Table 3 Dependence of telomere length (T/S) on SIRT1 and XRCC6 variantsa

variablesb Coefficient SE P-value R2 age ? SIRT1 rs7986005 (rec) -8.867 9 10-3 (age) 2.187 9 10-3 6.17 9 10-5 0.0571 1.73 9 10-1 (SIRT1) 6.553 9 10-2 8.52 9 10-3 XRCC6 rs132793 (add) 1.04 9 10-1 3.877 9 10-2 7.55 9 10-3 0.020 age ? XRCC6 rs132793 (add) -8.70 9 10-3 (age) 2.073 9 10-3 3.42 9 10-5 0.066 1.05 9 10-1 (XRCC6) 3.790 9 10-2 5.98 9 10-3 age ? SIRT1 rs7896005 (rec) -8.95 9 10-3 (age) 2.162 9 10-3 4.35 9 10-5 0.0816 XRCC6 rs132793 (add) 1.81 9 10-1 (SIRT1) 6.482 9 10-2 5.50 9 10-3 1.17 9 10-1 (XRCC6) 3.804 9 10-2 2.31 9 10-3

a Linear regression was performed on natural log-transformed T/S b SNPs are coded 0, 1, or 2 according to the inheritance mode of the minor alleles, recessive (rec) for rs7896005 and additive (add) for rs132793 (see Materials and methods)

SIRT1 and longevity

Table 4 Comparison of telomere length (T/S) in different XRCC6 genotypesa

Genotype Number Mean ± SD P-value GG 240 1.393 ± 0.829 AG 100 1.533 ± 0.877 AA 15 1.745 ± 0.894 AA ? AG 115 1.561 ± 0.878 GG vs. AG vs. 1.91 9 10-2 b AA GG vs. AA 4.36 9 10-2 c GG vs. 4.42 9 10-2 c (AA ? AG) a XRCC6 SNP rs132793

Because SIRT1 passed our criterion of association with the aging endophenotype of telomere length, we next examined the association of this gene with longevity. To test its association with longevity, we compared the allele and genotype frequencies of rs7896005 in Caucasian (European-origin) subjects of the LHAS study population (0.80 assignment probability). We found that the frequency of the minor allele of the SIRT1 SNP rs7896005 was higher in the long-lived cases (C90 years old) than in the young controls (20–59 years old) of the LHAS (Table 5). Logistic models testing effects of this SNP on case–control binary outcomes predicted that the odds of homozy- gotes of the minor allele being associated with the long-living case group is more than two-fold higher than the odds of other genotype carriers (Table 6). This recessive model for association of the SIRT1 variant with longevity mirrors the recessive genetic model for its association with telomere length (see text and Table 3).

c Wilcoxon rank sum test (with Bonferroni correction)

rs132793 are independent of age. Indeed, we didn’t see any statistically significant interaction between age and rs132793 (not shown).

To see whether the SIRT1 variant interacts with age or the XRCC6 SNP, we performed multiple linear regression (Table 3, model d). The SIRT1 SNP showed a significant association with T/S, and addition of this SNP to the model including age and the XRCC6 SNP rs132793 improved the model, as shown by the higher R2 values. However, we were not able to detect any statistical interactions between the predictors (not shown). These results suggest that SIRT1 and XRCC6 impinge on telomere length through separate pathways independently of age.

in LHAS, we replicated this analysis in a different population. Samples from the GCS were used. Cau- casian (European-origin) subjects of the GCS study population (0.80 assignment probability) were chosen to correspond to the LHAS sample. In this sample, the cases were centenarians (C98 years old) and the young controls were 20–59 years old. The LD block in SIRT1 of which rs7896005 is a part is similar in the GCS Caucasian population to the one in the LHAS population, though not identical (Online Resource 2).

Table 5 Contingency of longevity upon SIRT1 and XRCC6 variantsa

Gene (SNP) Subject group Genotype P-valueb Allele P-valueb SIRT1 AA AG GG 8.0 9 10-3 A G 5.6 9 10-3 (rs7896005) Casec 35 105 84 175 273 Controld 21 134 138 176 410 XRCC6 AA AG GG 0.74 A G 0.94 (rs132793) Casec 12 60 152 84 364 Controld 12 84 197 108 478

Table 6 Effect of XRCC6 on association of SIRT1 with longevitya

Sample Gene (SNP) OR 95% CI P-value LHASb SIRT1 (rs7896005)d 2.382 1.355–4.282 2.96 9 10-3 SIRT1 (rs7896005)d 2.385 1.356–4.287 2.94 9 10-3 ? XRCC6 (rs132793)e 1.029 0.755–1.398 8.53 9 10-1 GCSc SIRT1 (rs7896005)d 1.334 0.978–1.824 6.96 9 10-2

The logistic regression yielded odds one-third higher for cases based on homozygosity for the minor allele as compared to the other genotypes, which was near significant (Table 6), providing support for the asso- ciation of SIRT1 with longevity.

Discussion

Our study agrees with the consensus that telomere shortening occurs with age. We also found that function ability decreased with telomere shortening, which is likely due to the dependence of the function variables on age. There is a good deal of variability in the association of function and physiologic variables with telomere length in human studies, and additional examination of these associations is needed (Mather et al. 2011). Some of these discrepancies are likely due to the lack of adjustment for age. However, other differences include study design, population, function and disease status, and methods of assessment of telomere length and of function. We also made novel findings: (1) the SIRT1 and XRCC6 SNPs had significant effects on telomere length; (2) rs7896005 in SIRT1 had significant effects on longevity, whereas the XRCC6 SNP rs132793 did not; (3) the effects of the SNPs on telomere length were independent of age;

XRCC6 and longevity

Finally, we tested whether rs132793 is associated with longevity, because XRCC6 fit the criterion of associ- ation with the aging endophenotype of telomere length. To test association of XRCC6 with longevity, we compared the allele and genotype frequencies of rs132793 between nonagenarian cases and young controls of the LHAS population, but found no significant differences (Table 5). Incorporation of the XRCC6 SNP rs132793 in the same logistic model with the SIRT1 SNP rs7896005 showed that the association with longevity of SIRT1 persists indepen- dently of XRCC6 (Table 6).

(4) the effects of the SIRT1 and XRCC6 were independent of each other.