MUTATIONS AND POLYMORPHISMS OF FMS-LIKE TYROSINE KINASE 4

FIELD OF THE INVENTION

[0001] 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).

BACKGROUND OF THE INVENTION

[0002] Conventional medical approaches to diagnosis and treatment of disease is based on clinical data alone, or made in conjunction with a diagnostic test. Such traditional practices often lead to therapeutic choices that are not optimal for the efficacy of the prescribed drug therapy or to minimize the likelihood of side effects for an individual subject Therapy specific diagnostics (a. lea., theranostics) is an emerging medical technology field, which provides tests useful to diagnose a disease, choose the correct treatment regime and monitor a subject's response. That is, theranostics are useful to predict and assess drug response in individual subjects, i.e., individualized medicine. 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.

[0003] Progress in pharmacogenomics and pharmacogenetics, which establishes correlations between responses to specific drugs and the genetic profile of individual patients and/or their tumours, is foundational to the development of new theranostic approaches. As such, there is a need in the art for the evaluation of patient-to-patient variations and tumour mutations in gene sequence and gene expression. A common form of genetic profiling relies on the identification of DNA sequence variations called single nucleotide polymorphisms ("SNPs"), which are one type of genetic alteration leading to patient-to-patient variation in individual drug response. In addition, it is well established in the art that acquired DNA

changes (mutations) are responsible, alone or in part, for pathological processes. It follows that, there is a need art to identify and characterize genetic mutations and SNPs, which are useful to identify the genotypes of subjects and their tumours associated with drug responsiveness, side effects, or optimal dose.

[0004] 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. Lee et al, Proc. Nat. Acad. Sci. 93: 1988-1992, 1996. 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.

SUMMARY OF THE INVENTION

[0005] 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. In several embodiments, the cancer can be colon cancer.

[0006] The invention also provides an isolated polynucleotide having a sequence encoding an FLT-4 mutation. In several embodiments, the FLT-4 mutations are the previously- unidentified mutations listed in TABLE 1. Accordingly, 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.

[0007] 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. Then, an anti-cancer therapy is administered to the subject.

[0008] 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.

[0009] The invention also provides for the use of each of the mutations of the inventions as a drug target.

DETAILED DESCRIPTION OF THE INVENTION

[0010] It is to be appreciated that certain aspects, modes, embodiments, variations and features of the invention are described below in various levels of detail in order to provide a substantial understanding of the present invention. In general, such disclosure provides new FLT-4 mutations and SNPs that may be useful, alone or in combination, in the diagnosis and treatment of subjects in need thereof. Accordingly, 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. In other aspects, 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.

[0011] Definitions. The definitions of certain terms as used in this specification are provided below. Definitions of other terms may be found in the glossary provided by the U.S. Department of Energy, Office of Science, Human Genome Project (httpJ/www.ornl.gov/sci/techresources/Human Genome/glossary/).

[0012] As used herein, the term "allele" means a particular form of a gene or DNA sequence at a specific chromosomal location (locus).

[0013] As used herein, 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. [0014] As used herein, 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). [0015] As used herein, 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.

[0016] As used herein, 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. Typically, an effective amount of the compounds of the present invention, sufficient for achieving a therapeutic or prophylactic effect, range from about 0.000001 mg per kilogram body weight per day to about 10,000 mg per kilogram body weight per day. Preferably, 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. [0017] 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. Corless CL et al,, J. Clin. Oncol. 22(18):3813-25 (September 15, 2004); Verweij l etal, Lancet 364(9440): 1127- 34 (September 25, 2004); Kantarjian HM et al, Blood 104(7): 1979-88 (October 1, 2004). By

inhibiting multiple targets, Glivec® has potential as an anticancer therapy for several types of cancer, including leukaemia and solid tumours.

[0018] 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).

[0019] 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]). Oberg K, Chemotherapy 47 Suppl 2:40-53 (2001); Raderer M et al, Oncology 60(2):141-5 (2001); Aparicio T et al, Eur. J. Cancer 37(8):1014-9 (May 2001). Sandostatin® LAR® regulates hormones in the body to help manage diseases and their symptoms.

[0020] 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).

[0021] 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. 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).

[0022] The orally bioavailable rapamycin derivative everolimus inhibits oncogenic signalling in tumour cells. By blocking the mammalian target of rapamycin (mTOR)- mediated signalling, everolimus exhibits broad antiproliferative activity in tumour cell lines and animal models of cancer. Boulay A etal, Cancer Res. 64:252-261 (2004). In preclinical studies, everolimus also potently inhibited the proliferation of human umbilical vein endothelial cells directly indicating an involvement in angiogenesis. By blocking tumour cell

proliferation and 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).

[0023] 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.

[0024] 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 Orleans, 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.

[0025] 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). Weisberg E et al Cancer Cell 1:433-443 (2002); Fabbro D et al., Anticancer Drug Des. 15:17-28 (2000). In preclinical studies, midostaurin showed broad antiproliferative activity against various tumour cell lines, including those that were resistant to several other chemotherapeutic agents. [0026] The somatostatin analogue pasireotide is a stable cyclohexapeptide with broad somatotropin release inhibiting factor (SRIF) receptor binding. Bruns C et al, Eur. J. Endocrinol. 146(5):707-16 (May 2002); Weckbecker G et al, Endocrinology 143(10):4123- 30 (October 2002); Oberg K, Chemotherapy 47 Suppl 2:40-53 (2001).

[0027] LBH589 is a histone deacetylase (HDAC) inhibitor. By blocking the deacetylase activity of HDAC, HDAC inhibitors activate gene transcription of critical genes that cause apoptosis (programmed cell death). 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). [0028] 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.

[0029] 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. Weisberg E et al, Cancer Cell 7(2): 129-41 (February 2005); O'Hare T et al, Cancer Cell 7(2):1 17-9 (February 2005). AMN107 also shows activity against mutated variants of BCR-ABL. AMN 107 is currently being studied in phase I clinical trials.

[0030] As used herein, the term "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.

[0031 ] As used herein, "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.

[0032] As used herein, the term "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. [0033] As used herein, the term "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. As used herein, genotype includes a full- genotype and/or a sub-genotype.

[0034] As used herein, the term "locus" means a location on a chromosome or DNA molecule corresponding to a gene or a physical or phenotypic feature. [0035] As used herein, the term "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.

[0036] As used herein, 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.

[0037] As used herein, the term "nucleotide pair" means the two nucleotides bound to each other between the two nucleotide strands.

[0038] As used herein, the term "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%.

[0039] As used herein, the term "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. Also, 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. [0040] As used herein, the term "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. In addition, 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. In a particular embodiment, the polynucleotide contains polynucleotide sequences from the FLT-4 gene.

[0041 ] As used herein, the term "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. In a particular embodiment, the polypeptide contains polypeptide sequences from the FLT-4 protein.

[0042] As used herein, the term "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.

[0043] As used herein, the term "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.

[0044] As used herein, the term "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.

[0045] As used herein, 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). [0046] As used herein, the administration of an agent or drug to a subject or patient includes self-administration and the administration by another. It is also to be appreciated that the various modes of treatment or prevention of medical conditions as described are intended to mean "substantial", which includes total but also less than total treatment or prevention, and wherein some biologically or medically relevant result is achieved. [0047] The details of one or more embodiments of the invention are set forth in the accompanying description below. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods and materials are now described. Other features, objects, and advantages of the invention will be apparent from the description and the claims. In the specification and the appended claims, the singular forms include plural referents unless the context clearly dictates otherwise. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. All references cited herein are incorporated herein by reference in their entireties and for all purposes to the same extent as if each individual publication, patent, or patent application was specifically and individually incorporated by reference in its entirety for all purposes.

[0048] FLT-4 Mutations and Polymorphisms of the Invention. To investigate FLT-4 mutations in association with cancer DHPLC (Lilleberg S. L., Curr. Opin. Drug. Discov. Devel., 6(2):237-52 (2003)) analysis was conducted on blood samples from colon cancer patients. Five (5) missense mutations were identified as summarized below in TABLE 1 and TABLE 2 below. Computational analyses were designed to evaluate the effect of these mutations on FLT-4 function.

TABLE 1 FLT-4 Mutations Identified in Colon Cancer Patients

Allelic Unmutated Mutated

Exon Mutation/SNP Frequency Sequence Sequence

AAOGAC HeteroCTCTTGGTCAACAGGA CTCTTGGTCGACAGGA

Exon 4 N149D zygous AGGSEQ ID NO:1 AGGSEQ ID NO: 2

ATOGTG CTTCCGCGCCATGGTGG CTTCCGCGCCGTGGTGG Exon 21 M963V 0.55 AGCTCGSEQ ID NO : 3 AGCTCGSEQ ID NO: 4

CAGGCTGGATCGGAGG CAGGCTGGAGCGGAGG Exon 21 GAT>GAG

D970E 0.03 CGGCSEQ ID NO: 5 CGGC SEQ ID NO: 6 Exon 25 Ins 60bp 0.25 60 bp insertion 60 bp insertion

E1 123-A1143

GGGCCTCCCCGTACCCT GGGCCTCCTCGTACCCT Exon 25 CCG>TCG 0.2 P1 U4S GGGSEQ ID NO: 7 GGGSEQ ID NO : 8

[0050] As shown above in TABLE 1 and further summarized below in TABLE 2, five (5) missense mutations were identified were identified in the present invention.

TABLE 2

FLT-4 Mutations in Cancer Patients

Gene Cancer NT change Mutation/SNP AlIe. Obs.

Freq.

FLT-4 Colon AAOGAC N149D het 2 FLT-4 Colon ATG>GTG M963V 0.55 1 FLT-4 Colon GAT>GAG D970E 0.03 1 FLT-4 Colon Ins 60bp E1123-A1 143 0.25 1 FLT-4 Colon CCG>TCG P1114S 0.2 1

[0051] The 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.

[0052] Identification ofFLT-4 Mutations and Polymorphisms of the Invention in Human Cancers. Identification and Characterization of Gene Sequence Variation. 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.

[0054] 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., ψang et al., Science 280: 1077-1082 (1998)).

[0055] An association between SNP' s and/or mutations and a particular phenotype (e.g., cancer type) 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.

[0056] 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. For example, a sequence variant (e.g., 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 )).

[0057] In describing the polymorphic and mutant sites of the invention, reference is made to the sense strand of the gene for convenience. As recognized by the skilled artisan, however, 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. Thus, the invention also includes single-stranded polynucleotides and mutations that are complementary to the sense strand of the genomic variants described herein.

[0058] Identification and Characterization of SN Ps and Mutations. Many different techniques can be used to identify and characterize SNPs and mutations, including single- strand conformation polymorphism (SSCP) analysis, heteroduplex analysis by denaturing high-performance liquid chromatography (DHPLC), direct DNA sequencing and computational methods (Shi et al, CHn. Chem. 47:164-172 (2001)). There is a wealth of sequence information in public databases; computational tools useful to identify SNPs in silico by aligning independently submitted sequences for a given gene (either cDNA or genomic sequences). The most common SNP-typing methods currently include hybridization, primer extension, and cleavage methods. Each of these methods must be connected to an appropriate detection system. 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.

[0059] In a particularly preferred embodiment, the detection of polymorphisms and mutations is detected using INVADER™ technology (available from Third Wave Technologies Inc. Madison, Wisconsin USA). In this assay, 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.

[0060] The identity of 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)). Alternatively, 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)). A polymerase-mediated primer extension method may also be used to identify the polymorphisms/mutations. Several such methods have been described in the patent and scientific literature and include the "Genetic Bit Analysis" method (WO 92/15712) and the ligase/polymerase mediated genetic bit analysis (U.S. Pat. No. 5,679,524). Related methods are disclosed in WO 91/02087, WO 90/09455, WO 95/17676, and U.S. Pat. Nos. 5,302,509 and 5,945,283. Extended primers containing a polymorphism or mutation may be detected by mass spectrometry as described in U.S. Pat. No. 5,605,798. Another primer extension method is allele- specific PCR. Ruafio et al, Nucl Acids Res. 17: 8392 (1989); Ruafio et al, Nucl Acids Res. 19: 6877-6882 (1991); WO 93/22456; Turki et al, J. Clin. Invest. 95: 1635-1641 (1995). In addition, 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.

[0061 ] Haplotyping and Genotyping Oligonucleotides. The invention provides methods and compositions for haplotyping and/or genotyping the genetic polymorphisms (and possibly mutations) in an individual. As used herein, 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. [0062] The 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. [0063] 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). Such 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. [0065] 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.

[0066] Other 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". In a preferred embodiment, the 3 '-terminus of a primer-

extension oligonucleotide is a deoxynucleotide complementary to the nucleotide located immediately adjacent to the polymorphic/mutant site.

[0067] Direct Genotyping Method of the Invention. One embodiment of 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. As will be readily understood by the skilled artisan, the two "copies" of a germline gene in an individual may be the same on each allele or may be different on each allele. In a particularly preferred embodiment, the genotyping method comprises determining the identity of the nucleotide pair at each polymorphic and mutant site.

[0068] Typically, the nucleic acid mixture is isolated from a biological sample taken from the individual, such as a blood sample, tumour or tissue sample. Suitable 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. Furthermore, it will be understood by the skilled artisan that mRNA or cDNA preparations would not be used to detect polymorphisms or mutations located in introns or in 5' and 3' nontranscribed regions. 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.

[0069] Direct Haplotyping Method of the Invention. 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. As will be readily appreciated by those skilled in the art, 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. In a particularly preferred embodiment, the nucleotide at each polymorphic or mutant site is identified.

[0070] In a preferred embodiment, 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. In a particularly preferred embodiment, the haplotyping method comprises identifying the phased sequence of nucleotides at each polymorphic/mutant site in each copy of the gene. When haplotyping both copies 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. For example, if the 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.

[0071 ] In both the genotyping and haplotyping methods, 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. For example, where a SNP is known to be guanine and cytosine in a reference population, 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. Alternatively, the site may

be negatively determined to be not guanine (and thus cytosine/cytosine) or not cytosine (and thus guanine/guanine).

[0072] Indirect Genotyping Method using Polymorphic and Mutation Sites in Linkage Disequilibrium with a Target Polymorphism or Mutation. In addition, 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.

[0073] Amplifying a Target Gene Region. The target regions may be amplified using any oligonucleotide-directed amplification method, including but not limited to polymerase chain reaction (PCR). (U.S. Pat. No. 4,965,188), ligase chain reaction (LCR) (Barany et al, Proc. Natl. Acad Sci. USA 88:189-193 (1991); published PCT patent application WO 90/01069), and oligonucleotide ligation assay (OLA) (Landegren et al, Science 241: 1077-1080 (1988)). 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. Typically, 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.

[0074] Other known 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)). [0075] 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. In some embodiments, more than one polymorphic/mutant site may be detected at once using a set of allele-specific oligonucleotides or oligonucleotide pairs. Preferably, 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.

[0076] 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.

[0077] 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.

[0078] Determining Population Genotypes and Haplotypes and Correlating them with a Trait. 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).

[0079] In another aspect of the invention, 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.

[0080] In preferred embodiments, 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.

[0081] In another embodiment, 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. For example, the frequency data may be present in a database that is accessible by a computer. Once the frequency data are obtained, the frequencies of the genotypes or haplotypes of interest in the reference and trait populations are compared. In a preferred embodiment, 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. [0082] In a preferred embodiment, 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). Hardy- Weinberg equilibrium postulates that the frequency of finding the haplotype pair HiZH ₂ is equal to PH- _W {H _\ /Hi) = 2p(H _\ )p (H ₂ ) if H ₁ ≠ H ₂ and PH^W (HXIH ₂ ) =p (Hi)/? (H ₂ ) if Hi = H ₂ . 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 ηardy- 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 System™ 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)).

[0083] In one embodiment of this method for predicting a haplotype pair, 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. In rare cases, either no haplotypes in the reference population are consistent with the possible haplotype pairs, or alternatively, multiple reference haplotype pairs are consistent with the possible haplotype pairs. In such cases, the individual is preferably haplotyped using a direct molecular haplotyping method such as, for example, those discussed supra. [0084] In a preferred embodiment, 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. For statistical methods useful in the methods of the present invention, see Bailey NTJ, Statistical Methods in Biology,

3 ^rd Edition (Cambridge Univ. Press, Cambridge, 1997); Waterman MS, Introduction to Computational Biology (CRC Press, 2000) and Bioinformatics, Baxevanis AD & Ouellette BFF, eds. (John Wiley & Sons, Inc., 2001).

[0085] In a preferred embodiment of the method, 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.

[0086] Li another embodiment of the invention, 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. [0087] Correlating Subject Genotype or Haplotype to Treatment Response, In order to deduce a correlation between a clinical response to a treatment and a genotype or haplotype, 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. [0088] It is preferred that 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.

[0089] 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. Statistical analysis methods, which may be used, are described in Fisher LD & vanBelle G, Biostatistics: A Methodology for the Health Sciences (Wiley-lnterscience, New York, 1993). This analysis may also include a regression calculation of which polymorphic/mutation sites in the gene contribute most significantly to the differences in phenotype.

[0091 ] 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. 10 (McGraw-Hill, New York, 1991), standard gradient descent methods (Press et al, Numerical Recipes in C: The Art of Scientific Computing, Ch. 10 (Cambridge University Press, Cambridge, 1992), or other global or local optimization approaches (see discussion in Judson, supra) can also be used.

[0092] 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).

[0093] After the clinical, mutation and polymorphism data have been obtained, 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 polymoφhism/mutation group), and then the averages and standard deviations of clinical responses exhibited by the members of each polymorphism/mutation group are calculated.

[0094] From the analyses described above, the skilled artisan that predicts clinical response as a function of genotype or haplotype content may readily construct a mathematical model. The identification of an association between a clinical response and a genotype or haplotype (or haplotype pair) for the gene may be the basis for designing a diagnostic method to

deteπnine those individuals who will or will not respond to the treatment, or alternatively, will respond at a lower level and thus may require more treatment, i.e., a greater dose of a drug or suffer an adverse reaction. 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.

