BMPR2 gene rearrangements account for a significant proportion of mutations in familial and idiopathic pulmonary arterial hypertension

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

De Novo Mutations in the BMPR2 Gene in Patients with Heritable Pulmonary Arterial Hypertension

A substantial proportion of patients with pulmonary arterial hypertension (PAH) have mutations in the Bone Morphogenetic Protein Receptor type‐2 (BMPR2) gene. PAH due to BMPR2 mutations is inherited as an autosomal dominant trait with several unique features, including a wide variety of mutations, reduced penetrance, a skewed gender ratio, variable expressivity and genetic anticipation. To address the genetic background of these unique features of BMPR2 mutation, we conducted a systematic analysis of 15 PAH families with BMPR2 mutation. The exonic protein coding sequence of BMPR2 was amplified by polymerase chain reaction and the products were sequenced directly to detect point mutations in BMPR2. Parental identification was carried out to confirm the parental relationship using multiplex 15 loci analysis. Combining mutation detection in family members with parental identification, we described three cases of de novo mutation in the BMPR2 gene by different modes in a PAH family. These de novo mutations may account for the wide variety of mutations in BMPR2. Taken together with the juvenile onset of the disease, there is possibly some balance of de novo mutations and untransmittable mutations which keeps the frequency of PAH low in the general population.     

Connectivity map analysis of nonsense-mediated decay-positive BMPR2-related hereditary pulmonary arterial hypertension provides insights into disease penetrance

The molecular mechanisms underlying the reduced penetrance seen in the nonsense-mediated decay-positive (NMD+) BMPR2 mutation-associated hereditary pulmonary arterial hypertension (HPAH) remain unknown. We reasoned that the cellular and genetic mechanisms behind this phenomenon could be uncovered by combining expression profiling with Connectivity Map (cMap) analysis. Cultured lymphocytes from 10 patients with HPAH and 10 matched familial control subjects, all with NMD+ BMPR2 mutations, were subjected to expression analysis. For each group, the expression data were combined before analysis. This generated a signature of 23 up-regulated and 12 down-regulated genes in patients with HPAH compared with control subjects (the "PAH penetrance signature"). Although gene set enrichment analysis of this signature was not uniquely informative, cMap analysis identified drugs with expression signatures similar to the PAH penetrance signature. Several of these drugs were predicted to influence reactive oxygen species (ROS) formation. This hypothesis was tested and confirmed in the same cells initially subjected to the expression analysis using quantitative biochemical detection of ROS concentration. We conclude that expression of the PAH penetrance signature represents an increased risk of developing clinical HPAH and that ROS formation may play a role in pathogenesis of HPAH. These results provide the first molecular insights into NMD+ BMPR2 related HPAH penetrance and highlight the potential utility of cMap analyses in pulmonary research.     

Genetics in pulmonary arterial hypertension in a large homogeneous Japanese population

Pulmonary arterial hypertension (PAH) is a rare but serious disease with a grave prognosis. Bone morphogenetic protein type 2 receptor (BMPR2) gene is a strong pathogenic factor for PAH. As a collaborative team from Kyorin University and Keio University in Japan, we have analyzed the BMPR2 gene in 356 probands and more than 50 family members, including secondary patients. Importantly, the study population is a racially, ethnically, and socially homogeneous population. In PAH patients, there is a high incidence of unique mutations in BMPR2, and several mutations are frequently observed in the Japanese population, suggesting that these common and recurring mutations may be highly pathogenic or have high penetrance, explaining why they are found frequently throughout the world. We have also mapped each breakpoint of exonic deletions/duplications and found that most break and rejoining points are in the Alu elements. Reviewing the distribution of the reported mutations on each exon of BMPR2 revealed that the number and frequency of mutations are imbalanced among exons. The penetrance of BMPR2 gene mutations was 3‐fold higher in females than males. Full elucidation of BMPR2‐mediated pathogenic mechanisms in PAH requires persistent efforts to achieve precision or individualized medicine as a therapeutic strategy for PAH.     

