Introduction 1 2 The design of the Rotterdam Study 3 1 Fig. 1 Diagram of examination cycles of the Rotterdam Study (RS). RS1 refers to the baseline examination of the original cohort (pilot phase 07/1989-12/1989; cohort recruitment 01/1990-09/1993). RS2, RS3 and RS4 refer to re-examination of the original cohort members. RSPlus1 refers to the extension of the cohort with persons in the study district that became 55 years since the start of the study or those of 55 years or over that migrated into the study district. RSPlus2 refers to the re-examination of the extension cohort. RSYoung1 refers to the baseline examination of all persons aged 45 and over living in the study district that had not been examined (i.e. mainly comprising those aged 45–55 years)  In 1999, 3,011 participants (out of 4,472 invitees) who had become 55 years of age or moved into the study district since the start of the study were added to the cohort. In 2006, a further extension of the cohort was initiated in which about 6,000 subjects aged 45–54 years, living in the Ommoord district, were invited (expected number of participants about 4,000). By the end of 2008, the Rotterdam Study is therefore expected to comprise about 15,000 subjects aged 45 years or over. 1 The participants in the Rotterdam Study are followed for a variety of diseases that are frequent in the elderly (and many are also in the not so elderly): coronary heart disease, heart failure and stroke, Parkinson disease, Alzheimer disease and other dementias, depression and anxiety disorders, macular degeneration and glaucoma, diabetes mellitus and osteoporosis. The Rotterdam Study has been approved by the institutional review board (Medical Ethics Committee) of the Erasmus Medical Center and by the review board of the Netherlands Ministry of Health, Welfare and Sports. The approval has been renewed every 5 years. Separate approval has been obtained for the introduction of major new elements in the study (e.g. MRI investigations). In the remainder of this article the objectives and major findings will be presented with an update of the methods for cardiovascular diseases, neurological diseases, ophthalmologic diseases, psychiatric diseases, endocrine diseases, as well as for genetic and for pharmaco-epidemiologic studies. Cardiovascular diseases Objectives Research on the epidemiology of cardiovascular diseases focuses on three primary areas of interest: studies on risk factors for atherosclerosis and coronary heart disease, studies on the detection of subjects at high risk of coronary heart disease, and studies on cardiovascular conditions in older age. Two groups of putative risk factors for atherosclerosis and coronary heart disease are included. The first are endocrine factors, including estrogens and androgens, insulin and insulin-like growth hormone I, and thyroid gland and adrenal gland hormones. The second group contains factors involved in haemostasis, inflammation and endothelial function. Research also focuses on genetic factors in these areas in relation to risk of coronary heart disease. The ability of classical cardiovascular risk factors to identify subjects at high risk of coronary heart disease is limited. Risk stratification may be improved when based on the presence of atherosclerosis. To this end, repeated measurements of non-coronary atherosclerosis and measurements of coronary, carotid and aortic arch calcification have been included in the study. Another line of research focuses on cardiovascular diseases in the elderly that are in large part the consequence of ischemic heart disease, like heart failure and atrial fibrillation. An important topic in this area is the early diagnosis of heart failure using echocardiographic assessment of asymptomatic systolic and diastolic dysfunction of the left ventricle. Atrial fibrillation is another major chronic condition frequent at older age. Examination of the determinants and prognosis of atrial fibrillation is part of this research line. Major findings 4 5 6 7 8 6 9 10 11 12 13 14 15 Methods update 9 11 1 10 Determinants were assessed by physical examinations, collection of blood samples, and by questionnaires and interview. The role of genetic factors is studied using the candidate gene approach and more recently the genome wide association study. 