C10ORF2,HELQ和PRIM1基因在原发性卵巢功能不全中的作用和相关机制研究

发布时间：2018-05-05 04:29

本文选题：Perrault综合征 + C10ORF2　；参考：《山东大学》2016年博士论文

【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.1388GA(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.1388GA(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.1388GA(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.1388GA(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。
[Abstract]:Chapter 1 the role and mechanism of mitochondrial related gene C10ORF2 in the pathogenesis of familial primary ovarian dysfunction combined with sensorineural deafness (Perrault syndrome): the purpose of sequencing of the exon group of Perrault syndrome: primary ovarian dysfunction (POI) is a highly heterogeneous, complicated cause of the disease. Endocrine disorder, patients usually have amenorrhea before 40 years of age. The incidence of the disease is about 1%.. The disease is characterized by primary or secondary amenorrhea and with varying degrees of low estrogen symptoms. There is no reliable early diagnostic indicator and early and effective prevention of the occurrence and progress of the disease. It is very important to identify high risk people. Primary ovarian dysfunction is mainly divided into two types of syndrome type POI and non syndrome type POI, and multiple pathogenic genes are screened in two types. The found syndrome type POI includes crisp X syndrome, palpebral fissure syndrome (BPES), galactohyperglycemia, and carbohydrate deficiency Glycoprotein syndrome I, Perrault syndrome (PS) and so on. The pathogenetic genes of patients with Perrault syndrome, including HARS2, LARS2, C10ORF2, CLPP, and other patients with sensorineural deafness, were found in the Perrault syndrome by sequencing of the exon group. The female patients were accompanied by typical primary ovarian dysfunction. The whole exon group was sequenced and the other members of the family were sequenced in order to find a new POI pathogenic gene. Methods: the whole exon group was sequenced by Agilent SureSelect Human All Exon.V5 technique, and all the single base changes, insertion / deletion and public database were detected. The non synonymous mutation of the amino acid changes was detected and the candidate loci were screened according to the Mendel's law of heredity. Finally, the gene was screened according to the autosomal recessive way, and the common homozygous mutation site was screened in the patients. A missense mutation at the c.1388GA (p.R463Q) site of the C10ORF2 gene, which belongs to a new missense mutation that changes the amino acid from arginine to glutamine. The patient is a homozygous mutation and occurs in the line of the helicase of the.C10ORF2 gene of the encoded protein region of the protein. The replication of particles is essential. It is speculated that the changes in protein function caused by this mutation affect the mitochondrial function of the cells and lead to the occurrence of Perrault syndrome. Conclusion: the new missense mutation loci have been found in the spin region of the C10ORF2 gene by exons sequencing technology, and the discovery of the mutation is from the function of mitochondria. Research on the mechanism of POI provides a new direction. Second the mechanism of C10ORF2 gene in primary ovarian dysfunction. Objective: in the family of patients with sensorineural deafness (PS) with primary ovarian dysfunction, we found the missense mutation site c.1388 located on the C10ORF2 gene by the exon sequencing. GA (p.R463Q). In the previous study, it was found that the other amino acid changes at the same site resulted in the severe manifestation of the cerebellar cerebellar dysplasia in the infant. The.C10ORF2 gene encoded DNA helicase was involved in the process of mitochondrial replication in eukaryotic cells, and the mitochondria were regulated by the rich organelles in the granulosa and oocytes. The metabolism, cell cycle and signal transduction of granulosa cells and oocytes, but the relationship between the function of the gene and the ovary is not yet reported. This topic is to explore the pathogenesis of POI by C10ORF2 gene mutation. Method: by collecting the patients with Perrault syndrome. In the peripheral blood of the family, the peripheral blood DNA was extracted and the immortalized lymphocyte line of the C100RF2 gene c.1388GA (p.R463Q) mutation was established. The mitochondrial ATP production in the peripheral blood immortalized cells, the mitochondrial copy number and the formation of intracellular ROS were detected. The mitochondrial function changes caused by the mutation of the C10ORF2 gene were studied. The results were as follows: By extracting the peripheral blood of the patients and constructing immortalized lymphocytes, we found that the C10ORF2 gene mutation leads to the decrease of ATP in the cells and the increase of the ROS level, and the number of mitochondrial copies in the immortalized lymphocyte of the C10ORF2 gene mutation is obviously lower than that of the normal person. The changes seriously affect the activity of mitochondria in the cells and the level of oxidative phosphorylation, resulting in mitochondrial dysfunction. Conclusion: in the C10ORF2 gene, the c.1388GA (p.R463Q) mutation seriously affects the mitochondrial function of the cells. If this mutation occurs in the ovary, it will impede the normal energy metabolism of oocytes and granulosa cells in the egg nests. If the mitochondrial function is seriously defective, if the mitochondrial function is seriously defective, the decrease of ATP formation and the accumulation of ROS will lead to the death of the oocyte which can not be compensable. Therefore, the metabolic disorder in the mitochondria within the ovary caused by the C10ORF2 gene mutation may be one of the important mechanisms leading to the POI. The second chapter of the HELQ gene is in the original. Analysis of mutations in patients with ovarian dysfunction: the etiology of primary ovarian insufficiency (POI) is highly heterogeneous, and the cause of most patients is not yet clear. We found that POI, natural menopause age and premenopausal age have common genetic susceptibility, providing a new direction for the study of POI. Genomic association analysis found 13 sites associated with menopause age. In the subsequent bioinformatics analysis, 8 candidate