‘DBP (mmHg) ©SBP (mmHg) &MAP (mmHg) ©8070601451351251009080PP poe Hye Cee MyreGenotype Dominant Recessive Alleles

Japanese cohort, characterized by a large number of centenarians and semi-supercentenarians of homoge- neous ethnic origin, confirmed an enrichment of the G allele in centenarians and its association to longevity according to the recessive model. Combined analyses sustained the association at genotype level. Data of European and Japanese populations from 1000 Genomes and from HapMap databases as well as our results on the Italian group demonstrate that rs275653 is in complete linkage disequilibrium with rs422858. The absence of the rs275653 GG genotype in our Japanese controls diverges from the data of the small cohort of Japanese subjects in 1000 Genomes database (rs275653 GG genotype: one over 90 subjects). How- ever, comparison of the genotype prevalence in Japa- nese population of 1000 Genomes and in our Japanese controls failed to evidence any statistical difference both at genotype and at allele level.

variant of rs422858 belongs to an E-box element which is lost in the presence of the minor allele. Supportive evidence to our genetic data comes from ex vivo findings indicating that the longevity- associated variants have functional consequences on the expression of the corresponding receptor protein on the surface of circulating cells. Indeed, by FACS experiments, we demonstrated that PMNs of subjects homozygous for the minor rs275653 allele (and con- sequently for the C variant of rs422858) had a signif- icantly lower percentage of AT R-positive cells with 1 respect to subjects heterozygous and homozygous for the major allele. A similar difference was observed when the CD16+ PMN fraction was analysed. Consis- tently, previously reported transfection experiments in adrenal cortical and in vascular smooth muscle cells (Kumar et al. 2005) documented that reporter con- structs containing the A rs422858 variant had higher promoter activity than constructs with the minor C variant, suggesting that this SNP could affect the lev- els of AT R gene transcription. 1

Fig. 3 Blood pressure in Italian and Japanese centenarians according to rs275653 genotype. Data are represented as mean± SEM. a Diastolic blood pressure was significantly lower in cen- tenarians with GG genotype as compared to those AA genotype (additive model, P00.031) or to centenarians with AA and AG genotypes (recessive model, P00.040). G allele was also associ- ated to reduced DBP (P00.037). SBP and MAP (b and c, respec- tively) were statistically different in centenarians carrying the rs275653 G allele (P00.046 and P00.042, respectively)

the specific genetic variants involved are still largely unknown. Evidences in mammals point to a role of genes involved in the major causes of death, i.e. car- diovascular disease, cancer and brain disease. The vasoactive hormone AngII has been involved in organ senescence, and in previous studies, we demonstrated that disruption of the mouse gene encoding for AT R 1A markedly prolonged animal lifespan (Benigni et al. 2009) and protected mice from vascular and cardiac injury.

Our findings are in line with the hypothesis that SNP variations in the AGTR1 promoter can influence protein levels of the corresponding receptor and that a region including or linked to the AGTR1 promoter could play a role in modulating local RAS system. Since our sequencing-based screening study was con- fined to the proximal portion of AGTR1 promoter and to coding regions and intron/exon boundaries, the possibility that rs275653 and the linked rs422858

Here we demonstrate that in an Italian cohort, the C variant of rs422858 and the linked G-variant of rs275653 are overrepresented in centenarians with an OR of 4.55 according to the recessive model, in line with what should be expected for a polygenic trait like longevity. Replication of rs275653 genotyping in the

and rs2307085 are in linkage disequilibrium to var- iants in other regulatory elements in the promoter or in introns cannot be excluded.

2010). Other studies have found an association of ′ another SNP, the rs5186 (A1166C) in the 3 -UTR region of AGTR1, with hypertension and the metabolic syndrome (Mottl et al. 2008; Palatini et al. 2009). The A1166C SNP affects AT R expression (Sethupathy et 1 al. 2007) since the A variant is the target of the micro- RNA hsa-miR155. In untreated hypertensive young subjects, a correlation among protein expression levels and rs5186 genotype has been recently demonstrated (Ceolotto et al. 2011). In the Italian cohort, we failed to see any association of rs5186 with human longevity, suggesting that the mechanisms by which rs422858/ rs275653 or linked factors affect lifespan are not lim- ited to its effects on blood pressure. In both Italian and Japanese centenarians, however, we observed a statis- tically significant decreased systolic blood pressure in homozygous for GG rs275653 genotype suggesting that this polymorphism might influence blood pres- sure. In control subjects, we observed a reduction of SBP in subject homozygous for GG genotype; how- ever, this difference was not statistically significant. It is likely that the wide range of age in the control population may have masked the association between the G allele of rs275653 and lower blood pressure.

It is likely that the rs275653 and rs422858 minor allele variants or other linked elements in the AGTR1 promoter dampen cell surface levels of AT R protein 1 in PMNs as well as in their CD16+ subset. AT R levels 1 in several cell types including neutrophils have been shown to be affected by hypercholesterolemia or dys- lipidemia (Marino et al. 2007, 2009; Guasti et al. 2008). In the present study, blood cholesterol and lipids were not different among subjects with GG, GA or the AA genotypes of rs275653, thus ruling out that changes in blood lipid profile accounts for the differences in AT R expression. 1