APOLIPOPROTEIN B GENE POLYMORPHISMS IN PATIENTS FROM SERBIA WITH ISCHEMIC CEREBROVASCULAR DISEASE

The plasma concentration of apoB has recently been reported to be the best lipid predictor of coronary heart disease. The possible associations of genetic markers in the apolipoprotein B gene (XbaI, EcoRI, MspI, Ins/Del, and 4311 A/G polymorphisms) were evaluated in patients with ischemic cerebrovascular disease (ICVD) and controls of equivalent BMI. The odds ratio for ICVD in the X+X+ genotype was 2.22, 95% CI 1.24-3.96 (P<0.05), while that for ICVD in the Ins/Ins genotype was 2.82, 95% CI 1.57-5.06 (P<0.05). The patients had significantly higher frequency of the 4311A allele compared to the controls (P<0.01). Our results support the assumption that apoB gene polymorphisms may contribute to the extent of cerebrovascular disease risk.


INTRODUCTION
Most strokes are ischemic in origin; of these 80% are caused by arterial occlusion secondary to atherosclerosis (B a m f o r d et al., 1990).Evidence indicates that modifiable risk factors (such as lipids and lipoproteins) interact with genetic factors to cause stroke (E l b a z et al., 1999).Studies in twins, families, and animal models provide substantial evidence for a genetic contribution to ischemic stroke (J e f f s et al., 1997; S c h u l z et al., 2004).The genetic factors seem to be more important in large-vessel stroke and small vessel stroke than in cryptogenic stroke, and there is no epidemiological evidence for a genetic component in cardioembolic stroke (S c h u l z et al., 2004; J e r r a r d-D u n n e et al., 2003).This finding emphasizes the importance of stroke subtypes and lends support to the view that largevessel stroke and myocardial infarction share similar pathological mechanisms and genetic susceptibility (D i c h g a n s, 2007).al., 1994).It was also suggested that apoB gene polymorphisms may modulate plasma lipid/lipoprotein and glucose levels in patients with type 2 diabetes (D u m a n et al., 2006).The apoB gene has so far been mainly investigated in familiar hypercholesterolemia and coronary artery diseases.Accordingly, the aim of our study was to investigate the possible association of five polymorphisms in the apoB gene (XbaI, EcoRI, MspI, Ins/Del, and 4311A/G) with ischemic cerebrovascular disease in Serbia.

Sample
Blood samples were obtained from 60 patients who had suffered a completed stroke or a transient ischemic attack.These were proven by computer tomography or magnetic resonance of the brain.Atherosclerosis of the eye bottom as well as both carotids and vertebral arteries was assessed by ultrasound examination.The control group consisted of 245 unrelated healthy Serbian subjects whose annual health examination showed them to be free of cerebrovascular, cardiovascular, or chronic inflammatory disease.The control group was body mass index matched (BMI) with patients.Informed consent was obtained from each participant in the study.Personal data (age, sex, weight, height, and blood pressure) were obtained from all participants.All subjects with a personal or family history of diabetes and/or thyroid dysfunction were excluded, as well as individuals taking any lipid-lowering drugs.
Blood samples were collected from participants after 12 hours of fasting.The total plasma cholesterol (TC) and triglyceride (TG) levels were determined on a Monarch Plus apparatus (Instrumentation Laboratory, Lexington, USA) using enzymatic colorimetric methods.The HDL cholesterol (HDLC) was determined after dextran sulfate -Mg 2+ precipitation of VLDL and LDL, using the CHOD-PAP method.The LDL cholesterol (LDLC) was calculated using the Friedewald formula (F r i e d e w a l d et al., 1972) for participants with triglyceride levels <4.5 mmol/l.All reagent kits were from Instrumentation Laboratory (Lexington, USA).Serum apoA-I and serum apoB were quantified by immunonephelometry with reagents from Beckman Instruments (Fullerton, CA).

DNA analysis
Genomic DNA was isolated from whole blood cells by proteinase K digestion and phenol/chloroform extraction (K u n k e l et al., 1977).Genomic fragments containing apoB gene polymorphisms XbaI (codon 2488, exon 26), EcoRI (codon 4154, exon 29), MspI (codon 3611), point mutation A/G at nucleotide 12932 (codon 4311, exon 29), and Ins/Del (signal peptide) were amplified by the polymerase chain reaction (PCR) on a Touch Down TM thermal cycler (Hybaid, Teddington, UK).Genotypes were determined by RFLP and/or gel electrophoresis as previously described (G l i š i ć et al., 1995; G l i š i ć et al., 1997; G l i š i ć and A l a v a n t i ć, 1996; R a j p u t -W i l l i a m s et al., 1988) and visualized by the GDS8000 gel documentation system (Ultra Violet Products Inc., Upland, CA).

Statistical analysis
Conformance of the allele frequencies to Hardy-Weinberg equilibrium proportions was tested by the χ 2 test.Genotype and allele frequencies in different groups were compared by the gene counting method and chi-squared analysis.The unadjusted odds ratios and their 95% confidence intervals (CI) were also calculated.The Student t-test was used to compare differences between two means.If the distribution of quantitative variables was skewed, logtransformed values were used for the analysis.In all tests, differences with two-tailed alpha-probability (P) ≤0.05 were considered statistically significant.The correction for multiple testing was performed by multiplying the p value by the number of polymorphisms analyzed in the study.For the analysis, we used the Statistica software package (Version 5, Stat Soft Inc., 1997).

Description of the population
Descriptive statistics of concomitants and their lipid and lipoprotein variables are presented in Table 1.The patient group was generally older and had significantly lower HDL, but also higher triglyceride and apoB levels.Blood pressure values were significantly higher in the patients than in the controls.

