Characterization of the Human DihydropyrimidineDehydrogenase Gene

Xiaoxiong Wei,*,1,2 Guillermo Elizondo,*,1 Andrea Sapone,* Howard L. McLeod,†Hannu Raunio,‡ Pedro Fernandez-Salguero,*,3 and Frank J. Gonzalez*,4

*Laboratory of Metabolism, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20892;†Department of Medicine and Therapeutics, University of Aberdeen, Aberdeen, AB9 2ZD, United Kingdom;

and ‡Department of Pharmacology and Toxicology, University of Oulu, Oulu, Finland

Received November 3, 1997; accepted May 12, 1998

Dihydropyrimidine dehydrogenase (DPD) catabolizesendogenous pyrimidines and pyrimidine-based antime-tabolite drugs. A deficiency in human DPD is associatedwith congenital thymine-uraciluria in pediatric patientsand severe 5-fluorouracil toxicity in cancer patients. Thedihydropyrimidine dehydrogenase gene (DPYD) wasisolated, and its physical map and exon–intron organi-zation were determined by analysis of P1, PAC, BAC,and YAC clones. The DPYD gene was found to contain 23exons ranging in size from 69 bp (exon 15) to 961 bp (exon23). A physical map derived from a YAC clone indicatedthat DPYD is at least 950 kb in length with 3 kb of codingsequence and an average intron size of about 43 kb.The previously reported 5* donor splice site mutationpresent in pediatric thymine-uraciluria and cancer pa-tients can now be assigned to exon 14. All 23 exons weresequenced from a series of human DNA samples, andthree point mutations were identified in three racialgroups as G1601A (exon 13, Ser534Asn), A1627G (exon 13,Ile543Val), and G2194A (exon 18, Val732Ile). These stud-ies, which have established that the DPYD gene is un-usually large, lay a framework for uncovering new mu-tations that are responsible for thymine-uraciluria andtoxicity to fluoropyrimidine drugs. © 1998 Academic Press

INTRODUCTION

The human dihydropyrimidine dehydrogenase gene(DPYD)5 encodes dihydropyrimidine dehydrogenase

(DPD, EC 1.3.1.2), the first and rate-limiting enzyme inthe three-step metabolic pathway involved in the deg-radation of the pyrimidine bases uracil and thymine(Gonzalez and Fernandez-Salguero, 1995). In addition,this catabolic pathway is the only route for the synthe-sis of b-alanine in mammals (Wasternack, 1980). Inpediatric patients, DPD deficiency is associated withcongenital thymine-uraciluria, a complex hereditarydisease that presents a variety of symptoms such asdevelopmental delay, epilepsy, and microcephaly (Bak-keren et al., 1984; Brockstedt et al., 1985; Wadman etal., 1985; van Gennip et al., 1997). However, the clin-ical manifestation of congenital thymine-uraciluria ishighly variable, with some patients being severely af-fected while others appear essentially asymptomatic(Wilcken et al., 1985). Recent reports showing lack ofcorrelation between genotype and phenotype for twoDPD-deficient subjects in a family of Pakistani originand two sisters in one Finnish family suggested thatadditional genetic and/or environmental factors maybe required to trigger the clinical symptoms (Fernan-dez-Salguero et al., 1997; Holopainen et al., 1997). DPDis also responsible for detoxification of pyrimidine-based anti-metabolite analogues, such as 5-fluorouracil(5FU), a drug that is commonly used in the treatmentof solid tumors (Harris et al., 1991). Since over 80% ofthe administered 5FU is degraded by DPD (Heggie etal., 1987), the level of DPD catalytic activity in cancerpatients could affect the efficacy of 5FU treatment. Incancer patients with very low levels of DPD activity,toxic reactions were reported that in some cases werelife-threatening and sometimes fatal (Harris et al.,1991; Flemming et al., 1993; Lu et al., 1993). Drug–drug interactions between 5FU and sorivudine, a newantiviral agent, resulted in 5FU toxicity leading tonearly 20 deaths in Japan (David, 1994; Whitley, 1995;

1 Contributed equally to this work.2 Present address: Center for Drug Evaluation and Research, Food

and Drug Administration, Parklawn Building, Rockville, MD 20857.3 Present address: Laboratorio de Bioquimica y Biologia Molecu-

lar, Facultad de Ciencias, Universidad de Extremadura, 06080-Badajoz, Spain.

4 To whom correspondence should be addressed at Laboratory ofMetabolism, National Cancer Institute, Building 37, Room 3E-24,Bethesda, MD, 20892. Telephone: (301) 496-9067. Fax: (301) 496-8419. E-mail: fjgonz@helix.nih.gov.

5 Abbreviations used: DPYD, dihydropyrimidine dehydrogenasegene; DPD, dihydropyrimidine dehydrogenase; YACs, yeast artificial

chromosomes; PFGE, pulsed-field gel electrophoresis; CHEF, con-tour-clamped homogeneous electric field; 5FU, 5-fluorouracil; RFLP,restriction fragment length polymorphism; SSCP, single-strand con-formational polymorphism.

