At~wrosclerosb, 89 ( 1991) 69-X1 #B 1991 Elsevier Scientific Publishers .-IDONIS 002191509100137Y

ATHERO

69 Ireland,

Ltd. OO?l-9150/91/$03.50

04659

Variation of apolipoprotein B gene is associated with myocardial infarction and lipoprotein levels in Danes Anne Tybjaerg-Hansen

I-2, Barge G. Nordestgaard 3, Lars Ulrik Gerdes ’ and Steve E. Humphries

4,

’Charing Cross Sunley Research Centre. London (U.K. ), ’ Hagedorn Research Laboratory, Grntofte lDenmurk1 -’Department of Chemical Pathology and Metabolic Disorders, St. Thomas’ Hospital, Lfndon (U.K.), and ’ Department of Internal Medicine and Cardiology I, AOrhusCounty Hospital, Unir,ersity of Arhus, A’rhus (Denmark) (Received 15 January, 199 1) (Revised, received 21 March, 199 1) (Accepted 4 April, 1991)

Summary

Three DNA polymorphisms (XbaI, EcoRI, MspI) in the 3’-end of the apolipoprotein B gene were studied in relation to atherosclerosis, lipoprotein levels and age in three groups of atherosclerotic individuals and in nonatherosclerotic controls. The atherosclerotic groups comprised a postmyocardial infarction group with a mean age of 48 years, a group of individuals operated on for carotid stenosis with a mean age of 62 years, and a group of &year-olds with clinical coronary artery disease, peripheral arterial disease, or both. All 311 individuals were unrelated Caucasians of Danish ancestry. For the XbaI polymorphism, the X- allele was an independent predictor for myocardial infarction on multivariate analysis, but did not distinguish between patients and controls on univariate analysis. Additionally, this polymorphism was associated with variation in lipoprotein levels, but there was no clear evidence of a gene dosage effect. For the EcoRI polymorphism, the E- allele was associated with elevated levels of VLDL cholesterol, plasma triglycerides and VLDL triglycerides. Similar, but weaker associations were found for the MspI polymorphism. There were no significant differences in allele frequencies as a function of age for any of the DNA polymorphisms. In conclusion, while variation associated with the EcoRI polymorphism appears to be involved in the regulation of VLDL metabolism, variation associated with the XbaI polymorphism may determine susceptibility to coronary artery disease independent of other conventional risk factors, but it also appears to affect variation in lipoprotein levels.

Correspondence to: Dr. Anne Tybjaerg-Hansen. Department of Clinical Chemistry, Bispebjerg Hospital, University of Copenhagen. Bispebjerg Bakke, DK-2400 Copenhagen NV,

Denmark. Phone: + 45 35821450.

+45 35312639

(direct

line). ext. 2639; Fax:

70 Key words:

DNA polymorphisms; Coronary Lipids; Cholesterol; Triglycerides;

artery disease; Coronary heart Genetic markers; Chromosome

Introduction Apolipoprotein B-100 (apo B-100) is the major protein component of very low density lipoprotein (VLDL) and low density lipoprotein (LDL), and serves as the ligand for removal of LDL from the circulation by receptor-mediated endocytosis via the LDL receptor [ll. The mature apo B-100 peptide contains 4536 amino acids [2-51 and its structural gene, 43 kb long 161, resides in the short arm of chromosome 2 [7-111. So far, only one rare single base mutation in the apo B gene has been shown to affect binding to the LDL receptor [ 121 and cause hypercholesterolemia and premature coronary artery disease [13-141. A number of common DNA restriction fragment length polymorphisms (RFLPs) of the apo B gene have provided some preliminary evidence that common alleles of apo B may also be associated with altered lipid levels and/or atherosclerosis [15-301. These studies have been difficult to interpret for several reasons, which may suggest explanations for the often conflicting results. There is considerable variation in allele frequencies of these RFLPs among different ethnic groups [20,22,27], and associations between specific alleles and lipoprotein phenotype and/or atherosclerosis may be restricted to certain ethnic groups. Nonetheless, the majority of studies have been conducted in ethnically heterogeneous populations from the United States and the United Kingdom [15,17-21,26-27,291, and fewer in ethnically more homogeneous populations [ 16,2225,28,30]. The clinical criteria applied for the selection of study groups, especially control groups, have also varied greatly. Most studies have only included one population sample, and have not been able to address the question of reproducibility within the same population. Furthermore, all except one study 1171 have applied only univariate analysis to distinguish between allele frequencies in atherosclerotic and nonatherosclerotic groups. Such univariate differences in allele frequencies between patients and con-

disease: 2

Atherosclerosis;

trols could be due to an effect of the RFLP on atheroclerosis, on its own or through linkage disequilibrium with another gene, or to simple coassociation of the RFLP and a traditional risk factor such as LDL cholestrol level. Conversely, an actual effect of an RFLP on atherosclerosis could be masked by stronger traditional risk factors, predominant in such case/control studies. The aim of the present study was to determine whether three RFLPs (XbaI, EcoRI, MspI) in the 3’-end of the apo B gene were independent predictors (using multivariate analysis) for atherosclerosis, and/or associated with variation in lipid and lipoprotein levels. To evaluate whether the results were consistent (reproducible), these questions were addressed in three different samples from the ethnically relatively homogeneous Danish population. Materials

and methods

Subjects The three groups of atherosclerotic and nonatherosclerotic individuals were almost the same as previously described [31], except that subjects with diabetes mellitus were excluded from the present study. None of the participants were being treated with lipid lowering drugs, and individuals with familial hypercholesterolemia (FH) or secondary dyslipidemias were excluded from the study. The atherosclerotic groups comprised: a postmyocardial infarction group of men younger than 55 years of age (MI study, Table l>, a group of older men and women operated on for carotid stenosis (Carotid Study, Table 2), and a group of 85-year-olds with clinical coronary and/or peripheral arterial disease (85-year-olds Study, Table 3). The nonatherosclerotic controls were within the same age range as the patients. The 85-year-olds were originally selected as a representative population sample, and were later divided into atherosclerotic and nonatherosclerotic individuals. The examination procedure has been described elsewhere [31].

