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Original Research: Pulmonary Vascular Disease |

Characteristics of Pulmonary Arterial Hypertension in Affected Carriers of a Mutation Located in the Cytoplasmic Tail of Bone Morphogenetic Protein Receptor Type 2Mutation Affecting the Cytoplasmic Tail of BMPRII FREE TO VIEW

Barbara Girerd, PhD; Florence Coulet, PharmD, PhD; Xavier Jaïs, MD; Mélanie Eyries, PhD; Cathelijne Van Der Bruggen, BSc; Frances De Man, PhD; Arjan Houweling, MD, PhD; Peter Dorfmüller, MD, PhD; Laurent Savale, MD, PhD; Olivier Sitbon, MD, PhD; Anton Vonk-Noordegraaf, MD, PhD; Florent Soubrier, MD, PhD; Gérald Simonneau, MD; Marc Humbert, MD, PhD; David Montani, MD, PhD
Author and Funding Information

From the University Paris-Sud (Drs Girerd, Jaïs, Dorfmüller, Savale, Sitbon, Simonneau, Humbert, and Montani), Le Kremlin-Bicêtre, France; Assistance Publique-Hôpitaux de Paris (AP-HP), Service de Pneumologie (Drs Girerd, Jaïs, Dorfmüller, Savale, Sitbon, Simonneau, Humbert, and Montani), Centre de Référence de l’Hypertension Pulmonaire Sévère, DHU Thorax Innovation, Hôpital Bicêtre, Le Kremlin-Bicêtre, France; Institut National de la Santé et de la Recherche Médicale (INSERM) U999 (Drs Girerd, Jaïs, Dorfmüller, Savale, Sitbon, Simonneau, Humbert, and Montani), LabEx LERMIT, Centre Chirurgical Marie Lannelongue, Le Plessis Robinson, France; the Genetics Department (Drs Coulet, Eyries, and Soubrier), Hôpital Pitié-Salpêtrière, AP-HP, Paris, France; the ICAN Institute for Cardiometabolism and Nutrition (Drs Eyries and Soubrier), Paris, France; the Unité Mixte de Recherche en Santé (UMR_S 1166) (Drs Eyries and Soubrier), UPMC - Université Paris-Sorbonne, and INSERM, Paris, France; and the Departments of Pulmonary Medicine (Ms Van Der Bruggen and Drs De Man and Vonk-Noordegraaf), Institute for Cardiovascular Research, and the Department of Clinical Genetics (Dr Houweling), VU University Medical Center, Amsterdam, The Netherlands.

CORRESPONDENCE TO: David Montani, MD, PhD, Université Paris-Sud, Service de Pneumologie, Centre de Référence de l’Hypertension Pulmonaire Sévère, Hôpital de Bicêtre, 78 rue du Général Leclerc, 94275 Le Kremlin Bicêtre, France; e-mail: david.montani@bct.aphp.fr


FUNDING/SUPPORT: Dr Montani received a young investigator award for this work from the 5th World Symposium on Pulmonary Hypertension, February 27-March 1, 2013, Nice, France.

Reproduction of this article is prohibited without written permission from the American College of Chest Physicians. See online for more details.


Chest. 2015;147(5):1385-1394. doi:10.1378/chest.14-0880
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BACKGROUND:  Mutations in BMPR2 encoding bone morphogenetic protein receptor type 2 (BMPRII) is the main genetic risk factor for heritable pulmonary arterial hypertension (PAH). The suspected mechanism is considered to be a defect of BMP signaling. The BMPRII receptor exists in a short isoform without a cytoplasmic tail, which has preserved BMP signaling.

METHODS:  This cohort study compared age at PAH diagnosis and severity between patients carrying a BMPR2 mutation affecting the cytoplasmic tail of BMPRII and affected carriers of a mutation upstream of this domain.

RESULTS:  We identified 171 carriers affected with PAH with a mutated BMPR2. Twenty-three were carriers of a point mutation located on the cytoplasmic tail of BMPRII. This population was characterized by having an older age at diagnosis compared with other BMPR2 mutation carriers (43.2 ± 12.1 years and 35.7 ± 14.6 years, P = .040), a lower pulmonary vascular resistance (13.3 ± 3.5 and 17.4 ± 6.7, P = .023), and a higher proportion of acute vasodilator responders with a long-term response to calcium channel blockers (8.7% and 0%, P = .02). No statistically significant differences were observed in survival. An in vitro assay showed that mutations located in the cytoplasmic tail led to normal activation of the Smad pathway, whereas activation was abolished in the presence of mutations located in the kinase domain.

CONCLUSIONS:  Patients carrying a mutation affecting the cytoplasmic tail of BMPRII were characterized by an older age at diagnosis compared with other BMPR2 mutation carriers, less severe hemodynamic characteristics, and a greater chance of being a long-term responder to calcium channel blockers. Further investigations are needed to better understand the consequences of these BMPR2 mutations in BMPRII signaling pathways and their possible role in pulmonary arterial remodeling.