[0096] Patient Selection for Prophylactic Therapy Based Upon Polymorphisms and/or Mutations. The application of genotypes and/or haplotypes that correlate with a predisposition for disease will be used to select patients for preventative therapy. [0097] Computer System for Storing or Displaying Polymorphism and Mutation Data. 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. In a preferred embodiment, 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. In addition, 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.

[0098] Nucleic Acid-based Diagnostics, hi another aspect, 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. In certain preferred embodiments, 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. For example, under stringent hybridization conditions, 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. Alternatively, 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. In this embodiment, the presence or absence of amplification by PCR determines the haplotype of the SNP or the specific mutation. [0099] 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. Preferably, 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.

[0100] Kits of the Invention, 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. For example, 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.

[0101 ] In another embodiment, 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. Alternatively, where the oligonucleotides are to be used to amplify a target region, 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.

[0102] In a preferred embodiment, such kit may further comprise a DNA sample collecting means. In particular, the genotyping primer composition may comprise at least two sets of allele specific primer pairs. Preferably, the two genotyping oligonucleotides are packaged in separate containers.

[0103] For antibody-based kits, 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.

[0104] For oligonucleotide-based kits, 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. [0105] 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.

[0106] Making Polymorphisms and Mutations of the Invention. Effects of the polymorphisms and mutations identified herein on gene expression may be investigated by preparing recombinant cells and/or organisms, preferably recombinant animals, containing a polymorphic variant and/or mutation of the gene.

[0107] In one aspect, 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. Alternatively, 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. For example, for a nucleic acid sequence of about 20-40 bases, 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. 7.0 with 10 N NaOH), 0.1% SDS, and 100 mg/ml denatured salmon sperm DNA, (2) hybridization: incubate filters in prehybridization solution plus probe at 42°C for 14-48 hours, (3) wash; three 15 minutes washes in 6xSSC and 0.1% SDS at room temperature, followed by a final 1-1.5 minutes wash in 6xSSC and 0.1% SDS at 55 ⁰ C. Other equivalent procedures, e.g., employing organic solvents such as formamide, are well known in the art. Standard stringency conditions are well characterized in standard molecular biology cloning texts. See, for example, Sambrook, Fritsch & Maniatis, Molecular Cloning A Laboratory Manual, 2nd Ed., (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 1989); Glover DN, DNA Cloning, Volumes I and II , (1985); Oligonucleotide Synthesis, Gait MJ, ed. (1984); Nucleic Acid Hybridization, Hames BD & Higgins SJ, eds. (1984).

[0108] Recombinant Expression Vectors. Another aspect of the invention includes vectors containing one or more nucleic acid sequences encoding a mutant or polymorphic polypeptide. In practicing the present invention, 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. land 7/(1985); Oligonucleotide Synthesis, Gait, Ed. (1984); Nucleic Acid Hybridization, Hames & Higgins, eds. (1985); Transcription and Translation, Hames & Higgins, Eds. (1984); Animal Cell Culture, Freshney, ed. (1986); Immobilized Cells and Enzymes (IRL Press, 1986); Perbal, A Practical Guide to Molecular Cloning; the series Methods in EnzymoL, (Academic Press, Inc., 1984); Gene Transfer Vectors for Mammalian Cells, Miller & Calos, eds. (Cold Spring Harbor Press, Cold Spring Harbor Laboratory, New York, 1987); and Methods in Enzymology, VoIs. 154 and 155, Wu & Grossman, and Wu, Eds., respectively.

[0109] For recombinant expression of one or more the polypeptides of the invention, 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. [0110] In general, expression vectors useful in recombinant DNA techniques are often in the form of plasmids. In the present specification, "plasmid" and "vector" can be used interchangeably as the plasmid is the most commonly used form of vector. However, 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. 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).

[011 1] 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. Within a recombinant expression vector, "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).

[0112] The term "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.).

[0113] 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. To prepare a recombinant cell 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. In a preferred embodiment, 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.

[0114] The recombinant expression vectors of the invention can be designed for expression of mutant polypeptides in prokaryotic or eukaryotic cells. For example, mutant/polymorphic polypeptides can be expressed in bacterial cells such as Escherichia coli (E. coli), insect cells

-SO-

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). Alternatively, 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).

[01 15] Expression of polypeptides in prokaryotes is most often carried out in E. coli with vectors containing constitutive or inducible promoters directing the expression of either fusion or non fusion polypeptides. 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. Often, in fusion expression vectors, 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. Such 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. [0116] Examples of suitable inducible non fusion E. 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). [0117] 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. In another embodiment, the mutant/polymorphic polypeptide expression vector is a yeast expression vector.

[0118] Examples of vectors for expression in yeast Saccharomyces cerivisiae 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). Alternatively, mutant polypeptide can be expressed in insect cells using baculovirus expression vectors. Baculovirus vectors available for expression of polypeptides in cultured insect cells (e.g., SF9 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)). [0119] hi yet another embodiment, a nucleic acid of the invention is expressed in mammalian cells using a mammalian expression vector. Examples of mammalian expression vectors include pCDM8 (Seed, Nature 329: 842 846 (1987)) and pMT2PC (Kaufman et al, EMBOJ. 6: 187 195 (1987)). When used in mammalian cells, the expression vector's control functions are often provided by viral regulatory elements. For example, commonly used promoters are derived from polyoma, adenovirus 2, cytomegalovirus, and simian virus 40. For other 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\ά Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 1989). [0120] In another embodiment, 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. Nonlimiting examples of 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. 8: 729 733 (1989)) and immunoglobulins (Banerji et al, Cell 33: 729 740 (1983); Queen & Baltimore, Cell 33: 741 748 (1983)), neuron specific promoters (e.g., the neurofilament promoter; Byrne & Ruddle, Proc. Natl Acad. Sci. USA 86: 5473 5477 (1989)), pancreas specific promoters (Edlund et al, Science 230: 912 916 (1985)), and mammary gland specific promoters (e.g., milk whey promoter; U.S. Pat. No. 4,873,316 and European Application Publication No.

264,166). Developmentally regulated promoters are also encompassed, e.g., the murine hox promoters (Kessel & Gruss, Science 249: 374379 (1990)) and the α-fetoprotein promoter (Campes & Tilghman, Genes Dev. 3: 537 546 (1989)).

[0121] 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. For a discussion of the regulation of gene expression using antisense genes see, e.g., Weintraub et al, "Antisense RNA as a molecular tool for genetic analysis," Reviews Trends in Genetics, Vol. 1(1) (1986). [0122] Another aspect of the invention pertains to host cells into which a recombinant expression vector of the invention has been introduced. The terms "host cell" and "recombinant host cell" are used interchangeably herein. It is understood that such terms refer not only to the particular subject cell but also to the progeny or potential progeny of such a cell. Because certain modifications may occur in succeeding generations due to either mutation or environmental influences, such progeny may not, in fact, be identical to the parent cell, but are still included within the scope of the term as used herein. [0123] A host cell can be any prokaryotic or eukaryotic cell. For example, 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). Other suitable host cells are known to those skilled in the art.

[0124] Vector DNA can be introduced into prokaryotic or eukaryotic cells via conventional transformation or transfection techniques. As used herein, the terms "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. [0125] For stable transfection of mammalian cells, it is known that, depending upon the expression vector and transfection technique used, only a small fraction of cells may integrate the foreign DNA into their genome. In order to identify and select these integrants, a gene that encodes a selectable marker {e.g., resistance to antibiotics) is generally introduced into the host cells along with the gene of interest. 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. Cells stably transfected with the introduced nucleic acid can be identified by drug selection {e.g., cells that have incorporated the selectable marker gene will survive, while the other cells die). [0126] 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. In one embodiment, 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. In another embodiment, 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.

[0127] 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. [0128] 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)).

[0129] Determination of Target Gene Transcription. The determination of the level of the expression product of the gene in a biological sample, e.g., the tissue or body fluids of an individual, may be performed in a variety of ways. The term "biological sample" is intended to include tissues, cells, biological fluids and isolates thereof, isolated from a subject, as well as tissues, cells and fluids present within a subject. Many expression detection methods use isolated RNA. For in vitro methods, any 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. (John Wiley & Sons, New York, 1987-1999). [0130] In one embodiment, 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. In other more preferred embodiments, 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. Large numbers of tissue samples can readily be processed using techniques well-known to those of skill in the art, such as, e.g., the single-step RNA isolation process of U.S. Pat. No. 4,843,155. [0131] 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. [0132] In one format, 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). A skilled artisan can readily adapt known mRNA detection methods for use in detecting the level of mRNA encoded by the markers of the present invention. [0133] 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. ScL USA 87: 1874-1878 (1990)); transcriptional amplification system (Kwoh et al, Proc. Natl. Acad. Sci. USA 86: 1173-1177 (1989)); Q-Beta Replicase (Lizardi et al., Biol. Technology 6: 1197 (1988)); rolling circle replication (U.S. Pat. No. 5,854,033); or any other nucleic acid amplification method, followed by the detection of the amplified molecules using techniques well-known to those of skill in the art. These detection schemes are especially useful for the detection of the nucleic acid molecules if such molecules are present in very low numbers. As used herein, "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. [0134] Real-time quantitative PCR (RT-PCR) is one way to assess gene expression levels, e.g., of genes of the invention, e.g., those containing SNPs and mutations of interest. 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. One method for doing this is TAQMAN® (PE Applied Biosystems, Foster City, Calif., USA) and exploits the 5' nuclease activity of AMPLITAQ GOLD™ DNA polymerase to cleave a specific form of probe during a PCR reaction. This is referred to as a

TAQMAN™ probe. See Luthra et al, Am. J. Pathol. 153: 63-68 (1998); Kuimelis et al, Nucl. Acids Symp. Ser. 37: 255-256 (1997); and Mullah et al, Nucl. Acids Res. 26(4): 1026- 1031 (1998)). During the reaction, cleavage of the probe separates a reporter dye and a quencher dye, resulting in increased fluorescence of the reporter. The accumulation of PCR products is detected directly by monitoring the increase in fluorescence of the reporter dye. Heid et al, Genome Res. 6(6): 986-994 (1996)). The higher the starting copy number of nucleic acid target, the sooner a significant increase in fluorescence is observed. See Gibson, Heid & Williams et al, Genome Res. 6: 995-1001 (1996).

[0135] Other technologies for measuring the transcriptional state of a cell produce pools of restriction fragments of limited complexity for electrophoretic analysis, such as methods combining double restriction enzyme digestion with phasing primers (see, e.g., EP 0 534858 Al), or methods selecting restriction fragments with sites closest to a defined mRNA end. (See, e.g., Prashar & Weissman, Proc. Natl. Acad. Sci. USA 93(2) 659-663 (1996)). [0136] Other methods statistically sample 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).

[0137] 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. Examples of 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. Generally, 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)).

[0138] For the production of antibodies to a protein encoded by one of the disclosed genes, various host animals may be immunized by injection with the polypeptide, or a portion thereof. Such host animals may include, but are not limited to, rabbits, mice and rats. Various 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.

[0139] Monoclonal antibodies (mAbs), 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.

[0140] In addition, techniques developed for the production of "chimaeric antibodies" (see Morrison et al, Proc. Natl. Acad. Set USA 81: 6851-6855 (1984); Neuberger et al, Nature 312: 604-608 (1984); and Takeda et al, Nature 314: 452-454 (1985)), by splicing the genes

from a mouse antibody molecule of appropriate antigen specificity together with genes from a human antibody molecule of appropriate biological activity can be used. A chimaeric antibody is a molecule in which different portions 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.

[0141] Alternatively, techniques described for the production of single chain antibodies (U.S. Pat. No. 4,946,778; Bird, Science 242: 423-426 (1988); Huston et al, Proc. Natl. Acad. ScI USA 85: 5879-5883 (1988); and Ward et al, Nature 334: 544-546 (1989)) can be adapted to produce differentially expressed gene single-chain antibodies.

[0142] Techniques useful for the production of "humanized antibodies" can be adapted to produce antibodies to the proteins, fragments or derivatives thereof. Such techniques are disclosed in U.S. Pat. Nos. 5,932,448; 5,693,762; 5,693,761; 5,585,089; 5,530,101; 5,569,825; 5,625,126; 5,633,425; 5,789,650; 5,661,016; and 5,770,429. [0143] 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.

[0144] 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. As used herein, "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. [0145] 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. Preferably, 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.

As noted above, 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)). In a preferred embodiment, 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.

[0146] Detection of Polypeptides. 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)). [0147] Detection of Polypeptides. Mass Spectroscopy. The identity as well as expression level of target polypeptide can be determined using mass spectrocopy technique (MS). 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). 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. For ionization, numerous matrix/wavelength combinations (e.g., matrix assisted laser desorption (MALDI)) or solvent combinations (e.g., ESI) can be employed.

[0148] For mass spectroscopy (MS) analysis, the target polypeptide can be solubilised in an appropriate solution or reagent system. The selection of a solution or reagent system, e.g., an organic or inorganic solvent, will depend on the properties of the target polypeptide and the type of MS performed, and is based on methods well-known in the art. See, e.g., Vorm et al, Anal. Chem. 61 : 3281 (1994) for MALDI; and Valaskovic et al, Anal. Chem. 67: 3802 (1995), for ESI. MS of peptides also is described, e.g., in International PCT Application No. WO 93/24834 and U.S. Pat. No. 5,792,664. 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. 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).

[0149] The mass of a target polypeptide determined by MS can be compared to the mass of a corresponding known polypeptide. For example, where the target polypeptide is a mutant protein, the corresponding known polypeptide can be the corresponding non-mutant protein, e.g., wild-type protein. With 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)).

[0150] Matrix Assisted Laser Desorption (MALDI). 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. Methods for performing MALDI are well-known to those of skill in the art. See, e.g., Juhasz et al., Analysis, Anal. Chem. 68: 941-946 (1996), and see also, e.g., U.S. Pat. Nos. 5,777,325; 5,742,049; 5,654,545; 5,641,959; 5,654,545 and 5,760,393 for descriptions of MALDI and delayed extraction protocols. Numerous methods for improving resolution are also known. 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. See Bordeaux Mass Spectrometry Conference Report, Hillenkamp, Ed., pp. 354-362 (1988); Bordeaux Mass Spectrometry Conference Report, Karas & Hillenkamp, Eds., pp. 416-417 (1988); Karas & Hillenkamp, Anal. Chem. 60: 2299-2301 (1988); and Karas etal, Biomed. Environ. Mass Spectrum 18: 841-843 (1989). The use of laser beams in TOF-MS is shown, e.g., in U.S. Patent Nos. 4,694,167; 4,686,366, 4,295,046 and 5,045,694, which are incorporated herein by reference in their entireties. Other 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. [0152] Surfaces Enhanced for Laser Desorption/Ionization (SELDI). 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. Several types of new MS 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.

[0153] Within the general category of SELDI are three separate subcategories: (1) Surfaces Enhanced for Neat Desorption (SEND), where the probe element surfaces, i.e., sample presenting means, are designed to contain Energy Absorbing Molecules (EAM) instead of "matrix" to facilitate desorption/ionizations of analytes added directly (neat) to the surface;

(2) SEAC, where the probe element surfaces, i.e., sample presenting means, are designed to contain chemically defined and/or biologically defined 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); and

(3) Surfaces Enhanced for Photolabile Attachment and Release (SEPAR), where the probe element surfaces, i.e., sample presenting means, are designed or modified to contain one or

more types of chemically defined cross-linking molecules to serve as covalent docking devices. 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 (e.g., protein) 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).

[0154] Functionalizing Polypeptides. 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. For example, 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. The artisan will recognize, however, that such a modification, e.g., the incorporation of a biotin moiety, can affect the ability of a particular reagent to interact specifically with the polypeptide and, accordingly, will consider this factor, if relevant, in selecting how best to modify a polypeptide of interest. 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. For example, 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. Other 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). Thus, 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.

[0155] Thiol-Reactive Functionalities. 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. A variety of thiol-reactive functionalities are known in the art, including, 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). [0156] If desired, 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.

[0157] 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. 11 : 327 (1978)); and Merrifield linkers (see, e.g., Bodansky et ah, Peptide Synthesis, 2 ^nd Edition (Academic Press, New York, 1976)). For example, 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.

[0158] Cleavable Linkers. A linker can provide a reversible linkage such that it is cleaved under the select conditions. In particular, 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. 31 : 7095 (1990)); 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. 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. Brown et al, MoI Divers, pp. 4-12 (1995); Rothschild et al, Nucl Acids. Res. 24: 351-66 (1996); and U.S. Pat. No. 5,643,722. 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. , MoI Immunol 30: 379-396 (1993). [0159] Combinations of any linkers are also contemplated herein. For example, a 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. 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. Methods for the preparation of diisopropylysilyl linkages and analogues thereof are well-known in the art. See, e.g., Saha e/ α/., J. Org. Chem. 58: 7827-7831 (1993).

[0160] Use of a Pin Tool to Immobilize a Polypeptide. The immobilization of a polypeptide of interest to a solid support using a pin tool can be particularly advantageous. 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.

[0161] A pin tool in an array, e.g., a 4 x 4 array, can be applied to wells containing polypeptides of interest. Where 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). During the capture step, the pins can be kept in motion (vertical, 1-2 mm travel) to increase the efficiency of the capture. Where 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. When a voltage is applied to the pin tool, the polypeptides are attracted to the anode or the cathode, depending on their net charge. [0162] For more specificity, 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. For example, the pins can have nickel ions attached, such that only polypeptides containing a polyhistidine sequence are bound. Similarly, 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. [0163] 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.

[0164] 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. In addition, MS geometries can be adapted for accommodating a pin tool apparatus.

[0165] Other Aspects of the Biological State. In various embodiments of the invention, 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. Also, where only a functional activity is known, e.g., as in cell cycle control, performance of the function can be observed. However known and measured, the changes in protein activities form the response data analyzed by the methods of this invention. In alternative and non-limiting embodiments, 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.

