摘要

【背景】

肺动脉高压（pulmonary arterial hypertension, PAH）属致死性心血管疾病，其特征性肺小动脉病理重构可导致肺血管阻力持续升高，最终引发右心衰竭。该疾病呈现显著的流行病学异质性，好发于青壮年人群，其中女性患病率显著高于男性。遗传易感性在疾病发病机制中发挥关键作用，2022年ESC/ERS指南已将BMPR2、ACVRL1等14个基因明确列为疾病相关危险基因。这些基因的表型呈现复杂异质性，包括外显率不全及性别差异性外显。以BMPR2为例，其致病突变携带者终生外显率仅约20%，且女性外显风险是男性的2倍。

【目的】

本研究基于携带ACVRL1或BMPR2致病性突变且外显率不全的PAH家系队列，通过高通量测序筛选新的PAH相关危险基因，并对其功能进行探究。

【方法】

本研究的发现队列纳入了23个携带ACVRL1突变的PAH家系。验证队列包含两个独立亚组：6个携带BMPR2突变的肺动脉高压家系，以及108例散发病例（其中25例检出ACVRL1突变，83例携带BMPR2突变）。家系中所有先证者均符合PAH诊断标准，而其携带相同变异的父母表型正常。通过全外显子组测序对比先证者与无症状亲本的遗传背景，筛选只在先证者中存在而亲本携带者中不存在的变异位点，探寻新的可能参与疾病发生的候选基因。并在细胞、动物模型上验证这一基因的功能。

【结果】

通过全外显子组测序对比先证者与无症状亲本的遗传背景，筛选出5个特殊家系，其特征为：先证者除携带ACVRL1或BMPR2致病性变异外，还存在J/J-Receptor(JR)基因杂合变异，而亲本仅携带已知致病性危险基因变异。与健康对照组相比，J/JR基因变异频率在携带ACVRL1或BMPR2突变的PAH患者中显著增加。对患者血清J蛋白检测发现，在校正性别、年龄混杂因素后，其表达水平相较健康对照组显著降低。值得关注的是，在未携带任一已知疾病相关危险基因突变的特发性肺动脉高压患者中，血清J蛋白表达水平亦显著降低。在BMPR2点突变大鼠疾病模型中，肺组织JR蛋白表达呈现显著性别差异：相较野生型对照组，雄性突变大鼠肺组织JR蛋白表达水平明显增加，而雌性突变大鼠则明显降低。转录组测序分析显示，雌雄突变大鼠肺组织间差异表达基因在TGF-β信号通路显著富集，其功能注释主要涉及血管生成调控。体外实验证实，外源性给予J蛋白经JR介导，可显著增加人肺动脉内皮细胞中SMAD1/5/8磷酸化水平，并增强细胞血管生成能力、迁移能力及生存活性。体内实验表明，腺相关病毒介导的大鼠肺动脉内皮细胞JR蛋白过表达，可显著减缓低氧诱导的雌、雄大鼠疾病进展，降低右心室收缩压，改善右心室肥厚指数，血管重构程度减轻。

【结论】

本研究通过全外显子组测序，在携带ACVRL1或BMPR2致病性突变且外显率不全的PAH家系中发现了新的PAH相关基因J/JR。血清J蛋白表达水平在携带ACVRL1或BMPR2致病性突变患者、特发性PAH患者中均降低。体外及体内实验表明，J/JR能够改善肺动脉内皮细胞功能及活性，延缓低氧造模大鼠疾病进程。此外，J蛋白在性别分化中的生物学功能及其表达水平的性别差异，为PAH患病率的性别偏倚和外显率性别差异提供了潜在解释。

Abstract

Background 

Pulmonary arterial hypertension (PAH) is a lethal cardiovascular disorder characterized by pathological remodeling of the pulmonary arterioles, leading to persistently elevated pulmonary vascular resistance and eventual right heart failure. The disease exhibits marked epidemiological heterogeneity, predominantly affecting young and middle-aged populations with a significantly higher prevalence in females compared to males. Genetic predisposition plays a pivotal role in its pathogenesis, with the 2022 ESC/ERS guidelines explicitly identifying 14 disease-associated genes including BMPR2 and ACVRL1. These genes demonstrate complex phenotypic heterogeneity, manifesting incomplete penetrance and sexual dimorphism. For instance, carriers of pathogenic BMPR2 mutations exhibit only approximately 20% lifetime penetrance, with females demonstrating twice the penetrance risk of males.

Objectives

To identify novel PAH-associated gene through high-throughput sequencing of cohorts of PAH families carrying pathogenic mutations in ACVRL1 or BMPR2 but exhibiting incomplete penetrance, and functionally characterize the risk gene in cellular and animal models.

Methods

The discovery cohort comprised 23 PAH pedigrees carrying ACVRL1 mutations. The validation cohort consisted of two independent subgroups: 6 PAH pedigrees with BMPR2 mutations and 108 sporadic cases (25 harboring ACVRL1 mutations and 83 with BMPR2 mutations). All probands from these pedigrees met PAH diagnostic criteria, while their parents carrying identical variants stayed asymptomatic. By comparing the whole-exome sequencing data of the proband with that of asymptomatic parents, variants presented only in the proband but not in the parental carriers were screened to identify novel candidate genes potentially involved in disease pathogenesis. Functional validation was then performed in cellular and animal models.

Results

Comparative analysis of genetic backgrounds between probands and asymptomatic parent carriers using whole-exome sequencing (WES) revealed five distinct pedigrees. These families demonstrated that probands carried both heterozygous J/JR variants and known pathogenic mutations(ACVRL1 or BMPR2), whereas their parents retained only the known pathogenic variants. Further analysis revealed significantly higher frequencies of J/JR variants in patients harboring ACVRL1 or BMPR2 mutations compared to healthy controls. Quantification of serum J protein levels demonstrated a marked reduction in patients after adjustment for sex and age as confounding factors, compared to healthy controls. Notably, idiopathic PAH patients lacking known disease-associated pathogenic variants also exhibited significantly decreased serum J protein level. In the BMPR2 mutated rats, sexually dimorphic alterations in pulmonary JR protein expression were observed: male mutant rats showed significantly elevated JR protein levels in lung tissue compared to wild-type, whereas female mutants exhibited markedly reduced expression. Transcriptomic profiling further revealed sex-specific differentially expressed genes enriched in TGFβ signaling pathways, functionally linked to vascular angiogenesis. In vitro, JR-mediated exogenous J protein administration significantly augmented SMAD1/5/8 phosphorylation in human pulmonary arterial endothelial cells (hPAECs), concomitant with enhanced angiogenic capacity, migratory, and cell viability. Adenovirus-associated viral (AAV) mediated overexpression of JR in rats substantially attenuated hypoxia-induced disease progression in both male and female rats, as evidenced by reduced right ventricular systolic pressure, improved right ventricular hypertrophy index, and mitigated vascular remodeling.

Conclusions

This study identified J/JR as a novel PAH-associated gene through WES in PAH pedigrees carrying ACVRL1 or BMPR2 pathogenic mutations with incomplete penetrance. Serum J protein expression levels were significantly reduced in both patients harboring ACVRL1 or BMPR2 pathogenic mutations and those with idiopathic PAH. In vitro and in vivo experiments demonstrated that J/JR enhances hPAECs function and viability, as well as attenuated disease progression in hypoxia-induced rats. Additionally, the biological function of J protein in sex determination and its sex-specific expression patterns provide a potential explanation for the disease's epidemiological sex bias.