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

In summary, we provide strong correlative evidence that individuals in Ashkenazi Jewish families with exceptional lon- gevity have better maintenance of telomere length and that the telomerase genes may function as important genetic determi- nants of both human longevity and telomere length. Addition- ally, our data suggest that both telomere length and variants of fl telomerase genes may have a cumulative in uence on lower fi disease prevalence and a favorable lipid pro le in centenarians and their offspring. Additional comprehensive studies on genetic and genomic variation of centenarians and their offspring com- prising candidate genes, especially those known to play a role in telomere maintenance in model organisms, may reveal pre- viously undescribed genomic regions and molecules that are operative in human health and longevity.

Materials and Methods

Study Design and Subjects. A cohort of Ashkenazi Jews with exceptional lon- gevitywasrecruitedaspreviouslydescribed(34).Probandswererequiredtobe living independently at 95 years of age as a reflection of good health, al- thoughatthetimeofrecruitment,theycouldbeatanylevelofdependency.In addition, probands had to have a first-degree offspring who was willing to participate in the study. Birth certificates or dates of birth as stated on pass- ports were used to verify the participants’ ages. The Ashkenazi population is descended from a founder population (estimated to be several thousands) originatinginthe15thcentury.This“foundereffect”resultedinapopulation both culturally and genetically homogeneous, from which several disease- related genes have been successfully identified (45). A majority of these in- dividuals were born in the United States or moved there before World War II. Telomere measurements (see below) were made on 86 Ashkenazi probands (aged 97.4± 0.3years) defined ashaving exceptional longevity,175 offspring of parents with exceptional longevity (aged 67.8 ± 0.6 years), and 93 controls who were offspring of parents with usual survival (aged 71.8 ± 1 years). By definition, parents of controls survived to the age of 85 years or less. The control group consisted largely of spouses of the offspring group. Informed writtenconsentwasobtainedinaccordancewiththepolicyoftheCommittee on Clinical Investigations of the Albert Einstein College of Medicine.

Mutation Screening and Genotyping of hTERT and hTERC. We sequenced genomicDNAisolatedfrombloodofcentenariansandcontrolstodiscoverall possible genetic variations in the exonic regions and intron–exon boundaries of the telomerase genes (hTERT and hTERC) by a 2D gene scanning (TDGS) method (49). To increase specificity, PCR amplification was designed in two steps. To begin with, triple-multiplex long-distance PCR coamplifies eight large fragments encompassing the coding and splicing junction regions of hTERT and hTERC. These products serve as a template for quadruple-multi- plex short PCRs to amplify target regions in 18 fragments; 16 fragments for hTERT and two fragments for hTERC. Primers are listed in Table S2. The mixture of amplicons after heteroduplexing was separated on the basis of size and melting temperature in a 2D gel. Then, DNA fragments containing heterozygous sequence variation were reamplified from the genomic DNA and sequenced to identify the position and nature of sequence variation. For hTERT, exon 1 was analyzed by nucleotide sequencing and exons 2, 4, and 5 were separately analyzed by denaturing gradient gel electrophoresis (DGGE) because of ambiguous melting patterns on a TDGS gel. Genotypes of the common variants in the telomerase gene were determined by DGGE using the same primers designed for the TDGS assay.

For each subject, a detailed medical history, physical examination, and blood sample collection were performed as previously described (23, 32, 34). The presence of hypertension, the metabolic syndrome, and diabetes was determined using National Cholesterol Education Program (NCEP), Adult Treatment Panel III (ATP III) criteria (46). Scores on the MMSE (47) ≤25 were considered to indicate impaired cognitive function. Triglyceride, LDL- cholesterol (LDL), and HDL-cholesterol (HDL) levels and average particle sizes were determined by NMR spectroscopy at LipoScience, Inc., as previously described (34). Large lipoprotein particle sizes were defined as >8.9 nm for HDL and >21.3 nm for LDL.

Statistical Analyses. Two comparisons have been established: (i) between probands and controls for whom crude data were adjusted for gender and (ii) between offspring and controls for whom crude data were adjusted for age and gender. Individual phenotypes that were not normally distributed were log natural-transformed for analysis and back-transformed for the current presentation. Associations of telomere length with other variables were estimated using regression on an indicator variable for group, age, and gender. In defining strata for age, cutoff points of 65, 75, 85, 95, and 105 years were used (Fig. 1). Telomere length distribution in probands appears to be nonnormally distributed; thus, to test for statistical significance, we applied nonparametric statistics analysis, and pairwise crude comparisons of telomeres between the study groups were carried out using the Wilcoxon signed-rank test. Parameters were estimated by maximum likelihood methods. The significance of the parameter (association) was evaluated using the likelihood ratio test by comparing the model in which the