Discussion

In a recent case-control study of U.S. Caucasians, a two-SNP- haplotype in the MTP gene on chromosome 4q was implicated as a modifier of human life span (5). In the same publication, however, the authors reportedly failed to confirm their findings in a much larger sample from France, raising concerns about the validity of the original association. To critically evaluate these u>1 results, we have performed a large-scale study on 1,000 German nonagenarians and centenarians and 550 matched controls. The validity of our study population for longevity research has been supported by an examination of the apoli- MEE) 62 alert63  poprotein E (APOE) 2, 3, and 4 haplotypes, which revealed   an increased frequency of 2 in LLI when compared with   controls (0.118 vs. 0.085; OR [CI] 1.45 [1.12–1.86]) (OR, odds ratio; CI, confidence interval) and a concurrently decreased 64   frequency of 4 (0.071 vs. 0.145; OR [CI] 0.45 [0.36–0.57]). These results agree with other longevity studies and corroborate both the validity and efficacy of our study sample. Assuming an MTP risk factor frequency of 0.2 and a relative risk of 1.4 (characteristic values of the U.S. samples), our study sample >8 would have had a power 80% to replicate the reported

Table 3. Frequency of MTP ‘‘risk’’ haplotype rs2866164-G, MTP Q95 in German, French, and U.S. Caucasian LLI and controls

Sample (no. of haplotypes) Frequency Germans LLI (2,036) 0.217 Centenarians (728) 0.224 Controls (1,066) 0.195 French LLI (1,126) 0.202 Controls (1,121) 0.197 U.S. Caucasians LLI, sample 1 (346) 0.176* Controls, sample 1 (364) 0.286 LLI, sample 2 (488) 0.193* Controls, sample 2 (462) 0.253

Second, inappropriate matching of cases and controls under even modest levels of population structure not only can lead to false positives but may also cause false-negative findings (15). Although the German control individuals were selected to ensure they were as close as possible to the LLI in terms of their age and geographic origin, systematic genetic differences be- tween these samples cannot be completely excluded. Thus, latent population structure may have been responsible for the failure of the present study to confirm the association observed in the Americans, but this would of course not explain the negative finding in the French samples. Another reason for the lack of replication in both European samples could have been the higher proportion of males in the U.S. LLI. Men reach 100 years of age less often than women. It would therefore seem plausible that particular risk alleles impact more upon male than female

Data on French and U.S. Caucasian samples are from Geesaman et al. (5). *HaplotypefrequencydifferencesbetweenAmericancasesandcontrolswere reported as statistically significant by the same authors. The second U.S. Caucasian sample represents proactively matched LLI and controls.

mortality, so that these alleles are more strongly underrepre- sented in male than female centenarians. However, no gender- specific effect has been observed in the German samples.

however, currently available evidence suggests that the putative association observed in the Americans (5) was due to recent changes in the genetic structure of the U.S. Caucasian popula- tion as a whole, rather than to genetic effects upon survival to old age. Although the known function of MTP in lipid metab- olism renders it a plausible candidate for human longevity, the reported association appears likely to have been artifactual. The study by Geesaman et al. (5) thus highlights the potential pitfalls that arise when a case-control design is used as a means to map longevity genes in humans. Such studies would generally be hampered by the inherent difficulty of appropriately matching case and control samples simply because, if living individuals are compared, they were born generations apart. Our own study also emphasizes the importance of replicating initial association findings in very large samples from different populations, espe- cially for complex phenotypes in which genes with only weak or moderate effects are likely to play a role.

Third, the observed discrepancy between the American con- trol individuals and both European populations could reflect differences brought about by recent changes of the selective forces acting upon the MTP gene in the U.S. Caucasian popu- lation. During the first half of the 20th century, U.S. Caucasians became one of the most advanced societies in the world, with medical, technological, and sociological improvements that had not been experienced by earlier generations (represented by the U.S. LLI) or by contemporary Europeans. In large populations, even small changes of selective pressure can create considerable changes in allele frequencies over relatively short periods of time (16). Because it is not known whether MTP has functions (other than those in lipid metabolism, for instance, in reproductive or cognitive processes) that would render it a suitable target of natural selection, the type of selective pressure that might be involved remains undefined at present.

Fourth, as originally pointed out by Geesaman et al. (5), strong environmental factors, such as diet, that modify the influence of a risk allele in different populations may have contributed to the observed inconsistency between studies. However, because the main difference between the Americans and the Europeans applies to the MTP allele and haplotype frequency in the control individuals, this explanation seems unlikely.