The Rotterdam Study: 2012 objectives and design update

Research on the epidemiology of cardiovascular disease focuses on the etiology and prediction of coronary heart disease and on cardiovascular conditions at older age, like heart failure and atrial fibrillation. Putative risk factors include three groups, endocrine factors, factors involved in hemostasis, inflammation and endothelial function, and genetic risk factors. A major focus is on non-invasive assessment of atherosclerosis to improve prediction of coronary heart disease, including measurement of coronary calcium with electron-beam and multi-detection CT and carotid plaque characterization by high-resolution MRI. Cerebrovascular diseases, including stroke, are also investigated in the Rotterdam Study. They are described under Neurological diseases.

Dermatoepidemiologic research in the Rotterdam Study focuses on the frequency of the most common skin conditions as well as on genetic and environmental factors associated with these skin diseases. The emphasis of the skin component is on cutaneous malignancies such as basal and squamous cell carcinomas (BCC and SCC, respectively) and their precursor lesions, and inflammatory dermatoses such as eczema and psoriasis. In addition to skin diseases, we examine the contribution of genetics and environmental exposures to the skin fenotype (e.g., skin pigmentation, wrinkling and photodamage) of the cohort members.

The prevalence of single and multiple BCC was studied in the Rotterdam Study and showed that a total of 524 patients (4.8% of included population) had developed this type of keratinocytic cancer and that 31.1% had developed more than one tumor during observation [112]. A multi-failure survival model suggested that people with red hair and higher levels of education, and those who had their first BCC at younger age were significantly more likely to develop multiple malignancies. In a candidate gene approach, Vitamin D-binding protein (VDBP) genotype was not associated with (multiple) BCC development, except possibly in the youngest age-group (A/T variant of rs7041 was associated with; adjusted HR = 1.88, 95% CI 1.10–3.20), whereas homozygote Gc1s carriers had a significantly lower BCC risk; adjusted HR = 0.53, 95% CI 0.31–0.91) [113].

In a pharmaco-epdemiologic study, the hypothesis that (prior) exposure to drugs such as high-ceiling diuretics increase skin cancer risk due to enhanced photosensitivity of the skin was tested. In line with the hypothesis, use of potassium sparing and thiazide agents were not and use of high-ceiling diuretics was associated with an increased hazard of BCC (highest quartile compared to non users; HR 1.6, 95% CI 1.1–2.4).

The main objective of the programme of endocrine epidemiology research is to study frequency and etiology of major disorders of the endocrine glands (pituitary, reproductive, thyroid, parathyroid, adrenal, and neuro-endocrine pancreas) and the musculoskeletal system. These include endocrine and locomotor diseases, including diabetes mellitus, osteoporosis, osteoarthritis, growth-hormone deficiency, hypo- and hyper-thyroidism and parathyroidism. The evaluation of risk factors for the above mentioned conditions includes serum measurements (such as classical hormones and other endocrine molecules) and genetic determinants of endocrine factors and signalling pathways.

We have provided epidemiological documentation on the hormone, growth factor and biomarker profiles in the general population and determined the association with several diseases. Within the topics of locomotor diseases and disability we have reported that heart failure, COPD, diabetes mellitus and chronic disorders leading to locomotor complaints, are risk factors which contribute considerably to locomotor disability [114, 115]. In relation to osteoporosis we have determined the incidence of vertebral [116] and non-vertebral fractures [117], and the relationship between bone mineral density (BMD), BMD change and the occurrence of fracture [117], as well as with heel ultrasound measurements [118] and bone resorption markers [119]. We have also studied the relation between endogenous sex hormones and their binding factors, with fractures [120], and showed that increased homocysteine levels are a strong and independent risk factor for osteoporotic fractures [121]. We studied the relations between osteoporosis and other chronic diseases like osteoarthritis [122], cancer [123], atherosclerosis [124] and diabetes [125, 126], and provided indications for the treatment and diagnosis of osteoporosis. Lastly, we were part of several large consortia studying epidemiological risk factors for osteoporosis [127‐129]. For osteoarthritis (OA) we have shown how a new marker of disease (CTX-II), is associated with the prevalence and the progression of radiographic OA [130], independent of known clinical risk factors. In addition, we have studied different aspects of OA disease definition and classification [131], evaluation of disease progression [132] and determined the most prominent risk factors leading to OA [133, 134]. We have also studied inflammatory aspects of endocrine diseases like diabetes mellitus [135], and the relations of hypo/hyperthyroidism to cardiovascular and neurological disease [136]. We further examined the influence of genetic variation in endocrine genes influencing hormone levels [137, 138], interaction of genetic factors in relation to fracture risk [138, 139], to cardiovascular risk factors [140] and to neurological conditions [141]. Our team has played a leading role in bringing together the global GENOMOS consortium which has performed prospective meta-analyses across many epidemiological cohorts for the most prominent candidate genes for osteoporosis (see also “Genetic and biomarker studies”).

