2006年欧洲人类遗传学会议DHPLC技术报告

EUROPEAN HUMAN GENETICS

CONFERENCE 2006

saturday, may 6 – tuesday may 9, 2006

C15. the origin of EFNB1 mutations in craniofrontonasal syndrome: frequent somatic mosaicism and explanation of the paucity of carrier males

Craniofrontonasal syndrome (CFNS) is an X-linked disorder in which males, despite being less severely affected than females, are paradoxically under-represented in pedigrees. To understand the molecular basis for this, we have examined the EFNB1 mutations in 59 CFNS families (39 new, 20 previously published). We identified 27 novel intragenic mutations and 3 deletions, 2 of which completely delete EFNB1. All mutations support a partial or complete lossof- function mechanism. A key goal of the study was to define the parental origin of germline mutations. As mosaicism would confound this analysis we first screened samples using Wave-DHPLC, specific restriction digests and Pyrosequencing. Mosaicism was identified in 6cases (out of 53 informative for this study), including in an apparently unaffected parent with 2 affected children, a sporadic case, and an affected female with two unaffected children. The proportion of mutant allele was quantified by Pyrosequencing and varied widely in different subjects and tissues (2-48%). In the remaining samples a paternal origin of mutation was demonstrated in 13 out of 15 informative cases. The bias towards mutations arising in the paternal germline is probably the major determinant of the scarcity of males in CFNS pedigrees, as only affected females are produced in the first generation. Post-zygoticmutations, the reduced reproductive fitness of affected females, and the occurrence of two thirds of X chromosomes in females also contribute. Our results have important implications for genetic counselling in

CFNS: the possible origins of mutation must be taken into account when providing recurrence risks.

　

C32. Identification and Functional Analysis of CITED2 Mutations in Patients with congenital Heart Defects

We present for the first time functionally relevant mutations of CITED2 in patients with congenital heart defects (CHD) (Sperling et al. Human Mutation 26(6), 575-582, 2005). Recent reports have demonstrated that mice lacking the transcription factor Cited2 die in utero showing various cardiac malformations. CITED2 encodes a CBREBBP/EP300 interacting transcriptional modulator of HIF1A and TFAP2. To study the potential impact of sequence variations in CITED2 for CHD in human, we screened a cohort of 392 well-characterized patients and 192 control individuals using DHPLC, sequencing and Amplifluor. genotyping techniques. We identified 15 CITED2 nucleotide alterations, thereof seven alterations that were only found in CHD patients and not detected in controls including three mutations leading to alterations of the amino acid sequence (p.Ser170_Gly178del, p.Gly178_Ser179ins9, p.Ser198_Gly199del). All three of the amino acid changing mutations cluster in the serine-glycine-rich junction of the protein to which so far no functionality had been assigned. Here we show that these mutations significantly reduce the capacity of CITED2 to transrepress HIF1A, additionally the p.Ser170_Gly178del mutation significantly diminishes TFAP2C coactivation. This reveals a modifying role for the serine-glycine-rich region in CITED2 function. In summary, these observed mutations occurring in patients with septal defects indicate the causative impact of CITED2 in the development of CHD in human.

　

C33. mutations in Desmoglein-2 gene are associated to arrhythmogenic right ventricular cardiomyopathy

Arrhythmogenic right ventricular cardiomyopathy (ARVC) is a myocardial disease characterized by progressive myocyte loss and fibrous and fatty tissue replacement of the right ventricular free wall, which is the substrate for reentrant arrhythmias and sudden death. The primarily mode of inheritance is autosomal dominant with reduced and age-related penetrance. Five disease genes have been identified so far, three of them (desmoplakin, plakophilin-2 and plakoglobin) are involved in the desmosomal complex. We hypothesized that mutations in desmoglein-2 (DSG2), the only desmoglein isoform expressed in cardiac myocytes, may account for ARVC.Sixty ARVC probands were screened for DSG2 mutations by denaturing high-performance liquid chromatography (DHPLC) and direct sequencing. Ten DSG2 mutations have been identified in 9 patients (15%). Among the DSG2 mutations, six were missense (Y86C, G100R, N266S, K294E, E331K, V391I), two insertion-deletions(G678fsX681, E418fsX419), one a nonsense (Q558X) and one a splice site mutation (1181-2A>G). None of the detected nucleotide changes was found in 560 control chromosomes. An endomyocardial biopsy was obtained in five patients, showing extensive loss of myocytes with fibro-fatty tissue replacement. In three of them, electron microscopy investigation was performed, showing intercalated disc paleness, decreased desmosome number and intercellular gap widening.This is the first demonstration that mutations in DSG2 gene are associated to ARVC. Based upon current data, we confirm that many forms of ARVC are due to alterations in the desmosome complex.

　

P0100. Prevalence of Lim Domain-Binding 3 (LDB3) gene mutations in idiopathic dilated cardiomyopathy.

Familial Idiopathic dilated cardiomyopathy (IDCM) is a genetically heterogeneous. Disease. Among disease-causing genes, LDB3 (MIM+605906) mapping at 10q22.2-q23.3) has been recently reported as causally linked to both non-compaction left ventricle (NCLV) and IDCM. DB3 encodes a protein that is a component of the Z-line in both skeletal and cardiac muscle. Recent studies have demonstrated that LDB3 knock-out mice develop cardiomyopathy and that defects of the gene may cause familial IDCM. Six mayor cDNA isoforms of LDB3 have been identified in human striated muscle and are generated by alternative splicing of a single gene.We screened the LDB3 gene in 115 unrelated index patients diagnosed with IDCM according to WHO criteria. The gene screening was performed by denaturing high performance liquid chromatography(DHPLC) and bidirectional sequencing of hteroduplex amplicons.Five mutations were identified in six probands (5,21%): D117N (2unrelated probands), S196L (one proband), T358A (one proband),T357I (one proband), V588I (one proband) (known mutations are in bold). The Val588Ile, although reported as rare polymorphism,was absent in 120 healthy controls. None of the patients showed echocardiographic features suggestive for NCLV according to Chin et al. and McKenna et al. criteria. We confirm LDB3 gene as candidate gene for familial IDCM, independently on echocardiographic pattern of NCLV. The prevalence of the LDB3 gene mutations in a consecutive series of more than 100 patients is 5%. Despite the description of NCLV as specific feature associated with LDB3 gene mutation the clinical phenotype did not show specific clinical markers.

