Sources of vitamin-D come from dietary intake, mainly oily fish such as salmon, mackerel and sardines (Chen et al. 2007) and egg yolks (Lamberg- Allardt 2006), and from endogenous synthesis by photolytic reaction of vitamin-D precursors in the skin (Lips 2006). Both types of sources are strongly linked to environment, such as climate and latitude, and social behaviour, such as daily diet, sun exposure and clothing. In addition, demographic factors must be considered, such as culture habits, gender and age, which act in several ways to change social behaviour as well as race, which is related to melanin pigmen- tation (Chen et al. 2007). Genes coding for vitamin-D metabolic enzymes and downstream mediators of vitamin-D signalling constitute a complex gene network. Vitamin-D receptor (VDR) plays a pivotal role in this network, acting as a ligand-dependent transcription factor that modulates the expression of vitamin-D responsive genes (Pike 1991).

tant for designing association studies of complex phenotypes, as revealed by recent research on the linkage disequilibrium (LD) pattern of the human genome. LD refers to the non-random transmission of alleles of neighbouring loci that is high for closer loci and decreases with distance. In addition, recombina- tion rate is a determinant factor for decreasing LD. The underlying structure of LD in the human genome reveals a series of discrete blocks of high LD interrupted by hotspot recombination sites. LD blocks range from 5 to 50 kb and are characterized by a reduced diversity of allelic combinations (haplotypes) (Wall and Pritchard 2003). Studies of the VDR genomic region in Caucasians have revealed a

scattered LD pattern with rs1568820 and rs4516035 polymorphisms included in a single block of the VDR promoter and rs1544410 and rs17878969 polymor- phisms included in a 30UTR block. Blocks are separated by a hotspot recombination region that includes the rs10735810 polymorphism (Fang et al.

Madison, USA). VDR genotyping followed protocols described by de la Torre et al. (2008). For each subject we obtained genotype information from five polymorphic variants located in the VDR gene at: 50 promoter region, rs11568820 and rs4516035; coding region, rs10735810 and 30UTR, rs1544410 and rs17878969 (Fig. 1a). All markers were coded as biallelic, including the rs17878969 microsallite poly- morphism that shows a biphasic distribution of short (\18 adenosine repeats, S) and long (C18 adenosine repeats, L) allele populations (Ingles et al. 1997).

Several studies reveal that polymorphisms at VDR locus are associated with life-limiting diseases such as cancer, cardiovascular diseases and immune sys- tem disturbances (Uitterlinden et al. 2004b). There- fore, we propose VDR as a candidate gene involved in human longevity as a result of its protection from age-related diseases and modulation of the immune response. Taking advantage of the known haplotype block structure of the VDR genomic region, we performed an association study for longevity consid- ering both single genotypes and haplotypes.

Statistical analysis

We compared genotype and allele distribution between controls and octogenarians by contingency table analysis performed by the OpenEpi web resource (Sullivan et al. 2009). Fisher exact test P values were considered in each comparison. Correction for multi- ple testing was done applying the SNP spectral decomposition approach, which takes into account the linkage disequilibrium between markers (Nyholt 2004). The significance threshold required to maintain a Type I error rate of 5% was P = 0.036 for two- marker haplotypes and P = 0.011 for five-marker haplotypes. Pair-wise linkage disequilibrium was measured by the normalized D0 coefficient (Lewontin 1988) that was estimated using Haploview 4.2 software (Barrett et al. 2005). Absolute D0 values range from 0 (no disequilibrium), indicating that alleles are randomly distributed and all expected haplotypes are observed, to 1 (complete disequilib- rium), indicating a non random distribution of alleles with a less observed haplotypes than expected.

Materials and methods

Subjects

In total, 214 Caucasian subjects from the city of Lleida in northeast Spain were included between January and June 2004: 104 octogenarians ([85 years), 72 women (mean age 89.4 ± 3.6; range 85–100) and 32 men (mean age 89.4 ± 3.2; range 85– 97), and 110 sex-matched healthy young individuals, 75 women (mean age 28 ± 5.8; range 17–39) and 35 men (mean age 29 ± 8.6; range 17–40), were selected as cases and controls, respectively. A comprehensive medical history was obtained from each octogenarian, focussing on the absence of vitamin-D related illness such as osteoporosis, autoimmune diseases, immuno- deficiency and cancer. Written informed consent for enrolment in the study and for personal data manage- ment was obtained from all subjects in accordance with Spanish laws. The Ethics Committee for our institution approved the study.

We used PHASE 2.1.1 software (Stephens et al. 2001) to perform statistical inference of haplotypes from our sample data. Only data from fully geno- typed subjects, 86 octogenarians (25 males) and 101 controls (35 males), were considered. We used the ‘‘out_pair’’ PHASE output file to assign haplotypes for each individual, with their corresponding proba- bility. Haplotype counts were estimated by obtaining the cumulative probability of each haplotype. In brief, for an individual bearing haplotype A with probability 0.3 and haplotype B with probability 0.7, 0.3 and 0.7 were added to the corresponding counts of haplotypes A and B, respectively. Following the same rationale, we estimated the presence of 0, 1 or 2 copies of a specific haplotype in each individual.

DNA sources and genotyping