deletion polymorphism in a healthy Serbian population: no association with serum lipid levels

Clinica Chimica Acta 263 (1997) 57–65

Study of apoB gene signal peptide insertion / deletion polymorphism in a healthy Serbian population: no association with serum lipid levels ˇ ´ Jelena Prljic, ´ Natasa ˇ Radovanovic, ´ Dragan Alavantic´ * Sanja Glisic, ˇ Institute of Nuclear Science, Laboratory for Radiobiology and Molecular Genetics — 080, VINCA P.O. Box 522, 11001 Belgrade, Serbia Received 10 October 1996; revised 6 February 1997; accepted 13 February 1997

Abstract The apolipoprotein B (apoB) signal peptide polymorphism was studied in unrelated healthy individuals. A total of 232 women and 222 men were analyzed separately. The relative frequencies of Del allele in women and men were 0.42 and 0.37, respectively. More heterozygous individuals were detected in comparison with other populations, using a modified silver staining method on polyacrylamide gel for visualization of Ins and Del alleles. There was no statistically significant difference in mean lipid levels adjusted for age, BMI, smoking habit and blood pressure between the three Ins / Del genotypes in both samples (ANOVA). Therefore, no differences were shown in the genotype frequency distribution throughout the lipid quartiles.  1997 Elsevier Science B.V. Keywords: Lipids; apoB gene; Polymorphism; Signal peptide; PCR

1. Introduction Epidemiological studies have identified plasma lipid level as one of the risk factors for development of atherosclerosis [1]. Apolipoprotein B (apoB), as a major protein in chylomicrons, VLDL, IDL and LDL particles, is a ligand for the LDL receptor mediating internalization of LDL particles [2]. It is important in *Corresponding author. Tel. / fax: 1 381 11 4462232; e-mail: [email protected] 0009-8981 / 97 / $17.00  1997 Elsevier Science B.V. All rights reserved PII S0009-8981( 97 )06556-X

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the assembly and secretion of chylomicrons from the intestine and VLDL from the liver [3]. Because of its central role in lipid transport, examining the variations of this protein in the apoB gene could help to explain inter-individual variation in lipid levels and susceptibility to coronary heart disease. The ApoB gene has been well described [4]. It is 43 kb long, with an 81-bp leader sequence coding for a 27-amino acid signal peptide. The signal peptide is involved in translocation of the protein across the endoplasmic reticulum membrane. Boerwinkle and Chan [5] localized a genetic polymorphism with two alleles: the insertion allele (Ins or 5 9 b sp27 ) with 27 amino acids and deletion allele (Del or 5 9 b sp24 ) with 24 amino acids. Later, Boerwinkle and colleagues [6] reported the third allele for this polymorphism discovered in MexicanAmerican subjects, the insertion* (Ins* or 5 9 b sp29 ) allele with 29 amino acids. Three amino acids (14–16) are absent in the shorter allele product (24 amino acids). In the third allele product, two amino acids are incorporated between the 23rd and 24th of a 27-amino acid signal peptide, creating a protein product of the Ins* allele. Observed Del allele frequency varies from only 0.09 in the Javanese population [7] to 0.42 in Caucasians. We have studied this apoB gene signal peptide insertion / deletion polymorphism in a sample of healthy individuals from Belgrade. We report both genotype and allele frequencies of this polymorphism in both women and men. In addition, we tried to find whether there was any relation between Ins /Del genotypes and / or alleles with variation of lipid values. We also examined genotype frequency distribution through lipid quartiles of the wide normal lipid values range, assuming the hypothesis that there is a difference between the highest and the lowest values.

2. Materials and methods

2.1. Subjects Four hundred and fifty-four healthy Serbian subjects of both sexes (232 females, 222 males) with an average age of 37.6 years, took part in this study. All were living in the Belgrade area and were selected according to normal lipid values. All subjects with a personal or family history of cardiovascular disease were excluded. Individuals taking any drugs with lipid-lowering effects were also excluded. All persons gave informed consent prior to their inclusion in the study. Blood samples were collected in the morning, after overnight fasting. Total cholesterol (TC), high density lipoprotein cholesterol (HDLC) and triglycerides (TG) were measured by enzymatic colorimetric tests using commercially available kits (PAP-tests, Yugomedica, Kragujevac). Low density lipoprotein

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Table 1 The characteristic of female and male subjects used in the study

n Age (years) BMI (kg / m 2 ) High BP Smokers TC (mmol / l)a LDLC (mmol / l)a HDLC (mmol / l)a TG (mmol / l)a

X6S.D. X6S.D. n (%) n (%) X6S.D. X6S.D. X6S.D. X6S.D.

