Abstract
Genetic variants in GJB2 and GJB6 genes are the most frequent causes of hereditary hearing loss among several deaf populations worldwide. Molecular diagnosis enables proper genetic counseling and medical prognosis to patients. In this study, we present an update of testing results in a cohort of Argentinean non-syndromic hearing-impaired individuals. A total of 48 different sequence variants were detected in genomic DNA from patients referred to our laboratory. They were manually curated and classified based on the American College of Medical Genetics and Genomics/Association for Molecular Pathology ACMG/AMP standards and hearing-loss-gene-specific criteria of the ClinGen Hearing Loss Expert Panel. More than 50% of sequence variants were reclassified from their previous categorization in ClinVar. These results provide an accurately interpreted set of variants to be taken into account by clinicians and the scientific community, and hence, aid the precise genetic counseling to patients.
Keywords: GJB2, GJB6, genetic variants, curation, hearing loss, argentina
1. Introduction
Congenital hearing loss (HL) is the most common sensory disorder that affects approximately 1–2 of 1000 infants, with 50% of cases resulting from genetic factors [1]. In 70–80% of neonates who fail newborn hearing screening, no other distinguishing physical findings are present and the HL is classified as non-syndromic. The majority of non-syndromic cases are of autosomal recessive inheritance (80%), 12–15% autosomal dominant, 1–5% X-linked and 1–5% mitochondrial [2]. In general, autosomal recessive loci are related to a prelingual HL, while autosomal dominant loci to a postlingual HL phenotype [3]. A large number of genes are involved in hereditary HL. To date, a total of 121 non-syndromic causative genes have been described: 76 of recessive inheritance, 49 of dominant inheritance and five X-linked (some genes can cause recessive and dominant hearing impairment) [4]. This landscape illustrates the auditory system complexity, comprising a large number of proteins, which together participate in hearing physiology and development [5].
Despite the wide genetic heterogeneity of hearing impairment, the most commonly mutated genes in severe to profound autosomal recessive non-syndromic hearing loss (ARNSHL) are GJB2 and GJB6 (encoding connexin-26 and 30, respectively), accounting for nearly 50% of the cases in most populations around the Mediterranean Sea [6,7,8,9,10,11]. GJB2 and GJB6 (DFNB1) genes are part of a gene family that encode gap-junction proteins. It is well demonstrated that they are expressed in cochlear supporting cells, with a role in endolymph potassium recycling, inositol triphosphate (IP3) transfer and diffusion of different metabolites [12,13]. Connexins (Cx) are formed of four transmembrane domains, two extracellular loops and three cytoplasmic domains: the amino-terminus, a cytoplasmic loop and the carboxy-terminus domain [13].
Both GJB2 and GJB6 are located in chromosome 13q12. The GJB2 gene comprises two exons and the coding region is completely contained in the second exon, leading to a 2290-nucleotide mRNA (GeneBank: NM_004004.6). On the other hand, the GJB6 gene consists of five exons and the last one contains the entire coding sequence which is transcribed to a 2110 bp mRNA (GeneBank: NM_006783.4). In general, mutations which produce a loss of gap-junction channel function are related to non-syndromic ARSHL [14,15,16].
The most frequent mutation in GJB2 is c.35delG in the Caucasian population [7,9,17,18,19]. In addition, there are more than 300 pathogenic variants identified in GJB2 (Deafness Variation Database) [20]. In the case of GJB6, two large deletions of 309 and 232-kb, del(GJB6-D13S1830) and del(GJB6-D13S1854), respectively, in the 5′ region of the gene, along with other rarer and less studied deletions, have been described [10,21,22,23,24,25]. Previous studies have demonstrated that different cohorts of Argentinean patients carry similar frequent genetic variants in GJB2 and GJB6 [26,27,28,29,30,31].
Identifying the genetic etiology of hearing impairment can provide proper counseling, clinical management and accurate estimation of deafness odds recurrence within a family [29,30]. Moreover, molecular diagnosis contributes with valuable prognosis information: DFNB1-hearing loss is not related to other phenotypic symptoms nor to significant hearing loss progression over time, and in general, it is related to a congenital profound bilateral hearing loss [32]. Affected probands carrying two truncating/nonsense variants in GJB2 present a more severe degree of hearing loss than those who carry two missense variants [18,28,33,34]. Furthermore, patients with GJB2 genetic variants present excellent outcomes in speech perception/production skills after cochlear implantation [35,36,37]. Therefore, correct interpretation of the phenotypic consequences of genetic variants is crucial in genetic diagnosis, since discrepancies in sequence variant interpretation and classification has been reported to lead to serious impact in patient health maintenance [38,39,40]. Thus, the American College of Medical Genetics and Genomics (ACMG) and the Association for Molecular Pathology (AMP) has developed guidelines for clinical interpretation of genetic variants [41]. In addition, the ClinGen Hearing Loss Clinical Domain Working Group (HLWG) has adapted the ACMG/AMP guidelines for the classification of genetic variants in the hearing loss framework [28,42].
In the present study we aimed to identify causative mutations in GJB2 and GJB6 genes in Argentinean non-syndromic hearing-impaired patients and report an update of allele and genotype frequencies. Furthermore, we performed a thorough manual curation of sequence variants according to ACMG/AMP standards and applied rigorously the latest hearing loss gene-specific criteria of the ClinGen Hearing Loss Expert Panel (HL-EP) [41,42]. These findings clearly highlight the importance of genetic studies with the appropriate comprehensive analysis by experts in the field, with the goal of providing an accurate molecular diagnosis, and consequently, precise genetic counseling to the patients.
2. Materials and Methods
2.1. Part A: Identification of Variants in an Argentinean Cohort
2.1.1. Patients
This study includes a total of 600 Argentinean non-related patients (290 females and 310 males) with non-syndromic sensorineural hearing loss. Clinical evaluation was performed by a clinical geneticist and included: personal history, physical examination, audiometric information, age of hearing impairment onset, hearing thresholds, pedigree and genetic assessment. For each patient, a complete medical history was obtained to exclude the possibility of environmental causes of hearing impairment (e.g., ototoxic drugs, infectious diseases, acoustic trauma). All subjects gave their informed consent for inclusion before they participated in the study. The study was conducted in accordance with the Declaration of Helsinki, and the protocol was approved by the Ethics Committee of Administración Nacional de Laboratorios e Institutos de Salud (ANLIS) (1912–2018). The workflow is summarized in Figure 1A.
A total of 477 sporadic and 123 familial cases (80% and 20%, respectively) were sequentially referred to the Laboratory of Physiology and Genetic of Hearing, INGEBI, in Buenos Aires, Argentina from 2004 to March 2020. Familial cases were of a dominant and recessive form of inheritance (59 and 64/123). All patients were analyzed by an ear-nose-throat (ENT) specialist using standard methods. The severity of deafness was classified considering the following thresholds in decibels: mild (20 to 39 dB), moderate (40 to 69 dB), severe (70 to 89 dB) and profound (90 dB). The patient’s deafness severity was defined by the ear with the minor degree of hearing loss. Complete audiological history data were compiled from affected subjects in case of need. Overall, 479 of patients (47%) exhibited prelingual HL, while 121 (20%) a postlingual phenotype. The severity of HL was: 102 moderate, 106 severe and 392 profound. A total of 69 patients were cochlear implanted. This prospective study (2004–2020) includes data previously reported in Dalamón et al. 2013 (n = 476 patients), but that was not curated following HL-EP standards.
2.1.2. Samples
Genomic DNA was isolated from whole blood samples extracted with 5% ethylene-diamine tetraacetic acid (EDTA) (Sigma-Aldrich, St. Louis, MO, USA) using the cetyltrimethyl-ammonium bromide (CTAB) (Sigma-Aldrich, St. Louis, MO, USA) method [43]. DNA concentration and quality were measured by absorbance at 260 nm and by the A260 nm/A280 nm and A260 nm/A230 nm ratios, respectively (NanoDropTM) (Thermo Fisher Scientific, Wilmington, NC, USA). All samples were stored at −20 °C.
2.1.3. GJB2/GJB6 Molecular Studies
Genetic variants in GJB2 were studied by direct sequencing of the coding exon 2, non-coding exon 1 and intronic boundaries. The splice site variants c.-23+1G>A and c.-22-2A>C were included in the screening. Primers, protocols and cycling programs used were as previously reported [28]. Bidirectional DNA sequencing was performed on an automatic sequencer (3730xl DNA Analyzer, Applied Biosystems, Foster City, CA, USA). Sequences obtained were analyzed by CodonCodeAligner program [44] and the BLAST NCBI interface (Basic local alignment search tool) [45] using the consensus sequence of GJB2 gene (GeneBank NG_008358.1). To examine the large deletions in GJB6:del(GJB6-D13S1830) and del(GJB6-D13S1854), a GAP-PCR and subsequent analysis were performed according to reported protocols [10,21].
