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岁以上人群中的患病率超过1%，因此构成了人口老龄化的主要全球健康问题。该疾病的主要特征是黑质纹状体多巴胺能神经元的主要损失，但导致神经元细胞损失的遗传病因在很大程度上仍然未知。许多全基因组关联研究（GWAS）已经确定了单核苷酸多态性（SNP），指出超过50个基因组位点含有散发性PD的“风险变体”。然而，已确定的风险变异主要映射到基因组的非编码区，这阻碍了对这些遗传变异如何导致PD风险增加的功能和分子理解。重要的是，已经确定PD GWAS相关基因座通常是非编码的，并且介导对远端基因表达的等位基因特异性作用，这与破坏远端基因表达的核心作用一致。 增强子元件在PD发病机制中的功能人诱导多能干细胞（hiPSC）技术通过允许产生人患者来源的体细胞（例如中脑多巴胺能神经元），首次提供了研究遗传成分知之甚少的散发性疾病的独特机会，所述体细胞携带促成疾病发展的所有遗传改变。该项目的总体目标是 建立一个转化的范例，克服了iPS系统的实质性技术限制，并创建了一个遗传定义的实验体外系统，用于研究培养皿中散发性PD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”GWAS PD命中与基因组调控增强子元件联系起来，并建立将PD风险等位基因与疾病相关效应基因表达联系起来的功能测定。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PD特异性细胞表型的关键基因。为了鉴定功能相关的候选风险变体（PD-eQLT），我们将通过在培养的同质多巴胺能神经元以及来自人脑的分选的神经元核中建立增强子图谱，将PD相关风险等位基因与神经元中基因活性相关的基因组调控（增强子）元件相关联。深入了解这些相互作用将有助于设计合理的治疗策略。该实验性体外平台将用于定义环境风险因素与遗传决定因素的相互作用，这些遗传决定因素已从GWA研究中预测为易患PD（GxE）。

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).