CN113444789A - Glaucoma-associated biomarkers and uses thereof - Google Patents

Abstract

The invention discloses a biomarker related to glaucoma and application thereof. The invention discloses the expression of NME5, CTNNAL1 and LY96 in glaucoma patients is up-regulated, and based on the invention discloses the application of the biomarkers NME5, CTNNAL1 and/or LY96 in the preparation of a product for diagnosing glaucoma and the product for diagnosing glaucoma, which comprises the reagent for detecting the expression level of NME5, CTNNAL1 and/or LY 96.

Description

Glaucoma-associated biomarkers and uses thereof

Technical Field

The invention relates to the field of biomedicine, and relates to a glaucoma-related biomarker and application thereof.

Background

Glaucoma is one of the major diseases causing visual impairment and irreversible blindness worldwide, and is a complex type of optic nerve degenerative disease, leading to progressive degeneration of Retinal Ganglion Cells (RGCs), optic nerve degeneration and visual field defect, having the characteristics of high blinding rate, and seriously threatening the visual Health of human (Resnikoff S, Pascolini D, Etya' ale D, et al. Global data on visual impact in the year 2002[ J ] Bulletin of the World Health Organization, 2004; 82), (11): 844-851). Estimated by Tham et al, the Global glaucoma prevalence in 2013 (age 40-80) is approximately 6430 ten thousand, 3.5% of the world's total population, with 570 million people having visual impairment and 310 million people being blind, and is expected to increase to 7600 million in 2020 and 1.118 billion in 2040 (Tham Y C, Li X, Wong T Y, et al, Global prediction of glaucomas and projects of glaucomas garden 2040: a systematic review and meta-analysis [ J ] Ophthalmology, 2014;121 (11): 2081-. Glaucoma has no unified classification standard so far, and is clinically classified into three major categories including primary glaucoma, secondary glaucoma and congenital glaucoma according to etiology, angle of the atrium, tonography and the like. Primary Glaucoma is further classified into Primary Open-Angle Glaucoma (POAG) and Primary closed-Angle Glaucoma (Primary Angle-cloure Glaucoma PACG) according to the anatomical morphology of the anterior chamber Angle.

Most scholars consider that POAG is a multifactorial disease, and is a complex disease formed by the combined action of genetic factors and environmental factors, wherein the genetic factors play a main role, and genetic research on POAG is helpful to search the pathogenic genes and further explore the pathogenic mechanism. With the continuous development of medical treatment, imaging and visual function special inspection technologies provide a sensitive reference index for the early diagnosis of POAG, but people with POAG lesions can be diagnosed early, and the genetic research of POAG is indispensable and extremely important, as the people without POAG lesions or possibly developing POAG are not effective. The research and screening of pathogenic genes or susceptibility genes related to the POAG pathogenesis have important significance for the early prevention, early diagnosis and early treatment of the POAG.

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.

The invention provides the use of reagents for detecting biomarkers including NME5, CTNNAL1 and/or LY96 in the manufacture of a product for diagnosing glaucoma.

Further, when the expression level of NME5, CTNNAL1, or LY96 in the sample of the subject is up-regulated compared to normal humans, then the subject has or is at risk of developing glaucoma. Wherein 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.

Further, the reagent comprises:

an oligonucleotide probe that specifically recognizes NME5, CTNNAL1, or LY96 gene;

primers that specifically amplify NME5, CTNNAL1, or LY96 genes; or

A binding agent that specifically binds to a protein encoded by NME5, CTNNAL1, or LY 96.

The invention provides a product for diagnosing glaucoma comprising reagents for detecting the levels of the biomarkers NME5, CTNNAL1 and/or LY 96.

Further, the reagent is a reagent that detects the amount of mRNA transcribed by the biomarker.

Further, the reagent is a reagent that detects the amount of the polypeptide encoded by the biomarker.

Further, the reagent is an antibody, an antibody fragment, or an affinity protein.

Further, the product is in the form of an in vitro diagnostic product.

Further, the product is a diagnostic kit.

Furthermore, the product also comprises a total RNA extraction reagent, a reverse transcription reagent and/or a second generation sequencing reagent.

