Reduced Dihydropyrimidine Dehydrogenase Activity Research Articles

Severe fluoropyrimidine toxicity due to novel and rare DPYD missense mutations, deletion and genomic amplification affecting DPD activity and mRNA splicing

Dihydropyrimidine dehydrogenase (DPD) is the initial and rate-limiting enzyme in the catabolism of 5-fluorouracil (5FU). Genetic variations in DPD have emerged as predictive risk factors for severe fluoropyrimidine toxicity. Here, we report novel and rare genetic variants underlying DPD deficiency in 9 cancer patients presenting with severe fluoropyrimidine-associated toxicity. All patients possessed a strongly reduced DPD activity, ranging from 9 to 53% of controls. Analysis of the DPD gene (DPYD) showed the presence of 21 variable sites including 4 novel and 4 very rare aberrations: 3 missense mutations, 2 splice-site mutations, 1 intronic mutation, a deletion of 21 nucleotides and a genomic amplification of exons 9–12. Two novel/rare variants (c.2843T>C, c.321+1G>A) were present in multiple, unrelated patients. Functional analysis of recombinantly-expressed DPD mutants carrying the p.I948T and p.G284V mutation showed residual DPD activities of 30% and 0.5%, respectively. Analysis of a DPD homology model indicated that the p.I948T and p.G284V mutations may affect electron transfer and the binding of FAD, respectively. cDNA analysis showed that the c.321+1G>A mutation in DPYD leads to skipping of exon 4 immediately upstream of the mutated splice-donor site in the process of DPD pre-mRNA splicing. A lethal toxicity in two DPD patients suggests that fluoropyrimidines combined with other therapies such as radiotherapy might be particularly toxic for DPD deficient patients. Our study advocates a more comprehensive genotyping approach combined with phenotyping strategies for upfront screening for DPD deficiency to ensure the safe administration of fluoropyrimidines.

Phenotypic and clinical implications of variants in the dihydropyrimidine dehydrogenase gene

Dihydropyrimidine dehydrogenase (DPD) is the initial and rate-limiting enzyme in the catabolism of the pyrimidine bases uracil, thymine and the antineoplastic agent 5-fluorouracil. Genetic variations in the gene encoding DPD (DPYD) have emerged as predictive risk alleles for 5FU-associated toxicity. Here we report an in-depth analysis of genetic variants in DPYD and their consequences for DPD activity and pyrimidine metabolites in 100 Dutch healthy volunteers. 34 SNPs were detected in DPYD and 15 SNPs were associated with altered plasma concentrations of pyrimidine metabolites. DPD activity was significantly associated with the plasma concentrations of uracil, the presence of a specific DPYD mutation (c.1905+1G>A) and the combined presence of three risk variants in DPYD (c.1905+1G>A, c.1129-5923C>G, c.2846A>T), but not with an altered uracil/dihydrouracil (U/UH2) ratio. Various haplotypes were associated with different DPD activities (haplotype D3, a decreased DPD activity; haplotype F2, an increased DPD activity). Functional analysis of eight recombinant mutant DPD enzymes showed a reduced DPD activity, ranging from 35% to 84% of the wild-type enzyme. Analysis of a DPD homology model indicated that the structural effect of the novel p.G401R mutation is most likely minor. The clinical relevance of the p.D949V mutation was demonstrated in a cancer patient heterozygous for the c.2846A>T mutation and a novel nonsense mutation c.1681C>T (p.R561X), experiencing severe grade IV toxicity. Our studies showed that the endogenous levels of uracil and the U/UH2 ratio are poor predictors of an impaired DPD activity. Loading studies with uracil to identify patients with a DPD deficiency warrants further investigation.

Polymorphisms in MIR27A Associated with Early-Onset Toxicity in Fluoropyrimidine-Based Chemotherapy.

The microRNA miR-27a was recently shown to directly regulate dihydropyrimidine dehydrogenase (DPD), the key enzyme in fluoropyrimidine catabolism. A common polymorphism (rs895819A>G) in the miR-27a genomic region (MIR27A) was associated with reduced DPD activity in healthy volunteers, but the clinical relevance of this effect is still unknown. Here, we assessed the association of MIR27A germline variants with early-onset fluoropyrimidine toxicity. MIR27A was sequenced in 514 patients with cancer receiving fluoropyrimidine-based chemotherapy. Associations of MIR27A polymorphisms with early-onset (cycles 1-2) fluoropyrimidine toxicity were assessed in the context of known risk variants in the DPD gene (DPYD) and additional covariates associated with toxicity. The association of rs895819A>G with early-onset fluoropyrimidine toxicity was strongly dependent on DPYD risk variant carrier status (Pinteraction = 0.0025). In patients carrying DPYD risk variants, rs895819G was associated with a strongly increased toxicity risk [OR, 7.6; 95% confidence interval (CI), 1.7-34.7; P = 0.0085]. Overall, 71% (12/17) of patients who carried both rs895819G and a DPYD risk variant experienced severe toxicity. In patients without DPYD risk variants, rs895819G was associated with a modest decrease in toxicity risk (OR, 0.62; 95% CI, 0.43-0.9; P = 0.012). These results indicate that miR-27a and rs895819A>G may be clinically relevant for further toxicity risk stratification in carriers of DPYD risk variants. Our data suggest that direct suppression of DPD by miR-27a is primarily relevant in the context of fluoropyrimidine toxicity in patients with reduced DPD activity. However, miR-27a regulation of additional targets may outweigh its effect on DPD in patients without DPYD risk variants.

