Genetics and Functional Studies of Age-Related Macular Degeneration

年龄相关性黄斑变性的遗传学和功能研究

• 批准号: 7528250
• 负责人: KANG ZHANG
• 金额: $ 34.76万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN DIEGO
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-09-30 至 2013-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7528250
• 关键词: 10q26; Age; Age related macular degeneration; Angiography; Antibodies; Applications Grants; Biochemistry; Blindness; Choroidal Neovascularization; Data; Development; Diagnostic; Disease; Early Intervention; Elderly; Endopeptidases; Etiology; Extracellular Matrix; Genes; Genetic; Haplotypes; Histology; Histopathology; Human; Immunohistochemistry; Injection of therapeutic agent; Investigation; Knock-out; Knockout Mice; Lasers; Lead; Macular degeneration; Measures; Modeling; Molecular; Mutation; Ophthalmoscopy; Pathogenesis; Pathology; Pathway interactions; Peptide Hydrolases; Play; Prevalence; Proteins; Public Health; Retina; Retinal; Risk; Role; Sequence Analysis; Somatomedins; Transgenic Mice; Variant; Visual impairment; Western Blotting; Wild Type Mouse; aging population; angiogenesis; case control; cohort; disorder risk; in vivo; mutant

DESCRIPTION (provided by applicant): Age-related macular degeneration (AMD) is the most common cause of visual impairment of the elderly in the developed world. Despite the increase in its prevalence within the aging population, its etiology and pathogenesis are poorly understood and treatment options are limited. We and others have demonstrated that HTRA1 may play a major role in genetic susceptibilty to AMD. The objectives of this proposal are to further analyze the possible causative variant(s) in 10q26, define the normal function of HTRA1, and to elucidate the molecular mechanisms leading to macular degeneration. Our central hypothesis is that HTRA1 plays a key role in retinal development, angiogenesis, and extracellular matrix modeling. Guided by this hypothesis, we propose to conduct the following specific aims. Specific Aim 1: To identify all SNP variants associated with AMD in LOC387715/HTRA1 region and investigate their role in AMD in transgenic mice. Our preliminary results have defined a major disease haplotype spanning a 10 kb region in 10q26 that explains the major risk of AMD. This region contains previously identified SNP LOC387715/ARMS2 rs10490924 and HTRA1 rs11200638, already shown to be associated with AMD. We propose to identify all SNP variants in this region by comprehensive direct sequencing analysis. Newly discovered SNPs will be used in a case control association study to investigate their association with AMD in a large cohort. Functional studies will be performed in transgenic mice carrying these AMD associated SNPs. Specific Aim 2: To determine the role of HTRA1 in retinal development and pathology. HTRA1 is expressed in the retina and RPE. In order to differentiate the role it plays in the RPE independent of that in retina, we will generate conditional knockout mice which delete HTRA1 in either the retina or RPE. Pathology will be examined in the conditional knockout (KO) by ophthalmoscopy, histology, ERG, and HTRA1 expression will be determined by QPCR, western blot and immunohistochemistry (IHC). Loss of HTRA1 function will be determined by measuring the reduction of or inability for choroidal neovascularization (CNV) to take place in homozygous and heterozygous conditional KO mice. Specific Aim 3: To examine the role of HTRA1 in AMD pathogenesis in vivo. A. Inhibition of CNV by injection of HTRA1 antibody in a CNV model. Our preliminary data indicate that over-expression of HTRA1 may contribute to AMD pathogenesis in humans. To verify this, we plan to quantify HTRA1 expression and the extent to which an HTRA1 antibody can inhibit CNV in a laser induced CNV model. B. Transgenic mice expressing WT and mutant HTRA1 genes. HTRA1 is a multi functional protein. In order to delineate which function of HTRA1 (protease, TGF? inhibition, or IGF domain) is involved in AMD pathogenesis in vivo, we will generate transgenic mice expressing different HTRA1 mutations. Mutants will lack either protease activity (SA mutant), the PDZ domain (?PDZ), the Mac25 domain (?Mac), or constitutively active protease. We will quantify the development of AMD-like features in these HTRA1 transgenic mice by ophthalmoscopy, angiography, immunohsitochemistry, biochemistry, and histopathology and compare them to age-matched wild-type mice. The identification of genes that have substantial impact on the risk of the disease may define key molecular pathways involved in its pathogenesis. This may lead to therapies directed at the underlying cause and pre-symptomatic diagnostics to allow for earlier intervention and treatment. PUBLIC HEALTH RELEVANCE: This grant application focuses on investigation of a major gene (HTRA1) that causes age-related macular degeneration, the leading cause of blindness with a significant public health impact.

