JP6817608B2 - A method for obtaining an index for testing the onset or risk of developing bone marrow tumor in humans, a method for obtaining an index for predicting the presence or future occurrence of a somatic mutation of the DDX41 gene in humans, and for these tests or predictions. kit - Google Patents

Description

The present invention relates to a method for examining the onset or risk of developing bone marrow tumors such as myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML) in humans, a method for obtaining an index for the examination, and the body of the DDX41 gene in humans. The present invention relates to a method for predicting the existence or future development of a cell mutation, a method for obtaining an index for prediction, and a kit for these methods.

Myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML) are both classified as bone marrow tumors and are thought to transition to AML after the onset of MDS. Since both diseases develop at middle and old age, the gene mutation associated with the onset has been previously considered to be an acquired gene mutation (somatic mutation). However, the inventors conducted genetic analysis on MDS / AML patients of Caucasian race and healthy subjects, and found that some MDS / AML patients had a embryonic cell mutation of the DDX41 gene, and the mutation was inherited. Then, it is reported for the first time that MDS or AML develops in almost all cases (Non-Patent Document 1).

Polprasert et al., 2015, Cancer Cell 27, 658-670

As mentioned above, in MDS / AML, it has been considered that the gene mutation associated with the onset is an acquired gene mutation (somatic mutation), and by examining the embryonic cell mutation, the onset of MDS / AML or It has traditionally been considered impossible to test for the risk of developing the disease.

Non-Patent Document 1 reports that embryonic cell mutations specific to MDS / AML patients in Caucasian races are observed, but the mutation sites are the same in yellow races such as East Asian races. Not exclusively. In addition, while familial cases of MDS / AML are known in the Caucasian race, only sporadic cases of MDS / AML are known in the yellow race, and familial cases have not been known in the past. Therefore, MDS / AML in the Yellow race was considered to be different from MDS / AML in the Caucasian race. Moreover, unlike familial diseases, in sporadic diseases it is difficult to identify embryonic cell mutations that correlate with the disease. Therefore, it was not possible to apply the findings disclosed in Non-Patent Document 1 to investigating embryonic cell mutations in yellow races and examining the onset or risk of developing MDS / AML.

Therefore, an object of the present invention is to provide a means for examining the onset or risk of developing bone marrow tumors such as MDS and AML in humans, especially yellow races such as East Asian races. It is also an object of the present invention to provide a means for obtaining useful indicators for the treatment of bone marrow tumors such as MDS and AML.

The present invention provides the following invention as a means for solving the following problems.
(1) A method for obtaining an index for examining the onset or risk of developing bone marrow tumor in humans.
At position 500, position 259, position 363, position 364, position 3, position 7, position 35, and position 187 in the amino acid sequence shown in SEQ ID NO: 2 of the polypeptide encoded by the wild-type DDX41 gene. And a method characterized by detecting one or more embryonic cell mutations in the DDX41 gene that result in mutations in one or more amino acids selected from the group consisting of position 256.
(2) The mutation in the amino acid is in the amino acid sequence shown in SEQ ID NO: 2.
Frameshift mutation in the 500th place alanine,
Substitution of tyrosine at position 259 with cysteine,
Substitution of serine at position 363 and tyrosine at position 364 with one tyrosine, or deletion of serine at position 363,
Substitution of glutamic acid at position 3 with lysine,
Nonsense mutation in glutamic acid at position 7,
Substitution of 35th place proline with arginine,
The method according to (1), wherein the mutation is one or more selected from the group consisting of the substitution of lysine at position 187 with arginine and the substitution of glutamic acid at position 256 with lysine.
(3) A mutation in the DDX41 gene is used as the position in the cDNA base sequence of wild-type DDX41 shown in SEQ ID NO: 1.
Overlapping cytosine in 1496,
Substitution of adenine at position 776 with guanine,
Cytosine-cytosine-thymine deletion at positions 1088 to 1090 or thymine-cytosine-cytosine deletion at positions 1087 to 1089,
Substitution of 7th place guanine with adenine,
Substitution of guanine at position 19 with thymine,
Substitution of cytosine at position 104 with guanine,
Substitution of adenine at position 560 with guanine, and substitution of guanine at position 766 with guanine,
The method according to (1) or (2), which is one or more mutations selected from the group consisting of.
(4) The mutation in the amino acid is in the amino acid sequence shown in SEQ ID NO: 2.
Frameshift mutation in alanine at position 500, and substitution of tyrosine at position 259 with cysteine,
The method according to (2), which is one or more of the mutations.
(5) A mutation in the DDX41 gene is used as the position in the cDNA base sequence of wild-type DDX41 shown in SEQ ID NO: 1.
Overlapping cytosine at position 1496 and replacement of adenine at position 776 with guanine,
The method according to (3), which is one or more of the mutations.
(6) The method according to any one of (1) to (5), wherein the bone marrow tumor is myelodysplastic syndrome (MDS) and / or acute myeloid leukemia (AML).
(7) The method according to (6), wherein the onset or risk of developing MDS and / or AML to be examined is the onset or risk of developing advanced MDS and / or AML.
(8) The method according to any one of (1) to (7), further comprising detecting a mutation in the CUX1 gene.
(9) A method for obtaining an index for predicting the existence or future development of a somatic mutation in the DDX41 gene in humans.
At position 500, position 259, position 363, position 364, position 3, position 7, position 35, and position 187 in the amino acid sequence shown in SEQ ID NO: 2 of the polypeptide encoded by the wild-type DDX41 gene. And a method characterized by detecting one or more embryonic cell mutations in the DDX41 gene that result in mutations in one or more amino acids selected from the group consisting of position 256.
(10) A somatic mutation in the DDX41 gene
The method according to (9), which is a somatic mutation of the DDX41 gene that causes a mutation of arginine at position 525 in the amino acid sequence shown in SEQ ID NO: 2 encoded by the wild-type DDX41 gene.
(11) A kit used for at least one of a method for examining the onset or risk of developing bone marrow tumor in humans and a method for predicting the presence or future occurrence of a somatic mutation of the DDX41 gene in humans. ,
Of the DDX41 genes, when mutated, the amino acid sequence shown in SEQ ID NO: 2 of the polypeptide encoded by the wild-type DDX41 gene is position 500, position 259, position 363, position 364, position 3, and position 3. A primer set designed to amplify sites containing one or more bases that cause mutations in one or more amino acids selected from the group consisting of positions 7, 35, 187, and 256.
Of the DDX41 genes, when mutated, the amino acid sequence shown in SEQ ID NO: 2 of the polypeptide encoded by the wild-type DDX41 gene is position 500, position 259, position 363, position 364, position 3, and position 3. Probes designed to bind to sites containing one or more bases that mutate at one or more amino acids selected from the group consisting of positions 7, 35, 187, and 256, and probes.
Among the polypeptides translated by the DDX41 gene, the wild-type amino acid sequence shown in SEQ ID NO: 2 is located at positions 500, 259, 363, 364, 3, 7, and 35. , A kit containing one or more selected from the group consisting of antibodies that recognize sites containing amino acids corresponding to one or more amino acids selected from the group consisting of positions 187 and 256.
(12) The kit according to (11), further comprising a reagent for detecting a mutation in the CUX1 gene.

