KR20220067863A - Salivary biomarker for diagnosing xerostomia and use thereof - Google Patents

Abstract

Provided are a composition for diagnosing xerostomia, a kit, and a method for diagnosing xerostomia using the same. According to this, the present invention can be used to diagnose xerostomia easily, objectively, and with high accuracy and specificity. The composition for diagnosing xerostomia includes a formulation for measuring an expression level of a gene selected from a group including NUCB2, PDCD6IP, PTGR1, FAM3D, and ZG16B, or an expression level of a protein or a fragment thereof.

Description

Salivary biomarker for diagnosing xerostomia and use thereof for diagnosing dry mouth

To a salivary biomarker for diagnosing dry mouth, a composition and kit for diagnosing dry mouth using the same, and a method for detecting a biomarker using the same.

Salivary gland function is affected by several conditions. Dry mouth is a subjective feeling of dry mouth because there is no saliva in the mouth. When the function of the salivary glands decreases due to dry mouth and the function of lubricating or antibacterial action decreases, dental caries and periodontal disease may appear as secondary diseases in the oral cavity. Dry mouth disease is difficult to recognize in the early stage of decreased salivary function, and it is difficult to distinguish between temporary dry mouth and organic dry mouth. Geriatric dry mouth due to aging is also caused by organic salivary gland dysfunction. Dry mouth caused by salivary gland dysfunction is difficult to cure after the disease has progressed, and treatment focuses on alleviating symptoms rather than completely curing the disease.

Dry mouth can be caused by various causes, such as Sjogren's syndrome, anemia, diabetes, aging, drug use, and nervous system diseases. Recently, a method for diagnosing and treating Sjogren's syndrome by detecting a biomarker in a saliva sample of an individual having dry mouth has been developed (US Patent Publication No. 9,833,519B2). However, a biomarker for diagnosing dry mouth has not been developed.

Therefore, there is a need to develop a biomarker capable of diagnosing dry mouth disease quickly, objectively, simply and inexpensively, and with high diagnostic accuracy and specificity.

Provided is a composition for diagnosing dry mouth.

Provides a kit for diagnosing dry mouth.

A method for diagnosing dry mouth is provided.

One aspect is the expression level of a gene selected from the group consisting of NUCB2 (Nucleobindin-2), PDCD6IP (Programmed cell death 6-interacting protein), PTGR1 (Prostaglandin reductase 1), FAM3D, and ZG16B (Zymogen granule protein 16 homolog B); Or it provides a composition for diagnosing dry mouth disease comprising an agent for measuring the expression level of a protein or fragment thereof.

The term "xerostomia or dry mouth" refers to a symptom of dry mouth due to decreased salivation or various other causes. Dry mouth can also be referred to as salivary gland hypofunction. Dry mouth can be caused by Sjogren's syndrome, anemia, diabetes, nutritional deficiencies, aging, drug use (e.g., antihistamines, drugs that act on the nervous system, high blood pressure), nervous system disorders, mental disorders (e.g. depression), and radiation therapy. can be caused by When dry mouth is induced, it is difficult to swallow food due to the dryness itself, and you may feel discomfort that it is difficult to speak, the occurrence of tooth decay (dental caries) and tooth decay (periodontitis) increases and is easy to worsen, fungal infection in the mouth, It can cause tongue pain, bad breath, and taste abnormalities. Dry mouth can also affect the development of oral diseases such as oral ulcers. Dry mouth can be diagnosed by measurement of salivation (salivation volume at rest less than 0.1 mL/min), salivography, biopsy, or nuclear medicine examination. It can also be diagnosed through salivary gland biopsy, and the degree of regression of the salivary gland parenchyma in the salivary gland tissue can be diagnosed. In Sjögren's syndrome, the infiltration pattern of immune cells in the salivary gland tissue is measured as an evaluation of salivary gland dysfunction, and it can be classified into scores 0 to 6 according to the degree of infiltration of immune cells.

The composition may be for detection in a saliva sample. The expression level may be reduced compared to the expression level of the normal control.

