Pulmonary arterial hypertension (PAH) is clinically characterized by a sustained elevation in mean pulmonary artery pressure leading to significant morbidity and mortality. The disorder is typically sporadic, and in such cases the term idiopathic PAH (IPAH) is used. However, cases that occur within families (familial PAH (FPAH)) display similar clinical and histopathological features, suggesting a common etiology. Heterozygous mutations of a type II member of the TGF‐β cell signaling superfamily known as BMPR2 on chromosome 2q33 have been identified in many kindreds with FPAH, yet display both reduced penetrance and sex bias. This report presents the compilation of data for 144 distinct mutations that alter the coding sequence of the BMPR2 gene identified in 210 independent PAH subjects. This large data set characterizes the extent of sequence variation and reveals that the majority (71%) of mutations in FPAH and IPAH comprise nonsense, frameshift, and splice‐site defects, and gene rearrangements. These predict premature termination of the transcript with likely loss through the process of nonsense‐mediated decay (NMD). A total of 44 missense mutations were identified that substitute amino acid residues at highly conserved sites within recognized functional domains of the mature receptor. We assess this category of mutations in the context of their heterogeneous effects on cell signaling when assayed by in vitro cell‐based systems. Disease‐causing mutation hot‐spots within BMPR2 are summarized. Taken together, these observations are likely to aid in the development of targeted mutation detection strategies relevant for patient management. Finally, we examine the age‐ and sex‐dependent reduced penetrance of BMPR2 mutations by reviewing bmpr2 animal models and the requirement for additional genetic and/or environmental modifiers of disease. In conclusion, these data provide compelling genetic evidence that haploinsufficiency is the predominant molecular mechanism underlying disease predisposition, and support the concept of a critical threshold of signaling activity below which disease may be precipitated. Hum Mutat 27(2), 121–132, 2006.

Heterozygous germline mutations in the gene encoding the bone morphogenetic protein type II (BMPR-II) receptor underlie the majority (>70%) of cases of familial pulmonary arterial hypertension (FPAH), and dysfunction of BMP signaling has been implicated in other forms of PAH. The reduced disease gene penetrance in FPAH indicates that other genetic and/or environmental factors may also be required for the clinical manifestation of disease. Of these, the serotonin pathway has been implicated as a major factor in PAH pathogenesis. We investigated the pulmonary circulation of mice deficient in BMPR-II ( BMPR2 ^+/− mice) and show that pulmonary hemodynamics and vascular morphometry of BMPR2 ^+/− mice were similar to wild-type littermate controls under normoxic or chronic hypoxic (2- to 3-week) conditions. However, chronic infusion of serotonin caused increased pulmonary artery systolic pressure, right ventricular hypertrophy, and pulmonary artery remodeling in BMPR2 ^+/− mice compared with wild-type littermates, an effect that was exaggerated under hypoxic conditions. In addition, pulmonary, but not systemic, resistance arteries from BMPR2 ^+/− mice exhibited increased contractile responses to serotonin mediated by both 5-HT ₂ and 5-HT ₁ receptors. Furthermore, pulmonary artery smooth muscle cells from BMPR2 ^+/− mice exhibited a heightened DNA synthesis and activation of extracellular signal-regulated kinase 1/2 in response to serotonin compared with wild-type cells. In vitro and in vivo experiments suggested that serotonin inhibits BMP signaling via Smad proteins and the expression of BMP responsive genes. These findings provide the first evidence for an interaction between BMPR-II-mediated signaling and the serotonin pathway, perturbation of which may be critical to the pathogenesis of PAH.

Mutations of the BMPR2 gene predispose to pulmonary arterial hypertension (PAH), a serious, progressive disease of the pulmonary vascular system. However, despite the fact that most PAH families are consistent with linkage to the BMPR2 locus, sequencing only identifies mutations in some 55% of familial cases and between 10% and 40% of cases without a family history (idiopathic or IPAH). We therefore conducted a systematic analysis for larger gene rearrangements in panels of both familial and idiopathic PAH cases that were negative on sequencing of coding regions. Analysis of exon dosage across the entire gene using Multiplex Ligation‐dependent Probe Amplification identified nine novel rearrangements and enabled full characterization at the exon level of previously reported deletions. Overall, BMPR2 rearrangements were identified in 7 of 58 families and 6 of 126 IPAH cases, suggesting that gross rearrangements underlie around 12% of all FPAH cases and 5% of IPAH. Importantly, two deletions encompassed all functional protein domains and are predicted to result in null mutations, providing the strongest support yet that the predominant molecular mechanism for disease predisposition is haploinsufficiency. Dosage analysis should now be considered an integral of part of the molecular work‐up of PAH patients.

Planarian flatworms are able to both regenerate their whole bodies and continuously adapt their size to nutrient status. Tight control of stem cell proliferation and differentiation during these processes is the key feature of planarian biology. Here we show that the planarian homolog of the phosphoinositide 3-kinase-related kinase (PIKK) family member SMG-1 and mTOR complex 1 components are required for this tight control. Loss of smg-1 results in a hyper-responsiveness to injury and growth and the formation of regenerative blastemas that remain undifferentiated and that lead to lethal ectopic outgrowths. Invasive stem cell hyper-proliferation, hyperplasia, hypertrophy, and differentiation defects are hallmarks of this uncontrolled growth. These data imply a previously unappreciated and novel physiological function for this PIKK family member. In contrast we found that planarian members of the mTOR complex 1, tor and raptor, are required for the initial response to injury and blastema formation. Double smg-1 RNAi experiments with tor or raptor show that abnormal growth requires mTOR signalling. We also found that the macrolide rapamycin, a natural compound inhibitor of mTORC1, is able to increase the survival rate of smg-1 RNAi animals by decreasing cell proliferation. Our findings support a model where Smg-1 acts as a novel regulator of both the response to injury and growth control mechanisms. Our data suggest the possibility that this may be by suppressing mTOR signalling. Characterisation of both the planarian mTORC1 signalling components and another PIKK family member as key regulators of regeneration and growth will influence future work on regeneration, growth control, and the development of anti-cancer therapies that target mTOR signalling. Planarian flatworms have a remarkable ability to regenerate that has driven the curiosity of scientists for more than a century. They are also able to continuously grow or degrow their bodies, depending on food availability. Around 25% of the cells in the planarian body are adult stem cells, which are responsible for this incredible plasticity. The initial response of planarians to injury is characterised by a rapid increase in stem cell division. Subsequently planarians form a specialised new tissue called the regenerative blastema to replace missing tissues. Currently, very little is known about the molecular signals controlling the response to injury or the tight regulation of growth. Here we discovered that a gene called Smg-1 and the conserved mTOR signalling pathway, a central regulator of animal growth, are both regulators of this process. SMG-1 is required to limit and act as a brake on the initial response to injury and ensure that it does not run out of control, while in contrast mTOR signalling is required to drive this process forward. Loss of SMG-1 leads to hyperactive responses to injury and subsequent growth that continues out of control. Eventually, these animals form outgrowths, which display several hallmarks of human cancers. These opposing roles suggested that Smg-1 phenotype would require mTOR signalling, and by reducing mTOR signalling and SMG-1 activity at the same time we found that this was the case. We conclude that Smg-1 is a novel regulator of regeneration and animal growth with an antagonistic role to mTOR signalling in planarians.