Department of Pulmonology, University Medical Center Groningen, University of Groningen, Groningen, the Netherlands; GRIAC research institute Groningen, University Medical Center Groningen, University of Groningen, Groningen, the Netherlands; Department of Genetics, University Medical Center Groningen, University of Groningen, Groningen, the Netherlands.
Department of Women's and Children's Health, Karolinska Institutet, Stockholm, Sweden; Department of Biosciences and Nutrition, Karolinska Institutet, Stockholm, Sweden.
Lancet Respir Med. 2018 May;6(5):379-388. doi: 10.1016/S2213-2600(18)30052-3. Epub 2018 Feb 26.
DNA methylation profiles associated with childhood asthma might provide novel insights into disease pathogenesis. We did an epigenome-wide association study to assess methylation profiles associated with childhood asthma.
We did a large-scale epigenome-wide association study (EWAS) within the Mechanisms of the Development of ALLergy (MeDALL) project. We examined epigenome-wide methylation using Illumina Infinium Human Methylation450 BeadChips (450K) in whole blood in 207 children with asthma and 610 controls at age 4-5 years, and 185 children with asthma and 546 controls at age 8 years using a cross-sectional case-control design. After identification of differentially methylated CpG sites in the discovery analysis, we did a validation study in children (4-16 years; 247 cases and 2949 controls) from six additional European cohorts and meta-analysed the results. We next investigated whether replicated CpG sites in cord blood predict later asthma in 1316 children. We subsequently investigated cell-type-specific methylation of the identified CpG sites in eosinophils and respiratory epithelial cells and their related gene-expression signatures. We studied cell-type specificity of the asthma association of the replicated CpG sites in 455 respiratory epithelial cell samples, collected by nasal brushing of 16-year-old children as well as in DNA isolated from blood eosinophils (16 with asthma, eight controls [age 2-56 years]) and compared this with whole-blood DNA samples of 74 individuals with asthma and 93 controls (age 1-79 years). Whole-blood transcriptional profiles associated with replicated CpG sites were annotated using RNA-seq data of subsets of peripheral blood mononuclear cells sorted by fluorescence-activated cell sorting.
27 methylated CpG sites were identified in the discovery analysis. 14 of these CpG sites were replicated and passed genome-wide significance (p<1·14 × 10) after meta-analysis. Consistently lower methylation levels were observed at all associated loci across childhood from age 4 to 16 years in participants with asthma, but not in cord blood at birth. All 14 CpG sites were significantly associated with asthma in the second replication study using whole-blood DNA, and were strongly associated with asthma in purified eosinophils. Whole-blood transcriptional signatures associated with these CpG sites indicated increased activation of eosinophils, effector and memory CD8 T cells and natural killer cells, and reduced number of naive T cells. Five of the 14 CpG sites were associated with asthma in respiratory epithelial cells, indicating cross-tissue epigenetic effects.
Reduced whole-blood DNA methylation at 14 CpG sites acquired after birth was strongly associated with childhood asthma. These CpG sites and their associated transcriptional profiles indicate activation of eosinophils and cytotoxic T cells in childhood asthma. Our findings merit further investigations of the role of epigenetics in a clinical context.
EU and the Seventh Framework Programme (the MeDALL project).
与儿童哮喘相关的 DNA 甲基化谱可能为疾病发病机制提供新的见解。我们进行了一项全基因组关联研究，以评估与儿童哮喘相关的甲基化谱。
我们在过敏症发病机制的机制（MeDALL）项目中进行了一项大规模的全基因组关联研究（EWAS）。我们使用 Illumina Infinium Human Methylation450 BeadChips（450K）在 207 名年龄为 4-5 岁的哮喘儿童和 610 名对照者以及 185 名年龄为 8 岁的哮喘儿童和 546 名对照者的全血中进行了全基因组甲基化检测，采用横断面病例对照设计。在发现分析中鉴定出差异甲基化的 CpG 位点后，我们在来自六个额外欧洲队列的 247 名儿童（4-16 岁）和 2949 名对照者中进行了验证研究，并对结果进行了荟萃分析。接下来，我们调查了脐带血中复制的 CpG 位点是否可以预测 1316 名儿童以后的哮喘。随后，我们研究了鉴定的 CpG 位点在嗜酸性粒细胞和呼吸道上皮细胞中的细胞特异性甲基化及其相关基因表达特征。我们在 16 岁儿童通过鼻刷收集的 455 个呼吸道上皮细胞样本中以及从血液嗜酸性粒细胞（16 名哮喘患者，8 名对照者[年龄 2-56 岁]）中分离的 DNA 中研究了复制的 CpG 位点的细胞特异性哮喘关联，并将其与 74 名哮喘患者和 93 名对照者（年龄 1-79 岁）的全血 DNA 样本进行了比较。使用荧光激活细胞分选对全血单核细胞进行亚群分选后，使用 RNA-seq 数据注释与复制 CpG 位点相关的全血转录谱。
在发现分析中鉴定出 27 个甲基化 CpG 位点。在荟萃分析后，其中 14 个 CpG 位点复制且通过了全基因组显著性检验（p<1·14 × 10）。在哮喘患者中，从 4 岁到 16 岁的整个儿童时期，在所有相关基因座上均观察到一致的低甲基化水平，但在出生时的脐带血中没有观察到。在使用全血 DNA 的第二次复制研究中，所有 14 个 CpG 位点均与哮喘显著相关，并且与嗜酸性粒细胞强烈相关。与这些 CpG 位点相关的全血转录特征表明，嗜酸性粒细胞、效应和记忆 CD8 T 细胞和自然杀伤细胞的激活增加，而幼稚 T 细胞的数量减少。在呼吸道上皮细胞中，这 14 个 CpG 位点中的 5 个与哮喘相关，表明跨组织的表观遗传效应。
出生后获得的全血 DNA 甲基化程度降低与儿童哮喘密切相关。这些 CpG 位点及其相关的转录谱表明，在儿童哮喘中，嗜酸性粒细胞和细胞毒性 T 细胞被激活。我们的发现值得进一步研究表观遗传学在临床环境中的作用。
欧盟和第七框架计划（MeDALL 项目）。
