Compound BMPR2gene mutations in a malignant variant of idiopathic pulmonary arterial hypertension

Pulmonary arterial hypertension (PAH; MIM 600799) is frequently associated with concomitant diseases, including congenital heart disease. 6% of patients with PAH show a family history of the disease [hereditary PAH (HPAH)], with the major genetic determinants of HPAH being heterozygous germline mutations in the bone morphogenetic protein type II receptor ( BMPR2 ). We present the case of a 38-year-old woman of Indian descent; initially admitted with progressive dyspnea [New York Heart Association (NYHA) class III]. The results of the proband’s clinical assessments are presented here. Cardiac catheterization confirmed idiopathic PAH with severe right ventricular hypertrophy associated with pulmonary arteriopathy. Initial treatment com-prised the dual endothelin receptor antagonist, bosentan, furosemide, warfarin and intravenous infusion of prostaglandin I2 (PGI2) for 3 days. Despite this, the patient died of pulmonary hemorrhagic edema and cardiogenic shock after 6 days of intensive care. After rela-tives’ consent, post mortem assessments confirmed a diagnosis of PAH; the heart displayed significant right ventricular hypertrophy and it was particularly noted that the right atrial appendage had undergone extreme dilation. Pulmonary arteriopathy was characterized by medial hypertrophy, arterialization of muscu-lar arteries and muscularization of non-mus-cularized analyses revealed the presence of cis -muta-tions in the BMPR2 gene (p.Cys123Arg and p.Arg332X). Cosegregation studies were not available.Our findings suggest that mutations of the BMPR2 gene gave rise to the onset of PAH in this patient and that the severity of the onset and progression could be attributed to the presence of multiple mutations in a gene-dosage manner.


Introduction
Pulmonary arterial hypertension (PAH) ( Table 1), 1 is a rare, fatal disease of the pulmonary arterioles, 1,2 where vasoconstriction and vascular remodeling lead to a progressive increase in pulmonary arterial pressure resulting in right ventricular failure. 2,3 PAH has an estimated prevalence of 15-50 cases per million, 4 the majority of which arise as a result of concomitant diseases (associated PAH) which include congenital heart disease, connective tissue disorders and infection with the human immunodeficiency virus. Spontaneouslyoccurring, idiopathic PAH (IPAH) accounts for at least 40% of the remaining cases and typically affects around twice as many women as men with a median age at diagnosis of around 47 years, although the disease may occur at any age. [5][6][7] A genetic contribution to PAH has long been identified and 6% of patients with PAH have reported a family history of the condition [hereditary PAH (HPAH)]. 8,9 Heterozygous germline mutations in the bone morphogenetic protein type II receptor 2 gene (BMPR2) have been identified in 10 to 40% of patients with apparently sporadic IPAH [10][11][12][13] and in 58 to 74% of patients with HPAH. [14][15][16] BMPR2 is a member of the transforming growth factor-(TGF-) receptor superfamily; mutations in genes of several members of this family have been implicated in the onset of PAH, indicating that the pathways controlled by members of this family are important to the integrity of the pulmonary vasculature (Table 2). [17][18][19] Furthermore, the pore-forming subunit, Kv1.5, forms functional voltage-gated potassium channels (Kv) in the pulmonary artery smooth muscle cells and abnormal potassium channel Kv1.5 function encoded by KCNA5 mutations alone and associated with BMPR2 mutations have been reported in patients with IPAH. 20 In the present case report, we describe the clinical and molecular genetic assessment of a 38-year-old woman of Indian descent who was admitted to our hospital because of progressive dyspnea [New York Heart Association (NYHA) class III]. These assessments led to a diagnosis of PAH and the identification of a double heterozyogosity of the BMPR2 gene. The patient subsequently died of pulmonary hemorrhagic edema and cardiogenic shock after 6 days of intensive care. It can be speculated that the early onset, severity of progression and fatal outcome of the disease can be explained un the light of the two-hits hypothesis as reported by Wang et al. 20

Clinical assessments
Physical and cardiac examinations included chest X-ray, 12-lead electrocardiogram (ECG), echocardiography, computed axial tomography (CAT) (3D reconstruction) and laboratory tests. Transthoracic echocardiography examinations were performed using an iE33 xMATRIX echocardiography system equipped with 2.5-3.5 MHz probes (Phillips Healthcare, Surrey, UK) which provided a non-invasive assessment of pulmonary artery pressure and a useful means of monitoring the patients' condition.

