A moderate reduction in the intake of calories (also known as caloric restriction [CR]) is extremely effective in delaying age-related decline and increasing longevity in organisms ranging from yeast to mammals.16–19 In this regard, Bonkowski and colleagues observed a striking difference in the response of growth hormone (GH) receptor/GH-bind- ing protein knockout (GHRKO) and normal mice to an iden- tical regimen of CR.20 When male and female longevity data were analyzed separately, it became evident that the effects of CR on maximal longevity were sexually dimorphic in GHRKO mice but not in normal mice.

Materials and Methods

Subject features

This case–control study was carried on as a part of the SICS. The SICS (2002) assembled a large cohort of 600 DNA samples of nonagenarians/centenarians (age range 90–109 years) and 800 DNA samples of young controls (age range 18–48 years) from a isolated region of southern Italy east of Naples with a high prevalence of longevity and health and characterized by a high level of endogamy.23 All participants for the current study were well characterized; in fact, de- tailed phenotypic information about self-reported health his- tory and cognitive and physical functions measured through Blessed memory tests and Barthel scores of activity of daily living; demographics and exposure to common risk factors such as alcohol and smoking were also collected. For the aim of the present investigation, 281 long-lived men (age range 90–108 years) and 195 controls (age range 18–48 years) were genotyped (Table 1).

It should be noted that susceptibility to many common diseases, as well as longevity, may well be the result of com- plex interactions involving genes, environmental factors, and, intriguingly, gender. In this scenario, FOXO is a key component of the insulin pathway, free radical production, and human longevity.

In the prospective population-based Leiden 85 plus Study (866 females and 379 males, aged 85 years or more), the ef- fect of genetic variants in FOXO1A and FOXO3A were ana- lyzed on metabolic profiles, fertility, fecundity, age-related diseases (i.e., diabetes, myocardial infarction, etc.), and mor- tality.21 In detail, FOXO3A haplotypes showed no associa- tion with longevity and other investigated variables. One possible drawback of this finding is total absence of gender stratification. In fact, the hormonal environment as well as tissue-specific gene expression is known to differ signifi- cantly between genders in vertebrates. Hormones may dif- ferentially affect gene expression in somatic tissues, thus leading to the gender-specific susceptibility to a complex phenotype, such as longevity.20 In this regard, Willcox et al.22 performed a nested case–control study of five candidate longevity genes (ADIPOQ, FOXO1A, FOXO3A, SIRT, and COQ7) with links to the insulin–IGF signaling pathway (IIS). This nested case–control study was conducted in an ethnic Japanese population from the Island of Oahu, Hawaii, with 213 male longevity “cases” and 402 male “average-lived” controls. Three single-nucleotide polymorphisms (SNPs) were found to be associated with the “longevity” phenotype   at the locus of the FOXO3A gene: rs2764264 (p 0.0002),

The subjects were enrolled by an Italian subsidiary (As- sociazione Longevità) of Elixir Pharmaceuticals (former Cen- tagenetix Inc). All subjects donated blood samples for DNA study and gave written informed consent to the study. The study was conducted in accordance with the ethical princi- ples that have their origins in the Declaration of Helsinki.

Genotyping by the Illumina BeadChip system

Genotyping was carried out using the Infinium II Assay- HumanHap BeadChip 317K-duo system (Illumina) using standard protocols. In fact, Illumina’s Infinium II Whole-Ge- nome Genotyping Assay is designed to interrogate a large number of SNPs at unlimited levels of loci multiplexing. The HumanHap 317-Duo workflow can be divided in three main

TABLE 1. AGE (YEARS) DISTRIBUTION BY GENDER AND BY CENTENARIAN/CONTROL PHENOTYPE

Males Females   I+ Phenotype n Mean SD Min–Max n Mean SD Min–Max I+   Centenarians 281 94.96 2.97 90–108 199 98.05 3.25 90–109   Controls 195 33.53 7.33 18–48 140 31.39 7.31 18–48

n, number of individuals; SD, standard deviation; Min, minimum age; M, Maximum age.

segments: (1) sample preparation; (2) sample fragmentation and hybridization; (3) extension, staining, and scanning.

We used the OR and 95% CI for homozy- gous minor versus homozygous major alleles from the cur- rent analysis and from Willcox et al. study to calculate the natural logarithms of the OR (Log-OR) and its standard er- ror (SE). The Log-OR estimates were combined to obtain a summary OR following fixed26 and Der Simonian and Laird random effects27 models using inverse variance calculations. Between-dataset heterogeneity was identified using the I2 metric for inconsistency,28 and its statistical significance was assessed by means of the (cid:3)2 distributed Q statistic 29 (the   number of df is given by k 1, where k is the number of an- alyzed datasets). All calculations have been performed us- ing R v2.7.1 software.