Some recent evidence has linked cytokine poly- morphisms with longevity and differential longevity in males and females (8-11). A study performed on Italian centenarians reported that those individuals who are ge- netically predisposed to produce high levels of inter- leukin(IL)-6 during aging, i.e., C- men at IL-6 –174 C/G single nucleotide polymorphism (SNP), have a reduced capacity to reach the extreme limits of human life (8). Moreover, –1082G IL-10 SNP, associated with high production of the cytokine, is increased among Italian male centenarians (9, 10), whereas +874T SNP at interferon (IFN)-γ gene, part of the 12CA/+847T hap- lotype responsible for increased production of the cy- tokine, is found less frequently in female centenarians (11). These findings indicate that different alleles at dif- ferent cytokine gene codings for pro- (IL-6 or IFN-γ) or anti-inflammatory (IL-10) cytokines affect individual life span expectancy, influencing the type and intensi- ty of immune-inflammatory responses to environmen- tal stressors. These gender-related effects are difficult to explain. However, men and women have claimed to fol- low different strategies to reach longevity, and the number of centenarian women always outnumbers that of centenarian men, the ratio between women and men in centenarians ranging from 4/1 to 7/1, with the exception of Sardinia, where it is about 2.1/1 (12, 13).

Cytokine genotyping

Amplification of the –174C/G IL-6 promoter SNP was performed as previously described, and the presence of a –174C allele was revealed by the presence of the NlaIII cutting site (8). IL-10 –1082 G→A SNP was identified us- ing an amplification refractory mutational system (ARMS) PCR method, as previously described (9). T→A alleles at +874 of IFN-γ gene were identified using ARMS method- ology, as previously described (11, 20).

Statistical analysis

IL-6 –174G/C, IL-10 –1082 G/A, and IFN-γ +874T/A allele frequencies were evaluated by gene count. Data were tested for goodness of fit between ob- served and expected genotype values (χ2 test) and their fit to the Hardy-Weinberg equilibrium. χ2 tests (3 x 2 or 2 x 2 tables) were performed to calculate significant different genotype (or allele) distribution between centenarians and controls.

Since the above studies were performed with Italian centenarians, it is not known whether the results are valid in populations of other ethnic origin. Notably, in Finnish and Irish populations, discordant results have been ob- tained (14-16). However, the authors did not study cen- tenarians, but somewhat younger cohorts, which were less selective than the centenarians investigated in the Italian studies (8-11). In the present paper, we analyzed IL-6, IL- 10 and IFN-γ SNPs in a sample of Sardinian centenarians, to evaluate if the reported associations of these SNPs with longevity are also detectable in a population that shows a genetic background quite different from that of main- land Italy (17, 18).

RESULTS

Table 1 shows the genotypic distribution of IL-6, IL- 10 and IFN-γ SNPs among centenarians and controls, and Table 2 shows their allele frequencies. All 112 cen- tenarians and 137 controls were typed for IL-6 poly- morphisms; IL-10 and IFN-γ polymorphisms were de- termined in 87 centenarians and 85 controls and in 96 centenarians and 94 controls respectively. No significant differences were observed on analyzing IL-6, IL-10 and IFN-γ genotype and allele frequencies among cen- tenarians and controls, either together or when data were analyzed by gender (Tables 1, 2). It is rather sim- plistic to examine the influence of a single cytokine in isolation, since cytokines interact in networks in which the functions of one cytokine are modified, modulated or substituted by another one(s) (21). However, no significant interactions were found when the three polymorphisms were analyzed in combination (data not shown).

METHODS

Subjects and study design

The AKEA study of Sardinian centenarians (the name “AKEA” is derived from an expression in the Sardinian language which means “health and life for 100 years!”) was carried out between January 1 and December 31, 1997 (12). One hundred and forty-one centenarians underwent a full interview, 129 donated a blood sample, and 112 consented to the genetic study described here. Their dates of birth were checked by matching information from census records, the baptism

1 - Genotype frequencies for –174G→C IL-6 gene SNP, for –1082G→A IL-10 gene SNP, and for +874T→A IFN-γ-10 gene SNP centenarians and controls from Sardinia (percentages in brackets).

IL-6 –174G IL-10 –1082G IFN-γ +874 Genotypes GG GC CC GG GA AA TT TA AA Centenarians 57 47 8 16 68 3 11 72 13 (50.9%) (42.0%) (7.1%) (18.4%) (78.2%) (3.4%) (11.5%) (75%) (13.5%) Men 21 12 3 6 25 1 6 22 4 (58.3%) (33.3%) (8.3%) (18.8%) (78.1%) (3.1%) (18.8%) (68.7%) (12.5%) Women 36 35 5 10 43 2 5 50 9 (47.4%) (46.0%) (6.6%) (18.2%) (78.2%) (3.6%) (7.9%) (78.1%) (14.0%) Controls 65 63 9 19 60 6 14 65 15.0 (47.4%) (46.0%) (6.6%) (22.4%) (70.6%) (7.0) (14.9%) (69.1%) (16.0%) Men 32 32 4 6 22 3 6 21 9 (47.9%) (47.9%) (5.1%) (19.3%) (71.0%) (9.7%) (16.7%) (58.3%) (25.0%) Women 33 31 5 13 38 3 8 44 6 (47.8%) (44.9%) (7.2%) (24.1%) (70.4%) (5.5%) (13.8%) (75.9%) (10.3%)

All groups were in Hardy-Weinberg equilibrium (p>0.05). test was performed (3 x 2 contingency tables) to calculate significant different genetic between centenarians and controls. No significant differences were observed. Note that cytokine SNPs were respectively typed in112 centenarians and controls (IL-6), 87 centenarians and 85 controls (IL-10), and 96 centenarians and 94 controls (IFN-γ).

DISCUSSION