Retyping the 1994 centenarian cohort for the ACE poly- morphism revealed 17 inconsistencies confirmed on a second experiment leading to a 5 % difference with the original data. In the majority of the discordant results (68 %), the differences resulted from the detection of the I allele in the new data, not reported in the 1994 data. Rechecking confirmed the presence of the I allele indicat- ing that discrepancies were mostly due to technical diffi- culties experienced during the initial typing to identify a large-sized allele.

For centenarians and for matched controls, APOE allele distribution between males and females was not signifi- cantly different (P value equal to 0.71 and 0.98, respec- tively) as was genotypic distribution (P value equal to 0.32

Table III. Allele and genotype distributions of the ACE polymorphism.

Allele No. alleles/centenarians (frequency) No. alleles/controls (frequency) I 467 (41.7 %) 490 (43.8 %) D 653 (58.3 %) 630 (56.2 %) Genotype No. centenarians (frequency) No. controls (frequency) II 103 (18.4 %) 101 (18.1 %) ID 261 (46.6 %) 288 (51.4 %) DD 196 (35.0 %) 171 (30.5 %) Statistical analyses Hardy-Weinberg equilibrium P = 0.33 0.29 Comparison of allelic frequencies P = 0.33 Comparison of genotypic frequencies P = 0.22

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Centenarians of the 1994 cohort included about 20 % of individuals born out of France or with unknown birth- places. A new genotyping of these 184 centenarians revealed a higher D allele frequency (62 %) compared to that observed in the group of centenarians known to be born in France (748 individuals, D allele frequency = 57.6 %). Although these frequencies were not significantly different, this observation suggested heterogeneity in the geographic distribution of the ACE polymorphism and pointed to the importance of stringent matching of cente- narians and controls. Archival material for the 1994 con- trol group did not permit a similar test.

sidering centenarians and controls irrespective of gender. The significance of the association between APOE and longevity is more robust as compared to the 1994 study. This observation may be solely accounted for by an increased number of individuals tested (560 in the present study versus 323 in the 1994 study) since the allele and genotype frequencies in both groups were similar in the two studies. Clearly, the centenarian class continues to demonstrate deficits of the homozygous E4 genotype and of the heterozygous E3E4 genotype as compared to con- trols. Centenarians carrying at least one E2 allele or homozygous for E3 are more frequent than controls, sug- gesting that these genotypes favour longevity. Miyata et al. [24] showed that the APOE isoforms can be classified according to their anti-oxidant activity in the following > > decreasing order E2 E3 E4. It thus appears that the apparent effect of the APOE alleles on longevity may be related to their anti-oxidant activity. An increased risk for AD in females was reported for the E4 heterozygous genotype as compared to non-carriers [25]. Such suscep- tibility was not observed for males. This discrepancy may reflect a gender difference in the etiology of AD. In the present study, differences in the frequency of E3E4 geno- type between centenarians and controls were both signifi- cant and similar in the male and female groups. Taken together, these observations suggest that the E4E4 and E3E4 genotypes are associated with decreased longevity both in males and females. The influence of the E2E4 genotype on longevity remains uncertain due to the low frequency of this genotype.

4. Discussion

An initial report of association of a genetic polymor- phism with a phenotypic trait requires replication with an independent population sample to be generally accepted. This is especially so when the association appears to be paradoxical. The purpose of this study was to reassess the association of APOE and ACE I/D polymorphisms with the phenotypic trait of longevity in French centenarians [10]. In designing the present study, we analysed the 1994 report for potential flaws in design.

First, the initial study was based on cases and controls that had not been precisely geographically matched. The origins of the centenarians were dispersed throughout France. In contrast, the control population was composed for the most part of individuals recruited in Lille, Stras- bourg and Toulouse for the ECTIM study [6, 21] and to a lesser extent of non-related individuals from the CEPH families with no documented birthplaces. Matching cases and controls for geographic origin is essential, because regional variation of allele frequencies has been observed [22, 23]. Second, the laboratory that genotyped the ECTIM individuals constituting the major part of the control group of the 1994 study was different from the one that typed the centenarians. With the present laboratory settings, fre- quencies of genotype errors of less than 1 % are rare for projects involving the characterization of large groups of individuals (hundreds, thousands). Because the rate and type of genotyping errors may vary between different laboratories, it is desirable to genotype all cases and controls in the same place, and preferably at the same time.