Histology
Tissue samples collected during the postmortem examination were fixed in a 10% buffered formalin solution for approximately 48 h and subsequently embedded in paraffin tissue blocks using standard histopathology methods [formalin-fixed paraffin embedded (FFPE)]. Histological sections, 3 µm in thickness, were obtained from each block and stained with hematoxylin-eosin. Selected samples were also stained using the Weigert method for elastic fibers and immunohistochemically tested using anti-smooth-muscle actin, anti-CD3 and anti-CD20 primary antibodies in order to highlight the pulmonary artery tunica media or the potential inflammatory infiltrate.

Mutation analysis
After informed consent of both living parents, genomic DNA of the index case was extracted from FFPE by means of the Maxwell 16 TM platform (Promega). The BMPR2, SMAD, TGFBR1, TGFBR2, ACVR1, ACVRL1, BMP9/GDF2, KCNK3, KCNA5, CAV1, ENG and SMAD2 genes screening was performed on a PGM platform (IonTorrent, LifeTechnologies) equipped with a 318 chip (www.lifetechnologies.com); sequence variants have been subsequently confirmed by conventional forward and reverse Sanger Sequencing on the ABI PRISM 3130Xl.

Clinical findings
The patient had been treated for respiratory disease for almost 3 years but there was no history of syncope, palpitation, chest pain or hemoptysis. Cardiac examinations showed: i) the patient had a heart systolic murmur; ii) the 12-lead ECG revealed sinus tachycardia, right axis deviation and right ventricular hypertrophy with strain; iii) the chest X-ray revealed cardiomegaly and features of pulmonary hypertension and bilateral pleural effusion; iv) chest CAT (3D reconstruction) showed a pulmonary artery trunk diameter of 39 mm and a ground glass area without pulmonary thromboembolism. Of note, an enlarged right ventricle and smaller left ventricle were observed and the septum was pushed towards the left ventricle due to very high pressure inside the right ventricle ( Figure 1A and B).
Transthoracic echocardiogram revealed: i) right ventricular systolic pressure of 96 mm/Hg (the peak of gradient through a severe tricuspid regurgitation was 76+20 mmHg of right atrial pressure, by the dilatation without collapsing vena cava; Figure 1C and D); ii) dilatation of the right chambers (the right atrium had a diameter of 57 mm, a wall measurement of 10.5 mm and a pulmonary artery trunk measurement of up to 38 mm; Figure 1E).
Evidence for right ventricular dysfunction was shown by a low value of tricuspid annular post-systolic excursion (TAPSE 3: <10 mm, severely abnormal). The acceleration time of pulmonary outflow (ACTpo) was 40 ms. There was no evidence of left to right atrial shunt.
Laboratory investigations excluded infections and immunological diseases. Cardiac catheterization confirmed severe PAH: 115/15   Treatment for idiopathic pulmonary arterial hypertension

Review
Following a diagnosis of severe PAH, treatment was initiated with bosentan 125 mg/day, diuretic (furosemide 125 mg), warfarin and intravenous infusion of prostaglandin I2 (PGI2) infusion for 3 days and non-invasive ventilation therapy (using a continuous positive airway pressure mask). However, no benefit was seen following these treatments and the patient subsequently died of pulmonary hemorrhagic edema and cardiogenic shock after 6 days of intensive care.
In this case a massive pulmonary edema may be due to the very high values of pulmonary pressure that led to flooding of the pulmonary alveoli and by a respiratory distress syndrome favored by the presence of an acute bronchopneumonia.
A pulmonary veno-occlusive hypertension disease was excluded from high-resolution computed tomography and capillary pulmonary wedge pressure (PCWP) was below 15 mm/Hg.

Gross and microscopic appearances Lungs
At necropsy (Figure 2 A-D), both lungs (each 590 g) showed diffuse endoalveolar hemorrhage and marked changes in all of the pulmonary arterial branches especially in terms of wall thickening.

