KR20250058170A - Use of Lcn2 as biomarker for diagnosis of neuromyelitis optica - Google Patents

Abstract

The present invention relates to the use of Lcn2 as a biomarker for diagnosing neuromyelitis optica, and more particularly, the present invention relates to a biomarker composition for diagnosing early-stage neuromyelitis optica comprising an Lcn2 (lipocalin-2) gene or a protein expressed from the gene, a composition for diagnosing early-stage neuromyelitis optica comprising a substance for measuring an mRNA level of the Lcn2 gene or a protein level thereof, a diagnostic kit for early-stage neuromyelitis optica, a method for providing information for predicting and diagnosing early-stage neuromyelitis optica, and a method for detecting the influx of aquaporin-4 into blood, comprising a step of measuring an mRNA level of the Lcn2 gene or a protein expression level thereof from a biological sample isolated from a subject.

Description

{Use of Lcn2 as biomarker for diagnosis of neuromyelitis optica}

The present invention relates to the use of Lcn2 as a biomarker for diagnosing neuromyelitis optica.

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

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; aquaporin-4), a water transport channel protein mainly expressed in astrocytes. Neuromyelitis optica-specific antibodies are found in approximately 75% of patients, and aquaporin-4 protein is the main antigen of 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, and when NMO-IgG, a neuromyelitis optica-specific antibody, binds to the antigen aquaporin-4, it induces cytotoxicity, damaging astrocytes and causing neuromyelitis optica.

Currently, most treatments for neuromyelitis optica include anticancer drugs, immunosuppressants, immunomodulators, and blood purification therapies, and a safety clinical trial for an antibody called iculizumab is in progress. However, these immunosuppressants have strong toxicity and are accompanied by serious side effects such as central nervous system infection, leukemia, cardiac toxicity, or death. Therefore, it is very urgent and important to develop a treatment that can minimize these side effects and maximize efficacy.

However, because the exact pathogenesis 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 is also insufficient.

Therefore, it is important to diagnose the onset of neuromyelitis optica early to prevent it or prescribe appropriate treatment to suppress the progression of the disease. However, there is currently no effective diagnostic marker that can diagnose the early onset of neuromyelitis optica.

Korean Patent Registration No. 10-1715127 Korean Patent Registration No. 10-1977843

Accordingly, the inventors of the present invention completed the present invention by conducting research to discover a highly accurate and reliable biomarker for diagnosing early neuromyelitis optica based on blood, and by elucidating that lipocalin-2 (Lcn2) has an increased expression level in the blood when early neuromyelitis optica develops, thereby confirming that it can be used as a biomarker for diagnosing early neuromyelitis optica.

Therefore, the purpose of the present invention is to provide a biomarker composition for diagnosing early-stage neuromyelitis optica, comprising the Lcn2 (lipocalin-2) gene or a protein expressed from the gene.

Another object of the present invention is to provide a composition for diagnosing early-stage neuromyelitis optica, comprising a substance for measuring the mRNA or protein level of the Lcn2 (lipocalin-2) gene.

Another object of the present invention is to provide an early-stage neuromyelitis optica diagnostic kit comprising the composition of the present invention.

Another object of the present invention is to provide a method for providing information for predicting and diagnosing early-stage neuromyelitis optica, comprising the steps of: (a) measuring an mRNA level or protein expression level for the Lcn2 (lipocalin-2) gene from a biological sample isolated from a patient; and (b) measuring an mRNA or protein expression level of the gene from a normal control sample and comparing the result with the measurement result of step (a).

Furthermore, another object of the present invention relates to a method for detecting whether aquaporin-4 is introduced into blood, comprising a step of measuring the mRNA level of the Lcn2 (lipocalin-2) gene or the protein expression level thereof from a biological sample isolated from an individual.

To achieve the above purpose, the present invention provides a biomarker composition for diagnosing early-stage neuromyelitis optica, comprising the Lcn2 (lipocalin-2) gene or a protein expressed from the gene.

In one embodiment of the present invention, the expression level of the gene may be increased when neuromyelitis optica is developed compared to a normal group.

The present invention also provides a composition for diagnosing early-stage neuromyelitis optica, comprising a substance for measuring the mRNA or protein level of the Lcn2 (lipocalin-2) gene.

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

The present invention also provides a diagnostic kit for early-onset neuromyelitis optica, comprising the diagnostic composition for early-onset neuromyelitis optica of the present invention.