[0166] The following EXAMPLES are presented in order to more fully illustrate the preferred embodiments of the invention. These EXAMPLES should in no way be construed as limiting the scope of the invention, as defined by the appended claims.

EXAMPLE I

BIOINFORMATICS ANALYSIS OF FLT-4 MUTATIONS

[0168] Identification of FLT-4 Mutations in Colon Cancer. To determine FLT-4 mutations in association with cancer, DHPLC analysis (Lilleberg SL, Curr. Opin. Drug Discov. Devel.,

6(2): 237-52 (March 2003)) was conducted on test samples derived from human tissues, e.g., colon cancer (a.k.a., colorectal cancer), as summarized in TABLE 3 below. Specifically, blood tissue samples were analyzed from colon cancer patients. Six (6) FLT-4 missense mutations were identified as detailed below in TABLE 3 (see also, TABLE 1 and TABLE 2, supra).

TABLE 3

FLT-4 Mutations ; in Cancer Patients

Mutation Amino Acid Chanεe Amino Acid Position Identifier

AAC>GAC N>D 149 N149D

ATOGTG M>V 963 M963V

GAT>GAG D>E 970 D970E

Ins 60bp Insert 1123-1143 E1123-A1143

CCG>TCG P>S 1114 P1114S

[0169] Computational Analysis of FLT-4 Mutations. The FLT-4 mutations identified in human cancer were analyzed using computational analysis tools to determine the effect(s) of these mutations on FLT-4 function.

[0170] Comparison of Known FLT-4 Mutations and SNPs with the FLT-4 Mutations and SNPs of the Present Invention. As summarized in TABLE 4, there were fifteen (15) coding SNPs within the SNP database for FLT-4 (dbSNP: http://www.ncbi.nlm.nih.gov/SNP/index.html)). None of the mutations in TABLE 1 matches know non-synonymous SNPs. N149D has been identified as a SNP previously.

TABLE 4 Select FLT-4 SNPs cSNP Allele 1 Allele 2 RefSNP frequency frequency

R1189C C T rs744282

E1164D G C rsl049080

P1066P C T rsl 1949194

H890Q C: 0.65 G: 0.35 rs448012

T494A A: 0.85 G: 0.15 rs307826

Y448Y C T rs3736062

Q285Q G A rsl 049077

L250L G C rsl 049076

L169L G: 0.917 T: 0.083 rs3736061

N149D 445: A G Evans AL et al., J. Med. Genet. 40(9):697-703, 2003

Q890H 2670:G C Evans AL et al., J. Med. Genet. 40(9):697-703, 2003

P954S 2860:C T Evans AL et al., J. Med. Genet. 40(9):697-703, 2003

PI008L 3023:C T Evans AL et al., J. Med. Genet. 40(9):697-703, 2003

R1146H 3437:G A Evans AL et al., J. Med. Genet. 40(9):697-703, 2003

R1324L 3971 :G T Evans AL et al., J. Med. Genet. 40(9):697-703, 2003

[0171] There are 12 types of FLT-4 mutations listed within the Human Gene Mutation Database (HGMD; Krawczak et al, Trends Genet, 13(3):121-2 (1997); Cooper et al., Hum. Genet., 98(5):629 (1996)) as summarized below in TABLE 5. None of the mutations within this database match any of the FLT-4 mutations of the present invention.

TABLE 5 FLT-4 Mutations in Disease

Codon Nucleotide Amino acid Phenotvpe

G854S cGGC-AGC Gly-Ser Lymphoedema, primary

G857R cGGG-AGG Gly-Arg Lymphoedema, primary

A915P gGCG-CCG Ala-Pro Lymphoedema, primary

C916W TGCa-TGG Cys-Trp Lymphoedema, primary

G933R cGGC-CGC Gly-Arg Lymphoedema, primary

H1035R CAC-CGC His-Arg Lymphoedema, primary

R1041Q CGG-CAG Arg-Gln Lymphoedema, primary

R1041P CGG-CCG Arg-Pro Lymphoedema, primary

R1041W tCGG-TGG Arg-Trp Lymphoedema, primary

L1044P CTG-CCG Leu-Pro Lymphoedema, primary

P11 14L CCG-CTG Pro-Leu Lymphoedema, primary

P1137L CCG-CTG Pro-Leu Lymphoedema, primary

[0172] Analysis of the Effect of FLT-4 Mutations on FLT-4 Protein Domain Structure and Function. Pfant Analysis of the Potential Effect of the FLT-4 mutations on FLT-4 Protein Domain Structure The effect of the FLT-4 mutations on the protein domain structure of FLT-4 was analyzed using the Pfam computational analysis tool. Pfam is a large collection of multiple sequence alignments and hidden Markov models covering many common protein families based on the Swissprot 44.5 and SP-TrEMBL 27.5 protein sequence databases. A search using Pfam (Bateman et al., Nucl. Acids Res., Database Issue 32:D138-D141 (2004)) showed that FLT-4 contained ten (10) domains as summarized in TABLE 6.

TABLE 6

Pfam Analysis of FLT-4 Domain Structure

Model Seq-from Seα-to Description

I-set 230 328 Immunoglobulin I-set domain

V-set 231 328 Immunoglobulin V-set domain ig 245 312 Immunoglobulin domain ig 349 398 Immunoglobulin domain ig 571 655 Immunoglobulin domain

V-set 677 766 Immunoglobulin V-set domain

I-set 678 766 Immunoglobulin I-set domain ig 692 750 Immunoglobulin domain

Pkinase 845 1169 Protein kinase domain

Pkinase Tyr 845 1169 Protein tyrosine kinase

[0173] Sequence alignments of the wild-type human FLT-4 polypeptide sequence with the Pfam domain polypeptide sequences are summarized below in TABLE 7 through TABLE 16. M963, D970 and Pl 114 were observed as located in the protein tyrosine kinase domain. Sequence alignment of the wild-type human FLT-4 polypeptide sequence with the Pfam immunoglobulin I-set domain sequence is summarized below in TABLE 7. An immunoglobulin I-set domain was identified in the wild-type FLT-4 polypeptide at amino acid position 230 to 328 (TABLE 7; score 15.1. E = 4.6e-05).

TABLE 7

Sequence Alignment Comparison of Human FLT-4 from AA 230 to AA 328 with Pfam

Model of Immunoglobulin I-set Domain

*->PkFtqkp.kdveVqeGesarFeCkVtGt.. PePtVsWfKdGqplssv

+ I + I++I +M +++ | + [+ + + I ( |++ FLT-4 230 YDIQLLPrKSLELLVGEKLVLNCTVWAEfnSGVTFDWDYPGKQAERG 276

1. lpsrrfkvtydgGsgtytLtlsnvqpdDsGkYtCvAtNsaGeaeasae l + l I + I+++ + I M I I I 1 I l + l I + I l FLT-4 277 KwVPERRSQQTHTE—LSSILTIHNVSQHDLGSYVCKANNGIQRFRESTE 324

LtVq<-*

++I FLT-4 325 VIVH 328

SEQ ID NO: 9

[0174] Sequence alignment of the wild-type human FLT-4 polypeptide sequence with the Pfam immunoglobulin V-set domain sequence is summarized below in TABLE 8. An immunoglobulin V-set domain was identified in the wild-type FLT-4 polypeptide at amino acid position 231 to 328 (TABLE 8; score 22.7, E - 0.00017).

TABLE 8

Sequence Alignment Comparison of Human FLT-4 from AA 231 to AA 328 with Pfam

Model of Immunoglobulin V-set Domain

*->qsvvtqepprLLLASLfWptsvtvaeGgsvtLρCtysfddsssssgt ++++ + |+ +|+++ I + II++ + +M + FLT-4 231 DIQLLP—RK SLELLVGEKLVLNCTVW AEFNSGV 262 gsvyWyrqqppGekpeliiisfytyyspsegngtvserfkgrvrfsgnps

++ I + + | ++++ 1 + 1 I + ++ +

FLT-4 263 -TFDWDY-PGK QAERGKWVPERRSQQTHTELSSI 294 kgdfsLtlsnlqlsDsGtYyCa gsgtrLtVK-*

I I I + I+++ I M l +++ + ++I+++I + FLT-4 295 -LTIHNVSQHDLGSYVCKanngiqrfRESTEVIVH 328

SEQ ID NO: 10

[0175] Sequence alignment of the wild-type human FLT-4 polypeptide sequence with the Pfam immunoglobulin domain sequence is summarized below in TABLE 9. An immunoglobulin domain was identified in the wild-type FLT-4 polypeptide at amino acid position 245 to 312 (TABLE 9; score 29.7, E = 9e-06).

TABLE 9

Sequence Alignment Comparison of Human FLT-4 from AA 245 to AA 312 with Pfam

Model of Immunoglobulin Domain

*->GesvtLtCsv...gppdvtvtWlrngk.le.gvlvtssenngdstyq

M++ I + | + | ++++ ||+ I H+ l + l +++++++++++ FLT-4 245 GEKLVLNCTVwaeFNSGVTFDWDYPGKqAErG KWVPERRSQQTH 288 t LtitnvtρeDsgtYtCvv<-*

1+ ++ I I l + l I ++ i l + l I + FLT-4 289 TelssiLTIHNVSQHDLGSYVCKA 312

SEQ ID NO: 11

[0176] Sequence alignment of the wild-type human FLT-4 polypeptide sequence with the Pfam immunoglobulin domain sequence is summarized below in TABLE 10. An immunoglobulin domain was identified in the wild-type FLT-4 polypeptide at amino acid position 349 to 398 (TABLE 10; score 13.8, E = 0.0038).

TABLE 10

Sequence Alignment Comparison of Human FLT-4 from AA 349 to AA 398 with Pfam

Model of Immunoglobulin Domain

*->GesvtLtCsv. gppdvtvtWlrngk. Ie . gvlvtssenngdstyqtL I |+|+ + I+++++I+++I I I + ++ + I

FLT-4 349 DELVKLPVKLaAYPPPEFQWYKDGKaLSgRHSPHA L 384 titnvtpeDsgtYt<-* ++++I |+ +| I I I FLT-4 385 VLKEVTEASTGTYT 398

SEQ ID NO: 12

[0177] Sequence alignment of the wild-type human FLT-4 polypeptide sequence with the Pfam immunoglobulin domain sequence is summarized below in TABLE 11. An immunoglobulin domain was identified in the wild-type FLT-4 polypeptide at amino acid position 571 to 655 (TABLE 1 1; score 15.6, E = 0.15).

TABLE I l

Sequence Alignment Comparison of Human FLT-4 from AA 571 to AA 655 with Pfam

Model of Immunoglobulin Domain

*->GesvtLtCsv . gppdvtvtWlrngk . Ie . gvlvtssenngdstyqt . I ++ I I+I++++ + +++I+I + ++ ++ ++ +

FLT-4 571 GQPVLLSCQAdSYKYEHLRWYRLNLsTLhDAHGNPLL Ld 609 LtitnvtpeDsgtYtCvv<-*

++ + ++ + ++ ++ ++ + +| + | +| M++I I l + l FLT-4 610 cknvhlfatplaasleevapgarhatlsLSIPRVAPEHEGHYVCEV 655

SEQ ID NO: 13

[0178] Sequence alignment of the wild-type human FLT-4 polypeptide sequence with the Pfam immunoglobulin V-set domain sequence is summarized below in TABLE 12. A protein kinase domain was identified in the wild-type FLT-4 polypeptide at amino acid position 677 to 766 (TABLE 12; score 18.8, E = 0.00045).

TABLE 12

Sequence Alignment Comparison of Human FLT-4 from AA 677 to AA 766 with Pfam

Model of Immunoglobulin V-Set Domain

*->qsvvtqepprLLLASLfWptsvtvaeGgsvtLpCtysfddsssssgt + +Il + + |++ +I++++I + + + + FLT-4 677 APRLTQ-NLT DLLVNVSDSLEMQCLVA-GAH AP 707 gsvyWyrq.qppGekpeliiisfytyyspsegngtvserfkgrvrfsgnp I + I l ++++ + + I I ++ ++

FLT-4 708 -SIVWYKDeRLL EEKSGVDLADSN 730 skgdfsLtlsnlqlsDsGtYyCa gsgtrLtVK-*

I+I++++ +I I M + ++ |+ + |+ FLT-4 731 Q KLSIQRVREEDAGRYLCSvcnakgcvNSSASVAVE 766

SEQ ID NO: 14

[0179] Sequence alignment of the wild-type human FLT-4 polypeptide sequence with the Pfam immunoglobulin I-set domain sequence is summarized below in TABLE 13. An immunoglobulin I-set domain was identified in the wild-type FLT-4 polypeptide at amino acid position 678 to 766 (TABLE 13; score 52.1, E = I .6e-12).

TABLE 13

Sequence Alignment Comparison of Human FLT-4 from AA 678 to AA 766 with Pfam

Model of Immunoglobulin I-Set Domain

* ->PkFtqkpkc4veVqeGesarFeCkVtGtPePtVsWfKdGqplssvllp

|+ Il + |+ i++ +I++++I I I |++ l+l I + j + FLT-4 678 PRLTQNLTDLLVNVSDSLEMQCLVAGAHAPSIVWYKDERLLEE 720 srrfkvtydgGsgtytLtlsnvqpdDsGkYtCvAtNsaGeaeasaeLtVq + |+ + +++| I++! +1+1 I l + l I + +! |++ |+ FLT-4 721 —KSGVDLAD—SNQKLSIQRVREEDAGRYLCξVCNAKGCVNSSASVAVE 766

SEQ ID NO: 15

[0180] Sequence alignment of the wild-type human FLT-4 polypeptide sequence with the immunoglobulin domain sequence is summarized below in TABLE 14. An immunoglobulin domain was identified in the wild-type FLT-4 polypeptide at amino acid position 692 to 750 (TABLE 14; score 33.7, E = 5.6e-07).

TABLE 14

Sequence Alignment Comparison of Human FLT-4 from AA 692 to AA 750 with Pfam

Model of Immunoglobulin Domain

*->GesvtLtCsv. gppdvtvtWlrngk. Ie. gvlvtssenngdstyqtL +|+ ++| ! I +++ |+++ ||+ J+ +[ + I FLT-4 692 ^' SDSLEMQCLVaGAHAPSIVWYKDERlLEeKSGVDLADSNQK L 733 titnvtpeDsgtYtCvv<-* +I++I++I l+l I I I FLT-4 734 SIQRVREEDAGRYLCSV 750

SEQ ID NO: 16

[0181] Sequence alignment of the wild-type human FLT-4 polypeptide sequence with the Pfam protein kinase domain sequence is summarized below in TABLE 15. A protein kinase domain was identified in the wild-type FLT-4 polypeptide at amino acid position 845 to 1169 (TABLE 15; score 131.2, E - 2.5e-36).

TABLE 15

Sequence Alignment Comparison of Human FLT-4 from AA 845 to AA 1169 with Pfam

Model of Protein Kinase Domain

*->yelleklGsGsfGtV ykakhkgtgkiVAvKiikkrkekskkd

++!+++| I l+l l+l + + + ++ +++++I t | | + I +++ +++ FLT-4 845 LHLGRVLGYGAFGKVveasaFGIHKGSSCDTVAVKML-KEGATASEH 890 ktarrrsEiqilkrL . sHpNIvrlidvfedkdhly . lVlEycegGLrlld +++ I++I I ++++ I l+l I+++++ ++ + ++ | + | I FLT-4 891 RALMS—ELKILIHIgNHLNVVNLLGACTKPQGPLmVIVEFCKYG N 934

Lfdyls..d

|+ +|+ +++ ++ +++++++++ + + + ++++++++++ + FLT-4 935 LSNFLRakRdafspcaekspeqrgrframvelarldrrrpgssdrvlfar 984 trrkgpll . fsedeakkialQilrgleYLHsnlgi

++++++ ++ ++++ ++ + I ++++++++ ++ I ++ Il + I + J I + ++ FLT-4 985 fskteggarraspdqEAEDLWLSpLTMEDLVCYSFQVARGMEFLASR-KC 1033 iHRDLKPeNILldengvvdafIKiaDFGlAkkis . t . sss . lttfvGTpw I I I I I I I I l + l +1 I I l + l I I 11+ +++ + +++ + + FLT-4 1034 IHRDLAARNILLSESDW KICDFGLARDIYkDpDYVrKGSARLPLK 1079

YmAPEvllggngygtkvDvWSlGvilyellt . GkpPFsgevqdsldqlql

+ ι 111 + ++ i + i +ι I i i + i i + i + i ++ i ι++ι ++

FLT-4 1080 WMAPESIF-DKVYTTQξDVWSFGVLLWEIFSIGASPYPGV QINEE 1123 irkilgpplefdspewdsisysefrrlpnlseeakdLikkcLnkDPskRl +++ I + ++ I l + ++ + ++ + I + | j+ i+

FLT-4 1124 FCQRLRDGTRMRAPELAT PAIRRIMLNCWSGDPKARP 1160 taeeilqHPwf<-*

+ I +++ + FLT-4 1161 AFSELVEσ--L 1169

SEQ ID NO: 17

[0182] Sequence alignment of the wild-type human FLT-4 polypeptide sequence with the Pfam protein tyrosine kinase domain sequence is summarized below in TABLE 16. A protein tyrosine kinase domain was identified in the wild-type FLT-4 polypeptide at amino acid position 845 to 1169 (TABLE 16; score 515.8, E = 4e-152).

