High-resolution melting analysis of the common c.1905+1G>A mutation causing dihydropyrimidine dehydrogenase deficiency and lethal 5-fluorouracil toxicity

Abstract

Dihydropyrimidine dehydrogenase (DPD) deficiency is a pharmacogenetic syndrome associated with life-threatening toxicity following exposure to the fluoropyrimidine drugs 5-fluorouracil (5-FU) and capecitabine (CAP), widely used for the treatment of colorectal cancer and other solid tumors. The most prominent loss-of-function allele of the DPYD gene is the splice-site mutation c.1905+1G>A. In this study we report the case of a 73-year old woman with metastatic colorectal cancer who died from drug-induced toxicity after the first cycle of 5-FU-containing chemotherapy. Her symptoms included severe neutropenia, thrombocytopenia, mucositis and diarrhea; she died 16 days later despite intensive care measures. Post-mortem genetic analysis revealed that the patient was homozygous for the c.1905+1G>A deleterious allele and several family members consented to being screened for this mutation. This is the first report in Spain of a case of 5-FU-induced lethal toxicity associated with a genetic defect that results in the complete loss of the DPD enzyme. Although the frequency of c.1905+1G>A carriers in the white population ranges between 1 and 2%, the few data available for the Spanish population and the severity of this case prompted us to design a genotyping procedure to prevent future toxic effects of 5-FU/CAP. Since our group had previously developed a high-resolution melting (HRM) assay for the simultaneous detection of KRAS, BRAF, and/or EGFR somatic mutations in colorectal and lung cancer patients considered for EGFR-targeted therapies, we included the DPYD c.1905+1G>A mutation in the screening test that we describe herein. HRM provides a rapid, sensitive, and inexpensive method that can be easily implemented in diagnostic settings for the routine pre-therapeutic testing of a gene mutation panel with implications in the pharmacologic treatment.

Keywords: 5-FU; 5-fluorouracil; DPD; DPYD; HRM; capecitabine; dihydropyrimidine dehydrogenase; toxicity.

Figure 1

Pedigree of the index patient and family members carrying the DPYD c.1905+1G>A mutation. The index patient is indicated by an arrow. For family members who were alive, age at the time of the study is shown below in italics. For deceased members, designated by a diagonal line through the symbol, age and cause of death are annotated (CRC, colorectal cancer; AMI, acute myocardial infarction). Numbers inside a diamond are children of unspecified sex. DPYD genotypes are wild type (−/−), heterozygous (+/−) and homozygous (+/+) carriers of the mutation.

Figure 2

Sequence chromatograms of the index patient and family members analyzed for the DPYD c.1905+1G>A mutation. Trace sequences of DPYD exon 14 coding and flanking intron region including position c.1905 (indicated with an arrow). From top to bottom: c.1905+1G>A homozygous (index patient, II.2), heterozygous (sons, III.2 and III.3), and wild type (sister, II.5).

Figure 3

HRM analysis of the DPYD c.1905+1G>A mutation in a 239-bp amplicon. Normalized and temperature-shift melting curves (A) and differential plot (B) of mutant homozygous (II.2, blue), heterozygous (III.2, red), and wild-type (II.5, green) samples, assayed in triplicate. As all samples were spiked with wild-type DNA, homozygous and heterozygous mutants show similar left-shifted melting curves and can be easily identified, especially in the differential plot.