The Finnish Cardiovascular Study (FINCAVAS): characterising patients with high risk of cardiovascular morbidity and mortality

The participant pool consists of patients who undergo exercise stress tests at Tampere University Hospital. All the consecutive patients coming in for an exercise stress test and willing to participate in the study have been and will be recruited between October 2001 and December 2007. Our goal is to recruit roughly 5,000 patients. Currently, the research group is actively analysing the data on the 2,212 patients (1,400 men and 812 women) recruited by the end of 2004. The study protocol was approved by the Ethical Committee of the Hospital District of Pirkanmaa, Finland, and all patients have given informed consent prior to the interview and measurements as stipulated in the Declaration of Helsinki.

In addition, roughly one thousand patients scheduled for spiroergometer testing (SensorMedics VMax29c, VIASYS Healthcare Inc., Yorba Linda, CA, USA) with pre- and post-test spirometry will be recruited between December 2005 and the end of 2007.

No specific sample size calculations were made prior to study initiation. In general terms, if we want to find a 10% difference in the prevalence of certain genotypes with a significance level of p < 0.05 and to reach an 80% ability to find significant differences between the groups, we need hundreds of patients. Since the study population is often divided into subgroups based on, for example, age, sex and medication, the size of the cohort should preferably be at least one thousand.

After an informed consent has been signed, the medical history of each patient is gathered with a computer-based questionnaire form [27]. Thereafter, the exercise test is performed. Blood samples are drawn for DNA analyses. Some of the patients are examined with coronary angiography either before or after the exercise test.

Prior to the exercise stress test, the subject lie down in supine position for 10 minutes, and the resting ECG is digitally recorded. The exercise test is performed using a bicycle ergometer with electrical brakes. The lead system used is the Mason-Likar modification of the standard 12-lead system [28]. The initial workload varies from 20 W to 30 W, and the load is increased stepwise by 10–30 W every minute. During the test, HR is continuously registered with ECG, while the systolic (SAP) and diastolic (DAP) arterial pressures are measured with a brachial cuff every two minutes. Breathing frequency is measured and the patient is asked about the perceived exertion level every two minutes using Borg's scale with 15 categories [29].

Most of the exercise tests are sign- and symptom-limited maximal tests using the recommended criteria for termination, but some of the tests for evaluating the status after a myocardial infarction (MI) have an HR upper limit of 120–130 bpm. The recovery phase after the exercise is at least five minutes.

FINCAVAS has access to the laboratory tests performed on the study patients at Tampere University Hospital between October 1998 and the end of the study. Plasma triglycerides, total and high-density lipoprotein (HDL) cholesterol as well as sensitive C-reactive protein (CRP) concentrations are analysed by a Cobas Integra 700 automatic analyser with reagents and calibrators recommended by the manufacturer (Hoffmann-La Roche Ltd., Basel, Switzerland). Low-density lipoprotein (LDL) concentration is calculated using Friedewald's formula [30]. A typical detection limit for CRP is 0.25 mg/l [31]. Genomic DNA is extracted from peripheral blood leukocytes using a commercially available kit and Qiagen BioRobot M48 Workstation according to the manufacturer's instructions (Qiagen Inc., Hilden, Germany).

One of the central objectives of FINCAVAS is to examine the effects of polymorphisms known or suspected to alter the development of atherosclerosis, cardiovascular function, pathophysiology or related pharmacogenetics. Moreover, we aim to find new polymorphisms affecting these processes. In the first of our two genotyping strategies, the genes found to be expressed in the arterial wall – thus possibly directly affecting the development of atherosclerosis – are selected for the detailed genotype-haplotype analysis. The selection is based on the results of another ongoing project in which the expression of the entire genome, roughly 30 000 genes, is scanned from 30 freshly collected arterial tissue samples with morphometrically and immunohistochemically verified atherosclerotic lesions.

The expression analysis is performed using Illumina BeadArray™ technology (Illumina, Inc, San Diego, CA, USA). After selecting the genes statistically most significantly expressed in the arterial wall, we design an Illumina custom panel for up to 1536 markers for single nucleotide polymorphisms of the genes chosen. The advantage of this strategy is the low price per sample, which enables us to analyse all the participants of FINCAVAS. As the price of genotyping steadily decreases, we are technically equipped to perform even more genotyping using the Illumina BeadChip arrays.

In the second strategy, genetic variation in the following cascades and anatomical structures particularly relevant for FINCAVAS are studied: regulation of blood pressure, adrenergic signal transduction, cardiac ion channels as well as pharmacokinetics and pharmacodynamics of drugs used to treat hypertension and CHD.

In this strategy, DNA samples are genotyped by employing the 5' nuclease assay and fluorogenic allele-specific TaqMan MGB probes [32], using the ABI Prism 7900HT Sequence Detection System (Applied Biosystems, Foster City, CA, USA). The samples are pipetted using an automated TECAN Freedom EVO-100 pipetting robot (Tecan Group Ltd, Männedorf, Switzerland). The nucleotide sequences of the primers and probes used in the polymerase chain reaction (PCR) are deduced from published sequences deposited in the GenBank and Celera databases and synthesised by Applied Biosystems. The PCR reactions containing genomic DNA, 1 × Universal PCR Master Mix, 900 nM of each primer and 200 nM of each probe are performed in 384-well plates using the standard protocol in a total volume of 5 μl.

End-point fluorescence is measured and genotype calling carried out by the allelic discrimination analysis module after the PCR results in the identification of polymorphisms chosen. Negative and positive controls (known genotypes) and random duplicates are used as quality control. Genotypes are analysed in the Centre for Laboratory Medicine, Department of Clinical Chemistry, Laboratory of Atherosclerosis Genetics at Tampere University Hospital.

