Rapid detection of the DPYD IVS14+1G>A mutation for screening patients to prevent fluorouracil-related toxicity.

Background: Deficiency of dihydropyrimidine dehydrogenase (DPD) has been linked to severe or lethal fluorouracil (FU)-related toxicity. The most prominent mutation in the DPYD gene is the IVS14+1G>A mutation, which causes skipping of exon 14 in the messenger RNA (mRNA) and results in DPD enzyme deficiency. Several methods have been described to detect this mutation, but all are labor intensive and low throughput. OBJECTIVE Our aim was to develop a high-throughput real-time PCR assay to screen patients for the IVS14+1G>A mutation.

Methods: Primers and probes were developed and several reaction conditions were tested. In total, 165 individuals were screened for this mutation, with DNA sequencing as a reference method.

Results: Results of the real-time PCR assay and DNA sequencing were 100% identical. In total, eight heterozygous individuals were identified, of which six were patients with severe FU-related toxicity after FU or capecitabine treatment and two were healthy volunteers.

Conclusion: This new real-time PCR assay with a high throughput is particularly suitable for large-scale screening for the IVS14+1G>A mutation in patients selected for treatment with fluoropyrimidines in order to prevent severe FU-related toxicity.