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WO2023068622A1 - Composition de biomarqueur pour diagnostiquer une néphrite lupique chez un patient atteint d'un lupus érythémateux disséminé et procédé de fourniture des informations nécessaires au diagnostic de la néphropathie lupique l'utilisant - Google Patents

Composition de biomarqueur pour diagnostiquer une néphrite lupique chez un patient atteint d'un lupus érythémateux disséminé et procédé de fourniture des informations nécessaires au diagnostic de la néphropathie lupique l'utilisant Download PDF

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WO2023068622A1
WO2023068622A1 PCT/KR2022/015270 KR2022015270W WO2023068622A1 WO 2023068622 A1 WO2023068622 A1 WO 2023068622A1 KR 2022015270 W KR2022015270 W KR 2022015270W WO 2023068622 A1 WO2023068622 A1 WO 2023068622A1
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protein
patients
lupus nephritis
nephritis
systemic lupus
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Korean (ko)
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김용길
이은주
김경곤
염정훈
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Asan Foundation
University of Ulsan Foundation for Industry Cooperation
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University of Ulsan Foundation for Industry Cooperation
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/564—Immunoassay; Biospecific binding assay; Materials therefor for pre-existing immune complex or autoimmune disease, i.e. systemic lupus erythematosus, rheumatoid arthritis, multiple sclerosis, rheumatoid factors or complement components C1-C9
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/158—Expression markers
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/10—Musculoskeletal or connective tissue disorders
    • G01N2800/101—Diffuse connective tissue disease, e.g. Sjögren, Wegener's granulomatosis
    • G01N2800/104—Lupus erythematosus [SLE]
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/34—Genitourinary disorders
    • G01N2800/347—Renal failures; Glomerular diseases; Tubulointerstitial diseases, e.g. nephritic syndrome, glomerulonephritis; Renovascular diseases, e.g. renal artery occlusion, nephropathy

Definitions

  • biomarker composition for diagnosing lupus nephritis in patients with systemic lupus erythematosus and a method for providing necessary information for diagnosing lupus nephritis in patients with systemic lupus erythematosus using the biomarker composition.
  • SLE Systemic lupus erythematosus
  • LN lupus nephritis
  • biomarkers are selected based on LN-associated pathophysiological pathways, a biased approach that limits novel biomarker detection.
  • profiling multiple proteins using proteome analysis is an unbiased method for screening biomarkers.
  • Previous studies utilized mass spectrometry (MS) and electrospray ionisation quadrupole time-of-flight tandem MS14 for unbiased screening of biomarkers for LN.
  • MS mass spectrometry
  • electrospray ionisation quadrupole time-of-flight tandem MS14 for unbiased screening of biomarkers for LN.
  • affinity-based approaches eg, antibody or aptamer-based assays
  • MS-based approaches Unlike affinity-based approaches, MS-based approaches generally detect high-abundance proteins and may not detect low-abundance proteins.
  • sequential window acquisition of all theoretical mass spectra (SWATH LC-MS)-based proteome analysis combined with liquid chromatography a new MS-based approach, can detect low-abundance proteins.
  • the present inventors Using this proteome analysis platform, the present inventors recently discovered urine ⁇ -2-glycoprotein 1 (Apolipoprotein H, ApoH) as a diagnostic biomarker for SLE (Korean Patent Application No. 10-2021-0031196).
  • the present inventors used SWATH LC-MS-based proteomic analysis to detect healthy controls (HC) .
  • HC healthy controls
  • An object of the present invention is to provide a biomarker composition for diagnosing systemic lupus erythematous (SLE) that can accurately and early diagnose lupus nephritis (LN) in patients with systemic lupus erythematous (SLE) by a simple and convenient method.
  • Another object is to provide a kit for diagnosing lupus nephritis in systemic lupus erythematosus patients comprising the composition.
  • Another object is to provide a method for providing information necessary for diagnosing lupus nephritis in patients with systemic lupus erythematosus using the biomarker composition.
  • Another object is to provide the use of the biomarker for diagnosing lupus nephritis in patients with systemic lupus erythematosus.
  • Another object is to provide a method for diagnosing lupus nephritis in a patient with systemic lupus erythematosus from a biological sample obtained from the subject.
