- Email: [email protected]
C0171: Identification of a MicroRNA Profile Related to Hypertrophic Cardiomyopathy
S12
ORAL COMMUNICATIONS / Thrombosis Research 133S3 (2014) S1–S34
Hungarian population were also recruited (n = 200). Mutations within SERPINC1 were detected by direct DNA sequencing. To investigate the founder effect rs2227596, rs941989, rs2227612, rs5877 and rs5878 intragenic SNP’s were examined by real time PCR and melting point analysis on a LightCycler 480 instrument. Four microsatellite markers (SERPINC1-Alu8, D1S196, D1S218 and as a negative control F13A01) were analyzed by fragment analysis method on an ABI 3130 Genetic Analyzer. Allelic frequency distribution of the above-mentioned genetic markers was determined in ATBp3 carriers and healthy controls, then haplotype and family tree analysis were performed. Results: Among patients with AT deficiency 74% carried the ATBp3 mutation. ATBp3 was associated with the same SNP haplotype in all carriers. Different haplotypes were observed in healthy controls. The SERPINC1-Alu8 and the marker closer to SERPINC1 (D1S218 – 0.6 cM) represented linked inheritance with the AT Bp3. The marker farther to SERPINC1 (D1S196 – 6.3 cM) was not associated with AT Bp3. Family tree analysis suggested founder effect. Conclusions: The most frequent genetic abnormality in AT deficiency is ATBp3 in the Hungarian population. The high frequency of this mutation can be explained by a founder effect.
microRNAs IN THROMBOSIS AND HEMOSTASIS C0171 IDENTIFICATION OF A MICRORNA PROFILE RELATED TO HYPERTROPHIC CARDIOMYOPATHY 1 E. Plana1 , D. Domingo2 , L. Martos1 , E. Fernandez ´ , J. Sancho3 , ´ Y. Abellan3 , M. del Carmen Salvador3 , E. Zorio4 , M. Angel Arnau4 , A. Montero4 , F. Espana ˜ 1 , P. Medina1 . 1 Haemostasis, Thrombosis, Atherosclerosis and Vascular Biology Research Group, La Fe University and Polytechnic Hospital, Valencia, Spain; 2 Cardiology Department, La Fe University and Polytechnic Hospital, Valencia, Spain; 3 Pathology Department, Legal Medicine Institute, Valencia, Spain; 4 Cardiology Department, La Fe University and Polytechnic Hospital, Valencia, Spain
Background: Hypertrophic cardiomyopathy (HCM) represents a pro-arrythmic state that can lead to sudden cardiac death (SCD). A possible complication of HCM is atrial fibrillation (AF), which can trigger thromboembolic events. microRNAs (miRNAs) are small non-coding RNA molecules that regulate protein expression. miRNAs seem to regulate cardiac hypertrophy, although the SCD- or AF-related miRNAs in HCM patients are currently unknown. The aim of our study was to identify a miRNA profile distinctive of HCM patients. Methods: We created a collection of fresh (N = 5) and formalinfixed paraffin-embedded (N = 23) ventricular myocardium from HCM patients deceased of SCD, and from controls (N = 33 and N = 12, respectively) the latter victims of cerebral hemorrhages or car accidents with no cardiac affectation. We measured miRNA expression with the GeneChip miRNA 3.0 Array (Affymetrix) in 5 HCM patients and in 5 controls. We selected those miRNAs with a significantly different expression level in patients, and with targets such as sarcomeric proteins, ion channels, intercellular unions, or that participate in calcium signalling, hypertrophy or fibrosis, given their relation with HCM, AF and malignant ventricular arrhythmias. Finally, we confirmed by RT-qPCR these differences in expression in all our samples. Results: With the expression arrays we have identified a large number of dysregulated miRNAs in HCM patients with SCD. So far we have confirmed the different expression of 6 miRNAs by RT-qPCR in all our samples. The expression of miR-486–3p is 48% reduced in HCM patients compared to controls (P = 0.003; CI 95%, 0.32–0.72), miR-222–3p is 31% reduced (0.043; 0.55–0.83), miR-103a-2–5p a
