Application of methylation level of ALX4 gene in specific region in diagnosis and prediction of colorectal cancer

Abstract

The invention discloses application of a reagent for detecting the methylation level of a specific region of an ALX4 gene in preparation of a kit for diagnosing colorectal cancer or predicting the risk of the colorectal cancer, and belongs to the technical field of cancer molecular diagnosis. Wherein the specific region comprises at least one part of chr11: 44311452-44311645, and the specific region comprises at least one part of chr11: 44311452- The methylation level of a specific region of the ALX4 gene is provided as a marker for early diagnosis or prediction of colorectal cancer, and the choices of technicians in the field are enriched. The methylation level of a specific region of the ALX4 gene is detected by adopting a methylated DNA co-immunoprecipitation technology, the sensitivity is high, the specificity is strong, and the kit has very important clinical application value.

Description

Technical Field

[0001] The present invention belongs to the technical field of cancer molecular diagnosis. Specifically, it relates to the application of the methylation level of a specific region of the ALX4 gene in the diagnosis and prediction of colorectal cancer. Background Art

[0002] Colorectal cancer is one of the most common malignant tumors globally, and its incidence and mortality rates are showing an increasing trend year by year. In China, according to the data of the National Cancer Center in 2023, colorectal cancer has surpassed gastric cancer and become the second most common cancer in terms of incidence. Moreover, nearly 80% of patients are diagnosed at the middle or advanced stage, and nearly half of the patients have a survival period of less than 5 years. Therefore, reducing the incidence and mortality of colorectal cancer has become a major public health problem that urgently needs to be solved in China and even globally.

[0003] According to the multi-stage theory of the carcinogenesis process, the occurrence of colorectal cancer morphologically shows a staged evolution from normal mucosal hyperplasia, adenoma formation, adenoma carcinogenesis to invasion and metastasis. It takes 10 - 15 years for an adenoma to evolve into colorectal cancer. The cure rate for early detection of colorectal cancer can reach over 90%, while that for advanced cancer is less than 10%. Intervention through screening is an effective measure to reduce the incidence and mortality of colorectal cancer.

[0004] Colonoscopy is the gold standard for colorectal cancer screening. However, due to its strong invasiveness and cumbersome bowel preparation, the compliance of Chinese people with colonoscopy screening is relatively low. In addition, the demand for colonoscopy is large. With the aging of the population, the number of the elderly and people over 40 years old is increasing, and the number of colonoscopy examinations has shown a blowout growth. Large-scale application of colonoscopy as a screening method will also cause a great waste of resources. The traditional screening program adopts a two-step screening mode combining questionnaire surveys with two fecal occult blood tests (FIT). For those with any positive result, they are determined to be positive in the primary screening, indicating a high-risk group and requiring colonoscopy examination. This screening has problems such as too high false positives and low detection rate of colorectal cancer, and this screening mode results in insufficient human resources input in hospitals, thus the project progresses slowly.

[0005] For the colorectal cancer auxiliary diagnosis technology based on blood DNA detection, the currently marketed products generally have low sensitivity, all less than 85%, and cannot meet the clinical needs. Clinical practice shows that the main limitation of blood Septin9 methylation detection lies in its relatively low sensitivity in identifying both colorectal cancer and precancerous lesions (adenomas). The sensitivity for advanced adenomas is only 7.9% to 38.7%.

[0006] The colorectal cancer screening technology based on fecal DNA detection mainly targets the characteristics of gene mutations and / or methylation of colorectal exfoliated cells, overcoming the main defects of detecting microbleeding. There are single-target and multi-target schemes, and it can also be combined with FIT for detection. It has the advantages of no need for special equipment, no dietary restrictions, and non-invasiveness. Although there have been significant improvements in the sensitivity and specificity of colorectal cancer, the detection rate for 0-II early cancers is still below 90%, and the detection rate of advanced adenomas such as precancerous lesions and high-grade intraepithelial neoplasia is even lower, all less than 65% or even lower. Although fecal DNA testing can be achieved by sampling at home, fecal sampling is relatively private and inconvenient, which is different from the public's medical habits. It will take a very long time for the public to accept fecal DNA testing for health education, so there are still certain problems with testing compliance in the screening process.

