General background text Pharmacogenetics Dihydropyrimidine dehydrogenase (DPD)

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1 General background text Pharmacogenetics Dihydropyrimidine dehydrogenase (DPD) Last updated: 26 October 2017 Definitions in pharmacogenetics The genotype is the hereditary information about a specific characteristic of an individual. This information is located in the genes, in the DNA that consists of nucleotides. The piece of the DNA that carries information for one specific hereditary characteristic is called a gene. The DNA is divided into chromosomes, which usually occur in pairs. A person generally has two copies (alleles) of a gene, one on each of the chromosomes of a chromosome pair. The phenotype indicates what the final manifestation (phenotypic state) of a certain genotype is. This can involve the functionality of a protein (for example the enzyme or the receptor), but also the physical manifestation of a disease. The phenotype is a result of the genotype that a person possesses, the degree of expression of the gene in question and the combination with environmental factors such as co-medication, diet and disease conditions. Variations can exist in a population within the DNA that encodes a protein. Variations can result in alleles that encode for proteins with no or reduced activity. The simplest form of variations are single-nucleotide polymorphisms (SNPs), in which a certain part of a gene differs by only one nucleotide. If a gene variation occurs in at least 1% of the population, then this is referred to as a genetic polymorphism. Wild-type is the name given to the most common active allele. There can be a number of different polymorphisms for a certain allele. Names of genes are given in italics. In some cases the abbreviation of the name of the gene can differ from the name of the gene product. This is the case for dihydropyrimidine dehydrogenase, where the gene is abbreviated to DPYD and the enzyme to DPD. Altered metabolic capacity and clinical consequences Fluorouracil is mainly (> 80%) converted by dihydropyrimidine dehydrogenase (DPD) to the inactive metabolite dihydrofluorouracil. DPD is mainly present in the liver, but also in most other tissues. Lower metabolic activity of DPD leads to increased intracellular concentrations of fluorodeoxyuridine monophosphate, the active metabolite of fluorouracil and its prodrug capecitabine. This leads to an increased risk of side effects such as neutropenia, thrombopenia and hand-foot syndrome. Variations in the gene that encodes the DPD enzyme can result in reduced or absent enzyme activity. The population can be divided into five phenotypes, based on the metabolic capacity of DPD that is present: - : absent metabolic capacity (two inactive alleles) -.5: very strongly reduced metabolic capacity (one inactive and one reduced-activity allele) - score 1: strongly reduced metabolic capacity (one fully active and one inactive allele or two reduced-activity alleles) - score 1.5: reduced metabolic capacity (one fully active and one reduced-activity allele) - score 2: normal metabolic capacity (extensive metaboliser (EM), two fully active alleles) There is also variation in metabolic capacity within a group with a certain gene activity score. As the genotype only determines part of the metabolic capacity, the guidelines for dose adjustment based on the genotype are no more than a tool that can be used to achieve the desired intracellular concentration of the active metabolite. Pharmacokinetic dose adjustment (guided by steady-state plasma concentrations or AUC) may be useful for fluorouracil to optimise the dose. This is not used for capecitabine, as it is only converted to fluorouracil in tissue. Genotyping The process of genotyping is used to determine the genotype. It indicates which alleles of the gene for DPD are present in the tested individual. Each allele has a name that consists of a star (*) and a number, an example of a 1

2 possible DPD genotype is DPYD*1/*2A. Standard nomenclature is not available for all discovered alleles. This is why some alleles are still referred to by nucleotide numbers followed by nucleotide changes (or by amino acid number preceded by symbol of the amino acid in the wild-type protein followed by the symbol of the amino acid in the variant). Many variations exist for DPD; more than 30 different allele variations have been identified and described in the literature. The incidences of all variants are low and whether DPD activity is affected is not fully known for each variant; a reliable genotyping test for DPD is not available. A number of these alleles are listed in Table 1, including their functionality. Genotyping usually screens for only the most common variant alleles. This may result in less common variants being missed and incorrectly being designated the wild-type. If two different gene variations are found during genotyping of one patient, it is possible that both gene variations are on the same allele. This means that this patient has one wild-type allele and one reduced-activity allele and not two reduced-activity alleles. The total gene activity score is different in these two cases. Table 1. DPYD alleles and metabolic capacity Metabolic Reduced capacity fluorouracil clearance and toxicity in studies Fully functional (wild-type; gene activity score 1) Reduced functionality # (gene activity.5) fully (inactive or null allele; gene activity score 0) Association with toxicity in a study, but no association in other studies or no other studies known Toxicity in cases Toxicity in cases, but no association in studies and/or no reduced clearance or *1 *4 = 1601G>A *5 = 1627A>G *9A = 85T>C 2846A>T 1236G>A / C>G (hapb3) $ 496A>G IVS10-15T>C *6 = 2194G>A 1896C>T *3 = 1897delC *7 = delTCAT *8 = 703C>T *9B = 85T>C G>A *10 = 2983G>T *11 = 1003G>T *12 = 62G>A 1156G>T 1651G>A 1845G>T *2A = IVS14+1G>A *13 = 1679T>G % 300C>A & 1024G>A & 1025A>G & 1475C>T & 1774C>T & Duplication of exons Toxicity in cases does not mean that an allele has reduced functionality. Toxicity may also occur in patients with genotype *1/*1, i.e. in patients with other fully functional alleles. The fact that there was no association with increased risk of toxicity in studies and/or reduced clearance by or activity of DPD means that these alleles are fully functional. # The nature of the change in protein sequence in these variants suggests that the protein may still be partially functional (only 1 amino acid changed) or remaining activity is expected for other reasons. $ Variant 1236G>A, which does not result in a change in amino acids, is in complete linkage disequilibrium with variant C>G, which results in abnormal splicing of a part of the mrna, resulting in the formation of a premature stop codon. The resulting DPD activity accounts for approximately half of the normal activity. Both variants are part of haplotype B3. % For these variants, there is limited evidence from studies or from a case report that measured DPD activity, that the variants (amino acid substitution (Ile560Ser) for *13) lead to an inactive enzyme. & These variants have a very low enzyme activity in vitro. 2