P0150. Prevalence of telethonin encoding t-cap gene in a consecutive series of 200 patients diagnosed with Hypertrophic (Hcm) and Dilated cardiomyopathy (Dcm)

Telethonin is a sarcomeric protein of 19 kDa possibly localized to the Z-disc of adult striated skeletal and cardiac muscles, where it interacts with the protein titin. Mutations in the T-cap gene encoding telethonin cause LGMD2G, a relatively mild form of autosomal recessive LGMD.Mutations of the T-cap gene have been recently reported in autosomaldominant HCM and DCM without myopathy.We aimed at determining the prevalence of T-cap gene mutations in a consecutive series of 200 patients clinically diagnosed with familial and sporadic DCM (n = 100), and HCM (n = 100).The series includes 200 index patients diagnosed with HCM and DCM using WHO criteria that accepted to enter our clinical and genetic program on familial cardiomyopathies. The local Ethical Committee has formally approved the project. T-cap gene has been screened by DHPLC and bidirectional sequencing of heteroduplex amplicons.We identified five heterozygous T-cap gene mutations (5 of 200 patients, 2.5%), four in familial HCM [R106C in two unrelated probands,and 637_640 Del2G in further two unrelated probands] (4%) and one[R63C] in one familial DCM (1%). The mutations were absent in a series of 100 healthy controls.Telethonin encoding T-cap gene mutations are associated to inherited DCM and HCM: the prevalence is higher in HCM than in DCM. This study provides the genetic epidemiology basis for progressing with further investigations on the role of telethonin in myocardial diseases and definition of precise clinical phenotype associated with these mutations.

　

P0173. molecular evidence against a role of the NF1 gene mutations in LEOPARD syndrome

LEOPARD syndrome (LS) is a rare autosomal dominant condition,mainly characterised by facial dysmorphisms, congenital heart defect,in particular hypertrophic cardiomyopathy, multiple lentigines and cafè-au-lait spots (CLS). Up to 90% of LS patients present missense mutation in PTPN11 gene, encoding for the SRC homology 2 (SH2)domain-containing PTPase (SHP-2). Several clinical manifestations overlap those of Noonan syndrome (NS), which is due to different PTPN11 mutations. In paediatric LS patients, CLS and/or lentigines pose difficult differential diagnosis mostly with Neurofibromatosis type 1 (NF1) and NF/NS, largely caused by NF1 gene mutations. To date,no other gene mutation has been concretely related to LS, even if a mutation in NF1 gene was reported in a patient supposed to be affected by LS. Increasing evidence suggests that SHP-2 and neurofibromin,the NF1 gene product, play their modulatory role through a common pathway. This evidence prompted us to screen the NF1 gene in 4 full-blown LS individuals, in which PTPN11 gene mutations had been excluded by DHPLC and sequence analysis of the entire coding region.The NF1 gene analysis was carried out by DHPLC and sequencing.No pathogenic mutation was detected in our LS individuals. This result indicates that NF1 gene mutations are not related to LS, thus implying that the clinical overlap between LS and NF1-NFNS is not paralleled by a unique molecular event.

　

P0195. mutations in the ND5 subunit of complex i of the mtDNA are a frequent cause of OXPHOs disease

Objective: To determine the frequency of mutations in the mitochondrial(MT)-ND5 gene in patients with OXPHOS disease. Background:Mutation detection in the mitochondrial genome is usually limited to common mutations and the tRNA genes. However, mutations in the ND subunits of complex I can be an important cause of OXPHOS disease. Methods: mutation screening of the mtDNA was performed by DHPLC-analysis of120 patients with a mitochondrial disorder.Heteroplasmy levels in different tissues were determined using PCR with fluorescently labelled primers and mutation specific restriction enzymes. Results: We found a MT-ND5 mutation in 3,3% of the patients.Two mutations were new and two have been previously reported. The 13513G>A mutation, associated with MELAS and MELAS/Leigh/Leber hereditary optic neuropathy overlap syndrome, was found in a relative low percentage in two patients, one with a Leigh and one with a MELAS phenotype. The 13042G>A, once detected in a patient with a MELAS/MERRF phenotype, was now found in a patient with a Leigh-like/NARP phenotype. A new mtDNA mutation (12622G>A)was identified in three brothers, all with infantile encephalopathy(Leigh syndrome) fatal within the first 15 days of life and a novel point mutation (13511A>T) occurred in a patient with a Leigh-like syndrome. Conclusions: Mutation screening of the mitochondrial ND5 gene is dvised for routine diagnostics of patients with OXPHOS disease, especially MELAS- and Leigh-like patients.

　

P0208. Molecular testing in Neurofibromatosis type 1 (NF1):mutational spectrum, patterns of recurrence and correlation withclinical features in italy

Neurofibromatosis type 1 (NF1) is a common autosomal dominant disorder in humans affecting 1 in 3500 individuals. In this work we review the mutational reports on Denaturing high performance liquid chromatography (DHPLC) analysis of the NF1 gene published until now and the NF1 clinical and molecular findings in the Italian population by reporting our experience with multiple screening techniques in the analysis of the whole NF1 coding region in a panel of 468 consecutive NF1 patients from 8 Italian centres catering with NF1.The study of 468 NF1 patients has been performed using multiple screening techniques, viz. protein truncation test (PTT), heteroduplex analysis (HA), fluorescence in situ hybridisation (FISH), Southern blotting, cytogenetic analysis, and DHPLC. Particularly, our study performed by DHPLC on 374 patients revealed 187 novel mutations with a mutation detection rate (in the northern eastern Italian regions and in Sicily) of 83% (i.e., the highest so far reported). Seventy-four of the 374 unrelated NF1 patients harboured 35 different recurrent mutations. Genotype-phenotype analysis revealed a higher rate (75%) of missense mutations (vs. other mutations) in patients harbouring non-optic pathway cerebral gliomas and a higher degree of severity of pigmentation anomalies in the group of patients with missense mutations (89%). We also summarised the Italian NF1 microdeletion spectrum (n = 18), which by refined FISH characterisation could be classified as typical (n= 14) and atypical (n=4). This work confirms that DHPLC analysis for routine molecular diagnosis in NF1 is a simple and efficient methodology with a high mutation detection rate.

　

P0548. mutation analysis of the msH6 gene in 52 mLH1/msH2-negative, HNPcc suspect italian patients

P. Pazienza*1, M. Barberis*2, I. Borelli2,3, S. Regazzoni1, D. Giachino1, G.Casalis Cavalchini2, A. Allavena2, M. Micheletti2,4, A. Arrigoni5, M. Schena6, E.Grosso3, T. Venesio7, E. David4, B. Pasini2,3, N. Migone2, M. De Marchi1;1Università degli Studi di Torino, Dipartimento di Scienze Cliniche e Biologiche,Orbassano, Italy, 2Università degli Studi di Torino, Dipartimento di Genetica,Biologia e Biochimica, Torino, Italy, 3ASO S.Giovanni Battista, SCDU GeneticaMedica, Torino, Italy, 4ASO S.Giovanni Battista, SCDU Anatomia Patologica,Torino, Italy, 5ASO S.Giovanni Battista, SC Gastroenterologia, Torino, Italy,6ASO S.Giovanni Battista, SC Oncologia Medica, Torino, Italy, 7IRCC, AnatomiaPatologica, Candiolo, Italy.

