05 - IJBMRF20141522 Neda M. Bogari

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International Journal of Biological & Medical Research

Int J Biol Med Res. 2014; 5(2): 3981-3987

Apolipoprotein B (XbaI) allele frequencies in an Egyptian Population: impact on

blood lipidsa b c a b

Neda M. Bogari , Azza M. Abdel-Latif and Maha A. Hassan , Ahmed Fawzy ,aFaculty of Medicine, Department of Medical Genetics, Umm Al-Qura University, Makkah, Kingdom of Saudi ArabiabDivision of Human Genetics & Genome Researches, Department of Molecular Genetics and Enzymology, National Research Centre, Cairo, Egypt.cHolding Company for Biological products and Vaccines (VACSERA-Egypt).

A R T I C L E I N F O A B S T R A C T

Keywords:

Apolipoprotein BXbaI polymorphismAllele frequencyBlood lipidsEgypt

Original Article

Background: Apolipoprotein B (ApoB) is the major protein component of chylomicrons, low-density lipoproteins (LDL), very low density lipoproteins (VLDL) and intermediate-density lipoproteins (IDL) and is the ligand for the LDL receptor. ApoB plays an important role in the maintenance of cholesterol homeostasis and lipoprotein metabolism. Numerous restriction fragment length polymorphisms of the ApoB gene have been reported to be associated with variation in lipid levels, obesity and/or coronary artery disease. Purpose: This study assesses the effect of Apo B restriction fragment length polymorphism (XbaI) on lipid and lipoprotein concentrations in an Egyptian population. Methods: Blood samples from 355 unrelated individuals aged 19 – 70 years were analysed and genotyping was performed using the XbaI restriction enzyme after polymerase chain reaction (PCR) amplification. Results: Allele

- +frequencies obtained for X and X were 69 and 31 %, respectively. Presence of the X+ allele was - -associated with increased levels of total cholesterol, ApoB, and LDL. X /X homozygous

+ +individuals in our sample displayed the lowest mean levels and homozygous X / X the highest, with heterozygous individuals displaying intermediate levels. Conclusion: These findings confirm the impact of ApoB genetic variation on some lipid related factors levels in an Egyptian population.

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Introduction

Copyright 2010 BioMedSciDirect Publications IJBMR - All rights reserved.ISSN: 0976:6685.c

Atherosclerosis and coronary artery disease (CAD) are growing

health problems worldwide, affecting an increasingly young

population [1, 2]. Established risk factors for CAD include age,

hypertension, smoking, diabetes, elevated total cholesterol, and

decreased high-density lipoprotein (HDL) cholesterol [3,4]. The

variability of serum levels of lipoproteins is believed to be

important [5]. A number of candidate genes has been implicated in

the pathogenesis of CAD and atherosclerosis [6,7]. One of the

genetic variants is located in the apolipoprotein B (ApoB) gene. It

has been reported that ApoB impacts total atherogenic particle

number in plasma, and is superior to LDL cholesterol as an index of

the lipid-related risk of vascular disease and as a guide to the

adequacy of LDL-lowering therapy [8]. However, for historical

reasons, cholesterol, and more specifically LDL-cholesterol,

remain the accepted indicators for atherosclerosis risk. We

suggest that ApoB measurement should be included in guidelines

as an indicator of cardiovascular risk. This has become the position

of the three major Canadian guideline groups [9-11]. The latest

European guidelines acknowledge the value of ApoB, but highlight

the current problem of restricted laboratory availability [12].

ApoB, the largest of all human apolipoproteins, is a major

constituent of low-density lipoproteins and of the triglyceride-rich

lipoproteins. ApoB has a fundamental role in the transport and

metabolism of plasma cholesterol serving as a ligand for the

receptor-mediated uptake of low-density lipoproteins by various

cells [13 -15]. It occurs in plasma as two major immunologically

distinct isoforms, ApoB-100 and ApoB-48. ApoB-100 is

synthesized in the liver and plays a role in the packaging of very

low-density lipoprotein particles, whereas ApoB-48 is synthesized

in the intestine and plays a role in the formation of chylomicrons

[13 - 17]. The two isoforms share a common N-terminal sequence.

* Corresponding Author : Neda M. Bogari

Faculty of Medicine, Department of Medical Genetics, Umm Al-Qura University, Makkah, Kingdom of Saudi [email protected]

Copyright 2010 BioMedSciDirect Publications. All rights reserved.c

International Journal ofBIOLOGICAL AND MEDICAL RESEARCH

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Neda M. Bogari et.al Int J Biol Med Res. 2014; 5(2): 3981-3987

3982

levels that are associated with atherosclerosis and CVD [26-28].

