Genetically Predicted Cardiac Troponin I Concentrations and Risk of Stroke and Atrial Fibrillation

Background: Observational studies have shown that elevated circulating cardiac troponin I (cTnI) concentrations were associated with higher risk of stroke and atrial brillation, but the causality remains unclear. Therefore, we conducted a two-sample mendelian randomization study to evaluate the causal effects of cTnI concentrations on the risk of stroke subtypes and atrial brillation. Methods: The instrumental variables for circulating cTnI concentrations were selected from a genome-wide association study meta-analysis of 48,115 European individuals. Applying a 2-sample mendelian randomization approach, we examined the associations of circulating cTnI concentrations with stroke (40,585 cases and 406,111 controls), ischemic stroke (34,217 cases and 406,111 controls), ischemic stroke subtypes (cardioembolic, large artery, small vessel stroke), intracerebral hemorrhage (1,545 cases and 1,481 controls) and atrial brillation (60,620 cases and 970,216 controls). Results: Genetically predicted elevated circulating cTnI concentrations were associated with increased risk of cardioembolic stroke (odds ratio [OR], 1.80; 95% condence interval [CI], 1.20-2.68; P = 0.004). However, no signicant association was observed for cTnI concentrations with large artery stroke, small vessel stroke, total stroke, ischemic stroke and intracerebral hemorrhage. Additionally, we also found that elevated cTnI concentrations were associated with higher risk of atrial brillation (OR, 1.30; 95% CI, 1.10-1.53; P = 0.003). Conclusions: This study provides evidence that genetically predicted circulating cTnI concentrations are causally associated with increased risk of cardioembolic stroke and atrial brillation.


Introduction
Stroke is a common cause of death and disability worldwide. Although many risk factors for stroke have been established, a substantial proportion of stroke risk factors remain unknown. [16] Cardiac troponin I (cTnI) is a structural protein released during myocardial injury and is widely used for the diagnosis of myocardial infarction. [12] Elevated circulating cTnI concentrations have shown to be associated with a higher risk of cardiovascular disease and death in the general population. [2,11] Some observational studies investigated the association between cTnI concentrations and the risk of stroke, and these studies yielded inconsistent results. [8,9,18,20,26,28] Few studies investigated the association of cTnI concentrations with different ischemic stroke (IS) subtypes. Given that different IS subtypes has different pathologic mechanisms, it is necessary to further assess the effect of cTnI concentrations on different IS subtypes. Additionally, much of the available evidence for the causal association between cTnI concentrations and risk of stroke comes from observational studies, which may be affected by confounding and reverse causality. Thus, whether cTnI concentrations are causally associated with the risk of stroke remains unknown.

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Mendelian randomization (MR) is a genetic technique in which genetic variants are used as proxies for exposure to examine the causal relationship between an exposure and a disease outcome. [7,22] Because genetic variants are randomly allocated at conception and unlikely to be affected by disease in later life, MR can avoid the confounding and reverse causality in observational studies. Recently, a genome-wide association study (GWAS) has identi ed several single nucleotide polymorphisms (SNPs) that are independently associated with circulating cTnI concentrations in the general population, [17] providing potential tools for MR. In this study, we aimed to use a two-sample MR to evaluate whether genetically predicted circulating cTnI concentrations are causally associated with the risk of stroke, IS, IS subtypes (large artery stroke [LAS], small vessel stroke [SVS] and cardioembolic stroke [CES]), intracerebral hemorrhage (ICH). Atrial brillation (AF) is an important risk factor of stroke. Given that elevated cTnI concentrations has also shown to be associated with an increased risk of AF. [21,27] Therefore, the causal effects of cTnI concentrations on AF was also investigated in this MR study.

Data Sources
The public available GWAS summary statistics used in this study were summarized in Table 1. Because all data came from published GWASs, no additional ethical approval from an institutional review board was needed for this study. Atrial brillation 60,620 cases/970,216 controls European GWAS meta-analysis of 6 studies [19] GWAS, genome-wide association study; HUNT, Trøndelag Health Study; GS:SFHS, Generation Scotland Scottish Family Health Study; ISGC: International Stroke Genetics Consortium.

Genetic Instrument For Circulating Cardiac Troponin I Concentrations
Instrumental variables for circulating cTnI concentrations were selected from a GWAS meta-analysis of the Trøndelag Health Study ((HUNT, n = 29,839) and the Generation Scotland Scottish Family Health Study (GS:SFHS, n = 18,276) with 48,115 European individuals. [17] This GWAS identi ed 12 genome-wide signi cant (P < 5×10 − 8 ) SNPs independently associated with cTnI concentrations (Supplementary Table 1). These 12 SNPs explained 1.2% phenotypic variation of circulating cTnI concentrations. [17] Outcome Data Sources Treatment criteria (TOAST). [1] For ICH, we used the summary statistics from the International Stroke Genetics Consortium (ISGC) metaanalysis including 1,545 cases and 1,481 controls. [24] For AF, we used a GWAS meta-analysis of 6 studies (The Nord-Trøndelag Health Study, deCODE, the Michigan Genomics Initiative, DiscovEHR, UK Biobank, and the AFGen Consortium) including 60,620 cases and 970,216 controls of European ancestry. [19] For the SNPs were not available in the outcome dataset, the proxy SNPs (r2 > 0.9) were used to replace them by using the LDlink (http://ldlink.nci.nih.gov) [13] according to the CEU population data from 1,000 Genomes Project.

