Cord blood DNA methylome in newborns later diagnosed with autism spectrum disorder reflects early dysregulation of neurodevelopmental and X-linked genes. Cord blood DNA methylome in newborns later diagnosed with autism spectrum disorder reflects early dysregulation of neurodevelopmental and X-linked genes

Autism spectrum disorder (ASD) is a neurodevelopmental disorder with complex heritability and higher prevalence in males. The neonatal epigenome has the potential to reflect past interactions between genetic and environmental factors during early development and influence future health outcomes. We performed whole-genome bisulfite sequencing of 152 umbilical cord blood samples from the MARBLES and EARLI high-familial risk prospective cohorts to identify an epigenomic signature of ASD at birth. Samples were split into discovery and replication sets and stratified by sex, and their DNA methylation profiles were tested for differentially methylated regions (DMRs) between ASD and typically developing control cord blood samples. DMRs were mapped to genes and assessed for enrichment in gene function, tissue expression, chromosome location, and overlap with prior ASD studies. DMR coordinates were tested for enrichment in chromatin states and transcription factor binding motifs. Results were compared between discovery and replication sets and between males and females. We identified DMRs stratified by sex that discriminated ASD from control cord blood samples in discovery and replication sets. At a region level, 7 DMRs in males and 31 DMRs in females replicated across two independent groups of subjects, while 537 DMR genes in males and 1762 DMR genes in females replicated by gene association. These DMR genes were significantly enriched for brain and embryonic expression, X chromosome location, and identification in prior epigenetic studies of ASD in post-mortem brain. In males and females, autosomal ASD DMRs were significantly enriched for promoter and bivalent chromatin states across most cell types, while sex differences were observed for X-linked ASD DMRs. Lastly, these DMRs identified in cord blood were significantly enriched for binding sites of methyl-sensitive transcription factors relevant to fetal brain development. At birth, prior to the diagnosis of ASD, a distinct DNA methylation signature was detected in cord blood over regulatory regions and genes relevant to early fetal neurodevelopment. Differential cord methylation in ASD supports the developmental and sex-biased etiology of ASD and provides novel insights for early diagnosis and therapy. Overall design: Investigation of DNA methylation with whole-genome bisulfite sequencing in umbilical cord blood from two high-risk cohorts of newborns diagnosed with autism spectrum disorder (ASD) or typical development (TD) at 36 months (TD n = 76, ASD n = 76).

自闭症谱系障碍（Autism spectrum disorder, ASD）是一类具有复杂遗传特性且男性患病率更高的神经发育障碍。新生儿表观基因组能够反映早期发育阶段遗传与环境因素的既往交互作用，并对未来健康结局产生影响。为识别出生时即可检测到的ASD表观基因组特征，本研究对来自MARBLES与EARLI高家族风险前瞻性队列的152份脐带血样本开展全基因组亚硫酸氢盐测序（whole-genome bisulfite sequencing）。研究将样本划分为发现集与重复集，并按性别进行分层，随后检测ASD组与典型发育（typical development, TD）对照组脐带血样本间的差异甲基化区域（differentially methylated regions, DMRs）。将鉴定得到的DMR定位至对应基因，并评估其在基因功能、组织表达谱、染色体位置方面的富集情况，以及与既往ASD研究的重叠程度。同时对DMR的基因组坐标开展染色质状态与转录因子结合基序的富集分析。对比发现集与重复集、男性与女性群体间的分析结果。本研究成功鉴定出按性别分层的DMR，可在发现集与重复集中有效区分ASD组与对照组脐带血样本。在区域层面，男性的7个DMR与女性的31个DMR在两个独立受试者队列中得到验证重复；而男性的537个DMR相关基因与女性的1762个DMR相关基因通过基因关联分析得到重复验证。这些DMR相关基因显著富集于脑与胚胎组织的表达特征、X染色体定位，且在既往针对死后脑组织的ASD表观遗传研究中已有相关鉴定结果。在男性与女性群体中，常染色体ASD相关DMR在多数细胞类型中均显著富集于启动子与二价染色质状态，而X连锁ASD相关DMR则表现出显著的性别差异。最后，本研究在脐带血中鉴定的这些DMR显著富集于与胎儿脑发育相关的甲基敏感转录因子结合位点。在ASD确诊前的新生儿阶段，脐带血中即可检测到与早期胎儿神经发育相关调控区域及基因的独特DNA甲基化特征。ASD相关脐带血甲基化差异支持了ASD的发育起源与性别偏向性病因学，并为早期诊断与治疗提供了全新的研究视角。总体实验设计：对两个高风险新生儿队列的脐带血样本开展全基因组亚硫酸氢盐测序以分析DNA甲基化水平，这些新生儿在36月龄时被诊断为自闭症谱系障碍（ASD）或典型发育（TD）（TD组n=76，ASD组n=76）。