METHODS AND COMPOSITIONS FOR DETECTING DIHYDROPYRIMIDINE DEHYDROGENASE SPLICING MUTATIONS

Abstract: The present invention provides compositions, methods and kits for the detection of genetic polymorphisms or mutations related to dihydropyrimidine dehydrogenase deficiency (DPDD). The polymorphism or mutations generally occur in the dihydropyrimidine dehydrogenase DPD gene in chromosome 1. Also provided are mutant forms of DPD.

Description: METHODS AND COMPOSITIONS FOR DETECTING DIHYDROPYRIMIDINE DEHYDROGENASE SPLICING MUTATIONS TECHNICAL FIELD OF THE INVENTION The present invention relates to methods and compositions for detecting splicing defects in the dihydropyrimidine dehydrogenase gene. The methods and compositions are useful for identifying persons who are at risk of a toxic reaction to the commonly employed cancer chemotherapy agent, 5-fluorouracil. BACKGROUND OF THE INVENTION 5-Fluorouracil (5-FU) is commonly used in the treatment of cancers, including cancers of die breast, head, neck, and digestive system. The efficacy of 5-FU as a cancer treatment varies significantly among patients. Clinically significant differences in systemic clearance and systemic exposure of 5-FU are often observed. See, Grem, J.L. In Chabner, B.A. and J.M. Collins (eds.), Cancer Chemotherapy: Principles and Practice, pp. 180-224, Philadelphia, PA, Lippincott, 1990). Furthermore, 5-FU treatment is severely toxic to some patients, and has even caused death. See, Fleming et al. (1993) Eur. J. Cancer 29A: 740-744; Thyss et al. (1986) Cancer Chemother. Pharmacol. 16: 64-66; Santini et al. (1989) Br. J. Cancer 59: 287-290; Goldberg et al. (1988) Br. J. Cancer 57: 186-189; Trump et al. (1991) /. Clin. Oncol. 9: 2027-2035; and Au et al. (1982) Cancer Res. 42: 2930-2937. Patients in whom 5-FU is severely toxic typically have low levels of dihydropyrimidine dehydrogenase (DPD) activity. See, Tuchman et al. (1985) N. Engl. J. Med. 313: 245-249; Diasio et al. (1988) J. Clin. Invest. 81: 47-51; Fleming et al. (1991) Proc. Am. Λssoc. Cancer Res. 32: 179; Harris et al. (1991) Cancer (Phila.) 68: 499-501; Houyau et al. (1993) J. Nat'l. Cancer Inst. 85: 1602-1603; Lyss et al. (1993) Cancer Invest. 11: 239-240. Dihydropyrimidine dehydrogenase (DPD, EC 1.3.1.2) is the principal enzyme involved in the degradation of 5-FU, which acts by inhibiting thymidylate synthase. See, Heggie et al (1987) Cancer Res. 47: 2203-2206; Chabner et al. (1989) In DeVita et al. (eds.), Cancer - Principles and Practice of Oncology, pp. 349- 395, Philadelphia, PA, Lippincott; Diasio et al. (1989) Clin. Pharmacokinet. 16: 215- 237; Grem et al. , supra. The level of DPD activity also affects the efficacy of 5-FU treatments, as 5-FU plasma levels are inversely correlated with the level of DPD activity. See, Iigo et al. (1988) Biochem. Pharm. 37: 1609-1613; Goldberg et al , supra.', Harris et al. , supra. ; Fleming et al. , supra. In turn, the efficacy of 5-FU treatment of cancer is correlated with plasma levels of 5-FU. In addition to its 5-FU degrading activity, DPD is also the initial and rate limiting enzyme in the three-step pathway of uracil and thymine catabolism, leading to the formation of β-alanine and j3-aminobutyric acid, respectively. See, Wasternack et al. (1980) Pharm. Ther. 8: 629-665. DPD deficiency is associated with inherited disorders of pyrimidine metabolism, clinically termed thymine-uraciluria. See, Bakkeren et al. (1984) Clin. Chim. Acta. 140: 247-256. Clinical symptoms of DPD deficiency include a nonspecific cerebral dysfunction, and DPD deficiency is associated with psychomotor retardation, convulsions, and epileptic conditions. See, Berger et al. (1984) Clin. Chim. Acta 141: 227-234; Wadman et al (1985) Adv. Exp. Med. Biol. 165A: 109-114; Wilcken et al. (1985) J. Inherit. Metab. Dis. 8 (Suppl. 2): 115-116; van Gennip et al. (1989) Adv. Exp. Med. Biol. 253A: 111-118; Brockstedt et al. (1990) /. Inherit. Metab. Dis. 12: 121-124; and Duran et al. (1991) J. Inherit. Metab. Dis. 14: 367-370. Biochemically, patients having DPD deficiency have an almost complete absence of DPD activity in fibroblasts (see, Bakkeren et al, supra) and in lymphocytes (see, Berger et al., supra and Piper et al. (1980) Biochim. Biophys. Acta 633: 400-409. These patients typically have a large accumulation of uracil and thymine in their cerebrospinal fluid (see, Bakkeren et al., supra.) and urine (see, Berger et al., supra.', Bakkeren et al., supra.; Brockstedt et al., supra.; and Fleming et al. (1992) Cancer Res. 52: 2899-2902). Familial studies suggest that DPD deficiency follows an autosomal recessive pattern of inheritance. See, Diasio et al., (1988) supra. Up to three percent of the general human population are estimated to be putative heterozygotes for DPD deficiency, as determined by enzymatic activity in lymphocytes. See, Milano and Eteinne (1994) Pharmacogenetics. This suggests that the frequency of homozygotes for DPD deficiency may be as high as one person per thousand. DPD has been purified from liver tissue of rats (see, Shiotani and Weber (1981) J. Biol Chem. 256: 219-224; Fujimoto et al. (1991); J. Nutr. Set. Vitaminol. 37: 89-98], pig [Podschun et al (1989) Eur. J. Biochem. 185: 219-224), cattle (see. Porter et al. (1991) J. Biol Chem. 266: 19988-19994), and humans (see, Lu et al (1992) 7. Biol Chem. 267: 1702-1709). The pig enzyme contains flavins and iron-sulfur prosthetic groups and exists as a homodimer with a monomer Mr of about 107,000 (see, Podschun et al , supra.). Since the enzyme exhibits a nonclassical two-site ping-pong mechanism, it appears to have distinct binding sites for NADPH/NADP and uracil/5,6-dihydrouracil (see, Podschun et al. (1990) /. Biol Chem. 265: 12966-12972). An acid-base catalytic mechanism has been proposed for DPD (see, Podschun et al. (1993) J. Biol Chem. 268: 3407-3413). The DPD cDNA is described in copending U.S. application SN 08/304,309. Recently, DPD mRNA from patients lacking dihydrompyrimidine activity was found to lack an exon which encodes a 165 base pair sequence found in the wild-type DPD cDNA. See, Meinsma et al. (1995) DNA and Cell Biology 14(1): 1-6. Because an undetected DPD deficiency poses a significant danger to a cancer patient who is being treated with 5-FU, a great need exists for a simple and accurate test for DPD deficiency. Such a test will also facilitate diagnosis of disorders that are associated with DPD deficiency, such as uraciluria. The present invention provides such a test, thus fulfilling these and other needs. SUMMARY OF THE INVENTION Particular mutations in the dihydropyrimidine dehydrogenase gene are described herein which lead to loss of dihydropyrimidine dehydrogenase activity. The mutations result in a loss of the amino acids from 581-635 of the protein encoded by the gene due to a splicing defect. The splicing defect results in the loss of an exon encoding the missing amino acids. The assays and compositions of the invention detect the splicing defect in the genomic DNA which results in the loss of the exon. A variety of assays for detecting splicing defect mutations in patients are provided. The assays determine whether RNA encoded by genomic DNA is competent to be spliced to produce mRNA with nucleic acids encoding the exon which corresponds to amino acids from 581-635 of the wild-type protein. For example, sequencing, PCR, LCR, and oligonucleotide array based assays are used to detect the mutations. In one class of embodiments, the methods comprise the step of determining whether a nucleic acid encoding an mRNA for the dihydropyrimidine dehydrogenase gene has an exon corresponding to amino acids 581-635 of a corresponding wild-type dihydropyrimidine dehydrogenase mRNA. This determination is performed in sequencing, PCR, LCR, and oligonucleotide array based hybridization assays. For example, in one class of PCR based assays, the intronic genomic DNA encoding the dihydropyrimidine dehydrogenase in the region flanking the exon which encodes amino acids 581-635 is hybridized to a set of PCR primers for amplification and analysis of the intron-exon splice boundaries. Example primers which are used for amplifying the splice regions include DELF1 and DELR1. Similar primers which hybridize to the same sites, or to sites proximal to the primer binding sites are also used. One particular mutation which results in an abnormal dihydropyrimidine dehydrogenase gene phenotype is the conversion of a G to an A residue at the 3' GT splice site of the exon which encodes amino acids 581-635 of the corresponding wild-type protein. The 