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WO2007121017A2 - Mutations et polymorphismes de la tyrosine kinase 4 de type fms - Google Patents

Mutations et polymorphismes de la tyrosine kinase 4 de type fms Download PDF

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Publication number
WO2007121017A2
WO2007121017A2 PCT/US2007/064116 US2007064116W WO2007121017A2 WO 2007121017 A2 WO2007121017 A2 WO 2007121017A2 US 2007064116 W US2007064116 W US 2007064116W WO 2007121017 A2 WO2007121017 A2 WO 2007121017A2
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Prior art keywords
flt
cancer
kdr
polypeptide
flt4
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PCT/US2007/064116
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English (en)
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WO2007121017A3 (fr
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Kenneth Wayne Culver
Jian Zhu
Stan Lilleberg
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Novartis Ag
Novartis Pharma Gmbh
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Publication of WO2007121017A2 publication Critical patent/WO2007121017A2/fr
Publication of WO2007121017A3 publication Critical patent/WO2007121017A3/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/106—Pharmacogenomics, i.e. genetic variability in individual responses to drugs and drug metabolism

Definitions

  • This invention relates generally to the analytical testing of tissue samples in vitro, and more particularly to aspects of genetic mutations and polymorphisms of FMS-like tyrosine kinase 4 (FLT-4).
  • FMS-like tyrosine kinase 4 FLT-4
  • Theranostic tests are also useful to select subjects for treatments that are particularly likely to benefit from the treatment or to provide an early and objective indication of treatment efficacy in individual subjects, so that the treatment can be altered with a minimum of delay.
  • Theranostics are useful in clinical diagnosis and management of a variety of diseases and disorders, which include, but are not limited to, e.g., cardiovascular disease, cancer, infectious diseases, Alzheimer's disease and the prediction of drug toxicity or drug resistance.
  • Theranostic tests may be developed in any suitable diagnostic testing format, which include, but is not limited to, e.g., immunohistochemical tests, clinical chemistry, immunoassay, cell-based technologies, and nucleic acid tests.
  • FMS-like tyrosine kinase 4 (a.k.a., FLT-4; vascular endothelial growth factor receptor 3; VEGFR3) is a receptor-type tyrosine kinase. Galland et al., Genomics 13: 475- 478, 1992.
  • the deduced amino acid sequence of the intracellular portion of the molecule showed that it was strongly related to FLTl and KDR and to a lesser degree to members of the class III receptor-type tyrosine kinases: FMS, PDGFR, KIT, and FLT-3.
  • the protein product of the FLT-4 receptor tyrosine kinase cDNA is structurally similar to the FLT-I and KDR/FLK-1 receptors (Pajusola et al., 1992), but FLT-4 does not bind VEGF (Pajusola et al., Oncogene 9: 3545-3555, 1994).
  • Lee et al. (1996) identified and characterized a vascular endothelial growth factor-related protein (VEGFC) that specifically binds to the extracellular domain of FLT-4 and stimulates tyrosine phosphorylation and mitogenesis of endothelial cells.
  • VEGFC vascular endothelial growth factor-related protein
  • FLT-4 mutation has been associated with lymphedema (Irrthum et al., Am. J. Hum. Genet. 67: 295-301, 2000; Brice et al, J Med Genet. 42(2):98-102, 2005) and haemangioma (Walter et al, Genes Chromosomes Cancer 33: 295-303, 2002). Accordingly, there is a need in the art for additional information about the relationship between FLT-4 mutations and cancer.
  • the invention provides for the use of an FLT-4 modulating agent in the manufacture of a medicament for the treatment of cancer in a selected patient population.
  • the patient population is selected on the basis of the genotype of the patients at an FLT-4 genetic locus indicative of efficacy of the FLT-4 modulating agent in treating cancer.
  • the cancer can be colon cancer.
  • the invention also provides an isolated polynucleotide having a sequence encoding an FLT-4 mutation.
  • the FLT-4 mutations are the previously- unidentified mutations listed in TABLE 1.
  • the invention provides vectors and organisms containing the FLT-4 mutations of the invention and polypeptides encoded by polynucleotides containing the FLT-4 mutations of the invention.
  • the invention further provides a method for treating cancer in a subject.
  • the genotype or haplotype of a subject is obtained at an FLT-4 gene locus, so that the genotype and/or haplotype is indicative of a propensity of the cancer to respond to the drug.
  • an anti-cancer therapy is administered to the subject.
  • the invention provides a method for diagnosing cancer in a subject and a method for choosing subjects for inclusion in a clinical trial for determining efficacy of an FLT-4 modulating agent; in both these methods the genotype and/or haplotype of a subject is interrogated at an FLT-4 gene locus. Also provided by the invention are kits for use in determining a treatment strategy for cancer.
  • the invention also provides for the use of each of the mutations of the inventions as a drug target.
  • the various aspects of the present invention relate to polynucleotides encoding FLT-4 mutations and polymorphisms of the invention, expression vectors encoding the FLT-4 mutant polypeptides of the invention and organisms that express the FLT-4 mutant/polymorphic polynucleotides and/or FLT-4 mutant/polymorphic polypeptides of the invention.
  • the various aspects of the present invention further relate to diagnostic/theranostic methods and kits that use the FLT-4 mutations and/or polymorphisms of the invention to identify individuals predisposed to disease or to classify individuals and tumours with regard to drug responsiveness, side effects, or optimal drug dose.
  • the invention provides methods for compound validation and a computer system for storing and analyzing data related to the FLT-4 mutations and polymorphisms of the invention. Accordingly, various particular embodiments that illustrate these aspects follow.
  • allele means a particular form of a gene or DNA sequence at a specific chromosomal location (locus).
  • the term “antibody” includes, but is not limited to, polyclonal antibodies, monoclonal antibodies, humanized or chimaeric antibodies and biologically functional antibody fragments sufficient for binding of the antibody fragment to the protein.
  • the term “clinical response” means any or all of the following: a quantitative measure of the response, no response, and adverse response (i.e., side effects).
  • the term “clinical trial” means any research study designed to collect clinical data on responses to a particular treatment, and includes but is not limited to phase I, phase II and phase III clinical trials. Standard methods are used to define the patient population and to enrol subjects.
  • the term "effective amount" of a compound is a quantity sufficient to achieve a desired pharmacodynamic, toxicologic, therapeutic and/or prophylactic effect, for example, an amount which results in the prevention of or a decrease in the symptoms associated with a disease that is being treated, e.g., the diseases associated with FLT-4 mutant polypeptides and FLT-4 mutant polynucleotides identified herein.
  • the amount of compound administered to the subject will depend on the type and severity of the disease and on the characteristics of the individual, such as general health, age, sex, body weight and tolerance to drugs. It will also depend on the degree, severity and type of disease. The skilled artisan will be able to determine appropriate dosages depending on these and other factors.
  • an effective amount of the compounds of the present invention ranges from about 0.000001 mg per kilogram body weight per day to about 10,000 mg per kilogram body weight per day.
  • the dosage ranges are from about 0.0001 mg per kilogram body weight per day to about 100 mg per kilogram body weight per day.
  • the compounds of the present invention can also be administered in combination with each other, or with one or more additional therapeutic compounds.
  • Glivec® (Gleevec® ; imatinib) is a medication for chronic myeloid leukaemia (CML) and certain stages of gastrointestinal stromal tumours (GIST). It targets and interferes with the molecular abnormalities that drive the growth of cancer cells.
  • Glivec® has potential as an anticancer therapy for several types of cancer, including leukaemia and solid tumours.
  • the aromatase inhibitor FEMARA is a treatment for advanced breast cancer in postmenopausal women. It blocks the use of oestrogen by certain types of breast cancer that require oestrogen to grow. Janicke F, Breast 13 Suppl 1 :S10-8 (December 2004); Mouridsen H et al, Oncologist 9(5):489-96 (2004).
  • Sandostatin® LAR® is used to treat patients with acromegaly and to control symptoms, such as severe diarrhoea and flushing, in patients with functional gastro-entero- pancreatic (GEP) tumours ⁇ e.g., metastatic carcinoid tumours and vasoactive intestinal peptide-secreting tumours [VIPomas]).
  • GEP gastro-entero- pancreatic
  • Sandostatin® LAR® regulates hormones in the body to help manage diseases and their symptoms.
  • ZOMET A® is a treatment for hypocalcaemia of malignancy (HCM)I and for the treatment of bone metastases across a broad range of tumour types. These tumours include multiple myeloma, prostrate cancer, breast cancer, lung cancer, renal cancer and other solid tumours. Rosen LS et al, Cancer 100(12):2613-21 (June 15, 2004).
  • Vatalanib (l-[4-chloroanilino]-4-[4-pyridylmethyl] phthalazine succinate) is a multi- VEGF receptor (VEGF) inhibitor that may block the creation of new blood vessels to prevent tumour growth.
  • VEGF VEGF receptor
  • This compound inhibits all known VEGF receptor tyrosine kinases, blocking angiogenesis and lymphangiogenesis. Drevs J et al, Cancer Res. 60:4819-4824 (2000); Wood JM et al, Cancer Res. 60:2178-2189 (2000).
  • Vatalanib is being studied in two large, multinational, randomized, phase III, placebo-controlled trials in combination with FOLFOX- 4 in first-line and second-line treatment of patients with metastatic colorectal cancer.
  • Thomas A et al 37th Annual Meeting of the American Society of Clinical Oncology, San Francisco, CA, Abstract 279 (May 12-15, 2001).
  • everolimus inhibits oncogenic signalling in tumour cells.
  • mTOR mammalian target of rapamycin
  • everolimus exhibits broad antiproliferative activity in tumour cell lines and animal models of cancer. Boulay A etal, Cancer Res. 64:252-261 (2004).
  • everolimus also potently inhibited the proliferation of human umbilical vein endothelial cells directly indicating an involvement in angiogenesis.
  • everolimus may provide a clinical benefit to patients with cancer.
  • Everolimus is being investigated for its antitumour properties in a number of clinical studies in patients with haematological and solid tumours. Huang S & Houghton PJ, Curr. Opin. Investig. Drugs 3:295-304 (2002).
  • Gimatecan is a novel oral inhibitor of topoisomerase I (topo I). Gimatecan blocks cell division in cells that divide rapidly, such as cancer cells, which activates apoptosis. Preclinical data indicate that gimatecan is not a substrate for multidrug resistance pumps, and that it increases the drug-target interaction. De Cesare M et al, Cancer Res. 61:7189-7195 (2001). Phase I clinical studies indicate that the dose-limiting toxicity of gimatecan is myelosuppression.
  • Patupilone is a microtubule stabilizer.
  • Altmann K-H Curr. Opin. Chem. Biol. 5:424-431 (2001); Altmann K-H et al, Biochim. Biophys. Acta 470:M79-M91 (2000); O'Neill V et al, 36th Annual Meeting of the American Society of Clinical Oncology; May 19- 23, 2000; New La, LA, Abstract 829; Calvert PM et al Proceedings of the 11th National Cancer Institute-European Organization for Research and Treatment of Cancer/ American Association for Cancer Research Symposium on New Drugs in Cancer Therapy, November 7-10, 2000; Amsterdam, The Netherlands, Abstract 575.
  • Patupilone blocked mitosis and induced apoptosis greater than the frequently used anticancer drug paclitaxel. Also, patupilone retained full activity against human cancer cells that were resistant to paclitaxel and other chemotherapeutic agents.
  • Midostaurin is an inhibitor of multiple signalling proteins. By targeting specific receptor tyrosine kinases and components of several signal transduction pathways, midostaurin impacts several targets involved in cell growth (e.g., KIT, PDGFR, PKC), leukaemic cell proliferation (e.g., FLT3), and angiogenesis (e.g., VEGFR2).
  • KIT KIT
  • PDGFR PDGFR
  • PKC leukaemic cell proliferation
  • angiogenesis e.g., VEGFR2
  • midostaurin showed broad antiproliferative activity against various tumour cell lines, including those that were resistant to several other chemotherapeutic agents.
