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Hereditary pulmonary veno-occlusive disease (hPVOD) is a severe form of autosomal recessive pulmonary hypertension and is due to biallelic loss of function of the EIF2AK4 gene (alias GCN2) coding for GCN2. GCN2 is a stress kinase that belongs to the integrated stress response pathway (ISR). Three rat lines carrying biallelic Gcn2 mutation were generated and found phenotypically normal and did not spontaneously develop a PVOD-related disease. We submitted these rats to amino acid deprivation to document the molecular and cellular response of the lungs and to identify phenotypic changes that could be involved in PVOD pathophysiology. Gcn2−/− rat lungs were analyzed under basal conditions and 3 days after a single administration of PEG-asparaginase (ASNase). Lung mRNAs were analyzed by RNAseq and single-cell RNAseq (scRNA-seq), flow cytometry, tissue imaging, and Western blots. The ISR was not activated after ASNase treatment in Gcn2−/− rat lungs, and apoptosis was increased. Several proinflammatory and innate immunity genes were overexpressed, and inflammatory cells infiltration was also observed in the perivascular area. Under basal conditions, scRNA-seq analysis of Gcn2−/− rat lungs revealed increases in two T-cell populations, a LAG3+ T-cell population and a proliferative T-cell population. Following ASNase administration, we observed an increase in calprotectin expression involved in TLR pathway activation and neutrophil infiltration. In conclusion, under basal and asparagine and glutamine deprivation induced by asparaginase administration, Gcn2−/− rats display molecular and cellular signatures in the lungs that may indicate a role for Gcn2 in immune homeostasis and provide further clues to the mechanisms of hPVOD development.

Hereditary Hemorrhagic Telangiectasia (HHT) is a rare vascular disorder causing abnormal vessel formation and characterized by autosomal dominant transmission. The associated considerable variability in symptoms and clinical severity complicate the management of the disease. In clinical routine, 3 main genes,ACVRL1(also known asALK1),ENGandSMAD4are screened for pathogenic variants at the origin of HHT. About 80% of HHT cases are caused by pathogenic coding variants inACVRL1andENG. However, at least 15% remain with no molecular explanations in the 3 main genes. We here report the identification of 2 never reported variants, c.-79C>T and c.-68G>A, in the 5’UTR ofENGin 2 unrelated HHT patients. These 2 variants are predicted to create upstream AUGs (uAUGs) in the 5’UTR, which are in frame with a stop codon located within the CoDing Sequence (CDS), thus generating Overlapping upstream Open reading frames (uoORFs). In other cases, uAUGs can lead to fully upstream ORFs (uORFs) when associated with stop codons located within the 5’UTR or to elongated CDS (eCDS) when in frame with the CDS and associated with the main stop codon.In order to assess the pathogenicity of theseENGvariants, we performedin vitrofunctional assays based on the expression of wild-type and mutant constructs in human cells. We found that these 5’UTRENGvariants were associated with a decrease of protein levels in HeLa and HUVEC cells. They were also associated with a decreased ability to activate BMP9-stimulated ALK1 receptor in a BMP-response element (BRE) assay. This assay is a mandatory element before providing a definitive molecular diagnosis and has been so far applied only on codingENGvariants. We applied the same experimental workflow on 3 additional uoORF-creating variants (c.-142A>T, c.-127C>T and c.-10C>T) and one eCDS-creating variant (c.-9G>A) in the 5’UTR ofENGpreviously reported in HHT patients. We found that all the analysed uoORF-creating variants alter endoglin levels and function. A comparison of our experimental results with patients’ clinical characteristics suggests that uoORF-creating variants leading to ENG protein levels ≤ 40%in vitrowould be associated with HHT. Additional experiments relying on an artificial deletion in our mutated constructs show that created uAUGs predicted to create uoORFs are able to initiate the translation indicating that the associated effect is likely caused by an alteration of the translation mechanism.Overall, we here identified two never reported 5’UTRENGvariations in HHT patients and shed new lights on the role of upstream ORFs on ENG regulation. Our findings contribute to the amelioration of molecular diagnosis in HHT.

The phenotype of pulmonary arterial hypertension (PAH) patients carryingWe report the genetic analysis findings, characteristics and outcomes of patients with heritable PAH carryingTwenty patients and eight unaffected relatives were identified. The median (min-max) age at diagnosis was 17 years (2-53), with a female-to-male ratio of 1.5. At diagnosis, most of the patients (74%) were in functional class III or IV with severe hemodynamic compromise, including a median pulmonary vascular resistance (PVR) of 14.0 (4.2-31.5) Wood units (WU). An associated congenital heart disease (CHD) was found in 7 PAH patients (35%). Patients with CHD-associated PAH were significantly younger at diagnosis than PAH patients without CHD. Four patients (20%) suffered from recurrent haemoptysis requiring repeated arterial embolisations. Thirteen out of 16 patients (81%) of whom imaging was available displayed chest computed tomography abnormalities, including dilated, tortuous pulmonary vessels, ground-glass opacities as well as bronchial and non-bronchial arteries anomalies. After a median follow-up of 47 months (1-591 months), 10 patients underwent lung transplantation and one patient benefited from a heart-lung transplantation due to associated CHD. Histopathologic analysis of lung explants showed a congested lung architecture with severe pulmonary arterial remodelling, subpleural vessel dilation and numerous haemorrhagic foci.PAH due to

BackgroundInflammation and dysregulated immunity are important in the development of pulmonary arterial hypertension (PAH). Compelling preclinical data supports the therapeutic blockade of interleukin-6 (IL-6) signalling.MethodsWe conducted a phase 2 open-label study of intravenous tocilizumab (8 mg·kg−1) over 6 months in patients with group 1 PAH. Co-primary end-points were safety, defined by incidence and severity of adverse events, and change in pulmonary vascular resistance. Separately, a mendelian randomisation study was undertaken on 11 744 individuals with European ancestry including 2085 patients with idiopathic/heritable disease for the IL-6 receptor (IL6R) variant (rs7529229), known to associate with circulating IL-6R levels.ResultsWe recruited 29 patients (male/female 10/19; mean±sd age 54.9±11.4 years). Of these, 19 had heritable/idiopathic PAH and 10 had connective tissue disease-associated PAH. Six were withdrawn prior to drug administration; 23 patients received at least one dose of tocilizumab. Tocilizumab was discontinued in four patients owing to serious adverse events. There were no deaths. Despite evidence of target engagement in plasma IL-6 and C-reactive protein levels, both intention-to-treat and modified intention-to-treat analyses demonstrated no change in pulmonary vascular resistance. Inflammatory markers did not predict treatment response. Mendelian randomisation did not support an effect of the lead IL6R variant on risk of PAH (OR 0.99, p=0.88).ConclusionAdverse events were consistent with the known safety profile of tocilizumab. Tocilizumab did not show any consistent treatment effect.

