Biomarker for diagnosis of neuromyelitis optica and use thereof

Abstract

The present invention relates to a biomarker for diagnosing neuromyelitis optica and its use. Specifically, the present invention relates to a biomarker composition for diagnosing neuromyelitis optica comprising one or more genes selected from the group consisting of Plac8 (placenta-specific 8), Klra2 (killer cell lectin-like receptor, subfamily A, member 2) and Mcoln2 (mucolipin 2) or a protein expressed from said genes; a composition for diagnosing neuromyelitis optica comprising a substance for measuring the mRNA level of said genes or the protein level thereof; a diagnostic kit for neuromyelitis optica comprising said composition; and a method for providing information for predicting and diagnosing neuromyelitis optica.

Description

Technology Field

[0001] The present invention relates to Plac8, Klra2, and Mcoln2 as biomarkers for the diagnosis of neuromyelitis optica. It concerns the use of. Background Technology

[0002] Neuromyelitis optica (NMO) is an autoimmune disease of the central nervous system characterized by inflammatory demyelinating lesions in the spinal cord and optic nerves.

[0003] Neuromyelitis optica can be distinguished from other diseases by the presence or absence of NMO-IgG, a neuromyelitis optica-specific antibody against aquaporin-4 (AQP4), a water transport channel protein primarily expressed in astrocytes. This specific antibody is found in approximately 75% of patients. Aquaporin-4 is the primary antigen for NMO-IgG and is mainly distributed in astrocytes, which constitute the blood-brain barrier that protects the brain and central nervous system from inflammatory cells. The binding of NMO-IgG to aquaporin-4 is a key mechanism in the pathogenesis of neuromyelitis optica; as the specific antibody NMO-IgG binds to the antigen aquaporin-4, it induces cytotoxicity, damaging astrocytes and ultimately causing the onset of neuromyelitis optica.

[0004] Currently, most treatments for neuromyelitis optica consist of anticancer drugs, immunosuppressants, immunomodulators, and blood purification therapies. Although safety clinical trials for an antibody called iquelumab are underway, these immunosuppressants are highly toxic and accompanied by severe side effects such as central nervous system infections, leukemia, cardiotoxicity, or death. Therefore, it is urgent and important to develop a treatment that can minimize these side effects and maximize efficacy.

[0005] Nevertheless, since the precise pathological mechanism of neuromyelitis optica is still not clearly known, there is no basic treatment method, and the development of therapeutic agents with effective therapeutic activity for rare diseases such as neuromyelitis optica remains insufficient.

[0006] Therefore, currently, it is important to diagnose the onset of neuromyelitis optica early to prevent it or to prescribe appropriate treatments to suppress the progression of the disease; however, there is still a lack of effective diagnostic markers to detect the early onset of neuromyelitis optica. Prior art literature

[0007] Korean Registered Patent 10-1715127 The problem to be solved

[0008] Accordingly, while conducting research to discover a new biomarker capable of diagnosing neuromyelitis optica with high accuracy and reliability, the inventors identified that the expression levels of the Plac8, Klra2, and Mcoln2 genes are increased in tissues and blood at the onset of neuromyelitis optica, thereby confirming that they can be used as new biomarkers for diagnosing neuromyelitis optica and completing the present invention.

[0009] Accordingly, the objective of the present invention is to provide a biomarker composition for diagnosing neuromyelitis optica, comprising one or more genes selected from the group consisting of Plac8 (placenta-specific 8), Klra2 (killer cell lectin-like receptor, subfamily A, member 2) and Mcoln2 (mucolipin 2), or a protein expressed from said genes.

[0010] Another objective of the present invention is to provide a composition for diagnosing neuromyelitis optica, comprising a substance for measuring the mRNA or protein levels of one or more genes selected from the group consisting of Plac8 (placenta-specific 8), Klra2 (killer cell lectin-like receptor, subfamily A, member 2), and Mcoln2 (mucolipin 2).

[0011] Another objective of the present invention is to provide a diagnostic kit for neuromyelitis optica comprising the above-mentioned composition for diagnosing neuromyelitis optica.

