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Original Study

Pulmonary Hypertension in Patients With Neurofibromatosis Type I

Montani, David MD, PhD; Coulet, Florence PharmD, PhD; Girerd, Barbara MD, PhD; Eyries, Mélanie PhD; Bergot, Emmanuel MD, PhD; Mal, Hervé MD; Biondi, Giuseppina MD; Dromer, Claire MD; Hugues, Thomas MD; Marquette, Charles MD, PhD; O'Connell, Caroline MD; O'Callaghan, Dermot S. MD; Savale, Laurent MD, PhD; Jaïs, Xavier MD; Dorfmüller, Peter MD, PhD; Begueret, Hugues MD; Bertoletti, Laurent MD; Sitbon, Olivier MD, PhD; Bellanné-Chantelot, Christine PharmD, PhD; Zalcman, Gérard MD, PhD; Simonneau, Gérald MD; Humbert, Marc MD, PhD; Soubrier, Florent MD, PhD

Author Information
doi: 10.1097/MD.0b013e31821be2b7
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Abstract

INTRODUCTION

Neurofibromatosis type I (NF1) or von Recklinghausen disease (OMIM 162200) is a genetic disease with an incidence of approximately 1 per 3500 individuals, transmitted as an autosomal dominant and fully penetrant trait.38 NF1 is characterized by a number of distinct clinical features, including café au lait spots, neurofibromas, and axillary or groin freckling. Cutaneous and subcutaneous neurofibromas develop in early adolescence. In contrast, plexiform neurofibromas, which are usually present at birth, may enlarge and/or undergo malignant transformation. Other less common clinical features include bone deformities, learning disabilities, short stature, and macrocephaly. Malignant tumors such as pheochromocytomas, gliomas, and juvenile chronic myeloid leukemia can also occur in the course of the disease.38

Vascular lesions observed in NF1 involve medium- and large-sized arteries and veins and have been grouped under the term NF1 vasculopathy, an uncommon but under-recognized complication that is responsible for considerable excess morbidity and mortality.35 According to published series, the prevalence of vascular lesions varies between 0.4% and 6.4% overall. However, this likely represents an underestimation of the true prevalence of vascular involvement in NF1 since most cases are detected only when patients develop symptoms. The most frequently observed vascular lesions in NF1 are aortic valve stenosis; coarctation of the abdominal aorta; renal artery stenosis; and aneurysms of the cerebral, carotid, renal, vertebral arteries and of the aorta.35 Lie et al28 described 3 basic types of vascular lesions: zonal intimal vascular smooth muscle cell (VSMC) proliferation in large elastic arteries; intimal VSMC proliferation, with associated fibrosis and neoangiogenesis of medium-sized elastic and muscular arteries; and plexiform or angiomatoid intimal proliferation in small arteries and arterioles.

Precapillary pulmonary hypertension (PH) is a rare and extremely severe complication of NF1 initially described in patients with advanced interstitial lung disease. Subsequently, plexiform pulmonary arteriopathy similar to that observed in idiopathic pulmonary arterial hypertension (PAH) (OMIM 178600) was described in NF1.40,45 In the most recent revision of the PH clinical classification,17,43 NF1 is listed in Group 5, corresponding to a cause of PH with unclear and/or multifactorial mechanisms. Indeed, the relative contributions of genetic factors (NF1 gene mutation), associated interstitial lung disease, and other as yet unidentified factors that promote pulmonary arterial remodeling remain unclear. It is also unknown why only a minority of NF1 patients develop this complication. Heritable PAH, which belongs to Group 1 of the PH clinical classification, also has a genetic origin, arising as an autosomal dominant disease with incomplete penetrance (around 20%). In most cases, heritable PAH develops as a result of mutations of genes encoding members of the transforming growth factor (TGF)-β/bone morphogenic protein receptor (BMPR) family, including BMPR2 and more rarely activin A receptor type II-like kinase-1 (ACVRL1) or endoglin.8,18,29,46

In the current study, we present 8 patients with NF1-associated PH in whom the NF1 gene mutation was identified, together with complete clinical, functional, radiologic, and hemodynamic characteristics. Based on these observations, we discuss the pathophysiologic mechanisms of NF1-associated PH, as inferred from the known signaling effects of NF1 tumor suppressor gene product neurofibromin in endothelial and VSMC, and from in vitro and in vivo experiments.

PATIENTS AND METHODS

Patients

Patients were identified through the French Network of Pulmonary Hypertension, or after referral by their physicians. The diagnosis of PH was established by right heart catheterization according to the current European Respiratory Society/European Society of Cardiology guidelines.17 All conditions associated with PH, as summarized in the revised classification, were excluded.43 All 8 patients met the National Institutes of Health (NIH) Diagnostic Criteria for NF1.1 All patients were evaluated by routine medical history, clinical examination, and extensive investigations, including echocardiography, high-resolution computed tomography (HRCT) of the chest, ventilation/perfusion lung scan, abdominal ultrasound, and human immunodeficiency virus (HIV) serology. Associated obstructive sleep apnea was excluded on the basis of clinical history and physical examination. Patients underwent genetic counseling and signed written informed consent before being tested for NF1 and BMPR2 point mutation and large size rearrangements.18 All clinical characteristics at diagnosis and follow-up were stored in the Registry of the French Network of Pulmonary Hypertension. This registry was established in accordance with French bioethics laws (French Commission Nationale de l'Informatique et des Libertés),21 and all patients gave their informed consent.

