Blood transcriptomics: the future of profiling pulmonary hypertension?

By Pablo Otero Nunez and Chris Rhodes

Pulmonary arterial hypertension (PAH) is a rare but devastating disorder with survival rates estimated at 52-75% at 5-years, even with modern day therapy¹. Distal pulmonary arteries of PAH patients undergo endothelial damage, smooth muscle and fibroblast proliferation, and inflammatory processes; which cause vasoconstriction and occlusion of vessels. The resulting increase in pulmonary vascular resistance is, in many patients, the cause of right heart failure and death.

PAH has a complex, unclear pathogenesis. Genetic predisposition to pulmonary hypertension is well recognised as is patient heterogeneity which severely burdens the search for effective therapies, as well as current clinical management. The so-called ‘omics’ approach, or high-throughput screening of all molecules of a biological class in a given tissue sample, has gained a lot of momentum in recent years. This is because, as opposed to standard clinical phenotypes for patient characterisation, omics offers greater granularity and could improve initial risk stratification, treatment selection and monitoring, as well as providing insights into biological pathways not yet targeted by current therapies.

Transcriptome profiling through RNA sequencing permits a high-quality comprehensive analysis of gene expression unrestricted by the probes designed for arrays which can be scaled to large sample sizes. Our recent study of transcriptomics in PAH focused on whole blood RNA analysis, as this offers an alternative “liquid biopsy” to lung biopsy, which carries a high risk in PAH, and can be performed sequentially.

Adopting a three-stage analysis using 359 PAH patients and 72 healthy controls, we identified 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. We were able to externally validate this signature with a recent meta-analysis of prior PAH blood microarray studies² and a signature derived from a study of explanted lung tissues from late-stage PAH patients³. In this rare disease, the sample size we used was an order of magnitude above that used in prior studies, and thus adequately powered to produce a model that performed with 87% accuracy in our validation test sample.

In contrast with existing literature, we were unable to demonstrate a specific peripheral transcript signature in PAH patients who respond well to calcium antagonists (vasoresponders) that would separate them from non-responders. This signature, however, had been reported in a study which had again relied on much smaller patient numbers⁴.

AMD1, which encodes a key enzyme involved in polyamine biosynthesis, was the only gene consistently dysregulated in both external analyses and in our dataset, with lower levels in PAH samples in all three studies. This finding was in contrast with prior metabolomic evidence showing higher polyamine levels as being negatively associated with survival⁵, although negative feedback processes could account for such disparities.

Using data from an international GWAS of PAH⁶, of which we represented the largest dataset, we performed an exploratory Mendelian randomisation (MR) analysis to determine which genes were most likely to be causally linked to disease development. This showed two significant hits of expression quantitative trait loci (eQTL) for genes we had observed as being dysregulated in PAH patients, were themselves associated with PAH. One of those genes, SMAD5, is part of the TGFβ-BMP signalling pathway, acting as a downstream effector of BMPR2, a cellular receptor encoded by the gene most commonly mutated in heritable PAH patients. SMAD5 downregulation in PAH, independent of BMPR2 mutation status, highlights the dysregulation of a different facet of this pathway in PAH pathogenesis.

This study does not assess the role of epigenetic and proteomic differences or post-translational modifications in the pathogenesis of PAH, which could add further information to the circulating transcriptome. Current research at our lab is focusing on these other areas in the omics field. Integrating metabolomic, genomic and transcriptomic data could add further to the canvas of molecular signatures associated with PAH and bring about personalised medicine for the prevention, diagnosis and treatment of this devastating disorder.

References

1. Galie N, Humbert M, Vachiery JL, Gibbs S, Lang I, Torbicki A, Simonneau G, Peacock A, Vonk Noordegraaf A, Beghetti M, Ghofrani A, Gomez Sanchez MA, Hansmann G, Klepetko W, Lancellotti P, Matucci M, McDonagh T, Pierard LA, Trindade PT, Zompatori M, Hoeper M. 2015 ESC/ERS Guidelines for the diagnosis and treatment of pulmonary hypertension: The Joint Task Force for the Diagnosis and Treatment of Pulmonary Hypertension of the European Society of Cardiology (ESC) and the European Respiratory Society (ERS): Endorsed by: Association for European Paediatric and Congenital Cardiology (AEPC), International Society for Heart and Lung Transplantation (ISHLT). Eur Respir J 2015; 46: 903-975.

