A familial approach was used to elucidate the genetic determinants of profound and partial dihydropyrimidine dehydrogenase (DPD; EC 1.3.1.2) deficiency in an Alabama family. In 1988, our laboratory diagnosed profound DPD deficiency in a breast cancer patient with grade IV toxicity after cyclophosphamide/methotrexate/5-fluorouracil chemotherapy (R. B. Diasio et al., J. Clin. Investig., 81: 47–51, 1988). We now report the genetic analysis of archived genomic DNA that reveals that the proband was a compound heterozygote for two different mutations, one in each allele: (a) a G to A mutation in the GT 5′ splicing recognition sequence of intron 14, which results in a 165-bp deletion (corresponding to exon 14) in the DPD mRNA (DPYD*2A); and (b) a T1679G mutation (now designated DPYD*13), which results in a I560S substitution. Sequence analysis revealed segregation of both mutations with the son and the daughter each inheriting one mutation. Phenotype analysis (DPD enzyme activity) confirmed that both children were partially DPD deficient. Plasma uracil and DPD mRNA levels were found to be within normal limits in both children. We conclude that profound DPD deficiency in the proband resulted from a combination of two mutations (one mutation in each allele) and that heterozygosity for either mutation results in partial DPD deficiency. Lastly, we identified two variant alleles reported previously as being associated with DPD enzyme deficiency [T85C resulting in a C29R substitution (DPYD*9A) and A496G (M166V) in a family member with normal DPD enzyme activity]. These data suggest that both variant alleles are unrelated to DPD deficiency and emphasize the need to perform detailed familial genotypic and phenotypic analysis while characterizing this pharmacogenetic syndrome.

The antimetabolite 5-FU3 has been in clinical use for ∼45 years and has evolved as an important agent in the treatment of a large spectrum of tumors, including gastrointestinal, breast, and head and neck (1). Over the past 20 years, the pyrimidine catabolic pathway, in particular the first enzymatic step involving DPD, has been recognized as critical in determining the ultimate metabolism and, in turn, the pharmacology of 5-FU (2). Although the antitumor action (and host toxicity) of 5-FU depends on anabolism of the drug to cytotoxic nucleotides, studies from our laboratory have shown that 80–90% of an administered dose of 5-FU is rapidly converted into biologically inactive metabolites through the catabolic pathway (3). Increased DPD activity (with corresponding increased catabolism of 5-FU) has been correlated with resistance to 5-FU (4, 5). Conversely, decreased DPD activity (with corresponding decreased catabolism of 5-FU) has been shown to increase 5-FU half-life, thus increasing the amount of drug available for the anabolic (cytotoxic) pathway (6). Taken together, these data suggest that the variability of DPD activity in the normal population may account for observed differences in the pharmacokinetics and oral bioavailability of 5-FU (7).

The importance of DPD in 5-FU toxicity has been illustrated by patients with DPD enzyme deficiency. After administration of standard doses of 5-FU, these patients develop profound toxicity including mucositis, granulocytopenia, neuropathy, and (in some cases) death (8, 9, 10, 11). In 1988, our laboratory reported one of the first profoundly DPD-deficient patients who developed grade IV toxicity after CMF chemotherapy (8). The familial pedigree of this patient revealed that the proband’s son and daughter were partially DPD deficient (8). Later population studies in breast cancer patients have demonstrated that ∼5% are DPD deficient, with enzyme activity below the 95th percentile of a control population (12).

Since being identified as a pharmacogenetic disorder, there has been a steady increase each year in the number of case reports of DPD deficiency with severe toxicity secondary to treatment with 5-FU (13, 14). These reports, combined with the recent introduction of new generation fluoropyrimidine-based chemotherapy agents (e.g., capecitabine) have resulted in continued research to understand the molecular basis of DPD deficiency with the purification and characterization of the human protein (15), cloning and characterization of the DPYD gene (16) and the promoter (17), and the identification of 19 variant alleles in DPD-deficient patients (18, 19, 20).

This report describes the first compound heterozygote genotype in a profoundly DPD-deficient patient with segregation of mutations among immediate family members. The mutations responsible for profound DPD deficiency in this patient and partial DPD deficiency in the proband’s two children are identified, and the pattern of inheritance is elucidated. This comprehensive familial approach which uses both phenotype and genotype analysis should be used in future studies to determine which mutations in the DPYD gene result in DPD deficiency.

Proband and Immediate Family Members.

In 1988, a previously healthy 40-year-old white female began treatment for infiltrating ductal carcinoma with a left-modified radical mastectomy and adjuvant CMF chemotherapy. The complete toxicity profile was described previously in detail (8). Briefly, 11 days after the first cycle of CMF chemotherapy, the patient was found to be neutropenic (white blood count of 1,400 with 1% neutrophils and 1% bands). Twenty six days after cycle 1, the second cycle was administered at reduced doses. Fourteen days later, the patient returned to the clinic with a white blood count of 1,300. The patient received the third cycle on schedule but at further reduced doses. Fourteen days later, the patient was hospitalized with fever, neutropenia, and a 2-day history of ataxia. The patient continued to deteriorate neurologically over the next 7 days, eventually becoming completely unresponsive. Over the succeeding 2 months, she regained all function and demonstrated no neurological deficits. Approximately 1 year later, the patient died from progression of disease. Although the cDNA and the structure of the human DPYD gene was not known in 1988, genomic DNA was prepared from the proband’s PBM cells for future analysis.

