The frequency of Klotho KL-VS polymorphism was studied in a large Italian population, ranging from young subjects to centenarians, to determine the specific time windows for its effect. Laura Invidia, Stefano Salvioli, Serena Altilia, Michela Pierini, Maria P. Panourgia, Daniela Monti, Francesco De Rango, Giuseppe Passarino and Claudio conducted the study. The Klotho gene expression defect in mice causes aging-like phenotypes, including short lifespan, osteoporosis and atherosclerosis. Conversely, overexpression of the gene suppresses aging and extends lifespan. While the effect of KL-VS on human longevity has been conflicting in previous studies due to different age groups being analyzed, this study found a significant increase in the heterozygous Klotho genotype in elderly people compared to young controls. However, no difference was present between centenarians and young controls. Thus, this non-monotonic trajectory suggests that this KL-VS heterozygous genotype is favorable for survival in old people but its beneficial effect decreases thereafter and becomes no more evident at extreme ages. These unusual age-related changes in Klotho KL-VS genotype frequency support the hypothesis that alleles and genotypes involved in aging and longevity may exert their biological effect at specific time windows. The study analyzed a large Italian population sample including people from very young to very old age (centenarians). DNA was extracted from peripheral blood mononuclear cells (PBMCs) using phenol/chloroform according to standard procedures. Since all six SNPs occur in perfect linkage disequilibrium, a single variant, 1155G[A can be used to tag the haplotype. The presence of G (wild-type allele) and/or A (KL-VS allele) at position 1155 in exon 2 of the Klotho gene was determined by a PCR amplification using the relationship S(x) = PS (x) ? (1 - P)S (x), where S(x) is the survival function of the whole population and P (1 - P) is the initial frequency of carriers (non-carriers) in the population before the survival selection begins to operate consistently. The study found that Klotho gene variants were associated with some features of the aging process, such as osteopenia/osteoporosis and atherosclerosis.following primers: Fw 50-AggCTCATgCCAAAg TCTgg-30 and Rv 50-gTTCCATgATgAACTTTTT gAgg-30; PCR conditions: 4000 at 95°C, 4000 at 61°C and 2 min at 68°C, for 29 cycles. The PCR product was digested by Acs I enzyme (Roche Diagnostics) for 5 h at 50°C and visualized after electrophoresis on 2% MS gel (Roche Diagnostics).

Results
To compare genotype distributions of KL-VS variant, we subdivided our sample into three gender-specific age classes as described in materials and methods (Table 1). These classes have been previously introduced on the basis of a synthetic survival curve constructed using historic mortality data taken from the Italian population from year 1890 onward (Passarino et al. 2006; Cardelli et al. 2008). In each age class and in each gender, the observed genotype frequencies were in agreement with those expected at Hardy-Weinberg equilibrium (P < 0.05).

Genotype frequencies of Klotho variant were tested for Hardy-Weinberg equilibrium in all subjects using Arlequin (V 3.11) software to check whether our experimental sample was representative of the Caucasian population. Analyses were performed using the SPSS statistical package (V 13.0). Linear regression analysis was then applied to genotype frequency data. Statistical significance was set at P < 0.05.

The genetic-demographic analysis was carried out on the whole sample categorized according to the KL-VS polymorphism. Figure 1 shows the maximum likelihood estimates of the survival functions of heterozygous subjects (blue curve) and of wt homozygous subjects (red curve), compared to the general population (green curve). We noted that the survival function of heterozygous subjects crosses twice the survival function of the wt homozygous subgroup, roughly around the ages of 70 and 90 years. This trend suggests that the heterozygous genotype has a different effect on survival at different ages. In particular, it indicates that heterozygous subjects have a higher chance to survive beyond 70 years of age than the general population; on the other hand, among old subjects, the chance of heterozygous subjects to survive beyond 90 years seems to be lower than the rest of the population.

Discussion
The studies regarding the role of Klotho gene variants in human longevity performed by the groups of Dietz and Puca arrived at contrasting results. The group of Dietz basically reported a survival advantage for the KL-VS/wt heterozygous genotype in elderly people, while the group of Puca reported no difference between young and long-living subjects. In this study, we analyzed the distribution of Klotho KL-VS genotypes in a large group of Italian people of different ages, from young to centenarians. We predicted in a previous work that changes in cohort mortality may influence the results of a genetic analysis (Yashin et al. 1999), and consequently, we subdivided the study group into three age classes based on demographic data, as indicated by Passarino et al. (2006), who considered gender-specific life expectancies. This approach allowed us to compare genotype frequency without introducing arbitrary age classes, as in previous studies on TNF-alpha polymorphisms and human longevity (Cardelli et al. 2008).