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Updated: Jul 30, 2026
Single Droplet Digital Polymerase Chain Reaction for Comprehensive and Simultaneous Detection of Mutations in Hotspot Regions
Published on: September 25, 2018
Screening for mutations in exons encoding the ligand-binding domain of the LDL receptor gene using PCR-CFLP and
K Pongrapeeporn1, W Leowattana, W Nuchpramool
1Department of Biochemistry, Faculty of Medicine, Siriraj Hospital, Mahidol University, Bangkok, Thailand.
Insights
Genetic screening in Thai patients identified no familial defective apolipoprotein B-100 mutations. However, mutations in the LDL receptor gene were detected, suggesting a genetic basis for primary hypercholesterolemia in this population.
Area of Science:
- Genetics
- Cardiovascular Disease
- Biochemistry
Background:
- Primary hypercholesterolemia, including familial hypercholesterolemia (FH) and familial defective apolipoprotein B-100 (FDB), significantly increases the risk of premature coronary artery disease.
- FH and FDB result from mutations in the LDL receptor and apolipoprotein B-100 genes, respectively.
Purpose of the Study:
- To screen Thai patients with primary hypercholesterolemia for mutations in the LDL receptor and apolipoprotein B-100 genes.
- To investigate the genetic underpinnings of hypercholesterolemia in a Thai cohort.
Main Methods:
- Screening for the common R3500Q mutation in the apolipoprotein B-100 gene.
- Utilizing PCR-CFLP analysis to screen for mutations in the ligand-binding domain exons of the LDL receptor gene, particularly exon 4.
- Employing PCR-SSCP analysis to screen other exons (3, 5, and 6) of the LDL receptor gene.
Main Results:
- The R3500Q mutation in the apolipoprotein B-100 gene was not found in any of the 45 patients studied.
- PCR-CFLP analysis revealed 18 abnormal patterns in exon 4 of the LDL receptor gene among 45 patients, including the previously identified D151Y mutation.
- PCR-SSCP analysis identified abnormal patterns in exons 3, 5, and 6 of the LDL receptor gene in 20 patients.
Conclusions:
- Mutations in the LDL receptor gene are present in Thai patients with primary hypercholesterolemia.
- The common R3500Q mutation of familial defective apolipoprotein B-100 is not a significant cause of hypercholesterolemia in this Thai cohort.
- Further DNA sequencing and family studies are required to fully characterize the identified LDL receptor gene mutations.
Abstract:
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.
