Imperial College London

DrJohnWharton

Faculty of MedicineNational Heart & Lung Institute

Honorary Senior Lecturer
 
 
 
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Contact

 

+44 (0)20 7594 6819j.wharton

 
 
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Location

 

530ICTEM buildingHammersmith Campus

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Summary

 

Publications

Publication Type
Year
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286 results found

Liu B, Azfar M, Legchenko E, West JA, Martin S, Van den Haute C, Baekelandt V, Wharton J, Howard L, Wilkins MR, Vangheluwe P, Morrell NW, Upton PDet al., 2024, ATP13A3 variants promote pulmonary arterial hypertension by disrupting polyamine transport., Cardiovasc Res

AIMS: Potential loss-of-function variants of ATP13A3, the gene encoding a P5B-type transport ATPase of undefined function, were recently identified in pulmonary arterial hypertension (PAH) patients. ATP13A3 is implicated in polyamine transport but its function has not been fully elucidated. Here, we sought to determine the biological function of ATP13A3 in vascular endothelial cells and how PAH-associated variants may contribute to disease pathogenesis. METHODS AND RESULTS: We studied the impact of ATP13A3 deficiency and overexpression in endothelial cell (EC) models (human pulmonary ECs, blood outgrowth ECs (BOECs) and HMEC-1 cells), including a PAH patient-derived BOEC line harbouring an ATP13A3 variant (LK726X). We also generated mice harbouring an Atp13a3 variant analogous to a human disease-associated variant to establish whether these mice develop PAH.ATP13A3 localised to the recycling endosomes of human ECs. Knockdown of ATP13A3 in ECs generally reduced the basal polyamine content and altered the expression of enzymes involved in polyamine metabolism. Conversely, overexpression of wild-type ATP13A3 increased polyamine uptake. Functionally, loss of ATP13A3 was associated with reduced EC proliferation, increased apoptosis in serum starvation and increased monolayer permeability to thrombin. Assessment of five PAH-associated missense ATP13A3 variants (L675V, M850I, V855M, R858H, L956P) confirmed loss-of-function phenotypes represented by impaired polyamine transport and dysregulated EC function. Furthermore, mice carrying a heterozygous germ-line Atp13a3 frameshift variant representing a human variant spontaneously developed a PAH phenotype, with increased pulmonary pressures, right ventricular remodelling and muscularisation of pulmonary vessels. CONCLUSION: We identify ATP13A3 as a polyamine transporter controlling polyamine homeostasis in ECs, deficiency of which leads to EC dysfunction and predisposes to PAH. This suggests a need for targeted therapies to al

Journal article

Liley J, Newnham M, Bleda M, Bunclark K, Auger W, Barbera JA, Bogaard H, Delcroix M, Fernandes TM, Howard L, Jenkins D, Lang I, Mayer E, Rhodes C, Simpson M, Southgate L, Trembath R, Wharton J, Wilkins MR, Gräf S, Morrell N, Pepke Zaba J, Toshner Met al., 2024, Shared and distinct genomics of chronic thromboembolic pulmonary hypertension and pulmonary embolism, American Journal of Respiratory and Critical Care Medicine, ISSN: 1073-449X

Rationale: Chronic Thromboembolic Pulmonary Hypertension involves formation and non-resolution of thrombus, dysregulated inflammation, angiogenesis and the development of a small vessel vasculopathy. Objectives: We aimed to establish the genetic basis of chronic thromboembolic pulmonary hypertension to gain insight into its pathophysiological contributors. Methods: We conducted a genome-wide association study on 1907 European cases and 10363 European controls. We co-analysed our results with existing results from genome-wide association studies on deep vein thrombosis, pulmonary embolism and idiopathic pulmonary arterial hypertension. Measurements and Main Results: Our primary association study revealed genetic associations at the ABO, FGG, F11, MYH7B, and HLA-DRA loci. Through our co-analysis we demonstrate further associations with chronic thromboembolic pulmonary hypertension at the F2, TSPAN15, SLC44A2 and F5 loci but find no statistically significant associations shared with idiopathic pulmonary arterial hypertension. Conclusions: Chronic thromboembolic pulmonary hypertension is a partially heritable polygenic disease, with related though distinct genetic associations to pulmonary embolism and to deep vein thrombosis.

Journal article

Ulrich A, Wu Y, Draisma H, Wharton J, Swietlik EM, Cebola I, Vasilaki E, Balkhiyarova Z, Jarvelin M, Auvinen J, Herzig K-H, Coghlan JG, Lordan J, Church C, Howard L, Pepke-Zaba J, Toshner M, Wort S, Kiely D, Condliffe R, Lawrie A, Graf S, Morrell N, Wilkins M, Prokopenko I, Rhodes C, Rhodes Cet al., 2024, Blood DNA methylation profiling identifies cathepsin Z dysregulation in pulmonary arterial hypertension, Nature Communications, Vol: 15, ISSN: 2041-1723

Pulmonary arterial hypertension (PAH) is characterised by pulmonary vascular remodelling causing premature death from right heart failure. Established DNA variants influence PAH risk, but susceptibility from epigenetic changes is unknown. We addressed this through epigenome-wide association study (EWAS), testing 865,848 CpG sites for association with PAH in 429 individuals with PAH and 1226 controls. Three loci, at Cathepsin Z (CTSZ, cg04917472), Conserved oligomeric Golgi complex 6 (COG6, cg27396197), and Zinc Finger Protein 678 (ZNF678, cg03144189), reached epigenome-wide significance (p < 10−7) and are hypermethylated in PAH, including in individuals with PAH at 1-year follow-up. Of 16 established PAH genes, only cg10976975 in BMP10 shows hypermethylation in PAH. Hypermethylation at CTSZ is associated with decreased blood cathepsin Z mRNA levels. Knockdown of CTSZ expression in human pulmonary artery endothelial cells increases caspase-3/7 activity (p < 10−4). DNA methylation profiles are altered in PAH, exemplified by the pulmonary endothelial function modifier CTSZ, encoding protease cathepsin Z.

