Studies On The Pathogenic Mechanisms Of SOD1 Gene Variants In Familial Amyotrophic Lateral Sclerosis

Background: Amyotrophic lateral sclerosis(ALS)is a fatal degenerative disease of the nervous system,characterized by progressive dysfunction and selective loss of upper and lower motor neurons.The clinical presentation of ALS patients are atypical,with progressive muscle weakness,atrophy,dysarthria,and dysphagia.Due to the unknown cause of the disease and the lack of effective diagnosis and treatment,the survival period for ALS patients is usually less than four years.About 10%～15% of people with ALS have a family history of the disease,known as familial amyotrophic lateral sclerosis(FALS),and the remaining 85% of people with ALS have no family history of the disease,known as sporadic amyotrophic lateral sclerosis(SALS).Genetic factors play an important role in the pathogenesis of ALS.The identification and functional analysis of causative genes in FALS family will not only facilitate early diagnosis,genetic counselling,and prognostic assessment of ALS patients,but also promote the study of the pathogenesis of ALS and the development of targeted drugs.Objective: This study applied whole exome sequencing and Sanger sequencing to identify pathogenic genetic variants in three unrelated Chinese FALS families,analysed the association between the clinical phenotype and genotype of patients,in combination with in vitro experiments revealed the pathogenesis and provided directions for the clinical treatment of ALS.Methods: Three unrelated Chinese ALS families were collected.Clinical data were gathered from all members,and relevant clinical examinations were performed.Peripheral venous blood samples were obtained from a total of 59 members of the three families,and genomic DNA was extracted.Whole exome sequencing was performed on the genomic DNA samples of the probands to obtain nucleotide variation data.Sequencing data were mapped and analyzed for potential pathogenic genetic variants using the Single Nucleotide Polymorphism database,Exome Aggregation Consortium,Genome Aggregation Database,China Metabolic Analytics Project,and the in-house BGI exome databases(Shenzhen,China)with 2471 Chinese controls.Sanger sequencing was used to test candidate pathogenic variants in family members.Conservation analysis,functional prediction,and protein structure modeling of candidate pathogenic variants were performed by bioinformatics analysis software.The 2015 revised American College of Medical Genetics and Genomics(ACMG)guidelines were used to assess and classify the pathogenicity of variants.The expression plasmids of pathogenic gene variants were constructed and transfected into cells,respectively.The effects of pathogenic gene variants on reactive oxygen species level,cell viability and protein localization were detected by dihydroethidium(DHE)staining,Cell Counting Kit-8(CCK-8)assay,antioxidant N-acetylcysteine(NAC)treatment,and laser confocal microscopy,respectively.Results: All patients presented with spinal onset,predominantly lower motor neurons presentation,and absence of cognitive dysfunction.Three known heterozygous missense variants c.255G>C(p.L85F),c.335G>A(p.C112Y),and c.436G>A(p.A146T)of the superoxide dismutase 1 gene(SOD1)may be the pathogenic gene variants in each of the three FALS families,respectively.Bioinformatics software analysis showed that all three variants reduced the stability of SOD1 protein and changed the distance or number of hydrogen bonds.According to the ACMG guidelines,the SOD1 gene c.255G>C(p.L85F)variant was classified as “pathogenic”,and the SOD1 gene c.335G>A(p.C112Y)and c.436G>A(p.A146T)variants were classified as “likely pathogenic”.In vitro experiments showed that the three SOD1 gene variants caused the increase of intracellular reactive oxygen species(P < 0.05),the decrease of cell viability(P < 0.05),and the formation of abnormal intracellular aggregates(P < 0.05)compared to the wild-type SOD1 gene,and the antioxidant NAC could inhibit the decrease of cell viability caused by the variants(P < 0.05).Conclusions:(1)The SOD1 gene c.255G>C(p.L85F),c.335G>A(p.C112Y),and c.436G>A(p.A146T)variants may be the causative gene variants in three Chinese ALS families,respectively.(2)These three SOD1 gene variants may lead to neuronal death through both oxidative stress and abnormal protein aggregation pathways,ultimately causing ALS.(3)NAC may be a potential therapeutic agent for ALS patients with SOD1 gene variants.Figures 26,Tables 26,References 102...