DNA was isolated from cheek swabs and blood spots with the use of QIAamps DNA Mini Kit (Qiagen, Germany). The Taqman technology was used to genotype for the two polymorphisms related to MTP. Primers and probes were s designed using Primer Express software (Applied Bio- systems). Primers and probes used were rs2866164: sense 50-GTTTAAACAAGAAATCTCAAACCATATCTAC-30, antisense 50-CTTGTAAATATAGTATATTTACCATATCTTAGT AAGTAAA-30, C-probe 50-FAM-CTGTTTACCAGACTGCTT, G-probe 50-VIC-CTGTTTAGCAGACTGCT and MTP Q/H: sense 50-CTTTCTGTTACTCCAGATGAAGGATGT-30, anti- sense 50-TTTCCCATTATTTTAGATGGGCTTT-30, Q-probe 50-FAM-CTCTCCTCTCTGCTGATT, H-probe 50-VIC-CT CTCCTCTGTGCTGATT. All probes contain minor groove binder groups and a nonflourescent quencher attached to their 30 ends. The real-time analyses were performed at the ABI PRISM 7700 Sequence Detection System equipped with the allelic discrimination module (software version 1.7; Applied Biosystems). DNA was amplified in a total volume of 10ml containing 5ml Taqman Universal Master Mix (Applied Biosystems), 900nM of each primer (DNA Tech- nology), 200nM of each probe (Applied Biosystems) and B 10ng DNA at an annealing temperature of 601C and 40 PCR cycles. In total, 26 subjects from the 1905 cohort and 24 of the middle-aged twins could not be genotyped for one or both polymorphisms due to a poor quality of DNA. Only subjects that could be genotyped for both poly- morphisms are used in the statistical analysis. Four subjects gave unclear genotyping results for H95Q and exon three in the MTP gene was sequenced. A missense mutation under the probe, E98D, was found in these samples. Only three of the expected four haplotypes were seen as expected, since the rs2866164-C/H95 haplotype has not previously been detected.

It is important to point out that the finding by man et al (2003) was not confirmed in the French reported in the same paper. Given this discrepancy, there a need for independent studies to confirm the association between MTP and exceptional longevity large and well-defined samples and preferable studies. In the Danish 1905 cohort study, we assessed complete Danish 1905 cohort in 1998.10,11 In total, participated in the survey, which included an physical and cognitive tests, as well as collection biological material (1651 participants provided a sample). Hence, they were 92–93 years when they the study and have been followed regarding mortality January 2005. We examined the two reported SNPs in complete Danish 1905 cohort study. If MTP does play a role in human longevity, a longitudinal study F nonagenarians a population with a very high F and 6.5 years of follow-up should be able to detect mortality difference. To rule out that a selection has place before the age of 92 years, a younger control group 575 middle-aged Danish twins (mean age 53.7 years) included.

Statistical analyses

The participants were followed from the date of blood sampling and until emigration, death or end of study period (January 2005). Information on emigration or death was retrieved from the Danish Central Population Register, which is continuously updated. The Kaplan–Meier method was used to plot cumulative survival curves. For the survival analysis, the subjects were stratified by the

Methods

The participants were selected from two major surveys conducted by our group, the 1905 cohort, all Danes born in 1905 (1651 DNA samples)10 ascertained in 1998

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

The difference in distribution of the risk alleles/haplo- type (rs2866164-G, MTP-Q95 and the haplotype composed of rs2866164-G/MTP-Q95) between the two age cohorts was tested with a critical ratio (Z) test, which calculates a 95% confidence interval of the difference between the two populations and a two-sided P-value.13,14 Stata version 8 (Stata Corporation) and EpiCalc 2000 (version 1.02; Gilmann and Myatt, Brixton, England) were used for statistical analysis.

Results

The Kaplan–Meier cumulative survival curves for the nonagenarian males and females are shown in Figures 1 and 2. Age is not a confounding factor as all participants are born in 1905. There is no significant survival difference, as tested by the logrank test, despite the large sample size and although more than 83% deceased at the end of the follow-up period; the P-values are 0.56, 0.31 and 0.97 in the total population of nonagenarians, males and females, respectively.

Figure 2 Kaplan–Meier survival estimates in females from the 1905 cohort study (N¼1168). 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 (P¼0.97).

Table 1 Risk SNP allele counts in males

SNP Middle-aged 1905-cohort Survivors 1905 rs 2866164-C 394 (74.6%) 698 (76.4%) 82 (77.4%) rs 2866164-G 134 (25.4%) 216 (23.6%) 24 (22.6%) MTP Q95 515 (97.5%) 874 (95.6%) 104 (98.1%) MTP H95 13 (2.5%) 40 (4.4%) 2 (1.9%)

The genotyping results are shown in Tables 1–3. The distribution is in Hardy–Weinberg equilibrium for both SNPs and haplotypes in both age groups (PZ0.29). None of the prevalence differences of the suggested risk alleles (rs2866164-G and MTP Q95) and the combined risk haplotype is significant on a 0.05 level (Tables 1–3). Furthermore, there is no statistically different distribution between the subjects from the 1905 cohort alive at the last follow-up in January 2005 (6.5 years after recruitment) and those who died before January 2005.

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

SNP Middle-aged 1905-cohort Survivors 1905 rs 2866164-C 442 (77.0%) 1727 (73.9%) 322 (72.9%) rs 2866164-G 132 (23.0%) 609 (26.1%) 120 (27.1%) MTP Q95 546 (95.1%) 2236 (95.7%) 423 (95.7%) MTP H95 28 (4.9%) 100 (4.3%) 19 (4.3%)

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