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WO1997007201A1 - Identification de deux nouveaux alleles mutants de la thiopurine-s-methyltransferase humaine, et ses utilisations diagnostiques - Google Patents

Identification de deux nouveaux alleles mutants de la thiopurine-s-methyltransferase humaine, et ses utilisations diagnostiques Download PDF

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Publication number
WO1997007201A1
WO1997007201A1 PCT/US1995/010347 US9510347W WO9707201A1 WO 1997007201 A1 WO1997007201 A1 WO 1997007201A1 US 9510347 W US9510347 W US 9510347W WO 9707201 A1 WO9707201 A1 WO 9707201A1
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Prior art keywords
tpmt
cdna
fragment
polynucleotide molecule
thiopurine
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PCT/US1995/010347
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English (en)
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William E. Evans
Eugene Y. Krynetski
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St. Jude Children's Research Hospital
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Priority to PCT/US1995/010347 priority Critical patent/WO1997007201A1/fr
Priority to AU34912/95A priority patent/AU3491295A/en
Publication of WO1997007201A1 publication Critical patent/WO1997007201A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/1003—Transferases (2.) transferring one-carbon groups (2.1)
    • C12N9/1007—Methyltransferases (general) (2.1.1.)

Definitions

  • the present invention is in the field of cancer therapeutics, diagnostics, and drug metabolism.
  • the present invention relates to characterization of the genetic basis for thiopurine methyltransferase deficiency.
  • Three separate point mutations are, at least in part, responsible for severe hematopoietic toxicity in cancer patients who are treated with standard dosages of 6-mercaptopurine, 6-thioguanine or azathioprine.
  • Thiopurine methyltransferase (TPMT, E.C. 2.1.1.67) is a cytoplasmic enzyme that preferentially catalyzes the S-methylation of aromatic and heterocyclic sulfhydryl compounds, including the anticancer agents 6- mercaptopurine (6MP) and 6-thioguanine, and the immunosuppressant azathioprine.
  • TPMT activity exhibits genetic polymorphism, with approximately 89% of Caucasians and African- Americans having high TPMT activity, 11% intermediate activity (presumed heterozygotes), and approximately one in 300 inheriting TPMT-deficiency as an autosomal recessive trait. (Weinshilboum, R.M.
  • TPMT activity is typically measured in erythrocytes, as the level of TPMT activity in human liver, kidney, lymphocytes and leukemic lymphoblast correlates with that in erythrocytes (Van Loon, J.A. and Weinshilboum, R.M., Biochem. Genet. 20:631-658 (1982); Szumlanski, C.L., et al,
  • Mercaptopurine, thioguanine, and azathioprine are prodrugs with no intrinsic activity, requiring intracellular conversion to thioguanine nucleotides (TGN), with subsequent incorporation into DNA, as one mechanism of their antiproliferative effect (Lennard, L., Eur. J. Clin. Pharmacol 45:329-339 (1992)).
  • these drugs are metabolized to 6-methyl- mercaptopurine (MeMP) or 6-methyl-thioguanine (MeTG) by TPMT or to 6- thiouric acid (6TU) by xanthine oxidase; MeMP, MeTG, and 6TU are inactive metabolites.
  • TPMT assays are not widely available and newly diagnosed patients with leukemia or organ transplant recipients are frequently given erythrocyte transfusions, precluding measurement of their constitutive TPMT activity before thiopurine therapy is initiated.
  • PCR-based methods can be developed to determine TPMT genotype and prospectively predict phenotype, as is now possible for drug metabolizing enzymes such as debrisoquin-hydroxylase (Heim, M. and Meyer, U.A., Lancet 336:529-532 (1990)) and N-acetyltransferase (Grant, D.M., Pharmacogenetics 3:45-50 (1993)).
  • the invention relates to the discovery of three point mutations in exons of TPMT which cause substitutions in the amino acid sequence of TPMT.
  • the presence of these mutant alleles is directly correlated with potentially fatal hematopoietic toxicity when patients are treated with standard dosages of mercaptopurine, azathioprine, or thioguanine.
  • a method for detecting these inactivating mutations in genomic DNA isolated from individual patients (subjects), to make a diagnosis of TPMT-deficiency, or to identify heterozygous individuals (i.e. , people with one mutant gene and one normal gene), having reduced TPMT activity.
  • the present invention therefore, provides a diagnostic test to identify patients with reduced TPMT activity based on their genotype. Such diagnostic test to determine TPMT genotype of patients is quiet advantageous because measuring a patient's TPMT activity has many limitations.
  • a preferred embodiment of the present invention relates to the discovery of an intron sequence of the TPMT gene. Using the sequence of this intron, a primer was made and used to detect A460G mutation of the TPMTB allele in genomic DNA (see Example 2, Detection ofA460G mutation of the TPMTB allele in genomic DNA). Amplifying a fragment with intron sequences confirmed that the TPMTB mutations are in fact present in the actual TPMT gene and are not mutations in a pseudogene.
  • the invention relates to isolated polynucleotide molecules comprising a mutant allele of thiopurine S-methyltransferase (TPMT) or a fragment thereof, which is at least ten consecutive bases long and contains a point mutation in at least one of the cDNA positions 238, 460, or 719.
  • the point mutation at cDNA position 238 is a cytosine substitution for guanine and the whole polynucleotide has the sequence shown in Figure 11.
  • the point mutation at cDNA position 460 is an adenine substitution for guanine and the whole polynucleotide has the sequence shown in Figure 12.
  • the point mutation at cDNA position 719 is a guanine substitution for adenine and the whole polynucleotide has the sequence shown in Figure 13.
  • the invention also relates to an isolated polynucleotide molecule comprising a mutant allele of thiopurine S-methyltransferase (TPMT) or a fragment thereof, which is at least 260 consecutive bases long and contains a point mutation at cDNA position 460 and a point mutation at cDNA position 719.
  • the point mutation at position 460 is an adenine substitution for guanine and the point mutation at position 719 is a guanine substitution for adenine, and the sequence of the whole polynucleotide molecule is shown in Figure 14.
  • a different aspect of the invention relates to a diagnostic assay for determining thiopurine S-methyl-transferase (TPMT) genotype of a person which comprises isolating nucleic acid from said person; amplifying for a thiopurine S-methyltransferase (TPMT) PCR fragment from said nucleic acid, which includes at least one of cDNA positions 238, 460, or 719, thereby obtaining an amplified fragment; and sequencing the amplified fragment thereby determining the thiopurine S-methyltransferase (TPMT) genotype of said person.
  • TPMT thiopurine S-methyl-transferase
  • Another embodiment of the invention relates to a diagnostic assay for determining thiopurine S-methyl-transferase (TPMT) genotype of a person which comprises isolating nucleic acid from said person; amplifying for a thiopurine S-methyltransferase (TPMT) PCR fragment from said nucleic acid, which includes at least one of cDNA positions 238, 460, or 719, thereby obtaining an amplified fragment; and treating the amplified DNA fragment with CviRI in its corresponding restriction buffer to detect presence or absence of a point mutation at cDNA position 238, Mw ⁇ l in its corresponding restriction buffer to detect presence or absence of a point mutation at cDNA position 460, or Accl in its corresponding restriction buffer to detect presence or absence of a point mutation at cDNA position 719, thereby determining the thiopurine S-methyltransferase (TPMT) genotype of said person.
