ESHG Posters 21

Retinitis pigmentosa, metaphyseal chondrodysplasia, and brachyodactyly: an affected brother and sister

I. Durbala¹, I. Dimofte¹, L. C. Enache², D. P. Balaban³, A. Popa¹, V. Broasca Madar⁴;
¹Medical Genetics Department, Faculty of Medicine, Constanta, ROMANIA, ²Cell and Molecular Biology Deparment, UMF "Gr.T.Popa", Iasi, ROMANIA, ³Biochemistry Department, Faculty of Medicine, Constanta, ROMANIA, ⁴Management and Public Health Department, Faculty of Medicine, Constanta, ROMANIA.

A brother and sister, children of normal parents, are described. They had retinitis pigmentosa, causing near blindness as a result of very narrow fields of vision, associated with metaphyseal chondrodysplasia and marked shortening of the metacarpals and terminal phalanges.
Autosomal recessive inheritance is suggested with a common biochemical cause for all this defects.
This apparently new association of retinitis pigmentosa with a systemic bone dysplasia emphasis that this not common clinical diagnosis has a variety of different possible causes.

De novo translocation t (13; 18)(q14, q23) in a girl with disproportional short stature, congenital glaucoma, unusual face, persistent ductus arteriosus and muscular hypotonia

B. I. Dimitrov, D. M. Avdjieva, E. G. Simeonov, I. Boneva, A. Andreev;
University Pediatric Hospital, Section of Clinical Genetics, Sofia, BULGARIA.

Kamelia, K.S., a female patient, born at term(W-3000 gr.,L-46 cm.), from second uneventful pregnancy of unrelated young parents was investigated because of disproportional rhizomelic short stature and dysmorphic face, reminding hypo/ achondroplasia. The first pregnancy was aborted spontaneously. Otherwise, the family history was unremarkable.
At admission the patient manifested disproportional short stature with shortening of the long bones of limbs, high forehead with prominent tubera, depressed nasal bridge, low set dysplastic ears, hypertelorism, short neck, congenital bilateral glaucoma and congenital heart disease (persistent ductus arteriosus). Cytogenetic testing disclosed a de novo translocation t(13;18)(q14;q23) in the patient. Progressing shortening of long bones and initial metaphyseal changes of both ulnae (hypoplasia, irregularity, cupping of the ends) have been observed during 1 year follow-up.
Authors discuss the differential diagnosis and genetic basis of this unusual case of skeletal dysplasia, caused by structural chromosomal aberration.

Study Of The Wolfram Syndrome Gene (wfs1) In Spanish Patients With Diabetes Mellitus And Deafness

E. Domenech, M. Gomez Zaera, V. Nunes;
Cancer Research Institute, Hospitalet de Llobregat, SPAIN.

Wolfram syndrome (WS) is an autosomal recessive neurodegenerative disorder characterised by early onset diabetes mellitus and progressive optic atrophy, as well as other clinical features such as deafness, diabetes insipida, renal tract abnormalities and diverse psychiatric illnesses.
A gene (WFS1) localized on 4p16.1 has been described, encoding a putative 890 amino acid transmembrane protein, WFS1. Recently, a new locus for WS has been identified on 4q22-24, providing additional evidence for the genetic heterogeneity of this syndrome.
This work aims to describe possible nucleotide changes in the WFS1 gene in a Spanish population affected with diabetes mellitus (DM, n=38), neurosensorial deafness (F, n=48) or both conditions (DM+F, n=48) vs. control group (C) of 48 healthy individuals.
We have identified a total of 18 nucleotide changes in the WFS1 gene: 3 mutations (D729N, L751I and V871M, the latter previously described), 4 new polymorphisms (1294C>G, 1308C>G, 1364C>T and 2438T>C) and 11 previously known polymorphisms. The analysis of the association of the polymorphisms with diabetes mellitus or deafness has revealed, when comparing DM and DM+F groups vs. control group, statistical differences in the allelic and genotypic distribution of the following changes: 1185T>C, 1832G>A, 2433G>A and 2565A>G.
Our data suggest that some changes in heterozygosis in the WFS1 gene can contribute to the diabetes mellitus and deafness phenotype, in the population studied.
This work has been supported by the MCYT, SAF 99-0079. E. Domènech has a FPI grant from MCYT.

Brachydactyly-Symphalangism-Deafness Syndrome (BSDS). A clinical-genetic study of 18 patients in two families

M. Bembea¹, C. Jurca¹, S. Bembea², R. Spineanu¹, M. Barbu¹;
¹Clinical Children Hospital, Oradea, ROMANIA, ²Clinical Adults Hospital, ORL Department, Oradea, ROMANIA.

Introduction: (Multiple Synostoses with Brachydactyly; Brachydactyly-Symphalangism Syndrome 186500, MIM) is a rare entity, and the diverse names reflect the uncertainty in the definition of the syndrome. Objective: The comparison of clinical-genetic aspects between the patients of two families and a correlation with data obtained from review. Patients and method: Our clinical-genetic study comprises 18 cases personally examined in two not connected families (a family with 14 cases, another family with 4 cases). Results: The cases offer a complete and well-defined clinical picture of a rare syndrome that has as definitory elements: brachydactyly+symphalangism+progressive deafness+autosomal dominant inheritance pattern. The syndrome is characterized by complete penetrance and variable expressivity. They are surprisingly similar in what the extension of finger morphologic lesions is concerned, but different in what the onset age and the degree of hearing loss is concerned. The large number of cases, on more generations, with constant association of the three anomalies, offers a good opportunity for gene investigation by linkage analysis. The role of Nog gene is discussed. Conclusions: The description of a large series of 18 examined cases belonging to two families shows the association of three distinct pathologic changes: Brachydactyly-Symphalangism-Deafness (BSD Syndrome). This definition of the syndrome, including all three pathologic changes, is distinguishing this syndrome from other resembling entities classified in the MIM catalogue.

