中国汉族卵巢早衰患者的拷贝数变异分析及相关基因的研究

发布时间：2018-05-21 20:09

本文选题：卵巢早衰 + 染色体核型　；参考：《浙江大学》2016年博士论文

【摘要】：第一部分:中国汉族卵巢早衰患者的临床特征及染色体核型分析目的:明确染色体异常在病因不明POF患者中所占比重及遗传学病因筛查对POF患者的重要性。材料与方法:招募2012年10月至2014年6月在浙江大学医学院附属妇产科医院生殖内分泌科门诊就诊的156名汉族POF患者及391名卵巢功能正常的汉族对照妇女,比较两组间临床资料的特点。对POF患者行外周血淋巴细胞G显带法检测核型,分析异常核型与临床特点之间的关系。结果:POF组与对照组相比内分泌异常明显。从临床特征上看,POF患者中原发性闭经占10.9%(17/156);大部分患者在30岁前出现闭经症状。在156名患者中,原发性闭经患者17例,核型异常12例,异常率70.6%;继发性闭经患者139例,核型异常12例,异常率8.6%。原发性闭经患者的染色体核型异常率显著高于继发性闭经患者。X染色体的结构异常占到所有染色体异常的62.5%。结论:染色体核型异常是中国汉族POF患者的重要遗传学病因,X染色体的结构异常更为常见。在POF的患者中,尤其是以原发闭经为首要症状的患者中,染色体核型的检查是必要的。第二部分:卵巢早衰患者基因组拷贝数变异分析目的:发现新的POF遗传学致病因素,明确CNV与POF发病的关系及POF患者CNVs的整体分布特点,探索新的POF致病基因。材料与方法:利用Affymetrix CytoScan HD高通量SNP array平台对前期入组并且已排除核型异常的90例POF患者和90例卵巢功能正常的对照进行全基因组CNVs的检测。在全基因组范围内对POF组和正常对照组的CNVs基本特征及分布特点进行了统计及生物信息学的分析,同时基于病例-对照研究的基本策略与DGV数据库对CNVs与POF的发病进行了关联分析。结果:POF组与对照组CNVs在各染色体的分布无明显差异,但在POF组中,CNVs缺失的检出率高于对照组。人均CNVs的总长度在两组间无明显差异,但POF组人均缺失的CNVs总长度为1412.40 kb,显著大于对照组936.50kb的平均总长度。最终发现5个候选CNVs状态,涉及到4q35.2、7q36.1、22q11.22、17p13.2共 4 个位点,覆盖 ZNF862、ATP6V0E2-AS1、ATP6V0E2、TOP3B、SPNS3 共 5个基因。结论:中国汉族POF患者人群中不存在特殊的CNVs分布,但是POF患者人群中人均缺失的CNVs长度大于正常人群,缺失CNVs累积作用于整个基因组与致病相关。第三部分:卵巢早衰相关候选基因TOP3B拷贝数变异的验证及致病机制初探目的:验证TOP3B位点CNV与汉族POF发病的相关性,探寻其可能的致病机制。材料与方法:进一步扩大POF组与对照组的样本量,利用Taqman探针进行实时荧光定量PCR检测该位点CNV。同时,对该位点CNV缺失的POF患者利用aCGH平台进一步检测,明确其边界,并对该位点拷贝数缺失的患者进行了 TOP3B全外显子的测序分析,荧光定量PCR检测了外周血中TOP3BmRNA表达水平。利用显微注射siRNA及荧光共聚焦显微镜在下调GV期卵母细胞TOP3B的表达后观察卵母细胞的成熟度;利用体外刺激原代培养的人卵巢颗粒细胞,通过siRNA干扰,实时荧光定量PCR,Western blot,流式细胞术,CCK-8检测了TOP3 表达降低对颗粒细胞的影响。结果:中国汉族人群POF患者TOP3B基因拷贝数异常的发生频率(7/132,5.30%)显著高于汉族人群对照(1/391,0.26%,Fisher's确切概率法,P0.05)及DGV大样本人群对照(5/873,0.57%,Fisher's确切概率法P0.05)。aCGH平台检测结果与SNParray平台结果一致。两例缺失的患者在rs239927和rs239918位点存在同样的单体型,网站预测对TOP3B的表达起到降调节的作用,其外周血mRNA水平显著低于正常人群。干扰TOP3B的表达,对GV期卵母细胞的成熟能力无明显影响,但可抑制颗粒细胞的增殖于G1期并促进其凋亡,颗粒细胞上与FSH作用相关的基因及受体表达减弱。结论:22q11.22 位点 chr22:22311348～22578983 的 CNV 与 POF 的发病相关,其可通过剂量效应导致所覆盖的TOP3B基因的表达降低,并可通过抑制颗粒细胞的增殖及促进其凋亡来影响卵泡的发育。
[Abstract]:Part one: the clinical features and karyotype analysis of Chinese Han patients with premature ovarian failure: the specific gravity of chromosomal abnormalities in the POF patients with unknown etiology and the importance of genetic screening for POF patients. Materials and methods: from October 2012 to June 2014 at the maternity and obstetrics hospital affiliated to the Medical College of Zhejiang University. In the Department of Endocrinology, 156 Han POF patients and 391 women with normal ovarian function were compared. The clinical data between the two groups were compared. The G banding method was used to detect the karyotype of the peripheral blood lymphocytes in the patients with POF. The relationship between the abnormal karyotype and the clinical characteristics was analyzed. Results: the endocrine abnormalities were obvious in the POF group compared with the control group. The clinical features of POF patients were 10.9% (17/156); most of the patients had amenorrhea symptoms before 30 years of age. Among 156 patients, 17 cases of primary amenorrhea, 12 cases of abnormal karyotype, 70.6% abnormality, 139 cases of secondary amenorrhea, 12 cases of abnormal karyotype, and abnormal rate of chromosome karyotype in the abnormal rate of 8.6%. primary amenorrhea 62.5%. conclusion that the structural abnormalities of.X chromosomes in patients with secondary amenorrhea account for all chromosomal abnormalities: chromosomal abnormalities are important genetic causes of POF in Chinese Han people, and the structural abnormalities of the X chromosome are more common. In patients with POF, especially in patients with primary closure of the primary symptoms, chromosome karyotype examination The second part: the second part: analysis of genomic copy number variation in patients with premature ovarian failure: discover new POF genetic pathogenic factors, clarify the relationship between CNV and POF and the overall distribution characteristics of CNVs in POF patients, explore new POF pathogenic genes. Materials and methods: using Affymetrix CytoScan HD high throughput