Screening for mutations in exons encoding the ligand-binding domain of the LDL receptor gene using PCR-CFLP and PCR-SSCP.

Primary hypercholesterolemia includes both monogenic disorders and polygenic conditions. Two well defined monogenic disorders are familial hypercholesterolemia (FH) and familial defective apolipoprotein (apo) B-100 (FDB). Both disorders convey high risk of premature coronary artery disease. FH and FDB are caused by mutations in LDL receptor and apo B-100 genes, respectively. In the present study, mutations in both genes in Thai subjects with primary hypercholesterolemia were screened. For apo B-100 gene, a common mutation R3500Q was screened. This mutation was not observed in the patients (n = 45). For LDL receptor gene, mutations in the exons encoding the ligand-binding domain were screened. By PCR-CFLP analysis, 18 abnormal CFLP patterns in exon 4, the hot spot for mutations, were found in patients (n=45). One of the DNA samples with abnormal CFLP patterns was previously identified and reported as a possible disease-causing mutation, namely D151Y. For the other exons, the screening technique was PCR-SSCP. Abnormal SSCP patterns in DNA samples from patients (n=20) were found as follows, two in exon 3, one in exon 5 and another one in exon 6. Further characterization by DNA sequencing and family studies for these abnormal patterns are underway.

Inexpensive, rapid and convenient PCR-minigel SSCP protocol for polymorphisms and mutations analyses of LDL receptor gene.

Hypercholesterolemia has been recognized as a major risk factor of atherosclerosis and coronary artery disease. The elevation in plasma low density lipoprotein (LDL) cholesterol is frequently due to genetic alteration at the genetic locus specifying the LDL receptors, leading to defective catabolism of LDL. In order to facilitate the molecular diagnosis of LDL receptor disorder, single strand conformation polymorphism (SSCP) analysis of polymerase chain reaction (PCR) amplified genomic DNA fragments has become a simple and sensitive screening method for identification of DNA polymorphisms and mutations in LDL receptor gene prior to DNA sequencing. In addition, SSCP patterns can be detected by silver staining to avoid hazardous radioactive material or other costly nonradioactive detection techniques. However, the original SSCP protocol is generally large-formatted, which is both time and reagents consuming as well as cumbersome. Minigel SSCP protocols have thus been devised but they involve, although commercially available, costly precast gels. We describe here a nonradioactive PCR-minigel SSCP protocol which is sensitive, inexpensive, rapid, reproducible and manually convenient. The results in this study demonstrate that minigel-SSCP (gel size: 10 cm x 7.3 cm x 0.075 cm) can detect conformation polymorphisms in PCR-fragments with a comparative sensitivity to large gel SSCP (gel size: 30 cm x 40 cm x 0.04 cm) as exemplified by the SSCP analyses of exon 13 of the LDL receptor gene. For minigel SSCP, the reagents for gel components and silver staining are reduced approximately 9 times and 10 times, respectively. For electrophoresis, electrical power is also reduced 10 times. This improved technique can become routinely used for molecular diagnosis of LDL receptor defect as well as for other genetic disorders.

Serum cardiac troponin T in unstable angina pectoris patients.

Cardiac troponin T (cTnT) is a regulatory contractile protein not normally found in blood. Its detection in the circulation has been shown to be a sensitive and specific marker for myocardial cell damage. In this study, we used a second-generation enzyme immunoassay for cTnT to determine whether its presence in the serum of patients with unstable angina was a prognostic indicator. Thirty patients with unstable angina pectoris (UAP) and 30 patients with Q-wave acute myocardial infarction (AMI) were screened for serum CK-MB activity and cTnT at 6, 12, 24 and 48 hours after the onset of chest pain, All of the mean concentrations of CK-MB activity determined in UAP patients were less than the upper limit of normal (25 U/L). Meanwhile, the mean concentration of cTnT at 6, 12, 24 and 48 hours after onset of chest pain were higher than the cutoff values (0.1 microg/L), We found that one third of UAP patients had serum cTnT at the time of admission more than 0.1 microg/L and that these groups of patients were associated with a high risk for cardiac events. Our results suggested that patients with elevated serum cTnT could be considered as high-risk patients for developing myocardial infarction, Patients with normal cTnT levels and a low or intermediate clinical risk could be stabilized and further stratified noninvasively.

