Kit for detecting MYH10 gene methylation degree associated with female cerebral arteriovenous malformation and its application

Abstract

The present invention discloses a method for detecting cerebral arteriovenous malformations MYH10 A kit for measuring the degree of gene methylation and its application, characterized in that the kit includes a pair of MYH10 Gene DNA methylation specific amplification primers, the nucleotide sequences are shown in SEQ ID NO.1 and SEQ ID NO.2 and a MYH10 Gene DNA methylation specific sequencing primer, the nucleotide sequence is shown in SEQ ID NO.3; MYH10 The region where the gene DNA sequence was detected was a specific location within the CpG28 methylation island within the gene and the DNA methylation level was analyzed by pyrosequencing technology; MYH10 The gene DNA methylation level is positively correlated with the prevalence of cerebral arteriovenous malformations; the advantage is that the diagnostic kit can conveniently and quickly detect cerebral arteriovenous malformations at the molecular level, with high detection efficiency and strong specificity.

Description

Technical Field

[0001] The present invention relates to the technical field of auxiliary diagnosis of cerebral arteriovenous malformations, and in particular to a kit for detecting the methylation degree of MYH10 gene associated with female cerebral arteriovenous malformations and an application thereof. Background Art

[0002] Brain arteriovenous malformation (BAVM) is a congenital disease, mainly due to the mutation of cerebral vascular formation during embryonic development, forming a chaotic vascular mass. The most common and serious clinical symptom of BAVM is cerebral hemorrhage caused by rupture of malformed blood vessels, with a hemorrhage rate of 1.3–3.9%. Once the malformed blood vessels rupture and bleed, it will not only threaten the patient's life, but also have a high probability of leaving varying degrees of disability and sequelae. Because most patients are young and middle-aged people aged 20-40, BAVM not only makes them lose their ability to work, but also affects their ability to take care of themselves, placing a heavy burden on their families and society. Therefore, early diagnosis and timely treatment of BAVM are imperative and have important clinical significance. Studies in recent years have shown that the degree of DNA methylation of genes related to vascular development plays an important role in the occurrence and development of BAVM.

[0003] The myosin heavy chain 10 (MYH10) gene is located on chromosome 17. The protein it encodes is a member of the myosin superfamily and is a traditional non-muscle myosin. Myosin is an actin-dependent motor protein with multiple functions, including regulating cell division, cell movement and cell polarity. MYH10 is highly expressed in various tissues such as the myocardium, lungs, and arteries. Currently, many research results have shown that MYH10 may be a high-risk warning indicator for atherosclerosis. The expression of MYH10 is positively correlated with the number of vascular smooth muscle cells, and negatively correlated with the thickness of the intima-media layer in patients with atherosclerosis. MYH10 gene mutations may change the proliferation and migration of vascular cells in the body, leading to vascular development defects, and then causing vascular-related lesions. Through studies on mouse animal models, researchers have found that MYH10 plays an important role in the development of the mouse heart and the formation of the embryonic vascular system. In clinical studies, researchers have also found that the expression of MYH10 is significantly increased in abdominal aortic aneurysms and can be used as a potential molecular marker for abdominal aortic aneurysms. It can be seen that MYH10 is closely related to vascular-related lesions. Cerebral arteriovenous malformations are considered to be a disease caused by abnormal vascular development due to internal or external factors during embryonic development. Abnormal MYH10 expression is likely to have an impact in the early stages of vascular system formation, leading to the occurrence and development of the disease. The expression level of the MYH10 gene or the degree of DNA methylation in the blood of patients with cerebral arteriovenous malformations is likely to be related to the progression of the disease. At present, there are no public research reports on the relationship between MYH10 gene DNA methylation and cerebral arteriovenous malformations at home and abroad. Summary of the invention

[0004] The technical problem to be solved by the present invention is to provide a kit for detecting the degree of methylation of the MYH10 gene related to female cerebral arteriovenous malformations, which is convenient, highly targeted and highly accurate, and its application, wherein the DNA methylation level of the MYH10 gene is positively correlated with the prevalence of female cerebral arteriovenous malformations, and the MYH10 gene DNA methylation level can be detected to assist in the diagnosis of female cerebral arteriovenous malformations.

[0005] The technical solution adopted by the present invention to solve the above technical problems is: a kit for detecting the methylation degree of the MYH10 gene associated with female cerebral arteriovenous malformation, comprising a pair of MYH10 gene methylation-specific amplification primers and a MYH10 gene methylation-specific sequencing primer:

[0006] The nucleotide sequence of the upstream primer for MYH10 gene methylation-specific amplification was: 5′-ATTTTAGTAGGTTTTTTAGGGTGTTGAT-3′;

[0007] The nucleotide sequence of the downstream primer for MYH10 gene methylation-specific amplification was: 5′-Biotin-ATTCATATACCCTCCCCAAAAT-3′;

[0008] The nucleotide sequence of the MYH10 gene methylation-specific sequencing primer is: 5′-GTTGGTTTAGGTGGTATTATTTA-3′.

