OObjective A basis for studying the pathogenesis of Perrault syndrome (PRLTS) caused by mutations in the HARS2 gene by studying of the effect of mutated loci of HARS2 gene on HARS2 protein in deaf families.Methods Detection and validation of the HARS2 gene in the DNA of samples from members of this family line.Analysis of protein modeling using software to predict the effect of Asp117Asn and Leu303Pro mutations on the structural stability of HARS2 protein.Lentiviral recombinant plasmids containing Leu303Pro mutation and Asp117Asn mutation of HARS2 gene ,wild-type HARS2 gene and empty vector were transfected into HEK 293T cells (human embryonic kidney cells) to obtain WT cell group, Asp117Asn cell group, Leu303Pro cell group and NC cell group.Northern-blot for mitochondrial tRNAHis amylation level;Western-blot for HARS2 protein expression;immunofluorescence for HARS2 protein expression site;Results (1)The preexisting patient and his brother were deaf and had a compound heterozygous mutation of HARS2 349G>A (p.Asp117Asn) and 908T>C (p.Leu303Pro);his father had a heterozygous mutation of HARS2 c.908T>C (p. Leu303Pro) heterozygous mutation; his grandmother, mother, and uncle had a heterozygous mutation of HARS2 349G>A (p.Asp117Asn);.(2)Mutations may affect HARS2 protein stability.(3)The mitochondrial tRNAHis amylation level in WT cell group was higher than the remaining three groups (P<0.05); Asp117Asn cell group had higher level of mitochondrial tRNA His amylation than Leu303Pro cell group (P=0.016).(4) HARS2 proteins are all expressed in mitochondria.(5)HARS2 protein was not significantly expressed in the NC cell group; HARS2 protein expression in WT cell group was not significantly different from Asp117Asn cell group (p=0.356) and higher than Leu303Pro cell group (p=0.000).Conclusion 1.Two new mutant loci c.349G>A and c.908T>C of the HARS2 gene were identified in this family.2.The mutation may lead to reduced mitochondrial tRNAHis ammonia-acylation capacity through reduced HARS2 protein ammonia-acylation capacity and/or expression, which in turn leads to mitochondrial dysfunction and thus hearing loss.