KR102523964B1 - Biomarker for screening cardiovascular comorbidities in a psoriasis patient and use thereof - Google Patents

Abstract

As biomarkers for diagnosing cardiovascular comorbidities in psoriasis patients, PON3 (Serum paraoxonase/lactonase 3) and MENT (Protein MENT) and their uses are provided. According to this, it can be used to detect the possibility and prognosis of cardiovascular comorbidities in psoriasis patients objectively, quickly, with high accuracy and specificity.

Description

Biomarker for screening cardiovascular comorbidities in a psoriasis patient and use thereof}

It relates to a biomarker for diagnosing cardiovascular comorbidities in psoriasis patients, a composition and kit for diagnosing cardiovascular comorbidities in psoriasis patients using the same, and a method for providing information using the same.

Psoriasis, known as a representative chronic inflammatory disease of skin diseases, has a prevalence of around 1% in Asians including Korea and 1-3% worldwide. Clinically, erythematous plaques with silvery white scales can occur throughout the body, and they progress chronically with improvement and deterioration, requiring long-term management. In addition, the importance of psoriasis as a systemic inflammatory disease is emerging from a simple skin disease. It also plays an important role in cardiovascular diseases such as stroke.

Cardiovascular disease refers to diseases that occur in the heart and major arteries. Cardiovascular disease occurs when fibrosis occurs in the middle layer of blood vessels around the heart or cholesterol builds up on the walls of blood vessels, causing the inside of the blood vessels to gradually become narrower, causing blood circulation disorders and high blood pressure, and oxygen and nutrients are not properly delivered to various organs including the heart. known to occur. The cardiovascular disease is known as a disease with the second highest mortality rate after cancer.

In particular, vascular inflammation significantly increases in patients with severe psoriasis, and this vascular inflammation can cause various cardiovascular comorbidities, leading to a greater risk of major adverse cardiac events such as heart attack and stroke. can There is a continuous demand for the discovery of biomarkers that can predict cardiovascular events and rapidly diagnose cardiovascular comorbidities.

Therefore, there is a continuous demand for the discovery of biomarkers capable of predicting cardiovascular comorbidities in psoriasis patients.

One aspect provides a biomarker composition for diagnosing cardiovascular comorbidities in psoriasis patients.

Another aspect provides a composition for diagnosing cardiovascular comorbidities in psoriasis patients comprising an agent for measuring the expression level of the biomarker.

Another aspect provides a kit for diagnosing cardiovascular comorbidities in a psoriasis patient comprising the diagnostic composition.

Another aspect provides an information providing method for diagnosing cardiovascular comorbidities in a psoriasis patient comprising measuring the expression level of the biomarker.

One aspect is a biomarker composition for diagnosing cardiovascular comorbidities in psoriasis patients, comprising at least one protein selected from the group consisting of PON3 (Serum paraoxonase/lactonase 3) and MENT (Protein MENT), a fragment thereof, or a gene encoding the protein is to provide

As used herein, the term "psoriasis" refers to a disease characterized by the presence of erythema, which is a symptom of reddening of the skin, and scaling, which is a symptom of white dead skin cells, on the thickened skin together. Psoriasis is a chronic skin disease, and the cause of the disease may be an immune disorder. The lesion of psoriasis may be plaque psoriasis, intertriginous psoriasis, lacrimal psoriasis, pustular psoriasis, psoriatic erythroderma, or a combination thereof. The psoriasis may accompany psoriatic arthritis. In the case of psoriasis, the incidence of cardiovascular diseases such as metabolic syndrome, acute myocardial infarction, and stroke may be higher than that of the general public.

The psoriasis patient refers to an individual having a psoriasis lesion. The subject may be a mammal, such as a human, cow, horse, pig, dog, sheep, goat, or cat. The subject may be a subject suffering from psoriasis or suspected of having psoriasis.

As used herein, the term "cardiovascular comorbidity" refers to a chronic disease occurring in the heart and major arteries, coexisting with or found with other diseases. The cardiovascular comorbidity refers to a wider range of cardiovascular diseases, and includes general cardiovascular diseases and cardiovascular risk factors statistically related to cardiovascular diseases. Cardiovascular disease is rapidly increasing as the dietary habits of Korea become westernized and the incidence of chronic diseases such as hypertension and obesity rapidly increases. The cardiovascular comorbidity may include hypertension, dyslipidemia, ischemic heart disease, coronary artery disease, angina pectoris, myocardial infarction, arteriosclerosis, atherosclerosis, cerebrovascular disease, stroke, arrhythmia, or a combination thereof.

As used herein, the term "biomarker" refers to organic biomolecules such as polypeptides, proteins, nucleic acids, genes, lipids, glycolipids, glycoproteins, sugars, etc. All inclusive.