The HNPCC syndrome bears high cancer risk at colorectum endometrium and other organs, due to MMR defects. Testing in clinical contexts is frequently limited to MLH1/MSH2, since only a minority of cases are thought to depend on MSH6 and other genes. Within a program aimed to prevent inherited cancer in the Piedmont area,we have implemented the mutation analysis of MSH6 on a set of 88 suspect HNPCC probands, in which 52 were previously found to be MLH1 and MSH2 mut- negative, with the aim to define proper criteria for MSH6 testing. Cases were included on the basis of recognized clinical criteria and on preliminary analyses on the tumor for IHC expression of MMR proteins and microsatellite instability. All 10 MSH6 exons were amplified in 19 amplimers from leukocyte DNA, and analysed by DHPLC. Screening for major deletions was performed by MLPA. Two point mutations (K537fs and Y1286delinsIN), one deletion of exons 2-4 and two variants of unknown pathogenic significance were found.In all three MSH6-mut patients the tumor was MSI-H; in one of them we demonstrated a double-negative IHC pattern (MSH2- and MSH6-).Within the limits of our small sample, MSH6-mut patients showed less typical family history. The inclusion of MSH6 in HNPCC genetic testing can significantly improve sensitivity. A MSH6 and/or MSH2 negative IHC, better than any clinical feature, appears to efficiently pinpoint cases for MSH6 screening among MSH2-neg cases.Supported by Regione Piemonte and Compagnia di S.Paolo, Torino

　

P0575. mutation analysis of the MYH gene in Unrelated czech APc mutation-negative polyposis patients

M. ?ulová1, K. Zídková1, Z. Kleibl2, J. ?tekrová1, V. Kebrdlová1, M. Kohoutová1;1Institute of Biology and Medical Genetics of the First Faculty of Medicine andGeneral Teaching Hospital, Charles University, Prague, Czech Republic, 2Instituteof Biochemistry and Experimental Oncology, the First Faculty of Medicine,Charles University, Prague, Czech Republic, Prague, Czech Republic.

In 20-30% of patients with classical familial adenomatous polyposis(FAP) and up to 90% of those with attenuated polyposis (<100colorectal adenomas; AFAP), no pathogenic germline mutation in the adenomatous polyposis coli (APC) gene can be identified. Some of the APC negative FAP and AFAP cases have recently been found to be attributable to MYH associated polyposis (MAP). MAP is an autosomal recessive syndrome associated with 5-100 colorectal adenomas and caused by mutation in the MYH gene. The MYH protein plays an important role in the base-excision-repair system as an adeninespecific DNA glycosylase.We screened for germline MYH mutations in 90 APC-mutationnegative probands with classical and attenuated familial adenomatous polyposis. The entire coding region and intron-exon borders of the MYH gene were analyzed. As a prescreening to detect DNA sequence changes, denaturing high performance liquid chromatography (dHPLC)was performed using the WAVE nucleic acid fragment analysis system(Transgenomic). Samples showing unique profiles were sequenced in both directions on ABI Prism 310 Genetic Analyzer (Applied Biosystems).In majority of patients multiple genetic changes in MYH gene were found. Altogether 10 previously reported changes and 8 novel genetic alterations, mostly in intronic sequences were identified. We have detected compound heterozygotes for two the most common germline mutations c.494A>G (p.Y165C); c.1145G>A (p.G382D) too. These variants are established to be associated with adenomatous polyposis and colorectal cancer.

　

P0579. A novel mutation in Neurofibromatosis type 1 (NF1)

S. Bendova1, T. Marikova1, A. Krepelova1, B. Petrak2;1Institute of Biology and Medical Genetics, Charles University - 2nd MedicalSchool, Prague, Czech Republic, 2Department of Child Neurology, CharlesUniversity - 2nd Medical School, Prague, Czech Republic.

Neurofibromatosis type 1 is one of the most autosomal dominant common diseases with an incidience 1:3000 in all ethnic groups. It is a multisystem disorder with the variable clinical features as café au lait spots, Lisch nodules, multiple freckling, neurofibromas or plexiform neurofibromas, optical gliomas, distinct osseous lesions and learning disabilities. Other clinical findings associated with NF1 are high-signal intensity foci on the T2 weighted MR images of the brain. These areas are going to be observed on MRI and data presented.Various somatic and germinal mutations in tumor-suppressor NF1 gene are causes of the Neurofibromatosis type 1 disease, more than 650 different mutations have been found up to this time.The family with three clinical affected members have been examined by molecular genetic methods. We have used PCR and DHPLC methods for detection of small insertions, deletions, indels and nucleotide substitutions. The positive results were confirmed by sequencing analysis. The novel mutation c.1_2delATinsCC has been found at all family members in the start codon NF1 gene.Mutation probably prevents the start of transcription and causes haploinsufficiency. It consequently leads to the deficit of Neurofibromin protein and to rare phenotype manifestations.

　

P0612. mutation screening of the APc gene in 134 spanish Families.

M. Fernández Ulloa1, P. Cabello1, D. Rey1, E. Garcia-Galloway1, R. Fernández1,A. Alonso2, C. San Román1;1Hospital Ramon y Cajal, Madrid, Spain, 2Hospital Virgen del Camino, Pamplona,Spain.

Background: Familial adenomatous polyposis (FAP) is an inherited,autosomal dominant syndrome caused by germ-line mutations of the adenomatous polyposis coli (APC) gene. Affected individuals develop hundreds to thousands of colonic polyps. More than 700 mutations have been described until now along the gene and it was found that the same APC mutation could cause different clinical manifestations.However, it has been suggested the relationship between thelocalization of the mutation and specific phenotypes associated with the disease.

matherial and methods: We study 134 unrelated patients with diagnosis of FAP and their relatives. Genomic DNA were obtained from affected individuals after informed consent and mutation analysis of the whole coding sequence of APC gene was done by dHPLC .The results obtained were confirmed by DNA sequencing.

Results: Sequence changes of the APC gene have been detected in 45% of the affected patients We have identified twenty novel mutations:three missense mutations, nine small deletions and two indels that create a new stop codon and produce a truncated protein, and six nonsense mutations. Nineteen mutations corresponded to exon 15and one to exon 13. Four novel silent polymorphisms were founded in exon 15 and 13 of the gene.

conclusion: The study of mutation status in the affected members as well as in the healthy individuals of each family has confirm that mutations are causative. We also describe the patient’s phenotypes associated with these mutations. The description of every new mutation finding may contribute to a better knowledge of these severe disease.

　

P0645. Molecular findings in CAPN , DYSF and GNE related to various muscle diseases

M. Krahn1, C. Pécheux1, K. Nguyen1, G. Bassez2, C. Beroud3, B. Eymard2, E.Hammouda4, V. Labelle1, A. Urtizberea4, F. Leturcq5, R. Bernard1, N. Levy1,6, F.rench Network on LGMD7;1Departement de Genetique medicale, Hopital d’enfants La Timone, Marseille,France, 2Institut de Myologie, Groupe Hospitalier Pitié-Salpêtrière, Paris,France, 3Laboratoire de Génétique Moléculaire, Institut de Génétique Humaine,Montpellier, France, 4AFM et Généthon, Evry, France, 5Laboratoire de BiochimieGénétique, H?pital Cochin, Paris, France, 6Inserm U491 : ?GénétiqueMédicale et Développement?, Faculté de Médecine Timone, Marseille, France,7-, -, France.

