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.     

The novel R75Q mutation in the GJB2 gene causes autosomal dominant hearing loss and palmoplantar keratoderma in a Turkish family

Dominant mutations in the GJB2 gene encoding connexin 26 (Cx26) can cause non-syndromic hearing impairment alone or in association with palmoplantar keratoderma (PPK). We have identified the novel G224A (R75Q) mutation in the GJB2 gene in a four-generation family from Turkey with autosomal dominant inherited hearing impairment and PPK. The age of onset and progression of hearing loss were found to be variable among affected family members, but all of them had more severe impairment at higher hearing frequencies. Interestingly, the novel R75Q mutation affects the same amino acid residue as described recently in a small family (R75W) with profound prelingual hearing loss and PPK. However, the R75W mutation was also observed in a control individual without PPK and unknown hearing status. Therefore, the nature of the R75W mutation remains ambiguous. Our molecular findings provide further evidence for the importance of the conserved R75 in Cx26 for the physiological function of the inner ear and the epidermal cells of the skin.     

Hereditary palmoplantar keratoderma and deafness resulting from genetic mutation of Connexin 26

Gap junctions, which mediate rapid intercellular communication, consist of connexins, small transmembrane proteins that belong to a large family of proteins found throughout the species. Mutations in the GJB2 gene, encoding Connexin 26, can cause nonsyndromic autosomal recessive or dominant hearing loss with or without skin manifestations. A 3-yr-old Korean female and her mother presented to our clinic with diffuse hyperkeratosis of the palms and soles (May 3, 2007). Skin biopsies from the soles of both patients demonstrated histopathological evidence of palmoplantar keratoderma. The patient and a number of her maternal family members also had congenital hearing loss. The combination of congenital hearing loss and palmoplantar keratoderma, inherited as an autosomal dominant trait, led us to test for a mutation in the GJB2 gene in both patients. The results showed the R75W mutation of the GJB2 gene in both. In conclusion, the simultaneous occurrence of a GJB2 mutation in a mother and daughter suggests that R75W mutation cause autosomal dominant hearing loss presenting with palmoplantar keratoderma. To the best of our knowledge, this is the first report of a GJB2 mutation associated with syndromic autosomal dominant hearing loss and palmoplantar keratoderma in a Korean family.     

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.     

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 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.     

Cochlear expression of a dominant-negative GJB2R75W construct delivered through the round window membrane in mice

Development of a gene-delivery method to the inner ear is an essential step for studies of hearing function and gene therapy. Application of liposomes or adenoviral vectors onto the intact round window membrane (RWM) offers the possibility of atraumatic exogenous gene transfer. GJB2 encodes the gap junction protein Connexin26, which plays a crucial role in potassium recycling in the inner ear. The R75W allele of GJB is a well-characterized mutation that causes deafness at the DFNA3 through a dominant-negative mechanism of action. In this study, a plasmid vector, pGJB2(R75W)-eGFP, was lipocomplexed with N-[1-(2,3-Dioleoloxy)propyl]N,N,N-trimethylammonium methylsulfate: cholesterol and applied onto mouse RWM. At 3 days (3d) post-treatment, immunohistochemistry demonstrated GJB2(R75W)-eGFP transgene expression in the cochlea in: inner and outer pillar cells, outer hair cells, Claudius cells and, in the spiral limbus and ligament. Significant hearing loss was detected by auditory brainstem response testing after 1, 2 and 3d post-treatment; hearing levels returned to control levels at 5d post-treatment. These data confirm that GJB2(R75W) induces functional impairment in the mature cochlea through a dominant negative effect, and importantly, that RWM application of exogenous genes is a feasible method to test their impact on hearing.     

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.     

