A novel missense mutation in the connexin30 causes nonsyndromic hearing loss

Dysfunctional gap junctions caused by GJB2 (CX26) and GJB6 (CX30) mutations are implicated in nearly half of nonsyndromic hearing loss cases. A recent study identified a heterozygous mutation, c.119C>T (p.A40V), in the GJB6 gene of patients with nonsyndromic hearing loss. However, the functional role of the mutation in hearing loss remains unclear. In this study, analyses of cell biology indicated that a p.A40V missense mutation of CX30 causes CX30 protein accumulation in the Golgi body rather than in the cytoplasmic membrane. The tet-on protein expression system was used for further study of mutant proteins in CX30 and CX30A40V co-expressions and in CX26 and CX30A40V co-expressions. The p.A40V missense mutation exerted a dominant negative effect on both normal CX30 and CX26, which impaired gap junction formation. Moreover, computer-assisted modeling suggested that this p.A40V mutation affects the intra molecular interaction in the hydrophobic core of Trp44, which significantly alters the efficiency of gap junction formation. These findings suggest that the p.A40V mutation in CX30 causes autosomal-dominant nonsyndromic hearing loss. These data provide a novel molecular explanation for the role of GJB6 in hearing loss.     

Prevalence of GJB2 (CX26) gene mutations in south Iranian patients with autosomal recessive nonsyndromic sensorineural hearing loss

Hereditary hearing loss is a genetically heterogeneous disorder. Mutations in connexin 26 (CX26), are a major cause in many countries and are largely dependent on ethnic groups. The purpose of our study was to evaluate the prevalence of GJB2 mutations among affected individuals from south of Iran. Fifty patients presenting with autosomal recessive non-syndromic hearing loss from Fars, province in south of Iran, were studied for mutations in GJB2 gene and screened by direct sequencing. Mutations were detected in 15 out of 50 patients (30?%). Eight different mutations were identified; six of them were previously identified (35delG, V27I M34V, V153I, A149T, V198M). The remaining two alleles, L28I and N169T, were novel variants. The most common mutations were 35delG followed by V153I with an allele frequency of 7 and 6?%, respectively. In this study, 30?% of our subjects were found to have the causative variants or polymorphisms in GJB2 and the c.35delG mutation was the most common cause in our patients. However, more study with larger sample size as well as in vitro functional study for these new variants in Xenopus oocytes is required.     

Evaluation of the pathogenicity of GJB3 and GJB6 variants associated with nonsyndromic hearing loss

A number of genes responsible for hearing loss are related to ion recycling and homeostasis in the inner ear. Connexins (Cx26 encoded by GJB2, Cx31 encoded by GJB3 and Cx30 encoded by GJB6) are core components of gap junctions in the inner ear. Gap junctions are intercellular communication channels and important factors that are associated with hearing loss. To date, a molecular genetics study of GJB3 and GJB6 as a causative gene for hearing loss has not been performed in Korea. This study was therefore performed to elucidate the genetic characteristics of Korean patients with nonsyndromic sensorineural hearing loss and to determine the pathological mechanism of hearing loss by analyzing the intercellular communication function of Cx30 and Cx31 variants. Sequencing analysis of the GJB3 and GJB6 genes in our population revealed a total of nine variants, including four novel variants in the two genes. Three of the novel variants (Cx31-p.V27M, Cx31-p.V43M and Cx-30-p.I248V) and two previously reported variants (Cx31-p.V84I and Cx30-p.A40V) were selected for functional studies using a pathogenicity prediction program and assessed for whether the mutations were located in a conserved region of the protein. The results of biochemical and ionic coupling tests showed that both the Cx31-p.V27M and Cx31-p.V84I variants did not function normally when each was expressed as a heterozygote with the wild-type Cx31. This study demonstrated that two variants of Cx31 were pathogenic mutations with deleterious effect. This information will be valuable in understanding the pathogenic role of GJB3 and GJB6 mutations associated with hearing loss.     

