Biomarker for detecting BCOR mutation

Abstract

To provide a novel biomarker that can readily detect a BCOR gene mutation in pluripotent stem cells and a method using the same.SOLUTION: A biomarker for detecting a BCOR gene mutation in pluripotent stem cells comprises a transcription product or protein selected from the group consisting of PRAC1, PRAC2, TBX1, MT1F, ZIC1, PITX2, MT1G and FOXC1.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to a biomarker for detecting BCOR mutations in pluripotent stem cells, more specifically, to a biomarker consisting of a transcript or protein selected from the group consisting of PRAC1, PRAC2, TBX1, MT1F, ZIC1, PITX2, MT1G, and FOXC1, and a method for detecting BCOR mutations in pluripotent stem cells using the biomarker. [Background technology]

[0002] In Japan, ES cells, iPS cells, and other cells have rarely been used in humans, so when using cells that are considered to be high risk, both known and unknown, they must be examined by the Committee on Specially Certified Regenerative Medicine. The Committee on Specially Certified Regenerative Medicine examines the genomic findings of the pluripotent stem cells used as raw materials, which cannot be denied as tumorigenic. These findings include (1) karyotype abnormalities, (2) structural abnormalities including SNV / Indel and copy number abnormalities (CNV) of tumor-related genes, and (3) significant residual foreign factors that may promote tumorigenesis. One of the tumor-related genes in (2) above is the BCOR (BCL6 corepressor) gene.

[0003] BCOR is a component of polycomb complex 1.1 and functions as an epigenetic regulator. Polycomb complex 1.1 suppresses gene expression through ubiquitination of Lys119 of histone H2A (H2AK119) and acts as a regulator of embryonic development, stem cell function, and hematopoiesis. Mutations in somatic cells of the BCOR gene are found in various cancer types, such as sarcoma, medulloblastoma, retinoblastoma, and hematological malignancies. It has also been reported that iPS cells are prone to mutations in the BCOR gene, and that when iPS cells with even a slight loss of BCOR function due to gene mutation are mixed in, iPS cells with BCOR mutations are selected by several passages of culture (Non-Patent Document 1). [Prior art documents] [Non-patent literature]

[0004] [Non-Patent Document 1] Nature Genetics volume 54, pages 1406-1416 (2022) Summary of the Invention [Problem to be solved by the invention]

[0005] However, loss-of-function mutations in the BCOR gene do not have a specific pattern, and it is difficult to establish a detection system for the gene mutation in cells. Therefore, the present invention aims to provide a novel biomarker that can easily detect BCOR gene mutations in pluripotent stem cells and a method using the same. [Means for solving the problem]

[0006] The present inventors have found, through comprehensive analysis using RNA-seq analysis, several genes whose expression is increased in association with BCOR gene mutations in pluripotent stem cells. These genes were shown to be highly expressed in pluripotent stem cells having BCOR gene mutations, even when quantitative PCR was used, in agreement with the results of RNA-seq analysis. The present inventors have further studied based on these findings, and as a result, have completed the present invention.

[0007] That is, the present invention is as follows. [1-1] A biomarker for detecting BCOR gene mutations in pluripotent stem cells, comprising a transcript or protein selected from the group consisting of PRAC1, PRAC2, TBX1, MT1F, ZIC1, PITX2, MT1G and FOXC1. [1-2] Biomarker for pluripotent stem cells, induced pluripotent stem cells, [1-1]. [1-3] Biomarker for [1-1] or [1-2] where the pluripotent stem cells are of human origin. [1-4] The biomarker according to any one of [1-1] to [1-3], wherein the mutation in the BCOR gene is a loss-of-function mutation. [2] A method for determining the presence or absence of a mutation in the BCOR gene in a pluripotent stem cell population, the method comprising the step of detecting one or more of the biomarkers described in any one of [1-1] to [1-4] in the pluripotent stem cell population. [3] (1) measuring the abundance of one or more biomarkers according to any one of [1-1] to [1-4] in a target pluripotent stem cell population and a pluripotent stem cell population not having a mutation in the BCOR gene; and (2) determining that a pluripotent stem cell having a mutation in the BCOR gene is present in the subject pluripotent stem cell population when the value of at least one biomarker measured in the subject pluripotent stem cell population is higher than the value measured in the subject pluripotent stem cell population not having a mutation in the BCOR gene; The method according to claim 2, comprising: [4] (1) measuring the abundance of one or more biomarkers according to any one of [1-1] to [1-4] in a subject pluripotent stem cell population; and (2) determining that the pluripotent stem cell population contains pluripotent stem cells having a mutation in the BCOR gene when the value measured in step (1) exceeds a reference value. The method according to claim 2, comprising: [5] The method according to [4], wherein the reference value is a value based on the measurement value of a biomarker described in any one of [1-1] to [1-4] in a pluripotent stem cell population not having a mutation in the BCOR gene. [6-1] The method according to any one of [2] to [5], wherein the pluripotent stem cells are induced pluripotent stem cells. [6-2] The method according to any one of [2] to [6-1], wherein the pluripotent stem cells are derived from humans. [7] The method according to any one of [2] to [6-2], wherein the mutation in the BCOR gene is a loss-of-function mutation. [8] The method according to any one of [2] to [7], characterized in that the biomarker is detected using a nucleic acid probe and / or a nucleic acid primer that specifically recognizes a transcription product selected from the group consisting of PRAC1, PRAC2, TBX1, MT1F, ZIC1, PITX2, MT1G and FOXC1, or an antibody that specifically recognizes a protein encoded by the transcription product. [9-1] A determination kit for detecting BCOR gene mutations in pluripotent stem cells, comprising a nucleic acid probe and / or nucleic acid primer that specifically recognizes a transcription product selected from the group consisting of PRAC1, PRAC2, TBX1, MT1F, ZIC1, PITX2, MT1G and FOXC1, or an antibody that specifically recognizes a protein encoded by the transcription product. [9-2] The kit according to [9-1], further comprising one or more biomarkers according to any one of [1-1] to [1-4]. Effect of the Invention

[0008] According to the present invention, a method for easily detecting a mutation in the BCOR gene in a pluripotent stem cell is provided, which makes it possible to provide a pluripotent stem cell with a low risk of tumorigenesis. [Brief description of the drawings]