Hemodynamic Measurements

Mean pulmonary arterial pressure (mPAP), pulmonary capillary wedge pressure (PCWP), right atrial pressure (RAP), and mixed venous oxygen saturation (SvO2) were measured at right-heart catheterization. Cardiac output was measured by the standard thermodilution technique. Cardiac index (CI) was calculated as the cardiac output divided by body surface area. The indexed pulmonary vascular resistance (PVRi) was calculated as (mPAP-PCWP)/CI and the indexed total pulmonary resistance (TPRi) as mPAP/CI, both expressed in mm Hg/L/min per m2. Acute vasodilator challenge was performed through inhalation of nitric oxide, according to previously described methods.32,44

Clinical and Functional Assessment

Routine evaluation at baseline included medical history and physical examination. Age at diagnosis and clinical status as assessed by modified New York Heart Association (NYHA) functional class were recorded at diagnosis. A non-encouraged 6-minute walk test according to the American Thoracic Society recommendations was performed.

Molecular Methods

PCR Amplification and Sequencing of the Coding Regions and Exon-Intron Junctions of NF1 and BMPR2 Genes

All NF1 and BMPR2 exons were amplified by polymerase chain reaction (PCR) in a total volume of 30 μL of reaction mixture containing 25 ng of genomic DNA of each sample, 1X PCR buffer, 1.5 mM MgCl2, 0.1 mM dNTP, 0.2 μM of each primer designed with primer3 software (Frodo.wi.mit.edu/cgi-bin/primer3/primer3_www.cgi) (Tables 1 and 2) and 1U Ampli Taq Gold DNA polymerase (Applied Biosystems, Foster City, CA). Amplification was performed on a GeneAmp PCR system 9700 (Applied Biosystems). The PCR products were cleaned up on a MultiScreen 96-well PCR plate (MILLIPORE, Billerica, MA) and sequencing reactions were carried out using the BigDye Terminator v1.1 cycle sequencing kit (Applied Biosystems). The products of the sequencing reactions were cleaned up using the Sephadex G-50 (GE healthcare, Life Sciences) in a MultiScreen-HV 96-well filter plate (MILLIPORE), then run up on a ABI 3730 DNA sequencer (Applied Biosystems). The resulting sequence data were analyzed with the SeqScape software, version 2.5 (Applied Biosystems) in comparison with the reference sequences of NF1 and BMPR2 genes (Accession Number respectively: NM_001042492.2 and NM_001204.6).

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TABLE 1:
Clinical Characteristics and Outcome of the 8 Patients With Neurofibromatosis Type I-Associated Pulmonary Hypertension
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TABLE 2:
Hemodynamic Characteristics of the 8 Patients With NF1-Associated PH

BMPR2 and NF1 Screening for Large Size Rearrangements

The BMPR2 gene was screened for large size rearrangements either using the SALSA 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).41 The NF1 gene was screened using the SALSA MLPA kit P081 NF1 mix 1 and SALSA MLPA kit P082 NF1 mix 2 (MRC-Holland BV) according to the manufacturer's instructions.

CASE REPORTS

Patient 1

A 59-year-old woman with a personal and familial history of NF1 presented with a 5-year history of gradually worsening dyspnea on exercise associated with chest pain. At initial assessment she was in NYHA functional class III. Clinical evaluation revealed a raised jugular venous pressure, a loud second heart sound, hepatomegaly, ascites, and peripheral edema. Cutaneous examination found numerous neurofibromas and café au lait spots. Chest radiograph showed an enlarged right heart and pulmonary arteries, and bilateral pleural effusions. Echocardiography revealed dilated right heart chambers, right ventricular hypertrophy, and an estimated systolic pulmonary artery pressure of 65 mm Hg. V/Q lung scan and HRCT showed no evidence of pulmonary arterial thrombus. Pulmonary function tests (PFTs) revealed normal values except for a decreased diffusion capacity of carbon monoxide (DLCO) to 59% of theoretical values. Six-minute walk distance (6MWD) was 230 m. Arterial blood gas at room air was PaO2 9.7 kPa and PaCO2 4.2 kPa without any shunt while breathing 100% oxygen.

Right heart catheterization revealed a RAP of 16 mm Hg, mPAP 48 mm Hg, PCWP 6 mm Hg, CI 2.0 L/min per m2, PVRi 21 mm Hg/L/min per m2, SvO2 62%, and no evidence of portal hypertension. Acute vasodilator testing was negative.

The patient was treated with dual endothelin receptor antagonist, diuretics, and anticoagulants. Despite this therapeutic combination she deteriorated over a 4-month period, with syncope, anorexia, weight loss, and progression to NYHA functional class IV dyspnea. 6MWD decreased to 127 m. Repeat right heart catheterization revealed a RAP of 14 mm Hg, mPAP 47 mm Hg, PCWP 6 mm Hg, CI 2.85 L/min per m2, PVRi 14.4 mm Hg/L/min per m2, and SvO2 57%. In view of her clinical worsening, intravenous epoprostenol was added. The patient further deteriorated and died within weeks.

