Prioritizing autoimmune-associated genetic variants that alter regulatory element activity in B cells

摘要 全基因组关联研究发现，整个基因组中有数百个遗传位点 与自身免疫性疾病有关。然而，导致自身免疫性疾病的因果遗传变异 每个轨迹在很大程度上是未知的。很难确定每个基因座的因果变量(S)，因为1) 在因果变体和数十到数百个非因果变体之间经常存在紧密的连锁不平衡， 这使得很难确定哪个变异确实是因果关系，以及2)因为大多数遗传关联发生在 在非编码区，它们对疾病相关基因和细胞类型的作用很难确定。 然而，变异体富含在非编码候选顺式调节区中，这些区域可能调节基因 疾病相关基因的表达。自身免疫基因变异体高度富含B细胞可及染色质， 这表明这种细胞类型中顺式调节活性的变化可能在疾病中起重要作用 进步。确定可以改变顺式调节活性的变体的一种方法是通过在 大规模平行报告化验，可以在一次化验中测试数千个变种的改变能力 记者以等位基因特有的方式表达。这种方法非常有效，因为我们已经发现了 报告表达的改变使因果变异的含量增加了58倍(根据统计精细图谱)。在这 提议，我们将在B细胞中应用MPRA来测试约18,000个与1型糖尿病相关的变异，多个 硬化症、炎症性肠病、类风湿性关节炎和牛皮癣 表情。为了确保B细胞MPRA与自身免疫性疾病相关，我们将评估它们是否富含 根据统计精细映射的因果变量。我们将根据变异的等位基因偏向效应对其进行排序 调整大小，并选择3个高效变种，以进行进一步的机械性随访。对于3个变体中的每一个，我们将 将变异等位基因编辑到健康供者的原代幼稚B细胞中，观察对当地B细胞基因的影响 表达(变异体1Mb以内)、细胞因子分泌和B细胞分化标志物。对于1-2个变种 在我们发现碱基编辑可以改变B细胞表型的地方，我们将识别纯合子风险和非风险等位基因携带者 对于Benaroya研究所内的健康受试者和自身免疫性疾病受试者 生物储存库。我们将评估变异是否与改变的B细胞表型相关，测试风险与 健康和疾病非危险变异携带者在幼稚、记忆力、血浆- 母细胞和浆细胞B细胞群体，以及相应的基因表达差异，可能会突出 变异体下游与疾病相关的通路。在这项研究完成后，我们将详尽地 测试了它们改变B细胞MPRA顺式调节活性的能力，并对高效果进行了跟踪 以确定其与疾病的生物学相关性。对这些优先风险的机械剖析 变异体可以提供丰富的靶向途径，为自身免疫设计新的治疗方法。

SUMMARY Genome-wide association studies have discovered hundreds of genetic loci throughout the genome that associate with autoimmune diseases. However, the causal genetic variants that drive autoimmune disease in each locus are for the most part unknown. It is difficult to identify the causal variant(s) in each locus because 1) there is often tight linkage disequilibrium between causal variants and tens to hundreds of non-causal variants, making it difficult to determine which variant is indeed causal, and 2) because most genetic associations occur in non-coding regions, where their actions on disease-relevant genes and cell types are hard to determine. However, variants are enriched in non-coding candidate cis-regulatory regions that may modulate gene expression of disease-relevant genes. Autoimmune genetic variants enrich highly in B cell accessible chromatin, indicating that variant modulation of cis-regulatory activity in this cell types may be important for disease progression. One way to determine the variants that can alter cis-regulatory activity is through testing them in massively parallel reporter assays, which can test thousands of variants in one assay for their ability to alter reporter expression in an allele-specific fashion. This approach is highly effective, as we have found variants that altered reporter expression enrich up to 58-fold for causal variants (according to statistical fine-mapping). In this proposal, we will apply MPRA in B cells to test ~18,000 variants associated with type 1 diabetes, multiple sclerosis, inflammatory bowel disease, rheumatoid arthritis, and psoriasis for their ability to alter reporter expression. To ensure B cell MPRA hits are relevant for autoimmune disease, we will assess whether they enrich for causal variants according to statistical fine-mapping. We will rank variants according to their allelic bias effect size and choose 3 high effect variants for further mechanistic follow-up. For each of the 3 variants, we will base edit variant alleles into primary naïve B cells from healthy donors and observe effects on local B cell gene expression (within 1 Mb of variant), cytokine secretion, and markers of B cell differentiation. For 1-2 variants where we find base editing to alter B cell phenotypes, we will identify homozygous risk and non-risk allele carriers for both healthy subjects and subjects with autoimmune disease within the Benaroya Research Institute biorepository. We will assess whether variants correlate with altered B cell phenotypes, testing whether risk vs. non-risk variant carriers in health and disease have differences in the percentage of naïve, memory, plasma- blasts, and plasma cell B cell populations, and corresponding differences in gene expression that may highlight disease-relevant pathways downstream of the variant. Upon completion of this study, we will have exhaustively tested variants for their ability to alter cis-regulatory activity in MPRA in B cells and followed up on high effect variants to determine their biological relevance to disease. Mechanistic dissection of these prioritized risk variants could provide a wealth of targetable pathways for which to design new therapeutics for autoimmunity.