  • TPMT thiopurine S-methyl-transferase
  • controls are run parallel to the above- described reaction steps, wherein cDNA, which is wild-type for TPMT sequence, is amplified for a wild-type TPMT fragment, thereby obtaining a wild-type TPMT fragment; and treating the wild-type TPMT fragment with
  • a further aspect of the invention relates to a diagnostic assay for determining thiopurine S-methyl-transferase (TPMT) genotype of a person which comprises isolating nucleic acid from said person; making a first and a second PCR primer wherein the first PCR primer is complementary to a region 5' to one of three point mutation sites at cDNA positions 238, 460, or 719; and the second PCR primer is complementary to a region 3' to the same one of the three point mutation sites at cDNA positions 238, 460, or 719; amplifying the sequence in between the first and the second primers; thereby obtaining an amplified fragment; and treating the amplified fragment with CviRI in its corresponding restriction buffer to detect presence or absence of a point mutation at cDNA position 238, Mwol in its corresponding restriction buffer to detect presence or absence of a point mutation at cDNA position 460, or Accl in its corresponding restriction buffer to detect presence or absence of a point mutation at cDNA position 719
  • a preferred embodiment of the invention relates to a diagnostic assay for determining thiopurine S-methyl-transferase (TPMT) genotype of a person which comprises isolating nucleic acid from said person; amplifying for a thiopurine S-methyltransferase (TPMT) PCR fragment from said nucleic acid using a first and a second set of primers in a first and a second PCR reaction, respectively; wherein the first set of primers contains primer X and primer Y, and the second set of primers contains primer X and primer Z; wherein the Y primer is complementary to a region 5' to one of three point mutation sites at cDNA positions 238, 460, or 719, and includes the wild type nucleotide for said cDNA position; the Z primer is identical to the Y primer except that instead of the wild type nucleotide, it contains the respective mutant nucleotide at the respective cDNA positions 238, 460, or 719; and the X primer is complementary to
  • a diagnostic kit for determining thiopurine S-methyltransferase (TPMT) genotype of a person comprising a carrier means having in close confinement therein at least two container means, wherein a first container means contains a first polynucleotide molecule described above, which contains at least one of the point mutations at cDNA positions 238, 460, or 719 and which contains the whole or part of the sequence shown in Figures 11-14, and a second container means contains a second polynucleotide molecule encoding a wild-type allele of thiopurine S-methyltransferase (TPMT) or a fragment thereof which is at least ten consecutive bases long and contains at least one of cDNA positions 238, 460, or 719, corresponding to the first polynucleotide of the first container means.
  • TPMT thiopurine S-methyltransferase
  • a further aspect of the invention relates to an isolated polynucleotide molecule having a sequence shown in Figure 15, SEQ ID NO:5, or a fragment thereof which is at least ten bases long.
  • the polynucleotide molecule has the nucleotide sequence identified as SEQ ID NO:6.
  • the invention relates to an isolated polynucleotide molecule complementary to the polynucleotide molecule having a sequence shown in Figure 15.
  • Figure 1 depicts Northern blot analysis of poly (A) + RNA isolated from various human tissues and hybridized with wild-type TPMT cDNA, demonstrating the presence of multiple TPMT mRNAs. Each lane contained ⁇ 2 ⁇ g of poly(A) + RNA. Lanes: 1, heart; 2, brain; 3, placenta; 4, lung; 5, liver; 6, skeletal muscle; 7, kidney; 8, pancreas; 9, peripheral blood leukocytes.
  • Figures 2A, 2B, and 2C depict relative expression of TPMT mRNA and TPMT activity among individuals of differing phenotype.
  • Figure 2 A Autoradiographs of hybridizations with the wild-type TPMT cDNA (Lower), followed by the h28S ribosomal oligonucleotide probe (Upper), of total RNA from human liver (lane 1), leukocytes of unrelated individuals (controls, lanes 2 and 3), the TPMT-deficient patient (lane 5), and her father
  • Figures 3A, 3B and 3C depict the difference in the TPMT wild-type and mutant sequences.
  • Figures 3 A and 3B Nucleotide sequence analysis of the fragment of the wild-type and the mutant clones derived from reverse transcription-PCR products of total RNA isolated from leukocytes. The adenine residue in the initiation codon is number +1.
  • Figure 3C Wild-type and mutant TPMT cDNA sequence (nt 232-243) and deduced amino acid sequence of the protein encoded. The mutation site is underlined.
  • Figures 4A, 4B, and 4C depict Northern blot analysis of total RNA
  • TPMT enzymatic activity in yeast transformed with the wild-type TPMT cDNA-containing vector (left lanes), the mutant TPMT cDNA-containing vector (center lanes), and the yeast expression vector without any cDNA insert (right lanes).
  • Each lane contained » 20 ⁇ g of total RNA.
  • FIG. 4 A Hybridization with 18S rRNA-specific yl8S oligonucleotide labeled with [ ⁇ , - 32 P]ATP.
  • Figure 4B Hybridization with TPMT cDNA probe labeled with [ ⁇ - 32 P]ATP.
  • Figure 4C Mean TPMT activity in yeast lysates (duplicate experiments), wt, Wild type; mut, mutant; cont., control.
  • Figure 5 depicts mutation-specific PCR amplification analysis of genomic DNA from the TPMT-deficient patient (Pt., lanes 9 and 10), her mother (Mo, lanes 7 and 8), father (Fa, lanes 5 and 6), and brother (Bro, lanes 3 and 4) and an unrelated control subject with TPMT activity of 22.8 u/ml pRBC (lanes 1 and 2).
  • W amplification with primers specific to wild-type genotype
  • M amplification with mutant-specific primers (see Table 1 for primer sequences).
  • Figures 6A and 6B depict relative level of TPMT protein and activity in a wild-type and a deficient patient.
  • Figure 6 A depicts a Western blot of RBC lysates probed by anti-TPMT antibodies. Lysate equivalent to 2 x 10 6 RBC was loaded in lanes 2 and 3. Yeast lysate expressing wild-type TPMT was utilized for comparison (lane 1).
  • Figure 6B depicts relative levels of TPMT protein and activities in erythrocytes determined by densitometry on the Western blot and radiochemical methods, respectively.
  • Figures 7A-7F depict differences in the wild-type and mutant TPMT cDNA sequence and deduced amino acid sequence of the protein encoded.
  • Figures 8A, 8B, 8C, and 8D depict PCR-RFLP analysis of cDNAs from the patient and his family members.
  • a wild-type cDNA was included as the control.
