A novel missense mutation in the second extracellular domain of GJB2, p.Ser183Phe, causes a syndrome of focal palmoplantar keratoderma with deafness

Gap junctions, which consist of connexins, are intercellular channels that mediate rapid intercellular communication. In the skin, connexins are involved in the regulation of epidermal growth and differentiation. GJB2 encodes connexin26, which is an important skin-expressed gap junction protein. Mutations in GJB2 cause a wide variety of unique disorders, but despite extensive research, their mechanisms of action are poorly understood. The identification of novel diseases caused by mutations in GJB2 may help to illuminate the genotype-phenotype correlation and elucidate the function of different regions of the protein. Here, we report the first account of a family with a GJB2 missense mutation in the second extracellular domain (p.Ser183Phe) that causes skin abnormalities in addition to sensorineural hearing loss. Using fluorescent connexin26-EGFP fusion proteins, we showed that the mutation induces a partial protein transport defect that cannot be rescued by wild-type protein. Dye-transfer experiments using a parachute assay revealed channel functionality. Although p.Ser183Phe affects the second extracellular domain, mutations in the first extracellular domain also lead to focal palmoplantar keratoderma and likewise perturb protein transport in a dominant-negative manner. Therefore, we hypothesize that focal palmoplantar keratoderma in gap junction skin disease may be specifically associated with connexin trafficking defects as well as with mutations affecting its extracellular domains, thus broadening the spectrum of GJB2-associated diseases.     

Three novel GJB2 (connexin 26) variants associated with autosomal dominant syndromic and nonsyndromic hearing loss

Connexin 26 (Cx26), encoded by the GJB2 gene, is a key protein involved in the formation of gap junctions in epithelial organs including the inner ear and palmoplantar epidermis. Pathogenic variants in GJB2 are responsible for approximately 50% of inherited sensorineural deafness. The majority of these variants are associated with autosomal recessive inheritance; however, rare reports of dominantly co‐segregating variants have been published. Since we began offering GJB2 testing in 2003, only about 2% of detected GJB2 variants from our laboratory have been classified as dominant. Here we report three novel dominant GJB2 variants (p.Thr55Ala, p.Gln57_Pro58delinsHisSer, and p.Trp44Gly); two associated with syndromic sensorineural hearing loss and one with nonsyndromic hearing loss. In the kindred with the p.Thr55Ala variant, the proband and his father present with only leukonychia as a cutaneous finding of their syndromic hearing loss. This phenotype has been previously documented in conjunction with palmoplantar hyperkeratosis, but isolated leukonychia is a novel finding likely associated with the unique threonine to alanine change at codon 55 (other variants at this codon have been reported in cases of nonsyndromic hearing loss). This report contributes to the short list of GJB2 variants associated with autosomal dominant hearing loss, highlights the variability of skin and nail findings associated with such cases, and illustrates the occurrence of both syndromic and nonsyndromic presentations with changes in the same gene.     

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.     

New evidence for the correlation of the p.G130V mutation in the GJB2 gene and syndromic hearing loss with palmoplantar keratoderma

The GJB2 gene located on chromosome 13q12 and encoding the connexin 26 (Cx26) protein, a transmembrane protein involved in cell-cell attachment of almost all tissues, including the skin, causes autosomal recessive and sometimes dominant nonsyndromic sensorineural hearing loss. GJB2 mutations have also been identified in syndromic disorders exhibiting hearing loss associated with skin problems. Recently, a new mutation, p.G130V in the GJB2 gene has been reported as causative for Vohwinkel syndrome. In this case the p.G130V mutation was found in two patients (son and father) with palmoplantar keratoderma. The father also showed also skin constrictions of the 2nd and 3rd toes of the right foot. Here, we report on another family with palmoplantar keratoderma associated with a dominant form of hearing loss confirming the genotype-phenotype correlation between the mutation p.G130V and the skin abnormalities observed in syndromic disorders with hearing loss as described by [Snoeckx et al. (2005) Hum Mutat 26:60-65].     

