CLEC16A内含子遗传多态性在常见变异型免疫缺陷中的机制研究

Common variable immunodeficiency disorder (CVID) is a type of primary immunodeficiency, with a large proportion of patients suffering from autoimmune comorbidity. Such “paradox” phenomenon is not well understood. The genetic mechanism of CVID remains elusive, and there is no effective treatment for CVID patients with noninfectious inflammatory complications. Furthermore, the comorbidity of cancer is also frequently observed among CVID patients. Therefore, it is important and in urgent need to understand the genetic mechanism of CVID and to develop effective targeted therapeutic approach. By dense genotyping of autoimmune risk loci in the largest CVID cohort assembled to date, we uncovered the first non-HLA genome-wide significant risk locus. The signal is located in the intron 19 of the CLEC16A gene. Genetic studies implicate that this locus may represent the missing link between immune deficiency and autoimmunity. Functional genomics data and our preliminary experimental results suggest that CLEC16A intron 19 region may function as an enhancer regulating gene expression. In the current study, we aim to identify its targeted genes and the transcription factors which mediate the interaction between this enhancer region and its targeted genes. We expect results from these studies will uncover the molecular mechanism how CLEC16A intron 19 locus functions in the pathogenesis of CVID, and provide novel insights into the development of potential new therapeutic approaches to more effectively treat this suffering medical condition.

常见变异型免疫缺陷是一种原发性免疫疾病，大部分患者存在自身免疫等并发症。目前对这一看似悖论的现象仍然缺乏充分的理解。该病遗传机制很不清楚，治疗手段极为有限，而恶性肿瘤在患者中发生率高。因而阐明这一疾病的分子遗传机制，开发针对性的治疗手段极为迫切。通过对免疫疾病遗传位点高密度的多态性关联分析，我们首次发现全基因组水平上显著相关的HLA区域外的遗传位点——CLEC16A基因内含子19区域。遗传学证据表明其可能是介导免疫缺陷与自身免疫之间的桥梁。功能基因组学研究及初步实验数据揭示其可能为一转录调控的增强子。本课题中，我们拟通过无偏的高通量技术手段，结合小鼠动物模型的研究，发现CLEC16A内含子19区域转录调控的靶基因；鉴定介导该区域与靶基因之间的转录因子复合体。我们期望本研究阐明CLEC16A位点在常见变异型免疫缺陷疾病中的作用机制，帮助建立针对性的治疗方案。

研究背景：原发性免疫缺陷病和自身免疫性疾病的症状经常共存于病人，目前对这一看似悖论的现象仍然缺乏充分的理解。并且这两种疾病有共同的遗传位点，例如CLEC16A内含子19区域。然而，对于CLEC16A位点对疾病发病机制的影响，尚知之甚少。.研究内容：常见变异型免疫缺陷(CVID)是一种原发性免疫缺陷疾病，但大部分患者存在自身免疫等并发症。我们利用CVID的遗传队列，进行精细定位预测可能的因果变异；通过CRISPR-Cas9技术删除了人类免疫细胞系中潜在的因果变异，利用RNA-seq发现其靶基因。我们通过环状染色体构象捕获对CLEC16A基因座与其靶基因之间的相互作用进行鉴定；通过细胞培养物中氨基酸的稳定同位素标记确定染色质相互作用的转录因子复合物；并通过RNA-seq和反相蛋白阵列发现了CLEC16A基因座调节的多个分子途径。我们对其远程调控的一靶基因构建了基因敲除小鼠。.研究结果：我们的研究表明，CLEC16A基因座是一增强子，通过细胞类型特异性染色质相互作用调节多个靶基因的表达，包括远距离调控基因ATF7IP2的表达。不同的转录因子复合物以等位基因特异性方式介导染色质相互作用。CLEC16A内含子19因果变异区域的缺失会影响AKT的表达和磷酸化水平，并导致免疫、代谢途径的改变，以及CD4+T细胞对胰岛素和PMA刺激的分子反应。ATF7IP2小鼠表现出发育缺陷。.结论：通过多组学和实验方法，我们发现了CLEC16A内含子19所调控的新的靶基，初步阐明了CLEC16A位点介导自身免疫和原发性免疫缺陷关联的潜在分子机制，对于开发CVID精准治疗提供了新的思路。