Developing DPYD Genotyping Method for Personalized Fluoropyrimidines Therapy

Fluoropyrimidine drugs are widely used in chemotherapy to treat solid tumors. However, severe toxicity has been reported in 10% to 40% of patients. The DPYD gene encodes the rate-limiting enzyme dihydropyrimidine dehydrogenase responsible for fluoropyrimidine catabolism. The DPYD variants resulting in decreased or no enzyme activity are associated with increased risk of fluoropyrimidine toxicity. This study aims to develop a pharmacogenetic test for screening DPYD variants to guide fluoropyrimidine therapy.

A multiplex allele-specific polymerase chain reaction (AS-PCR) assay, followed by capillary electrophoresis, was developed to detect 5 common DPYD variants (c.557A > G, c.1129-5923C > G, c.1679T > G, c.1905 + 1G > A, and c.2846A > T). Deidentified population samples were used for screening positive controls and optimizing assay conditions. Proficiency testing samples with known genotypes were analyzed for test validation. All variants detected were confirmed by Sanger sequencing.

From the deidentified population samples, 5 samples were heterozygous for c.557A > G, 2 samples were heterozygous for c.1129-5923C > G (HapB3), and 1 sample was heterozygous for c.2846A > T. The 20 proficiency samples matched with their assigned genotypes, including 13 wild-type samples, 3 samples heterozygous for c.1679T > G, 2 samples heterozygous for c.1905 + 1G > A, and 2 samples heterozygous for c.2846A > T. One of the 3 patient samples was heterozygous for c.1129-5923C > G (HapB3). All the variants detected by the multiplex AS-PCR assay were concordant with Sanger sequencing results.

A robust multiplex AS-PCR assay was developed to rapidly detect 5 variants in the DPYD gene. It can be used for screening DPYD variants to identify patients with increased risk of toxicity when prescribed fluoropyrimidine therapy.