GJB2 and mitochondrial A1555G gene mutations in nonsyndromic profound hearing loss and carrier frequencies in healthy individuals

This study aimed to assess mutations in GJB2 gene (connexin 26), as well as A1555G mitochondrial mutation in both the patients with profound genetic nonsyndromic hearing loss and healthy controls. Ninety-five patients with profound hearing loss (>90 dB) and 67 healthy controls were included. All patients had genetic nonsyndromic hearing loss. Molecular analyses were performed for connexin 26 (35delG, M34T, L90P, R184P, delE120, 167delT, 235delC and IVS1+1 A-->G) mutations, and for mitochondrial A1555G mutation. Twenty-two connexin 26 mutations were found in 14.7% of the patients, which were 35delG, R184P, del120E and IVS1+1 A-->G. Mitochondrial A1555G mutation was not encountered. The most common GJB2 gene mutation was 35delG, which was followed by del120E, IVS1+1 A-->G and R184P, and 14.3% of the patients segregated with DFNB1. In consanguineous marriages, the most common mutation was 35delG. The carrier frequency for 35delG mutation was 1.4% in the controls. 35delG and del120E populations, seems the most common connexin 26 mutations that cause genetic nonsyndromic hearing loss in this country. Nonsyndromic hearing loss mostly shows DFNB1 form of segregation.     

Detection of the 35delG/GJB2 and del(GJB6-D13S1830) mutations in Venezuelan patients with autosomal recessive nonsyndromic hearing loss

Severe to profound hearing impairment affects 1 of every 1000 newborn children each year. Inheritance accounts for 60% of these cases, of which 70% are nonsyndromic. The most common cause of autosomal recessive nonsyndromic hearing loss (ARNSHL) is mutation in GJB2, a gene on chromosome 13, which encodes a gap junction protein named Connexin 26. Mutations in GJB2 are responsible for 40% of genetic childhood deafness. The most common mutation, 35delG, predominates in many ethnic groups. Some families with linkage to the DFNB1 locus have none or only one mutated allele in GJB2, however, some subjects can exhibit a large deletion in another connexin gene, GJB6, resulting in a monogenic or digenic pattern of inheritance in this complex DFNB1 locus that contains both genes (GJB2 and GJB6). The aim of the study was to determine (1) the frequency for the 35delG (27.5%), del(GJB6-D13S1830) (2.5%) and del(GJB6-D13S1854) (0.0%) mutations in a cohort of 40 Venezuelan patients with ARNSHL and (2) the carrier frequency 35delG (4%), del(GJB6-D13S1830) (0%) and del(GJB6-D13S1854) (0%) in the Venezuelan population with no familial history of hearing impairment. One patient (2.5%) was detected as double heterozygote for the deletion del(GJB6-D13S1830) and 35delG mutation. This result has direct clinical implications because we include the molecular detection of the deletion del(GJB6-D13S1830) during the evaluation of the diagnosis of deafness in the Venezuelan population.     

GJB2 mutations and degree of hearing loss: a multicenter study

Hearing impairment (HI) affects 1 in 650 newborns, which makes it the most common congenital sensory impairment. Despite extraordinary genetic heterogeneity, mutations in one gene, GJB2, which encodes the connexin 26 protein and is involved in inner ear homeostasis, are found in up to 50% of patients with autosomal recessive nonsyndromic hearing loss. Because of the high frequency of GJB2 mutations, mutation analysis of this gene is widely available as a diagnostic test. In this study, we assessed the association between genotype and degree of hearing loss in persons with HI and biallelic GJB2 mutations. We performed cross-sectional analyses of GJB2 genotype and audiometric data from 1,531 persons, from 16 different countries, with autosomal recessive, mild-to-profound nonsyndromic HI. The median age of all participants was 8 years; 90% of persons were within the age range of 0-26 years. Of the 83 different mutations identified, 47 were classified as nontruncating, and 36 as truncating. A total of 153 different genotypes were found, of which 56 were homozygous truncating (T/T), 30 were homozygous nontruncating (NT/NT), and 67 were compound heterozygous truncating/nontruncating (T/NT). The degree of HI associated with biallelic truncating mutations was significantly more severe than the HI associated with biallelic nontruncating mutations (P<.0001). The HI of 48 different genotypes was less severe than that of 35delG homozygotes. Several common mutations (M34T, V37I, and L90P) were associated with mild-to-moderate HI (median 25-40 dB). Two genotypes--35delG/R143W (median 105 dB) and 35delG/dela(GJB6-D13S1830) (median 108 dB)--had significantly more-severe HI than that of 35delG homozygotes.     