SNP array platform for early entry 90 POF patients who had excluded abnormal karyotype and 90 cases of normal control of ovarian function were tested for complete genomic CNVs detection. The basic characteristics and distribution characteristics of CNVs in group POF and normal control group were statistically analyzed and bioinformatics were analyzed in the whole genome range, based on the basic strategy of case control study and the number of DGV. According to the correlation analysis of the incidence of CNVs and POF, there was no significant difference in the distribution of the chromosomes between the POF group and the control group, but in the POF group, the detection rate of the CNVs deletion was higher than that of the control group. The total length of the per capita CNVs was not significantly different between the two groups, but the total length of the CNVs in POF group was 1412.40 KB, which was significantly greater than that of the control group 936.. The average total length of 50kb was found to be 5 candidate CNVs States, involving a total of 4 loci of 4q35.2,7q36.1,22q11.22,17p13.2, covering ZNF862, ATP6V0E2-AS1, ATP6V0E2, TOP3B, and SPNS3. Conclusion: there is no special CNVs distribution in the population of Chinese Han POF, but the length of CNVs in POF patients is greater than that of the positive. The cumulative effect of the absence of CNVs on the whole genome is associated with the pathogenesis of the whole genome. The third part: validation of the TOP3B copy number variation of the candidate genes for premature ovarian failure and the preliminary study of the pathogenesis mechanism: to verify the correlation between the TOP3B locus CNV and the incidence of POF in the Han nationality and to explore the possible pathogenesis. Materials and methods: to further expand the POF and control groups The Taqman probe was used to detect the site CNV. with real time fluorescence quantitative PCR, and the POF patients with CNV deletion at the site were further detected by aCGH platform, and their boundaries were identified. The TOP3B exons were sequenced and the TOP3BmRNA expression in the peripheral blood was detected by the fluorescence quantitative PCR. Level. The maturity of oocyte was observed after the expression of TOP3B in GV oocyte by microinjection of siRNA and fluorescence confocal microscope; the human ovarian granulosa cells were stimulated in vitro by stimulating the original human ovarian granulosa cells in vitro, and by siRNA interference, real-time fluorescent quantitative PCR, Western blot, flow cytometry, and CCK-8 detected TOP3 expression to reduce the fine particle size. Results: the frequency of abnormal copy number of TOP3B gene (7/132,5.30%) in Chinese Han population (7/132,5.30%) was significantly higher than that of Han population (1/391,0.26%, Fisher's exact probability, P0.05) and DGV large sample population control (5/873,0.57%, Fisher's exact probability method P0.05).ACGH platform detection results were in agreement with the SNParray platform results. Two The missing patients have the same haplotype at the rs239927 and rs239918 loci. The site prediction plays a decreasing role in the expression of TOP3B, and the level of mRNA in peripheral blood is significantly lower than that of the normal population. The interference of TOP3B expression has no significant effect on the maturation of GV oocytes, but it inhibits the proliferation of granular cells in G1 phase and promotes it. Apoptosis, the expression of genes and receptors associated with the action of FSH on granulosa cells is weakened. Conclusion: the CNV of chr22:22311348 ~ 22578983 of the 22q11.22 site is associated with the pathogenesis of POF, which can reduce the expression of the TOP3B gene overlay by the dose effect, and can affect the follicle's hair by inhibiting the proliferation of granulosa cells and promoting its apoptosis. Breeding.
【学位授予单位】：浙江大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R711.75

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