Humans with Pulmonary Arterial Hypertension display a global hypermethylation signature that worsens in patients who have a mutation in the gene encoding the methylation eraser, Tet Methylcytosine Dioxygenase 2 (TET2)

Epigenetic changes in gene expression due to DNA methylation are important physiologic and pathologic regulators of pulmonary vascular structure and function. Genetic or acquired alterations in DNA methylation or demethylation have been associated with the development and progression of pulmonary arterial hypertension (PAH). However, the DNA methylome signature of human PAH and its consequences on the PAH transcriptome are unknown. Reduced Representation Bisulfite Sequencing (RRBS) was used for epigenome-wide mapping of DNA methylation in whole peripheral blood of 10 healthy people and 20 age/sex matched PAH patients. RNA deep sequencing was performed in parallel on the same samples. We used whole-exome sequencing to identify two PAH cohorts, one free of mutations in the genes know to be associated with PAH patients and the second had mutations of TET2 , a newly identified PAH gene that encodes an enzyme that mediates DNA demethylation. We report an increased in global DNA methylation in the blood of PAH patients compared to healthy controls. Patients carrying the TET2 mutation had a further increase in DNA compared to mutation free PAH patients. We identified 1,069 unique Differentially Methylated Regions (DMR) in the blood of PAH patients with a TET2 mutation. When organized into functional groups, we observed an enrichment of genes involved in ‘immune’, ‘cell differentiation’, or ‘metabolic’. When we compared these genes to publicly available data from an independent study on blood from PAH patients compared to controls, we identified 218 mRNA transcripts that align with our data (e.g. are hypermethylated blood genes that are downregulated in PAH blood), and functional analysis of these genes reveals common enriched terms in ‘immune’, ‘cell differentiation’, or ‘metabolic’ function. We characterized DNA methylation changes associated with TET2 mutation in the blood of patients with PAH compared to controls. This data demonstrates that epigenetic regulation of genes by methylation is involved in altered immune function, cell differentiation and metabolism in PAH. ### Competing Interest Statement The authors have declared no competing interest. ### Funding Statement Source of funding. This study was supported in part by U.S. National Institutes of Health (NIH) grants NIH 1R01HL113003-01A1 (S.L.A.) NIH 2R01HL071115-06A1 (S.L.A) NIH R24HL105333 (W.C.N.) Canada Foundation for Innovation and the Queens Cardiopulmonary Unit (QCPU) 229252 and 33012 (S.L.A.) Tier 1 Canada Research Chair in Mitochondrial Dynamics and Translational Medicine 950-229252 (S.L.A.) Canadian Institutes of Health Research (CIHR) Foundation Grant CIHR FDN 143261 the William J. Henderson Foundation (S.L.A.) and Canadian Vascular Network Scholar Award (F.P.) and Paroian Family PH Research Scholarship from the pulmonary hypertension association of Canada (F.P.) and the lung and heart research institute of Quebec foundations grant. Further support was provided by an Ontario Molecular Pathology Research Network (OMPRN)/ Ontario Institute for Cancer Research (OICR) Cancer Pathology Translational Research Grant and a Canada Foundation for Innovation Grant (M.J.R.). ### Author Declarations I confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained. Yes The details of the IRB/oversight body that provided approval or exemption for the research described are given below: Patient samples were obtained following informed consent, approved by Queen's University Institutional Review Boards (#6016826) I confirm that all necessary patient/participant consent has been obtained and the appropriate institutional forms have been archived, and that any patient/participant/sample identifiers included were not known to anyone (e.g., hospital staff, patients or participants themselves) outside the research group so cannot be used to identify individuals. Yes I understand that all clinical trials and any other prospective interventional studies must be registered with an ICMJE-approved registry, such as ClinicalTrials.gov. I confirm that any such study reported in the manuscript has been registered and the trial registration ID is provided (note: if posting a prospective study registered retrospectively, please provide a statement in the trial ID field explaining why the study was not registered in advance). Yes I have followed all appropriate research reporting guidelines, such as any relevant EQUATOR Network research reporting checklist(s) and other pertinent material, if applicable. Yes Aggregate data can be shared with researchers at established Universities or Medical schools upon request.