Patient 2

A 63-year-old woman was diagnosed at the age of 46 years with NF1 after she had been admitted to another hospital with a left-sided pneumothorax requiring surgical pleural abrasion and bullectomy. Clinical examination at that time revealed diffuse cutaneous neurofibromas. HRCT showed moderate pulmonary fibrosis with a large bulla. At 60 years of age she began to complain of dyspnea on exercise. PFTs showed reduced forced expiratory volume in 1 second (FEV1) at 45% of theoretical values, forced vital capacity (FVC) at 52%, total lung capacity (TLC) at 76%, and DLCO at 27%. Long-term supplementary oxygen was prescribed due to associated chronic hypoxia. A trial of systemic corticosteroids failed to ameliorate the lung fibrosis. PH was subsequently diagnosed at time of evaluation for potential lung transplantation.

At time of assessment the patient was in NYHA functional class III. Right heart catheterization showed mPAP of 52 mm Hg, CI 1.89 L/min per m2, and PVRi 25.9 mm Hg/L/min per m2 without response to acute vasodilator testing. Spiral CT of the chest showed dilated pulmonary arteries, emphysematous bullae, ground glass lung parenchymal changes, and left-sided pleural thickening but no evidence of endovascular defects. Congenital heart disease was excluded by echocardiography. An arterial blood gas test while breathing room air revealed PaO2 7.7 kPa and PaCO2 5.1 kPa. The 6MWD was 370 m, with a lowest pulse arterial oxygen saturation (SpO2) of 80% while receiving O2 at 6 L/min. Although this patient had confirmed severe NF1-associated parenchymal lung disease, the severity of the associated PH was considered out of proportion to what would be expected in the setting of isolated hypoxic PH. Accordingly, dual endothelin receptor antagonist and anticoagulation were started. Four months later, right heart catheterization showed mPAP of 48 mm Hg, an increase in CI (2.25 L/min per m2), and a decrease in PVRi 20 mm Hg/L/min per m2. There was also a small improvement in symptoms but a reduced 6MWD (250 m with 2 stops and a desaturation at 72%). After 2 years, she experienced clinical deterioration, and phosphodiesterase type 5 inhibitor was added without clinical stabilization (NYHA class III). Eighteen months later, the patient had a further clinical and hemodynamic deterioration, and despite addition of inhaled iloprost, the patient failed to improve and died.

Patient 3

A 53-year-old woman with a personal and familial history of NF1 presented for progressive dyspnea on exercise for the previous 2 years. She presented in NYHA functional class III with associated chest pain. HRCT demonstrated bilateral bullae consistent with mild NF1-associated lung disease. PFTs were normal except for an isolated decreased DLCO (38%). Echocardiography did not show any evidence of congenital heart disease but revealed a pericardial effusion and severe PH, confirmed by right heart catheterization (RAP 3 mm Hg, PCWP 15 mm Hg, mPAP 47 mm Hg, CI 2.68 L/min per m2, and PVRi 11.9 mm Hg/L/min per m2). Acute vasodilator testing was negative. The patient was treated with dual endothelin receptor antagonist and oral anticoagulants.

The patient deteriorated over the subsequent 4 months with NYHA functional class IV symptoms. Right heart catheterization showed RAP 24 mm Hg, mPAP 50 mm Hg, PCWP 14 mm Hg, CI 2.2 L/min per m2, and PVRi 16.4 mm Hg/L/min per m2. The 6MWD was 153 m. There was no evidence of portal hypertension, chronic thromboembolic pulmonary disease, HIV infection, or autoimmune disease. HRCT showed mild bilateral interstitial lung disease and bullae. Intravenous epoprostenol was added to dual endothelin receptor antagonist therapy. At re-evaluation 4 months later, the patient was in NYHA functional class II, with no further syncope or signs of right heart failure. One year after the addition of epoprostenol, she remained in NYHA functional class II with significant functional and hemodynamic improvement. The 6MWD increased to 350 m without desaturation. Repeat right heart catheterization revealed a RAP of 1 mm Hg, mPAP 30 mm Hg, PCWP 3 mm Hg, CI 3.63 L/min per m2, and PVRi 7.4 mm Hg/L/min per m2.

After 3 years, the patient experienced a clinical deterioration (NYHA functional class III) and decrease in 6MWD to 225 m. Worsening hemodynamics were confirmed at right heart catheterization: RAP of 10 mm Hg, mPAP 53 mm Hg, CI 3.03 L/min per m2, PVRi 14.2 mm Hg/L/min per m2, and SvO2 61% (O2 3l/min). A third specific PAH therapy, phosphodiesterase type 5 inhibitor, was added. The patient continued to deteriorate despite this therapeutic modification and died 6 months later.