NUCB2 (Nucleobindin-2) is a calcium-binding protein capable of performing a function in calcium homeostasis, and may also be referred to as HEL-S-109 or NEFA. The NUCB2 protein may be a protein encoded by a NUCB2 gene. NUCB2 may comprise the amino acid sequence of Uniprot number P80303 in humans. NUCB2 may comprise the amino acid sequence of Uniprot number P81117 in a mouse.

Programmed cell death 6-interacting protein (PDCD6IP) is a protein that functions in endocytosis or programmed cell death, and may also be referred to as AIP1, ALIX, DRIP4, or HP95. Overexpression of PDCD6IP can block apoptosis. PDCD6IP may comprise the amino acid sequence of Uniprot number Q8WUM4 in humans. PDCD6IP may comprise the amino acid sequence of Uniprot number Q9WU78 in the mouse.

PTGR1 (Prostaglandin reductase 1) is a NAD(P)H-dependent oxidoreductase involved in the metabolic inactivation of inflammatory and anti-inflammatory eicosanoids of prostaglandin, leukotriene and lipoxin. to be. PTGR1 is PRG-1, 15-oxoprostaglandin 13-reductase, dithioethione-inducible gene 1 protein, DIG-1, leukotriene B4 12-hydro It may also be called leukotriene B4 12-hydroxydehydrogenase (LTB4DH), or NAD(P)H-dependent alkenal/one oxidoreductase (NAD(P)H-dependent alkenal/one oxidoreductase). PTGR1 may comprise the amino acid sequence of Uniprot number Q14914 in humans. and the amino acid sequence of Uniprot No. P48758 in PTGR1 mice.

FAM3D may function in cytokine activity and may be responsible for negative regulation in glucagon secretion or insulin secretion. FAM3D may comprise the amino acid sequence of Uniprot number Q96BQ1 in humans. FAM3D may comprise the amino acid sequence of Uniprot number P97805 in a mouse.

Zymogen granule protein 16 homolog B (ZG16B) may play a role in carbohydrate binding or retinal homeostasis. ZG16B may also be referred to as EECP, HRPE773, JCLN2, PAUF, PRO1567, or LOC124220. ZG16B may comprise the amino acid sequence of Uniprot number Q96DA0 in humans. ZG16B may comprise the amino acid sequence of Uniprot number A0A0G2K7Y9 in rats.

The fragment is a part of the protein and may be an immunogenic polypeptide.

The gene refers to a nucleic acid encoding the protein. The expression level of the gene may be the expression level of mRNA encoding the protein. The expression level of mRNA may be a relative amount or an absolute amount of mRNA. Measuring the expression level of the gene may be measuring the amount of mRNA.

The expression level of the protein or fragment thereof may be a relative amount or an absolute amount of the protein or fragment thereof. Measuring the expression level of the protein or fragment thereof may be measuring the amount of the protein or fragment thereof.

The agent may be an antibody or antigen-binding fragment thereof that specifically binds to the protein or fragment thereof, or an aptamer. The antibody may be a polyclonal antibody or a monoclonal antibody. The term “antibody” may be used interchangeably with the term “immunoglobulin”. The antibody may be a polyclonal antibody or a monoclonal antibody. The antibody may be a full-length antibody. The antigen-binding fragment refers to a polypeptide comprising an antigen-binding site. The antigen-binding fragment may be a single-domain antibody, Fab, Fab', or scFv. The antibody or antigen-binding fragment may be attached to a solid support. The solid support is, for example, a metal chip, a plate, or the surface of a well. The antibody or antigen-binding fragment may be an antibody or antigen-binding fragment. The aptamer may be a single-stranded nucleic acid (DNA, RNA or modified nucleic acid) or peptide that specifically binds to the protein or fragment thereof.

The agent may be a nucleic acid comprising a polynucleotide identical to or complementary to a polynucleotide encoding the protein or fragment thereof. The nucleic acid may be a primer, a probe, or an antisense oligonucleotide. The primer, probe, or antisense oligonucleotide may be labeled with a fluorescent material, chemiluminescent material, or radioactive isotope at the end or inside thereof.

