Design of the substudies Study

An observational cohort study of consecutive coronary angiography patients purposed to evaluate their risk-factor profiles, disease stability, and severity, as well as the revascularization strategy.

Study II

A prospective, observational cohort study to analyze the capability of GRSs to predict recurrent ACS and CAD mortality.

Study III

A GWAS to search for risk variants exclusively for STEMI/NSTEMI in a case-control setting. The finding was replicated in another case-control cohort and in a prospective cohort.

Study IV

A prospective, observational cohort study to evaluate outcome, cardiovascular risk factors, and other manifestations of atherosclerosis in patients with or without risk alleles on chromosome 1p13.3 (rs656843).

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4.2. Study cohorts

The basis of Studies I to IV is the Corogene cohort. All methods and results are from the Corogene study, unless otherwise indicated. Patients from the Tampere Acute Coronary Syndrome Study (TACOS) and FINRISK cohorts are included in Studies III to IV to further test the discoveries of the Corogene Study (Table 5). Selected individuals from FINRISK cohorts served also as controls. Participants in all of these cohorts gave their written informed consent. The Ethics Committee of Helsinki University Hospital, Internal Medicine approved the research protocol for the Corogene study. The Ethics Committee of the National Institute of Welfare and the Coordinating Ethics Committee of the Helsinki and Uusimaa Hospital District approved the protocols of the FINRISK studies. The Ethics Committee at Tampere University Hospital approved the TACOS protocol.

Table 5. Individuals included in Studies I-IV.

Corogene FINRISK TACOS

N 5 294 23 036 998

Study I 5 294

Study II 2 090 with ACS

Study III

Discovery sample: Corogene 1 863 with MI of which

1579 genotyped

1 576 matched controls

Replication I: TACOS 566 matched controls 564 genotyped

MI patients

Replication II: FINRISK 16 627 of which 848

with MI Study IV

Discovery sample: Corogene 1 768 genotyped ACS

patients

Replication: FINRISK 21 161 of which 439

with PAD

TACOS indicates the Tampere Acute Coronary Syndrome Study; ACS, acute coronary syndrome; MI, myocardial infarction; PAD, peripheral artery disease.

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4.2.1. Corogene (I-IV)

The basis of this study was the Corogene cohort, which comprised 5 294 consecutive angiography patients in Helsinki University Central Hospital between June 2006 and March 2008. The Hospital District of Helsinki and Uusimaa serves more than 1.5 million inhabitants. In the hospitals of this area, a total of over 4800 coronary angiograms are performed annually, and Helsinki University Central Hospital performs 75%.

After excluding patients with other than Finnish citizenship, repeated angiograms for patients already included in the study, and patients with low hemoglobin, recent blood transfusion, or previous heart transplantation (511, 8.8%), the cohort comprised 5 294 patients (91.2% of all angiography patients). The patients were treated with standard drug regimens and procedures.

Patient classification

According to the angiography findings and the symptoms, these patients were classified into four groups:

Group 1. No CAD in angiographic evaluation. The angiogram in these patients was required to reveal either normal or mildly (up to 50%) obstructed coronary arteries. (I)

Group 2. Stable CAD. The requirement was significant stenosis (more than 50%) in at least one coronary artery and stable symptoms. (I)

Group 3. ACS. Criteria for inclusion were chest pain typical for ischemia combined with typical ECG changes, biomarker measurements (TnT, CKMBm), and at least one significantly (more than 50%) obstructed coronary artery. (I to IV)

Group 4. Other ischemic events. Patients had elevated biomarkers, symptoms typical for ACS, and possible ECG changes, but no significant coronary artery stenosis. (I)

Data collection

The patient questionnaire provided information on height, symptoms of CAD, other CVD manifestations, and other diseases. Additionally, collected information covered family history for CAD, medications, smoking, alcohol intake, drug abuse, allergies, special diets, social situation, and home nursing. Current smoking was defined as smoking or having quit smoking within six months before study inclusion. Ex-smoking was considered as having quit smoking more than six months before the inclusion.