[0009] Furthermore, the region where the MYH10 gene DNA sequence is detected is within the CpG28 methylation island region Chr17:8,380,173-8,380,539 of the gene.

[0010] Furthermore, the specific locations of the 7 CpG sites in the Chr17:8,380,173-8,380,539 region are:

[0011] Furthermore, the MYH10 gene DNA detection sequence uses pyrophosphate sequencing technology to analyze the DNA methylation level of 7 CpG sites in the MYH10 gene.

[0012] The use of the above-mentioned kit for detecting the methylation degree of the promoter region of the MYH10 gene associated with cerebral arteriovenous malformation in the preparation of a reagent for detecting cerebral arteriovenous malformation or a reagent for auxiliary diagnosis of cerebral arteriovenous malformation.

[0013] Compared with the prior art, the advantages of the present invention are: the present invention discloses for the first time a kit for detecting the degree of DNA methylation of the MYH10 gene associated with female cerebral arteriovenous malformations and its application, and the MYH10 gene DNA methylation level is positively correlated with the prevalence of female cerebral arteriovenous malformations. Therefore, the detection kit based on the detection of the MYH10 gene DNA methylation level can conveniently and quickly detect female cerebral arteriovenous malformations at the molecular level, with high detection efficiency and strong pertinence. At the same time, the drug targeting the MYH10 gene methylation is an innovative use for auxiliary diagnosis, detection and screening of cerebral arteriovenous malformations.

[0014] In summary, the detection kit based on detecting the DNA methylation level of the MYH10 gene of the present invention can conveniently and quickly detect female cerebral arteriovenous malformations at the molecular level, with high detection efficiency and strong pertinence, which is conducive to the early detection and timely treatment of female cerebral arteriovenous malformations. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1The CpG28 methylation island region where the detected sequence of the MYH10 gene is located is located. The specific location is hg19, Chr17:8,380,173-8,380,539. The detected region contains 7 CpG methylation sites;

[0016] Figure 2 This is an example of the MYH10 gene methylation level test results. The percentages shown in the figure are the methylation levels of the corresponding CpG sites, which are CpG1, 100%; CpG2, 74%; CpG3, 81%; CpG4, 75%; CpG5, 79%; CpG6, 70%; CpG7, 87%;

[0017] Figure 3 The methylation of MYH10 gene in patients with cerebral arteriovenous malformations was significantly higher than that in the control group;

[0018] Figure 4 The difference in MYH10 gene methylation between cerebral arteriovenous malformations and the control group in different genders (the methylation level of MYH10 gene in female cerebral arteriovenous malformations was significantly higher than that in the control group, *: p<0.01);

[0019] Figure 5 The sensitivity and specificity of MYH10 gene methylation and diagnosis of cerebral arteriovenous malformation were analyzed (CpG6, AUC=0.78, p=0.002, the sensitivity at the optimal critical point was 71.4%, and the specificity was 28.6%). DETAILED DESCRIPTION

[0020] The present invention is further described in detail below with reference to the accompanying drawings. Specific embodiments

[0022] 1. Collection of research subjects

[0023] Volunteers willing to participate in the study were recruited. According to the international diagnostic criteria for cerebral arteriovenous malformations, the volunteers were divided into a case group and a control group. The general situation of the volunteers was investigated in the form of a questionnaire. At the same time, venous blood was taken for general blood biochemical tests, as follows: 21 patients with cerebral arteriovenous malformations confirmed by angiography were collected from a hospital (11 males; 10 females, average age 46.52±11.26), and 21 normal people with no history of cardiovascular and cerebrovascular diseases confirmed by CT were collected as a control group (11 males; 10 females, average age 47.00±12.42) with strict gender and age matching. Fasting blood was drawn from all research subjects to test general biochemical indicators such as blood lipids and blood sugar. At the same time, 3 ml of venous blood was drawn into an EDTA anticoagulant tube and centrifuged at 4℃, 3000rpm for 20 minutes. The supernatant plasma and the middle layer of leukocytes were stored in a -80℃ low-temperature refrigerator for unified extraction of genomic DNA from the specimens.

[0024] 2. Extraction of genomic DNA

[0025] The whole blood genomic DNA of the sample obtained in the above steps was extracted using a Lab-Aid 820 fully automatic nucleic acid extractor, and the concentration of the obtained DNA was tested using a nucleic acid protein analyzer for the detection of the DNA methylation level of the MYH10 gene.

