Amplification of APOE gene region encoding common APOE isoforms from genomic DNA was conducted in a DNA Thermal Cycler (Perkin Elmer Cetus % 9600) using oligonucleotide primers (forward primer: 5 -GGC ACG GCT GTC % % CAA GGA GCT-3 and reverse primer: 5 -GAT GGC GCT GAG GCC GCG % CT-3 ). Each amplification reaction contained 100 ng of genomic DNA, 8 pmol of m each primer, 5 l of 10(cid:29) PCR reaction buffer, 10% (v:v) dimethylsulfoxide m (Sigma), 200 mol:l of dATP, dTTP, dCTP and dGTP (Merk), 2 U of Taq m polymerase in a final volume of 50 l. Each reaction mixture was denatured at

Distributions of the subjects by age and gender

Age:group Female Male N Age N Age Youth 62 29.292.9 75 27.793.1 Middle 57 48.392.4 132 46.993.8 Old 122 71.693.3 1055 72.796.0 Very old 5 87.091.6 54 87.592.7

N, subjects number; Age, mean9S.D. years.

94°C for 2 min and subjected to five cycles of amplification by primer annealing (67°C for 10 s), extension (72°C for 80 s) and denaturation (94°C for 10 s). Immediately after completing this mini-cycle, the reaction mixture was continuously subjected to 30 cycles of amplification by primer annealing (60°C for 5 s), extension (72°C for 80 s) and denaturation (94°C for 10 s). The length of PCR product was 262 bp.

2.4. Analysis of amplified APOE product with RFLP

m After PCR amplification, 12 l of each unpurified PCR product was directly digested with 5 U of CfoI at 37°C for overnight. The digested product was loaded onto a 20% nondenaturing polyacrylamide gel (acrylamide:bisacrylamide(cid:30)29:1) with 1 mm thick(cid:29)7 cm long for 2.5 h running under constant voltage (150 V). After electrophoresis, the gel was treated with ethidium bromide (0.5 mg:l) for 6 min and DNA fragments were visualized by UV illumination. The sizes of CfoI fragments were determined by the marker (MspI-digested pUC18 DNA) (Hixon and Vernier, 1990; Wenham et al., 1991).

Although the total length of PCR product was different from that obtained by Hixon and Vernier (1990) due to the difference of reverse primer, the length for APOE typing was same as Hixon since the surplus of the base pair was cut off at another CfoI cleavage site contained in the reverse primer.

The data base was reconstructed by using Microsoft Excel software. Allele frequencies were calculated by allele counting. Hardy–Weinberg equilibrium was tested by a x2 goodness of fit test. The other data were compared by a x2 test. The computer software of Epi Info 5.01 Chinese edition was also used in the analysis.

3. Results

Table 2 shows distribution of APOE allele genotype in the different age groups (male group); Table 3 shows distribution of APOE allele genotype in the different age groups (female group); Table 4 shows distribution of APOE allele genotype in the different age groups (male(cid:27)female); Table 5 shows the distributions of APOE*2, APOE*3 and APOE*4 allele frequencies. The allele frequency of APOE*4 in the very old group is significantly lower than that in the youth, middle and old group (2.5% vs 8.4, 7.9 or 7.6%; x2 (cid:30)4.6, 4.2 or 4.2; P(cid:30)0.03, 0.04 or 0.04), respectively.

The allele and genotype frequencies were in Hardy–Weinberg equilibrium by \ comparison with that of the corresponding theoretical distribution (P 0.05, Table

Distribution of APOE allele genotype in the different male age groups

aie ee BV eee ee ee OEY OP Apo E genotype 2/2 2/3 2/4 3/3 3/4 4/4Age GroupYouth (n=75 ) 3 8 1 52 11 0Middle = ( n=132) 1 13 3 98 16 1Old (n=1055 ) 4 102 17 801 120 11Very old = (n=54) 0 9 0 42 3 0

The value for testing Hardy–Weinberg equilibrium in the subgroup is 8.83, 3.30, 7.38 and 0.90 in the youth, middle, old and very old age groups, respectively \ (data not shown). The df is five in each age group (P 0.05). Each subgroup is also in Hardy–Weinberg equilibrium.

4. Discussion

ApoE is one of the major protein constituents in very low density lipoprotein and plays a central role in lipid metabolism. It is the product of a single gene on : chromosome 19 with the molecular weight of 34000 kDa glycoprotein which is composed of 299 amino acid residues (Mahley, 1988).

The APOE allele frequencies have been found to associate with some kind of diseases. Three common alleles of APOE are APOE*2, APOE*3 and

Distribution of APOE allele genotype in the different female age groups

      Apo E genotype 2/2 2/3 2/4 3/3 3/4 4/4Age Group20-39 (n=62) 1 7 1 44 8 140 - 59 (n=57) 0 4 1 44 8 060 - 84 (n=122) 0 13 1 90 18 0285 (n=5) 0 1 0 4 0 0 

Distribution of APOE allele genotype in the different sex-mixed age groups

 Apo E genotype 2/2a20 - 39(n=137)40 - 59(n=189 )60 - 84(n=1177)285(n=59 )4(2.9)1(0.5)4(0.3)(0.0)2/3(11.5)(16.9)2/42(1.5)4(2.1)18(1.5)0(0.0)96(70.1)142(75.1)871(74.0)46(78.0)(13.9)24(12.7)138(1.7)6.1)4/41(0.7)1(0.5)iv(0.9)0(0.0)

(Utermann et al., 1980; Zannis and Breslow, 1981), which can be distinguished by the variation of positions at 112 and 158 in the receptor-binding region of apoE (Weisgraber et al., 1981). If the positions with 112 cys and 158 arg, it forms isoform APOE*3 which is the commonest isoform; If the positions with 112 arg and 158 arg, it forms isoform APOE*4 which is associated with Alzheimer’s disease (Strittmatter and Roses, 1996), vascular dementia (Shigeji et al., 1993), cognition in the very old (Altstiel et al., 1997), coronary artery disease (Moore et al., 1997), gallstones (Bertomeu et al., 1996), diabetes and atherosclerosis (Semenkovich and Heinecke, 1997), colon cancer (Kervinen et al., 1996) and so on; If the positions

Distribution of APOE allele frequencies in the different sex-mixed age groups

Age:group APOE2 APOE3 APOE4 F M T F M T F M T Youth 8.1 10.0 9.1 83.1 82.0 82.5 8.8 8.0 8.4 Middle 4.4 6.8 6.1 87.7 85.2 86.0 7.9 8.0 7.9 Old 5.7 6.0 6.8 86.5 86.4 85.6 7.8 7.6 7.6 Very old 10.0 8.3 8.5 88.9 90.0 89.0 0.0 2.8 2.5

Percentages (%).

for Hardy–Weinberg equilibrium among all individuals (n(cid:30)1562)