Women

Men

232 37.73611.41 23.9863.13 67 (28.9) 83 (35.8) 4.9060.69 2.8060.66 1.6260.37 1.0460.44

222 37.77611.83 25.3662.73 47 (21.1) 119 (53.4) 4.7860.21 2.6960.15 1.5860.07 1.1360.13

BMI, body mass index; BP, blood pressure; TC, total cholesterol; LDLC, low density lipoprotein cholesterol; HDLC, high density lipoprotein cholesterol; TG, triglycerides. a Non-adjusted values.

cholesterol was calculated according to Friedewald’s formula [8]. The general characteristics of the healthy Yugoslav individuals grouped according to sex are given in Table 1.

2.2. DNA isolation, amplification and visualization DNA samples were prepared from 0.5 to 5 ml whole blood applying two different methods, with or without phenol extraction [9,10], collected with sodium citrate as anticoagulant. Two oligonucleotide primers for polymerase chain reaction (PCR) were used for amplification of short sequences (93 or 84 bp) [5]. Fifteen ml of the PCR product was loaded on to 8% polyacrylamide gel and visualization performed by modified silver staining method as described earlier [11].

2.3. Statistical analysis The allelic frequencies (Ins and Del) were estimated by the gene counting method. Differences in genotype distribution from that expected for HardyWeinberg equilibrium were obtained by the chi-square ( x 2 )-test. Adjusted lipid parameters (obtained by regression procedure) for age, BMI, smoking habit and blood pressure, were compared for genotype classes by analysis of variance (ANOVA) in sex-separated groups. A 5% significance level was used in both testing procedures. The x 2 -test was applied for comparing genotype frequencies between quartiles.

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3. Results

3.1. Frequency of the polymorphic variants The frequencies observed for each of the apoB signal peptide alleles (Ins allele and Del allele) and three different genotypes in healthy female and male samples are given in Table 2. The frequencies of Ins and Del alleles in the female sample were 0.58 and 0.42, respectively. In the male sample, the frequencies were 0.63 and 0.37, respectively. There was no significant difference in allele frequencies between the two samples. Observed genotype frequency distribution in both female and male samples showed statistically significant differences compared to the expected frequency distribution for the HardyWeinberg equilibrium ( x 2 -test, P . 0.01). As Table 2 shows, there is a significantly higher frequency of heterozygous and significantly lower frequency of Del /Del and Ins /Ins homozygous persons than expected.

3.2. Analysis of serum lipid values variation by genotype Individuals from both samples were subdivided according to signal peptide genotype and lipid parameters averaged in each group (Table 3). The effects of age, BMI, blood pressure and smoking were accounted for, and the mean values of the adjusted data were calculated. There was no significant association, analyzed by ANOVA, between the genotypes and variation of any lipid parameter in either female or male samples. It was shown that homozygotes Del /Del had higher TC, LDLC and TG levels in both female and male samples, as well as lower HDLC than homozygotes Ins /Ins in female samples. Heterozygotes Ins /Del had intermediate values. However, these tendencies did not reach significance. Table 2 ApoB signal peptide insertion / deletion genotypes and Ins and Del allele frequencies in the healthy population sample

Women

Men

Total, n

Ins allele, n (%)

Del allele, n (%)

Ins /Del genotypes

Observed frequencies, n (%)

Expected frequencies, n (%)

464

270 (58.2)

194 (41.8)

Ins /Ins Ind /Del Del /Del Ins /Ins Ins /Del Del /Del

53 164 15 72 135 15

78.5 (33.9) 112.7 (48.6) 40.4(17.5) 87.8 (39.5) 103.7 (46.7) 30.6 (13.8)

444

279 (62.8)

165 (37.2)

(22.8) (70.7) (6.5) (32.4) (60.8) (6.8)

x 2 -test, P

, 0.01

, 0.01

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Table 3 Adjusted mean lipid levels in female and male samples divided into groups according to their Ins /Del genotypes

Women Total Ins /Ins Ins /Del Del /Del ANOVA Men Total Ins /Ins Ins /Del Del /Del ANOVA

n

TC (mmol / l)

HDLC (mmol / l)

LDLC (mmol / l)

TG a (mmol / l)

