Centenarians (cases)

During 2009–2010 we obtained DNA from saliva samples in 64 centenarians of both genders (57 female, 7 male; 10261 years, range: 100, 108) living in nursing residencies of the Spain central area (Meseta Castellana). The most prevalent diseases in the centenarians’ cohort were osteoarthritis (72%), hypertension (62.5%), dementia (50%) and coronary artery disease (30%). Three centenarians were free of any diagnosed disease.

Statistical analysis

We tested Hardy-Weinberg equilibrium using a test. All statistical analyses were performed using the PASW (v. 18.0 for WINDOWS, Chicago). Genotype/allele frequencies were com- pared among the four study groups (centenarians, controls, endurance and power athletes) using a x2 test with a set at 0.05. We used logistic regression analysis to analyse the association between alleles and longevity after adjusting for sex.

Elite endurance athletes

This sample included the best 50 professional male Spanish (2663 years) road cyclists in the last years, i.e. all Tour de France

Discussion

Results

There were no failures in sample collection, DNA acquisition or genotyping procedures, except for 5 centenarians, for which the amount of DNA gathered from saliva was insufficient to allow ACTN3 R577X genotype assessment. Parallel genotyping results showed 100% concordance between the two laboratories.

Although the allelic/genotypic frequency of the ACTN3 R577X polymorphism did not significantly differ between centenarians and the control population, a major finding of our study was that overall, the allelic/genotypic distribution of the ACTN3 R577X polymorphism in centenarians was closest to that of those humans who arguably show the most ‘extreme’ muscle endurance phenotypes, i.e. professional road cyclists able to excel in such demanding events as the Tour de France [10]. Notably, the frequency of the null XX genotype in our cohort of centenarians (24%) was the highest ever reported in non-athletic Caucasian populations, and is only surpassed by the values of the professional road cyclists we studied (26%) and the elite endurance athlete’s cohort (made up of various types of Olympic-class endurance athletes, included road cyclists) of a previous report by Yang et al on Australian athletes (30%) [9].

study groups (all P.0.1). Figures 1 and 2 show the genotype and allele frequencies respectively in the different study groups. The ACTN3 R577X genotype distributions in our healthy controls were overall similar to those previously reported in healthy Spanish adults [10] and European Caucasians, i.e. ,18% frequency of the XX genotype [7]. There were no differences in genotype or allele frequencies between centenarians, and controls or endurance athletes (all P.0.1). We repeated the analyses by comparing X carriers vs. non-X carriers, or R carriers and non-R carriers and found no significant differences between groups (all P.0.1). However, we observed significant differences in genotype and allele frequencies between centenar- ians and power athletes: the frequency of X carriers was higher in the centenarians compared with the power athletes group (Figure 1, P = 0.011), and the frequency of the X allele was also higher in centenarians than in power athletes (Figure 2, P = 0.019).

Though natural selection in the human genome accelerated during the past 40,000 years, probably less than 1% of our genes arose during the late Pleistocene and Holocene epochs (126,000– 8,000 years before present), that is, after the Paleolithic era [4]. Genetically, therefore, we are still citizens of the Paleolithic era living in the 21st century; our genetic makeup is shaped to support the balance between energy intake and energy expenditure that was common to Paleolithic hunter–gatherer societies [3]. Among these people, survival depended upon the obligatory link between energy intake (food consumption) and energy expenditure for ensuring food acquisition [18]. Thus, ‘long time tracking persistence’ (i.e. mostly endurance or ‘aerobic’ exercise, such as walking/running 20 or more km/day) was likely necessary for successful hunting and thus for survival [5,6]. In this context, genetic variants with a documented influence on exercise/muscle related phenotypes could also be associated with survival. For instance, the D allele of the 287 bp Ins(I)/Del(D) polymorphism [rs1799752] in intron 16 of the angiotensin converting enzyme (ACE) gene, which has been associated with several exercise

The odds ratio (OR) of being a centenarian if the subject had the X allele was not significant: 1.236 (95% confidence interval (CI): 0.484–1.803), 1.041 (95% CI: 0.486–2.230), and 1.678 (95% CI: 0.928–3.033) compared to the control, endurance athletes, and power athletes group, respectively, after adjusting for sex. Likewise, the OR of being a centenarian if the subject had the XX genotype did not reach statistical significance: 1.032 (95% confidence interval (CI): 0.863–1.234), 1.132 (95% CI: 0.784– 1.636), and 1.165 (95% CI: 0.945–1.437) compared to the control, endurance athletes, and power athletes group, respectively, after

Genotypes (%)   Controls(n=283)Centenarians Power Endurance(n=59) (n=63) (n=50)

1. Genotype distribution of the ACTN3R577X polymorphism in our study cohorts. Symbol: *P=0.011 for centenarians vs.

Figure 1. Genotype distribution of the ACTN3R577X polymorphism in our study cohorts. Symbol: *P=0.011 for centenarians vs. power

Alleles (%)656055504540353025201510     Controls Centenarians Power Endurance(n=283) (n=59) (n=63) (n=50)

Figure 2. Allele distribution of the ACTN3R577X polymorphism in our study cohorts. Symbol: *P=0.019 for centenarians vs. power

2. Allele distribution of the ACTN3R577X polymorphism in our study cohorts. Symbol: *P=0.019 for centenarians vs.