Incidence of connexin-26 mutation 35delG and sensorineural hearing loss with unknown reason.

T. G. Markova¹, I. A. Shagina², S. M. Megrelishvili¹, N. G. Zaitseva¹, A. V. Polykov²;
¹St.-Petersburg ENT and Speech Research Institute, St.-Petersburg, RUSSIAN FEDERATION, ²Moscow Research Center for Medical Genetics, Moscow, RUSSIAN FEDERATION.

Connexin-26 (Cx26) mutations account for 30-60% of non-syndromal hearing loss (NSHL) in European and American populations. Only one mutation 35delG is responsible for 70-80% of detectable Cx26 mutations. The hearing impairment has been described as severe or profound for these patients. Recently some studies confirmed a high carrier frequency, 2,14%, for 35delG mutation in Russia, but there was no data about frequency of this mutation between NSHL in the country.
We have studied 75 unrelated individuals with congenital NSHL in order to evaluate the prevalence 35delG mutation. Most of patients have had NSHL with unknown reason. We did not exclude patients with some risk factors for acquired hearing loss. In a total this mutation was found in 44% of the patients tested and was presented in 42% of sporadic cases.
The hearing loss in homozygous and heterozygous individuals varied from moderate to profound. No evidence of progression of the hearing impairment was found. The majority of homozygous for 35delG mutation have severe or profound hearing loss. Only one homozygous has significant asymmetrical hearing impairment. Two heterozygous patients have mild hearing impairment, but both of them are from deaf families.
We have confirmed that 35delG mutation of the Cx26 gene accounts for a large proportion of cases with congenital NSHL and especially in cases with unknown reason. More over hearing loss have considered to be acquired in some of 35delG homozygote before identification of this mutation. Thus molecular genetic analysis will allow appropriating genetic counseling. Other Cx26 mutations are under study.

Does molecular testing really improve genetic counselling ? example of Cx26 in non syndromic isolated deafness

R. Bernard, N. Levy, S. Sigaudy;
Département de Génétique Médicale, Marseille, FRANCE.

Hearing impairment is frequent : 2/1000 individuals present with deafness occuring in childhood. Half of which is now considered as having an hereditary origin. Around 70% of genetic cases are non-syndromic and most of them involve the connexin 26 gene. Here we present a multidisciplinary approach toward genetic counseling and molecular exploration in inherited deafness. Based on our experience, two types of situations are particularly critical and will thus be discussed.
One concerns the identification of a single mutation in CX26 among patients affected with profund, bilateral, prelingual deafness. In such a situation, genetic heterogeneity as well as a possible autosomal dominant mode of inheritance have to be considered. Besides, some mutations in CX26 have been reported as not being disease causing. Further explorations in non coding sequences (5’ UTR exon, promoter, introns, enhancer¼ ) have thus to be discussed. In addition, deletions in the Connexin 30 gene, adjacent to CX 26, have been reported in a digenic association in patients carrying heterozygous CX26 mutations.
The second point concerns the identification of disease causing mutations leading to a prenatal diagnosis request from parents. This aspect has to be carefully considered since the French National Ethic Comittee was unfavourable to termination of pregnancies in case of mutations in CX26. To date, in France, no pluridisciplinary center of prenatal diagnosis access to prenatal diagnosis in deafness. We wish to discuss the difficulties raised by the recent identification of genes in sensory disorders with regard to the improvement of genetic counseling.

Evaluation of Relation between Glaucoma and Antioxidative System (GST-M and GST-T) Polymorphisms

A. C. Ozkilic¹, M. Guven¹, M. Unal², K. Devranoglu², T. Ulutin¹;
¹Medical Biology, Cerrahpasa Medical Faculty,Istanbul University, Istanbul, TURKEY, ²Ophtalmology,Cerrahpasa Medical Faculty,Istanbul University, Istanbul, TURKEY.

Recent studies have been pointed out to the important role of oxidative mechanisms in the opthalmological diseases such as glaucome and cataract. Glutathione S-transferase (GST), one of the enzymes of antioxidative system, has an important function in defending against oxidative damages. m (GST-M) and q (GST-T) are the isoenzymes of GST whose polymorphisms are shown to affect the tendency toward different kinds of diseases. We searched the relation between glaucoma and GST-M and GST-T polymorphisms that play role in oxidative defense mechanisms. Results of 46 glaucoma patients (45-71 years of age) were compared with 53 normal control cases (48-68 years of age) lacking in an ophtalmological disease. Significant relation was obtained between GST-M null (with deletion) genotype and glaucoma (x² : 4.02 , p:0.04) whereas there was no statistical significance between GST-T null genotype and glaucoma (x²:0.13 , p:0.71) According to this study assessing the pre-results of a wide-spread research, GST-M null genotype in Turkish population have an effect on tendency toward glaucoma.

Linkage analysis of a three generation pedigree with Autosomal Dominant Vitreoretinochoroidopathy (ADVIRC).

N. A. Hart-Holden¹, G. C. M. Black², P. D. Watson¹, B. A. Lafaut³, L. M. Messiaen⁴, B. Loeys⁴, A. P. Read¹;
¹Dept. Medical Genetics, St Marys Hospital, Manchester, UNITED KINGDOM, ²Dept. Clinical Genetics, St. Marys Hospital, Manchester, UNITED KINGDOM, ³Dept. of Ophthalmology, Ghent University Hospital, Ghent, BELGIUM, ⁴Dept. of Human Genetics, Ghent University Hospital, Ghent, BELGIUM.