71 pAI

Luborutov methods Procedures for blood sampling, methods for isolation of VLDL and LDL, determination of lipids in plasma and in lipoprotein fractions, and the measurement of apolipoproteins Al, AI1 and B were as previously described [31]. HDL, cholesterol was isolated by ultracentrifugation as the d > 1.125 g/ml fraction. HDL, cholesterol was calculated as total HDL cholesterol minus HDL, cholesterol.

pAB 3

3 5c

IZ

!

Fig.

I. Organization

gene. The diagram

Total genomic DNA was prepared from the leukocytes of 10 ml of blood after lysis of red blood cells and was digested with the restriction enzymes XbaI. EcoRI or MspI according to the manufacturer’s specifications (Boehringer Mannheim). DNA fragments were separated according to size on 0.7%-1.0% agarose gels were (SeaKern ME, Interkemi, Allercld, Denmark) and transferred to nylon filters (Hybond-N, Amersham. Aylesbury, Buckinghamshire, U.K.) by Southern blotting [32]. Probes were isolated from plasmids, prepared by electrophoresis on low melting point agarose. and subsequently labeled with deoxycytidine 5’-a-[‘? Pltriphosphate in aqueous solution (n3’P-dCTP, 10 mCi/ml, specific activity 3000 Ci/mmol, Amersham) by oligolabeling [33]. Filters were hybridized at a concentration of l-2 X 10h cpm/ml hybridization buffer. and washed and the radioactive bands were detected by autoradiography. In XhnI digests fragments of 8.5 kilobases (kb) (X-l and 3.5 kb (X+) were detected using the unique genomic fragment pAB3.5C as a probe [11.19]. In EcoRI digests fragments of 12.5 kb (E-1 and 10.5 kb (E+) were detected with a 456 bp cDNA probe. pAB3 [ 1 1,191. and in M.spI digests fragments of 2.6 kb (M-J and of 2.3-2.4 kb CM+) were detected using the same probe. The three DNA polymorphisms used in this study arc shown in Fig. 1, and have been described previously [ 11,191. The XbaI RFLP involves the third base (wobble position) of threonine codon 248X (ACC * ACT) without changing the amino acid sequence [34]. The EcoRI RFLP changes codon 4154 from glutamic acid (GAA) to lysine (AAA) [34], but it is not known what functional significance this may have. The RFLP de-

of the 3’.end illustrates

of the apolipoprotein

the location

for the 3 enzymes used in this study and the relation gene

probes

apolipoprotein

to

these

sites.

B gene probes.

MYpI. * variable

restriction

pAB3.5~ X = .Yhul,

site. HVR

B

of restriction and

sites

of the 2 pAB3

E = EcoRI.

= hypervariahle

=

M = AT-

rich region. kb = kilohasea.

tected with MspI is due to insertion or deletion immediately 3’ to the apo B gene of a number of copies of an AT-rich minisatellite that consists primarily of a 30 bp tandem repeat sequence [20,35]. Statistical mulysis Statistical analysis of the data was carried out using MINITAB computer programs (Minitab, State College, PA, U.S.A.). We considered statistical significance to be at the 0.05 level. Because of skewness, triglycerides, VLDL triglycerides and VLDL cholesterol were logarithmically transformed to achieve approximately normal distributions in all three studies. Statistical tests were carried out on the transformed values. The t-test was used to compare the means of continuous variables between atherosclerotic and nonatherosclerotic individuals. The Xl-test was used to compare percent smokers, genotype distribution and allele frequencies (by gene counting) between atherosclerotic and nonatherosclerotic individuals, as well as to compare allele frequencies as a function of age in nonatherosclerotic individuals. The degree of linkage disequilibrium between two RFLPs (XbuI vs. EcoRI, EcoRI vs. MspI and XbuI vs. MspI) was estimated using the standardized disequilibrium statistic (correlation coefficient), J [36]. Delta values significantly different from zero, indicate linkage disequilibrium.

72 For subjects heterozygous for both polymorphisms, the relative frequency of the two possible haplotypes was determined from the frequency of the unambiguously observed haplotypes. The stepwise procedure (step-up method) for Fischer’s linear discriminant analysis [37,38], a multivariate analysis, was applied to identify the independent set of discriminators between atherosclerotic and nonatherosclerotic groups in the three studies; genotypes were included in these analyses. The best subset of predictors in each study was obtained by the ranking of variables whose T-ratios were significant. One-way analysis of variance was performed on the unadjusted lipid and lipoprotein levels, as well as on the same values adjusted for the influence of age and BMI by polynomial regression to test the null hypothesis that phenotypic variation is not associated with genetic variation in the 3’-end of the apo B gene. The bases for these

TABLE

adjustments were the relations between age and BMI and lipid levels found in the Copenhagen City Heart Study [39-401. Adjusting for age and BMI resulted in only minor differences in the mean lipid levels, and did not change the overall conclusions. We have chosen to show the unadjusted values. In these analyses of the effect of apo B polymorphisms on lipid levels, the P values were not corrected for the number of comparisons performed (see discussion). Results

Study populations The clinical and biochemical characteristics of the atherosclerotic and nonatherosclerotic groups in the three studies are shown in Tables l-3. Differences between the means and percentages of these same characteristics evaluated by univariate analysis (t-test and X*-test) are also shown.