[0009] [Figure 1] FIG. 1 shows quantitative PCR amplification curves of the TBX1 gene in BCOR gene mutation-positive and BCOR gene wild-type iPS cells. [Diagram 2] FIG. 1 shows quantitative PCR amplification curves of the PRAC1 gene in BCOR gene mutation-positive and BCOR gene wild-type iPS cells. [Diagram 3] FIG. 1 shows quantitative PCR amplification curves of the PRAC2 gene in BCOR gene mutation-positive and BCOR gene wild-type iPS cells. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0010] 1. Biomarkers for detecting BCOR mutations The present invention provides a biomarker for detecting a mutation in the BCOR (BCL6 corepressor) gene in pluripotent stem cells (hereinafter, also referred to as the "biomarker of the present invention"). Specific examples of the biomarker of the present invention include a transcript or protein selected from the group consisting of PRAC1, PRAC2, TBX1, MT1F, ZIC1, PITX2, MT1G, and FOXC1. Hereinafter, a transcript selected from the group consisting of PRAC1, PRAC2, TBX1, MT1F, ZIC1, PITX2, MT1G, and FOXC1 may be referred to as the "biomarker transcript of the present invention." In addition, a protein selected from the group consisting of PRAC1, PRAC2, TBX1, MT1F, ZIC1, PITX2, MT1G, and FOXC1 (i.e., a protein encoded by the biomarker transcript of the present invention) may be referred to as the "biomarker protein of the present invention."

[0011] In this specification, the term "transcription product" refers to RNA encoding a protein, and hereinafter refers to mRNA unless otherwise specified. Complementary DNA (cDNA) synthesized by reverse transcriptase using RNA as a template is also included in the transcription product. Some biomarkers of the present invention have isoforms, for example, isoform a and isoform b are known for PRAC2 protein. In this case, unless otherwise specified, isoform a and isoform b are collectively referred to as PRAC2 protein, and the transcript encoding isoform a and the transcript encoding isoform b are collectively referred to as PRAC2 transcript. The same applies to other proteins and transcription products.

[0012] For proteins with isoforms, each isoform or a transcript encoding the isoform can be used as a biomarker of the present invention, but the isoform most highly expressed in pluripotent stem cells or a combination of the isoform with another isoform is preferred as a biomarker of the present invention. For example, isoform b is preferred for PRAC2 protein, isoform D is preferred for TBX1 protein, isoform 1 is preferred for MT1F protein, isoform c is preferred for PITX2 protein, and isoform 2 is preferred for MT1G protein.

[0013] The biomarker transcripts of the present invention are known transcripts, and for example, the PRAC1 transcript is listed under NCBI Accession No.: NM_032391.3 (SEQ ID NO: 1), the PRAC2 transcript is listed under NCBI Accession No.: NM_001282276.1 (SEQ ID NO: 2) (isoform a) and NCBI Accession No.: NM_001282275.2 (SEQ ID NO: 3) (isoform b), the TBX1 transcript is listed under NCBI Accession No.: NM_080646.2 (SEQ ID NO: 4) (isoform A), NCBI Accession No.: NM_005992.1 (SEQ ID NO: 5) (isoform B), NCBI Accession No.: NM_080647.1 (SEQ ID NO: 6) (isoform C) and NCBI Accession No.: NM_001379200.1 (SEQ ID NO: 7) (isoform D), and the MT1F transcript is listed under NCBI Accession No.: NM_001379200.1 (SEQ ID NO: 8) (isoform D). No.: NM_005949.4 (SEQ ID NO: 8) (isoform 1) and NCBI Accession No.: NM_001301272.2 (SEQ ID NO: 9) (isoform 2), ZIC1 transcript as NCBI Accession No.: NM_003412.4 (SEQ ID NO: 10), PITX2 transcript as NCBI Accession No.: NM_001204399.1 (SEQ ID NO: 11) and NM_153427.3 (SEQ ID NO: 12) (isoform a), NCBI Accession No.: NM_001204397.2 (SEQ ID NO: 13), NM_001204398.1 (SEQ ID NO: 14) and NM_153426.3 (SEQ ID NO: 15) (isoform b), and NCBI Accession No.: The sequences of the MT1G transcript are disclosed as NCBI Accession No.: NM_000325.6 (SEQ ID NO: 16) (isoform c), the MT1G transcript as NCBI Accession No.: NM_005950.3 (SEQ ID NO: 17) (isoform 1) and NCBI Accession No.: NM_001301267.2 (SEQ ID NO: 18) (isoform 2), and the FOXC1 transcript as NCBI Accession No.: NM_001453.3 (SEQ ID NO: 19).In the present invention, each transcription product may be RNA containing a base sequence shown in any one of SEQ ID NOs: 1 to 19 (wherein T is replaced with U), or may be RNA containing a base sequence substantially identical to the base sequence.

[0014] Examples of base sequences substantially identical to the base sequences shown in any of SEQ ID NOs: 1 to 19 include base sequences that have an identity of 80% or more, preferably 90% or more, and more preferably 95% or more (e.g., 96%, 97%, 98%, 99% or more) to these base sequences and that encode proteins having substantially the same activity as the biomarker proteins of the present invention.

[0015] Furthermore, examples of base sequences substantially identical to the base sequences shown in any of SEQ ID NOs: 1 to 19 include base sequences in which one or more nucleotides (preferably about 1 to 300, preferably about 1 to 150, more preferably about 1 to 30, particularly preferably one to several (2, 3, 4, 5, 6, 7, 8, 9, or 10)) have been substituted, inserted, added, and / or deleted from these base sequences, and which encode proteins having substantially the same activity as the biomarker proteins of the present invention.

[0016] The biomarker transcripts of the present invention can be obtained, for example, by isolating and purifying from cells or biological samples containing the transcripts by methods known per se, or may be produced by chemical synthesis or by in vitro transcription (IVT) methods.

[0017] The biomarker proteins of the present invention are known proteins. For example, PRAC1 protein is listed under NCBI Accession No.: NP_115767.1 (SEQ ID NO: 20); PRAC2 protein is listed under NCBI Accession No.: NP_001269205.1 (SEQ ID NO: 21) (isoform a) and NCBI Accession No.: NP_001269204.1 (SEQ ID NO: 22) (isoform b); TBX1 protein is listed under NCBI Accession No.: NP_542377.1 (SEQ ID NO: 23) (isoform A), NCBI Accession No.: NP_005983.1 (SEQ ID NO: 24) (isoform B), NCBI Accession No.: NP_542378.1 (SEQ ID NO: 25) (isoform C) and NCBI Accession No.: NP_001366129.1 (SEQ ID NO: 26) (isoform D); MT1F protein is listed under NCBI Accession No.: NP_001366129.1 (SEQ ID NO: 27) (isoform D); No.: NP_005940.1 (SEQ ID NO: 27) (isoform 1) and NCBI Accession No.: NP_001288201.1 (SEQ ID NO: 28) (isoform 2), ZIC1 protein is NCBI Accession No.: NP_003403.2 (SEQ ID NO: 29), PITX2 protein is NCBI Accession No.: NP_001191328.1 and NP_700476.1 (all of which are the same amino acid sequence) (SEQ ID NO: 30) (isoform a), NCBI Accession No.: NP_001191326.1, NP_001191327.1 and NP_700475.1 (all of which are the same amino acid sequence).) (SEQ ID NO: 31) (isoform b), and NCBI Accession No.: NP_000316.2 (SEQ ID NO: 32) (isoform c); MT1G protein is disclosed as NCBI Accession No.: NP_005941.1 (SEQ ID NO: 33) (isoform 1) and NCBI Accession No.: NP_001288196.1 (SEQ ID NO: 34) (isoform 2); and FOXC1 protein is disclosed as NCBI Accession No.: NP_001444.2 (SEQ ID NO: 35). In the present invention, each protein may be a protein comprising an amino acid sequence shown in any one of SEQ ID NOs: 20 to 35, or may be a protein comprising an amino acid sequence substantially identical to the amino acid sequence.

