Other phenotypic variables

Collection of data for health deficit variables construction of the deficit index are essentially described (Jazwinski et al. 2010). Briefly, the index (DI) was derived as the un-weighted count of number of deficits divided by the total number possible deficits (N = 48) in an individual. The reported medical history questionnaire was tered to all study participants, and its components used to construct the deficit index. This was mented with items from an activities-of-daily (ADL) questionnaire. The continuous-scale function performance test involves the performance ten standardized tasks (CS-PFP10) representing ities of daily living including carrying donning clothes, sweeping the floor, loading a climbing stairs along with a 6-min timed walk et al. 1996, 2005). The Yale Physical Activity is to assess physical function and activity (Dipietro et al. 1993). We used one of its indices, Yale Energy Expenditure (YEE), which the time spent for each activity multiplied by intensity code, expressed as kilocalories per Mini-Mental State Examination (MMSE) was used screen for cognitive impairment (Folstein et al. Rovner and Folstein 1987). Age of subjects verified using both documentary evidence and graphic questionnaires. Mortality data were using Social Security Death Index search.

genomic DNA, 19 Applied Biosystems, Inc. (ABI) SYBR Green PCR Master Mix, 2.5 mM DTT, 100 nM Tel-1b primer (50-CGG TTT GTT TGG GTT TGG GTT TGG GTT TGG GTT TGG GTT-30) and 900 nM Tel-2b primer (50-GGC TTG CCT TAC CCT TAC CCT TAC CCT TAC CCT TAC CCT-30). The reaction was processed in an ABI 7300 real-time PCR machine for 1 cycle at 95(cid:3)C for 10 min, 30 cycles of 95(cid:3)C, 15 s, and 54(cid:3)C, 1 min. The b-globin reaction consisted of 15 ng genomic DNA, 19 ABI SYBR Green PCR Master Mix, 300 nM Hbb3 primer (50- TGT GCT GGC CCA TCA CTT TG-30) and 700 nM Hbb4 primer (50-ACC AGC CAC CAC TTT CTG ATA GG-30). The reaction was processed for 1 cycle at 95(cid:3)C for 10 min, followed by 40 cycles at 95(cid:3)C for 15 s, 58(cid:3)C for 20 s, and 72(cid:3)C for 28 s. All the reactions were done in triplicate. The relative T/S ratio was calculated by the comparative Ct (2-DDCt) method. To determine the PCR efficiency, a series of two-fold dilutions of the reference DNA was added to every plate, and a standard curve was generated. According to the slopes of the standard curves, PCR amplification efficiencies for the target and reference samples were very similar, ranging from 92 to 108%. In a given study, the relative T/S ratios correlate

Genotyping

The Illumina GoldenGate assay was performed according to the manufacturer’s instructions. SNPs were selected according to the Illumina Assay Design Tool. Following completion of the assay, all the samples were analyzed using Illumina GenomeStudio. Initial clustering was performed with the 10th percen- tile GenCall score set at 0.4 for both samples and SNPs. Samples with call rates below 90% were removed, and

well with the mean terminal-restriction-fragment

Hardy–Weinberg proportions was tested using the v2 goodness-of-fit method or an exact method for small sample sizes. A P-value \ 0.05 was considered statistically significant. All statistical tests of signif- icance reported in this study are two-sided. The genetic modes of inheritance for each SNP were based on the assumed effects of the minor allele. For example, additivity (add) of rs132793 assumes an increasing effect with the increasing dose of the minor allele A, whereas dominance (dom) and recessiveness (rec) were assumed when GA = AA and GG = GA, respectively. Survival analyses were performed using the Cox proportional hazards regression, with age, sex, and T/S as covariates. Multiple comparison correc- tions for the initial tests of association of alleles or genotypes with telomere length and longevity are inherent in the respective statistical tests or have been incorporated post hoc as indicated.

the remaining samples were re-clustered. SNPs not in Hardy–Weinberg proportions or with minor allele frequency lower than 1% were removed.

SNP rs7896005 (A/G; A is ancestral) is located in an intron of SIRT1. It is part of a large linkage disequilibrium (LD) block encompassing the entire gene (Online Resource 2). The A allele of rs7896005 shows a great ethnic variation in frequency: in the HapMap-CEU sample, its frequency is 0.288; whereas in Asian and African samples, it exceeds 0.8.The SNP rs132793 (G/A) is located in the intergenic region between the convergent XRCC6 and NHP2L1 genes on chromosome 22 (Online Resource 3). According to the dbSNP database, the G allele of rs132793 is ancestral with its frequency ranging from about 0.8 in the HapMap-CEU sample to almost unity in the HapMap-YRI. It is located about 3.6 kb downstream from the end of the last exon of XRCC6 and about 6.3 kb downstream from the end of the last exon of NHP2L1. XRCC6 is about 4,300 nucleotides long from the first exon to the last (including introns), and the NHP2L1 is about 1,500 nucleotides long. NHP2L1 is a human ortholog of the S. cerevisiae Nhp2 protein and binds to a stem-loop structure of an RNA component in the spliceosome assembly (Saito et al. 1996; Nottrott et al. 1999).

Results

Telomere length, age, and health status