CN116218991A - Application of RabGGTase in diagnosis of ALS disease severity and disease progression rate - Google Patents

Abstract

The invention discloses application of RabGGTase in diagnosis of ALS disease severity and disease progression rate. The invention analyzes the correlation between the expression level of RabGGTB and the disease severity and disease progression rate of ALS patients, and finds that the expression level of RabGGTB is obviously correlated with ALSFRS-R score, and the expression level and the disease course of RabGGTB are obviously correlated with DeltaFS. The results show that the higher the expression level of RabGGTB, the more serious the disease degree of the patient; the higher the expression level of RabGGTB, the shorter the course of the disease, and the faster the rate of disease progression.

Description

Application of RabGGTase in diagnosis of ALS disease severity and disease progression rate

Technical Field

The invention belongs to the field of biological medicine, and relates to application of RabGGTase in diagnosis of ALS disease severity and disease progression rate.

Background

Amyotrophic Lateral Sclerosis (ALS) is a progressive lethal neurological degenerative disease of adult origin, and is mainly characterized by selective upper and lower motor neuron lesions. This neuronal pathology causes progressive muscle weakness, muscle atrophy, and bulbar paralysis. Death may occur 1 to 5 years after diagnosis, but 20% of patients survive over 5 years, 10% survive over 10 years. About 2 out of every 10 tens of thousands of people are ill each year, usually occurring between 40-70 years of age. A significant proportion of cases also exhibit cognitive or behavioral abnormalities typical of frontotemporal dementia (FTD). The etiology and pathogenesis of amyotrophic lateral sclerosis remain undefined. About 90% of cases are considered sporadic (sALS). The remaining 10% are inherited predominantly in an autosomal dominant manner (fALS). Most cases of fALS can be explained by mutations in four major genes, including C9ORF72, SOD1, FUS and TARDBP. The clinical phenotype of ALS is generally classified according to the first symptomatic site, with about 2/3 patients suffering from amyotrophic lateral sclerosis having a disease in the limb, clinically manifested as progressive muscle weakness, muscle atrophy in the limb, and the remaining 1/3 patients suffering from bulbar paralysis, mainly manifested as dysphagia.

ALS is characterized by heterogeneity of the relative burden of the area of onset, rate of progression, disease spread pattern, upper Motor Neurons (UMN), lower Motor Neurons (LMN), and cognitive pathology. This phenotypic variability in ALS complicates the measurement of disease progression. With the advent of the age of targeted therapies in ALS, accurately differentiating ALS disease severity and measuring disease progression rates remains a key priority in facilitating efficient clinical trial design and enabling further understanding of disease pathogenesis to develop therapies for ALS.

This heterogeneity of ALS patient severity and rate of disease progression results in difficulty in accurately predicting progression of ALS disease by clinicians, and current diagnosis of biomarkers of ALS disease, delay in diagnosis time, difficulty in identifying ALS disease, so exploring biomarkers for diagnosis of ALS severity and rate of disease progression is of great significance, helping to discover new ALS diagnosis and treatment methods, evaluate drug efficacy, rate of disease progression, and disease prognosis by biomarkers.

Disclosure of Invention

To remedy the deficiencies of the prior art, it is an object of the present invention to provide biomarkers related to the severity and rate of progression of ALS and their use in diagnosing the severity and rate of progression of ALS.

In order to achieve the above purpose, the present invention adopts the following technical scheme:

in a first aspect, the invention provides the use of an agent for detecting the level of RabGGTase expression in a sample for the manufacture of a product for diagnosing an ALS condition.

Further, the ALS conditions include ALS severity and rate of progression.

Further, the agent is selected from:

an oligonucleotide probe that specifically recognizes the RabGGTase gene; or (b)

Primers for specifically amplifying the RabGGTase gene; or (b)

A binding agent that specifically binds to a protein encoded by the RabGGTase gene.

Further, the binding agent comprises an antibody, an antibody functional fragment, a conjugated antibody which specifically binds to a RabGGTase encoded protein.

Further, the sample is selected from tissue, blood, urine, cerebrospinal fluid, a cell line, a tissue culture, or interstitial fluid.

Further, the blood includes blood-derived cells, platelets, serum, plasma.

Further, the blood-derived cells include nucleated cells; the nucleated cells include monocytes, macrophages, dendritic cells, lymphocytes and granulocytes.

Further, the blood-derived cells are monocytes or macrophages.

Further, the macrophages are formed by differentiation of monocytes.

Further, the RabGGTase includes RabGGTA, rabGGTB.

Further, the RabGGTase is RabGGTB.

In a second aspect, the invention provides a product for diagnosing an ALS condition, the product comprising a formulation, chip, kit or nucleic acid membrane strip capable of detecting the expression level of RabGGTase.

Further, the ALS conditions include ALS severity and rate of progression.

Further, the chip comprises a gene chip and a protein chip, wherein the gene chip comprises an oligonucleotide probe aiming at RabGGTase gene for detecting the transcription level of RabGGTase gene, and the protein chip comprises a specific binding agent of RabGGTase protein; the kit comprises a gene detection kit and a protein detection kit, wherein the gene detection kit comprises a reagent or a chip for detecting the transcription level of the RabGGTase gene, and the protein detection kit comprises a reagent or a chip for detecting the expression level of the RabGGTase protein.

