Rapid detection of the DPYD IVS14+1G>A mutation for screening patients to prevent fluorouracil-related toxicity
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Tessa M Bosch1, Remko Bakker, Jan H M Schellens
1Department of Pharmacy and Pharmacology, The Netherlands Cancer Institute/Slotervaart Hospital, Amsterdam, The Netherlands.
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.
Insights
A new real-time PCR assay effectively screens for the IVS14+1G>A mutation, a common cause of dihydropyrimidine dehydrogenase (DPD) deficiency. This method helps prevent severe fluorouracil toxicity in patients.
Area of Science:
- Pharmacogenomics
- Molecular Diagnostics
Background:
- Dihydropyrimidine dehydrogenase (DPD) deficiency is linked to severe fluorouracil (FU) toxicity.
- The IVS14+1G>A mutation in the DPYD gene is a primary cause of DPD deficiency, leading to exon 14 skipping.
- Existing methods for detecting this mutation are labor-intensive and low-throughput.
Purpose of the Study:
- To develop a high-throughput real-time PCR assay for the IVS14+1G>A mutation.
- To enable efficient screening of patients for DPD deficiency.
Main Methods:
- Development and optimization of primers and probes for real-time PCR.
- Screening of 165 individuals using the developed assay.
- Validation against DNA sequencing as the reference method.
Main Results:
- The real-time PCR assay demonstrated 100% concordance with DNA sequencing.
- Eight heterozygous individuals were identified.
- Six of the identified individuals experienced severe FU-related toxicity, while two were healthy volunteers.
Conclusions:
- The developed real-time PCR assay is a high-throughput and suitable method for large-scale screening.
- This assay can identify patients with the IVS14+1G>A mutation.
- Screening can help prevent severe fluoropyrimidine-related toxicity.
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