Screening of families with autosomal recessive non-syndromic hearing impairment (ARNSHI) for mutations in GJB2 gene: Indian scenario

Several studies have reported that mutations in the GJB2 gene (coding for connexin26) are a common cause of recessive non-syndromic hearing impairment. A GJB2 mutant allele, 35delG, has been found to have a high prevalence in most ethnic groups. Though mutations in the GJB2 gene have been shown to cause autosomal recessive deafness in Indian families, the frequencies of the various mutations are still unknown. In the present study, we analyzed 45 Indian families belonging to three different states, namely, Karnataka, Tamil Nadu, and Delhi with non-syndromic hearing impairment and an apparently autosomal recessive mode of inheritance. All the families were initially screened for three mutations (W24X, W77X, and Q124X) by using allele-specific PCR primers; mutations were confirmed by DNA sequencing. Families that were heterozygous or negative for tested mutations of the GJB2 gene were sequenced directly to identify the complementary mutation and other mutations in GJB2. Four families were homozygous for W24X, constituting around 8.8%. In two families, the affected individuals were compound heterozygotes for W24X; one family (DKB16) carried 35delG with W24X while the other family (DKB7) carried R143W with W24X. We suggest that W24X is a common allele among the mutations screened, causing autosomal recessive non-syndromic hearing impairment (ARNSHI) in the Indian population.     

Frequencies of gap- and tight-junction mutations in Turkish families with autosomal-recessive non-syndromic hearing loss

Mutations in genes encoding gap- and tight-junction proteins have been shown to cause distinct forms of hearing loss. We have now determined the GJB2[connexin 26 (Cx26)] mutation spectrum in 60 index patients from mostly large Turkish families with autosomal-recessive inherited non-syndromic sensorineural hearing loss (NSSHL). GJB2 mutations were found in 31.7% of the families, and the GJB2-35delG mutation accounted for 73.6% of all GJB2 mutations. The carrier frequency of GJB2-35delG in the normal Turkish population was found to be 1.17% (five in 429). In addition to the described W24X, 233delC, 120delE and R127H mutations, we also identified a novel mutation, Q80R, in the GJB2 gene. Interestingly, the Q80R allele was inherited on the same haplotype as V27I and E114G polymorphisms. As little is known about the mutation frequencies of most other recently identified gap- and tight-junction genes as a cause for hearing loss, we further screened our patients for mutations in GJB3 (Cx31), GJA1 (Cx43), DeltaGJB6-D13S1830 (Cx30) and the gene encoding the tight-junction protein, claudin 14 (CLDN14). Several novel polymorphisms, but no disease-associated mutations, were identified in the CLND14 and GJA1 genes, and we were unable to detect the DeltaGJB6-D13S1830 deletion. A novel putative mutation, P223T, was found in the GJB3 gene in heterozygous form in a family with two affected children. Our data shows that the frequency of GJB2 mutations in Turkish patients with autosomal-recessive NSSHL and the carrier rate of the GJB2-35delG mutation in the Turkish population, is much lower than described for other Mediterranean countries. Furthermore, mutations in other gap- and tight-junction proteins are not a frequent cause of hearing loss in Turkey.     

Prevalence of Deafness‐Associated Connexin‐26 (GJB2) and Connexin‐30 (GJB6) Pathogenic Alleles in a Large Patient Cohort from Eastern Sicily

Mutations in the gene encoding the gap junction protein connexin 26 (GJB2) and connexin 30 (GJB6) have been shown to be a major contributor to prelingual, sensorineural, nonsyndromic deafness. The aim of this study was to characterize and establish the prevalence of GJB2 and GJB6 gene alterations in 196 patients affected by sensorineural, nonsyndromic hearing loss, from Eastern Sicily. We performed sequence analysis of GJB2 and identified sequence variants in 68 out of 196 patients (34.7%); (28 homozygous for c.35delG, 22 compound heterozygous and 11 with only one variant allele). We found 12 different allelic variants, the most prevalent being c.35delG, which was found on 89 chromosomes (65.5%), followed by other alleles with different frequencies (p.E47X, c.‐23+1G>A, p.L90P, p.R184W, p.M34T, c.167delT, p.R127H, p.M163V, p.V153I, p.W24X, and p.T8M). Importantly, for the first time we present the frequency and spectrum of GJB2 mutations in NSHL patients from Eastern Sicily. No alterations were found in the GJB6 gene, confirming that alterations in this gene are uncommon in our geographic area. Note that 65.3% and 23.5% of our patients, respectively were found to be negative or carriers by GJB2 molecular screening. This emphasizes the need to broaden the genetic analysis to other genes involved in hearing loss.     

