Functional analysis of AMD-associated HTRA1 promoter polymorphism

描述（由申请人提供）：本提案的目的是定义HTRA1启动子多态性的功能作用，该多态性最近被确定为与年龄相关性黄斑变性（AMD）易感性相关的变异，并开始了解调节HTRA1表达的机制。黄斑变性是发达国家老年人不可逆失明的主要原因，是一种病因复杂的疾病，由多种遗传和环境因素引起。连锁研究显示，两个染色体区域，1q31-32和10q26，可能含有AMD的易感基因。虽然补体因子H （CFH）似乎是1q32上的主要易感基因，但10q26上的关键区域一直存在争议。虽然有研究发现假设基因LOC387715中的单核苷酸多态性（SNP） rs10490924是10q26上的AMD相关变异，但其他研究表明SNP rs11200638 (G>A)，位于HTRA1基因的假设启动子区域的G to A变异，是10q26上真正的AMD相关序列变化，并导致HTRA1启动子中的rs11200638导致HTRA1表达升高，从而增加湿性AMD的风险。我们在小鼠RPE中使用体内电穿孔法对这一假设进行了分析，结果显示，具有风险相关A的HTRA1启动子的活性显著低于具有非风险相关G的启动子，这与假设的预期结果相反。我们的酵母单杂交筛选发现两个转录因子与G等位基因的诱饵结合，而不与A等位基因的诱饵结合。最近，HTRA1位点上的其他SNP，包括启动子中的SNP rs2672598 (C>T)，被报道为与AMD风险显著相关的变异，支持HTRA1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，我们提出对HTRA1启动子及其变体的详细分析如下。在Specific Aim 1中，HTRA1启动子的活性将通过瞬时转染在牛RPE原代细胞中分析，并通过体内电穿孔在小鼠RPE中分析。这些分析将涉及两个启动子变体rs11200638和rs2672598的HTRA1启动子-荧光素酶报告基因构建，以及一系列启动子缺失。在Specific Aim 2中，我们将通过酵母单杂交筛选，进一步鉴定与rs11200638区域结合的转录因子，并对已经鉴定的两个因子CGGBP1和SLC2A4RG进行分析。电泳迁移量转移法（EMSA）和染色质免疫沉淀法（ChIP）将分别用于分析这些因子在体外和体内的结合。用表达载体共转染和小干扰RNA （siRNA）转染将分别用于通过过表达和基因敲低来分析这些因子的功能获得和功能丧失。这些分析结果将有助于解决关于HTRA1启动子变异的争议，并深入了解AMD的发病机制。

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.