Control Group Healthy Aged Fisher’s (n ¼ 391) Individuals (n ¼ 80) Exact Test ApoE Alleles (%) (%) p Value e2 4.88 5.00 1.000 e3 86.54 91.90 .066 e4 8.58 3.10 .020

Statistical Analysis

The genotypic distribution of the controls was tested for the Hardy–Weinberg equilibrium (HWE) using the chi- square test. The association between groups and genotypes or alleles was tested using the one-tailed Fisher’s exact test. Statistical significance was assumed for p , .05. GraphPad InStat statistical package (version 3) for Windows was used.

Notes: The distribution of ApoE genotypes in the population sample was consistent with the Hardy–Weinberg equilibrium (v2 ¼ 5.93, p . .05). n ¼ Number of individuals genotyped; N ¼ number of individuals in each genotype; % ¼ relative frequency.

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R ESULTS

In the group selected randomly from the population, E3/3 was the most common genotype (relative frequency of 75.99%), followed by E3/4 (13.19%), E2/3 (7.92%), E4/4 (1.58%), E2/4 (0.79%), and E2/2 (0.53%). The distribution of ApoE genotypes in the controls was consistent with the HWE (v2 ¼ 5.93, p . .05), indicating the lack of structure.

a thorough physical examination, and a detailed personal medical history was drawn up. Participants on regular medication were asked to bring all of their medications with them. From each individual a blood sample was obtained for DNA extraction and APOE genotyping. APOE genotyping was done blindly in the participants in both groups. Outcome was assessed without knowledge of individual APOE genotype or health status, and clinical and genetic tests were submitted independently for statistical analysis.

In the group of 80 healthy aged individuals, the most common genotype was also E3/3 (87.5%), followed by E3/4 (5%), E2/3 (3.75), E2/2 (2.5%), and E2/4 (1.25%); the E4/4 genotype was not found. The frequency of genotypes in the healthy aged group was comparable to that in the control group.

The relative frequencies of APOE alleles in the control group and the healthy aged individuals are shown in Table 2. The e4 allele was significantly less frequent in healthy aged individuals compared to the population-based sample (3.1% vs 8.58%, respectively; p ¼ .020). The frequency of e2 allele was not different between the groups, whereas in the healthy aged individuals, the higher frequency of e3 allele (compared to that in the control group) did not attain statistical significance (91.90% vs 86.54%, respectively; p ¼ .066).

ApoE Genotyping

For ApoE genotyping, one pair of primers was designed: sense primer sequence: 59 ACAGAATTCGCCCCGGCC TGGTACAC 39; antisense sequence: 59 TAAGCTTGGCA CGGCTGTCCAAGGA39 (13). This pair, of 26 and 25 bp primers, amplifies a 244 bp fragment in exon 4 of the ApoE gene on chromosome 9. Polymerase chain reaction (PCR) was carried out on 50–100 ng of genomic DNA with 0.2 U of Taq polymerase (Gibco, Invitrogen Gmbh, Karlsruhe, Germany) in a 50lL final volume containing 20 mM Tris- HCl (pH 8.4), 50 mM KCl, 3 mM MgCl, 10 pM each primer, 0.2 mM dNTPs, 10% (vol/vol) dimethyl sulfoxide (DMSO; Merck, Darmstadt, Germany) over 35 cycles at 948C for 1 minute, then 608C for 2 minute (two steps PCR) on a thermocycler (Hybaid, Ashford, UK). PCR product was visualized under UV light on 1.5% agarose NuSieve (FMC Bioproducts, Rockland, ME). Digestion of the 244 bp PCR product with restriction enzyme Hhal (overnight at 378C) produced fragments of 91, 83, 72, 48, and 35 bp, which were readily resolved by vertical electrophoresis on 8% acrylamide (Acryl/Bis 19:1) and visualized under UV light (312 nm) after staining with ethidium bromide. APOE alleles from individuals from both groups were determined. Participants were classified as e2, e3, and e4 carriers. Our Ethics Committee approved the study protocol, and written informed consent was obtained from all participants.

D ISCUSSION

Recent studies have demonstrated that the relative frequencies of the three alleles (e2, e3, and e4) of APOE are variable among different populations. The e3 allele seems to be equally distributed among different populations (between 77% and 88%), with the exception of Africans who have a lower frequency (66%–67%) (14,15). Genotype E3/3 is used as the referent for the risk estimation of the different ApoE isoforms. The frequency of e3 allele in our randomly selected control group (n ¼ 391, median age 43 years) was in the upper range of the frequencies found in non-African populations.

In contrast to that of e3 allele, the distribution of e4 among different populations is not so uniform. European Caucasian populations tend to have a geographic cline from

north to south (15). Studies in Italians and French pop- ulations have shown that the frequency of e4 allele ranges between 9% and 12% (15–17), whereas in Northern Europeans (Finns and Germans) it ranges between 14% and 19% (15). Our data showed that the e4 allele frequency was lower (8.58%) than that found in French and Italian persons, and the same was observed regarding e2 allele (4.88%). The frequency of e2 allele in French and Italian populations is between 7% and 8% (16,17).

has not been proved in a recent population-based study (31). However, other recent data (32) are discrepant, suggesting a positive association.