肌萎缩侧索硬化的基因突变研究

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苷酸重复扩增突变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ABI3730 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.601-2 A>G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.601-2A>G）。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)_n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.601-2A>G，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。

关键词  肌萎缩侧索硬化；基因突变；C9ORF72基因；(CAG)_n重复扩增

Mutation Study of Amyotrophic Lateral Sclerosis

Background Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease characterized by progressive muscle weakness, atrophy, dysarthria and respiratory failure, which is resulted from loss of motor neurons in the motor cortex, brain stem and spinal cord. To date, more than 30 major genes have been associated with ALS. Mutations of SOD1, TARDBP, FUS and C9ORF72 have been considered as the most common mutations in familiar ALS (FALS) patients and sporadic ALS (SALS). And previous reports identified a hexanucleotide GGGGCC (G₄C₂) repeat expansion in a non-coding region of C9ORF72 as the major cause of ALS and frontotemporal dementia (FTD), particularly in western populations. Furtherrmore, an intermediate-length trinucleotide (CAG)_n repeat expansion (27–31 units) in a coding region of ATXN2 has been identified as a risk factor of ALS.

Objective 1. To screen mutations of ALS-caused genes in patients with FALS and SALS by targeted resequencing 2. To detect the frequency of noncoding G₄C₂ repeat expansions and rare coding mutations of C9ORF72 in Chinese ALS patients and to preliminarily analyze the role of C9ORF72 in ALS. 3. To determine whether (CAG)_n repeat expansions in the coding region of ATXN2, ATXN3 and AR and the noncoding region of ATXN8 were associated with Chinese ALS cases.

Methods 1.Targeted resequencing focused on 26 known ALS-caused genes was performed using the Ion PGM Sequencer. Filter candidate mutations and calculate the mutation frequency of common mutated genes in Chinese ALS cases. 2. The repeat expansion in C9ORF72 was amplified by repeat-primed PCR, which were separated on an ABI3730 DNA Analyzer and visualized by GENEMAPPER software. In vivo cDNA splicing assay and splicing Minigene assay were performed to evaluate the effect of the splicing variant (c.601-2 A>G). 3. The (CAG)_n repeat expansion in ATXN2、ATXN3、ATXN8 and AR was amplified and analyzed by previous methods. The distributions of the repeat lengths in Chinese ALS and ethnically-matched healthy controls were analyzed by statistical methods.

Results 1.We found 36 mutations in 7 FALS cases (38.89%, 7/18) and 33 SALS cases (18.54%, 33/178), including 1 splicing site mutation, 1 nonsense mutations and 34 missense mutations. All mutations carried in FALS cases were found in ALS-identified genes, including SOD1 (27.78%), UBQLN2 (5.56%) and VAPB (5.56%). Moreover, mutations of ALS-identified genes were found in 14 SALS subjects (7.87%, 14/178) and SOD1 (2.25%) had the highest frequency. And mutations of ALS-associated genes were found in 19 ALS patients (10.67%, 19/178) and NEFH (3.93%) was the most common. 2. The disease-associated repeat expansion (more than 30 units) was found in 2 (2/252; 0.79%) of SALS patients. No abnormal expansion was found in FALS cases (0/24; 0%). No significant difference was observed in the distributions of the repeat sizes between patients and control subjects (p=0.805). A splicing site mutation (c.601-2A>G) of C9ORF72 was found in a SALS case. The cDNA sequencing results and in vivo Minigene splicing assay showed that the novel A-to-G transition abolished the invariable consensus AG splice acceptor of intron 4 and activated a cryptic splicing site at 3^rd-4^th base of exon 5 (c.601_604 del ATAG). The mutation disrupted the reading frame and may have created a premature termination codon (p.I201fsX235). The cDNA sequencing results revealed degradation of the mutant C9ORF72 mRNA possibly through nonsense-mediated mRNA decay (NMD). 3. A significant association in ATXN2 long-length polyQ repeat alleles (≥32 CAG repeats) with FALS (p=0.0001) and SALS (p=0.047) was observed in the current cohort. The intermediate-length trinucleotide repeat expansions (29-67 repeats) showed higher disease risk either in FALS cases (p=0.0291) or in SALS (p=0.0276). The distribution of ATXN3 and AR repeat lengths did not differ between ALS probands and control subjects (with a p value of 0.274 and 0.657, respectively).

Conclusions 1. FALS and SALS differed in genetic landscape, and SOD1 has been identified as the most common causative gene in ALS patients in this study. 2. The G₄C₂ repeat expansion of C9ORF72 was an infrequent cause in Chinese ALS patients 3. A splicing site mutation of C9ORF72 has been found in a SALS case, supporting a loss-of-function mechanism for ALS. 4. Long-length repeat expansion in ATXN2 (≥32) and intermediate-length trinucleotide expansion in ATXN8 (29-67) would possibly increase ALS disease risk. Repeat expansions in ATXN3 and AR were not affirmative risk factors in Chinese ALS subjects.

Keywords: Amyotrophic lateral sclerosis, mutation, C9ORF72, (CAG)_n repeat expansion mutation