[0018] Examples of amino acid sequences substantially identical to the amino acid sequences shown in any of SEQ ID NOs: 20 to 35 include amino acid sequences having a similarity or identity of 80% or more, preferably 90% or more, and more preferably 95% or more (e.g., 96%, 97%, 98%, 99% or more) to these amino acid sequences. Here, "similarity" refers to the ratio (%) of identical and similar amino acid residues to all overlapping amino acid residues in optimal alignment (preferably, the algorithm can take into account the introduction of gaps into one or both of the sequences for optimal alignment) when two amino acid sequences are aligned using a mathematical algorithm known in the art. "Similar amino acids" refer to amino acids that are similar in physicochemical properties, and examples of such amino acids include aromatic amino acids (Phe, Trp, Tyr), aliphatic amino acids (Ala, Leu, Ile, Val), polar amino acids (Gln, Asn), basic amino acids (Lys, Arg, His), acidic amino acids (Glu, Asp), amino acids with hydroxyl groups (Ser, Thr), and amino acids with small side chains (Gly, Ala, Ser, Thr, Met) that are classified into the same group. It is predicted that substitution with such similar amino acids will not cause a change in the phenotype of the protein (i.e., it is a conservative amino acid substitution). Specific examples of conservative amino acid substitutions are well known in the art and are described in various documents (see, for example, Bowie et al., Science, 247: 1306-1310 (1990)). The similarity or identity of amino acid sequences in this specification can be calculated using the homology calculation algorithm NCBI BLAST (National Center for Biotechnology Information Basic Local Alignment Search Tool) under the following conditions (expectation value = 10; gaps allowed; matrix = BLOSUM62; filtering = OFF).

[0019] Furthermore, examples of amino acid sequences substantially identical to the amino acid sequences shown in any of SEQ ID NOs: 20 to 35 also include proteins containing these amino acid sequences in which one or more amino acids (preferably about 1 to 100, preferably about 1 to 50, more preferably about 1 to 10, and particularly preferably one to several (2, 3, 4, or 5)) have been substituted, inserted, added, and / or deleted.

[0020] The biomarker protein of the present invention can be produced according to known protein synthesis methods, such as solid-phase synthesis, liquid-phase synthesis, etc. The obtained protein can be purified and isolated by known purification methods, such as solvent extraction, distillation, column chromatography, liquid chromatography, recrystallization, a combination of these, etc. Also, it may be isolated and purified from a biological sample by a method known per se. Alternatively, the biomarker protein of the present invention can be produced by culturing a transformant containing a nucleic acid encoding it, and isolating and purifying the protein from the resulting culture. Such a nucleic acid may be DNA or RNA, or may be a DNA / RNA chimera, but is preferably DNA. The nucleic acid may be double-stranded or single-stranded.

[0021] The biomarker of the present invention can detect not only loss-of-function mutations (i.e., nonsense mutations or frameshift mutations) but also the presence or absence of attenuated mutations such as missense mutations, mutations accompanied by splicing abnormalities, and mutations in gene regulatory regions (e.g., promoters, enhancers) that are accompanied by a decrease in the function of BCOR and / or a decrease in the expression level of the BCOR gene product compared to cells in which the BCOR gene is wild-type. In addition, the BCOR gene is on the X chromosome, and the mutation of the BCOR gene that can be detected by the method of the present invention may be present in an allele that has not been inactivated or in both alleles when the pluripotent stem cells are of the XX type. Since it is not necessary to consider the allele, the pluripotent stem cells used in the present invention are preferably of the XY type, but it is also possible to detect the XX type in which a mutation is inserted into the active X chromosome, resulting in loss of function of the BCOR gene. In this specification, loss-of-function mutations are included in attenuated mutations.

[0022] Examples of the attenuating mutations in the BCOR gene that are the subject of detection by the biomarkers of the present invention include nonsense and frameshift mutations that result in a shorter amino acid sequence than normal, and missense mutations that result in amino acid substitutions that attenuate the function of the BCOR protein. Specific examples of frameshift mutations include, but are not limited to, a single base insertion mutation at chrX:40,064,431 (hg38) and a single base insertion mutation at chrX:40,057,258 (hg38).

[0023] As used herein, the term "pluripotent stem cell" refers to a stem cell that can differentiate into tissues or cells with various different morphologies and functions in the body and has the ability to differentiate into cells of any lineage of the three germ layers (endoderm, mesoderm, ectoderm). Examples of pluripotent stem cells used in the present invention include induced pluripotent stem cells (iPS cells), embryonic stem cells (ES cells), embryonic stem cells derived from cloned embryos obtained by nuclear transfer (ntES cells), multipotent germline stem cells (mGS cells), and embryonic germ cells (EG cells), but preferably iPS cells (more preferably human iPS cells). When the pluripotent stem cells are ES cells or any cells derived from human embryos, the cells may be cells produced by destroying the embryo or cells produced without destroying the embryo, but from an ethical point of view, the cells are preferably cells produced without destroying the embryo.

[0024] ES cells are stem cells that are established from the inner cell mass of early mammalian embryos (e.g., blastocysts) such as humans and mice, and have pluripotency and the ability to proliferate through self-renewal. ES cells were discovered in mice in 1981 (MJ Evans and MH Kaufman (1981), Nature 292:154-156), and subsequently ES cell lines were established in humans, monkeys, and other primates (JA Thomson et al. (1998), Science 282:1145-1147; JA Thomson et al. (1995), Proc. Natl. Acad. Sci. USA, 92:7844-7848; JA Thomson et al. (1996), Biol. Reprod., 55:254-259; JA Thomson and VS Marshall (1998), Curr. Top. Dev. Biol., 38:133-165). ES cells can be established by extracting the inner cell mass from the blastocyst of a fertilized egg of a target animal and culturing the inner cell mass on a fibroblast feeder. Alternatively, ES cells can be established using only a single blastomere from an embryo at the cleavage stage before the blastocyst stage (Chung Y. et al. (2008), Cell Stem Cell 2: 113-117), or from an embryo that has stopped development (Zhang X. et al. (2006), Stem Cells 24: 2669-2676.).

