Submissions for variant NM_004004.6(GJB2):c.-22-2A>C

gnomAD frequency: 0.00056  dbSNP: rs201895089

Total submissions: 21

Submitter	RCV	SCV	Clinical significance	Condition	Last evaluated	Review status	Method	Comment
ClinGen Hearing Loss Variant Curation Expert Panel	RCV000710316	SCV000840501	likely pathogenic	Nonsyndromic genetic hearing loss	2021-04-12	reviewed by expert panel	curation	The filtering allele frequency of the c.-22-2A>C variant in the GJB2 gene is 0.35% for Ashkenazi Jewish chromosomes in gnomAD v2.1.1 (95% CI of 47/10344), which meets the allele frequency threshold defined by the ClinGen Hearing Loss Expert Panel for considering strong evidence against pathogenicity for autosomal recessive hearing loss variants (BS1). However, based on the evidence outlined below, the ClinGen Hearing Loss Expert Panel believes that the evidence for the pathogenicity of this variant for nonsyndromic hearing loss outweighs its high allele frequency in population databases. Therefore, the BS1 code will not contribute to the overall classification. The c.-22-2A>C variant in the GJB2 gene has been detected in at least 9 patients with hearing loss in trans with the pathogenic c.35delG variant (PM3_Very Strong; PMID: 25401782, 24039984, 33096615; Invitae internal data, SCV000935680.2; Children’s Hospital of Philadelphia internal data, Molecular Otolaryngology and Renal Research Laboratories internal data) and one individual with the the p.Leu90Pro variant suspected in trans (PMID: 19814620). Phase was confirmed in at least 6 of these 10 observations.This variant has been reported to segregate with hearing loss in at least 2 family members (PP1; PMID: 24039984). It was also identified in the heterozygous state without a second variant in 8 individuals with hearing loss, and in the biallelic state (with c.35delG) in one individual with reportedly normal hearing (GeneDx, ARUP internal data, SCV000680741.2, SCV000603828.1). Of note, the severity of hearing loss is reported to be mild-moderate in affected probands, some with onset in the third to fourth decade, suggesting that this variant may be a hypomorphic allele. RNA analysis using patient cells demonstrated that the c.-22-2A>C variant leads to expression of a novel GJB2 transcript with a slightly longer 5' UTR but otherwise normal coding region. The alternate transcript was shown to have reduced expression, but it is not clear if the lower expression is sufficient to lead to a phenotype (PS3_Supporting; PMID: 24039984). In summary, the VCEP has used expert judgement to classify this variant as likely pathogenic for autosomal recessive hearing loss based on the ACMG/AMP criteria applied, as specified by the Hearing Loss Expert Panel: PM3_Very Strong, PP1, PS3_Supporting, BS1.
Knight Diagnostic Laboratories, Oregon Health and Sciences University	RCV000416425	SCV000494235	pathogenic	Autosomal recessive nonsyndromic hearing loss 1A	2016-04-08	criteria provided, single submitter	clinical testing	The c.-22-2A>C splice-acceptor variant in the GJB2 gene has been previously reported in 3 individuals within one generation of a single family affected with Deafness and Hearing Loss (Gandía et al., 2013). In all cases, this variant was observed in trans with the well-established pathogenic variant 35delG; however, these individuals experienced mild to moderate hearing loss that developed in the 3rd-4th decade of life (postlingual) (Gandía et al., 2013). Functional splicing studies have demonstrated this variant abolishes the canonical splice-acceptor site for intron 1 of GJB2. The loss of the canonical splice-site results in a longer transcript caused by an alternative splice-acceptor site that is within intron 1(Gandía et al., 2013). This variant is reported at low frequency in the population databases (Exome Sequencing Project = 0.058%; 1000 Genomes = 0%; and ExAC = 0.114%). Therefore, this collective evidence supports the classification of the c.-22-2A>C as a Pathogenic variant for Deafness and Hearing Loss. We have confirmed this finding in our laboratory using Sanger sequencing.
