DPYD Genotyping of Patients with Fluoropyrimidine Treatment: Results of Protocol Implementation and Outcomes of Patients Carrying Unusual DPYD Variants

Background/Objectives: The DPYD gene encodes the enzyme dihydropyrimidine dehydrogenase that metabolizes fluoropyrimidines. Genetic variants in DPYD have been associated with altered enzyme activity; therefore, accurate detection and interpretation is critical for individualized fluoropyrimidine therapy. The most common causal variant is c.1129-5923C>G (rs75017182) located in intron 10, which introduces a cryptic splice site. This variant is in high linkage disequilibrium (LD) in the HapB3 haplotype with a benign synonymous variant in exon 11, c.1236G>A (rs56038477). Since c.1129-5923C>G and c.1236G>A have been reported in LD, many commercial kits use c.1236G>A as a proxy for the function-altering intronic variant. Methods: A DPYD genotyping protocol was implemented following the quality regulations that apply to clinical laboratories (EN-ISO9001:2015 and EN-ISO15189:2022). NGS, MLPA and Sanger sequencing were used for validation purposes. Results: Over the last 5 years a total of 2007 patients have been analyzed at our department. The observed DPYD genotype frequencies aligned with those observed in European populations. Importantly, we have identified a patient harboring the c.1236G>A variant, but in the absence of the c.1129-5923C>G variant. This last result supports recently published findings suggesting that these two variants may not be in perfect LD, as previously assumed, and lead to suboptimal dosing for those patients carrying this allele. Finally, low frequency variants (c.496A>G, c.2194G>A, and c.1601G>A), not described in DPYD analysis guidelines recommendations, were found in two patients who required fluoropyrimidines dose adjustment. Conclusions: These findings highlight the limitations of relying on proxy variants for clinical decision-making, as incomplete linkage disequilibrium may lead to misclassification of patients’ metabolic capacity. Furthermore, in order to provide safer protocols for DPYD-based personalized treatment genetic panels should expand to include additional rare DPYD variants.