| Objective To screen the gout-associated genes and develop the polymerase chain reaction-high resolution melting(PCR-HRM)methods for genotyping the screened and reported genes.To explore the genetic susceptibilities to primary gout in Chinese population from Lanzhou region.Methods Gout-associated genes were screened using the technique of exome sequencing to analyze four clinical samples from a core family of hypertriglyceridemia,and the molecular diagnosis assays of PCR-HRM were developed through designing the primers of the screened and documented genes using online Primer-BLAST software.Sanger sequencing was taken as a gold standard to verify the correctness,and the methodology detection performance was assessed through calculating sensitivity,specificity,repeatability and reproducibility.The clinical applicability was evaluated by genotyping for 398 samples of patients with primary gout.Analysis of allele and genotype frequencies,linkage disequilibrium and haplotype were performed using online SHEsis software.The Chinese genotype data of 1000-genome database in NCBI were used as controls and the genetic susceptibility to primary gout in Chinese population from Lanzhou region was determined by conducting chi-square test using SPSS software.Binomial logistic regression analysis was performed to evaluate the association of the genetic polymorphisms with primary gout under different genetic models.Results(1)The developed PCR-HRM assays genotyping for 20 SNPs(rs5128 C>G,rs2070667G>A,rs2070666 T>A,rs76353203 C>T and rs121918382 A>G in APOC3,rs2234668 G>A and rs5104 T>C in APOA4,rs3135507 C>T,rs2266788 T>C,rs662799 T>C and rs2075291 G>T in APOA5,rs2302515 C>G in APOBEC1,rs1174657 A>G,rs1174658 A>G and rs10911391 G>A in APOBEC4,rs2231153 T>C in ABCG2,rs1260326 A>G in GCKR,rs3825018 C>T,rs3832794G>-and rs148845071 G>A in SLC22A12 gene)were validated by Sanger sequecing and successfully genotyped 398 clinical samples.(2)The results of performance evaluation showed that the sensitivity and specificity reached 100%in the study.For the homozygous melting curve,the coefficient of variation(CV)of the T_m values were less than 1%in the SNPs of rs76353203,rs121918382,rs2075291,rs3135507,rs2266788,rs662799,rs1174657,rs2231153,rs1260326 and rs3825018,and the T_m value differences between two homozygous were statistically significant(P<0.05)in SNPs of rs3135507,rs2266788,rs662799,rs1174657,rs2231153,rs1260326 and rs3825018,which indicated that the repeatability and reproducibility are well.(3)Except for the SNPs of rs76353203,rs121918382,rs2075291 and rs2234668,the statistical analysis of other 16SNPs showed that the genotype frequency of rs2070666,the allele and genotype frequencies of rs10911391 and rs3135507 were found statistical differences between case and control groups(P<0.05).(4)After having stratified the case according to TG,the allele and genotype frequencies of rs2266788 and rs662799,and the allele frequency of rs1174658 were found statistical differences between high-TG and TG-normal gout groups(P<0.05).The allele and genotype frequencies of rs1260326 and the allele frequency of rs3832794 were found statistical differences in high-TG gout and control groups(P<0.05).(5)Under three genetic models,binomial logistic regression analysis reveals that the wild type AA of rs2070666 was found difference between case and control groups under recessive and co-dominant models;The wild type GG of rs10911391 and CC of rs3135507 were found differences between the two groups under dominant model;The heterozygous mutation AG of rs10911391 was found difference between the two groups under co-dominant model(P<0.05).The wild type TT of rs2266788 and rs662799 were found differences between high-TG and TG-normal gout groups under dominant model;The heterozygous mutation CT of rs2266788 was found difference between the two groups under co-dominant model;The homozygous mutation CC of rs662799 was found difference between the two groups under recessive and co-dominant models(P<0.05).The wild type AA of rs1260326and GG of rs3832794 were found differences between high-TG gout and control groups under dominant model;The heterozygous mutation AG of rs1260326 was found difference between the two groups under co-dominant model(P<0.05).(6)The results of haplotype analysis showed that the haplotype TG of rs5104 and rs5128,the haplotype CC and TC of rs2266788 and rs662799were found differences between case and control groups(P<0.05).Conclusion(1)The developed molecular diagnostic methods of 20 SNPs from eight gout-associated genes are suitable for clinical routine testing.(2)The polymorphisms of APOC3,APOA4,APOA5,APOBEC4,GCKR and SLC22A12 genes are associated with the susceptibility to primary gout in Chinese population from Lanzhou region. |
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