Gene for diagnosing atherosclerosis susceptibility

Abstract

The invention discloses a gene for diagnosing atherosclerosis susceptibility. According to the application, through clinical research on a human histocompatibility complex, high-frequency occurrence of DRB1 * 07: 01 and DQB1 * 02: 02 alleles in an AS patient is found, and through research verification, the DRB1 * 07: 01 alleles, the DQB1 * 02: 02 alleles and the DRB1 * 07: 01-DQB1 * 02: 02 haplotypes can be used as a new marker and a potential immunotherapy target for early diagnosis of AS.

Description

Technical Field

[0001] The invention belongs to the field of biomedicine, and in particular relates to genes for diagnosing susceptibility to atherosclerosis. Background Art

[0002] Atherosclerosis (AS) is the underlying disease of coronary heart disease, ischemic stroke and peripheral large vessel disease, and is the leading cause of death worldwide. Studies have shown that AS occurs in early stages of human development from infancy, and after a long period of development, clinical symptoms usually begin to appear at the age of 50 to 60. AS is a genetically susceptible disease, and the combined effects of genetic and environmental factors lead to the development of the disease, of which genetic factors account for approximately 30-60%.

[0003] At present, there are no specific indicators for the diagnosis of AS in routine clinical examinations, and it mainly relies on imaging methods such as arterial angiography, CT examination, MRI examination and ultrasound examination, among which invasive arterial angiography is the gold standard for diagnosis. As a result, although AS undergoes a long-term pathological progression process in the body, it is often not diagnosed until clinical symptoms appear, which ultimately makes it difficult to completely reverse AS lesions, and then causes adverse outcomes such as acute attacks of AS-related cardiovascular and cerebrovascular diseases. Therefore, developing early diagnostic methods for AS and intervening as early as possible are crucial for the prevention and outcome of cardiovascular and cerebrovascular diseases. Summary of the invention

[0004] To make up for the deficiencies of the prior art, the present invention provides genes for diagnosing susceptibility to atherosclerosis.

[0005] To achieve the above purpose, the present invention adopts the following technical solution.

[0006] The first aspect of the present invention provides the use of a reagent for detecting genotypes in the preparation of a product for diagnosing susceptibility to atherosclerosis, wherein the genotypes include any one or more of the DRB1*07:01 allele, the DQB1*02:02 allele, and the DRB1*07:01-DQB1*02:02 haplotype.

[0007] Furthermore, the reagents include amplification primers or probes of the genotype.

[0008] Furthermore, the reagent also includes a detectable label.

[0009] Furthermore, the detectable labels include radioisotopes, nucleotide chromophores, enzymes, substrates, fluorescent molecules, chemiluminescent moieties, magnetic particles, and bioluminescent moieties.

[0010] The second aspect of the present invention provides a product for diagnosing susceptibility to atherosclerosis, the product comprising a reagent for detecting a genotype, the genotype comprising any one or more of the DRB1*07:01 allele, the DQB1*02:02 allele, and the DRB1*07:01-DQB1*02:02 haplotype.

[0011] Furthermore, the products include test kits, chips, and test strips.

[0012] Furthermore, the kit also includes a buffer solution.

[0013] Furthermore, the kit also includes instructions.

[0014] The third aspect of the present invention provides the use of any one or more genotypes of the DRB1*07:01 allele, the DQB1*02:02 allele, and the DRB1*07:01-DQB1*02:02 haplotype in constructing a computational model for diagnosing susceptibility to atherosclerosis.

[0015] The fourth aspect of the present invention provides the use of any one or more genotypes of the DRB1*07:01 allele, the DQB1*02:02 allele, and the DRB1*07:01-DQB1*02:02 haplotype in constructing a system / device for diagnosing susceptibility to atherosclerosis.