The present invention provides a device for diagnosing/predicting glaucoma, characterized in that it comprises:

a processor;

an input module for inputting a level of a biomarker in a biological sample, the biomarker selected from NME5, CTNNAL1, and/or LY 96;

a computer-readable medium containing instructions that, when executed by the processor, perform an algorithm on the input levels of the biomarkers; and

an output module that indicates whether the subject has or is at risk of developing glaucoma.

The invention provides the use of NME5, CTNNAL1 or LY96 in the manufacture of a pharmaceutical composition for the treatment of glaucoma.

Further, the pharmaceutical composition includes an inhibitor of NME5, CTNNAL1, and/or LY 96.

Further, the inhibitor reduces the expression level of NME5, CTNNAL1, and/or LY 96.

The invention has the beneficial effects that:

according to the invention, the diagnosis of glaucoma can be realized by detecting the expression level of NME5, CTNNAL1 and/or LY96, 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

Figure 1 shows NME5 gene differential expression profiles;

FIG. 2 shows a CTNNAL1 gene differential expression profile;

FIG. 3 shows a differential expression profile of LY96 gene;

figure 4 shows a ROC plot of NME5 gene diagnosis of glaucoma;

FIG. 5 shows a ROC plot of the CTNNAL1 gene for diagnosing glaucoma;

FIG. 6 shows a ROC plot of LY96 gene for diagnosis of glaucoma;

FIG. 7 shows a ROC plot of NME5+ CTNNAL1 gene for the diagnosis of glaucoma;

FIG. 8 shows a ROC plot of NME5+ LY96 gene diagnosis of glaucoma;

FIG. 9 shows a ROC plot of the CTNNAL1+ LY96 gene for diagnosing glaucoma;

figure 10 shows a ROC plot for NME5+ CTNNAL1+ LY96 combined 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 term "biomarker" refers to an indicator of a patient's phenotype (e.g., a pathological state or possible reactivity to a therapeutic agent) that can be detected in a biological sample from the patient. Biomarkers include, but are not limited to, DNA, RNA, proteins, carbohydrates, or glycolipid-based molecular markers.

In the present invention, the biomarker includes NME5, CTNNAL1 and/or LY 96. Biomarkers such as NME5 (NME/NM 23 family member 5, gene ID: 8382), CTNNAL1 (catenin alpha like 1, gene ID: 8727), LY96 (lymphoma antigen 96, gene ID: 23643); including genes and their encoded proteins and homologs, mutations, and isoforms. The term encompasses full-length, unprocessed biomarkers, as well as any form of biomarker that results from processing in a cell. The term encompasses naturally occurring variants (e.g., splice variants or allelic variants) of the biomarkers. The gene ID is available at https:// www.ncbi.nlm.nih.gov/gene/.

The term "amplification primer" or "primer" refers to a nucleic acid fragment comprising 5 to 100 nucleotides, preferably 15 to 30 nucleotides, capable of initiating an enzymatic reaction (e.g., an enzymatic amplification reaction).

The term "probe" refers to oligonucleotides and their analogs, and relates to a series of chemicals that recognize a polynucleotide target sequence by hydrogen bonding interactions with the nucleotide bases of the target sequence. The probe or target sequence may be single-or double-stranded RNA or single-or double-stranded DNA or a combination of DNA and RNA bases. The probe is at least 8 nucleotides long and less than the length of the whole gene. Probes can be 10, 20, 30, 50, 75, 100, 150, 200, 250, 400, 500, and up to 2000 nucleotides in length, as long as less than the full length of the target gene. The probe may comprise a modified oligonucleotide to have a label detectable by fluorescence, chemiluminescence, or the like. Probes may also be modified to have detectable labels and quenching molecules, such as Taqman and Molecular Beacon probes.

The probe may also be a mixture of any oligonucleotide analogue type together or in combination with natural DNA or RNA. Also, oligonucleotides and analogs thereof may be used alone or in combination with one or more additional oligonucleotides or analogs thereof.

