Molecular mechanisms of Axenfeld-Rieger syndrome

描述（由申请人提供）：本申请代表我们对to/co/cf相关转录因子PITX 2在人类发育过程中的作用和功能的研究的继续，特别是关于眼结构的形成。PITX 2基因的突变导致人类青光眼相关的Axenomyo-Rieger综合征（ARS），以及Pitx 2-/-敲除小鼠眼睛发育的停滞。在我们以前的研究中，我们的特点Pitx 2转录本和基因组序列在几个物种。在患有Axene-Rieger综合征和其他类似病症的患者中筛选人PITX 2序列的突变，并且已经鉴定出多种突变。我们还启动了基因敲入小鼠的产生，该小鼠在Pitx 2基因中携带与在患有严重形式的ARS的患者中发现的突变相同的显性阴性突变。这些研究为我们进一步研究PITX 2在发育中的作用奠定了基础，具体目的是：1）阐明眼发育过程中PITX 2调控的分子机制。在此目标下，参与调控Pitx 2的区域和与这些序列相互作用的因子将被鉴定; 2）通过表征在PITX 2基因中携带K88 E显性负突变的突变小鼠和人类细胞系来探索PITX 2失活的病理效应。建立与Pitx 2突变相关的ARS动物模型将有助于研究Pitx 2在发育中的作用机制，鉴定修饰剂和评估治疗药物。由于Pitx 2基因敲除小鼠通常不表现出Axenlav-Rieger特征，而纯合-/-动物由于多种缺陷而在出生前死亡，因此不可能在这些动物中进行Axenlav-Rieger异常和青光眼的全面研究。我们预期Pitx 2基因显性负突变的Pitx 2（K88 E）小鼠将成为研究A-R综合征的较好模型。将通过分析表达野生型和突变型PITX 2的细胞/组织中的差异基因表达和微阵列来鉴定PITX 2的下游靶标。 将对人类患者进行突变分析，以确定在人眼中发挥最关键作用的因素。我们认为，这些研究将推进我们的知识的机制，正常的眼睛发育和青光眼的分子水平的理解。

DESCRIPTION (provided by applicant): This application represents a continuation of our studies of a role and function of a to/co/cf-related transcription factor PITX2 during human development, particularly in respect to the formation of ocular structures. Mutations in the PITX2 gene cause Axenfeld-Rieger syndrome (ARS) associated with glaucoma in humans and arrest in eye development in Pitx2-/- knockout mice. In our previous studies we characterized Pitx2 transcripts and genomic sequences in several species. Human PITX2 sequences were screened for mutations in patients with Axenfeld-Rieger syndrome and other similar conditions and multiple mutations have been identified. We also initiated generation of a knock-in mouse carrying dominant-negative mutation in Pitx2 gene identical to the mutation found in a patient with a severe form of ARS. These studies form basis for our further investigation of PITX2 role(s) in development that are specifically aimed at: 1) To elucidate molecular mechanisms underlying Pitx2 regulation during ocular development. Under this aim, regions involved in regulation of Pitx2 and factors interacting with these sequences will be identified; 2) To explore pathological effects of PITX2 inactivation by characterization of mutant mice and human cell lines carrying K88E dominant-negative mutation in the PITX2 gene. Creation of ARS animal model associated with Pitx2 mutations will facilitate studies of mechanisms of Pitx2's action in development, identification of modifiers and evaluation of therapeutic agents. Because of the fact that Pitx2 knockout mice generally do not demonstrate Axenfeld-Rieger features while homozygous -/- animals die before birth due to multiple defects, comprehensive studies of Axenfeld-Rieger anomalies and glaucoma are not possible in these animals. We anticipate that Pitx2(K88E) mice carrying dominant-negative mutation in Pitx2 gene will result in a better model to study A-R syndrome. Identification of downstream targets of PITX2 will be performed by analysis of the differential gene expression in cells/ tissues expressing wild type and mutant forms of PITX2 and microarrays. Mutation analysis in human patients will be performed to identify factors playing the most critical role in the human eye. We contend that these studies will advance our knowledge of the mechanisms of normal eye development and understanding of glaucoma at the molecular level.