Finally, no gene found associated with a functional phenotype showed a corresponding association with survival. However, two nom- inally significant associations with survival have been found in the whole sample, in relation to LOX (p = 0.013), SOD2 (p = 0.012) and UQCRFS1 (p = 0.038) genes, which variability were found to positively (LOX) and negatively (SOD2, UQCRFS1) influence the survival at gene level in this Cohort. TXNRD1, although showing a significant association with survival at SNP level (see Supplementary Table 2S_2), was not found associated with survival at gene level in the whole sample.

For each analyzed gene, the gene-based analysis was carried out using the following algorithm:

perform single SNP association analysis as describe before;

compute the gene-based statistic as the average of these single SNP statistics;

permute the dataset

,

of times, keeping LD between SNPs con- stant (i.e. permute phenotype labels);

for each permuted dataset, repeat steps

to

and build the null- distribution;

obtain the empirical p-value for the analyzed gene by comparing the original statistics with the permutation-based null-distribution.

After adjusting for multiple comparisons, none of the reported in the whole sample studied hold the statistical (Bonferroni corrected threshold: p b 1.3 × 10−3).

Gene-based analysis stratified by sex was carried out for genes car- rying at least one SNP showing a sex specific effect (p-value b 0.05 in Tables 2S_1 and 2S_2) on the analyzed traits in single SNP analysis. Table 3 reports the genes showing significant association at a nominal level of 0.05 with at least one trait under study plus survival in nonage- narian males (a) and females (b).

ForthegeneswithatleastoneSNPshowingasignificantsex-specific effect (p b 0.05), gene-based analyses were also carried out by sex. The empirical p-values obtained in the gene-based analysis have beenadjustedformultiplecomparisonsusingtheBonferronicorrection. For all calculations, the R statistical environment was used

ForthegeneswithatleastoneSNPshowingasignificantsex-specific effect (p b 0.05), gene-based analyses were also carried out by sex.

In males, the most interesting result was observed for UQCRFS1 gene with ADLperformance (p= 0.002) and UCP2 with WS (p= 0.009), the first one borderline significant also after the adjustment for multiple comparisons. Other nominally significant associations were found for SOD3 (p = 0.031) and XDH (p = 0.020) with ADL, GCLC (p = 0.024) with HG, CP (p = 0.021) and CYC1 (p = 0.022) with CCS. Borderline sig- nificant to a nominal level were the association of GPX4 (p = 0.045) and NOS3 (p = 0.052) with ADL and MMSE, respectively. No association with survival was reported in males for genes belonging to the pro-oxidant pathway here analyzed.

(R Development Core Team, 2011; http://www.r-project.com).

3. Results

3.1. QC analysis

After the QC phase, the final number of samples was 1080 (311 males and 769 females) including high quality genotypes for a total of 293 SNPs. In particular, 9 individuals were excluded from the analysis because of a call fraction lower than 90%. One SNP was excluded from the analysis because of a MiF value lower than 90% and one on the basis of MAF criterion (b1%) (see Supplementary Table 1S for details).

In females, a higher number of nominally significant associations were reported. The one of GLRX with HG levels (p = 0.001) hold also the correction for multiple comparisons (p b 1.3 × 10−3). Borderline significant also after correction for multiple testing (p = 0.002) are the associations of TXNRD1 gene with ADL scores, MT1A gene with MMSE performances and UCP3 with HG, in line with those found in the whole sample and reflecting the higher number of females in this Cohort. Other notable associations were observed for TXN2 with ADL (p = 0.038), UCP2 with HG strength (p = 0.008), SOD2 (p = 0.005) and GCLC (p = 0.007) with WS performances.

3.2. Gene-based analysis

Gene-based associationanalysis was carried out among the38 genes in the pro–anti-oxidant pathway and the five parameters of functional status (ADL, HG, WS, MMSE, CCS) plus survival. Table 2 lists the 14 genes showing a nominally significant association with at least one trait under study in the whole sample (p b 0.05). For each of them, the SNP showing the higher contribution to the association has been re- ported as best SNP.

As for survival, four nominally significant association with life- span were found in the female cohort, concerning the genes NDUFS1 (p = 0.009), TXNRD1 (p = 0.023), SOD2 (p = 0.035) and UCP3 (p = 0.041). This result is consistent with their associations with physical functioning and suggests that the variability of genes in the pro–antioxidant pathway can influence survival through an effect on physical performances, at least in the analyzed cohort.

Association with ADL performance was found for the TXNRD1 gene only (p = 0.010), which reported 6 nominally significant associations (p b 0.05) with this phenotype (see Supplementary Table 2S_1) at sin- gle SNP level.

aging and neurodegeneration (Cotman and Engesser-Cesar, 2002; Dik et al., 2003).

Table 3

Genes showing significant association at a nominal level of 0.05 with at least one trait under study (ADL, HG, WS, MMSE, CCS) plus survival, in nonagenarian males (a) and fe- males (b) from the Danish 1905 Cohort.

Thus, physically active people should benefit from exercise-induced adaptation in cellular antioxidant defense systems, becoming less vul- nerable to acute injury and chronic inflammation, finally delaying the onset of chronic illness and improve their quality of life (Dato et al., 2013).