Phenotypic analysis revealed that the proband was profoundly DPD deficient (no detectable enzyme activity). Pharmacokinetic analysis of the proband demonstrated altered 5-FU metabolism characterized by prolonged half-life (150 versus 13 min for normal controls) and decreased clearance (70 versus 594 ml/min/m2 for normal controls; Ref. 8). Phenotypic analysis of the proband’s children revealed that both her son and daughter were partially DPD deficient. Both children were otherwise healthy without any characteristic phenotype.

Determination of DPD Activity.

DPD enzyme activity was originally determined in the proband, her father, two children, and available extended family members from PBM cells as described (8). In the present study, DPD enzyme activity was reevaluated in the PBM cells of the available surviving family members (the proband’s husband, two children, and three grandchildren) and controls using a recently described semiautomated radioassay for DPD enzyme activity (21). Both the proband and her father (tested in 1988) have died since publication of the original study. The proband’s nephew (identified as partially DPD deficient in 1988) was unavailable for participation in this study. Plasma uracil concentrations were also measured (in surviving family members and controls where DPD enzyme activity was obtained) as described previously (8, 9).

Quantitation of DPD mRNA.

The theoretical basis and validation of quantitating DPD mRNA using the ABI PRISM 7700 Sequence Detection System (Applied Biosystems, Foster City, CA) is described in detail elsewhere (22). Briefly, total RNA was isolated from PBM cells of the proband’s husband, two children, and three grandchildren, along with controls using RNAzol (Biotecx, Houston, TX), following the manufacturer’s instructions. Total RNA was diluted to a final concentration of 5 ng/μl in water containing 12.5 μg/ml total yeast RNA as a carrier. All samples were stored at −70°C until analysis. Final DPD forward, reverse, and probe concentrations were 100, 200, and 100 nm, respectively. Thermal cycling conditions were 30 min at 48°C, followed by 10 min at 95°C and 40 cycles of 15 s at 95°C and 1 min at 60°C. The absolute standard curve method was used to determine the copy number of DPD mRNA (22).

Genotypic Analysis.

In the present study, genomic DNA from the proband’s husband, two children, and three grandchildren was prepared from PBM cells using the Easy-DNA kit (Invitrogen, San Diego, CA), following the manufacturer’s instructions. Archived genomic DNA obtained from the proband in 1988 was maintained at −70°C until analysis. All 23 exons along with the flanking intronic regions of the DPYD gene were PCR amplified under conditions similar to those described previously by our laboratory (9). Amplification of exon 1 (along with 427 bp of the promoter region of the DPYD gene) was carried out with 1% DMSO (17). The primers used to amplify each exon are listed in Table 1. After amplification, PCR products were resolved on 2% agarose gels and purified using a Qiaquick Gel Extraction kit (Qiagen, Valencia, CA) according to the manufacturer’s instructions. Samples were sequenced on an ABI 310 automated DNA sequencer using the dideoxynucleotide chain termination method (Applied Biosystems, Foster City, CA). Sequence reactions were repeated three times in each direction (using the same primers that were used to amplify each specific PCR amplicon) and analyzed using MacVector 4.1 sequence analysis software (IBI, New Haven, CT).

DPD Enzyme Activity in Proband and Immediate Family Members.

Although the proband could not be retested for DPD enzyme activity, several separate determinations in the original study demonstrated no detectable enzyme activity in the PBM cells (8). In the present study, we examined the DPD enzyme activity in the proband’s husband, two children, and three grandchildren. These data are summarized in Fig. 1 and Table 2. The proband’s husband (II-6) demonstrated DPD enzyme activity within the normal range. However, both the daughter (III-6) and son (III-7) demonstrated partial DPD deficiency, as reported originally (8). The proband’s three grandchildren (IV-1, IV-2, and IV-3), along with seven unrelated controls, demonstrated normal DPD enzyme activity (Fig. 1 and Table 2). The proband’s father (I-1) and nephew (III-2) are illustrated as partially DPD deficient in Fig. 1. These data were obtained from the original examination of this family (8) because the proband’s father is now deceased and the nephew was unavailable for participation in this study.

Plasma Uracil Levels.

Examination of the proband’s plasma in 1988 demonstrated elevated (1500 ng/ml) uracil levels (8). In the present study, the plasma uracil levels in the proband’s husband (II-6), two children (III-6 and III-7), and three grandchildren (IV-1, IV-2, and IV-3) were examined and found to be in the normal range (Table 2).

Quantitation of DPD mRNA.

DPD mRNA levels were quantitated by real-time quantitative PCR in the PBM cells of the proband’s husband (II-6), two children (III-6 and III-7), and three grandchildren (IV-1, IV-2, and IV-3) and are summarized in Table 2. No archived RNA was available from the proband (II-5) or her father (I-1). DPD mRNA levels were also quantitated in the PBM cells of seven unrelated control individuals (Table 2) with normal DPD enzyme activity. DPD enzyme activity correlated (R2 = 0.98) with mRNA levels in family members with normal enzyme activity but not in the partially DPD-deficient children (III-6 and II-7; Fig. 2).

Genomic DNA Analysis.

