Analysis Of Telomere Homeostasis And DNA Repair Gene FAM175A,TLK1 In Premature Ovarian Insufficiency

Chapter I Impaired Telomere Length and Telomerase Activity in Peripheral Blood Leukocytes and Granulosa Cells in Patients with Biochemical Premature Ovarian InsufficiencyObjective:Premature ovarian insufficiency(POI)is a result of pathological reproductive aging and encompasses normal,occult,’biochemical and overt stages.POI is defined as hypergonadotropic amenorrhea due to cessation of ovarian function before the age of 40 years.Telomere homeostasis as an important indicator of aging,includes telomere length and telomerase activity.Telomere attrition has been considered to be involved in age-related disorders.Studies have indicated telomere homeostasis was associated with occult and overt stages of POI.But the association between telomere homeostasis and biochemical POI remains elusive.In this study,we explored whether telomere length in leukocytes and granulosa cells,and relative telomerase activity in granulosa cells were associated with biochemical POI.Method:A total of 120 patients with biochemical POI and 279 control women were recruited by the Center for Reproductive Medicine of Shandong University.The peripheral blood and follicular fluid were collected,the granulosa cells were extracted from the follicular fluid.Telomere length in peripheral blood leukocytes(LTL)and granulosa cells(GTL)was measured using a modified Quantitative Polymerase Chain Reaction technique.The relative telomerase activity(RTA)in granulosa cells was detected using a modified quantitative-telomeric repeat amplification protocol assay.Results:Telomere length was shorted with advanced age.After adjusting for age,patients with biochemical POI(n = 120)exhibited significantly shorter LTLs[0.75 ±0.09 vs.1.79±0.12,P<0.001;OR = 0.54,95%confidence interval(CI)= 0.43-0.68]and GTLs(0.78 ± 0.09 vs.1.90 ± 0.23,P<0.001;OR = 0.54,95%CI = 0.41-0.70)than the controls(n = 279 for LTLs;n = 90 for GTLs).Significantly diminished RTAs in granulosa cells were detected in patients with biochemical POI(n = 31)compared with the controls(n = 38)(1.57 ± 0.59 vs.4.63 ± 0.93,P = 0.025;OR = 0.84,95%CI= 0.72-0.98).Conclusion:Shortened telomere length and diminished telomerase activity were associated with biochemical POI.These findings suggest that telomere length and telomerase activity may be considered as indicators for progression of ovarian decline.Chapter II Mutation in Family with Sequence Similarity 175 Is Not Responsible for Premature Ovarian InsufficiencyObjective:Premature ovarian insufficiency(POI)is highly heterogeneous in etiology.The known causes of POI include chromosomal and genetic defects,autoimmune,infections and iatrogenic factors,but etiology remains to be elucidated in most cases(known as idiopathic).Genetic etiology is responsible for about 20-25%of idiopathic POI cases.However,until now,only a few candidate genes have been proved causative.Such as,genes exerting known hormonal effects,genes required for oogenesis and folliculogenesis,Pleiotropic Mendelian genes affected ovarian function and so on.Recently,genes related with DNA damage repair have been identified causative in POI pedigrees and idiopathic POI cases.Family with sequence similarity 175 member A(FAM175A)was involved in the DNA double-strand break(DSB)repair pathway.It was required for DNA damage resistance,DNA repair and G2-M checkpoint control.Both homozygous and heterozygous Faml75a knockout mice exhibited decreased survival and increased tumor incidence.A meta-analysis including 22 genome-wide association studies identified 13 novel loci associated with age at menopause.One of them rs4693089 was closely linked with gene FAM175A.However,the contribution of FAM175A in patients with idiopathic POI remains elusive.The objective of this study is to investigate whether mutations in FAM175A gene were present in patients with idiopathic POI.Method:A total of 400 patients with idiopathic POI and 400 control women with regular menstruation were included.Sanger sequencing was performed in patients with idiopathic POI,and potentially pathogenic variants were confirmed in matched controls.Functional experiments were performed to explore the deleterious effects of mutations identified.DNA damage was induced by mitomycin C(MMC)treatment,DNA repair capacity was evaluated by histoneH2AX phosphorylation level,and G2-M checkpoint activation was assessed by percent of mitotic cells.Result(s):One missense variants in FAM175A,c.C727G(p.L243V)was identified in two patients with idiopathic POI but absent in 400 controls(the upper 90%confidence limit for the proportion 2/400 is 7.8%).HeLa cells over-expressing mutant p.L243V showed the same sensitivity to MMC-induced damage compared with wild type.Moreover,there were no differences in repair capacity and G2/M checkpoint activation after treatment with MMC between two groups.Conclusion(s):Our result suggests mutations of FAM175A gene may not be a common cause of POI.The mutation p.L243V in the FAM175A gene is not responsible for idiopathic POI in Chinese patients.Chapter Ⅲ Variation Analysis of Tousled Like Kinase 1 Gene in Patients with Premature Ovarian InsufficiencyObjective:Tousled like kinase 1(TLK1)a member of DNA repair gene family,is involved in the regulation of chromatin assembly and play an important role by a direct function in processing the ends of a DSB via interaction with Rad9.A meta-analysis of 22 genome-wide association studies reported 13 novel age at natural menopause loci.Eight candidate genes implicated in DNA repair(EXO1,HELQ,UIMC1,FAM175A,FANC1,TLK1,POLG,PRIM1)were included.In the following study,many of these loci,including rs10183486(TLK1)were identified also associated with early menopause and POI in European women.However,the potential role of TLK1 in POI pathogenesis is still elusive.Then we designed this study to delineate whether mutations in TLK1 contribute to idiopathic POI.Method:A cohort of 192 patients with POI was enrolled.All exons and exon-intron boundaries of TLK1 were amplified and sequenced.Result(s):Six known single-nucleotide polymorphisms(SNP)were identified in both POI and control groups,including rs149844334,rs1 1553951,rs771244101,rs2277339,rs113416007 and rsl7283147.The four SNPs located on the exons were synonymous mutations.No novel mutation was identified.Conclusion(s):This study indicates that mutations in TLK1 gene might not be common cause of POI in Chinese women.