BMPR2 mutation in a patient with pulmonary arterial hypertension and suspected hereditary hemorrhagic telangiectasia

Pulmonary arterial hypertension (PAH) and hereditary hemorrhagic telangiectasia (HHT) are distinct clinical entities caused by germline mutations in genes encoding members of the TGFbeta/BMP superfamily: BMPR2 in PAH and ACVRL1, ENG, or SMAD4 in HHT. When PAH and HHT occasionally co-exist within the same family, ACVRL1 mutations predominate. We report a 36-year-old woman initially diagnosed with PAH at age 24. At 35, following massive hemoptysis, multiple pulmonary arteriovenous malformations were discovered, prompting evaluation for HHT. She met the Cura?ao diagnostic criteria for suspected HHT based on additional findings of nasal telangiectases and epistaxis. Mutation analysis of ACVRL1, ENG, and SMAD4 was normal, but a germline nonsense mutation in BMPR2 was identified. This is the first known report of HHT features, particularly pulmonary AVMs, associated with a BMPR2 mutation. It adds further weight to a common molecular pathogenesis in PAH and HHT, and highlights that BMPR2 gene analysis is indicated in patients affected with both HHT and PAH.     

Autophagy contributes to BMP type 2 receptor degradation and development of pulmonary arterial hypertension

Pulmonary arterial hypertension (PAH) is characterised by an increase in mean pulmonary arterial pressure which almost invariably leads to right heart failure and premature death. More than 70% of familial PAH and 20% of idiopathic PAH patients carry heterozygous mutations in the bone morphogenetic protein (BMP) type 2 receptor (BMPR2). However, the incomplete penetrance of BMPR2 mutations suggests that other genetic and environmental factors contribute to the disease. In the current study, we investigate the contribution of autophagy in the degradation of BMPR2 in pulmonary vascular cells. We demonstrate that endogenous BMPR2 is degraded through the lysosome in primary human pulmonary artery endothelial (PAECs) and smooth muscle cells (PASMCs): two cell types that play a key role in the pathology of the disease. By means of an elegant HaloTag system, we show that a block in lysosomal degradation leads to increased levels of BMPR2 at the plasma membrane. In addition, pharmacological or genetic manipulations of autophagy allow us to conclude that autophagy activation contributes to BMPR2 degradation. It has to be further investigated whether the role of autophagy in the degradation of BMPR2 is direct or through the modulation of the endocytic pathway. Interestingly, using an iPSC-derived endothelial cell model, our findings indicate that BMPR2 heterozygosity alone is sufficient to cause an increased autophagic flux. Besides BMPR2 heterozygosity, pro-inflammatory cytokines also contribute to an augmented autophagy in lung vascular cells. Furthermore, we demonstrate an increase in microtubule-associated protein 1 light chain 3 beta (MAP1LC3B) levels in lung sections from PAH induced in rats. Accordingly, pulmonary microvascular endothelial cells (MVECs) from end-stage idiopathic PAH patients present an elevated autophagic flux. Our findings support a model in which an increased autophagic flux in PAH patients contributes to a greater decrease in BMPR2 levels. Altogether, this study sheds light on the basic mechanisms of BMPR2 degradation and highlights a crucial role for autophagy in PAH. © 2019 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.     