As used herein, the term "diagnosis" means to identify or predict the presence or characteristics of a pathological condition, to determine a subject's susceptibility to a specific disease or condition, to determine whether a subject is currently suffering from a particular disease or condition. to determine whether a subject has a particular disease or condition, to determine the prognosis of a subject with a particular disease or condition, or to use therametrics (eg, monitoring a subject's condition to provide information about the efficacy of a treatment). include

The biomarker composition is capable of diagnosing cardiovascular comorbidities in psoriasis patients, and may specifically mean determining/predicting the possibility of developing cardiovascular comorbidities in psoriasis patients or predicting prognosis.

Serum paraoxonase/lactonase 3 (PON3) rapidly hydrolyzes lactones such as statin prodrugs (e.g. lovastatin) and has low activity towards organophosphate parasones and aromatic carboxylic acid esters. In addition, aromatic lactones and 5- or 6-membered ring lactones are hydrolyzed with aliphatic substituents, but simple lactones or lactones with polar substituents are not hydrolyzed. PON3 may be encoded by the PON3 gene. PON3 may include the amino acid sequence of Uniprot No. Q15166 in humans.

MENT (Protein MENT) is a protein involved in cell proliferation regulation and is an oncogenic regulator that contributes to the tumor suppression function of DNMT3B. MENT can be encoded by the MENT gene. MENT may include the amino acid sequence of Uniprot No. Q9BUN1 in humans.

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

The gene refers to a nucleic acid encoding any one selected from the group consisting of PON3 and MENT.

Another aspect is to provide a composition for diagnosing cardiovascular comorbidities in patients with psoriasis, including an agent for measuring the expression level of at least one protein selected from the group consisting of PON3 and MENT, a fragment thereof, or a gene encoding the protein. The same parts as described above also apply to the composition.

As used herein, the term "measurement of expression level" means measuring whether or not the expression level of a specific protein (peptide) or a fragment thereof, or a gene encoding the protein. 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 expression level of the gene may be the expression level of mRNA encoding the proteins. The expression level of mRNA can be a relative amount or an absolute amount of mRNA. Measuring the expression level of the gene may be measuring the amount of mRNA.

Methods for measuring the expression level of the protein include Western blotting, enzyme linked immunosorbent assay (ELISA), radioimmunoassay (RIA), radical immunodiffusion, and Okteroni immunodiffusion method ( Ouchterlony immunodiffusion), rocket immunoeletrophoresis, immunohistochemical staining, immunoprecipitation assay, complement fixation assay, immunofluorescence, immunochromatography ( immunochromatography), FACS analysis (fluorescence activated cell sorter analysis) or protein chip method (protein chip technology).

The method of measuring the expression level of the mRNA or gene is reverse transcriptase polymerization (RT-PCR), competitive reverse transcriptase polymerase reaction (competitive RT-PCR), real-time reverse transcriptase polymerase reaction (real time quantitative RT-PCR), It may be quantitative polymerase reaction (quantitative RT-PCR), RNase protection method, Northern blotting, or DNA chip technology.

As used herein, the term "agent for measuring the expression level" refers to a molecule that can be used to determine the expression level of a specific protein or a gene encoding the same, specifically detecting and/or amplifying the protein or the gene. It may be one containing an agent that can be.

The agent may be an antibody or antigen-binding fragment thereof, or an aptamer that specifically binds to the protein or fragment thereof. 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 containing an antigen-binding site. The antigen-binding fragment may be a single-domain antibody, Fab, F(ab'), F(ab') ₂ or Fv. The antibody or antigen-binding fragment may be attached to a solid support. The solid support is, for example, a metal chip, plate, or the surface of a well. The aptamer may be a single-stranded nucleic acid (DNA, RNA or modified nucleic acid) or a 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 acids may be primers, probes, or antisense oligonucleotides. The primer, probe, or antisense oligonucleotide may be labeled with a fluorescent material, a chemiluminescent material, or a radioactive isotope at the end or inside thereof.

As used herein, the term "primer" is a nucleic acid sequence having a free 3' hydroxyl group, capable of forming base pairs with a template complementary to a specific base sequence, and copying the template strand. refers to a nucleic acid sequence that serves as a starting point for Primers can initiate DNA synthesis in the presence of a reagent for polymerization (i.e., DNA polymerase or reverse transcriptase) and four different nucleoside triphosphates in an appropriate buffer and temperature. For example, PCR amplification is performed using sense and antisense primers having 7 to 50 nucleotide sequences as specific primers for the mRNA of the gene encoding the biomarker of the present invention to measure the amount of production of the desired product Psoriasis It is possible to predict the possibility of cardiovascular comorbidity of the affected individual. PCR conditions and lengths of sense and antisense primers can be appropriately selected according to techniques known in the art. The primer is 10 to 100, 15 to 100, 10 to 80, 10 to 50, 10 to 30, 10 to 20, 15 to 80, 15 to 50, 15 to 30, 15 to 20, 20 to 100, 20 to 80 , 20 to 50, or 20 to 30 nt.

