Characterization of GDF2 Mutations and Levels of BMP9 and BMP10 in Pulmonary Arterial Hypertension

Abstract

Rationale: Recently, rare heterozygous mutations in GDF2 were identified in patients with pulmonary arterial hypertension (PAH). GDF2 encodes the circulating BMP (bone morphogenetic protein) type 9, which is a ligand for the BMP2 receptor.Objectives: Here we determined the functional impact of GDF2 mutations and characterized plasma BMP9 and BMP10 levels in patients with idiopathic PAH.Methods: Missense BMP9 mutant proteins were expressed in vitro and the impact on BMP9 protein processing and secretion, endothelial signaling, and functional activity was assessed. Plasma BMP9 and BMP10 levels and activity were assayed in patients with PAH with GDF2 variants and in control subjects. Levels were also measured in a larger cohort of control subjects (n = 120) and patients with idiopathic PAH (n = 260).Measurements and Main Results: We identified a novel rare variation at the GDF2 and BMP10 loci, including copy number variation. In vitro, BMP9 missense proteins demonstrated impaired cellular processing and secretion. Patients with PAH who carried these mutations exhibited reduced plasma levels of BMP9 and reduced BMP activity. Unexpectedly, plasma BMP10 levels were also markedly reduced in these individuals. Although overall BMP9 and BMP10 levels did not differ between patients with PAH and control subjects, BMP10 levels were lower in PAH females. A subset of patients with PAH had markedly reduced plasma levels of BMP9 and BMP10 in the absence of GDF2 mutations.Conclusions: Our findings demonstrate that GDF2 mutations result in BMP9 loss of function and are likely causal. These mutations lead to reduced circulating levels of both BMP9 and BMP10. These findings support therapeutic strategies to enhance BMP9 or BMP10 signaling in PAH.

Keywords: BMP10; BMP9; GDF2; pulmonary arterial hypertension.

Figure 1.

Characterization of expressed BMP9 (bone morphogenetic protein type 9) mutant proteins. (A) Schematic of different potential species of BMP9. Numbers in parentheses correspond to labels on western blots. (B) Conditioned media from HEK-EBNA cells expressing Pro:BMP9 (prodomain-bound BMP9)–wild-type (WT) or missense Pro:BMP9 variants were serially diluted and assayed for BMP9 GFD (growth factor domain) levels by ELISA. Data are the mean ± SEM from three independently generated batches of media. Paired Dunnett’s multiple comparisons test: *P < 0.05 and **P < 0.01. (C) Western blots of variants in conditioned media in nonreducing conditions. The volume loaded was normalized according to concentration based on ELISA. Blots are representative of n = 3 separate expression batches. (D) The ratio of the band intensities of the growth factor and prodomain for each mutant was normalized to WT. The daggers indicate mutants for which GFD concentration was too low to quantify. Data are the mean ± SEM from three independently generated batches of media. Paired Dunnett’s multiple comparisons test: *P < 0.05 and **P < 0.01. (E) Western blotting of those variants not readily detected by ELISA. Cell lysates (30 μg of total protein) and conditioned media (3 μl of Pro:BMP9-WT or 45 μl of Pro:BMP9 mutants and media from the empty vector control) were loaded. CM = conditioned media.

Figure 2.

