Invasive Assessment of Coronary Artery Disease in Clonal Hematopoiesis of Indeterminate Potential.

BACKGROUND: Clonal hematopoiesis of indeterminate potential (CHIP) occurs due to acquired mutations in bone marrow progenitor cells. CHIP confers a 2-fold risk of atherosclerotic cardiovascular disease. However, there are limited data regarding specific cardiovascular phenotypes. The purpose of this study was to define the coronary artery disease phenotype of the CHIP population-based on coronary angiography. METHODS: We recruited 1142 patients from the Vanderbilt University Medical Center cardiac catheterization laboratory and performed DNA sequencing to determine CHIP status. Multivariable logistic regression models and proportional odds models were used to assess the association between CHIP status and angiography phenotypes. RESULTS: We found that 18.4% of patients undergoing coronary angiography had a CHIP mutation. Those with CHIP had a higher risk of having obstructive left main (odds ratio, 2.44 [95% CI, 1.40-4.27]; P=0.0018) and left anterior descending (odds ratio, 1.59 [1.12-2.24]; P=0.0092) coronary artery disease compared with non-CHIP carriers. We additionally found that a specific CHIP mutation, ten eleven translocase 2 (TET2), has a larger effect size on left main stenosis compared with other CHIP mutations. CONCLUSIONS: This is the first invasive assessment of coronary artery disease in CHIP and offers a description of a specific atherosclerotic phenotype in CHIP wherein there is an increased risk of obstructive left main and left anterior descending artery stenosis, especially among TET2 mutation carriers. This serves as a basis for understanding enhanced morbidity and mortality in CHIP.

Clonal hematopoiesis and inflammation in the vasculature: CHIVE, a prospective, longitudinal clonal hematopoiesis cohort and biorepository.

ABSTRACT: Clonal hematopoiesis (CH) is an age-associated phenomenon leading to an increased risk of both hematologic malignancy and nonmalignant organ dysfunction. Increasingly available genetic testing has made the incidental discovery of CH clinically common yet evidence-based guidelines and effective management strategies to prevent adverse CH health outcomes are lacking. To address this gap, the prospective CHIVE (clonal hematopoiesis and inflammation in the vasculature) registry and biorepository was created to identify and monitor individuals at risk, support multidisciplinary CH clinics, and refine taxonomy and standards of practice for CH risk mitigation. Data from the first 181 patients enrolled in this prospective registry recapitulate the molecular epidemiology of CH from biobank-scale retrospective studies, with DNMT3A, TET2, ASXL1, and TP53 as the most commonly mutated genes. Blood counts across all hematopoietic lineages trended lower in patients with CH. In addition, patients with CH had higher rates of end organ dysfunction, in particular chronic kidney disease. Among patients with CH, variant allele frequency was independently associated with the presence of cytopenias and progression to hematologic malignancy, whereas other common high-risk CH clone features were not clear. Notably, accumulation of multiple distinct high-risk clone features was also associated with cytopenias and hematologic malignancy progression, supporting a recently published CH risk score. Surprisingly, ∼30% of patients enrolled in CHIVE from CH clinics were adjudicated as not having clonal hematopoiesis of indeterminate potential, highlighting the need for molecular standards and purpose-built assays in this field. Maintenance of this well-annotated cohort and continued expansion of CHIVE to multiple institutions are underway and will be critical to understanding how to thoughtfully care for this patient population.

Cost-Effective and Scalable Clonal Hematopoiesis Assay Provides Insight into Clonal Dynamics.

Clonal hematopoiesis of indeterminate potential (CHIP) is a common age-related phenomenon in which hematopoietic stem cells acquire mutations in a select set of genes commonly mutated in myeloid neoplasia which then expand clonally. Current sequencing assays to detect CHIP mutations are not optimized for the detection of these variants and can be cost-prohibitive when applied to large cohorts or to serial sequencing. In this study, an affordable (approximately US $8 per sample), accurate, and scalable sequencing assay for CHIP is introduced and validated. The efficacy of the assay was demonstrated by identifying CHIP mutations in a cohort of 456 individuals with DNA collected at multiple time points in Vanderbilt University's biobank and quantifying clonal expansion rates over time. A total of 101 individuals with CHIP/clonal cytopenia of undetermined significance were identified, and individual-level clonal expansion rate was calculated using the variant allele fraction at both time points. Differences in clonal expansion rate by driver gene were observed, but there was also significant individual-level heterogeneity, emphasizing the multifactorial nature of clonal expansion. Additionally, mutation co-occurrence and clonal competition between multiple driver mutations were explored.

Cost-effective and scalable clonal hematopoiesis assay provides insight into clonal dynamics.

