Parkinson-associated SNCA enhancer variants revealed by open chromatin in mouse dopamine neurons

The progressive loss of midbrain (MB) dopaminergic (DA) neurons defines the motor features of Parkinson disease (PD) and modulation of risk by common variation in PD has been well established through GWAS. Anticipating that a fraction of PD-associated genetic variation mediates their effects within this neuronal population, we acquired open chromatin signatures of purified embryonic mouse MB DA neurons. Correlation with >2,300 putative enhancers assayed in mice reveals enrichment for MB cis-regulatory elements (CRE), data reinforced by transgenic analyses of six additional sequences in zebrafish and mice. One CRE, within intron 4 of the familial PD gene SNCA, directs reporter expression in catecholaminergic neurons of transgenic mice and zebrafish. Sequencing of this CRE in 986 PD patients and 992 controls reveals two common variants associated with elevated PD risk. To assess potential mechanisms of action, we identify proteins whose binding is impacted by these enhancer variants. Additional genotyping across the SNCA locus identifies a single PD-associated haplotype, containing the minor alleles of both of the aforementioned PD-risk variants. Our work posits a model for how common variation at SNCA may modulate PD risk and highlights the value of cell context-dependent guided searches for functional non-coding variation. Overall design: 6 ATAC-seq libraries and 8 RNA-seq samples from sorted mouse Th-EGFP dopaminergic neurons collected at E15.5 from two distinct brain regions (midbrain and forebrain)

中脑（midbrain, MB）多巴胺能神经元（dopaminergic, DA）的进行性丢失是帕金森病（Parkinson disease, PD）运动症状的核心致病特征，而帕金森病风险受常见遗传变异调控这一结论已通过全基因组关联研究（Genome-Wide Association Study, GWAS）得到充分验证。我们推测，部分与帕金森病相关的遗传变异通过该神经元群体介导其生物学效应，因此获取了纯化的胚胎小鼠中脑多巴胺能神经元的开放染色质特征谱。通过与小鼠中已验证的2300余个推定增强子进行关联分析，发现该特征谱显著富集于中脑顺式调控元件（cis-regulatory elements, CRE）；后续通过斑马鱼与小鼠体内6个额外序列的转基因实验，进一步巩固了这一发现。其中一个位于家族性帕金森病基因SNCA第4内含子区域的顺式调控元件，可在转基因小鼠与斑马鱼的儿茶酚胺能神经元中驱动报告基因表达。我们对986名帕金森病患者与992名对照个体的该顺式调控元件进行测序，鉴定出两个常见变异体与帕金森病风险升高显著相关。为探究该顺式调控元件的潜在作用机制，我们筛选出了受这些增强子变异体影响结合的蛋白质。通过对SNCA基因座的额外基因分型分析，我们发现一个包含上述两个帕金森病风险变异体次要等位基因的单倍型与帕金森病显著相关。本研究提出了SNCA基因座常见变异调控帕金森病风险的潜在模型，并凸显了基于细胞背景靶向筛选功能性非编码变异的研究价值。整体实验设计：从胚胎日15.5（E15.5）小鼠的两个不同脑区（中脑与前脑）中，分选得到Th-EGFP标记的多巴胺能神经元，分别构建了6个ATAC-seq文库与8个RNA-seq样本。