The Role And Mechanism Of FANCI And FANCL Genes In The Pathogenesis Of Premature Ovarian Insufficiency

Chapter Ⅰ The role and mechanism investigation of FANCI gene in the pathogenesis of POIBackgroundPremature ovarian insufficiency（POI）,is characterized by cessation of ovarian function before the age of 40 years,and manifests as menstrual disturbances with increased gonadotrophins and estradiol deficiency.The etiology of POI is highly heterogeneous and consists of genetic,autoimmune,infectious,and iatrogenic factors.Genetic causes play an important role in the pathogenesis of POI,and it explains approximately 20-25%of patients.Over the past few decades,numerous causative genes identified by whole exome sequencing（WES）and other genetics technologies,mainly enriched in DNA damage repair,homologous recombination,and meiosis,indicating that DNA damage repair defects is important for POI pathogenesis.Although causative gene list is growing,just the tip of the iceberg of POI genetic is seen and the majority remains to be elucidated.Identifying new pathogenic genes and elucidating the pathogenesis of POI are under intense and challenging.Fanconi anemia（FA）pathway containing 22 FA genes is a classic DNA damage repair pathway,and mainly involved in DNA interstrand crosslinks（ICLs）and maintaining stability of replication fork.POI-like phenotype was observed in multiple FA gene-deficient mice.Mutations in FANCD1/BRCA2,FANCM,and FANCU/XRCC2,have been identified in POI patients,thus suggesting the importance of the FA pathway in ovarian development and function.FANCI is an important member of the FA pathway,and FANCL cooperates with UBE2T to ubiquitinate the FANCI-D2 dimer,which is a crucial event in the activation of FA pathway.Genomewide association studies（GWAS）found that the FANCI was significantly associated with age at natural menopause,Early menopause and POI.Therefore,we speculate that FANCI participate in genesis and development of follicles and ovarian function maintenance,but its exact function and mechanism of action remain to be elucidated.ObjectiveIn the present study,Fanci knockout mice were used to investigate the role of FANCI in ovarian development and function maintenance.FANCI gene mutations was screened using WES in POI patients,and the effect of mutations on FANCI function was analyzed.We will elucidate the role of FANCI in the pathogenesis of POI by animal models and pathogenic mutations.Methods98bp deletion in exon 5 of the Fanci gene by CRISPR/Cas9,caused frmneshift mutations and protein truncation,and Fanci gene knockout mice were successfully constructed.Ovarian function was determined through fertility test,estrous cycle observation,serum hormone detection,and ovarian histological analysis,Germ cells observation in embryonic gonads and primordial germ cells（PGC）counting confirmed the effect of FANCI defect on the development of germ cells.PGC proliferation,apoptosis,cell cycle,and DNA damage were evaluated to determine the function of FANCI in PGC development and genome stability maintenance.The mechanism of FANCI maintaining genomic stability in mouse embryonic fibroblast（MEF）was further explored.MMC was used to induce ICLs,and aphidicolin（APH）and hydrouria（HU）were used to induce replication stress.Cell replication stress response and DNA damage evaluated by Western blot,replication fork stability detected by DNA fiber assay,and genome stability assessed with comet assay and micronucleus formation were conducted to elucidate the mechanism of FANCI participating in ICLs repair and genome stability maintainance.WES screening was performed in patients with idiopathic POI to screen FANCI gene mutations.Sanger sequencing and 10× genomics haplotype sequencing were used for mutations verification.Bioinformatics function prediction of mutations was conducted to clarify the role of FANCI gene mutations in the pathogenesis of POI.Results1、Dysplasia of primordial germ cells in Fanci knock-out miceGenotype identification and protein level verification of Fanci gene knock-out mice were performed.The Fanci-/-mice were born in expected mendelian ratios and grew normally.Adult Fanci-/-female mice showed a POI phenotype,and was characterized by