Sean Agbor-Enoh1,2,3, Michael A Solomon4,5, Samuel B Brusca4,6, Jason M Elinoff4, Yvette Zou4, Moon Kyoo Jang1,2, Hyesik Kong1,2, Cumhur Y Demirkale4, Junfeng Sun4, Fayaz Seifuddin1, Mehdi Pirooznia1, Hannah A Valantine2,7, Carl Tanba8, Abhishek Chaturvedi9, Grace M Graninger4, Bonnie Harper4, Li-Yuan Chen4, Justine Cole10, Manreet Kanwar11, Raymond L Benza12, Ioana R Preston8. 1. Division of Intramural Research, National Heart, Lung, and Blood Institute, Bethesda, MD (M.J., H.K., F.S., M.P., S.A.-E.). 2. Genomic Research Alliance for Transplantation (GRAfT), Bethesda, MD (M.J., H.K., H.A.V., S.A.-E.). 3. Department of Internal Medicine, Division of Pulmonary and Critical Care Medicine, Johns Hopkins University School of Medicine, Baltimore, MD (S.A.-E.). 4. Pulmonary Arterial Hypertension Section of the Critical Care Medicine Department (S.B.B., J.M.E., Y.Z., C.Y.D., J.S., G.M.G., B.H., L.-Y.C., M.A.S.), National Institutes of Health Clinical Center, Bethesda, MD. 5. Cardiology Branch, National Heart, Lung, and Blood Institute of the National Institutes of Health, Bethesda, MD (M.A.S.). 6. Department of Internal Medicine, Division of Cardiology, University of California, San Francisco (S.B.B.). 7. Department of Internal Medicine, Stanford University School of Medicine, Palo Alto, CA (H.A.V.). 8. Department of Internal Medicine, Division of Pulmonary, Critical Care, and Sleep Medicine, Tufts Medical Center, Boston, MA (C.T., I.R.P.). 9. Pauley Heart Center, Virginia Commonwealth University School of Medicine, Richmond (A.C.). 10. Department of Laboratory Medicine (J.C.), National Institutes of Health Clinical Center, Bethesda, MD. 11. Cardiovascular Institute at Allegheny Health Network, Pittsburgh, PA (M.K.). 12. Department of Internal Medicine, Division of Cardiovascular Medicine, The Ohio State University Wexner Medical Center, Columbus (R.L.B.).
Abstract
BACKGROUND: Cell-free DNA (cfDNA) is a noninvasive marker of cellular injury. Its significance in pulmonary arterial hypertension (PAH) is unknown. METHODS: Plasma cfDNA was measured in 2 PAH cohorts (A, n=48; B, n=161) and controls (n=48). Data were collected for REVEAL 2.0 (Registry to Evaluate Early and Long-Term PAH Disease Management) scores and outcome determinations. Patients were divided into the following REVEAL risk groups: low (≤6), medium (7-8), and high (≥9). Total cfDNA concentrations were compared among controls and PAH risk groups by 1-way analysis of variance. Log-rank tests compared survival between cfDNA tertiles and REVEAL risk groups. Areas under the receiver operating characteristic curve were estimated from logistic regression models. A sample subset from cohort B (n=96) and controls (n=16) underwent bisulfite sequencing followed by a deconvolution algorithm to map cell-specific cfDNA methylation patterns, with concentrations compared using t tests. RESULTS: In cohort A, median (interquartile range) age was 62 years (47-71), with 75% female, and median (interquartile range) REVEAL 2.0 was 6 (4-9). In cohort B, median (interquartile range) age was 59 years (49-71), with 69% female, and median (interquartile range) REVEAL 2.0 was 7 (6-9). In both cohorts, cfDNA concentrations differed among patients with PAH of varying REVEAL risk and controls (analysis of variance P≤0.002) and were greater in the high-risk compared with the low-risk category (P≤0.002). In cohort B, death or lung transplant occurred in 14 of 54, 23 of 53, and 35 of 54 patients in the lowest, middle, and highest cfDNA tertiles, respectively. cfDNA levels stratified as tertiles (log-rank: P=0.0001) and REVEAL risk groups (log-rank: P<0.0001) each predicted transplant-free survival. The addition of cfDNA to REVEAL improved discrimination (area under the receiver operating characteristic curve, 0.72-0.78; P=0.02). Compared with controls, methylation analysis in patients with PAH revealed increased cfDNA originating from erythrocyte progenitors, neutrophils, monocytes, adipocytes, natural killer cells, vascular endothelium, and cardiac myocytes (Bonferroni adjusted P<0.05). cfDNA concentrations derived from erythrocyte progenitor cells, cardiac myocytes, and vascular endothelium were greater in patients with PAH with high-risk versus low-risk REVEAL scores (P≤0.02). CONCLUSIONS: Circulating cfDNA is elevated in patients with PAH, correlates with disease severity, and predicts worse survival. Results from cfDNA methylation analyses in patients with PAH are consistent with prevailing paradigms of disease pathogenesis.
BACKGROUND: Cell-free DNA (cfDNA) is a noninvasive marker of cellular injury. Its significance in pulmonary arterial hypertension (PAH) is unknown. METHODS: Plasma cfDNA was measured in 2 PAH cohorts (A, n=48; B, n=161) and controls (n=48). Data were collected for REVEAL 2.0 (Registry to Evaluate Early and Long-Term PAH Disease Management) scores and outcome determinations. Patients were divided into the following REVEAL risk groups: low (≤6), medium (7-8), and high (≥9). Total cfDNA concentrations were compared among controls and PAH risk groups by 1-way analysis of variance. Log-rank tests compared survival between cfDNA tertiles and REVEAL risk groups. Areas under the receiver operating characteristic curve were estimated from logistic regression models. A sample subset from cohort B (n=96) and controls (n=16) underwent bisulfite sequencing followed by a deconvolution algorithm to map cell-specific cfDNA methylation patterns, with concentrations compared using t tests. RESULTS: In cohort A, median (interquartile range) age was 62 years (47-71), with 75% female, and median (interquartile range) REVEAL 2.0 was 6 (4-9). In cohort B, median (interquartile range) age was 59 years (49-71), with 69% female, and median (interquartile range) REVEAL 2.0 was 7 (6-9). In both cohorts, cfDNA concentrations differed among patients with PAH of varying REVEAL risk and controls (analysis of variance P≤0.002) and were greater in the high-risk compared with the low-risk category (P≤0.002). In cohort B, death or lung transplant occurred in 14 of 54, 23 of 53, and 35 of 54 patients in the lowest, middle, and highest cfDNA tertiles, respectively. cfDNA levels stratified as tertiles (log-rank: P=0.0001) and REVEAL risk groups (log-rank: P<0.0001) each predicted transplant-free survival. The addition of cfDNA to REVEAL improved discrimination (area under the receiver operating characteristic curve, 0.72-0.78; P=0.02). Compared with controls, methylation analysis in patients with PAH revealed increased cfDNA originating from erythrocyte progenitors, neutrophils, monocytes, adipocytes, natural killer cells, vascular endothelium, and cardiac myocytes (Bonferroni adjusted P<0.05). cfDNA concentrations derived from erythrocyte progenitor cells, cardiac myocytes, and vascular endothelium were greater in patients with PAH with high-risk versus low-risk REVEAL scores (P≤0.02). CONCLUSIONS: Circulating cfDNA is elevated in patients with PAH, correlates with disease severity, and predicts worse survival. Results from cfDNA methylation analyses in patients with PAH are consistent with prevailing paradigms of disease pathogenesis.
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