Childhood Cataractogenesis: Heterogeneity of Gene Expression

描述（由申请人提供）：白内障发生通常与年龄相关，阻碍光传输到视网膜，因此阻碍视力。通过用人造透镜手术替换老化的透镜以恢复视力，可以有效地控制与晶状体相关的白内障。然而，儿童白内障没有这样的成功结果。婴儿期/幼儿期的白内障会使人衰弱。他们的手术治疗需要终身随访和视力损害。大约50%的先天性白内障是遗传性的，即使有关于其遗传关联的知识，也很少有人知道导致白内障发病机制的起始事件的分子组成。热休克转录因子HSF 4的DNA结合结构域（DBD）突变与最常见的儿童早期白内障形式（板层性白内障）相关。使用细菌人工染色体（BAC）转基因与操纵的HSF 4（DBD），我们已经重新创建了板层白内障的病理转基因小鼠，因此，第一次提出了一个范式，否则将难以调查的人类病理学的研究。 在本申请中，我们提出（1）采用下一代RNA测序（RNA-Seq）分析转基因白内障透镜的转录组，（2）。在转基因和野生型透镜中，用ChIP-Seq（染色质免疫沉淀与下一代DNA测序）分析HSF 4的整体DNA结合模式和（3）。利用aims 1和aims 2中的信息，通过微流控技术研究单个透镜纤维细胞中的基因表达谱 qPCR和单细胞RNA测序。RNA-seq和ChIP-seq的方法以及在单纤维细胞水平上的分析将鉴定板层性白内障病理学特征的基因/基因表达模式。单个纤维细胞的拟议表征将揭示与纤维相关的分子异质性的性质，这些纤维变得白内障和透明。但这一提议的动机不仅仅是 我们的目标是编制一份基因清单，其动机是为知识库做出贡献，从而（a）探索儿童早期白内障的药物干预措施，以保护视轴，（B）建立可测试的分子特征，为外科医生做出摘除或保留婴儿透镜的关键决策提供信息。

DESCRIPTION (provided by applicant): Cataractogenesis, commonly associated with age, impedes transmission of light to the retina and therefore obstructs vision. Age-related cataracts are managed efficiently by surgical replacement of the aged lens by a man-made lens to restore vision. However, there are no such successful outcomes for the childhood cataracts. Cataracts in infancy/early childhood are debilitating. Their surgical management entails a life-long follow up and impairment of vision. About 50% of the congenital cataracts are genetic and even when there is knowledge about their genetic associations, very little is known about the molecular make-up of the initiating event(s) that lead to the pathogenesis of the cataract. Mutations in the DNA binding domain (DBD) of the heat shock transcription factor, HSF4 have been associated with the most prevalent form of early childhood cataract, the lamellar cataract. Using bacterial artificial chromosome (BAC) transgenesis with manipulated HSF4 (DBD) we have recreated the lamellar cataract pathology in the transgenic mice; thus, for the first time presenting a paradigm for the study of a human pathology that would otherwise be difficult to investigate. In this application we propose to (1) Analyze the transcriptome of the transgenic cataractous lens employing next generation RNA sequencing (RNA-Seq), (2). Analyze the global DNA binding patterns of HSF4 with ChIP-Seq (chromatin immunoprecipitation with next generation DNA sequencing) in the transgenic and the wild type lens and (3). Use the information accrued from aims 1 and 2 to investigate gene expression profiles in individual lens fiber cells by microfluidic qPCR and single cell RNA-sequencing. The approaches of RNA-seq and ChIP-seq and analyses at the single fiber cell level will identify gene(s)/gene expression patterns characteristc of the lamellar cataract pathology. The proposed characterization of individual fiber cells will reveal the nature of the molecular heterogeneity that is associated with fibers, which become cataractous and those that are transparent. But it is not the motivation of this proposal to merely produce a list of genes, the motivation is to contribute to the knowledge base that would lead to (a) exploring pharmaceutical intervention for early childhood cataracts for protecting the visual axis and (b) the establishment of a testable molecular signature that would inform a surgeon's critical decision making to remove or to keep an infant's lens.