30% (0.029; 0.54–0.86), miR-1 a 43% (0.002; 0.40–0.74), miR-133a a 36% (0.004; 0.50–0.78), and miR-133b a 40% (0.004; 0.47–0.74). Conclusions: This is the first study that identifies a miRNA profile characteristic of HCM patients. The dysregulation of these miRNAs reveals a new level of protein regulation which could facilitate a pro-arrythmic state in these patients in the ventricles (causing SCD) or in the atria (causing AF with thromboembolic risk). A profile of dysregulated miRNAs could be useful for the stratification of the risk of complications in HCM patients, or for the definition of potential new therapeutic targets. ISCIII (CP09/00065, PI11/00019), FEDER and Red RIC (RD12/0042/0029), Generalitat Valenciana (GE-034/11, Prometeo 2011/027), and Instituto de Investigacion ´ Sanitaria La Fe. Pilar Medina is a Miguel Servet Researcher (ISCIII-FIS CP09/00065). C0232 MICROVESICLE-ASSOCIATED MICRORNA-19A AS A POTENTIAL MODULATOR OF BLOOD-CELL DERIVED TISSUE FACTOR IN FAMILIAL HYPERCHOLESTEROLEMIA R. Suades1 , T. Padro´ 1 , R. Alonso2 , P. Mata2 , L. Badimon1 . Cardiovascular Research Center (CSIC-ICCC), IIBSantPau, Hospital Sant Pau, Barcelona, Spain; 2 Foundation Jimenez Diaz, Madrid, Spain
1
Background: Familial hypercholesterolemia (FH) confers lifelong vascular exposure to high LDL-cholesterol plasma levels and an early development of atherosclerotic lesions, which lead to premature CAD. MicroRNAs (miRs), short non-coding RNA, post-transcriptionally regulate gene expression and, thereby pathophysiological processes such as thrombosis. miR-19a has been shown to be directly involved in TF regulation. Circulating microvesicles (cMVs), released by activated cells, play important roles in atherothrombosis and have recently emerged as a novel transport delivery system for miRs in plasma. We aimed to study miR-19a, platelet activation markers and TF in cMVs of FHpatients that had been under lipid-lowering treatment (as per guidelines) and with direct evidence of atherosclerosis by imaging (MRI). The FH-specific cMV profile and atherosclerotic plaque type were investigated. An age/gender/treatment-matched group of nonFH hypercholesterolemic patients was included for comparative purposes in a case-control study design. Methods: cMVs of FH and non-FH patients (n = 37/group) from the SpAnish Familial hypErcHolEsterolaemiA cohoRT (SAFEHEART) were isolated from platelet-free plasma (PFP). RNA from cMVs was obtained with the Exo-MiR extraction kit and miRNAs were measured using Taqman miR Custom Array Cards. Further, cMVs were analyzed by flow cytometry using annexin-V and cellspecific monoclonal antibodies. cMV-associated-TF activity was measured by a functional assay determining the FVII-dependent FXa generation. Results: miR-19a was consistently detected in cMVs of all analyzed subjects. Plasma of FH patients had significantly lower miR-19acMVs than non-FH patients (P < 0.05). In contrast, FH patients had higher numbers activated platelet-derived cMVs (pMVs) as well as of TF-rich MVs (TF+-cMVs) than non-FH patients (P < 0.0001). TF+-cMPs exerted procoagulant activity showing that TF was functionally active. In the FH-patients, miR-19a-cMVs negatively associated to MRI-detected lipid-rich atherosclerotic plaques, while overall TF+-cMVs and activated pMVs were positively correlated with lipidic plaques and inversely correlated with calcified plaques. Conclusions: Decrease in microvesicle-associated miR-19a in FH patients with subclinical atherosclerosis correlates with increased levels of TF+-cMVs and, therefore, exacerbates the atherothrombotic risk profile of FH patients.
