sess linkage disequilibrium D, between pairs of markers at the three loci, and to test the null hypothesis, D = 0, in the whole sample. Maximum Likelihood Estimation (MLE) was used to estimate, in each age-class and for each sex, the frequencies of the haplotypes defined by markers in disequilibrium. The hypothesis of homo- geneity among haplotypic frequencies in sample groups of same sex and different age was tested by means of Arlequin 1.1 software (exact tests of population differ- entiation).

Table 1 Genotypic and allelic frequencies of the APOA1- MspI-RFLP in samples of individuals (males, 1a; females, 1b) 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

1a Males (n = 327) 18–45 years 46–80 years 81–109 years Genotypes Counts (120) HWE Counts (155) HWE Counts (52) HWE ± ± ± AA 7 (5.8 2.1) 8.5 13 (8.4 2.2) 16.4 3 (5.8 3.2) 1.7 ± ± ± AP 50 (41.7 4.5) 46.9 75 (48.4 4.0) 68.1 13 (25.0 6.0) 15.5 ± ± ± PP 63 (52.5 4.6) 64.5 67 (43.2 4.0) 70.5 36 (69.2 6.4) 34.7 Alleles Counts (240) Counts (310) Counts (104) A 64 (26.7) 101 (32.6) 19 (18.3) ± ± ± P 176 (73.3 2.9) 209 (67.4 2.7) 85 (81.7 3.8) 1b Females (n = 473) 18–45 years 46–80 years 81–109 years Genotypes Counts (184) HWE Counts (112) HWE Counts (177) HWE ± ± ± AA 7 (3.8 1.4) 8.3 11 (9.8 2.8) 8.3 8 (4.5 1.6) 11.4 ± ± ± AP 64 (34.8 3.5) 61.5 39 (34.8 4.5) 44.4 74 (41.8 3.7) 67.1 ± ± ± PP 113 (61.4 3.6) 114.3 62 (55.4 4.7) 59.3 95 (53.7 3.7) 98.4 Alleles Counts (368) Counts (224) Counts (354) A 78 (21.2) 61 (27.2) 90 (25.4) ± ± ± P 290 (78.8 2.1) 163 (72.8 3.0) 264 (74.6 2.3)

1.0 0.9 0.8 0.7 0.6 0.5 0.4 observed allele frequencies 0.3 frequency trajectory 0.2 upper envelope 0.1 lower envelope 0.0 0 10 20 30 40 50 60 70 80 90 100 110 age (years) ycneuqerf elella evitaler

observed allele frequencies frequency trajectory upper envelope lower envelope 0 10 20 30 40 50 60 70 80 90 100 110 age (years)

Figure 1 Age-related frequency trajectory of the APOA1 allele P analysed in males (18–25; 26–45; 46–65; 66–80; 81–109 year old subjects). The observed frequencies of the P allele in the five age-groups, the fitting curve (least squares method), and the lower and upper envelopes of 10,000 simulated trajectories are shown.

S. Garasto et al.

Table 2 Genotypic and allelic frequencies of the APOC3- SstI-RFLP in samples of individuals (males, 2a; females, 2b) 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

2a Males (n = 327) 18–45 years 46–80 years 81–109 years Genotypes Counts (120) HWE Counts (155) HWE Counts (52) ± ± ± AA 98 (81.7 3.5) 98.1 124 (80.0 3.2) 123.8 41 (78.8 5.7) ± ± ± AP 21 (17.5 3.5) 20.8 29 (18.7 3.1) 29.5 10 (19.2 5.5) ± ± ± PP 1 (0.8 0.8) 1.1 2 (1.3 0.9) 1.8 1 (1.9 1.9) 0.7 Alleles Counts (240) Counts (310) Counts (104) A 23 (9.6) 33 (10.6) 12 (11.5) ± ± ± P 217 (90.4 1.9) 277 (89.4 1.8) 92 (88.5 3.1) 2b Females (n = 473) 18–45 years 46–80 years 81–109 years Genotypes Counts (184) HWE Counts (112) HWE Counts (177) ± ± ± AA 144 (78.3 3.0) 145.4 96 (85.7 3.3) 95.6 151 (85.3 2.7) ± ± ± AP 39 (21.2 3.0) 36.4 15 (13.4 3.2) 15.7 26 (14.7 2.7) ± ± PP 1 (0.5 0.5) 2.3 1 (0.9 0.9) 0.6 0 0.0 Alleles Counts (368) Counts (224) Counts (354) A 41 (11.1) 17 (7.6) 26 (7.3) ± ± ± 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 in 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 (χ2 = 7.56 with 4 df., p = 0.109) or alleles (χ2 = 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 in females, as shown by > heterogeneity tests among age-classes (p 0.05 in all the cases).

APOA1 variability was significantly associated with the age of the cohort, and in a sex specific way. In order α α to shed light on these findings, the and average A P effects exerted by the A and P alleles of APOA1 on lipidemic parameters were estimated in the sample of 46–80 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 parame- ter. The α and α average effects are shown in Table 6a: A P 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 associa- tion was present between age and APOA1 variability, no

In order to verify if haplotypes were more informa- tive than single markers, linkage disequilibrium (D) be- tween pairs of markers was analysed in the whole sam- ple of 800 subjects. The disequilibrium was significant between APOA1 and APOC3 markers (χ2 with 1 d.f. = 11.46; p = 0.0007; Drel. = 60.0%), as well as between APOC3 and APOA4 markers (χ2 with 1 d.f. = 339.14; p = 0.0000; Drel. = 82.7%), while it was non-significant between APOA1 and APOA4 markers (χ2 with 1 d.f. = 1.98; p = 0.159; Drel. = 7.5%). The frequencies of the haplotypes defined by APOA1- APOC3 and APOC3-APOA4 markers in males and fe- males of different age-classes are shown in Tables 4 and 5.