we have shown that mRNA with exon R1 as the first exon is expressed in a variety of mouse tissues. Although we have shown that in the mouse exon Rl undergoes several patterns of RNA splicing, the pattern appears to differ from that shown in humans. Finally, we have shown that axon R1 may be used in other axon positions than the first, suggesting that axon R1 may be used in other isoforms of dystrophin.

2135 2136 A Proplolactone alters gene expression and could guarantee the safety of Characterisation of a mouse homologue of DSCI, a gene implicated in Down Inactivated vaccines produced on continous cell lines.D.M. Fathallah. K. syndrome. J.J. Fuentes, C. Pucharcos, M. Pritchard, M. Nadal, X. Estivill. Zerria. R. Barbouche & K.Dellad. Laboratory of Immunology, Molecular Genetics Group, Molecular Genetics Dept., IRO, Barcelona, Catalonia, Spain. Pasteur Institute, Tunis, Tunisia Down syndrome (OS), caused by trisomy of chromosome 21, is a major cause of mental Residual cellular DNA (rcDNA) present In vaccines produced on continous cell lines retardation and congenital heart defects. The most likely consequence of the presence of might carry potentialy oncogenk genetic material. Ilence a maximum level of 100 pgr three copies of chromosome 21 is the overexpression of several genes, some of which must of residual rcDNA per dose of vaccine Is strictly Imposed by WHO. is proplolactone be responsible for the various phenotypic features of DS. The contributions of individual (BPL) is an alkylating agent commonly used as viral inactivactor in vaccine genes to the phenotype can only be assessed by the isolation of each gene followed by preparations. Because RPL interacts with DNA and affects its structure, the estimation expression and functional analyses. We have reported a gene, DSC1, isolated from a of the amount of residual rcDNA in vaccine preparation can not be accurately achieved foetal brain cDNA library which maps to region 2tq22.t-q22.2. This gene is highly even by the most advanced techniques.To Investigate the effect of SPL on gene expressed in brain and heart and may therefore be of potential relevance in DS. We have expression, we used two expression systems. A procaryotic one, in which a plasmid isolated the mouse homologize (Dscl) using human USCI as a probe. FIStH analysis on l)NA (PGELM7) carrying the ampicillin resistance gene (expression of IL lactamase), was chromosome spreads from TsDn65, (the mouse partially trisomic for chromosome 16), treated with Increasing concentrations of IPL and Introduced In highly competent DIIS mapped Dscd to mouse chromosome 16, in the region syntesnic with human chromosome alpha E. colf-cells. The bacteria were then plated In presence of lOOpgr/ml of 21. Similar to the human gene, the mouse gene has three alternative first exons. The ampicillin. A negative correlation between the number of clones expressing the deduced ft peptide sequence shares 93.9% identity with the humaut protein. Current studies ampicillin gene and Increased SPL concentrations was reproducibly observed. are focused on determining the functional significance of the alternative exons. lactamase expression was abolished at a ILPI. concentration of 1/4000. For the eucaryotic system. the CDM8 expression plasmid vector carrying the rm cDNA under the control of a chimeric CMV-IIIV promotor was used. The plasmid DNA treated with Increasing concentration of ISPL was transfected Into Cos cell and expression of the CSS molecule at the cell surface monitored using an FITC labelled anti CD8 MAb. Expression was gradually diminished and abolished at a KPL concentration of 1/200. These findings show that SPL alters gene expression and suggest that If concentration of fPI. higher than 1/2000 could be used In vaccine preparations without loss of antigenicity, the potential oncogenic risk of rcDNA can be overcome. Furthermore , use of other cell lines such as the tumorogenic BHK21 could be considered to produce a more cost effective vaccines. Published Abstracts: Gene Structure and Function (cont.) A367 2137 2138 isolation and characterization of the reduced folate carrier Inthe murine The Fanconi Anemia Group C Gene Shares a Conserved system. K Greer', J. and R. H. Finnell'. Pledrahita'. B. Oetama',B. Barber'. Texas Domain With MECI. ((Kenneth F. Grossmann, James A. Hejna and A&M University, Dept. of Veterinary Anatomy and Public Health. Gollege Statfon,TX 77843'. Robb E. Moses)). Oregon Health Sciences University, Department of Defective dosure of the neural tube during embryogenesisresults In such serious Molecular and Medical Genetics, Portland, OR 97201. and common birth defects as spina bifida and anencephaly. Neural tube defects The human gene for Fanconi anemia complementation group C (NTDs) occurat an average rate of 1per 1000 live births with various ethnic groups (hFACC) shows little homology to other genes. The murine homologue having have shown a marked higher prevalence rates. Several research groups (mFACC) protein sequence corresponding to hFACC exon 8 has decreaseInthe occurrence and recurrence rates of NTDswith the administration of Intracellular folates, homology to a known protein from S. cerevrsiae, MECI/ESRI (52% supplemental folic acid prior to and throughout early pregnancy. identity, such as tetrahydrofolate and Its derivatives are possibly responsible for the decreased 70% positive). This homology was determined using a BEAUTY frequency of NTIs due to their role in facilitating single-carbon transfers In critical search - an enhanced version of the BLAST search protocol. MECI is a biochemical pathways leading to DNA synthesis. Alterations in the folate pathway member of a family of P13 kinases, one of which is the ataxia could compromise DNA synthesis,resulting in abnormal development. To this end,the alterations to that mechanism are telangiectasia gene product (ATM). ATM does not share an apparent mechanisms of folate internalization and possible exon 8 homology with mouse or that being investigated. The reduced folate carrier (RFC) plays an important role during the human FAC. We conclude hFACC, intemalization of folate into the cell. The delivery of 5-methyltetrahydrofolate from the mFACC, and MEC1 share a related domain. folate receptors to the cytoplasm of the cell Is believed to be mediated by the RFC. As a first step in defining the relationship between NTDs and aberrant folate Protein Alignment: Intemalization, we are isolating and characterizing the RFC gene In a murine system. with PCR-dedved RFC cDNA produced Primary screening of a murine genomic library MEC1 511 RPEAAGKSEXIRILHSNFL 526 eight positive clones, one of which was selected for subcloning. A positive clone of approximately 8Kb was subcloned into pBluescript and has subsequently been 1:11 1111:1 dividedinto smaller subclones. Utilizing each subclone independently and deriving mFAC 284 LPQAACQPAIFRIVHEMFR 300 sequencing primers from the known cDNA sequence of the gene, the entiregenomic 1I sequence isbeing elucidated. The gene contains at least four exons and is at least hFAC 2 83 LPQAACHPAIrRVVDEM-1111C81:111QIH11:1R 2991R 29 9 3.5Kb in length. Following the complete sequence determination and characterization of the RFC, more specific studies will beInitiated in order to determine any correlations between altered RFC, folate supplementation, and the occurrence of NTDs.

2139 2140 gene carried Mutagenesis of the 3-untranslated region of the CFTR Arginine 73 and histidine 125 in the sulfatase homology are essential for on a 610 kb YAC to facilitate the analysis of CFTR mRNA expression arylsulfatase A. Wuh-Liang Hwu, Chia-Cheng Wu, Yu-May Lee and Tso-Ren Wang. in human celis. K. A. Henning and M. A. Rosenfeld. National Center for Department of Medical Genetics, National a'iwan University Hospital, Taipei, Taiwan, Human Genome Research, National Institutes of Health, Bethesda, MD. ROC. In order to better understand the role of the cystic fibrosis Arylsuilfatase A is the enzyme responsible for a human disease called metachromatic transmembrane conductance regulator (CFTR) gene in the pathogenesis of leukodystrophy. In this study, mutants of arginine 73 and histidine 125 in the sulfatase cystic fibrosis (CF), we would like to study the expression of CFTR from homology (sequences conserved between many sulfatases) of arylsulfatase A were created its endogenous promoter following gene transfer to a variety of normal by site specific mutagenesis. Arylsulfatase A activity was increased by 30 folds by trans- human and CF cell lines. The CFTR gene spans 250 kb, making transfer fection of wild type cDNA in baby hamster kidney (BIlK) cells. However, the activities of arginine 73 mutants of the gene along with its own promoter and regulatory sequences (replaced by lysine or leucine) or histidine 125 mutants (replaced difficult using conventional vectors. In addition to the problem of by phenylalanine or glutamine) transfected cells were only slightly higher than the mock transfected cells. The mutant enzymes also showed delivery, the presence of endogenous human CFTR mRNA makes it a much decreased substrate affinities. Because both arginine and histidine has been shown time-consuming task to differentiate mRNA produced by the introduced by chemical modifications to ise essential for arylsulfatase A, the results of this paper point out specifically two residues gene. To circumvent problems, we have introduced two new these in the sulfatase homology involved in the active site of arylsulfatase A. restriction sites into the CFTR gene carried on a 610 kb YAC which has been retrofitted with a neo marker for selection in mammalian cells. This YAC has previously been shown to produce CFTR mRNA and functional protein in CHO cells (Mogayzel et al., unpublished). A three base pair insertion producing a Pstl site and a single base pair change producing an EcoRI site were introduced into the 3 -untranslated region of exon 24 of the CFTR gene by a combination of site-directed mutagenesis and two- step gene replacement in yeast. The three base pair insertion will allow specific amplification of DNA or mRNA from the CFTR gene carried on the YAC, and the single base change will lead to the presence of a new EcoRI site in the amplified products. We are currently transferring this YAC by spheroplast fusion to CF cell lines known to express the AFsus mutation. The modified YAC should allow any m}RNA produced to be definitively distinguished from any endogenous transcripts. We will test for the presence of CFTR mRNA carrying the introduced mutations and for functional correction of CF cellular phenotypes.

2141 2142 Human Renin Binding Protein: complete genomic structure and Tissue specificity in the processing of mitochondrial polycistronic RNA at functional assessment as derived by molecular association preliminary the tRNALe(UtI)l gene boundary. Y. Koga, S. Yano, A. Koga and II. Kato. studies A. Knol6l2, H. Schunkerti, D. Bauer2, M. Platzer2, G. Stein3, A. Rosenthal2. D)epartunent of Pediatrics and Child llealthF,Kirume University School of Medicine, 'University of Regensburg,2Institute of Molecular Biotechnology Jena,3University of Kiirume, Japan. Jena, Germany We described the novel unprocessed RNA species (RNA 19), corresponding to the 16S The role of renin binding protein (RnBP) in human pathophysiology, despite its bio. rRNA +tltNALe(SIUR) + ND I genes, in the cultured cells derived from MELAS patient chemical characterization, is yet unclear. Herein, a molecular genetic approach was having A3243G point mutation at tRNAeu(UUR) gene. In this study, we have analyzed employed to investigate if RnBP may have the potential to affect renin and prorenin the sleady state levels of unprocessedRNA intermediates at tRNALeulUUR) gene bound- levels, blood pressure. A large scale sequencing effort within the human Xq28 anid/or ary, in various human tissues from normal individluals and in biopsied skeletal muscle chromosomal region allowed us to determine the precise location and the full genomic se- from 4 unrelated MELAS patients having A3243G mutation, to know the biological qiience of the RnBP gene. To search for potential mutations, all 11 exons, intronic splice meaning of this novel mitochondrial transcripts. Normal tissues include skeletal muscle, sites, and the promotor region were sequenced in 20 patients with essential hypertension, brain, heart, kidney, liver, uterus, spleen, and cultured skin fibroblasts. Total RNA was and in four normotensive individuals. No mutations were found within these regions. isolated, electrophoresed, transferred, and hybridized with 16S rtNA, or tRNAL'uWUR), However, a single base exchange polymorphism was identified 61 basepairs up- (TV-C) or ND I probes. hybridized signal were quantitated by BAS 2000 image analyzer. The stream from the intron6/ exon7 boundary which did not affect mRNAsplicing. The RNA 19 is recognized in all human tissues including muscle, heart, brain, kidney, liver, allele as well as renin and prorenin levels, blood heart rate, and T61C status pressure, spleen and uterus, ranging 3.5%, 11.6%, 13.6%, 8.4%, 4.2%, 1.7% and 6.5% of their left ventricular mass were determined in 505 Caucasian individuals randomly selected total ND I signal, respectively. Using tRNA1-u(UUfl) as a probe, intermediate transcript from the general population. Out of 217 male individuals without medication, 35 were corresponding to tRNALe'(UUR) + ND I is a dominant molecules in heart muscle, liver hemizygote for the C-allele and were characterized by lower prorenin levels (196j15 kidney, however, transcript corresponding to 16S rRNA + tRNALe't(UuA) is dominant vs mU/I; p=0.05) and a higher renin/prorenin ratio vs 256±12 (10.7±1.5 7.7±0.3%; in cultured skin fibroblasts. Our date suggest that mitochondrial processing system at p=0.002), whereas circulating renin, blood pressure, heart rate, or left ventricular mass the tRNAL-(UUR) boundary may be influenced by tissue specific manner. Since their index were not associated with the C-allele. No significant association was observed in steady state levels of unprocessing intermediates in the tissues varied, it may contribute women, however, only one woman without medication was homozygos for the C-allele. the oxygen capacity of the tissues, which may play an important role of the mitochondrial Given that circulating renin levels are under tight control, the data are consistent with respiratory capacity in the tissues. the notion that the C-allele in men is associated with a lower reninsequestration in the kidney that is balanced partially by a negative feedback on its expression and, there- fore, reduction of constitutively secreted prorenin. In summary, although we obtained no evidence for RnBP gene mutations that might effect renin levels or blood pressure, we could associate a T/C polymorphism in intron 6 of theAnBP gene with reduced prorenin levels in males. A368 Published Abstracts: Gene Structure and Function (cont.) 2143 2144 rDNA clones isolated from chromosonme specific cosmid libraries Myotonic dystrophy: alternatively spliced forms of the DMPK messenger (i.e. chromosomes 13, 21 and 22). T.ILabelaI and H.Lchrach2. RNA. A-S. Lial, G. Gourdont, C. Duros , C. Junien'-2 and II. Radvanyi'-2. 1) I Institute of Protein Biochemistry and Enzymology (CNR), Napoli, Italy and INSERM U383, IHopital Necker, Universite Rene Descartes, Paris, France; 2) llopital 21mperial Cancer Research Fund, London, UK. Ambroise Pare, Boulogne, France. In the human genome, 300-400 rDNA genes are located in five clusters Myotonic dystrophy (DM) is an autosomal dominant multisystemic disorder on the short arms of the acroccntric chromosomes 13. 14, 15, 21 and 22. characterized by a highly variable clinical phenotype. The mutation has been identified rDNA genes are present in 44 kb tandem arrays, only 13 kb of which are as an unstable trinucleotide CTG repeat in the 3' untranslated region of the transcribed. The analysis of rDNA genes cloned in YAC vectors gave poor myotonin-protein kinase (DMPK) gene which sequence has been determined by several results because the YAC instability atid the chimeric YACs. In order to by- groups. Studies of RNA messengers level show that mRNAs are transcribed from both pass this problem, we screened the ICRF chromosome specific cosmid I)MPK alleles in healthy controls (Jansen et al 1992). In adult patients, a consistent libraries (chromosomes 13, 21 and 22) with a PCR amplified fragment from decrease of mRNA and protein level has been reported in muscle and cell lines, very the C region (Cx H) of rDNA cluster. We obtained 133 positive clones from likely through a post-transcriptional mechanism (Fu et al 1993, Krahe et al 1995, Wang chromosome 13 library, 75 clones from chromosome 21 and 109 clones from et al 1995). chromosome 22. Part or positive clones (twenty for each chromosome) were Since different spliced forms of DMPK mRNA have been described (Fu et al 1993, initially analysed by PCR and all tlese clones, except for cne, gave the Mahadevan et al 1993, Jansen et al 1992), we decided to study more precisely the tissue- expected amplified fragment (275 bp in the C region). A preliminary specific expression of these spliced forms during development in normal and affected analysis of all these clones by EcoRI digestion and hybridisation with samples. By PCR, we confirmed, in different human tissues samples the presence of the probes from all the canonical rDNA regions showed that almost all cosnmids eight spliced forms described by Fu (1993) and the existence of the first exon described gave positive hybridisation for at least three regions and indicated also a by Mahadevan (1993). In addition, preliminary data seeni to indicate that a new spliced possible polyntorphism its the different chromosomes. Anyway these form is located in the first exon described by Fu (1993). Differences in the expression of results require a more detailed analysis. the alternatively spliced forms are also studied in four tissues of a normal 2.5-weeks-old fetus and in the same tissues of a DM 16-weeks-old fetus. This analysis will be extended to different stages of development.

2145 2146 Lipoprotein lipase (LPL) gene influences plasma lipids in Indian newborns. Characterization of a pentamer repeat in the mouse prosaposin P.S. Low. N. Saha. J.S.H. Tay and S. Artlkumaran. National University of Singapore, Singapore. gene . L.Mintz . H.Levy-Sprecherl and M.Horowitz. Department of Cell Lipoprotein lipase (LPL) plays a critical role in the determination of the plasma Research and Immunology, Tel-Aviv University, Ramat-Aviv and lipid and lipoprotein profile. It is a rate-limiting enzyme in the hydrolysis of lDepartment of Biology, The Technion City, Haifa, Israel. triglyceride-rich lipoproteins and very low density lipoprotein which is further The degradation of sphingolipids with short hydrophilic lipolysed to low-density lipoproteins (LDL). Decreased levels of high-density head groups by lipoproteins (IHDL) and LDL have been observed in cases of LPL deficiency. lysosomal enzymes depends on sphingolipid activator proteins. Four such We investigated the influence of the LPL gene polymorphism (intron 6, PAull) activator proteins (saposins) are encoded by one gene, the prosaposin. The on cord plasma lipid in 110 male and 85 female Indian newborns. Plasma total, first 88 nucleotides of the mouse prosaposin cDNA have not been found in IIDL-, LDL-cholesterol, triglycerides (TG), apo A-I and apo B levels were estimated. The P+ allele was seen in 52.8%. The genotypes showed a significant the genome yet. In an effort to find these missing nucleotides, a 1213bp deviation from the Hardy Weinberg equilibrium (X2 = 4.57, p < 0.05) due to mouse genomic prosaposin fragment was sequenced. A pentamer (GGGCT) excess of P+ and P- homozygotes. Female newborns had significantly higher repeating thirteen times was found 500bp upstream the first known exon. plasma levels of HDL-cholesterol (p < 0.01) and apo A-I (p = 0.01) levels. The This birth weight, sex and birth order adjusted plasma levels of TG was significantly sequence is not polymorphic as demonstrated by amplification of DNA higher in P+P+ genotype compared to the P-P- genotype (0.44 ± 0.14 vs 0.38 samples from eight different mouse strains. There is binding of nuclear ± 0.12 mmol/L; p = 0.04). Individuals with P+ allele showed a trend towards protein(s) to a sense oligonucleotide containing this repeat. The single- lower level of LDL-cholesterol which failed to reach statistical significance (p = stranded motif, phosphorylated at its 5' end, binds to nuclear protein(s) 0.07). Pvull polytnorphism contributed 2.5% (R' x 100) of the sanple variance (P = 0.07) of plasma LDL-cholesterol levels. There was an apparent gene- isolated from several cell lines. The oligonucleotide repeat binds also dosage effect on LDL-cholesterol levels. P+P+ individuals also showed a trend nuclear proteins derived from human cells. Such a repeat exists in the towards lower HDL-cholesterol and apo A-I levels. human and mouse immunoglobulin heavy chain switch regions and the There is thus genotype specific influence of the LPL Pvull gene polymorphism on cord lipid profile of the Indian newborns studied. herpes simplex I inverted repeats (ITRs), suggesting a role in recombinatios events.

2147 2148 Structural similarities between promoter regions of the human DRPLA and Sequence homology between baboon and human HLA-DRB1 alleles. HD genes for triplet repeat disorders Yoshiro Nagao and Masao Yamada. Implication for xenotransplantation. L.M. Perelygina and R.D.Ilenkel. Department of Genetics, National Children's Medical Research Center Southwest Foundation for Biomedical Research, San Antonio, Texas. Japan Dentatorubral and pallidoluysian atrophy (DRPLA) and lluntington's disease Sequence homology between baboon and human HLA-DRBI alleles. Implication for (Hl)) are hereditary nurodegenerative disorders caused by expansion of triplet repeats xenotransplantation. L.M. Perelygina and R.D. Henkel. Southwest Foundation for in the respective gene. Although both genes are ubiquitously expressed, the anatomical Btiomsedical Research, San Antonio, Texas. Intro. by Rogers J. Genetic cross-matching area predominantly affected is distinctive to each of the disorders. We have determined of the major histocompatibility complex (MHC) class II antigens of bone marrow a nucleotide sequence at the 5' upstream region of the DRPL.A gene and defined the transplant donors and recipients is necessary to reduce graft versus host reactions and promoter region by CAT assays. The region is consisted of a high CC content and has prevent xenograft rejection. The ability to closely cross-match the HLA class II several possible SpI binding sites and an Ap-2 motif, which have also been detected antigens of baboon transplant donor and human recipient is an important step toward in the HD promoter region. Moreover, one GC-rich direct repeat exists in the DRPLA achieving the goal of using xenografts to treat human diseases. region while two similar repeats in the lID region. The promoter regions of the two genes Lymphocyte DNA from 23 unrelated baboons from the SFBR colony was used to have a quite similar structure. We are looking for possible stimulators and/or inhibitom determine the extent of allelic variation at the HLA-DRB1 homolog. Our approach in transcription and translation of the genes insing this experimental system in order to combined I'CR amplification of the second exon of this gene with human DRBI address the question of why the lesion is limited to a distinctive area of the brain. allele-specific primers, analysis of single-strand conformational polymorphism (SSCP) of PCR products and direct sequencing of PCR products representing different MHC alleles. We isolated a total of 11 different baboon DRBI alleles based on their distinctive electrophoretic pattern in SSCP gels. Inheritance of these sequences in baboon pedigrees showed Mendelian segregation. The nucleotide sequences of baboon alleles have been determined using direct sequencing of PCR products. Twenty five baboon specific amino acid substitution were found, but only seven of them occurred in peptide binding groove. A comparison between predicted baboon DRBI amino acid sequences and the human DRBI sequences demonstrates that there are several baboon alleles that closely resemble their human counterparts (tip to 98% homology) while several other alleles differ at many residues in the sequence (70-74% homology). Thus, some of the baboons are poor candidates for xenograft donation while other baboons may be much more compatible and may be recommended as possible transplant donors for human recipients. Published Abstracts: Gene Structure and Function (cont.) A369 2149 2150 Characterization of the Huntington disease gene promotor region in Identification of human LINE-I transcription factors by a human and rat. C. Holzmann. D. Petersohn. G. Thiel, J. T. Eoolen and 0. functional complementation scheme. F. Sheen and G. D. Riess. Molecular Human Genetics, Ruhr-University, Bochum 44780, and Swergold. Food and Drug Administration, Maryland, U.S.A. Institute of Genetics, University of Cologne, 50674 Cologne, Germany. The expression of human LINE-1 (L]Hs) elements is enhanced in a Huntington's disease (HD) is a neurodegenerative disorder caused by an expanded and unstable (CAG),37 repeat within a gene of unknown function. human-mouse hybrid cell line, M28, in which the only human Also, the pathobiochemical mechanism of the elongated glutamine stretch chromosome is isochromosome 12p. No expression is detected in the causing cell death exclusively in specific areas of the brain is unknown and progenitor mouse cell line, LMTK-. We hypothesize that one or more necessitates further investigation. We, therefore, cloned the promotor genes located on 12p enhance LIHs expression. A scheme to isolate regions of the rat (1.3 kb) and human (1.1 kb) huntingtin genes. Sequence these genes is presented. An expression library of M28 cDNA is analysis of the human, rat and mouse promotor reveals a highly conserved constructed in an Epstein Barr Virus (EBV)-based episomal vector, and region of 140 bp immediatly upstream the first ATG. The analyzed promotor is used to transfect an indicator-HeLa cell line which contains an regions in all three species do not contain TATA or CAAT elements but integrated LlHs-pac selectable marker, a puromycin-resistance gene harbour several potential binding sites for SPI, CRE and AP2 transcription (pac) driven by an Li Hs promoter. Cells transfected with cDNAs that factors. Furthermore, the rat sequence contains an uninterrupted stretch of enhance LlHs expression may become resistant to puromycin by turning 44 pyrimidines (position 1100) which is known to form H-DNA. To analyze on the the function of the promotor region of the rhd gene, a hybrid gene was expression of integrated LIHs-pac maker gene. We have screened constructed containing 1334 bp of the 5-flanking sequence from the rhd 2x 106 transfected HeLa cells, and have obtained >20 puromycin- gene fused to the CAT gene. This construct and five 5-deletion derivates resistant colonies. Hirt extracts from 3 colonies contain plasmids that were transfected into two neuronal and one non-neuronal cell lines. In confer puromycin resistance to the indicator cell line upon re- general, expression of the reporter gene showed good correlation with the transfection. We also delineate the construction of a new murine endogenous expression of rhd in neuronal cell lines. Deletion mutants of episomal expression vector. the rhd promotor revealed the presence of regulatory elements that cause downregulation of CAT expression in neuronal cell lines, whereas the effects are milder in non-neuronal cells. The rhd-GAT construct R-CAT7 extending from -331 to -15 showed the highest levels of expression. CAT activity rapidly decreases with shorter constructs, which may be a consequence of deletion of the transcription start site(s).

2151 2152 Expression of oxidative phosphorylation genes in renal tumors and tumoral MAGE-Xp2, a novel gene with strong homology to MAGE-Xp. L.-Q. Tai'. cell lines. H. Faure-Vignyt, A. Heddi2, S. Giraudl, and G. Stepien'. 'CNRS-UMR D. McCurdY2. J. Nguyen'. Z. Wang'. P. Concannon3. R. A. Gatti'. 5534, Universiti C. Bernard; 2Laboratoire de Biologie Appliquie, INSA, Villeurbanne, 'Department of Pathology and Laboratory Medicine, UCLA School of France. Medicine, Los Angeles, CA; 2Division of Rheumatology, Children's Hospital The expression of several mitochondrial and nuclear genes involved in ATP production Mason Research was examined in renal tumors of different types and in cell lines derived from renal tu- of Orange County, Orange, CA; 3Virginia Center, Seattle, mors. Striking differences were revealed among three types of tumors, their respective WA. controls, and the cultured renal cells. The levels of all mitochondrial transcripts were Immunoscreening of a HEp-2 (human epithelium) expression library lower in tumor biopsies and tumoral cell lines than in the normal cell types. More- recovered a transcript with strong homology to the MAGE-Xp gene on over, a higher transcript level of nuclear genes involved in oxidative phosphorylation was chromosome Xp2l. Sequencing of a 1.5 kb cDNA clone showed 77% observed in the oncocytomas and in the more malignant urothelial tumor. Different homology (77% identity) with MAGE-Xp and a 895 bp open reading frame transcript patterns were observed in each of the tumoral and transformed cell lines, ex- 1041 ORF for with the start codon for the MAGE-Xp2 tumors the tumoral or (versus bp MAGE-Xp) plaining the divergence in metabolism between the different and yet to be recovered. The novel gene also maps to Xp, as demonstrated by transformed cell lines. In particular, a high transcript level for the adenine nucleotide two translocator isoform 2 gene (ANT2), which is usually not expressed in differentiated cells, PCR screening of a panel of radiation hybrids, using primers against was observed in oncocytomas and malignant urothelial renal tumor. This phenomenon sequences of lowest homology between the two genes. Hybridization to was also observed in renal carcinoma cell lines and transformed cells. These data provide Northern blots showed a major transcript of 2.0 kb, very similar to the 1.9 and the first argument for the involvement of the ANT2 protein in glycolytic ATP uptake 1.8 kb transcripts of MAGE-Xp; because of the extensive homology with in cancer cell mitochondria, and suggest a possible ANT2 antisense strategy for cancer MAGE-Xp, this could reflect cross-hybridization. When the two sequences therapy. were aligned, we noted a 55 bp gap in MAGE-Xp2, deleting the 3' end of exon 3 and the 5' end of exon 4 of MAGE-Xp, and a region of lower homology in 3'UT region. Further efforts, using 5' RACE, are underway to recover and sequence the Send of the transcript.

2153 2154 A study of the carboxyl-globular domain of COL6A1 and its putative role in the Investigation of the Association of Myotonic Dystrophy Kinase (DMK) pathogenesis of congenital heart defects, in trisomy 21. D. Trikka. T. Davis. A.M. Kessling. with Itself and Other Proteins. J.D. Waring', T. Subissati', M.A. Narang2, Academic Unit of Medical and Community Genetics, Imperial College. School of Medicine, L.A. Sabourin2, R.D. Gietz3, R.G. Korneluk" 2. ISoulange Gauthier Karsh Molecular Kennedy Galton Centre, Northwick Park and St. Mark's NHS Trust, Harrow, HAI 3UJ, UK. Genetics Laboratory, Children's Hospital of Eastern Ontario Research Institute, (Intro. by: Professor P. Scambler) Ottawa, Canada, 2Department of Microbiology and Immunology, University of Ottawa, Although chromosome 21 has been napped extensively, some regions remain elusive; Ottawa, Canada, 3Department of Human Genetics, University of Manitoba, Winnipeg, notably the telomeric region is under-represented in the existing genomic libraries. Canada. Myotonic Dystrophy (DM) is a common dominant heritable disorder with varying The human gene for the al chain of collagen VI (COL6AI) is located within band and of It is characterized and is in fetal heart and has been in the of symptoms age omset. primarily by myotonia progressive 21q22.3. COL6AI expressed implicated pathogenesis muscle wasting. The disease is caused by the expansion of a (CTG),, trinucleotide congenital heart defects in trisomy 21. repeat located in the 3'-UTR of a serine-threonine kinase (DMK). The function of this We have screened a chromosome 21 bacteriophage library and isolated a clone positive gene remains unknown and the mechanism of disease is still obscure. Currently, the for COL6AI. localisation was confirmed by FISH. Sequence analysis of this clone has expansion is speculated to either down-regulate expression of transcripts, or to interfere revealed the exon-intron organisation of the carboxyl-globular domain of this gene. The with the transport and/or processing of transcripts, possibly in a trans-dominant structure of this domain differs from the corresponding domain of the recently evolutionary fashion. diverged COL6A2, also located within band 21q22.3. The first two COL6A) exons of the We are currently investigating the normal function and structure of DMK. The carboxyl-globular domain are a single exon in COL6A2, and show a high degree of homology. specification of self-association resides within the C-terminus of the protein. We have to localize the Exon 2 of COL6A1 is only 9bp in length and encodes two cysteine residues involved in created a number of deletion and site-directed mutants of DMK further to associate with an 150 kDa disulphide bonds between collagen VI monomers. Few of the other intron-exon boundaries are domain responsible. Also, DMK appears phosphoprotein in C2C12 myoblasts. We have therefore expressed the same mutants in myoblasts to conserved between the two genes. In addition, the COL6AI 3'UTR is encoded by one exon, investigate the possibility that this domain also specifies association with other unlike the COL6A2 3'UTR which is encoded by an exon also coding for the C2 subdomain. proteins. We have also detected phosphorylation of DMK in insect and COS cells. Previous studies have shown the presence of three RFLPs (Bamlll C, BanHl B and Work is currently underway to localize the site(s) of phosphorylation and the effect on Taql) within the CO1,6AI locus, which detect genetic variation associated With variability in kinase activity. the congenital heart defect phenotype. Determining the exon-intron structure will allow the One possible clue to the function of DMK comes from its relationship to the newly positioning of the Bantl il C RFLP site within the carboxyl-globular region and the identified ROKo gene (a rho-binding kinase). DMK may therefore be involved in the identification of associated functional mutations. This will facilitate studies on the pathogenesis regulation of cytoskeletal arrangement. We have identified two candidate interacting ofcongenital heart defects, in trisomy 21. genes using the yeast two-hybrid system, one of which has homology to a known rho-binding protein. We will report on our attempts to verify tie interaction of DMK with these proteins, and to localize the domains responsible. A370 Published Abstracts: Gene Structure and Function (cont.) 2155 2156 Expression of myotonic dystrophy protein kinase (DMPK). Genomic Structure, Sequence, and Mapping of Human FGF8, with C. L. Winchester1',, N. Carey2, K. J. Johnson'. tl)ivision of Molecular Genetics, IBLS, Exclusion of its Role in Craniosynostosis / Limb Defect Syndromes University of Glasgow, Scotland, UK, 2Charing Cross and Westminster Medical School, Koh-ichiro Yoshigral, Nancy J. Leysens', Jenny Chang3, Deeann Ward34, University of London, England, UK. Jeffrey C. Murray'2, and Maximilian Muenke34. Departments of Pediatrics, College Myotonic dystrophy (DM) is an autosomal dominant neuromuscular disorder, although of Medicine, and 2liological Sciences, The University of Iowa, Iowa City, Iowa; 3The characterised by myotonia and muscle weakness, it presents as a multisystemic disease. Children's Hospital of Philadelphia, Division of Human Genetics and Molecular A genetic defect associated with DM is the expansion of an unstable CTG repeat in the Biology, and 4Departnients of Pediatrics and Genetics, University of Pennsylvania 3' untranslated region of a protein kinase (DMPK). Expression of this gene has been School of Medicine, Philadelphia, Pennsylvania investigated at the RNA and the protein level and a number of conflicting reports on Fibroblast growth factor-8 (Fgl8) is a recently identified growth factor that stimulates expression levels, protein size and localization have been published. Previous analyses the androgen-dependent growth of mouse mammary carcinoma cells. We describe here on DMPK protein have largely relied upon the use of DMPK antibodies raised against the human FGF8 genomic sequence and demonstrate conservation between the human synthetic peptides and have given contradictory results. We report the production of and mouse sequences, including alternatively spliced exons in mouse. Mapping of FGF8 DMPK polyclonal antibodies raised against a murine DMPK cDNA recombinant pro- by genetic linkage using a SSCP variant identified in this study is also reported and refines tein, their characterisation and their use in investigating DMPK expression patterns by the FGF8 map location to 10q24. Since FGF8 maps to the same chromosomal region as western analysis and immunocytocheinistry. FGFR2, has indeed been shown to be a ligand for FGFR2, and has an expression pattern consistent with limb and craniofacial anomalies, we have screened two kindreds with Pfeiffer syndrome which were previously linked to markers from 10q24-25 and a large number of individuals with craniosynostosis and limb anomalies for mutations in the coding sequence of FGF8. While no such mutations were identified, a rare polymorphic variant consisting of an 18-base-pair (six-amino-acid) insertion into exon la is reported that apparently has no clinical effect. Our exclusionary data suggest that mutations in FGF8 would be, at best, an infrequent cause of such disorders.

