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•CD14+monocytes from patients with CH stimulate inflammation through increased cytokine expression.•T cells from CH are deficient in GIMAP expression, suggesting CH may impair T-cell differentiation.

•We present a practical method to ascertain CHIP that combines sequence-based and population-based filtering in the UK Biobank and All of Us.•Small changes in filtering parameters can have a large effect on the accuracy of CHIP variant classification.

We have developed a cost-effective DNA methylation sequencing assay to improve monitoring of clonal hematopoiesis. By inferring cell-type proportions, this method enhances interpretation of clonal trajectories compared with interpretation based on variant allele fraction only.

•Development of a genotyped and phenotyped cohort of patients with CH is required to establish clinical guidelines and translational research.•Initial data from a prospective registry and biorepository of patients with CH recapitulate findings derived from retrospective studies.

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 mutations are not optimized for the detection of these variants and can be cost-prohibitive when applied to large cohorts or serial sequencing. In this study, an affordable (∼$8 per sample), accurate, and scalable sequencing assay for CHIP is introduced and validated. The efficacy of the assay is demonstrated by identifying CHIP mutations in a cohort of 456 individuals with DNA collected at multiple timepoints in the Vanderbilt BioVU biobank and quantifying clonal expansion rates over time. A total of 101 individuals with CHIP/CCUS 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. Additionally, the study explores mutation co-occurrence and clonal competition between multiple driver mutations.

Pulmonary hypertension (PH) is a heterogenous condition characterized by high pulmonary vascular resistance and elevated arterial pressures. Clonal hematopoiesis of indeterminate potential (CHIP) occurs when a hematopoietic stem cell acquires a somatic mutation that confers a proliferative advantage. CHIP is associated with a range of cardiovascular diseases. Recent work in a murine model of PH demonstrated that JAK2 CHIP causes PH via neutrophil mediated vascular remodeling. Little is known about the association between CHIP and pulmonary pressures in humans. We hypothesized that CHIP would be associated with higher pulmonary pressure and a higher prevalence of clinical PH in patients referred for echocardiography. We evaluated patients in the Vanderbilt BioVU biobank for the presence or absence of several CHIP mutations (DNMT3A, TET2, ASXL1, PPM1D, and JAK2) and correlated this finding with standard echocardiography parameters extracted from the electronic health record. PH was defined as an estimated pulmonary artery systolic pressure (ePASP) >=40mmHg. We analyzed 12,007 patients who had a reported ePASP after the date of sequencing. Of those, 49% (5,883 of 12,007) were male, with a mean age of 55+/-19.2 years. CHIP mutations were present in 477 (3.9%). The mean ePASP was 34.5+/-13.9mmHg and 32% (3,842 of 12,007) had an ePASP >= 40mmHg. The ePASP was higher among JAK2 positive patients (42.3[39.4-36.5]mmHg vs. 34.6[34.4-34.2]mmHg; p = 0.0002). This association persisted after adjusting for age, race, and sex, (Beta coefficient 4.1[1.8-6.5]mmHg, p = 0.0005). We found no association between ePASP and other CHIP mutations. The presence of a JAK2 mutation was associated with a higher overall prevalence of PH (1.5% [180 of 12,007] vs. 0.9% [108 of 12,007]; p = 0.007). In a large echocardiographic referral population, JAK2 CHIP mutations are associated with higher ePASP and a higher prevalence of PH. Treatment implications and the association between CHIP mutations and clinical outcomes in patients with PH warrants further study.

No relevant conflicts of interest to declare.

There is increasing evidence that Endothelin-1 (ET-1) plays a significant role in the pathogenesis of complications of sickle cell disease (SCD. SCD patients have elevated levels of endothelin-1 both in plasma and urine and ET-1 has been implicated in SCN, pain modulation, vaso-occlusive episodes, and acute chest syndrome. In the kidney, ET-1 is a potent vasoconstrictor, anti-natriuretic, and hypertrophic factor when signaling through the ETA receptor. ET-1 is released in response to shear stress, hypoxia, thrombin activation, and inflammatory cytokines, all of which are prevalent in SCD. Gender differences exist in other disease models, in which ET-1 plays a contributory pathophysiological role, with males typically exhibiting increased ET-1 mRNA expression, exaggerated ETA-mediated effects, and higher levels of renal injury. Little research exists highlighting the differences between males and females in SCD. It has been observed that the level of Hb F, a major modifier of disease severity in SCD, is higher in females compared to males. In addition, one study examined responses to chronic transfusion therapy, finding males to have increased vascular resistance and resulting decrease in oxygen delivery in response to transfusion when compared to females. We hypothesized that male SCD mice have enhanced ETA mediated responses to ET-1, ultimately leading to renal injury. To test this, we used male and female ‘HbS only' knock in SCD mice to measure renal artery blood flow velocity (BFV) via laser Doppler flowmetry in response to an ET-1 bolus (1nm/kg in 100uL saline). Male mice had an exaggerated decrease in BFV relative to females at 1, 5, and 10 minutes post ET-1 infusion while renal artery diameter remained unchanged throughout the experiment (84.4±5.8 vs. 56±5.7% of BFV baseline at 10 minutes, p<0.05, n=6). A follow up experiment was performed to determine ET-1, ETA receptor, and ETB receptor mRNA expression in the cortex of male and female SCD mice via qPCR. Males and females surprisingly exhibited no significant difference in these parameters although there was a strong trend suggesting females have increased levels of ET-1 mRNA expression compared to males (1.62±0.51 fold increase vs. males, p=0.056, n=5-6). While the exaggerated sensitivity to an ET-1 bolus is partially explained by the relative decrease in ET-1 mRNA expression in males, we nevertheless expected to see differences in ETA/ETB receptor expression. However, receptor-ligand binding could perhaps offer an alternate explanation for this phenomenon. Sex hormones are heavily implicated in other sex differences studies and the potential protective role of estrogen and/or detrimental role for testosterone are both viable explanations for the dichotomy seen in SCD. These data demonstrate a novel sexual dimorphism for SCD, revealing new insights into sex-specific pathophysiology that could have broad effects on treatment strategies in sickle cell nephropathy.