Published ahead of print on March 20, 2008, doi:10.1164/rccm.200712-1807OC
© 2008 American Thoracic Society doi: 10.1164/rccm.200712-1807OC
Clinical Outcomes of Pulmonary Arterial Hypertension in Carriers of BMPR2 Mutation1 Université Paris-Sud 11, UPRES EA 2705, Centre National de Référence de l'Hypertension Artérielle Pulmonaire, Service de Pneumologie et Réanimation Respiratoire, Institut Paris-Sud Cytokines, Hôpital Antoine-Béclère, Assistance Publique des Hôpitaux de Paris, Clamart, France; and 2 Université Pierre et Marie Curie–Paris 6, Laboratoire d'Oncogénétique et Angiogénétique Moléculaire, Groupe Hospitalier Pitié-Salpêtrière, Paris, France Correspondence and requests for reprints should be addressed to Marc Humbert, M.D., Ph.D., Service de Pneumologie et Réanimation Respiratoire, Hôpital Antoine-Béclère, 157 rue de la Porte de Trivaux, 92140 Clamart, France. E-mail: marc.humbert{at}abc.aphp.fr
Rationale: Germline mutations in the gene encoding for bone morphogenetic protein receptor 2 (BMPR2) are a cause of pulmonary arterial hypertension (PAH). Objectives: We conducted a study to determine the influence, if any, of a BMPR2 mutation on clinical outcome. Methods: The French Network of Pulmonary Hypertension obtained data for 223 consecutive patients displaying idiopathic or familial PAH in whom point mutation and large size rearrangements of BMPR2 were screened for. Clinical, functional, and hemodynamic characteristics, as well as outcomes, were compared in BMPR2 mutation carriers and noncarriers. Measurements and Main Results: Sixty-eight BMPR2 mutation carriers (28 familial and 40 idiopathic PAH) were compared with 155 noncarriers (all displaying idiopathic PAH). As compared with noncarriers, BMPR2 mutation carriers were younger at diagnosis of PAH (36.5 ± 14.5 vs. 46.0 ± 16.1 yr, P < 0.0001), had higher mean pulmonary artery pressure (64 ± 13 vs. 56 ± 13 mm Hg, P < 0.0001), lower cardiac index (2.13 ± 0.68 vs. 2.50 ± 0.73 L/min/m2, P = 0.0005), higher pulmonary vascular resistance (17.4 ± 6.1 vs. 12.7 ± 6.6 mm Hg/L/min/m2, P < 0.0001), lower mixed venous oxygen saturation (59 ± 9% vs. 63 ± 9%, P = 0.02), shorter time to death or lung transplantation (P = 0.044), and younger age at death (P = 0.002), but similar overall survival (P = 0.51). Conclusions: BMPR2 mutation carriers with PAH present approximately 10 years earlier than noncarriers, with a more severe hemodynamic compromise at diagnosis.
Key Words: bone morphogenetic protein receptor 2 • genetics • hemodynamics • pulmonary arterial hypertension • survival
Pulmonary arterial hypertension (PAH) is a severe disease affecting small pulmonary arteries, with a progressive remodeling leading to elevated pulmonary vascular resistance and right ventricular failure (1). PAH may occur in different clinical contexts, depending on associated clinical conditions (1, 2). Idiopathic PAH corresponds to sporadic disease, without any familial history of PAH or known triggering factor (2). When PAH occurs in a familial context (2–5), germline mutations in the bone morphogenetic protein receptor 2 (BMPR2) gene are detected in at least 70% of cases (6–10). BMPR2 mutations can also be detected in 11 to 40% of apparently sporadic cases (10–15). The distinction between idiopathic and familial BMPR2 mutation carriers may in fact be artificial, as these subjects have an inherited condition and may all correspond to a potential familial disease. Recent expert discussion pleads in favor of the term "heritable" PAH to describe these genetic forms of the disease. In any case, BMPR2 mutation represents the major genetic predisposing factor for PAH (6–17). The BMPR2 gene encodes a type 2 receptor for bone morphogenetic proteins, which belong to the transforming growth factor-β superfamily (16). Among several biological functions, bone morphogenetic proteins are involved in the control of vascular cell proliferation (16). Heterozygous inactivating mutations of BMPR2 are responsible for functional haploinsufficiency of the bone morphogenetic proteins' transduction pathway, and lead to proliferation of smooth muscle cells within pulmonary arteries (17). However, the penetrance of BMPR2 mutations is low (around 20%), and neither the factors involved in the initiation of the disease in affected subjects nor the precise molecular mechanisms underlying the responsibility of BMPR2 haploinsufficiency in the disease are identified (5, 16, 17). Previous reports have suggested that the clinical and hemodynamic presentation of familial PAH was not different from idiopathic PAH, with similar pathologic lesions as well as molecular and cellular abnormalities described in both subtypes (16, 18). However, a significant proportion of so-called idiopathic cases were in fact associated with BMPR2 mutations and this might have given a biased comparison (11). Recent findings have indicated that BMPR2 mutation carriers with familial or idiopathic PAH were less likely to display vasoreactivity than noncarriers, raising the possibility that monoallelic BMPR2 mutation identifies patients who may respond poorly to long-term vasodilator therapy (19). We hypothesized that a mutated BMPR2 status is associated with distinct disease phenotypes. To test this hypothesis, the French Network of Pulmonary Hypertension obtained data on consecutive patients displaying idiopathic or familial PAH in whom point mutation and large size rearrangements of BMPR2 were screened for. Clinical, functional, and hemodynamic characteristics, as well as outcomes, were compared in BMPR2 mutation carriers and noncarriers.
