Marker combinations, reagents, methods, products and uses for hypertension risk assessment

Abstract

The application belongs to the technical field of biology and specifically relates to a marker combination, reagent, method, product and application for high blood pressure risk assessment. The marker combination is composed of four SNP sites of rs1801253, rs7305099, rs11191548 and rs699. The detection method comprises the following steps: obtaining a sample to be detected, extracting DNA, detecting the genotypes of the marker combination, calculating a weighted genetic risk score based on the effect amount of each SNP site, and determining a risk level according to the score. The application can simply and efficiently assess the risk of an individual suffering from high blood pressure by combining the four SNP sites covering sympathetic nerve regulation, drug metabolism, steroid hormone synthesis and renin-angiotensin system respectively with the weighted genetic risk score model, and has the advantages of low cost, simple operation and high prediction accuracy, and can be used for early screening and risk stratification of high-risk groups of high blood pressure.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to biomarker combinations, reagents, methods, products, and applications for hypertension risk assessment. Background Technology

[0002] Hypertension is a common cardiovascular disease and a major risk factor for cardiovascular disease. The pathogenesis of hypertension is extremely complex, resulting from the long-term interaction of multiple factors, genes, and environmental factors. Genetic factors play a central role in this pathological process. Numerous studies have shown that hypertension has a significant familial aggregation, meaning that genetic variations play a significant role in disease susceptibility. These genetic variations typically manifest as polymorphisms of minor genes, meaning that small genetic differences in multiple genes accumulate and collectively affect an individual's blood pressure regulation mechanisms. While the individual effects of these minor genes are limited, their combined effects can significantly alter an individual's susceptibility to hypertension.

[0003] Currently, studies have identified several single nucleotide polymorphism (SNP) sites associated with hypertension. A SNP refers to a variation between a single nucleotide base in a DNA sequence, and its frequency in the population is at least greater than 1%. Certain specific SNPs are associated with a risk of developing certain diseases. Using PCR technology to detect a specific set of hypertension-related SNP sites in the DNA of a target individual can assess their risk of developing hypertension.

[0004] Patent CN105002286A discloses the application of a reagent for detecting multiple single nucleotide polymorphism (SNP) sites in the preparation of diagnostic agents or devices for predicting the risk of hypertension and / or cardiovascular disease. This invention predicts the risk of hypertension and cardiovascular disease through the detection of multiple SNPs, integrating 22 SNP sites related to blood pressure variability in Asian populations. Genotyping was performed on a large sample of study subjects, and a genetic risk score (GRS) was calculated based on years of follow-up studies. By incorporating the genetic risk score into traditional risk factor models, the predictive ability for hypertension and cardiovascular events is improved.

[0005] Patent CN110106251A discloses a combination of single nucleotide polymorphism (SNP) sites, a reagent kit, and a chip for predicting the risk of coronary heart disease. The set of 303 SNP sites involved in this invention and its applications are accurate, flexible, rapid, and low-cost. Model validation has shown that this invention has significant auxiliary predictive value for the risk of coronary heart disease.

[0006] However, existing technologies detect a large number of single nucleotide polymorphism (SNP) sites, resulting in high detection costs and complex operations. Therefore, there is an urgent need to develop a simple and efficient gene detection method that can accurately assess an individual's risk of developing hypertension using only a small number of SNP sites. Summary of the Invention

[0007] To address the aforementioned shortcomings, this invention provides a method for detecting DNA single nucleotide polymorphism sites associated with hypertension using PCR technology. This method is simple to operate, low in cost, and can accurately assess an individual's risk of developing hypertension by detecting a small number of key sites, providing strong support for clinical diagnosis and personalized treatment.

[0008] Terminology Explanation: In this invention, the term "Forward Primer" refers to a primer that binds complementary to the 5' end of the target sequence in the template strand, and is used to initiate the synthesis and extension of the DNA strand.

[0009] In this invention, the term "Reverse Primer" refers to a primer that binds complementary to the 3' end of the target sequence in the template strand, and works in conjunction with the forward primer to achieve specific amplification of the target fragment.

[0010] In this invention, the term "PC" refers to a probe designed for a specific sequence related to cytosine (C) to detect gene fragments or mutations containing a specific cytosine site.

[0011] In this invention, the term "PG" refers to a probe designed for specific sequences related to guanine (G), used to detect gene fragments or mutations containing specific guanine sites. In this invention, the term "PT" refers to a probe designed for a specific sequence related to thymine (T) to detect gene fragments or mutations containing a specific thymine site.

[0012] In this invention, the term "PA" refers to a probe designed for a specific sequence related to adenine (A), which is typically used to detect gene fragments or mutations containing a specific adenine site.

[0013] In this invention, the term "CT value" refers to the amplification cycle number when the fluorescence signal just exceeds the background threshold in fluorescent PCR. The smaller the Ct, the higher the initial content of the target gene in the sample.

[0014] The technical solution of this invention is as follows: On one hand, the present invention provides a biomarker combination for hypertension risk assessment, the biomarker combination consisting of the following four single nucleotide polymorphism sites: rs1801253, rs7305099, rs11191548 and rs699.

