Germline and Somatic Mutations in DNA Methyltransferase 3A(DNMT3A)Predispose to Pulmonary Arterial Hypertension (PAH) in Humans and Mice:Implications for Associated PAH

Abstract

AbstractBackgroundMutations are found in 10-20% of idiopathic PAH (IPAH) patients, but none are consistently identified in connective tissue disease-associated PAH (APAH), which accounts for ∼45% of PAH cases.TET2mutations, a cause of clonal hematopoiesis of indeterminant potential (CHIP), predispose to an inflammatory type of PAH. We now examine mutations in another CHIP gene,DNMT3A, in PAH.MethodsWe assessedDNMT3Amutation prevalence in PAH Biobank subjects as compared with controls, first using whole exome sequencing (WES)-derived CHIP calls in 1832 PAH Biobank patients versus 7509 age-and sex-matched gnomAD controls. We then performed deep, targeted panel sequencing of CHIP genes on a subset of 710 PAH Biobank patients and compared the prevalence ofDNMT3Amutations therein to an independent pooled control cohort (N = 3645). In another cohort of 80 PAH patients and 41 controls,DNMT3AmRNA expression was studied in peripheral blood mononuclear cells (PBMCs). Finally, we evaluated the development of PAH in a conditional, hematopoietic,Dnmt3aknockout mouse model.ResultsDNMT3Amutations were more frequent in PAH cases versus control subjects in the WES dataset (OR 2.60, 95% CI: 1.71-4.27). Among PAH patients, 33 hadDNMT3Avariants, most of whom had APAH (21/33). While 21/33 had somatic mutations (female:male 17:4), germline variants occurred in 12/33 (female:male 11:1). Hemodynamics were comparable with and withoutDNMT3Amutations (mPAP=58±21 vs. 52±18 mmHg); however, patients withDNMT3Amutations were unresponsive to acute vasodilator testing. Targeted panel sequencing identified that 14.6% of PAH patients had CHIP mutations (104/710), withDNMT3Aaccounting for 49/104. There was a significant association between all CHIP mutations and PAH in analyses adjusted for age and sex (OR 1.40, 95% CI: 1.09-1.80), thoughDNMT3ACHIP alone was not significantly enriched (OR:1.15, 0.82-1.61).DNMT3Aexpression was reduced in patient-derived versus control PAH-PBMCs. Spontaneous PAH developed inDnmt3a-/-mice, and it was exacerbated by 3 weeks of hypoxia.Dnmt3a-/-mice had increased lung macrophages and elevated plasma IL-13. The IL-1β antibody canakinumab attenuated PAH inDnmt3a-/-mice.ConclusionsGermline and acquiredDNMT3Avariants predispose to PAH in humans.DNMT3AmRNA expression is reduced in human PAH PBMCs. Hematopoietic depletion of Dnmt3a causes inflammatory PAH in mice.DNMT3Ais a novel APAH gene and may be a biomarker and therapeutic target.