Figures in this Article

Pulmonary arterial hypertension (PAH) is a severe disease affecting small pulmonary arteries: Progressive remodeling leads to elevated pulmonary vascular resistance and right ventricular failure.1,2 Mutations in BMPR2 are the main genetic risk factor for PAH, and mutations in this gene are identified in approximately 70% of patients with a familial form of PAH and up to 25% of patients displaying a sporadic PAH.3 It has been previously demonstrated that patients carrying a BMPR2 mutation develop PAH about 10 years earlier than noncarriers, with a more severe hemodynamic compromise at diagnosis, and are less likely to respond to acute vasodilator testing (0‒4% in recent series) compared with patients with idiopathic PAH.46

Mutations are spread throughout the 13 exons of BMPR2, with the exception of exon 13, and include missense mutations, nonsense mutations, splice defects, deletions, and duplication. In a previous study, we found no influence of mutation type on clinical phenotype in affected BMPR2 mutation carriers. The bone morphogenetic protein-receptor type 2 (BMPRII) is expressed as a short isoform lacking the cytoplasmic tail domain caused by alternative splicing.7,8 It has been suggested that the short isoform of BMPRII and the truncated BMPRII that lacks the cytoplasmic tail maintain the ability to transduce BMP signals.9 Moreover, Nishihara et al10 showed that a mutated BMPRII receptor, carrying a nonsense mutation in the cytoplasmic tail domain, retained most of its biologic activity, including the ability to phosphorylate Smad5, but at a lower efficacy than a wild-type BMPRII (long or short isoform).

The aim of our study was to compare the age at diagnosis and severity (clinical, functional, hemodynamic characteristics, and prognosis) of patients with PAH carrying a BMPR2 mutation located on the cytoplasmic tail of BMPRII with patients carrying a mutation located on the signal peptide, the ligand domain, or the kinase domain of BMPRII. We also performed in vitro experiments to investigate the functional consequences of BMPR2 mutations located on the cytoplasmic tail of the BMPRII receptor.

Patients

In the French Referral Centre for Pulmonary Hypertension (Université Paris-Sud, Le Kremlin-Bicêtre, France) and in the VU University Medical Centre (Amsterdam, Netherlands), genetic counseling and BMPR2 screening were offered to all patients displaying a familial form of the disease, to all patients with a PAH considered to be idiopathic, and to all patients exposed to drugs, toxins, or both. In this study, we reviewed data from all affected carriers of a BMPR2 mutation tested between January 2003 and December 2013. Affected carriers had no associated disease that caused PH. Of note, nine affected carriers of a BMPR2 mutation were exposed to fenfluramine. Out of them, two are carriers of a mutation located in the cytoplasmic tail of BMPRII. The first patient was a man diagnosed at 52 years old, with a 1- or 2-month exposure to fenfluramine 15 years before PAH diagnosis. The second patient was a woman diagnosed at 39 years old with a 2-month exposure to fenfluramine 10 years before PAH diagnosis. Neither patient responded acutely to vasodilators.

Pulmonary hypertension was defined as a mean pulmonary arterial pressure (mPAP) of ≥ 25 mm Hg associated with a normal pulmonary capillary wedge pressure (≤ 15 mm Hg). Hemodynamic evaluation by right-sided heart catheterization was performed at baseline in all patients, according to our previously described protocol.1113 Acute vasodilator response was tested with inhaled nitric oxide, as previously published and defined as a decrease in mPAP of > 10 mm Hg, to reach a level of ≤ 40 mm Hg with normal or increased cardiac output.1,2 Long-term responders to calcium channel blockers (CCBs) corresponded to patients who had marked hemodynamic improvement after 3 to 4 months of CCB therapy and were in New York Heart Association (NYHA) functional class I or II after at least 1 year on CCBs.11,14

In accordance with the guidelines of the American College of Chest Physicians,15 patients tested for BMPR2 mutations gave their written informed consent and underwent genetic counseling. Screening for clinical manifestations of hereditary hemorrhagic telangiectasia and screening for pulmonary venoocclusive disease, based on high-resolution CT scan of the chest and pulmonary function tests, were systematically done to eliminate these from the diagnosis. However, mutations in KCNK3 and Caveolin-1, known to cause autosomal dominant familial PAH, were not screened in this BMPR2 population. Indeed, mutations in KCNK316 and Caveolin-117 are very rare. Moreover, BMPR2 mutations are also extremely rare in the general population. Thereby, because of the extremely low frequencies of mutations in PAH-predisposing genes, it seems highly unlikely that our BMPR2 patients will be carriers of two mutations in two different PAH-predisposing genes.

Site-Directed Mutagenesis and Construction of Microinterfering RNA Expression Vectors

Four BMPR2 mutants (p.Arg591X, p.Arg873X, p.Arg873Gln, p.Ala391Thr) were generated by polymerase chain reaction through site-directed mutagenesis of the BMPR2 WT expression vector18 using the Stratagene Quick Change kit, according to the manufacturer’s recommendations. Primers used are described in e-Table 1. We decided to analyze the two types of mutations localized in a potential hot spot of mutations: Arg873 (p.Arg873X and p.Arg873Gln). We arbitrarily chose another mutation localized in the cytoplasmic tail domain (p.Arg591X) to eliminate the specific effect of a mutation localized in Arg873. We also analyzed an arbitrary mutation localized in the kinase domain (p.Ala391Thr), where mutations are known to disrupt Smad signaling.19 Microinterfering RNA (miRNA) directed against murine BMPR2 and murine Activin A Receptor type IIA (ACVR2A) were designed and inserted into an expression vector using a BLOCK-iT Pol II miR RNA interference expression vector kit (Thermo Fisher Scientific Inc), according to the manufacturer’s recommendations.