Published Abstracts: Genetic Counseling and Genetic Education 2157 2158 PREMARITAL COUNSELING, AN EXPERIENCE FROM BAHRAIN Why shostld a basic course in genetics he required ror premedical and other health program students? C.E. Carrasco. University of Puerto Rico at Ponce. S S Al Arrayed, N Hafadh, S. Serafl, Genetic Department, Salmaslys Medical Biology Department, P.O. Box 7186, Ponce, PR 0)t)732-7186. Centre, Ministry of Health, Manama, Babrain Health professionals may be open to malpractice suits through ignorance or Introduction: Bahrain was the first country in the Arabian Gulf to recognize the burden misconceptions Of genetic ctmnditiolss in their patliens. Such is the case of the of blood genetic disorders. The studies shows that 1-2% of newborn have sickle cell orthodotitist who prescribed bracers for one of his patients without taking the titise to disease, 11-18% are carrier, while the carder rate for the beta thalasaenia is 2Y% In an check her pedigree. Two years latet. the hracers and the teeth camne off! The fact is attempt to reduce the incidence of babies born with these diseases, the genetic clinic that neither Genetics, nor Human Genetics are reqtiired undergraduate courses for Pie- slasted the service of (Premarital and in 1985. In 1992 the screening counseling) medical, Pre-odotntology, nor Pre-nursing students in Puerto Rico nor in the United Ministry of Health expanded this service to cover all the primary health care centres and States. are courses at identified couples at high risks were counseled and adviced material and methods: The Neither such part of the basic science curriculum schools of' services was preceded by an information and training course to all doctors and nurses in medicine in the Island noi in the US. Sonie topics of human genetics may be included health centres. This was followed by mass media campaign on the availability of such in their biochemistry, embryology and/or anatorny courses. There is little knowledge services. A special risk assessment sheet was designed, which included information amtd less concern for genetic conditions present in the Island's population such as about sex, age, education, occupation, consanguinity, medical and surgical history, albinism (1/15 are allele carriers) or for rare traits such as Jarcho-Levins. Doctors and infection sexually transmitted habits as history, disease, family genetic history, smoking, nurses fail to inform families of their genetic predisposition in an island where alcohol are asked for. Followed by general physical examination. Thse investigation conditions suggest a "founding fathers effect". Genetic counselors are virtually non- includes full blood count, blood groups, hemoglobin electrophoresis, Glucose 6 phosphate dehydrogenase deficiency, rubella AB, some cases are screened for venereal existing in Puerto Rico and scarce in the mainland. The establishment of a National disease hepatitis and Aids. On the second visit the results are discussed with the client Register of Genetic Diseases in Puerto Rico has been proposed. One of its purposes and counseling and management are given. Results: By studying and analyzing 300 would be to identify, screen and counsel the population who is at risk of having formaurfor 500 clientltaken at random: Male/Female ratio 52.5/47.5 the mean age at children with a particular hereditary illness. This would allow a cost-efficient health marriage for male is 26.5 year and for male 21.9 minimum age is 15 years and program to supply a service which is greatly needed. At present very few health maximum is 45. 23.2 were first cousins 1.5% were second Regarding Consanguinity: insurance cotupatmies will pay for the cost of genetic evaluation in pregnant women or cousins and 3% far relatives. Regarding blood diseases SCD was found in 1.6%, SCT- tewvbor babies. This is a for most health cate services in the US and PR is 13'/6, Beta thaI trait 2'/4, Average A2 in Beta that trait is 5.6%. G6pd deficiency was reality found in 26% of samples, 8% of attended couples were found tp be at rinsk of having no exceptiots. But health piofessionals hase to be aware of the presence and proportion affected offspring, The consanguinity rate among them was S1Y5 of genetic tare in the popstlation they serve. Certainly one, way is to require these Conclusion: All clients benefited from premarital counseling session especially the courses at least at the undergradtiate level. Learning genetics has been enhanced aiid couples at risk. ectjoyed by sitldetils svWlo ctilct living talcautis of replicatiot, tutitation, transcriptiosn. translation and/or pedigree tracing in real life situations. 2159 2160 A Carrier forFactor VIII Deficiency and Fragile X Syndrome: Reproductive transmission of undisclosed CFTR mutation from Case History and Genetic CounselingIssues infertile healthy male with Cystic Fibrosis. P.A. Kulch and D.M. Main, California Pacific Medical L. Hauckl, C. Cunniff 1 D.Quinn 1, A.Maddalena2, The University of Center, San Francisco, CA. Arizona College of Medicine, of and Obstetrics Studies suggest that up to 10% ofmales presenting with congenital Departments Pediatrics, bilateral absence of the vas deferens (CBAVD) are compound heterozygotes and Genetics and IVF Gynecology, Tucson1; Institute, Fairfax, VA.2 for Fibrosis These males are We here Cystic (CF). unique generally clinically present unique genetic counseling issues relating to a asymptomatic except for variable spermatogenesis. 40-60% of men with woman for both carrying genes factor VIII deficiency and fragile X CBAVD carry one known CFTR gene mutation and the remainder have no her first syndrome. During pregnancy, the proband presented with a common CFTR mutation. With the recent introduction of microsurgical family history of two brothers affected with factor VIII deficiency. epididymal sperm aspiration (MESA) combined with assisted reproductive Linkage analysis revealed with 98% accuracy that the proband was a technology (IVF and ICSI), carrier and compound heterozygote males with carrier on the maternal X for factor VIII Because of deficiency. strong CBAVD have the option of fathering children. A healthy male carrier for the feelings against termination of the pregnancy she chose not to pursue delta F508 mutation with CBAVD presented with his wife for amniocentesis prenatal diagnosis. Ultrasonography performed at approximately 20 because of her advanced maternal age. He had previously undergone 16 CF weeks gestation revealed a male fetus. A male was born at 40 weeks mutation screening analysis and normal sweat chloride testing following his gestation, and factor VIII activity testing was normal. At age three years diagnosis of CBAVD. His wife is a noncarrier for the 31 CF mutation panel; this boy was referred to pediatric 'neurology for severe global he declined screening with the additional 15 mutation panel. CF carrier developmental delay. Diagnostic evaluation demonstrated an expansion of testing of the fetus requested by this couple indicate that this male fetus is a the FMR locus consistent with full expression of the fragile X syndrome. carrier only for the R334W mutation. The possibilities exist that (M)this Because of the relatively close proximity of the FMR and factor VIII loci, CBAVD male is a compound heterozygote, (2)nonpaternity, (3)amniotic fluid initial concern was raised regarding the origin of the respective mutations. sample mixup (4)sperm/egg mixup (5)new mutation in the fetus. Pregnancy However, molecular genetic analysis performed on the mother, maternal management has not changed since the fetus is known only to be a carrier. grandmother and maternal grandfather revealed that the maternal This case report illustrates the strength and limitations of CFTR carrier grandfather is a normal transmitting male. Therefore, the mother was testing in the infertile male population in that CBAVD males initially counseled that all male offspring will likely be affected with either fragile X diagnosed as CF carriers may be in actuality compound heterozygotes. The syndrome or factor VIII deficiency, and all ofher daughters will be carriers implication of the revised genotype effects the reproductive risks and the for either one or the other. psychological, medical, and confidentiality issues addressed in preconceptual Unique counseling issues in this case include: a new marriage with a and prenatal counseling. strong desire to have more children, her Hispanic cultural background and strong feelings against termination of pregnancy. Published Abstracts: Genetic Counseling and Genetic Education (cont.) A371 2161 2162 Molecular diagnosis of DRPLA in a patient with clinical features of Issues in genetic testing of laboratory personnel. B.A. Zehnbauer. Huntington disease and IT15-CAG repeats alleles in the normal range Barnes-Jewish Hospital and Washington Univ. School of Medicine, St. Louis, MO. Aurora Sinchez (1), Victor Volpini (2), David Genis (3), Sergi Castellvi-Bel (1), A medical resident on a molecular diagnostic lab rotation, requested to perform direct Montserrat Mili (1), Jordi Corral (2), Dolores Jimenez (1) and Xavier Estivill (1, 2) mutation testing for mutant alleles of a relatively cosnmon, autosomal recessive disease 1. Genetics Service. Hospital Clinic, Barcelona, Spain 2. Genetics Department. for which the lab routinely screened clinical specimens. The trainee had considerable Institut de Recerca Oncologica, Barcelona, Spain 3. Neurology Service. Hospital Josep molecular expertise, wanted to experience the newer methodology, and performed the Trueta, Girona, Spain assayincluding l)NA isolated from his own blood, with direct supervision of a senior for the The unstable expansion of trinucleotide repeats might represent the most common ge- technologist. The results indicated that the trainee was a carrier of a mutation netic mechanism for dominantly inherited neurodegenerative disorders as Huntington's disorder. Positive and negative controls performed appropriately. The trainee showed disease (lID), spinocerebellar ataxia type I (SCAI), Machado-Joseph disease (MJD) and some distress at the findings, refused genetic counseling but agreed to repeat testing for dentatorubral-pallidoluysian atrophy (DRPLA). The clinical spectrum of these disorders confirmation. The mutation was detected by a lab technologist in a duplicate specimen A second mutant allele was not detected either overlaps. On the other hand, all these diseases are inherited as autosomal dominant traits smsing two different test formats. by with late onset, and the pathological expression of the disease is limited to specific regions method (90% alleles). Only after the carrier status was confirmed did the trainee disclose of this result as the basis of the initial motivation to be involved in the of the brain. They are clinically characterized by ataxia, chorea, dementia, and, in some a prior suspicion of them, psychiatric symptomatology, being these features due to neuronal loss. Genetic particular testing procedure. The trainee was again offered formal genetic counseling anticipation is present in all them, as well as a differential disease phenotype depending by another individual and refused. Complete pre-test education had taken place prior confidential because the trainee did not disclose on the sex of the transmitting parent. All these neurodegenerative diseases are caused by to the labwork but was not any prior was and the same kind of dynamic mnsitation (the expansion of a CAG trinucleotide) in different indications for genetic disease. Testing for additional family members requested is genes. We present here the case of a 42-year-old woman with chorea, myoclonic epilepsy pre-test counseling was offered to them. Whether existence of genetic predisposition of and psychyatric symptomatology. The magnetic resonance imaging (MRI) showed slight withheld or unknown, this example illustrates the concerns for privacy in disclosure brain atrophy. filer deceased father and two brothers had the same clinical traits. The molecular genetic findings. While the practice of using lab personnel as test controls is clinical diagnosis was HD and, therefore, the number of CAG repeats in the IT15 gene was not new, the number of relatively common diseases for which we know the genetic etiology studied. The proband showed two alleles in the normal range (20 and 22 repeats). After is increasing rapidly. The ELSI issues of the Human Genome Project are of concern to and this result and following our protocols for a negative result for a HD CAG expansion, staff and colleagues as well as patients. Standard lab guidelines for containment we studied other CAG trinucleotides in known genes causing neurodegenerative diseases. randomization of lab controls and maintaining objectivity in testing must be considered. Among them, we studied the CAG expansion causing DRPLA and, an expanded allele of 70 repeats was found in one of the proband chromosomes. This case highlights the im- portance of genetic analysis in neurodegenerative disorders and jts utility as a diagnostic tool.

Published Abstracts: Genetic Epidemiology and Population Genetics 2163 2164 Detection of two new mutations and various polymorphic sites at the Hinf I/Tsp5O9 I and BsoF I polymorphisms in the flanking LDL receptor locus of French Canadian hypercholesterolemic children. region of the human VNTR locus DISBO in gorilla, chimpanzee, and L A--soline'. E. Leitersdorf. M. Lambert'. A. Reshef. J-C. Feoli-Fonseca'. E. orangutan humans. Levvy..H~pital Ste-Justine and Universitd de Montrdal, Qudbec, Canada and K. Balamurugan5. G.T. Duncana. B. Budowle'. and N. L. Traceyv 7Hadassah University Hospital, Jerusalem, Israel. 1. Research Institute, Miami Children's Hospital, Miami, Familial hypercholesterolemia (FH), is the most common monogenic disorder in Florida, 2. Florida International University, Miami, Florida Western populations. The frequency of FH is at least two times more prevalent in 3. FBI, Forensic Science Research and Training Center, French Canadians from Quebec province. Five mutations at the low-density Quantico, Virginia 22135 lipoprotein (LDL) receptor locus accounted for 83% of mutant alleles found in 376 The minisatellite locus DlS80(1p35-p36) is a VNTR French-Canadian adults diagnosed with heterozygous FH. A recent study confirmed (Variable Number of Tandem Repeat) which is highly this high frequency of heterozygous FH in our pediatric population with polymorphic. The polymorphic nature and the relatively short hypercholesterolemia. However, the 5 established molecular defects did not account repeat sequence make this locus a useful tool for analyzing for the persistent increase in LDL cholesterol and the positive parental history of small biological samples which have been environmentally hyperlipidemia in 29% of French Canadian children that entered the study. In order insulted and/or aged (1). Recently a Hinf I (+)/Tsp509 I to probe for new mutations, 29 unrelated hypercholesterolemic children, with no polymorphism was reported in the 5' flanking region of this genetic defect defined, were investigated. SSCP analysis and DNA sequencing locus (2,3). We found another sequence polymorphism in the revealed the presence of 2 novel mutations: a) A 7bp duplication in exon 4 (681ins7) 3' flanking region of this locus located 32 bases upstream which causes a frameshift, the introduction of a stop at codon 208 and premature from the 3' end; it is the first nucleotide next to the last chain termination; b) An A to G change in exon 8 substituting a conserved tyrosine repeat in the array (3). This G>C transversion creates a for a cysteine at amino acid 354. The 681 ins7 mutation was found in 1 patient and BsoF I restriction site polymorphism. We have examined three the Y354C mutation in 2 other unrelated subjects. Two children carried the recently primate species, chimpanzee, gorilla and oranutang to reported Y468X mutation. Among all 29 children (58 chromosomes), various known determine if these two bases are also polymorphic in the polymorphism were observed: Frequency of the A2 allele for the exon 7 Smal flanking region of these species. We also sequenced two polymorphism was 24% (14/58 alleles); A2 allele for the exon 8 Stul polymorphism alleles from a western lowland gorilla to examine the was 15.5% (9/58); A2 allele for the exon 12 mincml polymorphism was 25.8% allelic structure as well as the flanking regions. We were (15/58); A2 allele for exon 13 Avail polymorphism was 18.9% (11/58); A2 allele for able to find five differences in the 5' flanking region and the exon 15 Mspl polymorphism was 25.8% (15/58). These frequencies are similar no dissimilarities in the 3' flanking region as compared to to those previously reported. Rapid molecular assays were developed to directly 25 human alleles. detect the 2 new mutations in order to offer direct detection lof mutations to a larger (1). Budowle B, et al; Am J Hum Genet 48:107-144, 1991. (2). fragment of the French Canadian population. Alonso A, et al; Int J Leg Med 107:216-218, 1995.(3) GT Duncan, K Balamurugan, B Budowle, and M L Tracey; Submitted for Publication 2165 2166 Evaluation of mental retardation in carbonic anhydrase 11-deficient patients from Identification of cancer predisposing loci by linkage disequilibrium D. southern Tunisia. D.M. Fathallah'. M. Beigoui'. K. Ouahchi'. W.S. Sly2 and K. analysis in French-Canadians. N. Benachenhou. Labuda. I. Gorska-Flioot Dellagij. 'Pasteur Institute, Tunis, Tunisia and 'St. Louis University School of and D. Sinnett. Centre de Cancdrologie Charles Bruneau, Centre de Recherche, H1pital Medicine, St. Louis, Missouri. Ste-Justine, Dipartement de Pddiatrie, and Centre de recherche de l'Hftel-Dieu, Universite de Canada. Mental retardation (MR) is a common feature in the syndrome of osteopetrosis with Montreal, Inactivation of for faithful transmission of the genetic information renal tubular acidosis and cerebral calcification, and a constant feature of patients with genes responsible results in a high frequency of replication errors (RER+) that eventually leads to cancer. the mutation in of Arabic descent. The that CA II splice junction patients discovery For example, germline mutations in mismatch-repair genes were causally related to a wealth of information on deficiency is the underlying cause of this disease provided some familial tumors, such as hereditary non-polyposis cancer (HINPCC). The RER+ the physiological role(s) of this isoenzyme including its implication in osteoclast- phenotype, readily detected as microsatellite instability, is characteristic for HNPCC mediated bone resorption. However, the MR is poorly documented and its cells and was also observed in non-familial cancers. pathogenesis poorly understood. Cerebral calcifications are observed in other diseases Since the RER+ phenotype reveals a deficiency in mismatch-repair, its expression in where no MR occurs. We studied MR in our series of 22 Tunisian patients from a non-familial cancers could be explained by an incomplete penetrance of germline homogeneous Arabic ethnic group, previously shown to have a unique mutation in the predisposing mutations in the corresponding genes. We are testing this hypothesis by CA If gene. We have used the Columbia test to measure the IQ of the patients and a linkage disequilibrium analysis in French-Canadians. Because of the presence of a control group of 150 healthy children. The control group was selected from the same number of rare genetic diseases due to founder effect in this population, we expect that. ethnic group and judged clinically to be free of any known diseases that might cause similarly, a cancer predisposing locus will be characterized by a single predominant mutation. Such a locus will be revealed MR. PCR analysis in this control group sample showed no instance of the mutation predisposing by linkage disequilibrium between RER+ tumors and candidate loci using intragenic and causing CA 11 deficiency in Arabic patients. The patients with CA 11 deficiency were mismatch-repair flanking polymorphisms. The results are tested by the likelihood approach of found to have an of 35 with mental retardation from average IQ ranging profound Terwilliger (1995). This study has been focused on the most frequent neoplasms: had a 20% rate of moderate (52%) to moderate and mild (48%). The control group lymphoblastic acute leukemia, lung, colon, prostate and breast cancers, and the MR. This rate was confirmed by a second test for IQ measurement: the Tony-2 test. candidate predisposing genes: hMLHI, hMSH2, hMSH3, hPMS1, hPMS2, GTBP Family studies in the control group showed frequent occurrence (80%) of inbreeding and Pol&. Our preliminary data on the totality of the available samples (RER+ and among families with more than one mentally retarded child. These findings confirm the RER-) did not demonstrate any linkage disequilibrium with hMLHL. As the founder association of CA 11 deficiency due to this mutation with severe MR. They also effect for mismatch-repair mutations could be expected in the subgroup of patients suggest that MR from other causes, independent of CA II deficiency, also occurs with a displaying a RER+ phenotype, we are now analyzing microsatellites instability between high frequency in this ethnic group, possibly explained by the high rate of normal and tumoral tissues. The linkage disequilibrium study is also continued for consanguinity. other candidate mismatch-repair loci. This work is supported by "Fondation Charles Bruneau". NB. has afellowship of the Medical Research Council of Canada. A372 Published Abstracts: Genetic Epidemiology and Population Genetics (cont.) 2167 2168

Non-independent data points in multiple linear regression analysis of A novel (CGG)6AGG insertion in the triple repeats of FMR I gene in Asians. parent-offspring pairs with Huntington disease. A.C.lush, N.G.Ranen, S.-H. Chen. J. Schoof. N. Buroker. C. R. Scott. University of Washington and C.A.Ross. The Johns Hlopkins University, Baltimore, Maryland. Children's Hospital and Medical Center. Seattle, WA. A multiple linear regression analysis was performed on 58 affected parent-offspring pairs One of the most important characteristics of FMR I gene is the polymorphic nature from 26 distinct Huntingtondisease pedigrees to determine the effect of trinucleotide repeat length on age at onset. Because the assumption of independence of data points is of CGG repeats in the 5' UTR ofthe gene. The repeat size varies from 5-52 CGGs violated with this type of family data, a resampling strategy was utilized to systematically in the general population with the most common one at 30 repeats. We and others draw sub-samples of 26 independent parent-offspring pairs. Sixty sub-samples (of a have evaluated an Asian population living in Seattle for their repeat sizes and found possible 3.6 million) were drawn to fit the regression model, which included triplet repeat the Asians having 29 repeats as the most common one and a less common one at 36 length, age at onset, and gender of the transmitting parent; and triplet repeat length repeats. Sequence analysis revealed a basic sequence of and age at onset of the affected offspring. The resampling regression analysis produced designated as 9/9/6/9 for the "36 a range of values for r-squared, indicating the fit of each model. In some fo the sixty (CGG)9AGG(CGG),AGG(CGG)6AGG(CGG)g regression models, one predictor variable (parental repeats) was not significant and would repeats". Eleven of the 145 (8%) Asian chromosomes had the (CGG)6AGG insert have been forced out of the model if stepwise regression had been used. For this reason, within their CGG repeat sequences of the FMR I gene. At least 6 different sequence regular regression was used to retain all the predictor variables in each model. The fit variations have evolved from the basic sequence found in the population. They are of the regression model based on all 58 parent-offspring pairs was well within the range 9/9/6/2/8, 9/9/6/11, 9/9/16, 9/9/15, 9/6/6/9 and 11/6/6/9. All individuals with the produced by the resampling analysis, and therefore demonstrates that models based on insertion had a single haplotype of DXS548/FRAXACl: 194/D (significantly datasets that violate the assumption of independence may, in fact, still be valid. deviating from the expected), indicating a common origin ofthe repeats.

When DNA samples from 38 American Indian chromosomes were screened, none of the 38 had the insert. Thus this insert appears to be an Asian specific marker.

2169 2170 Population variations in dystrophin gene polymorphisms in Singapore. Species specificity of the DiS80 AFLP and the CSF1 POITPOXITHOI STR LL Chiu'. PS Lai'. N Saha2. WL Lee3 and PS Low'. 'Department of multiplex In forensic casework validation. H. C. Coleman. T. H. Aulinskas Paediatrics, National University of Singapore, 2Division of Human Genetics, and G. R. Riley. GeneLex Corporation, Seattle, WA 98134, (800) 523-6487. University of Pittsburgh, USA, 3Department of Neurology, Tan Tock Seng We have evaluated the species specificity of the Perkin Elmer D1S80 and Hospital, Singapore. Promega CSFIPO/TPOX/THO1 (CTT) multiplex kits as part of our ongoing The molecular heterogeneity of pERT87-8/Taql, Cala/PstI, forensic and paternity casework validation program. In addition to interspecies Cf23a/Taq I, pERT87-15/BamHI and pERT87-15/XmnI polymorphisms in the testing, limits of detection were established for D1S80 and CTT. Newer systems dystrophin gene were studied in three ethnic groups in Singapore. Altogether were validated using samples from a series of adjudicated cases. Previous test a total of 454 chromosomes comprising 153 Chinese, 169 Malay and 135 results using generally accepted RFLP and PCR reverse dot blot methods were Indians were analysed for each marker. Frequencies of the rare allele ranged corroborated in all cases by the D1 S80 and CTT results. Preliminary evaluation from 0.18 (Cala/PstI) to 0.45 (pERT87-15/Xmnl). The frequency of all the rare was begun on the new F13AO1/FESFPS/vWF STR multiplex kit (Promega). alleles were closely similar in the three ethnic groups except for Cf23a/Taql The species specificity of the DIS80 and CTT kits were tested using DNA locus, where the Indians were observed to have a significantly lower frequency extracted from 27 different species, including: 14 non-human primates compared to Chinese and Malays (p<0.05). The allele frequencies of Taql+ composed of higher order primates and both Old and New World lower order (pERT87-8) and XmnI +(pERT87-15) were also closely similar to those reported primates, 8 common game, farm and household animal species, and 5 human in Caucasian populations but differs for the BamHI- allele (pERT87-15). The microbial pathogens or commensals. D1S80 and CTT crossreacted strongly for the later in all groups were more to frequencies three ethnic similar those DNAs: D1 crossreacted with three reported in Japanese populations. The observed heterozygosities in females with certain non-human primate S80 only one of varied between 0.19 to 0.52 at all the loci with Indians showing the lowest primates (chimpanzee, gibbon and orangutan), and at least system CTT crossreacted with all but two of the tested and heterozygosity rates. Genetic affinity of these three ethnic groups with Blacks primates (spider squirrel DNA from mixed and Whites were studied by genetic distance analysis. The genetic distance monkey). Standard validation specimens included specimens, matrix showed that Malays and Indians are nearest to each other and distinct partially degraded DNA, specimens from non-probative evidence and from the Chinese and White Americans while the Black Americans form a specimens from previously adjudicated cases. The ability to detect mixed separate distinct branch from the other cluster of four populations. The markers samples (those containing DNA from more than one individual) was evaluated used in this study should prove useful for population genetics and in formulating using both evidence specimens that were known to be mixed based on previous a genetic strategy of choice of markers for linkage analysis of dystrophin gene testing and a series of mixed neat DNAs. Assay sensitivity was tested both by in our local population. serial DNA dilution and by testing the ability to amplify partially degraded DNA.

2171 2172 A hypervariable region of the low density lipoprotein receptor gene for use Genotype analysis in Spanish phenylketonuria patients L.R.Desviat, in studies of human diversity. P. L. Deininger, A. R. Knight, B. Perez and M. Ugarte. Centro de Biologia Molecular Severo Ochoa. UAM. Madrid. hi. A. Batzer and D. HI. Kass. LSUI Medical Center, and Ochsner Medical Fosindation, 5pain. New Orleans, LA. Phenylketoniuria (PKU) due to a deficiency of hepatic phenylalanine hydroxylase (PAH) Previous evolutionary studies of a number of nuclear DNA loci among human popu- is the most common error of amino acid metabolism. Since the PAIl gene was cloned in lations have revealed exceedingly low nucleotide diversity. We have sequenced a 950 bp 1985, there has been a dramatic increase year after year in the number of identified siu- region of the low density lipoprotein receptor (LDLR) gene in 9 individuals from diverse tations, as new molecular approaches for mutation detection are employed. In an initial populations. The Alui repeat region has over 1% nucleotide diversity. This represents study of the molecular basis of PKU in Spain, 62% of the mutant alleles were identified over 50-fold increase in diversity relative to a typical neutrally evolving locus. The high- in 37 patients, after screening for known Eusropean mutations and direct sequencing after est level of sequence variation does not extend upstream of the Alu, but does extend SSCP analysis. Its order to complete the characterization of the PKU alleles in the Span- somewhat beyond the downstream end. Thus, there is about a 500 bp region of hyper- ish population we have now investigated 128 independent mutant chromosomes, using variability which tapers off somewhat for the next few hundred bases. However, even denaturing gradient gel electrophoresis (DGGE) and direct sequencing. At present, this the flanking regions are niuch more diverse than most of the human genome and this approach has identified 85% of the alleles, containing a total of 37 different mutations, diversity may extend throughout the whole LDLR locus. The variability is largely single apart from common and new polymorphisms. The mutational spectrum includes seven base point mutations, although there are also some small insertion/deletion mutations. novel mutations, D129G, P122Q, S196fsdel22bp, P147S, D151G, A165T and P407S. The sequence variations represent a mixture of common alleles present throughout mul- Seven mutations represent 44% of the Spanish PKU alleles, the most common being tiple populations and random changes seen in single individuals. Although the region of IVSIO (13%),165T (10%) and V388M (6%). The rest of the mutations are rare. These hypervariablity focuses around an Alu repeat, the lower variability seen in an adjacent data confirm the great heterogeneity expected from previous stsmdies. The mutation pro- Alu repeat, the relatively high variability in the region between the two Alu repeats, and file and relative frequencies, when compared to those in related European populations, oeir previous studies of other Alu loci, suggest that the variability is not specific to the show specific characteristics, stressing the need for performing the complete genotype Alus repeat. It seems most likely that this locus represents a hypermutable site for an, as analysis in each country. A systematic analysis of RFLP haplotypes, VNTR and STR yet, ussudetermnined reason. This would make it an outstanding locus for studies of human alleles associated with the identified mutations has also been conducted, providing valu- molecular anthropology and forensics. Alternatively, we cannot rule out the possibility able information for population genetic studies and investigation of the origins of the that this locus is highly diverse because it is less subject to fixation than other loci. mutations. IHowever, the entire region was fixed in the human genome following a gene conversion event that occurred after divergence from chimp and gorilla and diversity must have accumulated since that time. Published Abstracts: Genetic Epidemiology and Population Genetics (cont.) A373 2173 2174 Seasonality of twinning rates: temporal and geographical variations. Aldur W. Temporal changes in the seasonality pattern of births in populations in Eriksson and Johan Fellman, Folkhalsan Institute of Genetics. Population Genetics nosrthern Europe. J. FeIllna,/A. W._.Eriksson and H. Lundstrom. Folkhilsan Unit, P013 21 1. IIN-00251 I helsinki Finland. Institute of' (ienetics. Population Genetics Unit. P013 211, FIN-00251 Helsinki, There has been little agreemenit on whether twin matemities occur seasonally, Fituland: Central Bureau of Statistics, S-1 1581 Stockholm, Sweden. whichl may be a consequence of the fact that different populations have been studied Seasonal variations in the human birth rates are expected to be more marked in for various periods. [he statistics for twin maternities depend on different factors. regions with distinct geophysical periods as the changes between summer and winter e.g. the age and the birth order distribution of the mothers and the population on higher latitudes. We stud)y the cyclic trends and the monthly heterogeneity by specific twinning rates. which may vary between the populations and must be slightly modifying the method of Walter and Flwood (lrit. J. prey. soc. Med. 1975. considered. In the past. socioeconomic status. general health, including physical 29:18). The angle 0 is defined so that it is directly connected with the month(s) with condition. duration of day light. nutritional habits etc. may have had influence on the highest birth rate (one month 300). TIhe monthly heterogeneity is tested by the the seasonality of births and especially of twin births. To day family planning Walter and Elwood X2-test. influences the favorite timne of birth. Therefore. we find it valuable to study the sea- O)ur maits analysis is based onl birth data from Sweden, 1816-1950, first given by sonality of the twinning rate in different populations and for various time periods. D)aln (1974). By analysing subperiods of 10 years we observe a marked change in We consider the changing pattern of seasonal variation in twinning rates for two periods for Denmark (1855-94 and 1937-84). for Switzerland (1876-90), and for the seasonality pattern of the data. At the beginning the highest monthly birth rate is United Kingdons (England and Wales, 1952-59, 1963-75, north-eastern Scotland, observed around January (00 < 0< 300) and an isolated peak in September. After that tile month with the highest rate continuously moves from January towards April and northern Ireland, 1975-79). - For Denmark, 1855-94, the rate of twin maternities by months show highly significant sinusoidal departures with maximum (900<0<1200). The peak in September is still discemible. The monthly in April. For the period 1937-84 similar peaks can be distinguished but they are less heterogeneity starts from a rather high but nonsignificant level. Then it decreases to a accentuated and not significant. For Switzerland a similar seasonality can be seen mininuum whereupon it finally rises again. Subdata from southern and northern but it is not significant. 1 he series for England and Wales show statistically highly Sweden. from rural and urban areas show sinsilar temporal changes. Birth statistics significant seasotiality svith a peak in March to May. Similar pattern cannot be I'r Sweden for the period 1749-1757 (the Wargentin data) show a similar pattern as found in data from north-eastern Scotland and northern Ireland. for Sweden in the first half of the 19th century (O 60). Studies on Norwegian data Our results show seasonal sariations in twinning raics which was more for the period 1911-50 gives similar results as for Sweden for the same period. accentuated in the past. Studies of seasonality in twinning (lelnand large series and The observed seasonal pattern in the past may be the "natural" response of the this fact and the deseasonality can at least partly explain the conflicting results populatiots to the climatic conditions hut the development since the beginning of this earlier reported. centtury indicates an increased influence of socio-economic factors.

