Depression

The Geriatric Depression Scale (GDS) is an instru- ment designed to measure depression in the elderly consisting of a series of yes/no questions in reference to how they felt on the day of administration (Sheikh and Yesavage 1986). A score above five is suggestive of moderate depression, while a score above ten is suggestive of severe depression. In our analyses GDS scores were dichotomized as one if the subject showed depression symptoms (GDS [ 5) and zero otherwise.

applied to estimate how the variability of genes analyzed in the present study influences (i) the predisposition to human longevity (ii) the variability of functional parameters including cognitive function, functional performance and depression status. In particular, in the regression models used to test the association between the variability of analyzed genes and the human longevity, the variable sex was used as covariate In the association analyses involving the functional parame- ters, covariate adjustment included sex, age and height when HG scores were analyzed, only age and sex when disability and depression status were investigated. As mentioned before MMSE scores were normalized for age and educational status according to a standardized procedure reported in Grigoletto et al. (1999).

Cognitive performance

Mini mental state examination (MMSE) test was used to evaluate the cognitive performance in the analysed sample. It is a 30-item questionnaire that assesses orientation, episodic memory, attention, language, and construction functions (Folstein et al. 1975). Since the test is affected by age and educational status, the MMSE scores were normalized for these variables according to a standardized procedure (Grigoletto et al. 1999). In our analyses MMSE scores were dichotomized as 1 if the subject showed a normal cognitive function (MMSE C 23) and zero otherwise.

Interaction analysis

In order to explore the interaction effects between the analyzed polymorphisms on the analyzed phenotypes (longevity and functional parameters), the recently developed Model-based multifactor dimensionality reduction (MB-MDR) algorithm will be applied (Calle et al. 2010).

Statistical analyses

For each polymorphism of the NOS genes, allele frequencies were estimated by counting genes from the observed genotypes. The Hardy–Weinberg equi- librium (HWE) was tested using the exact test proposed by Wigginton et al. (2005). Standard errors for alleles were computed according to the hypothesis of the multinomial distribution. Pairwise measures of linkage disequilibrium (LD) between the analyzed loci were calculated with the Haploview 4.2 (Barrett et al. 2005). The amount of LD was quantified by Lewon- tin’s coefficient (D0).

Results

Genetic analyses

Table 2 summarizes the main characteristics of the analyzed sample stratified by age-group as previously defined. The age distributions in males and females are reported in supplementary Fig. 1.

Supplementary Table 1 shows the genotype fre- quencies in cases and controls. All SNPs followed the HWE in the control group (p [ 0.05). No linkage disequilibrium was detected at any of the loci.

Single-locus analysis

In order to test the association between the analysed phenotypes and the variability of the NOS genes the RobustSNP algorithm (a robust association test suit- able for both quantitative and binary traits which also takes into account covariates) recently proposed by So and Sham (2011) was adopted. This test was based on

Different genetic models (dominant, additive and recessive) were used to test association, using for each SNP the minor allele as reference. Table 3 shows

Table 2 Socio-demographic characteristics and functional parameters (ADL, HG, GDS and MMSE) in the total sample and stratified for group membership

Controls Cases Total (N = 442)a (N = 321)a (N = 763) Age Mean (SD) 64.9 (18.1) 96.2 (3.3) 78.2 (20.8) Range 19–91 89–107 19–107 Body mass index (BMI) Mean (SD) 27.1 (4.3) 23.3 (4.1) 25.1 (4.6) Range 15.01–45.35 12.98–40.55 12.98–45.35 Hand grip strengthb Mean (SD) 22.24 (9.40) 12.67 (6.47) 16.99 (9.24) Range 4.00–55.00 0.00–42.0 0.00–55.00 Activities of daily living (ADL)b [n (%)] Disable (\5) 55 (17.4) 220 (68.5) 275 (43.1) Non disable (=5) 262 (82.6) 101 (31.5) 363 (56.9) Geriatric Depression Scale (GDS)b [n (%)] Depressed (C5) 107 (33.8) 74 (23.1) 181 (28.4) Non depressed (\5) 210 (66.2) 247 (76.9) 457 (71.6) Mini mental state examination (MMSE)c [n (%)] Normal (C23) 202 (66.66) – Impaired (\23) 101 (33.33)

NOS gene polymorphisms and functional parameters

Subjects 65–91 years of age (N = 317) were further analyzed for examining the association between the variability of NOS genes and physical (HG and ADL) and cognitive (MMSE and GDS) abilities. Table 4 reports the results of the association tests for the three genetic models using the minor allele for each SNP as reference, and after adjusting for the appropriate parameters (see ‘‘Materials and Methods’’).

The NOS1 rs1879417 was found significantly associated with cognitive performance in an additive manner (p = 0.045) with subjects carrying the Model less frequent C allele showing lowest MMSE scores. However, after adjustment for multiple comparisons this association failed to reach statistical significance (p Adj = 0.093). In addition, we found that NOS1 rs2682826 variation significantly influenced the GDS performance (p = 0.015). Subjects with at least Model one T allele (less frequent allele) showed a significant higher probability to have depression symptoms than subjects homozygous for the allele G (dominant model). After adjustment for multiple comparisons this association remained statistically significant (p = 0.033). Adj

ADL scores were dichotomized as one if the subject was not independent in all 5 items and 0 otherwise. GDS scores were dichotomized as 1 if the subject showed depression symptoms (GDS[5) and 0 otherwise (see ‘‘Materials and Methods’’)