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.     

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.     

Frequency of the recessive 30delG mutation in the GJB2 gene in Northeast-Hungarian individuals and patients with hearing impairment.

Mutations in the GJB2 gene, which encodes a gap junction protein (connexin 26) account for up to 50% of cases of congenital autosomal recessive non-syndromic hearing impairment. A single mutation, 30delG, is responsible for 70% of this autosomal recessive hearing loss in Europe. This study describes the 30delG mutation analysis of 23 Hungarian families (64 individuals) with at least two subjects with congenital non-syndromic hearing defect and of 52 unrelated individuals from the Northeastern population of Hungary. In all patients, non-progressive hearing impairment varied from moderate to profound involving all frequencies. DNA was tested by PCR based restriction enzyme assay (BSiYI). Sixty-four percent of the patients displayed this one base deletion in GJB2. Out of these, 65.9% were homozygous for this mutation and 34.1% were heterozygotes. The latter showed compound heterozygosity since in these 14 patients, eight previously reported different nucleotide changes were observed on the second allele. The carrier frequency of the 30delG mutation among control group was one in 10.4 (9.6%). This high frequency of 30delG corresponds more to frequencies reported in Southern than in North Europeans.     

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).     

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.     

Frequencies of gap- and tight-junction mutations in Turkish families with autosomal-recessive non-syndromic hearing loss

Mutations in genes encoding gap- and tight-junction proteins have been shown to cause distinct forms of hearing loss. We have now determined the GJB2[connexin 26 (Cx26)] mutation spectrum in 60 index patients from mostly large Turkish families with autosomal-recessive inherited non-syndromic sensorineural hearing loss (NSSHL). GJB2 mutations were found in 31.7% of the families, and the GJB2-35delG mutation accounted for 73.6% of all GJB2 mutations. The carrier frequency of GJB2-35delG in the normal Turkish population was found to be 1.17% (five in 429). In addition to the described W24X, 233delC, 120delE and R127H mutations, we also identified a novel mutation, Q80R, in the GJB2 gene. Interestingly, the Q80R allele was inherited on the same haplotype as V27I and E114G polymorphisms. As little is known about the mutation frequencies of most other recently identified gap- and tight-junction genes as a cause for hearing loss, we further screened our patients for mutations in GJB3 (Cx31), GJA1 (Cx43), DeltaGJB6-D13S1830 (Cx30) and the gene encoding the tight-junction protein, claudin 14 (CLDN14). Several novel polymorphisms, but no disease-associated mutations, were identified in the CLND14 and GJA1 genes, and we were unable to detect the DeltaGJB6-D13S1830 deletion. A novel putative mutation, P223T, was found in the GJB3 gene in heterozygous form in a family with two affected children. Our data shows that the frequency of GJB2 mutations in Turkish patients with autosomal-recessive NSSHL and the carrier rate of the GJB2-35delG mutation in the Turkish population, is much lower than described for other Mediterranean countries. Furthermore, mutations in other gap- and tight-junction proteins are not a frequent cause of hearing loss in Turkey.     

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.     

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.     

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.     

Mutation analysis of Connexin 31 (GJB3) in sporadic non-syndromic hearing impairment

Mutations in GJB3, the gene encoding the gap junction protein Connexin 31 (CX31), have been pathogenically linked to erythrokeratodermia and non-syndromic autosomal dominant (DFNA2) or recessive hereditary hearing impairment (HHI). To determine the contribution of CX31 to sporadic deafness, we assessed 63 individuals with non-syndromic hearing impairment for CX31 mutations. Single coding exon of CX31 was amplified from genomic DNA and then sequenced. Single nucleotide sequence alteration was present in 15 out of 63 patients (24%), all of the positives being heterozygous for the four different single base pair changes that were detected: C94T, C201T, C357T and C798T. Of these, only C94T transition, identified in two patients, results in amino acid change, R32W, while the other three changes are single nucleotide polymorphisms (SNPs). The R32W substitution in CX31 has been previously documented and is speculated to manifest variable penetrance, similar to the polymorphic allele encoding CX26M34T. Over one-third of all samples were also screened with denaturing high-performance liquid chromatography (dHPLC). Seven out of 25 individuals screened were determined to be positive for CX31 sequence variation. Sequence analysis of the 25 individuals screened identified nucleotide alterations in all of the 7 'positives' and in none of the 16 'negatives' yielding a specificity and sensitivity of 100%. Thus, dHPLC represents a highly efficient CX31 screening technique. This study suggests that while sequence alterations are common, pathogenic mutations of CX31 are infrequent in sporadic non-syndromic hearing impairment.     