Further, the kit includes reagents for detecting the expression level of the RabGGTase gene or protein by RT-PCR, qRT-PCR, biochip assay, southern blotting, northern blotting, in situ hybridization, immunoblotting, immunofluorescence and immunohistochemistry, ELISA, transcriptomics, proteomics.

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

Further, the reagent for treating a sample includes a reagent for separating monocytes or a reagent for promoting differentiation of monocytes into macrophages.

Further, the mononuclear cell separation reagent comprises lymphocyte separation liquid and mononuclear cell separation liquid.

Further, the mononuclear cell separation reagent is lymphocyte separation liquid.

Further, the agent that promotes differentiation of monocytes into macrophages includes macrophage colony stimulating factor, granulocyte macrophage colony stimulating factor, phorbol ester.

Further, the agent that promotes differentiation of monocytes into macrophages is a macrophage colony stimulating factor.

Further, the RabGGTase includes RabGGTA, rabGGTB.

Further, the RabGGTase is RabGGTB.

In a third aspect, the invention provides the use of RabGGTase in constructing a computational model for diagnosing ALS conditions.

Further, the ALS conditions include ALS severity and rate of progression.

Further, the RabGGTase includes RabGGTA, rabGGTB.

Further, the RabGGTase is RabGGTB.

In a fourth aspect, the invention provides a system for diagnosing an ALS condition, the system comprising:

(1) The ALS disease condition evaluation device comprises a control unit and a storage unit, wherein the control unit and the storage unit are connected through an information communication terminal device which is in communication connection with each other and used for evaluating the disease condition of an ALS patient;

(2) Information communication terminal apparatuses that are communicatively connected to each other: for providing data concerning the expression level of RabGGTase in a sample from an ALS patient.

Further, the ALS conditions include ALS severity and rate of progression.

Further, the control unit of the ALS patient condition assessment device includes four units:

1) A data receiving unit: for receiving data about the expression level of RabGGTase in the sample transmitted from the information communication terminal device;

2) Discrimination value calculation unit: calculating a discrimination value based on discrimination of the expression level of RabGGTase in the sample received by the data receiving unit and the expression level of RabGGTase stored in the storage unit as an explanatory variable;

3) Discrimination value reference evaluation unit: evaluating the risk of developing the disease of the ALS patient based on the discrimination value calculated by the discrimination value calculation unit;

4) An evaluation result transmitting unit: which transmits the evaluation result of the ALS patient condition obtained by the discrimination value reference evaluation unit to the information communication terminal device.

Further, the RabGGTase includes RabGGTA, rabGGTB.

Further, the RabGGTase is RabGGTB.

The invention has the advantages and beneficial effects that:

the invention discovers a biomarker-RabGGTB gene related to the disease severity or disease progress rate of an ALS patient, and can judge the disease severity or disease progress rate of the ALS patient by detecting the expression level of RabGGTB in peripheral blood mononuclear-macrophages of the ALS patient, thereby guiding a clinician to provide a prevention scheme or a treatment scheme for the ALS patient.

Drawings

FIG. 1 is a statistical plot of the expression levels of RabGGTB for immunofluorescence detection of different rates of ALS progression.

FIG. 2 is a graph showing analysis of correlation between ALSFRS-R scores and RabGGTB expression levels.

FIG. 3 is a graph showing the correlation between ΔFS and the correlation index, wherein 3A is a graph showing the correlation between ΔFS and the expression level of RabGGTB, and 3B is a graph showing the correlation between ΔFS and the course of the disease.

FIG. 4 is a ROC graph of RabGGTB diagnosing the rate of progression of ALS.

Detailed Description

Through extensive and intensive studies, the invention discovers that the expression level of RabGGTB is related to the disease severity and the disease progress rate of ALS patients, and the higher the expression level of RabGGTB is, the higher the disease severity of ALS patients is, and the faster the disease progress of diseases is.

In the present invention, the term "biomarker" refers to an indicator, such as a predictive, diagnostic and/or prognostic indicator, that is detectable in a sample. The biomarker may be indicative of a particular subtype of a disease or disorder (e.g., ALS), characterized by a particular, molecular, pathological, histological, and/or clinical nature, and/or may be indicative of a particular cell type or state (e.g., epithelial cells, mesenchymal cells, etc.) and/or response to therapy. Biomarkers include, but are not limited to, polynucleotides (e.g., DNA and/or RNA), polynucleotide copy number changes (e.g., DNA copy number), polypeptides, and polynucleotide modifications (e.g., post-translational modifications), carbohydrates, and/or glycolipid-based biomarkers. The biomarker may be present in a sample obtained from the subject prior to the onset of a physiological or pathophysiological condition, including symptoms thereof. Thus, the presence of a biomarker in a sample obtained from a subject may indicate an increased risk of the subject developing a physiological or pathophysiological state or symptoms thereof. Alternatively or additionally, the biomarker may be expressed normally in the individual, but its expression may change (i.e., it increases (upregulates; overexpression) or decreases (downregulates; underexpression)) before onset of the physiological or pathophysiological state (including its symptoms). Thus, a change in the level of the biomarker may indicate an increased risk that the subject will develop a physiological or pathophysiological state or symptoms thereof. Alternatively or additionally, the change in biomarker levels may reflect a change in a particular physiological or pathophysiological state or symptom thereof in the subject, allowing the nature (e.g., severity) of the physiological or pathophysiological state or symptom thereof to be tracked over a period of time. Such means may be used, for example, to monitor a treatment regimen for assessing its effectiveness (or other aspects) in a subject.