Patients Patients were seen within the French Network of Pulmonary Hypertension between January 1, 2004, and June 1, 2007. In accordance with the guidelines of the American College of Chest Physicians (20), patients with idiopathic and familial PAH tested for BMPR2 mutations underwent genetic counseling and signed written, informed consent. Two hundred and thirty-three consecutive patients (195 idiopathic and 38 familial PAH) were tested for BMPR2 point mutation and large size rearrangements. This corresponded to 68 BMPR2 mutation carriers (40 idiopathic and 28 familial), 155 idiopathic PAH noncarriers, and 10 patients with familial PAH with no evidence of BMPR2 mutation. Patients with familial PAH with no evidence of BMPR2 mutation were excluded from the analysis to decrease the risk of misclassification in the BMPR2 mutation noncarrier group (Figure 1).
A diagnosis of PAH was established by means of right heart catheterization and acute vasodilator challenge was performed through inhalation of nitric oxide or intravenous injection of prostacyclin, according to previously described methods (1, 21, 22). Idiopathic PAH was recognized after ruling out all associated conditions of pulmonary hypertension, summarized in the revised classification, and by determining no existence of additional pulmonary hypertension cases in the family of the patient (2). Familial PAH was recognized if there was more than one confirmed case in the family (2). All clinical characteristics at PAH diagnosis and follow-up were stored in the Registry of the French Network of Pulmonary Hypertension. This registry was set up in agreement with French bioethics laws (French Commission Nationale de l'Informatique et des Libertés), and all patients gave their informed consent (23). The six-minute-walk distance as well as the percentage of reference values were studied (23, 24). All patients were treated according to international guidelines, which recommend a similar management in familial and idiopathic PAH (25, 26).
Molecular Methods
Screening of BMPR2 large size rearrangements. The BMPR2 gene was screened for large size rearrangements either using the SALSA multiplex ligation-dependent probe amplification (MLPA) P093 HHT probe mix kit (MRC-Holland BV, Amsterdam, The Netherlands) according to the manufacturer's instructions or by quantitative multiplex PCR of fluorescent short fragments (QMPSF) (27). In the latter case, BMPR2 exons were amplified (size of fragments ranging from 132 to 308 bp) in three multiplex PCRs and the forward primer of each pair was 5'-labeled with the 6-Fam fluorochrome. Fragment analysis of multiplex PCR (from MLPA or QMPSF) was performed on an ABI 3730 DNA analyzer and results were analyzed using GeneMapper software, version 4.0 (Applied Biosystems). Two DNA samples from unaffected individuals were used as controls in each experiment. Electrophoregrams were superimposed on those generated with a control DNA by adjusting to the same levels the peaks obtained for the control amplicons.
Statistical Analysis
Clinical and Functional Characteristics A BMPR2 mutation was identified in 40 of 195 idiopathic PAH (20.5%) and 28 of 38 familial cases (73.7%). We thus compared 68 idiopathic and familial BMPR2 mutation carriers with 155 idiopathic PAH noncarriers. Age at diagnosis of PAH was significantly younger in BMPR2 mutation carriers, as compared with noncarriers (34.5 ± 14.5 vs. 46.0 ± 16.1 yr, P < 0.0001; Table 2). No other clinical differences were found between the two subgroups at diagnosis, including a similar female predisposition to PAH, regardless of BMPR2 status (Table 3). Six-minute-walk distance at diagnosis was 351 ± 106 m in BMPR2 mutation carriers versus 328 ± 120 m in noncarriers (P = 0.21). Because patients with BMPR2 mutations were younger, we also expressed the six-minute-walk distance as a percentage of reference values (55 ± 14 in carriers vs. 60 ± 21% of predicted values in noncarriers, P = 0.17).
