METHODS OF DIAGNOSING MYELODYSPLASTIC SYNDROME (MDS) OR LEUKEMIA USING NUCLEIC ACIDS OR FRAGMENTS ENCODING FLT3 KINASE

Abstract

encoding a receptor protein kinase, wherein the nucleic acid has tandem duplication in a nucleotide sequence of a juxtamembrane and is useful for diagnosis of leukemia; a polypeptide encoded by the nucleic acid; an antibody capable of specifically binding to a region encoded by the nucleic acid having tandem duplication occurring in a nucleotide sequence of a juxtamembrane; a nucleic acid capable of specifically binding to the nucleic acid having tandem duplication occurring in a nucleotide sequence of a juxtamembrane; a method for detection of the nucleic acid encoding a receptor protein kinase; and a kit therefor. A nucleic acid encoding a receptor protein kinase, wherein the nucleic acid has tandem duplication in a nucleotide sequence of a juxtamembrane; a polypeptide encoded by the nucleic acid; an antibody capable of specifically binding to the portion of the polypeptide; a nucleic acid capable of specifically binding to the nucleic acid; a method for detection of the nucleic acid; and a kit for detection.

Description

[0001]This application is a Continuation of co-pending application Ser. No. 11/892,241, filed on Aug. 21, 2007. Application Ser. No. 11/892,241 is a Division of application Ser. No. 10/653,147, filed on Sep. 3, 2003, now abandoned, which is a division of application Ser. No. 09/942,711 filed on Aug. 31, 2001, which issued as U.S. Pat. No. 6,846,630 on Jan. 25, 2005, which is a division of application Ser. No. 09/284,654 filed on Apr. 16, 1999, now abandoned. Application Ser. No. 09/284,654 is the national phase of PCT International Application No. PCT/JP97/03667 filed on Oct. 13, 1997. This application also claims priority of Application No. 8-297329 filed in Japan on Oct. 18, 1996 under 35 U.S.C. §119. The entire contents of each of the above-identified applications are hereby incorporated by reference.

TECHNICAL FIELD

[0002]The present invention concerns a nucleic acid encoding a receptor protein kinase, which has tandem duplication in a nucleotide sequence of a juxtamembrane, a polypeptide, a method for detection of the above nucleic acid and a kit for detection.

BACKGROUND ART

[0003]Proliferation and differentiation of cells, and responses of cells to various stimuli are strictly regulated by various growth factors. These growth factors are known to act via receptors which are specific to the above growth factors (Nicola, N. A., Annu. Rev. Biochem. 58, 45, 1989; Lowenberg, B., Blood 81, 281, 1991). Of those receptors, the receptors containing a tyrosine kinase domain are classified as receptor tyrosine kinases (RTKs).

[0004]RTKs comprise an entracellular region, a transmembrane region, as well as an intracellular region containing a tyrosine kinase domain and a juxtamembrane between the transmembrane region and the tyrosine kinase domain, and further roughly classified into four types according to structural characteristics and amino acid sequence homology.

[0005]Type I receptors have a monomeric structure, with two cysteine-rich repeat sequences in their extracellular region, and are exemplified by the EGF receptor, HER2/neu and the like.

[0006]Type II receptors have a structure comprising two subunits each for α and β, which are bound via S--S bond, wherein the a chain is an extracellular region containing one cysteine-rich repeat sequence, and wherein the β chain has a transmembrane region, a juxtamembrane, and a tyrosine kinase domain. Examples are an insulin receptor and an IGF-1 receptor.

[0007]Type III receptors have a monomeric structure containing five immunoglobulin-like cysteine-rich sequences in their extracellular region and two tyrosine kinase domains interrupted by a kinase insert in their intracellular region. Examples are PDGF receptor, fms (CSF-1 receptor), kit (SLF receptor) and the like.

[0008]Type IV receptors resemble type III receptors but have three immunoglobulin-like repeat sequences, and are exemplified by FGF receptor.

[0009]fms-Like tyrosine kinase 3 (hereinafter abbreviated as FLT3; Matthews, W., Cell 55, 1143, 1991; Rosnet, O., Genomics 9, 380, 1991), which is expressed in leukemic cells etc., also referred to as fetal liver kinase 2 (FLK2) or STK-1, is known to as type III receptors (Small, D., Proc. Natl. Acad. Sci. USA 91, 459, 1993; Lyman, S. D., Oncogene 8, 815, 1993; Rosnet, O., Blood 82, 1110, 1993; Agnes, F., Gene 145, 283, 1994)

[0010]In these receptor tyrosine kinases, aggregation, such as dimerization, takes place upon binding of a ligand, such as a growth factor, to the extracellular region, thereby resulting in the activation of kinase. Although in these tyrosine kinases, their ligands have been first found and then their receptors in most cases, there are receptors of which ligands remain unknown.

[0011]Regarding FLT3, which has been remarked in proliferation mechanism of hematopoietic stem cells and leukemia, after finding the FLT3, the FLT3 ligand has been found (Lyman, S. D., Cell 75, 1157, 1993; Hannum, C., Nature 363, 643, 1994). Since the FLT3 ligand is expressed in almost all leukemic cells, it is assumed that cells are proliferated by a mechanism of autocrine stimulation in leukemia (Meierhoff, G., Leukemia 9, 1368, 1995). Also, FLT3 mRNA has been reported to be expressed in lymphatic leukemic cells and myelocytic leukemic cells (Birg, F. Blood 80, 2584, 1994; Da Silva, N., Leukemia 8, 885, 1994; Brasel, K., Leukemia 9, 1212, 1995; Drexler, H. G., Leukemia 10, 588, 1996). However, there remains unknown how the FLT3 mRNA expression is associated with the pathology of lymphatic leukemia and myelocytic leukemia.

[0012]A human FLT3 cDNA has been cloned, and a cDNA nucleotide sequence and the amino acid sequence of the FLT3 protein have been determined [O. Rosnet et al., Blood, 82(4), 1110-1119 (1993)]. The present situation, however, is that the structure and function of FLT during the hematopoietic stem cell differentiation and the malignant alterations to leukemic cells have not been analyzed well.

DISCLOSURE OF INVENTION

[0013]Accordingly, a first object of the present invention is to provide a nucleic acid encoding a receptor protein kinase, wherein the nucleic acid has tandem duplication in a nucleotide sequence of a juxtamembrane and is useful for diagnosis of leukemia, and to provide a nucleic acid encoding the above juxtamembrane. A second object of the present invention is to provide a polypeptide which is encoded by the above nucleic acid. A third object of the present invention is to provide an antibody capable of specifically binding to a portion encoded by a nucleic acid having tandem duplication occurring in a nucleotide sequence of a juxtamembrane. A fourth object of the present invention is to provide a nucleic acid capable of specifically binding to a nucleic acid having tandem duplication occurring in a nucleotide sequence of a juxtamembrane. A fifth object of the present invention is to provide a method for detection of the nucleic acid encoding a receptor protein kinase and a kit therefor.

[0014]Conventionally, as to the FLT3, the same receptor protein kinase is expressed, irrespective of kinds of cells and differentiation [O. Rosnet et al., Blood, 82(4), 1110-1119 (1993)]. As a result of the detailed investigation and intensive studies of the FLT3 expression in leukemic cells, however, the present inventors surprisingly have found a receptor protein kinase gene having novel tandem duplication in a juxtamembrane, and found that the above tandem duplication is somatic, and that the expression of FLT3 having the above tandem duplication is associated with leukemia malignancy and mal-consequence of prognosis, and the present invention has been completed thereby.

[0015]Accordingly, the gist of the present invention is as follows:

[1] a nucleic acid encoding a receptor protein kinase, wherein the nucleic acid has tandem duplication in a nucleotide sequence of a juxtamembrane;[2] the nucleic acid according to item [1] above, wherein the receptor protein kinase is a receptor tyrosine kinase;[3] the nucleic acid according to item [2] above, wherein the receptor tyrosine kinase is FMS-like tyrosine kinase 3 (FLT3);[4] the nucleic acid according to any one of items [1] to [3] above, wherein the nucleic acid comprises a nucleotide sequence encoding an amino acid sequence as shown by any one of SEQ ID NOs: 1 to 5 in Sequence Listing in a juxtamembrane;[5] the nucleic acid according to any one of items [1] to [3] above, wherein the nucleic acid comprises a nucleotide sequence as shown by any one of SEQ ID NOs: 6 to 15 in Sequence Listing in a juxtamembrane;[6] a nucleic acid encoding a tandem duplication mutant of FLT3 as shown by any one of SEQ ID NOs: 16 to 20 in Sequence Listing;[7] a nucleic acid comprising a nucleotide sequence encoding a tandem duplication mutant as shown by any one of SEQ ID NOs: 21 to 25 in Sequence Listing, or a nucleic acid capable of hybridizing thereto under stringent conditions, wherein the nucleic acid has tandem duplication in a nucleotide sequence encoding a juxtamembrane;[8] a nucleic acid having tandem duplication, wherein the nucleic acid encodes an amino acid sequence as shown by any one of SEQ ID NOs: 1 to 5 in Sequence Listing;[9] a nucleic acid as shown by any one of SEQ ID NOs: 6 to 15 in Sequence Listing, or a nucleic acid capable of hybridizing thereto under stringent conditions, wherein the nucleic acid has tandem duplication;[10] a polypeptide encoded by the nucleic acid according to any one of items [1] to [9] above;[11] a polypeptide comprising an amino acid sequence as shown by any one of SEQ ID NOs: 1 to 5, and 16 to 20 in Sequence Listing;[12] a polypeptide encoded by a nucleic acid having tandem duplication in a nucleotide sequence of a juxtamembrane, wherein the polypeptide results from at least one of deletion, substitution or addition of one or more amino acid residues in an amino acid sequence as shown by any one of SEQ ID NOs: 1 to 5, and 16 to 20;[13] an antibody capable of specifically binding to a region encoded by a nucleic acid having tandem duplication occurring in a nucleotide sequence of a juxtamembrane of a receptor protein kinase;[14] a nucleic acid capable of specifically binding to a nucleic acid having tandem duplication occurring in a nucleotide sequence of a juxtamembrane of a receptor protein kinase;[15] a method for detection of a nucleic acid encoding receptor protein kinase and having tandem duplication occurring in a nucleotide sequence of a juxtamembrane, comprising:step (a): preparing a human nucleic acid sample;step (b): subjecting the nucleic acid sample obtained instep (a) to gene amplification reaction to provide a nucleic acid fragment obtained by amplifying a region having tandem duplication in a juxtamembrane which can be found in a nucleic acid encoding a receptor protein kinase; andstep (c): examining the presence of tandem duplication for the nucleic acid fragment of step (b);[16] the method for detection according to item [15] above, characterized in that the method is utilized in diagnosis of M2, M4, or M5 based on the FAB (French-American-British) classification of acute myeloid leukemia;[17] a kit for detection of a nucleic acid encoding a receptor protein kinase and having tandem duplication in the nucleotide sequence of a juxtamembrane, characterized in that the kit comprises primers for amplifying a region having tandem duplication, wherein the region can be found in the receptor protein kinase gene;[18] the kit according to item [17] above, characterized in that the kit is utilized in diagnosis of M2, M4, or M5 based on the FAB (French-American-British) classification of acute myeloid leukemia; and[19] use of the nucleic acid according to any one of items [1] to [9] above for detection of a nucleic acid encoding a receptor protein kinase and having tandem duplication in a nucleotide sequence of a juxtamembrane.

BRIEF DESCRIPTION OF THE DRAWINGS

[0016]FIG. 1 is a view showing agarose gel electrophoresis of the case where RT-PCR is carried out with RNA obtained from leukemic cells derived from AML patients as a template. In the figure, lanes 1 to 5 respectively show results for Patients belonging to M1, M2, M3, M4 and M5 (M34 patients) on FAB classification, and lanes 6 to 9 respectively show results for M1, M2 (M155 patients), M3 and M4 (M162 patients).

[0017]FIG. 2 is a schematic view showing tandem duplication at exon 11 and exon 12 for M34, M155, M162, M810 and M839.

BEST MODE FOR CARRYING OUT THE INVENTION

[0018]The present invention will be explained below.

[0019]The nucleic acid encoding a receptor protein kinase of the present invention has tandem duplication in a region encoding a juxtamembrane. The nucleic acid of the present invention encoding a protein kinase can be a nucleic acid encoding either tyrosine kinase or serine-threonine kinase. For diagnosis of leukemia, preferred are nucleic acids encoding a receptor protein kinase, and nucleic acids encoding FMS-like tyrosine kinase 3 (FLT3) are preferably used.

[0020]In the present invention, the juxtamembrane is present between the transmembrane region and the kinase domain of the receptor protein kinase, and the juxtamembrane constructs an intracellular membrane region together with the kinase domain [O. Rosnet, at al., Blood, 82 (4), 1110-1119 (1993)].

[0021]In the present invention, tandem duplication refers to a nucleotide sequence in which an entire portion or partial portion of a nucleic acid encoding a juxtamembrane is repeated one or more times in the same orientation. The above repeat nucleotide sequences can be lined up one directly after another, or they can contain optional nucleotide sequences between each of the repeat nucleotide sequences. In addition, the number of duplicated base is not particularly limited. Furthermore, although mutations of deletion, substitution or addition of one or more bases can exist in a portion of a nucleotide sequence between the corresponding tandem duplications. In the tandem duplication of the present invention, the tandem duplication may be detected as length mutation. For example, the tandem duplication is contained in cDNA having nucleotide sequences of SEQ ID NOs: 6 to 10, and in genomic DNA having nucleotide sequences of SEQ ID NOs: 11 to 15 as nucleic acids encoding a juxtamembrane.

[0022]Nucleic acids (cDNA or genomic DNA) having tandem duplication which are newly found in a nucleotide sequence of the juxtamembrane of FLT3 are named M34 (SEQ ID NOs: 6 and 11), M155 (SEQ ID NOs: 7 and 12), M162 (SEQ ID NOs: 8 and 13), M810 (SEQ ID NOs: 9 and 14) and M839 (SEQ ID NOs: 10 and 15), respectively, and their schematic view is shown in FIG. 2. Incidentally, it is desired that the tandem duplication in the present invention takes place in-frame. Amino acid sequences encoded by the above SEQ ID NOs: 6 to 10 are shown in SEQ ID NOs: 1 to 5.

[0023]The nucleic acid of the present invention concerns a nucleic acid encoding a receptor protein kinase, wherein the nucleic acid has the tandem duplication as described above in a nucleotide sequence of a juxtamembrane, particularly a nucleic acid of FMS-like tyrosine kinase 3 (FLT3) mutant, wherein the nucleic acid has the tandem duplication in a nucleotide sequence of a juxtamembrane. The amino acid sequences of a juxtamembrane having the tandem duplication are shown by e.g. SEQ ID NOs: 1 to 5 as mentioned above, and the nucleic acids of the present invention are those comprising nucleotide sequences encoding these amino acid sequences. Concretely, for example, the present nucleic acids are those comprising nucleotide sequences shown by SEQ ID NOs: 6 to 15. More particularly, a nucleic acid of a tandem duplication mutant of FLT3 comprising a nucleic acid of a juxtamembrane includes, for example, nucleic acids encoding tandem duplication mutants of FLT3 shown by SEQ ID NOs: 16 to 20, more concretely, nucleic acids comprising nucleotide sequences encoding tandem duplication mutants of FLT3 shown by SEQ ID NOs: 21 to 25. In addition, they may be a nucleic acid capable of hybridizing to the above nucleic acid under stringent conditions, and having tandem duplication in a nucleotide sequence encoding a juxtamembrane.

