Results

Telomere length, age, and health status

Telomere length was determined in DNA from peripheral blood leukocytes as the T/S using a q-PCR assay (Cawthon 2002). The was confined to the Caucasian subjects in the LHAS as determined by analysis (0.80 assignment probability), because sample size was sufficient. Consistent with published studies, there was a significant correlation of T/S with age (q = -0.207; P = 8.09 10-5; Table 2; Fig. 1). On the other hand, T/S positively correlated with better health status, sented by the CS-PFP10, the YEE, the MMSE, collectively by the DI. Of these, CS-PFP10 was prominent in its significance (q = 0.261; P = 7.78 10-5). This association of CS-PFP10 with length was near significant (P \ 0.07) after ment for age; however, the correlations of remaining measures of healthy aging with telomeres did not survive this adjustment (Table indicating that they were related to the decline health status as a function of age. No differences in the mean T/S values were between male and female subjects (Online 1). However, males performed significantly better females in the CS-PFP10 (P = 1.59 9 10-4),

Pairwise LD comparison

LD blocks for SIRT1 and XRCC6 were examined using Haploview (http://www.broad.mit.edu/mpg/hap loview). D0/LOD values are designated by: bright red (D0 = 1, LOD C 2); blue (D0 = 1, LOD \ 2); shades of pink/red (D0 \ 1, LOD C 2); white (D0 \ 1, LOD \ 2). The numbers shown inside the boxes are D0 values 9 100 (empty boxes represent the D0 value of 1). Haplotype blocks are defined by the confidence bounds on D0. If the one-sided upper 95% confidence bound on D0 lies between 0.7 and 0.98, the SNP pairs involved are considered to be in strong LD (Gabriel et al. 2002).

Statistics

All the statistical analyses were performed in R (version R 2.11.1; R Development Team, 2008). Because of non-normality of the T/S data, either non- parametric tests or parametric tests on natural-log transformed T/S were performed. Agreement with

extensive LD block in the gene (Online Resource 2) was chosen for analysis of association with telomere length. No significant differences in genotype distri- bution of the SNP were observed between males and females, and the population sample was in Hardy– Weinberg proportions for variation at this locus (P >| 0.1). The mean T/S values varied significantly among the three genotypes at rs7896005: AA (N = 45; 1.69 ± 1.098 [mean ± SD]), AG (N = 167; 1.36 ± 0.724), and GG (N = 145; 1.47 ± 0.876) (P = 0.0134, Kruskal–Wallis test). This difference was especially evident between the minor-allele homozygotes (AA) and the other two genotypes. This pattern of phenotypic variation is best fit by assuming the recessivity of the minor allele. Adjustment for age in a multiple linear regression model showed that the association of SIRT1 with T/S is independent of age (Table 3, model a).

Table 2 Spearman correlation of telomere length (T/S) with healthy aging

Variable Coefficient P-value qa P-valuea (q) Age -0.207 8.09 9 10-5 n.a.b n.a.b CS- 0.261 7.78 9 10-5 0.125 6.12 9 10-2 PFP10c YEEd 0.105 4.94 9 10-2 0.026 6.33 9 10-1 MMSEe 0.159 2.93 9 10-3 0.044 4.15 9 10-1 DIf -0.092 8.23 9 10-2 -0.012 8.20 9 10-1

b Not applicable c Continuous

scale physical function performance test d Yale energy expenditure e Mini

mental state examination f

60 70 80 90 100 Age, years )S/T(nl ,htgnel eremoleT 0.25.10.15.00.05.0−0.1−5.1−

Association of XRCC6 with telomere length

We next assessed the potential impact of XRCC6 on the age-telomere length correlation. Ku70, encoded by XRCC6 is part of the Ku70/80 complex which acts in concert with SIRT1 in telomere maintenance. The XRCC6 SNP rs132793 in the 30 flanking region of this gene is in high LD (D0 = 1) with two SNPs in the promoter region and one in the first intron of XRCC6 (Online Resource 3). This SNP has been used as a marker for XRCC6 in several studies (Sobczuk et al. 2010; Li et al. 2011; Liu et al. 2007). No significant differences in genotype distribution of the SNP were observed between males and females, and the popu- lation sample was in Hardy–Weinberg proportions for variation at this locus (P >| 0.1).

Fig. 1 Plot of normal log-transformed telomere length (T/S) versus age with a linear regression slope. Each circle represents an individual subject

The mean T/S ratios significantly differed between the subjects grouped according to the genotypes of rs132793 (P = 1.91 9 10-2, Table 4). The mean T/S values increased as the number of copies of the minor allele A increased, resulting in significant differences between GG and AA groups (P = 4.36 9 10-2) and GG and AG ? AA combined groups (P = 4.42 9 10-2). The association of T/S with rs132793 was examined in detail using multiple linear regression (Table 3). The additive mode of inheritance for the genetic effect of the minor allele was statistically significance (model b), and the significance persisted after adjustment for age (model c). These results indicate that the shorter telomeres associated with

may well reflect the overall gender difference in health status, because males scored significantly better than females on the DI (P = 1.62 9 10-5). According to Cox regression, there was no association of T/S with survival (P SS | 0.1), but survival was highly dependent on age (P = 9.14 9 10-12).

SIRT1 and telomere length

We first examined SIRT1 as a potential modulator of the age-telomere length correlation. The SIRT1 SNP rs7896005, located in an intron and part of an

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