Molecular epidemiology of DFNB1 deafness in France

Anne-Franoise Roux 2 Nathalie Pallares-Ruiz 2 Anne Vielle 2 Valrie Faugre 2 Carine Templin 2 Dorothe Leprevost 2 Franoise Artires 1 Genevive Lina 0 Nicolas Molinari 5 Patricia Blanchet 4 Michel Mondain 3 Mireille Claustres 2 0 Service d'ORL, Hopital Edouard Herriot , Lyon , France 1 Service D'Audiophonologie, Institut St-Pierre , Palavas-Les-Flots , France 2 Laboratoire de Genetique Moleculaire , CHU Montpellier, IURC, Montpellier , France 3 Service d' ORL , CHU Montpellier , France 4 Departement de Genetique Medicale , CHU Montpellier , France 5 Laboratoire de Biostatistique, Epidemiolgie et Recherche Clinique , IURC, Montpellier Background: Mutations in the GJB2 gene have been established as a major cause of inherited non syndromic deafness in different populations. A high number of sequence variations have been described in the GJB2 gene and the associated pathogenic effects are not always clearly established. The prevalence of a number of mutations is known to be population specific, and therefore population specific testing should be a prerequisite step when molecular diagnosis is offered. Moreover, population studies are needed to determine the contribution of GJB2 variants to deafness. We present our findings from the molecular diagnostic screening of the GJB2 and GJB6 genes over a three year period, together with a population-based study of GJB2 variants. Methods and results: Molecular studies were performed using denaturing High Performance Liquid Chromatograghy (DHPLC) and sequencing of the GJB2 gene. Over the last 3 years we have studied 159 families presenting sensorineural hearing loss, including 84 with non syndromic, stable, bilateral deafness. Thirty families were genotyped with causative mutations. In parallel, we have performed a molecular epidemiology study on more than 3000 dried blood spots and established the frequency of the GJB2 variants in our population. Finally, we have compared the prevalence of the variants in the hearing impaired population with the general population. Conclusion: Although a high heterogeneity of sequence variation was observed in patients and controls, the 35delG mutation remains the most common pathogenic mutation in our population. Genetic counseling is dependent on the knowledge of the pathogenicity of the mutations and remains difficult in a number of cases. By comparing the sequence variations observed in hearing impaired patients with those sequence variants observed in general population, from the same ethnic background, we show that the M34T, V37I and R127H variants can not be responsible for profound or severe deafness. - Background The genetic origin of deafness is suspected in more than half of the congenital hearing loss cases. More than 400 syndromes can include hearing loss or deficient hearing functions as a component. However, non syndromic expression of deafness is observed in more than 70 % of cases. In the non syndromic forms of hearing loss (NSHL), familial or sporadic cases are observed and the transmission is predominantly autosomal recessive. Genetic heterogeneity has been established by linkage studies: more that 50 loci associated with NSHL (including dominant and recessive autosomal, and X-linked types of transmission) have been localized, making possible the identification of a number of causative deafness genes http://dnalab-www.uia.ac.be/dnalab/hhh/. Despite the extreme genetic heterogeneity, the recessive DFNB1 locus, mapping to chromosome 13q12, is by far the most prevalent. This locus contains the two Gap junction genes GJB2 and GJB6, encoding, respectively connexin 26 (CX26) and connexin 30 (CX30). These proteins associate in hexamers to form homo- and hetero-connexons [1,2]. Two connexons from adjacent cells dock to form a functional channel that will allow, among other small molecules, the diffusion of potassium ions critical for the normal sensory hair cell excitation [3]. The contribution of the GJB2 gene in NSHL varies from 0 to 40 % in diverse populations [4] and this genetic heterogeneity is also emphasized by the variation in frequency of specific mutations among different populations. More than 70 mutations in the GJB2 gene have been reported [5], and although the majority are rare or private, the prevalence of four mutations define specific ethnic origins. The 35delG mutation accounts for approximately 70 % of GJB2 mutant alleles in Northern and Southern European, as well as American Caucasian populations, with a carrier frequency of 2.3 % to 4 % [6-9]. The three other mutations, 167delT, 235delC or R143W represent the most common pathogenic alleles in Ashkenazi Jews [10], Asian [11-14] and Ghanian populations [15,16], respectively. Recently, we and other groups have identified a large 309 kb deletion that includes the 5' region of the GJB6 gene and most of its coding region [17]. It is unclear whether this deletion removes regulatory elements common to GJB6 and GJB2 resulting, in addition to the deletion of GJB6, in reduced expression of the wild type GJB2 gene [17-20]. This deletion also appears to have an ethnic specific origin as it is absent from the Siberian (manuscript in preparation), Chinese [21], Austrian and Italian populations [17,22]. In this report, when we refer to GJB6 mutations we will consider only this particular mutation (GJB6-D13S1830). Molecular diagnostic testing of non syndromic deafness was initiated in Montpellier in early 2000. This testing was carried out in parallel with a molecular epidemiology study of GJB2 variants in the Languedoc Roussillon region. Although many reports have estimated the carrier frequency in French and Mediterranean populations (mostly of the 35delG mutation), differences of frequency between samples are observed (0.0 to 2.7 % in France). This is essentially due to the size and composition of control samples [23]; for review see [4]. Assessment of GJB2 variant sequence distribution in Languedoc Roussillon was necessary, as we had observed significant differences in the distribution of CFTR mutations between several French regions [24]. In this study, we present our results from three years of molecular diagnostic testing of GJB2/GJB6 including the clinical and associated audiologic findings and also determine the prevalence and spectrum of DFNB1 mutations in the southern France population. In addition, we report the first screening of the most frequent GJB2 variants, on several thousand dried bloodspots (Guthrie cards) from newborns and thus re-evaluate the pathogenic status of some GJB2 variants. Methods Patients A total of 159 unrelated families, comprised of 184 patients with sensorineural hearing loss, were referred from the Genetic Counseling Department and/or the Ear, Nose and Throat specialized clinics (Centre Hospitalier Universitaire of Montpellier and Lyon). All patients had permanent hearing loss not caused by infections, exposure to drugs or other prenatal or perinatal etiology of deafness. Informed c (...truncated)