2.1.4. Data Analysis
In order to establish a genotype/phenotype correlation, presumably pathogenic identified GJB2 allele variants were classified as truncating (T) and non-truncating (NT) mutations [46]. Truncating mutations are loss-of-function (LoF) and include nonsense variants, insertions, deletions and duplications that introduce a shift in reading frame leading to a premature termination of protein translation, as well as the donor splice-site variant c.-23+1G>A leading to non-functional mRNA. Both del(GJB6-D13S1830) and del(GJB6-D13S1854) were also classified as truncating, because they lead to a nearly complete absence of Cx26 protein expression [10,47,48]. The group of non-truncating variants consists of missense variants (leading to amino acid substitutions) and the in-frame deletion (delGlu120). The acceptor splice-site variant c.-22-2A>C was defined as non-truncating since a residual expression of the wild type transcript due to the activation of an alternative acceptor splice site has been reported [49]. A chi square statistical analysis was performed in order to analyze the differences between groups.
2.2. Part B: Curation of Variants
GJB2-GJB6 Variant Curation
Manual curation of variants required information gathered from: population data, genotypes, segregation, phenotypic features and functional and experimental data of the reported variants. Nomenclature of sequence variants identified were achieved according to HGVS standards [50] and manually revised with the Mutalyzer name-checker tool [51]. Computational and predictive evidence was performed in silico through diverse strategies according to the type of variant analyzed: missense variants with REVEL [52] and Combined Annotation Dependent Depletion (CADD) [53] tools, splice site and silent variants with Human Splicing Finder [54] and MaxEntScan softwares [55] and loss of function variants (nonsense, frameshift and canonical splice site) following the ACMG/AMP recommendations [56]. REVEL, CADD and MaxEntScan scores were determined with the Variant Effect Predictor tool [57].
Reports in PubMed, as well as internal data from our laboratory, and seven different databases were used: 1. gnomAD [58], 2. dbSNP [59], 3. NHLBI-ESP’s EVS [60], 4. ClinVar [61], 5. LOVD [62], 6. Deafness Variation Database (DVD) [20] and 7. Database of Genomics Variants [63,64]. Variant filtering allele frequency was calculated by using inverse allele frequency [65]. More than 250 publications from PubMed were revised up to June 2020 to validate genetic variant interpretation. Clinical histories of patients were used to provide further information regarding segregation analysis and phenotypic features.
Collected information was manually assessed in order to organize and score the strength of evidence. Genetic variants were interpreted according to ACMG/AMP guidelines [41] and hearing loss gene specific criteria of the ClinGen HL-EP [42]. The final criteria score was manually assigned through the Varsome tool [66] in order to obtain the variant classification. Members of the Laboratory of Physiology and Genetics of Hearing discussed and reviewed final variant classification. A summary of information to be used by clinicians concerning variant specific criteria is detailed in Supplementary Table S1.
3. Results
3.1. General Genetic Findings
GJB2 and GJB6 single nucleotide variants (SNVs) and deletions del(GJB6-D13S1830) and del(GJB6-D13S1854) were studied in 600 NSHL Argentinean patients by Sanger Sequencing and GAP-PCR, respectively. Overall, 48 different sequence variants were identified in the 1200 alleles tested from the entire cohort of patients. The most frequent mutated alleles detected were: c.35delG (9.1%), p.Val27Ile (8.3%), p.Met34Thr (1.5%), c.167delT (1.16%) and the del(GJB6-D13S1830) (0.99%), followed by p.Val37Ile, p.(Glu47*), p.Arg143Trp, p.(Lys168Arg) and del(GJB6-D13S1854) with frequencies from 0.83% to 0.4%. Other variants were found less than five times in the cohort; their specific allele frequency is detailed hereafter.
A total of 229 patients, representing 38% of the studied cohort, exhibited genetic variants in GJB2/GJB6, either in heterozygous (n = 117) or homozygous states, two different variants in the same gene, or GJB6 deletions in combination with GJB2 variants. Familial segregation was performed in 36/97 cases, confirming the in trans occurrence. All other genotypes involving two known causative variants were presumed of biallelic inheritance. A total of 42 diverse biallelic genotypes were identified (Figure 2). The most prevalent genotype detected was the homozygous c. (35delG) variant (33.3% of the biallelic mutations), followed by the compound heterozygous c.(35delG);(167delT) (8.33%). Genotype (GJB2:c.35delG);(GJB6:del(GJB6-D13S1830) and (GJB2:c.35delG);(GJB6:del(GJB6-D13S1854) were detected in 5.2% and 4.16% of hearing-impaired individuals, respectively. Compound heterozygous involving one of the large deletions in GJB6 accounted for the 15.6% of the total detected genotypes (15/96).
Biallelic causative mutations were found in 36% of ARNSHL familial cases (23/64) and 15.5% of sporadic ones (74/477). Overall, 38.5% of the cases were compound heterozygous for the c.(35delG) variant in trans with different mutations, and other 30.2% carried two different non-35delG variants.
Additionally, two patients carried the mutations p.Arg75Trp and p.Arg75Gln with a dominant mode of inheritance. A summary of genotypes, phenotypes and segregation is detailed in Supplementary Table S2.
3.2. Genotype-Phenotype Characterization
In order to correlate the identified genotypes with audiological features, we categorized the genetic variants as truncating (T) or non-truncating (NT). The variants of the 97 positively genotyped patients with biallelic recessive GJB2, dominant GJB2 and/or compound GJB2/GJB6 variants were correlated with their HL severity (moderate, severe, profound).
A total of 42 different genotypes were categorized: 11 homozygous truncating (T/T), 23 heterozygous truncating/non-truncating (T/NT), six homozygous non-truncating (NT/NT) and two autosomal dominant NT (AD). Distribution of genotypes/phenotypes and relative frequencies of the degree of HL in the three groups are shown in Figure 2.
Biallelic T/T genotypes were mostly related to a worse degree of hearing impairment, since 83% of those patients exhibited profound HL, 12% severe and 5% moderate HL. In contrast, biallelic NT/NT genotypes showed a milder degree of hearing impairment since 60% of these cases had moderate HL and 20% severe/profound HL. Compound heterozygous T/NT genotypes ranged from moderate to profound with no clear trends: 43% with profound HL, 18% severe HL and 39% moderate HL (Figure 2 inner box). There were significant differences among the three groups, with X2 testing (p < 0.0001).
3.3. Variant Curation
In order to further analyze and validate the identified variants, we performed a manual revision of available evidence following the Guidelines and recommendations of the ACMG/AMP and The ClinGen Hearing loss Expert Panel.
A total of 48 sequence variants were identified in GJB2 and GJB6 in our study cohort and 44 were manually curated, since four variants had been already curated as pathogenic by the HL-EP group: c.35delG, p.Met34Thr, p.Val37Ile and c.167delT. Variant c.-22-2A>C was reported as Variant of Unknown Significance (VUS) by the HL-EP group but was reclassified with new available information in this work explained further below.
From the total of variants analyzed, 23 were classified as pathogenic (P), three likely pathogenic (LP), nine uncertain significance (VUS), four likely benign (LB) and five benign (B). The final classification of the 44 variants and their general information is shown in Table 1.
Table 1.
