Molecular mechanisms of Axenfeld-Rieger syndrome

描述(申请人提供)：本申请代表了我们对与to/co/cf相关的转录因子PITX2在人类发育过程中的作用和功能的研究的继续，特别是在眼睛结构的形成方面。PITX2基因的突变导致人类青光眼相关的阿森菲尔德-里格综合征(ARS)，以及Pitx2/-基因敲除小鼠的眼睛发育停滞。在我们之前的研究中，我们描述了几个物种的Pitx2转录本和基因组序列。对患有阿森菲尔德-里格综合征和其他类似疾病的患者的人类PITX2序列进行了突变筛查，并发现了多个突变。我们还启动了一种携带Pitx2基因显性-负突变的敲入小鼠的生成，该突变与在一名严重形式的ARS患者中发现的突变相同。这些研究为我们进一步研究PITX2(S)在发育中的作用奠定了基础，具体目的是：1)阐明眼睛发育过程中PITX2调控的分子机制。2)通过对携带PITX2基因K88E显性-负性突变的突变小鼠和人细胞株的鉴定，探讨PITX2失活的病理效应。建立与Pitx2基因突变相关的ARS动物模型将有助于研究Pitx2‘S在发育过程中的作用机制、寻找修饰剂和评价治疗药物。由于Pitx2基因敲除小鼠一般不表现出AXENFELD-Rieger特征，而纯合子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AXENFeld-Rieger异常和青光眼的全面研究。我们预计，携带Pitx2基因显性-负性突变的Pitx2(K88E)小鼠将为研究A-R综合征提供更好的模型。PITX2下游靶点的鉴定将通过分析表达野生型和突变型PITX2的细胞/组织和微阵列中的差异基因表达来完成。将对人类患者进行突变分析，以确定对人眼起最关键作用的因素。我们认为，这些研究将促进我们对正常眼睛发育机制的了解，并从分子水平上理解青光眼。

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.