CN113430263A - Biomarker-based product for diagnosing glaucoma and application thereof - Google Patents

Abstract

The invention discloses a product for diagnosing glaucoma based on biomarkers selected from DFNA5, MYH11 and/or KHDRBS1 and uses thereof. Compared with normal control, DFNA5 and KHDRBS1 show obvious up-regulation in glaucoma patients, MYH11 shows obvious down-regulation in glaucoma patients, and the diagnosis efficacy results show that DFNA5, MYH11 and/or KHDRBS1 have higher accuracy, sensitivity and specificity when applied to the diagnosis of glaucoma.

Description

Biomarker-based product for diagnosing glaucoma and application thereof

Technical Field

The invention relates to the field of biomedicine, and relates to a product for diagnosing glaucoma based on biomarkers and application thereof.

Background

Glaucoma (glaucoma) is a group of Retinal Ganglion Cells (RGCs); death is an important feature, and diseases represented by characteristic visual field defects and optic atrophy together; glaucoma, the second most largely blind eye disease worldwide today, causes over 7000 million people to become injured, severely threatening the visual health of humans (Casson RJ, Chidlow G, Wood JP, et al. Definition of glaucomma: clinical and experimental receptors [ J ]. Clip Exp Ophthalmol, 2012, 40(4): 341) 349.).

Glaucoma is clinically classified into three major types, i.e., congenital, Primary and secondary, in which the Primary Glaucoma is open or closed according to the state of the anterior chamber angle, and is classified into Primary open-angle Glaucoma (POAG) and Primary closed-angle Glaucoma (PACG) (Quigley HA. Glaucoma [ J ]. Lancet, 2011, 377(9774): 1367-1377.); the clinical features of POAG are represented by elevated intraocular pressure, but the anterior chamber angle is always open, which is the most common clinical type of glaucoma, and about 70% of the global glaucoma types are POAG (Beidoe G, mouse SA. Current primary open-angle glaucoma procedures and future directions [ J ]. Clin Ophthalmol, 2012, 6(1):1699-

POAG, an important type of glaucoma, is characterized by acquired optic atrophy, loss of RGC and its axons, and maintenance of the opening angle at all times with elevated intraocular pressure (the glaucoma group of the chinese medical society ophthalmology conference, consensus among experts in primary glaucoma diagnosis and treatment in china (2014) [ J ]. the chinese journal of ophthalmology, 2014,05 (00)), a chronically progressive optic neuropathy. Intraocular pressure is the only controllable factor in Glaucoma patients, and effective control is an important management of Glaucoma (Mantravadi A V, Vadhar N. Glaucoma [ J ]. Prim Care, 2015,42(3): 437 449.). However, the existence of normal intraocular POAG and the characteristic of POAG occult pathogenesis can cause missed diagnosis of part of patients, and irreversible damage to optic nerve usually exists when diagnosis is confirmed. Early diagnosis is of great significance in order to save visual function of POAG patients to the greatest extent possible.

Disclosure of Invention

In order to make up for the defects of the prior art, the invention researches biomarkers related to the occurrence and development of glaucoma based on the role of genetic factors in the occurrence and development of the glaucoma, thereby providing a new means for diagnosing and treating the glaucoma.

In a first aspect, the invention provides a product for diagnosing glaucoma, said product comprising reagents for detecting the level of the biomarkers DFNA5, MYH11 and/or KHDRBS1 in a sample.

Further, the level of DFNA5, MYH11 and/or KHDRBS1 in the sample is determined by measuring the protein level or mRNA level of DFNA5, MYH11 and/or KHDRBS1 in the sample.

Further, the protein levels of DFNA5, MYH11 and/or KHDRBS1 in the sample are measured by using immunostaining, immunofluorescence, western blot or ELISA.

Further, the mRNA levels of DFNA5, MYH11 and/or KHDRBS1 in the sample were measured by using microarray, RNA-seq, in situ hybridization, RNA-scope and conventional semi-quantitative or quantitative RT-PCR.