The composition is KV108 (Immunoglobulin kappa variable 1-8), GSLG1 (Golgi apparatus protein 1), FAM3B (Protein FAM3B), MUC21 (Mucin-21), RB11A (Ras-related protein Rab-11A), LYSC (Lysozyme C) , and CALL3 (Calmodulin-like protein 3) may further include an agent for measuring the expression level of a gene selected from the group consisting of, or the expression level of a protein or a fragment thereof.

KV108 (Immunoglobulin kappa variable 1-8) may be a variable region of an immunoglobulin light chain. KV108 may also be referred to as IGKV18, L9, or IGKV1-8. KV108 may comprise the amino acid sequence of Uniprot number A0A0C4DH67 in humans.

Golgi apparatus protein 1 (GSLG1) can bind to fibroblast growth factor and E-selectin. GSLG1 may also be referred to as CFR-1 (Cysteine-rich fibroblast growth factor receptor), ESL-1 (E-selectin ligand 1), or Golgi sialoglycoprotein MG-160. GSLG1 may comprise the amino acid sequence of Uniprot number Q92896 in humans. GSLG1 may comprise the amino acid sequence of Uniprot number Q61543 in a mouse.

Protein FAM3B (FAM3B) can induce apoptosis of alpha cells and beta cells in the pancreas. FAM3B may also be called Cytokine-like protein 2-21 or Pancreatic-derived factor (PANDER). FAM3B may comprise the amino acid sequence of Uniprot number P58499 in humans. FAM3B may comprise the amino acid sequence of Uniprot number Q9D309 in a mouse.

MUC21 (Mucin-21) may be a biomarker of lung cancer. MUC21 may also be referred to as Epiglycanin. MUC21 may comprise the amino acid sequence of Uniprot number Q5SSG8 in humans. MUC21 may comprise the amino acid sequence of Uniprot number D9N008 or A8QW50 in a mouse.

Ras-related protein Rab-11A (RB11A) may be a small GTPase Rab that regulates intracellular membrane migration. RB11A may also be referred to as Rab-11 or YL8. RB11A may comprise the amino acid sequence of Uniprot No. P62491 in humans. RB11A may comprise the amino acid sequence of Uniprot number P62492 in a mouse.

LYSC (Lysozyme C) is an enzyme that functions in hydrolysis and glycosylation. LYSC may also be referred to as 1,4-beta-N-acetylmuramidase C. LYSC may comprise the amino acid sequence of Uniprot number P61626 in humans. The LYSC may comprise the amino acid sequence of Uniprot number P17897 or P08905 in a mouse.

CALL3 (Calmodulin-like protein 3) can function as a specific light chain of myosin-10. CALL3 may also be referred to as CLP (CaM-like protein) or Calmodulin-related protein NB-1. CALL3 may comprise the amino acid sequence of Uniprot number P27482 in humans. CALL3 may comprise the amino acid sequence of Uniprot number in the mouse.

Another aspect provides a kit for diagnosing dry mouth comprising an agent for measuring the expression level of a gene selected from the group consisting of NUCB2, PDCD6IP, PTGR1, FAM3D, and ZG16B, or the expression level of a protein or fragment thereof.

NUCB2, PDCD6IP, PTGR1, FAM3D, ZG16B, and xerostomia are as described above.

The kit may further include a sample necessary for diagnosing dry mouth. The kit may include a solid support, a substrate for immunological detection of an antibody or antigen-binding fragment, a suitable buffer, a chromogenic enzyme, a secondary antibody labeled with a fluorescent material, or a chromogenic substrate. The kit may include a polymerase, a buffer, a nucleic acid, a coenzyme, a fluorescent material, or a combination thereof for nucleic acid detection. The polymerase is, for example, Taq polymerase.

The kit includes KV108 (Immunoglobulin kappa variable 1-8), GSLG1 (Golgi apparatus protein 1), FAM3B (Protein FAM3B), MUC2 (Mucin-21), RB11A (Ras-related protein Rab-11A), LYSC (Lysozyme C) , and CALL3 (Calmodulin-like protein 3) may further include an agent for measuring the expression level of a gene selected from the group consisting of, or the expression level of a protein or a fragment thereof.