2. Elinoff JM, Mazer AJ, Cai R, Lu M, Graninger G, Harper B, Ferreyra GA, Sun J, Solomon MA, Danner RL. Meta-analysis of Blood Genome-Wide Expression Profiling Studies in Pulmonary Arterial Hypertension. Am J Physiol Lung Cell Mol Physiol 2019.

3. Stearman RS, Bui QM, Speyer G, Handen A, Cornelius AR, Graham BB, Kim S, Mickler EA, Tuder RM, Chan SY, Geraci MW. Systems Analysis of the Human Pulmonary Arterial Hypertension Lung Transcriptome. Am J Respir Cell Mol Biol 2019; 60: 637-649.

4. Hemnes AR, Trammell AW, Archer SL, Rich S, Yu C, Nian H, Penner N, Funke M, Wheeler L, Robbins IM, Austin ED, Newman JH, West J. Peripheral blood signature of vasodilator-responsive pulmonary arterial hypertension. Circulation 2015; 131: 401-409; discussion 409.

5. Rhodes CJ, Ghataorhe P, Wharton J, Rue-Albrecht KC, Hadinnapola C, Watson G, Bleda M, Haimel M, Coghlan G, Corris PA, Howard LS, Kiely DG, Peacock AJ, Pepke-Zaba J, Toshner MR, Wort SJ, Gibbs JS, Lawrie A, Graf S, Morrell NW, Wilkins MR. Plasma Metabolomics Implicates Modified Transfer RNAs and Altered Bioenergetics in the Outcomes of Pulmonary Arterial Hypertension. Circulation 2017; 135: 460-475.

6. Rhodes CJ, Batai K, Bleda M, Haimel M, Southgate L, Germain M, Pauciulo MW, Hadinnapola C, Aman J, Girerd B, Arora A, Knight J, Hanscombe KB, Karnes JH, Kaakinen M, Gall H, Ulrich A, Harbaum L, Cebola I, Ferrer J, Lutz K, Swietlik EM, Ahmad F, Amouyel P, Archer SL, Argula R, Austin ED, Badesch D, Bakshi S, Barnett C, Benza R, Bhatt N, Bogaard HJ, Burger CD, Chakinala M, Church C, Coghlan JG, Condliffe R, Corris PA, Danesino C, Debette S, Elliott CG, Elwing J, Eyries M, Fortin T, Franke A, Frantz RP, Frost A, Garcia JGN, Ghio S, Ghofrani HA, Gibbs JSR, Harley J, He H, Hill NS, Hirsch R, Houweling AC, Howard LS, Ivy D, Kiely DG, Klinger J, Kovacs G, Lahm T, Laudes M, Machado RD, MacKenzie Ross RV, Marsolo K, Martin LJ, Moledina S, Montani D, Nathan SD, Newnham M, Olschewski A, Olschewski H, Oudiz RJ, Ouwehand WH, Peacock AJ, Pepke-Zaba J, Rehman Z, Robbins I, Roden DM, Rosenzweig EB, Saydain G, Scelsi L, Schilz R, Seeger W, Shaffer CM, Simms RW, Simon M, Sitbon O, Suntharalingam J, Tang H, Tchourbanov AY, Thenappan T, Torres F, Toshner MR, Treacy CM, Vonk Noordegraaf A, Waisfisz Q, Walsworth AK, Walter RE, Wharton J, White RJ, Wilt J, Wort SJ, Yung D, Lawrie A, Humbert M, Soubrier F, Tregouet DA, Prokopenko I, Kittles R, Graf S, Nichols WC, Trembath RC, Desai AA, Morrell NW, Wilkins MR, Consortium UNBRD, Consortium UPCS, Consortium UPB. Genetic determinants of risk in pulmonary arterial hypertension: international genome-wide association studies and meta-analysis. Lancet Respir Med 2019; 7: 227-238.