2175 2176 Birth Month and Overripeness Ovopathy, a Predisposing Factor for Program for a Clinical Genetics Database. R.R. Lebel and Insulin-dependent Diabetes? L.L. Field, R. Penner, S. Tran. University of Calgary, J.P. Gennuso. Genetics Services, SC, Elmhurst, IL. Calgary, Alberta, Canada. At least 8 genetic loci, including HILA, have now been identified as contributing to Employing commercially available software with the susceptibility to insulin-dependent diabetes mellitus (IDDM), but the less than 50% addition of features customized for a clinical genetics concordance rate in MZ twinls suggests that important non-genetic factors (which may service, we have developed a database of some 2000 four- interact with genetic factors) are also involved. Jongbloet et al. (l)iab.Res.9:51-58, generation pedigrees obtained at initial consultation, indicated 1988) reported that Dutch IDDM patients were more often born in the late win- ter usually by either pregnancy with maternal age over (January to March), and that this corresponded to conception during the spring 34, abnormal results of other testing during pregnancy, restoration of the mammalian ovulatory cycle when delayed ovulation could history of previous abnormal pregnancy outcomes, or The persons result in ova to tera- effects. In order to pediatric genetic problem. minimum number of theoretically overripe leading tologic investigat in each Jonghloet's hypothesis that seasonal overripeness ovopathy may be involved in the represented patient profile is, thus, 14 (patient, spouse, their parents and grandparents) for obstetric cases, etiology of IDDM, we analyzed the birth month of 587 diabetic children ascertained through Alberta Children's Hospital, 211 non-diabetic siblings, and 40,176 Alberta and 15 (the above plus the child proband) for pediatric cases. uncles and cousins further scope children born live during 1975-1990 using data obtained from Statistics Canada. The Aunts, expand the of the data, which can be considered to be prospectively results showed a birthrate in the from March to slightly higher general population ascertained for the purpose of any health problems not in The diabetic children were also more to have been September (highest May). likely involved in the reason for referral. of such born from March to in and the birth month of the smaller Thus, perusal August (highest July), peak a database can help in reaching epidemiologic conclusions sample of siblings was also in May. There were no significant differences between regarding the population incidence of complex traits. We diabetic children and their unaffected siblings in the monthly or quarterly distribution will demonstrate the function of the database with examples of birth months, providing no support to the theory that overripeness ovopathy of analysis of such traits in families and in populations. to We that Albertan families contributes IDDM etiology. recognize may plan The program also allows for assessment according to ethnic to summer a cultural factor which could obscure pregnancies produce spring/ births, background, geographic location (home zip code), or other any natural human seasonal fertility cycles. Nevertheless, there was no evidence that features of the consultation (e.g. diagnostic procedures children born uluring the late winter are more predisposed to IDDM. performed). Funded by the Medical Research Council of Canada.

2177 2178 13-Haplotypes in two normal Mexican populations: Mestizo and Huichol Demographic differences in participants in a national hearing impairment registry. A.R. Villalobos-Arcmbula. F. Rivas, L. Sandoval. R. Bustos. J.M. Canto and I B. Ibarra. Divisi6n de Gen6tica, Centro de Investigaci6n Biomedica de P. S. Ing and D. Strickland2., 1NIDCD HHIRR and 2Univ. of Nebraska AP CP 44310 Medical Center, Omaha, NE. Occidente, CMNO, IMSS, 1-3838, Guadalajara, Jal., Mexico. The National Institute on Deafness and Other Communication Disorders 13-haplotypes in 99 ,3 chromosomes from a Mexican Huichol American (NIDCD) established the Hereditary Hearing Impairment Resource Registry native group (79 observed and 20 infered from 45 unrelated subjects and 10 (HHIRR) to be a national resource for the biomedical community in the area of members from four families) and 97 from an urban Mestizo population were hereditary hearing impairment research. Families "participate" in the HHIRR analyzed for the following polymorphic sites: e(Hcll), OyAY (Hdlll), 5',3' *t by providing genetic, audiologic and medical information via paper (Hc1l), and (AvIl) at 5' subhaplotype (5'sHp), and 3'1 (Hfl) at 3'sHp. Among questionnaire. Individuals who have a hearing impairment can "respond" to Huichols, eight different 5'sHp were observed, being 1,13 and 2 the most requests for demographic information via paper or electronic survey; these frequent (.794, .093 and .041, respectively). In Mestizos, 17 5'sHp were data will determine the degree to which "participants" demographically reflect found, with haplotypes 1, 3, 2, 5 and 9 as the most frequent ones (.391, .172, the target population (nonparticipating "respondents" plus "participants") and .092, .069 and .046, respectively. 3'sHp frequency distributions were .454 the general population (NHANES). Twenty-eight questions in the demographic survey cover: age; sex; marital status; household status size and income: (++), .080 (+-) and .464 (-+) in Huichols and .563 (++), .149 (+-) and .287 educational level. type of school and assistance; ethnicity; and cause of the (-+) in Mestizos. Pairwise comparison for both, 5' and 3sHp distributions hearing impairment. between these two populations showed significant differences (Chi-square, 677 responses to the demographic survey were received between October p<0.00005 for 5'sHp and <0.04 for 3'sHp). Differences for 3'sHp were mainly 1994 and December 1995. 383 were from HHIRR participants: 294 were from given by an excess of the -+ haplotype. The 5'sHp distributions were also HHIRR non-participants. The primary difference between the two groups is compared with other data sets from 23 worldwide populations (including that participants tend to be more severely hearing impaired children with African, Amerindian, Asian, Caucasian and Pacific Islanders) from the parents who are hearing, whereas non-participating respondents tend to be literature. Huichol distribution were different at the 0.05 level from all other adults who use residual hearing as their primary mode of communication. populations except Koreans. Mestizo distribution was also different from Both groups are predominantly (-95%) White, obviating the need to design others, except three Caucasian samples (England, Germany, Algeria). strategies to enroll under-represented segments of both the general and Present observations regarding Sand 3'sHp are consistent with an important targeted populations. Asian contribution to the Huichol genome at this chromosomal region. The This work was supported by NIDCD Contract NOI-DC-3-2134. Mestizo data are explained by simple genetic admixture. A374 Published Abstracts: Genetic Epidemiology and Population Genetics (cont.) 2179 2180 The strength of linkage disequilibrium between two vitamin D receptor Consanguinity and common adult diseases in Israel Arab polymorphisms varies by ethnicity. S. A. Inglesi, R. W. Hlaile', B. E. Henderson', communities. L. Jaber2. T. Shohat*. J.I. Rotter and M. Shohat*. *Department of Medical L. N. Kolonel3, G. Nakaichi', G.-Y.Shi,4, M. C. Yu', R. K. Ross', G. A. Coetzee2. Genetics, Rabin Medical Center, Beilinson Campus, Petah Tiqva, Departments of Preventive Medicinel & Urology2, Norris Comprehensive Cancer 2Taybe Children's Center, University of Southern California School of Medicine. 3Cancer Research Center ledical Center, Taybe Tel Aviv University, Israel and Medical Genetics and Birth Defect Center, Cedars-Sinai of Hawaii, IJniversity of llawaii, llonolulu, 4Department of Community, Occupational, Medical Center and UCLA, Los and Family Medicine, National of Singapore. Angeles, CA, USA. University While consanguinity has been proven to have a To evaluate the adequacy of a commonly used Bsinl RFLP as a marker of the vitamin deleterious effect with regard to congenital malformation receptor locus, we 583 4 groups D (VDR) genotyped individuals from ethnic for this and rare autosomal recessive diseases, little information marker, as well as for a polymorphic site in the 3' untranslated region (3'UTR) of this exists on its role in multifactorial common adult gene. At the latter site, we identified 12 alleles, A13 to A24, of a poly-A microsatellite morbidity. The aim of this study was to investigate the located approximately 1 kb upstream from the 3' end of the 3.2 kb 3'UTR. Allele size effects of consanguinity on the prevalence of common followed a bimodal distribution with distinct short (AW3-A,7) and long (Als-A24) allele diseases in adulthood such as diabetes mellitus, populations. Poly-A allele frequency differed by ethnicity, with the frequency of short myocardial infarction, bronchial asthma and duodenal alleles being highest in non-Ilispanic whites (41%), intermediate in African-Americans ulcer. As part of a larger study aiming to investigate the and Hispanics (29% and 31%, respectively), and lowest in Asians (14%). In each of inbreeding coefficient in the Israeli-Arab community,we the four ethnic groups, both the Bsml and the poly-A markers were in llardy-Weinberg distributed questionnaires to parents of 4100 second-grade equilibrium, and some degree of linkage disequilibrium was observed with coupling be- students in 158 randomly chosen schools. Among the 3772 tween 13smil and short poly-A alleles and between BsmI b and long poly-A alleles. The responders (92%), 34.8% of the students' fathers and 31% strength of the linkage disequilibrium varied by ethnicity, with departures from complete of their mothers were found to be products of con- disequilibrium producing disagreement between the BsmI and poly-A genotypes. Geno- sanguineous matings. There was no difference in the typic disagreement was lowest in non-Hispanic whites (7%), intermediate in Asians and prevalence (males, females) between the offspring of Hispanics (16% and 19%, respectively), and highest among African Americans (37%), consanguineous versus non-consanguineous matings for indicating that BsmI is not a good marker for the VDR 3'UTR genotype in all popula- diabetes mellitus (consanguinity: 4.3%, 1.5% vs non- tions. consanguinity: 2.9%, 1.6%) myocardial infarction (2.7%, 0.03% vs 2.3%, 0.03%), bronchial asthma (2.4%, 2.0%, vs 3.7%, 2.3%) or duodenal ulcer (7.0%, 3.0% vs 7.8%, 2.9%), respectively.This study suggests that ever in a population with a high rate of consanguinity, there is no significant increase in the prevalence of these common adult diseases.

2181 2182 Overlapping genetic susceptibilities In IDDM and Cystic Fibrosis Lack of association of CHRNA4 S248F mutation with epileptic Complicated by Diabetes (CFIDM): Benefit of dual quantitative syndromes with presumed genetic etiology. W.L. Lee'. A.H.N. 2 assessments In related diseases. L.J. Krueger'. D.S. Hoisclaw. Jr'. H.L. P.S. Low3. L.Y. Wong2. H.T. Qng3 and U. Raian4. Tan Tock Seng Hospital1, Dorkin2 and M. Carringtor?. Dept of Pediatrics Med. College of PA & Institute of Molecular and Cell Biology, National University of Singapore2, Hahnemann University, Philadelphia, PA; Div. of Pediatric Pulmonology and National University Hospital3, School Health Services4. Allergy2, Tufts University SOM, Boston, MA & Biological Carcinogenesis and A missense mutation in the alpha4 subunit of the nicotinic acetylcholine Development Program3, National Cancer Institute, FCRDC, Frederick, MD. receptor (CHRNA4) has been found to be associated with autosomal The incidence of CF complicated by diabetes (CFIDM) is much higher dominant frontal lobe epilepsy. In addition, a nonsense mutation in than that found in Type insulin-dependent diabetes mellitus (IDDM) in the CHRNA4 has been reported to cosegregate with 20q-linked benign general population (12% versus 0.1%). In our previous study [J. Clin neonatal familial Immunol. 14(6):353-358, 1994J, the incidence DQa and genotypes in convulsions. DQ0I As part of an ongoing evaluation of neurotransmitter mutations in the CF/DM population (n=26) were in disequilibrium compared to the normal epilepsy, we investigated CHRNA4 as a candidate gene for various forms of control and CF/non-DM populations (n=42). In a pt.-matched, total epilepsies which are thought to be in A total of 101 ascertainment study of CF/DM, an increased allele frequency of the Asp57 genetic etiology. patients with one of were allele, DQB1*0201 was determined (40.4% versus 28.0% for CF/DM and the following epileptic syndromes evaluated: CF/non-DM). The IDDM protective Asp57* alleles were lower in the CF/DM benign focal epilepsy of childhood (31), childhood absence epilepsy (26), versus CF/non-DM (p c 0.025) or normal controls (p< 0.008). One juvenile absent epilepsy (2), juvenile myoclonic epilepsy (14), interpretations of the MHC results underscores a potential mechanistic photosensitive epilepsy (7), benign familial infantile epilepsy (5), benign conflict between CF/DM (a non-autoimmune disorder) and IDDM (an familial neonatal convulsions (1), primary generalized epilepsy with autoimmune disease). By assessing other known diabetogenic loci, this generalized tonic-clonic seizures (6). Nine patients had idiopathic epilepsy interpretation may be tested. -and strong family history of idiopathic epilepsy. However, identifying susceptibility genes in multifactorial diseases is Genomic DNA from lymphocytes was used in a PCR assay (Steinlein et difficult and even more difficult in CF/DM because of limiting pedigree size. al, 1995) which allowed for rapid detection of the missense mutation that In this ongoing multicenter trial (10 National CF Clinical Centers) of CF/DM replaces serine with phenylalanine at codon 248, a highly conserved amino using a matched-design [n=139 CF/DM (1x) and n=?00 CF/non-DM (2X)J, acid residue in the second transmembrane domain. All 101 samples we hope to establish that the investigation of related diseases will not only examined failed to show this mutation. confirm the identity of candidate IDDM susceptibility genes, but may provide The S248F mutation in CHRNA4 does not appear to be associated with fresh insight into the diabetogenic process encoded by these genes. the idiopathic epileptic syndromes examined in this study.

2183 2184 A mIinstion of HIA-oQAI owon 52 and HLA-i4 confers susctibility lfo fnsulin- PCR Amplification of Alleles at Locus D17S5: Population Genetic depdent diabetes eellitus. S.P. Lin , Y.J. Lee, F.S. Lo' and J.G. C, . Dept of Pediatrics, Mackay Memorial Hospital, Taipei. and ,Dept of Moecular Modcine. Taipei Study in Hong Kong Chinese and Detection of Allelic loss in patients Municipal Jen-Ai Hospital, Taipei, Taiwan. To date, more than twelve separate chromosome regions have been implicated in the with Hepatocellular Carcinoma. C.K.Mal, S.C.F.Tam, S.W.Ngs, developeent of human type I diabetes mellitus (IOOM). A sajor part of the genetic R.M.C.Wongg 2 and A.S.F.Lok 3 1 Dept. of Clinical Biochemistry, 2 Virus susceptibility to this disease in husane has been sapped to the husan leukocyte antigen (HLA) class II region (OR, OP. and OQ). We, therefore, analysed the HUA-OQA1 52 and Unit, Queen Mary I lospital, I long Kong and Dept. of Internal Medicine, HLA-OR4 between TIOOM children and healthy old control subjects. The method involved the selective amplification of a DNA fragment fro the HLA-OQAl and HLA-OR4 gene by using University of Michigan, Ann Arbor, U.S.A. the mutagenic primers. After PCR, we can accurately obtain the results after enzyme 1)17S5, a highly polymorphic, variable number of tandem digestion and electrophoresis. Sixty-seven IOOM patients and 69 nondiabetic control repeat subjects were analysed using this method. Statistical analysis wae evaluated using X (VN I R) locus, have proven a powerful tool for paternity testing, genetic tests with Yates correction when appropriate. About the result, please ee the tables. We concluded that susceptibility is confered by DQAl 52 (Arg) as well as DR4. disease mapping. individual identification in forensic science, and Individuals with two OQA1 52 (Arg) alleles and CR4 hosozygous or heterozygous did not have more relative risk for disease. Therefore, OQA1 52 (Arg) and CR4 alleles are deternmination of clonality in human tumors. A population genetic study independent risk factors. We suggest that other genes in this region may be responsible was to a for the disease. performed provide database for the probability calculation of Table 1 forensic identification and paternity testing. In addition, the application of OQA1 IDOM f Control f RR P CR4 10CM f Control f RR P the 1)17S5 locus in detection of loss of heterozygosity (LOH) in Arg/Arg 53 0.79 24 0.35 7.0 <0.05 o/v;v/- 33(1:32) 0.50 17(3;14) 0.24 3.16 <0.05 Arg/- 13 0.19 35 0.51 NS - 3-34 0.50 52 0.76 NS chromosome 17p in hepatocellular carcinoma (HCC) was demonstrated. 1 -/- 0.02 10 0.14 NS Total 67 69 In the population genetic study, a total of 102 genotypes corresponding to Table 2 19 alleles were found in a population of 953 Hong Kong Chinese OQA1 52 OR4 IOOM Control RR P most alleles 4>1>5>6. The (n-67) (n-69) individuals. The frequent were, allelic Arg/Arg ./../- 26 11 3.3 <0.05 frequency were significantly different from the Caucasian populations Arg/Arg -/- 27 13 2.9 <0.05 (p<0.OOI). Thc expected and observed heterozygosity were 89.5% and Arg/- v/v,./- 7 6 NS 90.3% respectively. The discrimination power was 0.98. The chance of Arg/- -/- 6 29 NS -/- of. 0 0 MS exclusion was 0.79. In the LOH study. 46 1ICC patients were studied, 39 -/- -/- 1 10 NS cases were found informative and 15 have loss ofheterozygosity(38.5%). Published Abstracts: Genetic Epidemiology and Population Genetics (cont.) A375 2185 2186 A Linkage Disequilibrium Mapping Study of Tourette Syndrome (TS) in Population stratification among Caucasians at the monoamine oxidase B locus: the Central Valley of Costa Rica CA Mathew.s', MMC DeMille', LD Herrera2. comparison of relative risk and haplotype relative risk data for bipolar affective VI Reus', M Spesny2, II Garrido2, JL Salas2, B van de Wetering3, and NB Freimer'. disorder. A. Parsian and R. D. Todd. Washington University School of Medicine, St. University of California at San Franciscol; Hospital National de Nifios, San Jose, Costa Louis, MO USA. Rica2; and University llopsital Rotterdam, Dijkzigt, the Netherlands3 There have been several reports of association of MAO-A gene polymorphisms Tourette Syndrome (TS) is a neuropsychiatric disorder that affects approximately and bipolar affective disorder. In order to determine the possible role of the MAO 1/3000 individuals world wide and consists of multiple motor and vocal tics, as well region in susceptibility to affective disorders, we have genotyped 83 probands of as occasional associated symptoms of obsessions, compulsions, and attentional deficits. bipolar affective disorder families, 56 sets of parents of bipolar probands, and 84 Although the hereditary basis of TS has been suggested by several of lines of evidence, normal controls for intronic simple sequence repeat polymorphisms of the MAO-A to date genetic linkage studies of this disorder have been unsuccessful. This is in part and MAO-B genes. For MAO-A there were no significant differences in allele because of the difficulty of collecting large pedigrees with sufficient numbers of affected frequencies between bipolar and normal control groups for both genders. However, individuals and in part because tic disorders (of which TS is the most severe) can be for MAO-B there were differences between for both genders. In difficult to diagnose due to the waxing and waning nature of the symptoms. In order to significant groups minimize the difficulties created by uncertain phenotypes and potentially small sample contrast, allele-wise haplotype relative risk analysis for the 56 bipolar proband- trios found no differences between transmitted and non- sizes, we are using linkage disequilibrium (LD) mapping to search for a gene or genes that parent significant allele for MAO-A or B. These data do not the predispose to TS. This approach is based on a search for shared chromosomal segments in transmitted frequencies support that distantly related affected individuals. Using this approach in an appropriate population it association of MAO-A or B with bipolar affective disorder but do demonstrate is possible to limit investigation to a relatively small number of individuals with narrowly undetected population stratification can be an important source of bias among defined (severe) phenotypes (recruited from hospital and clinic populations). A search North American Caucasians. for shared segments can be effectively performed with relatively small sample sizes. We have sampled patients who are severely affected with TS and who are from the Central Valley population of Costa Rica. This population is primarily descended from a limited number of founder families, and is thus potentially informative for LD analysis. We will soon initiate a complete genome scan (with markers at 5 cM intervals) in a search for shared segments.

2187 2188 Phenylketonuria in Venezuela: presence of two novel mutations. B.Perez, DNA studies of limb-girdle muscular dystrophy in the Northern Indiana Amish NI.De Luccal, L,.R.Desviati, J.Villegas2, S.Arias' and M.Ugarte'. 'Centro de Biologia show no evidence for a modifying gene. V.M. Pratt', C.E. Jackson', Molecular Severo Ochoa. UAM. Madrid.Spain. 2IDEA Caracas.Venezuela. 31VIC. W~aIaCe2, A. Feit', and G.L. Feldman'. 'Henry Ford Hospital, Detroit Ml and Caracas. Venezuela. 2Emory University School of Medicine, Atlanta GA. Phenylketonruria (PKU) is the most common error of amino acid metabolism caused Autosomal recessive limb-girdle muscular dystrophy (LGMD) is due to by a deficiency of the liver enzyme phenylalanine hydroxylase (P'AII). Neonatal mass mutations in at least 5 genes. The gene for LGMD type 2A codes calpain 3 screening for phenylketontiria has therefore been implemented in Western countries and, (CANP3). Because of the presence of multiple mutations in the CANP3 gene in more recently, in Latin American countries such as Venezuela. Up to now more than patients with LGMD2A from the genetic isolate of Isle of La Reunion and a low 250 mutations in the PAll gene have been described associated with specific ethnic and prevalence rate, a hypothesis was proposed that a second gene (nuclear or geographical groups. The goal of this study was to understand the molecular basis mitochondrial) was necessary for complete penetrance IRichard et al., 19951. in order to and treatment of the disorder in of PKU in Venezuela improve diagnosis We have studied another isolate, the Amish of northern Indiana, with LGMD2A Latin American countries. We are currently searching for mutations in patients from caused a R7690 mutation in CANP3 One proof of the Venezuela using a mutation scanning method based on PCR/D(GGE and subsequent by single (CQG-+CAG). would be the identification of unaffected R7690 direct sequencing. We have analyzed five Venezuelan PKU families using as source of hypothesis clinically l)NA whole blood and/or dried blood spots. In order to provide insight into the origin of homozygotes. We have examined >350 individuals for R7690. We identified the identified mutations we have analysed the haplotypes and polymorphic short tandem 46 homozygous wild-type couples, 80 couples with one spouse heterozygous for repeat (STR) alleles. We have found European PKU mutations such as IVSIOnt546, R769Q, 5 heterozygous couples, and studied over 60 other family individuals R243Q and Rt408W on the background of haplotylses 6.7, 1.8 and 2.3 respectively. The within this Amish community for this mutation. Results of testing for the haplotype-inutation association of these three mutations reflects the European origin children of 4 of the 5 carrier couples are as follows: 11 homozygous wild-type of these PKU mutations. We have found two new mutations, S349L detected in one and 14 R7690 heterozygotes; 3 R769Q homozygotes in 2 families were also homozygous patient and one C deletion in exon 9 (P314fsdelC). The mutation S349L has identified. The creatine kinase (CK) levels were markedly elevated in 2 of these been detected in chromosomes with haplotype 6.7 and the deletion on the background R7690 homozygotes. A CK level has not yet been obtained for the third R7690 of haplotype 4.3. The patient homozygous for S349L exhibits a classical phenotype. homozygote. The fifth couple declined testing for their children, but muscle The mutation P314fsdelC results in a frame-shift and a codon. The premature stop weakness suggestive of LGMD was observed in 2 children. Twelve separate patient bearing this mutation in combination with IVSlOnt546 also presents a classical phenotype. This study contributes to the enrichment of the knowledge about the origin maternal lineages were found for the 30 nuclear families with the R7690 and 4 distinct mitochondrial DNA were found in of PKU in Latin America. mutation, (mtDNA) haplotypes the 4 lineages studied, suggesting that a modifying mtDNA gene cannot explain the Reunion observations. The absence of non-manifestig R769Q homozygotes is evidence against a modifying gene within this population, permitting genetic counseling on the basis of mutation testing.

2189 2190 DYNAMICS OF SICKLE CELL GENE FREQUENCY IN RELLIS, First report on mutation analysis of Propionic Acidemia in Latin America A SUB DIVIDED POPULATION P.Rodriguez-Ponibol, C. Perez-Cerda', J. Iloenicka', L.R. Desviat', B. Perez', S. Murol, E. Richard', V. Cornejo2, R. Ciugliani3. M. XVajner4, and M. Ugarte'. RANANA GV, *BUSI BR**, MURTY JS* 'Centro de Bliologia Molecular Severo Ochoa. UAM-CSIC. Madrid. Spain. 2INTA, *Dept.of Genetics, Osmania Univ. Hyderabad-500007, India. Universidad de Chile. Santiago de Chile. 3Unidade de Genetica Medica. Hospital de **Dept.of Anthropology, Andhra tniv. Waltair-530 003. Clinicas de Porto Alegre. 4lnstituto de Biociencia, UFRGS. Porto Alegre. Brazil Propionic acidemia (PA) is an inborn error of organic acid metabolism caused by a Studies on the dynamics of gene frequency in subdivided deficiency of propionyl-CoA carboxylase (PCC), resulting from mutations in either of populations on polymorphisms influenced by natural the non-identical alpha and beta subunits. selection and genetic drift are8 very few. The present To date, twelve disease causing-mutations have been reported in Caucasian and examines the of Hb over study dynamics gene generations Japanese patients with beta subunits defects. Several of those are clustered around the of Andhra Pradesh a subdivided in Rellis recently I exon and there seems to exist an association between mutation and ethnic The behaviour of 548AA, 272AS Msp populations. reproductive so the which is the most mutation in Caucasian and individuals in the two indicates group, Ins/Del frequent populations 18SS sub-populations has never been described in The aim of this is the of of sicklers Japanese patients. study analysis both higher fertility and mortality suggesting the l'CCB mutations in Latin America. The includes 11 reproductive compensation for the of homozygous prevalent gene study patients, sloss 8 from 1 from Ecuador and 2 from Brazil. Most of them have been classified as children. However the Hb Chile, sickle cell gene frequency beta subunit deficient and beta exhibits decline over in both the by genetic complementation and/or alpha protein generations sub-groups for the most common Caucasian mutations was direct due to altered selection from heterotic analysis. Screening performed by apparently pattern of the DNA in all Our results shows a selection to directional selection under the decline of sequencing analysis genomic patients. presence of with a 35% of the alleles in a C1228T malaria and improved health conditions. The gene Ins/Del very high frequence, (7/20) study, transition in 5% (1/20) and an lnsll70T also in 5% (1/20). These three mutations frequency over generations is significantly higher in have been detected in PA One of them, Insll70T is a new Chachati Relli (0.3571, 0.2424, 0.2107) than in Relli previously Spanish patients. Kapu mutation that has been found to now in in (0.1667, 0.1428,' 0.1392) possibly due to a) founder only up Spain (manuscript preparation). As has been described for other diseases and in spite of the ethnical heterogeneity effect, b) more intense gene-environment interaction genetic in Latin America, we have found a of Caucasian mutations. the indicated or c) drift prevalence Although by greater morbidity larger genetic C1228T is a mutation first detected in a the presence of that mutation to smaller size. The thus Japanese patient, due population present study in Mediterranean Greece and and now in a Brazilian could out the of on patients (from Spain) patient brings importance changing selection pattern be a recurrence mechanism because a is involved. This the dynamics of gene frequency. explained by CpG dinucleotide work provides a starting point in the undertanding of the genetic basis of Propionic Acidemia in Latin America. A376 Published Abstracts: Genetic Epidemiology and Population Genetics (cont.) 2191 2192 ANGIOTENZIN-CONVERTING ENZYME (ACE) GENE Comparison of survival among African American women and European POLYMORPHISM AND CORONARY HEART DISEASE IN MOSCOW. American women with stage III and IV breast cancer in a Health Care M.I.Shadrina, P.A. Slominsky, N.V.Perova I, S.A. Limborska System. M. J. Worsham, C. C. Johnson. S. R. Wolman. J. Abrams. A. Institute of Molecular Genetics RAS., Moscow, Russia, Blount. U. Raju. S. D. Nathanson. Henry Ford Health System, Detroit, MI, and George Washington University School of Medicine, National Center for Preventive Medicine, Moscow, Russia Washington, D,C. Earlier it that plasma ACE level and has been shown Several studies have demonstrated poorer survival from breast cancer myocardial infarction risk is associated with ACE gene among African American (AA) women compared to European insertion/deletion (I/D) polymorphism. We study a possible American (EA) women. We identified all women with stage III and IV association between ACE gene polymorphism and CHD breast cancer diagnosed between Jan. 1988 and Jan. 1995 and followed development in Moscow population. For this purpose we through Nov. 1995 (N= 128) from the population of women served by compared allele and genotype frequencies for ACT gene l/D our health care system where treatment is relatively standardized. Using polymorphism in random sample of moscovites and a sample of residential addresses, we linked study subjects with block-level census CHD patients with lipid spectrum shift. We don't find any data to obtain information on income. Among the cases, 39% were AA significant differences in l/D alleles and Il/ID/DD genotypes and 61% were EA. Mean age at diagnosis was 61 years Crude 5-year frequency in control group and CHD patients. DD genotype survival was 31% for AA and 47% for EA. women, p=0.07. Median follow-up of those still alive was 5.5 years. Age at diagnosis, income, groups was are significantly frequency is 0.37 in both and histology, estrogen and progesterone receptor status and tumor size and groups higher, than in control from France, USA, Australia, and grade were evaluated for prognostic importance. In a proportional not differ significantly from those in CHD and myocardial hazards model, we assessed the simultaneous importance of factors infarction samples of above mentioned populations. Also we found to have univariate significance at p

2193 Epidemilologic risk factors associated with spina hifida in a Hispanic population. P. A. Zartnian'. D. A. Johnston5 and H. Notihrilo". 'The University of Texas Graduate School of Biomedical Sciences-Houston, Texas, 2MD Anderson Cancer Center, Houston, Texas and 'The University of Texas Medical Schlool-Houtston, Texas. Neural tube defects (NTDs) are one of the mnost cotomon birth defects in the United States. NTDs result from failure of the neural tube to close during embryogenesis. Approximately lmlf of NTI)s result itl anencephaly which is lethal with the other half resulting in spina bifida which causes serious handicaps but is usually not lethal. NTDs vary in incidence worldwide with Mexico having one of the highest incidences at 3.5/1,000. Individuals of Mexican descent living in the US have an intermediary incidence of NTDs. Our study focused on factors associated with spina bifida among Hispanic individuals of Mexican descent. Medical records of 60 children with isolated spina bifida were examined to determine the level of defect atid were classified according to both the "zipper" and "multisite closure" models. A control group of 37 children matched for age, sex, ethnicity and presence of a malformation recognized at or near birth was chosen. Mothers of both groups were questioned regarding their ethnicity and age, in addition to their family, educational, occupational, health, exposure and pregnancy histories. Similar questions were asked regarding the children's fathers. In the case-control study, the ntsmber of mothers and fathers bom in Mexico and of children conceived in Mexico were significantly larger in the spina bifida group. Also, the ntsmber of women having a miscarriage before the birth of the affected child was significantly higher in the spina bifida group. Increased patental age was associated with both high and Clostsre site I defects. The number of children conceived in Mexico was significantly higher in the Closure site I group. The results of the present study indicate that parental place of birth, pregnancy history, place of conception, and patenial age may contribute to the causal heterogeneity of spins bifida in the Hispanic population of Mexican descent.