Loss of activity in Pro:BMP9 (prodomain-bound BMP9 [bone morphogenetic protein type 9]) mutants predicted to be pathogenic. (A and B) C2C12 cells transfected with human ALK1, BRE-luciferase, and TK-Renilla plasmids were serum-starved, followed by treatment with Pro:BMP9 variants (10, 30, 100 pg/ml) for 6 hours, and luciferase assay. The Firefly:Renilla luciferase ratios were calculated for each sample and data normalized, with cells treated with 100 pg Pro:BMP9-WT designated as 100%, and serum-free media–treated cells designated as 0%. Data are mean ± SEM from three independently generated batches of media. van Elteren test: *P_adj < 0.05 and **P_adj < 0.01. (C and D) Transcriptional responses of blood outgrowth endothelial cells to Pro:BMP9-WT and variants based on quantification of the prodomains. Cells were starved in EBM2/0.1% fetal bovine serum (FBS) and then treated with Pro:BMP9-WT and variants (0.5, 1.5, 5 ng/ml) for 4 hours. The expression of (C) ID1 and (D) BMPR2 mRNA were normalized to B2M and are expressed as fold change relative to EBM2/0.1% FBS. Data are mean ± SEM from three independently generated batches of media. van Elteren test: *P_adj < 0.05, **P_adj < 0.01, and ***P_adj < 0.001. (E) Blood outgrowth endothelial cells were treated overnight with 5 ng/ml Pro:BMP9-WT or variants (normalized according to the amount of prodomain) in EBM2/2% FBS followed by induction of apoptosis by addition of TNFα (tumor necrosis factor-α) and cycloheximide for 3.5 hours. Cells were stained with annexin V-FITC and propidium iodide before flow cytometry analysis. Cells were assigned as healthy (AV⁻/PI⁻), early apoptotic (AV⁺/PI⁻), or late apoptotic/necrotic (AV⁺/PI⁺). Data are the mean ± SEM from three independently generated batches of media. Paired Dunnett’s multiple comparisons test: *P < 0.05 and **P < 0.01. (F and G) Pulmonary artery endothelial cells were starved overnight in EBM2/0.1% FBS to induce apoptosis in the presence of Pro:BMP9-WT or variants (0.5, 1 ng/ml). Each BMP9 treatment was supplemented with conditioned media from HEK-EBNA cells transfected with empty vector to equalize the total volume of conditioned media per well (2% vol/vol for 1 ng/ml, 1% vol/vol for 0.5 ng/ml). Apoptosis was measured by Caspase-GLO 3/7 assays. Data are mean ± SEM from three independent batches of media. van Elteren test: *P_adj < 0.05. GFD = growth factor domain; SFM = serum-free media; WT = wild type.

Figure 3.

Loss of active BMP9 (bone morphogenetic protein type 9) in patients with pulmonary arterial hypertension who carry putatively pathogenic GDF2 alleles. (A) BMP9 GFD (growth factor domain) concentration in ethylenediaminetetraacetic acid–plasma from diseased, GDF2 heterozygous females and healthy age-matched females was measured by ELISA. Data are mean ± SEM; one-way ANOVA: *P < 0.05 and **P < 0.01. (B) HMEC1-BRE cells quiesced in serum-free media overnight were treated with 5% plasma for 6 hours before luciferase activity was assessed. Data are mean ± SEM; t test, *P < 0.05 and **P < 0.01. (C) Identical to A except for pBMP10 concentration. **P < 0.01. (D) The concentration of pBMP10 plotted against the concentration of BMP9 GFD in ethylenediaminetetraacetic acid–plasma. Data are mean ± SEM; one-way ANOVA: *P < 0.05 and **P < 0.01. There were 8 healthy control subjects, 11 benign missense (m.s.) carriers, 4 pathogenic m.s. carriers, and 3 deletion carriers (one of which is an early truncation). B9 Ab = anti-BMP9 antibody; SFM = serum-free media.

Figure 4.

Plasma BMP9 (bone morphogenetic protein type 9) and pBMP10 levels are not reduced in pulmonary arterial hypertension (PAH), but a subset of patients with PAH exhibit reduced plasma BMP9 and pBMP10 levels. Plasma samples collected from control subjects and patients with PAH were assayed for (A) BMP9 and (B) pBMP10. Data are presented as median ± interquartile ranges (Kruskal-Wallis test: *P < 0.05, **P < 0.01, and ***P < 0.001). (C) Human aortic endothelial cells quiesced in 0.1% basal media were incubated with 3% ethylenediaminetetraacetic acid–plasma for 1 hour. Expression of ID mRNA was measured by quantitative PCR and normalized to B2M and data expressed as fold change relative to the EBM2/0.1% fetal bovine serum control (mean ± SEM; t test, *P < 0.05 and **P < 0.01). (D and E) Plasma BMP9 levels were plotted against plasma pBMP10 levels in control individuals (n = 120) (D) and patients with PAH (n = 187) (E). Dashed lines represent 25th and 75th percentiles. Spearman correlation is shown by a solid regression line.