Novel Mutations and Decreased Expression of the Epigenetic Regulator TET2 in Pulmonary Arterial Hypertension

Abstract

Background: Pulmonary arterial hypertension (PAH) is a lethal vasculopathy. Hereditary cases are associated with germline mutations in BMPR2 and 16 other genes; however, these mutations occur in <25% of patients with idiopathic PAH and are rare in PAH associated with connective tissue diseases. Preclinical studies suggest epigenetic dysregulation, including altered DNA methylation, promotes PAH. Somatic mutations of Tet-methylcytosine-dioxygenase-2 (TET2), a key enzyme in DNA demethylation, occur in cardiovascular disease and are associated with clonal hematopoiesis, inflammation, and adverse vascular remodeling. The role of TET2 in PAH is unknown.

Methods: To test for a role of TET2, we used a cohort of 2572 cases from the PAH Biobank. Within this cohort, gene-specific rare variant association tests were performed using 1832 unrelated European patients with PAH and 7509 non-Finnish European subjects from the Genome Aggregation Database (gnomAD) as control subjects. In an independent cohort of 140 patients, we quantified TET2 expression in peripheral blood mononuclear cells. To assess causality, we investigated hemodynamic and histological evidence of PAH in hematopoietic Tet2-knockout mice.

Results: We observed an increased burden of rare, predicted deleterious germline variants in TET2 in PAH patients of European ancestry (9/1832) compared with control subjects (6/7509; relative risk=6; P=0.00067). Assessing the whole cohort, 0.39% of patients (10/2572) had 12 TET2 mutations (75% predicted germline and 25% somatic). These patients had no mutations in other PAH-related genes. Patients with TET2 mutations were older (71±7 years versus 48±19 years; P<0.0001), were more unresponsive to vasodilator challenge (0/7 versus 140/1055 [13.2%]), had lower pulmonary vascular resistance (5.2±3.1 versus 10.5±7.0 Wood units; P=0.02), and had increased inflammation (including elevation of interleukin-1β). Circulating TET2 expression did not correlate with age and was decreased in >86% of PAH patients. Tet2-knockout mice spontaneously developed PAH, adverse pulmonary vascular remodeling, and inflammation, with elevated levels of cytokines, including interleukin-1β. Long-term therapy with an antibody targeting interleukin-1β blockade resulted in regression of PAH.

Conclusions: PAH is the first human disease related to potential TET2 germline mutations. Inherited and acquired abnormalities of TET2 occur in 0.39% of PAH cases. Decreased TET2 expression is ubiquitous and has potential as a PAH biomarker.

Keywords: CREST syndrome; DNA methylation; connective tissue diseases; epigenetics; pulmonary arterial hypertension; scleroderma, limited.

Figure 1.. Increased mutation and decreased TET2 gene expression in blood from PAH patients.

Topologic analysis and gene expression of the Human DNA tet methylcytosine dioxygenase 2 (TET2) in pulmonary arterial hypertension (PAH). A) Distribution of TET2 somatic and germline deleterious variants. B) Locations of rare deleterious PAH patient-derived TET2 variants within the two-dimensional protein structures. The numbers of variants at each amino acid position are indicated on the y-axis. Germline variants are shown above the protein schematic; mosaic variants are below. D-MIS, predicted damaging missense; LGD, likely-gene-disrupting (stopgain, frameshift). TET/JBP, TET/J-binding protein catalytic domain. Protein domain coordinates were modified according to UniProtKB). C) TET2 gene expression in peripheral blood mononuclear cells (PBMCs) from 41 healthy control, 30 idiopathic PAH (IPAH), 50 scleroderma associated PAH (SSc-PAH) and 19 scleroderma without PAH (SSc). Violin plot was used to visualise data distribution; black line is the median; red lines are the upper and lower quartile. one-way ANOVA. **P<0.01; ****P<0.0001; n.s. non-significant.

Figure 2.. TET2 mutation is associated with a pro-inflammatory phenotype in blood of PAH patients.

