Exploring the causal pathway from ischemic stroke to atrial fibrillation: a network Mendelian randomization study

Background and purpose Previous studies have found ischemic stroke is associated with atrial fibrillation. However, the causal association between ischemic stroke and atrial fibrillation is not clear. Furthermore, the network relationship among ischemic stroke, atrial fibrillation and its risk factors need further attention. This study aims to examine the potential causal association between ischemic stroke and atrial fibrillation and further to explore potential mediators in the causal pathway from ischemic stroke to atrial fibrillation. Methods Summary statistics from the ISGC (case = 10,307 and control = 19,326) were used as ischemic stroke genetic instruments, AFGen Consortium data (case = 65,446 and control = 522,744) were used for atrial fibrillation, and other consortia data were used for potential mediators (fasting insulin, white blood cell count, procalcitonin, systolic and diastolic blood pressure, body mass index, waist circumference, and height). Under the framework of network Mendelian randomization, two-sample Mendelian randomization study was performed using summary statistics from several genome-wide association studies. Inverse-variance weighted method was performed to estimate causal effect. Results Blood pressure mediates the causal pathways from ischemic stroke to atrial fibrillation. The total odds ratio of ischemic stroke on atrial fibrillation was 1.05 (95% confidence interval [CI], 1.02 to 1.07; P = 1.3 × 10−5). One-unit increase of genetically determined ischemic stroke was associated with 0.02 (DBP: 95% CI, 0.001 to 0.034, P = 0.029; SBP: 95% CI, 0.006 to 0.034, P = 0.003) upper systolic and diastolic blood pressure levels. Higher genetically determined systolic and diastolic blood pressure levels were associated with higher atrial fibrillation risk (DBP: RR, 1.18; 95% CI, 1.03 to 1.35; P = 0.012. SBP: RR, 1.18; 95% CI, 1.01 to 1.38; P = 0.04). Specially, we also found the bidirectional causality between blood pressure and ischemic stroke. Conclusions Our study provided a strong evidence that raised blood pressure in stroke patients increases the risk of atrial fibrillation and active acute blood pressure lowering can improve the outcome in ischemic stroke patients.

Ⅰ Basic characteristics of the ISGC (International Stroke Genetics Consortium) Table S2 Ⅱ Basic characteristics of the MAGIC (Meta-analyses of glucose and insulin-related traits consortium) Ⅲ Ⅳ Ⅰ Illustration of the MR analysis when a genetic variant is in linkage disequilibrium with a causal variant. Figure S2 Ⅱ Illustration of the MR analysis when the population stratification exists.
Figure S3 Ⅲ Illustration of pleiotropic in the MR analysis.

Figure S4
Ⅳ Scatter plots showing the per-allele association with AF plotted against the perallele association with IS before and after removing rs12646447. Figure S5 Ⅴ Scatter plots showing the per-allele association with DBP plotted against the perallele association with IS (a), SBP plotted against the per-allele association with IS (b), AF plotted against the per-allele association with DBP (c), AF plotted against the per-allele association with SBP (d), IS plotted against the per-allele association with DBP (e), IS plotted against the per-allele association with SBP(f), AF plotted against the per-allele association with IS(g).

Figure S6
Ⅵ Leave-one-out plots of MR estimate in causal effects of IS on AF before and after removing rs12646447. Figure S7Ⅶ Leave-one-out plots of MR estimate in causal effects of IS on DBP (a), IS on SBP (b), DBP on AF (c), SBP on AF (d), DBP on IS (e), SBP on IS (f) and AF on IS (g).

Inverse-variance weighted method
Inverse-variance weighted (IVW) estimator can be calculated from summarized data. To some extent, IVW method is a fixed-effect meta-analysis, where the IV-specific causal estimates are the study-specific estimates, and the weights are the inverse-variance weights. The causal estimate from the IVW method ( IVW  ) is calculated by the following equation: The standard error is estimated by:

MR-Egger method
MR-Egger method assume that the correlation between the genetic associations with the exposure ( IV X   ) and the direct effects of the genetic variants on the outcome ( IV Y   ) is zero, which refer as InSIDE (Instrument Strength Independent of Direct Effect). By fitting the linear model:  and E  are the coefficients in the regression model. The slope and intercept represent the causal effect of exposure on outcome and the average pleiotropic effect across the genetic variants (the average direct effect of a variant with the outcome), respectively. The estimate coefficients from Egger regression is calculated from the following equation:

Weak Instruments
The F statistic is a measure of instrument strength. It is related to the proportion of variance in the exposure explained by the genetic variants (R 2 ), sample size (n) and the number of instruments (k) by the formula The commonly cited rule-of-thumb is that F > 10 avoids bias in IV analysis.

Mediation analysis
The association of IS with AF was mediated by DBP and SBP were tested in an additional analysis after DBP and SBP were identified as potential mediators. The total effect (risk ratio: RR) of 1 SD increase of IS on AF was 1.04 [log(RR)= 0.047]. The effect of 1 SD increase of IS on DBP was 0.019, and 1 unit increase in DBP was associated with AF was 1.18[log(OR) = 0.169]. Thus, the mediated effect of DBP was 0.019×0.169=0.0032. The mediated proportion was 0.0032/0.047 = 6.8%. In a similar way, we obtain the mediated proportion of DBP in the causal pathway of IS on AF was 7.4%. In addition, the association of DBP with AF was mediated by IS and the association of SBP with AF was mediated by IS were tested in a post-hoc analysis. The mediated proportion of IS in the causal pathway of DBP on AF was 13.9%; the mediated proportion of IS in the causal pathway of SBP on AF was 14.1%. Details are listed in supplemental table 12.

Figure S1
Ⅰ Illustration of the MR analysis when a SNP is in linkage disequilibrium with a SNP.
Linkage disequilibrium (LD) is the correlation between allelic states at different loci within the population. In Mendelian randomization studies, assumptions are violated if the genetic variant being used as an instrument is in linkage disequilibrium with a genetic variant that is related to the confounders or outcome. ( Pleiotropy refers to a genetic variant having multiple functions. The Mendelian randomization study will result in the violation of the assumptions 3 if the variant is associated with pleiotropic effects (X1) that do not (other than via the modifiable exposure of interest) influence the outcome; genetic variants with pleiotropic effects that do influence the outcome will, however, invalidate the Mendelian randomization approach.

Figure S4
Ⅳ Scatter plots showing the per-allele association with AF plotted against the perallele association with IS before and after removing rs12646447.
(a) Before removing rs12646447 (b) After removing rs12646447 Figure S5Ⅴ Scatter plots showing the per-allele association with DBP plotted against the perallele association with IS (a), SBP plotted against the per-allele association with IS (b), AF plotted against the per-allele association with DBP (c), AF plotted against the per-allele association with SBP (d), IS plotted against the per-allele association with DBP (e), IS plotted against the per-allele association with SBP(f).