High frequency of 35delG GJB2 mutation and absence of del(GJB6-D13S1830) in Greek Cypriot patients with nonsyndromic hearing loss

Mutations in the GJB2 (Connexin 26) gene are responsible for more than half of all cases of prelingual, recessive, inherited, nonsyndromic deafness in Europe. This paper presents a mutation analysis of the GJB2 and GJB6 (Connexin 30) genes in 30 Greek Cypriot patients with sensorineural nonsyndromic hearing loss compatible with recessive inheritance. Ten of the patients (33.3%) had the 35delG mutation in the GJB2 gene. Moreover, 9 of these were homozygous for the 35delG mutation, whereas 1 patient was in the compound heterozygous state with the disease causing E47X nonsense mutation. Another patient with severe sensorineural hearing loss was heterozygous for the V153I missense mutation. Finally, no GJB6 mutations or the known del(GJB6-D13S1830) were identified in any of the investigated Greek Cypriot nonsyndromic hearing loss patients. This work confirms that the GJB2 35delG mutation is an important pathogenic mutation for hearing loss in the Greek Cypriot population. This finding will be used toward the effective diagnosis of nonsyndromic hearing loss, improve genetic counseling, and serve as a potential therapeutic platform in the future for the affected patients in Cyprus.     

Altered gating properties of functional Cx26 mutants associated with recessive non-syndromic hearing loss

Connexins (Cx) form gap junctions that allow the exchange of small metabolites and ions. In the inner ear, Cx26 is the major gap junction protein and mutations in the Cx26-encoding gene, GJB2, are the most frequent cause of autosomal recessive non-syndromic hearing loss (DFNB1). We have functionally analyzed five Cx26 mutations associated with DFNB1, comprising the following single amino-acid substitutions: T8M, R143W, V153I, N206S and L214P. Coupling of cells expressing wild-type or mutant Cx26 was measured in the paired Xenopus oocyte assay. We found that the R143W, V153I and L214P mutations were unable to form functional channels. In contrast, the T8M and N206S mutants did electrically couple cells, though their voltage gating properties were different from wild-type Cx26 channels. The electrical coupling of oocytes expressing the T8M and N206S mutants suggest that these channels may retain high permeability to potassium ions. Therefore, deafness associated with Cx26 mutations may not only depend on reduced potassium re-circulation in the inner ear. Instead, abnormalities in the exchange of other metabolites through the cochlear gap junction network may also produce deafness.     

Novel mutations in the connexin 26 gene (GJB2) that cause autosomal recessive (DFNB1) hearing loss.

Mutations in the connexin 26 (Cx26) gene (GJB2) are associated with the type of autosomal recessive nonsyndromic neurosensory deafness known as "DFNB1." Studies indicate that DFNB1 (13q11-12) causes 20% of all childhood deafness and may have a carrier rate as high as 2. 8%. This study describes the analysis of 58 multiplex families each having at least two affected children diagnosed with autosomal recessive nonsyndromic deafness. Twenty of the 58 families were observed to have mutations in both alleles of Cx26. Thirty-three of 116 chromosomes contained a 30delG allele, for a frequency of .284. This mutation was observed in 2 of 192 control chromosomes, for an estimated gene frequency of .01+/-.007. The homozygous frequency of the 30delG allele is then estimated at .0001, or 1/10,000. Given that the frequency of all childhood hearing impairment is 1/1,000 and that half of that is genetic, the specific mutation 30delG is responsible for 10% of all childhood hearing loss and for 20% of all childhood hereditary hearing loss. Six novel mutations were also observed in the affected population. The deletions detected cause frameshifts that would severely disrupt the protein structure. Three novel missense mutations, Val84Met, Val95Met, and Ser113Pro, were observed. The missense mutation 101T-->C has been reported to be a dominant allele of DFNA3, a dominant nonsyndromic hearing loss. Data further supporting the finding that this mutation does not cause dominant hearing loss are presented. This allele was found in a recessive family segregating independently from the hearing-loss phenotype and in 3 of 192 control chromosomes. These results indicate that 101T-->C is not sufficient to cause hearing loss.     