Pulmonary fibrosis (PF) and pulmonary hypertension (PH) are chronic diseases of the pulmonary parenchyma and circulation, respectively, which may coexist, but underlying mechanisms remain elusive. Mutations in the GCN2 gene (EIF2AK4) were recently associated with pulmonary veno-occlusive disease. This study aims to explore the involvement of the GCN2/eIF2α pathway in the development of PH during PF, in both human disease and in an experimental animal model. Lung tissue from PF patients with or without PH were collected at the time of lung transplantation, and controls were obtained from tumor resection surgery. Experimental lung disease was induced in either male wild-type or EIF2AK4-mutated Sprague-Dawley rats, randomly receiving a single intratracheal instillation of bleomycin or saline. Hemodynamic studies, as well as organ collection were performed 3 weeks post-instillation. Only significant results (p

To study the genotypes and phenotypes of cerebral arteriovenous fistulas that drain or do not drain through the vein of Galen, and true vein of Galen aneurysmal malformations, in order to determine whether genotyping could help improve classification of these malformations and their management. We carried out a retrospective review of genetic and phenotypic data in databases of four centers. All children with cerebral arteriovenous fistula or vein of Galen aneurysmal malformations aged below 18 years at onset were included. We recorded the nature of the genetic variant or absence of variant, age at onset, type of malformation, symptoms at onset (hemorrhage, neurological deficit, hydrocephalus, incidental, and heart failure), type of venous drainage and the long-term outcome. One hundred and fifteen children were included. Autosomal dominant variants were identified in 39% of patients. The most frequent variant affected was the gene (25%) followed by (8%) and the HHT-associated genes (5%). HHT gene variants were only observed in pial arteriovenous fistula not draining into the vein of Galen; on the contrary, variants were only seen in genuine vein of Galen aneurysmal malformation. variants were identified in all types of shunts. variants seem specific to the vein of Galen aneurysmal malformation, variants are associated with either pial arteriovenous fistulas or with genuine VGAM and HHT gene variants seem specific to pial arteriovenous fistulas. The genetic data helps to classify these malformations and to guide treatment toward lowest risk of post-operative cerebral ischemic-hemorrhagic complications.

Background Diagnostic ionizing radiation is a risk factor for breast cancer (BC). BC risk increases with increased dose to the chest and decreases with increased age at exposure, with possible effect modification related to familial or genetic predisposition. While chest X-rays increase the BC risk of BRCA1/2 mutation carriers compared to non-carriers, little is known for women with a hereditary predisposition to BC but who tested negative for a BRCA1 or BRCA2 (BRCA1/2) mutation. Methods We evaluated the effect of chest X-rays from diagnostic medical procedures in a dataset composed of 1552 BC cases identified through French family cancer clinics and 1363 unrelated controls. Participants reported their history of X-ray exposures in a detailed questionnaire and were tested for 113 DNA repair genes. Logistic regression and multinomial logistic regression models were used to assess the association with BC. Results Chest X-ray exposure doubled BC risk. A 3% increased BC risk per additional exposure was observed. Being 20 years old or younger at first exposure or being exposed before first full-term pregnancy did not seem to modify this risk. Birth after 1960 or carrying a rare likely deleterious coding variant in a DNA repair gene other than BRCA1/2 modified the effect of chest X-ray exposure. Conclusion Ever/never chest X-ray exposure increases BC risk 2-fold regardless of age at first exposure and, by up to 5-fold when carrying 3 or more rare variants in a DNA repair gene. Further studies are needed to evaluate other DNA repair genes or variants to identify those which could modify radiation sensitivity. Identification of subpopulations that are more or less susceptible to ionizing radiation is important and potentially clinically relevant.

Background Heritable pulmonary arterial hypertension (PAH) is most commonly due to heterozygous mutations of the BMPR2 gene. Based on expert consensus, guidelines recommend annual screening echocardiography in asymptomatic BMPR2 mutation carriers. The main objectives of this study were to evaluate the characteristics of asymptomatic BMPR2 mutation carriers, assess their risk of occurrence of PAH and detect PAH at an early stage in this high-risk population. Methods Asymptomatic BMPR2 mutation carriers underwent screening at baseline and annually for a minimum of 2 years (DELPHI-2 study; ClinicalTrials.gov: NCT01600898). Annual screening included clinical assessment, ECG, pulmonary function tests, 6-min walk distance, cardiopulmonary exercise testing, chest radiography, echocardiography and brain natriuretic peptide (BNP) or N-terminal (NT)-proBNP level. Right heart catheterisation (RHC) was performed based on predefined criteria. An optional RHC at rest and exercise was proposed at baseline. Results 55 subjects (26 males; median age 37 years) were included. At baseline, no PAH was suspected based on echocardiography and NT-proBNP levels. All subjects accepted RHC at inclusion, which identified two mild PAH cases (3.6%) and 12 subjects with exercise pulmonary hypertension (21.8%). At long-term follow-up (118.8 patient-years of follow-up), three additional cases were diagnosed, yielding a PAH incidence of 2.3% per year (0.99% per year in males and 3.5% per year in females). All PAH cases remained at low-risk status on oral therapy at last follow-up. Conclusions Asymptomatic BMPR2 mutation carriers have a significant risk of developing incident PAH. International multicentre studies are needed to confirm that refined multimodal screening programmes with regular follow-up allow early detection of PAH., Asymptomatic BMPR2 mutation carriers have a 2.3% per year risk of developing PAH. DELPHI-2 provides the platform for future international multicentre studies to refine multimodal screening algorithms in BMPR2 mutation carriers. http://bit.ly/3oi2KJ1

Contains fulltext : 237895.pdf (Publisher’s version ) (Open Access) BACKGROUND: Ovarian cancer risk in BRCA1 and BRCA2 mutation carriers has been shown to decrease with longer duration of oral contraceptive use. Although the effects of using oral contraceptives in the general population are well established (approximately 50% risk reduction in ovarian cancer), the estimated risk reduction in mutation carriers is much less precise because of potential bias and small sample sizes. In addition, only a few studies on oral contraceptive use have examined the associations of duration of use, time since last use, starting age, and calendar year of start with risk of ovarian cancer. OBJECTIVE: This study aimed to investigate in more detail the associations of various characteristics of oral contraceptive use and risk of ovarian cancer, to provide healthcare providers and carriers with better risk estimates. STUDY DESIGN: In this international retrospective study, ovarian cancer risk associations were assessed using oral contraceptives data on 3989 BRCA1 and 2445 BRCA2 mutation carriers. Age-dependent-weighted Cox regression analyses were stratified by study and birth cohort and included breast cancer diagnosis as a covariate. To minimize survival bias, analyses were left truncated at 5 years before baseline questionnaire. Separate analyses were conducted for each aspect of oral contraceptive use and in a multivariate analysis, including all these aspects. In addition, the analysis of duration of oral contraceptive use was stratified by recency of use. RESULTS: Oral contraceptives were less often used by mutation carriers who were diagnosed with ovarian cancer (ever use: 58.6% for BRCA1 and 53.5% BRCA2) than by unaffected carriers (ever use: 88.9% for BRCA1 and 80.7% for BRCA2). The median duration of use was 7 years for both BRCA1 and BRCA2 carriers who developed ovarian cancer and 9 and 8 years for unaffected BRCA1 and BRCA2 carriers with ovarian cancer, respectively. For BRCA1 mutation carriers, univariate analyses have shown that both a longer duration of oral contraceptive use and more recent oral contraceptive use were associated with a reduction in the risk of ovarian cancer. However, in multivariate analyses, including duration of use, age at first use, and time since last use, duration of oral contraceptive use proved to be the prominent protective factor (compared with 10 years [hazard ratio, 0.37; 95% confidence interval, 0.19-0.73]; P(trend)=.008). The inverse association between duration of use and ovarian cancer risk persisted for more than 15 years (duration of ≥10 years; BRCA1 15 years since last use [hazard ratio, 0.56; 95% confidence interval, 0.18-0.59]). Univariate results for BRCA2 mutation carriers were similar but were inconclusive because of limited sample size. CONCLUSION: For BRCA1 mutation carriers, longer duration of oral contraceptive use is associated with a greater reduction in ovarian cancer risk, and the protection is long term.