[0016] A fifth aspect of the present invention provides a system / device for diagnosing susceptibility to atherosclerosis, the system / device comprising: Acquisition unit: used to obtain the expression level of any one or more genotypes of DRB1*07:01 allele, DQB1*02:02 allele, and DRB1*07:01-DQB1*02:02 haplotype in the sample; Processing unit: used to obtain the result of diagnosing susceptibility to atherosclerosis according to the expression level of any one or more genotypes of DRB1*07:01 allele, DQB1*02:02 allele, and DRB1*07:01-DQB1*02:02 haplotype.

[0017] Furthermore, if the frequency of any one or more genotypes of the DRB1*07:01 allele, the DQB1*02:02 allele, and the DRB1*07:01-DQB1*02:02 haplotype increases, a diagnosis result is obtained that the sample is susceptible to atherosclerosis.

[0018] Advantages and beneficial effects of the present invention: Through clinical research on the human histocompatibility complex (HLA), this application found that DRB1*07:01 and DQB1*02:02 alleles appear frequently in AS patients. The research has verified that the DRB1*07:01 allele, DQB1*02:02 allele, and DRB1*07:01-DQB1*02:02 haplotype can be used as a new marker for the early diagnosis of AS and a potential immunotherapy target. DETAILED DESCRIPTION

[0019] The definitions of some terms used in this specification are provided below. Unless otherwise defined, all technical and scientific terms used herein generally have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0020] The present invention provides the use of a reagent for detecting genotypes in preparing a product for diagnosing susceptibility to atherosclerosis, wherein the genotypes include any one or more of the DRB1*07:01 allele, the DQB1*02:02 allele, and the DRB1*07:01-DQB1*02:02 haplotype.

[0021] The reagents include amplification primers or probes for the genotype.

[0022] In some embodiments, primer refers to a short nucleic acid molecule, such as a DNA oligonucleotide, such as a sequence of at least 15 nucleotides, which can anneal with a complementary target nucleic acid molecule by nucleic acid hybridization to form a hybrid between the primer and the target nucleic acid chain. The primer can be extended along the target nucleic acid molecule by a polymerase. Therefore, the primer can be used to amplify the target nucleic acid molecule, wherein the sequence of the primer is specific for the target nucleic acid molecule, and the primer hybridizes with the target nucleic acid molecule under very high stringency hybridization conditions.

[0023] Can be by PCR, real-time PCR or other nucleic acid amplification methods known in the art, use primer pair to amplify nucleic acid sequence.Upstream or forward primer is the primer in the 5' direction relative to the reference point on the nucleic acid sequence.Downstream or reverse primer is the primer in the 3' direction relative to the reference point on the nucleic acid sequence.In general, comprise at least one forward and one reverse primer in the amplification reaction.

[0024] In some embodiments, a probe refers to a molecule that can bind to a specific sequence or subsequence or other part of another molecule. Unless otherwise indicated, a probe generally refers to a polynucleotide probe that can bind to another polynucleotide (often referred to as a target polynucleotide) through complementary base pairing. Depending on the stringency of the hybridization conditions, the probe can bind to a target polynucleotide that lacks complete sequence complementarity with the probe. The probe can be labeled directly or indirectly. Hybridization methods include, but are not limited to, solution phase, solid phase, mixed phase, or in situ hybridization assays.

[0025] In some embodiments, primers or probes can be chemically synthesized using phosphoramidite solid phase support method or other well-known methods. Many means known in the art can also be used to modify. The limiting examples of these modifications include methylation, capping, displacement with one or more analogs of natural nucleotides and modification between nucleotides. For example, modify uncharged connectors (for example, methyl phosphate, phosphotriester, phosphoramidite, carbamate, etc.), or modify charged connectors (for example, phosphorothioate, phosphorodithioate, etc.).

[0026] The reagent also includes a detectable label.