GJB2 Mutations in Mongolia: Complex Alleles,Low Frequency,and Reduced Fitness of the Deaf

We screened the GJB2 gene for mutations in 534 (108 multiplex and 426 simplex) probands with non‐syndromic sensorineural deafness, who were ascertained through the only residential school for the deaf in Mongolia, and in 217 hearing controls. Twenty different alleles, including four novel changes, were identified. Biallelic GJB2 mutations were found in 4.5% of the deaf probands (8.3% in multiplex, 3.5% in simplex). The most common mutations were c.IVS1 + 1G > A (c.‐3201G > A) and c.235delC with allele frequencies of 3.5% and 1.5%, respectively. The c.IVS1 + 1G > A mutation appears to have diverse origins based on associated multiple haplotypes. The p.V27I and p.E114G variants were frequently detected in both deaf probands and hearing controls. The p.E114G variant was always in cis with the p.V27I variant. Although in vitro experiments using Xenopus oocytes have suggested that p.[V27I;E114G] disturbs the gap junction function of Cx26, the equal distribution of this complex allele in both deaf probands and hearing controls makes it a less likely cause of profound congenital deafness. We found a lower frequency of assortative mating (37.5%) and decreased genetic fitness (62%) of the deaf in Mongolia as compared to the western populations, which provides an explanation for lower frequency of GJB2 deafness in Mongolia.     

A novel missense mutation in GJB2 disturbs gap junction protein transport and causes focal palmoplantar keratoderma with deafness

Gap junctions are intercellular channels that mediate rapid intercellular communication. They consist of connexins, small transmembrane proteins that belong to a large family found throughout the animal kingdom. In the skin, several connexins are expressed and are involved in the regulation of epidermal growth and differentiation. One of the skin expressed gap junction genes is GJB2, which codes for connexin 26 and is associated with a wide variety of keratinisation disorders. Here, we report on a family with a novel GJB2 mutation (p.His73Arg) causing a syndrome of focal palmoplantar keratoderma with severe progressive sensorineural hearing impairment, a phenotype reminiscent of Vohwinkel syndrome. Using fluorescent connexin fusion proteins, we show that the mutation induces a transport defect similar to that found for the Vohwinkel syndrome mutation p.Asp66His. Co-transfection into cells expressing wild type connexin26 shows that the mutant has a dominant negative effect on connexin trafficking. We suggest that there may be a weak genotype-phenotype correlation for mutations in GJB2.     

Two Iranian families with a novel mutation in GJB2 causing autosomal dominant nonsyndromic hearing loss

Mutations in GJB2, encoding connexin 26 (Cx26), cause both autosomal dominant and autosomal recessive nonsyndromic hearing loss (ARNSHL) at the DFNA3 and DFNB1 loci, respectively. Most of the over 100 described GJB2 mutations cause ARNSHL. Only a minority has been associated with autosomal dominant hearing loss. In this study, we present two families with autosomal dominant nonsyndromic hearing loss caused by a novel mutation in GJB2 (p.Asp46Asn). Both families were ascertained from the same village in northern Iran consistent with a founder effect. This finding implicates the D46N missense mutation in Cx26 as a common cause of deafness in this part of Iran mandating mutation screening of GJB2 for D46N in all persons with hearing loss who originate from this geographic region.     

Connexin mutations associated with palmoplantar keratoderma and profound deafness in a single family

Recently, mutations in two gap junction genes, GJB2 and GJB3 (encoding Connexin 26 and Connexin 31, respectively), have been shown to underlie either inherited hearing loss and skin disease or both disorders. In this study, we have extended our analysis of a small family in which palmoplantar keratoderma and various forms of deafness is segregating. In addition to the previously described sequence variant M34T in GJB2, two other sequence variants were identified: D66H also in GJB2 and R32W in GJB3. As D66H segregated with the skin disease, it is likely to underlie the palmoplantar keratoderma. The other two gap junction variants identified may contribute to the type of hearing impairment and the variable severity of the skin disease in the family.     

