Application of substance for detecting mutation site of cloned hematopoietic gene

Abstract

The invention relates to the technical field of biological medicine, in particular to application of a substance for detecting a mutation site of a cloned hematopoietic gene, and the cloned hematopoietic gene comprises any one or a combination of at least two of DNMT3A, ASXL1, TET2, GNAS, JAK2, SF3B1, TP53 and BCL11B. In the application disclosed by the invention, the kit can realize relapse monitoring of the risk of the age-related vascular diseases by detecting peripheral blood of a subject, has the advantages of high throughput, stability and reliability, is beneficial to better understanding the pathophysiology of the age-related vascular diseases and improving the clinical service of a patient, and has a wide application prospect. The method has important guiding significance on clinical early intervention and treatment target discovery.

Description

Technical Field

[0001] The present disclosure relates to the field of biomedicine technology, and in particular to an application of a substance for detecting mutation sites of clonal hematopoietic genes, and in particular to an application of a substance for detecting mutation sites of clonal hematopoietic genes in the preparation of a clonal hematopoietic mutation gene site detection kit for recurrence monitoring of age-related vascular disease risks. Background Art

[0002] Vascular diseases such as hypertension, stroke, and atherosclerosis are characterized by high morbidity, mortality, and disability. The pathogenesis is not fully understood, but they are closely related to aging. In clinical practice, the treatment methods for each disease are different. For example, arterial thrombectomy and drug intervention are mainly used for ischemic stroke, and surgical removal of hematoma and conservative treatment are mainly used for hemorrhagic stroke. Although these methods can improve the prognosis of patients, they are prone to cause irreversible damage such as movement disorders, cognitive disorders, and hemiplegia, and have a high disability rate.

[0003] Studies have found that clonal hematopoiesis of indeterminate potential (CHIP) may be a new independent risk factor in addition to traditional risk factors such as hypertension, hyperlipidemia, age, hyperglycemia, smoking and body mass index. CHIP refers to a subtype of hematopoietic stem cells (HSCs) or other hematopoietic progenitor cells (HPCs) that develop into non-tumor-driven gene mutations in order to better adapt to the environment. The occurrence of CHIP is the inevitable result and objective existence of the combined effects of HSCs' intrinsic aging and external environmental factors. With age, the DNA gene mutation rate of HSCs increases. Most mutations have no effect on HSCs' function and do not induce disease, but some mutations give HSCs stronger survival ability and increase their role in producing blood cells. CHIP is HSCs with molecular genetic mutation characteristics. Through multi-lineage hematopoietic differentiation, they form terminally differentiated mature blood cells with reproducible biological markers.

[0004] It is currently believed that most CHIPs are caused by mutations in HSCs, and the mutated genes include DNMT3A, TET2, ASXL1, JAK2, TP53, etc. CHIP is significantly associated with the risk of blood tumors, coronary heart disease, and all-cause mortality, and is therefore considered an important precancerous state. CN114026254A discloses the detection of cancer, cancer-derived tissues, and / or a type of cancer cell, and provides a blood disorder (HD) test combination for targeted detection of methylation patterns or variations specific to various blood disorders, such as clonal hematopoiesis of uncertain potential (CHIP) and blood cancers, such as leukemia, lymphoma (e.g., lymphoma), multiple myeloma, and myeloid tumors. Further provided herein include: methods for designing, manufacturing, and using the HD test combination for detecting various blood disorders. However, the invention cannot be used for recurrence monitoring of vascular diseases associated with increased age. Summary of the invention

[0005] In order to solve the above technical problems, the present disclosure provides an application of a substance for detecting mutation sites of clonal hematopoietic genes in the preparation of a clonal hematopoietic mutation gene site detection kit for recurrence monitoring of age-related vascular disease risks, which can be used for recurrence monitoring of age-related vascular disease risks.

[0006] In a first aspect, the present disclosure provides an application of a substance for detecting mutation sites of clonal hematopoietic genes in the preparation of a clonal hematopoietic mutation gene site detection kit for recurrence monitoring of age-related vascular disease risks, wherein the clonal hematopoietic genes include any one or a combination of at least two of DNMT3A, ASXL1, TET2, GNAS, JAK2, SF3B1, TP53 and BCL11B.

[0007] This disclosure is based on the whole genome sequencing data of the Third China National Stroke Registry (CNSR-III) cohort and the whole genome sequencing data of the COSMIC (Catalogue of Somatic Mutations in Cancer) database, and conducts a whole genome association study (GWAS), as well as a risk score constructed by multifactor COX regression and a survival analysis of the Fine & Gray competing risk model, and selects 25 gene mutations at 23 sites of 8 high-risk mutation genes. The clonal hematopoietic mutation gene loci enrich the pathogenic mutation map of age-related vascular diseases, deeply explain the significant relationship between CHIP gene mutations and age-related vascular diseases, and provide a scientific basis for early pathogenic gene screening and intervention treatment of the disease.