[0015] In another aspect, the present invention provides a reagent for hypertension risk assessment, the reagent comprising a primer set and a probe set for amplifying the aforementioned biomarker combination.

[0016] Specifically, the primer set and probe set include primer pairs for amplifying the rs1801253 site as shown in SEQ ID NO:1-SEQ ID NO:2 and probe sequences as shown in SEQ ID NO:3-SEQ ID NO:4; primer pairs for amplifying the rs7305099 site as shown in SEQ ID NO:9-SEQ ID NO:10 and probe sequences as shown in SEQ ID NO:11-SEQ ID NO:12; primer pairs for amplifying the rs11191548 site as shown in SEQ ID NO:17-SEQ ID NO:18 and probe sequences as shown in SEQ ID NO:19-SEQ ID NO:20; and primer pairs for amplifying the rs699 site as shown in SEQ ID NO:25-SEQ ID NO:26 and probe sequences as shown in SEQ ID NO:27-SEQ ID NO:28.

[0017] More specifically, the reagent also includes a detection reagent for genotyping the combination of markers, the detection reagent including a fluorescently labeled probe.

[0018] Preferably, the fluorescent label in the fluorescently labeled probe is selected from one or more of FAM, VIC, HEX, ROX, CY3, and CY5.

[0019] Specifically, the reagent also includes one or more of DNA polymerase, dNTPs, reaction buffer, and magnesium ion source.

[0020] In another aspect, the present invention provides a kit for hypertension risk assessment, the kit comprising the aforementioned reagents.

[0021] Specifically, the kit also includes one or more of the following: a sample collector, a cell lysis reagent, a DNA extraction reagent, a positive control, a negative control, and an internal control.

[0022] Specifically, the method of using the kit includes: obtaining a sample to be tested; extracting DNA from the sample; detecting the genotype of the aforementioned biomarker combination in the DNA; and assessing the patient's risk of hypertension based on the detected genotype.

[0023] Specifically, the sample to be tested is selected from one or more of peripheral blood, saliva, oral mucosal cells, tissue samples, dried blood spots, urine, hair follicles, or amniotic fluid.

[0024] More specifically, the genotype detection is performed using polymerase chain reaction (PCR), and the amplification program includes: pre-denaturation at 90-95℃ for 1-5 minutes, 1 cycle; 90-95℃ for 5-15 seconds, 55-65℃ for 15-30 seconds, 8-15 cycles; 90-95℃ for 5-15 seconds, 55-65℃ for 15-30 seconds, 25-35 cycles, during which fluorescence signals are collected.

[0025] Preferably, the genotype detection is performed using polymerase chain reaction, with the following amplification program: 95℃ pre-denaturation for 2 minutes, 1 cycle; 95℃ for 10 seconds, 60℃ for 25 seconds, 10 cycles; 95℃ for 10 seconds, 60℃ for 20 seconds, 28 cycles, during which fluorescence signals are collected.

[0026] More specifically, the steps for assessing the risk of hypertension in the subject include: calculating a weighted genetic risk score based on the effect size of each SNP locus, using the formula: OR i SNP i The weight of the site; X i The number of risky bases; the risk level of the subject is determined based on the weighted genetic risk score.

[0027] Preferably, the risk level includes three levels: low risk, medium risk, and high risk, and is classified using an EDM model: the risk threshold is determined by using the arithmetic mean and standard deviation (sd) of the genetic risk score of the reference population as a benchmark, combined with a threshold coefficient (xs). A risk threshold below (mean...) is defined as... xs×sd) is low risk, between (mean) The risk level is considered medium if xs×sd) is between (mean+xs×sd) and (mean+xs×sd), and high if it is higher than (mean+xs×sd). The threshold coefficient xs is calculated based on the number of SNP sites N included in the project, using the formula xs=1+1.5 / (lnN+1.5). The SNP sites include rs1801253, rs11191548, rs699, and rs7305099, with a site count N=4 and a threshold coefficient xs=1.52.

[0028] In another aspect, the present invention provides the use of the aforementioned reagents or kits in the preparation of products for hypertension risk assessment, early screening, auxiliary diagnosis, or risk stratification.

[0029] Compared with the prior art, the present invention has the following beneficial effects: (1) Completeness of pathway coverage: The four sites cover sympathetic regulation (ADRB1), drug metabolism (CYP3A5), steroid hormone synthesis (CYP17A1) and renin-angiotensin system (AGT) respectively, with no functional redundancy and complementary information; (2) Adaptability to Chinese population: Site screening prioritizes the inclusion of data from Chinese population studies. The association of rs11191548 in the CYP17A1 gene is well-supported in the Han Chinese population. (3) Small sample verifiability: By weighted integration of large sample prior information from literature through meta-analysis, only 100-200 retrospective hospital samples are needed to complete clinical validation, significantly reducing R&D costs; (4) Prediction performance optimization: Example 2 uses Meta-weighted GRS and multi-model integration, with an AUC of 0.86, which is 0.07 higher than the unweighted model. The external validation AUC is 0.82, which confirms good generalization. Attached Figure Description

[0030] Figure 1 Comparison of ROC curves for risk assessment of hypertension trends in patients (primers and probes are both from the first group). Detailed Implementation

[0031] The present invention will be further clearly and completely illustrated below through embodiments. These embodiments are only some examples of the present invention and are not intended to limit the present invention, but are only for illustrating the present invention. Unless otherwise specified, the experimental methods used in the following embodiments are all conventional experiments, and the materials and reagents used in the following embodiments are commercially available unless otherwise specified.

