A novel hearing-loss-related mutation occurring in the GJB2 basal promoter

Mutations in the GJB2 gene are a major cause of non-syndromic recessive hearing loss in many countries. In a significant fraction of patients, only monoallelic GJB2 mutations known to be either recessive or of unclear pathogenicity are identified. This paper reports a novel GJB2 mutation, -3438C-->T, found in the basal promoter of the gene, in trans with V84M, in a patient with profound hearing impairment. This novel mutation can abolish the basal promoter activity of GJB2. These results highlight the importance of extending the mutational screening to regions outside the coding region of GJB2.     

Spectrum and frequencies of mutations in the GJB2 (Cx26) gene among 156 Czech patients with pre-lingual deafness

Mutations in the gene gap junction beta 2 (GJB2), the gene for the connexin 26, are the most common cause of pre-lingual deafness worldwide. The mutation 35delG within GJB2 is prevalent in Europe. To date, there are no data about GJB2 mutation spectrum and frequencies from the Czech population. We investigated and report here the spectrum and frequencies of mutations in the GJB2 gene among 156 unrelated, congenital deafness Czech patients. Allele-specific polymerase chain reaction, together with fluorescent fragment analysis, were used for the detection of the 35delG mutation. The entire coding region of the GJB2 was directly sequenced in all patients who were not homozygous for the 35delG. No pathogenic mutation was detected in 51.9% of patients. At least one pathogenic mutation was found in 48.1% of patients, and both pathogenic mutations were detected in 37.8% of patients. Single mutations in a heterozygous state were detected in 10.3% of patients. The mutation 35delG accounts for 82.8% of detected disease mutations, Trp24stop accounts for 9.7% of pathogenic alleles and was found in patients with gypsy heritage. Mutation 313del14 accounts for 3.7% of pathogenic alleles. The frequency of 35delG heterozygotes in the Czech Republic is 1 : 29.6. Testing for only the three most common mutations would detect over 96% of all pathogenic alleles in the Czech Republic.     

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.     

The novel R75Q mutation in the GJB2 gene causes autosomal dominant hearing loss and palmoplantar keratoderma in a Turkish family

Dominant mutations in the GJB2 gene encoding connexin 26 (Cx26) can cause non-syndromic hearing impairment alone or in association with palmoplantar keratoderma (PPK). We have identified the novel G224A (R75Q) mutation in the GJB2 gene in a four-generation family from Turkey with autosomal dominant inherited hearing impairment and PPK. The age of onset and progression of hearing loss were found to be variable among affected family members, but all of them had more severe impairment at higher hearing frequencies. Interestingly, the novel R75Q mutation affects the same amino acid residue as described recently in a small family (R75W) with profound prelingual hearing loss and PPK. However, the R75W mutation was also observed in a control individual without PPK and unknown hearing status. Therefore, the nature of the R75W mutation remains ambiguous. Our molecular findings provide further evidence for the importance of the conserved R75 in Cx26 for the physiological function of the inner ear and the epidermal cells of the skin.     

遗传性非综合征型耳聋患者GJB2基因编码序列分析

目的 对6个遗传性非综合征型耳聋家系成员的GJB2基因编码序列进行分析,寻找耳聋患者的致病基因突变,探讨GJB2基因突变致病的遗传模式.方法 提取患者及家系成员的外周血基因组DNA,扩增GJB2基因的编码序列,然后对扩增产物进行DNA测序,对出现重叠峰形的扩增产物进行TA克隆后再测序,确定基因突变是否存在于同一拷贝.结果 6个遗传性非综合征型耳聋家系中,4个家系是GJB2基因突变所致.患者的GJB2基因突变包括235delC、299-300delAT、79G→A+341A→G和109G→A.非致聋突变79G→A与341A→G组合具有致聋效应,109G→A和235delC的杂合突变可能也有致聋效应.结论 GJB2基因突变致聋具有明显异质性,非致聋突变并非完全不致聋,环境因素或其它基因可能参与GJB2基因突变所致耳聋.     

