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

Department of Biochemistry, Faculty of Medicine, Siriraj Hospital, Mahidol University, Bangkok, Thailand.
Journal of the Medical Association of Thailand = Chotmaihet thangphaet 01/2002; 84 Suppl 3:S619-27.
Source: PubMed


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.

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Available from: Wattana Leowattana, Oct 08, 2015
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    ABSTRACT: The low-density lipoprotein receptor (LDLR) is a key regulator of cholesterol homeostasis, and defects in the function of LDLR result in familial hypercholesterolemia (FH). In the present study, we performed structural analyses of two novel LDLR mutations, D151Y and M391T. Both mutations occurred in conserved residues of LDLR. The D151Y mutation, in the ligand binding domain, caused an elimination of a hydrogen bond in the calcium binding site, higher solvent accessibility and a loss of negative charge in the Y151 residue. On the other hand, the M391T mutation, in the beta-propeller of the epidermal growth factor (EGF) precursor homology domain, caused an additional hydrogen bond to form, higher solvent accessibility and a distortion of the beta-strand. These data suggest that the irregular structures of the mutated LDLRs are likely to cause the functional defect that contributes to the pathology of FH.
    Full-text · Article · Nov 2008 · Biochemical and Biophysical Research Communications