[0032] Example 1: Screening and Detection Methods for SNP Sites This embodiment describes in detail the screening process for the four SNP sites in this invention, which is based on a systematic literature review and meta-analysis.

[0033] 1. Literature search strategy Search databases: PubMed, Embase, Cochrane Library, CNKI, Wanfang Database.

[0034] Search period: from database creation to December 2024.

[0035] Search terms: (hypertension OR high blood pressure OR elevated bloodpressure) AND (SNP OR polymorphism OR variant) AND (genetic OR gene OR association).

[0036] Inclusion criteria: (1) The study type was a case-control study or a cohort study; (2) The study subjects were humans; (3) The OR value and 95% confidence interval of the association between SNP loci and hypertension were provided; (4) The sample size was ≥500 cases; (5) The P value was <0.05.

[0037] Literature exclusion criteria: (1) reviews, conference abstracts, case reports; (2) incomplete or unextractable data; (3) duplicate publications.

[0038] 2. Meta-analysis process Meta-analysis was performed using Stata 16.0 software. The odds ratios (ORs) of the included studies were pooled, and the pooled OR and its 95% confidence interval were calculated. I0.0 2 Statistics assess heterogeneity among studies, I 2 <50% adopt a fixed effects model, I 2 ≥50% adopt the random effects model.

[0039] 3. Site screening results Through systematic literature search, 156 SNP loci associated with hypertension were initially identified. After meta-analysis, the following four loci were ultimately selected as the target sites for detection: (1) rs1801253 site (ADRB1 gene) Meta-analysis results: pooled OR = 1.42, 95% CI: 1.28–1.58, P < 0.001. 2 =35.2%. This site is located in the coding region of the ADRB1 gene, encoding the β1-adrenergic receptor, which is mainly expressed in heart and kidney tissues. It is a key molecule regulating heart rate, myocardial contractility, and renin secretion, and directly participates in the regulation of blood pressure homeostasis. Polymorphism at the rs1801253 site affects the activation level of the renin-angiotensin system by altering the activity of the β1-adrenergic receptor, ultimately increasing the risk of hypertension.

[0040] (2) rs7305099 site (WNK1 gene) Meta-analysis results: pooled OR = 1.35, 95% CI: 1.21–1.51, P < 0.001. 2=42.8%. This site is located in the intron region of the WNK1 gene, which encodes WNK kinase 1. The WNK1 gene is mainly expressed in kidney tissue and is a core molecule regulating renal sodium and potassium ion transport and water-electrolyte balance, closely related to salt-sensitive hypertension. The polymorphism at the rs7305099 site affects the renal reabsorption efficiency of sodium ions by altering the expression or activity of WNK kinase 1, ultimately increasing the risk of hypertension.

[0041] (3) rs11191548 site (CNNM2 gene) Meta-analysis results: pooled OR = 1.38, 95% CI: 1.24–1.54, P < 0.001. 2 =38.5%. This site is located in the intron region of the CNNM2 gene, which encodes cyclic nucleotide-regulated ion channel regulator protein 2. It is mainly expressed in the kidneys and vascular smooth muscle tissue, participating in the maintenance of magnesium ion homeostasis and the regulation of vasodilation and vasoconstriction, and is an important molecule for maintaining stable blood pressure. The polymorphism at the rs11191548 site affects vascular elasticity and renal electrolyte regulation by altering the protein's ion transport function, ultimately increasing the risk of hypertension.

[0042] (4) rs699 site (AGT gene) Meta-analysis results: pooled OR = 1.45, 95% CI: 1.31–1.61, P < 0.001. 2 =31.6%. This site is located in the coding region of the AGT gene, which encodes angiotensinogen, an initiating substrate of the renin-angiotensin-aldosterone system (RAAS). Its hydrolysis products can induce vasoconstriction and are core molecules in regulating blood pressure. Polymorphism at the rs699 site affects the production of vasoconstriction-related substances by altering the synthesis level of angiotensinogen, ultimately increasing the risk of hypertension.

[0043] 4. Advantages of site combination The four SNP loci selected in this invention act on different blood pressure regulatory pathways: rs1801253 and rs699 mainly act on the renin-angiotensin system, rs7305099 mainly acts on the renal ion transport system, and rs11191548 mainly acts on the vasomotor regulation system. This combination of loci covering multiple pathways can comprehensively assess the genetic risk of hypertension and has higher predictive accuracy compared to single-pathway loci.

[0044] Example 2: Reagent Kit Preparation This embodiment describes in detail the preparation method of the hypertension gene detection kit of the present invention.

[0045] 1. Primer and probe design and synthesis Based on the genomic sequences of four SNP loci, TaqMan primers and probes were designed using Primer Express 3.0 software. Primer design parameters: length 18-22 bp, GC content 40%-60%, Tm value 58-62℃, amplified product length 80-150 bp. Probe design parameters: length 15-20 bp, Tm value 8-10℃ higher than primers, 5' end labeled with a fluorescent group, 3' end labeled with a quencher group.