  • Three samples were run for each cDNA, a cDNA fragment (nucleotide 323-806, 484 bp) amplified by PCR without restriction enzyme digestion (lane 1), PCR products digested by Accl to yield 398 bp and 86 bp fragments when the G719A mutation was present (lane 2), PCR products digested by Mwol to yield 340 bp and 144 bp fragments when the wild-type sequence was present at nucleotide 460 or one fragment of 484bp when the G460A mutation was present (lane 3), and a 123 bp DNA ladder (lane M).
  • Figure 8A depicts wild-type cDNA as control
  • Figure 8B depicts TPMT- deficient patient
  • Figure 8C depicts father
  • Figures 9A, 9B, and 9C depict comparison of TPMT mRNA, protein, and activity levels in yeast transformed with vector alone without any insert (lane 1), vector with wild-type cDNA (lane 2), vector with cDNA containing G460A (lane 3), vector with cDNA containing A719G (lane 4), vector with cDNA containing both G460A and A719G (TPMTB) (lanes 5 and 6).
  • Figure 9A depicts Northern blot of yeast total RNA hybridized with TPMT cDNA probe and subsequently stripped and reprobed with yl8S rRNA-specific oligonucleotide
  • Figure 9B depicts Western blot of yeast lysates (0.25 ⁇ g for lanes 1-5, 25 ⁇ g for lane 6) with anti-TPMT antibodies
  • Figure 9C depicts TPMT activities measured by in vitro incubation of yeast lysates with substrate concentrations of 10 ⁇ M 6MP and 1 mM SAM.
  • Figures 10A, 10B, 10C, and 10D depict in vitro stabilities of TPMT wild-type and mutant proteins.
  • Figure 10A depicts TPMT activities expressed
  • Figures 10B-10D depict immunoreactive protein with equal loading of wild-type.
  • Figure 10B depicts wild type protein;
  • Figure IOC depicts TPMT 460 ;
  • Figure 10D depicts TPMT m ; respectively.
  • FIGS.11A-11B depict the cDNA sequence of G238C mutant TPMT (TPMT A) .
  • Figures 12A-12B depict the cDNA sequence of G460A mutant TPMT.
  • Figures 13A-13B depict the cDNA sequence of A719G mutant TPMT.
  • Figures 14A-14B depict the cDNA sequence of TPMTB mutant.
  • Figure 15 depicts a partial sequence of a TPMTB intron.
  • Figure 16 depicts a primer comprised of an intron sequence.
  • TPMT thiopurine S-methyltransferase
  • TPMTA approximately 100-fold.
  • the allele containing this G238C mutation is designated as TPMTA.
  • TPMTB human TPMT-deficiency was identified and defined as (TPMTB).
  • This mutant allele involves two nucleotide transitions with amino acid changes at cDNA position 460 (G 60 -A), codon 154 (Ala 15 *Thr), and cDNA position 719 (A 719 -*G), codon 240 (Tyr ⁇ -Cys).
  • Heterologous expression established that either mutation in TPMTB alone leads to a reduction in catalytic activity, while both mutations lead to complete loss of activity.
  • the mutations at cDNA positions 238 ajid 460 eliminate the recognition site for Cv RI and Mwol, respectively, while A719G adds an_4ccl restriction site.
  • TPMTB was detected in genomic DNA of 18 out of 25 (72%) of Caucasians with heterozygous phenotypes, indicating that TPMTB is the most prevalent mutant allele associated with TPMT-deficiency.
  • PCR primers are constructed that are complementary to the region of the mutant allele encompassing the point mutation.
  • a primer consists of a consecutive sequence of polynucleotides complementary to any region in the allele encompassing the position which is mutated in the mutant allele.
  • PCR primers complementary to a region in the wild-type allele corresponding to the mutant PCR primers are also made to serve as controls in the diagnostic methods of the present invention.
  • the size of these PCR primers range anywhere from five bases to hundreds of bases. However, the preferred size of a primer is in the range from 10 to 40 bases, most preferably from 15 to 32 bases. As the size of the primer decreases so does the specificity of the primer for the targeted region.
  • primers to one or both sides of the targeted position i.e. the cDNA positions 238, 460, or 719, are made and used in a PCR amplification reaction, using known methods in the art (e.g. 2 Massachusetts General Hospital & Harvard Medical School, Current Protocols
  • an amplified fragment can be analyzed in several ways to determine whether the patient has a mutant allele of the TPMT gene.
  • the amplified fragment can be simply sequenced and its sequence compared with the wild- type cDNA sequence of TPMT. If the amplified fragment contains one or more of the point mutations described in the present invention, the patient is likely to have TPMT-deficiency or be a heterozygote (i.e. , reduced activity) and therefore, develop hematopoietic toxicity when treated with standard amounts of mercaptopurine, azathioprine, or thioguanine.
  • a combination of PCR fragment amplification and RFLP analysis is used to determine TPMT genotype of the individual.
  • a fragment of the genomic DNA sequence i.e. a sequence of the genomic sequence.
  • DNA of the patient is amplified by making a primer containing the mutation site, i.e. cDNA positions 238, 460, or 719.
  • a primer containing the mutation site i.e. cDNA positions 238, 460, or 719.
  • Each amplified fragment, as well as the fragments generated against the wild-type cDNA are treated with the corresponding restriction endonuclease (i.e. , CviRI for fragment amplified for cDNA position 238; Mwol for fragment amplified for cDNA position 460; Accl for fragment amplified for cDNA position 719) in the presence of the appropriate cutting buffer for each enzyme.
  • the preferred buffers are those recommended by the manufacturer of the restriction enzyme.
  • the fragments After the fragments have been incubated in the restriction reaction mixtures at the recommended temperatures, and restriction reactions have been allowed to proceed to completion, they are electrophoresed on a gel, e.g. a 2% agarose gel. The size of the fragments are measured using standard ladder size- markers. If the amplified fragments are not cut with either CviRI or Mwol, it indicates that the genomic DNA of the patient contains a mutation at cDNA positions 238 and/or 460, respectively. If the fragment is cut with the restriction enzyme Accl, however, it indicates that the genomic DNA which was amplified a the region encompassing cDNA position 719 contains the 719 mutation.
  • a single primer encompassing a mutation site it is preferred to (1) simply sequence the amplified fragment, (2) use conditions where PCR-amplification will occur only when one of the mutations is present in genomic DNA or the cDNA fragment (i.e. , mutation specific PCR (MSPCR)), or (3) use RFLP analysis to determine the TPMT genotype of a subject.
  • MSPCR mutation specific PCR
  • DNA of the patient, as well as a control is amplified separately, using a wild type and a mutation-containing primer. The content of each amplification vial (containing wild type or mutant type primer) is then examined for the presence of amplified DNA.
  • a method of visualization includes electrophoresing the contents of each amplification vial (i.e. , control DNA + wild type primer, control DNA + primer containing specific mutation, patient's genomic DNA + wild type primer, patient's genomic DNA + primer containing specific mutation), staining the electrophoresis gel with ethidium bromide, shining UN light on the gel, and looking for the presence or absence of an amplified band in each lane.