408P - Dpyd Genotyping As a Predictive Biomarker of Toxicity in Breast Cancer

ABSTRACT Aim: Capecitabine is an orally bioavailable pro-drug of 5-fluorouracil (5FU) used for the treatment of metastatic breast cancer (m-BC). Dihydropyrimidine dehydrogenase (DPD) is the rate-limiting enzyme in the catabolism of 5FU and eliminates more than 80% of the administered drug. DPD deficiency is an autosomal recessive disorder associated with significant morbidity and mortality in those with complete or relative deficiency (incidence 0.1% and 5% respectively). Our aim was to determine whether DPD testing could be used as a biomarker of toxicity. Methods: Consecutive patients with m-BC who received capecitabine in our institution from December 2012 to December 2013 were identified. Records were reviewed for 1) admission to hospital, 2) cost-efficiency analysis of DPD testing and 3) capecitabine-associated toxicities. For all our outcomes we used descriptive analysis. DPD testing was done according to our institution's guidelines with the genotyping of four DPYD sequence variants associated with reduced DPD activity. Patients were divided into two groups (A) those who had DPD deficiency and (B) those who did not or who were not tested. Results: Forty-eight patients received capecitabine, F:M (46:2), mean age 57 years (range, 35-83). Two patients in each group were admitted to hospital: Group A for 17 and 52 days, Group B for 2 and 5 days. Cost of hospitalization for patients in group A was 9 times that of group B. The cost of DPD testing is Conclusions: This analysis suggests that testing for DPD deficiency could be a potentially useful predictive biomarker of toxicity in patients with m-BC planned to receive capecitabine. Those with a genetic variant could either avoid capecitabine or start at a reduced dose. We plan to prospectively validate these results by implementing DPD testing for a period of one year in this patient group. Disclosure: All authors have declared no conflicts of interest.

Frequency of the DPYD * 2A allele in the Czech population

The pyrimidine analogue 5-fluorouracil (5-FU) is widely used for chemotherapy of many solid tumors, such as colorectal, breast, head/neck, ovarian and skin cancer. More than 80% of administered 5-FU is rapidly detoxified in the liver via a multistep metabolic pathway, involving dihydropyrimidine dehydrogenase (DPD) as the initial and rate-limiting enzyme. Reduced DPD activity can lead to the accumulation of active 5-FU metabolite (FdUMP), which leads to 5-FU toxicity. Individuals with low DPD activity (about 3-5% of patients) cannot effectively inactivate 5-FU and will develop severe to lethal hematological, gastrointestinal or neurological toxicities. Pharmacogenetic variation may contribute to risk of toxicity and/or altered therapeutic benefit. Enzyme DPD has a key function for 5-FU metabolism (degradation and inactivation) and is encoded by DPYD gene. In the majority of cases, a G to A mutation in the splicing recognition sequence of intron 14 (IVS14+1G>A) of the DPD-encoding gene DPYD was shown to be responsible. The mutant allele, designated DPYD*2A, produces a nonfunctional enzyme due to skipping of exon 14. Frequence of DPYD*2A allele in caucasion population is 0,91% and in the deficite of DPD is 52%. It was recommended that screening for DPYD*2A should be routinely carried out prior to start of 5-FU therapy, and that heterozygotes should receive limited dose of 5-FU only, while homozygotes, who are at high risk to develop severe complications, should be treated with alternative therapeutic drugs. 212 males and 210 females (the age of tested patients was 18-69 years) of the Czech population were tested. The material of DNA isolation was obtained oral mucosa by Flocked Swabs or by taking incoagulable peripheral venous blood. DNA was isolated by using QIAmp DNA Minikit or by the semi-automatic isolation DNA QuickGene 810. Mutation IVS14+1 G>A DPYD gene (DPYD * 2 allele) was tested by using certificated method strip assay (PGX-5FU) and by new created method High-Resolution Melting (HRM) (Figure​Figure1).1). Were compared to results of both methods with each other and assessed allele frequency in the Czech population. Figure 1 Analysis of mutation IVS14+1 G>A DPYD gene by methods DPYD * 2A allele was demonstrated in a heterozygous state in 3/422 (0.7%), in the homozygous state was not found. Czech frequency of DPYP * 2A allele 0.36% is 2.5 times lower than the reported frequency 0.91% in the European population. The results of both methods were completely identical. New cheaper method for detection DPYD * 2A was introduced and validated. Low frequency of DPYD * 2A allele in the Czech population assumes the presence of other mutations in the DPYD gene and in other genes affecting the metabolism of 5-FU (eg . TYMS, MTHFR). Genetic test is recommended prior to cancer treatment by 5-FU, because are suitable the lower doses in the heterozygotes and choose other chemotherapy for the homozygotes with risk of toxic reaction.

