In order to ensure external validity of genotyping methods, a subset of polymorphisms/samples was corroborated in a second laboratory using a different method (Chanock et al. 2007). As such, genotyping (polymorphisms K153R and E164K) of the samples of 134 centenarians (78% of total) was also performed in the genomics laboratory of Vall d'Hebron Hospital (Barcelona, Spain) during the spring of 2012 using a different methodology, i.e. custom-designed Taqman SNP genotyping assays (assay ID: C___282184_30 for the K153R variant and assay ID: C__57828961_10 for the E164K variant) (Applied Biosystems, Foster City, CA, USA). All samples were run in a 7500 Real-Time PCR System (Applied Biosystems, Foster City, CA, USA). The analysis had pre-read and post-read steps of the plate consisting of 1 min at 60°C before and after the PCR cycle. The cycle conditions were 10 min at 95°C, followed by 40 cycles of 15 s at 92°C and 1 min at 60°C.

Genotyping in the Spanish cohort was performed specifically for research purposes. The researchers in charge of genotyping were blinded to the participants' identities. Saliva samples were tracked solely with barcoding, and personal identities were only made available to the main study researcher who was not involved in actual genotyping. In all participants, genomic DNA was extracted from saliva samples using the classical phenol-chloroform DNA extraction protocol with alcoholic precipitation. Genomic DNA was suspended in 50 μl Milli-Q H2O and stored at -20°C.

In all samples (from both cases and controls groups), sequences corresponding to the E164K, I225T, K153R and P198A variants were amplified during 2009-2012 by polymerase chain reaction (PCR) in the Genetics Laboratory of the European University of Madrid (Spain). The resulting PCR products were genotyped by single-base extension (SBE) (Gonzalez-Freire et al. 2010). The primers used for E164K, I225T, K153R, and P198A were 5'-GAAAACCCAAATGTTGCTTC-3' and 5'-TGTCTAGCTTATGAGCTTAGGG-3'. The PCR conditions were as follows: initial denaturation at 95°C for 10 min; 35 cycles at 95°C for 1 min, 52°C for 45 s, and 72°C for 1 min; and a final extension at 72°C for 5 min.

In the Italian cohort, genomic DNA was purified from peripheral blood samples using the QiaAmp DNA Mini kit (QIAGEN, Hilden, Germany) according to the manufacturer's protocol. In order to obtain unlimited quantities of centenarians' DNA for different genotyping projects, an in vitro immortalization of genomic DNA was performed by means of whole-genome amplification using a commercially available kit (GenomiPhi, GE Healthcare, Piscataway, NJ,Controls. The genotype distributions of the MSTN K153R polymorphism met the Hardy-Weinberg equilibrium in both the control (P=0.168) and the centenarian groups (P=0.465). Figures 1 and 2 show the MSTN K153R allele and genotype frequencies, respectively, in the two study groups. In keeping with the results obtained in the Spanish cohort, the frequency of the R allele in Italian controls (3.0%) was also similar to that previously reported in European Caucasians. The frequency of the variant R allele and R allele carriers was significantly higher in centenarians (7.6% and 15.2%) than in controls (3.0% and 5.7%) (χ²=8.116, P=0.004 and χ²=7.059, P=0.008, respectively). No participant had the very rare RR genotype except one participant in the control group. The sex-adjusted OR of being an Italian centenarian if the subject had the R allele was 2.44 (95% CI 1.19-5.21, P=0.031), compared to the control group.

Results: Spanish cohort
The median (min, max) of the Barthel score in the centenarians group was 15 (0, 100), whereas the mean ± SD (min, max) of MMSE was 13±10 (0, 32). Approximately 13% of the centenarians were virtually independent during daily living without assistance from other persons; i.e., their Barthel score was 80 and their score in MMSE was >23 (Christensen et al., 2008). The highest Barthel and MMSE scores (95 and 28, respectively) corresponded to an individual aged 102 years.

Failure rate of genotyping (due in all cases to insufficient amount of DNA) was 3 of 387 (0.78%) in controls and 16 of 172 (9.3%) in centenarians with the SBE method (European University of Madrid) and 17 of 134 (12.7%) with the Taqman method (Vall d'Hebron Hospital). Successfully genotyped samples of centenarians showed 100% concordant results between the two laboratories. No subject carried a mutant allele of the E164K (rs35781413), I225T, and P198A variations.

Statistical power
Based on the observed prevalence of the R allele of the K153R polymorphism in the combined cohorts (Spanish and Italian subjects), our sample size had an 80% power to detect an OR of 1.92 for being a centenarian between R allele carriers and non-carriers with a significance level (α) of 0.05. Hence, the power calculations in the whole study sample imply that our data would have been sufficient to detect moderate (OR of 1.5-2.3) differences in allele and genotype frequencies between centenarians and controls.

Genotype distributions of the MSTN K153R polymorphism met the Hardy-Weinberg equilibrium in both the control (P=0.581) and the centenarian groups (P=0.135). Figures 1 and 2 show the MSTN K153R allele and genotype frequencies, respectively, in the two study groups. The frequency of the R allele in the Spanish controls (~3%) was similar to that previously reported in European Caucasians, i.e., 3-4%, with a frequency of mutant homozygotes (RR) below 1% (Corsi et al., 2002; Ferrell et al., 1999; Kostek et al., 2009). The frequency of the variant R allele and R allele carriers was significantly higher in centenarians (7.1% and 12.8%) than in controls (2.7% and 5.5%) (χ²=10.815, P=0.001 and χ²=8.546, P=0.003).