[0046] Table 1. Probe sequences for the rs1801253 site.

[0047] Table 2 Probe sequences for the rs7305099 site

[0048] Table 3 Probe sequences at rs11191548 site

[0049] Table 4 Probe sequences at the rs699 site

[0050] To determine the optimal primer-probe combination, this invention designed two sets of TaqMan primers and probes (set 1 and set 2) for each SNP site. Using the same human genomic DNA standard (concentration 10 ng / μL) as template, parallel amplification experiments were performed under completely identical qPCR reaction systems and amplification programs (95℃ pre-denaturation for 2 min; 95℃ for 10 s, 60℃ for 25 s, 10 cycles; 95℃ for 10 s, 60℃ for 20 s, 28 cycles). The Ct value (cycle threshold) was used as the primary evaluation index, while the fluorescence signal intensity of the amplification curve was also examined. A lower Ct value indicates higher primer-probe binding efficiency to the template and earlier amplification reaction initiation.

[0051] Each primer and probe group was performed in three independent replicates, and the mean and standard deviation were recorded. The Ct values ​​of the four groups of SNP site primers and probes are compared in Table 5.

[0052] (The amplification effect of the first group was better than that of the second group).

[0053] Table 5 Comparison of Ct values ​​for four groups of SNP site primers and probes

[0054] As shown in Table 5, under the same template input and reaction conditions, the average Ct values ​​of the first group of primers and probes for the four SNP sites were significantly lower than those of the second group, with the Ct value difference ranging from 2.1 to 2.3 cycles. Specifically, the average Ct value of the first group for the rs1801253 site was 17.3, reaching the fluorescence threshold 1.2 cycles earlier than the second group (18.7); the average Ct value of the first group for the rs7305099 site was 17.3, reaching the fluorescence threshold 1.8 cycles earlier than the second group (19.1); the average Ct value of the first group for the rs11191548 site was 20, reaching the fluorescence threshold 2.1 cycles earlier than the second group (22.1); and the average Ct value of the first group for the rs699 site was 15.15, reaching the fluorescence threshold 1.85 cycles earlier than the second group (17).

[0055] Three independent replicate experiments showed that the standard deviation of Ct values ​​in the first group was 0.15–0.35, which was lower than that in the second group (0.25–0.55), indicating that the amplification reaction of the first group of primers and probes had better repeatability and higher system stability. In qPCR quantitative detection, lower Ct values ​​and better repeatability directly reflect stronger binding affinity between the primers and probes and the target sequence, and higher amplification efficiency.

[0056] Based on the combined Ct values ​​and repeatability evaluation results, the first set of primers and probes exhibited higher amplification efficiency and better reaction stability in the detection of all four SNP sites. Therefore, this invention preferentially uses the first set of primers and probes as the primers and probes for the detection of each SNP site.

[0057] Fluorescent labeling scheme: rs1801253 site: VIC-labeled G allele probe, FAM-labeled C allele probe; rs7305099 locus: ROX-labeled G allele probe, CY5-labeled T allele probe; rs11191548 site: VIC-labeled T allele probe, FAM-labeled C allele probe; rs699 site: CY5-labeled A allele probe, ROX-labeled G allele probe.

[0058] Primers and probes were synthesized by Sangon Biotech (Shanghai) Co., Ltd., purified by HPLC, and stored at -20℃.

[0059] 2. Preparation of lysis buffer Lysis buffer formulation 1 (preferred formulation): 1M Tris-HCl (pH 8.0): 150 μL; 10% TWEEN 20: 5.5 μL; 1M DTT: 50 μL; 0.5M EDTA: 3 μL; 10% Triton X-100: 15 μL; DEPC H2O: 1276.5 μL; Total volume: 1500 μL.

[0060] Preparation method: In a clean workbench, add each component in the above proportions, vortex to mix, and dispense into 1.5mL centrifuge tubes, 500μL per tube, and store at -20℃.

[0061] Lysis buffer formulation 2 (control formulation): 1M Tris-HCl (pH 8.0): 100 μL; 10% TWEEN 20: 5 μL; 1M DTT: 60 μL; 10% Triton X-100: 15 μL; 10% SDS: 15 μL; 1M guanidine hydrochloride: 110 μL; DEPC H2O: 1195 μL; Total volume: 1500 μL.

[0062] Experimental results showed that lysis buffer formulation 1 had higher DNA extraction efficiency, and the qPCR amplification Ct value was on average 1.5 cycles earlier than that of formulation 2. Therefore, formulation 1 was selected as the final lysis buffer formulation.

[0063] 3. Preparation of reaction solution Pore ​​1 reaction solution (ADRB1+WNK1): 5× Buffer: 4 μL; 250 mM MgSO4: 0.24 μL; 25 mM dNTPs: 0.16 μL; Taq DNA polymerase (5 U / μL): 0.5 μL; ADRB1 and WNK1 primer-probe mixture: 0.54 μL; DEPC H2O: 9.56 μL; Template DNA: 5 μL; Total volume: 20 μL.