[0012] Another objective of the present invention is to provide a method for providing information for predicting and diagnosing neuromyelitis optica, comprising: (a) measuring the mRNA level or protein expression level for one or more genes selected from the group consisting of Plac8 (placenta-specific 8), Klra2 (killer cell lectin-like receptor, subfamily A, member 2) and Mcoln2 (mucolipin 2) from a biological sample isolated from a patient; and (b) measuring the mRNA or protein expression level of said genes from a normal control sample and comparing it with the measurement result of step (a). means of solving the problem

[0013] To achieve the above objective, the present invention provides a biomarker composition for diagnosing neuromyelitis optica, comprising one or more genes selected from the group consisting of Plac8 (placenta-specific 8), Klra2 (killer cell lectin-like receptor, subfamily A, member 2), and Mcoln2 (mucolipin 2), or a protein expressed from said genes.

[0014] In one embodiment of the present invention, the expression level of the gene may increase when neuromyelitis optica develops compared to the normal group.

[0015] In one embodiment of the present invention, the Plac8 (placenta-specific 8) gene may be composed of the nucleotide sequence of SEQ ID NO. 1, the Klra2 (killer cell lectin-like receptor, subfamily A, member 2) gene may be composed of the nucleotide sequence of SEQ ID NO. 2, and the Mcoln2 (mucolipin 2) gene may be composed of the nucleotide sequence of SEQ ID NO. 3.

[0016] In addition, the present invention provides a composition for diagnosing neuromyelitis optica, comprising a substance for measuring the mRNA or protein level of one or more genes selected from the group consisting of Plac8 (placenta-specific 8), Klra2 (killer cell lectin-like receptor, subfamily A, member 2), and Mcoln2 (mucolipin 2).

[0017] In one embodiment of the present invention, the material may be a primer, probe, or antibody that specifically binds to the gene or protein.

[0018] In addition, the present invention provides a diagnostic kit for neuromyelitis optica comprising the composition for diagnosing neuromyelitis optica according to the present invention.

[0019] Furthermore, the present invention provides a method for providing information for predicting and diagnosing neuromyelitis optica, comprising: (a) measuring the mRNA level or protein expression level of one or more genes selected from a group consisting of Plac8 (placenta-specific 8), Klra2 (killer cell lectin-like receptor, subfamily A, member 2), and Mcoln2 (mucolipin 2) from a biological sample isolated from a patient; and (b) measuring the mRNA or protein expression level of said genes from a normal control sample and comparing it with the measurement result of step (a).

[0020] In one embodiment of the present invention, the biological sample may be tissue, blood, serum, or plasma.

[0021] In one embodiment of the present invention, the method may further include a step of determining that neuromyelitis optica has developed when the mRNA or protein expression level of the gene is increased compared to a normal control group.

[0022] In one embodiment of the present invention, the Plac8 (placenta-specific 8) gene may be composed of the nucleotide sequence of SEQ ID NO. 1, the Klra2 (killer cell lectin-like receptor, subfamily A, member 2) gene may be composed of the nucleotide sequence of SEQ ID NO. 2, and the Mcoln2 (mucolipin 2) gene may be composed of the nucleotide sequence of SEQ ID NO. 3. Effects of the invention

[0023] When using the biomarkers for diagnosing neuromyelitis optica provided in the present invention, it is possible to rapidly and accurately diagnose the onset and severity of neuromyelitis optica using only blood samples as well as brain tissue. Brief explanation of the drawing

[0024] Figure 1 shows the results of analyzing changes in genomic expression levels in samples isolated from a normal group and a patient group at different stages of neuromyelitis optica progression. A shows the genomic changes between the normal group and the early stage of neuromyelitis optica (MOG D9), and B shows the genomic changes between the normal group and the significantly advanced stage of neuromyelitis optica (MOG D19). Figure 2 is a schematic diagram showing the process of manufacturing a mouse model of neuromyelitis optica in one embodiment of the present invention. Figure 3 shows the results of analyzing changes in the expression levels of each gene using qRT-PCR on samples (brain tissue and blood) from a normal group and a patient group at different stages of neuromyelitis optica, using primer sets for detecting each of the Plac8, Klra2, and Mcoln2 genes designed in the present invention. Specific details for implementing the invention

[0025] The present invention is characterized by providing a new biomarker capable of diagnosing early-stage neuromyelitis optica.

