Study of the Regulatory Mechanisms of Gene Expression in Venous Thromboembolic Disease: microRNAs

Abstract

Venous Thrombosis (VT) is a frequent complex disease that involves a disruption of the balance of the hemostatic system. microRNAs (miRNAs) are small non-coding RNAs that regulate gene expression. Promising findings in other complex diseases encourage their use as clinical biomarkers. However, up to now, the implication of miRNAs in VT has not been studied in-depth. The main objective of this Thesis was to study the regulatory mechanisms of the gene expression through miRNAs in VT. The specific objectives were (1) to identify a plasma miRNA profile associated with VT and to analyze its suitability as biomarker, (2) to dissect the interactions between biological layers in the biological context of VT and (3) to identify factors affecting either expression or quantification of miRNAs in plasma. In the first article, the differential expression of miRNAs in plasma was analyzed in the ‘Genetic Analysis of Idiopathic Thrombophilia’ 2 (GAIT-2) Project, which involves 935 individuals belonging to 35 extended Spanish families with idiopathic thrombophilia. First, we conducted a discovery phase, in which 752 miRNAs were measured in plasma by quantitative Polymerase Chain Reaction in 104 individuals of GAIT-2 (52 VT cases and 52 controls). Sixteen miRNAs were selected, which were measured in the entire GAIT-2 (n=935). Four of the miRNAs were significantly associated with VT (false discovery rate <0.1): hsa-miR-885-5p, hsa-miR-126-3p, hsa-miR-192-5p and hsa-miR-194-5p. All the four miRNAs returned significant odds ratio for VT, in the range 1.3-2.12. The discriminatory ability of the profile including the four miRNAs, age and sex retuned an area under the curve (AUC) of 0.7. In addition, significant correlations were found between the miRNAs and clinical VT phenotypes. In the second article, the four miRNAs identified above were integrated with the gene expression levels of 260 genes of the blood coagulation pathway and 14 clinical VT phenotypes. 51 VT cases and 51 controls of the GAIT-2 Project were included. Feature selection was conducted by the building of linear models for VT discrimination, which were then optimized using penalized regression. We obtained three models with AUC >0.7. The first model (VT ~ GATA2 + von Willebrand Factor) showed that the expression of GATA2 in blood was inversely correlated with the blood levels of von Willebrand Factor, and that the disruption of this relationship represents a prothrombotic phenotype. The second model (VT ~ Factor IX, ANXA2 + ENTPD1 + ILK + PDPK1 + PRKAR1A + STXBP3 +hsa-miR- 885-5p + hsa-miR-192-5p) represented an interaction between the fibrinolytic system and platelet activation through the αIIbβ3 signaling pathway. The third model (VT ~CSRP1+ LYN + hsa-miR-192-5p+ hsa-miR-885-5p) revealed the interaction between two group of genes involved in platelet activation, correlated with Protein S, and two miRNAs, in the biological context of VT. In the third article, we used the miRNA expression data of the discovery phase (103 miRNAs in 104 subjects) to analyze the effect of biological and technical factors in their expression. First, we found that the hemolysis marker represented ~10% of the shared variability of miRNA expression. Therefore, using this value as a continuous covariate, beyond as a categorical control, could help to increase consistency across miRNA studies. Second, we found that the expression of miRNAs in plasma was not biased by any blood cell count. Then, we identified 1,323 genetic variants associated with the expression of 16 miRNA genes, that represent 158 independent loci. Finally, we found that these loci were enriched in promoter regions from several tissues, though not in blood tissue. This finding is in agreement with the results regarding blood cell counts, and encourages the role of circulating miRNAs as biomarkers of tissue specific conditions.

El objetivo general de esta Tesis fue estudiar los mecanismos de regulación de la expresión génica mediante microRNAs (miRNAs) en la trombosis venosa (VT). En el primer artículo, se explora la expresión diferencial de miRNAs en VT utilizando la población del proyecto ‘Genetic Analysis of Idiopathic Thrombophilia’ 2 (GAIT-2), compuesta por 935 individuos de 35 familias con trombosis idiopática. El diseño experimental implicó una fase de descubrimiento en la que 752 miRNAs plasmáticos se cuantificaron en 104 individuos del GAIT2 (52 casos y 52 controles) y se seleccionaron 16 miRNAs para la fase de validación, en la que se cuantificaron en toda la población GAIT-2 (n=935). Cuatro de los miRNAs se asociaron significativamente con la enfermedad (false discovery rate <0.1): hsa-miR-885-5p, hsa-miR-126-3p, hsa-miR-192-5p y hsa-miR-194-5p. El segundo artículo supone la integración de los cuatro miRNAs identificados con los niveles de expresión en sangre de 260 genes de la vía de la coagulación y con 14 fenotipos clínicos de VT. Se incluyeron 102 individuos del GAIT-2 y se llevó a cabo mediante la construcción de modelos lineales para la discriminación de VT. El primer modelo reveló que los niveles de expresión de GATA2 están inversamente correlacionados con los niveles de Factor von Willebrand y que la disrupción de dicha relación representa un fenotipo protrombótico. El segundo modelo representó una interacción entre el sistema fibrinolítico y la activación plaquetaria mediante la vía de señalización αIIbβ3. El último modelo identificó dos grupos de genes implicados en la activación plaquetaria, correlacionados con la Proteína S, y dos miRNAs. En el tercer artículo, se analiza el efecto de variables biológicas y técnicas en la expresión de miRNAs en plasma. Primero, identificamos que utilizar el marcador de hemolisis como covariable continua podría ayudar a mejorar la concordancia entre estudios de miRNAs. Segundo, de nuestros datos se desprende que los conteos celulares no sesgan sistemáticamente la expresión de miRNAs en plasma. Por último, identificamos 1,323 variantes genéticas asociadas con la expresión de 16 genes de miRNAs, que suponen 158 loci independientes, enriquecidos en promotores de diversos tejidos, aunque no de sangre.