GJB2: the spectrum of deafness-causing allele variants and their phenotype

Genetic testing was completed on 1,294 persons with deafness referred to the Molecular Otolaryngology Research Laboratories to establish a diagnosis of DFNB1. Exon 2 of GJB2 was screened for coding sequence allele variants by denaturing high-performance liquid chromatography (DHPLC) complemented by bidirectional sequencing. If two deafness-causing mutations of GJB2 (encoding Connexin 26) were identified, further screening was not performed. If only a single deafness-causing mutation was identified, we screened for the g.1777179_2085947del (hereafter called del(GJB6-D13S1830); GenBank NT_024524.13) and mutations in the noncoding region of GJB2. Phenotype-genotype correlations were evaluated by categorizing mutations as either protein truncating or nontruncating. A total of 205 persons carried two GJB2 exon 2 mutations and were diagnosed as having DFNB1; 100 persons carried only a single deafness-causing allele variant of exon 2. A total of 37 of these persons were c.35delG carriers, and 51 carried other allele variants of GJB2. Persons diagnosed with DFNB1 segregating two truncating/nonsense mutations had a more severe phenotype than persons carrying two missense mutations, with mean hearing impairments being 88 and 37%, respectively (P < 0.05). The number of deaf c.35delG carriers was greater than expected when compared to the c.35delG carrier frequency in normal-hearing controls (P < 0.05), suggesting the existence of at least one other mutation outside the GJB2 coding region that does not complement GJB2 deafness-causing allele variants.     

Profound, prelingual nonsyndromic deafness maps to chromosome 10q21 and is caused by a novel missense mutation in the Usher syndrome type IF gene PCDH15

We studied a consanguineous family (Family A) from the island of Newfoundland with an autosomal recessive form of prelingual, profound, nonsyndromic sensorineural hearing loss. A genome-wide scan mapped the deafness trait to 10q21-22 (max LOD score of 4.0; D10S196) and fine mapping revealed a 16 Mb ancestral haplotype in deaf relatives. The PCDH15 gene was mapped within the critical region and was an interesting candidate because truncating mutations cause Usher syndrome type IF (USH1F) and two missense mutations have been previously associated with isolated deafness (DFNB23). Sequencing of the PCDH15 gene revealed 33 sequencing variants. Three of these variants were homozygous exclusively in deaf siblings but only one of them was not seen in ethnically matched controls. This novel c.1583 T>A transversion predicts an amino-acid substitution of a valine with an aspartic acid at codon 528 (V528D). Like the two DFNB23 mutations, the V528D mutation in Family A occurs in a highly conserved extracellular cadherin (EC) domain of PCDH15 and is predicted to be more deleterious than the previously identified DFNB23 missense mutations (R134G and G262D). Physical assessment, vestibular and visual function testing in deaf adults ruled out syndromic deafness because of Usher syndrome. This study validates the DFNB23 designation and supports the hypothesis that missense mutations in conserved motifs of PCDH15 cause nonsyndromic hearing loss. This emerging genotype–phenotype correlation in USH1F is similar to that in several other USH1 genes and cautions against a prognosis of a dual sensory loss in deaf children found to be homozygous for hypomorphic mutations at the USH1F locus.     

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

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

Performance of cochlear implant recipients with GJB2‐related deafness

Congenital profound hearing loss affects 0.05–0.1% of children and has many causes, some of which are associated with cognitive delay. For prelingually‐deafened cochlear implant recipients, the etiology of deafness is usually unknown. Mutations in GJB2 have been established as the most common cause of heritable deafness in the United States. In this report, we identify cochlear implant recipients with GJB2‐related deafness and examine the performance of these individuals. Cochlear implant recipients received a battery of perceptive, cognitive, and reading tests. Neither subjects nor examiners knew the etiology of deafness in these individuals. The implant recipients were then examined for mutations in GJB2 using an allele‐specific polymerase chain reaction assay, single‐strand conformation polymorphism analysis, and direct sequencing. GJB2 mutations were the leading cause of congenital deafness among the cochlear implant recipients screened. Cochlear implant recipients with GJB2‐related deafness read within one standard deviation of hearing controls better than other congenitally deaf cochlear implant recipients and non‐cochlear implant recipients. Individuals with congenital deafness should be offered GJB2 screening. Positive results establish an etiologic diagnosis and provide prognostic, genetic, and therapeutic information. Effective rehabilitation for profoundly deaf individuals with GJB2‐related deafness is possible through cochlear implantation. © 2002 Wiley‐Liss, Inc.     

