Statistical analysis

All statistical analyses were performed using the PASW (v. 18.0 for Windows, Chicago), except for statistical power, which we calculated with the

respectively). Two centenarians had the very rare RR genotype (vs. no participant of the control group). The odds ratio (OR) of being a Spanish centenarian if the subject had the R allele was 3.48 (95 % confidence – interval (CI) 1.67 7.28 P=0.001), compared to the con- trol group, after adjusting for sex.

StatMate software, version 2.0 (GraphPad, San Diego, CA, USA) (Emanuele et al. 2010; Minoretti et al. – 2006). We tested Hardy Weinberg equilibrium using a χ2 test. Genotype/allele frequencies were compared among the two study groups (centenarians and con- trols) within each cohort (Spanish and Italians) using the χ2 test with α set at 0.05. We used logistic regres- sion analysis to analyse the association between alleles and longevity within each of the two cohorts after adjusting for sex.

Italian cohort There were no failures in sample col- lection, DNA acquisition or genotyping procedures for both centenarians and young 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 previ- ously reported in European Caucasians. The frequency of the variant R allele and of 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 dur- ing daily living without assistance from other per- ≥ sons, 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 in- sufficient 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 be- tween the two laboratories. No subject carried a mu- tant 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 com- bined cohorts (Spanish and Italian subjects), our sam- ple size had an 80 % power to detect an OR of 1.92 for being a centenarian between R allele carriers and non- α carriers with a significance level ( ) 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 poly- – morphism 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 of 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,

Discussion

Our main finding was that the variant R allele of the MSTN K153R polymorphism is associated with ex- ceptional longevity in the two independent cohorts we studied, with a significantly higher proportion of this

K R Spanish cohort Replication (Italian) 100 90 80 70 60 50 40 30 20 ** * 10 0 Centenarians Controls Centenarians Controls (n=156) (n=384) (n=79) (n=316) )%( ycneuqerf elellA

are due to its potential to alter the function of the MSTN gene (Ferrell et al. 1999). Indeed, the Lys (K)153Arg(R) amino acid replacement is found within the active mature peptide of the myostatin protein and could influence proteolytic processing with its pro- peptide or affinity to bind with the putative endogenous receptor for myostatin, ActRIIB (Jiang et al. 2004; Lee et al. 2005). The fact that the Lys(K)153Arg(R) substi- tution is conserved in all avian and mammalian species suggests that it might affect myostatin function (Ferrell et al. 1999).