Analytical and clinical performance of two homogeneous assays for measuring of LDL-cholesterol.

A clinical laboratory currently estimates LDL-Cholesterol (LDL-C) concentration using the Friedewald calculation, which requires fasting specimens and is subject to error with increasing triglycerides levels. We evaluated the analytical and clinical performance of the direct LDL-C assay from two companies, Roche Diagnostics (LDL-C(Roche)) and Wako Pure Chemical (LDL-C(Wako)). Both methods meet current guidelines for precision with within-run coefficients of variation less than 3 per cent. The LDL-C(Roche) assay correlated well with the LDL-C from the Friedewald equation (LDL-C(Fried), r = 0.958, y = 0.85x + 17.08 mg/dL, n = 422). The LDL-C(Wako) assay also correlated with the LDL-C(Fried) (r = 0.946, y = 0.86x + 7.81 mg/dL, n = 422). In addition, at the medical decision cutoff points, LDL-C(Roche) assay and LDL-C(Wako) showed positive predictive values of 87.44 per cent and 69.67 per cent respectively. We conclude that the LDL-C(Roche) assay meets the currently established analytical and clinical performance, but LDL-C(Wako) assay meets only analytical performance. Clinical performance needs further evaluation.

Chlamydia pneumoniae antibodies and angiographically demonstrated coronary artery disease in Thailand.

Recent reports have suggested an association between Chlamydia pneumoniae and coronary artery disease. This study investigated the relationship between the presence of immunoglobulin G (IgG), immunoglobulin A (IgA) of C. pneumoniae in angiographically diagnosed coronary disease. Patients enrolled were 243 (178 male, 65 female, mean age 61 +/- 10 years) with angiographically proven at least one significant coronary artery stenosis. Fifty-eight patients (33 male, 25 female, mean age 57 +/- 11 years) with no angiographic evidence of coronary lesions were used as the normal coronary angiogram group. Control subjects (95 male, 92 female, mean age 58 +/- 17 years) were used as normal healthy persons who had no history of coronary artery disease. C. pneumoniae IgG and IgA antibodies were measured by ELISA method. We found that 179 out of 243 (73.7%) coronary artery disease (CAD) patients were positive for IgG and 132 out of 243 (54.3%) were positive for IgA. In 58 normal coronary angiogram patients, 23 (39.7%) cases were positive for IgG and 6 (10.3%) cases were positive for IgA. Among 187 healthy controls, 111 (59.4%) cases were positive for IgG and 83 (44.4%) were positive for IgA. When C. pneumoniae IgG antibodies were considered, there was significant difference between CAD patients and healthy controls (OR = 1.91, 95% CI = 1.27 - 2.88, p = 0.0018). In cases of positive IgA antibodies, significant difference was also found between CAD patients and healthy controls (OR = 1.49, 95% CI = 1.02 - 2.19, p = 0.0257). These findings were also found with higher odds ratio when we compared between CAD patients and normal coronary angiogram patients. The result suggested that C. pneumoniae infection is common in Thai people and chronic C. pneumoniae infection is more common in CAD patients. Chronic C. pneumoniae infection may be associated with the development of atherosclerotic coronary disease and treatment with antibiotics should be considered in ischemic heart disease.

Improved detection of radiofrequency current-induced minor myocardial injury by cardiac troponin T measurement.