Because only archived genomic DNA was available from the proband, DPYD gene-specific primers (located in bordering intronic regions and listed in Table 1) were used to amplify the 23 exons (along with intron/exon splice junctions) in the proband’s DPYD gene. Sequence analysis of purified PCR amplicons showed that the proband contained two heterozygote mutations: (a) a G to A mutation in the GT 5′ splicing recognition sequence of intron 14, which results in a 165-bp deletion (corresponding to exon 14) in the DPD mRNA (DPYD*2A); and (b) a T1679G mutation (designated DPYD*13), which results in a I560S substitution. These data are summarized in the family’s pedigree (Fig. 1) and in Table 2. There were no other mutations or polymorphisms detected in the proband’s DPYD gene.

The proband’s husband (II-6, and father to both children) demonstrated neither the DPYD*13 nor DPYD*2A mutation in his DPYD gene. However, complete sequence analysis of the father revealed that he was a heterozygote for both the T85C (C29R, DPYD*9A) and the A496G (M166V) mutations (as shown in Fig. 1 and Table 2).

Sequence analysis of the proband’s two children revealed a segregation of mutations, with the daughter (III-6) demonstrating a heterozygote DPYD*13 T1679G (I560S) genotype and the son (III-7) demonstrating a heterozygote DPYD*2A (intron 14 G1A) genotype. The proband’s daughter (III-6) also demonstrated a heterozygote A496G (M166V) mutation. All three of the proband’s grandchildren (IV-1, IV-2, and IV-3) demonstrated a heterozygote genotype for the A496G (M166V) mutation (as summarized in Fig. 1 and Table 2). The locations of the mutations (within the DPYD gene) characterized in this study are shown in Fig. 3.

In the present study, we describe the genotypic characterization of one the first known profoundly DPD-deficient cancer patients. Surviving family members were analyzed to elucidate the pattern of inheritance and to determine the role of each mutation in DPD deficiency. Several earlier studies of DPD-deficient patients have reported homozygote mutations resulting in complete DPD deficiency (9, 11, 19, 20, 23, 24); however, this is the first report of a compound heterozygote genotype with segregation of mutations among immediate family members. These analyses demonstrate a codominant pattern of inheritance for this pharmacogenetic disease. In addition, two previously reported DPD mutations [T85C (C29R, DPYD*9A) and A496G (M166V)] thought previously to result in DPD deficiency (19, 23, 24) are shown in this study to have no functional significance to DPD activity.

To determine the molecular basis for DPD deficiency in the proband, archived genomic DNA (isolated from the patient’s PBM cells in 1988) was used as a template, and all 23 exons (including the intron/exon junctions) were PCR amplified. Primers in the flanking introns were designed based on data from the characterization of the human DPYD gene (16), promoter (17), and recent studies examining variant DPYD alleles (25). Complete sequence analysis revealed two heterozygous mutations in the proband: (a) a G to A mutation in the GT 5′ splicing recognition sequence of intron 14 (DPYD*2A); and (b) a T1679G mutation (now designated DPYD*13), which results in a nonconserved I560S substitution. Complete sequence analysis demonstrated that these alleles represent the only sequence differences in this DPD-deficient patient. The DPYD*2A mutation remains the most characterized and frequently observed allele associated with DPD enzyme deficiency (9, 11, 25). However, the DPYD*13 missense mutation has been reported in only one other patient with reduced DPD enzyme activity (26).

The proband’s husband (and father to both partially DPD-deficient children) demonstrated neither the DPYD*2A nor the DPYD*13 mutation in his DPYD gene (as shown in Table 2). However, complete sequence analysis of the father revealed that he was heterozygote for both the DPYD*9A and the A496G (M166V) mutations. Both of these mutations have been reported previously to be associated with DPD deficiency (19, 23, 24). However, the father demonstrated normal DPD activity. Although the frequency of these alleles in the general population remains to be determined, their identification in an individual with normal enzyme activity suggests that both are nonfunctional polymorphisms. This conclusion is supported by a recently published study demonstrating normal DPD enzyme activity in individuals with the DPYD*9A polymorphism (26) and by the identification of the A496G (M166V) genotype in the proband’s three grandchildren with normal DPD enzyme activity (Fig. 1). These results (combined with the autosomal codominant pattern of inheritance demonstrated in the proband and her children, see below) confirm that neither of these mutations alters DPD enzyme activity.

Sequence analysis of the proband’s two children demonstrated segregation of both mutations (DPYD*2A and DPYD*13) identified in the proband. The son demonstrated a heterozygote DPYD*2A genotype whereas the daughter demonstrated a heterozygote DPYD*13 genotype. This unique segregation of mutations revealed that the proband was a compound heterozygote containing one variant allele on each DPYD gene. Furthermore, this genotype allowed the independent assessment of the effect of each mutation on DPD enzyme activity. As shown in Table 2, both children demonstrated partial DPD deficiency, thus demonstrating that both forms of the DPYD gene were expressed and that this pharmacogenetic syndrome follows a codominant pattern of inheritance, not autosomal recessive as originally thought (8).