Update of spectrum c.35delG and c.‐23+1G>A mutations on the GJB2 gene in individuals with autosomal recessive nonsyndromic hearing loss

Hearing loss (HL) is the most common birth defect and the most prevalent sensorineural condition worldwide. It is associated with more than 1,000 mutations in at least 90 genes. Mutations of the gap junction beta‐2 protein (GJB2) gene located in the nonsyndromic hearing loss and deafness (DFNB1) locus (chromosome 13q11‐12) are the main causes of autosomal recessive nonsyndromic hearing loss worldwide, but important differences exist between various populations. In the present article, two common mutations of the GJB2 gene are compared for ethnic‐specific allele frequency, their function, and their contribution to genetic HL in different populations. The results indicated that mutations of the GJB2 gene could have arisen during human migration. Updates on the spectrum of mutations clearly show that frequent mutations in the GJB2 gene are consistent with the founder mutation hypothesis.     

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.     

Mutations in the TMPRSS3 gene are a rare cause of childhood nonsyndromic deafness in Caucasian patients

Two loci for nonsyndromic recessive deafness located on chromosome 21q22.3 have previously been reported, DFNB8 and DFNB10. Recently a gene which encodes a transmembrane serine protease, TMPRSS3 or ECHOS1, was found to be responsible for both the DFNB8 and DFNB10 phenotypes. To determine the contribution of TMPRSS3 mutations in the general congenital/childhood nonsyndromic deaf population we performed mutation analysis of the TMPRSS3 gene in 448 unrelated deaf patients from Spain, Italy, Greece, and Australia who did not have the common 35delG GJB2 mutation. From the 896 chromosomes studied we identified two novel pathogenic mutations accounting for four mutant alleles and at least 16 nonpathogenic sequence variants. The pathogenic mutations were a 1-bp deletion resulting in a frameshift and an amino acid substitution in the LDLRA domain of TMPRSS3. From this and another study we estimate the frequency of TMPRSS3 mutations in our sample as 0.45%, and approximately 0.38% in the general Caucasian childhood deaf population. However, TMPRSS3 is still an important contributor to genetic deafness in populations with large consanguineous families.     

Targeted Droplet‐Digital PCR as a Tool for Novel Deletion Discovery at the DFNB1 Locus

Pathogenic variants at the DFNB1 locus encompassing the GJB2 and GJB6 genes account for 50% of autosomal‐recessive, congenital nonsyndromic hearing loss in the United States. Most cases are caused by sequence variants within the GJB2 gene, but a significant number of DFNB1 patients carry a large deletion (GJB6‐D13S1830) in trans with a GJB2 variant. This deletion lies upstream of GJB2 and was shown to reduce GJB2 expression by disrupting unidentified regulatory elements. First‐tier genetic testing for hearing loss includes GJB2 sequence and GJB6‐D13S1830 deletion analysis; however, several other deletions in this locus, each with distinct breakpoints, have been reported in DFNB1 patients and are missed by current panels. Here, we report the development of a targeted droplet digital polymerase chain reaction‐based assay for comprehensive copy‐number analysis at the DFNB1 locus that detects all deletions reported to date. This assay increased detection rates in a multiethnic cohort of 87 hearing loss patients with only one identified pathogenic GJB2 variant. We identify two deletions, one of which is novel, in two patients (2/87 or 2.3%), suggesting that other pathogenic deletions at the DFNB1 locus may be missed. Mapping the assayed DFNB1 deletions also revealed a ～95 kb critical region, which may harbor the GJB2 regulatory element(s).     

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

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

Mutations of ESPN cause autosomal recessive deafness and vestibular dysfunction

We mapped a human deafness locus DFNB36 to chromosome 1p36.3 in two consanguineous families segregating recessively inherited deafness and vestibular areflexia. This phenotype co-segregates with either of two frameshift mutations, 1988delAGAG and 2469delGTCA, in ESPN, which encodes a calcium-insensitive actin-bundling protein called espin. A recessive mutation of ESPN is known to cause hearing loss and vestibular dysfunction in the jerker mouse. Our results establish espin as an essential protein for hearing and vestibular function in humans. The abnormal vestibular phenotype associated with ESPN mutations will be a useful clinical marker for refining the differential diagnosis of non-syndromic deafness.     

