Genetic Study Of Amyotrophic Lateral Sclerosis 1. Study On The Characteristics Of Gene Mutations In Chinese Patients With Amyotrophic Lateral Sclerosis 2. Study On The Transcriptional Effects Of Amyotrophic Lateral Sclerosis-associated KIF5A Gene Mutation

Part 1 Exploration of genetic architecture in Chinese amyotrophic lateral sclerosis patientsObjective:To explore the mutation frequency of new ALS-causing genes including GLE1,ANXA11,TIA1,KIF5A,and GLT8D1 identified in European and American amyotrophic lateral sclerosis(ALS)patients in the Chinese population.We further explore the distinct genetic architecture in Chinese mainland ALS patients,to provide evidence-based evidence for future clinical genetic counseling and clinical gene detection in patients with ALS.Methods:From January 2015 to March 2019,Chinese mainland patients diagnosed as positive ALS,suspected ALS and laboratory-supported ALS as per E1 Escorial criteria were recruited in the present study.We use Sanger sequencing technique to detect GLE1 gene in 250 patients(FALS=20,SALS=230),ANXA11 gene in 383 patients(FALS=18?SALS=353,ALS-FTD=12),TIA1 gene in 588 patients(FALS=29,SALS=546,ALS-FTD=13),KIF5A gene in 693 patients(FALS=33,SALS=645,ALS-FTD=15),and GLT8D1 gene in 885 patients(FALS=44,SALS=821,ALS-FTD=20).The C9orf72 hexanucleotide(GGGGCC)n repeat analysis was performed in 20 ALS-frontotemporal dementia(ALS-FTD)patients by repeat PCR technique.Whole Exome Sequencing(WES)was used to screen a cohort of 302 unrelated patients(FALS=44,SALS=238,ALS-FTD=20).Results:Combined with Sanger sequencing and WES sequencing data(excluding repeated patients),it was found that the pathogenic mutation frequency of GLE1 gene,ANXA11 gene,TIA1 gene,KIF5A gene and GLT8D1 gene in our cohort was 0%(0/532),1.1%(7/655),0%(0/848),0.23%(2/885),and 0%(0/885),respectively.In our 44 FALS patients,the highest mutation frequency of known ALS gene was found in SOD1(50%),followed by FUS(7%),UBQLN2(2%),VAPB(2%),ANXA11(2%),and KIF5A(2%).In our 238 SALS patients,the highest mutation frequency of known ALS gene was also found in SOD1(4%),followed by FUS(1%),TARDBP(1%),TBK1(1%)OPTN(1%).Another genes frequency including SETX,NEFH,ANXAll,NEK1,DCTN1,and TUBA4A was less than 1%.Additionally,the frequency of ANXA11 gene,UBQLN2 gene and NEFH gene mutation in 20 ALS-FTD patients was 10%(2/20),5%(1/20),and 5%(1/20)respectively.No six nucleotide(GGGGCC)n repeat mutations of the C9orf72 gene were found in ALS-FTD patients.Conclusion:GLE1,TIA1,KIF5A,and GLT8D1 newly reported ALS pathogenic genes are rare in Chinese mainland ALS patients.The mutation frequency of ANXA 11 gene in ALS patients in China is similar to that in European patients.There is a significant difference ALS mutation spectrum between Chinese and European/North American population.SOD1 gene is the highest mutation frequency in Chinese patients,and ANXA11 gene may be a common pathogenic gene in ALS-FTD patients in China.Part 2 Exploration of the effect of ALS-related KIF5A mutations on gene splicingObjective:To explore the effect of all 10 different ALS-related KIF5A C-terminal mutant sites including c.2987de1A,c.2989delA,c.2993-3C>T,c.2993-1G>A,c.2996delA,c.3019A>G,c.3020G>A,c.3020+1G>A.c.3020+2T>A,c.3020+3A>G,on the normal transcription of KIF5A gene.Methods:A DNA fragment encompassing exon25 to exon28 and the flanking intronic sequences of KIF5A was directly amplified from the genomic DNA of a healthy individual.The PCR product was digested with the Bam HI and Mlu 1restriction enzymes and cloned into the pCAS2 vector,which had also been digested with Bam H1 and Mlu 1,to construct the recombinant plasmid of wild type pCAS2.Using wild-type pCAS2 recombinant plasmid as a template and site-directed mutagenesis technique,the mutant pCAS2 recombinant plasmids containing those mentioned above ten different mutation sites were amplified.The resulting constructs were transfected into HEK293T cells.Total cellular RNA was extracted after 24 hours,followed by RT-PCR analysis.Results:The c.2993-3C>T mutation does not affect the transcription of the terminal sequence of the KIF5A gene.The c.2993-3C>T,c.3019A>G,c.3020G>A,c.3020+1G>A,c.3020+2T>A,and c.3020+3A>G mutant sites all caused the skipping of exon 27,resulting in the same frameshift mutation p.Asn999Valfs*40.The c.2987delA mutation caused p.Asp996Alafs*52.The c.2989delA mutation caused?.Asn997Metfs*51.The c.2993-1G>A mutation caused p.Gly998Glufs*50.The?.2996delA mutation caused p.Asn999Metfs*49.Discussion:ALL ALS-related KIF5A C-terminal mutant sites,except c.2993-3C>T,resulted in almost identical results:frameshift mutations occurred after amino acids 996,997,998,and 999.Part 3 Preliminary study on the molecular mechanism of ANXA11 gene mutationObjective:To explore the effects of c.107C>G(p.P36R),c.1471G>A(p.G491R),and c.174-2A>G mutations identified in the first part of our study on the function and normal transcription of Annexin A11 protein.Methods:Total RNA from an ALS patient harboring c.174-2A>G mutation was isolated from blood lymphocytes.The reverse transcription(RT)reactions were performed.Complementary DNA sequences containing the splice site mutation were amplified by PCR.The resulting products were analyzed by Sanger sequencing.The triplex of the terminal terminating codon of normal wild-type ANXA11 gene cDNA was removed and cloned into pEGFP-N1 vector plasmid by restriction enzymes'digestion and ligation.The mutated recombinant plasmids with c.107C>G or c.1471 G>A mutation were constructed by site-directed mutagenesis technique and then transfected into HEK293T cells for 36 hours.The cells were fixed with paraformaldehyde and stained with DAPI dye.The expression and distribution of mutant proteins ANXA11-p.P36R and ANXA11-p.G491R were observed by fluorescence confocal microscope.The effects of p.P36R and p.G491R on the local three-dimensional spatial structure of Annexin All protein were analyzed by bioinformatics method.Results:The mutation c.174-2A>3 lead to the skipping of exon6,which resulted in a large fragment deletion p.A58_Q187del.p.P36R or p.G491R mutation increased Annexin A11 protein aggregation in the cytoplasm.Bioinformatics analysis suggested that p.P36R or p.G491R might destroy the local three-dimensional spatial structure of Annexin A11 protein.Discussion:Both p.P36R and p.G491R caused the abnormal aggregation of Annexin A11 protein in cytoplasm.Mutation c.174-2A>G changed the normal structure of Annexin All protein.