CN113667739B - Diagnostic marker combinations for coronary artery disease - Google Patents

Diagnostic marker combinations for coronary artery disease Download PDF

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CN113667739B
CN113667739B CN202110991622.5A CN202110991622A CN113667739B CN 113667739 B CN113667739 B CN 113667739B CN 202110991622 A CN202110991622 A CN 202110991622A CN 113667739 B CN113667739 B CN 113667739B
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mboat2
coronary artery
apba2
rfc2
expression level
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CN113667739A (en
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陈飞
黄柳
靳雅琼
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Second Hospital of Hebei Medical University
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Abstract

The invention relates to the field of biotechnology and biological medicine, in particular to a diagnosis marker combination comprising coronary artery diseases. The invention provides a marker combination for diagnosing coronary artery disease, which comprises MBOAT2, APBA2 and RFC2; preferably, the marker combination further comprises ARRB2 or ZNF33B. The invention also provides a product for diagnosing coronary artery diseases and application thereof.

Description

Diagnostic marker combinations for coronary artery disease
Technical Field
The invention relates to the field of biotechnology and biological medicine, in particular to a diagnosis marker combination comprising coronary artery diseases.
Background
The myocardium is constantly in need of oxygen-enriched blood. The coronary arteries (emanating from the coronary sinus of the aorta, which is connected to the heart) are responsible for the transport of this blood. Coronary artery disease can narrow one or more branches of the coronary arteries, thereby blocking blood flow, leading to chest pain (angina) or heart attacks (also known as myocardial infarction or MI). Coronary artery disease has been widely recognized as a male disease. Men develop on average about 10 years earlier than women, because women are protected by high levels of estrogen prior to menopause. After menopause, coronary artery disease becomes more common in women. Because women have a longer life, the prevalence of women is rather higher in the 75 year old and older population. Thus, appropriate treatment and lifestyle changes can effectively alleviate coronary artery disease and prevent problems that may occur in the future.
Angina is a feeling of chest discomfort, heaviness, pressure, pain, burning, swelling, squeezing, or pain. Angina pectoris is easily misdiagnosed as dyspepsia or heartburn. Such cramps may also occur in the shoulders, arms, neck, throat, jaw, or back. When pressure or inflammation causes aortic occlusion, and blood flow is greatly reduced, coronary artery disease suddenly worsens, and sudden pain and other symptoms can also occur. Other symptoms of coronary heart disease include: sudden cardiac arrest may occur from shortness of breath (shortness of breath), palpitations (arrhythmia), rapid heartbeat, weakness or dizziness, nausea, sweating, dizziness, weakness, debilitation, and sometimes the first symptoms.
At present, many methods for diagnosing coronary heart disease, such as electrocardiographic examination, echocardiography, CT and the like, can indirectly reflect whether the coronary artery has lesions or not. Coronary angiography is a diagnostic method capable of directly observing coronary lesion sites, stenosis degree, distal blood flow smoothness and the like and measuring left ventricular function. Therefore, coronary angiography is considered to be the most dominant and reliable method of diagnosing coronary heart disease, being the relative "gold standard".
Besides healthy diet, frequent exercise, healthy weight maintenance and emotion management, the susceptible population can also regularly check up and treat as soon as possible, so that the diagnosis marker combination provided by the invention has practical application value.
Disclosure of Invention
The inventor of the invention can efficiently and accurately diagnose the marker and the marker combination of the coronary artery disease by analyzing, screening, verifying and providing the data of the data set. Also provided are kits, systems for detecting coronary artery disease and uses thereof.