[0008] It is easy for a person skilled in the art to understand that the kit described in the present disclosure includes the substance for detecting the mutation site of the clonal hematopoietic gene.

[0009] Preferably, the substance for detecting the mutation site of the clonal hematopoietic gene includes a detection probe for detecting the mutation site of the clonal hematopoietic gene.

[0010] Preferably, the mutation sites of the clonal hematopoietic gene DNMT3A include any one of DNMT3Ac.2645G>A, DNMT3Ac.2645G>C, DNMT3Ac.2644C>T, DNMT3Ac.1741T>G, DNMT3Ac.958C>T, DNMT3Ac.2207G>A and DNMT3Ac.2206C>T, or a combination of at least two of them.

[0011] Preferably, the mutation sites of the clonal hematopoietic gene ASXL1 include ASXL1c.1210C>T and / or ASXL1c.1249C>T.

[0012] Preferably, the mutation site of the clonal hematopoietic gene TET2 includes any one of TET2c.662_663insA, TET2c.780delG and TET2c.3088C>T, or a combination of at least two of them.

[0013] Preferably, the mutation site of the clonal hematopoietic gene GNAS includes any one of GNASc.601C>G, GNASc.602G>A and GNASc.601C>T, or a combination of at least two of them.

[0014] Preferably, the mutation site of the clonal hematopoietic gene JAK2 includes JAK2c.1849G>T.

[0015] Preferably, the mutation sites of the clonal hematopoietic gene SF3B1 include any one of SF3B1c.1997A>C, SF3B1c.1997A>G, SF3B1c.1998G>C, SF3B1c.1998G>T and SF3B1c.1996A>C, or a combination of at least two of them.

[0016] Preferably, the mutation site of the clonal hematopoietic gene TP53 includes any one of TP53c.848G>A, TP53c.743G>A and TP53 c.328C>T, or a combination of at least two of them.

[0017] Preferably, the mutation site of the clonal hematopoietic gene BCL11B includes BCL11Bc.1786G>A.

[0018] Preferably, the nucleic acid sequence of the detection probe for the DNMT3Ac.2645G>A mutation gene site is as shown in SEQ ID NO: 1 and SEQ ID NO: 2;

[0019] The nucleic acid sequences of the detection probes for the DNMT3Ac.2645G>C mutation gene sites are shown in SEQ ID NO:3 and SEQ ID NO:4;

[0020] The nucleic acid sequences of the detection probes for the DNMT3Ac.2644C>T mutation gene sites are shown in SEQ ID NO:5 and SEQ ID NO:6;

[0021] The nucleic acid sequences of the detection probes for the DNMT3Ac.1741T>G mutation gene sites are shown in SEQ ID NO:7 and SEQ ID NO:8;

[0022] The nucleic acid sequences of the detection probes for the DNMT3Ac.958C>T mutation gene sites are shown in SEQ ID NO:9 and SEQ ID NO:10;

[0023] The nucleic acid sequences of the detection probes for the DNMT3Ac.2207G>A mutation gene sites are shown in SEQ ID NO: 11 and SEQ ID NO: 12;

[0024] The nucleic acid sequences of the detection probe for the DNMT3Ac.2206C>T mutation gene site are shown in SEQ ID NO:13 and SEQ ID NO:14.

[0025] Preferably, the nucleic acid sequence of the detection probe for the ASXL1c.1210C>T mutation gene site is as shown in SEQ ID NO: 15 and SEQ ID NO: 16;

[0026] The nucleic acid sequences of the detection probe for the ASXL1c.1249C>T mutation gene site are shown in SEQ ID NO:17 and SEQ ID NO:18.

[0027] Preferably, the nucleic acid sequence of the detection probe for the TET2c.662_663insA mutation gene site is as shown in SEQ ID NO: 19 and SEQ ID NO: 20;

[0028] The nucleic acid sequences of the detection probes for the TET2c.780delG mutation gene site are shown in SEQ ID NO: 21 and SEQ ID NO: 22;

[0029] The nucleic acid sequence of the detection probe for the TET2c.3088C>T mutation gene site is shown in SEQ ID NO:23 and SEQ ID NO:24.