RabGGTA includes wild-type, mutant or fragments thereof. The term encompasses full length, unprocessed RabGGTA, as well as any form of RabGGTA derived from processing in a cell. The term encompasses naturally occurring variants (e.g., splice variants or allelic variants) of RabGGTA. The term encompasses, for example, the RabGGTA gene, the human RabGGTA, and RabGGTA from any other vertebrate source, including mammals, such as primates and rodents (e.g., mice and rats). As a preferred embodiment, in the present invention, rabGGTA is a human gene and the gene ID is 5875.

RabGGTB includes wild type, mutant or fragments thereof. The term encompasses full length, unprocessed RabGGTB, as well as any form of RabGGTB derived from processing in a cell. The term encompasses naturally occurring variants (e.g., splice variants or allelic variants) of RabGGTB. The term encompasses, for example, the RabGGTB gene, the human RabGGTB, and RabGGTB from any other vertebrate source, including mammals, such as primates and rodents (e.g., mice and rats). As a preferred embodiment, in the present invention, rabGGTB is a human gene, and the gene ID is 5876.

The term "amyotrophic lateral sclerosis" is commonly abbreviated as "ALS", amyotrophic lateral sclerosis, also known as motor neuron disease (motor neuron disease, MND), the latter name commonly used in the united kingdom, also known as Charcot's disease, france, and us also known as Lu Galei (Lou Gehrig) disease, the above names being used interchangeably herein to refer to the same condition. The development or progression of familial ALS and sporadic ALS can be determined by the present method. All forms of ALS are contemplated herein. Subjects with ALS may have fast-progressive ALS or slow-progressive ALS, as these terms are well known in the art. Those skilled in the art will appreciate that the precise classification of slow progressive subjects and fast progressive subjects depends on the scoring system used to assess disease and disease progression. Exemplary scoring systems include, for example, an inspection-based Appel ALS (AALS) score and a questionnaire-based ALS function rating scale (ALSFRS-R) score. alsfr-R scoring is based on 12 questions divided into four regions (fine movement, large movement (gross movement), medulla oblongata and respiration), and ranges from 48 (normal) to 0 (lowest function). AALS scoring is based on objective testing of five categories (bulbar, respiratory function, arm and leg function, and muscle strength) and ranges from 30 (normal) to 164 (maximum impairment).

In the present invention, the ALS disease progression rate refers to an evaluation of the time required for an increase in the severity of ALS disease. ALS severity may be assessed by measuring the number of symptoms of the disease present in the patient and/or the severity of any one or more symptoms. A variety of scoring systems may be used to assess the severity of a disease, including an examination-based Appel ALS (AALS) score and a questionnaire-based ALS function scoring scale (ALSFRS-R) score. The rate of progression in such a case may be determined by calculating the change in score over time, for example the change in monthly ALSFRS-R score. As described herein, rabGGTB is closely related to the severity and rate of progression of ALS, and thus the level of RabGGTB can also be used to assess the rate of progression of ALS.

The methods of the invention may be used to assess the rate of progression of ALS in a subject, and may be used to determine whether a subject with ALS is likely to have slowly progressive ALS or is likely to have rapidly progressive ALS. This is accomplished by determining the level of one or more ALS progression biomarkers as described above and in the present invention and optionally comparing the level to an appropriate reference level. As will be appreciated, whether an increase (or elevation), decrease (or depression) or the same or a similar increase in biomarker level as compared to a reference level indicates that the subject may have slow progressive ALS or fast progressive ALS, or indicates what rate of progression the subject has, depending on the reference level used in the assessment.

The reference level may represent a healthy subject, a subject with slowly progressive ALS, a subject with rapidly progressive ALS, or a subject with a particular rate of progression. Thus, whether an increase, decrease, or no change in the biomarker level as compared to the reference level indicates that the subject may have slow-progressive ALS or fast-progressive ALS or have a particular rate of progression, depending on whether the reference level represents a healthy subject, a subject with slow-progressive ALS, a subject with fast-progressive ALS, or a subject with a particular rate of progression. For example, as described above and in the present disclosure, subjects with fast-progressive ALS typically have increased (or elevated) levels of RabGGTase in a biological sample (e.g., blood, serum, plasma, CSF, or urine) as compared to subjects with slow-progressive ALS. Also as demonstrated by the present invention, the level of RabGGTase is closely related to the rate of progression of ALS, with low levels of RabGGTase being related to lower rates of progression, rather than higher levels of RabGGTase being related thereto. Preferably, rabGGTase comprises RabGGTA, rabGGTB; more preferably, rabGGTase is RabGGTB.