Mutations in the connexin26/GJB2 gene are the most common event in non-syndromic hearing loss among the German population

Congenital sensorineural hearing loss affects approximately 1/1,000 live births. Mutations in the gene encoding connexin26 (GJB2) have been described as a major cause of genetic nonsyndromic hearing impairment. Additionally, another gap junction gene, connexin30 (GJB6), was found to be responsible for hereditary hearing loss. We have studied 134 patients with severe to profound hearing loss or deafness and 13 patients with mild to moderate nonsyndromic sensorineural hearing loss in order to evaluate the prevalence of connexin26 and connexin30 mutations in Germany. Mutations in the connexin26 gene were found in 30 patients (22%) with profound to severe hearing impairment whereas only one novel single nucleotide polymorphism (396G-->A) in the connexin30 gene was detected. Among the 13 patients with mild to moderate hearing loss neither mutations in the connexin26 nor in the connexin30 gene could be detected. These results demonstrate that mutations in the connexin26 gene are also a frequent cause of hereditary non-syndromic hearing loss in Germany. Therefore a screening of mutations in the connexin26 gene should be performed in every case of non-syndromic hearing loss of unknown origin.     

Different functional consequences of two missense mutations in the GJB2 gene associated with non‐syndromic hearing loss

Mutations in the GJB2 gene, which encodes the gap junction (GJ) protein connexin26 (Cx26), are the most common cause of inherited non‐syndromic hearing loss (NSHL). We identified two missense mutations, p.D46E (c.138T>G) and p.T86R (c.257C>G), of GJB2 in Korean HL families. The novel p.D46E mutation exhibited autosomal dominant inheritance, while the p.T86R mutation, which is exclusively found in Asians, segregated with an autosomal recessive pattern. Thus, we sought to elucidate the pathogenic nature of such different inherited patterns of HL. We studied protein localization and gap junction functions in cells transfected with wild‐type or mutant Cx26 tagged with fluorescent proteins, which allowed visual confirmation of homozygous or heterozygous mutant GJs. The Cx26‐D46E mutant was targeted to the plasma membrane, but this mutant protein failed to transfer Ca²⁺ or propidium iodide intercellularly, suggesting disruption of both ionic and biochemical coupling. Heterozygous GJs also showed dysfunctional intercellular couplings and hemichannel opening, confirming the dominant‐negative nature of the p.D46E mutation. The Cx26‐T86R mutant protein did not form GJs, since the mutated protein was confined in the cytoplasm and not transported to the cell membrane. When Cx26‐T86R was co‐expressed with Cx26‐WT, ionic and biochemical coupling was normal, consistent with the recessive nature of the mutation. These studies revealed distinct pathogenic mechanisms of two GJB2 mutations identified in Korean families. © 2009 Wiley‐Liss, Inc.     

A novel C202F mutation in the connexin26 gene (GJB2) associated with autosomal dominant isolated hearing loss

Mutations in the GJB2 gene encoding connexin26 (CX26) account for up to 50% of cases of autosomal recessive hearing loss. In contrast, only one GJB2 mutation has been reported to date in an autosomal dominant form of isolated prelingual hearing loss. We report here a novel heterozygous 605G→T mutation in GJB2 in all affected members of a large family with late childhood onset of autosomal dominant isolated hearing loss. The resulting C202F substitution, which lies in the fourth (M4) transmembrane domain of CX26, may impair connexin oligomerisation. Finally, our study suggests that GJB2 should be screened for heterozygous mutations in patients with autosomal dominant isolated hearing impairment, whatever the severity of the disease.


Keywords: C202F mutation; connexin26 gene (GJB2); autosomal dominant hearing loss     

Spectrum and frequencies of non GJB2 gene mutations in Czech patients with early non-syndromic hearing loss detected by gene panel NGS and whole-exome sequencing

Non-syndromic autosomal recessive hearing loss is an extremely heterogeneous disease caused by mutations in more than 80 genes. We examined Czech patients with early/prelingual non-syndromic, presumably genetic hearing loss (NSHL) without known cause after GJB2 gene testing. Four hundred and twenty-one unrelated patients were examined for STRC gene deletions with quantitative comparative fluorescent PCR (QCF PCR), 197 unrelated patients with next-generation sequencing by custom-designed NSHL gene panels and 19 patients with whole-exome sequencing (WES). Combining all methods, we discovered the cause of the disease in 54 patients. The most frequent type of NSHL was DFNB16 (STRC), which was detected in 22 patients, almost half of the clarified patients. Other biallelic pathogenic mutations were detected in the genes: MYO15A, LOXHD1, TMPRSS3 (each gene was responsible for five clarified patients, CDH23 (four clarified patients), OTOG and OTOF (each gene was responsible for two clarified patients). Other genes (AIFM1, CABP2, DIAPH1, PTPRQ, RDX, SLC26A4, TBC1D24, TECTA, TMC1) that explained the cause of hearing impairment were further detected in only one patient for each gene. STRC gene mutations, mainly deletions remain the most frequent NSHL cause after mutations in the GJB2.     

