CHPT1 gene methylation detection kit for assisting in diagnosing or detecting cerebral aneurysm

Abstract

The application discloses a kit for assisting in diagnosing or detecting a cerebral aneurysm CHPT1 The kit is characterized in that the kit comprises reagents for detecting CHPT1 a target sequence in a gene promoter region, CHPT1 the target sequence in the gene promoter region is shown as SEQ ID NO. 2: CGGCAGCCGGGCAGGCCGGCCTGACCTCGACCTCCGCCGTGCGGGCCCGACCGGTGAGTCCAGCCCGGCAGTCGCAGGACCCGGCCG, and the reagents comprise CHPT1 a methylation-specific amplification upstream primer, a methylation-specific amplification downstream primer and CHPT1 a methylation-specific sequencing primer, and further comprise CHPT1 RT-qPCR quantitative amplification primers for a transcriptome, so that the intracranial aneurysm can be detected at a molecular level in a convenient and fast manner, and the detection efficiency is high and the detection is targeted.

Description

Technical Field

[0001] This invention belongs to the field of biomedical detection and molecular diagnostic technology, specifically relating to a device for assisting in the diagnosis or detection of cerebral aneurysms. CHPT1 Gene methylation detection kit. Background Technology

[0002] Cerebral aneurysm rupture leading to subarachnoid hemorrhage is a type of acute cerebrovascular event with high mortality and disability rates. This disease has a sudden onset and is characterized by a high mortality rate (30%–45%) and a high disability rate (approximately 50%), and is considered a neurosurgical emergency. Despite continuous advancements in techniques such as microsurgical clipping and endovascular interventional therapy, the long-term prognosis for many patients remains unsatisfactory. Cerebral aneurysms commonly occur in people aged 40–66. Once a cerebral aneurysm ruptures and bleeds, it will cause death in one-third of patients, and leave varying degrees of sequelae in another third, imposing a huge economic burden on families and society. Cerebral aneurysms are localized abnormal dilation of the intracranial arterial wall; rupture leading to subarachnoid hemorrhage has an extremely high mortality and disability rate. Currently, early identification of unruptured cerebral aneurysms in clinical practice mainly relies on imaging examinations, including digital subtraction angiography (DSA), computed tomography angiography (CTA), and magnetic resonance angiography (MRA). However, these methods have limitations such as radiation exposure, contrast agent allergies, and invasive procedures, and are difficult to implement for large-scale screening of high-risk populations. Therefore, developing non-invasive molecular diagnostic biomarkers based on peripheral blood samples has significant clinical value and social implications for the early detection and prevention of rupture of cerebral aneurysms. In recent years, increasing evidence suggests that epigenetic modifications, especially DNA methylation, play an important role in regulating inflammatory responses and vascular pathology in cerebrovascular diseases. Multiple genome-wide methylation studies have shown that abnormal DNA methylation patterns are closely related to delayed cerebral ischemia, neuroinflammatory responses, vasospasm, and poor prognosis following cerebral aneurysm rupture.

[0003] Abnormal phospholipid metabolism is involved in the pathological processes of various vascular diseases. Choline phosphotransferase 1 (CHPT1) has attracted attention for its crucial role in catalyzing the conversion of diacylglycerol (DAG) to phosphatidylcholine (PC) in the Kennedy pathway. To date, there is no research on this topic both domestically and internationally. CHPT1 Studies linking gene methylation to brain aneurysms have not been reported, and there are no studies linking it to brain aneurysms. CHPT1 A technical approach for the combined use of methylation level and mRNA expression level in the diagnosis of cerebral aneurysms. Summary of the Invention

[0004] The technical problem to be solved by this invention is to provide a method that can achieve high sensitivity and high specificity based on peripheral blood samples for the auxiliary diagnosis or detection of cerebral aneurysms. CHPT1 Gene methylation detection kit.

[0005] The technical solution adopted by this invention to solve the above-mentioned technical problems is: a device for assisting in the diagnosis or detection of cerebral aneurysms. CHPT1 Gene methylation detection kit, including detection CHPT1 The reagent for the gene promoter region target sequence, as described CHPT1 The target sequence of the gene promoter region is shown in SEQ ID NO.2: CGGCAGCCGGGCAGGCCGGCCTGACCTCGACCTCCGCCGTGCGGGCCCGACCGGTGAGTCCAGCCCGGCAGTCGCAGGACCCGGCCG.