[0025] The nt ES cells are ES cells derived from cloned embryos produced by nuclear transfer technology, and have almost the same characteristics as ES cells derived from fertilized eggs (Wakayama T. et al. (2001), Science, 292:740-743; S. Wakayama et al. (2005), Biol. Reprod., 72:932-936; Byrne J. et al. (2007), Nature, 450:497-502). In other words, nt ES (nuclear transfer ES) cells are ES cells established from the inner cell mass of blastocysts derived from cloned embryos obtained by replacing the nucleus of an unfertilized egg with the nucleus of a somatic cell. To generate nt ES cells, a combination of nuclear transfer technology (Cibelli JB et al. (1998), Nature Biotechnol., 16:642-646) and ES cell generation technology (mentioned above) is used (Wakayama Sayaka et al. (2008), Experimental Medicine, Vol. 26, No. 5 (special issue), pp. 47-52). In nuclear transfer, the nucleus of a somatic cell is injected into an enucleated unfertilized egg of a mammal, and the egg can be initialized by culturing for several hours.

[0026] As the ES cell line used in the present invention, for example, various mouse ES cell lines established by inGenious targeting laboratory, RIKEN (RIKEN) and the like can be used in the case of mouse ES cells, and for example, various human ES cell lines established by University of Wisconsin, NIH, RIKEN, Kyoto University, National Center for Child Health and Development, Cellartis, ESI Bio, WiCell Research and the like can be used in the case of human ES cell lines. Specifically, for example, human ES cell lines include CHB-1 to CHB-12 strains, RUES1 strain, RUES2 strain, HUES1 to HUES28 strains, etc. distributed by ESI Bio, H1 strain, H9 strain, etc. distributed by WiCell Research, and KhES-1 strain, KhES-2 strain, KhES-3 strain, KhES-4 strain, KhES-5 strain, SSES1 strain, SSES2 strain, SSES3 strain, etc. distributed by RIKEN.

[0027] iPS cells are cells obtained by reprogramming mammalian somatic cells or undifferentiated stem cells by introducing specific factors (nuclear reprogramming factors). Currently, there are various types of iPS cells, including iPS cells established by Yamanaka et al. by introducing four factors, Oct3 / 4, Sox2, Klf4, and c-Myc, into mouse fibroblasts (Takahashi K, Yamanaka S., Cell, (2006) 126: 663-676); human-derived iPS cells established by introducing the same four factors into human fibroblasts (Takahashi K, Yamanaka S., et al. Cell, (2007) 131: 861-872.); Nanog-iPS cells established by selecting cells using the expression of Nanog as an indicator after introducing the above four factors (Okita, K., Ichisaka, T., and Yamanaka, S. (2007). Nature 448, 313-317.); and iPS cells created by a method that does not contain c-Myc (Nakagawa M, Yamanaka S., et al. Nature Biotechnology, (2008) 26, 101-106), and iPS cells established by introducing six factors using a virus-free method (Okita K et al. Nat. Methods 2011 May;8(5):409-12, Okita K et al. Stem Cells. 31(3):458-66.) can also be used. In addition, induced pluripotent stem cells established by introducing four factors, OCT3 / 4, SOX2, NANOG, and LIN28, produced by Thomson et al. (Yu J., Thomson JA. et al., Science (2007) 318: 1917-1920.), induced pluripotent stem cells produced by Daley et al. (Park IH, Daley GQ. et al., Nature (2007) 451: 141-146), induced pluripotent stem cells produced by Sakurada et al. (JP Patent Publication No. 2008-307007), etc. can also be used.

[0028] In addition, all published papers (e.g., Shi Y., Ding S., et al., Cell Stem Cell, (2008) Vol3, Issue 5,568-574; Kim JB., Scholer HR., et al., Nature, (2008) 454, 646-650; Huangfu D., Melton, DA., et al., Nature Biotechnology, (2008) 26, No 7, Any of the induced pluripotent stem cells known in the art and described in the above-mentioned publications (e.g., JP 2008-307007 A, JP 2008-283972 A, US2008-2336610, US2009-047263, J2007-069666, WO2008-118220, WO2008-124133, WO2008-151058, WO2009-006930, WO2009-006997, WO2009-007852) can be used.

[0029] As induced pluripotent stem cell lines, various iPS cell lines established by NIH, RIKEN, Kyoto University, Lonza, etc. are available. For example, human iPS cell lines include RIKEN's HiPS-RIKEN-1A strain, HiPS-RIKEN-2A strain, HiPS-RIKEN-12A strain, Nips-B2 strain, etc.; Kyoto University's 253G1 strain, 253G4 strain, 1201C1 strain, 1205D1 strain, 1210B2 strain, 1383D2 strain, 1383D6 strain, 201B7 strain, 409B2 strain, 454E2 strain, 606A1 strain, 610B1 strain, 648A1 strain, 1231A3 strain, FfI-01s04 strain, QHJI01s04 strain, Lonza's TC-1133HKK_05G strain, TC-1133HKK_06E strain, or iPS cell lines obtained by genetically modifying the above-mentioned iPS cell lines.

[0030] The species of origin of the pluripotent stem cells is not particularly limited, and may be, for example, cells from rodents such as rats, mice, hamsters, and guinea pigs, lagomorphs such as rabbits, ungulates such as pigs, cows, goats, and sheep, carnivores such as dogs and cats, and primates such as humans, monkeys, rhesus monkeys, marmosets, orangutans, and chimpanzees. The preferred species of origin is human.

[0031] 2. How to determine the presence or absence of BCOR gene mutations The present invention provides a method for determining the presence or absence of a mutation in the BCOR gene in a pluripotent stem cell population, comprising a step of detecting a biomarker of the present invention for the pluripotent stem cell population (hereinafter, sometimes referred to as the "determination method of the present invention"). In the determination method of the present invention, only one type of the biomarker of the present invention may be detected, or two or more types may be detected. When two or more types are detected, the combination of the biomarkers to be detected is not particularly limited, and different types of transcripts or proteins may be detected (e.g., PRAC1 transcripts and PRAC2 transcripts are detected, etc.), or the same type of transcripts or proteins may be detected (e.g., PRAC2 transcripts and PRAC2 proteins are detected, or PRAC2 isoform a and PRAC2 isoform b are detected, etc.). In one aspect, the determination method of the present invention is a method for determining the presence of a mutation in the BCOR gene in a pluripotent stem cell population when the biomarker of the present invention is detected in the pluripotent stem cell population.

