Since 1994, the number of new centenarians that have been included in the cohort initiated at CEPH has reached over 600. This new group of individuals provides an opportunity to reassess the proposed relationship between longevity and the ACE and APOE polymorphisms in the sample. Five hundred and sixty French centenarians were each paired with a younger individual of the same sex and geographic origin, and both groups were genotyped. In order to test the quality of the original genotypic data [10], DNA available from the initial cohort was genotyped again.

quencies between centenarians and controls were formed using (cid:1)2 contingency tests.

3. Results

Centenarians and controls were paired for two param- eters: sex and geographic origin. Both pairings were used to improve the likelihood that both members of the pair originate from the same population. Due to the large age difference (around 50 years) between cases and controls, it appeared desirable to match, whenever possible, the regional place of birth of the centenarian with that of both parents of the control individual. It was thus possible to match 397 centenarians and controls (71 % of the two groups) on the basis of administrative regions. When such pairing was not possible, the matching region was enlarged to one of the following five multiregional areas (Île-de- France, north-west, north-east, south-west and south-east France). This procedure enabled matching of the remain- ing 163 centenarians.

2. Materials and methods

2.1. Studied populations

Centenarians were recruited when they were in their 100th year or beyond, after informed consent. Among the 932 centenarians for whom DNA is available for genotyp- ing, each member of a group of 560 centenarians was matched for sex and geographic origin with a healthy control individual. Each group was composed of a total of 466 females and 94 males. The age of control individuals ranged from 18 to 70 years (mean age, 51.2 years). The mean age of centenarians was 103.1 years (age at death or actual age). The geographic origin in France of males and females of both groups is shown in table I.

The groups of centenarians and controls thus defined were genotyped for the APOE and ACE I/D polymor- phisms. In order to develop a set of high quality genotypic

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Table II. Genotype distribution of the APOE polymorphism and statistical analyses.

and 0.70, respectively). Thus, the data from both sexes for each group were pooled for further statistical analysis. Significant differences were observed between centenar- ians and controls for allelic and genotypic frequencies (P values both < 10–7). This statistically significant difference was also found when genotypes E2E2 and E2E3 on the one hand and E3E4 and E4E4 on the other hand were pooled to restrict bias due to the small size samples (P value < 10–8). The E4E4 genotype was under-represented in centenarians compared to controls (Fisher exact test, P value = 0.03). Among E4 heterozygotes, the ratio of E3E4 to E2E4 geno- type was smaller in centenarians compared to that of controls (4.2 % versus 17.6 %, P = 0.002).

Genotype Number of Number of controls centenarians (frequency) (frequency) E2, E2 4 (0.7 %) 2 (0.4 %) E2, E3 104 (18.6 %) 71 (12.7 %) E2, E4 13 (2.3 %) 7 (1.2 %) E3, E3 385 (68.8 %) 351 (62.7 %) E3, E4 54 (9,6 %) 123 (22.0 %) E4, E4 0 6 (1.0 %) Statistical analyses Hardy-Weinberg P = 0.06 P = 0.28 equilibrium Comparison of allelic P = 1.2 × 10–8 frequencies Comparison of genotypic P = 3.4 × 10–8 frequencies

Most of the centenarians belonging to the 1994 cohort (309 individuals [10]) were again genotyped for APOE. Comparing the new and previous data identified 18 incon- sistencies, which were rechecked and confirmed in the laboratory, leading to a discordance rate of 5.8 % for the centenarian data.

data, the following procedures were used. Control DNAs of known APOE and ACE genotypes were systematically added to each microtitre-plate of DNA from subjects and controls. Genotypes were scored from gel electrophoretic profiles, independently, by two different investigators, blinded to the status (subjects or controls) of the individu- als represented by the DNA samples. When misinterpre- tation of a specific allele was suspected, the experiment was repeated.

ACE allele frequencies were homogeneously distrib- uted between gender in both populations of centenarians and controls (P value for allele frequencies equals to 0.16 and 0.31, respectively) as were genotype frequencies (P value equals to 0.39 and 0.43, respectively). Thus, statis- tical analyses were performed using pooled data from both genders. Allele and genotype frequencies for the ACE I/D polymorphism are shown in table III. The relative frequen- cies of the II, ID and DD genotypes were not significantly different from values predicted by Hardy-Weinberg equi- librium in centenarians and controls (table III). Compari- son of allelic and genotypic frequencies failed to reveal a statistically significant difference between the centenarian and the control populations.

Genotype frequencies of the control group were in Hardy-Weinberg equilibrium for both loci (tables II and III for APOE and ACE, respectively). Allelic frequencies at the ACE and APOE loci observed in the control group were not significantly different from those reported in the ECTIM study (Étude cas témoins sur l’infarctus du myocarde, ’Case-control study for myocardial infarction’) a large epidemiological study performed in the French popula- tion [6, 21]. Genotype frequencies for the APOE locus are reported in table II. Distribution of genotypes in the cen- tenarian population was in agreement with the Hardy- Weinberg expectation although the P value was border- line (P = 0.06).