Molecular mechanisms of anterior segment disorders

项目摘要 眼前节发育不全（ASD）表型包括一组特征在于结构异常的病症。 涉及虹膜、角膜、虹膜角膜角和透镜的异常，并与高风险的视力相关。 青光眼、角膜混浊和其他并发症造成的损害。这组条件包括 Axenvan-Rieger异常（ARA）和综合征（ARS）、无虹膜、Peters异常/角膜混浊，以及 儿童青光眼（即使没有可见的ASD，也被认为与异常发育有关 流出道）。我们确定PITX 2突变是ARS的原因，然后扩大了与PITX 2突变的相关性。 第二个ARS基因FOXC 1随后也被证明可以解释不同的ASD表型。 ASD表型和最近的一项研究表明，它在POAG在多个群体。PITX 2和FOXC 1发挥着 在ASD中起主要作用，解释了40-60%的ARA/ARS，大多数阴性病例仍然无法解释。 对于更广泛的ASD谱，识别基因诊断的成功率在人群中各不相同，但是， 同样，仍然是不完整的（低于50%），需要确定新的机制。的 该项目的总体目标是通过以下方法揭示ASD和相关青光眼/视力损害的机制： 研究这种疾病的不同方面：人类表型，与PITX 2/FOXC 1相关的机制， 以及通过斑马鱼外显子组/基因组人类研究和CRISPR-Cas9编辑发现新基因。 具体来说，我们的目标是：1）揭示全方位的眼睛异常的“眼前节发育不全” 紊乱ASD患者的高分辨率成像将调查后段的可能性 缺陷及其对视力丧失的影响。这一目标将为人类疾病提供新的见解， 通过我们的初步数据，在两名（两名）ARS患者中发现了新的特征; 2）定义发育 FOXC 1和PITX 2的作用和途径，并探索它们对人类疾病的贡献。这一目标将 使用最近开发的细胞系鉴定斑马鱼中foxc 1的靶点，并探索其调控元件 基因我们从foxc 1调控缺失突变体之一的初步结果显示， 表型，不同于功能丧失系，并有可能成为第一个斑马鱼胚胎 青光眼突变体所获得的结果将与先前生成的pitx 2数据一起进行分析， 揭示了共同的路径。由于我们早期的数据表明WNT通路的失调是一个主要的 pitx 2缺陷的结果，其在眼睛表型中的作用将被进一步检查;最后，3）为了发现 利用家族外显子组/基因组数据研究在眼前节发育不全中起作用的新因素 在斑马鱼中进行ASD和基于基因组编辑的建模将对确定的因素进行评估， 利用我们以前开发的资源，在已知途径中定位。我们的应用程序的优势在于 在其创新的方法，我们产生的独特资源，强大的方法，和杰出的 我们的团队在脊椎动物遗传学，眼睛发育和ASD表型方面的专业知识。

PROJECT SUMMARY Anterior segment dysgenesis (ASD) phenotypes comprise a group of disorders characterized by structural abnormalities involving the iris, cornea, iridocorneal angle, and lens, and associated with a high risk of visual impairment through glaucoma, corneal opacification and other complications. This group of conditions includes Axenfeld-Rieger anomaly (ARA) and syndrome (ARS), aniridia, Peters anomaly/corneal opacities, and pediatric glaucoma (even in the absence of visible ASD, believed to be associated with abnormal development of the outflow tract). We identified mutations in PITX2 as the cause of ARS and then expanded associated phenotypes to other ASDs; the second ARS gene, FOXC1, was also subsequently shown to explain diverse ASD phenotypes and a recent study implicates it in POAG in multiple populations. PITX2 and FOXC1 play a major role in ASD, explaining 40-60% of ARA/ARS with the majority of negative cases remaining unexplained. For the broader ASD spectrum, the success rate for identifying a genetic diagnosis varies in populations but, likewise, continues to be incomplete (less than 50%) necessitating the identification of novel mechanisms. The overall goal of this project is to uncover mechanisms of ASD and associated glaucoma/visual impairment by investigating diverse aspects of this disease: human phenotype, mechanisms associated with PITX2/FOXC1, and novel gene discovery through exome/genome human studies and CRISPR-Cas9 editing in zebrafish. Specifically, we aim: 1) To reveal the full range of ocular anomalies in ‘anterior segment dysgenesis’ disorders. High-resolution imaging in individuals with ASD will investigate the possibility of posterior segment defects and their contribution to vision loss. This aim will provide new insight into human disease as suggested by our initial data which identified novel features in two (out of two) ARS patients; 2) To define developmental roles and pathways of FOXC1 and PITX2 and explore their contribution to human disease. This aim will identify targets of foxc1 in zebrafish using recently developed lines and explore regulatory elements of this gene. Our preliminary results from one of the foxc1 regulatory deletion mutants show a strong glaucoma phenotype, different from the loss-of-function line, and is likely to become the first zebrafish embryonic glaucoma mutant. The obtained results will be analyzed together with the previously generated pitx2 data to reveal common pathways. Since our earlier data indicate dysregulation of the WNT pathway as a major outcome of pitx2 deficiency, its role in the eye phenotype will be further examined; and finally, 3) To discover novel factors with a role in anterior segment dysgenesis by utilizing exome/genome data from families affected with ASD and genome editing-based modeling in zebrafish. The identified factors will be evaluated for their position in known pathways using our previously developed resources. The strength of our application is in its innovative approaches, the unique resources that we generated, robust approach, and the outstanding expertise of our team in vertebrate genetics, eye development and ASD phenotypes.