  • the somatostatin analogue pasireotide is a stable cyclohexapeptide with broad somatotropin release inhibiting factor (SRIF) receptor binding.
  • SRIF broad somatotropin release inhibiting factor
  • LBH589 By triggering apoptosis, LBH589 induces growth inhibition and regression in tumour cell lines. LBH589 is being tested in phase I clinical trials as an anticancer agent. See also, George P etal, Blood 105(4): 1768-76 (February 15, 2005).
  • AEE788 inhibits multiple receptor tyrosine kinases including EGFR, HER2, and VEGFR, which stimulate tumour cell growth and angiogenesis. Traxler P et ah, Cancer Res. 64:4931-4941 (2004). In preclinical studies, AEE788 showed high target specificity and demonstrated antiproliferative effects against tumour cell lines and in animal models of cancer. AEE788 also exhibited direct antiangiogenic activity. AEE788 is currently in phase I clinical development.
  • AMN 107 is an oral tyrosine kinase inhibitor that targets BCR-ABL, KIT, and PDGFR.
  • Preclinical studies have shown in cellular assays using Philadelphia chromosome- positive (Ph+) CML cells that AMN 107 is highly potent and has high selectivity for BCR- ABL, KIT, and PDGFR.
  • Pr+ Philadelphia chromosome- positive
  • AMN107 also shows activity against mutated variants of BCR-ABL.
  • AMN 107 is currently being studied in phase I clinical trials.
  • FLT-4 modulating agent is any compound that alters (e.g., increases or decreases).
  • the expression level or biological activity level of FLT-4 polypeptide compared to the expression level or biological activity level of FLT-4 polypeptide in the absence of the FLT-4 modulating agent.
  • FLT-4 modulating agent can be a small molecule, antibody, polypeptide, carbohydrate, lipid, nucleotide, or combination thereof.
  • the FLT-4 modulating agent can be an organic compound or an inorganic compound.
  • expression includes but is not limited to one or more of the following: transcription of the gene into precursor mRNA; splicing and other processing of the precursor mRNA to produce mature mRNA; mRNA stability; translation of the mature mRNA into protein (including codon usage and tRNA availability); and glycosylation and/or other modifications of the translation product, if required for proper expression and function.
  • gene means a segment of DNA that contains all the information for the regulated biosynthesis of an RNA product, including promoters, exons, introns, and other untranslated regions that control expression.
  • genotype means an unphased 5' to 3' sequence of nucleotide pairs found at one or more polymorphic or mutant sites in a locus on a pair of homologous chromosomes in an individual.
  • genotype includes a full- genotype and/or a sub-genotype.
  • locus means a location on a chromosome or DNA molecule corresponding to a gene or a physical or phenotypic feature.
  • mutant means any heritable or acquired variation from the wild-type that alters the nucleotide sequence thereby changing the protein sequence.
  • the term “mutant” is used interchangeably with the terms “marker”, “biomarker”, and “target” throughout the specification.
  • the term "medical condition” includes, but is not limited to, any condition or disease manifested as one or more physical and/or psychological symptoms for which treatment and/or prevention is desirable, and includes previously and newly identified diseases and other disorders.
  • nucleotide pair means the two nucleotides bound to each other between the two nucleotide strands.
  • polymorphic site means a position within a locus at which at least two alternative sequences are found in a population, the most frequent of which has a frequency of no more than 99%.
  • polymorphism means any sequence variant present at a frequency of >1% in a population.
  • the sequence variant may be present at a frequency significantly greater than 1% such as 5% or 10% or more.
  • the term may be used to refer to the sequence variation observed in an individual at a polymorphic site.
  • Polymorphisms include nucleotide substitutions, insertions, deletions and microsatellites and may, but need not, result in detectable differences in gene expression or protein function.
  • polynucleotide means any RNA or DNA, which may be unmodified or modified RNA or DNA.
  • Polynucleotides include, without limitation, single- and double-stranded DNA, DNA that is a mixture of single- and double-stranded regions, single- and double-stranded RNA, RNA that is mixture of single- and double-stranded regions, and hybrid molecules comprising DNA and RNA that may be single-stranded or, more typically, double-stranded or a mixture of single- and double-stranded regions.
  • polynucleotide refers to triple-stranded regions comprising RNA or DNA or both RNA and DNA.
  • the term polynucleotide also includes DNAs or RNAs containing one or more modified bases and DNAs or RNAs with backbones modified for stability or for other reasons.
  • the polynucleotide contains polynucleotide sequences from the FLT-4 gene.
  • polypeptide means any polypeptide comprising two or more amino acids joined to each other by peptide bonds or modified peptide bonds, i.e., peptide isosteres.
  • Polypeptide refers to both short chains, commonly referred to as peptides, glycopeptides or oligomers, and to longer chains, generally referred to as proteins.
  • Polypeptides may contain amino acids other than the 20 gene-encoded amino acids.
  • Polypeptides include amino acid sequences modified either by natural processes, such as post- translational processing, or by chemical modification techniques that are well known in the art. Such modifications are well described in basic texts and in more detailed monographs, as well as in a voluminous research literature.
  • the polypeptide contains polypeptide sequences from the FLT-4 protein.
  • small molecule means a composition that has a molecular weight of less than about 5 kDa and more preferably less than about 2 kDa.
  • Small molecules can be, e.g., nucleic acids, peptides, polypeptides, glycopeptides, peptidomimetics, carbohydrates, lipids, lipopolysaccharides, combinations of these, or other organic or inorganic molecules.
  • mutant nucleic acid means a nucleic acid sequence, which comprises a nucleotide that is variable within an otherwise identical nucleotide sequence between individuals or groups of individuals, thus, existing as alleles. Such mutant nucleic acids are preferably from about 15 to about 500 nucleotides in length.
  • the mutant nucleic acids may be part of a chromosome, or they may be an exact copy of a part of a chromosome, e.g., by amplification of such a part of a chromosome through PCR or through cloning.
  • the mutant probes according to the invention are oligonucleotides that are complementary to a mutant nucleic acid.
  • SNP nucleic acid means a nucleic acid sequence, which comprises a nucleotide that is variable within an otherwise identical nucleotide sequence between individuals or groups of individuals, thus, existing as alleles. Such SNP nucleic acids are preferably from about 15 to about 500 nucleotides in length.
  • the SNP nucleic acids may be part of a chromosome, or they may be an exact copy of a part of a chromosome, e.g., by amplification of such a part of a chromosome through PCR or through cloning.
  • the SNP nucleic acids are referred to hereafter simply as "SNPs".
  • the SNP probes according to the invention are oligonucleotides that are complementary to a SNP nucleic acid. In a particular embodiment, the SNP is in the FLT-4 gene.
  • the term "subject" means that preferably the subject is a mammal, such as a human, but can also be an animal, e.g., domestic animals (e.g., dogs, cats and the like), farm animals (e.g., cows, sheep, pigs, horses and the like) and laboratory animals (e.g., monkey (e.g., cynmologous monkey), rats, mice, guinea pigs and the like).
  • the administration of an agent or drug to a subject or patient includes self-administration and the administration by another.
  • missense mutations were located in the human FLT-4 gene (NT 077451). Bioinformatics analysis of the FLT-4 mutations of the invention are further detailed in EXAMPLE I.
  • Sequence variation in the human germline consists primarily of SNPs, the remainder being short tandem repeats (including micro-satellites), long tandem repeats (mini-satellites), and other insertions and deletions.
  • a SNP is the occurrence of nucleotide variability at a single position in the genome, in which two alternative bases occur at appreciable frequency (i.e., >1%) in the human population.
  • a SNP may occur within a gene or within intergenic regions of the genome.
  • SNPs Due to their prevalence and widespread nature, SNPs have the potential to be important tools for locating genes that are involved in human disease conditions. See e.g., ⁇ ang et al., Science 280: 1077-1082 (1998)).
  • An association between SNP' s and/or mutations and a particular phenotype does not necessarily indicate or require that the SNP or mutation is causative of the phenotype. Instead, an association with a SNP may merely be due to genome proximity between a SNP and those genetic factors actually responsible for a given phenotype, such that the SNP and said genetic factors are closely linked. That is, a SNP may be in linkage disequilibrium ("LD") with the "true" functional variant. LD exists when alleles at two distinct locations of the genome are more highly associated than expected. Thus, a SNP may serve as a marker that has value by virtue of its proximity to a mutation or other DNA alteration (e.g., gene duplication) that causes a particular phenotype.
  • LD linkage disequilibrium
  • SNPs and mutations that are associated with disorders may also have a direct effect on the function of the genes in which they are located.
  • a sequence variant e.g., SNP
  • SNP may result in an amino acid change or may alter exon-intron splicing, thereby directly modifying the relevant protein, or it may exist in a regulatory region, altering the cycle of expression or the stability of the mRNA (see, e.g., Nowotny et al, Current Opinions in Neurobiology, 11 :637-641 (2001 )).
  • nucleic acid molecules containing the gene may be complementary double stranded molecules and thus reference to a particular site on the sense strand refers as well to the corresponding site on the complementary antisense strand. That is, reference may be made to the same polymorphic or mutant site on either strand and an oligonucleotide may be designed to hybridize specifically to either strand at a target region containing the polymorphic and/or mutant site.
  • the invention also includes single-stranded polynucleotides and mutations that are complementary to the sense strand of the genomic variants described herein.
  • Detection technologies include fluorescent polarization (Chan et al., Genome Res. 9:492-499 (1999)), Iuminometric detection of pyrophosphate release (pyrosequencing) (Ahmadiian et al, Anal. Biochem. 280:103-10 (2000)), fluorescence resonance energy transfer (FRET)-based cleavage assays, DHPLC, and mass spectrometry (Shi, Clin Chem 47:164-172 (2001); U.S. Pat. No. 6,300,076 Bl). Other methods of detecting and characterizing SNPs and mutations are those disclosed in U.S. Pat. Nos. 6,297,018 Bl and 6,300,063 Bl.
  • the detection of polymorphisms and mutations is detected using INVADERTM technology (available from Third Wave Technologies Inc. Madison, Wisconsin USA).
  • INVADERTM technology available from Third Wave Technologies Inc. Madison, Wisconsin USA.
  • a specific upstream "invader” oligonucleotide and a partially overlapping downstream probe together form a specific structure when bound to complementary DNA template.
  • This structure is recognized and cut at a specific site by the Cleavase enzyme, resulting in the release of the 5' flap of the probe oligonucleotide.
  • This fragment then serves as the "invader” oligonucleotide with respect to synthetic secondary targets and secondary fluorescently labelled signal probes contained in the reaction mixture. This results in specific cleavage of the secondary signal probes by the Cleavase enzyme.
  • Fluorescent signal is generated when this secondary probe (labelled with dye molecules capable of fluorescence resonance energy transfer) is cleaved.
  • Cleavases have stringent requirements relative to the structure formed by the overlapping DNA sequences or flaps and can, therefore, be used to specifically detect single base pair mismatches immediately upstream of the cleavage site on the downstream DNA strand.
  • Ryan D et al Molecular Diagnosis 4(2): 135-144 (1999) and Lyamichev V et al. Nature Biotechnology 17: 292-296 (1999), see also U.S. Pat. Nos. 5,846,717 and 6,001,567.
  • polymorphisms and mutations may also be determined using a mismatch detection technique including, but not limited to, the RNase protection method using riboprobes (Winter et al, Proc. Natl. Acad. ScL USA 82:7575 (1985); Meyers et al, Science 230:1242 (1985)) and proteins which recognize nucleotide mismatches, such as the E. coli mutS protein (Modrich P 5 Ann Rev Genet 25:229-253 (1991)).
