TW202101002A - Use of protein biomarker for diagnosing kawasaki disease - Google Patents

Abstract

A protein biomarker for diagnosing Kawasaki Disease is used to solve the problem of the time-consuming conventional method with poor specificity. The protein biomarker is selected from a group consisting of protein S100A8, protein S100A9, protein S100A12, peroxiredon-2, α-1 acid glycoprotein 1 and neutrophil defensing 1.

Description

Use of protein biomarkers to diagnose Kawasaki disease

The present invention relates to the use of a protein biomarker, especially the use of a protein biomarker to diagnose Kawasaki disease.

Kawasaki disease, also known as mucocutaneous lymphnode syndrome, is a disease of inflammation of blood vessels throughout the body. Kawasaki disease is particularly prevalent in Taiwan, Japan, South Korea and other Asian countries, and it is more common in children under 5 years of age.

Generally speaking, the diagnosis of Kawasaki disease depends on the clinical symptoms of the patient. When the patient has a fever that lasts for more than five days, and has red and swollen hands and feet, pleomorphic skin rash, conjunctival bleeding without secretions, chapped lips, or strawberry tongue , Kawasaki disease can be diagnosed when four of the five standard diagnostic criteria such as 1.5 cm enlarged lymph glands appear in the neck.

However, the above-mentioned clinical symptoms are similar to those of streptococcus, adenovirus, enterovirus, etc., and about 15% of patients with Kawasaki disease are patients with atypical Kawasaki disease (the same symptoms of persistent fever, but the above five standard diagnosis There are less than four items in the requirements) must be combined with cardiac ultrasound examination to be able to confirm the diagnosis, so there is a possibility of misdiagnosis, and the patient may miss the golden period of treatment, which may increase the risk of coronary artery aneurysms (coronary artery aneurysms) Risks can even lead to the death of patients. In view of this, it is indeed necessary to provide a protein biomarker that can be used to diagnose Kawasaki disease to solve the above problems.

In order to solve the above problems, the purpose of the present invention is to provide a protein biomarker for the diagnosis of Kawasaki disease.

The protein biomarker of the present invention is used for diagnosing Kawasaki disease. The protein biomarker is selected from S100A8 protein, S100A9 protein, S100A12 protein, protein peroxidase-2, α-1-acid glycoprotein 1, and neutrophil leukocyte Cell defensins 1 group consisting of.

Accordingly, the protein biomarker of the present invention is used for diagnosing Kawasaki disease. Through the use of the protein biomarker, whether the suspected patient is a Kawasaki disease patient can be quickly diagnosed with good diagnostic accuracy and sensitivity.

Among them, the protein biomarkers include S100A8 protein, S100A9 protein, S100A12 protein, protein peroxidase-2, α-1-acid glycoprotein 1, and neutrophil defensin 1, which can further improve the accuracy of diagnosis And sensitivity.

In order to make the above and other objectives, features and advantages of the present invention more comprehensible, the following describes the preferred embodiments of the present invention in conjunction with the accompanying drawings in detail as follows:

The "screening population" mentioned in the present invention refers to 104 patients from Kaohsiung Veterans General Hospital, including 28 patients who did not suffer from Kawasaki disease, and 76 patients who were diagnosed with Kawasaki disease. The "screening population" is used for the screening of protein biomarkers of the present invention.

The "clinical pathological data" mentioned in the present invention is established based on the clinical data of the "screening group", including whether the patient has fever and other related clinical symptoms.

The "protein expression profile" of the present invention is the result of analyzing the protein content in serum samples by mass spectrometer combined with enzyme immunoassay method, and is established by comparing the "clinical pathological data" of the present invention. It contains and Protein related to Kawasaki disease.

In an embodiment of the present invention, the protein biomarker system for diagnosing Kawasaki disease may include proteins related to Kawasaki disease in the protein expression profile. In detail, the protein biomarker can include protein products of six genes, including S100A8, S100A9, S100A12, PRDX2, ORM1, or DEFA1. The protein biomarker can also include any combination of the aforementioned protein products, where the S100A8 gene has the following The nucleotide sequence shown in SEQ ID NO: 1, and its protein product (ie, S100A8 protein (protein S100A8)) has the amino acid sequence shown in SEQ ID NO: 2; the S100A9 gene has the amino acid sequence shown in SEQ ID NO: 3 The nucleotide sequence shown, the protein product (ie, S100A9 protein (protein S100A9)) has the amino acid sequence shown in SEQ ID NO: 4; the S100A12 gene has the nucleoside shown in SEQ ID NO: 5 Acid sequence, its protein product (ie, S100A12 protein (protein S100A12)) has the amino acid sequence shown in SEQ ID NO: 6; the PRDX2 gene has the nucleotide sequence shown in SEQ ID NO: 7, its protein The product (that is, peroxiredon-2) has the amino acid sequence shown in SEQ ID NO: 8; the ORM1 gene has the nucleotide sequence shown in SEQ ID NO: 9, which The protein product (that is, α-1-acid glycoprotein 1) has the amino acid sequence shown in SEQ ID NO: 10; the DEFA1 gene has the nucleus shown in SEQ ID NO: 11 The amino acid sequence, and its protein product (ie, neutrophil defensin 1) has the amino acid sequence shown in SEQ ID NO: 12.

