【作者】 王文婷；

【导师】 陈子江；

【作者基本信息】 山东大学 ， 妇产科学， 2016， 博士

【摘要】 第一章线粒体相关基因C10ORF2在家族性原发性卵巢功能不全合并感音神经性耳聋(Perrault综合征)发病中的作用和机制研究第—节Perrault综合征家系全外显子组测序研究目的：原发性卵巢功能不全(POI)是一种高度异质性、病因复杂的妇科内分泌疾病,患者通常在40岁以前就出现闭经,发病率约为1%。该疾病的特点是原发性或继发性闭经并且伴随不同程度的低雌激素症状。目前尚没有可靠的早期诊断指标,也不能早期有效的预防该疾病的发生和进展,因而病因研究和早期识别高风险人群是十分重要的。原发性卵巢功能不全主要分为综合征型POI和非综合征型POI两种,并在两种类型中分别筛选出了多个致病基因。其中已发现的综合征型POI包括脆X综合征、小睑裂综合征(BPES).半乳糖血症、碳水化合物缺乏糖蛋白综合征I型、Perrault综合征(PS)等,通过全外显子组测序发现Perrault综合征患者的致病基因包括HARS2、LARS2、C10ORF2、CLPP等。Perrault综合征患者在患有感音神经性耳聋的同时,女性患者还伴有典型的原发性卵巢功能不全症状。我们对先证者及家系其他成员进行全外显子组测序,以期发现新的POI致病基因。方法：对该Perrault综合征中先证者、患病姐妹以及父母采用Agilent SureSelect Human All Exon.V5技术对全外显子组进行测序,将检出的全部单碱基改变、插入／缺失与公共数据库比对排除已知多态,之后检出造成氨基酸改变的非同义突变,再根据孟德尔遗传定律筛出候选位点。最后按照常染色体隐性遗传的方式进行基因筛选,且在患者中筛选出共同的纯合突变位点。结果：全外显子组测序后发现了符合常染色体隐性遗传的一个错义突变位点,该突变位于C10ORF2基因的c.1388G>A(p.R463Q)位点,属于一个新发的错义突变,该突变使该位置的氨基酸由精氨酸变为谷氨酰胺。患者为纯合突变,并发生于编码蛋白的解旋酶区域。C10ORF2基因的解旋酶作用对线粒体的复制至关重要,推测该突变导致的蛋白功能改变影响了细胞线粒体功能,从而导致了Perrault综合征的发生。结论： 通过外显子测序技术在C10ORF2基因的解旋区域内发现了新发错义突变位点,该基因突变的发现为从线粒体功能方面探索POI的机制研究提供了新的方向。第二节C10ORF2基因在原发性卵巢功能不全中的作用机制研究目的：在原发性卵巢功能不全伴随感音神经性耳聋患者(PS)家系中,我们通过全外显子组测序发现了位于C10ORF2基因上的错义突变位点c.1388G>A(p.R463Q)。在既往研究中,发现位于同一位点的其他氨基酸改变导致患者婴儿脊髓小脑发育不全的严重表现。C10ORF2基因编码的DNA解旋酶参与了真核细胞内线粒体复制过程,而线粒体作为颗粒细胞和卵母细胞内含量丰富的细胞器,调节了颗粒细胞和卵母细胞的代谢、细胞周期和细胞信号转导。但目前该基因功能与卵巢的关系尚无报道。本课题通过研究C10ORF2突变导致细胞线粒体功能发生的变化,探讨(C10ORF2基因突变引发POI的致病机制。方法：通过收集Perrault综合征患者家系外周血,提取先证者外周血液DNA,建立C100RF2基因c.1388G>A(p.R463Q)位点突变的永生化淋巴细胞系,检测患者外周血永生细胞内线粒体ATP产生,线粒体拷贝数以及细胞内ROS的生成等,研究C10ORF2基因突变导致的细胞线粒体功能变化。结果：通过提取患者外周血并构建永生化淋巴细胞进行功能实验,我们发现C10ORF2基因突变导致了细胞内ATP产生减少,ROS水平升高,并发现C10ORF2基因突变的永生化淋巴细胞内线粒体拷贝数也明显低于正常人水平。这些实验结果表明该基因突变严重影响了细胞内线粒体的活力和氧化磷酸化水平,导致了线粒体功能障碍。结论：在C10ORF2基因中,c.1388G>A(p.R463Q)位点突变严重影响了细胞的线粒体功能。如果该突变发生在卵巢内,将阻碍卵巢内卵母细胞和颗粒细胞的正常能量代谢,使卵母细胞成熟障碍。如果线粒体功能严重缺陷,使细胞ATP生成减少及ROS累积增多,将导致卵母细胞无法代偿而发生死亡。因此,C10ORF2基因突变导致的卵巢内线粒体内能量代谢障碍可能是导致POI的重要机制之一。第二章HELQ基因在原发性卵巢功能不全患者中的突变分析目的：原发性卵巢功能不全(POI)病因具有高度异质性,多数患者病因尚不明确。我们发现POI和自然绝经年龄以及早绝经年龄具有共同的遗传易感性,为POI的病因学研究提供了新的方向。有研究通过全基因组关联分析发现了13个与绝经年龄相关的位点,在随后的生物信息学分析中发现8个候选基因与DNA损伤修复相关,其中HELQ基因具有较高的POI风险性,因此被列为POI的重要候选基因,它编码了蛋白HEL308,是一个DNA依赖性的ATP酶及DNA解旋酶,并且与RAD51同源蛋白一起参与了DNA链间交联反应的修复。链间交联反应的修复需要细胞分裂间期S期检查点和范可尼贫血途径的共同协同作用来完成。Helq-/-基因敲除小鼠生育能力降低,且卵巢组织切片呈现明显的萎缩现象。本研究旨在通过对汉族POI患者进行HELQ基因的外显子测序,明确该基因在POI患者中的突变情况,为POI病因学研究及临床遗传咨询与生育指导提供理论依据。方法：选取在2012至2014年就诊于山东大学附属生殖医院的中国汉族POI患者192例,将其血液DNA提取后,进行HELQ基因全部外显子及外显子与内含子邻接部位测序。所测结果与数据库中国汉族北方正常人群进行序列比对,用SPSS软件将SNP位点的基因型和等位基因频率进行统计分析。结果：在HELQ基因外显子测序中发现6个单核苷酸多态位点,其中rs1494961, rs2047210两个位点经过统计分析后发现,基因型和等位基因频率与对照组相比没有统计学差异,rs13141136,rs7665103,rs11099600,rs12645412与对照人群比较有统计学差异,但属于同义改变。结论：首次在中国汉族原发性卵巢功能不全患者中筛查了HELQ基因的编码序列,发现HELQ基因突变不是原发性卵巢功能不全的常见原因。第三章PRIM1基因在原发性卵巢功能不全患者中的突变分析及基因敲除小鼠研究第—节PRIM1基因在原发性卵巢功能不全患者中的突变分析目的：近年来越来越多的研究发现原发性卵巢功能不全和早绝经之间具有共同的遗传易感性,为探索POI的致病机制提供了一个新的方向。有研究通过全基因组关联分析,发现了13个与绝经年龄相关的位点,其中包括PRIM1基因。PRIM基因不仅与自然绝经年龄密切相关,并且后续在欧洲人群中的研究发现PRIM1基因的非同义多态性位点rs2277339与早绝经和POI也有显著相关性。PRIM1基因编码的DNA引发酶主要参与DNA复制过程,在合成冈崎片段时通过促进合成RNA引物来完成DNA的合成。本研究旨在通过对192例中国汉族POI患者进行PRIM1编码序列测定,从而确定该基因突变是否导致POI的发生。方法：选取2014年至2015年就诊于山东大学附属生殖医院的192例POI病人为研究对象,应用直接测序技术进行PRIM1基因编码区域测序即基因突变筛查,所获结果与Ensemble数据库中正常中国北方人群作为对照组进行对比,用SPSS软件将SNP位点的基因型和等位基因频率进行统计分析。结果：在POI病人中筛查出3个已知SNP位点rs2277339,rs1131514和rs1026565,分别位于外显子1,外显子10和内含子6中,它们的基因型和等位基因频率在POI和对照组数据中相比没有统计学差异。