3' wild-type splice site is recognized by restriction endonucleases which recognize the Mae II cleavage site. The ability of the cleavage site to be cleaved by restriction endonucleases which recognize die Mae II site is used to distinguish wild-type from abnormal dihydropyrimidine dehydrogenase genomic DNAs. For example, a region including the mutation is amplified by PCR, and the PCR products cleaved by Mae II. This results in the cleavage of nucleic acids amplified by the wild-type gene, but mutant sequences are not amplified. In addition to PCR detection methods, nucleic acid arrays are used to discriminate single base-pair mismatches, or to directly sequence DNA by hybridization to arrays. The sequence of the splice site is also determined by standard or PCR sequencing of the site, e.g., using primers which flank the site in a pol I or taq PCR based sequencing assay. In addition to methods and compositions for abnormal dihydropyrimidine dehydrogenase gene detection, the invention provides kits for practicing the methods. Typically, die kit contains a first PCR primer which binds to DNA 3' of a splice site in d e genomic DNA for dihydropyrimidine dehydrogenase gene for an exon encoding amino acids 581-635, and a second PCR primer which binds to DNA 5' of a splice site in d e genomic DNA for dihydropyrimidine dehydrogenase gene for an exon encoding amino acids 581-635. The kit optionally contains other components such as instructions for die detection of an abnormal dihydropyrimidine dehydrogenase gene, restriction enzymes such as Mae II, PCR or other in vitro amplification reagents (buffers, enzymes, salts and die like), oligonucleotide array detection chips and die like. DETAILED DESCRIPTION OF THE DRAWING Figure 1 provides genomic sequence from the DPD gene in the region of a splicing mutant which leads to die loss of an exon in die mRNA which encodes amino acids 581-635. The primer binding sites for DELF-l and DELRl are indicated. The primer binding site for DPD15F and DPD15R (see, Meinsma et al. (1995) DNA and Cell Biology 14(1): 1-6) are also indicated. The Mae II site at d e 3' splice junction is indicated. In a mutant form, die G nucleotide at the 3* splice junction (residue 434) is mutated to an A nucleotide. Figure 2 provides further details of PCR reaction components for amplifying the region of a splicing site mutant. The exon is underlined. The splice site which is polymorphic is in bold text. Figure 3 is a schematic of the splicing product of the mutant DPD gene. Figure 4 is a schematic representation of an example strategy for amplifying an exon deleted in mutant Dpd mRNA from die genomic DNA for die gene. The restriction endonuclease Mae II produces fragments of 278 bp and 131 bp in die wild-type allele. A point mutation at d e donor splicing consensus sequence (GT to AT) destroys d e restriction site, and thus, the mutant form remains undigested (409 bp). DEΠNΓΠONS Unless defined otherwise, all technical and scientific terms used herein have d e same meaning as commonly understood by one of ordinary skill in die art to which this invention belongs. Singleton et al (1994) Dictionary of Microbiology and Molecular Biology, second edition, John Wiley and Sons (New York); Walker (ed) (1988) The Cambridge Dictionary of Science and Technology, The press syndicate of die University of Cambridge, NY; and Hale and Marham (1991) The Harper Collins Dictionary of Biology Harper Perennial, NY provide one of skill with a general dictionary of many of die terms used in this invention. Although any methods and materials similar or equivalent to those described herein can be used in die practice or testing of die present invention, preferred methods and materials are described. For purposes of die present invention, d e following terms are defined below. The term "nucleic acid" refers to deoxyribonucleotides, ribonucleotides, and polymers diereof in eitiier single- or double-stranded form, and unless specifically limited, encompasses known analogues of natural nucleotides mat hybridize to nucleic acids in a manner similar to naturally occurring nucleotides. Unless otherwise indicated, a particular reference nucleic acid sequence implicitly encompasses conservatively modified variants thereof (i.e., sequence variants tiiat hybridize with d e reference nucleic acid, that encode die same amino acid sequence as d e reference nucleic acid, or that encode amino acid sequences containing conservative amino acid substitutions as compared to die amino acid sequence encoded by die reference nucleic acid), and complementary sequences and as well as die sequence explicitly indicated. The term "wild type," when applied to a gene, nucleic acid or gene product, especially a protein and/or biological property, denotes a gene, gene product, protein, or biological property predominantly found in nature. "Mutant" proteins, nucleic acids and the like occur less frequently and are often associated with loss or alteration of biological activity and a diseased state. The term "dihydropyrimidine dehydrogenase gene" refers to a naturally occurring nucleic acid that encodes a protein that has dihydropyrimidine dehydrogenase activity, or tiiat is specifically recognized by antibodies specific for dihydropyrimidine dehydrogenase. The term encompasses both wild-type and mutant genes. "Intron-exon boundary" denotes a sequence of a gene present in or derived from a genomic DNA, wherein die sequence comprises intron sequences tiiat are absent in a mature mRNA transcript, contiguous to exon regions that are present in d e mature mRNA transcript. A "splicing defect" is a failure to correcdy remove an intron or to correctiy connect two exons in d e mature mRNA transcript, which often are die result of sequence mutations in die intron-exon boundary. "Stringency" relates to d e conditions under which hybridization and annealing take place. Low stringency hybridization and annealing conditions permit d e annealing of complementary nucleic acids that contain mismatched nucleic acids. As die stringency is raised, annealing of sequences containing mismatched nucleic acids is disfavored. Conditions which result in low or high stringency levels are known in the art (e.g., increasing die annealing temperature raises d e stringency). An extensive guide to die hybridization of nucleic acids is found in Tijssen (1993) Laboratory Techniques in Biochemistry and Molecular Biology-Hybridization with Nucleic Acid Probes part I chapter 2 "overview of principles of hybridization and die strategy of nucleic acid probe assays", Elsevier, New York. Generally, highly stringent hybridization and wash conditions are selected to be about 5° C lower than the thermal melting point (TJ for d e specific sequence at a defined ionic strength and ph. The T a is the temperature (under defined ionic strength and pH) at which 50% of the target sequence hybridizes to a perfectly matched probe. Very stringent conditions are selected to be equal to d e T„ for a particular probe. Methods of alignment of sequences for comparison and determination of whedier two sequences bind die same target are well known in d e art. Optimal alignment of sequences for comparison may be conducted by d e local homology algorithm of Smith and Waterman (1981) Adv. Appl. Math. 2: 482; by d e homology alignment algorithm of Needleman and Wunsch (1970) J. Mol. Biol 48: 443; by die search for similarity method of Pearson and Liprnan (1988) Proc. Natl. Acad. Sci. USA 85: 2444; by computerized implementations of tiiese algoritiims (including, but not limited to CLUSTAL in die PC/Gene program by Intelligenetics, Mountain View, California, GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group (GCG), 575 Science Dr., Madison, Wisconsin, USA); die CLUSTAL program is well described by Higgins and Sharp (1988) Gene, 73: 237-244 and Higgins and Sharp (1989) CABIOS 5: 151-153; Corpet, et al. (1988) Nucleic Acids Research 16, 10881-90; Huang, et al. (1992) Computer Applications in the Biosciences 8, 155-65, and Pearson, et al. (1994) Methods in Molecular Biology 24, 307- 31. Alignment is also often performed by inspection and manual alignment. A "restriction endonuclease which recognizes a Mae II cleavage site" is a DNA endonuclease that recognizes and cleaves a nucleic acid which is cleaved by the restriction endonuclease Mae π (see also, Figure 1). A "PCR primer" is one of a pair of oligonucleotides complementary to a subregion of a nucleic acid diat, when annealed to d e nucleic acid, generates a double stranded sequence which supports nucleic