The terms "level of expression" or "expression level" are generally used interchangeably and generally refer to the amount of a polynucleotide or amino acid product or protein in a biological sample. "expression" generally refers to the process by which information encoded by a gene is converted into structures that are present and operational in a cell. Thus, "expression" of a gene as used herein refers to transcription into a polynucleotide, translation into a protein, or even post-translational modification of a protein. Transcribed polynucleotides, translated proteins, or fragments of post-translationally modified proteins are also considered to be expressed, whether they are derived from transcripts produced or degraded by alternative splicing, or from post-translational processing of proteins (e.g., by proteolysis). "expressed genes" include those that are transcribed into a polynucleotide (e.g., mRNA) and then translated into a protein, as well as those that are transcribed into RNA but not translated into a protein (e.g., transfer RNA and ribosomal RNA).

The term "diagnosis" is used herein to refer to the identification or classification of a molecular or pathological state, disease or disorder. For example, "diagnosing" can refer to identifying a risk of developing glaucoma, either by the involved tissue/organ (e.g., glaucoma), or by a molecular characteristic (e.g., expression characterized by a particular gene or one or a combination of the proteins encoded by the gene).

"aiding diagnosis" refers to a method of aiding in making a clinical decision as to the presence or nature of a particular type of symptom or condition. For example, a method of aiding diagnosis of glaucoma may include measuring the expression of certain genes in a biological sample from an individual.

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.

An "antibody fragment" comprises only a portion of an intact antibody, wherein the portion preferably retains at least one, preferably most or all, of the functions normally associated with the portion when present in an intact antibody. In one embodiment, the antibody fragment comprises the antigen-binding site of an intact antibody, thereby retaining the ability to bind antigen. In another embodiment, an antibody fragment (e.g., an antibody fragment comprising an Fc region) retains at least one of the biological functions normally associated with the Fc region when present in an intact antibody, such as FcRn binding, ADCC function, and complement binding. In one embodiment, the antibody fragment is a monovalent antibody having an in vivo half-life substantially similar to that of an intact antibody. For example, such an antibody fragment may comprise an antigen-binding arm linked to an Fc sequence capable of conferring in vivo stability to the fragment.

The term "monoclonal antibody" as used herein refers to an antibody obtained from a substantially homogeneous population of antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations that may be present in minor amounts. Monoclonal antibodies are highly specific, being directed against a single antigen. Furthermore, unlike polyclonal antibody preparations which typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen.

Monoclonal antibodies herein specifically include "chimeric" antibodies in which a portion of the heavy and/or light chain is identical with or homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular antibody class or subclass, and the remainder of one or more chains is identical with or homologous to corresponding sequences in antibodies derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, so long as they exhibit the desired biological activity.

The term "diabodies" refers to small antibody fragments having two antigen-binding sites, which fragments comprise a variable heavy domain (VH) linked to a variable light domain (VL) in the same polypeptide chain (VH-VL). By using a linker that is too short to allow pairing between the two domains on the same chain, the domains are forced to pair with complementary domains on the other chain and two antigen binding sites are created.

An "affinity matured" antibody is one that has one or more alterations in one or more hypervariable regions thereof which result in an improvement in the affinity of the antibody for an antigen compared to a parent antibody that does not have those alterations. In certain embodiments, an affinity matured antibody will have nanomolar or even picomolar affinity for the target antigen.

The detection of the gene expression level described herein can employ assay methods known in the art, including, but not limited to, methods that detect the amount of an RNA transcript of the biomarker or the amount of a polypeptide encoded by the biomarker.

The RNA transcript of the biomarker may be converted to cDNA complementary thereto by methods known in the art, and the amount of the RNA transcript may be obtained by determining the amount of complementary cDNA. The amount of RNA transcript of a biomarker, or cDNA complementary thereto, can be normalized to the amount of total RNA or total cDNA in a sample, or to the amount of RNA transcript of a panel of housekeeping genes, or cDNA complementary thereto.

In this context, RNA transcripts can be detected and quantified by methods such as hybridization, amplification, sequencing, including, but not limited to, methods of hybridizing RNA transcripts to probes or primers, methods of detecting the amount of RNA transcripts or their corresponding cDNA products by various quantitative PCR techniques or sequencing techniques based on the Polymerase Chain Reaction (PCR). The quantitative PCR techniques include, but are not limited to, fluorescent quantitative PCR, real-time PCR, or semi-quantitative PCR techniques. Such sequencing techniques include, but are not limited to, Sanger sequencing, second-generation sequencing, third-generation sequencing, single cell sequencing, and the like. Preferably, the sequencing technique is next generation sequencing, more preferably a targeted RNA-seq technique.