  • riboprobes Winter et al, Proc. Natl. Acad. ScL USA 82:7575 (1985); Meyers et al, Science 230:1242 (1985)
  • proteins which recognize nucleotide mismatches such as the E. coli mutS protein (Modrich P 5 Ann Rev Genet 25:229-253 (1991)).
  • variant alleles can be identified by single strand conformation polymorphism (SSCP) analysis (Orita et al, Genomics 5:874-879 (1989); Humphries et al, in Molecular Diagnosis of Genetic Diseases, Elles R, ed. (1996) pp. 321-340) or denaturing gradient gel electrophoresis (DGGE) (Wartell et al, Nucl Acids Res. 18:2699-2706 (1990); Sheffield et al, Proc. Natl. Acad. ScL USA 86: 232-236 (1989)).
  • SSCP single strand conformation polymorphism
  • DGGE denaturing gradient gel electrophoresis
  • a polymerase-mediated primer extension method may also be used to identify the polymorphisms/mutations.
  • multiple polymorphic and/or mutant sites may be investigated by simultaneously amplifying multiple regions of the nucleic acid using sets of allele-specific primers as described in WO 89/10414.
  • the invention provides methods and compositions for haplotyping and/or genotyping the genetic polymorphisms (and possibly mutations) in an individual.
  • the terms "genotype” and “haplotype” mean the genotype or haplotype containing the nucleotide pair or nucleotide, respectively, that is present at one or more of the novel polymorphic (or mutant) sites described herein and may optionally also include the nucleotide pair or nucleotide present at one or more additional polymorphic (or mutant) sites in the gene.
  • the additional polymorphic (and mutant) sites may be currently known polymorphic/mutant sites or sites that are subsequently discovered.
  • compositions contain oligonucleotide probes and primers designed to specifically hybridize to one or more target regions containing, or that are adjacent to, a polymorphic or mutant site.
  • Oligonucleotide compositions of the invention are useful in methods for genotyping and/or haplotyping a gene in an individual.
  • the methods and compositions for establishing the genotype or haplotype of an individual at the novel polymorphic/mutant sites described herein are useful for studying the effect of the polymorphisms and mutations in the aetiology of diseases affected by the expression and function of the protein, studying the efficacy of drugs targeting, predicting individual susceptibility to diseases affected by the expression and function of the protein and predicting individual responsiveness to drugs targeting the gene product.
  • Some embodiments of the invention contain two or more differently labelled genotyping oligonucleotides, for simultaneously probing the identity of nucleotides at two or more polymorphic or mutant sites. It is also contemplated that primer compositions may contain two or more sets of allele-specific primer pairs to allow simultaneous targeting and amplification of two or more regions containing a polymorphic or mutant site. [0064] Genotyping oligonucleotides of the invention may be immobilized on or synthesized on a solid surface such as a microchip, bead, or glass slide (see, e.g., WO 98/20020 and WO 98/20019).
  • Immobilized genotyping oligonucleotides may be used in a variety of polymorphism and mutation detection assays, including but not limited to probe hybridization and polymerase extension assays.
  • Immobilized genotyping oligonucleotides of the invention may comprise an ordered array of oligonucleotides designed to rapidly screen a DNA sample for polymorphisms and mutations in multiple genes at the same time.
  • An allele-specific oligonucleotide primer of the invention has a 3' terminal nucleotide, or preferably a 3' penultimate nucleotide, that is complementary to only one nucleotide of a particular SNP, thereby acting as a primer for polymerase-mediated extension only if the allele containing that nucleotide is present.
  • Allele-specific oligonucleotide (ASO) primers hybridizing to either the coding or noncoding strand are contemplated by the invention.
  • An ASO primer for detecting gene polymorphisms and mutations can be developed using techniques known to those of skill in the art.
  • genotyping oligonucleotides of the invention hybridize to a target region located one to several nucleotides downstream of one of the novel polymorphic or mutant sites identified herein. Such oligonucleotides are useful in polymerase-mediated primer extension methods for detecting one of the novel polymorphisms or mutations described herein and therefore such genotyping oligonucleotides are referred to herein as "primer- extension oligonucleotides”.
  • the 3 '-terminus of a primer- extension oligonucleotide is a deoxynucleotide complementary to the nucleotide located immediately adjacent to the polymorphic/mutant site.
  • a genotyping method of the invention involves isolating from an individual a nucleic acid mixture comprising at least one copy of the gene of interest and/or a fragment or flanking regions thereof, and determining the identity of the nucleotide pair at one or more of the polymorphic/mutant sites in the nucleic acid mixture.
  • the two "copies" of a germline gene in an individual may be the same on each allele or may be different on each allele.
  • the genotyping method comprises determining the identity of the nucleotide pair at each polymorphic and mutant site.
  • the nucleic acid mixture is isolated from a biological sample taken from the individual, such as a blood sample, tumour or tissue sample.
  • tissue samples include whole blood, tumour or as part of any tissue type, semen, saliva, tears, urine, faecal material, sweat, buccal smears, skin and hair.
  • the nucleic acid mixture may be comprised of genomic DNA, mRNA, or cDNA and, in the latter two cases, the biological sample must be obtained from an organ in which the gene may be expressed.
  • mRNA or cDNA preparations would not be used to detect polymorphisms or mutations located in introns or in 5' and 3' nontranscribed regions.
  • a gene fragment If a gene fragment is isolated, it must usually contain the polymorphic and/or mutant sites to be genotyped. Exceptions can include mutations leading to truncation of the gene where a specific polymorphism may be lost. In these cases, the specific DNA alterations are determined by assessing the flanking sequences of the gene and underscore the need to specifically look for both polymorphisms and mutations.
  • One embodiment of the haplotyping method of the invention comprises isolating from an individual a nucleic acid molecule containing only one of the two copies of a gene of interest, or a fragment thereof, and determining the identity of the nucleotide at one or more of the polymorphic or mutant sites in that copy.
  • the nucleic acid may be isolated using any method capable of separating the two copies of the gene or fragment.
  • any individual clone will only provide haplotype information on one of the two gene copies present in an individual. If haplotype information is desired for the individual's other copy, additional clones will need to be examined. Typically, at least five clones should be examined to have more than a 90% probability of haplotyping both copies of the gene in an individual.
  • the nucleotide at each polymorphic or mutant site is identified.
  • a haplotype pair is determined for an individual by identifying the phased sequence of nucleotides at one or more of the polymorphic/mutant sites in each copy of the gene that is present in the individual.
  • the haplotyping method comprises identifying the phased sequence of nucleotides at each polymorphic/mutant site in each copy of the gene.
  • the identifying step is preferably performed with each copy of the gene being placed in separate containers. However, if the two copies are labelled with different tags, or are otherwise separately distinguishable or identifiable, it is possible in some cases to perform the method in the same container.
  • first and second copies of the gene are labelled with different first and second fluorescent dyes, respectively, and an allele-specific oligonucleotide labelled with yet a third different fluorescent dye is used to assay the polymorphic/mutant sites, then detecting a combination of the first and third dyes would identify the polymorphism or mutation in the first gene copy, while detecting a combination of the second and third dyes would identify the polymorphism or mutation in the second gene copy.
  • the identity of a nucleotide (or nucleotide pair) at a polymorphic and/or mutant site may be determined by amplifying a target region containing the polymorphic and/or mutant sites directly from one or both copies of the gene, or fragments thereof, and sequencing the amplified regions by conventional methods. It will be readily appreciated by the skilled artisan that only one nucleotide will be detected at a polymorphic or mutant site in individuals who are homozygous at that site, while two different nucleotides will be detected if the individual is heterozygous for that site.
  • the polymorphism or mutation may be identified directly, known as positive-type identification, or by inference, referred to as negative-type identification.
  • a site may be positively determined to be either guanine or cytosine for all individuals homozygous at that site, or both guanine and cytosine, if the individual is heterozygous at that site.
  • the site may be negatively determined to be not guanine (and thus cytosine/cytosine) or not cytosine (and thus guanine/guanine).
  • the identity of the alleles present at any of the novel polymorphic/mutant sites of the invention may be indirectly determined by genotyping other polymorphic/mutant sites in linkage disequilibrium with those sites of interest. As described supra, two sites are said to be in linkage disequilibrium if the presence of a particular variant (polymorphism or mutation) at one site is indicative of the presence of another variant at a second site. See, Stevens JC, MoI, Diag. 4:309-317 (1999).
  • Polymorphic and mutant sites in linkage disequilibrium with the polymorphic or mutant sites of the invention may be located in regions of the same gene or in other genomic regions. Genotyping of a polymorphic/mutant site in linkage disequilibrium with the novel polymorphic/mutant sites described herein may be performed by, but is not limited to, any of the above-mentioned methods for detecting the identity of the allele at a polymorphic/mutant site.
  • the target regions may be amplified using any oligonucleotide-directed amplification method, including but not limited to polymerase chain reaction (PCR).
  • PCR polymerase chain reaction
  • LCR ligase chain reaction
  • OLA oligonucleotide ligation assay
  • Oligonucleotides useful as primers or probes in such methods should specifically hybridize to a region of the nucleic acid that contains or is adjacent to the polymorphic/mutant site.
  • the oligonucleotides are between 10 and 35 nucleotides in length and preferably, between 15 and 30 nucleotides in length. Most preferably, the oligonucleotides are 20 to 25 nucleotides long. The exact length of the oligonucleotide will depend on many factors that are routinely considered and practiced by the skilled artisan.
  • nucleic acid amplification procedures may be used to amplify the target region including transcription-based amplification systems (U.S. Pat. No. 5,130,238; EP 329,822; U.S. Pat. No. 5,169,766, published PCT patent application WO 89/06700) and isothermal methods (Walker et al, Proc. Natl Acad. Sci. USA 89: 392-396 (1992)).
  • a polymorphism or mutation in the target region may be assayed before or after amplification using one of several hybridization-based methods known in the art. Typically, allele-specific oligonucleotides are utilized in performing such methods.
  • the allele-specific oligonucleotides may be used as differently labelled probe pairs, with one member of the pair showing a perfect match to one variant of a target sequence and the other member showing a perfect match to a different variant.
  • more than one polymorphic/mutant site may be detected at once using a set of allele-specific oligonucleotides or oligonucleotide pairs.
  • the members of the set have melting temperatures within 5°C, and more preferably within 2°C, of each other when hybridizing to each of the polymorphic or mutant sites being detected.
  • Hybridizing Allele-Specific Oligonucleotide to a Target Gene Hybridization of an allele-specific oligonucleotide to a target polynucleotide may be performed with both entities in solution, or such hybridization may be performed when either the oligonucleotide or the target polynucleotide is covalently or noncovalently affixed to a solid support. Attachment may be mediated, for example, by antibody-antigen interactions, poly-L-Lys, streptavidin or avidin-biotin, salt bridges, hydrophobic interactions, chemical linkages, UV cross-linking, baking, etc.
  • Allele-specific oligonucleotide may be synthesized directly on the solid support or attached to the solid support subsequent to synthesis.
  • Solid-supports suitable for use in detection methods of the invention include substrates made of silicon, glass, plastic, paper and the like, which may be formed, for example, into wells (as in 96-well plates), slides, sheets, membranes, fibres, chips, dishes, and beads.
  • the solid support may be treated, coated or derivatised to facilitate the immobilization of the allele-specific oligonucleotide or target nucleic acid.
  • the genotype or haplotype for the gene of an individual may also be determined by hybridization of a nucleic sample containing one or both copies of the gene to nucleic acid arrays and subarrays such as described in WO 95/11995.
  • the arrays would contain a battery of allele-specific oligonucleotides representing each of the polymorphic or mutant sites to be included in the genotype or haplotype.
  • the present invention provides a method for determining the frequency of a genotype or haplotype in a population.
  • the method comprises determining the genotype or the haplotype for a gene present in each member of the population, wherein the genotype or haplotype comprises the nucleotide pair or nucleotide detected at one or more of the polymorphic sites in the gene and mutations identified in the region, and calculating the frequency at which the genotype or haplotype is found in the population.
