Statistics

Statistical analyses were performed using the SAS statistical package (SAS institute). Allele frequencies were determined by means of the gene-counting method. The significance of a frequency increase or decrease across the four age groups was assessed by the X2 test for trends (Mantel-Haenszel) (22). The association between the £-Iocus and the level of serum lipoproteins was assessed by linear regression us the number of £3 and £4 alleles in the genotype, with ad justment for gender and age (covariance analysis). Tg values were log-transformed before analysis in view of their skewed distribution. The average effect of the Apo E alleles (denoted aI, a2, (3) on plasma total Chol, lOl-Chol and Apo B levels (adjusted for age) in men and women was determined according to the following formulas (23):

IEF was carried out in a Bio-Phoresis TM Horizontal Electrophoresis Cell (Bio-Rad, Richmond, CA, U.S.A.), using a Model 3000/300 power unit. Paper wicks were soaked in cathode and anode solutions, 0.1 mollL NaOH and 0.04 mollL glutamiC acid respectively. Af ter prefocusing at 500 constant V for 30 minutes, the sample applicator was placed onto the gel surface near the cathode, and up to 20 samples (30 III each) were applied. Focusing was carried out under 12 W until con stant voltage was reached (usually 1 hour), and then un der l300 constant V for 20 minutes.

The immunoblotting procedure was substantially that described by Havekes et al. (21) with minor mo difications. After the run, the slab gel was washed for 15 minutes in 192 mmolll glycine, 25 mmolll Tris-HCl, and 20% methanol, pH 8.3, and the prote ins were then transferred overnight at room temper ature onto 0.45-llm pore size nitrocellulose mem brane by simple diffusion. After transfer, the nitro cellulose sheet was incubated in 0.15 mollL NaCI, 10 mmollL Tris-HCI, 0.2% Tween 20, and 4% bovine serum albumin, pH 7.4, for 1 hour at 3rC, and then washed twice for 15 minutes in 0.15 mollL NaCI, 10 mmolll Tris-HCI, 0.2% Tween 20, and 0.5% bovine serum albumin (washing buffer). The nitrocellulose was then incubated with sheep anti ApoE antiserum (1:500, v/v) (Immuno, Wien, Austria) in washing buffer for 1 hour at 3rc. After three washes (20 ml) in washing buffer, it was incubated

where 122,123 etc. are the expected phenotype frequencies assuming a Hardy-Winberg equilibrium; 1122, 1123 etc. are the phenotypic means, and 11 is the grand mean of the sample.

RESULTS

The mean age, Apo E phenotype distribution and allele frequency values observed in our population (to tal and by sex) are shown in Table 1. Serum lipid con-

Table 1 - Age (mean±SD), distribution of Apo E phenotypes and allele frequencies in 228 subjects (total and by sex).

N Age Phenotypes Allele Frequencies (years) 2\2 2\3 2\4 3\3 3\4 4\4 E*2 E*3 E*4 All 228 55±16 3 25 1 158 37 4 0.070 0.829 0.101 Males 124 56±13 1 14 1 87 20 1 0.068 0.839 0.093 Females 104 54±18 2 11 71 17 3 0.072 0.817 0.111

Table 2 - Apo E phenotype and serum lipid concentrations (mg/dL, mean + SD) in 228 subjects. 

N Age Tg Chol HDL-Chol LDL-Chol ApoB Total 228 55±16 152±82 211±48 55±13 126±42 117±33 E 2/2 3 42±21 69±40 177±18 76±10 93±5 91±19 E 2/3 25 58±14 147±92 199±53 60±17 112±49 110±30 E2/4 1 37 178 174 51 104 120 E 3/3 158 56±16 152±77 212±46 55±13 127±39 117±34 E3/4 37 53±15 156±99 216±54 55±13 130±47 118±34 E4/4 4 52±9 183±71 244±25 47±5 157±32 139±39

cholesterol metabolism in an Italian population are similar to those in the recent literature.

centrations (total and by phenotype) are reported in Table 2, where it can be seen that the highest total Chol, LDL-Chol and Apo B levels were found in the E4/4 subjects. As expected, E3/3 was by far the most common phenotype, followed by E3/4 and E2/3. Our £2 frequency was much the same as that re ported for Caucasian and other populations, whereas the £4 frequency (0.101) was lower than the 0.140- 0.227 range described in the literature (7, 19,24).

Our allele frequencies (£2=0.070, £3=0.829, £4=0.101), however, differ from those of other work ers. This is particularly true in the case of £4 fre quency. Indeed, population studies have revealed a lack of geographical uniformity in its distribution; the highest £4 frequency was observed in Northern Eu rope (0.227) (7), vs 0.149 and 0.175 in North Amer ica (24,25), and 0.139 in Germany (26). Our low find ing, however, is in line with another Italian study (27).

The Mantel-Haenszel X2 test showed an increase in £3 frequency with age (p=0.013), coupled with a de crease in £4 frequency (p=0.015) [fable 3), that was al so more marked in males (data not shown). The av erage effect of the £2 allele was to decrease total Chol, LDL-Chol and Apo B by 16.9, 18.5 and 7.3 mg/dL, respectively, in men and by 17.7,14.5 and 12.9 mg/dL, respectively, in women. By contrast, the average effect of the £4 allele was t() increase the previous lipid plasma levels by 9.3, S.3 and 9.1 mg/dL, respectively, in men and by 10.5,10.8 and 5.6 mg/dL, respectively, in women.

The three alleles are an important {actor in deter mining the plasma levels of the lipoproteins that transport cholesterol (9,28). In vitro and in vivo studies have identified the physiopathological mech anism responsible for the differences in allele behav ior, whereby £2 and £4 are respectively associated with the lowest and highest total Chol, LDL-Chol and Apo B levels (29). The average effect findings show that the values of these three parameters are re duced in £2 carriers, and increased in £4 carriers. Covariance analysis confirmed the positive correlation between Chol, LDL-Chol and Apo B levels and £4. This correlation had already been established in larg-