Long-Distance PCR-based Screening for Large Rearrangements of the LDL Receptor Gene in Korean Patients with Familial Hypercholesterolemia Sung Han Kim, 1 Ji Hyun Bae, 2 Jae Jin Chae, 2 Un Kyung Kim, 2 Seong-Joon Choe, 3 Yong Namkoong, 4 Hyo-Soo Kim, 3 Young-Bae Park, 3 and Chung Choo Lee 1* Background: The LDL receptor is a cell-surface protein that regulates plasma cholesterol by specific uptake of LDL particles from the blood circulation. Familial hy- percholesterolemia (FH) results from defective catabo- lism of LDL, which is caused by mutations in the LDL-receptor gene. Methods: For the rapid and reliable detection of large rearrangements in the LDL-receptor gene, we estab- lished a screening method based on long-distance PCR as an alternative to Southern-blot hybridization. Using long-distance PCR, 45 unrelated Korean subjects het- erozygous for FH were screened to assess the frequency and nature of major structural rearrangements in the LDL-receptor gene. Results: Two different deletion mutations, FH6 (same type as FH3 and FH311) and FH 32, were detected in four families by long-distance PCR. Detailed restriction mapping and sequence analysis showed that FH6 was a 5.71-kb deletion extending from intron 8 to intron 12 and that FH32 was a 2-kb deletion extending from intron 6 to intron 7. Sequence analysis for the breakpoints of all deletions detected in Korean FH patients showed that only the left arms of the Alu repetitive sequences were involved in the deletion event. Conclusions: The screening method based on long- distance PCR provides a powerful strategy for the de- tection of large rearrangements in the LDL-receptor gene and is a rapid and reliable screening alternative to Southern-blot hybridization. © 1999 American Association for Clinical Chemistry Familial hypercholesterolemia (FH) is a common autoso- mal dominant disorder caused by a defect in the LDL- receptor gene; the frequency of FH heterozygotes is 1 in 500 in most populations (1). FH is characterized by a selective increase of plasma LDL-cholesterol, giving rise to tendon and skin xanthomatosis and to premature coronary heart disease (1, 2). Mutations of the LDL-receptor gene are genetically heterogeneous, and a large number of mutations (to date, 300) including both large and small rearrangements have been reported in various populations and ethnic groups (1, 3–7 ). These mutations, which disrupt the syn- thesis, intracellular transport, ligand-binding capacity, internalization, and recycling ability of the LDL-receptor protein, have revealed many aspects of the structure- function relationship of the LDL-receptor gene (1, 8, 9). The vast majority of the large rearrangements of the LDL-receptor gene are deletions of various sizes along the entire length of the gene (10, 11). Most rearrangements have arisen by recombination between two Alu repeats oriented in the same direction, and these account for relatively high percentages of the mutations in the LDL- receptor gene for FH in some populations (1, 7, 12–16 ). In general, Southern-blot hybridization has been used in screening large rearrangements of various genes, in- cluding the LDL-receptor gene. However, standard PCR techniques can provide a rapid and reliable screening alternative to Southern-blot hybridization for the detec- tion of various large rearrangements in the LDL-receptor gene in FH patients. We therefore established a screening scheme for large rearrangements of the LDL-receptor 1 Department of Biology and SRC for Cell Differentiation, and 2 Depart- ment of Molecular Biology, Seoul National University, Seoul 151-742, Korea. 3 Department of Internal Medicine, Seoul National University Hospital, Seoul 110-799, Korea. 4 Department of Biology, Kangnung National University, Kangnung 210- 702, Korea. *Address correspondence to this author at: Department of Biology, College of Natural Science, Seoul National University, Seoul 151-742, Korea. Fax 82-02-872-1993; e-mail chunglee@plaza.snu.ac.kr. Received February 26, 1999; accepted June 4, 1999. Clinical Chemistry 45:9 1424 –1430 (1999) Lipids and Lipoproteins 1424 Downloaded from https://academic.oup.com/clinchem/article/45/9/1424/5643477 by guest on 26 June 2022