Further, the product also includes reagents for processing the sample.

In a second aspect, the invention provides the use of a product according to the first aspect of the invention in the manufacture of a means for diagnosing glaucoma.

In a third aspect, the invention provides the use of a reagent for detecting a biomarker in a sample, the biomarker comprising DFNA5, MYH11 and/or KHDRBS1, in the manufacture of a product for diagnosing glaucoma.

Further, the reagent comprises a reagent for detecting the level of the biomarker by a sequencing technology, a nucleic acid hybridization technology, a nucleic acid amplification technology and a protein immunity technology.

Further, the agent is selected from: a probe that specifically recognizes the biomarker; primers that specifically amplify the biomarkers; or an antibody that specifically binds to the biomarker.

Further, the sample includes, but is not limited to, tissue or fluid, such as tissue, blood, plasma, serum, lymph, urine, serosal cavity fluid, spinal fluid, synovial fluid, aqueous humor, tears, saliva, or components or treated materials thereof.

Preferably, the sample is selected from the group consisting of tissue, blood.

The invention has the beneficial effects that:

according to the invention, by detecting the expression levels of DFNA5, MYH11 and/or KHDRBS1, the diagnosis of glaucoma can be realized, the detection sensitivity is increased, the detection capability and efficiency are improved, and the capability of guiding the clinical treatment of glaucoma is improved.

Drawings

FIG. 1 shows a diagram of differential expression of the DFNA5 gene;

FIG. 2 shows a MYH11 gene differential expression profile;

FIG. 3 shows a graph of the differential expression of KHDRBS1 gene;

FIG. 4 shows a ROC plot of DFNA5 gene for diagnosing glaucoma;

FIG. 5 shows a ROC plot of MYH11 gene diagnosis for glaucoma;

FIG. 6 shows ROC plots of KHDRBS1 gene for diagnosing glaucoma;

FIG. 7 shows a ROC plot of DFNA5+ MYH11 gene diagnosis of glaucoma;

FIG. 8 shows a ROC plot of DFNA5+ KHDRBS1 gene for diagnosing glaucoma;

FIG. 9 shows ROC plots of MYH11+ KHDRBS1 gene diagnosis of glaucoma;

FIG. 10 shows ROC plots for DFNA5+ MYH11+ KHDRBS1 in combination for the diagnosis of glaucoma.

Detailed Description

The invention will be described in further detail below with the understanding that the terminology is intended to be in the nature of words of description rather than of limitation.

The terms "abundance," "level," and "content" are used interchangeably herein to refer to a quantitative content (e.g., weight or mole), a semi-quantitative content, a relative content (e.g., weight% or mole% within a grade), a concentration, and the like. Thus, these terms encompass the absolute or relative amounts or concentrations of disease treatment biomarkers in a sample.

The term "and/or" means and includes any and all possible combinations of one or more of the associated listed items, as well as the lack of combinations when interpreted in the alternative (or).

In the present invention, the term "biomarker" means a compound, preferably a gene, which is differentially present (i.e. increased or decreased) in a biological sample from a subject or a group of subjects having a first phenotype (e.g. having a disease) compared to a biological sample from a subject or a group of subjects having a second phenotype (e.g. no disease). The term "biomarker" generally refers to the presence/concentration/amount of one gene or the presence/concentration/amount of two or more genes.

The term "biomarker value" or "biomarker level" refers to a value measured or derived for at least one corresponding biomarker in a subject, and which is typically at least partially indicative of the abundance or concentration of the biomarker in a sample taken from the subject. Thus, a biomarker value may be a measured biomarker value, which is a biomarker value measured for a subject, or alternatively may be a derived biomarker value, which is a value derived from one or more measured biomarker values, for example, by applying a function to one or more measured biomarker values. The biomarker values may be in any suitable form, depending on the manner in which the values are determined. For example, biomarker values may be determined using high throughput techniques such as sequencing platforms, array and hybridization platforms, mass spectrometry, immunoassays, immunofluorescence, flow cytometry, or any combination of these techniques. In a preferred example, biomarker values relate to the abundance or activity level of an expression product or other measurable molecule, quantified using techniques such as quantitative RT-PCR, sequencing, and the like. In this case, the biomarker values may be in the form of amplification levels or cycle numbers, which are logarithmic representations of biomarker concentrations within the sample, as known to those skilled in the art. In other preferred examples, immunofluorescence of cells containing the expression product is used to quantify biomarker values.

