【作者】 焦雪；

【导师】 陈子江；

【作者基本信息】 山东大学 ， 妇产科学， 2014， 博士

【摘要】 第一部分中国汉族卵巢早衰患者染色体核型分析背景：卵巢早衰(POF)是一种临床特征高度异质、病因复杂的妇科内分泌疾病,40岁前发病率约为1%,近年来发病呈上升趋势。目前尚无可靠指标早期诊断,也无有效措施改善卵巢功能,因而病因研究及识别高风险人群十分必要。染色体畸变是POF目前已明确的病因之一,但其发生率报道不一。既往有多种染色体畸变类型在POF中报道,但大样本POF人群的核型研究较少见。最常见的异常核型是X染色体畸变,45,X和X短臂异常POF患者中,50%表现为原发性闭经和性腺发育不全,而45,X的嵌合体、Xq缺失及X-常染色体易位患者较多表现为继发性闭经。部分染色体畸变类型的POF携带者可将致病畸变传递给子代。因此,在大样本中国汉族POF患者中明确染色体畸变的发生率、异常核型的分布特征及与临床表型的相关性意义重大。目的：本研究旨在通过大样本中国汉族POF患者染色体核型分析,明确染色体畸变的发生率及其分布特征,阐明畸变核型与临床表型的相关性,为POF病因学研究及临床遗传咨询和生育指导提供理论基础。方法：募集2003年～2011年就诊的中国汉族POF患者531例,置备中期分裂相染色体进行G显带核型分析,明确染色体畸变的发生率及各不同异常核型的分布；识别染色体畸变发生的热点区域；按临床闭经类型分组,比较两组间染色体畸变的分布差异：结合临床表型及内分泌特征,分析染色体核型与临床表型的相关性。结果：研究发现染色体畸变的发生率为12.1%(64/531),X染色体异常约占93.7%(60/64)。最为常见的染色体畸变为X染色体结构异常,包括15例Xq缺失,2例Xp缺失,11例等臂染色体(6例短臂等臂,5例长臂等臂),1例环状染色体,1例倒位染色体,1例双着丝粒染色体和1例复杂重排。识别9例非嵌合X-常染色体易位,除1例外均涉及x染色体长臂(Xq22-Xq24)。不伴有X染色体结构异常的非整倍体19例,包括7例非嵌合45,X单体,9例45,X嵌合体,3例47,XXX三体。原发性闭经组染色体畸变率(21.4%,12/70)明显高于继发性闭经组(10.6%,49/461)(P=0.01)。45,X单体及其嵌合与原发性闭经表型关联性最高(46.7%)。3例含46,XY细胞系患者中,2例表现为继发性闭经。另检测到1例罕见的常染色体罗伯逊平衡易位。结论：首次大样本中国汉族POF患者染色体核型分析发现染色体畸变发生率为12.1%,X染色体异常率占93.7%,证实染色体异常,尤其是X染色体异常是POF的重要病因之一,有助于正确的生育指导和遗传咨询,为定位卵子发育相关区域及确定POF候选基因提供有价值的参考。第二部分生殖细胞特异性转录因子SOHLH2基因变异与卵巢早衰的相关性研究背景：始基卵泡池的大小及其耗竭速率是女性生殖寿命和生育潜能的重要决定因素。卵泡和卵子发生是多种信号通路交互协调的动态过程,生殖细胞特异性的关键基因准确有序地转录和表达至关重要,这需要生殖细胞特异性的转录调控因子的精细操纵。既往研究发现生殖细胞特异性转录因子FIGLA、NOBOX、LHX8、SOHLH1和SOHLH2等可形成独特而复杂的转录调控网络,调节生殖细胞众多关键基因(BMP15、GDF9、ZP1-3等)的时空特异性表达,维持卵泡发生发育和正常卵巢功能。SOHLH2基因编码早期精卵发生特异性的转录因子,在胚胎生殖细胞簇,始基卵泡和初级卵泡的卵母细胞限制性表达,在卵泡早期分化发育中发挥重要作用。Sohlh2+雌鼠卵巢内始基卵泡向初级卵泡过渡障碍,卵泡提早耗竭,导致不育,类似人非综合征型POF的临床表型。因此SOHLH2可作为POF的重要候选基因。目的：本研究通过对364例中国汉族及197例塞尔维亚POF患者进行SOHLH2基因突变.筛查,探讨SOHLH2基因变异与不同种族POF发病机制的相关性。方法：以364例中国汉族POF患者和197例塞尔维亚POF患者为研究对象,同时分别募集400例和200例相同种族的健康女性为正常对照。采用直接测序法筛查两个不同种族POF患者SOHLH2基因的突变情况,并利用多种预测软件对识别的新发变异进行序列比对和功能预测。结果：直接测序结果共发现11种SOHLH2基因的新发变异,其中错义变异包括在中国汉族POF患者中发现的p.Glu79Lys(2例)、p.Glu105Gly和p.Thr321Pro,和在塞尔维亚POF患者中发现的p.Leul20Phe(3例)和p.Leu204Phe。蛋白序列比对仅发现错义变异p.Glu79Lys和p.Glu105Gly涉及在哺乳动物中高度保守的氨基酸,功能预测为致病性。在中国汉族POF患者中发现1例c.-210G>T,该基因变异位于富集转录因子结合位点和CpG岛的核心启动子区。在塞尔维亚POF发现的新发变异中,预测内含子变异c.530+6T>G可能通过影响RNA剪切导致转录本无义降解,继而发挥致病作用。因此本研究在两个不同种族POF患者中识别的新发变异可能通过影响SOHLH2的转录、蛋白表达及同源／异源二聚体的形成而导致卵巢衰竭。结论：本研究在中国汉族和塞尔维亚两个不同种族POF患者中发现11种SOHLH2基因新发变异,提示SOHLH2基因变异是POF的致病机制之一,同时进一步印证转录因子SOHLH2在早期卵泡和卵子发生中发挥的重要作用。第三部分体细胞转录因子NR5A1基因突变在卵巢早衰发病机制中的作用研究背景：卵巢早衰(POF)病因具高度遗传异质性,多数患者病因尚不明确。体细胞转录因子的调控对卵泡和卵子发生至关重要。NR5A1基因编码类固醇生成因子SF1,是参与肾上腺、性腺发育,类固醇生成和生殖过程的关键转录因子。卵巢中SF1主要表达于颗粒细胞、卵泡膜细胞及黄体等体细胞,可调控STAR、CYP11A1、CYP19A1、 AMH、INHA及LHB转录,在卵巢类固醇生成、卵泡生长成熟过程中发挥重要作用。颗粒细胞特异性Nr5a1敲除雌鼠表现为不孕,卵巢内卵泡减少,缺乏黄体,提示NR5A1基因变异可能参与人卵巢早衰发病机制。目的：本研究旨在通过对400例中国汉族特发性非综合征型POF患者进行NR5A1基因突变筛查,并对识别的新发变异进行功能预测及体外功能实验验证,探讨该基因变异与POF发病的相关性。方法：以2003年-2012年就诊于山东大学附属生殖医院的400例中国汉族POF患者为研究对象,同时募集400例卵巢功能正常的育龄期健康女性为正常对照,应用直接测序技术对NR5A1基因编码区进行突变筛查。应用Align GVGD、Polyphen-2和SIFT进行功能预测,通过双荧光素酶报告系统检测转录活性,细胞免疫荧光染色技术检测蛋白表达及细胞定位三方面探讨新发变异的致病作用。结果：在400例中国汉族特发性非综合征型POF患者中发现1个新发变异c.13T>G(p.Tyr5Asp, p.Y5D),该杂合错义变异位于第二外显子。突变的酪氨酸位于DNA结合结构域上游,在各物种中高度保守。体外实验证实该错义变异影响NR5A1对下游调控基因Amh、Inhibin-a、Cypllal和Cyp19a1的转录活性,但不具有显性负效应；蛋白的表达及细胞定位无明显改变。结论：大样本中国汉族POF患者NR5A1基因突变筛查发现1例非功能域的杂合变异,该错义变异可能通过单倍剂量不足机制影响对下游调控基因的转录激活参与卵巢衰竭致病,但NR5A1基因变异不是中国汉族POF的常见致病机制。更多还原