This polymorphism arises from a single nucleotide change. A

cytosine base in the ''wild type'' allele becomes a thymine base in

the ''rare'' allele [28,29]. This base change creates a cutting site for

the restriction endonuclease XbaI and the polymorphism is known

as ApoB XbaI polymorphism, which is silent. This means that it does

not change the amino acid sequence of ApoB [27]. The aim of this

study was to determine the allele frequencies of the XbaI

polymorphism of ApoB in an Egyptian population. We then

investigated possible correlations with pathologies associated with

LDL-C, Chol, and ApoB levels and the risk of hyperlipidemia

associated with this gene.

Mice containing only one functional copy of the mApoB gene

show the opposite effect, being resistant to hypercholesterolemia.

Mice containing no functional copies of the gene are not viable [25].

A polymorphism on codon 2488 of exon 26 in the human ApoB gene

has been shown to influence the triglyceride (TG), LDL-C, and ApoB













Three hundred and fifty five unrelated subjects (215 men and

140 women), aged 19 – 70 years were randomly recruited in Cairo.

At study entry, all subjects completed a questionnaire covering data

on demographics, family status, education level, smoking habits,

history of cardiovascular disease, blood pressure, medications, age,

weight, height and sex. Written informed consent was obtained

from each subject. Body mass index (BMI; the body mass in

kilogram divided by the square of the individual's height in meters)

was calculated. Obesity was defined by a body mass index (BMI) >

30 kg/m2. Hypertension was defined as the existence of a systolic

blood pressure > 140 mmHg or blood pressure diastolic > 90 mmHg

(or receiving antihypertensive treatment according to WHO

recommendations) [30]. The presence of diabetes was determined

by fasting glucose > 125 mg/dl. Dyslipidemia was defined as total

cholesterol (TC) > 250 mg/dl and / or triglycerides (TG) > 87.5

mg/dl and a value of LDL- cholesterol (LDL- C)> 160 mg/dl.

The shorter ApoB-48 protein is produced after RNA editing of

the ApoB-100 transcript at residue 2180 (CAA->UAA), resulting in

the creation of a stop codon, and early translation termination. The

ApoB gene is located on the short (p) arm of chromosome 2

between positions 24 and 23. Human ApoB-100 cDNA is 14

kilobases in length and encodes a 4563-amino acid precursor

protein. This gene comprises 29 exons and 28 introns. The

distribution of introns is extremely asymmetrical, most of them

appearing in the 5' terminal one-third of the gene. Although most of

the exons fall within the normal size limits for mammalian genes,

two are unusually long: 1906 and 7572 base pairs. The latter exon is

by far the longest reported for a vertebrate gene. ApoB is the sole

protein component of LDL and VLDL [18-20]. The interaction of

ApoB-100 with LDL receptors mediates the uptake of LDL from

liver and peripheral cells; hence, it serves the function of

solubilizing cholesterol within LDL [21].

Sequencing of human ApoB revealed a number of

polymorphisms that affect lipid metabolism and cardiovascular

disease (CVD) [22]. Because of the central role of apolipoprotein in

lipid metabolism, even small changes in the protein structure or

function may be of physiological importance [23]. Mice

overexpressing mApoB have increased levels of LDL "bad

cholesterol" and decreased levels of HDL "good cholesterol"[24].

Mice containing only one functional copy of the mApoB gene show

the opposite effect, being resistant to hypercholesterolemia. Mice

containing no functional copies of the gene are not viable [25]. A

polymorphism on codon 2488 of exon 26 in the human ApoB gene














Mice containing only one functional copy of the mApoB gene

show the opposite effect, being resistant to hypercholesterolemia.

Mice containing no functional copies of the gene are not viable [25].

A polymorphism on codon 2488 of exon 26 in the human ApoB gene


Materials and methodsSubjects

3983

Blood samples were taken in EDTA tubes, in the morning after a

12-hour fast. Plasma was immediately separated from the cell pellet

after centrifugation at 250 g for 15 minutes at 4° C. Total cholesterol

(TC) and triglycerides (TG) were measured by enzymatic

colorimetric tests [31,32]. High-density lipoprotein cholesterol

(HDL-C) concentrations were determined by enzymatic assay after

phosphotungstic acid and magnesium precipitation [33]. Low-

density lipoprotein cholesterol (LDL-C) was calculated according to

Friedewald's formula and very-low-density lipoprotein cholesterol

(VLDLC) was calculated using the formula TG/5 [34]. Fasting blood

sugar was assayed by glucose oxidase-peroxidase method [35].

