Functional analysis of AMD-associated HTRA1 promoter polymorphism

AMD相关HTRA1启动子多态性的功能分析

• 批准号: 7639793
• 负责人: NORIKO ESUMI
• 金额: $ 20.5万
• 依托单位: JOHNS HOPKINS UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-09-01 至 2011-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7639793
• 关键词: 10q26; 1q31; 1q32; Age related macular degeneration; Alleles; Binding; Binding Proteins; Biochemical; Biological Assay; Blindness; Cattle; Cells; Chromosomes; Code; Complement Factor H; Complex; Developed Countries; Developing Countries; Disease; Elderly; Electrophoretic Mobility Shift Assay; Electroporation; Environmental Risk Factor; Exons; Genes; Genetic; Genetic Polymorphism; Human; Human Genome; Hybrids; In Vitro; Luciferases; Mus; Nucleic Acid Regulatory Sequences; Pathogenesis; Play; Positioning Attribute; Predisposition; Promoter Regions; Reporter; Reporting; Repression; Research; Reverse Transcriptase Polymerase Chain Reaction; Risk; Role; Series; Single Nucleotide Polymorphism; Small Interfering RNA; Susceptibility Gene; Testing; Transfection; Variant; Yeasts; base; cDNA Library; chromatin immunoprecipitation; expression vector; gain of function; in vivo; insight; loss of function; overexpression analysis; promoter; public health relevance; transcription factor; yeast two hybrid system

DESCRIPTION (provided by applicant): The objective of this proposal is to define the functional role of the HTRA1 promoter polymorphism that was recently identified as a variant associated with susceptibility to age-related macular degeneration (AMD), and to begin to understand the mechanism regulating HTRA1 expression. AMD is the leading cause of irreversible blindness among elderly people in the developed countries and an etiologically complex disease caused by multiple genetic and environmental factors. Linkage studies revealed two chromosomal regions, 1q31-32 and 10q26, that likely contain susceptibility genes for AMD. Although complement factor H (CFH) appears to be the major susceptibility gene on 1q32, the critical region on 10q26 has been controversial. While several studies identified single nucleotide polymorphism (SNP) rs10490924 in the hypothetical gene LOC387715 as the AMD-associated variant on 10q26, other studies suggested SNP rs11200638 (G>A), a G to A variant located in the putative promoter region of the HTRA1 gene, as the true AMD-associated sequence change on 10q26 and led to the hypothesis that the rs11200638 in the HTRA1 promoter causes higher HTRA1 expression, and thereby increases the risk for wet AMD. Our analyses to explore this hypothesis using in vivo electroporation in mouse RPE showed that the HTRA1 promoter with risk-associated A had a significantly lower activity than the promoter with non-risk-associated G, opposite to the result expected from the hypothesis. Our yeast one-hybrid screen identified two transcription factors that bound to bait with G allele, but not to bait with A allele. More recently, additional SNPs in the HTRA1 locus, including SNP rs2672598 (C>T) in the promoter, have been reported as variants significantly associated with the risk for AMD, supporting the likelihood of HTRA1 as the susceptibility gene for AMD. Based on these findings and our own results, detailed analyses of the HTRA1 promoter and its variants are proposed as below. In Specific Aim 1, HTRA1 promoter activity will be analyzed in bovine RPE primary cells by transient transfection and in mouse RPE by in vivo electroporation. These analyses will involve HTRA1 promoter- luciferase reporter constructs for the two promoter variants, rs11200638 and rs2672598, as well as a series of promoter deletions. In Specific Aim 2, additional transcription factors that bind to the rs11200638 region will be identified by yeast one-hybrid screen, and the two factors already identified, CGGBP1 and SLC2A4RG, will be analyzed. Electrophoretic mobility shift assay (EMSA) and chromatin immunoprecipitation (ChIP) will be used to analyze binding of these factors in vitro and in vivo, respectively. Cotransfection with an expression vector and small interfering RNA (siRNA) transfection will be used for gain-of-function analysis by overexpression and loss-of-function analysis by gene knockdown of these factors, respectively. Results of these analyses will help solve the controversy about the HTRA1 promoter variant and gain insights into the pathogenesis of AMD. PUBLIC HEALTH RELEVANCE: Age-related macular degeneration (AMD) is the leading cause of irreversible blindness among elderly people in the developed countries including the USA. Multiple genes and environmental factors seem to play important roles in developing the disease. A large-scale genetic studies have identified multiple sequence changes in the human genome that are associated with and possibly responsible for the risk of developing AMD, and several of such sequence variants are located in or near the HTRA1 gene. Therefore, the aim of this research is to experimentally analyze the two variants identified in the HTRA1 promoter region and determine their functional consequence on the expression of the HTRA1 gene.

