Impact of CYP2C9 and VKORC1 polymorphism on warfarin response during initiation of therapy / 中华心血管病杂志

<p><b>OBJECTIVE</b>To investigate potential contributions of genetic variants of cytochrome P-450 2C9 (CYP2C9) and vitamin K expoxide reductase (VKORC1) to the anticoagulation response during the initiation of warfarin therapy in the Han Chinese population.</p><p><b>METHODS</b>A total of 798 Han Chinese patients received long-term warfarin anticoagulant therapy orally after valve replacement in our hospital between 2000 and 2008 were included in this study. Nine single nucleotide polymorphism (SNP) loci [rs12572351 G > A, rs9332146 G > A, rs4917639 G > T, rs1057910 A > C (CYP2C9(*)3), rs1934967 G > T, rs1934968 G > A, rs9923231 C > T (VKORC1-1639 G > A), rs2359612 G > A and rs10871454 C > T] in 2 genes including CYP2C9 and VKORC1, which were possibly correlated with warfarin pharmacokinetics and pharmacodynamics through literature retrieval, were selected and analyzed. Warfarin steady-state dose requirement, time to the INR (the international normalized ratio) within the therapeutic range and percent of the INR of more than 3.5 were compared among genotype subgroups. SNaPshot technique was used to detect gene SNPs; Hardy-Weinberg genetic equilibrium test was used to test population representativeness.</p><p><b>RESULTS</b>CYP2C9(*)3 genotype did not affect the required warfarin dose while it was associated with increased risk of bleeding when treated with routine dosage regimen during the initiation of treatment. The allelic mutation frequency at VKORC1 gene rs10871454G > A and VKORC1-1639G > A SNP loci was 92.04% and 88.03%, respectively and rs10871454 was in perfect linkage disequilibrium with-1639. Patients with VKORC1 rs10871454 genetic mutation required lower warfarin dose in the first 28 days of therapy. VKORC1-1639 genetic polymorphism was also associated with shorter time to the INR within the therapeutic range and increased risk of over-anticoagulation.</p><p><b>CONCLUSION</b>Detecting genetic polymorphism of CYP2C9 and VKORC1 could guide clinical use of warfarin to reduce the risk of adverse reactions including bleeding in patients receiving chronic anticoagulation therapy.</p>

Clinical application of anticoagulation treatment with warfarin after prosthetic heart valve replacement: a single center-based survey / 南方医科大学学报

<p><b>OBJECTIVE</b>To investigate the clinical application of anticoagulation treatment with warfarin after prosthetic heart valve replacement and compare the effect and safety of different anticoagulant intensities.</p><p><b>METHODS</b>A total of 845 Chinese patients receiving oral warfarin for anticoagulant treatment after prosthetic heart valve replacement in Guangdong General Hospital between 2000 and 2008 were enrolled in this survey. The general data, clinical data, medications, international normalized ratio (INR) and results of echocardiogram of these patients were followed up to observe the incidence of complication of thrombo-embolism and such adverse effect as hemorrhage.</p><p><b>RESULTS</b>All the patients were of Han nationality, and Cantonese accounted for 88.04%. The daily mean maintenance dose of warfarin was 2.92∓0.88 mg in these patients with a median INR of 2.09∓0.39. Of these patients, 44.62% received low-intensity anticoagulant treatment with warfarin with the INR maintained between 1.5 and 2.0, and 56.45% had standard anticoagulant intensity with the INR maintained between 2.0 and 3.0. The total incidence of thrombo-embolism was 4.14%. Severe hemorrhage occurred in 14 cases (1.66%), most frequently in the alimentary tract. The events of hemorrhage were correlated to the type of prosthetic heart valve replacement, occurring more frequently in patients with mechanical prosthetic heart valve replacement than in those with biological ones. No significant difference was found in the incidence of thrombo-embolism and server hemorrhage between the two groups receiving low and standard intensity therapy anticoagulant.</p><p><b>CONCLUSION</b>The effect and safety of low-intensity anticoagulant treatment are comparable to that of standard intensity treatment in Chinese Han patients, and anticoagulation treatment with warfarin is effective and safe to maintain the INR between 1.8-3.0.</p>

High glucose up-regulates GRK2 gene expression in H9C2 cardiomyoblasts in vitro / 南方医科大学学报

<p><b>OBJECTIVE</b>To study the effect of high glucose on GRK2 gene expression in H9C2 cardiomyoblasts in vitro.</p><p><b>METHODS</b>H9C2 cardiomyoblasts were cultured for 72 h in the presence of 0, 5.5, 12.5, 25 or 33 mmol/L glucose (with the osmotic pressure adjusted with monnitol). Semi-quantitative detection of GRK2 gene expression in H9C2 cardiomyoblasts was carried out using RT-PCR and phosph-Akt (Ser473) protein level was measured by Western blotting.</p><p><b>RESULTS</b>Glucose in the culture medium (5.5 to 33 mmol/L) concentration-dependently increased the mRNA expression of GRK2 concentration and decreased phosphorylation Akt (ser473) level in in H9C2 cardiomyoblasts.</p><p><b>CONCLUSION</b>Increased GRK2 gene expression may play an important role in cardiac dysfunction in diabetes.</p>

Identification of the interactions between the truncated fragments of macrophage migration inhibitory factor and CD74 using a yeast two-hybrid system / 南方医科大学学报