232 53 164 15

4.9060.22 4.8960.19 4.9060.24 4.9160.16 NS

1.6260.08 1.6360.08 1.6260.07 1.6060.08 NS

2.8060.13 2.7960.12 2.8060.14 2.8260.12 NS

1.0460.14 1.0360.12 1.0460.15 1.0560.09 NS

222 72 135 15

4.7060.23 4.6860.20 4.7060.24 4.7460.26 NS

1.4860.05 1.4860.06 1.4860.05 1.4860.05 NS

2.6810.18 2.6660.16 2.6860.19 2.7260.20 NS

1.1910.14 1.1960.14 1.1960.15 1.2360.16 NS

TC, total cholesterol; HDLC, high density lipoprotein cholesterol; LDLC, low density lipoprotein cholesterol; TG, triglycerides. a Triglycerides do not have normal distribution and were log transformed.

3.3. Genotype /allele frequency distribution in the lipid quartiles For further investigation, female and male samples were divided according to lipid parameters adjusted for four risk factors: age, BMI, smoking habit and blood pressure. The proportion of Ins and Del alleles for insertion / deletion polymorphism in the lipid quartiles were counted and the genotype frequencies compared using the x 2 -test. There was no significant difference in the genotype frequencies between the quartiles for any lipid parameter (TC, HDL cholesterol, LDL cholesterol and TG) in both male and female subjects (Table 4).

4. Discussion The Del allele frequency observed in our population is similar to that published for a Caucasian population. In a Caucasian group of both genders it was 0.42 [5] and 0.35 [12], respectively. In a female and male control group from Southern Norway, Del allele frequencies were 0.36 and 0.37, respectively [13]. In male controls from the Strasbourg population, Del allele frequency was 0.29 [14]. Most of the authors found Del allele to be associated with elevated total cholesterol level [15,16] and LDL cholesterol level in control samples [13,14,17,18]. Boerwinkle and colleagues [19] discovered that the Del allele showed a slight relationship with high triglyceride levels in the sample from

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Table 4 Alleles and genotypes frequencies of insertion / deletion polymorphism in female and male subjects Lipids

x 2 -test

Allele frequency

Frequency, % (n)

Ins (%)

Del (%)

Ins /Ins

Ins /Del

Del /Del

I (56) II (55) III (56) IV (55) I (56) II (55) III (57) IV (54) I (55) II (56) III (56) IV (55) I (55) II (56) III (56) IV (55)

0.616 0.709 0.616 0.573 0.634 0.618 0.596 0.667 0.636 0.670 0.616 0.591 0.627 0.643 0.607 0.636

0.384 0.291 0.384 0.427 0.366 0.382 0.404 0.333 0.364 0.330 0.384 0.409 0.373 0.357 0.393 0.364

0.304 (17) 0.418 (23) 0.321 (18) 0.255 (14) 0.303 (17) 0.345 (19) 0.298 (17) 0.352 (19) 0.345 (19) 0.357 (20) 0.304 (17) 0.291 (16) 0.291 (16) 0.375 (21) 0.268 (15) 0.364 (20)

0.625 (35) 0.582 (32) 0.589 (33) 0.636 (35) 0.661 (37) 0.545 (30) 0.596 (34) 0.630 (34) 0.582 (32) 0.625 (35) 0.625 (35) 0.600 (33) 0.673 (37) 0.536 (30) 0.678 (38) 0.545 (30)

0.071 (4) 0.089 (5) 0.109 (6) 0.036 (2) 0.109 (6) 0.105 (6) 0.019 (1) 0.073 (4) 0.018 (1) 0.071 (4) 0.109 (6) 0.036 (2) 0.089 (5) 0.054 (3) 0.091 (5)

Females TC I (58) II (58) III (59) IV (57) HDLC I (58) II (58) III (58) IV (58) LDLC I (58) II (58) III (58) IV (58) TG I (59) II (58) III (57) IV (58)

0.595 0.595 0.576 0.588 0.534 0.578 0.629 0.586 0.595 0.569 0.595 0.569 0.576 0.612 0.544 0.595

0.405 0.405 0.424 0.412 0.466 0.422 0.371 0.414 0.405 0.431 0.405 0.431 0.424 0.388 0.456 0.405

0.224 (13) 0.224 (13) 0.237 (14) 0.228 (13) 0.172 (10) 0.190 (11) 0.310 (18) 0.241 (14) 0.241 (14) 0.207 (12) 0.259 (15) 0.207 (12) 0.186 (11) 0.293 (17) 0.210 (12) 0.224 (13)

