1. Introduction

individuals with controls and stratification in multiethnic populations. These difficulties are overcome by the trans- mission disequilibrium test (TDT) and related approaches [5]. However, this test most often requires genotyping of parents who are, as mentioned above, obviously unavail- able for the study of longevity. Thus, at present, attempts to localize or define functional loci involved in human lon- gevity are based on case-control association studies in order to demonstrate statistically significant differences in allele frequencies at candidate loci between groups of aged subjects and matched controls. Such experiments should be designed to minimize false positive results by a rigorous sampling procedure. Case-control studies are usually performed on genes that may be candidates involved in longevity, because of their known or postu- lated function. Such candidate genes include those that may contribute to the risk of cardiovascular disease, the main cause of mortality in western countries, especially among individuals over 80 years of age. Thus, the first candidate genes to be studied for association with longev- ity were ACE and APOE. Polymorphic alleles at the ACE and APOE loci are known to be associated with an increased risk for myocardial infarction [6] and for hyper- lipidemia and Alzheimer’s disease (AD), respectively [7–9]. As early as 1994, 338 centenarians and 164 control individuals aged between 20 and 70 years were compared for the allele frequencies of these genes [10]. The provoca-

Prolonged human life expectancy, with consequent increase in the number of aged individuals, has stimulated the search for genes involved in longevity and ageing. A population-based study of centenarians showed that their siblings have an increased probability of survival to advanced age, indicating a familial component to longev- ity [1]. In addition, a study of British aristocracy estimated heritability of longevity to be between 0.16 and 0.37, depending on sex and age at death of both parents [2]. Nevertheless, it is difficult to distinguish between shared environmental and genetic factors. Studies of twin regis- ters estimated heritability of the phenotypic trait longevity in Scandinavian populations to be approximately 0.25– 0.50 [3, 4]. These data suggest the existence of genetic factors associated with longevity and, consequently, of functional polymorphisms located in genes that remain to be identified. Two types of genetic analyses are presently available for studies of human multifactorial complex traits such as longevity. The first method is linkage analysis which is impractical for the study of longevity because of a) the low estimated heritability, b) the expected large number of implicated loci, and c) the unavailability of parents of ‘cases’, i.e. of aged individuals. The second method, involving association studies, is prone to biases due to well-identified factors, such as loose matching of

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Table I. Distribution of matched centenarians and controls.

frequencies for both genes between the two groups lated numerous attempts to replicate these observations different population samples.

Region of origin Number of females in Number of males in each population each population Île-de-France 39 13 North-east 103 22 North-west 113 12 South-east 155 27 South-west 56 20 Total 466 94

The association of the APOE4 allele with decreased longevity was confirmed in Finnish, Chinese and Japanese populations [11–13], thus suggesting that this allele is present in the corresponding populations with frequencies that lead to a significant population attributable risk despite notable environmental differences. Results of studies to confirm the association with the ACE polymorphism are less clear cut. Monitoring of potential progressive changes in allele frequencies of ACE as a function of age in a case-referent and in a retrospective cohort study of the Copenhagen population failed to reveal statistically sig- nificant changes, an observation which led the authors to the conclusion that ACE alleles do not influence longevity [14].

2.2. Genotyping

ACE I/D (insertion/deletion) polymorphism was geno- typed by the polymerase chain reaction (PCR) and subse- quent electrophoretic analysis. The two alleles of 190 bp (D, deletion) and 490 bp (I, insertion) in intron 16 were separated on a 2 % agarose gel as described [18]. APOE genotypes were determined by PCR-RFLP (restriction frag- ment length polymorphism). Amplicons were digested by HhaI and genotypes were determined after electrophore- sis on a 10 % polyacrylamide gel stained with ethidium bromide, as described [19].

It has, however, been suggested that the role of the ACE polymorphism in longevity may only be effective in very old individuals such as centenarians. The observed para- doxical effect of ACE D-containing genotypes on mortality has been interpreted as an antagonist pleiotropic gene action [15]. Galinsky et al. in a study of 282 British individuals aged over 84 years and 200 controls reported differences in ACE genotypic frequency in the male group of older subjects as compared to the female group and to controls [16]. However, the multiple statistical tests per- formed in this study and the near borderline level of significance (P = 0.03) observed for the association pre- clude definite conclusions.

2.3. Statistical analyses

Allele frequencies were computed from the genotype frequencies. Hardy-Weinberg equilibrium was tested in both populations of controls and centenarians by a (cid:1)2 test with one or three degree of freedom for loci with two (ACE) and three (APOE) alleles (HWE software, J. Ott, 1988 [20]). Comparison of allele frequencies and genotype fre-