Prevalent connexin 26 gene (GJB2) mutations in Japanese

The gene responsible for DNFB1 and DFNA3, connexin 26 (GJB2), was recently identified and more than 20 disease causing mutations have been reported so far. This paper presents mutation analysis for GJB2 in Japanese non-syndromic hearing loss patients compatible with recessive inheritance. It was confirmed that GJB2 mutations are an important cause of hearing loss in this population, with three mutations, 235delC, Y136X, and R143W, especially frequent. Of these three mutations, 235delC was most prevalent at 73%. Surprisingly, the 35delG mutation, which is the most common GJB2 mutation in white subjects, was not found in the present study. Our data indicated that specific combinations of GJB2 mutation exist in different populations.     

GJB2 mutations in Iranians with autosomal recessive non-syndromic sensorineural hearing loss

Hereditary hearing loss (HHL) is an extremely common disorder. About 70% of HHL is non-syndromic, with autosomal recessive forms accounting for approximately 85% of the genetic load. Although very heterogeneous, the most common cause of HHL in many different world populations is mutations of GJB2, a gene that encodes the gap junction protein connexin 26 (Cx26). This study investigates the contribution of GJB2 to the autosomal recessive non-syndromic deafness (ARNSD) load in the Iranian population. One hundred sixty eight persons from 83 families were studied. GJB2-related deafness was diagnosed in 9 families (4, 35delG homozygotes; 3, 35delG compound heterozygotes; 1, W24X homozygote; 1, non-35delG compound heterozygote). The carrier frequency of the 35delG allele in this population was approximately 1% (1/83). Because the relative frequency of Cx26 mutations is much less than in the other populations, it is possible that mutations in other genes play a major role in ARNSD in Iran.     

Frequencies of GJB2 mutations in German control individuals and patients showing sporadic non-syndromic hearing impairment

Mutations in the GJB2 gene encoding the gap-junction protein connexin 26 have been identified in many patients with childhood hearing impairment (HI). One single mutation, 35delG (30delG), accounts for up to 70% of all analyzed European patients with autosomal recessive inherited HI and 10% of patients with HI of unknown origin, respectively. We screened 188 control individuals and 342 German patients with non-syndromic sporadic HI for the 35delG, compound heterozygosity and other GJB2 mutations by PCR, restriction enzyme based screening, SSCP and sequencing. In all patients, non-progressive hearing impairment varied from moderate to profound involving all frequencies. This study revealed one novel silent mutation (438C/T), three novel gene variants resulting in amino acid substitutions (K112E, T123S, K223R) and two novel HI-related mutations (I82M, 313del14).     

Strong linkage disequilibrium for the frequent GJB2 35delG mutation in the Greek population

Approximately one in 1,000 children is affected by severe or profound hearing loss at birth or during early childhood (prelingual deafness). Up to 40% of congenital, autosomal recessive, severe to profound hearing impairment cases result from mutations in a single gene, GJB2, that encodes the connexin 26 protein. One specific mutation in this gene, 35delG, accounts for the majority of GJB2 mutations detected in Caucasian populations. Some previous studies have assumed that the high frequency of the 35delG mutation reflects the presence of a mutational hot spot, while other studies support the theory of a common founder. Greece is among the countries with the highest carrier frequency of the 35delG mutation (3.5%), and a recent study raised the hypothesis of the origin of this mutation in ancient Greece. We genotyped 60 Greek deafness patients homozygous for the 35delG mutation for six single nucleotide polymorphisms (SNPs) and two microsatellite markers inside or flanking the GJB2 gene. The allele distribution in the patients was compared to 60 Greek normal hearing controls. A strong linkage disequilibrium was found between the 35delG mutation and markers inside or flanking the GJB2 gene. Furthermore, we found a common haplotype with a previous study, suggesting a common founder for the 35delG mutation.     