[0006] Further, the reagent includes CHPT1 upstream primers for gene methylation-specific amplification CHPT1 Gene methylation-specific amplification downstream primers and CHPT1 Gene methylation-specific sequencing primers, as described CHPT1 The nucleotide sequence of the upstream primer for gene methylation-specific amplification is shown in SEQ ID NO.3: 5'-GGTAGAGTGGTTTTGTAGTGGTTAATA-3'; CHPT1 The nucleotide sequence of the downstream primer for gene methylation-specific amplification is shown in SEQ ID NO.4: 5'-Biotin-CCCCAACCCCAAAAACTATAA-3'; CHPT1 The nucleotide sequence of the gene methylation-specific sequencing primer is shown in SEQ ID NO.5: 5'-GGTTTTGTAGTGGTTAATATTG-3'.

[0007] Furthermore, it also includes CHPT1 Primers for RT-qPCR quantitative amplification of the transcriptome.

[0008] Furthermore, the aforementioned CHPT1 Primers for RT-qPCR quantitative amplification of the transcriptome include CHPT1 Forward amplification primers for the transcriptome and CHPT1 The reverse amplification primers for the transcriptome, as described CHPT1 The nucleotide sequence of the forward amplification primers for the transcriptome is shown in SEQ ID NO.7: 5'-CGCTCGTGCTCATCTCCTACTG-3'. CHPT1The nucleotide sequence of the reverse amplification primer for the transcriptome is shown in SEQ ID NO.8: 5'-AGTTTGTTCTTCTGGCTTGTTTCCC-3'.

[0009] The technical solution of the present invention also provides another method for assisting in the diagnosis or detection of cerebral aneurysms. CHPT1 Gene methylation detection kit, including detection CHPT1 Transcriptome reagents, as described CHPT1 The transcriptome sequence is shown in SEQ ID NO. 6: CGCTCGTGCTCATCTCCTACTGTCCCACGGCCACCGAAGAGGCACCATACTGGACATACCTTTTATGTGCACTGGGACTTTTTATTTACCAGTCACTGGATGCTATTGATGGGAAACAAGCCAGAAGAACAAACT, and the reagents include CHPT1 Primers for RT-qPCR quantitative amplification of the transcriptome.

[0010] Furthermore, the aforementioned CHPT1 Primers for RT-qPCR quantitative amplification of the transcriptome include CHPT1 Forward amplification primers for the transcriptome and CHPT1 The reverse amplification primers for the transcriptome, as described CHPT1 The nucleotide sequence of the forward amplification primers for the transcriptome is shown in SEQ ID NO.7: 5'-CGCTCGTGCTCATCTCCTACTG-3'. CHPT1 The nucleotide sequence of the reverse amplification primer for the transcriptome is shown in SEQ ID NO.8: 5'-AGTTTGTTCTTCTGGCTTGTTTCCC-3'.

[0011] Compared with the prior art, the advantages of the present invention are: the present invention is used to assist in the diagnosis or detection of cerebral aneurysms. CHPT1 Gene methylation detection kit, first discovery CHPT1 The association between gene promoter methylation and cerebral aneurysms: This study revealed the epigenetic feature of significantly elevated methylation levels at specific CpG sites (especially sites 1 and 3-13) in the CHPT1 promoter region in patients with cerebral aneurysms. CHPT1 is involved in phosphatidylcholine synthesis and platelet-activating factor metabolism, and its downregulation is closely related to loss of vascular wall integrity and inflammatory response, which has a clear pathophysiological basis. CHPT1The AUC for transcriptome mRNA expression level was 0.829 (p < 0.001), with a sensitivity of 60.0% and a specificity of 96.7%. The AUC for average methylation of DNA promoter regions was 0.925 (p < 0.001), with a sensitivity of 86.0% and a specificity of 94.0%. These results indicate high diagnostic value for cerebral aneurysms. CHPT1 The diagnostic value is higher when methylation and transcriptome expression levels are combined, with an AUC of 0.968, a sensitivity of 96.7%, and a specificity of 86.7%. Using peripheral blood samples avoids the risks and discomfort of invasive procedures such as DSA, making it suitable for large-scale screening and follow-up monitoring of high-risk populations.