[0032] In this specification, unless otherwise specified, "cell" includes "cell population". A cell population may be composed of one type of cell, or may be composed of two or more types of cells. Therefore, the term "pluripotent stem cell" includes not only a single pluripotent stem cell, but also a pluripotent stem cell population. Furthermore, a "pluripotent stem cell population" means a cell population containing one or more pluripotent stem cells, but unless otherwise specified, a pluripotent stem cell population is a cell population that mainly contains pluripotent stem cells (typically, a cell population in which the proportion of pluripotent stem cells in the cell population (number of pluripotent stem cells / total number of cells × 100) is 90% or more). Furthermore, a pluripotent stem cell population that does not have a mutation in the BCOR gene (hereinafter also referred to as a "wild-type cell population") means a cell population in which the proportion of cells having loss-of-function mutations in the cell population is less than 1% or is undetectable by amplicon sequencing analysis, as shown in the Examples below. Furthermore, in the determination method of the present invention, a pluripotent stem cell population is typically determined to have a BCOR gene mutation when the cell population contains one or more cells (preferably at a ratio of 1% or more) having a mutation in the BCOR gene.

[0033] Furthermore, as used herein, "detecting a biomarker" encompasses not only examining the presence or absence of the biomarker of the present invention in a pluripotent stem cell population (i.e., whether or not a transcript or protein is present in an amount equal to or greater than the detection limit of a detection method), but also measuring (quantifying) the amount of the biomarker present. Thus, the determination method of the present invention can also be carried out using the amount of the biomarker of the present invention as an index. Thus, in another aspect, the determination method of the present invention comprises: (1) measuring the abundance of one or more biomarkers of the present invention in a subject pluripotent stem cell population (hereinafter also referred to as a "subject cell population") and a wild-type cell population; and (2) determining that a mutation in the BCOR gene is present in the target cell population when the value measured in the target cell population is higher than the value measured in the wild-type cell population for at least one biomarker (preferably all of the measured biomarkers); The method includes:

[0034] In step (1) above, the abundance of a biomarker is typically measured in a cell population having the same number of cells.

[0035] The amount of BCOR gene present in the pluripotent stem cells of interest may be compared with a preset reference value to determine the presence or absence of a mutation in the BCOR gene in the pluripotent stem cells. (1') determining the abundance of one or more biomarkers of the invention in a subject cell population; and (2') determining that a mutation in the BCOR gene is present in the subject cell population when the value measured in step (1) exceeds a reference value. The method includes:

[0036] In one embodiment of the present invention, the reference value used in the above step (2') is a value based on the measurement value of the biomarker of the present invention in a wild-type cell population. Such a measurement value is typically measured in a cell population having the same number of pluripotent stem cells. As the "reference value" used in the present invention, for example, the average value, mode, or median of the measurements in multiple cell populations, or a value calculated from these values ​​by arithmetic operations, can also be used.

[0037] The above-mentioned reference value may be a cut-off value. A method for calculating the cut-off value is well known in the art. For example, the amount of the biomarker of the present invention is measured in a target cell population and a wild-type cell population, and the diagnostic sensitivity and diagnostic specificity at the measured values ​​are determined. Based on these values, a receiver operating characteristic (ROC) curve is created using commercially available analysis software. Then, the values ​​at which the diagnostic sensitivity and diagnostic specificity are as close to 100% as possible are determined, and the values ​​can be used as cut-off values.

[0038] In addition, in this specification, "determining the presence or absence of a mutation in the BCOR gene" includes not only determining whether or not a cell population has a mutation in the BCOR gene, but also analyzing (e.g., analyzing, evaluating, calculating, etc.) the proportion of cells having a mutation in the BCOR gene in the cell population and the type of mutation. For example, a calibration curve can be created by measuring the abundance of the biomarker of the present invention using multiple samples whose proportions of cells having a mutation in the BCOR gene are known, and based on the calibration curve, the proportion of cells having a mutation in the BCOR gene can be calculated from the abundance of the biomarker of the present invention in a cell population whose proportion is unknown.

[0039] The detection or quantification of the biomarkers of the present invention in a cell population can be examined by preparing an RNA (e.g., total RNA, mRNA) fraction from the sample and detecting the biomarker transcripts of the present invention contained in the fraction. Thus, in one embodiment, the determination method of the present invention comprises detecting or quantifying the biomarker transcripts of the present invention using a nucleic acid probe or a nucleic acid primer that can specifically recognize the biomarker transcripts of the present invention, respectively.

[0040] The RNA fraction can be prepared by known methods such as guanidine-CsCl ultracentrifugation and AGPC, and high-purity total RNA can be rapidly and easily prepared from a trace amount of sample using a commercially available RNA extraction kit (e.g., RNeasy Mini Kit; manufactured by QIAGEN, etc.). Means for detecting the biomarker transcripts of the present invention in the RNA fraction include, for example, a method using hybridization (Northern blot, dot blot, etc.) or a method using quantitative PCR (e.g., real-time PCRR, digital PCR, etc.).

[0041] In the case of Northern blot or dot blot hybridization, the detection or quantification of the biomarker transcripts of the present invention can be carried out, for example, using a nucleic acid probe capable of specifically recognizing each of the transcripts of the biomarkers of the present invention. Examples of such nucleic acid probes include nucleic acids that contain a sequence complementary to a continuous region of 15 or more bases, preferably 16 to 100 bases, more preferably 17 to 80 bases, and even more preferably 18 to 50 bases, in the base sequence of the known transcripts described above. The nucleic acid may be DNA or RNA, or may be a DNA / RNA chimera, but is preferably DNA. The nucleic acid used as a probe may be double-stranded or single-stranded. In the case of a double-stranded nucleic acid, it may be double-stranded DNA, double-stranded RNA, or a DNA:RNA hybrid. In the case of a single-stranded nucleic acid, one containing an antisense strand sequence can be used.

[0042] The nucleic acid probe is preferably labeled with a labeling agent to enable detection of the target nucleic acid. Examples of the labeling agent include radioisotopes, enzymes, fluorescent substances, and luminescent substances. Examples of the radioisotopes include 32 P], [ 3 H], [ 14 C], etc. are used. As the enzyme, those that are stable and have a large specific activity are preferred, for example, β-galactosidase, β-glucosidase, alkaline phosphatase, peroxidase, malate dehydrogenase, etc. are used. As the fluorescent substance, for example, fluorescamine, fluorescein isothiocyanate, etc. are used. As the luminescent substance, for example, luminol, luminol derivatives, luciferin, lucigenin, etc. are used. Furthermore, biotin-(strept)avidin can also be used to bind the probe and the labeling agent.