[0064] Pore ​​2 reaction solution (CNNM2+AGT): 5× Buffer: 4 μL; 250 mM MgSO4: 0.4 μL; 25 mM dNTPs: 0.16 μL; Taq DNA polymerase (5 U / μL): 0.5 μL; CNNM2 and AGT primer-probe mixture: 0.52 μL; DEPC H2O: 9.42 μL; Template DNA: 5 μL; Total volume: 20 μL.

[0065] Among them, 5× Buffer, MgSO4, dNTPs and Taq DNA polymerase were purchased from Yisheng Biotechnology (Shanghai) Co., Ltd.

[0066] 4. Reagent kit assembly Assemble the above-mentioned lysis buffer, reaction solution components, oral swab sampler, sample collection tube, qPCR reaction tube, and instruction manual into a complete kit. Store the kit at 2-8°C for 12 months.

[0067] Example 3 Experimental Procedure This embodiment describes in detail the complete experimental procedure for detecting the risk of hypertension using the kit of the present invention.

[0068] 1. Sample collection (1) Preparation before sampling: The person to be tested should avoid eating, drinking, and smoking within 30 minutes before sampling.

[0069] (2) Oral cleaning: Rinse your mouth with water 2-3 times to remove food residue in the mouth.

[0070] (3) Sampling procedure: Gently rub both sides of the cheeks for 10-15 seconds to remove oral mucosal cells; tear open the outer packaging of the oral swab, and do not touch the swab head with your hands; insert the swab into the oral cavity and gently scrape the inner walls of both sides of the oral cavity 10-15 times with the swab head; break off the swab head and put it into a sample tube containing 1500μL of lysis buffer, and tighten the tube cap.

[0071] 2. DNA extraction (1) Vortexing: Place the sample tube on the vortex apparatus and shake it vigorously for 2 minutes to allow the cells to fully lyse.

[0072] (2) Room temperature incubation: Place the sample tube at room temperature (20-25℃) for 10 minutes.

[0073] (3) Centrifugation: Place the sample tube in a centrifuge and centrifuge at 12,000 rpm for 10-15 seconds. Take the supernatant as a PCR template.

[0074] The extracted DNA can be used directly for qPCR detection without further purification.

[0075] 3. Preparation of qPCR reaction system (1) Prepare qPCR reaction tubes and label well 1 and well 2.

[0076] (2) According to the formula in Example 2, add reagent A (Taq DNA polymerase, MgSO4, dNTPs), primer probe mixture and reagent B (buffer components) in sequence.

[0077] (3) Add 5 μL of the lysed sample DNA template.

[0078] (4) Vortex mix for 5-10 seconds, let stand for 5-10 seconds, and vortex mix again for 5-10 seconds to ensure the reaction system is fully mixed.

[0079] 4. PCR amplification program Place the prepared qPCR reaction tubes into the qPCR instrument and perform amplification according to the following procedure: Pre-denaturation: 95℃, 2 minutes, 1 cycle; Phase 1 amplification: 95℃ for 10 seconds, 60℃ for 25 seconds, 10 cycles (no fluorescence signal acquisition); Second-stage amplification: 95℃ for 10 seconds, 60℃ for 20 seconds, 28 cycles (fluorescence signal acquisition).

[0080] Programmed heating rate: 6-9℃ / second; cooling rate: 6-8℃ / second.

[0081] Fluorescence acquisition channels: FAM, VIC, CY5, ROX.

[0082] 5. Genotyping interpretation Genotype can be determined based on the signal intensity of each fluorescence channel: rs1801253 site (VIC / FAM channel): VIC positive, FAM positive → GC genotype; VIC positive, FAM negative → GG genotype; VIC negative, FAM positive → CC genotype.

[0083] rs7305099 site (ROX / CY5 channel): ROX positive, CY5 positive → GT genotype; ROX positive, CY5 negative → GG genotype; ROX negative, CY5 positive → TT genotype.

[0084] rs11191548 site (VIC / FAM channel): VIC positive, FAM positive → TC genotype; VIC positive, FAM negative → TT genotype; VIC negative, FAM positive → CC genotype.

[0085] rs699 site (CY5 / ROX channel): CY5 positive, ROX positive → AG genotype; CY5 positive, ROX negative → AA genotype; CY5 negative, ROX positive → GG genotype.

[0086] Fluorescence signal threshold setting: rs1801253: VIC≥0.15, FAM≥0.1; rs7305099: ROX≥0.1, CY5≥0.1; rs11191548: VIC≥0.05, FAM≥0.05; rs699: CY5≥0.05, ROX≥0.05.

[0087] Example 4: Meta-analysis weighted GRS construction and AUC validation This embodiment details the construction method of the weighted genetic risk score (GRS) model based on meta-analysis, and the validation results of the kit's detection accuracy using a validation population of 100 people (50 hypertensive patients and 50 healthy controls).

[0088] 1. Construction of the weighted GRS model (1) GRS model construction method The GRS value of the project is calculated using the EDM model. Based on the normal distribution of the GRS value, the mean and standard deviation are used to classify the grades.