0.741 (43) 0.690 (40) 0.678 (40) 0.719 (41) 0.724 (42) 0.776 (45) 0.638 (37) 0.690 (40) 0.707 (41) 0.724 (42) 0.672 (39) 0.724 (42) 0.780 (46) 0.638 (37) 0.667 (38) 0.742 (43)

0.035 (2) 0.086 (5) 0.085 (5) 0.053 (3) 0.103 (6) 0.034 (2) 0.052 (3) 0.069 (4) 0.052 (3) 0.069 (4) 0.069 (4) 0.069 (4) 0.034 (2) 0.069 (4) 0.123 (7) 0.034 (2)

Males TC

HDLC

LDLC

TG

Quartile (n)

NS

NS

NS

NS

NS

NS

NS

NS

TC, total cholesterol; LD/ HDLC, low / high density lipoprotein cholesterol; TG, triglyceride; I, first quartile with the lowest values for TC, LDLC and TG, and the highest value for HDLC; II and III, second and third quartiles; IV, forth quartile with the highest values for TC, LDLC and TG, and the lowest value for HDLC. NS, non-significant.

Nancy, France. We found non-significantly elevated TC, LDLC and TG, and decreased HDLC, in individuals homozygous for the Del allele. In agreement with our study, Peacock et al. [20] did not find insertion / deletion genotype to

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affect lipoprotein level variation in age-matched male individuals from Sweden. We applied the quartile analysis to our healthy female and male groups to see if lipid extremes were related to allele and genotype frequencies. Similarly, Gaffney et al. [12] applied quintile analysis to their non-sex-separated group from Glasgow, but without detecting any significant difference in either allele or genotype frequencies. We performed a separate analysis of female and male samples, because of the obvious gender influence [21–24] on the type and degree of genotype-to-phenotype relationship, when considering variation in the lipoprotein concentrations. The genotype frequencies observed for both women and men in our study differed significantly from the expected ones, assuming Hardy-Weinberg equilibrium, as in the study of Visvikis et al. [25]. Suspecting technical reasons, we initially re-amplified over 70% of the individuals, but the result was the same. In addition, we typed two other two-allelic apoB polymorphic loci for the same population sample, and their genotype frequencies were in agreement with the Hardy-Weinberg expectations. We detected more heterozygous individuals with apoB insertion / deletion signal peptide polymorphism in the Serbian population compared to any other population. We assume that one of the reasons for these differences could be the previously unused, precisely modified silver staining method for visualization of the PCR products. This is important, especially if any of the products were synthesized in lower quantities (in about 25% samples, which were re-amplified three times). The basis of the unequal quantities of the two alleles for this polymorphic locus is not clear, but it is also observed in other studies. Xu et al. [26] and Boerwinkle et al. [19] detected heterozygotes with lower synthesis of either the Ins allele or the Del allele even by ethidium bromide staining. Problems appear when one of the synthesized alleles is represented by a more vague band. Recently, we showed that an impressive quantity difference could exist among alleles, when one of them is vague, by using silver staining compared to ethidium bromide staining. The presence of alleles represented by vague bands was confirmed by a radiolabeled probe [11]. The second possible explanation for the number of heterozygotes could be that this sample actually was not a random one: i.e., persons studied were generally selected as both healthy and normolipidemic. Further investigation of hyperlipidemic individuals would be a good test for these findings in a healthy Serbian population. Although genetic variations in apoB signal peptide could affect transport of the peptide across the endoplasm reticulum membrane, the exact mechanism of the Ins /Del influence on lipid metabolism is not completely understood. New light in understanding the Ins /Del influence was given by Pajukanta et al. [27], in a study of plasma lipid response during a low-fat and low-cholesterol diet in connection with Ins /Del polymorphism. Individuals homozygous for the Del allele exhibited small variations in VLDL and HDL 2 cholesterol but the LDL

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cholesterol level was elevated during dietary change. This effect probably could not be attributed only to the Ins /Del polymorphism, but helps to discover the point of synergistic effect for some of the apoB polymorphisms. In support of this, a strong linkage disequilibrium with XbaI apoB gene polymorphism for different populations was shown [7,27,28]. Thus, a linkage disequilibrium with some other functionally more important polymorphisms could offer a better explanation of the data observed in our study.

Acknowledgments This work was supported by Government Research Grants 0306 and 13M03. The authors thank Gordana Milenkovic´ for her technical assistance.

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