结论：PRIM1基因突变在中国汉族原发性卵巢功能不全患者中并不常见。虽然在欧洲人群中rs2277339位点与原发性卵巢功能不全相关,但在中国汉族POI妇女中并无意义,可能由种族差异性或本研究的样本量较少导致。PRIM1基因在POI发病中的作用尚需继续深入研究。第二节Prim1基因敲除小鼠的生育力分析目的：在原发性卵巢功能不全的遗传学研究中不仅需要基因组学的研究为寻找新的致病位点提供依据,而探索POI致病机制过程中更需要动物体内实验的生物学支持。因此,在进行PRIM1基因筛查的同时,为了评估PRIMl基因在体内的生理作用,我们构建了Prim1基因敲除小鼠,研究该基因的缺失是否影响小鼠生长发育及生殖功能。方法：采用CRISPR/Cas9技术,在该靶基因内寻找Cas9切点,设计、构建对应的gRNA序列,测试其核酸内切活性后,与Cas9-mRNA共同注射小鼠单细胞胚,繁育后取得F1代杂合子Prim1+/-小鼠。通过合笼后记录生育子代数,子代基因型比例,卵巢形态,大小和卵巢内各级卵泡数目等研究Prim1基因缺失对小鼠生殖系统的影响。结果：Prim1+/-小鼠合笼后没有纯合子Primr1-/-小鼠出生。在杂合子Prim1+/-小鼠研究中发现,其子代数目、卵巢大小及卵巢内各级卵母细胞数目等与野生型小鼠相比没有明显差异。结论:Prim1+/-小鼠与野生型小鼠比较没有明显生育力差异。没有Prim1纯合子子代出生,说明该基因的缺失可能严重影响了细胞的周期和DNA损伤修复过程而导致细胞凋亡的发生,使Prim1-/-小鼠胚胎死亡。在今后的研究中,可考虑构建条件性Prim1基因敲除小鼠,从而研究该基因对卵巢功能的影响。更多还原

【Abstract】 Chapter Ⅰ.The Effects and Mechanisms of Mitochondria-related gene C10ORF in the Pathogenesis of Perrault SyndromePart Ⅰ.Whole Exome Sequencing in Perrault Syndrome FamilyOBJECTIVE:Primary Ovarian Insufficiency is a highly heterogeneous complex disease. It is one of the pathogenesis for female infertility which characterized by secondary amenorrhoea, elevated gonadotrophin level (FSH> 40 IU/L) and declined estrogen to menopausal level. It is the end phase of primary ovarian insufficiency. The overall presumption of POI prevalence in general population was reported to be 1-2%. The disorder is heterogeneous, with a wide spectrum of causes, including genetic, autoimmune, metabolic, infectious and iatrogenic. Genetic causes are highly heterogeneous and include both isolated (nonsyndromic) POI and syndromic forms. However, the etiology remains to be elucidated in most cases. The development of genomics technology for illuminate disease susceptibility loci and candidate genes provides a more efficient and effective way. Perrault syndrome (PS) is an autosomal recessive ovarian dysgenesis associated with sensorineural deafness. The patients showed a typical primary ovarian insufficiency (POI) phenotype. In this study, we will perform the whole exome sequencing (WES) in the Perrault Syndrome Family with ovarian POI to identify the innovative genes responsible for POI.METHODS:The study participants comprised the perrault syndrome patients and virulence genes suspected carriers and direct family members suspected carriers, then sequencing the whole genome exons by the way of Agilent V5. We checked out all the single-base changes, insertion/deletion and compared with public databases, then eliminate known polymorphism. After the detection of amino acid change with non-synonymous mutations, then find out candidate loci according to the rules of Mendelian inheritance. We made the genetic screening by the rule of autosomal recessive heredity, and then find out the candidate loci lacated on C10ORF2 gene. RESULT:By directly sequence the whole genome exome, and analysis the result according to the phenotype and Mendelian inheritance. We find out a novel homozygous mutations C10ORF2 c.1388G>A (p.R463Q).CONCLUSION:With the application of WES in the PS famlily with POI, we identified a new loci in C10ORF2 gene. As C10ORF2 is closely related to the function of mitochondria, mutations of