infertility,disappearance of the estrous cycle,marked increase in serum FSH level and atrophy ovary with no follicles.Compared with wild-type mice,both the number of primordial follicles in the ovaries of 3 days old Fanci-/-female mice and spermatogonia in the testes of 3 days old male Fanci-/-mice significantly reduced,suggesting that germ cell dysplasia occurred in the embryonic gonad.Further observation of the gonads in Fanci-/-embryos and PGC counting revealed that decreased number of PGC in embryonic day 9.5（E9.5）Fanci-/-had emerged,and at E11.5 the PGC number reduction was more remarkable.Moreover,the PGC doubling time significantly prolonged.Yet,PGC migration in Fanci-/-embryos was not disturbed,and somatic cells in the gonads developed normally.2.Mechanism exploration of FANCI in the development of primordial germ cells An increased proportion of Cleaved PARP1 positive PGC was observed in E11.5 Fanci-/-embryo,indicating that apoptosis rate raised in Fanci knock-out primordial germ cells.Reduced ratio of PGC with EdU incorporation suggested that the proliferation capacity of PGC obviously decreased.A higher proportion of PGC with cyclin B1 cytoplasmic accumulation demonstrated that remarkable G2 arrest occurred.The declined FANCD2 expression in S-phase PGC of Fanci-/-embryo declared that the FA pathway of Fanci-/-embryo was inactivated.A greater proportion of PGCs showed elevated levels of double strand break markers,including γH2AX、53BP1,and increased proportion of phosphorylated p53 positive PGC were found,indicating that inactive FA pathway led to DNA damage accumulation in Fanci-/-embryo PGC.When ICLs and replication stress were induced in MEF using MMC and APH respectively,the expression of FANCD2 in Fanci-/-MEF decreased,the ubiquitination modification was absence,increased level of yH2AX,phosphorylated RPA2,phosphorylated p53,and an evident increase proportion of 53BP1 foci positive cells were observed.Deletion of Fanci resulted in FA pathway inactivation,failure of ICLs repair and replication stress resolution,and led to accumulated DNA damage and activation of p53 in MEF.APH or HU treatment shortened nascent DNA tract length of Fanci-/-MEF,denoting that replication forks instability induced abnormal resection of nascent DNA in Fanci-/-MEF cells.Fanci-/-MEF treated with APH or HU showed higher micronucleus formation rates,longer comet tail,which indicating replication stress resulted in genome instability in Fanci-/-MEF.3.FANCIgene mutations analysis in POI patientsThough whole genome sequencing,sanger sequencing and 10× genomics sequencing,two carriers with FANCI compound heterozygotic mutations were identified in 1030 idiopathic POI patients,including c.158-2A>G/c.959A>G（P.Q320R）and c.97C>T（p.L33F）/c.1865C>T（p.S622L）。Minigene examination revealed that c.158-2A>G caused splice variants,and resulted in frameshift mutation and truncated protein generation（p.S54Pfs*5）.The function prediction of the mutation showed that the amino acids of the four mutation sites were highly conserved among a range of species and located at important functional domains of FANCI.The function prediction software revealed that the four mutation sites might be pathogenic,indicating that pathogenic variation in the FANCI gene may be involved in the pathogenesis of POI.Conclusion（1）FANCI plays a key role in ovarian development and function maintenance by participating in ICLs repair and replication fork protection.（2）FA pathway inactivation and DNA damage accumulation caused by FANCI germline-deletion leads to abbreviated cell cycle progression and proliferation defect of PGC,insufficient production and premature depletion of follicles.