A multi-omics approach using a mouse model of cardiac malformations for prioritization of human congenital heart disease contributing genes [RNA-seq]

Congenital heart disease (CHD) is the most common type of birth defect, affecting ~1% of all live births. Malformations of the cardiac outflow tract (OFT) account for ~30% of all CHD and include a range of CHDs from bicuspid aortic valve (BAV) to tetralogy of Fallot (TOF). We hypothesized that transcriptomic profiling of a mouse model of CHD would highlight disease-contributing genes implicated in congenital cardiac malformations in humans. To test this hypothesis, we utilized global transcriptional profiling differences from a mouse model of OFT malformations to prioritize damaging, de novo variants identified from exome sequencing datasets from published cohorts of CHD patients. Notch1+/-;Nos3-/- mice display a spectrum of cardiac OFT malformations ranging from BAV, semilunar valve (SLV) stenosis to TOF. Global transcriptional profiling of the E13.5 Notch1+/-;Nos3-/- mutant mouse OFTs and wildtype controls was performed by RNA sequencing (RNA-Seq). Analysis of the RNA-Seq dataset demonstrated genes belonging to the Hif1α, Tgf-β, Hippo, and Wnt signaling pathways were differentially expressed in the mutant OFT. Mouse to human comparative analysis was then performed to determine if patients with TOF and SLV stenosis display an increased burden of damaging, genetic variants in gene homologs that were dysregulated in Notch1+/-; Nos3-/- OFT. We found an enrichment of de novo variants in the TOF population among the 1,352 significantly differentially expressed genes in Notch1+/-;Nos3-/- mouse OFT but not the SLV population. This association was not significant when compared to only highly expressed genes in the murine OFT and of de novo variants in the TOF population. These results suggest that transcriptomic datasets generated from the appropriate temporal, anatomic and cellular tissues from murine models of CHD may provide a novel approach for the prioritization of disease-contributing genes in patients with CHD. Global transcriptional profiling of the E13.5 Notch1+/-;Nos3-/- mutant mouse OFTs and wildtype controls was performed by RNA sequencing (RNA-Seq)

先天性心脏病（Congenital heart disease, CHD）是最常见的出生缺陷类型，约影响1%的活产新生儿。心脏流出道（cardiac outflow tract, OFT）畸形占所有CHD的约30%，涵盖从二叶主动脉瓣（bicuspid aortic valve, BAV）到法洛四联症（tetralogy of Fallot, TOF）等多种CHD亚型。我们提出假说：对CHD小鼠模型开展转录组分析，可揭示人类先天性心脏畸形中与疾病致病相关的基因。为验证该假说，我们利用心脏流出道畸形小鼠模型的全局转录谱差异，对已发表的CHD患者队列外显子组测序数据中鉴定出的有害新发变异进行优先级排序。Notch1+/-;Nos3-/-小鼠表现出从二叶主动脉瓣、半月瓣（semilunar valve, SLV）狭窄到法洛四联症的一系列心脏流出道畸形。我们对E13.5天的Notch1+/-;Nos3-/-突变小鼠心脏流出道组织及野生型对照样本进行了RNA测序（RNA-Seq），以开展全局转录谱分析。对该RNA测序数据集的分析显示，Hif1α、Tgf-β、Hippo及Wnt信号通路相关基因在突变型心脏流出道组织中存在差异表达。随后我们开展了小鼠-人类跨物种比较分析，以探究法洛四联症和半月瓣狭窄患者，其基因同源物的有害遗传变异负荷是否在Notch1+/-;Nos3-/-小鼠心脏流出道组织中失调的基因中升高。我们发现，在Notch1+/-;Nos3-/-小鼠心脏流出道组织中1352个显著差异表达的基因中，法洛四联症患者队列存在有害新发变异的富集现象，但半月瓣狭窄患者队列未观察到此现象。若仅以小鼠心脏流出道组织中高表达基因作为参照，该关联在法洛四联症患者队列的新发变异分析中并无统计学显著性。上述结果表明，利用合适的时间维度、解剖结构及细胞类型的小鼠CHD模型生成的转录组数据集，可为CHD患者疾病致病基因的优先级排序提供全新的研究思路。我们对E13.5天的Notch1+/-;Nos3-/-突变小鼠心脏流出道组织及野生型对照样本进行了RNA测序（RNA-Seq）。