[0043] The above nucleic acid probe can be obtained by amplifying a nucleic acid of a desired length by PCR using a primer set designed based on the sequence of the biomarker transcript of the present invention and a cell-derived cDNA or genomic DNA as a template, or by cloning the above gene or cDNA from the cDNA or genomic DNA library by colony or plaque hybridization or the like, and, if necessary, by forming a fragment of an appropriate length using a restriction enzyme, etc. Alternatively, it can also be obtained by chemical synthesis using a commercially available automatic DNA / RNA synthesizer, etc.

[0044] In the case of Northern hybridization, the RNA fraction prepared as described above is separated by gel electrophoresis, then transferred to a membrane such as nitrocellulose, nylon, polyvinylidene difluoride, etc., and specifically hybridized in a hybridization buffer containing the labeled probe prepared as described above, and the label bound to the membrane is detected for each band by an appropriate method, or the amount of label is detected or quantified for each band, thereby measuring the transcription amount of the biomarker of the present invention. In the case of dot blot, the membrane spotted with the RNA fraction is similarly subjected to a hybridization reaction, and the transcription or transcription amount of the biomarker of the present invention can be detected by detecting the label of the spot or measuring the amount of label of the spot.

[0045] In a preferred embodiment, quantitative PCR is used as a method for detecting or quantifying the biomarker transcripts of the present invention. Quantitative PCR can be performed by a known method, for example, by synthesizing cDNA with reverse transcriptase using total RNA as a template, and performing PCR in the presence of a set of nucleic acid primers specific to the target gene, DNA polymerase, and a dye or probe that can function as a DNA intercalator to quantify the expression level.

[0046] Examples of digital PCR include droplet digital PCR (ddPCR) and chip-based digital PCR (cdPCR). Digital PCR is performed, for example, by the following procedure. A reaction solution containing a probe set, a DNA sample, a PCR primer set, and DNA polymerase is set in a digital PCR device. Here, the mixing ratio of each reaction solution component can be appropriately selected and optimized within a known range, and can be appropriately changed depending on the primer set, probe set, etc. used.

[0047] The nucleic acid primers used in quantitative PCR can be a pair of primers that are present on the synthesized cDNA and are specific to the gene to be amplified. The primers can be designed taking into consideration the size of the amplified product (e.g., 80-150 bp is preferable), the size of the primer (e.g., 17-25 bases), the GC content (e.g., 40-60%), the sequence of the 3' end (e.g., the base at the 3' end should preferably be G or C, and primers with too much GC content near the 3' end should be avoided), the sequence gap (e.g., to avoid repeating the sequence), the sequence complementarity (e.g., to avoid 3 or more bases of complementarity within the upstream primer or between primers), the Tm value (e.g., the Tm value of the upstream primer and the downstream primer should be the same. The Tm value is 2(A+T)+4(G+C)), and the like, and can be designed using primer design software well known to those skilled in the art. The design of PCR primers can also be requested from Applied Biosystems Inc., etc. Such primers can be prepared or synthesized in the same manner as the nucleic acid probes.

[0048] A fluorescent dye can be bound to the probe used in quantitative PCR (the probe is also included in the "nucleic acid probe"). Various fluorescent dyes are commercially available, such as 6-FAM (fluorescein), HEX, TE, Quasar 670, Quasar 570, Quasar 705, Pulsar 650, TET, HEX, VIC, JOE, CAL Fluor Orange, CAL Fluor Gold, CAL Fluor Red, Texas Red, Cy, and Cy5. In addition, each probe is preferably further bound to a quencher capable of quenching the fluorescence from the fluorescent dye. The quencher is not particularly limited as long as it can quench the fluorescence from the fluorescent dye, and may be a fluorescent dye or a non-fluorescent dye, but a non-fluorescent dye is preferable from the viewpoint of detection accuracy. Specific examples of the quencher include 6-carboxytetramethylrhodamine (TAMRA), 6-carboxy-X-rhodamine (ROX), Eclipse Dark Quencher, Iowa black FQ (IBFQ), minor groove binder (MGB), non-fluorescent quencher (NFQ), etc. In a preferred embodiment, a fluorescent dye is bound to the 5' or 3' end of each probe, and a quencher is bound to the opposite end.

[0049] The detection or quantification of the biomarker proteins of the present invention can be carried out by preparing a protein fraction from a cell population and detecting or quantifying the proteins contained in the fraction. The detection or quantification of these proteins can be carried out by immunological assays (e.g., ELISA, FIA, RIA, Western blot, etc.) using antibodies that specifically recognize each protein, but is typically carried out by immunological assays, particularly ELISA. Alternatively, a method using the simple kit of the present invention described in 3 below may be used.

[0050] Antibodies capable of specifically recognizing each of the biomarker proteins of the present invention can be produced by existing general production methods using these proteins or partial peptides having epitopes as immunogens. In the present specification, the term "antibody" includes, but is not limited to, natural antibodies such as polyclonal antibodies and monoclonal antibodies (mAbs), chimeric antibodies that can be produced using genetic recombination techniques, humanized antibodies, single-chain antibodies, and binding fragments thereof. Preferably, the antibody is a polyclonal antibody, a monoclonal antibody, or a binding fragment thereof. The binding fragment means a partial region of the above-mentioned antibody having specific binding activity, and specifically includes, for example, F(ab')2, Fab', Fab, Fv, sFv, dsFv, sdAb, and the like (Exp. Opin. Ther. Patents, Vol.6, No.5, p.441-456, 1996). The class of the antibody is not particularly limited, and includes antibodies having any isotype such as IgG, IgM, IgA, IgD, or IgE. Preferably, it is IgG or IgM, and more preferably IgG in consideration of ease of purification, etc. In the present invention, it is also preferable to use a commercially available antibody or a kit or array containing an antibody as an antibody capable of specifically recognizing each of the biomarker proteins of the present invention.

