MATERIAL AND METHODS

Study Population

The population in this study was drawn from the 1997 and 1999 surveys of the longitudinal study of aging Danish twins (LSADT) which has been described in detail else- where (18). In brief, the LSADT began in 1995 and is an ongoing longitudinal study of twins born before 1929 and identified in The Danish Twin Registry. The Danish Twin Registry includes all twins born in Denmark between 1870 and 1910, and same-sex pairs born between 1911 and 1930. The LSADT participants are interviewed every second year in their homes by lay interviewers from the Danish Na- tional Institute of Social Research who have substantial ex- perience in interviewing the elderly. The 1997 survey comprised 2172 individuals corresponding to a participa- tion rate of 78.7 percent. We subsequently asked individu- als from pairs where both twins participated in the  interview study (n 974) to allow a trained technician to visit them in their homes and draw a sample of blood. Blood was sampled from 689 individuals (Fig. 1) and ACE genotype was determined in 554. For 130 monozygotic (MZ) twins, the ACE genotype was determined for one twin in the pair and was then assigned to the co-twin. Genotyping failed due to technical problems in 5 cases. The study population from the 1997 survey therefore con- sisted of 684 individuals of whom 547 were reassessed in 1999 (Fig. 1).

Physical performance is important for younger people but even more so among the elderly where physical perfor- mance together with cognitive function determine whether life can be lived independently. Cognitive impairment and dementia have also been reported to be associated with ACE genotype, although recent studies have come to oppo- site conclusions regarding ACE genotype and risk for Alzheimer’s disease (AD) (7, 8). An increased risk of AD was associated with the I allele in one study (7), but the D allele in another (8).

enced by ACE genotype since a German study found an in- creased frequency of the DD genotype in octogenarians (9). This finding could not, however, be confirmed in two large studies of centenarians (10, 11). Studies of elderly twins have found evidence of substantial genetic influence on both physical (14) and cognitive functioning (15, 16) as well as a moderate genetic influence on life span (17). Twin studies have mostly been used for determining the relative contribution of genes and environment to the variation of a phenotype but less so for identifying specific genetic vari- ants of importance. Based on the previous association stud- ies and the fact that the renin-angiotensin system is involved in cardiovascular and renal homeostasis and that ACE activity and angiotensin receptors can be demon- strated in various peripheral tissues including skeletal mus- cle (12) and brain (13) it seems conceivable that the I/D polymorphism that is associated with the circulating ACE activity could be associated with physical and cognitive function and survival at older ages. We therefore found it relevant to investigate the possible relations of ACE geno- type to physical and cognitive abilities in a large sample of older Danish twins who are being followed longitudinally. This twin cohort provided the evidence for a substantial ge- netic component to the phenotypes we study. Here we use the twin cohort as a longitudinal study of individuals, al- though we take advantage of and pay attention to the de- pendency between the two twins within a pair. Based on the previous literature, it could be hypothesised that the II ACE genotype is associated with better physical perfor- mance in the elderly. Previous studies were not consistent

As twin zygosity was determined through a self-report questionnaire, a method that has been shown to result in less than 5 percent misclassification (19), the number of misclassified MZ twins was very small.

Genotyping

The ACE genotyping was done according to Rigat et al (20). To avoid the earlier reported mistyping of ACE het- erozygotes (ID genotypes misclassified as DD) (21, 22), all samples typed as DD were reamplified as described by Shan- mugam et al (21) with another set of primers in the pres- ence of a positive control (ID/II).

Physical and Cognitive

Physical ability was assessed through self-report in 1997, and through both self-report and the measurement of per- formance in two physical tasks in 1999. The questions re-

'1 died before and 79 declinedarticipation in 1999 surve:  $$Longitudinal Study of AgingDanish Twins - 1997 survey2172 participating twins1198 singles and487 pairs (974 individuals)280 declined, Asked to give DNA samplesampling failed in 5Sufficient DNA sample obtainedfrom 689*ACE genotypedDirect: 554 / Indirect’: 130 Study population 1997N = 684      Reassessment population 1999N = 547   Interview including:Self-reported physicalabilities, health, life-style and cognitivetesting       Same instrument as in1997+physical tests                

FIGURE 1. Flow chart of 684 ACE genotyped twins from the Longitudinal Study of Aging Danish Twins - the 1997 and 1999 surveys.