Transcatheter radiofrequency current application in patients with cardiac arrhythmias was reported to be associated with a low rate of an increase in the activity of enzyme creatine kinase (CK) and CK-MB isoenzyme. As the novel heart-specific protein troponin T (cTnT) was shown to be superior to CK and CK-MB in detecting small damage to myocardial tissue in various clinical situations including unstable angina, a comparison of the diagnostic efficiency of these marker proteins to detect myocardial damage was made in 34 patients (mean age 38.3 +/- 15.6 years) undergoing radiofrequency (RF) catheter ablation of accessory pathways (n = 17) and atrioventricular nodal reentrant tachycardia (n = 17). Serial measurements of total CK and CK-MB activity before and every 8 hours for 24 hours after ablative procedure were performed with enzymatic and immunoinhibition method, respectively, using automated chemical analyzer Hitachi 717. Serum concentration of cTnT was determined by one-step sandwich ELISA performed on ES 300 analyzer (Boehringer Mannheim). With a median of 7.0 (range 1-39) RF current pulses only 12 (35%) and 10 (29%) of 34 patients showed an increase above the upper limit of normal CK and CK-MB activity, respectively. The peak activity of CK (mean peak = 285.8 +/- 517.7 IU/L) occurred at a variable time that infrequently coincided with those of peak CK-MB activity (23.1 +/- 8.0 IU/L). By contrast, all except 4 (88%) of 34 patients exhibited a distinct elevation of cTnT concentration (mean peak = 0.56 +/- 0.63 ng/ml), with almost all (33) of these 34 patients showed an early peak value at 8 hours postprocedural. There was, on the average, a small but distinct higher relative increase (5.6 times) in cTnT concentration from the upper limit of reference range compared with those of CK (1.5 times) and CK-MB peak activity (0.9 time). In conclusion, cTnT exhibited a minor but distinct elevation in its concentration and demonstrated a higher rate and magnitude of increase following radiofrequency current application than the conventional CK and CK-MB isoenzyme. Measurements of cTnT serum concentration may thus provide a useful test method for assessing the effect of the new transcatheter ablation procedures on myocardial tissue.

The efficiency of the siriraj blood warmer for in-line blood warming.

The efficiency of the Siriraj Blood Warmer invented by the investigators was evaluated. The warmer operates by heat exchange with a water-bath at 39.50 C. The blood is warmed during its passage through a 270-cm blood warming coil which is in the water-bath. Simulation of massive transfusion and exchange transfusion was performed by using water in a 500-ml bottle refrigerated at 40 C for 24 hours. A blood transfusion set was attached to the bottle and a blood warming coil. A three-way stopcock (the proximal stopcock) connected the blood warming coil to the female adaptor end of a 50-cm extension set. Another three-way stopcock (the distal stopcock) was placed to the male adaptor end of the extenaion set. A 2-ml syringe where the temperature of the water passing through was recorded was attached to each three-way stopcock. The Terumo Infusion Pump was used to control the flow rate at 200, 250, and 300 ml/hr for massive transfusion. Either a 5-ml or a 10-ml syringe was attached to the proximal three-way stopcock for the push-pull technique of exchange transfusion. The water temperature was recorded every minute for 15 minutes. The moan water temperatures at the proximal syringe were 37.98 + 0.030 C, 38.19 + 0.030 C, 38.21 + 0.50 C for the flow rates of 200, 300 ml/hr, respectively, and 36.2 + 0.20 C, 37.2 + 0.20 C for the flow when using the 5-ml and the 10ml syringes, respectively. The mean water temperatures at the distal syringe decreased to 32.1 + 0.10 C, 33.0 + 0.10 C, 33.8 + 0.10 C, 32.4 + 0.30 C, and 35.7 + 0.30 C, respectively. The water temperatures were directly related to the flow rates, but the mean differences between the water temperatures were inversely related to the flow rate. Fifteen pairs of blood samples drawn from 15 units of whole blood before and immediately after free haemoglobin were not statistically significant. The Siriraj Blood Warmer can work very efficiently in warming blood for massive and exchange transfusions and does not make a significant change in plasma potassium or increase haemolysis.