Germline and somatic variants in DNMT3A and other clonal haematopoiesis of indeterminate potential genes contribute to pulmonary arterial hypertension

Abstract

Background and aims: Multiple germline gene variants promote familial and idiopathic pulmonary arterial hypertension (PAH); however, none are consistently identified in associated PAH with connective tissue disease (APAH-CTD). Moreover, the role of somatic variants in genes mediating clonal haematopoiesis of indeterminate potential (CHIP) in PAH is unknown. Here, somatic and germline DNMT3A variants and CHIP gene variants in PAH were evaluated.

Methods: Exome sequencing (ES) was compared between PAH Biobank participants (n = 1832 European ancestry/2572 total), vs. gnomAD controls (7509 European ancestry/141 456 total). Subsequently, targeted panel sequencing (TPS) of 22 CHIP genes, including DNMT3A, was performed in PAH (n = 1659) vs. controls (n = 3644). Somatic CHIP variants in the UK Biobank using ES (controls = 448 239; PAH = 2559) were also assessed. DNMT3A mRNA expression was measured in peripheral blood mononuclear cells (PBMCs) of patients with scleroderma APAH-CTD (n = 50), idiopathic PAH (n = 30), scleroderma without PAH (n = 19), and healthy controls (n = 41). Hemodynamic were evaluated in haematopoietic Dnmt3a-knockout mice.

Results: Predicted deleterious germline DNMT3A variants were increased in subjects of European ancestry (6/1832) vs. controls (6/7509) (relative risk [RR] = 4.1, P = .018). In the entire PAH Biobank cohort (n = 2572), DNMT3A germline and somatic variants were further enriched (PAH: 1.28% vs. controls: .43%, P = 1.65 × 10-10). Eight DNMT3A mutations (.39%) were likely germline (female/male: 7/1) and 25 (.82%) likely somatic (female/male: 21/4), including 13/33 APAH-CTD participants. TPS identified CHIP in 242 PAH subjects (48% DNMT3A). DNMT3A- and all-CHIP variants were associated with PAH after correcting for age, sex, and age-CHIP interactions (odds ratio [OR]: 25.44, P = 4.50 × 10-5; OR: 23.35, P = 2.87 × 10-8, respectively). In the UK Biobank, CHIP mutations were increased in PAH (PAH = 5.35% vs. Control = 3.45%, P ≤ .0001). DNMT3A was reduced in PAH-PBMCs (area under curve [AUC] = .82) (P < .0001). Haematopoietic Dnmt3a-knockout in mice caused inflammatory PAH, which was attenuated by IL-1β antibody therapy.

Conclusions: Germline DNMT3A variants and somatic variants of DNMT3A and CHIP genes increase the risk of PAH, including APAH-CTD, promote inflammation, and constitute potential biomarkers and therapeutic targets.

Keywords: DNA (cytosine 5) Methyltransferase 3 Alpha (DNMT3A) • Associated pulmonary arterial hypertension; PPM1D • Somatic gene variant; Connective tissue disease; DNA methylation.