Genotyping was performed in the genomics laboratory of Universidad Europea de Madrid during the summer of 2012. Allelic discrimination analysis was performed with prede-  signed Applied Biosystems TaqMan SNP Genotyping As- says on demand for the five polymorphisms: PPARD A/G (rs2267668) (ID: C__15872729_10), PPARGC1A Gly482Ser (rs8192678) (ID: C___1643192_20), NRF1 A/G (rs6949152) (ID: C__29144830_10), NRF2 A/C (rs12594956) (ID: C__32072163_20), and TFAM Ser12Thr (rs1937) (ID: C___8975662_10). PCR amplification was performed using a StepOne(cid:4) Real-Time PCR System (Applied Biosystems, Foster City, CA), with a denaturation stage at 95(cid:2)C for 10 min, 50 cycles of denaturation at 92(cid:2)C for 15 sec, anneal- ing/extension at 60(cid:2)C for 1min, and a final extension stage of 30sec at 60(cid:2)C.

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Participants

Written consent was obtained from each participant. The study protocol was approved by the institutional ethics committee (Universidad Europea de Madrid, Spain) and was in accordance with the Declaration of Helsinki for Human Research of 1974 (last modified in 2008). All of the study participants were of the same Caucasian (Spanish) descent for three or more generations; the majority (*90%) of them were born and lived most of their lives and in the same areas of Spain (Meseta Castellana, *600 meters altitude).

To ensure proper internal control, for each genotype analysis, we used positive and negative controls from dif- ferent DNA aliquots that were previously genotyped by the same method according to recent recommendations for replicating genotype–phenotype association studies.40 All of the analyses were performed by 2 experienced independent investigators who were blinded to subject data.

Centenarians (cases)

During 2009–2011 we obtained DNA from saliva samples in 107 centenarians of both genders (89 women, 18 men; age range, 100–111) living in nursing residencies of the Spanish central area (Meseta Castellana). This cohort included the oldest European individual, recognized by the Gerontology Research Group, a man who recently died at the age of 111 years, and *10% of the cohort was aged ‡105 years. The most prevalent diseases were osteoarthritis (80.0%), hyper- tension (72.7%), dementia (53.6%), and coronary artery dis- ease (41.8%). Four centenarians were free of any diagnosed disease.

Following recent recommendations for replicating human genotype–phenotype association studies,40 a subset of ge- notype results (rs8192678 in 100 controls) was corroborated in a second laboratory (Progenika Biopharma, Parque Tec- nolo´gico de Zamudio, Derio-Vizcaya, Spain) using a differ- ent methodology, i.e., newly developed low-density DNA microarray based on allele-specific probes. The design, fab- rication, validation, and analysis of the arrays for the latter methodology were performed following the procedure de- tailed elsewhere45 with minor modifications. The PCR products were fluorescently labeled and hybridized to the DNA microarray using an automated platform (Tecan HS4800, Mannedorf, Switzerland). Finally, the microarrays were scanned (Innopsys S.A., Carbonne, France), and we determined variants using software that converts the inten- sity of the spots into the genotype of each variant.45

We evaluated the centenarians’ cognitive ability with the Mini-Mental State Examination (MMSE),41 as well as their independence/functional capacity with the Barthel index. The latter is an instrument widely used to measure the ca- pacity of a person for the execution of 10 basic activities in daily life, obtaining a quantitative estimation of the subject’s level of independency.42,43 The sum-score ranges from 0 (totally dependent) to 100 (totally independent). The median (min, max) of the MMSE and Barthel score was 11 (0, 32) and 15 (range, 0–00) respectively. Approximately 12% of the centenarians were virtually independent during daily living without assistance from other persons, i.e., their Barthel and MMSE score was ‡ 80 and > 23, respectively.44

Statistical analysis

All statistical analyses were performed using the PASW (v. 18.0 for Windows, Chicago, IL). We tested Hardy– Weinberg equilibrium using a chi-squared test. We com- pared allele and genotype frequencies in cases and controls using the chi-squared test with a set at 0.05. All statistical comparisons were corrected for multiple comparisons using the Bonferroni method, in which the threshold p value is obtained by dividing 0.05 by the number of polymorphisms

Young adults (controls)

Inclusion criteria for this group were: (1) Age £50 years, (2) free of any diagnosed cardiometabolic disease, and (3) having no known family history of high longevity (90+ years).

studied (i.e., 0.05/5 =0.01). We also used logistic regression to analyse the association between alleles and longevity after adjusting for sex.

of the CC genotype in the control group (0%) 2.8% in centenarians (2 men, 1 woman). This mar- association remained when studying only men in both i.e., v2 = 15.696, p < 0.001 for the between-group of the frequency of CC genotype, but not when only women (v2 = 2.940, p = 0.230). We found no odds ratio for any of the genotypes or alleles we

Results