Background We report two cases of RASA1-related capillary malformation-arteriovenous malformation (CM-AVM1) syndrome mimicking hereditary haemorrhagic telangiectasia (HHT).Methods and results A 28-year-old man, previously embolised for cerebral arteriovenous malformations (AVMs), presented with epistaxis and typical nasal telangiectasias of HHT. CT scan revealed a large portocaval shunt. The second patient was a 9-year-old girl presenting with cyanosis and several mucocutaneous telangiectasias, similar to those observed in typical cases of HHT. CT scan revealed a huge and complex pulmonary AVM of the right lower lobe and a hepatic AVM within the left lobe. HHT diagnosis was considered possible according to the Curaçao criteria for the two patients, with at least two criteria for each. Genetic tests did not find any mutation in the three classic genes (Endoglin, Activin receptor-like kinase 1 or Mothers against decapentaplegic homolog 4), but identified in both cases an RASA1 mutation, known to cause CM-AVM1 syndrome.Conclusions Pulmonary AVM and portocaval shunt, usually encountered in HHT, have not yet been described in the CM-AVM1 syndrome. RASA1 screening may be considered in case of HHT suspicion, particularly when mutations are not found in the usually affected genes.

BACKGROUND: Pulmonary arterial hypertension (PAH) is a lethal vasculopathy. Hereditary cases are associated with germline mutations in BMPR2 and 16 other genes. However, these mutations occur in under 25% of idiopathic PAH patients (IPAH) and are rare in PAH associated with connective tissue diseases (APAH). Preclinical studies suggest epigenetic dysregulation, including altered DNA methylation, promotes PAH. Somatic mutations of Tet-methylcytosine-dioxygenase-2 (TET2), a key enzyme in DNA demethylation, occur in cardiovascular disease and are associated with clonal hematopoiesis, inflammation and adverse vascular remodeling. The role of TET2 in PAH is unknown. METHODS: To test for a role of TET2, we utilized a cohort of 2572 cases from the PAH Biobank. Within this cohort, gene-specific rare variant association tests were performed using 1832 unrelated European PAH patients and 7509 non-Finnish European gnomAD subjects as controls. In an independent cohort of 140 patients, we quantified TET2 expression in peripheral blood mononuclear cells. To assess causality, we investigated hemodynamic and histologic evidence of PAH in hematopoietic Tet2-knockout mice. RESULTS: We observed an increased burden of rare, predicted deleterious, germline variants in TET2 in PAH patients of European ancestry (9/1832) compared to controls (6/7509; relative risk=6, p=0.00067). Assessing the whole cohort, 0.39% (10/2572) of patients had 12 TET2 mutations (75% predicted germline and 25% somatic). These patients had no mutations in other PAH-related genes. Patients with TET2 mutations were older (71±7 years versus 48±19 years, p86% of PAH patients. Tet2-knockout mice spontaneously developed PAH, adverse pulmonary vascular remodeling and inflammation, with elevated levels of cytokines, including IL-1β. Chronic therapy with an antibody targeting IL-1β blockade regressed PAH. CONCLUSIONS: PAH is the first human disease related to potential TET2 germline mutations. Inherited and acquired abnormalities of TET2 occur in 0.39% of PAH cases. Decreased TET2 expression is ubiquitous and has potential as a PAH biomarker.

Supplemental Digital Content is available in the text., Background: Approximately 25% of patients with pulmonary arterial hypertension (PAH) have been found to harbor rare mutations in disease-causing genes. To identify missing heritability in PAH, we integrated deep phenotyping with whole-genome sequencing data using Bayesian statistics. Methods: We analyzed 13 037 participants enrolled in the NBR study (NIHR BioResource—Rare Diseases), of which 1148 were recruited to the PAH domain. To test for genetic associations between genes and selected phenotypes of pulmonary hypertension, we used the Bayesian rare variant association method BeviMed. Results: Heterozygous, high impact, likely loss-of-function variants in the kinase insert domain receptor (KDR) gene were strongly associated with significantly reduced transfer coefficient for carbon monoxide (posterior probability=0.989) and older age at diagnosis (posterior probability=0.912). We also provide evidence for familial segregation of a rare nonsense KDR variant with these phenotypes. On computed tomographic imaging of the lungs, a range of parenchymal abnormalities were observed in the 5 patients harboring these predicted deleterious variants in KDR. Four additional PAH cases with rare likely loss-of-function variants in KDR were independently identified in the US PAH Biobank cohort with similar phenotypic characteristics. Conclusions: The Bayesian inference approach allowed us to independently validate KDR, which encodes for the VEGFR2 (vascular endothelial growth factor receptor 2), as a novel PAH candidate gene. Furthermore, this approach specifically associated high impact likely loss-of-function variants in the genetically constrained gene with distinct phenotypes. These findings provide evidence for KDR being a clinically actionable PAH gene and further support the central role of the vascular endothelium in the pathobiology of PAH.

Rationale: The cellular and molecular landscape and translational value of commonly used models of pulmonary arterial hypertension (PAH) are poorly understood. Single-cell transcriptomics can enhance molecular understanding of preclinical models and facilitate their rational use and interpretation.Objectives: To determine and prioritize dysregulated genes, pathways, and cell types in lungs of PAH rat models to assess relevance to human PAH and identify drug repositioning candidates.Methods: Single-cell RNA sequencing was performed on the lungs of monocrotaline (MCT), Sugen-hypoxia (SuHx), and control rats to identify altered genes and cell types, followed by validation using flow-sorted cells, RNA in situ hybridization, and immunofluorescence. Relevance to human PAH was assessed by histology of lungs from patients and via integration with human PAH genetic loci and known disease genes. Candidate drugs were predicted using Connectivity Map.Measurements and Main Results: Distinct changes in genes and pathways in numerous cell types were identified in SuHx and MCT lungs. Widespread upregulation of NF-κB signaling and downregulation of IFN signaling was observed across cell types. SuHx nonclassical monocytes and MCT conventional dendritic cells showed particularly strong NF-κB pathway activation. Genes altered in SuHx nonclassical monocytes were significantly enriched for PAH-associated genes and genetic variants, and candidate drugs predicted to reverse the changes were identified. An open-access online platform was developed to share single-cell data and drug candidates (http://mergeomics.research.idre.ucla.edu/PVDSingleCell/).Conclusions: Our study revealed the distinct and shared dysregulation of genes and pathways in two commonly used PAH models for the first time at single-cell resolution and demonstrated their relevance to human PAH and utility for drug repositioning.

