Data_Sheet_1_Somatic and de novo Germline Variants of MEDs in Human Neural Tube Defects.docx

BackgroundNeural tube defects (NTDs) are among the most common and severe congenital defects in humans. Their genetic etiology is complex and remains poorly understood. The Mediator complex (MED) plays a vital role in neural tube development in animal models. However, no studies have yet examined the role of its human homolog in the etiology of NTDs. MethodsIn this study, 48 pairs of neural lesion site and umbilical cord tissues from NTD and 21 case-parent trios were involved in screening for NTD-related somatic and germline de novo variants. A series of functional cell assays were performed. We generated a Med12 p.Arg1784Cys knock-in mouse using CRISPR/Cas9 technology to validate the human findings. ResultsOne somatic variant, MED12 p.Arg1782Cys, was identified in the lesion site tissue from an NTD fetus. This variant was absent in any other normal tissue from different germ layers of the same case. In 21 case-parent trios, one de novo stop-gain variant, MED13L p.Arg1760∗, was identified. Cellular functional studies showed that MED12 p.Arg1782Cys decreased MED12 protein level and affected the regulation of MED12 on the canonical-WNT signaling pathway. The Med12 p.Arg1784Cys knock-in mouse exhibited exencephaly and spina bifida. ConclusionThese findings provide strong evidence that functional variants of MED genes are associated with the etiology of some NTDs. We demonstrated a potentially important role for somatic variants in the occurrence of NTDs. Our study is the first study in which an NTD-related variant identified in humans was validated in mice using CRISPR/Cas9 technology.背景 神经管缺陷（Neural tube defects, NTDs）是人类最常见且最为严重的先天性缺陷之一，其遗传病因复杂且至今尚未完全阐明。中介体复合物（Mediator complex, MED）在动物模型的神经管发育过程中发挥关键作用，但目前尚无研究探讨其人类同源基因在NTD病因学中的功能。 方法 本研究纳入48对NTD患者的神经管病灶组织与脐带组织，以及21个病例-父母核心家系样本，用于筛选与NTD相关的体细胞及生殖系新发变异。本研究开展了一系列细胞功能实验，并利用CRISPR/Cas9技术构建Med12 p.Arg1784Cys敲入小鼠，以验证人类样本中的研究发现。 结果 本研究在1例NTD胎儿的病灶组织中检测到1个体细胞变异MED12 p.Arg1782Cys，该变异未在同1病例不同胚层的其他正常组织中检出。在21个病例-父母核心家系中，检测到1个新发无义变异MED13L p.Arg1760*。细胞功能实验结果显示，MED12 p.Arg1782Cys可降低MED12蛋白表达水平，并干扰MED12对经典Wnt信号通路的调控作用。构建的Med12 p.Arg1784Cys敲入小鼠表现出露脑畸形与脊柱裂表型。 结论 本研究结果为MED基因功能变异与部分NTD的病因学相关提供了有力证据，证实了体细胞变异在NTD发生过程中可能发挥的重要作用。本研究是首个利用CRISPR/Cas9技术在小鼠体内验证人类来源NTD相关变异的研究。