Screening for Mutations in Exons Encoding the Ligand- Binding Domain of the LDL Receptor Gene Using PCR- CFLP and PCR-SSCP
KLAI-UPSORN PONGRAPEEPORN, Ph.D.*, WATT ANA LEOWATTANA, M.D.***, WILAIRAT NUCHPRAMOOL, M.D.****, KW ANDOA KERDSAENG, B.Sc. *, PIKUN THEPSURIYANON, M.Sc.*****, SUDCHAREE KIARTIVICH, M.Sc. ***, PREYANUJ YAMWONG, M.D.**, SOMPONG ONG-AJYOOTH, M.Sc. *, ANCHALEE AMORNRATTANA, M.Sc. **, LUMPOON KASEMSUK, B.Sc.*, SIV ADEE LAUNGSUW AN, M.Sc. ***, KOSIT SRIBHEN, M.D.***
Affiliation : * Department of Biochemistry, ** Department of Preventive and Social Medicine, *** Department of Clinical Pathology, Faculty of Medicine, Siriraj Hospital, **** Department of Clinical Chemistry, Faculty of Medical Technology, Mahidol University, Bangkok, 10700, Thailand. ***** Amersham Pharmacia Biotech, South East Asia.
Primary hypercholesterolemia includes both monogenic disorders and polygenic condi- tions. Two well defined monogenic disorders are familial hypercholesterolemia (FH) and familial defective apolipoprotein (apo) B-1 00 (FDB). Both disorders convey high risk of premature coro- nary artery disease. FH and FDB are caused by mutations in LDL receptor and apo B-1 00 genes, respectively. In the present study, mutations in both genes in Thai subjects with primary hyper- cholesterolemia 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 Dl51Y. 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.
Keywords : Hypercholesterolemia, LDL Receptor Gene, Mutation. PCR-CFLP, PCR-SSCP
