本文選題：卵巢早衰 + 染色體核型�。� 參考：《浙江大學(xué)》2016年博士論文

【摘要】：第一部分:中國(guó)漢族卵巢早衰患者的臨床特征及染色體核型分析目的:明確染色體異常在病因不明POF患者中所占比重及遺傳學(xué)病因篩查對(duì)POF患者的重要性。材料與方法:招募2012年10月至2014年6月在浙江大學(xué)醫(yī)學(xué)院附屬婦產(chǎn)科醫(yī)院生殖內(nèi)分泌科門(mén)診就診的156名漢族POF患者及391名卵巢功能正常的漢族對(duì)照婦女,比較兩組間臨床資料的特點(diǎn)。對(duì)POF患者行外周血淋巴細(xì)胞G顯帶法檢測(cè)核型,分析異常核型與臨床特點(diǎn)之間的關(guān)系。結(jié)果:POF組與對(duì)照組相比內(nèi)分泌異常明顯。從臨床特征上看,POF患者中原發(fā)性閉經(jīng)占10.9%(17/156);大部分患者在30歲前出現(xiàn)閉經(jīng)癥狀。在156名患者中,原發(fā)性閉經(jīng)患者17例,核型異常12例,異常率70.6%;繼發(fā)性閉經(jīng)患者139例,核型異常12例,異常率8.6%。原發(fā)性閉經(jīng)患者的染色體核型異常率顯著高于繼發(fā)性閉經(jīng)患者。X染色體的結(jié)構(gòu)異常占到所有染色體異常的62.5%。結(jié)論:染色體核型異常是中國(guó)漢族POF患者的重要遺傳學(xué)病因,X染色體的結(jié)構(gòu)異常更為常見(jiàn)。在POF的患者中,尤其是以原發(fā)閉經(jīng)為首要癥狀的患者中,染色體核型的檢查是必要的。第二部分:卵巢早衰患者基因組拷貝數(shù)變異分析目的:發(fā)現(xiàn)新的POF遺傳學(xué)致病因素,明確CNV與POF發(fā)病的關(guān)系及POF患者CNVs的整體分布特點(diǎn),探索新的POF致病基因。材料與方法:利用Affymetrix CytoScan HD高通量SNP array平臺(tái)對(duì)前期入組并且已排除核型異常的90例POF患者和90例卵巢功能正常的對(duì)照進(jìn)行全基因組CNVs的檢測(cè)。在全基因組范圍內(nèi)對(duì)POF組和正常對(duì)照組的CNVs基本特征及分布特點(diǎn)進(jìn)行了統(tǒng)計(jì)及生物信息學(xué)的分析,同時(shí)基于病例-對(duì)照研究的基本策略與DGV數(shù)據(jù)庫(kù)對(duì)CNVs與POF的發(fā)病進(jìn)行了關(guān)聯(lián)分析。結(jié)果:POF組與對(duì)照組CNVs在各染色體的分布無(wú)明顯差異,但在POF組中,CNVs缺失的檢出率高于對(duì)照組。人均CNVs的總長(zhǎng)度在兩組間無(wú)明顯差異,但POF組人均缺失的CNVs總長(zhǎng)度為1412.40 kb,顯著大于對(duì)照組936.50kb的平均總長(zhǎng)度。最終發(fā)現(xiàn)5個(gè)候選CNVs狀態(tài),涉及到4q35.2、7q36.1、22q11.22、17p13.2共 4 個(gè)位點(diǎn),覆 蓋 ZNF862、ATP6V0E2-AS1、ATP6V0E2、TOP3B、SPNS3 共 5個(gè)基因。結(jié)論:中國(guó)漢族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長(zhǎng)度大于正常人群,缺失CNVs累積作用于整個(gè)基因組與致病相關(guān)。第三部分:卵巢早衰相關(guān)候選基因TOP3B拷貝數(shù)變異的驗(yàn)證及致病機(jī)制初探目的:驗(yàn)證TOP3B位點(diǎn)CNV與漢族POF發(fā)病的相關(guān)性,探尋其可能的致病機(jī)制。材料與方法:進(jìn)一步擴(kuò)大POF組與對(duì)照組的樣本量,利用Taqman探針進(jìn)行實(shí)時(shí)熒光定量PCR檢測(cè)該位點(diǎn)CNV。同時(shí),對(duì)該位點(diǎn)CNV缺失的POF患者利用aCGH平臺(tái)進(jìn)一步檢測(cè),明確其邊界,并對(duì)該位點(diǎn)拷貝數(shù)缺失的患者進(jìn)行了 TOP3B全外顯子的測(cè)序分析,熒光定量PCR檢測(cè)了外周血中TOP3BmRNA表達(dá)水平。利用顯微注射siRNA及熒光共聚焦顯微鏡在下調(diào)GV期卵母細(xì)胞TOP3B的表達(dá)后觀察卵母細(xì)胞的成熟度;利用體外刺激原代培養(yǎng)的人卵巢顆粒細(xì)胞,通過(guò)siRNA干擾,實(shí)時(shí)熒光定量PCR,Western blot,流式細(xì)胞術(shù),CCK-8檢測(cè)了TOP3 表達(dá)降低對(duì)顆粒細(xì)胞的影響。結(jié)果:中國(guó)漢族人群POF患者TOP3B基因拷貝數(shù)異常的發(fā)生頻率(7/132,5.30%)顯著高于漢族人群對(duì)照(1/391,0.26%,Fisher's確切概率法,P0.05)及DGV大樣本人群對(duì)照(5/873,0.57%,Fisher's確切概率法P0.05)。aCGH平臺(tái)檢測(cè)結(jié)果與SNParray平臺(tái)結(jié)果一致。兩例缺失的患者在rs239927和rs239918位點(diǎn)存在同樣的單體型,網(wǎng)站預(yù)測(cè)對(duì)TOP3B的表達(dá)起到降調(diào)節(jié)的作用,其外周血mRNA水平顯著低于正常人群。干擾TOP3B的表達(dá),對(duì)GV期卵母細(xì)胞的成熟能力無(wú)明顯影響,但可抑制顆粒細(xì)胞的增殖于G1期并促進(jìn)其凋亡,顆粒細(xì)胞上與FSH作用相關(guān)的基因及受體表達(dá)減弱。結(jié)論:22q11.22 位點(diǎn) chr22:22311348～22578983 的 CNV 與 POF 的發(fā)病相關(guān),其可通過(guò)劑量效應(yīng)導(dǎo)致所覆蓋的TOP3B基因的表達(dá)降低,并可通過(guò)抑制顆粒細(xì)胞的增殖及促進(jìn)其凋亡來(lái)影響卵泡的發(fā)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.
【學(xué)位授予單位】：浙江大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2016
【分類(lèi)號(hào)】：R711.75

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