Introduction: Pulmonary veno-occlusive disease (PVOD) is an orphan fatal disease. The identification of biallelic inactivating mutations of EIF2AK4 (encoding GCN2) as the major genetic cause of PVOD has clarified the nosology of the disease. On the other hand, BMP/BMPR2 axis is a major axis of pulmonary arterial hypertension pathobiology, a close but better understood form of severe pulmonary hypertension (PH) Objective: To investigate the functional relationship between GCN2 and the BMP/BMPR2 axis. Methods: We investigated GCN2 and the BMP/BMPR2 axis in human and animal models of PVOD, and in cultured human pulmonary microvascular endothelial cells (hPMEC) with interventional experiments (siRNA and pharmacological inhibitors/activators) Results: Pulmonary GCN2 protein expression was decreased in all forms of PVOD. We showed that GCN2 loss-of-function negatively regulates BMP-dependent SMAD1/5/9 signaling in hPMEC. This molecular relationship was confirmed in vivo, in the lungs of a newly created transgenic rat model knock out for Eif2ak4. We found a 4 fold decrease in SMAD1/5/9 phosphorylation in KO rats. We showed this regulation may be mediated through GCN2-dependent repression of chordin, an extracellular antagonist of BMP signaling. GCN2 inhibition induced a dramatic increase in hPMEC proliferation, which is highly relevant of PVOD genesis. BMP9 treatment was able to block this exuberant proliferation Conclusion: GCN2 loss-of-function negatively regulates SMAD1/5/9 phosphorylation. Despite this dampened BMP signaling, exogenous BMP9 was still able to reverse GCN2 inhibition-induced hPMEC proliferation. BMP9 may hence be considered as potential therapeutic options for PVOD

Introduction: Heritable pulmonary arterial hypertension (PAH) is most commonly due to heterozygous mutations of the BMPR2 gene (encoding Bone Morphogenetic Protein Receptor type II). Despite the lack of clear evidence, asymptomatic BMPR2 mutation carriers are recommended to undergo annual screening echocardiography for early detection of PAH. Objectives: To evaluate the utility of a comprehensive multimodal PAH screening program in asymptomatic adult BMPR2 mutation carriers. Methods: Asymptomatic BMPR2 mutation carriers underwent screening at baseline and annually for a minimum of two years (DELPHI-2 study, NCT01600898). Annual screening included clinical assessment, electrocardiogram, pulmonary function tests, 6-minute walk distance, cardiopulmonary exercise test (CPET), chest x-ray, echocardiography and N-terminal pro-brain natriuretic peptide level. Right heart catheterization (RHC) was performed based on predefined criteria. An optional RHC at rest and exercise was proposed at baseline. Results: Fifty-five subjects (26 males, median age 37 years) were included. At baseline, no PAH was suspected based on echocardiography and NT pro-BNP levels. All subjects accepted RHC at inclusion which identified two mild PAH cases (3.6%) and 12 subjects with exercise pulmonary hypertension of unknown significance (21.8%). At long term follow-up, three additional PAH cases were diagnosed, yielding a PAH incidence of 2.3%/year. All detected cases have remained at low risk status on oral PAH therapy at last follow-up. Conclusion: In adult BMPR2 mutation carriers, a multimodal screening program with regular follow-up allows early detection of PAH.

Pulmonary veno-occlusive disease (PVOD) occurs in humans either as a heritable form (hPVOD) due to biallelic inactivating mutations of EIF2AK4 (encoding GCN2) or as a sporadic form in older age (sPVOD). The chemotherapeutic agent mitomycin C (MMC) is a potent inducer of PVOD in humans and in rats (MMC-PVOD). Here, we compared human hPVOD and sPVOD, and MMC-PVOD pathophysiology at the histological, cellular, and molecular levels to unravel common altered pathomechanisms. MMC exposure in rats was associated primarily with arterial and microvessel remodeling, and secondarily by venous remodeling, when PVOD became symptomatic. In all forms of PVOD tested, there was convergent GCN2-dependent but eIF2α-independent pulmonary protein overexpression of HO-1 (heme oxygenase 1) and CHOP (CCAAT-enhancer-binding protein [C/EBP] homologous protein), two downstream effectors of GCN2 signaling and endoplasmic reticulum stress. In human PVOD samples, CHOP immunohistochemical staining mainly labeled endothelial cells in remodeled veins and arteries. Strong HO-1 staining was observed only within capillary hemangiomatosis foci, where intense microvascular proliferation occurs. HO-1 and CHOP stainings were not observed in control and pulmonary arterial hypertension lung tissues, supporting the specificity for CHOP and HO-1 involvement in PVOD pathobiology. In vivo loss of GCN2 (EIF2AK4 mutations carriers and Eif2ak4-/- rats) or in vitro GCN2 inhibition in cultured pulmonary artery endothelial cells using pharmacological and siRNA approaches demonstrated that GCN2 loss of function negatively regulates BMP (bone morphogenetic protein)-dependent SMAD1/5/9 signaling. Exogenous BMP9 was still able to reverse GCN2 inhibition-induced proliferation of pulmonary artery endothelial cells. In conclusion, we identified CHOP and HO-1 inhibition, and BMP9, as potential therapeutic options for PVOD. ispartof: AMERICAN JOURNAL OF RESPIRATORY CELL AND MOLECULAR BIOLOGY vol:63 issue:1 pages:118-131 ispartof: location:United States status: published

IntroductionTBX4 mutation causes small patella syndrome (SPS) and/or pulmonary arterial hypertension (PAH). The characteristics and outcomes of PAH associated with TBX4 mutations are largely unknown.MethodsWe report the clinical, functional, radiologic, histologic and haemodynamic characteristics and outcomes of heritable PAH patients carrying a TBX4 mutation from the French pulmonary hypertension (PH) network.Results20 patients were identified in 17 families. They were characterised by a median age at diagnosis of 29 years (0–76 years) and a female to male ratio of three. Most of the patients (70%) were in New York Heart Association (NYHA) functional class III or IV with a severe haemodynamic impairment (median pulmonary vascular resistance (PVR) of 13.6 (6.2–41.8) Wood units). Skeletal signs of SPS were present in 80% of cases. Half of the patients had mild restrictive or obstructive limitation and diffusing capacity of the lung for carbon monoxide (DLCO) was decreased in all patients. High-resolution computed tomography (HRCT) showed bronchial abnormalities, peri-bronchial cysts, mosaic distribution and mediastinal lymphadenopathies. PAH therapy was associated with significant clinical improvement. At follow-up (median 76 months), two patients had died and two had undergone lung transplantation. One-year, three-year and five-year event-free survival rates were 100%, 94% and 83%, respectively. Histologic examination of explanted lungs revealed alveolar growth abnormalities, major pulmonary vascular remodelling similar to that observed in idiopathic pulmonary arterial hypertension (IPAH) and accumulation of cholesterol crystals within the lung parenchyma.ConclusionPAH due to TBX4 mutations may occur with or without skeletal abnormalities across a broad age range from birth to late adulthood. PAH is usually severe and associated with bronchial and parenchymal abnormalities.