According to the present invention, it is possible to obtain an index for examining the onset or risk of developing bone marrow tumors such as MDS and AML in humans, particularly yellow races such as East Asian races. Each mutation is an embryonic cell mutation, and in general, bone marrow tumors such as MDS and AML develop at an early age and therefore have a high risk of being inherited to offspring. However, according to the present invention, bone marrow tumors are detected early from the presymptomatic stage. It is possible to lead to prevention.

According to another aspect of the present invention, it is possible to obtain an index for predicting the existence or future development of a somatic mutation of the DDX41 gene in humans, particularly yellow races such as East Asian races. Some of the somatic mutations in the DDX41 gene cause mutations in the amino acid sequence of the polypeptide encoded by the DDX41 gene, and such mutations in the amino acid sequence are effective targets for new drug development in bone marrow tumors. ..

It is a figure which shows the mutation of the DDX41 gene found in the Example in an MDS / AML patient. It is a figure which showed the ratio of the DDX41 gene mutation case in the MDS / AML case by race. It is a figure which shows the ratio which the DDX41 gene mutation case is CUX1 somatic mutation positive, and DDX41 wild type case is CUX1 somatic mutation positive among MDS / AML cases. It is a figure which shows the ratio of the DDX41 gene mutation positive case for each of the low-risk case and the high-risk case among MDS / AML cases. It is a figure for demonstrating the rate of embryonic cell mutation of the DDX41 gene in MDS / AML cases and healthy subjects. It is a figure which shows the geographical distribution of the DDX41 gene mutation.

Hereinafter, the present invention will be described in detail.
<Gene>
In the present invention, the "gene" includes polynucleotides such as genomic DNA, mRNA, and cDNA, and may be artificially prepared as well as naturally occurring ones. The terms "DDX41 gene" and "CUX1 gene" refer to polynucleotides that retain the genetic information of these genes, and are not limited to the sense strand that encodes the amino acid sequence, but antisense, which is the complementary strand (template strand) thereof. It may be a chain, not necessarily the whole, but a partial fragment. Further, the polynucleotide may be a double strand of DNA or a single strand of DNA or RNA.

The DDX41 (DEAD (Asp-Glu-Ala-Asp) box polypeptide 41, DEAD (Asp-Glu-Ala-Asp) box polypeptide 41) gene is located on human chromosome 5 and its representative cDNA base sequence ( The CDS of GenBank Accession No. NM_016222) is as shown in SEQ ID NO: 1, and the amino acid sequence of the polypeptide encoded by the nucleotide sequence shown in SEQ ID NO: 1 is as shown in SEQ ID NO: 2. In the present invention, the position of the base in the cDNA base sequence shown in SEQ ID NO: 1 is used to indicate the position of the base contained in the DDX41 gene, but the position is other than the cDNA base sequence shown in SEQ ID NO: 1 by the DDX41 gene. In the case of each polynucleotide such as a morphological gene, for example, genomic DNA, mRNA (which may be mRNA of another isoform), cDNA (which may be cDNA of another isoform), the SEQ ID NO: in each morphological gene. It means a base at a position corresponding to the position of the cDNA base sequence shown in 1. For example, when expressed as "overlapping of cytosine at position 1496 as the position in the cDNA base sequence of wild-type DDX41 shown in SEQ ID NO: 1," genomic DNA, mRNA (may be mRNA of another isoform), cDNA (others). At the position corresponding to the cytosine at position 1496 of the cDNA base sequence shown in SEQ ID NO: 1 in each polynucleotide such as (which may be the cDNA of the isoform of), the base corresponding to the cytosine on the cDNA (genomic DNA sense). It includes mutations in which two duplicates (guanine in genomic DNA antisense strands and cDNA antisense strands) are present, which is cytosine in strands, cDNA sense strands and mRNAs.

The CUX1 (cut-like homeobox 1) gene is located on human chromosome 7, and its typical cDNA base sequence (GenBank Accession No. NM_001202543 CDS) is as shown in SEQ ID NO: 3. Yes, the amino acid sequence of the polypeptide encoded by the nucleotide sequence shown in SEQ ID NO: 3 is as shown in SEQ ID NO: 4. As for the CUX1 gene, as in the DDX41 gene, the base position in the cDNA base sequence shown in SEQ ID NO: 3 is used to indicate the position of the base contained in the CUX1 gene.

<Method of obtaining an index for testing the onset or risk of developing bone marrow tumor in humans>
In the present invention, the "method for obtaining an index for examining the onset or risk of developing bone marrow tumor in humans" is "a method for obtaining data (or index) for determining the onset or risk of developing bone marrow tumor in humans", It can also be expressed as "a method of assisting (or supporting) the examination of the onset or risk of developing bone marrow tumor in humans".

The human being who is the target of the method of the present invention is not particularly limited, and may be a human who has already developed a bone marrow tumor or a human who has not developed a bone marrow tumor at the time of carrying out the method. Good. The human race is preferably the yellow race, more preferably the East Asian race. East Asians refer to people with ancestors from the eastern part of the Asian continent and its surroundings, including Japan, the Korean Peninsula, and China.

The method of the present invention can be carried out using a sample separated from the human to be inspected.
The method of the present invention is located at positions 500, 259, 363, 364, 3 and 7, in the amino acid sequence shown in SEQ ID NO: 2 of the polypeptide encoded by the wild-type DDX41 gene. It is characterized by detecting one or more embryonic cell mutations in the DDX41 gene that result in mutations in one or more amino acids selected from the group consisting of positions 35, 187 and 256. These mutations may be mutations that alter or abolish the function of the polypeptide encoded by the DDX41 gene.

It can be determined that the detected mutation is not a somatic mutation but an embryonic cell mutation, for example, by confirming the presence of the mutation in a sample obtained from the normal tissue of the subject, and more preferably from the normal tissue. It can be determined by confirming the presence of mutations in both the obtained sample and the sample obtained from the tumor tissue of the bone marrow tumor or the tissue suspected to be the tumor tissue. When using only samples obtained from normal tissue, multiple measurements are performed, and / or measurements are performed using samples obtained from multiple locations in normal tissue, and the mutation is confirmed by confirming the mutation. It is preferable to judge that it is a cell mutation.