For all participants DXA-based BMD measurements of the lumbar spine, dual hip and total body BMD, as well as determination of body composition parameters are assessed with a ProdigyTM total body fan-beam densitometer (GE Lunar Corp, Madison, WI, USA). Hip structural analysis [142] of DXA scans is available in a subset of participants, while hip strength indexes (software by GE Lunar) are determined for all scans. In the current follow-up cycle we have introduced since 2009 iDXA measurements (GE Lunar) which performs lumbar spine, dual hip and total body scans. Measurements include L1–L4 BMD, bilateral total hip and femoral neck BMD and total body BMD. From the total body scan, we measure lean mass and fat mass body composition, including total body, trunk, arm, legs, and android and gynoid regions of interest. X-ray examinations of vertebral bodies, hips, knees and hand/wrist are obtained by a digitalized Fuji FCR system (FUJIFILM Medical Systems) and assessed for the presence of fractures and/or degenerative changes of the joints. Vertebral fractures are assessed using the qualitative algorithm-based technique termed the ABQ method, an update to the quantitative McCloskey–Kanis method [143]. Incident clinical fractures are obtained from computerized records of the general practitioners and hospital registries which are regularly checked by research physicians who review and code the fracture information. Muscle strength is assessed in all participants with a hand grip dynamometer. The incidence and progression of OA is done using Kellgren scores obtained from X-rays of hip, knee, hands, en spine. The complete set of X-rays is also available in digitized form. Novel diagnostic assessments for OA are currently underway using Magnetic Resonance Imaging (MRI) on a large subset of the population. Several specific biomarker assessments in blood/serum/plasma and urine are done for the diagnosis and evaluation of risk factors of endocrine and metabolic diseases. Candidate gene and genome-wide association studies (GWAs) are actively pursued within the scope of our research for many of the above mentioned endocrine and locomotor traits and diseases. Finally, validated questionnaires evaluating nutrient intake (e.g., calcium and vitamins) and activities of daily living, allow to evaluate the role of environmental factors in endocrine conditions and locomotor diseases of the elderly. For recent references in EJE see [144‐172].

Fibrogenesis of the liver is most probably not only the result of well known liver diseases, such as viral hepatitis, alcoholic liver disease or non-alcoholic fatty liver disease (NAFLD), but rather a complex interaction between a genetic predisposition and these liver disorders. Liver research in the Rotterdam Study will concern the association between these known causes of liver disease and the occurrence, magnitude, and progression of fibrosis in combination with genetic and environmental factors. Additional research focus will be on NAFLD. NAFLD is considered the hepatic manifestation of the metabolic syndrome and has become the most common chronic liver disease in Western countries in parallel with epidemics of obesity and type II diabetes mellitus. We aim to study the occurrence and risk factors of NAFLD in a general population and generate insight into the association with cardiovascular morbidity and mortality.

The association between factors known to influence liver function and the occurrence of steatosis and fibrosis will be studied. Additionally the association of these conditions with age, gender, nutritional intake, concurrent alcohol intake, risk factors for viral hepatitis, BMI, waist-to-hip ratio, serum glucose, insulin, and diabetes mellitus, serum cholesterol and triglycerides will be studied. All clinical information will be obtained by interview (updated with liver specific questions) and clinical examination. For recent EJE references see [176‐183].