TABLE 16

Sequence Alignment Comparison of Human FLT-4 from AA 845 to AA 1169 with Pfam

Model of Protein Tyrosine Kinase Domain

*->l klgkkLGeGaFGeVykGtl kg . sgegt kikVAVKtLkeigasseei I + I I ++ I I l l l l + f +++ ++ l +++ + + I I I I + M I I I + + I

FLT-4 845 LHLGRVLGYGAFGKWEASAFGiHKGSSCDTVAVKMLKE-GATASE- 889 gredFlrEAsiMkklGdHpNiVrLlGvctkegePggpglyiVtEymegGd ++++++ ( ++ I + ++ I + I I + I + Il I + I i I +++ I I ++++ I ++++ I +

FLT-4 890 -HRALMSELKILIHIGNHLNWNLLGACTKPQGP LMVIVEFCKYGN 934

LldfLrkhr

I + I I I ++ I + ++ +++++++++ + + + ++++++++++ +

FLT-4 935 LSNFLRAKRdafspcaekspeqrgrframvelarldrrrpgssdrvlfar 984 egrpLtlkdLlsfalQiAkGMeYLesknfv

++++++ ++ +++++ ++ + + I I I ++| I+++++ l + l + I I l + l + |++++

FLT-4 985 fskteggarraspdqeaedlWLSPLTMEDLVCYSFQVARGMEFLASRKCI 1034

HRDLAARNcLVsenlvVKIsDFGLaRdiynddyYvrkkgggklPvkWmAP

I I i I I I I l + l + l 1+ i I I l + l I I I Il I I l + l +1 + I I+++I I I I I I I

FLT- 4 1035 HRDLAARNILLSESDWKICDFGLARDIYKDPDY-VRKGSARLPLKWMAP 1083

Esl kygkFtskSDVWSFGVlLWEiftlGeqPFYpgm. sneevlellyedG

I I + + + + + + I ++ I I I I I I I I I I I I j I + I I + + I I I I ++ M l + + + + I + I I

FLT- 4 1084 ESIFDKVYTTQSDVWSFGVLLWEIFSLGASP-YPGVqINEEFCQRL-RDG 1131 yRLprPenCPdelYdlMlqCWaedPedRPtFselverL<-*

|+ +| )++++ + ++Il M | |+ M I I I I I l + l

FLT-4 1132 TRMRAPELATPAIRRIMLNCWSGDPKARPAFSELVEIL 1169

SEQ ID NO: 18

[0183] Analysis of the Potential Effect ofFLT-4 mutations on FLT-4 Protein Regulatory Sites. NetPhos Analysis of the Effect of FLT-4 mutations on FLT-4 Protein Phosphorylation. Potential FLT-4 phosphorylation sites were identified by computational analysis using the NetPhos computational analysis tool. NetPhos produces neural network predictions for serine, threonine and tyrosine phosphorylation sites in eukaryotic proteins (Blom et ah, J. MoI. Biol, 294(5): 1351-1362, 1999). Potential FLT-4 phosphorylation sites predicted by NetPhos are summarized below in TABLE 17. To be considered a potential phosphorylation site a threshold score of 0.5 was required. The predicted phosphorylation sites marked in bold text represent possible mutation interference.

TABLE 17 FLT-4 Phosphorylation Sites Predicted by NetPhos

Phosphorylation Amino Acid Position

46, 48, 50, 75, 128, 173, 174, 239, 284, 301, 322, 334, 376, 380, 431, 439, 565, 582, 623, 660, 694, 722, 734, 773, 814, 871, Serine 947, 953, 976, 977, 986, 996, 1030, 1046, 1073, 1085, 1113,

1191, 1193, 1194, 1200, 1217, 1220, 1225, 1249, 1250, 1268, 1281, 1289, 1295 _τ , . , 43, 70, 78, 287, 323, 464, 493, 494, 506, 681, 1093, 1132, 1254,

Threonine ^ ^ ₅₄

„ . 109, 129, 230, 268, 308, 435, 583, 585, 651, 669, 812, 830, 932,

Tyrosme ₁₀₆₃ , 1068

[0184] 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. , Applied Bioinformatics, 1 : 107- 108 (2002)). Other potential sites for protein modification of FLT-4 polypeptide as predicted by ProSite analysis are summarized below in TABLE 18. Amino acid positions highlighted in bold text represent possible interference by mutations. Pl 114 was observed as close to a potential myristoylation site.

TABLE 18 Potential FLT-4 protein modification sites predicted by PRQSITE

Function Amino Acid Position

PSOOl 07|PROTEIN_KINASE_ATP (pattern) 851-879 Protein kinases ATP-binding region signature PS00240|RECEPTOR_TYR_KIN JII (pattern) 904-917 Receptor tyrosine kinase class III signature PSOO 109|PROTEIN_KINASE_TYR (pattern)

1033-1045 Tyrosine protein kinases specific active-site

4-9; 16-21; 20-25; 107-112; 122-127; 164-169;

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;

PS00005|PKC_PHOSPHO_SITE (pattern) 582-584; 977-979; 1030-1032; 1073-1075; 1249- Protein kinase C phosphorylation site 1251; 1264-1266

209-212; 225-228; 287-290; 301-304; 346-349; 493-496; 506-509; 553-556; 597-600; 623-626;

PS00006|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

PS00007|TYR_PHOSPHO_SITE (pattern)

739-746

Tyrosine kinase phosphorylation site

PS00004|CAMPJ ^> HOSPHO_SITE (pattern) cAMP- and cGMP-dependent protein kinase 993-996; 1070-1073 phosphorylation site

PS00003|SULFATION (rule) Tyrosine sulfation

823-837 site

[0185] 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. Chenna et ah, Nucleic Acids Res., 31 (13):3497-500 (2003). 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.

TABLE 19 Summary of Sequence Alignment of FLT-4 Sequences from Multiple Organisms