In the method of the present invention, among the group consisting of the 500th position, the 259th position, the 363rd position, the 364th position, the 3rd position, the 7th position, the 35th position, the 187th position and the 256th position of SEQ ID NO: 2. , At least one embryonic cell mutation of the DDX41 gene that results in a mutation at at least one amino acid may be detected, but the group is more preferably at positions 500, 259, 363 and 364. And 35th place, more preferably 500th place and 259th place, and most preferably 500th place. The present inventors have found that humans having these embryonic cell mutations have a particularly high risk of developing bone marrow tumors such as MDS and AML.

In the present invention, each mutation of an amino acid or a base in a gene in a polypeptide is specified by a predetermined expression of 1 or 2, but the same mutation may be specified by yet another expression, and they are all the same. Treat as a mutation in.

In the method of the invention, if a human subject has one or more of the embryonic mutations in the DDX41 gene at at least one of the allelic loci of the DDX41 gene, the human subject will develop a bone marrow tumor. It can be determined that there is a risk of developing the disease in the future even if the disease does not occur at this time. In addition, within the scope of the investigation by the present inventors shown in Examples, all DDX41 embryo cell mutation-positive patients have only one of the above DDX41 embryo cell mutations, and the mutation is an allele. Only one of the loci is allowed.

The mutations of each of the above amino acids in the amino acid sequence shown in SEQ ID NO: 2 of the polypeptide encoded by the wild-type DDX41 gene are missense mutation (mutation to another amino acid) and nonsense mutation (mutation to termination codon in the gene). ), Frame shift mutation (a mutation in which the amino acid corresponding to the mutation position of the base in the gene and / or the amino acid sequence downstream of it changes due to the insertion and / or deletion of the base in the gene), in-frame missing Includes loss mutation and the like. These amino acid mutations at each of the above positions of SEQ ID NO: 2 may involve mutations in the amino acid sequence at each position in SEQ ID NO: 2 as well as the amino acid sequence downstream thereof.

The mutation at each amino acid may be any of the above, but typically the mutation at the amino acid is in the amino acid sequence shown in SEQ ID NO: 2.
Frameshift mutation in the 500th place alanine,
Substitution of tyrosine at position 259 with cysteine,
Substitution of serine at position 363 and tyrosine at position 364 with one tyrosine, or deletion of serine at position 363,
Substitution of glutamic acid at position 3 with lysine,
Nonsense mutation in glutamic acid at position 7,
Substitution of 35th place proline with arginine,
One or more mutations selected from the group consisting of the substitution of lysine at position 187 with arginine and the substitution of glutamic acid at position 256 with lysine.
More preferably
In the amino acid sequence shown in SEQ ID NO: 2,
Frameshift mutation in the 500th place alanine,
Substitution of tyrosine at position 259 with cysteine,
One or more mutations in the substitution of serine at position 363 and tyrosine at position 364 with one tyrosine, or the deletion of serine at position 363, and the substitution of proline at position 35 with arginine.
Particularly preferably, in the amino acid sequence shown in SEQ ID NO: 2.
Frameshift mutation in alanine at position 500, and substitution of tyrosine at position 259 with cysteine,
One or more of these mutations.

The frameshift mutation of alanine at position 500 in SEQ ID NO: 2 may result from duplication of cytosine at position 1496 as a position in the cDNA sequence of wild-type DDX41 shown in SEQ ID NO: 1 in the DDX41 gene. Preferred but not limited to this. Here, "overlapping cytosine at position 1496" is expressed as the position in the cDNA base sequence of wild-type DDX41 shown in SEQ ID NO: 1 in which 1-base cytosine at position 1496 is replaced with 2-base cytosine-cytosine. It is a mutation that can be expressed by various expressions such as "insertion of cytosine between positions 1495 and 1496" and "overlap of cytosine at position 1495".

The substitution of tyrosine at position 259 with cysteine in SEQ ID NO: 2 is caused by the substitution of adenine at position 776 with guanine as the position in the cDNA base sequence of wild-type DDX41 shown in SEQ ID NO: 1 in the DDX41 gene. However, the present invention is not limited to this.

Substitution of thymine at position 363 and thymine at position 364 to one thymine in SEQ ID NO: 2 (or deletion of cytosine at position 363) in the DDX41 gene of the wild-type DDX41 shown in SEQ ID NO: 1. The position in the cDNA base sequence is preferably caused by a deletion of cytosine-cytosine-thymine at positions 1088 to 1090 (or a deletion of thymine-cytosine-thymine at positions 1087 to 1089). However, it is not limited to this.

The substitution of glutamic acid at position 3 with lysine in SEQ ID NO: 2 is caused by the substitution of guanine at position 7 with adenine as the position in the cDNA base sequence of wild-type DDX41 shown in SEQ ID NO: 1 in the DDX41 gene. However, the present invention is not limited to this.

The nonsense mutation of glutamic acid at position 7 in SEQ ID NO: 2 is caused by the replacement of guanine at position 19 with thymine as a position in the cDNA base sequence of wild-type DDX41 shown in SEQ ID NO: 1 in the DDX41 gene. It is preferable, but not limited to this.

The substitution of proline at position 35 with arginine in SEQ ID NO: 2 is caused by the substitution of cytosine at position 104 with guanine as the position in the cDNA base sequence of wild-type DDX41 shown in SEQ ID NO: 1 in the DDX41 gene. However, the present invention is not limited to this.

The substitution of lysine at position 187 with arginine in SEQ ID NO: 2 results from the substitution of adenine at position 560 with guanine as the position in the cDNA base sequence of wild-type DDX41 shown in SEQ ID NO: 1 in the DDX41 gene. However, the present invention is not limited to this.

The substitution of glutamic acid at position 256 with lysine in SEQ ID NO: 2 is caused by the substitution of guanine at position 766 with adenine as the position in the cDNA base sequence of wild-type DDX41 shown in SEQ ID NO: 1 in the DDX41 gene. However, the present invention is not limited to this.

In the present invention, when one or more of the above embryonic cell mutations is detected in a sample isolated from a subject, the subject has developed a bone marrow tumor, or even if it has not developed at this time, in the future. It can be used as an index for determining that there is a risk of developing the disease. In the present invention, "myeloproliferative neoplasm" refers to a neoplastic disease derived from myeloid cells, such as myelodysplastic syndrome (MDS), acute myeloid leukemia (AML), and MDS / MPN (myeloproliferative neoplasm). ), MPN (myeloproliferative neoplasm) and other diseases. The method of the present invention is particularly effective when the "bone marrow tumor" is MDS and / or AML. As shown in FIG. 4, the present inventors have particularly frequently performed the above-mentioned embryonic cell mutations in cases of advanced MDS in which myeloblasts exceed 5% in the bone marrow and cases of AML among MDS and / or AML cases. I found. From this, it is determined that the subject in which the embryonic cell mutation is detected by the method of the present invention has advanced MDS or AML, or is at risk of developing it in the future even if it does not develop at present. You can get the index to do.