In addition, the present invention provides a method for providing information for predicting and diagnosing early-stage neuromyelitis optica, comprising the steps of: (a) measuring an mRNA level or protein expression level for the Lcn2 (lipocalin-2) gene from a biological sample isolated from a patient; and (b) measuring an mRNA or protein expression level of the gene from a normal control sample and comparing the result with the measurement result of step (a).

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

If the mRNA or protein expression level of the above gene for diagnosing early-stage neuromyelitis optica is increased compared to a normal control group, a step of determining that it is early-stage neuromyelitis optica may be further included.

The present invention also provides a method for detecting whether aquaporin-4 is introduced into blood, comprising a step of measuring the mRNA level of Lcn2 (lipocalin-2) gene or the protein expression level thereof from a biological sample isolated from an individual.

In one embodiment of the present invention, if the mRNA level or protein expression level of the Lcn2 (lipocalin-2) gene is increased compared to a normal control group, it may be determined that aquaporin-4 has entered the blood and an aquaporin-4-related disease has developed.

In one embodiment of the present invention, the aquaporin-4 related disease may be neuromyelitis optica or cognitive impairment.

By using Lcn2 (lipocalin-2), a biomarker for diagnosing early neuromyelitis optica provided in the present invention, it is possible to quickly and accurately diagnose the onset of early neuromyelitis optica using only a blood sample.

Figure 1 shows the results of analyzing the changes in genome expression levels in samples isolated from the normal group and patient groups at different stages of neuromyelitis optica progression. A shows the genome changes confirmed in the normal group and the early stage of neuromyelitis optica (MOG D9), and B shows the genome changes confirmed in the normal group and the significantly advanced stage of neuromyelitis optica (MOG D19).
Figure 2 shows the results of RT-PCR analysis to detect the Lcn2 gene in samples (brain tissue and blood) derived from a normal group and a group of patients at each stage of progression of neuromyelitis optica using the Lcn2 detection primer set designed in the present invention.

The present invention is characterized by providing a novel biomarker capable of diagnosing neuromyelitis optica in its early stage.

The present inventors, while conducting research to discover a biomarker that can accurately and effectively diagnose and predict the onset of neuromyelitis optica in the early stage, identified genes that showed differences in expression levels in samples from the neuromyelitis optica group compared to the normal group, and confirmed that Lcn2 (lipocalin-2) can be used as a diagnostic marker for early neuromyelitis optica.

In one embodiment of the present invention, brain tissue and blood were separated from a normal mouse group and a group of mice with neuromyelitis optica at each stage of progression, and then genome analysis was performed to identify genes showing differences in expression levels between each experimental group. As a result, the Lcn2 (lipocalin-2) gene showing a specific difference in expression level in the group with neuromyelitis optica compared to the normal group was identified.

The expression level of the above Lcn2 (lipocalin-2) gene was found to be increased in the onset of neuromyelitis optica compared to the normal group, indicating that it can be used as a biomarker for the diagnosis of neuromyelitis optica.

In addition, in another embodiment of the present invention, a primer set capable of specifically detecting only the Lcn2 (lipocalin-2) gene was prepared, and using the primer set, RT-PCR analysis was performed on brain tissue and blood obtained from a normal group, an early-onset neuromyelitis optica group, and a severe-onset neuromyelitis optica group, respectively. In the normal group, a reaction product was not detected, but was detected in the blood sample of the early-onset neuromyelitis optica group, thereby confirming the expression level of the Lcn2 gene. In addition, when the primer set of the present invention was used, it was confirmed that the reaction product was also detected in the brain tissue sample of the neuromyelitis optica group, but the detection level was very low.

Through this, the inventors of the present invention were able to quickly and accurately detect and diagnose the onset of neuromyelitis optica through analysis of the expression level of Lcn2 (lipocalin-2) discovered in the present invention, and in particular, were able to confirm the onset of early neuromyelitis optica.

Therefore, the present invention can provide a biomarker composition for diagnosing early-stage neuromyelitis optica, comprising the Lcn2 (lipocalin-2) gene or a protein expressed from the gene.

In the present invention, the base sequence of the Lcn2 gene is shown in sequence number 1, and the amino acid sequence is shown in sequence number 2.

In addition, the present invention can provide a composition for diagnosing early-stage neuromyelitis optica, comprising a substance for measuring the mRNA level of the Lcn2 (lipocalin-2) gene or its protein level.