[0030] Preferably, the nucleic acid sequence of the detection probe for the GNASc.601C>G mutation gene site is as shown in SEQ ID NO:25 and SEQ ID NO:26;

[0031] The nucleic acid sequences of the detection probes for the GNASc.602G>A mutation gene sites are shown in SEQ ID NO:27 and SEQ ID NO:28;

[0032] The nucleic acid sequences of the detection probe for the GNASc.601C>T mutation gene site are shown in SEQ ID NO:29 and SEQ ID NO:30.

[0033] Preferably, the nucleic acid sequence of the detection probe for the JAK2c.1849G>T mutation gene site is as shown in SEQ ID NO:31 and SEQ ID NO:32.

[0034] Preferably, the nucleic acid sequence of the detection probe for the SF3B1c.1997A>C mutation gene site is as shown in SEQ ID NO:33 and SEQ ID NO:34;

[0035] The nucleic acid sequences of the detection probes for the SF3B1c.1997A>G mutation gene sites are shown in SEQ ID NO:35 and SEQ ID NO:36;

[0036] The nucleic acid sequences of the detection probes for the SF3B1c.1998G>C mutation gene sites are shown in SEQ ID NO:37 and SEQ ID NO:38;

[0037] The nucleic acid sequences of the detection probes for the SF3B1c.1998G>T mutation gene sites are shown in SEQ ID NO:39 and SEQ ID NO:40;

[0038] The nucleic acid sequences of the detection probe for the SF3B1c.1996A>C mutation gene site are shown in SEQ ID NO:41 and SEQ ID NO:42.

[0039] Preferably, the nucleic acid sequence of the detection probe for the TP53c.848G>A mutation gene site is as shown in SEQ ID NO:43 and SEQ ID NO:44;

[0040] The nucleic acid sequences of the detection probes for the TP53c.743G>A mutation gene sites are shown in SEQ ID NO:45 and SEQ ID NO:46;

[0041] The nucleic acid sequence of the detection probe for the TP53c.328C>T mutation gene site is shown in SEQ ID NO:47 and SEQ ID NO:48.

[0042] Preferably, the nucleic acid sequence of the detection probe for the BCL11Bc.1786G>A mutation gene site is as shown in SEQ ID NO:49 and SEQ ID NO:50.

[0043] Preferably, the detection probe is a Taqman probe, and the Taqman probe is modified with a fluorescent group.

[0044] Preferably, the kit further comprises any one or a combination of at least two of a DNA extraction reagent, a PCR reagent and a probe hybridization reagent.

[0045] In a second aspect, the present disclosure provides a system for recurrence monitoring of age-related vascular disease risk, comprising a clonal hematopoietic mutation gene site detection module, which detects mutation sites of the following clonal hematopoietic genes in a biological sample: any one or a combination of at least two of DNMT3A, ASXL1, TET2, GNAS, JAK2, SF3B1, TP53 and BCL11B.

[0046] Preferably, the biological sample comprises peripheral blood or genomic DNA derived from peripheral blood.

[0047] Compared with the prior art, the technical solution provided by the embodiments of the present disclosure has the following advantages:

[0048] Based on the whole genome sequencing data of the Third China National Stroke Registry (CNSR-III) cohort and the whole genome sequencing data of the COSMIC (Catalogue of Somatic Mutations in Cancer) database, the present invention conducts a genome-wide association study (GWAS), a risk score constructed by multivariate COX regression, and a survival analysis of the Fine & Gray competing risk model, and identifies clonal hematopoietic genes and corresponding mutation gene sites that can be used to monitor the risk of age-related vascular diseases.

[0049] A detection probe is designed based on the determined mutation gene site to prepare a kit for recurrence monitoring of the risk of age-related vascular diseases. The kit has the effects of high accuracy, high detection efficiency and reliable detection results, thereby realizing the recurrence monitoring of the risk of age-related vascular diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0050] The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate embodiments consistent with the present disclosure and, together with the description, serve to explain the principles of the present disclosure.

[0051] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, for ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative labor.

[0052] Figure 1 The present invention discloses a flow chart of a method for screening clonal hematopoietic mutation gene loci for age-related vascular diseases, including 8 high-risk mutation genes, namely DNMT3A, ASXL1, TET2, GNAS, JAK2, SF3B1, TP53 and BCL11B, and 25 gene mutation types at 23 loci of the 8 high-risk mutation genes.

[0053] Figure 2 The mutation site is DNMT3Ac.2645G>A (DNMT3A R882H ) of real-time fluorescence quantitative PCR results.

[0054] Figure 3 DNMT3Ac.2645G>C(DNMT3A R882P ) of real-time fluorescence quantitative PCR results.