In the present invention, the biomarker comprises DFNA5, MYH11 and/or KHDRBS 1.

In the present invention, DFNA5 (gasdermin E, gene ID: 1687) includes the DFNA5 gene and homologs, mutations, and isoforms thereof. The term encompasses full-length, unprocessed DFNA5, as well as any form of DFNA5 that results from processing in a cell. The term encompasses naturally occurring variants (e.g., splice variants or allelic variants) of DFNA 5.

MYH11 (myostatin latent chain 11, gene ID: 4629) includes the MYH11 gene and homologs, mutations, and isoforms thereof. The term encompasses full-length, unprocessed MYH11, as well as any form of MYH11 that results from processing in a cell. The term encompasses naturally occurring variants (e.g., splice variants or allelic variants) of MYH 11.

KHDRBS1 (KH RNA binding, signal transduction associated 1, gene ID: 10657) includes KHDRBS1 gene and its homologs, mutations, and isoforms. The term encompasses full length, unprocessed KHDRBS1, as well as any form of KHDRBS1 that results from processing in the cell. The term encompasses naturally occurring variants (e.g., splice variants or allelic variants) of KHDRBS 1.

The gene ID is available at https:// www.ncbi.nlm.nih.gov/gene/.

The term "primer" refers to an oligonucleotide that, when paired with a DNA strand, is capable of priming the synthesis of a primer extension product in the presence of a suitable polymerizing agent. The primer is preferably single-stranded for maximum amplification efficiency, but may also be double-stranded. The primer must be long enough to prime the synthesis of extension products in the presence of the polymerization agent. The length of the primer depends on many factors, including the application, the temperature to be used, the template reaction conditions, other reagents, and the source of the primer. For example, depending on the complexity of the target sequence, the primer can be at least about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 50, 75, 100, 150, 200, 300, 400, 500 to the 3 ' end of the primer a length of one base shorter than the template sequence to allow extension of the nucleic acid strand, although the 5 ' end of the primer can extend in length beyond the 3 ' end of the template sequence. In certain embodiments, the primer may be a large polynucleotide, such as about 35 nucleotides to several kilobases or more. The primer may be selected to be "substantially complementary" to a sequence on the template that is designed to hybridize thereto and serve as a synthesis initiation site. By "substantially complementary" is meant that the primers are sufficiently complementary to hybridize to the target polynucleotide. Ideally, a primer does not contain a mismatch to the template to which it is designed to hybridize, but this is not required. For example, a non-complementary nucleotide residue can be attached to the 5' end of the primer, while the remainder of the primer sequence is complementary to the template. Alternatively, a non-complementary nucleotide residue or a stretch of non-complementary nucleotide residues may be interspersed into the primer, provided that the primer sequence has sufficient complementarity with the template sequence to hybridize therewith, thereby forming a template for synthesizing a primer extension product.

The term "probe" refers to a molecule that binds to a particular sequence or subsequence or other portion of another molecule. Unless otherwise indicated, the term "probe" generally refers to a nucleic acid probe that binds to another nucleic acid (also referred to herein as a "target polynucleotide") by complementary base pairing. Probes can bind target polynucleotides that lack complementarity to the entire sequence of the probe, depending on the stringency of the hybridization conditions. Probes may be directly or indirectly labeled and include primers within their scope.

The term "antibody" is used herein in the broadest sense and includes monoclonal antibodies (e.g., full length or intact monoclonal antibodies), polyclonal antibodies, multivalent antibodies, multispecific antibodies (e.g., bispecific antibodies, so long as they exhibit the desired biological activity), and may also include certain antibody fragments (as described in more detail herein). The antibody may be a human, humanized and/or affinity matured antibody.