In the method of the present invention, the means for detecting the presence or absence of each mutation is not particularly limited, and any means can be adopted. Each mutation may be detected by detecting the presence or absence of a mutation in a base that causes a mutation in each amino acid in the DDX41 gene, or the presence or absence of a mutation in each amino acid in a polypeptide encoded by the DDX41 gene. It may be done by. In the present invention, the presence of a mutation in the base of the DDX41 gene that causes a mutation in one or more amino acids in SEQ ID NO: 2 means that a mutant poly in which a predetermined amino acid is mutated as a transcription / translation product of the mutated DDX41 gene. The peptide does not have to actually be present in the subject, but a mutation in the base of the DDX41 gene that gives rise to a mutant polypeptide in which a given amino acid is mutated, assuming that the mutant DDX41 gene is transcribed and translated. (The same applies to mutations in other genes). Methods for detecting mutations in the DDX41 gene include a method of nucleic acid amplification using a primer set and sequencing of the amplification product, a method of using a probe that specifically hybridizes to the mutation site, a method of using real-time PCR and a DNA chip, etc. Can be mentioned. Methods for detecting the presence or absence of mutations in each amino acid in the polypeptide encoded by the DDX41 gene include immunostaining and protein array methods.

Hereinafter, embodiments of detecting mutations using genomic DNA, mRNA (or cDNA prepared from mRNA), and polypeptide contained in a sample isolated from a human living body will be individually described.

(1) Embodiment of detecting mutation in genomic DNA When a mutation is detected using the genomic DNA contained in a sample isolated from human, the genomic DNA is extracted from the sample isolated from human. The extraction method is not particularly limited, and a known method can be used.

The sample isolated from humans is not particularly limited as long as it can extract genomic DNA. Specifically, for example, a pathological specimen (for example, a stomach for detecting Helicobacter pylori) when blood, bone marrow fluid, hair, nails, oral mucosa, skin, urine, stool, and other organs are collected for another purpose in the past. Mucosal biopsy, etc.) and the like. Alternatively, cells isolated from humans may be cultured and genomic DNA may be extracted from the proliferated cells.

In addition, the extracted genomic DNA is, for example, PCR (Polymerase Chain Reaction) method, LAMP (Loop-Mediated Isothermal Amplification) method, NASBA (Nucleic acid sequence based amplification) method, TMA (Transcription-mediated amplification) method, SDA (Strand). It may be amplified by a commonly used gene amplification method such as Displacement Amplification) method or ICAN (Isothermal and Chimeric primer-initiated Amplification of Nucleic acids) method. At this time, the region containing at least one mutation of the DDX41 gene to be analyzed may be amplified.

Since the mutation analyzed in the present invention is a mutation contained in the exon region of the DDX41 gene, a sample obtained by cutting out only the exon region from genomic DNA may be used as a detection target sample.

The method for detecting the presence or absence of mutation in the DDX41 gene using a sample containing the prepared genomic DNA is not particularly limited, but for example, a method for sequencing genomic DNA or an amplification product, specific. Oligonucleotide probe method, Oligonucleotide Ligation Assay method, PCR-SSCP method, PCR-CFLP method, PCR-PHFA method, Invader method, RCA (Rolling Circle Amplification) method, Primer Oligo Base Extension) method, etc. can be mentioned.

Each nucleic acid material such as a primer set and a probe used for detecting a mutation can be prepared by a DNA synthesizer or the like.

The presence or absence of mutation in the gene can also be detected by digesting the genomic DNA or the amplification product with an appropriate restriction enzyme and detecting the difference in the size of the cleaved fragments by Southern blotting or the like.

In this way, the genomic DNA contained in the sample isolated from the subject (human body) can be used to obtain an index for examining the onset or risk of developing bone marrow tumors such as MDS and AML in humans. .. Specifically, when a predetermined mutation is found, the index indicates that the subject has developed a bone marrow tumor such as MDS or AML, or will develop in the future even if it has not developed at present. It is an index for judging that there is a risk of.

(2) Embodiment of detecting mutation in mRNA or cDNA When detecting mutation using mRNA contained in a sample isolated from a subject (human body), usually, first, a sample isolated from a human body is usually first detected. Extract the mRNA from. The extraction method is not particularly limited, and a known method can be used.

The sample isolated from the subject is not particularly limited as long as it can extract mRNA and expresses or may express the DDX41 gene.

Next, cDNA is prepared from the extracted mRNA by reverse transcription reaction. Further, the obtained cDNA may be amplified by the gene amplification method as described above for genomic DNA, if necessary.

The method for detecting the presence or absence of a mutation in the DDX41 gene using a sample containing the prepared cDNA is not particularly limited, but is the same as, for example, in the case of detecting a gene mutation using the above genomic DNA. The presence or absence of mutation in the DDX41 gene can be detected using the above method.

In this way, using mRNA or cDNA contained in a sample isolated from a subject (human body), it is possible to obtain an index for examining the onset or risk of developing bone marrow tumors such as MDS and AML in humans. it can. Specifically, when a predetermined mutation is found, the index indicates that the subject has developed a bone marrow tumor such as MDS or AML, or will develop in the future even if it has not developed at present. It is an index for judging that there is a risk of.

(3) Embodiment of detecting mutation in polypeptide In embodiment of detecting mutation using a polypeptide (protein) contained in a sample separated from a subject (human body), first, separation from the subject is performed. The polypeptide is extracted from the sample. The extraction method is not particularly limited, and a known method can be used.

The sample isolated from the human body is not particularly limited, and a polypeptide can be extracted, and a translation product of the DDX41 gene (that is, the DDX41 protein) is expressed or may be expressed. Anything is fine.

The method for detecting the presence or absence of a mutation in the DDX41 gene using a sample containing the prepared polypeptide is not particularly limited, and for example, the polypeptide which is a translation product of the DDX41 gene is mentioned above. An antibody that specifically recognizes only a polypeptide having a predetermined amino acid mutation or only a polypeptide not having a predetermined amino acid mutation is prepared, and the mutation is detected by the ELISA method or Western blotting using the antibody. can do.

In addition, a translation product (polypeptide) of the DDX41 gene is isolated from the sample containing the polypeptide, cleaved directly or with an enzyme or the like as necessary, and mutations are detected using a protein sequencer or a mass spectrometer. can do.

Furthermore, a translation product (polypeptide) of the DDX41 gene can be isolated from a sample containing the polypeptide, and mutations can be detected based on the isoelectric point of the polypeptide.

