Frequencies of GJB2 mutations in German control individuals and patients showing sporadic non-syndromic hearing impairment

Mutations in the GJB2 gene encoding the gap-junction protein connexin 26 have been identified in many patients with childhood hearing impairment (HI). One single mutation, 35delG (30delG), accounts for up to 70% of all analyzed European patients with autosomal recessive inherited HI and 10% of patients with HI of unknown origin, respectively. We screened 188 control individuals and 342 German patients with non-syndromic sporadic HI for the 35delG, compound heterozygosity and other GJB2 mutations by PCR, restriction enzyme based screening, SSCP and sequencing. In all patients, non-progressive hearing impairment varied from moderate to profound involving all frequencies. This study revealed one novel silent mutation (438C/T), three novel gene variants resulting in amino acid substitutions (K112E, T123S, K223R) and two novel HI-related mutations (I82M, 313del14).     

Screening of families with autosomal recessive non-syndromic hearing impairment (ARNSHI) for mutations in GJB2 gene: Indian scenario

Several studies have reported that mutations in the GJB2 gene (coding for connexin26) are a common cause of recessive non-syndromic hearing impairment. A GJB2 mutant allele, 35delG, has been found to have a high prevalence in most ethnic groups. Though mutations in the GJB2 gene have been shown to cause autosomal recessive deafness in Indian families, the frequencies of the various mutations are still unknown. In the present study, we analyzed 45 Indian families belonging to three different states, namely, Karnataka, Tamil Nadu, and Delhi with non-syndromic hearing impairment and an apparently autosomal recessive mode of inheritance. All the families were initially screened for three mutations (W24X, W77X, and Q124X) by using allele-specific PCR primers; mutations were confirmed by DNA sequencing. Families that were heterozygous or negative for tested mutations of the GJB2 gene were sequenced directly to identify the complementary mutation and other mutations in GJB2. Four families were homozygous for W24X, constituting around 8.8%. In two families, the affected individuals were compound heterozygotes for W24X; one family (DKB16) carried 35delG with W24X while the other family (DKB7) carried R143W with W24X. We suggest that W24X is a common allele among the mutations screened, causing autosomal recessive non-syndromic hearing impairment (ARNSHI) in the Indian population.     

Genetic testing for hereditary hearing loss: connexin 26 (GJB2) allele variants and two novel deafness-causing mutations (R32C and 645-648delTAGA)

Mutations in GJB2 are the most common cause of hereditary congenital hearing loss in many countries and are found in about half of persons with severe-to-profound congenital autosomal recessive non-syndromic hearing loss (ARNSHL). We report the results of GJB2 mutation screening in 209 consecutive persons with congenital deafness of indeterminate etiology using an allele-specific polymerase chain reaction assay, single-strand conformational polymorphism analysis, and direct sequencing. GJB2 allele variants were detected in 74 of 209 deaf individuals (35%). Over one-fourth of screened individuals were either homozygous (n=31) or heterozygous (n=24) for the 35delG mutation. Of those with the 35delG mutation, 51 (92.7%) were diagnosed with GJB2-related deafness. Nineteen persons were identified with other GJB2 allele variants - two novel deafness-causing mutations (R32C, 645-648delTAGA), one mutation of unknown significance (E47K), and one benign polymorphism (I128I). While these data enable health care professionals to provide parents and patients with improved genetic counseling data, difficulty still exists is determining whether some missense mutations compromise auditory function and are deafness-causing.     

Strong linkage disequilibrium for the frequent GJB2 35delG mutation in the Greek population

Approximately one in 1,000 children is affected by severe or profound hearing loss at birth or during early childhood (prelingual deafness). Up to 40% of congenital, autosomal recessive, severe to profound hearing impairment cases result from mutations in a single gene, GJB2, that encodes the connexin 26 protein. One specific mutation in this gene, 35delG, accounts for the majority of GJB2 mutations detected in Caucasian populations. Some previous studies have assumed that the high frequency of the 35delG mutation reflects the presence of a mutational hot spot, while other studies support the theory of a common founder. Greece is among the countries with the highest carrier frequency of the 35delG mutation (3.5%), and a recent study raised the hypothesis of the origin of this mutation in ancient Greece. We genotyped 60 Greek deafness patients homozygous for the 35delG mutation for six single nucleotide polymorphisms (SNPs) and two microsatellite markers inside or flanking the GJB2 gene. The allele distribution in the patients was compared to 60 Greek normal hearing controls. A strong linkage disequilibrium was found between the 35delG mutation and markers inside or flanking the GJB2 gene. Furthermore, we found a common haplotype with a previous study, suggesting a common founder for the 35delG mutation.     