【Abstract】 PartⅠCytogenetic Analysis of Chinese Women with Premature Ovarian FailureBackground:Premature ovarian failure (POF) is a complex gynecological endocrinal disorder with significantly genetic and clinical heterogeneity. POF occurs in1%of the general female population prior to age40years; however, the incidence has being increasing in recent years. Given no reliable marker for early diagnosis or effective treatment for improvement of ovarian function, it is of necessity to conduct the etiology study and identify those who are at high risk for POF. Chromosomal abnormalities have long been recognized as a frequent and main cause of POF, with widely varying percentages in reported series. Although numerous different karyotypic anomalies have been found, few large cohorts of POF have been studied. The most common abnormality involved an X chromosome. One half of POF patients with45,X monosomy and anomalies in short arm of X chromosome presented with primary amenorrhea and gonadal dysgenesis. And the majority of patients with45,X mosaicism and anomalies in long arm of X chromosome presented with secondary amenorrhea. Specific abnormal karyotypes with less severe perturbations of ovarian function might be at risk of being transmitted to offspring. Therefore, it is of significance to determine the prevalence and distribution of cytogenetic anomalies in this ethnic group with POF.Objective:The aim of the present study is to analyze the karyotype of large-scale Chinese patients with POF, identify the prevalence and distribution of chromosomal abnormalities, and their association with clinical phenotype, and therefore provide theoretical basis to further etiological research, genetic counseling and family planning.Methods:A total of531women with POF were recruited from2003to2011. Karyotype analysis was performed on GTG-banded metaphase chromosomes using a standard protocol. Analysis of the prevalence and distribution of anomalies, identification of hot region with abnormalities were performed. Distribution differences were compared between women with primary and those with secondary amenorrhea. Association of karyotype with phenotype was determined according to corresponding clinical manifestation.Results:Chromosomal abnormalities were detected in64of531cases (12.1%). Of the64,60(93.7%) involved the X chromosome. The most common abnormality was X-structural aberrations. We detected17terminal deletions [15del(Xq);2del(Xp)],11isochromosomes [6i(Xp);5i(Xq)],1ring X,1inv X,1isodicentric and1complex X arrangement. Nine X-autosome translocations were detected. All but one involved Xq, usually in regions Xq22-Xq24. Aneuploidy without a structurally abnormal X was found in19cases, including745,X monosomy,945,X mosaicisms, followed by347,XXX. Karyotypic abnormalities were more frequent in patients with primary amenorrhea (15/70,21.4%) than those with secondary amenorrhea (49/461,10.6%)(P=0.01).45,X and45,X/46,XX mosaicism were the complements most frequently associated with primary amenorrhea (46.7%). Two of the three cases with46,XY or 45,X/46,XY