Materials and methods

human longevity, we analyzed a sample of Italian subjects divided into gender-specific age classes according to demographic information obtained from the survival function S(x) for the Italian population of the XX century (Passarino et al. 2006; Cardelli et al. 2008). In brief, the limit between the youngest class and the intermediate class has been chosen as the age at which the curvature of the survival function S(x) is negative and has the largest absolute value (formally, the age corresponding to the minimum value of the second derivative S00(x) of the survival function). Equivalently, the limit between the intermediate class and the oldest class was chosen as the age at which the curvature of the graph is positive and has the largest absolute value (formally, the age at which the second derivative S00(x) of the survival function attains the maximum value). The age classes defined according to this criterion are gender-specific and account for the different survival of men and women in the Italian population. For men [women], the first class consists of individuals aged\66 [\73] years, the second class of individuals aged 66–88 [73–91] years, the third class of individuals aged[88 [[91] years.

Human Klotho gene maps on chromosome 13q12, and its product is highly conserved (86% amino acid identity with the mouse Klotho protein) (Matsumura et al. 1998). A common variant, termed ‘‘KL-VS’’, composed of six single nucleotide polymorphisms (SNPs) in perfect linkage disequilibrium, spans exon 2 and its flanking sequence and is present in *15% of Caucasians (Arking et al. 2002). It has been suggested that the KL-VS allele influences the trafficking and catalytic activity of Klotho protein (Arking et al. 2002). In studies on humans, Klotho gene has been associated with some features of the aging process such as osteopenia/osteoporosis and atherosclerosis (Arking et al. 2002, 2003, 2005; Kawano et al. 2002; Low et al. 2005; Mullin et al. 2005; Yamada et al. 2005). Association studies of Klotho gene variants with

relationship S(x) = PS (x) ? (1 - P)S (x) where a b 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, that is before the survival selection begins to operate consistently (Vaupel and Yashin 1985). In the present case we analyzed the survival function of people carrying the KL-VS/wt heterozygous or KL-VS homozygous genotype in comparison with that of people carrying the wt homozygous genotype. The Matlab 6.1 package was used for genetic-demographic analyses.

A total of 1,089 (669 women and 420 men) unrelated individuals (age range: 19–109 years), born and residing in northern and central Italy, were recruited to participate in this study. Information about health status (based on their clinical history and on blood tests) was obtained for all participants, and no major pathological conditions were present (dia- betes, cancer, dementia, cardiovascular diseases). The sample included 238 centenarian subjects (191 women and 47 men), who were classified as healthy (categories A and B according to the criteria reported in Franceschi et al. 2000).

DNA was extracted from peripheral blood mono- nuclear 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 the exon 2 of the Klotho gene was determined by a PCR amplification using the following primers: Fw 50-AggCTCATgCCAAAg TCTgg-30 and Rv 50-gTTCCATgATgAACTTTTT gAgg-30; PCR conditions: 4000 at 95(cid:2)C, 4000 at 61(cid:2)C and 2 min at 68(cid:2)C, for 29 cycles. The PCR product was digested by Acs I enzyme (Roche Diagnostics) for 5 h at 50(cid:2)C and visualized after electrophoresis on 2% MS gel (Roche Diagnostics).

Results

To compare genotype distributions of KL-VS variant, we subdivide our sample into three gender-specific age classes as described in materials and methods (Table 1). These classes have been previously intro- duced on the basis of a synthetic survival curve constructed using historic mortality data taken from the Italian population from year 1890 onward (Pas- sarino 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).

quencies among the three above mentioned age classes. Univariate analysis indicates that in our sample the genotype frequencies are significantly different (P = 0.03). In particular the analysis shows a relevant increase of KL-VS heterozygous state in elderly subjects (age class 2) compared to young controls (age class 1). This result is in accordance with data previously published in Caucasian and Ashke- nazi-Jewish populations (Arking et al. 2002, 2005). On the contrary, no difference is present between long-

Genotype frequencies of Klotho variant were tested for Hardy–Weinberg equilibrium in all subjects using Arlequin (V 3.11) software in order to check whether our experimental sample was representative of the Caucasian population.

Analyses were performed using the SPSS statisti- cal package (V 13.0). Linear regression analysis was then applied to genotype frequency data. Statistical significance was set at P B 0.05.