Another aspect comprises the steps of measuring the expression level of a gene selected from the group consisting of NUCB2, PDCD6IP, PTGR1, FAM3D, and ZG16B, or the expression level of a protein or a fragment thereof in a saliva sample isolated from a subject suspected of dry mouth; comparing the measured expression level with the expression level of a normal control; And when the expression level is decreased compared to the expression level of the normal control, it provides a method of detecting a biomarker in order to provide information necessary for diagnosing dry mouth or dry mouth, comprising the step of determining dry mouth do.

NUCB2, PDCD6IP, PTGR1, FAM3D, ZG16B, and xerostomia are as described above.

The method comprises measuring the expression level of a gene selected from the group consisting of NUCB2, PDCD6IP, PTGR1, FAM3D, and ZG16B, or the expression level of a protein or a fragment thereof, in a saliva sample isolated from a subject suspected of dry mouth. .

The subject may be a mammal, such as a human, cow, horse, pig, dog, sheep, goat, or cat. The subject may be suffering from a dry mouth disorder or may be an individual suspected of suffering from dry mouth.

The saliva sample refers to a saliva sample obtained from the subject. The saliva sample may be a sample that has been subjected to pretreatment such as centrifugation and filtration. The saliva sample includes a protein sample or a nucleic acid sample obtained from the saliva sample.

The measuring may include incubating the saliva sample with an antibody or antigen-binding fragment thereof, or an aptamer that specifically binds to the protein or fragment thereof, or the same as or complementary to a polynucleotide encoding the gene. It may include incubating the specific polynucleotide.

The measuring step includes electrophoresis, immunoblotting, enzyme-linked immunosorbent assay (ELISA), polymerase chain reaction, Northern blotting, polymerase chain reaction (PCR). ), protein chip, immunoprecipitation, microarray, electron microscopy, or a combination thereof. The electrophoresis may be SDS-PAGE, isoelectric point electrophoresis, two-dimensional electrophoresis, or a combination thereof.

The method comprises comparing the measured expression level to the expression level of a normal control. The method may include determining whether the expression level of any one selected from the group consisting of NUCB2, PDCD6IP, PTGR1, FAM3D, and ZG16B is reduced compared to the expression level of a normal control. The normal control group is a group that does not have dry mouth and means a negative control group. In the method, the amount of the measured expression level of the saliva sample may be reduced compared to the expression level of the normal control. For example, when the amount of the detected biomarker is less than the amount of a normal control, the subject may be diagnosed as having or having a high probability of having dry mouth. Treatments such as oral care, control of drug administration, administration of saliva substitutes, administration of oral moisturizers, electrical stimulation of the oral cavity or acupuncture may be performed on an individual diagnosed with dry mouth.

The method is KV108 (Immunoglobulin kappa variable 1-8), GSLG1 (Golgi apparatus protein 1), FAM3B (Protein FAM3B), MUC2 (Mucin-21), RB11A (Ras-related protein Rab-11A), LYSC (Lysozyme C) The method may further include measuring the expression level of a gene selected from the group consisting of , and CALL3 (Calmodulin-like protein 3), or the expression level of a protein or a fragment thereof.

According to a composition and kit for diagnosing dry mouth disease, and a method for diagnosing dry mouth disease using the same according to an aspect, it can be used to diagnose dry mouth disease simply, objectively, with high accuracy and specificity.

1 is an SDS-PAGE gel image of saliva samples of a normal control group and a group of patients with dry mouth.
2 is a Venn diagram showing the number of proteins identified in a saliva sample and proteins having a p-value of less than 0.05 analyzed and statistically significant.
3a to 3e are graphs showing the expression levels of Nucleobindin-2, Programmed cell death 6-interacting protein, Prostaglandin reductase 1, Protein FAM3D, and Zymogen granule protein 16 homolog B according to the scores of the normal control group and the dry mouth patient group ( y-axis: fluorescence intensity, N: normal control, S1: score 1, S2: score 2, S345: score 3 or higher).

Hereinafter, it will be described in more detail through examples. However, these examples are for illustrative purposes of one or more embodiments, and the scope of the present invention is not limited to these examples.