karyotype presented with’secondary amenorrhea’. One balanced autosomal Robertsonian translocation was also detected.Conclusion:In summary, this report of the largest cohort of Chinese women yet studied found the prevalence of chromosomal abnormalities in POF to be12.1%, most frequent anomalies involving X chromosome (93.7%). This confirms a major role for X chromosome abnormalities in POF, and highlights the importance of routine assessment of chromosomal abnormalities. Chromosomal studies thus provide valuable clinical information for reproductive management and genetic counseling. In addition to providing an etiologic explanation for the individual patient with POF, the cases facilitate identification of genes responsible for POF. PartⅡ Germ Cell-specific Transcription Factor SOHLH2in Premature Ovarian FailureBackground:Establishment of the initial primordial follicle pool and subsequent maintenance is important determinant of female reproductive lifespan and fertility potential. Oogenesis occurs with folliculogenesis through concomitant crosstalk signaling in mammals. This well-orchestrated dynamics must be, in part, dictated by coordinated interactions of genes preferentially expressed in oocytes, which in turn are modulated by germ cell specific transcriptional regulators. Several transcriptional regulators preferentially expressed in oocytes, such as FIGLA, NOBOX, LHX8, SOHLH1, and SOHLH2, established complex specialized regulatory networks to regulate ovary-specific gene (e.g., BMP15, GDF9, ZP1-3) expression pivotal for early folliculogenesis. SOHLH2encodes the spermatogenesis-and oogenesis-specific bHLH transcription factor and plays pivotal roles in early oogenesis and folliculogenesis. Its expression is uniquely confined to germ cell clusters of embryonic ovary and oocytes of primordial to primary follicle stage. Sohlh2-null female mice exhibited defective primordial-to-primary follicle transition, atrophied ovaries devoid of follicles and infertility, mimicking human POF phenotype. Therefore, SOHLH2is an attractive candidate gene of POF.Obejective:The present study was designed to determine whether SOHLH2variants contribute to POF in a large cohort of non-syndromic POF patients of Chinese (n=364) and Serbian (n=197) origin.Methods:A total of561women with POF of Han Chinese (n=364) and Serbian (n=197) origin were recruited. Ethnically matched healthy women served as controls (400Chinese and200Serbian). All were screened for variants in the SOHLH2gene by Sanger sequencing. Novel Variants were sequence-aligned and functional predicted by various online tools.Results:Eleven distinct novel heterozygous variants were identified in cohorts of POF but were absent in matched controls. These included the non-synonymous variants p.Glu79Lys (n=2cases), p.Glu105Gly, and p.Thr321Pro, which were found among four Chinese POF cases, and p.Leu120Phe (n=3cases) and p.Leu204Phe, which were found among four Serbian women. Protein alignments reveal that p.Glu79Lys and p.Glu105Gly involve amino acids highly conserved among mammals, both of which are predicted to be deleterious. The c.