Development And Application Of Molecular Diagnosis Technologies Of SLC2A9, SLC22A12, SLC17A1 And GCKR Genetic Polymorphisms

Objective To develop rapid and routine molecular diagnosis technologies genotyping for the 11 single nucleotide polymorphisms(SNPs)of SLC2A9(rs3733591,rs3733589,rs1014290,rs3756231 and rs2276961),SLC22A12(rs7932775 and rs559946),SLC17A1(rs1183201)and GCKR(rs780093,rs780094 and rs6744393)genes,and then verify the susceptibility to gout of the above-mentioned SNPs in Han Chinese population from Lanzhou region.Methods The polymerase chain reaction(PCR)-high resolution melting(HRM)detection systems for above-mentioned 11 SNPs were developed through designing PCR primers with online Primer-BLAST software,and their genotyping performances were verified by Sanger sequencing.A case-control study was conducted by genotyping for 398 clinical samples from patients with gout using the developed PCR-HRM asaays and collecting the genotype data of 211 Han Chinese in Beijing(CHB)and Southern Han Chinese(CHS)from 1000 Genomes Project in NCBI as control groups to verify the susceptibility to gout of 11 SNPs in Chinese Han population from Lanzhou region.Results(1)The results of Sanger sequencing have verified that the self-developed PCR-HRM assays can correctly genotype the 11 SNPs.(2)The case-control study showed that: 1)The susceptibility to gout: the distribution of genotype and allele frequencies of the five(rs3733591,rs3733589,rs1014290,rs3756231 and rs7932775)in 11 SNPs are statistical differences between case and control groups(P<0.05).2)Binary logistic regression analysis of the risk of gout:(1)rs3733591: The CC homozygotic mutation increases the risk of gout compares with the TT wild genotype;the susceptibility to gout increases no matter under the dominant(CC+CT vs TT)or recessive(CC vs TT+CT)genetic models.(2)rs3733589: the AA homozygotic mutation decreases the risk of gout compares with the GG wild genotype;the susceptibility to gout decreases no matter under the dominant(AA+AG vs GG)or recessive(AA vs GG+AG)genetic models.(3)rs1014290: the GG homozygotic mutation decreases the risk of gout compares with the AA wild genotype;the susceptibility to gout decreases under the recessive(GG vs AA+AG)genetic model.(4)rs3756231: the GG homozygotic mutation decreases the risk of gout compares with the AA wild genotype;the susceptibility to gout decreases under the recessive(GG vs AA+AG)genetic model.(5)rs7932775: the CC homozygotic and CT heterozygous mutation increases the risk of gout compares with the TT wild genotype;the susceptibility to gout increases under the dominant(CC+CT vs TT)genetic models.3)Haplotype analysis:(1)SLC2A9:the CGAAT and TAAGC haplotypes constructed with rs3733591,rs3733589,rs1014290,rs3756231 and rs2276961 SNPs confer to the risk of gout,but the TAGGC haplotype decreases the one.(2)SLC22A12: the CC haplotype composed by rs7932775 and rs559946 add the risk of gout.4)Triglyceride(TG)stratification analysis in case group showed that the distribution of genotype and allele frequencies of the 11 SNPs were not statistical differences between case and control groups.(P>0.05).Conclusion The PCR-HRM molecular diagnosis methods genotyping for the 11 SNPs were successfully developed and correctly genotyped 398 clinical samples from gout patients,which can be applied to the rapidly and precisely routine genotyping detection.The genetic polymorphisms of SLC2A9(rs3733591,rs3733589,rs1014290,rs3756231 and rs7932775)and SLC22A12(rs7932775)are associated with gout development in Han Chinese population from Lanzhou region.