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.     

Moderate hearing loss and pseudodominant inheritance due to L90P/35delG mutations in the GJB2 (connexin 26) gene

Mutations in the GJB2 (connexin 26-Cx26) gene are responsible for 20-50% of cases with prelingual non-syndromic deafness in a large part of the world including Turkey. Although most of the cases with Cx26 deafness have a recessive mode of inheritance, a small group of families demonstrated dominant or pseudodominant inheritance. In this report we present a Turkish family in which the proband had congenital profound deafness and was found to be homozygous for the 35delG mutation, whereas the father and a paternal uncle who had milder, late-onset sensorineural hearing loss had compound heterozygous 35delG and L90P mutations. This family and previous reports with the L90P mutation demonstrate that the hearing loss associated with the L90P/35delG genotype is consistently milder than that of 35delG homozygotes. GJB2 gene screening should be considered in families with seemingly dominant inheritance and late-onset moderate hearing loss.     

Connexin-30 deletion analysis in connexin-26 heterozygotes

Mutations in the Connexin-26 gene (Cx 26, GJB2) are the most common cause of hereditary nonsyndromic sensorineural hearing loss (SNHL). DNA analysis of the Cx 26 gene in deaf or hard-of-hearing individuals frequently demonstrates heterozygosity despite the fact that most mutations are known to be recessive. A 342-kb deletion in a gene adjacent to Cx 26, the Connexin-30 gene (Cx 30, GJB6), has been reported to cause deafness in the homozygous state or in combination with heterozygous mutations in Cx 26 (digenic inheritance). We have analyzed deaf or hard-of-hearing Cx 26 heterozygotes and individuals with no mutations in Cx 26 for this Cx 30 deletion. We found that 4/20 (20%) of the Cx 26 heterozygotes are heterozygous for this deletion and that no individuals were homozygous for the Cx 30 deletion. Cx 30 deletion analysis is recommended for all individuals with nonsyndromic SNHL following Cx 26 sequencing that does not demonstrate two recessive mutations.     

Closing the gap on autosomal dominant connexin-26 and connexin-43 mutants linked to human disease

Cells within the vast majority of human tissues communicate directly through clustered arrays of intercellular channels called gap junctions. Gene ablation studies in mouse models have revealed that these intercellular channels are necessary for a variety of organ functions and that some of these genes are essential for survival. Molecular genetics has uncovered that germ line mutations in nearly half of the genes that encode the 21-member connexin family of gap junction proteins are linked to one or more human diseases. Frequently, these mutations are autosomal recessive, whereas in other cases, autosomal dominant mutations manifest as disease. Given the broad and overlapping distribution of connexins in a wide arrangement of tissues, it is hard to predict where connexin-linked diseases will clinically manifest. For instance, the most prevalent connexin in the human body is connexin-43 (Cx43), yet autosomal dominant mutations in the GJA1 gene, which encodes Cx43, exhibit modest developmental disorders resulting in a disease termed oculodentodigital dysplasia. Autosomal recessive mutations in the gene encoding Cx26 result in moderate to severe sensorineural hearing loss, whereas autosomal dominant mutations produce hearing loss and a wide range of skin diseases, including palmoplantar keratoderma. Here, we will focus on autosomal dominant mutations of the genes encoding Cx26 and Cx43 in relation to models that link genotypes to phenotypic outcomes with particular reference to how these approaches provide insight into human disease.     

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.     

De novo mutation of the connexin 26 gene associated with dominant non-syndromic sensorineural hearing loss

Mutations of the connexin 26 (Cx26) gene cause isolated recessive or dominant hearing loss or both sensorineural hearing impairment and keratoderma. We have identified the first de novo mutation of the Cx26 gene, R75 W, in a sporadic case of isolated profound hearing loss. R75 W has been previously observed in association with hearing impairment and keratoderma in one family and is thus thought to cause both syndromic and non-syndromic hearing loss. This case illustrates the risk of a possible erroneous diagnosis of autosomal recessive hearing loss in a sporadic case.     

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.     

