Approach for Phased Sequence-Based Genotyping of the Critical Pharmacogene Dihydropyrimidine Dehydrogenase ().

Pre-treatment genotyping of four well-characterized toxicity risk-variants in the dihydropyrimidine dehydrogenase gene () has been widely implemented in Europe to prevent serious adverse effects in cancer patients treated with fluoropyrimidines. Current genotyping practices are largely limited to selected commonly studied variants and are unable to determine phasing when more than one variant allele is detected. Recent evidence indicates that common variants modulate the functional impact of deleterious variants in a phase-dependent manner, where a - or a -configuration translates into different toxicity risks and dosing recommendations. is a large gene with 23 exons spanning nearly a mega-base of DNA, making it a challenging candidate for full-gene sequencing in the diagnostic setting. Herein, we present a time- and cost-efficient long-read sequencing approach for capturing the complete coding region of . We demonstrate that this method can reliably produce phased genotypes, overcoming a major limitation with current methods. This method was validated using 21 subjects, including two cancer patients, each of whom carried multiple variants. Genotype assignments showed complete concordance with conventional approaches. Furthermore, we demonstrate that the method is robust to technical challenges inherent in long-range sequencing of PCR products, including reference alignment bias and PCR chimerism.