新疆维、哈、汉民族SLC2A9基因功能区突变与痛风的关联研究和功能验证

Uric acid transporter protein mutation or abnormal expression can leads to uric acid reabsorption and excretion disorder, this maybe the mainly reason for hyperuricemia and gout.But the exact mechanism has not been elucidated. Facilitated glucose transporter protein-9 (SLC2A9) located in the renal proximal tubule epithelial cell basolateral membrane, plays an important role in the process of reabsorption of uric acid. Our research team first discovered the uric acid levels, incidence of hyperuricemia and gout were difference in different ethnic. We still found SLC2A9 gene promoter region SNPs were different too in these ethnic, the genetic mechanisms must be verified. So this study intends to detect SLC2A9 gene promoter region SNPs and tag SNPs use SNaPshot method in gout and countrol group of Han,Uygur and Hazarker nations firstly, then using site-directed mutagenesis and dual luciferase reporter gene system to detect the relationship between SNPs and gene transcription. Finally, apply RT-PCR and Western Blot technology to detect SLC2A9 protein levels of renal tubular epithelial cells in patients with gout from Renal biopsy and urine sediment sample. The aim is want to found the molecular pathogenesis mechanism of gout and ethnic heterogeneity from a gene funtion new perspective, give a new drug targets specific for treatment and prevention to different nations gout patient in future.

目前认为痛风主要与尿酸转运蛋白变异或表达异常导致尿酸重吸收增加和排泄减少有关，确切机制尚未阐明。葡萄糖易化转运蛋白-9（SLC2A9) 主要分布于肾脏近曲小管上皮细胞的基底外膜，与尿酸重吸收密切相关。本课题组首次发现新疆不同民族尿酸水平、高尿酸血症、痛风发病率存在差异，该基因启动子区SNPs小样本筛查也存在民族差异，差异是否与该基因功能区突变导致基因转录和蛋白表达异常有关有待查明。本研究拟利用SNaPshot方法检测不同民族痛风患者和对照人群SLC2A9基因功能区SNPs和tag SNPs，采用定点突变技术及双荧光素酶报告基因系统检测阳性SNPs位点对基因转录水平的影响，荧光定量PCR和Western Blot方法检测痛风患者肾活检组织和尿沉渣中脱落的肾小管上皮细胞中SLC2A9蛋白表达水平，从功能研究的角度探讨痛风发病分子机制及民族异质性的深层次机理，为不同民族痛风特异性防治提供新的靶点。

研究背景：痛风主要与尿酸转运蛋白变异或表达异常导致尿酸重吸收增加和排泄减少有关，确切机制尚未阐明。SLC2A9主要分布于肾脏近曲小管上皮细胞的基底外膜，与尿酸重吸收密切相关。本课题组首次发现新疆汉族、维吾尔族和哈萨克族血尿酸水平尿酸代谢紊乱存在差异，汉族高于维吾尔族，尤以哈萨克族最低。研究内容：(1)SLC2A9基因启动子区SNPs和tag SNPs 筛选;(2)新疆维、汉、哈三个民族男性痛风患者和正常对照人群SLC2A9基因启动子区筛选出的SNPs和tagSNPs的基因分型;(3)SLC2A9基因启动子区突变与痛风的关联性和民族差异分析;(4)SLC2A9基因启动子区突变对基因转录的影响及功能验证。重要结果：维吾尔族体重、BMI、腰围、臀位、臀比和血压值均高于其两个民族(P<0.05)。汉族、维吾尔族和哈萨克族血尿酸水平分别为371.91±126.33µmol/L、358.75±119.72µmol/L和274.21±118.68µmol/L，哈萨克族最低，具有民族差异。汉族高尿酸血症(40.0%)检出率明显高于维吾尔族(35.5%)，哈萨克族高尿酸血症检出率最低(18.8%)。不同民族糖脂代谢指标和糖脂代谢紊乱也表现出民族差异。汉、维、哈三个民族SLC2A9基因不同位点基因突变也具有民族异质性。rs13137343和rs13137074位点是汉族和维吾尔族高尿酸血症和痛风可能的遗传危险因素，携带rs13137074位点T等位基因和rs13137343位点的人群发生高尿酸血症的风险较高。rs13137343位点A等位基因也可能与哈萨克族高尿酸血症发生风险有关。SLC2A9基因位点突变对启动子激活能力的影响进行检测发现分别转染rs13137343和rs13137074位点突变型报告载体，与野生型载体比较，荧光素酶Luciferase的活性分别下调了42.51%和23.60%，提示这两个位点对SLC2A9基因的转录活性发生了重要作用。科学意义：(1)本研究发现SLC2A9基因不同SNPs与高尿酸血症和痛风的关联性以及民族差异，初步探讨了发现三个民族尿酸代谢的分子遗传学机制。(2)本研究对尿酸代谢关键基因研究热点区域进行探索性工作，探索了高尿酸血症的分子遗传学机理及民族异质性，同时补充完善中国人群SNP数据库，具有潜在的应用价值。