Ultrastructure of the Hair in Genetic Prelingual Deafness Associated with the 35delG Mutation in the Connexin 26 Gene (GJB2)

Ultrastructure of the surface of long hair in 77 subjects with a phenotype of childhood prelingual deafness was evaluated by scanning electron microscopy. The subjects were homozygous or heterozygous carriers of the 35delG mutation in the connexin 26 gene (GJB2). The presence of severe abnormalities in the marginal layer of the cuticular plate (fracture-like defects) is pathognomonic for homozygous carriers of the 35delG mutation. Ultrastructural characteristics of the hair in subjects with connexin-associated deafness signifi cantly differed from those in healthy volunteers (control group of the same age) and deaf people with nongenetic hearing impairment. Analysis of variance revealed no differences in hair thickness between deaf homozygous and heterozygous carriers of the 35delG GJB2 gene mutation and healthy volunteers.     

A large deletion including most of GJB6 in recessive non syndromic deafness: a digenic effect?

Congenital profound deafness has a known genetic origin in more than 50% of all cases. The majority of the non syndromic hearing loss (NSHL) show an autosomal recessive inheritance. Mutations in the GJB2 gene (connexin 26) account for more than 50% of the recessive non syndromic deafness (DFNB1) among 30 loci. Other connexin genes have been more rarely involved and attention was given here to the GJB6 gene (connexin 30). We show that homozygous deletion of a minimal 150 kb region encompassing this gene causes NSHL. More strikingly, association of this deletion in trans of the GJB2 gene 35delG or E47X mutations is also associated with NSHL.     

Clinical phenotype and mutations in connexin 26 (DFNB1/GJB2), the most common cause of childhood hearing loss

Mutations in the gene for connexin 26, GJB2, are the most common cause of hearing loss in American and European populations, with a carrier rate of about 3%-a rate similar to that for cystic fibrosis. A single mutation, 35delG, is responsible for most of this autosomal recessive hearing loss, DFNB1. A broad spectrum of mutations in GJB2 has been found to be associated with hearing loss, including another deletion mutation, 167delT, which has a carrier rate of about 4% in the Ashkenazi Jewish population. Mutations in GJB2 have also been found to be associated with dominant nonsyndromic hearing loss, DFNA3. Clinical studies have shown that the recessive hearing loss can vary from mild to profound, even within the same sibship. This type of hearing loss is nonsyndromic and is accompanied by normal vision, vestibular responses, and no malformations of the inner ear detectable by computed tomography scanning. Progressive and asymmetrical hearing loss has been noted in some cases, but it accounts for fewer than one-third of the cases of this type of hearing loss. The discovery of mutations in GJB2 that cause hearing loss has profound implications in the early diagnosis of hearing loss in general. The relative ease of diagnosis by genetic testing of Cx26 permits early identification of children with GJB2/DFNB1 hearing loss. This testing, coupled with hearing loss diagnosed by infant auditory brainstem response audiometry, will ensure that hearing-impaired children and their parents receive proper medical, audiologic, genetic, and educational counseling. Am. J. Med. Genet. (Semin. Med. Genet.) 89:130-136, 1999.     

Exploring the clinical and epidemiological complexity of GJB2-linked deafness

GJB2 mutation analysis was performed in 179 unrelated subjects with sporadic or familial hearing loss (HL). Among 57 families, 18 showed a vertical transmission of HL, the disease being present in two or three generations. Besides 155 nonsyndromic cases, 24 patients presenting with extra-auditory clinical signs were included in the molecular study. GJB2 mutation analysis was also performed in 19 subjects with an anamnestic history of perinatal risks factors for acquired HL. The 35delG mutation accounted for 22.1% of analyzed chromosomes in sporadic cases and 39.4% in familial cases; 35delG prevalence reached 41% in autosomal recessive and 44.4% in pseudodominant pedigrees. Two novel GJB2 mutations were identified in compound heterozygosity with 35delG allele (D159V, 284ins/dup[CACGT]). Two 35delG homozygous subjects were identified among HL cases classified as environmental in origin. Four patients 35delG heterozygous (35delG/V95M, 35delG/L90P, 35delG/167delT, and 35delG/?) and two homozygous presented with extra-auditory clinical signs involving different organs (skin, vascular system, hemopoietic lineages, and thyroid). In a high proportion of 35delG heterozygous HL patients (52%), no second GJB2 mutation was detected. The reported data highlight the complexity of the genetic epidemiology of GJB2-linked deafness, further enlarging the spectrum of situations in which GJB2 mutation analysis should be performed. The presence of extra-auditory signs in a significant portion of GJB2-mutated patients suggests the possibility that GJB2 loss of function could contribute to clinical phenotypes presenting in association with deafness. This hypothesis deserves further investigation. The failure to identify a presumed partnering GJB2 mutation in a high proportion of deaf patients remains a challenging problem to be clarified.     