1

CHARACTERISTICS FARCTION STUDY

OF

Characteristics

No. of subjects Age (years) BMI (kg/m*) Systolic BP (mm Hg) Diastolic BP (mm Hg) Triglycerides (mM) a VLDL triglycerides (mM) a Cholesterol (mMI VLDL cholesterol (mM) a LDL cholesterol (mM) HDL cholesterol (mM) HDL, cholesterol (mM) HDL, cholesterol (mM) LDL chol/HDL chol fipolipoprotein B (g/l) Apolipoprotein AI (g/l) Apolipoprotein AI1 (g/l) Glucose (mM)

ATHEROSCLEROTIC

AND

NONATHEROSCLEROTIC

Atherosclerotic

Nonatherosclerotic

50 48.2kO.7 26.7 f 0.6 125+3 85+1 2.19*0.17 1.57kO.16 6.38kO.16 0.99+0.12 4.3lkO.13 1.08+0.03 0.22 f 0.02 0.86 f 0.03 4.16kO.18 1.21 kO.05 1.30 f 0.03 0.53 f 0.01 5.2OkO.10

39 48.5 * 0.6 25.5 i 0.4 129+2 85+2 1.63+0.17 1.00+0.15 5.82kO.15 0.60 + 0.08 3.97kO.14 1.25 + 0.05 0.23 f 0.02 1.03*0.04 3.36kO.17 1.11 kO.04 1.48 + 0.04 0.59 + 0.02 5.10*0.10

MEN

IN THE

MYOCARDIAL

IN-

t-test (PI

NS NS NS NS < 0.01 < 0.001 < 0.05 < 0.01 NS ( < 0.10) < 0.01 NS i 0.001 < 0.01 NS ( < 0.101 < 0.001 < 0.05 NS *‘-test

Smokers

(%I

74

59

NS

Values in upper part of table are means f SEM. NS = P > 0.05: BMI = body mass index; BP = blood pressure: a Statistics based on log-transformed values; untransformed values are shown.

chol = cholesterol;

73 TABLE

7

CHARACTERISTICS STUDY

OF ATHEROSCLEROTIC

Characteristics

No. of subjects Age (years) BMI (kg/m’) Systolic BP (mm HgJ Diastolic BP (mm HgJ Triglycerides (mMJ a VLDL triglycerides (mM) ’ Cholesterol (mM) VLDL cholesterol (mM) (’ LDL cholesterol (mMJ HDL cholesterol (mMJ LDL chol/HDL chol Apolipoprotein B (g,/i) Apolipoprotein AI (g/l) Glucose (mM)

AND NONATHEROSCLEROTIC

MEN AND WOMEN

IN THE CAROTID

Women

Men Atherosclerotic

Nonatherosclerotic

52 62.9-t I.1 26.2 + 0.5 16Ok3 96*2 1.52~0.10 0.94 * 0.08 6.27 * 0.15 0.70 f 0.08 4.31 kO.14 1.26 * 0.04 3.6OkO.17 1.23+0.04 1.31 + 0.03 5.6rJkO.18

54 63.0 * 1.0 26.2 + 0.4 14x+3 85i_ I 1.44&0.10 0.88 * 0.08 5.71+0.13 0.57 * 0.05 3.87+0.11 I .27 * 0.05 3.30*0.17 1.14*0.04 1.38 + 0.04 fl.o5*0.12

t-test (PJ

NS NS < 0.05 < 0.001 NS NS < 0.01 NS (p < 0.10) < 0.05 NS NS NS ( < 0.10) NS < 0.05

t-test

Atherosclerotic

Nonatherosclerotic

25 59.J+ I.5 24.5 i_ 0.X 151+4 88+2 I .X3 + 0.24 1.11+0.1X 7.44 f 0.47 0.96 * 0.25 5.07 i 0.39 1.41 + 0.07 3.80 ir 0.3 I 1.31 kO.08 1.43 * 0.05 5.31*0.1X

28 59.9i I.4 24.2 rf-0.9 145k4.5 83&2 1.32~0.17 0.73 k IJ. 1 I 6.29 i 0.23 0.51 rf-0.10 4.10 i_ 0.23 1.68 i 0.09 2.62 _t 0.70 1.17+0.07 I .8X +0.0x 5.Y8+0.11

NS

61

60

68

NS

NS ( < 0.10) NS ( < 0.10) NS ( < 0.10) < 0.05 < 0.01 < 0.05 < 0.05 < 0.01 NS < 0.001 < 0.01

chol = cholesterol.

3

CHARACTERISTICS OF X5-YEAR-OLDS STUDY Characteristics

Number of subjects Age (years) BMI (kg/m’) Systolic BP (mm HgJ Diastolic BP (mm IIgJ Triglycerides (mMJ “ Cholesterol (mMJ LDL cholesterol (mMJ HDL cholesterol (mM) LDL chol/HDL chol Glucose (mM)

Smokers

NS

NS

Values in upper part of table are means i SEM. NS = P > 0.05: BMI = body mass index; BP = blood pressure: ,’ Statistics based on log-transformed values; untransformed values are shown.

