Arya Mani

Olujimi Ajijola

eLife assessment
In this small study involving patients with a history of myocardial infarction, Fawaz et al. found no significant contribution of clonal hematopoiesis and mosaic loss of the Y chromosome to the incidence of myocardial infarction and atherosclerosis. Although the evidence provided by the study is incomplete due to its small sample size, the findings are valuable for guiding future larger studies that will further investigate this significant and controversial subject.

anonymous

Reviewer #2 (Public review):
Summary:
The preprint by Fawaz et al. presents the findings of a study that aimed to assess the relationship between somatic mutations associated with clonal hematopoiesis (CHIP) and the prevalence of myocardial infarction (MI). The authors conducted targeted DNA sequencing analyses on samples from 149 MI patients and 297 non-MI controls from a separate cohort. Additionally, they investigated the impact of the loss of the Y chromosome (LOY), another somatic mutation frequently observed in clonally expanded blood cells. The results of the study primarily demonstrate no significant associations, as neither CHIP nor LOY were found to be correlated with an increased prevalence of MI. The null findings regarding CHIP are partly in conflict with several larger studies in the literature. However, it must be noted that the authors did find trends to an association between CHIP and a higher incidence of MI during follow-up among those without a history of MI at baseline, which is more consistent with previous research work. The association with incident MI reached statistical significance in men, particularly in those not showing LOY, suggesting potential interactions between different clonally-expanded somatic mutations.
Strengths:
Overall, this is a useful research work on an emerging risk factor for cardiovascular disease (CVD). The use of a targeted sequencing approach is a strength, as it offers higher sensitivity than the whole exome sequencing approaches used in many previous studies. Reporting null findings is definitely relevant in an emerging field such as the role of somatic mutations in cardiovascular disease.
Weaknesses:
The study suffers from important limitations, which cast some doubts onto the authors' conclusions, as detailed below:
(1) The small sample size of the study population is a critical limitation, particularly when reporting null findings that conflict (partly) with positive findings in much larger studies, totaling hundreds of thousands of individuals (e.g. Zekavat et al, Nature CVR 2023, Vlasschaert et al, Circulation 2023; Zhao et al, JAMA Cardio 2024). The authors claim that they have 90% power to detect an effect size of CHIP on MI comparable to that in previous reports (a hazard ratio of 1.7, mainly based on the findings by Jaiswal et al, NEJM 2014,2017). However, this analysis is simply based on the predicted prevalence of CHIP in MI(+) and MI(-) patients, and it does not consider the complex relationship between age CHIP and atherosclerotic disease. More advanced approaches to calculate statistical power may have provided a more accurate estimation. It must also be noted that recent work in much larger populations suggest that the overall effect of CHIP on atherosclerotic CVD is smaller than 1.7, most likely due to the heterogeneity of effects of different mutated genes (e.g. Zekavat et al, Nature CVR 2023, Vlasschaert et al, Circulation 2023; Zhao et al, JAMA Cardio 2024). In addition, several analyses in the current manuscript are conducted separately in MI(+) (n= 149) and MI(-) (N=297) individuals, further limiting statistical power. Power is even lower in the investigation of the effects of LOY and its interaction with CHIP, as only men are included in these analyses. Overall, I believe the study is underpowered from a statistical point of view, so the authors' findings need to be interpreted with caution.
(2) Related to the above, it is widely accepted that the effects of CHIP on CVD are highly heterogeneous, as some mutated genes appear to have a strong impact on atherosclerosis, whereas the effect of others is negligible (e.g. Zekavat et al, Nature CVR 2023, Vlasschaert et al, Circulation 2023, among others). TET2 mutations are frequently considered a "positive control", given the multiple lines of evidence suggesting that these mutations confer a higher risk of atherosclerotic disease. However, no association with MI or related variables was found for TET2 mutations in the current work, which likely reflects the limited statistical power of the study to assess accurately the effects of CHIP mutations on atherosclerotic disease.
(3) One of the most essential features of CHIP is the tight correlation with age. In this study, the effect of age on CHIP (e.g. Supp. Tables S5, S6) is statistically significant, but substantially milder than in previous studies. Given the relatively modest effect size of age on CHIP here, it is not surprising that no association with MI or atherosclerotic disease was found, considering that this association would have a much smaller effect size. It must be considered, however, that the advanced age of the population may have confounded the analysis of these relationships, as acknowledged by the authors.
(4) CHIP represents just one type of clonal hematopoiesis (e.g. see <a href="https://doi.org/10.1182/blood.2023022222">https://doi.org/10.1182/blood.2023022222</a>). In this context, it must be noted that the mutated genes included in the definition of "CHIP" here are markedly different than in most previous studies, particularly when considering specifically the studies that demonstrated an association between CHIP and atherosclerotic CVD. For instance, the definition of CHIP in this manuscript includes genes such as ANKRD26, CALR, CCND2, DDX41... that are not prototypical CHIP genes. This is unlikely to have major impact on the main results, as the vast majority of mutations detected are indeed in bona fide CHIP genes, but it needs to be considered when interpreting the authors' findings. Furthermore, the strategy used here for CHIP variant calling and curation is substantially different than that used in previous studies. This is important, because such differences in the definition of CHIP and the curation of variants are at the basis of most conflicting findings in the literature regarding the effects of this condition. The authors estimate that the effect of these discrepancies on the definition of CHIP is limited, but small differences can have substantial impact in a study with limited sample size.
(5) A major limitation of the current study is the cross-sectional design of most of the analyses. For instance, it is not surprising that no association is found between CHIP and prevalent atherosclerosis burden by ultrasound imaging, considering that many individuals may have developed atherosclerosis years or decades before the expansion of the mutant clones, limiting the possible effect of CHIP on atherosclerosis burden. Similarly, the analysis of the relationship between CHIP and a history of MI may be confounded by the potential effects of MI on the expansion of mutant clones. In this context, it is noteworthy that the only positive results here are found in the analysis of the relationship between CHIP at baseline and incident MI development over follow-up. A larger sample size in these longitudinal analyses would provide deeper insights into the relationship between CHIP and MI.

Evaluation of Clonal Hematopoiesis and Mosaic Loss of Y Chromosome in Cardiovascular Risk: an analysis in prospective studies

Figures and data

Characteristics of the population at the time of inclusion

CHIP, mLOY and their combination are as frequent in MI(+) and MI(-) subjects

CHIP and mLOY are not associated with increased hsCRP level

CHIP and mLOY are not associated with an increased atherosclerotic burden

CHIP and mLOY do not increase significantly the risk of incident MI, but could accelerate it in male subjects.

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