Danish study population German study population Cases Controls Cases Controls Number, LLI 1383/1089a 736 1613 1104 Women, LLI (%) 70.7/71.3a 49.6 73.0 74.4 Age at intake, LLI 92–93 46–55 95–110 60–75 Number, centenarians 190/143a — 748 — Women, centenarians (%) 80.0/79.0a — 80.5 —

Study approvals were received from the Danish National Committee on Biomedical Research Ethics and the Ethics Committee of the University Hospital Schleswig-Holstein, respectively.

Abbreviation: LLI, long-lived individuals. aThe number to the left applies to rs3803304.

Genotyping

The primary SNP for association testing was rs3803304. In addition to this, six supplementary variants, which had previously been selected for coverage of the common genetic variation in AKT1 using data from the International HapMap

As rs3803304 was previously found to be associated with longevity,9 it was investigated more thoroughly using a genotypic, dominant and recessive model. No significant associations were found with any of the models applied (data not shown).

Genotyping of rs3803304 in the Danish study population and of rs3803304, rs2494731, rs2494732, rs2498796, rs2494738 and rs1130214 in the German study population was performed using predesigned TaqMan SNP genotyping assays (Applied Biosystems, Pleasonton, CA, USA). The SNP rs2494748 could not be genotyped in the German population because of technical difficulties.

To scrutinize for potential effects of variation in AKT1 on extreme survival, the analysis was next restricted to include centenarians only (Table 3), resulting in the ability to detect effects of size OR¼1.25 (for rs2494738, OR¼1.4) in the combined study population with a power of at least 80%. One nominally significant indication (P¼0.03) of a disadvantageous effect of rs3803304 on longevity was found in Danish men, and this indication was supported when applying genotypic and dominant models (P¼0.02 and 0.01, respectively).

Genotyping of rs2494731, rs2494732, rs2498796, rs2494738, rs2494748 rs1130214 in the Danish study population was part of a previous study was performed using the Illumina GoldenGate Technology (Illumina Inc., Diego, CA, USA) as described by Soerensen et al.13

Statistical analysis

In addition to applying a cross-sectional approach, the effects of the seven SNPs on survival during old age were investigated in the Danish 1905 Birth Cohort. Regardless of the genetic model applied, no associations with survival during the follow-up of 412 years were found. Restriction of analysis to include centenarians only did not change this result (data not shown).

All analyses were carried out using the statistical software Stata (Stata version 11.2; Stata Corporation, College Station, TX, USA). Logistic regression was used to assess the association between genotype and longevity with dose of minor allele coded as 0, 1 and 2. An additive model was applied to all of the seven AKT1 SNPs; however, for rs3803304, the potential association with longevity was further explored using a genotypic, dominant and recessive model.

Mortality analysis in the Danish 1905 Birth Cohort was performed on all of the seven AKT1 SNPs using a left-truncated Cox proportional hazards model to adjust for late entry into the data set according to age.

DISCUSSION

AKT1 has recently been suggested as a third universal longevity gene, together with APOE and FOXO3. To further elucidate this potential role of AKT1, case–control studies involving Danish and German long-lived individuals and younger controls were performed. In addition to the previously described candidate variation rs3803304,9 our study was expanded to include a number of additional SNPs to ensure better coverage of the known allelic variance in AKT1.

To account for the multiple testing of seven SNPs, a Bonferroni-corrected

Power calculations were performed using the freely available software Quanto (Quanto version 1.2.4, http://hydra.usc.edu/gxe/), assuming a 1 in 20 chance of becoming 92–93 years old, a 1 in 400 chance of becoming a centenarian and a Bonferroni-corrected significance level of 0.007.

RESULTS

The main finding of this study was a lack of formal replication of the association of rs3803304 with longevity as previously found in three independent populations.9 In the initial report by Pawlikowska et al,9 they found that the association with longevity appeared driven by the minor allele homozygous genotype, as subjects homozygous for the minor allele were underrepresented among long-lived cases. In line with this, restricting the analysis to centenarians revealed a trend of supportive evidence in Danish men, with the same direction of effect as identified before.9 It should be noted, though, that the sample size of the refined group of centenarian men was very limited (n¼ 37), and when applying a Bonferroni correction for multiple statistical testing, the association did not remain significant.

Characteristics of the Danish and German study populations summarized in Table 1.

All genotyped SNPs were in Hardy–Weinberg equilibrium in both control groups, except for rs2494738, which displayed an excess of heterozygotes in the Danish control group (P¼0.02).

The set of tested AKT1 SNPs included the candidate rs3803304 and six additional SNPs, of which three are tagging (rs2494731, rs2494738 and rs1130214). The remaining (rs2494732, rs2498796 and rs2494748) are distributed evenly the gene and were added to enable a more comprehensive coverage the gene.

To facilitate a more exhaustive exploration of the possible relevance AKT1 variations in longevity, six additional SNPs available from a study13 were included and tested in both the Danish and study population. Of note, one of these, rs2494731, is in linkage disequilibrium with the AKT1 SNP rs2498804, which recently found to be associated with lifespan in a population Dutch nonagenarians.3 Nevertheless, we did not replicate an with longevity, whether applying a cross-sectional