TABLE

NS

y’-test

*‘-test 75

(P 1

(921

ATHEROSCLEROTIC

AND

NONATHEROSCLEROTIC

Men

AND

WOMEN

IN

THE

Women

Atherosclerotic

Nonatherosclerotic

10 x5.0*0.0 24.1 f 0.X 129+7 69+5 0.95+0.13 6.25 * 0.31 4.23 f 0.26 1.59+0.12 2.80 + 0.27 4.47io.15

I4 85.0 3.0.0 24. I t- 0.7 I145i5 JO+3 0.95*0.10 6.04 * 0.22 4.07 * 0.22 1.53*0.11 2.88 I 0:28 4.89 * 0.20

50

MEN

29

f-test (P)

NS NS NS ( < 0.10) NS NS NS NS NS NS NS

NS

Atherosclerotic

Nonatherosclerotic

15 85.OkO.O 27.2+ 1.9 143+4 65 ? 2 1.2X*0.15 7.15+0.33 5.00+0.31 1.57 * 0.08 3.35 + 0.30 4.79 + 0.31

24 85.0 * 0.0 26.6 _t 1.0 139+3 66k2 1.03 * 0.07 7.30 i 0.23 5.00 * 0.23 1.84+0.07 2.86 f 0.22 4.71 +0.14

13

I3

Values in upper part of table are means i SEM. NS = P > 0.05; BMI = body mass index: BP = blood pressure; ” Statistics based on log-transformed values; untransformed values are shown.

r-test(P)

NS NS NS NS NS NS NS < 0.05 NS NS

NS chol = cholesterol;

74 TABLE

3

RELATIVE

ALLELE x&I

FREQUENCIES (x-1

Atherosclerotic MI study (48 years) Men 0.51 (50) Carotid

OF RFLPs

OF THE APOLIPOPROTEIN EcoRI

B GENE

(E-1

MspI (M-1

Nonatherosclerotic

x2

Atherosclerotic

Nonatherosclerotic

x’

Atherosclerotic

Nonatherosclerotic

x’

0.43 (38)

NS

0.26 (50)

0.18 (39)

NS

0.26 (50)

0.16 (38)

NS

study (62 years)

Men

0.43 (50)

0.45 (54)

NS

0.16 (48)

Women All

0.52 (24) 0.46 (74)

0.46 (28) 0.46 (82)

NS NS

0.13 (23) 0.15 (71)

0.13 (51) 0.16 (28) 0.14 (79)

NS NS NS

0.19 (49) 0.11 (231 0.17 (72)

0.16 (51) 0.16 (281 0.16 (79)

NS NS NS

0.54 (14) 0.44 (24) 0.47 (38)

NS NS NS

0.00 (8) 0.17 (9) 0.09 (17)

0.18 (11) 0.18 (17) 0.18 (28)

NS NS NS

0.19 (8) 0.17 (9) 0.18 (17)

0.18 (11) 0.13 (16) 0.15 (27)

NS NS NS

X5-year-olds Men Women All

study 0.55 (10) 0.43 (15) 0.48 (25)

Allele frequencies are frequencies of alleles lacking the cutting site for XhaI and EcoRI RFLPs, and frequency of 2.6 kb allele for MspI RFLP. RFLP = restriction fragment length polymprphism. MI = myocardial infarction. NS = P > 0.05. Number of individuals in the groups are shown in parentheses. Discrepancies between number of individuals in this table and in Tables l-3 are. for a few, due to technical error, but for the majority in the 85.year-olds study due to lack of DNA (for details see legends to Tables 6-Y).

Apolipoprotein B polymorphisms in atherosclerotic and nonatherosclerotic indkliduals None of the genotype distributions, in atherosclerotic or in nonatherosclerotic individuals, differed significantly from that expected for a sample in Hardy-Weinberg equilibrium (data not shown). On univariate analysis (X2-test) there were no significant differences (P > 0.05) in allele frequencies between atherosclerotic and nonatherosclerotic men or women in any of the 3 studies

TABLE

(Table 41. Noteworthy however, the frequencies of the X-, E- and M- alleles were all higher in men with MI compared to nonatherosclerotic controls. Taking the three samples together, the standardized linkage disequilibrium statistic CA) and 95% confidence intervals for the three pairwise combinations of the three RFLPs were as follows: XbaI vs. EcoRI, A = 0.30 (0.224-0.374), EcoRI vs. MspI, A = 0.52 (0.459-0.5801, XbaI vs. MspI,

5

RANKING OF INDEPENDENT ATE ANALYSIS

DISCRIMINATORS

Rank 2

Rank 1 MI study Men

HDL,

Carotid study Men Women

Diastolic BP Apolipoprotein

85.year-olds Men Women

cholesterol

PREDICTING

ATHEROSCLEROSIS

BY STEPWISE

MULTIVARI-

Rank 4

Rank 3

(inv.)

VLDL

triglycerides

Presence

of X

AI (inv.)

Plasma Plasma

cholesterol glucose finv.)

Apolipoprotein Diastolic BP

allele

AI (inv.)

_

LDL chol/HDL

chol (inv.)

study -

_ _

_ _

_ _

The characteristics shown in Tables l-3 simultaneously with the genotypes shown in Table 4 were ranked according to importance as independent discriminators; multivariate analysis using a step-up procedure was used; only discriminators where the T-ratio was significant (P < 0.051, were ranked. chol = cholesterol. inv. = inversely related to atherosclerosis.

