Analysis Of The Acetaldehyde Dehydrogenase 2 (ALDH2) Rs671 Single Nucleotide Polymorphism And Alternative Splicing

Objective: To investigate the changes of acetaldehyde dehydrogenase 2(ALDH2)at m RNA level of individuals with carrying ALDH2 rs671 single nucleotide polymorphism(SNP),and Alternative splicing(AS)occurs in ALDH2 m RNA in gastric cancer cells.Methods:(1)The peripheral anticoagulant whole blood was collected from the physical examination population(aged 20-40 years old,24 males and 24 females)in the affiliated Hospital of Guizhou Medical University;The primers that can specifically amplify the polymorphic sites of ALDH2 rs671 gene were designed for polymerase chain reaction(PCR)amplification;The DNA of peripheral blood leukocytes(WBC)was extracted as a template,and the PCR products were purified and Sanger sequencing was performed to identify the ALDH2rs671 gene polymorphism of the subjects.(2)Using WBC complementary DNA(c DNA)as a template,primers specifically amplified ALDH2 rs671c(m RNA site corresponding to ALDH2 rs671 SNP site,c:c DNA)were used for PCR amplification.The PCR products was purified and Sanger sequencing was performed to identify the sequence of ALDH2 rs671 c site.(3)The reverse transcription-PCR(RT-PCR)products of subjects with ALDH2 rs671(AA)were TA cloned and sequenced,and the proportion of c DNA expressed as "A" at ALDH2 rs671 c site was confirmed.(4)The total RNA of gastric cancer cell line AGS was used to detect the transcriptional initiation site of ALDH2 m RNA by rapid amplification of c DNA ends(RACE).(5)ALDH2 gene specific primers were designed to amplify the RT-PCR products of gastric cancer cell lines(AGS,HGC-27,MKN45),normal gastric mucosal epithelial cell line GES-1 and WBC,and then analysis the AS of ALDH2 m RNA.(6)BLAST tool was used for nucleotide sequence alignment analysis.(7)The open reading frame(ORF)of ALDH2 transcript 1(Vatiant1,V1)was analyzed by DNAStar software.Results:(1)Among the 48 subjects,the gene frequency of ALDH2rs671(GG,GA,AA)genotypes were 58.3%,39.6% and 2.1%,respectively,and the sequencing results of c DNA at ALDH2 rs671 c site were consistent,all showed as "G".(2)Subjects with genotype ALDH2 rs671(AA)showed "G" approximate85.7%(6/7)and "A" approximate 14.3%(1/7)at ALDH2 rs671 c site.(3)Thirteen newtranscriptional initiation sites of ALDH2 in gastric cancer cell line AGS were detected using 5’RACE.(4)AS was found at the 3’-terminal of exon 2 of V1 from5’RACE products.(5)AS occurs at three different sites in the 3’-terminal of exon 1 of V1 and the 5’-terminal of exon 3.(6)AS also occurs in the 3’-terminal of exon 2 of V1 and the 5’-terminal of exon 4.(7)Intron 1 of V1 retains an exon with a length of116 nucleotides.(8)Bioinformatics analysis showed that V1 contained 8 ORF with a length of more than 100 amino acids.Conclusion:(1)Among the 48 subjects in our study,individuals with genotypes of ALDH2 rs671(AA)may occurs RNA editing(A-to-I)in ALDH2 m RNA;In 19 individuals with ALDH2 rs671(GA)genotype,the ALDH2 rs671 c site of c DNA showed "G".(2)There are 13 new transcriptional initiation sites of ALDH2 m RNA in gastric cancer cell AGS,and exist 6 new AS forms in gastric cancer cells(AGS,HGC-27,MKN45)and human WBC,suggesting that it exist complex expression regulation and function.