W44C mutation in the connexin 26 gene associated with dominant non-syndromic deafness

Although more than 50% of recessive non-syndromic deafness is attributed to mutations in the connexin 26 (Cx26) gene, only a few reported families have shown dominant transmission of the trait. The W44C mutation was originally reported in two families from the same geographic region of France, which exhibited dominant non-syndromic hearing loss. In this report, we describe a third family with early-onset severe-to-profound non-syndromic hearing loss segregating with the W44C mutation. Our observation places W44C among recurrent mutations in the Cx26 gene and emphasizes the importance of screening for this as well as other Cx26 mutations in autosomal dominant families.     

Mutation R184Q of connexin 26 in hearing loss patients has a dominant-negative effect on connexin 26 and connexin 30

Hearing impairment is the most common sensory disorder worldwide. In a recent study, the authors have shown that a heterozygous missense mutation, p.R184Q, in the connexin 26 (Cx26) is causally related to hearing loss. However, the functional change in the Cx26R184Q mutant remains unknown. This study compared the intracellular distribution and assembly of mutant Cx26R184Q with that of the wild-type (WT) Cx26 and Cx30WT in tet-on HeLa cells and the effect that the mutant protein had on those cells. Fluorescent localization assay of WT Cx26 showed the typical punctuate pattern of gap junction channel between neighboring expression cells. Conversely, the p.R184Q missense mutation resulted in accumulation of the Cx26 mutant protein in the Golgi apparatus rather than in the cytoplasmic membrane. Cx26R184Q coexpressed with either Cx26WT or Cx30WT showed perinuclear localization by bidirectional tet-on expression system, suggesting the impairment of the ability of both WT proteins to intracellular trafficking and targeting to the plasma membrane. Therefore, we proposed that Cx26R184Q has a dominant-negative effect on the function of WT Cx26 and Cx30.     

Postnatal development of the organ of Corti in dominant-negative Gjb2 transgenic mice

Hereditary hearing loss is one of the most prevalent inherited human birth defects, affecting one in 2000. A strikingly high proportion (50%) of congenital bilateral nonsyndromic sensorineural deafness cases have been linked to mutations in the GJB2 coding for the connexin26. It has been hypothesized that gap junctions in the cochlea, especially connexin26, provide an intercellular passage by which K(+) are transported to maintain high levels of the endocochlear potential essential for sensory hair cell excitation. We previously reported the generation of a mouse model carrying human connexin26 with R75W mutation (R75W+ mice). The present study attempted to evaluate postnatal development of the organ of Corti in the R75W+ mice. R75W+ mice have never shown auditory brainstem response waveforms throughout postnatal development, indicating the disturbance of auditory organ development. Histological observations at postnatal days (P) 5-14 were characterized by i) absence of tunnel of Corti, Nuel's space, or spaces surrounding the outer hair cells, ii) significantly small numbers of microtubules in inner pillar cells, iii) shortening of height of the organ of Corti, and iv) increase of the cross-sectional area of the cells of the organ of Corti. Thus, morphological observations confirmed that a dominant-negative Gjb2 mutation showed incomplete development of the cochlear supporting cells. On the other hand, the development of the sensory hair cells, at least from P5 to P12, was not affected. The present study suggests that Gjb2 is indispensable in the postnatal development of the organ of Corti and normal hearing.     

连接蛋白基因一个新致聋突变体p.Y155X及功能分析

目的 观察连接蛋白(connexin 26,CX26)基因的一个新致聋突变c.465T→A,P.Y155X,在体外表达细胞功能的改变,以探讨其致聋的可能机理.方法 常染色体隐性遗传耳聋家系的先证者外周血抽提DNA,DNA直接测序法分析CX26基因突变.将在该家系发现的突变c.465T→A,P.Y155X和野生型CX26(wtCX26)定向克隆到pEGFP-N1质粒,构建CX26 p.Y155X-EGFP及wtCX26-EGFP融合蛋白表达载体,转染HeLa细胞,Western印迹分析蛋白的表达,共聚焦显微镜观察突变蛋白和野生型CX26在HeLa细胞的定位及有无间隙连接斑形成,染料转移实验分析间隙连接的功能.结果 在该耳聋家系发现CX26基因一个新的致聋突变:c.465T→A,P.Y155X.CX26 P.Y155X突变体在HeLa细胞表达的突变蛋白的分子量小于野生型蛋白分子量;突变蛋白在细胞质表达,不能分布到细胞膜和形成间隙连接,无染料转移.野生型表达于细胞膜并形成间隙连接斑,能转移染料.结论 CX26 P.Y155X突变体在翻译后不能从细胞内转运到细胞膜,不能形成间隙连接通道.CX26基因c.465T→A,P.Y155X导致常染色体隐性遗传性聋.     

