DNA sources and genotyping

Genomic DNA from controls was extracted from peripheral blood lymphocytes using a KG-Midi extraction kit (Camgen, Cambridge, UK). DNA from octogenarians was obtained from buccal swabs using the BuccalAmp DNA extraction kit (Epicentre,

5' regulatory region10126134516035 (A/G)E “)uf Je laid 1bB Controls =I «PolyA (S/U)1517878969 (A..5/A.,.)Bsmi (B/b)151844410 (N/6)_4 63 5 79bedOctogenarians

Fig. 1 Gene structure of the VDR locus and linkage disequi- librium (LD) pattern of populations analysed. (a) Graphical representation of the VDR gene with the location of polymor- phisms selected. Polymorphisms are named according to rs code and alias. Small open arrows indicate alternative VDR promoters that incorporate alternative exon 1 (1a–1f). Grey boxes correspond to untranslated exons, empty boxes to coding exons. UTR untranslated region. All markers were coded as biallelic including the rs17878969 microsallite polymorphism that shows a biphasic distribution of short (\18 adenosine repeats, S) and long (C18 adenosine repeats, L) allele

populations (Ingles et al. 1997). (b) Graphical representation of the LD pattern in Controls and Octogenarians. D0 values were calculated and plotted by Haploview 4.2 software (http:// www.broad.mit.edu/mpg/haploview). Numbers inside boxes correspond to D0 9100. Block structure was considered for marker pairs showing D0 [0.8, following the ‘‘solid-spine’’ block definition implemented in Haploview. In controls, two blocks were identified, block-1 at the 50 regulatory region including markers rs4516035–rs11568820 and block-2 at the 30UTR region including markers rs17878969–rs1544410. No block pattern was observed in octogenarians

Comparison of global haplotype distribution between controls and octogenarians was done by the ‘‘product of approximate conditionals’’ (PAC)-likelihood test implemented in PHASE 2.1.1 (Li and Stephens 2003). Differences between groups for specific hap- lotypes were evaluated by 2 9 2 contingency table analysis performed by the OpenEpi web resource.

differences in genotype and allele distribution between octogenarians and controls, considering all subjects, gender between groups and gender within each group (Table 1). No statistical differences were observed for single-marker genotype association modelled as dominant (common allele homozygotes and heterozygotes versus rare allele homozygotes), recessive (rare allele homozygotes versus common allele homozygotes and heterozygotes) and codomi- nant (cross genotype comparison). Allele distribution did not differ in any comparison performed (Table 1). Linkage disequilibrium pattern in the control

Results

The genotype and allele distribution conform to Hardy–Weinberg equilibrium for all markers in both the control and octogenarian samples. We evaluated

Linkage disequilibrium pattern in the control sample was in accordance with available data for Caucasian populations (Fang et al. 2005) and block

structure (D0 [ 0.8) was observed for markers rs11568820–rs4516035 (block-1, Fig. 1b) and mark- ers rs1544410–rs17878969 (block-2, Fig. 1b). Exon- 2 polymorphism (rs10735810) did not show any significant D0 with the 50 or 30UTR markers. In contrast, a modest D0 value (D0 \ 0.68) was observed in the octogenarian sample (Fig. 1b).

markers (block 1 and 2, respectively). No statistical differences were observed for 50UTR haplotype distribution (rs11568820 and rs4516035, block 1) between controls and octogenarians. However, sta- tistically significant differences were observed for global 30UTR haplotype distribution (rs1544410 and rs17878969, block 2) between controls and octoge- narians (PHASE PAC-likelihood test P = 0.01) (Fig. 2a), mainly due to differences in male subjects

According to the observed LD pattern, were independently inferred from 50 to

Table 1 Genotype and allele distribution