HURAT1 Gene Promoter Region-87C / T SNP With Primary Gout Associated And Function

Objective:human urate transporter 1(hURAT1) is one of the most important transporters which plays an important role in maintaining the uric acid level. According to our previous report, we found -87 C/T SNP in the promoter showed a statistically significant association with hyperuricacid. Further, the luciferase assay results demonstrated that transcriptional activity of the promoter of hURATl gene containing the C allele was significantly higher in comparison to the T-allele. As the genetic factors of gout and hyperuricacid are not all the same, we want to confirm whether this SNP is associated with gout of Han males in Shandong Province. Additionally, this study is also designed to discuss the mechanism of the different transcription activities for the SNP.Methods:We included 335 Han gout males in Shandong Province and 376 healthy controls. Total genome DNA was prepared from human total blood. Then DNA was subjected to PCR amplification containing-87C/T SNP. The PCR products were analysised by DNA sequencing. X2 test was used for comparing genotype and allele frequencies, which can further explain whether there is association between the SNP and gout. In addition, we used Transfac 6.0 and Alibaba 2.1 software to predict the different transcriptional factors between-87C/T SNP. Nuclear extracts were extracted from OK cells and probes containing-87C/T SNP were labeled with ddUTP. Then EMSA assay was used for observing protein-probe interaction for this SNP. Furthermore, we estimated the possible transcription factors involved for the SNP by cold competition assay. The unlabeled probes used were CREB, GR and USF.Results:1. hURATl polymorphisms of -87C/T SNP is significantly associated with the risk of gout (P=0.004). Carriers of the "C" allele has a 1.43 fold increased risk of gout. 2. The Transfac Database shows that the C-allele of-87C/T SNP may increase a binding site of USF and the T-allele may increase a Glucocorticoid Receptor (GR) binding site. The Alibaba2.1 predicts that C allele of the SNP may be in the CREB binding site. Additionally, besides the different quantities of transcriptional factors, different combined activities above may be involved for the SNP.3. By EMSA assay there are two bands for both alleles of the SNP, but a dramatic reduction in the above band for the C allele. Cold competition assay shows that the specific bands are successfully competed for the cons GR probe and cons CREB probe, but not the cons USF probe.Conclusions:-87 C/T SNP in the promoter of hURAT1 gene is associated with risk of gout of Han males in Shandong Province. The different transcriptional activities for this SNP is due to different combined affinity of GR.