Two different connexin 26 mutations in an inbred kindred segregating non-syndromic recessive deafness: implications for genetic studies in isolated populations

Non-syndromic recessive deafness (NSRD) is the most common form of prelingual hereditary hearing loss. To date, 10 autosomal NSRD loci (DFNBs) have been identified by genetic mapping; at least three times as many additional loci are expected to be identified. We have performed linkage analyses in two inter-related inbred kindreds, comprised of >50 affecteds, from a single Israeli-Arab village segregating NSRD. Genetic mapping by two-point and multi-point linkage analysis in 10 candidate regions identified the segregating gene to be on human chromosome 13q11 (DFNB1). Haplotype analysis, using eight microsatellite markers spanning 15 cM in 13q11, suggested the segregation of two different mutations in this kindred: affected individuals were homozygotes for either haplotype or compound heterozygotes. The gene for the connexin 26 gap junction protein, recently shown to be mutant in both dominant and recessive deafness, maps to this locus. We identified two distinct mutations, W77R and Gdel35, both of which likely inactivate connexin 26. The Gdel35 change likely occurs at a mutational hotspot within the connexin 26 gene. The recombination of marker alleles at the polymorphisms studied in 13q11, at known map distances from the mutations, allowed us to estimate the age of the mutations to be 3-5 generations (75-125 years). This study independently confirms the identity of connexin 26 as an NSRD gene. Importantly, we demonstrate that in small populations with high rates of consanguinity, as compared with large outbred populations, recessive mutations may have very recent origin and show allelic diversity.      

Prelingual deafness: high prevalence of a 30delG mutation in the connexin 26 gene

Prelingual non-syndromic (isolated) deafness is the most frequent hereditary sensory defect. In >80% of the cases, the mode of transmission is autosomal recessive. To date, 14 loci have been identified for the recessive forms (DFNB loci). For two of them, DFNB1 and DFNB2, the genes responsible have been characterized; they encode connexin 26 and myosin VIIA, respectively. In order to evaluate the extent to which the connexin 26 gene (Cx26) contributes to prelingual deafness, we searched for mutations in this gene in 65 affected Caucasian families originating from various countries, mainly tunisia, France, New Zealand and the UK. Six of these families are consanguineous, and deafness was shown to be linked to the DFNB1 locus, 10 are small non consanguineous families in which the segregation of the trait has been found to be compatible with the involvement of DFNB1, and in the remaining 49 families no linkage analysis has been performed. A total of 62 mutant alleles in 39 families were identified. Therefore, mutations in Cx26 represent a major cause of recessively inherited prelingual deafness since according to the present results they would underlie approximately half of the cases. In addition, one specific mutation, 30delG, accounts for the majority (approximately 70%) of the Cx26 mutant alleles. It is therefore one of the most frequent disease mutations so far identified. Several lines of evidence indicate that the high prevalence of the 30delG mutation arises from a mutation hot spot rather than from a founder effect. Genetic counseling for prelingual deafness has been so far considerably impaired by the difficulty in distinguishing genetic and non genetic deafness in families presenting with a single deaf child. Based on the results presented here, the development of a simple molecular test could be designed which should be of considerable help.      