Detection of biomarker nucleic acids

In some embodiments, the biomarker is assessed by determining biomarker nucleic acid transcript levels. In an illustrative nucleic acid-based assay, nucleic acids are isolated from cells contained in a biological sample according to standard methods (Sambrook et al, 1989, supra; and Ausubel et al, 1994, supra). The nucleic acid is typically fractionated or whole cell RNA. In some embodiments, the nucleic acid is amplified by a template-dependent nucleic acid amplification technique. A variety of template-dependent methods can be used to amplify the disease treatment biomarker sequences present in a given template sample. An exemplary nucleic acid amplification technique is the polymerase chain reaction (referred to as PCR).

In certain advantageous embodiments, the template-dependent amplification involves transcript quantification in real time. For example, real-time PCR techniques can be used to quantify RNA or DNA. By determining the concentration of the target DNA amplification product in a PCR reaction that completes the same number of cycles and is within its linear range, the relative concentration of a particular target sequence in the original DNA mixture can be determined. If the DNA mixture is cDNA synthesized from RNA isolated from different tissues or cells, the relative abundance of the particular mRNA from which the target sequence is derived can be determined for each tissue or cell. Real-time PCR is typically performed using any PCR instrument available in the art. In general, instruments for real-time PCR data collection and analysis include a thermal cycler, optics for fluorescence excitation and emission collection, and optionally a computer and data acquisition and analysis software.

In certain embodiments, the target nucleic acid is quantified using blotting techniques, which are well known to those skilled in the art. Southern blotting involves the use of DNA as a target, whereas Northern blotting involves the use of RNA as a target. Each providing different types of information, although in many respects, cDNA blots are similar to blots or RNA material. Briefly, probes are used to target DNA or RNA species that have been immobilized on a suitable substrate, often a nitrocellulose filter. The different substances should be spatially separated to facilitate analysis. This is usually done by gel electrophoresis of the nucleic acid material, followed by "blotting" onto the filter. Subsequently, the blotted target is incubated with a probe (usually labeled) under conditions that promote denaturation and rehybridization. Removing the unbound probe to complete the detection. After detection/quantification, the results observed in a given subject can be compared to a reference group or control subject population that is statistically significant herein. In this way, the amount of disease biomarker nucleic acid detected can be correlated to the progression or severity of the disease.

Chip hybridization utilizes biomarker-specific oligonucleotides attached to a solid substrate, which may consist of a particulate solid phase designed as a microarray, such as a nylon filter, glass slide, or silicon chip. Microarrays are known in the art and consist of a surface on which probes with sequences corresponding to gene products (e.g., cdnas) can be specifically hybridized or bound at known locations to detect biomarker gene expression.

Quantification of hybridization complexes is well known in the art and can be accomplished by any of several methods. These methods are typically based on the detection of labels or markers, such as any radioactive, fluorescent, biological or enzymatic labels or tags used as standard in the art. Labels may be applied to the oligonucleotide probes or to RNA derived from the biological sample.

In general, mRNA quantification can be suitably performed together with a calibration curve to achieve accurate mRNA determination. Furthermore, it is preferred to quantify the transcripts originating from the biological sample by comparison with a control sample, said sample being characterized by a known expression pattern of the transcripts examined.

Detection of biomarker proteins

In some embodiments, disease treatment biomarkers are assessed at the protein expression level by demonstrating the presence of the protein (isolated or one or in the cell), or by one or more known functional properties of the biomarker. For example, anti-DFNA 5, MYH11, KHDRBS1 antibodies for use in protein assays specific for DFNA5 or specific for MYH11 or specific for KHDRBS1 are known in the art, are commercially available, and can also be readily produced by one of skill in the art. Antibodies and antigen-antibody complexes can be detected by several assays well known in the art, including immunofluorescence assays, immunohistochemistry, Fluorescence Activated Cell Sorting (FACS) analysis, enzyme-linked immunosorbent assays (ELISA), Radioimmunoassays (RIA), light emission immunoassays, and western blot analysis.