3 Translation from genotype to phenotype When an individual s genotype has been determined and one wants to know what the metabolic capacity for DPD is, then the genotype needs to be translated to the phenotype. A complication in this process is that in very rare cases with two or more different s genotyping does not provide conclusive information about whether these variants are located on the same allele or on different alleles. Therefore, in some cases, genotyping does not provide sufficient information to predict the metabolic capacity for DPD and therefore the phenotype. For some patients, additional therefore determine the phenotype and the associated gene activity score. As the genotyping laboratory is responsible for determining the genotype, in the ideal situation the phenotype (i.e. the gene activity score) should be given to the healthcare provider. If the genotyping laboratory only provides the variants determined in the patient, you will find information about the translation to a phenotype below and/or whether extra information is required from the laboratory. The three tables below describe the relationship between the genotyping result and the predicted phenotype for various situations, or the tables state which possible genotypes and predicted phenotypes a patient with this genotyping result can have. Table 2. Relationship between genotyping result and predicted phenotype for 0 or 1 that results in decreased DPD activity Genotyping result Genotype (given as functionality Predicted phenotype of both alleles) No (*1/*1) Fully functional/fully functional score 2 a reduced functional gene Fully functional/reduced score 1.5 variant (*1/2486T or *1/1236A) functionality a fully gene Fully functional/inactive score 1 variant (*1/*2A or *1/*13) a reduced functional gene Reduced functionality/reduced score 1 variant (2486T/2486T or 1236A/1236A) functionality a fully gene Inactive/inactive variant (*2A/*2A or *13/*13) Table 3. Relationship between genotyping result and predicted phenotypes in 2 different s that result in decreased DPD activity Genotyping result Possible Explanation predicted phenotypes two different reduced functional s (2486T/1236A or *1/2486T+1236A) score (50-75% of the If the two s are located on different alleles, this results in a gene activity score of 1. If the two s are located on the same allele, the gene activity score depends on the effect that the two s have on each other. This effect is not known. If the second has no effect, the gene activity score of the allele is the same as without the second, i.e. 0.5 and the gene activity score of the genotype 1.5. If the two s amplify each other in such a way that the allele becomes fully, the gene activity score of the allele is 0 and the gene activity score of the genotype is 1. As the s 2486T and 1236A reduce the DPD activity via various mechanisms (an amino acid substitution (Asp949Val) and a splicing defect in a part of the mrna respectively), the most probable scenario is that they exert their effect on the activity of the allele independently. This would result in a gene activity score for the allele of 0.25 (halving of the allele activity by both s) and thus a gene activity score of 1.25 for the genotype. This is a problem, because there are no recommendations for a gene activity score of However, factors other than gene variations in the gene for DPD can also influence the DPD activity. For this reason, it is recommended to link the patient to a gene activity score of 1 if the measured DPD activity is approximately equal to 50% of the activity and to a gene activity score of 1.5 if the measured DPD activity is approximately equal to 75% of the normal enzyme activity. If the measured DPD 3