The molecular basis for DPD deficiency is best understood in the DPYD*2A mutation. A previous study by our laboratory demonstrated that a homozygote DPYD*2A genotype results in a 165-bp deletion (corresponding to exon 14) in the DPD mRNA (9). Western blot analysis demonstrated that the aberrant DPD mRNA was translated into a nonfunctional polyubiquitinated DPD protein. Thus, enhanced proteolysis provides a hypothetical mechanism for loss of DPD catalytic activity, similar to what has been reported with human thiopurine S-methyltransferase deficiency (27). In contrast, the DPYD*13 mutation results in a single nonconserved I560S substitution. Although this substitution does not occur in any recognized functional domain, amino acids located in regions critical for enzyme structure and/or catalytic function would be expected to be conserved across species. A recent report examining the conservation of the DPD enzyme demonstrates that the I560 position is 100% conserved across all five mammalian species (human, mouse, rat, bovine, and pig) examined (28) and suggests that this position is important in maintaining DPD enzyme activity.

The phenotypic characterization of this family demonstrating partial DPD deficiency together with a known genotype prompted us to examine other types of analysis (plasma uracil and DPD mRNA levels) that may, in the future, be used to identify DPD deficiency before treatment with 5-FU. Analysis of the partially DPD-deficient individuals (the proband’s daughter and son) showed plasma uracil levels in the normal range (<50 ng/ml; Table 2). Elevated uracil levels were detected only in the proband, who demonstrated profound (no detectable enzyme activity) DPD deficiency. These analyses suggest that only profoundly, not partially, DPD-deficient individuals have elevated plasma uracil levels. This is particularly important because a recent study reports that most DPD-deficient patients in a population of patients who demonstrated unanticipated toxicity secondary to treatment with 5-FU are partially deficient (31% partial versus 12% profound deficiency; Ref. 10). These data illustrate the potential limitations of determining plasma uracil levels for the identification and characterization of partially DPD-deficient patients.

In addition to uracil levels, DPD mRNA levels were also evaluated (Fig. 2 and Table 2). DPD mRNA levels quantitated from the proband’s husband and three grandchildren (individuals in the family having normal DPD activity) correlated with enzyme activity (Fig. 2). These data agree with previous studies from our laboratory demonstrating a linear relationship between DPD mRNA and activity levels (22). The partially DPD-deficient family members (III-6 and III-7) also demonstrated DPD mRNA levels in the normal range, suggesting that neither mutation (DPYD*2A nor DPYD*13) affects DPD mRNA levels. This is not particularly surprising because neither mutation would directly interfere with RNA transcription, nor do they occur in regions known to affect RNA stability (29). Although several recent pharmacogenomic studies have used DPD mRNA as a surrogate marker for DPD enzyme activity to predict 5-FU efficacy (30, 31), the present studies suggest that quantitation of DPD mRNA cannot be used to identify DPD-deficient patients with this genotype. Taken collectively, these data indicate that phenotypic analysis of DPD enzyme activity remains the most reliable method for the identification and characterization of this pharmacogenetic syndrome.

In summary, comparative phenotypic and genotypic analysis of this family has allowed us to conclude that: (a) a new compound heterozygote genotype resulting in profound DPD deficiency has been identified; (b) DPD deficiency exhibits an autosomal codominant pattern of inheritance, not autosomal recessive as thought originally; (c) two mutations reported previously to be associated with DPD deficiency [T85C (C29R, DPYD*9A) and A496G (M166V)] have no functional significance on DPD enzyme activity. Further analysis suggests that neither uracil nor DPD mRNA levels can be used to predict partial DPD deficiency for the mutations examined in this study. This approach demonstrates the usefulness of familial genotypic and phenotypic analyses to determine the functional significance DPYD mutations.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1

Supported by USPHS Grant CA 62164.

3

The abbreviations used are: 5-FU, 5-fluorouracil; DPD, dihydropyrimidine dehydrogenase; CMF, cyclophosphamide/methotrexate/5-FU; PBM, peripheral blood mononuclear.

Fig. 1.

Pedigree of a family demonstrating both profound and partial DPD deficiency. Fully shaded symbols (•) represent complete (profound) DPD deficiency. Half-shaded symbols (, ╡) indicate partial DPD deficiency. Unshaded symbols (○, □) indicate normal DPD activity. Hatched symbols (, ) represent individuals not tested. Deceased family members are designated by a diagonal line in the symbol. The genotype for each family member tested is listed below each symbol. Consanguinous marriage is shown by the double line in the proband’s (II-5, indicated by a solid arrow) parents (I-1 and I-2). Phenotypic data for the proband’s parents (I-1 and I-2), brother (II-4), niece and nephew (III-1 and III-2) were extrapolated from Diasio et al.(8).

Fig. 1.

Pedigree of a family demonstrating both profound and partial DPD deficiency. Fully shaded symbols (•) represent complete (profound) DPD deficiency. Half-shaded symbols (, ╡) indicate partial DPD deficiency. Unshaded symbols (○, □) indicate normal DPD activity. Hatched symbols (, ) represent individuals not tested. Deceased family members are designated by a diagonal line in the symbol. The genotype for each family member tested is listed below each symbol. Consanguinous marriage is shown by the double line in the proband’s (II-5, indicated by a solid arrow) parents (I-1 and I-2). Phenotypic data for the proband’s parents (I-1 and I-2), brother (II-4), niece and nephew (III-1 and III-2) were extrapolated from Diasio et al.(8).

Close modal
Fig. 2.