[0027] In some embodiments, a detectable label refers to a composition that can generate a detectable signal indicating the presence of a target polynucleotide in a sample for determination. Suitable detectable labels include any composition that can be detected by fluorescence, spectroscopy, photochemistry, biochemistry, immunology, electricity, optics or chemical means. Including but not limited to UV-Vis labels, near infrared labels, fluorescent groups, phosphorescent groups, magnetic spin resonance labels, photosensitizers, photocleavable moieties, chelating centers, heavy atoms, radioisotopes, isotope detectable spin resonance labels, paramagnetic moieties, chromophores, luminophores.

[0028] In some embodiments, the fluorescent group includes but is not limited to FAM (Carboxyfluorescein, green fluorescence), FITC (fluorescein isothiocyanate), TET (tetrachloro-6-carboxyfluorescein), HEX (hexachloro-6-methylfluorescein), JOE (2,7-dimethyl-4,5-dichloro-6-carboxyfluorescein), rhodamine (Rhodamine dyes such as R110, TAMRA, Texas Red, etc.), ROX, AlexaFluor dyes (such as Alexa 350, Alexa 405, Alexa 430, Alexa 488, Alexa 500, Alexa 514, Alexa 532, Alexa 546, Alexa 555, Alexa 568, Alexa594, Alexa 610, Alexa 633, Alexa 635, Alexa 647, Alexa 660, Alexa 680, Alexa 700, Alexa 750, Alexa 790), ATTO dyes (such as ATTO 390, ATTO 425, ATTO 465, ATTO 488, ATTO 495, ATTO 514, ATTO 520, ATTO 532, ATTO Rho6G, ATTO 542, ATTO 550, ATTO 565, ATTO Rho3B, ATTO Rho11, ATTO Rho12, ATTO Thio12, ATTO Rho101, ATTO 590, ATTO 594, ATTO Rho13, ATTO 610, ATTO 620, ATTO Rho14, ATTO 633, ATTO 647, ATTO 647N, ATTO 655, ATTO Oxa12, ATTO 665, ATTO 680, ATTO 700, ATTO 725, ATTO 740), DyLight dyes, cyanine dyes (such as Cy2, Cy3, Cy3.5, Cy3b, Cy5, Cy5.5, Cy7, Cy7.5), FluoProbes dyes, SulfoCy dyes, Seta dyes, IRIS dyes, SeTau dyes, SRfluor dyes, Square dyes.

[0029] The present invention provides a product for diagnosing susceptibility to atherosclerosis, the product comprising a reagent for detecting a genotype, the genotype comprising any one or more of a DRB1*07:01 allele, a DQB1*02:02 allele, and a DRB1*07:01-DQB1*02:02 haplotype.

[0030] The products include test kits, chips, and test strips.

[0031] In some embodiments, the chip is also referred to as an array, which refers to a solid support comprising a connected nucleic acid or peptide probe. The array typically comprises a plurality of different nucleic acid or peptide probes connected to the substrate surface according to different known positions. These arrays, also referred to as "microarrays", can typically be produced using mechanical synthesis methods or light-guided synthesis methods that incorporate a combination of photolithography and solid phase synthesis methods. The array can comprise a flat surface, or can be a nucleic acid or peptide on a bead, gel, polymer surface, fiber such as an optical fiber, glass, or any other suitable substrate. The array can be packaged in a certain manner to allow for diagnosis of a fully functional device or manipulation of other methods.

[0032] In some embodiments, the kit may further include a fluorescent pigment, and a variety of known fluorescent pigments may be used. For example, a method using an intercalator having a labeling function, a method using a probe that binds a fluorescent substance to a nucleotide that specifically hybridizes to a relatively amplified DNA sequence, etc. may be cited. As intercalators, ethidium bromide and SYBR Green I as unsaturated fluorescent pigments, and Resolight and EvaGreen as saturated fluorescent pigments may be cited. As for the amount used, it is recommended by the manufacturer and seller of the fluorescent pigment used.

