Life Span

During the last decade a variety of twin studies have shown that approximately 25 percent of the variation in life span is caused by genetic differences. This seems to be a rather consistent finding in various Nordic countries in different time periods and even so among other species not living in the wild (Herskind et al., 1996; Iachine et al., 1999; Finch and Tanzi, 1997).

Reproduction

Kohler et al. (1999) studied the genetic dispositions influencing fertility and fertility-related behavior using Danish twins born in the period 1870-1964. It was found that genetic influences on fertility exist, and that their relative magnitude and pattern depend on sex and on the socioeconomic environment experienced by successive birth cohorts. Genetic effects were most pronounced in periods with consciously controlled fertility, suggesting that the genetic disposition primarily affects fertility behavior and motivation for having children. Analyses of fertility motivation in some of the more recent twin cohorts, measured by age at first attempt to have children, supported this interpretation.

Health and Diseases

For a number of diseases which occur in early or midlife, such as insulin-dependent diabetes and schizophrenia, twin studies have demonstrated the existence of a significant genetic component (Kyvik et al., 1995; Plomin et al., 1994). Genetic factors also influence cardiovascular diseases which occur in early or midlife, while for cardiovascular diseases occurring late in life there is little evidence of a genetic effect (Marenberg et al., 1994). Dementia has a very strong genetic component, not only with regard to early-onset monogenic types but also to late-onset dementia (Breitner et al., 1993; Gatz et al., 1997). Alternatively, twin studies provide little evidence of genetic factors in the etiology of Parkinson disease (Tanner et al., 1999). Twin studies of general physical functioning such as strength (younger individuals) or functional abilities (elderly individuals) show evidence of a considerable genetic influence, an influence that seems to increase with age (Christensen et al., 2000b).

Psychology and Behavior

The genetic component of physical abilities and diseases is usually broadly accepted. Similar claims regarding psychological and behavioral phenotypes often meet resistance, although a very large body of evidence suggests that heritabilities consistently range from 30 to 50 percent for personality, vocational interests, general intelligence, and scholastic achievement (Plomin et al., 1994). Depression symptomatology (Kendler et al., 1987; McGue and Christensen, 1997) and even proclivity to divorce (McGue and Lykken, 1992) have also been shown to have substantial genetic contributions to their etiology.

Cohort Studies

This design uses the entire study population of persons at risk for the outcome of interest and follows this cohort over time. The cohort study design is feasible if the cost of genotyping (e.g., of APOE variants) is inexpensive. In this case, the APOE genotype can be determined for all participants who have provided a biological sample, and all outcomes studied can be stratified not only on sex and age but also on APOE genotype. For example, if one wanted to study cardiovascular diseases, survey participants within a reasonable age range and free of cardiovascular diseases at intake would be genotyped and followed over time. Traditional survival analysis could then be used to assess the effect of the different APOE genotypes on cardiovascular disease risk. In cohort studies, the assessment of gene-environment interactions can be done by comparing the disease-free survival of individuals stratified on genotype (e.g., +/- APOE e4) and exposure to the environmental factor (+/-), or by using standard techniques such as Cox regression analysis.

Nested Case-Control Studies

This design is an attractive option if the outcome of interest is a rare disease/condition or if the assessment of the gene variant is expensive (which today is often the case, but this is likely to change within a few years). In the nested case-control study, all new cases of the phenotype of interest (e.g., cardiovascular diseases) are included as well as one to four controls selected from the study base. Including more than four controls results in a limited increase in statistical power (Rothman, 1986). The method for selection of controls from the survey population is essential, and must include consideration of factors such as the ethnic background of the cases and the controls (for more details, see Rothman and Greenland, 1998, and Khoury et al., 1993).

Only the biological samples from the cases and the one to four controls per case are analyzed for the gene variants of interest. From the frequencies of the gene variants in the case and the control group, the odds ratio can be estimated, which is approximately equal to the relative risk associated with an allele if the outcome studied is not too common (i.e., less than 5 percent of the population studied will get the disease/condition). Gene-environment interactions are assessed by stratifying on genotype; i.e., the relative risk associated with the exposure is estimated for each genotype separately—most often using a multivariate logistic regression analysis with interaction terms (Hosmer and Lemeshow, 1989). However, as seen in Figure 3-4, several hundred cases and controls are required to detect modest interaction terms, and some researchers argue that in some scenarios the estimated sample size requirement in Figure 3-4 may be underestimated (Garcia and Lubin, 1999). Household surveys, therefore, will often be one of the best methods to fulfill sample size requirements.

FIGURE 3-4

Number of cases required for 80 percent power at a 5 percent Type I error in a case-control study designed to detect gene-environment interaction with two controls per case over a series of frequencies of exposure (e.g., head trauma) and proportions of (more...)

