Signatures of cardioembolic and large-vessel ischemic stroke

Abstract

Objective: The cause of stroke remains unknown or cryptogenic in many patients. We sought to determine whether gene expression signatures in blood can distinguish between cardioembolic and large-vessel causes of stroke, and whether these profiles can predict stroke etiology in the cryptogenic group.

Methods: A total of 194 samples from 76 acute ischemic stroke patients were analyzed. RNA was isolated from blood and run on Affymetrix U133 Plus2.0 microarrays. Genes that distinguish large-vessel from cardioembolic stroke were determined at 3, 5, and 24 hours following stroke onset. Predictors were evaluated using cross-validation and a separate set of patients with known stroke subtype. The cause of cryptogenic stroke was predicted based on a model developed from strokes of known cause and identified predictors.

Results: A 40-gene profile differentiated cardioembolic stroke from large-vessel stroke with >95% sensitivity and specificity. A separate 37-gene profile differentiated cardioembolic stroke due to atrial fibrillation from nonatrial fibrillation causes with >90% sensitivity and specificity. The identified genes elucidate differences in inflammation between stroke subtypes. When applied to patients with cryptogenic stroke, 17% are predicted to be large-vessel and 41% to be cardioembolic stroke. Of the cryptogenic strokes predicted to be cardioembolic, 27% were predicted to have atrial fibrillation.

Interpretation: Gene expression signatures distinguish cardioembolic from large-vessel causes of ischemic stroke. These gene profiles may add valuable diagnostic information in the management of patients with stroke of unknown etiology though they need to be validated in future independent, large studies.

Figure 1

Figure 1A. Hierarchical cluster plot of the 40 genes that were significantly different for cardioembolic stroke compared to large vessel stroke. Genes are shown on the y-axis and subjects are shown on the x-axis. Red indicates a high level of gene expression and blue indicates a low level of gene expression. Subjects cluster by diagnosis. One group of genes has a high level of expression in cardioembolic stroke and a low level of expression in large vessel stroke. A separate group of genes has a low level of expression in cardioembolic stroke and a high level of expression in large vessel stroke. The cardioembolic group may cluster into two subgroups. Figure 1B. Principal Components Analysis (PCA) of the 40 genes found to differentiate cardioembolic stroke from large vessel stroke. Each sphere represents a single subject. The ellipsoid surrounding the spheres represents two standard deviations from the group mean.

Figure 2

Leave one out cross-validation prediction analysis of the 40 genes found to differentiate cardioembolic stroke from large vessel stroke. The probability of the predicted diagnosis is shown on the y-axis. The actual diagnosis is shown on the x-axis, where patients with known cardioembolic stroke are shown on the left, and patients with known large vessel stroke are shown on the right. The probability of a predicted diagnosis of cardioembolic stroke is indicated by the green diamonds, and the probability of a predicted diagnosis of large vessel stroke is indicated by the orange squares. Subjects with known cardioembolic stroke were predicted to have cardioembolic stroke for 69 out of 69 samples (100% correct prediction). Subjects with known large vessel stroke were predicted to have large vessel stroke for 29 out of 30 samples (96.7% correct prediction). A sample is considered misclassified if the predicted class does not match the known class with a probability greater than 0.5.

Figure 3

Figure 3A. Hierarchical cluster analysis of the 37 genes that were significantly different in subjects with cardioembolic stroke due to atrial fibrillation compared to those with non-atrial fibrillation causes. Genes are shown on the y-axis and subjects are shown on the x-axis. Red indicates a high level of gene expression and blue indicates a low level of gene expression. Subjects cluster by diagnosis. One group of genes has a high level of expression in cardioembolic stroke due to atrial fibrillation and a low level of expression in non-atrial fibrillation causes. Another group of genes has a low level of expression in cardioembolic stroke due to atrial fibrillation and a high level of expression in non-atrial fibrillation causes. Figure 3B. Principal Components Analysis of the 37 genes found to differentiate cardioembolic stroke due to atrial fibrillation from non-atrial fibrillation causes. Each sphere represents a single subject. The ellipsoid surrounding the spheres represents two standard deviations from the group mean.

Figure 4

Leave one out cross-validation prediction analysis of the 37 genes found to differentiate cardioembolic stroke due to atrial fibrillation from non-atrial fibrillation causes. The probability of the predicted diagnosis is shown on the y-axis. The actual diagnosis is shown on the x-axis, where patients with known atrial fibrillation are on the left, and patients with known non-atrial fibrillation stroke are on the right. The probability of a predicted diagnosis of stroke due to atrial fibrillation is indicated by the green diamonds, and the probability of a predicted diagnosis of non-atrial fibrillation stroke is indicated by the orange squares. Subjects with known cardioembolic stroke due to atrial fibrillation were predicted to have atrial fibrillation as a cause of stroke in 30 out of 30 samples (100% correct prediction). Subjects with cardioembolic stroke due to non-atrial fibrillation causes were correctly predicted in 22 out of 24 samples (91.7% correct prediction). A sample is considered misclassified if the predicted class does not match the known class with a probability greater than 0.5.