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MicroRNAs as antiphospholipid syndrome biomarkers
Figure 4. Antiphospholipid syndrome (APS) patients show a specific miRNA profile distinct from both non-autoimmune patients with previous thrombotic events and aPL-negative systemic lupus erythematosus (SLE) patients. Twenty-three thrombotic non-antiphospholipid syndrome patients (non-APS) and 25 aPL-negative SLE patients were included, and the circulating microRNA (miRNA or miR) signatures of APS were compared. One-way ANOVA was used for statistical comparisons. A Bonferroni correction was applied for multiple testing. P<0.05 was considered statistically significant. Beeswarm plot of each differentially expressed miRNA ratio is shown along with mean, Standard Deviation and P-value. n.s.: no significant statistical differences.
Supplementary Figure S2B). Thus, our data support the theory that there is a specific circulating miRNA signa- ture in APS which remains stable over time.
APS patients show a specific miRNA profile different from both non-autoimmune patients with previous thrombotic events and aPL-negative SLE patients
To assess the specificity of the miRNA signature found in APS patients, and in order to analyze whether the altered expression of the circulating miRNAs evaluated was linked to their thrombophilic status, an additional dis- ease group including 23 patients with thrombosis in the absence of an associated autoimmune disease was evalu- ated. In these patients, the ratios formed by the expression levels of the 11 selected miRNAs were significantly differ- ent from those described in APS patients, except for the ratios miR-19b/miR-15a and miR-19b/miR-145 which exhibited non-significant differences (Figure 4). To evalu- ate if the altered expression of the miRNA signature was a sign of an autoimmune status, an additional disease group, including 25 SLE patients negative for aPL, was analyzed. In this SLE cohort, the ratios produced by the selected circulating miRNAs were significantly different from those found in APS patients, except for the ratios miR-19b/miR-34a, miR-20a/miR-374a and miR-124/miR- 296 which exhibited non-significant differences (Figure 4).
Potential influence of standard therapy on the profile of circulating miRNAs in APS
APS patients were classified into two groups based on the treatment received, including 30 primary APS patients treated with antiplatelet agents and 62 primary APS patients treated with anticoagulant drugs. The statistical comparison between patients treated with antiplatelet and anticoagulant agents showed no significant differ-
ences in miRNA signature, except for the ratio miR- 20a/374 (Online Supplementary Figure S3).
Circulating miRNAs are associated with clinical features of APS and show potential as biomarkers for the development of atherosclerosis
The levels of some circulating miRNA ratios that inte- grate the signature in APS were associated with the ocur- rence of fetal losses in these patients, including elevated levels of miR-19b/miR-124 and miR-20a/miR-374, and reduced levels of miR-124/miR-296 (Figure 5A). Associations between miRNA ratios and the type of thrombosis suffered by APS patients were also identified. Thus, elevated levels of ratios miR-20a/miR-145 and miR- 20a/miR-374 were significantly associated with the ocur- rence of arterial thrombosis in APS patients (Figure 5B). Furthermore, the ratios of miR-19b/miR-124 and miR- 124/miR-296 were also found to be associated with the presence of a pathological CIMT in these patients (Figure 5C). To accurately evaluate their relevance as biomarkers of early atherosclerosis, we conducted combined ROC analyses of these miRNA ratios. The combination of both circulating miRNA ratios as a panel showed an evident accuracy, with an AUC of 0.76 at a sensitivity of 67% and specificity of 78% from a cut-off value of 0.41 (Figure 5D).
Cluster analysis
Hard clustering analysis in the APS cohort differentiated 3 clusters representing different thrombotic risk profile groups. Clinical and laboratory parameters of each cluster are resumed (Figure 6A). Briefly, cluster 1 (50% of the clus- tered cohort) was characterized by lower prevalence of cardiovascular risk factors and aPL multiple positivity. Conversely, cluster 1 shows a higher rate of venous thrombotic event when compared to the other clusters.
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