DFNB16 is a frequent cause of congenital hearing impairment: implementation of STRC mutation analysis in routine diagnostics

Increasing attention has been directed toward assessing mutational fallout of stereocilin (STRC), the gene underlying DFNB16. A major challenge is due to a closely linked pseudogene with 99.6% coding sequence identity. In 94 GJB2/GJB6‐mutation negative individuals with non‐syndromic sensorineural hearing loss (NSHL), we identified two homozygous and six heterozygous deletions, encompassing the STRC region by microarray and/or quantitative polymerase chain reaction (qPCR) analysis. To detect smaller mutations, we developed a Sanger sequencing method for pseudogene exclusion. Three heterozygous deletion carriers exhibited hemizygous mutations predicted as negatively impacting the protein. In 30 NSHL individuals without deletion, we detected one with compound heterozygous and two with heterozygous pathogenic mutations. Of 36 total patients undergoing STRC sequencing, two showed the c.3893A>G variant in conjunction with a heterozygous deletion or mutation and three exhibited the variant in a heterozygous state. Although this variant affects a highly conserved amino acid and is predicted as deleterious, comparable minor allele frequencies (MAFs) (around 10%) in NSHL individuals and controls and homozygous variant carriers without NSHL argue against its pathogenicity. Collectively, six (6%) of 94 NSHL individuals were diagnosed with homozygous or compound heterozygous mutations causing DFNB16 and five (5%) as heterozygous mutation carriers. Besides GJB2/GJB6 (DFNB1), STRC is a major contributor to congenital hearing impairment.     

Mutational Spectrum of MYO15A and the Molecular Mechanisms of DFNB3 Human Deafness

Deafness in humans is a common neurosensory disorder and is genetically heterogeneous. Across diverse ethnic groups, mutations of MYO15A at the DFNB3 locus appear to be the third or fourth most common cause of autosomal‐recessive, nonsyndromic deafness. In 49 of the 67 exons of MYO15A, there are currently 192 recessive mutations identified, including 14 novel mutations reported here. These mutations are distributed uniformly across MYO15A with one enigmatic exception; the alternatively spliced giant exon 2, encoding 1,233 residues, has 17 truncating mutations but no convincing deafness‐causing missense mutations. MYO15A encodes three distinct isoform classes, one of which is 395 kDa (3,530 residues), the largest member of the myosin superfamily of molecular motors. Studies of Myo15 mouse models that recapitulate DFNB3 revealed two different pathogenic mechanisms of hearing loss. In the inner ear, myosin 15 is necessary both for the development and the long‐term maintenance of stereocilia, mechanosensory sound‐transducing organelles that extend from the apical surface of hair cells. The goal of this Mutation Update is to provide a comprehensive review of mutations and functions of MYO15A.     

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

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

Large deletion of the GJB6 gene in deaf patients heterozygous for the GJB2 gene mutation: genotypic and phenotypic analysis

Recent investigations identified a large deletion of the GJB6 gene in trans to a mutation of GJB2 in deaf patients. We looked for GJB2 mutations and GJB6 deletions in 255 French patients presenting with a phenotype compatible with DFNB1. 32% of the patients had biallelic GJB2 mutations and 6% were a heterozygous for a GJB2 mutation and a GJB6 deletion. Biallelic GJB2 mutations and combined GJB2/GJB6 anomalies were more frequent in profoundly deaf children. Based on these results, we are now assessing GJB6 deletion status in cases of prelingual hearing loss.     

湖州地区聋哑学校68名聋哑学生耳聋易感基因突变分析研究

目的研究4个耳聋易感基因GJB2、GJB3、SLC26A4、线粒体12SrRNA在湖州市聋哑学校68名聋哑学生中的突变类型分布情况。方法应用飞行时间质谱技术,对68名聋哑学生进行GJB2、GJB3、SLC26A4、线粒体12SrRNA 4个耳聋易感基因检测,检测位点包含以上基因的20个热点突变。结果68名聋哑学生中共检出耳聋基因突变27例,阳性率39.71％,其中GJB2基因突变19例,占70.37％;GJB3基因突变l例,占3.7％;SLC26A4基因突变5例,占18.52％;线粒体12SrRNA基因突变2例,占7.41％。结论在湖州市聋哑学校中,GJB2是最常见的耳聋突变基因,235delC是GJB2基因最常见的突变位点。     