Nucleotide Change | Protein Variant | Mutated Alleles/ Total Alleles Tested (Percentage) |
Reference | ClinVar | Final Classification (ACMG/AMP HL-EP) |
Rules Applied |
---|---|---|---|---|---|---|
c.-23+1G>A | - | 2/1200 (0.16%) |
[68] | Pathogenic | Pathogenic | PM2, PVS1, PM3_VS, PS3_P |
c.-22-12C>T | - | 1/1200 (0.083%) |
[69] | Benign | Benign | BA1, BP4 |
c.-22-2A>C | - | 1/1200 (0.083%) |
[70] | VUS * | Likely Pathogenic | PM3_M, PP1_S, PS3_P, BS1. |
c.-15C>T | - | 1/1200 (0.083%) |
[69] | Benign/ Likely Benign |
Benign | BA1, BP4 |
c.23C>T | p.Thr8Met | 1/1200 (0.083%) |
[71] | Conflicting Interpretation |
VUS | PM2_P, PS3_P, PM3 |
c.24G>A | p.(Thr8=) | 1/1200 (0.083%) |
[72] | Conflicting Interpretation |
VUS | PP3, PM2 |
c.29T>C | p.Leu10Pro | 1/1200 (0.083%) |
[73] | absent | VUS | PM2, PP3, PS3_M |
c.35G>T | p.Gly12Val | 2/1200 (0.16%) |
[74] | Pathogenic/ Likely Pathogenic | Pathogenic | PM2_M, PM3_VS, PP3, PS3_M |
c.35delG | p.Gly12Valfs*2 | 111/1200 (9.25%) |
[75] | Pathogenic * | Pathogenic | already curated by HL-EP |
c.56G>C | p.Ser19Thr | 1/1200 (0.083%) |
[74] | Likely Pathogenic | Pathogenic | PM2, PM3_VS, PP1_M, PS3_M |
c.59T>C | p.Ile20Thr | 4/1200 (0.34%) |
[76] | Pathogenic/ Likely Pathogenic | Pathogenic | PM2, PM3, PP1_P, PP3, PS3_M |
c.79G>A | p.Val27Ile | 100/1200 (8.34%) |
[77] | Benign | Benign | BA1, BP2, BS3_P |
c.101T>C | p.Met34Thr | 19/1200 (1.58%) |
[78] | Pathogenic * | Pathogenic | already curated by HL-EP |
c.109G>A | p.Val37Ile | 10/1200 (0.84%) |
[77] | Pathogenic * | Pathogenic | already curated by HL-EP |
c.139G>T | p.(Glu47*) | 6/1200 (0.5%) |
[79] | Pathogenic | Pathogenic | PVS1, PM2_P, PM3_VS |
c.167delT | p.Leu56Argfs*81 | 14/1200 (1.16%) |
[75] | Pathogenic * | Pathogenic | already curated by HL-EP |
c.223C>T | p.Arg75Trp | 1/1200 (0.083%) |
[80] | Pathogenic | Pathogenic | PM2, PS2_VS, PP1_P, PP3, PS3 |
c.224G>A | p.Arg75Gln | 1/1200 (0.083%) |
[81] | Pathogenic | Pathogenic | PM2, PS4_M, PP1_M, PM5, PP3, PS3_M |
c.229T>C | p.Trp77Arg | 1/1200 (0.083%) |
[82] | Pathogenic | Pathogenic | PM2_P, PM3_VS, PP3, PS3_M |
c.232dupG | p.(Ala78Glyfs*24) | 1/1200 (0.083%) |
[83] | Likely Pathogenic | Pathogenic | PM2, PVS1, PM3 |
c.232G>T | p.(Ala78Ser) | 1/1200 (0.083%) |
[28] | absent | VUS | PM2, PP3, PM5 |
c.246C>G | p.Ile82Met | 1/1200 (0.083%) |
[84] | Likely Pathogenic | Pathogenic | PM2, PP1_M, PM3_VS, PP3, PS3_M |
c.249C>G | p.Phe83Leu | 2/1200 (0.16%) |
[85] | Benign/ Likely Benign |
Likely Benign | BS1_P, BP2, BS3_P |
c.250G>C | p.Val84Leu | 1/1200 (0.083%) |
[77] | Pathogenic/ Likely Pathogenic | Pathogenic | PM2, PP3, PM3_VS, PP1_P |
c.269T>C | p.Leu90Pro | 3/1200 (0.25%) |
[68] | Conflicting Interpretation | Pathogenic | BS1_P, PP3, PM3_VS, PS3_M |
c.269dup | p.(Val91Serfs*11) | 1/1200 (0.083%) |
[68] | Pathogenic | Pathogenic | PM2, PVS1, PM3_VS, PP1_M |
c.283G>A | p.(Val95Met) | 2/1200 (0.16%) |
[77] | Pathogenic/ Likely Pathogenic | Pathogenic | PM2, PM3_VS, PP1_P, PP3 |
c.313_326del14 | p.(Lys105Glyfs*5) | 2/1200 (0.16%) |
[79] | Pathogenic | Pathogenic | PM2_P, PVS1, PM3_VS. |
c.326G>T | p.Gly109Val | 1/1200 (0.083%) |
[86] | absent | Likely Pathogenic | PM2, PM3, PS3_M |
c.334_335delAA | p.(Lys112Glufs*2) | 2/1200 (0.16%) |
[77] | Pathogenic /Likely Pathogenic | Pathogenic | PM2, PVS1, PM3_VS, PP1_M |
c.358_360delGAG | p.Glu120del | 2/1200 (0.16%) |
[79] | Pathogenic | Pathogenic | PM2_P, PM4, PM3_VS, PS3_M |
c.380G>A | p.Arg127His | 1/1200 (0.083%) |
[87] | Conflicting Interpretation | Benign | BA1, BS2, BS4, PM3_P |
c.384C>T | p.(Ile128=) | 1/1200 (0.083%) |
[75] | Likely Benign | Likely Benign | PM2, BP2, BP4, BP7 |
c.385G>A | p.(Glu129Lys) | 1/1200 (0.083%) |
[71] | Conflicting Interpretation | VUS | PM2, PM3 |
c.427C>T | p.Arg143Trp | 5/1200 (0.42%) |
[88] | Pathogenic | Pathogenic | PM2_P, PM3_VS, PP1_P |
c.439G>A | p.(Glu147Lys) | 1/1200 (0.083%) |
[89] | Pathogenic/ Likely Pathogenic | Pathogenic | PM2, PM3_VS, PP1_Mod, PP3 |
c.457G>A | p.Val153Ile | 3/1200 (0.25%) |
[90] | Benign/ Likely Benign |
Benign | BA1, BS2 |
c.478G>A | p.(Gly160Ser) | 2/1200 (0.16%) |
[85] | Conflicting Interpretation | Likely Benign | BS1_P, PP3, BP2, PM3 |
c.487A>G | p.Met163Val | 2/1200 (0.16%) |
[90] | VUS | VUS | PM2_P, PP3, PS3_M, BS2 |
c.487A>C | p.Met163Leu | 1/1200 (0.083%) |
[91] | Pathogenic | VUS | PM2, PS3_P |
c.503A>G | p.(Lys168Arg) | 5/1200 (0.42%) |
[92] | Conflicting Interpretation | VUS | PM2_P, PP3 |
c.551G>C | p.Arg184Pro | 4/1200 (0.34%) |
[79] | Conflicting Interpretation | Pathogenic | PM2, PM3_VS, PP3, PS3_M |
c.569T>A | p.(Val190Asp) | 1/1200 (0.083%) |
[28] | absent | VUS | PM2, PM3, PP3 |
c.617A>G | p.Asn206Ser | 4/1200 (0.34%) |
[90] | Pathogenic | Pathogenic | PM2_P, PP3, PP1_M, PM3_VS, PS3_M |
c.632G>A | p.(Cys211Tyr) | 1/1200 (0.083%) |
[28] | absent | Likely Pathogenic | PM2, PM3,PP3, PP1_P |
c.*1C>T (3′UTR) | - | 2/1200 (0.16%) |
[93] | Conflicting Interpretation | Likely Benign | BS1_P, BP4 |
del(GJB6-D13S1830) | - | 12/1200 (1%) |
[10] | Pathogenic | Pathogenic | PS3, PS4, PM2_P, PM3_VS |
del(GJB6-D13S1854) | - | 5/1200 (0.42%) |
[21] | Pathogenic | Pathogenic | PM2, PS3, PS4, PM3_VS |
Genetic variants’ distribution spanned the entire length of Cx26 and involved almost all protein domains (Figure 3).
Interestingly, based on the specific criteria applied during our curation procedure, 59% of sequence variants evaluated in this study changed their previous category submitted to ClinVar: 36% with considerable re-interpretation and 23% with resolution of similar categories (for instance, P/LP submission for p.Gly12Val variant was confirmed as pathogenic or B/LB submission for p.Val153Ile variant was classified as benign) (Figure 4).
After the curation procedure, the pathogenic final classification represented a total of 23 variants, of which 11 changed their previous status in ClinVar, and 12 remained in the same category (Figure 4). In addition, nine variants previously considered as conflicting interpretation of pathogenicity were reclassified as: pathogenic (two cases), benign or likely benign (three cases) and uncertain significance (four cases). The c.269T>C, p.Leu90Pro and c.551G>C, p.Arg184Pro variants were reinterpreted as pathogenic, since both mutations had strong evidence concerning allelic data (PM3_VeryStrong) and functional studies demonstrating a deleterious effect (PS3). The c.380G>A, p.(Arg127His); c.478G>A, p.(Gly160Ser) and the 3′UTR variant c.*1C>T were reclassified to likely benign and benign mostly based on their high population frequencies (BA1 and BS1 rules). The last four variants with conflicting interpretations in ClinVar: c.23C>T, p.Thr8Met; c.24G>A, p.(Thr8=); c.385G>A, p.(Glu129Lys); c.503A>G, p.(Lys168Arg), were reclassified as being of uncertain significance since available information was not sufficient to determine their pathogenicity. The c.487A>C, p.Met163Leu variant listed in ClinVar as pathogenic for dominant HL, was reinterpreted in this work as being of uncertain significance, due to the lack of strong evidence: absent in population database (PM2), low REVEL score, functional study with only supporting evidence (PS3_P) and only reported three times (PS4_P).