The above material may be a primer, probe or antibody that specifically binds to the gene or protein, and in one embodiment of the present invention, a primer set of sequence numbers 3 and 4 specific to the Lcn2 gene was designed.

In the present invention, the biomarker includes organic biomolecules such as polypeptides or nucleic acids (e.g., mRNA, etc.), lipids, glycolipids, glycoproteins, sugars (monosaccharides, disaccharides, oligosaccharides, etc.), etc., which show an increase or decrease in expression in tissues, cells, or blood when neuromyelitis optica is developed. In particular, the biomarker provided in the present invention may be the Lcn2 gene, or a protein expressed by the gene, the expression amount of which increases in a sample of an individual with neuromyelitis optica compared to a normal group.

In the present invention, the expression level of a gene preferably means the mRNA level at which the gene is expressed, i.e., the amount of mRNA, and a material capable of measuring the level may include a primer or probe specific to the gene. In the present invention, the primer or probe specific to the gene may be a primer or probe capable of specifically amplifying the entire gene or a specific region of the gene, and the primer or probe may be designed by a method known in the art.

In the present invention, the term "primer" means a single-stranded oligonucleotide which can act as an initiation point of template-directed DNA synthesis under suitable conditions (i.e., four different nucleoside triphosphates and a polymerization enzyme) in a suitable temperature and a suitable buffer. The suitable length of the primer may vary depending on various factors, such as temperature and the use of the primer. In addition, the sequence of the primer does not need to have a sequence that is completely complementary to a part of the sequence of the template, and it is sufficient if it has sufficient complementarity within a range that can hybridize with the template and perform the primer's unique function. Therefore, the primer in the present invention does not need to have a sequence that is completely complementary to the nucleotide sequence of the template gene, and it is sufficient if it has sufficient complementarity within a range that can hybridize with the gene sequence and perform the primer function. In addition, it is preferable that the primer according to the present invention can be used for a gene amplification reaction.

The above amplification reaction refers to a reaction for amplifying a nucleic acid molecule, and amplification reactions of such genes are well known in the art, and may include, for example, polymerase chain reaction (PCR), reverse transcription polymerase chain reaction (RT-PCR), ligase chain reaction (LCR), electron-mediated amplification (TMA), nucleic acid sequence substrate amplification (NASBA), etc.

In the present invention, the term probe means a linear oligomer of natural or modified monomers or linkages, which comprises deoxyribonucleotides and ribonucleotides and can specifically hybridize to a target nucleotide sequence, and which exists naturally or is artificially synthesized. The probe according to the present invention may be single-stranded, and preferably may be an oligodeoxyribonucleotide. The probe of the present invention may include natural dNMPs (i.e., dAMP, dGMP, dCMP and dTMP), nucleotide analogues or derivatives. In addition, the probe of the present invention may also include ribonucleotides. For example, the probes of the present invention may be used to probe backbone modified nucleotides, such as peptide nucleic acids (PNA) (M. Egholm et al., Nature, 365:566-568(1993)), phosphorothioate DNA, phosphorodithioate DNA, phosphoroamidate DNA, amide-linked DNA, MMI-linked DNA, 2'-O-methyl RNA, alpha-DNA and methyl phosphonate DNA, sugar modified nucleotides, such as 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, such as C-5 substituted pyrimidines (substituents may be fluoro-, bromo-, chloro-, 7-deazapurine having a C-7 substituent (including iodo-, methyl-, ethyl-, vinyl-, formyl-, ethidyl-, propynyl-, alkynyl-, thiazolyl-, imidazolyl-, pyridyl-), inosine and diaminopurine.

In addition, in the present invention, the material capable of measuring the level of the protein 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.

The above “antibody” can be used as manufactured by a technique known to those skilled in the art, and the manufacturing of the antibody can be produced by a method well known in the art, for example, in the case of a polyclonal antibody, by 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 manufactured from any animal species host, such as a goat, rabbit, sheep, monkey, horse, pig, cow, or dog. In the case of a monoclonal antibody, the antibody can be manufactured using a hybridoma method well known in the art (Kohler et al., European Jounral of Immunology, 6, 511-519, 1976) or can be manufactured 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 a functional fragment of an antibody molecule. The functional fragment of an antibody molecule means a fragment having at least an antigen-binding function, and includes Fab, F(ab'), F(ab') 2, and Fv, etc. In addition, the present invention can provide an early-stage neuromyelitis optica diagnostic kit comprising the early-stage neuromyelitis optica diagnostic biomarker or the diagnostic composition according to the present invention.