Clonal hematopoiesis of indeterminate potential (CHIP) is a common age-related phenomenon that occurs when hematopoietic stem cells acquire mutations in a select set of genes commonly mutated in myeloid neoplasia which then expand clonally. Current sequencing assays to detect CHIP are not optimized for the detection of these variants and can be cost-prohibitive when applied to large cohorts or serial sequencing. Here, we present and validate a CHIP targeted sequencing assay that is affordable (∼$8/sample), accurate and highly scalable. To demonstrate the utility of this assay, we detected CHIP in a cohort of 456 individuals with DNA collected at multiple timepoints in the Vanderbilt BioVU biobank and quantified clonal expansion rates over time. A total of 101 individuals with CHIP were identified, and individual-level clonal expansion rate was calculated using the variant allele fraction (VAF) at both timepoints. Differences in clonal expansion rate by driver gene were observed, but there was also significant individual-level heterogeneity, emphasizing the multifactorial nature of clonal expansion. We further describe the mutation co-occurrence and clonal competition between multiple driver mutations.

Blockade of IL-6 signaling alleviates atherosclerosis in Tet2-deficient clonal hematopoiesis.

Clonal hematopoiesis (CH) increases the risk of atherosclerotic cardiovascular disease possibly due to increased plaque inflammation. Human studies suggest that limitation of interleukin-6 (IL-6) signaling could be beneficial in people with large CH clones, particularly in TET2 CH. Here we show that IL-6 receptor antibody treatment reverses the atherosclerosis promoted by Tet2 CH, with reduction of monocytosis, lesional macrophage burden and macrophage colony-stimulating factor 1 receptor (CSF1R) expression. IL-6 induces expression of Csf1r in Tet2-deficient macrophages through enhanced STAT3 binding to its promoter. In mouse and human Tet2-deficient macrophages, IL-6 increases CSF1R expression and enhances macrophage survival. Treatment with the CSF1R inhibitor PLX3397 reversed accelerated atherosclerosis in Tet2 CH mice. Our study demonstrates the causality of IL-6 signaling in Tet2 CH accelerated atherosclerosis, identifies IL-6-induced CSF1R expression as a critical mechanism and supports blockade of IL-6 signaling as a potential therapy for CH-driven cardiovascular disease.

Genetic modification of inflammation- and clonal hematopoiesis-associated cardiovascular risk.

Clonal hematopoiesis of indeterminate potential (CHIP) is associated with an increased risk of cardiovascular diseases (CVDs), putatively via inflammasome activation. We pursued an inflammatory gene modifier scan for CHIP-associated CVD risk among 424,651 UK Biobank participants. We identified CHIP using whole-exome sequencing data of blood DNA and modeled as a composite, considering all driver genes together, as well as separately for common drivers (DNMT3A, TET2, ASXL1, and JAK2). We developed predicted gene expression scores for 26 inflammasome-related genes and assessed how they modify CHIP-associated CVD risk. We identified IL1RAP as a potential key molecule for CHIP-associated CVD risk across genes and increased AIM2 gene expression leading to heightened JAK2- and ASXL1-associated CVD risk. We show that CRISPR-induced Asxl1-mutated murine macrophages had a particularly heightened inflammatory response to AIM2 agonism, associated with an increased DNA damage response, as well as increased IL-10 secretion, mirroring a CVD-protective effect of IL10 expression in ASXL1 CHIP. Our study supports the role of inflammasomes in CHIP-associated CVD and provides evidence to support gene-specific strategies to address CHIP-associated CVD risk.

A practical approach to curate clonal hematopoiesis of indeterminate potential in human genetic data sets.

Clonal hematopoiesis of indeterminate potential (CHIP) is a common form of age-related somatic mosaicism that is associated with significant morbidity and mortality. CHIP mutations can be identified in peripheral blood samples that are sequenced using approaches that cover the whole genome, the whole exome, or targeted genetic regions; however, differentiating true CHIP mutations from sequencing artifacts and germ line variants is a considerable bioinformatic challenge. We present a stepwise method that combines filtering based on sequencing metrics, variant annotation, and population-based associations to increase the accuracy of CHIP calls. We apply this approach to ascertain CHIP in ∼550 000 individuals in the UK Biobank complete whole exome cohort and the All of Us Research Program initial whole genome release cohort. CHIP ascertainment on this scale unmasks recurrent artifactual variants and highlights the importance of specialized filtering approaches for several genes, including TET2 and ASXL1. We show how small changes in filtering parameters can considerably increase CHIP misclassification and reduce the effect size of epidemiological associations. Our high-fidelity call set refines previous population-based associations of CHIP with incident outcomes. For example, the annualized incidence of myeloid malignancy in individuals with small CHIP clones is 0.03% per year, which increases to 0.5% per year among individuals with very large CHIP clones. We also find a significantly lower prevalence of CHIP in individuals of self-reported Latino or Hispanic ethnicity in All of Us, highlighting the importance of including diverse populations. The standardization of CHIP calling will increase the fidelity of CHIP epidemiological work and is required for clinical CHIP diagnostic assays.