Correlation between DPD enzyme activity and mRNA levels. DPD mRNA level (X axis) was plotted against the DPD enzyme activity (Y axis) and demonstrates a linear correlation coefficient of 0.986 in four family members [proband’s husband (II-6) and three grandchildren (IV-1, IV-2, and IV-3)] with normal enzyme activity (•). However, although both partially DPD-deficient family members (III-6 and III-7) demonstrate normal DPD mRNA levels, these levels do not correlate with enzyme activity (□).

Fig. 2.

Correlation between DPD enzyme activity and mRNA levels. DPD mRNA level (X axis) was plotted against the DPD enzyme activity (Y axis) and demonstrates a linear correlation coefficient of 0.986 in four family members [proband’s husband (II-6) and three grandchildren (IV-1, IV-2, and IV-3)] with normal enzyme activity (•). However, although both partially DPD-deficient family members (III-6 and III-7) demonstrate normal DPD mRNA levels, these levels do not correlate with enzyme activity (□).

Close modal
Fig. 3.

Location of mutations within the DPYD gene. A schematic map of the human DPD gene (with exons 1 to 23 represented by solid numbered boxes) is shown with the locations of the mutations characterized in this study. A heterozygote genotype for the DPYD*2A or DPYD*13 mutations was shown to result in decreased DPD enzyme activity.

Fig. 3.

Location of mutations within the DPYD gene. A schematic map of the human DPD gene (with exons 1 to 23 represented by solid numbered boxes) is shown with the locations of the mutations characterized in this study. A heterozygote genotype for the DPYD*2A or DPYD*13 mutations was shown to result in decreased DPD enzyme activity.

Close modal
Table 1

Oligonuclotides used to amplify genomic DNA

ExonDPD primers5′ Primer sequence3′ Amplicon (bp)
1a Forward 124 ACTTGGTGGATGGATGGAGGAACATCTAC 832 
 Reverse 130 TCCTGAAATCTCTTCCGAAGTAAACAG  
Forward 125 GTTTAAACAAATGCCAACATATTTCC 270 
 Reverse 131 TGTACTTTAATACCTTATTTCTAAGTG  
Forward 127 TTCTCAGGATCTTAGAGAATTAAGC 264 
 Reverse 133 TCTCTCCACTGACAAATTAATACC  
Forward 126 ACACGGACTCTGAATGAGTATAAGG 242 
 Reverse 132 CCACAGATAATAGAGAACGAAGATC  
5b Forward 132 GTTTGTCGTAATTTGGCTG 284 
 Reverse 138 ATTTGTGCATGGTGATGG  
Forward 130 CGGGCTGGTAAAACAAGAATTCG 485 
 Reverse 135 TATTGCTTCAAGCCACCTGCAAA  
7b Forward 133 GTCCTCATGCATATCTTGTGTG 360 
 Reverse 139 GCTACATCAGGCAGAAGC  
8b Forward 134 GCCCCACATCGTGCTATGAACA 459 
 Reverse 140 CCAGAATGACTGCCTTCAGAC  
9b Forward 135 CCCTCCTCCTGCTAATG 278 
 Reverse 141 CTCAGCAGCACATTGTTC  
10b Forward 136 GAGAGTGACACTTCATCCTGG 342 
 Reverse 142 GGAGTTGTACACCAACAG  
11b Forward 137 ACTGGTAACTGAAACTCAG 442 
 Reverse 143 CTAGCTTTCAGGGAATTG  
12b Forward 138 TTCCTGTATGTGAGGTGTA 453 
 Reverse 144 GAAGCACTTATCCATTGG  
13 Forward 128 GATGTAATATGAAACCAAGTATTGG 296 
 Reverse 134 ACGATAGACATTTCTATATGACTTC  
14 Forward 115 GTGAGAAGGACCTCATAAAATATTGTC 342 
 Reverse 92 GAATTGGATGTTTAAATAAACATTCACCAAC  
15b Forward 139 TATCTTTGTGTACAACTGGA 391 
 Reverse 145 TGTGAAATCCAAGGGACC  
16b Forward 140 AACGGTGAAAGCCTATTGG 223 
 Reverse 146 TAGTAACTATCCATACGGGGG  
17b Forward 141 CACGTCTCCAGCTTTGCTGTTG 238 
 Reverse 147 CGGGCAACTGATTCAAGTCAAG  
18b Forward 142 TGGGATGTGAGGGGGTGAATG 246 
 Reverse 148 TTCAGCAACCTCCAAGAAAGCCA  
19b Forward 143 TGTCCAGTGACGCTGTCATCAC 300 
 Reverse 149 CATTGCATTTGTGAGATGGAG  
20b Forward 144 GAGAAGTGAATTTGTTTGGAG 424 
 Reverse 150 CACAGACCCATCATATGGCTG  
21b Forward 145 TCTGACCTAACATGCTTC 230 
 Reverse 151 CCAGTAAAGTAGGCATAC  
22b Forward 146 GAGCTTGCTAAGTAATTCAGTGG 291 
 Reverse 152 AGAGCAATATGTGGCACC  
23 Forward 147 GGGGACAATGATGACCTATGTGG 1091 
 Reverse 39 ATTAGTTGCTATAATCATGAAGG  
ExonDPD primers5′ Primer sequence3′ Amplicon (bp)
1a Forward 124 ACTTGGTGGATGGATGGAGGAACATCTAC 832 
 Reverse 130 TCCTGAAATCTCTTCCGAAGTAAACAG  
Forward 125 GTTTAAACAAATGCCAACATATTTCC 270 
 Reverse 131 TGTACTTTAATACCTTATTTCTAAGTG  
Forward 127 TTCTCAGGATCTTAGAGAATTAAGC 264 
 Reverse 133 TCTCTCCACTGACAAATTAATACC  
Forward 126 ACACGGACTCTGAATGAGTATAAGG 242 
 Reverse 132 CCACAGATAATAGAGAACGAAGATC  
5b Forward 132 GTTTGTCGTAATTTGGCTG 284 
 Reverse 138 ATTTGTGCATGGTGATGG  
Forward 130 CGGGCTGGTAAAACAAGAATTCG 485 
 Reverse 135 TATTGCTTCAAGCCACCTGCAAA  
7b Forward 133 GTCCTCATGCATATCTTGTGTG 360 
 Reverse 139 GCTACATCAGGCAGAAGC  
8b Forward 134 GCCCCACATCGTGCTATGAACA 459 
 Reverse 140 CCAGAATGACTGCCTTCAGAC  
9b Forward 135 CCCTCCTCCTGCTAATG 278 
 Reverse 141 CTCAGCAGCACATTGTTC  
10b Forward 136 GAGAGTGACACTTCATCCTGG 342 
 Reverse 142 GGAGTTGTACACCAACAG  
11b Forward 137 ACTGGTAACTGAAACTCAG 442 
 Reverse 143 CTAGCTTTCAGGGAATTG  
12b Forward 138 TTCCTGTATGTGAGGTGTA 453 
 Reverse 144 GAAGCACTTATCCATTGG  
13 Forward 128 GATGTAATATGAAACCAAGTATTGG 296 
 Reverse 134 ACGATAGACATTTCTATATGACTTC  
14 Forward 115 GTGAGAAGGACCTCATAAAATATTGTC 342 
 Reverse 92 GAATTGGATGTTTAAATAAACATTCACCAAC  
15b Forward 139 TATCTTTGTGTACAACTGGA 391 
 Reverse 145 TGTGAAATCCAAGGGACC  
16b Forward 140 AACGGTGAAAGCCTATTGG 223 
 Reverse 146 TAGTAACTATCCATACGGGGG  
17b Forward 141 CACGTCTCCAGCTTTGCTGTTG 238 
 Reverse 147 CGGGCAACTGATTCAAGTCAAG  
18b Forward 142 TGGGATGTGAGGGGGTGAATG 246 
 Reverse 148 TTCAGCAACCTCCAAGAAAGCCA  
19b Forward 143 TGTCCAGTGACGCTGTCATCAC 300 
 Reverse 149 CATTGCATTTGTGAGATGGAG  
20b Forward 144 GAGAAGTGAATTTGTTTGGAG 424 
 Reverse 150 CACAGACCCATCATATGGCTG  
21b Forward 145 TCTGACCTAACATGCTTC 230 
 Reverse 151 CCAGTAAAGTAGGCATAC  
22b Forward 146 GAGCTTGCTAAGTAATTCAGTGG 291 
 Reverse 152 AGAGCAATATGTGGCACC  
23 Forward 147 GGGGACAATGATGACCTATGTGG 1091 
 Reverse 39 ATTAGTTGCTATAATCATGAAGG  
a