Beyond the major gene BMPR2, several new genes predisposing to PAH have been identified during the last decade. Recently, preliminary evidence of the involvement of the KDR gene was found in a large genetic association study.We prospectively analysed the KDR gene by targeted panel sequencing in a series of 311 PAH patients referred to a clinical molecular laboratory for genetic diagnosis of PAH.Two index cases with severe PAH from two different families were found to carry a loss-of-function mutation in the KDR gene. These two index cases were clinically characterised by low diffusing capacity for carbon monoxide adjusted for haemoglobin (DLCOc) and interstitial lung disease. In one family, segregation analysis revealed that variant carriers are either presenting with PAH associated with low DLCOc, or have only decreased DLCOc, whereas non-carrier relatives have normal DLCOc. In the second family, a single affected carrier was alive. His carrier mother was unaffected with normal DLCOc.We provided genetic evidence for considering KDR as a newly identified PAH-causing gene by describing the segregation of KDR mutations with PAH in two families. In our study, KDR mutations are associated with a particular form of PAH characterised by low DLCOc and radiological evidence of parenchymal lung disease including interstitial lung disease and emphysema.

International audience; Rationale: Recently, rare heterozygous mutations in GDF2 were identified in patients with pulmonary arterial hypertension (PAH). GDF2 encodes the circulating BMP (bone morphogenetic protein) type 9, which is a ligand for the BMP2 receptor.Objectives: Here we determined the functional impact of GDF2 mutations and characterized plasma BMP9 and BMP10 levels in patients with idiopathic PAH.Methods: Missense BMP9 mutant proteins were expressed in vitro and the impact on BMP9 protein processing and secretion, endothelial signaling, and functional activity was assessed. Plasma BMP9 and BMP10 levels and activity were assayed in patients with PAH with GDF2 variants and in control subjects. Levels were also measured in a larger cohort of control subjects (n = 120) and patients with idiopathic PAH (n = 260).Measurements and Main Results: We identified a novel rare variation at the GDF2 and BMP10 loci, including copy number variation. In vitro, BMP9 missense proteins demonstrated impaired cellular processing and secretion. Patients with PAH who carried these mutations exhibited reduced plasma levels of BMP9 and reduced BMP activity. Unexpectedly, plasma BMP10 levels were also markedly reduced in these individuals. Although overall BMP9 and BMP10 levels did not differ between patients with PAH and control subjects, BMP10 levels were lower in PAH females. A subset of patients with PAH had markedly reduced plasma levels of BMP9 and BMP10 in the absence of GDF2 mutations.Conclusions: Our findings demonstrate that GDF2 mutations result in BMP9 loss of function and are likely causal. These mutations lead to reduced circulating levels of both BMP9 and BMP10. These findings support therapeutic strategies to enhance BMP9 or BMP10 signaling in PAH.

International audience; INTRODUCTION:TBX4 mutation cause small patella syndrome (SPS) and/or pulmonary arterial hypertension (PAH). The characteristics and outcomes of PAH associated with TBX4 mutations are largely unknown.METHODS:We report the clinical, functional, radiologic, histologic and haemodynamic characteristics and outcomes of heritable PAH patients carrying a TBX4 mutation from the French PH Network.RESULTS:Twenty patients were identified in 17 families. They were characterised by a median age at diagnosis of 29 (0-76) year-old and a female to male ratio of 3. Most of the patients were in NYHA functional class III or IV (70%) with a severe hemodynamic impairment (median pulmonary vascular resistance of 13.6 [6.2-41.8] Wood Units). Skeletal signs of SPS were present in 80% of cases. Half of the patients had mild restrictive or obstructive limitation and diffusing capacity for carbon monoxide was decreased in all patients. High-resolution computed tomography showed bronchial abnormalities, peri-bronchial cysts, mosaic distribution and mediastinal lymphadenopathies. PAH therapy was associated with significant clinical improvement. At follow-up (median 76 months), two patients died and two underwent lung transplantation. One-, three- and five-year event-free survival rates were 100%, 94% and 83%, respectively. Histologic examination of explanted lungs revealed alveolar growth abnormalities, major pulmonary vascular remodelling similar to that observed in idiopathic PAH, and accumulation of cholesterol crystals within the lung parenchyma.CONCLUSION:PAH due to TBX4 mutations may occur with or without skeletal abnormalities across a broad age range from birth to late adulthood. PAH is usually severe and associated with bronchial and parenchymal abnormalities.

Pulmonary arterial hypertension (PAH) is a rare disease that leads to premature death from right heart failure. It is strongly associated with elevated red cell distribution width (RDW), a correlate of several iron status biomarkers. High RDW values can signal early-stage iron deficiency or iron deficiency anaemia. This study investigated whether elevated RDW is causally associated with PAH. A two-sample Mendelian randomisation (MR) approach was applied to investigate whether genetic predisposition to higher levels of RDW increases the odds of developing PAH. Primary and secondary MR analyses were performed using all available genome-wide significant RDW variants (n=179) and five genome-wide significant RDW variants that act via systemic iron status, respectively. We confirmed the observed association between RDW and PAH (OR 1.90, 95% CI 1.80–2.01) in a multicentre case–control study (cases n=642, disease controls n=15 889). The primary MR analysis was adequately powered to detect a causal effect (odds ratio) between 1.25 and 1.52 or greater based on estimates reported in the RDW genome-wide association study or from our own data. There was no evidence for a causal association between RDW and PAH in either the primary (ORcausal 1.07, 95% CI 0.92–1.24) or the secondary (ORcausal 1.09, 95% CI 0.77–1.54) MR analysis. The results suggest that at least some of the observed association of RDW with PAH is secondary to disease progression. Results of iron therapeutic trials in PAH should be interpreted with caution, as any improvements observed may not be mechanistically linked to the development of PAH., Mendelian randomisation using genetic data from the largest-to-date PAH cohort does not support red cell distribution width or iron deficiency as a cause of PAH, which is important when interpreting iron replacement trials in this condition http://bit.ly/2PPaa88

International audience; Background: Splenic artery aneurysm (SAA) is a rare but potentially fatal condition. Rupture results in 25% mortality up to 75% in pregnant women with 95% fetal mortality. Brief reports suggest an increased risk of developing SAA in patients with HHT.Methods: We analyzed enhanced multidetector CT data in 186 HHT patients matched (gender and ± 5 year old) with 186 controls. We screened for SAA and recorded diameter of splenic and hepatic arteries and hepatic, pancreatic and splenic parenchymal involvements. We determined by univariate and multivariate analysis, the relationship with age, sex, genetic status, cardiovascular risk factors (CVRF) and visceral involvement.Results: SAA concerned 24.7% of HHT patients and 5.4% of controls, p

We report the clinical, functional, radiologic, histologic and haemodynamic characteristics and outcomes of heritable PAH patients carrying a20 patients were identified in 17 families. They were characterised by a median age at diagnosis of 29 years (0-76 years) and a female to male ratio of three. Most of the patients (70%) were in New York Heart Association (NYHA) functional class III or IV with a severe haemodynamic impairment (median pulmonary vascular resistance (PVR) of 13.6 (6.2-41.8) Wood units). Skeletal signs of SPS were present in 80% of cases. Half of the patients had mild restrictive or obstructive limitation and diffusing capacity of the lung for carbon monoxide (PAH due to

OBJECTIVE To assess the spectrum of genetic anomalies in a cohort of children presenting at least one cerebral or spinal pial arteriovenous fistula (AVF), and to describe their clinical characteristics. METHODS From 1988 to 2016, all consecutive patients with at least one cerebral or spinal pial AVF were screened for genetic disease. All patients aged