Hemodynamics As compared with noncarriers, BMPR2 mutation carriers were characterized by a more severe hemodynamic compromise at diagnosis, with a significantly higher mean pulmonary artery pressure (  ) (64 ± 13 vs. 56 ± 13 mm Hg, P < 0.0001), a lower cardiac index (2.13 ± 0.68 vs. 2.50 ± 0.73 L/min/m2, P = 0.0005), a higher pulmonary vascular resistance (17.4 ± 6.1 vs. 12.7 ± 6.6 mm Hg/L/min/m2, P < 0.0001), and a lower mixed venous oxygen saturation (53 ± 9 vs 59 ± 9%, P = 0.02). Mutation carriers were less likely to exhibit a significant response to acute vasodilatory test during right heart catheterization (defined by a fall in of least 10 mm Hg, reaching an absolute value of under 40 mm Hg, associated with no change or an increase in cardiac index) (Table 3) (19, 22, 25, 28).
Medical Management
Survival
BMPR2 Mutations Germline BMPR2 mutations were located throughout the BMPR2 gene, affecting regions of the gene encoding for the four main parts of the protein (ligand binding, transmembrane region, kinase domain, and cytoplasmic tail). Five long deletions have been identified in the present series, corresponding to two exon 10 deletions, one large deletion from exons 1 to 4, one deletion of exons 11 to 13, and a deletion of the whole allele (Table 4).
We have studied a group of 223 patients with familial and idiopathic PAH with or without germline BMPR2 mutations and treated according to the best standard of care in a national pulmonary hypertension network. Our results indicate that there is a significantly increased severity of hemodynamic status in BMPR2 mutation carriers at diagnosis when compared with noncarriers, as demonstrated by higher , lower cardiac index, higher total pulmonary resistance, and lower mixed oxygen saturation. Although there was no difference in overall survival after diagnosis, BMPR2 mutation carriers had a significantly shorter time to death or lung transplantation, indicating that they were more likely to undergo lung transplantation. Because lung transplantation is the ultimate alternative for severe PAH cases who cannot be managed medically (25), time to death or transplantation is an indicator of disease severity in this patient population (29). In addition, BMPR2 mutation carriers developed the disease earlier with fatal events occurring at a younger age. Therefore, the overall findings plead in favor of a more severe disease state in patients with BMPR2 mutation. In recent registries, including the French registry (23), no difference between familial and idiopathic PAH could be evidenced in terms of severity, presumably because of the substantial proportion of BMPR2 mutation carriers in each group, which in fact biased the comparison. The present comprehensive analysis of well-defined BMPR2 mutation carriers and noncarriers allows avoiding such a bias. The observation that subjects with BMPR2 mutation develop the disease earlier and with a more severe hemodynamic compromise illustrates the constitutive susceptibility conferred by the mutation on the course of the disease. Although the penetrance of BMPR2 mutation is low, in agreement with data from mice with heterozygous inactivation of BMPR2, which only shows a susceptibility to inflammatory stress or excess serotonin (30, 31), BMPR2 haploinsufficiency determines a pulmonary vascular status that leads to PAH in an accelerated fashion. The younger age of onset is also observed for other diseases where an inherited genetic factor confers a strong predisposition, as in hereditary forms of breast and colon cancer. In these cases, a heterozygous germline mutation in a DNA repair gene or tumor suppressor gene favors a second genetic event, or second hit, which in turn leads to a series of somatic mutations in specific cells and to the development of cancer (32). In the case of BMPR2 mutation, the gene is involved in proliferation of pulmonary artery vascular smooth muscle cells under the control of bone morphogenetic proteins. Indeed, a reduced apoptotic response to bone morphogenetic proteins was shown in pulmonary artery vascular smooth muscle cells from pulmonary hypertensive patients with a BMPR2 mutation, as compared with control cells, suggesting a change in phenotype secondary to genetic events (33, 34). Somatic genetic events were investigated in smooth muscle cells from affected pulmonary arteries, such as DNA microsatellite instability, a hallmark of genome instability, or loss of heterozygosity attesting chromosomal deletion, but negative results do not plead in favor of the hypothesis of a second genetic somatic event (35). Although other kinds of somatic genetic or epigenetic events cannot be eliminated, their nature remains elusive. Any familial disease may lead to an earlier diagnosis in subsequent similar cases. However, a majority of BMPR2 mutant carriers from the present study displayed idiopathic PAH (40 of 68, 59%). In