In one aspect the invention provides a marker combination for diagnosing coronary artery disease, comprising MBOAT2, APBA2 and RFC2.
Preferably, the marker combination further comprises ARRB2 or ZNF33B.
Preferably, the MBOAT2 is highly expressed in the patient.
Preferably, the APBA2 is under-expressed in the patient.
Preferably, the RFC2 is highly expressed in a patient.
Preferably, the ARRB2 is highly expressed in the patient.
Preferably, the ZNF33B is highly expressed in a patient.
"MBOAT2" as used herein refers to a nucleic acid encoding all or part of the MBOAT2 protein or substantially identical to all or part of the nucleic acid sequence or analog thereof, having a Gene ID of 129642.
As used herein, "APBA2" refers to a nucleic acid encoding all or part of the MBOAT2 protein or substantially identical to all or part of the nucleic acid sequence or analog thereof, which has a Gene ID of 321.
"RFC2" as used herein refers to a nucleic acid encoding all or part of the MBOAT2 protein or substantially identical to all or part of the nucleic acid sequence or analog thereof, with a Gene ID of 5982.
"ARRB2" as used herein refers to a nucleic acid encoding all or part of an MBOAT2 protein or substantially identical to all or part of a nucleic acid sequence or analog thereof, having a Gene ID of 409.
"ZNF33B" as used herein refers to a nucleic acid encoding all or part of the MBOAT2 protein or substantially identical to all or part of the nucleic acid sequence or analog thereof, having a Gene ID of 7582.
Preferably, high expression means that the expression level of the marker in the patient is at least 1.1 fold higher, in particular at least 1.1 fold, 1.2 fold, 1.3 fold, 1.4 fold, 1.5 fold, 1.6 fold, 1.7 fold, 1.8 fold, 1.9 fold, 2.0 fold, 2.1 fold, 2.2 fold, 2.3 fold, 2.4 fold, 2.5 fold, 2.6 fold, 2.7 fold, 2.8 fold, 2.9 fold, 3.0 fold, 3.1 fold, 3.2 fold, 3.3 fold, 3.4 fold or 3.5 fold or more, relative to the control expression level in a healthy control population.
Preferably, low expression means that the expression level of the marker in the patient is less than the expression level in a healthy control population, e.g. about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% of the control expression level.
In another aspect, the invention provides a kit for detecting the expression level of at least any one of the aforementioned marker combinations.
Preferably, the at least any one includes two, three, four or more.
Preferably, the expression level includes an mRNA expression level and/or a protein expression level.
Preferably, the kit includes a reagent for detecting the mRNA expression level and/or the protein expression level.
Preferably, the reagent for detecting the expression amount of mRNA includes a reagent used in the following method: quantitative detection methods based on PCR, southern hybridization, northern hybridization, dot hybridization, fluorescence In Situ Hybridization (FISH), DNA microarrays, ASO methods, high throughput sequencing platforms.
Preferably, the reagent for detecting mRNA expression level includes a specific primer and/or a probe.
Preferably, the probe may be a DNA, RNA, DNA-RNA chimera, PNA or other derivative. The length of the probe is not limited, and any length may be used as long as it specifically hybridizes to the target nucleotide sequence and binds thereto. The probe may be as short as 25, 20, 15, 13 or 10 bases in length. Also, the probes can be as long as 60, 80, 100, 150, 300 base pairs or more in length, even for the entire gene. Since different probe lengths have different effects on hybridization efficiency and signal specificity, the probe length is usually at least 14 base pairs, and the length complementary to the nucleotide sequence of interest is optimally 15-25 base pairs, with the longest length generally not exceeding 30 base pairs. The probe self-complementary sequence is preferably less than 4 base pairs to avoid affecting hybridization efficiency.
Preferably, the reagent for detecting the protein expression level includes a reagent used in the following method: western Blot (Western Blot), enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), sandwich assay, immunohistochemical staining, mass spectrometry, immunoprecipitation assay, complement fixation assay, flow cytometry fluorescence assay, and protein chip.
Preferably, the reagent for detecting the expression amount of the protein includes a specific antibody.