[0007] Therefore, developing new colorectal cancer markers with higher sensitivity based on blood samples with the highest clinical user compliance can become an urgent issue to be solved. Summary of the invention

[0008] In order to solve the above technical problems, the inventors of the present invention have made great efforts to develop effective colorectal cancer methylation markers in the blood based on non-bisulfite technology, which markers make early diagnosis of cancer and cancer risk possible. Unexpectedly, it was found that a specific region of the ALX4 gene is methylated in colorectal cancer cells. Using this gene as a biomarker, the methylation level of this gene is detected by a non-bisulfite-treated methylation detection system, which has high sensitivity and can diagnose colorectal cancer, thereby completing the present invention.

[0009] In a first aspect, the present invention provides use of a detection reagent for the methylation level of a specific region of the ALX4 gene in the preparation of a kit for diagnosing colorectal cancer or predicting the risk of colorectal cancer.

[0010] The ALX homeobox 4 (ALX4) gene encodes a paired-like homeodomain transcription factor that is expressed in the mesenchyme of developing skeleton, limbs, hair, teeth, and mammary gland tissue. Mutations in this gene cause parietal foramen 2 (PFM2); an autosomal dominant disorder characterized by insufficient ossification of the parietal bones. Mutations in this gene also cause a form of frontonasal dysplasia with alopecia and hypogonadism; suggesting a role for this gene in craniofacial development, mesenchymal-epithelial communication, and hair follicle development. Deletion of a segment of chromosome 11 containing del(11)(p11p12) of this gene causes Potocki-Shaffer syndrome (PSS): a syndrome characterized by male craniofacial dysmorphism, cognitive impairment, multiple exostoses, and genital anomalies. In mice, this gene has been shown to use dual translation start sites separated by 16 codons.

[0011] Almost all tumors are caused and promoted by genetic alterations and epigenetic variations. By comparing tumor cells and normal cells, a large number of epigenetic abnormalities have been discovered and reported, among which DNA methylation is the most common epigenetic effect.

[0012] Methylation of cytosine on DNA is a covalent DNA "epigenetic" modification. DNA methylation is carried out by DNA cytosine methyltransferases (DNMTs). DNMTs can transfer a methyl group from an S-adenosylmethionine to the C-5 position of cytosine. DNA methylation occurs almost specifically at CpG dinucleotide positions, and CpG dinucleotides are unevenly distributed in the human genome. Regions with concentrated enrichment are generally called CpG islands. Such CpGs exist in repetitive sequences of the human genome and regulatory regions at the 5' end of many genes. DNA methylation abnormalities in tumors include hypomethylation (or demethylation) and hypermethylation. Hypermethylation occurs in, but is not limited to, tumor suppressor genes, and hypomethylation occurs in, but is not limited to, proto-oncogenes.

[0013] All aspects of tumorigenesis and development may be related to DNA methylation changes, involving cell cycle regulation, DNA damage repair, biochemical metabolism of carcinogenic compounds, apoptosis, and angiogenesis. Different types of tumors may have a specific group of tumor suppressor genes hypermethylated and proto-oncogenes hypomethylated, that is, there is a specific methylation map for a cancer type, and the type of cancer may be confirmed based on the methylation map.

[0014] In the present invention, through long-term exploration and verification with a large number of clinical samples, the inventors unexpectedly found that the methylation level of a specific region of the ALX4 gene is significantly different between colorectal cancer and non-colorectal cancer.

[0015] In some embodiments of the present invention, the methylation region of the specific region of the ALX4 gene is obtained in colorectal cancer population samples and normal samples using a probe combination, and the coverage information of each probe in the probe combination is as follows:

[0016] 。

[0017] In some specific embodiments of the present invention, the specific region includes at least a part of chr11:44311452-44311645, and there are 11 methylation sites in chr11:44311452-44311645, which are chr11:44311462-44311463, chr11:44311480-44311481, chr11:44311517-44311518, chr11:44311520-44311521, chr11:44311522-44311523, chr11:44311557-44311558, chr11:44311570-44311571, chr11:44311574-44311575, chr11:44311581-44311582, chr11:44311608-44311609, and chr11:44311642-44311643 respectively.