[0024]Furthermore, the nucleic acid of the present invention concerns a nucleic acid encoding a juxtamembrane and having tandem duplication, the nucleic acid including, for example, a nucleic acid having tandem duplication, wherein the nucleic acid encodes amine acid sequences shown by SEQ ID NOs: 1 to 5, concretely, nucleic acids shown by SEQ ID NOs: 6 to 15, or a nucleic acid has tandem duplication capable of hybridizing to those nucleic acids under stringent conditions.

[0025]Here, hybridizing under stringent conditions refers to hybridization with the nucleic acids, wherein the hybridization comprises, for example, incubating a nucleic acid-immobilized membrane with a probe at 50° C. for 12 to 20 hours in 6×SSC, wherein 1×SSC indicates 0.15 M NaCl, 0.015 M sodium citrate, pH 7.0, containing 0.5% SDS, 0.1% bovine serum albumin (BSA), 0.1% polyvinyl pyrrolidone, 0.1% Ficol 400, and 0.01% denatured salmon sperm DNA, but not limited to the above conditions.

[0026]The nucleic acid of the present invention can be obtained by, e.g. the following method.

[0027]First, cells in which length mutation takes place are detected by synthesizing cDNA by a reverse transcriptase with RNA as a template, the RNA purified from various pathologic cells, particularly leukemia cells, thereafter carrying out DNA amplification reaction using primers which are targeted to a region encoding a juxtamembrane of a desired receptor protein kinase, and comparing the length of the amplified DNA fragments by means of an electrophoresis method. Further, it is possible to identify whether or not a mutation is tandem duplication by determining a nucleotide sequence of the obtained amplified DNA fragment.

[0028]Next, cDNA, encoding a receptor protein kinase, the cDNA having novel tandem duplication of the present invention can be obtained by synthesizing cDNA by a reverse transcriptase with RNA obtained from cells in which tandem duplication takes place, thereafter carrying out DNA amplification reaction using primers which can specifically amplify cDNA of a desired receptor protein kinase.

[0029]The nucleic acid of the present invention can be also obtained by using genomic DNA as a template, the genomic DNA purified from pathologic cells.

[0030]In the present invention, leukemia cells are selected as the pathologic cells, and FLT3 is preferably targeted as the receptor protein kinase.

[0031]An FLT3 gene comprises 21 exons, and alternatively, the juxtamembrane is encoded in 18 by at 3'-side of exon 10 and 117 by at 5'-side of exon 11 [O. Rosnet, at al., Oncogene, 6, 1641-1650 (1991)]. Primers covering the region of exon 11 and exon 12 can be selected as primers used in DNA amplification reaction. Examples of the nucleotide sequences are shown in SEQ ID NOs: 26 and 27. Incidentally, exon 12 and 16 by at 3'-side of exon 11 encode a partial portion of the tyrosine kinase domain.

[0032]DNA amplified fragments as shown by SEQ ID NOs: 6 to 10 are obtained when RNA is used as a template for DNA amplification reaction, and DNA amplified fragments as shown by SEQ ID NOs: 11 to 15 are obtained when genomic DNA is used as a template. As a result, it is confirmed that these resulting fragments have in-frame tandem duplication within exon 11 or exons 11 to 12.

[0033]Alternatively, nucleotide sequences of cDNA encoding a whole length of FLT3 and having the above in-frame tandem duplication are shown in SEQ ID NOs: 21 to 25.

[0034]The polypeptide of the present invention is a polypeptide encoded by the above nucleic acids. Concretely, there can be exemplified a polypeptide comprising amino acid sequences of SEQ ID NOs: 1 to 5, and tandem duplication mutants of FLT3 as shown by SEQ ID NOs: 16 to 20.

[0035]The polypeptide of the present invention can be obtained by purifying from cells expressing the polypeptide, and can be also obtained by employing a conventional gene engineering procedures. A tandem duplication mutant of FLT3, for example, can be obtained by inserting the above nucleic acids into a suitable expression vector, and then expressing the product in a suitable host. In addition, a polypeptide with only a juxtamembrane of a receptor protein kinase of the present invention can be obtained by inserting a DNA fragment encoding a juxtamembrane alone to the above expression vector.

[0036]Further, the polypeptide of the present invention can be expressed as a fusion protein. For instance, in order to increase amounts of expression of a desired protein, N-terminal peptide chain derived from other protein is added to N-terminus of the desired protein, or a suitable peptide chain is added to N-terminus or C-terminus of the desired protein to express the resulting polypeptide, so that purification of the desired protein using a resin carrier having affinity to the peptide chain can be facilitated.

[0037]The present polypeptide also encompasses a polypeptide encoded by a nucleic acid having tandem duplication in a nucleotide sequence of a juxtamembrane, wherein the polypeptide results from at least one of deletion, substitution or addition of one or more amino acid residues in amino acid sequences of the present invention, e.g. SEQ ID NOs: 1 to 5, 16 to 20 in Sequence Listing. In other words, there can be a case where no mutations take place in amino acid sequences in the region encoded by tandemly duplicated nucleic acids, and deletion, substitution or addition of amino acid residues takes place in other portions of amino acid sequences; or a case where deletion, substitution or addition of amino acid residues takes place in amino acid sequences of the region encoded by, tandemly duplicated nucleic acids. Introduction of deletion, substitution or addition of the amino acid residues can be easily carried out by introducing mutation into the desired nucleic acid sequence by a method using restriction endonucleases, nucleases and the like, or a method for performing site-directed mutagenesis [W. Ito, et al., Gene, 102, 67-70 (1991)] etc., thereby incorporating the mutated nucleic acid sequence into an expression vector to express the product in a suitable host cell.

[0038]In the present invention, the antibody refers to an antibody capable of specifically binding to a region encoded by a nucleic acid having tandem duplication occurring in a nucleotide sequence of a juxtamembrane of the receptor protein kinase. In order to obtain the antibody, for example, the antibody is obtained as anti-serum by immunizing animals with a peptide having amino acid sequences of SEQ ID NOs: 1 to 5 together with adjuvant by conventional method. In addition, the antibody can be obtained as a monoclonal antibody by a method described in G. Galfare, et al., Nature, 266, 550-552 (1997).

[0039]In the present invention, the nucleic acid capable of specifically binding to nucleic acids having tandem duplication occurring in a nucleotide sequence of a juxtamembrane of the receptor protein kinase is not to be particularly limited, and is exemplified by antisense DNA of double stranded DNA having tandem duplication or RNA corresponding to the antisense DNA.

[0040]The method for detection of a nucleic acid of the present invention comprises the following steps:

step (a): obtaining a human nucleic acid sample;step (b): subjecting the nucleic acid sample obtained in the above step (a) to gene amplification reaction to provide a nucleic acid fragment obtained by amplifying a region having tandem duplication in a juxtamembrane, wherein the region can be found in a nucleic acid encoding a receptor protein kinase; andstep (c): examining the presence of tandem duplication for the nucleic acid fragment of the above step (b).

[0041]First, step (a) will be described. The human nucleic acid sample usable in the present invention is not to be particularly limited, as long as it is a nucleic acid encoding a receptor protein kinase, the nucleic acid having tandem duplication in a nucleotide sequence of a juxtamembrane, such as genomic DNA, cDNA or mRNA. The human nucleic acid sample can be prepared by conventionally performed known method, including, for instance, a method described in Molecular Cloning: A LABORATORY MANUAL, 2nd eds, (T. Maniatis at al., Cold Spring Harbor Laboratory Press, published in 1989).

[0042]Secondly, step (b) will be described. The nucleic acid sample and suitable primers are used to amplify a nucleic acid encoding a region containing mutation site which can be found in a juxtamembrane of the receptor protein kinase of the interest to obtain a desired nucleic acid fragment. A method for performing DNA amplification reaction usable in this step is not particularly limited, as long as it is a method capable of amplifying the above region, and there can be utilized nucleic acid amplification methods, such as a nucleic acid amplification method utilizing RT-PCR method, PCR method, or RNA polymerases (Japanese Patent Laid-Open Nos. Hei 2-5864 and Hei 7-203999), or strand substitution amplification method (Japanese Examined Patent Publication No. Hei 7-114718, and Japanese Patent Laid-Open No Hei 7-88242). Among them, the RT-PCR method or PCR method is preferably used.

[0043]The region to be amplified having tandem duplication in a juxtamembrane includes, for example, in case of FLT3, a region containing a whole or partial portion of the region from 18 by at 3'-side of exon 10 to 117 by at 5'-side of exon 11, without being particularly limited thereto as long as the region contains an exon 11 site.

[0044]The primers used in RT-PCR method or PCR method are not particularly limited as long as they are primers capable of amplifying a DNA fragment containing the above mutation site. Concretely, there can be exemplified a primer pair having nucleotide sequences as shown in SEQ ID NOs: 26 and 27 in Sequence Listing. In addition, PCR conditions are not particularly limited, and conventionally performed known conditions can be used on PCR reaction.

[0045]Thirdly, step (c) will be described. In this step, the presence of tandem duplication for the nucleic acid fragment obtained in step (b) is examined. The method for detection of the presence of tandem duplication is not particularly limited, and it is preferable that a method of comparing lengths of amplified DNA fragments by means of agarose gel electrophoresis method is used.

[0046]In addition, a method for examining single strand conformation polymorphism (SSCP) can be used as a method for detection of mutation which is usable in this step. The method comprises examining the differences of a higher-order structure as the differences of mobility in electrophoresis, wherein the high-order structure is dependent on a nucleotide sequence in which single-stranded DNA is formed by intramolecular interaction (Proc. Natl. Aced. Sci. USA, 86: 2766-2770, 1989). The presence or absence of mutation can be detected by subjecting the nucleic acid fragment obtained in step (b) to electrophoresis under conditions described in the above-mentioned publication, and comparing its mobility with that of a nucleic acid fragment derived from a normal receptor protein kinase.

[0047]Other detection methods include a method in which the above step (c) is altered to Other method for detection of mutation. For the detection of mutation, there can be used a known method for detection of mutation, such as hybridization method using a suitable DNA fragment containing a mutation site as a probe, or DGGE method [Val C. Sheffield et al., Proc. Natl. Acad. Sci. USA 86, 232-236 (1989)]. In addition, a method for detection of mutation using a MutS protein is known (Japanese Patent Laid-Open No. Hei 7-327698).

[0048]The mutation can be identified by sequencing the nucleotide sequence for DNA fragment in which a length mutation is confirmed by means of the above-mentioned method. For sequencing the nucleotide sequence, a conventionally used method can be employed, including, for example, a method comprising cloning an amplified DNA fragment into a suitable vector and determining the nucleotide sequence, or a method for determining the nucleotide sequence using an amplified fragment per se as a template.

[0049]As described above, the present invention provides a use of a nucleic acid of the present invention described above for detection of a nucleic acid encoding a receptor protein kinase, wherein the nucleic acid has tandem duplication in a nucleotide sequence of a juxtamembrane.

[0050]The method for detection of a nucleic acid of the present invention can be utilized in diagnosis of M2, M4, and M5 based on the FAB (French-American-British) classification of acute myeloid leukemia (AML). Based on the FAB (French-American-British) classification, pathologic types of AML are classified into six classes as M1 (myeloblastic, no maturation potential), M2 (myeloblastic, with maturation potential), M3 (promyelocytic), M4 (myelomonocytic), M5 (monocytic), and M6 (erythroleukemia) (Shin Rinsho Kensa Gishi Koza, 10, Ketsuekigaku, 75, Igaku-Shoin).

[0051]It is understood that patients harboring an FLT3 gene having the tandem duplication of the present invention belong to classes M2, M4, and M5 above, and that the patients relapse into symptom to death even with transient symptom remission, so that their prognosis leads to mal-consequence. Therefore, according to the detection method of the present invention, there can be provided a method for examination useful to the pathologic judgment of AML.

[0052]Incidentally, of the above patients, the detection of mutation using genomic DNA from myelocyte of a patient obtained at the time of symptom remission is carried out, and as a result, tandem duplications in a juxtamembrane are not found. This mutation is therefore assumed to be a somatic mutation.

[0053]Also, the nucleic acid of the present invention, which has tandem duplication, serves as a marker for myelodysplastic syndrome (MDS), which develops in the pre-stage of leukemia, AML with dysplasia, and the like, as well as AML as classified based on the FAB classification. The detection method of the present invention therefore is a method for examination which is useful for the pathologic judgment of these diseases.

[0054]By utilizing the above detection method, there can be provided a kit for detection of a nucleic acid of the present invention. Concretely, there is a kit for detection of a nucleic acid by the above described detection method, the nucleic acid encoding a receptor protein kinase and having tandem duplication in the nucleotide sequence of a juxtamembrane, characterized in that the kit comprises primers for amplifying a region having tandem duplication, wherein the region can be found on the receptor protein kinase gene.

[0055]The diagnosis of the above AML etc. can be easily carried out by using such a kit.

[0056]In the present invention, a polypeptide encoded by a nucleic acid having tandem duplication as described above can be further detected by the steps shown below:

step 1: obtaining a human protein sampler andstep 2: examining the presence of tandem duplication in the nucleotide sequence of a juxtamembrane of the protein sample obtained in the. above step 1.

[0057]First, step 1 will be described. The human protein sample can be prepared by preparing a membrane protein from a cell which is assumed to have the polypeptide of the present invention expressed therein (e.g., leukemic cell, in case of FLT3),

[0058]Second, step 2 will be described. The method for detection of tandem duplication mutations is not particularly limited, and can be carried out by using a labeled antibody capable of specifically binding to the juxtamembrane encoded by a nucleic acid having a tandem duplication mutation.

[0059]This step can, for example, be carried out by a method comprising subjecting the protein sample obtained in step 1 to SDS-PAGE to separate proteins, and subsequently detecting the desired protein by immunoblotting method.

[0060]In another embodiment of the present invention, there can be provided a method for regulating the proliferation, immune response and signal information transmission of leukemic cells, hematopoietic stem cells, etc. using the above nucleic acids or polypeptides, or nucleic acids or antibodies capable of specifically binding thereto.

