Homozygous DNA variants in exon 9 of the LDL receptor gene in a Thai patient with primary hypercholesterolemia phenotype.

Mutation in low density lipoprotein (LDL) receptor gene causes an inherited primary hypercholesterolemia namely familial hypercholesterolemia (FH). In this study, 46 Thai patients with primary hypercholesterolemia were screened for mutations in exon 9 of the LDL receptor gene by polymerase chain reaction-restriction fragment length polymorphism (PCR - RFLP). The analysed fragment was 224 bp in length. According to the published cDNA sequence, exon 9 of the LDL receptor gene contains several hypermutable CpG dinucleotides. Three of these sites are Hpa II recognition sites. PCR product of exon 9 obtained from amplification of wild-type DNA sample would yield four fragments after Hpa II digestion. The expected sizes of these restriction fragments were 15, 30, 40 and 139 bp. All normocholesterolemic subjects (n = 33) showed normal RFLP. However, in one patient (72 year old female), abnormal RFLP from Hpa II digestion of the amplified exon 9 was observed, i.e., a fragment of 70 bp and another one smaller than 139 bp. Such RFLP reflects that exon 9 of both alleles of the LDL receptor gene in this patient lost one and gained one Hpa II site. It is interesting that this patient, eventhough harbouring two mutations on both alleles of the LDL receptor gene (presumably homozygous genotype of FH), apparently revealed lipid levels of heterozygous phenotype of FH without symptoms of coronary artery disease. It has yet to be proved whether these genetic variations are disease-related mutations or presumably common DNA polymorphisms.

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.

Mutation analysis of exon 9 of the LDL receptor gene in Thai subjects with primary hypercholesterolemia.

The low density lipoprotein (LDL) receptor plays an important role in cholesterol homeostasis. A mutation in this gene causes an autosomal codominant disorder, namely familial hypercholesterolemia (FH). In this study, single strand conformation polymorphism (SSCP) analysis was used to screen for mutations in exon 9 of the LDL receptor gene in a group of 45 Thai patients (11 males and 34 females) with primary hypercholesterolemia. The peptide encoded by exon 9 belongs to the epidermal growth factor (EGF) precursor homology domain which is highly conserved in the LDL receptor protein. An abnormal SSCP pattern was observed in one female patient. The same screening strategy was also used to screen DNA samples from 33 normolipidemic subjects. All of these samples showed normal SSCP pattern. By direct DNA sequencing, the underlying mutation in the DNA with abnormal SSCP pattern was identified. The index subject was heterozygous for a T to C transition at nucleotide 1235. This transition would cause a nonconservative substitution of a nonpolar side chain amino acid "methionine" at codon 391, with an uncharged polar side chain amino acid "threonine", note M391T. From multiple amino acid sequence alignment in six species, the amino acid at codon 391 and the others nearby are completely conserved. Such nonconservative substitution of an amino acid residue in a highly conserved region could consequently result in a functional and/or structural defect in the receptor protein. In conclusion, we propose that M391T is likely to be the cause of hypercholesterolemia in this index subject.

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.