Pulmonary Arterial Hypertension: A Current Perspective on Established and Emerging Molecular Genetic Defects

Pulmonary arterial hypertension (PAH) is an often fatal disorder resulting from several causes including heterogeneous genetic defects. While mutations in the bone morphogenetic protein receptor type II (BMPR2) gene are the single most common causal factor for hereditary cases, pathogenic mutations have been observed in approximately 25% of idiopathic PAH patients without a prior family history of disease. Additional defects of the transforming growth factor beta pathway have been implicated in disease pathogenesis. Specifically, studies have confirmed activin A receptor type II‐like 1 (ACVRL1), endoglin (ENG), and members of the SMAD family as contributing to PAH both with and without associated clinical phenotypes. Most recently, next‐generation sequencing has identified novel, rare genetic variation implicated in the PAH disease spectrum. Of importance, several identified genetic factors converge on related pathways and provide significant insight into the development, maintenance, and pathogenetic transformation of the pulmonary vascular bed. Together, these analyses represent the largest comprehensive compilation of BMPR2 and associated genetic risk factors for PAH, comprising known and novel variation. Additionally, with the inclusion of an allelic series of locus‐specific variation in BMPR2, these data provide a key resource in data interpretation and development of contemporary therapeutic and diagnostic tools.     

An homozygous mutation in KCNK3 is associated with an aggressive form of hereditary pulmonary arterial hypertension

Pulmonary arterial hypertension (PAH) is a rare devastating disease characterized by a high genetic heterogeneity with several related genes recently described, including BMPR2,TBX4 and KCNK3. The association between KCNK3 and PAH has been recently identified, but the prognosis and phenotype associated with these mutations have been poorly described. We studied a series of 136 idiopathic and hereditary PAH Spanish patients for BMPR2, TBX4 and KCNK3 mutations. We report the results of KCNK3 in which we were able to describe two new mutations (p.Gly106Arg and p.Leu214Arg) in three patients. The first one was found in a patient belonging to a consanguineous Romani family, who carried a homozygous mutation in KCNK3 and developed a severe and early form of the disease. To the best of our knowledge, this is the first time that a homozygous mutation in KCNK3 is reported in a PAH patient. The second one was found in a patient who presented at the young adult age a severe form of the disease. The present report supports the contribution of KCNK3 mutations to the genetic etiology of PAH and strongly suggests that mutations in KCNK3 follow incomplete dominance with worsening of the clinical features in homozygous patients.     

Synergistic heterozygosity for TGFβ1 SNPs and BMPR2 mutations modulates the age at diagnosis and penetrance of familial pulmonary arterial hypertension

PurposeWe hypothesized that functional TGFβ1 SNPs increase TGFβ/BMP signaling imbalance in BMPR2 mutation heterozygotes to accelerate the age at diagnosis, increase the penetrance and SMAD2 expression in familial pulmonary arterial hypertension.MethodsSingle nucleotide polymorphism genotypes of BMPR2 mutation heterozygotes, age at diagnosis, and penetrance of familial pulmonary arterial hypertension were compared and SMAD2 expression was studied in lung sections.ResultsBMPR2 mutation heterozygotes with least active -509 or codon 10 TGFβ1 SNPs had later mean age at diagnosis of familial pulmonary arterial hypertension (39.5 and 43.2 years) than those with more active genotypes (31.6 and 33.1 years, P = 0.03 and 0.02, respectively). Kaplan-Meier analysis also showed that those with the less active single nucleotide polymorphisms had later age at diagnosis. BMPR2 mutation heterozygotes with nonsense-mediated decay resistant BMPR2 mutations and the least, intermediate and most active -509 TGFβ1 SNP genotypes had penetrances of 33, 72, and 80%, respectively (P = 0.003), whereas those with 0–1, 2, or 3–4 active single nucleotide polymorphism alleles had penetrances of 33, 72, and 75% (P = 0.005). The relative expression of TGFβ1 dependent SMAD2 was increased in lung sections of those with familial pulmonary arterial hypertension compared with controls.ConclusionsThe TGFβ1 SNPs studied modulate age at diagnosis and penetrance of familial pulmonary arterial hypertension in BMPR2 mutation heterozygotes, likely by affecting TGFβ/BMP signaling imbalance. This modulation is an example of Synergistic Heterozygosity.     