[0061]Among them, a preferred embodiment includes an application to immunotherapy for tumors. Conventionally, it has been known that tumor-specific peptides of proteins specifically expressed in tumor cells serve as targets if T cell immune responses to tumor cells. In a method for performing the application, the techniques described in the following reports, for example, can be utilized. Concretely, CD4+T cells restricted to HLA-DR are isolated, the cells specifically reacting with ras peptide resulting from substitution of 12th amino acid glycine with another amino acid in the human T cells (Jung, S. J. Exp. Med. 173, 273, 1991), and a CTL (cytotoxic T lymphocyte) recognizing a peptide consisting of eight amino acids including the mutation site for 61th amino acid mutation can be derived from a mouse immunized with a recombinant vaccinia virus capable of producing ras protein, which has mutation at 61th amino acid (Skipper, J., J. Exp. Med. 177, 1493, 1993). In addition, in a mouse immunized with a soluble mutant ras protein prepared by gene recombination, the in vivo proliferation of tumor cells having the same mutation is suppressed (Fenton, R. G., J. Natl. Cancer Inst. 85, 1294, 1993), and a CTL showing cytotoxic activity against tumor cells expressing the same mutant ras can be obtained from splenocytes sensitized with the mutant ras peptide (Peace, D. J., J. Exp. Med. 179, 473, 1994). On the other hand, the bcr-abl chimeric protein, which is often detected in chronic myelocytic leukemia, possesses high tyrosine kinase activity and plays a key role in the onset of leukemia and the proliferation of leukemic cells. By immunizing with a peptide in the vicinity of the fusion site of this fusion protein, T cells reactive to this fusion protein can be obtained (Chen, W., Proc. Natl. Aced. Sci. USA 89, 1468, 1992). Moreover, antisense DNA or RNA corresponding to the fusion gene is capable of suppressing the proliferation of tumors expressing this gene in vivo (Skorski, T., Proc. Natl. Acad. Sci. USA 91, 4504, 1994).

[0062]It is therefore possible to obtain T cells reactive to a receptor protein kinase comprising the peptide of the present invention, wherein the peptide is encoded by a nucleic acid having tandem duplication occurring in the nucleotide sequence of a juxtamembrane, and to regulate the proliferation of cells that express the above kinase by immunizing with the above peptide.

[0063]Also, when the presence of the tandem duplication of the present invention is involved in cell proliferation regulation, it is possible to regulate the signaling mechanism with antisense DNA or RNA for the above gene to regulate cell proliferation.

[0064]When binding a ligand to an extracellular region, the receptor protein kinase undergoes a conformational change to form a dimer, resulting in increased kinase domain activity in the intracellular region, whereby self-phosphorylation or phosphorylation of a substrate of the above kinase takes place. In these steps, various signaling molecules are involved, and the information transmitted into cells causes various biological phenomena, such as cell morphological change, cell movement, morphogenesis, cell proliferation, malignant alteration, differentiation, and apoptosis. Acute myelocytic leukemic cells of high malignancy have been reported to possess strong affinity to the FLT3 ligand and promote cell proliferation (Piacibello, W., Blood 36, 4105, 1995; Lisovsky, M., Blood 36, 22a, 1995; McKenna, H., J. Exp. Hematol. 24, 378, 1996; Dehmel, U., Leukemia 10, 261, 1996). In cells expressing the FLT3 tandem duplication mutant of the present invention, it is expected that the system for signaling from the FLT3 ligand is highly activated. Hematopoietic stem cells that express the mutant are therefore provided as a source of hematopoietic stem cells possessing strong proliferation potential. By comparing the hematopoietic stem cells with cells expressing the normal FLT3, materials and methods suitable for screening for various drugs can be provided.

[0065]As described above, by utilizing a method of the present invention, it is applicable to the examination and treatment of blood cell diseases, hematopoietic stem cell diseases, and other diseases.

[0066]The present invention will be hereinafter described in more detail by means of examples, but the present invention is not limited by those examples.

Example 1

[0067]1) Analysis of FLT3 Gene Expression Pattern

[0068]On 80'cases of acute leukemia patients (50 cases of child ALL, 30 cases of adult AML), analysis of FLT3 gene expression was carried out by RT-PCR method. The primers used were designed to have nucleotide sequences as shown by SEQ ID NOs: 26 and 27 in Sequence Listing, and to completely cover and amplify a transmembrane region through a juxtamembrane. By using the above primer pair, the resulting amplified DNA product is 366 by in length, when normal FLT3 has been transcribed.

[0069]A total RNA was extracted from a peripheral blood or myelocyte derived from the above patient with a Trizol reagent (manufactured by LifeTech), followed by DNA amplification reaction using an RT-PCR kit (manufactured by Takara Shuzo Co., Ltd.) and Thermal Cycler (manufactured by Takara Shuzo Co., Ltd.) under following conditions. cDNA was synthesized from a total RNA using a reverse transcriptase. In 50 μl of a reaction mixture containing 1 μl of the cDNA (equivalent to 40 ng of a total RNA), 200 μM dNTP mixture, 1×PCR buffer, 2 U of Taq DNA polymerase, and 20 pmol each of the above-described primers, the above reaction mixture was heated at 94° C. for 5 minutes, and thereafter repeated 35 times of a thermal cycle consisting of 64° C. for 30 seconds, 72° C. for 45 seconds, and 94° C. for 30 seconds, and then finally treated at 72° C. for 5 minutes. To check quality of RNA, RT-PCR was carried out in the same manner except that a pair of the primers shown by SW. ID NOs: 28 and 29 in Sequence Listing were used, with β-actin as the target. The amplified DNA products thus obtained were subjected to electrophoresis on 2 to 3% agarose gel (manufactured by FMC) containing ethidium bromide, and detected under UV irradiation. One example of electrophoresis pattern is shown in FIG. 1, and the results are shown in Table 1.

TABLE-US-00001 TABLE 1 Number of positive Length FAB Number of mRNA expression of mutation subtype cases examined FLT3 (%) (%) AML total 30 22 (73%) 5 (17%) M1 3 2 (67%) 0 M2 9 7 (78%) 1 (14%) M3 8 5 (68%) 0 M4 5 4 (80%) 2 (40%) M5 4 3 (75%) 2 (50%) M6 1 1 (100%) 0 ALL total 50 39 (78%) 0 cALL 27 24 (89%) 0 pre-B ALL 13 11 (85%) 0 B-ALL 1 0 0 T-ALL 9 4 (44%) 0

[0070]It is found from Table 1 that the transcription product of the FLT3 gene was found in 35 cases (78%) of the 50 ALL cases and 22 cases (73%) of the 30 AML cases. Among them, the amplified DNA product longer than the expected 366 by was detected in 5 cases (23%) of the 22 FLT3-positive AML cases, so that a length mutation in FLT3 gene was observed. Incidentally, in four cases (M34, M155, M810, and M839), the expected 366 by band and a longer band than the expected were detected. In one case (M162), the 366 by band was not detected, and a longer band alone was detected.

[0071]2) Analysis of Nucleotide Sequence of Length Mutation Product of FLT3 Gene

[0072]To examine in more detail length mutations in the gene in the above 5 cases, the amplified DNA product was purified from agarose gel, and nucleotide sequences of the exon 11 and exon 12 regions were determined. As a result, it was confirmed that these length mutations resulted from tandem duplications in nucleotide sequences of the respective juxtamembrane. Concretely, a 39 by or 60 by tandem duplication within exon 11 was found in cases M34, M162, and M839; and a 26 by tandem duplication including a 4 by (GGCA) insert was found in case M810. In addition, case M155 was found to have a 63 by tandem duplication comprising the first 16 by of exon 12 immediately after exon 11, one cytosine residue insert, and the last 46 by of exon 11. The nucleotide sequences obtained are shown in SEQ ID NOs: 6 to 10 in Sequence Listing, and a schematic view of these tandem duplications is shown in FIG. 2.

[0073]Characteristically, these tandem duplications occur in-frame, and these mutations are reflected in the actually expressed polypeptides. The amino acid sequences encoded by these nucleotide sequences are shown in SEQ ID NOs: 1 to 5.

[0074]3) Analysis of Nucleotide Sequence of Genomic DNA

[0075]Amplified DNA products obtained by PCR with FLT3 genomic DNA derived from myelocytes from the above 5 cases of patients as templates were analyzed. PCR reaction was carried out under amplification conditions such that 2 U of Taq DNA polymerase (Takara Shuzo Co., Ltd.) was added to a PCR buffer containing 50 ng of genomic DNA, 200 μM of a dNTP mixture, and 20 pmol of each of primers, to make up a total volume of 50 μl. For exons 10 to exon 19, each exon was individually subjected to amplification DNA reaction. The length mutation was observed in same manner as that when mRNA was analyzed in the case where exon 11 and exon 12 were amplified with primers of SEQ ID NOs: 30 and 31, and primers of SEQ ID NOs: 32 and 33 in Sequence Listing as pairs. When the PCR products were purified using QIAEXII (QIAGEN), cloned into pCRII vector (Invitrogen), and subjected to nucleotide sequence analysis, similar results to those with the nucleotide sequences of cDNA (SEQ ID NOs: 21 to 25) were obtained. The results are collectively shown in FIG. 2.

Example 2

[0076]Analysis of Mutations in Juxtamembrane of Receptor Protein Kinase and their Pathologic Relationship

[0077]To analyze mutations in a juxtamembrane of the receptor protein kinase and their pathologic relationship, the relationship between the pathologic classification of symptoms and FLT3 gene is shown in Table 1. Five cases showing tandem duplication in the nucleotide sequence of a juxtamembrane belonged to M2 (myeloblastic, with maturation potential), M4 (myelomonocytic), or M5 (monocytic) based on the FAB classification, and all of them were cases in which patients relapse into symptom to death even with transient symptom remission.

[0078]The nucleotide sequences of exon regions encoding tyrosine kinase domain were also analyzed, and no mutations were found in these regions.

[0079]Therefore, it was suggested that the in-frame tandem duplications in gene region encoding a juxtamembrane of FLT3 were associated with AML with monocyte growth was suggested.

[0080]Also, since such length mutations were not detected in DNA samples from myelocytes collected from three cases (M34, M162, and M810) at the time of complete remission, the tandem duplication of the present invention was found to be a somatic mutation.

INDUSTRIAL APPLICABILITY

[0081]According to the present invention, there can be provided a novel receptor protein kinase having tandem duplication mutation in the nucleotide sequence of a juxtamembrane, and its nucleotide sequence and amino acid sequence information. In addition, there can be provided pathological diagnoses, a method for examination of leukemia etc. utilizing the present invention, a kit and a reagent for examination related thereto. Furthermore, there can be provided a method for regulating and analyzing conditions of proliferation and differentiation, malignant alteration, immune response, and signalling for cells represented by hematopoietic stem cells and leukemia cells utilizing the present invention, and a kit and a reagent related thereto.