As used herein, the term "probe" means a nucleic acid fragment such as RNA or DNA capable of specifically binding to a target nucleic acid, for example, mRNA, and confirms the presence, content, and expression level of a specific mRNA. It may be labeled so that it can be The probe may be manufactured in the form of an oligonucleotide probe, a single strand DNA probe, a double strand DNA probe, an RNA probe, or the like. For example, by performing hybridization using a probe having a nucleic acid sequence complementary to mRNA of a gene encoding the biomarker of the present invention, and measuring the expression level of mRNA through the degree of hybridization, cardiovascular comorbidities in individuals with psoriasis. probability of occurrence can be predicted. Selection of an appropriate probe and hybridization conditions can be appropriately selected according to techniques known in the art. 10 to 100, 15 to 100, 10 to 80, 10 to 50, 10 to 30, 10 to 20, 15 to 80, 15 to 50, 15 to 30, 15 to 20, 20 to 100, 20 to 80 , 20 to 50, or 20 to 30 nt.

In addition, the primer or probe may be chemically synthesized using a phosphoramidite solid support synthesis method or other well-known methods. In addition, such nucleic acid sequences can be modified through various methods known in the art. Examples of such modifications are methylation, capping, substitution of one or more homologs of the natural nucleotide, or modifications between nucleotides, e.g., uncharged linkages (e.g., methyl phosphonates, phosphotriesters, phosphoroamidates, carbamates). etc.) or charged linkages (eg phosphorothioates, phosphorodithioates, etc.). Primers or probes can also be modified with labels that can directly or indirectly provide a detectable signal. Examples of the label may include a radioactive isotope, a fluorescent molecule, or biotin.

Another aspect is to provide a kit for diagnosing cardiovascular comorbidities in psoriasis patients, including the composition for diagnosing cardiovascular comorbidities in psoriasis patients. The same parts as described above also apply to the kit.

The kit has an effect of diagnosing or predicting the possibility and prognosis of cardiovascular comorbidities in psoriasis patients by measuring the expression level of the biomarker. The kit may further include samples necessary for predicting the onset of cardiovascular comorbidities in patients with psoriasis. The kit may include a solid support, a substrate for immunological detection of the antibody or antigen-binding fragment, a suitable buffer, a chromogenic enzyme, a fluorescently labeled secondary antibody, or a chromogenic substrate. The kit may include a polymerase, a buffer, a nucleic acid, a coenzyme, a fluorescent substance, or a combination thereof to detect nucleic acids. The polymerase is, for example, Taq polymerase.

Another aspect is to treat cardiovascular comorbidities in patients with psoriasis, comprising measuring the expression level of at least one protein selected from the group consisting of PON3 and MENT, a fragment thereof, or a gene encoding the protein, from a biological sample isolated from the subject. It is to provide a method of providing information for diagnosis. The same parts as described above are also applied to the method.

As used herein, the term "individual" refers to all organisms that have or are likely to develop cardiovascular comorbidities, and specific examples include mammals such as humans, cows, horses, pigs, dogs, sheep, goats, Or it could be a cat.

The biological sample refers to a sample obtained from the subject. The biological sample may be, for example, blood, plasma, serum, bone marrow fluid, lymph fluid, saliva, tear fluid, mucosal fluid, amniotic fluid, or a combination thereof. The biological sample includes a protein sample or a nucleic acid sample obtained from the sample.

Measuring the expression level may include incubating the biological sample with an antibody or antigen-binding fragment thereof that specifically binds to the protein or fragment thereof, or an aptamer. The measuring step is performed by electrophoresis, immunoblotting, enzyme-linked immunosorbent assay (ELISA), protein chip, immunoprecipitation, microarray, electron microscopy, or a combination thereof. It can be. The electrophoresis may be SDS-PAGE, isoelectric focusing, two-dimensional electrophoresis, or a combination thereof.

Measuring the expression level may include incubating the biological sample with a polynucleotide identical to or complementary to a polynucleotide encoding the protein or fragment thereof. The measuring step may be performed by Northern blotting or polymerase chain reaction (PCR). The PCR may be real-time PCR or reverse transcription PCR.

The method may further include comparing the measured expression level of the subject with that of a control group.

The control group may be an individual who does not have cardiovascular risk factors or cardiovascular comorbidities in patients with psoriasis.

The method may further include determining that the subject has a cardiovascular comorbidity or predicting a high risk of developing the subject when the expression level measured in the subject is reduced compared to the expression level of the control group. there is.

Among psoriasis patients, life therapy or drug treatment may be performed on individuals who are predicted to have a high probability of developing cardiovascular comorbidities. The lifestyle therapy may be maintenance of a normal weight, regular exercise, smoking cessation, abstinence from alcohol, restriction of salt intake, low-fat diet, or a combination thereof. These drugs include angiotensinconverting enzyme (ACE) inhibitors, angiotensin II receptor blockers, beta blockers, calcium channel blockers, diuretics, and alpha blockers. (alpha blocker), alpha-beta blocker, vasodilator (vasodilator), HMG-CoA reductase inhibitor (statin), bile acid binding resin, fibric acid and its derivatives, nicotinic acid and its derivatives, omega -3, or a combination thereof.