ORAL COMMUNICATIONS / Thrombosis Research 133S3 (2014) S1–S34
Hungarian population were also recruited (n = 200). Mutations within SERPINC1 were detected by direct DNA sequencing. To investigate the founder effect rs2227596, rs941989, rs2227612, rs5877 and rs5878 intragenic SNP’s were examined by real time PCR and melting point analysis on a LightCycler 480 instrument. Four microsatellite markers (SERPINC1-Alu8, D1S196, D1S218 and as a negative control F13A01) were analyzed by fragment analysis method on an ABI 3130 Genetic Analyzer. Allelic frequency distribution of the above-mentioned genetic markers was determined in ATBp3 carriers and healthy controls, then haplotype and family tree analysis were performed. Results: Among patients with AT deficiency 74% carried the ATBp3 mutation. ATBp3 was associated with the same SNP haplotype in all carriers. Different haplotypes were observed in healthy controls. The SERPINC1-Alu8 and the marker closer to SERPINC1 (D1S218 – 0.6 cM) represented linked inheritance with the AT Bp3. The marker farther to SERPINC1 (D1S196 – 6.3 cM) was not associated with AT Bp3. Family tree analysis suggested founder effect. Conclusions: The most frequent genetic abnormality in AT deficiency is ATBp3 in the Hungarian population. The high frequency of this mutation can be explained by a founder effect.
microRNAs IN THROMBOSIS AND HEMOSTASIS C0171 IDENTIFICATION OF A MICRORNA PROFILE RELATED TO HYPERTROPHIC CARDIOMYOPATHY 1 E. Plana1 , D. Domingo2 , L. Martos1 , E. Fernandez ´ , J. Sancho3 , ´ Y. Abellan3 , M. del Carmen Salvador3 , E. Zorio4 , M. Angel Arnau4 , A. Montero4 , F. Espana ˜ 1 , P. Medina1 . 1 Haemostasis, Thrombosis, Atherosclerosis and Vascular Biology Research Group, La Fe University and Polytechnic Hospital, Valencia, Spain; 2 Cardiology Department, La Fe University and Polytechnic Hospital, Valencia, Spain; 3 Pathology Department, Legal Medicine Institute, Valencia, Spain; 4 Cardiology Department, La Fe University and Polytechnic Hospital, Valencia, Spain
Background: Hypertrophic cardiomyopathy (HCM) represents a pro-arrythmic state that can lead to sudden cardiac death (SCD). A possible complication of HCM is atrial fibrillation (AF), which can trigger thromboembolic events. microRNAs (miRNAs) are small non-coding RNA molecules that regulate protein expression. miRNAs seem to regulate cardiac hypertrophy, although the SCD- or AF-related miRNAs in HCM patients are currently unknown. The aim of our study was to identify a miRNA profile distinctive of HCM patients. Methods: We created a collection of fresh (N = 5) and formalinfixed paraffin-embedded (N = 23) ventricular myocardium from HCM patients deceased of SCD, and from controls (N = 33 and N = 12, respectively) the latter victims of cerebral hemorrhages or car accidents with no cardiac affectation. We measured miRNA expression with the GeneChip miRNA 3.0 Array (Affymetrix) in 5 HCM patients and in 5 controls. We selected those miRNAs with a significantly different expression level in patients, and with targets such as sarcomeric proteins, ion channels, intercellular unions, or that participate in calcium signalling, hypertrophy or fibrosis, given their relation with HCM, AF and malignant ventricular arrhythmias. Finally, we confirmed by RT-qPCR these differences in expression in all our samples. Results: With the expression arrays we have identified a large number of dysregulated miRNAs in HCM patients with SCD. So far we have confirmed the different expression of 6 miRNAs by RT-qPCR in all our samples. The expression of miR-486–3p is 48% reduced in HCM patients compared to controls (P = 0.003; CI 95%, 0.32–0.72), miR-222–3p is 31% reduced (0.043; 0.55–0.83), miR-103a-2–5p a
