Results

Genotype and allele frequencies

The FOXO3A SNPs investigated in this study and their relative positions and linkage disequilibrium (LD) blocks are shown in Fig. 1. All 15 FOXO3A SNPs were in Hardy–Weinberg equilib- rium in the control group (P > 0.05⁄15 SNPs) (data not shown). The genotype and allele frequencies of the 15 SNPs for middle- aged controls, nonagenarians, centenarians, and the combined group of oldest old (nonagenarians + centenarians) are listed in Supporting information Table S1. The frequencies of the rare alleles and rare genotypes generally tended to be higher in the oldest old when compared to the middle-aged controls. One exception was rs13220810, where the frequency of the most common allele and genotype was higher in the oldest old than in the controls. The frequencies for the nonagenarians were in general in between the frequencies for the middle-aged control group and the centenarians, supporting an increase in allele and genotype frequencies with age. Stratification by sex indicated that these tendencies were most pronounced for males (data not shown).

To correct for multiple testing, while taking into account the LD between the individual SNPs, permutation analysis was per- formed using the Plink software. Data found to be nominally sig- nificant, and the corresponding corrected values are listed in Supporting information Table S3, while the results holding for correction for multiple testing are listed in Table 2. Of the nomi- nally significant findings (both genders combined), the PCCG values for rs7762395 did hold for correction for multiple testing, so did the PCCGs for rs12206094, rs13217795, rs13220810, rs2764264, rs479744, rs7762395, rs9400239, and rs9486902 when restricting to males. None of the nominally significant

haplotypes and longevity - Case–control

Three haplotype blocks were predicted in the gene region analyzed (see Fig. 1); block 1: rs9486902, rs10499051, rs12206094, rs2802292, and rs13220810, block 2: rs2764264, rs7762395, rs12207868, rs13217795, rs9400239, rs12212067, and rs9398172, and block 3: rs3800232. To test the association with longevity of these haplotype blocks in our sample, we used these haplotype definitions in Plink and found nominally significant differences for block 1 between the oldest old and the controls; haplotype CACTC, P = 0.03 and haplotype TACTT, P = 0.008 (both genders combined). To

FOXO3A SNPs and longevity – Case–control analysis

P-values for the allelic frequency case–control comparisons (PCCA) and the genotype frequency case–control comparisons (PCCG) between the middle-aged and oldest old cohort are listed in Supporting information Table S2; results for nominally significant effects (P < 0.05) are listed in Table 1. Nominally sig- nificant differences in allele frequencies were found for the males for five SNPs (rs12206094, rs13220810, rs2802292, rs3800231, and rs9486902), while with respect to genotype

Block 1 Block 2 Block 3 )¤( 2096849sr )*◊/‡(15099401sr 49060221sr )¥/*◊/‡/#( 2922082sr )◊(01802231sr )‡/#( 4624672sr )◊/*¤/*‡( 5932677sr )*◊( 86870221sr )‡/#( 59771231sr )◊( 9320049sr 76021221sr )* ¤( 2718939sr )◊(1320083sr 2320083sr )◊( 447974sr a re ees ee es Oa tactic LL. L£ Bhai 

Males and females Males N (oldest old) = 1089, N (oldest old) = 313, N (controls) = 736 N (controls) = 371 No. dbSNP ID PCCG PCCG PCCA OR 95%CI 1 rs9486902 0.040† 0.0083 0.0118 1.40 1.07–1.83 3 rs12206094 0.032† 0.0038 0.0076 1.38 1.08–1.75 4 rs2802292 >0.05 0.0211 0.0078 1.34 1.08–1.67 5 rs13220810 0.046(cid:2) 0.0133 0.0223 0.75 0.58–0.96 6 rs2764264 >0.05 0.0078 >0.05 7 rs7762395 0.012 0.0031 >0.05 9 rs13217795 >0.05 0.0215 >0.05 10 rs9400239 >0.05 0.0098 >0.05 12 rs9398172 >0.05 0.0435 >0.05 13 rs3800231 >0.05 0.0373 0.0495 1.26 1.00–1.59 15 rs479744 0.048 0.0026 >0.05

Only nominally (P < 0.05) significant PCCA (P-values obtained from allele based case-control comparison) and PCCG (P-values obtained from genotype based case-control comparison) are included in the table. N, number of individuals in group; OR, odds ratio; 95% CI, 95% confidence interval; NA, not analyzed (since no homozygotes (rare genotype) were found). †When assuming a recessive model. (cid:2)When assuming a dominant model.

Males and females Males N (oldest old) = 1089, N (oldest old) = 313, N (controls) = 736 N (controls) = 371 PCCG uncorrected PCCG corrected PCCG uncorrected PCCG Genotype model 3 rs12206094 >0.05 0.0038 0.0180 6 rs2764264 >0.05 0.0078 0.0309 7 rs7762395 0.012 0.080 0.0031 0.0219 10 rs9400239 >0.05 0.0098 0.0320 15 rs479744 0.048 0.294 0.0026 0.0070 Recessive model 1 rs9486902 0.04 0.219 0.0060 0.0269 3 rs12206094 0.032 0.186 0.0011 0.0029 6 rs2764264 >0.05 0.0020 0.0059 7 rs7762395 0.003 0.024 0.0006 0.0049 9 rs13217795 >0.05 0.0050 0.0249 10 rs9400239 >0.05 0.0020 0.0069 15 rs479744 0.008 0.095 0.0006 0.0019 Dominant model 5 rs13220810 0.046 0.985 0.004 0.026

rs10499051, rs12006094, rs2802292; rs12006094, rs2802292, rs132202810 and 2802292, rs132202810, rs2764264, respec- tively). When stratifying by gender, males showed, in addition to window 1–4, also significance for window 5 (rs13220810, rs2764264, and rs7762395) and 13 (rs3800231, rs3800232, and rs479744). As before, no effects were nominally significant for the females.

investigate the haplotype structure in more detail, the ‘sliding window’ feature in the Plink software was applied (using a window of three SNPs). Haplotype frequencies and P-values from the haplotype frequency based case-control comparison (PCCH) between the middle-aged and the oldest old are listed in Supporting information Table S4, whereas data being nomi- nally significant and data passing correction of multiple testing are shown in Table 3.

When correcting for multiple testing, one window (and two haplotypes) did hold for correction: window 1 (rs9486902, rs10499051, and rs12206094) haplotypes TAC and CAC were found to be significant for males only (see Table 3).