Journal article

Chen C-N, Hajji N, Yeh F-C, Rahman S, Ali S, Wharton J, Baxan N, Zhao L, Xie C-Y, Chen Y-G, Frid MG, Chelladurai P, Pullamsetti SS, Stenmark KR, Wilkins MR, Zhao Let al., 2023, Restoration of Foxp31 regulatory T cells by HDAC-dependent epigenetic modulation plays a pivotal role in resolving pulmonary arterial hypertension pathology, American Journal of Respiratory and Critical Care Medicine, Vol: 208, Pages: 879-895, ISSN: 1073-449X

Rationale: Immune dysregulation is a common feature of pulmonary arterial hypertension (PAH). Histone deacetylase (HDAC)-dependent transcriptional reprogramming epigenetically modulates immune homeostasis and is a novel disease-oriented approach in modern times. Objectives: To identify a novel functional link between HDAC and regulatory T cells (Tregs) in PAH, aiming to establish disease-modified biomarkers and therapeutic targets. Methods: Peripheral blood mononuclear cells (PBMCs) were isolated from idiopathic PAH (IPAH) patients and rodent models of pulmonary hypertension (PH): monocrotaline (MCT), Sugen5416-hypoxia (SuHx) rats and Treg-depleted mice. HDAC inhibitor Vorinostat (SAHA) was used to examine the immune modulatory effects in vivo, ex vivo and in vitro. Measurements and Main Results: Increased HDAC expression was associated with reduced Foxp3+ Tregs and increased programmed cell death-1 (PD-1) signalling in PBMCs from IPAH patients. SAHA differentially modified a cluster of epigenetic-sensitive genes and induced Foxp3+ Treg conversion in IPAH T cells. Rodent models recapitulated these epigenetic aberrations and T cell dysfunction. SAHA attenuated PH phenotypes and restored FOXP3 transcription and Tregs in PH rats; interestingly, the effects were more profound in female rats. Selective depletion of CD25+ Tregs in SuHx mice neutralized the effects of SAHA. Furthermore, SAHA inhibited endothelial cytokine/chemokine release upon stimulation and subsequent immune chemotaxis. Conclusions: Our results indicated HDAC aberration was associated with Foxp3+ Treg deficiency and demonstrated an epigenetic-mediated mechanism underlying immune dysfunction in PAH. Restoration of Foxp3+ Tregs by HDACi is a promising approach to resolve PH pathology, highlighting the potential benefit of developing "epigenetic therapies” for PAH.

Journal article

Prapa M, Lago-Docampo M, Swietlik EM, Montani D, Eyries M, Humbert M, Welch CL, Chung WK, Berger RMF, Bogaard HJ, Danhaive O, Escribano-Subias P, Gall H, Girerd B, Hernandez-Gonzalez I, Holden S, Hunt D, Jansen SMA, Kerstjens-Frederikse W, Kiely DG, Lapunzina P, McDermott J, Moledina S, Pepke-Zaba J, Polwarth GJ, Schotte G, Tenorio-Castano J, Thompson AAR, Wharton J, Wort SJ, Megy K, Mapeta R, Treacy CM, Martin JM, Li W, Swift AJ, Upton PD, Morrell NW, Graf S, Valverde Det al., 2022, First Genotype-Phenotype Study in TBX4 Syndrome Gain-of-Function Mutations Causative for Lung Disease, AMERICAN JOURNAL OF RESPIRATORY AND CRITICAL CARE MEDICINE, Vol: 206, Pages: 1522-1533, ISSN: 1073-449X

Journal article

Kariotis S, Jammeh E, Swietlik EM, Pickworth JA, Rhodes CJ, Otero P, Wharton J, Iremonger J, Dunning MJ, Pandya D, Mascarenhas TS, Errington N, Thompson AAR, Romanoski CE, Rischard F, Garcia JGN, Yuan JX-J, An T-HS, Desai AA, Coghlan G, Lordan J, Corris PA, Howard LS, Condliffe R, Kiely DG, Church C, Pepke-Zaba J, Toshner M, Wort S, Graf S, Morrell NW, Wilkins MR, Lawrie A, Wang Det al., 2022, Biological heterogeneity in idiopathic pulmonary arterial hypertension identified through unsupervised transcriptomic profiling of whole blood, Nature Communications, Vol: 13, Pages: 1-1, ISSN: 2041-1723

Journal article

Wojciak-Stothard B, Ainscough AJ, Smith TJ, Haensel M, Rhodes CJ, Fellows A, Whitwell HJ, Vasilaki E, Grey K, Freeman A, Howard LS, Wharton J, Dunmore B, Upton PD, Wilkins MR, Edel Jet al., 2022, An organ-on-chip model of pulmonary arterial hypertension identifies a BMPR2-SOX17-prostacyclin signalling axis, Communications Biology, Vol: 5, Pages: 1-15, ISSN: 2399-3642

Pulmonary arterial hypertension (PAH) is an unmet clinical need. The lack of models of human disease is a key obstacle to drug development. We present a biomimetic model of pulmonary arterial endothelial-smooth muscle cell interactions in PAH, combining natural and induced bone morphogenetic protein receptor 2 (BMPR2) dysfunction with hypoxia to induce smooth muscle activation and proliferation, which is responsive to drug treatment. BMPR2- and oxygenation-specific changes in endothelial and smooth muscle gene expression, consistent with observations made in genomic and biochemical studies of PAH, enable insights into underlying disease pathways and mechanisms of drug response. The model captures key changes in the pulmonary endothelial phenotype that are essential for the induction of SMC remodelling, including a BMPR2-SOX17-prostacyclin signalling axis and offers an easily accessible approach for researchers to study pulmonary vascular remodelling and advance drug development in PAH.

