肌萎缩侧索硬化的遗传学和基础研究

【作者】 邹漳钰；

【导师】 崔丽英；

【作者基本信息】 北京协和医学院 ， 神经病学， 2013， 博士

【摘要】 第一部分中国肌萎缩侧索硬化患者的遗传学研究背景：肌萎缩侧索硬化(ALS)是一种由于大脑皮质、脑干和脊髓前角的上下运动神经元进行性变性,导致肌肉无力、萎缩,言语、吞咽、呼吸功能障碍的神经变性疾病。迄今为止,全世界已经发现了17种与ALS相关的突变基因。其中C9orf72基因、铜/锌超氧化物歧化酶1(SOD1)基因、反式激活反应-DNA结合蛋白(TARDBP)基因和肉瘤熔合(FUS)基因被认为是ALS最常见的4种突变基因。目的：本研究旨在对一个大样本的中国ALS患者进行系统的遗传学研究,明确我国ALS患者常见的突变基因及其突变率,并系统回顾国内外有关ALS患者SOD1、ANG、 TARDBP、FUS、C9orf72基因突变的研究,进行基因突变型—临床表型分析。方法：提取北京协和医院神经科就诊的324例SALS患者和20个家族性ALS(FALS)家系,以及255例健康对照的全基因组DNA,对SOD1基因全部5个外显子,ANG基因第2号外显子,TARDBP基因第2-6号外显子,FUS基因第3-6、12-15号外显子,VCP基因第4、5、10、14号外显子,PFNl基因全部3个外显子进行PCR扩增；对20例FALS先证者,除以上外显子外,还扩增FUS基因其他7个外显子,VCP基因其他13个外显子,PCR产物直接测序。使用重复引物聚合酶链反应扩增C9orf72基因非编码外显子1a和1b间的内含子上的GGGGCC核苷酸六聚体重复序列,产物行毛细管电泳分析产物荧光片断长度,确定重复序列数目。检索国内外对ALS患者进行SOD1、ANG、TARDBP、FUS、C9orf72基因突变筛查的研究,计算各研究的基因突变率,汇总后分别计算不同国家、亚欧北美三大洲及全世界ALS患者中这些基因的突变率,并结合文献进行不同基因突变型—临床表型的关联分析。结果：在FALS中发现5例SOD1突变和2例FUS突变,在SALS中发现6例FUS突变、3例SOD1突变,3例TARDBP突变和1例ANG突变,所有患者均未发现C9orf72突变、VCP突变和PFN1突变。除ANG基因的p.V103I突变为新突变外,其余突变国内外文献均有报道。4例携带FUS基因的SALS患者表现为特殊的少年型ALS,其中1例p.G504Wfs*14和1例p.R495X突变经证实为新发突变。SALS患者中PFN1基因rs13204T等位基因的频率明显高于对照组,携带T等位基因者发生SALS的风险是携带C等位基因者的1.58倍(95%可信区间为1.15-2.15)。SOD1基因p.H46R突变是亚洲人种多见的较为良性的突变类型,患者均以下肢起病,病情进展缓慢,生存期长；而FUS基因p.P525L, p.R495X和移码突变的临床表型为发病年龄小,病情进展迅速、生存期短。结论：SOD1基因和FUS基因为中国大陆FALS患者突变率排名前两位的基因(分别为26.9%和10.0%)；FUS基因(1.9%)、SOD1基因(1.3%)和TARDBP基因(0.9%)分别为SALS患者排名前三位的基因。某些特定的基因突变有其特征性的临床表型。第二部分肉瘤熔合基因突变与肌萎缩侧索硬化神经元损害的相关性研究背景：肉瘤熔合(FUS)基因突变可引起肌萎缩侧索硬化(ALS)。病理资料显示野生型FUS蛋白主要位于神经元的细胞核内,而FUS基因突变的ALS患者神经元细胞质内出现大量的FUS蛋白聚集并可形成阳性包涵体。