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RESEARCH PRODUCT

Validating a Rapid Method for Detecting Common Polymorphisms in the APOA5 Gene by Melting Curve Analysis Using LightTyper

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The recently identified apolipoprotein A-V gene (APOA5) has been shown to play an important role in hypertriglyceridemia (1). Genetic variation in APOA5 has been consistently associated with plasma triglyceride concentrations in several studies (2)(3)(4). Moreover, some studies have demonstrated additional associations with lipoprotein subclasses, remnant-like particles, and cardiovascular disease risk (4)(5)(6). Several single-nucleotide polymorphisms (SNPs) in the human APOA5 gene have been detected with differing frequencies depending on the population analyzed (7)(8), and Klos et al.(7) have also suggested context-dependent associations in different populations. Overall, 5 common SNPs, −1131T>C, −3A>G, 56C>G, IVS3 + 476G>A, and 1259T>C, have been widely reported. Apart from the 56C>G SNP, the other SNPs are in strong linkage dysequilibrium, producing 3 haplotypes (11111, 22122, and 11211) representing ∼98% of the population in Caucasians (5)(9); therefore, 2 independent APOA5 SNPs (56C>G and −1131T>C) can be analyzed in association studies as indicators of the corresponding haplotypes. The former consists of a nonsynonymous substitution, changing codon 19 from serine to tryptophan (S19W), whereas the latter is a T-to-C substitution 1131 nucleotides upstream of the initiation codon. To date, in most published reports, these SNPs have been genotyped by PCR with restriction fragment length polymorphism (RFLP) analysis (3). A system for high-throughput genotyping using fluorescence melting curve analysis, the LightTyper™ (Roche), has recently become commercially available. This instrument (10) offers higher throughput than the original LightCycler™ (11). The LightTyper is designed explicitly for melting curve analysis to perform rapid, straightforward, reliable allelic discrimination. The system, which uses 384-well plates, provides postamplification genotyping within 10–15 min and performs genotyping automatically. A variety of probe chemistries are compatible for genotyping, including single-labeled probes and fluorescence resonance energy transfer probes (10). SimpleProbes™ are designed to specifically hybridize to a …