Copy Number Variants Analysis And The Related Gene In Chinese Han Women With Premature Ovarian Failure

Part ? Clinical characteristics and karyotype analysis in Chinese Han women with POFObject:To identify the proportion of chromosomal abnormalities in Chinese Han women with POF and the importance of genetic screen in POF.Materials and Methods:156 Chinese Han women with POF and 391 normal controls from Women Hospital,Zhejiang University were recruited for this study.We examined and compared the clinical characteristics between these two groups.G-banding method was performed to determine the peripheral blood lymphocyte karyotype of POF.And we analyzed the association between abnormal karyotype and clinical features.Results:POF patients were obviously abnormal in endocrine.POF patients with primary amenorrhea accounted for 10.9%(17/156);most patients showed amenorrhea before the age of 30.In 17 patients with primary amenorrhea,abnormal karyotype were detected in 12 cases,abnormal rate was 70.6%;12 cases of 139(8.6%)patients with secondary amenorrhea were detected abnormal karyotype.The rate of abnormal chromosome karyotype in primary amenorrhea patients is significantly higher than patients with secondary amenorrhea.62.5%of all chromosomal abnormalities were structure abnormalities in X chromosome.Conclusion:Abnormal karyotype is important in genetic causes of POF.For POF patients,especially who with primary amenorrhea,karyotype examination is necessary.X chromosome structural abnormalities are more common in POF.Part ? Genomic copy number variations analysis in premature ovarian failureObject:To found new genetic risk factors and disease-causing genes of POF,identify distribution characteristics of CNVs in POF population and the association between CNVs and POF.Materials and Methods:Affymetrix CytoScan HD SNP array platform were performed to detect genome-wide CNVs in 90 cases and 90 controls.CNVs basic features and distribution characteristics were analyzed by statistics and bioinformatics method.With the basic strategy of case-control study,we did association analysis between CNVs loci and POF.Results:There was no significant difference in the chromosomal distribution of CNVs between POF and controls,the largest proportion of CNVs were detected in X chromosome.The proportion of loss CNVs to total in POF was higher than control.The average total length of CNVs spanned of each case was longer than that of each control.Eventually,we found five candidate CNVs state,involves q35.2,7q36.1,22q11.22,17p13.2 four loci,covering ZNF862,ATP6V0E2-AS1,ATP6V0E2,TOP3B,SPNS3 five genes.Conclusion:There was no special CNVs distribution in the Chinese Han population with POF,but the average total length of loss CNVs in POF is greater than control.Cumulative effect of CNVs on the entire genome is associated with POF.Part ? Validation of candidate gene TOP3B in POF and preliminary mechanism study on itObject:To verify the association between TOP3B CNV and POF and to explore the possible pathogenic mechanism.Materials and Methods:We enlarge the sample size of POF and control,using qPCR in Taqman method to detect TOP3B CNV.We performed aCGH(Array comparative genomic hybridization,CGH)to identify its boundaries in positive cases.All of TOP3B exons were analyzed by sequencing,the peripheral blood mRNA expression levels were detected by qPCR.Oocytes in GV stage were injected siRNA-targeting TOP3B by microscopic injection,and then the maturation abilities were estimated.We knockdown the expression of TOP3B by siRNA interference,detected the proliferation,apoptosis and reaction of human ovarian granulosa cells by qPCR,Western blot,flow cytometry and CCK-8.Results:The frequency of abnormal TOP3B copy number in POF cases(7/132,5.30%)was higher than both our controls(1/391,0.26%,Fisher exact test P=0.000364).Furthermore,DNA sequencing also found rs239918 and rs239927 are consistent in TOP3B copy number loss cases;their mRNA levels were down regulated.aCGH had the same result to SNP array platform.After interferencing TOP3B expression,maturation abilities were not changed in oocyte.In granulosa cell it inhibited proliferation,promoted apoptosis and regulated the expression of related genes and receptors related to FSH reduced.Conclusion:CNVs in 22q11.22 were found to be associated with POF for the first time in Chinese population;it can affect the expression of TOP3B by dose-effect.TOP3B can inhibit granulosa cell proliferation and promote its apoptosis to affect the development of follicles.