Example 1. Identification of differentially expressed biomarkers in saliva samples of dry mouth patients

1. Preparation of saliva samples from dry mouth patients

Saliva samples were collected from a normal control group (n=3) and a patient diagnosed with dry mouth from Hanyang University. Dry mouth was diagnosed by evaluating salivary gland function in clinical examinations of salivary gland secretion and oral mucosa condition, and salivary gland scans. Salivary gland function was classified into a score of 1 to 5 depending on the condition of the salivary gland tissue and the infiltration of lymphocytes around the parenchyma of the salivary gland by performing a salivary gland biopsy. Saliva sampling was performed by classification into 3 groups of score 1, score 2, and score 3 to 5.

In order to prevent proteolysis or modification of the saliva sample, cOmplete as a protease inhibitor, Mini EDTA-free protease inhibitor cocktail (Roche) and PhosSTOP (Roche) as a phosphatase inhibitor were added to the saliva sample obtained immediately after the serum was obtained. added. The inhibitor was dissolved in 200 μl of phosphate buffered saline (PBS) according to the manufacturer's protocol to make 50 X, and then added to each sample so that the final concentration of the inhibitor was 1.5 X. After quantification of the saliva sample to which the inhibitor was added, it was stored at -80°C until used in the experiment.

2. Concentration and Quantification of Saliva Samples

A total of 12 saliva samples from 4 groups were concentrated using a 3k centrifugal filter (Amicon Ultra, Millipore). A filter was prepared by adding 400 μl of water (HPLC grade, J.T Baker) twice and 400 μl of 10 mM Tris buffer (pH 8.0) containing 0.1% (w/v) SDS twice to a 3k centrifugal filter. .

The saliva sample was diluted 2-fold with 10 mM Tris buffer containing 0.1% (w/v) SDS, and the precipitate was removed by vortexing. After adding the diluted sample to the prepared filter, the sample was concentrated by centrifugation at a speed of 14000x g at 10°C for about 10 minutes. After that, the filter containing the sample was washed 5 times with 10 mM Tris buffer containing 0.05% (w/v) SDS. After washing in a new tube, the filter containing the concentrated sample was turned upside down, and the concentrated sample was recovered by centrifugation at a speed of 3000x g at 10°C for about 2 minutes.

The recovered sample was quantified by bicinchoninic acid (BCA) analysis.

3. Preparation of samples for mass spectrometry and in-gel digestion

For proteome analysis, 55 μg of 12 individual samples from 4 groups obtained in 2. were separated according to molecular weight using NuPAGE gels (Bis-Tris gels from 4% to 12%, Invitrogen, Carlsbad, CA, USA). did The gel was stained with Coomassie Brilliant Blue R-250 (FIG. 1). The stained gel for 12 individual samples was divided into 12 slices, and each gel slice was transferred to a microcentrifuge tube. Proteins contained in each gel piece were reduced by disulfide bonds at 56°C, then alkylated in a dark environment at 25°C, and digested with trypsin overnight. Then, the peptide was extracted from a solution of 67% (v/v) acetonitrile (ACN)/5% (v/v) formic acid (FA) (Wako Pure Chemicals, Osaka, Japan). The extracted peptides were dried in Speedvac and dissolved in 20 μl of 0.4% (v/v) acetic acid.

4. Mass Spectrum Analysis

10 μl of each sample obtained in 3. was injected onto a reversed-phase Magic C18AQ column (15 cm X 75 μm) on an Eksigent MDLC system (Eksigent Technologies Dublin, CA, USA). The column was mixed with 95% Buffer A (100% (v/v) water containing 0.1% (v/v) formic acid) and 5% (v/v) Buffer B (0.1% (v/v) of 100% (v/v) ACN) containing formic acid) was equilibrated with a mixed buffer. The working flow rate was 0.35 μl/min (min) under the following gradient conditions:

100% Buffer A from 0 min to 5 min; 92% Buffer A and 8% Buffer B from 5 min to 85 min; 70% Buffer A and 30% Buffer B from 85 to 90 minutes; 30% Buffer A and 70% Buffer B from 90 min to 95 min; 30% Buffer A and 70% Buffer B from 95 to 100 minutes; and 98% Buffer A and 2% Buffer B from 100 to 110 minutes.

