An iPSC based platform for functionally assessing genetic and environmental risk

描述(申请人提供)：帕金森病(PD)是全球第二常见的慢性进行性神经退行性疾病，在60岁以上的人口中患病率超过1%，因此构成了老龄化人口的主要全球健康问题。该病的主要特征是黑质纹状体多巴胺能神经元的大量丢失，但导致神经元细胞丢失的遗传病因在很大程度上仍不清楚。大量的全基因组关联研究(GWAS)已经确定了单核苷酸多态(SNPs)，这些SNPs指向50多个包含散发性帕金森病“危险变异”的基因组基因座。然而，已识别的风险变异主要映射到基因组中知之甚少的非编码区，这阻碍了对这些遗传变异如何导致帕金森病风险增加的功能和分子理解。重要的是，已经确定PD GWASs相关的基因座通常是非编码的，并且中介等位基因特异性的远端基因表达效应，这与干扰基因表达的中心作用是一致的。 增强子元件在帕金森病发病机制中的作用人类诱导多能干细胞(HiPSC)技术首次为研究基因成分鲜为人知的散发性疾病提供了独特的机会，允许产生人类患者来源的体细胞，如中脑多巴胺能神经元，这些细胞携带导致疾病发展的所有基因变化。该项目的总体目标是建立一种转化范式，它克服了iPS系统的实质性技术限制，并创建了一个基因定义的体外实验系统，用于研究盘中散发性帕金森病的分子和生物学机制。为了实现这一目标，我们将建立一个新的实验框架，将“描述性的”GWASPD HITS与基因组调节增强子元件联系起来，并建立功能分析，将PD风险等位基因与疾病相关效应基因的表达联系起来。我们的目的是从分子水平上了解多个遗传风险等位基因之间的复杂相互作用，并确定其表达水平影响帕金森病特异性细胞表型的关键基因。为了识别功能相关的候选风险变异(PD-eQLTs)，我们将通过在培养的同源多巴胺能神经元以及从人脑中分离的神经元细胞核中建立增强子图谱，将PD相关的风险等位基因与与神经元基因活性相关的基因组调控(增强子)元件联系起来。对这些相互作用的洞察将有助于设计合理的治疗策略。这个体外实验平台将被用来定义环境风险因素与从GWA研究中预测的易患帕金森病(GxE)的遗传决定因素的相互作用。

DESCRIPTION (provided by applicant): Parkinson's disease (PD) is the second most common chronic progressive neurodegenerative disease worldwide, with a prevalence of more than 1% in the population over the age of 60, thus constituting a major global health problem of the aging population. The disease is primarily characterized by a major loss of nigrostriatal dopaminergic neurons but the genetic etiology leading to the neuronal cell loss has largely remained unknown. Numerous genome-wide association studies (GWAS) have identified single nucleotide polymorphisms (SNPs) that point to more than 50 genomic loci containing "risk variants" for sporadic PD. However the identified risk variants predominantly map to poorly understood non-coding regions of the genome, which has impeded functional and molecular understanding of how these genetic variants contribute to the increased risk for PD. Importantly, it has been established that PD GWAS associated loci are typically non-coding and mediate allele-specific effects on distal gene expression, consistent with a central role for disruption on enhancer element function in PD pathogenesis Human induced pluripotent stem cell (hiPSC) technology offers for the first time the unique opportunity to study sporadic diseases whose genetic components are poorly understood, by allowing for the generation of human patient-derived somatic cells such as midbrain dopaminergic neurons which carry all the genetic alterations that contributed to the development of the disease. The overall goal of this project is to establish a transformational paradigm, which overcomes the substantial technical limitations of the iPS system and creates a genetically defined experimental in vitro system for studying the molecular and biological mechanisms of sporadic PD in the dish. To achieve this, we will establish a novel experimental framework to link "descriptive" GWAS PD hits to genomic regulatory enhancer elements and establish functional assays for connecting PD risk alleles to the expression of disease relevant effector genes. Our aim is to gain molecular understanding of the complex interactions between multiple genetic risk alleles and to identify key genes of which the expression level affects the PD specific cellular phenotype. To identify functional relevant candidate risk variants (PD-eQLTs) we will link PD associated risk alleles to genomic regulatory (enhancer) elements that are relevant for gene activity in neurons by establishing an enhancer map in cultured homogenous dopaminergic neurons as well as in sorted neuronal nuclei from human brain. Insights into these interactions will facilitate devising rational therapeutic strategies. This experimental in vitro platform will be used to define the interactions of environmental risk factors with the genetic determinants that have been predicted from GWA studies to predispose to PD (GxE).