The c.1A>G (p.Met1Val) variant in GJB2 may cause a truncated or absent protein by altering the start codon of the coding sequence and is predicted to lead to the omission of a critical region of the protein. There have been multiple pathogenic variants observed in the region between this site and the next expected start codon (PVS1; ClinVar Variation IDs: 21387, 188758). There are also multiple pathogenic/likely pathogenic start-loss variants at this position which may indicate that this residue is critical to the function of the protein (ClinVar Variation IDs: 550716, 2070085, 371781, 551915). The highest population minor allele frequency of the variant is 0.02% (5/30616) in the South Asian population in gnomAD v.2.1.1, which does not meet any population codes based on the thresholds defined by the ClinGen Hearing Loss Expert Panel (BA1/BS1/PM2_Supporting not met). Intercellular diffusion of microinjected neurobiotin in coupling-deficient HeLa cells transfected with Connexin 26 cDNAs was analyzed. The transfectant M1V showed reduced neurobiotin transfer compared to wild-type and equivalent to background levels indicating that this variant impacts protein function (PS3_Moderate; PMID: 12189493). This variant has been detected in at least 6 individuals with nonsyndromic hearing loss (4.5 PM3_Very Strong points). Two individuals with nonsyndromic hearing loss were homozygous for the variant (PMID: 26188157, 18941476). One individual with bilateral sensorineural hearing loss was compound heterozygous with a pathogenic variant c.167delT, p.Leu56fs (ClinVar ID: 17010, PMID: 20146813). Another three individuals with nonsyndromic hearing loss were compound heterozygous (one confirmed trans, the other two assumed trans) with the pathogenic c.35delG, p.Gly12fs variant (ClinVar ID: 17004, PMID: 9482292, 10218527, 23555729). In summary, this variant meets criteria to be classified as pathogenic for autosomal recessive hearing loss based on the ACMG/AMP criteria applied as specified by the Hearing Loss Expert Panel: PVS1, PM3_Very Strong, PS3_Moderate (VCEP specifications version 2; 10.24.2023). intercellular diffusion of microinjected neurobiotin in coupling-deficient HeLa cells transfected with Connexin 26 cDNAs was analyzed. The transfectant M1V showed reduced neurobiotin transfer compared to wild-type and equivalent to background levels indicating that this variant impacts protein function (PS3_Moderate; PMID: 12189493). This variant has been detected in at least 6 individuals with nonsyndromic hearing loss (4.5 PM3_Very Strong points). Two individuals with nonsyndromic hearing loss were homozygous for the variant (PMID: 26188157, 18941476). One individual with bilateral sensorineural hearing loss was compound heterozygous with a pathogenic variant c.167delT, p.Leu56fs (ClinVar ID: 17010, PMID: 20146813). Another three individuals with nonsyndromic hearing loss were compound heterozygous (one confirmed trans, the other two assumed trans) with the pathogenic c.35delG, p.Gly12fs variant (ClinVar ID: 17004, PMID: 9482292, 10218527, 23555729). In summary, this variant meets criteria to be classified as pathogenic for autosomal recessive hearing loss based on the ACMG/AMP criteria applied as specified by the Hearing Loss Expert Panel: PVS1, PM3_Very Strong, PS3_Moderate (VCEP specifications version 2; 10.24.2023).