Protein Biomarkers and Risk Scores in Pulmonary Arterial Hypertension Associated with Ventricular Septal Defect: Integration of Multi-Omics and Validation

The molecular mechanisms underlying pulmonary arterial hypertension (PAH) in congenital ventricular septal defects (VSD) are unclear. We aimed to reveal molecular pathways and potential biomarkers by multi-omics analysis in VSD-PAH. Plasma from 160 children, including 120 VSD patients with/without PAH and 40 healthy children was studied by integrated proteomics, metabolomics, and bioinformatics analyses. Proteomics (iTRAQ) identified 107 differential proteins (DPs) between patients with/without PAH including significantly increased ADIPO, DBH, ANPEP, TRF1, GP1BA and decreased GNAS in VSD-PAH. Metabolomics discovered 191 differential metabolites between patients with/without PAH including elevation of 5-HT, taurine, creatine, sarcosine, and 2-oxobutanoate and decrease of vanillylmandelic acid, 3,4-dihydroxymandelate, 15-Keto-PGF2𝛼, fructose-6-phosphate , L-glutamine, dehydroascorbate, hydroxypyruvate, threonine, L-cystine, and 1-aminocyclopropane-1-carboxylate. The DPs differential proteins were validated in a new cohort of patients (N=80). Integrated analyses identified key pathways including cAMP, ECM-receptor interaction, AMPK, HIF-1, PI3K-Akt signaling pathways, and amino acid metabolisms. Increased plasma protein levels of DBH, ADIPO, and ANPEP were found to be independently associated with the occurrence of PAH with a new total risk score from these three proteins developed for clinical diagnosis. In this integrated multi-omics analysis in VSD-PAH patients, we have for the first time found that VSD-PAH patients present important differential proteins, metabolites, and key pathways. We have developed a total-risk-score (based on the plasma concentration of DBH, ANPEP and ADIPO) as predictor of development of PAH in CHD-VSD patients. Therefore, these proteins may be used as biomarkers and the new total-risk-score has significant clinical implications in the diagnosis of PAH.