Autosomal dominant vitreoretinochoroidopathy (ADVIRC) is a rare condition, which was first described by Kaufman et al. (1982). Lafaut et al reported a three-gene pedigree with ADVIRC, on which we have performed linkage analysis. A number of vitreoretinal disorders have been localised to a region of chromosome 11 (11p14-11q23) and include dominant familial exudative vitreoretinopathy 1 and 3 (EVR1 and EVR3), dominant inflammatory vitreoretinopathy (VMD2) and dominant neovascular inflammatory vitreoretinopathy (VRNI). Due to the relative richness of this region in retinal genes with overlapping phenotypes, initial analysis was performed for markers D11S1902 (11q13.3) and D11S527 (11q13.5). Further linkage analysis was performed yielding two recombinants. Evidence that the ADVIRC locus is centromeric to marker D11S929 was provided with a crossover in individual 5, in which segregation occurred with marker D11S922 (11p15.5) but not with D11S929 (11p14). A crossover in individual 10 at D11S4139 (11q13.3) indicates that the locus is centromeric to this marker. In this individual segregation occurred with D11S1362 (11q14.1) but not with D11S4139. Of the number of candidate genes in this region, the most likely to be associated with ADVIRC is PAX6. Normally associated with aniridia, PAX6 has been implicated broadly in human anterior segment malformations (Hanson et al 1994, Mirzayans et al 1995).

Nance-Horan syndrome: refinement of the gene localization on Xp22.13 and analysis of 5 candidate genes

A. Toutain¹, B. Dessay¹, N. Ronce¹, D. Trump², C. Moraine¹, B. Franco³;
¹Genetics Department, Tours, FRANCE, ²Department of Medical Genetics, Cambridge, UNITED KINGDOM, ³Telethon Institute of Genetics and Medicine, Naples, ITALY.

Nance-Horan syndrome (NHS) is an X-linked condition characterized by congenital cataracts, dental abnormalities, dysmorphic features, and mental retardation in 30% cases. Previous studies have mapped the disease gene to a 2 cM interval on Xp22.2 between DXS43 and DXS999. We report additional linkage data resulting from the analysis of 11 independent NHS families. A maximum Lod score of 9.94 (theta = 0.00) was obtained at the RS1 locus and a recombination with the locus DXS1195 on the telomeric side was observed in two families, thus refining the location of the gene to an interval of around 1 Mb on Xp22.13. Direct sequencing or SSCP analysis of the coding exons of 5 genes (SCML1, SCML2, STK9, RS1 and PPEF1), considered as candidate genes on the basis of their location in the critical interval, failed to detect any mutation in 12 unrelated NHS patients thus making it highly unlikely that these genes are implicated in NHS.

Linkage study and detection of a new mutation in PNR gene in large family with inherited retinitis pigmentosa

A. Polyakov¹, V. Fedotov², O. Khlebnikova³, N. Vasserman¹, A. Dergacheva¹, S. Tverskaya¹;
¹Research Centre for Medical Genetics, Moscow, RUSSIAN FEDERATION, ²Genetic Counseling Department of Diagnostic Center, Voronezh, RUSSIAN FEDERATION, ³Institute of eye diseases, Moscow, RUSSIAN FEDERATION.

Retinitis pigmentosa (RP) is a heterogeneous group of eye diseases, which includes some autosomal recessive and autosomal dominant inherited types of disorders caused by mutations in different genes. As usual the clinical history of RP starts within the first decade with night blindness, followed during the second and the third decade by slow reduction of the visual field. Here we are reported about large families from Voronezh province with inborn night blindness, hemeralopia, cataract and retinal pigmentary degeneration detected on first year of life. The parents are not consanguine and have 15 children, 6 of them are affected and one of these is newborn. We have carried out the linkage analysis with candidate genes and revealed the LOD score of 3.53 on chromosome 15 between D15S123 and D15S979 in 38 cM interval. The absence of recombination did not allow to refine this interval. The photoreceptor cell-specific nuclear receptor gene (PNR) is a candidate gene from this locus. The mutations in it are known in some cases of RP with late onset. We have screened PNR and detected the delA481 in maternal chromosome, which segregated with affected status. It is a first frame shift mutation in PNR; other reported mutations were in frame. We have not found the second mutation in this family in this gene during sequencing the coding part of paternal chromosome.

Linkage to 18qter differentiates two clinically overlapping syndromes: Congenital Cataracts Facial Dysmorphism Neuropathy and Marinesco-Sjögren syndromes.

C. Lagier-Tourenne¹, D. Chaigne², J. Gong³, J. Flori⁴, D. Christmann⁵, J. Flament³, J. L. Mandel¹, M. Koenig¹, H. Dollfus⁶;
¹IGBMC, Illkirch, FRANCE, ²Clinique Sainte Odile, Strasbourg, FRANCE, ³Service d'ophtalmologie, Hopitaux universitaires de Strasbourg, FRANCE, ⁴Centre médico-chirurgical et ostétrical, Schiltigheim, FRANCE, ⁵Service de radiologie, Hopitaux universitaires de Strasbourg, FRANCE, ⁶Fédération de génétique, Hopitaux universitaires de Strasbourg, FRANCE.

The Marinesco –Sjögren (MS) syndrome is an autosomal recessive disorder characterized by somatic and mental retardation, congenital cataracts and cerebellar ataxia. Peripheral neuropathy and acute rhabdomyolysis have been also occasionally described. CCFDN syndrome (Congenital Cataracts, Facial Dysmorphism and Neuropathy) is a recently reported condition considered to be a differential diagnosis of MSS (Tournev et al, 1999). CCFDN has been diagnosed, so far, only in a specific Gypsy group in Bulgaria. This disorder was mapped by linkage analysis to 18qter (Angelicheva et al, 1999).
We performed the clinical and molecular study of two consanguineous families with patients initially reported to have MS syndrome. The patients of the first family, originating from Turkey, had a classical MS syndrome especially with a pronounced cerebellar atrophy on MRI and an evocative muscle pathology. Linkage to 18qter was ruled out.
The three affected siblings of the second family, of Gypsy origin, differed clinically from the previous family by the presence of microcornea, marked peripheral neuropathy and a discrete cerebellar atrophy. The molecular findings were consistent with the diagnosis of CCFDN syndrome. Indeed, all three affected siblings were homozygous over four consecutive 18qter markers (D18S1122 to D18S70), confirming linkage to the CCFDN locus and excluding the diagnosis of MS syndrome.
This study emphasizes the clinical overlap between the MS and CCFDN syndromes which are however distinct genetic entities. Further molecular studies should lead to a better delineation of these syndromes.

