 whereas it was absent in unexposed individuals (red dashed box in Figure 2A and 2B). However, as the number of CH mutations in this last subgroup analysis is low, the corresponding cosine value indicates these results should be interpreted with caution.
PWH with a CH mutation were more likely to have had at least once a viral load of >50 copies/mL 1 year prior to study visit (19.6% vs 8.2%, P =.052), suggesting a more active HIV reservoir. In virally suppressed PWH, the HIV-1 CA-DNA roughly equals total integrated proviral DNA, while HIV-1 CA-RNA is associated with HIV-1 transcriptional activity [27]. Although these parameters of HIV reservoir strongly intercorrelate, HIV CA-RNA was increased in CH mutation carriers and HIV CA-DNA was not ( Figure 3B). The discrepancy between CA-RNA and CA-DNA suggests an effect on transcriptional activity [27]. As a result, PWH with viral load between 50 and 200 copies/mL (viral blip) 1 year prior to study visit (10.6%) had higher levels of CA-RNA (P <.01) but no differences in CA-DNA. Although the CA-RNA difference disappeared after correction for CD4 nadir and age (Supplemental Table 7), the ratio of HIV-1 CA-RNA to CA-DNA, which represent relative viral transcription level [27], was increased in CH mutation carriers (OR, 5.563; 95% CI, 1.768-45.970; P =.017; Supplementary Table 7), independent of CD4 nadir, age, and CD4/CD8 T-cell ratio.

DISCUSSION
In the current study, we show that CH driver mutations in PWH on long-term successful cART are common and are independently associated with older age, low CD4 nadir, and increased CD4/CD8 T-cell ratio, as well as with markers of coagulation. Furthermore, we show that CH is linked to increased relative HIV-1 transcription level (ie, increased HIV-1 CA-RNA to HIV-1 CA-DNA ratio). Age is a known CH predictor in the general population [3,4,16], and an association was present in the current study, supporting the validity of our data. CH may be important in PWH as their presence has been associated with hematological (pre)malignancies, as well as with CVD and all-cause mortality in HIV-uninfected individuals [3]. Recently, a large-scale A, CH mutation prevalence increased with age in PWH. The stacked bars represent the percentage of PWH carrying a CH mutation per age category (the CH mutation with highest variant allele frequency is plotted for individuals carrying multiple mutations). B, The proportion of CH mutations in genes other than DNMT3A in PWH aged ≥55 years (n = 90) was larger compared to HIV-uninfected overweight controls (n = 296). C, CH mutation size nonsignificantly increased in PWH as compared to HIV-uninfected overweight controls. D, CH mutation size was significantly larger in PWH as compared to population controls. C and D, Each dot represents a CH mutation (the number of mutations is annotated on the x-axis and the size on the y-axis), with a horizontal line and number to the right indicating the mean CH mutation size and the Wilcoxon-rank sum test P value at the top.
CH study by Bick et al reported increased prevalence of large clones, or CHIP, in PWH as compared to control individuals [11]. In line with this, the probability of large clones, adjusted for age, was increased in PWH as compared to HIV-uninfected overweight controls, and the probability of all non-DNMT3A clones (small and large) was intriguingly driven by something other than age in PWH as compared to population controls in the present study. Consistent with the latter, and the hypothesis that time is needed for a clone to grow, clone size was significantly increased in PWH as compared to age-and sex-matched population controls. The absence of this in our PWH versus overweight controls comparison is most likely due to the differences in age and BMI, as we have recently shown that clone size may be increased in obese individuals [16]. Our observation that something other than age drives CH prevalence in PWH was substantiated by the fact that the mutational processes underlying the identified CH mutations may be different between PWH and controls. Only in PWH, a ROS mutational signature could be assigned to some of the observed CH mutations. ROS production is indeed increased in PWH and, interestingly, has been linked to nucleoside reverse transcriptase inhibitor (NRTI)-induced mitochondrial toxicity [3,4]. The enhanced ROS signature found in CH mutations from PWH compared to HIV-uninfected overweight controls could therefore mirror increased mitochondrial dysfunction in PWH. We could not assess whether either HIV or cART use relates to CH mutation development, as all PWH were on cART and with plasma HIV RNA levels <200 copies/mL. However, our data indicate that the ROS signature may contribute to mutations in PWH with prior exposure to zidovudine, whereas it was absent in unexposed individuals. Zidovudine, an NRTI, has been extensively linked to hematological toxicity [28].