Published Abstracts: Inborn Errors of Metabolism and Biochemical Genetics 2194 2195 Measurement of citrulline by tandem mass spectrometry (MS/MS) allows Over-expression of a functionally active human Prolidase in Escherichia coli neonatal detection of argininosuccinic acid (ASA) synthetase deficiency and purification to homogeniety C.Carriles and P.Hechtman. McGill University, (ASD) and argininosuccinic acid lyase deficiency (ALD). J.E. Abdenur, Montreal, Canada. imin- A. Schenone, A. Fuertes, A.Guinle and N.A. Chamoles. Fundacion para el Estudio de Prolidase Deficiency, a rare autosomal recessive disorder, is marked by massive las Enfermedades Neurometabolicas, Buenos Aires, Argentina odipeptiduria, severe skin ulcers, mild mental retardation and recurrent infections. Mu- result in a clinical disorder with variable MS/MS has been recognized as an important tool for diagnosis of several amino acid tations in the PEIPD gene (chr 19pl3.2) severity which the leads to the mani- (AA) disorders. However, its use for the neonatal detection of urea cycle defects has and age of onset. The mechanism by enzymic deficiency not understood. Since excreted must not been reported. We present a case of ASD and one of ALD detected in the neonatal fested symptoms is iminodipeptides pass through period by MS/MS. the plasma, the disorder is potentially treatable by replacement of deficient enzyme in the In this the interconversion between the form of prolidase Both patients presented at 48-72 hr of age with lethargy and progressed into coma. circulation. regard, apo-enzyme metal cofactor) and the holo-enzyme (Mn-associated) form may be critical for Samples for metabolic studies were obtained on the 6th day of life. In the ASD patient (without success of treatment. Iluman Prolidase cDNA was cloned into the bacterial expression we also analyzed, retrospectively, a sample obtained at 48 hr for PKU newborn vector pRSET C. The plasmid encodes a chimeric protein with a hexahistidine tag and screening (NBS). Blood dots in filter paper were analyzed by MS/MS (VG an enterokinase cleavage site at the N-terminus. When fully induced, the recombinant Quatro-Il-Fisons,UK). Citrulline (m/z 232 - neutral loss 119) was measured as its ratio accounts for up to 5% of soluble E. coli protein. The purified enzyme was >98% with deuterated ornithine (C/O*). ASA was identified in urine scanning by MS at m/z protein free of contaminants by SDS-PAGE and had a specific activity of 15,000 jsmol/h/mg. 23 459. All results were confirmed running the same samples with a Beckman AA mg of purified enzyme were obtained per liter of culture. Purified prolidase is activated analyzer. 20 fold by incubation with Mn. In addition to its catalytic role. Mn also appears to Results: Both patients had abnormally elevated C/O*. ASA was markedly elevated stabilize the structure of prolidase. The T1/2 at 60C for the apoenzyme is 6 min and 52 in the ALD patient (Table 1): min for the holoenzyme. Normal ASD-NBS ASD ALD NH4 (ug/dl) < 80 - >700 1,149 C/O < 1.1 53.3 77.4 7.21 AGA ND ND ND +++ We conclude that citrulline measurement by MS/MS is useful for the diagnosis of ASD and ALD. These results also suggest that the detection of these diseases is possible for NBS programs using MS/MS. Published Abstracts: Inborn Errors of Metabolism and Biochemical Genetics (cont.) A377 2196 2197 Packaging cells expressing the Adenovirus (Ad) El, polymerase, and Improvement of gene transfer in cultured renal epithellal cells by preterminal proteins to allow the growth of a new class of replication DNA/peptide/liposome complex. D. Chan. Y. H. Lien. and L Lai. Department of defective Ad- vector for use in Duchenne muscular dystrophy (DMD) Pediatrics and Medicine, University of Arizona, Tucson, Arizona, USA. J.S. Chamberlain' and A. Amalfitano 2. University of Michigan, Depts. of 'Human Liposome-mediated gene therapy is limited by low efficiency in DNA transport Genetics and 2Pediatric Genetics, Ann Arbor, MI. Adenovirus (Ad) based vectors have been extensively utilized to transfer genes into into the nucleus. Polycations, such as polylysine and the SV40 nuclear localization mammalian cells in vitro and in vivo. however, the in vivo efficacy of current Ad vectors signal, reportedly enhance liposome-mediated gene transfer in vitro (Gao and is severely limited, especially when considering the transfer of the full length dystrophin Huang, Biochem. 35:1027, 1996). We tested the effect of a synthetic "Plus B" cDNA (>1ikb). These limitations include: 1) a sub-optimal carrying capacity; 2) the Peptide (provided by Life Technologies) on liposome-mediated gene transfer in generation of replication-competent Ad (RCA), which contaminate Ad vector prepara- renal epithelial cell lines. 10 ;.g of Plus B was incubated with 0.4 fig of tions; and 3) the expression of viral epitopes by current Ad vectors, despite deletion of pCMVLacZ plasmid, which expresses 8-galactosidase, then mixed with 4 jtg of the El genes. The latter problem limits in vivo gene transfer and expression to only LipofectAMINE or Lipofectin. The mixture was added to cells grown in a 24-well transient periods of time, secondary to host immune responses directed (in-part) against the viral epitopes present in infected cells. To address each of these problems, we have plate and 8-galactosidase activity measured with X-gal stain 48 h later. The % constructed a new packaging cell line which coexpresses three Ad gene products; the El, increase in transfection efficiency of each cell lines compared with experiments polymerase, and preterminal proteins. These cells allow the growth of El, polymerase, without Plus B is summarized as follows: and preterminal protein mutant Ads. Each of these proteins have a pivotal role in the life Cell lines % increase Cell lines % increase cycle of Ad, because in their absence viral replication and protein expression is severely LLC-PK1 71% MDCK 66% disabled. A new class of Ad vector, containing deletions of all three of the aforemen- MTC 244% CA(+)1 84% tioned gene regions can now he isolated. These vectors will have an increased carrying CA(-)1 91% CA(+)2 312% capacity (approaching 13 kb) a dramatically decreased ability to generate RCA, and (most importantly) a decreased amount of viral protein expression. This class of vector CA(-)2 39% CA(+)3 92% is predicted to allow for a substantially longer duration of transduced gene expression in LLC-PK1 is a pig proximal tubular cell lines; MDCK, a dog distal tubular cell target tissues and will be useful in the therapy of DMD as well as many other genetic lines; MTC, a mouse proximal tubular cell lines; and CA(+ )/(-) are renal tubular diseases. epithelial cell lines developed from normal and carbonic anhydrase II deficient mice, respectively. Plus B had no effect on Lipofectin-mediated gene transfer. We conclude that the Plus B Peptide enhances lipofectAMINE-, but not Lipofectin-, mediated gene transfer and may be potentially useful for improving liposome- mediated gene therapy in vivo.

2198 2199 Optimizing adenoviral gene therapy vectors: tissue specificity and immune Methyl histidinuria In children with autism. R.E. Hillman. M. Muskopf. C. modulation. M.A. Hauser', A.M. Saulino', S.D. llauschka2, and J.S. Chamberlainl. Jones and J. Miles. University of Missouri, Division of Medical Genetics, 'Department of Human Genetics, University of Michigan Medical School, Ann Arbor, Columbia, Missouri. MI 48109-0618; 2Department of Biochemistry SJ-70, University of Washington, Seattle, WA 98195. Our clinic has divided children with autism and autistic behavior based Current-generation adenoviral vectors suiffer froni the limitation that they give rise to on their dysmorphology examination into those who are dysmorphic and long-term expression of recombinant genes only when administered to immunodeficient those with normal examinations or with minimal findings consistent with or immunotolerant animals. In immunocompetent adult animals, expression begins to their families. As part of the evaluation urine is obtained for amino acids. decline after one week and is usually undetectable by 3-4 weeks. This has been shown Quite unexpectedly there was a high incidence of methyl histidinuria in to be due in part to a cytotoxic T-lymphocyte (CTL) response to virally transduced this population. The rate was quite different for the dysmorphic children cells. We are pursuing two strategies to reduce this CTI, response. Firstly, we are than for the non-dysmorphic children and both were than seen in testing muscle-specific regulatory cassettes based on the mouse muscle creatine kinase higher promoter and enhancer. This promoter has been extensively characterized, is relatively other children screened in our laboratory. In the non-dysmorphic small, and exhibits a high degree of muscle specificity. Expression of transgenes only in children, 18 of 60 (30%) had increased secretion of methyl histidine. In muscle tissue will reduce the overall level of antigen presentation and will thus reduce the dysmorphic children, only 1 of 16 (17%). (Chi square= 4.15, the systemic level of virally-induced inflammation. These cassettes also contain the lacZ p<.05). In other children screened the rate was less than 2%. Non- reporter gene, the SV40 late polyadenylation signal, and an artificial intron derived from dysmorphic but not dysmorphic children had a rate significantly different adenoviral sequences. Testing by transient transfection into cultured mouse myoblasts as well as by direct intramuscular injection of plasmid DNA indicates that the best of from other children screened for a variety of diseases. The folklore of these expression cassettes is approximately 10% as active as the CMV promoter. Our autism is replete with vitamin, mineral, and dietary treatments directed second approach to reducing the antiviral CfrL response entails engineering vectors that primarily towards the methionine pathway and homocystinuria has been express immunomodulatory activities in additions to the transgene. We will describe in reported in a small number of language delayed children. Although the vivo testing of a first generation adenovirus that overexpresses the adenoviral E3 protein present findings are unexplained, they suggest that methylation processes gpl4.7. should be examined in the non-dysmorphic children with autism.

2200 2201 Cortical blindness following hyperammonemic coma in patients with Use of Recombinant Granulocyte Colony stimulating Factor (G-cSF) in ornithine a patient with Glycogen Storage Disease type IB (GSD 3B), acute trpnscarbamoylase (OrC) deficiency. 0. Hurkol, bilateral otitis media and epididysitis. A Iglesias. P Ashton-Prolla A.F. Lewandalp2, and S.W. Brusilowl. The Johns Hopkins University C Sansarica. Department of Human Genetics, Mount Sinai Hospital, New School of Medicine, Baltimore, MD ZChildren's National Medical Ce*ter, York City, New York. Washington, DC. Granulocyte Colony Stimulating Factor (G-CSF) was administered to Cortical blindness is the loss of visual perception and optokinetic a 10 year-old male with GSD IB who presented with acute bilateral nystagmus (OKN) with preservation of anterior pupillary reflexes and otitis media and epididymitis. The patient had a history of frequent urinary tract infections, respiratory tract infections and retinal anatomy. We report 4 pediatric patients with arc deficiency cutaneous infections with chronic neutropenia (absolute neutrophil (1 female and 3 males ranging in age from 9 months to 5 years) who count 0.4-0.7 x 1,000/mm3). On admission, there was a history of developed temporary cortical blindness following a hyperammonemic minor trauma to the testicular area. The patient had fever, episode. Each was hospitalized for treatment of hyperammonemic abdominal pain, nausea, vomiting, dysuria, and was limping. On encephalopathy, with peak plasma ammonium levels of 230-666 uM, physical exam, bilateral otitis media was noted. Edema, erythema, loss increased temperature and tenderness over the left scrotal area were Visual was suggested by the inability to fix gaze, follow the found. Testicular torsion was ruled-out by sonogram. Urine culture parent's face, or locate objects, and was usually unsuspected until at was positive for Staphylococcus aureus. The diagnosis of acute least 24-48 hours after awakening from coma. The patients were epididymitis was made. The leukocyte count on admission was unusually quiet, with decreased physical activity and interaction with 2,500/mm3 with 15% of segmented cells and 2% of bands (absolute parents until their vision returned, at which time they again became neutrophil count 375). The patient was successfully treated with interactive. Bedside testing included. the child's ability to focus oxacillin 100 mg/kg/day and 5 mcg/kg/day of G-CSF for two weeks. During the four initial days of treatment the leukocyte count on his/her reflection in a mirror or to demonstrate OKN. All increased from 2,530/mm' to 6,6oe/mm', and the total neutrophil count patients had documented abnormalities on further testing (visual from 425/mm' to 3,029/mm' with rapid resolution of all clinical evoked response, MRI, and/or CT). These data suggest that cortical signs. No metabolic derangement was noted during this admission. blindness may occur more frequently than is recognized following an One week after the treatment was ended, the absolute leukocyte count of returned to its basal level. G-CSF, which has sporadically been episode hyperammonemic encephalopathy. Physicians caring for these is indicated for serious infections patients should be watchful for signs of visual loss (fixed gaze, used in patients with GSD 1B, and mainly on a short term basis. Our case showed that in failure to track, absent OKN, inability to focus on a mirrored combination with the proper antibiotic, by promoting production of reflection) and consider formal testing of the visual cortex. leukocytes, G-CSF has effectively contributed to the resolution of the acute infections of our patient. A378 Published Abstracts: Inborn Errors of Metabolism and Biochemical Genetics (cont.) 2202 2203 Prenatal treatment of fraternal twins discordant for tyrosinemia type 1. Complementatlon of APRT Phenotype In A9 Cells by C-R Scott E.Y Cheng K. Feldman R. Jack S Lindstedt University of Washington, Electroporatlon of a BAC. ((Patrick L. Johnstone, Susan L. Kohler, Petra Seattle, Washington. M. Jakobs and Robb E. Moses)). Oregon Health Sciences University, Tyrosinemia type 1,, due to a deficiency in hepatic fumarylacetoacetase results in Department of Molecular and Medical Genetics, Portland, OR 97201. derangements of hepatic, renal, and bone marrow functions and hepatocellular The transfer of a bacterial artificial chromosome (BAC), 64-A15 carcinoma. In some cases, the metabolic derangements have been successfully corrected (Research Genetics), approximately 120 kb in size, containing the human with oral ingestion of 2-(2-nitro-4-trifluoro-methybenzoyl)-l,3-cyclohexanedione adenosine phosphonbosyltransferase (APRT) gene into a mouse A9 cell line in deficient in de novo synthesis of nucleotides has been observed to correct (NTBC). There is little information about the prenatal onset of liver damage for the deficiency. The BAC was identified by PCR screening using primers tyrosinemia type 1. We present a case of in utero treatment of fraternal twins in which derived from the APRT sequence in GenBank. Mouse fibroblasts grown to one is affected with tyrosinemia type I. The at risk pregnancy was identified because 80% confluency were electroporated using a BioRad GenePulser unit set at a previous child with tyrosinemia type I who is currently being treated with NTBC. a capacitance of 960 I1F and 300 volts in a 0.4 cm gap width cuvette. Twin B was found to be affected based on linkage studies and amniotic fluid Selection was performed using aminopterin, an antifolate, which acts to determination ofsuccinylacetone (Succ-Ac). The pregnancy was dichorionic and effectively block de novo production of purines and dTMP. Adenine, a diamniotic with separate placentation sites. At 35.5 weeks gestation, after IRB precursor of APRT-dependent production of purine tibonucleotides, was also approval, amniocenteses and cordocenteses of Twin A (unaffected) and Twin B were provided to offset the lack of de novo purine ribonucleotide production. completed to determine the level of hepatic dysfunction in Twin B. Five milligrams of Positive colonies could be observed at approximately 14 days. The NTBC was then instilled into the amniotic fluid ofTwin B. Each fetus was sampled electroporation of five micrograms of APRTcontaining BAC DNA Into again 3 days later. The following summarizes some of the metabolic analyses before an approximately one million mouse cells resulted In greater than 500 corrected after in utero treatment with NTBC. colonies. This study supports the use of genomic DNA in a BAC to Twin A Twin B Mother functionally complement metabolic deficiencies. Additional studies are Succ-Ac under way to test the APRTcomplementation system in human primary 16 weeks ND 167 NA fibroblasts. Pre-Rx ND 54 NA Post-Rx ND 52 NA Tyrosine Pre-Rx 106 109 46 Post-Rx 464 424 237 Birth 220 210 NA

2204 2205 Enzyme replacement therapy for Gaucher disease in Canada. J. J. MacKenzie' and A biochemical anomaly In mothers of singleton ornithine transcarbamylase J.T.R. Clarke2 1. Division of Medical Genetics, Queen's University, Kingston, Ontario, (OTC) deficient probands. NE Maestri', CR Lord', M Glynn2, A Bale2, SW Canada, and 2. Division of Clinical Genetics, The Hospital for Sick Children and the Brusilow'. tJohns Hopkins Medical Institutions, Baltimore, MD. and 2Yale University ofToronto, Toronto, Ontario, Canada. University School of Medicine, New Haven, CT. Gaucher disease is the most common ofthe lysosomal storage diseases with an incidence OTC deficiency is an X-linked disorder of urea synthesis; the phenotype of ofup to 1/40,000 in the general population. Because ofthe commitment ofprovincial state- heterozygous females ranges from apparent normality to profound neurological operated health insurance plans to provide equal access ofpatients to accepted health care In impairment. The allopurinol (AP) test was developed to identify the carrier the face of the extremely high cost of this form of therapy, we undertook to examine how status of asymptomatic, at-risk adult females based on 24-hour [orotidine + treatment was being financially supported in each ofthe 10 provinces ofthe country. orotatel excretion (OOE) following ingestion of 300 mg AP. Discriminant A total of 25 patients with type I Gaucher disease (I5 children and 10 adults of diverse specific and 85% sensitive in classifying 41 ethnic origins) were on enzyme replacement at the time of our study. The indications for analysis, using log[OOEJ, was 100% and obligate carriers and 36 population-based female controls. To study phenotypic treatment included massive splenomegaly, growth failure, and bony, hematologic variability among heterozygous OTC females, we proposed using the AP test to pulmonary complications of the disease. Treatment regimes varied markedly, from 12U to We hypothesized 120 units ofenzyme/fg/month. The administration frequency varied from 3 times weekly to identify carrier status of mothers of OTC deficient probands. biweekly. Outcomes of therapy were assessed on the basis of the patients' and/or parents' that the distribution of OOE among non-carrier mothers would be the same as subjective impression of therapeutic response and by measurement of generally accepted the distribution among controls. 185 females from 97 families completed a indicators of objective response to treatment (hematologic indices, splenic volume, number medical/family history questionnaire and an AP test. Of 34 mothers of singleton ofbony crises etc.). Subjective and objective reports demonstated benefit from therapy. female probands, the AP test clearly showed 4 to be heterozygous carriers; among The financial support for therapy varied markedly from one province to another. No the 30 mothers identified as non-carriers, the mean log[OOE] (1.71 pg) was patient with severe Gaucher disease was denied therapy as a result ofinability to pay for the significantly higher than among 41 population-based controls (1.60 ug, p = 0.01), medication. Both federal and provincial governments, private insurance carriers, and the but not in the carrier range. DNA from white blood cells was used for mutation commercial supplier of the enzyme participated in providing financial support for the analysis in 12 non-carrier mothers of singleton females to verify that AP testing treatment. However, in only one province (Ontario) had government health care officials had correctly determined their carrier status. Mutations could be identified in 9 accepted the development, by a multidisciplinary panel of medical experts, of formal singleton females; none of their mothers carried the mutation. Although this guidelines for determining eligibility for reimbursement for cnzynsc replacement treatment. indicates that these non-carrier mothers do not have an OTC mutation in their Eligibility was based on medical criteria. The model is currently being examined by health WBCs, somatic mosaicism involving a portion of the liver or a variant of OTC care officials for application to ensure equitable access to other high cost therapies. deficiency caused by a mutation at another locus could account for the anomalous AP test results.

2206 2207 Neonatal and delayed onset liver Involvement In disorders of oxidative KERNICTERUIS-ASSOCIATrED G6PD DEFICIENCY: D. Chrdtien. A. Rotig. P. Rustin. C. Tl'E IMI'ORTANCE OF PREVENTION phosphorylatlon. V. Cormier-Daire. 2 and INSERM U-393. LQoraigean-G0a0nesL Leo Levin MD,2 Robert Gall MD Dubuisson*. 0. Bernard*. A. Munnich. Dept of Genetics and Ernest Beutler MD3 H11pital des Enfants Malades and *Hfpital de Bicetre. Paris, France. I.Genetics Department, North York General hospital, North York, ON Canada Reviewing a series of 22 patients, we have identified two clinical profiles in 2. Markhami-Stoullville Ilospital, Markham, ON Canada 3. The Scripps Research hepatic failure ascribed to genetic defects of oxidative phosphorylation. The Institute, La Jolla, CA 92037-1037 USA Two cases ofkernictens-associated G6PD deficiency are described. Case I$ BU, neonatal type (9/22) had an early onset (before one week), a rapidly fatal course and delivered at term to a 30 year old Filipino primigravida developed feeding problems and a frequent neurological involvement. The other type ( 13/22) had a delayed onset (2 lethargy on day 3. On day 5 his total serum bilinubin was 880umoVL. He died on day months-4 years), a milder clinical course, an inconstant neurological involvement 9, in spite of aggressive treatment G61PD deficiency was diagnosed by red cell enzyme delivered a WU, who also had G6PD fatal outcome. Elevated and/or CSF lactate with assay. The couple subseqtientlv healthy male, and an occasionally plasma deficiency. I Ic was trealed with early pholotlerapy, fluid supplementation and had a abnormal aspect of the liver (steatosis. cirrhosis) were consistent features of the peak serum hilinibin level of 223umol/l, (day 3). Casge_2: MA was the third son disease. Respiratory enzyme deficiency in the liver involved complex I, complex IV delivered to a healthy 28 year old Afghani fiemale. tie was small for gestational age and generalized enzyme deficiency in both subtypes. Liver and brain were the most (birth weight 4 Ibs, I0 oz at 36 5 weeks), developed feeding problems and jaundice on day 3. His peak serum bilirubin level on day 5 was 640umol/L and he had a double severely (if not the only) affected organs in our series. The delayed onset forth volume exchange transfusion A red cell G6PD assay revealed he had a G6PD (leserves intensive efforts as several cases spontaneously recovered (3/22) or deficiency. At 6 weeks he had severe feeding problems, irritability, hypertonia, and markedly improved following ursodesoxycholic therapy and liver transplantation seizure-like episodes. Early diagnosis of hyperbilirubinemia characteristic ofG6PD of and death. (4/22). Frequency of consanguineous tnatings in our series (6/22) makes autosomnal deficiency can result in prevention sequelae including neurologic damage The bilirtubin in GMPI) deficient neonates occurs between day 3 and 5. It is recessive inheritance likely but in the absence of information regarding the disease peak serulm unlikely timat hospitalizalion ol'a newborn can be extended to cover this period. Cord causing genes, prenatal diagnosis can rely only on the expression of enzyme blood screening of neonates for GOlD deficiency would facil tate appropriate deliciency in cultured fibroblasts of the proband (4/22). management and prevention of neurologic damage and death. Published Abstracts: Inborn Errors of Metabolism and Biochemical Genetics (cont.) A379 2208 2209 Carnitine palmitoyltransferase II (CPT II) deflency presenting with ARSACS: Possibly a lysosomal storage disease? A. Richtert . K. Morgan2. JP. Bouchard3. J. Mathieu4i J. Lamarche5. J.Rioux6, 1 Hudson6, SB. Melanconl. hyperammonemia in an infant B. Ross, A.L. Zuckerberg,.M . Gerahty. Center 'Genetique, Hop. Sainte-Justine; 2Genetics, MGH, Montreal; 3Neurologie, Hop. for Medical Genetics, t)epartment of Pediatrics, Johns Hopkins School o Medicine, Enfant-Jesus, Quec; 4 Neurologie, Ho'p. Chicoutimi, 5Pathologie, CUSE, Baltimore, MD 21287-4922. Sherbrooke, Canada; 6Whitehead Inst.,Cent. for Genome Res, MIT, Cambrid&e Mass. Two types of carnitine palmitoyltransferase 11 (CPT II) defiency have been described. Autosomal recessive spastic ataxia of Charlevoix-Saguenay (ARSACS) is a The more common presentation is in adults, with muscular weakness and recurrent myo- homogeneous, early onset disease with specific ocular manifestations, restricted in globinturia. Pediatric presentations are much more rare and only 7 cases of CPT II have occurrence to individuals with origins in the Charlevoix-Saguenay region in Eastern been described in the literature. A lethal neonatal form, which may be associated with Quebec. Disease cause is unknown. We aim to map and clone the gene. dysmorphism has been seen and a later infantile form, presenting with hypoketotic hy- Using 8 informative families we undertook a first round genome screen at poglycemnia and cardiomyopathy is also described. We describe the presentation and Genethon with 120 highly polymorphic loci (ave. interval 30cM). It did not provide course of this rare disorder in an infant. A 3 month old male infant presented with en- evidence of linkage. Results of a second screen (300 loci, aver. interval 10cM) cephalopathy and seizures following a 2 day history of vomiting. Past medical and family performed at the Whitehead Institute's high-throughput mapping facility are currently being analyzed. history were unremarkable. Physical examination was significant for encephalopathy and Two autopsies revealed swollen neurons characteristic of storage diseases. The hepatomegaly. Laboratory tests showed a hypoketotic hypoglycemia associated with a majority of cells had dense, lipofuscin-like granules.prompting a detailed study of 18 hyperchloremic metabolic acidosis. Liver function tests were elevated. Plasma lactate lysosomal enzyme activities. We compared values obtained from 12 ARSACS skin levels were normal. However plasma ammonium was 520 mM/L (normal< 50). Plasma fibroblast cell strains with controls. Although the reduction in enzyme levels seen in amino acids showed normal levels of urea cycle intermediates with paradoxical low levels ARSACS fibroblasts did not approach the magnitude of the lysosomal storage diseases of glutamine (121 mnM/L; range 337-673). Despite the elevated ammonium, glutamine specific for each enzyme, highly significant reduction (p <0.001) was observed in the levels were also low in urine and CSF. Urine organic acids revealed large amounts of mean values of a-glucosidase activity. Statistically significant reductions (p <0.05) adipic, suberic, sebacic, octenedioic and decenedioic acids. Ketone bodies and orotic were observed for mean activity of tI-glucosidase, arylsulfatase B, sphingomyelinase, acid were not found. Initial measurements showed very low levels of free carnitine. After acid lipase and N-acetylglucosamine-6-sulfate sulfatase. A single cell strain tested had carnitine replacement there was an increase in the ratio of long chain to total (39%), normal level of esterification of intracellular cholesterol, while 8 strains examined for while the the ratio of free to total was reduced (50%). A plasma acylcarnitine profile filipin-cholesterol staining had normal patterns. No differences were seen in the levels showed very low acetylcarnitine levels consistent with CPT 11 or carnitine translocase of II others. We are completing our studies by determining the urinary levels of deficiency. Enzyme analysis in fibroblasts showed a reduction in CPT 11 activity to 6% glycosaminoglycans, oligosaccharides and sialic acid. Ultrastructural investigation of of normal. The patient respnded rapidly to IV Dextrose and enteral carnitine. The skin punch biopsy material and cultured fibroblasts is under study.Whether ARSACS patient is now 1 year old and has normal growth and development. lie remains on is in fact caused by mild deficiencies of one or a combination of lysosomal enzymes is carnitine supplementation. Echocardiogram has revealed an asympomatic hypertrophic still unclear. Our continued approach targeting the neuronal pathology, the possible cardiomyopathy. enzyme defect combined with large scale gene mapping will yield the cause of ARSACS. Supported by MDA-C, FRSQ-RGMA , CGDN-NCE, MRC & NIH.