Remarkably, we propose that the splice site variant c.-22-2A>C interpreted as being of uncertain significance by the HL-EP group should be reclassified as likely pathogenic according to new uncovered data. Thus, due to the usage of an alternative variant nomenclature c.-24A>C (not HGVS), a previous report by [70] had not been taken into account for variant classification. After consultation with the author [70], correct nomenclature was confirmed, thus, the allelic data of the report was considered, strengthening the PM3 criteria, and hence, the variant pathogenicity. Final classification and comment on clinical significance for each variant was submitted to ClinVar.
The most frequently applied rules were PM2 and PM3 (20% each), corresponding to population and allelic data, respectively, followed by the PS3 standard, which included functional studies demonstrating a deleterious effect (12%) (Figure 5A). The frequency of rules used among the three main categories (B/LB, VUS and LP/P) is shown in Figure 5B. In this regard, PM3_VeryStrong and PM2 rules together with computational evidence suggesting a damaging impact of the mutation to the protein (PP3) and deleterious effect demonstrated by functional assays (PS3) were the most frequent criteria applied, particularly for the classification of pathogenic and likely pathogenic variants. High frequency of variants in the general populations (BA1), neutral impact predicted by in silico analysis (BP4) and allelic data (BP2) were associated with benign or likely benign categories. In the case of variants interpreted as uncertain significance, damaging computational evidence was almost always applied along with low frequency in the general population. However, this evidence was not strong enough to determine its pathogenicity. Complete information about the criteria applied and variant interpretation is detailed in Supplementary Table S3.
4. Discussion
Autosomal Recessive Non-Syndromic Hearing Loss (ARNSHL) is a heterogeneous condition that affects millions of individuals worldwide. Genetic variants in GJB2 and GJB6 genes are the most prevalent genetic causes of HL among several populations, and consequently, are the focus of universal newborn hearing screening programs [77,94,95]. Identifying mutations in those genes becomes crucial in molecular strategy approaches, as they provide valuable prognostic information for medical intervention. This study provides an update and extension of our previous reports and states that sequence variants in GJB2 and GJB6 genes are frequent in Argentinean patients with non-syndromic sensorineural HL [26,27,28,96].
Molecular diagnosis due to GJB2 and GJB6 variants was more successful in family cases with ARNSHL than in sporadic ones (36% vs. 15.5%). As reported previously, our data confirms c.35delG as the most frequent GJB2 mutation causing non-syndromic hearing loss in the Argentinean population with a prevalence of 9.25% of the detected GJB2-mutated alleles [26,27,28,30]. These results are in concordance with Caucasian population frequencies [73,75,87,97]. On the other hand, since 26/98 (26.5%) genotyped patients resulted in GJB2 compound heterozygous for non-c.35delG alleles, appropriate molecular diagnosis requires the complete sequencing of the gene including the untranslated exon 1. Interestingly, both dominant mutations in GJB2 were detected in the same protein residue (Arg75) and led to a profound HL phenotype, in accordance with the dominant negative effect of the two variants demonstrated by functional studies [98,99,100]. Of note, both variants del(GJB6-D13S1830) and del(GJB6-D13S1854) accounted for 15.3% of genotyped patients, which resulted in similar frequencies reported in Spain but greater than other European countries [6,7,8,21,97,101,102,103]. Our cohort of patients exhibited non-syndromic moderate-to-profound hearing loss due to biallelic GJB2 mutations and compound GJB2/GJB6 mutations. In accordance with previous studies truncating variants were mostly related to profound HL, while non-truncating variants to a milder degree of hearing impairment, reinforcing the notion that inactivating variants lead to a severe degree of HL [8,34,46,97].
Regarding variant curation, allelic and population data along with computational evidence were the most used information in variant assessment. The PM3 and PM2 rules accounted for 20% each of the total parameters applied. The PM3 criteria gathers the information regarding the compound heterozygous variants in HL patients. Since GJB2 variants are mostly related to an autosomal recessive mode of inheritance, the identification of a second mutation resulted essential and conclusive in variant interpretation. In addition, the absence or low frequency of the sequence variant in the general population (defined by PM2 rule) represented an important evidence during data analysis.
The curation of variants performed in the present work highlights the importance of specialized guidelines to analyze and interpret variants for the clinical use of databases. Moreover, it indicates the need of scientific community interaction and data sharing to avoid or reduce difficulties in variant curation [104,105]. Manual curation, although time consuming is strictly needed, as shown for the p.Met163Leu variant, which was originally interpreted as pathogenic for autosomal dominant HL by one submitter, and now reclassified to uncertain significance. An additional example is that of the splice site variant c.-22-2A>C, classified as of uncertain significance and now reinterpreted to likely pathogenic as a result of the clarification of nomenclature issues, and hence, strengthening the criteria applied. Likewise, more than half of the mutations were reclassified after the curation procedure, and satisfyingly, this reduced the number of “conflicting interpretation” categorization submitted to ClinVar.
In summary, the present work provides a set of variants that lead to hearing loss with an accurate interpretation of their phenotypic consequence. Moreover, it demonstrates the importance of a comprehensive analysis of sequence data performed by experts in the hearing field in order to provide reliable data to be used by clinicians in patient diagnosis and genetic counseling.
Acknowledgments
We acknowledge Ezequiel Surace for proofreading the manuscript carefully and Bruno de Brasi for their comments and suggestions that have greatly helped in improving the quality of the report. Paula Buonfiglio is a CONICET fellow. We thank all families for their kind contribution to this study.
Supplementary Materials
The following are available online at https://www.mdpi.com/2073-4425/11/10/1233/s1, Table S1: Summary of HL-EP specifications, Table S2: Summary of genotypes detected and Table S3: Curated variants procedure.
Author Contributions
P.B. performed the molecular genetic studies and curation of variants and wrote the manuscript. C.D.B. contributed with graphic design and variant curation. L.L. and F.G. contributed with bioinformatic analysis. V.L., S.M. and B.P. recruited patients and acquired the clinical data. A.B.E. raised funding and assured the general supervision of the research group. V.D. designed the genetic study and wrote the manuscript. All authors have read and agreed to the published version of the manuscript.
Funding
This study was supported by an International Research Scholar grant from the Grand Prix Fondation Pour L’Audition and from the “Agencia Nacional de Promoción Científica y Tecnológica” PICT-2018-00539 to A.B.E., and a grant for Medical Research from Fundación Alberto J. Roemmers to P.B.
Conflicts of Interest
The authors declare no conflict of interest.
Footnotes
Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.
References
- 1.Morton C.C., Nance W.E. Newborn hearing screening—A silent revolution. N. Engl. J. Med. 2006;354:2151–2164. doi: 10.1056/NEJMra050700. [DOI] [PubMed] [Google Scholar]
- 2.Morton N.E. Genetic Epidemiology of Hearing Impairment. Ann. N. Y. Acad. Sci. 1991;630:16–31. doi: 10.1111/j.1749-6632.1991.tb19572.x. [DOI] [PubMed] [Google Scholar]
- 3.Shearer A.E., Hildebrand M.S., Smith R.J.H. Hereditary Hearing Loss and Deafness Overview. In: Adam M.P., Ardinger H.H., Pagon R.A., Wallace S.E., Bean L.J.H., Stephens K., Amemiya A., editors. GeneReviews. University of Washington, Seattle; Seattle, WA, USA: 1999. [Google Scholar]
- 4.Van Camp G., Smith R.J.H. Hereditary Hearing Loss Homepage. [(accessed on 16 October 2020)]; Available online: https://hereditaryhearingloss.org.
