Mette Soerensen, The Danish Aging Research Center, Epidemiology, Institute of Public Health, University of Southern Denmark, J.B. Winsloews Vej

B,

Odense C, Denmark. Tel.:

; fax:

; e-mail: msoerensen@health.sdu.dk Accepted for publication

November

categories, that is, age < 80 (N = 578) and age ‡ 80 (N = 435), as well as age < 80 (N = 578) compared to octogenarians (N = 177), nonagenari- ans (N = 131), and centenarians (N = 127), showed a significant increase in minor allele frequency (MAF) of rs3772190 (A) in centenarians com- pared to the < 80 age group (OR = 1.46, P = 0.016).

Results

The characteristics of the study cohorts are summarized in Table 1 and in the Supporting Information, while lists of all the TERC and TERT SNPs tested in the study are provided in the Supporting Information. Genotype distributions of all SNPs were in agreement with Hardy–Weinberg equilib- rium (data not shown). For all statistical analyses, only genotype and hap- lotype groups with a frequency above 3% are presented.

In contradiction to this finding, mortality analysis of samples with avail- able longitudinal data (N = 773) indicated a significantly reduced survival in the rs3772190 A allele carriers ‡ 80 years (see Table 3). Compared to the rs3772190 GG group, the AG heterozygotes showed reduced survival [hazard rate (HR) = 1.31, P = 0.009], whereas the homozygotes AA showed the same but nonsignificant tendency (HR = 1.32, P = 0.112). Combining the AG and AA groups strengthened the finding (HR = 1.31 P = 0.006). No significant associations were observed for rs12696304, but the effect estimates were comparable to the effect of rs3772190 in both size and direction (data not shown), as would be expected because of the high LD between the two TERC SNPs.

LTL association study

First, we analyzed the two TERC candidate SNPs for association with LTL using participants from the Unilever Twin Cohort (UT), the Longitudinal Study of Aging Danish Twins (LSADT), the Danish 1905 Birth Cohort Study (1905 cohort), and the Danish Longitudinal Centenarian Study (DLCS) for which LTL data were available (N = 864). The minor alleles for both SNPs were found to be negatively correlated with LTL. Applying an additive model, as was performed in the previous studies, the age- and gender adjusted regression coefficient for rs3772190 was )0.08 (P = 0.011). Because a test for interaction between rs12696304 and sex indicated a significant effect (P = 0.012), the regression analysis was stratified by sex. When applying an additive model, the regression coeffi- cient for rs12696304 was )0.13 (P = 0.014) for males. The two TERC SNPs were in high, although not in perfect, linkage disequilibrium (LD) in the study population (r2 = 0.88). Accordingly, the results were correlated, although a slight discrepancy in effect was noticed, most likely as a conse- quence of the imperfect LD. Next, we investigated the four TERT candi- date SNPs; however, none of the TERT SNPs, nor their haplotypes, showed association with LTL (data not shown). The data are summarized in Table 2. Correction for multiple testing by the Bonferroni step-down approach left the two estimates borderline significant: P = 0.066 and P = 0.07 for rs3772190 and rs12696304, respectively.

Next, we analyzed the association of the four TERT candidate SNPs with longevity (N = 1069). Allele, genotype, and haplotype frequencies were compared between individuals grouped into the same age categories as for the TERC SNPs. None of the SNPs or haplotypes showed association with longevity (data not shown). Prospective mortality analysis using sam- ples in which longitudinal data were available (N = 850) did not reveal association of any of the four SNPs with longevity (data not shown). How- ever, individuals heterozygote (AG) for rs33954691 had a decreased mor- tality risk at an age younger than 85 (HR = 0.50, P = 0.011) compared to the most frequent homozygote (GG) group.

When correcting for multiple testing using the Bonferroni step-down approach, none of the associations with longevity remained significant (data not shown).

Finally, for an exhaustive evaluation of TERT variation and longevity, we also investigated 11 TERT tagging SNPs covering the common variation in TERT in Caucasians in 1089 members of the 1905 cohort and 736 mid- dle-aged controls (see the Supporting Information). Neither single-marker comparisons and haplotype comparisons (‘Sliding window’ of three con- secutive SNPs at a time) nor prospective mortality analysis showed any associations (data not shown). A similar evaluation of TERC was not possi- ble because no tagging SNPs were known for Caucasians in the TERC encoding region at the time of conducting this study.

Longevity association study

First, we analyzed the association of the two TERC candidate SNPs with longevity using participants from the UT, LSADT, 1905, and DLCS cohorts (N = 1013). Comparison of allele and genotype frequencies between age

Table 1 Characteristics of the study cohorts investigated for the two TERC and four TERT candidate SNPs

The Cohorts UT LSADT 1905 DLCS Mean age at intake [years (range)] 68.2 (58.8–80.9) 78.8 (73.3–94.2) 93.2 (92.4–93.7) 100 (99.1–100.3) Females (%) 100 66.1 82.3 77.1 N (2 TERC candidate SNPs) 213 547 125 128 MAF of rs12696304 0.28 0.28 0.28 0.33 MAF of rs3772190 0.26 0.26 0.25 0.33 N (4 TERT candidate SNPs) 213 549 175 132 MAF of rs2853691 0.32 0.37 0.39 0.35 MAF of rs33954691 0.11 0.11 0.12 0.11 MAF of rs2736098 0.32 0.24 0.25 0.25 MAF of rs2853669 0.36 0.28 0.29 0.29 N (mTRFL) 214 468 119 63 Mean age at mTRFL assessment (years) 68.2 79.3 99.7 102.2 mTRFL ± SD 6.25 ± 0.042 5.71 ± 0.023 5.20 ± 0.043 5.18 ± 0.06