Genetics And Basic Research For Amyotrophic Lateral Sclerosis

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.