Background: Pulmonary arterial hypertension (PAH) is a rare disease with an emerging genetic basis. Heterozygous mutations in the gene encoding the bone morphogenetic protein receptor type 2 ( BMPR2 ) are the commonest genetic cause of PAH, whereas biallelic mutations in the eukaryotic translation initiation factor 2 alpha kinase 4 gene ( EIF2AK4 ) are described in pulmonary veno-occlusive disease/pulmonary capillary hemangiomatosis. Here, we determine the frequency of these mutations and define the genotype-phenotype characteristics in a large cohort of patients diagnosed clinically with PAH. Methods: Whole-genome sequencing was performed on DNA from patients with idiopathic and heritable PAH and with pulmonary veno-occlusive disease/pulmonary capillary hemangiomatosis recruited to the National Institute of Health Research BioResource–Rare Diseases study. Heterozygous variants in BMPR2 and biallelic EIF2AK4 variants with a minor allele frequency of sorting intolerant from tolerant predictions) were identified as potentially causal. Phenotype data from the time of diagnosis were also captured. Results: Eight hundred sixty-four patients with idiopathic or heritable PAH and 16 with pulmonary veno-occlusive disease/pulmonary capillary hemangiomatosis were recruited. Mutations in BMPR2 were identified in 130 patients (14.8%). Biallelic mutations in EIF2AK4 were identified in 5 patients with a clinical diagnosis of pulmonary veno-occlusive disease/pulmonary capillary hemangiomatosis. Furthermore, 9 patients with a clinical diagnosis of PAH carried biallelic EIF2AK4 mutations. These patients had a reduced transfer coefficient for carbon monoxide (K co ; 33% [interquartile range, 30%–35%] predicted) and younger age at diagnosis (29 years; interquartile range, 23–38 years) and more interlobular septal thickening and mediastinal lymphadenopathy on computed tomography of the chest compared with patients with PAH without EIF2AK4 mutations. However, radiological assessment alone could not accurately identify biallelic EIF2AK4 mutation carriers. Patients with PAH with biallelic EIF2AK4 mutations had a shorter survival. Conclusions: Biallelic EIF2AK4 mutations are found in patients classified clinically as having idiopathic and heritable PAH. These patients cannot be identified reliably by computed tomography, but a low K co and a young age at diagnosis suggests the underlying molecular diagnosis. Genetic testing can identify these misclassified patients, allowing appropriate management and early referral for lung transplantation.

RESUME La decouverte de nouveaux genes de susceptibilite a l’hypertension pulmonaire a bouleverse nos connaissances sur la physiopathologie de la maladie. A cote des mutations du gene BMPR2 qui sont, par leurs frequences et leurs effets, les principales mutations responsables des formes heritables de l’hypertension arterielle pulmonaire (HTAP) isolee, des mutations ont ete decrites dans le gene KCNK3 (TASK1) codant pour un canal potassium, le gene SMAD8 codant, comme BMPR2, pour une molecule de signalisation de la voie BMP, le gene CAV1 codant pour la caveoline-1. Les mutations des genes de l’endogline et du recepteur ACVRL1 s’inscrivent dans le cadre de la maladie de Rendu-Osler et la survenue de l’HTAP en aggrave la severite, et les mutations du gene TBX4 sont egalement responsables de formes syndromiques d’HTAP compliquant de facon rare le Small Patella Syndrome. Les mutations du gene EIF2AK4 (GCN2) sont responsables d’une forme rare et severe d’hypertension pulmonaire, la maladie veino-occlusive pulmonaire appelee aussi hemangiomatose capillaire pulmonaire. L’analyse des mecanismes d’action et de regulation de ces genes et des consequences fonctionnelles des mutations conduit a la conception de nouveaux traitements a visee etiologique de l’hypertension pulmonaire.

SummaryBackground Hereditary hemorrhagic telangiectasia (HHT) is an autosomal dominant genetic disorder that is caused by mutations in mainly two genes, that is ENG, encoding endoglin (HHT1), or ACVRL1, encoding activin receptor-like kinase 1 (ALK-1/HHT2). HHT is characterized by recurrent epistaxis, mucocutaneous telangiectasia, and vascular visceral dysplasia responsible for visceral arteriovenous malformations (AVM). Aim to report the experience of two university hospitals (Trousseau, Paris, and CHIC, Creteil) with screening children for HHT and pulmonary AVM (PAVM) using high resolution computed tomography (HRCT). Methods parents with confirmed HHT were offered to have their children screened for the mutation identified in their family, and informed consent was obtained. Children carrying the same mutation as their parents underwent HRCT of the chest without contrast. Results between 2008 and 2015, 99 children were screened for HHT mutations. Mutations were identified in 59 patients, that is 24 HHT1 and 35 HHT2. Radiologic and clinical screening was possible in 52 patients (21 HHT-1 and 31 HHT-2). Among those, PAVM was identified in 13 patients (25%; n = 8 HHT1; n = 5 HHT2), and four of them required embolization therapy. Conclusion This study highlights the usefulness of genetic screening in children with known HHT family. It also suggests that a non-invasive protocol such as HRTC is an efficient approach to detect non-symptomatic lesions that are present early on in children carrying the ENG (HHT1), but also the ACVRL1 mutations (HHT2). Pediatr Pulmonol. 2017;52:642–649. © 2017 Wiley Periodicals, Inc.

Beyond the major gene

Rationale: Recently, rare heterozygous mutations in GDF2 were identified in patients with pulmonary arterial hypertension (PAH). GDF2 encodes the circulating BMP (bone morphogenetic protein) type 9, which is a ligand for the BMP2 receptor. Objectives: Here we determined the functional impact of GDF2 mutations and characterized plasma BMP9 and BMP10 levels in patients with idiopathic PAH. Methods: Missense BMP9 mutant proteins were expressed in vitro and the impact on BMP9 protein processing and secretion, endothelial signaling, and functional activity was assessed. Plasma BMP9 and BMP10 levels and activity were assayed in patients with PAH with GDF2 variants and in control subjects. Levels were also measured in a larger cohort of control subjects (n = 120) and patients with idiopathic PAH (n = 260). Measurements and Main Results: We identified a novel rare variation at the GDF2 and BMP10 loci, including copy number variation. In vitro, BMP9 missense proteins demonstrated impaired cellular processing and secretion. Patients with PAH who carried these mutations exhibited reduced plasma levels of BMP9 and reduced BMP activity. Unexpectedly, plasma BMP10 levels were also markedly reduced in these individuals. Although overall BMP9 and BMP10 levels did not differ between patients with PAH and control subjects, BMP10 levels were lower in PAH females. A subset of patients with PAH had markedly reduced plasma levels of BMP9 and BMP10 in the absence of GDF2 mutations. Conclusions: Our findings demonstrate that GDF2 mutations result in BMP9 loss of function and are likely causal. These mutations lead to reduced circulating levels of both BMP9 and BMP10. These findings support therapeutic strategies to enhance BMP9 or BMP10 signaling in PAH.