- 5.Richardson G.P., de Monvel J.B., Petit C. How the Genetics of Deafness Illuminates Auditory Physiology. Annu. Rev. Physiol. 2011;73:311–334. doi: 10.1146/annurev-physiol-012110-142228. [DOI] [PubMed] [Google Scholar]
- 6.Cama E., Melchionda S., Palladino T., Carella M., Santarelli R., Genovese E., Benettazzo F., Zelante L., Arslan E. Hearing loss features in GJB2 biallelic mutations and GJB2/GJB6 digenic inheritance in a large Italian cohort. Int. J. Audiol. 2009;48:12–17. doi: 10.1080/14992020802400654. [DOI] [PubMed] [Google Scholar]
- 7.Del Castillo I., Moreno-Pelayo M.A., Del Castillo F.J., Brownstein Z., Marlin S., Adina Q., Cockburn D.J., Pandya A., Siemering K.R., Chamberlin G.P., et al. Prevalence and evolutionary origins of the del(GJB6-D13S1830) mutation in the DFNB1 locus in hearing-impaired subjects: A multicenter study. Am. J. Hum. Genet. 2003;73:1452–1458. doi: 10.1086/380205. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 8.Feldmann D., Denoyelle F., Chauvin P., Garabédian E.-N., Couderc R., Odent S., Joannard A., Schmerber S., Delobel B., Leman J., et al. Large deletion of the GJB6 gene in deaf patients heterozygous for the GJB2 gene mutation: Genotypic and phenotypic analysis. Am. J. Med. Genet. A. 2004;127A:263–267. doi: 10.1002/ajmg.a.20588. [DOI] [PubMed] [Google Scholar]
- 9.Pampanos A., Economides J., Iliadou V., Neou P., Leotsakos P., Voyiatzis N., Eleftheriades N., Tsakanikos M., Antoniadi T., Hatzaki A., et al. Prevalence of GJB2 mutations in prelingual deafness in the Greek population. Int. J. Pediatr. Otorhinolaryngol. 2002;65:101–108. doi: 10.1016/S0165-5876(02)00177-5. [DOI] [PubMed] [Google Scholar]
- 10.del Castillo I., Villamar M., Moreno-Pelayo M.A., del Castillo F.J., Alvarez A., Tellería D., Menéndez I., Moreno F. A deletion involving the connexin 30 gene in nonsyndromic hearing impairment. N. Engl. J. Med. 2002;346:243–249. doi: 10.1056/NEJMoa012052. [DOI] [PubMed] [Google Scholar]
- 11.Hilgert N., Smith R.J.H., Van Camp G. Forty-six genes causing nonsyndromic hearing impairment: Which ones should be analyzed in DNA diagnostics? Mutat. Res. 2009;681:189–196. doi: 10.1016/j.mrrev.2008.08.002. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 12.Rabionet R., Gasparini P., Estivill X. Molecular genetics of hearing impairment due to mutations in gap junction genes encoding beta connexins. Hum. Mutat. 2000;16:190–202. doi: 10.1002/1098-1004(200009)16:3<190::AID-HUMU2>3.0.CO;2-I. [DOI] [PubMed] [Google Scholar]
- 13.Meşe G., Richard G., White T.W. Gap Junctions: Basic Structure and Function. J. Investig. Dermatol. 2007;127:2516–2524. doi: 10.1038/sj.jid.5700770. [DOI] [PubMed] [Google Scholar]
- 14.Bruzzone R., Veronesi V., Gomès D., Bicego M., Duval N., Marlin S., Petit C., D’Andrea P., White T.W. Loss-of-function and residual channel activity of connexin26 mutations associated with non-syndromic deafness. FEBS Lett. 2003;533:79–88. doi: 10.1016/S0014-5793(02)03755-9. [DOI] [PubMed] [Google Scholar]
- 15.Palmada M., Schmalisch K., Böhmer C., Schug N., Pfister M., Lang F., Blin N. Loss of function mutations of the GJB2 gene detected in patients with DFNB1-associated hearing impairment. Neurobiol. Dis. 2006;22:112–118. doi: 10.1016/j.nbd.2005.10.005. [DOI] [PubMed] [Google Scholar]
- 16.White T.W. Functional analysis of human Cx26 mutations associated with deafness. Brain Res. Rev. 2000;32:181–183. doi: 10.1016/S0165-0173(99)00079-X. [DOI] [PubMed] [Google Scholar]
- 17.Gasparini P., the Genetic Analysis Consortium of GJB235delG. Rabionet R., Barbujani G., Melchionda S., Petersen M., Brøndum-Nielsen K., Metspalu A., Oitmaa E., Pisano M., et al. High carrier frequency of the 35delG deafness mutation in European populations. Eur. J. Hum. Genet. 2000;8:19–23. doi: 10.1038/sj.ejhg.5200406. [DOI] [PubMed] [Google Scholar]
- 18.Cryns K. A genotype-phenotype correlation for GJB2 (connexin 26) deafness. J. Med. Genet. 2004;41:147–154. doi: 10.1136/jmg.2003.013896. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 19.Lucotte G. High prevalences of carriers of the 35delG mutation of connexin 26 in the Mediterranean area. Int. J. Pediatri. Otorhinolaryngol. 2007;71:741–746. doi: 10.1016/j.ijporl.2007.01.010. [DOI] [PubMed] [Google Scholar]
- 20.Azaiez H., Booth K.T., Ephraim S.S., Crone B., Black-Ziegelbein E.A., Marini R.J., Eliot Shearer A., Sloan-Heggen C.M., Kolbe D., Casavant T., et al. Genomic Landscape and Mutational Signatures of Deafness-Associated Genes. Am. J. Hum. Genet. 2018;103:484–497. doi: 10.1016/j.ajhg.2018.08.006. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 21.del Castillo F.J. 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. J. Med. Genet. 2005;42:588–594. doi: 10.1136/jmg.2004.028324. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 22.Wilch E., Azaiez H., Fisher R.A., Elfenbein J., Murgia A., Birkenhäger R., Bolz H., Da Silva-Costa S.M., Del Castillo I., Haaf T., et al. A novel DFNB1 deletion allele supports the existence of a distant cis-regulatory region that controls GJB2 and GJB6 expression. Clin. Genet. 2010;78:267–274. doi: 10.1111/j.1399-0004.2010.01387.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 23.Feldmann D., Le Maréchal C., Jonard L., Thierry P., Czajka C., Couderc R., Ferec C., Denoyelle F., Marlin S., Fellmann F. A new large deletion in the DFNB1 locus causes nonsyndromic hearing loss. Eur. J. Med. Genet. 2009;52:195–200. doi: 10.1016/j.ejmg.2008.11.006. [DOI] [PubMed] [Google Scholar]
- 24.Bliznetz E.A., Makienko O.N., Okuneva E.G., Markova T.G., Polyakov A.V. New recurrent large deletion, encompassing both GJB2 and GB6 genes, results in isolated sensorineural hearing impairment with autosomal recessive mode of inheritance. Russ. J. Genet. 2014;50:415–420. doi: 10.1134/S1022795414020045. [DOI] [PubMed] [Google Scholar]
- 25.Tayoun A.N.A., Abou Tayoun A.N., Mason-Suares H., Frisella A.L., Bowser M., Duffy E., Mahanta L., Funke B., Rehm H.L., Amr S.S. Targeted Droplet-Digital PCR as a Tool for Novel Deletion Discovery at the DFNB1 Locus. Hum. Mutat. 2016;37:119–126. doi: 10.1002/humu.22912. [DOI] [PubMed] [Google Scholar]
- 26.Dalamón V., Lotersztein V., Béhèran A., Lipovsek M., Diamante F., Pallares N., Francipane L., Frechtel G., Paoli B., Mansilla E., et al. GJB2 and GJB6 Genes: Molecular Study and Identification of Novel GJB2 Mutations in the Hearing-Impaired Argentinean Population. Audiol. Neurotol. 2010;15:194–202. doi: 10.1159/000254487. [DOI] [PubMed] [Google Scholar]
- 27.Dalamón V., Béhèran A., Diamante F., Pallares N., Diamante V., Elgoyhen A.B. Prevalence of GJB2 mutations and the del(GJB6-D13S1830) in Argentinean non-syndromic deaf patients. Hear. Res. 2005;207:43–49. doi: 10.1016/j.heares.2005.04.012. [DOI] [PubMed] [Google Scholar]
- 28.Dalamón V., Florencia Wernert M., Lotersztein V., Craig P.O., Diamante R.R., Barteik M.E., Curet C., Paoli B., Mansilla E., Elgoyhen A.B. Identification of four novel connexin 26 mutations in non-syndromic deaf patients: Genotype-phenotype analysis in moderate cases. Mol. Biol. Rep. 2013;40:6945–6955. doi: 10.1007/s11033-013-2814-x. [DOI] [PubMed] [Google Scholar]
- 29.Gravina L.P., Foncuberta M.E., Estrada R.C., Barreiro C., Chertkoff L. Carrier frequency of the 35delG and A1555G deafness mutations in the Argentinean population. Int. J. Pediatri. Otorhinolaryngol. 2007;71:639–643. doi: 10.1016/j.ijporl.2006.12.015. [DOI] [PubMed] [Google Scholar]
- 30.Gravina L.P., Foncuberta M.E., Prieto M.E., Garrido J., Barreiro C., Chertkoff L. Prevalence of DFNB1 mutations in Argentinean children with non-syndromic deafness. Report of a novel mutation in GJB2. Int. J. Pediatr. Otorhinolaryngol. 2010;74:250–254. doi: 10.1016/j.ijporl.2009.11.014. [DOI] [PubMed] [Google Scholar]
- 31.Reynoso R.A., Hendl S., Barteik M.E., Curet C.A., Nicemboin L., Moreno Barral J., Rodríguez Ballesteros M., Del Castillo I., Moreno F. Genetic study of hearing loss in families from Argentina. Rev. Fac. Cien. Med. Univ. Nac. Cordoba. 2004;61:13–19. [PubMed] [Google Scholar]
- 32.Smith R.J.H., Jones M.K.N. Nonsyndromic Hearing Loss and Deafness, DFNB1. 28 September 1998 [Updated 18 August 2016] In: Adam M.P., Ardinger H.H., Pagon R.A., Wallace S.E., Bean L.J.H., Stephens K., Amemiya A., editors. GeneReviews®. University of Washington, Seattle; Seattle, WA, USA: 1993. [Google Scholar]
- 33.D’Andrea P., Veronesi V., Bicego M., Melchionda S., Zelante L., Di Iorio E., Bruzzone R., Gasparini P. Hearing loss: Frequency and functional studies of the most common connexin26 alleles. Biochem. Biophys. Res. Commun. 2002;296:685–691. doi: 10.1016/S0006-291X(02)00891-4. [DOI] [PubMed] [Google Scholar]