Somatic Mutations in Cardiovascular Disease.

Advances in population-scale genomic sequencing have greatly expanded the understanding of the inherited basis of cardiovascular disease (CVD). Reanalysis of these genomic datasets identified an unexpected risk factor for CVD, somatically acquired DNA mutations. In this review, we provide an overview of somatic mutations and their contributions to CVD. We focus on the most common and well-described manifestation, clonal hematopoiesis of indeterminate potential. We also review the currently available data regarding how somatic mutations lead to tissue mosaicism in various forms of CVD, including atrial fibrillation and aortic aneurism associated with Marfan Syndrome. Finally, we highlight future research directions given current knowledge gaps and consider how technological advances will enhance the discovery of somatic mutations in CVD and management of patients with somatic mutations.

Reversible, regional ST-segment elevation due to chylothorax.

Chylothorax is an uncommon complication of thoracic surgery and, to our knowledge, has never been documented as a cause of dynamic ST-segment elevation (STE). A 63-year-old woman with history of right pneumonectomy presented with chest pain and regional STE on 12-lead electrocardiogram (ECG). Normal troponin-I and a computed tomography (CT) scan showing a large right hemithoracic fluid collection indicated the unique cause of STE, which resolved after thoracentesis, was pericardial inflammation and cardiac compression from chylothorax. This case emphasizes nuances of ECG interpretation in the context of regional STE and explores the pathophysiology that links chylothorax with acute pericarditis.

Real-world experience using hydroxyurea in children with sickle cell disease in Lilongwe, Malawi.

INTRODUCTION: Sickle cell disease (SCD) is among the most common inherited hematologic diseases in sub-Saharan Africa (SSA). Historically, hydroxyurea administration in SSA has been restricted due to limited region-specific evidence for safety and efficacy. METHODS: We conducted a prospective observational cohort study of pediatric patients with SCD in Malawi. From January 2015 to November 2017, hydroxyurea at doses of 10-20 mg/kg/day was administered to children with clinically severe disease (targeted use policy). From December 2017 to July 2018, hydroxyurea was prescribed to all patients (universal use policy). RESULTS: Of 187 patients with SCD, seven (3.7%) died and 23 (12.3%) were lost to follow-up. The majority (135, 72.2%) were prescribed hydroxyurea, 59 (43.7%) under the targeted use policy and 76 (56.3%) under the universal use policy. There were no documented severe toxicities. Under the targeted use policy, children with SCD demonstrated absolute decreases in the rates of hospitalization (-4.1 per 1000 person-days; -7.2, -1.0; P = .004), fevers (-4.2 per 1000 person-days; -7.2, -1.1; P = .002), transfusions (-2.3 per 1000 person-days; 95% confidence interval: -4.9, 0.3; P = .06), and annual school absenteeism (-51.2 per person-year; -60.1, -42.3; P < .0001) within 6 months of hydroxyurea commencement. CONCLUSION: We successfully implemented universal administration of hydroxyurea to children with SCD at a tertiary hospital in Malawi. Similar to recently reported trials, hydroxyurea was safe and effective during routine programmatic experience, with clinical benefits particularly among high-risk children. This highlights the importance of continued widespread scale-up of hydroxyurea within SCD programs across SSA.

Nephrin as a biomarker of sickle cell glomerulopathy in Malawi.

BACKGROUND: Glomerulopathy is an increasingly identified complication in young patients with sickle cell disease (SCD). Hyperfiltration and albuminuria followed by declining glomerular filtration rates and eventual end-stage renal disease (ESRD) is assumed to be the typical progression of glomerular disease. There are only a few reported biomarkers to identify early-stage renal disease in SCD. PROCEDURES: We detail the renal profile of 101 children with SCD in Malawi and propose a novel urinary biomarker for the identification of early renal disease. RESULTS: Among children with sickle cell anemia, 24.8% had a urine albumin-creatinine ratio of 30 mg/g or above. In univariate analysis, only patients with higher urinary nephrin, a urinary marker of glomerular injury, had significantly greater odds of having albuminuria. In multivariable analysis, nephrin remained significantly associated with albuminuria. A nephrin-creatinine ratio (NCR) cut-point of 622 ng/mg, the 50th percentile, was associated with a 45.8 times greater odds of having albuminuria in children with nephrinuria above this value. Further analysis revealed this urinary NCR cut-point to have 96% sensitivity, 64% specificity, 47% positive predictive value, and 98% negative predictive value for the presence of albuminuria. CONCLUSIONS: These data suggest that a substantial number of children with SCD in Malawi have renal disease and could be at risk for worsening nephropathy and ESRD as they age. Our data suggest that urinary nephrin could be utilized as an early marker of glomerular disease in SCD.