Transient Transfection Assays

Mouse embryonic endothelial cells were plated into 24-well plates at 4 × 104 cells per well. They were transfected the following day with 0.3 μg of BMPR2WT or BMPR2 mutant vectors, 0.3 μg of each miRNA expression vector to inhibit endogenous murine BMPR2 and ACVR2A expression, and 50 ng of a specific reporter for Smad-1/5 activation, BRE2-Luc,20 using Lipofectamine transfection reagent (Thermo Fisher Scientific Inc) according to the manufacturer’s recommendations. In all reporter assays, Renilla luciferase gene vector (Promega Corporation) cotransfection (50 ng) was used as an internal control to normalize transfection efficiency. Cells were incubated overnight with the transfection mix and then serum depleted (0.4%) and treated or not with BMP4 (50 ng/mL) for 24 h. Luciferase activity was measured by luminometry using a Dual-Luciferase revelation system (Promega Corporation), according to the manufacturer’s recommendations.

Statistical Analyses

The main objective of this study was to compare age at PAH diagnosis between affected carriers of a mutation localized in the cytoplasmic tail domain and affected carriers of a mutation upstream of this domain. The secondary end point included comparisons of hemodynamic parameters, NYHA functional class, and 6-min walking distance between these two groups.

Data were compared using the χ2 test, the χ2 test with a Yates correction, and Student t test or the Mann-Whitney test, as appropriate. Statistical analyses of the transfection assay were performed with XLSTAT software (Addinsoft) using one-way analysis of variance for all data, followed by a Student Newman-Keuls post hoc analysis for multiple tests. A P value < .05 was considered to indicate statistical significance.

Clinical and Hemodynamic Characteristics

One hundred seventy-one BMPR2 mutation carriers (145 from the French population and 26 from the Dutch PAH population), from 139 different families, were identified. Studied subjects were mainly Caucasian Europeans (158); 10 were of Afro-Caribbean origin, and three were of other origins (Indian and Asian). Of the total, 126 patients (from 101 families) had a point mutation and, of these, 23 patients (18 families) carried a mutation located on the cytoplasmic tail of BMPRII (Fig 1, Table 1). Fifteen patients (from 11 families) carried a splice-site mutation in intron 2, 3, 6, 7, 9, or 10. Finally, in 30 patients (27 families), a large rearrangement (deletion or a duplication) of BMPR2 was identified. All splice defects and large rearrangements involved at least the signal peptide, the ligand domain, or the kinase domain of BMPRII.

Figure Jump LinkFigure 1 –  A, B, Frequency of BMPR2 point mutations identified in distinct families with pulmonary arterial hypertension (PAH). COOH = carboxy-terminus.Grahic Jump Location
Table Graphic Jump Location
TABLE 1 ]  Mutations Affecting the Cytoplasmic Tail of BMPRII and Identified in Unrelated Patients With PAH

BMPRII = bone morphogenetic protein-receptor type 2; PAH = pulmonary arterial hypertension.

Affected carriers of a mutation located only on the cytoplasmic tail (n = 23) had broadly similar clinical, functional, and hemodynamic characteristics at diagnosis when compared with patients carrying a mutation of the signal peptide, the ligand, or the kinase domain of BMPRII (n = 148), except for those with an older age at diagnosis (44.4 ± 14.0 and 36.8 ± 15.0 years, respectively; P = .025) (Table 2). To avoid bias caused by inclusion of multiple affected family members, we subsequently compared these characteristics by including only the first patient with PAH diagnosed in each family. In accordance with results from the whole cohort, unrelated affected carriers of a mutation affecting the cytoplasmic tail of BMPRII were significantly older at PAH diagnosis compared with unrelated affected carriers of a mutation upstream of the cytoplasmic tail domain (43.2 ± 12.1 and 35.7 ± 14.6 years, respectively; P = .040) (Table 3). Affected carriers of a mutation in the cytoplasmic tail have a significantly lower pulmonary vascular resistance (PVR) (13.3 ± 3.5 mm Hg/L/min vs 17.4 ± 6.7 mm Hg/L/min; P = .023) (Table 3). Familial forms of PAH caused by a mutation affecting the cytoplasmic tail domain are presented in Figure 2.

Table Graphic Jump Location
TABLE 2 ]  Clinical, Functional, and Hemodynamic Characteristics at Diagnosis of PAH in Affected Carriers of the BMPR2 Mutation

mPAP = mean pulmonary artery pressure; NYHA = New York Heart Association; PcwP = pulmonary capillary wedge pressure; PVR = pulmonary vascular resistance; RAP = right atrial pressure; Svo2 = mixed venous oxygen saturation. See Table 1 legend for expansion of other abbreviation.

Table Graphic Jump Location
TABLE 3 ]  Clinical, Functional, and Hemodynamic Characteristics at Diagnosis of PAH in Unrelated Affected Carriers of the BMPR2 Mutation

See Table 1 and 2 legends for expansion of abbreviations.

Figure Jump LinkFigure 2 –  Familial forms of PAH caused by a mutation affecting the cytoplasmic tail of bone morphogenetic protein-receptor type 2 (BMPRII). Ages indicate age at PAH, age at death, or age at last evaluation for unaffected carriers of the familial BMPR2 mutation. See Figure 1 legend for expansion of other abbreviation.Grahic Jump Location

At the last follow-up, among the 23 patients carrying a mutation affecting the cytoplasmic tail, 15 patients were still alive after a median follow-up of 98 months (minimum-maximum: 8-350), five patients had died after a median period of 39 months (minimum-maximum: 11-108), and three had received a transplant (after, respectively, 39, 77, and 105 months). Survival analyses of patients carrying a mutation located on the cytoplasmic tail and the overall population of BMPR2 mutation carriers are presented in Figure 3; there were no differences between the two populations.