4 a variant with reduced a fully (*2A/2846T or *1/*2A+2486T; *13/2846T or *1/* T; *2A/1236A or *1/*2A+1236A; *13/1236A or *1/* A) two different fully s (*2A/*13 or *1/*2A+*13) one homozygous for the other (2486T/2486T+ 1236A or 1236A/2486T+ 1236A) one heterozygous for a fully (2846T/*2A+2846 T or 2846T/* T or 1236A/*2A+1236 A or 1236A/* A).5 or 1 (25 or 50% of the or 1 (0 or 50% of the score of (25-50% of the.5 activity lies between these values (approximately 63% of the normal DPD activity) as a precautionary measure and because it is recommended to adjust the initial dose after starting based on toxicity and effectiveness the recommendation is to link the patient to a gene activity score of 1. The patient file should then state that the patient actually has a gene activity score of If the two s are located on different alleles, this results in a gene activity score of 0.5 (one allele one inactive allele). If the two s are located on the same allele, the gene activity score is 1 (one fully functional and one inactive allele). If the two s are located on different alleles, this results in a gene activity score of 0 (two inactive alleles). If the two s are located on the same allele, the gene activity score is 1 (one fully functional and one inactive allele). For one of the alleles, the gene activity score is 0.5. For the other allele, the gene activity score is not known, but lies between 0 and 0.5 (refer to the explanation for heterozygous for two different s with reduced functionality). The recommendation is to link the patient to a gene activity score of 0.5 if the measured DPD activity is approximately equal to 25% of the activity and to a gene activity score of 1.0 if the measured DPD activity is approximately equal to 50% of the normal enzyme activity. If the measured DPD activity lies somewhere in between these values (approximately 38% of the normal DPD activity) as a precautionary measure and because it is recommended to adjust the initial dose after starting based on toxicity and effectiveness the recommendation is to link the patient to a gene activity score of 0.5. The patient file should then state that the patient actually has a gene activity score of For one of the alleles, the gene activity score is 0.5, for the other it is 0. Therefore, the gene activity score for the genotype is

5 a homozygous for a fully (*2A/*2A+2846T or *2A/*2A+1236A or *13/* T or *13/* A) one fully and heterozygous for the other (*2A/*2A+*13 or *13/*2A+*13) two different gene variants with reduced functionality (2486T+1236A/ 2486T+1236A) a a fully (*2A+2486T/*2A+ 2486T or * T/* T or *2A+1236A/*2A+ 1236A or * A/* A) two different fully s (*2A+*13/*2A+ -1 (0-50% of the The gene activity score is not known for either allele, but lies between 0 and 0.5 (refer to the explanation for heterozygous for two different s functionality). The recommendation is to link the patient to a gene activity score of 0 if the measured DPD activity is approximately equal to 0% of the activity and to a gene activity score of 1.0 if the measured DPD activity is approximately equal to 50% of the normal enzyme activity. If the measured DPD activity lies somewhere in between these values (approximately 25% of the the recommendation is to link the patient to a gene activity score of 0.5. *13) Phenotyping: At the moment, the determination of the DPD enzyme activity in peripheral mononuclear blood cells appears to be the most reliable phenotyping method. In other words, the phenotype determined using this method correlates most accurately to the maximum dose tolerated by the patient. The average Caucasian DPD activity determined using this method is 9.9 ± 0.95 nmol/hour per mg protein [Kuilenburg, 2000]. 5

6 Table 4. Relationship between genotyping result and predicted phenotype for 3 or more different s that result in reduced DPD activity Genotyping result Explanation General As patients with 3 or more different s are rare, only a general explanation is provided here about the determination of the predicted phenotype should it occur. If it does occur, it is important to examine how the variants that have been found can be distributed over the two alleles and whether this results in different predicted phenotypes. As there are only two validated s that result in an allele activity, an allele with 3 or more s will always contain a fully gene variant and therefore always has a gene activity score of 0. The gene activity scores for alleles with 0, 1 or 2 s are listed in tables 2 and 3. If all the potential distributions of the s over the alleles result in the same gene activity score for the genotype (i.e. the sum of the scores for the alleles), this is the gene activity score for the patient. If different distributions result in genotypes with different gene activity scores, determine which genotype the patient has (refer to table 3 for more information). Phenotyping The process of used to determine the phenotype, which means: measuring or estimating the activity of the DPD enzyme. Possible phenotyping methods are analysing DPD enzyme activity in peripheral mononuclear blood cells or measuring uracil concentration in plasma or urine [Ploylearmsaeng, 2006]. The average Caucasian DPD enzyme activity is 9.9 ± 0.95 nmol/hour per mg protein [Kuilenburg, 2000]. Methods that are less commonly used nowadays are the 2-13 C-uracil breath test [Mattison, 2004] or analysis of plasma uracil/dihydrouracil ratios [Ciccolini, 2006; Zhou, 2007]. The first test measures 13 CO2 formation following breakdown of 2-13 C-uracil mediated by DPD and other enzymes in the pyrimidine catabolic route. The second test measures conversion of the endogenous uracil substrate. The activity of the DPD enzyme can also be determined by analysing conversion of a single dose of uracil [van Staveren, 2013]. All existing phenotyping methods have limited applicability for either accuracy or sensitivity reasons or because of the feasibility of screening large numbers of patients (costs and time commitment). Ethnic variation in prevalence of phenotypes and allele frequency In the Caucasian population, approximately 3-5% have partial DPD deficiency and % have complete DPD deficiency. Approximately 8% of the African American population have partial DPD deficiency. The frequency of occurrence of the various DPD alleles and the different phenotypes appears to vary significantly between nations and ethnic groups. The 1236A allele and the *2A allele are the most common cause of DPD deficiency in the population in the Netherlands, accounting for a 25% and 50% reduction in DPD activity respectively. Allele frequencies and phenotypes per population are given in table 5. Table 5. Ethnic variation in prevalence of phenotypes and allele frequency Prevalence of phenotype Allele frequency (%) (%) Ethnicity Country PM # IM # EM # *2A *13 A>T G>A Caucasian Asian African- American The Netherlands South West Asia East Asia