Sequence CWU 1

33197PRTHomo sapiens 1Gln Phe Arg Tyr Glu Ser Gln Leu Gln Met Val Gln Val Thr Gly Ser1 5 10 15Ser Asp Asn Glu Tyr Phe Tyr Val Glu Ser Gln Leu Gln Met Val Gln 20 25 30Val Thr Gly Ser Ser Asp Ser Glu Tyr Phe Tyr Val Asp Phe Arg Glu 35 40 45Tyr Glu Tyr Asp Leu Lys Trp Glu Phe Pro Arg Glu Asn Leu Glu Phe 50 55 60Gly Lys Val Leu Gly Ser Gly Ala Phe Gly Lys Val Met Asn Ala Thr65 70 75 80Ala Leu Glu Leu Ala Lys Gln Glu Ser Gln Ser Arg Leu Pro Ser Lys 85 90 95Cys2100PRTHomo sapiens 2Gln Phe Arg Tyr Glu Ser Gln Leu Gln Met Val Gln Val Thr Gly Ser1 5 10 15Ser Asp Asn Glu Tyr Phe Tyr Val Asp Phe Arg Glu Tyr Glu Tyr Asp 20 25 30Leu Lys Trp Glu Phe Pro Arg Glu Asn Leu Glu Phe Gly Lys Val Leu 35 40 45Gly Ser Glu Tyr Asp Leu Lys Trp Glu Phe Pro Arg Glu Asn Leu Glu 50 55 60Phe Gly Lys Val Leu Gly Ser Gly Ala Phe Gly Lys Val Met Asn Ala65 70 75 80Thr Ala Tyr Gly Ile Ser Lys Thr Gly Val Ser Ile Gln Val Ala Val 85 90 95Lys Met Leu Lys 100392PRTHomo sapiens 3Gln Phe Arg Tyr Glu Ser Gln Leu Gln Met Val Gln Val Thr Gly Ser1 5 10 15Ser Asp Asn Glu Tyr Phe Tyr Val Asp Phe Arg Glu Tyr Glu Tyr Asp 20 25 30Leu Lys Trp Glu Phe Asp Phe Arg Glu Tyr Glu Tyr Asp Leu Lys Trp 35 40 45Glu Phe Pro Arg Glu Asn Leu Glu Phe Gly Lys Val Leu Gly Ser Gly 50 55 60Ala Phe Gly Lys Val Met Asn Ala Thr Ala Tyr Gly Ile Ser Lys Thr65 70 75 80Gly Val Ser Ile Gln Val Ala Val Lys Met Leu Lys 85 90489PRTHomo sapiens 4Gln Phe Arg Tyr Glu Ser Gln Leu Gln Met Val Gln Val Thr Gly Ser1 5 10 15Ser Asp Asn Glu Tyr Phe Tyr Val Asp Phe Arg Glu Tyr Glu Tyr Asp 20 25 30Leu Lys Trp Glu Phe Pro Arg Glu Asn Trp His Lys Trp Glu Phe Pro 35 40 45Arg Glu Asn Leu Glu Phe Gly Lys Val Leu Gly Ser Gly Ala Phe Gly 50 55 60Lys Val Met Asn Ala Thr Ala Tyr Gly Ile Ser Lys Thr Gly Val Ser65 70 75 80Ile Gln Val Ala Val Lys Met Leu Lys 85592PRTHomo sapiens 5Gln Phe Arg Tyr Glu Ser Gln Leu Gln Met Val Gln Val Thr Gly Ser1 5 10 15Ser Asp Asn Glu Tyr Phe Tyr Val Asp Phe Arg Gly Ser Ser Asp Asn 20 25 30Glu Tyr Phe Tyr Val Asp Phe Arg Glu Tyr Glu Tyr Asp Leu Lys Trp 35 40 45Glu Phe Pro Arg Glu Asn Leu Glu Phe Gly Lys Val Leu Gly Ser Gly 50 55 60Ala Phe Gly Lys Val Met Asn Ala Thr Ala Tyr Gly Ile Ser Lys Thr65 70 75 80Gly Val Ser Ile Gln Val Ala Val Lys Met Leu Lys 85 906296DNAHomo sapiens 6caatttaggt atgaaagcca gctacagatg gtacaggtga ccggctcctc agataatgag 60tacttctacg ttgaaagcca gctacagatg gtacaggtga ccggctcctc agatagtgag 120tacttctacg ttgatttcag agaatatgaa tatgatctca aatgggagtt tccaagagaa 180aatttagagt ttgggaaggt actaggatca ggtgcttttg gaaaagtgat gaacgcaaca 240gctttggaat tagcaaaaca ggagtctcaa tccaggttgc cgtcaaaatg ctgaaa 2967300DNAHomo sapiens 7caatttaggt atgaaagcca gctacagatg gtacaggtga ccggctcctc agataatgag 60tacttctacg ttgatttcag agaatatgaa tatgatctca aatgggagtt tccaagagaa 120aatttagagt ttgggaaggt actaggatcc gaatatgatc tcaaatggga gtttccaaga 180gaaaatttag agtttgggaa ggtactagga tcaggtgctt ttggaaaagt gatgaacgca 240acagcttatg gaattagcaa aacaggagtc tcaatccagg ttgccgtcaa aatgctgaaa 3008276DNAHomo sapiens 8caatttaggt atgaaagcca gctacagatg gtacaggtga ccggctcctc agataatgag 60tacttctacg ttgatttcag agaatatgaa tatgatctca aatgggagtt tgatttcaga 120gaatatgaat atgatctcaa atgggagttt ccaagagaaa atttagagtt tgggaaggta 180ctaggatcag gtgcttttgg aaaagtgatg aacgcaacag cttatggaat tagcaaaaca 240ggagtctcaa tccaggttgc cgtcaaaatg ctgaaa 2769267DNAHomo sapiens 9caatttaggt atgaaagcca gctacagatg gtacaggtga ccggctcctc agataatgag 60tacttctacg ttgatttcag agaatatgaa tatgatctca aatgggagtt tccaagagaa 120aattggcaca aatgggagtt tccaagagaa aatttagagt ttgggaaggt actaggatca 180ggtgcttttg gaaaagtgat gaacgcaaca gcttatggaa ttagcaaaac aggagtctca 240atccaggttg ccgtcaaaat gctgaaa 26710276DNAHomo sapiens 10caatttaggt atgaaagcca gctacagatg gtacaggtga ccggctcctc agataatgag 60tacttctacg ttgatttcag aggctcctca gataatgagt acttctacgt tgatttcaga 120gaatatgaat atgatctcaa atgggagttt ccaagagaaa atttagagtt tgggaaggta 180ctaggatcag gtgcttttgg aaaagtgatg aacgcaacag cttatggaat tagcaaaaca 240ggagtctcaa tccaggttgc cgtcaaaatg ctgaaa 27611386DNAHomo sapiens 11caatttaggt atgaaagcca gctacagatg gtacaggtga ccggctcctc agataatgag 60tacttctacg ttgaaagcca gctacagatg gtacaggtga ccggctcctc agatagtgag 120tacttctacg ttgatttcag agaatatgaa tatgatctca aatgggagtt tccaagagaa 180aatttagagt ttggtaagaa tggaatgtgc caaatgtttc tgcagcattt cttttccatt 240ggaaaatctt taaaatgcac gtactcacca tttgtctttg cagggaaggt actaggatca 300ggtgcttttg gaaaagtgat gaacgcaaca gctttggaat tagcaaaaca ggagtctcaa 360tccaggttgc cgtcaaaatg ctgaaa 38612480DNAHomo sapiens 12caatttaggt atgaaagcca gctacagatg gtacaggtga ccggctcctc agataatgag 60tacttctacg ttgatttcag agaatatgaa tatgatctca aatgggagtt tccaagagaa 120aatttagagt ttggtaagaa tggaatgtgc caaatgtttc tgcagcattt cttttccatt 180ggaaaatctt taaaatgcac gtactcacca tttgtctttg cagggaaggt actaggatcc 240gaatatgatc tcaaatggga gtttccaaga gaaaatttag agtttggtga gaatggaatg 300tgccaaatgt ttctgcagca tttcttttcc attggaaaat ctttaaaatg cacgtactca 360ccatttgtct ttgcagggaa ggtactagga tcaggtgctt ttggaaaagt gatgaacgca 420acagcttatg gaattagcaa aacaggagtc tcaatccagg ttgccgtcaa aatgctgaaa 48013366DNAHomo sapiens 13caatttaggt atgaaagcca gctacagatg gtacaggtga ccggctcctc agataatgag 60tacttctacg ttgatttcag agaatatgaa tatgatctca aatgggagtt tgatttcaga 120gaatatgaat atgatctcaa atgggagttt ccaagagaaa atttagagtt tggtaagaat 180ggaatgtgcc aaatgtttct gcagcatttc ttttccattg gaaaatcttt aaaatgcacg 240tactcaccat ttgtctttgc agggaaggta ctaggatcag gtgcttttgg aaaagtgatg 300aacgcaacag cttatggaat tagcaaaaca ggagtctcaa tccaggttgc cgtcaaaatg 360ctgaaa 36614357DNAHomo sapiens 14caatttaggt atgaaagcca gctacagatg gtacaggtga ccggctcctc agataatgag 60tacttctacg ttgatttcag agaatatgaa tatgatctca aatgggagtt tccaagagaa 120aattggcaca aatgggagtt tccaagagaa aatttagagt ttggtaagaa tggaatgtgc 180caaacgtttc tgcagcattt cttttccatt ggaaaatctt taaaatgcac gtactcacca 240tttgtctttg cagggaaggt actaggatca ggtgcttttg gaaaagtgat gaacgcaaca 300gcttatggaa ttagcaaaac aggagtctca atccaggttg ccgtcaaaat gctgaaa 35715366DNAHomo sapiens 15caatttaggt atgaaagcca gctacagatg gtacaggtga ccggctcctc agataatgag 60tacttctacg ttgatttcag aggctcctca gataatgagt acttctacgt tgatttcaga 120gaatatgaat atgatctcaa atgggagttt ccaagagaaa atttagagtt tggtaagaat 180ggaatgtgcc aaatgtttct gcagcatttc ttttccattg gaaaatcttt aaaatgcacg 240tactcaccat ttgtctttgc agggaaggta ctaggatcag gtgcttttgg aaaagtgatg 300aacgcaacag cttatggaat tagcaaaaca ggagtctcaa tccaggttgc cgtcaaaatg 360ctgaaa 36616665PRTHomo sapiens 16Met Pro Ala Leu Ala Arg Asp Ala Gly Thr Val Pro Leu Leu Val Val1 5 10 15Phe Ser Ala Met Ile Phe Gly Thr Ile Thr Asn Gln Asp Leu Pro Val 20 25 30Ile Lys Cys Val Leu Ile Asn His Lys Asn Asn Asp Ser Ser Val Gly 35 40 45Lys Ser Ser Ser Tyr Pro Met Val Ser Glu Ser Pro Glu Asp Leu Gly 50 55 60Cys Ala Leu Arg Pro Gln Ser Ser Gly Thr Val Tyr Glu Ala Ala Ala65 70 75 80Val Glu Val Asp Val Ser Ala Ser Ile Thr Leu Gln Val Leu Val Asp 85 90 95Ala Pro Gly Asn Ile Ser Cys Leu Trp Val Phe Lys His Ser Ser Leu 100 105 110Asn Cys Gln Pro His Phe Asp Leu Gln Asn Arg Gly Val Val Ser Met 115 120 125Val Ile Leu Lys Met Thr Glu Thr Gln Ala Gly Glu Tyr Leu Leu Phe 130 135 140Ile Gln Ser Glu Ala Thr Asn Tyr Thr Ile Leu Phe Thr Val Ser Ile145 150 155 160Arg Asn Thr Leu Leu Tyr Thr Leu Arg Arg Pro Tyr Phe Arg Lys Met 165 170 175Glu Asn Gln Asp Ala Leu Val Cys Ile Ser Glu Ser Val Pro Glu Pro 180 185 190Ile Val Glu Trp Val Leu Cys Asp Ser Gln Gly Glu Ser Cys Lys Glu 195 200 205Glu Ser Pro Ala Val Val Lys Lys Glu Glu Lys Val Leu His Glu Leu 210 215 220Phe Gly Thr Asp Ile Arg Cys Cys Ala Arg Asn Glu Leu Gly Arg Glu225 230 235 240Cys Thr Arg Leu Phe Thr Ile Asp Leu Asn Gln Thr Pro Gln Thr Thr 245 250 255Leu Pro Gln Leu Phe Leu Lys Val Gly Glu Pro Leu Trp Ile Arg Cys 260 265 270Lys Ala Val His Val Asn His Gly Phe Gly Leu Thr Trp Glu Leu Glu 275 280 285Asn Lys Ala Leu Glu Glu Gly Asn Tyr Phe Glu Met Ser Thr Tyr Ser 290 295 300Thr Asn Arg Thr Met Ile Arg Ile Leu Phe Ala Phe Val Ser Ser Val305 310 315 320Ala Arg Asn Asp Thr Gly Tyr Tyr Thr Cys Ser Ser Ser Lys His Pro 325 330 335Ser Gln Ser Ala Leu Val Thr Ile Val Gly Lys Gly Phe Ile Asn Ala 340 345 350Thr Asn Ser Ser Glu Asp Tyr Glu Ile Asp Gln Tyr Glu Glu Phe Cys 355 360 365Phe Ser Val Arg Phe Lys Ala Tyr Pro Gln Ile Arg Cys Thr Trp Thr 370 375 380Phe Ser Arg Lys Ser Phe Pro Cys Glu Gln Lys Gly Leu Asp Asn Gly385 390 395 400Tyr Ser Ile Ser Lys Phe Cys Asn His Lys His Gln Pro Gly Glu Tyr 405 410 415Ile Phe His Ala Glu Asn Asp Asp Ala Gln Phe Thr Lys Met Phe Thr 420 425 430Leu Asn Ile Arg Arg Lys Pro Gln Val Leu Ala Glu Ala Ser Ala Ser 435 440 445Gln Ala Ser Cys Phe Ser Asp Gly Tyr Pro Leu Pro Ser Trp Thr Trp 450 455 460Lys Lys Cys Ser Asp Lys Ser Pro Asn Cys Thr Glu Glu Ile Thr Glu465 470 475 480Gly Val Trp Asn Arg Lys Ala Asn Arg Lys Val Phe Gly Gln Trp Val 485 490 495Ser Ser Ser Thr Leu Asn Met Ser Glu Ala Ile Lys Gly Phe Leu Val 500 505 510Lys Cys Cys Ala Tyr Asn Ser Leu Gly Thr Ser Cys Glu Thr Ile Leu 515 520 525Leu Asn Ser Pro Gly Pro Phe Pro Phe Ile Gln Asp Asn Ile Ser Phe 530 535 540Tyr Ala Thr Ile Gly Val Cys Leu Leu Phe Ile Val Val Leu Thr Leu545 550 555 560Leu Ile Cys His Lys Tyr Lys Lys Gln Phe Arg Tyr Glu Ser Gln Leu 565 570 575Gln Met Val Gln Val Thr Gly Ser Ser Asp Asn Glu Tyr Phe Tyr Val 580 585 590Glu Ser Gln Leu Gln Met Val Gln Val Thr Gly Ser Ser Asp Ser Glu 595 600 605Tyr Phe Tyr Val Asp Phe Arg Glu Tyr Glu Tyr Asp Leu Lys Trp Glu 610 615 620Phe Pro Arg Glu Asn Leu Glu Phe Gly Lys Val Leu Gly Ser Gly Ala625 630 635 640Phe Gly Lys Val Met Asn Ala Thr Ala Leu Glu Leu Ala Lys Gln Glu 645 650 655Ser Gln Ser Arg Leu Pro Ser Lys Cys 660 66517994PRTHomo sapiens 17Met Pro Ala Leu Ala Arg Asp Ala Gly Thr Val Pro Leu Leu Val Val1 5 10 15Phe Ser Ala Met Ile Phe Gly Thr Ile Thr Asn Gln Asp Leu Pro Val 20 25 30Ile Lys Cys Val Leu Ile Asn His Lys Asn Asn Asp Ser Ser Val Gly 35 40 45Lys Ser Ser Ser Tyr Pro Met Val Ser Glu Ser Pro Glu Asp Leu Gly 50 55 60Cys Ala Leu Arg Pro Gln Ser Ser Gly Thr Val Tyr Glu Ala Ala Ala65 70 75 80Val Glu Val Asp Val Ser Ala Ser Ile Thr Leu Gln Val Leu Val Asp 85 90 95Ala Pro Gly Asn Ile Ser Cys Leu Trp Val Phe Lys His Ser Ser Leu 100 105 110Asn Cys Gln Pro His Phe Asp Leu Gln Asn Arg Gly Val Val Ser Met 115 120 125Val Ile Leu Lys Met Thr Glu Thr Gln Ala Gly Glu Tyr Leu Leu Phe 130 135 140Ile Gln Ser Glu Ala Thr Asn Tyr Thr Ile Leu Phe Thr Val Ser Ile145 150 155 160Arg Asn Thr Leu Leu Tyr Thr Leu Arg Arg Pro Tyr Phe Arg Lys Met 165 170 175Glu Asn Gln Asp Ala Leu Val Cys Ile Ser Glu Ser Val Pro Glu Pro 180 185 190Ile Val Glu Trp Val Leu Cys Asp Ser Gln Gly Glu Ser Cys Lys Glu 195 200 205Glu Ser Pro Ala Val Val Lys Lys Glu Glu Lys Val Leu His Glu Leu 210 215 220Phe Gly Thr Asp Ile Arg Cys Cys Ala Arg Asn Glu Leu Gly Arg Glu225 230 235 240Cys Thr Arg Leu Phe Thr Ile Asp Leu Asn Gln Thr Pro Gln Thr Thr 245 250 255Leu Pro Gln Leu Phe Leu Lys Val Gly Glu Pro Leu Trp Ile Arg Cys 260 265 270Lys Ala Val His Val Asn His Gly Phe Gly Leu Thr Trp Glu Leu Glu 275 280 285Asn Lys Ala Leu Glu Glu Gly Asn Tyr Phe Glu Met Ser Thr Tyr Ser 290 295 300Thr Asn Arg Thr Met Ile Arg Ile Leu Phe Ala Phe Val Ser Ser Val305 310 315 320Ala Arg Asn Asp Thr Gly Tyr Tyr Thr Cys Ser Ser Ser Lys His Pro 325 330 335Ser Gln Ser Ala Leu Val Thr Ile Val Gly Lys Gly Phe Ile Asn Ala 340 345 350Thr Asn Ser Ser Glu Asp Tyr Glu Ile Asp Gln Tyr Glu Glu Phe Cys 355 360 365Phe Ser Val Arg Phe Lys Ala Tyr Pro Gln Ile Arg Cys Thr Trp Thr 370 375 380Phe Ser Arg Lys Ser Phe Pro Cys Glu Gln Lys Gly Leu Asp Asn Gly385 390 395 400Tyr Ser Ile Ser Lys Phe Cys Asn His Lys His Gln Pro Gly Glu Tyr 405 410 415Ile Phe His Ala Glu Asn Asp Asp Ala Gln Phe Thr Lys Met Phe Thr 420 425 430Leu Asn Ile Arg Arg Lys Pro Gln Val Leu Ala Glu Ala Ser Ala Ser 435 440 445Gln Ala Ser Cys Phe Ser Asp Gly Tyr Pro Leu Pro Ser Trp Thr Trp 450 455 460Lys Lys Cys Ser Asp Lys Ser Pro Asn Cys Thr Glu Glu Ile Thr Glu465 470 475 480Gly Val Trp Asn Arg Lys Ala Asn Arg Lys Val Phe Gly Gln Trp Val 485 490 495Ser Ser Ser Thr Leu Asn Met Ser Glu Ala Ile Lys Gly Phe Leu Val 500 505 510Lys Cys Cys Ala Tyr Asn Ser Leu Gly Thr Ser Cys Glu Thr Ile Leu 515 520 525Leu Asn Ser Pro Gly Pro Phe Pro Phe Ile Gln Asp Asn Ile Ser Phe 530 535 540Tyr Ala Thr Ile Gly Val Cys Leu Leu Phe Ile Val Val Leu Thr Leu545 550 555 560Leu Ile Cys His Lys Tyr Lys Lys Gln Phe Arg Tyr Glu Ser Gln Leu 565 570 575Gln Met Val Gln Val Thr Gly Ser Ser Asp Asn Glu Tyr Phe Tyr Val 580 585 590Asp Phe Arg Glu Tyr Glu Tyr Asp Leu Lys Trp Glu Phe Pro Arg Glu 595 600 605Asn Leu Glu Phe Gly Lys Val Leu Gly Ser Glu Tyr Asp Leu Lys Trp 610 615 620Glu Phe Pro Arg Glu Asn Leu Glu Phe Gly Lys Val Leu Gly Ser Gly625 630 635 640Ala Phe Gly Lys Val Met Asn Ala Thr Ala Tyr Gly Ile Ser Lys Thr 645 650 655Gly Val Ser Ile Gln Val Ala Val Lys Met Leu Lys Glu Lys Ala Asp 660 665 670Ser Ser Glu Arg Glu Ala Leu Met Ser Glu Leu Lys Met Met Thr Gln 675 680 685Leu Gly Ser His Glu Asn Ile Val Asn Leu Leu Gly Ala Cys Thr Leu 690 695