GENOMICS 51, 391–400 (1998)ARTICLE NO. GE985379

3910888-7543/98 $25.00

Copyright © 1998 by Academic PressAll rights of reproduction in any form reserved.

Okuda et al., 1997). This was due to irreversible inhi-bition of DPD by (E)-5-(2-bromovinyl)uracil, a sorivu-dine metabolite (Ogura et al., 1997).

Population studies on DPD catalytic activity indi-cates that at least 3% of the general population couldbe partially deficient for DPD enzyme activity (Lu etal., 1993; Milano and Etienne, 1994). These individualshave no symptoms in the absence of drug treatment.However, they could be at risk for developing toxicity ifexposed to 5FU during chemotherapy (Tuchman et al.,1985; Diasio and Lu, 1994). The human DPD cDNAwas isolated (Yokota et al., 1994) and used in an initialcharacterization of the DPYD gene that led to the iden-tification of a GT3 AT point mutation at a 59 splicingdonor consensus sequence that is found in a mutantallele designated allele DPYD*2 (Meinsma et al., 1995;Wei et al., 1996; Vreken et al., 1996; Fernandez-Salguero et al., 1997). This mutation leads to faultypre-mRNA splicing and no DPD activity in subjectsthat are homozygous (Meinsma et al., 1995). When themutation is present in a heterozygote, half the meanactivity of normal subjects is found, which is sufficientto lead to severe 5FU toxicity in cancer patients (Wei etal., 1996; van Kuilenburg et al., 1997).

In the present study, a physical map of the humanDPYD gene was determined, the exon–intron bound-aries were identified, and a restriction map of the genewas developed, revealing that it spans at least 950 kbof chromosome 1 (Takai et al., 1994). Primers weredesigned and used for PCR amplifications and sequenc-ing of all 23 exons of family members of a cancerpatient who was identified as the proband with splicemutation in previous report (Wei et al., 1996), andadditional point mutations were found. The elucidationof the structure of the human DPYD gene will aid inthe identification of novel mutations and prevention of5FU toxicity and diagnosis of thymine-uraciluria.

MATERIALS AND METHODS

Screening of human genomic libraries. A human phage librarywas screened using the human DPD cDNA as a probe. Human P1and PAC libraries were screened using PCR and pairs of primersdistributed along the exons of the DPD cDNA (Genome System, St.Louis, MO), and five clones that contained different regions of theDPYD gene were isolated. A human genomic YAC library wasscreened using the DPD cDNA as a probe (Genome Systems). Ahuman BAC library was also screened by using the human DPDcDNA (Research Genetics, Huntsville, AL).

Determination of the exon–intron junctions. The majority of theexon–intron junction sequences were obtained through direct se-quencing of P1, PAC, and BAC clones. DNA was extracted followingthe methods recommended by the manufacturers (Genome Systemand Research Genetics) and further purified using Qiagen affinitycolumns (Chatsworth, CA). YAC clones were grown in AHC selectivemedium at 30°C for 24 to 48 h (Rose et al., 1990; Brownstein et al.,1989). YAC DNA was isolated according to the manufacturer’s in-structions. Oligonucleotide primers, designed from the human DPDcDNA, were used for primer extension PCRs to sequence these clonesdirectly to determine the intron sequence surrounding the exons. Ina few cases, exon–intron boundaries located at gaps between theseclones were isolated using the Genomewalk system (Clontech Labo-

ratories, Inc., Palo Alto, CA), in which suppression PCR was applied(Siebert et al., 1995). Briefly, genomic DNA was digested withEcoRV, ScaI, DraI, PvuII, and SspI, respectively. A specially de-signed adapter was ligated with the end of these DNA fragments,and the ligated products were used as a template in a PCR in whichadapter primers and gene-specific primers, designed from the humanDPD cDNA sequence (Table 1), were used. Amplification was carriedout as follows: initial denaturation at 94°C for 2 min, then 94°C for25 s and 65 to 74°C for 4 min for annealing and extension for 7 cycles,followed by 32 cycles of 94°C for 25 s and 63 to 72°C for 4 min forannealing and extension. The PCR-amplified fragments were di-rectly sequenced using the same primers and dye terminators (Per-kin-Elmer, Norwark, CT) and an ABI 377 DNA sequencer (AppliedBiosystems, Foster City, CA). The primers used in this study weresynthesized using an Applied Biosystems Model 394 DNA Synthe-sizer. Sequences were analyzed using the MacVector program (Ox-ford Molecular Group, Inc., Campbell, CA).

Primer extension. Primer E1R, 59-TCGATGTCCGCGGAGTC-CTTA-39, was used for 59 primer extension. The primer was purifiedon a 20% acrylamide gel before being labeled with [g-P32]ATP. Hy-bridization was carried out at 65°C with 10 mg of mRNA isolatedfrom a frozen human liver specimen, and the primer extension reac-tion was carried out at 42°C for 1 h. Single-strand phage DNA wasused as molecular ladder, and 240 forward primer was used forsequencing reactions (Amersham Life Science, Arlington Heights,IL). Samples were dissolved in loading dye, heated at 90°C, andsubjected to electrophoresis on a 9% acrylamide/7 M urea gel. The gelwas dried and exposed to X-ray film with aid of an intensifyingscreen.

