Genetic and Functional Characterization of <i>DPYD</i> Exon 4 Deletion Common in the Finnish Population

The DPYD gene encodes the dihydropyrimidine dehydrogenase (DPD) enzyme, which metabolizes fluoropyrimidine drugs such as 5-fluorouracil and its prodrugs capecitabine and tegafur. These are commonly used in chemotherapy regimens for treatment of solid tumours, especially in colon, breast, and head and neck cancers, in both the metastatic and adjuvant settings. Myelosuppression, oral mucositis, gastrointestinal toxicity and neurotoxicity are the most common adverse effects of fluoropyrimidine therapy. Certain genetic variants in DPYD have been shown to reduce DPD activity, impairing fluoropyrimidine metabolism and significantly increasing the risk of severe fluoropyrimidine-associated toxicity [1]. DPYD germline variants account for 50%–75% of the cases of grade ≥ 3 fluoropyrimidine-related toxicities and explain most of lethal toxicities. The Clinical Pharmacogenetics Implementation Consortium guideline recommends a 50% fluoropyrimidine dose reduction for patients with heterozygous nonfunctional DPYD germline variants or 25%–50% in those with two partially inactivating variants [1]. To date, four decreased function DPYD variants—c.1905 + 1G > A (rs3918290, also known as DPYD*2A, DPYD:IVS14 + 1G > A), c.1129–5923C > G (rs75017182, HapB3), c.1679 T > G (rs55886062, DPYD*13, p.I560S) and c.2846A > T (rs67376798, p.D949V)—are the most commonly tested, due to their well-established population frequencies and known impacts on enzyme function and risk of toxicity. The exon-skipping variant DPYD*2A and the missense variant c.1679 T > G result in a completely nonfunctional DPD-enzyme, whereas the c.2846A > T missense variant and HapB3 splice site variant partially disrupt the protein function. Despite preemptive genotyping and genotype-guided dosing, around 20%–30% of patients may still experience severe or even lethal fluoropyrimidine treatment-related adverse effects [2], suggesting there are unidentified variants affecting the DPD activity. A recent study using multiplex ligation-dependent probe amplification described a novel exon 4 deletion causing DPD deficiency in four out of 167 Finnish cancer patients with planned capecitabine treatment [3]. Additionally, a study performed among 125 Canadian patients with severe fluoropyrimidine toxicity identified one individual with a heterozygous DPYD exon 4 germline deletion [4]. The rarity of this deletion in the Canadian population suggests that this copy number variation (CNV) may be more prevalent in the isolated Finnish population. Here, we investigated the DPYD exon 4 deletion in a cohort of 356 unrelated healthy Finnish volunteers in high-throughput next-generation sequencing data and quantitative PCR (qPCR) to study the population frequency of this variation in Finland and further characterize its breakpoints for genetic testing. Moreover, we studied the effects of this variant on DPD activity. The study was conducted in accordance with the Basic & Clinical Pharmacology & Toxicology policy for experimental and clinical studies [5]. The study included 356 unrelated healthy Finnish volunteers (aged 18–40) from previous pharmacogenetics studies, and their blood samples were collected as previously described [6, 7]. Genomic DNA was extracted from ethylenediaminetetraacetic acid (EDTA) blood samples with the Maxwell 16 LEV Blood DNA Kit on a Maxwell 16 Research automated nucleic acid extraction system (Promega, Madison, WI, United States). All study participants gave written-informed consent. The study protocols were approved by the Coordinating Ethics Committee of the Hospital District of Helsinki and Uusimaa. The following workflow has been illustrated in Figure 1. Out of the 356 study participants, 201 samples were previously sequenced with targeted paired-end capture-based next generation sequencing (NGS) using an Illumina HiSeq2000 sequencing system (Illumina, San Diego, CA, United States) in collaboration with the Institute for Molecular Medicine Finland (FIMM) Technology Centre, University of Helsinki [6]. Structural variants and indels in the DPYD gene were identified from the targeted sequencing data using the Manta 1.6 program, which can predict exact breakpoint sites with confidence estimates [8]. DPYD exon 4 CNVs that were identified were further quantified with a Custom TaqMan® Copy Number Assay targeting the DPYD exon 4 with