[0018] In some preferred embodiments of the present invention, the specific region may include one or more of the above methylation sites, which may be continuous or spaced. In some more preferred embodiments of the present invention, the specific region includes 5 methylation sites, namely chr11:44311570-44311571, chr11:44311574-44311575, chr11:44311581-44311582, chr11:44311608-44311609, and chr11:44311642-44311643. In some most preferred embodiments of the present invention, the specific region includes chr11:44311570-44311643. Further, the methylation region can be extended by several bases on this basis. In some specific embodiments of the present invention, the specific region includes chr11:44311570-44311644.

[0019] In some embodiments of the present invention, the detection reagent includes a methylation region enrichment or methylation treatment reagent, and also includes a qPCR detection reagent.

[0020] Methylation enrichment technology is an analytical method for studying methylation modifications on DNA. DNA methylation is an important epigenetic modification, which involves the addition of a methyl group to the cytosine ring in the DNA molecule. This modification plays a key role in regulating biological processes such as gene expression, cell differentiation, and genomic stability. Therefore, understanding the status of DNA methylation is very important for understanding biological processes and the occurrence and development of diseases.

[0021] Common methylation enrichment techniques include methylation-specific PCR (MSP), methylation-sensitive restriction enzyme digestion, MeDIP-Seq (Methylated DNA Immunoprecipitation sequencing), and MBD-Seq (Methyl-CpG Binding Domain sequencing), among which:

[0022] MSP uses methylation-specific primers to selectively amplify methylated DNA fragments by PCR. It is simple, fast, and suitable for the analysis of specific CpG sites, but it cannot provide genome-wide methylation information and is only applicable to pre-determined target regions.

[0023] Methylation-sensitive restriction enzyme digestion utilizes the sensitivity differences of restriction enzymes to DNA sequences to distinguish methylated and non-methylated DNA regions. This method is based on the principle that DNA methylation affects the sensitivity of base pairs on the cytosine ring to restriction enzymes. This technique does not require the use of expensive sequencing technologies and can be analyzed by methods such as gel electrophoresis. However, it cannot provide high-resolution information on individual CpG sites and usually provides the methylation status of the overall region. At the same time, it is limited by the specificity of the selected restriction enzyme, and some methylated sites may be missed or over-detected. Additionally, it cannot directly distinguish 5-methylcytosine from other forms of DNA modification.

[0024] MeDIP-Seq uses methylated DNA antibodies to selectively enrich methylated DNA fragments, and then analyzes the enriched material through high-throughput sequencing technology. It can enrich the entire methylated genomic region and is suitable for genome-wide methylation analysis. However, it cannot provide high-resolution information on individual CpG sites.

[0025] MBD-Seq uses methylated DNA-binding proteins (such as MBD2 or MBD3) to enrich methylated DNA fragments, and then analyzes them through sequencing. It can provide a relatively high enrichment efficiency and is suitable for genome-wide methylation analysis. However, similar to MeDIP-Seq, it cannot provide high-resolution information on individual CpG sites.

[0026] In the present invention, the methylation treatment is also referred to as methylation conversion. Common methylation treatment sequencing techniques include bisulfite sequencing (BS-seq). BS-seq uses bisulfite to treat DNA, converting unmethylated cytosine into uracil, while methylated cytosine remains unaffected, and then analysis is performed through sequencing. It can provide high-resolution information on individual CpG sites and can perform methylation analysis on the entire genome. However, the experimental steps are relatively cumbersome.

[0027] In some embodiments of the present invention, the methylated DNA immunoprecipitation (MeDIP) technique is used to enrich methylated fragments. The methylated DNA antibody is selected from one of the 5-methylcytidine antibody, 5-methylcytosine (5-mC) antibody, 5-hydroxymethylcytosine (5-hmC) antibody, 5-formylcytosine (5-fC) antibody, and 5-carboxylcytosine (5-caC) antibody.

[0028] In some preferred embodiments of the present invention, the methylation region enrichment reagent includes a 5-methylcytosine antibody.

[0029] Furthermore, the qPCR detection reagent includes a primer pair and a probe targeting the specific region.

