processing their data.

Abstract:
The study investigates genetic variations in genes that regulate energy homeostasis, damage repair, and signaling response to environmental changes or external signals that are critical in modulating the lifespan of organisms. The two-stage case-control study was performed in two independent sets of long-lived individuals from Calabria (Italy). In stage 1, 317 SNPs in 104 genes were analyzed in 78 cases (median age 98 years) and 71 controls (median age 67 years). In stage 2, 310 candidate SNPs identified in stage 1 were analyzed in an independent sample composed of 288 cases (median age 92 years) and 554 controls (median age 67 years). Two SNPs, rs282070 located in intron 1 of the MAP3K7 gene, and rs2111699 located in intron 1 of the GSTZ1 gene, were significantly associated with longevity in stage 2. Both genes are implicated in the cellular response to internal and external environmental changes, playing a crucial role in inflammation processes that accompany aging. The study confirms that long-lived subjects have genetic variants that allow them to optimize cellular responses and better deal with environmental and internal stresses.

Introduction:
Over the decades, developed societies have experienced a gradual increase in life expectancy, sharply increasing the percentage of elderly people in the population. This has led to an interest in understanding how individuals achieve a long and healthy life. Most gene-longevity association studies have so far focused on a single or a few candidate genes. However, given the complexity of the longevity trait, a single-locus approach may not be suitable as each locus is likely to exert a small effect on the trait. Thus, approaches have been developed that take into account complex traits caused by an intricate pattern of genetic variants where epistatic effects may be not negligible. Recent technological improvements in genotyping technology have made whole genome genotyping more accessible. Genome-wide association studies (GWAS) are at present widely used to find genetic variants contributing to variation in human lifespan. Along with GWAS, studies that consider candidate biochemical pathways consisting of many biologically related genes and a minimal number of tagging SNPs that efficiently capture all the common genetic variation in the assayed genomic region are also being routinely used. Family-based studies have demonstrated the existence of an important familial and genetic component of longevity. The offspring of long-lived subjects have a significant survival advantage and a higher probability to have been or to become long-lived. Twin studies found that approximately 25% of the overall variation in human lifespan can be attributed to genetic factors, which become more relevant at advanced ages. 

The study analyzed 317 SNPs in 104 selected genes involved in longevity pathways, including DNA repair, apoptosis and cell cycle regulation, metabolism of xenobiotics, insulin/IGF-1, and neuromodulation. A first sample (stage 1) was used to test all selected SNPs, and only those showing some hint of association were subsequently retested in a second sample (stage 2), thus reducing the number of association tests.

Materials and methods:
The study recruited a stage 1 sample between 1994 and 1997 in Calabria (southern Italy) that included 149 unrelated subjects of which 78 were considered as cases (age ≥90 years; median age, 98 years; 36 males and 42 females) and 71 as controls (age <90 years; median age, 67 years; 39 males and 32 females). A stage 2 sample was recruited subsequently between 2005 and 2008, also in Calabria, that included 842 unrelated subjects of which 288 were cases (age ≥90 years; median age, 92 years; 125 males and 163 females) and 554 were controls (age <90 years; median age, 67 years; 277 males and 277 females). The chromosomal region encompassing the candidate genes was identified through databases. The gene region was defined as the region from up to 5 kbp upstream of the transcription start site to up to 5 kbp downstream of the transcription stop site, allowing no overlap with the neighboring genes. In the choice of candidate SNPs, different online databases were used. Younger subjects were contacted through general physicians, while subjects older than 90 years were identified by screening population registers in different municipalities distributed across the entire Calabria region. All participants gave written informed consent before processing their data.