In particular embodiments, immunofluorescence or immunocytochemistry is performed to detect the protein. Cells, such as diseased tissue cells, can be isolated or enriched by methods known in the art. Isolation or enrichment of cells refers to a process in which the percentage of specific cells (e.g., cells of a diseased tissue) is increased relative to the percentage in the sample prior to the enrichment procedure. Purification is an example of enrichment. In other embodiments, antibodies to surface markers on cells can be linked to a solidOn a support to perform the separation. Procedures for separation may include the use of antibody magnetic beads (e.g., Miltenyi)^TMBeads), affinity chromatography, "panning" using antibodies attached to a solid matrix, or any other convenient technique, such as Laser Capture Microdissection. Other techniques that provide particularly accurate separation include FACS. Once deposited on the slide, the cells can be fixed and probed with labeled antibodies to detect disease diagnostic biomarkers.

Antibodies specific for disease biomarkers can be directly conjugated to fluorescent markers including fluorescein, FITC, rhodamine, Texas Red, Cy3, Cy5, Cy7, and other fluorescent markers and the filters observed under a fluorescent microscope equipped with appropriate filters. The antibody may also be conjugated to an enzyme that initiates the reaction upon addition of an appropriate substrate, thereby providing a colored precipitate on the cells with the biomarker protein detected. The slide can then be viewed by a standard optical microscope. Alternatively, a primary antibody specific for a disease diagnostic biomarker may be further bound to a secondary antibody conjugated to a detectable moiety.

Immunohistochemistry is in principle very similar to immunofluorescence or immunocytochemistry, however, for example, in contrast to cell suspensions, tissue specimens are probed with antibodies specific for disease treatment biomarkers. The biopsy specimen is fixed and processed and optionally embedded in paraffin and, if necessary, sectioned to provide a cell or tissue slide for subsequent detection with heparanase-specific antibodies. Alternatively, frozen tissue cryostats can be sectioned and then antibody probed to avoid fixation-induced antigen masking. Antibodies, as in immunofluorescence or immunocytochemistry, are coupled to a fluorescent or enzyme-linked detectable moiety and used to probe tissue sections by methods described for immunofluorescence, and then viewed by fluorescence or confocal microscopy depending on the detection method used. After the reaction product is formed, visualization of the reaction product precipitate can be observed by standard optical microscopy if an enzymatically detectable moiety is utilized.

In other embodiments, assays such as ELISA and RIA are used, which follow similar principles for detecting specific antigens. As an illustrative example, DFNA5, MYH11 or KHDRBS1 can be measured by using RIA with DFNA5, MYH11 or KHDRBS1 specific antibodies, typically radiolabeled with 125I. Antibodies specific for DFNA5, MYH11 or KHDRBS1 were chemically linked to the enzyme in an ELISA assay. Capture antibodies specific for DFNA5, MYH11 or KHDRBS1 are immobilized on a solid support. Unlabeled samples, such as protein extracts from biological samples, are then incubated with the immobilized antibodies under conditions in which non-specific binding is blocked, and unbound antibodies and/or proteins are removed by washing. Bound DFNA5, MYH11 or KHDRBS1 was detected by labeling an antibody specifically with a second DFNA5, MYH11 or KHDRBS 1. In RIA, antibody binding is measured directly by measuring radioactivity, whereas in ELISA binding is detected by the reaction of a colorless substrate to a colored reaction product as a function of the activity of the linked enzyme. Thus, the change can be easily detected by spectrophotometry.