In this way, the polypeptide contained in the sample isolated from the subject (human body) can be used to obtain an index for examining the onset or risk of developing bone marrow tumors such as MDS and AML in humans. .. Specifically, when a predetermined mutation is found, the index indicates that the subject has developed a bone marrow tumor such as MDS or AML, or will develop in the future even if it has not developed at present. It is an index for judging that there is a risk of.

Hereinafter, the description of the method of the present invention will be returned.
The method of the present invention more preferably further comprises detecting a somatic mutation in the CUX1 gene in addition to the embryonic mutation in the DDX41 gene. We have previously reported that somatic mutations in the CUX1 gene cause loss-of-function mutations or haplo deletions in bone marrow tumors such as MDS / AML as tumor suppressor genes in poor prognosis types. There is. Therefore, in the case where the embryonic cell mutation of the DDX41 gene was found in the subject, the mutation of the CUX1 gene was further detected, and if the mutation of the CUX1 gene was detected, the poor prognosis of the bone marrow tumor in the subject was determined. Can be predicted. The position of the mutation in the CUX1 gene is not particularly limited, but for example, one or more selected from the group consisting of positions 142, 439, 796, 815 and 890 in the amino acid sequence of SEQ ID NO: 4. One or more mutations in the CUX1 gene that result in mutations in amino acids. The CUX1 gene mutation can be detected by the same method as the above-mentioned method for detecting the mutation in the DDX41 gene. The mutation in the CUX1 gene detected in this case is usually a somatic mutation. A mutation in the CUX1 gene can be determined to be a somatic mutation by confirming the presence of the mutation in a sample obtained from a tumor tissue of a bone marrow tumor or a tissue suspected to be a tumor tissue, and more preferably, a bone marrow tumor. If the presence of a mutation can be confirmed in a sample obtained from a tumor tissue or a tissue suspected to be a tumor tissue, and the presence of a mutation cannot be confirmed in a sample obtained from a normal tissue, the mutation is a somatic mutation. It can be determined that it is a cell mutation. This embodiment of the present invention is particularly useful for subjects who have been previously identified as developing bone marrow tumors.

<Method of obtaining an index for predicting the existence or future development of somatic mutations in the DDX41 gene in humans>
Another embodiment of the invention is in a subject at positions 500, 259, 363, 364, 3 in the amino acid sequence shown in SEQ ID NO: 2 of the polypeptide encoded by the wild-type DDX41 gene. One or more embryonic cell mutations in the DDX41 gene that result in mutations in one or more amino acids selected from the group consisting of positions, 7, 35, 187 and 256 were detected and the mutations It relates to a method of obtaining an index for predicting that a somatic mutation of the DDX41 gene is present or may occur in the subject when it is detected.

At this time, the "somatic mutations in the DDX41 gene" that predict the existence or future development include positions 525, 227, and 232 in the amino acid sequence shown in SEQ ID NO: 2 encoded by the wild-type DDX41 gene. 1 or more selected from the group consisting of 1st, 191st, 183rd, 530th, 592th, 321st, 35th, 499th, 370th, 219th and 377th Somatic mutations in the DDX41 gene that result in mutations in the amino acids, more preferably mutations in one or more amino acids selected from the group consisting of positions 525, 227 and 232, are particularly preferred. In our study shown in the Examples, subjects with bone marrow tumors having one of the above DDX41 embryonic mutations in one of the allelic loci have a high probability of being in the other of the allelic loci. It was revealed that it has one of the somatic mutations of the above DDX41 gene. The mutation of arginine at position 525 in the amino acid sequence shown in SEQ ID NO: 2 includes, but is not limited to, substitution with histidine. The mutation of threonine at position 227 in the amino acid sequence shown in SEQ ID NO: 2 includes, but is not limited to, substitution with methionine or alanine. The mutation of threonine at position 232 in the amino acid sequence shown in SEQ ID NO: 2 includes, but is not limited to, substitution with alanine. As a "somatic mutation of the DDX41 gene" that predicts its existence or future development, a somatic mutation of the DDX41 gene that results in the substitution of arginine at position 525 with histidine is particularly preferable. Since such amino acid mutations in the DDX41 protein are effective as pharmaceutical targets for bone marrow tumors such as MDS and AML, the presence or future of somatic mutations in the DDX41 gene can be achieved by the method of the present invention. Predicting its presence aids in the selection of drugs for effective treatment or prevention of the subject.

<Kit>
The kit of the present invention predicts the method for examining the onset or risk of developing bone marrow tumors such as MDS and AML in humans as described above, and the presence or future occurrence of somatic mutations in the DDX41 gene in humans. A kit used for at least one of the methods.

The kit comprises one or more selected from the group consisting of the following primer sets, probes and antibodies.
The primer set included in the kit is the 500th, 259th, and 363rd positions in the amino acid sequence shown in SEQ ID NO: 2 of the polypeptide encoded by the wild-type DDX41 gene when it is mutated among the DDX41 genes. Specific sites containing one or more bases that mutate at one or more amino acids selected from the group consisting of positions, 364, 3, 7, 7, 35, 187, and 256. It is a primer set designed so that it can be amplified.

The base in the DDX41 gene is the position in the cDNA base sequence of wild-type DDX41 shown in SEQ ID NO: 1.
One or both bases of cytosine at position 1495 and cytosine at position 1496,
776th place adenine,
1-3 bases of cytosine at position 1088, cytosine at position 1089 and thymine at position 1090, or thymine at position 1087, cytosine at position 1088 and cytosine at position 1089 1-3 Bases,
7th place guanine,
19th place guanine,
104th place cytosine,
A base at a position corresponding to at least one base selected from the group consisting of adenine at position 560 and guanine at position 766 is particularly preferred.

Each primer in the primer set typically contains DNA having 10 or more bases, preferably 15 or more, 50 or less, preferably 30 or less, and is amplified, detected, labeled, immobilized, etc. as necessary. A DNA region for various purposes, a labeled molecule, or the like may be linked.

The primer set may be designed so that the site containing the base can be amplified in both the wild type and the mutant type of the DDX41 gene, and the DDX41 gene may be designed to amplify the base. It may be designed so that the site containing the base can be amplified only when it is a variant with respect to the base, or the site containing the base can be amplified only when the DDX41 gene is a wild type with respect to the base. It may be designed to.

As a template for performing an amplification reaction using the primer set, genomic DNA containing the DDX41 gene, cDNA prepared from mRNA, fragments thereof, or the like can be used.

The kit of the present invention containing the primer set can contain other components required for nucleic acid amplification reaction and other components required for nucleic acid sequencing, and may include the following probes.

The probe included in the kit is located at positions 500, 259, 363, and 363 of the amino acid sequence shown in SEQ ID NO: 2 of the polypeptide encoded by the wild-type DDX41 gene when mutated among the DDX41 genes. It can specifically bind to a site containing one or more bases that mutate with one or more amino acids selected from the group consisting of positions 364, 3, 7, 35, 187 and 256. It is a probe designed to.