[0055] Figure 4 JAK2 c.1849G>T (JAK2 V617F ) of real-time fluorescence quantitative PCR results. DETAILED DESCRIPTION

[0056] In this disclosure, the term "recurrence monitoring" and related terms refer to a description of the likely outcome of a given condition (eg, age-related vascular disease).

[0057] Embodiments of the present disclosure include "monitoring" a subject who may be at risk for developing age-related vascular disease. The subject may be a patient who has not been diagnosed with age-related vascular disease, but may be at risk for developing age-related vascular disease due to various clinical or medical assessments.

[0058] In the present disclosure, the method for detecting whether a subject's biological sample contains a mutated gene site includes nucleic acid extraction, real-time fluorescence quantitative PCR, and real-time fluorescence quantitative analysis. Exemplarily, the above method can be used to detect gene site mutations.

[0059] In the present disclosure, the 23 mutant gene sites that can be used for diagnosis are all known gene sites in the prior art, and can be detected by methods known in the prior art for detecting mutant gene sites, such as by preparing corresponding gene chips.

[0060] The molecular biology methods used in the present disclosure can all refer to the corresponding methods recorded in public publications such as "Current Protocols in Molecular Biology (published by Wiley)".

[0061] Unless otherwise specified, all reagents used in the examples can be purchased from commercial sources.

[0062] In order to more clearly understand the above-mentioned objectives, features and advantages of the present disclosure, the scheme of the present disclosure will be further described below. It should be noted that the embodiments of the present disclosure and the features in the embodiments can be combined with each other without conflict.

[0063] In the following description, many specific details are set forth to facilitate a full understanding of the present disclosure, but the present disclosure may also be implemented in other ways different from those described herein; it is obvious that the embodiments in the specification are only part of the embodiments of the present disclosure, rather than all of the embodiments.

[0064] Example 1

[0065] Based on the whole genome sequencing data of the Third China National Stroke Registry (CNSR-III) cohort and the whole genome sequencing data of the COSMIC (Catalogue of Somatic Mutations in Cancer) database, a genome-wide association study (GWAS) was performed, as well as a risk score constructed by multivariate COX regression and a survival analysis using the Fine & Gray competing risk model. Twenty-five gene mutations at 23 loci of eight high-risk mutation genes were selected. The analysis methods and results are shown in Figure 1 .

[0066] Taqman MGB probes were designed based on the above-mentioned clonal hematopoietic mutation gene loci of age-related blood vessels:

[0067] The nucleic acid sequences of the Taqman MGB probe for detecting the DNMT3Ac.2645G>A mutation gene site are shown in SEQ ID NO: 1 and SEQ ID NO: 2;

[0068] The nucleic acid sequences of the Taqman MGB probe for detecting the DNMT3Ac.2645G>C mutation gene site are shown in SEQ ID NO:3 and SEQ ID NO:4;

[0069] The nucleic acid sequences of the Taqman MGB probe for detecting the DNMT3Ac.2644C>T mutation gene site are shown in SEQ ID NO:5 and SEQ ID NO:6;

[0070] The nucleic acid sequences of the Taqman MGB probe for detecting the DNMT3Ac.1741T>G mutation gene site are shown in SEQ ID NO:7 and SEQ ID NO:8;

[0071] The nucleic acid sequences of the Taqman MGB probe for detecting the DNMT3Ac.958C>T mutation gene site are shown in SEQ ID NO:9 and SEQ ID NO:10;

[0072] The nucleic acid sequences of the Taqman MGB probe for detecting the DNMT3Ac.2207G>A mutation gene site are shown in SEQ ID NO: 11 and SEQ ID NO: 12;

[0073] The nucleic acid sequences of the Taqman MGB probe for detecting the DNMT3Ac.2206C>T mutation gene site are shown in SEQ ID NO: 13 and SEQ ID NO: 14;

[0074] The nucleic acid sequences of the Taqman MGB probe for detecting the ASXL1c.1210C>T mutation gene site are shown in SEQ ID NO: 15 and SEQ ID NO: 16;

[0075] The nucleic acid sequences of the Taqman MGB probe for detecting the ASXL1c.1249C>T mutation gene site are shown in SEQ ID NO: 17 and SEQ ID NO: 18;

[0076] The nucleic acid sequences of the Taqman MGB probe for detecting the TET2c.662_663insA mutation gene site are shown in SEQ ID NO: 19 and SEQ ID NO: 20;

[0077] The nucleic acid sequences of the Taqman MGB probe for detecting the TET2c.780delG mutation gene site are shown in SEQ ID NO: 21 and SEQ ID NO: 22;

[0078] The nucleic acid sequences of the Taqman MGB probe for detecting the TET2c.3088C>T mutation gene site are shown in SEQ ID NO:23 and SEQ ID NO:24;