the gene may made an obstacles in the cell mitochondrial energy metabolism of ovaries of oocyte and its support cells. Resulting in most patients accompanied with a typical primary ovarian premature aging signs. The new discovery provide a new research orientation in the mechanism research of POI.Part Ⅱ. Functional Study of C10ORF2 gene in Primary Ovarian InsufficiencyOBJECTIVE:In a familial Aggregation Analysis of Perrault syndrome with a typical phenotype of POI, we found a new gene closely related to function of mitochondria through the whole exome sequencing (WES) and identified a new mutation loci in C10ORF2 c.1388G>A(p.R463Q). This gene encodes a hexameric DNA helicase which unwinds short stretches of double-stranded DNA in the 51 to 3’ direction, along with mitochondrial single-stranded DNA binding protein and mtDNA polymerase gamma, is thought to play a key role in mtDNA replication. Mutations in this gene cause infantile onset spinocerebellar ataxia (IOSCA) and progressive external ophthalmoplegia (PEO) and are also associated with several mitochondrial depletion syndromes. Alternative splicing results in multiple transcript variants encoding distinct isoforms. Perrault syndrome is characterized by POI in females and progressive hearing loss in both females and males. The objective of our study was to investigate the effect of mutation in the immortalized cell line, and illustrate the gene function in the mitochondrial, which may offer a new thought in searching the mechanism of POI.METHODS:Genomic DNA samples from the probands with POI and from their unaffected parents were evaluated in our laboratory by the whole genome exome sequence according to previously published methods, extract the blood of recessive hereditary probands, and create immortalized cell line. The proband presented with primary amenorrhea and postmenopausal levels of follicle-stimulating hormone and luteinizing hormone. Her ovaries were not visualized on abdominal ultrasound. We test the function of mitochondria in the mutant immortalized cell, including the mtDNA copy number, detect the transcripts of mitochondrial genes, ATP and ROS level was measured in conducted immortalized cell line,and test the mitochondria membrane potential.RESULT: Compared with other control group, the function of mitochondria in mutant cell was functionally impaired, the mutant cell showed a turbulence of OXPHOS in the cell, accompanied with decrease ATP and increased ROS production, decreased transcripts of mitochondrial genes and mtDNA copy number. However, mitochondria exist in the cell plentifully, and possess a vital role in the cell energy supply, especially in oocyte, the potential role of C10ORF2 gene is unneglectable in the maintenance of female fertility mediated by functional of mitochondria.CONCLUSION:C10ORF2 gene plays a key role in mitochondrial replication, it functions as an energy supplier factor in oocyte cells. Mutations in C10ORF2 gene are associated with familial POI with Perrault Syndrome, which mediated by the impaired function of mitochondria. These studies provide evidence for the pitoval role of mitochondria in POI.Chapter II. The Screening of