Abstract:ObjectiveTo determine the pathogenicity of rare, missense variants in known causative recessive deafness genes by sequencing of unaffected family members of the deaf patients.MethodsA total of 800 probands with hearing impairment were collected to screen the deafnesscausing genes GJB2 and/or SLC26A4 by PCR and Sanger sequencing, including the coding exons and flanking sequences. When probands harbored the pathogenic mutations and if possible, some unaffected family members of probands were recruited to further analyze the mutations in the corresponding genes.ResultsTwo rare missense variants were found in three unaffected family members in different families, including p.T123N (n=2) in GJB2 and p.A434T (n=1) in SLC26A4, with the opposite allele to a known pathogenic recessive mutation.Conclusionp.T123N in GJB2 and p.A434T in SLC26A4 are rare but benign variants for deafness. Sequencing of unaffected family members of the affected patients may be an efficient approach to clarify the pathogenicity of some rare variants for recessive genetic disorders with high penetrance. More reliable data will be provided for deafnesscausing gene diagnosis and counseling through screening plentiful samples.