According to the composition and kit for diagnosing cardiovascular comorbidities in psoriasis patients according to one aspect, and a method for providing information for diagnosing cardiovascular comorbidities in psoriasis patients using the same, the occurrence and prognosis of cardiovascular comorbidities in psoriasis patients are objectively determined. , and can be used to detect rapidly, with high accuracy and specificity.

1 is a diagram showing the results of liquid chromatography analysis of a depleted plasma sample (x axis: time (minutes), y axis: mass absorbance unit (mAU)).
2 is a diagram showing a process of analyzing a serum sample in the present invention.
3 is a diagram showing a heatmap obtained through hierarchical clustering analysis of proteins that are differentially expressed more than twice in psoriasis patients and psoriasis patients with cardiovascular comorbidities.

It will be described in more detail through the following examples. However, these examples are for illustrative purposes only and the scope of the present invention is not limited to these examples.

Example 1: Sample collection

Serum samples were collected from 7 psoriasis patients with cardiovascular comorbidities such as hypertension and dyslipidemia and 4 psoriasis patients without cardiovascular comorbidities from Hanyang University. Serum samples from 3 matched healthy adults were obtained as negative controls. In order to prevent proteolysis or modification of the serum sample, complete, Mini EDTA-free protease inhibitor cocktail (Roche) and PhosSTOP (Roche) as a phosphatase inhibitor were added to the serum obtained immediately after the serum was obtained. did The inhibitor was dissolved in 200 μl of phosphate buffered saline (PBS) according to the manufacturer's protocol to make 50X, and then added to each sample to a final concentration of 1.5X. Serum samples to which inhibitors were added were quantified and stored at -80°C until use in experiments.

Example 2: Depletion of Serum Samples

In order to remove proteins present in large amounts in serum, such as albumin and immunoglobulin G, the obtained serum samples were depleted.

Specifically, 10 mg of each serum sample obtained in Example 1 was prepared according to the quantitative value for each sample, and the experiment was performed under the same conditions. The prepared serum sample was diluted 6-fold with the buffer provided by the manufacturer, and the diluted sample was filtered through a 0.22 μm spin filter. 100 μl of the filtered sample was injected into a Multiple Affinity Removal System (MARS) 14 column (Agilent), and high-performance liquid chromatography was performed according to the manufacturer's manual to deplete the sample. 14 abundant proteins (albumin, immunoglobulin G, alpha-1-antitrypsin, immunoglobulin A, transferrin, haptoglobin, fibrinogen, alpha-2-macroglobulin, alpha-1-acidic glycoprotein, immune Globulin M, apolipoprotein A-I, apolipoprotein A-II, complement protein C3, and transthyretin) were removed from the filtrate, respectively. In order to confirm whether the amount of protein in the filtrate collected for each serum sample was similar, the UV absorbance was measured in real time for the obtained filtrate, and the results of the reduced serum sample in one of the samples are shown in FIG. 1 ( Green line: indicates the point at which filtrate collection begins Red line: indicates the point at which filtrate collection ends).

Example 3: Concentration and quantification of depleted serum samples

Filters were prepared by adding 400 μl of water (HPLC grade, J.T Baker) twice and 400 μl of 10 mM HEPES buffer (pH 8.0) twice to a 3k centrifugal filter (Amicon Ultra, Millipore).

에서 10분 동안 원심분리하여 시료를 농축하였다. 그 후 시료가 포함된 필터를 10 mM HEPES 완충액으로 5회 세척하였다. 새로운 튜브에 세척 후 농축된 시료가 함유된 필터를 뒤집어서 끼운 후, 3000x g의 속도로 10℃에서 2분 동안 원심분리하여 농축된 시료를 회수(recovery)하였다.The serum sample depleted in Example 2 was diluted by adding 1.5-fold volume of 10 mM HEPES buffer. After adding the diluted sample to the prepared filter, 10

Samples were concentrated by centrifugation for 10 min. Thereafter, the filter containing the sample was washed 5 times with 10 mM HEPES buffer. After washing in a new tube, the filter containing the concentrated sample was turned upside down and inserted, and the concentrated sample was recovered by centrifugation at 10 ° C. for 2 minutes at a speed of 3000x g.

The collected samples were quantified by bicinchoninic acid (BCA) analysis.