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     

Prevalence of Deafness‐Associated Connexin‐26 (GJB2) and Connexin‐30 (GJB6) Pathogenic Alleles in a Large Patient Cohort from Eastern Sicily

Mutations in the gene encoding the gap junction protein connexin 26 (GJB2) and connexin 30 (GJB6) have been shown to be a major contributor to prelingual, sensorineural, nonsyndromic deafness. The aim of this study was to characterize and establish the prevalence of GJB2 and GJB6 gene alterations in 196 patients affected by sensorineural, nonsyndromic hearing loss, from Eastern Sicily. We performed sequence analysis of GJB2 and identified sequence variants in 68 out of 196 patients (34.7%); (28 homozygous for c.35delG, 22 compound heterozygous and 11 with only one variant allele). We found 12 different allelic variants, the most prevalent being c.35delG, which was found on 89 chromosomes (65.5%), followed by other alleles with different frequencies (p.E47X, c.‐23+1G>A, p.L90P, p.R184W, p.M34T, c.167delT, p.R127H, p.M163V, p.V153I, p.W24X, and p.T8M). Importantly, for the first time we present the frequency and spectrum of GJB2 mutations in NSHL patients from Eastern Sicily. No alterations were found in the GJB6 gene, confirming that alterations in this gene are uncommon in our geographic area. Note that 65.3% and 23.5% of our patients, respectively were found to be negative or carriers by GJB2 molecular screening. This emphasizes the need to broaden the genetic analysis to other genes involved in hearing loss.     

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.     

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.     

Pyrosequencing for detection of mutations in the connexin 26 (GJB2) and mitochondrial 12S RNA (MTRNR1) genes associated with hereditary hearing loss

Hereditary hearing loss (HHL) is one of the most common congenital disorders and is highly heterogeneous. Mutations in the connexin 26 (CX26) gene (GJB2) account for about 20% of all cases of childhood deafness, and approach 50% in documented recessive cases of non-syndromic hearing loss. In addition, a single mitochondrial DNA mutation, mt1555A>G, in the 12S rRNA gene (MTRNR1), is associated with familial cases of progressive deafness. Effective screening of populations for HHL necessitates rapid assessment of several of these potential mutation sites. Pyrosequencing links a DNA synthesis protocol for determining sequence to an enzyme cascade that generates light whenever pyrophosphate is released during primer strand elongation. We assessed the ability of Pyrosequencing to detect common mutations causing HHL. Detection of the most common CX26 mutations in individuals of Caucasian (35delG), Ashkenazi (167delT), and Asian (235delC, V37I) descent was confirmed by Pyrosequencing. A total of 41 different mutations in the CX26 gene and the mitochondrial mt1555A>G mutation were confirmed. Genotyping of up to six different adjacent mutations was achieved, including simultaneous detection of 35delG and 167delT. Accurate and reproducible results were achieved taking advantage of assay flexibility and experimental conditions easily optimized for a high degree of standardization and cost-effectiveness. The standardized sample preparation steps, including target amplification by PCR and preparation of single-stranded template combined with automated sequence reaction and automated genotype scoring, positions this approach as a potentially high throughput platform for SNP/mutation genotyping in a clinical laboratory setting. .     

Factors that affect hearing level in individuals with the mitochondrial 1555A>G mutation

The mitochondrial 1555A>G mutation is one of the most common mutations responsible for hearing loss in Asians. Although the association with aminoglycoside exposure is well known, there is great variation in the severity of hearing loss. We analyzed hearing levels in 221 Japanese individuals with this mutation and attempted to identify relevant covariants including (i) age, (ii) aminoglycoside exposure, (iii) heteroplasmy ratio, and (iv) other gene mutations. At every age, average hearing levels were worse than those in normal subjects, suggesting that mitochondrial function itself may affect the severity of hearing loss. Although the hearing loss in individuals with the 1555A>G mutation progressed with age, the rate did not differ from that of the normal subjects. Those who had reported aminoglycoside exposure had moderate-to-severe hearing impairment regardless of age, confirming that such exposure is the most important environmental variable. We also confirmed the presence of heteroplasmy, which is known to modify the expression of other mitochondrial diseases, but found no evidence for a significant correlation with hearing impairment. A high prevalence of GJB2 heterozygous mutations was noted, indicating that these mutations may exhibit epistatic interaction with the 1555A>G mutation.     