The nano HPLC system was mounted on an LTQ XL-Orbitrap mass spectrometer (Thermo Scientific, San Jose, CA, USA). The spray voltage was set at 2.5 kV, and the temperature of the heated capillary was set at 250°C. Survey full-scan MS spectra (300-1,800 m/z) were obtained with 1 microscan and a resolution of 60,000 to select precursors and charge states A preview mode for measurement was set. MS/MS spectra for the 10 most potent ions from the preview irradiation scan were obtained in the ion trap for the full scan with the following options: isolation width, 2 m/z (mass versus charge); normalized collision energy, 35%; dynamic exclusion duration, 360 seconds (sec). Lead materials with +1 charge and undetermined charge states were discarded during data-dependent acquisition.

Each LC-MS/MS file was analyzed using the Andromeda algorithm of MaxQunat1.5.8.3. MS and MS/MS data were compared against SwissProt human database (January 2019). The investigation was limited to accepting tryptic peptides with two missed cleavages. Carboamidomethylation of cysteine was set as a fixed modification (+57.021 Da) and methionine oxidation as a fluid modification (+15.995 Da). Mass tolerance was set at 0.5 Da for MS/MS data and 15 ppm for MS data. For decoy database research, two target values were applied: a strict false discovery rate (FDR) of 0.01 and a relaxed FDR of 0.05. All proteins were identified using two or more matching peptides.

5. Label-free quantitative analysis

Based on the label-free quantification intensity (LFQ intensity), the relative abundance of proteins in a total of 12 samples in which 3 saliva samples were collected from 3 groups of the normal control group and the dry mouth patients group calculated. The intensity was extracted using MaxQunat1.5.8.3. Then, the LFQ intensity for each protein was analyzed for relative quantitative analysis using Perseus. Even when no protein was present or below the detection limit, null values were compensated. In order to calculate the fold difference between the normal control group and the group of patients with dry mouth, the missing values were filled in by downshifting the width by 0.3 and 1.8 from the normal distribution. Statistical significance was performed on the LFQ intensity obtained from the ANOVA test with three separate samples of the normal control group and the patient group with dry mouth, and a p-value of less than 0.05 was considered statistically significant.

6. Identification of salivary proteins related to patient groups according to dry mouth and identification of differentially expressed proteins

From the LFQ intensity analysis, a total of 703 proteins were identified in 12 individual samples of the normal control group and the dry mouth group. Among the identified proteins, 51 proteins with a p-value of less than 0.05 were identified. Fig. 2 shows the results of analyzing the identified proteins and proteins having a p-value of less than 0.05 and showing the number as a Venn diagram.

In addition, Table 1 shows proteins that were statistically significant with a p-value of less than 0.05.