[0051] When applying each immunological detection method or quantification method to the determination method of the present invention, it is not necessary to set special conditions, operations, etc. A detection or quantification system for the biomarker of the present invention can be constructed by adding ordinary technical considerations of a person skilled in the art to the usual conditions and operations of each method. For details of these general technical means, reviews, books, etc. can be referred to. For example, "Radioimmunoassay" edited by Irie Hiroshi (Kodansha, published in 1974), "Radioimmunoassay 2" edited by Irie Hiroshi (Kodansha, published in 1979), "Enzyme Immunoassay" edited by Ishikawa Eiji et al. (Igaku Shoin, published in 1978), "Enzyme Immunoassay" edited by Ishikawa Eiji et al. (2nd edition) (Igaku Shoin, published in 1982), "Enzyme Immunoassay" edited by Ishikawa Eiji et al. (3rd edition) (Igaku Shoin, published in 1987), "Methods in ENZYMOLOGY" Vol. 70 (Immunochemical Techniques (Part A)), Vol. 73 (Immunochemical Techniques (Part B)), Vol. 74 (Immunochemical Techniques (Part C)), Vol. 84 (Immunochemical Techniques (Part D: Selected Immunoassays)), Vol. 92 (Immunochemical For example, reference can be made to Vol. 121 (Immunochemical Techniques (Part I: Hybridoma Technology and Monoclonal Antibodies)) of the same book (both published by Academic Press).

[0052] In addition, the detection or quantification of the biomarkers of the present invention can be performed using iTRAQ, which is capable of high-throughput protein detection or quantitative analysis. TM Detection or quantification may also be performed using proteomic analysis in combination with a reagent (ABI) and a mass spectrometer.

[0053] In view of the above, in one aspect, the measurement method of the present invention is a method characterized by detecting the biomarkers using a nucleic acid probe and / or a nucleic acid primer that specifically recognizes one or more of the biomarker transcription products of the present invention, or an antibody that specifically recognizes a protein encoded by the transcription product.

[0054] 3. BCOR gene mutation detection kit Furthermore, the present invention provides a determination kit for detecting BCOR gene mutations in pluripotent stem cells (hereinafter, the determination kit of the present invention). The determination kit of the present invention preferably contains a nucleic acid probe and / or a nucleic acid primer that specifically recognizes a transcription product selected from the group consisting of PRAC1, PRAC2, TBX1, MT1F, ZIC1, PITX2, MT1G, and FOXC1, or an antibody that specifically recognizes a protein encoded by the transcription product. Only one type of these may be contained, or multiple types may be contained.

[0055] In one embodiment, the determination kit of the present invention is also provided as a kit (sometimes referred to as "the simple kit of the present invention" in this specification) that can detect the presence or absence of the biomarker of the present invention in a sample by simply contacting the sample with a substrate using ELISA. The simple kit of the present invention includes a substrate on which an antibody (hereinafter also referred to as "first antibody") that specifically recognizes the biomarker protein of the present invention is immobilized, and typically, the antibody is a labeled antibody. In addition, the substrate included in the simple kit of the present invention preferably includes an antibody (hereinafter also referred to as "second antibody") that specifically recognizes the biomarker protein of the present invention, which is different from the above antibody, and in this embodiment, the biomarker of the present invention can be detected by the sandwich ELISA method. In addition, it is also preferable that an antibody (e.g., anti-IgG antibody or anti-IgM antibody) that recognizes the first antibody is further immobilized on the substrate included in the simple kit of the present invention, and such an antibody can be used to confirm that the determination by the kit has been performed normally.

[0056] When the determination kit of the present invention includes the above-mentioned nucleic acid probe or nucleic acid primer (also simply referred to as "nucleic acid") as a component, the same nucleic acids as those exemplified in the determination method of the present invention in the above 2. can be used. These nucleic acids can be provided as solids in a dried or alcohol precipitated state, or can be provided in a dissolved state in water or a suitable buffer solution (e.g., TE buffer, etc.). When used as a labeled probe, the nucleic acid can be provided in a state in which it has been previously labeled with any of the above-mentioned labeling substances, or can be provided separately from the labeling substances and labeled when used. Alternatively, the nucleic acid can be provided in a state in which it has been fixed (also referred to as being supported or solid-phased) on a suitable substrate. Examples of substrates include, but are not limited to, glass, silicon, plastic, nitrocellulose, nylon, polyvinylidene difluoride, etc. Examples of immobilization methods include, but are not limited to, a method in which a functional group such as an amino group, an aldehyde group, an SH group, or a biotin group is first introduced into the nucleic acid, and a functional group that can react with the nucleic acid (e.g., an aldehyde group, an amino group, an SH group, or a streptavidin group) is also introduced onto the substrate, and the substrate and the nucleic acid are crosslinked by a covalent bond between the two functional groups, or, for polyanionic nucleic acids, a method in which the substrate is coated with polycations and the nucleic acid is immobilized by utilizing electrostatic bonds.

[0057] When the determination kit of the present invention contains the above-mentioned antibodies as a component, these antibodies include the same antibodies as those exemplified in the determination method of the present invention in 2. above.

[0058] The determination kit of the present invention may contain, in addition to the above-mentioned nucleic acid or antibody, other substances necessary for the reaction for detecting or quantifying the expression of the biomarker of the present invention. These other substances may be provided in a state where they coexist with the nucleic acid or antibody, etc., or may be provided together with a separate reagent, so long as they do not adversely affect the reaction. For example, when the reaction for detecting or quantifying the expression of the biomarker of the present invention is PCR, examples of the other substances include a reaction buffer, dNTPs, a heat-resistant DNA polymerase, etc. When quantitative PCR is used, competitor nucleic acid and a fluorescent reagent (the above-mentioned intercalator, fluorescent probe, etc.) may further be included. When the reaction for detecting or quantifying the expression of the biomarker of the present invention is an antigen-antibody reaction, examples of the other substances include a reaction buffer, a competitor antibody, a labeled secondary antibody (for example, when the primary antibody is a rabbit antibody, mouse anti-rabbit IgG labeled with peroxidase, alkaline phosphatase, etc.), a blocking solution, an ELISA plate, etc. In addition, the determination kit of the present invention may contain an instruction manual that describes the method of using the kit and the reagents, the criteria for determination, etc. Furthermore, the above-mentioned determination kit may contain one or more biomarkers of the present invention for use as, for example, a positive control. The types, specific examples, and usage methods of the reagents used in the determination kit of the present invention are all incorporated by reference in the contents of "2. Method for determining the presence or absence of a BCOR gene mutation."

[0059] The present invention will be described in more detail below with reference to examples, but the present invention is not limited to these in any way. EXAMPLES

[0060] Genetic analysis of BCOR gene mutation-positive iPS cells iPS cells were maintained in culture with iMatrix-511MG (matrixosome, Catalogue No. 892-005) at 0.5 μg / cm 2A culture dish coated with iMatrix-511MG and StemFit (registered trademark) AK03N medium (Ajinomoto) were used. At the time of subculture, cells were detached using TrypLE Select (Gibco, Catalogue No. A1285901) diluted 2-fold with EDTA (Nacalai Tesque, Catalogue No. 13567-84), suspended in StemFit (registered trademark) AK03N medium containing 10 μM Y-27632 (Fujifilm Wako Pure Chemical Industries, Catalogue No. 039-24591), and seeded on a culture dish coated with iMatrix-511MG. One day after seeding, the medium was changed to StemFit (registered trademark) AK03N medium without Y-27632, and the culture was continued until the cells approached confluence.