KDR_mouse

KDR_rat

KDR_human

KDR_chicken

FLT4_rat_2

FLT4_rat

FLT4_mouse

FLT4_human

VEGFR3_chicken

FLTl_mouse

FLTl_rat

FIiTl_human

FLTl_chicken

VEGFR2_ctucken MSRFLPRLSLGVEGESTPREKEPGAFPLQSRALSPAAHWRFTEIRQHTHQCVTAAQELRF

KDR_fly

KDR_mouse

KDR_rat

KDR_human

KDR_chicken

FLT4_rat_2

FLT4_rat

FLT4_mouse

FLT4_human

VEGFR3_chxcken

FLTl_raouse

FLTl_rat

FLTl_human

FLTl_chicken

VEGFR2_chicken QEQHCLPALSVLQVPAWSIPSTHPTI FEKYVSIYRGQNVVYQNSAPLSKFSNDREGTGTG

KDR_fly

KDR_mouse MESKALLA

KDR _^ rat MESRALLA

KDR_human M _Q SKVLLA

KDR_chicken HELGPLRV

FLT4_rat_2 _' MQPGAALN

FLT4_rat MQPGAALH

FLT4_mouse M _Q PGAALN

FLT4_huraan MQRGAALC

VEGFR3_chicken MTVRSTVLMEKSξESNRYFEMFVHE

FLTl_mouse MVSCWDTA

FLTl_rat __. MVSCWDTA

FLTl_human MVSYWDTG

FLTl_chicken MPRQLLSG

VEGFR2_chicken SLQξHPVSLNCCLEASLSASGGKGDCKGDTKGTLARQPIAAQKEHQDRKAF

KDR_fly MTPLKTξVKAF

KDR_mouse —VALWFCVETRAAS VGLPGDFLHPPKLST

KDR_rat —VALWFCVETRAAS VGLPGDξLHPPKLξT

KDR_huπιan --VALWLCVETRAAS VGLPξVSLDLPRLSI

KDR_chicken —LTVLLCLPPVFTG LFIS MDQPTLSI

FLT4_rat_2 —LRLWLCLGLLQG LANGYSMTPPTLNI

FLT4_rat —RRLWLCLGLLQG LANGYSMTPPTLNI

FLT4_mouse —LRLWLCIJGLLQG LANGYSMTPPTLNI

FLT4_human —LRLWLCLGLLDG LVξGYSMTPPTLNI

VEGFR3_chicϊcen QLARCVSCTACRRY LVξξYSMTPPTLSI

FLTl_mouse —VLPYALLGCLLLT GYGSGSKLKVPELSL

FLTl_rat —VLPCALLGCLLLT GYCSGξKLKGPELSL

FLTI _n human —VLLCALLSCLLLT GSSSGξKLKDPELSL

FLTl_chicken —TVLLG—AAFLLA GSTSGSKLKVPVLSV

VEGFR2_chicken --ADTDVAIAALAEHTGKGRTPREEGRAVRRSGEWRERFNRSQQVGAVRASRξSRPAAR S

KDR_fly —FTLHVLFSCISH GLVEGSRLPDPQLLP

KDR_mouse QK DILTILANT

KDR_rat QK DILTILANT

KDR_human QK DILTIKANT

KDR_chicken QK SVLTITTND

FLT4_rat_2 TE DSYVIDTGD

FLT4_rat TE DSYVIDTGD

FLT4_mouse TE DSYVIDTGD

FLT4_huraan TE ESHVIDTGD

VEGFR3_chicfcen TE EEKVINAKD

FLTl_mouse KG TQHVMQAGQ

FLTl_rat KG TQHVMQAGQ

FLTl_human KG ^■ TQHIMQAGQ

FLTl_chicken NG RQHVVQAGQ

VEGFR2_chicken DGPKETSPRAAPAMLGGPSAALAACALQALLLTLGIAEDEFFEKPVLSIAKDHIVILPGE

KDR_fly DG DTHLQHVGE

KDR_mouse TLQITCR GQRD LDWLWPNAQ-

KDR_rat TLQITCR GQRD LDWLWPNTP-

KDR_human TLQITCR GQRD LDWLWPNNQ-

KDR_chicken TLNITCS GQRA VYWSWPNNQ-

FLT4_rat_2 SLSISCR- -GQHP- -LEWTWPGAQEVLTTGGK

FLT4_rat ELSISCR- -GQHP- -LEWTWRGAQEVLTTGGK

FLT4_mouse SLSISCR- -GQHP- -LEWTWPGAQEVLTTGGK

FLT4_human SLSISCR- -GQHP- -LEWAWPGAQEAPATGDK

VEGFR3_chicken TLTITCRTGDLCRξIPPDTDHARAGWKPQRCFDRGMWHSRVKGLVDTYSGRKEVFDPGA L

FLTl_mouse TLFLKCR GEAA HSWSLPTTV S

FLTl_rat TLFLKCR GEAA HSWSLPTTV S

FLTl_human TLHLQCR GEAA HKWSLPEMV S

FLTl_chicken TLNLTCR GEML HSWSLPEAL S

VEGFR2_ctαcken DLKIKCR GARP -VSWLLGEAQ R

KDR_fly TLTLICR GSTA LHWRL—AS R

KDR_mouse DS--EERVLVTECGG-GDξIFCKTLTIPRVVGNDTGAYKCSYR DVDIAξTVYVY

KDR_rat DS-EERVLVTECG DSIFCKTLTVPRVVGNDTGAYKCFYR DTDVSSIVYVY

KDR_human GS-EQRVEVTECξ DGLFCKTLTIPKVIGNDTGAYKCFYR ETDLASVIYVY

KDR_chicken NA-EKRLAVTGCS DGPFCKTLTLLRVIGNDTGDYRCLYG DSQAATTIYVY

FLT4_rat_2 DS—EDTQVVQDCEGTEARPYCKVLSLAQTHANNTGSYYCYYKYIKARIEGTTAASTYV F

FLT4_rat DS—EDTQVVQDCEGTEARPYCKVLSLAQTHANNTGSYYCYYKYIKARIEGTTAAξTY VF

FLT4_mouse DS"EDTRVVHDCEGTEARPYCKVLLLAQTHANNTGSYHCYYKYIKARIEGTTAASTYVF

FLT4_human DS-EDTGVVRDCEGTDARPYCKVLLLHEVHANDTGSYVCYYKYIKARIEGTTAASSYVF

VEGFR3_chicken LAFAEEMSAVTGMEEPEDQ-AVPCHMAPQWCLGTYHTCCSFLRVCDCWVPLCCPPTAYSR

FLTl_mouse QEDKRLSITPPSACGRDNRQFCSTLTLDTAQANHTGLYTCRYLPTSTSKKKKAESSIYIF

FLTl_rat QEDKKLSVTR-SACGRNNRQFCSTLTLNMAQANHTGLYSCRYLPKSTSKEKKMESAIYIF

FLTl_human KESERLξITK-SACGRHGKQFCSTLTLNTAQANHTGFYSCKYLAVPTSKKKETESAIYI F

FLTl_chiclten KDξKRLNVTK-YACGRNGTQFCSTLTLSRTQANDTGRYSCRYPTξPVKKKR—ESIV YVF

VEGFR2_chicken KDP-RVDISS CRNNSRQSCSTLLLRNVTASDTGYLTCVHDDAPS—REELTAKIYVF

KDR_fly NVS-SVRIES CEERLHKHCSKLVIHNLRHNDTGIYSCSHKKSSD—HEVST YVF

KDR_mouse VR- DYREPFIASVSDQHGIVYITENKNKTVVIPCRGSISNLNVSLCARYPE

KDR_rat VQ- DHRSPFIASVSDEHGIVYITENKNKTVVIPCRGSISNLNVSLCARYPE

KDR_human VQ- DYRSPFIASVSDQHGVVYITENKNKTVVIPCLGSISNLNVξLCARYPE

KDR_chicken VQ- DYRSPFVTSVGDQLGIVYIT—KNKTWVPCLGTVSNLNVSLHAKYPE

FLT4__rat_2 VR- DFEQPFINKP DTLLVNRKDSHWVPCLVSIPGLNITLRSQξS-

FLT4_rat VR- DFEQPFINKP DTLLVNRKDSMWVPCLVξIPGLNITLRSQSS-

FLT4_mouse VR- DFKHPFINKP DTLLVNRKDSMWVPCLVξIPGLNITLRξQSS-

FLT4_human VR- DFEQPFINKP DTLLVNRKDAMWVPCLVSIPGLNVTLRξQSξ-

VEGFR3_chicken IQQDKRVLSPGξNFEQPFINKF ETLLliKKENTWVPCLVSIPDLNVTLISQNS-

FLTl_mouse VS DAGSPFIEMHTDIPKLVHMTEGR—QLIIPCRVTSPNVTVTLK-KFPF

FLTl_rat VS DAGSPFIEMHSDIPKLVHMTEGR--ELIIPCRVTSPNITVTLK-KFPF

FLTl_human IS DTGRPFVEMYSEIPEIIHMTEGR—ELVIPCRVTSPNITVTLK-KFPL

FLTl_chicken IN DTSNPFVEKHSDIPKIIHMTVGK—EMIIPCRVTAPNIAVTLK-KIPR

VEGFR2_chicken VE DYRNPFVEFYPEHPQIIYLLEAKK-TVVIPCRVTξPDIRPQLM-QYPH

KDR_fly VK DPHHPFVEAY-SLPHPLFAYRNDP-YFWPCRTTYPNQNVILE-TQMN

KDR_mouse KRFVPDGNRISWDSEIGFTLPSYMISYAGMVFCEAKINDETYQξIMYIVVWGYRIYDVI

KDR_rat KRFVPDGNRISWDSEKGFTIPSYMISYAGMVFCEAKINDETYQSIMYIVLWGYRIYDVV

KDR_human KRFVPDGNRISWDSKKGFTIPSYMISYAGMVFCEAKINDESYQSIMYIVVVVGYRIYDW

KDR_chiCken KVFVPDGKSIξWDNKKGFTIPSHLISYAGMVFCEAKIDNESYQSVIYIVAVVGYRIYDL T

FLT4_rat_2 -VLHPDGQEVLWDDRRGMRVPTLLLRDALYLQCETTWGDQDFLSNPFLVHITGNELYDIQ

FLTA_rat -VLHPDGQEVLWDDRRGMRVPTLLLRDALYLQCETTWGDQDFLSNPFLVHITGNELYDIQ

FLT4_mouse -ALHPDGQEVLWDDRRGMRVPTQLLRDALYLQCETTWGDQNFLSNLFVVHITGNELYDIQ

FLT4_human -VLWPDGQEVVWDDRRGMLVSTPLLHDALYLQCETTWGDQDFLSNPFLVHITGNELYDIQ

VEGFR3_chicken -LIHPDRKAIFWDNKKGMQVPTQLIRDξLFVQCETVIDKKVFKSNFFIIHIAGIELYDI Q

FLTl_mouse DTLTPDGQRITWDSRRGFIlANATYKEIGLLNCEATVNGHLYQT-NYLTHRQTNTILDVQ

FLTl_rat DALTPDGQRIAWDξRRGFIIANATYKEIGLLTCEATVNGHLYQT-SYLTHRQTNTILDV Q

FLTl_human DTLIPDGKRIIWDSRKGFIIξNATYKEIGLLTCEATVNGKLYKT-NYLTHRQTNTIIDV Q

FLTl_chicken ETLIPDGKTIIWDNMRGFRIPEATYRFIGLLSCETTIGGHKYST-KYLTHRETNTIFDIK

VEGFR2_chicken REKLKFKK-MVWDPKRGFVI PξPAFTYξGVLTCTAGINGSVFTS-YYLVQKLEDRVQNLA

KDR_fly PMADDVKRGVQWDPKKGFTVPLKPYDSYHLITCLTRVDNAEFSξ-VYLLKRLTMEIKNL A

KDR_mouse LSPPHEIELSAGEKLVLNCTARTELNVGLDFTWHSPPξKSHHKKIVNRDVKPFPGTVAK M

KDR_rat LSPPHEIELSAGEKLVLNCTARTELKVGLDFSWQFPESKHQHKKIVNRDVKSLPGTVAKM

KDR_human LSPSHGIELξVGEKLVLNCTARTELNVGIDFNWEYPSSKHQHKKLVNRDLKTQSGSEMK K

KDR_chicken MNPHYQVELAVGEKLVLNCTVRTELNVGIDFKWDYPSIK—EKRATIRDLKTTAG-EIK T

FLT4_rat_2 LYPKKSLELLVGEKLVLNCTVWAEFDSGVTFDWDYPGKQAERAKWVP ERRSQQTHTE

FLT4_rat LYPKKSLELLVGEKLVLNCTVWAEFDSGVTFDWDYPGKQAERAKWVP ERRSQQTHTE

FLT4_mouse LYPKKSMELLVGEKLVLNCTVWAEFDSGVTFDWDYPGKQAERAKWVP ERRSQQTHTE

FLT4_human LLPRKSLELLVGEKLVLNCTVWAEFNSGVTFDWDYPGKQAERGKWVP ERRSQQTHTE

VEGFR3_chicken LYPKKAMELLVGEKLVLNCTVWAEFNSGVRFQWTYPGKQMQRAVIES ERRSLQTHTE

FLTl_mouse IRPPξPVRLLHGQTLVLNCTATTELNTRVQMξWNYPGKATKRASIRQRI --DRSHSHNNV

FLTl_rat ISPPSPVRFLRGQTLVLNCTVTTDLNTRVQMSWNYPGKATKRASIRQRI --DQξNPHSNV

FLTl_human 1STPRPVKLLRGHTLVLNCTATTPLNTRVQMTWξYPDEKNKRASVRRRI—DQSNSHA NI

FLTl_chicken LSTPRLVKLLKGDSLAINCTVKAAWNTRVQMTWTYPGEAMKRGSVTQRI—DQKNREAN V

VEGFR2_chicken LK-ATPKKLLVGETLYLECKAETFINGRIEFIWTCPNGKHPYPRRGMD RSKPVYK

KDR_fly IE-PERPRVLVGDTLILNCξAETTYNGRIHFEWEFHKERINRPHHFSTT--PVQLAQIM V

KDR_mouse FLξTLTIESVTKSDQGEYTCVAξSGRMIKRNRTFVRVHTKPFIAFGSGMKξLVEATV G-S

KDR_rat FLSTLTIDξVTKSDQGEYTCTAYSGLMTKKNKTFVRVHTKPFIAFGSGMKSLVEATVG- S

KDR_huπian FLξTLTIDGVTRSDQGLYTCAASSGLMTKKNSTFVRVHEKPFVAFGξGMESLVEATVG -E

KDR_chiCken FVSTLTIDSVNLξDKGRYTCAASSGRMNHKNξξYFIIHEEPFIHLEK-MENVVEHKL G-D

FLT4_rat_2 LSSILTIHNVSQHDLGPYVCEANNGIQRFRESTEVIVHEKPFIξVEWLKGPVLEATAGD E

FLT4_rat LSSILTIHNVξQHDLGPYVCEANNGIQQFREξTEVIVHEKPFIξVEWLKGPVLEATA GDE

FLT4_mouse LSSILTIHNVSQNDLGPYVCEANNGIQRFRESTEVIVHEKPFIξVEWLKGPVLEATAGD E

FLT4_human LSSILTIHNVSQHDLGSYVCKANNGIQRFRESTEVIVHENPFISVEWLKGPILEATAGDE

VEGFR3_chicken LSSILTLHNVSQQDLGRYTCTATNGAQMLEESTDVIVHEKPFINVEWRKGPVIEATAGDE

FLTl_mouse FHSVLKINNVESRDKGLYTCRVKSGSSFQEFNTSVHVYEKGFISVKHRKQPVQETTAGRR

FLTl_rat FHξVLKINNVESRDKGLYTCRVKξGξSFRTFNTSVHVYEKGFISVKHRKQQVQETIA GKR

FLTl_hιunan FYSVLTIDKMQNKDKGLYTCRVRSGPSFKSVNTSVHIYDKAFITVKHRKQQVLETVAGKR

FLTl_chιcken FYSILVIDKVRDIDKGQYACHVKSGPSNKLVNTTVIVYDKRFINLKRRRKTMLEAVAGRK

VEGFR2_Chxcken FGSSLTIENVTMEDGGLYICR-TFNLTSLEANVTVTVYDKPFLEVSHKKGLYYEITSGHK

KDR_fly MSKALIVPNVTMEDKGTYTCTGSIEFKKLQMSTKVIVYEHPFLNVTHNKRKFTSTVEGRR

KDR_mouse QVRIPVKYLξYPAPD-IKWYRNGRPIESNYTMIVG DELTIMEVTERDAGNYTVILT

KDR_rat QVRI PVKYLSYPAPD-IKWYRNGRPIESNYTMIVG DELTIMEVSERDAGNYTVILT

KDR_human RVRIPAKYLGYPPPE-IKWYKNGIPLESNHTIKAG HVLTIMEVξERDTGNYTVILT

KDR_chicken TVSI PVKFKGYPξPE-AKWYKNGKVMNANHTVKLG YTLVITDATEKDAGKYTVVLT

FLT4_rat_2 MVKLPVKLAAYPPPE-FQWYKDRKAVTGRHNPHA LVLKEVTEASAGVYTLALW

FLT4_rat HVKLPVKLAAYPPPE-FQWYKDRKAVTGRHNPHA LVLKEVTEASAGVYTLALW

FLT4_mθuse LVKLPVKLAAYPPPE-FQWYKDRKAVTGRHNPHA LVLKEVTEASAGVYTLALW

FLT4_human LVKLPVKLAAYPPPE-FQWYKDGKALSGRHSPHA LVLKEVTEASTGTYTLALW

VEGFR3_chicken AVKLPVKWAYPPPD-FQWYKDGKLIP-KQSQSS MQIKDVAEHHAGTYTLVLR

FLTl_mouse SYRLSMKVKAFPSPE-IVWLKDGSPATLKSARYLV—HGYSLIIKDVTTEDAGDYTILL G

FLTl_rat ξHRLSMKVKAFPξPE-WWLKDGVPATEKSARYξV--HGYSLIIKDVTAEDAGDYTIL LG

FLTl_human SYRLSMKVKAFPξPE-WWLKDGLPATEKSARYLT--RGYSLIIKDVTEEDAGNYTILL� �

FLTl_chicken SYRLPMKVKAFPSPE-VTWLKDGLPAAEKCARYMV--KNYSLIIKDVAEEDAGNYTIILS

VEGFR2_chicken SLKLAAKVDAFPRPT-VTWYKDGKLIKDNQSCYEPDPAKYRLGIRDVRPKHAGNYTIVLS

KDR_fly -VQFEPRVNAVPAPDRVLWYKDGVAISENSTCYET—AGYNLTIKQVRQKDAGIFTIAL S

* * * .. . . . .* .+. +

KDR mouse NPISKEKQSHHVSLWNVPPQIGEKALI --ξPMDSYQYGTKQTLTCTVYANPPLHHIQWY

KDR_rat NPISKEKQSHMVSLWNVPPQIGEKALI—SPMDSYQYGTHQTLTCTVYANPPLHHIQWY

KDR_human NPISKEKQSHVVSLVVYVPPQIGEKSLI—SPVDSYQYGTTQTLTCTVYAIPPPHHIHW Y

KDR_chicken NPTNKMQKRHTFTLLVNVPPQIGENALM—APVDSYKYGSTQTLTCTIYAVPPPAAVLW Y

FLT4_rat_2 NξAAGLRQNISLELVVNVPPHIHEKEAS—SPS-IYSRHSRQTLTCTTYGVPQPLSVQ WH

FLT4_rat NSAAGLRQNISLELVVNVPPHIHEKEAS—ξPξ-IYSRHSRQTLTCTTYGVPQPLSV QWH

FLT4_mouse NSAAGLRQNISLELVVNVPPHIHEKEAε--εPS-IYSRHSRQTLTCTAYGVPQPLSVQ WH

FLT4_huraan NξAAGLRRNISLELVVNVPPQIHEKEAS—ξPS-IYSRHξRQALTCTAYGVPLPL� �IQWH

VEGFR3_chicken NRLVGLEKHIξLQLIVNVPPRIHEKETS—SPS-IYSRRSPQALTCTVYGIPAPEVIQ WQ

FLTl_mouse IKQSRLFKNLTATLIVNVKPQIYEKSVξξLPSPPLYPLGSRQVLTCTVYGIPRPT-IT WL

FLTl_rat IKQSKLFRNLTATLIVNVKPQIYEKSVSSLPSPPLYPLGSRQVLTCTVYGIPQPT-IKWL

FLTl_human IKQ3NVFKNLTATLIVNVKPQIYEKAVξξFPDPALYPLGSRQILTCTAYGIPQPT-IK WF

FLTl_chicken LRQWNLξKNLTVTLKVNVKPQIYENAVSSFPDPNLYLLSξKQVLTCTVYGIPPPK-IT WM

VEGFR2_chicken NAKHGLYKNLTMQLWTERPKIYEKETD-FDKIERVELRSEHKIQCTVSGNPDPK-VEWK

KDR_fly NQERGLYRNISYKLEVRVKPKI FEEDVA-PAGPQTFRYDQRHKLTCTAFGIPMPN _' ITWF

KDR_mouse WQLEEACSYRPGQ TξPYACKEWRHVEDFQGGNKIEVTKNQYALIEG

KDR_rat WQLEEACξYRPξQ TNPYTCKEWRHVKDFQGGNKIEVTKNQYALIEG

KDR_human WQLEEECANEPSQAVS VTNPYPCEEWRSVEDFQGGNKIEVNKNQFALIEG

KDR_chicken WQLEEECTFξPQKVRL GANPYACRKWKVISERKGGNQVEIKQR-VVTIAG

FLT4_rat_2 WRPWTPCKTFAQRSLR RRQPRDGMPQCRDWKEVTTQDAVNPIESLDTWTESVEG

FLT4_rat WRPWTPCKTFAQRSLR RRQPRDGMPQCRDWKEVTTQDAVNPIESLDTWTESVEG

FLT4_mouse WRPWTPCKTFAQRSLR RRQQRDGMPQCRDWKEVTTQDAVNPIESLDSWTEFVEG

FLT4_human WRPWTPCKMFAQRξLR RRQQQDLMPQCRDWRAVTTQDAVNPIESLDTWTEFVEG

VEGFR3_chicken WRPWMPCRMFSRRSLNSRHRAARRHQRDRMPECKDWKDVSQQDAVNPIESIDTWVEFVEG

FLTl_raouse WHPCHHNHξKERYDFCT ENEESFILDPSξNLGNRIESISQRMTVIEG

FLTl_rat WHPCHYNHξKERNDFCF GSEESFILDSSSNIGNRIEGITQRMMVIEG

FLTl_human WHPCNHNHξEARCDFCS NNEESFILDADSNMGNRIESITQRMAI IEG

FLTl_chicken WYPCRQNHSKTRRGFCξ RTDGSFKLKTGSNIGNRIQSIIERTAI IEG

VEGFR2_chicken WQPCSLTD—TRCNPK GQKIVVQ-ENMFIGNKIKξIENITMKHGD

KDR_fly WQPCDPSANLTECKLY TDPLPIENVDDFPQNPIKDVHξKVGLLKS

KDR_mouse LVIQAANVSALYKCEAINKAGRGERVISFHVIRGPE ITVQPAAQPTEQESVSLLCTA

KDR_rat LVIQAAΫVSALYKCEAINKAGRGERVISFHVIRGPE ITVQPftTQPTERESMSLLCTA

KDR_human LVIQAAWVSALYKCEAVNKVGRGERVISFHVTRGPE ITLQPDMQPTEQESVSLWCTA

LVIQAAHVSALYRCHATNRAGSξERVISFHVTRGLE INLQPRξQLTEKDNTSLQCTA

LVIQDANVSAMYKCVVFNKVGQDERLIYFYVTTIPDGFSIESEPSEDPLSGQSVRLSCRA LVIQDANVSAMYKCWFNKVGQDERLIYFYVTTIPDGFSIESEPSEDPLEGQSVRLSCRA LVIQDANVSAMYKCVVVNKVGQDERLIYFYVTTIPDGFSIESEPSEDPLEGQξVRLSCR A LVIQKANVSAMYKCVVSNKVGQDERLIYFYVTTIPDGFTIESKPSEELLEGQPVLLSCQA LAIQEANVSAMYKCIASNKVGRDERLIYFYVTTIPDGFEIESQPSEEPIEGQDLQLξCN A LVVADSQTPGIYSCRAFNKIGTVERNIKFYVTDVPNG—FHVξLEKMPAEGEDLKLSC W LVVADSRTPGSYSCKAFHKIGTVERDIRFYVTDVPNG—FHVSLEKIPTEGEDLKLξC VV LVVADSRISGIYICIASNKVGTVGRNISFYITDVPNG—FHVNLEKMPTEGEDLKLSCT V LVVAEAKSSGIYSCVASNKVGKAERNVSFLVTDVPSG—FHISLEKVPIEGENLVLSCS A LTMENSSTSGIYYCVASNKVGEEERSIEFYVSDVPSG--LQTAPQVTAIAGSTAQLTCRA LWKTANVξGVYSCTARNELGNRTHRIPFYVDDKPQP—FEIEP-STAVAGDDITLTCR G