Shestopal et al.(17).

b

van Kuilenburg et al.(23).

Table 2

Phenotypic and genotypic analysis of proband, family members, and controls

SubjectDPD activity (nmol/min/mg)aPlasma uracil (ng/ml)DPD mRNA (copies/ng RNA)Heterozygote genotypeEffect of genotype
II-5 probandb Undetectableb 1500b Not determinedc Intron 14 GIA (DPYD*2ADeletion of exon 14 
    T1679G (DPYD*131560S 
II-6 husband 0.18 <50 1.3 ± 0.10 T85C (DPYD*9AC29R 
    A496G M166V 
III-6 daughter 0.01 <50 1.9 ± 0.20 T1679G (DPYD*131560S 
    A496G M166V 
III-7 son 0.06 <50 2.3 ± 0.33 Intron 14 GIA (DPYD*2ADeletion of exon 14 
IV-1 grandson 0.25 <50 2.6 ± 0.31 A496G M166V 
IV-2 grandson 0.24 <50 2.5 ± 0.18 A496G M166V 
IV-3 granddaughter 0.21 <50 2.0 ± 0.06 A496G M166V 
Controls (n = 7; ±SD) 0.18 ± 0.03 <50 1.87 ± 0.58 Not determined  
SubjectDPD activity (nmol/min/mg)aPlasma uracil (ng/ml)DPD mRNA (copies/ng RNA)Heterozygote genotypeEffect of genotype
II-5 probandb Undetectableb 1500b Not determinedc Intron 14 GIA (DPYD*2ADeletion of exon 14 
    T1679G (DPYD*131560S 
II-6 husband 0.18 <50 1.3 ± 0.10 T85C (DPYD*9AC29R 
    A496G M166V 
III-6 daughter 0.01 <50 1.9 ± 0.20 T1679G (DPYD*131560S 
    A496G M166V 
III-7 son 0.06 <50 2.3 ± 0.33 Intron 14 GIA (DPYD*2ADeletion of exon 14 
IV-1 grandson 0.25 <50 2.6 ± 0.31 A496G M166V 
IV-2 grandson 0.24 <50 2.5 ± 0.18 A496G M166V 
IV-3 granddaughter 0.21 <50 2.0 ± 0.06 A496G M166V 
Controls (n = 7; ±SD) 0.18 ± 0.03 <50 1.87 ± 0.58 Not determined  
a

Normal DPD enzyme activity ranges from 0.11 to 0.75 in 99% of the normal population (7).

b

DPD enzyme activity and uracil levels were determined in a previous study prior to death (8).

c

Archived total RNA was not available for analysis.