QuantStudio™ 12 K Flex Real-Time PCR System following the manufacturer's instructions (ThermoFisher Scientific, Waltham, MA, United States) among the 201 sequenced samples. The remaining 155 samples were also genotyped using this assay. RNAseP (Ribonuclease P RNA component H1 gene, RPPH1) was used as the reference assay. Four replicates of each sample were studied. Copy numbers were calculated with CopyCaller™ Software v2.1 (Applied Biosystems®) according to the manufacturer's instructions. Relative quantification was used to determine the heterozygosity of the samples with RQ ~ 1.00 considered as wildtype, RQ ~ 0.5 as deletion and RQ ~ 1.5 as duplication. GnomAD database (dataset SVs v4.1.0) was used to study the frequency of the DPYD exon 4 CNV (named as DEL_CHR1_3418253E) across different populations. The frequencies of the functionally significant DPYD single nucleotide variants c.1236G > A (rs56038477, marker for HapB3), c.1679 T > G (rs55886062), c.1905 + 1G > A (rs3918290) and c.2846A > T (rs67376798) were investigated in the sequencing data from 201 participants and in genome wide genotype data for the remaining 155 participants that were previously genotyped with the Illumina HumanCoreExome-24v1-1_A BeadChip (Illumina, San Diego, CA, United States) [6, 7]. The effects of DPYD exon 4 deletion and the c.1236G > A and c.1905 + 1G > A variants on DPD enzyme activity were estimated by measuring the uracil and dihydrouracil concentrations in the fasting plasma samples of all identified variant allele carriers and a total of 50 noncarriers. The samples were stored at −70°C until analysis. Uracil and dihydrouracil were quantified using liquid chromatography–tandem mass spectrometry as described previously [9] with minor modifications. Prior to analysis, stably isotope labelled internal standards (uracil-d2 and 5,6-dihydrouracil-13C, 15 N2) were added to 300 μL of plasma, and plasma proteins were precipitated by adding 900 μL of methanol:acetonitrile (50:50 v/v). Supernatants were then evaporated using Genevac miVac centrifugal concentrator (Thermo Fisher Scientific), and the dry extracts were reconstituted in 100 μL of 0.1% formic acid. The chromatographic separation was achieved on an Acquity UPLC HSS T3 column (100 × 2.1 mm ID, particle size 1.8 μm; Waters, Milford, MA, United States) protected by a VanGuard precolumn of the same material. Mobile phases A and B consisted of 0.1% formic acid in water and acetonitrile, respectively. Column temperature was set at 32°C, the flow rate was maintained at 0.3 μL/min, and the gradient profile was 0% B for 3.0 min, 90% B for 1 min and 0% B from 3 min. A QTrap 5500 mass spectrometer interfaced with ESI ion source (AbSciex, Toronto, ON, Canada) was operated in the negative ion multiple reaction monitoring (MRM) mode for quantification of uracil and in the positive ion mode for dihydrouracil [9]. The quantification limit for uracil and dihydrouracil were 1.0 and 8.0 ng/mL, respectively. The precision and accuracy were below 12% and within 7% for both analytes at relevant concentrations. Before statistical analysis, the uracil and dihydrouracil concentrations and the dihydrouracil/uracil concentration ratio were logarithmically transformed and compared between the genotypes using multivariate general linear model. A p value of below 0.05 was considered statistically significant. The DPYD exon 4 deletion was found in our dataset with a frequency of 1.7%, with six identified heterozygous carriers among 356 participants (minor allele frequency 0.8%). A total of 13 (3.7%) and 6 (1.7%) participants were heterozygotes for the c.1905 + 1G > A and c.1236G > A variants, respectively (minor allele frequencies 1.8% and 0.8%). The c.1679 T > G and c.2846A > T single nucleotide variations were not found among the study participants. In the GnomAD database, the heterozygous DPYD exon 4 deletion was identified in 50 individuals among Finnish genetic ancestry group including 3238 individuals (population frequency 1.55%) and in only one non-Finnish European person among 29 544 individuals. Based on the Manta analysis, we identified an identical 13 849 bp deletion in five samples spanning a region that includes the DPYD exon 4, with high-confidence breakpoint position prediction (chr1:97734782-97748631, hg38 build) among the 201 sequenced samples. In all cases, the breakpoint start and end positions showed a two-base pair uncertainty downstream of the predicted