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.     

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     

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     

Connexin-26–associated deafness: Phenotypic variability and progression of hearing loss

PurposeTo evaluate genotype-phenotype correlation over time for a cohort of children with connexin-26 (GJB2)–associated autosomal recessive hearing loss.MethodsFifty-two children were identified from a database of individuals with homozygous or compound heterozygous mutations in GJB2 and subjected to chart review of their otolaryngologic and serial audiometric evaluations. Genotype-phenotype correlations were identified among the members of this group by appropriate statistical analyses.ResultsHearing loss was most severe in individuals with two truncating mutations in GJB2 and mildest in those with two nontruncating mutations. Progressive hearing loss was seen directly by serial audiometry in 24% of all subjects, and suggested in a total of 28% when those with normal newborn hearing screens and subsequent hearing loss were included. Progression was particularly common among carriers of the p.V37I allele either in homozygosity or in compound heterozygosity with a truncating allele; these children are primarily of Asian descent and demonstrate mild, slowly progressive hearing loss.ConclusionsPhenotype in GJB2-associated hearing loss is correlated with genotype, with truncating mutations giving rise to more severe hearing loss. Progression of hearing loss is not uncommon, especially in association with the p.V37I allele. These results suggest that close audiometric follow-up is warranted for patients with GJB2-associated recessive hearing loss.     

The ACE I allele is associated with increased risk for ruptured intracranial aneurysms

Genetic and environmental factors play roles in the aetiology of ruptured intracranial aneurysms. Hypertension has been reported as a risk factor for intracranial aneurysm haemorrhage. We have tested if genotypes at the angiotensin converting enzyme (ACE) gene locus are associated with ruptured intracranial aneurysms. The insertion/deletion polymorphism in the ACE gene was genotyped in 258 subjects presenting in East Anglia with ruptured intracranial aneurysms (confirmed at surgery or angiographically) and 299 controls from the same region. ACE allele frequencies were significantly different in the cases and the controls (alleles χ²₁=4.67, p=0.03). The I allele was associated with aneurysm risk (odds ratio for I allele v D allele = 1.3 (95% CI=1.02-1-65); odds ratio for II v DD genotype = 1.67 (95% CI=1.04-2.66)). The I allele at the ACE locus is over-represented in subjects with ruptured intracranial aneurysms. These data are supported by non-significant trends in the same direction in two previous smaller studies. Thus, this allele may be associated with risk for ruptured intracranial aneurysms.


Keywords: ACE I allele; ruptured intracranial aneurysms     

M34T and V37I mutations in GJB2 associated hearing impairment: evidence for pathogenicity and reduced penetrance

Despite research the role of the M34T and V37I variants of GJB2 in causing hearing impairment (HI) remains controversial. Our purpose was to test a hypothesis that M34T and V37I are pathogenic but have distinct features resulting in a reduced penetrance. We screened for known GJB2/GJB6 mutations 233 Polish consecutive unrelated subjects with non-syndromic, sensorineural HI who were previously found to carry 35delG mutation on one chromosome. The most frequent mutations were also analyzed in approximately 1,000 controls. We found that M34T and V37I were significantly (P < 10(-6)) overrepresented among patients, but their penetrance was estimated as 1/10 relative to mutations of undisputed pathogenicity. This finding apparently could not be explained by low degree of HI associated with M34T and V37I since another mutation causing comparably mild HI (L90P) did not have reduced penetrance. Subsequent analyses showed that the patients with M34T/35delG and V37I/35delG had significantly later onset of HI than patients with other genotypes (P < 10(-6)) including the L90P/35delG (P = 0.006). Also, among these patients (but not others) a strong correlation between the degree of HI and its duration was found (r = 0.79, P < 10(-5)). We tentatively suggest that M34T and V37I might cause mild HI characterized by relatively late onset and progression.     

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.     

Pathogenetic role of the deafness-related M34T mutation of Cx26

Mutations in the GJB2 gene, which encodes the gap junction protein connexin26 (Cx26), are the major cause of genetic non-syndromic hearing loss. The role of the allelic variant M34T in causing hereditary deafness remains controversial. By combining genetic, clinical, biochemical, electrophysiological and structural modeling studies, we have re-assessed the pathogenetic role of the M34T mutation. Genetic and audiological data indicate that the majority of heterozygous carriers and all five compound heterozygotes exhibited an impaired auditory function. Functional expression in transiently transfected HeLa cells showed that, although M34T was correctly synthesized and targeted to the plasma membrane, it inefficiently formed intercellular channels that displayed an abnormal electrical behavior and retained only 11% of the unitary conductance of the wild-type protein (HCx26wt). Moreover, M34T channels failed to support the intercellular diffusion of Lucifer Yellow and the spreading of mechanically induced intercellular Ca2+ waves. When co-expressed together with HCx26wt, M34T exerted dominant-negative effects on cell-cell coupling. Our findings are consistent with a structural model, predicting that the mutation leads to a constriction of the channel pore. These data support the view that M34T is a pathological variant of Cx26 associated with hearing impairment.     

