Changes in the connexin 26 gene (<Emphasis Type="Italic">GJB2</Emphasis>) in Russian patients with hearing loss: Results of long-term molecular diagnostics of hereditary nonsyndromic hearing loss

Molecular testing for mutations in the connexin 26 gene (GJB2) is a routine diagnostic analysis for subjects with hereditary hearing loss worldwide. However, till now there is no assessment of the diagnostic significance of this analysis for Russian patients, and there are difficulties in interpretation of the results of DNA diagnostics. In the present study, a sample of 705 patients with nonsyndromic autosomal recessive hearing loss from different regions of Russian Federation was investigated. A portion of DFNB1 hearing loss caused by mutations in the GJB2 gene among the sample was 46%. The frequency of DFNB1 hearing loss was 1:1000, that is, the frequency of isolated autosomal recessive hearing loss 1:500 in the population. It was found that each sixteenth individual in Russia is a heterozygous carrier of the mutation in the GJB2 gene. Totally, 20 pathological GJB2 alleles were detected; among them, a c.35delG mutation with the allelic frequency 81% prevails. Six most frequent mutations (c.35delG, c.313_326del14, c.23+1G>A (IVS1+1G>A), c.235delC, c.167delT, and p.Glu120del), which account for 95% of pathological GJB2 alleles, were detected. Mutations previously not described in the GJB2 gene (c.129delG, p.Gly200Arg, and c[Arg127His, Gly160Ser]) were found. An optimal algorithm of molecular testing of Russian patients which detects up to 100% of mutations in the GJB2 gene was suggested. Data concerning a clinical significance of p.Met34Thr and p.Val37Ile mutations are confirmed in the study. Eight polymorphic substitutions in the GJB2 gene which do not have clinical significance (p.Val27Ile, c.*3C>A, p.Val153Ile, p.Gly160Ser, c.Arg127His, p.Glu114Gly (c.341A>G), c.-45C>A, and p.Ala149Thr) were also detected.     

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 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.     

Evaluation of dHPLC for CX26 mutation screening in patients from southern France with sensorineural deafness

The GJB2 gene (or CX26 for connexin 26) is one of the major genes causing nonsyndromic sensorineural hearing loss (NSSNHL). More than 50 sequence variations have been identified as polymorphisms or associated with autosomal or recessive forms of deafness. Though a major mutation, 35delG, is easily detectable by PCR digest; it is often present in the compound heterozygous state in our population in trans with recurrent, but less frequent, mutations. The CX26 gene is composed of a single coding exon that facilitates sequencing strategies. However, for mutation screening purposes, it is necessary to use high-throughput and cost-effective genotyping methods. Therefore, we have assessed denaturing high-performance liquid chromatography (dHPLC) in patients with known mutations in the CX26 gene. We conclude that dHPLC analysis is suitable for rapid and reliable scanning of the gene in deaf patients.     

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.     

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.     

<Emphasis Type="Italic">GJB2</Emphasis> and <Emphasis Type="Italic">GJB6</Emphasis> gene mutations found in Indian probands with congenital hearing impairment

Genetically caused deafness is a common trait affecting one in 1000 children and is predominantly inherited in an autosomal-recessive fashion. Several mutations in the GJB2 gene and a deletion of 342 kb in GJB6 gene (delGJB6-D13S1830) have been identified worldwide in patients with hearing impairment. In the present study, 303 nonsyndromic hearing-impaired patients (140 familial; 163 sporadic) were examined clinically and screened for mutations in GJB2 and GJB6 genes. Mutations in GJB2 gene were found in 33 (10.9%) patients of whom six (18.2%) were carriers for the mutant allele. The most frequent mutation was p.W24X accounting for 87% of the mutant alleles. In addition, six other sequence variations were identified in the GJB2 gene viz., c.IVS1+1G>A, c.167delT, c.235delC, p.W77X, p.R127H (polymorphism), p.M163V. None of the samples showed del(GJB6-D13S1830) or any point mutations in GJB6 gene.     

Changes in the connexin 26 (GJB2) gene in Russian patients with hearing disorders: results of long-term molecular diagnostics of hereditary nonsyndromic deafness

Search for mutations in the connexin 26 gene (GJB2) is a routine molecular-genetic analysis ofthe hereditary deafness worldwide. However, till now there is no assessment of the diagnostic significance of this analysis for Russian patients, and there are difficulties in interpretation of the results of DNA diagnostics. In the present study, a sample of 705 patients with nonsyndromic autosomal recessive deafness from different regions of Russian Federation was investigated. A portion of deafness like DFNB1 caused by mutations in the GJB2 gene among the sample was 46%. The frequency of deafness of such genetic type was 1:1000, that is, the frequency of isolated autosomal recessive deafness was 1:500 in the population. It was found that each sixteenth individual in Russia is a heterozygous carrier of the mutation in the GJB2gene. Totally, 20 pathological GJB2 alleles were detected; among them, a c.35delG mutation with the allelic frequency 81% prevails. Six most frequent mutations (c.35delG, c.313_326de114, c.-23+1G>A (IVS1+1G>A), c.235delC, c.167delT, and p.Glul20del), which account for 95% of pathological GJB2 alleles, were detected. Mutations previously not described in the GJB2 gene (c.129delG, p.Gly200Arg, and c[Arg127His, Gly160Ser]) were found. An optimal algorithm of molecular investigation of Russian patients which detects up to 100% of mutations in the GJB2 gene was suggested. Data concerning a clinical significance of p.Met34Thr and p.Va137Ile mutations are confirmed in the study. Eight polymorphic substitutions in the GJB2gene which do not have clinical significance (p.Va127Ile, c.*3C>A, p.Va115311e, p.Gly160Ser, c.Arg127His, p.Glull4Gly (c.341A>G), c.-45C>A, and p.Ala149Thr) were also detected.     