<p><b>OBJECTIVE</b>To investigate the interaction domains between macrophage migration inhibitory factors (MIF) and the extracellular segment of type-II trans-membrane protein CD74 using a yeast two-hybrid system.</p><p><b>METHODS</b>By using molecular cloning techniques, the DNA fragments encoding MIF, MIF(50-65) and MIF(1-50/65-115) were introduced into the pGBKT7 vector to construct the corresponding recombinant bait plasmids, and the DNA fragments encoding CD74(73-232), CD74(73-109), CD74(1109-149) and CD74(149-232) into the pGADT7 vector to construct the recombinant activation domain (AD) plasmids. PEG/LiAC method was employed to transform the above 3 recombinant bait plasmids paired with each of the 4 recombinant AD plasmids into the chemical competent yeast AH109 cells. The transformed yeast AH109 cells were screened consecutively on SD/-Trp-Leu and SD/-Trp-Leu-Ade-His/X-alpha-gal nutritional media.</p><p><b>RESULTS</b>The results of restriction endonuclease digestion and DNA sequencing verified the correct construction of all the recombinant plasmids. The yeast AH109 cells transformed with each of the 3 recombinant bait plasmids could grow on SD/-trp nutritional media without autonomous activation effect on the reporter gene MEL1. The cells transformed with each of the 4 recombinant AD plasmids could also grow on SD/-leu nutritional media without activation of the reporter gene MEL1. Only the yeast AH109 cells co-transformed with MIF, MIF(50-65), or MIF(1-50/65-115) plasmid and CD74(73-232) plasmid could grow on SD/-Trp-Leu-Ade-His nutritional media with transcription activation of the reporter gene MEL1.</p><p><b>CONCLUSION</b>MIF interacts with the intact extracellular segment of CD74 (CD74(73-232)) independent of the functional domain of MIF(50-65).</p>

An efficient method for screening effective siRNAs using dual-luciferase reporter assay system / 南方医科大学学报

<p><b>OBJECTIVE</b>To establish an efficient method for screening effective small interference RNA (siRNA) using dual-luciferase reporter assay system.</p><p><b>METHODS</b>Based on the siRNA expression vector pSilencer-4.1, 3 candidate green fluorescence protein (GFP) gene siRNA expression plasmids, namely pSi-GFPsiRNA1, pSi-GFPsiRNA2, and pSi-GFPsiRNA3, along with the negative control pSi-Negative, were constructed. Using the pGL3-promoter vector, the GFP-luciferase (GFP-LUC) expression plasmid pGL3-GFPf was constructed with the same Kozak consensus translation initiation site and start codon ATG for GFP-LUC coding sequence. The GFP fragment containing the target sequences of 3 GFP siRNAs was introduced into the 3' untranslate region of LUC in the modified pGL3-promoter vector to construct the plasmid pGL3-GFPp. The GFP siRNAs expression plasmids and Renilla luciferase reporter vector pRL-TK were co-transfected with pGL3-GFPf or pGL3-GFPp into the HEK293 cells, respectively. The luciferase activities were determined by dual-luciferase reporter assay, and the GFP mRNA expressions were detected by real-time quantitative PCR.</p><p><b>RESULTS</b>In the groups cotransfected with GFP siRNAs expression plasmids and pGL3-GFPf, the luciferase activities were reduced obviously, and the reduction was more significant in cells transfected with GFPsiRNA1 compared with the control cells (P<0.01).GFP mRNA levels were also markedly lowered in cells transfected with GFPsiRNA1 as shown by real-time PCR (P<0.01). In addition, the results of dual-luciferase reporter assay and real-time PCR showed that among the groups cotransfected with GFP siRNAs expression plasmids and pGL3-GFPp, the GFP expression was inhibited most obviously by GFPsiRNA1 (P<0.01).</p><p><b>CONCLUSION</b>The dual-luciferase reporter assay system provides a useful method for screening effective siRNAs targeting specific genes.</p>

Association of the polymorphism of macrophage migration inhibitory factor gene with coronary heart disease in Chinese population / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>Inflammation is involved in the process of coronary heart disease (CHD). Macrophage migration inhibitory factor (MIF) is a proinflammatory cytokine which can inhibit the random migration of macrophages and concentrate macrophages at the inflammatory site, and is thought to play an important role in cell mediated immunity. The present study is to investigate the association of the -173 G/C polymorphism of MIF gene with the outcome of the CHD.</p><p><b>METHODS</b>One hundred and thirty-eight patients with coronary angiography (CAG) proved CHD were studied, and 163 healthy matched controls in Guangdong were studied. Patients and controls were genotyped for a single nucleotide polymorphism in the 5'-flanking region at position -173 of the MIF gene, using PCR-RFLP analysis, followed by DNA sequencing identification.</p><p><b>RESULTS</b>Only MIF -173G/G and MIF -173G/C genotypes were detected in CHD patients and controls. The MIF -173 G allele was detected in 0.966 of normal controls and 0.917 of patients, while MIF -173 C allele was detected in 0.034 of normal controls and 0.083 of patients. Individuals possessing a MIF-173*C genotype have an increased risk of CHD (16.7% versus 6.8%) (OR: 2.764, 95% CI: 1.295-5.899; P= 0.007).</p><p><b>CONCLUSION</b>These results suggest that MIF -173G /C polymorphism was associated with CHD in Chinese population, the MIF -173C allele might be a risk factor for CHD in Chinese Han nationality.</p>