The expression level of the aforementioned protein biomarker is positively correlated with the diagnosis of Kawasaki disease. Therefore, a doctor or examiner can obtain a test specimen from a suspected patient and determine the performance of the protein biomarker in the test specimen The amount of the protein biomarker is compared with a reference value (for example, the expression amount of the protein biomarker in a normal sample). When the expression amount of the protein biomarker in the test sample is higher or lower than the reference value, It shows that the suspected patient is a Kawasaki disease patient.

In detail, the specimen to be tested may be a body fluid specimen, for example, a blood specimen, preferably a plasma specimen (excluding the blood specimen (Obtained from the blood cells) or a serum sample (obtained after removing the anticoagulant factors in the plasma sample). In this embodiment, the sample to be tested is the serum sample. By removing the blood cells and anticoagulant factors in the blood sample, it is possible to avoid receiving blood cells when measuring the expression level of the protein biomarker. And the interference of anticoagulant factors.

In addition, the doctor or the examiner can extract the total protein (total protein) in the test specimen, and then determine the expression level of the protein biomarker in the test specimen, for example, using enzyme-linked immunosorbent method (enzyme-linked immunosorbent assay). The linked immunosorbent assay, referred to as ELISA, is paired with an antibody specific to the protein biomarker to determine the expression level of the protein biomarker in the test specimen, and the protein standard product with a certain concentration provided by the ELISA reagent , As a quality control (internal control), the absolute expression of each protein biomarker is obtained after quantification. In addition, in addition to the above-mentioned enzyme-linked immunosorbent assay, doctors or inspectors can also detect proteins using Western blot assay, two dimensional electrophoresis, mass spectrometry, etc. Method to determine the expression level of the protein biomarker in the test specimen.

It is worth noting that when the protein biomarker is a single protein, the reference value can refer to the expression level of the protein biomarker in the normal specimen. Taking S100A8 protein as an example, doctors or examiners can determine S100A8 separately The expression level of the protein in the test specimen, and the expression level of the S100A8 protein in the normal specimen is measured, and then the two expression levels are compared. When the expression level of the S100A8 protein in the test specimen is higher At the time, it can be known that the suspected patient is a Kawasaki disease patient. Moreover, when the protein biomarker is a combination of several proteins, each protein can have a different weight coefficient, and the doctor or examiner can multiply the relative weight coefficient by the expression of each protein of the test specimen. The sum of several risk values (that is, the total value of the sample to be tested), and the sum of several risk values obtained by multiplying the expression of each protein in the normal sample and the relative weight coefficient (that is, , The total value of the normal specimen), when the total value of the test specimen is greater than the total value of the normal specimen, the doctor or examiner can know that the suspected patient is a Kawasaki disease patient.

In this embodiment, the SVM computer algorithm (support vector machine (libsvm package)) is used to make each protein have different weight coefficients. In detail, the expression level of each protein of the test subject from the screening population is used as the training cohort, and the weight coefficient of each protein is constructed using the conventional computer algorithm to construct a good The diagnostic panel (diagnostic panel).

In order to prove that the protein biomarker of the present invention can indeed be applied to the protein biomarker for diagnosing Kawasaki disease, and when the protein biomarker is used to diagnose Kawasaki disease, it has good diagnostic accuracy and sensitivity, so the following tests were performed:

(A) The protein product of a single gene

In this test, the enzyme-linked immunosorbent assay was used to measure the expression level of S100A8 protein in the test specimens from the verified population, and to compare patients who did not suffer from Kawasaki disease (ie, FC group) with patients who were diagnosed with Kawasaki disease (ie, KD group) expression level, the results are shown in Figure 1a, which are consistent with the analysis results of the protein expression level map. The S100A8 protein expression level of the test specimen from patients diagnosed with Kawasaki disease is higher than that from those who did not suffer from Kawasaki. The expression level of S100A8 protein in the test specimen of the patient with the disease.

In addition, the S100A8 protein expression level of the test specimen from the verification group is brought into the diagnostic module constructed by the screening group for judgment, and compared with the clinical pathological data, it can be learned The sensitivity of S100A8 protein expression as a protein biomarker is 65%, and the diagnostic accuracy is 93%.