A new large deletion in the DFNB1 locus causes nonsyndromic hearing loss

Mutations in the GJB2 gene encoding the gap junction protein connexin 26 are responsible for up to 30% of all cases of autosomal recessive nonsyndromic hearing impairment (HI) with prelingual onset in most populations. The corresponding locus DFNB1, located on chromosome 13q11–q12, is also affected by three distinct deletions. These deletions extended distally to GJB2, which remains intact.We report a novel large deletion in DFNB1 observed in a patient presenting profound prelingual HI. This deletion was observed in trans to a GJB2 mutated allele carrying the p.Val84Met (V84M) mutation and was shown to be associated with hearing loss. The deletion caused a false homozygosity of V84M in the proband. Quantification of alleles by quantitative fluorescent multiplex PCR (QFM-PCR) enabled us to study the breakpoints of the deletion. The deleted segment extended through at least 920 kb and removed the three connexin genes GJA3, GJB2 and GJB6. The distal breakpoint inside intron 2 of CRYL1 gene differed from the breakpoints of the known DFNB1 deletions. This case highlights the importance of screening for large deletions in molecular studies of GJB2.     

Consensus interpretation of the p.Met34Thr and p.Val37Ile variants in GJB2 by the ClinGen Hearing Loss Expert Panel

PurposePathogenic variants in GJB2 are the most common cause of autosomal recessive sensorineural hearing loss. The classification of c.101T>C/p.Met34Thr and c.109G>A/p.Val37Ile in GJB2 are controversial. Therefore, an expert consensus is required for the interpretation of these two variants.MethodsThe ClinGen Hearing Loss Expert Panel collected published data and shared unpublished information from contributing laboratories and clinics regarding the two variants. Functional, computational, allelic, and segregation data were also obtained. Case–control statistical analyses were performed.ResultsThe panel reviewed the synthesized information, and classified the p.Met34Thr and p.Val37Ile variants utilizing professional variant interpretation guidelines and professional judgment. We found that p.Met34Thr and p.Val37Ile are significantly overrepresented in hearing loss patients, compared with population controls. Individuals homozygous or compound heterozygous for p.Met34Thr or p.Val37Ile typically manifest mild to moderate hearing loss. Several other types of evidence also support pathogenic roles for these two variants.ConclusionResolving controversies in variant classification requires coordinated effort among a panel of international multi-institutional experts to share data, standardize classification guidelines, review evidence, and reach a consensus. We concluded that p.Met34Thr and p.Val37Ile variants in GJB2 are pathogenic for autosomal recessive nonsyndromic hearing loss with variable expressivity and incomplete penetrance.     

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.     

Identification of a novel in-frame deletion in KCNQ4 (DFNA2A) and evidence of multiple phenocopies of unknown origin in a family with ADSNHL

Autosomal dominant sensorineural hearing loss (ADSNHL) is extremely genetically heterogeneous, making it difficult to molecularly diagnose. We identified a multiplex (n=28 affected) family from the genetic isolate of Newfoundland, Canada with variable SNHL and used a targeted sequencing approach based on population-specific alleles in WFS1, TMPRSS3 and PCDH15; recurrent mutations in GJB2 and GJB6; and frequently mutated exons of KCNQ4, COCH and TECTA. We identified a novel, in-frame deletion (c.806_808delCCT: p.S269del) in the voltage-gated potassium channel KCNQ4 (DFNA2), which in silico modeling predicts to disrupt multimerization of KCNQ4 subunits. Surprisingly, 10/23 deaf relatives are non-carriers of p.S269del. Further molecular characterization of the DFNA2 locus in deletion carriers ruled out the possibility of a pathogenic mutation other than p.S269del at the DFNA2A/B locus and linkage analysis showed significant linkage to DFNA2 (maximum LOD=3.3). Further support of genetic heterogeneity in family 2071 was revealed by comparisons of audio profiles between p.S269del carriers and non-carriers suggesting additional and as yet unknown etiologies. We discuss the serious implications that genetic heterogeneity, in this case observed within a single family, has on molecular diagnostics and genetic counseling.     