Example 4: Sample preparation for mass spectrometry and in-solution digestion

For proteomic analysis, 300 μg of each sample obtained in Example 3 was digested in a solution state. Specifically, a 300ug sample was prepared and the volume of the sample was diluted to 100ul using 25mM ammonium bicarbonate. Using urea (UREA) powder, it was added to the sample so as to become 8M urea (UREA). The disulfide bonds were reduced at 37°C with 0.5 μmol of tris(2-carboxyethyl)phosphine (TCEP), and then alkylated with 1 μmol of 2-Iodoacetamide (IAA) at 25°C in the dark. After that, 8M urea (UREA) was further added with 25mM ammonium bicarbonate to make 1M urea (UREA). LysC, an endoprotease, was added and digested at 30°C for 2 hours, followed by digestion with trypsin at 37°C overnight. Then, it was purified using a SepPak® tC18 cartridge (Sep-Pak tC18 1cc Vac Cartrige, 100 mg, Waters) and dried by vacuum drying with a SpeedVac. After dissolving the rolled peptide in 100 mM tetraethylammonium bromide (TEAB), the peptide was quantified.

Example 5: Tandem Mass Tag (TMT) labeling of peptide samples

TMT labeling reagent (Thermo Fisher Scientific) was dissolved in 60 μl of acetonitrile, and 30 μl of TMT labeling reagent was added to 30 μg of peptide sample in each of the samples prepared in Example 4. The reaction was incubated at 20°C for 1 hour to label the peptide, and then 5 µl of 5% (w/v) hydroxylamine was added and incubated at 20°C for 15 minutes to terminate the labeling reaction. TMT-labeled samples were combined into one tube, purified with a SepPak® tC18 cartridge and vacuum dried using a SpeedVac.

The dried sample was dissolved in 10 mM ammonium formate, and Pierce?? Peptides were fractionated into 96 fractions using a High pH Reversed-phase Peptide Fractionation Kit. After combining 96 fractions into 24 fractions, the fractions were vacuum dried using a SpeedVac (FIG. 2).

Example 6: Peptide Mass Spectral Analysis

3 μl of each sample obtained was injected onto a reverse phase PepMap RSLC C18 column (50 cm×75 μm) on an Ultimate 3000 system. The column was equilibrated with 100% Buffer A (100% water containing 0.1% (v/v) formic acid). Buffer B is 100% acetonitrile containing 0.1% (v/v) formic acid. The working flow rate was 300 nL/min with the following gradient conditions:

- 0 min: 95% Buffer A and 5% Buffer B

- 0 to 4 minutes: 5% Buffer B

- 4 min to 13 min: 5% to 10% Buffer B

- 13 min to 150 min: 10% to 25% Buffer B

- 150 min to 155 min: 25% to 28% Buffer B

- 155 to 160 minutes: 28% to 40% Buffer B

- 160 to 165 minutes: 40% to 80% Buffer B

- 165 min to 170 min: 80% Buffer B

- 170 min to 170.1 min: 80% to 5% Buffer B

- 170.1 min to 180 min: 5% Buffer B

The Nano LC system was mounted on a Tribrid Orbitrap Eclipse. A full-scan MS spectra (350 ~ 2,000 m/z) was obtained with 1 microscan and a resolution of 120,000 to select a precursor and measure the charge state. set the preview mode for MS/MS spectra for the 20 most intense ions from the preview survey scan were obtained in the ion trap for the full scan with the following options:

- isolation width, 0.5 m/z; CID collision energy, 35%; Dynamic exclusion duration, 30 seconds (sec).

Thereafter, fragment ions were separated from the MS/MS spectra under the following conditions. MS/MS spectra (Survey full-scan MS spectra) are obtained with 1 microscan, and MS3 spectra are performed for up to 10 fragment ions including intact mass tags by SPC (synchronous precursor selection) method Obtained with options such as:

- isolation width, 0.4 m/z; HCD collision energy, 55%, scan range (m/z), 100-1000.

Example 7: Data processing for protein identification and label-based quantitative analysis

Each LC-MS/MS file was analyzed using the SEQUEST algorithm of Proteome Discoverer 2.4. MS and MS/MS data were compared against the SwissProt human database (containing 20376 proteins, updated May 2020). The investigation was limited to allowing two missed cleavages tryptic peptides. Carboamidomethylation of cysteine (+57.021 Da) and TMT labeling of lysine and peptide N-terminus (+304.207 Da) fixed modification, methionine oxidation by fluid modification (+15.995 Da) set up Mass tolerance was set at 0.6 Da for MS/MS data and 10 ppm for MS data. For the decoy database search, two target values were applied: a strict False Discovery Rate (FDR) of 0.01 and a relaxed FDR of 0.05.

The relative amounts of proteins present in the three groups of psoriasis patient samples with cardiovascular comorbidity (7), psoriasis patient samples without cardiovascular comorbidity (4), and negative control samples (3) were derived from each sample. It was calculated based on the signal to noise value of the labeled peptide. Signal-to-noise values of reporter ions of each peptide were extracted using Proteome Discoverer 2.4. In order to find statistically significant proteins, an ANOVA test was performed on the abundance values based on signal-to-noise for three groups (psoriasis with cardiovascular comorbidity, psoriasis without comorbidity, and normal control group) (IBM SPSS Statistics 25.0 software). Afterwards, statistical significance was confirmed between each group through Scheffe's post hoc test, and a p-value of less than 0.05 was considered statistically significant.

