The ACTN3 gene encodes for the synthesis of a-actinin-3 in skeletal-muscle fibres, a sarcomeric protein necessary for producing ‘explosive’ powerful contractions. A premature stop codon polymor- phism [Arg(R)577Ter(X), rs1815739] in ACTN3 was first described by North et al. [7]. This genetic variation, which probably preceded

finishers one or more time, including stage winners and finishers [10]. Their VO was 73.166.2 ml?kg21?min21. 2max

The main purpose of the present study was to compare the allelic and genotypic frequency of the ACTN3 R577X polymor- phism between Spanish centenarians (aged 100–108 years), and a group of healthy young adults (controls, aged ,40 years) of the same ethnic origin. A secondary aim was to compare the data obtained in centenarians with those of two groups of humans who show the two ‘extreme’ phenotypes of the exercise performance continuum, i.e. elite (world-class) athletes excelling in endurance and power sports respectively. We hypothesized that: (i) the XX genotype tends to be overrepresented in centenarians compared with the general population; (ii) the ACTN3 R577X allelic/ genotypic frequency distribution of centenarians is similar to that of endurance athletes; and (iii) the XX genotype will be overrepresented in centenarians compared with the power athlete group.

Elite power athletes

This sample included 63 elite power (track and field) Spanish male athletes (2763 years), including the best Spanish jumpers (n= 18) and sprinters in recent years (n= 45). Thirteen of them were Olympians during the period 2000–2008. Their VO was 2max 60.365.5 ml?kg21?min21.

Genotype assessment of the ACTN3 R577X polymorphism

Genotyping was performed specifically for research purposes. The researchers in charge of genotyping were totally blinded to the participants’ identities, i.e. saliva samples were tracked solely with bar-coding and personal identities were only made available to the main study researcher who was not involved in actual genotyping.

Methods

The study was designed and carried out in accordance with the recommendations for the human genotype-phenotype association studies recently published by the NCI-NHGRI Working Group on Replication in Association Studies [14]. These recommendations include among others, the following items: indicating time period and location of subject recruitment, success rate for DNA acquisition, sample tracking methods, and genotyping with a second technology in a second laboratory.

‘Reference’ genotyping

Genotyping was originally performed during 2008–2010 in the genetics laboratory of UEM (Madrid) following previously described methodology [10]. The polymerase chain reaction (PCR) was performed in order to amplify the sequence containing the mutation. A fragment of 291 bp was amplified with the following primers: ACTN3-F 59- CTGTTGCCTGTGGTA- AGTGGG labelled a 59 with VIC and ACTN3-R 59- TGGTCA- CAGTATGCAGGAGGG. The PCR conditions were as follows: initial denaturing at 95uC 5 min; 35 cycles at 95uC 30 s, 60uC 30 s, 72uC 30 s and a final extension at 72uC 10 min. ACTN3 genotypes were established by enzymatic digestion of amplicons with Dde I. The R577X change creates a restriction site resulting in fragments of 108, 97 and 86 bp. Digestion of the R577 allele results in fragments of 205 and 86 bp, and digestion of the 577X allele results in fragments of 108, 97 and 86 bp. The digestion products detected by capillary electrophoresis (ABI Prism 310 genetic analyzer; Applied Biosystems, Foster City, CA) were those labelled with VIC, i.e. 108 bp for 577X, and 205 bp for R577.

Participants

Written consent was obtained from each participant. The study protocol was approved by the institutional ethics committee (Universidad Europea de Madrid (UEM). Spain) and was in accordance with the Declaration of Helsinki for Human Research of 1974 (last modified in 2000).

All the study participants were of the same Caucasian (Spanish) descent for $3 generations. The majority (,90%) of them lived most of their lives and were born in the same areas of Spain (Meseta Castellana, ,600 m altitude).

Young adults (controls)

aged #40 years, (ii) free of any diagnosed cardiometabolic disease, (iii) having no known family history of high longevity (90+ years). During year 2008, we extracted genomic DNA from saliva samples of 283 University students of both genders (67 females, 216 males; mean6SD age: 2162 years) who met the aforemen- tioned criteria and were randomly selected from two Universities of the same city (Madrid, Spain): Universidad Polit´ecnica (N = 200) and UEM (n= 83). Their maximal oxygen uptake (VO ) was 2max 48.064.3 ml?kg21?min21. These controls served as subjects in previous research from our group [15,16].

Reliability assessment of genotype analysis in a second laboratory

Following recent recommendations [14], genotype results of the ACTN3 R577X polymorphism were replicated in a different laboratory (Progenika Biopharma, Parque Tecnolo´gico de Zamudio, Derio- Vizcaya, Spain) using a different technology, i.e. newly developed low-density DNA microarray based on allele-specific probes. The design, fabrication, validation and analysis of the arrays were performed following the procedure described elsewhere [17]. In brief, the PCR products were fluorescently labelled and hybridized to the DNA microarray in an automated platform (Ventana Medical Systems, Inc., Tucson, AZ, USA). The microarrays were scanned (Innopsys S.A., Carbonne, France) and we determined variants using a developed software that converts the intensity of the spots into the genotype of each variant.

Centenarians (cases)