acid syntiiesis by primer extension in a PCR reaction. DETAILED DISCUSSION OF THE INVENTION The present invention provides compositions, mediods and kits for detecting point mutations in DPD nucleic acid samples. Genetic amplification techniques are used to detect point mutations in a splicing region of the DPD gene which results in die deletion of an exon in die DPD gene product, and causes one form of DPDD (DPD deficiency). Isolation of nucleic acid samples To amplify a target intron-exon boundary in a sample, die nucleic acid encoding die sequence is made accessible to die components of the amplification system. In general, tiiis accessibility is ensured by isolating d e nucleic acids from d e sample, however, isolation is optional (mediods for amplifying nucleic acids, e.g., by PCR from whole cells are known and appropriate). A variety of techniques for extracting nucleic acids from biological samples are known in the art. For example, see those described in Rotbart et al. , 1989, in PCR Technology (Erlich ed., Stockton Press, New York) and Han et al., 1987, Biochemistry 26:1617-1625. The methods described by Fries et al., Am. J. Med. Genet., 46:363-368 (1993), are also useful. Nucleic acids are isolated from cell or tissue samples from patients, and from cell culture. The culture of cells used in conjunction with die present invention, including cell lines and cultured cells from tissue or blood samples, including stem cells is well known in die art. Freshney (Culture of Animal Cells, a Manual of Basic Technique, third edition Wiley-Liss, New York (1994)) and d e references cited therein provides a general guide to die culture of cells. See also, Kuchler et al. (1977) Biochemical Methods in Cell Culture and Virology, Kuchler, R.J., Dowden, Hutchinson and Ross, Inc, and Inaba et al. (1992) J. Exp. Med. 176, 1693-1702. The mediods of die present invention are employed for analyzing nucleic acid from a variety of different tissues, such as tissues samples (e.g., fetal and amniotic fluid tissue) and patient-derived tissue culture cell lines (e.g., fibroblast cell lines). Where blood cells are used, whole blood should be drawn with an anticoagulant in a sealed vacuum tube kept separated from otiier samples and handled with clean gloves. For best results, blood is processed immediately after collection; if this is impractical, it is kept in a sealed container until use. Cells in otiier physiological fluids, such as amniotic fluid, are also optionally assayed. When using any of these fluids, die cells in d e fluid are typically separated from tile fluid component by centrifugation. Several mediods are particularly well suited for use in amplifying target regions. The choice between these mediods is typically governed by d e size of die sample which is available for testing. One method is crude extraction which is useful for relatively large samples. Briefly, mononuclear cells from samples of blood, buffy coat, amniocytes from amniotic fluid, cultured chorionic villus cells, or die like are isolated by standard procedures (see, Freshney, supra). If testing is not performed immediately after sample collection, aliquots of cells can be pelletted in sterile Eppendorf tubes and the dry pellet frozen at -20 °C or colder until use. The blood cells are resuspended (typically from 10 to about 10* nucleated cells per 100 μl to 1 ml) in a buffer of 50 mM Tris-HCl (pH 8.3), 50 mM KC1 1.5 mM MgCl 2 , 0.5% Tween 20, 0.5% NP40 supplemented with 100 μg/ml of proteinase K. After incubating at 56°C for 2 hr, the cells are heated to 95 °C for 10 min to inactivate the proteinase K and immediately moved to wet ice (snap-cool). If gross aggregates are present, anotiier cycle of digestion in die same buffer should be undertaken. Ten μl of this extract is generally used for amplification. When extracting DNA from tissues, e.g. , chorionic villus cells or confluent cultured cells, die amount of die above mentioned buffer with proteinase K varies according to die size of d e tissue sample. The extract is incubated for 4-10 hrs at 50°- 60°C and then at 95 °C for 10 minutes to inactivate die proteinase. During longer incubations, fresh proteinase K should be added after about 4 hr at the original concentration. When the sample contains a small number of cells, extraction may be accomplished, e.g., by mediods as described in Higuchi, "Simple and Rapid Preparation of Samples for PCR", in PCR Technology, Ehrlich, H.A. (ed.), Stockton Press, New York, which is incorporated herein by reference. A relatively easy procedure for extracting DNA for amplification is a "salting out" procedure adapted from die method described by Miller et al., Nucleic Acids Res. , 16:1215 (1988). Mononuclear cells are separated on a Ficoll-Hypaque gradient. The cells are resuspended in 3 ml of lysis buffer (10 mM Tris-HCl, 400 mM NaCl, 2 mM Na 2 EDTA, pH 8.2). Fifty μl of a 20 mg/ml solution of proteinase K and 150 μl of a 20% SDS solution are added to die cells and tiien incubated at 37° C overnight. Rocking die tubes during incubation will improve he digestion of die sample. If die proteinase K digestion is incomplete after overnight incubation (fragments are still visible), an additional 50 μl of die 20 mg/ml proteinase K solution is mixed in die solution and incubated for anodier night at 37°C on a gently rocking or rotating platform. Following adequate digestion, one ml of a 6M NaCl solution is added to the sample and vigorously mixed. The resulting solution is centrifuged for 15 minutes at 3000 rpm. The pellet contains d e precipitated cellular proteins, while d e supernatant contains d e DNA. The supernatant is removed to a 15 ml tube that contains 4 ml of isopropanol. The contents of d e tube are mixed gently until die water and die alcohol phases have mixed and a white DNA precipitate has formed. The DNA precipitate is removed and dipped in a solution of 70% edianol and mixed gentiy. The DNA precipitate is removed from d e ethanol and air dried. The precipitate is placed in distilled water and dissolved. Kits are also commercially available for die extraction of high-molecular weight (i.e., genomic) DNA. These kits include Genomic Isolation Kit A.S.A.P. (Boehringer Mannheim, Indianapolis, IN), Genomic DNA Isolation System (GIBCO BRL, Gaithersburg, MD), Elu-Qui DNA Purification Kit (Schleicher & Schuell, Keene, NH), DNA Extraction Kit (Stratagene, La Jolla, CA), TurboGen Isolation Kit (Invitrogen, San Diego, CA), and the like. Use of these kits according to d e manufacturer's instructions is generally acceptable for purification of DNA when practicing die mediods of d e present invention. The concentration and purity of the extracted DNA may be determined by spectrophotometric analysis of die absorbance of a diluted aliquot at 260 nm and 280 nm. Eitiier 10 μl of crude extract, or 1 μg of purified DNA by d e alternate mediods are used for amplification. PCR Amplification of the Intronr£xon Boundaries of the DPD gene The nucleic acids at intron-exon boundaries are typically amplified when deteπruning whetiier a splicing defect is present in a sample. In a preferred embodiment, amplification is performed by die PCR metiiod. Although die PCR process is well known in the art (see, U.S. Patent Nos. 4,683,195; 4,683,202; and 4,965,188, each of which is incorporated herein by reference) and altiiough commercial vendors, such as Roche Molecular Systems, sell PCR reagents and publish PCR protocols, some general PCR information is provided below for purposes of clarity and full understanding of die invention for tiiose unfamiliar with die PCR process. In brief, a DNA sample is heat denatured to separate the individual complementary strands and annealed in die presence a pair of oligonucleotide primers which are complementary to sequences present on opposite stands and located on either side of the sequence to be detected. The PCR mixture is then subjected to repeated cycles of DNA synthesis, denaturation and reannealing. The first step of each cycle of die PCR involves de separation of die nucleic acid duplex formed by die primer extension. Once die strands are separated, d e next step in PCR involves hybridizing die separated strands with primers diat flank die target sequence. The primers are then extended to form complementary copies of the target strands. For successful PCR amplification, die primers are designed so d at die position at which each primer hybridizes along a duplex sequence is such diat an extension product syndiesized from one primer, when separated from die template (complement), serves