- 34.Azaiez H., Parker Chamberlin G., Fischer S.M., Welp C.L., Prasad S.D., Thomas Taggart R., del Castillo I., Van Camp G., Smith R.J.H. GJB2: The spectrum of deafness-causing allele variants and their phenotype. Hum. Mutat. 2004;24:305–311. doi: 10.1002/humu.20084. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 35.Green G.E., Scott D.A., McDonald J.M., Teagle H.F.B., Tomblin B.J., Spencer L.J., Woodworth G.G., Knutson J.F., Gantz B.J., Sheffield V.C., et al. Performance of cochlear implant recipients withGJB2-related deafness. Am. J. Med. Genet. 2002;109:167–170. doi: 10.1002/ajmg.10330. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 36.Bauer P.W., Geers A.E., Brenner C., Moog J.S., Smith R.J.H. The Effect of GJB2 Allele Variants on Performance After Cochlear Implantation. Laryngoscope. 2003;113:2135–2140. doi: 10.1097/00005537-200312000-00015. [DOI] [PubMed] [Google Scholar]
- 37.Usami S.-I., Nishio S.-Y., Moteki H., Miyagawa M., Yoshimura H. Cochlear Implantation from the Perspective of Genetic Background. Anat. Rec. 2020;303:563–593. doi: 10.1002/ar.24360. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 38.Amendola L.M., Jarvik G.P., Leo M.C., McLaughlin H.M., Akkari Y., Amaral M.D., Berg J.S., Biswas S., Bowling K.M., Conlin L.K., et al. Performance of ACMG-AMP Variant-Interpretation Guidelines among Nine Laboratories in the Clinical Sequencing Exploratory Research Consortium. Am. J. Hum. Genet. 2016;99:247. doi: 10.1016/j.ajhg.2016.06.001. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 39.Booth K.T., Kahrizi K., Babanejad M., Daghagh H., Bademci G., Arzhangi S., Zareabdollahi D., Duman D., El-Amraoui A., Tekin M., et al. Variants in CIB2 cause DFNB48 and not USH1J. Clin. Genet. 2018;93:812–821. doi: 10.1111/cge.13170. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 40.Harrison S.M., Dolinsky J.S., Knight Johnson A.E., Pesaran T., Azzariti D.R., Bale S., Chao E.C., Das S., Vincent L., Rehm H.L. Clinical laboratories collaborate to resolve differences in variant interpretations submitted to ClinVar. Genet. Med. 2017;19:1096–1104. doi: 10.1038/gim.2017.14. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 41.Richards S., Aziz N., Bale S., Bick D., Das S., Gastier-Foster J., Grody W.W., Hegde M., Lyon E., Spector E., et al. Standards and guidelines for the interpretation of sequence variants: A joint consensus recommendation of the American College of Medical Genetics and Genomics and the Association for Molecular Pathology. Genet. Med. 2015;17:405–423. doi: 10.1038/gim.2015.30. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 42.Oza A.M., DiStefano M.T., Hemphill S.E., Cushman B.J., Grant A.R., Siegert R.K., Shen J., Chapin A., Boczek N.J., Schimmenti L.A., et al. Expert specification of the ACMG/AMP variant interpretation guidelines for genetic hearing loss. Hum. Mutat. 2018;39:1593–1613. doi: 10.1002/humu.23630. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 43.Murray M.G., Thompson W.F. Rapid isolation of high molecular weight plant DNA. Nucleic Acids Res. 1980;8:4321–4326. doi: 10.1093/nar/8.19.4321. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 44.Sequence Assembly and Alignment Software-CodonCode. [(accessed on 16 October 2020)]; Available online: https://www.codoncode.com/
- 45.Altschul S.F., Gish W., Miller W., Myers E.W., Lipman D.J. Basic local alignment search tool. J. Mol. Biol. 1990;215:403–410. doi: 10.1016/S0022-2836(05)80360-2. [DOI] [PubMed] [Google Scholar]
- 46.Snoeckx R.L., Huygen P.L.M., Feldmann D., Marlin S., Denoyelle F., Waligora J., Mueller-Malesinska M., Pollak A., Ploski R., Murgia A., et al. GJB2 Mutations and Degree of Hearing Loss: A Multicenter Study. Am. J. Hum. Genet. 2005;77:945–957. doi: 10.1086/497996. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 47.Shahin H., Walsh T., Sobe T., Lynch E., King M.-C., Avraham K.B., Kanaan M. Genetics of congenital deafness in the Palestinian population: Multiple connexin 26 alleles with shared origins in the Middle East. Hum. Genet. 2002;110:284–289. doi: 10.1007/s00439-001-0674-2. [DOI] [PubMed] [Google Scholar]
- 48.Del Castillo F.J., Del Castillo I. DFNB1 Non-syndromic Hearing Impairment: Diversity of Mutations and Associated Phenotypes. Front. Mol. Neurosci. 2017;10:428. doi: 10.3389/fnmol.2017.00428. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 49.Gandía M., Del Castillo F.J., Rodríguez-Álvarez F.J., Garrido G., Villamar M., Calderón M., Moreno-Pelayo M.A., Moreno F., del Castillo I. A novel splice-site mutation in the GJB2 gene causing mild postlingual hearing impairment. PLoS ONE. 2013;8:e73566. doi: 10.1371/journal.pone.0073566. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 50.den Dunnen J.T., Dalgleish R., Maglott D.R., Hart R.K., Greenblatt M.S., McGowan-Jordan J., Roux A.-F., Smith T., Antonarakis S.E., Taschner P.E.M. HGVS Recommendations for the Description of Sequence Variants: 2016 Update. Hum. Mutat. 2016;37:564–569. doi: 10.1002/humu.22981. [DOI] [PubMed] [Google Scholar]
- 51.Wildeman M., van Ophuizen E., den Dunnen J.T., Taschner P.E.M. Improving sequence variant descriptions in mutation databases and literature using the Mutalyzer sequence variation nomenclature checker. Hum. Mutat. 2008;29:6–13. doi: 10.1002/humu.20654. [DOI] [PubMed] [Google Scholar]
- 52.Ioannidis N.M., Rothstein J.H., Pejaver V., Middha S., McDonnell S.K., Baheti S., Musolf A., Li Q., Holzinger E., Karyadi D., et al. REVEL: An Ensemble Method for Predicting the Pathogenicity of Rare Missense Variants. Am. J. Hum. Genet. 2016;99:877–885. doi: 10.1016/j.ajhg.2016.08.016. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 53.Rentzsch P., Witten D., Cooper G.M., Shendure J., Kircher M. CADD: Predicting the deleteriousness of variants throughout the human genome. Nucleic Acids Res. 2019;47:D886–D894. doi: 10.1093/nar/gky1016. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 54.Desmet F.-O., Hamroun D., Lalande M., Collod-Béroud G., Claustres M., Béroud C. Human Splicing Finder: An online bioinformatics tool to predict splicing signals. Nucleic Acids Res. 2009;37:e67. doi: 10.1093/nar/gkp215. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 55.Yeo G., Burge C.B. Maximum entropy modeling of short sequence motifs with applications to RNA splicing signals. Burge. J. Comput. Biol. 2004;11:377–394. doi: 10.1089/1066527041410418. [DOI] [PubMed] [Google Scholar]
- 56.Abou Tayoun A.N., Pesaran T., DiStefano M.T., Oza A., Rehm H.L., Biesecker L.G., Harrison S.M., ClinGen Sequence Variant Interpretation Working Group (ClinGen SVI) Recommendations for interpreting the loss of function PVS1 ACMG/AMP variant criterion. Hum. Mutat. 2018;39:1517–1524. doi: 10.1002/humu.23626. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 57.Shamsani J., Kazakoff S.H., Armean I.M., McLaren W., Parsons M.T., Thompson B.A., O’Mara T.A., Hunt S.E., Waddell N., Spurdle A.B. A plugin for the Ensembl Variant Effect Predictor that uses MaxEntScan to predict variant spliceogenicity. Bioinformatics. 2019;35:2315–2317. doi: 10.1093/bioinformatics/bty960. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 58.Karczewski K.J., Francioli L.C., Tiao G., Cummings B.B., Alföldi J., Wang Q., Collins R.L., Laricchia K.M., Ganna A., Birnbaum D.P., et al. The mutational constraint spectrum quantified from variation in 141,456 humans. Nature. 2020;581:434–443. doi: 10.1038/s41586-020-2308-7. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 59.Sherry S.T. dbSNP: The NCBI database of genetic variation. Nucleic Acids Res. 2001;29:308–311. doi: 10.1093/nar/29.1.308. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 60.Exome Variant Server, NHLBI GO Exome Sequencing Project (ESP), Seattle, WA. [(accessed on 16 October 2020)]; Available online: http://evs.gs.washington.edu/EVS/
- 61.Landrum M.J., Lee J.M., Benson M., Brown G.R., Chao C., Chitipiralla S., Gu B., Hart J., Hoffman D., Jang W., et al. ClinVar: Improving access to variant interpretations and supporting evidence. Nucleic Acids Res. 2018;46:D1062–D1067. doi: 10.1093/nar/gkx1153. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 62.Fokkema I.F.A.C., Ivo F.A., Taschner P.E.M., Schaafsma G.C.P., Celli J., Laros J.F.J., den Dunnen J.T. LOVD v.2.0: The next generation in gene variant databases. Hum. Mutat. 2011;32:557–563. doi: 10.1002/humu.21438. [DOI] [PubMed] [Google Scholar]
- 63.MacDonald J.R., Ziman R., Yuen R.K.C., Feuk L., Scherer S.W. The Database of Genomic Variants: A curated collection of structural variation in the human genome. Nucleic Acids Res. 2014;42:D986–D992. doi: 10.1093/nar/gkt958. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 64.EMBL-EBI Database of Genomic Variants Archive. [(accessed on 16 October 2020)]; Available online: https://www.ebi.ac.uk/dgva/
- 65.Frequency Filter. [(accessed on 16 October 2020)]; Available online: https://www.cardiodb.org/allelefrequencyapp/
- 66.Kopanos C., Tsiolkas V., Kouris A., Chapple C.E., Aguilera M.A., Meyer R., Massouras A. VarSome: The human genomic variant search engine. Bioinformatics. 2019;35:1978–1980. doi: 10.1093/bioinformatics/bty897. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 67.6601 Free Vector Icons of Webpage. [(accessed on 16 October 2020)]; Available online: https://www.flaticon.com/free-icons/webpage.