  • a DNA intercalating dye such as ethidium bromide
  • control DNA from an individual who is known to be homozygote for TPMT wild type is amplified as described above and the results are analyzed as follows. Presence of a band in the lane containing control DNA + wild type and absence of a band in the lane containing control DNA + primer indicates that the particular mutation, which is encompassed in the sequence of the mutant type primer, does not exist on either of the TPMT alleles of the control DNA as expected.
  • a sequence of non-TPMT derived polynucleotides (not complementary to the TPMT gene) is added to the end of the primer.
  • the non-TPMT derived sequence of polynucleotides is added to the 5' end of the primer.
  • the size of the amplified fragment is sufficiently increased so that if the fragment is cut with a restriction enzyme, the sub-fragments generated are sufficiently large to be detected.
  • two common primers are used, each of which is complementary to either side of the mutation site. Common primers are those which do not encompass the mutation sites, i.e.
  • the primers are elongated in opposite directions so that they amplify a relatively large fragment encompassing the site of mutation. This fragment is subsequently analyzed by RFLP analysis. As described above, presence of G238C or G460A will destroy the CviRI and Mwol recognition sites, hence, a fragment containing these mutations does not cut with the corresponding restriction enzyme. On the other hand, presence of A719G results in the creation of a recognition site for Accl, hence, a fragment containing this mutation is cut with this enzyme.
  • PCR conditions and primers are developed which amplify only when the target mutation is present (Figure 5), or when only the wild-type sequence is present at the mutation site (i.e. , allele specific PCR (ASPCR) amplification or mutation specific PCR (MSPCR) amplification).
  • ASPCR allele specific PCR
  • MSPCR mutation specific PCR
  • the mutation sites on the genomic DNA are amplified separately by using wild-type and mutant primers. If only a wild-type or a mutant-type fragment is amplified, the individual is homozygous for the wild-type or the particular mutant-type TPMT. However, presence of more than one type of fragment indicates that the individual is heterozygous for TPMT allele.
  • An example of a diagnostic assay that is carried out according to the present invention to determine the TPMT genotype of a person is as follows. This example is provided for illustrative purposes and is not meant to be limiting.
  • Tissue containing DNA (e.g. , not red blood cells) from the subject is obtained.
  • tissue include white blood cells, mucosal scrapings of the lining of the mouth, epithelial cells, pancreatic tissue, liver, et cetera.
  • Genomic DNA of the individual subject is isolated from this tissue by the known methods in the art, such as phenol/chloroform extraction.
  • Six vials, numbered 1-6, are set up with each containing an equal aliquot of the genomic DNA of the subject.
  • PCR primers encompassing cDNA positions 238 (both wild-type and G238C mutant), 460 (both wild-type and G460C mutant), and 719 (both wild-type and A719G mutant) are synthesized.
  • the primers are preferably 10-40 bases long, most preferably 15-31 bases long.
  • Each type of primer pair wild-type and mutant is added to only one of the vials 1-6, and using a standard PCR procedure, a TPMT fragment in each of the six vials is amplified. Next, the content of each vial is analyzed by the various methods described above, which include RFLP analysis, sequencing, mutation-specific amplification, or a combination of such methods.
  • the mutant alleles of the present invention are used to express mutant proteins.
  • the mutant proteins are produced in an expression system such as yeast, bacterial, or mammalian cell systems.
  • recombinant plasmids are constructed that contain yeast GAL10-CYC1 promoter and mutant form of TPMT cDNA, and a PGK terminator.
  • GAL10-CYC1 promoter is induced by galactose.
  • TPMT protein is obtained from the yeast cell lysates.
  • the proteins may be purified by known methods in the art, such as DEAE ion exchange chromatography (Van Loon, J.A., and R.M.
  • the mutant proteins are used in a variety of diagnostic assays and methods. For example, they are used to test whether a given therapeutic drug can be metabolized by the mutant proteins.
  • This assay allows development of medicaments which, like 6-mercaptopurine, 6-thioguanine, or azathioprine, are effective against a given cancer or useful in preventing rejection of a transplant, yet do not cause the severe toxicity which is brought about by said drugs in patients who have TPMT-deficiency.
  • the drug which is being tested is incubated under simulated physiological conditions (for example in isolated body fluid such as plasma or blood and at body temperature) for various lengths of time. At various time-points aliquots are removed and analyzed for presence or absence of the drug or its expected byproduct(s) to determine whether and when the drug is properly metabolized.
  • the mutant proteins are used to raise antibodies according to known methods routinely used by the artisans.
  • antibody refers both to monoclonal antibodies which have a substantially homogeneous population and to polyclonal antibodies which have heterogeneous populations. Polyclonal antibodies are derived from the antisera of animals immunized with the analyte. Monoclonal antibodies to specific TPMT mutants may be obtained by methods known in the art. See, for example Kohler and Milstein, Nature 256:495-491 (1975). Such antibodies may be of any immunoglobulin class including IgG, IgM, IgE, IgA, IgD and
  • SUBSTTTUTE SHEET any subclass thereof.
  • antibody is meant to include both intact molecules as well as fragments thereof, such as Fab and F(ab') 2 , which are capable of binding antigen.
  • analyte in this context refers to not only the intact mutant TPMT protein but also to any fragment of the protein which contains an antigenic site capable of binding to an antibody and which antigenic site is present in the mutant protein but lacking from the wild-type protein.
  • Such analytes are prepared by a routine method in which a series of shortened peptides are expressed recombinantly, for example in the same way that the whole mutant proteins are expressed in yeast.
  • the shortened peptides are made by for example, progressively deleting one codon, either on the 5 ' or the 3' end of the coding region of the mutant cDNA, yet preserving the mutated codon, before it is inserted into the expression vector.
  • a number of peptides are produced that are progressively smaller in size by one amino acid, yet contain the mutation.
  • Antibodies are raised against these peptides as well as the whole mutant protein.
  • the antibodies are tested for their ability to distinguish wild-type TPMT from the mutant TPMT proteins by a standard immunoassay method such as ELISA (2 Massachusetts General Hospital & Harvard Medical School, Current Protocols In Molecular Biology, Chapter 11 (Green Publishing Associates and Wiley-Interscience 1991)), using recombinantly expressed wild- type TPMT and mutant TPMT proteins.
  • mutant cDNA as well as shortened mutant cDNA is expressed using expression vector pGEX-2T (Pharmacia Biotech, Uppsala, Sweden) containing the DNA fragment encoding glutathione S-transferase from Schistosoma japonicum to construct a recombinant plasmid with an insert for the uninterrupted coding frame of GST-mutant TPMT fusion protein.
  • Anti-mutant TPMT antibodies are raised in rabbits by immunization with GST-mutant TPMT fusion protein (Rockland Corp., Gilbertsville, PA) and then purified by affinity chromatography. The antibodies are purified in sequence (by affinity chromatography) on sepharose with immobilized GST and GST-mutant TPMT. Antibodies so obtained are used in a variety of assays and methods.