Endothelin-1 contributes to the progression of renal injury in sickle cell disease via reactive oxygen species.

BACKGROUND AND PURPOSE: Endothelin-1 (ET-1) is increased in patients with sickle cell disease and may contribute to the development of sickle cell nephropathy. The current study was designed to determine whether ET-1 acting via the ETA receptor contributes to renal injury in a mouse model of sickle cell disease. EXPERIMENTAL APPROACH: Adult, humanized HbSS (homozygous for sickle Hb) mice had increased ET-1 mRNA expression in both the cortex and the glomeruli compared with mice heterozygous for sickle and Hb A (HbAS controls). In the renal cortex, ETA receptor mRNA expression was also elevated in HbSS (sickle) mice although ETB receptor mRNA expression was unchanged. Ligand binding assays confirmed that sickle mice had increased ETA receptors in the renal vascular tissue when compared with control mice. KEY RESULTS: In response to PKC stimulation, reactive oxygen species production by isolated glomeruli from HbSS sickle mice was increased compared with that from HbSA controls, an effect that was prevented by 1 week in vivo treatment with the selective ETA antagonist, ABT-627. Protein and nephrin excretion were both elevated in sickle mice, effects that were also significantly attenuated by ABT-627. Finally, ETA receptor antagonism caused a significant reduction in mRNA expression of NADPH oxidase subunits, which may contribute to nephropathy in sickle cell disease. CONCLUSIONS AND IMPLICATIONS: These data support a novel role for ET-1 in the progression of sickle nephropathy, specifically via the ETA receptor, and suggest a potential role for ETA receptor antagonism in a treatment strategy.

Endothelin-1 as a master regulator of whole-body Na+ homeostasis.

The current study was designed to determine whether vascular endothelial-derived endothelin-1 (ET-1) is important for skin Na(+) buffering. In control mice (C57BL/6J), plasma Na(+) and osmolarity were significantly elevated in animals on high- vs. low-salt (HS and LS, respectively) intake. The increased plasma Na(+) and osmolarity were associated with increased ET-1 mRNA in vascular tissue. There was no detectable difference in skin Na(+):H2O in HS fed mice (0.119 ± 0.005 mM vs. 0.127 ± 0.007 mM; LS vs. HS); however, skin Na(+):H2O was significantly increased by blockade of the endothelin type A receptor with ABT-627 (0.116 ± 0.006 mM vs. 0.137 ± 0.007 mM; LS vs. HS; half-maximal inhibitory concentration, 0.055 nM). ET-1 peptide content in skin tissue was increased in floxed control animals on HS (85.9 ± 0.9 pg/mg vs. 106.4 ± 6.8 pg/mg; P < 0.05), but not in vascular endothelial cell endothelin-1 knockout (VEET KO) mice (76.4 ± 5.7 pg/mg vs. 65.7 ± 7.9 pg/mg; LS vs. HS). VEET KO mice also had a significantly elevated skin Na(+):H2O (0.113 ± 0.007 mM vs. 0.137 ± 0.005 mM; LS vs. HS; P < 0.05). Finally, ET-1 production was elevated in response to increasing extracellular osmolarity in cultured human endothelial cells. These data support the hypothesis that increased extrarenal vascular ET-1 production in response to HS intake is mediated by increased extracellular osmolarity and plays a critical role in regulating skin storage of Na(+).

Variable reactive hyperemia in normotensive strains of rat.

Previous studies from our laboratory report variation in nitric oxide (NO)-dependent arterial pressure within the same strain of normotensive Sprague-Dawley rat dependent upon the commercial vendor supplying the rats. Clinical assessment of endothelial NO activity and endothelial function in general has used postocclusion, flow-mediated dilation (FMD). Therefore, this study was conducted to determine whether the reactive hyperemic response was different between two normotensive strains from two different suppliers, Sprague-Dawley (SD) and Wistar-Kyoto (WKY) rats from Charles River (CR) and Harlan Laboratories (H), respectively. Rats were anesthetized and the femoral artery was occluded for 5 min, with femoral blood flow measured continuously by use of an ultrasonic perivascular flow probe. The average area under the reactive hyperemic response curve (3-min duration) was not different between SD rats from CR (80 ± 23 mL/min∙s; n = 6) and H (94 ± 16 mL/min∙s; n = 6). As previously reported, blood pressures were higher in the SD rats from H versus CR. WKY rats from both suppliers had significantly larger hyperemia; 371 ± 67 versus 281 ± 71 mL/min∙s (n = 5) for the CR and H WKY rats, respectively, but again, were not different between vendors. Blood pressures in WKY rats were similar between vendors. These results suggest that differences in NO bioactivity are not discernable with an adapted FMD protocol in the rat and that normotensive strains of rat can have large differences in reactive hyperemia despite having similar blood pressures.