Patient 4

A 68-year-old woman with known personal and familial history of NF1 was admitted for evaluation of a 1-year history of worsening dyspnea. Her NF1 medical history revealed cerebral glioma and hepatic neurofibroma. She was in NYHA functional class III, associated with chest pain, asthenia, and weight loss. Echocardiography showed dilated right heart chambers, a hypertrophic and akinetic right ventricule, and an estimated systolic PAP of 110 mm Hg without any evidence of congenital heart disease. Arterial blood gases showed PaO2 8.9 kPa. HRCT demonstrated mild interstitial lung disease. The only abnormality revealed by PFTs was a DLCO at 39%. The 6MWD was 180 m with 5 stops, and a lowest SpO2 of 86% was recorded. Right heart catheterization showed severe hemodynamic impairment with mPAP of 54 mm Hg, PCWP 8 mm Hg, CI 2.28 L/min per m2, and PVRi 20.2 mm Hg/L/min per m2. No acute vasodilator response was observed. She was initiated on dual endothelin receptor antagonist. After 3 months, the patient remained in NYHA functional class III, and the 6MWD had decreased to 103 m (−77 m). In the absence of improvement, phosphodiesterase type 5 inhibitor was added. After 6 months of combination therapy, the patient showed clinical (NYHA functional class II, 6MWD of 286 m) and hemodynamic improvement. After a further 2 years, she experienced progressive clinical deterioration to NYHA functional class III and severe hemodynamic impairment (mPAP 50 mm Hg, CI 2.06 L/min per m2, and PVRi 23.3 mm Hg/L/min per m2). The possibility of additional prostacyclin therapy was proposed but the patient refused. At most recent assessment the patient was noted to be in NYHA functional class IV and had experienced 2 episodes of acute right heart failure, necessitating intensive care unit admission for inotropic support.

Patient 5

A 66-year-old patient, a former smoker (40 pack-years) with known NF1, was admitted for progressive dyspnea (NYHA class III). Transthoracic echocardiography revealed a severe tricuspid regurgitation with right ventricular dilatation and an estimated right ventricular systolic pressure of 80 mm Hg. Right heart catheterization confirmed the presence of severe PH with mPAP 43 mm Hg, PCWP 6 mm Hg, CI 2.1 L/min per m2, and PVRi 17.6 mm Hg/L/min per m2. No acute vasodilator response was observed. HRCT showed mosaic perfusion pattern with mild emphysema. PFTs were within the normal range (FEV1 104% and TLC 117% predicted) with the exception of decreased DLCO (47%). The 6MWD was 200 m. Eight months after initial evaluation, the patient was receiving dual oral specific PAH therapy (phosphodiesterase type 5 inhibitor and dual endothelin receptor antagonist) with some improvement in both symptoms (increase in 6MWD to 350 m) and pulmonary hemodynamics (mPAP 28 mm Hg, RAP 5 mm Hg, PVR 2.8 mm Hg/L/min, and CI 3.6 L/min).

Patient 6

A 63-year-old woman in whom the diagnosis of NF1 had been established in childhood based on the presence of cutaneous neurofibromas, café au lait spots, and Lisch nodules was referred for evaluation of progressive dyspnea. Because she reported marked dyspnea with mild exercise, she was determined to be in NYHA functional class III. HRCT revealed enlarged pulmonary arteries, bilateral pulmonary interstitial opacities, and widespread cystic changes. PFTs showed TLC at 125% predicted, FEV1 at 69% predicted, FVC at 85% predicted, FEV1/FVC ratio at 69%, and DLCO of only 23% predicted. PaO2 and PaCO2 values at room air were 6.8 kPa and 4.3 kPa, respectively. Her 6MWD with 3 L/min of supplemental oxygen was 375 m with a profound arterial desaturation from 91% to 72%. Transthoracic echocardiography revealed normal left ventricular function, moderate dilation of the right ventricle associated with an estimated systolic pulmonary pressure at 70 mm Hg. Right heart catheterization confirmed severe PH: RAP 4 mm Hg, mPAP 36 mm Hg, PCWP 4 mm Hg, CI 3.27 L/min per m2, and PVRi 9.8 mm Hg/L/min per m2. Dual endothelin receptor antagonist and long-term oxygen therapy were initiated. Despite this management, the patient remained in NYHA functional class III. After 18 months, right heart catheterization showed RAP 3 mm Hg, PAP 38 mm Hg, CI 4.3 L/min per m2, and PVRi 7.9 mm Hg/L/min per m2. The 6MWD was 360 m (−15 m). The patient was listed for double lung transplantation.

Patient 7

A 53-year-old woman with familial NF1 diagnosed 20 years earlier and severe chronic obstructive pulmonary disease (COPD) was referred for consideration for lung transplantation or lung volume reduction surgery. Long-term oxygen had been prescribed for the preceding 7 years. Her past history was remarkable for a neurofibrosarcoma 15 years previously treated by surgery and radiotherapy. At the time of admission, clinical examination revealed several neurofibromas and café au lait spots on the abdomen, thorax, and neck. She was in NYHA functional class III. HRCT showed numerous bilateral low attenuation areas, predominantly located in the apexes, suggestive of cystic lung disease, without any evidence of chronic thromboembolic disease. PFTs showed a pattern of severe obstructive lung disease (FEV1 29% predicted, FVC 83% predicted, FEV1/FVC 29%). Measurements of TLC and DLCO were not possible. Arterial blood gas results with 4 L/min supplemental oxygen were PaO2 10.5 kPa and PaCO2 6.4 kPa. Transthoracic echocardiography showed normal left ventricular systolic function but dilatation of right heart chambers and an estimated value of the sPAP of 44 mm Hg. The 6MWD with 6 L/min oxygen was 60 m. Right heart catheterization disclosed the following results: RAP 10 mm Hg, mPAP 31 mm Hg, PCWP 10 mm Hg, CI 4.7 L/min per m2, and PVRi 4.5 mm Hg/L/min per m2. The patient was not selected for lung volume reduction because the lung disease was not classified as emphysema but rather as cystic lung disease; she was being evaluated for lung transplantation.