The base in the DDX41 gene is the position in the cDNA base sequence of wild-type DDX41 shown in SEQ ID NO: 1.
One or both bases of cytosine at position 1495 and cytosine at position 1496,
776th place adenine,
1-3 bases of cytosine at position 1088, cytosine at position 1089 and thymine at position 1090, or thymine at position 1087, cytosine at position 1088 and cytosine at position 1089 1-3 Bases,
7th place guanine,
19th place guanine,
104th place cytosine,
A base at a position corresponding to at least one base selected from the group consisting of adenine at position 560 and guanine at position 766 is particularly preferred.

The probe typically contains DNA having 10 or more bases, preferably 15 or more, 50 or less, preferably 40 or less, more preferably 30 or less, and is amplified, detected, labeled, and immobilized as needed. A DNA region for various purposes such as, or a labeled molecule or the like may be linked. The probe may also be immobilized on a solid phase carrier.

The probe may be designed to be capable of binding to a site containing the base in both wild-type and mutant forms of the DDX41 gene with respect to the base. It may be designed so that it can bind to the site containing the base only when it is a mutant type, or it can bind to the site containing the base only when the DDX41 gene is a wild type with respect to the base. It may be designed.

As the DDX41 gene to which the probe binds, genomic DNA, mRNA, cDNA, fragments thereof and the like can be used, and the genomic DNA and cDNA can be used for binding to the probe in an appropriately single-strand state. Can be done.

The kit of the present invention containing the probe can contain other components necessary for detecting a conjugate of the probe and the DDX41 gene. The kit can include the primer set and the probe in combination.

The antibody is located at positions 500, 259, 363, 364, 3 and 7 in the wild-type amino acid sequence shown in SEQ ID NO: 2 among the polypeptides which are translation products of the DDX41 gene. , An antibody that specifically recognizes a site containing an amino acid corresponding to one or more amino acids selected from the group consisting of positions 35, 187 and 256.

If necessary, the antibody may have a tag portion for various purposes such as detection, labeling, and immobilization, or a labeled molecule or the like linked thereto. The antibody may also be immobilized on a solid phase carrier.

The antibody may be designed so that a site containing the amino acid can be recognized only when the amino acid is a mutant type in the polypeptide, and the amino acid may be recognized only when the amino acid is a wild type. It may be designed so that the site containing the above can be recognized.

Variants of the amino acid in the polypeptide include a frame-shifted mutation of arginine at position 500, substitution of tyrosine at position 259 with cysteine, serine at position 363 and the wild amino acid sequence shown in SEQ ID NO: 2. Substitution of tyrosine at position 364 to one tyrosine (or deletion of serine at position 363), substitution of glutamic acid at position 3 with lysine, nonsense mutation of glutamic acid at position 7, proline at position 35 One or more variants selected from the group consisting of substitution of arginine, substitution of lysine at position 187 with arginine, and substitution of glutamic acid at position 256 with lysine.

The kit of the present invention containing the antibody can contain components necessary for a detection method such as an ELISA method and a Western blotting method.

The kit of the present invention preferably further contains a reagent for detecting a mutation in the CUX1 gene. The mutation of the CUX1 gene is preferably a mutation that causes a mutation in the amino acid sequence of the polypeptide encoded by the CUX1 gene, and the position of the mutation of the CUX1 gene is not particularly limited. For example, the mutation in the amino acid sequence of SEQ ID NO: 4 is the first. A mutation in the CUX1 gene that causes a mutation in one or more amino acids selected from the group consisting of positions 142, 439, 796, 815 and 890.

As a reagent for detecting a mutation in the CUX1 gene, a primer set designed to specifically amplify a site containing one or more bases that cause the above mutation in the CUX1 gene, or a primer set designed to specifically amplify the above-mentioned mutation in the CUX1 gene is generated. Examples include probes designed to specifically bind to sites containing one or more bases, and antibodies that recognize sites containing amino acids that correspond to amino acids that can cause mutations in polypeptides that are translation products of the CUX1 gene. Be done.

<Method of testing the onset or risk of developing bone marrow tumor in humans>
Other embodiments of the present invention
A method of examining the onset or risk of developing bone marrow tumors in humans.
At position 500, position 259, position 363, position 364, position 3, position 7, position 35, and position 187 in the amino acid sequence shown in SEQ ID NO: 2 of the polypeptide encoded by the wild-type DDX41 gene. And one or more embryonic cell mutations of the DDX41 gene that result in mutations in one or more amino acids selected from the group consisting of position 256.

Mutations of each amino acid and base, a detection method, a preferred embodiment, and the like in this embodiment are the same as those described in the column of <Method for obtaining an index for testing the onset or risk of developing bone marrow tumor in humans>. Is.

<Method of predicting the presence or future development of somatic mutations in the DDX41 gene in humans>
Other embodiments of the present invention
A method for predicting the presence or future development of somatic mutations in the DDX41 gene in humans.
At position 500, position 259, position 363, position 364, position 3, position 7, position 35, and position 187 in the amino acid sequence shown in SEQ ID NO: 2 of the polypeptide encoded by the wild-type DDX41 gene. And one or more embryonic cell mutations of the DDX41 gene that result in mutations in one or more amino acids selected from the group consisting of position 256.

For mutations of each amino acid and base, detection method, preferred embodiment, etc. in this embodiment, <method for obtaining an index for examining the onset or risk of developing bone marrow tumor in humans> and <body of DDX41 gene in humans>. It is the same as described in the column of> Method for obtaining an index for predicting the existence or future development of a cell mutation.

In order to deepen the understanding of the present invention, the present invention will be described below with reference to Examples, but it goes without saying that the present invention is not limited to these Examples.

<Identification of gene mutations in myelodysplastic syndromes (MDS) and acute myeloid leukemia (AML)>
In this example, for patients diagnosed with MDS or AML and for whom informed consent was obtained, a sample for the present invention was collected and covered according to a protocol approved by the Ethics Committee of Kyoto University. Genetic mutation analysis was performed. The above patients investigated were all Japanese of East Asian race.

In this example, targeted deep sequence analysis was performed using normal tissue-derived specimens and tumor tissue-derived specimens collected from 1070 MDS or AML patients, which are important for the development of DDX41 and other MDS / AML. A gene mutation analysis was performed. Based on the above, we attempted to elucidate the embryonic and somatic gene abnormalities of DDX41.

