subcloning and haploid DNA sequencing. Pairwise linkage disequilibrium D was calculated using the
equation D = h − pq [8], where h is the frequency of the haplotype with the rare alleles at both sites, p is the
frequency of the rare allele at one site and q at the second site. Significance of the linkage disequilibrium
between two sites was determined by x
2
analysis based upon differences between observed and expected num-
bers of haplotypes [9]. A general linear model GLM analysis was used to determine the association of geno-
types and other independent variables with plasma quantitative traits. Those lipid and apolipoprotein val-
ues that were not normally distributed were trans- formed logarithm or square root. The independent
variables considered were the linear effect of age, cigarette smoking and body mass index BMI. Signifi-
cance of the genotypic effects was based on the F-test conditional on all other variables being in the model
i.e. the type 3 sum of squares as reported by SAS. A nominal significance level of a B 10 was used in the
discussion. The contribution of each polymorphism to
Fig. 3. Restriction digestion patterns for four APOC4 mutant sites in exons 1, 2 and 3. The enzyme used to cut each fragment is given at
far right of each gel picture. Genotypes are given above each sample. M indicates the markers used and the sizes of corresponding bands of
the marker are given in bp on the left-hand side.
Fig. 2. DNA sequence flanking the mutation sites in APOC4 exons 1, 2, and 3. Exon 1, a wild type sequence for positions 609 and 620, b
mutant type sequence; Exon 2, a wild type sequence for position 3139, b mutant type sequence for position 3139, c wild type
sequence for position 3187, d mutant type sequence for position 3187. Exon 3, a wild type sequence for position 3568, b mutant
type for position 3568. The position of each variable site is indicated by an arrow and the number of the nucleotide.
variation in the dependent quantitative traits was esti- mated as described elsewhere [10]. All calculations were
done in SAS version 6.08.
3. Results
3
.
1
. DNA sequence 6ariation A total of 902 bp DNA sequence, including 614 bp of
all three exons and 288 bp of flanking intronic sequence were amplified from 50 individuals 100 chromosomes
and subjected to SSCP analyses. PCR amplification followed by SSCP analyses identified two variant pat-
terns each in exon 1 and exon 2, and one variant pattern in the first half of exon 3 data not shown.
Comparison of the DNA sequencing data of exon 1 with the published sequence [1] identified two point
mutations at nucleotide positions 609 A G and 620 G A Fig. 2. Both mutations were located in the
non-coding region of exon 1. While the nucleotide change at position 620 eliminated the MnLI restriction
site Fig. 3, the nucleotide change at 609 position did not create or eliminate any restriction site.
DNA sequencing of SSCP variant patterns seen in exon 2 identified two point mutations at nucleotide
positions 3139 T C and 3187 G A Fig. 2. The 3139 mutation affected codon 36 and predicts the re-
placement of residue Leu CTG with ProCCG in the mature apoC4 protein. Similarly, the nucleotide change
at position 3187 affected codon 52 and would result in the substitution of AspGAC for Gly GGC. The
missense mutations at codons 36 and 52 created restric- tion sites for A6aI and BsmFI, respectively Fig. 3. The
variant pattern of exon 3 corresponded to a missense mutation T G Fig. 2 at codon 96 nucleotide
position 3568, which changed LeuCTC to ArgCGC. This mutation also created a restriction site for HhaI
Fig. 3. Thus, of the 902 bp of sequence analyzed, five variable sites were identified with a frequency of about
one mutation per 180 bp.
3
.
2
. Population frequency of 6ariable sites Following the identification of five variable sites in 50
individuals, all 592 subjects were screened to examine the population distribution of these mutations Table
2. The frequencies of three mutant alleles at exon 1 A609G, G620A and exon 2 Gly52Asp were observed
at a low frequency of about 1 each. However, vari- ants observed in exon 2 Leu36Pro and exon 3
Leu96Arg were common polymorphisms. The fre- quencies of the Leu36Pro alleles were almost identical
0.4980.502. The frequencies of the Leu96Arg alleles were 0.6430.357. The average heterozygosities for these
two polymorphisms were 0.50 and 0.46, respectively, which is at or near the maximum heterozygosity attain-
able by a diallelic system.
3
.
3
. Linkage disequilibrium The three amino acid polymorphisms in exons 2 and
3 are contained in a single 845 bp fragment that could be subcloned and the haploid DNA inserts could then
be sequenced to determine unequivocally the phase of double heterozygotes. There was one double het-
erozygote at the codon 36 TC and codon 52 GA sites, two double heterozygotes at the codon 52 GA
and codon 96 TG sites and 205 double heterozygotes at the codons 36 and 96 sites. The 845 bp fragment was
amplified from the three individuals double het- erozygotes at codons 3652 and codons 5296 and from
six double heterozygote individuals at codons 3696. Subcloning and sequencing of the double heterozygote
individual at codons 3652 revealed the presence of the TG and CA haplotypes. The two double heterozygotes
at codons 5296 showed the presence of two GT, AG of the four possible haplotypes AT, AG, GT and GG,
indicating a non-random association between the two sites. The six double heterozygotes sequenced at codons
3696 demonstrated the presence of three haplotypes including, CG, CT and TT. The fourth possible haplo-
type, TG, was not observed. A random combination of six alleles at codons 36 TC, 52 GA and 96 TG
would result into eight possible haplotypes: H1 = TGT, H2 = CAG,
H3 = CGT, H4 = CGG,
H5 = TGG, H6 = TAT, H7 = TAG and H8 = CAT. However, only
the H1, H2, H3 and H4 haplotypes were observed in this study, which strongly indicate that the three amino
acid polymorphisms are in linkage disequilibrium.