High prevalence of V37I genetic variant in the connexin-26 (GJB2) gene among non-syndromic hearing-impaired and control Thai individuals

Hearing loss is highly prevalent with a worldwide incidence of 1-2 per 1000 newborns. Several previous studies have demonstrated that mutations of connexin 26 (Cx26 or GJB2) are responsible for most cases of the recessive non-syndromic sensorineural hearing loss (NSSHL). Certain mutations have been described frequently among various populations, which include 35delG, 167delT, and 235delC. Recently, a missense mutation, V37I, was reported as a pathogenic change in East Asian affected individuals. To identify genetic variants associated with NSSHL in Thai population, we performed mutation analysis of Cx26 in 166 unrelated probands with NSSHL and 205 controls. We identified seven novel genetic variants in Cx26. We also identified a high prevalence of the V37I mutation among both affected probands (11.1%) and control subjects (8.5%), which suggests that the pathologic role of V37I may be modified by other genes. Our data support previous studies that show heterogeneity in the frequencies and types of mutations in Cx26 within populations and among ethnicities and that before clinical significance and causality can be attributed to a genetic variant, functional characterization is necessary.     

A familial case of Keratitis-Ichthyosis-Deafness (KID) syndrome with the GJB2 mutation G45E

Keratitis-Ichthyosis-Deafness (KID) syndrome (OMIM 148210) is a congenital ectodermal defect. KID consists of an atypical ichthyosiform erythroderma associated with congenital sensorineural deafness. A rare form of the KID syndrome is a fatal course in the first year of life due to severe skin lesion infections and septicaemia. KID appears to be genetically heterogeneous and may be caused by mutations in connexin 26 or connexin 30 genes. GJB2 mutations in the connexin 26 gene are the main cause of the disease. Most of the cases caused by GJB2 mutations are sporadic, but dominant transmission has also been described. To date, the rare lethal form of the disease has been only observed in two Caucasian sporadic patients with the GJB2 mutation, with the p.Gly45Glu (G45E) arising de novo. We have reported an African family with dizygotic twins suffering from a lethal form of KID. The dizygosity of the twins was confirmed by microsatellite markers. The two patients were heterozygous for the G45E mutation of GJB2, whereas the mutation was not detected in the two parents. The unusual transmission of the disease observed in this family could be explained by the occurrence of a somatic or more probably a germinal mosaic in one of the parents.     

Lack of correlation between the type of COL1A1 or COL1A2 mutation and hearing loss in osteogenesis imperfecta patients

Osteogenesis imperfecta (OI) is caused by mutations in COL1A1 and COL1A2 that code for the alpha1 and alpha2 chains of type I collagen. Phenotypes correlate with the mutation types in that COL1A1 null mutations lead to OI type I, and structural mutations in alpha1(I) or alpha2(I) lead to more severe OI types (II-IV). However, correlative analysis between mutation types and OI associated hearing loss has not been previously performed. A total of 54 Finnish OI patients with previously diagnosed hearing loss or age 35 or more years were analyzed here for mutations in COL1A1 or COL1A2. Altogether 49 mutations were identified, of which 41 were novel. The 49 mutations represented the molecular genetic background of 41.1% of the Finnish OI population. A total of 38 mutations were in COL1A1 and 11 were in COL1A2. Of these, 16 were glycine substitutions and 16 were splicing mutations in alpha1(I) or alpha2(I). In addition, 17 null allele mutations were detected in COL1A1. A total of 32 patients (65.3%) with a mutation had hearing loss. That is slightly more than in our previous population study on Finnish adults with OI (57.9%). The association between the mutation types and OI type was statistically evident. Patients with COL1A1 mutations more frequently had blue scleras than those with COL1A2 mutations. In addition, patients with COL1A2 mutations tended to be shorter than those with COL1A1 mutations. However, no correlation was found between the mutated gene or mutation type and hearing pattern. These results suggest that the basis of hearing loss in OI is complex, and it is a result of multifactorial, still unknown genetic effects.