Functional consequences of novel connexin 26 mutations associated with hereditary hearing loss

In a study of 530 individuals with non-syndromic, sensorineural hearing loss, we identified 18 mutations at connexin 26 (Cx26), four of which are novel (-23G>T, I33T, 377_383dupTCCGCAT, W172R) and the remaining 14 (ivs1+1G>A, M1V, 35delG, W24X, I35S, V37I, R75W, W77X, 312del14, E120del, Q124X, Y136X, R143W, R184P) being mutations previously described. To gain insight into functional consequences of these mutations, cellular localization of the mutant proteins and their ability to permit lucifer yellow transfer between cells was studied in seven of them (W24X, I33T, I35S, R75W, E120del, W172R and R184P). I35S and R184P showed impaired trafficking of the protein to the plasma membrane. I33T, R75W, E120del and W172R showed predominantly membrane localization but did not form functional gap junction channels. Surprisingly, W24X, a protein-truncating mutation, apparently permits formation of a full-length protein, perhaps due to a stop codon read-through mechanism. These results provide further evidence that Cx26 mutations affect gap junction activity by mis-regulation at multiple levels.     

Mutations in GJB2, GJB6, and mitochondrial DNA are rare in African American and Caribbean Hispanic individuals with hearing impairment

Autosomal recessive nonsyndromic sensorineural hearing impairment (ARNSHI) comprises 80% of familial hearing loss cases. Approximately half result from mutations in the connexin 26 (Cx26) gene, GJB2, in Caucasian populations. Heterozygous mutations in GJB2 occasionally co-occur with a deletion of part of GJB6 (connexin 30; Cx30). It is estimated that approximately 1% of deafness is maternally inherited, due to mutations in mitochondrial DNA (mtDNA). Few studies have focused on the frequency of mutations in connexins or mtDNA in African American (AA) and Caribbean Hispanic (CH) admixture populations. In this study, we performed bidirectional sequencing of the GJB2 gene and polymerase chain reaction (PCR) screening for the common GJB6 deletion, as well as PCR/RFLP analysis for three mutations in mtDNA (A1555G, A3243G, A7445G), in 109 predominantly simplex AA and CH individuals. Variations found were a 101T > C (M34T; 1/101 cases), 109G > A (V37I; 1/101), 35delG (mutation; 4/101, (3/4) of non-AA/CH ethnicity), 167delT (mutation; 1/101), 139G > T (mutation; E47X; 1/101 homozygote, consanguineous), -15C > T (1/101), 79G > A (V27I; 9/101), 380G > A (R127H; 4/101; Guyana, India, Pakistan ethnicity), 670A > C (Indeterminate; K224Q; 1/101), 503A > G (novel; K168R; 3/101) and 684C > A (novel; 1/101). All but one of the AA and CH patients had monoallelic variations. There were no hemizygous GJB6 deletions in those with monoallelic GJB2 variations. We also did not identify any patients with the three mutations in mtDNA. Bidirectional sequencing of the GJB2 gene was performed in 187 AA and Hispanic healthy individuals. Our results reveal that GJB2 mutations, GJB6 deletions, and mtDNA mutations may not be significant in these minority admixture populations.     

