Results

the Italian cohort, we sequenced exons and putative regions of AGTR1 to determine if any gene which may alter the amino acidic sequence or regulation, is differently represented in vs. younger controls. We identified eight previously reported variants (Fig. 1 and Online 2), including a non-synonymous variant leading to amino acid substitution at posi- 298 (p.Asn298Ser), which was found in a centenar- and not in controls. This change occurs in the transmembrane domain of AT R and is pre- 1 to be damaging in silico. In addition to the rare we have also observed ten known SNPs during sequencing of AGTR1, and therefore, we have eval- their distribution in centenarians and in controls 1; Table 1). Of the ten SNPs observed, two were in HW equilibrium and were removed from further The rs422858 is a dinucleotide change (AG/ but for practical reasons here, the major allele AG “ ” “ ” defined as A and the CC allele as C (Erdmann al. 1999). Single marker analysis in the eight SNPs passed the HW equilibrium test revealed signifi- different rs422858 and rs275653 genotype distri- 0 between centenarians and controls (P 0.004 for markers, Table 1). Rs422858 and rs275653, locat- − in the promoter region of AGTR1 at 174/173 bp and − 113 bp, respectively, are in complete linkage dis- Under the recessive model, the minor of rs422858 and rs275653 were significantly 0 to extreme old age (unadjusted P 0.003; 0 0 P 0.021; OR 4.55) (Table 1). significant difference was observed for rs422858 rs275653 also under the dominant and the additive but this difference did not pass the Bonferroni

Statistical analysis

Quantitative variables were expressed as mean±SEM. ANOVA and T test were used to compare continuous variables. Categorical variables were analysed by chi- square test. If n<5, exact Fisher test statistics was “ ” used. In the presence of a category with 0 frequency, the statistical analyses were performed using Elrm software for R (Zamar et al. 2007). Markers deviating – from the Hardy Weinberg (HW) equilibrium in the control population (P<0.05) were excluded from the analysis. Odds ratios (OR) and 95 % confidence inter- vals were analysed using SISA statistics.

Haploview software v4.1 (Barrett et al. 2005) was used for blocks definition, haplotypes estimation and allelic association tests. Nominal P values were reported and were corrected for multiple testing by permutation 100,000 times. The Bonferroni correction was applied to adjust for multiple testing when appro- priate. Logistic regression analysis was performed using MedCalc software. Level of significance was set at a P value <0.05 assuming a two-tailed model.

Bioinformatic analysis

Rs2307085, located on exon 1, was nominally sig- 0 nificantly associated to longevity at both genotypic (P 0 0.022) and allele levels (P 0.023) (Table 1). Rs422858 and rs275653 together with rs2307085 were found to be in a single Haplotype block (Fig. 1, Online Resource 3).

The sequence variants found in AGTR1 promoter were analysed for potential transcription factors binding sites using TESS (http://www.cbil.upenn.edu/cgi-bin/ tess/tess). The effect of base substitution on protein

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0 0 to longer lifespan (P 0.0092; OR 1.56) even after 0 correction for gender (P 0.00083). For each SNP ex- amined, there was no significant difference in the allelic and genotypic distribution between males and females.

In this block, the haplotype A-A-C was associated with 0 0 a reduced lifespan (nominal P 0.007, permuted P 0.024); consistently, the minor C-G-C haplotype was 0 nominally associated with longevity (P 0.028) (Online Resource 3). Stepwise logistic regression analysis showed a significantly different distribution of the rs275653 (and the linked rs422858) genotypes among 0 centenarians and controls (P 0.0066), while no signif- icance was observed for all the other SNPs. After adjust- ing for gender, rs275653 exhibited a stronger effect than 0 without adjustment (P 0.00095).

These results prompted us to validate the association data between variants of rs275653 and longevity in a Japanese population of 589 centenarians/semi-supercentenarians and in younger individuals whose age was comparable to that of Italian controls. We chose to study a population of different ethnic origin because the Japanese population is

Logistic regression of the allele distributions of all markers confirmed the previous findings, with only the minor allele of rs275653 being significantly associated