Journal article

Constantine A, Rhodes CJ, Ricci P, Li W, Price LC, Mccabe C, Wharton J, Wilkins MR, Howard LS, Dimopoulos K, Wort SJet al., 2022, Correlation between right ventricular dysfunction and plasma protein profile in pulmonary hypertension, Publisher: EUROPEAN RESPIRATORY SOC JOURNALS LTD, ISSN: 0903-1936

Conference paper

Errington N, Kariotis S, Jammeh E, Fong Y, Lihan Z, Chen H, Jatkoe T, Bridges C, Vener T, Wharton J, Thompson R, Toshner M, Howard LS, Rhodes CJ, Wilkins M, Wang D, Lawrie Aet al., 2022, Unsupervised Clustering of PH Using Circulating miRNA - Towards Molecular Classification of PH?, International Conference of the American-Thoracic-Society, Publisher: AMER THORACIC SOC, ISSN: 1073-449X

Conference paper

Rhodes C, Wharton J, Swietlik E, Harbaum L, Girerd B, Coghlan G, Lordan J, Church C, Pepke-Zaba J, Toshner M, Wort SJ, Kiely D, Condliffe R, Lawrie A, Graf S, Montani D, Boucly A, Sitbon O, Humbert M, Howard LS, Morrell NW, Wilkins MRet al., 2022, Using the plasma proteome for risk stratifying patients with pulmonary arterial hypertension, American Journal of Respiratory and Critical Care Medicine, Vol: 205, Pages: 1102-1111, ISSN: 1073-449X

Rationale: N-terminal pro-brain natriuretic peptide (NT-proBNP), a biomarker of cardiac origin, is used to risk stratify patients with pulmonary arterial hypertension (PAH). Its limitations include poor sensitivity to early vascular pathology. Other biomarkers of vascular or systemic origin may also be useful in the management of PAH.Objectives: Identify prognostic proteins in PAH which complement NT-proBNP and clinical risk scores.Methods: An aptamer-based assay (SomaScan-V4) targeting 4,152 proteins was used to measure plasma proteins in patients with idiopathic, heritable or drug-induced-PAH from the UK National Cohort of PAH (n=357) and the French EFORT study (n=79). Prognostic proteins were identified in discovery-replication analyses of UK samples. Proteins independent of 6-minute walk distance (6-MWD) and NT-proBNP entered LASSO modelling and the best combination in a single score was evaluated against clinical targets in EFORT.Measurements and Main Results: Thirty-one proteins robustly informed prognosis independent of NT-proBNP and 6-MWD in the UK Cohort. A weighted combination score of 6 proteins was validated at baseline (5-year mortality, AUC:0.73, 95%CI:0.63-0.85) and follow-up in EFORT (AUC:0.84, 95%CI:0.75-0.94, p=9.96x10-6). The protein score risk-stratified patients independent of established clinical targets and risk equations. The addition of the 6-protein model score to NT-proBNP improved prediction of 5-year outcomes from AUC:0.762 (0.702-0.821) to 0.818 (0.767-0.869) by ROC analysis (p=0.00426 for difference in AUC) in the UK replication and French samples combined. Conclusions: The plasma proteome informs prognosis beyond established factors in PAH and may provide a more sensitive measure of therapeutic response.

Journal article

Kariotis S, Jammeh E, Swietlik EM, Rhodes CJ, Errington N, Thompson R, Wharton J, Coghlan G, Lordan J, Corris P, Howard LS, Condliffe RA, Kiely D, Church A, Pepke-Zaba J, Toshner M, Wort J, Graf S, Morrell NW, Wilkins M, Wang D, Lawrie Aet al., 2022, Longitudinal Analysis of Three Major Risk-Associated Transcriptomic Subgroups Within the IPAH Classification, International Conference of the American-Thoracic-Society, Publisher: AMER THORACIC SOC, ISSN: 1073-449X

Conference paper

Harbaum L, Rhodes CJ, Wharton J, Lawrie A, Karnes JH, Desai AA, Nichols WC, Humbert M, Montani D, Girerd B, Sitbon O, Boehm M, Novoyatleva T, Schermuly RT, Ghofrani HA, Toshner M, Kiely DG, Howard LS, Swietlik EM, Gräf S, Pietzner M, Morrell NW, Wilkins MRet al., 2022, Mining the plasma proteome for insights into the molecular pathology of pulmonary arterial hypertension., American Journal of Respiratory and Critical Care Medicine, Vol: 205, Pages: 1-12, ISSN: 1073-449X

RATIONALE: Pulmonary arterial hypertension (PAH) is characterized by structural remodelling of pulmonary arteries and arterioles. Underlying biological processes are likely reflected in a perturbation of circulating proteins. OBJECTIVES: To quantify and analyse the plasma proteome of PAH patients using inherited genetic variation to inform on underlying molecular drivers. METHODS: An aptamer-based assay was used to measure plasma proteins in 357 patients with idiopathic or heritable PAH, 103 healthy volunteers and 23 relatives of PAH patients. In discovery and replication subgroups, the plasma proteomes of PAH and healthy individuals were compared and the relationship to transplantation-free survival in PAH determined. To examine causal relationships to PAH, protein quantitative trait loci (pQTL) that influenced protein levels in the patient population were used as instruments for Mendelian randomisation (MR) analysis. MEASUREMENTS AND MAIN RESULTS: From 4,152 annotated plasma proteins, levels of 208 differed between PAH patients and healthy subjects and 49 predicted long-term survival. MR based on cis-pQTL located in proximity to the encoding gene for proteins that were prognostic and distinguished PAH from health estimated an adverse effect for higher levels of netrin-4 (odds ratio [OR] 1.55, 95%-confidence interval [CI] 1.16-2.08) and a protective effect for higher levels of thrombospondin-2 (OR 0.83, 95%-CI 0.74-0.94) on PAH. Both proteins tracked the development of PAH in previously healthy relatives and changes in thrombospondin-2 associated with pulmonary arterial pressure at disease onset. CONCLUSIONS: Integrated analysis of the plasma proteome and genome implicates two secreted matrix-binding proteins, netrin-4 and thrombospondin-2, in the pathobiology of PAH.