[0012] In summary, the detection kit based on the detection of CHPT1 gene methylation level in this invention can conveniently and rapidly detect cerebral aneurysms at the molecular level. It boasts high detection efficiency and strong specificity, facilitating early detection and timely treatment of cerebral aneurysms. CHPT1 Drugs targeting gene methylation levels represent an innovative application for the auxiliary diagnosis, detection, and screening of cerebral aneurysms. Attached Figure Description

[0013] Figure 1 To show the distribution of different types of methylation on each chromosome through genome-wide methylation alignment analysis; Figure 2 To compare the differences in the amount of DNA methylation in the whole genome between the case group and the control group samples; Figure 3 To screen samples from the case group and the control group CHPT1 Comparison of methylation rates in genes, * indicates P value < 0.05; Figure 4 For the selected location CHPT1 The target sequence of the gene promoter region and the distribution of 13 CpG methylation sites; Figure 5 The DNA methylation sequencing profile shows that the methylation levels of CpG1 to CpG13 are 21%, 17%, 15%, 21%, 20%, 20%, 22%, 15%, 13%, 22%, 22%, 24%, and 22%, respectively. Figure 6 To verify the difference between the cerebral aneurysm group and the control group CHPT1 Differences in DNA methylation are compared, *** indicates P < 0.001; Figure 7 for CHPT1 ROC curve analysis of methylation at 13 CpG sites in the gene for the diagnosis of cerebral aneurysms; Figure 8 Plasma in the cerebral aneurysm group and the control group CHPT1Transcriptome mRNA expression level analysis, *** indicates P < 0.001; Figure 9 For the comparative analysis of plasma CHPT1 protein blots in the cerebral aneurysm group and the control group, *** indicates P < 0.001; Figure 10 for CHPT1 ROC curve analysis of the combined diagnosis of cerebral aneurysms using average methylation and transcriptome expression levels. Detailed Implementation

[0014] The present invention will be further described in detail below with reference to the accompanying drawings and embodiments.

[0015] Specific Example 1: Discovery from Whole Genome Methylation Sequencing CHPT1 Significant differences in methylation were observed.

[0016] 1. Clinical data of volunteers: This study collected data from 5 patients (3 males, 2 females) with cerebral aneurysms hospitalized in the neurosurgery department of a hospital between January and July 2018, and 6 age- and sex-matched controls (3 males, 3 females). The control group was carefully matched for sex and age, and excluded patients with acute cerebrovascular disease; primary diagnoses included trigeminal neuralgia and hemifacial spasm. Exclusion criteria included: severe cardiovascular and cerebrovascular diseases; end-stage or severe hepatic and renal insufficiency; malignant tumors; and other diseases leading to multiple organ failure. The diagnosis of cerebral aneurysms was based on cranial CT or MRI results, with digital subtraction angiography (DSA) used when necessary. Initial assessment was performed by a physician, followed by review by a senior physician to ensure consistency between diagnosis and classification. This study has been approved by the ethics committee of a hospital. After obtaining informed consent from all participants, 4 mL of peripheral venous blood was collected from patients within 6 hours of admission and placed in an anticoagulant tube. The upper plasma layer and the middle leukocyte layer were carefully collected after centrifugation at 3000 rpm for 15 minutes at 4°C.

[0017] 2. DNA extraction and quality testing Genomic DNA was extracted from peripheral blood leukocytes using the Qiagen Genomic DNA Extraction Kit (Qiagen, Germany). DNA sample quality was determined using Nanodrop, and samples typically met the following requirements: OD260 / OD280 ≥ 1.8, OD260 / OD230 ≥ 1.5.

[0018] 3. Whole-genome DNA methylation sequencing (1) DNA fragmentation: The extracted genomic DNA was processed using an enzymatic fragmentation method (AIRT™ DNAFragmentation Kit, Bioo Scientific, USA) to obtain randomly distributed small DNA fragments.

[0019] (2) End Repair and Fragment Screening: T4 DNA polymerase and Klenow enzyme were used to blunt the ends of the fragmented DNA. Klenow enzyme has a dual function: its 3′→5′ exonuclease activity removes the 3′ overhang, and its polymerase activity fills in the 5′ recess. Simultaneously, T4 polynucleotide kinase (T4 PNK) phosphorylates the 5′ end of the DNA fragment. After end repair, Agencourt AMPure XP magnetic beads were used to enrich and purify DNA fragments in the 300-400 bp range.