Figure Jump LinkFigure 3 –  A, Time to death in affected carriers of a BMPR2 mutation in the cytoplasmic tail and other BMPR2 mutation carriers. B, Time to death or lung transplantation in affected carriers of a BMPR2 mutation in the cytoplasmic tail and other BMPR2 mutation carriers.Grahic Jump Location

Two acute vasodilator responders were identified among the 23 patients carrying a mutation affecting the cytoplasmic tail (8.7%) and none in other BMPR2 mutation carriers. These two patients carried two different BMPR2 mutations affecting the cytoplasmic tail of BMPRII: respectively, c.2618G > A, p.Arg873Gln and c.2617C > T, p.Arg873X in exon 12, located on the same amino acid, Arg873. One of these patients fulfilled the criterion of having a long-term response to CCBs. The patient who responded to long-term CCB was still alive and still received CCBs after 350 months.

In our cohort of 171 affected BMPR2 mutation carriers, we identified nine additional patients with PAH from seven different families carrying one of the mutations identified in acute vasodilator responders (eight carriers of mutation c.2617C > T, p.Arg873X and one carrier of mutation c.2618G > A, p.Arg873Gln). None of these patients had evidence of an acute vasodilator response. We identified 101 point mutations in the BMPR2 gene in patients with PAH from different families. Analyses of the frequencies of point mutations in BMPR2 suggest that Arg873 may be a hot spot for mutations: An Arg873 mutation was identified in eight different families out of the 101 carriers of a point mutation (7.9%) (Fig 1, Table 1). Thus, a founder effect could be evoked. However, in one patient, the mutation Arg873X was a de novo mutation (the mutation was not detected in either parent, and paternity was confirmed by the analysis of informative markers) (Fig 2, Family ABH058), which can rule out the hypothesis of a founder effect. Moreover, Arg873 mutations have been previously reported.21 One patient carrying a mutation affecting Arg873 has subsequently received a transplant, and pathologic assessment has confirmed pulmonary arterial hypertension with severe arterial remodeling (Fig 4).

Figure Jump LinkFigure 4 –  Lung histology from a patient with PAH with an Arg873 mutation in BMPR2 (hematoxylin eosin saffron). A, Pulmonary artery (top) displaying important thickening of the muscular tunica media. B, Remodeled pulmonary artery (left) harboring a proliferative cell-rich lesion (center) that appears to involve the periphery of the vessel wall; note the congestive dilation lesions (filled with bright red erythrocytes). C, Small pulmonary artery adjacent to a terminal bronchiole, showing eccentric loose intimal fibrosis with two small lumina, thus corresponding to an organized thrombotic lesion. D, Microvessel displaying muscularization and fibrous constriction. See Figure 1 legend for expansion of abbreviation.Grahic Jump Location
Functional Analysis of Mutations Affecting the Cytoplasmic Tail

We used an in vitro reporter-system assay to investigate the functional consequences of BMPR2 mutations located on the cytoplasmic tail of the BMPRII receptor. Four different mutations were studied: p.Arg591X, p.Arg873X, and p.Arg873Gln located in the cytoplasmic tail of BMPRII, and p.Ala391Thr located in the kinase domain. We analyzed the ability of these BMPR2 mutants to activate BRE2-Luc, a specific reporter for Smad1/5 activation,20 in response to BMP4 treatment (50 ng/μL).

Cells transfected with p.Arg591X, Arg873X, and Arg873Gln BMPR2 mutants showed a similar activation of the SMAD pathway as the wild-type BMPR2 (fold change of luciferase activity in response to BMP4 of 1.25 ± 0.15, 1.25 ± 0.17, 0.78 ± 0.03, and 0.65 ± 0.04, respectively). However, this response was abolished by the p.Ala391Thr mutation (0.18 ± 0.027) located in the kinase domain of BMPRII (Fig 5). These results indicate that the BMPRII protein, encoded by BMPR2 with a mutation localized in the cytoplasmic tail, was able to activate the BMP signaling pathway in a similar way as wild-type BMPRII.

Figure Jump LinkFigure 5 –  Functional analysis of BMPR2 mutants. Mouse embryonic endothelial cells were transfected with 0.3 μg of wild-type BMPR2 (BMPR2 WT) or BMPR2 mutant (BMPR2 mut) vectors, 0.3μg of miRNA expression vectors to inhibit endogenous murine BMPR2 and ACVR2A expression, and 50 ng of a specific reporter for Smad-1/5 activation, BRE2-Luc. After transfection, cells were treated or not with BMP4 (50 ng/mL) for 24 h, and luciferase activity was measured. Data are expressed as the ratio between luciferase activity normalized with Renilla luciferase. Data are expressed as the means of three independent experiments ± SD *P < .05 relative to untreated cells for each transfection condition. ACVR2A = activin receptor II; miRNA = microinterfering RNA.Grahic Jump Location

In a large cohort of BMPR2 mutation carriers, we have demonstrated that patients carrying a BMPR2 mutation localized on the cytoplasmic tail may develop severe PAH even if their mutations do not affect Smad signaling. No differences were observed in the NYHA functional class, the 6-min walk test, or survival time compared with other BMPR2 mutation carriers. However, patients carrying a BMPR2 mutation that affected the cytoplasmic tail were significantly older at diagnosis than patients with a mutation located upstream of the cytoplasmic tail, with lower pulmonary vascular resistance at diagnosis.