BMPR2 mutations have short lifetime expectancy in primary pulmonary hypertension

In a nationwide study, we identified a total of 59 patients diagnosed with primary pulmonary hypertension (PPH) in Finland between the years 1987 and 1999. These data support a minimum estimate for a PPH population prevalence of 5.8 cases/million with an incidence of 0.2-1.3 cases/million/year. The male-to-female ratio among the patients was 1:4, while 7% (4/59) of the PPH probands had a known family history of the disorder. Familial or sporadic PPH showed no geographic clustering to any region of Finland. Sequencing of the coding regions and exon-intron boundaries of the bone morphogenetic protein receptor type 2 (BMPR2) identified heterozygous BMPR2 mutations in 12% (3/26) of the sporadic and 33% (1/3) of the familial patients. All four mutations were different, and two of those have been previously reported in other populations. Pathogenic defects in BMPR2 include a novel missense mutation (c.2696G>C encoding R899P), located within the receptor intracellular cytoplasmic domain whose function has been poorly characterized. Our analysis demonstrates that this mutant, while localizing to the cell surface, does not impact on SMAD-mediated (mothers against decapentaplegic homolog) intracellular signaling, but leads to constitutive activation of the p38(MAPK) pathway. The absence of a founder mutation in a genetically homogeneous population, such as the Finns, suggests that all identified BMPR2 mutations have to be rather young while the ancestral (if any) mutations have been lost either due to repetitive genetic bottlenecks or due to significant negative selection. Hum Mutat 26(2), 1-6, 2005. (c) 2005 Wiley-Liss, Inc.     

Somatic frameshift mutations of bone morphogenic protein receptor 2 gene in gastric and colorectal cancers with microsatellite instability

Mounting evidence exists that perturbation of bone morphogenic protein (BMP) signaling is involved in cancer development, especially in gastrointestinal cancers. However, somatic mutations of the genes encoding BMP and BMP receptors have not yet been discovered in human cancer tissues. By analyzing a public database, we found that BMP receptor 2 (BMPR2) and BMP1 genes had mononucleotide repeats in their coding sequences that could be mutation targets in cancers with microsatellite instability (MSI). In this study, we analyzed the mutation of BMPR2 and BMP1 genes in gastric (GC) and colorectal cancers (CRC) with MSI [31 GC with high MSI (MSI-H), 13 GC with low MSI (MSI-L), 38 CRC with MSI-H and 15 CRC with MSI-L] by single-strand conformation polymorphism analysis and DNA sequencing. Overall, we found seven frameshift mutations in the BMPR2 gene, but not in the BMP1 gene. The mutations were an identical deletion mutation of one base in the repeats (c.1748delA) that would result in premature stops of the amino acid synthesis (p.Asn583ThrfsX44). The BMPR2 mutations were detected in 6.5% of GC and 13.2% of CRC with MSI-H. All the cancers with the BMPR2 mutation showed loss of BMPR2 expression. Our data indicate that frameshift mutation of BMPR2 gene occurs in GC and CRC with MSI-H, and suggest that the BMPR2 mutation might contribute to cancer pathogenesis by inactivating BMPR2-mediated BMP signaling.     

A novel R486Q mutation in BMPR1B resulting in either a brachydactyly type C/symphalangism-like phenotype or brachydactyly type A2

Heterozygous missense mutations in the serine-threonine kinase receptor BMPR1B result typically in brachydactyly type A2 (BDA2), whereas mutations in the corresponding ligand GDF5 cause brachydactyly type C (BDC). Mutations in the GDF inhibitor Noggin (NOG) or activating mutations in GDF5 cause proximal symphalangism (SYM1). Here, we describe a novel mutation in BMPR1B (R486Q) that is associated with either BDA2 or a BDC/SYM1-like phenotype. Functional investigations of the R486Q mutation were performed and compared with the previously reported BDA2-causing mutation R486W and WT BMPR1B. Overexpression of the mutant receptors in chicken micromass cultures resulted in a strong inhibition of chondrogenesis with the R486Q mutant, showing a stronger effect than the R486W mutant. To investigate the consequences of the BMPR1B mutations on the intracellular signal transduction, we used stably transfected C2C12 cells and measured the activity of SMAD-dependent and SMAD-independent pathways. SMAD activation after stimulation with GDF5 was suppressed in both mutants. Alkaline phosphatase induction showed an almost complete loss of activation by both mutants. Our data extend the previously known mutational and phenotypic spectrum associated with mutations in BMPR1B. Disturbances of NOG-GDF5-BMPR1B signaling cascade can result in similar clinical manifestations depending on the quantitative effect and mode of action of the specific mutations within the same functional pathway.     

