Genotyping of the Apolipoprotein B R3500Q Mutation Using Immobilized Locked Nucleic Acid Capture Probes

Abstract

Hyperlipidemia and coronary heart disease (CHD) are associated with genetic variation in the apolipoprotein B (apoB) gene ( 1). Nonexchangeable apolipoprotein B-100 (ApoB-100) is an important determinant of LDL-cholesterol in plasma; it plays a central role in cholesterol transport by its association to LDL particles as a ligand for the LDL receptor ( 2). One of the first mutations in the apoB gene to be discovered was the ApoB-100 R3500Q (apoB_R3500Q) mutation ( 3), a single nucleotide transition, CGG to CAG, in exon 26. This mutation reduces the affinity to the LDL receptor by at least 95% ( 4) and is the major cause of familial defective ApoB-100 (FDB). The frequency of the mutation is 1:500 to 1:700 in Caucasians ( 5)( 6). Because the cholesterol concentration is often within the reference interval in FDB patients, the only reliable way of detecting FDB is by genotyping. At present, genotyping of the apoB_R3500Q mutation is based on PCR ( 7)( 8)( 9), but other methods, such as heteroduplex analysis and real-time PCR, have also been applied ( 10)( 11)( 12). In general, these methods are time-consuming and need to be optimized. Here we describe a simple, rapid, and sensitive assay for genotyping the apoB_R3500Q mutation that is suitable for the 96-well microtiter plate format and relies on hybridization of PCR amplicons to allele-specific locked nucleic acid (LNA) capture probes ( 13)( 14).