The maximum-likelihood pairwise linkage disequi- librium between all APOC4 polymorphisms and be-
tween APOC4 and two APOE codons 112 and 158 polymorphisms is presented in Table 3. We chose the
APOE gene because of its close proximity to the APOC4 gene. Among the APOC4 polymorphisms link-
age disequilibrium was observed between five pairs: nt. 620codon 36, nt. 620codon 96, codon 36codon 96,
codon 36codon 52 and codon 52codon 96. None of the APOC4 polymorphisms showed linkage disequi-
librium with either of the two APOE polymorphisms. However, the two APOE polymorphisms were in non-
random association with each other in this sample population.
Table 2 Genotype, allele frequencies and heterozygosities H
of APOC4 polymorphisms
Polymorphic position nt. or amino Genotype
Allele acid changerestriction enzyme
Exon 1: nucleotide 609 A G A = 0.990
AA = 580 98.0 AG = 12 2.0
G = 0.010 Total = 592
H =0.020
x 2 = 0.003,
d.f.1, P\0.95 GG = 546 92.2
Exon 1: nucleotide 620 G AMnLI
GA = 46 7.8 G = 0.961
A = 0.039 Total = 592
H =0.075
x 2 = 0.01, d.f.1,
P\0.90 Exon 2: nucleotide 3139 codon 36
TT = 136 23.6 T C Leu ProA6aI
T = 0.498 TC = 302 52.4
CC = 138 24.0 C = 0.502
Total = 576 H
=0.500 x
2 = 1.36, d.f.1, P\0.25
G = 0.997 Exon 2: nucleotide 3187 codon 52
GG = 572 99.3 GA = 4 0.7
G A Gly AspBsmFI A = 0.003
Total = 576 H
=0.006 x
2 = 0.07, d.f.1, P\0.70
T = 0.643 TT = 232 40.1
Exon 3: nucleotide 3568 codon 96 T G Leu ArgHhaI
TG = 279 48.3 G = 0.357
GG = 67 11.6 H
=0.459 Total = 578
x 2 = 1.49, d.f.1,
P\0.25
Table 3 Pairwise linkage disequilibrium D between five APOC4 and two APOE variable sites
APOC4codon 36 APOC4codon 52
APOC4codon 96 APOEcodon 112 APOEcodon 158
APOC4nt. 620 −
0.002 NS
a
0.0 NS
a
APOC4nt.609 0.005 NS
a
0.001 NS
a
0.009 NS
a
0.009 NS
a
APOC4nt. 620 −
0.005 P = 0.04 0.0 NS
a
− 0.014 P = 0.007
− 0.006 NS
a
− 0.0001 NS
a
− 0.002 P = 0.046
APOC4codon 36 −
0.080 P = 0.00001 −
0.007 NS
a
− 0.004 NS
a
APOC4codon 52 −
0.001 P = 0.007 0.0 NS
a
− 0.0004 NS
a
APOC4codon 96 −
0.004 NS
a
0.005 NS
a
− 0.007
APOEcodon 112 P = 0.024
a
NS, non-significant.
3
.
4
. Association with quantitati6e traits A general linear model GLM was used to analyze
the association of the five APOC4 polymorphisms simultaneously with the two APOE polymorphisms.
Since this analysis takes into account multiple compari- sons, the significant levels do not need to be adjusted.
The APOE polymorphisms of codon 112 E3E4 and codon 158 E3E2 were included in the analysis be-
cause of their known effects on plasma cholesterol levels. A summary of the P-values from the GLM is
given in Table 4. APOC4codon 36 and codon 96 polymorphisms showed a significant association with
triglyceride levels in women. The Leu36 and Arg96 alleles were associated with elevated triglyceride levels
Table 5. The codon 36 and codon 96 polymorphisms explained 2.1 and 1.5 of the variation in triglyceride
levels, respectively in women. The codon 36 polymor- phism also showed significant association with apoA-I
levels in women. The APOC4nt. 620 variable site was associated with HDL-cholesterol levels in men and
Lpa levels in women Table 5.
Previously we have described the association of the APOE polymorphism with plasma cholesterol levels in
this population [11]. In that analysis, variation in the APOE gene codons 112 and 158 was treated as one
locus with three alleles APOE2, APOE3 and APOE4. However, in this paper we have analyzed the
contribution of the codons 112 and 158 polymorphisms separately. The APOE4 codon 112 and APOE2
codon 158 polymorphisms showed the expected asso- ciations with cholesterol and LDL-cholesterol levels
Table 4. The APOE codon 112 codon 158 polymor- phism explained 0.9 4.2 and 1.1 4.5 of the
variation in cholesterol, and LDL-cholesterol, respec- tively. The greatest effect of the APOE polymorphism
was on apoB levels where it accounted for 1.5 codon 112 and 6.5 codon 158 of the apoB variation. To
investigate the possibility whether an interaction exists between the APOE and APOC4 polymorphisms in
affecting cholesterol levels, a pairwise interaction was examined between all polymorphisms, but no signifi-
cant interaction was found data not shown.
4. Discussion