[0020] (3) Adapter ligation: A single adenine (A) base is added to the 3′ end of the purified DNA fragment to form a sticky end, thereby reducing inter-fragment self-ligation during the adapter ligation process. The methylated sequencing adapter (with a single thymine T base at its 3′ end) is specifically ligated to the above DNA fragment, wherein the adapter sequence matches the amplification primers on the flow cell of the subsequent sequencing platform, and the purified ligation product is recovered.

[0021] (4) Sulfite conversion: The ligation product is treated with bisulfite to deaminate unmethylated cytosine (C) and convert it to uracil (U), while methylated cytosine (5mC) remains unchanged.

[0022] (5) Library amplification and quality control: The transformed product was amplified by PCR to construct the final sequencing library. The library concentration was initially determined using Qubit 2.0, and after dilution to 1 ng / μL, the insert size was detected using an Agilent 2100 Bioanalyzer. After confirming that the fragment size met expectations, the effective concentration of the library was accurately quantified by RT-qPCR (requirement >2 nM).

[0023] (6) Sequencing: The qualified sequencing libraries were pooled according to the effective concentration and target data volume requirements, and the HiSeq 4000 sequencing platform was used for paired-end sequencing.

[0024] 4. Whole-genome methylation sequencing results Genome alignment analysis revealed significant differences in the distribution of different types of methylation in human chromosomes. For example... Figure 1 As shown, CpG methylation was the most abundant across different chromosomes, significantly higher than CHG and CHH methylation, and the differences in CpG methylation were the most pronounced. Meanwhile, as... Figure 2 As shown, we also found through whole-genome methylation sequencing that the number of hypomethylated sites was significantly higher than the number of hypermethylated sites between cerebral aneurysms and the control group. Figure 3 As shown, through screening, we found that in the cerebral aneurysm group... CHPT1 The methylation rate in the DNA sequence was significantly higher than that in the control group. And as... Figure 4 As shown, CHPT1

[0025] Previous research has shown that hypermethylation in gene promoter regions may participate in the pathological process of diseases by regulating gene expression. To investigate... CHPT1 The correlation between gene epigenetic modifications and cerebral aneurysms Figure 4 As shown, we selected CHPT1 The methylation islands in the gene promoter region were sequenced using the sequence chr12:101697267-101698588. Methylation sequencing was performed using a Pyromark Q24 pyrosequencing system (Qiagen). After evaluation using the Pyromark Assay Design software scoring system, we ultimately selected... CHPT1 The promoter region fragment chr12:101697692-101697778 has the nucleotide sequence shown in SEQ ID NO.2: CGGCAGCCGGGCAGGCCG GCCTGACCTCGACCTCCGCCGTGCGGGCCCGACCGGTGAGTCCAGCCCGGCAGTCGCAGGACCCGGCCG Further research was conducted. PyroMark Assay Design software identified 13 CpG sites in this region for quantitative methylation analysis. The specific locations of the 13 CpG sites near the CHPT1 gene promoter region are shown in Table 1: Table 1. Locations of 13 CpG sites near the CHPT1 gene promoter region

[0026] Specific Implementation Example 2: Expanded Clinical Sample Validation CHPT1 Significant differences in methylation were observed.

[0027] 1. Clinical data of volunteers: This study included 80 patients with cerebral aneurysms (40 males, 40 females, mean age 52.79 ± 9.472 years, p > 0.05) and 80 age- and sex-matched controls (40 males, 40 females, mean age 53.21 ± 10.47 years, p > 0.05) who were hospitalized in the neurosurgery department of a certain hospital from January to December 2024 (Table 2). The inclusion and exclusion criteria for this study were consistent with those used in the first phase of clinical sample collection. The initial diagnosis was made by the attending physician and subsequently confirmed by a senior physician. Written informed consent was obtained from all participants, and the study protocol was approved by the ethics review committee of the hospital. After obtaining informed consent from all participants, 4 mL of peripheral venous blood was collected from patients within 6 hours of admission and placed in an anticoagulant tube. The blood was then carefully extracted after centrifugation at 3000 rpm for 15 minutes at 4°C, followed by the removal of the upper plasma layer and the middle leukocyte layer. Routine biochemical indicators, including total cholesterol, triglycerides, low-density lipoprotein, high-density lipoprotein, lipoprotein a, apolipoprotein A1, apolipoprotein B, and apolipoprotein E, were examined using an automated biochemical analyzer (Olympus AU2700, Japan). The results are shown in Table 2.