Molecular interaction of connexin 30.3 and connexin 31 suggests a dominant-negative mechanism associated with erythrokeratodermia variabilis

Connexins are homologous four-transmembrane-domain proteins and major components of gap junctions. We recently identified mutations in either GJB3 or GJB4 genes, encoding respectively connexin 31 (Cx31) or 30.3 (Cx30.3), as causally involved in erythrokeratodermia variabilis (EKV), a mostly autosomal dominant disorder of keratinization. Despite slight differences, phenotypes of EKV Mendes Da Costa (Cx31) and EKV Cram-Mevorah (Cx30.3) show major clinical overlap and both Cx30.3 and Cx31 are expressed in the upper epidermal layers. These similarities suggested to us that Cx30.3 and Cx31 may interact at a molecular level. Indeed, expression of wild-type Cx30.3 in HeLa cell resulted only in minor amounts of protein addressed to the plasma membrane. Mutant Cx30.3 was hardly detectable and disturbed intercellular coupling. In sharp contrast, co-expression of both wild-type proteins led to a gigantic increase of stabilized heteromeric gap junctions. Furthermore, co-expressed wild-type Cx30.3 and Cx31 coprecipitate, which demonstrates a physical interaction. Inhibitor experiments revealed that this interaction begins in the endoplasmic reticulum. These results not only provide new insights into epidermal connexin synthesis and polymerization, but also allow a novel molecular explanation for the similarity of EKV phenotypes.     

Assessment of the genetic causes of recessive childhood non-syndromic deafness in the UK - implications for genetic testing

Approximately one in 2000 children is born with a genetic hearing impairment, mostly inherited as a non-syndromic, autosomal recessive trait, for which more than 30 different genes have been identified. Previous studies have shown that one of these genes, connexin 26 (GJB2), accounts for 30-60% of such deafness, but the relative contribution of the many other genes is not known, especially in the outbred UK population. This lack of knowledge hampers the development of diagnostic genetic services for deafness. In an effort to determine the molecular aetiology of deafness in the population, 142 sib pairs with early-onset, non-syndromic hearing impairment were recruited. Those in whom deafness could not be attributed to GJB2 mutations were investigated further for other mapped genes. The genetic basis of 55 cases (38.7%) was established, 33.1% being due to mutations in the GJB2 gene and 3.5% due to mutations in SLC26A4. None of the remaining 26 loci investigated made a significant contribution to deafness in a Caucasian population. We suggest that screening the GJB2 and SLC26A4 genes should form the basis of any genetic testing programme for childhood deafness and highlight a number of important issues for consideration and future work.     

An alpha-tectorin gene defect causes a newly identified autosomal recessive form of sensorineural pre-lingual non-syndromic deafness, DFNB21

In our efforts to identify new loci responsible for non-syndromic autosomal recessive forms of deafness, DFNB loci, we have pursued the analysis of large consanguineous affected families living in geographically isolated areas. Here, we report on the study of a Lebanese family comprising nine members presenting with a pre-lingual severe to profound sensorineural isolated form of deafness. Linkage analysis led to the characterization of a new locus, DFNB21, which was assigned to chromosome 11q23-25. Already mapped to this chromosomal region was TECTA. This gene encodes alpha-tectorin, a 2155 amino acid protein which is a component of the tectorial membrane. This gene recently has been shown to be responsible for a dominant form of deafness, DFNA8/12. Sequence analysis of the TECTA gene in the DFNB21- affected family revealed a G to A transition in the donor splice site (GT) of intron 9, predicted to lead to a truncated protein of 971 amino acids. This establishes that alpha-tectorin mutations can be responsible for both dominant and recessive forms of deafness. Comparison of the phenotype of the DFNB21 heterozygous carriers with that of DFNA8/12-affected individuals supports the hypothesis that the TECTA mutations which cause the dominant form of deafness have a dominant-negative effect. The present results provide genetic evidence for alpha-tectorin forming homo- or heteromeric structures.      

LRTOMT: a new tone in understanding the symphony of non-syndromic deafness

Mutations of LRTOMT, a fusion gene with alternative reading frames, cause nonsyndromic deafness in humans
Ahmed et al. (2008)
Nature Genetics 40: 1335–1340.     

