A Refined Study of <em>FCRL</em> Genes from a Genome-Wide Association Study for Graves’ Disease

To pinpoint the exact location of the etiological variant/s present at 1q21.1 harboring FCRL1-5 and CD5L genes, we carried out a refined association study in the entire FCRL region in 1,536 patients with Graves’ disease (GD) and 1,516 sex-matched controls by imputation analysis, logistic regression, and cis-eQTL analysis. Among 516 SNPs with P<0.05 in the initial GWAS scan, the strongest signals associated with GD and correlated to FCRL3 expression were located at a cluster of SNPs including rs7528684 and rs3761959. And the allele-specific effects for rs3761959 and rs7528684 on FCRL3 expression level revealed that the risk alleles A of rs3761959 and C of rs7528684 were correlated with the elevated expression level of FCRL3 whether in PBMCs or its subsets, especially in CD19+ B cells and CD8+ T subsets. Next, the combined analysis with 5,300 GD cases and 4,916 control individuals confirmed FCRL3 was a susceptibility gene of GD in Chinese Han populations, and rs3761959 and rs7528684 met the genome-wide association significance level (Pcombined = 2.27×10−12 and 7.11×10−13, respectively). Moreover, the haplotypes with the risk allele A of rs3761959 and risk allele C of rs7528684 were associated with GD risk. Finally, our epigenetic analysis suggested the disease-associated C allele of rs7528684 increased affinity for NF-KB transcription factor. Above data indicated that FCRL3 gene and its proxy SNP rs7528684 may be involved in the pathogenesis of GD by excessive inhibiting B cell receptor signaling and the impairment of suppressing function of Tregs.为精准定位1q21.1区域内携带有FCRL1-5与CD5L基因的致病变异位点的确切位置，本研究针对1536名格雷夫斯病（Graves’ disease, GD）患者及1516名性别匹配对照人群的全FCRL基因区域，采用基因型插补分析、逻辑回归和顺式表达数量性状位点（cis-eQTL）分析开展了精细化关联研究。在初始全基因组关联研究（Genome-Wide Association Study, GWAS）筛查得到的516个P<0.05的单核苷酸多态性（Single Nucleotide Polymorphism, SNP）中，与GD显著关联且与FCRL3表达水平相关的最强信号位点，聚集于包含rs7528684与rs3761959的SNP簇内。针对rs3761959与rs7528684的等位基因特异性效应分析显示，rs3761959的风险等位基因A与rs7528684的风险等位基因C，均可上调FCRL3的表达水平，且该效应在外周血单个核细胞（Peripheral Blood Mononuclear Cells, PBMCs）及其细胞亚群中均存在，尤以CD19阳性B细胞与CD8阳性T细胞亚群中最为显著。随后，本研究联合5300例GD病例与4916名对照个体开展验证分析，证实FCRL3为中国汉族人群GD的易感基因，且rs3761959与rs7528684均达到全基因组关联显著性水平（联合分析P值分别为2.27×10^−12与7.11×10^−13）。此外，携带rs3761959风险等位基因A与rs7528684风险等位基因C的单倍型，与GD发病风险显著相关。最后，表观遗传学分析表明，rs7528684的疾病相关风险等位基因C可增强其与核因子κB（Nuclear Factor-Kappa B, NF-κB）转录因子的结合亲和力。上述研究结果提示，FCRL3基因及其代理SNP rs7528684，可能通过过度抑制B细胞受体信号通路以及削弱调节性T细胞（Regulatory T cells, Tregs）的免疫抑制功能，参与GD的发病机制。