Connexin 26 gene (GJB2) mutation modulates the severity of hearing loss associated with the 1555A→G mitochondrial mutation

We report a high prevalence of GJB2 heterozygous mutations in patients bearing the 1555A→G mitochondrial mutation, and describe a family in which potential interaction between GJB2 and a mitochondrial gene appears to be the cause of hearing impairment. Patients who are heterozygotes for the GJB2 mutant allele show hearing loss more severe than that seen in sibs lacking a mutant GJB2 allele, suggesting that heterozygous GJB2 mutations may synergistically cause hearing loss when in the presence of a 1555A→G mutation. The present findings indicate that GJB2 mutations may sometimes be an aggravating factor, in addition to aminoglycoside antibiotics, in the phenotypic expression of the non‐syndromic hearing loss associated with the 1555A→G mitochondrial mutation. © 2001 Wiley‐Liss, Inc.     

DNA sequence analysis of GJB2, encoding connexin 26: observations from a population of hearing impaired cases and variable carrier rates, complex genotypes, and ethnic stratification of alleles among controls

Mutations in GJB2 are associated with hereditary hearing loss. DNA sequencing of GJB2 in a cohort of hearing impaired patients and a multi-ethnic control group is reported. Among 610 hearing impaired cases, 43 DNA sequence variations were identified in the coding region of GJB2 including 24 mutations, 8 polymorphisms, 3 unclassified variants (G4D, R127C, M163V), 1 controversial variant (V37I), and 7 novel variants (G12C, N14D, V63A, T86M, L132V, D159, 592_600delinsCAGTGTTCATGACATTC). Sixteen non-coding sequence variations were also identified among cases including the IVS1+1A>G mutation, 2 polymorphisms, and 13 novel variants. A diagnosis of GJB2-associated hearing loss was confirmed for 63 cases (10.3%). Heterozygous mutations were found in 39 cases (6.4%). Eleven cases carrying novel or unclassified variants (1.8 %) and 18 cases carrying the controversial V37I variant were identified (3%). In addition, 294 control subjects from 4 ethnic groups were sequenced for GJB2. Thirteen sequence variations in the coding region of GJB2 were identified among controls including 2 mutations, 6 polymorphisms, 2 unclassified variants (G4D, T123N), 1 controversial variant (V37I), and 2 novel variants (R127L, V207L). Nine sequence variations were identified among controls in the non-coding regions in and around GJB2 exon 2. Of particular interest among controls were the variability in carrier rates and ethnic stratification of alleles, and the complex genotypes among Asians, 47% of whom carried two to four sequence variations in the coding region of GJB2. These data provide new information about carrier rates for GJB2-based hearing loss in various ethnic groups and contribute to evaluation of the pathogenicity of the controversial V37I variant.     

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.     

Identification of a novel psoriasis susceptibility locus at 1p and evidence of epistasis between PSORS1 and candidate loci

The pathogenesis of all forms of psoriasis remains obscure. Segregation analysis and twin studies together with ethnic differences in disease frequency all point to an underlying genetic susceptibility to psoriasis, which is both complex and likely to reflect the action of a number of genes. We performed a genome wide analysis using a total of 271 polymorphic autosomal markers on 284 sib relative pairs identified within 158 independent families. We detected evidence for linkage at 6p21 (PSORS1) with a non-parametric linkage score (NPL)=4.7, p=2 × 10^-6 and at chromosome 1p (NPL=3.6, p=1.9 × 10^-4) in all families studied. Significant excess (p=0.004) paternal allele sharing was detected for markers spanning the PSORS1 locus. A further three regions reached NPL scores of 2 or greater, including a region at chromosome 7 (NPL 2.1), for which linkage for a number of autoimmune disorders has been reported. Partitioning of the data set according to allele sharing at 6p21 (PSORS1) favoured linkage to chromosomes 2p (NPL 2.09) and 14q (NPL 2.0), both regions implicated in previous independent genome scans, and suggests evidence for epistasis between PSORS1 and genes at other genomic locations. This study has provided linkage evidence in favour of a novel susceptibility locus for psoriasis and provides evidence of the complex mechanisms underlying the genetic predisposition to this common skin disease.