KDR_mouse DRNTFENLTWYKLGSQATSVHMGESLTPVCKNLDALWK LNGTMFξ-NξTNDILIVAF

KDR_rat DRNTFENLTWYKLGSQATSVHMGEξLTPVCKNLDALWK LNGTVFξ-NξTNDILIVAF

KDR_human DRξTFENLTWYKLGPQPLPIHVGELPTPVCKNLDTLWK LNATMFξ-NξTHDILIHEL

KDR _^ chicken DKFTFEKLξWYKLSAHALQTPFGGLPMPVCKNLDALQK LNATVSNTNGENVTLELIL

FLT4_rat_2 DNYTYEHLRWYRLNLSTLHDAQGNPLLLDCKNVHLFATPLEANLEEAEPGARHATLSLNI

FLT4_rat DNYTYEHLRWYRLNLSTLHDAQGNPLLLDCKNVHLFATPLEANLEEAEPGARHATLSLNI

FLT4_mσuse DNYTYEHLRWYRLNLξTLHDAQGNPLLLDCKNVHLFATPLEANLEEAEPGARHATLSLN I

FLT4_human DSYKYEHLRWYRLNLξTLHDAHGNPLLLDCKNVHLFATPLAAξLEEVAPGARHATLSL SI

VEGFR3_chicken DNYTYENLQWYRLKLξKLHDEEGNPLVLDCKNVHHYATKMQGELR-FQPDSNDATLLLT I

FLTl_mouse NKFLYRDITWILLRTVHNRTMHHS ISKQKMATTQDYξITLNLVI

FLTl_rat SKFLYRDITWILLRTVNNRTMHHS ISKQKMATTQDYSITLNLVI

FLTl_human NKFLYRDVTWILLRTVNNRTMHYS ISKQKMAITKEHSITLNLTI

FLTl_chicken NKFMYKDISWILPRTVTN QTKARKALNKEYSITLTLTI

VEGFR2_chiCken SRYIYSNLAWSYPSLGAAH GξSLTRSADAYSIξLTLII

KDR_fly TRYLYDRLTWYDPLGYKVP KDETTLRIEPYTIξLξIKL

KDR_mouse QNAξLQDQGDYVCSAQDKKTK-KRHCLVKQLIILERMAPMITGNLENQTTTIGETIEVT C

KDR_rat QNASLQDQGNYVCSAQDKKTK-KRHCLVKQLVILERMAPMITGNLENQTTTIGETIEVVC

KDR_human KNASLQDQGDYVCLAQDRKTK-KRHCVVRQLTVLERVAPTITGNLENQTTSIGESIEVξ C

KDR_chicken RNISLQDGGDYVCIAQDKKAK-TQHCLVKHLTVQEPMAPTLVGNLENQTTNIGETIEVξ C

FLT4_rat_2 PRVAPEDEGDYVCEVQDRRNQ-DKHCHKKYLSVQALEAPRLTQNLTDLLVNVSDξLEMR C

FLT4_rat PRVAPEDEGDYVCEVQDRRξQ-DKHCHKKYLSVQALEAPRLTQNLTDLLVNVRTξLEM RC

FLT4_mouse PRVAPEDEGDYVCEVQDRRSQ-DKHCHKKYLSVQALEAPRLTQNLTDLLVNVSDSLEMRC

FLT4_human PRVAPEHEGHYVCEVQDRRξH-DKHCHKKYLSVQALEAPRLTQNLTDLLVNVSDξLEM QC

VEGFR3_chicken PNISLGEEGDYVCEVQNRKTR-EKHCHKKYISVQALEIPRLKQNLTDIWVNVSDSIEMRC

FLTl_raouse KNVSLEDSGTYACRARNIYTG-EDILRKTEVLVRDSEAPHLLQNLSDYEVξIξGSTTL DC

FLTl_rat KNVSLEDξGTYACRARNIYTG-EEILRKTEVLVRDLEAPLLLQNLSDKEVSISGSTTLD C

FLTl_huraan MNVSLQDSGTYACRARNVYTG-EEILQKKEITIRDQEAPYLLRNLSDHTVAIξSSTTLD C

FLTl_chicken RNVSLAHSGTYTCRARNIFTG-KEVLQKKDVSIRAQEAPALLRQLMDQTVNTSNSAMLEC

VEGFR2_chicken ANVTEEHSGLYKCRAQHQHNGξDTLEQHTRLLIRAKAAPYVLQNLTDLEVNISGKI ILEC

KDR_fly PNVξRNHTLGYECQALNINTN-KVVNVTSALTIDERQGPWLMQNLTNQDVNSSSTLTLA C

KDR_mouse PASGNPTPHITWFKDKETLVEDξGIVLRDGNRNLTIRRVRKEDGGLYTCQACRVLGCAR A

KDR_rat FTSGNPTPLITWFKDNETLVEDSGIVLKDGNRNLTIRRVRKEDGGLYTCQACNVLGCARA

KDR_huπιan TAξGNPPPQIMWFKDNETLVEDSGIVLKDGNRNLTIRRVRKEDEGLYTCQACSVLGCAK V

KDR_chicken TVNGIPPPNITWFKNGETLFEDξGIVLKDGNKTLTIRRVRKEDGGLYTCLACNILGCKK A

FLT4_rat_2 PVAGAHVPSIVWYKDERLLEKESGIDLADSNQRLSIQRVREEDAGRYLCSVCNAKGCVNS

FLT4_rat PVAGAHVPSIVWYKDERLLEKESGIDLADSNQRLξIQRVREEDAGRYLCSVCNAKGCVN S

FLT4_mouse PVAGAHVPSIVWYKDERLLEKESGIDLADξNQRLSIQRVREEDAGRYLCSVCNAKGCVN S

FLT4_human LVAGAHAPSIVWYKDERLLEEKSGVDLADξNQKLξIQRVREEDAGRYLCSVCNAKGCV NS

VEGFR3_chicken KVDGNHVPDIξWYKDEKLVEEVSGIDLADFNQRLSIQRVREEDAGLYLCξVCNAKGCV NS

FLTl_mouse QARGVPAPQITWFKNNHKIQQEPGIILGPGNSTLFIERVTEEDEGVYRCRATNQKGAVES

FLTl_rat QARGVPAPQITWFKNNHKIQQEPGIILGPGNSTLFIERVTEEDEGVYRCRATNQKGVVES

FLTl_human HANGVPEPQITWFKNNHKIQQEPGI ILGPGSξTLFIERVTEEDEGVYHCKATNQKGSVES

FLTl_chicken QVHGIPEPQITWFKNHEEIQQESGI ILGPGSRMLFIERVKEEDEGLYQCIATNLKGSVEξ

VEGFR2_chicken RVSGTPEPQVTWSKDGYPISAASGISMENN—TLVIERVKKDDEGLYECKATNDMGQDS T

KDR_fly LAYGVPAPFITWYKDKTPVTEGPGITLKDDG-TLI IERVKKDDEGIYECRAξNDGGEAKT

KDR_raouse ETLFIIEGAQEKTNLEVIILVGTAVIAMFFWLLLVIVLRTVKRAREGELKT-GYLSIVMD

KDR_rat ETLFIIEGVQEKTNLEVIILVGTAVIAMFFWLLLVILVRTVKRANξGELKT-GYLSIVM D

KDR_human EAFFIIEGAQEKTNLEIIILVGTAVIAMFFWLLLVIILRTVKRANGGELKT-GYLSIVMD

KDR_chicken EAYFSVEGAEEKTNLELIILVGTAVIAMFFWLLLVIILRTVKRANGGDMKT-GYLSIIMD

FLT4_rat_2 SASVAVEGSEDKGSMEIVILIGTGVIAVFFWVLLLLIFCNMKRPAHADIKT-GYLξIIM D

FLT4_rat SASVAVEGSEDKGSMEIVILIGTGVIAVFFWVLLLLIFCNMKRPAHADIKT-GYLξIIM D

FLT4_mouse SASVAVEGSEDKGSMEIVILIGTGVIAVFFWVLLLLIFCNMKRPAHADIKT-GYLSIIMD

FLT4_human SASVAVEGξEDKGξMEIVILVGTGVIAVFFWVLLLLIFCNMRRPAHADIKT-GYLξI IMD

VEGFR3_chicken SASVSVEGSDDKTNVEIVILIGTGVIAVFFWILLILIFCNIKRPAHADIKT-GYLSIIMD

FLTl_mouse AAYLTVQGTSDKSNLELITLTCTCVAATLFWLLLTLFIRKLKR-SξξEVKT-DYLSII MD

FLTl_rat SAYLTVQGTξDKSNLELITLTCTCVAATLFWLLLTLFIRKLKR-ξξSEVKT-DYLSI IMD

FLTl_human SAYLTVQGTSDKξNLELITLTCTCVAATLFWLLLTLLIRKMKR-ξSSEIKT-DYLSII MD

FLTl_chicken TAYVTVQGTVERSNLELITLTCTCVAATLFWLLLTLFIRKLKRPYFSETKTNHYLSIIMD

VEGFR2_chicken SAFIKIQGSEEKSNIEVI ILVCTGLAATLFWLLLTLFIRKLRKPDATDIKT-GYLSIIMD

KDR_fly SAVITVVGEDGKPNIEVI ILVSTGAAATFLWIMLILFIRKLRKPξSADLKT-GYLξIIMD

+***.**

KDR_mouse PDELPLDERCERLPYDASKWEFPRDRLKLGKPLGRGAFGQVIEADAFGIDKTATCKTVAV

KDR_rat PDELPLDERCERLPYDASKWEFPRDRLKLGKPLGRGAFGQVIEADAFGIDKTATCKTVAV

KDR__human PDELPLDEHCERLPYDASKWEFPRDRLKLGKPLGRGAFGQVIEADAFGIDKTATCRTVAV

KDR_chicken PDEVPIDEHCERLPYDASKWEFPRDRLKLGKPLGRGAFGQVIEADAFGIDKTATCRTVAV

FLT4_rat_2 PGEVPLEEQCEYLSYDVSQWEFPRERLHLGRVLGHGAFGKVVEASAFGINKGSSCDTVAV

FLT4_rat PGEVPLEEQCEYLSYDVSQWEFPRERLHLGRVLGHGAFGKVVEASAFGINKGξSCDTVA V

FLT4_mouse PGEVPLEEQCEYLSYDASQWEFPRERLHLGRVLGHGAFGKVVEASAFGINKGSξCDTVA V

FLT4_human PGEVPLEEQCEYLξYDASQWEFPRERLHLGRVLGYGAFGKVVEASAFGIHKGSξCDTV AV

VEGFR3_Chicken PGEVPLEEQCEYLPYDξSKWEFPRDRLRLGKVLGHGAFGKVVEASAFGINKSNξCETV AV

FLTl_mouse PDEVPLDEQCERLPYDASKWEFARERLKLGKSLGRGAFGKVVQASAFGIKKSPTCRTVAV

FLTl_rat PDEVPLDEQCERLPYDAξKWEFARERLKLGKSLGRGAFGKVVQASAFGIKKξPTCRTV AV

FLTl_human PDEVPLDEQCERLPYDASKWEFARERLKLGKξLGRGAFGKWQASAFGIKKSPTCRTVAV

FLTl_chicken PDEVPLDEQCECLPYDASKWEIARERLKLGKSLGHGAFGKVVQASAFGIKKSPTCRIVAV

VEGFR2_chicken PEEMPLDEQCDRLPYDSSKWEFPRDRLRLGKTLGHGAFGKVVEASAFGIDKSSTCKTVAV

KDR_fly PEQMPLDEQCDRLPYDSNKWEFPQDRLRLGKTLGHGAγGKVVEASAFGIDKIξTCKTV AV

KDR_mouse KMLKEGATHSEHRALMSELKILIHIGHHLNVVNLLGACTKPGGPLMVIVEFCKFGNLξT Y

KDR_rat KMLKEGATHSEHRALMSELKILIHIGHHLNVVNLLGACTKPGGPLMVIVEFCKFGNLξT Y

KDR_human KMLKEGATHSEHRALMSELKILIHIGHHLNVVNLLGACTKPGGPLMVIVEFCKFGNLSTY

KDR_chicken KMLKEGATHSEHRALMSELKILIHIGHHLNVVNLLGACTKPGGPLMVIVEYCKFGNLξA Y

FLT4_rat_2 KMLKEGATASEHRALMSELKILIHIGNHLNVVNLLGACTKPNGPLMVIVEFCKYGNLξN F

FLT4_rat KMLKEGATAξEHRALMSELKILIHIGNHLNVVNLLGACTKPNGPLMVIVEFCKYGNLSN F

FLT4_raouse KMLKEGATASEHRALMSELKILIHIGNHLNVVNLLGACTKPNGPLMVIVEFCKYGNLξN F

FLT4_human KMLKEGATAξEHRALMSELKILIHIGNHLNVVNLLGACTKPQGPLMVIVEFCKYGNLSN F

VEGFR3_chicken KMLKEGATASEHKALMSELKILIHIGNHLNWNLLGACTKPNGPLMVIVEFCKYGNLξNY

FLTl_raouse KMLKEGATASSYKALMTELKILTHIGHHLNVVNLLGACTKQGGPLMVIVEYCKYGNLSNY

FLTl_rat KMLKEGATASEYKALMTELKILTHIGHHLNVVNLLGACTKQGGPLMVIVEYCKYGNLSNY

FLTl_human KMLKEGATAξEYKALMTELKILTHIGHHLNVVNLLGACTKQGGPLMVIVEYCKYGNLSN Y

FLTl_chicken KMLKEGATASEYKALMTELKILIHIGHHLNIVNLLGACTKNGGPLMVIVEYCKYGNLSNY

VEGFR2_chicken KMLKECATTNECKALMSELKILIHIGHHLNVVNLLGACTKAGGPLMVIVEYCKYGNLSNY

KDR_fly KMLKVGATNNEWRALMSELKILIHIGHHLNVVNLLGACTKRGGPLMIIVEFCKYGNLSNY

KDR_mouse LRGKRNEFVPYKξKGARFRQGKD YVGELSVDLKRRLDξITSSQSSAξSGFVEEKSLS

KDR_rat LRGKRNEFVPYKSKGARFRSGKD YVGELSVDLKRRLDSITSSQSSASSGFVEEKξLS

KDR_huraan LRSKRNEFVPYKTKGARFRQGKD YVGAIPVDLKRRLDξITξξQξSASSGFVEEKSLS

KDR _^ chicken LRSKRSEFVPYKTKSARFRQGKEN—YIGDISTDLKQRLDSITSSQSSTξSGFVEERS LS

FLT4_rat_2 LRVKRETFDPYAEKSPEQRRRFRA—MVEGAKADRR RLGSTDRALFTRFLMGKGSA

FLT4_rat LRVKRETFDPYAEKSPEQRRRFRA—MVEGAKADRR RLGSTDRALFTRFLMGKGSA

FLT4_mouse LRVKRDTFNPYAEKSPEQRRRFRA—MVEGAKADRR RPGSSDRALFTRFLMGKGSA

FLT4 _m human LRAKRDAFSPCAEKSPEQRGRFRA—MVELARLDRR RPGSSDRVLFARFSKTEGGA

VEGFR3_chicken LRTKREGFSPYREKSPRLRIQVQS—IVEAVRADRRξ RSGTSDSAIFNRFLMHKS—

FLTl_mouse LKSKRDLFCLNKDAALHMELKKES—LEPGLEQGQKPRLDSVξSS-SVTSSSFPEDRS VS

FLTl_rat LKSKRDFFCLNKDAALHMEPKKEK—LEPDLEQDQKPRLDSVSSSESFTSSGFQEDKξ VS

FLTl_human LKSKRDLFFLNKDAALHMEPKKEK—MEPGLEQGKKPRLDSVTSSESFASSGFQEDKξ LS

FLTl_chicken LKSKRNFFSPTKDPSLQGELMKDK—KGIEPVEGKKQRLASVTSSESFASSGFQEDKSL ξ

VEGFR2_chicken LRGKRGDFIAYKSQENSGQAEKSLDESSSDLTELIKRRLESVASTGSSASSGFIEDKSYS

KDR_fly LRξKRGDFWYKSQD—GKAVRSξ—SGCDLSELIKRRLESVASTGSSASSGFIEDK ξYC

KDR_raouse DVEEEEAS-EELYKDFLTLEHLICYSFQVAKGMEFLASRKCIHRDLAARNILLSEKNVVK

KDR_rat DVEEEEAS-EELYKDFLTLEHLICYSFQVAKGMEFIASRKCIHRDLAARNILLSEKNVVK

KDR_human DVEEEEAP-EDLYKDFLTLEHLICYξFQVAKGMEFLAξRKCIHRDLAARNILLSEKNW K

KDR_chicken DVEEEDAGSEDLCKNPLTMEDLICYSFQVARGMEFLAξRKCIHRDLAARNILLSDNHVV K

FLT4_rat_2 RRAPFVQEAEDLWLSPLTMEDLVCYSFQVALGMEFLASRKCIHRDLAARNILLSEξDIV K

FLT4_rat RRAPFVQEAEDLWLSPLTMEDLVCYSFQVARGMEFLAξRKCIHRDLAARNILLSESDIV K

FLT4_mouse RRAPLVQEAEDLWLξPLTMEDLVCYξFQVARGMEFLASRKCIHRDLAARNILLξESD IVK

FLT4_hwnan RRAξPDQEAEDLWLSPLTMEDLVCYξFQVARGMEFLASRKCIHRDLAARNILLSESDV VK

VEGFR3_chicken QTAQPIQEVDDLWQξPLTMEDLICYSFQVARGMEFLASRKCIHRDLAARNILLξENNV VK

FLTl_mouse DVEGDE-DYSEISKQPLTMEDLIξYSFQVARGMEFLξSRKCIHRDLAARNILLξENN VVK

FLTl_rat DVEGGE-DYξEISKQPLTMEDLISYSFQVARGMEFLSSRKCIHRDLAARNILLξENNV VK

FLTl_human DVEEEE-DSDGFYKEPITMEDLISYξFQVARGMEFLSSRKCIHRDIAARNILLξENNV VK

FLTl_chicken DAEEDEEDAAELYKLPLTMEDLISYSFQVARGMEFLξSRKCIHRDLAARNILLSENNWK

VEGFR2_chicken DSEEDEEDAEDLYKRPLTLEDLICYξFQVAKGMEFLASRKCIHRDLAARNILLξENNW K

KDR_fly DSEEEEEEQEDLYKKVLTLEDLICYSFQVAKGMEFLAξRKCIHRDLAARNILLSENNWK

KDR_mouse ICDFGLARDIYKDPDYVRKGDARLPLKWMAPETIFDRVYTIQSDVWSFGVLLWEIFSLGA

KDR_rat ICDFGLARDIYKDPDYVRKGDPRLPLKWMAPETIFDRIYTIQSGVWSFGVLLWEIFSLGA

KDR_human ICDFGLARDIYKDPDYVRKGDARLPLKWHAPETIFDRVYTIQSDVWSFGVLLWEIFSLGA

KDR_chicken ICDFGLARDIYKDPDYVRKGDARLPLKWMAPETIFDRVYTIQSDVWξFGVLLWEIFSLG A

FLT4_rat_2 ICDFGLARDIYKDPDYVRKGSARLPLKWMAPESIFDKVYTTQSDVWSFGVLLWEIFSLGA

FLT4_rat ICDFGLARDIYKDPDYVRKGSARLPLKWMAPESIFDKVYTTQSDVWξFGVLLWEIFSLG A

FLT4_mouse ICDFGLARDIYKDPDYVRKGSARLPLKWMAPESIFDKVYTTQSDVWSFGVLLWEIFSLGA

FLT4 human ICDFGLARDIYKDPDYVRKGSARLPLKWMAPESIFDKVYTTQξDVWξFGVLLWEIFSL GA

VEGFR3_chicken ICDFGLARDIYKDPDYVRKGSARLPLKWMAPESIFDKVYTTQSDVWξFGVLLWEIFSLG A

FLTl_mouse ICDFGLARDIYKNPDYVRRGDTRLPLKWMAPESIFDKVYSTKξDVWSYGVLLWEIFSLG G

FLTl_rat ICDFGLARDIYKNPDYVRRGDTRLPLKWMAPESIFDKVYξTKξDVWξYGVLLWEIFS LGG

FLTl_human ICDFGLARDIYKNPDYVRKGDTRLPLKWMAPESIFDKIYSTKSDVWSYGVLLWEIFSLGG

FLTl_chicken ICDFGLARDIYKNPDYVRKGDARLPLKWMAPESIFDKIYNTKSDVWSYGVLLWEIFSLGA

VEGFR2_chicken ICDFGLARDIYKDPDYVRKGDARLPLKWMAPEAIFDKIYTTQSDVWSFGVLLWEIFξLG A

KDR_fly ICDFGLARDVYKDPDYVRKGDARLPLKWMAPEAIFDKIYTTQSDVWSFGVLMWEIFSLGA

KDR _^ mouse SPYPGVKIDEEFCRRLKEGTRMRAPDYTTPEMYQTMLDCWHEDPNQRPSFSELVEHLGNL

KDR_rat SPYPGVKIDEKFCRRLKEGTRMRAPDYTTPEMYQTMLDCWHEDPNQRPAFSELVEHLGNL

KDR_human ξPYPGVKIDEEFCRRLKEGTRMRAPDYTTPEMYQTMLDCWHGEPSQRPTFSELVEHLGN L

KDR_chicken SPYPGVKIDEEFCRRLKEGTRMRAPDYTTPEMYQTMLDCWHGDPKQRPTFξELVEHLGN L

FLT4_rat_2 ξPYPGVQINEEFCQRLKDGTRMRAPELATPAIRHIMQSCWSGDPKARPAFSDLVEILGD L

