DNA methyltransferase 3B deficiency unveils a new pathological mechanism of pulmonary hypertension.

DNA methylation plays critical roles in vascular pathology of pulmonary hypertension (PH). The underlying mechanism, however, remains undetermined. Here, we demonstrate that global DNA methylation was elevated in the lungs of PH rat models after monocrotaline administration or hypobaric hypoxia exposure. We showed that DNA methyltransferase 3B (DNMT3B) was up-regulated in both PH patients and rodent models. Furthermore, Dnmt3b -/- rats exhibited more severe pulmonary vascular remodeling. Consistently, inhibition of DNMT3B promoted proliferation/migration of pulmonary artery smooth muscle cells (PASMCs) in response to platelet-derived growth factor-BB (PDGF-BB). In contrast, overexpressing DNMT3B in PASMCs attenuated PDGF-BB-induced proliferation/migration and ameliorated hypoxia-mediated PH and right ventricular hypertrophy in mice. We also showed that DNMT3B transcriptionally regulated inflammatory pathways. Our results reveal that DNMT3B is a previously undefined mediator in the pathogenesis of PH, which couples epigenetic regulations with vascular remodeling and represents a therapeutic target to tackle PH.

The features of rare pathogenic BMPR2 variants in pulmonary arterial hypertension: Comparison between patients and reference population.

BACKGROUND: Mutations in the gene encoding bone morphogenetic protein receptor type 2 (BMPR2) are the most common genetic risk factors underlying pulmonary arterial hypertension (PAH). However, the features of PAH-related BMPR2 rare variants remain unclear. We propose that the discrepancy of BMPR2 rare variants landscape between patients with PAH and reference population would be important to address the genetic background of PAH-related variants. METHODS: We genotyped BMPR2 rare variants in 670 Chinese patients with pulmonary arterial hypertension. The BMPR2 rare variants were screened in 10,508 reference people from two exome databases. RESULTS: The prevalence of rare BMPR2 variants in patients with PAH was significantly higher compared to the reference population (21.5%, 144/670 vs 0.87%, 91/10508, p = 1.3 × 10-118). In patients with PAH, 49% of identified BMPR2 rare variants were loss-of-function or splicing. These BMPR2 rare variants were only observed in 1% of the reference population (p = 9.0 × 10-12). Arg491, which is absent in the reference population, represented as hot-spot site (14.6%, 21/144) in PAH patients. BMPR2 missense mutations in PAH patients were more likely distributed in extracellular ligand-binding domain (ECD, 29.7% vs 11.1%, p < 0.001). Compared with Non-PAH-related variations, PAH-related missense variants tend to alter the amino acid electric status (51.4% vs 23.3%, p < 0.001). CONCLUSIONS: BMPR2 variants located in extracellular ligand-binding domain or altered the amino acid electric status are more pathogenic.

Association of Rare PTGIS Variants With Susceptibility and Pulmonary Vascular Response in Patients With Idiopathic Pulmonary Arterial Hypertension.

Importance: Idiopathic pulmonary arterial hypertension (IPAH) is a fatal disease with high heritability; however, the bone morphogenetic protein receptor 2 (BMPR2) gene only accounts for 17% of IPAH. The genetic basis of IPAH needs further investigation. Objective: To identify novel IPAH susceptibility genes other than BMPR2. Design, Setting, and Participants: This 2-stage, case-control genetic association study enrolled 230 patients with IPAH from 2 referral pulmonary hypertension centers in China. Eligible patients had no BMPR2 variants and were compared with 968 healthy control participants. Data were collected from January 1, 2000, to July 31, 2015, and analyzed from August 1, 2015, to May 30, 2018. Exposures: PTGIS rare variants. Main Outcomes and Measures: Whole-genome sequencing was performed to identify putative IPAH genes in a discovery cohort, with validation in an independent referral cohort. Correlation of genotype and hemodynamic characteristics was then evaluated at baseline and after pulmonary vasodilator testing. Functional assessments were conducted to analyze the effects of identified genetic variants on transcript splicing, enzymatic activity, and endothelial cell phenotypes. Results: Among 230 patients with IPAH (164 female [71.3%]; mean [SD] age, 34 [18] years), an enrichment of rare variants in a gene encoding prostacyclin synthase (PTGIS) was identified in the discovery cohort. The association of PTGIS rare variants with IPAH was confirmed in the replication cohort. In the combined data set, PTGIS rare variants were found in 14 of 230 cases (6.1%) and 8 of 968 controls (0.8%) (odds ratio, 7.8; 95% CI, 3.2-18.8; P = 5 × 10-6, logistic regression). Compared with patients without PTGIS variants, inhaled iloprost induced a more significant decrease of pulmonary vascular resistance (difference in the least square mean, -21.7%; 95% CI, -31.4% to -12.0%; P < .001, linear regression model) and an increase of cardiac index (difference in the least square mean, 18.3%; 95% CI, 8.8%-27.8%; P < .001, linear regression model) in patients with PTGIS variants. The minigene assay indicated that the c.521 + 1G>A variant resulted in aberrant messenger RNA transcripts. The functional studies showed that the 2 missense rare variants (R252Q and A447T) resulted in a decrease in prostacyclin production and increased cell death of pulmonary microvascular endothelial cells. Conclusions and Relevance: This study identified 3 rare loss-of-function variants in the PTGIS gene from 2 independent cohorts with IPAH. The genetic variants of PTGIS predispose pulmonary vascular responses to the iloprost stimulation. These findings suggest that PTGIS variants may be involved in the pathogenesis of IPAH.

