Analysis For Meiotic Genes Mutation And Prospective Cohort Study Of Primary Ovarian Insufficiency

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Meiosis Genes EXO1,RAD51 and PUM1 in Premature Ovarian InsufficiencyBackgroundPremature ovarian insufficiency(POI)is characterized by cessation of menstruation before 40 years of age and elevated serum level of follicle stimulating hormone(FSH)(>25IU/L).Approximately 1%-5%of women are affected.With the increasing population of women who postpone childbearing until 30 years of age or later,POI attracted more attention due to its irreversible impairment of either fertility or well-being of the affected women.The etiology of POI is heterogeneous,and genetic factors play an important role in its occurrence and development.Exploring new pathogenic genes and pathogenesis are important directions of POI etiology research.Meiosis is a key event of the formation of primordial follicles pool that underpins reproductive success.During the prophase I,homologous recombination(HR)is initiated by programmed DNA double-strand breaks(DSBs).In mice,sustained DSBs induced by impaired recombination may trigger pachytene arrest and result in oocyte apoptosis,followed by ovarian dysfunction and infertility after birth.Recently,through whole exome sequencing(WES)in sporadic or familial patients with POI,a few genes involved in meiotic recombination have been found,such as HFM1,STAG3,MSH5,MSH4,MCM8,MCM9 and BRCA2,which introduced a new mechanism receiving more and more attention for ovarian function.ObjectiveTo investigate the role of meiotic homologous recombination genes in the pathogenesis of POI.MethodsFifty POI patients with primary amenorrhea(PA)were recruited from the Reproductive Hospital Affiliated to Shandong University.A total of 200 females with regular menstrual cycle and normal hormone levels were recruited as control.Genomic DNA of the participants were extracted from peripheral blood leukocyte and DNA sequencing was performed on the Illumina platform.Reads were aligned against the GRCh37(hg19)reference human genome.The mutant yeast with the homologous variants identified in human were constructed to detect meiotic nuclear divisions and spore viability.The mutations leading to abnormal meiosis progress of yeast were further explored in human cell line.Human HEK293 cells were transfected by wild-type or mutant plasmids and exposed to Etoposide(ETO)to induce DSBs,and the DNA damage marker yH2AX was evaluated by Western blot.Meanwhile,the efficiency of HR was measured by GFP-based HR reporter assay.In addition,a cohort of 196 Han Chinese women with idiopathic POI were recruited,and a total of 192 females with normal ovarian function were recruited as control group.The entire UTR sequence,exon sequence and intron-exon boundaries of the PUMl gene were sequenced by Sanger sequencing.Calculation of genotype of frequencies and allele frequencies of variants,and statistical analysis were used to consider whether there is any difference between POI patients and controls.The online software was used to assess the possible functional impact of variant.Results1.Novel missense variants of HR genes identified in POI patients with PAThrough WES in 50 POI patients with primary amenorrhea,8 heterozygous missense variants in homologous recombination genes were identified in 9 patients:EXO1 p.Thr52Ser,EXO1 p.Gly223Asp,RAD51 p.Glu68Gly,RMI1 p.Arg622Trp,MSH5 p.Arg422Cys,MSH2 p.Pro622A1a,MSH6 p.Met1184Lys and MLH1 p.Thr45Ala.2.Exol p.Thr52Ser impairs the spore viability and HR repairThe specific mutant yeast under SKI background were generated to explore the effect of variants on meiosis.Compared with the wild type strain,the Exol p.Thr52Ser strain had dramatically reduced spore viability,20%lower 24h nuclear division,and significantly fewer Rpa and Rad51 foci during meiosis.Moreover,we overexpressed mutant and wild-type EXO1 in HEK293 cells.We observed significantly lower efficiency of HR and higher concentration of yH2AX in EXO1 p.Thr52Ser transfected cells.3.RAD51 p.Glu68Gly impairs localization and HR repair in human cellsThe RAD51 protein was fused with GFP and observed by fluorescence microscopy after 24 hours transfection in HEK293 cells.Strikingly,the wild-type protein was mainly localized in nucleus,while the mutant RAD51 p.Glu68Gly existed in cytoplasm exclusively.We also found the concentration of yH2AX was significantly higher in cells overexpressing mutant RAD51 compared with the cells overexpressing wild-type RAD51 after ETO treatment.Moreover,the HR efficiency of mutant RAD51 was significantly lower than that of wild-type.4.Variation analysis of PUM1Fourteen variants of PUM1 identified in 196 women with POI,including seven novel