PCR amplification of coding exons. From the exon–intron bound-aries and flanking sequences, specific primers (Table 2) were devel-oped to amplify all 23 coding exons and flanking intronic regions ofthe DPYD gene. Two fragments corresponding to exons and the firstand last exons had one primer located within the coding sequencesdue to the existence of highly repetitive sequences in the correspond-ing flanking intronic regions. PCR amplification of exons and flank-ing intronic fragments was carried out in a 100-ml reaction mixturecontaining 10 mM Tris–HCl, pH 8.3 or 9.0, 50 mM KCl, 0.5 mM ofeach primer, 1.5 mM MgCl2, 0.2 mM of each dNTP, 2.5 units of Taqpolymerase, and 500 ng of genomic DNA, by denaturing at 94°C for1 min, annealing at 50 to 60°C for 1 min, and extending at 72°C for1 min for 31 cycles. All PCR products were purified using Wizardcolumns (Promega Corp., Madison, WI) and sequenced as indicatedabove. The sequencing primers were derived from the cDNAs, andthe sequences of these primers are available from the authors uponrequest.

DNA analysis by PFGE. High-molecular-weight YAC DNA fromyeast clones was prepared in agarose plugs using the CHEF genomicDNA plug kit (Bio-Rad, Hercules, CA). Total digestion with restric-tion endonucleases was carried out on one-third of each plug (con-taining about 20 3 106 yeast cells) by overnight incubation with 50units of the appropriate enzymes. Partial digestion was performed onone-third of each plug by using 2.5 units of SfiI or 5 units of EagI andNruI and incubation times of 5, 10, 20, 30, and up to 60 min. Therestriction fragments were separated by pulsed-field gel electro-phoresis (PFGE) in a 1% SeaKem GTG agarose gel (Sigma ChemicalCo., St. Louis, MO). The gel was run at 200 V and 12°C in 0.53 TBEbuffer for 24 h using a PC 500 SwitchBack Pulse Controller (HoeferScientific Instruments, San Francisco, CA) and transferred to a GeneScreen Plus nylon membrane, baked at 80°C for 1 h under vacuum,and hybridized with 32P-labeled probes at 42°C for 12 to 18 h. Blotswere washed in 23 SSC/0.5% SDS (13 SSC is 15 mM NaCl and 1.5mM sodium citrate) at 65°C for 5 min, moved to room temperature,and then exposed to autoradiographic films overnight at 280°C. Thefilters were hybridized with different probes after being strippedwith 4 N NaOH at 42°C for 30 min and neutralization with 0.2 mMTris–HCl, pH 7.5. All probes were generated by PCR amplification oftwo or three exons from the cDNA. Some single exons were amplifiedand labeled as probes to determine further the orientation of the

392 WEI ET AL.

restriction fragments. Mapping was also performed on P1, PAC, andBAC clones using partial digestion and PFGE as described above.

Gene copy number analysis. Human genomic DNA 5 (mg) andYAC3759, in amounts that were equivalent to multiples of a singlecopy as calculated by Pinkert (1994), were digested with HindIII andapplied to a 0.8% agarose gel. Amounts of 1, 2, 5, and 10 ng ofYAC3759 DNA were calculated to represent 1, 2, 5, and 10 copies ofthe DPYD gene, respectively. The DNA was transferred to a nylonmembrane and hybridized with a DPD cDNA probe labeled with[32P]dCTP. The hybridization signals were scanned with a MolecularDynamics Storm PhosphorImager, and the density of the bands wasdetermined and compared.

Genotyping of the mutant alleles by PCR-RFLP and SSCP. APCR-RFLP method was used to detect a mutant allele with aG2194A substitution (Val732Ile) in exon 18. A pair of primersDPD98F and DPD99R (Table 2) was used to amplify a 253-bp frag-ment that can be digested by MaeIII to yield 156- and 97-bp frag-ments, respectively. A point mutation of A to G will eliminate thisrestriction site, and the 250-bp fragment will remain intact. Detec-tion of a splicing mutation at the 59 donor site of the exon 14–intron14 boundary was performed using PCR-RFLP with MaeII digestion,as previously described (Wei et al., 1996).

