Yoshida Yu, Uchida Keiko, Kodo Kazuki, Shibata Hironori, Furutani Yoshiyuki, Nakayama Tomotaka, Sakai Satoshi, Nakanishi Toshio, Takahashi Takao, Yamagishi Hiroyuki
Department of Pediatrics, Keio University School of Medicine, Tokyo, Japan.
Department of Pediatrics, Keio University School of Medicine, Tokyo, Japan; Health Center, Keio University, Kanagawa, Japan.
J Mol Cell Cardiol. 2022 Oct;171:105-116. doi: 10.1016/j.yjmcc.2022.07.002. Epub 2022 Jul 29.
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.
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.
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.
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.
肺动脉高压（PAH）是一种致命疾病，约10%的病例与基因变异有关。近期的基因研究报告了PAH患者中TBX4基因的致病变异，尤其是在儿童期发病的患者中，但由TBX4变异导致PAH的发病机制尚未完全阐明。
我们采用基于聚合酶链反应的双向测序方法，对75例日本散发性或家族性PAH患者的TBX4基因进行了分析。使用计算机分析以及体外分析（包括荧光素酶测定、免疫细胞化学和染色质免疫沉淀法（ChIP））来评估检测到的变异是否具有功能改变。我们还通过抑制Tbx4表达的小鼠胚胎肺外植体分析了TBX4的功能。
在3例患者（4.0%）中检测到推定的致病变异。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，而鉴定出的TBX4变异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的体外抑制导致肺形态发生不足，同时Tie2和Kruppel样因子4表达特异性下调。
我们的结果表明，TBX4变异是日本人群中一部分PAH的遗传病因。TBX4变异可能通过破坏TBX4介导的对FGF10信号的直接调节以及涉及PAH相关分子的肺血管内皮功能障碍，导致肺形态发生不足，从而引发PAH。