Stoichiometric imbalance in the receptor complex contributes to dysfunctional BMPR-II mediated signalling in pulmonary arterial hypertension

Heterozygous germline defects in a gene encoding a type II receptor for bone morphogenetic proteins (BMPR-II) underlie the majority of inherited cases of the vascular disorder known as pulmonary arterial hypertension (PAH). However, the precise molecular consequences of PAH causing mutations on the function of the receptor complex remain unclear. We employed novel enzymatic and fluorescence activity based techniques to assess the impact of PAH mutations on pre-mRNA splicing, nonsense-mediated decay (NMD) and receptor complex interactions. We demonstrate that nonsense and frameshift mutations trigger NMD, providing further evidence that haplo-insufficiency is a major molecular consequence of disease-related BMPR2 mutations. We identified heterogeneous functional defects in BMPR-II activity, including impaired type I receptor phosphorylation, receptor interactions and altered receptor complex stoichiometry leading to perturbation of downstream signalling pathways. Importantly, these studies demonstrate that the intracellular domain of BMPR-II is both necessary and sufficient for receptor complex interaction. Finally and to address the potential for resolution of stoichiometric balance, we investigated an agent that promotes translational readthrough of a BMPR2 nonsense reporter construct without interfering with the NMD pathway. We propose that stoichiometric imbalance, due to either haplo-insufficiency or loss of optimal receptor-receptor interactions impairs BMPR-II mediated signalling in PAH. Taken together, these studies have identified an important target for early therapeutic intervention in familial PAH.     

Bone morphogenetic protein receptor type II C-terminus interacts with c-Src: implication for a role in pulmonary arterial hypertension

Mutations of bone morphogenetic protein receptor type II (BMPR-II) have been associated with familial and idiopathic pulmonary arterial hypertension (PAH). BMPR-II is a member of the transforming growth factor-beta receptor superfamily. It consists of extracellular, transmembrane, and kinase domains, and a unique C-terminus with mostly unknown function. However, a number of PAH-causing mutations are predicted to truncate the C-terminus, suggesting that this domain plays an important role in the homeostasis of pulmonary vessels. In this study, we sought to elucidate the functional role of this C-terminus by seeking its interacting partners. Using yeast two-hybrid screening, we identified c-Src tyrosine kinase as a binding partner of this C-terminus. In vitro co-immunoprecipitation confirmed their interaction. Mutations truncating the C-terminus disrupted their interaction, while missense mutation within kinase domain reduced their interaction. In addition, BMPR-II and c-Src tyrosine kinase colocalized within intracellular aggregates when overexpressed in HEK293 cells. Moreover, mutations truncating the C-terminus disrupted their colocalization, whereas missense mutation within kinase domain had no effect on their colocalization. Furthermore, BMP ligand stimulation decreased c-Src-activating phosphorylation at Tyrosine 418 in pulmonary smooth muscle cells in both time- and concentration-dependent manners. Mutations that truncated the C-terminus abolished this response. Taken together, these results suggest a model in which proliferative effect of c-Src by vasoactive molecules is balanced by opposing effect of BMP signaling in basal state, and the loss of this balance due to BMPR2 mutations leads to increased c-Src activity and subsequently cell growth.