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 3rd-4th base of exon 5 （c.601₆04 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.