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.     

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.     

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.     

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.     

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.     

氟西汀对BMPR2表达的影响以及对野百合碱诱导大鼠肺动脉高压的预防作用

目的探讨氟西汀对骨形态生成蛋白受体2(bone morphogenetic protein receptor 2,BMPR2)表达的影响以及对野百合碱(monocrotaline,MCT)诱导大鼠肺动脉高压(pulmonary arterial hypertension,PAH)的预防作用。方法将24只Wistar大鼠随机分成三组:对照组、MCT组和氟西汀处理组。采用多导生理记录仪测量血流动力学相关指标,HE染色方法观察肺动脉的形态学改变,以及利用RT-PCR方法检测肺动脉BMPR2的表达。结果与对照组相比,MCT组肺动脉压力、肺动脉中膜厚度百分比以及右心肥厚指数均明显升高,BMPR2在肺动脉上的表达明显减少(<0.01)。给予氟西汀处理后,氟西汀明显抑制了MCT诱发的肺动脉压力的升高、肺动脉重构和右心肥厚,并逆转了BMPR2的表达(<0.05)。结论肺动脉的构型重建可能与BMPR2的表达减少有关。氟西汀可能通过逆转BMPR2的表达有效地预防MCT诱导的PAH。     

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.     

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.     

内皮-间充质转化在肺动脉高压发病机制中的研究进展

正肺动脉高压(pulmonary hypertension,PH)是由多种已知和未知原因引起的肺循环血压异常升高的一种病理生理综合症,主要累及心血管及呼吸系统~([1-2])。根据2015年最新PH的临床分型,PH分为动脉型肺动脉高压(pulmonary arterial hypertension,PAH)、左心疾病所致PH、肺部疾病或缺氧所致PH、     

Dysfunctional intracellular trafficking in the pathobiology of pulmonary arterial hypertension

Discussions of the initiation of pulmonary arterial hypertension (PAH) in man and in experimental models have centered around intimal and medial proliferation in medium-sized pulmonary arteries. The histologic events are thought to include disordered proliferation of enlarged, vacuolated endothelial cells, neo-muscularization of the affected blood vessels, and vascular pruning. The discovery of the association of familial and sporadic PAH with mutations in BMPR2 has generated intense interest in cytokine receptor trafficking and function in the endothelial cell and how this might be disrupted to yield an enlarged proliferative cell phenotype. Nevertheless, considerations of the subcellular machinery of membrane trafficking in the endothelial cell and consequences of the disruption of this outward and inward membrane trafficking are largely absent from discussions of the pathobiology of PAH. Long-standing electron microscopy data in the PAH field has demonstrated marked disruptions of intracellular membrane trafficking in human and experimental PAH. Further, a role of the membrane-trafficking regulator Nef in simian HIV-induced PAH in macaques and in HIV-induced PAH in man is now evident. Additionally, monocrotaline and hypoxia are known to disrupt the function of Golgi tethers, SNAREs, SNAPs, and N-ethylmaleimide-sensitive factor ("the Golgi blockade hypothesis"). These results, along with recent reports demonstrating the trapping of PAH-associated human BMPR2 mutants in the Golgi, highlight the implications of disrupted intracellular membrane trafficking in the pathobiology of PAH. The purpose of this review is to present a brief overview of the molecular basis of intracellular trafficking and relate these considerations to the pathobiology of PAH.     

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.     

Defining the clinical validity of genes reported to cause pulmonary arterial hypertension

PurposePulmonary arterial hypertension (PAH) is a rare, progressive vasculopathy with significant cardiopulmonary morbidity and mortality. Genetic testing is currently recommended for adults diagnosed with heritable, idiopathic, anorexigen-, hereditary hemorrhagic telangiectasia–, and congenital heart disease–associated PAH, PAH with overt features of venous/capillary involvement, and all children diagnosed with PAH. Variants in at least 27 genes have putative evidence for PAH causality. Rigorous assessment of the evidence is needed to inform genetic testing.MethodsAn international panel of experts in PAH applied a semi-quantitative scoring system developed by the NIH Clinical Genome Resource to classify the relative strength of evidence supporting PAH gene-disease relationships based on genetic and experimental evidence.ResultsTwelve genes (BMPR2, ACVRL1, ATP13A3, CAV1, EIF2AK4, ENG, GDF2, KCNK3, KDR, SMAD9, SOX17, and TBX4) were classified as having definitive evidence and 3 genes (ABCC8, GGCX, and TET2) with moderate evidence. Six genes (AQP1, BMP10, FBLN2, KLF2, KLK1, and PDGFD) were classified as having limited evidence for causal effects of variants. TOPBP1 was classified as having no known PAH relationship. Five genes (BMPR1A, BMPR1B, NOTCH3, SMAD1, and SMAD4) were disputed because of a paucity of genetic evidence over time.ConclusionWe recommend that genetic testing includes all genes with definitive evidence and that caution be taken in the interpretation of variants identified in genes with moderate or limited evidence. Genes with no known evidence for PAH or disputed genes should not be included in genetic testing.     

Copy-number variation in BMPR2 is not associated with the pathogenesis of pulmonary arterial hypertension

Background  

Copy-number variations (CNVs) are structural variations in the genome involving 1 kb to 3 mb of DNA. CNV has been reported within intron 1 of the BMPR2 gene. We propose that CNV could affect phenotype in familial and/or sporadic pulmonary arterial hypertension (PAH) by altering gene expression.