[0026] While researching to discover biomarkers capable of accurately and rapidly diagnosing and predicting the onset of neuromyelitis optica, the inventors identified genes in samples from a group with neuromyelitis optica that showed differences in expression levels compared to a normal group. Among these, they confirmed that the expression levels of Plac8 (placenta-specific 8), Klra2 (killer cell lectin-like receptor, subfamily A, member 2), and Mcoln2 (mucolipin 2) genes were significantly increased in the group with neuromyelitis optica compared to the normal group, thereby determining that these markers could be used as biomarkers for the diagnosis of neuromyelitis optica.

[0027] In one embodiment of the present invention, brain tissue and blood were isolated from a normal mouse group and a group of mice with neuromyelitis optica at different stages of progression, respectively, and then genomic analysis was performed to identify genes showing differences in expression levels between the experimental groups. As a result, three genes, Plac8, Klra2, and Mcoln2, were identified as genes that specifically showed differences in expression levels in the group with neuromyelitis optica compared to the normal group.

[0028] In addition, in another embodiment of the present invention, a primer set capable of detecting Plac8, Klra2, and Mcoln2 genes specifically for each gene was designed and prepared. Using this, brain tissue and blood were obtained from a normal group, an early neuromyelitis optica group (MOG Day-9), and a severe neuromyelitis optica group (MOG Day-19), respectively, and qPCR was performed. As a result, it was found that reaction products, which were hardly detected in the normal group, were detected in the samples from the neuromyelitis optica group, and it was confirmed that the amount of reaction products increased as the disease progressed.

[0029] Through this, the inventors found that the onset of neuromyelitis optica can be rapidly and accurately detected and diagnosed by analyzing the expression levels of the biomarker genes Plac8, Klra2, and Mcoln2 discovered in this invention.

[0030] Accordingly, the present invention can provide a biomarker composition for diagnosing neuromyelitis optica, comprising one or more genes selected from the group consisting of Plac8 (placenta-specific 8), Klra2 (killer cell lectin-like receptor, subfamily A, member 2) and Mcoln2 (mucolipin 2), or a protein expressed from said genes.

[0031] In the present invention, the Plac8 (placenta-specific 8) gene may be composed of the nucleotide sequence of SEQ ID NO. 1, the Klra2 (killer cell lectin-like receptor, subfamily A, member 2) gene may be composed of the nucleotide sequence of SEQ ID NO. 2, and the Mcoln2 (mucolipin 2) gene may be composed of the nucleotide sequence of SEQ ID NO. 3.

[0032] In addition, the present invention may provide a composition for diagnosing neuromyelitis optica comprising a substance for measuring the mRNA or protein level of one or more genes selected from the group consisting of Plac8 (placenta-specific 8), Klra2 (killer cell lectin-like receptor, subfamily A, member 2) and Mcoln2 (mucolipin 2).

[0033] The above material may be a primer, probe, or antibody that specifically binds to the gene or protein. In one embodiment of the present invention, a primer set of SEQ ID NOs. 4 and 5 was devised as a primer capable of specifically detecting the Plac8 (placenta-specific 8) gene, a primer set of SEQ ID NOs. 6 and 7 was devised as a primer capable of specifically detecting the Klra2 (killer cell lectin-like receptor, subfamily A, member 2) gene, and a primer set of SEQ ID NOs. 8 and 9 was devised as a primer capable of specifically detecting the Mcoln2 (mucolipin 2) gene.