Statistical analysis
The primary MR analysis was performed by using inverse-variance weighted (IVW) method, [6] which assumes that all genetic variants are valid genetic instruments (e.g., no directional pleiotropy). Given that the IVW estimate may be biased by the directional pleiotropy, weighted median [4] and MR-Egger [3] methods were also used to test the robustness of the IVW estimate. The weighted median method can provide a consistent estimate if at least 50% of the instrumental variables are valid. The MR-Egger approach can detect and adjust for the bias due to directional pleiotropy.
Several methods were conducted to evaluate the potential pleiotropy. First, we used Mendelian Randomization Pleiotropy Residual Sum and Outlier (MR-PRESSO) [23] method to detect potential pleiotropic outlier (P < 0.1), and the IVW estimate was re-calculated if the pleiotropic outlier was identi ed.
Second, the heterogeneity across different SNPs was assessed by using the Cochran's Q statistics (P < 0.05 indicates signi cant). Third, we performed MR-Egger intercept test to examine the directional pleiotropy, and the signi cant MR-Egger intercept test (P < 0.05) indicates the presence of directional pleiotropy. Finally, leave-one-out (LOO) analysis was used to test whether a single SNP drive the association.

Results
Genetically predicted circulating cTnI concentrations and risk of stroke A SNP rs151313792 for cTnI concentrations was not available in the stroke (total stroke, IS, IS subtypes) outcome dataset, and no proxy SNP (r 2 > 0.8) can be used to replace it. The SNP rs8039472 was used as a proxy for rs8024538 (r 2 = 0.92), because rs8024358 was not available in the stroke ((total stroke, IS, IS subtypes and ICH) outcome dataset. For ICH, a total of 4 SNPs were not available and no proxy SNP. Additionally, MR-PRESSO identi ed a outlier for CES outcome (Supplementary Table 2), and no outlier was identi ed for other stroke outcome (LAS, SVS, stroke, IS and ICH).
As shown in Fig. 1, after remove the outlier, the IVW estimates indicated that genetically predicted elevated cTnI concentrations were associated with an increased risk of CES (OR, 1.80; 95%CI, 1.20-2.68; P = 0.004; Fig. 1). Similar effect estimates were observed in weighted median and MR-Egger estimates, although with broader CIs due to lower statistical power (Fig. 1) Fig. 1).

Discussion
To the best of our knowledge, this was the rst MR study to investigate the relationship between circulating cTnI concentrations and risk of stroke, IS, IS subtypes, ICH and AF using a two-sample MR approach. Our ndings indicate that genetically predicted elevated cTnI concentrations were causally associated with an increased risk of CES but not LAS, SVS, stroke, ischemic stroke and ICH. Additionally, we also found that elevated cTnI concentrations were associated with higher risk of AF.
Recently, a meta-analysis of 12 studies [5] performed in general population indicated that elevated highsensitivity assayed troponins is associated with an increased risk of stroke. However, this meta-analysis has a high heterogeneity, and stroke subtypes were not provided in this study. [5] Another study [8] conducted in 9 European community-based cohorts demonstrated that elevated hsTnI concentrations were associated with increased risk of stroke, IS and ICH. However, these studies are observational studies that are easily in uenced by its inherent limitation, such as confounding and reverse bias. Therefore, in order to provide causal evidence for the effects of cTnI concentrations on the risk of stroke, we conducted this MR study to reduce the possibility of confounding and reverse causation. In this MR study, we found that circulating cTnI concentrations were only associated with increased risk of CES but not with LAS, SVS, total stroke, IS and ICH. A possible explanation for the observed signi cant association between cTnI concentrations and CES is that elevated cTnI concentrations may be related with asymptomatic arrhythmias, in particular, AF. [15] Several previous studies have reported that elevated cTnI concentrations were associated with an increased risk of AF in the general population. [21,27] Hence, using the MR approach, we also evaluate the association of genetically predicted cTnI concentrations with AF, and found that genetically predicted cTnI concentrations were causally associated with an increased risk of AF. Therefore, the observed association of cTnI concentrations with CES might be related to AF. Further studies are still needed to investigate the mechanisms by which elevated cTnI concentrations increased the risk of CES.
The major strength of this study is the two-sample MR study design, which can reduce the confounding factors and reverse causality in observational studies. Other important strengths are the large samples of stroke cases and that associations with IS subtypes can be further investigated. However, several limitations still needed to be noticed. The major limitation of MR study is potential pleiotropy, which may bias the IVW estimates. Therefore, several approaches were conducted to evaluate and adjust for potential pleiotropy. The potential outliers identi ed by MR-PRESSO method were excluded before IVW estimates. Weighed median and MR-Egger estimates that are relatively robust to pleiotropy, were also performed. LOO analysis and forest-plot were also used to evaluate whether a single SNP drive the association. The results of these analyses were consistent, indicating that this observed association was unlikely due to the pleiotropy. Additionally, our results were based on the samples of European ancestry.
Thus, these ndings in this study might not be extrapolated to other population.
To conclude, this MR study provided evidence to support that genetically predicted elevated cTnI concentrations are causally associated with increased risk of CES and AF.
Page 8/12 Declarations Figure 1 Mendelian randomization estimates of circulating cardiac troponin I concentrations and risk of stroke and atrial brillation.

Supplementary Files
This is a list of supplementary les associated with this preprint. Click to download. Supplementarymeterial.pdf