[0033] The kit also includes instructions and buffer. The instructions may include instructions for obtaining and processing samples. In addition, the kit may include genomic DNA of bacteria used as a positive control for PCR and sterile water used as a negative control.

[0034] In some embodiments, the components of the test kit can be packaged in the form of an aqueous medium or in a lyophilized form. Suitable containers in the test kit usually include at least a vial, test tube, flask, PET bottle, syringe or other container, in which a component can be placed, and preferably, can be appropriately divided. When there is more than one component in the test kit, the test kit will usually also include a second, third or other additional container, in which additional components are placed separately. However, the components of different combinations can be contained in a vial. The test kit of the application will usually also include a container for accommodating reactants, sealed for commercial sale. This container can include a plastic container of injection molding or blow molding, in which required vials can be retained.

[0035] The solid support of the kit can be, for example, plastic, silicon, metal, resin, glass, membrane, particle, precipitate, gel, polymer, sheet, sphere, polysaccharide, capillary, film, plate or slide.

[0036] The present invention provides a system / device for diagnosing susceptibility to atherosclerosis, the system / device comprising: Acquisition unit: used to obtain the expression level of any one or more genotypes of DRB1*07:01 allele, DQB1*02:02 allele, and DRB1*07:01-DQB1*02:02 haplotype in the sample; Processing unit: used to obtain the result of diagnosing susceptibility to atherosclerosis according to the expression level of any one or more genotypes of DRB1*07:01 allele, DQB1*02:02 allele, and DRB1*07:01-DQB1*02:02 haplotype.

[0037] In some embodiments, the implementation of the system may include performing or completing the selected tasks manually, automatically, or in combination thereof. Moreover, actual instruments and devices according to embodiments of the system of the present invention may implement multiple selected tasks using an operating system by hardware, by software, or by firmware, or by a combination thereof.

[0038] For example, the hardware for performing the selected task can be a chip or a circuit. As software, the selected task can be implemented as a plurality of software instructions executed by a computer using any suitable operating system. In the present invention, one or more tasks according to the exemplary embodiments of the method and / or system as described in the present invention can be performed by a processing unit, such as a computing platform for executing a plurality of instructions. Optionally, the processing unit includes a volatile memory for storing instructions and / or data, and / or a non-volatile memory for storing instructions and / or data, for example, a magnetic hard disk and / or a removable medium. Optionally, a network connection is also provided. A display and / or a user input device such as a keyboard or a mouse are also optionally provided.

[0039] The invention will be further described below in conjunction with specific examples. It should be understood that the specific embodiments described herein are presented by way of example and are not intended to limit the invention. The main features of the invention may be used in a variety of embodiments without departing from the scope of the invention.

[0040] Example

[0041] 1. Materials and Methods 1) Research subjects This study collected 102 clinical samples from the Department of Cardiology, Liaoyuan Central Hospital, Jilin Province from October 2023 to August 2024. All clinical samples were divided into training set and validation set, of which there were 35 cases in the training set, 18 patients susceptible to coronary atherosclerosis and 17 non-susceptible controls; there were 67 cases in the validation set, 22 patients susceptible to coronary atherosclerosis and 45 non-susceptible controls.

[0042] Susceptible population criteria: patients aged 30 to 60 years old with stenosis of the main coronary arteries (left anterior descending coronary artery, left circumflex branch and left main trunk, right main trunk) greater than or equal to 50% as diagnosed by CTA.

[0043] Criteria for non-susceptible population: people aged 30 to 60 years old, with main coronary artery stenosis (left anterior descending coronary artery, right main trunk, left circumflex branch and left main trunk) less than 20% as diagnosed by CTA.

[0044] This study has been approved by the Ethics Committee, and all subjects signed informed consent and accepted epidemiological investigation.