In the present invention, the term "sample" generally means any biological sample obtained from an individual, body fluid, body tissue, cell line, tissue culture or other source. This term includes body fluids, e.g., blood (e.g., peripheral or venous blood), urine, and other samples of biological origin, e.g., fat aspirates and solid tissue biopsy samples, e.g., biopsy samples (e.g., tissue biopsy samples), or tissue cultures or cells derived from tissue cultures and their progeny. This definition also includes samples obtained from sources after any treatment, such as by reagent treatment, solubilization, or enrichment of certain components (e.g., proteins or polynucleotides). This definition also encompasses clinical samples, and also includes cells in culture, cell supernatants, cell lysates, serum, plasma, biological fluids, and tissue samples. The source of the sample may be solid tissue, such as an organ or tissue sample or biopsy sample or aspirate derived from fresh, frozen, and/or preserved; blood or any blood component, such as blood-derived cells; body fluids such as cerebrospinal fluid, amniotic fluid, ascites, or intercellular fluid; cells derived from any developmental stage of the subject. The biological sample may contain compounds that are not naturally found in natural tissue, such as preservatives, anticoagulants, buffers, fixatives, nutrients, antibiotics, or the like. The sample may be used in a diagnostic assay or a monitoring assay. Preferably, the blood derived cells include, but are not limited to, monocytes, macrophages into which monocytes differentiate, dendritic cells, lymphocytes, granulocytes; in a specific embodiment of the invention, the sample is macrophages into which monocytes differentiate.

In the present invention, "detecting the level of RabGGTase expression in a sample" refers to determining the amount or presence of RNA expression products of the RabGGTase gene. Methods of detecting the expression profile of RabGGTase include methods based on polynucleotide hybridization analysis, methods based on polynucleotide sequencing, immunohistochemical methods, and methods based on proteomics. These methods generally detect the expression product (e.g., mRNA) of RabGGTase in the present invention. In preferred embodiments, PCR-based methods, such as reverse transcription PCR (RT-PCR), and array-based methods, such as microarrays, are used. "microarray" refers to an ordered arrangement of hybridizable array elements, such as, for example, polynucleotide probes, on a substrate. The term "probe" refers to a molecule that selectively binds to a specifically contemplated target biomolecule, such as a nucleotide transcript or protein encoded by or corresponding to an intrinsic gene. The probes may be synthesized by one skilled in the art or may be derived from a suitable biological preparation. Probes can be specifically designed to label them. Examples of molecules that may be used as probes include, but are not limited to, RNA, DNA, proteins, antibodies, and/or organic molecules.

In the present invention, the oligonucleotide probe against the RabGGTase gene may be a DNA, RNA, DNA-RNA chimera, PNA or other derivative. The length of the probe is not limited, and any length may be used as long as it specifically hybridizes to the target nucleotide sequence and binds thereto. The probe may be as short as 25, 20, 15, 13 or 10 bases in length. Also, the probes can be as long as 60, 80, 100, 150, 300 base pairs or more in length, even for the entire gene. Since different probe lengths have different effects on hybridization efficiency and signal specificity, the probe length is usually at least 14 base pairs, and the length complementary to the nucleotide sequence of interest is optimally 15-25 base pairs, with the longest length generally not exceeding 30 base pairs. The probe self-complementary sequence is preferably less than 4 base pairs to avoid affecting hybridization efficiency.

The term "primer" refers to a single stranded polynucleotide capable of hybridizing to a nucleic acid and allowing polymerization of the complementary nucleic acid (typically by providing a free 3' -OH group).

The term "binding agent" refers to any entity that binds to a target of interest as described herein. In many embodiments, the binding agent of interest is one that specifically binds to its target, as it distinguishes its target from other potential binding partners in a particular interaction environment. In general, the binding agent may be or include an entity of any chemical class (e.g., polymer, non-polymer, small molecule, polypeptide, compound, lipid, nucleic acid, etc.). In some embodiments, the binding agent is a single chemical entity. In some embodiments, the binding agent is a complex of two or more discrete chemical entities that associate with each other by non-covalent interactions under relevant conditions. For example, one of skill in the art will appreciate that in some embodiments, a binding agent may include a "universal" binding moiety (e.g., one of biotin/avidin/streptavidin and/or a class-specific antibody) and a "specific" binding moiety (e.g., an antibody or aptamer with a particular molecular target) linked to a partner of the universal binding moiety. In some embodiments, this approach may allow for molecular assembly of multiple binding agents through the ligation of different specific binding moieties to the same universal binding moiety partner. In some embodiments, the binding agent is or comprises a polypeptide (including, for example, an antibody or antibody fragment). In some embodiments, the binding agent is or comprises a small molecule. In some embodiments, the binding agent is or comprises a nucleic acid. In some embodiments, the binding agent is an aptamer (aptamer). In some embodiments, the binding agent is a polymer; in some embodiments, the binding agent is not a polymer. In some embodiments, the binders are non-polymeric in that they lack polymeric moieties. In some embodiments, the binding agent is or includes a compound. In some embodiments, the binding agent is or comprises a lectin. In some embodiments, the binding agent is or comprises a peptide mimetic. In some embodiments, the binding agent is or comprises a scaffold protein. In some embodiments, the binding agent is or comprises a mimotope. In some embodiments, the binding agent is or includes a stapled peptide (stapledpeptide). In certain embodiments, the binding agent is or comprises a nucleic acid, such as DNA or RNA.

