Premature Coronary Artery Disease - Clinical and Molecular Genetic Aspects

Purposes Study part 1: Quantify the occurrence of risk factors of atherosclerosis among patients with very premature CAD in the Central Denmark Region and investigate to what degree patients are treated according to national guidelines.

Study part 2: Investigate the occurrence of CAD among middle-aged 1st degree relatives of established patients having very premature CAD.

Study part 3: Evaluate possible genetic "risk factors" in families with a high CAD prevalence using exome sequencing analysis.

Hypotheses Hypothesis 1: Patients with very premature CAD have modifiable risk factors, which are inadequately treated relative to current national guidelines.

Hypothesis 2: The occurrence of very premature CAD is associated with familial accumulation of the disease.

Hypothesis 3: The use of exome sequencing in selected families with phenotypic severe premature CAD will identify disease-causing genetic variants.

Method Study part 1. Patients suffering from premature CAD who have had a percutaneous coronary intervention (PCI) or coronary artery bypass graft operation (CABG) done before the age of 40 years will be selected from the West Denmark Heart Registry (VDHD). Specifically, patients treated at Aarhus University Hospital (AUH) in Skejby for myocardial infarction (MI), stable or unstable angina pectoris in the period of January 1st 2007 - December 31st 2013 will be recruited and the occurrence of registered risk factors be evaluated (sample indicates app. 170 patients per year). After informed consent is obtained, they will participate in this research study. Participants will be interviewed in the cardiac outpatient clinic at the Dep. of Cardiology, AUH, Skejby. The following information about cardiovascular risk factors will be collected; family history of atherosclerosis (a pedigree analysis will be performed and cases of diagnosed CVD will be identified), prior medical history (i.e. hypertension, dyslipidemia, diabetes, chronic kidney disease, vascular disease), smoking status and number of pack years, alcohol consumption and vascular symptoms (i.e. angina pectoris, intermittent claudication). Gathered information will be compared to the registered information in patient records and VDHD. In addition, information about localization of the invasive treatment, the degree of CAD, and the latest estimate of the left ventricular ejection fraction (LVEF) will be collated. All patients will be examined regarding height and weight, abdominal girth and a blood sample is collected. An automated office blood pressure measurement (Bp-TRU device) will be performed. Levels of creatinine (e-GFR), total cholesterol, LDL cholesterol, HDL cholesterol, triglycerides and HbA1c will be measured. Further, a blood sample will be saved for prospective DNA analysis (below). The investigators will evaluate to what degree the treatment targets have been met in accordance with national guidelines. Analysis will be corrected to reflect changes in these treatment guidelines, which have occurred in recent years.

Study part 2. Patients from study 1 (index patients) are requested to establish contact to 1st degree relatives between 30 and 65 years of age for the purpose of participating in Study part 2. The form of contact follows regular department guidelines for contact to family members. 1st degree relatives who give their informed consent will be included in study part 2 (participants). Patient records from participants who are diagnosed with atherosclerosis will be obtained. Exclusion criteria will be former diagnosis of CAD by CTCA or percutaneous transluminal coronary angiography, chronic atrial fibrillation, renal failure (e-GFR < 30ml/min), obesity (BMI > 30 is indicative but body geometry plays a role), former allergic contrast reaction and pregnancy. A traditional risk profile and blood samples will be collected as in study part 1. Prior to the CTCA, LVEF is estimated by echocardiography. A CTCA is performed according to the standard protocol in the department. Initially the CT-scan is performed without contrast in order to estimate the coronary calcification by Agatstons method. Afterwards a scan is performed with 70 ml of iodine-containing contrast. ECG modulation and radiation-reduced protocols will be used so that an average participant (70 kg) may expect to receive a radiation dose below 3 mSv. The analysis of the contrast study will focus on automated- and semi-automated plaque quantification. Non-cardiac findings will be described according to department standard procedures by the Dep. of Radiology, AUH, Skejby, and medical intervention performed as clinically indicated.

Study part 3. Families from study part 1 and 2, who are considered severely affected by atherosclerosis, evaluated on a basis of family size, number of affected and severity of disease, will be selected. Family members (index patients, 1st degree relatives and if relevant also their 1st degree relatives) who consent,will be included in study part 3. Contact with relatives who have not yet participated in the project will be attempted as in study part 2. A traditional risk profile and a blood sample for DNA analysis (exome sequencing) will be requested if it has not already been obtained. The analysis takes place at Department of Molecular Medicine (MOMA), AUH, Skejby, who have the required equipment, software and laboratory expertise in performing the tests as well as the bioinformatic analysis. First, genomic DNA is purified and fragmented to a size of 200-500 base pairs. Illumina TruSeq libraries (to be used with the exome sequencing) will be produced at a Caliper Sciclone-robot where the exome enrichment with Nimblegens EZ in solution Exome v3 kit will also take place. The dataanalysis will be taking place at MOMA under the guidance of the MOMA staff. First sequences will be trimmed and aligned to the humane genome. Second, alignments will be fine-tuned before variants will be called, including single nucleotide variants and insertions/deletions of various size. The interpretation of the data will be taking place in the software program Cartagenia, which gives access to an abundance of public and commercial databases and also offers the opportunity of integrating private databases. Variants will be filtered on a basis of frequency, gene position, existing knowledge and different in-silico prediction tools. Possible significant findings will be validated by Sanger sequencing.

Statistical analysis At the found genotypes from study part 3, coupling analysis is performed to identify regions in the genome shared by disease-affected individuals. The use of pedigree analysis and coupling analysis substantially reduce the candidate intervals in the genome, thereby increasing the statistical power to identify disease-causing variants even in small families. Because the precise genetic architecture and family structure of the included families is not known, exact calculation of statistical power will not be possible. In case of a dominant monogenetic disease, a filtration only based on localization (coupling) reduces the potential variants by app. 80% within a pair of affected siblings and by more than 95% for monogenetic recessive diseases, if affected individuals from inbred families.

Research Biobank A research biobank will be established under The Institute of Clinical Medicine, Health, Aarhus University. A sample of 2x4 ml (BD Vacutainer K3E 7,2mg Ref. 368860) and 1x4ml (BD Vacutainer SST II Advance, Ref. 367957) blood from all participants in studies 1-3 will be submitted to the biobank for further analysis. The sampling is performed at AUH, Skejby or a glass and a return envelope will sent to the patient for the drawing of blood by their family physician. The sample is cryo-preserved and exome sequencing is performed on the participants of study part 3. The biobank will be carried on after the project has ended for the purpose of future research. In case of future analysis on the preserved material The Research Ethics Committee and the participant (if still alive) will be noticed before hand. Permission to store the biological material will be applied continuously according to specified intervals by The Danish Data Protection Agency. All participants may demand their biological material destroyed at any time.