Journal article

Howard LSGE, He J, Watson GMJ, Huang L, Wharton J, Luo Q, Kiely DG, Condliffe R, Pepke-Zaba J, Morrell NW, Sheares KK, Ulrich A, Quan R, Zhao Z, Jing X, An C, Liu Z, Xiong C, Robbins PA, Dawes T, de MA, Rhodes CJ, Richter MJ, Gall H, Ghofrani HA, Zhao L, Huson L, Wilkins MRet al., 2022, Supplementation with Iron in Pulmonary Arterial Hypertension: Two Randomized Crossover Trials (vol 18, pg 981, 2021), ANNALS OF THE AMERICAN THORACIC SOCIETY, Vol: 19, Pages: 703-703, ISSN: 1546-3222

Journal article

Novoyatleva T, Rai N, Kojonazarov B, Veeroju S, Ben-Batalla I, Caruso P, Shihan M, Presser N, Gotz E, Lepper C, Herpel S, Manaud G, Perros F, Gall H, Ghofrani HA, Weissmann N, Grimminger F, Wharton J, Wilkins M, Upton PD, Loges S, Morrell NW, Seeger W, Schermuly RTet al., 2022, Deficiency of Axl aggravates pulmonary arterial hypertension via BMPR2 (vol 4, 1002, 2021), COMMUNICATIONS BIOLOGY, Vol: 5

Journal article

Kariotis S, Jammeh E, Swietlik EM, Pickworth JA, Rhodes CJ, Otero P, Wharton J, Iremonger J, Dunning MJ, Pandya D, Mascarenhas TS, Errington N, Thompson AAR, Romanoski CE, Rischard F, Garcia JGN, Yuan JX-J, An T-HS, Desai AA, Coghlan G, Lordan J, Corris PA, Howard LS, Condliffe R, Kiely DG, Church C, Pepke-Zaba J, Toshner M, Wort S, Graf S, Morrell NW, Wilkins MR, Lawrie A, Wang D, Bleda M, Bleda M, Hadinnapola C, Haimel M, Auckland K, Tilly T, Martin JM, Yates K, Treacy CM, Day M, Greenhalgh A, Shipley D, Peacock AJ, Irvine V, Kennedy F, Moledina S, MacDonald L, Tamvaki E, Barnes A, Cookson V, Chentouf L, Ali S, Othman S, Ranganathan L, Gibbs JSR, DaCosta R, Pinguel J, Dormand N, Parker A, Stokes D, Ghedia D, Tan Y, Ngcozana T, Wanjiku I, Polwarth G, Mackenzie Ross RV, Suntharalingam J, Grover M, Kirby A, Grove A, White K, Seatter A, Creaser-Myers A, Walker S, Roney S, Elliot CA, Charalampopoulos A, Sabroe I, Hameed A, Armstrong I, Hamilton N, Rothman AMK, Swift AJ, Wild JM, Soubrier F, Eyries M, Humbert M, Montani D, Girerd B, Scelsi L, Ghio S, Gall H, Ghofrani A, Bogaard HJ, Noordegraaf AV, Houweling AC, Veld AHI, Schotte Get al., 2021, Biological heterogeneity in idiopathic pulmonary arterial hypertension identified through unsupervised transcriptomic profiling of whole blood, Nature Communications, Vol: 12, Pages: 1-14, ISSN: 2041-1723

Idiopathic pulmonary arterial hypertension (IPAH) is a rare but fatal disease diagnosed by right heart catheterisation and the exclusion of other forms of pulmonary arterial hypertension, producing a heterogeneous population with varied treatment response. Here we show unsupervised machine learning identification of three major patient subgroups that account for 92% of the cohort, each with unique whole blood transcriptomic and clinical feature signatures. These subgroups are associated with poor, moderate, and good prognosis. The poor prognosis subgroup is associated with upregulation of the ALAS2 and downregulation of several immunoglobulin genes, while the good prognosis subgroup is defined by upregulation of the bone morphogenetic protein signalling regulator NOG, and the C/C variant of HLA-DPA1/DPB1 (independently associated with survival). These findings independently validated provide evidence for the existence of 3 major subgroups (endophenotypes) within the IPAH classification, could improve risk stratification and provide molecular insights into the pathogenesis of IPAH.