FLT4_rat SPYPGVQINEEFCQRLKDGTRMRAPELATPAIRKIMQSCWSGDPKARPAFSDLVEILGDL

FLT4_mouse ξPYPGVQINEEFCQRLKDGTRMRAPELATPAIRHIMQξCWξGDPKARPAFξDLVEI LGDL

FLT4_human SPYPGVQINEEFCCRLRDGTRMRAPELATPAIRRIMLNCWSGDPKARPAFSELVEILGDL

VEGFR3_chicken SPYPGVQINEEFCQRLKDGTRMRAPEYATAEIYRIMLSCWHGDPKERPTFSDLVEILGNL

FLTl_mouse SPYPGVQMDEDFCSRLKEGMRMRTPEYATPEIYQIMLDCWHKDPKERPRFAELVEKLGDL

FLTl_rat ξPYPGVQMDEDFCξRLKEGMRMRTPEYATPEIYQIMLDCWHKDPKERPRFAELVEKLG DL

FLTl_human SPYPGVQMDEDFCξRLREGMRMRAPEYSTPEIYQIMLDCWHRDPKERPRFAELVEKLGD L

FLTl_chicken SPYPGVQIDEDFCξKLKEGTRMRAPEQATEEIYQIMLDCWRξNPNERPWFSELVKRLG DL

VEGFR2_chicken SPYPGVQIDEDFCRRLKEGTRMRξPEYSTPEVYQTMLDCWHGVPTERPTFTELVERLGD L

KDR_fly SPYPGLHIDEEFCCRLKEGTRMKAPEYSSSEIYQTMLDCWHGEPSQRPTFTELVERLGDL

KDR_mouse LQAN-AQQDGKDYIVLPMξETLSMEEDSGLSLPTξPVSCMEEEEVCDP KFHY

KDR_rat LQAN-AQQDGKDYIVLPMξETLSMEEDξGLξLPTSPVSCMEEEEVCDP KFHY

KDR_human LQAN-AQQDGKDYIVLPISETLSMEEDSGLξLPTξPVSCMEEEEVCDP KFKY

KDR_chicken LQAN-VRQDGKDYVVLPLξVSLNMEEDSGLSLPTξPASCKEEEEVCDP KFHY

FLT4_rat_2 LQGGGWQEEEEECMAL—HSSQSSEEDGFMQASTTALHITEADAESSPPξMHCHSLAA RY

FLT4 rat LQGGGWQEEEEECMAL—HSSQξξEEDGFMQASTTALHITEADAESSPPSMHCHξL AARY

FLT4_mouse LQGGGWQEEEEERMAL—HSξQSSEEDGFMQASTTALHITEADADDSPPSMHCHSLAA RY

FLT4_hωnan LQGRGLQEEEEVCMAP—RSξQξξEEGSFSQVSTMALHIAQADAEDSPPSLQRHξ LAARY

VEGFR3_chicken LQEN-VQQEGKDYIPL—NDSHSSEDDGFSQVPξξAQ—QNξDEEDFDMRIRCHS LAARY

FLTl_jnouse LQAN-VQQDGKDYIPL—NAILTRNξGFTYSTPTFSEDLFKDGFADPHFH

FLTl_rat LQAN-VQQDGKDYIPL—NAILTRNSGFTYξVPTFξEDFFKDGFTDPKFH

FLTl__huraan LQAN-VQQDGKDYIPI—NAILTGNSGFTYSTPAFSEDFFKξSISAPKFN

FLTl_chicken LQAS-VQQEGKDYIPL—DTIFTAESGF PPAξDPLCKEKFPVPSPN

VEGFR2_chicken LQAN-VQQDGKDYIPL--NITLCPDGEPKSKTCPVEENLNKCVNRWSAVE

KDR_fly LQAS-VQQEGKHYI PI--NTALLTKADP-SNQSPTEETξTRPVS-LRDSG

KDR_mouse DNTAGISHYLQNSKRKξRPVSVKTFEDIPLEEPEVKVIPDDξQTDSGMVLASEELKTL ED

KDR_rat DNTAGISHYLQNξKRKξRPVSVKTFEDIPLEEPEVKVIPDDξQTDSGMVLASEELKT LED

KDR_human DNTAGISQYLQNξKRKSRPVSVKTFEDIPLEEPEVKVIPDDNQTDSGMVLASEELKTLE D

KDR_chicken DNTAGIξQYRQGSKRKSRPVSVKTFEDIPLVTT-VKWQEENQTDξGMVLASEELKTLE E

FLT4_rat_2 YNCVSFPGRLVRGTKAPGSSRMKTFEELPMTPTTYKASVD-NQTDSGMVLASEEFEQIES

FLT4_rat YNCVξFPGRLVRGTKAPGSξRMKTFEELPHTPTTYKAξVD-NQTDSGMVLASEEFEQ IES

FLT4_mouse YNCVSFPGRLARGTKTPGSSRMKTFEELPMTPTTYKASMD-NQTDξGMVLASEEFEELE S

FLT4_human YNWVSFPGCLARGAETRGSSRMKTFEEFPMTPTTYKGξVD-NQTDSGMVLASEEFEQIE ξ

VEGFR3_Chicken YNCVSFPGCLTGGNQIRCξSRIKTFEEFPMTHTMYKftHPD-NQTDSGMVLAξEEFER IEN

FLTl_πiouse -ξGξSDDVRYVHAFKFMSLERIKTFEELξPNS TξMFEDYQLDTSTLLGSPLLKRFTW

FLTl_rat -SGSSDDVRYVNAFKFMξLERIKTFEELSPNA TSMFEDYHLDTSξLLTSPLLKRFTW

FLTl_huπιan -SGSSDDVRYVNAFKFMSLERIKTFEELLPNA TξMFDDYQGDSSTLLAξPMLKRFTW

FLTl_chicken -CRSTERARYINTFKIKPPQRIKTFEELPIKE KLVFNDYQADSGMVLASEELKRFTW

VEGFR2_chiCken -TGSGSKRR PLSVKTFDEVSVGK-EKVMHE--EξDξGMVLTSDEIKSPKR

KDR fly -TAWNIKIR PESVKTFDEVILENG-TNKIHEGGQξDSGIGLSSDDLKTLKR

KDR_mouse RNK LξPξFGGMMPSKSRESVASEGSNQTξGYQSGYHξDDTDTTVYSSDEAGLLKM

KDR_rat RNK LSPξFGGMMPSKSRESVASEGξNQTSGYQSGYHSDDTDTTVYSSDEAGLLKL

KDR_human RTK LSPSFGGMVPSKSRESVASEGSKQTSGYQSGYHSDDTDTTVYSSEEAELLKL

KDR_chicken RDKQ VKIPFSTLAPSKξNESVMSEASNQTSGYQSGYHSDDMDTMVCSSEDTELLCA

FLT4_rat_2 RHR QEGSFSRKDPGQHMD-ISRGHPDLQGRRRRPTQGAQGGKVFYNNEYGEVSQP

FLT4_rat RHR QEGSFξRKDPGQHMD-ISRGHPDLQGRRRRPTQGAQGGKVFYNNEYGEVSQP

FLT4_mouse RHR PEGSFSCKGPGQHMD-1PRGHPDPQGRRRRPTQGAQGGKVFYNNEYGEVSQP

FLT4_human RHR QESGFR

VEGFR3_chicken RHR KξGGFSSKGPNRTAE-LSAEQξDLRGRCRPξYGSQVGGQTFYNSEYGELξEH

FLTl_mouse TETK—PKAξMKIDLRIASKSKEAGLξDLPRP-SFCFξSCGHIR-PVQ DDES—ELG

FLTl_rat TETK--PKASMKIDLRITSKSKEAGLSDLPGP-SFCFSSCGHIR-PVRQEDEDDP—EL G

FLTl_human TDSK—PKASLKIDLRVTSKSKESGLSDVSRP-SFCHSSCGHVSEGKRRFTYDHA--EL E

FLTl_chicken TGSK—QKWTL-FGMKGVSRSKESGLSGITKPRSFCSFSCDQLSESKRRYTYGNT--VL E

VEGFR2_chicken LEIRξWPYGIMALARRAVNKSKESILSDHEREAVKYQPAVQVEEDTMDFPLEDSVLLPM D

KDR_fly LESLARPRSFMSRAMK—RKSKESVL—LEGEMDKYPPLVP ξLξLEDS SLD

KDR_mouse VDAAVHA DSGTTLRSPPV SEQ ID NO:19

KDR_rat VDVAGHV DξGTTLRSSFV SEQ ID NO:20

KDR_Jiuman IEIGVQTGSTAQILQPDSGTTLSSPPV SEQ ID NO: 21

KDR_chicken QEASPTLPRVHG-LVHDξPAPLVSPPL SEQ ID NO:22

FLT4_rat_2 CTEGDCCP SAGSTFFADξNY SEQ ID NO: 23

FLT4_rat CTEGDCCP SAGSTFFADSHY SEQ ID NO: 24

FLT4_mouse CTEGDCCP SAGSTFFADSξY SEQ ID NO: 25

FI/T4 human SEQ ID NO:26

VEGFR3_chicken SEDGξCTPPGEGASPTALHTSFFSEQY SEQ ID NO:27

FLTl_mouse -KEξCCSPPPD YNSVVLYξSPPA SEQ ID KO:28

FLTl_rat -KESCCSPPPD YNSVVLYSSPPA SEQ ID NO:29

FLTl_huraan RKIACCSPPPD YNξVVLYSTPPI SEQ ID NO: 30

FLTl_chicken KMKACHSPPPD YSSVVHYSQPSI SEQ ID NO:31

VEGFR2_chiCken PξLECHSPPPD YNSVVQCSAPPV SEQ ID NO:32

KDR_fly ξEMECHSPPPD YNYWRYSTPPV SEQ ID NO:33

[0187] A summary of the sequence alignment of FLT-4 sequences from multiple organisms is shown in TABLE 20 below.

TABLE 20 Summary of Sequence Alignment of FLT-4 Sequences from Multiple Organisms

Mutation Comment N149D Conserved in FLT4 and KDR M963V Conserved in FLT4

D970E Conserved in FLT4 and KDR

P1114S Conserved in FLT4, FLTl and KDR

[0188] Analysis of the Potential Effect ofFLT-4 mutations on FLT-4 Protein Secondary Structure. The Effect of FLT-4 Mutation on Amino Acid Property. The change of amino acid property observed by FLT-4 mutation is summarized in TABLE 21 (Valdar WS, Proteins 48(2): 227-41 (2002)).

TABLE 21 Influence of FLT-4 Mutations on FLT-4 Amino Acid Property

Mutation Property change

N149D None predicted.

M963V None predicted.

D970E None predicted.

P1114S None predicted.

[0189] 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.

[0190] All TABLES {e.g., TABLE 23; TABLE 25; TABLE 27; TABLE 29; and TABLE 31) in this section that summarize the FLT-4 protein secondary structure as predicted by nnPredict use "H", "E" and a dash "-" as identifiers, which are defined as follows. A helix element is designated by the letter "H". A strand element is designated by the letter "E". No prediction is designated by a dash ("-")• Grey shading represents polypeptide regions where mutation was identified.

[0191] The amino acid sequence of wild-type FLT-4 polypeptide (SEQ ID NO:26) is shown below in TABLE 22. The position of the mutated amino acid residues is highlighted in bold, underlined text.

TABLE 22

MQRGAALCLRLWLCLGLLDGLVSGYSMTPPTLNITEESHVIDTGDSLSISCRGQHPL EWA WPGAQEAPATGDKDξEDTGWRDCEGTDARPYCKVLLLHEVHANDTGSYVCYYKYIKARI EGTTAASSYVFVRDFEQPFINKPDTLLVNRKDAMWVPCLVSIPGLNVTLRSQSSVLWPDG QEVVWDDRRGMLVSTPLLHDALYLQCETTWGDQDFLSNPFLVHITGNELYDIQLLPRKSL ELLVGEKLVLNCTVWAEFNSGVTFDWDYPGKQAERGKWVPERRSQQTHTELSSILTIHNV SQHDLGSYVCKANNGIQRFRESTEVIVHENPFISVEWLKGPILEATAGDELVKLPVKLAA YPPPEFQWYKDGKALSGRHSPHALVLKEVTEASTGTYTLALWNSAAGLRRNISLELWNV PPQIHEKEASSPξIYSRHSRQALTCTAYGVPLPLSIQWHWRPWTPCKMFAQRSLRRRQQ Q DLMPQCRDWRAVTTQDAVNPIESLDTWTEFVEGKNKTVSKLVIQNANVSAMYKCVVSNKV GQDERLIYFYVTTIPDGFTIESKPSEELLEGQPVLLSCQADSYKYEHLRWYRLNLSTLHD AHGNPLLLDCKNVHLFATPLAASLEEVAPGARHATLSLSIPRVAPEHEGHYVCEVQDRRS HDKHCHKKYLSVQALEAPRLTQNLTDLLVNVSDSLEMQCLVAGAHAPSIVWYKDERLLEE KSGVDLADSNQKLSIQRVREEDAGRYLCSVCNAKGCVNSSASVAVEGSEDKGSMEIVILV GTGVIAVFFWVLLLLIFCNMRRPAHADIKTGYLSIIMDPGEVPLEEQCEYLSYDASQWEF PRERLHLGRVLGYGAFGKVVEASAFGIHKGSSCDTVAVKMLKEGATASEHRALMSELKIL IHIGNHLNVVNLLGACTKPQGPLMVIVEFCKYGNLSNFLRAKRDAFSPCAEKSPEQRGRF RAMVELARLDRRRPGSSDRVLFARFSKTEGGARRASPDQEAEDLWLSPLTMEDLVCYSFQ VARGMEFLASRKCIHRDLAARNILLSESDVVKICDFGLARDIYKDPDYVRKGSARLPLKW MAPESIFDKVYTTQSDVWSFGVLLWEIFSLGASPYPGVQINEEFCQRLRDGTRMRAPELA TPAIRRIMLNCWSGDPKARPAFSELVEILGDLLQGRGLQEEEEVCMAPRSSQSSEEGSFS QVSTMALHIAQADAEDSPPSLQRHξLAARYYNWVSFPGCLARGAETRGSSRMKTFEEFP M TPTTYKGSVDNQTDSGMVLASEEFEQIESRHRQESGFR

SEQ ID NO: 26

[0192] A schematic representation of the secondary structure of wild-type FLT-4 polypeptide (SEQ ID NO:26) predicted using nnPredict analysis is shown below in TABLE 23. The position of the mutated amino acid residues are identified with grey shaded text.

TABLE 23 HHHHHHHHHHH E EEE EEE £ _EE HHHHH _H H _HH EEEEEH—E _EEE

E— ^' EEEEE EH@ EEE

—EE EE HH-HHHHE EEEE HHHH—H—H

HHHHH-HHEEEH-EEHE E E—EEEEEE— EEH EEE EEEE EEE EEH HHHHHHHH EHHHHHHH EEEHHHHHHH-H-H EEE _{EEE EEE E} _ _{E HHH} HHHHH HEHEH-H HHHEEE HHEEEEEE EE HHH EEE HHHHHHHHHH-HH-H— HH HH-HHHHHHHHH-HH-HHH EEE H—HHH-H—HHHH—HHHEE HHHHHHH EEEH HHHH- HH EE HEEE E EEE EEEEEEE EEEEHHHHHHHHHHH EEEE HHHHHH

—HHHH EE EHEHHHHHHE HHHHHHHHH HHHHHHHHHHHHHE

EHH HHHEH EEEEHH HHHHH

HH|HHHHH-| HEHEE —HHHHH HHHHHHHH

—HHHHHHH--HH HHHHHH EEEH H H _EE EEH-HEEEHHH g HHHHHH

-HHHHHHEE HHHHHHHHHHH HHHHH ^' -—

EHHHHHHHHH HH-HHHHHEE _EEEH — _HHH H _HH

[0193] The amino acid sequence of FLT-4 mutant polypeptide Nl 49D (SEQ ID NO:34) is shown below in TABLE 24. The position of the mutated amino acid residue is highlighted in bold underlined text.

TABLE 24

MQRGAALCLRLWLCLGLLDGLVSGYSMTPPTLNITEESHVIDTGDSLSISCRGQHPL EWA WPGAQEAPATGDKDSEDTGVVRDCEGTDARPYCKVLLLHEVHANDTGSYVCYYKYIKARI EGTTAASSYVFVRDFEQPFINKPDTLLVDRKDAMWVPCLVSIPGLNVTLRSQSSVLWPDG QEVVWDDRRGMLVSTPLLHDALYLQCETTWGDQDFLSNPFLVHITGNELYDIQLLPRKSL ELLVGEKLVLNCTVWAEFNSGVTFDWDYPGKQAERGKWVPERRSQQTHTELSSILTIHNV SQHDLGSYVCKANNGIQRFRESTEVIVHENPFISVEWLKGPILEATAGDELVKLPVKLAA YPPPEFQWYKDGKALSGRHSPHALVLKEVTEASTGTYTLALWNSAAGLRRNISLELVVNV PPQIHEKEASSPSIYSRHSRQALTCTAYGVPLPLSIQWHWRPWTPCKMFAQRSLRRRQQQ DLMPQCRDWRAVTTQDAVNPIESLDTWTEFVEGKNKTVSKLVIQNANVSAMYKCVVSNKV GQDERLIYFYVTTIPDGFTIESKPSEELLEGQPVLLSCQADSYKYEHLRWYRLNLSTLKD AHGNPLLLDCKNVHLFATPLAASLEEVAPGARHATLSLSIPRVAPEHEGHYVCEVQDRRS HDKHCHKKYLSVQALEAPRLTQNLTDLLVNVSDSLEMQCLVAGAHAPSIVWYKDERLLEE KSGVDLADξNQKLSIQRVREEDAGRYLCSVCNAKGCVNSSASVAVEGSEDKGSMEIVIL V GTGVIAVFFWVLLLLIFCNMRRPAHADIKTGYLSIIMDPGEVPLEEQCEYLSYDASQWEF PRERLHLGRVLGYGAFGKVVEASAFGIHKGSSCDTVAVKMLKEGATASEHRALMSELKIL IHIGNHLNVVNLLGACTKPQGPLMVIVEFCKYGNLSNFLRAKRDAFSPCAEKSPEQRGRF RAMVELARLDRRRPGSSDRVLFARFSKTEGGARRASPDQEAEDLWLSPLTMEDLVCYSFQ VARGMEFLASRKCIHRDLAARNILLSESDVVKICDFGLARDIYKDPDYVRKGSARLPLKW MAPESIFDKVYTTQSDVWSFGVLLWEIFSLGASPYPGVQINEEFCQRLRDGTRMRAPELA TPAIRRIMLNCWSGDPKARPAFSELVEILGDLLQGRGLQEEEEVCMAPRSSQSSEEGSFS QVSTMALHIAQADAEDSPPSLQRHSLAARYYNWVSFPGCLARGAETRGSSRMKTFEEFPM TPTTYKGSVDNQTDSGMVLASEEFEQIESRHRQESGFR

SEQ ID NO: 34

[0194] A schematic representation of the secondary structure of FLT-4 mutant polypeptide N149D (SEQ ID NO:34) predicted using nnPredict analysis is shown below in TABLE 25. The position of the mutated amino acid residue is identified by grey shaded text.