6 12 13 Neurological diseases Objectives Neuroepidemiologic research in the Rotterdam Study focuses on the frequency, etiology and early recognition of the most frequent neurologic diseases in the elderly, including dementia (in particular Alzheimer disease), Parkinson disease and stroke. In neurodegenerative and cerebrovascular disorders clinical symptoms typically become manifest late in the disease course, the occurrence of clinical disease does not reflect the underlying spectrum of disease-related pathology, and most of the clinical syndromes are etiologically heterogeneous. Therefore, an additional research focus is on the causes and consequences of pre-symptomatic brain pathology that can be assessed with non-invasive imaging modalities. Major findings 16 17 18 19 20 21 22 23 25 26 27 28 29 29 30 31 32 Methods update Assessment of dementia and Alzheimer disease 16 18 Assessment of parkinsonism and Parkinson disease 17 19 Assessment of stroke and stroke subtypes 20 28 Assessment of cognitive function Global cognitive function is measured through the Mini Mental State Examination (MMSE) in all surveys. From the third survey onwards we added a 30 min test battery that was designed to assess executive function and memory function, and which includes a Stroop test, a Letter Digit Substitution Task, a Word Fluency Test, and a 15 words Word List Learning test. Rotterdam Scan Study: brain imaging within the Rotterdam Study 33 31 Psychiatric diseases Objectives The aim of the psychiatric research in the Rotterdam Study is to investigate the determinants, correlates and consequences of common psychiatric problems in the elderly. The focus has been on depressive disorders but anxiety disorders, sleep disturbances and complicated grief are also being studied. Major findings The one-month prevalence of both major and minor depression at baseline was 1.5%, the prevalence of anxiety disorder was 8.2%. 34 35 36 37 39 40 41 Methods update 1 Assessment of determinants 34 35 37 40 Assessment of outcomes 42 43 44 45 46 Ophthalmologic diseases Objectives The primary objectives of the ophthalmological part of the Rotterdam Study are to study frequency and risk factors of common and invalidating eye diseases with emphasis on age-related macular degeneration (AMD) and primary open angle glaucoma (POAG). Another line of research focuses on the associations between retinal vessel abnormalities and cardiovascular and neurologic diseases. Major findings 47 48 50 ε 51 52 53 54 55 56 57 58 59 60 61 62 62 63 64 65 Methods update Repeated ophthalmic measurements include best corrected ETDRS visual acuity, refractive error, Goldmann applanation tonometry, keratometry, slitlamp examination of the anterior segment and visual field testing. In pharmacological mydriasis we make colour photographs of the lens, 35 degree photography of the macular area, and simultaneous stereoscopic imaging of the optic disc and macular area. Digital photography of the macular area and optic disc are added since the fourth follow-up examination. Scanning laser ophthalmoscopy (HRT II) measurements of the optic disc, macular pigment density measurements and optical coherence tomography of the macular area and optic disc were added during follow-up. 66 57 58 67 Genetic studies Objectives The first objective of the laboratory team is to collect, store and manage the biological tissues sampled in the Rotterdam Study. The second objective of the group concerns genotyping and assessment of biomarkers. Major findings 68 69 70 5 71 72 Methods update Data collection, storage and management At each examination, blood, serum, plasma (citrate, heparine and EDTA based), sputum, and urine are collected. Fasting blood samples are collected along with challenged samples as part of a glucose tolerance test. Sputum is collected before and after a dexamethasone-suppression test. Sputum is frozen at −196°C before and after the challenge and stored at −80°C. To obtain serum and plasma, tubes are centrifuged according to a protocol standardising time and conditions from the drawing of blood to centrifugation. All samples are snap frozen at −196°C using liquid nitrogen and stored at −80°C. RNA is isolated from blood within 5 h after sampling and stored at −20°C. DNA is isolated from blood and extraction has been recently automated using a Hamilton STAR pipetting platform and AGOWA magnetic bead technology. DNA sample storage is in Matrix 2D-barcode tubes in 96-well format. Overnight urine samples are collected, frozen at −196°C and stored at −80°C. For data management, an in-house customized laboratory management system has been developed. Sample retrieval will be automated with an in-house customized laboratory track and trace system. Blood assessments For all participants, serum cholesterol, HDL, LDL, triglycerides, glucose and glucose levels are assessed. In urine, micro albumin and creatinine are