ARUP Laboratories, Molecular Genetics and Genomics, ARUP Laboratories	RCV000507029	SCV000603828	pathogenic	not specified	2016-12-01	criteria provided, single submitter	clinical testing
GeneDx	RCV000579320	SCV000680741	pathogenic	not provided	2024-08-08	criteria provided, single submitter	clinical testing	Published functional studies using patient cells demonstrate the use of two alternative cryptic splice acceptor sites in lieu of the destroyed canonical splice acceptor site, resulting in a longer 5'UTR and reduced expression of the alternate transcript; however, the coding regions remain intact and it is not clear if the lower expression is sufficient to have a clinical effect (PMID: 24039984); In silico analysis supports a deleterious effect on splicing; This variant is associated with the following publications: (PMID: 34428318, 30455902, 30311386, 29754767, 34426522, 34062854, 29016196, 19814620, 33096615, 34652575, 31053783, 31980526, DiStefano2020[paper], 24039984, 26778469, 34325055, 34308104, 36651276, 36979683, 36837553)
Women's Health and Genetics/Laboratory Corporation of America, LabCorp	RCV000416425	SCV000698235	likely pathogenic	Autosomal recessive nonsyndromic hearing loss 1A	2023-07-10	criteria provided, single submitter	clinical testing	Variant summary: GJB2 c.-22-2A>C is located in a canonical splice-site and is predicted to affect mRNA splicing resulting in a significantly altered protein due to either exon skipping, shortening, or inclusion of intronic material. Several computational tools predict a significant impact on normal splicing: Five predict the variant abolishes the canonical 3' acceptor site. At least one publication reports experimental evidence that this variant leads to alternative splicing and two novel transcripts which may express some level of wild type GJB2 (Gandia_2013). However, the tissue type used in this study, saliva, may not represent the most optimal tissue type to base evidence supporting residual transcript expression in an affected tissue. A cochlear tissue, or another ear expressed tissue type may be more informative. The variant allele was found at a frequency of 0.00061 in 251058 control chromosomes. This frequency is not significantly higher than estimated for a pathogenic variant in GJB2 causing Autosomal Recessive Non-Syndromic Hearing Loss (0.00061 vs 0.025), allowing no conclusion about variant significance. c.-22-2A>C has been reported in the literature as a compound heterozygous genotype with c.35delG in individuals predominantly reported to have postlingual mild-moderate hearing loss (example, Gandia_2013, Burke_2016, Stanghellini_2014, Cabanillas_2018, Buonfigilio_2020). Although one conference abstract showed that there was no significant difference in allelic or genotypic frequencies between patient and control groups in an Italian subpopulation, the authors did state that the disease-causing effect of this variant when in trans with a truncating mutation cannot be excluded (Gandia_2013). Furthermore, at-least one recent study re-evaluated this variant as "Likely Pathogenic" reporting the ACMG/AMP criteria coupled with hearing-loss-gene-specific criteria of the ClinGen hearing loss expert panel (Buonfigilio_2020). These data indicate that the variant is likely to be associated with disease. To our knowledge no direct experimental evidence evaaluating a variant specific impact on protein function and assembly have been reported. The following publications have been ascertained in the context of this evaluation (PMID: 33096615, 26778469, 29986705, 24039984, 27057829, 35864128, 34652575, 31195736, 34062854, 25401782). Multiple clinical diagnostic laboratories and an expert panel (ClinGen Hearing Loss Variant Curation Expert Panel) have submitted clinical-significance assessments for this variant to ClinVar after 2014. Multiple submitters reported the variant with conflicting assessments (Pathogenic, n=2; Likely pathogenic, n=6; Benign, n=1; Likely benign, n=1, VUS, n=2 to include the expert panel). Some submitters cite overlapping evidence utilized in the context of this evaluation. Based on the evidence outlined above, the variant was classified as likely pathogenic for postlingual mild-moderate hearing loss.
Illumina Laboratory Services, Illumina	RCV000416425	SCV000915630	likely pathogenic	Autosomal recessive nonsyndromic hearing loss 1A	2018-08-16	criteria provided, single submitter	clinical testing	The GJB2 c.-22-2A>C variant occurs in a canonical splice site (acceptor) and is therefore predicted to disrupt or distort the normal gene product. The c.-22-2A>C variant has been identified in a compound heterozygous state in four individuals with hearing loss. Three of these individuals were from a single Spanish family and exhibited mild, postlingual hearing impairment, with onset in the third to fourth decade of life (Gandía et al. 2013). The fourth individual was of Italian origin and displayed moderate hearing impairment of unspecified onset (Stanghellini et al. 2014). In all four cases, the c.-22-2A>C variant was found in trans with a well-known pathogenic null variant, c.35delG. The Spanish pedigree also included two normal hearing heterozygous individuals, consistent with a recessive pattern of inheritance. The variant was absent from 92 normal hearing control individuals and is reported at a frequency of 0.004539 in the Ashkenazi Jewish population of the Genome Aggregation Consortium. Quantitative RT-PCR experiments indicated that the variant abolishes the canonical acceptor splice site for intron 1 of GJB2 but that a reduced amount of transcript is produced via an alternative acceptor splice site (Gandía et al. 2013). Based on the evidence, the c.-22-2A>C allele is classified as likely pathogenic for autosomal recessive nonsyndromic hearing loss. This variant was observed by ICSL as part of a predisposition screen in an ostensibly healthy population.