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 modalities, which include MR-imaging, neuropsychological testing and more recently gait assessment.

Neurodegenerative and cerebrovascular diseases are highly frequent in the elderly. The prevalence increases from age 55 to 65 years to age 90 years and above from less than 1% to over 40% for dementia [184], from less than 0.5% to more than 4% for Parkinson disease [185], and from approximately 1% to nearly 10% for stroke [186]. The incidence figures follow this pattern of a strong increase with age over the entire age range, with the age-specific incidence of dementia being identical for men and women at least until the age of 85 [187] but with men having a higher age-specific incidence of both stroke and Parkinson disease than women throughout the age range [186, 188].

Vascular pathology and vascular risk factors are associated with worse cognitive performance [189], which also translates in people with vascular pathology or risk factors for vascular disease having an increased risk of dementia, including Alzheimer disease [190]. Moreover, several life style factors are associated with the risk of dementia and Alzheimer disease [191‐193], suggesting that onset of dementia may at least partly be delayed or prevented. Commonly used drugs may have a role in this [194]. Similar risk factor profiles also underlie cognitive decline prior to the clinical diagnosis of dementia [195, 196].

The classical risk factors for stroke also predict risk of stroke in the Rotterdam Study [197]. Novel risk factors, including inflammatory markers, may be etiologically relevant but thus far add little to the identification of people at risk http://​www.​ncbi.​nlm.​nih.​gov/​pubmed/​17015791 [198]. Possibly underlying this is that a large amount of stroke goes clinically undetected [199]. Nearly 20% of elderly people have at least one silent brain infarct, and thereby a nearly fourfold increased risk of clinical stroke, a more than doubled risk of dementia including Alzheimer disease, and an increased risk of depression [199].

With the advent of genome-wide association studies, the Rotterdam Study has contributed to large-scale collaborations and contributed to the identification of novel genes underlying the risk of Alzheimer disease and stroke [200, 201].

Neuroimaging reveals that brain pathology is widespread [202] and can go clinically undetected for a long time. In addition to the silent infarcts, many apparently healthy elderly have ischemic changes in their cerebral white matter, i.e. white matter lesions, that are associated with an increased risk of dementia, stroke and depression. Also brain atrophy, especially of the hippocampus, is already present years before onset of even the earliest sign of cognitive impairment or subjective complaints. This emphasizes the need to shift the attention in etiologic research of neurodegenerative and cerebrovascular disease to the causes of pre-symptomatic and underlying brain changes. Technological advances in image acquisition, optimized imaging sequences and automated post-processing of multispectral MR data are major drivers of the rapid developments in this field. With our current imaging protocol we can now not only investigate established markers of brain pathology, such as infarcts, white matter lesions, and atrophy, but also extend towards novel markers, such as cerebral microbleeds and diffusion tensor imaging (see further section on “Population imaging”).

At baseline and follow-up examinations participants undergo ophthalmic measurements including best-corrected ETDRS visual acuity, refractive error, Goldmann applanation tonometry, keratometry, slit lamp examination of the anterior segment and visual field testing. In pharmacological mydriasis we made 35° color photographs of the macular area, and 20° simultaneous stereoscopic imaging of the optic disc and macular area. Since the fifth follow-up, 35° stereoscopic color photographs of the optical disc and the macular area were made (RS-I-5). Analog fundus photography was replaced by stereoscopic digital imaging of the macular area and optic disc since the third follow-up examination. Optic nerve head analysis with a Heidelberg Retina Tomograph, macular pigment density, and melanin optical density measurements were added during the third follow-up (RS-I-3). At fifth follow-up examination, fourier domain optical coherence tomography of the macular area and optical disc, axial length and width measurements of cornea, anterior chamber, lens, posterior chamber and retina measured with Lensstar; and fundus autofluorescence, infra-red and red-free measurements were added (RS-I-5).

Classification of AMD, POAG, and retinal vessel diameters remained unchanged; refractive error was evaluated as spherical value + half cylindrical value, following clinical standards.