-210G>T found in the Chinese POF cohort lies in the core promoter region, which is enriched with transcription factor binding sites and CpG islands. In the Serbian cohort, the variant most likely to have a deleterious effect is c.530+6T>G, which is predicted to affect RNA splicing and result in nonsense mediated decay of transcripts. Disturbing the expression, transactivation or homo-/hetero-dimerization of the SOHLH2protein could result in ovarian failure.Conclusion:Our identification of novel variants in the SOHLH2gene, in women with POF of both Chinese and Serbian origin, suggests a contribution to the etiology of POF. This indicates the pivotal role of the transcription factor SOHLH2in early oogenesis and folliculogenesis. PartⅡSomatic Transcription Factor NR5A1Mutation in Premature Ovarian FailureBackground:The etiology of premature ovarian failure (POF) is genetically heterogenous, and no predominant explanation has been documented. Nuclear receptor subfamily5, group A, member1(NR5A1) encodes steroidogenic factor1(SF1), a nuclear receptor involved in adrenal and gonadal development, steroidogenesis, and reproduction. SF1serves as a master transcriptional regulator of multiple genes, including STAR, CYP11A1, CYP19A1, AMH, INHA, and LHB. Human SF1protein is mainly expressed in ovarian somatic cells of growing follicles and corpus lutea. Granulosa cell-specific Nr5al knockout mice exhibit infertility, hypoplastic ovaries with decreased follicles and absence of corpora lutea. These findings indicate a critical role of NR5A1in ovarian development and function and its disruption might involve in the pathogenesis of human POF.Objective:The present study was designed to determine whether NR5A1variants account for women with nonsyndromic POF in Han Chinese.Methods:Mutation screening in the NR5A1gene in a large cohort of Chinese women with non-syndromic POF (400cases) were performed with direct sequencing. Subsequently, functional prediction using Align GVGD, Polyphen-2and SIFT, transactivation activity assay using Dual-Luciferase Reporter Assay System, and protein expression and nuclear localization with immunofluorescence were conducted to determine the causative role of the novel variants identified in vitro.Results:A novel heterozygous missense variant-c.13T>G (p.Tyr5Asp) in exon2was identified. The mutated tyrosine, located seven residues upstream of the DNA-binding domain (DBD), was highly conserved among SF1orthologs. This non-synonymous variant impaired transcriptional activation on Amh, Inhibin-a, Cypl1al and Cypl9al gene, as shown by transactivation assays. However, no dominant negative effect was observed, nor was there impact on protein expression and nuclear localization.Conclusion:A novel heterozygous variant p.Tyr5Asp, in a novel non-domain region of NR5A1, was identified in the large cohort of Chinese women with POF. This missense variant is presumed to result in haploinsufficiency and impairment of transactivation activity on different SF1target genes. Irrespectively, perturbation in NR5A1is not a common explanation for POF in Chinese.更多还原