Am. J. Hum. Genet. 47:429-439, 1990

Variation at the Apolipoprotein (apo) Al-CiII-AIV Gene Cluster and apo B Gene Loci Is Associated with Lipoprotein and Apolipoprotein Levels in Italian Children C.-F. Xu,* M. N. NanjeeJ J. Savill,$ P. J. Talmud,* F. Angelico,§ M. Del Ben,§ R. Antonini,§ B. Mazzarella,§ N. Millert and S. E. Humphries* *Charing Cross Sunley Research Centre, Hammersmith, London; tBowman-Gray School of Medicine, Winston-Salem; *Laboratory of Microbiology, Department of Health, Brisbane; and §lstituto di Terapia Medica Sistematica dell 'Universita, La Sapienza di Roma, Rome

Summary We have used RFLPs of the apolipoprotein (apo) B gene and apo AI-CIII-AIV gene cluster to estimate the genetic contribution of variation at these loci to the variability of plasma lipid, lipoprotein, and apolipoprotein levels in 209 children from Sezze in central Italy. The sample was randomly divided into group I (107 children) and group 11 (102 children). Four site polymorphisms (PvuII, XbaI, MspI, and EcoRI) of the apo B gene and five site polymorphisms (XmnI, PstI, SstI, PvuII-CIII, and PvuII-AIV) of the apo AlCIII-AIV gene cluster were examined in group I children. After adjustment for gender, age, and body-mass index, polymorphisms at both gene loci (PvuII-B, PvuII-CIII, and PvuII-AIV) were associated with significant effects on the levels of plasma apo Al, apo B, or high-density lipoprotein-cholesterol. RFLPs that showed significant effects in group I were genotyped in group II. All three polymorphisms were associated with similar effects on apolipoprotein levels, though for all RFLPs the magnitude of the effects was smaller in the group II children and only statistically significant for the effect of the PvuII-B genotype on apo Al levels. In the total sample of 209 children 7.4% of the sample variance in apo Al levels was explained by variation associated with the apo B PvuII-B RFLP. In addition, the PvuII-B RFLP was associated with significant effects on plasma apo B levels and explained 5.7% of the sample variance. The PvuII-CIII and PvuII-AIV polymorphisms were both associated with differences in apo Al levels, explaining 3.7%-5.7% of the sample variance. Taken together, the three PvuII polymorphisms explained 17.7% of the phenotypic variance in apo Al levels. There was significant evidence for an effect of nonlinearity of the PvuII-CIII genotypes on apo Al levels, with the individuals heterozygous for the polymorphism having the highest apo AI levels. No evidence of interaction between genotype and gender, age, and body-mass index was shown by covariance analysis. The molecular explanation of this effect is unclear. Our data show that variation at both the apo AI-CIII-AIV and apo B loci are associated with lipoprotein and apolipoprotein levels in this sample of Italian children. Introduction Various environmental nutritional, and genetic factors'

ease

diet contribute to interindividual differences in the levels of plasma lipid, lipoprotein, and apolipoprotein are i d e(Ehnholm et al. 1982; Lewis et al. 1987; Nordoy and Goodnight 1990), path analysis and twin study have shown that the heritability of many of these variables is high (Berg 1979; Hamsten et al. 1986; Austin et al.

(CHolved Altpathougenvsiron t actronar st ais-

Received September27, 1989; final revision receivedMay 15, 1990. Address for correspondence and reprints: Chun-fang Xu, Charing Cross Sunley Research Centre, Lurgan Avenue, Hammersmith, London W6 8LW, England. '

C) 1990 by The American Society of Human Genetics. All rights reserved.

0002-9297/90/4703-0007$02.00

1987; Hasstedt et al. 1987; Pairitz et al. 1988). Primary disorders of lipoprotein metabolism, although rare, are known to be (Goldstein be

major genetic determinants for

to Brown etal.a183 for CHD (Goldstein et al. 1973; Brown et al. 1983; Brown and Goldstein 1984). For example, defects in the low-