Herein, the amount of polypeptide can be detected by, for example, proteomics or reagents. Preferably, the agent is an antibody, an antibody fragment or an affinity protein. Any suitable protein quantification method may be used in the methods provided herein. In certain embodiments, an antibody-based method is used. Exemplary methods that can be used include, but are not limited to, immunoblotting (western blot), enzyme-linked immunosorbent assay (ELISA), immunohistochemistry, flow cytometry bead arrays, and mass spectrometry. Some types of ELISA are commonly used, including direct ELISA, indirect ELISA, and sandwich ELISA.

Applications of

The present invention relates to the use of reagents for determining biomarkers in a sample, including NME5, CTNNAL1 and/or LY96, in the manufacture of a diagnostic product for glaucoma.

Preferably, the diagnostic product comprises a reagent that detects the expression level of NME5, CTNNAL1 and/or LY96 in the sample.

As an alternative embodiment, the reagent comprises:

an oligonucleotide probe that specifically recognizes NME5, CTNNAL1, or LY96 gene;

primers that specifically amplify NME5, CTNNAL1, or LY96 genes; or

A binding agent that specifically binds to a protein encoded by NME5, CTNNAL1, or LY 96.

As alternative embodiments, binding agents for proteins include, but are not limited to, peptides, peptide mimetics, aptamers, spiegelmers, dappin, ankyrin repeat proteins, Kunitz-type domains, antibodies, single domain antibodies, and monovalent antibody fragments.

As a preferred embodiment, the binding agent for the protein is an antibody.

As an alternative embodiment, the sample comprises plasma, serum or blood extract, brush, biopsy or surgically excised tissue or fluid sample from the subject.

(diagnostic) product

The invention relates to a product for diagnosing glaucoma, comprising reagents for detecting the biomarkers NME5, CTNNAL1 and/or LY 96.

In one embodiment, the agent is an agent that detects the level of expression of the biomarker.

In one embodiment, the diagnostic product is in the form of an in vitro diagnostic product.

In a specific embodiment, the diagnostic product is a diagnostic kit.

In one embodiment, the reagent is a reagent that detects the amount of RNA, particularly mRNA, transcribed from the biomarker. In yet another embodiment, the reagent is a reagent that detects the amount of cDNA complementary to the mRNA.

In a preferred embodiment, the diagnostic product further comprises a total RNA extraction reagent, a reverse transcription reagent and/or a secondary sequencing reagent.

The total RNA extraction reagent can be a total RNA extraction reagent which is conventional in the field.

The reverse transcription reagent may be a reverse transcription reagent conventional in the art, and preferably includes a dNTP solution and/or an RNA reverse transcriptase.

The second-generation sequencing reagent may be a reagent conventionally used in the art as long as it can satisfy the requirement of second-generation sequencing of the resulting sequence. The second generation sequencing reagents may be commercially available products, examples of which include, but are not limited to, Illumina MIseq. Reagent Kit v3 (150 cycles) (MS-102. sup. 3001), TruSeq. Targeted RNA Index KitA-96 Inds (384 Samples) (RT-402. sup. 1001). Secondary sequencing is a technique conventional in the art, such as targeted RNA-seq technology. Thus, the second generation sequencing reagents may also include reagents that can be tailored for constructing a library Illumina targeting RNA-seq, such as the Targeted RNA Custom Panel Kit (96 Samples) (RT-102-1001).

In a preferred embodiment, the agent is a probe or primer.

In alternative embodiments, the reagent is a reagent that detects the amount of the polypeptide encoded by the biomarker.

In particular embodiments, the agent is an antibody, antibody fragment, or affinity protein.

A "kit" is an article of manufacture (e.g., a package or container) containing probes for specifically detecting the biomarker genes or proteins of the present invention. In certain embodiments, the article of manufacture is marketed, distributed, or sold as a unit for performing the methods of the present invention.