  • the population may be a reference population, a family population, a same sex population, a population group, or a trait population (e.g., a group of individuals exhibiting a trait of interest such as a medical condition or response to a therapeutic treatment).
  • a trait population e.g., a group of individuals exhibiting a trait of interest such as a medical condition or response to a therapeutic treatment.
  • frequency data for genotypes and/or haplotypes found in a reference population are used in a method for identifying an association between a trait and a genotype or a haplotype.
  • the trait may be any detectable phenotype, including but not limited to cancer, susceptibility to a disease or response to a treatment.
  • the method involves obtaining data on the frequency of the genotypes or haplotypes of interest in a reference population and comparing the data to the frequency of the genotypes or haplotypes in a population exhibiting the trait.
  • Frequency data for one or both of the reference and trait populations may be obtained by genotyping or haplotyping each individual in the populations using one of the methods described above.
  • the haplotypes for the trait population may be determined directly or, alternatively, by the predictive genotype to haplotype approach described above.
  • the trait is susceptibility to a disease, severity of a disease, the staging of a disease or response to a drug.
  • Such methods have applicability in developing diagnostic tests and therapeutic treatments for all pharmacogenetic applications where there is the potential for an association between a genotype and a treatment outcome, including efficacy measurements, PD measurements, PK measurements and side effect measurements.
  • the frequency data for the reference and/or trait populations are obtained by accessing previously determined frequency data, which may be in written or electronic form.
  • the frequency data may be present in a database that is accessible by a computer.
  • the frequencies of the genotypes or haplotypes of interest in the reference and trait populations are compared.
  • the frequencies of all genotypes and/or haplotypes observed in the populations are compared. If a particular genotype or haplotype for the gene is more frequent in the trait population than in the reference population at a statistically significant amount, then the trait is predicted to be associated with that genotype or haplotype.
  • the haplotype frequency data for different ethnogeographic groups are examined to determine whether they are consistent with Hardy- Weinberg equilibrium.
  • Hartl DL et al. Principles of Population Genomics, 3rd Ed. (Sinauer Associates, Sunderland, MA, 1997).
  • a statistically significant difference between the observed and expected haplotype frequencies could be due to one or more factors including significant inbreeding in the population group, strong selective pressure on the gene, sampling bias, and/or errors in the genotyping process. If large deviations from ⁇ ardy- Weinberg equilibrium are observed in an ethnogeographic group, the number of individuals in that group can be increased to see if the deviation is due to a sampling bias. If a larger sample size does not reduce the difference between observed and expected haplotype pair frequencies, then one may wish to consider haplotyping the individual using a direct haplotyping method such as, for example, CLASPER SystemTM technology (U.S. Pat. No. 5,866,404), SMD, or allele-specific long-range PCR (Michalotos-Beloin et al, Nucl. Acids Res. 24: 4841-4843 (1996)).
  • CLASPER SystemTM technology U.S. Pat. No. 5,866,404
  • SMD SMD
  • allele-specific long-range PCR
  • the assigning step involves performing the following analysis. First, each of the possible haplotype pairs is compared to the haplotype pairs in the reference population. Generally, only one of the haplotype pairs in the reference population matches a possible haplotype pair and that pair is assigned to the individual. Occasionally, only one haplotype represented in the reference haplotype pairs is consistent with a possible haplotype pair for an individual, and in such cases the individual is assigned a haplotype pair containing this known haplotype and a new haplotype derived by subtracting the known haplotype from the possible haplotype pair.
  • the individual is preferably haplotyped using a direct molecular haplotyping method such as, for example, those discussed supra.
  • statistical analysis is performed by the use of standard ANOVA tests with a Bonferoni correction and/or a bootstrapping method that simulates the genotype phenotype correlation many times and calculates a significance value. When many polymorphisms and/or mutations are being analyzed, a calculation may be performed to correct for a significant association that might be found by chance.
  • the trait of interest is a clinical response exhibited by a patient to some therapeutic treatment, for example, response to a drug targeting or to a therapeutic treatment for a medical condition.
  • a detectable genotype or haplotype that is in linkage disequilibrium with a genotype or haplotype of interest may be used as a surrogate marker.
  • a genotype that is in linkage disequilibrium with another genotype is indicated where a particular genotype or haplotype for a given gene is more frequent in the population that also demonstrates the potential surrogate marker genotype than in the reference population. If the frequency is statistically significant, then the marker genotype is predictive of that genotype or haplotype, and can be used as a surrogate marker.
  • genotype or haplotype data is obtained on the clinical responses exhibited by a population of individuals who received the treatment, hereinafter the "clinical population". This clinical data may be obtained by analyzing the results of a clinical trial that has already been previously conducted and/or by designing and carrying out one or more new clinical trials.
  • the individuals included in the clinical population be graded for the existence of the medical condition of interest. This grading of potential patients could employ a standard physical exam or one or more lab tests. Alternatively, grading of patients could use genotyping or haplotyping for situations where there is a strong correlation between haplotype pair and disease susceptibility or severity.
  • the therapeutic treatment of interest is administered to each individual in the trial population, and each individual's response to the treatment is measured using one or more predetermined criteria. It is contemplated that in many cases, the trial population will exhibit a range of responses, and that the investigator may choose more than one responder groups (e.g., low, medium, high) made up by the various responses. In addition, the gene for each individual in the trial population is genotyped and/or haplotyped, which may be done before or after administering the treatment. [0090] These results are then analyzed to determine if any observed variation in clinical response between polymorphism/mutation groups is statistically significant.
  • a second method for finding correlations between genotype and haplotype content and clinical responses uses predictive models based on error-minimizing optimization algorithms, one of which is a genetic algorithm.
  • Judson R Genetic Algorithms and Their Uses in Chemistry, in Reviews in Computational Chemistry, Vol. 10, Lipkowitz KB & Boyd DB, eds. (VCH Publishers, New York, 1997) pp. 1-73.
  • Simulated annealing Press et al, Numerical Recipes in C: The Art of Scientific Computing, Ch. 10 (Cambridge University Press, Cambridge, 1992)
  • neural networks (Rich E & Knight K, Artificial Intelligence, 2nd Edition, Ch.
  • Correlations may also be analyzed using analysis of variation (ANOVA) techniques to determine how much of the variation in the clinical data is explained by different subsets of the polymorphic and mutant sites in the gene.
  • ANOVA is used to test hypotheses about whether a response variable is caused by or correlates with one or more traits or variables that can be measured (Fisher & vanBelle, supra, Ch. 10).
  • correlations between individual response and genotype or haplotype content are created. Correlations may be produced in several ways. In one method, individuals are grouped by their genotype or haplotype (or haplotype pair) (also referred to as a polymo ⁇ hism/mutation group), and then the averages and standard deviations of clinical responses exhibited by the members of each polymorphism/mutation group are calculated.
  • the diagnostic method may take one of several forms: for example, a direct DNA test (i.e., genotyping or haplotyping one or more of the polymorphic/mutant sites in the gene), a serological test, or a physical exam measurement. The only requirement is that there be a good correlation between the diagnostic test results and the underlying genotype or haplotype. In a preferred embodiment, this diagnostic method uses the predictive genotyping/haplotyping method described above. [0095] Patient Selection for Therapy Based Upon Polymorphisms and/or Mutations. The application of genotypes and/or haplotypes that correlate with efficacious drug responses will be used to select patients for therapy of existing diseases. Genotypes and haplotypes that correlate with adverse consequences will be used to either modify how the drug is administered (e.g., dose, schedule or in combination with other drugs) or eliminated as an option.
  • a direct DNA test i.e., genotyping or haplotyping one or more of the polymorphic/mutant sites in the gene
  • serological test i.
  • the invention also provides a computer system for storing and displaying polymorphism and mutation data determined for the gene.
  • the computer system comprises a computer processing unit, a display, and a database containing the polymorphism/mutation data.
  • the polymorphism/mutation data includes the polymorphisms, mutations, the genotypes and the haplotypes identified for a given gene in a reference population.
  • the computer system is capable of producing a display showing haplotypes organized according to their evolutionary relationships.
  • a computer may implement any or all analytical and mathematical operations involved in practicing the methods of the present invention.
  • the computer may execute a program that generates views (or screens) displayed on a display device and with which the user can interact to view and analyze large amounts of information relating to the gene and its genomic variation, including chromosome location, gene structure, and gene family, gene expression data, polymorphism data, mutation data, genetic sequence data, and clinical population data (e.g., data on ethnogeographic origin, clinical responses, genotypes, and haplotypes for one or more populations).
  • the polymorphism and mutation data described herein may be stored as part of a relational database (e.g., an instance of an Oracle database or a set of ASCII flat files). These polymorphism and mutation data may be stored on the computer's hard drive or may, for example, be stored on a CD-ROM or on one or more other storage devices accessible by the computer. For example, the data may be stored on one or more databases in communication with the computer via a network.
  • a relational database e.g., an instance of an Oracle database or a set of ASCII flat files.
  • the invention provides SNP and mutation probes, which are useful in classifying subjects according to their types of genetic variation.
  • the SNP and mutation probes according to the invention are oligonucleotides, which discriminate between SNPs or mutations and the wild-type sequence in conventional allelic discrimination assays.
  • the oligonucleotides according to this aspect of the invention are complementary to one allele of the SNP/mutant nucleic acid, but not to any other allele of the SNP/Mutant nucleic acid. Oligonucleotides according to this embodiment of the invention can discriminate between SNPs and mutations in various ways.
  • an oligonucleotide of appropriate length will hybridize to one SNP or mutation, but not to any other.
  • the oligonucleotide may be labelled using a radiolabel or a fluorescent molecular tag.
  • an oligonucleotide of appropriate length can be used as a primer for PCR, wherein the 3' terminal nucleotide is complementary to one allele containing a SNP or mutation, but not to any other allele.
  • the presence or absence of amplification by PCR determines the haplotype of the SNP or the specific mutation.
  • Genomic and cDNA fragments of the invention comprise at least one novel polymorphic site or mutation identified herein, have a length of at least 10 nucleotides, and may range up to the full length of the gene.
  • a fragment according to the present invention is between 100 and 3000 nucleotides in length, and more preferably between 200 and 2000 nucleotides in length, and most preferably between 500 and 1000 nucleotides in length.
  • kits of the Invention provides nucleic acid and polypeptide detection kits useful for haplotyping and/or genotyping the genes in an individual. Such kits are useful for classifying individuals for the purpose of classifying individuals. Specifically, the invention encompasses kits for detecting the presence of a polypeptide or nucleic acid corresponding to a marker of the invention in a biological sample, e.g., any tissue or bodily fluid including, but not limited to, serum, plasma, lymph, cystic fluid, urine, stool, cerebrospinal fluid, ascites fluid or blood, and including biopsy samples of body tissue.
  • a biological sample e.g., any tissue or bodily fluid including, but not limited to, serum, plasma, lymph, cystic fluid, urine, stool, cerebrospinal fluid, ascites fluid or blood, and including biopsy samples of body tissue.
  • the kit can comprise a labelled compound or agent capable of detecting a polypeptide or an mRNA encoding a polypeptide corresponding to a marker of the invention in a biological sample and means for determining the amount of the polypeptide or mRNA in the sample, e.g., an antibody which binds the polypeptide or an oligonucleotide probe which binds to DNA or mRNA encoding the polypeptide.
  • Kits can also include instructions for interpreting the results obtained using the kit.
  • the invention provides a kit comprising at least two genotyping oligonucleotides packaged in separate containers.
  • the kit may also contain other components such as hybridization buffer (where the oligonucleotides are to be used as a probe) packaged in a separate container.
  • the kit may contain, packaged in separate containers, a polymerase and a reaction buffer optimized for primer extension mediated by the polymerase, such as in the case of PCR.
  • such kit may further comprise a DNA sample collecting means.
  • the genotyping primer composition may comprise at least two sets of allele specific primer pairs.