The diagnostic kit of the present invention may include primers, probes or antibodies capable of measuring the expression level of the marker gene or the amount of protein expressed from the gene, the definitions of which are as described above.

When the diagnostic kit of the present invention is applied to a PCR amplification process, the kit of the present invention may optionally include reagents necessary for PCR amplification, such as a buffer, a DNA polymerase (e.g., a thermostable 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 when 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 labeled substrate.

Furthermore, the kit according to the present invention may be manufactured with a plurality of separate packagings or compartments containing the reagent components described above.

In addition, the present invention can provide a microarray for diagnosing early neuromyelitis optica, which includes the biomarker for diagnosing early neuromyelitis optica.

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

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

Furthermore, the present invention can provide a method for providing information for predicting and diagnosing early-stage neuromyelitis optica, preferably comprising the steps of: (a) measuring an mRNA level or protein expression level for the Lcn2 (lipocalin-2) gene from a biological sample isolated from a patient; and (b) measuring an mRNA or protein expression level of the gene from a normal control sample and comparing the result with the measurement result of step (a).

The method for measuring the expression level of a gene or the amount of a protein as described above can be performed by including a known process for isolating mRNA or protein from a biological sample using a known technique.

In the present invention, the "biological sample" refers to a sample collected from a living body, in which the expression level of the gene or the level of the protein is different from that of the normal control group according to the degree of occurrence or progression of neuromyelitis optica, and the sample may include, but is not limited to, blood, serum, plasma, saliva, urine, etc., and preferably, plasma.

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

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

The measurement of the above protein level can utilize antibodies, in which case the marker protein in the biological sample and the antibody specific thereto form a complex, i.e. an antigen-antibody complex, and the amount of the antigen-antibody complex formed can be quantitatively measured through the size of the signal of the detection label. The detection label can be selected from the group consisting of enzymes, fluorophores, ligands, luminescent substances, microparticles, redox molecules, and radioisotopes, but is not limited thereto. Analytical methods for measuring the protein level include, but are not limited to, Western blot, ELISA, radioimmunoassay, radioimmunodiffusion, orchite immunodiffusion, rocket immunoelectrophoresis, tissue immunostaining, immunoprecipitation assay, complement fixation assay, FACS, protein chips, etc.

Therefore, the present invention can confirm the mRNA expression amount or protein amount of a marker gene in a control group and the mRNA expression amount or protein amount of a marker gene in a patient with neuromyelitis optica or a patient suspected of neuromyelitis optica through the above detection methods, and by comparing the degree of the expression amount with that of the control group, it is possible to predict and diagnose the onset, progression stage, or prognosis of neuromyelitis optica.

More specifically, the method for predicting or diagnosing the onset of the above-mentioned neuromyelitis optica can be determined to be caused by neuromyelitis optica if the expression level of the marker gene according to the present invention or the amount of its expressed protein is increased compared to a normal control sample, and particularly, if the expression level of the gene or protein in the present invention is confirmed to be increased compared to the control, it can be determined to be an early-stage neuromyelitis optica.

Furthermore, the present invention can provide a method for detecting whether aquaporin-4 is introduced into blood, comprising a step of measuring the mRNA level of the Lcn2 (lipocalin-2) gene or the protein expression level thereof from a biological sample isolated from an individual.

Aquaporin-4 (AQP4) is a protein belonging to the aquaporin family of integral membrane proteins that conduct water across cell membranes. Aquaporins AQP1, 3, 4, 5, 8, 9, and 11 have been found in the central nervous system (CNS), and AQP1, 4, and 9 are mainly found in the brain and spinal cord. Of these, AQP4 is most abundant in the cerebellum and spinal gray matter, and is highly expressed in astrocytes around microvessels.

Meanwhile, when aquaporin-4, which exists in astrocytes, enters the bloodstream, it affects the development of various diseases including neuromyelitis optica. In the case of neuromyelitis optica, the specific antibody for neuromyelitis optica (NMO-IgG) binds to aquaporin-4, a water channel protein, inducing an immune response, causing inflammation in the optic nerve and spinal cord, leading to the development of the disease.