1
Grem J. L. Fluoropyrimidines Ed. 2 Chabner B. A. Longo D. L. eds. .
Cancer Chemotherapy and Biotherapy
,
:
149
-197, Lippincott-Raven Philadelphia  
1996
.
2
Diasio R. B., Harris B. E. Clinical pharmacology of 5-fluorouracil.
Clin. Pharm.
,
16
:
215
-237,  
1989
.
3
Heggie G. C., Sommadossi J. P., Cross D. S., Huster W. J., Diasio R. B. Clinical pharmacokinetics of 5-fluorouracil and its metabolites in plasma, urine, and bile.
Cancer Res.
,
47
:
2203
-2206,  
1987
.
4
Diasio R. B., Johnson M. R. Dihydropyrimidine dehydrogenase (DPD). Its role in 5-FU clinical toxicity and tumor resistance.
Clin. Cancer Res.
,
5
:
2672
-2673,  
1999
.
5
Milano G., McLeod H. L. Can dihydropyrimidine dehydrogenase impact 5-fluorouracil-based treatment? Eur.
J. Cancer
,
36
:
37
-42,  
2000
.
6
Baker S. D., Diasio R. B., O’Reilly S., Lucas V. S., Khor S. P., Sartorius S. E., Donehower R. C., Grochow L. B., Spector T., Hohneker J. A., Rowinsky E. K. Phase I and pharmacologic study of oral fluorouracil on a chronic daily schedule in combination with the dihydropyrimidine dehydrogenase inactivator eniluracil.
J. Clin. Oncol.
,
4
:
915
-926,  
2000
.
7
Lu Z., Zhang R., Diasio R. B. Dihydropyrimidine dehydrogenase activity in human peripheral blood mononuclear cells and liver: population characteristics, newly identified patients, and clinical implication in 5-fluorouracil chemotherapy.
Cancer Res.
,
53
:
5433
-5438,  
1993
.
8
Diasio R. B., Beavers T. L., Carpenter J. T. Familial deficiency of dihydropyrimidine dehydrogenase. Biochemical basis for familial pyrimidinemia and severe 5-fluorouracil-induced toxicity.
J. Clin. Investig.
,
81
:
47
-51,  
1988
.
9
Johnson M. R., Hageboutros A., Wang K., High L., Smith J. B., Diasio R. B. Life-threatening toxicity in a dihydropyrimidine dehydrogenase-deficient patient following treatment with topical 5-fluorouracil.
Clin. Cancer Res.
,
5
:
2006
-2011,  
1999
.
10
Johnson M. R., Diasio R. Importance of dihydropyrimidine dehydrogenase(DPD) deficiency in patients exhibiting toxicity following treatment with 5-fluorouracil.
Adv. Enzyme Regul.
,
41
:
151
-157,  
2001
.
11
Wei X., McLeod H. D., McMurrough J., Gonzalez F. J., Salguero P. F. Molecular basis of the human dihydropyrimidine dehydrogenase deficiency and 5-fluorouracil toxicity.
J. Clin. Investig.
,
98
:
610
-615,  
1996
.
12
Lu Z., Zhang R., Carpenter J., Diasio R. B. Decreased dihydropyrimidine dehydrogenase activity in a population of patients with breast cancer—implication for 5-fluorouracil-based chemotherapy.
Clin. Cancer Res.
,
4
:
325
-329,  
1998
.
13
Morrison G. B., Bastian A., Dela-Rosa T., Diasio R. B., Takimoto C. H. Dihydropyrimidine dehydrogenase deficiency: a pharmacogenetic defect causing severe adverse reactions to 5-fluorouracil-based chemotherapy.
Oncol. Nursing Forum
,
24
:
83
-88,  
1997
.
14
van Kuilenburg A. B., Haasjes J., Richel D. J., Zoetekouw L., Van Lenthe H., De Abreu R. A., Maring J. G., Vreken P., Gennip A. H. Clinical implications of dihydropyrimidine dehydrogenase (DPD) deficiency in patients with severe 5-fluorouracil-associated toxicity: identification of new mutations in the DPD gene.
Clin. Cancer Res.
,
12
:
4705
-4712,  
2000
.
15
Lu Z., Zhang R., Diasio R. B. Purification and characterization of dihydropyrimidine dehydrogenase from human liver.
J. Biol. Chem.
,
267
:
17102
-17109,  
1992
.
16
Johnson M. R., Wang K., Tillmanns S., Albin N., Diasio R. B. Structural organization of the human dihydropyrimidine dehydrogenase gene.
Cancer Res.
,
57
:
1660
-1663,  
1997
.
17
Shestopol S. A., Johnson M. R., Diasio R. B. Molecular cloning and characterization of the human dihydropyrimidine dehydrogenase promoter.
Biochim. Biophys. Acta
,
1494
:
162
-169,  
2000
.
18
McLeod H. L., Collie-Duguid E. S., Vreken P., Johnson M. R., Wei X., Sapone A., Diasio R. B., Fernandez-Salguero P., van Kuilenberg A. B., van Gennip A. H., Gonzalez F. J. Nomenclature for human DPYD alleles.
