Childhood Cataractogenesis: Heterogeneity of Gene Expression

儿童白内障发生：基因表达的异质性

• 批准号: 8984890
• 负责人: SURAJ P BHAT
• 金额: $ 38.5万
• 依托单位: UNIVERSITY OF CALIFORNIA LOS ANGELES
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-01-01 至 2018-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8984890
• 关键词: Address; Age; Bacterial Artificial Chromosomes; Binding; Biogenesis; Cataloging; Catalogs; Cataract; Cells; ChIP-seq; Child; Childhood; Chromatin; Clinical; Crystalline Lens; DNA Binding; DNA Binding Domain; DNA Sequence; Decision Making; Development; Diet; Disease; Environment; Event; Excision; Eye; Fiber; Gene Expression; Gene Expression Profile; Gene Transfer Techniques; Genes; Genetic; Genomic Segment; Health; Heterogeneity; Human; Human Pathology; Individual; Infant; Intervention; Knowledge; Lead; Left; Lens Fiber; Life; Maps; Methodology; Microfluidics; Modeling; Molecular; Molecular Profiling; Motivation; Mutation; Nature; Operative Surgical Procedures; Ophthalmologist; Outcome; Pathogenesis; Pathology; Pathway interactions; Pattern; Pharmaceutical Solutions; Pharmacologic Substance; Phenotype; Property; Retina; Risk Factors; Social status; Sorting - Cell Movement; Stream; Surgeon; Surgical Management; Therapeutic Intervention; Time; Transgenic Mice; Transgenic Organisms; Vision; Visual; Visual impairment; age related; aged; base; chromatin immunoprecipitation; congenital cataract; disease phenotype; early childhood; fiber cell; follow-up; genetic association; heat shock transcription factor; hybrid gene; infancy; knowledge base; lens; light transmission; man; mouse model; mutant; neurodevelopment; next generation; next generation sequencing; transcriptome; transcriptome sequencing

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.

描述（由申请人提供）：白内生生成通常与年龄相关，阻碍了视网膜传播，因此阻碍了视力。通过人造镜头恢复视力，通过手术替换老年镜头来有效地管理与年龄相关的白内障。但是，儿童白内障没有这样的成功结果。婴儿期/幼儿时期的白内障令人衰弱。他们的手术管理需要终身跟进和视力障碍。大约50％的先天性白内障是遗传性的，即使对它们的遗传关联有所了解，对导致白内障发病机理的起始事件的分子组成知之甚少。热休克转录因子的DNA结合结构域（DBD）中HSF4的突变与最普遍的儿童白内障形式（层状白内障）相关。使用操纵的HSF4（DBD），我们使用细菌性人造染色体（BAC）转基因，我们在转基因小鼠中重新创建了层状白内障病理。因此，首次提出一个范式来研究人类病理，原本很难研究。 在此应用中，我们建议（1）分析采用下一代RNA测序（RNA-Seq）的转基因性白内障晶状体的转录组，（2）。用转基因和野生型透镜和（3）中的ChIP-Seq（染色质免疫沉淀和下一代DNA测序）分析HSF4的全局DNA结合模式（染色质免疫沉淀）。使用AIMS 1和2的信息来研究微流体细胞中单个晶状体纤维细胞中的基因表达谱 qPCR和单细胞RNA - 测序。 RNA-SEQ和CHIP-SEQ的方法以及单纤维细胞水平的分析将识别层状白内障病理学的基因/基因表达模式。所提出的单个纤维细胞的表征将揭示与纤维相关的分子异质性的性质，这些纤维变成了白内障和透明的纤维。但这不是这一提议的动力 产生基因列表，其动机是为知识库做出贡献，这将导致（a）探索针对保护视觉轴的幼儿白内障的药物干预措施，以及（b）建立可测试的分子签名，以告知外科医生的关键决策以取消或保持婴儿的镜头。