Non-Coding Autoimmune Risk Variant Defines Role for ICOS in T Peripheral Helper Cell Development

Our fine-mapping of 76 non-MHC loci associated with risk for rheumatoid arthritis (RA, 11,475 cases, 15,870 controls) has recently identified rs117701653, a non-coding single nucleotide polymorphism (SNP) in the CTLA4/CD28/ICOS locus, as the variant most likely to modulate RA risk within this region, with the minor allele C showing disease protection compared to the major allele A. We found that this SNP exhibits allele-specific protein binding and transcriptional modulation, further supporting its regulatory nature. Here, we aimed to determine whether the functional non-coding variant rs117701653 modulates the expression of genes within the CD28/CTLA4/ICOS locus. To address this question, we employed low-input RNA sequencing using the Illumina SmartSeq2 platform on resting total CD4+ T cells from the blood of 24 healthy subjects (8 AA, 8 AC, 8 CC) recruited based on genotype from the Mass General Brigham biobank. We performed a targeted cis-eQTL mapping analysis for protein coding genes with transcription start sites (TSS) within a 1MB window of SNP rs117701653. These genes included WDR12, NBEAL1, CYP20A1, ABI2, RAPH1, CD28, CTLA4, ICOS, and PARD3B. Surprisingly, allelic variation at rs117701653 correlated strongly with expression of ICOS. This suggests that individuals carrying the protective allele C at rs117701653 express lower levels of ICOS, implicating ICOS as a plausible target gene influenced by this protective allele. The raw sequence data from the 24 healthy individuals are available through dbGAP. Gene expression matrices have been deposited to GEO accession GSE235868.]]>我们针对与类风湿关节炎（rheumatoid arthritis, RA，病例11475例、对照15870例）风险相关的76个非主要组织相容性复合体（non-MHC）位点进行精细定位，近期鉴定出rs117701653——这是CTLA4/CD28/ICOS基因座上的一种非编码单核苷酸多态性（single nucleotide polymorphism, SNP）——为该区域内最有可能调控RA风险的变异体；相较于主要等位基因A，次要等位基因C可发挥疾病保护作用。我们发现该SNP呈现等位基因特异性蛋白结合与转录调控特性，进一步佐证了其调控属性。本研究旨在探究该功能性非编码变异体rs117701653是否会调控CD28/CTLA4/ICOS基因座内的基因表达。为解答该问题，我们基于基因型从马萨诸塞州总医院布里格姆分校（Mass General Brigham）生物样本库中招募了24名健康受试者（8名AA基因型、8名AC基因型、8名CC基因型），并采用Illumina SmartSeq2平台对其血液中的静息总CD4+ T细胞开展低起始量RNA测序。我们针对SNP rs117701653上下游1Mb范围内、转录起始位点（transcription start sites, TSS）处于该区域的蛋白编码基因开展了靶向顺式表达数量性状位点（cis-eQTL）定位分析。所涉基因包括WDR12、NBEAL1、CYP20A1、ABI2、RAPH1、CD28、CTLA4、ICOS及PARD3B。令人意外的是，rs117701653的等位基因变异与ICOS的表达水平呈现显著相关性。这表明携带rs117701653保护性等位基因C的个体，其ICOS表达水平更低，提示ICOS为该保护性等位基因所调控的潜在靶基因。本研究中24名健康受试者的原始测序数据可通过基因型与表型数据库（dbGAP）获取；基因表达矩阵已提交至基因表达综合数据库（Gene Expression Omnibus, GEO），收录号为GSE235868。