Keywords: psoriasis; PSORS1; epistasis     

The ACE gene and Alzheimer's disease susceptibility

A recent study suggested that the insertion (I) allele in intron 16 of the angiotensin converting enzyme gene (ACE) is associated with Alzheimer's disease (AD) risk. In our series of 239 necropsy confirmed late onset AD cases and 342 elderly non-demented controls aged >73 years, we found significantly different ACE genotype distributions in the case and control groups (p=0.007). Homozygotes for both the I and D alleles were associated with a higher risk compared to DI heterozygotes. While the APOE ε4 allele was strongly associated with AD risk in our series, we found no evidence for an interaction between the APOE and ACE loci. In addition, no interactions were observed between ACE and gender or age at death of the AD cases. A meta-analysis of all published reports (12 case-control series in total) suggested that both the II and ID ACE genotypes are associated with increased AD risk (odds ratio (OR) for II v DD 1.36, 95% confidence interval (CI)=1.13-1.63, OR for DI v DD 1.33, 95% CI=1.14-1.53, p=0.0002).


Keywords: Alzheimer's disease; ACE gene; I allele     

Evaluation of newborn screening bloodspot-based genetic testing as second tier screen for bedside newborn hearing screening

PurposeBedside newborn hearing screening is highly successful in identifying deaf or hard-of-hearing infants. However, newborn hearing screening protocols have high loss to follow-up rates. We propose that bloodspot-based genetic testing for GJB2 alleles can provide a means for rapid confirmation in a subset of infants who fail bedside newborn hearing screening.MethodsWe performed a case-control study comparing the prevalence of common GJB2 mutations from deidentified bloodspots designated as “refer” by newborn hearing screening and contemporaneously selected randomly chosen controls designated as “pass.” Between March 2006 and December 2007, 2354 spots were analyzed for common alleles, c.35delG, c.167delT, c.235delC, and p.V37I in GJB2 with a subset reanalyzed by conventional Sanger sequencing to search for additional alleles.ResultsThe prevalence of biallelic GJB2 mutations in bloodspots from infants who referred by newborn hearing screening is approximately 1 in 50 (23/1177). In contrast, one bloodspot from an infant who passed newborn hearing screening was identified to harbor biallelic GJB2 mutations.ConclusionsThese findings show that when a newborn refers by newborn hearing screening, there is a significant chance that GJB2-related hearing loss is present. Bloodspot-based genetic testing for common GJB2 alleles should be considered as second tier testing for bedside newborn hearing screening.     

Apolipoprotein E and familial longevity

Exceptional longevity is associated with substantial heritability. The ?4 allele in apolipoprotein E and the linked G allele in rs2075650 of TOMM40 have been associated with increased mortality and the ?2 allele with decreased mortality, although inconsistently. Offspring from long-lived families and spouse controls were recruited at 3 sites in the United States and Denmark. We used generalized estimating equations to compare the likelihood of carrying risk alleles in offspring (n = 2307) and spouse controls (n = 764), adjusting for age, sex, level of education, and family membership. The likelihood of carrying an APOE ?4 allele or a G allele in rs2075650 was lower (odds ratio [OR], 0.75; p = 0.005 and OR, 0.70; p = 0.002) and the likelihood of carrying an APOE ?2 allele was higher (OR, 1.5; p = 0.007) among family members in the offspring generation than among their spouse controls. Our findings support the hypothesis that both reduction in the frequency of the ?4 allele and increase in the frequency of the ?2 allele contribute to longevity.     

Coincidence of mutations in different connexin genes in Hungarian patients

Mutations in the GJB2 gene are the most common cause of hereditary prelingual sensorineural hearing impairment in Europe. Several studies indicate that different members of the connexin protein family interact to form gap junctions in the inner ear. Mutations in different connexin genes may accumulate and, consequently lead to hearing impairment. Therefore, we screened 47 Hungarian GJB2- heterozygous (one mutation in coding exon of the GJB2 gene) patients with hearing impairment for DNA changes in two further connexin genes (GJB6 and GJB3) and in the 5' non-coding region of GJB2 including the splice sites. Eleven out of 47 GJB2-heterozygous patients analyzed carried the splice site mutation -3170G>A in the 5'UTR region of GJB2. One out of these 11 patients showed homozygous -3170G>A genotype in combination with p.R127H. Next to the GJB2 mutations we noted 2 cases of deletion in GJB6 [Delta(GJB6-D13S1830)] and 3 (2 new and 1 described) base substitutions in GJB3 [c.357C>T, c.798C>T and c.94C>T (p.R32W)] which are unlikely disease-causing. Our results suggest the importance of routine screening for the rather frequent -3170G>A mutation (in addition to c.35delG) in patients with hearing impairment.