We have previously demonstrated that patients with PAH without an identified BMPR2 mutation are characterized by having an older age at PAH diagnosis compared with BMPR2 mutation carriers (46.0 ± 16.1 years vs 36.5 ± 14.5 years, P < .0001) and having a less severe hemodynamic profile with a lower mPAP (56 ± 13 mm Hg vs 64 ± 13 mm Hg, P < .0001) and lower PVR (12.7 ± 6.6 mm Hg/L/min vs 17.4 ± 6.1 mm Hg/L/min, P < .0001).4,22 Interestingly, even though patients carrying a BMPR2 mutation affecting the cytoplasmic tail had broadly similar clinical, functional, and hemodynamic characteristics at diagnosis compared with other BMPR2 mutation carriers, these patients were diagnosed at the same age as noncarriers of BMPR2 mutations, and they had a similar hemodynamic profile (mPAP = 57.2 ± 11.3 mm Hg, PVR = 13.3 ± 3.5 mm Hg/L/min) (Table 3).

A long-term response to CCBs is rare among patients with PAH and is associated with better long-term survival.11 It has been demonstrated that an acute vasodilator response is observed in 12.5% of patients with idiopathic PAH and a long-term response to CCBs in 6.8%.11 It is widely accepted that patients with PAH carrying a BMPR2 mutation are less likely to respond to acute vasodilator testing (respectively, 0% to 4% in a recent series). No data are currently available concerning the long-term response to CCBs in this population.46 Interestingly, in our cohort of patients with a mutation affecting the cytoplasmic tail of BMPRII, 8.7% responded to acute vasodilators and 4.3% were long-term responders to CCBs. Once again, carriers of a mutation in the cytoplasmic tail of BMPRII seem to have a similar profile as patients with idiopathic PAH. Thus, a long-term response to CCBs could be observed in patients carrying a BMPR2 mutation affecting the cytoplasmic tail, underlying the importance of performing an acute vasodilator test in all patients with sporadic or familial PAH, regardless of family history.

As previously reported,19 we have demonstrated that BMPR2 mutations in the cytoplasmic tail (p.Arg873X, p.Arg873Gln, p.Arg591X) do not affect the ability to activate Smad signaling. All cytoplasmic tail mutants failed to inhibit BMP4-induced activation of the BMP/Smad pathway, suggesting that the deleterious effect of these mutants is caused by the impairment of another function of BMPRII. The cytoplasmic tail has several poorly understood functions, including regulation of p38 and p42/44 MAPK19,23 and interaction with LIMK,24 c-Src,25 Tctex,26 and Trb3.27 Taken together, these results suggest that the cytoplasmic tail of BMPRII is not essential for the transduction of BMP signaling through Smads but may have an unidentified function responsible for the development of PAH. p38MAPK is a well-known alternative Smad-independent signaling pathway downstream of BMPRII. Rudarakanchana et al19 observed a gain of function potentiating p38MAPK signaling when BMPRII was mutated in the cytoplasmic tail. Moreover, it has been demonstrated in mice carrying a mutant BMPR2 allele, which encodes a receptor lacking the cytoplasmic tail domain, that the cytoplasmic tail of BMPRII is essential for embryogenesis and inhibits ALK2-mediated BMP7 signaling in pulmonary-artery smooth muscle cells.28

One can hypothesize that these cases of PAH correspond to a coincidental association with a polymorphism in the cytoplasmic tail. However, no changes in this region have been reported in control databases of rare variants in the Exome Sequencing Project (8,600 white and 4,406 black) or 1000genome (1,092 human exomes), whereas mutations affecting the cytoplasmic tail were identified in 13.5% of patients with heritable PAH. Moreover, we observed that 39% of patients had the familial form of the disease in carriers of a mutation that affected the cytoplasmic tail (seven familial forms out of 18 distinct families). In six familial forms, we tested two patients with PAH per family: the mutation segregated with the disease (in five families patients with PAH were first-degree relatives, and in one family patients with PAH were second-degree relatives) (Fig 2). Mutations were frequent on Arg873; they were involved in eight unrelated patients out of 101 different families who were carriers of a point mutation (8%). A founder effect was not supported because we identified a de novo mutation at this location, and recurrent mutations at this hot spot are the most likely reason. Altogether, these observations demonstrate that mutations affecting the cytoplasmic tail of BMPRII are deleterious and predispose to PAH.

As observed in Figure 2, we identified unaffected carriers of BMPR2 mutations in families with a mutation affecting the cytoplasmic tail of BMPRII but also in other BMPR2 families. These relatives were not included in the present study because the absence of PAH in any of these mutation carriers may be explained by the incomplete time of observation, their age, the incomplete penetrance of BMPR2 mutation, and the small size of the cohort of unaffected relatives.

In this study we demonstrated a genotype-phenotype link in BMPR2 mutation carriers. Indeed, patients with PAH who are carriers of a mutation located in the cytoplasmic tail of BMPRII are characterized by an older age at diagnosis compared with other BMPR2 mutation carriers and less severe hemodynamic characteristics and can be long-term responders to CCBs. Moreover, we confirmed that mutations located in the cytoplasmic tail led to normal activation of the Smad pathway. Further investigations are needed to better understand the consequences of mutations affecting the cytoplasmic tail in the BMPRII signaling pathways, their possible role in pulmonary arterial remodeling, and the balance between vasoconstriction and vasodilation.

Author contributions: D. M. is guarantor of the article. B. G., M. H., and D. M. contributed to conceiving and designing this study; B. G., X. J., L. S., O. S., G. S., M. H., and D. M. contributed to performing clinical phenotyping of French patients with PAH; C. V. D. B., F. D. M., and A. V.-N. contributed to performing clinical phenotyping of patients with PAH from the Netherlands; P. D. contributed to performing lung histology from a patient with PAH with an Arg873 mutation of the BMPR2 gene; M. E., A. H., and F. S. contributed to performing genetic testing; F. C., M. E., and F. S. contributed to performing functional analyses of the BMPR2 mutants; B. G. and D. M. contributed to analyzing the clinical data and wrote the manuscript; and all authors contributed to reviewing the manuscript.