Genetic testing for hereditary hearing loss: connexin 26 (GJB2) allele variants and two novel deafness-causing mutations (R32C and 645-648delTAGA)

Mutations in GJB2 are the most common cause of hereditary congenital hearing loss in many countries and are found in about half of persons with severe-to-profound congenital autosomal recessive non-syndromic hearing loss (ARNSHL). We report the results of GJB2 mutation screening in 209 consecutive persons with congenital deafness of indeterminate etiology using an allele-specific polymerase chain reaction assay, single-strand conformational polymorphism analysis, and direct sequencing. GJB2 allele variants were detected in 74 of 209 deaf individuals (35%). Over one-fourth of screened individuals were either homozygous (n=31) or heterozygous (n=24) for the 35delG mutation. Of those with the 35delG mutation, 51 (92.7%) were diagnosed with GJB2-related deafness. Nineteen persons were identified with other GJB2 allele variants - two novel deafness-causing mutations (R32C, 645-648delTAGA), one mutation of unknown significance (E47K), and one benign polymorphism (I128I). While these data enable health care professionals to provide parents and patients with improved genetic counseling data, difficulty still exists is determining whether some missense mutations compromise auditory function and are deafness-causing.     

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.     

Mutations in the KCNQ4 gene are responsible for autosomal dominant deafness in four DFNA2 families.

We have previously found linkage to chromosome 1p34 in five large families with autosomal dominant non-syndromic hearing impairment (DFNA2). In all five families, the connexin31 gene ( GJB3 ), located at 1p34 and responsible for non-syndromic autosomal dominant hearing loss in two small Chinese families, has been excluded as the responsible gene. Recently, a fourth member of the KCNQ branch of the K+channel family, KCNQ4, has been cloned. KCNQ4 was mapped to chromosome 1p34 and a single mutation was found in three patients from a small French family with non-syndromic autosomal dominant hearing loss. In this study, we have analysed the KCNQ4 gene for mutations in our five DFNA2 families. Missense mutations altering conserved amino acids were found in three families and an inactivating deletion was present in a fourth family. No KCNQ4 mutation could be found in a single DFNA2 family of Indonesian origin. These results indicate that at least two and possibly three genes responsible for hearing impairment are located close together on chromosome 1p34 and suggest that KCNQ4 mutations may be a relatively frequent cause of autosomal dominant hearing loss.     

Connexin 26 gene (GJB2) mutation modulates the severity of hearing loss associated with the 1555A→G mitochondrial mutation

We report a high prevalence of GJB2 heterozygous mutations in patients bearing the 1555A→G mitochondrial mutation, and describe a family in which potential interaction between GJB2 and a mitochondrial gene appears to be the cause of hearing impairment. Patients who are heterozygotes for the GJB2 mutant allele show hearing loss more severe than that seen in sibs lacking a mutant GJB2 allele, suggesting that heterozygous GJB2 mutations may synergistically cause hearing loss when in the presence of a 1555A→G mutation. The present findings indicate that GJB2 mutations may sometimes be an aggravating factor, in addition to aminoglycoside antibiotics, in the phenotypic expression of the non‐syndromic hearing loss associated with the 1555A→G mitochondrial mutation. © 2001 Wiley‐Liss, Inc.     

一个同时携带线粒体DNA A1555G突变和GJB2235delC单杂合突变家系的基因型与听力表型

目的 调查一个同时携带线粒体DNA A1555G突变和GJB2 235delC突变的非综合征型耳聋家系,分析其基因型和听力表型的关系.方法 对家系成员进行临床听力测试,收集家系中8名成员的外周静脉血样本,从白细胞中提取DNA,聚合酶链反应扩增GJB2基因和线粒体DNA(mitochondric DNA,mtDNA)目的 片段,对扩增片段直接测序进行GJB2基因、mtDNA 12S rRNA及tRNASer(UCN)基因突变分析.结果 此家系先证者存在mtDNA A1555G突变和GJB2 235delC杂合突变,听力表型为极重度感音神经性耳聋.其他母系成员携带mtDNA A1555G突变,未发现tRNASer(UCN)基因突变,家系中其他母系成员听力表型为双侧对称高频下降或听力正常.结论 GJB2 235delC单杂合突变可能参与了mtDNA A1555G的听力损害.     

