have signifi cantly longer telomeres than unrelated controls and that this trait is strongly heritable. Moreover, we have demon- strated that offspring of centenarians do not show an appreciable age-related decline in telomere length as is observed in our un- related control population as well as in other cross-sectional < studies (5). Because telomere length in younger persons ( 75 fi years of age) is not signi cantly different between offspring of centenarians and unrelated controls, these results suggest that families with exceptional longevity have superior telomere length maintenance. Interestingly, unrelated controls older than 86 years of age exhibit longer telomeres than younger individuals (controls), suggesting that the rate of telomere attrition may be an important determinant of overall survival in the general population. The relatively older group may be affected by sur- vival bias that selected out individuals with aging-related disease or those who would have died before reaching the age of 85 years, leaving the survivors with relatively longer telomeres.

Telomere length was adjusted for group, gender, and age at recruitment.

hTERT hTERC of common or variants with telomere length in- fi dicates no signi cant association (Table S1). However, of the two haplotypes that are enriched in centenarians compared with controls, Hap 3 shows a positive association with telomere length P after adjusting for age and gender ( = 0.007), whereas Hap 6 fi P does not reach a signi cant threshold ( =0.59)becauseof itslow frequencies in the tested population (Table 6). In contrast, a centenarian-depleted haplotype, Hap 1, shows no association with telomere length (Table 5).

Because shorter telomere length is associated with diseases of aging, including hypertension, the metabolic syndrome, and dementia (10), we studied the association between telomere length, major age-related diseases, and lipid profi les in cen- tenarians, their offspring, and controls. Lipid profi les are known predictors of age-related diseases, and we have previously demonstrated that centenarians and their offspring have sig- nifi cantly larger HDL and LDL particle sizes and that these are heritable phenotypes of healthy aging associated with a lower prevalence of morbidity. We found that longer telomeres are indeed associated with lower prevalence of hypertension, the metabolic syndrome, type 2 diabetes, and better cognitive func- fi tion as well as with healthier lipid pro les.

Discussion

Several studies have demonstrated a close relation between telomere length and life span in humans, including long-lived humans (4). However, interpretations of results from these studies are often confounded because of a lack of adequate controls. We employed a unique study design to overcome this shortcoming in a cohort of Ashkenazi Jewish individuals with exceptional longevity (centenarians), their offspring (approx- imate age of 70 years), and age- and gender-matched controls without a family history of unusual longevity. The use of off- spring of individuals with exceptional longevity and their matched controls provides a powerful approach to identify ge-

 3039 C>THis 1013 His 

Fig. 3. 2D gene scanning of human telomerase gene (hTERT and hTERC). The coding regions and exon–intron junctions of the hTERT and hTERC were amplified by 2-step PCR. Eighteen PCR fragments were displayed in a 2D gel according to their size and melting temperature. (A) 2D gene scanning pattern from a centenarian subject with the fragment identification number and a heteroduplex band in exon 14 of the hTERT. (B) Common genetic variation in exon 14 was identified as 3097 C > T (His-1013 His) by nucleotide sequencing.

Centenarians Nucleotide change Protein change (n = 100) (n = hTERT Non-synonymous 835 G > A Ala 279 Thr 0 1 3148 G > A Ala 1050 Thr 0 1 Synonymous 915 G > A Ala 305 Ala 43 27 1812 A > G Ala 604 Ala 1 1 1849 C > T Leu 618 Leu 1 1 2355 C > G Ser 785 Ser 1 0 2481 G > A Thr 827 Thr 3 0 2739 C > T His 913 His 4 1 3097 C > T His 1013 His 28 11 Intronic IVS1-211G > A 2 0 IVS1-187 T > C 52 36 IVS4+10 C > T 4 0 IVS4+21 C > T 1 0 IVS16+64 C > T 2 0 IVS16+99 C > T 30 12 hTERC Intronic IVS-99 C > G 1 0 IVS+12 A > G 1 0 IVS+63 T > C 30 27

Previously unknown unique variants are indicated in bold.

Telomere length, as a quantitative trait, is fairly well studied fl (19), but genetic factors that in uence it are not well understood. hTERT Recent studies suggest that heterozygote mutation in hTERC and genes, which are the essential components of telo- merase, show defective phenotypes in several diseases, indicating that half the usual dose of telomerase is inadequate for main- tenance of telomeres with normal length (35, 36). Because living to 100 years of age is a rare phenotype in humans, with a prev- alence of 1 in 10,000 individuals in the general population (37), we hypothesized that centenarians may harbor rare gain-of- fl function mutations in the telomerase genes that may also in u-

hTERT ence the length of telomeres. Sequence analysis of and hTERC genes revealed that rare synonymous or intronic variants hTERT n in are enriched in centenarians ( = 19) compared with n P controls ( = 3) (Table 2; = 0.041). In contrast, centenarians are completely devoid of nonsynonymous variants, whereas there are two control individuals who carry heterozygote non- > > synonymous variants, 893 G A (Ala-279 Thr) and 3148 G A (Ala-1050 Thr). Although these nonsynonymous changes are predicted to be functionally benign, they are also found in idi- fi > opathic pulmonary brosis (IPF) patients with a MAF 5% (38), raising the possibility that these variants may have negative

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Fig. 4. Schematic representation of exons, introns, UTRs, and variants discovered. (A) hTERT. (B) hTERC. Green blocks, coding exons; white blocks, 5′ and 3′

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