Financial/nonfinancial disclosures: The authors have reported to CHEST the following conflicts of interest: Dr Dorfmüller has received speaker fees from Actelion Pharmaceuticals Ltd. Dr Vonk-Noordegraaf reports receiving lecture fees from Actelion Pharmaceuticals Ltd, Bayer AG, GlaxoSmithKline, Eli Lilly and Company, and Pfizer Inc; serving on the industry advisory board for Actelion Pharmaceuticals Ltd and Bayer AG; and serving on steering committees for Actelion Pharmaceuticals Ltd, Bayer AG, and Pfizer Inc. Drs Girerd, Coulet, Jaïs, Eyries, De Man, Houweling, Savale, Sitbon, Soubrier, Simonneau, Humbert, and Montani and Ms Van Der Bruggen have reported that no potential conflicts of interest exist with any companies/organizations whose products or services may be discussed in this article.

Role of sponsors: The sponsor had no role in the design of the study, the collection and analysis of the data, or the preparation of the manuscript.

Other contributions: We thank K. Miyazono, MD, PhD, and P. Ten Dijke, PhD, for kindly providing the BMPR2WT expression vector and the BRE2-Luc vector, respectively. We also thank Newmed Publishing for reading the manuscript.

Additional information: The e-Table can be found in the Supplemental Materials section of the online article.

ACVR2A

activin receptor II

BMPRII

bone morphogenetic protein-receptor type 2

CCB

calcium channel blocker

miRNA

microinterfering RNA

mPAP

mean pulmonary arterial pressure

NYHA

New York Heart Association

PAH

pulmonary arterial hypertension

PVR

pulmonary vascular resistance

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Montani D, Savale L, Natali D, et al. Long-term response to calcium-channel blockers in non-idiopathic pulmonary arterial hypertension. Eur Heart J. 2010;31(15):1898-1907. [CrossRef] [PubMed]
 
McGoon M, Gutterman D, Steen V, et al; American College of Chest Physicians. Screening, early detection, and diagnosis of pulmonary arterial hypertension: ACCP evidence-based clinical practice guidelines. Chest. 2004;126(1_suppl):14S-34S. [CrossRef] [PubMed]
 
Ma L, Roman-Campos D, Austin ED, et al. A novel channelopathy in pulmonary arterial hypertension. N Engl J Med. 2013;369(4):351-361. [CrossRef] [PubMed]
 
Austin ED, Ma L, LeDuc C, et al. Whole exome sequencing to identify a novel gene (caveolin-1) associated with human pulmonary arterial hypertension. Circ Cardiovasc Genet. 2012;5(3):336-343. [CrossRef] [PubMed]
 
Hanyu A, Ishidou Y, Ebisawa T, Shimanuki T, Imamura T, Miyazono K. The N domain of Smad7 is essential for specific inhibition of transforming growth factor-beta signaling. J Cell Biol. 2001;155(6):1017-1027. [CrossRef] [PubMed]
 
Rudarakanchana N, Flanagan JA, Chen H, et al. Functional analysis of bone morphogenetic protein type II receptor mutations underlying primary pulmonary hypertension. Hum Mol Genet. 2002;11(13):1517-1525. [CrossRef] [PubMed]
 
Korchynskyi O, ten Dijke P. Identification and functional characterization of distinct critically important bone morphogenetic protein-specific response elements in the Id1 promoter. J Biol Chem. 2002;277(7):4883-4891. [CrossRef] [PubMed]
 
Pfarr N, Szamalek-Hoegel J, Fischer C, et al. Hemodynamic and clinical onset in patients with hereditary pulmonary arterial hypertension andBMPR2mutations. Respir Res. 2011;12:99. [CrossRef] [PubMed]
 
Sztrymf B, Coulet F, Girerd B, et al. Clinical outcomes of pulmonary arterial hypertension in carriers ofBMPR2mutation. Am J Respir Crit Care Med. 2008;177(12):1377-1383. [CrossRef] [PubMed]
 
Yang X, Long L, Southwood M, et al. Dysfunctional Smad signaling contributes to abnormal smooth muscle cell proliferation in familial pulmonary arterial hypertension. Circ Res. 2005;96(10):1053-1063. [CrossRef] [PubMed]
 
Foletta VC, Lim MA, Soosairajah J, et al. Direct signaling by the BMP type II receptor via the cytoskeletal regulator LIMK1. J Cell Biol. 2003;162(6):1089-1098. [CrossRef] [PubMed]
 
Wong WKP, Knowles JA, Morse JH. Bone morphogenetic protein receptor type II C-terminus interacts with c-Src: implication for a role in pulmonary arterial hypertension. Am J Respir Cell Mol Biol. 2005;33(5):438-446. [CrossRef] [PubMed]
 
Machado RD, Rudarakanchana N, Atkinson C, et al. Functional interaction between BMPR-II and Tctex-1, a light chain of Dynein, is isoform-specific and disrupted by mutations underlying primary pulmonary hypertension. Hum Mol Genet. 2003;12(24):3277-3286. [CrossRef] [PubMed]
 
Chan MC, Nguyen PH, Davis BN, et al. A novel regulatory mechanism of the bone morphogenetic protein (BMP) signaling pathway involving the carboxyl-terminal tail domain of BMP type II receptor. Mol Cell Biol. 2007;27(16):5776-5789. [CrossRef] [PubMed]
 