700Ser Gly Pro Ile Tyr Leu Ile Phe Glu Tyr Cys Cys Tyr Gly Asp Leu705 710 715 720Leu Asn Tyr Leu Arg Ser Lys Arg Glu Lys Phe His Arg Thr Trp Thr 725 730 735Glu Ile Phe Lys Glu His Asn Phe Ser Phe Tyr Pro Thr Phe Gln Ser 740 745 750His Pro Asn Ser Ser Met Pro Gly Ser Arg Glu Val Gln Ile His Pro 755 760 765Asp Ser Asp Gln Ile Ser Gly Leu His Gly Asn Ser Phe His Ser Glu 770 775 780Asp Glu Ile Glu Tyr Glu Asn Gln Lys Arg Leu Glu Glu Glu Glu Asp785 790 795 800Leu Asn Val Leu Thr Phe Glu Asp Leu Leu Cys Phe Ala Tyr Gln Val 805 810 815Ala Lys Gly Met Glu Phe Leu Glu Phe Lys Ser Cys Val His Arg Asp 820 825 830Leu Ala Ala Arg Asn Val Leu Val Thr His Gly Lys Val Val Lys Ile 835 840 845Cys Asp Phe Gly Leu Ala Arg Asp Ile Met Ser Asp Ser Asn Tyr Val 850 855 860Val Arg Gly Asn Ala Arg Leu Pro Val Lys Trp Met Ala Pro Glu Ser865 870 875 880Leu Phe Glu Gly Ile Tyr Thr Ile Lys Ser Asp Val Trp Ser Tyr Gly 885 890 895Ile Leu Leu Trp Glu Ile Phe Ser Leu Gly Val Asn Pro Tyr Pro Gly 900 905 910Ile Pro Val Asp Ala Asn Phe Tyr Lys Leu Ile Gln Asn Gly Phe Lys 915 920 925Met Asp Gln Pro Phe Tyr Ala Thr Glu Glu Ile Tyr Ile Ile Met Gln 930 935 940Ser Cys Trp Ala Phe Asp Ser Arg Lys Arg Pro Ser Phe Pro Asn Leu945 950 955 960Thr Ser Phe Leu Gly Cys Gln Leu Ala Asp Ala Glu Glu Ala Met Tyr 965 970 975Gln Asn Val Asp Gly Arg Val Ser Glu Cys Pro His Thr Tyr Gln Asn 980 985 990Arg Arg18986PRTHomo sapiens 18Met Pro Ala Leu Ala Arg Asp Ala Gly Thr Val Pro Leu Leu Val Val1 5 10 15Phe Ser Ala Met Ile Phe Gly Thr Ile Thr Asn Gln Asp Leu Pro Val 20 25 30Ile Lys Cys Val Leu Ile Asn His Lys Asn Asn Asp Ser Ser Val Gly 35 40 45Lys Ser Ser Ser Tyr Pro Met Val Ser Glu Ser Pro Glu Asp Leu Gly 50 55 60Cys Ala Leu Arg Pro Gln Ser Ser Gly Thr Val Tyr Glu Ala Ala Ala65 70 75 80Val Glu Val Asp Val Ser Ala Ser Ile Thr Leu Gln Val Leu Val Asp 85 90 95Ala Pro Gly Asn Ile Ser Cys Leu Trp Val Phe Lys His Ser Ser Leu 100 105 110Asn Cys Gln Pro His Phe Asp Leu Gln Asn Arg Gly Val Val Ser Met 115 120 125Val Ile Leu Lys Met Thr Glu Thr Gln Ala Gly Glu Tyr Leu Leu Phe 130 135 140Ile Gln Ser Glu Ala Thr Asn Tyr Thr Ile Leu Phe Thr Val Ser Ile145 150 155 160Arg Asn Thr Leu Leu Tyr Thr Leu Arg Arg Pro Tyr Phe Arg Lys Met 165 170 175Glu Asn Gln Asp Ala Leu Val Cys Ile Ser Glu Ser Val Pro Glu Pro 180 185 190Ile Val Glu Trp Val Leu Cys Asp Ser Gln Gly Glu Ser Cys Lys Glu 195 200 205Glu Ser Pro Ala Val Val Lys Lys Glu Glu Lys Val Leu His Glu Leu 210 215 220Phe Gly Thr Asp Ile Arg Cys Cys Ala Arg Asn Glu Leu Gly Arg Glu225 230 235 240Cys Thr Arg Leu Phe Thr Ile Asp Leu Asn Gln Thr Pro Gln Thr Thr 245 250 255Leu Pro Gln Leu Phe Leu Lys Val Gly Glu Pro Leu Trp Ile Arg Cys 260 265 270Lys Ala Val His Val Asn His Gly Phe Gly Leu Thr Trp Glu Leu Glu 275 280 285Asn Lys Ala Leu Glu Glu Gly Asn Tyr Phe Glu Met Ser Thr Tyr Ser 290 295 300Thr Asn Arg Thr Met Ile Arg Ile Leu Phe Ala Phe Val Ser Ser Val305 310 315 320Ala Arg Asn Asp Thr Gly Tyr Tyr Thr Cys Ser Ser Ser Lys His Pro 325 330 335Ser Gln Ser Ala Leu Val Thr Ile Val Gly Lys Gly Phe Ile Asn Ala 340 345 350Thr Asn Ser Ser Glu Asp Tyr Glu Ile Asp Gln Tyr Glu Glu Phe Cys 355 360 365Phe Ser Val Arg Phe Lys Ala Tyr Pro Gln Ile Arg Cys Thr Trp Thr 370 375 380Phe Ser Arg Lys Ser Phe Pro Cys Glu Gln Lys Gly Leu Asp Asn Gly385 390 395 400Tyr Ser Ile Ser Lys Phe Cys Asn His Lys His Gln Pro Gly Glu Tyr 405 410 415Ile Phe His Ala Glu Asn Asp Asp Ala Gln Phe Thr Lys Met Phe Thr 420 425 430Leu Asn Ile Arg Arg Lys Pro Gln Val Leu Ala Glu Ala Ser Ala Ser 435 440 445Gln Ala Ser Cys Phe Ser Asp Gly Tyr Pro Leu Pro Ser Trp Thr Trp 450 455 460Lys Lys Cys Ser Asp Lys Ser Pro Asn Cys Thr Glu Glu Ile Thr Glu465 470 475 480Gly Val Trp Asn Arg Lys Ala Asn Arg Lys Val Phe Gly Gln Trp Val 485 490 495Ser Ser Ser Thr Leu Asn Met Ser Glu Ala Ile Lys Gly Phe Leu Val 500 505 510Lys Cys Cys Ala Tyr Asn Ser Leu Gly Thr Ser Cys Glu Thr Ile Leu 515 520 525Leu Asn Ser Pro Gly Pro Phe Pro Phe Ile Gln Asp Asn Ile Ser Phe 530 535 540Tyr Ala Thr Ile Gly Val Cys Leu Leu Phe Ile Val Val Leu Thr Leu545 550 555 560Leu Ile Cys His Lys Tyr Lys Lys Gln Phe Arg Tyr Glu Ser Gln Leu 565 570 575Gln Met Val Gln Val Thr Gly Ser Ser Asp Asn Glu Tyr Phe Tyr Val 580 585 590Asp Phe Arg Glu Tyr Glu Tyr Asp Leu Lys Trp Glu Phe Asp Phe Arg 595 600 605Glu Tyr Glu Tyr Asp Leu Lys Trp Glu Phe Pro Arg Glu Asn Leu Glu 610 615 620Phe Gly Lys Val Leu Gly Ser Gly Ala Phe Gly Lys Val Met Asn Ala625 630 635 640Thr Ala Tyr Gly Ile Ser Lys Thr Gly Val Ser Ile Gln Val Ala Val 645 650 655Lys Met Leu Lys Glu Lys Ala Asp Ser Ser Glu Arg Glu Ala Leu Met 660 665 670Ser Glu Leu Lys Met Met Thr Gln Leu Gly Ser His Glu Asn Ile Val 675 680 685Asn Leu Leu Gly Ala Cys Thr Leu Ser Gly Pro Ile Tyr Leu Ile Phe 690 695 700Glu Tyr Cys Cys Tyr Gly Asp Leu Leu Asn Tyr Leu Arg Ser Lys Arg705 710 715 720Glu Lys Phe His Arg Thr Trp Thr Glu Ile Phe Lys Glu His Asn Phe 725 730 735Ser Phe Tyr Pro Thr Phe Gln Ser His Pro Asn Ser Ser Met Pro Gly 740 745 750Ser Arg Glu Val Gln Ile His Pro Asp Ser Asp Gln Ile Ser Gly Leu 755 760 765His Gly Asn Ser Phe His Ser Glu Asp Glu Ile Glu Tyr Glu Asn Gln 770 775 780Lys Arg Leu Glu Glu Glu Glu Asp Leu Asn Val Leu Thr Phe Glu Asp785 790 795 800Leu Leu Cys Phe Ala Tyr Gln Val Ala Lys Gly Met Glu Phe Leu Glu 805 810 815Phe Lys Ser Cys Val His Arg Asp Leu Ala Ala Arg Asn Val Leu Val 820 825 830Thr His Gly Lys Val Val Lys Ile Cys Asp Phe Gly Leu Ala Arg Asp 835 840 845Ile Met Ser Asp Ser Asn Tyr Val Val Arg Gly Asn Ala Arg Leu Pro 850 855 860Val Lys Trp Met Ala Pro Glu Ser Leu Phe Glu Gly Ile Tyr Thr Ile865 870 875 880Lys Ser Asp Val Trp Ser Tyr Gly Ile Leu Leu Trp Glu Ile Phe Ser 885 890 895Leu Gly Val Asn Pro Tyr Pro Gly Ile Pro Val Asp Ala Asn Phe Tyr 900 905 910Lys Leu Ile Gln Asn Gly Phe Lys Met Asp Gln Pro Phe Tyr Ala Thr 915 920 925Glu Glu Ile Tyr Ile Ile Met Gln Ser Cys Trp Ala Phe Asp Ser Arg 930 935 940Lys Arg Pro Ser Phe Pro Asn Leu Thr Ser Phe Leu Gly Cys Gln Leu945 950 955 960Ala Asp Ala Glu Glu Ala Met Tyr Gln Asn Val Asp Gly Arg Val Ser 965 970 975 Glu Cys Pro His Thr Tyr Gln Asn Arg Arg 980 98519983PRTHomo sapiens 19Met Pro Ala Leu Ala Arg Asp Ala Gly Thr Val Pro Leu Leu Val Val1 5 10 15Phe Ser Ala Met Ile Phe Gly Thr Ile Thr Asn Gln Asp Leu Pro Val 20 25 30Ile Lys Cys Val Leu Ile Asn His Lys Asn Asn Asp Ser Ser Val Gly 35 40 45Lys Ser Ser Ser Tyr Pro Met Val Ser Glu Ser Pro Glu Asp Leu Gly 50 55 60Cys Ala Leu Arg Pro Gln Ser Ser Gly Thr Val Tyr Glu Ala Ala Ala65 70 75 80Val Glu Val Asp Val Ser Ala Ser Ile Thr Leu Gln Val Leu Val Asp 85 90 95Ala Pro Gly Asn Ile Ser Cys Leu Trp Val Phe Lys His Ser Ser Leu 100 105 110Asn Cys Gln Pro His Phe Asp Leu Gln Asn Arg Gly Val Val Ser Met 115 120 125Val Ile Leu Lys Met Thr Glu Thr Gln Ala Gly Glu Tyr Leu Leu Phe 130 135 140Ile Gln Ser Glu Ala Thr Asn Tyr Thr Ile Leu Phe Thr Val Ser Ile145 150 155 160Arg Asn Thr Leu Leu Tyr Thr Leu Arg Arg Pro Tyr Phe Arg Lys Met 165 170 175Glu Asn Gln Asp Ala Leu Val Cys Ile Ser Glu Ser Val Pro Glu Pro 180 185 190Ile Val Glu Trp Val Leu Cys Asp Ser Gln Gly Glu Ser Cys Lys Glu 195 200 205Glu Ser Pro Ala Val Val Lys Lys Glu Glu Lys Val Leu His Glu Leu 210 215 220Phe Gly Thr Asp Ile Arg Cys Cys Ala Arg Asn Glu Leu Gly Arg Glu225 230 235 240Cys Thr Arg Leu Phe Thr Ile Asp Leu Asn Gln Thr Pro Gln Thr Thr 245 250 255Leu Pro Gln Leu Phe Leu Lys Val Gly Glu Pro Leu Trp Ile Arg Cys 260 265 270Lys Ala Val His Val Asn His Gly Phe Gly Leu Thr Trp Glu Leu Glu 275 280 285Asn Lys Ala Leu Glu Glu Gly Asn Tyr Phe Glu Met Ser Thr Tyr Ser 290 295 300Thr Asn Arg Thr Met Ile Arg Ile Leu Phe Ala Phe Val Ser Ser Val305 310 315 320Ala Arg Asn Asp Thr Gly Tyr Tyr Thr Cys Ser Ser Ser Lys His Pro 325 330 335Ser Gln Ser Ala Leu Val Thr Ile Val Gly Lys Gly Phe Ile Asn Ala 340 345 350Thr Asn Ser Ser Glu Asp Tyr Glu Ile Asp Gln Tyr Glu Glu Phe Cys 355 360 365Phe Ser Val Arg Phe Lys Ala Tyr Pro Gln Ile Arg Cys Thr Trp Thr 370 375 380Phe Ser Arg Lys Ser Phe Pro Cys Glu Gln Lys Gly Leu Asp Asn Gly385 390 395 400Tyr Ser Ile Ser Lys Phe Cys Asn His Lys His Gln Pro Gly Glu Tyr 405 410 415Ile Phe His Ala Glu Asn Asp Asp Ala Gln Phe Thr Lys Met Phe Thr 420 425 430Leu Asn Ile Arg Arg Lys Pro Gln Val Leu Ala Glu Ala Ser Ala Ser 435 440 445Gln Ala Ser Cys Phe Ser Asp Gly Tyr Pro Leu Pro Ser Trp Thr Trp 450 455 460Lys Lys Cys Ser Asp Lys Ser Pro Asn Cys Thr Glu Glu Ile Thr Glu465 470 475 480Gly Val Trp Asn Arg Lys Ala Asn Arg Lys Val Phe Gly Gln Trp Val 485 490 495Ser Ser Ser Thr Leu Asn Met Ser Glu Ala Ile Lys Gly Phe Leu Val 500 505 510Lys Cys Cys Ala Tyr Asn Ser Leu Gly Thr Ser Cys Glu Thr Ile Leu 515 520 525Leu Asn Ser Pro Gly Pro Phe Pro Phe Ile Gln Asp Asn Ile Ser Phe 530 535 540Tyr Ala Thr Ile Gly Val Cys Leu Leu Phe Ile Val Val Leu Thr Leu545 550 555 560Leu Ile Cys His Lys Tyr Lys Lys Gln Phe Arg Tyr Glu Ser Gln Leu 565 570 575Gln Met Val Gln Val Thr Gly Ser Ser Asp Asn Glu Tyr Phe Tyr Val 580 585 590Asp Phe Arg Glu Tyr Glu Tyr Asp Leu Lys Trp Glu Phe Pro Arg Glu 595 600 605Asn Trp His Lys Trp Glu Phe Pro Arg Glu Asn Leu Glu Phe Gly Lys 610 615 620Val Leu Gly Ser Gly Ala Phe Gly Lys Val Met Asn Ala Thr Ala Tyr625 630 635 640Gly Ile Ser Lys Thr Gly Val Ser Ile Gln Val Ala Val Lys Met Leu 645 650 655Lys Glu Lys Ala Asp Ser Ser Glu Arg Glu Ala Leu Met Ser Glu Leu 660 665 670Lys Met Met Thr Gln Leu Gly Ser His Glu Asn Ile Val Asn Leu Leu 675 680 685Gly Ala Cys Thr Leu Ser Gly Pro Ile Tyr Leu Ile Phe Glu Tyr Cys 690 695 700Cys Tyr Gly Asp Leu Leu Asn Tyr Leu Arg Ser Lys Arg Glu Lys Phe705 710 715 720His Arg Thr Trp Thr Glu Ile Phe Lys Glu His Asn Phe Ser Phe Tyr 725 730 735Pro Thr Phe Gln Ser His Pro Asn Ser Ser Met Pro Gly Ser Arg Glu 740 745 750Val Gln Ile His Pro Asp Ser Asp Gln Ile Ser Gly Leu His Gly Asn 755 760 765Ser Phe His Ser Glu Asp Glu Ile Glu Tyr Glu Asn Gln Lys Arg Leu 770 775 780Glu Glu Glu Glu Asp Leu Asn Val Leu Thr Phe Glu Asp Leu Leu Cys785 790 795 800Phe Ala Tyr Gln Val Ala Lys Gly Met Glu Phe Leu Glu Phe Lys Ser 805 810 815Cys Val His Arg Asp Leu Ala Ala Arg Asn Val Leu Val Thr His Gly 820 825 830Lys Val Val Lys Ile Cys Asp Phe Gly Leu Ala Arg Asp Ile Met Ser 835 840 845Asp Ser Asn Tyr Val Val Arg Gly Asn Ala Arg Leu Pro Val Lys Trp 850 855 860Met Ala Pro Glu Ser Leu Phe Glu Gly Ile Tyr Thr Ile Lys Ser Asp865 870 875 880Val Trp Ser Tyr Gly Ile Leu Leu Trp Glu Ile Phe Ser Leu Gly Val 885 890 895Asn Pro Tyr Pro Gly Ile Pro Val Asp Ala Asn Phe Tyr Lys Leu Ile 900 905 910Gln Asn Gly Phe Lys Met Asp Gln Pro Phe Tyr Ala Thr Glu Glu Ile 915 920 925Tyr Ile Ile Met Gln Ser Cys Trp Ala Phe Asp Ser Arg Lys Arg Pro 930 935 940Ser Phe Pro Asn Leu Thr Ser Phe Leu Gly Cys Gln Leu Ala Asp Ala945 950 955 960Glu Glu Ala Met Tyr Gln Asn Val Asp Gly Arg Val Ser Glu Cys Pro 965 970 975His Thr Tyr Gln Asn Arg Arg 98020986PRTHomo sapiens 20Met Pro Ala Leu Ala Arg Asp Ala Gly Thr Val Pro Leu Leu Val Val1 5 10 15Phe Ser Ala Met Ile Phe Gly Thr Ile Thr Asn Gln Asp Leu Pro Val 20 25 30Ile Lys Cys Val Leu Ile Asn His Lys Asn Asn Asp Ser Ser Val Gly 35 40 45Lys Ser Ser Ser Tyr Pro Met Val Ser Glu Ser Pro Glu Asp Leu Gly 50 55 60Cys Ala Leu Arg Pro Gln Ser Ser Gly Thr Val Tyr Glu Ala Ala Ala65 70 75 80Val Glu Val Asp Val Ser Ala Ser Ile Thr Leu Gln Val Leu Val Asp 85 90 95Ala Pro Gly Asn Ile Ser Cys Leu Trp Val Phe Lys His Ser Ser Leu 100 105 110Asn Cys Gln Pro His Phe Asp Leu Gln Asn Arg Gly Val Val Ser Met 115 120 125Val Ile Leu Lys Met Thr Glu Thr Gln Ala Gly Glu Tyr Leu Leu Phe 130 135 140Ile Gln Ser Glu Ala Thr Asn Tyr Thr Ile Leu Phe Thr Val Ser Ile145 150 155 160Arg Asn Thr Leu Leu Tyr Thr Leu Arg Arg Pro Tyr Phe Arg Lys Met 165 170 175Glu Asn Gln Asp Ala Leu Val Cys Ile Ser Glu Ser Val Pro Glu Pro 180 185 190Ile Val Glu Trp Val Leu Cys Asp Ser Gln Gly Glu Ser Cys Lys Glu 195