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, addiction to smoking, and complicated grief are also being studied.

In the first years of the Rotterdam Study (RS-I-1) psychiatric data collection was very limited. However, in the second visit most participants were screened for depressive symptoms and from the third examination onwards, which began in 1997, depressive symptoms and disorders have been ascertained in all participants. An assessment of anxiety disorders, sleeping disturbances and complicated grief were added in the fourth examination and have been included in all follow-up visits of the Rotterdam Study I and II, and in the baseline of the Rotterdam Study III. Recent additions to the protocol include a screening for psychotic symptoms and, starting with the third examination of the Rotterdam Study II, ambulatory polysomnography.

Psychiatric research in the Rotterdam Study focuses on biological risk factors. The vascular depression hypothesis was tested with different measures of atherosclerosis, arterial stiffness and cerebral blood flow [230]. We also examined whether blood levels of vitamins and fatty acids, immune parameters, and markers of folate metabolism increased the likelihood of depression [231]. In one ongoing project, diurnal patterns of cortisol secretion are related to psychiatric and other disorders such as subclinical atherosclerosis [232]. Studies of genetic polymorphisms and brain morphology are underway [233]. Current data collection includes a dexamethasone suppression test to measure hypothalamic–pituitary–adrenal axis activity in all participants, which is unique in a population-based study. Also, psychiatric problems and psychological traits such as happiness, sleep duration and depression are increasingly studied as determinants of health and mortality [234, 235].

Information on depression is obtained from (a) psychiatric examinations, (b) self-reported histories of depression, (c) medical records, and (d) registration of antidepressant use [236]. The psychiatric examination during follow-up visits consists of a screening with the Center for Epidemiologic Studies Depression Scale (CES-D), and in the screen-positive participants a semi-structured interview performed by a trained clinician (Schedules for Clinical Assessment in Neuropsychiatry, [237]). To continuously monitor incidence of depression throughout follow-up, trained research-assistants scrutinize the medical records of the general practitioners and copy the information about possible depressive episodes.

The following anxiety disorders are assessed with a slightly adapted Munich version of the Composite International Diagnostic Interview: generalized anxiety disorder, specific and social phobia, agoraphobia without panic disorder, and panic disorder [238, 239].

Sleep quality and disturbance is measured with the Pittsburgh Sleep Quality Index. In addition, sleep duration and fragmentation are assessed with actigraphy, a method that infers wakefulness and sleep from the presence or absence of limb movement [240]. In total, nearly 2,000 persons participated in this actigraphy study: they wore an actigraph and kept a sleep diary for, on average, six consecutive nights. In 2011 we have begun to follow these participants up with another 6 day assessment using actigraphy. In addition, 1,000 persons will receive one night of polysomnography in their home setting.

The Inventory of Complicated Grief is used to identify traumatic grief [241]. This is a condition distinct from normal grief and bereavement-related depression, characterized by symptoms like disbelief about the death and searching for the deceased.

The objectives are to study the incidence of chronic obstructive pulmonary disease (COPD), to investigate genetic and environmental risk factors for COPD, and to study the effect of COPD on mortality. COPD is defined as a disease state characterized by airflow limitation that is not fully reversible. The airflow limitation is usually both progressive and associated with an abnormal inflammatory response of the lungs to noxious particles or gases such as tobacco smoke [260]. COPD is a worldwide leading and still increasing cause of chronic morbidity and mortality that will change from the sixth to the third most common cause of death worldwide by 2020, whilst rising from fourth to third in terms of morbidity [261].