High frequency hearing loss correlated with mutations in the GJB2 gene

Genetic hearing impairment affects approximately 1/2000 live births. Mutations in one gene, GJB2, coding for connexin 26 cause 10%-20% of all genetic sensorineural hearing loss. Mutation analysis in the GJB2 gene and audiology were performed on 106 families presenting with at least one child with congenital hearing loss. The families were recruited from a hospital-based multidisciplinary clinic, which functions to investigate the aetiology of sensorineural hearing loss in children and which serves an ethnically diverse population. In 74 families (80 children), the aetiology was consistent with non-syndromic recessive hearing loss. Six different connexin 26 mutations, including one novel mutation, were identified. We show that GJB2 mutations cause a range of phenotypes from mild to profound hearing impairment and that loss of hearing in the high frequency range (4000-8000 Hz) is a characteristic feature in children with molecularly diagnosed connexin 26 hearing impairment. We also demonstrate that this type of audiology and high frequency hearing loss is found in a similar-sized group of deaf children in whom a mutation could only be found in one of the connexin 26 alleles, suggesting connexin 26 involvement in the aetiology of hearing loss in these cases. In our study of the M34T mutation, only compound heterozygotes exhibited hearing loss, suggesting autosomal recessive inheritance.     

Hearing loss: frequency and functional studies of the most common connexin26 alleles

Mutations in the GJB2 gene, encoding the gap-junction channel protein connexin 26, account for the majority of recessive forms and some of the dominant cases of deafness. Here, we report the frequency of GJB2 alleles in the Italian population affected by hearing loss and the functional analysis of six missense mutations. Genetic studies indicate that, apart from the common 35delG, only few additional mutations can be detected with a significant frequency in our population. Transfection of communication-incompetent HeLa cells with Cx26 missense mutations revealed three distinct classes of functional deficits in terms of protein expression, subcellular localisation and/or functional activity. Moreover, the M34T mutant acted as a dominant inhibitor of wild-type Cx26 channel activity when the two proteins were co-expressed in a manner mimicking a heterozygous genotype. These data support the hypothesis of a functional role for M34T as a dominant allele and represent a further step towards a complete understanding of the role of GJB2 in causing hearing loss.     

Screening for mutations in the GJB3 gene in Brazilian patients with nonsyndromic deafness

Deafness is a complex disorder that is affected by a high number of genes and environmental factors. Recently, enormous progress has been made in nonsyndromic deafness research, with the identification of 90 loci and 33 nuclear and 2 mitochondrial genes involved (http://dnalab-www.uia.ac.be/dnalab/hhh/). Mutations in the GJB3 gene, encoding the gap junction protein connexin 31 (Cx31), have been pathogenically linked to erythrokeratodermia variabilis and nonsyndromic autosomal recessive or dominant hereditary hearing impairment. To determine the contribution of the GJB3 gene to sporadic deafness, we analysed the GJB3 gene in 67 families with nonsyndromic hearing impairment. A single coding exon of the GJB3 gene was amplified from genomic DNA and then sequenced. Here we report on three amino acid changes: Y177D (c.529T > G), 49delK (c.1227C > T), and R32W (c.144-146delGAA). The latter substitution has been previously described, but its involvement in hearing impairment remains uncertain. We hypothesize that mutations in the GJB3 gene are an infrequent cause of nonsyndromic deafness.     