Connexin26 gene ( GJB2): prevalence of mutations in the Chinese population

The connexin26 gene ( GJB2) has been shown to be responsible for DFNB1 and DFNA3 (Autosomal Recessive Hereditary Nonsyndromic Deafness Locus 1 and Autosomal Dominant Hereditary Nonsyndromic Deafness Locus 3). Two hundred ten independently ascertained Chinese probands with nonsyndromic hearing loss (NSHL) were evaluated for mutations in GJB2, including 43 probands from families with more than one sib with NSHL, likely indicating dominant inheritance, and sporadic cases of NSHL, compatible with recessive inheritance. Of the 210 probands, 43 (20%) were homozygous or heterozygous for mutations in GJB2. Four different mutations were identified: 35delG, 109G-A, 235delC, and 299-300delAT. It was confirmed that GJB2 mutations are an important cause of hearing loss in this population. Of these four mutations, 235delC was the most prevalent at 93%; yet the 35delG mutation, which is the most common GJB2 mutation in Caucasian subjects (Europeans and Americans), was found in low frequency in the present study. It appears from our limited data and reports from other East Asians that 235delC is the most prevalent GJB2 mutation in these populations. GJB2 mutations are consistent with ethnic predilections.     

Transgenic expression of a dominant-negative connexin26 causes degeneration of the organ of Corti and non-syndromic deafness

Hereditary deafness affects about 1 in 2000 children and mutations in the GJB2 gene are the major cause in various ethnic groups. GJB2 encodes connexin26, a putative channel component in cochlear gap junction. However, the pathogenesis of hearing loss caused by the GJB2 mutations remains obscure. The generation of a mouse model to study the function of connexin26 during hearing has been hampered by the fact that Gjb2 knockout mice are embryonic lethal. To establish viable model mice we generated transgenic mice expressing a mutant connexin26 with R75W mutation that was identified in a deaf family with autosomal-dominant inheritance. The previous expression analysis revealed that the R75W connexin26 inhibited the gap channel function of the co-expressed normal connexin26 in a dominant-negative fashion. We established two lines of transgenic mice that showed severe to profound hearing loss, deformity of supporting cells, failure in the formation of the tunnel of Corti and degeneration of sensory hair cells. Despite robust expression of the transgene, no obvious structural change was observed in the stria vascularis or spiral ligament that is rich in connexin26 and generates the endolymph. The high resting potential in cochlear endolymph essential for hair cell excitation was normally sustained. These results suggest that the GJB2 mutation disturbs homeostasis of cortilymph, an extracellular space surrounding the sensory hair cells, due to impaired K(+) transport by supporting cells, resulting in degradation of the organ of Corti, rather than affecting endolymph homeostasis in mice and probably in humans.     

Connexin 26 mutations in cases of sensorineural deafness in eastern Austria

Mutations in the connexin 26 (Cx26) gene (GJB2) are associated with autosomal nonsyndromic sensorineural hearing loss. This study describes mutations in the Cx26 gene in cases of familial and sporadic hearing loss (HL) by gene sequencing and identifies the allelic frequency of the most common mutation leading to HL (35delG) in the population of eastern Austria. For this purpose we have developed and applied a molecular beacon based real-time mutation detection assay. Mutation frequencies in the Cx26 gene of individuals from affected families (14 out of 46) and sporadic cases (11 out of 40) were 30.4% and 27.5%, respectively. In addition to known disease related alterations, a novel mutation 262 G-->T (A88S) was also identified. 35delG accounted for almost 77% of all Cx26 mutations detected and displayed an allelic frequency in the normal hearing population of 1.7% (2 out of 120). The high prevalence of the 35delG mutation in eastern Austria would therefore allow screening of individuals and family members with Cx26 dependent deafness by a highly specific and semi-automated method.     

Molecular genetics of hearing impairment due to mutations in gap junction genes encoding beta connexins

