Statistical analysis

Quality control: Individuals showing a genotyping rate <93% and SNPs with a genotyping rate <95% were excluded from the dataset. We screened for contaminations and evaluated relationships among individuals using identity by descent (IBD) estimations. Samples showing extreme heterozygosis and related individuals were removed. X chromosome data were used to check for discordances in terms of gender assignment. SNPs with a minor allele frequency (MAF) <0Degree of freedom (df) was used to apply the Pearson chi-squared test (χ2) and Fisher exact test (if cell counts were less than 5) to compare genotype frequencies between cases and controls. Logistic regression was also performed under an additive model and by comparing homozygous minor versus homozygous major alleles. Correction for population stratification was performed using logistic regression under an additive model adjusting along the top four statistically significant principal components. Allele frequencies, genotype counts, odd ratios (OR), and 95% confidence intervals (95% CI) were estimated for each SNP. Logistic regression analyses were also performed assuming an additive model and by comparing homozygous minor versus homozygous major alleles on a set of SNPs located at the FOXO3A locus (Table 2).

To confirm the null influence of population structure on our results, we also performed association tests correcting along the top four principal components by means of logistic regression assuming an additive effect of allele dosage on the same SNP set. We first focused on rs2802288, showing linkage disequilibrium (LD) with rs2802292 (r2 ≥ 1) in the HapMap CEU data panel. Particularly, allelic association tests highlighted significant association for rs2802288 in males (MAF 0.49; p 0.003; OR 1.51; 95% CI, 1.15-1.98). Moreover, this association was also confirmed by logistic regression under an additive model (p 0.0026; OR 1.53; 95% CI, 1.16-2.03) and by comparing homozygous minor versus homozygous major alleles (p 0.0023; OR 2.44; 95% CI, 1.37-4.34) (Table 2). Using this analysis, multimarker association tests showed a slightly significant association (p 0.03) for a 24kb haplotype (Fig. 2, block 1; Table 3) comprising three SNPs carrying the minor allele (A) for rs2802288, and defining boundaries of a locus affected by a potential causative mutation for the extreme longevity phenotype. As can be observed in Fig. 2, the LD plot defines a locus that includes the 5'-untranslated regions and the first coding region of the investigated gene.

Subsequently, to confirm the gender-specificity of the FOXO3A association, we also studied 199 female longevity cases (mean age 98 years) and 140 female controls (mean age 31 years) drawn from the same population (88% power to detect the association). No differences in terms of allele frequencies between centenarians and controls were detected for rs2802288 in female individuals (176 centenarians and 113 controls that pass the quality control filtering criteria) (MAF <0.48; p >0.3975; OR <0.86; 95% CI, 0.62-1.21) (Table 2). The absence of association in the female gender has been further confirmed by logistic regression assuming an additive model (p >0.4034; OR 0.87; 95% CI, 0.62-1.21) and by comparing homozygous minor versus homozygous major alleles (p >0.4048; OR 0.75; 95% CI, 0.39-1.47) (Table 2).

Imputation analysis was performed using IMPUTE2 software to represent the 6q21 region better. Interestingly, the imputed rs2802292 (MAF 0.47) that was previously indicated by Willcox et al. as the best association showed r2 ≥ 0.79 with rs2802288 (MAF 0.49), similar to that observed in the HapMap CEU panel. In fact, our imputation analysis confirmed that rs2802292, unrepresented on the BeadChip, was responsible for the strongest association effect in this region (MAF 0.468; p 0.0028; OR, 1.53; CI 95%, 1.16-2.03, allelic association test). Additionally, we confirmed this significant association by logistic regression under additive model (p 0.0022; OR, 1.57; CI 95%, 1.17-2.10) and by comparing homozygous minor versus homozygous major alleles (p 0.0019; OR, 2.58; CI 95%, 1.42-4.70) (Table 2).