[0079] The nucleic acid sequences of the Taqman MGB probe for detecting the GNASc.601C>G mutation gene site are shown in SEQ ID NO:25 and SEQ ID NO:26;

[0080] The nucleic acid sequences of the Taqman MGB probe for detecting the GNASc.602G>A mutation gene site are shown in SEQ ID NO:27 and SEQ ID NO:28;

[0081] The nucleic acid sequences of the Taqman MGB probe for detecting the GNASc.601C>T mutation gene site are shown in SEQ ID NO:29 and SEQ ID NO:30;

[0082] The nucleic acid sequences of the Taqman MGB probe for detecting the JAK2c.1849G>T mutation gene site are shown in SEQ ID NO:31 and SEQ ID NO:32;

[0083] The nucleic acid sequences of the Taqman MGB probe for detecting the SF3B1c.1997A>C mutation gene site are shown in SEQ ID NO:33 and SEQ ID NO:34;

[0084] The nucleic acid sequences of the Taqman MGB probe for detecting the SF3B1c.1997A>G mutation gene site are shown in SEQ ID NO:35 and SEQ ID NO:36;

[0085] The nucleic acid sequences of the Taqman MGB probe for detecting the SF3B1c.1998G>C mutation gene site are shown in SEQ ID NO:37 and SEQ ID NO:38;

[0086] The nucleic acid sequences of the Taqman MGB probe for detecting the SF3B1c.1998G>T mutation gene site are shown in SEQ ID NO:39 and SEQ ID NO:40;

[0087] The nucleic acid sequences of the Taqman MGB probe for detecting the SF3B1c.1996A>C mutation gene site are shown in SEQ ID NO:41 and SEQ ID NO:42;

[0088] The nucleic acid sequences of the Taqman MGB probe for detecting the TP53c.848G>A mutation gene site are shown in SEQ ID NO:43 and SEQ ID NO:44;

[0089] The nucleic acid sequences of the Taqman MGB probe for detecting the TP53c.743G>A mutation gene site are shown in SEQ ID NO:45 and SEQ ID NO:46;

[0090] The nucleic acid sequences of the Taqman MGB probe for detecting the TP53c.328C>T mutation gene site are shown in SEQ ID NO:47 and SEQ ID NO:48;

[0091] The nucleic acid sequences of the Taqman MGB probe for detecting the BCL11Bc.1786G>A mutation gene site are shown in SEQ ID NO:49 and SEQ ID NO:50.

[0092] The nucleic acid sequences of the Taqman MGB probes for each mutation gene site are shown in Table 1 ;

[0093] Table 1

[0094]

[0095]

[0096]

[0097]

[0098] Example 2

[0099] In the study of recurrence surveillance patients in the Third China National Stroke Registry (CNSR-III) cohort, we randomly selected 202 samples from peripheral blood leukocytes of patients undergoing recurrence surveillance in the CNSR-III cohort to investigate whether there are clonal hematopoietic mutation loci associated with age-related vascular disease in these patients.

[0100] The genomic DNA of leukocytes from peripheral blood was extracted by centrifugal adsorption column method which can specifically bind DNA:

[0101] Add 1 mL of cell lysis buffer to the sample, mix by inversion, centrifuge at 10,000 rpm for 1 min, remove the supernatant, leaving the cell nuclear pellet, add 200 μL of buffer to the cell nuclear pellet, and shake until thoroughly mixed; add 200 μL of GB buffer and 20 μL of Proteinase K premixed solution to the above 200 μL sample, and mix by inversion thoroughly; place in a 56°C metal bath for 10 min, invert and mix several times during the period until the solution becomes clear; leave at room temperature for 5 min;

[0102] Add 350 μL BD buffer and mix thoroughly by inverting; put the above solution into the adsorption column, centrifuge at 12,000 rpm for 30 seconds, and discard the waste liquid in the collection tube; add 500 μL GDB buffer to the adsorption column, centrifuge at 12,000 rpm for 30 seconds, and discard the waste liquid in the collection tube; add 600 μL PWB rinse solution to the adsorption column, centrifuge at 12,000 rpm for 30 seconds, and discard the waste liquid in the collection tube; repeat the rinse once; centrifuge at 12,000 rpm for 2 minutes, and discard the waste liquid in the collection tube;

[0103] The adsorption column was placed at room temperature for 2 min and then placed in a 1.5 mL centrifuge tube;

[0104] 30 μL of TB elution buffer preheated to 65°C was added dropwise to the middle of the adsorption membrane of the adsorption column. After standing at room temperature for 2 minutes, the mixture was centrifuged at 12,000 rpm for 2 minutes, and the extracted peripheral blood genomic DNA was collected in a centrifuge tube.