Problems in Gene-Environment Studies within Household Surveys

Household surveys are most often conducted with lay interviewers, which places certain constraints on which phenotypes can be studied. For example, in medically oriented surveys, cognitive abilities and depression symptomatology can be assessed, but a dementia or depression diagnosis according to internationally recognized criteria can be obtained only in follow-up studies with the assessment done by specialists.

The biggest challenge to gene-environment interaction studies in the years to come will probably be a multiple comparison problem. With 50,000-100,000 genes being identified, many of which are likely to have several variants, an enormous number of possible gene-environment interactions can be studied. To choose a significance level in statistical testing of gene-environment interaction seems difficult. The most reasonable strategy probably will be testing of biologically plausible interactions and verification of positive findings in large studies, which again points towards the important role that household surveys can play in future gene-environment studies.

The Longitudinal Study of Aging Danish Twins (LSADT)

In 1995, LSADT began by assessing all cooperating Danish twins aged 75 years and older (a sample of nearly 2,500 individuals). The assessment was repeated in 1997 by including all twins who participated in 1995 as well as a sample of previously unassessed twin pairs who were between 73 and 76 years old in 1997. In 1999, we included all Danish twins aged 70 years and older. By 1999, over 5,000 individuals aged 70 years and older had completed the LSADT intake assessment, nearly 3,500 individuals had completed a two-year follow-up assessment, and nearly 1,000 individuals had completed both a two- and four-year follow-up assessment. Additional assessments are planned for 2001 and 2003.

The first two waves (1995 and 1997) included anthropometric measures and questions on sociodemographic factors, lifestyle habits, self-rated health, diseases, sensory deficit, symptoms, medications, physical abilities, depression symptomatology, family history, and social life. Cognitive abilities were assessed using the Mini Mental State Examination (Folstein et al., 1975) and a number of other tests. The 1997 wave used the same survey instrument. In the 1999 wave, the survey instrument was expanded to include physical performance tests, measurement of lung functioning (spirometry), and sampling of DNA by self-administered finger prick or cheek brushes (see details about DNA sampling in a later section of this chapter) (Christensen et al., 1996, 1998, 1999, 2000a, 2000b; Yashin et al., 1998; Andersen-Ranberg et al., 1999; Kohler et al., 1999).

The Longitudinal Study of Middle-Aged Twins

To complement the LSADT studies with a twin study of middle-aged twins, we identified a random sample of twin pairs born between 1931 and 1952 through the Danish Twin Registry (Kyvik et al., 1996; Skytthe et al., 1998). In 1999, more than 5,000 twins aged 46-67, including monozygotic twins and fraternal twins of same and opposite sex, were invited to participate in an extensive face-to-face interview conducted by lay interviewers, which included tests of physical and cognitive function. The questionnaire, which was based on the LSADT questionnaire, included items on the twins' current and childhood socioeconomic status, social network, self-rated health, diseases, use of medications, lifestyle habits, physical activity at work and during leisure time, reproductive history, and a brief food frequency questionnaire (Gaist et al., 2000). The same procedure used in LSADT was implemented for DNA sampling.

The Danish 1905-Cohort Survey

In order to study nonagenarians, all Danes born in 1905 were invited in 1998 to participate in a home-based two-hour multidimensional interview that included cognitive and physical performance tests. The interview instrument was similar to the LSADT instrument and carried out by the same lay interviewers. Population-based registers were used to evaluate representativeness. Participants and nonparticipants were highly comparable with regard to marital status, institutionalization, and hospitalization patterns, but men and rural residents were more likely to participate than women and urban residents. Despite the known difficulties of conducting surveys among the very old, the study showed that it was possible to conduct a nationwide survey of more than 2,000 fairly representative nonagenarians using lay interviewers. Again, the LSADT procedure was used for DNA sampling (Nybo et al., in press).

The Longitudinal Danish Centenarian Study

This study is a nationwide survey of all persons living in Denmark who celebrated their 100th birthday during the period April 1, 1995, to May 31, 1996. The residence of all centenarians in the study population was identified through the Civil Registration System by the personal identification number of each centenarian. Approximately two weeks after their 100th birthday, all centenarians received a letter explaining the study, and their permission was sought to allow a geriatrician and a geriatric nurse to visit in order to interview them and carry out a physical examination including phlebotomy. More than 200 centenarians participated (Andersen-Ranberg et al., 1999).