HELQ Gene in Chinese Patients with Primary Ovarian InsufficiencyOBJECTIVE:A meta-analysis of 22 genome-wide association studies has reported 13 novel loci associated with age at menopause. In the following biological pathway analysis, eight candidate genes (EXO1, HELQ, UIMC1, FAM175A, FANCI, TLK1, POLG, PRIM1) identified are related to DNA damage repair and replication. Among all of these genes, HELQ encodes the protein HEL308, which is a DNA-dependent ATPase and DNA helicase and required for repair of DNA interstrand crosslinks through the association with RAD51 paralogs. Helq-/- mouse displayed subfertility and dysgenesis or atrophy of ovary was observed in histological sections, mimicking the human POI phenotype and suggesting HELQ’s critical role in germ cell maintenance. Therefore, in this study, the potential role of HELQ mutations in women with POI was explored by sequencing the coding region of HELQ gene.METHODS:The study comprised 192 Chinese Han women with secondary amenorrhoea who were recruited. All the patients recruited were non-syndromic and idiopathic. Genomic DNA was extracted from peripheral blood samples. The 18 exons and exon-intron boundaries of HELQ gene were amplified by using polymerase chain reaction (PCR). The PCR products were purified using the method of polyethylene glycol precipitation and sequenced directly on an automated sequencer (ABI3100; Applied Biosystems) using the ABI-Prism big-dye Terminator Cycle Sequencing Ready Reaction Kit (Applied Biosystems). All sequence variants were confirmed in triplicate with forward and reverse sequencing.RESULT: Six known single-nucleotide polymorphisms (SNP) were identified in the HELQ gene. Three synonymous SNP located in the exon regions, including rs1494961(exon 2), rs13141136 (exon 3) and rs7665103 (exon6). The other three SNP were in the exon-intron boundaries, including rs 11099600 (intron 4), rs2047210 (intron 10) and rs 12645412 (intron 11). For genotype frequencies of these 6 SNP, no significant difference was found between cases and controls.CONCLUSION:Although difference in the allele frequency was shown in the other four SNP, rs13141136, rs7665103, rs11099600 and rs12645412, no amino acid was altered and thus they are unlikely to be causative for POI. In conclusion, although no mutations were found in the HELQ gene, genes pivotal for DNA repair could not be fully ruled out in the candidates for POI, which is characterized by genetic heterogeneity with distinct loci in different ethnicities. A specific molecular perturbation in a given gene may vary, even within a given thnic group. Therefore, future studies in different ethnic populations are necessary to determine the role of HELQ in POI.Chapter Ⅲ. Variation of PRIM1 Gene in Chinese Patients with Primary Ovarian Insufficiency and Reproductive Phenotype Analysis in Prim1+/- MousePart Ⅰ. Variation Analysis of PRIM1 Gene in Chinese Patients with Primary Ovarian InsufficiencyOBJETIVE: A meta-analysis of 22 genome-wide association studies reported 13 novel loci associated with AANM, many of which have been replicated in other ethnicities. Candidate genes located at these loci are implicated in either