[0105] Real-time fluorescence quantitative PCR and high-throughput gene chips were used to detect clonal hematopoietic mutation gene loci in age-related blood vessels in randomly sampled samples:

[0106] Take 2 μL of the extracted peripheral blood genomic DNA sample and Taq PCR Mix premix, mix them, and add them to a 384-well plate; seal the plate with a sealing film, shake and mix, and centrifuge at 1000 rpm for 1 min to remove bubbles;

[0107] The samples in the 384-well plate were added to a gene chip containing 25 groups of gene probes (SEQ ID NO: 1 to SEQ ID NO: 50) using an automatic sample loading instrument; the gene chip was detected using real-time fluorescence quantitative PCR.

[0108] The test results are shown in Table 2. We found DNMT3A in the random sample of the CNSR-III cohort. R882H These results are consistent with the genome sequencing results of the CNSR-III cohort, indicating that this mutation is indeed present in these samples.

[0109] Specifically, we detected DNMT3A in individuals with sample numbers 0520032 and 0740027. R882H Mutation, which means that the DNMT3A gene of these two individuals has a specific mutation at this position. This consistency verifies the accuracy of our detection method and further confirms the presence of this mutation in the CNSR-III cohort.

[0110] These findings provide further evidence for a role for this clonal hematopoietic mutation in age-associated vascular disease and its association with recurrent vascular events.

[0111] Table 2

[0112] Sample No. CNSR-III Example 1 0520032 <![CDATA[DNMT3A R882H Mutation occurs]]> <![CDATA[DNMT3A R882H Mutation occurs]]> 0740027 <![CDATA[DNMT3A R882H Mutation occurs]]> <![CDATA[DNMT3A R882H Mutation occurs]]>

[0113] Example 3

[0114] The clonal hematopoietic mutation gene loci were detected in the community population samples of age-related vascular diseases (basic characteristics are shown in Table 3).

[0115] Table 3

[0116]

[0117] Collect and store samples from people with age-related vascular diseases in the community. Collect peripheral blood in purple tubes containing the anticoagulant EDTA or heparin and store them at -80°C. Avoid repeated freezing and thawing of samples.

[0118] The peripheral blood genomic DNA was extracted using a centrifugal adsorption column method that can specifically bind to DNA:

[0119] Add 2 times the volume of cell lysis buffer to the blood sample, mix by inversion, centrifuge at 10,000rpm for 1min, remove the supernatant, leaving the cell nuclear pellet, add 200μL buffer to the cell nuclear pellet, shake until thoroughly mixed; add 200μL GB buffer and 20μL Proteinase K premix solution to the above 200μL sample, mix by inversion thoroughly; place in a 56℃ metal bath for 10min, mix by inversion several times during the period, until the solution becomes clear; place at room temperature for 5min;

[0120] Add 350 μL BD buffer and mix thoroughly by inverting; put the above solution into the adsorption column, centrifuge at 12,000 rpm for 30 seconds, and discard the waste liquid in the collection tube; add 500 μL GDB buffer to the adsorption column, centrifuge at 12,000 rpm for 30 seconds, and discard the waste liquid in the collection tube; add 600 μL PWB rinse solution to the adsorption column, centrifuge at 12,000 rpm for 30 seconds, and discard the waste liquid in the collection tube; repeat the rinse once; centrifuge at 12,000 rpm for 2 minutes, and discard the waste liquid in the collection tube;

[0121] The adsorption column was placed at room temperature for 2 min and then placed in a 1.5 mL centrifuge tube;

[0122] 30 μL of TB elution buffer preheated to 65°C was added dropwise to the middle of the adsorption membrane of the adsorption column. After standing at room temperature for 2 minutes, the mixture was centrifuged at 12,000 rpm for 2 minutes, and the extracted peripheral blood genomic DNA was collected in a centrifuge tube.

[0123] Real-time fluorescence quantitative PCR and high-throughput gene chips were used to detect clonal hematopoietic mutation gene loci in the community population with age-related vascular diseases:

[0124] Take 2 μL of the extracted peripheral blood genomic DNA sample and Taq PCR Mix premix, mix them, and add them to a 384-well plate; seal the plate with a sealing film, shake and mix, and centrifuge at 1000 rpm for 1 min to remove bubbles;

[0125] The samples in the 384-well plate were added to a gene chip containing 25 sets of gene probes (SEQ ID NO: 1 to SEQ ID NO: 50) using an automatic sample loading instrument; the gene chip was tested using real-time fluorescence quantitative PCR. R882H )、DNMT3Ac.2645G>C(DNMT3A R882P )、JAK2 c.1849G>T(JAK2 V617F The real-time fluorescence quantitative PCR results of Figure 2 , Figure 3 and Figure 4 .