Journal article

Novoyatleva T, Rai N, Kojonazarov B, Veeroju S, Ben-Batalla I, Caruso P, Shihan M, Presser N, Goetz E, Lepper C, Herpel S, Manaud G, Perros F, Gall H, Ghofrani HA, Weissmann N, Grimminger F, Wharton J, Wilkins M, Upton PD, Loges S, Morrell NW, Seeger W, Schermuly RTet al., 2021, Deficiency of Axl aggravates pulmonary arterial hypertension via BMPR2, COMMUNICATIONS BIOLOGY, Vol: 4

Journal article

Errington N, Iremonger J, Pickworth JA, Kariotis S, Rhodes CJ, Rothman AM, Condliffe R, Elliot CA, Kiely DG, Howard LS, Wharton J, Thompson AAR, Morrell NW, Wilkins MR, Wang D, Lawrie Aet al., 2021, A diagnostic miRNA signature for pulmonary arterial hypertension using a consensus machine learning approach, EBioMedicine, Vol: 69, ISSN: 2352-3964

BACKGROUND: Pulmonary arterial hypertension (PAH) is a rare but life shortening disease, the diagnosis of which is often delayed, and requires an invasive right heart catheterisation. Identifying diagnostic biomarkers may improve screening to identify patients at risk of PAH earlier and provide new insights into disease pathogenesis. MicroRNAs are small, non-coding molecules of RNA, previously shown to be dysregulated in PAH, and contribute to the disease process in animal models. METHODS: Plasma from 64 treatment naïve patients with PAH and 43 disease and healthy controls were profiled for microRNA expression by Agilent Microarray. Following quality control and normalisation, the cohort was split into training and validation sets. Four separate machine learning feature selection methods were applied to the training set, along with a univariate analysis. FINDINGS: 20 microRNAs were identified as putative biomarkers by consensus feature selection from all four methods. Two microRNAs (miR-636 and miR-187-5p) were selected by all methods and used to predict PAH diagnosis with high accuracy. Integrating microRNA expression profiles with their associated target mRNA revealed 61 differentially expressed genes verified in two independent, publicly available PAH lung tissue data sets. Two of seven potentially novel gene targets were validated as differentially expressed in vitro in human pulmonary artery smooth muscle cells. INTERPRETATION: This consensus of multiple machine learning approaches identified two miRNAs that were able to distinguish PAH from both disease and healthy controls. These circulating miRNA, and their target genes may provide insight into PAH pathogenesis and reveal novel regulators of disease and putative drug targets.

Journal article

Zhu N, Swietlik EM, Welch CL, Pauciulo MW, Hagen JJ, Zhou X, Guo Y, Karten J, Pandya D, Tilly T, Lutz KA, Martin JM, Treacy CM, Rosenzweig EB, Krishnan U, Coleman AW, Gonzaga-Juaregui C, Lawrie A, Trembath RC, Wilkins MR, Morrell NW, Shen Y, Graf S, Nichols WC, Chung WKet al., 2021, Rare variant analysis of 4241 pulmonary arterial hypertension cases from an international consortium implicates FBLN2, PDGFD, and rare de novo variants in PAH, Genome Medicine: medicine in the post-genomic era, Vol: 13, Pages: 1-18, ISSN: 1756-994X

BackgroundPulmonary arterial hypertension (PAH) is a lethal vasculopathy characterized by pathogenic remodeling of pulmonary arterioles leading to increased pulmonary pressures, right ventricular hypertrophy, and heart failure. PAH can be associated with other diseases (APAH: connective tissue diseases, congenital heart disease, and others) but often the etiology is idiopathic (IPAH). Mutations in bone morphogenetic protein receptor 2 (BMPR2) are the cause of most heritable cases but the vast majority of other cases are genetically undefined.MethodsTo identify new risk genes, we utilized an international consortium of 4241 PAH cases with exome or genome sequencing data from the National Biological Sample and Data Repository for PAH, Columbia University Irving Medical Center, and the UK NIHR BioResource – Rare Diseases Study. The strength of this combined cohort is a doubling of the number of IPAH cases compared to either national cohort alone. We identified protein-coding variants and performed rare variant association analyses in unrelated participants of European ancestry, including 1647 IPAH cases and 18,819 controls. We also analyzed de novo variants in 124 pediatric trios enriched for IPAH and APAH-CHD.ResultsSeven genes with rare deleterious variants were associated with IPAH with false discovery rate smaller than 0.1: three known genes (BMPR2, GDF2, and TBX4), two recently identified candidate genes (SOX17, KDR), and two new candidate genes (fibulin 2, FBLN2; platelet-derived growth factor D, PDGFD). The new genes were identified based solely on rare deleterious missense variants, a variant type that could not be adequately assessed in either cohort alone. The candidate genes exhibit expression patterns in lung and heart similar to that of known PAH risk genes, and most variants occur in conserved protein domains. For pediatric PAH, predicted deleterious de novo variants exhibited a significant burden compared to the background mutation rate (2.45×

Journal article

Howard LSGE, He J, Watson GMJ, Huang L, Wharton J, Luo Q, Kiely DG, Condliffe R, Pepke-Zaba J, Morrell NW, Sheares KK, Ulrich A, Quan R, Zhao Z, Jing X, An C, Liu Z, Xiong C, Robbins PA, Dawes T, de Marvao A, Rhodes CJ, Richter MJ, Gall H, Ghofrani HA, Zhao L, Huson L, Wilkins MRet al., 2021, Supplementation with iron in pulmonary arterial hypertension: two randomized crossover trials., Annals of the American Thoracic Society, Vol: 18, Pages: 981-988, ISSN: 1546-3222