ACE gene insertion-deletion polymorphism and risk of advanced retinopathy of prematurity in Kuwaiti Arabs

M. Z. Haider, M¹, L. V. Devarajan, M¹, M. Al-Essa, M¹, H. Kumar, M²;
¹Kuwait University, Safat, KUWAIT, ²Ibn Sina Hospital, Ophthalmology Dept, KUWAIT.

Retinopathy of prematurity (ROP) is a disease characterized by neovascularization which occurs in infants with short gestational age and low birth weight and leads to retinal detachment and blindness. In a proportion of ROP cases, the disease progresses to advanced stages despite rigorous intervention. The genotypes for angiotensin converting enzyme (ACE) gene insertion/deletion (I/D) polymorphism were determined in 181 Kuwaiti premature infants by using a polymerase chain reaction (PCR) method. The incidence of I/D genotypes was compared in ROP cases (n = 74) and controls (n = 107) and within the two subgroups of ROP patients; one in which ROP regressed spontaneously (stage 1-3; n = 53) and two, in which it progressed to advanced stages (stage 4-5; n = 21). When the ROP cases were considered collectively, the incidence of DD genotype was almost identical to that in controls. The incidence of heterozygous ID genotype was higher in controls and the incidence of II genotype was higher in ROP cases compared to controls (p < 0.01). In contrast to this, when ROP cases were divided in two subgroups the incidence of DD genotype was significantly higher in advanced stage ROP cases compared to spontaneously regressed ROP cases (p < 0.04). The incidences of ID and II genotypes were not significantly different amongst the two subgroups of ROP patients. The data suggests that the presence of DD genotype of ACE gene I/D polymorphism in Kuwaiti premature infants is associated with a higher risk of progression of ROP to advanced stages.

F. Di Leva¹, L. Strollo², A. P. D'Adamo³, M. Carella³, A. Caravelli¹, G. Auletta², W. Livi⁴, A. Renieri⁵, M. D'Urso¹, P. Gasparini³, E. Marciano², A. Franzè¹;
¹IIGB-CNR, Naples, ITALY, ²Institute of Audiology, Department of Neuroscience and Behavioural Sciences, University “ Federico II “, Naples, ITALY, ³TIGEM, Via P. Castellino 111, Naples, ITALY, ⁴Institute of Otorhinolaryngological Sciences, University “Le Scotte, Siena, ITALY, ⁵Institute of Medical Genetics, University University “Le Scotte”, Siena, ITALY.

Otosclerosis is the single most common cause of hearing impairment among white adults with a prevalence of 0.2-1%. Mean age of onset is in the third decade and 90% of affected persons are under 50 years of age at the time of diagnosis. Long-term follow up suggests that about 10% of these persons ultimately develop a profound neurosensorial hearing loss across all frequencies. The etiology of otosclerosis is unknown and its genetics is poorly understood. The majority of studies indicate autosomal dominant inheritance with reduced penetrance or heterogeneity or digenic recessive inheritance or digenic inheritance, but of an X-linked dominant gene and an autosomal recessive gene. Other studies suggest that otosclerosis may be a xenogenetic disease that requires a specific host genotype and exposure to a specific viral pathogen for the disease phenotype to be expressed.
To elucidate the pathogenesis of the otosclerosis, identification of the responsible genes is essential. Until now, genetic linkage in otosclerosis has been demonstrated only in two families. The first OTSC locus was identified on chromosomes 15q25-q26, and recently the second on chromosome 7q34-q36. Both OTSC loci are very large of 14.5 and 16 cM, respectively. We have selected Italian families with otoslerosis and we have realized a linkage exclusion/association analysis. Based on this strategy, we have excluded the linkage of some families to known OTSC loci suggesting the existence of at least one new OTSC locus. We have also evidence that one of our families is linked to OTSC1 locus.

Spectrum of connexin 26 gene (GJB2) mutations in families from Bashkortostan with inhereted non-syndromic hearing loss.

L. U. Dzhemileva, I. M. Khidiatova, E. K. Khusnutdinova;
Institute of biochemistry and genetics, Ufa, RUSSIAN FEDERATION.

Recent findings that a high proportion of non-syndromic hereditary sensorineural hearing loss is due to mutations in the gene for connexin 26 indicate the crucial role that the gene product plays for normal functioning of the cochlea. Mutations in the GJB2 gene account for the large proportion of pre-lingual hearing impairment with a prevalence up to 50% in autosomal recessive cases and still undefined prevalence in sporadic cases.
Ninety-seven subjects affected by non-syndromic sensorineural hearing impairment were unrolled in the study. The patients had either a family history of childhood hearing deficit or represented sporadic cases. Cx26 mutations we found in 58% of subjects. We identified the prevalence of the 35delG allele in the patients of sporadic cases with non-syndromic hereditary sensorineural hearing loss from Bashkortostan to be 52% of chromosomes screened. Screening the patients of 97 subjects we identified 66% 35delG mutation homozygotes and 20% heterozygotes. In addition to above mutations several types of mutations - del235G, 313-314delAA, 360delG, del314A were identified. The 35delG mutation was present in 86% of all Cx26 mutations identified. Also we observed two new mutations del314A and 360delG in the patients. The possible implication of the connexin genes mutations in the pathophysiology of some progressive adult deafness opens new prospects in the fine diagnostic of the ear diseases and eventually may lead to new therapeutic strategies applied to the cochlea.

Mutations in CLDN14 are a rare cause of non-syndromic recessive childhood deafness.

