Venous Thromboembolism in Myotonic Dystrophy Type 1 (DM1-VTE)

Investigators identified a high risk of deep vein thrombosis and pulmonary embolism in patients presenting myotonic dystrophy type 1 treated in our hospital, 10 times higher than general population matched on age and sex. These venous thromboembolic events were frequently severe and lethal. Investigators suspect that this high risk of venous thromboembolism is due to coagulation abnormalities specific to myotonic dystrophy type 1. The purpose of this study is to determine: 1/ if there is a hypercoagulable state in myotonic dystrophy type 1 by testing patient's coagulation, and 2/ if genes encoding factors involved in coagulation have modified expression resulting in this hypercoagulable state. Understanding the pathophysiology will help preventing venous thromboembolism in these patients. It is the first study to describe this specific issue.

Investigators have identified in the cohort of 1084 patients presenting myotonic dystrophy type 1 (DM1) a 10% prevalence of venous thromboembolism (VTE) and a 7‰ annual incidence, which is 10-fold higher than in the general population and 3-fold compared to patients with other myopathies. Patients' clinical presentations were very similar to those observed in patients with severe hypercoagulable states caused by mutations in genes encoding factors involved in coagulation, fibrinolysis or their regulation and represented a frequent cause of death. To Investigator's knowledge, this association between VTE and DM1 has never been reported to date and no competing project has been initiated on this topic by any other team. Investigators hypothesize that VTE in DM1 may be related to a hypercoagulable state resulting from an imbalance between coagulation processes and fibrinolysis properties. Because the expression of pathogenic CTG repeats in DM1 leads to a RNA gain-of-function mechanism, Investigators propose these abnormalities may be the consequence of alternative splicing misregulation and/or abnormal gene expression of coagulation, fibrinolysis factors or other factors involved in their regulation. Investigators applied a candidate gene strategy to screen splicing profiles of 33 genes coding for haemostasis factors in a DM1 context. Using expression large-scale datasets of DM1 tissue samples from skeletal muscle and heart, Investigators identified splicing defects in 4 genes involved in haemostasis, in particular 3 involved in the fibrinolytic system: PLAT, PLAU and SERPINE1. These preliminary analyses were however not performed on liver samples whereas genes coding for haemostasis factors are synthesized and mainly expressed in liver. Analysis of liver and monocytes/megacaryotes RNA samples appears to be an essential step. The objectives are to (i) study haemostatic properties of a cohort of patients with DM1 with and without personal history of VTE, in order to determine whether a hypercoagulable state may be associated with DM1 and whether it may be correlated to the occurrence of VTE, and (ii) to perform a global transcriptomic approach using massive parallel sequencing (RNAseq) on liver and monocytes/megacaryotes. RNA samples obtained from DM1 patients will help identify candidate genes with altered expression or alternative splicing misregulation that may underlie VTE in DM1. The study will include two complementary parts in DM1 patients with and without personal history of VTE and healthy volunteers: haemostasis tests, and transcriptomic analysis on RNA samples isolated from liver biopsies and blood monocytes/megacaryocytes samples.Three teams will be involved in the study: Team 1 (clinical study), Team 2 (haematology) and Team 3 (transcriptomic analysis). Haemostasis tests will be performed by Team 1 and analysed by Team 2 in (i) 100 patients prospectively enrolled in Investigator's Institution by Team 1, aged >18 years, with genetically proven DM1, after providing written informed consent, without any current antithrombotic treatment, including 80 without and 20 with personal history of VTE and (ii) 30 healthy volunteers matched on age and sex. RNAseq will be performed on (i) liver tissue issued from 3 patients with DM1 and 6 controls and (ii) monocytes/megacaryocytes isolated from the blood of 15 patients with DM1 (7 with and 8 without VTE) and 15 controls prospectively enrolled in the study. Inclusion and exclusion criteria will be similar to those of patients included in the study of haemostasis properties. Liver tissues are already available in a tissue bank. Blood will be prospectively collected in DM1 patients and controls by Team 1. Monocytes and megacaryocytes will be isolated and cultured by Team 2. Team 3 will extract total RNA from liver samples, monocytes and megacaryocytes to the Centre National de Génotypage for RNA sequencing and perform the analysis of the data and the validation of the newly identified candidates. The estimated study duration is 72 months, including (i) 66 months for patient inclusions, coagulation testsand monocytes/megacaryocytes isolations, and (ii) 6 months for fibrinolysis tests, RNA extraction and sequencing, data analysis, validation. Investigators believe the study will have important implications for the clinical management of patients with DM1. Specific strategies will be proposed for the prevention of VTE with potentially larger indications for prophylactic anticoagulant treatments and longer duration after a first episode of VTE. Regarding the high mortality associated with VTE and the identification in the registry of pulmonary embolism as a frequent cause of death, sudden and non-sudden, in DM1 patients, the modification of these treatment strategies may be associated with an important clinical benefit.The identification of haemostasis abnormalities by coagulation and fibrinolysis tests could be useful for risk stratification in patients with DM1 and could allow targeted treatments.

Myotonic dystrophy 1, Venous thromboembolism, Pulmonary embolism, Deep vein thrombosis, Hypercoagulability, Thrombophilia, RNA alternative splicing

Two populations: Population n°1: prospective, with 3 arms Population n°2: retrospective, with 2 arms

Myotonic dystrophy type 1 patients with a history of venous thromboembolism (pulmonary embolism and/or deep vein thrombosis)

Venipuncture of 30 milliliters of blood. The following tests will be performed: thromboelastography (TEG®), standard tests of coagulation, genetic thrombophilia, lupus anticoagulant, fibrinolysis markers (Alpha-2-antiplasmin, amidolytic activity, plasmin anti-plasmin complexes, Plasminogen Activator Inhibitor-1 (PAI-1) antigen, plasminogen amydolytic activity), and a global test of fibrinolytic activity.

Venipuncture of 60 milliliters of blood. Monocytes and megacaryocytes culture. RNA extraction from monocytes and megacaryocytes.

Results of prothrombin time (PT) and activated partial thromboplastin time (APPT) in the 3 arms of population n°1

Testing for: Antithrombin III mutation C protein mutation S protein mutaiton Activated C protein resistance mutation Factor II G20210 mutation Lupus anticoagulant. Results given in: presence or absence (yes or no)

Results of the following fibrinolytic markers: alpha-2-antiplasmine, amidolytic activity, PAI-1 antigen, plasminogen amydolytic activity in the 3 arms of population n°1

Evaluation of coagulation and/or fibrinolysis genes' expression and alternative splicing in the 3 arms of population n°1 and in the 2 arms of population n°2

Bioanalysis of the patients' transcriptomes after global RNA sequencing, focusing on expression or alternative splicing misregulation of coagulation and/or fibrinolysis genes. Results given in : gene name(s) and description

Inclusion Criteria: Population N°1 Age over 18 years Patient living in France and with medical insurance Patient having given his informed and written consent DM1 groups: genetically proven DM1 VTE groups: at least 1 history of VTE (PE and/or DVT) Healthy volunteers: patient without any medical history (no DM1, no VTE, no thrombophilia), and without taking any anti-thrombotic medication Population N°2 Liver tissue of patients with genetically proven DM1 (tissue bank) Liver tissue of patients without DM1 or any history of VTE (tissue bank) Exclusion Criteria: Patient opposed to data collection and analysis 1. Population N°1 Genetically proven thrombophilia Anti-thrombotic medication Hemoglobin levels < 7 g/dL Hemoglobin levels < 9 g/dL in case of cardiac of respiratory condition 2. Population N°2 Liver tissue quality insufficient for RNA extraction and analysis

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