Genetically predicted lower FLT3L levels increase the risk of hypertrophic cardiomyopathy partly mediated by phosphate: Evidence from a 2-step Mendelian randomization analysis

Abstract

We performed a 2-step Mendelian randomization (MR) study to investigate the associations of Fms-related tyrosine kinase 3 ligand (FLT3L) and phosphate levels with the risk of hypertrophic cardiomyopathy (HCM). Genetic instruments for 75 circulating inflammatory factors were obtained from the NHGRI-EBI GWAS Catalog, while summary statistics for circulating phosphate and HCM were derived from the UK Biobank and FinnGen, respectively. Univariable MR analysis using the inverse-variance weighted method indicated that genetically predicted higher phosphate levels were associated with an increased risk of HCM (OR = 1.36, P = 4.82 × 10⁻²). Among the inflammatory markers, FLT3L emerged as a significant candidate and showed inverse associations with phosphate levels (β = -0.05, P = 1.70 × 10⁻⁹) and HCM (OR = 0.79, P = 4.10 × 10⁻²). Bidirectional MR analyses did not support a causal effect of phosphate on FLT3L. Mediation analysis suggested that phosphate levels accounted for an estimated 12.05% of the total effect of FLT3L on HCM. Genetic liability to lower FLT3L levels is associated with a higher risk of HCM, and this relationship may be partially mediated through circulating phosphate levels.

1. Introduction

Hypertrophic cardiomyopathy (HCM) is one of the most common inherited cardiomyopathies, with an estimated prevalence of 0.2% to 0.5% in the general population.^[1] Characterized by unexplained myocardial hypertrophy, HCM is typically inherited and can lead to a range of adverse cardiovascular outcomes, including heart failure and atrial fibrillation.^[2] Beyond its clinical impact, HCM also imposes substantial psychosocial and societal burdens on affected families.^[3]

HCM is a leading cause of sudden cardiac death in the young, with pathogenesis incompletely understood beyond sarcomeric gene mutations.^[4–6] Emerging evidence implicates inflammation as a key driver of HCM progression; for instance, elevated interleukin-6 levels independently predict adverse outcomes, and multi-omics analyses have identified immune-related signatures in HCM myocardium.^[7–10] Separately, disturbances in phosphate homeostasis, a critical regulator of cardiovascular function, have been linked to myocardial remodeling and vascular dysfunction, suggesting a potential role for phosphate in HCM pathology.^[11,12] While these lines of evidence suggest independent roles for inflammation and phosphate in HCM, it remains unclear whether they act through distinct pathways or whether they are mechanistically linked. Specifically, could inflammation contribute to HCM risk partly by altering phosphate metabolism? Although phosphate is known to promote inflammation in conditions like chronic kidney disease,^[13] the reverse direction, whether systemic inflammation perturbs phosphate homeostasis to induce cardiac remodeling, remains largely unexplored in the context of HCM. This knowledge gap complicates causal inference from observational studies.

To address this question, we applied a 2-step Mendelian randomization (MR) framework to investigate the causal relationships among inflammatory factors, serum phosphate levels, and HCM. 2-step MR enables the evaluation of potential mediation pathways by using summary statistics from genome-wide association studies (GWAS).^[14–16]

2. Methods

2.1. Study design

A 2-step MR framework was applied using multiple GWAS summary statistics datasets to assess the mediating role of phosphate in the relationship between inflammatory factors and HCM. In the first stage, univariable MR was conducted to evaluate the causal effects of 75 inflammatory factors (Fig. 1) on both phosphate levels and HCM. The causal influence of phosphate on these traits was also assessed to enable mediation analyses by excluding bidirectional causality between the exposure and the mediator. Inflammatory factors that exhibited significant causal effects on both phosphate and HCM, but were not themselves affected by phosphate, were selected for further analysis. In the second stage, multivariable MR was performed using the selected inflammatory factors as exposures and phosphate as a covariate to estimate their direct effects on HCM. Indirect effects mediated by phosphate were calculated by multiplying the estimated causal effect of each inflammatory factor on phosphate (from univariable MR) by the estimated causal effect of phosphate on HCM (from multivariable MR). This framework allows phosphate to serve as a key mediator, enabling decomposition of total effects into direct effects (independent of phosphate) and indirect effects (mediated by phosphate).

Figure 1.

Flow chart of the study. IVs = instrumental variables.

2.2. Data sources

Summary-level data for 75 circulating inflammatory factors were obtained from the NHGRI-EBI GWAS Catalog, corresponding to the published proteome-wide GWAS by Zhao et al.^[17] Genetic summary statistics for circulating phosphate and HCM were obtained from the UK Biobank (UKBB) and FinnGen consortium, respectively (Supplementary Table 1, Supplemental Digital Content 1).