PCR-single-strand conformational polymorphism (SSCP) analysiswas used to determine the frequencies of mutant alleles G1601A(exon 13, Ser534Asn) and A1627G (exon 13, Ile543Val) (Broly et al.,1995). A 255-bp PCR fragment corresponding to the mutated regionof exon 13 was amplified from human genomic DNA using a forwardprimer DPD70F (Table 2) and a reverse primer 59-AGCTCTTC-GAATCATTGATG-39. PCR was carried out as described above withthe addition of 0.7 mCi of [a-32P]dCTP (.800 Ci mmol21) for label-ing. SSCP analysis was performed using the following conditions: 3ml of the PCR product was mixed with 3 ml of a solution containing10 mM NaOH, 20 mM EDTA, 95% formamide, 0.05% bromophenol

blue, and 0.5% xylene cyanol. The mixture was heated at 95°C for 5min followed by quick cooling on ice. The samples were then loadedon a 5% acrylamide gel prepared with a vinyl polymer, HR-1000 40%concentrate (Genomic Corp., Foster City, CA). Electrophoresis wascarried out at room temperature, in 89 mM Tris, 89 mM boric acid,pH 8.0, and 2 mM EDTA using a Model STS 45 sequencing gelapparatus from International Biotechnologies, Inc. (New Haven, CT)at 60 W constant power. The gel was run until the bromophenol bluemarker reached the bottom, and the gel was transferred to filterpaper, dried, and subjected to autoradiography for 12–24 h. Samplesthat were previously sequenced and determined to be homozygous,heterozygous, and wildtype for the two exon 13 mutations were usedas references to compare with samples for the population study.

Family and participants. Blood samples from 16 members from aBritish family were collected after the proband was found to bepartially DPD deficient after 5FU toxicity developed. The study wasapproved by the local institutional review boards, and informedconsent was obtained from all participants. The pedigree was shownin a previous report (Wei et al., 1996). To determine mutation fre-quencies in the human DPYD gene, random blood samples were alsocollected from Finland (90 subjects), Taiwan (131 subjects), Japan(50 subjects), and the United States (105 subjects), representingdifferent racial groups.

RESULTS

Screening of YAC, BAC PAC, P1, and l PhageLibraries

Four l clones (6, 10, 17, and C 13B), two P1 clones(P1 1602 and P1 2304), three PAC clones (PAC5916,PAC5917, and PAC5945), and two BAC clones

TABLE 1

Sequences of Acceptor and Donor Splice Sites in DPYDa

Adherence toconsensus

(%)b Exon 39 Acceptor 59 Donor

Adherence toconsensus

(%)

1 CGAGgtacggac 8483 2 tttatgctgtctctttagAGTA CTTTgtaagtac 7977 3 tttttaaatatgttgcagAATT TGAGgtaagtct 10086 4 gttattttatttccctagATGC CAAGgtaaattc 8690 5 tttgtattaattttgcagAACT TGAGgtatgata 8387 6 caattgatttccccgtagGTAT TAAGgtaatgcc 8194 7 tttctcctttcttttcagTACT AAAGgtaaatga 8676 8 agcaacggtctctcgtagATAA ATAGgtgagtag 9778 9 ataaatttgattacttagGTTT GCAGgtataaca 6995 10 tttcctttcatcattcagGAAT GGAGgtaaaatg 8085 11 tgttctgttttgttttagATGG AAAGgtacagtg 7093 12 tttcacttgttttttcagTAAA ACAGgtaggcat 8394 13 ttggtttgtattttgcagTCAC TAAGgtaagaaa 9493 14 tctttactctttcatcagGACA CAACgtaagtgt 8684 15 ttggatttctttttaaagATTG TGAGgtaatggt 8187 16 ttcctttcttgtttaaagGATT GCAGgtaaggac 9582 17 accgacctatttgaacagAGCT GAAGgtaagaac 9482 18 ttgatgtgtcttgcatagGTGG TCTGgtaggtgt 8181 19 tattctgattttgtgtagGACA CCAGgtaggtgt 8987 20 ctgctctgtcttttctagGTAT CAAGgtatgtgc 8983 21 tgtgtgttttccttttagAAAC CAAGgtaagaat 9474 22 ttcagtggctatttttagGATG CCAGgtaagaat 9498 23 ctctatttctgtttgcagGCTA

a Exon and intron sequences are shown in upper- and lowercase, respectively. The AG and GT consensus sequences for the different intronsare in boldface print.

b Consensus scores determined by the method of Shapiro and Senapathy (1987), indicating adherence to consensus acceptor and donorsplice sites, are shown in the left and right columns, respectively.

393GENE STRUCTURE OF DPYD

(BAC2-9 and BAC6-3) were isolated using the DPDcDNA and PCR primers derived from the cDNA se-quence. The length of these clones ranged from 15 kb(l phage) to 120 kb (P1, PAC, and BAC). However,each clone contained only a small part of the gene.Since numerous gaps remained after these cloneswere characterized, a human YAC library wasscreened in an attempt to isolate the completegene. Three YAC clones (YAC3759, YAC3760, andYAC3051) that contained the DPYD gene as deter-mined by hybridization with probes derived from theDPD cDNA were isolated.

