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

总结 全基因组关联研究已经发现了整个基因组中的数百个遗传位点， 与自身免疫性疾病有关。然而，导致自身免疫性疾病的遗传变异， 每个位点大部分是未知的。很难确定每个基因座中的因果变异，因为1） 在致病变异体和数十至数百个非致病变异体之间经常存在紧密连锁不平衡， 这使得很难确定哪种变异确实是因果关系，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，变异体富含非编码候选顺式调控区，其可以调节基因表达。 疾病相关基因的表达。自身免疫遗传变体高度富集B细胞可接近的染色质， 这表明在这种细胞类型中顺式调节活性的变异调节可能对疾病的发生很重要， 进展确定可以改变顺式调节活性的变体的一种方法是通过在 大规模平行报告基因测定，可以在一次测定中测试数千种变异体改变 以等位基因特异性方式表达报告基因。这种方法非常有效，因为我们已经发现了 改变的报告基因表达使致病变体富集高达58倍（根据统计学精细作图）。在这 我们将在B细胞中应用MPRA来测试约18，000种与1型糖尿病相关的变异， 硬化症、炎症性肠病、类风湿性关节炎和银屑病，因为它们能够改变报告基因， 表情为了确保B细胞MPRA命中与自身免疫性疾病相关，我们将评估它们是否富集了 根据统计精细映射的因果变量。我们将根据其等位基因偏倚效应对变体进行排序 选择3种高效变体进行进一步的机制随访。对于这3个变量中的每一个，我们将基于 将变异等位基因编辑到来自健康供体的原代幼稚B细胞中，并观察对局部B细胞基因的影响 表达（在变体的1 MB内）、细胞因子分泌和B细胞分化的标志物。对于1-2个变体 当我们发现碱基编辑改变B细胞表型时，我们将鉴定纯合子风险和非风险等位基因携带者 Benaroya研究所内的健康受试者和自身免疫性疾病受试者 生物储藏室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 健康和疾病的非风险变异携带者在天真，记忆，血浆- 原始细胞和浆细胞B细胞群，以及相应的基因表达差异， 变异体下游的疾病相关通路。在完成这项研究后，我们将全面 测试了变体改变B细胞中MPRA顺式调节活性的能力， 变异以确定其与疾病的生物学相关性。对这些优先风险的机械解剖 变异体可以提供丰富的靶向途径，用于设计新的自身免疫疗法。

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.