mutations and seven single-nucleotide polymorphisms(SNPs).The online prediction tools were used to predict the functions of the variations and the results showed that no variations alter RNA splicing,microRNA binding or the function of PUM1 protein.ConclusionWe firstly identified novel potentially pathogenic variants in EXO1 and RAD51,which adversely effected meiotic recombination,finally causing POI.The present study further highlighted the contribution of DNA repair genes.Meanwhile,we also conclude that the variants in PUM1 may not contribute to POI in Han Chinese women;the exact role of PUM1 in human POI needs future studies in larger cohorts and other ethnic populations may determineChapter ? Analysis for Next-Generation Sequencing Panel of 500 Patients with POIBackgroundCurrently,more than 70 genes have been reported to be associated with POI,which are related to gonad development,DNA replication and repair,follicular development,hormone signaling,immunity and metabolism.With the development of next-generation sequencing(NGS),multiple POI candidate genes can be screened for mutations simultaneously through genetic panel.Since 2015,several studies have selected different ovarian function related genes to detect candidate variants in patients with POI by NGS,which indicate that the gene panel is expected to become a new method of genetic etiology diagnosis of POI.However,there are some deficiencies in the previous research,such as the candidate genes lack of conclusive evidence of POI pathogenesis or the single-method for mutation screening.ObjectiveTo identify potential causative variations for POI using a targeted gene panel,which will expand the spectrum of POI causing genes and reveal novel genetic architecture of POI.MethodsBased on previously researches,28 causative genes with confirmative functional evidence were included in the target panel,including 11 meiosis genes(HFM1,MSH4,MSH5,SPIDR,SMC1B,SYCE1,,STAG3,MCM8,MCM9,NUP107 and CSB-PGBD3),8 ligands and receptors associated genes(AMH,AMHR2,BMP15,BMPR2,FSHR,GDF9,PGRMC1 and KHDRBS1)and 9 transcription factors preferentially expressed in the ovary(FIGLA,FOXL2,NOBOX,NR5A1,POLR2C,SOHLH1,WT1,NANOS3,and LHX8).Five hundred patients with POI were recruited from the Reproductive Hospital Affiliated to Shandong University and screened with the designed genetic panel.We referred to all nonsynonymous variations and rare variations.The potential variants were classified as pathogenic(P),likely pathogenic(LP),and variations of uncertain significance(VUS)according to the guidelines proposed by the American College of Medical Genetics(ACMG).Pathogenic variations(P)referred to nonsense or frameshift variations,variations located in the canonical splice site(?2 intronic base pairs from the intron/exon boundary),and those previously reported to be pathogenic.Likely pathogenic variations referred to non-synonymous missense variants with a bioinformatics pathogenicity prediction of "Deleterious" by metaSVM combined with a CADD score>3 or a DANN score>0.95.The filtered variants were confirmed by Sanger sequencing.The parents of POI patients carrying compound heterozygous variations or multigenic variants were also sequenced when their DNAs were available.Result1.Genetic analysisIn the cohort of 500 patients with POI,a total of 14.4%(72/500)of the patients carried 61 pathogenic or likely pathogenic variations in 19 of the genes in the panel.Interestingly,only three of the variations had been reported in previous studies(p.R355H in NOBOX,p.R313H in NR5A1,and p.R351G in MSH5),58 variations were firstly identified in patients with POI.The 19 genes include 6 meiosis genes(HFM1,SPIDR,SMC1B,MSH5,MSH4,CSB-PGBD3),6 transcription factors(SOHLH1,POLR2C,FIGLA,NOBOX NR5A1,FOXL2)and 7 genes involved in ligands and receptors(AMH,AMHR2,GDF9,BMP15,FSHR,BMPR2,PGRMC1).FOXL2 harbored the highest mutation frequency.Sixteen patients carried FOXL2 heterozygous variations(16/500,3.20%),13 of which with variant c.1045C>G(p.R349G)(13/500,2.60%).The frequency of p.R349G in POI patients was significantly higher than that in 1000 Genomes database(0.08%,p=0.001)and East Asians of the ExAC database(0.24%,p<0.001).2.Clinical characteristics of patients carrying P or LP variationsAmong the patients carrying digenic or multigenic variations,a higher percentage of primary amenorrhea(44.44%vs.19.05%),earlier onset of POI(20.10± 6.81 years vs.24.97± 4.67 years),and later menarche(15.82± 1.50 years vs.13.95± 2.56 years)were observed compared with women carrying monogenic variations.However,these differences did not reach statistical significance(p>0.05).3.Analysis of genetic model of POI candidate genesPrevious studies have suggested that NOBOX p.R355H heterozygous