Exon–Intron Junctions and Sequence Analysis

The P1, PAC, and BAC clones were directly se-quenced to determine the exon–intron consensus splicesequences using primers synthesized from the cDNAand walking toward the 59 and 39 intronic flankingregions of each exon. From the intronic sequences, thelocation of each individual exon was determined bysequencing the consensus donor and acceptor splicesite sequences. The consensus score for these splicingsequences was calculated according to the method ofShapiro and Senapathy (1987) (Table 1). All donor and

TABLE 2

PCR Amplification of DPYD Coding Exons

Exon Primers Sequence Fragment (bp) cDNA position

1 DPDE1F 59-GCTGTCACTTGGCTCTCT-39 183 281–39DPD111R1 59-CACCTACCCGCAGAGCA-39

2 DPD108F 59-GTGACAAAGTGAGAGAGACCGTGTC-39 285 40–150DPD109R 59-GCCTTACAATGTGTGGAGTGAGG-39

3 DPD112F 59-GAATGCTACCCAATTAAAGTGG-39 269 151–233DPD113Rd 59-CCTACCACCATCCTGTGACTG-39

4 DPD96F 59-GGTAGAAAATAGATTATCTC-39 142 234–321DPD97R 59-CAATACTTGTGATGAATG-39

5 DPD94F 59-GTTTGTCGTAATTTGGCTG-39 287 322–483DPD95R 59-ATTTGTGCATGGTGATGG-39

6 DPD92F 59-GAGGATGTAAGCTAGTTTC-39 350 484–680DPD93R1 59-CCATTTGTGTGCGTGAAGTTC-39

7 DPD90F 59-GTCCTCATGCATATCTTGTGTG-39 361 681–762DPD121R 59-GCTTCTGCCTGATGTAGC-39

8 DPD122F 59-GCCCCACATCGTGCTATGAACA-39 461 763–850DPD91R1 59-GTCTGAAGGCAGTCATTCTGG-39

9 DPD88F 59-CCCTCCTCCTGCTAATG-39 149 851–958DPD89R 59-TGCTTTACTGCCTTTGG-39

10 DPD86F 59-GATAGTGACACTTCATCCTGG-39 340 959–1128DPD87R 59-CTGTTGGTGTACAACTCC-39

11 DPD84 59-ACTGGTAACTGAAACTCAG-39 443 1129–1339DPD85R4 59-CAATTCCCTGAAAGCTAG-39

12 DPD82F1 59-ACGACTCACTATAGGGCA-39 436 1340–1524DPD83R1 59-GAAGCACTTATCCATTGG-39

13 DPD70F 59-CGGATGACTGTGTTGAAGTG-39 434 1525–1740DPD71R 59-TGTGTAATGATAGGTCGTGTC-39

14 DPDdelF 59-TGCAAATATGTGAGGAGGGACC-39 409 1741–1905DPDdelR 59-CAGCAAAGCAACTGGCAGATTC-39

15 DPD72F 59-ATTGTGATTGCTAGCATTATG-39 294 1906–1974DPD119R 59-TGTGAAATCCAAGGGACC-39

16 DPD116F 59-AACGGTGAAAGCCTATTGG-39 223 1975–2058DPD117R 59-TAGTAACTATCCATACGGGGG-39

17 DPD114F 59-CACGTCTCCAGCTTTGCTGTTG-39 238 2059–2179DPD115R 59-CGGGCAACTGATTCAAGTCAAG-39

18 DPD98F 59-TGGGATGTGAGGGGGTGAATG-39 253 2180–2299DPD99R 59-TTCAGCAACCTCCAAGAAAGCCAC-39

19 DPD100F 59-TGTCCAGTGACGCTGTCATCAC-39 300 2300–2442DPD80R 59-CATTGCATTTGTGAGATGGAG-39

20 DPD78F 59-GAGAAGTGAATTTGTTTGGAG-39 399 2443–2622DPD79R1 59-CACAGACCCATCATATGGCTG-39

21 DPD76F 59-TCTGACCTAACATGCTTC-39 187 2623–2766DPD77R 59-CTTGATGGTAGGAATAGG-39

22 DPD74F 59-GAGCTTGCTAAGTAATTCAGTGGC-39 288 2767–2908DPD75R 59-AGAGCAATATGTGGCACC-39

23 DPDASPF 59-GGGGACAATGATGACCTATGTGG-39 269 2909–3060DPDASPR 59-GGTGACATGAAAGTTCACAGCAAC-39

Note. All 23 pairs of primers are designed from respective introns with four exceptions: the forward primer for exon 1 (E1F) and the reverseprimer for exon 23 (ASPR) as well as primers DPD72F and DPD77R, which are also derived from the cDNA. The assignment of positions forindividual exons was based on the cDNA sequence by Yokota et al. (1994).

394 WEI ET AL.

acceptor splice sites had a 70–100% adherence to thecorresponding consensus sequences. In addition, allthe exon–intron boundaries in the DPYD gene con-curred exactly with the GT/AG rule (Robberson et al.,1990).

Primer Extension

To determine the start site of transcription, primerextension analysis was carried out. One major bandwas identified at 137 bp 59 to the splice site of exon 1(Fig. 1), which is 17 bp 59 to the 59 terminal base of theDPD cDNA (Yokota et al., 1994). A few additional faintbands that may result from minor transcription startsites were detected.