RATIONALE: Iron deficiency, in the absence of anaemia, is common in patients with idiopathic and heritable pulmonary arterial hypertension (PAH) and is associated with a worse clinical outcome. Oral iron absorption may be impeded by elevated circulating hepcidin levels. The safety and benefit of parenteral iron replacement in this patient population is unclear. OBJECTIVES: To evaluate the safety and efficacy of parenteral iron replacement in pulmonary arterial hypertension. METHODS: In two randomised, double blind, placebo-controlled 12 week crossover studies, 39 patients in Europe received a single infusion of ferric carboxymaltose (Ferinject®) 1000 mg (or 15 mg/kg if weight < 66.7Kg) or saline as placebo and 17 patients in China received iron dextran (Cosmofer®) 20 mg iron/kg body weight or saline placebo. All patients had idiopathic or heritable PAH and iron deficiency at entry as defined by: a serum ferritin < 37 µg/l or iron < 10.3 µmol/l or transferrin saturations < 16.4%. RESULTS: Both iron treatments were well tolerated and improved iron status. Analysed separately and combined, there was no effect on any measure of exercise capacity (using cardiopulmonary exercise testing or 6 minute walk test) or cardio-pulmonary haemodynamics, as assessed by right heart catheterisation, cardiac magnetic resonance or plasma NT-proBNP, at 12 weeks. CONCLUSION: Iron repletion by administration of a slow release iron preparation as a single infusion to PAH patients with iron deficiency without overt anaemia was well tolerated but provided no significant clinical benefit at 12 weeks. Clinical trial registered with ClinicalTrials.gov (NCT01447628).

Journal article

Wu Y, Wharton J, Walters R, Vasilaki E, Aman J, Zhao L, Wilkins MR, Rhodes CJet al., 2021, The pathophysiological role of novel pulmonary arterial hypertension gene SOX17., European Respiratory Journal, Vol: 57, ISSN: 0903-1936

Pulmonary arterial hypertension (PAH) is a progressive disease predominantly targeting pre-capillary blood vessels. Adverse structural re-modelling and increased pulmonary vascular resistance result in cardiac hypertrophy and ultimately failure of the right ventricle. Recent whole genome and exome sequencing studies have identified SOX17 as a novel risk gene in PAH, with a dominant mode of inheritance and incomplete penetrance. Rare deleterious variants in the gene and more common variants in upstream enhancer sites have both been associated with the disease and a deficiency of SOX17 expression may predispose to PAH. This review aims to consolidate the evidence linking genetic variants in SOX17 to PAH and explores the numerous targets and effects of the transcription factor, focussing on the pulmonary vasculature and the pathobiology of PAH.

Journal article

Swietlik EM, Greene D, Zhu N, Megy K, Cogliano M, Rajaram S, Pandya D, Tilly T, Lutz KA, Welch CCL, Pauciulo MW, Southgate L, Martin JM, Treacy CM, Penkett CJ, Stephens JC, Bogaard HJ, Church C, Coghlan G, Coleman AW, Condliffe R, Eichstaedt CA, Eyries M, Gall H, Ghio S, Girerd B, Grünig E, Holden S, Howard L, Humbert M, Kiely DG, Kovacs G, Lordan J, Machado RD, Mackenzie Ross RV, McCabe C, Moledina S, Montani D, Olschewski H, Pepke-Zaba J, Price L, Rhodes CJ, Seeger W, Soubrier F, Suntharalingam J, Toshner MR, Vonk Noordegraaf A, Wharton J, Wild JM, Wort SJ, Lawrie A, Wilkins MR, Trembath RC, Shen Y, Chung WK, Swift AJ, Nichols WC, Morrell NW, Gräf Set al., 2021, Bayesian inference associates rare KDR variants with specific phenotypes in pulmonary arterial hypertension., Circulation: Genomic and Precision Medicine, Vol: 14, Pages: 57-70, ISSN: 2574-8300

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 NIHR BioResource - Rare Diseases (NBR) study, of which 1,148 were recruited to the PAH domain. To test for genetic associations between genes and selected phenotypes of pulmonary hypertension (PH), 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 (KCO, posterior probability (PP)=0.989) and older age at diagnosis (PP=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 five 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 Vascular Endothelial Growth Factor Receptor 2 (VEGFR2), 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.

Journal article

Harbaum L, Rhodes CJ, Otero-Núñez P, Wharton J, Wilkins MRet al., 2021, The application of 'omics' to pulmonary arterial hypertension, British Journal of Pharmacology, Vol: 178, Pages: 108-120, ISSN: 0007-1188

Recent genome-wide analyses of rare and common sequence variations have brought greater clarity to the genetic architecture of pulmonary arterial hypertension and implicated novel genes in disease development. Transcriptional signatures have been reported in whole lung tissue, pulmonary vascular cells and peripheral circulating cells. High-throughput platforms for plasma proteomics and metabolomics have identified novel biomarkers associated with clinical outcomes and provided molecular instruments for risk assessment. There are methodological challenges to integrating these datasets, coupled to statistical power limitations inherent to the study of a rare disease, but the expectation is that this strategy will reveal novel druggable targets and biomarkers that will open the way to personalised medicine. Here we review the current state-of-the-art and future promise of "omics" in the field of translational medicine in pulmonary arterial hypertension.

Journal article

Ulrich A, Otero-Núñez P, Wharton J, Swietlik E, Graf S, Morrell N, Wang D, Lawrie A, Wilkins M, Prokopenko I, Rhodes C, on behalf of The NIHR BioResourceRare Diseases Consortium, and UK PAH Cohort Study Consortiumet al., 2020, Expression quantitative trait locus mapping in pulmonary arterial hypertension, Genes, Vol: 11, ISSN: 2073-4425

Expression quantitative trait loci (eQTL) can provide a link between disease susceptibility variants discovered by genetic association studies and biology. To date, eQTL mapping studies have been primarily conducted in healthy individuals from population-based cohorts. Genetic effects have been known to be context-specific and vary with changing environmental stimuli. We conducted a transcriptome- and genome-wide eQTL mapping study in a cohort of patients with idiopathic or heritable pulmonary arterial hypertension (PAH) using RNA sequencing (RNAseq) data from whole blood. We sought confirmation from three published population-based eQTL studies, including the GTEx Project, and followed up potentially novel eQTL not observed in the general population. In total, we identified 2314 eQTL of which 90% were cis-acting and 75% were confirmed by at least one of the published studies. While we observed a higher GWAS trait colocalization rate among confirmed eQTL, colocalisation rate of novel eQTL reported for lung-related phenotypes was twice as high as that of confirmed eQTL. Functional enrichment analysis of genes with novel eQTL in PAH highlighted immune-related processes, a suspected contributor to PAH. These potentially novel eQTL specific to or active in PAH could be useful in understanding genetic risk factors for other diseases that share common mechanisms with PAH.