  • HBD Hemoglobin subunit delta
  • SERPINC1 Antithrombin-III protein or a combination thereof
  • systemic erythematosus containing an agent capable of measuring the expression level of the gene encoding them
  • a biomarker composition for diagnosing lupus nephritis (LN) in patients with systemic lupus erythematous (SLE).
  • HBD Hemoglobin subunit delta
  • SERPINC1 Antithrombin-III
  • AT3, AT3D, ATIII, THPH7, serpin family C member 1, ATIII-R2, ATIII-T2 or ATIII-T1 refers to a small protein molecule in plasma that inactivates thrombin .
  • SERPINC1 is a glycoprotein produced in the liver, composed of 432 amino acids, and is known to include three disulfide bonds and a total of four possible glycosylation sites.
  • systemic lupus erythematous refers to a disease in which an inflammatory response due to autoimmunity occurs in various tissues of the body. Depending on the tissue involved, various symptoms such as skin rash, photosensitivity, arthritis, mouth ulcer, nephritis, cytopenia, vasculitis, and serositis appear. Although the exact cause of systemic lupus erythematosus is not known, it is known that genetic factors are involved in the pathogenesis. In addition to genetic factors, certain viral infections, exposure to ultraviolet rays, excessive stress, some drugs including antibiotics, or environmental factors caused by several hormones have also been reported to play an important role in the pathogenesis.
  • LN lupus nephritis
  • lupus nephritis refers to nephritis caused by systemic lupus erythematosus affecting the kidneys. It is known that various autoantibodies or immune complexes present excessively in the blood are deposited in the glomeruli of the kidneys, and inflammatory cells infiltrate the glomeruli to cause inflammation and damage to the glomeruli of the kidneys. As a symptom of lupus nephritis, kidney tissue is destroyed and proteinuria, microscopic hematuria (sometimes gross hematuria) appear in urine examination, and findings such as red blood cells, white blood cells, and various cellular casts appear in urine sediment.
  • hematuria sometimes gross hematuria
  • lupus nephritis which causes symptoms such as hypertension, hyperlipidemia, and hyperglycemia.
  • Lupus nephritis accounts for 30-60% of adult systemic lupus erythematosus patients, occurs in up to 70% in children, is the most fatal cause of death among systemic lupus erythematosus complications, and is related to patient prognosis.
  • Glomerulonephritis is the most common type of lupus nephritis, but necrotizing vasculitis and acute interstitial nephritis also occur.
  • diagnosis means confirming the presence or character of a pathological condition.
  • biomarker may also be used as a diagnostic marker, and refers to a substance capable of diagnosing whether a patient with systemic lupus erythematosus develops lupus nephritis in a biological sample.
  • Polypeptides or nucleic acids e.g., mRNA, etc.
  • lipids lipids
  • organic biomolecules such as glycolipids, glycoproteins or sugars (eg, monosaccharides, disaccharides, oligosaccharides, etc.); and the like.
  • the biomarker composition is ORM1 (Alpha-1-acid glycoprotein 1), CP (Ceruloplasmin), AFM (Afamin), ORM2 (Alpha-1-acid glycoprotein 2), SERPINA3 (Alpha-1-antichymotrypsin), SERPINA1, A1BG ( Alpha-1B-glycoprotein; P04217-2), A1BG (Alpha-1B-glycoprotein; P04217), CA1 (Carbonic anhydrase 1), SERPINA6 (Corticosteroid-binding globulin), MSH2 (DNA mismatch repair protein Msh2), RNF149 (E3 ubiquitin -protein ligase RNF149), HP (Haptoglobin), HMCN1 (Hemicentin-1), HBA1 (Hemoglobin subunit alpha), HBB (Hemoglobin subunit beta), LRG1 (Leucine-rich alpha-2-glycoprotein), RCN1 (Reticulocalbin-1) ,
  • the biomarker composition is selected from the group consisting of ORM1, CP, AFM, ORM2, SERPINA3, SERPINA1, A1BG, CA1, SERPINA6, MSH2, RNF149, HP, HMCN1, HBA1, HBB, LRG1, RCN1, TF, ALB and AZGP1 It may further include an agent capable of measuring the expression level of one or more proteins or genes encoding them.
  • Alpha-1-acid glycoprotein 1 also called AGP-A, AGP1, HEL-S-153w, ORM, or orosomucoid 1
  • ORM a member of the acute phase protein family, is known to activate monocytes, induce T-cell proliferation, and promote secretion of pro-inflammatory cytokines such as tumor necrosis factor ⁇ , interleukin (IL)-1 and IL-6.
