非综合征型视网膜色素变性家系PRPF31基因突变的筛查及功能鉴定

发布时间：2018-05-01 12:08

本文选题：视网膜色素变性 + PRPF31　；参考：《郑州大学》2017年硕士论文

【摘要】：研究背景和目的视网膜色素变性(Rentinitis pigmentosa,RP,OMIM:268000)是光感受器细胞(包括视杆细胞和视锥细胞)异常而导致的遗传性视网膜疾病。RP在世界范围内的发病率约为1/5000-1/3500;在我国,RP的发病率也在逐年上升,约为l/l000,是引起失明的重要原因之一。根据是否同时伴随其他眼部症状,RP可以分为综合征型视网膜色素变性(Syndromic retinitis pigmentosa)和非综合征型视网膜色素变性(Nonsyndromic retinitis pigmentosa,NSRP),其中NSRP约占RP的65%。RP的发病机制异常复杂,涉及多种不同的生物代谢途径。其致病基因所编码的蛋白主要参与光传导、感光细胞结构的维持以及m RNA剪接等过程。不同生物途径中各种蛋白的编码基因突变而使蛋白功能受损,都可能导致光感受器细胞的异常,引起RP的发生。2012年,许菲等在一个常染色体显性遗传RP家系的研究中发现PRPF31基因的7号外显子上有一个c.544_618del75bp的新发突变。但是,许菲等的研究选取的家系并不完整,只是该家族大家系中的一小部分;另外由于RP具有极强的临床和遗传异质性,可能存在新的尚未发现的致病突变位点;并且许菲等的研究没有对该c.544_618del75bp缺失突变具体机制和功能进行深入探索。因此,我们对该家系进行补充和完善,对许菲的研究结果在整个大家系中进行验证,同时寻找新的致病突变,进一步阐明基因型和表型的关系,并对c.544_618del75bp缺失突变的功能进行初步研究,为RP的分子遗传机制及临床诊断和治疗提供理论依据。研究对象对许菲等研究的RP家系进行补充和完善,并对该家系成员进行了详细的病史采集和眼科检查,对家系内患者进行了确诊。详细了解该家系成员的信息后绘制家系系谱。抽取该家系成员外周静脉血5mL。此外,随机选取100名健康个体作为正常对照,抽取外周血5mL。本研究通过了郑州大学伦理委员会的审批,所有受试者均知情同意。研究方法1.本研究采用全血基因组提取试剂盒提取了外周血基因组DNA,对该RP家系成员提取了RNA,并反转录成cDNA。2.对该家系中全部成员和100名健康对照通过sanger测序进行PRPF31基因c.544_618del75bp突变位点的测序筛查验证,鉴定该基因型与表型在家系中的共分离。并且对家系内患者的基因组cDNA进行c.544_618del75bp突变位点的sanger测序验证。3.对该突变位点进行生物信息学功能分析,并通过SWISS MODEL软件预测野生型和突变型PRPF31基因编码蛋白的三维结构。4.采用实时荧光定量RT-PCR技术检测该家系中患者和正常人的外周血中PRPF31基因mRNA的表达水平。构建携带野生型和突变型PRPF31基因的过表达载体并采用质粒提取试剂盒进行提取和纯化,其后转染293T细胞,通过Western blot检测野生型和突变型基因编码蛋白在293T细胞内是否有表达。运用RT-PCR技术在转染野生型和突变型过表达载体的293T细胞中检测PRPF31基因mRNA的表达水平,验证突变对PRPF31基因功能的影响。5.通过查阅文献及The Human Protein Atlas数据库筛选和mRNA剪切相关并且在人类外周血中有表达的RP9、ROM1、SNRNP200和TOPORS等基因。运用实时荧光定量RT-PCR技术在该家系患者和正常人的外周血中检测这些基因mRNA的表达水平,并在转染野生型和突变型过表达载体的293T细胞中验证这些基因的表达情况。6.采用SPSS21.0统计软件进行数据分析。家系内患者与健康对照之间mRNA的表达水平采用独立样本t检验进行统计分析,所有定量资料用均数±标准差(mean±SD)表示,运用Bivariate相关性分析,对家系内所有成员中PRPF31基因的mRNA表达水平与其相关基因的mRNA表达水平分别进行相关性分析。P0.05具有统计学意义。结果1.该家系内大部分患者都在10岁前发病,均以夜盲为首发症状,伴有视力有下降、视野缺损等症状,眼底检查表明视盘颜色相对正常,但有不同程度的视网膜色素细胞萎缩。2.经过sanger测序发现所有患者的DNA和cDNA中都携带有PRPF31基因c.544_618del75bp突变。除1例外显不全成员外,家系内正常人和100例健康对照中则没有检测到该突变。本研究结果与许菲等人的研究结果一致。此外,我们在PRPF31基因上发现了一个IVS6-78_IVS6-75del4CACA的缺失突变,但所有患者中均未发现该突变。这两个缺失突变同时存在于家系中的1例外显不全的成员中,并且位于不同的染色体上。家系内正常人中IVS6-78_IVS6-75del4CACA缺失突变发生率为31.4%,且都为杂合突变。100例健康对照有38%的人有IVS6-78_IVS6-75del4CACA突变并且均为杂合突变,其等位基因的频率为21.5%。3.利用SWISS MODEL对c.544_618del75bp突变型PRPF31基因所编码的蛋白进行三维结构的预测,结果提示蛋白的结构发生了比较明显的缺失改变。利用Mutation Taster在线软件对PRPF31基因上的c.544_618del75bp突变位点和IVS6-78_IVS6-75del4CACA突变位点进行功能预测,结果显示c.544_618del75bp缺失是一种致病突变,能够造成氨基酸序列的改变和剪切位点的改变,进而可能造成蛋白质结构的改变。而IVS6-78_IVS6-75del4CACA位点能够使剪切位点发生改变,可能改变蛋白质的结构。4.通过实时荧光定量RT-PCR技术在家系内16例患者与26例正常对照之间对PRPF31基因mRNA表达水平进行比较,发现患者外周血中PRPF31基因的mRNA表达水平(0.65±0.40)显著低于正常对照(1.35±1.15),差异有统计学意义(P0.05)。构建携带野生型和突变型PRPF31基因的过表达载体并转染293T细胞后,Western blot检测到突变型和野生型PRPF31的过表达载体在293T细胞内能够正常表达,采用实时荧光定量RT-PCR检测PRPF31基因的mRNA表达水平,发现野生型和突变型PRPF31基因转染组PRPF31基因mRNA表达水平显著高于阴性对照组(P0.001),且野生型PRPF31基因的mRNA表达水平显著高于突变型(P0.001)。5.采用实时荧光定量RT-PCR在家系内16例患者与26例正常对照之间检测RP9、ROM1、SNRNP200和TOPORS等基因的mRNA的表达水平,发现患者外周血中RP9和ROM1基因mRNA的表达水平(分别是0.52±0.34和0.79±0.67)显著低于正常对照(分别是1.50±1.13和1.74±1.72),差异有统计学意义(P0.05)。运用Bivariate相关性分析,对家系内39例成员(16例患者与26例正常对照者)的PRPF31基因的mRNA表达水平与RP9、ROM1基因的mRNA表达水平分别进行相关性分析,结果显示:PRPF31基因与RP9基因的mRNA表达水平呈显著的正相关(r=0.71,P=0.000)。在转染后的293T细胞中对RP9、ROM1、SNRNP200和TOPORS基因的表达水平进行体外验证,发现突变型PRPF31转染组中RP9的表达水平低于野生型转染组,但是差异没有统计学意义。结论1.PRPF31基因的杂合突变c.544_618del75bp可能是该视网膜色素变性家系的致病突变,而IVS6-78_IVS6-75del4CACA缺失突变可能是一个多态位点。2.PRPF31基因c.544_618del75bp致病突变能够降低该基因的mRNA表达水平,这可能是PRPF31基因c.544_618del75bp突变导致RP发生的重要机制。3.PRPF31基因c.544_618del75bp突变能够使ADRP相关基因RP9的表达水平显著降低,表明PRPF31基因的c.544_618del75bp突变可能通过影响RP9的正常功能导致RP的发生。