Preferably, the antibodies include monoclonal antibodies, polyclonal antibodies.
Preferably, the antibody comprises an intact antibody molecule, any fragment of an antibody or an antibody with modifications, in particular, the antibody comprises a chimeric antibody, scFv, fab, F (ab') 2, fv, etc. Provided that the fragment is capable of retaining the ability to bind to the protein. The preparation of antibodies for detecting protein levels is well known to those skilled in the art, and any method may be used in the present invention to prepare the antibodies.
Preferably, the kit may further include an mRNA expression level auxiliary detection reagent, a protein expression level auxiliary detection reagent, an mRNA expression level auxiliary detection instrument, and a protein expression level auxiliary detection instrument.
Preferably, the mRNA expression level-aiding detection reagent includes, but is not limited to: reagents for visualizing the amplicon corresponding to the primer, for example, reagents for visualizing the amplicon by agarose gel electrophoresis, enzyme-linked gel method, chemiluminescence method, in situ hybridization method, fluorescence detection method, or the like; an RNA extraction reagent; a reverse transcription reagent; cDNA amplification reagents; preparing a standard substance used for a standard curve; a positive control; negative control.
Preferably, the protein expression level-assisted detection reagent includes, but is not limited to: blocking solution, antibody diluent, washing buffer, chromogenic stop solution and standard substance for preparing standard curve.
In another aspect, the invention provides a system for diagnosing coronary artery disease, comprising a computing device for deriving a diagnostic result based on the expression level of at least any one of the above combinations of markers.
Preferably, the system may further comprise a detection device for detecting the expression level of the marker.
Preferably, the detection device comprises a real-time quantitative PCR instrument, a high-throughput sequencing platform, a detection chip and a chip signal reader.
Preferably, the chip includes a probe for detecting the expression level of the marker.
Preferably, the chip further comprises an internal reference probe.
Preferably, the internal reference comprises GAPDH or β -action.
Preferably, the chip comprises a protein chip and/or a gene chip.
Preferably, the system further includes an evaluation result transmitting unit that can transmit an evaluation result of the subject to an information communication terminal device that the patient or the medical staff can refer to.
In another aspect, the invention provides a method of diagnosing coronary artery disease comprising detecting the amount of expression of at least any one of the foregoing combinations of markers.
Preferably, the method further comprises the step of collecting information from the subject.
The term "subject" herein means any animal, and also refers to human and non-human animals.
The term "non-human animal" herein includes all vertebrates, e.g., mammals, such as non-human primates (particularly higher primates), sheep, dogs, rodents (e.g., mice or rats), guinea pigs, goats, pigs, cats, rabbits, cattle, and any domestic animals or pets; and non-mammals such as chickens, amphibians, reptiles, and the like.
In a preferred embodiment, the subject is a human.
Preferably, the sample comprises: tissue, blood, urine, saliva, semen, milk, cerebrospinal fluid, tears, sputum, mucus, lymph, cytosol, ascites, pleural effusions, amniotic fluid, bladder irrigation fluid and bronchoalveolar lavage fluid.
Preferably, the blood comprises: serum, plasma, whole blood.
Preferably, the blood is whole blood.
Preferably, the whole blood is derived from peripheral blood.
In another aspect, the invention provides reagents for detecting the expression level of at least any one of the MBOAT2, APBA2, RFC2, ARRB2, ZNF33B and marker combinations, the kit and the application of the system in preparing products for diagnosing whether a subject suffers from coronary artery diseases.
Implementation of the "method, system for diagnosing coronary artery disease" described herein may include performing or completing selected tasks manually, automatically, or a combination thereof.