Patient 8

A 61-year-old woman with personal history of NF1, diagnosed when she was aged 23 years, was referred with increasing dyspnea. A diagnosis of COPD had been made previously, for which she was treated with inhaled corticosteroids and bronchodilators. As a result of progressive deterioration, continuous oxygen was subsequently prescribed. She was a former cigarette smoker with a 20 pack-year history of tobacco exposure stopped 6 years earlier. At the time of assessment, she was in NYHA functional class III. There was clinical evidence of right heart failure on physical examination. Chest radiograph showed enlarged right heart and pulmonary arteries and interstitial syndrome on the bottom with hyperclarity of the apexes. HRCT of the chest confirmed the presence of widespread cystic changes in both upper lung zones. PFTs suggested a distal obstructive defect (forced expiratory flow rate 25-75 at 46% of theoretical values) with a severely reduced DLCO at 24% of predicted. Arterial blood gas analysis confirmed severe hypoxemia at 5.3 kPa without any shunt while breathing 100% oxygen. Echocardiography showed moderate dilatation of the right ventricle and an estimated systolic pulmonary pressure of 61 mm Hg with mild pericardial effusion. Right heart catheterization confirmed PAH with RAP of 4 mm Hg, mPAP 37 mm Hg, PCWP 8 mm Hg, CI 2.3 L/min per m2, PVRi 12.6 mm Hg/L/min per m2, and SvO2 with 6 L/min of oxygen of 71%. No acute vasodilator response was observed. The 6MWD was 115 m with 4 stops. The patient was treated with dual endothelin receptor antagonists, diuretics, and oral anticoagulants. Three months later, there was no improvement in her clinical status. Repeat right heart catheterization showed no hemodynamic improvement: mPAP was 43 mm Hg, PCPW 6 mm Hg, CI 3.1 L/min per m2, and PVRi 11.9 mm Hg/L/min per m2. Because of associated ankle pain she was unable to repeat the 6MWD test. In view of her persistent symptoms, continuous infusion of epoprostenol was initiated and she was listed for double lung transplantation. She was successfully transplanted after 4 months. Pathologic assessment of explanted lungs showed interstitial fibrosis with partial loss of parenchymal architecture, associated with pronounced vascular remodeling, mostly intimal fibrosis of pulmonary arteries and muscularization of small arterioles (Figure 1).

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FIGURE 1:
Pathologic assessment of lungs obtained at the time of transplantation of a patient displaying NF1-associated PH (Patient 8). A. Interstitial fibrosis with partial loss of parenchymal architecture is associated with pronounced arterial remodeling; note a muscular artery totally occluded by intimal fibrosis (*). Magnification × 40, hematoxylin-eosin (HE) staining. B. Pulmonary artery (A) and its adjacent bronchiole (B). The arterial lumen is markedly narrowed by eccentric thickening of the intimal layer (arrow). Magnification × 100, HE staining. C. Most small pulmonary arterioles display uniform wall thickening through hyperplasia of pericytes/smooth muscle cells (arrows). Magnification × 200, HE staining. D. Widespread interstitial remodeling with fibrotic thickening of alveolar septa and numerous intraalveolar macrophages/giant cells. Magnification × 100, HE staining. [See figure in color online at http://www.md-journal.com.]

RESULTS AND DISCUSSION

We identified 8 patients with NF1 and precapillary PH (defined by a mPAP ≥25 mm Hg and PCWP ≤15 mm Hg) from the French Network of Pulmonary Hypertension. Anthropometric data, medical history, clinical and hemodynamic parameters, and clinical outcomes of the 8 patients studied are summarized in Tables 1-3. NF1 mutations are listed in Table 4. Although NF1 affects male and female patients without sex predominance, there were 7 females but only 1 male in the current series, suggesting a possible female predominance in NF1-associated PH, as it is classically observed in idiopathic and heritable PAH.18 This female predominance confirms previous reports in NF1-associated PH,45 supporting a possible role of estrogen metabolism, as previously described in idiopathic PAH.2 It is noteworthy that PH developed late in the course of NF1, with a median age at diagnosis of 62 years (range, 53-68 yr). None of these patients had identified risk factors of PAH, including anorexigen use, conditions associated with PAH (portal hypertension, HIV infection, connective tissue diseases, congenital cardiopathy), or chronic thromboembolic disease. This late onset is in sharp contrast with heritable PAH, which is characterized by a younger age at diagnosis (mean, 35.7 yr in BMPR2 mutation carriers and 21.8 yr in ACVRL1 mutation carriers) when compared to PAH patients without identified mutation (mean age, 47.6 yr).18 NF1 is transmitted as an autosomal dominant and usually fully penetrant trait in early adolescence. The median delay between diagnosis of PAH and NF1 was 44.5 years (range, 17-55 yr).