2210 2211 Non-invasive molecular testing for carnitine palmitoyl transferase deficiency. Inductions of Cu/Zn Superoxide Dismutase- and Nitric Oxide Synthase- Like G.D. Vladutiu and S.Srivastriva State Univ. of New York at Buffalo and Children's Immunoreactivities in Rabbit Spinal Cord after Transient Ischemia: M. Hospital of Buffalo, Buffalo, NY. Watanabe"3, M. Sakurai2. K. Abe', M. Aoki', M. Shoji', K. Mizushima3, K. Okamoto3 Carnitine palmitoyl transferase (EC 2 3.1.21; CPT) plays an important role in the and Y. Itoyarna'. transport of long-chain fatty acids within mitochondria. Two distinct forms of the Departments of 'Neurology, and 2Thoracic and Cardiovascular Surgery, Tohoku University School of Medicine, Sendai; 3Gunma University School of Medicine, enzyme, CPTI and CPT2, are located on the inner surface of the outer and inner Maebashi, Japan. mitochondrial membranes, respectively. CPT) deficiency is a rare infantile disorder Arnyotrophic lateral sclerosis (ALS) is a motor netiron disease characterized by presenting with fasting hypoglycemia and inappropriately low ketogenesis. CPT2 relatively selective degeneration of motor neurons in the spinal cord, brainstem and deficiency, also rare, is a lethal systemic disorder in children and newborns presenting motor cortex. About 10% of ALS is familial, and the remaining sporadic. Recently, with hypoketotic hypoglycemia, carnitine deficiency, cardiomyopathy, and early death. mutations in the Cu/Zn superoxide disinutase (SOD) gene have been identified in a Myopathic CPT2 deficiency is the most common disorder of muscle lipid metabolism number of patients with familial ALS. ALS is currently thought to occur by cytotoxicity and the most cause of of mutant Cu/Zn SOD protein or "gain of function" in patients with the SOD mutations. frequent hereditary myoglobinuria Symptomatic patients have Although the exact mechanism is still unknown, a hypothesis of the interaction between been reported as CPT-deficient with a wide range of residual activity in skeletal muscle the mtstant protein and nitric oxide (NO) is suspected. from <10% to 60% of normal reference activity. The application of molecular testing in In order to understand 1) a possible interaction between Cu/Zn SOD and NO under cases of CPT deficiency will help to determine whether or not patients with high a stressful condition that leads to death of motor neurons, 2) the distributions and residual activity actually have deleterious mutations in CPT. Four mutant CPT2 alleles properties of NO synthase (NOS) under a normal state, the distributions and inductions are known which can be used to the molecular defect in >95% of of Cu/Zn SOD, neuronal and endothelial NOS (nNOS and eNOS), and nitrotyrosine collectively identify (NT) were immunohistochemically examined in rabbit spinal cords after 5 and 15 affected patients. We used restriction enzyme analysis ofPCR-amplified genonmic minutes of transient ischemia. The neurons in the anterior horns (AH) were selectively DNA, to study a CPT-deficient patient who proved to be a compound heterozygote for lost 7 days after 1 5-min ischemia as compared with those of sham-operated controls. In the most common mutant allele, SerI 13Leu, and an unidentified second mutant allele. the normal spinal cords, a number of neurons in the AHs were positive for the nNOS, The studies were performed initially in DNA isolated from peripheral blood leukocytes atid ottly slightly positive for the Cu/Ztn SOD and the eNOS. Immunoreactivities for and confirmed in buccal cells collected with brtshes. studies were then the proteins were indttced at 8-24 hours both after 5- and 15-mnm ischernia. In contrast, cytology Family NT-like immunoreactivity was negative both in the normal and postischemic spinal performed using buccal cell DNA to identify carriers. Buccal cell analysis provides a cords. These results suggest that Cu/Zn SOD- and nNOS-, and eNOS-like cost-effective, non-invasive method for the preparation ofDNA, thus facilitating family immunoreactivities are induced, but that, even though an interaction of Cu/Zn SOD studies in CPT deficiency disorders This technique will now bp, used to study with NO could be present, NT was not detected in the motor neurons in the rabbit individuals who have complete or partial CPT2 deficiencies, screen infants at risk for spinal cords after tranisient ischernia. Other factors could be required for NT formation the lethal infantile CPT2 deficiency, and search for new mutations in patients with CPT found in degenerative motor neuron death in AL-S. The mutant Cu/Zn SOD protein deficiency who have uncharacterized mutations. might play an important role in this process.

2212 Conmptiterized issethods in differentiating Mucopolysaccharidoses (MPIS) types tising TIFF files. K. Yanamandra. B.Y. Lee. S. Krueger. and T.F. Thurmon. Department of Pediatrics, LSU Medical School, Shreveport, Louisiana. Turbidimetric methods were used for initial mucopolysaccharidoses screening on specimens received in our laboratory. Specimens that tested positive were then subjected to TLC procedure on cellulose plates to classify them into different MPS types such as Httrler syndrome, Hunter syndrome, San Filippo, Morquio, etc. Like many other laboratories, we often found it difficult to identify individual mucopolysaccharides, which resulted in ambiguous typing. This, in turn, restlted in more expensive enzyme testing since several enzymes then had to be tested. The present investigation was undertaken to determine if we could improve the TLC procedure with computer imaging. Following TLC, we subjected the chromatogratmss for computer imaging and converted the images into TIFF files which were ttsed to analyze the individual mtucopolysaccharides on SigmaPlots. Our TIFF/Sigmaplot method significantly improved resolution of the mucopoly- saccharides. In the process, we also found that the method was efficientt in qttantitating the chromatographic bands and that the images could be archived for fttture comparative analysis. A380 Published Abstracts: Linkage Mapping and Polymorphisms 2213 2214 Genes linked to systemic hypertension (HTN) and non-Insuiin dependent Genetic mapping of the MUL gene for Mulibrey nanism. K. Avela"2, diabetes meilitus (NIDDM) are not associated with Primary Open Angle M. Lipsanen-Nyman3, J. Perheentupa3, C. Wallgren-Pettersson" 2, S. Marchand4, l Glaucoma (POAG). R.R. Allinaham1, JL. Wiaas2, K.F. Daih1, L. Hemd S. Fauri4, P. Sistonen', A. de la Chapelle'2 and A.-E. Lehesjoki'2. Department of Youn1, M. RPitchardl, D.A. Tallettl, K.H. Jones, E.A. Del Bono2, R. Medical Genetics, University of Ilelsinki, Finland, 2Folkhailsan Institute of Genetics, Abdulmessil M. Ter-minassian3, J.L. Haines3, and M.A. Pericak-Vancel. Duke Itelsinki, Finland, 3Childrens Hospital, University of Helsinki, Finland, 4G4nithon, University Medical Center, Durham, NC1, New England Medical Center, Boston, Evry, France, 5Finnish Red Cross Blood Transfusion Service, Helsinki, Finland. MA1, Massachusetts General Hospital, Boston, MA1. Mulibrey nanism (MUscle-Llver-lBRain-EYe nanism; MUL; MIM No. 253250), is a POAG is a complex genetic trait whose etiology Is unknown but is likely rare auutosomal recessively inherited disorder enriched in the Finnish population. The due to a combination of genetic and environmental factors. HTN and NIDDM basic metabolic defect is not known. The syndrome is restricted to tissues of have been identified as risk factors for POAG. Polymorphisms within or flanking mesodermal origin. MUL. is characterized by growth failure of prenatal onset, genes for angiotensinogen (AGT), glucagon receptor (GCGR), a locus on 20q, hypotonia of the muscles, constrictive pericarditis, hepatomegaly due to passive and glucokinase (GCK) have been linked to HTN, NIDDM, and MODY 1 and 2 congestion, enlarged brain ventricles, yellowish dots in the ocular fundi, and typical (maturity onset diabetes of the young, variants of NIDDM), respectively. Genetic facial features. Hypoplasia of various endocrine glands causing hormonal deficiencies is studies were performed on families using these markers as candidate genes to a common finding. The diagnosis is based on the phenotype. The constrictive determine if any are associated with POAG. Fourteen families with a total of 44 pericarditis is the most important factor in determining the quality of life of the sampled affected individuals were ascertained for POAG by Duke and New patients as well as the prognosis of the disease. We conducted a genome-wide systematic search for linkage in five Finnish multiplex England Medical Centers. POAG was defined as mm Hg lOP>21 and families including 10 affected individuals. So far, 240 microsatellite markers have been glaucomatous optic nerve and visual field loss in one or both eyes. Individuals analyzed. Lod scores suggestive of linkage to markers on chromosome 17q were recently with other forms of glaucoma were excluded from analysis. All family members obtained. We are currently confirming these findings and refilling the localization of the were genotyped using polymorphic intragenic and/or flanking markers for AGT MUL gene by analyzing more markers in the region in an exteded family panel. (1q42-43), GCGR (17q25), the MODY 1 locus (20q12-13), and GCK (7p). Linkage analysis was performed using LOD score and sibpair methodology. LOD score analysis excluded tight linkage in these data when assuming either a dominant or recessive genetic model. In addition, there was no evidence of excess allele sharing among affected sibpairs. POAG does not appear to be associated with AGT, GCGR, the MODY I locus, or GCK indicating that these genes do not confer major risk for POAG. This does not exclude the possibility that other genes associated with HTN or NIDDM may be associated with POAG.

2215 2216 Effect of incorporating simulated loss of heterozygosity data on expected Non-medullary thyroid cancer: attempts to locate susceptibility genes by linkage LOD scores in familial prostate cancer. M.D. Badzioch', R. Pack1, in two Canadian families with goitre and non-medullari thyroid cancer. and M.D. reare". 'UT MD Anderson Cancer Center, Houston TX and 2Institute of G. Bignell'. S. Sun2. P. R. J. Rosenblatt P. H. T. Public Health, Cambridge, UK. Biggs'. Hamoudil. Buu'. Druker2. Hudson3. R.Houlston'. S. Narod4. M. Stratton' and W. Foulkes2 'Institute of Cancer The aggregation of prostate cancer (PC) in families is consistent with autosomal dominant inheritance (Carter, 1992, PNAS). As the gene(s) responsible for the major Research, Sutton, Surrey, UK, 2Department of Medicine, McGill University, Montreal, portion of this complex trait have not been identified, we have collected blood and Quebec, Canada, The Whitehead Institute, Cambridge, MA, 4Women's College tumor samples from 29 affected men and 16 relatives in 10 families for genetic linkage Hospital, Toronto, Ontario, Canada. analysis. To estimate statistical power to detect a major gene we applied a marker Non-medullary thyroid cancer (NMTC) has a genetic component. Case-control simulation and analysis technique which includes tumor loss of heterozygosity (LOl) studies have showed that there are elevated relative risks for NMTC (range 5-9) observations in the likelihood calculations through three additional parameters, A and associated with a family history of NMTC. We are studying two Canadian families t, the probabilities of observing loss of one or both marker alleles in the tumor, with multiple cases of non-toxic goitre and/or NMTC. Family 236, originally described respectively, and p, the probability that the allele retained did not come from the same parent as the disease-predisposing one (Teare, 1994, J Med Genet and Rohde, 1995, by Couch in 1986, is a large pedigree of 80 related members, 19 of whom have had Hum Hered). We defined six age classes for the men and all women were set as non-toxic goitre. Many have had thyroid surgery followed by replacement therapy. One phenotype unknown. A and e were set to 0.7 and 0.05, respectively. individual has also developed microinvasive NMTC. Family 152 is a smaller pedigree The average simulated LOD score using an 8 allele marker locus with allele frequencies of but all four individuals in the third generation have been affected by goitre and/or either 0.1 or 0.3 for the ten pedigrees was 1.32 with a maximum LOD score of 4.7. These NMTC. In addition, one individual died of an extramedullary PNET at the age of 20. results reflect assumptions of complete genetic homogeneity and close meiotic linkage, 0 In total, 7/14 related members of the pedigree have had goitre and/or NMTC. = 0.0. Two values of p, 0.0 and 0.1, were used in simulating and analyzing the LOll. With p = 0.1, the average LOD score was 1.66 and the maximum was 5.8. These figures Linkage analysis has been carried out at several candidate gene loci, including increased to 2.13 and 6.06, respectively, when p was set to 0.0. Simulation of a single 7SII-R, RET, thyroglobulin and THRAI. The lod scores at these loci are shown below a score affected father-son pair resulted in maximum of 0.59 when tumors were assumed locus narkers LOD score (pd 236 only available from both individuals. Overall, the expected advantage in collecting tumor Thyroglobulin DBSS57.DSS284 -2.83,-2.07 @0=0.1 samples was found in this assessment of familial prostate cancer pedigrees. The increase RET stcl.2 4.78*@O=0.0l in LOD scores was especially significant for isolated father-son pairs when tumors were TFIRAl - -2.36*wO=0.0l available from both individuals. However, due to the complex nature of the disease TSH-R D14SlO00 -1.93* (@=0. 1: 0.17) it will likely require several-fold more families than presented here to localize the loci As a result of these negative findings, we are now proceeding to a genomic search responsibility for inherited predisposition. (This work was supported, in part, by an NCI using 240 markers evenly spaced the genome. R25 Predoctoral Fellowship in Cancer Prevention to MDB.) througout This will be completed and presented.

2217 2218 Assessment of the possible contribution of the hearing impairment loci Refining the location of the BRIC/PFIC sene through identification of DFNA1 10 and DFNB1 9 in two populations; the United Arab Emirates shared haplotypes. L.N. BIull, J.A. deYoung, N. Stricker', V.E.Hl. Carlton', and the British Pakistani population. K.A. Brownl, G. Karbani2, G. Parry3, S. Baharloo', R. Sinke2, S. Sela-Hlerman', P.F. Whitington3, N. Lomri'l L.M. Moynihan'. A.H. Janjua4, L.I. Al-Gazali5, V. Newton4, A.F. Markham', B. Scharschmidt',R}.H.J. Houwen2, A.S. Knisely4 and N.B. Freimer'. 'University of R.F. Mueller2. 'Molecular Medicine Unit, University of Leeds, Leeds, UK. 2Yorkshire California, San Francisco; 2Wilhelmina Children's Hospital, Utrecht, The Netherlands; Regional Genetics Service, St James's University Hospital, Leeds, UK. 3Child 3University of Chicago; 4Children's Hospital of Pittsburgh and University of Development Centre, St Luke's Hospital, Bradford, UK. 4Department of Audiology and Pittsburgh. Education of the Deaf, Manchester University, Manchester, UK. 5Department of Loci for two autosomal recessive liver diseases, benign recurrent intrahepatic Paediatrics, Faculty of Medicine and Health Sciences, United Arab Emirates cholestasis (BRIC) and progressive familial intrahepatic cholestasis (PFIC), have been University, Al-Ain, United Arab Emirates. mapped to 18q21-q22 in Dutch and Amish families, respectively (Ilouwen et al., 1994; To date ten loci for autosomal dominant hearing impairment (DFNA1 10) and nine Carlton et al., 1995), suggesting that these diseases may both be due to mutations in - loci for autosomal recessive hearing loss (DFNBI 9) have been identified, the latter in the same gene. To refine the localization of the BRIC/PFIC geuse, we have searched for a number of genetically isolated populations by autozygosity mapping. Each shared haplotypes in affected individuals, using microsatellite markers from the region. genetically isolated population is expected to have a unique subset of genes involved in We identified marker haplotypes conserved between PFIC-affected individuals in inherited hearing impairment. However, a number of the genes in that subset will also several Amish and non-Amish families. The pattern of haplotype sharing localizes the be involved in genetic hearing impairment in other populations. In addition, a locus PFIC gene to a region of about two cM. We also found evidence for locus heterogeneity implicated in dominant hearing loss in one population may contribute to recessive among Amish as well as non-Amish families. forms of hearing loss in other populations. We examined the inheritance ofBRIC in Dutch and non-Dutch families. We identifed To compare the relative frequencies of the known hearing impairment loci in two two distinct extended haplotypes among affected individuals from these families. The populations of interest we have analysed, by autozygosity mapping, 27 consanguineous pattern of haplotype sharing places the BRIC gene in the same two cM region as the families from the British population originating from the Mirp1sr region of Pakistan PFIC gene, supporting our initial hypothesis that the two disorders are caused by and 12 consanguineous families from the United Arab Emirates (UAE), all of which mutations in the same gene. segregate non-syndromal autosomal recessive hearing impairment. We are using additional microsatellite markers to refine the localization of the Preliminary data suggest that the UAE population segregates the recessive loci DFNB4, IIRIC/PFIC gene, through mapping of the borders of known shared haplotypes, and DFNB8 and DFNB9, with one family putatively linked in each case, whilst the British identification of shared haplotypes in additional individuals. Pakistani population segregates the loci DFNB1, DFNB3, DFNB4 and DFNB5 with 4, 1, 3 and I fanuilies potentially linked in each instance. In addition, our data also suggest that dominantloci may be implicated in autosomal recessive hepring impairment in both popuilations; D)FNA2 and DFNA8 in one and two faniilies rdpectively from the UAE and DFNA5 in one family from the British Pakistani population. Published Abstracts: Linkage Mappi ng and Polymorphisms (cont.) A381 2219 2220 Investigation of genetic variability in the human serotonin 5-HT5A receptor Clinical and linkage studies in three large Belgian families with affective disorder. gene S. Claes'. P. Raevmaekers' 2, G. Verheven2. M. van den Broeck2. S. Diegendaele3. S Cichont,J Erdmannt.4MM N 1then D Shimron-AbarbanellJ, M Rietsche2, M Al- A. Tanahe3. J. Godderis'. JJ. Cassiman' and C. Van Broeckhoven2. 'Center for b3, M Borrmann3 W Maiera Franzek4. J KmrnegZmB R Fiminersa Human Genetics and ' Department of Psychiatry, University of Leuven, Belgium. Wcig& 2Laboratory for Neurogenetics, Born-Bunge Foundation, University of Antwerp, 1Propping' Belgium. 3Psychiatric group practice, Brugge, Belgium. (1) Inst Hum Genet and (2) Dept Psychiatriy, Univ Bonn, Germany (3) Mental State Three large Belgian families with one BPI proband and several relatives with Hospital Haar, Germany; Depts of Psychiatry (4) Univ Wtrzburg (5) TU Dresden, affective disorders were sampled. All family members were administered a Germany (6) Dept Medical Statistics, Univ Bonn, Germany structured interview, and diagnoses were determined using a consensus procedure. Serotonin (5-hydroxytryptamine, 5-HT) is a neurotransmitter in the central and peri- In total, 4 individuals with a diagnosis of bipolar , 6 with bipolar 11 and 19 with pheral nervous system mediating a wide variety of sensory, motor, and cortical recurrent major depression were considered as affected. Healthy relatives were functions through multiple 5-HT receptor subtypes. Since disturbances of serotoner- divided according to age in 4 penetrance classes. Linkage simulations studies gic pathways have been strongly implicated in a variety of neuropsychiatric disorders showed that under a homogeneity model, an average maximal LOD score of 5 can be expected. However, each of the families has a of genetic variation in genes coding for serotonin receptor proteins might well be invol- separately power nearly 50% to reach a LOD score of 2. to We have to ved in the genetic predisposition the diseases. sought identify genetic Chromosome 18 was selected as a candidate region, because of recent positive variation in the 5-HT5A receptor gene by screening genomic DNA samples from 137 linkage findings by several groups. One marker on 1 8p and seven markers on 1 8q unrelated subjects (including 45 schizophrenic, 46 bipolar affective and 46 healthy were analyzed. With the 18p marker, negative LOD-scores were found in all controls). Fragments encompassing the whole coding sequence (1047bp) as well as families. In the investigated interval on 1 8q, small positive LOD-scores were found intron-exon boundaries (101 bp) and 5' (51bp) and 3' (l9bp) untranslated regions, with marker D18S51 in family H and with marker D18S41 in family C. However, were amplified tising the polymerase chain reaction (PCR). PCR products were sub- multipoint linkage analysis excludes linkage with this region in these two families. sequently screened for sequence variation using single strand conformation analysis. Other candidate regions that were investigated are chromosomes 4p, 1 2q1 4-q24, We identified two silent mutations in the coding region (A-sT at nucleotide position 21q22.3 and 22q1 1. LOD scores were negative in all families for all markers, making the presence of a major disease gene in these candidate regions unlikely. (nt) +12, and C-+T at nt +789) and two nucleotide substitutions in the 5' untranslated Apparently, genetic factors in other chromosomal regions determine the region (G-+C at nt -19, and C-*T at nt -18). Allele frequencies did not differ signifi- vulnerability for affective disorders in the presented families. cantly between bipolar affective, schizophrenic, and control groups. In conclusion, our study does not provide evidence that the 5-HT 5A receptor gene plays either a major or a minor role in the genetic predisposition to schizophrenia or bipolar af- fective disorder.

2221 2222 Searching for a chromosome 6 association with late onset Alzheimer Disease. Linkage studies of bipolar disorder with chromosome 18 markers J. S. Collins. R. T. Perry. B. Watson. S. S. Bassett. D. A. Myers. M. S. Albert. R. N. Craddock, I'. Bowen, C. Kirov, M. Gill2, C. Spurlock, II. Valladal, .1. Powell', R, Tanzi. L. Rodes. J. L. Haines. D. Blacker. and R. C. P. Go. University of Murray', P. McGuflin, D. Collier', M.J.Oweii. University of Wales College of Medicine, Alabama at Birmingham, Birmingham, Alabama, Johns Hopkins University, Cardiff, UK; 'Institute of Psychiatry, London, UK; 2Trinity College, Dublin, Eire. Baltimore, Maryland, and Massachusetts General Hospital, Boston, Evidence consistent with the existence of one or more bipolar disorder susceptibility Massachusetts. loci on chronmosome 18 has been reported by IBerrettini et al (1994) and Stineet al (1995) Alzheimer Disease (AD) is a dementing disease which has been estimated to in the pericentromneric region, Freimer et al (1996) on distal 18q and Stine et al (1995) on affect 50% of the U.S. population ages 85 and older. There are currently four inre proxitnal 18q. Somiie analyses indicated greater evislence for linkage in pedigrees in autosomal dominant types of AD described in the literature. Three are early onset AD which have which paternal transmission of disease occurs. We have undertaken linkage analyses using forms of been mapped to chromosomes 1, 14, and 21. The ApoE 16 highly pnlyniorphic markers spanning both "Iterrettini" and "Freimer" regions in our e4 allele on chromosome 19 is a risk factor for a late onset form of AD. We sample of 48 UK bipolar pedigrees. There was no significant evidence for using propose that a region on chromosome 6 is also associated with late onset AD in linkage families. AD has been previously associated with the HLA class I antigen alleles either paranietric or non-parametric analyses although small positive lod scores (max. A2, B7, B15, B16, and Cw3, the class It antigen allele DR3, and the class III 0.37 with D18S469) were found with some markers in the "Freimer" region. Evidence complement marker allele C4B2. Information was gathered by the NIMH for linkage was sot significantly increased by analyses that allowed for heterogeneity or Genetics Initiative AD Study Group as part of a genome wide scan for late onset for differing recombination fractions in males and females, nor by analysing the subset AD genes. This study consisted of researchers and patients from the University of of pedigrees consistent with paternal transmission. Alabama at Birmingham, Johns Hopkins, and Massachusetts General Hospital. The eight markers on chromosome six used in these analyses were D6S285, D6S251, D6S254, D6S943, D6S105, D6S259, D6S263, D6S271. Sib-Pair analysis encompassed 275 families with two or more siblings affected with late onset AD. Families were also grouped by ApoE allele status and 85 families contained one or more siblings homozygous for the ApoE e4 allele. The SAGE program SIBPAL was used to test that the proportion of genes identical by descent (IBD) is greater than the expected 0.50 in concordant affected sibs. In the subset of families with ApoE e4 homozygous individuals, marker D6S259 was significant at p=0.05 at a mean of 0.54. This marker is located about 17 cM telomeric to the HLA region and additional markers in thig area of interest will be genotyped. If confirmed, this finding may signify a new region on chromosome 6p involved in late onset AD and possibly related to ApoE allele status.

2223 2224 Exclusion of timp3 as a candidate locus in age-related macular Identification of Five SSCP-polymorphisms in the human glucocorticoid degeneration. MA DeLaPaz', JM Seddon2, RM Heinis', F Lennon', .L llaines3, receptor gene. R.H. de Rijk. G.G. Turner*. P. Emery#. E.M. Sternberg. P.W. MA Pericak-Vancel. iDuke University Medical Center, Durham, N.C.; 2Massachusetts Gold and S. Detera-Wadleigh* Clinical Neuroendocrinology and Clinical Eye and Ear Infirmary and 3Massachusetts General Hospital, Boston; Neuro-genetics Branches (*), NIMH, NIH, Bethesda, MD, USA and Age-related macuilar degeneration (AMD) is a genetically complex disorder. The Rheumatology and Rehabilitation Research Unit (#), University of Leeds, U.K. tissue inhibitor of metalloproteinases-3 (TIMP3) gene on chromosome 22 has been During inflammation or immune challenge the adrenal glands release identified as the gene that is mutated in Sorsby's fundus dystrophy, a rare macular increasing amounts of glucocorticoids which are the most powerful endogenous dystrophy which resembles clinically AMD. The purpose of this study was to determine whether TIMP3 is a susceptibility gene for the AMD phenotype. inhibitors of immune functions. Animal models resembling human autoimmune 45 multiplex AMD families were identified in Massachusetts and North Carolina. diseases, suggest a role for these hormones in the pathogenesis of human Individuals with extensive intermediate drusen, any large drumsen, geographic atrophy, autoimmunity. Since the efficacy of glucocorticoid action is mediated trough the or evidence of exudative maculopathy were coded as affected for the purpose of the glucocorticoid receptor (GR), we screened the GR-gene in patients with analysis. Linkage analysis was performed using both model dependent (Lod score) and Systemic Lupus Erythematosis (n=40), Rheumatoid Arthritis (n=30) and in model independent sibpair methods. For the Lod score analysis, both autosomal matched controls (n=25). Using single stranded conformation polymorphism dominant as well as recessive low penetrance "affecteds only" analyses were examined. (SSCP) and sequencing according to the method of Sanger we identified five Three markers, D22S280, D22S529, D225268, tightly linked and flanking the TIMP3 hot-spots for polymorphism of the 9 exons (exons 2-8, exon 9a & 9P) locus, were chosen for the analysis. Association studies were performed by examining examined. In intron 5 we identified a transversion from G/C to T/A in several one randomly chosen affected individual per family and comparing the AMD patients patients. Two polymorphisms were identified in exon 9a. The first at position with a series of age, sex, and ethnically matched controls with no known history of 2430, a C/G transition from T/A in tome RA- and SLE-patients and in controls AMI). This polymorphism is in the coding region but does not result in an amino acid Lod score analysis excluded linkage for approximately 10 cM around TIMP3 for all change. The second in the 3' untranslated region, in all three groups, showing a models tested. In addition no significant findings were observed with the sibpair stretch of A's ranging from 16-19. In addition, in exon 9t also two analyses. In conclusion, there was no evidence of linkage or association between AMD and TIMP3, suggesting that although clinically similar, the genetic etiologies of these polymorphism were found in the 3' untranslated region. The first was identified two disorders have of different etiologies. in several RA-patients, shows a G/C to T/A transversion, but this was also found in SLE-patients and controls. The second, seen in some SLE- and RA-patients, was a transition from A/T to G/C, as well in several controls. The clinical implications of these polymorphism with respect to susceptibility in autoimmune disorders remains to be investigated. A382 Published Abstracts: Linkage Mlapping and Polymorphisms (cont.) 2225 2226 AN ID)IOPANrHC CLUBFOOT FAMILY DOES NOT SHOW LINKAGE TO THE St. Louis Breast Cancer Tissue Registry and the Cooperative Breast CI IROMOSOME REGION LINKED TO DISTAL ARTIIROGRYPOSIS Cancer Tissue Registry (CBCTR): A combined resource for marker 1. F.R. Di tzLT.R.Rebbeck2D.D.Blake K.D.Mathewsa. 'University of Iowa, 2University of validation studies. W. G. pillay , M. Schaller ,John Mc2, C. Helms Rod Pennsylvania3. The etiology of congenital clubfoot is unknown. Idiopathic clubfoot may have one or several H. Doniss-Kelle Div. Hum. Mol. Genet., Washington Univ. Sch. Med., St. causes. Clubfoot deformity occurs with syndromes, neuromuscular disorders and chromosomal Louis. MO, St. Lukc's Hospital, Chesterfield, MO. abnormalities; althotigh most clubfeet occur as an isolated anomaly. Distal arthrogryposis (XA) The St. Louis Breast Cancer Tissue Registry is a collaborative effort among 7 St. is an autosomal dominant disorder characterized by clubfeet and arthrogrypotic hand deformities Louis area hospitals (Barnes-Jewish, DePaul, St. Anthony's, St. Louis University, in othersvise normal individuals. DA I has been localized to a 65 centimorgan area of the St. Luke's, St. John's) and is designed to assemble a large set of well characterized pericentromeric region ofchromosome 9 in one large family (Bamshad et al, 1994). We paraffin-embedded tissues from breast cancer cases along with associated clinical evaluated a large family with idiopathic clubfoot for evidence of linkage to this region of and pathological data including at least 5 years of follow-up information. The St. chromosome 9 to test the hypothesis that clubfoot, as an isolated deformity, is afornfirusle of Louis project is part of the National Cancer Institute sponsored CBCTR, which also DA includes 3 other participating institutions: Fox Chase Cancer Center (Philadelphia), 1. Kaiser Fotundation Research Institute (Portland, OR), and the Univ of Miami Our family consisted of 4 generations with 28 members available for genotyping ofwhich I I Jackson Hospital (Miami). The purpose of the CBCTR is to provide the resource have cltubfeet. Genotyping was done using four polymorphic short tandom repeats (STR's) to qualified researchers interested in large-scale testing of potential tumor markers spanning the region ofchromosome 9 liked to distal arthrogryposis. Analysis was performed for prognostic significance. Thus far, the St. Louis project has contributed more using the MLINK subroutine of LINKAGE v 5.1. Parameter estimates for clubfoot heritability than 500 cases to the combined CBCTR database which is available for online were obtained from our prior segregation analysis of idiopathic clubfoot [Rebbeck et al, 1993]. searching access over the internet. More than 4,000 cases are resident in the Model and Assumptions: Maximum likelihood linkage analyses were undertaken using the CBCTR database and it is expected that an additional 4,000 cases will be added method described in Lathrop et al 119841, and implemented in the software package FASTLINK within a year. Each case submitted to the CBCTR database has undergone a recent [Cottitigham et al 1993]. For this pilot analysis, a single multigeneration family was analyzed pathological review according to standardized criteria established by the CBCTR. using four polymorphic markers chromosome 9. The clinical and outcome data include demographic information, diagnosis, tumor 1 here was no evidence of linkage to any ofthe tested loci on chromosome 9. There was size, histopathology (including carcinoma in situ), extent of disease (including 9 the markers for value q<. 1. In involved lyniph nodes and distant metastases), treatment modalities, time-to- evidence of exclusion (i.e.lod <-2) to chromosome at two of recurrence, survival time and vital status. Patient anonymity has been maintained as general, two-point lod score naps are more robust in initial tests of the presence of linkage in a identifiers are not included and only categorical information is available over the specific chromosomnal region [Risch 19901. These results imply that the gene responsible for the internet. Researchers interested in utilizing the CBCTR resource can obtain hereditary pattern ofclubfoot in this single family is not located onichromosome 9. application information by contacting the CBCTR Web site Both DA and congenital clubfoot are probably heterogeneous in their etiology. Our finding (http://wwwicic.nci.nih.gov/cbctr/). does not exclude etiological overlap between some clubfoot and some DA. It does exclude all clubfoot from being a variant of this form of DA.