Example 8: Identification of proteins differentially expressed in psoriasis patients with psoriasis and cardiovascular comorbidities

As described in Example 7, from the results of the identification of 3 groups of proteins present in psoriasis patients with cardiovascular comorbidities, psoriasis patients without comorbidities, and normal control samples, a total of 1079 proteins were identified as unique peptide 2 It was confirmed that more than one was identified. Among the identified proteins, 1041 proteins had signal-to-noise values that could be quantitatively analyzed based on labeling. ANOVA test for 3 groups (psoriasis with cardiovascular comorbidity, psoriasis without comorbidity, negative control) for abundance values based on signal-to-noise values for 1041 proteins to find statistically significant proteins. , it was possible to obtain 184 proteins with a p-value of less than 0.05, and a post-hoc test using the Scheffe method for comparison between each group revealed a statistical difference in expression level between the psoriasis group with and without cardiovascular comorbidities. Table 1 shows 142 significant proteins.

UniProt Accession No. protein name ANOVA p-value Q92854 Semaphorin-4D 1.65E-06 O00754 Lysosomal alpha-mannosidase 6.72E-06 P08514 Integrin alpha-IIb 1.16E-05 P07996 Thrombospondin-1 5.05E-05 O60234 Glia maturation factor gamma 5.45E-05 Q9HCN6 Platelet glycoprotein VI 6.16E-05 P21926 CD9 antigen 1.20E-04 Q6ZRP7 Sulfhydryl oxidase 2 1.25E-04 Q5SQ64 Lymphocyte antigen 6 complex locus protein G6f 1.41E-04 P16278 Beta-galactosidase 1.52E-04 P08648 Integrin alpha-5 1.54E-04 Q15942 Zyxin 1.77E-04 Q86YW5 Trem-like transcript 1 protein 2.15E-04 P16109 P-selectin 2.22E-04 P35590 Tyrosine-protein kinase receptor Tie-1 2.54E-04 P29122 Proprotein convertase subtilisin/kexin type 6 2.55E-04 Q8NI99 Angiopoietin-related protein 6 3.24E-04 P01008 Antithrombin-III 3.68E-04 Q14847 LIM and SH3 domain protein 1 3.71E-04 P02671 Fibrinogen alpha chain 5.40E-04 P30044 Peroxiredoxin-5, mitochondrial 6.09E-04 Q9UJU6 Drebrin-like protein 6.09E-04 P46109 Crk-like protein 6.38E-04 P40197 Platelet glycoprotein V 8.43E-04 P31431 Syndecan-4 9.26E-04 P05106 Integrin beta-3 9.58E-04 Q05682 Caldesmon 1.01E-03 P08581 Hepatocyte growth factor receptor 1.12E-03 P04275 von Willebrand factor 1.13E-03 Q9Y251 Heparanase 1.17E-03 P54727 UV excision repair protein RAD23 homolog B 1.50E-03 Q06481 Amyloid-like protein 2 1.56E-03 Q9NRW1 Ras-related protein Rab-6B 1.66E-03 Q9H1B5 Xylosyltransferase 2 1.79E-03 P06702 Protein S100-A9 1.96E-03 P10619 Lysosomal protective proteins 1.99E-03 P23528 Cofilin-1 2.05E-03 Q15166 Serum paraoxonase/lactonase 3 2.18E-03 P58215 Lysyl oxidase homolog 3 2.21E-03 Q15555 Microtubule-associated protein RP/EB family member 2 2.40E-03 P10586 Receptor-type tyrosine-protein phosphatase F 2.45E-03 Q9UKU6 Thyrotropin-releasing hormone-degrading ectoenzyme 2.57E-03 P21291 Cysteine and glycine-rich protein 1 2.58E-03 P04424 Argininosuccinate lyase 2.61E-03 P51149 Ras-related protein Rab-7a 2.62E-03 P19021 Peptidyl-glycine alpha-amidating monooxygenase 3.26E-03 P10599 Thioredoxin 3.30E-03 Q15389 Angiopoietin-1 3.88E-03 O43852 Calumenin 3.92E-03 Q14247 Src substrate cortactin 3.99E-03 Q02763 Angiopoietin-1 receptor 4.30E-03 Q99439 Calponin-2 4.64E-03 P62491 Ras-related protein Rab-11A 4.66E-03 O75563 Src kinase-associated phosphoprotein 2 5.29E-03 O95866 Megakaryocyte and platelet inhibitory receptor G6b 5.37E-03 Q76LX8 A disintegrin and metalloproteinase with thrombospondin motifs 13 5.40E-03 Q13201 Multimerin-1 5.53E-03 O75368 SH3 domain-binding glutamic acid-rich-like protein 5.57E-03 