| BackgroundCoronary artery disease (CAD) is one of the commonest cardiovascular diseases. Coronary artery occlusion is mainly caused by thrombus, and platelet play an important role on the process of thrombosis. Most platelet membrane glycoprotein (GP) are protein complex with two or more polypeptide subunits non-covalently associated with the platelet membrane. GPIb-IX-V complex is composed of four subunits: GPIbα, GPIbβ, GPIX and GPV, which arise from different genes. GPIbα is disulfide linked to GPIbβ forming the heterodimer GPIb, which is non-covalently associated with GPIX and GPV. GPIba is the largest polypeptide in the GPIb-IX-V complex and contains within its N-terminus the region that binds von Willebrand factor (vWF). This polypeptide also contains a high-affinity binding site for thrombin that is necessary for platelet activation at low thrombin concentrations. In the arterial circulation, Occlusive thrombus is almost exclusively initiated by plaque rupture and adhesion of platelets to subendothelial vWF by its specific platelet receptor, the GPIba chain of the GPIb-IX-V complex.Three relatively frequent polymorphisms in GPIba have been described in the general population: Kozak sequence -5T/C polymorphism, variable number of tandem repeats (VNTR) polymorphism and HPA-2a/2b polymorphism. It has been reported that Kozak-5T/C polymorphism could influence the efficiency of mRNA translation and the expression density of the GPIb/IX/V complex on the platelet surface. This Kozak sequence C allele is said to be more efficient at translation.Therefore, the GPIba Kozak sequence -5T/C polymorphism is a target for investigation in patients with CAD. Recently, some reports have concluded that Kozak -5T/C polymorphism is significantly associated with CAD and myocardial infarction, but there are also some conflicting reports. The role of this polymorphism for the development and progression of CAD is still under discussion.We investigated the GPIba Kozak sequence -5T/C polymorphism in 196 patients undergoing coronary angiography by polymerase chain reaction and restriction fragment length polymorphism (PCR-RFLP) assay. Our main purpose is to explore the distribution of Kozak-5T/C polymorphism in Chinese people and the relationship between this gene polymorphism and CAD.Material and Methods1. Subjects:196 Han ethnic persons without blood relationship, aged 37-85 years, were recruited to the study. All of the person were performed selected coronary angiography and from the Cardiovascular Department of the Second Affiliated Hospital, Medical College of Zhejiang University. Among them, 105 CAD patients (76male and 29 female, aged 64.64±9.54 years);91 control subjects (50 male and 41 female, aged 59.02±10.73 years) were from patients with normal angiography.According to their clinical manifestation, the patients were also divided into two groups using criteria defined by the World Health Organization (WHO). The ACSgroup consisted 59 patients (40male and 19 female, aged 64.12±9.51 years);the non-ACS group consisted 128 patients (79male and 49 female, aged 60.60±10.87 years).2. Methods:2.1 Collection of clinical features: All subjects were enquired in detail for history of smoking, hypertension, diabetes, PT, INR and major biochemical data were also determinated. Selecting coronary angiography was done using standard techniques. Coronary artery disease was defined on the basis of angiographic criteria as at least 50% stenosis in one of the major coronary arteries or major branches.2.2 Extraction of genomic DNA: Leucocytes were collected from EDTA anticoagulated peripheral blood, genomic DNA was extracted by the salting out method and stored at -72 °C.2.3GPIba Kozak-5T/C genotypes was determined by PCR-RFLP: (1) The primers of Douglas H are referenced, the forward primer: 5'-GGGAGTAGGGAGGACAGGAG-3', and the reverse primer: 5'-AGTGTAAGGCATCAGGGTTG-3'. (2) Touchdown PCR was carried out using a 25ul reaction mix containing 200ng of genomic DNA, lOpmol/L of each primer, 200umol/L of each dNTP, 1.5mmol/L of MgCI2, 10 X buffer 2.5ul, 1.5U of Taq DNA polymerase, The Touchdown PCR cycles were modified as follows: 5 min initial at 95 °C, followed by 2 cycles of 1 min of denaturation at 95 °C and 1 min of annealing at 64 °C and 2 min of extension at 72 °C, followed by 6 cycles with annealing temperature descending from 64 °C to 59°C, followed by 30 cycles of 1 min of denaturation at 95 °C and 1 min of annealing at 59 °C and 2 min of extension at 72 °C, and then followed by a final 10 min extension step at 72 °C. (3) Following amplification, a restriction digestion was performed to detect the Kozak-5T/C polymorphism with the restriction endonuclease Ava II. (4) Electrophoresis: The PCR products (348bp) and these digestion products were separated usingelectrophoresis through 2% agarose gel, and staining with ethidium bromide and visualized under UV light. (5) Fragments of 129bp and 219bp were detected in the presence of the T allele, and 348bp band was indicated in the presence C allele.3. Statistical analysis:Using the SPSS 10.0 for windows statistical package. Count data were performedby Student's t-test and measure data were performed by chi-squared test. Genotypic andallelic frequencies between groups were analyzed by the chi-squared test. Statisticalsignificance was taken as P<0.05.Results1. General characteristics of the patients in the studyThe CAD group was elder than the control group (PO.05). No significant difference was found between the groups with regard to sex, total cholesterol, HDLc, LDLc, history of smoking, hypertension and diabetes.2. Genotypic distribution and allelic frequency of the Kozak-5T/C polymorphism in the study groupThe genotypic frequencies in the study group were 51.5% for TT, 40.3% for CT and 8.2% for CC;and the frequencies of T and C allele were 71.7% and 28.3%. Genotypic distribution was accordance with Hardy-Weinberg equilibrium.3. Kozak-5T/C polymorphism and CADThe frequencies of Kozak-5T/C genotypes and alleles did not show significant differences in the presence and absence of CAD ( P > 0.05 ). The genotypic frequencies of CC, CT, TT were 9.5%, 41.9%, 48.6% and 6.6%, 38.5%, 54.9% in the CAD group and the controls, respectively. The allelic frequencies of C and T were 30.5%, 69.5% and 25.8%, 74.2% in the CAD group and the controls, respectively.4. Kozak-5T/C polymorphism and the number of stenosed coronary vessels.There was no significant difference in the genotypic frequencies and allelicfrequencies, between the one stenosed vessel subgroup and more stenosed vessels subgroup (P > 0.05 ).5. Kozak-5T/C polymorphism and ACSThe frequencies of Kozak-5T/C genotypes and alleles did not show significant differences in the presence and absence of ACS ( P > 0.05 ). The genotypic frequencies of CC, CT, TT were 8.5%, 40.7%, 50.8% and7.8%, 39.1%, 53.1% in the ACS group and the non-ACS group, respectively. The allelic frequencies of C and T were 28.8%, 71.2% and 27.3%, 72.7% in the ACS group and the non-ACS group, respectively.There was no significant difference of Kozak-5T/C polymorphism between the premature (under 60-year-old) ACS subgroup and the non-ACS group.6. Kozak-5T/C polymorphism and INRThere was no significant difference of the prothrombin time international normalized ratio among the three groups of CC, CT and TT genotypes ( P > 0.05 ).ConclusionThere is Kozak-5T/C polymorphism in Han ethnic group;the C and T alleles frequency are 28.3% and 71.7% respectively. No relationship was found between the platelet GPIba gene Kozak-5T/C polymorphism and the risk of CAD and ACS.
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