Heart
The heart (470 g) displayed significant right ventricular hypertrophy; the right atrial appendage had undergone extreme dilation to the extent that it was incorporated into the main right atrial chamber. Histopathology ( Figure 3A-D) revealed pulmonary arteriopathy which was characterized primarily by medial hypertrophy, arterialization of muscular arteries and muscularization of non-muscularized distal arteries. Focal intimal fibrosis and mild plaques were detectable throughout the lung parenchyma. There were neither thrombotic lesions nor signs of pulmonary veno-occlusive disease.

Molecular findings
Molecular investigations on the index case identified two BMPR2 mutations; the first was a missense mutation in exon #3 (p.Cys123Arg) and the second was a stop codon mutation in     It shares with other type I receptors a high degree of similarity in serine-threonine kinase subdomains, a glycine-and serine-rich region (called the GS domain) precedingthe kinase domain, and a short C-terminal tail. The encoded protein, sometimes termed ALK1, shares similar domain structures with other closely related ALK or activin receptor-like kinase proteins that form a subfamily of receptor serine/threonine kinases. Mutations in this gene are associated with hemorrhagic telangectasia type 2, also known as Rendu-Osler-Weber syndrome 2. 18q21 601366 SMAD2 AD* The protein encoded by this gene belongs to the SMAD, a family of proteins similar to the gene products of the Drosophila gene 'mothers against decapentaplegic' (Mad) and the C. elegans gene Sma. SMAD proteins are signal transducers and transcriptional modulators that mediate multiple signaling pathways. This protein mediates the signal of the transforming growth factor (TGF)-β, and thus regulates multiple cellular processes, such as cell proliferation, apoptosis, and differentiation. This protein is receptor activation (SARA) protein. In response to TGF-β signal, this protein is pho phorylated by the TGF-β receptors. The phosphorylation induces the dissociation of this protein with SARA and the association with the family member SMAD4. The assciation with SMAD4 is important for the translocation of this protein into the nucleus, where it binds to target promoters and forms a transcription repressor complex with other cofactors. This protein can also be phosphorylated by activin type 1 receptor kinase, and mediates the signal from the activin. Alternatively spliced transcript variants have been observed for this gene.  exon #8 (p.Arg332X) ( Figure 4). Both mutations have previously been associated with the IPAH phenotype (Table 3) 21,22 and were absent in 250 healthy controls (500 chromosomes); in silico analysis PolyPhen-2/Sift) confirmed the pathogenicity of the C123R mutation (data not shown). Additionally, the phase analysis by NGS revealed that both mutations were present on the same allele (cis-segregation; Figure 4).

Discussion
Heterozygous germline mutations in the BMPR2 gene have previously been implicated in the onset of PAH 8 where they have been identified in approximately 10 to 40% of patients with cases of IPAH previously thought to have arisen spontaneously 10,11 and in 58 to 74% of patients with HPAH. [14][15][16] In this case study of a 38-year-old woman with a history of respiratory disease who presented with severe dyspnea, we identified two mutations in the BMPR2 gene which were in cis configuration. After 6 days of intensive care the patient had a severe fatal hemodynamic compromise which we feel could be attributed to the presence of the double/compound mutation of the BMPR2 gene identified. We speculate that gene dosage could have affected the severity of the PAH phenotype, which has been previously reported for other inherited cardiovascular diseases. 23 Girolami et al. described the clinical outcome of four patients with triple mutations in genes coding for sarcomere proteins and concluded that the multiple mutations conferred an increased risk of end-stage progression and ventricular arrhythmia. 23 The major limitation of the present study is the family cascade screening: we could not have access either to the medical records and genetic material of parents and siblings. The presence of cis-mutations of the BMPR2 sug-gests that they might arise from one parental allele. However since both of them are living and self-reporting healthy, it can be speculated that one mutation is inherited and the second happened de novo in the germ line.
Carrying a single BMPR2 mutation per se does not imply developing PAH since heterozygous carriers of disease-causing BMPR2 mutations only have 20% chance of developing PAH over life times. 24 Thus multiple hits (including compound/double heterozygous mutations) are required to cause disease onset and progression in the BMPR2 mutations' carriers.
Taken together, these cardiac, genetic and pathological findings -although limited to the index case -provide further evidence of the role of the BMPR2 gene in PAH and its potential as a target for novel therapeutic interventions. In addition, the detection of these mutations may allow preclinical diagnosis of family members of the patient we observed who are currently asymptomatic but at risk of the onset of PAH disease symptoms.