Abstract Body (Do not enter title and authors here): Background: Epigenetic changes in gene expression due to DNA methylation regulate pulmonary vascular structure and function. Genetic or acquired alterations in DNA methylation or demethylation promote development of pulmonary arterial hypertension (PAH). However, the DNA methylome signature of human PAH and its transcriptomic consequences are unknown.

Methods: Reduced Representation Bisulfite Sequencing was used for epigenome-wide mapping of DNA methylation in whole peripheral blood of 10 healthy people and 20 age/sex matched PAH patients from the PAH Biobank. We used whole-exome sequencing to identify two PAH cohorts, one free of mutations of know PAH-associated gene variants, and a second with mutations of TET2, a newly identified PAH gene that encodes TET2, which mediates DNA demethylation.

Results: Patients carrying TET2 mutations had greater DNA CpG methylation compared to mutation-free PAH patients, who themselves had increased methylation compared to controls. Differentially Methylated Regions (DMR) were more common in patients with PAH with TET2 mutations (1,135 DMR) than without mutations (260 DMR). In a public, blood-derived, PAH transcriptomic dataset, we assayed for transcript downregulation of genes that were hypermethylated in our cohort. 161 TET2 DMRs (38 of which were common to PAH without TET2 mutation) corresponded to downregulated transcripts. Functional analysis of the hypermethylated genes in TET2 patients revealed similar conserved functions, related to T cell differentiation and metabolism. Functional comparison of the 38 hypermethylated genes found in both PAH cohorts and the 161 hypermethylated genes unique to TET2 mutation also revealed regulation of T cell pathways. The most hypermethylated and downregulated genes was transcription factor 7 (TCF7), a regulator of T cell differentiation, which would be predicted to promote inflammation.

Conclusion: Pan-chromosomal hypermethylation in PAH is greatest in patients with TET2 mutations. Gene hypermethylation may contribute to hallmarks of PAH, including alterations in immune function, T cell differentiation, and metabolism.