  • the two genotyping oligonucleotides are packaged in separate containers.
  • the kit can comprise, e.g., (1) a first antibody, e.g., attached to a solid support, which binds to a polypeptide corresponding to a marker or the invention; and, optionally; (2) a second, different antibody which binds to either the polypeptide or the first antibody and is conjugated to a detectable label.
  • the kit can comprise, e.g., (1) an oligonucleotide, e.g., a detectably-labelled oligonucleotide, which hybridizes to a nucleic acid sequence encoding a polypeptide corresponding to a marker of the invention; or (2) a pair of primers useful for amplifying a nucleic acid molecule corresponding to a marker of the invention.
  • the kit can also comprise, e.g., a buffering agent, a preservative or a protein- stabilizing agent.
  • the kit can further comprise components necessary for detecting the detectable-label, e.g., an enzyme or a substrate.
  • the kit can also contain a control sample or a series of control samples, which can be assayed and compared to the test sample.
  • Each component of the kit can be enclosed within an individual container and all of the various containers can be within a single package, along with instructions for interpreting the results of the assays performed using the kit.
  • the present invention includes one or more polynucleotides encoding mutant or polymorphic polypeptides, including degenerate variants thereof.
  • the invention also encompasses allelic variants of the same, that is, naturally occurring alternative forms of the isolated polynucleotides that encode mutant polypeptides that are identical, homologous or related to those encoded by the polynucleotides.
  • non-naturally occurring variants may be produced by mutagenesis techniques or by direct synthesis techniques well known in the art. Accordingly, nucleic acid sequences capable of hybridizing at low stringency with any nucleic acid sequences encoding mutant polypeptide of the present invention are considered to be within the scope of the invention.
  • a typical prehybridization, hybridization, and wash protocol is as follows: (1) prehybridization: incubate nitrocellulose filters containing the denatured target DNA for 3-4 hours at 55°C in SxDenhardt's solution, 6xSSC (2OxSSC consists of 175 gNaCl, 88.2 g sodium citrate in 800 ml H 2 O adjusted to pH.
  • Recombinant Expression Vectors Another aspect of the invention includes vectors containing one or more nucleic acid sequences encoding a mutant or polymorphic polypeptide.
  • many conventional techniques in molecular biology, microbiology and recombinant DNA are used. These techniques are well known and are explained in, e.g., Current Protocols in Molecular Biology, VoIs. I-III, Ausubel, ed. (1997); Sambrook et al., Molecular Cloning: A Laboratory Manual, 2 nd Edition. (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 1989); Glover DN, DNA Cloning: A Practical Approach, VoIs.
  • the nucleic acid containing all or a portion of the nucleotide sequence encoding the polypeptide is inserted into an appropriate cloning vector, or an expression vector (i.e., a vector that contains the necessary elements for the transcription and translation of the inserted polypeptide coding sequence) by recombinant DNA techniques well known in the art and as detailed below.
  • an expression vector i.e., a vector that contains the necessary elements for the transcription and translation of the inserted polypeptide coding sequence
  • expression vectors useful in recombinant DNA techniques are often in the form of plasmids.
  • plasmid and “vector” can be used interchangeably as the plasmid is the most commonly used form of vector.
  • the invention is intended to include such other forms of expression vectors that are not technically plasmids, such as viral vectors (e.g., replication defective retroviruses, adenoviruses and adeno-associated viruses), which serve equivalent functions.
  • viral vectors e.g., replication defective retroviruses, adenoviruses and adeno-associated viruses
  • Such viral vectors permit infection of a subject and expression in that subject of a compound. Becker et al., Meth. Cell Biol. 43: 161 89 (1994).
  • the recombinant expression vectors of the invention comprise a nucleic acid encoding a mutant or polymorphic polypeptide in a form suitable for expression of the nucleic acid in a host cell, which means that the recombinant expression vectors include one or more regulatory sequences, selected on the basis of the host cells to be used for expression that is operatively linked to the nucleic acid sequence to be expressed.
  • "operably linked" is intended to mean that the nucleotide sequence of interest is linked to the regulatory sequences in a manner that allows for expression of the nucleotide sequence (e.g., in an in vitro transcription/translation system or in a host cell when the vector is introduced into the host cell).
  • regulatory sequence is intended to include promoters, enhancers and other expression control elements (e.g., polyadenylation signals). Such regulatory sequences are described, for example, in Goeddel, Gene Expression Technology: Methods In En ⁇ ymology (Academic Press, San Diego, Calif., 1990). Regulatory sequences include those that direct constitutive expression of a nucleotide sequence in many types of host cell and those that direct expression of the nucleotide sequence only in certain host cells (e.g., tissue specific regulatory sequences). It will be appreciated by those skilled in the art that the design of the expression vector can depend on such factors as the choice of the host cell to be transformed, the level of expression of polypeptide desired, etc.
  • the expression vectors of the invention can be introduced into host cells to thereby produce polypeptides or peptides, including fusion polypeptides, encoded by nucleic acids as described herein (e.g., mutant polypeptides and mutant-derived fusion polypeptides, etc.).
  • mutant and Polymorphic Polypeptide-Expressing Host Cells Another aspect of the invention pertains to mutant and polymorphic polypeptide-expressing host cells, which contain a nucleic acid encoding one or more mutant/polymorphic polypeptides of the invention.
  • the desired isogene may be introduced into a host cell in a vector such that the isogene remains extrachromosomal. In such a situation, the gene will be expressed by the cell from the extrachromosomal location.
  • the isogene is introduced into a cell in such a way that it recombines with the endogenous gene present in the cell.
  • Such recombination requires the occurrence of a double recombination event, thereby resulting in the desired gene polymorphism or mutation.
  • Vectors for the introduction of genes both for recombination and for extrachromosomal maintenance are known in the art, and any suitable vector or vector construct may be used in the invention. Methods such as electroporation, particle bombardment, calcium phosphate co-precipitation and viral transduction for introducing DNA into cells are known in the art; therefore, the choice of method may lie with the competence and preference of the skilled practitioner.
  • mutant/polymorphic polypeptides can be expressed in bacterial cells such as Escherichia coli (E. coli), insect cells -SO-
  • baculovirus expression vectors fusing baculovirus expression vectors
  • fungal cells e.g., yeast, yeast cells or mammalian cells. Suitable host cells are discussed further in Goeddel, Gene Expression Technology: Methods In En ⁇ ymology (Academic Press, San Diego, Calif., 1990).
  • the recombinant expression vector can be transcribed and translated in vitro, for example using T7 promoter regulatory sequences and T7 polymerase.
  • the SMP2 promoter is useful in the expression of polypeptides in smooth muscle cells, Qian et al, Endocrinology 140(4): 1826 (1999).
  • Fusion vectors add a number of amino acids to a polypeptide encoded therein, usually to the amino terminus of the recombinant polypeptide.
  • Such fusion vectors typically serve three purposes: (i) to increase expression of recombinant polypeptide; (ii) to increase the solubility of the recombinant polypeptide; and (iii) to aid in the purification of the recombinant polypeptide by acting as a ligand in affinity purification.
  • a proteolytic cleavage site is introduced at the junction of the fusion moiety and the recombinant polypeptide to enable separation of the recombinant polypeptide from the fusion moiety subsequent to purification of the fusion polypeptide.
  • enzymes, and their cognate recognition sequences include Factor Xa, thrombin and enterokinase.
  • Typical fusion expression vectors include pGEX (Pharmacia Biotech Inc; Smith and Johnson, Gene 67: 31 40 (1988)), pMAL (New England Biolabs, Beverly, Mass.) and pRIT5 (Pharmacia, Piscataway, N.J.) that fuse glutathione S transferase (GST) 5 maltose E binding polypeptide, or polypeptide A, respectively, to the target recombinant polypeptide.
  • GST glutathione S transferase
  • coli expression vectors include pTrc (Amrann et al, Gene 69:301 315 (1988)) and pET l id (Studier et al, Gene Expression Technology: Methods In En ⁇ ymology (Academic Press, San Diego, Calif., 1990) pp. 60-89).
  • One strategy to maximize recombinant polypeptide expression in E. coli is to express the polypeptide in host bacteria with an impaired capacity to proteolytically cleave the recombinant polypeptide. See, e.g., Gottesman, Gene Expression Technology: Methods In Enzymology (Academic Press, San Diego, Calif., 1990) 119 128.
  • Another strategy is to alter the nucleic acid sequence of the nucleic acid to be inserted into an expression vector so that the individual codons for each amino acid are those preferentially utilized in the expression host, e.g., E. coli (see, e.g., Wada et al., Nucl. Acids Res. 20: 2111-2118 (1992)).
  • Such alteration of nucleic acid sequences of the invention can be carried out by standard DNA synthesis techniques.
  • the mutant/polymorphic polypeptide expression vector is a yeast expression vector.
  • yeast Saccharomyces cerivisiae examples include pYepSecl (Baldari et al, EMBOJ. 6: 229 234 (1987)), pMFa (Kurjan & Herskowitz, Cell 30: 933 943 (1982)), pJRY88 (Schultz et al, Gene 54: 113 123 (1987)), pYES2 (InVitrogen Corporation, San Diego, Calif., USA), and picZ (InVitrogen Corp, San Diego, Calif., USA).
  • mutant polypeptide can be expressed in insect cells using baculovirus expression vectors.
  • Baculovirus vectors available for expression of polypeptides in cultured insect cells include the pAc series (Smith et al, MoI. Cell. Biol. 3: 2156 2165 (1983)) and the pVL series (Lucklow & Summers, Virology 170: 31 39 (1989)).
  • a nucleic acid of the invention is expressed in mammalian cells using a mammalian expression vector.
  • mammalian expression vectors include pCDM8 (Seed, Nature 329: 842 846 (1987)) and pMT2PC (Kaufman et al, EMBOJ. 6: 187 195 (1987)).
  • the expression vector's control functions are often provided by viral regulatory elements.
  • promoters are derived from polyoma, adenovirus 2, cytomegalovirus, and simian virus 40.
  • suitable expression systems for both prokaryotic and eukaryotic cells see, e.g., Chapters 16 and 17 of Sambrook, et al., Molecular Cloning: A Laboratory Manual, 2nd Ed.(Co ⁇ Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 1989).
  • the recombinant mammalian expression vector is capable of directing expression of the nucleic acid preferentially in a particular cell type ⁇ e.g., tissue specific regulatory elements are used to express the nucleic acid).
  • tissue specific regulatory elements are known in the art.
  • suitable tissue specific promoters include the albumin promoter (liver specific; Pinkert, et al, Genes Dev. 1 : 268 277 (1987)), lymphoid specific promoters (Calame & Eaton, Adv. Immunol. 43: 235 275 (1988)), in particular promoters of T cell receptors (Winoto & Baltimore, EMBO J.
  • promoters are also encompassed, e.g., the murine hox promoters (Kessel & Gruss, Science 249: 374379 (1990)) and the ⁇ -fetoprotein promoter (Campes & Tilghman, Genes Dev. 3: 537 546 (1989)).
  • the invention further provides a recombinant expression vector comprising a DNA molecule of the invention cloned into the expression vector in an antisense orientation. That is, the DNA molecule is operatively linked to a regulatory sequence in a manner that allows for expression (by transcription of the DNA molecule) of an RNA molecule that is antisense to a mutant polypeptide mRNA. Regulatory sequences operatively linked to a nucleic acid cloned in the antisense orientation can be chosen that direct the continuous expression of the antisense RNA molecule in a variety of cell types, for instance viral promoters and/or enhancers, or regulatory sequences can be chosen that direct constitutive, tissue specific or cell type specific expression of antisense RNA.
  • the antisense expression vector can be in the form of a recombinant plasmid, phagemid or attenuated virus in which antisense nucleic acids are produced under the control of a high efficiency regulatory region, the activity of which can be determined by the cell type into which the vector is introduced.
  • a high efficiency regulatory region the activity of which can be determined by the cell type into which the vector is introduced.