In the first cohort of the Rotterdam Study (RS-I) of 7,983 participants, 648 cases were identified with incident COPD after a median follow-up time of 11 years. This resulted in an overall incidence rate of 9.2/1,000 person-years (PY) (95% CI, 8.5–10.0). The incidence rate of COPD was higher among men (14.4/1,000 PY; 95% CI, 13.0–16.0) than among women (6.2/1,000 PY; 95% CI, 5.5–7.0) and higher in smokers than in never-smokers (12.8/1,000 PY; 95% CI, 11.7–13.9 and 3.9/1,000 PY; 95% CI, 3.2–4.7, respectively). Remarkable was the high incidence in the youngest females in the age category of 55–59 years (7.4/1,000 PY; 95% CI, 4.1–12.6). For a 55 year-old man and woman, still free of COPD at cohort entry, the risk to develop COPD over the coming 40 years was 24 and 16%, respectively [260]. Since COPD is not only affecting the lungs, but is also characterised by extrathoracic manifestations, another line of research focuses on the role of systemic inflammation in the pathogenesis of COPD and its comorbidities. High levels of hsCRP (3 mg/l), a marker of systemic inflammation, were associated with a significantly increased risk of incident COPD (hazard ratio (HR), 1.7; 95% confidence interval (95% CI), 1.16–2.49) compared with persons with low CRP levels (<1 mg/l). The risk remained increased after adjustment for potential confounders and introduction of a potential latency period of 3 years. The risk was most pronounced for former smokers (HR, 2.2; 95% CI, 1.12–3.74). No CRP single nucleotide polymorphism or haplotype was associated with a significantly increased or decreased COPD risk [262].

Spirometric measures of lung function are heritable traits that reflect respiratory health and predict morbidity and mortality. We meta-analyzed genome-wide association studies for two clinically important lung-function measures: forced expiratory volume in the first second (FEV₁) and its ratio to forced vital capacity (FEV₁/FVC), an indicator of airflow obstruction. This meta-analysis included 20,890 participants of European ancestry from four CHARGE Consortium studies: Atherosclerosis Risk in Communities, Cardiovascular Health Study, Framingham Heart Study and Rotterdam Study. We identified eight loci associated with FEV₁/FVC (HHIP, GPR126, ADAM19, AGER-PPT2, FAM13A, PTCH1, PID1 and HTR4) and one locus associated with FEV₁ (INTS12-GSTCD-NPNT) at or near genome-wide significance [P < 5 × 10(−8)] in the CHARGE Consortium dataset [263]. The Hedgehog signalling pathway plays an important role in lung morphogenesis and cellular responses to lung injury. A genome-wide association study has demonstrated that two single nucleotide polymorphisms (SNPs) near the Hedgehog-interacting protein (Hip) gene, SNP identifiers rs1828591 and rs13118928, are associated with risk of chronic obstructive pulmonary disease (COPD). We investigated the association between genetic variation near the Hip gene and COPD, and whether risk estimates were modified by smoking behaviour in the Rotterdam Study. Both SNPs were significantly associated with risk of COPD (OR 0.80; 95% CI 0.72–0.91). Homozygosity for the minor G allele resulted in a decreased risk of COPD of approximately 40% (95% CI 0.47–0.78). There was a significant interaction with the number of pack-years of smoking (P = 0.004). The meta-analysis yielded an odds ratio for COPD of 0.80 per additional G allele [P = 3.4 × 10(−9)]. Genetic variation near the Hedgehog-interacting protein gene was significantly associated with risk of COPD, depending on the number of pack-years of smoking [264].

The first objective of the laboratory team is to collect, store and manage the biological tissues mainly blood and urine sampled in the Rotterdam Study. The second objective of the group concerns genotyping and assessment of biomarkers.

