Genotype distribution differences between elderly and controls showed statistical significance for Int1.1T/C polymorphism (P = 0.03), but not for CCR5D32 or (cid:2)403G/A polymorphisms (Table 1). Sex-based differences between cohorts for the CCR5D32 poly- morphism were not significant. Nevertheless, differences between elderly and control males were observed for the (cid:2)403G/A genotype (P = 0.029) and allele distribution (OR = 3.2; 95%CI = 1.2–8.5; P = 0.012) (Table 1). In addition, differences were observed when (cid:2)403A allele was modeled as dominant (OR = 4; 95%CI = 1.4–11.8; P = 0.01). We also found a genotype association for Int1.1C allele un- der recessive model (Peto OR = 8.8; 95%CI = 1.7–44.7; P = 0.008) that was mainly contributed by differences between elderly and control females (Peto OR = 8.4; 95%CI = 1.4–49.9; P = 0.02).

Our findings on RANTES allele/haplotype distribution among elderly suggest a RANTES genetic determinant that is prone to a pro-inflammatory phenotype in males and an anti-inflammatory phenotype in females, which could contribute to the known sex- specific differences in aging. Sexual dimorphism in immune responsiveness has been reported with respect to genetic back- ground. Some evidence of an increase in frequency of the IL-10 anti-inflammatory cytokine gene in long-lived males has been reported [12] and early work suggested that sexual dimorphism existed in immune responsiveness in the HLA supratype A1B8Cw7DR3 [13].

Haplotypes for RANTES gene markers were inferred by pLink software (pLink web resource available at http://pngu.mgh.har- vard.edu/~purcell/plink/). Haplotype distribution between elderly and controls in all subjects, males and females, showed significant differences (Omnibus test P = 0.02, P = 0.05 and P = 0.04, respec-

Table 1

Allele, genotype and haplotype distribution of CCR5D32, RANTES (cid:2)403G/A and RANTES Int1.1T/C polymorphisms in Spanish controls and elderly subjects.

rs code, (alias) Genotype allele haplotype Controls Elderly All subjects n (%) Males n (%) Females n (%) All subjects n (%) Males n (%) Females n (%) rs333, (CCR5D32) Genotypes wtwt 88 (80) 29 (82.9) 59 (78.7) 79 (76) 22 (68.8) 57 (79.2) wtD32 22 (20) 6 (17.1) 16 (21.3) 21 (20.2) 9 (28.1) 12 (16.7) D32D32 0 (0) 0 (0) 0 (0) 3 (2.9) 1 (3.1) 2 (2.8) Alleles wt 198 (90) 64 (91.4) 134 (89.3) 179 (86.1) 53 (82.8) 126 (87.5) D32 22 (10) 6 (8.6) 16 (10.7) 27 (13) 11 (17.2) 16 (11.1) rs2107538 (RANTES (cid:2)403G/A) Genotypes GG 75 (68.2) 28 (80) 47 (62.7) 66 (63.5) 16 (50)a 50 (69.4) GA 34 (30.9) 7 (20) 27 (36) 35 (33.7) 15 (46.8) 20 (27.8) AA 1 (0.9) 0 (0) 1 (1.3) 3 (2.9) 1 (3.1) 2 (2.8) A carriers 35 (31.8) 7(20) 28 (37.3) 38 (36.6) 16 (49.9)a 22 (30.6) Alleles G 184 (83.6) 63 (90) 121 (80.7) 167 (80.3) 47 (73.4)a 120 (83.3) A 36 (16.4) 7 (10) 29 (19.3) 41 (19.7) 17 (26.6) 24 (16.7) rs2280789 (RANTES Int1.1T/C) Genotypes TT 86 (78.9) 29 (85.3) 57 (76) 71 (73.2)b 21 (77.8) 50 (71.4) TC 23 (21.1) 5 (14.7) 18 (24) 20 (20.6) 5 (18.5) 15 (21.4) CC 0 (0) 0 (0) 0 (0) 6 (6.2)b 1 (3.7) 5 (7.2)b Alleles T 195 (89.5) 63 (92.7) 132 (88) 162 (83.5) 47 (87) 115 (82.1) C 23 (10.5) 5 (7.3) 18 (12) 32 (16.5) 7 (13) 25 (17.9) rs2107538 and rs2280789 (RANTES haplotype)c Haplotype counts GT 182 (83.3) 61 (89.6) 121 (80.5) 148 (76.2) 40 (74)d 108 (77) AC 23 (10.4) 5 (7.3) 18 (11.9) 24 (12.3) 7 (12.9) 17 (12.1) AT 13 (6.1) 2 (3.1) 11 (7.6) 14 (7.3) 7 (13.1)e 7 (5.1) GC 0 (0) 0 (0) 0 (0) 8 (4.2)f 0 (0) 8 (5.8)f

a Statistical differences between elderly and control males for global genotype distribution (P = 0.029); RANTES (cid:2)403A modeled as dominant (Odds Ratio (OR) = 4.0; confidence interval (CI) = 1.4–11.8; P = 0.01) and allele distribution (OR = 3.2; 95%CI = 1.2–8.5; P = 0.014). b Statistical differences between elderly and controls for global genotype distribution (P = 0.03) and RANTES Int1.1C under recessive model (Peto OR = 8.8; 95%CI = P = 0.008), mainly attributable to an overrepresentation in females (Peto OR = 8.4; 95%CI = 1.4–49.9; P = 0.02). c Statistical differences between elderly and controls for global haplotype distribution for all subjects (P = 0.02), males (P = 0.05) and females (P = 0.04). d GT haplotype was underrepresented in elderly males (OR = 0.3; 95%CI = 0.12–0.88; P = 0.02). e AT haplotype was overrepresented in elderly males (OR = 4.9; 95%CI = 0.97–24.7; P = 0.03). f GC haplotype was overrepresented in elderly subjects (P = 0.04) due to the overrepresentation in females (Peto OR = 8.3; 95%CI = 2–34; P = 0.005).

40% 40% CONTROLS I35% Males 36% Females @ELOERLY.30% Poon 30%8 25% 25%<2 20% 20%s P=0.024® 15% 15% 10a 8 710% 10%29 9i. ° ° ~ ° I]0% + 0% +Carriers of Carriers of Carriers of Carriers ofanti-inflammatory pro-inflammatory anti-inflammatory pro-inflammatoryhaplotype (GC) haplotype (AT) haplotype (GC) haplotype (AT) 

Fig. 1. Prevalence of carriers of RANTES anti-inflammatory (cid:2)403G-Int1.1C (GC) and pro-inflammatory (cid:2)403A-Int1.1T (AT) haplotypes between elderly and controls in males (left) and females (right). The numbers within each haplotype combination for RANTES genotypes are shown at the top of each bar.

It has been previously described that the inflammation index, calculated as the sum of positive indicators such as C-reactive pro- tein, plasma fibrinogen and urinary albumin, shows sex differences and varies with age [14]. Women have a higher inflammation in- dex during all life stages compared with men but slower rates of increase in inflammation with age. This can contribute to the de- crease in the inflammation differences between the sexes in the el- derly population (>80 year-old) [14].