OR odds ratio, CI confidence interval, nr not retained in the model

complete linkage disequilibrium with rs422858. Thus, we sought to investigate whether genetic variants in AGTR1 promoter could affect AT R receptor pres- 1 ence on peripheral blood leukocytes by FACS. Due to the low frequency of subjects homozygous for the minor alleles of rs422858 and rs275653, we could identify one Italian centenarian with this ge- notype alive at the time of laboratory determina- tions, so we decided to extend the observation of AT R presence to younger controls. Three groups 1 of subjects, each including one centenarian and four younger controls and differing for their geno- type at rs275653 (GG, AG and AA), were evalu- ated. The three groups of subjects were comparable for total cholesterol (GG 227±48 mg/ dL; AG 198±30 mg/dL; AA 185±24 mg/dL) and lipids. By FACS analysis, we found specific ex- pression of AT R both on PMNs (57.5±14 %) and 1 on PBMC (17.3±8.9 %), which include mono- cytes, B and T cells. A representative flow cyto- metric analysis of AT R on PMNs and on their 1 CD16+ subset is shown in Fig. 2a, b. In PMNs of subjects homozygous for the minor rs275653 var- iant, significantly less cells positive for AT R (GG: 1 mean 42.8±8.8 %, median 43.5 %) were found as compared to heterozygous (AG: mean 68.9±10.9 %, 0 median 66.8 %, P 0.0024) or homozygous individuals for the common allele (AA: mean 60.7±10.4 %, median 61.0 %, P0 0.0185) (Fig. 2c). Analysis of AT R cell 1 surface presence in the PMN CD16+ fraction showed significantly lower percentage of AT R-positive cells in 1 subjects homozygous for the minor rs275653 allele (GG: mean 68.0±8.1 %, median 64.4 %) in respect to heterozygous (AG: mean 87.0±6.1 %, median 87.4 %, 0 P 0.0030) or homozygous subjects for the common

0 allele (AA: mean 81.1±4.5 %, median 80.5 %, P 0.0134) (Fig. 2d). AT R protein levels in PBMCs 1 showed a similar trend as in PMNs; however, differ- ences among rs275653 genotype subgroups did not reach statistical significance (AT R-positive cells in 1 PBMC according to genotype GG: mean 16.9±5.7 %; AG: mean 20.5±7.7 %; AA: mean 19.3±7.1 %). Failure to find a statistical difference in the percentage of AT R-positive cells among genotypes is likely attribut- 1 able to the high individual variability along with the small number of subjects analysed.

Association of rs275653 and blood pressure

High blood pressure is associated with severe compli- cations, including heart, kidney disease and stroke, which reduce life expectancy in humans. The renin– angiotensin system is involved in blood pressure reg- ulation and vasoconstriction to the extent that knock- ing out the AT R gene causes hypotension in mice 1 (Oliverio et al. 1998). Based on this observation, we evaluated whether rs275653 was associated with changes in blood pressure.

Centenarians with GG genotype of rs275653 had lower blood pressure, either systolic (SBP), diastolic (DBP) or mean arterial pressure (MAP) with respect to centenarians with AA genotype. This difference was statistically significant for DBP under recessive (P0 0.040) and additive model (P0 0.031) as well as at allele analysis (P0 0.037) (Fig. 3). A significant difference was observed for SBP and MAP at allele analysis. In control subjects, systolic blood pressure levels were slightly reduced in subjects with rs275653 GG geno- type; however, the difference did not reach a statistical significance (not shown).

18275653count Al     b€ ;oj 4Q aj° ‘a 7a TASnegative _—— FITCcontrol T,Rc d *» 100 * » 100) —#*#Zz “ici o= 80 O 80Oo6 60 <3 60aw 40 kz 40ke =< 20 & 20* 0 x oOrs275653 GG AG AA GG AG AAn=5 n=5 n=5 n=5 n=5 n=52. AT,R expression in PMNs and in the CD16" fraction ofPMNs. Overall differences were evaluated by |

PMNs. Overall differences were evaluated by ANOVA 0.0036); individuals with GG genotype had significantly lower expression of AT R as compared to those with AG (P00.0024) 1 and AA (P00.0185) genotypes. No statistically significantly dif- ference was observed between AG and AA genotypes. d AT R 1 surface expression (percentage of positive cells) in CD16+ fraction of PMNs. Overall there was a statistically significant difference among groups (P 00.0016). AT R expression in the group ANOVA 1 of subjects homozygous for the minor variant of rs275653 was lower in respect to AG (P00.0030) and AA (P00.0134) groups. Each column shows the mean±SEM

Fig. 2 AT R expression in PMNs and in the fraction of 1 PMNs of subjects according to rs275653 genotype. Evaluation of AT R expression in controls was done twice to correct for 1 inter-assay variability, and the means of the two evaluations were considered in the statistical analysis. Centenarians were analysed once. Representative flow cytometry analysis of AT1R expression on total PMNs (a) and CD16+ fraction of PMNs (b) in subjects with different genotypes for rs275653 locus: homozygous for minor (GG) and major (AA) alleles and heterozygous (AG). On the left graphs, the fluorescence of negative controls is shown. c Percentage of cells positive for AT R surface expression in 1

findings are sustained by data obtained in the cohort of centenarians and semi-supercentenarians of Asian de- scent, thus ensuring a validation on a genetically di- verse population.

Discussion

Here we have identified a region in the promoter of human AGTR1 associated to extreme longevity. Stud- ies on a test group of Italian centenarians and controls provided evidence of a significant association between SNP variants of AGTR1 and human lifespan. These

Although studies in short-lived organisms and in human isolated populations with exceptional lifespan have clearly shown that genes play a role in longevity,

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