Protein biomarker expression can also be detected by luminescence immunoassay. Much like ELISA and RIA, in a luminescent immunoassay, the biological sample/protein extract to be tested is immobilized on a solid support and probed with a specific label (labeled antibody). The label is luminescent again and upon binding emits light as an indication of specific recognition. Luminescent labels include substances that emit light when activated by electromagnetic radiation, electrochemical excitation, or chemical activation, and may include fluorescent and phosphorescent substances, scintillators, and chemiluminescent substances. The label may be part of a catalytic reaction system, such as an enzyme, enzyme fragment, enzyme substrate, enzyme inhibitor, coenzyme, or catalyst; a part of a chromogen system, such as a fluorophore, dye, chemiluminescent, luminescent or sensitizing agent; dispersible particles (which may be non-magnetic or magnetic), solid supports, liposomes, ligands, receptors, hapten radioisotopes, and the like.

Western blot analysis is another method for assessing the amount of a disease biomarker polypeptide in a biological sample. Protein extracts from biological samples of cells (e.g., cells of diseased tissue) are lysed in a denaturing ionization environment and aliquots are applied to a polyacrylamide gel matrix. As it migrates toward the anode, the proteins will separate based on molecular size characteristics. The antigen is then transferred to a nitrocellulose, PVDF or nylon membrane, and then membrane blocking is performed to minimize non-specific binding. The membrane is probed with an antibody directly coupled to the detectable moiety or subsequently probed with a secondary antibody containing the detectable moiety. Typically, horseradish peroxidase or alkaline phosphatase is conjugated to an antibody and the activity is visualized using a chromogenic or luminescent substrate.

In particular embodiments, protein capture arrays are used that allow for the simultaneous detection and/or quantification of large numbers of proteins. For example, low density protein arrays on filter membranes, it is now possible to use protein arrays to analyze protein expression in body fluids, such as serum of healthy or diseased subjects and in subjects before and after drug treatment. Exemplary protein capture arrays include arrays comprising spatially addressed antigen binding molecules, commonly referred to as antibody arrays, which can facilitate extensive parallel analysis of a variety of proteins defining a proteome or a sub-proteome. Antibody arrays have been shown to have desirable specificity and acceptable background characteristics.

Diagnostic product

The invention provides a product for diagnosing glaucoma, comprising reagents for detecting the biomarkers of the invention in a sample; and instructions for using the product to assess whether the subject is suffering from or susceptible to glaucoma.

The diagnostic product may also optionally include suitable reagents for detecting the marker, positive and negative controls, wash solutions, blotting membranes, microtiter plates, dilution buffers, and the like. For example, a protein-based assay diagnostic product can include (i) at least one disease biomarker polypeptide; and (ii) an antibody that specifically binds to a disease treatment biomarker polypeptide. Alternatively, the nucleic acid-based test kit can comprise (i) a disease treatment biomarker polynucleotide; and (ii) specifically hybridize to a disease treatment biomarker polynucleotideThe primer or probe of (1). Enzymes suitable for amplifying nucleic acids may also be included, including various polymerases (reverse transcriptase, Taq, Sequenase^TMDNA ligase, etc.), deoxyribonucleotides and buffers to provide the reaction mixture required for amplification. Such kits will also typically contain a different container for each individual reagent and enzyme, and each primer or probe, in a suitable manner.

Any form of sample assay capable of detecting a sample biomarker described herein may be used. Typically, the assay will quantify the biomarkers in the sample to an extent, for example whether their concentration or amount is above or below a predetermined threshold. Such kits may take the form of test strips, dipsticks, cartridges, chip-based or bead-based arrays, multi-well plates, or a series of containers, and the like. One or more reagents are provided to detect the presence and/or concentration and/or amount of a selected sample biomarker. The sample from the subject may be dispensed directly into the assay or indirectly from a stored or previously obtained sample.

The present invention will be described in further detail with reference to the accompanying drawings and examples. The following examples are intended to illustrate the invention only and are not intended to limit the scope of the invention. The experimental procedures, in which specific conditions are not specified in the examples, are generally carried out under conventional conditions or conditions recommended by the manufacturers.

Examples genetic markers associated with glaucoma diagnosis

1. Data and preprocessing

Downloading gene expression data of a data set GSE27276 of glaucoma and comparison thereof from a GEO database, annotating the gene expression data by using an annotation file, taking an average value of a plurality of probes corresponding to the same gene as an expression quantity of the gene expression data, and then obtaining a gene expression matrix file.