30% (0.029; 0.54–0.86), miR-1 a 43% (0.002; 0.40–0.74), miR-133a a 36% (0.004; 0.50–0.78), and miR-133b a 40% (0.004; 0.47–0.74). Conclusions: This is the first study that identifies a miRNA profile characteristic of HCM patients. The dysregulation of these miRNAs reveals a new level of protein regulation which could facilitate a pro-arrythmic state in these patients in the ventricles (causing SCD) or in the atria (causing AF with thromboembolic risk). A profile of dysregulated miRNAs could be useful for the stratification of the risk of complications in HCM patients, or for the definition of potential new therapeutic targets. ISCIII (CP09/00065, PI11/00019), FEDER and Red RIC (RD12/0042/0029), Generalitat Valenciana (GE-034/11, Prometeo 2011/027), and Instituto de Investigacion ´ Sanitaria La Fe. Pilar Medina is a Miguel Servet Researcher (ISCIII-FIS CP09/00065). C0232 MICROVESICLE-ASSOCIATED MICRORNA-19A AS A POTENTIAL MODULATOR OF BLOOD-CELL DERIVED TISSUE FACTOR IN FAMILIAL HYPERCHOLESTEROLEMIA R. Suades1 , T. Padro´ 1 , R. Alonso2 , P. Mata2 , L. Badimon1 . Cardiovascular Research Center (CSIC-ICCC), IIBSantPau, Hospital Sant Pau, Barcelona, Spain; 2 Foundation Jimenez Diaz, Madrid, Spain
1
Background: Familial hypercholesterolemia (FH) confers lifelong vascular exposure to high LDL-cholesterol plasma levels and an early development of atherosclerotic lesions, which lead to premature CAD. MicroRNAs (miRs), short non-coding RNA, post-transcriptionally regulate gene expression and, thereby pathophysiological processes such as thrombosis. miR-19a has been shown to be directly involved in TF regulation. Circulating microvesicles (cMVs), released by activated cells, play important roles in atherothrombosis and have recently emerged as a novel transport delivery system for miRs in plasma. We aimed to study miR-19a, platelet activation markers and TF in cMVs of FHpatients that had been under lipid-lowering treatment (as per guidelines) and with direct evidence of atherosclerosis by imaging (MRI). The FH-specific cMV profile and atherosclerotic plaque type were investigated. An age/gender/treatment-matched group of nonFH hypercholesterolemic patients was included for comparative purposes in a case-control study design. Methods: cMVs of FH and non-FH patients (n = 37/group) from the SpAnish Familial hypErcHolEsterolaemiA cohoRT (SAFEHEART) were isolated from platelet-free plasma (PFP). RNA from cMVs was obtained with the Exo-MiR extraction kit and miRNAs were measured using Taqman miR Custom Array Cards. Further, cMVs were analyzed by flow cytometry using annexin-V and cellspecific monoclonal antibodies. cMV-associated-TF activity was measured by a functional assay determining the FVII-dependent FXa generation. Results: miR-19a was consistently detected in cMVs of all analyzed subjects. Plasma of FH patients had significantly lower miR-19acMVs than non-FH patients (P < 0.05). In contrast, FH patients had higher numbers activated platelet-derived cMVs (pMVs) as well as of TF-rich MVs (TF+-cMVs) than non-FH patients (P < 0.0001). TF+-cMPs exerted procoagulant activity showing that TF was functionally active. In the FH-patients, miR-19a-cMVs negatively associated to MRI-detected lipid-rich atherosclerotic plaques, while overall TF+-cMVs and activated pMVs were positively correlated with lipidic plaques and inversely correlated with calcified plaques. Conclusions: Decrease in microvesicle-associated miR-19a in FH patients with subclinical atherosclerosis correlates with increased levels of TF+-cMVs and, therefore, exacerbates the atherothrombotic risk profile of FH patients.