as a template for die extension of die other primer. The cycle of denaturation, hybridization, and extension is repeated as many times as necessary to obtain d e desired amount of amplified nucleic acid. In a preferred embodiment of die PCR process, strand separation is achieved by heating the reaction to a sufficiently high temperature for an sufficient time to cause the denaturation of d e duplex but not to cause an irreversible denaturation of die polymerase (see U.S. Patent No. 4,965,188). Typical heat denaturation involves temperatures ranging from about 80°C to 105°C for times ranging from seconds to minutes. Strand separation, however, can be accomplished by any suitable denaturing metiiod including physical, chemical, or enzymatic means. Strand separation may be induced by a helicase, for example, or an enzyme capable of exhibiting helicase activity. For example, die enzyme RecA has helicase activity in die presence of ATP. The reaction conditions suitable for strand separation by helicases are known in die art (see Kuhn Hoffman-Berling, 1978, CSH-Quantitative Biology 43:63-67; and Radding, 1982, Ann. Rev. Genetics 16:405-436, both of which are incorporated herein by reference). Template-dependent extension of primers in PCR is catalyzed by a polymerizing agent in the presence of adequate amounts of four deoxyribonucleoside triphosphates (typically dATP, dGTP, dCTP, and dTTP) in a reaction medium comprised of die appropriate salts, metal cations, and pH buffering system. Suitable polymerizing agents are enzymes known to catalyze template-dependent DNA syntiiesis. In some instances, DPD-encoding RNA may be used as die initial template for primer extension is RNA. Polymerizing agents suitable for synthesizing a complementary, DNA (cDNA) sequence from die RNA template are reverse transcriptase (RT), such as avian myeloblastosis virus RT, Moloney murine leukemia virus RT, or Thermits thermophilus (Tth) DNA polymerase, a tiiermostable DNA polymerase with reverse transcriptase activity marketed by Roche Molecular Systems Typically, the RNA template is heat degraded during d e first denaturation step after the initial reverse transcription step leaving only DNA template. Suitable polymerases for use with a DNA template include, for example, E. co DNA polymerase I or its Klenow fragment, T 4 DNA polymerase, 7ϊh polymerase, and Taq polymerase, a heat-stable DNA polymerase isolated from Thermus aquaticus and commercially available from Roche Molecular Systems The latter enzyme is widely used in d e amplification and sequencing of nucleic acids. The reaction conditions for using Taq polymerase are known in die art and are described in Innis, supra. When RNA is amplified, an initial reverse transcription (RT) step is carried out to create a DNA copy (cDNA) of the RNA. PCT patent publication No. WO 91/09944, published July 11, 1991, incorporated herein by reference, describes high-temperature reverse transcription by a tiiermostable polymerase diat also functions in PCR amplification. High-temperature RT provides greater primer specificity and improved efficiency. A "homogeneous RT-PCR" in which d e same primers and polymerase suffice for both d e reverse transcription and the PCR amplification steps, and die reaction conditions are optimized so diat botii reactions occur without a change of reagents is also available. Thermus thermophilus DNA polymerase, a tiiermostable DNA polymerase diat can function as a reverse transcriptase, is used for all primer extension steps, regardless of template. Both processes can be done without having to open die tube to change or add reagents; only die temperature profile is adjusted between die first cycle (RNA template) and die rest of die amplification cycles (DNA template). The PCR metiiod can be performed in a step-wise fashion, where after each step new reagents are added, or in a fashion where all of die reagents are added simultaneously, or in a partial step-wise fashion, where fresh or different reagents are added after a given number of steps. For example, if strand separation is induced by heat, and die polymerase is heat-sensitive, then die polymerase is added after every round of strand separation. However, if, for example, a helicase is used for denaturation, or if a thermostable polymerase is used for extension, then all of d e reagents are added initially, or, alternatively, if molar ratios of reagents are of consequence to die reaction, die reagents may be replenished periodically as they are depleted by die synthetic reaction. Those skilled in the art will know d at die PCR process is most usually carried out as an automated process with a thermostable enzyme. In tiiis process, die temperature of die reaction mixture is cycled through a denaturing region, a primer annealing region, and an extension reaction region. Alternatively, die annealing and extension temperature can be die same. Reverse transcriptase-PCR uses such a two-step temperature cycling. A machine specifically adapted for use with a tiiermostable enzyme is commercially available from Roche Molecular Systems. Those skilled in die art will also be aware of the problems of contamination of a PCR by the amplified nucleic acid from previous reactions and nonspecific amplification. Methods to reduce these problems are provided in PCT patent application Serial No. 91/05210, filed July 23, 1991, incorporated herein by reference. The method allows the enzymatic degradation of any amplified DNA from previous reactions and reduces nonspecific amplification. The PCR amplification is carried out in die presence of dUTP instead of dTTP. The resulting double-stranded, uracil-containing product is subject to degradation by uracil N-glycosylase (UNG), whereas normal thy mine-containing DNA is not degraded by UNG. Adding UNG to d e amplification reaction mixture before the amplification is started degrades all uracil-containing DNA diat might serve as target. Because die only source of uracil-containing DNA is d e amplified product of a previous reaction, this method effectively sterilizes die reaction mixture, eliminating die problem of contamination from previous reactions (carry-over). UNG itself is rendered temporarily inactive by heat, so the denaturation steps in the amplification procedure also serve to inactivate the UNG. New amplification products, therefore, though incorporating uracil, are formed in an effectively UNG-free environment and are not degraded. Those practicing die present invention should note that, although the preferred embodiment incorporates PCR amplification, amplification of target sequences in a sample may be accomplished by any known metiiod, such as ligase chain reaction (LCR), transcription amplification, and self-sustained sequence replication, each of which provides sufficient amplification so diat the target sequence can be detected by nucleic acid hybridization to a probe. Persons of skill will appreciate that in methods such as LCR, primers that are complementary to the specific polymorphism or mutation are used. In this instance amplification occurs when the polymorphism (i.e., point mutation) is present in the nucleic acid sample. Alternatively, methods that amplify the probe to detectable levels can be used, such as Qø-replicase amplification. The term "probe" encompasses, inter alia, die sequence specific oligonucleotides used in die above procedures; for instance, die two or more oligonucleotides used in LCR are "probes" for purposes of the present invention, even though some embodiments of LCR only require ligation of the probes to indicate die presence of an allele. Examples of techniques sufficient to direct persons of skill through such in vitro amplification methods, including d e polymerase chain reaction (PCR) die ligase chain reaction (LCR), Q3-replicase amplification and other RNA polymerase mediated techniques (e.g. , NASBA) are found in Berger and Kimmel, Guide to Molecular Cloning Techniques, Mediods in Enzymology volume 152 Academic Press, Inc., San Diego, CA (Berger); Sambrook et al. (1989) Molecular Cloning - A Laboratory Manual (2nd ed.) Vol. 1-3, Cold Spring Harbor Laboratory, Cold Spring Harbor Press, NY, (Sambrook); and Current Protocols in Molecular Biology, F.M. Ausubel et al., eds., Current Protocols, a joint venture between Greene Publishing Associates, Inc. and John Wiley & Sons, Inc., (1994 Supplement) (Ausubel), and in Muilis et al. , (1987) U.S. Patent No. 4,683,202; PCR Protocols A Guide to Methods and Applications (Innis et al. eds) Academic Press Inc. San Diego, CA (1990) (Innis); Amheim & Levinson (October 1, 1990) C&EN 36-47; The Journal Of N1H Research (1991) 3, 81-94; (Kwoh et al. (1989) Proc. Natl Acad. Sci. USA 86, 1173; Guatelli et al. (1990) Proc. Natl Acad. Sci. USA 87, 1874; Lomell et al. (1989) J. Clin. Chem 35, 1826; Landegren et al, (1988) Science 241, 1077-1080; Van Brunt (1990) Biotechnology 8, 291-294; Wu and Wallace, (1989) Gene 4, 560; Barringer et al. (1990) Gene 89, 117, and Sooknanan and Malek (1995) Biotechnology 13: 563-564. Improved mediods of cloning in vitro amplified nucleic acids are described in Wallace et a\., U.S. Pat. No. 5,426,039. The present invention provides, inter alia, a polymorphic form of DPD. In particular, die invention provides a DPDD polymorphism wherein the G at position 434 of the gene sequence shown in Figure 1 is replaced by an A. This particular polymorphism is detected by a variety of amplification techniques, preferably PCR as described supra. To detect this polymorphism by PCR, die PCR reaction is performed in die presence of primers that are complimentary to opposite strands of die genomic DNA, wherein die complementary sequences are located on either side of die point mutation. The precise sequences recognized by die primers are not critical. Typically, any pair of primers can be used as long as they (1) bracket the polymorphism, (2) are reasonably near to die polymorphism (while die primer binding sequence may be as far from die polymorphism as can support a PCR reaction, i.e. , 1 to about 10 kb, it is preferable that the binding sequence be within about 500 nucleotides or less, and more preferable diat the binding sequence be within 100 nucleotides of die exon- intron boundary to be assayed), and (3) bind die primers with an adequate degree of specificity. It is preferable that the sequence be unique to die gene of interest. Such sequences are identified by comparing sequences as described herein. Smaller primers have a higher probability of recognizing sites outside of die desired binding site, whereas very large primers are more expensive to make; generally, a primer of about 15-20 nucleotides is adequate, and therefore preferred. Example primers used herein are DELF1, which hybridizes to nucleic acids corresponding to nucleotide 154 to nucleotide 175 of die DPD gene sequence, and DELRl, which hybridizes to nucleic acids corresponding to nucleotide 563 to nucleotide 542 of die DPD gene sequence as described in Figure 1. It is important to note diat other 15-20 nucleotide sequences within 500 nucleotides of the point mutation are also acceptable. Given the sequence of die affected exon, adjacent introns, and adjacent additional exons provided herein, selecting and making alternate primers is within die skill of a skilled practitioner in the relevant art. M Λ WO 97/35034 16 The present invention also provides kits for die detection of genetic polymorphisms or mutations associated with DPDD. The kits comprise a vial containing amplification primers that span a DPDD-associated polymorphism or mutation in the gene encoding DPD. For example, die vial optionally contains DELF-l and DELR-1. The kits optionally contain a vial containing a thermostable polymerase, genetic size markers for gels, amplification reagents, instructions and the like. Sequence analysis of Amplification Products A variety of methods are employed to analyze die nucleotide sequence of die amplification products. Several techniques for detecting point mutations following amplification by PCR have been described in Chehab et al. , Methods in Enzymology, 216:135-143 (1992); Maggio et al , Blood, 81(l):239-242 (1993); Cai and Kan, Journal of Clinical Investigation, 85(2):550-553 (1990) and Cai et al. , Blood, 73:372-374 (1989). One particularly useful technique is analysis of restriction enzyme sites following amplification. In this method, amplified nucleic acid segments are subjected to digestion by restriction enzymes. Identification of differences in restriction enzyme digestion between corresponding amplified segments in different individuals identifies a point mutation. Differences in die restriction enzyme digestion is commonly determined by measuring the size of restriction fragments by electrophoresis and observing differences in die electrophoretic patterns. Generally, the sizes of the restriction fragments is determined by standard gel electrophoresis techniques as described in Sambrook, and, e.g. , in Polymeropoulos et al, Genomics, 12:492-496 (1992). The size of die amplified segments obtained from affected and normal individuals and digested with appropriate restriction enzymes are analyzed on agarose or poiyacrylamide gels. Because of die high discrimination of die poiyacrylamide gel electrophoresis, differences of small magnitude are easily detected. Other mutations resulting in DPDD-related polymorphisms of DPD encoding genes also add unique restriction sites to die gene diat are determined by sequencing DPDD-related nucleic acid sequences and comparing diem to normal sequences. Another useful method of identifying point mutations in PCR amplification products employs oligonucleotide probes specific for different sequences. The oligonucleotide probes are mixed with amplification products under hybridization conditions. Probes are either RNA or DNA oligonucleotides and optionally contain not only naturally occurring nucleotides but also analogs such as digoxygenin dCTP, biotin dCTP, 7-azaguanosine, azidotiiymidine, inosine, or uridine. The advantage of using nucleic acids comprising analogs include selective stability, resistance to nuclease activity, ease of signal attachment, increased protection from extraneous contamination and an increased number of probe-specific colored labels. For instance, in preferred embodiments, oligonucleotide arrays are used for die detection of specific point mutations as described below. Probes are typically derived from cloned nucleic acids, or are synthesized chemically. When cloned, die isolated nucleic acid fragments are typically inserted into a replication vector, such as lambda phage, pBR322, M13, pJB8, c2RB, pcoslEMBL, or vectors containing die SP6 or T7 promoter and cloned as a library in a bacterial host. General probe cloning procedures are described in Arrand J.E., Nucleic Acid Hybridization A Practical Approach, Hames B.D., Higgins, S.J., Eds., IRL Press 1985, pp. 17-45 and Sambrook, J., Fritsch, E.F., Maniatis, T., Molecular Cloning A Laboratory Manual, Cold Spring Harbor Press, 1989, pp. 2.1-3.58, both of which are incorporated herein by reference. Oligonucleotide probes and primers are synthesized chemically with or without fluorochromes, chemically active groups on nucleotides, or labeling enzymes using commercially available mediods and devices like die Model 380B DNA syndiesizer from Applied Biosystems, Foster City, California, using reagents supplied by die same company. Oligonucleotides for use as probes, e.g., in in vitro amplification mediods, or for use as gene probes are typically synthesized chemically according to the solid phase phosphoramidite triester metiiod described by Beaucage and Caruthers (1981), Tetrahedron Letts. , 22(20): 1859-1862, e.g., using an automated synthesizer, as described in Needham-VanDevanter et al. (1984) Nucleic Acids Res., 12:6159-6168. Oligonucleotides can also be custom made and ordered from a variety of commercial sources known to persons of skill. Purification of oligonucleotides, where necessary, is typically performed by eitiier native acrylamide gel electrophoresis or by anion-exchange HPLC as described in Pearson and Regnier (1983) J. Chrom. 