Autosomal Dominant Hearing Loss resulting from p.R75Q Mutation in the GJB2 Gene: Nonsyndromic presentation in a South Indian Family

Mutations in the GJB2 gene encoding the gap junction protein Connexin 26 have been associated with autosomal recessive as well as dominant nonsyndromic hearing loss. Owing to the involvement of connexins in skin homeostasis, GJB2 mutations have also been associated with syndromic forms of hearing loss showing various skin manifestations. We report an assortatively mating hearing impaired family of south Indian origin with three affected members spread over two generations, having p.R75Q mutation in the GJB2 gene in the heterozygous condition. The inheritance pattern was autosomal dominant with mother and son being affected. Dermatological and histopathologic examinations showed absence of palmoplantar keratoderma. To the best of our knowledge, this is the first report from India on p.R75Q mutation in the GJB2 gene with nonsyndromic hearing loss.     

一个同时携带线粒体DNA A1555G突变和GJB2235delC单杂合突变家系的基因型与听力表型

目的 调查一个同时携带线粒体DNA A1555G突变和GJB2 235delC突变的非综合征型耳聋家系,分析其基因型和听力表型的关系.方法 对家系成员进行临床听力测试,收集家系中8名成员的外周静脉血样本,从白细胞中提取DNA,聚合酶链反应扩增GJB2基因和线粒体DNA(mitochondric DNA,mtDNA)目的 片段,对扩增片段直接测序进行GJB2基因、mtDNA 12S rRNA及tRNASer(UCN)基因突变分析.结果 此家系先证者存在mtDNA A1555G突变和GJB2 235delC杂合突变,听力表型为极重度感音神经性耳聋.其他母系成员携带mtDNA A1555G突变,未发现tRNASer(UCN)基因突变,家系中其他母系成员听力表型为双侧对称高频下降或听力正常.结论 GJB2 235delC单杂合突变可能参与了mtDNA A1555G的听力损害.     

一个同时携带线粒体DNA A1555G突变和GJB2235delC单杂合突变家系的基因型与听力表型

目的 调查一个同时携带线粒体DNA A1555G突变和GJB2 235delC突变的非综合征型耳聋家系,分析其基因型和听力表型的关系.方法 对家系成员进行临床听力测试,收集家系中8名成员的外周静脉血样本,从白细胞中提取DNA,聚合酶链反应扩增GJB2基因和线粒体DNA(mitochondric DNA,mtDNA)目的 片段,对扩增片段直接测序进行GJB2基因、mtDNA 12S rRNA及tRNASer(UCN)基因突变分析.结果 此家系先证者存在mtDNA A1555G突变和GJB2 235delC杂合突变,听力表型为极重度感音神经性耳聋.其他母系成员携带mtDNA A1555G突变,未发现tRNASer(UCN)基因突变,家系中其他母系成员听力表型为双侧对称高频下降或听力正常.结论 GJB2 235delC单杂合突变可能参与了mtDNA A1555G的听力损害.     

High prevalence of the W24X mutation in the gene encoding connexin-26 (GJB2) in Spanish Romani (gypsies) with autosomal recessive non-syndromic hearing loss

Molecular testing for mutations in the gene encoding connexin-26 (GJB2) at the DFNB1 locus has become the standard of care for genetic diagnosis and counseling of autosomal recessive non-syndromic hearing impairment (ARNSHI). The spectrum of mutations in GJB2 varies considerably among the populations, different alleles predominating in different ethnic groups. A cohort of 34 families of Spanish Romani (gypsies) with ARNSHI was screened for mutations in GJB2. We found that DFNB1 deafness accounts for 50% of all ARNSHI in Spanish gypsies. The predominating allele is W24X (79% of the DFNB1 alleles), and 35delG is the second most common allele (17%). An allele-specific PCR test was developed for the detection of the W24X mutation. By using this test, carrier frequencies were determined in two sample groups of gypsies from different Spanish regions (Andalusia and Catalonia), being 4% and 0%, respectively. Haplotype analysis for microsatellite markers closely flanking the GJB2 gene revealed five different haplotypes associated with the W24X mutation, all sharing the same allele from marker D13S141, suggesting that a founder effect for this mutation is responsible for its high prevalence among Spanish gypsies.     