描述（由申请人提供）：本提案的目的是确定HTRA 1启动子多态性的功能作用，该多态性最近被鉴定为与年龄相关性黄斑变性（AMD）易感性相关的变体，并开始了解调节HTRA 1表达的机制。AMD是发达国家老年人不可逆失明的主要原因，是由多种遗传和环境因素引起的病因复杂的疾病。连锁研究揭示了两个染色体区域，1 q31 -32和10 q26，可能含有AMD的易感基因。虽然补体因子H（CFH）似乎是1 q32上的主要易感基因，但10 q26上的关键区域一直存在争议。虽然有几项研究将假设基因LOC 387715中的单核苷酸多态性（SNP）rs 10490924确定为10 q26上的AMD相关变体，但其他研究表明SNP rs 11200638（G>A），一种位于HTRA 1基因推定启动子区域的G至A变体，由于10 q26上真正的AMD相关序列改变，并导致HTRA 1启动子中的rs 11200638引起更高的HTRA 1表达，从而增加湿性AMD的风险的假设。我们的分析，以探讨这一假设，在小鼠RPE体内电穿孔显示，HTRA 1启动子与风险相关的A有一个显着较低的活性比启动子与非风险相关的G，相反的结果预期从假设。我们的酵母单杂交筛选确定了两个转录因子与G等位基因的诱饵结合，但不与A等位基因的诱饵结合。最近，HTRA 1基因座中的其他SNP，包括启动子中的SNP rs 2672598（C>T），已被报道为与AMD风险显著相关的变体，支持HTRA 1作为AMD易感基因的可能性。基于这些发现和我们自己的结果，HTRA 1启动子及其变体的详细分析如下。在特定目标1中，将通过瞬时转染在牛RPE原代细胞中分析HTRA 1启动子活性，并通过体内电穿孔在小鼠RPE中分析HTRA 1启动子活性。这些分析将涉及两种启动子变体rs 11200638和rs 2672598的HTRA 1启动子-荧光素酶报告基因构建体，以及一系列启动子缺失。在特异性目的2中，将通过酵母单杂交筛选鉴定与rs 11200638区域结合的其他转录因子，并分析已经鉴定的两个因子CGGBP 1和SLC 2A 4 RG。电泳迁移率变动试验（EMSA）和染色质免疫沉淀（ChIP）将分别用于分析这些因子在体外和体内的结合。与表达载体共转染和小干扰RNA（siRNA）转染将分别用于通过这些因子的过表达进行的功能获得分析和通过基因敲减进行的功能丧失分析。这些分析的结果将有助于解决HTRA 1启动子变异的争议，并深入了解AMD的发病机制。 公共卫生关系：视网膜相关性黄斑变性（AMD）是包括美国在内的发达国家中老年人不可逆失明的主要原因。多种基因和环境因素似乎在这种疾病的发生中发挥着重要作用。一项大规模的遗传学研究已经确定了人类基因组中的多个序列变化，这些变化与AMD的发生风险相关并可能导致AMD的发生，其中几个序列变异位于HTRA 1基因中或附近。因此，本研究的目的是实验分析在HTRA 1启动子区域中鉴定的两种变体，并确定它们对HTRA 1基因表达的功能后果。