The identification of mutations in large-sized genes is challenging on a routine basis in clinical diagnostic settings. Methods for mutation screening are particularly adapted to this task.Here, we report our experience in mutation screening using DHPLC analysis and subsequent sequencing of detected variants, in the largesized genes encoding calpain-3 (CAPN3, 15q15.1-q21.1, 24 coding exons), dysferlin (DYSF, 2p13.1-p13.3, 55 coding exons) and UDP-Nacetylglucosamine 2-epimerase/N-acetylmannosamine kinase (GNE,9p12-13, 11 coding exons), in three cohorts of respectively 48, 93, and 31 myopathic patients.Mutations in CAPN3 and DYSF cause the most frequent forms of autosomal recessive Limb Girdle Muscular Dystrophies (LGMD2),respectively LGMD2A and LGMD2B. The most common form of autosomal recessive (AR) hereditary inclusion-body myopathy (HIBM) is caused by mutations in GNE.Patients were included after initial clinical and pathological assessment,allowing to select the subsequent molecular analyses. A resume of the results of the molecular analyses are presented in the table below. At least one pathogenic allele was detected in >85% of patients included for CAPN3 and DYSF analysis. The lower mutation detection rate observed for GNE may be correlated to genetic heterogeneity and/or difficulties in defining phenotypic criteria for the inclusion of patients. Mutations identified in the CAPN3 and DYSF genes were compiled in the locus-specific databases UMD-CAPN3 and UMD-DYSF, adapted from the generic software UMD (Universal Mutations Database).

　

P0660. molecular analysis of the COH1 gene in cohen syndrome.

C. Pescucci1, E. Katzaki1, F. Mari1, C. Speciale1, F. Ariani1, M. Zeviani2, M. Bugiani3,

M. B. Petersen4, P. Gasparini5, M. Bedeschi6, P. Veggiotti7, R. Fischetto8,R. Grasso9, R. Ghilardi10, A. Selicorni10, D. Milani10, M. Di Rocco11, M. Mantovan12,M. Priolo13, A. Mendicino14, R. Tenconi15, A. Renieri1;1Medical Genetics, Siena, Italy, 2Molecular Neurogenetics Division, IstitutoBesta, Milano, Italy, 3Neurology, Istituto Besta, Milano, Italy, 4Dept. of Genetics,Aghia Sophia Children’s Hospital, Athens, Greece, 5Medical Genetics, Trieste,Italy, 6Medical Genetics, ICP, Clinica Mangiagalli, Milano, Italy, 7Child NeuropsychiatryDepartment, Neurological Institute Casimiro Mondino FoundationIRCCS University of Pavia, Pavia, Italy, 8Metabolic Disorders- Medical GeneticsDivision, Ospedale Regionale Pediatrico Giovanni XXIII, Bari, Italy, 9ScientificInstitute Eugenio Medea, Bosisio Parini, Lecco, Italy, 10Clinical Genetics Ambulatory,Clinica Pediatrica de Marchi, Milano, Italy, 11Pediatrics II, Istituto Gaslini,Genova, Italy, 12Child Neuropsychiatry, Azienda Ospedaliera Careggi, Firenze,Italy, 13Medical Genetics, Azienda Ospedaliera Bianchi-Melacrino- Morelli, ReggioCalabria, Italy, 14Genetics, AUSL RME, Roma, Italy, 15Medical Genetics,Dept. Pediatrics, Universiy of Padova, Padova, Italy.

In 1973, Cohen et al. described a new syndrome whose main features were truncal obesity, hypotonia, mental retardation of variable degree,characteristic craniofacial dysmorphisms ( down slanting palpebral fissures, short philtrum, open mouth, prominent upper central incisors,prominent nose) and abnormalities of the hands and feet. Beside the facial gestalt, major diagnostic criteria of Cohen syndrome include retinal dystrophy and neutropenia. This syndrome is transmitted as an autosomal recessive trait, with considerable variability of expression.Recently, mutations in COH1 gene (locus 8q22-q23) have been reported in patients with Cohen syndrome.We have collected a cohort of 21 patients, originating from different countries. A diagnostic rating of ‘certain’ (10 patients), ‘probable’(5) and ‘possible’ (6) was assigned on the basis of clinical criteria.DHPLC mutation analysis of the COH1 gene is ongoing. Until now,we have analyzed 20 exons out of 62, identifying mutations in four patients: three point mutations in genetic compound state and one homozygous intragenic deletion spanning from exon 6 to exon 16. In addition, mutation analysis revealed three intronic variants of unknown significance (IVS 2-60 A>G, IVS 17+61 C>A, IVS 21-86 A>T). At present, mutations in the COH1 gene were identified in patients mutations in the COH1 gene were identified in patients clinically classified as ‘certain’ and ‘probable’, while no mutations were not found in patients classified as ‘possible’.

　

P0667. A large deletion involving the connexin 26 gene

D. Feldmann1,2, L. Jonard1, F. Fellmann3, P. Thierry4, R. Couderc1,2, E. N. Garabédian5,2,F. Denoyelle5,2, S. Marlin6,2;1Laboratoire de Biochimie et de Biologie Moléculaire, CHU Trousseau, AP-HP,Paris, France, 2INSERM U587, Unité de génétique des déficits sensoriels,Institut Pasteur, Paris, France, 3Service de Génétique, H?pital Saint Jacques,Besan鏾n, France, 4Service de Pédiatrie, CH Vesoul, Vesoul, France, 5Serviced’ORL et de Chirurgie Cervico-Faciale, CHU Trousseau, AP-HP, Paris, France,6Unité de Génétique Clinique, CHU Trousseau, AP-HP, Paris, France.

Mutations in the GJB2 gene coding connexin 26 are the most frequent cause of congenital non-syndromic hearing impairment (NSHI) in Caucasian populations. Recently two large deletions involving the connexin 30 gene were described in NSHI patients with a GJB2 mutation in trans. We report here for the first time a deletion of GJB2 in a patient with congenital, profound hearing impairment associated with developmental delay. Screening for GJB2 mutations was carried out by denaturing highperformance liquid chromatography (DHPLC) followed by sequencing.The patient appeared to be homozygous for a new GJB2 mutation c.250G>A, p.Val84Met. Known GJB6 deletions analyzed by specific PCR were absent and no mutation was founded in the first non-coding exon of GJB2. The mutation p.Val84Met is potentially deleterious because Valine 84 is evolutionarily highly conserved and was not observed in 200 chromosomes of normal-hearing individuals. The mutation was heterozygous in the patient’s father, but absent in the mother and in the maternal grand parents. Uniparental disomy was excluded by genotyping with a set of 8 markers from the long arm of chromosome 13. A set of 6 microsatellite markers in 13q12-11 analysed in the family confirmed a large deletion involving GJA3 (CX46), GJB2 (CX26) and GJB6 (CX30) in the patient and his mother.Our results indicated that the patient was compound heterozygous for [pVal84Met]+[del(GJA3-GJB2-GJB6)] and that this genotype can induce deafness. This observation shows the importance of GJB2 analysis patients with hearing impairment and the segregation study of the GJB2 mutations.