Labcorp Genetics (formerly Invitae), Labcorp	RCV000579320	SCV000935680	likely benign	not provided	2024-01-25	criteria provided, single submitter	clinical testing
Illumina Laboratory Services, Illumina	RCV001109959	SCV001267343	benign	Ichthyosis, hystrix-like, with hearing loss	2017-08-23	criteria provided, single submitter	clinical testing	This variant was observed as part of a predisposition screen in an ostensibly healthy population. A literature search was performed for the gene, cDNA change, and amino acid change (where applicable). No publications were found based on this search. Allele frequency data from public databases was too high to be consistent with this variant causing disease. Therefore, this variant is classified as benign.
Illumina Laboratory Services, Illumina	RCV001109960	SCV001267344	benign	Autosomal dominant nonsyndromic hearing loss 3A	2017-08-23	criteria provided, single submitter	clinical testing	This variant was observed as part of a predisposition screen in an ostensibly healthy population. A literature search was performed for the gene, cDNA change, and amino acid change (where applicable). Publications were found based on this search. The evidence from the literature, in combination with allele frequency data from public databases where available, was sufficient to rule this variant out of causing disease. Therefore, this variant is classified as benign.
INGEBI, INGEBI / CONICET	RCV000710316	SCV001433672	likely pathogenic	Nonsyndromic genetic hearing loss	2020-08-31	criteria provided, single submitter	clinical testing	Based on ACMG/AMP guidelines and Hearing Loss Expert Panel specific criteria: the filtering allele frequency of the c.-22-2A>C variant in the GJB2 gene is 0.35% (of 47/10344 alleles Ashkenazi Jewish with chromosomes with 95% CI) from Genome Aggregation Database calculated by using inverse allele frequency at https://www.cardiodb.org/allelefrequencyapp/),applying to BS1 rule. The c.-22-2A>C variant has been detected in at least 4 patients with hearing loss in trans with the pathogenic c.35delG variant (PMID: 25401782, 24039984; Invitae internal data, SCV000935680.2; Laboratory of Physiology and Genetics of Hearing internal data described by ClinGen HL-EP). In addition to this, new evidence is considered since the c.-22-2A>C variant was reported in trans with p.Leu90Pro in a hearing impaired patient (PMID: 19814620, nomenclature issue checked with author: the variant called c.-24A>C in that paper is indeed c.-22-2A>C). In all those genotypes detected, patients exhibited postlingual mild-moderate hearing loss. Since this variant has a high frequency in population databases, the strength of PM3 rule was downgraded from very strong to moderate (PM3_Moderate). The c.[-22-2A>C];[35delG] genotype segregated in two affected and two unaffected members of the family (PP1_Strong; PMID: 24039984). RNA analysis showed that patients with the c.-22-2A>C variant used two alternative splice sites leading to slightly longer 5' UTR transcripts containing normal coding region but with alternated expression. This variant is suspected to be a hypomorphic allele resulting in a postlingual mild-moderate hearing loss phenotype (PS3_Supporting; PMID: 24039984). Therefore, this variant meets criteria to be classified as likely pathogenic for autosomal recessive non-syndromic hearing loss (BS1, PM3_Moderate, PP1_Strong, PS3_Supporting).
Neurogenetic Laboratory, Second Faculty of Medicine, Charles University	RCV000416425	SCV001571591	uncertain significance	Autosomal recessive nonsyndromic hearing loss 1A	2021-03-30	criteria provided, single submitter	clinical testing
Fulgent Genetics, Fulgent Genetics	RCV002481293	SCV002789084	likely pathogenic	Autosomal recessive nonsyndromic hearing loss 1A; Mutilating keratoderma; Ichthyosis, hystrix-like, with hearing loss; Autosomal dominant keratitis-ichthyosis-hearing loss syndrome; Palmoplantar keratoderma-deafness syndrome; Knuckle pads, deafness AND leukonychia syndrome; Autosomal dominant nonsyndromic hearing loss 3A; X-linked mixed hearing loss with perilymphatic gusher	2022-02-18	criteria provided, single submitter	clinical testing
Revvity Omics, Revvity	RCV000579320	SCV003834596	likely pathogenic	not provided	2023-05-11	criteria provided, single submitter	clinical testing
CeGaT Center for Human Genetics Tuebingen	RCV000579320	SCV004136783	pathogenic	not provided	2023-10-01	criteria provided, single submitter	clinical testing	GJB2: PM3:Very Strong, PP1:Strong, PM2:Supporting, PS3:Supporting
Center for Genomic Medicine, King Faisal Specialist Hospital and Research Center	RCV000507029	SCV004232558	pathogenic	not specified	2024-01-18	criteria provided, single submitter	research
Laboratory of Medical Genetics, National & Kapodistrian University of Athens	RCV000416425	SCV005051791	likely pathogenic	Autosomal recessive nonsyndromic hearing loss 1A	2024-02-01	criteria provided, single submitter	curation