Expression of 30 pro-inflammatory cytokines were assessed in blood of 9 healthy patients, 10 age/sex-matched PAH TET2 variant carriers and 10 matched PAH TET2 non-carriers patients. A) TET2 variant carriers show increased levels of 28 cytokines (IFNa2; IP-10; IL-12p40; IL12p70; IL-6; IL-1β; IL-2; Fractalkine; IFNy; IL-15; IL-1a; IL-18; IL-3; G-CSF; IL-7; TNFa; IL-17A; MIP-1a; MIP-1B; MDC; TNFB; IL-5; Flt-3L; MCP-3; IL-8; RANTES; GRO alpha) and decreased levels of 2 cytokines (MCP-1; Eotaxin) compared to matched non-carriers patients. Values are expressed as fold change compared to healthy patients. B) TET2 carriers display overall increased levels of pro-inflammatory markers measured by an increase of the area under the curve. One-way ANOVA. Values are expressed as mean±SEM. ***P<0.001

Figure 3.. TET2 depletion in hematopoietic cells and pulmonary hypertension in a murine model.

Haemodynamic assessment of pulmonary hypertension using right heart catheterization and echocardiography of 6–15 Tet2^−/− and age-sex matched Tet2^f/f mice show A) increased right ventricular systolic pressure (RVSP), B) decreased pulmonary artery acceleration time (PAAT) and, C) increased total pulmonary resistance (TPR) in Tet2^−/− mice compared to Tet2^f/f animals. D) Pulmonary arterial vascular remodelling was blindly quantified by the percent of wall thickness: (total diameter−internal diameter)/total diameter by immunofluorescence (smooth muscle actin); 10 arteries per mice on 5 mice per group. Pulmonary vascular wall thickness is increase in Tet2^−/− mice compared to Tet2^f/f. Perfusion of pulmonary vessels has been assessed in Tet2^−/− and Tet2^f/f mice (FITC albumin perfusion, 2 photon microscopy). Perfused vessels have been clustered according to their volumes in 3 categories: small (15–225μm³), intermediate (225–3347μm³) and big (3347–50000μm³). Tet2^−/− mice display decreased numbers of E) small and F) intermediate (P=0.08) perfused vessels whilst G) number of large vessels perfused remains the same compared to Tet2^f/f (number of vessels/1e⁶ μm3). H) Representative pictures of vessels perfused by FITC-albumin in the lung of Tet2^−/− and Tet2^f/f (500X550μm). I) Compared to Tet2^f/f animals, lungs from mutated mice (Tet2^−/−) show an elevated macrophages population measured by fluorescence activated cell sorting (FACS; F4/80; CD11b; n=6 Tet2^−/− and age-sex matched Tet2^f/f). J) Quantification of inflammatory cytokines and chemokines in total lung of 3 Tet2^−/− and age-sex matched Tet2^f/f mice displays up-regulation of Il1b, Cxcr2, Csf3r, C5ar1, Fpr2, Amica1, Ccr1, Mmp9, Cd33, Itgam, H2-Q10, Arg2, Clec4n, Il1rn, Rsad2, Il1r2 and downregulation of Ccr6 gene expression in mutated animals. Results show change >1.8 fold Log2-transformed normalized NanoString mRNA counts. Unpaired t- test. Values are expressed as mean±SEM. n.s.: non-significant; *P<0.05; **P<0.01; ***P<0.001.

Figure 4.. IL-1β blockade reverses PH in Tet2 mutated mice.

10, 7-monthTet2^−/− mice were treated with an antibody against IL-1β (Tet2^−/− + IL-1β ab; IP; 10mg/kg/week; 6weeks) or IgG2a (Tet2^−/− + IL-1β ab IgG2a; IP; 10mg/kg/week; 6 weeks). Tet2^−/− + IL-1β ab mice showed A) significant increased pulmonary artery acceleration time (PAAT); B) decreased right ventricular systolic pressure (RVSP); C) decreased mean pulmonary arterial hypertension (mPAP); D) reduced Total pulmonary resistance (TPR) normalized by body weight associated. E) IL-1β antibody treatment significantly prevents weight loss observed in Tet2^−/− mice treated with IgG2a. n=5 per group. One-way ANOVA; mixed effect ANOVA. Values are expressed as mean±SEM. *P<0.05, **P<0.01, ***P<0.001.