一精神分裂症家系致病基因克隆的初步研究

发布时间：2018-06-07 06:17

本文选题：精神分裂症 + 全基因外显子组测序　；参考：《中南大学》2013年硕士论文

【摘要】：背景：精神分裂症是一类最为常见的严重的精神疾病,其发病率在1%左右,主要特征表现为思维、情感与行为障碍以及精神活动与环境的不协调。在以往精神分裂症易感基因的研究中,采用的主要方法包括连锁分析和关联研究两种。但传统的实验方法存在时间长、操作繁琐、人力物力损耗大等缺点,最近发展起来的外显子组测序(whole-exome sequencing,WES)能够在较短的时间内检测出患者基因组所有外显子及其侧翼序列中的微小序列变异,无论是普通变异还是罕见变异都表现出很高的灵敏度,为克隆疾病的致病基因提供了新的研究策略。目的：对一精神分裂症家系的2名患者行外显子组测序,结合全基因组连锁分析和全基因组CNV芯片分析的结果,以期寻找和克隆其致病基因。方法：在前期工作中,我们收集了一来自湖南的精神分裂症家系,包括3代成员,有患者8人,遗传模式符合常染色体显性遗传或X染色体显性遗传。(1)选取家系内两名患者(Ⅱ：4、Ⅲ：1)和一名家系内非患者(Ⅱ：3)行全基因组CNV芯片分析,通过查询DGV数据库(Database of Genomic Variants),确定有无新发CNV变异。(2)将家系内采集到血标本的12名成员(包括6名患者,6名家系内非患者)的gDNA行全基因组扫描,分别按照常染色体显性遗传和X染色体显性遗传进行连锁分析。(3)选取家系内2名患者(Ⅱ.3、Ⅲ：3)进行外显子组测序,先过滤掉同义突变、公共遗传突变数据库正常人携带的突变以及纯和突变,然后在2名患者变异集合中取交集,再通过连锁定位区间进一步减少候选致病基因变异的数量。(4)通过查询UCSC、OMIM、Uniprot等数据库对候选致病基因的生物信息学进行分析。(5)采用Western blotting的方法从蛋白水平检测候选致病基因X在该精神分裂症家系患者和非患者、正常对照的血浆中是否存在表达差异。结果：(1)全基因组CNV芯片分析的结果显示46个两名患者带有而家系内非患者不带有的CNV变异均为非致病性多态改变,排除了CNV在该家系致病的可能。(2)通过全基因组连锁分析,分别在4号染色体rs2014158和rs335077之间(4q32.2-34.3)、7号染色体rs1024516和rs2056865之间(7q21.11-31.2)的区域取得连续分布正LOD值区间。4号染色体区间在4q34.3处取得最高LOD值2.017,7号染色体区间在7q22.3处取得最高LOD值2.017。(3)通过外显子组测序,发现两名患者共同带有的杂合变异共410个(包括387个SNPs和23个Indels),位于连锁分析定位区间4q32.2-34.3和7q21.11-31.2的变异共有6个,其中位于7q21.11-31.2区间内的2个变异SLC25A13(c.1889TG)和X(c.9575CG),在家系内与临床表型完全共分离。(4)通过查询文献、数据库等对SLC25A13基因及X基因的生物信息进行分析,提示变异SLC25A13(c.1889TG)为一罕见多态的可能性大,而X基因为散发精神分裂症的已知的易感基因。(4)Western blotting的结果显示血浆中X蛋白的表达量在该家系内患者与非患者及正常对照间未见明显差异。结论：(1)通过全基因组扫描连锁分析,将该精神分裂症家系的致病基因定位到染色体4q32.2-34.3和7q21.11-31.2。(2)通过外显子组测序,发现X基因可能为该精神分裂症家系的候选致病基因。
[Abstract]:Background: schizophrenia is one of the most common serious mental diseases. Its incidence is about 1%. The main features are thinking, emotional and behavioral disorders and incoordination between mental activity and the environment. In the previous study of susceptibility genes of schizophrenia, the main methods used include two types of linkage analysis and association studies. The experimental methods have long time, complicated operation and great loss of human and material resources. The recently developed exons sequence (whole-exome sequencing, WES) can detect the small sequence variation in all exons and flanking sequences of the patient genome in a short time, whether common variation or rare mutation table High sensitivity has provided a new research strategy for the pathogenic genes of cloned diseases.
Objective: 2 patients with schizophrenia were sequenced by exons, combined with the results of whole genome linkage analysis and whole genome CNV chip analysis, in order to find and clone the pathogenic genes.
Methods: in the previous work, we collected a schizophrenic family from Hunan, including 3 generations and 8 patients. The genetic pattern accords with autosomal dominant inheritance or X chromosome dominant inheritance. (1) two patients in the family (II: 4, III: 1) and a family non patient (II: 3) were selected to analyze the whole genome CNV chip analysis. DGV database (Database of Genomic Variants) was querying to determine whether there were new CNV variants. (2) complete genomic scans of 12 members of the family (including 6 patients, 6 family non patients) were collected in the family line, and the linkage analysis was carried out according to the autosomal dominant inheritance and the dominant inheritance of X chromosome. (3) 2 in the family system were selected. Patients (II.3, 3) were sequenced exons, first filtering out synonymous mutations, mutation and pure and mutation of normal people in the common genetic mutation database, and then intersecting in 2 patients' variation sets, and further reducing the number of candidate gene mutations through the linkage location interval. (4) by querying UCSC, OMIM, Uniprot and so on The bioinformatics of the candidate genes were analyzed by the library. (5) Western blotting was used to detect the difference in the expression of the candidate gene X in the plasma of the schizophrenic family and non patients.
Results: (1) the total genomic CNV chip analysis showed that 46 two patients had non pathogenic CNV mutations in the family non patients and excluded the possibility of CNV in the family. (2) through the whole genome linkage analysis, between rs2014158 and rs335077 (4q32.2-34.3) and chromosome 7, chromosome 4, chromosome 7, respectively. The region between rs1024516 and rs2056865 (7q21.11-31.2) obtained the continuous distribution of the positive LOD range, the interval of chromosome.4, the highest LOD value of the LOD value 2.017,7, the maximum LOD value of the LOD value 2.017. (3) was sequenced by exons, and two patients were found to have a total of 410 heterozygous variations (including 387 SNPs and 23). Indels), there were 6 variations in 4q32.2-34.3 and 7q21.11-31.2, of which 2 variations of SLC25A13 (c.1889TG) and X (c.9575CG) in the 7q21.11-31.2 interval were completely separated from the clinical phenotype in the family. (4) the biological information of the SLC25A13 and X genes was analyzed by query literature, database and so on. It is suggested that the variant SLC25A13 (c.1889TG) is a rare polymorphism, and the X gene is a known susceptibility gene for sporadic schizophrenia. (4) the result of Western blotting shows that the expression of X protein in the plasma is not significantly different between the patients in the family and the non patients and the normal controls.
Conclusion: (1) through the whole genome scan linkage analysis, the pathogeny genes of the schizophrenic family were located to chromosome 4q32.2-34.3 and 7q21.11-31.2. (2) by exon sequencing. It was found that the X gene might be a candidate gene for the schizophrenia family.
【学位授予单位】：中南大学
【学位级别】：硕士
【学位授予年份】：2013
【分类号】：R749.3

【共引文献】