M. Wattenhofer¹, V. Falciola¹, A. Pampanos², R. Rabionet³, N. Scamuffa¹, C. Rossier¹, M. B. Petersen², X. Estivill³, S. E. Antonarakis¹, A. Reymond¹;
¹Medical Genetics, Geneva University, Medical School, Geneva, SWITZERLAND, ²Institute of Child Health, Athens, GREECE, ³Medical and Molecular Genetics Center, Institut de Recerca Oncologica, Barcelona, SPAIN.

Approximatively 50% of early-onset deafness cases are genetic and 50% of recessive cases are due to mutations in the GJB2 gene. The claudin CLDN14 gene on chromosome 21, encoding a member of the tight junction protein family, is mutated in two DFNB29 Pakistani families. To determine if CLDN14 mutations are an important contributor to the etiology of childhood deafness, we screened the CLDN14 single-coding exon on 366 chromosomes of non-syndromic recessive deaf patients from Spain and Greece, negative for GJB2 gene mutations. This study allowed us to define multiple polymorphisms in the CLDN14 gene.
An amino-acid substitution, G101R, found in a Greek patient in heterozygozity and leading to G101R is likely to be pathogenic. This variant has not been found in the remaining 365 chromosomes and affects a conserved residue of the claudin family. Gly101 is within the second transmembrane domain of this tight junction protein family and is preserved in 18 of the 20 described CLDN proteins. Unlike the modification observed in our patient the two remaining CLDN present small hydrophobic residues at this position (Ala and Val). R101 has been transmitted by the hearing father and cannot therefore be considered as a dominant mutation. We have investigated newly identified 5’UTR exons for a potential maternally-inherited mutation in this patient and found no potential second mutation. We are currently examining the pathogenicity of the G101R substitution in a functional assay. Our results indicate that mutations in CLDN14 do not substantially contribute to the non-syndromic deafness in the Mediterranean population.

H. Caria¹^,2, T. Matos¹, I. Galhardo³, M. Simão³, A. O'Neill⁴, Ó. Dias³, M. Andrea³, C. Correia¹, G. Fialho¹;
¹Center of Genetics and Molecular Biology, Lisbon, PORTUGAL, ²High Scholl of Health, Politecnic Institute of Setubal, Setubal, PORTUGAL, ³Center of ORL, University of Lisbon, Lisbon, PORTUGAL, ⁴Center of ORL, Hospital Egas Moniz, Lisbon, PORTUGAL.

Mitochondrial DNA mutations have been implicated in hearing loss, existing several mutations already reported in deaf families. However, despite the increasing number of reports implicating mtDNA mutations in hearing impairment, the frequency of these mutations as cause of nonsyndromic sensorineural hearing impairment (NSSHI) remains unknown. Some of those mutations, namely A1555G and A7445G, have been describe in different ethnic populations raising the possibility that such mutations are more frequent than initially thought. Screening for mtDNA mutations might thus be worthwhile in many cases of familial hearing impairment.
In the present study, we have analysed 30 NSSHI families with possible maternal inheritance, as well as 500 unrelated normal hearing individuals, for the presence of A1555G and A7445G mutations, by using restriction digestion and when relevant, direct sequencing of PCR products. Three other mutations affecting mitochondrial tRNASer (UCN) gene at nt 7472, 7510 and 7511 were also studied. The results obtained represent an additional contribution for the genetic characterization of the portuguese population.

Mutations in the connexin 26 gene in German, Hungarian and Polish patients with hearing impairment

B. Haack¹^,2, T. Tóth³, M. Wróbel⁴, I. Sziklai³, W. Szyfter⁴, H. Zenner², N. Blin¹, M. Pfister², S. Kupka¹^,2;
¹Department of Anthropology and Human Genetics, University of Tübingen, GERMANY, ²Department of Otolaryngology, University of Tübingen, GERMANY, ³Medical and Health Science Center, Department of Otolaryngology, University of Debrecen, HUNGARY, ⁴Department of Otolaryngology, University of Medical Sciences, Poznan, POLAND.

Mutations in the connexin 26 gene (GJB2) encoding a gap-junction protein have been identified in many patients with childhood hearing impairment (HI). One single mutation, 35delG, accounts for up to 70% of all analyzed European patients with HI inherited in autosomal recessive manner and 10% of patients with HI of unknown origin, respectively. Therefore we screened our collectives containing 291 German, 55 Polish and 90 Hungarian patients and corresponding control subjects for this and other connexin 26 mutations by PCR, SSCP and sequencing. For German patients with sporadic hearing impairment the 35delG frequency was 0.1 and for Polish cases 0.43, respectively. For Hungarian individuals the allele frequency was 0.41. All patients showing heterozygosity for 35delG or conspicous SSCP-results were sequenced. This study revealed several new patient-related mutations (e.g. G59V, D66A, I82M, K112E, R127H) and new gene variants resulting in amino acid substitutions. In summary, more than 20 new allelic changes were detected and for most of them, patterns of inheritance were documented.

Association of the T14709C mutation of mitochondrial DNA with maternally inherited diabetes mellitus and deafness in an Italian Family.

L. Rigoli¹, D. C. Salpietro¹, R. A. Caruso², C. Di Bella¹, G. Calabro'¹, V. Procopio¹, M. Rigoli¹, I. Barberi³;
¹Unità Operativa di Genetica ed Immunologia Pediatrica, Messina, ITALY, ²Dpt.Patologia Umana, Messina, ITALY, ³Dpt. Scienze Pediatriche Mediche e Chirurgiche, Messina, ITALY.