Journal article

Swietlik EM, Ghataorhe P, Zalewska K, Wharton J, Howard LS, Taboada D, Cannon JE, Morrell NW, Wilkins MR, Toshner M, Pepke-Zaba J, Rhodes Cet al., 2020, Plasma metabolomics exhibit response to therapy in chronic thromboembolic pulmonary hypertension, European Respiratory Journal, Vol: 57, Pages: 1-14, ISSN: 0903-1936

Pulmonary hypertension is a condition with limited effective treatment options. Chronic thromboembolic pulmonary hypertension (CTEPH) is a notable exception with pulmonary endarterectomy (PEA) often proving curative. This study investigated the plasma metabolome of CTEPH patients, estimated reversibility to an effective treatment and explored the source of metabolic perturbations.We performed untargeted analysis of plasma metabolites in CTEPH patients compared to healthy controls and disease comparators. Changes in metabolic profile were evaluated in response to PEA. A subset of patients were sampled at three anatomical locations and plasma metabolite gradients calculated.We defined and validated altered plasma metabolite profiles in patients with CTEPH. 12 metabolites were confirmed by ROC analysis to distinguish CTEPH and both healthy (AUCs 0.64–0.94, all p<2×10−5) and disease controls (AUCs 0.58–0.77, all p<0.05. Many of the metabolic changes were notably similar to those observed in idiopathic pulmonary arterial hypertension (IPAH). Only five metabolites (5-methylthioadenosine, N1-methyladenosine, N1-methylinosine, 7-methylguanine, N-formylmethionine) distinguished CTEPH from chronic thromboembolic disease or IPAH. Significant corrections (15–100% of perturbation) in response to PEA were observed in some but not all metabolites. Anatomical sampling identified 188 plasma metabolites, with significant gradients in tryptophan, sphingomyelin, methionine, and Krebs cycle metabolites . Metabolites associated with CTEPH and gradients also showed significant associations with clinical measures of disease severity.We identified a specific metabolic profile that distinguishes CTEPH from controls and disease comparators, despite the observation that most metabolic changes were common to both CTEPH and IPAH patients. Plasma metabolite gradients implicate cardiopulmonary tissue metabolism of metabolites associated with PH and metabolites t

Journal article

Otero-Nunez P, Rhodes C, Wharton J, Swietlik E, Kariotis S, Harbaum L, Dunning M, Elinoff J, Errington N, Thomson R, Iremonger J, Coghlan G, Corris P, Howard L, Kiely D, Church C, Pepke-Zaba J, Toshner M, Wort S, Desai A, Humbert M, Nichols W, Southgate L, Tregouet D-A, Trembath R, Prokopenko I, Graf S, Morrell N, Wang D, Lawrie A, Wilkins Met al., 2020, Multi-omic profiling in pulmonary arterial hypertension, Publisher: EUROPEAN RESPIRATORY SOC JOURNALS LTD, ISSN: 0903-1936

Conference paper

Swietlik E, Ghataorhe P, Zalewska K, Wharton J, Howard LS, Taboada D, Cannon JE, Morrell NW, Wilkins MR, Toshner MR, Pepke-Zaba J, Rhodes CJet al., 2020, Plasma metabolomics in chronic thromboembolic pulmonary hypertension, Publisher: EUROPEAN RESPIRATORY SOC JOURNALS LTD, ISSN: 0903-1936

Conference paper

Rhodes C, Otero-Núñez P, Wharton J, Swietlik E, Kariotis S, Harbaum L, Dunning M, Elinoff J, Errington N, Roger T, Iremonger J, Coghlan G, Corris P, Howard L, Kiely D, Church C, Pepke-Zaba J, Toshner M, Stephen W, Desai A, Humbert M, Nichols W, Southgate L, Tregouet D-A, Trembath R, Prokopenko I, Graf S, Morrell N, Wang D, Lawrie A, Wilkins Met al., 2020, Whole blood RNA profiles associated with pulmonary arterial hypertension and clinical outcome, American Journal of Respiratory and Critical Care Medicine, Vol: 202, Pages: 586-594, ISSN: 1073-449X

Rationale: Idiopathic and hereditary pulmonary arterial hypertension (PAH) are rare but comprise a genetically heterogeneous patient group. RNA-sequencing linked to the underlying genetic architecture can be used to better understand the underlying pathology by identifying key signalling pathways and stratify patients more robustly according to clinical risk. Objectives: Using a three-stage design of RNA discovery, RNA validation/model construction and model validation to define a set of PAH-associated RNAs and a single summarising RNA model score. To define genes most likely to be involved in disease development, we performed Mendelian randomisation (MR) analysis. Methods: RNA-sequencing was performed on whole blood samples from 359 patients with idiopathic, heritable and drug-induced PAH and 72 age- and sex-matched healthy volunteers. The score was evaluated against disease severity markers including survival analysis using all-cause mortality from diagnosis. MR used known eQTL and summary statistics from a PAH GWAS. Measurements and Main Results: We identified 507 genes with differential RNA expression in PAH patients compared to controls. A model of 25 RNAs was able to distinguish PAH with 87% accuracy (AUC 95% CI: 0.791-0.945) in model validation. The RNA model score was associated with disease severity and long-term survival (p=4.66x10-6) in PAH. MR detected an association between SMAD5 levels and PAH disease susceptibility (OR:0.317, 95%CI:0.129-0.776, p=0.012). Conclusions: A whole blood RNA signature of PAH, which includes RNAs relevant to disease pathogenesis, associates with disease severity and identifies patients with poor clinical outcomes. Genetic variants associated with lower SMAD5 expression may increase susceptibility to PAH.

