In contrast, the excess of the D allele for French cente- narians, initially reported in 1994 [10], was not confirmed in the present study. Since 1994, the ACE association with longevity has been examined by several authors [14, 16, 26]. In a Danish population sample, relative frequencies of the DD genotype and the D allele were not associated with age in either women or men [14]. A study of a sample from a British population composed of 100 males and 170 females aged 84 years and older [16] suggested a weak association between ACE and longevity which was restricted to males. However, as discussed by the authors, the absence of Hardy-Weinberg equilibrium for the ACE locus in the control population hinders firm conclusions. Finally, Faure-Delanef et al. [26] genotyped ACE polymor- phism on 84 French centenarians who had not previously been studied. The data, when merged with those of the 1994 study, decreased the significance of the ACE geno- typic association.

For the present study, cases and controls were matched for sex and for geographic origin. The centenarian popu- lation is on average 50 years older than that of the control group. Thus, for each case, a control was defined as an individual whose parents were both born in the same region as that of the centenarian. Such a procedure samples the population at a time period when population mobility was low and minimizes the effect of population immigra- tion.

In contrast to the association of APOE with longevity, which relies on a two-fold difference in the E4 allele frequency in centenarians compared to controls, the initial report of the association of ACE with longevity in French centenarians was based on a 6 % difference in the D allele frequency (i.e. 0.61 in centenarians as compared to 0.55 in controls). Failures to replicate the association may by attributed to several non-exclusive factors. First, within France, the D allele frequency varies among regions by more than 3 % [6]. This variation may increase when

Using these matched case-control groups, the associa- tion of APOE with longevity was confirmed. As there were no sex differences, analyses were initially performed con-

133

Conclusion

different European regions are compared (e.g. 7 % differ- ence between Belfast and Toulouse [6]). Thus, it is man- datory to finely match control and centenarians on geo- graphic origin. Such matching was crudely performed in the 1994 study [10]. It could not be performed for 20 % of the centenarians who were of unknown or foreign origin in that study. Such a proportion may have contributed to the unusually high D allele frequency observed. Second, accurate genotyping of the I/D polymorphism of ACE requires reliable amplification of two DNA fragments markedly different for molecular size (190 and 490 bp for the D and I allele, respectively). It is well known that sub-optimal PCR conditions lead to the misclassification of I/D heterozygotes as D/D homozygotes, thus resulting in the overestimation of the D allele frequency [27]. Repeat genotyping by us of the centenarians reported in the 1994 publication [10] revealed discrepancies in 17 of 338 individuals. In the initial typing, the D allele fre- quency in these 17 cases was 79 %, suggesting that errors in DNA identification may not be the only cause of these discrepancies. Strikingly, 12 of these cases were initially typed as D/D homozygotes. Repeat typing classified 11 of them as I/D heterozygotes. Thus, it is likely that genotyp- ing errors in the 1994 cohort may have led to overestima- tion of the D allele frequency in the centenarian popula- tion.

The present discussion emphasizes the risk of reporting false positive association when rigorous experimental design is compromised. Matching of cases and controls is essential. Robust and standardized genotyping protocols should be used on large numbers of samples. Cases and controls should be interspersed during the genotyping procedures, thus ensuring an equivalent contribution to the genotypic data of both groups not only by each col- laborating laboratory but also by each individual set of experiments. The present study, which incorporated these recommendations, confirms the association of the APOE polymorphism with longevity. However, the association of the ACE D allele with longevity in the French population is not replicated. Although we may not exclude that such an association exists in this population, it is likely to be considerably weaker than initially claimed and would thus require much larger groups to be reliably demon-

Acknowledgements. We thank F. Péron for the recruit- ment of the elderly and H. Cann for helpful discussions about the manuscript. This study was funded by the ‘Ministère de l’Éducation nationale, de l’Enseignement supérieur et de la Recherche’.

[11] Castro E., Ogburn C.E., Hunt K.E., Tilvis R., Louhija J., Pent- tinen R., Erkkola R., Panduro A., Riestra R., Piussan C., Deeb S.S., Wang L., Edland S.D., Martin G.M., Oshima J., Polymorphisms at the Werner locus: I. Newly identified polymorphisms, ethnic variability of 1367Cy/Arg, and its stability in a population of Finnish centenarians, Am. J. Med. Genet. 82 (1999) 399–403.

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