genes were found to be associated with DNA damage repair. The HELQ gene had a high POI risk, so it was listed as an important candidate for POI. It encodes a protein HEL308, a DNA dependent ATP enzyme and DNA solution. The reparation of the interchain crosslinking reaction of DNA is involved with the RAD51 homologous protein. The repair of interchain crosslinking reaction requires the joint synergy of the S phase checkpoint of the cell division interval and the Vankoni anemia pathway to reduce the fertility of the.Helq-/- knockout mice, and the ovarian tissue section presents a obvious atrophy. The purpose of this study is to determine the mutation of the gene in the HELQ gene of the Han POI patients, and to provide a theoretical basis for the study of POI etiology and the guidance of clinical genetic counseling and reproductive guidance. Methods: to select 192 cases of POI patients of Han Chinese in the affiliated reproductive Hospital of Shandong University from 2012 to 2014. After the blood DNA was extracted, all the exons and exons of the HELQ gene were sequenced and the adjacent parts of the intron were sequenced. The results were compared with the normal population of the northern Han population in the database of China, and the genotype and allele frequencies of the SNP loci were analyzed by SPSS software. The results showed that 6 mononuclear nuclei were found in the exon sequencing of the HELQ gene. The two loci of rs1494961 and rs2047210 were statistically analyzed, and the genotype and allele frequencies were not statistically different from those of the control group. Rs13141136, rs7665103, rs11099600 and rs12645412 were statistically different from those of the control population, but they were synonymous changes. The coding sequence of HELQ gene is screened in patients with ovarian insufficiency, and the mutation of HELQ gene is not a common cause of primary ovarian insufficiency. Third the mutation analysis of the PRIM1 gene in the patients with primary ovarian insufficiency and the study of the gene knockout mice, the first part of the PRIM1 base in the patients with primary ovarian insufficiency Variable analysis objective: in recent years, more and more studies have found common genetic susceptibility between primary ovarian dysfunction and premenopause, which provides a new direction for exploring the pathogenesis of POI. A total of 13 sites associated with menopause age were found through the whole genome association analysis, including the PRIM1 gene.PRIM. The gene is not only closely related to the natural menopause age, and subsequent studies in the European population have found that the non synonymous polymorphic loci of the PRIM1 gene, rs2277339, and the early menopause and POI also have significant correlation with the.PRIM1 gene encoded DNA priming enzymes involved in the DNA replication process, and the synthesis of RNA primers to complete D by promoting the synthesis of RNA primers in the synthesis of kazaki fragments NA synthesis. The purpose of this study was to determine whether the mutation of the gene could lead to the occurrence of POI by measuring the PRIM1 coding sequence of 192 cases of POI patients in the Han nationality of China. Methods: 192 cases of POI patients who were diagnosed in the affiliated reproductive Hospital of Shandong University from 2014 to 2015 were selected as the research object, and the PRIM1 gene encoding was encoded by direct sequencing technology. The regional sequencing is the gene mutation screening, the results were compared with the normal Chinese northern population in the Ensemble database, and the genotype and allele frequencies of the SNP loci were statistically analyzed with SPSS software. Results: 3 known SNP loci rs2277339, rs1131514 and rs1026565 were screened in POI patients, respectively. The genotype and allele frequencies of the exons 1, exon 10 and intron 6 were not statistically different between the POI and the control group. Conclusion: the PRIM1 gene mutation is not common in the patients with primary ovarian dysfunction in Han Chinese. Although the rs2277339 loci in the European population are associated with primary ovarian dysfunction, but in the European population, There is no significance in the Chinese Han POI women. It is possible that the role of ethnic differences or less samples of this study leads to the further study of the role of the.PRIM1 gene in the pathogenesis of POI. Second the objective of Fertility Analysis in Prim1 knockout mice: the genetic study of primary ovarian dysfunction requires not only genomics. The study provides a basis for finding new pathogenicity sites, and it is more necessary to explore the biological support of animal experiments in the process of POI pathogeny. Therefore, in order to assess the physiological role of the PRIMl gene in the body, we constructed a Prim1 gene knockout mouse to investigate whether the deletion of the gene affects mice. Growth and reproduction function. Methods: CRISPR/Cas9 technique was used to find the Cas9 point of the target gene, design and construct the corresponding gRNA sequence. After testing its nucleic acid endonuclease activity, the mouse single cell embryo was injected with Cas9-mRNA, and the F1 generation heterozygote Prim1+/- mouse was obtained after breeding. The subgeneration subalgebra and the progeny genotypes were recorded by the cage. The effect of Prim1 gene deletion on the reproductive system in mice. Results: no homozygote Primr1-/- mice were born after Prim1+/- mice were caged. In the study of heterozygote Prim1+/- mice, the subalgebra, the size of the ovary and the number of oocytes at all levels in the ovary There was no significant difference in the birth type mice. Conclusion: there was no significant difference in fertility between the Prim1+/- mice and the wild type mice. No Prim1 homozygote offspring was born, indicating that the deletion of the gene may seriously affect the cell cycle and the process of DNA damage repair and lead to the apoptosis of the cells, causing the death of the Prim1-/- mouse embryo in the future. In the study, conditional Prim1 knockout mice could be considered to study the effect of the gene on ovarian function.

【学位授予单位】：山东大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R711.75

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