DNA Methylation Analysis in Acute Coronary Syndrome and Atrial Fibrillation: DIANA Clinical Trial

Study population will include 20 healthy controls admitted to electrocardiographic control without further cardiological, electrocardiographic and clinical evidence of cardiac pathologies, 20 patients with ACS, and 40 patients with ACS affected or not by AF, recruited at the UTIC of the UOC of Cardiology at the "AORN A. Cardarelli" (Naples, Italy) (Director of the UOC: Dr. Ciro Mauro, as Head of Unit of the study at AORN Cardarelli; assisted by Dr. Antonio Ruocco, Medical Director of Cardiology).

Patients with clinically diagnosed of AF and ACS, in particular unstable angina pectoris (UAP), acute myocardial infarction without ST elevation (NSTEMI) and acute myocardial infarction with ST elevation (STEMI), based on clinical history, symptoms, ECG, biomarkers of damage cardiac, coronary angiography, risk factors and/or other clinical tests according to the guidelines for UAP/ NSTEMI and STEMI, will be recruited at the UTIC of the "AORN A. Cardarelli" (Naples, Italy). All recruited subjects will sign a written informed consent for a blood sampling for non-profit research purposes. Pharmacological therapy, diagnostic information and clinical examination data and medical history will be collected from medical records. Blood samples will be collected during normal clinical practice without taking additional samples from the first day of admission before the use of drugs such as heparin and contrast agents (> 90% of the samples will be collected within a day of acute event). Patients with primary cardiomyopathies, congenital heart disease, valvular diseases, stroke, diabetes mellitus, autoimmune diseases, acute infections, chronic lung infections, COPD, emphysema, pneumoconiosis, asthma, chronic bronchitis, tuberculosis, pleurisy, chronic liver disease and kidney disease, hyperthyroidism or subjects whom will claim an acute febrile illness within 2 weeks, will be excluded from the study.

The collection of whole blood will be carried out from patients and controls during the normal clinical practice. Biological samples will be immediately processed and stored at +4 °C at the regional reference biobank of the U.O.C. of Clinical Immunology and Immunohematology, Transfusion Medicine and Transplantation Immunology with annexed Single Regional Reference Laboratory for Organ Transplant Immunology (LIT) at the Department of Internal Medicine at the University of Campania "Luigi Vanvitelli" (Naples, Italy).

A total of 25 mL of peripheral venous blood will be collected in EDTA tubes and usually processed within 1 hour. Peripheral blood mononuclear cells (PBMNCs) will be isolated by Ficoll gradient using Histopaque®-1077 (Sigma-Aldrich) according to manufacturer's instructions.

CD4+ and CD8+ T lymphocytes will be purified from PBMCs using EasySep™ Human CD4+ T and Cell Isolation Kit and EasySep™ Human CD4+ T Cell Isolation Kit (STEMCELL Technologies), starting by 5x107/mL of PBMNCs, respectively.

Genomic DNA of purified lymphocite subset from each study participant will be isolated immediately after cell isolation using DNeasy Blood \& Tissue Kit (Qiagen) according to manufacturer's protocol. DNA concentration and purity will be determined by using NanoDrop spectrophotometer ND-2000 (Thermo Scientific) through the evaluation of the absorbance ratio A260/A280 and DNA integrity checked on 1% agarose gel.

RRB Sequencing and bioinformatic analyses will be performed by an external Service.