Apolipoproteins A1 and B (ApoA1 and ApoB) were determined by

immunoturbidimetric methods. Coefficients of variation (CV) for

total cholesterol, HDL-C, and triglyceride measurements were

below 5%.

Genomic DNA was isolated from peripheral blood. The isolated

DNA was of good quality (absorbance 260 nm/280 nm, ratio >

1.75). PCR was used to amplify a 230 bp fragment in the ApoB gene

using the oligonucleotide primers F: 5'-AAATAACCTTAA

TCATCAATTGGT -3' as upstream primer and R: 5'- GGTTCTTAG

CAGCAAGAGTC -3' as downstream primer (36). Each amplification

was performed using 100 ng of total genomic DNA in a final volume

of 25 μL containing 40 pmol of each oligonucleotide, 0.2 mmol/L of

each dNTP, 1.5 mmol/L MgCl2, 10 mmol/L Tris (pH 8.4), and 0.25

units of Taq polymerase (Fermentase Co. Canada). Hybridization

was carried out in a DNA thermal cycler (Bio-Rad Laboratories;

Foster City, California, USA) in which DNA templates were odenatured at 95 C for 5 min, amplification consisting of 35 cycles in

o 0 o95 C for 45s, 60 C for 1 min, and 72 C. Products were subjected to orestriction enzyme analysis by digestion at 37 C for 2 h with 10

U/mL of XbaI restriction endonuclease in each 10 μL of PCR sample

in the buffer recommended by the manufacturer of the

endonuclease (Roche Co. Mannheim, Germany). The fragments

separated by electrophoresis on 2% agarose gels. After

electrophoresis, gels were stained with 1 mg/dl ethidium bromide

solution for 10 min, and DNA fragments were visualized by gel

documentation (Bio-Rad Laboratories; Foster City, California, USA).

DNA samples were quantified by absorbance measurement and this

allowing samples concentration to be normalised to produce

consistent results. The genotypes for all samples were reassessed

twice to confirm the results and ensure reducibility. Some

suspected genotypes were validated by purifying the PCR products

using GeneJET gel extraction and DNA cleanup kit (Thermo Fisher

Scientific Inc. USA) and were directly sequenced in an ABI Prism

310 Automated Sequencer, using the ABI Prism Big Dye Terminator

Cycle Sequencing Ready Reaction Kit (PE Applied Biosystems).

The following nomenclature has been used to specify the

genotypes at ApoB XbaI:

· X-/X- samples contain 230 bp fragments (wild-type, CC, or

homozygous absence of restriction site)

· X-/X+ samples contain 230, 160, 70 bp fragments (CT,

heterozygous absence of restriction site deletion)

· X+/X+ samples contain 160 and 70 bp fragments: (rare-type,

TT, or homozygous presence of restriction site).

All statistical calculations for the biochemical data were

performed using Statistical Package for the Social Sciences (SPSS)

version 11.5 (IBM Corporation). Data were expressed as mean ± SD,

whereas categorical variables were summarized as frequencies and

percentages. Data was evaluated by t test and one-way analysis of

variance. Allele and genotypic frequencies for ApoB XbaI were

calculated with the gene counting method. For XbaI polymorphism,

deviation from Hardy–Weinberg equilibrium and allele frequency

were tested by using power marker software [38]. For all tests, a

two-sided p value less than 0.05 was used as the level of

significance.

A summary of the clinical, biochemical characteristics and

genotype frequencies of the 355 studied individuals is presented:

Table 1 shows that our sample of Egyptian women has a

significantly increased prevalence of obesity, levels of total

cholesterol, HDL cholesterol and ApoA1 compared to men. Smoking

levels were much higher in men than women.

Table 2 shows allele frequencies obtained for X- and X+ were

72.7 and 27.3%, respectively; the X-/X- genotype presented the

highest frequency (54.6%), X+/X- (36.2%) and the X+/X+ genotype

had the lowest frequency (9.2%). The genotype frequencies did not

deviate significantly from the Hardy-Weinberg equilibrium.

The mean values of BMI and average concentrations of lipid

parameters by genotype are shown in Table 3. A non-significant

effect is observed on the BMI (p = 0.069).