　

P0673. Novel large rearrangement in the cFtR gene in cystic fibrosis patients from Reunion Island

J. Nextoux1, M. P. Audrezet2, M. Viel3, C. Leroy3, F. Cartault4, O. Raguenes2, C.Ferec2, J. F. Lesure5, M. Renouil6, T. Bienvenu1;1Institut Cochin, U567, Paris, France, 2U613, CHU Brest, Brest, France, 3Laboratoirede Biochimie et Genetique Moleculaire, Hopital Cochin, Paris, France,4Service de Genetique, CHD Felix Guyon, St Denis, Reunion, France, 5Servicedes Petits Enfants, Hopital d’Enfants, St Denis, Reunion, France, 6Service dePediatrie, CHS Sud Reunion, France.

The Reunion Island (RI) is a French province, 800 kms to the east of Madagascar, and 200 kms to the west of Mauritius. In RI, the birth prevalence of the Cystic fibrosis (CF) is particularly high in the population of European origin. In a previous study we have demonstrated that the screening of the 27 exons of the CF transmembrane conductance regulator (CFTR) gene by DHPLC allowed the detection of 93% of the molecular defects present in RI. Unidentified CF mutations may lie in introns or in regulatory regions, or correspond to gene rearrangements at the heterozygous state which escape detection using current PCR based techniques. Using a combination of different methods, 6 of the 13 unidentified CF alleles were found to harbor a novel deletion of 5288 bp, spanning the exons 17a, 17b and 18. This accounts for 46% of unidentified alleles. Identification and examination of the breakpoint sequences showed that this deletion is different from sequences showed that this deletion is different from the 3120+1Kbdel8.6Kb previously found in the Palestinian arabs.The IVS16+3316_IVS18+644del5288 bearing chromosomes had a common intragenic haplotype. This mutation causes an in frame deletion of 160 amino acids that are part of the transmembrane domain 2 of the CFTR protein. Clinical evaluation of homozygotes (n=2) and compound heterozygotes (n=2) indicate that this deletion represents a severe mutation associated with positive sweat test,pancreatic insufficiency and early age at diagnosis. In conclusion, we have shown that this novel gross genomic rearrangement is the fourth most common mutation in CF patients from RI.

　

P0681. Detection of mtDNA mutations in 12s rRNA gene (mtRNR1) and tRNA-ser(UNc) gene (mtts1) using DHPLc in patients with nonsyndromic hereditary hearing impairment

P. Primignani1, L. C. Trotta1, P. Castorina1, F. Lalatta1, U. Ambrosetti1,2, L. Garavelli3,D. Degiorgio1, F. Sironi1, M. Travi1, D. A. Coviello1;1Fondazione Policlinico Ospedale Maggiore, Mangiagalli e Regina Elena, Milan,Italy, 2ENT Audiology Department University of Milan, Milan, Italy, 3MedicalGenetic Service - Arcispedale S. Maria Nuova, Reggio Emilia, Italy.

Since 2001 we have analysed 601 subjects, affected by neurosensorial deafness with various degrees of hearing loss. Mutations in the GJB2 gene, encoding for the gap-junction protein Connexin26 (Cx26), are responsible for the majority of non-syndromic recessive deafness and among these the 35delG allele is the most common in the Mediterranean population. In our cohort of patients we identified 169 subjects affected by Cx26 and/or delta(GJB6-D13S1830) deafness;among these 102 (60,3%) exhibited a 35delG homozygous genotype,47 (27,8%) were compound heterozygous 35delG/not-35delG while16 patients (9,5%) were compound heterozygous not-35delG and 4 showed a dominant mutation.Mutations in MTRNR1 gene (A1555G, 961delT), encoding 12S ribosomal RNA, account for most of the cases of hereditary deafness induced by amino glycoside’s administration. We screened all the affected patients, with one or without Cx26 recessive mutations, for the A1555G substitution by DHPLC, and the positive samples were subjected to sequencing analysis. We found 5 patients carrying the A1555G and the subsequent family analysis led to the identification of this mutation in 6 relatives (4 of these were pre-symptomatic). Mutations in the MTTS1 gene, encoding the Serine tRNA, are an additional cause for nonsyndromic maternally inherited hearing impairment with onset in childhood. Until now at least 4 MTTS1 mutations have been described (A7445G, 7472insC, T7510C, T7511C). We will discuss the results of the analysis of this hot spot region in our 421 unresolved cases using the DHPLC screening method in order to investigate the prevalence of these mutations in our population.

　

P0694. Phenotype-genotype correlations in filaminopathies A

C. Goizet1,2, G. Sole1,3, I. Coupry1, C. Rooryck-Thambo1,2, C. Marchal3, V. Michel3,

S. De-Bruxelles3, N. Phillip4, L. Olivier-Faivre5, O. Boute6, A. Dieux-Coesler6,H. Journel7, G. Viot8, A. Toutain9, A. David10, M. C. Addor11, L. Villard4, D.Lacombe1,2, B. Arveiler1,2;1Laboratoire de Génétique Humaine, Développement et Cancer, Université VictorSegalen Bordeaux 2, Bordeaux, France, 2Service de Génétique Médicale,H?pital Pellegrin-Enfants, Bordeaux, France, 3Département de Neurologie,H?pital Pellegrin-Tripode, Bordeaux, France, 4Département de Génétique Médicale,H?pital Timone-Enfants, Marseille, France, 5Centre de Génétique, H?pitald’Enfants, Dijon, France, 6Service de Génétique Clinique, H?pital Jeanne deFlandre, Lille, France, 7Unité de Génétique Clinique, CH Bretagne Atlantique,Vannes, France, 8Consultation de Génétique Médicale, H?pital Cochin, Paris,France, 9Service de Génétique Médicale, H?pital Bretonneau, Tours, France,10Unité de Génétique Clinique, H?pital Mère-Enfant, Nantes, France, 11Servicede Génétique Médicale, CHU Vaudois, Lausanne, Switzerland.