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.     

Deficient membrane integration of the novel p.N14D-GJB2 mutant associated with non-syndromic hearing impairment

Mutations in GJB2, the gene encoding for the Gap Junction protein Connexin 26 (Cx26), have been established as the major cause of hereditary, non-syndromic hearing impairment (HI). We report here the identification of a novel point mutation in GJB2, c.40A>G [p.N14D], detected in compound heterozygosity with the c.35delG mutation in two brothers with moderate non-syndromic sensorineural HI. The mother who carried one wildtype and a p.N14D allele displayed normal hearing. The mutation leads to substitution of the neutral amino acid asparagine (N) by the negatively charged aspartic acid (D) at amino acid number 14, a position that is conserved among Cx26 of different organisms and among many other connexin isoforms. To investigate the impact of this mutation on protein function, Cx26 activity was measured by depolarization activated hemichannel conductance in non-coupled Xenopus laevis oocytes. Oocytes injected with the p.N14D mutant cRNA showed strongly reduced currents compared to wildtype. Coinjection of wildtype and mutant cRNA at equimolar levels restored the conductive properties supporting the recessive character of this mutation. Total Cx26 protein expression and cell surface abundance examined by western blotting and by quantitative immunoassays revealed that the hemichannel was properly synthesized but not integrated into the plasma membrane. In this study we have shown that the GJB2 mutation p.N14D is associated with recessively inherited HI and exhibits a defective phenotype due to diminished expression at the cell surface.     

Double Heterozygosity with Mutations Involving both the GJB2 and GJB6 Genes is a Possible, but very Rare, Cause of Congenital Deafness in the Czech Population

Mutations in the GJB2 gene are the most common cause of prelingual, autosomal recessive, sensorineural hearing loss worldwide. Nevertheless, 10% to 50% of patients with prelingual nonsyndromic deafness only carry one mutation in the GJB2 gene. Recently a large 342 kb deletion named Δ(GJB6‐D13S1830) involving the GJB6 gene was reported in Spanish and French deafness patients, either in a homozygous state or in combination with a monoallelic GJB2 mutation. No data have been reported about the frequency of this mutation in central Europe. Thirteen Czech patients with prelingual nonsyndromic sensorineural deafness carrying only one pathogenic mutation in the GJB2 gene were tested for the presence of the Δ(GJB6‐D13S1830) mutation. One patient with a GJB2 mutation (313del14) also carried the Δ(GJB6‐D13S1830). This is the first reported Czech case, and probably also the first central European case, of prelingual deafness due to mutations involving both the GJB2 and GJB6 genes. In addition, the Δ(GJB6‐D13S1830) was not detected in 600 control chromosomes from Czech individuals with normal hearing. We show that in the Czech Republic the Δ(GJB6‐D13S1830) is not the second most common causal factor in deafness patients heterozygous for a single GJB2 mutation, and that Δ(GJB6‐D13S1830) is very rare in central Europe compared to reports from Spain, France and Israel.     

Recurrence of reported CDH23 mutations causing DFNB12 in a special cohort of South Indian hearing impaired assortative mating families – an evaluation

Mutations in CDH23 are known to cause autosomal‐recessive nonsyndromic hearing loss (DFNB12). Until now, there was only one study describing its frequency in Indian population. We screened for CDH23 mutations to identify prevalent and recurring mutations among South Indian assortative mating hearing‐impaired individuals who were identified as non‐DFNB1 (GJB2 and GJB6). Whole‐exome sequencing was performed in individuals found to be heterozygous for CDH23 to determine whether there was a second pathogenic allele. In our study, 19 variants including 6 pathogenic missense mutations were identified. The allelic frequency of pathogenic mutations accounts to 4.7% in our cohort, which is higher than that reported previously; three mutations (c.429+4G>A, c.2968G>A, and c.5660C>T) reported in the previous Indian study were found to recur. DFNB12 was found to be the etiology in 3.4% of our cohort, with missense mutation c.2968G>A (p.Asp990Asn) being the most prevalent (2.6%). These results suggest a need to investigate the possibility for higher proportion of CDH23 mutations in the South Indian hearing‐impaired population.     