[0026] 3. DNA methylation level determination

[0027] In this study, pyrosequencing was used to methylate the CpG28 island region within the MYH10 gene, specifically the seven CpG sites within hg19, Chr17:8,380,173-8,380,539 (e.g. Figure 1 The basic principle of this technology is to treat the DNA sample with bisulfite and then amplify it with polymerase chain reaction (PCR), which can keep the methylated cytosine (C) base unchanged and convert the unmethylated C into uracil (U). Then, PCR sequencing is performed using sequencing primers to determine which site is methylated. In this study, PyroMarkAssay Design software was used for primer design. The PCR amplification primers and sequencing primers used in the experiment are as follows:

[0028] The nucleotide sequence of the MYH10 gene DNA methylation-specific forward primer (Forward primer) SEQ ID NO.1 is as follows: 5'-ATTTTAGTAGGTTTTTTAGGGTGTTGAT-3';

[0029] The nucleotide sequence of the MYH10 gene DNA methylation-specific downstream primer (Reverse primer) SEQ ID NO. 2 is as follows: 5'-Biotin-ATTCATATACCCTCCCCAAAAT-3';

[0030] The nucleotide sequence of the MYH10 gene DNA methylation-specific sequencing primer (Sequencing primer) SEQ ID NO. 3 is as follows: 5'-GTTGGTTTAGGTGGTATTATTTA-3'.

[0031] Specific steps for determining DNA methylation levels:

[0032] Step 1: Sulfite conversion. The sample DNA was subjected to bisulfite conversion using a bisulfite treatment kit (EpiTech Bisulfite Kits; Qiagen; #59104). The converted DNA was obtained by following the steps of loading, washing, and elution according to the instructions.

[0033] Step 2: PCR reaction. Take 20 ng of the converted DNA sample in the first step and add it to the Pyromark PCR Kit (Pyromark PCR Kit; Qiagen; #978703), and add the above pair of MYH10 gene methylation-specific amplification primers to perform PCR amplification. The amplification conditions are: first denaturation at 95°C for 3 min; then annealing reaction at 94°C for 30 s, 56°C for 30 s, and 72°C for 1 min, for a total of 40 cycles; then extension reaction at 72°C for 5 min.

[0034] Step 3: Preliminary preparation for pyrophosphate sequencing: pre-add 45 μl of annealing buffer (PyroMark Annealing Buffer; Qiagen; #979009) containing 0.3 μM of the above-mentioned methylation-specific sequencing primers to a 96-well plate; transfer the total amount of mixed agarose beads to be used (3 μl per sample) to an Eppendorf tube; add binding buffer (PyroMark Binding Buffer; Qiagen; #979006) to the agarose beads so that the average volume per sample is about 50 μl, and mix the mixture; add the above mixture to the PCR product (50 μl reaction volume), 50 μl per sample; mix the PCR product at room temperature for 10 minutes to allow the magnetic beads to bind to biotin; in the vacuum preparation workstation, add 180 ml of high-purity water, 70% ethanol, washing buffer (PyroMark Wash Buffer; Qiagen; #979008) and 120 ml of denaturing buffer (PyroMark Denaturation Solution; Qiagen; #979007); turn on the pump of the vacuum preparation workstation and rinse the vacuum preparation tool in high-purity water for 30 seconds; then move the vacuum preparation tool (PyroMark Vacuum Prep Filter Probes; Qiagen; #979010) to the PCR plate and grab the agarose beads (complete this operation within three minutes after the magnetic beads bind to the PCR product); pick up the PCR plate and check whether most of the magnetic beads are adsorbed on the vacuum preparation tool; put the vacuum preparation tool in 70% ethanol for 5 seconds; then move it to denaturation buffer for 5 seconds; then move it to wash buffer for 5-10 seconds; turn off the pump; put the vacuum preparation tool into the plate containing sequencing primers and shake it to release the agarose beads (sequencing primers can also be added last); rinse the vacuum preparation tool with high-purity water; place the PSQ96 plate with samples on a heating plate and heat it to 80℃ for 2 minutes, then cool it to room temperature, and then perform pyrophosphate sequencing reaction.

[0035] Step 4: Pyrophosphate sequencing: The samples in the PSQ96 plate in step 3 were sequenced using the Pyromark Gold Q24 Reagents (Qiagen; #978802) on a PyroMark Q24 pyrophosphate sequencer, and the results were then analyzed for methylation using the PyroMark CpG software (see methylation level detection results for details). Figure 2 ).