DNA damage repair and replication or immune function. According to the Meta analysis, the nonsynonymous SNP rs2277339 in the PRIM1 gene is closely related to AANM. EM and POI in European women. During discontinuous DNA replication, PRIM1 (primase) plays a key role in the process of DNA synthesis initiation by synthesizing RNA primers for Okazaki fragments. However, the potential role of PRIM1 in POI pathogenesis has not been determined. Therefore, we examined 192 Han Chinese women with idiopathic POI by sequencing the coding region of PRIM1 gene to determine whether variants in this gene contribute to human POI.METHODS:Between June 2014 and September 2015, a number of 192 Han Chinese women with secondary amenorrhea were recruited. All the patients recruited were non-syndromic and idiopathic. Genomic DNA was extracted from peripheral blood samples. All the 13 exons and exon-intron boundaries of PRIM1 gene were sequenced. The sequence variants were confirmed by three independent PCR runs, followed by sequencing in both forward and reverse directions.RESULT: Three known SNPs, rs2277339 in exon 1, rs1131514 in exon 10, and rs1026565 in intron 6, were identified. Comparisons of genotype and allele frequencies showed no significant differences between POI cases and the general population. No plausible mutations were identified.CONCLUSION:Mutations in the coding region of PRIM1 are not common in Chinese women with POI. The exact role of PRIM1 in POI pathogenesis needs to be further explored in larger cohorts from Chinese and other ethnic populations.Part II.Reproductive Ability Analysis in Prim1+/- MouseOBJECTIVE: as a candidate gene in the POI, Priml was reported to be implicated in either DNA repair or replication according to the prior genome-wide association studies. It is functions as a primase in the process of DNA synthesis initiation by synthesizing RNA primers for Okazaki fragments. It involved the process of DNA repair but the functions in the oocyte not clear, the study aimed to construct Prim1 mutant mouse to investigate the phenotype of fertility and illustrate how C10ORF2 gene defects lead to the pathogenesis of POI.METHODS:Use the CRISPR/Cas9 technique to construct Prim1+/- mouse, after the mating test with wild type to ensure the reproductive capacity of Prim1+/- mouse, we keep the record of the offspring of every female mutant mouse, make ovarian tissue section with 3 weeks old mutant mouse to study the difference of oocyte at different levels.RESULT: Obviously there is no Prim1+/- offsprings after the mating of Prim1+/- mouse, E5, E1, E14, E16 days’embryos included, we guess the homozygous genes lead to an embryonic death in early days especially in the cleavage stage. But the Prim1+/-mouse shows a normal reproductive phenotype compared with the wild type.CONCLUSION:Priml gene participate in the DNA replication, impaired function of the gene are associated with the DNA repair pathway, the loss of the function would induce cell to death, theses study provided us the lethality of the systemic knockout gene in the embryogenesis, and showed us a further study with a purpose knockout in oocyte maturation process.更多还原