UniProt Accession No. gene name protein name p-value P04217 A1BG Alpha-1B-glycoprotein 8.1694E-07 P02763 A1AG1 Alpha-1-acid glycoprotein 1 0.0004 P27797 CALR Calreticulin 0.001 P40199 CEAM6 Carcinoembryonic antigen-related cell adhesion molecule 6 0.002 A0A0C4DH67 KV108 Immunoglobulin kappa variable 1-8 0.002 P01008 ANT3 Antithrombin-III 0.004 Q8TAX7 MUC7 Mucin-7 0.005 Q16778 H2B2E Histone H2B type 2-E 0.005 P04196 HRG Histidine-rich glycoprotein 0.005 Q14914 PTGR1 Prostaglandin reductase 1 0.007 P02751 FINC Fibronectin 0.007 O75131 CPNE3 Copine-3 0.008 P0DOX5 IGG1 Immunoglobulin gamma-1 heavy chain 0.009 P60842 IF4A1 Eukaryotic initiation factor 4A-I 0.009 P25705 ATPA ATP synthase subunit alpha, mitochondrial 0.009 Q92896 GSLG1 Golgi apparatus protein 1 0.012 P00747 PLMN Plasminogen 0.014 P58499 FAM3B Protein FAM3B 0.016 Q16610 ECM1 Extracellular matrix protein 1 0.016 P08133 ANXA6 Annexin A6 0.016 O15511 ARPC5 Actin-related protein 2/3 complex subunit 5 0.018 P01009 A1AT Alpha-1-antitrypsin 0.020 Q05315 LEG10 Galectin-10 0.021 A0A0A0MS15 HV349 Immunoglobulin heavy variable 3-49 0.022 Q96DA0 ZG16B Zymogen granule protein 16 homolog B 0.025 P20591 MX1 Interferon-induced GTP-binding protein Mx1 0.025 O75083 WDR1 WD repeat-containing protein 1 0.026 P30740 ILEU Leukocyte elastase inhibitor 0.028 Q99880 H2B1L Histone H2B type 1-L 0.028 Q5SSG8 MUC21 Mucin-21 0.030 P02765 FETUA Alpha-2-HS-glycoprotein 0.032 Q12792 TWF1 Twinfilin-1 0.033 P62491 RB11A Ras-related protein Rab-11A 0.035 P13473 LAMP2 Lysosome-associated membrane glycoprotein 2 0.036 Q8WUM4 PDC6I Programmed cell death 6-interacting protein 0.037 P62244 RS15A 40S ribosomal protein S15a 0.038 A0A0C4DH33 HV124 Immunoglobulin heavy variable 1-24 0.039 Q12913 PTPRJ Receptor-type tyrosine-protein phosphatase eta 0.039 P41218 MNDA Myeloid cell nuclear differentiation antigen 0.042 P14780 MMP9 Matrix metalloproteinase-9 0.042 P80188 NGAL Neutrophil gelatinase-associated lipocalin 0.042 P80303 NUCB2 Nucleobindin-2 0.043 P30046 DOPD D-dopachrome decarboxylase 0.043 Q9BWD1 THIC Acetyl-CoA acetyltransferase, cytosolic 0.043 Q92876 KLK6 Kallikrein-6 0.043 P16401 H15 Histone H1.5 0.043 P61626 LYSC Lysozyme C 0.044 P22894 MMP8 Neutrophil collagenase 0.047 Q96BQ1 FAM3D Protein FAM3D 0.048 Q15365 PCBP1 Poly(rC)-binding protein 1 0.049 P27482 CALL3 Calmodulin-like protein 3 0.049

Table 2 shows the proteins whose expression level decreased compared to the normal control, among the proteins with a p-value of less than 0.05, which were statistically significant.

UniProt Accession No. gene name protein name Differential expression ratio
(Score1/Normal group) Differential expression ratio
(Score 2/Normal group) Differential expression ratio
(Score 3 or higher/Normal group) P80303 NUCB2 Nucleobindin-2 0.38 0.06 0.11 Q8WUM4 PDC6I Programmed cell death 6-interacting protein 0.00 (detected only in the normal group) 0.00 (detected only in the normal group) 0.00 (detected only in the normal group) Q14914 PTGR1 Prostaglandin reductase 1 0.00 0.00 0.13 Q96BQ1 FAM3D Protein FAM3D 0.22 0.00 0.12 Q96DA0 ZG16B Zymogen granule protein 16 homolog B 0.87 0.34 0.53 A0A0C4DH67 KV108 Immunoglobulin kappa variable 1-8 0 (detected only in the normal group) 0
(detected only in normal group) 0
(detected only in normal group) Q92896 GSLG1 Golgi apparatus protein 1 0.46373 0 0.346157 P58499 FAM3B Protein FAM3B 0.580505 0 0 Q5SSG8 MUC21 Mucin-21 0.053745 0 0.239191 P62491 RB11A Ras-related protein Rab-11A 0.531124 0.802295 0.512411 P61626 LYSC Lysozyme C 0.577288 0.394294 0.332109 P27482 CALL3 Calmodulin-like protein 3 0.433622 0.859402 0.341424

Expression levels of the proteins listed in Table 2 according to the scores of the normal control group and the dry mouth disease patient group are shown in FIGS. 3A to 3E (N: normal control group, S1: score 1, S2: score 2, S345: score 3 or higher).