Among the biomarker analyses our study documenting the relationship between homocysteine and osteoporosis was novel [121] and has since been widely replicated. Across all research lines in the Rotterdam Study, several candidate gene studies have also yielded new insights coming from both exploratory studies as well as from collaborative replication efforts. A unique feature of the Rotterdam Study is exploited by studying the relationship between pleiotropic gene variants and multiple diseases and disease related endpoints. For example, the studies on the promoter region of the IGF-1 gene revealed a series of consistent associations ranging from birth weight to diabetes [280], while other consistent associations involve the estrogen receptor alpha (ESR1) gene in relation to osteoporosis [281], osteoarthritis, height, myocardial infarction [282], age-at-menopause, and depression. Rotterdam Study investigators are playing leading roles in the emerging large global consortia focussed on assessing the contribution of complex disease gene variants by prospective meta-analysis across many epidemiological cohorts [283], such as CHARGE, ENGAGE and the GENOMOS/GEFOS [284, 285]. Since 2005 the genome wide association study (GWAs) has changed the field of complex genetics, and identified an ever growing list of common variants contributing to disease risk and explaining genetic variance of traits. Initial findings in the Rotterdam Study from individual collaborations replicating early GWAs hits included CFH in age-related macula degeneration [286], NOS1AP in QT interval [287], and several SNPs involved in height, type 2 diabetes, and breast cancer (collaboration with WTCCC investigators). The Rotterdam Study has generated GWAs data for almost the complete dataset summing to over 11,000 DNA samples, and is involved as a major collaborative centre for meta-analysis studies of GWAs data, including national programs (RIDE, NGI-NCHA), EU-funded projects (GEFOS, TREATOA, ENGAGE), and voluntary collaborations (GIANT, MAGIC, CHARGE). Especially, from the CHARGE consortium (the Rotterdam Study together with the Framingham Study, AGES, CHS, and ARIC) many important publications have emerged on a wide variety of phenotypes and diseases from all major research lines in the Rotterdam Study [288‐292].

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.

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, abeta42/40 and thyroid hormones (TSH).

Affiliated laboratory facilities include a medium/high-throughput platform for candidate genes studies and GWAs 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 9 384), an ABI7900HT Taqman machine (running 1 ng gDNA in 2l reactions), a WAVE 3500HT dHPLC, Sequenom iPlex, and two ABI3100 sequencing machines. DNA sample handling is centred on 384-well plates. Candidate gene studies are done mostly using Taqman and Sequenom 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. GWAs genotyping studies are based on 500 K Affymetrix arrays (a pilot project of 450 women) and 550 and 610 K Illumina arrays for the complete Rotterdam Study cohort encompassing over 11,000 DNA samples. The in-house GWAs genotyping facility has been partly sponsored by NWO investment grants (911-03-012; 175.010.2005.011), is part of the Erasmus Medical Center Biomics core facility, and serves as knowledge center for polymorphism analysis attracting national and international interested parties, both academic and industrial.

We have genotyped over 300 individual polymorphisms as part of candidate gene studies across the complete cohort and conducted a large number of candidate gene studies in the Rotterdam Study. These mostly concern individual potentially functional single nucleotide polymorphisms (SNPs) per gene, but sometimes also haplotype tagging (delete space) SNPs (e.g., ESR1, ESR2, HSD11B1, fibrinogen), and also high density SNP screening (e.g., the vitamin D receptor gene). The candidate genes studied include the apolipoprotein E gene (APOE), the angiotensin-converting enzyme (ACE), the gene encoding angiotensinogen (AGT), angiotensin II type 1 receptor (AT1R) gene, G protein beta3 (GNB3), adducine gene, Cholesteryl Ester Transfer Protein (CETP), Hepatic Lipase, Phosphodiesterase 4D (PDE4D), ALOX5AP encoding 5-lipoxygenase activating protein, a polymorphism in the regulatory region of the Insulin-like Growth Factor 1 (IGF-1) gene, the hemochromatosis (HFE) gene, Complement factor H gene (CFH), and several polymorphisms in genes from the estrogen-, thyroid-, cortisol-, vitamin D-, IGF-, and Wnt signalling pathways, the homocysteine pathway, and several matrix molecules.

Genome Wide Association studies (GWAs) are based on genotyping epidemiological cohorts with ultra-high density SNP arrays with up to 1 million SNPs. The method has been shown to successfully identify common genetic factors for hundreds of traits and diseases (see www.​genome.​gov/​ GWAstudies). Through a large grant from the Dutch research organisation NWO one of the world’s largest GWAs datasets has been facilitated involving over 11,000 DNA samples from the Rotterdam Study cohorts. This GWAs dataset is based on the Illumina 550 and 610 K arrays and will be useful for all research lines within the Rotterdam Study. In addition, it will also serve as a control GWAs dataset for other research centers in and outside The Netherlands for both SNP frequencies as well as copy number variations (CNVs). In addition our group has also been active in developing new software for GWAs analyses [292].