(1) Identification of Mutant Genes by Targeted Deep Sequence Analysis First, targeted deep sequence analysis of DNA was performed on samples derived from normal tissues and tumor tissues of 1070 MDS and AML patients. Among the genomic regions, the exon region encoding the DDX41 protein was concentrated using SureSelect Target Enrichment (Agilent Technologies, Inc.) and sequenced using HiSeq2000 (Illumina). Since embryonic cell mutations are contained in both normal tissue-derived DNA and tumor tissue-derived DNA, somatic cell mutations are contained only in tumor tissue-derived DNA, so that the base sequence of the tumor tissue-derived DNA and the normal tissue-derived DNA are derived. By comparing the base sequences of the DNAs of the genes, embryonic mutations and somatic mutations of the genes were confirmed. The base sequence of the exon region encoding the DDX41 protein (GenBank Accession No. NM_016222) in the wild-type DDX41 genomic DNA, that is, the cDNA sequence is shown in SEQ ID NO: 1, and the amino acid sequence of the DDX41 protein encoded by the cDNA base sequence of SEQ ID NO: 1. Is shown in SEQ ID NO: 2.

The results of the targeted deep sequence analysis for the DDX41 gene are shown in the table below and FIG. In this example, regarding embryo cell gene mutations, only mutations detected in specimens derived from two or more patients were treated as embryo cell gene mutations.

By targeted deep sequence analysis, we detected 44 (about 4%) embryonic gene mutations and 23 (about 2%) somatic mutations in 1070 sporadic MDS / AML. The data for "East Asian race" in Figure 2 is from the above study, and the data for "Caucasus race" shown for comparison is reported in Polprasert et al., 2015, Cancer Cell 27, 658-670. It is a thing.

(2) Identification of mutation frequency of embryonic cell mutation and somatic mutation and analysis of mutation pattern by target sequence analysis 44 cases in which one or both of embryonic cell gene mutation and somatic gene mutation were found by searching our cohort. Of these, approximately 50% of cases had DDX41 embryonic and somatic mutations simultaneously (Fig. 2, “East Asian race”). It was clarified by a subcloning method that almost no somatic mutations in DDX41 were found in DDX41 embryonic mutation-negative cases, and that DDX41 embryonic mutations and somatic mutations significantly occur at allelic loci. In addition, within the scope of this study, it is subcloning that all DDX41 embryo cell mutation-positive patients have only one DDX41 embryo cell mutation and that mutation is present in only one of the allelic loci. It became clear by the method of. From the above, the mutation form of DDX41 regarding the onset of MDS / AML is explained by the two-hit theory (two mutations in the same gene are the cause).

As shown in FIG. 1, as a result of targeted deep sequence analysis of the DDX41 gene, hotspot frameshift mutation A500fs (22 cases) (2.2% in total) and hotspot were frequently observed as embryonic cell mutations of DDX41 at least multiple times. Missense mutation Y259C (6 cases), E256K (2 cases), K187R (2 cases), P35R (4 cases), E3K (2 cases) (2% total), inframe deletion mutation 363_364del (4 cases) (0.4 cases total) %) And nonsense mutation E7X (2 cases) (0.2% in total) were observed.

DDX41 somatic mutations include hotspot missense mutation R525H (8 cases), T227A / M (3 cases in total), T232A (2 cases), A191fs, F183S, G530D, I592F, P321L, P35R, P499S, Q370X. , R219H, T377N (1 case each). The following table shows the correspondence between base mutations and amino acid mutations for R525H, T232A, and T227A / M in which mutations were found in multiple cases among these mutations.

These somatic mutations were frequently observed in patients with positive embryonic cell mutations (Fig. 2), and exhibited a 2-hit mutation form. From the above, in MDS / AML, searching for somatic mutations as well as embryonic mutations in DDX41 is useful for clarifying more detailed pathological conditions. In addition, the presence or future development of somatic mutations such as the R525H mutation of the DDX41 protein can be predicted using the presence of embryonic cell mutations in DDX41 as an index.

Searching for somatic mutations in the target deep sequence for genes other than DDX41 for all MDS / AML cases (1070) revealed a significantly higher frequency of CUX1 somatic mutations in cases with DDX41 mutations (44). (Fig. 3). The base sequence of the exon region encoding the CUX1 protein (GenBank Accession No. NM_001202543) in the wild-type CUX1 genomic DNA, that is, the cDNA sequence is shown in SEQ ID NO: 3, and the amino acid sequence of the CUX1 protein encoded by the cDNA base sequence of SEQ ID NO: 3. Is shown in SEQ ID NO: 4. The somatic mutations of the CUX1 gene confirmed in the case with DDX41 mutation (44) are summarized in the table below.

We have previously reported that CUX1 causes a loss-of-function mutation or haplo deletion in myelodysplastic tumors such as MDS / AML as a tumor suppressor gene in a disease type with a poor prognosis. Therefore, in cases where DDX41 mutation is observed, poor prognosis can be predicted by confirming whether CUX1 mutation has occurred.

In fact, as a result of analyzing the mutation frequency for each disease type in all MDS / AML cases, the frequency is higher in cases where myeloblasts (juvenile myeloid cells) exhibit a progressive disease type of more than 5% in the bone marrow. DDX41 embryonic cells or somatic mutations were found in (Fig. 4). This result suggests that searching for the DDX41 mutation may be useful for differentiating the type of MDS / AML and predicting the prognosis. Depending on the case, it is not possible to evaluate whether it is a high-risk type or a low-risk type, and Fig. 4 shows the proportion of DDX41 mutation-positive patients only in the cases for which the risk could be evaluated.

(3) Analysis of DDX41 embryo cell mutation enrichment rate and regional differences Confirm the frequency of all DDX41 embryo cell mutations A500fs, Y259C, E256K, K187R, P35R, E3K, del363_364, and E7X shown in Fig. 1 in healthy subjects. did. The presence or absence of mutations in the DDX41 gene in healthy individuals was estimated using the Exome Aggregation Consortium (ExAC) database (Broad Institute) (URL: http://exac.broadinstitute.org) (Caucasus race: n = 33,362). , East Asian Race: n = 4,327). Mutations in A500fs (1 case), Y259C (3 cases), and K187R (3 cases) were detected in 4327 East Asians, but E256K, P35R, E3K, del363_364, and E7X were rarely observed. It was.

Figure 5 shows the number of cases in which each DDX41 embryo cell mutation and D140 frame-shift mutation were observed in 1070 MDS / AML cases of the East Asian race determined in the above study, and Polprasert et al., 2015, Cancer. The number of cases in which each of the above DDX41 embryo cell mutations and D140 frame-shift mutations was observed in 1045 MDS / AML cases of the Caucasian race reported in Cell 27, 658-670, and Caucasian from the above database. The number of cases of each DDX41 embryo cell mutation in each race and East Asian race is shown.