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.     

Molecular genetics of hearing impairment due to mutations in gap junction genes encoding beta connexins

Deafness is a complex disorder that involves a high number of genes and environmental factors. There has been enormous progress in non-syndromic deafness research during the last five years, with the identification of over 50 loci and 15 genes. Among these, three genes, GJB2, GJB3, and GJB6, encode for connexin proteins (Connexin26, Connexin31, and Connexin30, respectively). Another connexin (Connexin32, encoded by GJB1) is involved in X-linked peripheral neuropathy and hearing impairment. Mutations in these genes cause autosomal recessive (GJB2 and GJB3), autosomal dominant (GJB2, GJB3, and GJB6) or X-linked (GJB1) hearing impairment, both syndromic (GJB2, keratoderma; GJB3 erythrokeratodermia variabilis; and GJB1, peripheral neuropathy), and non-syndromic (GJB2, GJB3, and GJB6). Among these genes, mutations in GJB2 account for about 50% of all congenital cases of hearing impairment. Three mutations in GJB2 (35delG, 167delT, and 235delC) are particularly common in specific populations (Caucasoid, Jewish Ashkenazi, and Oriental, respectively), leading to carrier frequencies between one in 30 and one in 75. Over 50 mutations have been identified in the GJB2 gene, of which some missense changes (M34T, W44C, G59A, D66H, and R75W) have a negative dominant action in hearing impairment, with partial to full penetrance. Functional studies for some missense mutations in connexins 26, 30, and 32 have indicated abnormal gap junction conductivity. Expression patterns in mouse and rat cochlea indicate that Connexin26 and Connexin30 are expressed in the supportive cells of the cochlea, suggesting a potential role in endolymph potassium recycling. The high prevalence of mutations in GJB2 in some populations provides the tools for molecular diagnosis, carrier detection, and prenatal diagnosis of congenital hearing impairment.     

Deafness heterogeneity in a Druze isolate from the Middle East: novel OTOF and PDS mutations, low prevalence of GJB2 35delG mutation and indication for a new DFNB locus

About 60% of congenital hearing impairment cases in developed countries are due to genetic defects. Data on the molecular basis of hereditary hearing reflects vast genetic heterogeneity. There are >400 disorders in which hearing impairment is one of the characteristic traits of a syndrome. Linkage studies have identified more than 40 human chromosomal loci associated with non-syndromic hearing loss. So far, 16 of these 40 non-syndromic hearing impairment genes have been identified. We have studied the molecular basis of hearing impairment in four Druze families from the same village in Northern Galilee. The Druze are a small, isolated population in the Middle East practising endogamous marriage. Thus it was expected that a single mutation would account for hearing impairments in all these families. Our results show that at least four different genes are involved. Hearing impairment was caused in one family by a novel mutation in the recently identified OTOF (the DFNB9 gene), by a novel Pendred syndrome mutation (Thr193Ile) in another family, and by a GJB2 mutation (35delG also known as 30delG) in the third family. In the fourth family linkage was excluded from all known hearing impairments loci (recessive and dominant) as well as from markers covering chromosomes 11-22, pointing therefore to the existence of another non-syndromic recessive hearing loss (NSRD) locus on chromosomes 1-10.     

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.     

Deafness resulting from mutations in the GJB2 (connexin 26) gene in Brazilian patients

Congenital deafness occurs in approximately 1 in 1000 live births. In developed countries about 60% of hearing loss is genetic. However, in Brazil most cases of hearing loss are due to environmental factors, such as congenital infections (mainly rubella), perinatal anoxia, kernicterus and meningitis. Recently, it has been demonstrated that the GJB2 gene is a major gene underlying congenital sensorial deafness. Mutations in this gene cause 10-20% of all genetic sensory hearing loss. One specific mutation, 35delG, accounts for the majority of mutant alleles. The extent of the hearing impairment varies from mild/moderate to profound, even within the patients homozygous for the common 35delG mutation. There may also be progression with age. Mutation analysis in the GJB2 gene was performed on 36 families (group A) presenting with at least one individual with non-syndromic deafness (NSD). An unselected series of 26 deaf individuals referred by other services where the environmental factors were not completely excluded was also part of the study (group B). Mutations in the GJB2 gene were found in 22% (eight patients) of the families tested in group A, and 11.5% (three patients) of individuals within group B. This finding should facilitate diagnosis of congenital deafness and allow early treatment of the affected subjects.     