Introduction: Pulmonary hypertension associated with neurofibromatosis type 1 (PH-NF1) is a rare but severe complication of NF1. The largest study about PH-NF1 described 8 patients and there are few data about outcomes on PH treatment. Methods: We report clinical, functional, radiologic, and hemodynamic characteristics and outcomes of patients with pre-capillary PH-NF1 identified within the French PH Registry. Computed tomography (CT) at PH diagnosis were analysed by a pneumologist and a radiologist. Results: We identified 39 NF1-PH cases, characterized by a female predominance (77%) and a median age at PH diagnosis of 63 [min-max 35-82] y-o. At diagnosis, 90% were in NYHA functional class III or IV. Median 6-min walk distance was 209 [50-460] m. Pulmonary function tests showed normal or mild abnormalities except low DLCO (29 [20-58] %). PH was severe with a mean pulmonary artery pressure of 45 [29-71] mmHg, a cardiac index of 2.3 [1.2-3.6] L/min/m2 and pulmonary vascular resistance of 9.8 [4.6-20.1] WU. CT showed pulmonary cysts (72%), ground glass opacities (67%) and emphysema (49%). 38 patients received PAH-specific therapy. Transplant-free survival rates at 1, 3 and 5 years were 92%, 64% and 44%, respectively. Four patients underwent lung transplantation. Conclusions: PH-NF1 was characterized by female predominance, advanced age, severe hemodynamic impairment and poor prognosis.

International audience; Background Heritable forms of pulmonary arterial hypertension (PAH) and pulmonary veno-occlusive disease/pulmonary capillary haemangiomatosis (PVOD/PCH) diverge by lung histopathological lesions, clinical and para-clinical presentation, their responsible genes, and mode of transmission. Since the identification of the BMPR2 gene in families affected by PAH, mutations in several other genes have been discovered for both forms. The mutation landscape in these new genes is not yet well known.Methods We set up a next-generation sequencing-based targeted sequencing gene panel allowing known genes for PAH and PVOD/PCH to be analysed simultaneously. Genetic analysis was prospectively performed on 263 PAH and PVOD/PCH patients (adult and paediatric cases).Results Pathogenic mutations were identified in 19.5% of sporadic PAH patients (n=180), 54.5% of familial PAH patients and 13.3% of PVOD/PCH patients. BMPR2 was the most frequently mutated gene, followed by TBX4 in both paediatric and adult PAH. BMP9 mutations were identified in 1.2% of adult PAH cases. EIF2AK4 biallelic mutations were restricted to PVOD/PCH. A truncating mutation and a predicted loss-of-function variant were also identified in BMP10 in two severely affected sporadic PAH female patients.Conclusion Our results confirm that mutations are found in genes beyond BMPR2 in heritable PAH, emphasise the role of TBX4 and BMP9, and designate BMP10 as a new PAH gene.

International audience; In 2017, we implemented CTNNA1 germline analysis in probands suspected of having hereditary diffuse gastric cancer. Here, we report the results from a retrospective series of 41 cases, including the identification of a new family with a CTNNA1 mutation and the first prophylactic total gastrectomy in an asymptomatic carrier after a normal upper endoscopy. Diffuse gastric cancer foci with loss of catenin alpha-1 expression were seen in the resected tissue, suggesting that CTNNA1 and CDH1 germline mutations behave in a similar manner. Life-changing prophylactic total gastrectomy should therefore also be considered in CTNNA1 mutation carriers.

The impact of bone morphogenetic protein receptor 2 (BMPR2) gene mutations on vascular remodelling in pulmonary arterial hypertension (PAH) is unknown. We sought to identify a histological profile of BMPR2 mutation carriers.Clinical data and lung histology from 44 PAH patients were subjected to systematic analysis and morphometry.Bronchial artery hypertrophy/dilatation and bronchial angiogenesis, as well as muscular remodelling of septal veins were significantly increased in PAH lungs carrying BMPR2 mutations. We found that patients displaying increased bronchial artery remodelling and bronchial microvessel density, irrespective of the mutation status, were more likely to suffer from severe haemoptysis. History of substantial haemoptysis (>50 mL) was significantly more frequent in BMPR2 mutation carriers. 43.5% of BMPR2 mutation carriers, as opposed to 9.5% of noncarriers, displayed singular large fibrovascular lesions, which appear to be closely related to the systemic lung vasculature.Our analysis provides evidence for the involvement of the pulmonary systemic circulation in BMPR2 mutation-related PAH. We show that BMPR2 mutation carriers are more prone to haemoptysis and that haemoptysis is closely correlated to bronchial arterial remodelling and angiogenesis; in turn, pronounced changes in the systemic vasculature correlate with increased pulmonary venous remodelling, creating a distinctive profile in PAH patients harbouring a BMPR2 mutation.

RESUME Les interventions ayant pour but de modifier le genome de la descendance sont proscrites depuis 1994 en France mais le developpement de methodes comme CRISPR-Cas9 conduit a s’interroger sur leurs utilisations potentielles sur les cellules germinales et l’embryon humains. La seule indication medicale acceptable serait d’eviter la transmission d’une pathologie genique a l’enfant mais les conditions, notamment celles relatives a l’efficacite et a l’innocuite de ces methodes, ne sont pas actuellement reunies pour envisager leur utilisation clinique. De plus il existe d’autres moyens permettant aux couples concernes de realiser leur projet parental. Les questions ethiques suscitees par ces technologies incitent a recommander l’ouverture d’une reflexion pluridisciplinaire qui devrait etre menee dans le cadre d’un debat plus large portant sur l’ensemble des interventions medicales realisees lors de de l’assistance medicale a la procreation, pouvant avoir des consequences sur le genome des enfants a naitre et eventuellement sur celui des generations suivantes. En revanche, les recherches, y compris sur les cellules germinales et l’embryon humains, devraient pouvoir etre menees quand elles sont scientifique- ment et medicalement justifiees.

Pulmonary veno-occlusive disease (PVOD) is a rare form of pulmonary hypertension (PH) characterised by preferential remodelling of the pulmonary venules. In the current PH classification, PVOD and pulmonary capillary haemangiomatosis (PCH) are considered to be a common entity and represent varied expressions of the same disease. The recent discovery of biallelic mutations in the EIF2AK4 gene as the cause of heritable PVOD/PCH represents a major milestone in our understanding of the molecular pathogenesis of PVOD. Although PVOD and pulmonary arterial hypertension (PAH) share a similar clinical presentation, with features of severe precapillary PH, it is important to differentiate these two conditions as PVOD carries a worse prognosis and life-threatening pulmonary oedema may occur following the initiation of PAH therapy. An accurate diagnosis of PVOD based on noninvasive investigations is possible utilising oxygen parameters, low diffusing capacity for carbon monoxide and characteristic signs on high-resolution computed tomography of the chest. No evidence-based medical therapy exists for PVOD at present and lung transplantation remains the preferred definitive therapy for eligible patients.