Moreover, the actual instrumentation and equipment of the embodiments of the methods, systems of the present invention could implement the various selected tasks could be implemented by hardware, by software, or by firmware or by a combination thereof using an operating system.
Drawings
FIG. 1 is a diagram of the differential box of differential genes in GSE12288 database, A is MBOAT2, B is APBA2, C is RFC2, D is ARRB2, E is ZNF33B, F is PRKX, G is FN3KRP, and H is SULT1B1.
FIG. 2 is a diagram of the differential box of differential genes in GSE20680 database, A is MBOAT2, B is APBA2, C is RFC2, D is ARRB2, E is ZNF33B, F is PRKX, G is FN3KRP, and H is SULT1B1.
FIG. 3 is a ROC curve of MBOAt2+APBA2+RFC2+ARRB2 as diagnosed in database GSE 12288; the ordinate is sensitivity, and the abscissa is specificity.
FIG. 4 is a ROC curve of MBOAt2+APBA2+RFC2+ZNF33B as diagnosed in database GSE 12288; the ordinate is sensitivity, and the abscissa is specificity.
FIG. 5 is a ROC curve of MBOAt2+PRKX+FN3KRP+ZNF33B diagnosed in database GSE 12288; the ordinate is sensitivity, and the abscissa is specificity.
FIG. 6 is a ROC curve of APBA2_RFC2 as diagnosed in database GSE 12288; the ordinate is sensitivity, and the abscissa is specificity.
FIG. 7 is a ROC curve of MBOAt2+APBA2+RFC2 as diagnosed in database GSE 12288; the ordinate is sensitivity, and the abscissa is specificity.
FIG. 8 is a ROC curve of MBOAt2+PRKX+SULT1B1 diagnosed in database GSE 12288; the ordinate is sensitivity, and the abscissa is specificity.
Detailed Description
The present invention is further described in terms of the following examples, which are given by way of illustration only, and not by way of limitation, of the present invention, and any person skilled in the art may make any modifications to the equivalent examples using the teachings disclosed above. Any simple modification or equivalent variation of the following embodiments according to the technical substance of the present invention falls within the scope of the present invention.
Example 1 screening and verifying diagnostic Effect of marker combinations
Data from databases GSE12288 (110 patient+112 control), GSE20680 (143 patient+52 control) were downloaded, and differential expression analysis was performed using R-packets, screening for genes differentially expressed in patient and control (p < 0.05). And comparing the screening results of the two databases, screening markers with consistent expression quantity changes in the two databases, wherein the analysis results show that the MBOAT2, APBA2, RFC2, ARRB2, ZNF33B, PRKX, FN3KRP and SULT1B1 are consistent in the two databases, and the difference analysis is shown in figure 1.
To improve the accuracy of diagnosis, MBOAT2, APBA2, RFC2, ARRB2, ZNF33B, PRKX, FN KRP, SULT1B1 were combined, ROC values of each combination and the accompanying drawings are shown in table 1 below:
table 1, marker combinations and AUC values thereof
Marker and marker combination AUC Drawings Optimal threshold (specificity, sensitivity)
MBOAT2 0.632711039
APBA2 0.618506494
RFC2 0.592288961
ARRB2 0.594034091
ZNF33B 0.578246753
PRKX 0.589772727
FN3KRP 0.581737013
SULT1B1 0.635308442
MBOAT2+APBA2+RFC2+ARRB2 0.722402597 3 0.566(0.818,0.545)
MBOAT2+APBA2+RFC2+ZNF33B 0.720373377 4 0.533(0.782,0.598)
MBOAT2+PRKX+FN3KRP+ZNF33B 0.646590909 5 0.585(0.809,0.438)
APBA2_RFC2 0.654626623 6 0.532(0.718,0.571)
MBOAT2+APBA2+RFC2 0.70625 7 0.499(0.727,0.643)
MBOAT2+PRKX+SULT1B1 0.635308442 8 0.499(0.455,0.759)
From the data in table 1, it can be seen that it is difficult to accurately diagnose disease with either a single marker or a combination of two markers, and when three markers are combined, especially the mboat2+apba2+rfc2 combination, the AUC value reaches 0.7, providing an accurate diagnostic marker combination. Further increasing the number of markers in the mboat2+apba2+rfc2 combination can continue to improve the accuracy of diagnosis.

Claims (2)

1. Use of an agent that detects the expression level of any one of the following sets of marker combinations in the manufacture of a product for diagnosing whether a subject has coronary artery disease:
1）MBOAT2+APBA2+RFC2+ARRB2，
2）MBOAT2+APBA2+RFC2+ZNF33B，
3）MBOAT2+APBA2+RFC2；
the expression level is mRNA expression level.
2. The use of claim 1, wherein the diagnosis further entails collecting a sample from the subject, the sample being blood.
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