[0034] In the present invention, the term "biomarker" includes organic biomolecules such as polypeptides or nucleic acids (e.g., mRNA, etc.), lipids, glycolipids, glycoproteins, sugars (monosaccharides, disaccharides, oligosaccharides, etc.) that show an increase or decrease in expression in tissues, cells, or blood upon the onset of neuromyelitis optica. In particular, the biomarker provided in the present invention may be the marker gene of the present invention or the protein in which said gene is expressed, the expression level of which increases in samples from individuals with neuromyelitis optica compared to a normal group.

[0035] In the present invention, the expression level of a gene preferably refers to the level of mRNA in which the gene is expressed, i.e., the amount of mRNA, and a substance capable of measuring said level may include a primer or probe specific to said gene. In the present invention, the primer or probe specific to said gene may be a primer or probe capable of specifically amplifying the entire gene or a specific region of the gene, and said primer or probe may be designed through methods known in the art.

[0036] In the present invention, the term "primer" refers to a single-stranded oligonucleotide capable of acting as a starting point for template-directed DNA synthesis under suitable conditions (i.e., four different types of nucleoside triphosphates and polymerases) within a suitable temperature and a suitable buffer. The suitable length of the primer may vary depending on various factors, such as temperature and the application of the primer. Furthermore, the sequence of the primer does not need to be perfectly complementary to a portion of the template sequence; it is sufficient to have sufficient complementarity within the range where it can hybridize with the template and perform its inherent function. Therefore, the primer in the present invention does not need to have a sequence perfectly complementary to the nucleotide sequence of the template gene; it is sufficient to have sufficient complementarity within the range where it can hybridize with this gene sequence and perform its function as a primer. Additionally, it is preferable that the primer according to the present invention be used in a gene amplification reaction.

[0037] The above amplification reaction refers to a reaction that amplifies nucleic acid molecules, and such gene amplification reactions are well known in the art and may include, for example, quantitative polymerase chain reaction (qPCR), polymerase chain reaction (PCR), reverse transcription polymerase chain reaction (RT-PCR), ligase chain reaction (LCR), electron-mediated amplification (TMA), nucleic acid base sequence substrate amplification (NASBA), etc.

[0038] In the present invention, the term "probe" refers to a linear oligomer of natural or modified monomers or linkages, comprising deoxyribonucleotides and ribonucleotides, capable of specifically hybridizing to a target nucleotide sequence, and is said to be naturally occurring or artificially synthesized. The probe according to the present invention may be a single strand, preferably an oligodeoxyribonucleotide. The probe of the present invention may include natural dNMP (i.e., dAMP, dGMP, dCMP, and dTMP), nucleotide analogs, or derivatives. Additionally, the probe of the present invention may also include ribonucleotides. For example, the probe of the present invention comprises backbone-modified nucleotides, e.g., peptide nucleic acid (PNA) (M. Egholm et al., Nature, 365:566-568 (1993)), phosphothioate DNA, phosphodithioate DNA, phosphoroamidate DNA, amide-linked DNA, MMI-linked DNA, 2'-O-methyl RNA, alpha-DNA and methylphosphonate DNA, sugar-modified nucleotides, e.g., 2'-O-methyl RNA, 2'-fluoro RNA, 2'-amino RNA, 2'-O-alkyl DNA, 2'-O-allyl DNA, 2'-O-alkynyl DNA, hexose DNA, pyranosyl RNA and anhydrohexitol DNA, and nucleotides having base modifications, e.g., C-5 substituted pyrimidines (substituents are fluoro-, bromo-, chloro-, iodo-, It may include methyl-, ethyl-, vinyl-, formyl-, ethityl-, propynyl-, alkynyl-, thiazoryl-, imidazoryl-, pyridyl-), 7-deazpurine having a C-7 substituent (substituents include fluoro-, bromo-, chloro-, iodo-, methyl-, ethyl-, vinyl-, formyl-, alkynyl-, alkenyl-, thiazoryl-, imidazoryl-, pyridyl-), inosine, and diaminopurine.

[0039] In addition, the material capable of measuring the level of the protein in the present invention may include antibodies such as polyclonal antibodies, monoclonal antibodies, and recombinant antibodies that can specifically bind to the protein expressed from the marker gene of the present invention.