[Abstract]:Background and objective retinal pigment degeneration (Rentinitis pigmentosa, RP, OMIM:268000) is an abnormal genetic retinal disease caused by the abnormalities of photoreceptor cells (including rod cells and cone cells) and the incidence of.RP in the world is about 1/5000-1/3500; in China, the incidence of RP is also rising year by year, about l/l000, which is a cause of loss. One of the important reasons of Ming is that RP can be divided into Syndromic retinitis pigmentosa (retinitis pigmentosa) and non syndrome type retinal pigment degeneration (Nonsyndromic retinitis pigmentosa, NSRP) according to whether it is accompanied by other ocular symptoms at the same time. The same biological metabolic pathway. The protein encoded by its pathogenic gene mainly participates in the process of light conduction, the maintenance of photosensitive cell structure and the splicing of M RNA. The mutation of the encoding genes of various proteins in different biological pathways may cause the damage of the protein function, which may cause the abnormal of the photoreceptor cells, cause the occurrence of RP in.2012 years, and so on. In the study of an autosomal dominant hereditary RP family, a new mutation of c.544_618del75bp was found in exon 7 of the PRPF31 gene. However, the studies selected by xanphi were not complete, only a small part of the family family. In addition, because of the strong clinical and genetic heterogeneity of RP, there may be new yet new ones. The findings of the found mutation site, and the study of Xu Fei, did not explore the specific mechanism and function of the c.544_618del75bp deletion mutation. Therefore, we supplemented and perfected the family. The results of the study were verified in the whole family, and a new pathogenic mutation was found, and the genotypes and tables were further clarified. The function of c.544_618del75bp deletion mutation was preliminarily studied in order to provide a theoretical basis for the molecular genetic mechanism and clinical diagnosis and treatment of RP. The research subjects supplemented and perfected the RP families of the study of Xu Fei, and carried out a detailed history collection and ophthalmology examination for the family members of the family. The family members of the family were given a detailed understanding of the family genealogy. In addition to the peripheral venous blood 5mL. of the family members, 100 healthy individuals were selected as normal controls, and the 5mL. of the peripheral blood was selected for examination and approval of the Zhengzhou University ethics committee. All the subjects were informed consent. The study method was adopted in 1. studies. The whole blood genome Extraction Kit extracted genomic DNA from peripheral blood, extracted RNA from the members of the RP family, and reverse transcriptional cDNA.2. to all members of the family and 100 healthy controls by sequencing the c.544_618del75bp mutation site of the PRPF31 gene by Sanger sequencing to identify the genotype and phenotype in the family. And the Sanger sequencing of the c.544_618del75bp mutation site of the genome cDNA in