PCR amplification of coding exons. Primers weredeveloped to amplify each exon and flanking intronicsequences specifically (Table 2). The amplified frag-ments are shown in Fig. 2, and each could yield a cleansequence. In some cases, two additional minor bandswere obtained upon amplification of exon 8 usingDPD122F and DPD121R1. However, clean sequencewas always obtained from this PCR product even whenthere was a mixture indicating that the additionalbands were derived from a non-DPYD portion of thegenome. The human DPYD gene was found to contain23 exons ranging from 69 bp (exon 15) to 961 bp (exon23). The first and last exons contain 59 untranslatedand 39 noncoding regions, respectively. The first exoncontains 137 bp with 39 bp of coding region. The lastexon, having the stop codon TAA, contains 168 bp ofcoding sequence and at least 1230 bp of noncodingDNA that extends to a putative poly(A) addition signalcorresponding to that described in the human lympho-cyte-derived DPD (GenBank Accession No. U20938)that extended beyond the sequence derived from thehuman liver DPD cDNA (Yokota et al., 1994). A secondpoly(A) addition site was found 29 bp upstream of thissite. However, only a single mRNA of about 3 kb isfound in human liver (data not shown), indicating thatthere may only be one transcript that results from asingle polyadenylation site or perhaps two sites in closeproximity. Attempts to determine intron size by use ofLong PCR failed.

FIG. 1. Primer extension analysis. Poly(A)1 mRNA (10 mg) wasprepared from frozen human liver and used as template for reversetranscription using the primer E1R that starts with the last basepairof the first DPD exon. The mark indicates the band corresponding tothe major transcription start site.

FIG. 2. PCR amplification of DPYD exons. Genomic DNA was isolated from white blood cells, and PCRs were performed. PCR productsfrom 23 exons amplified from human genomic DNA were electrophoresed on a 4% agarose gel. A 1-kb DNA ladder was used as size marker(Gibco BRL, Gaithersburg, MD). Each pair of primers used to amplify individual exons is listed in Table 2.

395GENE STRUCTURE OF DPYD

Mapping and Alignment of YAC and Other Clones

To determine the approximate size of the humanDPYD gene, restriction mapping was conducted by di-gesting YAC3759 with SacII, SfiI, EagI, MluI, andNruI, respectively. This YAC clone was hybridizedwith probes derived from the 59 flanking region andexon 23, indicating that it contained the completeDPYD gene. A representative PFGE separation of thedigested YAC3759 is shown in Fig. 3A. A correspond-ing Southern blot using a probe derived from full-length DPD cDNA is shown in Fig. 3B. The size ofYAC3759 was estimated by PFGE to be 1100 kb. Theminimal size of the DPYD gene is estimated to be atleast 950 kb, discounting the first and last fragmentsderived from restriction enzyme digestions. YAC3760did not hybridize with exons 1–3 on the 59 end but

hybridized with exon 23. YAC3051 hybridized withexons 10 and 23, respectively. The restriction maps forthe P1, PAC, and BAC clones were coincident with themap corresponding to YAC3759, although they covereda small portion of the gene, usually about 1 to 3 exons.The average size for these smaller clones was about100 kb. Based on these results, a restriction map ofhuman DPYD was generated, and YAC3759 was foundto contain the complete gene (Fig. 4). The exons foundin each restriction fragment are shown at the top of thefigure. This was determined by hybridization with se-lected regions of the cDNA prepared by PCR and ran-dom primer labeling with [32P]dATP.

Gene Copy Number Analysis

Southern blot analysis using the full-length DPDcDNA as a probe revealed multiple bands in HindIII-digested human genomic and YAC3759 DNAs (Fig. 5).The slight difference in migration between the YACand human DNA is due to load effects. Linear regres-sion analysis of the density of one of the bands fromYAC3759 was used to determine the DPYD copy num-ber, which was found to correspond to one copy ofYAC3759. This indicates that DPYD is present as asingle-copy gene.

Analysis of DPYD Exons

To search for mutations, all 23 DPYD exons andflanking regions were amplified and sequenced fromgenomic DNAs of 16 members of a single family. Twopoint mutations were found in exon 13, G1601A andA1627G, that result in the amino acid substitutionsSer534Asp and Ile543Val, respectively. In this family,A1627G was found only in subjects having the 59 donorsplice mutation at the exon 14–intron 14 junction (Weiet al., 1996). The G2194A in exon 18, which leads toVal732Ile, was found in two family members. A silentmutation, C1897T in exon 14, was found in severalsubjects.