Laboratory of Prof. Karen Avraham, Tel Aviv University	RCV000416425	SCV005073750	pathogenic	Autosomal recessive nonsyndromic hearing loss 1A	2024-05-08	criteria provided, single submitter	research	In ClinVar: 5 'Likely pathogenic' and 2 'Pathogenic' submissions
Equipe Genetique des Anomalies du Developpement, Université de Bourgogne	RCV000416425	SCV005395944	likely pathogenic	Autosomal recessive nonsyndromic hearing loss 1A	2024-09-18	criteria provided, single submitter	clinical testing
Victorian Clinical Genetics Services, Murdoch Childrens Research Institute	RCV000416425	SCV005400138	likely pathogenic	Autosomal recessive nonsyndromic hearing loss 1A	2023-07-16	criteria provided, single submitter	clinical testing	Based on the classification scheme VCGS_Germline_v1.3.4, this variant is classified as Likely pathogenic. Following criteria are met: 0102 - Loss of function is a known mechanism of disease in this gene and is associated with both deafness and skin conditions (OMIM). Dominant negative is also a suggested mechanism (PMID: 28428247). (I) 0108 - This gene is associated with both recessive and dominant disease. The autosomal dominant diseases are commonly associated with pathogenic missense variants. The autosomal recessive disease is associated with bi-allelic loss-of-function variants and includes missense and protein truncating variants (NIH Genetics Home Reference, PMID: 12792423). (I) 0112 - The condition associated with this gene has incomplete penetrance (PMID:31160754). (I) 0115 - Variants in this gene are known to have variable expressivity. Severity can range from mild to profound with intrafamilial variability also commonly seen. Commonly, truncating variants are associated to a more severe hearing loss (PMID: 20301449). (I) 0209 - Splice site variant proven to affect splicing of the transcript with uncertain effect on protein sequence. RT-PCR performed on patient saliva RNA demonstrated that this variant abolishes the use of the WT acceptor splice site and introduces the use of two alternate acceptor splice sites, both of which are predicted to keep the coding region intact and are expressed at lower levels compared to the WT transcript. The residual expression of these two alternate transcripts has been hypothesized to explain for the milder phenotype observed in affected individuals although the exact effect is currently unknown. Additionally, as specific protein studies were not performed, the effect of the variant on protein sequence is still unclear (PMID: 24039984). (SP) 0251 - This variant is heterozygous. (I) 0304 - Variant is present in gnomAD (v2) <0.01 for a recessive condition (162 heterozygotes, 0 homozygotes). (SP) 0801 - This variant has very strong previous evidence of pathogenicity in unrelated individuals. This variant has been classified as likely pathogenic by the ClinGen Hearing Loss Variant Curation Expert Panel and associated with mild and moderate autosomal recessive non-syndromic hearing loss (ClinVar, PMID: 31053783). (SP) 0903 - This variant has limited evidence for segregation with disease. This variant has been shown to co-segregate with the NM_004004.6(GJB2):c.35del; p.(Gly12Valfs*2) variant in three siblings with mild postlingual hearing loss (PMID: 24039984). (SP) 1208 - Inheritance information for this variant is not currently available in this individual. (I) Legend: (SP) - Supporting pathogenic, (I) - Information, (SB) - Supporting benign
Counsyl	RCV000416425	SCV001132402	uncertain significance	Autosomal recessive nonsyndromic hearing loss 1A	2019-04-01	no assertion criteria provided	clinical testing
PreventionGenetics, part of Exact Sciences	RCV004530519	SCV004120794	likely pathogenic	GJB2-related disorder	2024-02-06	no assertion criteria provided	clinical testing	The GJB2 c.-22-2A>C variant is located in the 5' untranslated region. This variant has been reported in at least four patients with mild to moderate hearing loss from two families in the compound heterozygous state with a second pathogenic variant (Gandía et al. 2013. PubMed ID: 24039984; Stanghellini et al. 2014. PubMed ID: 25401782). This variant has also been shown to disrupt the canonical splice acceptor site, although some transcripts with an intact coding region were detected (Gandía et al. 2013. PubMed ID: 24039984). This variant is reported in 0.45% of alleles in individuals of Ashkenazi Jewish descent in gnomAD, which is higher than expected for a fully penetrant autosomal recessive pathogenic variant in this gene. This variant is classified by the ClinGen Hearing Loss Variant Curation Expert Panel as likely pathogenic for autosomal recessive hearing loss, noting the mild to moderate phenotype in some patients (https://www.ncbi.nlm.nih.gov/clinvar/variation/375406/). This variant is interpreted as likely pathogenic.