the family of families verifies that.3. has a bioinformatics function analysis on the mutation site, and the SWISS MODEL software is used to predict the three dimensional structure of the wild and mutant PRPF31 gene encoding proteins by the real-time fluorescent quantitative RT-PCR technology for the detection of the family. The expression level of PRPF31 gene mRNA in the peripheral blood of the patients and normal people. The overexpression vector carrying the wild type and the mutant PRPF31 gene was constructed and the Plasmid Extraction Kit was used to extract and purify it. Then the 293T cells were transfected, and the Western blot was used to detect whether the wild type and mutant gene encoded proteins were in 293T cells. The expression level of PRPF31 gene mRNA was detected by RT-PCR technique in 293T cells transfected with wild type and mutant overexpressed vector, and the effect of mutation on the function of PRPF31 gene was verified by screening the literature and The Human Protein Atlas database screening and mRNA shear correlation and expressed in human peripheral blood. P200 and TOPORS genes. The expression of these genes was detected in the peripheral blood of the family and normal people by real time fluorescence quantitative RT-PCR, and the expression of these genes was verified in the 293T cells transfected with wild type and mutant overexpressed vector..6. was used for data analysis by SPSS21.0 statistics software. The expression level of mRNA was statistically analyzed by independent sample t test, and all quantitative data were expressed with mean mean + standard deviation (mean + SD), and Bivariate correlation analysis was used to analyze the mRNA expression level of PRPF31 gene in all members of the family and the mRNA expression level of related genes respectively. 05 the results were statistically significant. 1. most of the patients in the family were onset before the age of 10, with night blindness as the first symptom, accompanied by visual loss, visual field defect and other symptoms. Fundus examination showed that the color of the optic disc was relatively normal, but a different degree of retinal pigment cell atrophy.2. was found in all patients' DNA and cDNA through Sanger sequencing. The PRPF31 gene c.544_618del75bp mutation was carried. The mutation was not detected in normal people in the family and in 100 healthy controls except for 1 exceptions. The results of this study were in accordance with the results of Xu Fei et al. In addition, we found a IVS6-78_IVS6-75del4CACA deletion mutation in the PRPF31 gene, but all the patients were found to have a deletion mutation. The two deletion mutations were found in 1 exceptions in the family and on different chromosomes. The incidence of IVS6-78_IVS6-75del4CACA deletion mutations in normal families was 31.4%, and all of the.100 cases with heterozygous mutations had a IVS6-78_IVS6-75del4CACA mutation in 38% of the healthy controls. A heterozygous mutation, the frequency of its allele is 21.5%.3. using SWISS MODEL to predict the three-dimensional structure of the protein encoded by the c.544_618del75bp mutant PRPF31 gene. The results suggest that the structure of the protein has a distinct deletion change. Mutation Taster online software is used for the c.544_618del75bp process on PRPF31 gene. The