Genetic Analysis In Chinese Patients With Familial Or Young-onset Amyotrophic Lateral Sclerosis

Objective:The aim of our study was to investigate the genetic characteristics in familial or young-onset amyotrophic lateral sclerosis(ALS)patients in a Chinese center.Methods:Patients with familial or young-onset(age of onset < 45 years old)ALS,admitted to the Department of Neurology,First Hospital,Shanxi Medical University from January2017 to December 2020,were reviewed.The clinical data was collected.Whole exome sequencing(WES)was performed to identify the disease-associated variants.Single nucleotide variants and small insertions/deletions(INDELs)were further predicted with silico tools and compared to the Single Nucleotide Polymorphism Database(db SNP),the1000 Genomes Project,and Exome Aggregation Consortium.Repeat-primed polymerase chain reaction(RP-PCR)was used for C9ORF72 or NOTCH2 NLC hexanucleotide repeat expansions(HRE)for patients without variants in WES.The evolutionary conservations were estimated and the structures of proteins were constructed by Swiss-Model server.Immunohistochemistry was conducted to confirm the misfolded SOD1 protein.Immunofluorescence staining was conducted with an anti-p62 antibody and an anti-NOTCH2 NL antibody to detect positive deposits in the intranuclear inclusions.Results:9 patients,including 4 familial ALS and 6 young-onset ALS were enrolled,one of them was young-onset ALS as well as a familial ALS.Genetic analysis identified related missense mutations of SOD1(4/6,66.7%),FUS(1/6,16.7%),and NEK1(1/6,16.7%)in6 patients.NEK1 c.290G>A mutation(NM＿012224.2 exon4)in a patient with familial ALS and SOD1 c.362A>G mutation(NM＿000454 exon5)in a young-onset ALS patient were novel.The novel mutations were predicted to be deleterious,affected evolutionarily highly conserved amino acid residue.The number and position of hydrogen bonds between the mutated site and its surrounding amino acid residues changed.Misfolded SOD1 protein was identified in patient with SOD1 c.362A>G mutation.One familial ALS patient carried a NOTCH2 NLC HRE and the P62 positive protein and NOTCH2 NLC protein deposits in the intranuclear inclusions were identified in this patient through immunofluorescence staining.Conclusion:Two novel mutations were detected in our patients.Patients with familial or young-onset ALS often carried related gene mutations and genetic sequencing should be thus routinely performed.