由此推测FUS基因突变引起入核转运障碍而导致FUS蛋白在细胞质中的异位分布,由于FUS蛋白在细胞质的过度堆积而获得了毒性,引起运动神经元的损伤。目的：本实验旨在观察野生型FUS与不同的FUS突变蛋白在神经元细胞质中的分布差异,并分析神经元细胞质中FUS蛋白的分布是否与神经元凋亡率存在相关性,是否与携带突变的ALS患者的临床表型具有相关性。方法：在Neuro2a细胞中过表达野生型FUS基因、p.R521H和p.P525L突变FUS基因,通过免疫荧光测定野生型FUS与不同的FUS突变蛋白在神经元细胞质中的分布,通过Western blotting检测转染野生型和不同突变FUS基因的神经元中PARP蛋白的表达,通过流式细胞仪测定神经元的凋亡率,观察FUS蛋白在神经元细胞质中的分布差异,并测定神经元凋亡率。结果：野生型FUS蛋白主要分布在细胞核中,仅少许位于细胞质中,p.R521H突变FUS蛋白除分布于细胞核外,在细胞质中也有明显的分布,而p.P525L突变FUS蛋白则主要分布中细胞质中,细胞核内仅有少许分布,无论转染野生型FUS还是突变型FUS的细胞中均未见FUS阳性包涵体形成；转染p.P525L突变和转染p.R521H突变的细胞其细胞质中荧光强度所占比例均显著高于野生型FUS的细胞(P均＜0.01),转染p.P525L突变的细胞其细胞质中荧光强度所占比例也明显高于转染p.R521H突变的细胞(P<0.05),Western blotting检测发现FUS突变并未明显促进PARP蛋白的表达,流式细胞仪检测显示FUS突变未引起细胞凋亡率的明显增加。结论：不同的FUS基因突变对入核转运的影响程度不同,而且其入核转运障碍的程度与携带突变的ALS患者临床病情进展程度相关,即入核转运障碍程度重的FUS突变引起较严重的临床表型；过表达FUS突变可能并不直接导致神经元的凋亡。第三部分亚甲基四氢叶酸还原酶基因多态性与肌萎缩侧索硬化的关系：荟萃分析目的：亚甲基四氢叶酸还原酶(MTHFR)基因c.677C>T和c.1298A>C单核苷酸多态性与肌萎缩侧索硬化(ALS)发病风险是否存在关联存在争议,因此我们就相关的研究进行了荟萃分析。方法：对PubMed, EmBase, ISI, HuGe,和CNKI数据库2012年9月30日以前的文献进行检索,有3个病例对照研究符合纳入标准。对于入选的每个研究进行Hardy-Weinberg遗传平衡检验。再计算等位基因模型、纯合模型、杂合模型、显性模型和隐性模型下的合并OR值及相应的P值。使用Q统计方法评价研究间的异质性。结果：MTHFR基因c.677C>T或者c.1298A>C多态性和ALS总体发病风险没有明显相关性。然而,亚组分析提示在基于医院的研究中c.677C>T在纯合模型(比值比(OR)=1.54,95%可信区间(CI)=1.03-2.31,P=0.03)和隐性遗传模型(OR=1.50,95%CI=1.02-2.21,P=0.04)下与ALS易感性存在明显相关性；c.677C>T在杂合模式下可降低45岁前起病的患者ALS发病风险(OR=0.68,95%CI=0.50-0.93,P=0.02)。结论：荟萃分析的结果提示MTHFR c.677CT基因型对于45岁起病的ALS患者具有保护作用。研究的设计、起病年龄、种族和地域都是研究异质性的可能来源；因此对于这个荟萃分析的结果的解释应谨慎。今后需要有大样本、设计合理、以人群为基础的病例对照研究来探索基因—基因和基因—营养—环境之间的相互作用对于MTHFR基因多态性和ALS发病风险的影响。更多还原

