Page 284 - Personalised medicine of fluoropyrimidines using DPYD pharmacogenetics Carin Lunenburg

Chapter 11
Phasing in compound heterozygous DPYD carriers
Three databases were used to identify compound heterozygous DPYD variant allele carriers and determine the phasing, i.e. allelic location, of variants.
Genome of the Netherlands Datasets
The Genome of the Netherlands (GoNL) trio datasets contain information of related fathers, mothers, and children, and phasing information is therefore available. Datasets were previously processed and phased using trio-aware variant calling.34 After the exclusion of children, phased variant call format (VCF) files for 496 subjects (fathers and mothers) were obtained from the GoNL repository. The toolset Bedtools (v2) was used to extract all variants found in the DPYD locus (chr1:97,543,300─98,386,615). Next, for all individuals, the carrier status of DPYD*2A, DPYD*13, c.1236G>A and c.2846A>T was examined. Individuals who carry at least one of the four actionable DPYD variants were identified and, using a custom Python script,35 the phasing of variants was assessed for individuals with multiple variants.
1000 Genomes Database
The 1000 Genomes Project is the largest publicly available catalogue of human variation and genotyped phased data. It originally ran from 2008 until 2015, and thereafter it was maintained and expanded by the IGSR (International Genome Sample Resource).36 On 27 October 2016, phased data of the DPYD gene (chr1: 97,543,300─98,386,615) was downloaded from the 1000 Genomes ftp server (phase 3; GRCh37; chr1: 97,543,300─98,386,615) using Tabix (v1.1).37 The statistical program R (v3.2.5)38 was used to select the genotypes at four DPYD risk alleles in unrelated individuals of Caucasian descent.
Exome Trios Leiden University Medical Centre Database
This diagnostic database of the clinical genetics department of LUMC contains 433 complete exome trios (father, mother, and child). The exome was enriched by the Agilent sureselect v5 kit and sequenced using various Illumina (San Diego, CA, USA) sequencers (Hiseq 2000, 2500, 4000, Nextseq). Carrier status of the abovementioned DPYD variants was established by querying the trio VCF files. We also investigated all samples with sufficient coverage of this region to obtain a reliable frequency estimate. In the case of trios, only parents were taken into account.
Results
Patient cases and clinical implications
Details of the demographics and clinical characteristics of the seven cases are described in the Supplementary Material (patient cases). All patients received treatment with fluoropyrimidines and were identified as compound heterozygous DPYD variant allele carriers, either prior to the start of treatment or retrospectively. Table 1 shows an overview of the cases. Table 2 shows all genotype and phenotype results. With additional genetic testing phasing could be determined in four out of seven patients. In three patients, the variants were located in trans and one patient carried the variants in cis. With the phasing information available, it is possible to calculate a dose recommendation using publicly
282