Molecular screening of patients with nonsyndromic hearing loss from Nanjing city of China

Hearing loss is the most frequent sensory disorder involving a multitude of factors,and at least 50% of cases are due to genetic etiology.To further characterize the molecular etiology of hearing loss in the Chinese population,we recruited a total of 135 unrelated patients with nonsyndromic sensorineural hearing loss (NSHL) for mutational screening of GJB2,GJB3,GJB6,SLC26A4,SLC26A5 IVS2-2A>G and mitochondrial 12SrRNA,tRNA Ser(UCN) by PCR amplification and direct DNA sequencing.The carrier frequencies of deafness-causing mutations in these patients were 35.55% in GJB2,3.70% in GJB6,15.56% in SLC26A4 and 8.14% in mitochondrial 12SrRNA,respectively.The results indicate the necessity of genetic screening for mutations of these causative genes in Chinese population with nonsyndromic hearing loss.     

Cx26 deafness: mutation analysis and clinical variability

Mutations in the gap junction protein connexin 26 (Cx26) gene (GJB2) seem to account for many cases of congenital sensorineural hearing impairment, the reported prevalence being 34-50% in autosomal recessive cases and 10-37% in sporadic cases. The hearing impairment in these patients has been described as severe or profound. We have studied 53 unrelated subjects with congenital non-syndromic sensorineural hearing impairment in order to evaluate the prevalence and type of Cx26 mutations and establish better genotype-phenotype correlation. Mutations in the Cx26 gene were found in 53% of the subjects tested, 35.3% of the autosomal recessive and 60% of the sporadic cases in our series. Three new mutations were identified. The hearing deficit varied from mild to profound even in 35delG homozygotes within the same family. No evidence of progression of the impairment was found.Alterations of the Cx26 gene account for a large proportion of cases of congenital non-syndromic sensorineural deafness, so it seems appropriate to extend the molecular analysis even to subjects with mild or moderate prelingual hearing impairment of unknown cause.     

Connexin mutations associated with palmoplantar keratoderma and profound deafness in a single family

Recently, mutations in two gap junction genes, GJB2 and GJB3 (encoding Connexin 26 and Connexin 31, respectively), have been shown to underlie either inherited hearing loss and skin disease or both disorders. In this study, we have extended our analysis of a small family in which palmoplantar keratoderma and various forms of deafness is segregating. In addition to the previously described sequence variant M34T in GJB2, two other sequence variants were identified: D66H also in GJB2 and R32W in GJB3. As D66H segregated with the skin disease, it is likely to underlie the palmoplantar keratoderma. The other two gap junction variants identified may contribute to the type of hearing impairment and the variable severity of the skin disease in the family.     

A connexin 26 mutation causes a syndrome of sensorineural hearing loss and palmoplantar hyperkeratosis (MIM 148350)

We report a missense mutation in the connexin 26 gene (GJB2) in a family with an autosomal dominant syndrome of hearing loss and hyperkeratosis. The affected family members have high frequency, slowly progressive, bilateral, sensorineural hearing loss and palmoplantar hyperkeratosis. The mutation causes an amino acid substitution (G59A), which may disrupt a reverse turn in the first extracellular loop of connexin 26. Connexin 26 mutations have been reported in syndromes of deafness and palmoplantar keratoderma. These data provide additional evidence for the role of connexin 26 in syndromes of this type.     

