# Patent

## Title
Nucleic acid encoding receptor type protein kinase

## Abstract

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; 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.

## Background
This application is a divisional of application Ser. No. 09/284,654, filed on Apr. 16, 1999, now abandoned and for which priority is claimed under 35 U.S.C. §120, application Ser. No. 09/284,654 is the national phase of PCT International Application No. PCT/JP97/03667 filed on Oct. 13, 1997 under 35 U.S.C. §371. The entire contents of each of the above-identified applications are hereby incorporated by reference. This application also claims priority of application Ser. No. 8-297329 filed in Japan on Oct. 18, 1996 under 35 U.S.C. §119.


TECHNICAL FIELD
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
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).
RTKs comprise an extracellular 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.
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.
Type II receptors have a structure comprising two subunits each for α and β, which are bound via S—S bond, wherein the α 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.
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.
Type IV receptors resemble type III receptors but have three immunoglobulin-like repeat sequences, and are exemplified by FGF receptor.
fms-Like tyrosine kinase 3 (hereinafter abbreviated as FLT3; Matthews, W., Cell 65, 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).
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.
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 368, 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.
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
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.
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.
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 in step (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; and
        step (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
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).
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
The present invention will be explained below.
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.
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, et al., Blood, 82 (4), 1110-1119 (1993)].
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.
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.
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.
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 amino 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.
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.
The nucleic acid of the present invention can be obtained by, e.g. the following method.
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.
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.
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.
In the present invention, leukemia cells are selected as the pathologic cells, and FLT3 is preferably targeted as the receptor protein kinase.
An FLT3 gene comprises 21 exons, and alternatively, the juxtamembrane is encoded in 18 bp at 3′-side of exon 10 and 117 bp at 5′-side of exon 11 [O. Rosnet, et 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 bp at 3′-side of exon 11 encode a partial portion of the tyrosine kinase domain.
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.
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.
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.
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.
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.
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.
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).
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.
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; and
    step (c): examining the presence of tandem duplication for the nucleic acid fragment of the above step (b).


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 et al., Cold Spring Harbor Laboratory Press, published in 1989).
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.
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 bp at 3′-side of exon 10 to 117 bp at 5′-side of exon 11, without being particularly limited thereto as long as the region contains an exon 11 site.
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.
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.
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. Acad. 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.
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).
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.
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.
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).
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.
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.
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.
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.
The diagnosis of the above AML etc. can be easily carried out by using such a kit.
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 sample; and
        step 2: examining the presence of tandem duplication in the nucleotide sequence of a juxtamembrane of the protein sample obtained in the above step 1.
    
    


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).
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.
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.
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.
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 of 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. Acad. 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).
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.
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.
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 86, 4105, 1995; Lisovsky, M., Blood 86, 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.
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.
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
1) Analysis of FLT3 Gene Expression Pattern
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 bp in length, when normal FLT3 has been transcribed.
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 SEQ 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 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 (63%)
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









It is found from Table 1 that the transcription product of the FLT3 gene was found in 39 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 bp 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 bp band and a longer band than the expected were detected. In one case (M162), the 366 bp band was not detected, and a longer band alone was detected.
2) Analysis of Nucleotide Sequence of Length Mutation Product of FLT3 Gene
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 bp or 60 bp tandem duplication within exon 11 was found in cases M34, M162, and M839; and a 26 bp tandem duplication including a 4 bp (GGCA) insert was found in case M810. In addition, case M155 was found to have a 63 bp tandem duplication comprising the first 16 bp of exon 12 immediately after exon 11, one cytosine residue insert, and the last 46 bp 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.
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.
3) Analysis of Nucleotide Sequence of Genomic DNA
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
Analysis of Mutations in Juxtamembrane of Receptor Protein Kinase and Their Pathologic Relationship
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.
The nucleotide sequences of exon regions encoding tyrosine kinase domain were also analyzed, and no mutations were found in these regions.
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.
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
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.

## Claims

1. A method for detecting diagnostically a nucleic acid encoding FLT3 kinase and having a tandem duplication mutation present in a nucleotide sequence of a juxtamembrane domain, comprising:
step (a) preparing a human nucleic acid sample; 
step (b): subjecting the nucleic acid sample obtained in step (a) to a gene amplification reaction, wherein a nucleic acid fragment comprising exon 11 or exons 11 to 12 of the FLT3 gene and having a tandem duplication mutation in the juxtamembrane domain is amplified; and 
step (c): detecting the presence of the tandem duplication mutation in the nucleic acid fragment of said step (b), 
wherein the presence of a tandem duplication mutation is indicative of a disease selected from the group consisting of leukemia or Myelodysplastic syndrome, wherein said disease is associated with tandem duplication mutation in a nucleotide sequence of the juxtamembrane domain.

2. The method according to claim 1, further comprising:
step (d): comparing the amplified nucleic acid fragment obtained in step (b) to a sequence derived from a normal receptor protein kinase, thereby detecting the presence of tandem duplication mutation in the juxtamembrane domain.

3. The method according to claim 1, wherein the tandem duplication mutation is a length mutation.

4. The method according to claim 1, wherein the gene amplification reaction of step (b) is carried out 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.

5. The method according to claim 1, wherein the tandem duplication mutation is not found in a wild-type gene.

6. A kit for detection of a nucleic acid encoding a FLT3 kinase and having tandem duplication mutation in the nucleotide sequence of a juxtamembrane, comprising primers 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 for amplifying a region comprising exon 11 or exons 11. to 12 of the PLT3 gene and having tandem duplication mutation, wherein the region can be found in the FLT3 kinase gene.