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TABLE 3:
Six-Minute Walk Distance and Pulmonary Function Tests in the 8 Patients With NF1-Associated PH
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TABLE 4:
NF1 Mutations of the 8 Patients With NF1-Associated PH

In the current series, all 8 patients met the NIH diagnostic criteria for NF1 with typical clinical presentation including at least several neurofibromas and café au lait spots.1 Median age at diagnosis of NF1 was 13 years (range, 8-46 yr), older than that observed in the large series of DeBella et al,10 in which 97% of patients met diagnostic criteria by the age of 8 years. Because of the retrospective design of the study, however, we were unable to determine precisely the age at which diagnostic criteria were first established. It is therefore not known whether these patients had a late-onset form of NF1. Dyspnea and signs of right heart failure were the major features prompting evaluation for associated PH. All patients had severe hemodynamic impairment at diagnosis, with a low CI (median, 2.3 L/min per m2; range, 1.9-4.7) and high levels of mPAP (median, 47.5 mm Hg; range, 31-54) and PVRi (median, 15.1 mm Hg/L/min per m2; range, 4.5-25.9). Of the 8 patients, 6 underwent vasodilator testing with inhaled nitric oxide, and none of them had a vasodilator response, as previously described.44 Acute vasodilator response with nitric oxide is reported in about 10% of idiopathic PAH, and is associated with long-term response to calcium channel blockers and an excellent prognosis.44 However, we have recently shown that the proportion of acute vasodilator response was low in heritable PAH18 as well as in PAH associated with various conditions.32 Furthermore, all patients were in NYHA functional class III at the time of diagnosis and had severe exercise limitation; median 6MWD was low with a median value of 180 m (range, 60-375 m).

Even if it was rarely observed, it has been recently demonstrated that NF1 may be associated with parenchymal lung involvement.50 Zamora et al,50 in an exhaustive review of the literature, found 64 patients with diffuse lung disease associated with neurofibromatosis, including the 3 cases they reported in their study. The mean age of these patients (50 yr) was broadly similar to our series of NF1-associated PH. However, a male predominance was observed (69%), in contrast to the female predominance we observed in association with PH.50 In these patients with diffuse lung disease, PFTs showed obstructive pattern in 43%, restrictive pattern in 37%, and a mixed pattern in 17%, with a decreased DLCO in the vast majority of cases.50 On HRCT of the chest, the most frequent radiologic findings observed were reticular abnormality, bullae, ground-glass opacities, cysts, and emphysema.50 In the current series of NF1-associated PH, lung involvement was observed in 5 patients and was in accordance with data reported by Zamora et al.50 Lung involvement was variously manifested by lung cysts (n = 4), large bullae (n = 1), pneumothorax (n = 1), infiltrates (n = 1), and moderate pulmonary fibrosis (n = 1). One can hypothesize that precapillary PH observed in these NF1 patients might have been due to associated parenchymal lung disease. However, 3 patients with confirmed severe PH had no significant lung involvement on HRCT of the chest, and had normal PFTs. Furthermore, in most patients, measurements of spirometry and lung volumes were in the normal range: median FEV1 was 99% (range, 29%-109%) and median TLC was 96% (range, 76%-125%). In contrast, DLCO was markedly decreased in all patients (median, 27%; range, 23%-59%), suggesting significant pulmonary vascular involvement. This pattern of mild lung parenchyma abnormality (mainly characterized by cystic lesions) and associated disproportionate pulmonary vascular involvement in NF1 is similar to the well-described association of pulmonary Langerhans cell histiocytosis and PH.15 One patient (Patient 8) had lung transplantation and therefore had a complete pathologic assessment. However, this patient also displayed severe associated parenchymal lung disease with diffuse interstitial lung disease and widespread cystic changes. Pathologic assessment of explanted lungs confirmed interstitial fibrosis with partial loss of parenchymal architecture (Figure 1A), but also showed pronounced pulmonary arterial remodeling (Figure 1A-C). Pulmonary vascular involvement included arterial remodeling with eccentric thickening of the intimal layer and uniform wall thickening through hyperplasia of pericytes/smooth muscle cells in most of the small pulmonary arterioles (Figure 1C). To our knowledge, pathologic assessment of pulmonary vasculopathy in NF1-associated PH has been reported in only 2 cases, 1 characterized by an extensive irregular thickening of the intima of pulmonary arteries,40 and 1 with association of plexiform lesions.45 These observations reinforce the hypothesis of a disproportionate pulmonary vascular involvement in the context of a diffuse lung disease.

In the current series, all patients received conventional therapy for PH (oxygen if needed, anticoagulation, diuretics). In addition, 7 patients received specific PH therapies, including endothelin receptor antagonists (n = 7), phosphodiesterase type 5 inhibitors (n = 5), and prostanoids (n = 4). The outcomes of these patients were characterized by a limited response to specific PH therapy and poor outcome (3 deaths, 2 patients awaiting or evaluated for lung transplantation, and 1 patient transplanted). These results were in accordance with data published by Stewart et al,45 who reported 4 cases of NF1-associated PH and updated previously published reports. Of the 7 patients for whom natural history was known, 5 patients died within 3 years of presentation.45 This limited response to specific PH therapies and the poor prognosis of NF1-associated PH emphasize the importance of referral for lung transplantation assessment early in the course of the disease in eligible patients.

Both the BMPR2 and the NF1 gene were screened for small size mutations and large gene rearrangements. No BMPR2 gene abnormality was detected in any of the patients studied. By contrast, heterozygous germline mutations of the NF1 gene were identified in all cases. NF1 mutations were of different types, including short deletions, nonsense and missense mutations, and a complete deletion of the gene (Table 4). Two of these mutations, located on exon 16 and 21 (exon numbering is according to the nomenclature of NM_001042492.2), have been already reported by Fahsold et al.14 The single missense mutation located in exon 21 was described by Maynard et al31 and is located within the cysteine-rich domain. Five others are novel and are located in various exons of the genes (in exon 10, 16, 21, 27, and 37), interrupting the gene at the site of the mutation or by a frameshift. No relationship was previously found between the position of the truncating mutation and the features of the NF1 phenotype,14 and, similarly, we did not find any relationship between the occurrence of PH and a specific position of the mutation.