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.     

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.     

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.     

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.     

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.

     

GJB2 mutations in patients with non-syndromic hearing loss from Northeastern Hungary

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, c.35delG, accounts for the majority of mutations in Caucasian patients with HI. In the present study we screened 500 healthy control individuals and a group of patients with HI from Northeastern Hungary for GJB2 mutations. The patients' group consisted of 102 familial from 28 families and 92 non-familial cases. The most common mutation in the Hungarian population is the c.35delG, followed by the c.71G>A (p.W24X) mutation. 34.3% of the patients in the familial group were homozygous, and 17.6% heterozygous for 35delG. In the non-familial group the respective values were 37% and 18% (allele frequency: 46.2%). In the general population an allele frequency of 2.4% was determined. Several patients were identified with additional, already described or new GJB2 mutations, mostly in heterozygous state. The mutation c.380G>A (p.R127H) was formerly found only in heterozygous state and its disease relation was controversial. We demonstrated the presence of this mutation in a family with three homozygous patients and 4 heterozygous unaffected family members, a clear indication of recessively inherited HI. Furthermore, we provided evidence for the pathogenic role of two new mutations, c.51C>A (p.S17Y) and c.177G>T (p.G59V), detected in the present study. In the latter case the pattern of inheritance might be dominant. Our results confirm the importance of GJB2 mutations in the Hungarian population displaying mutation frequencies that are comparable with those in the Mediterranean area.     

Low prevalence of Connexin 26 (GJB2) variants in Pakistani families with autosomal recessive non-syndromic hearing impairment

The Pakistani population has become an important resource for research on autosomal recessive non-syndromic hearing impairment (ARNSHI) due to the availability of large extended and highly consanguineous pedigrees. Here is presented the first report on the prevalence of gap junction beta-2 (GJB2) variants in Pakistan. One hundred and ninety-six unrelated Pakistani families with ARNSHI were recruited for a study on the genetics of NSHI. DNA sequencing of the GJB2 coding region was done on two affected individuals per family. Evolutionary conservation and predicted effect on the protein product were studied in order to hypothesize whether or not a variant was potentially deleterious. Homozygous putatively functional GJB2 variants were identified in 6.1% of families. None of the putatively functional GJB2 variants were observed in the compound heterozygous state. The six putatively causative variants noted were 231G > A(W77X), 71G > A(W24X), 167delT, 95G > A(R32H), 358-360delGAG(delE120), and 269T > C(L90P), with 231G > A(W77X) and 71G > A(W24X) being the most common. In addition, five benign polymorphisms, 380G > A(R127H), 457G > A(V153I), 493C > T(R165W), 79G > A(V27I), and 341 A > G(E114G), were identified within this population. In a few individuals, benign polymorphisms were observed to occur on the same haplotype, namely [457G > A(V153I); 493C > T(R165W)] and [79G > A(V27I); 341 A > G(E114G)]. The spectrum of GJB2 sequence variants in Pakistan may reflect shared origins of hearing impairment alleles within the Indian subcontinent. The high degree of consanguinity within Pakistan may have maintained the GJB2 prevalence at a much lower rate than within India and other populations.     

Mutations in the KCNQ4 gene are responsible for autosomal dominant deafness in four DFNA2 families.

We have previously found linkage to chromosome 1p34 in five large families with autosomal dominant non-syndromic hearing impairment (DFNA2). In all five families, the connexin31 gene ( GJB3 ), located at 1p34 and responsible for non-syndromic autosomal dominant hearing loss in two small Chinese families, has been excluded as the responsible gene. Recently, a fourth member of the KCNQ branch of the K+channel family, KCNQ4, has been cloned. KCNQ4 was mapped to chromosome 1p34 and a single mutation was found in three patients from a small French family with non-syndromic autosomal dominant hearing loss. In this study, we have analysed the KCNQ4 gene for mutations in our five DFNA2 families. Missense mutations altering conserved amino acids were found in three families and an inactivating deletion was present in a fourth family. No KCNQ4 mutation could be found in a single DFNA2 family of Indonesian origin. These results indicate that at least two and possibly three genes responsible for hearing impairment are located close together on chromosome 1p34 and suggest that KCNQ4 mutations may be a relatively frequent cause of autosomal dominant hearing loss.