[0040] The “antibody” mentioned above may be one manufactured using a technique known to a person skilled in the art. For example, in the case of a polyclonal antibody, the antibody may be produced by a method widely known in the art, which involves injecting an antigen of the protein into an animal and collecting blood from the animal to obtain serum containing the antibody. Such polyclonal antibodies can be produced from any animal species host, such as goats, rabbits, sheep, monkeys, horses, pigs, cattle, and dogs. In the case of a monoclonal antibody, it may be manufactured using a hybridoma method widely known in the art (Kohler et al., European Journal of Immunology, 6, 511-519, 1976) or by using a phage antibody library technique (Clackson et al., Nature, 352, 624-628, 1991; Marks et al., J. Mol. Biol., 222:58, 1-597, 1991). In addition, the antibody according to the present invention may include not only a complete form having two full-length light chains and two full-length heavy chains, but also functional fragments of the antibody molecule. A functional fragment of the antibody molecule refers to a fragment that possesses at least an antigen-binding function, and includes Fab, F(ab'), F(ab') 2, and Fv, etc.

[0041] In addition, the present invention may provide a diagnostic kit for neuromyelitis optica comprising a biomarker for diagnosing neuromyelitis optica according to the present invention or a diagnostic composition.

[0042] The diagnostic kit of the present invention may include a primer, a probe, or an antibody capable of measuring the expression level of the marker gene or the amount of protein expressed from the gene, and the definitions thereof are as described above.

[0043] If the diagnostic kit of the present invention is applied to a PCR amplification process, the kit of the present invention may optionally include reagents required for PCR amplification, such as a buffer, a DNA polymerase (e.g., a heat-stable DNA polymerase obtained from Thermus aquaticus (Taq), Thermus thermophilus (Tth), Thermus filiformis, Thermis flavus, Thermococcus literalis, or Pyrococcus furiosus (Pfu)), a DNA polymerase cofactor, and dNTPs; and if the diagnostic kit of the present invention is applied to an immunoassay, the kit of the present invention may optionally include a secondary antibody and a substrate of a label.

[0044] Furthermore, the kit according to the present invention may be manufactured into a plurality of separate packages or compartments containing the above-mentioned reagent components.

[0045] In addition, the present invention may provide a microarray for diagnosing neuromyelitis optica comprising the biomarker for diagnosing neuromyelitis optica.

[0046] In the microarray of the present invention, a primer, probe, or antibody capable of measuring the expression level of the marker protein or the gene encoding it is used as a hybridizable array element and is immobilized on a substrate. A preferred substrate may be a suitable rigid or semi-rigid support, such as a membrane, filter, chip, slide, wafer, fiber, magnetic or non-magnetic bead, gel, tubing, plate, polymer, microparticle, and capillary. The hybridizable array element is arranged and immobilized on the substrate, and such immobilization may be performed by a chemical bonding method or a covalent bonding method such as UV. For example, the hybridizable array element may be bonded to a glass surface modified to include an epoxy compound or an aldehyde group, and may also be bonded by UV on a polylysine coated surface. Additionally, the hybridizable array element may be bonded to the substrate via a linker (e.g., ethylene glycol oligomer and diamine).

[0047] Meanwhile, if the sample applied to the microarray of the present invention is nucleic acid, it may be labeled and hybridized with array elements on the microarray. Hybridization conditions may vary, and the detection and analysis of the degree of hybridization may be performed in various ways depending on the labeling material.

[0048] Furthermore, the present invention may provide a method for providing information for predicting and diagnosing neuromyelitis optica, preferably comprising: (a) a step of measuring the mRNA level or protein expression level of one or more genes selected from a group consisting of Plac8 (placenta-specific 8), Klra2 (killer cell lectin-like receptor, subfamily A, member 2) and Mcoln2 (mucolipin 2) from a biological sample isolated from a patient; and (b) a step of measuring the mRNA or protein expression level of said genes from a normal control sample and comparing it with the measurement result of step (a).