[0126] The results of the detection of clonal hematopoietic mutation loci in community-based age-related vascular disease population samples are shown in Table 4. In the community-based age-related vascular disease population, clonal hematopoietic mutations at three high-frequency loci were detected. These loci include c.2645G>A (DNMT3A R882H )、c.2645G>C(DNMT3A R882P), and c.1849G>T of JAK2 gene (JAK2 V617F ). For the test results of DNMT3Ac.2645G>A site, 48 people (accounting for 50.53% of the total samples) carried this mutation gene site; for the test results of DNMT3Ac.2645G>C site, 50 people (accounting for 52.63% of the total samples) carried this mutation gene site; and for the test results of JAK2 c.1849G>T site, 53 people (accounting for 55.79% of the total samples) carried this mutation gene site.

[0127] "These results suggest that the prevalence of these clonal hematopoietic mutations in the population is relatively high, suggesting that they may play a role in the pathogenesis of the disease. These findings will help further research and clinical diagnosis to better understand the association between these genetic variants and vascular disease," said Dr.

[0128] Table 4

[0129]

[0130] It should be noted that, in this article, relational terms such as "first" and "second" are only used to distinguish one entity or operation from another entity or operation, and do not necessarily require or imply any such actual relationship or order between these entities or operations. Moreover, the terms "include", "comprise" or any other variants thereof are intended to cover non-exclusive inclusion, so that a process, method, article or device including a series of elements includes not only those elements, but also other elements not explicitly listed, or also includes elements inherent to such process, method, article or device. In the absence of further restrictions, the elements defined by the sentence "comprise a ..." do not exclude the existence of other identical elements in the process, method, article or device including the elements.

[0131] The above description is only a specific embodiment of the present disclosure, so that those skilled in the art can understand or implement the present disclosure. Various modifications to these embodiments will be apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the present disclosure. Therefore, the present disclosure will not be limited to the embodiments described herein, but will conform to the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. Use of a substance for detecting mutation sites of clonal hematopoietic genes in the preparation of a clonal hematopoietic mutation gene site detection kit for recurrence monitoring of age-related vascular disease risk, characterized in that: The clonal hematopoietic genes include any one or a combination of at least two of DNMT3A, ASXL1, TET2, GNAS, JAK2, SF3B1, TP53 and BCL11B.

2. The use according to claim 1, characterized in that: The substance for detecting the mutation site of the clonal hematopoietic gene includes a detection probe for detecting the mutation site of the clonal hematopoietic gene.

3. The use according to claim 1 or 2, characterized in that: The mutation sites of the clonal hematopoietic gene DNMT3A include any one or a combination of at least two of DNMT3Ac.2645G>A, DNMT3Ac.2645G>C, DNMT3Ac.2644C>T, DNMT3Ac.1741T>G, DNMT3Ac.958C>T, DNMT3Ac.2207G>A and DNMT3Ac.2206C>T; Preferably, the mutation sites of the clonal hematopoietic gene ASXL1 include ASXL1c.1210C>T and / or ASXL1c.1249C>T; Preferably, the mutation site of the clonal hematopoietic gene TET2 includes any one or a combination of at least two of TET2c.662_663insA, TET2c.780delG and TET2c.3088C>T; Preferably, the mutation site of the clonal hematopoietic gene GNAS includes any one or a combination of at least two of GNASc.601C>G, GNASc.602G>A and GNASc.601C>T; Preferably, the mutation site of the clonal hematopoietic gene JAK2 includes JAK2c.1849G>T; Preferably, the mutation site of the clonal hematopoietic gene SF3B1 includes any one or a combination of at least two of SF3B1c.1997A>C, SF3B1c.1997A>G, SF3B1c.1998G>C, SF3B1c.1998G>T and SF3B1c.1996A>C; Preferably, the mutation site of the clonal hematopoietic gene TP53 includes any one or a combination of at least two of TP53c.848G>A, TP53c.743G>A and TP53 c.328C>T; Preferably, the mutation site of the clonal hematopoietic gene BCL11B includes BCL11Bc.1786G>A.

