Hela Azaiez

A novel deletion involving the connexin-30 gene, del(GJB6-d13s1854), found in trans with mutations in the GJB2 gene (connexin-26) in subjects with DFNB1 non-syndromic hearing impairment

Hearing impairment is a common and highly heterogeneous sensory disorder. Genetic causes are thought to be responsible for more than 60% of the cases in developed countries.1 In the majority of cases, non-syndromic hearing impairment is inherited in an autosomal recessive pattern.2 Thirty eight different loci and 20 genes for autosomal recessive non-syndromic hearing impairment (ARNSHI) have been identified to date.3 In many populations, up to 50% of all cases of ARNSHI are caused by mutations in the DFNB1 locus (MIM 220290) on 13q12.4 This locus contains the GJB2 gene (MIM 121011), encoding connexin-26 (Cx26),5 which belongs to a family of transmembrane proteins with about 20 members in humans. Hexamers of connexins (connexons) are displayed in the plasma membrane. Docking of connexons on the surfaces of two adjacent cells results in the formation of intercellular gap junction channels.6 Several different connexins, including Cx26, have been shown to participate in the complex gap junction networks of the cochlea.7,8 It has been postulated that these networks play a key role in potassium homeostasis, which is essential for the sound transduction mechanism.9 Given the high prevalence of DFNB1 deafness, molecular testing for GJB2 mutations has become the standard of care for the diagnosis of patients with non-syndromic hearing impairment of unknown cause.10 However, the finding of a large number of affected subjects with only one GJB2 mutant allele complicates the molecular diagnosis of DFNB1 deafness. In different studies, these have accounted for 10–50% of deaf subjects with GJB2 mutations.4 It was hypothesised that there could be other mutations in the DFNB1 locus but outside the GJB2 gene. This hypothesis gained support by the finding of a deletion in the DFNB1 locus outside GJB2 but truncating the neighbouring GJB6 gene (MIM 604418), which …

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.

Utilizing Ethnic-Specific Differences in Minor Allele Frequency to Recategorize Reported Pathogenic Deafness Variants

Ethnic-specific differences in minor allele frequency impact variant categorization for genetic screening of nonsyndromic hearing loss (NSHL) and other genetic disorders. We sought to evaluate all previously reported pathogenic NSHL variants in the context of a large number of controls from ethnically distinct populations sequenced with orthogonal massively parallel sequencing methods. We used HGMD, ClinVar, and dbSNP to generate a comprehensive list of reported pathogenic NSHL variants and re-evaluated these variants in the context of 8,595 individuals from 12 populations and 6 ethnically distinct major human evolutionary phylogenetic groups from three sources (Exome Variant Server, 1000 Genomes project, and a control set of individuals created for this study, the OtoDB). Of the 2,197 reported pathogenic deafness variants, 325 (14.8%) were present in at least one of the 8,595 controls, indicating a minor allele frequency (MAF) > 0.00006. MAFs ranged as high as 0.72, a level incompatible with pathogenicity for a fully penetrant disease like NSHL. Based on these data, we established MAF thresholds of 0.005 for autosomal-recessive variants (excluding specific variants in GJB2) and 0.0005 for autosomal-dominant variants. Using these thresholds, we recategorized 93 (4.2%) of reported pathogenic variants as benign. Our data show that evaluation of reported pathogenic deafness variants using variant MAFs from multiple distinct ethnicities and sequenced by orthogonal methods provides a powerful filter for determining pathogenicity. The proposed MAF thresholds will facilitate clinical interpretation of variants identified in genetic testing for NSHL. All data are publicly available to facilitate interpretation of genetic variants causing deafness.