Genotyping for Cx26 and Cx30 mutations in cases with congenital hearing loss

Hearing loss is the most frequent sensory defect in human being. The 13q11-q12 region contains the GJB2 and GJB6 genes, which code connexin 26 (CX26) and connexin 30 (CX30) proteins, respectively. The 35delG, 167delT, and 235delC mutations in the Cx26 gene are the main cause for sporadic nonsyndromic hearing loss (NSHL) in many populations. The 342-kb deletion [del(GJB6-D13S1830)] of the Cx30 gene is the second most common connexin mutation after the 35delG mutation in some NSHL populations. In our study 47 hearing-impaired students were included. The Cx26 gene and the Cx30 gene were analyzed for presence of the 35delG, 167delT, and 342-kb deletion [del(GJB6-D13S1830)]. Genotyping were performed for detecting 35delG, 167delT, and del(GJB6-D13S1830) mutations using the PCR-ELISA techniques. According to the results obtained from 47 cases, the 35delG mutation was detected in 7 cases ( approximately 14.9%). Four of these mutations were determined as homozygote mutant ( approximately 8.5%), and three were determined as heterozygote mutant ( approximately 6.4%). However, 167delT and del(GJB6-D13S1830) mutations were not detected in the study group. These results support the overwhelming majority of 35delG in our study group from deafness school in our study. In conclusion, the 35delG mutation was determined as the most frequently shown mutation that leads to congenital hearing loss as in previous studies from Turkey.     

Low incidence of GJB2, GJB6 and mitochondrial DNA mutations in North Indian patients with non-syndromic hearing impairment

Mutations at the DFNB1 locus which encode connexin 26 (CX26) and connexin 30 (CX30) proteins, respectively, are main cause for sporadic and familial non-syndromic hearing impairment (NSHI) in many populations. 342-kb deletion [del (GJB6-D13S1830)] of Cx30 gene is second most common connexin mutation. Specific mitochondrial DNA (mtDNA) mutations have been found to be associated with NSHI. In this study, we screened 210 NSHI patients for GJB2 mutations, ΔGJB6-D13S1830 deletion and three point mutations in mtDNA (A1555G, A3243G, A7445G) using PCR, DHPLC and sequencing in North Indian cohort. 35delG was found to be the most common mutation (10.9%), followed by W24X (3.8%) and W77X (1.9%) mutations. We did not observe GJB6-D13S1830 deletion and three mitochondrial point mutations in our cohort. Most of patients (50/58) carried monoallelic variations. Our results reveal different spectrum of GJB2 mutations specific to North Indian cohort, with 35delG being most prevalent. These results suggest that different types of GJB2 mutations affect autosomal recessive NSHI according to ethnic background.     

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.     

Digenic inheritance of non-syndromic deafness caused by mutations at the gap junction proteins Cx26 and Cx31

Mutations in the genes coding for connexin 26 (Cx26) and connexin 31 (Cx31) cause non-syndromic deafness. Here, we provide evidence that mutations at these two connexin genes can interact to cause hearing loss in digenic heterozygotes in humans. We have screened 108 GJB2 heterozygous Chinese patients for mutations in GJB3 by sequencing. We have excluded the possibility that mutations in exon 1 of GJB2 and the deletion of GJB6 are the second mutant allele in these Chinese heterozygous probands. Two different GJB3 mutations (N166S and A194T) occurring in compound heterozygosity with the 235delC and 299delAT of GJB2 were identified in three unrelated families (235delC/N166S, 235delC/A194T and 299delAT/A194T). Neither of these mutations in Cx31 was detected in DNA from 200 unrelated Chinese controls. Direct physical interaction of Cx26 with Cx31 is supported by data showing that Cx26 and Cx31 have overlapping expression patterns in the cochlea. In addition, by coimmunoprecipitation of mouse cochlear membrane proteins, we identified the presence of heteromeric Cx26/Cx31 connexons. Furthermore, by cotransfection of mCherry-tagged Cx26 and GFP-tagged Cx31 in human embryonic kidney (HEK)-293 cells, we demonstrated that the two connexins were able to co-assemble in vitro in the same junction plaque. Together, our data indicate that a genetic interaction between these two connexin genes can lead to hearing loss.     

Connexin 26 (GJB2) mutations in the Turkish population: implications for the origin and high frequency of the 35delG mutation in Caucasians

Mutations in the Connexin 26 (GJB2/Cx26) gene are responsible for more than half of all cases of prelingual non-syndromic recessive deafness in many Caucasian populations. To determine the importance of Cx26 mutations as a cause of deafness in Turks we screened 11 families with prelingual non-syndromic deafness, seven (64%) of which were found to carry the 35delG mutation. We subsequently screened 674 Turkish subjects with no known hearing loss and found twelve 35delG heterozygotes (1.78%; 95% confidence interval: 0.9%-3%) but no examples of the 167delT mutation. To search for possible founder effects, we typed chromosomes carrying the 35delG mutation for closely linked polymorphic markers in samples from Turkey and United States and compared the allele frequencies with those of hearing subjects. The data showed a modest degree of disequilibrium in both populations. Analyses of two pedigrees from Turkey demonstrated both conserved and different haplotypes, suggesting possible founder effects and multiple origins of the 35delG mutation.     