2.3. Statistical methods

In the univariable MR analysis, instrumental variables (IVs) were chosen according to several criteria: the IVs exhibited significant associations with the traits or phenotypes; independence of the IVs was ensured by clumping them within a 10 Mb window and applying a linkage disequilibrium threshold of R² <0.001, using the European population from the 1000 Genomes Project as the reference panel^[18]; biallelic variants with a minor allele frequency above 0.01^[19]; and each instrument comprised at least 3 variants. The inverse-variance weighted method was utilized as the primary approach in univariable MR analyses, with weighted median and MR-Egger methods^[20] applied for sensitivity testing, as previously reported.^[21–23] Additionally, MR-PRESSO was used to detect horizontal pleiotropic outliers.^[24] Furthermore, assessments of heterogeneity were incorporated using the Cochran's Q test,^[25] and pleiotropy was assessed through the MR-Egger intercept test. Finally, we conducted a leave-one-out sensitivity analysis to assess the impact of each IV on the outcome, ensuring the robustness of the results.^[26–28] All univariable MR analyses were carried out in R, utilizing the TwoSampleMR package. In the multivariable MR analysis, IVs were selected based on the same criteria as in the univariable MR analysis and were harmonized across exposure and mediator datasets. The multivariable MR analysis was performed in R using the MVMR package.

3. Results

Univariable MR analysis of the 75 circulating inflammatory factors was conducted, with the complete results for all markers visually presented in Figure 2. Following our prespecified screening criteria, we identified significant associations between 8 inflammatory factors and phosphate levels, and 3 inflammatory factors with HCM, among the 75 markers analyzed (Fig. 2). Notably, Fms-related tyrosine kinase 3 ligand (FLT3L) emerged as the only inflammatory factor demonstrating causal effects on both phosphate levels and HCM. Bidirectional univariable MR indicated that FLT3L was not causally influenced by phosphate (Figs. 3 and 4), supporting its selection for subsequent causal mediation analyses. After removing outlier variants identified by MR-PRESSO (Supplementary Table 2, Supplemental Digital Content 2), subsequent sensitivity analyses revealed no evidence of residual heterogeneity or horizontal pleiotropy (Figure 5 and Supplementary Tables 3 and 4, Supplemental Digital Content 3). Leave-one-out analysis further confirmed that no single FLT3L-associated single nucleotide polymorphism drove the association with phosphate levels (Supplementary Fig. 1, Supplemental Digital Content 4).

Figure 2.

Univariable MR analysis of the causal associations between 75 inflammatory factors, hypertrophic cardiomyopathy, and phosphate levels.

Figure 3.

Univariable MR analyses. (A) Causal effects of FLT3L and phosphate levels on HCM, assessed using IVW, MR-Egger, and WM methods, shown as ORs with 95% CIs. (B) Bidirectional effects between FLT3L and phosphate levels using the same MR methods, expressed as effect estimates (β) with 95% CIs. CIs = confidence intervals, FLT3L = Fms-related tyrosine kinase 3 ligand, HCM = hypertrophic cardiomyopathy, IVW = inverse-variance weighted, MR = Mendelian randomization, ORs = odds ratios, WM = weighted median.

Figure 4.

Scatter plots illustrating the causal relationships among FLT3L, HCM, and phosphate levels. (A) Scatter plot with FLT3L as the exposure and HCM as the outcome; (B) Scatter plot with FLT3L as the exposure and phosphate as the outcome; (C) Scatter plot with phosphate as the exposure and HCM as the outcome; (D) Scatter plot with phosphate as the exposure and FLT3L as the outcome. FLT3L = Fms-related tyrosine kinase 3 ligand, HCM = hypertrophic cardiomyopathy, MR = Mendelian randomization, SNP = single nucleotide polymorphism.

Figure 5.

Funnel plots in the analyses of the causal relationships among FLT3L, hypertrophic cardiomyopathy (HCM), and phosphate levels. (A) Funnel plot with FLT3L as the exposure and HCM as the outcome; (B) Funnel plot with FLT3L as the exposure and phosphate as the outcome; (C) Funnel plot with phosphate as the exposure and HCM as the outcome; (D) Funnel plot with phosphate as the exposure and FLT3L as the outcome. FLT3L = Fms-related tyrosine kinase 3 ligand, IV = instrumental variable, MR = Mendelian randomization, SE = standard error.

Figure 6 illustrates the total effect of the exposure on the outcome, the indirect effect mediated by phosphate, and the direct effect of the exposure on the outcome. Overall, the mediation analysis indicates that the causal effect of FLT3L on HCM is partially mediated through phosphate, with approximately 12.05% of the total effect attributable to this indirect pathway.

Figure 6.

Mediation analysis of the effect of phosphate between Fms-related tyrosine kinase 3 ligand and hypertrophic cardiomyopathy. CIs = confidence intervals, OR = odds ratio.

4. Discussion

In this MR analysis, we assessed potential causal effects of 75 inflammatory factors on HCM and identified an inverse causal relationship between FLT3L and HCM. Further mediation analysis indicated that this association may be partially mediated through circulating phosphate levels.

