肌萎缩侧索硬化的基因突变研究

发布时间：2018-04-13 04:37

本文选题：肌萎缩侧索硬化 + 基因突变　；参考：《北京协和医学院》2016年硕士论文

【摘要】：背景肌萎缩侧索硬化(amyotrophic lateral sclerosis, ALS)是一种致死性的神经退行性疾病,通常累及大脑皮质、脑干和脊髓前角的上、下运动神经元,导致肌肉无力和萎缩、言语困难及呼吸功能障碍。目前,全世界已发现的ALS致病基因主要有30余个,SOD1、FUS、TARDBP和C9ORF72基因最为常见。其中,C9ORF72基因非编码区的六核菅酸重复扩增突变GGGGCC (G4C2)是西方国家ALS与额颞叶痴呆(frontotemporal dementia, FTD)的最主要原因。此外,ATXN2基因编码区的三核苷酸(CAG)n重复扩增也是ALS发病的风险因素之一。目的(1)应用高通量测序技术检测ALS目标基因突变,明确ALS患者常见的突变基因及其突变率。(2)检测C9ORF72基因的非编码区G4C2突变和编码区突变,初步分析C9ORF72基因在ALS中发挥的作用。(3)明确ATXN2, ATXN3.,, AR基因编码区和ATXN8基因非编码区的(CAG)n重复扩增与ALS是否有关。方法(1)应用Ion Torrent测序平台进行靶向重测序,目标基因panel包括26个ALS明确致病基因及相关易感基因的全部外显子区域。筛选ALS候选致病基因突变及初步分析其致病性,明确常见突变基因及突变频率。(2)采用Repeat-primed PCR扩增C9ORF72基因G4C2重复扩增区域,使用AB13730 DNA Analyzer仪器和GeneMapper软件分析G4C2重复次数。通过体内cDNA测序和体外Minigene检测两种方法确定C9ORF72基因的剪接位点变异c.601-2AG的致病性。(3)扩增ATXN2、ATXN3、ATXN8和AR基因的(CAG)n重复扩增区域,确定(CAG)n在ALS患者和健康对照中的重复次数,通过统计学方法分析(CAG)n重复扩增突变与ALS是否有关。结果(1)我们在7例FALS患者(38.89%,7/18)和33例SALS患者(18.54%,33/178)中检测到36种基因突变类型,包括1个剪接位点突变,1个无义突变和34个错义突变。FALS患者携带的基因突变分布在ALS明确致病基因SOD1、UBQLN2和VAPB中,其中突变频率最高的基因为SOD1(27.78%)。SALS患者中14例(7.87%,14/178)携带ALS明确致病基因的突变,SOD1突变频率最高。其余19例SALS患者(10.67%,19/178)携带ALS相关基因的突变,其中频率最高的是NEFH(3.93%)。(2)我们发现2例SALS患者(0.79%,2/252)携带C9ORF72基因的G4C2重复扩增突变(大于30次)。统计学分析结果显示该重复扩增突变在中国ALS患者和健康对照中的分布情况没有显著性差异(p=0.805)。此外,我们发现了C9ORF72基因的一个剪接位点突变(c.601-2AG)。体内cDNA测序及体外MiniGene实验结果均显示出该突变破坏了第4内含子中的剪接受体位点,使第5外显子中隐匿剪接受体位点激活,可能导致成熟mRNA中缺失四个核苷酸(c.601_604 del ATAG),读码框发生移位并提前出现终止密码(p.I201fsX235); cDNA测序结果还显示出无义介导的mRNA降解(Nonsense-Mediated mRNA Decay, NMD)。该突变为C9ORF72基因编码区突变的首次发现,也为Loss-of-Function致病机制提供新证据。(3)我们发现ATXN2基因的(CAG)。长片段重复(≥32次)在ALS患者和正常人中分布有显著性差异(p=0.028),而中间片段(24-31次)没有(p=0.839)；ATXN8基因的(CAG)n中间片段(29-67次)和ALS疾病有关(p=0.0130)；ATXN3和AR基因的长片段、中间片段和短片段重复均和ALS无关。结论(1)本研究发现FALS和SALS患者中突变频率最高的基因均为SOD1,FALS和SLAS突变谱存在明显差异。(2) C9ORF72基因的六核苷酸重复扩增突变G4C2不是中国ALS患者的常见基因突变类型。(3)发现一个C9ORF72基因剪接位点突变c.601-2AG,为ALS的Loss-of-Function致病机制增加新证据。(4) ATXN2基因的(CAG)n长片段(≥32次)和ATXN8基因的中间片段(29-67次)可能与ALS有关。ATXN3和AR基因的(CAG)n突变与ALS无关。
[Abstract]:The background of amyotrophic lateral sclerosis (amyotrophic lateral, sclerosis, ALS) is a fatal neurodegenerative disease, usually involving the cerebral cortex, brainstem and spinal cord anterior horn, motor neurons, leading to muscle weakness and atrophy, speech difficulties and respiratory dysfunction. At present, there are more than 30 ALS, the main pathogenic gene the whole world has been found in SOD1, FUS, TARDBP and C9ORF72 genes are the most common. Among them, six non nuclear acid C9ORF72 gene encoding region Kan repeat amplification mutation GGGGCC (G4C2) in western countries, ALS and frontotemporal dementia (frontotemporal dementia, FTD) of the main reason. In addition, the ATXN2 gene encoding region of trinucleotide (CAG one of the risk factors of n repeat amplification) is the pathogenesis of ALS. (1) ALS target detection using high-throughput sequencing gene mutation, clear common in patients with ALS mutation and mutation rate. (2) the detection of C9ORF72 gene Mutations encoding G4C2 mutation and encoding region, preliminary analysis of the C9ORF72 gene play a role in ALS. (3) clear ATXN2, ATXN3., AR, encoding gene and ATXN8 gene in non encoding region (CAG) of N and ALS. If the repeat amplification method (1) using Ion Torrent sequencing platform targeting weight sequencing of target gene panel, including all 26 ALS specific virulence genes and susceptibility