  • the antibodies of the present invention are used in a variety of protein assay methods (such as standard radioimmunoassay using labelled antibodies against protein bound to a membrane, ELISA, et cetera), to determine whether a given individual has a given mutant phenotype.
  • tissue lysate from the patient is obtained from blood, liver, pancreas, or any other tissue expressing TPMT.
  • the presence or absence of the mutant protein is detected using the detectably labelled antibodies of the present invention.
  • the lysate may be crude or purified to various extent.
  • an intron sequence of the TPMT gene is disclosed (see Figure 16).
  • probes are made to clone the genomic sequence and define the gene, hence, defining all of the introns in the TPMT gene.
  • the sequence and the location of the other introns, as well as the whole structure of the chromosomal gene is obtained.
  • a useful method for such determination is PCR-based method of DNA walking (Siebert, P.D. et al. , Nucleic Acids Research 23:1087-1088 (1995)).
  • This method allows walking from a known sequence (e.g. , an intron) to uncloned DNA fragments. In this way, sequence information on DNA fragments adjacent to that already known is generated.
  • Another more common technique involves using the DNA fragment encompassing the intron sequence for screening the genomic library (see, e.g., 1 Massachusetts General Hospital and Harvard Medical School, Current Protocols in Molecular Biology, Chapter 6 (Green Publishing Associates and Wiely-Interscience 1991). Hence, the complete genomic sequence of TPMT is so determined. The chromosomal location of the gene is determined, using known methods in the art (Lee, D. et al, Drug Metab. Disp. 23:398-405 (1995)). It should be noted that the presence of multiple TPMT-like pseudogene sequences in the human genome precludes using the known cDNA sequence for direct cloning of TPMT functional gene.
  • TPMT thiopurine S-methyltransferase
  • oligonucleotide primers for intron sequences are synthesized and PCR-based methods are developed that are specific for the human gene which encodes TPMT (versus pseudogenes). Accordingly, more specific diagnostic tests are developed to detect the presence of mutations or wild-type TPMT sequences in genomic DNA. Since the intron and exon structure of human TPMT can now be detected, efficient methods to detect mutations at the human TPMT locus, such as single-strand conformation polymorphism (SSCP) analysis, are developed, thus facilitating the identification of new mutations responsible for loss of TPMT activity. Finally, by defining the 5' and 3' untranslated regions of the TPMT gene, it is now possible to understand the genetic regulation of this gene, and thus analyze and predict changes in TPMT protein levels and activity.
  • SSCP single-strand conformation polymorphism
  • Blood samples were also obtained from two healthy female volunteers having erythrocyte TPMT activities consistent with the homozygous wild-type genotype (11 and 19 u/ml pRBC) and from the mother and father of the propositus, who had TPMT activities indicative of a heterozygous genotype (5.6 and 3.6 u/ml PRBC, respectively).
  • Total leukocyte RNA was extracted by the method of Chomczynski and Sacchi (Chomczynski and Sacchi, Anal.
  • TPMT phenotype was assigned on the basis of erythrocyte TPMT activity, according to the criteria of Weinshilboum and Sladek (Weinshilboum and Sladek, Am. J. Hum. Genet. 32:651-662 (1980)). The studies were approved by the institutional review board for clinical trials at St. Jude
  • First-strand cDNA was synthesized, essentially as described (Schuetz et al. , J. Clin. Invest. 42: 1018-1024 (1993)), from 2 ⁇ g of total cellular RNA.
  • the reaction mixture (100 ul) contained 10 mM Tris-HCl (pH 8.3 at 20°C), 50 mM KCl, 1.5 mM MgCl 2 , 0.001 % (wt vol) gelatin, 0.2 mM dNTPs, 20 units of RNasin, 200 ng of the random hexamers, and 200 units of Moloney murine leukemia virus reverse transcriptase (Superscript; GEBCO/BRL) and was incubated at 42 °C for 60 min.
  • PCR primers were synthesized on the basis of the published colon carcinoma TPMT cDNA sequence ( Figure 11, SEQ ID NO: ; Honchel et al , Mol. Pharmacol. 43:818-881 (1993)).
  • Table 1 The sequences of primers used for the first round of amplification and all subsequent PCR amplifications are given in Table 1.
  • Each cycle of amplification consisted of denaturation at 94 °C for 1 min, annealing at 55 °C for 30 sec, and primer extension at 72 °C for 2 min (35 cycles).
  • TPMT PCR products were made blunt and the product was cloned into the Sma I site of plasmid pGEM-7Zf(+) (Promega).
  • the inserts present in positive clones were sequenced by automated fluorescence sequencing.
  • Oligonucleotide h28S (5'-GCA-CAT-ACA- CCA-AAT-GTC-TGA-ACC-TGC-GGT-3') was homologous to the GenBank
  • Northern blot analysis was performed on human and yeast total RNA or multiple tissue Northern blots MTN I and II (Clontech). Membranes were hybridized with the radiolabeled TPMT cDNA, stripped, then reprobed with either the h28S or yl8S oligonucleotide, in the case of total RNA analysis, or with the human. ⁇ -actin cDNA (Clontech), in the case of poly(A) + MTN blots.
  • PCR conditions were as described above except that 100 ng of the cDNA clone served as a template with primers 3 and 4 (Table 1) and 2.5 units of Pyrococcus furiosum DNA polymerase for amplification.
  • the coding region of the wild-type or the mutant TPMT cDNA was ligated into pYeDP yeast expression vector. Authenticity of the PCR products was confirmed by sequencing.
  • Transformation of the yeast strain 2805 was carried out by treatment with lithium acetate (Becker and Lundblad in Current Protocols in Molecular Biology, eds. Ausbel et al (Green & Wiley Interscience, New York), Vol. 2, pp. 13.7.1-13.7.10 (1993)).
  • Yeast cells transformed with recombinant expression vectors were grown on galactose-containing medium, and the lysate and total RNA were prepared as described (Krynetski et al. , FEBS Lett.
  • Erythrocyte lysates were analyzed for TPMT activity by the non-chelated radiochemical assay of Weinshilboum et al. (Weinshilboum et al. ,
  • Mutation-specific primers and reaction conditions were developed to detect the presence or absence of the G ⁇ -C TPMT mutation in the propositus and her family. Five hundred nanograms of genomic DNA was amplified by
  • PCR using primers 5 and 6 (Table 1). PCR conditions were as follows: denaturation at 94°C for 1 min, annealing at 64°C for 45 sec, and elongation at 72°C for 1 min for 35 cycles. Amplification products were then diluted 1 : 10 with water and aliquots were separately reamplified by using (i) primers 5 and 7 for wild-type sequence, or (ii) primers 5 and 8 for the mutant sequence
  • TPMT cDNA synthesized by reverse transcription-PCR was used as a template in PCR amplification with primers 5 and 6, for 30 cycles of denaturation at 94°C for 1 min, annealing at 62°C for 45 sec, and elongation at 72 °C for 1.5 min.
  • the synthesized DNA fragments were treated with CviRI restriction endonuclease (Megabase Research Products, Lincoln, NB) and analyzed by non-denaturing 8% PAGE.