7s 3 = 0.27 (0.192-0.344) indicating linkage disequilibrium for all three pairs of polymorphisms. The ranked significant independent discriminators predicting atherosclerosis by stepwise multivariate analysis are shown in Table 5. In the MI Study, the X-- allele was the third most important predictor after HDL, cholesterol and VLDL triglycerides. None of the three RFLPs were independent predictors for carotid atherosclerosis or for atherosclerosis in %-year-olds in the present study. There were no significant differences in allele frequencies as a function of age in nonatheroscle-

TABLE

0

MEAN

PLASMA

I.IPIDS.

LIPOPROTEINS

rotic individuals for any of the three RFLPs ied. in men or in women (data not shown).

stud-

Apolipoproteitz B polytnorphisms atzd lipid lel>els Xhal polymorphism. In nonatherosclerotic men in the MI and Carotid studies as well as in 85year-old men, the heterozygotes (X Xf ) had the lowest mean plasma cholesterol levels, although these differences only reached statistical significance in the MI Study (Table 6). Nonatherosclerotic men with the genotype X-Xhad the highest apo AI levels; this difference was significant in the MI Study (P < 0.05)

AND ,\POLIPOPROTEINS

IN MEN WITH

DIFFERENT

.YhrlI CiENOTYPES

__-. Nonatherosclerotic

Atherosclerotic x-x

MI stud\ Pla\ma cholesterol VLDL cholesterol LDL ch~~lesterol 1IDL cholesterol Plasma triglyceridtzs VLDL triglyceride5 Apolipoprotein B Apolipoprotcin Al

II = 12 (mM) 6.21 kO.22 CmM)” 1.30~0.31 CmM) 3.84+0.25 (mM) I .07 + 0.07 (mM) ,’2.77+ 0.53 (mM)“Z.14+0.4Y l.ll+o.ox (g/l) (F/l) I.36 * 0.06

(‘arotid \tud) Plasma cholesterol VLDL cholesterol LDL cholesterol tlDL cholesterol Plasma triglycerides VLDI, triglyceride Apolipoprotein B Apolipoprotein izl

I, = 7 (mM) 6.12~0.22 CmM) ‘I 0.55 +O.lO CmM) 4.31 i 0.30 CmM) 1.77+O.I4 CmM) ‘I 1.55+0.2Y CmM) .’O.YXi-O.22 (g/l) 1.21 io.09 I.3 i 0.09 (g/l)

x-

Xi

I? = 27 6.33 + 0.24 O.YO+O.l7 4.33+0.17 I .09 + 0.05 2.00_+ 0.18 1.3X+0.14 1.22fO.06 1.77 + 0.04

x+x+

,I = I1 h.77 * 0.34 0.X3+0.17 4.7X*0.28 l.O’J i 0.05 2.00~0.30 1.39+0.28 1.2Y*o.l0 1.33 f 0.05

ANOVA (P)

NS NS = 0.05 NS NS NS NS NS

X- X-

X

II = 8 h.2S * 0.25 0.73 + 0.21 3.19+0..2 1.37 * 0.10 1.90+0.51 1.09*0.39 1.24+o.oh l.h7+0.10

rr-17 5.27 rf 0.70 0.45 f 0.05 3.56+0.21

12 5.80 * 0.35 0.51 i-o.12 3.YS * 0.32 I.J3+0.14 I .4ls f 0.25 O.YOf 0.20 l.lhiO.11 I 35 IO.1 I

I,=

NS NS NS NS NS NS NS NS

Population Xs-yrar-clld~-\tudy Plasma chole~lerol LDL cholesterol I IDL choloterol Plasma triglycerides

(mM) (mM) CmM) CmM) ,’

II = 7 h.l3i_O.l7 3.OY + 0. I4 1.6X+0.118 0.7Y~O.IO

Xi

xi

?(.

Ii i 0.23 i 0. I7 +0.x I .26* 11.08 I. IS + 0.07 I.ix*o.l3 l.X1+(1.3h O.X2rt_O.l7 1.72_c(l.33 o.Yx+o.oh 1.18~0.06 I..iYiO.Oh I.JX+O.llh

,I

:=

2j

5.61 10.17 (1.56 k 0.00 3.X5*0.15 1.20 k O.Oh 1.33~0.12 0.80~O.ll 1.17_tO.O4 I .33 + 0.05 sample

,I= 6.14 0.74 4.31

ANOVA (I’)

< 0.0 I NS = 0.05 NS NS NS < o.n5 c 11.05

II’17 5.72+0.1(1 O.h3 i 0.0X 3.ShtO.l(> I .73 + I).117 1.57iO.lX 0.“)‘)+1).1-1 I.14iO.Oh I.34 * 0.05

NS NS NS NS NS NS NS NS

I, = 5 h.53+0.64 3.54 + 0.5X 1.51+0.20 1.05+0.20

NS NS NS NS

h

n = I2 5.iJh+O.73 4.00~022 l.SOi0.l.i 1.01 iO.12

Value\ are means+SEM. X = allele lacking variable xhul restriction site. Xi = allele with variable .YhrlI rrstriction site. ANOVA = analysis of variance. II = number of individuals with that specific genotype: three individual\ were not typed for technical reasons. NS = P > 0.0.5. ” Statistics based on log-transformed values: untransformed values are shown. ” Same indiciduals as atherosclerotic + nonatherosclerotic men in Table 3.