429

430

density-lipoprotein (LDL)-receptor gene cause familial hypercholesterolemia (Brown and Goldstein 1984). Recently it was reported that a rare amino acid substitution (Arg-Gln) at residue 3500 of the apo B gene alters receptor binding ability in patients with familial defective apo B100 (Soria et al. 1989), causing hypercholesterolemia which in some patients is associated with premature atherosderosis (Tybjaerg-Hanssen et al. 1990). Epidemiological studies have established that elevated plasma levels of LDL and apo B (Kannel et al. 1979; Sniderman et al. 1980; Whayne et al. 1981) and depressed plasma levels of high-density lipoprotein (HDL) and apo AI (Miller and Miller 1975; Castelli et al. 1977; Avogaro et al. 1980; Maciejko et al. 1983) are correlated with increased risk of CHD. Apo B100, which is the sole protein in LDL, mediates the endocytosis of cholesterol by binding to the LDL-receptor on the cell surface (Brown et al. 1981), while apo Al, which is the major apoprotein of HDL, is an activator of lecithin:cholesterol acyltransferase (LCAT) (Fielding et al. 1973). The gene for apo AI is closely linked to the genes for apo CIII and apo AIV, clustered on the long arm of chromosome 11 (Lawet al. 1984; Karathanasis 1985). Difference in function or level of expression of both apo B and the apoproteins on chromosome 11 may therefore be involved in the development of CHD. A number of RFLPs of the apo B gene and of the apo AI-CIII-AIV gene cluster have been identified (Kessling et al. 1985; Priestly et al. 1985; Shoulders et al. 1985; Talmud et al. 1985; Barni et al. 1986; Coleman et al. 1986; Oettgen et al. 1986). Several studies have shown a correlation between serum lipid levels and the apo B XbaI RFLP (Berg 1986; Law et al. 1986; Talmud et al. 1987). Population associations showing that genetic variation is involved in the development of hypertriglyceridemia have been reported with use of RFLPs of the apo AI-CIII-AIV gene cluster detected with the enzymes SstI (Rees et al. 1985) and XmnI (Kessling et al. 1986), and associations between the PstI RFLP of the apo AI-CIII-AIV gene cluster and HDL cholesterol and apo AI levels have been reported (Ordovas et al. 1986; Kessling et al. 1988a; Wile et al. 1989). In Italy, CHD rates have steadily increased in this century (Research Group ATS-RF2 of the Italian National Research Council 1981; Research Group ATSRF2-Ob43 of the Italian National Research Council 1987), possibly as a result of changes in environmental factors such as diet and smoking (Research Group ATSRF2-Ob43 of the Italian National Research Council 1987; Angelico et al. 1989). Children have less exposure to some of the environmental factors which may con-

Xu et al.

tribute to differences in plasma lipid levels in adults, such as smoking and alcohol consumption, although they may be more influenced by developmental factors, such as hormone levels, than are adults. Since CHD risk-factor variables are related to the initial stages of atherosclerosis in the young (Newman et al. 1986), it is important to study the genetic and environmental factors determining serum lipoproteins in children. The distribution of plasma lipid, lipoprotein, and apolipoprotein levels in children from population studies in the United States are currently available (Lauer et al. 1975; Srinivasan et al. 1976; Ellefson et al. 1978; Tamir et al. 1981), and some of the studies show that the means of serum cholesterol concentration remained relatively constant during age 6-11 years. However, few association studies between apoprotein RFLPs and plasma lipid traits have been carried out in children. We present data from a study of 209 Italian children, aged 8-11 years, by using RFLPs of the apo B gene and of the apo AlCIII-AIV gene cluster to examine whether variations at these loci contribute to the interindividual differences in plasma lipid, lipoprotein, and apolipoprotein levels. Material and Methods Subjects The present study was carried out in an elementary school in Sezze Romano, south of Rome, where a comprehensive community control program for chronic disease is also in progress. A total of 209 children (113 boys and 96 girls), aged 8-11 years, who were attending the third and forth year of the school were recruited in May 1986. Screening operations were performed in the school's health office by trained personnel over a 3-d period. Blood was drawn after a 10-14-h fast. Children were semirecumbent, and blood was obtained with minimal venous stasis. Only children who did not go to school during these 3 d, a few who refused blood sampling, and two in whom venipuncture could not be performed because of difficulties in taking blood were excluded. More than 90% of eligible children participated in the study. The children enrolled in the study were ethnically homogenous. All children had been born in the Sezze area in central Italy, and according to a questionnaire record their families were natives of Sezze. No immigration has occurred in this small town in recent history. Lipid Measurement

Total plasma cholesterol and triglycerides (TG) were measured by standard enzymatic-colorimetric method

Apolipoprotein in Italian Children

(Boehringer-Biochemia). HDL cholesterol was determined after precipitation of apo B-containing lipoproteins by heparin-Mg+ +. HDL3 cholesterol was evaluated after precipitation of HDL2 with dextran-sulfate according to the method of Gidez et al. (1982), and HDL2 cholesterol was calculated. LDL cholesterol was calculated using the Friedewald et al. formula (1972). apo Al and apo B were determined by radial immunodiffusion using Nor-partigen plates (Behing-Werke AG). DNA Analysis