functional prediction of variable sites and IVS6-78_IVS6-75del4CACA mutation sites shows that c.544_618del75bp deletion is a kind of pathogenic mutation, which can cause changes in the amino acid sequence and the shear site, and may cause the change of protein structure. The IVS6-78_IVS6-75del4CACA site can change the shear site and may be possible to change the shear site. The structure of protein.4. was compared with the mRNA expression level of the PRPF31 gene between 16 patients in the family and 26 normal controls by real-time fluorescence quantitative RT-PCR. It was found that the mRNA expression level of the PRPF31 gene in the peripheral blood of the patients was significantly lower than that of the normal control (1.35 + 1.15), and the difference was statistically significant (P0.05). After carrying the overexpressed vector of the wild type and mutant PRPF31 gene and transfecting 293T cells, Western blot detected that the overexpressed vector of mutant and wild type PRPF31 could be expressed normally in 293T cells. The mRNA expression level of PRPF31 gene was detected by real-time quantitative RT-PCR, and PR and mutant PRPF31 gene transfection group PR was found. The expression level of mRNA in the PF31 gene was significantly higher than that in the negative control group (P0.001), and the mRNA expression level of the wild type PRPF31 gene was significantly higher than that of the mutant type (P0.001).5. using real-time fluorescent quantitative RT-PCR in 16 patients in the family and 26 normal controls to detect the expression level of RP9, ROM1, SNRNP200, and TOPORS. The expression level of RP9 and ROM1 gene mRNA in blood (0.52 + 0.34 and 0.79 + 0.67 respectively) was significantly lower than that of normal controls (1.50 + 1.13 and 1.74 + 1.72 respectively). The difference was statistically significant (P0.05). The mRNA expression level of the PRPF31 gene in 39 members of the family (16 patients and 26 normal controls) by Bivariate correlation analysis and RP9, RO The correlation analysis of the mRNA expression level of the M1 gene showed that the mRNA expression level of the PRPF31 gene was positively correlated with the mRNA expression level of the RP9 gene (r=0.71, P=0.000). The expression level of RP9, ROM1, SNRNP200 and TOPORS genes in the transfected 293T cells was verified in vitro, and the expression level of the mutant transfection group was found. It is lower than the wild type transfection group, but the difference is not statistically significant. Conclusion the heterozygous mutation c.544_618del75bp of the 1.PRPF31 gene may be a pathogenic mutation of the retina pigmented family, and the IVS6-78_IVS6-75del4CACA deletion mutation may be a polymorphic locus of the.2.PRPF31 gene c.544_618del75bp pathogenic mutation to reduce the mR of the gene. The expression level of NA, which may be an important mechanism of the mutation of the PRPF31 gene c.544_618del75bp, causes the.3.PRPF31 gene c.544_618del75bp mutation to significantly reduce the expression level of the ADRP related gene RP9, indicating that the c.544_618del75bp mutation of the PRPF31 gene may lead to the occurrence of RP by affecting the normal function of RP9.

【学位授予单位】：郑州大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R774.13

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