mutation can cause POI disease in Caucasian women.In this study,both POI-52 and its sister with POI carried NOBOX compound heterozygous mutations p.L558FS and p.R355H,which was inherited from their father and mother separately,suggesting that there may be racial differences in the NOBOX gene inheritance pattern.In addition,here we firstly reported a POI patient with both MSH4 p.M740FS and MSH5 p.R351G mutations.The MSH4-MSH5 heterodimer plays an important role in homologous recombination repair of DNA double strand breaks,which is essential for meiosis,indicating that not only one subunit deficiency,but also dysfunctional MSH4-MSH5 interaction or cumulative haploinsufficiency of both subunits,may disrupt homologous recombination during meiosis,finally causing POI.ConclusionIn this study,a self-designed targeted gene panel covering 28 causative genes of POI used in 500 patients expanded the variation spectrum and genetic architecture of POI.Specific variations in pleiotropic genes may result in isolated POI;whereas polygenic defects could exert cumulative deleterious effect on severity of POI phenotype.A fuller understanding of POI genetics would contribute to the individualized prediction,diagnosis,and intervention for women with POI or at high risk for developing POI.Chapter ? Progression and outcome of premature ovarian insufficiency:a prospective cohort studyBackgroundThe phenotypes of POI are highly heterogeneous,and the disease progression and outcome are unclear.In 2008,American Society of Reproductive Medicine(ASRM)divided the evolution of POI into three stages:occult,biochemical,and overt POI.However,the current studies on POI are all cross-sectional studies,lack of evidence-based medical evidence for disease staging.Besides,no unified diagnosis standard for occult and biochemical POI.Thus,it is difficult for clinicians to make early diagnosis and prognosis evaluation of POI.ObjectiveThe purpose of our study was to establish a POI follow-up cohort to observe the progression and outcome of POI,explore the progression characteristics of POI diseases with different etiologies,and find out the early warning markers and intervention nodes of ovarian function decline.MethodsThe study was an observational prospective cohort study conducted by the Reproductive Hospital Affiliated to Shandong University.All participants were presented with FSH>15 IU/L at least twice over an interval of one month.The cohort of patients were grouped into three subgroups based on the basic FSH level:group B was 15 IU/L<FSH?25 IU/L,group F was 25 IU/L<FSH?40 IU/L,and group R was FSH>40 IU/L.Follow-up was conducted every 3-6 months for a total of three years.The changes of menstrual cycle and characteristics of the patients were recorded in each follow-up visit,and endocrine hormones,antimullerian hormone(AMH),inhibit B(INHB),thyroid function,liver function,blood lipid levels and ovarian ultrasound were also performed.The baseline data have been analyzed,and the follow-up results will be analyzed at the end of the 3-year follow-up.ResultsA total of 553 patients have been enrolled,reaching the expected sample size.1.Etiology analysis:The rate of chromosome abnormal was 12.8%,iatrogenic factor was 11.0%,autoimmune factor was 22.6%,and the cause of disease was unknown in 54.1%of patients.2.Lipid metabolic analysis:no significant correlations were found between serum FSH level and lipid metabolism indexes,but the levels of triglyceride and cholesterol were significantly higher in patients with amenorrhea than those without amenorrhea.3.cohort follow-up analysis:the current follow-up results showed that basic FSH level fluctuated more obviously in group F,increased in a fluctuating way in group B,and maintained a high level in group R.About 15%of patients in group B and 20%of patients in group F enter to POI/POF within 2 years.4.Pregnancy rate:a total of 12.4%of the patients achieved clinical pregnancy during the 2-year follow-up period,among which the rate of natural pregnancy and IVF pregnancy was 8.0%,and the rate of donor pregnancy was 4.4%.ConclusionsThe follow-up works are still in progress.The current data show that FSH level in early POI patients fluctuates greatly,and there is still a probability of successful pregnancy assisted by natural pregnancy or autologous IVF.Therefore,attention should be paid to the monitoring of ovarian function in such patients and timely fertility guidance.With the increase of follow-up nodes and the continuous accumulation of data,it is expected to depict the characteristics of POI disease development and outcome more clearly,and provide evidence for early diagnosis,timely intervention and long-term prevention of complications in patients with POI.