Rationale: Pulmonary arterial hypertension is characterized by vascular remodeling and neomuscularization. PW1 + progenitor cells can differentiate into smooth muscle cells (SMCs) in vitro. Objective: To determine the role of pulmonary PW1 + progenitor cells in vascular remodeling characteristic of pulmonary arterial hypertension. Methods and Results: We investigated their contribution during chronic hypoxia–induced vascular remodeling in Pw1 nLacZ+/− mouse expressing β-galactosidase in PW1 + cells and in differentiated cells derived from PW1 + cells. PW1 + progenitor cells are present in the perivascular zone in rodent and human control lungs. Using progenitor markers, 3 distinct myogenic PW1 + cell populations were isolated from the mouse lung of which 2 were significantly increased after 4 days of chronic hypoxia. The number of proliferating pulmonary PW1 + cells and the proportion of β-gal + vascular SMC were increased, indicating a recruitment of PW1 + cells and their differentiation into vascular SMC during early chronic hypoxia–induced neomuscularization. CXCR4 inhibition using AMD3100 prevented PW1 + cells differentiation into SMC but did not inhibit their proliferation. Bone marrow transplantation experiments showed that the newly formed β-gal + SMC were not derived from circulating bone marrow–derived PW1 + progenitor cells, confirming a resident origin of the recruited PW1 + cells. The number of pulmonary PW1 + cells was also increased in rats after monocrotaline injection. In lung from pulmonary arterial hypertension patients, PW1-expressing cells were observed in large numbers in remodeled vascular structures. Conclusions: These results demonstrate the existence of a novel population of resident SMC progenitor cells expressing PW1 and participating in pulmonary hypertension–associated vascular remodeling.

SUMMARY Next generation sequencing (NGS) technologies, which allow high speed automated DNA sequencing, DNA sequence analysis and comparisons using big data algorithms are being used more and more in medical diagnosis, to provide prognosis information and for choosing a treatment that best fits the patient. The French “Academie nationale de medecine ” (ANM) and “ Academie des technologies “ (NATF) jointly stress the scientific and medical importance of these technologies and call for bringing together strengths of the national computer science industry and the medical community in a public-private consortium in order to build a Demonstrator and Center network filling up the growing gap between France and the most advanced countries in this field with great potential. They underline also common pitfalls which need to be addressed in technic, legal, economic, education and ethics issues, in order to make this complex project a success.

Genetic causes of pulmonary arterial hypertension (PAH) and pulmonary veno-occlusive disease (PVOD) have been identified, leading to a growing need for genetic counselling.Between 2003 and 2014, genetic counselling was offered to 529 PAH and 100 PVOD patients at the French Referral Centre for Pulmonary Hypertension.Mutations in PAH-predisposing genes were identified in 72 patients presenting as sporadic PAH (17% of cases; 62 mutations in BMPR2, nine in ACVRL1 (ALK1) and one in ENG) and in 94 patients with a PAH family history (89% of cases; 89 mutations in BMPR2, three in ACVRL1 (ALK1) and two in KCNK3). Bi-allelic mutations in EIF2AK4 were identified in all patients with a family history of PVOD (n=19) and in seven patients (8.6%) presenting as sporadic PVOD. Pre-symptomatic genetic diagnosis was offered to 272 relatives of heritable PAH patients, identifying mutations in 36.4% of them. A screening programme is now offered to asymptomatic mutation carriers to detect PAH in an early phase and to identify predictors of outcomes in asymptomatic BMPR2 mutation carriers. BMPR2 screening allowed us to offer pre-implantation diagnosis to two couples with a BMPR2 mutation.Genetic counselling can be implemented in pulmonary hypertension centres.

Introduction Pulmonary veno-occlusive disease (PVOD) occurs in humans as heritable form due to biallelic inactivating mutations of EIF2AK4 (encoding GCN2), or as a sporadic form. The pathobiology of this orphan fatal disease is currently unknown. However, the identification of EIF2AK4 as the major genetic cause of PVOD has clarified the nosology of the disease. On the other hand, BMP/BMPR2 axis is a major axis of pulmonary arterial hypertension pathobiology, a close but better understood form of severe pulmonary hypertension (PH). Objective To investigate the functional relationship between GCN2 and the BMP/BMPR2 axis. Methods We investigated GCN2 and the BMP/BMPR2 axis in human and animal models of PVOD, and in cultured human pulmonary microvascular endothelial cells (hPMEC) with interventional experiments (siRNA and pharmacological inhibitors/activators). Results Pulmonary GCN2 protein expression was decreased in all forms of PVOD. We showed that GCN2 loss-of-function negatively regulates BMP-dependent SMAD1/5/9 signaling in hPMEC. This molecular relationship was confirmed in vivo, in the lungs of a newly created transgenic rat model knock out for Eif2ak4 (Δ152Ex1/Δ152Ex1). We found a 4 fold decrease in SMAD1/5/9 phosphorylation in KO rats. Since those rats don’t have spontaneous PH, the decrease in SMAD1/5/9 phosphorylation is not the mere consequence of high pulmonary artery pressures. We showed this regulation may be mediated through GCN2-dependent repression of chordin, an extracellular antagonist of BMP signaling. GCN2 inhibition induced a dramatic increase in hPMEC proliferation, which is highly relevant of PVOD genesis. BMP9 treatment was able to block this exuberant proliferation. Conclusion GCN2 loss-of-function negatively regulates SMAD1/5/9 phosphorylation. Despite this dampened BMP signaling, exogenous BMP9 was still able to reverse GCN2 inhibition-induced hPMEC proliferation. BMP9 may hence be considered as potential therapeutic options for PVOD.

Introduction L’hypertension pulmonaire associee a la neurofibromatose de type 1 (HTP-NF1) est une complication rare mais severe de la NF1. Les donnees disponibles dans la litterature sont limitees a des cas isoles ou de petites series ( Methodes Nous rapportons les caracteristiques cliniques, fonctionnelles, radiologiques et hemodynamiques ainsi que l’evolution de patients souffrant d’HTP-NF1, issues du registre francais de l’HTP. Une analyse systematique des TDM thoraciques (n = 36) et des prelevements histologiques disponibles (3 poumons explantes) a ete realisee. Les resultats sont exprimes en mediane (min–max). Resultats Nous avons identifie 41 cas d’HTP-NF1, caracterises par une predominance feminine avec un ratio femme/homme de 3,6 et un âge au diagnostic d’HTP de 64 ans [min–max 35–82]. Au diagnostic, 90 % des patients etaient en classe fonctionnelle NYHA III ou IV. La distance parcourue lors du test de marche de 6 minutes etait de 225 m [50–460]. Alors que les EFR montraient des volumes pulmonaires normaux ou legerement diminues, il existait une DLCO effondree a 30 % [18–62] et une hypoxemie severe (PaO2 en air ambiant 56 [38–79] mmHg). L’hemodynamique retrouvait une HTP severe : pression arterielle pulmonaire moyenne 43[29–71] mmHg, index cardiaque 2,3[1,2–3,7] L/min/m2 et resistances vasculaires pulmonaires (RVP) 10,5 [4,5–20,1] UW. L’analyse des TDM thoraciques montrait des kystes pulmonaires (81 %), des opacites en verre depoli (75 %), de l’emphyseme (53 %) et des reticulations (44 %). Des traitements specifiques de l’HTP ont ete inities chez 35 patients avec une amelioration significative fonctionnelle et hemodynamique a la premiere evaluation. Lors du suivi, 4 patients ont ete transplantes et la survie sans transplantation a 1, 3 et 5 ans etait de 88 %, 56 % et 41 %. L’etude histolologique a montre des lesions de pneumopathie interstitielle desquamative et de pneumopathie interstitielle non specifique associees a un important remodelage vasculaire chez les 3 patients. Conclusion La NF1 peut se compliquer d’HTP severe le plus souvent disproportionnee a l’atteinte parenchymateuse. Le pronostic de l’HTP-NF1 est severe malgre l’initiation de traitement specifique de l’HTP. La transplantation pulmonaire represente une option therapeutique pour les patients eligibles.