The second line of research aims to identify new genetic factors involved in complex genetic disorders using family as well as population-based approaches. These studies focus on neuropsychiatric diseases, cardiovascular disease and metabolic diseases. Several studies using linkage and sibpair analysis are ongoing. The focus of research within this line of research is on genetic isolated populations. These studies are primarily conducted in an isolated population in the South West of the Netherlands comprising 20,000 inhabitants. Genome screens concerning several disorders are ongoing.

The third line of research specfically targets methods and statistical analysis of genetic epidemiologic research. This line of research is embedded within the emperical studies conducted in the first two lines of research. Specifically sib-pair studies and research in isolated populations is targetted. Further, the education for the MSc, DSc and PhD programme is embedded in this research line. Several courses on the design and statistical analysis of genetic studies are taught as part of the research programme organised by the Netherlands Institute of Health Sciences (NIHES).

There is a close collaboration within the Erasmus Medical Center with the Department of Clinical Genetics. This has resulted in a joint Genetic Epidemiologic Unit which included expertise in clinical, molecular biologic and statistical aspects of genetic research. Further, there is a collaboration with several clinical groups including internal medicine, neurology and childhood psychiatry.

The challenge for the near future for genetic epidemiological research will be the identification of genes involved in the etiology of common late-onset disorders, like diabetes, cardiovascular disease and dementia. In these “complex” genetic disorders the risk associated with a mutation may depend for a large part on interaction with other genetic or environmental risk factors. There is increasing interest in population based studies of these disorders. These concern primarily candidate gene studies.

Involvement of a particular gene in the pathogenesis of disease may be suspected based on the gene product, the protein, or homology of the gene or protein to a gene or protein that is known to be involved in the disease. To determine whether a candidate gene is involved, DNA variations in the gene can be studied. The rationale of this approach is that if there is a causal relationship, a particular mutation should at least be present more often in cases than in unaffected relatives or unrelated controls.

Genetic epidemiological studies are extremely valuable in distinguishing between causes and consequences of disease, because genetic information remains stable over time. The role of several common genes in the pathogenesis of disease is investigated in the setting of the Rotterdam Study. This is a single-centre prospective follow-up study in which 7,983 residents aged 55 years and over of the Rotterdam suburb Ommoord participate (response rate 78%). The aim of the study is to investigate determinants of chronic and disabling cardiovascular, neurodegenerative, locomoter and ophthalmologic diseases. Further, the pathway through which a gene is involved in a disease is examined. Specific genes that are currently being investigated in the Rotterdam study are: the apolipoprotein E gene (APOE), the gene for Insulin-Like growth factor I ( IGF-I), the hemochromatosis susceptibility gene (HFE), the angiotensine-converting enzyme gene (ACE) and the gene for Nitric-oxide synthethase (eNOS).

Insulin-like growth factor-I, one of the somatomedines, belongs to the family of peptide hormones and has high structural similarity with pro-insulin. IGF-I is produced in liver, bone cells and other tissues and stimulates somatic growth and is involved in cell growth and metabolism. IGF-I serum levels are involved in pathogenesis of diseases, such as diabetes, cardiovascular diseases, cancer and osteoporosis. Levels are influenced by growth hormone (GH), insulin and nutrition, but these factors only partly explain the wide variation in the levels. studies on the role of IGF-I have been hampered by the fact that circulating IGF-I levels do not necessarily reflect the local production of IGF-I in specific tissues, such as pancreatic beta cells, myocardium or bone cells. Over 90% is bound to specific IGF binding proteins, of which IGF-BP3 is the most frequent. a genetic polymorphism in the promoter region of IGF-I has been identified, which may influence the IGF-I production. In the Rotterdam Study 7,012 subjects have been typed to study the functionality of the IGF-I gen.

In our research projects we assess:

• Relation of polymorphisms to serum levels and body height
• Relation of genetic variation to vascular pathology, diabetes, cancer, neurologic disease, bone disorders and mortality
• Interaction between IGF-I polymorphisms and other genetic and non-genetic factors.

Hemochromatosis is a common recessive disorder in populations of Caucasian origin. The disease is characterized by iron accumulation in the tissues of many organs throughout the body. Increased iron stores have been associated with a wide spectrum of disease including cardio and cerebro-vascular diseases, diabetes, cancer, bone and liver diseases. Several mutations have been described; the most common ones are the C282Y and H63D mutations of the HFE gene located on chromosome 6. There are scarcity of reports on the penetrance, clinical expression and pathology of the know HFEmutations in the elderly. Moreover, not all hemochromatosis patients carry the known mutations, suggesting that other genes are involved.

We are studying the frequency, penetrance and clinical expression of the known HFE mutations through genotyping participants in a population-based study: The Rotterdam Study. The Rotterdam Study is a population-based study of 7,893 subjects aged 55 years and over. In this study, cardiovascular, neurological and endocrine disorders have been studied in relation to their genetic and non-genetic risk factors.

Studies done:

• A population-based study of the effect of the HFE C282Y and H63D mutations on serum iron levels in the elderly
• HFE gene mutations and liver function in the elderly
• Does serum bilirubin projects against HFE related pathologies?

