Myostatin is a skeletal muscle-specific secreted peptide that essentially modulates myoblast prolifera- tion and thus muscle mass/strength (McPherron et al. 1997). Myostatin loss or inhibition has proven effec- tive in ameliorating symptoms of weakness in several animal models of muscle injury, atrophy and disease (Benny Klimek et al. 2010; Bogdanovich et al. 2002; Lee and McPherron 2001; Liu et al. 2008; Morrison et al. 2009; Murphy et al. 2010; Qiao et al. 2009; Siriett et al. 2006; Tsuchida 2008; Wagner et al. 2008; Zhu et al. 2007). Myostatin inhibition, as well as variations in the MSTN gene, can also have functional consequen- ces in humans.

Identifying candidate gene variants associated with longevity is possible by studying the genotype of cente- narians (Martin et al. 2007). This group of people are the – survival tail of population, with a lifespan of at least 15 20 years higher than the average westerner. Centenarians are also a model of healthy ageing, as the onset of disability in these individuals is generally delayed until

group of controls were (1) age 50 years, (2) free of any diagnosed cardiometabolic disease and (3) having no known family history of high longevity (90+ years). – During years 2008 2009, we extracted genomic DNA from saliva samples of 387 individuals (170 women, – 217 men). During 2009 2012, we obtained DNA from saliva samples in 172 centenarians of both genders (142 – women, 30 men; age range 100 111 years) living main- ly in nursing residencies of the Spanish central area (Meseta Castellana). This cohort included the oldest European individual (111 years) alive in June 2012 (http://www.grg.org/Adams/E.HTM) and ~9 % of the ≥ cohort was aged 105 years. The most prevalent dis- eases were osteoarthritis (67 %), hypertension (57 %), dementia (49 %) and cardiovascular disease (29 %). Two centenarians were free of any diagnosed disease.

The main purpose of this study was to compare the allele and genotype frequency of the MSTN K153R polymorphism between Spanish centenarians (aged – 100 111 years) and a group of healthy younger adults (controls, aged <50 years) of the same ethnic origin. We also genotyped the very infrequent MSTN exonic variants E164K (rs35781413), I225T and P198A, ow- ing to the fact that they can also cause amino acid replacements in the gene product (myostatin) expressed in human skeletal muscle (Saunders et al. 2006). The results in the Spanish population were independently replicated in a sample of Italian cente- narians. Based on the aforementioned studies, partic- ularly those suggesting an inhibitor role of myostatin on the mTOR pathway, we hypothesised that the var- iant R allele of the MSTN K153R polymorphism could be more frequent in centenarians compared with the general population.

’ We evaluated the centenarians cognitive ability with the Mini-Mental State Examination (MMSE) (Folstein et al. 1975), as well as their independence/functional capacity with the Barthel Index. The latter is an instru- ment widely used to measure the capacity of a person for the execution of ten basic activities in daily life, obtain- ’ ing a quantitative estimation of the subject s level of independency (Collin et al. 1988; Mahoney and Barthel 1965). The sum score ranges from 0 (totally dependent) to 100 (totally independent).

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 (Chanock et al. 2007). These rec- ommendations 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 tech- nology in a second laboratory.

cation of the results obtained in the Spanish popu- lation in an unrelated Italian cohort. Two groups of Italian subjects born and living in Northern Italy were investigated: 79 healthy centenarians (40 – women, 39 men; age range 100 104 years) and a control group of 316 healthy young subjects aged< – 50 years (165 women, 151 men; age range 28 ’ 50 years). The participants ages were defined by the dates of birth as stated on identity cards. All patients and controls in the Italian cohort were Caucasian whites ascertained to be of Italian de- ‘ ’ scent. The criterion of Italian descent was met ’ when an individual s parents and four grandparents originated from Italy. The history of past and cur- rent diseases was accurately collected, checking the ’ centenarians medical documentation and the cur- rent drug therapy. Italian centenarians did not have major age-related disease (i.e. severe cognitive im- pairment, clinically evident cancer, ischemic heart disease, renal insufficiency, severe physical impair- ment), although part of this group had decreased

Participants

Written consent was obtained from each participant. The study protocol was approved by the corresponding institutional ethics committees (European University of Madrid, Spain and University of Pavia, Italy) and was in accordance with the Declaration of Helsinki for Human Research of 1974 (last modified in 2000).

Spanish cohort All the Spanish 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). Inclusion criteria for the