Analysis of Trafficking,Stability and Function of Human Connexin 26 Gap Junction Channels with Deafness-Causing Mutations in the Fourth Transmembrane Helix

Human Connexin26 gene mutations cause hearing loss. These hereditary mutations are the leading cause of childhood deafness worldwide. Mutations in gap junction proteins (connexins) can impair intercellular communication by eliminating protein synthesis, mis-trafficking, or inducing channels that fail to dock or have aberrant function. We previously identified a new class of mutants that form non-functional gap junction channels and hemichannels (connexons) by disrupting packing and inter-helix interactions. Here we analyzed fourteen point mutations in the fourth transmembrane helix of connexin26 (Cx26) that cause non-syndromic hearing loss. Eight mutations caused mis-trafficking (K188R, F191L, V198M, S199F, G200R, I203K, L205P, T208P). Of the remaining six that formed gap junctions in mammalian cells, M195T and A197S formed stable hemichannels after isolation with a baculovirus/Sf9 protein purification system, while C202F, I203T, L205V and N206S formed hemichannels with varying degrees of instability. The function of all six gap junction-forming mutants was further assessed through measurement of dye coupling in mammalian cells and junctional conductance in paired Xenopus oocytes. Dye coupling between cell pairs was reduced by varying degrees for all six mutants. In homotypic oocyte pairings, only A197S induced measurable conductance. In heterotypic pairings with wild-type Cx26, five of the six mutants formed functional gap junction channels, albeit with reduced efficiency. None of the mutants displayed significant alterations in sensitivity to transjunctional voltage or induced conductive hemichannels in single oocytes. Intra-hemichannel interactions between mutant and wild-type proteins were assessed in rescue experiments using baculovirus expression in Sf9 insect cells. Of the four unstable mutations (C202F, I203T, L205V, N206S) only C202F and N206S formed stable hemichannels when co-expressed with wild-type Cx26. Stable M195T hemichannels displayed an increased tendency to aggregate. Thus, mutations in TM4 cause a range of phenotypes of dysfunctional gap junction channels that are discussed within the context of the X-ray crystallographic structure.     

The role of the cytoskeleton in the formation of gap junctions by Connexin 30

Mutations in the genes that encode Connexin 26 (GJB2) and Connexin 30 (GJB6) are the most common known cause of hereditary nonsyndromic sensorineural deafness. Cx26 and Cx30 share a similar protein structure, as well as the same expression distribution pattern in the cochlea. Cx26 has different intracellular trafficking properties compared to those of Cx43 and Cx32, whose trafficking manner is consistent with the classical membrane protein secretory pathway. Until now, however, the trafficking patterns of Cx30 have not been studied. By means of an immunofluorescence staining approach, we found that the targeting of Cx30 to gap junctions in transfected HeLa cells is not affected by brefeldin A, suggesting a Golgi-independent feature, similar to Cx26. Nocodazole had a minimal effect on assembly and distribution of Cx30 gap junctions. Cytochalasin B-induced actin filament depolymerization, however, affected both the pattern and the distribution of Cx30 gap junctions. Co-localization with and/or interaction between Cx30 and microtubules and cortical actin filaments, but not with the tight/adherens junction protein ZO-1, was confirmed by immunofluorescence and/or immunoprecipitation methods. The results suggest that the cytoskeleton, and especially actin filaments, are important components in the processes of assembly, trafficking and stabilization of Cx30 gap junctions.     

Unexpected heterogeneity due to recessive and de novo dominant mutations of GJB2 in an Iranian family with nonsyndromic hearing loss: implication for genetic counseling

Mutations in the GJB2 gene are the most common cause of nonsyndromic autosomal recessive sensorineural hearing loss (HL). A few mutations in GJB2 have also been reported to cause dominant nonsyndromic HL. Here we report a large inbred family including two individuals with nonsyndromic sensorineural hearing loss. A dominant GJB2 mutation, c.551G>A (p.R184Q), was detected in the proband, yet his parents were negative for the mutation. The second affected person had heterozygous c.35delG mutation, which was inherited from his father. Large deletions of the GJB6 gene were not detected in this family. This study highlights the importance of mutation analysis in all affected cases within a pedigree.     

Forty-six genes causing nonsyndromic hearing impairment: which ones should be analyzed in DNA diagnostics?