2. Differential expression analysis

Differential expression analysis was performed using the "limma" package in the R software, with a differential gene screening criterion of Pvalue < 0.05.

The analysis results show that DFNA5 and KHDRBS1 show significant up-regulation, MYH11 shows significant down-regulation, and the expression profiles are shown in fig. 1-3, wherein: p < 0.05; **: p < 0.01; ***: p < 0.001.

3. Diagnostic efficacy analysis

The AUC value, sensitivity and specificity of the differentially expressed gene as a detection variable are analyzed by using an R package 'pROC' ROC curve, and the diagnostic efficacy is judged. When the diagnostic efficacy of each gene was judged, the expression level of the gene was directly used for analysis. Calling a pROC package, reading in an expression quantity matrix constructed by a target gene, and running a command for drawing an ROC curve, wherein the command adopts for circulation and simultaneously relates to a command for adding AUC, thres (threshold value) and smooth (fitted curve). When the diagnosis efficiency of gene combination is judged, firstly, glmnet is used for conducting Logistic regression on genes, the established Logistic regression model is utilized, the influence of a certain prediction variable on the result probability at each level is observed by using a prediction function, the prediction probability is calculated, and an ROC curve of the prediction result is drawn.

The results are shown in table 1 and fig. 4-10, and it can be seen from the table that DFNA5, MYH11, KHDRBS1 and their combinations have high accuracy in diagnosing glaucoma, and particularly the combination of the three has high accuracy, sensitivity and specificity.

TABLE 1 differential expression Gene diagnostic potency analysis

The preferred embodiments of the present application have been described in detail with reference to the accompanying drawings, however, the present application is not limited to the details of the above embodiments, and various simple modifications can be made to the technical solution of the present application within the technical idea of the present application, and these simple modifications are all within the protection scope of the present application.

It should be noted that, in the foregoing embodiments, various features described in the above embodiments may be combined in any suitable manner, and in order to avoid unnecessary repetition, various possible combinations are not described in the present application.

In addition, any combination of the various embodiments of the present application is also possible, and the same should be considered as disclosed in the present application as long as it does not depart from the idea of the present application.

Claims (10)

1. A product for diagnosing glaucoma, comprising reagents for detecting the level of the biomarkers DFNA5, MYH11 and/or KHDRBS1 in a sample.

2. The product of claim 1, wherein the level of DFNA5, MYH11 and/or KHDRBS1 in the sample is determined by measuring the protein level or mRNA level of DFNA5, MYH11 and/or KHDRBS1 in the sample.

3. The product of claim 2, wherein the protein levels of DFNA5, MYH11 and/or KHDRBS1 in the sample are measured by using immunostaining, immunofluorescence, western blot or ELISA.

4. The product of claim 3, wherein the mRNA levels of DFNA5, MYH11 and/or KHDRBS1 in the sample are measured using microarray, RNA-seq, in situ hybridization, RNA-scope, and conventional semi-quantitative or quantitative RT-PCR.

5. The product of any one of claims 1-4, further comprising a reagent for processing the sample.

6. Use of a product according to any one of claims 1 to 5 for the manufacture of a means for diagnosing glaucoma.

7. Use of a reagent for the detection of a biomarker in a sample for the manufacture of a product for the diagnosis of glaucoma, wherein the biomarker comprises DFNA5, MYH11 and/or KHDRBS 1.

8. The use of claim 7, wherein the reagents comprise reagents for detecting biomarker levels by sequencing techniques, nucleic acid hybridization techniques, nucleic acid amplification techniques, protein immunization techniques.

9. Use according to claim 8, wherein the agent is selected from: a probe that specifically recognizes the biomarker; primers that specifically amplify the biomarkers; or an antibody that specifically binds to the biomarker.

10. The use according to any one of claims 7 to 9, wherein the sample is selected from the group consisting of tissue, blood.

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