Therefore, if the expression level of Lcn2 in a sample obtained from an individual is increased compared to normal, it can be determined that aquaporin-4 has entered the blood and caused an aquaporin-4-related disease.

Here, the aquaporin-4 related disease may be neuromyelitis optica or cognitive impairment.

As described above, the novel diagnostic and predictive biomarkers of neuromyelitis optica identified in the present invention are expressed more highly in samples of early-stage neuromyelitis optica, particularly in blood. 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.

Hereinafter, the present invention will be described in more detail by way of examples. These examples are only intended to explain the present invention more specifically, and it will be apparent to those skilled in the art that the scope of the present invention is not limited to these examples.

<Example 1>

Analysis of genetic expression changes according to the progression of neuromyelitis optica

In order to discover a marker capable of diagnosing the onset of neuromyelitis optica, the present inventors conducted an experiment using a normal mouse group and a neuromyelitis optica-induced mouse group. The neuromyelitis optica-induced mice were animals in which neuromyelitis optica was induced by administration of oligodendrocyte glycoprotein (MOG), and the diseased animals were divided into groups of MOG D9 (day 9 of MOG administration; early stage of neuromyelitis optica) and MOG D19 (day 19 of MOG administration; severe stage showing severe lesions) based on the time when the lesions of neuromyelitis optica began due to MOG administration. Blood-derived plasma and brain tissue were extracted from each of the above mouse groups, and highly pure RNA (260/280 ratio = 1.8-2.0) was extracted from each sample using a trizol reagent known in the art. Specifically, first, the sample obtained from each experimental mouse was mixed with the same volume of Trizol in a 1:1 ratio, and then incubated at room temperature for about 1 hour. Then, high-purity chloroform corresponding to 1/5 of the reaction solution volume was added to extract RNA. Afterwards, 100% isopropanol was added to isolate RNA when purifying RNA for the final analysis, and the purity of the purified RNA was measured using Nano drop. The results showed that the purity of the RNA extracted from each sample had a 260/280 ratio of 1.8 to 2.0.

Thereafter, a whole genome analysis was performed on the total RNA extracted through the above method. A library for whole genome analysis was produced using the TruSeq Stranded Total RNA LT Kit known in the art, and the expression changes of the genome in the sample were analyzed through Illumina HiSeq 4000 Next Generation sequencing analysis, and the results are shown in Fig. 1.

As a result of the analysis, as shown in Figure 1, we analyzed the genomes that showed differences in expression between the normal group and the neuromyelitis optica disease group, and performed sequencing analysis on the genomes whose expression levels were significantly increased in the disease group compared to the normal group.

Sequencing analysis results showed that the lipocalin-2 (Lcn2) gene had a significantly increased expression level in the neuromyelitis optica disease group compared to the normal group, and in particular, the expression level was found to be significantly increased in the MOG D19 sample, which is the early stage of neuromyelitis optica.

Through this, the inventors of the present invention were able to determine that lipocalin-2 (Lcn2) can be used as a biomarker for the diagnosis of neuromyelitis optica, and in particular, that it can be used for the diagnosis of early neuromyelitis optica.

<Example 2>

Preparation of primers for detection of lipocalin-2 (Lcn2)

In order to easily detect Lcn2, which was newly discovered as a diagnostic marker for neuromyelitis optica in the above <Example 1>, a primer set was created as follows. To this end, cDNA was synthesized using oligodT primer and reverse transcriptase to obtain completely processed mRNA from the total RNA extracted in the above Example 1, and 100 ng of template cDNA was used for RT-PCR to perform reverse transcription-polymerase chain reaction (RT-PCR) using the synthesized cDNA. Based on RNA obtained from the normal group and the neuromyelitis optica mouse group, a region that does not overlap with other genes and is easy to detect was selected for the expression of the Lcn2 gene, and a primer set that can produce a PCR amplification product of about 100 bp was designed. In addition, all primers were designed to have the same Tm value for effective PCR performance. The primer sets that the present inventors use to specifically detect only the Lcn2 gene are as follows. When the following primer set is used, an amplification product for the DNA region from base 403 to 502 of the Lcn2 gene sequence of sequence number 1 can be obtained.