76

and borderline significant in the Carotid Study (P < 0.10) (Table 6). Among atherosclerotic men in the MI Study, individuals with the genotype X+X+ had the highest LDL cholesterol level, individuals with the genotype X-X- had the lowest LDL cholesterol, while men with genotype X+X- had an intermediate LDL level (Table 6). Among atherosclerotic women in the Carotid study (Table 71, X+X+ women had the highest, X-X- women the lowest, and X+X- women an intermediate level of plasma and VLDL triglycerides. In 85year-old women, X-X- women had the lowest triglyceride levels as well. EcoRl polymorphism. Since E-Eindividuals were few, E-E- and E-E+ individuals were grouped together. In the MI Study, nonatherosclerotic men with the E-E - or E-E + genotypes had significantly higher VLDL cholesterol levels than E+ Ef men (Table 8). The same trend was found for nonatherosclerotic men in the Carotid study (VLDL cholesterol was not measured in the

TABLE

7

MEAN TYPES

PLASMA

LIPIDS,

LIPOPROTEINS,

AND

study of 85year-olds). Nonatherosclerotic women in the Carotid study showed the same association (Table 91, as well as a similar significant relationship with levels of plasma triglycerides and VLDL triglycerides, possibly reflecting an association between cholesterol and triglycerides in VLDL particles (correlation coefficient between VLDL cholesterol and VLDL triglycerides was 0.92, P < 0.001). In the two studies in which VLDL cholesterol was measured, similar trends in levels of VLDL cholesterol, plasma triglycerides and VLDL triglycerides were found for atherosclerotic individuals (Tables 8 and 9). Atherosclerotic women in the Carotid Study with the E-E-or E-E+ genotypes had significantly higher plasma cholesterol and LDL cholesterol than women with the E+E+ genotype (Table 9). A similar trend was found for plasma cholesterol and LDL cholesterol in the 85yearold women. Mspl polymorphism. The effects associated with the MspI polymorphism on lipid and

APOLIPOPROTEINS

Atherosclerotic x-x-

Carotid Study Plasma cholesterol VLDL cholesterol LDL cholesterol HDL cholesterol Plasma triglycerides VLDL triglycerides Apolipoprotein B Apolipoprotein AI

n=5 (mM) 8.17+ 1.51 (mM) a 0.36 f 0.07 (mM) 6.19* 1.54 (mM1 1.62 k 0.27 (mM) a 0.90 i 0.12 (mM1 a 0.41+ 0.07 (g/l) 1.38 + 0.30 (g/l) 1.46kO.08

IN WOMEN

(mM1

(mM) (mM) (mM) a

DIFFERENT

XbaI

GENO-

Nonatherosclerotic x-x+

’

n = 15 7.63 + 0.59 1.09 f 0.40 5.18+0.36 1.36 +O.OS 1.97kO.34 1.15+0.24 1.34 f 0.08 1.43 * 0.07

x+x+ n=4 6.13 kO.31 1.14 + 0.27 3.65 + 0.21 1.34k0.15 2.16kO.56 1.59 + 0.54 1.12kO.10 1.33+0.13

ANOVA (P) NS NS NS NS < 0.05 < 0.05 NS NS

x-x-

x-x+

X+X’

n=4 5.51 f 0.78 0.36 f 0.05 3.60+ 0.50 1.55kO.29 1.12+0.09 0.56+0.10 0.97kO.10 1.77kO.30

n = 18 6.25 f 0.30 0.59 f 0.15 3.91 f 0.30 1.76+0.11 1.40k0.18 0.82+0.16 1.16kO.09 1.93kO.10

n=6 6.92* 0.32 0.37 f 0.05 4.99+ 0.30 1.56+0.15 1.20+0.13 0.57i70.09 1.34+0.10 1.81~0.12

NS NS NS NS NS NS NS NS

n = 13 7.29 f 0.22 5.00+0.23 1.79+0.11 1.10+0.14

NS NS NS 0.05. a Statistics based on log-transformed values; untransformed values are shown. ’ Same individuals as atherosclerotic + nonatherosclerotic women in Table 3.

77 TABLE MEAN

X PLASMA

LIPIDS,

LIPOPROTEINS

AND APOLIPOPROTEINS

IN MEN WITH

or E-E’

E-E+

ANOVA (P)

MI study Plasma cholesterol VLDL cholesterol LDL cholesterol HDL cholesterol Plasma triglycerides VLDL triglycerides Apolipoprotein B Apolipoprotein AI

n = 23 (mM) 6.31 + 0.22 (mM) a 1.08&0.21 (mM) 4.10+0.17 1.13?0.06 (mM) (mM) ” 2.32 k 0.32 (mM) a 1.64+0.28 1.13+0.06 (g/l) 1.35 * 0.05 (g/l)

n = 27 6.45 + 0.23 0.91 20.12 4.50+0.19 1.04 f 0.03 2.0750.18 1.50+0.17 1.28 i 0.07 1.26 k 0.03

NS NS NS NS NS NS NS NS

Carotid study Plasma cholesterol VLDL cholesterol LDL cholesterol HDL cholesterol Plasma triglycerides VLDL triglycerides Apolipoprotein B Apolipoprotein AI

n = 15 (mM) 6.27 f 0.26 (mM) a 0.75kO.11 (mM) 4.25 + 0.24 1.27 k 0.08 (mM) (mM) * 1.61 +0.16 (mM) a 1.00+0.15 I .25 & 0.05 (g/l) 1.32 * 0.07 (g/l)

,1= 33 6.34 k 0.20 0.69li_O.11 4.41 kO.18 1.24*0.04 1.48+0.13 0.92kO.10 1.24 _L0.05 1.27 k 0.03

NS NS NS NS NS NS NS NS

X5-year-oids study Plasma cholesterol LDL cholesterol HDL cholesterol Plasma triglycerides

EcoRI

GENOTYPES

Nonatherosclerotic

Atherosclerotic E-E-

DIFFERENT

(mM) (mM) (mM) (mM) a

E-E-

or E-E’