255:137-149. The sequence of the synthetic oligonucleotides can be verified using die chemical degradation method of Maxam and Gilbert (1980) in Grossman and Moldave (eds.) Academic Press, New York, Methods in Enzymology 65:499-560. Oligonucleotide probes and primers are selected using commercially available computer programs to compare known DNA sequences from gene sequences found in gene libraries, such as Genebank and EMBL, and die sequences described herein. The programs identify unique nucleotide sequences within die gene of interest. One such program is Eugene. Oligonucleotide sequences for PCR of a unique genomic DNA such as a chromosome subsequence are chosen optimally by choosing sequences according to previously established protocols or by computer programs diat choose die degree of homology desired along with die length of the probe. Sequences are chosen to avoid technical problems such as primer dimers resulting from amplification of hybridized primers. Primers and probes are optionally labeled with fluorophores or enzymes diat generate colored products. This allows simultaneous use of probes to different DPDD- related polymorphisms or mutations. Identification of hybridization of a specifically labelled primer provides a means for determining which polymorphism or mutation is present in the nucleic acid of die sample. The primers used in the assay are labeled with more than one distinguishable fluorescent or pigment color. Primers are labeled with Texas red, rhodamine and its derivatives, fluorescein and its derivatives, dansyl, umbelliferone and die like or with horse radish peroxidase, alkaline phosphatase, biotin, avidin, or the like. Primers and probes are labeled directly or indirectly. The common indirect labeling schemes covalentiy bind a ligand to die nucleotide and prepare labeled probe by incorporating die ligand using random priming or nick translation. The ligand tiien binds an anti-ligand which is covalently bound to a label. Ligands and anti-ligands vary widely. When a ligand has an anti-ligand, e.g. , biotin, thyroxine, or cortisol, die ligand is used in conjunction with die labelled naturally-occurring anti-ligand. Alternatively, a hapten or antigen may be used in combination with an antibody, which is optionally labeled. Sequence specific oligonucleotide probes hybridize specifically with a particular segment of he target polymorphism or mutation amplification products and have destabilizing mismatches with die sequences from other polymorphisms or mutations. Under sufficiently stringent hybridization conditions, die probes hybridize specifically only to exactly complementary sequences. The stringency of die hybridization conditions can be relaxed to tolerate varying amounts of sequence mismatch. Detection of the amplified product utilizes this sequence-specific hybridization to insure detection of only die correct amplified target, thereby decreasing die chance of a false positive caused by die presence of homologous sequences from related polymorphisms or mutations. Specific DPD-encoding polymorphisms or mutations are also identified by sequencing die amplification products or restriction fragments diereof. Sequencing is performed by a variety of mediods well known in die art. For example, the sequence of the amplified nucleic acid segments may be determined by die Maxam-Gilbert chemical degradation metiiod as described in Sambrook. Generally, Sanger dideoxy-mediated sequencing is employed as described in Sambrook, or sequencing by hybridization is performed as described below. Detection of Splicing Site Defects Using Nucleic acid Arrays In one preferred class of embodiments, splicing site defects at intron-exon boundaries are detected by hybridization of amplification products which include die splicing site to oligonucleotide arrays which discriminate single base-pair mismatches. In this embodiment, primers are used to amplify an intron-exon boundary in a PCR reaction, resulting in PCR amplicons which comprise the intron-exon boundary. The sequence of die entire PCR amplicon, or any subsequence diereof can be determined by labeling die PCR amplicon (typically with biotin or a fluorescent label) and hybridization to an array of oligonucleotide probes. In these hybridization mediods single base pair mismatches in labeled nucleic acids to probes in die array are distinguished. Preferably in this class of embodiments, die oligonucleotide arrays are designed to sequence nucleic acids at die intron-exon boundary. More preferably, the arrays are designed to discriminate whether a particular nucleotide is altered relative to the wild-type sequence. This is done by constructing an array with two or more oligonucleotide probe sets which differ by a single nucleotide. Hybridization to he known probe sequence by a target nucleic acid under conditions where a single mismatch does not bind indicates die presence of a fully complementary nucleic acid. Sequencing by hybridization to arrays of oligonucleotides is described in U.S. Patent No. 5,202,231, to Drmanac et al. and, e.g., in Drmanac et al. (1989) Genomics 4: 114-128. Mediods of constructing and designing arrays for sequencing and detection of single nucleotide alterations is known in die art. The development of very large scale immobilized polymer synthesis (VLSIPS™) technology provides mediods for arranging large numbers of oligonucleotide probes for die detection and sequencing of nucleic acids in very small arrays. See, WO 90/15070 and 92/10092; Pirrung et al. , U.S. Patent No. 5,143,854 (see also PCT Application No. WO 90/15070); McGall et al. , U.S. Patent No. 5,412,087; and US Pat. No. 5,384,261. See also, Fodor et al. (1991) Science, 251: 767-777 and Sheldon et al. (1993) Clinical Chemistry 39(4): 718-719. The oligonucleotide arrays are typically placed on a solid surface such as a glass slide with an area less than 1 inch 2 , although much larger surfaces are optionally used. Mechanical and Light directed oligonucleotide array construction methods are used for die construction of oligonucleotide arrays. Light directed mediods are die most common, and are found, e.g., in U.S. Patent No. 5,143,854. The light directed mediods discussed in die '854 patent typically proceed by activating predefined regions of a substrate or solid support and then contacting die substrate with a preselected monomer solution. The predefined regions are activated with a light source, typically shown through a photolithographic mask. Otiier regions of die substrate remain inactive because tiiey are blocked by die mask from illumination. Thus, a light pattern defines which regions of die substrate react with a given nucleic acid reagent. By repeatedly activating different sets of predefined regions and contacting different reagent solutions with the substrate, a diverse array of oligonucleotides is produced on the substrate. Outer steps, such as washing unreacted reagent solutions from die substrate, are used as necessary. The surface of a solid support is typically modified with linking groups having photolabile protecting groups and illuminated through a photolithographic mask, yielding reactive hydroxyl groups in die illuminated regions. For instance, during oligonucleotide synthesis, a 3'-O- phosphoramidite (or otiier nucleic acid synthesis reagent) activated deoxynucleoside (protected at die 5'-hydroxyl with a photolabile group) is tiien presented to die surface and coupling occurs at sites diat were exposed to light in die previous step. Following capping, and oxidation, die substrate is rinsed and die surface iUvminated through a second mask, to expose additional hydroxyl groups for coupling. A second S'-protected, 3'-O-phosphoramidite activated deoxynucleoside (or other monomer as appropriate) is then presented to die resulting array. The selective photodeprotection and coupling cycles are repeated until the desired set of oligonucleotides (or other polymers) is produced. The PCR amplicons detected on die arrays are labeled with a composition detectable by spectroscopic, photochemical, biochemical, immunochemical, or chemical means. For example, useful labels include 32P, 35S, fluorescent dyes, chromophores, electron-dense reagents, enzymes (e.g., as commonly used in an ELISA), biotin, dioxigenin, or haptens and proteins for which anϋsera or monoclonal antibodies are available. In preferred embodiments, die label is detectable spectroscopically, i.e. , is chromogenic. Suitable chromogens include molecules and compounds which absorb light in a distinctive range of wavelengths so diat a color may be observed, or emit light when irradiated with radiation of a particular wavelengtii or wavelength range (e.g., a fluorescent label). EXAMPLES The following examples are offered by way of illustration and not limitation. One of skill will readily recognize a variety of parameters and conditions which can be changed or modified to yield essentially identical results. The following example pinpoints a polymoφhism or "mutation" of die gene encoding DPD diat is associated with DPDD, and provides a novel metiiod for die efficient detection of this DPDD splicing mutation. Exampfø 1 The PCR reaction is performed using human genomic DNA obtained from fibroblast cultures of 5 members of a family, one of which was afflicted with DPDD. The PCR primers were selected so as to bracket die exon diat is not present in die DPDD gene and part of the two introns on either side of this exon. Primers were synthesized using an Applied Biosystems 