1.00 0.75 0.50 0.25 0.00 0 500 1000 1500 2000 2500 analysis time haplotype = 11 haplotype = 12 haplotype = 22

Shown are counts (frequencies) for rs2866164 and MTP Q/H.

Discussion

In contrast with the previous publication in PNAS by Geesaman et al,7 our data fail to provide evidence for a role for MTP in human longevity. There is no increased mortality among carriers of the ‘risk haplotype’ composed of the rs2866164-G and the Q95 allele in the longitudinal study of 1651 nonagenarians and, furthermore, there is no significant difference between the distribution of the two SNPs and the ‘risk haplotype’ in the two tested age cohorts. The paper by Geesaman et al,7 included a confirmatory study of a French centenarian cohort compared to younger French controls. There was no significant difference in allele frequencies with respect to the risk haplotype. However, the risk haplotype was found to be out of

Figure 1 Kaplan–Meier survival estimates in males from the 1905 cohort study (N¼457). Haplotype 2 is the suggested risk haplotype composed of the rs2866164-G and the Q95 allele and haplotype 1 is all other haplotypes. The analysis time is days. The difference is not significant (P¼0.31).

Table 3 Haplotype counts and frequencies

Middle-aged 1905 cohort Survivors 1905 Allele Rs2866164-C rs2866164-G rs2866164-C rs2866164-G rs2866164-C rs2866164-G Males Mean age: 55.5 years Age 92–93 years Age 99 years Q95 394 (74.6%) 120 (22.7%) 698 (76.4%) 176 (19.2%) 82 (77.4%) 22 (20.8%) H95 0 14 (2.7%) 0 40 (4.4%) 0 2 (1.9%) Females Mean age: 52.1 years Age 92–93 years Age 99 years Q95 442 (77.0%) 104 (18.1%) 1727 (73.9%) 509 (21.8%) 322 (72.9%) 101 (22.9%) H95 0 28 (4.9%) 0 100 (4.3%) 0 19 (4.3%)

Shown are counts (frequencies) for the two-SNP haplotype formed by combining alleles of the two SNP’s in MADT and the 1905 cohort. The 1905 survivors are the participants still alive January 2005. Only three of the four haplotypes were observed.

Hardy–Weinberg equilibrium with the other haplotypes in the centenarians, but not in the controls. We find frequencies in our middle-aged population very close to F the French control group the frequency is 24.1% for rs2866164-G compared to 26.4% in the French population and for the ‘risk haplotype’ the frequency is 20.1% compared to 19.6%. The risk haplotype is in Hardy– Weinberg equilibrium in our cohorts for both the indivi- dual SNPs and the combined haplotypes.

Our population is slightly younger than that of Geesa- man et al.7 Their long-lived individuals had a mean age of 100.8 years. Participants in our study population were 92–93 years when they entered the study and 99 years at the end of follow-up - and intuitively this seems to be an already much selected population, close to becoming centenarians. However, being 92–93 is only ‘half way to becoming a centenarian’ in terms of selection: only about one in 20 of the 1905 cohort made it of age 92–93, but it is also only about one in 20 of these survivors to age 92–93 who will celebrate a 100-year birthday. Hence, the Danish 1905 cohort provides a powerful opportunity to test for selection against a genotype marker and even though our cases are slightly younger, this large study of 1651 subjects aged 92–93 years should be able to detect a significant difference. As can be seen from Figures 1 and 2, there is no significant survival difference between those carrying the suggested risk allele and those without this allele. In males F F there is even a small not significant tendency to a better survival of those homozygous for the suggested risk allele.

One explanation for the discrepancy between the study by Geesaman et al,7 and our data could be that the American population is less homogeneous than ours. A major problem in genetic association studies is the failure to adequately match the genetic backgrounds of the cases and controls, and in the study of Geesaman et al, the cases and controls were born more than 50 years apart. The Danish population is known to be genetically and ethnically very homogeneous and therefore well suited for association studies in different age groups. The middle- aged twins were born and grew up in Denmark. Very little immigration has taken place in Denmark in the years between 1905 and 1960. The minimal immigration into Denmark in the cohorts comprised by these surveys, the high response rate in both surveys (63% in the 1905 cohort study and 80% in the middle-aged twins) and the high genetic homogeneity in the Danish population, minimize population stratification as well as selection bias within the population. Furthermore, we have the ability to conduct a longitudinal study on nonagenarians as we have followed the 1905 cohort for 6.5 years, which is not affected by any population stratification. And the results all point in the F same direction no haplotype-dependent mortality differ- ence in the 1905 cohort and no significant difference in frequency of the tested SNPs between the middle-aged population and the 1905 cohort. However, although the Danish population is very homogeneous, no cross-genera- tional comparisons are necessarily immune from stratifica- tion and it is therefore possibleF but very unlikelyF that our results are false-negative due to stratification.