The follow-up data consists of information on major cardiovascular events (hospitalisation due to angina pectoris, MI or stroke), coronary procedures (angioplasties and bypass operations) and causes of deaths (Fig. 1). The follow-up data will be gathered at 2, 5 and 10 years. Information on clinical events will be collected from the Finnish National Hospital Discharge Register and data on mortality from the Causes of Death Register; these sources have been shown to be reliable [33]. Information on operative procedures will be received from the Hospital Information System of Tampere University Hospital.

History of prior MI is based on patient interviews, resting ECG recordings and hospital records. Data on the existence of hypertension, valvular diseases, cardiomyopathies, other heart diseases and diabetes (types 1 and 2) is based on interviews and hospital records. The presence of abnormal atrio-ventricular conduction, right (RBBB) and left (LBBB) bundle branch blocks, delta waves, pathological Q waves and long QT syndrome are based on resting ECGs and hospital records.

The material in the FINCAVAS database is woven together from many data sources (Fig. 1). The patient history data is based on both patient interview and hospital records; the data is recorded with a computer-based form [20]. Data on demographics (i.e., age, sex, weight, height, body mass index, waist to hip ratio, occupation) and lifestyle (alcohol consumption, physical exercise at work and leisure time) is collected using the same software before the exercise test. Furthermore, exposure to classic cardiovascular risk factors (i.e., smoking, hypertension, diabetes, occurrence of hyperlipidemia and family history) is covered extensively.

Cardiovascular symptoms (including the functional capacity classification developed by the New York Heart Association, NYHA) and diseases are recorded in detail, with special emphasis on CHD and prior MIs. All the medications used by the patient are entered into the database, including the length of possible pause before the exercise test.

The actual exercise tests generate the majority of the data. Values for HR, SAP, DAP, breathing frequency, physical load, perceived exertion level, peak expiratory flow before and after the exercise as well as oxygen saturation in a finger tip, if measured, are gathered on the software mentioned earlier.

Continuous ECG is recorded at 500 Hz with CardioSoft exercise ECG system (Version 4.14, GE Healthcare, Freiburg, Germany) and analysed by Modified CASE software (GE Healthcare, Freiburg, Germany). The system recognises ventricular escape beats, premature ventricular complexes (PVC), premature supraventricular complexes, pauses of 1 or 2 missed beats, ventricular bigeminy, ventricular couplets (2 PVCs), ventricular runs (> 2 PVCs), ventricular tachycardia, atrial fibrillation/flutter, ventricular fibrillation and asystole. In addition, the following parameters are assessed and recorded: amplitudes of P, Q, R, S and T; durations of P, Q, R, S, RP and QT; levels of J-point and ST-segments at 60 ms; ST integral; ST slope; T wave alternans; ST-segment depression/heart rate (ST/HR) index; ST/HR slope; ST/HR hysteresis [34, 35] and, finally, HR recovery and noise level. These parameters are determined from 14 ECG leads: aVL, I, -aVR, II, aVF, III, V1 to V6, CM5 and V4R. Furthermore, all these parameters are determined at 14 points in time: before exercise, seven times during the exercise (start, 2 min, 4 min, 6 min, 8 min, 10 min and maximal load), and six times during the rest after the exercise (1 min, 2 min, 3 min, 4 min, 5 min and 6 min). Therefore, more than 3,500 ECG parameters are determined for each exercise test and stored into a separate database. However, any other points in time besides the ones specified here as well as additional parameters can be analysed as needed based on the continuous digital ECG recordings.

Raw data on laboratory tests is received from Tampere University Hospital. This bulk of data covers all the laboratory tests performed on the study patients. Minimum and maximum values for each test on each patient are searched using database queries and recorded into the FINCAVAS database.

The data on operative procedures is collected from the Hospital Information System of Tampere University Hospital. For each patient, an angiography is linked to an exercise test if they were a maximum of 6 months apart, and if no coronary bypass operations or coronary angioplasties are performed between them.

All the coronary angiographies are analysed by the same dedicated cardiologist (MN). TIMI (thrombolysis in myocardial infarction) flow, diameter and %-stenosis in the ostial, proximal, middle and distal parts of the left main (LM), the left anterior descending (LAD), the left circumflex (LCX), and the right (RCA) coronary arteries are registered. Furthermore, the same parameters are assessed for major branches: the diagonal branches I and II as well as the first septal branch of the LAD; the left obtuse marginal I and II as well as posterior descending branches of the LCX; posterior descending, right ventricular and posterior left ventricular branches of the RCA; and, finally, the left intermediate marginal branch. The stenoses are graded into seven categories: 0%, <50%, 50–74%, 75–89%, 90–98%, 99% and 100%.

With the exception of ECG parameters, the data depicted above is combined into a single SPSS file (release 12.0.1 for Windows, SPSS Inc, Chicago, Illinois, USA). The file comprises of 510 variables. Due to the enormous amount of ECG data, the ECG parameters are stored in separate files and will be added to the SPSS file based on the requirements of each subproject.

Laboratory and ECG data are entered into source databases or data files electronically and automatically. The other sources necessitate manual input. However, transferring the data from separate databases to the FINCAVAS database is done electronically using database tables and queries. Adequate cross-checks are done to ensure the accuracy of the data. Furthermore, data is validated by checking missing data and the plausibility of the ranges of values, as well as by inspecting the consistency of related data fields. Errors identified by these procedures result in checking the raw data when possible or eliminating the incorrect piece of data.