A maternal effect in the transmission of non-insulin dependent diabetes mellitus (NIDDM) suggests the involvement of genetic factors encoded by mitochondrial DNA (mtDNA) in its pathogenesis. In this report, the np 3243, np 7445 and np 14709 mt mutations, and the 10.4 Kb deletion in 10 Italian families (Southern Italy) with maternally inherited diabetes mellitus and/or deafness have been analysed.The np 3243, np 7445 mutations and the 10.4 KB deletion were not found in any of the subjects of our study. Instead,in a family the mutation T14709C was found in three probands and in their diabetic mother. In this pedigree, a 16-year-old boy became deaf at the age of 12. The sister, who was diagnosed as having deafness at the age of 6, was affected by a severe myopathy and complained of progressive muscle weakness and exercise intolerance. A diagnosis of mitochondriopathy was based on a deltoid muscle biopsy that showed a large number of ragged-red fibres. The brother, a 10-year-old boy became deaf and developed diabetes at the age of 5 and 8 years, respectively.Their mother, a 60-year-old female was diagnosed as having NIDDM at the age of 57 years. The clinical phenotypes of our subjects carrying the T14709C mtDNA mutation are different. Identification of all mtDNA alterations or of interactions of mtDNA with nuclear genes would be required for the correct evaluation of the pathogenic mechanism in maternally inherited diabetes mellitus and deafness.

Prevalence of GJB2 and mtDNA mutations in childhood deafness in the Greek population.

A. Pampanos¹, M. Grigoriadou¹, T. Iliades², N. Voyiatzis², J. Economides³, P. Leotsakos³, P. Neou⁴, M. Tsakanikos⁴, T. Antoniadi⁵, I. Konstantopoulou⁶, D. Yannoukakos⁶, K. Gronskov⁷, K. Brondum-Nielsen⁷, E. Papadopoulou⁸, J. Gyftodimou¹, A. Skevas⁹, M. B. Petersen¹;
¹Institute of Child Health, Athens, GREECE, ²Aristotle University of Thessaloniki, Thessaloniki, GREECE, ³"Aghia Sophia" Children's Hospital, Athens, GREECE, ⁴"P. & A. Kyriakou" Children's Hospital, Athens, GREECE, ⁵"MITERA" Maternity and Surgical Center, Athens, GREECE, ⁶National Center for Scientific Research "Demokritos", Athens, GREECE, ⁷The John F. Kennedy Institute, Glostrup, DENMARK, ⁸University General Hospital of Crete, Heraklion, GREECE, ⁹University of Ioannina, Ioannina, GREECE.

Mutations in the GJB2 gene have been shown as a major contributor to prelingual, sensorineural, non-syndromic deafness. Mitochondrial DNA (mtDNA) mutations have been reported in several unrelated families, but a more precise estimate of the prevalence of these mutations as causes of non-syndromic, childhood hearing impairment has not been well established. In the present study, patients were examined by an extensive questionnaire to exclude syndromic forms and environmental causes of deafness. The 35delG mutation was found in 42.2% of the chromosomes in 45 familial cases of prelingual, non-syndromic deafness (18 homozygotes and 2 heterozygotes) and in 30.6% of the chromosomes in 165 sporadic cases (45 homozygotes and 11 heterozygotes). Patients heterozygous for the 35delG mutation were analyzed by direct genomic sequencing of the coding region of the GJB2 gene, which revealed the W24X (2 alleles), L90P (2 alleles), 291insA (1 allele), and R184P (1 allele) mutations. The patients were also screened for the known mtDNA mutations A1555G, A7445G, 7472insC, and A3243G. The homoplasmic A1555G mutation was detected in two cases, one sporadic case and one family with hearing disabled members in both paternal and maternal lineages. The A7445G mutation was detected in one sporadic case of mild hearing loss. We conclude that GJB2 mutations are responsible for a large proportion of prelingual, non-syndromic deafness in the Greek population, and that routine screening for mtDNA mutations in congenital/childhood onset deafness is warranted.

A. Roux¹, N. Pallares-Ruiz¹, M. Mondain², P. Blanchet³, M. Claustres¹;
¹Laboratoire de Genetique Moleculaire, CHU, Montpellier, FRANCE, ²ORL Pediatrique, CHU, Montpellier, FRANCE, ³Service de Genetique Medicale, CHU, Montpellier, FRANCE.

Congenital profound deafness has a known genetic origin in more than 50% of the cases. If the majority of the non syndromic hearing impairment (NSHI) show an autosomal recessive inheritance associated to more than 25 loci, abnormal pattern in the GJB2 gene (connexin 26) is most commonly observed. Other connexin genes have been more rarely involved and attention was given to GJB6 gene (connexin 30). We have shown that a homozygous deletion of a minimal 250 kb region encompassing this gene causes NSHI. More strikingly, real time quantitative PCR has evidenced that the association of this deletion in trans of the GJB2 gene 35delG or E47X mutations is also associated with NSHI. Finding this deletion in several unrelated families prompt us to suggest that this mutation is not rare. To assess the origin of this deletion, haplotype analysis was performed in the DFNB1 region (encompassing the GJB2 and GJB6 genes): the preliminary data do not favor the hypothesis of an ancestral mutation. The cloning of the deletion borders is under progress. Sequence analysis of these regions should help to elucidate whether this deletion arise from a hotspot mutational event.

High frequency of 35delG mutation in Connexin 26 gene in Czech population and among Czech recipients of cochlear implantate.

P. Seeman¹, P. Lesny², D. Groh², V. Boleslavská³, E. Seemanová, Prof², D. Rasková³, Z. Kabelka, Doc²;
¹Charles University Prague, Praha 5, CZECH REPUBLIC, ²Charles University Prague, Praha, CZECH REPUBLIC, ³University Hospital Motol, Praha, CZECH REPUBLIC.