Deafness is a complex disorder that involves a high number of genes and environmental factors. There has been enormous progress in non-syndromic deafness research during the last five years, with the identification of over 50 loci and 15 genes. Among these, three genes, GJB2, GJB3, and GJB6, encode for connexin proteins (Connexin26, Connexin31, and Connexin30, respectively). Another connexin (Connexin32, encoded by GJB1) is involved in X-linked peripheral neuropathy and hearing impairment. Mutations in these genes cause autosomal recessive (GJB2 and GJB3), autosomal dominant (GJB2, GJB3, and GJB6) or X-linked (GJB1) hearing impairment, both syndromic (GJB2, keratoderma; GJB3 erythrokeratodermia variabilis; and GJB1, peripheral neuropathy), and non-syndromic (GJB2, GJB3, and GJB6). Among these genes, mutations in GJB2 account for about 50% of all congenital cases of hearing impairment. Three mutations in GJB2 (35delG, 167delT, and 235delC) are particularly common in specific populations (Caucasoid, Jewish Ashkenazi, and Oriental, respectively), leading to carrier frequencies between one in 30 and one in 75. Over 50 mutations have been identified in the GJB2 gene, of which some missense changes (M34T, W44C, G59A, D66H, and R75W) have a negative dominant action in hearing impairment, with partial to full penetrance. Functional studies for some missense mutations in connexins 26, 30, and 32 have indicated abnormal gap junction conductivity. Expression patterns in mouse and rat cochlea indicate that Connexin26 and Connexin30 are expressed in the supportive cells of the cochlea, suggesting a potential role in endolymph potassium recycling. The high prevalence of mutations in GJB2 in some populations provides the tools for molecular diagnosis, carrier detection, and prenatal diagnosis of congenital hearing impairment.     

GJB2 mutations in patients with non-syndromic hearing loss from Northeastern Hungary

Mutations in the GJB2 gene encoding the gap-junction protein connexin 26 have been identified in many patients with childhood hearing impairment (HI). One single mutation, c.35delG, accounts for the majority of mutations in Caucasian patients with HI. In the present study we screened 500 healthy control individuals and a group of patients with HI from Northeastern Hungary for GJB2 mutations. The patients' group consisted of 102 familial from 28 families and 92 non-familial cases. The most common mutation in the Hungarian population is the c.35delG, followed by the c.71G>A (p.W24X) mutation. 34.3% of the patients in the familial group were homozygous, and 17.6% heterozygous for 35delG. In the non-familial group the respective values were 37% and 18% (allele frequency: 46.2%). In the general population an allele frequency of 2.4% was determined. Several patients were identified with additional, already described or new GJB2 mutations, mostly in heterozygous state. The mutation c.380G>A (p.R127H) was formerly found only in heterozygous state and its disease relation was controversial. We demonstrated the presence of this mutation in a family with three homozygous patients and 4 heterozygous unaffected family members, a clear indication of recessively inherited HI. Furthermore, we provided evidence for the pathogenic role of two new mutations, c.51C>A (p.S17Y) and c.177G>T (p.G59V), detected in the present study. In the latter case the pattern of inheritance might be dominant. Our results confirm the importance of GJB2 mutations in the Hungarian population displaying mutation frequencies that are comparable with those in the Mediterranean area.     

Evidence for single origins of 35delG and delE120 mutations in the GJB2 gene in Anatolia

Eighteen different sequence changes, including three novel alterations, were detected in GJB2, encoding connexin 26, in 371 Turkish probands with non-syndromic sensorineural hearing loss. Two frequently detected mutations, 35delG and delE120, were shown to have single origins based on the conserved genotypes of two closely linked microsatellite and five single nucleotide polymorphism markers. Carrier frequencies of 35delG and delE120 in Egypt and Turkic populations of the Near East provide insights about the origin of these two mutations.     

M34T and V37I mutations in GJB2 associated hearing impairment: evidence for pathogenicity and reduced penetrance

Despite research the role of the M34T and V37I variants of GJB2 in causing hearing impairment (HI) remains controversial. Our purpose was to test a hypothesis that M34T and V37I are pathogenic but have distinct features resulting in a reduced penetrance. We screened for known GJB2/GJB6 mutations 233 Polish consecutive unrelated subjects with non-syndromic, sensorineural HI who were previously found to carry 35delG mutation on one chromosome. The most frequent mutations were also analyzed in approximately 1,000 controls. We found that M34T and V37I were significantly (P < 10(-6)) overrepresented among patients, but their penetrance was estimated as 1/10 relative to mutations of undisputed pathogenicity. This finding apparently could not be explained by low degree of HI associated with M34T and V37I since another mutation causing comparably mild HI (L90P) did not have reduced penetrance. Subsequent analyses showed that the patients with M34T/35delG and V37I/35delG had significantly later onset of HI than patients with other genotypes (P < 10(-6)) including the L90P/35delG (P = 0.006). Also, among these patients (but not others) a strong correlation between the degree of HI and its duration was found (r = 0.79, P < 10(-5)). We tentatively suggest that M34T and V37I might cause mild HI characterized by relatively late onset and progression.