of population structure on our results, we also performed as- sociation tests correcting along the top four principal com- ponents by means of logistic regression assuming an addi- tive effect of allele dosage on the same SNP set. We first focused on rs2802288, showing linkage disequilibrium (LD) with rs2802292 (r2 —e 1) in the HapMap CEU data panel.33 Particularly, allelic association tests highlighted significant  om) = ( 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  ~“o model (p 0.0026; OR 1.53; 95% CI, 1.16–2.03) and by comparing homozygous minor versus homozygous major oO) = ( mS 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 haplo- type (Fig. 2, block 1; Table 3) comprising three SNPs carry- ing the minor allele (A) for rs2802288, and defining bound- aries 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 -untrans- lated 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 de- tected for rs2802288 in female individuals (176 centenarians and 113 controls that pass the quality control filtering crite- ria) (MAF week’ 0.48; p me) = ( 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 y= ws an additive model (p 0.4034; OR 0.87; 95% CI, 0.62–1.21) and by comparing homozygous minor versus homozygous Oo”) = ( oey major alleles (p 0.4048; OR 0.75; 95% CI, 0.39–1.47) (Table 2).

pute25 software to represent the 6q21 region better. Interest-   ingly, the imputed rs2802292 (MAF 0.47) that was previ- ously indicated by Willcox et al.22 as the best association, showed r2 — 0.79 with rs2802288 (MAF  0.49), similar to that observed in the HapMap CEU panel.33 In fact, our im- putation analysis confirmed that rs2802292, unrepresented on the BeadChip, was responsible for the strongest associa-   mo) = ( tion 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 ho- »oo)= mozygous major alleles (p 0.0019; OR, 2.58; CI 95%, 1.42–4.70) (Table 2).

Furthermore, we undertook a meta-analysis to explore the significance of the rs2802292 association with longevity by combining the association results of the current study and the findings coming from the Willcox et al. investigation. We combined the logarithm of the OR estimates and 95% CI for homozygous minor versus homozygous major alleles from G‘G the current analysis (OR 2.44; 95% CI, 1.37–4.34) and from  the Willcox et al. study (OR 2.75; 95% CI, 1.51–5.02) to ob- tain a summary OR using fixed26 and Der Simonian and Laird random effects models.27 Fixed effects models assume that the effect of a risk allele has the same value in each dataset whereas random effects models assume that the risk allele effects for each study vary around some overall aver- age effect.35 The inconsistency metric I2,28 was 0%, while the Q statistic29 was not statistically significant (p   0.88), indi- cating a null between-study heterogeneity evidence. Because no evidence of between-study heterogeneity has been proven, fixed effects26 and Der Simonian and Laird random effects27 coincide, generating identical summary point esti- mates (OR  2.66; 95% CI, 1.74–4.07; p see= 0.0001) (Fig. 3).

To evaluate a possible joint effect of the FOXO3A gene and the environmental exposure (such as smoke, etc.) or FOXO3A and the nongenetic attributes (such as clinical-cog- nitive variables), we performed a covariates analysis. No sig- nificant association of genotypes with cognitive variables (Barthel score, cognitive score, anxiety, depression) or clini- cal phenotypes (diabetes mellitus, glaucoma, high blood pressure, kidney diseases, osteoporosis, smoking history, cancer) was detected. As a result, we could hypothesize that FOXO3A has an effect on longevity independently of the tested variables. Subsequently, we generated a LD plot with the software HaploView v4.0.30 LD blocks are delineated by black lines in Fig. 2 and defined using the Four Gamete Rule, a variant on the algorithm described in previously published study,31 as implemented in HaploView software.34



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