  • CP Ceruloplasmin
  • caeruloplasmin is also called caeruloplasmin, and refers to a2-glycoprotein that binds to copper in plasma.
  • CP is a major protein that transports copper in the blood and is known to play an important role in iron metabolism.
  • the expression level of the protein or the gene encoding it can be measured in a urine sample of an individual.
  • HC healthy controls
  • SLE systemic lupus erythematosus patients without nephritis
  • n-LN systemic lupus erythematosus patients diagnosed with lupus nephritis
  • the four urine biomarkers were significantly higher in n-LN patients than in SLE patients without nephritis. It can be confirmed that the expression is increased. In addition, some n-LN patients showed non-significant proteinuria (UPCR ⁇ 500mg/g) symptoms, but in the urine samples of these patients, it was confirmed that the expression of the four urine biomarkers was increased compared to SLE patients without nephritis. there is. Therefore, urine ORM1, SERPINC1, CP, and HBD can be usefully utilized to detect early lupus nephritis even before significant proteinuria develops in patients with systemic erythematosus.
  • the lupus nephritis may be proliferative lupus nephritis or non-proliferative lupus nephritis.
  • urine HBD shows high accuracy in distinguishing between proliferative and non-proliferative LN.
  • proliferative LN concomitant administration of high-dose steroids and immunosuppressants is required, and in the case of non-proliferative LN, a general SLE treatment method is selected. Therefore, since proliferative LNs and non-proliferative LNs differ in treatment methods, urine HBD can accurately distinguish proliferative LNs from non-proliferative LNs and enable selection of an appropriate treatment method.
  • urine HBD shows a significant positive correlation with histological activity index. Histological activity index after treatment is important for LN prognosis. However, it is difficult to estimate the activity index without performing a kidney biopsy because clinical renal parameters do not accurately reflect the histological activity index. On the other hand, since kidney biopsy is an invasive procedure and is often difficult to perform repeatedly, urine HBD can be usefully used to estimate histological activity index in a simple and convenient way instead.
  • Measurement of the expression level of the protein is a process of confirming the presence and expression level of the ApoH protein in a biological sample of an individual in order to diagnose lupus nephritis in a patient with systemic lupus erythematosus. It is to check the amount of protein using molecules.
  • Agents capable of measuring the expression level of the protein include monoclonal antibodies, polyclonal antibodies, chimeric antibodies, ligands, peptide nucleic acids (PNAs), aptamers ( aptamer) and nanoparticles, but may be at least one selected from the group consisting of, but is not limited thereto.
  • Analysis methods for this include Western blotting, enzyme linked immunosorbent assay (ELISA), radioimmunoassay (RIA), radioimmunodiffusion, Ouchterlony immunodiffusion method, rocket ( rocket) immunoelectrophoresis, immunoprecipitation assay, immunohistochemical analysis, complement fixation assay, FACS (Fluorescence activated cell sorter) or protein chip, etc. It is not limited thereto.
  • Measurement of the expression level of the gene encoding the protein is a process of confirming the presence and expression level of the gene in a biological sample of an individual in order to diagnose the onset of lupus nephritis in a systemic lupus erythematosus patient. It is to check the amount of a gene using a molecule that binds.
  • the agent capable of measuring the expression level of the gene encoding the protein is at least one selected from the group consisting of a primer pair, a probe, and an antisense nucleotide that specifically binds to the gene. It may be, but is not limited thereto.
  • RT-PCR reverse transcriptase-polymerase chain reaction
  • RPA RNase protection assay
  • Another aspect provides a kit for diagnosing lupus nephritis in systemic lupus erythematosus patients comprising the biomarker composition.
  • the kit can diagnose whether a systemic lupus erythematosus patient has lupus nephritis by using a biological sample isolated from the subject. Specifically, the kit can diagnose whether a patient with systemic lupus erythematosus has lupus nephritis by using a urine sample collected from the subject.
  • a urine sample that can be easily collected from an individual, an early diagnosis of lupus nephritis in a patient with systemic lupus erythematosus may be possible in a simpler and more convenient way without a kidney biopsy.
  • the kit may include tools and reagents commonly used in immunological analysis, as well as preparations for measuring the expression level of the protein or gene.
