Placental methylome reveals a 22q13.33 brain regulatory gene locus associated with autism [WGS]

Most autism spectrum disorder (ASD) cases involve complex genetics interacting with perinatal environment, complicating the discovery of common genetic risk. The epigenetic layer of DNA methylation shows dynamic developmental changes and molecular memory of in utero experiences, particularly in placenta, a fetal tissue discarded at birth. However, current array-based methods to identify novel ASD risk genes lack coverage of the most structurally and epigenetically variable regions of the human genome. Here we used whole genome bisulfite sequencing (WGBS) in placenta samples from prospective ASD studies to discover a previously uncharacterized ASD risk gene in a co-methylated block at 22q13.33. Differentially methylated region (DMR) analysis identified 139 DMRs common to ASD in placental samples, including a high-confidence 118 kb hypomethylated block at 22q13.33 that replicated in two additional cohorts and identified the novel transcript NHIP as neuronal hypoxia inducible, placenta associated (gene name approved by HGNC). NHIP characterized by high expression in brain, increased expression following neuronal differentiation or hypoxia in vitro, and minimal expression in ASD placenta. Transient NHIP overexpression in cell lines increased cellular proliferation and altered expression of genes regulating synapses and neurogenesis in response to hypoxia, significantly overlapping with NHIP-associated transcript levels in ASD brain and known ASD risk genes. A common Alu insert near a fetal brain enhancer correlated with NHIP placental and brain expression levels and ASD risk, demonstrating a common genetic influence on DNA methylation levels. Together, these results demonstrate a novel environmentally-responsive ASD risk gene relevant to brain development in a hitherto uncharacterized region of the human genome. WGBS, WGS, RNAseq on brain, and cell

绝大多数自闭症谱系障碍（autism spectrum disorder, ASD）病例涉及与围产期环境相互作用的复杂遗传机制，这为常见遗传风险因子的发现增添了极大难度。DNA甲基化的表观遗传层兼具动态发育变化与子宫内暴露经历的分子记忆特征，这一特性在出生即被丢弃的胎儿组织胎盘中尤为突出。然而，当前用于识别新型ASD风险基因的基于芯片的检测方法，无法覆盖人类基因组中结构与表观遗传变异程度最高的区域。本研究对前瞻性ASD研究的胎盘样本开展全基因组亚硫酸氢盐测序（whole genome bisulfite sequencing, WGBS），在22q13.33区域的共甲基化区块中发现了此前未被表征的ASD风险基因。差异甲基化区域（differentially methylated region, DMR）分析共鉴定出胎盘样本中与ASD相关的139个DMR，其中包括位于22q13.33的118 kb高可信度低甲基化区块，该区块在另外两个独立队列中得到验证，并鉴定出新型转录本NHIP，其功能为神经元缺氧诱导且胎盘相关（基因名称已获人类基因命名委员会（HGNC）批准）。NHIP在大脑中呈高表达，在体外进行神经元分化或缺氧刺激后表达上调，而在ASD患者胎盘组织中表达量极低。在细胞系中瞬时过表达NHIP可促进细胞增殖，并改变突触与神经发生相关基因在缺氧应答中的表达模式，该表达谱与ASD患者大脑中NHIP相关转录水平以及已知ASD风险基因的表达模式显著重叠。位于胎儿脑增强子附近的常见Alu插入序列（Alu insert）与NHIP在胎盘及大脑中的表达水平以及ASD风险相关，这证实了遗传变异对DNA甲基化水平的普遍调控作用。综上，本研究在人类基因组此前未被表征的区域中发现了一种新型环境响应型ASD风险基因，该基因与大脑发育密切相关。本研究还使用了WGBS、全基因组测序（WGS）、大脑RNA测序（RNA-seq）以及细胞