Then, the expression level of S100A8 protein of each test specimen is measured, and compared with the clinical pathological data of the screening population, and then the receiver operating characteristic curve (receiver operating characteristic curve, ROC curve) of the S100A8 protein is drawn. , And calculate the area under the curve of ROC to be 0.76.

In addition, according to the same procedure described above, the expression level of S100A9 protein, the expression level of S100A12 protein, the expression level of protein peroxidase-2, the expression level of α-1-acid glycoprotein 1, and the defensin of neutrophil cells The expression level of 1 is the result of the discrimination of protein biomarkers. The difference in expression level of each protein is shown in Figures 1b to 1f. The S100A9 protein, S100A12 protein, and α- 1-Acid glycoprotein 1 and neutrophil defensin 1 have higher expression levels, while the test specimens from patients diagnosed with Kawasaki disease have lower expression levels of protein peroxidase-2. The analysis results of the protein expression profile are consistent.

After comparing with the clinical pathological data, the sensitivity and diagnostic accuracy are calculated, and the results are shown in Table 1.

Table 1. Discrimination results of each protein biomarker. gene Sensitivity (%) Diagnostic accuracy (%) S100A9 61% 92% S100A12 61% 88% PRDX2 89% 92% ORM1 65% 84% DEFA1 84% 90%

Then draw the operator acceptance characteristic curve of each protein, and calculate the area under the ROC curve, which are 0.69, 0.76, 0.90, 0.74, and 0.87, respectively, showing that the above five protein biomarkers have good predictive value.

(B) The combination of six proteins

This test is carried out according to the same procedure as above, but the protein biomarkers include S100A8 protein, S100A9 protein, S100A12 protein, protein peroxidase-2, α-1-acid glycoprotein 1, and neutrophil defensin The combination of 1, the result of comparison between the discriminant result after bringing into the diagnostic module and the clinical pathological data, the sensitivity is 94%, and the diagnostic accuracy is 86%.

Then, the operator who also drew the six protein combination acceptance characteristic curve (as shown in Figure 2), the area under the ROC curve was calculated to be 0.94, which shows that compared to a single protein, the six protein combination It has better predictive value when used as a protein biomarker.

In summary, the protein biomarker of the present invention is used for diagnosing Kawasaki disease. Through the use of the protein biomarker, whether the suspected patient is a Kawasaki disease patient can be quickly diagnosed with good diagnostic accuracy and sensitivity.

Although the present invention has been disclosed using the above-mentioned preferred embodiments, it is not intended to limit the present invention. Anyone who is familiar with the art without departing from the spirit and scope of the present invention may make various changes and modifications relative to the above-mentioned embodiments. The technical scope of the invention is protected. Therefore, the scope of protection of the invention shall be subject to the scope of the attached patent application.

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[Picture 1a] In the test (A), a histogram of the expression level of S100A8 protein between patients without Kawasaki disease and patients with Kawasaki disease diagnosed ( P <0.00001). [Picture 1b] In the test (A), a bar graph comparing the expression level of S100A9 protein between patients without Kawasaki disease and patients diagnosed with Kawasaki disease ( P <0.00001). [Picture 1c] In the test (A), a histogram of the expression level of S100A12 protein between patients without Kawasaki disease and patients with Kawasaki disease ( P <0.0001). [Picture 1d] In the test (A), the histogram of the expression level of protein peroxidase-2 between patients without Kawasaki disease and patients with Kawasaki disease diagnosed ( P <0.05). [Figure 1e] In the test (A), a histogram of the expression level of α-1-acid glycoprotein 1 between patients without Kawasaki disease and patients diagnosed with Kawasaki disease ( P <0.00001). [Figure 1f] In the test (A), a bar graph comparing the expression levels of defensin 1 in neutrophil cells between patients without Kawasaki disease and patients diagnosed with Kawasaki disease ( P <0.00001). [Picture 2] In test (B), a combination of S100A8 protein, S100A9 protein, S100A12 protein, protein peroxidase-2, α-1-acid glycoprotein 1, and neutrophil defensin 1 was used The operator accepts the characteristic curve when diagnosing Kawasaki disease.

Claims (2)

Use of a protein biomarker for diagnosing Kawasaki disease. The protein biomarker is selected from S100A8 protein, S100A9 protein, S100A12 protein, protein peroxidase-2, α-1-acid glycoprotein 1, and neutrophil cell defense A group consisting of element 1. The use of the protein biomarker described in item 1 of the scope of patent application for diagnosing Kawasaki disease, wherein the protein biomarker includes S100A8 protein, S100A9 protein, S100A12 protein, protein peroxidase-2, α-1-acid Glycoprotein 1 and neutrophil defensin 1.

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