4. The use according to claim 3, characterized in that: The nucleic acid sequences of the detection probes for the DNMT3Ac.2645G>A mutation gene sites are shown in SEQ ID NO: 1 and SEQ ID NO: 2; The nucleic acid sequences of the detection probes for the DNMT3Ac.2645G>C mutation gene sites are shown in SEQ ID NO:3 and SEQ ID NO:4; The nucleic acid sequences of the detection probes for the DNMT3Ac.2644C>T mutation gene sites are shown in SEQ ID NO:5 and SEQ ID NO:6; The nucleic acid sequences of the detection probes for the DNMT3Ac.1741T>G mutation gene sites are shown in SEQ ID NO:7 and SEQ ID NO:8; The nucleic acid sequences of the detection probes for the DNMT3Ac.958C>T mutation gene sites are shown in SEQ ID NO:9 and SEQ ID NO:10; The nucleic acid sequences of the detection probes for the DNMT3Ac.2207G>A mutation gene sites are shown in SEQ ID NO: 11 and SEQ ID NO: 12; The nucleic acid sequences of the detection probe for the DNMT3Ac.2206C>T mutation gene site are shown in SEQ ID NO:13 and SEQ ID NO:

14.

5. The use according to claim 3 or 4, characterized in that: The nucleic acid sequences of the detection probes for the ASXL1c.1210C>T mutation gene sites are shown in SEQ ID NO: 15 and SEQ ID NO: 16; The nucleic acid sequences of the detection probes for the ASXL1c.1249C>T mutation gene sites are shown in SEQ ID NO: 17 and SEQ ID NO: 18; Preferably, the nucleic acid sequence of the detection probe for the TET2c.662_663insA mutation gene site is as shown in SEQ ID NO: 19 and SEQ ID NO: 20; The nucleic acid sequences of the detection probes for the TET2c.780delG mutation gene site are shown in SEQ ID NO: 21 and SEQ ID NO: 22; The nucleic acid sequence of the detection probe for the TET2c.3088C>T mutation gene site is shown in SEQ ID NO:23 and SEQ ID NO:

24.

6. The use according to any one of claims 3 to 5, characterized in that: The nucleic acid sequences of the detection probes for the GNASc.601C>G mutation gene sites are shown in SEQ ID NO:25 and SEQ ID NO:26; The nucleic acid sequences of the detection probes for the GNASc.602G>A mutation gene sites are shown in SEQ ID NO: 27 and SEQ ID NO: 28; The nucleic acid sequences of the detection probes for the GNASc.601C>T mutation gene sites are shown in SEQ ID NO:29 and SEQ ID NO:30; Preferably, the nucleic acid sequence of the detection probe for the JAK2c.1849G>T mutation gene site is as shown in SEQ ID NO:31 and SEQ ID NO:

32.

7. The use according to any one of claims 3 to 6, characterized in that: The nucleic acid sequences of the detection probes for the SF3B1c.1997A>C mutation gene sites are shown in SEQ ID NO:33 and SEQ ID NO:34; The nucleic acid sequences of the detection probes for the SF3B1c.1997A>G mutation gene sites are shown in SEQ ID NO:35 and SEQ ID NO:36; The nucleic acid sequences of the detection probes for the SF3B1c.1998G>C mutation gene sites are shown in SEQ ID NO:37 and SEQ ID NO:38; The nucleic acid sequences of the detection probes for the SF3B1c.1998G>T mutation gene sites are shown in SEQ ID NO:39 and SEQ ID NO:40; The nucleic acid sequences of the detection probe for the SF3B1c.1996A>C mutation gene site are shown in SEQ ID NO:41 and SEQ ID NO:

42.

8. The use according to any one of claims 3 to 7, characterized in that: The nucleic acid sequences of the detection probes for the TP53c.848G>A mutation gene sites are shown in SEQ ID NO:43 and SEQ ID NO:44; The nucleic acid sequences of the detection probes for the TP53c.743G>A mutation gene sites are shown in SEQ ID NO:45 and SEQ ID NO:46; The nucleic acid sequences of the detection probes for the TP53c.328C>T mutation gene sites are shown in SEQ ID NO:47 and SEQ ID NO:48; Preferably, the nucleic acid sequence of the detection probe for the BCL11Bc.1786G>A mutation gene site is as shown in SEQ ID NO:49 and SEQ ID NO:

50.

9. The use according to any one of claims 2 to 8, characterized in that: The detection probe is a Taqman probe, and the Taqman probe is modified with a fluorescent group; Preferably, the kit further comprises a DNA extraction reagent, a PCR reagent and a probe hybridization reagent.

10. A system for recurrence monitoring of age-related vascular disease risk, characterized in that: It comprises a clonal hematopoietic mutation gene site detection module, which detects the mutation sites of the following clonal hematopoietic genes in the biological sample: any one or a combination of at least two of DNMT3A, ASXL1, TET2, GNAS, JAK2, SF3B1, TP53 and BCL11B; preferably, the biological sample comprises peripheral blood or genomic DNA derived from peripheral blood.

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