394 DNA & RNA syndiesizer. The forward primer was DELF1, which encompassed nucleotide 154 to nucleotide 175 of die DPFD gene sequence and had the sequence TGCAAATATGTGAGGAGGGACC (see also Figure 1 and Figure 2). The reverse primer was DELRl, which encompassed nucleotide 563 to nucleotide 542 of die DPFD gene sequence and had the sequence CAAAGCAACTGGCAGATTC (see also Figure 1 and Figure 2). PCR was carried out in 50 μl of a reaction mixture consisting of 10 mM Tris-HCl pH 8.3, 50 mM KC1, 2.5 mM MgCl 2 , 0.5 mM dNTPs, 1 μM of each primer, and 2.5 units Taq polymerase (Roche Molecular Systems) for 30 cycles denaturing at 96°C for 1 min, annealing at 55 °C for 1 min, and extending at 72°C for 2 min. The amplified products were extracted witii 1 volume of chloroform and purified by filtration through Centricon 100 filter units. Typically, one-fifth of the purified PCR product was used for sequence analyses witii an Applied Biosystems 373A automated sequencer and fluorescent dye-deoxy terminator chemistry. Sequence data were analyzed using the MacVector sequence analysis software (International Biotechnology). The PCR fragments were analyzed by electrophoresis in a 1 % agarose gel stained with etiiidium bromide. For Southern blot analysis, die DNA in the gels were depurinated by a 20-min incubation in 200 mM HCI and denatured by a 20-min incubation in 0.5 M NaOH. The DNA was transferred to Gene Screen Plus nylon membranes (New England Nuclear) overnight in 0.5 M NaOH, and was fixed to die filter by baking for 90 min at 80°C. Prehybridization was done in a solution of 6 x SSC, Denhardt's reagent, 0.5% NaDodSO 4 , and 0.2 mg/ml sonicated salmon sperm DNA at 65 °C for 3 hr, tiien hybridization was carried out overnight at 65 °C in the same solution containing 1.5 z 10* cpm/ml of 32 P random priming-labeled human DPD, cDNA. After washing at 65 β C for 20 min in 2 x SSC, 0.5% NaDodSO 4 , and 45 min in 0.1 x SSC, 0.5% NaDodSO 4 at 65 °C, the membranes were exposed to X-ray film (Eastman Kodak Co.) at -80°C for 30 min. The results of sequencing die various genomic DNAs reveal die presence of a G to A mutation at position 434. This mutation was located within die splice site. The DPDD patient was homozygous for this mutation. Both parents and one sibling were heterozygous and another was homozygous for die normal allele. The G to A mutation resulted in die destruction of a restriction site recognized by restriction endonucleases which recognize die Mae II binding sequence, ACGT. Consequendy, after PCR amplification of genomic DNA using die primers DELF1 and DELRl, digestion of die resulting PCR product with a restriction endonuclease diat cleave die Maell site yields fragments having die following sizes: Normal allele Mutant allelg 278 + 131 bp 408 bp Example 2: Identification of a Polymorphism which causes DPDD Family pgHfrreβ Blood samples from a British family were collected after a family member displayed excessive 5-FU toxicity during chemotherapy. This family is unrelated to any family with DPD deficiency diat has previously been reported. To screen for die presence of die mutation leading to the deficiency in die normal population, genomic DNA was collected from healthy subjects representing die following ethnic groups: Caucasians (English and Finnish), Asians (Japanese and Taiwanese) and African-Americans. DPD catalytic activity. DPD catalytic activity for the family under study was determined from peripheral blood mononuclear cells using a previously described HPLC method (McMurrough et al. (1996) Br. J. Clin. Pharmacol). In brief, peripheral blood mononuclear cells were purified from die patient's blood in a density gradient using Ficoll- Hypaque (Pharmacia, Upsala) and incubated for 90 min with [ C]-5 FU. The production of 5-FU metabolites were then quantified by HPLC analysis by radiodetection. Isolation of RNA and RT-PCR. Total RNA was isolated from peripheral blood mononuclear cells by die guanidinium thiocyanate-phenol-chloroform metiiod (Chomezynski et al (1987) Anal. Biochem. 162: 156-159). The RNA solutions were dissolved in DEPC-treated water and stored at -80'C until use. RT-PCR was performed as described (Meinsma et al. (1995) DNA Cell. Biol. 14: 1-6) and die products were analyzed by electrophoresis in 0.8% agarose gels and visualized by staining with ethidium bromide. Cloning and characterization of the genomic fragment encoding the deleted exon. The DPD cDNA was used as a probe to isolate a PI clone containing about 100 kbp of the human DPD gene (PAC 5945) from a high density PAC human genomic library (Genome Systems, St. Louis, MO). Southern blotting was used to confirm diat die PI clone contained die deleted exon using a probe located within the deleted fragment. This probe was syndiesized from die DPD cDNA by using d e primers: DPD 15 (forward): 5' TTGTGACAAATGTTTCCC 3' and DPD15R (reverse): 5' AGTCAGCCTTTAGTTCAGTGACAC 3' to specifically amplify the putative exon. PCR conditions were as indicated below but extension was carried out at 72 β C for 1 min. This PCR fragment was purified using a Wizard PCR purification kit (Promega, Madison, WI), labeled with [o^Pj-dCTP and hybridized with the clone PAC 5945. DNA was purified from this genomic clone using Qiagen columns ( iagen, Chatsworth, CA), and die 5' and 3' ends of die deleted exon and adjacent intronic regions were sequenced by chromosome walking from witiύn die deleted exon using dideoxy terminator chemistry and an ABI 373A DNA sequencer (Applied Biosystems, Foster City, CA). The intronic sequences obtained allowed die selection of appropriate primers (e.g., delFl and delRl) to amplify from genomic DNA die complete exon and immediate flanking intronic sequences. All primers used in this study were syndiesized with a 391 DNA&RNA synthesizer (Applied Biosystems, Foster City, CA). Genotyping of the mutant DPD allele. A 409 bp PCR genomic fragment corresponding to the deleted exon (from G1822 to C1986 in Yokota, et al. (1994) J. Biol. Chem. 269: 23192-23196) plus the flanking intronic sequences containing the AG and GT splicing consensus sequences was amplified from human genomic DNA using die primers delFl (forward) 5' TGCAAATATGTGAGGAGGGACC 3' and delRl (reverse) 5' CAGCAAAGCAACTGGCAGATTC 3'. PCR amplification was carried out in a 100 μl reaction mixture containing 10 mM Tris-HCl (pH 8.3), 50 mM KC1, 1.5 mM MgCl 2 , 0.2 mM of each dNTP, 0.5 μM of each primer, 2.5 units of Taq Polymerase (Roche Molecular Systems) and 500 ng of genomic DNA template for 31 cycles by denaturing at 94°C for 1 min, annealing at 60°C for 1 min and extending at 72°C for 2 min. Subjects identified as wild type, heterozygous or homozygous for die splicing mutation could be distinguished by digesting die PCR product with the restriction endonuclease Mae U (Boehringer, Mannheim, Indianapolis, IN) and electrophoresis in 1% regular, 3% NuSieve agarose gels (FMC Bioproducts, Rockland, ME). The genotypes obtained were verified by sequencing the 409 bp PCR product. The sources of the genomic DNA samples for the different ethnic groups correspond to those previously described (Fernandez-Salguero et al. (1995) Am. J. Hum. Genet. 57: 651-660). DPD catalytic activity. To determine DPD catalytic activities in die proband and family members, lymphocytes were isolated and subjected to analysis by HPLC (McMurrough et al. (1996) Br. J. Clin. Pharmacol). Activities ranged between 24% to 70% of the mean DPD activity determined in die normal population (155 pmole/min/mg protein) (Table 1). It should be noted diat all die subjects in die pedigree presented a relatively low catalytic activity (below 70% of die mean in die normal population). Subject II, a cancer patient who exhibited 5-FU toxicity, had catalytic activity which was 24% of die mean population. RT-PCR analysis. RT-PCR was performed on total RNA isolated from peripheral blood mononuclear cells corresponding to each of die members of die pedigree shown in Figure I. The full length coding region of the DPD cDNA was amplified in tiiree fragments of 1.5 kbp, 906 bp and 919 bp. The 1.5 kbp and 906 bp PCR fragments were detected in all die members of die family and correspond to die normal mRNA as expected from die cDNA sequence. A 741 bp band corresponded to the deleted exon and was heterozygous with respect to die wild type 906 bp fragment. Sequencing of this 741 bp PCR fragment confirmed diat a 165 bp deletion has occurred that precisely corresponds to diat previously reported in a Dutch family (Meinsma et al. (1995) DNA Cell Biol 14: 1-6). No homozygous mutant