PH was diagnosed in all patients described in the current series after the age of 50 years. This contrasts with our recent observation of a younger age at PH diagnosis in BMPR2 or ACVRL1 mutation carriers, as compared with noncarriers.18,46 Therefore, this genetic context does not result in a similar early onset of PH, although the cellular signaling abnormalities caused by NF1 mutation are well defined and would promote VSMC growth. The frequency of NF1-associated PH is markedly lower than BMPR2 mutation-associated PH, since the usual penetrance is considered, in the latter, to be around 20%,29,30 even if it varies between families. In addition, it should be emphasized that familial PH has never been reported in the setting of NF1 mutations; this also contrasts with the frequent observations in BMPR2 mutation carriers, and more rarely in ACVRL1 mutation carriers.

Neurofibromin has a guanosine triphosphatase (GTPase)-activating protein (GAP) domain that is responsible for decreasing the level of Ras bound to guanosine triphosphate (GTP) by hydrolyzing GTP bound to small monomeric GTP-bound Ras.3 This GTPase activity therefore acts as a negative regulator of signal transmitted by Ras.5,11 Indeed, Ras is at the center of several pathways, including the MAP kinase pathway ending by ERK activation and transcriptional activation of target genes.12

GTP-Ras is also able to induce activation of the PI3kinase-AKT pathway.39 An important intermediate of this pathway is the TSC1-TSC2 complex.19 In its active nonphosphorylated form, this complex acts as a repressor of mammalian target of rapamycin (mTOR), a serine/threonine protein kinase that regulates cell growth and proliferation.19 Because Ras persistent activation, through a deficient neurofibromin activity, can phosphorylate and inactivate TSC2, neurofibromin deficiency leads to mTOR activation and phosphorylation of 2 targets important for translation regulation, S6kinase 1 (S6K1) and eukaryote initiation factor E4E binding protein 1 (4EBP), which will in turn increase vascular endothelial growth factor (VEGF) and HIF-1α production and other proteins.7,13,22-24 Neurofibromin was also shown to modulate adenylate-cyclase activity through a Ras independent mechanism48 (Figure 2). Banerjee et al4 reported an activation of activator of transcription-3 (STAT3) as an intermediate of NF1/mTOR signaling, which was identified through the screening of molecules inhibiting growth of NF1-deficient cells.

F2-7
FIGURE 2:
Schematic representation of the main signaling pathways modulated by NF1. NF1 decreases the rate of activated Ras by its guanosine triphosphatase activity, and its inactivation leads to downstream activation of the pathway. The drugs that can be used for treatment are shown in boxes. The 3 drugs inhibit key signaling molecules that are activated in excess in NF1-mutated cells. Abbreviations: 4EBP = 4E-binding protein, mTOR = mammalian target of rapamycin, NF1 = neurofibromin, PDGFR = platelet-derived growth factor receptor, PI3K = phosphatidylinositol 3-kinase, Rheb = Ras homolog enriched in brain, S6K1 = p70 ribosomal protein S6 kinase 1, TSC = tuberous sclerosis protein.

It is interesting that other multiple congenital anomaly syndromes that are due to Ras/MAPK activation by germline mutations, such as Noonan syndrome, Costello syndrome, and Leopard syndrome, are also associated with PH.6,36,47 The mechanisms that promote PH in these disorders and in NF1 are likely to be similar.

The molecular mechanisms underlying cell proliferation associated with monoallelic NF1 inactivation have been analyzed in depth. As expected from the GAP function of neurofibromin, the level of activated Ras was found to be increased in endothelial cells transduced with a shRNA knocking down NF1.33 Endothelial cells with NF1 knockdown display an increased proliferation and migration, which is dependent on ERK activation. ERK is also activated in endothelial cells isolated from patients' peripheral blood. The increased proliferation and migration observed in peripheral blood endothelial cells from NF1 patients was blocked by an MEK inhibitor. In addition, NF1−/− Schwann cell conditioned medium is able to induce an angiogenic response in NF1+/− endothelial cells, an effect that is not observed with wildtype Schwann cells.33

VSMC is also a major cellular component involved in pulmonary artery vessel wall hypertrophy. In NF1smKO mice, in which the NF1 gene is selectively inactivated in VSMCs, an increased phosphorylated ERK, induced by an elevation of activated Ras, and an upregulation of phosphorylated mTOR and its effector S6K were observed.49

VSMCs from NF1+/− mice respond with increased proliferation and migration to PDGF-BB as compared with wildtype cells.27 PDGF signaling is increased in VSMC from NF1+/− mice, as illustrated by an increased ERK activation in response to PDGF-BB, an effect which was abolished by cell transduction with an NF1 expressing vector. This increased response was also observed in human VSMCs with an NF1 knockdown.27 These observations made in both endothelial cells and VSMCs identify the PDGF-Ras pathway as critically important for VSMC proliferation and migration. This pathway could also be important in pericytes, since these cells (which express the PDGF-R) exert a major role in vascular wall homeostasis and are considered progenitors of more differentiated vascular cells when expressing specific progenitor markers.9