addition, 5 of 28 familial cases of BMPR2 mutant carriers were the first identified case in these families. Therefore, only 23 BMPR2 mutant carriers (34%) had a familial history of PAH at diagnosis. Thus, heightened suspicion of PAH was present in a minority of BMPR2 mutant carriers. To further analyze this population, we have compared delay between onset of symptoms and diagnosis in patients with or without familial history of PAH. As expected, patients with a history of PAH had a shorter delay between onset of symptoms and diagnosis (1.5 ± 2.3 vs. 0.6 ± 0.6 yr, P = 0.036). This could not explain the differences in disease presentation, such as the more than 10-year age difference at diagnosis and death between BMPR2 mutation carriers and noncarriers. The later onset in noncarriers could imply that, compared with patients with a germline BMPR2 haploinsufficiency, additional events are required for the disease to develop, resulting in a significant older age of onset. The nature of these events is unknown, and they are probably partially similar to those involved in BMPR2 mutation carriers. For example, a down-regulation of the bone morphogenetic protein pathway is observed in both types of smooth muscle cells, either from BMPR2 mutation carriers or noncarriers. In this latter case, the down-regulation origin is unknown (36). The worse hemodynamic parameters at diagnosis in BMPR2 mutation carriers might reflect an accelerated process of the disease, but this is not confirmed by the overall survival. Nevertheless, time to death or lung transplantation was shorter in BMPR2 mutation carriers as compared with noncarriers, further suggesting that BMPR2 mutation confers a more severe phenotype. The disequilibrium in the female/male ratio was previously reported for familial and idiopathic PAH (5, 23, 37, 38) and many hypotheses have been raised to explain it, including a disadvantage of male embryos carrying a BMPR2 mutation (5). Interestingly, the female/male ratio was identical in BMPR2 mutation carriers and noncarriers, suggesting that this disequilibrium does not result from a meiotic drive or embryonic lethality but more likely from specific sex-dependent factors acting later in life and favoring the occurrence of the disease in females. We have excluded from the analysis the 10 patients with familial PAH who did not carry BMPR2 mutations. It is possible, however, that these patients carry mutations in some unexplored parts of the BMPR2 gene. As mentioned in METHODS, we have performed amplification of all coding exons and intronic junctions of the BMPR2 gene and may thus miss alterations in other intronic regions or other mechanisms, such as epigenetic alterations of this gene that have not been recognized at this time. This possibility has been partly evaluated by Aldred and colleagues who analyzed part of the 5'-untranslated region and promoter of the gene in familial PAH (39). DNA upstream of the coding region was analyzed by direct sequencing in 16 families. In one family, a mutation predicted to form a cryptic translational start site was identified. This mutant transcript contains a premature stop codon (39). These results further emphasize the possibility that current techniques could miss BMPR2 mutations in some patients. In conclusion, BMPR2 mutation carriers with PAH present approximately 10 years earlier than noncarriers with a more severe hemodynamic compromise at diagnosis. A better understanding of the mechanisms by which BMPR2 mutations define a subclass of patients with more severe disease is critical for improving our knowledge of PAH.
The authors thank Dr. Grégory Raux (Service de Génétique CHU Charles Nicolle ROUEN) for the design of QMPSF primers and Ms. Rebecca Honan for reading the manuscript.
Supported in part by grants from the Ministère de l'Enseignement Supérieur et de la Recherche and the Université Paris-Sud 11. R.S. is supported by a grant from European Respiratory Society. Originally Published in Press as DOI: 10.1164/rccm.200712-1807OC on March 20, 2008 Conflict of Interest Statement: None of the authors has a financial relationship with a commercial entity that has an interest in the subject of this manuscript. Received in original form December 10, 2007; accepted in final form March 20, 2008
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ABSTRACT
AT A GLANCE COMMENTARY
2, Fisher's exact test, Mann-Whitney test, or t test, as appropriate. Both the time to death (patients undergoing lung transplantation were considered as censored at the date of transplantation) and the time to death or lung transplantation (patients undergoing lung transplantation were considered as an event at the date of transplantation) were described using Kaplan-Meier curves, and compared with the use of the log-rank test. A P value of less than 0.05 was considered to indicate statistical significance. 
P = 0.003.