[0049] The method for measuring the expression level of a gene or the amount of protein described above may be performed by including a known process for isolating mRNA or protein from a biological sample using known technology.

[0050] In the present invention, the term "biological sample" refers to a sample taken from a living organism in which the expression level of the gene or the level of the protein differs from that of a normal control group according to the degree of occurrence or progression of neuromyelitis optica. The sample may include, for example, but not limited to, blood, serum, plasma, saliva, and urine.

[0051] The measurement of the expression level of the above gene is preferably the measurement of the mRNA level, and methods for measuring the mRNA level include, but are not limited to, quantitative polymerase chain reaction (qPCR), reverse transcription polymerase chain reaction (RT-PCR), real-time reverse transcription polymerase chain reaction, RNase protection assay, Northern blot and DNA chip.

[0052] In addition, methods for measuring the amount or activity of a protein can be performed using various methods known in the art, such as, for example but not limited to, Western blot, Northern blot, ELISA (enzyme-linked immunosorbent assay), radioimmunoassay (RIA), radioimmunodiffusion, and immunoprecipitation assay.

[0053] The measurement of the above protein level may utilize antibodies. In this case, the marker protein in the biological sample and the antibody specific to it form a conjugate, i.e., an antigen-antibody complex, and the amount of antigen-antibody complex formed can be quantitatively measured through the magnitude of the signal of a detection label. Such detection labels may be selected from a group consisting of enzymes, fluorescent dyes, ligands, luminescent materials, microparticles, redox molecules, and radioisotopes, but are not limited thereto. Analytical methods for measuring protein levels include, but are not limited to, Western blot, ELISA, radioimmunoassay, radioimmunodiffusion, Ouchteroni immunodiffusion, rocket immunoelectrophoresis, tissue immunostaining, immunoprecipitation assay, complement fixation assay, FACS, protein chips, etc.

[0054] Accordingly, through the detection methods described above, the present invention can confirm the mRNA expression amount or protein amount of a marker gene in a patient with neuromyelitis optica or a patient suspected of having neuromyelitis optica with the mRNA expression amount or protein amount of a marker gene in a control group, and can predict and diagnose the onset, stage of progression, or prognosis of neuromyelitis optica by comparing the degree of expression amount with that of a control group.

[0055] More specifically, the method for predicting or diagnosing the onset of neuromyelitis optica according to the present invention can determine that neuromyelitis optica has been induced if the expression level of the marker gene or the amount of the expressed protein is increased compared to a normal control sample.

[0056] As described above, the biomarkers for the diagnosis and prediction of neuromyelitis optica identified in the present invention show increased expression in samples, particularly in blood, in which neuromyelitis optica has developed in the early stages; therefore, by measuring the expression levels of these markers, the progression stage of neuromyelitis optica can be accurately and rapidly predicted and diagnosed in the early stages.

[0058] The present invention will be explained in more detail below through examples. These examples are merely for the purpose of explaining the present invention more specifically, and it will be obvious to those skilled in the art that the scope of the present invention is not limited to these examples.

[0060] <Example 1>

[0061] Analysis of changes in genomic expression based on the onset of neuromyelitis optica

[0062] The inventors conducted experiments using a normal mouse group and a mouse group with neuromyelitis optica to identify markers capable of diagnosing the onset of neuromyelitis optica. In this case, the mice with neuromyelitis optica were prepared by administering oligodendrocyte glycoprotein (MOG) mixed with a CFA adjuvant to activate T cells recognizing MOG, thereby inducing neuromyelitis optica. The diseased animals were classified into two groups based on the time when neuromyelitis optica lesions began following MOG administration: MOG D9 (9th day after MOG administration; early stage of neuromyelitis optica, showing symptoms where the hind legs are usually extended but no tail movement is observed when the tail is lifted) and MOG D19 (19th day after MOG administration; severe stage showing serious lesions, showing symptoms of weight loss accompanied by limping hind legs and complete paralysis of the tail) (see Fig. 2).