Biochemical changes in serum lipids were compared with the

genotype status. A small but significant increase in cholesterol

levels is associated with the X+ allele (X+/X+: 186 ± 17.27 vs., X-/X-:

175 ± 15.21 mg/dl p 0.041), LDL-C (X+/X+: 115 ± 32.43 vs., X-/X-:

110 ± 31.39 mg/dl p 0.021), and ApoB (X+/X+: 119 ± 11.47 vs., X-

/X-: 107 ± 11.35 mg/dl p 0.019) levels. The ApoB concentration in

the presence of the X+/X+ genotype is elevated by comparison to X-

/X+ and X-/X-.

ApoB gene XbaI polymorphism status

Statistical analysis

Result

Biochemical analysis of blood

DNA isolation and genotyping


3984

Age (years)

Body mass index (BMI) (kg/m2)

Cigarette smokers (%)

Diabetes type 2 (%)

Dyslipidemia%

Obesity%

Blood Glucose (mmol/L)

TC (mg/dl)

HDL-C (mg/dl)

LDL-C (mg/dl)

TG (mg/dl)

Apolipoprotein A1 (mg/dl)

Apolipoprotein B (mg/dl)

Males

(n=215)

Females

(n=140)

Total

Population

(n= 355)

49.46 ± 9.34

27.01 ± 6.21

8.9

17.5

17.8

51.5

6.04 ± 3.55

192.1 ± 23.4

46.1 ± 11.5

114 ± 26.3

191 ± 29.3

141 ± 21.6

115 ± 19.5

CC

(%)

117

(54.4)

64

(45.7)

181

(51)

CT

(%)

78

(36.3)

50

(35.7)

128

(36.1)

51.11± 9.48

29.67 ± 6.43

18.6

15.8

15.7

39.5

6.04 ± 2.49

178± 32.5

42.2 ± 19.40

114 ± 21.7

189 ± 36.5

121 ± 25.9

117 ± 12.5

48.36 ± 10.17

25.52 ± 4.51

0.5

10.7

21.4

69.5

6.04 ± 4.77

193 ± 24.8

48.9 ± 11.9

113 ± 29.2

192 ± 22.8

149 ± 28.3

113 ± 13.5

TT

(%)

20

(9.3)

26

(18.6)

46

(12.9)

C

0.73

0.64

0.69

T

0.27

0.36

0.31

NS

<0.001

<0.001

NS

<0.001

<0.001

NS

<0.001

<0.001

NS

<0.001

<0.001

NS

Parameter

ApoB

Population(n= 355)

Genotype

Males (n=215)

Allele

Females (n=140)

P value

Table 1: Clinical characteristics and biochemical parameters of the sample population

Table 2: Numbers and frequency of subjects with the ApoB XbaI gene polymorphism X-/X- (CC), X-/X+ (CT) and X+/X+ (TT) genotype and X- (C) and X+ (T) alleles of the sample population


3985

X-/X- (CC)

(n=181)X-/X+ (CT)

(n= 128)X+/X+ (TT)

(n= 46)P value

(ANOVA)

27.2±5.6

28.5±4.5

29.3±3.6

0.069

175 ± 15.21

179 ± 16.23

186 ± 17.27

0.041

123 ± 18.65

112 ± 19.65

134 ± 12.63

0.131

42.6 ± 21.26

44.3 ± 22.29

43.5 ± 23.25

0.167

104 ± 33.39

110 ± 31.39

115 ± 32.43

0.021

131 ± 11.25

131 ± 13.23

132 ± 15.29

0.91

105 ± 10.35

107 ± 11.37

119 ± 11.47

0.019

Parameter Body mass index 2(kg/m )

TC (mg/dl)

TG(mg/dl)

HDL-C (mg/dl)

LDL-C (mg/dl)

Apo A1 (mg/dl)

Apo B(mg/dl)

Table 3: Body mass index and Lipid profile variables according to genotypes in the total population

Discussion

TC: Total cholesterol; HDL-C: High density lipoprotein cholesterol; LDL-C: Low-density lipoprotein cholesterol; TG: Triglyceride. NS: not significant. ANOVA: analysis of variance statistical test

This study is the first to examine the relationship between the

ApoB XbaI polymorphisms and plasma lipid parameters in the

Egyptian population. We have shown that Apo XbaI polymorphisms

are linked in a statistically significant manner to cholesterol, ApoB

protein, and LDL-C levels. The genotype frequencies observed in

this study were in Hardy-Weinberg equilibrium and the allele

frequencies found were similar to those described in other

populations [39-41]. The frequency of the rare allele X+ XbaI

polymorphism observed in our population (0.45) is similar to that

observed in Finland (0.38), Italy (0.37) and Brazil (0.40) [42 - 44].