  • the TPMT cDNA prepared from leukocyte RNA from the TPMT-deficient patient (Evans et al , J. Pediatr.
  • the TPMT coding region of the cDNA was reamplified by using primers 3 and 4, designed to introduce BamHI and Ec ⁇ RI restriction sites at the
  • SUBSTITUTE SHEET ends of the cDNA fragment and to add an AAA sequence just before the initiation codon, to increase efficiency of expression in yeast (Krynetski et al. , Pharmacogenetics 5:27-31 (1995)).
  • Two recombinant plasmids were constructed that contained yeast GAL10-CYC1 promoter, either the wild-type or mutant form of TPMT cDNA, and a PGK terminator. After introduction of the vectors into the yeast cells, GALIO-CYCI promoter was induced by galactose.
  • Northern blot analysis demonstrated that only yeast which contained either the wild-type or the mutant TPMT cDNA synthesized TPMT mRNA, in comparable quantities ( Figure 4).
  • TPMT Activity of the wild-type TPMT was 113-123 nmol/hr per mg of protein compared with ⁇ 1.0 nmol/hr per mg of protein for mutant TPMT; no TPMT activity was found in the yeast transformed with control plasmid (i.e., no cDNA insert).
  • the first step involved amplification of a 232-bp TPMT fragment which included nt 238 (primers 5 and 6; Table 1).
  • the amplification product was diluted and used thereafter as a template for the second round of amplification.
  • two sets of primers were used, fully coinciding with either the wild-type or the G ⁇ '-C mutant TPMT (see Table 1).
  • the perfectly matched pair of primers gave the desired 130-bp product. In this way, it was possible to detect the presence or absence of the G 238 ⁇ C mutation in the entire family of the
  • TPMT-deficient patient Figure 5
  • the mutation-specific PCR amplification indicated that the TPMT allele containing the inactivating G 238 - ⁇ mutation is present in genomic DNA of the patient and her mother, but not in her father or brother.
  • the patient is heterozygous for G 238 -»C, indicating that her other allele carries a different defect.
  • SUBSTITUTE SHEET (RULE 25) development of PCR-based methods to determine TPMT genotype and prospectively predict phenotype, as is now done for drug-metabolizing enzymes such as debrisoquin hydroxylase (Heim and Meyer, Lancet 336:529-532 (1990)) and N-acetyltransferase (Grant, Pharmacogenetics 3:45-50 (1993)).
  • drug-metabolizing enzymes such as debrisoquin hydroxylase (Heim and Meyer, Lancet 336:529-532 (1990)) and N-acetyltransferase (Grant, Pharmacogenetics 3:45-50 (1993)).
  • debrisoquin hydroxylase Heim and Meyer, Lancet 336:529-532 (1990)
  • N-acetyltransferase Grant, Pharmacogenetics 3:45-50 (1993)
  • the nucleotide sequence of cD ⁇ A isolated from leukocyte R ⁇ A of a patient with TPMT activity of 8.3 u/ml pRBC was identical to the previously published wild-type colon carcinoma TPMT cD ⁇ A sequence.
  • the open reading frame of cD ⁇ A clones obtained from total liver R ⁇ A was also identical to the published wild-type colon carcinoma TPMT cD ⁇ A, which was not unexpected since TPMT activities in colon, leukocytes, and liver are correlated ( Pacifici et al , Xenobiotica 23:671-679 (1993)).
  • SUBSTITUTE SHEET (RULE 25) nature of hematopoietic toxicity when full dosages of mercaptopurine or azathioprine are given to TPMT-deficient patients (Evans et al. , J. Pediatr. 119:985-989 (1991); McLeod et al, Lancet 1341:1151 (1993); Lennard et al , Arch. Dis. Child. 69:511-519 (1993)), which can be fatal (Schutz et al.
  • Total leukocyte RNA was isolated (Chomczynski, P. and Sacchi, N., Anal. Biochem. 162:156 (1987)) from normal leukocytes of an 5-year-old boy with acute lymphocytic leukemia in complete remission, who had developed severe hematopoietic toxicity on standard dosages of 6MP (50 mg/m 2 /day). At the initial presentation of toxicity, his erythrocyte concentration of TGNs was > 15 fold higher than the population median (4400 versus 280 pmol/ml pRBC). Subsequently, he was documented to have TPMT-deficiency (0.6 unit/ml of packed red blood cells).
  • First-strand cDNA was synthesized from 2 ⁇ g of total RNA and then amplified to obtain TPMT coding region as previously described (Krynetski, E.Y. et al, Proc. Natl. Acad. Sci. (USA) 92:949-953 (1995)).
  • the PCR fragments were either made blunt and cloned into the Sma I site of plasmid pGEM-7Zf ( + ) (Promega, Madison, Wisconsin) , or directly cloned into
  • PCRTMII Invitrogen, San Diego, California. Plasmids were purified with Qiagen kits (Qiagen, Chatsworth, California) and sequenced with an automated sequencer, using the cycle sequencing reaction employing fluorescence-tagged dye terminators (PRISM, Applied Biosystems, Foster City, California).
  • membranes were stripped and reprobed with yl8S oligonucleotide, washed with 5 x SSC 3 times at room temp for 5 min each time and finally with 5 x SSC at 65 °C for 5 min, and then exposed to x-ray film with intensifying screen at -70°C for 1 hr.
  • the wild type and mutant cDNA clones were used as templates for site- directed mutagenesis. PCR conditions were as described above except that annealing temperature was changed to 50 °C and 1.3 units of Pyrococcus furiosus DNA polymerase (Stratagene, La Jolla, California) was used. After amplification, the PCR products were ligated into pYeDP 1/8-2 yeast expression vector as previously described (Krynetski E.Y., et al, Proc. Natl
  • telomere sequence was also ligated into the expression vector.
  • Recombinant plasmids were constructed that contained galactose-inducible GALIO-CYCI promoter (Cullin, C. and Pompon, D, Gene 65:203-211 (1988)), either the wild-type or mutant forms of TPMT cDNA, and a PGK terminator. Nucleotide structures of all cDNAs were confirmed by sequencing.
  • Transformation of the yeast strain 2805 was carried out by treatment with lithium acetate (Becker, D.M., and Lundblad, V., Current Protocols in
  • Yeast cells transformed with recombinant expression vectors or the vector without TPMT cDNA (control) were grown on galactose-containing medium for 24 hr at 30°C.
  • Yeast cells were treated with Lyticase (Sigma, St. Louis, MO), sedimented after washing with a buffer (pH 6.2) containing 20 mM 2-[N-morphol_no]ethanesulfonic acid
  • MES MES
  • Tris-HCl buffer pH 7.8 containing 100 KIU/M1 aprotinin and 1 mM phenylmethylsulfonyl fluoride.