The odds ratios for the purpose of evaluating the enrichment of embryonic cell mutations in DDX41 in MDS / AML for healthy individuals were A500fs (odds ratio = 91; 95% confidence interval = 15-3746 (p <0.0001)), respectively. Y259C (odds ratio = 8.0; 95% confidence interval = 1.7-50 (p = 0.0004)), E256K (odds ratio = 20; 95% confidence interval = 0.97-422 (p = 0.004)) (*), K187R (odds) Ratio = 2.7; 95% confidence interval = 0.23-24 (p = 0.26)), P35R (odds ratio = 36.5; 95% confidence interval = 2.0-679 (p <0.0001)) (*), E3K (odds ratio = 20) 95% confidence interval = 0.97-422 (p = 0.004)) (*), 363_364del (odds ratio = 36.5; 95% confidence interval = 2.0-679 (p <0.0001)) (*), E7X (odds ratio = 20) The 95% confidence interval was 0.97-422 (p = 0.004)) (*) (Fig. 5). (*) Refers to mutations with Woolf-Haldane correction. The odds ratios for these DDX41 embryonic mutations identified in East Asians are the odds ratios for each mutation in MDS / AML cases to healthy individuals in East Asians. On the other hand, for the DDX41 embryonic cell mutation D140fs confirmed in the Caucasian race, the odds ratio in MDS / AML cases to healthy subjects in the Caucasian race was calculated. The odds ratio = 19, 95% confidence interval = 5.8-59. It was (<p = 0.001) (Fig. 5).

All DDX41 embryonic mutations A500fs, Y259C, E256K, K187R, P35R, E3K, del363_364, E7X are calculated together to show the odds ratio of having DDX41 embryonic mutations in MDS / AML cases to healthy individuals in East Asian races. Was 15; 95% confidence interval = 2.3-961 (p = 0.0001). Embryonic cell mutations in DDX41 were significantly enriched in MDS / AML overall.

Furthermore, in a study of 33362 Caucasian races, E256K (1 case) and E3K (2 cases) were found, but A500fs, Y259C, del363_364, E7X, K187R, P35R were not found, and distribution with East Asian races. Was different.

From the above, it was confirmed that A500fs and Y259C are mutations endemic to East Asian races, and E256K and E3K are embryonic cell mutations that are distributed over a wider area and are distributed worldwide including the Caucasus Asian races. Was done (Fig. 6).

These results indicate that different mutations are expected depending on which region of the globe the DDX41 is sequenced, not just in Japan. When investigating mutations in East Asia, it is recommended to prioritize A500fs and Y259C, followed by other mutations.

(4) Clinical significance of embryonic cell mutation of DDX41
According to the report of Polprasert et al., 2015, Cancer Cell 27, 658-670, the embryonic cell mutation of DDX41 develops in an autosomal dominant inheritance, so there is a 50% chance that it will be inherited in children and cause leukemia. .. Therefore, if a familial elderly-onset MDS / AML family is found, it may be due to a mutation in the embryonic cells of DDX41. Therefore, the DDX41 gene should be sequenced to confirm the presence or absence of mutations.

Furthermore, even in sporadic cases of MDS / AML with an unknown family history, if a mutation in DDX41 is found, there is a high probability that MDS / AML will be found in the family in the future. If the carrier's family died of other illnesses at a young age, or if the child was still young, the family case might not be clear. The DDX41 mutation is inherited in the offspring with a 50% chance, and there is a 50% chance that there are positives in siblings with the same parents. With this information, the risk of otherwise undiscovered disease can be estimated years to decades before the onset.

Searching for embryonic cell mutations in DDX41 is also important when considering hematopoietic stem cell transplantation for MDS / AML. The DDX41 mutation should be searched for in MDS / AML cases, and if positive, the potential donor sibs, parents, and offspring should be searched for similar mutations.

If a candidate hematopoietic stem cell transplant donor in the family is positive for the DDX41 mutation, the probability of post-transplant donor-derived leukemia increases if transplantation is performed from that donor. Therefore, the priority as a donor candidate should be lowered and unrelated donor candidates should be considered.

Claims (8)

A method for obtaining an index for examining the onset or risk of developing bone marrow tumor in humans.
In a sample obtained from the normal tissue of the subject, in the amino acid sequence shown in SEQ ID NO: 2 of the polypeptide encoded by the wild-type DDX41 gene,
# 500 of alanine of a frame-shift mutation
A method characterized by detecting one or more mutations in the DDX41 gene that results in. A mutation in the DDX41 gene is used as the position in the cDNA base sequence of wild-type DDX41 shown in SEQ ID NO: 1.
Duplicate of the 1496 position of cytosine
In a method according to claim 1. The method of claim 1 or 2 , wherein the bone marrow tumor is myelodysplastic syndrome (MDS) and / or acute myeloid leukemia (AML). The method of claim 3 , wherein the risk of developing or developing MDS and / or AML to be tested is the risk of developing or developing progressive MDS and / or AML. The method according to any one of claims 1 to 4 , further comprising detecting a mutation in the CUX1 gene. A method for obtaining an index for predicting the presence or future development of a somatic mutation in the DDX41 gene in humans.
In a sample obtained from the normal tissue of the subject, in the amino acid sequence shown in SEQ ID NO: 2 of the polypeptide encoded by the wild-type DDX41 gene,
# 500 of alanine of a frame-shift mutation
Cause, detecting one or more mutations of the DDX41 gene and somatic mutation of the DDX41 gene, in the amino acid sequence shown in SEQ ID NO: 2 encoded by the wild-type DDX41 gene, a mutation of # 525 arginine A method characterized by a somatic mutation in the resulting DDX41 gene. A kit used for at least one method of testing the onset or risk of developing bone marrow tumors in humans and predicting the presence or future occurrence of somatic mutations in the DDX41 gene in humans.
The somatic mutation of the DDX41 gene is a somatic mutation of the DDX41 gene that causes a mutation of arginine at position 525 in the amino acid sequence shown in SEQ ID NO: 2 encoded by the wild-type DDX41 gene.
Of DDX41 gene, when mutated, so that it can amplify a region comprising a base which affords a mutation at the 500 position of the amino acid in the amino acid sequence shown in SEQ ID NO: 2 of the polypeptide encoded by the wild-type DDX41 gene 1 or more Designed primer set,
Of DDX41 gene, when mutated, so that it can be combined with site comprising wild-type DDX41 gene by a base which affords a mutation at the 500 position of the amino acid in the amino acid sequence shown in SEQ ID NO: 2 of the polypeptide encoded one or more Designed probe and
Among the polypeptides that are translation products of the DDX41 gene, one or more selected from the group consisting of antibodies that recognize the site containing the amino acid corresponding to the amino acid at position 500 in the wild-type amino acid sequence shown in SEQ ID NO: 2 Contains,
The mutation in the amino acid is in the amino acid sequence shown in SEQ ID NO: 2.
# 500 of alanine of a frame-shift mutation
In it, kit. The kit according to claim 7 , further comprising a reagent for detecting a mutation in the CUX1 gene.

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