[0089] Based on the genetic data of the samples, a machine learning model was selected to construct a model for assessing the quality of life risk of diabetes based on the sample genetic data. Specifically, the GRS value of the items was calculated using an EDM model (the sum of the logarithms with the weights of the relevant loci and a constant e as the base is called GRS). Based on the normal distribution of the GRS values, the mean and standard deviation were used to classify the levels. The formula for calculating the GRS value is as follows:

[0090] Among them OR i SNP i Site weights (see Table 6); Xi represents the number of critical bases; The project rank is calculated using the GRS, mean, and sd values ​​according to the formula. The mean is the arithmetic mean of the GRS values ​​of all samples involved in the calculation; and the sd is the standard deviation of the GRS values ​​of the samples involved in the calculation.

[0091] Taking the rs1801253 locus as an example: the number of risk alleles for the GG genotype is 0, for the GC genotype it is 1, and for the CC genotype it is 2.

[0092] Table 6 Weight Information

[0093] (2) Risk stratification criteria Level 1 (Low Risk): ; Level 2 (Medium Risk): ; Level 3 (High Risk): ; The formula for calculating the threshold coefficient xs is: ; In the formula, N is the number of sites included in the project; mean is the arithmetic mean of all GRS values ​​of the samples involved in the calculation; and sd is the standard deviation of the GRS values ​​of the samples involved in the calculation.

[0094] 2. Verification Experimental Design (1) Validation population One hundred participants were recruited, including 50 patients with hypertension (case group) and 50 healthy controls (control group).

[0095] Inclusion criteria for the case group: aged 18-75 years, meeting the diagnostic criteria of the Chinese Guidelines for the Prevention and Treatment of Hypertension (systolic blood pressure ≥140 mmHg and / or diastolic blood pressure ≥90 mmHg), without secondary hypertension, and having signed an informed consent form.

[0096] Inclusion criteria for the control group: age 18-75 years, systolic blood pressure <120 mmHg and diastolic blood pressure <80 mmHg, no family history of hypertension, and signed informed consent form.

[0097] (2) Detection method The kit of this invention was used to perform genotyping of all subjects at four SNP loci, calculate the GRS value for each subject, and determine the risk level according to the risk stratification criteria.

[0098] (3) Statistical analysis Statistical analysis was performed using SPSS 26.0 software. Sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV), and area under the ROC curve (AUC) were calculated. The chi-square test was used to compare the differences in genotype distribution between the case group and the control group.

[0099] 3. Verification Results (1) Genotype distribution The frequency of risk alleles was significantly higher in the case group than in the control group: rs1801253: 62% in the case group and 38% in the control group (P<0.001); rs7305099: 58% in the case group and 35% in the control group (P<0.001); rs11191548: 60% in the case group and 36% in the control group (P<0.001); rs699: 64% in the case group and 40% in the control group (P<0.001).

[0100] (2) Risk stratification and distribution Case group: 4 low-risk cases (8%), 18 medium-risk cases (36%), and 28 high-risk cases (56%). Control group: 29 cases (58%) were low-risk, 19 cases (38%) were medium-risk, and 2 cases (4%) were high-risk.

[0101] The differences in risk stratification distribution between the two groups were statistically significant (P<0.001).

[0102] (3) Validation of the stepwise distribution of clinical risk factors To verify the association between risk stratification of this kit and the actual prevalence trend of hypertension, the distribution of clinical risk factors in subjects at different risk levels was analyzed. The results showed that as the GRS risk level increased, each clinical risk factor exhibited a significant stepwise increasing trend, specifically as follows: Blood pressure levels: The mean systolic blood pressure was (125.3±8.2) mmHg in the low-risk group, (138.6±10.4) mmHg in the medium-risk group, and (152.4±11.7) mmHg in the high-risk group; the mean diastolic blood pressure was (78.5±5.3) mmHg, (86.2±6.8) mmHg, and (94.7±7.5) mmHg, respectively. The differences among the three groups were statistically significant (P<0.001), and pairwise comparisons between adjacent groups showed P<0.01.

[0103] Positive family history rate: The positive family history rate of hypertension was 15.2% (5 / 33) in the low-risk group, 48.6% (18 / 37) in the medium-risk group, and 76.7% (23 / 30) in the high-risk group. Trend test (Cochran-Armitage trend test) χ²=24.36, P<0.001.

[0104] BMI: The average BMI was (22.6±2.2) kg / m² for the low-risk group, (24.9±2.6) kg / m² for the medium-risk group, and (27.3±3.1) kg / m² for the high-risk group. The differences among the three groups were statistically significant (P<0.001).

[0105] The above results indicate that the risk stratification of this kit is highly consistent with the traditional clinical risk factors for hypertension. Low-risk individuals correspond to low exposure and low phenotypic characteristics, while high-risk individuals correspond to high exposure and high phenotypic characteristics. Each clinical indicator shows a clear dose-response relationship, proving that this kit can accurately reflect the hypertension prevalence trend of the subjects.

[0106] (4) Scoring criteria for clinical risk factors: According to the "Guidelines for the Prevention and Treatment of Hypertension in China (2024 Revised Edition)," the diagnostic criteria for hypertension in adults should continue to be systolic blood pressure ≥140 mmHg and / or diastolic blood pressure ≥90 mmHg; systolic blood pressure ≥120 and / or <140, and diastolic blood pressure ≥80 and / or <90 are defined as high-normal values; systolic blood pressure <120 and diastolic blood pressure <80 are defined as normal blood pressure; according to the standard, hypertension is scored as 2 points, high-normal as 1 point, and normal blood pressure as 0 points.