  • the tools or reagents may include, but are not limited to, suitable carriers, labeling substances capable of generating detectable signals, chromophores, solubilizers, detergents, buffers, and stabilizers.
  • labeling substance is an enzyme, it may include a substrate capable of measuring enzyme activity and a reaction terminating agent.
  • the carrier includes a soluble carrier and an insoluble carrier
  • an example of the soluble carrier is a physiologically acceptable buffer known in the art, such as PBS
  • an example of the insoluble carrier is polystyrene, polyethylene, polypropylene, polyester, poly polymers such as acrylonitrile, fluororesin, crosslinked dextran, polysaccharide, latex-coated magnetic microparticles, other paper, glass, metal, agarose, and combinations thereof.
  • the biomarker composition is as described above.
  • Another aspect is information necessary for diagnosing lupus nephritis in a patient with systemic lupus erythematosus, comprising measuring the expression level of HBD protein, SERPINC1 protein or a combination thereof, or a gene encoding the same in a biological sample obtained from the subject. provides a method for providing
  • the method comprises one or more selected from the group consisting of ORM1, CP, AFM, ORM2, SERPINA3, SERPINA1, A1BG, CA1, SERPINA6, MSH2, RNF149, HP, HMCN1, HBA1, HBB, LRG1, RCN1, TF, ALB and AZGP1
  • a step of measuring the expression level of the protein or the gene encoding it may be further included.
  • the method is to diagnose systemic lupus erythematosus patients with lupus nephritis when the expression level of the protein or the gene encoding it is higher than the expression level measured in a biological sample obtained from a normal individual or a systemic lupus erythematosus patient without nephritis. Additional steps may be included.
  • biological sample refers to any sample obtained from an individual capable of measuring the expression level of the 23 proteins or genes encoding them according to one aspect.
  • the biological sample may be blood, plasma, serum, urine, saliva, sputum, cerebrospinal fluid, cell culture fluid, tissue extract, or tumor tissue, but is not limited thereto.
  • the biological sample may be urine.
  • the biological sample may be prepared by treatment by a method commonly used in the art.
  • biomarker comprising an agent capable of measuring the expression level of HBD protein, SERPINC1 protein or combination thereof, or a gene encoding the same, for diagnosing lupus nephritis in patients with systemic lupus erythematosus to provide.
  • Another aspect is a method for diagnosing lupus nephritis in a patient with systemic lupus erythematosus, comprising measuring the expression level of HBD protein, SERPINC1 protein or a combination thereof, or a gene encoding the same in a biological sample obtained from the subject provides
  • Redundant content is omitted in consideration of the complexity of the present specification, and terms not otherwise defined in the present specification have meanings commonly used in the technical field to which the present invention belongs.
  • the body through a non-invasive and simple method of measuring the expression level of proteins such as HBD, SERPINC1 or the gene encoding them in the subject's urine sample Since it is possible to accurately diagnose the onset of lupus nephritis in patients with lupus erythematosus, it is possible to detect lupus nephritis in the early stages without significant proteinuria, and it is possible to distinguish between proliferative and non-proliferative types, so the mortality rate of systemic lupus erythematosus patients It will be useful in the diagnosis and treatment of lupus nephritis, a major side effect that increases
  • HCs healthy controls
  • SLE systemic lupus erythematosus patients
  • Volcano plot showing protein expression (proteins ranked according to q-value; Log10 scale, y-axis) and relative abundance (Log2 fold change, x-axis) to identify ⁇ 1).
  • Figure 2 is differentially expressed in urine samples of systemic lupus erythematosus patients without nephritis (SLE) and systemic erythematosus patients newly diagnosed with lupus nephritis (n-LN) using the SWATH LC-MS platform according to one aspect.
  • Volcano showing proteins (FDR: ⁇ 0.05, log2 fold change ⁇ 1) as protein expression (proteins ranked according to q-value; Log10 scale, y-axis) and relative abundance (Log2 fold change, x-axis) it's a plot
  • Figure 3 shows the urinary ORM1 (A), urinary SERPINC1 (B), urinary CP (C), urinary HBB (D) and urinary HBD (E) of HCs, SLE patients without nephritis and n-LN patients according to one aspect. This is the result of performing ELISA-based verification for each. Values shown in the figures are mean ⁇ SEM. *p ⁇ 0.05, **p ⁇ 0.01, ***p ⁇ 0.001.