Studies in progress:

• HFE gene mutations, hypertension, myocardial infarction and atherosclerosis.
• HFE gene mutations and risk of arthropathies and joint pains
• Mutations in the HFE gene and risk of neurological disorders: Alzheimer's disease, vascular dementia and parkinsonism
• Survival in patients with the HFE C282Y and H63D gene mutations
• HFE and cancer

Angiotensin Converting Enzyme (ACE) has been involved in several common disorders including hypertension, cardiovascular disease and Alzheimer's disease. Studies on the role of the common insertion/deletion (I/D) polymorphism in the ACE gene in the development of diease have been hampered by small sample size. Further, the role of ACE in different chronic disorders has not been approached simultaneously. We are studying the functional and clinical effects on the I/D polymorphism in ACE in several disorders in the Rotterdam Study.

We assess:

• The relation of polymorphism to serum levels of ACE
• The relation of genetic variation to vascular pathology, diabetes, neurologic disease, and mortality
• The interaction between the ACE polymorphism and other genetic and non-genetic factors.

For more information on this project:

The most successful approach to identify new genes in disorders has been a search of the full genome in families. Linkage anlaysis has been a powerful approach for genome screening. In the past decade considerable progress has been made in unravelling the etiology of important single gene diseases such as Huntington's disease and cystic fibrosis. Within the genetic epidemiologic unit several linkage projects are ungoing in collaboration with the Department of Clinical Genetics. These studies concern families in which a disease transmitted as a Mendelian trait. The challenge for the near future in genetic research will be to unravel the genetic etiology of common diseases such as cardiovascular diseases, diabetes mellitus, osteoporosis, osteoarthtitis and unipolar depression. Findings on the genetics of these complex disorders have often been equivocal. There may be several explanations for this. Firstly, different genes may play a role in different families and different disease genes may even be involved in a single family, which may result in false exclusion of linkage. Secondly, false negative findings may occur when the disease is the result of the interplay  of different genetic and enviromental factors. An alternative approach is to examine affected sib-paires. Within the unit, affect sib-pair studies of osteoarthritis are in progress. We are further studying families with Alzheimer's disease, Parkinson's disease and hematochromatosis.

To unravel the genetics of a complex disease requires a different strategy. In recent years there has been growing interest in mapping disease genes in genetically isolated populations. Complex traits are expected to be more homogeneous in these populations. Due to the small number of founders, the total gene pool and therewith the number of different genes involved in a trait is limited. This increases the chances of success of genetic research considerably. Genetic variation will be reduced further as a consequence of genetic drift. For this reason, it has been suggested to focus on populations of prolonged isolation such as the Finnish. Although there is an ongoing debate on the question whether these old populations are suitable for genetic association studies, several studies of complex disorders including multkiple sclerosis, hypercholesteremia and osteoarthritis have proven to be highly successful.

In 1995 we have started our research program "Genetic Research in Isolated Populations" (GRIP). This program is conducted in a genetically isolated population in the Southwest of the Netherlands. As part of the GRIP program, we have studied several complex genetic disorders including diabetes mellitus type 1 and 2, Parkinson's disease and Alzheimer's disease. Using municipal records and genealogical databases of this isolated population of 20,000 residents, we were able to link most of the patients of each disorder to a common ancestor. For two disorders, diabetes type II and Parkinson's disease, we have successfully finished the genome screen while the analysis in Alzheimer patients and diabetes type 1 are still ongoing. For diabetes type 2 we were able to localise a region. This region was associated with increased fasting glucose levels in first degree relatives, suggesting this segment harbours most likely a gene involved in the pathogenesis of diabetes in the GRIP population. Also for Parksinson's disease, a disorder with a relatively low heritability, we have been able to localise a susceptibility region in GRIP.

The study of complex genetic traits necessitates the development of new statistical methods. A genetic trait is complex when several genetic and environmental factors are involved. Often, the contribution of a genetic factor is small and therefore, genes are difficult to detect. Moreover interaction between a gene and an enviromental factor may exist. Thus finding a good link function between trait values and covariates, choosing the right study design and adjusting for the covariates is very important to have enough power to detect genes.

The traits studied may be binary or quantitative. An example of a binary trait is myocardial infarction and of a quantitative trait is blood pressure. Often quantitiative factors are involved in the etiology of diseases, e.g. levels of cholesterol may predict myocardial infarction. For late onset diseases such as Alzheimer's disease, detection of genes is even more difficult, because not much family information is available and because of competing risks with other diseases.

In Rotterdam we study genetic factors in samples of families, of isolated populations and of the general poulation (Rotterdam Study). The amount of relatedness decreases from a family, via the isolated population to the general population. Therefore different samples can be used in different stages of the analysis of identifying genetic factors. We are mainly interested in two aspects of genetic epidemiology, namely estimation of the location of a gene and estimation of the relation between a candidate gene and the trait.

Estimation of the location of a gene can be carried out using family data. These may  concern large pedigrees or sibpairs. Here the segregation of marker alleles and the trait in relatives is studied. If a marker is linked to the trait gene, they will tend to segregate together. However, fine mapping of genes involved in complex diseases using linkage methods requires a huge number of families and therefore unrelated individuals from isolated or general population are often used for this purpose. The latter studies may often concern studies of candidate genes.

Candidate genes are genes for which evidence exists that they are involved in etiology of the trait. They may be located in candidate regions or there may be biological reasons to consider a gene as a candidate gene. Estimation and verification of the relation between a candidate gene and a trait in a certain population requires genetic knowledge of this population. Here we enter the field of population genetics. An issue playing a role here is the possibility that the population is a mixture of subpopulation with different genetic background. Note that this may also play a role in linkage disequilibrium mapping.