[0107] According to the "Adult Weight Determination" standard issued by the National Health Commission of China, a BMI of 18.5 ≤ BMI < 24 is assigned a score of 0; an overweight BMI of 24 ≤ BMI < 28 and an underweight BMI < 18.5 are assigned a score of 1, and 1 point is assigned for monitoring metabolic indicators such as blood pressure and blood sugar; a BMI ≥ 28 is highly correlated with metabolic diseases such as hypertension and is assigned a score of 2. Individuals with a family history of hypertension (positive) were assigned a score of 1, while individuals without a family history of hypertension (negative) were assigned a score of 0. The clinical indicators of 100 subjects were analyzed (the subjects and the genetic testing sources were the same).

[0108] Table 7 Score Results

[0109] (5) Risk assessment and discrimination performance Using clinical blood pressure measurements and comprehensive clinical assessments as a reference, ROC curve analysis was employed to evaluate the ability of the kit of this invention to distinguish continuous trends in hypertension.

[0110] Area under the ROC curve (AUC): 0.894 (95% CI: 0.829–0.959).

[0111] The AUC value reflects the probability that the model ranks hypertensive patients ahead of healthy individuals. It does not depend on the specific cut-off point selection for risk stratification and is independent of the EDM three-level classification system, but the results are complementary.

[0112] The above indicators demonstrate that the kit of the present invention has excellent ability to distinguish the prevalence trend of hypertension.

[0113] In summary, the kit of this invention demonstrated good assessment accuracy in a validation population of 100 people, with an AUC value of 0.894. It can effectively distinguish between hypertensive patients and healthy individuals and has high clinical application value.

[0114] Comparative Example (1) Comparative Example 1: Detection scheme lacking rs1801253 site The difference between this comparative example and the embodiment is that the detection site only includes three SNP sites: rs7305099, rs11191548, and rs699, and lacks the rs1801253 site of the ADRB1 gene.

[0115] The same kit preparation method and experimental procedure as in the example were used, except that the primer probe at the rs1801253 site in well 1 was replaced with DEPC H2O.

[0116] The same validation population of 100 people was used for testing, and the results are as follows: AUC value: 0.834 (95% CI: 0.752-0.916); Compared with the previous example, the AUC value of this comparative example decreased by 0.060, the sensitivity decreased by 2.0%, and the specificity decreased by 1.0%. The results indicate that the rs1801253 locus makes an important contribution to the prediction of hypertension risk, and the absence of this locus reduces the accuracy of detection.

[0117] (2) Comparative Example 2: Detection scheme lacking rs11191548 site The difference between this comparative example and the embodiment is that the detection site only includes three SNP sites: rs1801253, rs7305099, and rs699, and lacks the rs11191548 site of the CNNM2 gene.

[0118] The same kit preparation method and experimental procedure as in the example were used, except that the primer probe at the rs11191548 site in well 2 was replaced with DEPC H2O.

[0119] The same validation population of 100 people was used for testing, and the results are as follows: AUC value: 0.845 (95% CI: 0.766-0.924); Compared with the previous example, the AUC value of this comparative example decreased by 0.049, while the sensitivity and specificity remained essentially the same. The results indicate that the rs11191548 locus also makes a significant contribution to the prediction of hypertension risk; the absence of this locus reduces detection accuracy.

[0120] (3) Comparative Example 3: Detection scheme for the newly added rs4762 site The difference between this comparative example and the embodiment is that, based on the four core SNP sites, the rs4762 site of the AGTR1 gene (wild type: A, risk base T, wild type weight 1, heterozygous mutation weight 1.02, homozygous mutation weight 1.12) is added for detection.

[0121] The rs4762 locus is located in the coding region of the AGTR1 gene, encoding the angiotensin II type 1 receptor. Meta-analysis results showed that the association between this locus and hypertension was 1.12 (95% CI: 0.98-1.28, P=0.089), which did not reach the level of statistical significance.

[0122] Table 8. Sequences of the rs4762 site gene probe

[0123] Table 9 Ct values

[0124] Using the same kit preparation method as in the examples, a primer probe at the rs4762 site was added to well 2. Due to fluorescence channel limitations, an additional reaction well was required.

[0125] The same validation population of 100 people was used for testing, and the results are as follows: AUC value: 0.887 (95% CI: 0.812-0.962); Compared to the previous examples, the AUC value of this comparative example was slightly lower (a decrease of 0.007), and the detection cost increased by approximately 25% due to the addition of one reaction well, increasing operational complexity. The results indicate that adding the rs4762 site does not significantly improve detection accuracy; instead, it increases detection cost and operational complexity.

[0126] (4) Comparative Example 4: Detection scheme for the newly added rs1799983 site The difference between this comparative example and the previous example is that, based on the four core SNP sites, the rs1799983 site of the NOS3 gene (wild type: G, risk base T, wild type weight 1, heterozygous mutation weight 1, homozygous mutation weight 1.05) was added for detection.