Mutation analysis of the GJB2 (connexin 26) gene in Egypt

Fifty to eighty percent of autosomal recessive deafness is due to mutations in the GJB2 gene encoding connexin 26. Among Caucasians, the c.35delG mutation in this gene accounts for up to 30 to 70% of all cases with early childhood deafness. In this study, we present the analysis of the GJB2 gene in 159 Egyptians from 111 families with non-syndromic mild to profound hearing impairment. An additional family with Vohwinkel syndrome, a combination of hearing impairment and palmoplantar keratoderma with constriction of the digits, was also included. We used direct sequencing analysis to detect all possible coding GJB2 variants in this population. The presence of the g.1777179_2085947del mutation (hereafter called del(GJB6-D13S1830)) was also investigated as it was shown to be the second most common mutation causing non-syndromic prelingual hearing impairment in Spain. Sequencing analysis of one randomly chosen individual per family revealed that the c.35delG mutation was present in 24 out of 222 chromosomes (10.8%), making it the most frequent mutation in the GJB2 gene in Egypt. Five other mutations were already described previously [p.Thr8Met, p.Val37Ile, p.Val153Ile, c.333_334delAA, c.1-3172G>A (commonly designated as IVS1+1G>A)]. This study also revealed three other novel gene variants resulting in amino acid substitutions (p.Phe142del, p.Asp117His, p.Ala148Pro). In contrast with most populations, the del(GJB6-D13S1830) mutation upstream of the GJB2 gene was not present in this Egyptian population. A dominant mutation at a highly conserved residue, p.Gly130Val, was found in the family with Vohwinkel syndrome.     

Different functional consequences of two missense mutations in the GJB2 gene associated with non‐syndromic hearing loss

Mutations in the GJB2 gene, which encodes the gap junction (GJ) protein connexin26 (Cx26), are the most common cause of inherited non‐syndromic hearing loss (NSHL). We identified two missense mutations, p.D46E (c.138T>G) and p.T86R (c.257C>G), of GJB2 in Korean HL families. The novel p.D46E mutation exhibited autosomal dominant inheritance, while the p.T86R mutation, which is exclusively found in Asians, segregated with an autosomal recessive pattern. Thus, we sought to elucidate the pathogenic nature of such different inherited patterns of HL. We studied protein localization and gap junction functions in cells transfected with wild‐type or mutant Cx26 tagged with fluorescent proteins, which allowed visual confirmation of homozygous or heterozygous mutant GJs. The Cx26‐D46E mutant was targeted to the plasma membrane, but this mutant protein failed to transfer Ca²⁺ or propidium iodide intercellularly, suggesting disruption of both ionic and biochemical coupling. Heterozygous GJs also showed dysfunctional intercellular couplings and hemichannel opening, confirming the dominant‐negative nature of the p.D46E mutation. The Cx26‐T86R mutant protein did not form GJs, since the mutated protein was confined in the cytoplasm and not transported to the cell membrane. When Cx26‐T86R was co‐expressed with Cx26‐WT, ionic and biochemical coupling was normal, consistent with the recessive nature of the mutation. These studies revealed distinct pathogenic mechanisms of two GJB2 mutations identified in Korean families. © 2009 Wiley‐Liss, Inc.     

Sensorineural hearing loss and the incidence of Cx26 mutations in Austria

A clinical evaluation and Cx26 mutation analysis was performed in 92 consecutive patients with sensorineural hearing loss in order to delineate the spectrum of genetically caused hearing loss. Among patients of Austrian origin, 53% were classified with hereditary hearing loss. Cx26 mutations were found in 26% of NSHL patients (40% of familial vs 18% of sporadic cases). The mutation 35delG accounted for 52.8% of all presumed GJB2 disease alleles. The second most frequent mutation was L90P (16.7%) having been reported with a prevalence of 0.7-3.5% in other populations. Three novel mutations were found. The novel mutation, R143Q, was associated with dominant high-frequency hearing loss. Pseudodominant transmission of NSHL was seen in four families with Cx26 mutations. A mutation 35delG carrier rate of 0.9% was observed among 672 controls from West-Austria. Cx26 mutations were found associated with mild to profound, and with asymmetric hearing impairment.     

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.