200 205Glu Ser Pro Ala Val Val Lys Lys Glu Glu Lys Val Leu His Glu Leu 210 215 220Phe Gly Thr Asp Ile Arg Cys Cys Ala Arg Asn Glu Leu Gly Arg Glu225 230 235 240Cys Thr Arg Leu Phe Thr Ile Asp Leu Asn Gln Thr Pro Gln Thr Thr 245 250 255Leu Pro Gln Leu Phe Leu Lys Val Gly Glu Pro Leu Trp Ile Arg Cys 260 265 270Lys Ala Val His Val Asn His Gly Phe Gly Leu Thr Trp Glu Leu Glu 275 280 285Asn Lys Ala Leu Glu Glu Gly Asn Tyr Phe Glu Met Ser Thr Tyr Ser 290 295 300Thr Asn Arg Thr Met Ile Arg Ile Leu Phe Ala Phe Val Ser Ser Val305 310 315 320Ala Arg Asn Asp Thr Gly Tyr Tyr Thr Cys Ser Ser Ser Lys His Pro 325 330 335Ser Gln Ser Ala Leu Val Thr Ile Val Gly Lys Gly Phe Ile Asn Ala 340 345 350Thr Asn Ser Ser Glu Asp Tyr Glu Ile Asp Gln Tyr Glu Glu Phe Cys 355 360 365Phe Ser Val Arg Phe Lys Ala Tyr Pro Gln Ile Arg Cys Thr Trp Thr 370 375 380Phe Ser Arg Lys Ser Phe Pro Cys Glu Gln Lys Gly Leu Asp Asn Gly385 390 395 400Tyr Ser Ile Ser Lys Phe Cys Asn His Lys His Gln Pro Gly Glu Tyr 405 410 415Ile Phe His Ala Glu Asn Asp Asp Ala Gln Phe Thr Lys Met Phe Thr 420 425 430Leu Asn Ile Arg Arg Lys Pro Gln Val Leu Ala Glu Ala Ser Ala Ser 435 440 445Gln Ala Ser Cys Phe Ser Asp Gly Tyr Pro Leu Pro Ser Trp Thr Trp 450 455 460Lys Lys Cys Ser Asp Lys Ser Pro Asn Cys Thr Glu Glu Ile Thr Glu465 470 475 480Gly Val Trp Asn Arg Lys Ala Asn Arg Lys Val Phe Gly Gln Trp Val 485 490 495Ser Ser Ser Thr Leu Asn Met Ser Glu Ala Ile Lys Gly Phe Leu Val 500 505 510Lys Cys Cys Ala Tyr Asn Ser Leu Gly Thr Ser Cys Glu Thr Ile Leu 515 520 525Leu Asn Ser Pro Gly Pro Phe Pro Phe Ile Gln Asp Asn Ile Ser Phe 530 535 540Tyr Ala Thr Ile Gly Val Cys Leu Leu Phe Ile Val Val Leu Thr Leu545 550 555 560Leu Ile Cys His Lys Tyr Lys Lys Gln Phe Arg Tyr Glu Ser Gln Leu 565 570 575Gln Met Val Gln Val Thr Gly Ser Ser Asp Asn Glu Tyr Phe Tyr Val 580 585 590Asp Phe Arg Gly Ser Ser Asp Asn Glu Tyr Phe Tyr Val Asp Phe Arg 595 600 605Glu Tyr Glu Tyr Asp Leu Lys Trp Glu Phe Pro Arg Glu Asn Leu Glu 610 615 620Phe Gly Lys Val Leu Gly Ser Gly Ala Phe Gly Lys Val Met Asn Ala625 630 635 640Thr Ala Tyr Gly Ile Ser Lys Thr Gly Val Ser Ile Gln Val Ala Val 645 650 655Lys Met Leu Lys Glu Lys Ala Asp Ser Ser Glu Arg Glu Ala Leu Met 660 665 670Ser Glu Leu Lys Met Met Thr Gln Leu Gly Ser His Glu Asn Ile Val 675 680 685Asn Leu Leu Gly Ala Cys Thr Leu Ser Gly Pro Ile Tyr Leu Ile Phe 690 695 700Glu Tyr Cys Cys Tyr Gly Asp Leu Leu Asn Tyr Leu Arg Ser Lys Arg705 710 715 720Glu Lys Phe His Arg Thr Trp Thr Glu Ile Phe Lys Glu His Asn Phe 725 730 735Ser Phe Tyr Pro Thr Phe Gln Ser His Pro Asn Ser Ser Met Pro Gly 740 745 750Ser Arg Glu Val Gln Ile His Pro Asp Ser Asp Gln Ile Ser Gly Leu 755 760 765His Gly Asn Ser Phe His Ser Glu Asp Glu Ile Glu Tyr Glu Asn Gln 770 775 780Lys Arg Leu Glu Glu Glu Glu Asp Leu Asn Val Leu Thr Phe Glu Asp785 790 795 800Leu Leu Cys Phe Ala Tyr Gln Val Ala Lys Gly Met Glu Phe Leu Glu 805 810 815Phe Lys Ser Cys Val His Arg Asp Leu Ala Ala Arg Asn Val Leu Val 820 825 830Thr His Gly Lys Val Val Lys Ile Cys Asp Phe Gly Leu Ala Arg Asp 835 840 845Ile Met Ser Asp Ser Asn Tyr Val Val Arg Gly Asn Ala Arg Leu Pro 850 855 860Val Lys Trp Met Ala Pro Glu Ser Leu Phe Glu Gly Ile Tyr Thr Ile865 870 875 880Lys Ser Asp Val Trp Ser Tyr Gly Ile Leu Leu Trp Glu Ile Phe Ser 885 890 895Leu Gly Val Asn Pro Tyr Pro Gly Ile Pro Val Asp Ala Asn Phe Tyr 900 905 910Lys Leu Ile Gln Asn Gly Phe Lys Met Asp Gln Pro Phe Tyr Ala Thr 915 920 925Glu Glu Ile Tyr Ile Ile Met Gln Ser Cys Trp Ala Phe Asp Ser Arg 930 935 940Lys Arg Pro Ser Phe Pro Asn Leu Thr Ser Phe Leu Gly Cys Gln Leu945 950 955 960Ala Asp Ala Glu Glu Ala Met Tyr Gln Asn Val Asp Gly Arg Val Ser 965 970 975Glu Cys Pro His Thr Tyr Gln Asn Arg Arg 980 985212978DNAHomo sapiens 21atgccggcgt tggcgcgcga cgcgggcacc gtgccgctgc tcgttgtttt ttctgcaatg 60atatttggga ctattacaaa tcaagatctg cctgtgatca agtgtgtttt aatcaatcat 120aagaacaatg attcatcagt ggggaagtca tcatcatatc ccatggtatc agaatccccg 180gaagacctcg ggtgtgcgtt gagaccccag agctcaggga cagtgtacga agctgccgct 240gtggaagtgg atgtatctgc ttccatcaca ctgcaagtgc tggtcgatgc cccagggaac 300atttcctgtc tctgggtctt taagcacagc tccctgaatt gccagccaca ttttgattta 360caaaacagag gagttgtttc catggtcatt ttgaaaatga cagaaaccca agctggagaa 420tacctacttt ttattcagag tgaagctacc aattacacaa tattgtttac agtgagtata 480agaaataccc tgctttacac attaagaaga ccttacttta gaaaaatgga aaaccaggac 540gccctggtct gcatatctga gagcgttcca gagccgatcg tggaatgggt gctttgcgat 600tcacaggggg aaagctgtaa agaagaaagt ccagctgttg ttaaaaagga ggaaaaagtg 660cttcatgaat tatttgggac ggacataagg tgctgtgcca gaaatgaact gggcagggaa 720tgcaccaggc tgttcacaat agatctaaat caaactcctc agaccacatt gccacaatta 780tttcttaaag taggggaacc cttatggata aggtgcaaag ctgttcatgt gaaccatgga 840ttcgggctca cctgggaatt agaaaacaaa gcactcgagg agggcaacta ctttgagatg 900agtacctatt caacaaacag aactatgata cggattctgt ttgcttttgt atcatcagtg 960gcaagaaacg acaccggata ctacacttgt tcctcttcaa agcatcccag tcaatcagct 1020ttggttacca tcgtaggaaa gggatttata aatgctacca attcaagtga agattatgaa 1080attgaccaat atgaagagtt ttgtttttct gtcaggttta aagcctaccc acaaatcaga 1140tgtacgtgga ccttctctcg aaaatcattt ccttgtgagc aaaagggtct tgataacgga 1200tacagcatat ccaagttttg caatcataag caccagccag gagaatatat attccatgca 1260gaaaatgatg atgcccaatt taccaaaatg ttcacgctga atataagaag gaaacctcaa 1320gtgctcgcag aagcatcggc aagtcaggcg tcctgtttct cggatggata cccattacca 1380tcttggacct ggaagaagtg ttcagacaag tctcccaact gcacagaaga gatcacagaa 1440ggagtctgga atagaaaggc taacagaaaa gtgtttggac agtgggtgtc gagcagtact 1500ctaaacatga gtgaagccat aaaagggttc ctggtcaagt gctgtgcata caattccctt 1560ggcacatctt gtgagacgat ccttttaaac tctccaggcc ccttcccttt catccaagac 1620aacatctcat tctatgcaac aattggtgtt tgtctcctct tcattgtcgt tttaaccctg 1680ctaatttgtc acaagtacaa aaagcaattt aggtatgaaa gccagctaca gatggtacag 1740gtgaccggct cctcagataa tgagtacttc tacgttgaaa gccagctaca gatggtacag 1800gtgaccggct cctcagatag tgagtacttc tacgttgatt tcagagaata tgaatatgat 1860ctcaaatggg agtttccaag agaaaattta gagtttggga aggtactagg atcaggtgct 1920tttggaaaag tgatgaacgc aacagctttg gaattagcaa aacaggagtc tcaatccagg 1980ttgccgtcaa aatgctgaaa gaaaaagcag acagctctga aagagaggca ctcatgtcag 2040aactcaagat gatgacccag ctgggaagcc acgagaatat tgtgaacctg ctgggggcgt 2100gcacactgtc aggaccaatt tacttgattt ttgaatactg ttgctatggt gatcttctca 2160actatctaag aagtaaaaga gaaaaatttc acaggacttg gacagagatt ttcaaggaac 2220acaatttcag tttttacccc actttccaat cacatccaaa ttccagcatg cctggttcaa 2280gagaagttca gatacacccg gactcggatc aaatctcagg gcttcatggg aattcatttc 2340actctgaaga tgaaattgaa tatgaaaacc aaaaaaggct ggaagaagag gaggacttga 2400atgtgcttac atttgaagat cttctttgct ttgcatatca agttgccaaa ggaatggaat 2460ttctggaatt taagtcgtgt gttcacagag acctggccgc caggaacgtg cttgtcaccc 2520acgggaaagt ggtgaagata tgtgactttg gattggctcg agatatcatg agtgattcca 2580actatgttgt caggggcaat gcccgtctgc ctgtaaaatg gatggccccc gaaagcctgt 2640ttgaaggcat ctacaccatt aagagtgatg tctggtcata tggaatatta ctgtgggaaa 2700tcttctcact tggtgtgaat ccttaccctg gcattccggt tgatgctaac ttctacaaac 2760tgattcaaaa tggatttaaa atggatcagc cattttatgc tacagaagaa atatacatta 2820taatgcaatc ctgctgggct tttgactcaa ggaaacggcc atccttccct aatttgactt 2880cgtttttagg atgtcagctg gcagatgcag aagaagcgat gtatcagaat gtggatggcc 2940gtgtttcgga atgtcctcac acctaccaaa acaggcga 2978222982DNAHomo sapiens 22atgccggcgt tggcgcgcga cgcgggcacc gtgccgctgc tcgttgtttt ttctgcaatg 60atatttggga ctattacaaa tcaagatctg cctgtgatca agtgtgtttt aatcaatcat 120aagaacaatg attcatcagt ggggaagtca tcatcatatc ccatggtatc agaatccccg 180gaagacctcg ggtgtgcgtt gagaccccag agctcaggga cagtgtacga agctgccgct 240gtggaagtgg atgtatctgc ttccatcaca ctgcaagtgc tggtcgatgc cccagggaac 300atttcctgtc tctgggtctt taagcacagc tccctgaatt gccagccaca ttttgattta 360caaaacagag gagttgtttc catggtcatt ttgaaaatga cagaaaccca agctggagaa 420tacctacttt ttattcagag tgaagctacc aattacacaa tattgtttac agtgagtata 480agaaataccc tgctttacac attaagaaga ccttacttta gaaaaatgga aaaccaggac 540gccctggtct gcatatctga gagcgttcca gagccgatcg tggaatgggt gctttgcgat 600tcacaggggg aaagctgtaa agaagaaagt ccagctgttg ttaaaaagga ggaaaaagtg 660cttcatgaat tatttgggac ggacataagg tgctgtgcca gaaatgaact gggcagggaa 720tgcaccaggc tgttcacaat agatctaaat caaactcctc agaccacatt gccacaatta 780tttcttaaag taggggaacc cttatggata aggtgcaaag ctgttcatgt gaaccatgga 840ttcgggctca cctgggaatt agaaaacaaa gcactcgagg agggcaacta ctttgagatg 900agtacctatt caacaaacag aactatgata cggattctgt ttgcttttgt atcatcagtg 960gcaagaaacg acaccggata ctacacttgt tcctcttcaa agcatcccag tcaatcagct 1020ttggttacca tcgtaggaaa gggatttata aatgctacca attcaagtga agattatgaa 1080attgaccaat atgaagagtt ttgtttttct gtcaggttta aagcctaccc acaaatcaga 1140tgtacgtgga ccttctctcg aaaatcattt ccttgtgagc aaaagggtct tgataacgga 1200tacagcatat ccaagttttg caatcataag caccagccag gagaatatat attccatgca 1260gaaaatgatg atgcccaatt taccaaaatg ttcacgctga atataagaag gaaacctcaa 1320gtgctcgcag aagcatcggc aagtcaggcg tcctgtttct cggatggata cccattacca 1380tcttggacct ggaagaagtg ttcagacaag tctcccaact gcacagaaga gatcacagaa 1440ggagtctgga atagaaaggc taacagaaaa gtgtttggac agtgggtgtc gagcagtact 1500ctaaacatga gtgaagccat aaaagggttc ctggtcaagt gctgtgcata caattccctt 1560ggcacatctt gtgagacgat ccttttaaac tctccaggcc ccttcccttt catccaagac 1620aacatctcat tctatgcaac aattggtgtt tgtctcctct tcattgtcgt tttaaccctg 1680ctaatttgtc acaagtacaa aaagcaattt aggtatgaaa gccagctaca gatggtacag 1740gtgaccggct cctcagataa tgagtacttc tacgttgatt tcagagaata tgaatatgat 1800ctcaaatggg agtttccaag agaaaattta gagtttggga aggtactagg atccgaatat 1860gatctcaaat gggagtttcc aagagaaaat ttagagtttg ggaaggtact aggatcaggt 1920gcttttggaa aagtgatgaa cgcaacagct tatggaatta gcaaaacagg agtctcaatc 1980caggttgccg tcaaaatgct gaaagaaaaa gcagacagct ctgaaagaga ggcactcatg 2040tcagaactca agatgatgac ccagctggga agccacgaga atattgtgaa cctgctgggg 2100gcgtgcacac tgtcaggacc aatttacttg atttttgaat actgttgcta tggtgatctt 2160ctcaactatc taagaagtaa aagagaaaaa tttcacagga cttggacaga gattttcaag 2220gaacacaatt tcagttttta ccccactttc caatcacatc caaattccag catgcctggt 2280tcaagagaag ttcagataca cccggactcg gatcaaatct cagggcttca tgggaattca 2340tttcactctg aagatgaaat tgaatatgaa aaccaaaaaa ggctggaaga agaggaggac 2400ttgaatgtgc ttacatttga agatcttctt tgctttgcat atcaagttgc caaaggaatg 2460gaatttctgg aatttaagtc gtgtgttcac agagacctgg ccgccaggaa cgtgcttgtc 2520acccacggga aagtggtgaa gatatgtgac tttggattgg ctcgagatat catgagtgat 2580tccaactatg ttgtcagggg caatgcccgt ctgcctgtaa aatggatggc ccccgaaagc 2640ctgtttgaag gcatctacac cattaagagt gatgtctggt catatggaat attactgtgg 2700gaaatcttct cacttggtgt gaatccttac cctggcattc cggttgatgc taacttctac 2760aaactgattc aaaatggatt taaaatggat cagccatttt atgctacaga agaaatatac 2820attataatgc aatcctgctg ggcttttgac tcaaggaaac ggccatcctt ccctaatttg 2880acttcgtttt taggatgtca gctggcagat gcagaagaag cgatgtatca gaatgtggat 2940ggccgtgttt cggaatgtcc tcacacctac caaaacaggc ga 2982232958DNAHomo sapiens 23atgccggcgt tggcgcgcga cgcgggcacc gtgccgctgc tcgttgtttt ttctgcaatg 60atatttggga ctattacaaa tcaagatctg cctgtgatca agtgtgtttt aatcaatcat 120aagaacaatg attcatcagt ggggaagtca tcatcatatc ccatggtatc agaatccccg 180gaagacctcg ggtgtgcgtt gagaccccag agctcaggga cagtgtacga agctgccgct 240gtggaagtgg atgtatctgc ttccatcaca ctgcaagtgc tggtcgatgc cccagggaac 300atttcctgtc tctgggtctt taagcacagc tccctgaatt gccagccaca ttttgattta 360caaaacagag gagttgtttc catggtcatt ttgaaaatga cagaaaccca agctggagaa 420tacctacttt ttattcagag tgaagctacc aattacacaa tattgtttac agtgagtata 480agaaataccc tgctttacac attaagaaga ccttacttta gaaaaatgga aaaccaggac 540gccctggtct gcatatctga gagcgttcca gagccgatcg tggaatgggt gctttgcgat 600tcacaggggg aaagctgtaa agaagaaagt ccagctgttg ttaaaaagga ggaaaaagtg 660cttcatgaat tatttgggac ggacataagg tgctgtgcca gaaatgaact gggcagggaa 720tgcaccaggc tgttcacaat agatctaaat caaactcctc agaccacatt gccacaatta 780tttcttaaag taggggaacc cttatggata aggtgcaaag ctgttcatgt gaaccatgga 840ttcgggctca cctgggaatt agaaaacaaa gcactcgagg agggcaacta ctttgagatg 900agtacctatt caacaaacag aactatgata cggattctgt ttgcttttgt atcatcagtg 960gcaagaaacg acaccggata ctacacttgt tcctcttcaa agcatcccag tcaatcagct 1020ttggttacca tcgtaggaaa gggatttata aatgctacca attcaagtga agattatgaa 1080attgaccaat atgaagagtt ttgtttttct gtcaggttta aagcctaccc acaaatcaga 1140tgtacgtgga ccttctctcg aaaatcattt ccttgtgagc aaaagggtct tgataacgga 1200tacagcatat ccaagttttg caatcataag caccagccag gagaatatat attccatgca 1260gaaaatgatg atgcccaatt taccaaaatg ttcacgctga atataagaag gaaacctcaa 1320gtgctcgcag aagcatcggc aagtcaggcg tcctgtttct cggatggata cccattacca 1380tcttggacct ggaagaagtg ttcagacaag tctcccaact gcacagaaga gatcacagaa 1440ggagtctgga atagaaaggc taacagaaaa gtgtttggac agtgggtgtc gagcagtact 1500ctaaacatga gtgaagccat aaaagggttc ctggtcaagt gctgtgcata caattccctt 1560ggcacatctt gtgagacgat ccttttaaac tctccaggcc ccttcccttt catccaagac 1620aacatctcat tctatgcaac aattggtgtt tgtctcctct tcattgtcgt tttaaccctg 1680ctaatttgtc acaagtacaa aaagcaattt aggtatgaaa gccagctaca gatggtacag 1740gtgaccggct cctcagataa tgagtacttc tacgttgatt tcagagaata tgaatatgat 1800ctcaaatggg agtttgattt cagagaatat gaatatgatc tcaaatggga gtttccaaga 1860gaaaatttag agtttgggaa ggtactagga tcaggtgctt ttggaaaagt gatgaacgca 1920acagcttatg gaattagcaa aacaggagtc tcaatccagg ttgccgtcaa aatgctgaaa 1980gaaaaagcag acagctctga aagagaggca ctcatgtcag aactcaagat gatgacccag 2040ctgggaagcc acgagaatat tgtgaacctg ctgggggcgt gcacactgtc aggaccaatt 2100tacttgattt ttgaatactg ttgctatggt gatcttctca actatctaag aagtaaaaga 2160gaaaaatttc acaggacttg gacagagatt ttcaaggaac acaatttcag tttttacccc 2220actttccaat cacatccaaa ttccagcatg cctggttcaa gagaagttca gatacacccg 2280gactcggatc aaatctcagg gcttcatggg aattcatttc actctgaaga tgaaattgaa 2340tatgaaaacc aaaaaaggct ggaagaagag gaggacttga atgtgcttac atttgaagat 2400cttctttgct ttgcatatca agttgccaaa ggaatggaat ttctggaatt taagtcgtgt 2460gttcacagag acctggccgc caggaacgtg cttgtcaccc acgggaaagt ggtgaagata 2520tgtgactttg gattggctcg agatatcatg agtgattcca actatgttgt caggggcaat 2580gcccgtctgc ctgtaaaatg gatggccccc gaaagcctgt ttgaaggcat ctacaccatt 2640aagagtgatg tctggtcata tggaatatta ctgtgggaaa tcttctcact tggtgtgaat 2700ccttaccctg gcattccggt tgatgctaac ttctacaaac tgattcaaaa tggatttaaa 2760atggatcagc cattttatgc tacagaagaa atatacatta taatgcaatc ctgctgggct 2820tttgactcaa ggaaacggcc atccttccct aatttgactt cgtttttagg atgtcagctg 2880gcagatgcag aagaagcgat gtatcagaat gtggatggcc gtgtttcgga atgtcctcac 2940acctaccaaa acaggcga 2958242949DNAHomo sapiens 24atgccggcgt tggcgcgcga cgcgggcacc gtgccgctgc tcgttgtttt ttctgcaatg 60atatttggga ctattacaaa tcaagatctg cctgtgatca agtgtgtttt aatcaatcat 120aagaacaatg attcatcagt ggggaagtca tcatcatatc ccatggtatc agaatccccg 180gaagacctcg ggtgtgcgtt gagaccccag agctcaggga cagtgtacga agctgccgct 240gtggaagtgg atgtatctgc ttccatcaca ctgcaagtgc tggtcgatgc cccagggaac 300atttcctgtc tctgggtctt taagcacagc tccctgaatt gccagccaca ttttgattta 360caaaacagag gagttgtttc catggtcatt ttgaaaatga cagaaaccca agctggagaa 420tacctacttt ttattcagag tgaagctacc aattacacaa tattgtttac agtgagtata 480agaaataccc tgctttacac attaagaaga ccttacttta gaaaaatgga aaaccaggac 540gccctggtct gcatatctga gagcgttcca gagccgatcg tggaatgggt gctttgcgat 600tcacaggggg aaagctgtaa agaagaaagt ccagctgttg ttaaaaagga ggaaaaagtg 660cttcatgaat tatttgggac ggacataagg tgctgtgcca gaaatgaact gggcagggaa 720tgcaccaggc tgttcacaat agatctaaat caaactcctc agaccacatt gccacaatta 780tttcttaaag taggggaacc cttatggata aggtgcaaag ctgttcatgt gaaccatgga 840ttcgggctca cctgggaatt agaaaacaaa gcactcgagg agggcaacta ctttgagatg 900agtacctatt caacaaacag aactatgata cggattctgt ttgcttttgt atcatcagtg 960gcaagaaacg acaccggata ctacacttgt tcctcttcaa agcatcccag tcaatcagct 1020ttggttacca tcgtaggaaa gggatttata aatgctacca attcaagtga agattatgaa 1080attgaccaat atgaagagtt ttgtttttct gtcaggttta aagcctaccc acaaatcaga 1140tgtacgtgga ccttctctcg aaaatcattt ccttgtgagc aaaagggtct tgataacgga

