We propose a pulmonary vascular disease (PVD) NOMICS study to systemically characterize WHO Groups 1- 5 pulmonary hypertension (PH) patients utilizing clinical, biochemical, imaging, physiological and pathological assessments combined with genomic and RNA technology to improve our mechanistic and pathobiological understanding of the pulmonary vascular disease process. With a new OMIC typing we ultimately hope to better target the right patient for the right therapeutic intervention Specifically, we will develop an algorithmic approach for the evaluation and classification of all PH patients inclusive of clinical testing; we will assess genomics across WHO Groups 1-5 classification cohorts; we will assess miRNA across WHO Groups 1-5 classification cohorts in available tissue; we will assess dynamic circulating miRNA across WHO Groups 1-5 classification cohorts at two intervals; we will utilize advanced analytics of miRNA, DNA and bioinformatics modeling strategies to classify patients into a new grid based upon the above findings whereby patients can be studied together across the traditional WHO Group categories and ultimately assess disease specific modalities and therapeutic targets. We will genetically characterize all patients by sequencing them to screen for mutations in all the known genes for PH (BMPR2, ACVRL1, CAV1, ENG, SMAD9, KCNK3, EIF2AK4). We will make libraries of all candidate genes for resequencing using a custom Haloplex kit targeting all exons and adjacent splice sites and conserved regulatory sequences followed by indexing and multiplexing to decrease costs. We will assess small RNA deep-sequencing using our own RNA deep-sequencing (RNAseq) protocols, our RNAseq cDNA library preparation and our bioinformatics pipeline. To capture longer RNA transcripts we will use a HydroRNAseq protocol, a method which is based on the sRNAseq with partial alkaline hydrolysis for longer sequences. Since the lifespan of a transcript and the translational efficiency of protein-coding transcripts is determind by regulatory protein complexes associated with RNA from transcription to degradation, we developed a cross-linking and immunoprecipitation-sequencing protocol that uses photoactivatable nucleoside analogous to increase the cross-linking efficiency between RNA and binding-proteins. We will be measuring circulating miRNAs, as purely non-structural components which might have better kinetics than established protein biomarkers and which may respond to systemic changes. Because they are widely expressed with/or without additional stimuli, they might be potential biomarkers, may help coordinate responses to hypoxia, ischemia, flow or stretch mediated changes, inflammation or epigenetic factors, may reflect tissue-specific expression and may play a role in transcriptional regulation. Although generally perceived to be less stable than miRNAs, they can reliably be measured in circulation, in some cases to correlate with the tissue expression, and thus may be a unifying and improved biomarker pathway. Ultimately, these techniques will enable novel characterization of PVD into pathobiologic substrates.
The current WHO Group classification of pulmonary hypertension has been a useful clinical tool, has led to many therapeutic advances for the WHO Group 1 patients, more recently for Group 4 patients, and has provided significant outcome and epidemiological evidence of differences in survival amongst the patients. However, it has become increasingly evident that this system is flawed with regard to a deeper understanding of pathobiologic subtypes of pulmonary vascular disease. At the NYP- Columbia and Cornell PH Centers, broad expertise and the ability to carry out key novel testing will enable us to help re-define pulmonary vascular disease in terms of pathobiologic substrates.
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