A Deafness-Associated Mutant Human Connexin 26 Improves the Epithelial Barrier In Vitro

A large proportion of recessive nonsyndromic hearing loss is due to mutations in the GJB2 gene encoding connexin 26 (Cx26), a component of a gap junction. Within different ethnic groups there are specific common recessive mutations, each with a relatively high carrier frequency, suggesting the possibility of heterozygous advantage. Carriers of the R143W GJB2 allele, the most prevalent in the African population, present with a thicker epidermis than noncarriers. In this study, we show that (R143W)Cx26-expressing keratinocytes form a significantly thicker epidermis in an organotypic coculture skin model. In addition, we show increased migration of cells expressing (R143W)Cx26 compared to (WT)Cx26-overexpressing cells. We also demonstrate that cells expressing (R143W)Cx26 are significantly less susceptible to cellular invasion by the enteric pathogen Shigella flexneri than (WT)Cx26-expressing cells. These in vitro studies suggest an advantageous effect of (R143W)Cx26 in epithelial cells. The first two authors contributed equally to this work.     

BAAV mediated GJB2 gene transfer restores gap junction coupling in cochlear organotypic cultures from deaf Cx26Sox10Cre mice

The deafness locus DFNB1 contains GJB2, the gene encoding connexin26 and GJB6, encoding connexin30, which appear to be coordinately regulated in the inner ear. In this work, we investigated the expression and function of connexin26 and connexin30 from postnatal day 5 to adult age in double transgenic Cx26(Sox10Cre) mice, which we obtained by crossing connexin26 floxed mice with a deleter Sox10-Cre line. Cx26(Sox10Cre) mice presented with complete connexin26 ablation in the epithelial gap junction network of the cochlea, whereas connexin30 expression was developmentally delayed; immunolabeling patterns for both connexins were normal in the cochlear lateral wall. In vivo electrophysiological measurements in Cx26(Sox10Cre) mice revealed profound hearing loss accompanied by reduction of endocochlear potential, and functional experiments performed in postnatal cochlear organotypic cultures showed impaired gap junction coupling. Transduction of these cultures with a bovine adeno associated virus vector restored connexin26 protein expression and rescued gap junction coupling. These results suggest that restoration of normal connexin levels by gene delivery via recombinant adeno associated virus could be a way to rescue hearing function in DFNB1 mouse models and, in future, lead to the development of therapeutic interventions in humans.     

Extrinsic innervation of ileum and pelvic flexure of foals with ileocolonic aganglionosis

Globally 360 million people have disabling hearing loss and, of these, 32 million are children. Human hearing relies on 15,000 hair cells that transduce mechanical vibrations to electrical signals in the auditory nerve. The process is powered by the endo-cochlear potential, which is produced by a vascularized epithelium that actively transports ions in conjunction with a gap junction (GJ) system. This “battery” is located “off-site” in the lateral wall of the cochlea. The GJ syncytium contains the GJ protein genes beta 2 (GJB2/connexin26 (Cx26)) and 6 (GJB6/connexin30 (Cx30)), which are commonly involved in hereditary deafness. Because the molecular arrangement of these proteins is obscure, we analyze GJ protein expression (Cx26/30) in human cochleae by using super-resolution structured illumination microscopy. At this resolution, the Cx26 and Cx30 proteins were visible as separate plaques, rather than being co-localized in heterotypic channels, as previously suggested. The Cx26 and Cx30 proteins thus seem not to be co-expressed but to form closely associated assemblies of GJ plaques. These results could assist in the development of strategies to treat genetic hearing loss in the future.     