Such kits may comprise carrier means compartmentalized to receive, in close confinement, one or more container means (e.g., vials, tubes, etc.), each container means comprising one of the separate components to be used in the method. For example, one of the container means may comprise a probe that carries or can carry a detectable label. Such probes may be polynucleotides specific for polynucleotides of one or more genes comprising gene expression characteristics. Where the kit utilizes nucleic acid hybridization to detect a target nucleic acid, the kit can also have a container containing one or more nucleic acids for amplifying the target nucleic acid sequence and/or a container containing a reporter means, such as a biotin-binding protein, such as avidin or streptavidin, bound to a reporter molecule, such as an enzymatic, fluorescent, or radioisotope label.

A kit will generally comprise the above-described container and one or more additional containers containing commercially and user-desired materials, including buffers, diluents, filters, needles, syringes, and package inserts containing instructions for use. A label may be present on the container to indicate that the composition is for a particular therapeutic or non-therapeutic application, and may also indicate the direction of in vivo or in vitro use, such as those described above. Other optional components of the kit include one or more buffers (e.g., blocking buffer, wash buffer, substrate buffer, etc.), other reagents (e.g., substrate chemically altered by enzymatic labeling), epitope retrieval solutions, control samples (positive and/or negative controls), control sections, and the like.

Device for measuring the position of a moving object

The present invention provides a device for diagnosing/predicting glaucoma, the device comprising:

a processor;

an input module for inputting a level of a biomarker in a biological sample, the biomarker selected from NME5, CTNNAL1, and/or LY 96;

a computer-readable medium containing instructions that, when executed by the processor, perform an algorithm on the input levels of the biomarkers; and

an output module that indicates whether the subject has or is at risk of developing glaucoma.

An apparatus as applied herein shall at least comprise the above-mentioned modules. The modules of the device are operatively connected to each other. How the modules are operatively linked will depend on the type of module contained in the device.

The processor may execute a series of machine-readable instructions, which may be embodied in a program or software. The instructions may be stored in a memory location, such as a memory. Instructions may be directed to a processor that may then program or otherwise configure the processor to implement the present disclosure. Examples of operations performed by a processor may include read, decode, execute, and write-back.

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 NME5, CTNNAL1 and LY96 show significant up-regulation, and the expression of the NME5, the CTNNAL1 and the LY96 is shown in figures 1 to 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 predict () 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, from which it can be seen that NME5, CTNNAL1, LY96 and their combinations have high accuracy in diagnosing glaucoma, especially the combination of the three, which 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. Use of a reagent for the detection of a biomarker in the manufacture of a product for the diagnosis of glaucoma, wherein the biomarker is selected from NME5, CTNNAL1 and/or LY 96.

2. The use of claim 1, wherein the subject has glaucoma when the expression level of NME5, CTNNAL1, or LY96 is up-regulated in the sample from the subject as compared to normal humans.

3. Use according to claim 1 or 2, wherein the agent comprises:

an oligonucleotide probe that specifically recognizes NME5, CTNNAL1, or LY96 gene;

primers that specifically amplify NME5, CTNNAL1, or LY96 genes; or

A binding agent that specifically binds to a protein encoded by NME5, CTNNAL1, or LY 96.

4. The use of claim 1, wherein the agent is an agent that detects the amount of RNA transcribed from the biomarker.

5. The use of claim 4, wherein the agent is an agent that detects the amount of mRNA transcribed from the biomarker.

6. The use of claim 1, wherein the agent is an agent that detects the amount of a polypeptide encoded by the biomarker.

7. The use of claim 6, wherein the agent is an antibody, an antibody fragment or an affinity protein.

8. Use according to any one of claims 1 to 7, wherein the product is in the form of an in vitro diagnostic product.

9. Use according to claim 8, wherein the product is a diagnostic kit.

10. A device for diagnosing/prognosing glaucoma, characterized in that it comprises:

a processor;

an input module for inputting a level of a biomarker in a biological sample, the biomarker selected from NME5, CTNNAL1, and/or LY 96;

a computer-readable medium containing instructions that, when executed by the processor, perform an algorithm on the input levels of the biomarkers; and

an output module that indicates whether the subject has or is at risk of developing glaucoma.

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