Table 5 Interaction between APOE and ACE in centenarians 

ACE has functions besides its role on the renin-angiotensin and kallikrein-kinin systems. Its ability to cleave neuropeptides such as enkephalin, substance P, and LHRH, and its regional distribution in the brain point to neuroendocrine functions. Furthermore, an increase with age in brain cortical ACE activity suggests that an adaptive response to increasing needs may occur during ageing. ACE may also function as an immunomodulator by helping to process endogenous peptides within the MHC class I complex in cytotoxic T lymphocytes, and its level is also associated with the I/D polymorphism in these cells. These data indicate potential roles of ACE outside the cardiovascular system that may well influence survival. Insofar as the quantity of ACE enzyme is a significant parameter in these other functions, they provide a rationale for understanding the differential influence on survival of the ACE-I/D polymorphism. However, the association with longevity may also stem from linkage disequilibrium with another influential polymorphism in a nearby gene. 

In this regard, it is notable that the gene for human growth hormone, which may have an important role in senescence, shows strong linkage to ACE, on chromosome 17q23 (ref. 26).

The frequencies of APOE alleles are shown in parenthesis for each genotype at the ACE locus. The visible trend of negative correlation between the e2 and D alleles does not reach significance (p=0.09 for a comparison of the 2 allele frequency between DD and II genotypes), Framingham (USA)* and Minster (West Germany). Although a decrease in the 4 allele frequency was found in Canadian octogenarians, it was not as marked as in our centenarians, and there was no significant change in the 2 allele frequency; on the other hand, the increase of the 3 allele frequency in females was less pronounced in our group of centenarians. Another study*> reported a decreased frequency of 4 in older female diabetic patients.

The well-documented impact of ApoE-e4 on cardiovascular risk is commonly attributed to its hypercholesterolaemic effect; the 2 allele has opposite effects on total and LDL-cholesterol, which has led to the suggestion that it might protect against atherosclerosis. However, it also has a hypertriglyceridaemic effect and may be a risk factor for ischaemic heart disease in certain environments. Thus, while the lower frequency of ApoE-e4 in centenarians is consistent with its risk factor status for heart disease, the increase in 2 allele frequency is a more unexpected result. If 2 and 3 were neutral, with respect to survival, the decrease in 4 frequency would result in an evenly distributed increase in the two other allele frequencies, whereas in fact, the entire increase is borne by 2, adding to the evidence for a protective effect of this allele.

The same questions arise for ApoE, which lies in the apolipoprotein cluster in chromosome 19q13.2 (ref. 32). There is compelling evidence for a direct effect of the e4 allele: ApoE plays a role in nerve development and repair; it is expressed in large quantities in the brain, where it binds to B-amyloid with an affinity that is highest for the e4 isozyme; and the 4 allele has an increased frequency in late-onsetIn order to examine possible associations in populations of different ethnic backgrounds residing in different environments, it is important to verify the following methods. Firstly, the ACE diallelic polymorphism was genotyped by amplifying the variable segment and resolving the 190 and 490 bp alleles on a 1.5% agarose gel. Secondly, APOB polymorphisms were analyzed by amplification of the two DNA segments, direct visualization of the amplified product on a 10% non-denaturing polyacrylamide gel for ins/del, and visualization of the Xbal digestion products on a 1.5% agarose gel for the Thr substitution. Thirdly, Southern hybridization with probe 674 and Accl digested samples revealed the polymorphism at D4S95. Fourthly, BCL-2 microsatellite alleles were typed using primers 5'-TGTGTGTGTGTAGCGCGTGT-3' and 5'-CTGGCCGTGTGAGTGTGTGT-3' for amplification in a reaction mix containing 200 ng genomic DNA, 10 pmoles of each primer, and 0.75 U of Taq polymerase in a final volume of 50 pl. The alleles were revealed by Southern hybridization with a (CA) probe after resolution of the fragments on a 10% denaturing polyacrylamide gel. 

Methodology: Centenarians were recruited from all over France (regional distribution: 40% Paris region, 18% central, 4% northern, 5% eastern, 15% western, 18% southern), at their home or in institutions, through media advertising and retirement organizations. They were all Caucasians residing in France. The criterion for inclusion in the study was being at least 99 years old on the day of blood collection. The population of 338 centenarians was composed of 11% men and 89% women with a mean age of 100.71 + 0.13. Controls were chosen among unrelated normal individuals from French CEPH pedigrees.

Acknowledgements: We thank Fondation IPSEN, ARRCO, AGIRC, and the participating clinicians as well as E. Snard and J. Boirot for help in collecting the centenarians, F. Lethrosne, F. Dufour, C. Habita, M. Legrand, L. Gressin, C. Massart, C. Billon for technical assistance, M.R. Hayden for probe 674; F. Cambien, T. Kirkwood, and R. Moulias for support and discussion.

Genotypes: APOE genotypes were analyzed by Hhal digestion of a 244 bp PCR-amplified fragment containing the two polymorphic sites, with the restriction fragments resolved on a 10% non-denaturing polyacrylamide gel. 

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