Clinical evidence of the nonpathogenic nature of the M34T variant in the connexin 26 gene

Mutations in GJB2 are the most common cause of congenital nonsyndromic hearing loss. The controversial allele variant M34T has been hypothesized to cause autosomal dominant or recessive nonsyndromic hearing impairment and some in vitro data has been consistent with this hypothesis. In this report, we present the clinical and genotypic study of 11 families (seven familial forms of nonsyndromic sensorineural hearing loss (NSSNHL) and four sporadic cases) in which the M34T GJB2 variant has been identified. The M34T mutation did not segregate with the deafness in six of the seven familial forms of NSSNH. Eight persons with normal audiogram presented a heterozygous M34T variation and five normal hearing individuals were composite heterozygous for M34T and another GJB2 mutation. Four normal hearing individuals with a documented audiogram were M34T/35delG and one was M34T/(GJB6-D13S1830)del. Screening a French control population of 116 subjects we have found an M34T allele frequency of 1.72%. This percentage was not significatively different from the prevalence of the M34T allele in the deaf population, which was 2.12%. All these data suggest that the M34T variant is not clinically significant in human and is a frequent polymorphism in France.     

Did the GJB2 35delG mutation originate in Iran?

Mutations in GJB2 are a major cause of autosomal recessive non-syndromic hearing loss (ARNSHL) in many populations. A single mutation of this gene (35delG) accounts for approximately 70% of GJB2 mutations that are associated with ARNSHL in Caucasians in many European countries and also in Iranian. In this study, we used PCR and restriction digestion to genotype five single nucleotide polymorphisms (SNPs) that define the genetic background of the 35delG mutation over an interval of 98 Kbp that includes the coding and flanking regions of GJB2. Two microsatellite markers, D13S175 and D13S141, were also analyzed in patients and controls. These data suggest that the 35delG mutation originated in northern Iran.     

GJB2 mutations in Iranians with autosomal recessive non-syndromic sensorineural hearing loss

Hereditary hearing loss (HHL) is an extremely common disorder. About 70% of HHL is non-syndromic, with autosomal recessive forms accounting for approximately 85% of the genetic load. Although very heterogeneous, the most common cause of HHL in many different world populations is mutations of GJB2, a gene that encodes the gap junction protein connexin 26 (Cx26). This study investigates the contribution of GJB2 to the autosomal recessive non-syndromic deafness (ARNSD) load in the Iranian population. One hundred sixty eight persons from 83 families were studied. GJB2-related deafness was diagnosed in 9 families (4, 35delG homozygotes; 3, 35delG compound heterozygotes; 1, W24X homozygote; 1, non-35delG compound heterozygote). The carrier frequency of the 35delG allele in this population was approximately 1% (1/83). Because the relative frequency of Cx26 mutations is much less than in the other populations, it is possible that mutations in other genes play a major role in ARNSD in Iran.     

GJB2 mutations: passage through Iran

Hereditary hearing loss (HHL) is a very common disorder. When inherited in an autosomal recessive manner, it typically presents as an isolated finding. Interestingly and unexpectedly, in spite of extreme heterogeneity, mutations in one gene, GJB2, are the most common cause of congenital severe-to-profound deafness in many different populations. In this study, we assessed the contributions made by GJB2 mutations and chromosome 13 g.1777179_2085947del (the deletion more commonly known as del (GJB6-D13S1830) that includes a portion of GJB6 and is hereafter called Delta(GJB6-D13S1830)) to the autosomal recessive non-syndromic deafness (ARNSD) genetic load in Iran. Probands from 664 different nuclear families were investigated. GJB2-related deafness was found in 111 families (16.7%). The carrier frequency of the 35delG mutation showed a geographic variation that is supported by studies in neighboring countries. Delta(GJB6-D13S1830) was not found. Our prevalence data for GJB2-related deafness reveal a geographic pattern that mirrors the south-to-north European gradient and supports a founder effect in southeastern Europe.     

