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

regulation. However, it could be indirectly involved in telomere metabolism by its post-transcriptional func- tion. In a two-hybrid assay, a physical interaction between Ku70 and NHP2L1 was found (Stelzl et al. 2005), raising the possibility of such an indirect effect. Considering all these data and information together, we regard XRCC6 as the gene directly responsible for the phenotypes of rs132793 described in this article.

SNP functions

The SIRT1 SNP is in an intron. It is 35 nucleotides away from a nearby exon/intron junction and may affect splicing, though we couldn’t find any such putative function using the SNPinfo Web Server (Xu and Taylor 2009). However, using another web search tool, Pupasuite 3 (http://pupasuite.bioinfo.cipf.es) incorporated in GeneCards Version 3 (www.gene cards.org), we found that the SNP is in the close proximity of other potentially functional SNPs. For example, rs7896005 is only 334 nucleotides down- stream of rs11327756, which is embedded in a DNA triplex forming sequence. Triplex DNA structures have been suggested as regulatory regions for con- trolling gene expression (Goni et al. 2004), and SNPs located in these sequences can affect triplex formation and hence gene expression (Duca et al. 2008).

Telomere length maintenance

is potentially harmful because it doesn’t involve sequence homology. Fusion of an exposed terminus with a DSB end in the middle of a would be catastrophic. Telomeric fusion two chromatids would also be catastrophic if the dicentric chromatids segregate inappropri- during mitosis. This questions the utility of this pathway as an alternative telomere length- mechanism. In any case, inactivation of either of Ku subunits results in telomere shortening in mouse primary cell types (d’Adda di Fagagna al. 2001). Our findings indicate that telomere is more effective in homozygotes of the allele of rs132793. Therefore, we conclude that A allele of rs132793 is coupled with upregulated of XRCC6 that results in more efficient length maintenance. Regarding the SIRT1 increased expression of SIRT1 correlates with DSB repair through HR and longer telomeres, not with NHEJ (Oberdoerffer et al. 2008; Palacios al. 2010). Therefore, we suggest that the intron SNP is coupled with upregulation of SIRT1 that in more efficient HR-mediated telomere main- Thus, we consider different pathways of maintenance, involving either SIRT1 or even though these genes seem to overlap each other in some of their functions.

As for rs132793, this SNP lies between XRCC6 and NHP2L1 (Online Resource 3); see Materials and Methods for details. Upstream of NHP2L1 (farther downstream of XRCC6) lies a gene called MEI1, and we found that a few SNPs in MEI1 are also in high LD with rs132793 (not shown). This gene is expressed specifically in testis and is required for normal meiotic chromosome synapsis (Libby et al. 2003; Sato et al. 2006). Because we didn’t see any sex-specific pheno- types of rs132793, we rule out MEI1 as a gene responsible for the effects of rs132793 described in this article. SNP rs132793 is in high LD with four SNPs in or near XRCC6. Of these, according to our database search, rs132770 in the 50 promoter region of XRCC6 has regulatory potential. It is located 30 nucleotides upstream of the start of the first exon, and the locus is part of a number of transcription factor binding sites, with the profile of predicted binding factors and their affinities substantially varying depending on the alleles present.

The possibility that SIRT1 and XRCC6 are in different pathways for telomere maintenance could explain the lack of any statistical interaction between the two genes observed in this study. However, we cannot rule out the possibility that our study was not powered enough to detect such interactions due to either insufficient sample sizes or suboptimal statisti- cal modeling. In this regard, we note the high variability of T/S values throughout the age range (Fig. 1), which resulted in the low R2 values in all the models of linear regression. Thus, the rate of telomere

Online Resource 3 also shows that rs132793 is in LD with NHP2L1. NHP2L1 encodes a nuclear protein that is known to bind to the 50-stem-loop of U4 snRNA, a component of the U4/U6 snRNP in the pre- mRNA splicing machinery (Nottrott et al. 1999; Watkins et al. 2002). Based on its exclusive affinity for RNA molecules, we exclude the possibility of NHP2L1 being directly involved in telomere length

shortening is likely to vary among different individ- uals of the same age group, and this suggests additional factors, such as individual-specific envi- ronments, are in operation. It is also important to acknowledge the imperfect measure of global telo- mere shortening that leukocyte telomere length pro- vides. Among the factors contributing to this limitation is the superposition of the state of the leukocyte population itself, which can undergo peri- odic fluctuations.

statistically significant effect of the size seen, the GCS was not, and this is reflected in its near significance (P = 0.0696). The genetic architecture of the two populations differs, as evidenced even by the LD profile in the SIRT1 gene (Online Resource 2). Thus, the complex genetic and environmental nature of exceptional longevity must be taken into account. This complexity includes potential gene–gene and gene- environment interactions, as seen in other studies (Jazwinski et al. 2010; Bergman et al. 2007). This remains an area open to further investigation.