1200tacagcatat ccaagttttg caatcataag caccagccag gagaatatat attccatgca 1260gaaaatgatg atgcccaatt taccaaaatg ttcacgctga atataagaag gaaacctcaa 1320gtgctcgcag aagcatcggc aagtcaggcg tcctgtttct cggatggata cccattacca 1380tcttggacct ggaagaagtg ttcagacaag tctcccaact gcacagaaga gatcacagaa 1440ggagtctgga atagaaaggc taacagaaaa gtgtttggac agtgggtgtc gagcagtact 1500ctaaacatga gtgaagccat aaaagggttc ctggtcaagt gctgtgcata caattccctt 1560ggcacatctt gtgagacgat ccttttaaac tctccaggcc ccttcccttt catccaagac 1620aacatctcat tctatgcaac aattggtgtt tgtctcctct tcattgtcgt tttaaccctg 1680ctaatttgtc acaagtacaa aaagcaattt aggtatgaaa gccagctaca gatggtacag 1740gtgaccggct cctcagataa tgagtacttc tacgttgatt tcagagaata tgaatatgat 1800ctcaaatggg agtttccaag agaaaattgg cacaaatggg agtttccaag agaaaattta 1860gagtttggga aggtactagg atcaggtgct tttggaaaag tgatgaacgc aacagcttat 1920ggaattagca aaacaggagt ctcaatccag gttgccgtca aaatgctgaa agaaaaagca 1980gacagctctg aaagagaggc actcatgtca gaactcaaga tgatgaccca gctgggaagc 2040cacgagaata ttgtgaacct gctgggggcg tgcacactgt caggaccaat ttacttgatt 2100tttgaatact gttgctatgg tgatcttctc aactatctaa gaagtaaaag agaaaaattt 2160cacaggactt ggacagagat tttcaaggaa cacaatttca gtttttaccc cactttccaa 2220tcacatccaa attccagcat gcctggttca agagaagttc agatacaccc ggactcggat 2280caaatctcag ggcttcatgg gaattcattt cactctgaag atgaaattga atatgaaaac 2340caaaaaaggc tggaagaaga ggaggacttg aatgtgctta catttgaaga tcttctttgc 2400tttgcatatc aagttgccaa aggaatggaa tttctggaat ttaagtcgtg tgttcacaga 2460gacctggccg ccaggaacgt gcttgtcacc cacgggaaag tggtgaagat atgtgacttt 2520ggattggctc gagatatcat gagtgattcc aactatgttg tcaggggcaa tgcccgtctg 2580cctgtaaaat ggatggcccc cgaaagcctg tttgaaggca tctacaccat taagagtgat 2640gtctggtcat atggaatatt actgtgggaa atcttctcac ttggtgtgaa tccttaccct 2700ggcattccgg ttgatgctaa cttctacaaa ctgattcaaa atggatttaa aatggatcag 2760ccattttatg ctacagaaga aatatacatt ataatgcaat cctgctgggc ttttgactca 2820aggaaacggc catccttccc taatttgact tcgtttttag gatgtcagct ggcagatgca 2880gaagaagcga tgtatcagaa tgtggatggc cgtgtttcgg aatgtcctca cacctaccaa 2940aacaggcga 2949252958DNAHomo sapiens 25atgccggcgt tggcgcgcga cgcgggcacc gtgccgctgc tcgttgtttt ttctgcaatg 60atatttggga ctattacaaa tcaagatctg cctgtgatca agtgtgtttt aatcaatcat 120aagaacaatg attcatcagt ggggaagtca tcatcatatc ccatggtatc agaatccccg 180gaagacctcg ggtgtgcgtt gagaccccag agctcaggga cagtgtacga agctgccgct 240gtggaagtgg atgtatctgc ttccatcaca ctgcaagtgc tggtcgatgc cccagggaac 300atttcctgtc tctgggtctt taagcacagc tccctgaatt gccagccaca ttttgattta 360caaaacagag gagttgtttc catggtcatt ttgaaaatga cagaaaccca agctggagaa 420tacctacttt ttattcagag tgaagctacc aattacacaa tattgtttac agtgagtata 480agaaataccc tgctttacac attaagaaga ccttacttta gaaaaatgga aaaccaggac 540gccctggtct gcatatctga gagcgttcca gagccgatcg tggaatgggt gctttgcgat 600tcacaggggg aaagctgtaa agaagaaagt ccagctgttg ttaaaaagga ggaaaaagtg 660cttcatgaat tatttgggac ggacataagg tgctgtgcca gaaatgaact gggcagggaa 720tgcaccaggc tgttcacaat agatctaaat caaactcctc agaccacatt gccacaatta 780tttcttaaag taggggaacc cttatggata aggtgcaaag ctgttcatgt gaaccatgga 840ttcgggctca cctgggaatt agaaaacaaa gcactcgagg agggcaacta ctttgagatg 900agtacctatt caacaaacag aactatgata cggattctgt ttgcttttgt atcatcagtg 960gcaagaaacg acaccggata ctacacttgt tcctcttcaa agcatcccag tcaatcagct 1020ttggttacca tcgtaggaaa gggatttata aatgctacca attcaagtga agattatgaa 1080attgaccaat atgaagagtt ttgtttttct gtcaggttta aagcctaccc acaaatcaga 1140tgtacgtgga ccttctctcg aaaatcattt ccttgtgagc aaaagggtct tgataacgga 1200tacagcatat ccaagttttg caatcataag caccagccag gagaatatat attccatgca 1260gaaaatgatg atgcccaatt taccaaaatg ttcacgctga atataagaag gaaacctcaa 1320gtgctcgcag aagcatcggc aagtcaggcg tcctgtttct cggatggata cccattacca 1380tcttggacct ggaagaagtg ttcagacaag tctcccaact gcacagaaga gatcacagaa 1440ggagtctgga atagaaaggc taacagaaaa gtgtttggac agtgggtgtc gagcagtact 1500ctaaacatga gtgaagccat aaaagggttc ctggtcaagt gctgtgcata caattccctt 1560ggcacatctt gtgagacgat ccttttaaac tctccaggcc ccttcccttt catccaagac 1620aacatctcat tctatgcaac aattggtgtt tgtctcctct tcattgtcgt tttaaccctg 1680ctaatttgtc acaagtacaa aaagcaattt aggtatgaaa gccagctaca gatggtacag 1740gtgaccggct cctcagataa tgagtacttc tacgttgatt tcagaggctc ctcagataat 1800gagtacttct acgttgattt cagagaatat gaatatgatc tcaaatggga gtttccaaga 1860gaaaatttag agtttgggaa ggtactagga tcaggtgctt ttggaaaagt gatgaacgca 1920acagcttatg gaattagcaa aacaggagtc tcaatccagg ttgccgtcaa aatgctgaaa 1980gaaaaagcag acagctctga aagagaggca ctcatgtcag aactcaagat gatgacccag 2040ctgggaagcc acgagaatat tgtgaacctg ctgggggcgt gcacactgtc aggaccaatt 2100tacttgattt ttgaatactg ttgctatggt gatcttctca actatctaag aagtaaaaga 2160gaaaaatttc acaggacttg gacagagatt ttcaaggaac acaatttcag tttttacccc 2220actttccaat cacatccaaa ttccagcatg cctggttcaa gagaagttca gatacacccg 2280gactcggatc aaatctcagg gcttcatggg aattcatttc actctgaaga tgaaattgaa 2340tatgaaaacc aaaaaaggct ggaagaagag gaggacttga atgtgcttac atttgaagat 2400cttctttgct ttgcatatca agttgccaaa ggaatggaat ttctggaatt taagtcgtgt 2460gttcacagag acctggccgc caggaacgtg cttgtcaccc acgggaaagt ggtgaagata 2520tgtgactttg gattggctcg agatatcatg agtgattcca actatgttgt caggggcaat 2580gcccgtctgc ctgtaaaatg gatggccccc gaaagcctgt ttgaaggcat ctacaccatt 2640aagagtgatg tctggtcata tggaatatta ctgtgggaaa tcttctcact tggtgtgaat 2700ccttaccctg gcattccggt tgatgctaac ttctacaaac tgattcaaaa tggatttaaa 2760atggatcagc cattttatgc tacagaagaa atatacatta taatgcaatc ctgctgggct 2820tttgactcaa ggaaacggcc atccttccct aatttgactt cgtttttagg atgtcagctg 2880gcagatgcag aagaagcgat gtatcagaat gtggatggcc gtgtttcgga atgtcctcac 2940acctaccaaa acaggcga 29582621DNAArtificial SequenceDescription of Artificial Sequence Synthetic DNA 26tgtcgagcag tactctaaac a 212722DNAArtificial SequenceDescription of Artificial Sequence Synthetic DNA 27atcctagtac cttcccaaac tc 222818DNAArtificial SequenceDescription of Artificial Sequence Synthetic DNA 28cttcctgggc atggagtc 182920DNAArtificial SequenceDescription of Artificial Sequence Synthetic DNA 29cgctcaggag gagcaatgat 203019DNAArtificial SequenceDescription of Artificial Sequence Synthetic DNA 30caatttaggt atgaaagcc 193119DNAArtificial SequenceDescription of Artificial Sequence Synthetic DNA 31caaactctaa attttctct 193221DNAArtificial SequenceDescription of Artificial Sequence Synthetic DNA 32tgtctttgca gggaaggtta c 213320DNAArtificial SequenceDescription of Artificial Sequence Synthetic DNA 33gtacctttca gcattttgac 20

