The aim of our study was to support the potential role of FOXO3A as a key component that influences longevity with a case-control study. We screened 480 long-living cases (281 males and 199 females) and 335 young controls (195 males, 140 females) from an isolated and homogeneous population of southern Italy (Southern Italian Centenarian Study, SICS) with an Illumina BeadChip 300K system and we focused on the FOXO3A gene. As previously described, we hypothesized that gender might influence the association between FOXO3A and longevity; thus, we analyzed males and. Infarction, etc.), and mortality.21 In detail, FOXO3A haplotypes showed no association with longevity and other investigated variables. One possible drawback of this finding is the total absence of gender stratification. In fact, the hormonal environment as well as tissue-specific gene expression is known to differ significantly between genders in vertebrates. Hormones may differentially affect gene expression in somatic tissues, thus leading to 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 (Associazione Longevit) of Elixir Pharmaceuticals (former Centagenetix Inc). All subjects donated blood samples for DNA study and gave written informed consent for the study. The study was conducted in accordance with the ethical principles 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-Genome 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 into three main segments: (1) sample preparation; (2) sample fragmentation and hybridization; (3) extension, staining, and scanning. We used the odds ratio (OR) and 95% confidence interval (CI) for homozygous minor versus homozygous major alleles from the current analysis and from Willcox et al. study to calculate the natural logarithms of the OR (Log-OR) and its standard error (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 chi-squared distributed Q statistic29 (the number of df is given by k - 1, where k is the number of analyzed datasets). All calculations have been performed using R v2.7.1 software.

The BeadChips were imaged using a two-color confocal laser system with 0.8-m resolution. The bead intensities were extracted, and genotypes were calculated using an Illumina-supplied cluster file, which is based on a set of reference samples. The normalization algorithm adjusts for nominal offset, cross-talk, and intensity variations observed in the two-color channels. The data for each BeadChip were self-normalized using information contained within the array. This normalization algorithm removes outliers, adjusts for channel-dependent background and global intensity differences, and also scales the data. The X and Y color channels undergo an affine coordinate transformation to make the data appear as canonical as possible, with the homozygotes lying along the transformed x and y axes. All genotypes were evaluated using a quantitative quality score called GenCall score. A GenCall score ranges from 0 to 1 and reflects the proximity within a cluster plot of the intensities of that genotype to the centroid of the nearest cluster.

Haplotype analyses

We used HaploView 4.030 for block definition, haplotype estimation, and association tests. We defined haplotype blocks using the Four Gamete Rule, a variant on the algorithm described in the Wang study,31 and we estimated haplotypes using the default accelerated expectation-maximization (EM) algorithm.30 Differences in haplotype frequencies between centenarians and controls were tested by means of chi-squared tests; haplotypes with a frequency <0.01 were excluded from the analysis.

Statistical analysis