Introduction: Filamin A, encoded by the FLNA gene located on chromosome Xq28, is a cytoskeletal protein that cross-links actin in a regulated fashion. Mutations in FLNA have been associated with various phenotypes including periventricular heterotopia (PH),oto-palato-digital syndrome type 1 and 2 (OPD), Melnick-Needles syndrome (MNS) and frontometaphyseal dysplasia (FMD). We have analyzed the FLNA gene in 48 patients who presented clinical and/or brain MRI signs that belong to the spectrum of filaminopathies A.Material and Methods: Thirty-two patients had PH, ten had OPD, two had MNS and four had complex congenital malformation disorders. We used the dHPLC technology for an indirect search of mutation in the entire coding region (47 exons), the 5’ and 3’ UTR regions, and in the intron-exon boundaries. All variants identified using dHPLC were secondary sequenced.Results: We have identified nine novel different mutations in ten patients with PH. The p.Pro207Leu missense mutation was observed in six members of a large OPD family, and two novel missense mutations (p.N187Ser and p.Ser1199Leu) in two unrelated OPD patients. The previously known p.Ala1188Thr mutation was observed in the two unrelated patients with MNS. Discussion: We have identified eleven novel mutations in FLNA.Our data corroborate previous findings concerning the existence of phenotype-genotype correlations in filaminopathies A. Although PH is largely due to loss-of-function mutations, OPD and MNS are caused by mutations leading to the production of a full length protein and providing a gain-of-function effect. The later mutations clustered within a few regions and some are highly recurrent.

　

P0727. Genetic study of spanish families affected by hypertrophic cardiomiopathy

P. Cabello, E. Garcia-Galloway, J. Moya, G. Moreno, E. Ramos, C. Moro, C.San Román;Ramon y Cajal hospital, Madrid, Spain.

Background Hypertrophic Cardiomyopathy is typically an autosomaldominant disease characterized by left ventricular hypertrophy and myofibrllar disarray. The clinical and pathological manifestations are diverse, and range from asymptomatic clinical courses to severe heart failure and sudden cardiac death. Genetic data suggest that 50-60% of the adults with HCM have mutations in one of eigth genes that encode different components of the cardiac sarcomere: β-myosin heavy chain,cardiac Troponin-T, cardiac Troponin-I, α-tropomyosin, cardiac myosin binding protein C, essential and regulatory myosin light chains and cardiac actin.

Results Here, we present the preliminary results of frequencies and types of mutations in 58 unrelated patients with typical phenotype of HCM. In all patients a systematic mutation screening was performed in three sarcomeric genes for which HCM mutations have been described most frequently (MYH7, TNNT2 and MYBPC3) using the DHPLC and sequence analysis. A population of 50 healthy individuals were also included.We report three novel mutations and one previously describe mutation present in 9 patients and affecting two genes (MYH7 and TNNT2). Six of our patients harboured the same novel mutation and the other three had different mutation.

conclusions Most of the sarcomeric protein gene abnormalities are missense mutations that results in a single amino acid substitution within or close to important functional domains. The mutations describe in our study are agree with that, but the frequency distribution of mutations founded in this work suggest the need for a more extensive HCM mutation screening in Spanish population.

Acknowledgments Microelectrónica Espa?ola and FIS Project RTIC05.

　

P0766. congenital myasthenic syndrome (cms): A review of molecular Genetic Analysis to date

A. Jani1, D. Beeson2, S. Man1, P. J. Clouston1, A. Seller1;1Genetics Laboratories, The Churchill, Oxford, United Kingdom, 2NeurosciencesGroup, Weatherall Institute of Molecular Medicine, Oxford, United Kingdom.

Congenital Myasthenic Syndromes (CMS) are a group of clinically and genetically heterogeneous conditions caused by defective transmission at the neuromuscular junction, resulting in fatigable muscle weakness.Identification of the causative mutations is important for clinical and family management.The Oxford Myasthenia centre, supported by the National Specialist Commissioning Advisory Group (Department of Health) provides a National Clinical and Genetic service for the diagnosis of CMS. Full mutation screening is offered for the genes encoding the α, β, δ andε subunits of the acetylcholine receptor (AChR), rapsyn, choline acetyltransferase (ChAT) and the collagen-like tail subunit (ColQ). Our strategy involves clinical assessment followed by targeted mutation screening by dHPLC WAVE. and sequencing. Functional analysis to determine pathogenicity of novel mutations is also available.To date we have received 334 referrals, 247 of which are affected probands. Pathogenic changes have been found in 19% (47 cases,of which 8 are novel mutations). In addition 56 confirmation tests and 49 carrier tests have been performed. We will present a review of data, including a breakdown of the numbers, types and outcomes of referrals, produced to date.

　

P0819. Founder mutation s133t (p.ser133thr) of the sLc26A4 gene in turkish families with Pendred syndrome

L. Jonard1, F. Denoyelle2,3, D. Feldmann1,3, N. Lucidarme4, H. Dollfus5, C. Petit3,E. N. Garab¨|dian2, R. Couderc1,3, S. Marlin6,3;1Laboratoire de Biochimie, H.pital Armand Trousseau, AP-HP, Paris, France,de Biochimie, H.pital Armand Trousseau, AP-HP, Paris, France,2Service d?ˉORL et de Chirurgie Cervico-Faciale, H.pital Armand Trousseau,AP-HP, Paris, France, 3INSERM U587, Unité de génétique des déficits sensoriels,Institut Pasteur, Paris, France, 4Service de pédiatrie, endocrinologieinfantile, H.pital Jean Verdier, AP-HP, Bondy, France, 5Service de GénétiqueMédicale, H.pital de Hautepierre, Strasbourg, France, 6Unité de GénétiqueMédicale, H.pital Armand Trousseau, AP-HP, Paris, France.

The gene SLC26A4 encodes a chloride/iodide and chloride/formate transporter implicated in various forms of human hereditary hearing impairment.Mutations of SLC26A4 were first identified in Pendred Syndrome by Everett in 1997, and have since been further associated with the nonsyndromic autosomal form of deafness DFNB4 and with Enlarged Vestibular Aqueduct Syndrome.We here report three unrelated non-consanguineous Pendred families from Turkey in which all patients presented with profound to severe deafness and goiter. Molecular analysis of the 20 coding exons of SLC26A4 with denaturing high-performance liquid chromatography (DHPLC) followed by sequencing elicited the same homozygous p.Ser133Thr (c.398T>A) mutation in all patients. This mutation affects an amino acid conserved in all vertebrate species and located in the SLC26A transporters signature domain. It was not observed in 100 chromosomes of normal-hearing individuals. Since Borck (2003) reported the p.Ser133Thr mutation at the homozygous level in another Turkish family, and since all families analysed here originated from either east or south of Turkey, we set out to examine microsatellite and SNP markers linked to the SLC26A4 locus in 7q31.1 whether a founder effect or recurrent mutation might be at cause.A single haplotype was shared by all patients analysed, suggesting that p.Ser133Thr is a founder mutation in Turkish patients with Pendred syndrome.

　

P0834. investigation of TSC genes mutation spectrum in tuberous sclerosis families in taiwan

D. Chu, M. Huang;Graduate Institution of Medical Biotechnology, Chang Gung University, Tao-Yuan, Taiwan Republic of China.