Claims

1. A method of detecting mutations in FLT3 kinase indicative of a disease state, comprising:obtaining a human nucleic acid sample;amplifying a portion of a nucleic acid encoding a polypeptide having FLT3 kinase activity from the sample, thereby generating an amplicon;ascertaining the presence of a length mutation in the amplicon, wherein the length mutation is within a region defined by SEQ ID NO: 26 and SEQ ID NO: 33;wherein the presence of the length mutation is a prognostic for myelodysplastic syndrome (MDS) or leukemia.

2. The method of claim 1, wherein the ascertaining step comprises comparing the length of the amplicon to the length or expected length of an amplicon lacking a length mutation.

3. The method of claim 1, wherein the ascertaining step comprises sequencing the amplicon.

4. The method of claim 1, wherein the nucleic acid sample is selected from the group consisting of genomic DNA, cDNA, or mRNA.

5. The method of claim 1, wherein in the nucleic acid sample is obtained from a patient diagnosed with leukemia.

6. The method of claim 1, wherein the length mutation is not found in a wild-type gene.

7. The method of claim 1, wherein the amplifying step is performed with a primer pair selected from the group consisting of SEQ ID NOs:26 and 27, SEQ ID NOs:30 and 31, and SEQ ID NOs:32 and 33.

8. The method of claim 1, wherein the length mutation is a tandem duplication.

9. The method of claim 1, wherein the leukemia is acute myeloid leukemia.

10. The method of claim 9, wherein the acute myeloid leukemia is sub-classified as M2, M4, or M5 based on the French-American-British (FAB) classification system.

11. A method of screening a human subject having myelodysplastic syndrome (MDS) or leukemia, comprising:obtaining a nucleic acid sample from the subject; andascertaining whether the sample includes a length mutation in a region of the FLT3 gene defined by SEQ ID NO: 26 and SEQ ID NO: 33; and wherein the presence of said length mutation is indicative of poor prognosis.

12. The method of claim 11, wherein the length mutation is comprised of one or more tandem duplications of nucleotides.

13. The method of claim 12, wherein the length mutation further comprises one or more nucleotide insertions.

14. The method of claim 11, wherein the nucleic acid sample is selected from the group consisting of genomic DNA, cDNA, or RNA.

15. The method of claim 11, wherein the mutation is detected in a target region defined by a primer pair selected from the group consisting of SEQ ID NOs: 26 and 27, SEQ ID NOs: 30 and 31, and SEQ ID NOs: 32 and 33.

16. The method of claim 11, wherein the presence of a mutation is detected by gel electrophoresis or sequencing.

17. The method of claim 11, wherein the leukemia is acute myeloid leukemia (AML).

18. The method of claim 11, wherein the region of the FLT3 gene is defined by SEQ ID NO: 26 and SEQ ID NO: 27.

19. A kit for detecting mutations in FLT3 kinase indicative of a disease state comprising nucleic acid oligomers that are capable of priming the amplification of at least a portion of a nucleic acid encoding a polypeptide having FLT3 kinase activity from a human nucleic acid sample, wherein the primers direct the amplification of at least a portion of FLT3 kinase DNA defined by SEQ ID NO: 26 and SEQ ID NO: 33, and wherein the mutations are characterized by one or more tandem duplications.

20. The kit of claim 19, wherein the nucleic acid oligomers are selected to amplify a region defined by at least one of the following primer pairs: SEQ ID NOs:26 and 27, SEQ ID NOs:30 and 31, and SEQ ID NOs:32 and 33.

21. A kit for detecting mutations in FLT3 kinase indicative of a disease state comprising nucleic acid oligomers that are capable of priming the amplification of a portion of a nucleic acid encoding a polypeptide having FLT3 kinase activity from a human nucleic acid sample, wherein the amplified portion of FLT3 kinase nucleic acid corresponds to the region defined by SEQ ID NO: 26 and SEQ ID NO: 27 or a partial portion thereof, and wherein the mutations are characterized by one or more tandem duplications.

Images