Whole Exome Sequencing Of 1030 Patients With Premature Ovarian Insufficiency

Premature ovarian insufficiency(POI)refers to the loss of ovarian function in women before the age of 40.The clinical manifestations are amenorrhea or oligomenorrhea and elevated gonadotropin levels(FSH>25IU/L).POI not only causes reproductive disorders but also seriously affects women's physical and mental health.Due to the irreversibility of ovarian function decline,there is currently no effective method to restore ovarian function.Therefore,early warning,early diagnosis and early intervention for POI patients are of paramount importance.The etiologies of POI include genetic defects(chromosomal abnormalities and gene mutations),autoimmune diseases,iatrogenic factors(radiotherapy,chemotherapy,and ovarian surgery),and environmental factors.However,the cause of most patients is still unclear(namely,idiopathic POI).Genetic factors have always been the focus of research on the etiology of POI.On the one hand,a determined molecular diagnosis facilitates the identification of pathogenesis of patients,possible complications,and the risk of genetic defects transmitted to the offspring,which is helpful for clinical intervention and health management.On the other hand,genetic screening can also recognize high-risk groups,thereby achieving risk warnings.Although dozens of POI pathogenic genes have been identified so far,only a small proportion of patients could be explained(<5%),indicating that the genetic factors of POI still needs to be explored.Concerning the high heterogeneity of POI,it is necessary to study in larger sample size and explore the genetic mechanism of POI.Previous studies concerning NGS on sporadic POI were based on limited sample size,and the high genetic heterogeneity of POI makes it challenging to determine causative genes among patients.Hence,we performed the largest scale exome sequencing of sporadic POI to systematically explore the genetic background of POI.In the first chapter of this paper,we performed whole-exome sequencing(WES) on 1030 patients with idiopathic POI.The control cohorts in present study comprise HUABIAO whole-exome sequencing project and the East Asian population of gnomAD public database.First,we strictly evaluated the pathogenicity of variants and according to ACMG guideline and determined pathogenic/likely pathogenic(P/LP)variants.Next,based on a gene list with 719 genes related to ovarian function,we performed collapsing analysis about P/LP vairants,and identified 27 genes with P/LP variants significantly enriched in POI.Of this 27 genes,14 genes had never been reported in POI with pathogenic vairants,including CCDC155,CPEBI,HMMR,HSD17B1,LGR4,MCMDC2,NUP43,PPM1B,PRDM1,PTCH1,SPATA22,STRA8,ZAR1 and ZP3.Then,we conducted in vitro experimental validation about vairants in in two novel genes(STRA8 and MCMDC2)and verified that all variants could impair the protein activity.These variants may compromise the processes of meiosis initiation and homologous recombination repair,which led to the occurrence of POI.The results of functional experiments also indicated that our mutation screening and classification methods are effective,and the 14 newly identified candidate genes are new potential pathogenic genes for POI.We further explored the genetic characteristics of POI and tried to determine the explanation rate of POI based on those 14 newly identified POI pathogenic genes and 88 known POI pathogenic genes.After combinding functional experiments' results on variants of uncertain significance(VUS),we identified 231 pathogenic variants in 223 POI patients,indicating that the contribution rate of genetic variation on POI can reach 21.65%.In the pathogenic pattern analysis,we found that monoallelic,biallelic and multiple-hits variants accounts for 78.92%(176/223),12.56%(28/223),and 8.52%(19/223)separately.Subsequent analysis of the interpretation rate of each gene found that a single gene only can explain up to 11 cases(NR5A1,GALT,MCM9),and the contribution rate of a single gene is less than 1.2%,confirming the high genetic heterogeneity of POI.In the phenotypic analysis regarding amenorrhea age,we found that the genetic variation contribution rate of primary amnorrhea(PA)was higher than that of secondary amnorrhea(SA)(25.0%vs 20.6%),and that the contribution of biallelic variants(6.7%vs 2.2%)and multi-hits variants(2.5%vs 1.8%)of PA were also higher than SA.These results indicated that PA has a stronger genetic component.Furthermore,gene-phenotype tendency analysis suggested that the enrichment of FSHR on PA is more significant.Last but not least,gene set analysis showed that pathways implicated in meiosis,DNA damage repair,mitochondrial function,fatty acid metabolism,steroidogenesis and oxidoreductase activity were significantly enriched in POI.The presence of multiple patients in POI family indicated the existence of shared genetic pathogenic factors.Therefore,in the second chapter,we performed WES and genetic analysis on the family of a familial POI,and identifed a new POI candidate gene,C3orf33.Two sisters suffered from POI in a POI pedigree all carried C3orf33 biallelic nonsense mutations from their parents:paternal p.Arg85Ter and maternal p.Glu172Ter.Subsequently,we screened the C3orf33 gene mutation in 1,030 sporadic patients,and found that another patient carrying a homozygous nonsense C3orf33 mutation p.Arg85Ter.All patients with biallelic C3orf33 variants presented with PA.No homozygous loss-of-function mutations of this gene were found in the control data of HUABIAO and gnomAD database.These genetic evidence suggests that C3orf33 may be a potential pathogenic gene for POI,but there are few studies on the function of this gene,and its correlation with ovarian function is still unclear.In order to further expore the pathogenicity of C3orf33 on POI,we firstly analyzed the expression profile and confirmed that C3orf33 is highly expressed in mouse ovaries,both in oocytes and granulosa cells.Subsequently,we used the CRSPR/Cas9 technology to construct the C3orf33 knockout mice.Through the reproductive phenotype analysis of C3orf33-/-mice,however,we found no abnormalities on the ovarian morphology and fertility of C3orf33-/-,and that the number and morphology of follicles in each stage are normal.Considering that mice and humans have differences in time scale and mono-vs.poly-ovulation,we could not exclude C3orf33 as a possible POI candidate gene.In summary,we identified 14 novel potential causative genes for POI based on exome sequencing of 1030 patients with POI.We also detected at least one pathogenic variant in 21.65%of 1030 POI patients,indicating a strong genetic component of POI etiologies.In addition,exome sequencing on a POI pedigree revealed that C3orf33 was a potentially novel candidate gene for POI,which was recurrent in 1,030 sporadic cases.Our results expand the mutation spectrum of POI pathogenic genes,and provide clues for molecular diagnosis and risk warning of POI.