UT, Unilever Twin Cohort Study, LSADT, the Longitudinal Study of Aging Danish Twins, 1905: the Danish 1905 Birth Cohort Study, DLCS, the Danish Longitudinal Centenarian Study, MAF, minor allele frequency, mTRFL, mean terminal restriction fragment length (LTL), SD, standard deviation; TERC, telomerase RNA component; TERT, telomerase reverse transcriptase.

Table 2 Regression analysis of LTL by TERC candidate SNP genotypes

severalreportshaveprovidedevidencethatLTLisassociatedwithincreased risk of age-related disease and mortality in humans (Bakaysa et al., 2007; Kimura et al., 2008; Fitzpatrick et al., 2011). One puzzling observation was, however,that cross-sectional comparison of genotypefrequencies in predefined age groups suggested that the MAF of rs3772190 was signifi- cantly increased in the centenarians, in an apparent conflict with the mortality analysis using follow-up data. However, this cross-sectional estimatewasbasedontherathersmallsamplesizeofcentenarians(127of 1013studyparticipants);hence,itmightsimplybeachancefinding.More- over, repeating the mortality analysis for rs3772190 with exclusion of the centenarian subgroup did not change the results, while performing the mortalityanalysisofthecentenariansseparatelyeliminatedtheassociation. Hence, it appears that the centenarian subgroup did not contribute to the mortalityriskestimateoffollow-updata.

Coefficient P-value 95% CI rs3772190 (N = 864) )0.08 0.011 )0.15; )0.01 rs12696304 (N = 860) )0.06 0.046 )0.12; )0.001 rs12696304 males (N = 187) )0.13 0.014 )0.24; )0.02 rs12696304 females (N = 673) )0.04 0.248 )0.12; 0.03

95% CI, 95% confidence interval, P-values below 0.05 are shown in bold. An additive model was applied for the regression analyses. LTL, leukocyte telomere length; TERC, telomerase RNA component.

Table 3 Cox regression analysis by TERC rs3772190 genotypes

Genotype HR P-value 95% CI rs3772190 AG 1.31 0.009 1.070; 1.611 (age > 80, N = 720) AA 1.32 0.112 0.936; 1.862 AG + AA 1.31 0.006 1.081; 1.598

Finally, despite the very thorough examination of the genetic variation in the tert encoding locus in the present study, we found no evidence for association of TERT SNPs or haplotypes with longevity. Applying both a cross-sectional and a longitudinal study approach, we investigated the association of longevity to common genetic variation in TERT by examin- ing the four candidate SNPs investigated by Atzmon et al. (2010) in 1069 individuals in the age range of 58–100 plus years, as well as 11 tagging SNPs in 736 middle-aged and 1089 oldest-old individuals. Nonetheless, we cannot rule out that a putative effect of TERT variation on longevity might be population specific, that is, relevant in a population of Ashke- nazi Jews, but not in Danes.

HR, hazard rate; 95% CI, 95% confidence interval; TERC, telomerase RNA component. P-values below 0.05 are shown in bold.

Discussion

In conclusion, we have replicated associations of genetic variation in the TERC locus with LTL and, moreover, have found association of varia- tion in the TERC locus with longevity. Hence, our study suggests that TERC is associated with both LTL and human longevity.

In the present study, we investigated genetic variations in the TERT and TERC loci, and the possible association with LTL and longevity. The results point to association of TERC variants with both LTL and mortality at advanced ages.

We observed that rs3772190 and rs12696304 of TERC were associated with a significantly shorter LTL, in line with the results recently observed by Levy et al. (2010), Codd et al. (2010), and Shen et al. (2011), although in our study the association of rs12696304 with LTL was restricted to males. Correcting for multiple testing by the Bonferroni step-down approach left the estimates borderline significant (P-corrected: 0.066 for rs3772190 and 0.07 for rs12696304). If instead applying the less conser- vative Benjamini and Hochberg correction method, the effect of rs12696304 remains significant (P = 0.042), illustrating the difference in the use of diverse correction methods. We found no significant associa- tion of the TERT candidate SNPs rs2853669, rs2736098, rs33954691, and rs2853691 or their haplotypes with LTL or longevity, that is, we did not replicate the findings by Atzmon et al. (2010). Our sample size of 865 was much larger than that of Atzmon et al. (74 centenarians and 49 con- trols), thus giving our study a higher statistical power. A lack of associa- tion of TERT variants to LTL is also supported by two recent genome-wide association studies (Codd et al., 2010; Levy et al., 2010) and one candi- date study (Mirabello et al., 2010). The underlying reasons for the dis- crepancy in the findings might, however, relate to differences between the populations (Ashkenazi Jews vs. Danes) and perhaps different LTL measurement methods. We have employed Southern blot analysis, while the Atzmon et al. (2010) used qPCR to measure LTL.

Experimental procedures

Subjects

For the investigation of the two TERC and four TERT candidate SNPs, DNA from participants from the 1905 Cohort, the DLCS, the LSADT, and the UT Cohort Study were used. Briefly, the prospective follow-up studies of the 1905 and DLCS cohorts of oldest old were initiated when the partici- pants were 92 and 100 years of age, respectively (Andersen-Ranberg et al., 2001; Nybo et al., 2001). The LSADT study includes all Danish twins age 70 plus (Skytthe et al., 2002), and the UT cohort includes 220 female twin pairs, aged 59–81 years (Gunn et al., 2009). Participants were followed with respect to vital status until January 1st 2010 or until death, whichever came first. The information on vital status was retrieved from the Danish Central Population Register (Pedersen et al., 2006). Per- missions to collect blood samples and the usage of register-based infor- mation were granted by the Danish National Committee on Biomedical Research Ethics.

Genotyping