The formulations, chips, kits or membrane strips described herein may be used to detect the expression levels of a plurality of genes including the RabGGTB gene and their expression products (e.g., a plurality of genes associated with ALS conditions). A plurality of markers of the ALS disease condition are detected simultaneously, so that the accuracy of diagnosis of the ALS disease condition can be greatly improved.

The term "chip" also referred to as an "array" refers to a solid support comprising attached nucleic acid or peptide probes. The array typically comprises a plurality of different nucleic acid or peptide probes attached to the surface of a substrate at different known locations. These arrays, also known as "microarrays," can generally be produced using mechanical synthesis methods or light-guided synthesis methods that combine a combination of photolithographic methods and solid-phase synthesis methods. The array may comprise a planar surface or may be a bead, gel, polymer surface, fiber such as optical fiber, glass or any other suitable nucleic acid or peptide on a substrate. The array may be packaged in a manner that allows for diagnosis or other manipulation of the fully functional device.

A "microarray" is an ordered arrangement of hybridization array elements, such as polynucleotide probes (e.g., oligonucleotides) or binding agents (e.g., antibodies), on a substrate. The substrate may be a solid substrate, for example, a glass or silica slide, beads, a fiber optic binder, or a semi-solid substrate, for example, a nitrocellulose membrane. The nucleotide sequence may be DNA, RNA or any arrangement thereof.

In certain embodiments, provided herein are kits for detecting mRNA levels of one or more biomarkers. In certain embodiments, the kit comprises one or more probes that specifically bind to mRNA of one or more biomarkers. In certain embodiments, the kit further comprises a wash solution. In certain embodiments, the kit further comprises reagents for performing hybridization assays, mRNA isolation or purification means, detection means, and positive and negative controls. In certain embodiments, the kit further comprises instructions for using the kit. The kit may be customized for use at home, clinically, or for research.

In certain embodiments, provided herein are kits for detecting protein levels of one or more biomarkers. In certain embodiments, the kit comprises a test strip coated with an antibody that recognizes a protein biomarker, a wash solution, reagents for performing the assay, protein isolation or purification means, detection means, and positive and negative controls. In certain embodiments, the kit further comprises instructions for using the kit. The kit may be customized for use at home, clinically, or for research.

In the present invention, the kit may employ, for example, a test strip, a film, a chip, a disk, a test strip, a filter, a microsphere, a slide, a multi-well plate, or an optical fiber. The solid support of the kit may be, for example, a plastic, a silicon wafer, a metal, a resin, a glass, a membrane, particles, a precipitate, a gel, a polymer, a sheet, a sphere, a polysaccharide, a capillary, a film, a plate, or a slide. The biological sample may be, for example, a cell culture, a cell line, a tissue, an oral tissue, a gastrointestinal tissue, an organ, a cellular organelle, a biological fluid, a blood sample, a urine sample, or skin.

In the present invention, a nucleic acid membrane strip comprises a substrate and an oligonucleotide probe immobilized on the substrate; the substrate may be any substrate suitable for immobilization of oligonucleotide probes, such as nylon membrane, nitrocellulose membrane, polypropylene membrane, glass sheet, silica gel wafer, micro magnetic beads, etc.

The term "monocytes" refers to cells having a single core in the blood, including lymphocytes and monocytes, as well as hematopoietic stem cells and progenitor cells.

The term "macrophage" refers to macrophages produced by the differentiation of monocytes, macrophages of any differentiated or undifferentiated phenotype (including M1 macrophages) and any cells contained within the mononuclear phagocyte system, including monocytes. Likewise, the term "macrophage" as used herein includes, but is not limited to, adipose tissue macrophages, monocytes, cooper cells, sinus tissue cells, alveolar macrophages (dust cells), tissue macrophages resulting in giant cells (tissue cells), langerhans cells, microglial cells, huo Fubao mole cells, mesangial cells (intraglomerular mesangial cell), osteoclasts, epithelial-like cells, erythroid macrophages (redpulp macrophag) (Dou Nachen cells), peritoneal macrophages, lysoMac.

The term "lymphocyte separation medium" may be a lymphocyte separation medium known in the art, and may be, for example, soy baby LTS1077. In the art, the composition of lymphocyte separation fluid is consistent, as in the embodiment of the present invention, a mixture of dextran and diatrizosamine, a commonly used reagent for separation and purification of cells by gravity acceleration generated by centrifugation according to cell density difference, with a density of 1.077g/mL.

The term "macrophage colony-stimulating factor" or short form "M-CSF" refers to secreted hematopoietic growth factors that are involved in the proliferation, differentiation and survival of monocytes, macrophages and myeloid progenitor cells. It binds to colony stimulating factor 1 receptor. In some examples, the concentration of M-CSF in the differentiation medium is about 1 to about 500ng/ml, or about 10 to about 450ng/ml, or about 20 to about 400ng/ml, or about 30 to about 350ng/ml, or about 40 to about 300ng/ml, or about 50 to about 250ng/ml, or about 60 to about 200ng/ml, or about 70 to about 180ng/ml, or about 80 to about 160ng/ml, or about 90 to about 140ng/ml, or about 100 to about 120ng/ml, or about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 220, 240, 260, 280, 300, 320, 340, 360, 380, 400, 420, 440, 460, 480, or 500ng/ml. In a specific embodiment of the invention, the concentration of M-CSF in the differentiation medium is 20ng/ml.