[0063] Brain tissue was extracted from each mouse group, and high-purity RNA (260 / 280 ratio = 1.8–2.0) was extracted from each sample using Trizol, a reagent known in the industry. Specifically, first, an equal volume of Trizol was mixed with the sample obtained from each experimental mouse in a 1:1 ratio, incubated at room temperature for about 1 hour, and then high-purity chloroform equivalent to 1 / 5 of the reaction volume was added to extract RNA. Subsequently, 100% isopropanol was added to separate the RNA for final analysis, and the purity of the purified RNA was measured using a Nano drop. As a result, the purity of the RNA extracted from each sample was all found to have a 260 / 280 ratio between 1.8 and 2.0.

[0064] Subsequently, whole-genome analysis was performed on the total RNA extracted by the above method. A library for whole-genome analysis was prepared using the TruSeq Stranded Total RNA LT Kit known in the art, and changes in genome expression within the sample were analyzed through Illumina HiSeq 4000 Next Generation sequencing analysis, and the results are shown in Figure 1.

[0066] As a result of the analysis, as shown in Figure 1, genomes showing differences in expression between the normal group and the neuromyelitis optica disease group were analyzed, and sequencing analysis was performed on genomes in which the expression level was significantly increased in the disease group compared to the normal group.

[0067] Sequencing analysis revealed that the expression levels of Plac8 (placenta-specific 8), Klra2 (killer cell lectin-like receptor, subfamily A, member 2), and Mcoln2 (mucolipin 2) genes were significantly increased in the neuromyelitis optica group compared to the normal group.

[0069] Through this, the inventors were able to find that Plac8 (placenta-specific 8), Klra2 (killer cell lectin-like receptor, subfamily A, member 2) or Mcoln2 (mucolipin 2) can be used as new biomarkers for the diagnosis of neuromyelitis optica.

[0071] <Example 2>

[0072] Preparation of primers for detecting biomarkers for the diagnosis of neuromyelitis optica

[0073] To facilitate the detection of the Plac8, Klra2, and Mcoln2 genes, which were newly discovered as diagnostic markers for neuromyelitis optica in <Example 1> above, primer sets capable of specifically detecting each gene were constructed as shown in Table 1 below. To this end, cDNA was synthesized using oligodT primers and reverse transcriptase to obtain fully processed mRNA from the total RNA extracted in Example 1, and 100 ng of template cDNA was used for RT-PCR to perform reverse transcription gene amplification analysis (RT-PCR) using the synthesized cDNA. Based on RNA obtained from the normal group and the neuromyelitis optica mouse group, regions where the expression of the Plac8, Klra2, or Mcoln2 genes did not overlap with other genes and were easy to detect were selected, and primer sets for detecting each gene were designed. In addition, to ensure effective PCR performance, the primers were designed so that the Tm value was between 55 and 65°C and the CG content was between 50 and 55%.

[0074]

[0076] <Example 3>

[0077] Diagnosis of neuromyelitis optica through marker detection using the primer set devised in the present invention

[0078] The inventors obtained brain tissue and blood samples, respectively, from the neuromyelitis optica-induced disease mice prepared in Example 1 using the primer sets for detecting each of the Plac8, Klra2, and Mcoln2 genes designed in Example 2, and then performed qPCR to determine whether the onset of neuromyelitis optica could be diagnosed by analyzing the expression levels of each corresponding marker gene.

[0079] First, qPCR was performed using the primer set designed in the present invention on brain tissue and blood obtained from the normal mouse group and the neuromyelitis optica mouse group (MOG D9, MOG D19) according to disease progression used in the above example. The amount of amplified product was measured in real time by measuring the fluorescence intensity using the SYBR-Green fluorescent dye for detection of the qPCR product, and the conditions for performing qPCR are as shown in Table 2 below, and the analysis results are shown in Figure 3.

[0080]

[0081] As shown in Figure 3, the analysis of brain tissue revealed increased expression levels of the Plac8, Klra2, and Mcoln2 genes in MOG Day-9 and MOG Day-19 optic neuromyelitis mouse models compared to the control group, and the expression of these markers was found to increase as the disease progressed.