TABLE 25 HHHHHHHHHHH E EEE EEE _EEE HHHHHHHHH EEEEEH--EEEE

E EEEEE EH| EEE

—EE EE HH-HHHHE EEEE HHHH—H—H

HHHHH-HHEEEH-EEHE E E—EEEEEE— EEH EEE EEEE EEE EEH HHHHHHHH EHHHHHHH EEEHHHHHHH-H-H EEE _{EEE EEE E} _ _{E HHH} HHHHH HEHEH-H HHHEES HHEEEEEE EE HHH EEE HHHHHHHHHH-HH-H— HH HH-HHHHHHHHH-HH-HHH EEE H—HHH-H-HHHH—HHHEE HHHHHHH EEEH HHHH- HH EE HEEE E EEE EEEEEEE EEEEHHHHHHHHHHH EEEE HHHHHH

—HHHH EE EHEHHHHHHE HHHHHHHHH HHHHHHHHHHHHHE

EHH HHHEH EEEEHH HHHHH

HHHHHHHH HEHEE HHHHH HHHHHHHH

--HHHHHHH--HH HHHHHH EEEH H H _EE EEH-HEEEHHH HHHHHH

-HHHHHHEE HHHHHHHHHHH HHHHH

EHHHHHHHHH HH-HHHHHEE _EEEH —HHHHHH

[0195] The amino acid sequence of FLT-4 mutant polypeptide M963 V (SEQ ID NO:35) is shown below in TABLE 26. The position of the mutated amino acid residue is highlighted in bold underlined text.

TABLE 26

MQRGAALCLRLWLCLGLLDGLVSGYSMTPPTLNITEESHVIDTGDSLSISCRGQHPL EWA WPGAQEAPATGDKDSEDTGVVRDCEGTDARPYCKVLLLHEVHANDTGSYVCYYKYIKARI EGTTAASSYVFVRDFEQPFINKPDTLLVNRKDAMWVPCLVSIPGLNVTLRSQSSVLWPDG QEVVWDDRRGMLVSTPLLHDALYLQCETTWGDQDFLSNPFLVHITGNELYDIQLLPRKSL ELLVGEKLVLNCTVWAEFNSGVTFDWDYPGKQAERGKWVPERRSQQTHTELSSILTIHNV SQHDLGSYVCKANNGIQRFRESTEVIVHENPFISVEWLKGPILEATAGDELVKLPVKLAA YPPPEFQWYKDGKALSGRHSPHALVLKEVTEASTGTYTLALWNSAAGLRRNIξLELVVN V PPQIHEKEASSPSIYSRHSRQALTCTAYGVPLPLSIQWHWRPWTPCKMFAQRSLRRRQQQ DLMPQCRDWRAVTTQDAVNPIESLDTWTEFVEGKNKTVSKLVIQNANVSAMYKCVVSNKV GQDERLIYFYVTTIPDGFTIESKPSEELLEGQPVLLSCQADSYKYEHLRWYRLNLSTLHD AHGNPLLLDCKNVHLFATPLAASLEEVAPGARHATLSLSIPRVAPEHEGHYVCEVQDRRS HDKHCHKKYLSVQALEAPRLTQNLTDLLVNVSDSLEMQCLVAGAHAPSIVWYKDERLLEE KSGVDLADSNQKLSIQRVREEDAGRYLCSVCNAKGCVNSSASVAVEGSEDKGSMEIVILV GTGVIAVFFWVLLLLIFCNMRRPAHADIKTGYLSIIMDPGEVPLEEQCEYLSYDASQWEF PRERLHLGRVLGYGAFGKVVEASAFGIHKGSSCDTVAVKMLKEGATASEHRALMSELKIL IHIGNHLNWNLLGACTKPQGPLMVIVEFCKYGNLSNFLRAKRDAFSPCAEKSPEQRGRF RAVVELARLDRRRPGSSDRVLFARFSKTEGGARRASPDQEAEDLWLSPLTMEDLVCYSFQ VARGMEFLASRKCIHRDLAARNILLSESDVVKICDFGLARDIYKDPDYVRKGSARLPLKW MAPESIFDKVYTTQSDVWSFGVLLWEIFSLGASPYPGVQINEEFCQRLRDGTRMRAPELA TPAIRRIMLNCWSGDPKARPAFSELVEILGDLLQGRGLQEEEEVCMAPRSSQSSEEGSFS QVSTMALHIAQADAEDSPPSLQRHSLAARYYNWVSFPGCLARGAETRGSSRMKTFEEFPM TPTTYKGSVDNQTDSGMVLASEEFEQIESRHRQESGFR

SEQ ID NO: 35

[0196] A schematic representation of the secondary structure of FLT-4 mutant polypeptide M963V (SEQ ID NO:35) predicted using nnPredict analysis is shown below in TABLE 27. The position of the mutated amino acid residue is identified by grey shaded text.

TABLE 27 HHHHHHHHHHH E EEE EEE _EEE HHHHHHHHH EEEEEH-- _E E _EE

E EEEEE EH EEE

—EE EE HH-HHHHE EEEE HHHH—H—H

HHHHH-HHEEEH-EEHE E E—EEEEEE— EEH EEE EEEE EEE EEH HHHHHHHH EHHHHHHH EEEHHHHHHH-H-H EEE _{EEE EEE E} _ _{E HHH} HHHHH H _EHEH - _{H HHHEEE} HHEEEEEE EE HHH EEE HHHHHHHHHH-HH-H— HH HH-HHHHHHHHH-HH-HHH EEE H—HHH-H—HHHH-HHHEE HHHHHHH EEEH HHHH- HH EE HEEE E EEE EEEEEEE EEEEHKHHHHHHHHH EEEE HHHHHH

--HHHH EE EHEHHHHHHE HHHHHHHHH HHHHHHHHHHHHHE

EHH HHHEH EEEEHH HHHHH

EH|HHHHH HEHEE HHHHH HHHHHHHH

--HHHHHHH—HH HHHHHH EEEH H H _EE EEH-HEEEHHH HHHHHH

-HHHHHHEE HHHHHHHHHHH HHHHH

EHHHHHHHHH HH-HHHHHEE _EEEH — _HHHHHH

[0197] The amino acid sequence of FLT-4 mutant polypeptide D970E (SEQ ID NO:36) is shown below in TABLE 28. The position of the mutated amino acid residue is highlighted in bold underlined text.

TABLE 28

MQRGAALCLRLWLCLGLLDGLVSGYSMTPPTLNITEEξHVIDTGDSLSISCRGQHP LEWA WPGAQEAPATGDKDSEDTGVVRDCEGTDARPYCKVLLI-HEVHANDTGSYVCYYKYIKAR I EGTTAASSYVFVRDFEQPFINKPDTLLVNRKDAMWVPCLVSIPGLNVTLRSQSSVLWPDG QEVVWDDRRGMLVSTPLLHDALYLQCETTWGDQDFLSNPFLVHITGNELYDIQLLPRKSL ELLVGEKLVLNCTVWAEFNSGVTFDWDYPGKQAERGKWVPERRSQQTHTELSSILTIHNV SQHDLGSYVCKANNGIQRFRESTEVIVHENPFISVEWLKGPILEATAGDELVKLPVKLAA YPPPEFQWYKDGKALSGRHSPHALVLKEVTEASTGTYTLALWNSAAGLRRNISLELVVNV PPQIHEKEASSPSIYSRHSRQALTCTAYGVPLPLSIQWHWRPWTPCKMFAQRSLRRRQQQ DLMPQCRDWRAVTTQDAVNPIESLDTWTEFVEGKNKTVSKLVIQNANVSAMYKCWSNKV GQDERLIYFYVTTIPDGFTIESKPSEELLEGQPVLLSCQADSYKYEHLRWYRLNLSTLHD AHGNPLLLDCKNVHLFATPLAASLEEVAPGARHATLSLSIPRVAPEHEGHYVCEVQDRRS HDKHCHKKYLSVQALEAPRLTQNLTDLLVNVSDSLEMQCLVAGAHAPSIVWYKDERLLEE KSGVDLADSNQKLSIQRVREEDAGRYLCSVCNAKGCVNSSASVAVEGSEDKGSMEIVILV GTGVIAVFFWVLLLLIFCNMRRPAHADIKTGYLSIIMDPGEVPLEEQCEYLSYDASQWEF PRERLHLGRVLGYGAFGKVVEASAFGIHKGSSCDTVAVKMLKEGATASEHRALMSELKIL IHIGNHLNVVNLLGACTKPQGPLMVIVEFCKYGNLSNFLRAKRDAFSPCAEKSPEQRGRF RAMVELARLERRRPGSSDRVLFARFSKTEGGARRASPDQEAEDLWLSPLTMEDLVCYSFQ VARGMEFLAS-RKCIHRDLAARNILLSESDVVKICDFGLARDIYKDPDYVRKGSARLPLK W MAPESIFDKVYTTQSDVWSFGVLLWEIFSLGASPYPGVQINEEFCQRLRDGTRMRAPELA TPAIRRIMLNCWSGDPKARPAFSELVEILGDLLQGRGLQEEEEVCMAPRSSQSSEEGSFS QVSTMALHIAQADAEDSPPSLQRHSLAARYYNWVSFPGCLARGAETRGSSRMKTFEEFPM TPTTYKGξVDNQTDSGMVLASEEFEQIESRHRQESGFR

SEQ ID NO:36

[0198] A schematic representation of the secondary structure of FLT-4 mutant polypeptide D970E (SEQ ID NO:36) predicted using nnPredict analysis is shown below in TABLE 29. The position of the mutated amino acid residue is identified by grey shaded text.

TABLE 29 HHHHHHHHHHH E EEE EEE _EEE HHHHHHHHH EEEEEH—EEEE

E EEEEE EH EEE

-EE EE HH-HHHHE EEEE HHHH--H—H

HHHHH-HHEEEH-EEHE E E—EEEEEE— _{EEH EEE EE} EE _E EE gen HHHHHHHH EHHHHHHH EEEHHHHHHH-H-H EEE _{EEE EEE E} _ _{E HHH} HHHHH HEHEH-H HHHEEE HHEEEEEE EE HHH EEE HHHHHHHHHH-HH-H— HH HH-HHHHHHHHH-HH-HHH EEE H—HHH-H—HHHH—HHHEE HHHHHHH EEEH HHHH- HH EE HEEE E EEE EEEEEEE EEEEHHHHHHHHHHH EEEE HHHHHH

—HHHH EE EHEHHHHHHE HHHHHHHHH HHHHHHHHHHHHHE

EHH HHHEH EEEEHH HHHHH

HHHHHHHHH§ HEHEE HHHHH HHHHHHHH

—HHHHHHH--HH HHHHHH EEEH H H _EE EEH-HEEEHHH HHHHHH

-HHHHHHEE HHHHHHHHHHH HHHHH

EHHHHHHHHH HH-HHHHHEE _EEEH —HHHHHH

[0199] The amino acid sequence of FLT-4 mutant polypeptide Pl 114S (SEQ ID NO:37) is shown below in TABLE 30. The position of the mutated amino acid residue is highlighted in bold underlined text.

TABLE 30

MQRGAALCLRLWLCLGLLDGLVSGYSMTPPTLNITEESHVIDTGDSLSISCRGQHPL EWA WPGAQEAPATGDKDSEDTGWRDCEGTDARPYCKVLLLHEVHANDTGSYVCYYKYIKARI EGTTAASSYVFVRDFEQPFINKPDTLLVNRKDAMWVPCLVSIPGLNVTLRSQSSVLWPDG QEVVWDDRRGMLVSTPLLHDALYLQCETTWGDQDFLSNPFLVHITGNELYDIQLLPRKSL ELLVGEKLVLNCTVWAEFNSGVTFDWDYPGKQAERGKWVPERRSQQTHTELSSILTIHNV SQKDLGSYVCKANNGIQRFRESTEVIVHENPFISVEWLKGPILEATAGDELVKLPVKLAA YPPPEFQWYKDGKALSGRHSPHALVLKEVTEASTGTYTLALWNSAAGLRRNISLELVVNV PPQIHEKEASSPSIYSRHSRQALTCTAYGVPLPLSIQWHWRPWTPCKMFAQRSLRRRQQQ DLMPQCRDWRAVTTQDAVNPIESLDTWTEFVEGKNKTVSKLVIQNANVSAMYKCWSNKV GQDERLIYFYVTTIPDGFTIESKPSEELLEGQPVLLSCQADSYKYEHLRWYRLNLSTLHD AHGNPLLLDCKNVHLFATPLAASLEEVAPGARHATLSLSIPRVAPEHEGHYVCEVQDRRS HDKHCHKKYLSVQALEAPRLTQNLTDLLVNVSDSLEMQCLVAGAHAPSIVWYKDERLLEE KSGVDLADSNQKLSIQRVREEDAGRYLCSVCNAKGCVNSSASVAVEGSEDKGSMEIVILV GTGVIAVFFWVLLLLIFCNMRRPAHADIKTGYLSIIMDPGEVPLEEQCEYLSYDASQWEF PRERLHLGRVLGYGAFGKVVEASAFGIHKGSSCDTVAVKMLKEGATASEHRALMSELKIL IHIGNHLNVVNLLGACTKPQGPLMVIVEFCKYGNLSNFLRAKRDAFSPCAEKSPEQRGRF RAMVELARLDRRRPGSSDRVLFARFSKTEGGARRASPDQEAEDLWLSPLTMEDLVCYSFQ VARGMEFLASRKCIHRDLAARNILLSESDVVKICDFGLARDIYKDPDYVRKGSARLPLKW MAPESIFDKVYTTQSDVWSFGVLLWEIFSLGAξSYPGVQINEEFCQRLRDGTRMRAPEL A TPAIRRIMLNCWSGDPKARPAFSELVEILGDLLQGRGLQEEEEVCMAPRSSQSSEEGSFS QVSTMALHIAQADAEDSPPSLQRHSLAARYYNWVSFPGCLARGAETRGSSRMKTFEEFPM TPTTYKGSVDNQTDSGMVLASEEFEQIESRHRQESGFR

SEQ ID NO: 37

[0200] A schematic representation of the secondary structure of FLT-4 mutant polypeptide Pl 114S (SEQ ID NO:37) predicted using nnPredict analysis is shown below in TABLE 31. The position of the mutated amino acid residue is identified by grey shaded text.

TABLE 31 HHHHHHHHHHH E EEE EEE _EEE HKHHHHHHH EEEEEH—EEEE

E EEEEE EH EEE

—EE EE HH-HHHHE EEEE HHHH--H--H

HHHHH-HHEEEH-EEHE E E—EEEEEE— EEH EEE EEEE EEE EEH HHHHHHHH EHHHHHHH EEEHHHHHHH-H-H EEE _{EEE EEE E} _ _E HHH HHHHH HEHEH-H HHHEEE HHEEEEEE EE HHH EEE HHHHHHHHHH-HH-H— HH HH-HHHHHHHHH-HH-HHH EEE H—HHH-H-HHHH—HHHEE HHHHHHH EEEH HHHH- HH EE HEEE E EEE EEEEEEE EEEEHHHHHHHHHHH EEEE HHHHHH

—HHHH EE EHEHHHHHHE HHHHHHHHH HHHHHHHHHHHHHE

EHH HHHEH EEEEHH HHHHH

HHHHHHHH HEHEE HHHHH HHHHHHHH

—HHHHHHH—HH HHHHHH EEEH H H EE EEH-HEEEHHHH β E-HHHHHH

-HHHHHHEE HHHHHHHHHHH HHHHH

EHHHHHHHHH HH-HHHHHEE _EEEH — _HHHHHH

[0201] The influence of FLT-4 mutations on FLT-4 protein secondary structure is summarized below in TABLE 32.

TABLE 32 Influence of FLT-4 Mutations on FLT-4 Protein Secondary Structure

Mutation Predicted Protein Secondary Structure Change

N149D No change

M963V Affects 1 amino acid

D970E Extend upstream helix by 2 amino acids

P1114S Affects structure

[0202] General Overview Analysis. A summary of the results of computational analysis of the effect of the FLT-4 mutations and SNPs identified in the present invention on select features of wild-type FLT-4 is provided below in TABLE 33.

TABLE 33

Evaluation of FLT-4 Mutations by Sequence Features

Mutation Protein Phospho- Other AA AA Secondary domain rvlation modification conservation property Structure change

N149D -H-

M963V -H- + +

D970E -H- -H- ++

P1114S ++ + + +++ +

+: the effect of mutation on protein function is low ++: the effect of mutation on protein function is medium the effect of mutation on protein function is high

[0203] Four 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.

EXAMPLE II

ANALYSIS OF FLT-4 MUTATION FOR THERANOSTIC CANCER TREATMENT IN A

SUBJECT

[0204] In this invention, an agent that modulates FLT-4 biological activity (i.e., FLT-4 modulating agent, e.g., FLT-4 antagonist) is administered to a patient with cancer, e.g., colon cancer, when the patient has a single nucleotide polymorphism (SNP) pattern indicative of an

FLT-4 mutation that correlates with the disease. In one embodiment, the SNP is selected from the group consisting of the FLT-4 mutation summarized in TABLE 1 and TABLE 2.

[0205] .

EQUIVALENTS

[0206] The present invention is not to be limited in terms of the particular embodiments described in this application, which are intended as single illustrations of individual aspects of the invention. Many modifications and variations of this invention can be made without departing from its spirit and scope, as will be apparent to those skilled in the art. Functionally equivalent methods and apparatuses within the scope of the invention, in addition to those enumerated herein, will be apparent to those skilled in the art from the foregoing descriptions. Such modifications and variations are intended to fall within the scope of the appended claims. The present invention is to be limited only by the terms of the appended claims, along with the full scope of equivalents to which such claims are entitled.