A common founder for the 35delG GJB2 gene mutation in connexin 26 hearing impairment

Fifty to eighty percent of autosomal recessive congenital severe to profound hearing impairment result from mutations in a single gene, GJB2, that encodes the protein connexin 26. One mutation of this gene, the 35delG allele, is particularly common in white populations. We report evidence that the high frequency of this allelic variant is the result of a founder effect rather than a mutational hot spot in GJB2, which was the prevailing hypothesis. Patients homozygous for the 35delG mutation and normal hearing controls originating from Belgium, the UK, and the USA were genotyped for different single nucleotide polymorphisms (SNPs). Four SNPs mapped in the immediate vicinity of GJB2, while two were positioned up to 76 kb from it. Significant differences between the genotypes of patients and controls for the five SNPs closest to GJB2 were found, with nearly complete association of one SNP allele with the 35delG mutation. For the most remote SNP, we could not detect any association. We conclude that the 35delG mutation is derived from a common, albeit ancient founder.


Keywords: connexin 26; GJB2; 35delG; founder effect     

High prevalence of the W24X mutation in the gene encoding connexin-26 (GJB2) in Spanish Romani (gypsies) with autosomal recessive non-syndromic hearing loss

Molecular testing for mutations in the gene encoding connexin-26 (GJB2) at the DFNB1 locus has become the standard of care for genetic diagnosis and counseling of autosomal recessive non-syndromic hearing impairment (ARNSHI). The spectrum of mutations in GJB2 varies considerably among the populations, different alleles predominating in different ethnic groups. A cohort of 34 families of Spanish Romani (gypsies) with ARNSHI was screened for mutations in GJB2. We found that DFNB1 deafness accounts for 50% of all ARNSHI in Spanish gypsies. The predominating allele is W24X (79% of the DFNB1 alleles), and 35delG is the second most common allele (17%). An allele-specific PCR test was developed for the detection of the W24X mutation. By using this test, carrier frequencies were determined in two sample groups of gypsies from different Spanish regions (Andalusia and Catalonia), being 4% and 0%, respectively. Haplotype analysis for microsatellite markers closely flanking the GJB2 gene revealed five different haplotypes associated with the W24X mutation, all sharing the same allele from marker D13S141, suggesting that a founder effect for this mutation is responsible for its high prevalence among Spanish gypsies.     

High carrier frequency of the 35delG deafness mutation in European populations. Genetic Analysis Consortium of GJB2 35delG

Congenital deafness accounts for about 1 in 1000 infants and approximately 80% of cases are inherited as an autosomal recessive trait. Recently, it has been demonstrated that connexin 26 (GJB2) gene is a major gene for congenital sensorineural deafness. A single mutation (named 35delG) was found in most recessive families and sporadic cases of congenital deafness, among Caucasoids, with relative frequencies ranging from 28% to 63%. We present here the analysis of the 35delG mutation in 3270 random controls from 17 European countries. We have detected a carrier frequency for 35delG of 1 in 35 in southern Europe and 1 in 79 in central and northern Europe. In addition, 35delG was detected in five out of 376 Jewish subjects of different origin, but was absent in other non-European populations. The study suggests either a single origin for 35delG somewhere in Europe or in the Middle East, and the possible presence of a carrier advantage together with a founder effect. The 35delG carrier frequency of 1 in 51 in the overall European population clearly indicates that this genetic alteration is a major mutation for autosomal recessive deafness in Caucasoids. This finding should facilitate diagnosis of congenital deafness and allow early treatment of the affected subjects.     

Molecular screening of deafness in Algeria: High genetic heterogeneity involving DFNB1 and the Usher loci,DFNB2/USH1B,DFNB12/USH1D and DFNB23/USH1F

A systematic approach, involving haplotyping and genotyping, to the molecular diagnosis of non-syndromic deafness within 50 families and 9 sporadic cases from Algeria is described.Mutations at the DFNB1 locus (encompassing the GJB2 and GJB6 genes) are responsible for more than half of autosomal recessive prelingual non-syndromic deafness in various populations. A c.35delG mutation can account for up to 85% of GJB2 mutations and two large deletions del(GJB6-D13S1830) and del(GJB6-D13S1854) have also been reported in several population groups.In view of the genetic heterogeneity a strategy was developed which involved direct analysis of DFNB1. In negative familial cases, haplotype analysis was carried out, where possible, to exclude DFNB1 mutations. Following this, haplotype analysis of five Usher syndrome loci, sometimes involved in autosomal non-syndromic hearing loss, was carried out to identify cases in which Usher gene sequencing was indicated. When homozygosity was observed at a locus in a consanguineous family, the corresponding gene was exhaustively sequenced.Pathogenic DFNB1 genotypes were identified in 40% of the cases. Of the 21 cases identified with 2 pathogenic mutations, c.35delG represented 76% of the mutated alleles. The additional mutations were one nonsense, two missense and one splicing mutation. Four additional patients were identified with a single DFNB1 mutation. None carried the large deletions.Three families with non-syndromic deafness carried novel unclassified variants (UVs) in MYO7A (1 family) and CDH23 (2 families) of unknown pathogenic effect.Additionally, molecular diagnosis was carried out on two Usher type I families and pathogenic mutations in MYO7A and PCDH15 were found.     

