identification of the major cardiovascular risk factors; cardiology consultation including check of baseline blood pressure; peripheral venous blood sample for the investigation of the presences of RAGE −374T/A polymorphism. The cardiovascular risk factors were defined as follows: hypertension (systolic pressure > 140 mmHg or diastolic pressure >90 mmHg or antihypertensive therapy), tobacco smoke, hyperlipidemia, and diabetes mellitus. With regard to cigarette smoking, subjects were grouped under the headings “always” or “never”: in the former group were included subjects who had smoked daily for at least 1 year. We considered dyslipidemic patients with cholesterol levels above 200 mg/dL or in treatment with lipid-lowering drugs. The diagnosis of diabetes mellitus has been placed in patients previously treated with dietary treatments, who had received oral antidiabetic agents or insulin or had a fasting plasma glucose value higher than 126 mg/dL.

All subjects signed informed consent before study. The study was conducted in accordance with the guidelines of the Declaration of Helsinki for human research and the guidelines of our local ethics committee.

Maxwell 16 Blood DNA purification kit (Promega, Madison, WI, USA). The −374T/A polymorphism of the RAGE promoter was analyzed by polymerase chain reaction (PCR) restriction fragment length polymorphisms (RFLPs). In brief, a section of the RAGE promoter that contains the −374T/A variant was amplified by PCR. The sequences were 5'-CCTGGGTTTAGTTGAGATTTTTT-3' for upstream and 5'-GAAGGCACTTCCTCGGGTTCT-3' for downstream primers. The conditions of amplification were: 94 °C for 2 min; 30 cycles at 94 °C for 30 s, 58 °C for 30 s, 72 °C for 1 min; and finally 10 min at 72 °C. The PCR product (671 bp) was then subjected to Tsp509 I (New England Biolabs, Beverly, MA, USA) digestion for 4 h at 65 °C, whereas the PCR product was resolved by electrophoresis on 3% agarose gels. Genotypes were scored according to the patterns of DNA bands. Treatment of the T allele with Tsp509 I gave rise to five fragments of 284 bp, 217 bp, 110 bp, 44 bp, and 16 bp. In the case of the A allele, digestion resulted in four fragments of 327 bp, 284 bp, 44 bp, and 16 bp. To reduce the possibility of genotyping errors, all results were scored by two independent investigators

The hypothesis of normal distribution for continuous variables was tested according to Kolmogorov-Smirnov statistics. Continuous variables are expressed as mean ± standard deviation in the case of Gaussian distribution and as median in the case of variables with non-normal distribution. The data were analyzed using the Student t test for paired. All categorical variables are reported as absolute (n) and relative (%) frequencies. The associations between the categorical variables were evaluated using the chi-square test. Statistical significance was defined as p < 0.05.

5. Conclusions

Our research showed a higher prevalence of the AA genotype in male population of subjects aged ≥90 years who have reached that age in good state of health. These results confirm the hypothesis that longevity is the result of a good functioning of the immune system and a presumable hyper-expression of variants of anti-inflammatory genes of immunity. The differences in the genetic regulation of inflammatory processes may influence the presence of age-related disorders. Surprisingly, the prevalence of the AA genotype is lower in the long-living female population than in the male one, in which this genotype would appear to have a more protective role.

Thus, AA genotype would not seem to have a role in determining a better aging in the female gender. In the general population, women are more long-living than men, so one wonders if the endocrine system cannot have a protective role even in those subjects without this genotype. Further research is needed to identify other agents involved in healthy aging.

Conflicts of Interest

The authors declare no conflict of interest.

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