Modeling Alzheimer's disease genetic variants in hiPSC

阿尔茨海默病(AD)是一种毁灭性的神经退行性疾病，没有有效的治疗方法。最新的基因组- 对阿尔茨海默病的广泛关联研究(GWAS)已经确定了20个可重复的风险基因，为 了解AD生物学的新方面，并开发有效的干预措施。然而，每个GWA基因座 通常跨越几个基因和许多同等关联的全基因组显着指数/代理单一 处于强烈连锁不平衡的核苷酸多态(SNPs)；因此，识别仍然具有挑战性 哪些基因和风险SNPs与AD的发病密切相关。大多数GWAS风险变量都是非编码的 并有可能调控基因表达。因为可获得或开放的染色质与顺式调控的染色质重叠 序列，我们假设许多AD风险变异体调节染色质对转录的可及性 因子，从而改变与阿尔茨海默病相关的分子和细胞表型。我们最近展示了开放的 人类诱导多能干细胞(HiPSCs)来源神经元的染色质图谱有助于区分 调节性GWA是精神分裂症的风险变种。通过直接比较开度的定量测量 同一样本中杂合子SNP的两个等位基因之间的染色质，即等位基因特异性开放 染色质(ASOC)分析，我们进一步表明精神分裂症GWA风险变异经常在 HiPSC-神经元。更重要的是，我们发现神经元ASOC变异体在AD Gwas中高度丰富 风险变量，可以很容易地告知三个主要AD风险基因(BIN1、CD2AP)的假定监管风险变量 和CLU)。在这里，我们将ASOC方法扩展到其他HiPSC衍生的AD相关细胞类型，还 利用我们在CRISPR基因组/表观基因组编辑和AD细胞生物学方面的专业知识，解决三个具体的问题 问题：(1)哪些AD Gwas风险变种是功能性的？为此，我们将通过atac-seq映射开放染色质 在谷氨酸和GABA能神经元、星形胶质细胞和小胶质细胞中，并寻找AD风险的调节性变异体 存在ASOC；(2)哪些基因受推测具有功能的AD GWAS变异体调控，因此 有可能是因果关系吗？为此，我们将经济有效地分析所有假定的AD风险变种的监管效果 在HiPSC来源的神经元、星形胶质细胞和小胶质细胞中，通过结合多路CRISPR/Cas9表观基因组编辑 单细胞RNA-seq；(3)推测原因AD引起的细胞表型改变是什么 变种/基因？为此，我们将研究BIN1、CD2AP和CLU中调控变体的顺式调控效应 (和其他优先基因)对CRISPR工程的HiPSC中AD相关生化和细胞表型的影响 模特们。该项目将影响该领域，因为它超越了GWA，以破译因果机制和 开发有效的治疗方法。

Alzheimer’s disease (AD) is a devastating neurodegenerative disorder without an effective cure. Recent genome- wide association studies (GWAS) on AD have identified >20 reproducible risk loci, providing an opportunity for understanding novel aspects of AD biology and developing effective interventions. However, each GWAS locus typically spans several genes and many equally associated genome-wide significant index/proxy single- nucleotide polymorphisms (SNPs) that are in strong linkage disequilibrium; it thus remains challenging to identify which genes and risk SNPs are causally involved in AD pathogenesis. Most GWAS risk variants are noncoding and likely regulate gene expression. Because accessible or open chromatin overlaps with cis-regulatory sequences, we hypothesize that many causal AD risk variants modulate chromatin accessibility to transcription factors, thereby altering molecular and cellular phenotypes relevant to AD. We have recently shown that open chromatin profiles in neurons derived from human induced pluripotent stem cells (hiPSCs) can help prioritize regulatory GWAS risk variants of schizophrenia. By directly comparing the quantitative measurements of open chromatin between the two alleles of a heterozygous SNP within the same sample, i.e., allele-specific open chromatin (ASoC) assay, we further showed that schizophrenia GWAS risk variants frequently exhibit ASoC in hiPSC-neurons. More importantly, we found that the neuronal ASoC variants are highly enriched for AD GWAS risk variants and can readily inform putatively regulatory risk variants at three leading AD risk loci (BIN1, CD2AP and CLU). Here, we will extend the ASoC approach to other hiPSC-derived AD-relevant cell types and also harness our expertise in CRISPR genome/epigenome editing and AD cell biology to address three specific questions: (1) Which AD GWAS risk variants are functional? For this, we will map open chromatin by ATAC-seq in glutamatergic and GABAergic neurons, astrocytes and microglia, and search for regulatory AD-risk variants that present ASoC; (2) Which genes are regulated by the putatively functional AD GWAS variants and are thus likely to be causal? For this, we will cost-effectively assay the regulatory effects of all putative AD-risk variants in hiPSC-derived neurons, astrocytes and microglia by combining multiplexed CRISPR/cas9 epigenome editing with single-cell RNA-seq; (3) What are the cellular phenotypic changes caused by the putatively causal AD variants/genes? For this, we will study the cis-regulatory effects of regulatory variants in BIN1, CD2AP and CLU (and other prioritized genes) on AD-relevant biochemical and cellular phenotypes in CRISPR-engineered hiPSC models. This project would impact the field by moving beyond GWAS to decipher causal mechanisms and develop effective treatments.