Dihydropyrimidine Dehydrogenase Gene as a Major Predictor of Severe 5-Fluorouracil Toxicity

The importance of polymorphisms in the dihydropyrimidine dehydrogenase (DPD) gene (DPYD) for the prediction of severe toxicity in 5-fluorouracil (5-FU) based chemotherapy has been controversially debated. As a key enzyme in the catabolism of 5-FU, DPD is the top candidate for pharmacogenetic studies on 5-FU toxicity, since a reduced DPD activity is thought to result in an increased half-life of the drug, and thus, an increased risk of toxicity. Here, we review the current knowledge on well-known and frequently studied DPYD variants such as the c.1905+1G>A splice site variant, as well as the recent discoveries of important functional variation in the noncoding regions of DPYD. We also outline future directions that are needed to further improve the risk assessment of 5-FU toxicity, in particular with respect to metabolic profiling and in the context of different combination therapeutic regimens, in which 5-FU is used today.

Investigation of Ivs14+1G>A Polymorphism of Dpyd Gene in a Group of Bosnian Patients Treated with 5-Fluorouracil and Capecitabine

Adverse drug reactions still pose an important clinical problem. Dihydropyrimidine dehydrogenase (DPD) is an enzyme that regulates 5-FU quantities available for anabolic processes and hence affects its pharmacokinetics, toxicity and efficacy. There are several studies describing a hereditary (pharmacogenetic) disorder in which individuals with absent or significantly reduced DPD activity may even develop a life-threatening toxicity following exposure to 5-FU. The most common mutation is known as the DPYD*2A or as the splice-site mutation (IVS14 + 1G A) leading to creation of a dysfunctional protein. An objective behind the study was to ascertain existence of the IVS14 + 1G A mutation among the population of Bosnia and Herzegovina. Our research has undeniably attested to existence of one heterozygote for the DPYD gene mutation, i.e. one heterozygote for IVS14 + 1 G > A, DPYD*2A mutation.

Pharmacokinetics, a main actor in a many‐sided approach to severe 5‐FU toxicity prediction

Pharmacokinetics, a main actor in a many‐sided approach to severe 5‐FU toxicity prediction

Increased Prevalence of Dihydropyrimidine Dehydrogenase Deficiency in African-Americans Compared with Caucasians

African-American patients with colorectal cancer were observed to have increased 5-fluorouracil (5-FU)-associated toxicity (leukopenia and anemia) and decreased overall survival compared with Caucasian patients. One potential source for this disparity may be differences in 5-FU metabolism. Dihydropyrimidine dehydrogenase (DPD), the initial and rate-limiting enzyme of 5-FU catabolism, has previously been shown to have significant interpatient variability in activity. Several studies have linked reduced DPD activity to the development of 5-FU toxicity. Although the distribution of DPD enzyme activity and the frequency of DPD deficiency have been well characterized in the Caucasian population, the distribution of DPD enzyme activity and the frequency of DPD deficiency in the African-American population are unknown. Healthy African-American (n=149) and Caucasian (n=109) volunteers were evaluated for DPD deficiency using both the [2-(13)C]uracil breath test and peripheral blood mononuclear cell DPD radioassay. African-Americans showed significantly reduced peripheral blood mononuclear cell DPD enzyme activity compared with Caucasians (0.26+/-0.07 and 0.29+/-0.07 nmol/min/mg, respectively; P=0.002). The prevalence of DPD deficiency was 3-fold higher in African-Americans compared with Caucasians (8.0% and 2.8%, respectively; P=0.07). African-American women showed the highest prevalence of DPD deficiency compared with African-American men, Caucasian women, and Caucasian men (12.3%, 4.0%, 3.5%, and 1.9%, respectively). These results indicate that African-Americans, particularly African-American women, have significantly reduced DPD enzyme activity compared with Caucasians, which may predispose this population to more 5-FU toxicity.