Patient records, a cardiological data log, and the cardiac-surgery database provided information on height, risk factors, prior cardiac disease and operations, comorbidities, medication, chest x-ray, ECG, echocardiography, angiography, PCI, and CABG. Hypertension, dyslipidemia, and diabetes (types 1 and 2) were defined as fulfilling the criteria for the medication for these disorders.

Blood samples for serum and plasma analyses were drawn from the arterial line during the angiography. Laboratory values collected included complete blood count, CRP, blood lipids, glucose, HbA1c, liver enzymes, bilirubin, activated partial thromboplastin time, renal values (urea, creatinine), electrolytes, CK, cardiac markers

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such as b-type n-terminal natriuretic propeptide (proBNP), TnT, CKMBm, and D-dimer. Blood samples additionally served for extraction of deoxyribonucleic acid (DNA) and ribonucleic acid (RNA). Glomerular filtration rate (GFR) for each patient was estimated from serum creatinine by the Modification of Diet in Renal Disease Study (MDRD) equation.114

Patients of the Corogene Study were followed up until death or by the end of the year 2012. Statistics Finland (Tilastokeskus) provided the mortality data. The National Hospital Discharge Registry provided the information on ACS recurrence rate, and incidence of PAD, cerebral infarction, and atherosclerotic aortic disease.

4.2.2. TACOS (III)

The TACOS Study comprises ACS patients admitted to Tampere University Hospital between January 2002 and March 2003.160 _{The diagnostic criteria for ACS in the TACOS}

and Corogene studies were analogous, except for the use of TnI (TnI>0.2 mg/L) in TACOS whereas Corogene used TnT and CKMBm. Patients transferred from another department within the hospital or initially treated for ACS in other hospitals were excluded. The study sample comprised 998 MI patients, of whom 343 had STEMI, and 655 had NSTEMI. Of these, 564 MI patients had adequate clinical and adequate genotypic information for analysis in Study III.

4.2.3 FINRISK (III-IV)

FINRISK is a repetitive cohort study for which a new cohort is collected once every five years. The FINRISK cohorts reveal trends in cardiovascular risk factors in Finland.237,238

A random sample of 25- to 74-year-old inhabitants, representing different regions, genders, and age-groups, participated in each cohort by invitation. Altogether 23 036 individuals took part in the FINRISK 1992, 1997, 2002, and 2007 cohorts.

FINRISK cohorts provided both cases and controls for Studies III and IV. Additionally, the gene expression levels were tested from 513 individuals recruited during 2007 for the Dietary, Lifestyle, and Genetic determinants of Obesity and Metabolic syndrome (DILGOM) study, an extension of the FINRISK 2007 study. Of these samples, 372 served also as controls for the Corogene MI cases in the GWAS in Study III.

In Study III, controls for the Corogene MI patients (discovery sample) were selected from among participants living in the Helsinki-Vantaa region from the FINRISK 1997, 2002, and 2007 studies by use of risk set sampling. Independently for each Corogene MI case, two sex- and birth cohort (+- 5 years) -matched controls were chosen from all FINRISK participants. Individuals were suitable as controls if they were free of CVD at the time of ACS diagnosis of the chosen matched case. One FINRISK participant could serve as a control several times. For TACOS MI patients (replication sample I), controls came from the FINRISK 1992, 1997, 2002, and 2007 cohorts. The controls were required to be born in Pirkanmaa to match the geographical region of the cases in TACOS.

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FINRISK participants with an MI diagnosis in The National Hospital Discharge Registry and with their genotype data available were the selected cases for replication sample II. All the other genotyped FINRISK patients were included in the analysis of prospective setting, with the exception of participants with prevalent MI, insufficient data on phenotype, or who were among the individuals serving as controls in the discovery or in replication sample I. Replication sample II eventually comprised 16 627 participants, with 163 cases of incident NSTEMI, 99 cases of incident STEMI and 222 cases of incident MI with unspecified ST-segment status.

Study IV included FINRISK participants with both genotype and PAD phenotype information available, the individuals with incident PAD serving as cases and other subjects as controls, with the exception of prevalent PAD cases. The National Hospital Discharge Registry provided the information on prevalence and incidence of PAD.