[0028] Table 2. Comparison of clinical data between the clinical cerebral aneurysm case group and the control group.

[0029] As shown in Table 2, there were no significant differences between the two groups in terms of demographic characteristics (age, sex). However, in terms of blood biochemical indicators, patients with cerebral aneurysms had significantly elevated blood glucose levels and significantly decreased apolipoprotein A1 levels, reflecting more pronounced metabolic stress and the burden of unhealthy lifestyles.

[0030] 2. Genomic DNA methylation level determination: For quantitative DNA methylation analysis, genomic DNA was extracted from peripheral blood leukocytes using the Qiagen Genomic DNA Extraction Kit (Qiagen, Germany). After DNA concentration was measured using a nucleic acid protein analyzer, DNA was converted to bisulfite using the QIAGEN EpiTect Bisulfite Treatment Kit (Qiagen, Germany). Primer design was performed using PyroMark Assay Design 2.0 software. CHPT1 The specific primer sequences for pyrosequencing were designed, and the PCR amplification primers and sequencing primers used in the experiment are as follows: CHPT1 The nucleotide sequence of the gene DNA methylation-specific forward primer is shown in SEQ ID NO.3: 5'-GGTAGAGTGGTTTTGTAGTGGTTAATA-3'; CHPT1 The nucleotide sequence of the gene DNA methylation-specific downstream primer is as follows (SEQ ID NO.4): 5'-Biotin-CCCCAACCCCAAAAACTATAA-3'; CHPT1 The nucleotide sequence of the DNA methylation-specific sequencing primer is SEQ ID NO.5 as follows: 5'- GGTTTTGTAGTGGTTAATATTG-3'.

[0031] Figure 5 Showing CHPT1 Example of gene methylation level detection results. Figure 5 The percentages shown represent the methylation level of the corresponding CpG sites. The methylation levels of CpG1 to CpG13 are 21%, 17%, 15%, 21%, 20%, 20%, 22%, 15%, 13%, 22%, 22%, 24%, and 22%, respectively.

[0032] Figure 6 shows that, compared with the control group, the methylation levels of methylation sites 1, methylation sites 3-13, and the average methylation level were significantly increased in patients with cerebral aneurysms (P<0.001). Figure 7 The results of receiver operating characteristic (ROC) curve analysis showed that CHPT1Methylation showed good predictive power for cerebral aneurysms, specifically as follows: Methylation site 1, AUC=0.8380, sensitivity 68.0%, specificity 90.0%; Methylation site 2, AUC=0.5752, sensitivity 44.0%, specificity 78.0%; Methylation site 3, AUC=0.8224, sensitivity 74.0%, specificity 88.0%; Methylation site 4, AUC=0.8084, sensitivity 82.0%, specificity 68.0%; Methylation site 5, AUC=8954, sensitivity 76.0%, specificity 94.0%; Methylation site 6, AUC=9070, sensitivity 78.0%, specificity 90.0%; Methylation site 7, AUC=0.7770, sensitivity 82.0%, specificity 66.0%; Methylation site 8, AUC=0.9136, sensitivity 80.0%, specificity 94.0%; Methylation site 9, AUC=0.9050, sensitivity 84.0%, specificity 86.0%; Methylation site 10, AUC=0.8394, sensitivity 90.0%, specificity 66.0%; Methylation site 11, AUC=0.8962, sensitivity 88.0%, specificity 84.0%; Methylation site 12, ... AUC=0.9298, sensitivity 84.0%, specificity 92.0%; methylation site 13, AUC=0.8872, sensitivity 76.0%, specificity 90.0%.