start and end breakpoints. Among the five samples, the breakpoint positions were supported by 6–20 confidently mapped read pairs. No other large CNVs were observed in the gene. The detected exon 4 deletion was confirmed in all five samples by qPCR, and one additional exon 4 deletion carrier was found among the additional set of 155 volunteers. The fasting plasma dihydrouracil/uracil ratio was 51% lower in exon 4 heterozygotes than in noncarriers of the variants (p = 5.1 × 10−6) (Table 1 and Figure 2). Similarly, the ratio was decreased by 46% and 33% in c.1905 + 1G/A and c.1236G/A heterozygotes compared to noncarriers (p = 1.8 × 10−7 and p = 0.0068, respectively). Functionally deficient germline variants in the DPYD gene result in impaired DPD activity, leading to an increased risk of toxicity in cancer patients treated with fluoropyrimidine-based chemotherapy. Despite preemptive DPYD testing, several patients experience life-threatening adverse effects, suggesting that there may be other variants in the DPYD gene affecting toxicity in addition to the most commonly tested single nucleotide variants. In 2021, Saarenheimo et al. [3] reported a novel exon 4 deletion in the DPYD gene with a high frequency (2.4%) in a cohort of 167 cancer patients in Finland in whom fluoropyrimidine medication was planned. The deletion was found to result in a nonfunctional DPD enzyme by DPD activity and plasma uracil measurements. Our findings support these functional data, as the plasma dihydrouracil/uracil concentration ratio was reduced to about half in heterozygous carriers of the variant. Our findings among healthy Finnish individuals and data from the GnomAD database are also consistent with the frequency of this variant, suggesting a 1%–2% population frequency in Finland. The consistent data on loss-of-function and the relatively high frequency of the variation strongly support including it in preemptive DPYD testing protocols for Finnish patients. Since March 2020, the European Medicines Agency has recommended testing DPD deficiency before administering fluoropyrimidine, a practice currently implemented in the Finnish healthcare. Phenotyping of DPD activity with pretreatment uracil measurements has been used as an alternative to DPYD genotyping in clinical practice. However, phenotyping with uracil measurements is prone to preanalytical errors [10]. In the present study, the samples were collected in two clinical trials between 2012 and 2017. The sample collection and preparation were standardized, and the samples were stored at −70°C until analysis, which minimized variation due to preanalytical issues. CNV in DPYD is not extensively studied, yet the gene is known to include a common fragile site, FRA1E. Such structures are unstable and prone to breakage during replication [11]. Overall, CNVs ranging from ~1 kb to ~3 Mb are proposed as an additional important source of phenotype differences affecting pharmacogene functions and consequently drug treatments. Structural variation can erase enzyme function or set it on overdrive [12]. Around 20 different DPYD CNVs have been described (both heterozygous deletions and duplications) [4]. Complete DPD deficiency has been associated with intellectual disability, seizures, autism-like symptoms, speech delays, and obesity, but many DPD deficient individuals are completely healthy [1]. Similar symptoms have been reported in individuals with deletions in the DPYD gene. In our study, all six DPYD exon 4 deletion carriers were heterozygotes for the variant and confirmed to be healthy by medical history, clinical examination, and laboratory tests [6, 7]. As a conclusion, the DPYD exon 4 deletion can be detected using conventional laboratory methods as well as high throughput NGS methods, facilitating its inclusion in NGS-based gene panels. This deletion, although rare in other populations, is prevalent in the Finnish population, results in loss of DPD activity, and warrants testing prior to fluoropyrimidine treatment. Overall, large structural variations are an important source of individual differences in drug response. The authors wish to thank Eija Mäkinen-Pulli and Lisbeth Partanen for skilful technical assistance and the Institute for Molecular Medicine Finland (FIMM) Technology Centre for providing the sequencing services. The authors declare no conflicts of interest. Research data are not shared.

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