<Primer Set>

Forward primer (F): 5’-ACGGACTACAACCAGTTCGC-3’

Reverse primer (R): 5’-GGGACAGCTCCTTGGTTCTT-3’

<Example 3>

Diagnosis of neuromyelitis optica using the primer set for detecting Lcn2 designed in the present invention

The present inventors performed an experiment to confirm whether the onset of neuromyelitis optica can be diagnosed by analyzing the expression level of Lcn2 in brain tissue and blood samples using the Lcn2 detection primer set designed in Example 2 above.

First, RNA was isolated from tissues and blood obtained from the normal mouse group and the neuromyelitis optica mouse group (MOG D9, MOG D19) according to disease progression obtained in the above example, and then RT-PCR was performed using the primer set designed in the present invention. At this time, the RT-PCR performance conditions are as shown in Table 1 below, and the RT-PCR analysis results are shown in Fig. 2.

RT-PCR conditions No Temperature (℃) hour Cycle 1 95 5 minutes 1 2 95 20 seconds 32 3 60 30 seconds 4 72 15 seconds 5 72 5 minutes 1 6 4

As a result of the analysis, as shown in Fig. 2, while no RT-PCR reaction product was detected in the normal group samples, the RT-PCR reaction product was confirmed in the samples of the neuromyelitis optica group. In addition, uniquely, while the RT-PCR reaction product was very slightly confirmed in the tissue samples, the reaction product was very distinct in the blood samples, and a prominent RT-PCR reaction product was confirmed in the blood sample of MOG D9, the early stage of neuromyelitis optica.

Through these results, the inventors of the present invention were able to find out that the Lcn2 gene discovered in the present invention can be used as a biomarker for the diagnosis of neuromyelitis optica. In particular, since it was confirmed through blood samples that the expression of the Lcn2 gene is significantly increased from the early stage of neuromyelitis optica, it was found that the Lcn2 marker can be easily used for the diagnosis of early neuromyelitis optica based on blood samples.

The present invention has been described with reference to preferred embodiments thereof. Those skilled in the art will appreciate that the present invention may be implemented in modified forms without departing from the essential characteristics of the present invention. Therefore, the disclosed embodiments should be considered from an illustrative rather than a restrictive perspective. The scope of the present invention is indicated by the claims, not the foregoing description, and all differences within the scope equivalent thereto should be interpreted as being included in the present invention.

Attach electronic file of sequence list

Claims (11)

A biomarker composition for diagnosing early-stage neuromyelitis optica, comprising the Lcn2 (lipocalin-2) gene or a protein expressed from the gene. In the first paragraph,
A biomarker composition for diagnosing early-stage neuromyelitis optica, characterized in that the expression level of the gene increases when neuromyelitis optica develops compared to a normal group. A composition for diagnosing early-stage neuromyelitis optica, comprising a substance for measuring the mRNA or protein level of the Lcn2 (lipocalin-2) gene. In the third paragraph,
A composition for diagnosing early-stage neuromyelitis optica, characterized in that the substance is a primer, probe or antibody that specifically binds to the gene or protein. A diagnostic kit for early-onset neuromyelitis optica, comprising the composition of claim 3. (a) a step of measuring the mRNA level or protein expression level for the Lcn2 (lipocalin-2) gene from a biological sample isolated from a patient; and
(b) a step of measuring the mRNA or protein expression level of the gene from a normal control sample and comparing it with the measurement result of step (a).
Methods for providing information to predict and diagnose early-onset neuromyelitis optica. In Article 6,
A method for providing information for predicting and diagnosing early-onset neuromyelitis optica, characterized in that the biological sample is tissue, blood, serum or plasma. In Article 6,
A method for providing information for predicting and diagnosing early-stage neuromyelitis optica, characterized by further including a step of determining that it is early-stage neuromyelitis optica if the mRNA or protein expression level of the gene is increased compared to a normal control group. Comprising a step of measuring the mRNA level of Lcn2 (lipocalin-2) gene or its protein expression level from a biological sample separated from an object.
A method for detecting the influx of aquaporin-4 into the blood. In Article 9,
A method for detecting whether aquaporin-4 has entered the blood, characterized in that if the mRNA level or protein expression level thereof of the Lcn2 (lipocalin-2) gene is increased compared to a normal control group, it is determined that aquaporin-4 has entered the blood and an aquaporin-4-related disease has developed. In Article 10,
A method for detecting whether aquaporin-4 enters the blood, wherein the above aquaporin-4-related disease is characterized by neuromyelitis optica or cognitive impairment.