DNA was prepared from frozen whole blood by the Triton X-100 lysis method (Kunkel et al. 1977). Five micrograms of DNA was digested with each of the restriction enzymes PvuII, XbaI, MspI, EcoRI, XmnI, PstI, and SstI by using 5-10 units of enzyme/ pg of DNA, according to the manufacturer's recommended conditions (Anglian Biotech). Digested DNA was size separated on 0.7%-1.0% agarose gels and transferred to Hybond-N filters (Amersham) by Southern blotting according to a method described elsewhere (Kessling et al. 1988a). The apo B gene probes used were a unique genomic EcoRI fragment (pAB 3.5c) (Talmud et al. 1987; Xu et al. 1989) to detect the XbaI and MspI RFLPs, a 2.0-kb HindIII genomic fragment (BH2.0) of the 3' flanking region to detect the EcoRI RFLP (Talmud et al. 1988), and a 5' cDNA 959 to detect the PvuII-B RFLP (Darnfors et al. 1986). The apo AI-CIIIAIV gene-cluster probes used were a unique 2.2-kb PstI fragment of the apo AI gene to detect the XmnI, PstI, and SstI RFLPs and a 1.0-kb PvuII fragment and 1.05-kb PstI fragment (Kessling et al. 198 8b) to detect the PvuIICIII RFLP and PvuII-AIV RFLP, respectively. The labeling of probes and the hybridization, filter washing, and autoradiography procedures were those described elsewhere (Barni et al. 1986). Statistical Analysis The 209 samples were randomly divided into two groups for analysis. t-tests showed no significant difference between the mean values of the physical characters (age, sex, and body-mass index [BMI]) and plasma lipid, lipoprotein, and apolipoprotein concentrations of the two groups (table 1). The percentage phenotypic variance of lipid, lipoprotein, and apolipoprotein levels that was explained by age, gender, and BMI was estimated by linear regression. An initial study was performed on the first group of 107 children (61 boys and 46 girls), and genotypes for all the RFLPs described above were determined. x2 analysis was used to compare the allele frequencies of group I children both with

431 Table I Mean ± SD Lipid, Lipoprotein, and Apolipoprotein Levels

LEVEL (mg/dl), R2 x 100 Group I (N = 107) Group II (N = 102)

Cholesterol

.....

LDL .......... HDL .......... HDL2 ......... HDL3 ......... TG ........... apoAl ......... apo B .........

175 ± 25, 1.4 93 ± 22, .1 64 ± 10, 2.3 23 4, 4.1 41 7, 1.3 89 33, 18.7 164 ± 24, 3.1 91 ± 17, .6

179 ± 30, 2.9 96 ± 25, 4.6 65 ± 12, 1.9 24 8, 1.5 42 8, 3.1 92 37, 9.3 164 ± 27, 1.3 92 ± 22, 5.3

NOTE. -R2 X 100 = percentage of phenotypic variance of lipid, lipoprotein, and aplipoprotein levels explained by age, gender, and BMI.

U.K. population samples and with group II children. The correlation coefficient (delta) (Chakravarti et al. 1984) was used to estimate the pairwise linkage disequilibrium of RFLPs. The 95% confidence limits of delta were calculated using the z transformation (Sokal and Rohlf 1983). After adjustment of all the lipid traits by linear regression for age, gender, and BMI, a oneway analysis of variance was performed on the adjusted lipid levels to test the null hypothesis that phenotypic variation is not associated with genetic variation of the apo B gene and of the apo AI-CIII-AIV gene cluster. The percentage phenotypic variance of lipid traits explained by the genetic variations was estimated by regression. We considered statistical significance to be at the P < .05 level, except for the multiple comparisons presented in table 4A, where a level of P< .01 was used. Only RFLPs which were associated with a significant contribution (P < .01) to the phenotypic variation in traits in group I were examined in group II children (52 boys and 50 girls). Similar statistical analyses were carried out both in group II and in the entire sample of 209 children. Linear and nonlinear regression analysis was carried out using RFLP genotypes as dependent variables in situations where individuals heterozygous for the polymorphic site had the highest or lowest value compared with the other genotype classes. The linear effect was estimated by regression after coding genotype xlxl = 0, xlx2 = 1, and x2x2 = 2 (where x is an RFLP allele). The difference between the sum of squares regression due to genotype and the sum of squares due to the linear effect had 1 df and was a measure of the nonlinear effect of genotype on the dependent variable. The significance of the difference was estimated by an F-test.

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Apolipoprotein in Italian Children

433

Table 2 Linkage Disequilibrium Detected between RFLPs of apo B Gene and the apo Al-CIII-AIV Gene Cluster

apo BB

-po apo AI-CIII-AIV XbaI and MspI XbaI andEcoRl XbaI and PvuII PvuII-CIII and PvuII-AIV Delta ..42 Confidence limit

...