4.1. FLT3L and HCM

FLT3L is the ligand for the class III receptor tyrosine kinase FLT3 and functions as an early-acting hematopoietic cytokine.^[29,30] Notably, FLT3 is also expressed in cardiac tissue and has been shown to regulate the cardiac side population, a resident pool of cardiac progenitor cells,^[30] although the precise physiological role of the FLT3/FLT3L signaling axis in the heart remains incompletely understood. Experimental evidence provides preliminary support for a cytoprotective role of this pathway. Pfister et al^[29,31] first reported that activation of FLT3 signaling exerts a cytoprotective effect on cardiomyocytes and improves post-myocardial infarction cardiac remodeling. More recent studies have further supported this notion, demonstrating that pharmacological inhibition of FLT3 increases cardiomyocyte apoptosis and promotes maladaptive remodeling following myocardial infarction,^[32] whereas FLT3 activation mitigates mitochondrial fragmentation and improves cardiac function during cardiac remodeling.^[33] In addition, FLT3 signaling has been implicated in the regulation of cardiac progenitor cell populations.^[30] An MR study further suggested that circulating FLT3L may have a causal role in coronary artery disease in humans,^[34] highlighting the potentially complex role of FLT3 signaling in cardiovascular diseases. Collectively, these observations suggest that FLT3 signaling may contribute to cardiac cellular resilience and structural adaptation, which is broadly consistent with our MR findings showing a protective association between higher circulating FLT3L levels and reduced risk of HCM.

4.2. The role of phosphate levels in mediating the impact of FLT3L on HCM

The MR findings of this study indicate that genetically determined higher FLT3L levels are associated with lower phosphate levels, which in turn are linked to a reduced risk of HCM. This observation suggests that FLT3L may exert cardioprotective effects through mechanisms related to phosphate homeostasis. Existing mechanistic studies provide a plausible biological context for this association. Experimental studies in cellular models have shown that FLT3 ligand stimulation can increase the expression of hypoxia-inducible factor-1α (HIF-1α).^[35] HIF-1α is a key transcription factor involved in cellular responses to hypoxia and inflammation and has been implicated in the regulation of fibroblast growth factor 23 (FGF23) expression.^[36] Upon stabilization, HIF-1α forms heterodimers with HIF-1β and binds to hypoxia-responsive elements, thereby promoting FGF23 transcription.^[36–38] As a central endocrine regulator of phosphate metabolism, FGF23 plays a critical role in maintaining systemic phosphate balance through its actions on renal phosphate handling and vitamin D metabolism. Circulating phosphate concentrations are tightly controlled by coordinated endocrine and renal regulatory mechanisms. The kidney functions as a key phosphate-sensing organ and contributes to systemic phosphate homeostasis through adaptive metabolic and transport processes.^[39] Disturbances in phosphate metabolism have been increasingly implicated in cardiovascular remodeling and vascular disease.^[40] In addition to its endocrine role in phosphate homeostasis, accumulating evidence suggests that FGF23 may also exert direct effects on the myocardium. Experimental studies have demonstrated that FGF23 can activate fibroblast growth factor receptor 4 signaling in cardiomyocytes, triggering hypertrophic growth and myocardial remodeling through a Klotho-independent mechanism.^[41,42] These findings highlight a potential mechanistic intersection between phosphate metabolism and cardiac structural changes. Taken together, these interconnected biological pathways suggest that the cardioprotective association observed for FLT3L in our MR analyses may partially involve modulation of phosphate-related endocrine signaling. While this hypothesis remains speculative and requires further experimental validation, the present findings provide population-level genetic evidence supporting a potential causal pathway linking FLT3L, phosphate metabolism, and susceptibility to hypertrophic cardiomyopathy.

A major strength of our study lies in the application of MR, which improves causal inference by reducing confounding and minimizing reverse causation inherent to observational research. Our study had several limitations. Firstly, although we selected genetic variants strongly associated with FLT3L, these variants explain only a modest proportion of the variance in the exposure. Secondly, our outcome data were predominantly drawn from individuals of European descent, which could limit the extent to which our findings are applicable to populations with different ancestral backgrounds.

5. Conclusion

In this MR analysis, genetically lower FLT3L levels were associated with a higher risk of HCM. These findings provide evidence consistent with a potential causal relationship between FLT3L and HCM and suggest that higher FLT3L expression may conferprotective effects, potentially mediated through phosphate-lowering pathways. This mechanistic link underscores the possible role of phosphate regulation in modulating HCM risk.

Author contributions

Writing – original draft: Yanhui Li, Rumeng Chen, Chunyan Hou, Zhiwei Zheng, Shuling Xu, Xiaoqian Zheng, Menghua Liu, Xueqi Liao.

Writing – review & editing: Meihua Bao, Chunxia Huang, Sen Li.