As shown in FIGS. 3A to 3E , Nucleobindin-2, Programmed cell death 6-interacting protein, Prostaglandin reductase 1, Protein FAM3D, and Zymogen granule protein 16 homolog B were significantly reduced in expression levels in xerostomia and xerostomia. It was confirmed that the expression level varies according to the score of

Claims (13)

the expression level of a gene selected from the group consisting of Nucleobindin-2 (NUCB2), Programmed cell death 6-interacting protein (PDCD6IP), Prostaglandin reductase 1 (PTGR1), FAM3D, and Zymogen granule protein 16 homolog B (ZG16B), or a protein or A composition for diagnosing dry mouth disease, comprising an agent for measuring the expression level of a fragment thereof. The composition according to claim 1, for detection in a saliva sample. The composition of claim 1, wherein the expression level is reduced compared to the expression level of the normal control. The method according to claim 1, wherein the agent is
an antibody or antigen-binding fragment thereof, or an aptamer that specifically binds to the protein or fragment thereof; or
The composition is a nucleic acid comprising a polynucleotide identical to or complementary to the polynucleotide encoding the protein or fragment thereof. The composition of claim 4, wherein the antibody is a polyclonal antibody or a monoclonal antibody. The composition of claim 4, wherein the nucleic acid is a primer, a probe, or an antisense oligonucleotide. The method according to claim 1, KV108 (Immunoglobulin kappa variable 1-8), GSLG1 (Golgi apparatus protein 1), FAM3B (Protein FAM3B), MUC21 (Mucin-21), RB11A (Ras-related protein Rab-11A), LYSC (Lysozyme) C), and CALL3 (Calmodulin-like protein 3), the composition further comprising an agent for measuring the expression level of a gene selected from the group consisting of, or the expression level of a protein or a fragment thereof. A kit for diagnosing dry mouth, comprising an agent for measuring the expression level of a gene selected from the group consisting of NUCB2, PDCD6IP, PTGR1, FAM3D, and ZG16B, or the expression level of a protein or fragment thereof. The method according to claim 8, KV108 (Immunoglobulin kappa variable 1-8), GSLG1 (Golgi apparatus protein 1), FAM3B (Protein FAM3B), MUC2 (Mucin-21), RB11A (Ras-related protein Rab-11A), LYSC (Lysozyme) C), and CALL3 (Calmodulin-like protein 3), the kit further comprising an agent for measuring the expression level of a gene selected from the group consisting of, or the expression level of a protein or a fragment thereof. Measuring the expression level of a gene selected from the group consisting of NUCB2, PDCD6IP, PTGR1, FAM3D, and ZG16B, or the expression level of a protein or a fragment thereof in a saliva sample isolated from a subject suspected of dry mouth;
comparing the measured expression level with the expression level of a normal control; and
When the expression level is decreased compared to the expression level of the normal control, comprising the step of determining dry mouth,
A method of detecting a biomarker to provide information necessary for the diagnosis of dry mouth. The method according to claim 10, wherein the measuring comprises incubating the saliva sample with an antibody or antigen-binding fragment thereof, or an aptamer that specifically binds to the protein or fragment thereof, or a polynucleotide encoding the gene and incubating the same or complementary polynucleotide thereto. The method according to claim 10, wherein the measuring step is electrophoresis, immunoblotting, enzyme-linked immunosorbent assay (ELISA), polymerase chain reaction, Northern blotting, polymerase amplification reaction ( polymerase chain reaction: PCR), protein chip, immunoprecipitation, microarray, electron microscopy, or a method that is performed by a combination thereof. The method according to claim 10, KV108 (Immunoglobulin kappa variable 1-8), GSLG1 (Golgi apparatus protein 1), FAM3B (Protein FAM3B), MUC2 (Mucin-21), RB11A (Ras-related protein Rab-11A), LYSC (Lysozyme) C), and CALL3 (Calmodulin-like protein 3), the method further comprising the step of measuring the expression level of a gene selected from the group consisting of, or the expression level of a protein or a fragment thereof.

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