  • SDS-polyacrylamide gel electrophoresis was carried out following the method of Laemmli (Laemmli, U.K. , Nature 227:680-685 (1970)) using 15 % acrylamide slab gels. Proteins were then - ⁇ :trophoretically transferred to a nitrocellulose membrane and reacted with a polyclonal rabbit antiserum against human TPMT. This antibody was produced by immunizing rabbits with GST- TPMT fusion protein (McLeod, H.L. et al, Pharmacogenetics 5. ⁇ n press (1995)) and purified in sequence by affinity chromatography on sepharose with immobilized GST and GST-TPMT.
  • TPMT content in yeast lysate expressing TPMT cDNA was estimated by this analysis using the standard of purified GST-TPMT fusion protein treated by thrombin.
  • Erythrocyte lysates were analyzed for TPMT activity by the non-chelated radiochemical assay of Weinshilboum and coworkers (Weinshilboum, R.M., et al, Clin. Chim. Acta .55:323-333 (1978)).
  • the enzymatic reaction of TPMT was carried out at 37°C in a 1-ml mixture containing 0.1 M Tris-HCl, pH 7.5, yeast cytosol expressing
  • TPMT various concentrations of 6MP (4 ⁇ M), and DTT (250 ⁇ M). These reaction conditions differ from that previously published (Krynetski, E.Y. et al, Mol. Pharmacol. 47:1141-1147 (1995)); i.e. incubation at 37 C instead of 21.5 C, analysis of MeMP production formation instead of substrate disappearance, and the use of higher substrate concentrations in the present study.
  • the amount of TPMT in each reaction was made the same (0.57 ⁇ g TPMT) by adjusting the amount of yeast cytosol according to TPMT protein levels detected by the Western-blot analysis. For kinetic studies of 6MP, 1 mM of SAM was used, whereas for studies of SAM, 2 mM of 6MP was utilized. The reaction was started by the addition of yeast cytosol or 6MP (in 10 ⁇ l of
  • SUBSTITUTE SHEET (RULE 25) formation of the methylated metabolite, MeMP, using a gradient system essentially as described in Example 1 (Krynetski, E.Y., et al, Mol. Pharmacol. 47:114-1147 (1995)).
  • Non-linear least-squares regression was used to estimate Vmax and Km by fitting a Michaelis-Menten model to the non-transformed data as described in Example 1 (Krynetski, E.Y., et al, Mol.
  • Yeast cytosols expressing wild-type TPMT 460 , or TPMT 719 protein were incubated in 0.1 M Tris-HCl (pH 7.5) at 37°C (after an equilibration time of 3 min from 0°C to 37°C) for different lengths of time before being added to an assay mixture similar to that described above except using fixed concentrations of 2 mM 6MP and 1 mM SAM. The assay of TPMT activity was then allowed to proceed for 15 min at 37°C, and MeMP was measured as described above.
  • Total protein concentrations of yeast lysate in the incubation mixture were 0.09, 0.37, 0.08 mg/ml for the wild-type, TPMT ⁇ , and TPMT m , respectively, to give equal amount of TPMT in the incubation.
  • the same assay mixture without yeast lysate served as the blank, and the background values for non- enzymatic methylation ( ⁇ 10%) were subtracted from all values obtained.
  • An aliquot of sample at each time point was taken for Western blot analysis.
  • the samples at 0°C, 0 hr served as controls for the blots with TPMT 460 and
  • TPMT cDNA synthesized by reverse transcription-PCR was used as a template in PCR amplifications with primer D (5'-CAGGCTTTAGCATAATTTTCAATTCCTC-3', 779-806) and primer E
  • SUBSTITUTE SHEET (RULE 25) (5'-CAGAAGAACCAATCACCG-3', 323-340), for 30 cycles of denaturation at 94 °C for 1 min, annealing at 50 °C for 45 sec, and elongation at 72 °C for 1.5 min.
  • the synthesized DNA fragments were digested with Accl and Mwol restriction endonuclease (New England Biolabs, Beverly, MA) and analyzed by 2.5% MetaPhor agarose (EMC, Rockland, ME).
  • primer A ATG TAA TAC GAC TCA CTA TAA CCT GGA TTA ATG GCA AC, 466 ⁇ .83
  • primer B ATA ACA GAG TGG GGA GGC TGC, 408-428 of intron sequence
  • buffer A Invitrogen, San Diego, CA
  • Amplification conditions were: cycle 1, 80°C - for 1 min, 94°C for 2 min. 5 ⁇ l of 10 mM dNTP were added after heating to 80°C ("Hot start" protocol).
  • the reaction proceeded at 94°C for 1 min, 55 °C for 2 min and 72°C for 1 min for 35 cycles more and was accomplished with incubation for 7 min at 72 °C.
  • the products of the reaction were digested with Mwol restriction endonuclease (New England Biolabs, Beverly, MA) according to manufacturer's instructions and analyzed by electrophoresis in 2.5% Metaphor agarose (EMC, Rockland, ME).
  • EMC Methoxyribonuclease
  • EMC Metaphor agarose
  • 123 bp DNA Ladder length markers were used to estimate the fragments' size. Mwol digestion of wild-type DNA yielded a fragment of 265bp, whereas DNA containing the A460G mutation was not digested, yielding a fragment of 303bp.
  • First-strand cDNA was synthesized from total RNA of a TPMT- deficient patient whose erythrocyte TPMT activity and protein levels were 20- 30 fold less than wild-type patients (Fig. 6), and clones containing the TPMT open-reading frame were obtained from six independent PCR reactions. These clones were sequenced and revealed two distinct cDNAs, nine clones each.
  • TPMTA One sequence contained only the point mutation G 238 -C, described above, a mutant allele designated TPMTA.
  • the other sequence ( Figures 7A-7F) contained two point mutations, G 460 -A (G460A) and A 719 ⁇ G (A719G), leading to amino acid substitutions at codon 154 (Ala 154 ⁇ Thr) and codon 249 (Tyr ⁇ -Cys), designated TPMTB.
  • the equal abundance of cDNA clones for these two sequences suggests they are from two alleles of the TPMT gene expressed at comparable levels in this patient. Detection of TPMTB in Propositus Family Members
  • restriction fragment length polymorphism was used to identify the TPMTB allele in the propositus and his family members.
  • the wild-type cE>NA was cut by Mwol, but not by Accl, whereas the deficient patient's cDNA was heterozygous with respect to these restriction sites (Fig. 8), consistent with this patient having two different mutant TPMT alleles (i.e. TPMTA and TPMTB). Furthermore, the mother's TPMTB restriction pattern was the same as the propositus, while the father did not have the
  • TPMTB allele indicating that this patient inherited the TPMTB allele from his mother.
  • TPMT mRNA levels were similar in yeast expressing the wild-type and each of the three mutant cDNAs (i.e. TPMT m with only the G460A, TPMT m with only the A719G, and TPMTB with both mutations), suggesting that either of the point mutations alone or in combination did not alter transcription of TPMT cDNAs.
  • TPMT mRNA was not detected with yeast expressing the vector alone.
  • TPMT protein levels were similar between the wild-type and the TPMT ll9 mutant cDNA, but protein levels for the TPMT M and TPMTB were 4-fold and 400-fold less than the wild-type, respectively (Fig. 9B).
