Childhood Cataractogenesis: Heterogeneity of Gene Expression

描述(申请人提供)：白内障的发生，通常与年龄有关，阻碍光线传输到视网膜，因此阻碍视力。通过人工晶状体置换老化的晶状体以恢复视力，可以有效地治疗老年性白内障。然而，对于儿童白内障，目前还没有这样的成功结果。婴儿期/幼儿期的白内障会使人虚弱。他们的手术治疗需要终生随访和视力障碍。大约50%的先天性白内障是遗传的，即使知道它们之间的遗传关联，也对导致白内障发病的起始事件(S)的分子组成知之甚少。热休克转录因子HSF4的DNA结合域(DBD)突变与最常见的儿童早期白内障-板层性白内障有关。利用细菌人工染色体(BAC)转基因和操纵的HSF4(DBD)，我们在转基因小鼠中重建了板层白内障病理；因此，第一次为研究人类病理提供了一个范例，否则将很难研究。在这个应用中，我们建议(1)使用下一代RNA测序(RNA-Seq)来分析转基因白内障晶状体的转录组，(2)。用染色质免疫沉淀结合下一代DNA测序技术分析转基因晶状体和野生型晶状体中HSF4的整体DNA结合模式。利用AIMS 1和AIMS 2获得的信息，用微流控技术研究单个晶状体纤维细胞的基因表达谱 定量聚合酶链式反应和单细胞RNA测序。RNA-SEQ和CHIP-SEQ方法以及单纤维细胞水平的分析将识别板层状白内障病理的基因(S)/基因表达谱特征。所提出的对单个纤维细胞的表征将揭示与纤维相关的分子异质性的本质，这些纤维会变成白内障和透明的纤维。但这项提议的动机不仅仅是 为了制作一份基因清单，其动机是对知识库作出贡献，从而导致(A)探索对儿童早期白内障进行药物干预以保护视轴，以及(B)建立一个可测试的分子签名，为外科医生摘除或保留婴儿晶状体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.