The term "granulocyte macrophage colony-stimulating factor" or short form "GM-CSF" refers to cytokines that function as leukocyte growth factors. GM-CSF stimulates stem cells to produce granulocytes (neutrophils, eosinophils and basophils) and monocytes. Monocytes leave the circulation and migrate into the tissue, where they begin to mature into macrophages. Thus, activation of small numbers of macrophages can rapidly lead to their increase in number through a part of the innate immune/inflammatory cascade, a key process to combat infection.

The term "phorbol ester" refers to a natural, plant-derived organic compound that is a member of the tigrinan family of diterpenes.

The present invention provides for the use of RabGGTase in constructing a computational model for diagnosing ALS conditions, as will be appreciated by the skilled artisan, the step of correlating marker levels with a certain likelihood or risk may be performed and implemented in different ways. Preferably, the measured concentrations of the marker and one or more other markers are mathematically combined and the combined values are correlated with the underlying diagnostic problem. The determination of the marker values may be combined by any suitable prior art mathematical method.

In the present invention, the term "diagnosis" refers to any kind of procedure intended to obtain useful information in assessing whether a patient is or is likely to be or is more likely to suffer from a certain disease or condition in the past, at diagnosis or at the time of the future than an average or comparison subject (the latter preferably having similar symptoms) to find out how the disease is progressing or is likely to progress in the future or to assess the responsiveness of the patient to a certain treatment (e.g. administration of a suitable drug). In other words, the term "diagnosis" includes not only aiding in diagnosis, but also efforts to prognosis and/or monitor the progress of a disease or disorder.

The present invention provides a system programmed to implement the method of the present invention. The system is programmed or otherwise configured to analyze sequence data, construct an expression level matrix of genes. The system may regulate various aspects of the sequence analysis of the present disclosure, such as, for example, matching data against known sequences. The system may be the user's electronic device or a computer system remotely located relative to the electronic device. The electronic device may be a mobile electronic device.

AUC measurements are useful for comparing classifier accuracy across the entire data range. Classifiers with higher AUC have a higher ability to correctly classify between two target groups (e.g., ovarian cancer samples and normal or control samples) is not known. ROC curves are useful for characterizing the performance of a particular feature (e.g., any biomarker described herein and/or any entry of additional biomedical information) when distinguishing between two populations (e.g., individuals responding to a therapeutic agent and not responding). Typically, feature data is selected across the entire population (e.g., cases and controls) in ascending order based on the values of individual features. Then, for each value of the feature, the true and false positive rates of the data are calculated. The true positive rate is determined by counting the number of cases above the value of the feature and dividing by the total number of cases. False positive rates were determined by counting the number of controls above the value of the feature and dividing by the total number of controls. Although the definition refers to the case where the characteristic is increased in the case compared to the control, the definition also applies to the case where the characteristic is lower in the case compared to the control (in this case, a sample below the value of the characteristic will be counted). The ROC curve may be generated with respect to individual features and may be generated with respect to other individual outputs, for example, a combination of two or more features may be mathematically combined (e.g., added, subtracted, multiplied, etc.) to provide an individual sum value, and the individual sum value may be plotted in the ROC curve. In addition, any combination of features, the combination of which results from separate output values, may be plotted in the ROC curve. These combinations of features may include testing. ROC curves are plots of true positive rate (sensitivity) of the test versus false positive rate (1-specificity) of the test.

The invention will now be described in further detail with reference to the drawings and examples. The following examples are only illustrative of the present invention and are not intended to limit the scope of the invention. Simple modifications of the invention in accordance with the essence of the invention are all within the scope of the invention as claimed.

EXAMPLE 1 Effect of RabGGTB on ALS severity and progression

1. Sample source

We collected data from ALS patients diagnosed in the department of Utility second hospital neurology at university of Hebei medical science, 1 st 2021 nd 2022 nd 10 th, including detailed medical history, physical examination, laboratory examination, electrophysiological examination, etc., all in compliance with revised ALS diagnostic criteria. All selected cases exclude acute or chronic inflammatory diseases, such as acute pneumonia, rheumatoid arthritis, etc., and all sign informed consent. ALS function rating scale revision (ALSFRS-R) is used to assess disease severity, and disease progression rate (Δfs=48-initial ALSFRS-R score/time to onset to visit) is used to assess disease progression rate. Sample data for ALS patients are shown in Table 1.

2. Serum index

All blood indexes are provided by a second hospital clinical laboratory of Hebei university of medical science, the normal range of adult IL-6 is 0-7pg/ml, and the normal range of adult CRP is 0-6mg/L.