Leyton PA, Beppu H, Pappas A, et al. Deletion of the sequence encoding the tail domain of the bone morphogenetic protein type 2 receptor reveals a bone morphogenetic protein 7-specific gain of function. PLoS ONE. 2013;8(10):e76947. [CrossRef] [PubMed]
 

Figures

Figure Jump LinkFigure 1 –  A, B, Frequency of BMPR2 point mutations identified in distinct families with pulmonary arterial hypertension (PAH). COOH = carboxy-terminus.Grahic Jump Location
Figure Jump LinkFigure 2 –  Familial forms of PAH caused by a mutation affecting the cytoplasmic tail of bone morphogenetic protein-receptor type 2 (BMPRII). Ages indicate age at PAH, age at death, or age at last evaluation for unaffected carriers of the familial BMPR2 mutation. See Figure 1 legend for expansion of other abbreviation.Grahic Jump Location
Figure Jump LinkFigure 3 –  A, Time to death in affected carriers of a BMPR2 mutation in the cytoplasmic tail and other BMPR2 mutation carriers. B, Time to death or lung transplantation in affected carriers of a BMPR2 mutation in the cytoplasmic tail and other BMPR2 mutation carriers.Grahic Jump Location
Figure Jump LinkFigure 4 –  Lung histology from a patient with PAH with an Arg873 mutation in BMPR2 (hematoxylin eosin saffron). A, Pulmonary artery (top) displaying important thickening of the muscular tunica media. B, Remodeled pulmonary artery (left) harboring a proliferative cell-rich lesion (center) that appears to involve the periphery of the vessel wall; note the congestive dilation lesions (filled with bright red erythrocytes). C, Small pulmonary artery adjacent to a terminal bronchiole, showing eccentric loose intimal fibrosis with two small lumina, thus corresponding to an organized thrombotic lesion. D, Microvessel displaying muscularization and fibrous constriction. See Figure 1 legend for expansion of abbreviation.Grahic Jump Location
Figure Jump LinkFigure 5 –  Functional analysis of BMPR2 mutants. Mouse embryonic endothelial cells were transfected with 0.3 μg of wild-type BMPR2 (BMPR2 WT) or BMPR2 mutant (BMPR2 mut) vectors, 0.3μg of miRNA expression vectors to inhibit endogenous murine BMPR2 and ACVR2A expression, and 50 ng of a specific reporter for Smad-1/5 activation, BRE2-Luc. After transfection, cells were treated or not with BMP4 (50 ng/mL) for 24 h, and luciferase activity was measured. Data are expressed as the ratio between luciferase activity normalized with Renilla luciferase. Data are expressed as the means of three independent experiments ± SD *P < .05 relative to untreated cells for each transfection condition. ACVR2A = activin receptor II; miRNA = microinterfering RNA.Grahic Jump Location

Tables

Table Graphic Jump Location
TABLE 1 ]  Mutations Affecting the Cytoplasmic Tail of BMPRII and Identified in Unrelated Patients With PAH

BMPRII = bone morphogenetic protein-receptor type 2; PAH = pulmonary arterial hypertension.

Table Graphic Jump Location
TABLE 2 ]  Clinical, Functional, and Hemodynamic Characteristics at Diagnosis of PAH in Affected Carriers of the BMPR2 Mutation

mPAP = mean pulmonary artery pressure; NYHA = New York Heart Association; PcwP = pulmonary capillary wedge pressure; PVR = pulmonary vascular resistance; RAP = right atrial pressure; Svo2 = mixed venous oxygen saturation. See Table 1 legend for expansion of other abbreviation.

Table Graphic Jump Location
TABLE 3 ]  Clinical, Functional, and Hemodynamic Characteristics at Diagnosis of PAH in Unrelated Affected Carriers of the BMPR2 Mutation

See Table 1 and 2 legends for expansion of abbreviations.

References

Galiè N, Hoeper MM, Humbert M, et al; Task Force for Diagnosis and Treatment of Pulmonary Hypertension of European Society of Cardiology (ESC); European Respiratory Society (ERS); International Society of Heart and Lung Transplantation (ISHLT). Guidelines for the diagnosis and treatment of pulmonary hypertension. Eur Respir J. 2009;34(6):1219-1263. [CrossRef] [PubMed]
 
Galiè N, Hoeper MM, Humbert M, et al; ESC Committee for Practice Guidelines (CPG). Guidelines for the diagnosis and treatment of pulmonary hypertension: the Task Force for the Diagnosis and Treatment of Pulmonary Hypertension of the European Society of Cardiology (ESC) and the European Respiratory Society (ERS), endorsed by the International Society of Heart and Lung Transplantation (ISHLT). Eur Heart J. 2009;30(20):2493-2537. [CrossRef] [PubMed]
 
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Girerd B, Montani D, Coulet F, et al. Clinical outcomes of pulmonary arterial hypertension in patients carrying an ACVRL1 (ALK1) mutation. Am J Respir Crit Care Med. 2010;181(8):851-861. [CrossRef] [PubMed]
 
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Rosenzweig EB, Morse JH, Knowles JA, et al. Clinical implications of determiningBMPR2mutation status in a large cohort of children and adults with pulmonary arterial hypertension. J Heart Lung Transplant. 2008;27(6):668-674. [CrossRef] [PubMed]
 
Kawabata M, Chytil A, Moses HL. Cloning of a novel type II serine/threonine kinase receptor through interaction with the type I transforming growth factor-beta receptor. J Biol Chem. 1995;270(10):5625-5630. [CrossRef] [PubMed]
 