[0045] 2) Specimen collection and processing Vacuum EDTA anticoagulation blood collection tubes were used to collect 2 ml of fasting venous blood from the case group and the control group in the morning. After mixing, the blood was divided into 500 μl / tube and stored in a -80 ℃ refrigerator. The blood genomic DNA extraction kit of Tiangen Biochemical Technology Co., Ltd. was used to extract whole blood genomic DNA in strict accordance with the kit instructions.

[0046] 3) HLA-DRB1 and HLA-DQB1 genotyping Whole blood genomic DNA was delivered to Beijing Bofurui Medical Testing Laboratory Co., Ltd. for HLA-DRB1 and HLA-DQB1 genotyping using the NGS method.

[0047] 4) Statistical analysis SPSS 17.0 statistical software was used for data analysis, and the distribution frequencies of HLA-DRB1 and HLA-DQB1 gene loci between the two groups were compared using χ2. 2The OR value and 95% confidence interval (95% CI) of the OR value were calculated to estimate the relative risk. The difference was considered statistically significant when P < 0.05.

[0048] 2. Experimental results 1) Clinical characteristics of AS patients and controls This study investigated the distribution of HLA-DRB1 and HLA-DQB1 alleles in 40 patients with coronary atherosclerosis. There were 31 males in the patient group, accounting for 77.5%, and 9 females, accounting for 22.5%. The average age was 52.3±7.1 years, ranging from 32 to 60 years. Among them, 20 cases had single coronary artery disease, accounting for 50%; 14 cases had two-vessel disease, accounting for 35%; and 6 cases had three-vessel disease, accounting for 15%. Among the 40 AS patients, 15 had hypertension, accounting for 37.5%; 9 had diabetes, accounting for 22.5%; 22 had hypertriglyceridemia, accounting for 55%; and 7 had high LDL-C, accounting for 17.5%.

[0049] There were 23 males in the control group, accounting for 37.1%, and 39 females, accounting for 62.9%. The average age was 51.4±7.4 years old, ranging from 30 to 60 years old. Among the 62 control subjects, 15 had hypertension, accounting for 37.5%; 9 had diabetes, accounting for 22.5%; 22 had hypertriglyceridemia, accounting for 55%; and 7 had high LDL-C, accounting for 17.5%.

[0050] 2) Analysis of HLA-DRB1 and HLA-DQB1 alleles and HLA-DR / DQ haplotype differences In the training set, the frequencies of DRB1*07:01 genotype (HLA-DRB1 allele), DQB1*02:02 genotype (HLA-DQB1 allele), and DRB1*07:01-DQB1*02:02 haplotype (HLA-DR / DQ haplotype) were higher in the AS susceptible group than in the control group ( Table 1 ).

[0051] In the validation set, the frequencies of DRB1*07:01 genotype, DQB1*02:02 genotype and DRB1*07:01-DQB1*02:02 haplotype in the AS susceptible group were significantly higher than those in the control group (Table 2).

[0052] Table 1 Frequency of differentially expressed genes in the training set (n=35)

[0053]

[0054] Table 2 Differential gene frequencies in the validation set (n=67)

[0055]

[0056] 3) Analysis of the diagnostic efficacy of HLA-DRB1 and HLA-DQB1 alleles and HLA-DR / DQ haplotypes

[0057] The diagnostic efficacy data analysis of the HLA-DRB1*07:01 genotype is shown in Tables 3 and 4. The HLA-DRB1*07:01 genotype has a high diagnostic efficacy for AS susceptibility.

[0058] The diagnostic efficacy data analysis of the HLA-DQB1*02:02 genotype is shown in Tables 5 and 6. The HLA-DQB1*02:02 genotype has a high diagnostic efficacy for AS susceptibility.

[0059] The diagnostic efficacy data analysis of the HLA-DRB1*07:01-DQB1*02:02 haplotype is shown in Tables 7 and 8. The HLA-DRB1*07:01-DQB1*02:02 haplotype has a high diagnostic efficacy for AS susceptibility.