O43505 Beta-1,4-glucuronyltransferase 1 5.59E-03 P23381 Tryptophan--tRNA ligase, cytoplasmic 5.67E-03 P14543 Nidogen-1 5.99E-03 Q3ZCW2 Galectin-related proteins 6.25E-03 P33241 Lymphocyte-specific protein 1 6.38E-03 Q8IWL2 Pulmonary surfactant-associated protein A1 6.52E-03 O14773 Tripeptidyl-peptidase 1 6.53E-03 P06681 Complement C2 6.63E-03 P58546 Myotrophin 6.74E-03 Q9NZN3 EH domain-containing protein 3 7.07E-03 P30740 Leukocyte elastase inhibitor 7.31E-03 Q6YHK3 CD109 antigen 8.09E-03 P63241 Eukaryotic translation initiation factor 5A-1 8.42E-03 P50552 Vasodilator-stimulated phosphoprotein 8.69E-03 Q08380 Galectin-3-binding protein 8.71E-03 P29279 CCN family member 2 8.76E-03 P14618 Pyruvate kinase PKM 9.06E-03 Q14019 Coactosin-like protein 9.20E-03 O14960 Leukocyte cell-derived chemotaxin-2 9.27E-03 P80303 Nucleobidin-2 9.47E-03 O15117 FYN-binding protein 1 9.50E-03 P04278 Sex hormone-binding globulin 9.58E-03 Q08830 Fibrinogen-like protein 1 9.68E-03 O15335 Chondroadherin 9.73E-03 Q71U36 Tubulin alpha-1A chain 9.81E-03 P36871 Phosphoglucomutase-1 9.86E-03 Q9UJC5 SH3 domain-binding glutamic acid-rich-like protein 2 1.02E-02 Q9ULZ3 Apoptosis-associated speck-like protein containing a CARD 1.06E-02 Q9BYE9 Cadherin-related family member 2 1.09E-02 P23284 Peptidyl-prolyl cis-trans isomerase B 1.10E-02 P68036 Ubiquitin-conjugating enzyme E2 L3 1.18E-02 P37802 Transgelin-2 1.18E-02 P52907 F-actin-capping protein subunit alpha-1 1.21E-02 Q8WZA1 Protein O-linked-mannose beta-1,2-N-acetylglucosaminyltransferase 1 1.25E-02 P62993 Growth factor receptor-bound protein 2 1.26E-02 P52566 Rho GDP-dissociation inhibitor 2 1.28E-02 P10720 Platelet factor 4 variant 1.30E-02 P04004 Vitronectin 1.34E-02 P60660 Myosin light polypeptide 6 1.34E-02 P20160 Azurocidin 1.42E-02 P26641 Elongation factor 1-gamma 1.50E-02 O14950 Myosin regulatory light chain 12B 1.51E-02 P00568 Adenylate kinase isoenzyme 1 1.53E-02 P02766 Transthyretin 1.55E-02 P68366 Tubulin alpha-4A chain 1.60E-02 Q96PD5 N-acetylmuramoyl-L-alanine amidase 1.61E-02 P29350 Tyrosine-protein phosphatase non-receptor type 6 1.68E-02 P05109 Protein S100-A8 1.75E-02 O00194 Ras-related protein Rab-27B 1.83E-02 O43157 Plexin-B1 1.86E-02 P06733 Alpha-enolase 1.89E-02 Q9BYH1 Seizure 6-like protein 2.04E-02 O95810 Caveolae-associated protein 2 2.05E-02 P61960 Ubiquitin-fold modifier 1 2.07E-02 Q92896 Golgi apparatus protein 1 2.15E-02 P04080 Cystatin-B 2.18E-02 O00299 Chloride intracellular channel protein 1 2.19E-02 P05067 Amyloid-beta precursor protein 2.20E-02 Q9H4M9 EH domain-containing protein 1 2.25E-02 Q13822 Ectonucleotide pyrophosphatase/phosphodiesterase family member 2 2.27E-02 O00151 PDZ and LIM domain protein 1 2.47E-02 P06753 Tropomyosin alpha-3 chain 2.53E-02 Q9BUN1 Protein MENT 2.60E-02 P01019 Angiotensinogen 2.64E-02 Q13418 Integrin-linked protein kinases 2.69E-02 Q9Y6R7 IgGFc-binding protein 2.84E-02 Q86UX7 Fermitin family homolog 3 2.85E-02 P09493 Tropomyosin alpha-1 chain 2.86E-02 P30086 Phosphatidylethanolamine-binding protein 1 2.95E-02 P35579 Myosin-9 3.00E-02 Q8IWV2 Contactin-4 3.01E-02 P12111 Collagen alpha-3(VI) chain 3.06E-02 Q9Y490 Talin-1 3.30E-02 P28906 Hematopoietic progenitor cell antigen CD34 3.35E-02 P41218 Myeloid cell nuclear differentiation antigen 3.58E-02 P37840 Alpha-synuclein 3.64E-02 P60842 Eukaryotic initiation factor 4A-I 3.71E-02 P07437 Tubulin beta chain 3.72E-02 Q86U17 Serpin A11 3.77E-02 Q92859 Neogenin 3.89E-02 P07339 Cathepsin D 3.91E-02 Q00013 55 kDa erythrocyte membrane protein 3.99E-02 P67936 Tropomyosin alpha-4 chain 4.04E-02 Q9NY97 N-acetyllactosaminide beta-1,3-N-acetylglucosaminyltransferase 2 4.67E-02