Finger Prick

The blood spots were made by the participants themselves, guided by the interviewer, using a sterile automated incision device (Tenderfoot^®) with a standardized incision depth of 1.0 mm and length of 2.5 mm. This device has a safety clip, which prevents the premature release of the blade, and a retracting blade, which improves safety by protecting both participant and interviewer from injury due to an exposed blade contaminated with blood (Figure 3-5). The participant chose one of the three lateral fingers on either the right or left hand, and the chosen finger was warm, dry, and clean. The Tenderfoot safety clip was removed and the device was positioned longitudinally at the site of the distal part of the chosen finger and triggered. The first drop of blood was wiped away and the next blood drops distributed to the blood spot card. On the blood spot card there are five roundels, and we aimed at collecting three or four drops of blood on each roundel (in total 5 cm²) (Figure 3-6). The blood spot card was air dried and sent to a laboratory the same day, after being marked with the date, the interview subject number, and the number of the interviewer. Only the blood spot was sent to the laboratory, so that the identity of the participant was unknown to the laboratory, ensuring that all analyses and genotyping were done blinded. To avoid catastrophic sample loss, the blood spots were divided in two and are kept in two locked locations. The blood spot cards are stored in boxes at room temperature. The utensil cost was $2-3 per person and the interview time averaged 5-10 minutes depending on age, but with large variation.

FIGURE 3-5

Utensils for obtaining blood spots by finger prick in connection with household surveys. The blood spots can be made by the participants guided by the interviewer using the sterile automated incision device (e.g., Tenderfoot^R) with a standardized incision. (more...)

FIGURE 3-6

To avoid sample loss, the blood spots are divided in two and kept in two different locations.

Cheek Brushes

Alternatively, the DNA sample was taken by means of cheek brushes (Figure 3-7). Three cheek brushes were used for each subject. Again the samples were self-collected by the participant, guided by the interviewer. The brush was inserted in the mouth and twirled firmly against the inner cheek for 30 seconds. This process collects cells from which to obtain the DNA. The brush was placed in the original tube and the cap replaced. A new place on the inner side of the cheek was chosen for each brush. Every brush was labeled with the date, the participant number, and the number of the interviewer, and sent to the laboratory the same day (arrived the next day). In the laboratory the brush head was placed in a tube and kept at −80° C until the isolation of DNA took place (within one month). The utensil cost was $3-4 per person and the interview time averaged 2-5 minutes depending on age, but with large variation.

FIGURE 3-7

Brushes used to obtain biological samples from the inner cheek in connection with household surveys. The samples can be collected by the participant guided by the interviewer. The brush is inserted in the mouth and twirled firmly against the inner cheek (more...)

Participation in DNA Sampling

Naturally, the respondents in these surveys could choose to participate only in the interview and not the biological sampling (Table 3-2). The reason for lower participation in the DNA sampling among the oldest participants is proxy interviews: when a person in the study population was unable to participate (most often due to dementia), we tried to identify a relative who could answer on behalf of that person (a proxy interview). For ethical reasons we did not sample DNA from the participant in connection with proxy interviews.

TABLE 3-2

Surveys Conducted at the Aging Research Center at the University of Southern Denmark and the Danish Center for Demographic Research in the Period 1998-1999, Which Included Collection of Biological Material by a Finger Prick or Cheek Brush.

For nonproxy participants, the difference in participation rate between the surveys was modest: 97 percent in the survey of middle-aged twins (mean age 57) versus 90 percent in both LSADT-97 (mean age 76) and the 1905-Cohort Study. As seen in Table 3-2, the percentage of cheek brush samples is highly dependent on age: nearly all middle-aged participants provided DNA by finger prick, while among the oldest-old a fifth of the DNA samples were cheek brushes.

Storage and DNA Quality Control

The success of any data-intensive project is dependent on the ability to store and retrieve data. It is important to have a facility that is used to handling large numbers of biological samples. In the surveys mentioned above, the Department of Clinical Biochemistry at Odense University Hospital takes care of the registration, storage, and retrieval of the samples. This department handles 3 million biochemical analyses each year, and therefore an addition of some 10,000 samples did not impose major logistic problems. As mentioned, the biological samples are kept anonymously and apart from the information obtained during the interview, but the information is linkable through the participants' ID numbers. For quality control we have determined APOE gene variants on a subsample of the blood spots and the cheek brushes with a success rate of 99 percent. Based on our experience, the DNA isolated from cheek brushes is of a sufficient quantity and quality to perform at least 200 genotypings for each brush, while each blood spot card can provide DNA for approximately 2,000 genotypings. DNA can be obtained from blood spot cards stored for decades at room temperature (Strobel et al., 1998; Makowski et al., 1996). Although experiences with storing cheek brushes for longer time periods are few, it seems likely that storage at −80° C will preserve the DNA for decades.