Searching for evidence of DFNB2

Deafness is the most common form of sensory impairment in humans, affecting about 1 in 1,000 births in the United States. Of those cases with genetic etiology, approximately 80% are nonsyndromic and recessively inherited. Mutations in several unconventional myosins, members of a large superfamily of actin-associated molecular motors, have been found to cause hearing loss in both humans and mice. Mutations in the human unconventional Myosin VIIa (MYO7A), located at 11q13.5, are reported to be responsible for both syndromic and nonsyndromic deafness. MYO7A mutations are responsible for Usher syndrome type Ib, the most common genetic subtype of Usher I. Usher I is clinically characterized by congenital profound deafness, progressive retinal degeneration called retinitis pigmentosa (RP), and vestibular areflexia. Although a wide spectrum of MYO7A mutations have been identified in Usher Ib patients, four mutations have been reported to cause DFNB2, a recessive deafness without retinal degeneration, and one mutation has been implicated in a single case of dominant nonsyndromic hearing loss (DFNA11). Our study attempts to ascertain additional DFNB2 families to investigate the disparate nonsyndromic phenotype and alleged causative mutations. Data from both linkage and heterogeneity analyses on 36 selected autosomal recessive nonsyndromic deafness (RNSD) families, all previously excluded by mutational analysis from GJB2 (Cx26), the leading cause of nonsyndromic deafness, showed no evidence of DFNB2 within the sample. These negative results and the isolated reports of DFNB2 bring into question whether certain MYO7A mutations produce nonsyndromic recessive hearing loss.     

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.     

SLC26A4 gene is frequently involved in nonsyndromic hearing impairment with enlarged vestibular aqueduct in Caucasian populations

Sensorineural hearing loss is the most frequent sensory deficit of childhood and is of genetic origin in up to 75% of cases. It has been shown that mutations of the SLC26A4 (PDS) gene were involved in syndromic deafness characterized by congenital sensorineural hearing impairment and goitre (Pendred's syndrome), as well as in congenital isolated deafness (DFNB4). While the prevalence of SLC26A4 mutations in Pendred's syndrome is clearly established, it remains to be studied in large cohorts of patients with nonsyndromic deafness and detailed clinical informations. In this report, 109 patients from 100 unrelated families, aged from 1 to 32 years (median age: 10 years), with nonsyndromic deafness and enlarged vestibular aqueduct, were genotyped for SLC26A4 using DHPLC molecular screening and sequencing. In all, 91 allelic variants were observed in 100 unrelated families, of which 19 have never been reported. The prevalence of SLC26A4 mutations was 40% (40/100), with biallelic mutation in 24% (24/100), while six families were homozygous. All patients included in this series had documented deafness, associated with EVA and without any evidence of syndromic disease. Among patients with SLC26A4 biallelic mutations, deafness was more severe, fluctuated more than in patients with no mutation. In conclusion, the incidence of SLC26A4 mutations is high in patients with isolated deafness and enlarged vestibular aqueduct and could represent up to 4% of nonsyndromic hearing impairment. SLC26A4 could be the second most frequent gene implicated in nonsyndromic deafness after GJB2, in this Caucasian population.     

Connexin26 mutations associated with the most common form of non- syndromic neurosensory autosomal recessive deafness (DFNB1) in Mediterraneans

Non-syndromic neurosensory autosomal recessive deafness (NSRD) is the most common form of genetic hearing loss. Previous studies defined at least 15 human NSRD loci. Recently we demonstrated that DFNB1, located on the long arm of chromosome 13, accounts for approximately 80% of cases in the Mediterranean area. Further analysis with additional markers now identifies several recombinants which narrow the candidate region to approximately 5 cM, encompassed by markers D13S141 and D13S232 and including several ESTs and candidate genes, including the connexin26 (GJB2) gene. Analysis of PCR products from our affected patients' DNA shows two frameshift mutations in the connexin26 gene. Deletion of a G within a stretch of six Gs at position 35 of the GJB2 cDNA (mutation 35delG) leads to premature chain termination and is present in 63% of NSRD chromosomes, demonstrating linkage to chromosome 13. Deletion of a T at position 167 of GJB2 (mutation 167delT), also resulting in premature chain termination, was detected in another patient. Four neutral sequence polymorphisms were also identified. These findings are in agreement with a recent study showing that mutations in the connexin26 gene are associated with genetic forms of deafness in three Pakistani families and that GJB2 is DFNB1. Connexin26 is a member of a large family of proteins involved in formation of gap junctions, which are involved in electrical synapses and the direct transfer of small molecules and ionic currents between neighboring cells. The identification of GJB2 as the DFNB1 gene should provide a better understanding of the biology of normal and abnormal hearing, help form the basis for diagnosis and may facilitate development of strategies for treatment of this common genetic disorder.