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.

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.