Genotypes and susceptibility to ICVD
The genotype and allele frequencies of apoB gene polymorphisms XbaI, EcoRI, MspI, Ins/Del, and 4311 A/G in the patients with ICVD and controls are shown in Table 2.The observed genotype frequencies did not significantly differ from expected values according to the Hardy-Weinberg equilibrium, except for the Ins/Del polymorphism ones in the controls (G l i š i ć et al., 1997).The frequency of the apoB X+ allele was significantly higher in the ICVD patients than in the controls (P<0.05).The odds ratio (OR) for ICVD in the X+X+ genotype was 2.22, 95% CI 1.24-3.96.Frequency of the apoB Ins allele was also significantly higher in the ICVD patients compared to the controls (P<0.05).The OR for ICVD in the Ins/Ins genotype was 2.82, 95% CI 1.57-5.06.The patients with ICVD had significantly higher frequency of the apoB 4311A allele compared to the controls (P<0.01).We did not calculate the OR for carriership of the A allele, since all investigated patients had carrier status.No significant differences of genotype and allele frequency distribution for either the EcoRI or the MspI polymorphism at the apoB gene were observed between the patients and the controls.

DISCUSSION
The plasma concentration of apoB has recently been reported to be the best lipid predictor of coronary heart disease (S n i d e r m a n and M a r c o v i n The point mutation (A/G) at codon 4311 of the apoB gene was less thoroughly examined in recent reports.In the control group, frequency of the allele G (0.37) is among the highest in Caucasian populations.Rare homozygotes (GG) were not present in the Serbian ICVD group.A similar trend of lower GG frequency was observed in patients with myocardial infarction compared to controls (M o r e e l et al., 1992).The 4311 polymorphism had a significant effect on high density lipoprotein (HDL) cholesterol levels in a study of young myocardial infarction survivors and healthy population-based individuals (P e a c o c k et al., 1992).In our study, patients with ICVD had a significantly higher frequency of the A allele, which remained significant after correction for multiple testing.
We did not observe significant differences of genotype distribution and relative allele frequencies for the EcoRI and MspI polymorphisms in the apoB gene.These data are in accordance with some of previous reports (D e l g h a n d i et al., 1999; S a l a z a r et al., 2000).Others found the common M+ allele of the MspI RFLP polymorphism more frequently present in CAD patients than in controls, but no significant differences of allele frequencies were observed for the XbaI and EcoRI polymorphisms (S t e p a n o v et al., 1998).
It is very important that the control group in the present study was matched with patients according to BMI values, since recent findings showed that increased BMI is a risk factor for both total and ischemic stroke (H u et al., 2007).In addition to the different design of the previous studies, inconsistencies of the results might be attributable to different apoB haplotype distributions in the different populations studied.Also, most previous studies were focused on the effect of apoB gene polymorphisms on changes in lipid levels.There is a possibility that some of the polymorphisms may act through mechanisms not directly related to influence on measured lipid traits.Also, the significant effect of certain polymorphisms that we found could be due to linkage disequilibrium with other functional genetic markers.
There are no previous data on genotype distribution, allele frequencies or correlation of the apoB Ins/Del, EcoRI, MspI, XbaI, and 4311 A/G polymorphisms with ICVD in Serbian patients.Our study suggests association between polymorphisms in the apoB gene and ICVD in subjects of Serbian origin and supports the assumption that apoB polymorphisms may contribute to the extent of cerebrovascular risk.Insight into the genetic profile of affected subjects before the onset of ICVD clinical symptoms could have immediate clinical and public health benefits in predicting ICVD risk.Future studies on larger and independent samples and in different populations could confirm our results and elucidate these relations with a higher power of clarification.
Apolipoprotein B (apoB) plays a central role in lipoprotein metabolism.It is a component of chylomicrons, low-density lipoproteins (LDL), very low-density lipoproteins (VLDL), and intermediatedensity lipoproteins (IDL), as well as the ligand for the LDL receptor (M a h l e y et al., 1984).The given lipoprotein has numerous polymorphic sites.Among them are polymorphisms assigned according to the presence/absence of the cutting site of the restriction enzymes XbaI, EcoRI, and MspI; the 4311 Asn→Ser substitution; and an insertion/deletion of nine base pairs (I/D) in the signal peptide (R e n g e e s et al., 1992).ApoB gene polymorphisms XbaI, EcoRI, and MspI has been previously linked with variability of serum lipid levels and the risk of coronary atherosclerosis in several populations (H u m p h r i e s , 1988; K a m e r e e r et al., 1996; H a n s e n et al., 1994; D e l g h a n d i et al.; 1999, S t e p a n o v et al., 1998).I/D polymorphism has also been linked with variations in plasma cholesterol and CAD risk (H u m p h r i e s, 1988; K a m e r e e r et al., 1996; P e a c o c k et al., 1992, P e a c o c k et al., 1994).The X-, R-, and I alleles of the above-mentioned loci have been reported as risk factors for CAD (B o h n et APOLIPOPROTEIN B GENE POLYMORPHISMS IN PATIENTS FROM SERBIA WITH ISCHEMIC CEREBROVASCULAR DISEASE al.,1993; H u m p h r i e s, 1988; H e g e l e and B r es l o w, 1987; P e a c o c k et al., 1992, P e a c o c k et

Table 1 .
Study subject characteristics Values are expressed as means ± SD; # analyses were performed with log transformed values; P value from t-test; *χ 2 -test; NS nonsignificant.

Table 2 .
Genotype and allele frequencies of apolipoprotein B-100 gene polymorphisms XbaI, EcoRI, MspI, Ins/Del, and 4311 A/G in ICVD patients and controls