subjects were found in this pedigree. The generation of the mutant mRNA is illustrated in Figure 3. Faulty splicing is die mechanism for the generation of the shorter mRNA, which due to die absence of 55 amino acids would be unable to be translated into an intact DPD enzyme. Genomic DNA sequence analysis of the mutant DPD allele. A clone containing die deleted exon was isolated in a recombinant PI phage. Sequence of die intronic regions flanking he deleted exon enabled die development of a PCR reaction to sequence die exon and across die 5' and 3' splicing sites. Sequence of die 409 bp PCR genomic fragment clearly indicated die presence of a G to A point mutation (GT to AT) at die 5* donor splice consensus sequence in die British pedigree in subjects II, 112, TJ3, JJ4, 117, 118, JJ9, III6, HT8 and UJ9. Subjects πi, H.6, mi, HT2, IH.4 and IH7 were found to have two normal alleles. The subject previously reported to have die 165 bp deletion present in both alleles and his heterozygote brotiier (subjects 1 and 2 in Meinsma et al reference) were also analyzed and found to possess die splicing mutation „ M Λ WO 97/35034 26 present in both alleles for subject 1 and in one allele for subject 2. The G to A mutation correlates 100% with the corresponding RT-PCR results for die deletion of die entire exon, indicating that the CT to AT mutation in the 5' splicing consensus sequence of die DPD gene leads to skipping of die entire preceding exon. The genotypes and catalytic activities for this 5' -splicing mutation in the pedigree are summarized in Table I. Genotyping test for the mutant DPD allele and population screening. The 409 bp genomic PCR fragment containing the deleted exon has a restriction site for die endonuclease Mae II ( A-CGTϊ which produces two fragments of 278 bp and 131 bp from die wild type allele. This site is eliminated when a G to A mutation occurs at die S'-splicing site ( A-CAT). A diagrammatic representation of die strategy developed for PCR amplification of the 409 bp fragment and die restriction enzyme pattern expected with MAE II is shown in Figure 4. The reliability of die genotyping assay was confirmed by sequencing die 409 bp PCR product in these samples. By analyzing normal subjects witiiin different ethnic groups (Table II), we found diat this mutant allele is present in die Finnish (4.4%) and Taiwanese (5.4%) populations where heterozygotes were detected. However, within die samples tested to date, no mutant alleles were found in 35 Japanese or 20 African-American subjects. All heterozygous subjects found by restriction enzyme analysis were also confirmed by genomic DNA sequence analysis. TABLE I: Relationship between phenotype and genotype for the DPD catalytic activity and the presence of the 5 '-splicing mutation in the pedigree analyzed. Family Member DPD activity (%)' Genotype' 11 24 +/- πi 57 +/+ m 39 +/- m 36 +/- m 42 +/- us n/d n/d 116 35 +/+ TΠ 58 +/- 118 22 +/- 119 28 +/- mi 55 +/+ IH2 60 +/+ UJ3 n/d n/d IH4 60 +/+ m5 n/d n/d IH6 36 +/- I1T7 70 +/+ m8 31 +/- m9 56 +/- * DPD activity is expressed as percentage of the mean activity in the normal population. ' Genotype for the GT to AT splice mutation: +/+: wild type; +/-: heterozygote; /: homozygote; n/d: not determined TABLE YL'Allelic frequency for the presence of the 5 '-splicing mutation in different ethnic groups from the normal population Wild type Heterozygote Homozygote Subject* (+/+) (-/-) Caucasian British 30 100 (30)* 0 (0)* 0 (0)' Finnish 45 95.6 (43) 4.4 (2) 0 (0) Asian Taiwanese 37 94.6 (35) 5.4 (2) 0 (0) Japanese 35 100 (35) 0 (0) 0 (0) African- 20 100 (20) 0 (0) 0 (0) American The allelic frequency is indicated as a percentage of die total number of subjects analyzed. The absolute number of heterozygote subjects are indicated in parenthesis. All publications, patents and patent applications mentioned in this specification are herein incorporated by reference into die specification to the same extent as if each individual publication, patent or patent application was specifically and individually indicated to be incorporated herein by reference. Altitough the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, it will be obvious diat certain changes and modifications may be practiced within the scope of die appended claims.

Claims: WHAT IS CLAIMED IS: 1. A method of detecting a splicing defect in a dihydropyrimidine dehydrogenase gene, comprising determining whetiier a genomic DNA encoding die dihydropyrimidine dehydrogenase gene has a wild-type intron-exon boundary for an exon which encodes amino acids 581-635 of a corresponding wild-type dihydropyrimidine dehydrogenase protein. 2. The metiiod of claim 1, wherein die method comprises die step of amplifying intronic genomic DNA encoding die dihydropyrimidine dehydrogenase in die region flanking the exon which encodes amino acids 581-635. 3. The method of claim 2, wherein the method comprises amplifying die genomic DNA with a primer which hybridizes to a dihydropyrimidine dehydrogenase intronic nucleic acid which hybridizes to a primer selected from the group of primers consisting of DELF1, and DELRl under stringent conditions. 4. The method of claim 2, wherein DNA amplified widi the primers is cleaved with a restriction endonuclease which recognizes a Mae II cleavage site. 5. The method of claim 1 , wherein die sequence of die intron-exon boundary is determined using an oligonucleotide array. 6. A metiiod of screening patients for sensitivity to 5-fluorouricil, comprising isolating a genomic DNA from the patient which encodes die dihydropyrimidine dehydrogenase gene and determining whether the gene has a wild-type intron-exon boundary for an exon which encodes amino acids 581-635 of a corresponding wild-type dihydropyrimidine dehydrogenase protein. 7. The method of claim 6, wherein the method comprises die step of amplifying intronic genomic DNA encoding the dihydropyrimidine dehydrogenase in die region flanking die exon which encodes amino acids 581-635. 8. The metiiod of claim 7, wherein die metiiod comprises amplifying die genomic DNA with a primer which hybridize to a dihydropyrimidine dehydrogenase intronic nucleic acid which hybridizes to a primer selected from the group of primers consisting of DELF1 or DELRl under stringent conditions. 9. The method of claim 7, wherein DNA amplified with the primers is cleaved with a restriction endonuclease which recognizes a Mae II cleavage site. 10. A composition comprising a first PCR primer which binds to DNA 3' of a splice site in the genomic DNA for dihydropyrimidine dehydrogenase gene for an exon encoding amino acids 581-635, and a second PCR primer which binds to DNA 5' of a splice site in die genomic DNA for dihydropyrimidine dehydrogenase gene for an exon encoding amino acids 581-635. 11. The composition of claim 10, wherein the first PCR primer binds to intronic dihydropyrimidine dehydrogenase DNA. 12. The composition of claim 10, wherein the second PCR primer binds to intronic dihydropyrimidine dehydrogenase DNA. 13. The composition of claim 10, wherein the first PCR primer hybridizes under stringent conditions to a nucleic acid complementary to DELFl. 14. The composition of claim 10, wherein die second PCR primer hybridizes under stringent conditions to a nucleic acid complementary to DELRl. 15. A kit comprising a container, a first PCR primer which binds to DNA 3' of a splice site in die genomic DNA for dihydropyrimidine dehydrogenase gene for an exon encoding amino acids 581-635, and a second PCR primer which binds to DNA 5* of a splice site in die genomic DNA for dihydropyrimidine dehydrogenase gene for an exon encoding amino acids 581-635. 16. The kit of claim 15, wherein the kit further comprises instructions for the detection of splicing site defects in the dihydropyrimidine dehydrogenase gene. 17. The kit of claim 15, wherein die kit further comprises Mae II. 18. The composition of claim 15, wherein die first PCR primer hybridizes under stringent conditions to a nucleic acid complementary to DELFl. 19. The composition of claim 15, wherein die second PCR primer hybridizes under stringent conditions to a nucleic acid complementary to DELRl.

Inventors: FERNANDEZ-SALGUERO PEDRO, GONZALEZ FRANK J, FERNANDEZ-SALGUERO, PEDRO, GONZALEZ, FRANK, J.
Applicants: FERNANDEZ SALGUERO PEDRO, GONZALEZ FRANK J, US HEALTH, FERNANDEZ-SALGUERO, PEDRO, GONZALEZ, FRANK, J., THE GOVERNMENT OF THE UNITED STATES OF AMERICA, REPRESENTED BY THE SECRETARY, DEPARTMENT OF HEALTH AND HUMAN SERVICES
Application year: 1997
Main classification: C12Q1
IPC codes: C12Q1