[0127] Table 10 Sequences of the rs1799983 locus gene probe

[0128] Table 11 Ct values

[0129] The rs1799983 locus is located in the coding region of the NOS3 gene, encoding endothelial nitric oxide synthase. Meta-analysis results showed that the association between this locus and hypertension was 1.15 (95% CI: 1.02-1.30, P=0.024), which reached statistical significance, but the effect size was small. Furthermore, it showed some functional correlation with the rs1801253 locus (both are associated with vascular function regulation).

[0130] Using the same kit preparation method as in the examples, a primer probe at the rs1799983 site was added to well 1. Due to fluorescence channel limitations, an additional reaction well was required.

[0131] The same validation population of 100 people was used for testing, and the results are as follows: AUC value: 0.891 (95% CI: 0.817-0.965); Compared to the previous example, the AUC value of this comparative example was almost the same (a decrease of 0.003), but the addition of one reaction well increased the detection cost by approximately 25%. The results indicate that the addition of the rs1799983 site provides very limited improvement in detection accuracy and does not meet the cost-effectiveness principle.

[0132] (5) Summary of comparison between the examples and comparative examples The verification results of the examples were compared with those of the four comparative examples, and the results are shown in Table 12 (the primers and probes are all from the first group).

[0133] As can be seen from Table 12: (1) The AUC value of the example (the present invention) was the highest (0.894), which was significantly better than all comparative examples, indicating that the combination of the four SNP sites selected in the present invention has the best diagnostic accuracy.

[0134] (2) Comparative Example 1 and Comparative Example 2 lacked the rs1801253 and rs11191548 sites, respectively, and the AUC values ​​decreased to 0.834 and 0.845, respectively, indicating that these two sites make important contributions to the detection accuracy and are indispensable core sites.

[0135] (3) Comparative Examples 3 and 4 added rs4762 and rs1799983 sites respectively, with AUC values ​​of 0.887 and 0.891 respectively. Compared with the examples, there was almost no improvement, but the detection cost increased by about 25%, which did not meet the cost-effectiveness principle.

[0136] In summary, the four SNP loci (rs1801253, rs7305099, rs11191548, rs699) selected by this invention through systematic literature review and meta-analysis are the optimal combination. This combination can ensure high detection accuracy while taking into account detection cost and ease of operation, and has good clinical application value and market promotion prospects.

[0137] Table 12 Comparison of verification results between the examples and four comparative examples

[0138] The above detailed description is a specific illustration of one feasible embodiment of the present invention, and this embodiment is not intended to limit the patent scope of the present invention. It should be noted that all equivalent implementations or modifications made without departing from the present invention should be included within the scope of the technical solution of the present invention. Therefore, the protection scope of the present invention should be determined by the appended claims.

Claims

1. A combination of biomarkers for hypertension risk assessment, characterized in that, The biomarker combination consists of the following four single nucleotide polymorphism sites: rs1801253, rs7305099, rs11191548, and rs699.

2. A reagent for hypertension risk assessment, characterized in that, The reagent comprises a primer set and a probe set for amplifying the biomarker combination of claim 1.

3. The reagent according to claim 2, characterized in that, The primer and probe sets include primer pairs for amplifying the rs1801253 site as shown in SEQ ID NO:1-SEQ ID NO:2 and probe sequences as shown in SEQ ID NO:3-SEQ ID NO:4; primer pairs for amplifying the rs7305099 site as shown in SEQ ID NO:9-SEQ ID NO:10 and probe sequences as shown in SEQ ID NO:11-SEQ ID NO:12; primer pairs for amplifying the rs11191548 site as shown in SEQ ID NO:17-SEQ ID NO:18 and probe sequences as shown in SEQ ID NO:19-SEQ ID NO:20; and primer pairs for amplifying the rs699 site as shown in SEQ ID NO:25-SEQ ID NO:26 and probe sequences as shown in SEQ ID NO:27-SEQ ID NO:

28.

4. The reagent according to claim 2 or 3, characterized in that, The reagent also includes a detection reagent for genotyping the combination of biomarkers, the detection reagent including fluorescently labeled probes.

5. The reagent according to claim 4, characterized in that, The fluorescent label in the fluorescently labeled probe is selected from one or more of FAM, VIC, HEX, ROX, CY3, and CY5.

6. The reagent according to claim 2, characterized in that, The reagent also includes one or more of DNA polymerase, dNTPs, reaction buffer, and magnesium ion source.

7. A kit for hypertension risk assessment, characterized in that, The kit comprises the reagent according to any one of claims 2-6.

8. The reagent kit according to claim 7, characterized in that, The kit also includes one or more of the following: a sample collector, a cell lysis reagent, a DNA extraction reagent, a positive control, a negative control, and an internal control.

9. The reagent kit according to claim 7, characterized in that, The test samples for the kit are selected from one or more of peripheral blood, saliva, oral mucosal cells, tissue samples, dried blood spots, urine, hair follicles, or amniotic fluid.

10. The use of the reagent according to any one of claims 2-6, or the kit according to any one of claims 7-9, in the preparation of products for hypertension risk assessment, early screening, auxiliary diagnosis, or risk stratification.

Images