Molecular mechanisms of Axenfeld-Rieger syndrome

描述（由申请人提供）：本申请代表了我们在人类发育过程中，特别是在眼部结构形成方面，对to/co/cf相关转录因子PITX2的作用和功能的研究的延续。PITX2基因突变导致与人类青光眼相关的Axenfeld-Rieger综合征（ARS），并在PITX2 -/-敲除小鼠中导致眼睛发育停滞。在我们之前的研究中，我们在几个物种中鉴定了Pitx2转录本和基因组序列。在Axenfeld-Rieger综合征和其他类似疾病患者中筛选了人类PITX2序列的突变，并确定了多个突变。我们还开始培养携带Pitx2基因显性阴性突变的敲入小鼠，该突变与一名严重ARS患者中发现的突变相同。这些研究为我们进一步研究PITX2在发育中的作用奠定了基础，我们的具体目标是：1)阐明PITX2在眼发育过程中调控的分子机制。在此目标下，将确定参与Pitx2调控的区域以及与这些序列相互作用的因子；2)通过对PITX2基因携带K88E显性负突变的突变小鼠和人细胞系进行表征，探讨PITX2失活的病理影响。建立与Pitx2突变相关的ARS动物模型将有助于研究Pitx2在发育、鉴定修饰剂和评价治疗剂中的作用机制。由于Pitx2基因敲除小鼠通常不表现出Axenfeld-Rieger特征，而纯合子-/-动物由于多种缺陷在出生前死亡，因此无法对这些动物进行Axenfeld-Rieger异常和青光眼的综合研究。我们预计携带Pitx2基因显性阴性突变的Pitx2（K88E）小鼠将为研究a - r综合征提供更好的模型。鉴定PITX2的下游靶点将通过分析表达野生型和突变型PITX2和微阵列的细胞/组织中的差异基因表达来进行。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促进我们对正常眼睛发育机制的认识，并在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.