Dihydropyrimidine dehydrogenase deficiency in an Indian population

Dihydropyrimidine dehydrogenase (DPD) deficiency is prevalent in 3-5% of the Caucasian population; however, the frequency of this pharmacogenetic syndrome in the Indian population and other racial and ethnic groups remains to be elucidated. We describe an Indian patient who presented to clinic for the treatment of gastric adenocarcinoma with 5-flurouracil (5-FU) therapy who subsequently was diagnosed with DPD deficiency by using the peripheral blood mononuclear cell (PBMC) DPD radioassay. This observation prompted us to examine the data generated from healthy (cancer-free) Indian subjects who were enrolled in a large population study to determine the sensitivity and specificity of the uracil breath test (UraBT) in the detection of DPD deficiency. Thirteen Indian subjects performed the UraBT. UraBT results were confirmed by PBMC DPD radioassay. The Indian cancer patient demonstrated reduced DPD activity (0.11 nmol/min/mg protein) and severe 5-FU toxicities commonly associated with DPD deficiency. Of the 13 Indian subjects [ten men and three women; mean age, 26 years (range: 21-31 years)] enrolled in the UraBT, 12 Indian subjects demonstrated UraBT breath profiles and PBMC DPD activity within the normal range; one Indian subject demonstrated a reduced breath profile and partial DPD deficiency. DPD deficiency is a pharmacogenetic syndrome which is also present in the Indian population. If undiagnosed, the DPD deficiency can lead to death. Future epidemiological studies would be helpful to determine the prevalence of DPD deficiency among racial and ethnic groups, allowing for the optimization of 5-FU chemotherapy.

Known variant DPYD alleles do not explain DPD deficiency in cancer patients

Dihydropyrimidine dehydrogenase (DPD) degrades over 80% of administered 5-fluorouracil (5FU), thereby regulating the efficacy of this commonly used anticancer agent. DPD activity is highly variable (8-21-fold) and individuals with reduced activity have a high risk of 5FU toxicity. DPYD encodes DPD protein and 13 different mutations have been reported in DPD-deficient subjects. However, the contribution of these variant genotypes to polymorphic DPD activity in vivo is not clear. The previously described DPYD mutations are contained in 10 exons. These 10 exons were sequenced in a cohort of cancer patients with reduced (n = 23) or normal (n = 14) DPD activity to determine the contribution of each variant allele to low DPD activity in vivo. Eight of the 13 previously defined DPYD mutations (G62A, delta TCAT295-298, C703T, G1003T, G1156T, delta C1897, G2657A, and G2983T) were not detected. A previously defined exon 13 mutation (G1601A) was detected in three individuals with reduced DPD activity. An exon 14 splice donor site mutation (intron14 G1A) was detected in a normal DPD activity individual. It was demonstrated that T85C, A1627G and G2194A are common polymorphisms. A novel exonic mutation (T1679G) was detected in a patient with reduced DPD activity and 5FU toxicity. In addition, three novel common polymorphisms were detected in introns 10 and 13. Only three patients did not have any mutations and 30 had multiple DPYD mutations in the regions examined. However, only 17% (4/23) of the patients with a low DPD phenotype have a molecular basis for reduced activity. Although novel DPYD variants have been identified in this study, the 17 DPYD mutations now described do not entirely explain polymorphic DPD activity and toxic response to 5FU. These data emphasize the complex nature of the molecular mechanisms controlling polymorphic DPD activity in vivo.

Cloning and Initial Characterization of the Human DPYD Gene Promoter

Dihydropyrimidine dehydrogenase (DPD) is the rate-limiting enzyme in the degradation of pyrimidine bases and pyrimidine-based antimetabolites. Reduced DPD activity is associated with toxicity to 5-fluorouracil (5FU) therapy in cancer patients and with neurological abnormalities in paediatric patients. Although variant DPYD alleles have been identified in DPD-deficient patients, they do not adequately explain polymorphic DPD activity or associated clinical phenotypes in vivo. DPD may be transcriptionally regulated as mRNA levels correlate with activity and are differentially regulated in human tissues. A 1.85 kb 5′ flanking region of the human DPYD gene was cloned and has transcriptional activity in cultured cells. Analysis of this 5′ flanking region in rhesus and cynomolgus monkeys demonstrated conservation (>96%) between humans and primates. Putative binding sites for ubiquitous and cell-specific factors were identified. A polymorphism that disrupts a putative γ-interferon response element was identified in a cancer patient with reduced DPD activity and severe 5FU toxicity. Further insight into regulation of DPD expression may identify new avenues for the treatment of clinical problems associated with DPD deficiency.