Functional analysis of a dominant mutation of human connexin26 associated with nonsyndromic deafness

Cx26 has been implicated in dominant (DFNA3) and recessive (DFNB1) forms of nonsyndromic sensorineural deafness. While most homozygous DFNB1 Cx26 mutations result in a simple loss of channel activity, it is less clear how heterozygous mutations in Cx26 linked to DFNA3 cause hearing loss. We have tested the ability of one dominant mutation (W44C) to interfere with wild-type human Cx26 (HCx26wt). HCx26wt induced robust electrical conductance between paired oocytes, and facilitated dye transfer between transfected HeLa cells. In contrast, oocyte pairs injected with only W44C were not electrically coupled above background levels, and W44C failed to dye couple transfected HeLa cells. Moreover, W44C dramatically inhibited intercellular conductance of HCx26wt when co-expressed in an equal ratio, and the low levels of residual conductance displayed altered gating properties. A nonfunctional recessive mutation (W77R) did not inhibit the ability of HCx26wt to form functional channels when co-injected in the same oocyte pairs, nor did it alter HCx26wt gating. These results provide evidence for a functional dominant negative effect of the W44C mutant on HCx26wt and explain how heterozygous Cx26 mutations could contribute to autosomal dominant deafness, by resulting in a net loss, and/or alteration, of Cx26 function.     

Mutations in the WFS1 gene that cause low-frequency sensorineural hearing loss are small non-inactivating mutations

Hereditary hearing impairment is an extremely heterogeneous trait, with more than 70 identified loci. Only two of these loci are associated with an auditory phenotype that predominantly affects the low frequencies (DFNA1 and DFNA6/14). In this study, we have completed mutation screening of the WFS1 gene in eight autosomal dominant families and twelve sporadic cases in which affected persons have low-frequency sensorineural hearing impairment (LFSNHI). Mutations in this gene are known to be responsible for Wolfram syndrome or DIDMOAD (diabetes insipidus, diabetes mellitus, optic atrophy, and deafness), which is an autosomal recessive trait. We have identified seven missense mutations and a single amino acid deletion affecting conserved amino acids in six families and one sporadic case, indicating that mutations in WFS1 are a major cause of inherited but not sporadic low-frequency hearing impairment. Among the ten WFS1 mutations reported in LFSNHI, none is expected to lead to premature protein truncation, and nine cluster in the C-terminal protein domain. In contrast, 64% of the Wolfram syndrome mutations are inactivating. Our results indicate that only non-inactivating mutations in WFS1 are responsible for non-syndromic low-frequency hearing impairment.     

Cellular mechanisms of mutant connexins in skin disease and hearing loss

It has been demonstrated that distinct germline mutations within four connexin (Cx) genes, Cx26, Cx30, Cx31, and Cx30.3, underlie hearing loss and/or epidermal disease. Here, we describe two Cx26 mutations associated with skin disease. With the goal of understanding the mechanism(s) of Cx-associated human disease and how different mutations within the same Cx protein can result in different disorders, we performed a number of functional analyses investigating the cellular effects of disease-associated Cx mutations in keratinocytes and other cell types. Epidermal disease-associated proteins studied were primarily cytoplasmic with limited trafficking ability. FACS analysis of WT and mutant EGFP-Cx31 transfected keratinocytes revealed a high percentage of cell death associated with the skin disease-associated mutant Cx31 proteins.     

Cellular Mechanisms of Mutant Connexins in Skin Disease and Hearing Loss

It has been demonstrated that distinct germline mutations within four connexin (Cx) genes, Cx26, Cx30, Cx31, and Cx30.3, underlie hearing loss and/or epidermal disease. Here, we describe two Cx26 mutations associated with skin disease. With the goal of understanding the mechanism(s) of Cx-associated human disease and how different mutations within the same Cx protein can result in different disorders, we performed a number of functional analyses investigating the cellular effects of disease-associated Cx mutations in keratinocytes and other cell types. Epidermal disease-associated proteins studied were primarily cytoplasmic with limited trafficking ability. FACS analysis of WT and mutant EGFP-Cx31 transfected keratinocytes revealed a high percentage of cell death associated with the skin disease-associated mutant Cx31 proteins.     

Connexin 26 (GJB2) mutations, causing KID Syndrome, are associated with cell death due to calcium gating deregulation

The autosomic dominant KID Syndrome (MIM 148210), due to mutations in GJB2 (connexin 26, Cx26), is an ectodermal dysplasia with erythematous scaly skin lesions, keratitis and severe bilateral sensorineural deafness. The Cx26 protein is a component of gap junction channels in epithelia, including the cochlea, which coordinates the exchange of molecules and ions. Here, we demonstrate that different Cx26 mutants (Cx26D50N and Cx26G11E) cause cell death in vitro by the alteration of intra-cellular calcium concentrations. These results help to explain the pathogenesis of both the hearing and skin phenotypes, since calcium is also a potent regulator of the epidermal differentiation process.