Frequency of the recessive 30delG mutation in the GJB2 gene in Northeast-Hungarian individuals and patients with hearing impairment.

Mutations in the GJB2 gene, which encodes a gap junction protein (connexin 26) account for up to 50% of cases of congenital autosomal recessive non-syndromic hearing impairment. A single mutation, 30delG, is responsible for 70% of this autosomal recessive hearing loss in Europe. This study describes the 30delG mutation analysis of 23 Hungarian families (64 individuals) with at least two subjects with congenital non-syndromic hearing defect and of 52 unrelated individuals from the Northeastern population of Hungary. In all patients, non-progressive hearing impairment varied from moderate to profound involving all frequencies. DNA was tested by PCR based restriction enzyme assay (BSiYI). Sixty-four percent of the patients displayed this one base deletion in GJB2. Out of these, 65.9% were homozygous for this mutation and 34.1% were heterozygotes. The latter showed compound heterozygosity since in these 14 patients, eight previously reported different nucleotide changes were observed on the second allele. The carrier frequency of the 30delG mutation among control group was one in 10.4 (9.6%). This high frequency of 30delG corresponds more to frequencies reported in Southern than in North Europeans.     

Mutations in the gene for connexin 26 (GJB2) that cause hearing loss have a dominant negative effect on connexin 30

Mutations in the gene (GJB2) encoding connexin 26 (cx26) have been linked to sensorineural hearing loss either alone or as part of a syndrome. Here we compare the properties of four cx26 mutants derived from point mutations associated with dominantly inherited hearing loss, either non-syndromic (W44S, R75W) or with various skin disorders (G59A, D66H, R75W). Since cx26 and cx30 are co-localized within the inner ear the effect of the dominant cx26 mutations on both of these wild-type proteins was determined. Communication-deficient HeLa cells were transiently transfected with the various cDNA constructs by microinjection. Dye transfer studies using the gap junction permeant tracer Cascade Blue demonstrated a disruption to the intercellular coupling for all four of the mutant proteins. Immunostaining of the transfected cells revealed that for the G59A and D66H mutants this correlated with impaired intracellular trafficking and targeting to the plasma membrane, as both proteins had a perinuclear localization. The impaired trafficking was rescued by oligomerization both with cx26 and with cx30, suggesting that cx26 and cx30 can form heteromeric connexons. Significantly reduced dye transfer rates were observed between cells co-expressing either cx26 or cx30 together with W44S or R75W compared with the wild-type proteins alone. The dominant actions of the G59A and D66H mutants were only on cx30 and cx26, respectively. We suggest that cx26 and cx30 form heteromeric connexons in vivo, within the inner ear, with particular properties essential for hearing. Disruption of these heteromeric channels by certain mutations may underlie the non-syndromic nature of the deafness.     

The frequency of GJB2 and GJB6 mutations in the New York State newborn population: feasibility of genetic screening for hearing defects

In the US, approximately one in every 1000 children has hearing loss sufficiently severe to interfere with the acquisition of normal speech [Ann NY Acad Sci 630 (1991) 16]. The causes of non-syndromic hearing loss (NSHL) are known to be heterogeneous, with genetic factors accounting for 50-75%[Am J Med Genet 46 (1993) 486]. Often individuals with NSHL thought to be caused by mutations in GJB2 have only one detectable mutant allele [Am J Hum Genet 62 (1998) 792, Hum Mol Genet 6 (12) (1997) 2173]. Another gene that has been identified as a possible cause of NSHL is GJB6 that codes for the gap junction protein, connexin 30. A consecutive series of anonymous newborn dried blood specimens (n = 2089) was tested for two GJB2 mutations: (i) 35delG, a pan-ethnic mutation; and (ii) 167delT, a mutation more frequently found in individuals of Ashkenazi Jewish and Mediterranean descents. Mutation detection was validated using allele-specific oligonucleotide hybridization in single wells. Once the positive samples had been identified, the samples were pooled and retested. All positives in the individual experiment were correctly identified in the pooled experiment. The same random set of anonymous newborn dried blood specimens plus some additional samples were tested (n = 2112) for the 342-kb deletion in the GJB6 gene.