A borderline association was observed between NOS2 rs2297518 and HG scores (p = 0.049), Model with subjects homozygous for the less frequent allele A showing higher HG scores than those carrying at least one copy of the wild type G allele.

Finally, a significant association was found between NOS3 rs10277237 and ADL performance (p = 0.002) with subjects homozygous for the Model minor allele A (recessive model) showing significantly lower probability to be disable than those carrying at least one G allele. After adjustment for multiple comparisons this association remained statistically significant (p = 0.004). Adj

results obtained using the RobustSNP algorithm. After adjusting for sex, we found that NOS1 rs1879417 and NOS2 rs2297518 were significantly associated with the longevity phenotype. The additive model for rs1879417 and the recessive one for rs2297518 resulted to be the most likely genetic models, in which the presence of the relevant alleles (allele C for rs1879417 and allele A for rs2297518 varia- tion) decreases the probability to attain longevity (p = 0.008 and p = 0.031, respectively). Model Model After adjusting for multiple testing (due to the three different tested genetic models), the NOS1 rs1879417 remained statistically significant (p = 0.018), Adj

Interaction analysis

By using the MB-MDR approach, we did not find any significant interaction effect among the NOS gene polymorphisms and the analysed phenotypes.

Table 3 Results of the RobustSNP association test with the longevity phenotype in the analyzed sample

SNP Gene MAF z-score p Model p Model Adj rs1879417 NOS1 49.0 -2.659 0.008 Additive 0.018 rs2682826 37.0 0.919 0.358 Recessive 0.589 rs10459953 NOS2 29.5 -1.022 0.307 Additive 0.522 rs2297518 25.3 -2.163 0.031 Recessive 0.066 rs10277237 NOS3 29.0 -0.574 0.566 Dominant 0.805 rs1799983 32.5 0.743 0.457 Dominant 0.703

Model refers to the most likely genetic model among the dominant (DOM), recessive (REC) and additive (ADD) ones. p refers to model the p value for the most likely genetic model. p refers to the p value adjusted for multiple comparisons (due to the three different Adj tested genetic models). z-score represents the z-statistics for the regression analyses

different cognitive brain areas of aged rats and senescence accelerated mice (SAM), a murine model of age-related cognitive impairment and deterioration of learning and memory (Law et al. 2002; Colas et al. 2006; Han et al. 2010). It is also of interest that the rs1879417 is located in the promoter region of NOS1, upstream of a fairly complex transcriptional regulatory region (Bros et al. 2006). Polymorphisms in this region, which affect gene expression and neuronal transcriptome, have been associated with neurological disorders such as schizophrenia and Alzheimer’s disease (Reif et al. 2011a, b). Based on this, it is conceivable that the association observed might reflect the linkage disequilibrium between the rs1879417 and these functional variants.

Discussion

Nitrosative stress is now widely recognized as a significant causal factor in the physiological decline that characterizes the aging process in many tissues. Age-related alterations in nitric oxide synthase activ- ity and/or expression and the consequent alterations in NO production and bioactivity are likely to contribute to this decline.

In the present study, we provide evidence that the variability of NOS genes affects common age related phenotypes and longevity. We found an association between NOS1-rs1879417 (p = 0.008; p = Model Adj 0.018) and NOS2-rs2297518 (p = 0.031; p = Model Adj 0.066) and longevity, indicating that variation in these genes may influence human aging and lifespan.

As for NOS2 gene, we also found that the rs2297518 has a modest impact on HG strength, one of the most effective death predictor in the elderly.

range 65–91 years showed that the same variants were also weakly associated with geriatric parameters that are predictors of the age related physiological decline. In particular, the NOS1 rs1879417-C allele, that we found associated with a lower probability of survival to very old age, was also associated with lower cognitive performance (MMSE score \ 23). This association is in line with several evidences. First, it is well established that the glutamate–NO–cGMP pathway plays a role in learning and memory processes and cognitive performance during aging (Domek- Łopacin´ska and Strosznajder 2010; Paul and Ekamba- ram 2011). Second, recent studies indicate that nos1 knockout mice have impaired cognitive functions (Kirchner et al. 2004; Weitzdoerfer et al. 2004; Zoubovsky et al. 2011). Third, changes in the expres- sion and/or activity of NOS1 have been observed in

that the NOS2/NO/ONOO– overproduction induced by TNFa via NF-jB may be one of the underlying causes of sarcopenia (Hall et al. 2011). Our finding that the rs2297518-A allele, which reduces the chance of survival to very old age, is associated with higher HG scores seems somehow in contrast with the idea that higher HG strength is predictive of increased survival in the elderly population. This result is just weakly significant, and certainly needs to be verified. On the other hand it may reflect pleiotropic contrasting effects and functions of the iNOS NO in different cell types In this view, it can be hypothesized that the possible harmful effect exerted by the rs2297518-A allele in some cell-types (for instance a low NO production in macrophages) is more relevant for

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