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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.

Rationale: Familial pulmonary arterial hypertension results from heterozygous inactivating mutations of the BMPR2 gene. Traditional mutation analysis identifies pathogenic mutations in some 70% of linked families. We hypothesized that the apparent shortfall is due to mutations located in the promoter region of the gene, resulting in abnormal gene regulation., Objectives: To identify mutations in untranslated sequence regulating BMPR2 transcription., Methods: DNA upstream of the coding region was analyzed by direct sequencing in 16 families. Reverse transcription-polymerase chain reaction analysis and rapid amplification of cDNA ends of normal human lung RNA were used to investigate transcription of this region. Transcript levels were assessed by allele-specific expression analysis and inhibition of nonsense-mediated decay in lymphoblastoid cell lines., Measurements and Main Results: The wild-type transcriptional start site of BMPR2 was defined, 1,148 bp upstream of the ATG. Within this region, we identified a double-substitution mutation, predicted to form a cryptic translational start site, in one family. The mutant transcript contains a premature stop codon predicted to trigger nonsense-mediated decay. Expression analysis in the patient's cell line indeed showed reduced expression of the mutant transcript that could be restored to normal by inhibiting nonsense-mediated decay., Conclusions: Activation of a cryptic translation initiation site is a novel mutational mechanism in this disorder. These results demonstrate that the 5'-untranslated region of BMPR2 is considerably longer than previously thought, emphasizing the need to fully characterize the BMPR2 promoter and the importance of analyzing noncoding regions in patients with pulmonary arterial hypertension who are negative for mutations within the coding region and intron-exon junctions.

Rationale: The bone morphogenetic receptor type II gene is the major genetic determinant for the inherited form of pulmonary arterial hypertension. However, deleterious mutations of this gene are not observed in the majority of subjects who develop the condition spontaneously and familial disease displays age- and sex-dependent penetrance, indicating the requirement for additional environmental and/or genetic modifiers for disease development., Methods: We investigated polymorphic variation of the serotonin transporter gene, a biological candidate for predisposition to this vascular disorder., Results: No significant evidence of association between alleles of the serotonin transporter gene and pulmonary hypertension was detected, nor did we observe a relationship with age of onset in familial and idiopathic disease., Conclusions: Variation of the serotonin transporter gene appears unlikely to confer significant susceptibility to pulmonary arterial hypertension. This study emphasizes the need for adequately powered cohorts for association analyses to identify not only genetic determinants of disease susceptibility but also inherited modifiers for disease development.