Materials and Methods

In the Italian group AGTR1 exons, their flanking regions, the 5'- and 3'-UTR regions (including 250 bp of the promoter upstream 5'-UTR) were amplified from genomic DNA and sequenced in the two directions on anABI 3730 DNA analyzer (Applied Biosystems) was used in this study. Primers (Online Resource 1) designed according to the AGTR1 genomic reference NC_000003, built 37.1, at the National Center for Biotechnology Information. Variants are referred to transcript variant 4 (NM_031850) RNA reference sequence and numbering begins with the ATG start codon. For quality control purposes, a 5% random sample was sequenced twice. Moreover, subjects carrying very rare genotypes (<5%) were confirmed by repeating PCR reactions, followed by resequencing of both DNA strands. Additionally, the authors demonstrate that allelic variants in the promoter of AGTR1 are associated with reduced AT R 1 receptor expression levels in peripheral blood neutrophils. In addition, screening of the whole coding region of AGTR1 in an Italian cohort disclosed a novel mutation leading to amino acid substitution in one centenarian. Finally, an association between variants in AGTR1 promoter and diastolic blood pressure in centenarians was found.
 
Methods: For the replication study in the Japanese group, SNP genotyping was performed by TaqMan SNP assay (Applied Biosystems). The genotyping conditions for a cohort of 173 Italian centenarians (between 99 and 106 years of age, mean 100.91 ± 1.7 years) living in Italy were used. Analysis of AT R presence in peripheral leukocytes: Fresh blood samples were processed for peripheral blood mononuclear cells (PBMC) and polymorphonuclear cells (PMNs) purification as described (Noris et al., 1999; Noris et al., 1996), labeled with an anti-human AT R antibody (Anti AT-1, N-10, Santa Cruz), followed by incubation with fluorescein isothiocyanate goat F(ab)2 fragment anti-rabbit IgG (eBioscience). To identify the various leukocyte subsets, cells were stained with fluorochrome-conjugated mAb antibodies against CD45, CD16, CD14, CD3, CD4 (BD Bioscience and Biolegend). The samples were analyzed by fluorescence-activated cell sorting (FACS) analysis using FACSAria cytometer (BD Bioscience). Results were expressed as a percentage of AT R 1 positive cells (percent).
 
Results: In the Italian cohort, exons and putative regions of AGTR1 were sequenced to determine if any gene which may alter the amino acidic sequence or regulation is differently represented in vs. younger controls. Eight previously reported variants (Fig. 1 and Online 2), including a non-synonymous variant leading to amino acid substitution at position 298 (p.Asn298Ser), which was found in a centenarian and not in controls, were identified. This change occurs in the transmembrane domain of AT R and is predicted to be damaging in silico. In addition to the rare variants, ten known SNPs were observed during sequencing of AGTR1, and therefore, their distribution in centenarians and controls was evaluated (Table 1). Of the ten SNPs observed, two were in HW equilibrium and were removed from further analysis. The rs422858 is a dinucleotide change (AG/CC), but for practical reasons here, the major allele AG is defined as A and the CC allele as C (Erdmann et al., 1999). Single-marker analysis in the eight SNPs passed the HW equilibrium test and revealed significantly different rs422858 and rs275653 genotype distributions between centenarians and controls (P<0.004 for both markers, Table 1). Rs422858 and rs275653, located in the promoter region of AGTR1 at 174/173 bp and 113 bp, respectively, are in complete linkage disequilibrium. Under the recessive model, the minor alleles of rs422858 and rs275653 were significantly associated with extreme old age (unadjusted P<0.003; OR 4.55) (Table 1). A significant difference was observed for rs422858 and rs275653 also under the dominant and the additive models, but this difference did not pass the Bonferroni correction.
 
Statistical analysis: Quantitative variables were expressed as mean ± SEM. ANOVA and T-tests were used to compare continuous variables. Categorical variables were analyzed by chi-square test. If n<5, exact Fisher test statistics were 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 intervals were analyzed using SISA statistics.The processed text is:

AA.svGA+GG PIC%59ROFAMPIC%59ROPIC%59ROPIC%59ROPGGGAAAnoitalupoP 700.01.2 1.155.1522.0100.09.41 7.199.4830.02.2 0.194.1300.05.51 5.155.4400.0850.0533.0706.0eCsnailatI 851.0310.0092.0796.0oC 555.05.1 8.010.1290.0 a740.0 0.136.6000.14.1 7.00.1 a640.0 20.128.6770.0210.0061.0928.0eCesenapaJ 480.0000.0861.0238.0oC 797.03.1 8.030.

Introduction:
One of the most long-lived populations is the Japanese, with a high number of centenarians, and the rs275653 and rs422858 SNPs are in complete Linkage Disequilibrium (LD) in both Japanese and Italian subjects.

Results:
The minor allele frequency of rs275653 was lower in the Japanese population (0,084) compared to Italian controls (0,158). The GG genotype was significantly associated with longevity under the recessive and additive models in the Japanese cohort but not with the dominant model or at the allele level.
The GG genotype was enriched in both centenarians' groups from Italy and Japan compared to controls.
FACS analysis showed that individuals homozygous for rs275653 minor allele had significantly lower AT R surface expression on peripheral blood leukocytes than heterozygous or homozygous individuals for the common allele.
Centenarians with GG genotype had lower blood pressure, either systolic (SBP), diastolic (DBP) or mean arterial pressure (MAP) with respect to centenarians with AA genotype.