Tuberous sclerosis (TS) is an autosomal dominant disorder characterized by the development of hamartomatous growth in many organs. In this study, twenty-seven Taiwanese families including 25 familial and 2 sporadic cases suffering from this disease were tested for mutations in the TSC1 and TSC2 genes. We use RT-PCR method to detect the presence of hamartin and tuberin mRNA from patients with TS and their parents. Mutation screening of the TSC1 and TSC2 genes of the TSC patients was performed with denaturing high-performance liquid chromatography (dHPLC), then DNA sequence variations were confirmed with direct sequencing. Data indicated that genetic lesions were found in 16 patients among the 27 patients. Three possible pathogenic mutations were found in the TSC1 gene, including one missense, one nonsense and one frame shift mutation. In the TSC2 gene analysis ten possible pathogenic mutations in the sporadic cases were found, including two in-frame deletion, seven deletions, one nonsense mutation, and two missense mutations. In addition, real-time quantitative PCR protocol was conducted to determine the quantities of hamartin and tuberin mRNA in those patients with mutations.Comparing patients with mutation and their parents, various levels of hamartin and tuberin mRNA were found. Besides, no significant correlation between particular clinical features and these mutations were found. In conclusion, it seemed that no mutation hot spot was present in Taiwanese TS patients. More data need to be collected to further support this finding.

　

P0859. Plakophilin-2 mutations are the major determinant of familial arrhytmogenic right ventricular cardiomyopathy in the Netherlands

J. D. H. Jongbloed1, M. Entius2, Z. Bhuiyan3, R. Jongbloed4, J. P. van Tintelen1,A. C. P. Wiesfeld5, J. van der Smagt6, L. G. Boven1, M. M. A. M. Mannens3,R. M. W. Hofstra1, I. M. van Langen3, L. C. Otterspoor2, P. A. F. M. Doevedans2,L. M. Rodriguez7, A. A. M. Wilde8, I. C. van Gelder4, R. N. W. Hauer2;1Department of Clinical Genetics, University Medical Center Groningen, TheNetherlands, 2Heart Lung Center, University Medical Center Utrecht, The Netherlands,3Department of Clinical Genetics, Academic Medical Center Amsterdam,The Netherlands, 4Department of Clinical Genetics, University HospitalMaastricht, The Netherlands, 5Department of Cardiology, University MedicalCenter Groningen, The Netherlands, 6Department of Clinical Genetics, UniversityMedical Center Utrecht, The Netherlands, 7Department of Cardiology,University Hospital Maastricht, The Netherlands, 8Department of ExperimentalCardiology, Academical Medical Center Amsterdam, The Netherlands.

In at least 50% of cases Arrhythmogenic Right Ventricular Cardiomyopathy (ARVC) is a familial disease with an autosomal dominant mode of inheritance. Diagnosis is based on clinical criteria proposed by a Task Force in 1994. Recent identification of mutations in the plakophilin-2 (PKP2) gene in 27% of ARVC patients suggests an important role for this gene in this disorder. This will facilitate early identification of persons at risk. Our goal was to assess the prevalence of PKP2 mutations in patients with (supposedly) ARVC in four Dutch tertiary referral centers.A total of 96 index patients were analyzed; 56 patients fulfilled ARVC criteria whereas 40 did not. The PKP2 gene was screened for mutations using DGGE/DHPLC and direct sequencing. In 24 out of 56 index patients (43%) fulfilling the criteria, mutations were identified. Four different mutations were found more than once: 2386T>C, 235C>T,397C>T and 2489+1G>A. Haplotype analyses revealed that all of these were founder mutations. Of the 14 different mutations identified,4 were missense, 7 nonsense, 2 insertion-deletion and 1 splice-site mutation. Eleven of these were novel. Mutations were found in 17/23(73%) proven familial ARVC patients, whereas 11 proven non-familial cases, were non-carriers. In 2 out of 40 patients (5%) not fulfilling ARVC criteria, mutations were identified.In conclusion, PKP2 mutations can be identified in up to 43% of Dutch patients fulfilling Task Force criteria for ARVC. Identical mutations occur frequently. These results will facilitate early recognition of persons at risk and future studies of potential genotype-phenotype relationships.

　

P0955. An audit of a diagnostic service for Hypertrophic cardiomyopathy and Dilated cardiomyopathy.

K. L. Thomson1,2, E. Blair3,2, H. Watkins4,2, A. Seller1,2;1Oxford Genetics Laboratories, Oxford, United Kingdom, 2Oxford GeneticsKnowledge Park, Oxford, United Kingdom, 3Clinical Genetics, Oxford, UnitedKingdom, 4Department of Cardiovascular Medicine, Oxford, United Kingdom.

Since April 2004 the Oxford Genetics Knowledge Park, UK, has funded a molecular genetic service for Hypertrophic Cardiomyopathy (HCM) and Dilated Cardiomyopathy (DCM). Mutation screening by dHPLC and sequencing is available for the MYH7, MYBPC3, and TNNT2 genes. To date, we have identified pathogenic variants in 38/76 HCM and 5/13 DCM patients. Mutation testing has been undertaken in 78 relatives, of whom 61% were found to have the familial mutation. An audit of the analysis performed and the mutations and polymorphisms identified will be presented. Implementation of new strategies to increase detection rate will be considered as will the need for high throughput technologies for the delivery of an effective cardiomyopathy service.

　

P0998. mYH mutations in Belgian polyposis families.

M. Spaepen1, B. Biesmans1, B. Vankeirsbilck1, S. Tejpar2, G. Matthijs1;1Center for Human Genetics, University Hospital of Leuven, Belgium, 2Departmentof Gastroenterology, University Hospital of Leuven, Belgium.

Individuals with homozygous or compound heterozygous mutations in the base excision repair gene MYH are predisposed to develop multiple colorectal carcinomas and classic adenomatous polyposis. To evaluate the contribution of the MYH-mutations in Belgian polyposis families, mutation analysis has been performed by DHPLC on 179 unrelated patients in whom no mutations were identified in the APC- and MMR-genes or from whom the tumors were microsatellite stable. Six patients were found with biallelic mutations of the MYH gene: p.Y165C/p.Y165C, p.G382D/c.1249-1263del15bp, p.Y90X/p.P391L and, in three patients, p.Y165C/p.P391L. It is remarkable that four of these six patients were compound heterozygous for the p.P391L mutation what suggests a founder mutation. This missense mutation has previously also been found in 14% of the patients with biallelic mutations in the Netherlands (1). The c.1249-1263del15bp mutation has not been reported before. Further, seven patients were heterozygous carriers of only one pathogenic MYH mutation. This observation fits with the emerging evidence that monoallelic MYH mutation confer a potentially low-penetrant risk of colorectal cancer (2). Finally, the presence of four novel missense mutations (p.A208V, p.A226S, p.R295C, p.A359V) in a heterozygous state in the patient group and their complete absence in 200 healthy controls may also add to this feature. In conclusion: 10 % of the FAP/AFAP phenotypes (6/58) were characterized as MAP (MYH Associated Polyposis) from which more than half were (compound) heterozygous for p.P391L, a potential Belgian/Dutch founder mutation.

