背景:肺动脉高压(PAH)是一种致命的疾病,大约10%的病例与遗传变异有关。最近的遗传研究报道了PAH患者TBX4基因的致病变异,特别是在儿童发病的患者中,但TBX4变异导致PAH的发病机制尚未完全揭示。
方法:我们使用基于PCR的双向测序方法分析了75例日本散发性或家族性PAH患者的TBX4基因。使用计算机模拟分析以及包括荧光素酶测定在内的体外分析来评估检测到的变体。免疫细胞化学和染色质免疫沉淀(ChIP)是否具有改变的功能。我们还使用抑制Tbx4表达的小鼠胚胎肺外植体分析了TBX4的功能。
结果:在3例(4.0%)中检测到推定的致病性变异。我们的体外功能分析显示,TBX4直接调节成纤维细胞生长因子10(FGF10)的转录活性,而鉴定的TBX4变体蛋白由于核定位信号的破坏或DNA结合亲和力差而未能激活FGF10基因。此外,Tbx4的离体抑制导致肺形态发生不足以及Tie2和Kruppel样因子4表达的特异性下调。
结论:我们的结果提示TBX4变异是日本人群中PAH的遗传原因。TBX4中的变体可能通过破坏TBX4介导的FGF10信号传导的直接调节和涉及PAH相关分子的肺血管内皮功能障碍,通过不足的肺形态发生导致PAH。
BACKGROUND: Pulmonary arterial hypertension (PAH) is a fatal disease, with approximately 10% of cases associated with genetic variants. Recent genetic studies have reported pathogenic variants in the TBX4 gene in patients with PAH, especially in patients with childhood-onset of the disease, but the pathogenesis of PAH caused by TBX4 variant has not been fully uncovered.
METHODS: We analysed the TBX4 gene in 75 Japanese patients with sporadic or familial PAH using a PCR-based bidirectional sequencing method. Detected variants were evaluated using in silico analyses as well as in vitro analyses including luciferase assay, immunocytochemistry and chromatin immunoprecipitation (ChIP) whether they have altered function. We also analysed the function of TBX4 using mouse embryonic lung explants with inhibition of Tbx4 expression.
RESULTS: Putative pathogenic variants were detected in three cases (4.0%). Our in vitro functional analyses revealed that TBX4 directly regulates the transcriptional activity of fibroblast growth factor 10 (FGF10), whereas the identified TBX4 variant proteins failed to activate the FGF10 gene because of disruption of nuclear localisation signal or poor DNA-binding affinity. Furthermore, ex vivo inhibition of Tbx4 resulted in insufficiency of lung morphogenesis along with specific downregulation of Tie2 and Kruppel-like factor 4 expression.
CONCLUSIONS: Our results implicate variants in TBX4 as a genetic cause of PAH in a subset of the Japanese population. Variants in TBX4 may lead to PAH through insufficient lung morphogenesis by disrupting the TBX4-mediated direct regulation of FGF10 signalling and pulmonary vascular endothelial dysfunction involving PAH-related molecules.