Genotyping of Mutant DPYD Alleles and PopulationSurvey

To detect the 59 donor splice mutation (Wei et al.,1996) and the G2194A change in exon 18, PCR-RFLPwas used. An example of the latter genotyping resultsis shown in Fig. 6, where the different genotypes can bedistinguished by amplification using primers DPD98Fand DPD99R followed by digestion with MaeIII. SSCPanalysis was used to detect simultaneously two pointmutations in exon 13 (Fig. 7). The G1601A mutation inthe heterozygous state is seen in subjects 45, 53, and54. Subject 60 is a homozygote. Subjects 46 and 60,having the highest mobility fragment, are heterozy-gous for exon 13 A1627G. Subject 46 is also heterozy-gous for a base change in intron 12, and this may giverise to the band that migrates slightly faster than theband associating with the G1601A allele. Overexpo-

FIG. 3. (A) A representative PFGE of total digestion of YAC3759with six rare-cutting restriction enzymes. PFGE was performed us-ing the following conditions: pulse controller at 1–60 s of pulse (ratioof forward/back set at 3:1) at 200 V and 12°C for 24 h. (B) Southernblot of YAC3759. PFGE was transferred to a nylon membrane andhybridized with a [32P]dCTP-labeled probe prepared with the full-length DPD cDNA. The autoradiograph was developed by exposureto film overnight at 280°C with the aid of an intensifying screen.

396 WEI ET AL.

sure of the gel revealed that subjects 50 and 61 bothhad the lowest mobility band that correlates with thewildtype allele DPYD*1.

A total of 386 subjects were analyzed in this study.Only two heterozygotes for the 59 donor splice mutationof exon 14 were found in the Finnish population. TheA1627G mutation was found in all populations rangingfrom 35.2% in Japanese to 7.2% in the Finnish popu-lation (Table 3). G1601A and G2194A were present atfrequencies ranging from 0 to 6.7%.

DISCUSSION

Restriction mapping of a YAC clone using PFGErevealed that the DPYD gene is significantly largerthan that recently reported (Johnson et al., 1997). Inthis report, the size of the DPYD gene was determinedto be about 150 kb, with intron sizes ranging from lessthan 1 kb to over 20 kb. Differences between the re-sults of the present study and those of Johnson et al.

(1997) were also found in two exon–intron junctions atexons 16 (8 bp) and 18 (1 bp), which resulted in differ-ent sizes of these exons (Table 1 and Johnson et al.,1997). Johnson et al. apparently screened the same P1human genomic library as we did and obtained threepositive clones that contained only the last 3 exons.The majority of intron–exon junctions in their reportwere determined from use of the Genomewalk system(Clontech Laboratories, Inc.). Our results from thescreening of different P1, PAC, and BAC humangenomic libraries and subsequent mapping of theseclones revealed that most clones contained 1 to 3 exonswith an average insert size of 100 kb. Further mappingof YAC3759 demonstrated that the size of the DPYDgene is at least 950 kb, which is far larger that thanreported by Johnson et al. (1997). Copy number anal-ysis and comparison of restriction fragment sizes be-tween human DNA and the YAC clone revealed noevidence for variants or multiple copies of the DPYDgene in the human genome. The introns are so large,averaging at least 43 kb, that we were not able toamplify any of them by use of exon to exon primers,

FIG. 6. Detection of the G2194A allele (exon 18, Val732Ile). Inthe wildtype allele, MaeIII cleaves a 253-bp fragment into 156- and97-bp fragments, respectively, whereas the mutant allele remainsundigested. Subject 1 is homozygous (2/2), subject 2 is heterozygous(2/1), and the remaining subjects are wildtype (1/1).

FIG. 4. Restriction map of human DPYD. YAC3759 DNA was digested in agarose plugs, subjected to PFGE, and hybridized with thefull-length DPD cDNA and individual exons. Five rare-cutting restriction enzymes, SacII (Sa), SfiI (S), MluI (M), EagI (E), and NruI (N), wereused. P1, PAC, and BAC clones are also indicated.

FIG. 5. Analysis of DPYD copy numbers. Preparation of humangenomic DNA and YAC3759 DNA and determination of number ofcopies were as described under Materials and Methods. Lane H,human genomic DNA (5 mg), lanes 2–4, YAC3759 DNA (1, 2, 5, and10 ng). The blot was developed using the DPD cDNA as a probe.

397GENE STRUCTURE OF DPYD

even with the Long PCR methods currently available.It is noteworthy that during the course of these exper-iments, single bands for some introns such as introns 5,9, and 22, having sizes similar to those reported byJohnson et al. (1997), were obtained. However, se-quencing of these fragments obtained by using theprimers described by Johnson et al. (1997) revealedthat only one end had sequence similarity to the DPDcDNA from which the primer was made, while theother end was unrelated to the cDNA (exonic se-quence). These data suggest that these fragments donot reflect the true size of introns 5, 9, and 22 aspreviously reported.

The determination of the exon–intron junctions inthe DPYD gene provides an insight into the relation-ship between gene structure and protein function (Fig.8). In a previous study, functional domains in the DPDprotein were found that included putative consensusbinding sites for NADPH/NADP1 and FAD/FMN, auracil binding site, and (4Fe-4S) clusters (Yokota et al.,1994). The NADPH binding motif beginning at Val-335and ending at Ala-351 can now be assigned to exons 9and 10. The sequence encoding the FAD/FMN bindingdomain was located in exons 11 and 12, and the uracilbinding site was assigned mainly to exon 15. Putativebinding sites at the COOH-terminal region of the hu-

man DPD were identified for (4Fe-4S) clusters betweenresidues 953 and 964 and between residues 986 and997, respectively, which corresponded to exon 22.