  • Figure 4 shows urine ORM1 (A), urine SERPINC1 (B), urine CP (C), urine HBD (D), serum C3 (E), serum C4 (F), serum anti-dsDNA Ab (G ), a combination of urine ORM1, SERPINC1, CP and HBD (H) and a combination of serum C3 and urine ORM1 (I) are shown ROC curves for differentiating SLE patients without nephritis from n-LN patients.
  • SLE patients included (i) 22 SLE patients without nephritis (isolated microscopic hematuria or pyuria are not considered nephritis), (ii) 22 SLE patients with newly diagnosed LN. classified into groups. All LN diagnoses were confirmed by renal biopsy according to Society of Nephrology/Renal Pathology Society classification. In addition, to include very early LN, kidney biopsies were also performed in cases of decreased renal function and 500 mg/g > urine protein/creatinine ratio (UPCR) ⁇ 300 mg/g ⁇ active urine sediment. .
  • UPCR urine protein/creatinine ratio
  • the sex and mean age of patients with SLE were (i) 18 female and 4 male SLE patients without nephritis, 51.0 years (37.5-56.3 years), and (ii) 14 female SLE patients with newly diagnosed LN. 1, 3 males, 43.0 years old (23.0-49.5 years).
  • 20 subjects matching sex and age with the patient group were selected.
  • a 500 ⁇ l urine sample was lyophilized to reduce the volume in a centrifugal vacuum concentrator (LABCONCO, CentriVap, Kansas City, MO, USA).
  • the dried sample was reconstituted with 100 ⁇ L lysis buffer by mixing 5% sodium dodecyl sulfate and 50 mM triethylammonium bicarbonate (pH 7.55, Thermo Fisher Scientific). Samples were subjected to S trap-based trypsin digestion according to the previously described method (Lupus 2021, Vol. 30(8) 1306-1313) except for the trypsin/LysC mixture (Promega, Madison, WI, USA).
  • the dried peptides were reconstituted with 0.1% formic acid, and the absorbance at 205 nm wavelength was measured on a NanoDrop One spectrophotometer (ThermoFisher Scientific) to measure the peptide concentration. 40 ⁇ g peptides from each sample were assigned to individual assays and dried. All remaining peptides were combined for spectroscopic library generation .
  • Fractions of the combined samples were analyzed at 0.5 mL/min on an XBridge C18 column (4.6 mm i.d., 250 mm length, pore size 130 ⁇ particle size 5 ⁇ m; Waters Corporation, USA) using a Shimadzu Prominence HPLC instrument (Shimadzu, Japan). was performed at flow rate. 10 mM triethylammonium bicarbonate (TEAB, pH 8.5) as mobile phase A and 10 mM TEAB in 90% acetonitrile (pH 10) as mobile phase B were used for peptide fractionation. The sample was dissolved in 200 ⁇ L mobile phase A and then injected into a 2,100 ⁇ L sample loop.
  • TEAB triethylammonium bicarbonate
  • LC-MS/MS liquid chromatography-tandem mass spectrometry
  • eluent A and eluent B (0.1% formic acid in 100% acetonitrile) at 5 ⁇ l/min in 87 min increments (eluent B was 3-25% over 68 min, 25-25% over 5 min). 35%, 35-80% over 2 minutes, 80% hold for 3 minutes, 80-3% hold for 1 minute, 3% hold for 8 minutes).
  • the mass spectrometer undergoes data dependent acquisition (DDA) with 250 ms and 50 ms acquisition times, and 15 s dynamic exclusion for MS1 (400 to 1250 m/z) and MS2 (100 to 1500 m/z) scans, respectively. It worked in top30 mode.
  • DDA data dependent acquisition
  • Rolling collision energies were used for fractionation with charge states from +2 to +5. Other parameters were set as follows: ion source gas 1 (GS1) 15; ion source gas 2 (GS2) 20; curtain gas (CUR) 30; temperature (TEM) 250 °C; Ion Spray Voltage Plot (ISVF) 5500.
  • GS1 ion source gas 1
  • GS2 ion source gas 2
  • CUR curtain gas
  • TEM temperature
  • ISVF Ion Spray Voltage Plot
  • SWATH Sequential window acquisition of all theoretical mass spectra
  • LC setup was performed under the same conditions except for the gradient times described above.
  • the gradient of eluent B was 3-25% over 38 min, 25-32% over 5 min, 32-80% over 2 min, 80% hold over 3 min, 80-3% over 1 min, 3 min. An 80% retention was applied.