Liu F, Ventura F, Doody J, Massagué J. Human type II receptor for bone morphogenic proteins (BMPs): extension of the two-kinase receptor model to the BMPs. Mol Cell Biol. 1995;15(7):3479-3486. [PubMed]
 
Nohe A, Hassel S, Ehrlich M, et al. The mode of bone morphogenetic protein (BMP) receptor oligomerization determines different BMP-2 signaling pathways. J Biol Chem. 2002;277(7):5330-5338. [CrossRef] [PubMed]
 
Nishihara A, Watabe T, Imamura T, Miyazono K. Functional heterogeneity of bone morphogenetic protein receptor-II mutants found in patients with primary pulmonary hypertension. Mol Biol Cell. 2002;13(9):3055-3063. [CrossRef] [PubMed]
 
Sitbon O, Humbert M, Jaïs X, et al. Long-term response to calcium channel blockers in idiopathic pulmonary arterial hypertension. Circulation. 2005;111(23):3105-3111. [CrossRef] [PubMed]
 
Sitbon O, Humbert M, Nunes H, et al. Long-term intravenous epoprostenol infusion in primary pulmonary hypertension: prognostic factors and survival. J Am Coll Cardiol. 2002;40(4):780-788. [CrossRef] [PubMed]
 
Kovacs G, Avian A, Olschewski A, Olschewski H. Zero reference level for right heart catheterisation. Eur Respir J. 2013;42(6):1586-1594. [CrossRef] [PubMed]
 
Montani D, Savale L, Natali D, et al. Long-term response to calcium-channel blockers in non-idiopathic pulmonary arterial hypertension. Eur Heart J. 2010;31(15):1898-1907. [CrossRef] [PubMed]
 
McGoon M, Gutterman D, Steen V, et al; American College of Chest Physicians. Screening, early detection, and diagnosis of pulmonary arterial hypertension: ACCP evidence-based clinical practice guidelines. Chest. 2004;126(1_suppl):14S-34S. [CrossRef] [PubMed]
 
Ma L, Roman-Campos D, Austin ED, et al. A novel channelopathy in pulmonary arterial hypertension. N Engl J Med. 2013;369(4):351-361. [CrossRef] [PubMed]
 
Austin ED, Ma L, LeDuc C, et al. Whole exome sequencing to identify a novel gene (caveolin-1) associated with human pulmonary arterial hypertension. Circ Cardiovasc Genet. 2012;5(3):336-343. [CrossRef] [PubMed]
 
Hanyu A, Ishidou Y, Ebisawa T, Shimanuki T, Imamura T, Miyazono K. The N domain of Smad7 is essential for specific inhibition of transforming growth factor-beta signaling. J Cell Biol. 2001;155(6):1017-1027. [CrossRef] [PubMed]
 
Rudarakanchana N, Flanagan JA, Chen H, et al. Functional analysis of bone morphogenetic protein type II receptor mutations underlying primary pulmonary hypertension. Hum Mol Genet. 2002;11(13):1517-1525. [CrossRef] [PubMed]
 
Korchynskyi O, ten Dijke P. Identification and functional characterization of distinct critically important bone morphogenetic protein-specific response elements in the Id1 promoter. J Biol Chem. 2002;277(7):4883-4891. [CrossRef] [PubMed]
 
Pfarr N, Szamalek-Hoegel J, Fischer C, et al. Hemodynamic and clinical onset in patients with hereditary pulmonary arterial hypertension andBMPR2mutations. Respir Res. 2011;12:99. [CrossRef] [PubMed]
 
Sztrymf B, Coulet F, Girerd B, et al. Clinical outcomes of pulmonary arterial hypertension in carriers ofBMPR2mutation. Am J Respir Crit Care Med. 2008;177(12):1377-1383. [CrossRef] [PubMed]
 
Yang X, Long L, Southwood M, et al. Dysfunctional Smad signaling contributes to abnormal smooth muscle cell proliferation in familial pulmonary arterial hypertension. Circ Res. 2005;96(10):1053-1063. [CrossRef] [PubMed]
 
Foletta VC, Lim MA, Soosairajah J, et al. Direct signaling by the BMP type II receptor via the cytoskeletal regulator LIMK1. J Cell Biol. 2003;162(6):1089-1098. [CrossRef] [PubMed]
 
Wong WKP, Knowles JA, Morse JH. Bone morphogenetic protein receptor type II C-terminus interacts with c-Src: implication for a role in pulmonary arterial hypertension. Am J Respir Cell Mol Biol. 2005;33(5):438-446. [CrossRef] [PubMed]
 
Machado RD, Rudarakanchana N, Atkinson C, et al. Functional interaction between BMPR-II and Tctex-1, a light chain of Dynein, is isoform-specific and disrupted by mutations underlying primary pulmonary hypertension. Hum Mol Genet. 2003;12(24):3277-3286. [CrossRef] [PubMed]
 
Chan MC, Nguyen PH, Davis BN, et al. A novel regulatory mechanism of the bone morphogenetic protein (BMP) signaling pathway involving the carboxyl-terminal tail domain of BMP type II receptor. Mol Cell Biol. 2007;27(16):5776-5789. [CrossRef] [PubMed]
 
Leyton PA, Beppu H, Pappas A, et al. Deletion of the sequence encoding the tail domain of the bone morphogenetic protein type 2 receptor reveals a bone morphogenetic protein 7-specific gain of function. PLoS ONE. 2013;8(10):e76947. [CrossRef] [PubMed]
 
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