[0033] 3. CHPT1 Clinical relevance to patients with cerebral aneurysms: Subsequently, we further clarified CHPT1Transcriptome expression in the blood of clinical patients. In a control group of 80 patients, we selected 30 age- and sex-matched pairs of patients with cerebral aneurysms and control groups. Total RNA was extracted from blood using the RNA Blood Mini Kit (Qiagen, Germany). The simplified steps are as follows: Total RNA was extracted from 200 μL of fresh peripheral blood samples, strictly following the kit instructions. The concentration and purity of the extracted RNA were determined using a NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific, USA) (A260 / A280 ratio between 1.8 and 2.0). 500 ng of total RNA was used for reverse transcription using the PrimeScript RT Reagent Kit with gDNA Eraser (TaKaRa, Japan). First, the gDNA Eraser was added and incubated at 42°C for 2 min to remove genomic DNA contamination. Then, the reverse transcription mixture was added, and the reaction was terminated at 37°C for 15 min, followed by inactivation at 85°C for 5 sec. The resulting cDNA was stored at -20°C for later use.

[0034] Primer Primer 6 software was used to analyze... CHPT1 Primer design for RT-qPCR amplification of the transcriptome (NM_020244.3). Selection of gene promoter regions. CHPT1 The specific sequence of the fragment in the 455-589 region of the transcriptome (NM_020244.3) is shown in SEQ ID NO.6: CGCTCGTGCTCATCTCCTACTGTCCCACGGCCACCGAAGAGGCACCATACTGGACATACCTTTTATGTGCACTGGGACTTTTTATTTACCAGTCACTGGATGCTATTGATGGGAAACAAGCCAGAAGAACAAACT.

[0035] CHPT1 The nucleotide sequence of the forward amplification primers for the transcriptome is shown in SEQ ID NO.7: 5'-CGCTCGTGCTCATCTCCTACTG-3'. CHPT1 The nucleotide sequence of the reverse amplification primer for the transcriptome is shown in SEQ ID NO.8: 5'-AGTTTGTTCTTCTGGCTTGTTTCCC-3'.

[0036] Commercially available quantitative RT-qPCR kits [SYBR Green PCR Master Mix (Roche, USA)] were used, and qRT-PCR amplification was performed on a LightCycler 480 real-time PCR instrument according to the manufacturer's instructions. PCR results were analyzed using 2... ΔΔCT The comparison was performed as follows: A = CT(target gene, test sample) - CT(internal control gene, test sample); B = CT(target gene, control sample) - CT(internal control gene, control sample); K = AB; fold change = 2 -K As shown in Figure 8, RT-qPCR analysis indicated... ， Compared with the control group, patients with cerebral aneurysms CHPT1 Transcriptome expression was significantly reduced (P<0.001).

[0037] To further elucidate the changes in CHPT1 protein levels in patient blood, Western blot analysis was performed on plasma protein concentrations. The plasma protein detection procedure was simple as follows: Blood samples were centrifuged at 3000 rpm for 10 minutes at 4°C to collect the supernatant plasma. Protein quantification was performed on the plasma samples using a BCA protein assay kit (Beyotime, China). An equal volume of protein (30 μg / lane) was mixed with 5×SDS loading buffer and denatured at 95°C for 10 min. Protein samples were separated by 10% SDS-polyacrylamide gel electrophoresis (SDS-PAGE) under the following conditions: constant voltage of 80 V for 30 min, followed by constant voltage of 120 V for 60 min. Proteins were transferred to a polyvinylidene fluoride membrane (PVDF membrane, Millipore, USA) using a wet transfer method under the following conditions: constant current of 300 mA for 120 min. After transfer, the membrane was blocked with 5% skim milk (dissolved in TBST buffer) at room temperature for 1 h. Add CHPT1 primary antibody (1:1000 dilution, Santa Cruz, USA, Cat.SC-515577) or Transferrin internal control antibody (1:5000, Affinity, Cat.BF0720), and incubate overnight at 4°C. Wash the membrane three times with TBST buffer, 10 min each time. Add horseradish peroxidase (HRP)-labeled secondary antibody (1:5000 dilution), and incubate at room temperature for 1 h. Wash the membrane three times with TBST buffer, 10 min each time. Develop using enhanced chemiluminescent substrate (ECL, Neo-Syneema, China), and acquire images using a gel imaging system (Bio-Rad, USA). Quantitative analysis of the band gray values ​​was performed using ImageJ software, and the relative protein expression level of CHPT1 was expressed as the CHPT1 / Transferrin gray ratio. The results are shown in Figure 9. Compared with the control group, the CHPT1 protein level in patients with cerebral aneurysms was significantly reduced (P<0.001). Figure 10 The results of the ROC curve analysis show that, CHPT1 Transcriptome mRNA expression levels (AUC = 0.829, p < 0.001, sensitivity 60.0%, specificity 96.7%) and average methylation of DNA promoter regions (AUC = 0.925, p < 0.001, sensitivity 86.0%, specificity 94.0%) have high diagnostic value for cerebral aneurysms. CHPT1 The diagnostic value is higher when methylation and transcriptome expression levels are combined (AUC=0.968, sensitivity 96.7%, specificity 86.7%).