  • a host cell can be any prokaryotic or eukaryotic cell.
  • mutant polypeptide can be expressed in bacterial cells such as E. coli, insect cells, yeast or mammalian cells (such as Chinese hamster ovary cells (CHO) or COS cells).
  • bacterial cells such as E. coli, insect cells, yeast or mammalian cells (such as Chinese hamster ovary cells (CHO) or COS cells).
  • mammalian cells such as Chinese hamster ovary cells (CHO) or COS cells.
  • Other suitable host cells are known to those skilled in the art.
  • Vector DNA can be introduced into prokaryotic or eukaryotic cells via conventional transformation or transfection techniques.
  • transformation and “transfection” are intended to refer to a variety of art recognized techniques for introducing foreign nucleic acid ⁇ e.g., DNA) into a host cell, including calcium phosphate or calcium chloride co precipitation, DEAE dextran mediated transfection, lipofection, or electroporation. Suitable methods for transforming or transfecting host cells can be found in Sambrook, et ah, Molecular Cloning: A Laboratory Manual, 2nd ed. (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 1989), and other laboratory manuals.
  • a gene that encodes a selectable marker ⁇ e.g., resistance to antibiotics
  • Various selectable markers include those that confer resistance to drugs, such as G418, hygromycin and methotrexate.
  • Nucleic acid encoding a selectable marker can be introduced into a host cell on the same vector as that encoding mutant polypeptide or can be introduced on a separate vector.
  • a host cell that includes a compound of the invention such as a prokaryotic or eukaryotic host cell in culture, can be used to produce ⁇ i.e., express) recombinant mutant/polymorphic polypeptide.
  • the method comprises culturing the host cell of invention (into which a recombinant expression vector encoding mutant/polymorphic polypeptide has been introduced) in a suitable medium such that mutant polypeptide is produced.
  • the method further comprises the step of isolating mutant/polymorphic polypeptide from the medium or the host cell.
  • Purification of recombinant polypeptides is well known in the art and includes ion exchange purification techniques, or affinity purification techniques, for example with an antibody to the compound. Methods of creating antibodies to the compounds of the present invention are discussed below.
  • Transgenic Animals Recombinant organisms, i.e., transgenic animals, expressing a variant gene of the invention are prepared using standard procedures known in the art. Transgenic animals carrying the constructs of the invention can be made by several methods known to those having skill in the art. See, e.g., U.S. Pat. No. 5,610,053 and "The Introduction of Foreign Genes into Mice" and the cited references therein, in: Recombinant DNA, Watson JD, Gilman M, Witkowski J & Zoller M, eds. (W.H. Freeman and Company, New York) pp. 254-272.
  • Transgenic animals stably expressing a human isogene and producing human protein can be used as biological models for studying diseases related to abnormal expression and/or activity, and for screening and assaying various candidate drugs, compounds, and treatment regimens to reduce the symptoms or effects of these diseases.
  • Characterizing Gene Expression Level Methods to detect and measure mRNA levels (i.e., gene transcription level) and levels of polypeptide gene expression products (i.e., gene translation level) are well-known in the art and include the use of nucleotide microarrays and polypeptide detection methods involving mass spectrometers, reverse-transcription and amplification and/or antibody detection and quantification techniques. See also, Strachan T & Read A, Human Molecular Genetics, T 4 Edition. (John Wiley and Sons, Inc. Publication, New York, 1999)).
  • RNA isolation technique that does not select against the isolation of mRNA can be utilized for the purification of RNA from cells. See, e.g., Ausubel etal., Ed., Curr. Prot. MoI. Biol.
  • the level of the mRNA expression product of the target gene is determined.
  • Methods to measure the level of a specific mRNA are well-known in the art and include Northern blot analysis, reverse transcription PCR and real time quantitative PCR or by hybridization to a oligonucleotide array or microarray.
  • the determination of the level of expression may be performed by determination of the level of the protein or polypeptide expression product of the gene in body fluids or tissue samples including but not limited to blood or serum.
  • the isolated mRNA can be used in hybridization or amplification assays that include, but are not limited to, Southern or Northern analyses, PCR analyses and probe arrays.
  • One preferred diagnostic method for the detection of mRNA levels involves contacting the isolated mRNA with a nucleic acid molecule (probe) that can hybridize to the mRNA encoded by the gene being detected.
  • the nucleic acid probe can be, e.g., a full-length cDNA, or a portion thereof, such as an oligonucleotide of at least 7, 15, 30, 50, 100, 250 or 500 nucleotides in length and sufficient to specifically hybridize under stringent conditions to an mRNA or genomic DNA encoding a marker of the present invention.
  • Other suitable probes for use in the diagnostic assays of the invention are described herein. Hybridization of an mRNA with the probe indicates that the marker in question is being expressed.
  • the probes are immobilized on a solid surface and the mRNA is contacted with the probes, for example, in an Affymetrix gene chip array (Affymetrix, Calif. USA).
  • An alternative method for determining the level of mRNA corresponding to a marker of the present invention in a sample involves the process of nucleic acid amplification, e.g., by RT-PCR (the experimental embodiment set forth in U.S. Pat. No. 4,683,202); ligase chain reaction (Barany et al, Proc. Natl. Acad. Sci. USA 88:189-193 (1991)) self-sustained sequence replication (Guatelli et al, Proc. Natl. Acad.
  • amplification primers are defined as being a pair of nucleic acid molecules that can anneal to 5' or 3' regions of a gene (plus and minus strands, respectively, or vice-versa) and contain a short region in between. In general, amplification primers are from about 10-30 nucleotides in length and flank a region from about 50-200 nucleotides in length.
  • RT-PCR Real-time quantitative PCR
  • the RT- PCR assay utilizes an RNA reverse transcriptase to catalyze the synthesis of a DNA strand from an RNA strand, including an mRNA strand.
  • the resultant DNA may be specifically detected and quantified and this process may be used to determine the levels of specific species of mRNA.
  • TAQMAN® PE Applied Biosystems, Foster City, Calif., USA
  • cDNA pools such as by sequencing sufficient bases, e.g., 20-50 bases, in each of multiple cDNAs to identify each cDNA, or by sequencing short tags, e.g., 9-10 bases, which are generated at known positions relative to a defined mRNA end pathway pattern. See, e.g., Velculescu, Science 270: 484-487 (1995).
  • the cDNA levels in the samples are quantified and the mean, average and standard deviation of each cDN A is determined using by standard statistical means well-known to those of skill in the art. Norman TJ. Bailey, Statistical Methods In Biology, 3rd Edition (Cambridge University Press, 1995).
  • Detection of Polypeptides Immunological Detection Methods.
  • Expression of the protein encoded by the genes of the invention can be detected by a probe which is detectably labelled, or which can be subsequently labelled.
  • the term "labelled", with regard to the probe or antibody is intended to encompass direct-labelling of the probe or antibody by coupling, i.e., physically linking, a detectable substance to the probe or antibody, as well as indirect- labelling of the probe or antibody by reactivity with another reagent that is directly- labelled.
  • indirect labelling include detection of a primary antibody using a fluorescently- labelled secondary antibody and end-labelling of a DNA probe with biotin such that it can be detected with fluorescently-labelled streptavidin.
  • the probe is an antibody that recognizes the expressed protein.
  • a variety of formats can be employed to determine whether a sample contains a target protein that binds to a given antibody.
  • Immunoassay methods useful in the detection of target polypeptides of the present invention include, but are not limited to, e.g., dot blotting, western blotting, protein chips, competitive and non-competitive protein binding assays, immunohistochemistry, enzyme-linked immunosorbant assays (ELISA), fluorescence activated cell sorting (FACS), and others commonly used and widely- described in scientific and patent literature, and many employed commercially.
  • a skilled artisan can readily adapt known protein/antibody detection methods for use in determining whether cells express a marker of the present invention and the relative concentration of that specific polypeptide expression product in blood or other body tissues.
  • Proteins from individuals can be isolated using techniques that are well-known to those of skill in the art. The protein isolation methods employed can, e.g., be such as those described in Harlow & Lane, Antibodies: A Laboratory Manual (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 1988)).
  • various host animals may be immunized by injection with the polypeptide, or a portion thereof.
  • host animals may include, but are not limited to, rabbits, mice and rats.
  • adjuvants may be used to increase the immunological response, depending on the host species including, but not limited to, Freund's (complete and incomplete), mineral gels, such as aluminium hydroxide; surface active substances, such as lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, keyhole limpet haemocyanin and dinitrophenol; and potentially useful human adjuvants, such as bacille Camette-Guerin (BCG) and Corymbacterium parvum.
  • BCG Bacille Camette-Guerin
  • Monoclonal antibodies which are homogeneous populations of antibodies to a particular antigen, may be obtained by any technique that provides for the production of antibody molecules by continuous cell lines in culture. These include, but are not limited to, the hybridoma technique of Kohler & Milstein, Nature 256: 495-497 (1975); and U.S. Pat. No. 4,376,110; the human B-cell hybridoma technique of Kosbor et ah, Immunol. Today 4: 72 (1983); Cole et al, Proc. Natl. Acad. ScL USA 80: 2026-2030 (1983); and the EBV- hybridoma technique of Cole et al., Monoclonal Antibodies and Cancer Therapy (Alan R. Liss, Inc., 1985) pp. 77-96.
  • chimaeric antibodies are derived from different animal species, such as those having a variable or hypervariable region derived form a murine mAb and a human immunoglobulin constant region.
  • Antibodies or antibody fragments can be used in methods, such as Western blots or immunofluorescence techniques, to detect the expressed proteins. In such uses, it is generally preferable to immobilize either the antibody or proteins on a solid support.
  • Suitable solid phase supports or carriers include any support capable of binding an antigen or an antibody.
  • Well-known supports or carriers include glass, polystyrene, polypropylene, polyethylene, dextran, nylon, amylases, natural and modified celluloses, polyacrylamides, gabbros and magnetite.
  • a useful method for ease of detection, is the sandwich ELISA, of which a number of variations exist, all of which are intended to be used in the methods and assays of the present invention.
  • sandwich assay is intended to encompass all variations on the basic two-site technique. Immunofluorescence and EIA techniques are both very well- established in the art. However, other reporter molecules, such as radioisotopes, chemiluminescent or bioluminescent molecules may also be employed. It will be readily apparent to the skilled artisan how to vary the procedure to suit the required use.
  • Whole genome monitoring of protein i.e., the "proteome” can be carried out by constructing a microarray in which binding sites comprise immobilized, preferably monoclonal, antibodies specific to a plurality of protein species encoded by the cell genome.
  • binding sites comprise immobilized, preferably monoclonal, antibodies specific to a plurality of protein species encoded by the cell genome.
  • antibodies are present for a substantial fraction of the encoded proteins, or at least for those proteins relevant to testing or confirming a biological network model of interest.
  • methods for making monoclonal antibodies are well-known. See, e.g., Harlow & Lane, Antibodies: A Laboratory ManuaF (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 1988)).
  • monoclonal antibodies are raised against synthetic peptide fragments designed based on genomic sequence of the cell. With such an antibody array, proteins from the cell are contacted to the array and their binding is measured with assays known in the art.
  • Two-Dimensional Gel Electrophoresis Two-dimensional gel electrophoresis is well-known in the art and typically involves isoelectric focusing along a first dimension followed by SDS-PAGE electrophoresis along a second dimension. See, e.g., Hames et al , Gel Electrophoresis of Proteins: A Practical Approach (IRL Press, New York, 1990); Shevchenko et al., Proc. Natl Acad. Sci. USA 93: 14440-14445 (1996); Sagliocco et al., Yeast 12: 1519-1533 (1996); and Lander, Science 274: 536-539 (1996)).
  • MS-based analysis methodology is useful for analysis of isolated target polypeptide as well as analysis of target polypeptide in a biological sample.