A genotype-phenotype correlation for GJB2 (connexin 26) deafness

Introduction: Mutations in GJB2 are the most common cause of non-syndromic autosomal recessive hearing impairment, ranging from mild to profound. Mutation analysis of this gene is widely available as a genetic diagnostic test.

Objective: To assess a possible genotype-phenotype correlation for GJB2.

Design: Retrospective analysis of audiometric data from people with hearing impairment, segregating two GJB2 mutations.

Subjects: Two hundred and seventy seven unrelated patients with hearing impairment who were seen at the ENT departments of local and university hospitals from Italy, Belgium, Spain, and the United States, and who harboured bi-allelic GJB2 mutations.

Results: We found that 35delG homozygotes have significantly more hearing impairment, compared with 35delG/non-35delG compound heterozygotes. People with two non-35delG mutations have even less hearing impairment. We observed a similar gradient of hearing impairment when we categorised mutations as inactivating (that is, stop mutations or frame shifts) or non-inactivating (that is, missense mutations). We demonstrated that certain mutation combinations (including the combination of 35delG with the missense mutations L90P, V37I, or the splice-site mutation IVS1+1G>A, and the V37I/V37I genotype) are associated with significantly less hearing impairment compared with 35delG homozygous genotypes.

Conclusions: This study is the first large systematic analysis indicating that the GJB2 genotype has a major impact on the degree of hearing impairment, and identifying mild genotypes. Furthermore, this study shows that it will be possible to refine this correlation and extend it to additional genotypes. These data will be useful in evaluating habilitation options for people with GJB2 related deafness.

     

Prevalent connexin 26 gene (GJB2) mutations in Japanese

The gene responsible for DNFB1 and DFNA3, connexin 26 (GJB2), was recently identified and more than 20 disease causing mutations have been reported so far. This paper presents mutation analysis for GJB2 in Japanese non-syndromic hearing loss patients compatible with recessive inheritance. It was confirmed that GJB2 mutations are an important cause of hearing loss in this population, with three mutations, 235delC, Y136X, and R143W, especially frequent. Of these three mutations, 235delC was most prevalent at 73%. Surprisingly, the 35delG mutation, which is the most common GJB2 mutation in white subjects, was not found in the present study. Our data indicated that specific combinations of GJB2 mutation exist in different populations.     

V37I connexin 26 allele in patients with sensorineural hearing loss: evidence of its pathogenicity

Sensorineural hearing loss (SNHL) is the most common inherited sensory disorder, reported in 1-3 of every 1,000 births. It has been estimated that 50% of all cases of prelingual SNHL are genetically determined. There is tremendous genetic heterogeneity, with multiple dominant and recessive loci. Mutations of the gap junction beta-2 gene (GJB2) emerge as a leading cause of autosomal recessive non-syndromic SNHL. Over 90 sequence alterations have been reported, the pathogenicity of some of them being unknown or unclear. The status of the V37I allele of connexin 26 (GJB2 amino acid product) with regards to its association with SNHL has been controversial. This study examines the pathogenicity of V37I by comparing the frequency of this allele in 40 patients with SNHL of Chinese and Caucasian descent with the frequency of the allele in 100 anonymized, ethnically matched controls. The V37I allele was identified in 43.75 and 11.5% of the patient and control alleles of Chinese ethnicity, respectively, but was not found in either Caucasian cohort. We also compiled the audiograms of 15 individuals with SNHL homozygous for the V37I allele, and showed that these individuals present with a mild to moderate SNHL. These results indicate that (1) the V37I allele is common in individuals of Chinese descent but rarely present in individuals of Caucasian decent; and (2) the V37I allele is pathogenic, but produces milder hearing loss compared to nonsense mutations of connexin 26 such as the 35delG mutation.