  • the SWATH parameters were set as follows: lower limit m/z 400; upper limit m/z 1250; window overlap (Da) 1.0; CES was set at 5 for the small windows, 8 for the large windows, and 10 for the largest windows.
  • MS2 spectra were collected in the 100-1500 m/z range for 2.5 ms in high-sensitivity mode, with a total cycle time of 2.8 seconds.
  • SWATH-MS data for individual samples were processed using DIA-NN 1.7.10 with an in-house urine proteome spectroscopy library. Spectra were retrieved with default settings, but the m/z range was 400-1250 for precursors and 100-1500 for fragment ions. Protein quantification values were obtained using the MaxLFQ algorithm (PMID: 24942700) implemented in the diann R package (https://github.com/vdemichev/diann-rpackage). Protein intensity values were filtered for precursor and protein groups passing a q-value greater than or equal to 0.01. Normalization of protein intensity values was performed in the following way:
  • Creatine concentration was calculated by multiplying the protein strength value by the protein percentage value and dividing this value.
  • the protein ratio is the total quantified peptide amount divided by the amount of peptide prepared for MS analysis.
  • 143 protein groups (FDR ⁇ 0.05, log2 fold change ⁇ 1) were increased in SLE patients without nephritis compared to HCs (Fig. 1), and 67 protein groups were significantly different from those in SLE patients without nephritis. In comparison, it increased in n-LN patients (FIG. 2).
  • n-LN patients (FIG. 2).
  • 23 protein groups common between the two comparative analyzes were identified, as described in Table 1 below. The expression of these proteins was increased in SLE patients without nephritis rather than HC, and increased in n-LN patients than in SLE patients without nephritis.
  • SLE is systemic lupus erythematosus
  • n-LN is systemic lupus erythematosus newly diagnosed as lupus nephritis
  • Anti-dsDNA Abs include anti-double stranded DNA antibodies
  • ESR erythrocyte sedimentation rate
  • C-reactive protein C-reactive protein
  • UPCR urine protein creatinine ratio
  • RBCs red blood cells
  • HPF high power field
  • WBCs White blood cells
  • SLEDAI Systemic lupus erythematosus disease activity index
  • ISN/RPS International Society of Nephrology/Renal Pathology Society
  • ISN/RPS International Society of Nephrology/Renal Pathology Society
  • urine HBD can be used to accurately distinguish between proliferative and non-proliferative LN, select an appropriate treatment method, and estimate the histological activity index after treatment, which is important for LN prognosis.

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Abstract

La présente invention concerne une composition de biomarqueur pour le diagnostic de la néphropathie lupique chez des patients atteints de lupus érythémateux disséminé et un procédé de fourniture des informations nécessaires pour le diagnostic de la néphropathie lupique chez des patients atteints de lupus érythémateux disséminé, l'utilisant. Dans le procédé, un diagnostic précis peut être effectué d'une manière non invasive et simple de mesure du niveau d'expression de protéines telles que HBD et SERPINC1 ou des gènes codant pour celles-ci dans des échantillons d'urine d'individus pour voir si la néphropathie lupique se déclenche chez un patient atteint d'un lupus érythémateux disséminé. Une néphropathie lupique précoce dans laquelle une quantité significative de protéinurie n'a pas encore été générée peut être détectée et il est possible de distinguer si la néphropathie lupique précoce est proliférative ou non proliférative. Par conséquent, le procédé peut être avantageusement utilisé dans le diagnostic et le traitement de la néphropathie lupique qui est un effet secondaire principal augmentant la mortalité des patients atteints du lupus érythémateux disséminé.
PCT/KR2022/015270 2021-10-20 2022-10-11 Composition de biomarqueur pour diagnostiquer une néphrite lupique chez un patient atteint d'un lupus érythémateux disséminé et procédé de fourniture des informations nécessaires au diagnostic de la néphropathie lupique l'utilisant Ceased WO2023068622A1 (fr)

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KR1020210140135A KR102608933B1 (ko) 2021-10-20 2021-10-20 전신 홍반성 루푸스 환자의 루푸스 신염 진단용 바이오마커 조성물 및 이를 이용한 루푸스 신염 진단에 필요한 정보를 제공하는 방법
KR10-2021-0140135 2021-10-20

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