Study On Pathogenic Mechanism Of Perrault Syndrome With Deafness As Clinical Manifestation Caused By HARS2 Gene Mutation

Objective:In this study,use the second-generation high-throughput gene sequencing was performed on the propositus,and the candidate mutation loci were verified by Sanger sequencing in the DNA of the family samples.construct stably transfected HEK 293T cell lines stably overexpressing wild-type and mutant HARS2 based on the screened HARS2 mutation loci by second-generation high-throughput gene sequencing of the preceding witnesses and Sanger sequencing verification in DNA of homeotic samples of candidate mutant loci,and to verify the functional effects of mutations on HARS2 protein for the study of this will provide a basis for studying the pathogenesis of Perrault syndrome caused by mutations in the HARS2.According to the HARS2gene mutation sites screened out,stable transfected HEK 293T cell lines overexpressing wild-type and mutant HARS2 genes were constructed,and to verify the functional effects of mutations on HARS2 protein for the study of this will provide a basis for studying the pathogenesis of Perrault syndrome caused by mutations in the HARS2.Methods:DNA from three generations of family members totaling 10individuals was first tested and validated for the HARS2 based on the results of second-generation high-throughput gene sequencing of the preceding witnesses.The functional effects of mutations on HARS2 protein were predicted by bioinformatics analysis.Lentiviral recombinant plasmids expressing Asp117Asn mutation,Leu303Pro mutation and wild HARS2 were constructed and transfected into HEK 293T cells by lentiviral packaging system to obtain HEK293T cell lines stably transfected with Asp117Asn mutation HARS2（Asp117Asn cell group）,stably transfected with HEK 293T cell line stably transfected with Leu303Pro mutant HARS2（Leu303Pro cell group）,HEK 293T cell line stably transfected with wild-type HARS2（WT cell group）,and HEK293T cell line stably transfected with empty viral vector（NC cell group）were also established.Western blot was used to detect the expression of HARS2 protein in each of the above cell lines.Immunofluorescence was used to observe the difference of HARS2 protein localization in cells between WT cell group and Asp117Asn cell group and Leu303Pro cell group.Northern blot was used to detect the level of mitochondrial tRNA^His in NC cell group,WT cell group,Asp117Asn and Leu303Pro cell group,and compare the differences between the groups.Results:（1）Genetic sequencing of the Perrault syndrome presentee and family members showed that the presentee and sibling brother in this family were patients with HARS2 c.349G>A（p.Asp117Asn）and HARS2 c.908T>C（p.Leu303Pro）complex heterozygous mutations,their mother,grandmother,and uncle were HARS2 c.349G>A（p.Leu303Pro）heterozygous mutation,and his father is HARS2 c.908T>C（p.Asp117Asn）heterozygous mutation.（2）Both HARS2 p.Asp117Asn and HARS2 p.Leu303 locate in the catalytic region of HARS2 protein,there are highly conserved among different species,and the mutations may affect the catalytic function of HARS2 protein.（3）Western blot results showed that there was no significant expression of HARS2 protein in NC cell group,and significant expression of HARS2 protein was seen in WT cell group,Asp117Asn cell group and Leu303Pro cell group;the expression of HARS2 protein in Leu303Pro cell group was significantly lower than that in WT cell line,and the difference was statistically significant（P<0.001）;HARS2protein expression in Asp117Asn cell group was not significantly different from WT cell group（P>0.05）.（4）Immunofluorescence results showed that HARS2protein was localized in mitochondria in WT cell group.Immunolocalization results of HARS2 protein in Asp117Asn cell group and Leu303Pro cell group were consistent with WT cell group,localized in mitochondria,and mutation did not affect the intracellular localization of HARS2 protein.（5）Northern blot results showed:the level of mitochondrial tRNA^His amylation in WT cell group was increased compared with NC cell group,and the difference was statistically significant（P<0.05）.Compared with WT cell group;the mitochondrial tRNA^His aminoacylation levels were significantly lower in Asp117Asn and Leu303Pro cell lines,and the difference was statistically significant（P<0.001）;there was no significant difference in mitochondrial tRNAHis aminoacylation levels between Asp117Asn and Leu303Pro cell lines（P>0.05）.The mitochondrial tRNA^His aminoacylation levels in Leu303Procell group was lower than that in Asp117Asn cell group,and the difference was statistically significant（P<0.05）.Conclusion:（1）In this study,two pathogenic mutation sites of HARS2c.349G>A and c.908T>C were identified in Perrault syndrome families,enriching the mutation spectrum of HARS2.Bioinformatics analysis showed that HARS2 c.349G>A and c.908T>C were located in the catalytic region of HARS2 protein,and mutations may affect the catalytic function of HARS2protein;（2）We successfully constructed a HEK 293T cell line stably transfected which overexpressed wild-type and mutant HARS2,which laid a foundation for further research on the effect of mutation on the function of HARS2protein.（3）HARS2 c.908T>C mutation resulted in decreased expression of HARS2 protein.HARS2 c.349G>A and c.908T>C mutations resulted in decreased levels of mitochondrial tRNA^Hisaminoylation,suggesting that the mutations affected the aminoylation capacity of HARS2 protein.