| BackgroundEndothelia lipase (EL) is the most recently discovered member of the lipoprotein lipase (LPL ) or triglyceride (TG) lipase, gene family. The EL gene was cloned independently by Hirata and Jaye using distinct methods. The amino acide sequence of EL shares 45%, 40%, and 27% homology with LPL, hepatic lipase (HL) and pancreatic lipase (PL), respectively.EL is synthesized by endothelial cells and primarily acts as a phospholipase. Total of 17 variants of the EL gene were indentified by Delemos, six of these including Thr111Ile and Gly26Ser were potentially functional. EL activity indirectly influences the metabolism of high-densityliporotein cholesterol (HDL-c) level, HDL-c leves was markedly reduced in mice injected with an adenoviral vector encoding human EL. These results suggested that EL may play a central role in HDL metabolism. Furthermore, plasma HDL-c levels are an important independent risk factor for coronary heart disease (CHD).We investigated the EL Thrllllle and Gly26Ser polymorphism using polymerase chain reaction assay-restriction fragment length polymorphism (PCR-RELP) technique. Our purpose is to known the association of two polymorphisms with lipoprotein and CHD in Chinese. Meterial and Methods: 1. Subjects:438 Han ethnic persons without blood relationship 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, 242 CHD patients (180 male and 62 female, the average age is 64.7±9.52) at least 50% obstruction of one major coronary vessel, according with criteria defined by the World Health Organisation (WHO);196 control subjects (112 male and 82 female, the average age is 60.35±11.23) were from patients with normal angiography.2. Methods:2.1 Determination of lipoprotein: plasma level of lipoprotein was determined enzymatically with Olympus AUlOO auto detecting instrument.2.2 Isolation of DNA. Cells were collected from EDTA anticoagulated blood and stored at -72 °C, pending extraction by the salting out method.2.3 Thrl 11 He and Gly26Ser genotypes were determined by PCR-RELP.(1) The primers of Delemos are referenced. Thrl 1 llle genotype the forward primer: 5'- GCC TGT AAC CCA GTC ACT CTG GAG -3', and the reverse primer: 5' - CTA CAT TGG CGT CTT TCT CTC AT -3' . Gly26Ser genotypes the forward primer: 5'- AAG GTG TGA CCA ATC AGA GCC C -3', and the reverse primer: 5'- CAG CACAGAGAAGTGGCTGGG-3' .(2) PCR reactions containing 200 ng of DNA template, 1 uL of each lOumol/L primer solution, 200umol/L of each dNTP, 1.5 mmol/L MgCl2, 10 mmol/L Tris-HCl, pH 9.0, 50 mmol/L KC1, and 1.5 U of Taq DNA polymerase were amplified in a final volume of 25uL while undergoing denaturation at 95° C for5minutes, followed by 35cycles (95°Cforlminute, 61.5°Cfor 30 seconds, and 72° C for 1 minute), and an extension at 72° C for 10 minutes.(3) Diagnostic restriction enzyme for Thrl 1 llle and Gly26Ser wasNdel and Avail respectively. Total digestion volumes were lO.Oul and included 5.0ul of PCR product. Thrill He variant was digested overnight in a 37°C water bath, Gly26Ser digestion took place at 37°C for 2 hours in a thermal cycler. (4) PCR and digested products were resolved by electrophoresis in 2%agarose gels. Gels were visualized by ultraviolet ray. 3. Statistical analysis:Using the SPSS 13.0 for windows statistical package. Count data were performed by Student's t-test and measure data were performed by chi-squared test. Genotype and allele frequencies between groups were analysed by the chi-squared test. The association between the polymorphism and lipoprotein or CHD was analysed by Logistic regression analysis. Statistical significance was taken as p<0.05. Results 1. Frequence of Thrl 1 Hie genotype and allele:C, T allele and CC, CT genotype can be found in CHD and control group. TT genotype was not found. The fragment length of allele C and T were 282bp and 26bp. The genotype frequency of CC and CT were 76.7% and 23.3%. The frequence of C and T allele were 88.3% and 11.7%. Genotype distribution was accordance with Hardy-Weinberg equilibrium.The frequence of CC, CT in CHD and control were 76.86%, 23.14% and 76.53%, 23.47%. There are no association between Thrllllle genotype and CHD (x2=0.007, p>0.05)2. Demographic and biochemical characteristics of CC and CT groupCompared CC with CT genotype, CT group showed higher plasma level of HDL-c (/?<0.05)3. Logistic regression analysis between Thrl 1 Hie polymorphism and lipoproteinCT group showed significantly higher plasma level of HDL-c than CC group ( x2=5.77, OR=1.04, p<0.05) on logistic regression analysis.4. Logistic regression analysis between CHD and Thrl 1 Hie polymorphismBut logistic regression analysis revealed that there was no significant difference between CHD group and control group for Thrllllle polymorphism (x2=0.01, OR=0.96, /?>0.05) .5. Frequence of Gly26Ser genotype and allele:G allele and GG genotype can be found in CHD and control group. T allele and GT, TT genotype was not found. There was no Gly26Ser mutation in this study. ConclusionThe polymorphism of Thrllllle is present in patients with CHD inChinese, and T allele was related to high HDL-c level. There was no significant association between the polymorphism of Thrllllle and CHD. The Gly26Ser mutation was not found in this study. |
PDF Full Text Request