Oklahoma Medical Research Foundation, Oklahoma City.
Peking University First Hospital, Peking University, Ministry of Health of China, Beijing, China.
Arthritis Rheumatol. 2021 Dec;73(12):2303-2313. doi: 10.1002/art.41799. Epub 2021 Oct 29.
In a recent genome-wide association study, a significant genetic association between rs34330 of CDKN1B and risk of systemic lupus erythematosus (SLE) in Han Chinese was identified. This study was undertaken to validate the reported association and elucidate the biochemical mechanisms underlying the effect of the variant.
We performed an allelic association analysis in patients with SLE, followed by a meta-analysis assessing genome-wide association data across 11 independent cohorts (n = 28,872). In silico bioinformatics analysis and experimental validation in SLE-relevant cell lines were applied to determine the functional consequences of rs34330.
We replicated a genetic association between SLE and rs34330 (meta-analysis P = 5.29 × 10 , odds ratio 0.84 [95% confidence interval 0.81-0.87]). Follow-up bioinformatics and expression quantitative trait locus analysis suggested that rs34330 is located in active chromatin and potentially regulates several target genes. Using luciferase and chromatin immunoprecipitation-real-time quantitative polymerase chain reaction, we demonstrated substantial allele-specific promoter and enhancer activity, and allele-specific binding of 3 histone marks (H3K27ac, H3K4me3, and H3K4me1), RNA polymerase II (Pol II), CCCTC-binding factor, and a critical immune transcription factor (interferon regulatory factor 1 [IRF-1]). Chromosome conformation capture revealed long-range chromatin interactions between rs34330 and the promoters of neighboring genes APOLD1 and DDX47, and effects on CDKN1B and the other target genes were directly validated by clustered regularly interspaced short palindromic repeat (CRISPR)-based genome editing. Finally, CRISPR/dead CRISPR-associated protein 9-based epigenetic activation/silencing confirmed these results. Gene-edited cell lines also showed higher levels of proliferation and apoptosis.
Collectively, these findings suggest a mechanism whereby the rs34330 risk allele (C) influences the presence of histone marks, RNA Pol II, and IRF-1 transcription factor to regulate expression of several target genes linked to proliferation and apoptosis. This process could potentially underlie the association of rs34330 with SLE.
在最近的一项全基因组关联研究中，发现 CDKN1B 的 rs34330 与汉族人群系统性红斑狼疮（SLE）的风险之间存在显著的遗传关联。本研究旨在验证该报道的关联，并阐明该变异影响的生化机制。
我们在 SLE 患者中进行了等位基因关联分析，然后进行了一项元分析，评估了 11 个独立队列的全基因组关联数据（n=28872）。应用计算机生物信息学分析和 SLE 相关细胞系的实验验证来确定 rs34330 的功能后果。
我们复制了 SLE 与 rs34330 之间的遗传关联（元分析 P=5.29×10-8，优势比 0.84[95%置信区间 0.81-0.87]）。后续的生物信息学和表达数量性状基因座分析表明，rs34330 位于活性染色质中，并且可能调节多个靶基因。通过荧光素酶和染色质免疫沉淀实时定量聚合酶链反应，我们证明了显著的等位基因特异性启动子和增强子活性，以及 3 种组蛋白标记（H3K27ac、H3K4me3 和 H3K4me1）、RNA 聚合酶 II（Pol II）、CCCTC 结合因子和关键免疫转录因子（干扰素调节因子 1[IRF-1]）的等位基因特异性结合。染色体构象捕获显示 rs34330 与邻近基因 APOLD1 和 DDX47 的启动子之间存在长程染色质相互作用，并且通过基于成簇规律间隔短回文重复（CRISPR）的基因组编辑直接验证了对 CDKN1B 和其他靶基因的影响。最后，基于 CRISPR/失活 CRISPR 相关蛋白 9 的表观遗传激活/沉默证实了这些结果。基因编辑细胞系也显示出更高的增殖和凋亡水平。
综上所述，这些发现表明，rs34330 风险等位基因（C）通过影响组蛋白标记、RNA Pol II 和 IRF-1 转录因子的存在来调节与增殖和凋亡相关的多个靶基因的表达，从而影响 SLE 的发生。这一过程可能是 rs34330 与 SLE 相关的潜在机制。