Journal article

Hodgson J, Swietlik EM, Salmon RM, Hadinnapola C, Nikolic I, Wharton J, Guo J, Liley J, Haimel M, Bleda M, Southgate L, Machado RD, Martin JM, Treacy CM, Yates K, Daugherty LC, Shamardina O, Whitehorn D, Holden S, Bogaard HJ, Church C, Coghlan G, Condliffe R, Corris PA, Danesino C, Eyries M, Gall H, Ghio S, Ghofrani H-A, Gibbs JSR, Girerd B, Houweling AC, Howard L, Humbert M, Kiely DG, Kovacs G, Lawrie A, MacKenzie Ross RV, Moledina S, Montani D, Olschewski A, Olschewski H, Ouwehand WH, Peacock AJ, Pepke-Zaba J, Prokopenko I, Rhodes CJ, Scelsi L, Seeger W, Soubrier F, Suntharalingam J, Toshner MR, Trembath RC, Vonk Noordegraaf A, Wort SJ, Wilkins MR, Yu PB, Li W, Gräf S, Upton PD, Morrell NWet al., 2020, Characterization of GDF2 mutations and levels of BMP9 and BMP10 in pulmonary arterial hypertension, American Journal of Respiratory and Critical Care Medicine, Vol: 201, Pages: 575-585, ISSN: 1073-449X

OBJECTIVES: Recently, rare heterozygous mutations in GDF2 were identified in patients with pulmonary arterial hypertension (PAH). GDF2 encodes the circulating bone morphogenetic protein, BMP9, which is a ligand for the BMP type 2 receptor (BMPR2). Here we determine the functional impact of GDF2 mutations and characterised 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 signalling and functional activity was assessed. Plasma BMP9 and BMP10 levels and activity were assayed in PAH patients with GDF2 mutations, and controls. Levels were also measured in a larger cohort of controls (n=120) and idiopathic PAH patients (n=260). MAIN RESULTS: We identified novel rare variation at the GDF2 and BMP10 loci, including copy number variation. In vitro, BMP9 missense proteins demonstrated impaired cellular processing and secretion. PAH patients carrying 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 PAH patients and controls, BMP10 levels were lower in PAH females. A subset of PAH patients 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 signalling in PAH.

Journal article

Ulrich A, Wharton J, Thayer T, Swietlik E, Assad T, Desai A, Graf S, Harbaum L, Humbert M, Morrell N, Nichols W, Soubrier F, Southgate L, Tregouet D-A, Trembath R, Brittain E, Wilkins M, Prokopenko I, Rhodes Cet al., 2020, Mendelian randomization analysis of red cell distribution width in pulmonary arterial hypertension, European Respiratory Journal, Vol: 55, Pages: 1-9, ISSN: 0903-1936

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 if elevated RDW is causally associated with PAH.A two-sample Mendelian randomization (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 multi-centre case-control study (N cases = 642, N disease controls = 15,889). The primary MR analysis was adequately powered to detect a causal effect (OR) from between 1.25-1.52 or greater based on estimates reported in the RDW GWAS 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.Take home message – Mendelian randomization using genetic data from the largest-to-date pulmonary arterial hypertension (PAH) cohort do not support RDW or iron deficiency as a cause of PAH, which is important when interpreting iron replacement trials in this condition.

Journal article

Howard L, He J-G, Wharton J, Watson G, Gall H, Ghofrani H, Huang L, Kiely D, Luo Q, Sheares K, Zhao L, Wilkins Met al., 2019, Late Breaking Abstract - Supplementation of iron in pulmonary hypertension (SIPHON): results from a randomised controlled crossover trial, European-Respiratory-Society (ERS) International Congress, Publisher: European Respiratory Society, Pages: 1-2, ISSN: 0903-1936

Background: Iron deficiency in the absence of anaemia is common in patients with idiopathic pulmonary arterial hypertension (IPAH). It has been associated with inappropriately raised levels of hepcidin, the key regulator of iron homeostasis and correlates with disease severity and worse clinical outcomes. Oral iron absorption may be impeded by elevated hepcidin levels. The safety and benefits of parenteral iron replacement in IPAH are unknown.Methods: Iron-deficient patients (defined as ferritin <37µg/L, serum iron <10.3µmol/L, transferrin saturation <16.4% or soluble transferrin receptor >28.1nmol/L) were randomised to receive a single infusion of ferric carboxymaltose (FCM) 15 mg/kg up to 1000mg or saline placebo with cross-over after 12 weeks of treatment. The primary outcome was change in endurance time on cardio-pulmonary exercise testing (CPET) at 12 weeks. Secondary measures included resting haemodynamics, incremental CPET, iron indices, 6 minute walk distance, WHO functional class, quality of life score, NT-proBNP and cardiac anatomy and function from MRI.Results: 40 patients enrolled and 39 provided data for analysis. FCM improved iron indices, with a significant increase in ferritin (P=0.0003) and reduction in soluble transferrin receptor levels (P<0.0001) at 12 weeks, and was well tolerated. There was no significant change in endurance time or in any other secondary endpoint. Data from a meta-analysis including a parallel study from Fuwai Hospital, China, will be available at the time of presentation.Conclusion: Iron repletion through a single infusion of FCM improves iron indices and is well tolerated but does not improve exercise capacity in PAH.

Conference paper

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