Clonal Hematopoiesis in Chronic Thromboembolic Pulmonary Hypertension

Abstract

Background: The cause of chronic thromboembolic pulmonary hypertension (CTEPH) remains largely unknown. Recently, clonal hematopoiesis (CH) has been reported to be associated with cardiovascular and thromboembolic diseases. Here, we investigated the prevalence and clinical impact of CH in patients with CTEPH.

Methods and results: Whole-exome sequencing and deep-panel sequencing were performed in 214 patients with CTEPH. Clinical data before and after treatment were compared between patients with and without CH. RNA sequencing and serum analysis were performed to explore the pathogenesis that CH contributes to CTEPH. Among the enrolled patients, 20.1%, notably 44.4% who were 80 to 89 years old, had variants in CH-associated genes. In regard to clinical impact, B-type natriuretic peptide levels and home oxygen therapy rate were significantly higher, and 6-minute walk distance was significantly shorter after treatment in patients with CH than in those without CH. Moreover, novel clot reformation in the pulmonary artery despite the use of anticoagulants and additional angioplasty events after treatment completion were more frequent in patients with CH. RNA sequencing analysis revealed that blood coagulation and neutrophil extracellular trap formation pathways were enriched in patients with CH. Additionally, serum citrullinated histone H3 levels were higher in patients with CH than those without CH. These results were consistent in the subgroup of patients who did not have the history of hematological disorders.

Conclusions: The findings in this study raise the possibility that CH will induce a more prothrombotic state through neutrophil activation and neutrophil extracellular trap formation, contributing to pathogenesis and poor treatment response in patients with CTEPH.

Keywords: balloon pulmonary angioplasty; chronic thromboembolic pulmonary hypertension; clonal hematopoiesis; clot formation; neutrophil extracellular trap.

Figure 1. Prevalence of CH in patients with CTEPH.

The frequency of CH in patients with CTEPH detected using WES and deep panel sequencing is shown by age. The details on the analysis based on the previous reports and explored variants are described in Tables S1, S4. The error bars represent 95% CIs. CH indicates clonal hematopoiesis; CTEPH, chronic thromboembolic pulmonary hypertension; and WES, whole‐exome sequencing.

Figure 2. Analysis of variants identified in CH‐associated genes.

A, The number of variants identified in each of the CH‐associated genes is listed in order of frequency. B, The number of patients based on the number of variants identified per patient is shown in order of frequency. C, The distribution of types of variants is summarized. D, The number of accumulated variants identified in patients with CTEPH is displayed in order of frequency. E, The number of variants based on VAF is summarized. F, The number of variants with VAF of >10% in each CH‐associated gene is listed in order of frequency. ASXL1 indicates additional sex combs like 1; CH, clonal hematopoiesis; CTEPH, chronic thromboembolic pulmonary hypertension; DNMT3A, DNA methyltransferase 3A; GNAS, guanine nucleotide binding protein, α stimulating; JAK2, Janus kinase 2; PHIP, pleckstrin homology domain interacting protein; PPM1D, protein phosphatase, Mg²⁺/Mn²⁺ dependent 1D; SF3B1, splicing factor 3b subunit 1; SRSF2, serine and arginine rich splicing factor 2; TET2, tet methylcytosine dioxygenase 2; TP53, tumor protein p53; VAF, variant allele frequency; and ZRSR2, zinc finger CCCH‐type, RNA binding motif and serine/arginine rich 2.

Figure 3. Changes in clinical parameters at baseline and after treatment.

The hemodynamics, log₁₀ BNP, and 6MWD measured at baseline and after treatment in patients with CH (CH+) and those without CH (CH–) are shown. The Mann‐Whitney U test was used for differences between patients with and without CH, and the Wilcoxon signed rank test was used for comparisons between baseline and posttreatment data. A 2‐way repeated measures analysis of variance was also used to assess the differences in the changes from baseline to follow‐up between patients with and without CH. Boxplot's box indicates the 25th and 75th percentile, with the center bar as the median value. 6MWD indicates 6‐minute walk distance; BNP, B‐type natriuretic peptide; CH, clonal hematopoiesis; CO, cardiac output; PAP, pulmonary arterial pressure; PVR, pulmonary vascular resistance; and RAP, right atrial pressure.

Figure 4. Kaplan‐Meier event‐free analysis for the events of novel thrombus in the pulmonary artery.

Kaplan‐Meier event‐free analysis demonstrated that the novel thrombotic events were significantly more frequent in the CH‐positive group than in the CH‐negative group. CH indicates clonal hematopoiesis.

Figure 5. RNA sequencing analysis and NET formation assay.

The volcano plot (A), the enrichment analysis using the metascape (B), and the KEGG pathway analysis using the GAGE method (C) comparing patients with CH and those without CH. D, Levels of serum citrullinated histone H3 and cell‐free DNA, which are the common NET markers, in patients with CH and those without CH. Data are shown as the mean±SEM. CH indicates clonal hematopoiesis; FDR, false discovery rate; GAGE, generally applicable gene‐set enrichment; KEGG, Kyoto Encyclopedia of Genes and Genomes; and NET, neutrophil extracellular trap.

Figure 6. Potential pathogenesis of CTEPH medicated with CH.

The findings in this study suggest that CH induces a more prothrombotic state possibly through neutrophil activation and the NET formation pathway, contributing to thrombus formation, and when these thrombi are washed ashore and attach to the pulmonary arteries, which are the end of systemic venous flows, the pathogenesis of CTEPH is generated. CH indicates clonal hematopoiesis; CTEPH, chronic thromboembolic pulmonary hypertension; NET, neutrophil extracellular trap; and RBC, red blood cell.