The reproducibility between the two measurements was high (r = 0.94, P < 0.001). Results obtained 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 subtelomeric lengths. The telomerase gene may play a significant role in human longevity and the maintenance of telomere length. In summary, we provide strong correlative evidence that individuals in Ashkenazi Jewish families with exceptional longevity have better maintenance of telomere length, and that the telomerase genes may function as important genetic determinants of both human longevity and telomere length. Additionally, our data suggest that both telomere length and variants of the fl telomerase genes may have a cumulative influence on lower disease prevalence and a favorable lipid profile in centenarians and their offspring. Additional comprehensive studies on genetic and genomic variation of centenarians and their offspring comprising candidate genes, especially those known to play a role in telomere maintenance in model organisms, may reveal previously 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 longevity was recruited as previously described (34). Proband were required to be living independently at 95 years of age as a reflection of good health, although at the time of recruitment, they could be at any level of dependency. 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 passports were used to verify the participants' ages. The Ashkenazi population is descended from a founder population (estimated to be several thousands) originating in the 15th century. This founder effect resulted in a population both culturally and genetically homogeneous, from which several disease-related genes have been successfully identified (45). A majority of these individuals 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 ± 0.3 years) defined as having 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 written consent was obtained in accordance with the policy of the Committee on Clinical Investigations of the Albert Einstein College of Medicine.

Mutation Screening and Genotyping of hTERT and hTERC

We sequenced genomic DNA isolated from blood of centenarians and controls to discover all 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 co-amplifies eight large fragments encompassing the coding and splicing junction regions of hTERT and hTERC. These products serve as a template for quadruple-multiplex 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 re-amplified 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.