A splicing site mutation was described in pediatricpatients with congenital thymine-uraciluria (Meinsmaet al., 1995; Vreken et al., 1996; Fernandez-Salguero etal., 1997) and in a cancer patient who developed severetoxicity after 5FU treatment (Wei et al., 1996). Withthe DPYD gene structure now available, this splicingmutation, GT to AT, can be assigned to the 59 splicingdonor consensus sequence of the exon 14–intron 14junction. Screening for the presence of this allele, des-ignated DPYD*2 (McLeod et al., 1998), in samples fromFinnish, Taiwanese, Japanese, and African-Americansubjects revealed only two unrelated heterozygotes in theFinnish population (90 subjects, 180 alleles analyzed).Although a large-scale population study has not yet beenreported, these data indicate that this splicing mutationmay not be as common as previously thought (Wei et al.,1996; Ridge et al., 1998). The DPYD*2 allele frequency inthe Finnish populations is about 1%, which is still belowthe estimation of DPD deficiency based on the activitydeterminations, suggesting that up to 3% of the gen-eral population may be deficient in DPD (Harris et al.,1990; Milano and Etienne, 1994). At a 1% allele fre-quency for DPYD*2, the predicted frequency of ho-

FIG. 7. PCR-SSCP detection of the alleles G1601A and A1627G. Subjects 45, 53, and 54 are heterozygotes (1/2), and Subject 60 is ahomozygote (2/2) for G1601A. Subjects 46 and 61 are heterozygotes (1/2) for A1627G. Subject 46 also has a point mutation in intron 12.All mutations were confirmed by sequencing. SSCP analysis of mutant alleles revealed that the point mutations G1601A and A1627G arelocated on different alleles.

TABLE 3

DPYD Mutant Allelic Frequency in Different Populations

PopulationNo. of alleles

analyzed

Allelic frequency (%)

Exon 14-intron 14GT 3 AT splicing

mutation

Exon 13G1601A

Ser534Asn

Exon 13A1627G

Ile543Val

Exon 18G2194A

Val732Ile

Taiwanese 262 0.0 0.0 21.0 1.4Japanese 100 0.0 1.1 35.2 4.4Finnish 180 1.1 3.3 7.2 6.7African-Americans 210 0.0 0.5 22.7 1.9

Note. The positions of these mutations were assigned using the start codon ATG as base 1.

398 WEI ET AL.

mozygotes would be 1 homozygote in 10,000, based onthe Finnish population and using the Hardy-Weinburgequation. A recent report revealed that 8 of 11 patientswith complete DPD deficiency were found to be ho-mozygous for this splice mutation (van Kuilenburg etal., 1997). Thus this allele may be the most common ofthe defective DPYD alleles. Its prevalence in humansrequires genotypic analysis of larger populations.

Although the point mutations identified in this re-port are common in all populations, the amino acidchanges resulting from these base changes, A1627G(Ile543Val) and G2194A (Val732Ile), are conservativeexcept for G1601A (Ser534Asn) in exon 13. The pres-ence of these alleles did not correlate with the in-creased or reduced DPD catalytic activity in the singlelarge family analyzed, and a recent study revealed thatthere was no correlation between these point muta-tions and DPD catalytic activity in colorectal cancerpatients (Ridge et al., 1998). However, homozygoteswould have to be identified and carefully studied forlymphocyte DPD activity or cDNA expression studiesperformed using the correctly modified cDNAs.

DPD deficiency may be a consequence of different yetuncharacterized mutations, which contribute to theobserved phenotype in pediatric thymine-uraciluriapatients and cancer patients exhibiting toxicity to flu-oropyrimidine-based drugs. Recently, new mutationsin patients with thymine-uraciluria were reported thatconsisted of a 4-base deletion (delTCAT296-299) in exon4 and a single base deletion C1897 in exon 14, whichcaused a frameshift leading to a premature stop codonshortly thereafter (Vreken et al., 1997a,b). To facilitatethe search for mutations in DPYD, specific primers forall 23 exons were developed (Table 2) to amplify indi-vidual exons. These can be used for direct sequencingor for SSCP analysis to identify new alleles and facili-tate investigations into the genetic mechanisms of thedisease status of congenital thymine-uraciluria andthe pharmacogenetic syndrome related to DPD defi-ciency. In particular, it is still a mystery why certainsubjects that are totally deficient in enzyme activityand homozygous for DPYD*2 do not develop deleteri-ous phenotypes (Fernandez-Salguero et al., 1997; Hol-opainen et al., 1997).

ACKNOWLEDGMENTS

We thank Drs. Jin-ding Huang and William E. Evans for theircollection of DNA samples for the population study. The efforts ofJulieann Sludden in collecting the blood samples in the family studyare also greatly appreciated.

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