Hearing impairment is the most common sensory disorder, present in 1 of every 500 newborns. With 46 genes implicated in nonsyndromic hearing loss, it is also an extremely heterogeneous trait. Here, we categorize for the first time all mutations reported in nonsyndromic deafness genes, both worldwide and more specifically in Caucasians. The most frequent genes implicated in autosomal recessive nonsyndromic hearing loss are GJB2, which is responsible for more than half of cases, followed by SLC26A4, MYO15A, OTOF, CDH23 and TMC1. None of the genes associated with autosomal dominant nonsyndromic hearing loss accounts for a preponderance of cases, although mutations are somewhat more frequently reported in WFS1, KCNQ4, COCH and GJB2. Only a minority of these genes is currently included in genetic diagnostics, the selection criteria typically reflecting: (1) high frequency as a cause of deafness (i.e. GJB2); (2) association with another recognisable feature (i.e. SLC26A4 and enlarged vestibular aqueduct); or (3) a recognisable audioprofile (i.e. WFS1). New and powerful DNA sequencing technologies have been developed over the past few years, but have not yet found their way into DNA diagnostics. Implementing these technologies is likely to happen within the next 5 years, and will cause a breakthrough in terms of power and cost efficiency. It will become possible to analyze most - if not all - deafness genes, as opposed to one or a few genes currently. This ability will greatly improve DNA diagnostics, provide epidemiological data on gene-based mutation frequencies, and reveal novel genotype-phenotype correlations.     

Autosomal recessive nonsyndromic neurosensory deafness at DFNB1 not associated with the compound-heterozygous GJB2 (connexin 26) genotype M34T/167delT

Previous studies of the gap-junction beta-2 subunit gene GJB2 (connexin 26) have suggested that the 101T-->C (M34T) nucleotide substitution may be a mutant allele responsible for recessive deafness DFNB1. This hypothesis was consistent with observations of negligible intercellular coupling and gap-junction assembly of the M34T allele product expressed in Xenopus oocytes and HeLa cells. The results of our current study of a family cosegregating the 167delT allele of GJB2 and severe DFNB1 deafness demonstrate that this phenotype did not cosegregate with the compound-heterozygous genotype M34T/167delT. Since 167delT is a null allele of GJB2, this result indicates that the in vivo activity of a single M34T allele is not sufficiently reduced to cause the typical deafness phenotype associated with DFNB1. This observation raises the possibility that other GJB2 missense substitutions may not be recessive mutations that cause severe deafness and emphasizes the importance of observing cosegregation with deafness in large families to confirm that these missense alleles are mutant DFNB1 alleles.     

Connexin mutations in hearing loss,dermatological and neurological disorders

Gap junctions are important structures in cell-to-cell communication. Connexins, the protein units of gap junctions, are involved in several human disorders. Mutations in beta-connexin genes cause hearing, dermatological and peripheral nerve disorders. Recessive mutations in the gene encoding connexin 26 (GJB2) are the most common cause of childhood-onset deafness. The combination of mutations in the GJB2 and GJB6 (Cx30) genes also cause childhood hearing impairment. Although both recessive and dominant connexin mutants are functionally impaired, dominant mutations might have in addition a dominant-negative effect on wild-type connexins. Some dominant mutations in beta-connexin genes have a pleiotropic effect at the level of the skin, the auditory system and the peripheral nerves. Understanding the genotype-phenotype correlations in diseases caused by mutations in connexin genes might provide important insight into the mechanisms that lead to these disorders.     

Two Novel Missense Mutations in the Connexin 26 Gene in Turkish Patients with Nonsyndromic Hearing Loss

Most nonsyndromic hearing losses are caused by mutations in the GJB2 gene, and studies have revealed that the forms and frequencies of these mutations are largely dependent on ethnic origin. In the present study, we aimed to characterize the mutation profiles of 151 patients with hearing loss in Turkey. The entire coding region of the GJB2 was directly sequenced in all patients. We found 35 (23.2%) individuals carrying GJB2 mutations. Seven different mutations were identified, five of which were previously known (35delG, delE120, R184P, M163V, L90P), the remaining two being novel variants (M34V, L205V). The most common mutation was 35delG followed by delE120. The 35delG mutation was homozygous in 22 cases (14.5%) and heterozygous in 4 cases (2.6%). Compound heterozygosity for 35delG was also observed. The delE120 mutation was found in three patients in homozygous form. A homozygous L90P and heterozygous mutations M163V and M34V were found in single cases.