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

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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 (apoBR3500Q) 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 apoBR3500Q 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 apoBR3500Q 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).

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