RANTES (cid:2)403G/A and Int1.1T/C and CCR5D32 polymorphisms were genotyped by standard procedures (Supplementary Table 1) in a cohort of 214 subjects. A detailed description of cohorts can be found in Ref. [8], briefly: 110 sex-matched healthy young indi- viduals as a control group, 75 women (mean age 28 ± 5.8; range 17–39) and 35 men (mean age 29 ± 8.6; range 17–40), and 104 elderly subjects (>85 years), 72 women (mean age 89.4 ± 3.6; range 85–100) and 32 men (mean age 89.4 ± 3.2; range 85–97). Subjects were enrolled from the city of Lleida in northeast Spain, between January and June 2004. Elderly subjects were recruited from atten- dants to Primary Care Services (25%) and Nursing Home residents (75%). A comprehensive medical history was obtained from each elderly subject. Most frequent co-morbidities were hypertension (9.6%), stroke (7.7%), chronic obstructive pulmonary disease (4.8%) and heart disease (2.9%). A written informed consent for enrolling in the study and for personal data management was ob- tained from all subjects (elderly and controls) in accordance with Spanish laws. The Ethics Committee of our institution approved the study.

Carrier distribution in males and females of pro- and anti- inflammatory haplotypes is shown in Fig. 1. As revealed by haplo- type-specific test, elderly males were carriers of the pro-inflamma- tory AT haplotype, which was more prevalent in elderly than in controls (26% and 6%, respectively; OR = 5.6; 95%CI = 1.06–29.7; P = 0.038), while carriers of the AT haplotype were equally distrib- uted in elderly and control females (10% and 13%, respectively). In contrast, elderly females were carriers of the anti-inflammatory GC haplotype that was absent in control females (11.4% vs. 0%, respec- tively; Peto OR = 8.8; 95%CI = 2.1–36.5; P = 0.0024).

Our genetic study of Spanish elderly does not reveal any signif- icant association between the CCR5D32 polymorphism and longev- ity. The role of CCR5 in age-related diseases has been largely reviewed [9]. Assuming the hypothesis that alleles favouring inflammation-related diseases are detrimental for longevity and considering the inflammatory CCR5-mediated action, we could ex- pect an increased prevalence of defective CCR5D32 alleles, as well as CCR5D32 carriers, in long-lived individuals. The CCR5D32 poly- morphism has been studied in relation to diseases reducing life expectancy, such as acute myocardial infarction and prostate can- cer using centenarians as the reference population [10,11]. An overrepresentation of the CCR5D32 allele was reported in cente- narians compared to cases, assigning to this polymorphism a role in promoting longevity. In these studies the comparison of extreme phenotype populations (centenarians vs. affected cases) probably confers an increased power to detect association. However, the CCR5D32 prevalence in centenarians does not differ from that ob- served in the healthy control population which agrees with results obtained in our study. This could reflect a weak association of CCR5 with longevity that needs a more highly powered test to be established.

3. Results and discussion

Genotype and allele distributions of RANTES (cid:2)403G/A and CCR5D32 polymorphisms conform to Hardy–Weinberg equilibrium in both the control and elderly cohorts. In contrast, RANTES Int1.1T/C departs from Hardy–Weinberg equilibrium in the elderly cohort. Comparisons of genotype and allele distributions between controls and elderly were done by contingency table analysis performed by the OpenEpi web resource (http://www.openepi.com/OE2.3/Menu/ OpenEpiMenu.htm). Differences were evaluated considering all subjects and by sex between cohorts. For comparisons including zero values, odds ratio and 95%CI were calculated based on the Peto odds ratio method by using the on-line calculator http://www.hut- chon.net/ConfidORnulhypo.htm.Thisapproachhastheadvantageof accepting zero results without generating infinity. P values <0.05 were considered statistically significant. In addition, a modified Bonferroni-corrected nominal threshold of P = 0.05/N was used, where N is the ‘‘effective number of independent marker loci’’ after consideration of linkage disequilibrium between markers. N was calculated using the SNP (single nucleotide polymorphism) spectral decomposition approach web-based program SNPSpD (http:// gump.qimr.edu.au/general/daleN/SNPSpD/). This yields an experi- ment-wide significance threshold of 0.02 to keep Type I error rate at 5%.

The association of RANTES polymorphisms with longevity has not yet been described. Our results denote a sex-specific associa- tion with functional RANTES polymorphisms. Pro-inflammatory RANTES alleles/haplotypes were positively associated with elderly males. In contrast, anti-inflammatory RANTES alleles/haplotypes showed positive association with elderly females.

Elevation of circulating levels of RANTES has been described in unstable angina pectoris patients as predictive of future cardiovas- cular adverse events [9]. Furthermore, a significant elevation of major C-C chemokines, including RANTES, has been described dur- ing the course of acute myocardial infarction that actively contrib- utes to the pathophysiology of the disease. Finally, association studies of functional RANTES polymorphisms have been performed highlighting the role of RANTES in age-related diseases [4].