The Function And Mechanism Study Of HELQ Gene And Its Mutation In Women With Premature Ovarian Insufficiency

Objective:To explore the possible genetic variation of screened HELQ gene in patients with premature ovarian insufficiency（POI）,and to conduct the functional mechanism of the previously discovered recessive homozygous mutation of HELQ gene,so as to explore its mechanism in the pathogenesis in POI.Methods:Collected 50 cases of patients diagnosed as Premature ovarian Insufficiency（POI）in our center,recorded clinical data in detail,took its peripheral blood,and verified whether the patients carried new variation of HELQ gene by Sanger sequencing.DNA was extracted from peripheral blood cells,and primer design software Prime was used5.0 Primers for gene exons were designed.DNA extracted from peripheral blood of patients was used as template to amplify HELQ exons by polymerase chain reaction（PCR）.PCR products were purified and sequenced.PCR products with target bands were purified and sequenced,and the graphic files generated by sequencing were used Chromas.2.31 software.The chromosome stability and transcription level of HELQ gene were detected between patients with HELQ gene mutation and normal control group,and then in vitro functional experiments were performed on HEK293 T cells to verify the relationship between HELQ gene mutation and POI.To study the mechanism of HELQ recessive homozygous mutation（c.781A>G;p.R261G）previously screened: Lymphocytes were cultured from fresh peripheral blood to detect the chromosome stability of POI patients carrying mutations and normal control,and the transcription expression level in peripheral lymphocytes was detected by real-time quantitative PCR.HEK293 T cell lines were used as cell models to conduct DNA damage repair experiments: plasmids carrying mutant site（c.781A>G;p.R261G）were constructed,and the mutant sites were transferred into the genomes of HEK293 T cell lines by transient transfection technology.The cell lines carrying wild-type and mutant types were cultured for 24 hours and then DNA damage was performed with mitomycin C.Cell immunofluorescence technique was used to observe the protein localization of protein HELQ and DNA damage factor γH2AX after 6 hours of MMC treatment and 2 hours of recovery culture.The protein expression level of HELQ and DNA damage factor γH2AX was detected by immunoprecipitation technique.Results:Only five known SNP loci were found in peripheral blood DNA of 50 patients with POI: rs6831595（exon 1）,rs563102563（exon 1）,rs1494961（exon 2）,rs7665103（exon 6）and rs148668655（exon 7）,no pathogenic mutation was found.In previous studies,we found homozygous missense mutation of HELQ gene（c.781A>G;p.R261G）in POI families.By testing the chromosome stability of the patients and the normal control group,it was found that the patients with different concentrations of MMC had higher chromosome breaking points than the control group.The transcription of peripheral blood gene HELQ in patients was lower than that in control group.Immunofluorescence results showed that there was no significant change in the localization and expression of HELQ protein in the mutant HEK293 T cells,and there was no difference in the localization and expression of DNA damage moleculeγH2AX.Western blotting showed that there was no difference in the expression of HELQ in HEK293 T cells with mutated mutation after treatment with MMC for 6 h,but the expression of γH2AX in HEK293 T cells with mutated mutation was significantly increased after treatment with MMC for 6 h and then resumed culture for6 h still higher than wild group.Discussion:No harmful mutation was found in the exon region of HELQ gene in 50 sporadic patients with POI,and homozygous missense mutation may occur in HELQ gene with very low mutation frequency in Han POI patients（c.781A>G;p.R261G）may not affect HELQ localization but may lead to POI by affecting DNA damage repair.