The X+ frequency is lower than that reported in France (0.47 to

0.46), Denmark (0.49), Switzerland (0.55) and England (0.57) [45-

49], and very much greater than that reported in Nigeria (0.15),

China (0.01) and in Japan (0.05) [8, 50-52]. BMI is the parameter

most often used to evaluate the degree of obesity. In our study, the

increase in BMI was not significant (p = 0.09) in subjects with X+/X+

and this in agreement with the results obtained in a Caucasian and

Indian population [53 - 55].

Cholesterol has been the most discussed lipoprotein-related

proatherogenic risk variable. However, risk appears more directly

related to the number of circulating atherogenic particles that

contact and enter the arterial wall than to the measured

concentration of cholesterol in these lipoprotein fractions. The

atherogenic lipoprotein particles contain a single molecule of ApoB,

so the concentration of ApoB provides a direct measure of the

number of circulating atherogenic lipoproteins. The plasma levels

of ApoB are also associated with CVD. Evidence is accumulating that

ApoB might be a better risk indicator for cardiovascular events than

LDL-C [55] and the monitoring of ApoB gene polymorphisms could

be helpful for disease prediction. Numerous studies have attempted

to relate the presence of a XbaI restriction polymorphism of the

gene for ApoB to lipid abnormalities and atherosclerosis. The

results, so far, are inconsistent with studies on Caucasian

populations [44, 56-58] or Asian [51, 59, 60] finding no association

between lipid parameters and X+ allele. Other studies on healthy

subjects [61-63] and on patients with non-insulin dependent

diabetes [64] concluded the presence of an association. Zaman et al.

in a Japanese population showed that XbaI polymorphism was not

significantly associated with total cholesterol and HDL-C levels

[65]. The allele X+ has also been associated with elevated

triglycerides [54, 66, 67]. A previous study on 2166 Caucasians

showed similar effects of ApoB XbaI polymorphism on LDL-C and

cholesterol levels [68]. A study in a North Indian population showed

that the mean value of total serum cholesterol and ApoB was

significantly higher in CVD patients carrying homozygous X+ genotypes as compared to controls [39].

The mechanisms explaining the association of XbaI

polymorphism to lipid disturbances are unknown. Since ApoB is an

important component of each LDL-C particle, genetic variations

that affect either the level or the structure of ApoB are most likely to

act in a co-dominant fashion [69]. Our results of different XbaI

genotypes that were associated with total cholesterol, LDL-C, and

ApoB levels are in agreement with this suggestion. However, it is

noticeable that the XbaI polymorphism in ApoB results from a

mutation in exon 26 at the third base of the triplet (ACC → ACT)

encoding the threonine located at position 2488 of the

apolipoprotein B without changing the sequence of amino acid.

Therefore, the effect of this polymorphism on lipid concentrations

is probably not due to the ApoG gene product itself. Many authors

hypothesize a linkage disequilibrium between the XbaI

polymorphism and a causal mutation not yet identified and

responsible for these variations. In other words the observed

associations probably result from co-segregation with one or more


3986

functional variants in the ApoB gene or a gene located nearby

[70,71]. Confirmation of this hypothesis could partly explain the

inconsistency of results observed between different ethnic or

geographical populations. Furthermore, Demant et al. [72] suggest

that ApoB produced by the homozygous X+X+ may have a slightly

modified structure that would reduce its ability to interact with the

LDL receptor. The causal mutation in linkage disequilibrium with

the XbaI locus could be located in the region of the gene encoding

the ApoB binding site of the LDL receptor. Altering the rate of

clearance of LDL can cause disturbances to lipoprotein metabolism,

thus explaining the variations observed, not only at the level of LDL-

cholesterol and ApoB but also, total cholesterol, triglycerides,

ApoA1 concentrations and deposition of triglycerides in adipose

tissue from the lipoprotein particles containing ApoB (VLDL). CVD,

a leading cause of death in most industrialized countries, is a

multifactorial disease [72] and in future, more studies on the

association of apolipoprotein polymorphisms with lipid factors is

suggested. It may help to identify high-risk groups for CVD and may

be of use in genetic screening for CVD patients of different

genetic/ethnic background.

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