Despite tremendous insights gained in recent years into the pathophysiology of pulmonary vascular disease, there remains a significant deficit in our understanding of the genetic and molecular mechanisms underlying the development of PH in the context of NF1. There is a prevailing consensus that in order for cancers to develop in the context of NF1, a second somatic genetic "hit" is necessary. In this regard, NF1 mutations have been identified on the nonmutated allele of NF1 tumors.42 Similarly, signaling abnormalities were found in both NF1+/− and NF1−/− cells, with more marked levels of signaling dysregulation evident in the latter. Johannessen et al23 showed a codominant-like effect of NF1 mutation on cell phenotype, when Schwann cells from cutaneous and from a plexiform neurofibroma, homozygous or heterozygous for NF1 inactivation, were separated and analyzed. These authors showed that S6K1, AKT, and tuberin phosphorylation was higher in homozygous than in heterozygous cells. To date, no such analysis has been possible in cells from pulmonary arteries from patients with NF1-associated PH, though animal models provide interesting clues with respect to proliferation of VSMCs as a consequence of NF1 mutation. Indeed, mice with a NF1 gene inactivation targeted in VSMC (NF1smKO mice) develop normally and have no phenotypical abnormality of the vessel wall at the basal state.49 The level of NF1 expression in these animals remains at 14% of the levels measured in wildtype animals. However, this value likely represents an overestimation as a result of contamination by other cell types in which the gene expression is not abolished. This level of expression remains intermediate between heterozygous and homozygous cells for NF1 mutation. After carotid artery ligation, an abnormal response was observed in vessels, characterized by exaggerated remodeling with an increase in intimal thickening and both intima-to-media and intima-to-lumen ratios compared with wildtype mice. The increased neointimal proliferation was essentially composed of VSMCs. In this model, the increased VMSC proliferation secondary to NF1 loss was detectable only after artery stress.

It is unlikely that homozygous NF1 inactivation is a prerequisite for PH-associated VSMC proliferation, as evidenced in animal models with partial NF1 inactivation. Moreover, it is unlikely that the mechanisms responsible for the diffuse cellular proliferation of small and medium-sized arteries typical of PH also drive the development of localized tumors such as neurofibromas. More likely, there is an as yet unidentified circulating mitogenic factor that acts on vascular cells, which, in the setting of deregulated RAS signaling, causes aberrant cellular proliferation. Previous experimental evidence has shown the proliferation of endothelial cells and VSMCs in primary PH to be monoclonal.26 It would be of interest to determine whether a similar mechanism of cellular expansion occurs in NF1 PAH, as this might suggest that a mitogenic factor could similarly act on a monoclonal population of progenitor cells that are either circulating or natively associated with the vascular wall. For this pathologic process to be initiated, a trigger is necessary, as is the case in PAH patients carrying a BMPR2 mutation. Additional unidentified factors, be they inflammatory, infectious, or autoimmune, may also play a role.20 However, PH is an uncommon complication among patients with NF1, implying that the occurrence of initiating factors in this context is rare.

From the known effects of neurofibromin on growth regulation signaling, some therapeutic avenues can be envisioned. In this regard, several currently available agents have been suggested as potentially beneficial in NF1-associated PH. However, to the best our knowledge, no treatments have been tested for this particular indication. Indeed, drugs that can inhibit the dysregulation induced by neurofibromin deficiency have already been approved. Rapamycin is an mTOR inhibitor that has been shown to attenuate PH and neointimal formation in rats34 (see Figure 2). It was also proposed as a therapeutic option for NF1, and may be worthy of pursuit as a therapeutic alternative in NF1-associated PH.23 After carotid injury, NF1+/− mice display an increased VSMC neointimal proliferation at 21 days postinjury, compared with wildtype mice, associated with an increased ERK activation.25 Imatinib mesylate (Gleevec, Novartis, Basel, Switzerland), a potent inhibitor of the PDGF-BB axis, was given to NF1+/− injured mice and induced a 4-fold reduction in intima/media ratio compared with saline-treated NF1+/− mice, but had no effect in injured wildtype mice. These data are consistent with the involvement of the PDGF-BB axis in this model and its interaction with the NF1-Ras-ERK pathway. PDGF and PDGF receptors are also overexpressed in human PAH,37 and imatinib mesylate (which is currently undergoing evaluation in PAH) may also be of therapeutic potential in NF1-associated PH. Attenuation of the vascular wall proliferation that characterizes NF1 using statins (3-hydroxy-3-methylglutarylcoenzyme A reductase inhibitors) is another possible approach, since this class of agent is known to inhibit Ras activity through prevention of its lipid modification16 (see Figure 2).

In conclusion, PH represents a rare but severe complication of NF1 that is characterized by a late onset, female predominance, severe functional and hemodynamic impairment, and poor outcome. Specific PH therapies seem to have only a modest effect in these patients. PH should be recognized as a rare but severe complication of NF1, requiring an early referral of eligible patients for lung transplantation. This study demonstrates that NF1-associated PH may occur in patients with mild or absent parenchymal lung disease (mainly characterized by cystic lesions), reinforcing the hypothesis of an associated disproportionate pulmonary vascular involvement. The association of NF1 gene mutation and PH is of major significance in terms of advancing our understanding of the physiopathology of both NF1 and pulmonary vascular disease in general. Insights gained from the mechanisms underlying this devastating complication of NF1 should help identify possible novel therapeutic approaches in PH.

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