[0036] 4. Data Analysis

[0037] GraphPad Prism 8 was used to organize and analyze the data in this study. The primers designed in this study can detect the methylation of 7 CpG sites in the CpG28 methylation island hg19, Chr17:8,380,173-8,380,539 region within the MYH10 gene. By comparing the case and control groups, we found that the MYH10 gene CpG5 (p=0.03), CpG6 (p=0.011) and average methylation level (p=0.023) in patients with cerebral arteriovenous malformations were lower than those in the control group ( Figure 3 The results of gender group analysis showed that the average methylation level of MYH10 gene in male volunteers was significantly higher than that in female volunteers (p = 0.031, Figure 4 ), and the average methylation level of MYH10 gene in female patients with cerebral arteriovenous malformation was significantly higher than that in the control group (p = 0.037). The ROC curve analysis results showed that the average methylation level of MYH10 gene in all research subjects was below the line area AUC = 0.72, p = 0.018; CpG5 methylation AUC area was 0.75, p = 0.006; and CpG6 methylation AUC area was 0.78, p = 0.002 ( Figure 5 ), the sensitivity at the optimal cut-off point was 71.4% and the specificity was 28.6%.

[0038] The above research results show that the DNA methylation of the MYH10 gene in the blood of patients with brain arteriovenous malformations is significantly higher than that of the control group, and this difference only occurs in females. Therefore, the detection of the degree of DNA methylation of the MYH10 gene is expected to be a predictive factor for the early diagnosis of brain arteriovenous malformations.

[0039] In summary, the present invention provides a kit for detecting the degree of DNA methylation of the MYH10 gene and its application, which has the advantages of accuracy, reliability, flexibility, rapidity and economy in the detection kit for auxiliary diagnosis of cerebral arteriovenous malformation. The present invention uses the above-mentioned kit to detect the DNA methylation level of the MYH10 gene, which can quickly and reliably provide reference for auxiliary diagnosis, detection or screening of drugs for cerebral arteriovenous malformation.

[0040] The above description is not intended to limit the present invention, and the present invention is not limited to the above examples. Any changes, modifications, additions or substitutions made by a person skilled in the art within the spirit and scope of the present invention shall also fall within the protection scope of the present invention. Sequence Listing <110> Ningbo First Hospital <120> Kit for detecting MYH10 gene methylation degree associated with female cerebral arteriovenous malformation and its application <160> 3 <170> PatentIn version 3.1 <210> 1 <211> 28 <212> DNA <213> Artificial sequence <223> MYH10 gene methylation-specific amplification upstream primer (5'-ATTTTAGTAGGTTTTTTAGGGTGTTGAT-3' ) <400> 1 <210> 2 <211> twenty two <212> DNA <213> Artificial sequence <223> MYH10 gene methylation-specific amplification downstream primer (5'-Biotin-ATTCATATACCCTCCCCAAAAT-3') <400> 2 <210> 3 <211> twenty three <212> DNA <213> Artificial sequence <223> MYH10 gene methylation-specific sequencing primer (5'-GTTGGTTTAGGTGGTATTATTTA-3') <400> 3

Claims

1. A kit for assisting in detecting the methylation level of the MYH10 gene associated with female cerebral arteriovenous malformations, characterized in that It comprises a pair of MYH10 gene methylation-specific amplification primers and a MYH10 gene methylation-specific sequencing primer, wherein the nucleotide sequence of the MYH10 gene methylation-specific amplification upstream primer is shown in SEQ ID NO.1: 5'-ATTTTAGTAGGTTTTTTAGGGTGTTGAT-3'; The nucleotide sequence of the downstream primer for MYH10 gene methylation-specific amplification is shown in SEQ ID NO.2: 5'-Biotin-ATTCATATACCCTCCCCAAAAT-3'; The nucleotide sequence of the MYH10 gene methylation-specific sequencing primer is shown in SEQ ID NO.3: 5'-GTTGGTTTAGGTGGTATTATTTA-3'.

2. The kit for assisting in detecting the methylation degree of the MYH10 gene associated with female cerebral arteriovenous malformation according to claim 1, characterized in that: The region where the MYH10 gene DNA sequence is detected is within the CpG28 methylation island Chr17:8,380,173-8,380,539 region of the gene.

3. The kit for assisting in detecting the methylation degree of the MYH10 gene associated with female cerebral arteriovenous malformation according to claim 2, characterized in that: The specific locations of the 7 CpG sites in the Chr17:8,380,173-8,380,539 region are:

4. The kit for assisting in detecting the methylation degree of the MYH10 gene associated with female cerebral arteriovenous malformation according to claim 3, characterized in that: The MYH10 gene DNA detection sequence uses pyrophosphate sequencing technology to analyze the DNA methylation level of 7 CpG sites in the MYH10 gene.

5. Use of a kit for assisting in detecting the methylation level of the promoter region of the MYH10 gene associated with female cerebral arteriovenous malformations in the preparation of reagents for assisting in the diagnosis of cerebral arteriovenous malformations.

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