4. Association of hTERT common alleles/genotypes with exceptional longevity

Nature of variation Controls (n = 49) Centenarians (n = 73) P value† Allele, % TERT Exon 1 IVS1–187 T > C C 36.7 42.5 0.3717 Exon 2 973 G > A (A 305 A) A 30.6 34.9 0.4834 Exon 14 3097 C > T (H 1013 H) T 8.1 21.9 0.0049 Exon 16 IVS16+99 C > T T 11.2 16.4 0.2556 TERC IVS+63 T > C C 18.5 18.8 0.9402 Genotype, % TERT Exon 1 IVS1–187 T > C TT 40.8 31.5 0.4494 CT 44.9 52.1 CC 14.3 16.4 Exon 2 973 G > A (A 305 A) GG 53.1 43.8 0.5313 GA 32.7 42.5 AA 14.3 13.7 Exon 14 3097 C > T (H 1013 H) CC 85.7 63.9 0.035 CT 12.2 26.8 TT 2 8.5 Exon 16 IVS16+99 C > T CC 81.6 68.5 0.0972 CT 14.3 30.1 TT 4.1 1.4 TERC IVS+63 T > C TT 64.4 66.2 0.5653 CT 34.2 29.9 CC 1.4 3.9

† Adjusted for age and gender.

fi functional effects. In addition, although we did not nd a single hTERC coding variation in the gene, we found two rare intronic variants only in centenarians. The enrichment of both synon- ymous and intronic variants of hTERT and hTERC genes in centenarians is intriguing because these variants are known to play a functional role in the regulation of gene expression through modulation of mRNA stability, mRNA secondary fi structure, alternative splicing, or translational ef ciency (39, 40).

noncarriers after adjusting for age and gender [telomere repeat copy number to single-copy gene copy number (T/S) ratio of 0.62:0.41, crude difference = 0.21; P = 0.02 by Student t test, P = 0.037 by Mann-Whitney U test], raising the possibility that these rare variants may have a positive functional impact on telomere maintenance. Our study also revealed that common fl genetic variations in hTERT may in uence the telomere length maintenance associated with longer telomeres in families with exceptional longevity.

The expression level of hTERT is a major determinant of telomerase activity (41). Recent studies indicate that telomer- ase activation can be regulated by environmental interventions such as lifestyle changes or stress management (42). There are fl functional hTERT promoter variants known to in uence telo- merase expression and telomere length (43). However, because hTERT there is very little LD in the gene, it is not likely that the centenarian-enriched rare variants are proxies of functional hTERT variants in the promoter. It is tempting to speculate fl hTERT that the rare variants might in uence expression of in response to environmental stresses such as infl ammation or oxidative stress, which then promotes better maintenance of telomere length. Indeed, mean telomere length is much greater among the carriers of the rare variant compared with the

hTERT Haplotypes of the gene derived from four common variants showed associations with longevity and/or telomere – > length. These are two intronic variants, IVS1 187 T C and > > IVS16+99 C T, and two synonymous variants, 973 G A (Ala- > 305 Ala) and 3097 C T (His-1013 His). These common variants hTERT may contribute to the regulation of gene expression. A hTERT common intronic variant shown to be associated with susceptibility to IPF may also affect the expression levels of hTERT (44). Taken together, our study demonstrates that cen- tenarians may harbor individually rare but collectively more common genetic variations in genes involved in the telomere maintenance pathway, implicating a role of regulatory variants in

Table 5. hTERT haplotypes and association with exceptional longevity

Haplotype frequency, %

Hap IVS1–187 T > C 973 G > A (A305A) 3097 C > T (H1013H) IVS16+99 C > T Centenarians (n = 74) Controls (n = 49) P 1 T G C C 34.9 53.1 0.008 2 C A C C 21.9 23.5 0.322 3 T G C T 13.7 9.2 0.007 4 C A T C 10.3 5.1 0.392 5 C G C C 6.2 5.1 0.749 6 T G T C 6.8 1.0 0.024 7–9 Others 6.2 3.1 0.203

*Adjusted for age and gender.

Table 6. hTERT haplotypes and association with telomere length

Haplotype Hap Increase (+) or decrease (−) in telomere length (T/S ratio) P value T G C C 1 −0.0307 0.742 C A C C 2 −0.0759 0.447 T G C T 3 0.4040 0.007 C A T C 4 −0.0222 0.899 C G C C 5 −0.1280 0.648 T G T C 6 0.1235 0.591 Rare 7–9 −0.0837 0.340

using 2 μL of each DNA dilution were carried out in a 20-μL volume using the LightCycler FastStart DNA Master SYBR Green kit (Roche Diagnostics), with MgCl 2 added to a final concentration of 3 mM. Primers for telomeres and the single-copy gene HGB (β-globin gene) were added to final concen- trations of 0.1 μM per 0.9 μM and 0.3 μM per 0.7 μM, respectively. The primer sequences are TEL 1b, 5′-CGGTTTGTTTGGGTTTGGGTTTGGGTT-TGGGTTT- GGGTT-3′; TEL 2b, 5′-GGCTTGCCTTACCCTTACCCTTACCCTTACCCTT-ACCCT-3′; HGB1, 5′-GCTTCTGACACAACTGTGTTCACTAGC-3′; and HGB2, 5′-CACCAACTT- CATCCACGTTC-ACC-3′ (48). The enzyme was activated at 95°C for 10 min, followed by 30 cycles at 95°C for 5 s, 58°C for 10 s, and 72°C for 40 s for the HGB reaction or for 20 cycles at 95°C for 5 s, 56°C for 10 s, and 72°C for 60 s for the telomere reaction. All transition rates were set to 20°C/s with the ex- ception of the annealing transition rate in the telomere reaction, which was set to 4°C/s. Forty samples (mixed samples from probands, offspring, and controls) were analyzed by qPCR, both in our laboratory and by one of the authors (R.M.C.) for validation purposes, yielding a correlation of 0.89 (P < 0.001). In addition, to ensure reproducibility, when measuring previously un- analyzed samples, and to account for assay variability between batches, we used an anchor sample that was previously measured. The reproducibility between the two measurements was high (r = 0.94, P < 0.001). Results ob- tained using this qPCR were highly correlated with those obtained by the traditional terminal restriction fragment length approaches (48). One T/S ratio unit is equivalent to a mean telomere length of 4,270 bp in the leukocytes. The qPCR telomere length determination does not include subtelo- meric lengths.

the telomerase gene in human longevity and maintenance of telomere length.