determined in all participants. There have been a large number of specific blood/serum/plasma-based biomarker assessments including steroids (e.g. estrogens, androgens, vitamin D, cortisol), interleukins, CRP, IGF1, insulin, iron-parameters (iron, ferritin and transferrin saturation), fibrinogen, homocysteine, folic acid, riboflavine, pyridoxine, SAM/SAH ratio, cobalamine, Lp-PLA2, Fas/Fas-L, vitamins, a-beta42/40 and thyroid hormones (TSH). Genotyping facilities Affiliated laboratory facilities include a medium/high-throughput platform for candidate gene studies and genome wide association (GWA) analyses. The facilities use high-end automated machinery including a Caliper/Zymark ALH 3000 pipetting robot (including a TwisterII, and integrated plate sealer, plate reader (OD 260/280), a Tecan EVO 150 Freedom pipetting robot, a Deerac Equator NS808 nanoliter liquid dispenser, 15 electronic PCR machines (ABI 9700, 2 × 384), an ABI7900HT Taqman machine (running 1 ng gDNA in 2 μl reactions), a WAVE 3500HT dHPLC, and two ABI3100 sequencing machines. DNA sample handling is centred on 384-well plates. Candidate gene studies are done mostly using Taqman genotyping with throughputs at 30,000 genotypes per day. Continuous efforts are focussed on reducing the required amount of genomic DNA, which is now down to 1 ng per genotype. Genome-wide genotyping studies are based on 500/1,000 K Affymetrix arrays and 317, 550 and 1,000 K Illumina arrays with throughputs at 100–300 arrays per week. The genotyping facility has been partly sponsored by NWO investment grants (911-03-012; 175.010.2005.011), is part of the ErasmusMC Biomics core facility, and serves as knowledge centre for polymorphism analysis attracting national and international interested parties, both academic and industrial. Candidate gene studies 73 Genome wide association (GWA) studies 74 Pharmaco-epidemiologic studies Objectives A major objective of the pharmaco-epidemiologic studies is to investigate the role of drugs as determinants of disease in the Rotterdam Study. This includes studying efficacy and effectiveness of drugs, as well as adverse reactions to drugs. Major findings 75 26 76 78 79 77 78 79 80 81 82 83 91 Methods update For several reasons, a drug is a highly attractive determinant in clinical epidemiology. First, drugs are probably the most important therapeutic intervention in health care. Despite rigorous clinical research before registration, many important effects of drugs are discovered after marketing. Second, all marketed drugs have proven biological activity, meaning that it concerns a determinant which really matters. Third, and as a consequence of the availability of complete medication histories in Dutch health care, the role of drug exposure can be assessed in a detailed way. In the Rotterdam Study, there is an almost complete coverage of the population as of 1 January, 1991 thanks to the fact that all pharmacies which serve the Ommoord region are on one computer network. To date, almost 3 million prescriptions have been delivered to the population of the Rotterdam Study and of each prescription, details are available about the product name and contents, ATC-code, dosage and duration of drug therapy. Drugs are a group of determinants, which can be studied in association with a large variety of diseases. In the Rotterdam Study, there is a strong interest in the association between drugs and the cardiovascular, neurological, endocrinological and ophthalmological diseases, which have been the main topics since starting. However, there is also important information about the association with psychiatric diseases, cancer, and chronic obstructive pulmonary disease. Moreover, important information about secondary outcomes, such as drug blood levels, other laboratory information, and information about hospital discharge diagnoses, is gathered on a continuous basis to facilitate pharmaco-epidemiological studies. Management of the Rotterdam Study The Rotterdam Study is directed by a Management Team comprising Jan Heeringa, MD, study coordinator, Eric Neeleman, head IT, Frank van Rooij, head data-management, and the scientific principal investigators Albert Hofman (PI Rotterdam Study, chairman), Monique Breteler (PI Neurological diseases), Cornelia van Duijn (PI Genetic studies), Gabriel Krestin (PI Radiology), Huibert Pols (PI Endocrinology), Bruno Stricker (PI Pharmaco-epidemiology), Henning Tiemeier (PI Psychiatric diseases), André Uitterlinden (PI Genome wide analysis), Johannes Vingerling (PI Ophthalmologic diseases) and Jacqueline Witteman (PI Cardiovascular diseases).