Copy number variants are a common cause of non-syndromic hearing loss

BackgroundCopy number variants (CNVs) are a well-recognized cause of genetic disease; however, methods for their identification are often gene-specific, excluded as ‘routine’ in screens of genetically heterogeneous disorders, and not implemented in most next-generation sequencing pipelines. For this reason, the contribution of CNVs to non-syndromic hearing loss (NSHL) is most likely under-recognized. We aimed to incorporate a method for CNV identification as part of our standard analysis pipeline and to determine the contribution of CNVs to genetic hearing loss.MethodsWe used targeted genomic enrichment and massively parallel sequencing to isolate and sequence all exons of all genes known to cause NSHL. We completed testing on 686 patients with hearing loss with no exclusions based on type of hearing loss or any other clinical features. For analysis we used an integrated method for detection of single nucleotide changes, indels and CNVs. CNVs were identified using a previously published method that utilizes median read-depth ratios and a sliding-window approach.ResultsOf 686 patients tested, 15.2% (104) carried at least one CNV within a known deafness gene. Of the 38.9% (267) of individuals for whom we were able to determine a genetic cause of hearing loss, a CNV was implicated in 18.7% (50). We identified CNVs in 16 different genes including 7 genes for which no CNVs have been previously reported. CNVs of STRC were most common (73% of CNVs identified) followed by CNVs of OTOA (13% of CNVs identified).ConclusionCNVs are an important cause of NSHL and their detection must be included in comprehensive genetic testing for hearing loss.

A NOVEL DFNB1 DELETION ALLELE SUPPORTS THE EXISTENCE OF A DISTANT CIS-REGULATORY REGION THAT CONTROLS GJB2 AND GJB6 EXPRESSION

Wilch E, Azaiez H, Fisher RA, Elfenbein J, Murgia A, Birkenhäger R, Bolz HJ, da Silva‐Costa SM, del Castillo I, Haaf T, Hoefsloot L, Kremer H, Kubisch C, Le Marechal C, Pandya A, Sartorato EL, Schneider E, Van Camp G, Wuyts W, Smith RJH, Friderici KH. A novel DFNB1 deletion allele supports the existence of a distant cis‐regulatory region that controls GJB2 and GJB6 expression.

TBC1D24 Mutation Causes Autosomal-Dominant Nonsyndromic Hearing Loss

Hereditary hearing loss is extremely heterogeneous. Over 70 genes have been identified to date, and with the advent of massively parallel sequencing, the pace of novel gene discovery has accelerated. In a family segregating progressive autosomal‐dominant nonsyndromic hearing loss (NSHL), we used OtoSCOPE® to exclude mutations in known deafness genes and then performed segregation mapping and whole‐exome sequencing to identify a unique variant, p.Ser178Leu, in TBC1D24 that segregates with the hearing loss phenotype. TBC1D24 encodes a GTPase‐activating protein expressed in the cochlea. Ser178 is highly conserved across vertebrates and its change is predicted to be damaging. Other variants in TBC1D24 have been associated with a panoply of clinical symptoms including autosomal recessive NSHL, syndromic hearing impairment associated with onychodystrophy, osteodystrophy, mental retardation, and seizures (DOORS syndrome), and a wide range of epileptic disorders.

High-Throughput Genetic Testing for Thrombotic Microangiopathies and C3 Glomerulopathies

The thrombotic microangiopathies (TMAs) and C3 glomerulopathies (C3Gs) include a spectrum of rare diseases such as atypical hemolytic uremic syndrome, thrombotic thrombocytopenic purpura, C3GN, and dense deposit disease, which share phenotypic similarities and underlying genetic commonalities. Variants in several genes contribute to the pathogenesis of these diseases, and identification of these variants may inform the diagnosis and treatment of affected patients. We have developed and validated a comprehensive genetic panel that screens all exons of all genes implicated in TMA and C3G. The closely integrated pipeline implemented includes targeted genomic enrichment, massively parallel sequencing, bioinformatic analysis, and a multidisciplinary conference to analyze identified variants in the context of each patients specific phenotype. Herein, we present our 1-year experience with this panel, during which time we studied 193 patients. We identified 17 novel and 74 rare variants, which we classified as pathogenic (11), likely pathogenic (12), and of uncertain significance (68). Compared with controls, patients with C3G had a higher frequency of rare and novel variants in C3 convertase (C3 and CFB) and complement regulator (CFH, CFI, CFHR5, and CD46) genes (P<0.05). In contrast, patients with TMA had an increase in rare and novel variants only in complement regulator genes (P<0.01), a distinction consistent with differing sites of complement dysregulation in these two diseases. In summary, we were able to provide a positive genetic diagnosis in 43% and 41% of patients carrying the clinical diagnosis of C3G and TMA, respectively.

