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P 327 (88.9 1.6) 207 (92.4 1.8) 328 (92.7 1.4) Exact tests applied to this data did not reveal age-related heterogeneity either in males or females (A1-C3 haplotypes: p = 0.296 in males, p = 0.126 in females; C3-A4 haplotypes: p = 0.157 in males, p = 0.095 in females). The association between age and APOA1 variability was absent in females (Table 1b), where either genotypes (2=7.56 with 4 df., p=0.109) or alleles (2=3.22 with 2 df., p=0.200) did not discriminate among age-classes. As for APOC3 and APOA4 variability (Tables 2 and 3 respectively), no age-related variation in the gene pool was observed either in males or females, as shown by heterogeneity tests among age-classes (p>0.05 in all cases). 

APOA1 variability was significantly associated with the age of the cohort, and in a sex-specific way. In order to shed light on these findings, the average effects exerted by the A and P alleles of APOA1 on lipidemic parameters were estimated in the sample of 4680-year-old males, where the A and P alleles reached maximum and minimum age-related frequency values respectively (see Table 1a). Due to the low value of the standard deviation (SD) of the BMI (25.62 average value with 2.87 SD), data were not corrected for this parameter. The average effects are shown in Table 6a: the alleles A/P significantly decreased/increased serum average LDL-C but had non-significant effects on TC, HDL-C, TG. Interestingly, when the same analysis was carried out in the corresponding group of females (26.08 average BMI with 3.49 SD), where no association was present between age and APOA1 variability, no significant effect was found on any of the lipidemic parameters (Table 6b). 

In order to verify if haplotypes were more informative than single markers, linkage disequilibrium (D) between pairs of markers was analyzed in the whole sample of 800 subjects. The disequilibrium was significant between APOA1 and APOC3 markers (2 with 1 df.=11.46; p=0.0007; Drel.=60.0%), as well as between APOC3 and APOA4 markers (2 with 1 df.=339.14; p=0.0000; Drel.=82.7%), while it was non-significant between APOA1 and APOA4 markers (2 with 1 df.=1.98; p=0.159; Drel.=7.5%). The frequencies of the haplotypes defined by APOA1-APOC3 and APOC3-APOA4 markers in males and females of different age-classes are shown in Tables 4 and 5.

Table 3 shows genotypic and allelic frequencies of the APOA4-HincII-RFLP in samples of individuals (males, 3a; females, 3b) of different ages. Relative frequencies standard errors (100) are given in parentheses. A/B denotes Absence/Presence of the restriction site. HWE denotes values expected at Hardy-Weinberg Equilibrium.

Table 4 shows haplotypes of APOA1-MspI-RFLP (5) and APOC3-3!) SstI-RFLP (3) in samples of individuals of different ages. A/P indicates Absence/Presence of the restriction site. Haplotypic frequencies (100) are estimated by Maximum Likelihood method. Standard errors are in parentheses.

Table 5 shows haplotypes of APOC3-SstI-RFLP (5) and APOA4-ae3/) HincII-RFLP (3) in samples of individuals of different ages. A/P indicates Absence/Presence of the restriction site. Haplotypic frequencies (100) are estimated by Maximum Likelihood method. Standard errors are in parentheses.

Table 6 shows allele average effects and estimated for APOA1 A and P alleles respectively in 4580-year-old subjects. Average effects A/P are expressed in mg/dConfirming that genetic risk factors can be present and over-represented in the centenarians gene pool, several studies have shown that a genotype can be risky in middle-age but protective at very advanced age. It has also been suggested that cholesterol metabolism may differ between younger and older populations, as high serum total cholesterol concentration has been associated with increased survival in very old people. Furthermore, aging could be viewed as a side-effect of adaptive events controlled by genes causing damage, and allele P may be beneficial at very old age by repressing some causes of age-related damage.

The present study reveals two main conclusions. Firstly, the APOA1 allele P tends to increase its frequency as the cohort selected for best survival emerges from the population. Secondly, gene-sex interaction seems to be an important phenomenon in the genetic modulation of human lifespan. This analysis of the APOA1-APOC3-APOA4 gene cluster's variability reveals another paradox of centenarians and confirms the complexity of the human longevity phenotype.