2227 2228 Pelizaeus-Merzbacher disease (PMD): microsatellite markers, proteolipid Ascertainment of multiplex families with bipolar affective disorder and protein gene (PLP) intragenic polymorphism, carrier detection and Genomic screen of chromosomes 3, 5, 15, 16, 17, and 22. H.J. Edenberg. prenatal diagnosis. S.R. Dlguhy and M.E. Hodes. Indiana University School T. Foroud. P.M. Conneally. J.Numberger. J.R. DePaulo. C. Stein. D. Meyers. A. Goate. J. Rice, T. Reich, E. Gershon, S. Detera-Wadleigh. L. Goldin. M. Blehar. of Medicine, Indianapolis, Indiana. Indiana Univ. School of Medicine, Indianapolis IN; The Johns Hopkins University, PMD is an X-linked disorder of the central nervous system. For some cases, Baltimore, MD; Washington Univ., St. Louis, MO; NIMH, Bethesda, MD. a mutation has been found in the PLP protein coding region. In a large As part of a four-center study supported by NIMH, we have ascertained 161 kindred (originally described by Watanabe et al., 1965), affecteds present with families with multiple cases of major affective disorder. Each family was ascertained nystagmus and poor head control in the early postnatal period. Studies of this through a Bipolar I proband and includes at least one Bipolar I or Schizoaffective (BP family by us and others failed to find a mutation in the PLP coding region type) first degree relative. The total number of affected subjects is 744. A DIGS (Trofatter et al., 1989; Hudson, et al. 1989) or in putative 5' control elements. interview was administered to 1458 persons; cell lines were stored from 1396 subjects. X- We have carried out a preliminary screen of chromosomes 3, 5, 15, 16, 17, and 22, To provide a basis for counseling, we conducted linkage studies with as part of the NIMH-sponsored initiative on the genetics of bipolar disorder. chromosome microsatellite markers in a 15-member subset of the family. The Gcnotyping was performed on a set of 540 DNAs from 97 families, enriched for overall results suggested that the disease-causing gene was located in or affected relative pairs and parents where available, and including sorne common near the PLP region. A lod score of 2.76 (theta 10-6) was obtained with relatives. The sample included 31 individuals with SABP, 231 with BPI, 70 with BPII DXS178. Other markers gave varying amounts of recombination with the and 80 with MDDR. We examined a set of three hierarchical diagnoses of affected individuals: model I included schizoaffective, bipolar type (SABP) and bipolar I (BP I), disease-causing defect. A female of the family contacted us concerning model II included model I plus bipolar II (BP II), model III included model II plus carrier detection. Haplotype analysis with some of the previously tested unipolar recurrent depression (MDDR). Under model I, there were 118 affected sibling "flanking' markers suggested that she was not a carrier. However, the pairs, and 227 affected relative pairs. Model II contained 193 affected sibling pairs and woman's brother was recombinant for MFD66, and two of her maternal uncles 324 affected relative pairs and model III included 269 affected sibling pairs and 412 were recombinant for DXS424 and MFD79. We then tested a polymorphism affected relative pairs. To date, forty-five markers have been typed on this set of et in the first intron of PLP. The disease DNAs. Initial analysis of nonparametric affected sibling pairs, using the SIBPAL (reported by Mimault al., 1995) program to lest for excess allele sharing, did not provide strong evidence for linkage. segregated concordantly with an uncommon allele of the polymorphism (even Only one marker was significant under all three models, with 0.05

2229 2230 Evidence for genetic heterogeneity among individuals with clinical Identification of chromosome band specific dinucleotide polymorphic markers diagnoses of Congenital Fibrosis of the Extraocular Muscles. E. C. from microdissection/microcloning library of 4q35 Enle'. R . Johnson'. M. H. Jabakt. A. Assaf'. R. S. Hepler5. T. Zwasn3. A. H Chaohong Fan 2, Xiaoxuan He 2, Han-Xiang Deng', Teepu Siddique' ggggs'. 'Children's Hospital and Harvard Medical School, Boston. MA; Childrens 'Dept. of Neurology, Northwestern University Medical School Orthopedic Hospital and University of Washington Medical Center, Seattle, WA; "King 2 C.P.Li Biomedical Research Corporation Khalcd Eye Specialist Hospital, Riyadh, Kingdom of Saudi Arabia; 4Stoke Mandeville Human chromosome band 4q35 is an area of interest as the gene Hospital, Aylesbury, England; "Jules Stein Eye Institute, Los Angeles, CA. responsible for facioscapulohumeral muscular dystrophy (FSHD) is regionally 'Classic' congenital fibrosis of the extraocular muscles (CFEOM) is a genetically localized where few DNA polymorphic markers are currently available. To homogeneous. fully penetrant disorder that maps to the centromeric region of facilitate mapping and eventually isolating this gene, we have constructed a band- chromosome 12 and is characterized clinically by bilateral ptosis, restrictive infraductive specific genomic DNA library derived from 4q35 with microdissection and external ophthalmoplegia, and markedly limited and aberrant residual eye movements. microcloning techniques and identified novel subregional microsatellite markers. We have studied three families with congenital, static, and restrictive eye movement The dissected DNA from human chromosome band 4q35 were amplified disorders diagnosed as CFEOM that we believe are clinically and genetically distinct. with a universal primer by polymerase chain reaction (PCR) followed by the The first family has two affected siblings and demonstrates consanguineous autosomal digestion of amplified products with EcoRl and then cloning into pUCI9 plasmid recessive inheritance. Both children have bilateral plosis and total (external and vector. The chromosomal localization of PCR amplified microdissected products internal) ophthalmoplegia. In one the eyes are supraducted and in the second they are were confirmed by fluorecent in situ hybridization to normal human metaphase midline. The second family includes four affected individuals and one obligate carrier, chromsome spreads using the PCR products as probe. The generated 4q35 band- demonstrating autosomal dominant inheritance with partial penetrance. Two pecific plasmid library was screened for dinucleotide repeat using a radiolabeled individuals are classically affected, one is unilaterally affected, and one is bilaterally probe containing (CA),, repeats. A total number of 20,000 plasmid recombinants affected with the globes fixed in a supraducted position. In the third family, a mother were screened and 43 positive clones were isolated. Thirty five positive clones and one of her two children are affected. The mother's findings are classic, whereas were identified by sequening as containing (CA)n/(GT)n repeats. Three of them the affected child is only unilaterally affected. exhibiting over 20 (CA)/(GT) units were further characterized for their Linkage studies demonstrate that the first family's disease gene is not linked to the polymorphisms. The chromosomal specificity of these repeats were further classic CFEOM locus. Results from the second and third families are more ambiguous. confirmed by human-rodent hybrid cell panel analysis. Allele sizes and In each family all affected members share the same haplotypes for makers spanning the frequencies were determined in 50 unrelated individuals for each repeat. The CFEOM critical region on chromosome 12. However, several clinically unaffected observed helterozygosities for these three repeats were 0.61, 0.77 and 0.81 with family members have also inherited these haplotypes. Therefore, these families must observed respective allelic variants as 8,9,and 14. These three novel polymorpphic either have mutations at other loci, or their disorders exibit markedly reduced markers should assist in the fine mapping and molecular cloning of the gene for penetrance, a finding in sharp contrast to the fully penetrant inheritance pattern found in FSHD and those on 4q35. classic CFEOM families. (GenBamik accession nsmmbers are G16212, G16213, and G 16214) Acknowledgements: This work was supported by NIH grant #GM48291-03. Published Abstracts: Linkage Mapping and Polymorphisms (cont.) A383 2231 2232 Mutation screening of the ar,-adrenergic receptor gene (aOAR) in psychiatric patients. Genetic analysis of IgA nephropathy (IgA(;N), using whole genome screening and J. Feng'. J. L. Sobell'. D. Goldman'. E. Cook'. L. L. Heston'. S. S. Somner'. 'Mayo linkage analysis. Tatsuo Fukushima. Shinsuke Nomura. Shinichiro Kawai. Gengo Clinic/Foundation, Rochester, MN; 'NIAAA, Rockville, MD; 'University of Chicago, Osawa. Div of Nephrology, Dept of Medicine, Kawasaki Medical School, Kurashiki, Chicago, IL; 'University of Washington, Seattle, WA. Japan. The adrenergic receptors (AR), members of the G protein-coupled receptors superfamily mediate the diverse metabolic and neuroendocrine actions of epinephrine and norepinephrine. Background: The cause and pathogenesis of the most common glomeniar disease, There are three major types of AR: a,, o%, and A; each of these types has at least three IgACGN, are not clearly understood, though it has been suggested that this disease might subtypes. All AR types are expressed in the adult brain, with certain subtypes highly be a polygenic disorder because of the racial difference in its prevalence and familial expressed during fetal development. In various studies of psychiatric patients, alterations in clustering. Recently, we proposed a clue for determination of the genetic factor of the adrenergic receptor expression or function have been suggested (e.g., alterations in 13, and disease by genetic analysis (Nomura et al. Nephron: 73: 104: 1996). This report of increased concentration 1, AR expression in limbic brain structures schizophrenics; ,AR suggests the possibility of homozygosity mapping to detect the susceptible loci if this in prefrontal cortex of suicide victims; efficacy of %AAR agonists in amelioration of method is applied to inbred IgAGN patients. Therefore, we searched for regions with hyperarousal phenotypes in attention deficit hyperactivity disorder [ADHDI and autism). One high homozygosity-by-descent (HBD) among the whole genome of inbred IgAGN mechanism by which ARs may be involved in the pathogenesis of psychiatric disorders could patients, and carried out linkage analysis using the DNA markers of these loci. be through sequence changes that altered protein function. To begin to test this hypothesis, AMaterial & Method: Study I (HBD screening): To detect the HBD, we investigated the the asAR gene was screened in patients with either schizophrenia (n= 95), ADHD or autism homozygote of microsatellite markers prepared at every 15cM on all chromosomes (n=35), or alcoholism (n=44) (total = 174). The entire coding region was examined for among 16 inbred patients with IgAGN. Study 2 (linkage analysis): We used linkage sequence changes, using restriction endonuclease fingerprinting (REF). REF is a screening analysis among 20 familial IgAGN patients to investigate each locus which considered method that can detect virtually 100% of mutations in DNA fragments as large as 2 kb (Liu to be a candidate locus as a result of study 1. and Sommer 1995). Of the 174 patients screened (278 kb of total sequence), 4 different Result & Discussion: We found high prevalence of HBD sharing at the loci shown in abnormal electrophoretic fingerprints were found. By direct sequencing, two mutations have the following table. Linkage analysis was used in only 2q region. However, no positive been identified thus far. The first change, C74G, results in an A25G missense mutation. LOD score has been obtained. It is suggested that there are causative genes close to One Finnish alcoholic patient was found to be heterozygous for this change. The second these regions. identified change, C1091A, is silent. Among schizophrenic, autistic, or ADHD caucasian (g; not vet) patients of primarily western European descent, the frequency of the minor allele is .035. CHR Marker HBD share Linkage CHR Marker HBD share Linkage A tenfold greater frequency (.362) for the minor allele was observed in Native American Ip DIS 551 63% # 6p D6S 273 50% # (Pima) alcoholic patients, whereas none of the Finnish alcoholics carried the A allele. Iq DIS549 50% # 6q D6S264 81% 1 Additional studies are being conducted to assess the association of the missense change with 2p D2S 405 44% not linked l4q D14S 257 50% 1 disease and to identify the sequence changes underlying the remaining two aberrant fingerprints. *Liu Q, Sommer SS. BioTech 18: 470-77; 1995.

2233 2234 Analysis of candidate gene pigment epithelium-derived factor (PEDF)for The dopamine DRD2 receptor: no linkage of Ser3lICys and other DRD2 the autosomal dominant retinitis pigmentosa(adRP)locus RP13 on human markers to alcoholism or substance abuse in Southwestern American Indians. chromosome 17p13.3. D.D.Geyerl,2, C.lleinzinann23, G.E.Tiller', I.R.Rodriguez5, D. Goldman*. M.Urbanek. D. Guenther. R. Robin. J. Long Lab. of Neurogenetics, K.Mazuruk5, T.L.Kojis3, P.Flodman6, M.A.Spence', G.J.Lhaders, J.R.lleckenlively3, National Institute on Alcohol Abuse and Alcoholism, Rockville, MD 20852. and of Children's Ser3l ICys is the most abundant of three naturally occurring DRD2 amino acid J.B.Batemanl,23. Dept. Ophthalmology,'The Hospital, UCHSC, substitutions. It is also the only DRD2 polymorphism thought to affect receptor Denver, CO; Eleanor Roosevelt Institute,2Denver, CO; Jules Stein Eye Institute,3Dept. function lCravchik et al, AJHG, 57: A189, 1995). of 'Vanderbilt School of (impairing signal transduction) of Ophthalmology, 3UCLA, CA; Dept. Pediatrics, University an we observed that 1 is in of Using allele-specific assay, Cys3l surprisingly frequent Medicine, Nashville, TN; National Eye Institute, 5NIt, Bethesda, MD; Dept. a Southwestern American Indian population: 0.16 as compared to 0.03 in Pediatrics, 6UCI, CA Caucasians. Association studies to the TaqI "Al" allele had implicated DRD2 in Retinitis pigmentosa (RP) is a genetically heterogeneous group of disorders. Seven loci alcoholism and in "Reward Deficiency Syndrome." However, a substantial volume have been implicated in autosomal dominant RP (adRP). We reported RP segregating of evidence is not only contradictory but provides alternative explanations. These in a large seven generation family (UCLA-RPO9) and linked to markers on distal 17p include 2-4 fold population differences in TaqIA "Al" allele frequencies. The (Kojis et al.,Am. J. Hum. Genet. 58:347-355, 1996). The pigment epithelium-derived abundance of Cys3lI in this particular population enables us to directly test the factor (PEDF) locus has been mapped to 17pt3.1 by Tombran-Tink and colleagues hypothesis that functional genetic variation of DRD2 is associated with alcoholism (Genomics 19:266-272, 1994) and provides an intriguing candidate gene for adRP. Using or "Reward Deficiency Syndrome." We studied 460 psychiatrically interviewed YAC clones and genetic linkage analysis in CEPH families, we mapped PEDF to a more subjects who were members of three interrelated superpedigrees. Cys3 1I, the distal location on 17p in the region of the UCLA-RPO9 locus. Linkage between RP TaqIA polymorphism, and the intron-2 STR were genotyped. There was no and PEDF was assessed in this family initially using newly identified SSCP's in exon linkage (by SIBPAL) or association (by chisquare analysis) of Cys3l 1, the TaqIA 4 of the PEDF locus, which was relatively uninformative. Subsequent screening of two downstream marker or the intron-2 STR to DSMIIIR alcoholism or substance polymorphisms in intron 6 and exon 7 of PEDF, by sequence analysis, yielded significant abuse. As shown, the proportion of Ser3l ICys alleles identical by descent (i.b.d.) positive lod scores over 4.0 as calculated by LIPED. Two of 46 genotyped family members did not differ in sibpairs that were unaffected, discordant or concordant for were apparent recombinants; however, their sample integrity and or clinical status is in alcoholism. Nor did they differ for substance abuse. Frequencies of alcoholism and question. Accordingly, we conclude that PEDF must remain an attractive candidate substance abuse in the fifteen Cys3 I l/Cys31 1 homozygotes were equivalent to gene for adRP because of its expression pattern, function and chromosomal location. population prevalences for these disorders. NIH EY08282 Sibpair Type # of Pairs-EtOH Alleles i.b.d. # of Pairs-Drug Alleles i.b.d. Unaffected 41 0.511 148 0.490 Discordant 143 0.486 167 0.504 Affected 189 0.488 58 0.447

2235 2236 Generation of valid genotypic configurationss for multi-allelic loci and Mapping of human SATBI to chromosome 3 Hung W-Y., Fan C., Deng H-X., cornplex pedigrees. S.C.1leath. Ultiversity of W'ashington, Seattle, Washington. Siddique T. Department of Neurology, Northwestern University Medical School. Mdarkov chain Monte Carlo and Gibbs Special A-T-rich binding protein 1 (SATBI), a sequence context-specific bind- (MCMC) methods, particularly sampling, binds to nuclear of DNA become for studies with nmulti-allelic loci ing protein, selectively matrix/scaffold-associated regions have popslar genetic mapping dealing (MARs/SARs). The SATBt binding sites consist of a special AT-rich sequence context in and/or complex pedigrees because in these situations, conventional likelihood cal- which one strand is well-mixed A's, T's, and C's, excluding G's (ATC sequences). Cheris- culations are often infeasible. They allow the sampling of genotypic configurations ical interference assays show that SATBI binds along the minor groove with very little from the conditional distribution of genotypes given the data so that Moitte Carlo contact with the bases, suggesting that SATBI recognizes the ATC sequence indirectly the altered backbone structure in the double-stranded estimates of likelihoods or posterior parameter densities cats be made. The pro- through sugar-phosphate present DNA. MARs or SARs have an size of 500 are about every 30 kb, and cedure an initial genotypic cotfigstsation, consistent with observed data, average bp, spaced requires are control elements maintaining independent realms of gene activity. As a DNA binding the getteration of which is not trivial with complex pedigrees and multi-allelic loci. protein, SATBI has motif consisting of 15 glutamine repeats, a feature related to some A inetitod to generate feasible genotype configurations in these circumstances neurodegenerative diseases. human SATBI gene has been cloned but the location on is l)resented. Thie method as presented works with issdividslal loci but could be chromosome remains unknown. In order to map this gene, we synthesized DNA primers extended to handle multiple loci simultaneously, which is itoportant for multipoint based on cDNA sequence to amplify human specific DNA fragment from human-rodent cell The results that SATBI is located on chromosome 3. Several linkage analysis. The method is illustrated by generating feasible genotype con- hybrid panels. suggest genomic DNA clones ranging 18kbto 22kb in size have been isolated from bacteriophage a allele marker on a human figurations for 16 (lILA-B) complex tiulti-generation genomnic DNA labrary. Regional mapping of this gene is under way. pedigree of 1373 Newfoundland individuals, of which :z: 50% were typed. A384 Published Abstracts: Linkage Mapping and Polymorphisms (cont.) 2237 2238 Linkage and association studies of the Natural Resistance Associated Possible involvement of a region on chromosome 8 in the genetic susceptibility to Macrophage Protein (NRAMP1) locus in rheumatoid arthritis. John S, schizophrenia in the UK and Iceland. Marlow A, Hajeer A, Ollier W, Silman A, Worthington J. ARC ERU, University of G. Kalsi. J. Brynjolfsson.Th. Sigmundsson. D. Curtis. R. Butler. T. Read. P. Murphy. H. Manchester, Manchester UK. Petursson. H. Gurlina, Molecular Psychiatry Laboratory, Academic Department of Psychiatry, A candidate gene for a role in susceptibility to RA is NRAMPI, the human homo- University College London Medical School, Windcyer Building, 46 Cleveland Street, London W I P logue of the murine macrophage resistance gene Ity/Lsh/Bcg, which controls recessive 6DB. Department of Psychiatry, Borgaspitalinn, Reykjavik, Iceland. Joint Academic Department susceptibility to intracellular pathogens such as mycobacterium bovis. The gene acts ofPsychological Medicine, London Hospital Medical College, 3rd Floor Alexandra Wing, Turer early in the macrophage activation pathway, leading to upregulation of TNF ILl in- Street, London El 2AD. ducible nitric oxide synthase (NOS2) and MHC class II expression. These proteins have important roles in the induction and maintenance of inflammatory pathways in RA. Possible genetic linkage to schizophrenia was found by Pulver et al (1994) who studied sixteen Linkage analysis and association studies have been carried out to determine if there is linkage markers on chromosome 8p22-21 as part of a genome wide search. They found a any evidence of linkage between NRAMP1, and RA Two dinucleotide markers, D2S1471 maximum admixture lod of2.35 at D8S136 for a dominant model and 2.20 for a recessive model. a highly polymorphic marker, within 160Kb of NRAMP1, and a less polymorphic marker in the promoter region of NRAMP1, have been analysed in 115 affected sib-pair RA fam- We have attempted to reproduce this potential linkage il a sample ofmultiply affected Icelandic and families selected for their family size and their to resolve ilies (35 with 2 parents, 18 with 1 parent ) from the ARC National Repository and an English schi7ophirenia large power additional 85 probands from the ARC National Twin Study and 96 controls. Individuals heterogeneity of linkage. Using the disease models we have adopted since 1988 (Sherrington et were typed for both markers using a PCR, fluorescence based semi-automated analysis. al 1988) we also found maximum lod scores above 2.00 in a sample of 23 multiplex families with No evidence of increased allele sharing in affected sib-pairs was obtained using IBD, IBS the marker DRS136. Usingthe programme MFLINK (Curtis et al 1995) the maximised lod score and MLS-IBD analysis for the whole data set. A significant increase in allele sharing over models was 2.10 with a gene frequency of 0.1314 and with penetrances: 1O=0.0138, fl was observed with marker D2S1471, (LOD 0.74, p 0.05) in a HLA discordant subgroup =0.0138 and f2=0.2138. Using MFLINK the maximised admixture lod score over models was using 1131 and MLS-IBD. No significant difference in allele frequencies for any of the 2.170 with the proportion offamilies linked (alpha) found to be 70%. The disease gene frequency markers in the association study In a subset of sib-pairs which shared one or zero lILA was 0.1314 with penctrances fO)-0.0104, fl=0.0104 and 12 =0.4104. Further analyses using haplotypes, LOD scores suggestive of linkage were observed. This supprts a potential multipoint lod scores using additional markers also supported linkage in this region. We conclude role for an NRAMP polymorphism in RA susceptibility. that it is possible that this region on chromosome 8 contains a mutation increasing susceptibility to schizophrenia. References: Pulver A.E. et al American Journal of Medical Genetics. 60:252-60 (1995). Sherrington, R., et al (1988) Nature, 336, 164-167. Curtis D, Sham PC (1995): Am J Human Genet 57 (3): 703-716. Acknowledgements: The research wagfunded by MRC project grant no. G880473N, the Icelandic Science Council, Schizophrenia A National Emergency (SANE), the Joseph Levy Charitable Foundation, the Wellcome Trust and the Priory Hospital.

2239 2240 Search for additional genes causing autosomal dominant inclusion body Genetic dissection of complex diseases in isolated populations: linkage myopathies. D. L. Koller', W. C. Nichols2, M. R. Farlow3, P. M. Conneally', disequilibrium mapping of two systemic lupus erythematosus (SLE) D. Ginsburg2, and T. Foroudl. Departments of IMedical and Molecular Genetics and candidate susceptibility genes in the Finnish population. R. Krahe, 3Neurology, Indiana University School of Medicine, Indianapois, and 2Howard Hughes 11. Julkunen2, J. Ignatius', and J. Kere'. 'Dept. of Medical Genetics, Univ. of Medical Institute, University of Michigan, Ann Arbor. Helsinki, 2 Peijas-Rekola Hospital, Vantaa, Finland. We are studying a large kindred which is segregating an apparently unique inclusion SLE is a multi-systemic, chronic autoimmune disease of unknown cause. In addition body myopathy in an auitosomal dominant fashion. Termed spheroid body myopathy to environmental factors, genetic, epidemiological and animal nsodel studies have after the histological appearance of the inclusion bodies, this disorder is highly clearly implicated predisposing genes in the etiology of SLE. Identification of complex penetrant but has substantial variability in severity and age of onset. Clinical features disease genes is complicated by the polygenic nature of susceptibility, as well as locus include proximal weakness of the lower extremities detectable by early adulthood, and allele heterogeneity, so that parametric linkage analysis is often impractical. This progressing to the upper extremities and including other findings such as difficulty in problem is inherent to complex diseases and cannot be solved by changing the research swallowing and a nasal quality of speech. strategy, but by the choice of study population: in isolated founder populations, such The pedigree contains 91 individuals, 34 of whom are affected with the disorder. as the Finnish, these confounding factors are commonly less severe. For a number of Simulation studies have shown that this family has reasonable power for detection Mendelian disorders, linkage disequilibrium (LD) based strategies in the Finnish (mean LOD score=2.99) and exclusion (mean LOD=-2.63) of a disease gene with population have proven instrumental in the mapping and identification of the genes age-dependent penetrance, assuming a 10 cM map of markers with heterozygosity of 75 and their underlying mutations. There is no reason to believe that genes predisposing percent. to complex diseases would not similarly show LD in isolated patient populations. Approximately 200 highly polymorphic microsatellite markers have been typed to We have collected 208 (175 simplex, 33 multiplex) SLE families from Finland. Extensive date. The highest LOD scores achieved have been 1.77 and 1.42 for markers D5S1457 genealogical studies showed a slightly increased prevalence of SLE in the eastern part and D8S1104, respectively. Additional markers in these two regions have not provided of Finland, suggesting one or a few founder mutations in that sub-population. More evidence of linkage. Multipoint analysis of markers on chromosome lq and proximal Ip than half of these families have been linked as distant relatives by tracing church and has strongly excluded the region containing the alpha-tropomyosin gene (TPM3), muta- community records as far back as the early 17th century. Physiological studies of SLE pa- tions in which are known to be associated with autosomal dominant nemaline inclusion tients and mice have shown the interleukin 10 (ILO0) and interferon A and B (IFNA/B) myopathy. levels to be dysregulated. Genetic studies of murine SLE models have also implicated the respective genomic regions to contain major susceptibility loci. Therefore, the hu- man homologous regions on chromosome 1 (iLIO ) and 9p22 (IFNA/B cluster) have been examined for shared genomic segments indicative of LD with intragenic (promoter- associated) and flamikinig microsatellite markers. As part of this ptudy, ILIO was mapped to an 8 cM region in 1q31, and the phyical order of the genes And their flanking linkage markers in Iq31 and 9p22 has been refined further.

2241 2242 A highly informative polymorphism in the human neurotensin receptor gene on Allelic association study of NlaIII and Mspi polymorphisms of chromosome 20. F. Le. X. P. Zeng. and E. Richelson. Laboratory of catechol-O-methyltransferase gene and schizophrenia Y.-R. Lee' Neuropsychopharmacology, Mayo Clinic Jacksonville, 4500 San Pablo Road, C.-H. Cheni2 3, M.-Y. Liul4, F.-C. Wei5, lt.-G. ltwu', K.-J. Hsiaol"4. 'Institute of Jacksonville, FL32224, U.S.A. Genetics, 2Division of Neuropsychiatry, School of Medicine, Yang Ming University; The growing evidence suggests that neurotensin and the neurotensin receptor play 3Division of Psychiatry, Cheng llsin Rehabilitation and Medical Center; 4Department important roles in the etiology of some neurological and psychiatric disorders, of Medical Restarch, Veterans General Hospital-Taipei; 5Hung Chi Psychiatric including Parkinson's disease and schizophrenia. Thus, the gene for the NTR, which Hospital, Taipei; 6Department of Psychiatry, Taiwan University Hospital, Taipei, was mapped by others to chromosome 20q 13, may be used as a candidate gene for Taiwan. genetic analysis of the association between the neurotensin receptor and Catechol-O-methyltransferase (COMT) catalyzes transmethylation from S- neuropsychiatric diseases. We reported a highly informative dotible tetranucleotide adenosylmethionine to catecholamine neurotransmitters, and was thought to be involved microsatellite repeat polymorphism in the neurotensin receptor gene. This repeat in the pathogenesis of mental disorders in light of biogenic amine hypothesis. Recent was found among an almost exclusive CT region (-360 bp), in which there actually studies also reported suggestive linkage of schizophrenia with chromosome 22qll-13, to existed two tetranticleotide microsatellite repeats. The first microsatellite had nine which COMT gene was mapped. To elucidate if COMT gene is a susceptible gene of perfect "CCTT" repeats and the second had seventeen perfect "CTTT" repeats. schizophrenia, we carried out a case-control association study in a Chinese population Further study found that this microsatellite repeat was very polytiorphic, with 23 from Taiwan. The allelic and genotypic frequencies of two restriction fragment length alleles found in DNA samples from 105 unrelated individuals, which we examined. polymorphic (RFIP) markers of COMT gene, namely Nlall at exon 4 and MapI at Many alleles had very low frequencies, among which eleven were below 2%, eight exon 5 were compared between patients and normal controls. The NIalII RFLP at exon were between and four were over 10% 11.90 - The 2-8%, only (range 12.86%). 4 alters amino acid from valine to methionine at codon 158, and is associated with ge- estimated heterozygosity was 0.914 and PIC (polymorphism information content) netically determined and enzyme activity of COMT. The Mspl RFLP is inheritance has been demonstrated in two thermostability value 0.906. Additionally, Mendelian at exon 5 is a novel silent mutation at codon 199, which was identified recently in our for this this is a three-generation pedigrees polymorphism. Therefore, highly laboratory. No differences of allelic frequencies and genotypic frequencies of Nlall and imformative which will be very useful as a marker for polymorphismt genetic MspI polymorphisms were detected between schizophrenic patients (n=177) and normal genetic study of association between the neurotensin receptor and some controls (n=99). Our results suggest that the NIalIl polymorphism at exon 4, and the ncuropsychiatric disorders. (Supported by Mayo Foundation for Medical Education do not underlie the and Rescarch, and grant MH27692 from N.l.M.HI.) Mspl polymorphism at exon 5 of COMT gene genetic susceptibility to schizophrenia. Published Abstracts: Linkage Mapping and Polymorphisms (cont.) A385 2243 2244 Homozigosity mapping of an autosomal recessive form of A pilot genetic study of polycystic kidney disease in Poland. M. Lemmens'. N. demyelinating Charcot-Marie-Tooth disease. E. LeGuern" 2. Bogdanova'. B. Lenicke'. B. Dworniczak'. P. Jasik2. A. Krasniak2. W. Sulowicz2. J. Horst'. 'Institute for Human Genetics, Munster, Germany; 2Chair and Department A. Guilbot'*. M. Kessali. N.Ravisd'. J. Tassin'. T.Maisonobe'.Y. of Nephrology, Jagiellonian University, Cracow, Poland. Agid"2. D. Grid' and A. Bnce 2. both authors contributed equally to Autosomal dominant polycystic kidney disease (PKD) is a common genetic disorder this work.'INSERM U289, 2Fediration de Neurologie, 'Laboratoire de whose frequency in Poland has been estimated at 0,08%. One gene (PKDI) causing this disease is located on the short arm of chromosome 16 and was in la identified Neuropathologie R. Escourolle, H6pital de Salpetriere, Paris, France; 1994. The second gene (PKD2) has been mapped on the long arm of chromosome 4 4Service de Neurologie, CHU Mustapha, Alger, Algeria. in 1993 and it accounts for about 14% of the PKD families in Europe. The third locus is not mapped tip to date and this is a very rare form of Charcot-Marie-Tooth (CMT) disease is the most frequent inherited the disease. Here we report data on the first pilot genetic investigation of PKD in Poland. A peripheral motor and sensory neuropathy characterised by chronic distal total of 187 individuals (102 affected, 62 unaffected and 23 spouses) from 14 Polish weakness with progressive muscular atrophy and sensory loss of the PKD families are undergoing genetic investigation. Linkage analysis with the distal extremities. The dominant form of the disease is genetically microsatellite marker D16S291 has been performed on 10 of the 14 families. The study showed that in 7 pedigrees the disease is caused by mutations in the PKD1 heterogeneous but only one locus has been identified on chromosome gene and linkage to PKDI was unambigously excluded in one pedigrees. In two 8q 3-q21.1 for autosomal recessive CMT. By homozygosity mapping in families clear conclusions about linkage could not be reached. Linkage analysis with gene a large Algerian kindred, we have assigned a second locus for autosomal additional markers from the PKD1 region, namely D4S283, SM6, CW2, CW3 and KG8 as well as microsatellites flanking the PKD2 gene are planned. same locus is recessive CMT. Linkage analysis demonstrated that the These preliminary data reveal a proportion of about lO"/s non-PKDI families in the involved in a second Algerian family with a demyelinatinggMT. Polish population, which is in agreement with the reported one for Europe. Haplotype reconstruction and determination of the minimal region of Collection of new affected pedigrees and completing the linkage analysis for the first two disease loci is needed to confirm this conclusion. homozygosity restricts the candidate region to a 4 cM interval.

