The Model-based multifactor dimensionality reduction (MB-MDR) algorithm was applied to explore interaction effects between analyzed polymorphisms on phenotypes.

For each polymorphism of the NOS genes, allele frequencies were estimated by counting genes from observed genotypes. The Hardy-Weinberg equilibrium (HWE) was tested using the exact test proposed by Wigginton et al. (2005). Pairwise measures of linkage disequilibrium (LD) between the analyzed loci were calculated with Haploview 4.2 (Barrett et al. 2005). The amount of LD was quantified by Lewontin's coefficient (D0).

Table 2 summarizes the main characteristics of the analyzed sample stratified by age-group as previously defined. Supplementary Table 1 shows the genotype frequencies in cases and controls. All SNPs followed HWE in the control group (p [0.05). No linkage disequilibrium was detected at any of the loci.

The RobustSNP algorithm was adopted to test the association between the analyzed phenotypes and NOS genes' variability. Different genetic models (dominant, additive, and recessive) were used to test the association using for each SNP the minor allele as reference. Table 3 shows results obtained using the RobustSNP algorithm. The NOS1 rs1879417 and NOS2 rs2297518 were significantly associated with longevity after adjusting for sex. The additive model for rs1879417 and the recessive one for rs2297518 resulted to be the most likely genetic models in which the presence of relevant alleles decreases the probability to attain longevity.

The variability of NOS genes affects common age-related phenotypes and longevity. NOS1 rs1879417-C allele was associated with a lower probability of survival to very old age and lower cognitive performance (MMSE score \ 23). This association is in line with several evidences. First, it is well established that nitrosative stress is a significant causal factor in physiological decline characterizing aging in many tissues. Second, age-related alterations in nitric oxide synthase activity and/or expression and the consequent alterations in NO production and bioactivity contribute to this decline.The glutamate-NO/cGMP pathway plays a role in learning and memory processes, cognitive performance during aging, and depression (Domek-opacinska and Strosznajder 2010; Paul and Ekamba-ram 2011). Recent studies indicate that nos1 knockout mice have impaired cognitive functions (Kirchner et al. 2004; Weitzdoerfer et al. 2004; Zoubovsky et al. 2011). Changes in the expression and/or activity of NOS1 have been observed, suggesting 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 somewhat in contrast with the idea that higher HG strength predicts increased survival in the elderly population. This result is weakly significant and needs to be verified. On the other hand, it may reflect pleiotropic contrasting effects and functions of 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, low NO production in macrophages) is more relevant for some phenotypes than for others.

For two of the six variants analyzed, we found an association with geriatric conditions but not with longevity. Specifically, the SNP rs2682826 in the NOS1 gene and the SNP rs10277237 in the NOS3 gene were respectively associated with GDS (p = 0.015; p = 0.033) and Activity of Daily Living (p = 0.002; p = 0.004), two of the main factors affecting quality of life in older individuals. Depression is a complex disorder where inflammation, oxidative/nitrosative stress, and reduced hippocampal neurogenesis contribute substantially (Maes et al. 2009). It has been found that NOS1-derived NO negatively affects neurogenesis and that the overexpression of NOS1 in the hippocampus following chronic mild stress exposure suppresses hippocampal neurogenesis and induces depression (Zhou et al. 2007). We found that carriers of the minor rs2682826-T allele have a higher probability of being depressed. In line with our observation, the rs2682826-T allele has been found to be a susceptibility factor for recurrent depressive disorder and for traits where depression is a comorbidity factor such as Parkinson disease (Gaecki et al. 2011; Hancock et al. 2008). The rs2682826 is located in the 3'UTR of exon 29, downstream from the translation termination site of NOS1 (C276T). Differences in protein production elicited by this variation, which lies close to several miRNA binding sites (Ibarrola-Villava et al. 2011), could be at the basis of the observed association.