Spectrum of the GJB2 mutations in Belarussian patients with hearing loss. Findings of pilot genetic screening of hearing impairment in newborns

A total of 111 unrelated probands and their 8 sibs from Grodno oblast (Belarus) with bilateral isolated sensorineural hearing impairment were studied for the presence of mutations in the connexin 26 (GJB2) gene. Mutations were detected in 51 probands (46% of the sample). A significantly higher frequency of the GJB2 gene mutations was observed in familial cases of the disease with the autosomal recessive mode of inheritance (in 78% of families). Detected characteristics of the GJB2 gene mutation spectrum demonstrated that the using the algorithm, which was designed for Russian patients, is optimal for the molecular study of patients from Belarus. In the sample of patients with hearing loss, the highest (among other similar samples studied in the world) allele frequency of c.313_326del14 mutation (7% of all pathological GJB2 alleles) was registered; Polish origin of this deletion was suggested. It was demonstrated that detection of the GJB2 gene mutation on one patient’s chromosome only is insufficient to confirm a molecular genetic diagnosis of hearing loss of the DFNB1 genetic type (autosomal recessive hearing loss caused by the GJB2 gene mutations). Pilot screening for the GJB2 gene mutations in newborns from Grodno oblast was performed. The material from 235 children was studied during the screening; nine heterozygous carriers of the mutation were found. The c.35delG mutation was detected in a homozygous state in a single newborn (hearing loss of moderate severity was subsequently audiologically confirmed in this child).     

The c.242G>A mutation in LRTOMT gene is responsible for a high prevalence of deafness in the Moroccan population

Congenital hearing impairment (HI) affects one in 1,000 newborns and has a genetic cause in 50?% of the cases. Autosomal recessive non-syndromic hearing impairment is responsible for 70–80?% of all hereditary cases of HI. Recently, it has been demonstrated that, mutations of LRTOMT are associated with profound nonsyndromic hearing impairment at the DFNB63 locus. The objective of this study is to evaluate the carrier frequency of c.242G>A mutation in LRTOMT gene and define the contribution of this gene in the etiology of deafness in Moroccan population. We screened 105 unrelated Moroccan families with non-syndromic HI and 120 control individuals for mutation in the exon 8 of the LRTOMT gene, by sequencing and PCR-RFLP. The Homozygous c.242G>A mutation was found in 8.75?% of the families tested and in 4.16?% of control in the heterozygous state. Our results show that after the GJB2 gene mutation in LRTOMT gene is the second cause of congenital hearing impairment in Moroccan patients. This finding should facilitate diagnosis of congenital deafness of the affected subjects in Morocco.     

Functional defects of Cx26 resulting from a heterozygous missense mutation in a family with dominant deaf-mutism and palmoplantar keratoderma

Mutations in GJB2 encoding the gap junction protein connexin-26 (Cx26) have been established as the basis of autosomal recessive non-syndromic hearing loss. The involvement of GJB2 in autosomal dominant deafness has also been proposed, although the putative mutation identified in one family with both deafness and palmoplantar keratoderma has recently been suggested to be merely a non-disease associated polymorphism. We have observed a similar phenotype in an Egyptian family that segregated with a heterozygous missense mutation of GJB2, leading to a non-conservative amino acid substitution (R75W). The deleterious dominant-negative effect of R75W on gap channel function was subsequently demonstrated in the paired oocyte expression system. Not only was R75W alone incapable of inducing electrical conductance between adjacent cells, but it almost completely suppressed the activity of co-expressed wildtype protein. The Cx26 mutant W77R, which has been implicated in autosomal recessive deafness, also failed to form functional gap channels by itself but did not significantly interfere with the function of wildtype Cx26. These data provide compelling evidence for the serious functional consequences of Cx26 mutations in dominant and recessive deafness. Received: 22 June 1998 / Accepted: 15 July 1998     

<Emphasis Type="Italic">GJB2</Emphasis> deafness gene shows a specific spectrum of mutations in Japan,including a frequent founder mutation

Mutations in the GJB2 gene (connexin 26) are the major cause of autosomal recessive non-syndromic hearing impairment in many populations. In contrast to the volume of information regarding the involvement of GJB2 mutations in hearing impairment in populations of European ancestry, less is known regarding other ethnic groups. In this study, we analyzed the GJB2 gene for mutations in 1227 hearing-impaired Japanese individuals. This revealed a unique spectrum of GJB2 mutations, different from that found in the Caucasian population. The most frequent mutation in Japanese, 235delC, has never been reported in Caucasians. To investigate a possible founder effect for the 235delC mutation, we analyzed single nucleotide polymorphisms in the vicinity of the GJB2 gene. Results were consistent with inheritance of the 235delC mutation from a common ancestor. The results of this study have important implications for genetic diagnostic testing for deafness in the Japanese population.     