EfE*

--_ II = 12 6.01 _t 0.29 0.76 i 0.13 4.07 + 0.24 1.18+0.06 1.94 * 0.32 1.20f0.31 1.19+0.07 1.41 + 0.07 n = 14 5.55 + 0.30 0.70+0.14 3.70 _+0.25 1.14+0.06 1.52t 0.22 0.9t+0.19 1.10&0.07 1 31 * 0.04 ~-Popuiation sample tl=4 6.00t0.18 3.88 + 0.20 1.6Ot_0.26 1.15 +0.20 -

n = 27 5.73+0.17 0.53 * 0.09 3.92kO.18 1.28 k 0.06 1.48 2 0.20 0.91 i0.18 1.07 i 0.05 1.51 fO.05

ANOVA (P) NS < 0.05 NS NS NS NS NS NS

,, = 37 5.77+0.13 0.52 & 0.05 3.94kO.12 1.31+0.06 1.41+0.11 0.85 + 0.09 l.i5 +0.04 1.41+0.05

NS NS NS NS NS NS NS NS

n = 15 6.25 + 0.28 4 23 -t 0.23 1.58+0.10 0.96*1).11

NS NS NS NS

’

Values are means+SEM. E- = allele lacking variable EcoRI restriction site. E+ = allele with variabie EcoRl restriction site. ANOVA = analysis of variance. n = number of individuals with that specific genorype: iwo individuals were nor typed for technical reasons, and 10 were not typed due to lack of DNA. NS ==P > 0.05. ‘!Sratisttcs based on log-transformed values: untransformed vaiues are shown, ’ Same individuals as atherosclerotic+ nonatherosclerotic men in Table 3.

lipoprotein Ievels were similar to, but weaker than those found for the EcoRI polymorphism idata not shown). Discussion _@olipoprotein B polymorphisms in atherosclerotic and nonatherosclerotic indilliduals The three atherosclerotic groups in the present study had their primary atherosclerotic disease in different parts of the arterial system: in the coronary arteries, in the carotid arteries, or a mixture as in the 85year-olds study. Since it is well known that the conventional risk factors for coronary. carotid and aortodistal atherosclerosis

are not identical as also iilustrated in ‘Tables l-3 and 5, it is conceivable that this may also be true for genetic risk factors or RFLPs in linkage disequilibrium with such risk factors. Consequently, it is not surprising that the X- allele is an independent predictor for myocardial infarction in the MI study, but not for atherosclerosis in the other two studies. Seven studies in Caucasian populations, including the present, have compared frequencies of X- alleles in atherosclerotic patients versus controls: in patients with coronary artery disease (CAD) and controls [1?,18,27-29; this study], or in patients with aortodistal atherosclerosis and controls [21]. Significantly higher frequencies of

78 TABLE

9

MEAN TYPES

PLASMA

LIPIDS,

LIPOPROTEINS

AND

APOLIPOPROTEINS

Atherosclerotic E-E-

Carotid study Plasma cholesterol VLDL cholesterol LDL cholesterol HDL cholesterol Plasma triglycerides VLDL triglycerides Apolipoprotein B Apolipoprotein AI

(mM) (mM) a (mM1 (mM1 (mM1 a (mM1 a (g/l) (g/l)

orE-E’

n=5 10.19+ 1.46 1.72+ 1.19 7.09* 1.13 1.39*0.15 2.18+ 1.02 1.21 + 0.59 1.45 + 0.27 1.46 k 0.05

IN WOMEN

WITH

EcoRI

GENO-

Nonatherosclerotic E+E+

n = 18 6.86 + 0.35 0.76+0.12 4.66 f 0.36 1.44 + 0.09 1.71+0.19 1.06kO.19 1.28+0.08 1.42 + 0.07

ANOVA (P) < 0.01 NS < 0.05 NS NS NS NS NS

E-E-

orE-EC

(mM1 (mM1 (mM) fmM1 a

E+E+

ANOVA (P)

n=9 6.26 + 0.46 0.80 k 0.27 3.93 f 0.45 1.53&0.14 1.69kO.25 1.09+0.23 1.20+0.12 1.s4*0.13 Population

85year-olds study Plasma cholesterol LDL cholesterol HDL cholesterol Plasma’triglycerides

DIFFERENT

n=8 7.89 + 0.44 5.59 + 0.49 1.81 kO.17 1.07*0.15

sample

n = 19 6.30 + 0.28 0.37 + 0.06 4.18kO.27 1.76+0.11 l.l4+0.12 0.56 f 0.10 1.15+0.08 1.91~0.10

NS < 0.05 NS NS < 0.05 = 0.01 NS NS

n=18 7.18kO.24 5.00 k 0.22 1.69 k 0.06 1.07 k 0.07

NS NS NS NS

b

site. Values are means f SEM. E- = allele lacking variable EcoRI restriction site. E +.= allele with variable EcoRI restriction ANOVA = analysis of variance. n = number of individuals with that specific genotype; one individual was not typed for technical reasons, and 14 were not typed due to lack of DNA. NS = P > 0.05. a Statistics based on log-transformed values: untransformed values are shown. women in Table 3. ’ Same individuals as atherosclerotic + nonatherosclerotic