描述（由申请人提供）：年龄相关性黄斑变性（AMD）是发达国家老年人视力障碍的最常见原因。尽管其在老龄化人口中的患病率有所增加，但其病因和发病机制仍知之甚少，治疗选择也有限。我们和其他人已经证明 HTRA1 可能在 AMD 的遗传易感性中发挥重要作用。本提案的目的是进一步分析10q26中可能的致病变异，定义HTRA1的正常功能，并阐明导致黄斑变性的分子机制。我们的中心假设是 HTRA1 在视网膜发育、血管生成和细胞外基质建模中发挥关键作用。在这一假设的指导下，我们建议实现以下具体目标。具体目标 1：鉴定 LOC387715/HTRA1 区域中与 AMD 相关的所有 SNP 变异，并研究它们在转基因小鼠 AMD 中的作用。我们的初步结果定义了一个跨越 10q26 10 kb 区域的主要疾病单倍型，这解释了 AMD 的主要风险。该区域包含先前鉴定的 SNP LOC387715/ARMS2 rs10490924 和 HTRA1 rs11200638，已显示与 AMD 相关。我们建议通过全面的直接测序分析来识别该区域的所有 SNP 变异。新发现的 SNP 将用于病例对照关联研究，以在大型队列中研究它们与 AMD 的关联。将在携带这些 AMD 相关 SNP 的转基因小鼠中进行功能研究。具体目标 2：确定 HTRA1 在视网膜发育和病理学中的作用。 HTRA1 在视网膜和 RPE 中表达。为了区分它在 RPE 中的作用与在视网膜中的作用无关，我们将生成条件敲除小鼠，在视网膜或 RPE 中删除 HTRA1。将通过检眼镜检查条件性敲除 (KO) 中的病理学、组织学、ERG，并通过 QPCR、蛋白质印迹和免疫组织化学 (IHC) 确定 HTRA1 表达。 HTRA1 功能的丧失将通过测量纯合和杂合条件 KO 小鼠中脉络膜新生血管 (CNV) 发生的减少或无法发生来确定。具体目标 3：研究 HTRA1 在体内 AMD 发病机制中的作用。 A. 在 CNV 模型中注射 HTRA1 抗体抑制 CNV。我们的初步数据表明，HTRA1 的过度表达可能有助于人类 AMD 发病机制。为了验证这一点，我们计划量化 HTRA1 表达以及 HTRA1 抗体在激光诱导 CNV 模型中抑制 CNV 的程度。 B.表达WT和突变HTRA1基因的转基因小鼠。 HTRA1 是一种多功能蛋白。为了阐明 HTRA1 的哪些功能（蛋白酶、TGFβ 抑制或 IGF 结构域）与 AMD 体内发病机制有关，我们将产生表达不同 HTRA1 突变的转基因小鼠。突变体将缺乏蛋白酶活性（SA突变体）、PDZ结构域（?PDZ）、Mac25结构域（?Mac）或组成型活性蛋白酶。我们将通过检眼镜、血管造影、免疫组织化学、生物化学和组织病理学来量化这些 HTRA1 转基因小鼠中 AMD 样特征的发展，并将它们与年龄匹配的野生型小鼠进行比较。鉴定对疾病风险有重大影响的基因可能会定义参与其发病机制的关键分子途径。这可能会导致针对根本原因的治疗和症状前诊断，以便及早干预和治疗。公共健康相关性：这项拨款申请的重点是研究导致年龄相关性黄斑变性的主要基因 (HTRA1)，黄斑变性是导致失明的主要原因，对公共健康产生重大影响。