2245 2246 Genetic heterogeneity of autosomal recessive non-syndromic hearing Refined mapping of the locus for non-epidermolytic palmoplantar impairment in a Caucasian population. X. C. Li', E. Ives2, B. Ploplis', keratoderma, type Bothnia. L. K. Lind, A. Lundstrbm' and G. Holmgren. S. Lusconbe2, J. F. Battey' and E. R. Wilcoxl. 'Laboratory of Molecular Genetics, Departments of Clinical Genetics/Cell and Molecular Biology and 'Dermatology, Ume! National Institute on Deafness and Other Communication Disorders, National Institute University, Umeg, Sweden. of Health, Rockville, Maryland, U.S.A., 2Medical Genetics Program, Janeway Child I'almoplantar keratoderina (PPK) comprise a heterogeneous group of skin disorders Health Center, St. John's, Newfoundland, Canada. characterized by hyperkeratosis (thickening of the uppermost layer of the epidermis, Iereditary hearing impairment is estimated to cause 50% of all congenital hearing the stratum corneum) primarily of the palins and soles. PPK can be divided into either loss, one of the most common human sensory defects affecting approximately 1 per epiderniolytic (EPPK) or non-epidermolytic (NEPPK), forms that are clinically similar 1,000 live births. Autosomal recessive non-syndromic hearing impairment or over 75% butt microscopically distinguishable. An astosomal dominant variant of NEPPK with of all inherited hearing impairment and is highly heterogeneous. A higher degree of diffuse skin lesions is a frequent disorder in northern Sweden. This form of NEI'PK is genetic heterogeneity in the Caucasian population has been demonstrated at both the characterized by hyperkeratosis, but also by frequent dermatophyte infections of palms chromosomal and allelic levels by a linkage study in a large isolated consanguineous and soles, a complication rarely seen among other PPK patients. Caucasian kindred with non-syndromic hearing impairntent. At the chromosomal level, Previously, we have examined two Swedish families with NEPPK and localized the there are at least two loci on different chromosomes that are linked to the defective genes causative genetic defect to chromosome 12q11-q13, a region known to contain the type in this family. The hearing impairment gene among one subgroup of this family is linked II keratin gene cluster. We have now tested these families for mutations in the keratin 1 to chromosome 7q31, the DFNB4 locus. Pendred syndrome, an autosomal recessive gene and havefound several polymorphisms, bult none linked to the disorder. In contrast, syndromic deafness, is also linked to this locus. If the two diseases share the same the keratin I gene was found to be unlinked to NEPPK. Further linkage mapping has defective gene with different mutations, this information and the clinical correlation will delimited the region of interest to an approximately 11 cM interval on chromosome be very helpful for understanding the function of the gene, once it is cloned. The defective 12ql1-q13, centromeric to the type II keratin gene cluster. In addition, the haplotypes gene among another subgroup of this family is linked to a new chromosomal location, constructed for the affected family members were identical over some but not all of the but the size of the subgroup is not large enough to support a statistically significant LOD linked markers which would imply a common ancestral mutation for the disease in the score. At the allelic level, there are three alleles segregating independently within the two families. The shared part of the haplotype indicates that the region of interest can be subgroup that links to the DFNB4 loc'is. This suggests that mutations in the DFNB4 further delimited to 3 cM. We are now expanding the study to additional PPK families gene are abundant in the Caucasian population. in order to decrease this region further at the same time as we characterize the region in more detail.

2247 2248 Identification of a polymorphism upstream of the PAX 6 gene Collapsing strategies for optimizing the power of the TDT test. alternatively spliced exon 5a by SSCP and CFLP analysis. L.O. addox'. E.R. Martin'2, N.L. Kaplan' and B.S.VWeir2. ' Statistics and Biomathematics Branch, M. S. Cogen4. ansn M Descartes'5. 'Laboratory of Medical Genetics and Desartments NIEHS, P.O. Box 12233, Research Triangle Park, NC 27709; 2Program in Statistical of 'Pediatrics and Ophthalmology, University of Alabama at Birmingham; Alabama Genetics, Department of Statistics, North Carolina State University, Raleigh, NC Eye Foundation Hospital, Birmingham, Alabama. 27695-8203. Paired bo2L (PAX) genes, a family of transcription factors, perform a critical role in Because of concerns about asymptotic assumptions for multiple-allele TDT tests, it gene expression by interacting with DNA regulatory sequences. The PAX gene family has been thought necessary to collapsemnarker data to two alleles. Although we have currently consists ofnine genes, PAX I to PAX 9. Renal-coloboma syndrome and recently shown that the chi-square approximation for multiallel e TDT-like tests aniridia have been associated with mutations in the PAX 2 and 6 genes, respectively. proposed in the literature is, in fact, robust for a wide range of parameter values and The focus of the present study has been to better define the mutational spectrum and to under a variety of models of inheritance, or could even be avoided by the use of identify new ocular phenotypes associated with alteration of the PAX 2 and 6 genes. permutation tests, collapsing may still be a consideration with regard to optimality SSCP and a novel mutation detection assay CLEAVASE fragment length polymorphism issues. (CFLP) analysis have been used to identify sequence variants in the PAX 2 and 6 genes We use a multiallele test for marginal homogeneity of transmitted and nontransmitted of pediatric patients with developmental eye anomalies. SSCP, which has been shown alleles, which has been shown to be one of the most powerful TDT-like and to be highly dependent on the electrophoresis conditions, has 70% sensitivity for the tests, investigate the effects of two commonly sised collapsing strategies on power. We find detection of mutations in DNA fragments up to 300 basepairs. The CFLP assay utilizes that they can lead to more powerful tests in some situations. This us to ask the CLEAVASE I enzyme which has single-strand endonuclease activity to cleave at the prompted if we could devise a strategy for choosing the a 5' end of hairpin structures. The new CFLP technique has greater than 98% sensitivity, optimal collapsing pattern for particular comparable to direct sequencing, for the detection of basepair substitutions, small marker. We recently showed that, for rare deletions and insertions. CFLP will allow longer fragments of DNA (0.1-2.0 kb) to be diseases, we can use data from a case-control study to analyzed. predict the power of the marginal test. These data provide an estimate of the noncen- trality parameter of the test In a comparison of SSCP and CFLP analysis of the alternatively spliced 42 bp PAX 6 statistic. Powver can then be calculated from the noncentral chi-square distribution. we can the for data set. gene exon 5a, a patient with optic atrophy produced an abnormal migrating band. Exon Similarly, predict power any collapsed 5a was directly sequenced revealing that the child is heterozygous for the deletion of I IHence, we can collapse in all possible ways, including not collapsing at all, and choose the pattern that we are adenine nucleotide approximately 50 hp upstream of exon Sa. The patient's father did has the largest predicted power. With this optimal collapsing not have the variant SSCP or CFLP pattern. Direct sequencing of the mother's PAX 6 guaranteed a test that will be at least as powerful as simply applying the marginal test to the exonSa revealed that she is homozygous for the I bp deletion indicating that the finding original data. Although this increase in power is somewhat diminished when we is a neutral polymorphism. The results for other presumably heterozygous siblings are estimate the optimal collapsing scheme from case-control data, our results show that, pending. The identification of this previously unpublished polymorphism is noteworthy with adequate sample sizes, we generally obtain a more powerful test. as it may be encountered by others. A386 Published Abstracts: Linkagea Mapping and Polymorphisms (cont.) 2249 2250 Genetic analysis of congenital myasthenic syndrome. My.M.nold.M_ Refined genetic mapping of Sade' Y_ Goldhammer' IBlatt' F. Lennon L-H Yamanka, _MVance Autosomal Dominant Cone Degeneration. and MIA Pericak-Vanc. Duke University Medical Center, Durham, NC, L Mullen'. K Forsman2. B Lee1' J Heckenlivelyv and K Small,. 'The Chaim Sheba Medical Center, Tel-Aviv University, Israel. The Macula Center at the Jules Stein Eye Institute, UCLA, Congenital myasthenic syndromes (CMS) are a group of inherited disorders in Los Angeles, CA" Dept. of Clinical Genetics, University which neuromuscular transmission is compromised. They are usually inherited Hospital, Umea, Sweden2. as autosomal recessive disorders, although dominant forms do exist. Autosomal Dominant Cone Degeneration (ADCD) is a retinal One type of CMS has been attributed to an inherited abnormality of the ion degeneration that consists of photophobia, dyschromatopsia, channel of the acetylcholine receptor (AChR). Recent reports have shown that and decreased central vision. We have previously reported two separate point mutations in the M2 domain of the c-subunit (the linkage of this disorder to human chromosome 17p13. We transmembrane domain that forms the AChR ion channel) were present in both have ascertained additional family members from the original a familial (n=3 siblings) and an isolated case of CMS. We have obtained DNA family and one additional unrelated family in order to refine from 7 families of Iraqi and Iranian Jewish descent. These families, many of the linkage map. We studied 132 individuals from the original which are inbred, are likely to represent a single CMS mutation. family and 30 individuals from the new family. We now DNA was screened using allele-specific PCR, where one of the primers present refinement of linkage in the original family as well as contained a specific point mutation in exon 8 of the e-subunit. Using this in the unrelated multigenerational family. Recombinations technique, none of the affected individuals was shown to carry the published suggest that the gene is flanked by D17S796 and D17S786, mutation. In order to exclude this candidate gene, we performed linkage markers separated by 10 cM. We have no evidence to analysis with markers tightly-linked and flanking the CHRNE (e-subunit gene) suggest genetic heterogeneity at this point in time. We have locus. D17S1 175, the closest marker to CHRNE (2 Kb), excluded linkage (Z.- been able to exclude recoverin (RCVI) from causing ADCD, 2.00) ± 10 cM of CHRNE. Investigation into the P-subunit, using an but PITPN, PEDF, GUC2D, and I-arrestin-2 are currently intragenic, chromosome 17 polymorphic marker (CHRNB1) similarly excluded being evaluated as possible candidates for this disease. linkage (± 5 cM). Linkage studies of the other subunits (a, y and 6) are Supported in part by: ongoing and will be presented. If all AChR subunits are excluded, linkage The Foundation Fighting Blindness (KWS) studies will be initiated using DNA pooling strategies in drder to identify the The McCone Endowment (KWS) location of the CMS gene in this population.

2251 2252 Recessive Schwartz.-Jampel syndrome: exclusion of two candidate genes by HLA class 11 haplotype and DPB1 effects in multiplex IDDM families. LA, radiation hybrid mapping. S. Nicole and B. Fontaine. INSERM U 134 and Noble' A. M. Valdes2. M. Cook'. W. G. and H. A. Feddration de Neurologie, HOpital de la Piti6-Salpetriere, Paris, France. Klitz2. Thomson2. Erlich., Schwartz-Jampel syndrome (SJS) is a rare autosomal recessive disorder 'Roche Molecular Systems, Alameda, CA and Children's Hospital Oakland characterized by generalized myotonia resulting in a particular facies and Research Institute, Oakland, CA. 2Department of Integrative Biology, osteoarticular abnormalities. Using homozygosity mapping in five families, we University of California, Berkeley, CA. have localized the SJS locus to chromosome lp35-p36.1 in a 8 cM genetic interval We have analyzed HLA Class II typing data generated from 180 multiplex, (Nicole et al., 1995, Hum. Mol. Genet. 4:1633-1636). The study of eight new Caucasian families suggested genetic homogeneity and allowed us to reduce the SJS interval IDDM families. The Human Biological Data Interchange (HBDI, from 8 to 3 cM. No founder effect was demonstrated, even in families of identical Philadelphia, PA) provided 166 of these families. DRBI1, DQA1, DQB1, and ethnic origin (North-Africa) (Fontaine et al., Hum. Genet. In press). Low clinical DPB1 genotypes were determined using PCRISSOP methods. As expected, variability was found except for osteoarticular deformities. Several candidate our data indicate an increase in DR3IDR4, DR3IDR3, and DR4/DR4 genes implicated in osteoarticular and muscular functions are contained within genotypes in patients compared to controls. DR1/DR4, DR1/DR3, and chromosome lp Collagen genes Coll6AI and Col8A2, the Human Paired Domain gene Pax 7 and the gene coding for Agrin. Using the Genebridge 4 DR4/DR8 genotypes were also increased. The protective effect of DR2 is radiation hybrid panel and human specific STSs, we excluded the Pax 7 and Agrin limited to the common DRBI*1501-DQB1*0602 haplotype. Genotype-specific genes from the SJS locus. Col 16AI and Col8A2 could not be placed due to high effects observed include increased frequency of DQB1*0302 on DR4 sequence homology among the group of collagen genes. We are currently haplotypes in DR3/DR4 but not in DRI/DR4 patients. performing the physical map of the SJS locus using the "CEPH" YAC library. The role of the DPB1 locus in influencing disease susceptibility was This map will permit us to reduce the SJS locus by characterizing new genetic examined in these data. < markers. The reduction of SJS interval will allow us to study possible DPBI1*0301 is increased in patients (p 0.001) and microrearrangments in patients suffering from SJS and to identify the SJS gene by appears to be predisposing whether or not it is carried on a DR4 haplotype. positional cloning. (Supported by grants from the French Ministry of Research DPB1*0402 is decreased in patients (p < 0.025), with the protective effect and the AFM). most apparent on DR3 haplotypes. DPB1*0101 is only slightly increased in patients (p < 0.1); however, maternal transmissions to affected children of DR3 haplotypes carrying DPB1*0101 occur twice as frequently as do paternal transmissions. Transmission of DR3 haplotypes carrying other DPB1 alleles occurs at approximately equal maternal and patemal frequencies. Gender bias in transmission of four-locus haplotypes is the subject of further study on a larger number of IDDM families.

2253 2254 Progress in the search for expanded CAG/CTG repents associated with Search for the predisposing loci to severe bipolar disorder in the isolated psychosis. M.C. O'Donovan, C Guy, T Bowen, G Speight, L Jones, A Cardn stibpopulations of Finland. P. Pekkaninen. T. Partonen. J. Suvisaari. P-E. F Middle, P McGuffin, N Craddoc-kTM Owen. Departments of Psychological Medicine Bredbacka. L. Muhonen, J. SepVlai. J. Terwillinger. J. Lonnqvist & L. Peltonen. and Medical Genetics, University of Wales College of Medicine, Cardiff, Wales National Public Health Institute, Helsinki, Finland. Recent studies by ourselves and others using repeat expansion detection (RED) have Bipolar disorder (BPD) is a common psychiatric disorder with high heritability (-0.8) suggested an association between expanded CAG/CTG repeats and both schizophre- biut nmode of inheritance. We have collected 113 families, where I - tO nia and disorder. now complex bipolar We present RED data from an enlarged sample of 152 or manic 143 and 160 controls for full list inidividtials are affected by BPD schizoaffective disorder. type. Majority of schizophrenics, bipolar probands (see acknowledgements these families in Eastern or central of Finland. which have been shown of contributors) which confirmed the earlier findings. However, we found no correlation orginate parts with RED product size and age at onset or severity of phenotype. Furthermore, there to he populated relatively late (starting from the 17th century) by a small group of was no association between large CAG/CTG repeat size and any model of subclassi- settlers. TItis family collection could be powerftl in identifymig some selected fication of either disorder.Because of difficulties directly cloning expanded repeats, to vulnerability genes to BPD, since probably fewer genes are behind the BPD phenotype take this work forward, we have used the recently published screening set of Gastier and thai in more heterogexseosis populations. colleagues to examine 85 CAG/CTG loci which were described as polymorphic or which We have started the search for predisposing loci by studying the candidate regions in were mapped to candidate chromosomes for either schizophrenia or bipolar disorder. the chromosomes 4', 62, 132, 152, 18, 214 and X6, which have been suggested to So far, no alleles have been detected which are within the repeat size range identified contain vitinerahility loci to BPI). Each of these areas will he covered witlt multiple as of interest by RED. In a further attempt to corroborate the RED data, we have micosatellite markers and data will be sib and tried to in the analyzed by using linkage-. pair haplotype identify proteins containing expanded glutamine sequences brains and risk - methods. of from either disorder. western blots relative lymphoblasts patients suffering However, probed We earlier foutid evidence for locus in with an antibody (kindly donated by J-L Mandel) which recognises expanded glutamine have already asi X-chrotsmosoinal predisposing sequences have failed to reveal any such proteins (other than the TATA binding pro- one extended Finnish famitily'. In the prelintinary analyses of chroimiosotnal regions of 6, tein). Thus in summary, we have obtained data which supports the earlier findings of 13, 15, 18, 21, we have found support for linkage only on the chromosome 15. an association between psychoses and enlarged CAG/CTG repeats. However, to date, we have been unable to independently verify this at a locus specific level or by exami- 1. Blackwood DIIR, ei al. Nature Geitet. 12, 427-430 (1996). nation of protein from the tissues of affected individuals. Acknowledgements:We world 2. Ginits El, ct al. Nature Genet. 12, 431-435 (1996). like to acknowledge groups headed by the following collaborators. P McKeon (Ireland), 3. Berrettini WI]. et al. Proc. Nati. Acad. Sci. USA 91, 5918-5921 (1994). M Azevedo (Portugal), W Maier (Germany), D Wildenauer (Germany), Harald N. As- chauer Sandro Sorbi Elenor Feldman 4. Freinier NB. et al. Nature Genet. 12, 436-441 (1996). (Austria), (Italy), (UK), 5. Straub RE, et al. Nature Genet. 8. 291-296 (1994). 6. Pekkarinen P. et al. Gettinie Research 5. 105-114 (1995). Published Abstracts: Linkage Mapping and Polymorphisms (cont.) A387 2255 2256 Genetic determinant of human D2 dopamine receptor binding Refined mapping of a gene for Split hand\split foot malformation (SHFM3) to characteristics in vivo. T. Pohjalainen', J. Rinne2, K. Nigren3, P. Lehikoinen 3, chromosome lOq25. K. Anttilal, E.Syvalahtil, J. Ilietala'. Departments of IPharmacology and Clinical A.Raas-Rothschild', S.Manouvrier2, M.Gonzales3, SLyonnet, A.Munnich'. Pharmacology, 2Neurology and 'Psychiatry, University of Turku, 20520 Turku, 1.Ypartement de Gdnktique,INSERM U393.H6pital des Enfants Malades,149 rue de Finland, 3Turku University Cyclotron/PET Center, 20520 Turku, Finland. Sevres,Paris 75743 Cedex. 2Service de Pddiatrie, H6pital Huriez ,Lille.3Laboratoire The neurotransmitter dopamine has a central role in the regulation for movement, d'Embryologie pathologique, tJ6pital Saint-Antoine,Paris 75012,France. reward and hormone secretion. Although, some regulatory sequences and promoter Split hand\split foot malformation is a heterogeneous limb developmental defect regions have been identifiedl for human Dt, D5 and rat D2 receptors within the characterized the absence of and of the 5'flanking region. the regulatory factors of D2 dopamine receptor density in man are by digital rays syndactyly remaining digits. largely unknown. However, the significant interindividual variability in the striatal D2 The disorder is genetically heterogeneous with three loci described to date: SHFMI dopamine receptor density in vivo suggests the existence of genetic/regulatory factors. on chromosome 7q22.1,SHFM2 on Xq26,and SHFM3 on chromosome 1Oq25. We We determined striatal dopamine D2 recpetor density (Bmax) and affinity (Kd) in 33 report the mapping of the disease gene on chromosome 1Oq25 in two unrelated Finnish niale volunteers using positron emission tomography (PET) and ["Clraclopride. french families. The highest pairwise lod score value was obtained with probe The relationship between a Tflanking marker (TaqlA RFLP) of the D2 receptor gene afm249wc5 at locus DIOS222 (Zmax=6.62 at 0=0%). Haplotype analysis allowed us and receptor binding parameters were evaluated. A statistically significant reduction in to reduce the SHFM3 critical region from 19cM to 9cM between loci DIOS 1709 and the adjusted Bmax was observed in the Al/A2 genotype group compared to the A2/A2 DlOS1663. This interval overlaps with those reported respectively by Nunes et al, group. The Kd was not different between the two groups. The gene adjusted regulatory and Gurrieri et al. allowed exclusion of the FGFR2 which element may be in linkage disequilibrium with the TaIA polymorphism. Haplotype analysis gene also maps to lOq25.3-26 while Pax-2,a paired box gene could not be excluded( Zmax =5.35 at 0=0%). Fibroblast growth factor 8 (FGF8), retinol binding protein(RBP), Zinc finger protein (ZNF32), HMX-2, HOX II are good candidate genes both by their position and by their function.

2257 2258 JUVENILE GLAUCOMA LINKED TO GLC1A IN A FAMILY OF The Serotonin 7 (5-lIT7) Receptor Gene: Genomic Organization and Systematic SPANISH ORIGIN. J. E. Richards 2, P. R. Lichter', M. Boehnke3, M. Othman', Mutation Screening in Schizophrenia B. D. Cameron', H. M. Stringham3, C. A. Downs', S. M. Boyd Lewis', B. F. Boyd', and Bipolar Affective Disorder A. T. Johnson5. Departments of 1) Ophthalmology, 2) Epidemiology, and 3) Biostatistics, University of Michigan, Ann Arbor, MI, Clinica Boyd, Panama City, M Rietschel', MM NJtben' J Erdnmann2 D Shimron-Abarbanell2jM Albusi. Panana, 5) Department of Ophthalmology, University of Iowa, Iowa City. E Franzek, W Maierl- Proppng2 Purpose: To carry out clinical and genetic studies of autosomal dominant juvenile- (1) Dept Psychiatry and (2) Inst Hum Genet, Univ Bonn, Germany, (3) Mental State onset primary open-angle glaucoma (POAG) in a family of Spanish ancestry. Methods: Hospital Haar, Germany, (4) Dept Psychiatry, Univ Worzburg, Germany Twenty-two members of a six-generation Panamanian family of Spanish ancestry under- went an ophthalmologic evaluation. DNA samples from family members were screened We evaluated the possible contribution of genetic variation of the serotonin 5-HT7 via polymerase chain reaction assay of microsatellite repeat markers. Results: Eleven receptor to the development of schizophrenia and bipolar affective disorder. Cloning living family members covering four generations were diagnosed as affected with open- and characterization of exon-flanking intronic sequences enabled us to investigate angle glaucoma of primarily juvenile onset. Linkage analysis using selected microsatellite the whole coding region and the exon-intron boundaries of the human 5-HT7 repeat markers in the tq21-q31 region revealed strong evidence for linkage to the GLCIA SSCA we screened for of DNA variation in gene with a maximum lod score of 3.75 for markers DIS431 at a recombination fraction receptor gene. Using presence sequence of 0.00. Flanking markers D1S305 and D1S306 identify a large genetic inclusion interval a sample of 137 unrelated individuals including 45 schizophrenic and 46 bipolar which does not further serve to reduce the D1S445-AT3 genetic inclusion interval de- affective patients, as well as 46 healthy controls. We detected two rare naturally fined by recombination events in other GLCIA families we are studying. Conclusions: occurring receptor variants (Pro-279-Leu, Thr-92-Lys) and a silent substitution. The The most likely interpretation of our data is that a mutation in the GLCiA gene is re- occurrence of the Pro-279-Leu and Thr-92-Lys substitutions was studied in an for POAG in this of This the sponsible juvenile-onset family Spanish ancestry. expands extended of and controls The Leu-279 variant was countries of origin where this gene has been found to exist and indicates that this form sample patients (n=462) (n=335). of glaucoma may be distributed widely among people of European ancestry. It remains found in similar frequency in all groups, indicating that presence of this variant is to be seen what role this gene may play in families of other origins. Supported in part not causally related to the development of schizophrenia or bipolar affective by NIhI grants EY09580(JER) and EY07003(CORE) disorder. The Lys-92 variant was found in a single individual who suffered from bipolar affective disorder. Investigation of the patient's family revealed independent segregation between the Lys-92 variant and psychiatric illness. Our data suggests that genetic variation of the 5-HT7 receptor does not play a major role in the development of bipolar affective disorder and schizophrenia.

2259 2260 Linkage studies of the N-methyl-D-aspartate receptor subunit gene regions and Hereditary Cardioskeletal Myopathy Daniel R. Rosen (1), Patrick Mullaly (1), schizophrenia in southern African Bantu-speaking families. B. P. Riley, E. ahir, Steven 11. Horowitz (2). (1) Wadsworth Center, NY State Dept. of Health, Empire S. Raiagpnalan, M. Moguddi-Carter1, T. Jenkins2, and R. Williamson. Dept. of State Plaza, Albany, NY 12201-0509 (2) Division of Neurology, University of Missouri Biochemistry, Imperial College School of Medicine at St. Mary's, London, UK, School of Medicine, One Hospital Drive, Columbia, MO 65212 1. Psychiatry Department, Baragwanath Hospital, Soweto, RSA, 2. Department of We are studying the genetics of a large, six generation family, members of which are Genetics, School of Pathology, SAIMR, University of the Witwatersrand, affected by a cardiomyopathy in combination with progressive distal myopathy. The distal myopathy frequently begins in the leg, affecting the distal flexor and extensor Johannesburg, RSA muscles, especially the tibialis anterior (AT) muscle. Cardiomyopathy in affected indi- Both direct and indirect evidence implicate excitatory amino acid neurotransmission vidulals frequently occurs as disruption of the cardiac electrical system, including bundle in the aetiology of schizophrenia. The data are particularly suggestive for N-methyl- branch blocks, atrial flutter, and post-ventricular contractions (PVCs). Age of onset D-aspartate (NMDA) neurotransmission. We have studied the segregation and allelic is usually 25-35. The disorder appears to be passed as an autosomal dominant trait association of polymorphic markers around four ofthe six genes coding for subunits of with high penetrance. We have initiated genetic linkage analysis of this family, with the the neuronal NMDA receptor in a sample of southern African Bantu-speaking families goal of mapping and identifying the gene causing the cardioskeletal myopathy. We have genetically excluded one region of chromosome 14, where a gene for a similar cardioskele- multiply affected with DSM-I1-R schizophrenia. This population was chosen because tal myopathv was recently mapped, indicating that this disorder is heterogeneous. The anthropological and linguistic data suggest that it has diverged from a small initial latest results of the genetic mapping will be presented. population within the last 1,000 years, making shared genetic aetiology much more likely. We find positive two-point LOD score maxima of 0.876 at marker D9S1838 (9q34.3) near the NMDARI central subunit gene and 0.758 at marker D17S784 (1 7q25) near the NMDAR2C secondary subunit gene with no evidence for heterogeneity when analysing affected individuals only. Affected Pedigree Member (APM) analysis yields significantly increased allele sharing in both of these regions (empirical p=0.028 for D9S1826, 0.025 for D17S802). GENEHUNTER multipoint affected relative pair analysis supports the increased allele sharing on 9q34.3 (p=0.O.842 at D9S1838), but not that on 17q25: We propose that the NMDA receptor may be involved in the genetic predisposition lo schizophrenia in this population through variation in one or more of the sub-units, which is consistent With the genetic models for the inheritance of the disease. We suggest that the NMDA receptor loci should be tested for linkage and association in other foundet populatlns. A388 Published Abstracts: Linkage IMapping and Polymorphisms (cont.) 2261 2262 Mapping of a Crohn's disease susceptibility locus to chromosome 1. CONGENITAL RECESSIVE ICHTHYOSES UNLINKED TO A. L. Rothman and E. H. Epstein. Jr. Department of Dermatology, University TRANSGLUTAMINASE-1,-2, OR -3. L.J. Russell. M.L. Lee. J.G. of California, San Francisco. Compton. J.J. DiGiovanna.1 and S.J. Bale. Genetic Studies Section, A susceptibility locus for Crohn's disease (CD), a prevalent form of chronic LSB and IDerm Clin Res Unit, NIAMS, NIH, Bethesda, MD. inflammatory bowel disease, has been identified on chromosome 16 using the Congenital Recessive Ichthyosis (CRI) is a heterogeneous group of microsatellite markers D16S409 and D16S419 (Hugot et al. NATURE 379:821-3). genodermatoses displaying a spectrum of clinical features. Severe Although their results did not reach statistical significance, Hugot et al. also lamellar ichthyosis (LI) lies at one pole of the spectrum a n d detected a possible CD susceptibility locus on chromosome I with the marker congenital ichthyosiform erythroderma (CIE) at the other. A D1S236. We looked for linkage of CD to these same markers: DIS236, D16S409, number of other presentations cannot be categorized as either LI or D16S419, in a large, multiple generation, previously described (Katz et al. Am. J. CIE and constitute an intermediate phenotype. Using pedigrees with of Med. 44:483-7) CD family. We have analyzed data from 11 of the 19 family severe LI, we recently mapped the LI gene to chromosome 14q II, a members with a reasonable probability of a positive diagnosis. Several region bearing the gene (TGM1) for transglutaminase (Tg)- I. we and with was uninformative due Subsequently, others identified mutations in TGMI in LI recombinants were detected D16S419; D16S409 largely families. We now report results of linkage analysis in two families to a large number of homozygotes. The inheritance of D1S236 alleles is consistent with intermediate CRI phenotypes. Linkage analysis for the region with linkage of CD in this family to chromosome 1. These results suggest the of chromosome 14 containing TGMI allowed exclusion of >12cM in presence of a second CD susceptibility locus on chromosome 1. The existence of each family. TgI irreversibly cross-links epidermal proteins in order susceptibility loci on chromosomes 1 and 16 would indicate that CD exhibits to form the protective stratum coreum, a structure which is- genetic heterogeneity. abnormal in CRI. This vital role of Tgl suggests that CRI may be caused by abnormalities in other epidermally-expressed Tgs. Therefore, we performed multipoint linkage analysis over a 1 2cM interval of chromosome 20q containing TGM2 and TGM3. Again, each Tg locus was excluded. These results eliminate the three candidate Tg loci in these two families and suggest that other epidermally-expressed genes are involved. This data supports the hypothesis that the clinical variation in CRI reflects its genetic heterogeneity.

2263 2264 Linkage mapping of Charcot-Marie-Tooth disease type 2 (CMT2A) to No association between spolipoprotein E and advanced age-related macular chromosome ip. M. Saito, Y. Hayashi, T. Suzuki, II. Tanaka and S. Tsuji. degeneration. J Seddon'. M De La Paz2. K Clementse. M Pericak-Vance4. J Hainess. Department of Neurology, Brain Research Institute, Niigata University, Japan. Depts. of Ophthalmology'-3, Epidemiology', Molecular Neurogenetics3, Biostatistics2, Charcot-Marie-Tooth disease (CMT) is the most common form of inherited motor and Harvard Medical School & School of Public Health, Boston, MA; & Depts. of sensory neuropathy. On the basis of nerve conduction velocities and pathological findings Ophthalmology2 and Medicine4, Duke University Medical Center, Durham, NC. of peripheral nerve, two types of CMT with autosomal dominant inheritance, CMT type I is the cause of irreversible (CMTI) and CMT type 2 (CMT2), have been established. CMT2 is characterized by the Age-related macular degeneration (AMD) leading nerve conduction velocity of median nerve > 38 m/sec and is a genetically heterogeneous blindness among the elderly. Epidemiologic studies have demonstrated an association disorder with at least two loci identified; CMT2A (1p35-36) and CMT2B (3q13-22). Only of advanced AMD with cardiovascular risk factors, including an increased risk with 4 families with CMT2A have ever been described and clinical features of CMT2A have elevated cholesterol levels. Allelic variation in the apolipoprotein E (apoE) gene is not been described in detail. In this study, we performed linkage analysis of 2 Japanese related to cholesterol and apolipoproteins have been implicated in the pathogenesis of CMT2 families (family 693 and family 694) using the markers near CMT2A and CMT2B some forms of retinal degenerations. We tested the hypothesis that apoE allele loci. Highest muiltipoint lod scores of 2.01 at D1S244 and 1.93 at D1S228 were obtained frequencies were different between with and without and that for family 694 and family 693, respectively. The highest cumulative multipoint lod score subjects AMD, specific of 3.69 was obtained at D1S244. Obligate recombination events at DlS160 and D1S228 apoB genotypes were associated with increased risk of disease. as well as the multipoint linkage analysis indicate that the gene for CMT2A is likely to be We studied 143 sporadic cases with geographic atrophy and exudative disease, localized within a 14.7 cM region (7.1 cM centromeric and 7.6 cM telomeric to DIS244) types of AMD associated with visual loss. The age range was 50 to 91 and cases had as a 95% confidence interval. Linkage analysis sising D3S1551 and D3S1290 excluded no known family history of AMD. Two comparison groups consisted of 29 elderly the CMT2B locus iu our families. Clinical characteristics of the two CMT2A families subjects in the same age range with no evidence for AMD, and 220 unrelated include the following; 1) the posterior tibial muscles are often as severely involved as the individuals (age range 65 to 92). We also evaluated 38 families with at least two anterior tibial muscles, 2) generalized areflexia is infrequent, 3) peripheral nerves are not siblings with advanced AMD. No significant difference in allele frequency was seen clinically thickened and 4) sensory loss is infrequent. between cases and controls for any allele. Frequencies for apoE2, apoE3, and apoE4 among cases were 0.08, 0.79, and 0.13, respectively, which were almost identical to the frequencies in both control groups. In addition, genotypes for both AMD cases and controls were in Hardy-Weinberg equilibrium. The frequencies of the 3/3 and 3/4 genotypes in the cases were 0.66 and 0.16, respectively, compared to 0.59 and 0.24 in the 29 control individuals, and 0.59 and 0.25 in the population controls. Linkage analysis, using an autosomal dominant, low penetrance model, revealed negative lod scores (-0.57 at theta=O,-0.42 at theta=O.O05,-.30 at theta=0.10). Thus, there was no evidence for association or linkage of apoB with advanced AMD.

