Genotyping of Eight Thiopurine Methyltransferase Mutations: Three-Color Multiplexing, “Two-Color/Shared” Anchor, and Fluorescence-quenching Hybridization Probe Assays Based on Thermodynamic Nearest-Neighbor Probe Design

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Abstract
Background: The inherited deficiency of thiopurine methyltransferase (TPMT) leads to severe myelosuppression in homozygous patients treated with thiopurine derivatives. One in 300 Caucasians has a homozygous TPMT deficiency with no measurable enzyme activity. To date, eight single-point mutations have been characterized; one group (TPMT*3) accounts for 75% of these. Methods: We used four LightCyclerTM capillaries to investigate all eight mutations. The three mutations on exon 10 were detected in one capillary with a single “shared” anchor labeled 5′ with Cy5.5 and 3′ with fluorescein. A wild-type-compatible 3′-fluorescein-labeled probe 5′ adjacent to the anchor covered the TPMT*7 mutation, and a 5′-LC-RED640-labeled probe 3′ adjacent to the anchor covered the TPMT*3C mutation. For TPMT*4, the forward amplification primer was internally labeled with a fluorescence quencher [6-carboxytetramethylrhodamine (TAMRA)], and a 3′-fluorescein-labeled antisense wild-type-compatible probe was placed at the mutation. For TPMT*2 and TPMT*3D, located on exon 5, a shared anchor approach was chosen. TPMT*3B and TPMT*6 were detected in multiplex technique and TPMT*5 in conventional manner. Anchors and probes were designed using a thermodynamic nearest-neighbor model. Results: All mutations were detected using four capillaries with one amplification protocol in 40 min. The concentrations of the shared anchors had to be decreased to reduce their intrinsic fluorescence resonance energy transfer signals. The quenching approach using TAMRA produced a very reproducible upside-down-shaped melting curve in channel 1 of the LightCycler. Deviations from wild type were easily detected because the smallest melting point shift for any possible mutation under the core of the probes was 1.5 °C. Conclusions: This total TPMT genotyping approach shows that it is possible to use double site-labeled anchor oligonucleotides, that channel 1 of the LightCycler can be used as detection channel for mutations using a quenching design, and that the designed probes enable detection of wild types with 100% likelihood.