[0082] Furthermore, in the analysis performed on blood samples, it was possible to detect that the expression levels of the Plac8, Klra2, and Mcoln2 genes were all rapidly increased in MOG Day-9 and MOG Day-19 neuromyelitis optica mouse models compared to the control group when the primer set of the present invention was used.

[0084] Through these results, the inventors were able to determine that the Plac8, Klra2, and Mcoln2 genes discovered in this invention can be used as biomarkers for the diagnosis of neuromyelitis optica, and in particular, that these markers can be usefully employed to diagnose the onset and severity of the disease using only blood samples as well as tissue samples.

[0086] The present invention has been described above with reference to its preferred embodiments. Those skilled in the art will understand that the present invention may be embodied in modified forms without departing from the essential characteristics of the invention. Therefore, the disclosed embodiments should be considered in an illustrative rather than a restrictive sense. The scope of the invention is defined by the claims, not by the foregoing description, and all variations within the scope of the claims should be interpreted as being included in the invention.

Claims

Claim 1 A biomarker composition for diagnosing neuromyelitis optica, comprising one or more genes selected from the group consisting of Plac8 (placenta-specific 8), Klra2 (killer cell lectin-like receptor, subfamily A, member 2), and Mcoln2 (mucolipin 2), or a protein expressed from said genes. Claim 2 A biomarker composition for diagnosing neuromyelitis optica, wherein, in claim 1, the expression level of the gene increases when neuromyelitis optica develops compared to a normal group. Claim 3 A biomarker composition for diagnosing neuromyelitis optica, characterized in that, in claim 1, the Plac8 (placenta-specific 8) gene is composed of the nucleotide sequence of SEQ ID NO. 1, the Klra2 (killer cell lectin-like receptor, subfamily A, member 2) gene is composed of the nucleotide sequence of SEQ ID NO. 2, and the Mcoln2 (mucolipin 2) gene is composed of the nucleotide sequence of SEQ ID NO.

3. Claim 4 A composition for diagnosing neuromyelitis optica, comprising a substance for measuring the mRNA or protein levels of one or more genes selected from the group consisting of Plac8 (placenta-specific 8), Klra2 (killer cell lectin-like receptor, subfamily A, member 2), and Mcoln2 (mucolipin 2). Claim 5 A composition for diagnosing neuromyelitis optica, characterized in that, in claim 4, the substance is a primer, probe, or antibody that specifically binds to the gene or protein. Claim 6 A diagnostic kit for neuromyelitis optica comprising the composition of claim 4. Claim 7 (a) a step of measuring the mRNA level or protein expression level for one or more genes selected from the group consisting of Plac8 (placenta-specific 8), Klra2 (killer cell lectin-like receptor, subfamily A, member 2) and Mcoln2 (mucolipin 2) from a biological sample isolated from a patient; and (b) a step of measuring the mRNA or protein expression level of said gene from a normal control sample and comparing it with the measurement result of step (a), comprising a method for providing information for predicting and diagnosing neuromyelitis optica. Claim 8 A method for providing information for predicting and diagnosing neuromyelitis optica, characterized in that, in claim 7, the biological sample is tissue, blood, serum, or plasma. Claim 9 A method for providing information for predicting and diagnosing neuromyelitis optica, characterized in that, in claim 7, it further includes the step of determining that neuromyelitis optica has developed when the mRNA or protein expression level of the gene is increased compared to a normal control group. Claim 10 A method for providing information for predicting and diagnosing neuromyelitis optica, characterized in that, in claim 7, the Plac8 (placenta-specific 8) gene is composed of the nucleotide sequence of SEQ ID NO. 1, the Klra2 (killer cell lectin-like receptor, subfamily A, member 2) gene is composed of the nucleotide sequence of SEQ ID NO. 2, and the Mcoln2 (mucolipin 2) gene is composed of the nucleotide sequence of SEQ ID NO. 3.

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