Page 283 - Personalised medicine of fluoropyrimidines using DPYD pharmacogenetics Carin Lunenburg

Compound heterozygous DPYD variant allele carriers
Dihydropyrimidine dehydrogenase enzyme activity measurements
For all patients, DPD enzyme activity was determined. This could be either prior to treatment or retrospectively after the occurrence of severe toxicity. DPD enzyme activity measurement in peripheral blood mononuclear cells (PBMCs)24,25 was used as a reference to assess DPD activity, and has been used previously to determine dosages in DPYD variant carrying patients.21,26 A validated method27 was used, containing radiolabeled thymine as a substrate and consisting of high-performance liquid chromatography (HPLC) with online radioisotope detection using liquid scintillation counting. Normal values for healthy volunteers are 9.9±2.8 nmol/(mg*h), for DPD deficient patients are 4.8±1.7 nmol/(mg*h), and reference values range from 5.9 to 14 nmol/(mg*h).28 Dose reductions based on DPD enzyme activity were performed in a one-to-one ratio, as was previously described by Henricks et al.21 Thereafter, toxicity-guided dosing was used.
Molecular methods for estimation of phasing
In regard to the size of the DPYD gene, the location of the variants, and the material available (DNA, RNA) from the patients, three molecular methods to determine the phasing of the variants could be used in this study. In four patients, we could execute one or more of these methods. These methods are explained and illustrated in the Supplementary Material (Figure S1). Details on these techniques have been published elsewhere.29-31
Frequencies of compound heterozygous DPYD carriers
To investigate the incidence of compound heterozygous DPYD variant allele carriers (of the four genotyped DPYD variants) large databases were queried.32,33 The incidence was calculated using minor allele frequencies (MAFs) of each variant identified in the databases separately. Since the determined variants are not in the same haplotype, it was assumed that the inheritance of these individual DPYD variants is independent. All genotypes from the databases were calculated to be in Hardy-Weinberg equilibrium, except for DPYD*2A and c.1236G>A for the Exome Aggregation Consortium (ExAC)32 and Genome Aggregation Database (gnomAD)33 due to a slight overrepresentation of homozygous cases. The calculated frequencies were compared to frequencies from databases in which phasing could be determined.
Exome Aggregation Consortium and Genome Aggregation Database
Both the ExAC32 and gnomAD33 databases collect exome sequencing data and aggregate the data for public use. The ExAC dataset (v.0.3.1) contains sequenced data of 60,706 unrelated individuals. The gnomAD dataset (v.2.0) contains sequenced data of 123,136 exomes and 15,496 genomes from unrelated individuals. In ExAC, 2,791 DPYD variants, and in gnomAD, 2,190 DPYD variants were found. MAFs of DPYD variants from these databases reflect those of the population due to the large group size in the databases. Since both ExAC and gnomAD do not contain individual matched or phased data, it is not possible to search for compound heterozygous patients in these databases.
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