  • MS formats for use in analyzing a target polypeptide include ionization (I) techniques, such as, but not limited to, matrix assisted laser desorption (MALDI), continuous or pulsed electrospray ionization (ESI) and related methods, such as ionspray or thermospray, and massive cluster impact (MCI).
  • I ionization
  • MALDI matrix assisted laser desorption
  • ESI electrospray ionization
  • MCI massive cluster impact
  • Such ion sources can be matched with detection formats, including linear or non-linear reflectron time of flight (TOF), single or multiple quadrupole, single or multiple magnetic sector Fourier transform ion cyclotron resonance (FTICR), ion trap and combinations thereof such as ion-trap/TOF.
  • TOF linear or non-linear reflectron time of flight
  • FTICR magnetic sector Fourier transform ion cyclotron resonance
  • ion trap and combinations thereof such as ion-trap/TOF.
  • numerous matrix/wavelength combinations e.g., matrix assisted laser desorption (MALDI)
  • solvent combinations e.g., ESI
  • the target polypeptide can be solubilised in an appropriate solution or reagent system.
  • a solution or reagent system e.g., an organic or inorganic solvent
  • MS of peptides also is described, e.g., in International PCT Application No.
  • a solvent is selected that minimizes the risk that the target polypeptide will be decomposed by the energy introduced for the vaporization process.
  • a reduced risk of target polypeptide decomposition can be achieved, e.g., by embedding the sample in a matrix.
  • a suitable matrix can be an organic compound such as a sugar, e.g., a pentose or hexose, or a polysaccharide such as cellulose. Such compounds are decomposed thermolytically into CO 2 and H 2 O such that no residues are formed that can lead to chemical reactions.
  • the matrix also can be an inorganic compound, such as nitrate of ammonium, which is decomposed essentially without leaving any residue.
  • an inorganic compound such as nitrate of ammonium, which is decomposed essentially without leaving any residue.
  • Use of these and other solvents is known to those of skill in the art. See, e.g., U.S. Pat. No. 5,062,935.
  • Electrospray MS has been described by Fenn et al., J. Phys. Chem. 88: 4451-4459 (1984); and PCT Application No. WO 90/14148; and current applications are summarized in review articles. See Smith et al., Anal. Chem. 62: 882-89 (1990); and Ardrey, Spectroscopy 4: 10-18 (1992).
  • the mass of a target polypeptide determined by MS can be compared to the mass of a corresponding known polypeptide.
  • the corresponding known polypeptide can be the corresponding non-mutant protein, e.g., wild-type protein.
  • ESI the determination of molecular weights in femtomole amounts of sample is very accurate due to the presence of multiple ion peaks, all of which can be used for mass calculation.
  • Sub-attomole levels of protein have been detected, e.g., using ESI MS (Valaskovic et al, Science 273: 1199-1202 (1996)) and MALDI MS (Li et al, J. Am. Chem. Soc. 118: 1662-1663 (1996)).
  • Matrix Assisted Laser Desorption The level of the target protein in a biological sample, e.g., body fluid or tissue sample, may be measured by means of mass spectrometric (MS) methods including, but not limited to, those techniques known in the art as matrix-assisted laser desorption/ionization, time-of-flight mass spectrometry (MALDI- TOF-MS) and surfaces enhanced for laser desorption/ionization, time-of-fiight mass spectrometry (SELDI-TOF-MS) as further detailed below.
  • MS mass spectrometric
  • Methods for performing MALDI are well-known to those of skill in the art. See, e.g., Juhasz et al., Analysis, Anal. Chem.
  • MALDI-TOF-MS has been described by Hillenkamp et al., Biological Mass Spectrometry, Burlingame & McCloskey, eds. (Elsevier Science Publ., Amsterdam, 1990) pp. 49-60. [0151] A variety of techniques for marker detection us ing mass spectroscopy can be used.
  • MS techniques allow the successful volatilization of high molecular weight biopolymers, without fragmentation, and have enabled a wide variety of biological macromolecules to be analyzed by mass spectrometry.
  • SMDI Surfaces Enhanced for Laser Desorption/Ionization
  • Other techniques are used which employ new MS probe element compositions with surfaces that allow the probe element to actively participate in the capture and docking of specific analytes, described as Affinity Mass Spectrometry (AMS). See SELDI patents U.S. Pat. Nos. 5,719,060; 5,894,063; 6,020,208; 6,027,942; 6,124,137; and U.S. Patent application No. U.S. 2003/0003465.
  • SEAC probe elements have been designed with Surfaces Enhanced for Affinity Capture (SEAC). See Hutchens & Yip, Rapid Commun. Mass Spectrom. 7: 576-580 (1993).
  • SEAC probe elements have been used successfully to retrieve and tether different classes of biopolymers, particularly proteins, by exploiting what is known about protein surface structures and biospecific molecular recognition.
  • the immobilized affinity capture devices on the MS probe element surface, i.e., SEAC determines the location and affinity (specificity) of the analyte for the probe surface, therefore the subsequent analytical MS process is efficient.
  • SELDI Surfaces Enhanced for Neat Desorption
  • the probe element surfaces i.e., sample presenting means
  • EAM Energy Absorbing Molecules
  • probe element surfaces i.e., sample presenting means
  • affinity capture devices to facilitate either the specific or non-specific attachment or adsorption (so-called docking or tethering) of analytes to the probe surface, by a variety of mechanisms (mostly non-covalent);
  • SEPAR Photolabile Attachment and Release
  • the probe element surfaces i.e., sample presenting means
  • the analyte e.g., protein
  • the chemical specificities determining the type and number of the photolabile molecule attachment points between the SEPAR sample presenting means (i.e., probe element surface) and the analyte may involve any one or more of a number of different residues or chemical structures in the analyte (e.g., His, Lys, Arg, Tyr, Phe and Cys residues in the case of proteins and peptides).
  • a polypeptide of interest also can be modified to facilitate conjugation to a solid support.
  • a chemical or physical moiety can be incorporate into the polypeptide at an appropriate position.
  • a polypeptide of interest can be modified by adding an appropriate functional group to the carboxyl terminus or amino terminus of the polypeptide, or to an amino acid in the peptide, (e.g., to a reactive side chain, or to the peptide backbone.
  • a naturally-occurring amino acid normally present in the polypeptide also can contain a functional group suitable for conjugating the polypeptide to the solid support.
  • a cysteine residue present in the polypeptide can be used to conjugate the polypeptide to a support containing a sulfhydryl group through a disulfide linkage, e.g., a support having cysteine residues attached thereto.
  • bonds that can be formed between two amino acids include, but are not limited to, e.g., monosulfide bonds between two lanthionine residues, which are non- naturally-occurring amino acids that can be incorporated into a polypeptide; a lactam bond formed by a transamidation reaction between the side chains of an acidic amino acid and a basic amino acid, such as between the y-carboxyl group of GIu (or alpha carboxyl group of Asp) and the amino group of Lys; or a lactone bond produced, e.g., by a crosslink between the hydroxy group of Ser and the carboxyl group of GIu (or alpha carboxyl group of Asp).
  • a solid support can be modified to contain a desired amino acid residue, e.g., a GIu residue, and a polypeptide having a Ser residue, particularly a Ser residue at the N-terminus or C-terminus, can be conjugated to the solid support through the formation of a lactone bond.
  • the support need not be modified to contain the particular amino acid, e.g., GIu, where it is desired to form a lactone-like bond with a Ser in the polypeptide, but can be modified, instead, to contain an accessible carboxyl group, thus providing a function corresponding to the alpha carboxyl group of GIu.
  • a thiol-reactive functionality is particularly useful for conjugating a polypeptide to a solid support.
  • a thiol-reactive functionality is a chemical group that can rapidly react with a nucleophilic thiol moiety to produce a covalent bond, e.g., a disulfide bond or a thioether bond.
  • thiol-reactive functionalities include, e.g., haloacetyls, such as iodoacetyl; diazoketones; epoxy ketones, alpha- and beta-unsaturated carbonyls, such as alpha-enones and beta-enones; and other reactive Michael acceptors, such as maleimide; acid halides; benzyl halides; and the like. See Greene & Wuts, Protective Groups in Organic Synthesis, 2 nd Edition (John Wiley & Sons, 1991).
  • the thiol groups can be blocked with a photocleavable protecting group, which then can be selectively cleaved, e.g., by photolithography, to provide portions of a surface activated for immobilization of a polypeptide of interest.
  • Photocleavable protecting groups are known in the art (see, e.g., published International PCT Application No. WO 92/10092; and McCray etal., Ann. Rev. Biophys. Biophys. Chem. 18: 239-270 (1989)) and can be selectively de-blocked by irradiation of selected areas of the surface using, e.g., a photolithography mask.
  • Linkers A polypeptide of interest can be attached directly to a support via a linker. Any linkers known to those of skill in the art to be suitable for linking peptides or amino acids to supports, either directly or via a spacer, may be used. For example, the polypeptide can be conjugated to a support, such as a bead, through means of a variable spacer.
  • Linkers include, Rink amide linkers (see, e.g., Rink, Tetrahedron Lett. 28: 3787 (1976)); trityl chloride linkers (see, e.g., Leznoff, Ace Chem. Res.
  • linkers see, e.g., Bodansky et ah, Peptide Synthesis, 2 nd Edition (Academic Press, New York, 1976)
  • trityl linkers are known. See, e.g., U.S. Pat. Nos. 5,410,068 and 5,612,474.
  • Amino trityl linkers are also known. See, e.g., U.S. Pat. No. 5,198,531.
  • Other linkers include those that can be incorporated into fusion proteins and expressed in a host cell. Such linkers may be selected amino acids, enzyme substrates or any suitable peptide.
  • the linker may be made, e.g., by appropriate selection of primers when isolating the nucleic acid. Alternatively, they may be added by post-translational modification of the protein of interest.
  • Linkers that are suitable for chemically linking peptides to supports include disulfide bonds, thioether bonds, hindered disulfide bonds and covalent bonds between free reactive groups, such as amine and thiol groups.
  • Cleavable Linkers A linker can provide a reversible linkage such that it is cleaved under the select conditions.
  • selectively cleavable linkers including photocleavable linkers (see U.S. Pat. No. 5,643,722), acid cleavable linkers (see Fattom et al, Infect. Immun. 60: 584-589 (1992)), acid-labile linkers (see Welhoner et al, J. Biol Chem. 266: 4309-4314 (1991)) and heat sensitive linkers are useful.
  • a linkage can be, e.g., a disulfide bond, which is chemically cleavable by mercaptoethanol or dithioerythrol; a biotin/streptavidin linkage, which can be photocleavable; a heterobifunctional derivative of a trityl ether group, which can be cleaved by exposure to acidic conditions or under conditions of MS (see Koster et al, Tetrahedron Lett.
  • a levulmyl-mediated linkage which can be cleaved under almost neutral conditions with a hydrazinium/acetate buffer; an arginine-arginine or a lysine-lysine bond, either of which can be cleaved by an endopeptidase, such as trypsin; a pyrophosphate bond, which can be cleaved by a pyrophosphatase; or a ribonucleotide bond, which can be cleaved using a ribonuclease or by exposure to alkali condition.
  • an endopeptidase such as trypsin
  • a pyrophosphate bond which can be cleaved by a pyrophosphatase
  • a ribonucleotide bond which can be cleaved using a ribonuclease or by exposure to alkali condition.
  • a photolabile cross -linker such as 3-amino-(2-nitrophenyl)propionic acid can be employed as a means for cleaving a polypeptide from a solid support.
  • Other linkers include RNA linkers that are cleavable by ribozymes and other RNA enzymes and linkers, such as the various domains, such as CHi, CH 2 and CH 3 , from the constant region of human IgGl . See, Batra et al.
  • linker that is cleavable under MS conditions, such as a si IyI linkage or photocleavable linkage, can be combined with a linker, such as an avidin biotin linkage, that is not cleaved under these conditions, but may be cleaved under other conditions.