Next, among 142 statistically significant proteins, proteins showing a differential expression of more than 2-fold in the psoriasis group with cardiovascular comorbidity were additionally selected. The results of hierarchical clustering analysis on the selected proteins are shown in FIG. 3 . As shown in FIG. 3, 97 proteins that were differentially expressed more than twice in the psoriasis group having cardiovascular comorbidity compared to psoriasis were identified.

Among 97 proteins, biomarkers that were expressed more than twice as low in the psoriasis group with cardiovascular comorbidity compared to the psoriasis group without cardiovascular comorbidity were identified and additionally selected, and the results are shown in Table 2.

UniProt Accession No. protein name ratio
(Psoriasis group with/without cardiovascular disease) p-value: Scheffe method (psoriasis group with/without cardiovascular comorbidities) Q15166 Serum paraoxonase
/lactonase 3 (PON3) 0.35 0.003 Q9BUN1 Protein MENT (MENT) 0.32 0.028

Therefore, it can be seen that the biomarkers listed in Table 2 are significant biomarkers predicting cardiovascular comorbidities in psoriasis patients.

The above description of the present invention is for illustrative purposes, and those skilled in the art can understand that it can be easily modified into other specific forms without changing the technical spirit or essential features of the present invention. will be. Therefore, the embodiments described above should be understood as illustrative in all respects and not limiting.

Claims (14)

A biomarker composition for diagnosing cardiovascular comorbidities in psoriasis patients, comprising Serum paraoxonase/lactonase 3 (PON3) protein, a fragment thereof, or a gene encoding the protein,
Wherein the cardiovascular comorbidity is hypertension, dyslipidemia, or a combination thereof. The method according to claim 1, wherein the composition further comprises a MENT (Protein MENT) protein, a fragment thereof, or a gene encoding the protein, the composition. A composition for diagnosing cardiovascular comorbidities in patients with psoriasis, comprising an agent for measuring the expression level of a PON3 protein, a fragment thereof, or a gene encoding the protein,
Wherein the cardiovascular comorbidity is hypertension, dyslipidemia, or a combination thereof. The method of claim 3, wherein the formulation
an antibody or antigen-binding fragment thereof, or an aptamer that specifically binds to the protein or fragment thereof; or
A composition that is a nucleic acid comprising a polynucleotide identical to or complementary to a polynucleotide encoding the protein or fragment thereof. The method according to claim 3, wherein the composition further comprises an agent for measuring the expression level of the MENT protein, a fragment thereof, or a gene encoding the protein. The composition of claim 4, wherein the nucleic acid is a primer, probe, or antisense oligonucleotide. A kit for diagnosing cardiovascular comorbidities in psoriasis patients, comprising the composition of any one of claims 3 to 6,
The cardiovascular comorbidity is hypertension, dyslipidemia, or a combination thereof, the kit. An information providing method for diagnosing cardiovascular comorbidities in psoriasis patients, comprising measuring the expression level of PON3 protein, a fragment thereof, or a gene encoding the protein from a biological sample isolated from the subject,
Wherein the cardiovascular comorbidity is hypertension, dyslipidemia, or a combination thereof. The method according to claim 8, wherein the method further comprises measuring the expression level of the MENT protein, a fragment thereof, or a gene encoding the protein. The method according to claim 8, wherein the step of measuring the expression level comprises incubating the biological sample with an antibody or antigen-binding fragment thereof that specifically binds to the protein or fragment thereof, or an aptamer. . The method according to claim 8, wherein the step of measuring the expression level comprises incubating the biological sample with a polynucleotide identical to or complementary to a polynucleotide encoding the protein or fragment thereof. The method according to claim 8, wherein the step of measuring the expression level is electrophoresis, immunoblotting, enzyme-linked immunosorbent assay (ELISA), protein chip, immunoprecipitation, microarray, electron microscopy (electron microscopy) ), or a combination thereof. The method according to claim 8 , wherein the method further comprises comparing the expression level of the measured subject to that of a control group. The method according to claim 13, wherein the method adds a step of determining that the subject has a cardiovascular comorbidity or predicting a high level of risk of developing the subject when the expression level measured in the subject is reduced compared to the expression level of the control group. To include, the method.

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