The Role And Mechanism Of Homologous Recombination Gene HSF2BP And FBH1 In The Pathogenesis Of Premature Ovarian Insufficiency

Chapter Ⅰ The role and mechanism of HSF2BP gene variants in the pathogenesis of POIBackground:Premature Ovarian Insufficiency(POI)is defined by depletion of ovarian function before the age of 40 years,characterized by amenorrhea or oligomenorrhea and elevated serum level of follicle stimulating hormone(FSH).The etiology of POI is heterogenous.Genetic disorders and non-genetic factors account for nearly 50%of the patients,remaining half of the cases are idiopathic.Genetic disorders are responsible for 20-25%of POI.Therefore,genetic disorder is an indispensable factor in POI pathogenesis.Recently,with the widespread use of whole exome sequencing(WES)in POI pedigrees,DNA repair genes and meiosis genes have been enriched in the spectrum of causative genes of POI.The results suggest that DNA damage repair pathways and meiosis genes have important roles in the establishment and maintenance of ovarian reserve.The first meiotic division is a crucial process in oogenesis.During the prophase of first meiotic division,DNA double strand breaks(DSBs)are generated by SPO11.After the modification,the 3’-tailed single strand DNA(ssDNA)of DSBs are recognized by BRCA2,RAD51 and DMC1,which execute the critical step of strand invasion,followed with homologous recombination(HR)and crossover formation.HSF2BP is a recently identified meiosis-specific protein,which interacted with BRCA2 and BRME1 to form a complex to mediate the recruitment of RAD51 and DMC1 to the meiotic recombination sites and facilitate the strand invasion.Hsf2bp knockout female mice exhibited subfertility due to disruption of the recruitment of RAD51 and DMC1 onto ssDNA and delayed synapsis.Recently,a homozygous missense variation of HSF2BP was identified in a consanguineous family with POI.However,the role of HSF2BP variations in sporadic POI pathogenesis was still unknown.Objective:In this study we screened for HSF2BP variations in POI-WES database to investigate the role and mechanism of HSF2BP gene in the pathogenesis of sporadic POI.Methods:Variations in HSF2BP were screened and analyzed through our own POI-WES database.The pathogenic effects of HSF2BP variations and potential mechanism were further explored by functional studies in HeLa cells and KGN cells.The distribution of mutant protein in cells was observed by fluorescence assay to analyze the effect of variants on HSF2BP protein localization.DNA damage assays were performed to elucidate whether the variations impaired the DNA repair capacity of HSF2BP.Results:Through the variations screening of HSF2BP in the POI-WES database,two missense homozygous variations were identified:c.382T>C;p.C128R and c.557T>C;p.L186P.The wild-type HSF2BP protein was expressed in both cytoplasm and nucleus of HeLa and KGN cells.However,the signals of two mutant proteins localizing in the nucleus were significantly lower than that of wild-type protein,which suggested that the variations impaired the nuclear location of HSF2BP.The results of DNA damage assays showed that the level of yH2AX in HeLa cells overexpressing wild-type HSF2BP decreased to the untreated level after recovery.Whereas,the yH2AX level in cells overexpressing mutant HSF2BP was significantly higher than that in wild-type group after recovery,indicating the mutant HSF2BP had impaired DNA repair capacity.Conclusions:In the present study,two novel pathogenic homozygous variations of HSF2BP were identified in two patients with sporadic POI,highlighting the essential role of meiotic HR genes in the pathogenesis of sporadic POI.Chapter Ⅱ The function of FBH1 gene in the development and functional maintenance of ovarian and in the pathogenesis of POIBackground:Meiosis is a process in which diploid primordial germ cells convert into haploid gametes,which is essential for maintaining the genetic stability.During the meiotic prophase I,homologous recombination uses homologous DNA sequences as a template to repair DSBs.Aberrant meiotic recombination leads to meiotic process arrest and DSBs repairment defect,which often leads to gametogenic failure or produces aneuploid gametes resulting in infertility,miscarriage or birth defects.FBH1,also known as FBXO18,is a member of the UvrD family.FBH1 contains an F-box domain and forms an SCF ubiquitin ligase complex with SKP1 and CUL1.The SCF complex limits the assembly of RAD51 nucleofilaments in the process of meiosis of Schizosaccharomyces pombe.Deletion of Fbh1 in fission yeast results in low levels of spore viability,which correlate with accumulation of RAD51 foci on DNA and a failure in proper chromosome segregation.Loss of Skp1,a constitutive subunit of SCF complex,leads to a failure in synapsis and infertility of both male and female mice.However,the role of FBH1 in mammalian meiotic progress was still unknown.Objective:In this study,we used Fbh1 knockout mice to investigate the role of FBH1 in ovarian development and function maintenance.We used clinical POI cases to elucidate the role of FBH1 in the pathogenesis of POI.Methods:Fbh1 gene knockout mice were constructed by CRISPR/Cas9.Ovarian function was determined through fertility test and ovarian histological analysis.Variations in FBH1 were screened and analyzed through POI-WES database.Results:Fbh1 gene knockout mice were successfully constructed by CRISPR/Cas9.Genotype identification of Fbh1 gene knockout mice was performed.Through the fertility test,we found no abnormalities on the mean number of litters per month and the mean number of pups per litter of Fbh1-/-female mice.And the number and morphology of follicles in each stage of Fbh1-/-mice were normal.The defect of Fbh1 gene did not affect ovarian development in mice.Through the variations screening of FBH1 in the POI-WES database,a missense homozygous variation with high pathogenic risk was identified:c.1212G>T;p.R404S.The mutation was located in the first base of exon 7 of FBH1 gene.Such mutations resulted in errors during the splicing process and thus caused alterations of the open reading frame.Conclusions:Fbh1 gene knockout mice confirmed that the deletion of Fbh1 gene did not affect the fertility and ovarian development in mice,suggesting that Fbh1 is dispensable for the ovarian function maintenance in mice.The long-term effect of Fbh1 defect on the ovarian function needs to be observed.Although a missense homozygous variation with high pathogenic risk was screened from POI patients,the pathogenicity of FBH1 in human POI needs to be further investigated.