3. Isolation of peripheral blood mononuclear cells

Human peripheral blood lymphocyte isolates (Soleibao LTS 1077) were added to a PBMC high efficiency centrifuge tube (601002 Tianjin, ministry of the ocean biol technology Co., ltd.) and centrifuged at 200g for 2min at room temperature, the collected peripheral blood specimens were added, centrifuged at 800g for 30min, the intermediate white-film mononuclear cell layer was carefully aspirated into a fresh centrifuge tube, centrifuged at 300g for 13min, the supernatant was aspirated, 1 XPBS was repeatedly washed, PBMC resuspended in a frozen stock solution containing 90% v/v fetal bovine serum (cellmax, SA 211.02) and 10% dimethyl sulfoxide (sigma, D2650-100 ML), and transferred to a 1.8ML frozen stock tube overnight at-80℃and transferred to liquid nitrogen the next day for storage.

4. Peripheral blood mononuclear cell induced differentiation macrophage culture

Frozen peripheral blood mononuclear cells were thawed into RPMI-1640 complete medium (gibco, C11875500BT, 10% foetal calf serum cellmax, SA211.02,1% penicillin-streptomycin), centrifuged at 500g for 5min, the supernatant was aspirated, the cell pellet resuspended in RPMI-1640 complete medium, 20ng/ml macrophage colony stimulating factor (peprotech, 300-25-10) was added to the medium, the cells were seeded in 48 well plates, incubated in an incubator at 37℃for 7 days, and the culture was changed every 3 days. On day 7, cell culture broth was collected and cells were fixed in 4% paraformaldehyde fixative.

5. Immunofluorescence and confocal microscopy

Cells were fixed on 48-well plate slides with 4% paraformaldehyde in PBS, permeabilized with 0.3% Triton-X100 in PBS for 15min, blocked with 10% sheep serum blocking solution at room temperature for 1h, and then incubated overnight with primary antibodies CD68 (abcam, ab 53444), F4/80 (ab 6640), rabGGTB (GTX 105874). After overnight, cells were washed 3 times with 1 XPBS and then incubated with secondary antibodies Alexa Fluor488-conjugated Goat anti-Rabbit secondary antibody (1:1000,Thermo Fisher, #A-11034), alexaFluor 594-conjugated Goat anti-Mouse secondary antibody (1:1000,Thermo Fisher, #A-11032), alexa Fluor 647-conjugated Donkey anti-Goat secondary antibody (1:1000,Thermo Fisher, #A 211447) and DAPI for 1h at room temperature. Cells were washed 3 more times with 1 XPBS and blocked with blocking agents. Photographs were taken with a Zeiss laser confocal microscope. The parameters of the microscope are set at the beginning of each individual imaging procedure and remain constant throughout the imaging procedure.

6. Single factor analysis and multiple factor analysis

Single factor analysis: the counting data adopts: linear regression analysis, classification data using: logistic regression analysis. Multi-factor analysis: the counting data adopts: linear regression analysis, classification data using: logistic regression analysis. All statistical analyses were performed using GraphPad Prism 9 (GraphPad software) and SPSS software. * P <0.05 is considered statistically significant.

7. Diagnostic efficacy analysis

And analyzing the expression level of RabGGTB in the ALS rapid-progress group and the ALS slow-progress group, and performing ROC curve analysis, wherein the AUC value is the diagnosis efficacy, and the greater the AUC value is, the stronger the diagnosis efficacy is.

8. Statistical analysis

All count data are expressed as mean ± standard deviation and are normally tested using Shapiro-Wilk normal test. For data conforming to normal distribution, the differences between the two groups were examined by unpaired t-test and correlation analysis by Pearson correlation coefficient. For data that do not fit the normal distribution, the differences between the two groups were checked using the Mann-Whitney U test and the correlation analysis was checked using the Spearman rank correlation coefficient. All statistical analyses were performed using GraphPad Prism 9 (GraphPad software) and SPSS software. * P <0.05 is considered statistically significant.

9. Experimental results

The immunofluorescence method has the detection results shown in Table 1 and FIG. 1, and the results show that the expression level of RabGGTB in macrophages is 17.53+/-8.363; there was a statistical difference in the RabGGTB expression levels in the slow-and fast-progressing groups, and there was a statistical difference in the RabGGTB expression levels between the general and fast-progressing groups, which indicated that the higher the RabGGTB expression level in ALS patients, the faster the disease progression.

The single factor analysis and the multi-factor analysis are shown in table 2, table 3, fig. 2 and fig. 3, the single factor analysis finds that the RabGGTB is obviously related to the ALSFRS-R score (p=0.0067), the multi-factor analysis finds that the RabGGTB is obviously related to the ALSFRS-R score (p=0.041), and the result shows that the higher the expression level of the RabGGTB is, the more serious the disease degree of the patient is; the single factor analysis shows that the disease course (time to diagnosis), rabGGTB and DeltaFS (P < 0.01) are obviously related, the multi-factor analysis shows that the clear disease course, rabGGTB expression level and DeltaFS are obviously related (P is 0.001 and 0.016 respectively), and the result shows that the higher the RabGGTB expression level, the shorter the disease course, the faster the disease progress of ALS patients is.

The ROC curve results are shown in fig. 4, and the results show that AUC is 0.8214 (sensitivity= 0.7778, specificity= 0.7857), and RabGGTB can diagnose the disease progression of ALS patients.

