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Abstract：
BACKGROUND: Congenital heart defects (CHDs) affect approximately half of individuals with Down syndrome (DS), but the molecular reasons for incomplete penetrance are unknown. Previous studies have largely focused on identifying genetic risk factors associated with CHDs in individuals with DS, but comprehensive studies of the contribution of epigenetic marks are lacking. We aimed to identify and characterize DNA methylation differences from newborn dried blood spots (NDBS) of DS individuals with major CHDs compared to DS individuals without CHDs.
METHODS: We used the Illumina EPIC array and whole-genome bisulfite sequencing (WGBS) to quantitate DNA methylation for 86 NDBS samples from the California Biobank Program: (1) 45 DS-CHD (27 female, 18 male) and (2) 41 DS non-CHD (27 female, 14 male). We analyzed global CpG methylation and identified differentially methylated regions (DMRs) in DS-CHD versus DS non-CHD comparisons (both sex-combined and sex-stratified) corrected for sex, age of blood collection, and cell-type proportions. CHD DMRs were analyzed for enrichment in CpG and genic contexts, chromatin states, and histone modifications by genomic coordinates and for gene ontology enrichment by gene mapping. DMRs were also tested in a replication dataset and compared to methylation levels in DS versus typical development (TD) WGBS NDBS samples.
RESULTS: We found global CpG hypomethylation in DS-CHD males compared to DS non-CHD males, which was attributable to elevated levels of nucleated red blood cells and not seen in females. At a regional level, we identified 58, 341, and 3938 CHD-associated DMRs in the Sex Combined, Females Only, and Males Only groups, respectively, and used machine learning algorithms to select 19 Males Only loci that could distinguish CHD from non-CHD. DMRs in all comparisons were enriched for gene exons, CpG islands, and bivalent chromatin and mapped to genes enriched for terms related to cardiac and immune functions. Lastly, a greater percentage of CHD-associated DMRs than background regions were differentially methylated in DS versus TD samples.
CONCLUSIONS: A sex-specific signature of DNA methylation was detected in NDBS of DS-CHD compared to DS non-CHD individuals. This supports the hypothesis that epigenetics can reflect the variability of phenotypes in DS, particularly CHDs.
METHODS: We used the Illumina EPIC array and whole-genome bisulfite sequencing (WGBS) to quantitate DNA methylation for 86 NDBS samples from the California Biobank Program: (1) 45 DS-CHD (27 female, 18 male) and (2) 41 DS non-CHD (27 female, 14 male). We analyzed global CpG methylation and identified differentially methylated regions (DMRs) in DS-CHD versus DS non-CHD comparisons (both sex-combined and sex-stratified) corrected for sex, age of blood collection, and cell-type proportions. CHD DMRs were analyzed for enrichment in CpG and genic contexts, chromatin states, and histone modifications by genomic coordinates and for gene ontology enrichment by gene mapping. DMRs were also tested in a replication dataset and compared to methylation levels in DS versus typical development (TD) WGBS NDBS samples.
RESULTS: We found global CpG hypomethylation in DS-CHD males compared to DS non-CHD males, which was attributable to elevated levels of nucleated red blood cells and not seen in females. At a regional level, we identified 58, 341, and 3938 CHD-associated DMRs in the Sex Combined, Females Only, and Males Only groups, respectively, and used machine learning algorithms to select 19 Males Only loci that could distinguish CHD from non-CHD. DMRs in all comparisons were enriched for gene exons, CpG islands, and bivalent chromatin and mapped to genes enriched for terms related to cardiac and immune functions. Lastly, a greater percentage of CHD-associated DMRs than background regions were differentially methylated in DS versus TD samples.
CONCLUSIONS: A sex-specific signature of DNA methylation was detected in NDBS of DS-CHD compared to DS non-CHD individuals. This supports the hypothesis that epigenetics can reflect the variability of phenotypes in DS, particularly CHDs.
摘要：
背景：先天性心脏缺陷（CHDs）影响大约一半的唐氏综合征（DS）患者，但是不完全外显率的分子原因是未知的。以前的研究主要集中在确定与DS患者CHD相关的遗传风险因素。但是缺乏对表观遗传标记贡献的全面研究。我们旨在鉴定和表征与没有CHD的DS个体相比，患有主要CHD的DS个体的新生儿干血点（NDBS）的DNA甲基化差异。
方法：我们使用IlluminaEPIC阵列和全基因组亚硫酸氢盐测序（WGBS）定量来自加利福尼亚生物库计划的86个NDBS样品的DNA甲基化：（1）45个DS-CHD（27个女性，18男）和（2）41DS非冠心病（27女，14男）。我们分析了全球CpG甲基化，并确定了DS-CHD与DS非CHD比较（性别组合和性别分层）中的差异甲基化区域（DMRs），采血年龄,和细胞类型的比例。CHDDMRs被分析在CpG和基因环境中的富集，染色质状态，通过基因组坐标进行组蛋白修饰，并通过基因作图进行基因本体论富集。还在复制数据集中测试DMR,并将其与DS和典型发育(TD)WGBSNDBS样品中的甲基化水平进行比较。
结果：我们发现DS-CHD男性与DS非CHD男性相比，全球CpG低甲基化，这归因于有核红细胞水平升高，在女性中没有见过。在区域一级,我们在性别组合中确定了58、341和3938个CHD相关DMRs，只有女性,只有男性群体，分别,并使用机器学习算法选择19个可以区分CHD和非CHD的仅男性基因座。所有比较中的DMRs都富集了基因外显子，CpG群岛,和二价染色质，并定位到与心脏和免疫功能相关的术语富集的基因。最后,与背景区域相比，在DS和TD样品中，CHD相关的DMR差异甲基化的百分比更高。
结论：与DS非CHD个体相比，在DS-CHD的NDBS中检测到DNA甲基化的性别特异性特征。这支持了表观遗传学可以反映DS表型变异性的假设，尤其是CHD。
方法：我们使用IlluminaEPIC阵列和全基因组亚硫酸氢盐测序（WGBS）定量来自加利福尼亚生物库计划的86个NDBS样品的DNA甲基化：（1）45个DS-CHD（27个女性，18男）和（2）41DS非冠心病（27女，14男）。我们分析了全球CpG甲基化，并确定了DS-CHD与DS非CHD比较（性别组合和性别分层）中的差异甲基化区域（DMRs），采血年龄,和细胞类型的比例。CHDDMRs被分析在CpG和基因环境中的富集，染色质状态，通过基因组坐标进行组蛋白修饰，并通过基因作图进行基因本体论富集。还在复制数据集中测试DMR,并将其与DS和典型发育(TD)WGBSNDBS样品中的甲基化水平进行比较。
结果：我们发现DS-CHD男性与DS非CHD男性相比，全球CpG低甲基化，这归因于有核红细胞水平升高，在女性中没有见过。在区域一级,我们在性别组合中确定了58、341和3938个CHD相关DMRs，只有女性,只有男性群体，分别,并使用机器学习算法选择19个可以区分CHD和非CHD的仅男性基因座。所有比较中的DMRs都富集了基因外显子，CpG群岛,和二价染色质，并定位到与心脏和免疫功能相关的术语富集的基因。最后,与背景区域相比，在DS和TD样品中，CHD相关的DMR差异甲基化的百分比更高。
结论：与DS非CHD个体相比，在DS-CHD的NDBS中检测到DNA甲基化的性别特异性特征。这支持了表观遗传学可以反映DS表型变异性的假设，尤其是CHD。