the X- alle1e.m the patient groups were found on univariate analysis in three studies [17,21,27: in CAD patients vs. normocholesterolemic controls], and the same trend was found in two other studies [28; this study], but not in the studies by Deeb et al. and Genest et al. [18,29]. In agreement with the results of Hegele et al. [17], we found that the X- allele is anindependent discriminator for MI, that is, independent of lipid and lipoprotein levels, suggesting that some attribute of apo B other than its ability_ to bind to the LDL receptor may determine susceptibility to CAD. Seven studies have compared frequencies of the E- allele of the EcoRI RFLP in atherosclerotic patients and controls I17,18,21,27-29; this study]. Five of these found a significantly higher frequency of the E- allele in patients with coronary [17,27-291 or peripheral [21] atherosclerosis on univariate analysis, and similar trends were found for coronary atherosclerosis in the remaining two studies [18, this study]. On multivariate

analysis [17, this study] the EcoRI E- allele was not found to be an independent predictor for MI or peripheral atherosclerosis, suggesting that the E-allele may predispose to coronary and aortodistal atherosclerosis by coassociation with other risk factors, such as lipoprotein levels. Five studies have examined the frequency of the alleles of the MspI RFLP [17,18,29,30; this study]. On univariate analysis, one study [171 found a significantly higher frequency of the Mallele (ID1 allele) in MI patients, and a similar trend was apparent in our MI study. Deeb et al. [18], distinguishing 5 instead of 2 alleles, noted a significantly higher frequency of a 2.2 kb allele (allele 5) in CAD patients, and a similar trend was found by others [29]. Using a high resolution method [30] alleles containing 38, 44, 46 or 48 hypervariable elements showed an association with coronary heart disease, and with elevated lipoprotein levels. On multivariate analysis [17, this study], when biochemical and genetic vari-

79 ables were included, the MspI RFLP was not an independent predictor for atherosclerosis in any of these studies. Apolipoprotein

B polymorphisms

and lipid

lervls

The XbaI polymorphism (X’ allele) has been associated with elevated levels of plasma apo B [16,23], plasma cholesterol [15,16,19,24,281, LDL cholesterol [2X] and/or plasma triglycerides but not in all studies [1.5,19,20] in some, [17.18,21,22,25,27,29]. In a recent study 1411 the fractional catabolic rate of LDL cholesterol was highest in moderately hyperlipidemic individuals with the X-Xgenotype; this is compatible with observations that X~~X- individuals have the lowest levels of plasma cholesterol. The results from the present study do seem to suggest that the XhaI polymorphism is associated with variation in plasma cholesterol, LDL cholesterol and plasma apo B in men, and with plasma triglycerides and VLDL triglycerides in women, presumably through linkage disequilibrium with genetic variation elsewhere in the apo B gene or possibly in another gene. However, as has also been noted by others [28], associations are not the same in patients and controls. Thus. our data either do not support a simple codominant action of the XbaI alleles on the regulation of lipid and lipoprotein levels, or a second factor, genetic or environmental, interacts with variation at the apo B gene locus to determine an individuals plasma lipid and lipoprotein levels. In the present study an association between the E allele of the EcoRI polymorphism and high levels of VLDL cholesterol, plasma triglycerides and VLDL triglycerides was found in nonatherosclerotic women, with similar trends in nonatherosclerotic men, as well as in atherosclerotic men and women. This is in accordance with the results of a recent Austrian study [28] in an ethnically homogeneous sample of CAD patients and controls very similar to our MI study. The point mutation which gives rise to the EcoRI RFLP causes a change in the primary structure of the apo B protein altering amino acid 4154 from glutamic acid to lysine [34], and it is possible that this change in itself affects lipid and lipoprotein levels. Alternatively, the EcoRI polymorphism may be in linkage disequilibrium with another

common mutation of apo B that has this effect. The similarities between the results for the EcoRI and MspI RFLPs in the present study reflect the fact that the two RFLPs are in strong linkage disequilibrium. In this respect, our data are similar to those of others [17,29.30]. With 56 comparisons for each RFLP (men + women) one would expect a maximum of 2.8 f 1.63 significant associations (at P < 0.05) per RFLP by chance alone, but only if all lipid values and RFLPs were independent. This is not the case, since many of the lipid values are highly correlated and the RFLPs are in linkage equilibrium; the expected number of associations by chance is therefore considerably lower. Furthermore, as discussed above, for most associations consistent observations in other independent population samples, in the present or in other studies, suggest that these associations are not chance events. In conclusion, in the present study the Emallele of the EcoRI polymorphism in the 3’-end of the apo B gene was associated with elevated levels of VLDL cholesterol, plasma triglycerides and VL.DL triglycerides. The XhaI polymorphism. X- allele, was an independent predictor of myocardial infarction, but did not distinguish between patients and controls on univariate analysis. Additionally, this polymorphism was associated with variation in lipoprotein levels in a manner suggesting complex interaction with environmental or other genetic factors. Acknowledgement We thank Dagny Jensen for excellent technical assistance, and Bodil Kristiansen and Merete Appleyard, of the Copenhagen City Heart Study for practical help. We also thank Richard Morris, Department of Community Medicine, Guys Hospital, London, U.K., for statistical advice and Birthe Briiel for typing the tables. Dr. Jette Ingerslev is thanked for access to the 85year-olds. Anne Tybjaerg-Hansen was supported by Gerda and Aage Haensch’s Fund, the Danish Heart Foundation, a Wellcome-Carlsberg Travelling Research Fellowship, the Danish Medical Research Council, and Nordisk Insulinlaboratorium. B@rge G. Nordestgaard was supported by the

80 Danish Heart Foundation and the Danish Medical Research Council. Lars Ulrik Gerdes was a tostdoctoral Fellow at the University of Arhus, Arhus, Denmark. Steve E. Humphries was supported by the British Heart Foundation and the Charing Cross Sunley Research Trust.

IO

11

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