Mutations in Connexin 26 gene (Cx26) are the most common known genetic cause of sensorineural prelingual deafness. The mutation 35delG was reported to be by far the most common in Cx26. Most European patients with prelingual nonsyndromic deafness are homozygotes for 35delG.
No data about the heterozygotes frequency of this common mutation in Czech population were available befor our study.
In order to estimate the carrier frequency of 35delG in Cx26 gen in Czech population with normal hearing, we screened 503 randomly choosen DNA samples from healthy relatives of our neurologic patients and from patients with different neuromuscular disorders without hearing loss. Screening was done by allele specific PCR and positive samples were confirmed by fluorescent fragment analysis.
17 individuals out of 503 were heterozygous for 35delG, which represents a frequency of 1:29,6.
Further we tested 29 Czech patients - recipients of cochlear implantate, with nonsyndromic congenital - prelingual deafness with normaly hearing parents. 14 of them were homozygous for 35delG and 2 were heterozygotes, which represents that 51,7 % of investigated alleles were 35delG. Direct sequencing of all these 29 patients is in progress. First prenatal diagnosis for Cx26 mutation in Czech Republic was performed in a family with a congenitaly deaf child.
Our data are consistent with most European countries. To our knowledge these are first data about frequencies of Cx26 35delG mutation in patients with congenital deafness which received cochlear implantation. Supported by IGA and by VZ 11100003.

P. Primignani¹, F. Sironi¹, C. Curcio¹, P. Castorina², U. Ambrosetti³, D. A. Coviello¹;
¹Laboratorio di Genetica Medica, Dipartimento di Medicina di Laboratorio, Istituti Clinici di Perfezionamento, Milano, ITALY, ²Settore Specialistico di Genetica Medica, Istituti Clinici di Perfezionamento, Milano, ITALY, ³Dipartimento di Scienze Otorinolaringologiche, IRCSS Ospedale Policlinico di Milano, Milano, ITALY.

Hereditary deafness occurs in about 1:2000 newborns and approximately 70% of the cases are non syndromic and the major proportion is due to autosomal recessive inheritance (80%) (Non Syndromic Recessive Deafness - NSRD). GJB2 is the gene more often involved and encodes the gap-junctions protein connexin 26 (Cx26). More than 60 different Cx26 mutations are described but one is particularly common, the 35delG, a deletion of a guanine within a stretch of six Gs residues that starts at position 30 of the coding region. The 35delG mutation accounts for about 60% of the mutated GJB2 alleles.
We analysed by direct sequencing the GJB2 the gene in 138 NSRD patients and identified mutations in 88/276 chromosomes analysed; 26.1% (72/276) showed 35delG mutation, while the remaining showed 12 different mutations (31del14, G12V, W77R, E47X, V95M, 310del14, 35insG, M34T, 167delT, L90P, D179N and H100L). We also found two allele variants: V27I and V153I (http://www.iro.es/cx26deaf.html). The 35delG was present in about 68% of all Cx26 mutations identified. Two out of twelve were novel mutations but while H100L was identified in a family with a recessive form of hearing loss, the D179N mutation was present in a subject where the subsequent family history showed a dominant hearing loss segregation.
In conclusion our findings confirm that the 35delG mutation of the GJB2 gene is the most common cause of NSRD in our population, but many other mutations are also present indicating that the complete sequence is needed for an appropriate molecular diagnosis and genetic counselling.

Mutations of TMPRSS3, the transmembrane serine protease causing deafness DFNB8/10 fail to activate the amiloride-sensitive epithelial sodium channel (ENaC) in vitro

M. Guipponi¹^,2, G. Vuagniaux³, M. Wattenhofer², K. Shibuya⁴, M. Vasquez⁵, L. Dougherty¹, N. Scamuffa², E. Guida⁶, M. Okui⁴, J. Kudoh⁴, C. Rossier², M. Hancock¹, E. Hummler³, P. Marzella⁶, P. Marzella⁶, N. Shimizu⁴, N. Shimizu⁴, H. S. Scott¹, S. E. Antonarakis², B. C. Rossier³;
¹WEHI, Melbourne, AUSTRALIA, ²Division of Medical Genetics, CMU, Geneva, SWITZERLAND, ³Institut de Pharmocologie et de toxicologie, Lausanne, SWITZERLAND, ⁴Department of Molecular Biology, Tokyo, JAPAN, ⁵Department of Morphology, CMU, Geneva, SWITZERLAND, ⁶Department of Otolaryngology, Melbourne, AUSTRALIA.

The TMPRSS3 gene which is mutated in autosomal recessive deafness DFNB8/10 encodes a transmembrane serine protease also containing LDLRA and SRCR domains. To investigate the expression pattern of TMPRSS3 we isolated the mouse ortholog. The mouse Tmprss3 gene maps to MMU17 and encodes a protein with 88% identity to the human ortholog. RT-PCR on rat and RNA in situ hybridisation on mouse cochlea, respectively, revealed that it is principally expressed in the spiral ganglion and epithelial cells supporting the organ of Corti and semi-quantitative RT-PCR on mouse tissues showed strong expression in the stomach. Transient expression of a TMPRSS3-ECFP protein in HeLa cells showed a primarily localization within the endoplasmic reticulum. The amiloride-sensitive sodium channel (ENaC) is regulated by channel activating serine proteases (CAPs). ENaC is expressed in many sodium reabsorbing tissues including the inner ear where it colocalizes with TMPRSS3 and is likely to be involved in maintenance of low sodium concentrations in the endolymph. Functional studies in a Xenopus oocytes expression system demonstrated that TMPRSS3 increased ENaC currents and that a significant fraction of TMPRSS3 is catalytically processed. In contrast, six TMPRSS3 missense mutations causing deafness that fall in all identified domains except the TM and a S401A mutant in the catalytic triad of TMPRSS3 all failed to activate ENaC and to undergo proteolytic cleavage. TMPRSS3-related deafness and these data indicate that important signalling pathways in the inner ear are controlled by proteolytic cleavage and that this may lead to abnormal sodium homeostasis of the inner ear.
guipponi@wehi.edu.au

N. López-Bigas¹^,2, L. Simonneau³, M. Arbonés², X. Estivill²;
¹CGMM-IRO, L'hospitalet (Barcelona), SPAIN, ²Genetic Regulation Center, Barcelona, SPAIN, ³3- Laboratoire de Neurobiologie de l'Audition-Plasticité synaptique, INSERM U254, Montpellier, FRANCE.

