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: 7894625
• 负责人: KANG ZHANG
• 金额: $ 34.41万
• 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/7894625
• 关键词: 10q26; Age; Age related macular degeneration; Angiography; Antibodies; Applications Grants; Biochemistry; Blindness; Choroidal Neovascularization; Data; Development; Diagnostic; Disease; Early treatment; Elderly; 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; Transgenic Mice; Variant; Visual impairment; Western Blotting; Wild Type Mouse; aging population; angiogenesis; case control; cohort; disorder risk; in vivo; mutant; public health relevance

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）：与年龄相关的黄斑变性（AMD）是发达国家老年人视觉障碍的最常见原因。尽管其在老龄化人群中的患病率有所提高，但其病因和发病机理的理解很少，治疗方案受到限制。我们和其他人已经证明，Htra1可能在对AMD的遗传敏感性中起主要作用。该提案的目标是进一步分析第10季度的可能因果变体，定义了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起关键作用。在这一假设的指导下，我们建议进行以下特定目标。具体目标1：确定LOC387715/HTRA1区域中与AMD相关的所有SNP变体，并研究它们在转基因小鼠中的作用。我们的初步结果定义了一种主要的疾病单倍型，该单倍型跨越了10季度的10 kb区域，这解释了AMD的主要风险。该区域包含先前确定的SNP LOC387715/ARMS2 RS10490924和HTRA1 RS11200638，已经显示与AMD相关。我们建议通过全面的直接测序分析来识别该区域中的所有SNP变体。新发现的SNP将用于案例控制关联研究中，以调查其与AMD在大型队列中的关联。功能研究将在携带这些与AMD相关的SNP的转基因小鼠中进行。特定目的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中独立于视网膜中的作用，我们将生成有条件的敲除小鼠，从而在视网膜或RPE中删除Htra1。病理将在条件敲除（KO）中通过眼镜检查，组织学，ERG和HTRA1表达在QPCR，Western blot和Immunoychovemistry（IHC）中确定。 HTRA1功能的丧失将通过测量脉络膜新生血管形成（CNV）的降低或无能为力来确定。特定目的3：检查HTRA1在体内AMD发病机理中的作用。 A.通过在CNV模型中注入HTRA1抗体来抑制CNV。我们的初步数据表明，HTRA1的过表达可能有助于人类的AMD发病机理。为了验证这一点，我们计划量化HTRA1表达以及HTRA1抗体在激光诱导的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。为了描绘HTRA1（蛋白酶，TGF？抑制或IGF结构域）的哪种功能参与体内AMD发病机理，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域（？MAC）或组成性活性蛋白酶。我们将通过眼镜检查，血管造影，免疫肌化学，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。对疾病风险具有重大影响的基因的鉴定可能会定义与其发病机理有关的关键分子途径。这可能会导致针对根本原因和症状前诊断的疗法，以允许早期干预和治疗。公共卫生相关性：该赠款申请的重点是对造成年龄相关的黄斑变性的主要基因（HTRA1）的调查，这是失明的主要原因，具有重大的公共卫生影响。