Genome-wide DNA hypomethylation and RNA:DNA hybrid accumulation in Aicardi-Goutières syndrome. Homo sapiens

Aicardi-Goutières syndrome (AGS) is a severe childhood inflammatory disorder that shows clinical and genetic overlap with systemic lupus erythematosus (SLE). AGS is thought to arise from the accumulation of incompletely metabolized endogenous nucleic acid species owing to mutations in nucleic acid degrading enzymes TREX1 (AGS1), RNase H2 (AGS2, 3 and 4) and SAMHD1 (AGS5). However, the identity and source of such immunogenic nucleic acid species remain undefined. Using genome-wide approaches, we show that fibroblasts from AGS patients with AGS1-5 mutations are burdened by excessive loads of RNA:DNA hybrids. Using MethylC-seq, we show that AGS fibroblasts display pronounced and global loss of DNA methylation and demonstrate that AGS-specific RNA:DNA hybrids often occur within DNA hypomethylated regions. Altogether, our data suggest that RNA:DNA hybrids may represent a common immunogenic form of nucleic acids in AGS and provide the first evidence of epigenetic perturbations in AGS, furthering the links between AGS and SLE. Overall design: DNA-RNA immunoprecipitation (DRIP-seq) was performed on control 1, control 3, AGS1 P1, AGS1 P2, AGS2 P1, AGS2 P2, AGS4 P1, AGS4 P2, AGS5 P1 and AGS5 P2. MethylC-seq was performed on control 1, control 2, AGS1 P1, AGS2 P1, AGS4 P1 and AGS5 P1. Reduced representation bisulfite sequencing (RRBS) was performed on GM12697 and AGS2 LCL cell lines. RNA-seq was performed on 2 biological replicates each of control 1, AGS1 P1, AGS2 P1, AGS2 P2, AGS4 P1, AGS4 P2 and AGS5 P1.

Aicardi-Goutières综合征（Aicardi-Goutières syndrome, AGS）是一种严重的儿童炎性疾病，其临床表型与遗传特征与系统性红斑狼疮（systemic lupus erythematosus, SLE）存在重叠。现有研究认为，AGS的发病机制为核酸降解酶（包括TREX1（对应AGS1型）、RNase H2（对应AGS2、3及4型）与SAMHD1（对应AGS5型））发生突变后，内源性未完全代谢的核酸物质异常蓄积。然而，这类具有免疫原性的核酸物质的具体种类与来源仍未明确。本研究通过全基因组级别的分析方法证实，携带AGS1-5型突变的AGS患者成纤维细胞中，RNA:DNA杂交体（RNA:DNA hybrids）的负荷显著升高。通过甲基化C测序（MethylC-seq），我们发现AGS成纤维细胞呈现出显著且广泛的全基因组DNA甲基化丢失，并证实AGS特异性的RNA:DNA杂交体常富集于DNA低甲基化区域内。综合以上结果，本研究数据提示RNA:DNA杂交体可能是AGS中一类常见的免疫原性核酸形式，同时首次为AGS存在表观遗传扰动提供了证据，进一步强化了AGS与SLE之间的病理关联。本研究的整体实验设计如下：对对照1、对照3、AGS1 P1、AGS1 P2、AGS2 P1、AGS2 P2、AGS4 P1、AGS4 P2、AGS5 P1及AGS5 P2样本进行DNA-RNA免疫沉淀测序（DRIP-seq）；对对照1、对照2、AGS1 P1、AGS2 P1、AGS4 P1及AGS5 P1样本进行甲基化C测序（MethylC-seq）；对GM12697与AGS2淋巴母细胞样细胞系（lymphoblastoid cell line, LCL）进行简化代表性亚硫酸氢盐测序（reduced representation bisulfite sequencing, RRBS）；对对照1、AGS1 P1、AGS2 P1、AGS2 P2、AGS4 P1、AGS4 P2及AGS5 P1各设置2个生物学重复样本进行RNA测序（RNA-seq）。