【Abstract】 Part1. Genetics study of amyotrophic lateral sclerosis patients of Chinese originBackgrounds:Amyotrophic lateral sclerosis is a fatal neurodegenerative disease characterized by progressive muscle weakness, atrophy, dysarthria, dysphagia, and respiratory failure, which is resulted from loss of motor neurons in the motor cortex, brain stem, and spinal cord. To date,17genes have been associated with ALS. Mutations in SOD1, TARDBP, FUS, and C9ORF72are supposed to be the most common mutations in ALS patients.Objective:To determine the mutation rate of the most common mutated genes and genotype-phenotype associations in a large cohort of Chinese ALS patients.Methods:Screening for mutations of SOD1, ANG, TARDBP, FUS, VCP, C9orf72, and PFN1genes was consecutively carried out in20index FALS patients,324SALS patients, and245healthy controls admitted to Peking Union Medical College Hospital. Mutation rates of SOD1, ANG, TARDBP, FUS, C9orf72gene in ALS patients in different countries, continents and total mutation rate in the world were calculated, genotype-phenotype associations were analyzed by reviewing all published studies screening for mutations in these genes in ALS patients.Results:SOD1mutations and FUS mutations were found in five and two out of20index FALS patients, respectively. FUS, SOD1, TARDBP and ANG mutations were detected in six, three, three, and one out of324SALS patients, respectively. All of the mutations were reported previously except the mutations in ANG, p.V103I, which is novel. Four SALS patients carrying FUS mutations were juvenile ALS. The p.G504Wfs*14mutation and one p.R495X mutation was proved to be de novo mutation. Although no mutations was detected in VCP, C9orf72, and PFN1gene in either FALS patients or SALS patients, there was an increase in the frequency of the T allele of the rs13204single nucleotide polymorphism in PFN1in SALS patients compared with that in controls, individuals carrying the T allele had a greater risk of SALS than those carrying the C allele (odds ratio=1.58,95%confidence interval=1.15to2.15). Patients with p.H46R mutation in SOD1gene always manifested with weakness in the legs, the lower motor neuron signs usually dominate the clinical presentation and the disease progresses very slowly, with a mean survival of more than17years. Patients with mutations of p.P525L, p.R495X and nonsense mutations in FUS gene are associated with an early onset, a rapid disease progression, and short lifespan.Conclusions:Mutations in major ALS-related genes are present in approximately35.0%(95%CI,14.1%-55.9%) and4.0%(95%CI,1.9%-6.1%) of Chinese FALS and SALS patients, respectively. SOD1(25.0%) and FUS (10.0%) are the most frequently mutated genes in FALS patients, while FUS(1.9%), SOD1(0.9%) and TARDBP(0.9%) are the most common mutated gene in SALS patients in China. Some characterized clinical phenotypes are associated with some specific gene mutations. Part2. Correlation between FUS mutations and the injury of neurons in amyotrophic lateral sclerosisBackground:Mutations in fused in sarcoma (FUS) gene have been reported to cause a subset of amyotrophic lateral sclerosis (ALS) cases. Wild-type FUS is mostly localized in the nuclei of neurons, whereas the ALS mutants are partly mislocalized in the cytoplasm and can form inclusions. We proposed that mutations in FUS may impair nuclear import and lead to the cytoplasmic mislocalization of FUS, which will further cause the injury of neurons due to gain of toxicity.Aims:We aimed to observe the difference in the cytoplasmic localization of FUS protein in neuron transfected with wild type and mutant FUS, and analyze if the cytoplasmic localization of FUS protein is correlated with the apoptosis rate of neuron and the phenotypes of the mutation carriers.Methods:Wild type, p.R521H and p.P525L mutant FUS tagged with an N-terminal flag tag was transfected in cultured Neuron2a cells. Quantitative immunofluorescence analysis of wild type and mutant FUS in Neuron2a cell was performed48hours after transfection. Western blotting was used to analyze the expression of apoptotic protein PARP in transfected cells. Quantification of neuronal apoptosis as the percentage of Annexin V-EGFP/PI-positive neurons in transfected cells was also performed by Flow Cytometry.Results:Wild type FUS mainly localize in the nucleus, while both mutations showed a varying degree of cytosolic accumulation, with an intermediate mislocalization for p.R521H (p<0.01when compared with wild type FUS) and a severe mislocalization for p.P525L (p<0.001and P<0.05when compared with wild type FUS and p.R521H, respectively), which is correlated with the phenotype of the mutations. No stress granules was seen in cells transfected with both mutations. Western blotting showed that the expression of apoptotic protein PARP in cells transfected with mutant FUS was not higher than that in cells transfected with wild type FUS (both P>0.05). No significant difference in neuronal apoptosis rate was observed between cells transfected with mutant and wild type FUS (both P>0.05).Conclusion:Different degree of the cytosolic mislocalization of mutant FUS caused by FUS mutations is correlated with the phenotype of the mutations carriers, that is, the higher degree of cytosolic mislocalization of the mutant FUS, the more severe phenotype of the mutation carrier. Overexpression of FUS mutations may not directly lead to apoptosis of neuron. Part3. Association of methylenetetrahydrofolate reductase polymorphisms with amyotrophic lateral sclerosis:a meta-analysisObjective:Associations between methylenetetrahydrofolate reductase(MTHFR) c.677C>T or c.1298A>C polymorphisms and amyotrophic lateral sclerosis (ALS) risk are controversial. We conducted a meta-analysis of available published studies.Methods:A literature search was performed of PubMed, EmBase, ISI, HuGe, and CNKI databases up to September30,2012. Three case-control studies met the inclusion criteria. For the control group of each study, the distribution of genotypes was evaluated for agreement with Hardy-Weinberg equilibrium. The pooled ORs and the corresponding P value were estimated at the allele level, the homozygote model, the heterozygote model, the dominant model, and recessive model. Heterogeneity between studies was assessed by the Q statistic.Results:No significant association was found between c.677C>T or c.1298A>C and ALS risk for the overall population. However, subgroup analyses indicated c.677C>T was significantly associated with ALS susceptibility under homozygote (odds ratio (OR)=1.54,95%confidence interval (CI)=1.03-2.31, P=0.03) and recessive models (OR=1.50,95%CI=1.02-2.21, P=0.04) in hospital-based studies, and was protective against ALS under the heterozygote model (OR=0.68,95%CI=0.50-0.93, P=0.02) in patients with onset before age45.Conclusions:This meta-analysis suggests that MTHFR c.677CT genotype protects against ALS in patients with onset before age45. Study design, age of onset, ethnicity, and geography are potential sources of heterogeneity; our conclusions should be interpreted with caution. Large-scale, well-designed, population-based, case-control studies are necessary to investigate gene-gene and gene-nutrition-environment interactions between MTHFR polymorphisms and ALS.更多还原