ClinVar Genomic variation as it relates to human health

Variant summary: GJB2 c.229T>C (p.Trp77Arg) results in a non-conservative amino acid change located in the connexin, N-terminal domain (IPR013092) of the encoded protein sequence. Five of five in-silico tools predict a damaging effect of the variant on protein function. The variant allele was found at a frequency of 4.1e-05 in 246400 control chromosomes (gnomAD). This frequency is not higher than expected for a pathogenic variant in GJB2 causing Autosomal Recessive Non-Syndromic Hearing Loss (4.1e-05 vs 0.025), allowing no conclusion about variant significance. c.229T>C has been reported in the literature in multiple homozygous and compound heterozygous individuals affected with Autosomal Recessive Non-Syndromic Hearing Loss (Alvarez 2005, Carrasquillo 1997, Snoeckx 2005), and the variant was also found to segregate with the disease in one of these reported families (Carrasquillo 1997). These data indicate that the variant is very likely to be associated with disease. Functional studies have shown to have >99% reduction in junctional conductance in comparison to wild-type (Bruzzone_2003). Three clinical diagnostic laboratories have submitted clinical-significance assessments for this variant to ClinVar after 2014 without evidence for independent evaluation. All laboratories classified the variant as pathogenic. Based on the evidence outlined above, the variant was classified as pathogenic.

A homozygous missense variant was identified, NM_004004.5(GJB2):c.229T>C in exon 2 of the GJB2 gene. This substitution is predicted to create a major change from a tryptophan to an arginine at position 77, NP_003995.2(GJB2):p.(Trp77Arg). The tryptophan at this position has very high conservation (100 vertebrates, UCSC). In silico software predicts this variant to be disease causing. It is situated in the second transmembrane domain and functional studies have shown that it causes loss of channel activity. (Martin, PE. et al. (1999), Bruzzone, R. et al. (2002)). This variant is present in the gnomAD population database at a frequency of 0.0041% (10 in 246208, 0 homozygotes). It is one of the more frequent GJB2 variants and has been previously reported in patients with autosomal recessive deafness (Cryns, K. et al. (2004), Snoeckx, RL. et al. (2005), ClinVar). Based on current information, this variant has been classified as PATHOGENIC.

NM_004004.5(GJB2):c.229T>C(W77R) is classified as pathogenic in the context of GJB2-related DFNB1 nonsyndromic hearing loss and deafness. Sources cited for classification include the following: PMID 15967879, 22785241, 16380907, 10556284 and 12505163. Classification of NM_004004.5(GJB2):c.229T>C(W77R) is based on the following criteria: This is a well-established pathogenic variant in the literature that has been observed more frequently in patients with clinical diagnoses than in healthy populations. Please note: this variant was assessed in the context of healthy population screening.

Based on ACMG/AMP guidelines and Hearing Loss Expert Panel specific criteria: The c.229T>C, p.Trp77Arg variant has a filtering allele frequency of 0,00949% (7/34590 of Latino alleles with 95% CI) from Genome Aggregation Database v2.1.1 (http://gnomad.broadinstitute.org; calculated by using inverse allele frequency at https://www.cardiodb.org/allelefrequencyapp/), which meets the PM2_supporting criteria. This variant has been identified in trans with different pathogenic variants in at least four patients: (PMID: 9328482, 11935342, 15964725, 16088916, 16380907, 22785241, PMID:16467727, 16545002, 19371219) applying to PM3_VeryStrong. Computational evidence showed a damage impact of the mutation to the protein (REVEL: 0.934) meeting PP3 rule. Functional studies in HeLa cells and Xenopus laevis oocytes demonstrated a deleterious effect of the mutant without dominant effect to Human CX26 by dye transfer and junctional conductance measurements assays. In both cases the levels of dye transfer and conductance did not exceed background levels (PMID: 10556284, PMID: 12064630, PMID: 12505163) applying to PS3_Moderate rule. Therefore, the c.229T>C variant meets criteria to be classified as pathogenic for autosomal recessive non-syndromic hearing loss (PM2_Supporting, PM3_VeryStrong, PP3 and PS3_Moderate).

GJB2 c.229T>C, p.W77R alters a residue that is completely conserved in all sequenced vertebrates. The variant was previously reported to be deficient in formation of junctional channels (PMID: 18941476). The variant is homozygous in a Palestinian child with severe to profound pre-lingual hearing loss (Abu Rayyan 2020). It is not present in 1300 Palestinian controls and is present in 10/251404 alleles on gnomAD, all heterozygotes.

Published functional studies demonstrate that the W77R variant did not form homotypic junctional channels, had impaired intercellular coupling, was inefficiently targeted to the plasma membrane, and was retained in intracellular stores (Martin et al., 1999; Bruzzone et al., 2003); In silico analysis supports that this missense variant has a deleterious effect on protein structure/function; This variant is associated with the following publications: (PMID: 12064630, 22975760, 14985372, 12505163, 10556284, 9328482, 25388846, 30139988, 15967879, 15964725, 10982180, 11102979, 16380907, 11935342, 22785241, 21726435, 31163360, 31827275, 32747562, 33096615, 31589614, 33105617)

This sequence change replaces tryptophan, which is neutral and slightly polar, with arginine, which is basic and polar, at codon 77 of the GJB2 protein (p.Trp77Arg). This variant is present in population databases (rs104894397, gnomAD 0.02%). This missense change has been observed in individuals with autosomal recessive non-syndromic deafness (PMID: 16467727, 16545002, 19371219). It has also been observed to segregate with disease in related individuals. ClinVar contains an entry for this variant (Variation ID: 17003). Advanced modeling of protein sequence and biophysical properties (such as structural, functional, and spatial information, amino acid conservation, physicochemical variation, residue mobility, and thermodynamic stability) performed at Invitae indicates that this missense variant is expected to disrupt GJB2 protein function with a positive predictive value of 95%. Experimental studies have shown that this missense change affects GJB2 function (PMID: 10556284, 12064630, 12505163). For these reasons, this variant has been classified as Pathogenic.

The p.Trp77Arg variant has been reported in many individuals with hearing loss and was absent from many controls (Carrasquillo 1997 PMID: 9328482, Dalamón 2005 PMID: 15964725, Marlin 2005 PMID: 15967879, Prasad 2000 PMID: 11102979, Rabionet 2000 PMID: 10982180, Shahin 2002 PMID: 11935342, Snoeckx 2005 PMID: 16380907). It was also found to segregate with hearing loss in one large kindred (Carrasquillo 1997 PMID: 9328482). In summary, this variant meets our criteria to be classified as pathogenic. ACMG criteria applied: PM3_VeryStrong, PP1_Strong, PP3, PM2_Supporting.

The GJB2 c.229T>C variant is predicted to result in the amino acid substitution p.Trp77Arg. This variant was reported in the homozygous and compound heterozygous states in individuals with hearing loss (Carrasquillo et al. 1997. PubMed ID: 9328482; Raymond et al. 2019. PubMed ID: 31163360; Downie et al. 2019. PubMed ID: 31827275). This variant is reported in 0.020% of alleles in individuals of Latino descent in gnomAD. This variant is interpreted as pathogenic.