TABLE 1ALS patient data

TABLE 2 correlation of ALSRFS-R scores with correlation indicators

TABLE 3 correlation of ΔFS with correlation index

The above description of the embodiments is only for the understanding of the method of the present invention and its core ideas. It should be noted that it will be apparent to those skilled in the art that several improvements and modifications can be made to the present invention without departing from the principle of the invention, and these improvements and modifications will fall within the scope of the claims of the invention.

Claims (10)

1. Use of a reagent for detecting the expression level of RabGGTase in a sample for the preparation of a product for diagnosing an ALS condition;

preferably, the ALS condition includes the severity of ALS condition and the rate of progression of the condition.

2. The use according to claim 1, wherein the agent is selected from the group consisting of:

an oligonucleotide probe that specifically recognizes the RabGGTase gene; or (b)

Primers for specifically amplifying the RabGGTase gene; or (b)

A binding agent that specifically binds to a protein encoded by the RabGGTase gene;

preferably, the binding agent comprises an antibody, functional fragment of an antibody, conjugated antibody that specifically binds to a RabGGTase encoded protein.

3. The use according to claim 1, wherein the sample is selected from the group consisting of tissue, blood, urine, cerebrospinal fluid, cell lines, tissue cultures, and interstitial fluid;

preferably, the blood comprises blood-derived cells, platelets, serum, plasma;

preferably, the blood-derived cells are selected from nucleated cells; the nucleated cells comprise monocytes, macrophages, dendritic cells, lymphocytes and granulocytes;

preferably, the blood-derived cells are monocytes or macrophages.

Preferably, the RabGGTase comprises RabGGTA, rabGGTB;

preferably, the RabGGTase is RabGGTB.

4. A product for diagnosing an ALS condition, the product comprising a preparation, chip, kit or nucleic acid membrane strip capable of detecting the expression level of RabGGTase;

preferably, the ALS condition includes the severity of ALS condition and the rate of progression of the condition.

5. The product of claim 4, wherein the chip comprises a gene chip comprising an oligonucleotide probe for the RabGGTase gene for detecting the transcription level of the RabGGTase gene, a protein chip comprising a specific binding agent for the RabGGTase protein; the kit comprises a gene detection kit and a protein detection kit, wherein the gene detection kit comprises a reagent or a chip for detecting the transcription level of the RabGGTase gene, and the protein detection kit comprises a reagent or a chip for detecting the expression level of the RabGGTase protein.

6. The product of claim 5, wherein the kit comprises reagents for detecting the level of RabGGTase gene or protein expression by RT-PCR, qRT-PCR, biochip assay, southern blotting, northern blotting, in situ hybridization, immunoblotting, immunofluorescence and immunohistochemistry, ELISA, transcriptomics, proteomics.

7. The product of claim 4, further comprising a reagent for processing a sample;

preferably, the reagent for treating a sample comprises a reagent for isolating monocytes or a reagent for promoting differentiation of monocytes into macrophages;

preferably, the monocyte separating reagent comprises lymphocyte separating liquid and monocyte separating liquid;

preferably, the monocyte-separating agent is lymphocyte-separating liquid;

preferably, the agent that promotes differentiation of monocytes into macrophages comprises macrophage colony stimulating factor, granulocyte macrophage colony stimulating factor, phorbol ester;

preferably, the agent that promotes differentiation of monocytes into macrophages is a macrophage colony stimulating factor.

Preferably, the RabGGTase comprises RabGGTA, rabGGTB;

preferably, the RabGGTase is RabGGTB.

Application of RabGGTase in constructing a calculation model for diagnosing ALS disease;

preferably, the ALS condition includes ALS severity, rate of progression;

preferably, the RabGGTase comprises RabGGTA, rabGGTB;

preferably, the RabGGTase is RabGGTB.

9. A system for diagnosing an ALS condition, the system comprising:

(1) The ALS disease condition evaluation device comprises a control unit and a storage unit, wherein the control unit and the storage unit are connected through an information communication terminal device which is in communication connection with each other and used for evaluating the disease condition of an ALS patient;

(2) Information communication terminal apparatuses that are communicatively connected to each other: for providing data concerning the expression level of RabGGTase in a sample from an ALS patient;

preferably, the ALS condition includes the severity of ALS condition and the rate of progression of the condition.

10. The system of claim 9, wherein the control unit of the ALS patient condition assessment device comprises four units:

1) A data receiving unit: for receiving data about the expression level of RabGGTase in the sample transmitted from the information communication terminal device;

2) Discrimination value calculation unit: calculating a discrimination value based on discrimination of the expression level of RabGGTase in the sample received by the data receiving unit and the expression level of RabGGTase stored in the storage unit as an explanatory variable;

3) Discrimination value reference evaluation unit: evaluating the risk of developing the disease of the ALS patient based on the discrimination value calculated by the discrimination value calculation unit;

4) An evaluation result transmitting unit: transmitting the evaluation result of the ALS patient condition obtained by the discrimination value reference evaluation unit to the information communication terminal device;

preferably, the RabGGTase comprises RabGGTA, rabGGTB;

preferably, the RabGGTase is RabGGTB.

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