Absence of mutations in GJB2 (Connexin-26) gene in an ethnic group of southwest Iran

BACKGROUND:

The common GJB2 gene mutation (35delG) has been previously reported from Iranian patients that were affected with nonsyndromic autosomal recessive deafness. We, therefore, for the first time, investigated the prevalence and frequency of the GJB2 gene mutation in the Iranian deaf population with Arabian origins.

MATERIALS AND METHODs:

We amplified and sequenced the entire coding sequence of the GJB2 gene from 61 deaf patients and 26 control subjects.

RESULT:

None of the analyzed samples revealed deafness-associated mutation.

CONCLUSION:

This finding differs from several reports from Iran as we have focused on the GJB2 gene that possesses various mutations as the cause of congenital recessive deafness.     

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.     

Marital Structure,Genetic Fitness,and the <Emphasis Type="Italic">GJB2</Emphasis> Gene Mutations among Deaf People in Yakutia (Eastern Siberia,Russia)

Autosomal recessive deafness type 1A (DFNB1A) caused by mutations in the GJB2 gene (Cx26) is the main cause of nonsyndromic hearing impairment in many populations worldwide. It is considered that widespread prevalence of DFNB1A can be due to the long tradition of intermarriages between deaf people (assortative marriages) combined with their increased social adaptation and genetic fitness after widespread introduction of sign language. For the first time, the data on mating structure and reproduction of deaf people living in Yakutia (Eastern Siberia, Russia) are presented in comparison with contribution of the GJB2 gene mutations to the etiology of hearing impairment. The relative fertility of deaf people compared to their hearing siblings is 0.78 (mean number of children 1.76 ± 0.10 and 2.24 ± 0.09 to deaf and their hearing siblings, respectively, p = 0.0018). The rate of assortative marriages among deaf people is 77.1% (81 of 105 marriages). Biallelic mutations in the GJB2 gene were found in 42.2% (43 of 102) of examined deaf people, which corresponded to diagnosis DFNB1A for these patients. A comparison of deaf marital partners by GJB2 status revealed a proportion of noncomplementary marriages (24%) in which hearing loss in both partners was caused by the presence of biallelic GJB2 gene mutations resulting in the birth of only deaf children in such couples. Thus, the set of obtained data including a relatively high genetic fitness (expressed as relative fertility) of deaf people in Yakutia in combination with a high rate of assortative marriages among them and high incidence of DFNB1A indicates a possible weakening of selection against such trait as “deafness” and a possible increase in the frequency of GJB2 mutant alleles in subsequent generations.     

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.     

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.     

The Relationship between the p.V37I Mutation in GJB2 and Hearing Phenotypes in Chinese Individuals

The most common cause of nonsyndromic autosomal recessive hearing loss is mutations in GJB2. The mutation spectrum and prevalence of mutations vary significantly among ethnic groups, and the relationship between p.V37I mutation in GJB2 and the hearing phenotype is controversial. Among the 3,864 patients in this study, 106 (2.74%) had a homozygous p.V37I variation or a compound p.V37I plus other GJB2 pathogenic mutation, a frequency that was significantly higher than that in the control group (600 individuals, 0%). The hearing loss phenotype ranged from mild to profound in all patients with the homozygous p.V37I variation or compound p.V37I plus other GJB2 pathogenic mutation. There was no difference in the distribution of the hearing level in the group with the homozygous p.V37I variation and the group with the compound p.V37I variation plus pathogenic mutation. Most patients (66.04%) with the V37I-homozygous variation or p.V37I plus other pathogenic mutation had a mild or moderate hearing level. This study found a definite relationship between p.V37I and deafness, and most patients who carried the pathogenic combination with p.V37I mutation had mild or moderate hearing loss. Therefore, otolaryngologists should consider that the milder phenotype might be caused by the GJB2 p.V37I mutation.     

Contribution of GJB2 Mutations to Hearing Loss in the Hazara Division of Pakistan

Mutations of GJB2, which encodes connexin 26, are the most common cause of hereditary hearing loss in many human populations. This study was initiated to determine the prevalence of GJB2 mutations in individuals with hearing loss from the Hazara Division in Pakistan. We recruited 70 participants with nonsyndromic deafness segregating as an apparently recessive trait and directly sequenced the GJB2 coding region from their DNA. The homozygous mutations c.71 G→A (p.W24X), c.104 T→G (p.I35S), and c.35delG (p.G12VfsX1) were identified as the cause of hearing loss in three participants (4.28%); in populations from other areas of Pakistan, frequencies of 6–7% have been observed. The mutations c.104 T→G and c.35delG were identified in Pakistan for the first time. These results confirm the low prevalence of GJB2 mutations in Hazara and suggest that mutations in other genes may play a significant role in the etiology of deafness in this 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.