Comprehensive simulation and interpretation of single nucleotide substitutions in GJB2 reveals the genetic and phenotypic landscape of GJB2-related hearing loss

Genetic variants in the GJB2 gene are the most frequent causes of congenital and childhood hearing loss worldwide. In addition to nonsyndromic hearing loss, GJB2 pathogenic variants are also correlated with syndromic phenotypes, showing high genetic and phenotypic heterogeneity. To comprehensively delineate the genetic and phenotypic landscape of GJB2 variants, we interpreted and manually curated all the 2043 possible single-nucleotide substitution (SNS) coding variants in this gene following the hearing loss-specific ACMG/AMP guidelines. As a result, 61 (3.0%), 188 (9.2%), 1487 (72.8%), 301 (14.7%) and 6 (0.3%) variants were classified as pathogenic, likely pathogenic, variant of uncertain significance, likely benign and benign, respectively. Interestingly, 54% (84/156) of pathogenic/likely pathogenic missense variants were not recorded in ClinVar. Further analysis showed that the second transmembrane domain (TM2) and the 310 helix are highly enriched for pathogenic missense variants. The N-terminal tail and the extracellular loop (E1) showed a high density of variants that are associated with syndromic or dominant nonsyndromic hearing loss. On the other hand, the intracellular loops (CL and CT) were extremely tolerant to variation. Based on this new information, we propose refinements of the guidelines for variant interpretation in GJB2. In summary, our study interpreted all possible SNS variants in the coding region of the GJB2 gene, characterized novel clinically significant (N = 249) and benign or likely benign (N = 307) in this gene, and revealed significant genotype-phenotype correlations at this common hearing loss locus. The interpretation of GJB2 SNS variants in the coding region provides a prototype for genes with similarly high genetic and phenotypic heterogeneity.