Pharmacogenetics
,
6
:
455
-459,  
1998
.
19
Vreken P., Van Kuilenburg A., Meinsma R., van Gennip A. Identification of novel point mutations in the dihydropyrimidine dehydrogenase gene.
J. Inher. Metab. Dis.
,
20
:
335
-338,  
1997
.
20
Vreken P., Van Kuilenburg A., Meinsma R., Van Gennip A. Dihydropyrimidine dehydrogenase (DPD) deficiency: identification and expression of missense mutations C29R, R886H and R235W.
Hum. Genet.
,
101
:
333
-338,  
1997
.
21
Johnson M. R., Yan J., Shao L., Albin N., Diasio R. B. Semi-automated radioassay for determination of dihydropyrimidine dehydrogenase (DPD) activity. Screening cancer patients for DPD deficiency, a condition associated with 5-fluorouracil toxicity.
J. Chromatogr. B
,
696
:
183
-191,  
1997
.
22
Johnson M. R., Wang K., Smith J. B., Heslin M. J., Diasio R. B. Quantitation of dihydropyrimidine dehydrogenase expression by real-time reverse transcription polymerase chain reaction.
Anal. Biochem.
,
278
:
175
-184,  
2000
.
23
van Kuilenburg A. B., Haasjes J., Richel D. J., Zoetekouw L., Van Lenthe H., De Abreu R. A., Maring J. G., Vreken P., van Gennip A. H. Clinical implications of dihydropyrimidine dehydrogenase (DPD) deficiency in patients with severe 5-fluorouracil-associated toxicity: identification of new mutations in the DPD gene.
Clin. Cancer Res.
,
12
:
4705
-4012,  
2000
.
24
Ridge S. A., Sludden J., Brown O., Robertson L., Wei X., Sapone A., Fernandez-Salguero P. M., Gonzalez F. J., Vreken P., van Kuilenburg A. B., van Gennip A. H., McLeod H. L. Dihydropyrimidine dehydrogenase pharmacogenetics in Caucasian subjects.
Br. J. Clin. Pharmacol.
,
2
:
151
-156,  
1998
.
25
van Kuilenburg A. B., Muller E. W., Haasjes J., Meinsma R., Zoetekouw L., Waterham H. R., Baas F., Richel D. J., Gennip A. H. Lethal outcome of a patient with a complete dihydropyrimidine dehydrogenase (DPD) deficiency after administration of 5-fluorouracil: frequency of the common IVS14+1G>A mutation causing DPD deficiency.
Clin. Cancer Res.
,
5
:
1149
-1153,  
2001
.
26
Collie-Duguid E. S., Etienne M. C., Milano G., McLeod H. L. Known variant DPYD alleles do not explain DPD deficiency in cancer patients.
Pharmacogenetics
,
10
:
217
-223,  
2000
.
27
Tai H. L., Fessing M. Y., Bonten F. J., Yanishevsky Y., d’Azzo A., Krynetski E. Y., Evans W. E. Enhanced proteosomal degradation of mutant human thiopurine S-methyltransferase (TPMT) in mammalian cells: mechanism for TPMT protein deficiency inherited by TPMT*2, TPMT*3A, TPMT*3B or TPMT*3C.
Pharmacogenetics
,
9
:
641
-650,  
1999
.
28
Mattison, L. K., Johnson, M. R., and Diasio, R. B. A comparative analysis of translated dihydropyrimidine dehydrogenase (DPD) cDNA: conservation of functional domains and relevance to genetic polymorphisms. Pharmacogenetics, in press, 2002.
29
Goswami P., Sheren J., Albee L., Parsian A., Sim J., Ridnour L., Higashikubo R. Cell cycle-coupled variation in topoisomerase IIα mRNA is regulated by the 3′-untranslated region. Possible role of redox-sensitive protein binding in mRNA accumulation.
J. Biol. Chem.
,
275
:
38384
-38392,  
2000
.
30
Salonga D., Danenberg K. D., Johnson M., Metzger R., Groshen S., Tsao-Wei D. D., Lenz H. J., Leichma C. G., Leichman L., Diasio R. B., Danenberg P. V. Colorectal tumors responding to 5-fluorouracil have low gene expression levels of dihydropyrimidine dehydrogenase, thymidylate synthase, and thymidine phosphorylase.
Clin. Cancer Res.
,
4
:
1322
-1327,  
2000
.
31
Takenoue T., Kitayama J., Takei Y., Umetani N., Matsuda K., Nita M. E., Hatano K., Tsuruo T., Nagawa H. Characterization of dihydropyrimidine dehydrogenase on immunohistochemistry in colon carcinoma and correlation between immunohistochemical score and protein level or messenger RNA expression.
Ann. Oncol.
,
3
:
273
-279,  
2000
.