.30-.52

-

-.30 .17-.42

For the RFLPs showing a nonlinear relationship between traits and genotype, covariance analysis was carried out to look for interaction between genotype and gender or age. The significance of the interaction seen was established using an F-test. Results

In the initial analysis we determined genotypes for nine RFLPs of the apo B gene and of the apo Al-CIIIAIV gene cluster in 107 children. The variable sites of the restriction enzymes and the probes used for the apo B gene and the apo AI-CIII-AIV gene cluster are summarized in figure 1 and figure 2, respectively. For the RFLPs, the alleles are defined by the presence or absence of the variable cutting sites as shown in table 3. Linkage disequilibrium was detected between the apo B XbaI polymorphic site and the other three apo B RFLPs examined (table 2), while there was apparent linkage equilibrium between the RFLP pairs PvuII-BMspI, PvuII-B-EcoRI, and MspI-EcoRI (Delta = - .14,

-

-.33 .20-.45

.40 .26-.52

.14, and -.06 respectively). For the RFLPs of the ATCIII-AIV gene cluster, in agreement with other reports (Kessling et al. 1988b), apparent linkage equilibrium was observed, with the exception of the PvuII-CIII and PvuII-AIV RFLP pair, which are in a strong linkage disequilibrium (table 2). In table 3 we compared the allele frequencies of RFLPs observed in the Italian sample with allele frequencies reported by others for healthy normolipidemic individuals in the United Kingdom. Only the frequency of the apo B XbaT RFLP (X+ [site present] = .41] was significantly different from the allele frequency reported in the United Kingdom (X + = .53, P < .05) (Myant et al. 1989). There is evidence from a number of studies that the XbaI allele frequency differs in different ethnic groups (Aburatani et al. 1988). For the apo AT-CIIIAIV gene cluster, the allele frequencies of the RFLPs in this sample were not significantly different than frequencies found in the United Kingdom (Kessling et al. 1988b). After adjustment for gender, age, and BMI, one-way

Table 3 Estimated Allele Frequencies in the Italian Sample (N = 107) RARE ALLELE FREQUENCY GENE AND ENZYME

CUTTING SITE STATUS

Italy

+

.13

+

.41*

United Kingdom (sourcea)

apo B:

PvuII-B XbaI ......... MspI .........

........

EcoRI ........ apo AI-CIII-AIV XmnI PstI ..........

.........

SstI PvuII-CIII

...........

PvuII-AIV *

a

......

.....

+ + +

+

.11 .11

.08 .53 .15 .15

.13 .11 .08 .27 .07

.13 .10 .09 .27 .05

x2 p < .05. 1 = Myant et al. (1989); 2 = Xu et al. (1989); 3 = Kessling et al. (1988b).

(1) (1) (2) (1)

(3) (3)

(3) (3) (3)

Xu et al.

434

Table 4 A. Percentage of Sample Variance (R2 x 100) of Adjusted Lipid, Lipoprotein, and Apolipoprotein Levels That Is Explained by apo B and apo AI-CIII-AIV Gene Cluster in Group I Children apo AI-CIII-AIV

apo B

Cholesterol LDL .......... HDL .......... HDL2 HDL3 TG ........... AI ........... apo B ......... .....

.........

.........

PvuII-B

XbaI

MspI

EcoRI

XmnI

PstI

SstI

PvuII-CIII

PvuII-AIV

1.2 1.3 .2 .1 .2 .0 12.5** 7.6**

.7 .8 .9 .2 1.3 3.0 1.0 2.3

.3 .1 .3 2.0 .7 3.9 1.1 .2

.3 .1 .3 .5 .7 2.8 3.0 .6

3.8 .3 6.2

.1 .1 1.5 .0 3.4 .1 1.7

2.0 .8 2.8 3.6 2.0

1.2 3.2 4.2 2.0

.7 .3 8.6** 4.3 9.S** .1

7.5 4.5 4.6 5.3 2.8

2.5

5.3* 1.0 .3

4.5 6.1 10.9** .0

2.5 1.0

B. Percentage of Phenotypic Variance (R2 x 100) of Adjusted Lipid, Lipoprotein, and Apolipoprotein Levels That Is Explained by RFLPs of apoB and apo AI-CIII-AIV Gene Cluster in Group II (N = 102)

Cholesterol LDL HDL

HDL2 HDL3 TG apo AI apo B *

PvuII-B

PvuII-CIII

(rare allele frequency = .076)

(rare allele frequency = .261)

2.9 3.4 .4 .2 .6 1.1

.6 1.4 .5 .5 1.3 S.1 4.3 2.5

6.5* 4.5

PvuII-AIV (rare allele frequency = .066) .0 .4 1.2 1.1 1.0 .0

5.2* 1.5

P