HURAT1 Single Nucleotide Polymorphism And Genetic Risk For Hyperuricemia

Objective:hURAT1 is the mian gene of regulating surem uric acid,and it is one of the most important candidate genes of primary hyperuricemia.The present case-control study was designed to find SNPS and mutations on hURAT1,and to analyze whether SNPS and mutations on hURAT1 associated with primary hyperuricemia.Methods:DNA samples from 227 individuals with primary hyperuricemia and from 341 controls were sequenced.Promotor region,all exons and flanking introns of the hURAT1 gene in patients and control individuals were screened for mutations by polymerase chain reaction(PCR) and subsequent forward and reverse sequencing.SNPs and mutations on hURAT1 were recogonized by Genestar software;To evaluate chooseing samples to fit random principle and LD between every two snps,all snps of subjects were computed by haploview software;To identificate related polymorphisms on hURAT1 gene with hyperuricemia,genotype and allele frequencies between groups compared by X²test;To discriminate susceptive haplotypes on hURAT1 gene with hyperuricemia,individual haplotypes from related polymorphisms were estimated by the Phase software;to confirm the risk or prostective polymorphisms,all snps were analyzed by Logistic regression analysis,and by comparing clinical features.Results:1.hURAT1 gene sequencing resultBy sequencing promotor region,all exons,including exon-intron boundaries of hURAT1 gene in 568 individuals,thirteen SNPs were identified.Five were located in the promotor region:-454A＞T,-434T＞C,-382C＞T,-87C＞T,+118G＞A;Three werelocated in exon:C258T,C426T,T1309C.Five SNPs was located in exon-intron boundary:+2,271A＞G,+2,277C＞T.+2,278A＞G,+3,11G＞A,-8,103A＞G.+3.11G＞A was a novle polymorphism.2.Hardy-Weinberg equilibrium test and linkage disequilibrium(LD) analyzeNo deviations from Hardy-Weinberg equilibrium were observed for 13 polymorphisms of 568 individuals,indicated all samples were chosen randomly.Pairwise linkage disequilibrium analysis displayed a significant linkage disequilibrium between these polymorphisms:-454A＞T,-434T＞C,-382C＞T,-87C＞T,+118G＞A,C258T,C426T,+2,271A＞G,+2,277 C＞T and +2,+278A＞G(D'＞0.8);there was absolute linkage disequilibrium between -8,103G＞A and T1309C(D'=1).However,+3.11G＞A showed very little LD with next to each other,and created breaks in the pattern of LD.This phenomenone indicated +3,11G＞A was significantly associated primary hyperuricemia.3.Allele and genotype frequencies in cases and controls.Allele A of hURAT1 intron 3(+11 G＞A) was found significantly increased in the group of hperuricemia patients,being detected in 6.28%hperuricemia patients alleles and in 1.7%of healthy control alleles(P=0.003)The frequency of the AG_AA genotypes was significantly higher among hperuricemia patients than among healthy control subjects(12.11%VS 3.40%,P=8.21×10^-5).By correlate analysis for the 13 SNPs,the strongest association with hyperuricimia could be demonstrated for the +3,11G＞A polymorphism(by allele frequence OR=3.88;by genotype frequence OR=3.92).Furthermore,the -8,103G/A and T1309C polymorphisms were significantly associated with hyperuricemia(OR=1.43).The allele frequence distribution of -454A＞T,-434T＞C,-382C＞T,-87C＞T and +118G＞A in the promoter region were very different between two groups(P＜0.05),but no different by genotype frequence.No association was found for the other polymorphisms.4.Construction of hURAT1 haplotypesWe estimated the hURAT1 haplotypes of 8 SNPS(-454A＞T,-434T＞C,-382C＞T, -87C＞T,+118G＞A,+3,11 G＞A,-8,103A＞G,T1309C) in the cases and controls, separately.The 8 SNPS composed with 14 haplotypes,in which 10 haplotypes had afrequency of less than 2%were pooled into a single group and included in the haplotype analysis.Therefore,4 haplotypes coverd 98%people were received.The Ht4 haplotype,which was consistant of all mutational allele,was associated with a significantly increased risk of hyperuricedmia(OR=4.08,95%CI=1.95-8.55,P= 5.93×10^-5).Of note,only the Ht4 haplotype of the four haplotypes carrying the +3,11A allele was associated with a significantly increased risk of hyperuricedmia.Therefore,+3, 11 G＞A polymorphism may be associated with susceptibility ofhyperuricedmia5.logistic regression analysesTo confirm the causal variants,we tested the 13 SNP_s by multivariate logistic regression analysis.Regression modelling indicated that the +3,11G＞A was the causal variant because once the most-associated marker(+3,11G＞A) was included in the model,other markers did not improve the model,whereas the marker improved a model with any one of others.Individuals with allele A of +3,11G＞A polymorphism have higher levels of serum uric acid(by allele VS by genotype:496.35±169.41 VS 418.87±134.77,P=0.005).All these results indicated +3,11G＞A polymorphism is associated with hyperuricedmia.Conclusions:All these results suggest SNPS on hURAT1 confers susceptibility to primary hyperuricemia.+3,11G＞A polymorphism is the causal variant,Ht4 haplotype is the causal haplotype.