RAS系統和TGF-β1的基因多型性與原發性VUR疾病發生和腎病進行的相關性研究

Abstract

膀胱輸尿管逆流(VUR)為常見的小兒泌尿道疾病，部分病患會進行至末期腎病(ESRD)。VUR的發生與家族遺傳具高度相關性，且患者間的腎病進行差異很大。RAS系統和TGF-β1參與了泌尿系統的發育過程，且可能與腎臟疾病的病程進行相關。本研究以PCR及直接定序、限制酶切割或單股核酸構形多型性分析等技術，檢視了16位末期腎病與58位非末期腎病之原發性VUR病童和117位正常人，其RAS系統相關基因的AGT、ACE、AT1R及TGF-β1基因共25個多型性，和原發性VUR疾病發生及腎病進行的相關性。結果發現AGT基因之C-18T和TGF-β1基因之A-880G、G-800A、R25P、T263I等多型性並不存在於台灣族群中，並於ACE基因上檢視到未曾正式報導的A-3692C多型性。在族群遺傳分析方面，正常人族群中各多型性基因座的遺傳情形均處於哈溫平衡。AGT基因之C-532T與G-217A、A-20C與T174M、G-6A與M235T多型性間呈現強烈的連鎖不平衡現象；ACE基因啟動子上T-5491C、A-5466C、T-3892C、A-3692C、A-240T與介入子16內的I/D多型性間亦呈現非逢機的組合，顯示基因內重組率相當低；AT1R基因之A-1138T、T-810A、T-713G、C-521T、A-214C/G-213C及A-153G多型性間亦呈現強烈的連鎖不平衡。若針對原發性VUR病患與正常人族群之多型性對偶基因頻率進行相關性分析，僅TGF-β1基因之C-509T多型性具顯著性差異，其中對偶基因T與原發性VUR疾病發生有關。若以末期腎病的有無將原發性VUR患者分為二群時，則ACE基因上連鎖之多型性與腎病進行相關，其中T-A-T-A-A-I之單套型形式可視為病程惡化的危險因子。在多型性啟動子的轉錄活性分析方面，本研究構築了RAS系統相關基因之多型性單套型啟動子重組質體，以報導基因luciferase於293細胞株中進行體外基因表現。結果顯示包含A-240T及T-93C多型性之1.2 kb長(-1207 ~ +4)的ACE基因啟動子片段，與包含A-1138T、T-810A、T-713G、C-521T、A-214C/G-213C及A-153G多型性之1.4 kb長(-1332 ~ +45)的AT1R基因啟動子片段，均不影響報導基因的表現量；而包含C-532T、G-217A、G-152A、A-20C及G-6A多型性之0.7 kb長(-567 ~ +116)的AGT基因啟動子片段，含對偶基因-152G的啟動子轉錄活性達顯著差異，其中對偶基因G較A約有2.4倍高的轉錄活性。Vesicoureteral reflux (VUR) is a common pediatric disease that may lead to severe end-stage renal disease (ESRD) in part of patients. The development of VUR is highly familial inherited and the disease progression is variable. The renin-angiotensin system (RAS) and transforming growth factor-b1 (TGF-b1) involve in the development of urinary system and may also be the potential candidate prognostic factors in the progression of ESRD. In this study, the RAS related angiotensinogen (AGT), angiotensin-converting enzyme (ACE), and angiotensin II type 1 receptor (AT1R) as well as TGF-b1 gene polymorphisms were investigatedfor association with VUR susceptibility and progression in 74 Taiwanese children, including 16 with ESRD, and 117 normal controls. By polymerase chain reaction and directing sequencing, restriction enzyme digestion, or single strand conformation polymorphism analysis, a total of 25 polymorphisms within the AGT, ACE, AT1R, and TGF-b1 genes were studied. The novel ACE A-3692C was not formally reported, and no TGF-b1 A-880G, G-800A, R25P and T263I polymorphisms were detected. All the polymorphisms examined were in Hardy-Weinberg equilibrium. In the AGT gene, strong linkage disequilibrium between C-532T and G-217A, A-20C and T174M, A-6G and M235T were observed. The strong non-random association among the ACE gene T-5491C, A-5466C, T-3892C, A-3692C, A-240T, and Alu I/D polymorphisms spanning promoter to intron 16 suggests low levels of intragenic recombination within the ACE gene. Similar strong linkage disequilibrium was also seen in the AT1R gene A-1138T, T-810A, T-713G, C-521T, A-214C/G-213C, and A-153G polymorphisms. A statistically significant difference between primary VUR patients and normal controls was observed at the TGF-b1 gene -509 site, with T allele associated with primary VUR development. Furthermore, a significant allele association with ESRD was observed for the ACE loci, with the linked T-A-T-A-A-I haplotype as a risk factor for primary VUR progression. To assess the effect of polymorphism on gene expression, fragments containing the polymorphic haplotypes were fused to firefly luciferase reporter construct and transiently expressed in 293 cells. Within the 1.2 kb ACE and 1.4 kb AT1R promoter fragments, no appreciable effect on the gene expression was observed for the linked ACE A-240T and T-93C as well as the linked AT1R polymorphisms. However, reporter construct containing the AGT -152 G allele drove 2.4 times transcriptional activity compared with the -152 A allele.