Germline BMP9 mutation causes idiopathic pulmonary arterial hypertension.

BACKGROUND: Idiopathic pulmonary arterial hypertension (IPAH) is a rare disease with high heritability. Although several predisposing genes have been linked to IPAH, the genetic aetiology remains unknown for a large number of IPAH cases. METHODS: We conducted an exome-wide gene-based burden analysis on two independent case-control studies, including a total of 331 IPAH cases and 10 508 controls. Functional assessments were conducted to analyse the effects of genetic mutations on protein biosynthesis and function. RESULTS: The gene encoding human bone morphogenetic protein 9 (BMP9) was identified as a novel genetic locus displaying exome-wide association with IPAH in the discovery cohort (OR 18.8; p=1.9×10-11). This association was authenticated in the independent replication cohort (p=1.0×10-5). Collectively, the rare coding mutations in BMP9 occurred in 6.7% of cases, ranking this gene second to BMPR2, comprising a combined significance of 2.7×10-19 (OR 21.2). Intriguingly, the patients with BMP9 mutations had lower plasma levels of BMP9 than those without. Functional studies showed that the BMP9 mutations led to reduced BMP9 secretion and impaired anti-apoptosis ability in pulmonary arterial endothelial cells. CONCLUSION: We identify BMP9 as an IPAH culprit gene.

Elevated levels of plasma transforming growth factor-ß1 in idiopathic and heritable pulmonary arterial hypertension.

BACKGROUND: Aberrant transforming growth factor (TGF)-ß signaling is involved in the pathogenesis of pulmonary arterial hypertension (PAH). We aimed to investigate the predictive value of the upstream ligand of TGF-ß signaling (TGF-ß1) on long-term mortality and the clinical characteristics of patients with idiopathic pulmonary arterial hypertension (IPAH) and heritable PAH (HPAH). METHODS AND RESULTS: Plasma TGF-ß1 levels were measured in 151 IPAH and 65 HPAH patients retrospectively enrolled between January 2008 and March 2013, and compared to 61 healthy subjects. Data for mortality over time were obtained from hospital databases and from telephone follow-ups. The main outcome was all-cause death. Plasma TGF-ß1 was significantly higher in IPAH/HPAH patients compared to control subjects (4.74 vs. 2.61ng/mL, respectively; P<0.001). Mean follow-up time was 3.4±1.8years, during which 86 patients died. ROC curves were utilized to determine TGF-ß1 cutoff values. Compared to patients with TGF-ß1 of <3.74ng/mL, heart function was significantly impaired (percentage of patients with WHO functional class III/IV, 51.4% vs. 65.5%, P=0.043) and mortality risk was elevated (P=0.009) for patients with TGF-ß1>3.74ng/mL. However, the difference in mortality rate between patients with higher and lower TGF-ß1 levels was only statistically significant for female patients (P=0.004), despite a similar trend for male patients. Multivariate analyses revealed that TGF-ß1 (HR after log transformation base of 10: 2.623; 95%CI: 1.228-5.603; P=0.013) emerged as the independent predictor for all-cause mortality. CONCLUSION: High circulating levels of TGF-ß1 were an independent predictor of a poor outcome for IPAH/HPAH patients.