  • Linker such as an avidin biotin linkage
  • Acid-labile linkers are particularly useful chemically cleavable linkers for mass spectrometry, especially for MALDI-TOF, because the acid labile bond is cleaved during conditioning of the target polypeptide upon addition of a 3-HPA matrix solution.
  • the acid labile bond can be introduced as a separate linker group, e.g., an acid labile trityl group, or can be incorporated in a synthetic linker by introducing one or more silyl bridges using diisopropylysilyl, thereby forming a diisopropylysilyl linkage between the polypeptide and the solid support.
  • the diisopropylysilyl linkage can be cleaved using mildly acidic conditions, such as 1.5% trifluoroacetic acid (TFA) or 3-HPA/l% TFA MALDI-TOF matrix solution.
  • TFA trifluoroacetic acid
  • Methods for the preparation of diisopropylysilyl linkages and analogues thereof are well-known in the art. See, e.g., Saha e/ ⁇ /., J. Org. Chem. 58: 7827-7831 (1993).
  • Pin tools include those disclosed herein or otherwise known in the art. See, e.g., U.S. Application Serial Nos. 08/786,988 and 08/787,639; and International PCT Application No. WO 98/20166.
  • a pin tool in an array can be applied to wells containing polypeptides of interest.
  • the pin tool has a functional group attached to each pin tip, or a solid support, e.g., functionalized beads or paramagnetic beads are attached to each pin, the polypeptides in a well can be captured (1 pmol capacity).
  • the pins can be kept in motion (vertical, 1-2 mm travel) to increase the efficiency of the capture.
  • a reaction such as an in vitro transcription is being performed in the wells
  • movement of the pins can increase efficiency of the reaction. Further immobilization can result by applying an electrical field to the pin tool.
  • the pin tool (with or without voltage) can be modified to have conjugated thereto a reagent specific for the polypeptide of interest, such that only the polypeptides of interest are bound by the pins.
  • the pins can have nickel ions attached, such that only polypeptides containing a polyhistidine sequence are bound.
  • the pins can have antibodies specific for a target polypeptide attached thereto, or to beads that, in turn, are attached to the pins, such that only the target polypeptides, which contain the epitope recognized by the antibody, are bound by the pins.
  • Captured polypeptides can be analyzed by a variety of means including, e.g., spectrometry techniques, such as UV/VIS, IR 5 fluorescence, chemiluminescence, NMR spectroscopy, MS or other methods known in the art, or combinations thereof. If conditions preclude direct analysis of captured polypeptides, the polypeptides can be released or transferred from the pins, under conditions such that the advantages of sample concentration are not lost. Accordingly, the polypeptides can be removed from the pins using a minimal volume of eluent, and without any loss of sample. Where the polypeptides are bound to the beads attached to the pins, the beads containing the polypeptides can be removed from the pins and measurements made directly from the beads.
  • spectrometry techniques such as UV/VIS, IR 5 fluorescence, chemiluminescence, NMR spectroscopy, MS or other methods known in the art, or combinations thereof. If conditions preclude direct analysis of captured polypeptides, the polypeptides can be
  • Pin tools can be useful for immobilizing polypeptides of interest in spatially addressable manner on an array. Such spatially addressable or pre-addressable arrays are useful in a variety of processes, including, for example, quality control and amino acid sequencing diagnostics.
  • the pin tools described in the U.S. Application Nos. 08/786,988 and 08/787,639 and International PCT Application No. WO 98/20166 are serial and parallel dispensing tools that can be employed to generate multi-element arrays of polypeptides on a surface of the solid support.
  • the array surface can be flat, with beads or geometrically altered to include wells, which can contain beads.
  • MS geometries can be adapted for accommodating a pin tool apparatus.
  • aspects of the biological activity state, or mixed aspects can be measured in order to obtain drug and pathway responses.
  • the activities of proteins relevant to the characterization of cell function can be measured, and embodiments of this invention can be based on such measurements.
  • Activity measurements can be performed by any functional, biochemical or physical means appropriate to the particular activity being characterized. Where the activity involves a chemical transformation, the cellular protein can be contacted with natural substrates, and the rate of transformation measured. Where the activity involves association in multimeric units, e.g., association of an activated DNA binding complex with DNA, the amount of associated protein or secondary consequences of the association, such as amounts of mRNA transcribed, can be measured.
  • response data may be formed of mixed aspects of the biological state of a cell.
  • Response data can be constructed from, e.g., changes in certain mRNA abundances, changes in certain protein abundances and changes in certain protein activities.
  • test samples derived from human tissues e.g., colon cancer (a.k.a., colorectal cancer), as summarized in TABLE 3 below.
  • colon cancer a.k.a., colorectal cancer
  • blood tissue samples were analyzed from colon cancer patients.
  • FLT-4 missense mutations were identified as detailed below in TABLE 3 (see also, TABLE 1 and TABLE 2, supra).
  • N149D 445 A G Evans AL et al., J. Med. Genet. 40(9):697-703, 2003
  • V-set 231 328 Immunoglobulin V-set domain ig 245 312 Immunoglobulin domain ig 349 398 Immunoglobulin domain ig 571 655 Immunoglobulin domain
  • PROSlTE Analysis of the Potential Effect of FLT-4 mutations on Other FLT-4 Protein Regulatory Sites.
  • the effect of the FLT-4 mutations on other protein regulatory sites was analyzed using the PROSITE computational analysis tool.
  • PROSITE is a database of protein families and domains. It consists of biologically significant sites, patterns and profiles that help to reliably identify to which known protein family (if any) a new sequence belongs as well as to identify potential sites for protein modification (HuIo N. et ah, Nucl. Acids Res., 32:D134-D137 (2004); Sigrist C.J.A. et al, Brief Bioinform., 3:265-274 (2002); Gattiker A. et al.
  • PS00008JMYRISTYL pattern N-myristoylation 190-195; 306-31 1 ; 407-412; 630-635; 723-728; site 755-760; 866-871; 884-889; 904-909; 933-938; 991-996; 1117-1 122; 1267-1272; 1276-1281 33-36; 104-107; 166-169; 251-254; 299-302;
  • PSOOOO 11 ASN_GL YCOSYLATION (pattern) N- 411-414; 515-518; 527-530; 594-597; 683-686; glycosylation site 690-693; 758-761; 934-937; 1271-1274 50-52; 168-170; 376-378; 473-475; 537-539;
  • PKC_PHOSPHO_SITE pattern 582-584; 977-979; 1030-1032; 1073-1075; 1249- Protein kinase C phosphorylation site 1251; 1264-1266
  • CK2_PHOSPHO_SITE (pattern) Casein 722-725; 836-839; 871-874; 886-889; 986-989; kinase II phosphorylation site 1010-1013; 1046-1049; 1085-1088; 1093-1096; 1 193-1196; 1254-1257
  • ClustalW Polypeptide Alignment and Sequence Analysis to Estimate the Potential Effect ofFLT-4 mutation on FLT-4 Function ClustalW polypeptide alignment and sequence analysis was used to estimate the effect of FLT-4 mutation on FLT-4 biological function.
  • Known FLT-4 sequences of various organisms including, e.g., mouse KDR; rat KDR; human KDR; chicken KDR; rat_2 FLT4; rat FLT4; mouse FLT4; human FLT4; chicken VEGFR3; mouse FLTl ; rat FLTl ; human FLTl ; chicken FLTl ; chicken VEGFR2; and fly KDR sequences were obtained from GenBank and aligned using ClustalW.
  • ClustalW is a general purpose multiple sequence alignment program for DNA or proteins. It produces biologically meaningful multiple sequence alignments of divergent sequences. It calculates the best match for the selected sequences, and lines them up so that the identities, similarities and differences can be seen. [0186] For the position with the mutation reported, the mutated residue was inspected for its occurrence in organisms other than human. It was hypothesized that if the mutated residue was present in the wild-type sequence of another species in the corresponding position, the amino acid change may not have any adverse effect on the protein function. The results of the Clustal W comparison analysis are summarized below in TABLE 19 and TABLE 20. The mutated amino acid residues identified in the present studies is highlighted in bold, underlined text.
  • FLT4_mouse LQGGGWQEEEEERMAL H ⁇ QSSEEDGFMQASTTALHITEADADDSPPSMHCHSLAARY
  • FLTl_mouse TETK PKA ⁇ MKIDLRIASKSKEAGL ⁇ DLPRP-SFCF ⁇ SCGHIR-PVQ DDES—ELG
  • nnPredict Method Analysis of the Wild-type FLT-4 Secondary Structure Secondary structure predictions of wild-type FLT-4 (TABLE 22) and mutant FLT-4 polypeptides (TABLE 24; TABLE 26; TABLE 28; and TABLE 30) were performed by nnPredict.
  • the basis of the prediction is a two-layer, feed-forward neural network.
  • the network weights were determined by a separate program -- a modification of the Parallel Distributed Programming suite of McClelland and Rumelhart (MIT Press, Cambridge MA.1, Vol. 3, pp 318-362 (1988)). Complete details of the determination of the network weights is found in Kneller et. al. (J. MoI. Biol, (214): 171-182 (1990)).
  • the output is a secondary structure prediction for each position in the sequence.
  • FLT-4 mutations were identified in colorectal cancer.
  • the N149D had been identified previous as a SNP.
  • M963V, D970E and Pl 114S were found in the protein tyrosine kinase domain.
  • Pl 114 was highly conserved in FLT-4 as well as homologues, FLTl and KDR.
  • D970E was predicted to affect the protein secondary structure by extending an upstream helix in FLT-4 polypeptide.
  • M963V and Pl 114S were also predicted to alter the polypeptide's secondary structure.
  • an agent that modulates FLT-4 biological activity i.e., FLT-4 modulating agent, e.g., FLT-4 antagonist
  • FLT-4 modulating agent e.g., FLT-4 antagonist
  • cancer e.g., colon cancer
  • the SNP is selected from the group consisting of the FLT-4 mutation summarized in TABLE 1 and TABLE 2.

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Abstract

L'invention concerne de manière générale le test analytique d'échantillons tissulaires in vitro, et plus particulièrement des aspects de polymorphismes et de mutations génétiques du gène FLT 4. L'invention concerne des nouvelles mutations et des nouveaux SNP du gène FLT 4, utiles dans le diagnostic et le traitement de sujets nécessitant un tel diagnostic et un tel traitement. Les divers aspects de l'invention concernent ainsi des polynucléotides codant pour les mutations du gène FLT 4 de l'invention, des vecteurs d'expression codant pour les polypeptides mutants du gène FLT 4 de l'invention et des organismes qui expriment les polynucléotides mutants et polymorphes du gène FLT 4 et/ou les polypeptides mutants/polymorphes du gène FLT 4 de l'invention. Les divers aspects de l'invention concernent en outre des méthodes diagnostiques/théranostiques et des kits utilisant les mutations et les polymorphismes du gène FLT 4 de l'invention pour identifier des individus prédisposés à une maladie ou pour classer des individus en fonction de leur réponse à un médicament, des effets secondaires, ou d'une dose de médicament optimale.
PCT/US2007/064116 2006-03-20 2007-03-16 Mutations et polymorphismes de la tyrosine kinase 4 de type fms WO2007121017A2 (fr)

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Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
LILLEBERG STAN L: "IN-DEPTH MUTATION AND SNP DISCOVERY USING DHPLC GENE SCANNING" CURRENT OPINION IN DRUG DISCOVERY AND DEVELOPMENT, CURRENT DRUGS, LONDON, GB, vol. 6, no. 2, March 2003 (2003-03), pages 237-252, XP008078438 ISSN: 1367-6733 cited in the application *
WALTER JEFFREY W ET AL: "Somatic mutation of vascular endothelial growth factor receptors in juvenile hemangioma" GENES CHROMOSOMES AND CANCER, vol. 33, no. 3, March 2002 (2002-03), pages 295-303, XP002473439 ISSN: 1045-2257 cited in the application *

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