Supportive evidence for a role of NOSs in healthy aging and lifespan extension has come from studies with knockout models. It has been shown, for instance, that in mice lacking all three NOS isoforms the survival rate is reduced by 80 % (Tsutsui et al. 2009). Moreover, a reduction in survival rate of about 50 %, and an attenuation of caloric restriction-induced life extension has been observed in nos3 knockout mice (Dere et al. 2002; Nisoli et al. 2005).

Given this background, the present paper addresses the hypothesis that NOS genetic variants affect human aging and longevity. We analyzed common and potentially functional NOS SNPs in a sample group of 763 individuals from the south of Italy ranging from 19 to 107 years of age. Moreover, since the increase with age in ROS/RNS-mediated stress is accompanied by the deterioration of cognitive and physical abilities, which is one of the most important components of the quality of life in the elderly that adversely affects successful aging and longevity, we also explored the impact of NOSs variability on cognitive and physical performances. In this case, subjects aged 65–91 years, that is the age range where a significant increase of overall impairments occur, were analyzed.

Oxidative stress is a well established hallmark of the aging process and there is strong evidence for a causal role of NO and RNS therein (McCann et al. 1998; Drew and Leeuwenburgh 2002; Torregrossa et al. 2011). Markers of nitrosative stress injury, such as nitrosyla- tion and nitration of proteins, have been shown to increase as a function of age (Toprakci et al. 2000; Kanski et al. 2005; Kim et al. 2006). Moreover, a growing number of studies indicate that the availability and biological activity of NO is impaired during aging,

Materials and methods

Sample

The sample analyzed in the present study included 763 (417 females and 346 males) 19–107 years old subjects (median age 81 years). All subjects were born in Calabria (southern Italy) and their parents and grandparents were native of the same area.

The sample was collected during several campaigns of recruitment (De Rango et al. 2011). Subjects older than 65 years went through a multidimensional geri- atric assessment carried out by a geriatrician. In addition, each of these subjects was submitted to a home based interview by a trained operator, with the administration of a structured questionnaire for the collection of socio-demographic information, evalua- tion of physical, cognitive, depressive status, sensory deficits, medications, and self-reported health status. Subjects with dementia and/or neurologic disorders were not included. White blood cells (WBC) from blood buffy coats were used as source of DNA.

Table 1 Polymorphisms within the NOS genes analysed in the present study

Gene dbSNP ID Allele Physical Function symbol (major/ location annotation minor) NOS1 rs1879417 T/C Promoter rs2682826 C/T 30UTR NOS2 rs10459953 G/C 50UTR rs2297518 G/A Exon 16 Ser608Leu NOS3 rs10277237 G/A Promoter rs1799983 G/T Exon 7 Asp298Glu

Geriatric assessment

sample into two sex- and age-specific classes obtained according to the survival functions of the Italian population from 1890 onward (Passarino et al. 2006). In particular, the graphs of the second derivative of these survival functions indicated that at 88 years for males and 91 for females a significant change in the slope of these functions occurs. We used these two age and sex-specific cut-offs to categorize the sample into rationally defined ‘‘thresholds of longevity’’. For this reason in the present study men younger than 88 and women younger than 91 years will be defined as controls (N = 442), while men older than 88 and women older than 91 years will be defined as cases (N = 321).

The variability of genes that we tested for association with longevity was also investigated in relation to accepted biomarkers of the aging process that evaluate physical, cognitive, and psychological status in the elderly population. To this purpose a subgroup of the control sample whose subjects underwent a geriatric assessment (subjects 65–91 years of age) was further analyzed (N = 317).

Functional activity

The management of activities of daily living (bathing, dressing, toileting, transfer from bed to chair and feeding) was assessed using a modification of the Katz’ Index of ADL (Katz et al. 1970). The assessment was based on what the subject was able to do at the time of the visit. The score is given counting the number of activities in which the participant is dependent or independent at the time of the visit. In our analyses ADL scores were dichotomized as one if the subject was not independent in all five items and zero otherwise.

All the subjects gave their written informed consent to use their anonymous genetic and phenotypic data for genetic studies on aging and longevity. The recruit- ment campaigns received the approval of the Ethical committee of the University of Calabria.

Physical performance

SNPs selection

Hand grip (HG) strength was measured by using a handheld dynamometer (SMEDLEY’s dynamometer TTM) while the subject was sitting with the arm close to his/her body. The test was repeated three times with the stronger hand. The maximum of these values was used in the analyses. Since HG strength is affected by age, sex and height, the scores were corrected for these factors. When a test was not carried out, it was specified if it was due to physical disabilities or because the subject refused to participate.

from literature data, and using information from public databases (http://www.ncbi.nih.gov/, http://www.hap map.org/). The a priori selection was based on the following criteria: minor allele frequency (MAF) [10 % in Caucasians, putative functional significance (non-synonymous SNPs, SNPs located in the 50- and 30-UTR regions), SNPs previously investigated in association studies. For each gene the selected SNPs are reported in Table 1.

Depression