Postnatal development of the rat organ of Corti

Summary The development of the rat organ of Corti was studied during the first postnatal weeks. The temporal and the spatial patterns of cochlear development were investigated between 4 and 24 days after birth by means of semi-thin sections at approx. ten equidistant positions along the entire cochlear duct. At all examined positions width, thickness and cross sectional area of basilar membrane, cross-sectional area of tectorial membrane, of cells of Hensen, Claudius and Boettcher and of the organ of Corti were quantitatively analyzed. The most conspicuous maturational changes occur between 8 and 12 days after birth. These are the detachment of the tectorial membrane, the first appearance of filaments within the basilar membrane, the formation of the tunnel of Corti and the opening of the inner spiral sulcus. Quantitative analysis revealed that structures of a given position along the cochlear duct do not develop synchronously. Width of the basilar membrane and cross-sectional area of the tectorial membrane are already mature at the onset of hearing (10–12 days after birth). Length, thickness and cross-sectional area of the basilar membrane as well as cross-sectional area of the organ of Corti and of the cells of Hensen, Claudius and Boettcher still develop after the onset of hearing (up to 20–24 days after birth). We suggest that basic cochlear function is established by structures which are mature before the onset of hearing. Cochlear structures which develop after the onset of hearing might be involved in this improvement during this period.     

Linkage of a gene for dominant non-syndromic deafness to chromosome 19

Inherited hearing impairment can occur either in the presenceof other clinical features (syndromic hearing loss, SHL) orin isolation (non-syndromic hearing loss, NSHL). The latteris more common and is highly heterogeneous. To date, six NSHLloci have been mapped. We report the identification of a seventhlocus (DFNA4) on chromosome 19q13 and suggest DM kinase as apossible candidate gene.     

Properties of connexin26 gap junctional proteins derived from mutations associated with non-syndromal heriditary deafness.

Three point mutations of the connexin26 (GJB2) gene associated with hereditary deafness were studied using in vitro expression systems. Mutation M34T results in an amino acid substitution in the first transmembrane domain of the connexin protein, W77R is located in the second transmembrane domain and W44C is in the first extracellular loop. Wild-type and mutated connexin vectors were constructed and transfected into communication-deficient HeLa cells to obtain transient expression of the connexin proteins. Intercellular coupling was subsequently assessed by examining transfer of Lucifer yellow between cells. All three mutations resulted in impaired intercellular coupling. The mechanistic reasons for the functional inadequacies of the mutated proteins were investigated. First, intracellular trafficking and targeting of the expressed connexins were determined by immunohistochemistry. Mutation W77R was inefficiently targeted to the plasma membrane and retained in intracellular stores whereas the other two were targeted to the plasma membrane. Oligomerization assays showed that connexins M34T and W77R failed to assemble efficiently into hexameric gap junction hemichannels, but the W44C mutation did so. A cell-free translation system showed that the mutated proteins were inserted into microsomal membranes but the mutations have different effects on the post-translational properties of the expressed proteins. The results point to the conclusion that mutations in the transmembrane domains of connexin proteins influence gap junction assembly.     

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.     

Cochlear outer hair cells in a dominant-negative connexin26 mutant mouse preserve non-linear capacitance in spite of impaired distortion product otoacoustic emission

Mutations in the connexin26 gene (GJB2) are the most common genetic cause of congenital bilateral non-syndromic sensorineural hearing loss. Transgenic mice were established carrying human Cx26 with the R75W mutation that was identified in a deaf family with autosomal dominant negative inheritance [Kudo T et al. (2003) Hum Mol Genet 12:995–1004]. A dominant-negative Gjb2 R75W transgenic mouse model shows incomplete development of the cochlear supporting cells, resulting in profound deafness from birth [Inoshita A et al. (2008) Neuroscience 156:1039–1047]. The Cx26 defect in the Gjb2 R75W transgenic mouse is restricted to the supporting cells; it is unclear why the auditory response is severely disturbed in spite of the presence of outer hair cells (OHCs). The present study was designed to evaluate developmental changes in the in vivo and in vitro function of the OHC, and the fine structure of the OHC and adjacent supporting cells in the R75W transgenic mouse. No detectable distortion product otoacoustic emissions were observed at any frequencies in R75W transgenic mice throughout development. A characteristic phenotype observed in these mice was the absence of the tunnel of Corti, Nuel's space, and spaces surrounding the OHC; the OHC were compressed and squeezed by the surrounding supporting cells. On the other hand, the OHC developed normally. Structural features of the lateral wall, such as the membrane-bound subsurface cisterna beneath the plasma membrane, were intact. Prestin, the voltage-dependent motor protein, was observed by immunohistochemistry in the OHC basolateral membranes of both transgenic and non-transgenic mice. No significant differences in electromotility of isolated OHCs during development was observed between transgenic and control mice. The present study indicates that normal development of the supporting cells is indispensable for proper cellular function of the OHC.