[0060] Table 3 Diagnostic significance of HLA-DRB1*07:01 genotype in the validation set (n=67)

[0061]

[0062] Table 4 Diagnostic efficacy data of HLA-DRB1*07:01 genotype for validation set

[0063]

[0064] Table 5 Diagnostic significance of HLA-DQB1*02:02 genotype for the validation set (n=67)

[0065]

[0066] Table 6 Diagnostic efficacy data of HLA-DQB1*02:02 genotype for validation set

[0067]

[0068] Table 7 Diagnostic significance of HLA-DRB1*07:01-DQB1*02:02 haplotype for validation set (n=67)

[0069]

[0070] Table 8 Diagnostic efficacy data of HLA-DRB1*07:01-DQB1*02:02 haplotype on the validation set

[0071]

[0072] 4) Correlation analysis between differential alleles / haplotypes and clinical characteristics

[0073] Next, the correlation between the differential alleles DRB1*07:01, DQB1*02:02, and DRB1*07:01-DQB1*02:02 haplotypes and hypertension, hypertriglyceridemia, high LDL-C, low HDL-C, and vascular disease status in 40 AS patients was analyzed. The results are shown in Table 9. Unfortunately, no correlation was found between DRB1*07:01, DQB1*02:02, and DRB1*07:01-DQB1*02:02 in AS patients and these clinical characteristics.

[0074] Table 9 Relationship between DRB1 and DQB1 alleles and haplotypes and clinical characteristics of patients with atherosclerosis

[0075]

[0076]

[0077] The description of the above embodiments is only used to understand the method and core idea of ​​the present invention. It should be pointed out that, for those skilled in the art, several improvements and modifications can be made to the present invention without departing from the principle of the present invention, and these improvements and modifications will also fall within the scope of protection of the claims of the present invention.

Claims

1. Use of a reagent for detecting genotypes in the preparation of a product for diagnosing susceptibility to atherosclerosis, wherein the genotypes include any one or more of the DRB1*07:01 allele, the DQB1*02:02 allele, and the DRB1*07:01-DQB1*02:02 haplotype.

2. The use according to claim 1, characterized in that: The reagents include amplification primers or probes for the genotype.

3. The use according to claim 1, characterized in that: The reagent also includes a detectable label.

4. The use according to claim 3, characterized in that: The detectable labels include radioisotopes, nucleotide chromophores, enzymes, substrates, fluorescent molecules, chemiluminescent moieties, magnetic particles, bioluminescent moieties.

5. A product for diagnosing susceptibility to atherosclerosis, characterized in that: The product includes a reagent for detecting a genotype, and the genotype includes any one or more of a DRB1*07:01 allele, a DQB1*02:02 allele, and a DRB1*07:01-DQB1*02:02 haplotype.

6. The product according to claim 5, characterized in that The products include test kits, chips, and test strips.

7. The product according to claim 6, characterized in that The kit also includes a buffer; Preferably, the kit further comprises instructions.

8. Application of any one or more genotypes of the DRB1*07:01 allele, the DQB1*02:02 allele, and the DRB1*07:01-DQB1*02:02 haplotype in constructing a computational model for diagnosing susceptibility to atherosclerosis.

9. Application of any one or more genotypes of the DRB1*07:01 allele, the DQB1*02:02 allele, and the DRB1*07:01-DQB1*02:02 haplotype in constructing a system / device for diagnosing susceptibility to atherosclerosis.

10. A system / device for diagnosing susceptibility to atherosclerosis, characterized in that: The system / device comprises: Acquisition unit: used to obtain the expression level of any one or more genotypes of DRB1*07:01 allele, DQB1*02:02 allele, and DRB1*07:01-DQB1*02:02 haplotype in the sample; Processing unit: used to obtain the result of diagnosing susceptibility to atherosclerosis according to the expression level of any one or more genotypes of DRB1*07:01 allele, DQB1*02:02 allele, and DRB1*07:01-DQB1*02:02 haplotype.

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