7 # : PM (poor metaboliser), IM (intermediate metaboliser) and EM (extensive metaboliser) in the table above mean the following: PM: 0 fully functional alleles (gene activity -1) IM: 1 fully functional allele (gene activity score 1-1.5) EM: 2 fully functional alleles (gene activity score 2) Literature - Mattison LK et al. Rapid identification of dihydropyrimidine dehydrogenase deficiency by using a novel 2-13C-uracil breath test. Clin Cancer Res 2004;10: Ciccolini J et al. A rapid and inexpensive method for anticipating severe toxicity to fluorouracil and fluorouracil-based chemotherapy. Ther Drug Monit 2006;28: Ploylearmsaeng SA et al. How may anticancer chemotherapy with fluorouracil be individualised? Clin Pharmacokinet. 2006;45(6): Van Kuilenburg AB et al. Pitfalls in the diagnosis of patients with a partial dihydropyrimidine dehydrogenase deficiency. Clin Chem 2000;46: Zhou ZW et al. The dihydrouracil/uracil ratios in plasma and toxicities of 5-fluorouracil-based adjuvant chemotherapy in colorectal cancer patients. Chemotherapy 2007;53: van Staveren MC et al. Evaluation of predictive tests for screening for dihydropyrimidine dehydrogenase deficiency. Pharmacogenomics J 2013;13: Tuchman M et al. Familial pyrimidinemia and pyrimidinuria associated with severe fluorouracil toxicity. N Engl J Med 1985;313: Mattison LK et al. Increased prevalence of dihydropyrimidine dehydrogenase deficiency in African- Americans compared with Caucasians. Clin Cancer Res 2006;12: Van Kuilenburg ABP et al. Lethal outcome of patient with complete dihydropyrimidine dehydrogenase (DPD) deficiency after administration of 5-fluorouracil: frequency of the common IVS14 + 1G > A mutation causing DPD deficiency. Clin Cancer Res 2001;7: Sulzyc-Bielicka V et al. 5-Fluorouracil toxicity-attributable IVS14 + 1G > A mutation of the dihydropyrimidine dehydrogenase gene in Polish colorectal cancer patients. Pharmacol Rep 2008;60: Morel A et al. Clinical relevance of different dihydropyrimidine dehydrogenase gene single nucleotide polymorphisms on 5-fluorouracil tolerance. Mol Cancer Ther 2006;5: Seck K et al. Analysis of the DPYD gene implicated in 5-fluorouracil catabolism in a cohort of Caucasian individuals. Clin Cancer Res Aug 15;11(16): Yamaguchi K et al. Germline mutation of dihydropyrimidine dehydrogenese gene among a Japanese population in relation to toxicity to 5-fluorouracil. Jpn J Cancer Res 2001;92: Deenen MJ et al. Standard-dose tegafur combined with uracil is not safe treatment after severe toxicity from 5-fluorouracil or capecitabine. Ann Intern Med 2010;153: Rosmarin D et al. A candidate gene study of capecitabine-related toxicity in colorectal cancer identifies new toxicity variants at DPYD and a putative role for ENOSF1 rather than TYMS. Gut 2015;64: Deenen MJ et al. Relationship between single nucleotide polymorphisms and haplotypes in DPYD and toxicity and efficacy of capecitabine in advanced colorectal cancer. Clin Cancer Res 2011;17: Kristensen MH et al. Variants in the dihydropyrimidine dehydrogenase, methylenetetrahydrofolate reductase and thymidylate synthase genes predict early toxicity of 5-fluorouracil in colorectal cancer patients. J Int Med Res 2010;38: Lunenburg CA et al. Evaluation of clinical implementation of prospective DPYD genotyping in 5-fluorouracilor capecitabine-treated patients. Pharmacogenomics 2016;17: Henricks LM et al. Capecitabine-based treatment of a patient with a novel DPYD genotype and complete dihydropyrimidine dehydrogenase deficiency. Int J Cancer 2017 Sep 20 [Epub ahead of print]. 7