　

P1005. Prevalence of GJB2, GJB6 and A1555G mutations in the Italian population

P. Paterini1, D. Bastia1, M. Seri2, D. Turchetti2, C. Graziano2, C. Bergonzoni3, M.Montaguti3, V. Mantovani1,2, G. Romeo2;1CRBA Policlinico S.Orsola-Malpighi, Bologna, Italy, 2U.O. Genetica MedicaPoliclinico S.Orsola-Malpighi, Bologna, Italy, 3U.O. Otorinolaringoiatria PoliclinicoS.Orsola-Malpighi, Bologna, Italy.

Mutations in the connexin 26 encoding gene (GJB2) have been described as major cause of non-syndromic hearing loss (NSHL).Deletions involving connexin 30 (GJB6) were also associated with NSHL; A1555G mutation in mitochondrial (mt)DNA has been shown to predispose to aminoglycoside ototoxicity. Aim of our study was to evaluate the prevalence of these molecular defects in the Italian population. 133 patients with mild to profound,familiar or apparently sporadic NSHL were studied. 513 unaffected controls were also screened to evaluate the carrier frequency.Mutation screening by DHPLC and sequencing for GJB2, multiplex PCR for 309kb and 232kb deletions in GJB6 and restriction analysis for A1555G mutation were performed. 38 patients (28,6%) showed molecular variants of GJB2. In addition to known mutations two novel defects (G109W and 153delT) were found in compound heterozygotes with 35delG. Six novel variants (G>C - 3277, IVS1-6T>C, IVS1-2A>C, Y158Y, K221N, N62N) were also detected. One patient (0.8%) carried the 309kb GJB6 deletion and one showed homoplasmic A1555G mtDNA mutation.61 controls (12%) showed molecular variants of GJB2; 25 (1:20) were carriers of disease-causing mutations. Four novel variants (IVS1-6T>C, IVS1-2A>C, A/G at -8, D159N) were also detected.In conclusion, GJB2 mutations resulted responsible for almost one third of the NSHL in our patients and a high prevalence of mutation carriers was shown in our population. The low frequency of GJB6 deletions and A1555G mtDNA mutation suggests that the occurrence of these defects is restricted to specific populations.

　

P1032. P53 variants and recurrent spontaneous abortions

M. Kaare1, J. N. Painter1, V. Ulander2, R. Kaaja2, K. Aittom.ki1,3;1Folkh.lsan Institute of Genetics, Helsinki, Finland, 2Department of Obstetricsand Gynecology, Helsinki, Finland, 3Department of Clinical Genetics, Helsinki,Finland.

Recent studies indicate that p53 may regulate the response of embryonic cells to diverse environmental stresses. Moreover, it appears that maintaining a fine balance of p53 protein levels within embryonic cells is important for optimal development as both over-and underexpression can lead to different malformations or embryonic lethality. Because altered levels and activity of the p53 protein may be caused by mutations or polymorphisms in both the coding and noncoding regions of the gene we aimed to investigate if variations in p53 are associated with susceptibility for recurrent spontaneous abortion (RSA). We screened 86 Finnish patients (40 couples and 6 women) with unexplained RSA and 96 controls using DHPLC and sequencing. The six intronic and three exonic variations detected have been previously reported and are predicted to be common polymorphisms. When comparing the genotypes and allele frequencies of these variations between patients and controls, the C11992A polymorphism in intron 3, located 29 bp upstream of exon 4, was shown to be significantly more frequent in the patients than in the controls. Women carrying the rarer A allele have a more than two-fold increased risk of miscarriage (p=0.0193, OR 2.333, CI 1.1305-4.816), indicating that the A allele is associated with RSA. Further studies are, however, necessary to define whether this polymorphism has functional consequences. If the variation has a phenotypic effect, the C11992A variation could be one of the of genetic factors increasing susceptibility to RSA.

P1053. tRAPs mutations (tNF Receptor gene tNFRsF1A Associated Periodic syndrome) are frequent in rheumatoid arthritis families but show no evidence for association nor linkage with the disease

P. Dieud¨|1,2, D. Tchernitchko3,4, L. Michou1,5, E. Glikmans1, M. Goossens3,4, F.Corn¨|lis1,5, f. ECRAF1;1GenHotel-EA3886, Evry, France, 2Bichat Hospital AP-HP, Paris, France, 3INSERM U654, Creteil, France, 4Mondor Hospital, AP-HP, Cr¨|teil, France, 5Lariboisi¨¨re Hospital, AP-HP, Paris, France.

TNFRSF1A mutations cause TRAPS [OMIM*191190]. A recent study suggested that the R92Q mutation was associated with chronic polyarthritis. OBJECTIVE : We aimed at searching for TNFRSF1A mutations in RA, to be tested for linkage. MATERIAL AND METHODS: the 386 DNA of 100 trio families (one RA case and both parents) and 86 index cases of RA affected sib-pair (ASP) families from the French Caucasian population were investigated by dHPLC (denatured highperformance liquid chromatography) for TNFRSF1A mutations in exons 2 to 4. The test for association compared cases and . virtual controls .(derived in the trio families from un-transmitted parental chromosomes).The test for linkage relied on the transmission disequilibrium test (TDT)in trio families and cosegregation in ASP families. RESULTS : Only the R92Q mutation was detected, in 2 of the 100 index cases of trio families(including one de novo mutation) and 5 (6%) of the index cases of ASP families, but also 6% of the controls, showing no association with the disease. No RA linkage evidence was found : a) out of 7 heterozygous parents in the trio families, only 1 transmission of the mutation was observed ; b) among the 5 RA sibs of the mutated index cases from ASP families, only one carried the mutation. CONCLUSION : This TNFRSF1A investigation in RA from the French Caucasian population showed only the R92Q mutation, with a frequency of 4%, but no evidence for RA association nor linkage to the disease.

　

P1059. screening of the tsc1 and tsc2 genes for tuberous sclerosis patients

R. L. Touraine, A. Combes, M. Gilet;CHU de Saint Etienne, Saint etienne, France.

We did molecular testing of the TSC1 and TSC2 genes in 90 Tuberous Sclerosis (TS) families, using MLPA, DHPLC, sequencing and microsatellite analysis. A mutation has been identified in 51 cases,analysis is still in progress for 35 cases, and no mutation was found in four cases.We designed a strategy to screen novel cases, permitting to find more than half of TSC2 mutants and more than 80% of TSC1 mutants in a shorter time than previously.Two third of the mutations were de novo. TSC2 was more frequently involved than TSC1 (83% versus 17%), specially in those de novo cases (91% versus 9%). In familial cases, TSC1 and TSC2 were almost equally involved.Only one third of the 51 mutations were previously described. Most of the mutations were truncating mutations. Two large deletions of TSC2,were found.A better prognosis is ascribed to TSC1 mutations and to familial cases.In one of our families, all the persons with TS were very mildly affected.Unfortunately, clinical severity may vary from person to person in a same family. Nevertheles, half of de novo cases had a favorable outcome.For cases detected in utero, prognosis was not worse than for cases diagnosed postnataly. We believe it may be better, due to a sharper follow-up.In summary, even if turn around time can be improved, molecular testing in TS is long and laborious. Therefore, clinical diagnosis of TS is still a prerequisite to molecular testing.