| Objectives To develop a molecular diagnostic technology for the genotyping of 13 SNPs in ABCG2 gene,including rs3114018,r3114020,rs2231146,rs2231164,rs2725252,rs55976258,rs12641369,rs34455506,rs1448784,rs10011796,rs2725244,rs34472643 and rs3114015.To validate the relationship of ABCG2 polymorphisms with the susceptibility of primary gout in the Lanzhou region of China.MethodsUsing Primer-BLAST software designed the specific primers for 13 SNPs of ABCG2 gene,we established the PCR-HRM molecular diagnostic system for clinical routine detection,the genotyping results of the established methods were validated by the Sanger sequencing.Then we evaluated the sensitivity and specificity,repeatability and reproducibility of this method and the clinical applicability was evaluated for 398 samples of primary gout patients.In addition,a case-control analysis was performed to verify the susceptibility of primary gout in the Lanzhou region of China and the control group was from the 1000-genome database.The chi-square test and binomial logistic regression analysis were performed for the susceptibility of primary gout with ABCG2 polymorphisms,besides the Linkage disequilibrium(LD)and haplotype analysis were performe to evaluate the gout risk of haplotype.Results(1)Sequencing confirmed that the self-developed PCR-HRM method could genotype 13 SNPs in ABCG2 accurately and 398 samples were accomplished successfully.The performance evaluation results showed when this diagnostic system was used to detect the 13SNPs,its sensitivity and specificity can reach 100%.The coefficient of variation(CV)of the T_m values were less than 1%in all homozygous melting curves,and the T_m value differences between two homozygous were statistically significant(P<0.05)in each SNPs.It indicated that the PCR-HRM method has good repeatability and reproducibility.(2)For all SNPs which satisfied with the genetic equilibrium except for rs1448784,its case-control analysis showed that allele and genotype frequency distribution of the 11 SNPs in ABCG2 gene are statistical difference(P<0.05),they are rs2231164,rs34455506,rs34472643,rs2231146,rs12641369,rs2725252,rs3114018,rs2725244,rs311402,rs10011796 and rs55976258.(3)Further triglyceride(TG)stratification analysis in the case group revealed that the genotype frequencies and allele frequencies of the 11SNPs were not significantly different between high-TG group and normal-TG group(P>0.05).(4)After the binomial logistic regression analysis under four genetic models,we found that wild type of the 11 SNPs can increase the risk of gout under the dominant model and its homozygous mutation can reduce gout risk under the recessive model.The wide type of all 12 SNPs can increase the gout risk but not rs34455506 and rs34472643 under additive model.If we take the wild type as the reference,following genotypes can decrease the gout risk including the AA of rs2231164,the CT of rs34455506,the AG of rs34472643,the AG/GG of rs223116,the AG/AA of rs12641369,the GT/TT of rs2725252,the AC/AA of rs3114018,the AG/AA of rs2725244,the CT/TT of rs3114020,the CT/CC of rs10011796,the CT/TT of rs55976258 under co-dominant model.(5)LD analysis revealed that rs34455506 and rs34472643,rs2725244 and rs3114020 in ABCG2 gene have strong linkage disequilibrium relationships because their D′≧0.8 and r~2≧0.8.(6)The haplotypes“ACGAAGAATCC”and“ATAGAGAATCT”constructed from 11 SNPs(rs2231164,rs34455506,rs34472643,rs2231146,rs12641369,rs2725252,rs3114018,rs2725244,rs3114020,rs10011796 and rs55976258)can increase the risk of gout.ConclusionsThe PCR-HRM molecular diagnostic method established in this study can be used for the genotyping of the13 SNPs in ABCG2 gene,and it has good sensitivity,specificity,repeatability and reproducibility.The polymorphisms of ABCG2 gene are associated with the susceptibility of primary gout in the Lanzhou region of China. |
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