（3）FANCI mutations may cause the occurrence of POI.Chapter Ⅱ The role and mechanism investigation of FANCL gene mutation in the pathogenesis of POIBackgroundGene defects in DNA damage repair have received growing attention in study of POI genetics etiology.The FA pathway,which is a classical DNA repair pathway,orchestrates ICLs repair in the genome.Recently,mutations in FA genes have been identified in patients with POI,suggesting the importance of the FA pathway in follicle genesis and development and ovarian function maintenance.Ubiquitination of the FANCI-D2 complex is a key event in this pathway,and thus failure of ubiquitination due to impaired function of FANCL will prevent the activation of the FA pathway.Interestingly,deletion of the mouse Fancl gene exclusively results in a POI-like phenotype characterized by less oocytes and ovarian atrophy in adult female mice,which is attributed to reduced proliferation of primordial germ cells（PGCs）during embryonic development.Nevertheless,it remains unknown whether FANCL mutations contribute to the pathogenesis of human POI.ObjectivePotential pathogenic mutations in the FANCL gene were screened in Chinese patients with idiopathic POI.We will elucidate the role of FANCL gene in the pathogenesis of POI though analyzing functional change of the mutant FANCL.Methods200 Chinese patients with idiopathic POI and 200 matched controls were enrolled.All exons of the human FANCL gene were amplified by polymerase chain reaction（PCR）and sanger sequencing was used to identify the underlying causative variants of the FANCL gene.Wild-type and mutant FANCL plasmids were constructed and transfected into wild-type HEK293 cells,and the localization of mutant FANCL were analyzed.Wild type or FANCL-/-HEK293 cells were transfected with the desired FANCL plasmids and the cells were exposed to MMC to induce ICLs,and the expression of ubiquitinated FANCD2 and y H2AX were evaluated by Western blot.We also detected whether FANCL knock-down in HEK293 cells altered the expression of ubiquitinated FANCD2 and γ H2AX using Western blot after MMC treatment.Results1.Identification of two novel heterozygous frameshift mutations in the FANCL geneThough sanger sequencing,11 variants in the FANCL gene were identified in 200 Chinese idiopathic POI patients.Two novel heterozygous frameshift mutations,c.1048₁051delGTCT（p.Q350Vfs*18）and c.739dupA（p.M247Nfs*4）,were found in exon 13 and exon 9,respectively,and neither of them was present in any of the 200 controls.The remaining nine variants were previously identified as single-nucleotide polymorphisms（SNPs）.None of the SNPs showed significant differences between POI patients and the control population in terms of genotype or allelic frequencies.2.Mislocalization of the mutant FANCL proteinsThe overexpression of the FANCL protein in HEK293 was confirmed,and the p.Met247Asnfs*4 mutation resulted in a truncated protein weighing about 35 kDa,while the p.Gln350Valfs*18 mutation had no noticeable effect on the molecular weight of FANCL.In contrast to the nuclear localization of wild-type FANCL,the two mutant proteins were retained in the cytoplasm and were absent in the nuclei.3.Impaired ubiquitin-ligase activity and DNA repair capacity of the mutant FANCL proteinsThe HEK293 cells were transfected with FANCL plasmids,the level of ubiquitinated FANCD2 was increased,the γH2AX level dropped to nearly basal levels quickly in cells overexpressing wild-type FANCL after MMC treatment.But,the abundance of ubiquitinated FANCD2 in the cells overexpressing either the p.Gln350Valfs*18 or p.Met247Asnfs*4 mutation was comparable to that of the control group.In addition,the recovery of yH2AX expression delayed,suggesting that the two FANCL mutants led to compromised ubiquitin-ligase activity and DNA repair capacity.An restore of the ubiquitinated form of FANCD2 but less yH2AX were observed in FANCL-/-HEK293 cells complemented with wild-type FANCL,while no ubiquitinated FANCD2 but more yH2AX were seen in the cells complemented with FANCL mutants,implying that the mutations abolished the ubiquitin-ligase activity and impaired DNA damage repair ability of the FANCL protein.Also,reduced abundance of ubiquitinated form of FANCD2 but increased expression of γH2AX were detected in the FANCL-knockdown HEK293 cells,suggesting that haploinsufficiency of FANCL protein compromised ubiquitin-ligase activity and DNA repair ability.ConclusionWe identified two novel heterozygous frameshift mutations of the FANCL gene in POI patients,p.Q350Vfs*18 and p.M247Nfs*4.Mutant FANCL showed mislocalization and abolished ubiquitin-ligase activity,and functional haploinsuficiency caused by FANCL gene mutation attenuated the DNA repair capacity,which might be causative for POI.