Mutations in Grxcr1 Are The Basis for Inner Ear Dysfunction in the Pirouette Mouse

Recessive mutations at the mouse pirouette (pi) locus result in hearing loss and vestibular dysfunction due to neuroepithelial defects in the inner ear. Using a positional cloning strategy, we have identified mutations in the gene Grxcr1 (glutaredoxin cysteine-rich 1) in five independent allelic strains of pirouette mice. We also provide sequence data of GRXCR1 from humans with profound hearing loss suggesting that pirouette is a model for studying the mechanism of nonsyndromic deafness DFNB25. Grxcr1 encodes a 290 amino acid protein that contains a region of similarity to glutaredoxin proteins and a cysteine-rich region at its C terminus. Grxcr1 is expressed in sensory epithelia of the inner ear, and its encoded protein is localized along the length of stereocilia, the actin-filament-rich mechanosensory structures at the apical surface of auditory and vestibular hair cells. The precise architecture of hair cell stereocilia is essential for normal hearing. Loss of function of Grxcr1 in homozygous pirouette mice results in abnormally thin and slightly shortened stereocilia. When overexpressed in transfected cells, GRXCR1 localizes along the length of actin-filament-rich structures at the dorsal-apical surface and induces structures with greater actin filament content and/or increased lengths in a subset of cells. Our results suggest that deafness in pirouette mutants is associated with loss of GRXCR1 function in modulating actin cytoskeletal architecture in the developing stereocilia of sensory hair cells.

Characterising the spectrum of autosomal recessive hereditary hearing loss in Iran

Background Countries with culturally accepted consanguinity provide a unique resource for the study of rare recessively inherited genetic diseases. Although hereditary hearing loss (HHL) is not uncommon, it is genetically heterogeneous, with over 85 genes causally implicated in non-syndromic hearing loss (NSHL). This heterogeneity makes many gene-specific types of NSHL exceedingly rare. We sought to define the spectrum of autosomal recessive HHL in Iran by investigating both common and rarely diagnosed deafness-causing genes. Design Using a custom targeted genomic enrichment (TGE) panel, we simultaneously interrogated all known genetic causes of NSHL in a cohort of 302 GJB2-negative Iranian families. Results We established a genetic diagnosis for 67% of probands and their families, with over half of all diagnoses attributable to variants in five genes: SLC26A4, MYO15A, MYO7A, CDH23 and PCDH15. As a reflection of the power of consanguinity mapping, 26 genes were identified as causative for NSHL in the Iranian population for the first time. In total, 179 deafness-causing variants were identified in 40 genes in 201 probands, including 110 novel single nucleotide or small insertion–deletion variants and three novel CNV. Several variants represent founder mutations. Conclusion This study attests to the power of TGE and massively parallel sequencing as a diagnostic tool for the evaluation of hearing loss in Iran, and expands on our understanding of the genetics of HHL in this country. Families negative for variants in the genes represented on this panel represent an excellent cohort for novel gene discovery.

PDZD7 and hearing loss: more than just a modifier

Deafness is the most frequent sensory disorder. With over 90 genes and 110 loci causally implicated in non‐syndromic hearing loss, it is phenotypically and genetically heterogeneous. Here, we investigate the genetic etiology of deafness in four families of Iranian origin segregating autosomal recessive non‐syndromic hearing loss (ARNSHL). We used a combination of linkage analysis, homozygosity mapping, and a targeted genomic enrichment platform to simultaneously screen 90 known deafness‐causing genes for pathogenic variants. Variant segregation was confirmed by Sanger sequencing. Linkage analysis and homozygosity mapping showed segregation with the DFNB57 locus on chromosome 10 in two families. Targeted genomic enrichment with massively parallel sequencing identified causal variants in PDZD7: a homozygous missense variant (p.Gly103Arg) in one family and compound heterozygosity for missense (p.Met285Arg) and nonsense (p.Tyr500Ter) variants in the second family. Screening of two additional families identified two more variants: (p.Gly228Arg) and (p.Gln526Ter). Variant segregation with the hearing loss phenotype was confirmed in all families by Sanger sequencing. The missense variants are predicted to be deleterious, and the two nonsense mutations produce null alleles. This report is the first to show that mutations in PDZD7 cause ARNSHL, a finding that offers addition insight into the USH2 interactome. We also describe a novel likely disease‐causing mutation in CIB2 and illustrate the complexity associated with gene identification in diseases that exhibit large genetic and phenotypic heterogeneity.