- 68.Denoyelle F., Marlin S., Weil D., Moatti L., Chauvin P., Garabédian E.N., Petit C. Clinical features of the prevalent form of childhood deafness, DFNB1, due to a connexin-26 gene defect: Implications for genetic counselling. Lancet. 1999;353:1298–1303. doi: 10.1016/S0140-6736(98)11071-1. [DOI] [PubMed] [Google Scholar]
- 69.Gasmelseed N.M.A., Schmidt M., Magzoub M.M.A., Macharia M., Elmustafa O.M., Ototo B., Winkler E., Ruge G., Horstmann R.D., Meyer C.G. Low frequency of deafness-associated GJB2 variants in Kenya and Sudan and novel GJB2 variants. Hum. Mutat. 2004;23:206–207. doi: 10.1002/humu.9216. [DOI] [PubMed] [Google Scholar]
- 70.Sansović I., Knezević J., Musani V., Seeman P., Barisić I., Pavelić J. GJB2 mutations in patients with nonsyndromic hearing loss from Croatia. Genet. Test. Mol. Biomark. 2009;13:693–699. doi: 10.1089/gtmb.2009.0073. [DOI] [PubMed] [Google Scholar]
- 71.Kenna M.A., Wu B.L., Cotanche D.A., Korf B.R., Rehm H.L. Connexin 26 studies in patients with sensorineural hearing loss. Arch. Otolaryngol. Head Neck Surg. 2001;127:1037–1042. doi: 10.1001/archotol.127.9.1037. [DOI] [PubMed] [Google Scholar]
- 72.Matos T.D., Simões-Teixeira H., Caria H., Gonçalves A.C., Chora J., Correia M.D.C., Moura C., Rosa H., Monteiro L., O’Neill A., et al. Spectrum and frequency of GJB2 mutations in a cohort of 264 Portuguese nonsyndromic sensorineural hearing loss patients. Int. J. Audiol. 2013;52:466–471. doi: 10.3109/14992027.2013.783719. [DOI] [PubMed] [Google Scholar]
- 73.Pandya A., Arnos K.S., Xia X.J., Welch K.O., Blanton S.H., Friedman T.B., Garcia Sanchez G., Liu MD X.Z., Morell R., Nance W.E. Frequency and distribution of GJB2 (connexin 26) and GJB6 (connexin 30) mutations in a large North American repository of deaf probands. Genet. Med. 2003;5:295–303. doi: 10.1097/01.GIM.0000078026.01140.68. [DOI] [PubMed] [Google Scholar]
- 74.Rabionet R., Zelante L., López-Bigas N., D’Agruma L., Melchionda S., Restagno G., Arbonés M.L., Gasparini P., Estivill X. Molecular basis of childhood deafness resulting from mutations in the GJB2 (connexin 26) gene. Hum. Genet. 2000;106:40–44. doi: 10.1007/s004390051007. [DOI] [PubMed] [Google Scholar]
- 75.Zelante L., Gasparini P., Estivill X., Melchionda S., D’Agruma L., Govea N., Milá M., Monica M.D., Lutfi J., Shohat M., et al. Connexin26 mutations associated with the most common form of non-syndromic neurosensory autosomal recessive deafness (DFNB1) in Mediterraneans. Hum. Mol. Genet. 1997;6:1605–1609. doi: 10.1093/hmg/6.9.1605. [DOI] [PubMed] [Google Scholar]
- 76.Löffler J., Nekahm D., Hirst-Stadlmann A., Günther B., Menzel H.J., Utermann G., Janecke A.R. Sensorineural hearing loss and the incidence of Cx26 mutations in Austria. Eur. J. Hum. Genet. 2001;9:226–230. doi: 10.1038/sj.ejhg.5200607. [DOI] [PubMed] [Google Scholar]
- 77.Kelley P.M., Harris D.J., Comer B.C., Askew J.W., Fowler T., Smith S.D., Kimberling W.J. Novel mutations in the connexin 26 gene (GJB2) that cause autosomal recessive (DFNB1) hearing loss. Am. J. Hum. Genet. 1998;62:792–799. doi: 10.1086/301807. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 78.Kelsell D.P., Dunlop J., Stevens H.P., Lench N.J., Liang J.N., Parry G., Mueller R.F., Leigh I.M. Connexin 26 mutations in hereditary non-syndromic sensorineural deafness. Nature. 1997;387:80–83. doi: 10.1038/387080a0. [DOI] [PubMed] [Google Scholar]
- 79.Denoyelle F., Weil D., Maw M.A., Wilcox S.A., Lench N.J., Allen-Powell D.R., Osborn A.H., Dahl H.H., Middleton A., Houseman M.J., et al. Prelingual deafness: High prevalence of a 30delG mutation in the connexin 26 gene. Hum. Mol. Genet. 1997;6:2173–2177. doi: 10.1093/hmg/6.12.2173. [DOI] [PubMed] [Google Scholar]
- 80.Richard G., White T.W., Smith L.E., Bailey R.A., Compton J.G., Paul D.L., Bale S.J. Functional defects of Cx26 resulting from a heterozygous missense mutation in a family with dominant deaf-mutism and palmoplantar keratoderma. Hum. Genet. 1998;103:393–399. doi: 10.1007/s004390050839. [DOI] [PubMed] [Google Scholar]
- 81.Uyguner O., Tukel T., Baykal C., Eris H., Emiroglu M., Hafiz G., Ghanbari A., Baserer N., Yuksel-Apak M., Wollnik B. The novel R75Q mutation in the GJB2 gene causes autosomal dominant hearing loss and palmoplantar keratoderma in a Turkish family. Clin. Genet. 2002;62:306–309. doi: 10.1034/j.1399-0004.2002.620409.x. [DOI] [PubMed] [Google Scholar]
- 82.Carrasquillo M.M., Zlotogora J., Barges S., Chakravarti A. Two different connexin 26 mutations in an inbred kindred segregating non-syndromic recessive deafness: Implications for genetic studies in isolated populations. Hum. Mol. Genet. 1997;6:2163–2172. doi: 10.1093/hmg/6.12.2163. [DOI] [PubMed] [Google Scholar]
- 83.Putcha G.V., Bejjani B.A., Bleoo S., Booker J.K., Carey J.C., Carson N., Das S., Dempsey M.A., Gastier-Foster J.M., Greinwald J.H., Jr., et al. A multicenter study of the frequency and distribution of GJB2 and GJB6 mutations in a large North American cohort. Genet. Med. 2007;9:413–426. doi: 10.1097/GIM.0b013e3180a03276. [DOI] [PubMed] [Google Scholar]
- 84.Kupka S., Braun S., Aberle S., Haack B., Ebauer M., Zeissler U., Zenner H.-P., Blin N., Pfister M. Frequencies of GJB2 mutations in German control individuals and patients showing sporadic non-syndromic hearing impairment. Hum. Mutat. 2002;20:77–78. doi: 10.1002/humu.9044. [DOI] [PubMed] [Google Scholar]
- 85.Scott D.A., Kraft M.L., Carmi R., Ramesh A., Elbedour K., Yairi Y., Srisailapathy C.R., Rosengren S.S., Markham A.F., Mueller R.F., et al. Identification of mutations in the connexin 26 gene that cause autosomal recessive nonsyndromic hearing loss. Hum. Mutat. 1998;11:387–394. doi: 10.1002/(SICI)1098-1004(1998)11:5<387::AID-HUMU6>3.0.CO;2-8. [DOI] [PubMed] [Google Scholar]
- 86.Dalamón V., Lotersztein V., Lipovsek M., Bèherán A., Mondino M.E., Diamante F., Pallares N., Diamante V., Elgoyhen A.B. Performance of speech perception after cochlear implantation in DFNB1 patients. Acta Otolaryngol. 2009;129:395–398. doi: 10.1080/00016480802566295. [DOI] [PubMed] [Google Scholar]
