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


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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.

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