[0038] The foregoing description is not intended to limit the invention, nor is the invention limited to the examples given. Any changes, modifications, additions, or substitutions made by those skilled in the art within the scope of the invention should also be considered within the protection scope of the invention.

Claims

1. Used to assist in the diagnosis or detection of cerebral aneurysms. CHPT1 Gene methylation detection kit, characterized in that: Including detection CHPT1 The reagent for the gene promoter region target sequence, as described CHPT1 The target sequence of the gene promoter region is shown in SEQ ID NO.2: CGGCAGCCGGGCAGGCCGGCCTGACCTCGACCTCCGCCGTGCGGGCCCGACCGGTGAGTCCAGCCCGGCAGTCGCAGGACCCGGCCG.

2. The device for assisting in the diagnosis or detection of cerebral aneurysms as described in claim 1. CHPT1 Gene methylation detection kit, characterized in that: The reagent includes CHPT1 upstream primers for gene methylation-specific amplification CHPT1 Gene methylation-specific amplification downstream primers and CHPT1 Gene methylation-specific sequencing primers, as described CHPT1 The nucleotide sequence of the upstream primer for gene methylation-specific amplification is shown in SEQ ID NO.3: 5'-GGTAGAGTGGTTTTGTAGTGGTTAATA-3'; CHPT1 The nucleotide sequence of the downstream primer for gene methylation-specific amplification is shown in SEQ ID NO.4: 5'-Biotin-CCCCAACCCCAAAAACTATAA-3'; CHPT1 The nucleotide sequence of the gene methylation-specific sequencing primer is shown in SEQ ID NO.5: 5'-GGTTTTGTAGTGGTTAATATTG-3'.

3. The device for assisting in the diagnosis or detection of cerebral aneurysms as described in claim 2. CHPT1 Gene methylation detection kit, characterized in that: Also includes CHPT1 Primers for RT-qPCR quantitative amplification of the transcriptome.

4. The device for assisting in the diagnosis or detection of cerebral aneurysms as described in claim 3. CHPT1 Gene methylation detection kit, characterized in that: The aforementioned CHPT1 Primers for RT-qPCR quantitative amplification of the transcriptome include CHPT1 Forward amplification primers for the transcriptome and CHPT1 The reverse amplification primers for the transcriptome, as described CHPT1 The nucleotide sequence of the forward amplification primers for the transcriptome is shown in SEQ ID NO.7: 5'-CGCTCGTGCTCATCTCCTACTG-3'. CHPT1 The nucleotide sequence of the reverse amplification primer for the transcriptome is shown in SEQ ID NO.8: 5'-AGTTTGTTCTTCTGGCTTGTTTCCC-3'.

5. Used to assist in the diagnosis or detection of cerebral aneurysms. CHPT1 Gene methylation detection kit, characterized in that: Including detection CHPT1 Transcriptome reagents, as described CHPT1 The transcriptome sequence is shown in SEQ ID NO.6, and the reagents include... CHPT1 Primers for RT-qPCR quantitative amplification of the transcriptome.

6. The device for assisting in the diagnosis or detection of cerebral aneurysms as described in claim 5. CHPT1 Gene methylation detection kit, characterized in that: The aforementioned CHPT1 Primers for RT-qPCR quantitative amplification of the transcriptome include CHPT1 Forward amplification primers for the transcriptome and CHPT1 The reverse amplification primers for the transcriptome, as described CHPT1 The nucleotide sequence of the forward amplification primers for the transcriptome is shown in SEQ ID NO.7: 5'-CGCTCGTGCTCATCTCCTACTG-3'. CHPT1 The nucleotide sequence of the reverse amplification primer for the transcriptome is shown in SEQ ID NO.8: 5'-AGTTTGTTCTTCTGGCTTGTTTCCC-3'.

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