out following the Declaration of Helsinki, and was approved by the ethics committees of the participating institutions. Written informed consent was obtained from all subjects or their legal representatives.

We compared the allele/genotype distributions of the exonic MSTN variants K153R (rs1805086), E164K (rs35781413), I225T, and P198A in Spanish centenarians (cases, n=156; 132 women, age range 100-111 years) and younger adults (controls, n=384; 167 women, age <50 years). No subject in either group carried a mutant allele of E164K, I225T or P198A. The frequency of the variant R allele was significantly higher in centenarians (7.1%) than in controls (2.7%) (P=0.001). The odds ratio of being a centenarian if the subject had the R allele was 3.48 (95% confidence interval 1.67-7.28, P=0.001), compared to the control group after adjusting for sex. The results were replicated in an Italian cohort (centenarians, n=79 (40 women), age range 100-104 years; younger controls, n=316 (155 women), age <50 years), where a higher frequency of the R allele in centenarians (7.6%) compared to controls (3.0%) (P=0.004) was independently confirmed.

Myostatin inhibitors, such as MYO-029, have an adequate safety margin and are able to improve muscle strength/function or muscle contractile properties in some patients with muscular dystrophy (Krivickas et al., 2009; Wagner et al., 2008). Of the identified MSTN variations in humans, the Lys(K)153Arg(R) polymorphism located in exon 2 (rs1805086, 2379 A>G replacement) is one candidate to influence skeletal muscle phenotypes (Ferrell et al., 1999). The Lys(K)153Arg(R) amino acid replacement is found within the active mature peptide of the myostatin protein. It could influence proteolytic processing with its pro-peptide or affinity to bind with the extracellular activin type II receptor (ActRIIB). The MSTN K153R polymorphism is thus a candidate to influence skeletal muscle phenotypes, including in older people.

One of the most robust and well-studied improvements in longevity is caused by starvation and genetic down-regulation of a nutrient-sensing pathway, namely mammalian target of rapamycin (mTOR) (Fontana et al., 2010; Kenyon, 2010). The mTOR pathway is an evolutionarily conserved nutrient-sensing pathway that adjusts metabolism and growth to amino acid availability, growth factors, energy status, and stress (Fenton and Gout, 2011; Sengupta et al., 2010). A number of studies have demonstrated that myostatin acts as a negative regulator of mTOR-directed signaling (Amirouche et al., 2009; Langley et al., 2002; Lipina et al., 2010; McFarlane et al., 2006; Rios et al., 2004; Sartori et al., 2009; Trendelenburg et al., 2009), which provides support for a potential role of myostatin inhibition in longevity. Myostatin deletion is beneficial for bone density, insulin sensitivity, and heart function in senescent mice (Morissette et al., 2009). Inhibition of the myostatin receptor ActRIIB increased survival (by 17%) in myotubularin-deficient mice (Lawlor et al., 2011). An extension of lifespan (+30%) was also observed after therapy with follistatin (a natural antagonist of myostatin) in a mouse model of spinal muscular atrophy (Rose et al., 2009).

Human lifespan is a partly heritable phenotype, with some alleles contributing to longevity assurance because they enhance the structure and function of the organism (Martin et al., 2007). As we age, we inevitably experience age-associated declines in the systems and organs that determine physical fitness, notably in the skeletal muscle tissue, i.e. sarcopenia. Accelerated age loss of muscle strength is associated with higher mortality risk (Metter et al., 2004; Ruiz et al., 2008); as such, genes potentially associated with higher preservation of muscle function at late life could be associated not only with healthy aging and lower disability risk but also with longevity. One such candidate is the myostatin (MSTN or growth differentiation factor 8) gene (Huygens et al., 2004).

Myostatin is a skeletal muscle-specific secreted peptide that modadult controls. The study followed the recommendations for human genotype-phenotype association studies recently published by the NCI-NHGRI Working Group on Replication in Association Studies (Chanock et al. 2007). One of the recommendations was to specify the 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. 

The study investigated two groups of Italian subjects born and living in Northern Italy: 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). 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 of Italian descent. The criterion for Italian descent was met when an individual's parents and four grandparents originated from Italy.

The history of past and current diseases was accurately collected by checking the centenarians' medical documentation and the current drug therapy. Italian centenarians did not have major age-related diseases like severe cognitive impairment, clinically evident cancer, ischemic heart disease, renal insufficiency, or severe physical impairment, although part of this group had decreased cognitive and physical function.

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