The new development in the basic sciences will be to move to transciptomic studies and proteomic studies. With this view, the data collection protocol has been adjusted, standardizing blood collection. For recent EJE references see [290‐310].

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.

Important findings have been published on pharmaco-epidemiological topics concerning the main outcomes in the Rotterdam Study. Studies about the association between dementia, and antihypertensive drugs [293] and NSAIDs [194] have strongly suggested a protective effect of both groups of drugs. However, beta-blockers were associated with an increased risk of depression [294]. Several studies have been performed on cardiovascular topics [62, 295, 296]. In one of these studies, NSAIDs were associated with an increased risk of heart failure. A recent study demonstrated that short-term use of NSAIDs is associated with transient impairment of echocardiographic parameters [297]. In line with the suspicion that QTc-prolonging drugs may cause sudden cardiac death, it was demonstrated in the Rotterdam Study that a prolonged QTc is indeed an important risk factor [62]. Non-cardiovascular drugs which inhibit hERG-encoded potassium channels were associated with an increased risk of sudden cardiac death [298]. Furthermore, in one study it was demonstrated that high-dose corticosteroids increase the risk of atrial fibrillation [296]. In how far this effect is modified by susceptibility markers on chromosome 4q25 [299] or on KCNN3, a gene of which variants were associated with lone atrial fibrillation in a meta-analysis in the Rotterdam Study and several cohorts from the USA [300], is currently being investigated. Other important susceptibility genes for PR-interval [59], resting heart rate [301] and QRS duration [302] may prove to be important effect modifiers for drug effects. In the important area of locomotor diseases, studies have demonstrated that thiazide diuretics protect against hip fracture [303] and that statins reduce the risk of vertebral fracture [304]. On the other hand, the risk that long-term use of certain NSAIDs may aggravate signs of osteoarthritis has been emphasized [305]. In the area of ophthalmic diseases, a protective effect of cholesterol-lowering agents on macular degeneration has been studied [306, 307]. In other areas, such as pharmacogenetics and other causes of interactions between drugs, several important findings have been published [287, 308‐328]. In this regard, there proved to be important genetic variations in OCT1 and MATE1 transporters with consequences for metformin response in diabetics [329]. Genetic variation in the OCT1 transporter was also associated with response and survival time in users of drugs against Parkinsonism [330]. Interesting were the results of a GWA in users of the anticoagulant phenprocoumon which confirmed the important genetic variant for VKORC1, CYP2C9 and 4F2 [331]. An increasingly important topic is the association between long-term drug use and cancer. In the Rotterdam Study, high-ceiling diuretics were associated with an increased risk of basal cell carcinoma [332]. The importance of cytochrome P450 enzymes as effect modifiers was underlined by 2 studies, one of which showed increased survival in breast cancer patients on tamoxifen with a CYP2C19*2 polymorphism [333, 334].

For several reasons, a drug is a highly attractive determinant in clinical epidemiologic research. 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 January 1, 1991, thanks to the fact that all pharmacies which serve the Ommoord district are on one computer network. To date, more than three 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. This facilitates the use of detailed analyses with the drug as a time-varying determinant [335]. 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, endocrine, and ophthalmic diseases which have been the main topics since its start. 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. Further EJE references can be found in [336‐342].

Biomedical imaging allows for non- or minimally-invasive assessment of structural and functional changes that may reflect specific pathology. Recent developments in image data acquisition and analysis enable to use these techniques on a large scale. The Population Imaging Unit within the Rotterdam Study aims to assess imaging biomarkers of disease in a pre-symptomatic phase at the population level. Advantages of imaging measures include that they mark early disease, can be assessed reliably and reproducibly, and are quantitative rather than qualitative which makes them more powerful than most conventional outcome measures such as clinical phenotypes. The main imaging modalities that are currently being applied in the Population Imaging Unit are multidetector computed tomography (MDCT) and magnetic resonance imaging (MRI).

The Rotterdam Study research lines currently applying imaging within the Population Imaging Unit are those on neurological diseases and cardiovascular diseases.