Genetically testing mechanisms of ocular development and disease

描述（由申请人提供）：IV型胶原α 1基因（Col4a1）突变可导致小鼠前段发育不良（ASD）和眼压升高。最近，在ASD、高眼压和青少年青光眼的一个家族中发现了COL4A1突变，然而，COL4A1并没有被广泛认为是ASD的候选基因。我们假设COL4A1及其结合伙伴COL4A2的突变是尚未确定突变的患者ASD的基础。在Aim 1中，我们将通过对已知没有其他ASD致病基因突变的ASD患者进行突变分析，直接验证我们的假设。此外，我们试图确定Col4a1突变在何处以及通过何种细胞机制导致ASD。我们已经确定COL4A1在发育过程中普遍存在于眼基底膜中，这使得从继发性病理中确定原发性发病部位的任务变得复杂。为了克服这一障碍，我们开发并验证了Col4a1的条件突变等位基因。在Aim 2中，我们将仅在发育中的晶状体或仅在神经嵴起源的细胞中表达突变，以确定它们对ASD的相对贡献。最后，为了了解Col4a1突变如何导致眼部发育不良，并开始组装发育和致病途径，我们将确定ASD的遗传修饰因子。我们已经成功地绘制了一个位点，它能够强烈地拯救ASD，并允许突变小鼠拥有几乎正常发育的眼睛。在目标3中，我们将对两种不同的遗传背景进行系统的大规模筛选，以确定额外的显性或隐性修饰位点和基因。了解基因修饰物如何拯救疾病，可以为有针对性的治疗干预提供有价值的见解。据我们所知，目前没有其他小组评估COL4A1或COL4A2在眼部发育和疾病中的作用。本文所概述的实验利用了宝贵而独特的资源，将为正常眼部发育的机制和致病途径提供重要的新见解。

DESCRIPTION (provided by applicant): Mutations of the type IV collagen alpha 1 gene (Col4a1) can cause anterior segment dysgenesis (ASD) and elevated intraocular pressure in mice. Recently, a COL4A1 mutation was identified in a family with ASD, ocular hypertension and juvenile glaucoma, however, COL4A1, has not widely been considered a candidate gene for ASD. We hypothesize that mutations of COL4A1 and its binding partner COL4A2 underlie ASD in patients for whom mutations have not yet been identified. In Aim 1, we will test our hypothesis directly by performing mutational analysis on ASD patients that are known not to have mutations in other ASD-causing genes. Moreover, we seek to determine where, and by what cellular mechanism(s), Col4a1 mutation leads to ASD. We have determined that COL4A1 is ubiquitously present in ocular basement membranes during development, which complicates the task of determining sites of primary pathogenesis from secondary pathology. To overcome this obstacle we have developed and validated a conditional mutant allele of Col4a1. In Aim 2, we will express the mutation only in the developing lens or only in cells of neural crest origin to determine their relative contributions to ASD. Finally, to understand how Col4a1 mutations lead to ocular dysgenesis and to begin to assemble developmental and pathogenic pathways, we will identify genetic modifiers of ASD. We have already successfully mapped a locus that is able to strongly rescue ASD and permit mutant mice to have nearly normally developed eyes. In Aim 3, we will perform systematic large-scale screens of two distinct genetic backgrounds to identify additional dominant or recessive modifier loci and genes. Understanding how genetic modifiers rescue disease could provide valuable insight for how targeted therapeutic interventions might do the same. To our knowledge, no other group is currently evaluating the role of COL4A1 or COL4A2 in ocular development and disease. The experiments outlined in this proposal take advantage of valuable and unique resources and will provide important new insights into the mechanisms of normal ocular development and pathogenic pathways. PUBLIC HEALTH RELEVANCE: This application seeks to identify a novel genetic cause of ocular dysgenesis that leads to early onset and aggressive glaucoma. The study will use unique genetic tools to understand how these genes lead to disease and which cellular pathways might be targeted with novel therapeutic interventions.