[0061] RNA-seq analysis was performed on iPS cells (2 strains) containing BCOR gene mutations and iPS cells (6 strains) containing the wild-type BCOR gene. RNA was extracted from various iPS cells using RNeasy Mini kit (QIAGEN, Catalogue No. 74104). cDNA containing an adapter sequence was synthesized from the obtained RNA by the SMART (Switching Mechanism At 5'End of RNA Template) method, and sequence analysis was performed using Novaseq6000 (Illumina). The obtained read sequence was mapped to the genome sequence using DRAGEN Bio-IT Platform (Illumina), and the expression level was quantified on a gene-by-gene basis. The logFC was calculated for the obtained RNA-seq data using edgeR, which is an expression variation gene extraction software in the R language. Table 1 shows genes whose expression was increased by 32 times or more (logFC > 5) in BCOR gene mutation-positive iPS cells compared to the wild-type. All iPS cells used in this example were derived from human males.

[0062] [Table 1] EXAMPLES

[0063] Expression of TBX1, PRAC1 and PRAC2 genes in BCOR gene mutation-positive iPS cells Next, gene expression was examined by quantitative PCR for some of the genes in Table 1 (TBX1, PRAC1, PRAC2). iPS cell lines in which loss-of-function mutations in the BCOR gene were detected in 1% or more of the cells by amplicon sequencing analysis were designated as "BCOR gene mutation positive," and iPS cell lines in which this was less than 1% were designated as "BCOR gene wild type." The cell lines used are shown in Table 2. All iPS cells used in this example were derived from human males.

[0064] [Table 2]

[0065] After extracting RNA from various iPS cells, the RNA concentration was measured using an ultra-microspectrophotometer (Thermo Fisher Scientific, Catalogue No. ND-ONE-W). The obtained RNA was mixed with 500 ng and 0.5 μg of Oligo dt Primer (Promega, Catalogue No. C110A) and 10 nmol of dNTP Mix (Invitrogen, Catalogue No. 18427-013), and then heated at 65°C for 5 minutes. The obtained sample was subjected to reverse transcription reaction using SUPERase-InTM Rnase Inhibitor (Invitrogen, Catalogue No. AM2696) and Super Script IV (Invitrogen, Catalogue No. 18090050) to synthesize cDNA.

[0066] Quantitative PCR was performed using the obtained cDNA, Taqman Fast Advanced Master Mix (Thermo Fisher Scientific, Catalogue No. 4444557), and the QuantStudio 5 Real-Time PCR System (Thermo Fisher Scientific, Catalogue No. A28138). Taqman Probe (Thermo Fisher Scientific) was used as the primer and probe for quantitative PCR (Table 3).

[0067] [Table 3]

[0068] The quantitative PCR amplification curves for each gene are shown in Figures 1 to 3. In addition, the ΔCt value for each gene was calculated using GAPDH as an endogenous control (Table 4).

[0069] In TBX1 and PRAC1, the amplification curves of BCOR gene mutation-positive iPS cell lines (samples 1, 2, and 3) showed a faster rise and lower ΔCt values ​​than those of BCOR gene wild-type iPS cell lines (samples 4, 5, and 6) (Figures 1 and 2) (Table 3). In PRAC2, the amplification curves of BCOR gene mutation-positive iPS cell lines (samples 1, 2, and 3) showed a rise, but the curves of BCOR gene wild-type iPS cell lines (samples 4, 5, and 6) did not show a rise (Figure 3).

[0070] [Table 4]

[0071] These results demonstrated that the expression of TBX1, PRAC1, and PRAC2 genes was increased in iPS cell lines with BCOR gene mutations. It is also strongly suggested that the expression of other genes listed in Table 1 is also increased in iPS cell lines. [Industrial Applicability]

[0072] According to the present invention, a method for easily detecting a mutation in the BCOR gene in pluripotent stem cells is provided, which makes it possible to provide pluripotent stem cells with a low risk of tumorigenesis, and is particularly useful in the field of regenerative medicine.

Claims

1. A biomarker for detecting BCOR gene mutations in pluripotent stem cells, which consists of a transcript or protein selected from the group consisting of PRAC1, PRAC2, TBX1, MT1F, ZIC1, PITX2, MT1G, and FOXC1.

2. A method for determining the presence or absence of a mutation in the BCOR gene in a pluripotent stem cell population, the method comprising the step of detecting one or more of the biomarkers described in claim 1 in the pluripotent stem cell population.

3. (1) measuring the abundance of one or more biomarkers according to claim 1 in a target pluripotent stem cell population and a pluripotent stem cell population that does not have a mutation in the BCOR gene; and (2) determining that pluripotent stem cells having a mutation in the BCOR gene are present in the subject pluripotent stem cell population when the value of at least one biomarker measured in the subject pluripotent stem cell population is higher than the value measured in the subject pluripotent stem cell population without a mutation in the BCOR gene; The method of claim 2 , comprising:

4. (1) measuring the abundance of one or more biomarkers of claim 1 in a subject's pluripotent stem cell population; and (2) determining that the pluripotent stem cell population contains pluripotent stem cells having a mutation in the BCOR gene when the value measured in step (1) exceeds a reference value; The method of claim 2 , comprising:

5. The method according to claim 4, wherein the reference value is a value based on the measurement value of the biomarker described in claim 1 in a pluripotent stem cell population that does not have a mutation in the BCOR gene.

6. The method of claim 2, wherein the pluripotent stem cells are induced pluripotent stem cells.

7. The method of claim 2, wherein the mutation in the BCOR gene is a loss-of-function mutation.

8. The method according to any one of claims 2 to 7, characterized in that the biomarker is detected using a nucleic acid probe and / or nucleic acid primer that specifically recognizes a transcription product selected from the group consisting of PRAC1, PRAC2, TBX1, MT1F, ZIC1, PITX2, MT1G, and FOXC1, or an antibody that specifically recognizes a protein encoded by the transcription product.

9. A determination kit for detecting BCOR gene mutations in pluripotent stem cells, comprising a nucleic acid probe and / or nucleic acid primer that specifically recognizes a transcription product selected from the group consisting of PRAC1, PRAC2, TBX1, MT1F, ZIC1, PITX2, MT1G and FOXC1, or an antibody that specifically recognizes a protein encoded by the transcription product.

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