- 87.Estivill X., Fortina P., Surrey S., Rabionet R., Melchionda S., D’Agruma L., Mansfield E., Rappaport E., Govea N., Milà M., et al. Connexin-26 mutations in sporadic and inherited sensorineural deafness. Lancet. 1998;351:394–398. doi: 10.1016/S0140-6736(97)11124-2. [DOI] [PubMed] [Google Scholar]
- 88.Brobby G.W., Müller-Myhsok B., Horstmann R.D. Connexin 26 R143W mutation associated with recessive nonsyndromic sensorineural deafness in Africa. N. Engl. J. Med. 1998;338:548–550. doi: 10.1056/NEJM199802193380813. [DOI] [PubMed] [Google Scholar]
- 89.Frei K., Lucas T., Ramsebner R., Schöfer C., Baumgartner W.-D., Weipoltshammer K., Erginel-Unaltuna N., Wachtler F.J., Kirschhofer K. A novel connexin 26 mutation associated with autosomal recessive sensorineural deafness. Audiol. Neurootol. 2004;9:47–50. doi: 10.1159/000074186. [DOI] [PubMed] [Google Scholar]
- 90.Marlin S., Garabédian E.N., Roger G., Moatti L., Matha N., Lewin P., Petit C., Denoyelle F. Connexin 26 gene mutations in congenitally deaf children: Pitfalls for genetic counseling. Arch. Otolaryngol. Head Neck Surg. 2001;127:927–933. doi: 10.1001/archotol.127.8.927. [DOI] [PubMed] [Google Scholar]
- 91.Matos T.D., Caria H., Simões-Teixeira H., Aasen T., Dias O., Andrea M., Kelsell D.P., Fialho G. A novel M163L mutation in connexin 26 causing cell death and associated with autosomal dominant hearing loss. Hear. Res. 2008;240:87–92. doi: 10.1016/j.heares.2008.03.004. [DOI] [PubMed] [Google Scholar]
- 92.Samanich J., Lowes C., Burk R., Shanske S., Lu J., Shanske A., Morrow B.E. Mutations in GJB2, GJB6, and mitochondrial DNA are rare in African American and Caribbean Hispanic individuals with hearing impairment. Am. J. Med. Genet. A. 2007;143A:830–838. doi: 10.1002/ajmg.a.31668. [DOI] [PubMed] [Google Scholar]
- 93.Frei K., Ramsebner R., Lucas T., Hamader G., Szuhai K., Weipoltshammer K., Baumgartner W.-D., Wachtler F.J., Kirschhofer K. GJB2 mutations in hearing impairment: Identification of a broad clinical spectrum for improved genetic counseling. Laryngoscope. 2005;115:461–465. doi: 10.1097/01.mlg.0000157855.47143.71. [DOI] [PubMed] [Google Scholar]
- 94.Oliveira C.A., Maciel-Guerra A.T., Sartorato E.L. Deafness resulting from mutations in the GJB2 (connexin 26) gene in Brazilian patients. Clin. Genet. 2002;61:354–358. doi: 10.1034/j.1399-0004.2002.610506.x. [DOI] [PubMed] [Google Scholar]
- 95.Morell R.J., Kim H.J., Hood L.J., Goforth L., Friderici K., Fisher R., Van Camp G., Berlin C.I., Oddoux C., Ostrer H., et al. Mutations in the Connexin 26 Gene (GJB2) among Ashkenazi Jews with Nonsyndromic Recessive Deafness. N. Engl. J. Med. 1998;339:1500–1505. doi: 10.1056/NEJM199811193392103. [DOI] [PubMed] [Google Scholar]
- 96.Dalamon V., Fiori M.C., Figueroa V.A., Oliva C.A., del Rio R., Gonzalez W., Canan J., Elgoyhen A.B., Altenberg G.A., Retamal M.A. Gap-junctional channel and hemichannel activity of two recently identified connexin 26 mutants associated with deafness. Pflügers Arch. Eur. J. Physiol. 2016;468:909–918. doi: 10.1007/s00424-016-1788-7. [DOI] [PubMed] [Google Scholar]
- 97.Primignani P., Trotta L., Castorina P., Lalatta F., Sironi F., Radaelli C., Degiorgio D., Curcio C., Travi M., Ambrosetti U., et al. Analysis of the GJB2 and GJB6 Genes in Italian Patients with Nonsyndromic Hearing Loss: Frequencies, Novel Mutations, Genotypes, and Degree of Hearing Loss. Genet. Test. Mol. Biomark. 2009;13:209–217. doi: 10.1089/gtmb.2008.0086. [DOI] [PubMed] [Google Scholar]
- 98.Kudo T. Transgenic expression of a dominant-negative connexin26 causes degeneration of the organ of Corti and non-syndromic deafness. Hum. Mol. Genet. 2003;12:995–1004. doi: 10.1093/hmg/ddg116. [DOI] [PubMed] [Google Scholar]
- 99.Marziano N.K. Mutations in the gene for connexin 26 (GJB2) that cause hearing loss have a dominant negative effect on connexin 30. Hum. Mol. Genet. 2003;12:805–812. doi: 10.1093/hmg/ddg076. [DOI] [PubMed] [Google Scholar]
- 100.Piazza V., Beltramello M., Menniti M., Colao E., Malatesta P., Argento R., Chiarella G., Gallo L.V., Catalano M., Perrotti N., et al. Functional analysis of R75Q mutation in the gene coding for Connexin 26 identified in a family with nonsyndromic hearing loss. Clin. Genet. 2005;68:161–166. doi: 10.1111/j.1399-0004.2005.00468.x. [DOI] [PubMed] [Google Scholar]
- 101.Neocleous V., Aspris A., Shahpenterian V., Nicolaou V., Panagi C., Ioannou I., Kyamides Y., Anastasiadou V., Phylactou L.A. High Frequency of 35delG GJB2 Mutation and Absence of del(GJB6-D13S1830) in Greek Cypriot Patients with Nonsyndromic Hearing Loss. Genet. Test. 2006;10:285–289. doi: 10.1089/gte.2006.10.285. [DOI] [PubMed] [Google Scholar]
- 102.Günther B., Steiner A., Nekahm-Heis D., Albegger K., Zorowka P., Utermann G., Janecke A. The 342-kb deletion inGJB6is not present in patients with non-syndromic hearing loss from Austria. Hum. Mutat. 2003;22:180. doi: 10.1002/humu.9167. [DOI] [PubMed] [Google Scholar]
- 103.Uyguner O., Emiroglu M., Uzumcu A., Hafiz G., Ghanbari A., Baserer N., Yuksel-Apak M., Wollnik B. Frequencies of gap- and tight-junction mutations in Turkish families with autosomal-recessive non-syndromic hearing loss. Clin. Genet. 2003;64:65–69. doi: 10.1034/j.1399-0004.2003.00101.x. [DOI] [PubMed] [Google Scholar]
- 104.Pandey K.R., Maden N., Poudel B., Pradhananga S., Sharma A.K. The Curation of Genetic Variants: Difficulties and Possible Solutions. Genom. Proteom. Bioinform. 2012;10:317–325. doi: 10.1016/j.gpb.2012.06.006. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 105.Fokkema I.F.A.C., Ivo F.A., Velde K.J., Slofstra M.K., Ruivenkamp C.A.L., Vogel M.J., Pfundt R., Blok M.J., Lekanne Deprez R.H., Waisfisz Q., et al. Dutch genome diagnostic laboratories accelerated and improved variant interpretation and increased accuracy by sharing data. Hum. Mutat. 2019;40:2230–2238. doi: 10.1002/humu.23896. [DOI] [PMC free article] [PubMed] [Google Scholar]
Associated Data
This section collects any data citations, data availability statements, or supplementary materials included in this article.