  • TPMTB protein was detectable only when 100-fold more of yeast lysate protein was loaded on the gel (Fig. 9B, lane 6), whereas no protein binding to anti-TPMT-specific antibodies was detected with yeast expressing vector without cDNA (Fig. 9B), even with loading 150-fold more of the yeast lysate (36 ⁇ g). Thus, neither point mutation alone altered TPMT protein levels comparable to the cDNA with both mutations.
  • TPMTB mutant allele
  • TPMT-deficiency was due to low levels of TPMT protein. This is consistent with previous immunotitration studies demonstrating that the immunoreactive protein of TPMT is correlated with enzymatic activity (Woodson, L.C., et al, J. Pharmacol. Exp. Ther. 222:174-181 (1982)).
  • the residual TPMT protein in this patient may be from his TPMTA allele, which is associated with a 20- fold reduction in TPMT protein when expressed in yeast.
  • TPMTB cDNA Heterologous expression of the TPMTB cDNA in yeast produced TPMT mRNA levels comparable to wild-type, indicating that these mutations have no significant impact on transcription in yeast. However, the TPMT protein level was about 400-fold less in yeast expressing TPMTB compared to the wild-type cDNA, indicating a posttranscriptional mechanism for the loss of TPMT activity.
  • the TPMTB cDNA contains two transition mutations (G460A and A719G), which differ from the single nucleotide transversion responsible for loss of activity in the TPMTA cDNA described above (Krynetski, E.Y., et al, Proc. Natl. Acad. Sci. (USA) 92:949-953 (1995)).
  • TPMT 46Q G460A mutation
  • TPMT n9 the A719G mutation
  • TPMT n9 the A719G mutation
  • TPMT mRNA levels were comparable for wild-type, TPMTB, TPMT m , and TPMT ll9 (Fig. 9A).
  • TPMT protein levels were 4-fold lower for TPMT m and about 400-fold lower for TPMTB, compared to wild-type, while TPMT 119 had protein levels comparable to wild-type (Fig. 9A).
  • the G460A transition was associated with a marked increase in Km for both 6MP (46-fold) and the co-substrate SAM (200-fold, such that the intrinsic clearance for 6MP methylation (Vmax/Km) was > 10-fold lower than wild-type protein.
  • Vmax/Km the Vmax/Km ratio for heterologously expressed TPMTA, described above
  • TPMT-deficient patients with either TPMTA/TPMTB, or TPMTB/TPMTB genotypes have now been documented. While additional TPMT mutations will likely be discovered, the present investigation has identified the major mutant allele at the human TPMT locus in Caucasians.
  • the DNA-base method of the present invention for prospectively diagnosing TPMT-deficiency should niinimize the risk of potentially life-threatening hematopoietic toxicity in these patients.

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Abstract

Cette invention décrit des mutants de thiopurine-S-méthyltransférase (TPMT). Le mutant TPMTA possède un point de mutation situé à la position 238 (G238→C) de l'ADNc et le mutant TPMTB implique deux transitions nucléotidiques aux positions 460 (G460→A) et 719 (A719→G) de l'ADNc. Le TPMTB est l'allèle mutant prédominant associé à la déficience en TPMT humaine qui peut causer une toxicité potentiellement fatale, lorsque des patients sont traités avec de la mercaptopurine, de l'azathioprine ou de la thioguanine. Font l'objet de cette invention ces allèles mutants ainsi que des fragments PCR (réactions en chaîne à la polymérase), des protéines mutantes et des anticorps pour ces allèles, ainsi que des kits et des procédés pour analyser le génotype de TPMT d'individus nécessitant un traitement.
PCT/US1995/010347 1995-08-14 1995-08-14 Identification de deux nouveaux alleles mutants de la thiopurine-s-methyltransferase humaine, et ses utilisations diagnostiques WO1997007201A1 (fr)

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Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2003066892A1 (fr) * 2002-02-04 2003-08-14 Epidauros Biotechnologie Ag Polymorphismes du gene humain de la tpmt et utilisations de ceux-ci dans des applications diagnostiques et therapeutiques
US6946258B2 (en) 2002-03-04 2005-09-20 Biologix Diagnostics, Llc Rapid, immunochemical process for measuring thiopurine methyltransferase
US7807359B2 (en) * 2006-12-01 2010-10-05 Quest Diagnostics Investments Incorporated Methods of detecting TPMT mutations
US8039794B2 (en) 2008-12-16 2011-10-18 Quest Diagnostics Investments Incorporated Mass spectrometry assay for thiopurine-S-methyl transferase activity and products generated thereby
CN101333560B (zh) * 2007-06-29 2012-01-04 上海裕隆生物科技有限公司 嘌呤类药物敏感基因检测试剂盒
CN119932178A (zh) * 2025-03-10 2025-05-06 浙江大学医学院附属第二医院 检测人tpmt基因和nudt15基因多态性的组合物、试剂盒及方法

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
DRUG METABOLISM AND DISPOSITION, Volume 23, Number 3, issued 1995, LEE et al., "Thiopurine Methyltransferase Pharmacogenetics: Cloning of Human Liver cDNA and a Processed Pseudogene on Human Chromosome 18q21.1", pages 398-405. *
MOLECULAR PHARMACOLOGY, Volume 43, Number 6, issued 1993, HONCHEL et al., "Human Thiopurine Methyltransferase: Molecular Cloning and Expression of T84 Colon Carcinoma Cell cDNA", pages 878-887. *
PROC. NATL. ACAD. SCI. U.S.A., Volume 92, issued February 1995, KRYNETSKI et al., "A Single Point Mutation Leading to Loss of Catalytic Activity in Human Thiopurine S-Methyltransferase", pages 949-953. *

Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2003066892A1 (fr) * 2002-02-04 2003-08-14 Epidauros Biotechnologie Ag Polymorphismes du gene humain de la tpmt et utilisations de ceux-ci dans des applications diagnostiques et therapeutiques
US6946258B2 (en) 2002-03-04 2005-09-20 Biologix Diagnostics, Llc Rapid, immunochemical process for measuring thiopurine methyltransferase
US7807359B2 (en) * 2006-12-01 2010-10-05 Quest Diagnostics Investments Incorporated Methods of detecting TPMT mutations
CN101333560B (zh) * 2007-06-29 2012-01-04 上海裕隆生物科技有限公司 嘌呤类药物敏感基因检测试剂盒
US8039794B2 (en) 2008-12-16 2011-10-18 Quest Diagnostics Investments Incorporated Mass spectrometry assay for thiopurine-S-methyl transferase activity and products generated thereby
US8497471B2 (en) 2008-12-16 2013-07-30 Quest Diagnostics Investments Incorporated Mass spectrometry assay for thiopurine-S-methyl transferase activity and products generated thereby
CN119932178A (zh) * 2025-03-10 2025-05-06 浙江大学医学院附属第二医院 检测人tpmt基因和nudt15基因多态性的组合物、试剂盒及方法

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