Objective To study the pathogenesis of Perrault syndrome (PRLTS) caused by mutations in HARS2 gene by investigating the effect of mutated loci of the HARS2 gene on HARS2 protein in a deaf family.Methods The HARS2 gene in the DNA samples from members of this family line was detected and validated. Protein modeling was analyzed 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 (WT) 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 normal control (NC) cell group. Northern blot was adopted for mitochondrial tRNAHis aminoacylation level, Western blot for HARS2 protein expression and immunofluorescence for HARS2 protein expression site.Results ①The 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). His grandmother, mother, and uncle had a heterozygous mutation of HARS2 349G>A (p.Asp117Asn). ②Mutations might affect HARS2 protein stability. ③The mitochondrial tRNAHis aminoacylation level in WT cell group was higher than those in the remaining three groups (P<0.05). Asp117Asn cell group had higher level of mitochondrial tRNAHis aminoacylation than Leu303Pro cell group (P=0.016). ④HARS2 proteins were all expressed in mitochondria. ⑤HARS2 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 that in Leu303Pro cell group (P=0.000).Conclusion Two new mutant loci c.349G>A and c.908T>C of the HARS2 gene are identified in this family. The mutation may lead to reduced mitochondrial tRNAHis aminoacylation capacity through reducing HARS2 protein aminoacylation capacity and/or expression, which in turn leads to mitochondrial dysfunction and thus hearing loss.