Several connexin genes (GJB2, GJB3, GJA1, GJB1 and GJB6) have been found mutated in patients with non-syndromic and/or syndromic deafness. GJB1, the gene encoding connexin 32 (Cx32) cause X-linked Charcot-Marie-Tooth syndrome, which is characterized by peripheral neuropathy, and in some cases progressive hearing loss. In order to understand better the physiology and the function of the connexins in the inner ear we have analyzed the expression of Connexin 32 (Cx32) by RNA in situ hybridization and by immunohistochemistry during the mouse cochlea organogenesis from early otocyst (E12) up to the mature organ in adult.
As previously described in the literature for other connexins, such as connexin 26, (GJB2), connexin 30 (GJB6), connexin 43 (GJA1) and connexin 31 (GJB3), various subtypes of fibrocytes, either in the spiral limbus or along the spiral ligament, differentially express Cx32. Also, we describe different levels of Cx32 expression within the sensory cells and within the various supporting cells that compose the organ of Corti. A huge expression is also present in the basilar membrane cells and in the Reissner membrane cells of ectodermal origin, as in subsets of the cellular elements of the cochlear ganglion. These data are discussed in relation to the cochlear development

Mutation screening of the CYP-1B1 gene in patients with primary congenital glaucoma, and among healthy Gypsies.

L. Kalmar¹, A. Bors¹, H. Andrikovics¹, V. Homolya¹, K. Senyi², G. Hollo², A. Tordai¹;
¹Institute of Haematology and Immunology, National Medical Center, Budapest, HUNGARY, ²1st Institute of Ophtalmology, Semmelweis University, Budapest, HUNGARY.

Primary congenital glaucoma (PCG) is an autosomal recessive disorder associated with unknown developmental defect(s) in the anterior chamber, which leads to a severe eye disease. In the majority of PCG-cases, the disease causing mutation is located in the CYP-1B1 gene (GLCA3, cytochrom-P450-1B1). The type of the mutation shows large heterogenity in the Caucasian population. Recently a single ancestral mutation was found in an isolated Slovak Gypsy population of PCG-patients. All of the patients were homozigous for the Glu387Lys (G1505A) mutation in the CYP-1B1 gene, the carrier frequency was 10% among healthy Gypsies. The aim of our study was to identify the disease causing mutations in Hungarian PCG-patients. To predict the significance of the Glu387Lys mutation in Hungary, a population study was performed in two healthy Gypsy groups (Northern Hungary [NH]: n=207, Southern Hungary [SH]: n=150) by PCR-RFLP. We found significant differences between carrier frequencies of the two healthy Gypsy groups (NH:2,9%, SH:0%). We analysed 24 PCG-samples (11 Gypsies, 13 non-Gypsies) for the presence of the Glu387Lys mutation and, found 10/11 homozygous Gypsy patients, while this mutation was not present among non-Gypsy patients. In the absence of the Glu387Lys mutation, direct sequencing of the coding regions are performed to identify the disease causing mutation. In conclusion, we found the Glu387Lys mutation in Hungary with significant geografical differences, which contradicts the Slovakian endemic theory, but confirms the Northern (Slovakian) Gypsy origin of the mutation. Among the Hungarian Gypsy PCG-patients, the most common disease causing gene defect is the Glu387Lys mutation.

M. Trigueros¹, A. Giménez², D. Valverde¹;
¹Universidad de Vigo, departamento de genética, Vigo, SPAIN, ²Fundación Jiménez Díaz, Madrid, SPAIN.

Nail patella syndrome is inherited in an autosomal dominant manner. The syndrome is characterized by dysplasia of the nails, hypoplasia of the patella, elbow dysplasia, progressive kidney disease and open angle glaucoma.
Families have been studied whit OAG and NPS, but the results suggest that the NPS and OAG phenotypes in the families studied result from mutations in a single gene, lmx1b.
The gene lmx1b encoding the Lim homeodomain protein lmx1b plays a central role in dorso-ventral patterning of the vertebrate limb. The observation of a phenotype similar to NPS in mice prompted to consider lmx1b as a candidate gene for NPS.By the moment is not known the exactly mechanism of this gene.
We have studied samples of 92 patients by PCR and SSCP and identified three mutations in the coding region of the gene. />The mutations found are the following,in the exon 3 an insertion of a T in position 434 that leads to a stop codon. In exon 8, an heterozygous G--C transversion in position 1051 and in other sample, one insertion of G in position 997 leading to a stop codon.

Implications of the exons 1 and 2 of the MYOC gene in patients affected by open angle glaucoma

M. Saura¹, S. Bernal², D. Valverde¹;
¹Universidad de Vigo, Departamento de Genética, Vigo, SPAIN, ²Hospital San Paul, Barcelona, SPAIN.

PURPOSE: The myocilin/trabecular meshwork-inducible glucocorticoid response (MYOC/TIGR) gene was identified as a gene that cused open-angle glaucoma. Glaucoma is a group of disorders characterized by progressive excavation of the optic nerve head with associated loss of the visual field. Glaucoma is a higly prevalent disorder and is estimated to be the third most common cause of blindness worldwide. The mode of inheritance is autosomal dominant with reduced penetrance. The MYOC gene has been mapped at 1q23-q25. The MYOC gene consists of three exons; most known mutations map to the exon 3.
OBJECTIVES: Mutation identification in the exons 1 and 2. Implication of mutation in this patology.
METHODS: DNA from 92 patients with primary open-angle glaucoma was screened for PCR-SSCP analysis. Nucleotide sequence was determinated by automated sequencing.
RESULTS: Five mutations were identified in the exon 1, some of them have been mapped in the promoter region. In the exon 2 no mutations were present.