2265 2266 Refining the BRIC locus to an 8 cM interval between D18S69 and D18S64. Genetic linkage analysis in an Icelandic/British family sample fails to exdude the putative R.J. Sinker2, V.C. Cariton3, L. BU113, jiA. Juijn2, M. van Eijk2, R. Berger2, chromosome 13ql4.l-q32 schizophrenia susceptibility locus. P.L. Pearson', N. Freimer3 and R.H.J. Houwen2. 'Department of Human Genetics, G. Kalsi. C.-H. Chen, C. Smyth. Barniolfason. Th. Sigmundsson. D. Curtis. R. Butler. T. Utrecht University, Utrecht, The Netherlands, 2Wilhelmina Childrens Hospital Utrecht, Read. P. Murphy. H. Petursson. H. GtCing, Molecular Psychiatry Laboratory, Department of The Netherlands, and 3Neurogenetics Laboratory, UCSF, San Francisco, United States. isychially, University College London Medical School, Windeyer Building, 46 Cleveland Street, Benign Recurrent Intrahepatic Cholestasis (BRIC, MIM#243300) is a rare autosomal London WIP 6DB. DePartment of Psychiaty, Borgaspitalinn, Reykjavik, Iceland. Joint Academic recessive liver disease characterized by intermittent attacks of cholestasis, but without Department of Psychological Medicine, London Hospital Medical College, 3rd Floor Alexandra progression to chronic liver disease. The gene for this disease was mapped by Wing, Turner Street, London El 2AD. identification of a shared chromosomal segment in three distantly related patients from an isolated community (Nature Genet. 1994;8:380-6). The shared region extended from Possible genetic linkageto a genetic subtype of schizophrenia was found by Lin et al (I995) who D18S363 to D18S55, a distance of more than 20 cM. We report a refinement of this studied thirteen large families with markers on chromosome 13q14.1-q32 a region of the localization based on linkage analysis of the original pedigrees along with an additional chromosome which includes the gene encoding the 5HT receptor HTR2A. Lin et al found a 12 families containing 21 patients mainly of Dutch origin. maximum admixture lod of 1.61 with the marker D13SI44 at zero recombination fraction Two obligate recombinants were identified in the region, distal to D18S69 and assuming heterogeneity with the proportion of families linked estimated to be 50%/s. We have proximal to D18S64 respectively, thereby reducing the region of interest to 8cM. atteupted toreproduce this potential linkage in a sampleof multiply affected Icelandic and English Markers within this recombinant region (D18S39, D18S41, D18S1152, D18S1144, schizophrenia families selected for their large family sizeand their power to resolve heterogeneity = D18S1129, D18S1155) demonstrated a maximum lod score in excess of 3 at 0 0.00 of linkage. Using the disease we (6.74, 5.23, 7.89, 7.73, 7.74, 4.41 respectively). Further, linkage disequilibrium was models we have adopted since 1981 (Sherrington et al 1918) found for D18S1129 . The other markers are under investigation for linkage found amaximumlod score of 108with the marker D13S144 usinga spectrum definition ofbeing disequilibrium. affected ina sampleof23 multiplex families. The recombination fraction for this lod was 10%/.and By investigating more families with BRIC with all available markers a further reduction the proportion of families possibly linked to D13S144was 45%. We conclude that further linkage of the region of interest is anticipated, which will facilitate isolation of the causative gene analysis ofthis locus in nevsamples is warranted. References: Lin, M.-W. et al (1995) Psychiatric by positional cloning or by analysis of candidate genes mapped to this genomic interval. Genetics, 5, 117-126. Sherrington, R., et al (1988) Nature, 336, 164-167. Acknowledgements: The research was funded by MRC project grant no. G880473N, the Icelandic Science Council, Schi7ophrenin A National Emergency (SANE), the Joseph Levy Charitable Foundation, the Weilcome Trust and the Priory Hospital. Published Abstracts: Linkage Mapping and Polymorphisms (cont.) A389 2267 2268 Investigations of candidate genes in lumbosacral Confirmation of linkage of oculopharyngeal muscular dystrophy to myelomeningocele. M.C. Soeer. G. Worley. C.M. Wolpert. chromosome 14q11.2-q13 suggests evidence for a second mutation K. Viles. M. Beatv. J.F. Mackey. A.S. Avlsworthl. S.G. JM Stajich', JM Gilchrist2, F Lennonl, A Lee', L Yamaoka', B Rosil, PC Gaskell', Albriaht'. A. Lucas2. H.E. Fuchs. T. Georce. M.A. Pericak- Pritchard', L Donald2, AD Roses', JM Vance', MA Pericak-Vance. IDuke Vance. Duke University Medical Center, Durham, NC; University Medical Center, Durham, NC, 2Rhode Island Hospital, Brown University, University of North Carolina at Chapel Hill, Chapel Providence Hill, NC; 2Carolinas Medical Center, Charlotte, NC. Ocmilopharyngeal muscular dystrophy (OPMD)) is a late-onset, dominant disorder char- Neural tube defects (NTDs) are among the most common acterized by progressive ptosis, dysphagia, and extremity weakness with onset umsuially of birth defects and have a well established genetic after age 50. Linkage of Ol'MI) to I4ql t.2-q13 was reported in a series of French Cana- component. Since periconceptional folic acid dian families (Brais et al.,1995). laplotype analysis of the 2 cM flanking region in the supplementation reduces the risk for NTD births,a strong French Canadian OPMD population showed a single rare disease chromosome (tBrais et one or more interaction between environmental factors and al., 1996), most likely the result of a founder effect. We have sampled five mmiltigenera- genes is suggested. The NTDs have been subject to tional American OPMD) families (59 sampled individuals, 21 affecteds). Four of the five in and extensive scrutiny human and experimental systems, families are of French Canadian ancestry while the fifth is of English/Scottish origin. We have been identified. a number of candidate genes confirm linkage of OPMD to chromosome 14q in these families with no evidence of ge- We and 33 American Caucasian ascertained sampled netic The location score obtained was 6.30 over all and their Affected heterogeneity. peak multipoint (loglo) singleton patients parents. families. The American French Canadian OPMD patients showed the same disease hap- individuals have lumbosacral myelomeningocele and no as families evidence for a founder other family history of NTD. None of the mothers of the lotype configuration the French Canadian supporting folic acid effect. Haplotype analysis in the English/Scottish pedigree (z=2.78) showed an allelic probands reported periconceptional for the disease chromosome > .000005 supplementation for the NTD pregnancy. We investigated configuration segregating (equilibrium frequency in not found in either the American families of French Canadian or the four candidate genes for allelic transmission distortion controls) origin French Canadian families of Brais et al. These data that the including two genes in the folic acid pathway (MTHFR and original (1995). suggest MTHFD), NCAM (neural cell adhesion molecule), and the HLA American family of English/Scottish descent represents a second independent mutation system within the candidate in this disorder. The ethnic groups were compared with regard to frequency and severity by genotyping polymorphisms of extraocular muscle and weakness. No gene itself or by genotyping tightly linked, highly ptosis, dysphagia, (EOM) restriction, proximal polymorphic markers. We used the transmission differences between groups were noted for ptosis or dysphagia. A three-fold increase disequilibrium test (TDT) with Bonferroni correction for in the percentage of individuals with EOM restriction (66% versus 20%) and proximal multiple comparisons. No region showed evidence for weakness (89% versus 30%) was found in patients of English/Scottish origin compared mutation linkage disequilibrium suggesting that none represents a to those of French Canadian descent. The finding of an independent OPMD major locus for lumbosacral myelomeningocele. can play a critical role in the identification of the OPMD gene.

2269 2270 A genome screen for bipolar disorder: Chromsomes 2, 22, 23, 14 & X. O.C. Mapping a gene for Leber Congenital Amaurosis. QW Stockon. K L Stine', L. Chen'. J. Xu', D.A. Meyers', D. MacKitmon', F.J. McMahton', S. Anderson. R A Lewis & J R LuDski. Baylor College of Medicine, Houston, TX. Simpson', J.R. DePaulo'. E.S. Gershon2. J.1. Nurnbergerl, T. Reich4, M.C. Blehar2, Leber Congenital Amaurosis (LCA) is a rare autosomal recessive retinal and M.G. Mcliinis'. 'School of Medicine, Johns Baltimore, dystrophy. It is, however, one of the most common genetic causes of Hopkins University. blindness in children. It is a genetically heterogeneous group of disorders MD, 2NIMII, MD, 3Indiana University, Indianapolis, IN, 'Washington University, characterized by severe visual impairment from birth or early childhood, St. Louis, MO. infantile nystagmus, various ophthalmoscopic features, non-recordable ERG As part of a NIMII collaborative study on the genetics of bipolar (BP) affective responses and occasional systemic associations. disorder, we have performed an initial screen using highly polymorphic DNA markers. Recently the LCA1 locus was narrowed to a 1 cM region on 17p13 Each of the four sites screened approximialely 1/4 of the genome. We report here on (Camuzat, et al., Hum. Genet. 97:798-801, 1996). Initial data from our chromosomes 2. 11, 13, 14 and X. collection of families from the Kingdom of Saudi Arabia and North America = = Of the 54t) individuals genotyped, 31 had scliizoaffective(SA) BP. 231 had BP 1. 70h show exclusion of this region with a Z -14.59 at 0 0.1 for marker DI 7S804. We are attempting to identify and characterize one or more genes causing had BP IL1 and 81) had najor depression recurrent type (MDI)R). Data from 49 markers LCA. Individuals from 39 large inbred Saudi Arabian families and 38 North (as of 5/15/96) were analyzed. The marker order and intermnarker distances (average American families with LCA have been examined and blood samples collected. spacing of 12 to 13 cM) were calculated using CRIMAP. Linkage between thie Using a set of 314 evenly spaced highly polymorphic short tandem repeat anonymous DNA markers and two definitions of affected status were calculated stsing (STR) markers a genome wide screen is 52% complete on the inbred Saudi SAGE. LINKAGE and MAPMAKER/SIBS. The two affection status models considered Arabian families by a strategy of pooled-sample homozygosity mapping. I) SABP. BP 1. BP II- or 2) SABP, BP 1, BP 11. MDDR as affected. Using SAGE. two Potentially interesting loci will be evaluated using standard sib pair linkage point affected sib-pair analyses on the autosomes reveal a single locus (DI I 1998) with analysis. Three such loci have been identified and excluded. Once has been demonstrated several will be used to excess sharing (inodel 2, p

2271 2272 Evaluation of D8S131 polymorphism in normal studies and epileptic Lithium responsive bipolar disorder and chromosome 18 G.Turecki', M. Aida , Cavazzoni2, A. G. Rouleau' 'Centre for patients. A.H.N. Tay'. W.L. Lee2J L.Y. Wong'. P.S. Low3.T. QOg and U. P. Grof2, P. Duffy2. E.eGro.P, It. Martin!, Research in McGill University, of Raian4. Institute of Molecular and Cell Biology, National University of Neuroscience, Montreal, Canada,2Department Singapore', Tan Tock Seng Hospital2, National University Hospital3, School Psychiatry, University of Ottawa, Ottawa, Canada. Recent publications suggest that a major locus for bipolar disorder (BD) may be Health Services4. located on chromosome 18, however, there remains some controversy because the The human neuronal nicotinic cholinergic alpha2 receptor (CHRNA2) chromosomal region with positive findings was not coincident between studies. In locus has been mapped to chromosome 8 p21 at the D8S131 locus. Two addition, other studies have excluded linkage with the entire chromosome 18. simple sequence repeat (SSR) polymorphisms have been identified in the Conflicting results have been a major problem in linkage studies of BD and same locus (S Wood, personal communication). heterogeneity has been largely identified as one of the main reasons for the lack of excellent lithium in BD as a method to Since abnormalities in cholinergic signalling may be involved in epilepsy, replication. Studying long-term response reduce heterogeneity, we conducted a linkage study using 11 markers that span we investigated the possibility of allelic association between epilepsy and chromosome 18. Diagnoses were made using the SADS-L with RDC criteria, and D8S131 polymorphism. The frequency of D8S131 alleles in our local individuals with BD, schizoaffective disorder and recurrent major depression were population was first determined in 50 volunteers. considered affected. To be considered lithium responsive, all patients must have had a Genomic DNA from lymphocytes was used in the polymerase chain high risk of recurrence and must have been maintained on lithium exclusively, with no reaction (PCR) with fluorescent primers flanking the D8S131 C further episodes for a minimum of three years. Nineteen families with 59 genotyped affected individuals were investigated, assuming a recessive mode of inheritance with SSR. The PCR were on an ABI373 polymorphism products analysed sex specific penetrance consistent with the parameters obtained from segregation sequencer with Genescan software. The D8S131 C polymorphism was analysis in this population. Our results do not support the hypothesis that a major found to be highly informative in the Chinese population, with 11 different locus on chromosome 18 is involved in the etiology of BD in this population. alleles. We then examined the distribution of D8S1 31 'C' polymorphic alleles in 100 epileptic subjects to determine if there was any allele segregating preferentially with epilepsy. There was no significant difference in the allelic distribution between normal subjects and epileptic patients. A390 Published Abstracts: Linkage Mapping and Polymorphisms (cont.) 2273 2274 A second family with non-syndromic hearing lossis linked to chromosome A limb girdle muscular dystrophy locus in Manitoba Hutterites does not 6q (DFNA10). K. Verhoeven', G. Van Camp', W. Balemans', L. Van Laeri, map to known LGMD loci. T. Weilerl, C.R. Greenbergl, T. Zelinski', K. Morgan2, F. De Clau2. R. Smith3, 1. Schattemanl, P. Van de Ileyning2 and P. J. Willemsi. E. Nylen', B. Nickel ', K. Wrogemann'. 'University of Manitoba, Winnipeg, MB, 'Dept. of Medical Genetics, University of Antwerp, Belgium 2ENT Dept., University of Canada. 2Mc~iI University, Montreal, PQ, Canada. Antwerp, Belgium 3Dept. of Otolaryngology, University of Iowa, Iowa City, Iowa We have identified 17 males and females from three large Ilutterite kindreds with a 52242, USA. slowly progressive limb girdle muscular dystrophy (LGMD). In two of three kindreds, Hearing impairment of a significant degree involves nearly 10% of the population at onset of weakness was noted from mid-childhood to early adulthood. Distribution of the age of 65 and almost one-half of the population by the age of 80. Although most weakness is predominantly proximal with distal involvement also apparent. Facial mus- cases of hearing loss are probably the consequence of an interaction of genetic and cles are spared and there is no evidence of muscle pseudohypertrophy. EMGs show a environmental factors, families with pure genetic hearing loss have been described. A mixed pattern (myogenic and neurogenic) and muscle biopsies show a myopathic process. Belgian family compromising 14 affected individuals with progressive, autosomal, Dystrophin expression is normal. Parents of all affected individuals are asymptomatic dominant hearing loss was investigated. The hearing loss starts in the first to third and musculoskeletal examinations were normal. In the third kindred, pseudohypertro- decade, and progresses to a severe hearing loss by the sixth decade. The audiograms phy of the calves is evident, cardiomyopathy with sudden death has been documented generally showed a bilateral sensorineural hearing loss, involving all frequenies. and CK elevation is much more pronounced than in the other kindreds. Because of We investigated linkage to 10 known dominant loci (DFNAI-DFNA1O) and 9 known possible genetic heterogeneity, data from only one kindred with ten affected individuals recessive loci (DFNBI-DFNB2) for hereditary hearing loss. The markers that were and two consanguinity loops were used in linkage analysis to the seven known LGMD used for the linkage analysis, are listed in the Hereditary Hearing loss Homepage loci (i.e. LGMDIA on chr. 5q, LGMDIA on chr. 15, LGMD*B on chr. 2p, LGMDSC (http://alt-www.uia.ac.be/u/dnalab/hhh.html). Because the uncertain affection status on chr. 13q, LGMD2D on chr. 17q, LGMD2E on chr. 4q and LGMDtF on chr. 5q). of three family members, the family was divided in three different subfamilies, A, B and All of these loci have been excluded as the responsible locus with lod scores less than C. Lod scores were calculated separately for each subfamily, as well as for the family as -2.0 suggesting that there is another locus causing the LGMD in this kindred. Simulated a whole. Marker D6S407 gave a maximum lod score of 3.01 for subfamily A and a linkage analysis with Linkage 5.1 indicates that this large family is sufficient to establish maximum lod score of 1.51 for subfamily B at 0% recombination distance. Subfamily C linkage. If other candidate loci are excluded, we plan to perform a full genome scan. gave negative lod scores. For the family as a whole a maximum lod score of 3.11 was obtained with marker D)6S407. Haplotype analysis and analysis of key-recombinants placed DFNA10 in a 15 cM interval between the markers D6S287 and D6S270. Until now, for only a single dominant locus (DFNA2) more than one linked family has been reported. Our results show that, also for DFNA10 more than one family is linked, and confirm the involvement of chromosome 6q in the Identification of the DFNA10 gene and elucidation its function in the hearinghearingIprocess.process may contribute to a better insight in the physiology of hearing, and may provide a model for monogenic hearing loss in general.

2275 2276 Homozygosity Mapping of Late-Infantile Neuronal Ceroid P2-adrenergic receptor (PAR) polymorphisms and asthma in an inbred Lipofuscinosis. R. B. Wteelal. J. D. ShW1. M. Savukoski2. 1. Airvel,2, population. S. Willadsen- E.Summerhill H. Gidley. N.J. Cox C. Ober.The University L. Pelton2. R. M. GardinerI and R. E. Williams'l, IDept of Paediatrics, ofChicago. UCLMS, London 2Dept of Human Molecular Genetics, National Public Health Asthma is a multifactorial disorder characterized by inflammation ofthe airways and Institute, Helsinki, Finland. bronchial hyperresponsiveness (BHR). The increased incidence ofasthma among first The neuronal ceroid lipofuscinoses (NCL) are a group of autosomal recessive degree relatives and the greater concordance rates among monozygotic vs. dizygotic neurodegenerative disorders characterised by the accumulation of an twins suggest a genetic component to asthma. To facilitate the search for genes autofluorescent lipopigment in the CNS and other tissues. Three childhood conferring susceptibility to this complex disorder, we have studied asthma phenotypes forms of the disease exist: infantile (INCL), late-infantilc (LINCL) and juvenile in an inbred population, the Hlutterites. This population lives communally and prohibits (JNCL). In addition, a number of variants of the late infantile form have also smoking. As a result, exposure to first- or second-hand cigarette smoke is minimal. In been identified. The genes for the infantile (CLNI) and the juvenile (CLN3) addition to a genome-wide search for all genes which influence asthma, we studied diseases have been mapped to chromosomes Ip32 and 16pl 2.1 respectively microsatellite markers near genes previously reported to show linkage to asthma or the and have subsequently been isolated and cloned. A Finnish variant of LINCL asthma-associated phenotypes, BHR and atopy. One such region is the cytokine gene (CLN5) has also been mapped recently to chromosome 13q21.1-q32. The gene cluster on Sq3 1-33, which was previously reported to be linked to atopy or BHR. In for classical LINCL (CLN2) is as yet unmapped and it is clear that at least one the Hutterites, the 5q3 1-33 marker D5S820 showed modest evidence of linkage to further locus will account for this form of the disease. BHR (likelihood ratio x2, p0. 10). Using the transmission disequilibrium test, we found no the biochemical basis and significance of the NCLs and may also allow evidence that alleles at this locus are associated with BHR (p>0. 10). Therefore these prenatal screening of at risk families. data suggest that the linkage with DSS820 in the Hutterites is Aot related to polymorphisms in the OAR gene. Currently, we are investigating other candidates in this region. Supported by NIH grants HLUAI 49596 and HL07605.

2277 2278 Linkage analysis of candidate gene loci in rheumatoid arthritis (RA) Detection of DNA polymorphism of a simple repeat sequence families Worthingon J, John S. Myerscough A, Marlow A, SaA, Ollier W. ARC TAAA In the human CFTR gene. Z. Xu and D.C. Gruenert. University of ERU, University of Manchester, Oxford Rd, Manchester, UK. California, San Francisco, California. Family and Twin studies suggest genetic and environmental factors are implicated in Cystic fibrosis (CF) Is a fatal autosomal recessive disorder with a the aetiology of RA. Mathematical analysis has suggested that a single gene is unlikely 1:2500 frequency in the Caucasian population. A variety of different to explain the non-ItILA genetic susceptibility of RA. The pathology of RA indicates mutations have been identified in the gene responsible for CF, the cystic a number of disease genes, including inflammatory mediators and immune recognition fibrosis transmembrane conductance regulator (CFTR). The most frequent molecules. A set of 11 dinucleotide microsatellite markers with reported heteroxygosity mutation observed In the CFTR gene is a three base-pair phenylalanine ranging from 0.6-0.9, within or less than 2cM from 14 candidate genes (BCL2, CD40, deletion in codon 508 (AF508). This mutation is located in exon 10 of CFTR. CD401., IL6, IFNA, IFNG, IL.IA, IIlAB, ILIRN, 112, 11.5R, NOS3, PI) has been cosnpiled exon for use in a PCR fluorescence based semi-automated analysis. 115 affected sibling pair We have recently sequenced a 2908-bp genomic segment comprising RlA families (35 with 2 living parents and a further 18 with I parent), from the ARC 10 and flanking introns. A simple repeat sequence, TAAA, was Identified in National Repository for family material, were genotyped for the 11 markers. Standard intron 9 and appears to be dimorphic, with either nine (TAAA)g or eleven sib-pair analysis techniques [Identity by descent (IBD), Identity by State (IBS) and (TAAA) I1 copies on different chromosomes. To determine whether the maximum likelihood methods (MLS IBD)J were used to establish if there was an excess TAM repeat is polymorphic with respect to the AF508 mutation, we have of allele sharing within affected sib-pairs for each marker. For the whole data set allele screened DNA samples from patients using polymerase chain reaction and sharing was found to be consistent with expected sharing for all the markers. As HLA direct sequencing. Initial analysis showed that all AF508 chromosomes is known to be a major locus in RA, sib-pairs were sub-divided into two groups by IBD (n=20) contained 11 repeats (TAAA)I 1. Of 10 samples with one AF508 and status at HLA. Families showing stronger evidence of linkage to HLA i.e. concordant one non-AF508 chromosome, 12 chromosomes were associated with 11- for lILA were compared with those discordant for HLA. Only four markers generated and 8 with of 15 DNA an effective sample size of 20 or more. No evidence of increased sharing was seen for repeat 9-repeat (TAAA)g. Analysis non-AF508 these markers. No evidence of linkage between RA and the candidate genes was found, samples revealed that 9 chromosomes were linked with 11-repeat and however a larger data set would be required to completely exclude these genes. A larger another 21 chromosomes were associated with 9-repeat. It is not known number of families will also be necessary to investigate the effect of lILA. whether the non-AF508 chromosomes contain other CFTR mutations. The polymorphism of TAAA repeats Identified in this study may serve as an additional DNA marker for genetic diagnosis of CF and analysis of the evolution of the AF508 mutation. (supported by NIH DK46002, DK 47766 and the Cystic Fibrosis Foundation) Published Abstracts: Linkage Mapping and Polymorphisms (cont.) A391 2279 A new French family with the North Carolina Macular Dystrophy phenotype maps to the MCDR1 locus. S Yelchit B Puech L Mullen' and K Small'. The Macula Center at the Jules Stein Eye Institute, UCLA, Los Angeles, CA', Centre Hospitalier Regional Universitaire de Lille, Lille, France.2 A four generation family from northern France was ascertained with a macular degeneration clinically similar to North Carolina Macular Dystrophy (MCDRI). Previously, studies using microsatellite markers linked to the MCDR1 locus were uninformative in this family. A new set of markers in the MCDR1 region were used to determine if this genealogically different family maps to the MCDR1 locus at chromosome 6q1 6. 34 members of the family were examined, and 12 were affected. Genotyping with di- and tetranucleotide repeat polymorphisms was performed according to the Weber and May protocol. Two-point linkage analysis generated a significantly positive LOD score with D6S424 of 3.3 with a theta of 0.08 for the small French family. The results indicate that the macular degeneration in this French family maps to the MCDR1 locus. There is now no evidence of genetic heterogeneity between the French family and other MCDR1 families. Supported in part by: NEI / NIH ROI EY10239 (kWS) The McCone Endowment (KWS)

Published Abstracts: Molecular Genetics of Disease 2280 2281 Haplotype diversity and new mutations in the myosin-VIIA gene in USHIB Studies of candidate regions in lithium-responsive bipolar disorder. Israeli families. A. Adatol, 11 Kalinsky', Y. Pel-Orl, D. Weil2, C. Petit2, M. Alda', G. Turecki2, P. Cavazzonil, A. Duffy', E. Grof., R.B. Martin', G. Rouleaus, and B. Bonne-Tamirl. IDepartment of Human Genetics, Sackler Faculty of Medicine, P. Grofl. 'Department of Psychiatry, University of Ottawa, Ontario, Canada; 2Centre Tel-Aviv University, Ramat-Aviv 69978, Israel. 2Unite de Genetique Moleculaire for Research in Neuroscience, McGill University, Montreal, Quebec, Canada. Ilumaine, Centre National de la Recherche Scientifique Unite de Recherche Associee We have been systematically studying families of bipolar patients responsive to 1968, Institut Pasteur, 25 rue dim Docteur Rouix, 75724 Paris Cedex 15, France. lithium. Previous research indicates that lithium responders represent a distinct, more Usher syndrome type I (USHl) is an autosomal recessive disorder characterized by con- homogeneous subtype of bipolar illness characterized by strong presence of genetic genital sensorineural deafness, retinitis pigmentosa and vestibular dysfunction. Linkage factors. Segregation analyses of two independent samples of 71 and 25 families studies show that at least four loci (A, B, C and D) are responsible for USHT. USHIB respectively were both compatible with a major-gene effect. is localized to I1q13.5 and known to account for 75% of all USIII cases. Twenty-seven We have now collected clinical data and DNA samples from 130 genetically unrelated Israeli USIIl families from 12 different ethnic origins were typed for 7 microsatellite patients and from members of 19 families (151 subjects included to date). The candi- markers in the region of the USIITB locus. Considerable variation was observed among date regions are being selected based on assumed mechanisms of action of lithium (e.g. haplotypes of affected individuals from different ethnic origins and even within families interaction with the second messenger system), on biological findings in this subtype of from the same ethnic group. The human myosin VITA gene has been identified as the bipolar illness (e.g. periodic positivity of dexamethasone suppression test) and on pre- gene responsible for US111B based on nonsense and missense mutations and small dele- vionsly published linkage results. The studies use association and linkage strategies. To tions detected in five affected French families (Weil et al. 1995, Nature. 374:60). Several date, we have analyzed markers on chromosomes 11, 18, 21, and X. None of the markers other nonsense and missense mutations and one small insertion were reported in affected tested showed association with the illness. On the other hand, in an analysis of a separate American families (Weston et al. 1995, ASHG A232). Using SSCP and sequencing sample of 186 bipolar patients we have detected a strong deviation from Hardy-Weinberg techniques, 42 USIII affected individuals and their family members were screened for equilibrium in the 4q28-q31 region. This finding could implicate a locus on chromosome mutations in 15 out of the 48 myosin VITA exons. Two new missense mutations - one 4 associated with the mechanism of action of lithium. heterozygous for hlis>Tyr transition in a family of East-European origin and another homozygous for Gly>Arg transition in a family of Moroccan origin, were detected. In a family of Tunisian origin the affected sisters were found to be homozygous for a deletion of at least two exons. Other changes leading to silent polymorphisms were also identi- fied in these 15 myosin VITA exons. Additional SSCP shifts were observed in families of Iranian, Algerian and Polish-German origins. (Supported in part by grants from Is- rael Academy of Science & Humanities 01140042 (B.B.T.), Ministry of health 01140092 (B.B.T.) and the Appelbaum Foundation.

2282 2283 Molecular screening of the Rhodopsin gene in T5 Retinitis Pigm~ntosa Identification of novel transcripts around the Myotonic Dystrophy locus. patients fr~m the Bagque Country.IA.I.AlvareZM.DuranM.Molina, Madawi Alwazzan, Marion Ilamshere, and J. David Brook. Department of Genetics, E.Arostegui,R.Martin,M.L.Onaindia,M.I.Tejada: 1)Gnetics Unit,2)Ophthai University of Nottingham, U.K. mology Dept,3)Neurophisiology Dept.Basurto Hospital.Bilbao.Spain. Myotonic dystrophy is caused by an unstable polymorphic CTG trinucleotide repeat in Retinitis Pigmentosa(RP) is the term that defines a progressive the 3' untranslated region (UTR) of the DMPK gene. In order to explain the multisvs- degeneration of the photoreceptors,leading to night blindness andloss temic effects of DM we are currently examining the genomic organization and expression of visual field.Among all the loci involved,Rhodopsin(RHO) gene is the pattern(s) of genes in the vicinity of the DMPK locus. The expansion of the triplet repeat most common being related to around 20% of ADRP cases and to some may influence the chromatin configuration and the transcriptional regulation of neigh- ARRP and SCRP.We screened this gene in 65 families(15 ADll AR,37 SC bouring genes resulting in altered gene expression. Conventional hybridization methods and 2 Non Classified(NC))from the Basque Country using PCR,DGGE and as well as exon trapping allowed for the identification of novel transcripts around the Restriction Enzyme digestion plus Sequencing in the altered DGGE triplet repeat. We have cloned and characterised two genes ( 59 and 5C/2C ) which patterns.We found abnormal DGGE in 5 AD,1 AR,1 NC and 10 SC.In ADRP, map upstream of DMPK and two other genes ( 4W and 20D7 ) downstream of DMPK. we saw a mutation in RHO(S) located in the splice region in one fam., Other putative genes 9T9 and 23B were also identified. The transcriptional orientation another family had a silent mutation in exon 2 and a His211Arg of each of these transcripts and putative transcripts has been determined. In addition, mutation in exon 3;the other 3 ADRP had the polymorphisms C3982T in we have identified a number of exons which map to a region immediately downstream RHO(3)and C5321A in RHO(5)The NC fam. also showed the polymorphism in of DMPK. These constitute part of a recently identified homeodomain-encoding gene RHO(3).Regarding the SC group, we found a DNA alteration in RHO(1A) (DMAIIP). The partial cDNA sequence of 59 has been extended 5' indicating that the in one fam. and another change in RHO(2) in another family; the 8 full transcript is almost 3.4 kb. The putative transcriptional start site and possible tran- remaining had the C3982T polymorphism and a change in RHO(5)caused in scription factor binding sequences have been found in the promoter region of 59. Two 4 of the cases by the 5321 polymorphism and being the other 4 families cDNAs corresponding to 20D7 have been characterized and 1.5kb of sequence has been together with the ones showing the RHO(1A) and the RHO(2) alteration derived from a 2.4kb mesage. DNA database searches of these novel transcripts fail to sequenced at present.Up to now,the DNA changes found in 2 ADRP identify similar sequences and their precise function remains unknown. families are responsible for the disease which accounts for 13.3% of ADRP cases compared to 20% described.We can say that 33.33% of ADRP show any alteration in RHO gene.Concerning the SCRP families,21.62% of them have the C3982T polymorphism together with 20% of the ADRP. A total of 18.16% of the affected population has the polymorphism in RHO(3) in comparison to 10% of normal population as described.This data encourage a further study of the RHO gene tol elucidate a little more of the RP disease.