gene exon. Screening and mutation of ALS candidate genes to analyze its pathogenicity, clear common mutation gene and mutation frequency. (2) by Repeat-primed PCR amplification of C9ORF72 gene G4C2 repeat amplification region, the use of AB13730 DNA Analyzer instrument and GeneMapper software analysis of G4C2 repeat number. To determine the pathogenicity of c.601-2AG splice site variants of the C9ORF72 gene by cDNA sequencing in vivo and in vitro Minigene two methods. (3) ATXN3, ATXN8 and ATXN2 amplification, AR based Because of the (CAG) n repeat amplification region (CAG), to determine the number of n repeats in ALS patients and healthy controls, through statistical analysis (CAG) n repeat amplification and ALS mutation is related. Results (1) in 7 FALS patients (38.89%, 7/18) and 33 SALS patients (18.54%, 33/178) was detected in 36 mutations, including 1 splice site mutations, 1 nonsense mutations and 34 missense mutations in patients with.FALS gene mutation distribution in ALS clear pathogenic gene SOD1, UBQLN2 and VAPB, the mutation frequency was the highest in SOD1 (27.78%).SALS patients in 14 cases (7.87%, 14/178) mutations in the ALS gene SOD1 mutation clearly, the highest frequency. The remaining 19 cases of patients with SALS (10.67%, 19/178) mutations in the ALS gene, which is the highest frequency of NEFH (3.93%). (2) we found 2 cases of SALS patients (0.79%, 2/252) with C9ORF72 gene G4C2 repeat amplification process Change (above 30). Statistical analysis showed no significant difference between the repeat amplification mutation distribution in Chinese in ALS patients and healthy controls (p=0.805). In addition, we found a splice site mutation of C9ORF72 gene (c.601-2AG) in vivo and in vitro. CDNA sequencing results of MiniGene showed the mutation destroy the fourth intron splice acceptor site, the exon fifth splice acceptor sites in occult activation may lead to mature mRNA four nucleotide deletion (c.601_604 del ATAG), reading frame shift and advance the termination codon (p.I201fsX235); cDNA sequencing results also showed no degradation of mRNA mediatedrna (Nonsense-Mediated mRNA, Decay, NMD). The mutation for the encoding region of C9ORF72 gene was found for the first time, but also provide new evidence for the pathogenesis of Loss-of-Function. (3) we found that the ATXN2 gene (CAG). Repeat fragment (over 32 times) in ALS patients and normal people in the distribution had significant difference (p=0.028), and the middle segment (24-31) no (p=0.839); ATXN8 gene (CAG) n middle segment (29-67) and ALS disease (p=0.0130); long ATXN3 and AR gene. The middle and short fragments repeat and ALS were unrelated. Conclusion (1) this study found that FALS and SALS in patients with the highest frequency of mutations were SOD1, FALS and SLAS mutation spectrum had obvious difference. (2) C9ORF72 gene six nucleotide repeat amplification gene mutations of G4C2 is not China patients with ALS mutation type. (3) found a C9ORF72 gene splice site mutation c.601-2AG, adding new evidence for the pathogenesis of ALS. Loss-of-Function (4) gene ATXN2 (CAG) n long fragment (over 32 times) the middle fragment and ATXN8 gene (29-67) may be associated with ALS.ATXN3 and AR (CAG) gene N mutation with no ALS Close.

【学位授予单位】：北京协和医学院
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：R744.8

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