The fact that our data suggest that in PWH other processes significantly reduce the correlation of CH prevalence with age is intriguing. Associations between CH and cART duration as well as with age and coronary artery disease were recently reported [11]. However, no correlations with other HIV-related parameters, such as CD4 T-cell count, HIV medication, and HIV reservoir parameters, were reported. We identified that various other HIV-related parameters correlated with CH independent of age, such as CD4 nadir and CD4/CD8 T-cell ratio. CD4 nadir is a marker for the severity of prior immunosuppression and HIV infection course. The link with CD4 nadir suggests prior immunosuppression due to HIV infection is associated with an increased CH prevalence in PWH. Studies have also linked CH to chronic infection and infection risk [28,29]. Longitudinal studies are needed to further investigate the link between HIV infection and CH. While in HIVuninfected controls a link between CH and inflammation has been shown [3,30], no association was found between CH and general inflammation parameters in our cohort of PWH. Our sample size restricted our ability to dissect whether inflammation independent of CD4 nadir has an effect. In contrast, new factors such as HIV reservoir and markers of coagulation-ddimer and vWF-were independently associated with CH. Besides inflammatory markers, these coagulation factors have also been linked to mortality and CVD and are increased in PWH [27,31]. The interplay between coagulation, inflammation, and CH requires future mechanistic studies. The link with HIV reservoir is interesting, considering that several of the most important CH driver genes encode for epigenetic modifiers-genes that modify the epigenome through, for example, DNA methylation-with suggested downstream effects of altered gene expression and increased inflammation [30]. HIV transcription is known to rely on epigenetic modification, cell differentiation, and activation but also low-grade inflammation [1]. It is intriguing to speculate that increased relative HIV-1 transcription in individuals with CH could be the result of an aberrant epigenetic profile, causing alterations in the transcriptional program and cell differentiation [15]. Future studies using single-cell transcriptome analyses in a longitudinal setting could provide mechanistical insight into CH and HIV reservoir dynamics on the single-cell level. These studies could elucidate new mechanisms for (deep) HIV latency and influence future shock and kill strategies in HIV cure [32].
Limitations of the current study include the chosen control populations. The 300-Obese control individuals were processed with the exact same assay and in parallel with PWH, preventing possible confounding due to assay or batch differences, but they were generally older and showed an increased BMI. Although, at the same time, the inclusion of these controls with additional cardiovascular risk factors and older age could underestimate the effect of HIV infection itself on CH, because both age and obesity have been found to be associated with increased prevalence of CH [16]. The comparisons between PWH individuals and population controls are more appropriate due to age-and . People with HIV (PWH) clonal hematopoiesis (CH) mutation carriers present with significantly lower CD4 nadir, and higher cell-associated HIV RNA and coagulation markers. A, Carriers (n = 46) presented with significantly lower CD4 nadir as compared to noncarriers (n = 171). B, Carriers (n = 45) showed significantly increased RNA levels as compared to noncarriers (n = 169), and nonsignificantly (ns) increased DNA levels as compared to noncarriers (n = 167). C, Carriers (n = 46) presented with significantly increased coagulation markers d-dimer and von Willebrand factor (vWF) as compared to noncarriers (n = 171). D, Carriers (n = 45) showed comparable high-sensitivity C-reactive protein (hsCRP) as compared to noncarriers (n = 167) and carriers (n = 46) showed comparable soluble CD14 (sCD14) levels as compared to noncarriers (n = 170). In each plot the mean of noncarriers and CH mutation carriers is annotated with a horizontal bar and value; at the top the Wilcoxon-rank sum test P value is given.
sex-matching, but the assay was slightly different and as such we could only validate findings for non-DNMT3A mutations. Additionally, our limited sample size restricted the ability to link CH to non-AIDS-related comorbidities, further examine signature analyses, or perform sex-specific analyses. Furthermore, although the reported associations are interesting, the data do not allow us to draw causal inferences. The purpose of this study was