[0030] Furthermore, the specific region includes at least a part of chr11:44311452 - 44311645. Preferably, the methylation region is chr11:44311570 - 44311644. The primer pair is as shown in SEQ ID No.1 and SEQ ID No.2, and the probe is as shown in SEQ ID No.3.

[0031] In some other embodiments of the present invention, the methylation treatment reagent includes bisulfite.

[0032] Furthermore, the qPCR detection reagent includes a primer pair and a probe targeting the sequence after methylation treatment.

[0033] Even further, the methylation region includes at least a part of chr11:44311452 - 44311645. Preferably, the methylation region is chr11:44311570 - 44311644. The primer pair is as shown in SEQ ID No.4 and SEQ ID No.5, and the probe is as shown in SEQ ID No.6.

[0034] It is possible to achieve early detection of cancer by performing relevant methylated DNA analysis on a certain type of cancer. The current mainstream methylation analysis method is bisulfite treatment, which includes denaturation, deamination and desulfonation. DNA is first denatured into a single strand, and then subjected to high temperature, high salt, acidic and alkaline environments, encountering two extremes of ice and fire. The morphology of the converted DNA obtained is: mainly single strand, double strand mixed, fragment nicks, gap damage, and uracil state nucleotides. This process generally causes 90% of the DNA template to be lost, and a large amount of methylation information cannot be detected by subsequent processes. At the same time, during the base conversion treatment, there is an incomplete sequence conversion or over-conversion, which produces artificial bias, and the subsequent PCR amplification will further amplify it, resulting in inaccurate signals. Therefore, the methylation markers obtained based on bisulfite treatment generally have the problem of low sensitivity, especially in blood samples. After the limited number of free DNA fragments are treated with bisulfite, the difficulty of detecting methylation levels is greatly increased.

[0035] Although the sensitivity for colorectal cancer can be improved by increasing the number of methylated genes based on bisulfite treatment, such as the combined detection of 3 colorectal cancer gene methylation by Beijing Aikelun Medical Technology Co., Ltd., which was approved by the National Medical Products Administration NMPA in 2022. The sensitivity of clinical trials using blood sample cfDNA treated with bisulfite was 84.75% (328 / 387), but the sensitivity still cannot meet the actual clinical needs. Therefore, trying to discover colorectal cancer-related gene methylation markers in human blood samples based on non-bisulfite treatment methods and effectively detect changes in their methylation levels has also become the most urgent need for early cancer screening.

[0036] The second aspect of the present invention provides the use of a detection reagent for the methylation level of a specific region of the ALX4 gene in the preparation of a kit for diagnosing colorectal cancer or predicting the risk of colorectal cancer based on the following method:

[0037] S1, obtain cfDNA samples from biological samples of subjects;

[0038] S2, enriching methylated regions of cfDNA samples or performing methylation treatment;

[0039] S3, using the product enriched or treated in step S2 as a template, and using a primer pair and a probe targeting the untreated or treated specific region to perform qPCR amplification;

[0040] If there is a typical amplification curve and the Ct value is not greater than the preset threshold, the test subjects are diagnosed with colorectal cancer or have a risk of colorectal cancer.

[0041] The specific region includes at least a part of chr11:44311452-44311645. Preferably, the methylation region includes at least a part of chr11:44311570-44311644.

[0042] An amplification curve is a graph that detects the accumulation of products during the polymerase chain reaction (PCR). It is generated by monitoring the increase in fluorescence signal in the reaction solution. The following are the characteristics of a typical PCR amplification curve:

[0043] Initial stage:

[0044] Threshold Cycle (Ct) value: In the early stage of the PCR reaction, the fluorescence signal may be low, but as the PCR products accumulate, the fluorescence signal gradually increases. The Ct value refers to the number of cycles required in the PCR reaction for the fluorescence signal to rise above a pre-set threshold. A lower Ct value indicates a higher starting amount of the target DNA in the sample.

[0045] Exponential growth stage:

[0046] Exponential phase: In the middle stage of the PCR reaction, the PCR products increase exponentially. At this time, the increase rate of the Ct value will accelerate, reflecting the exponential growth of the target DNA in the PCR reaction.

[0047] Plateau stage:

[0048] Plateau stage: In the later stage of the PCR reaction, the accumulation of PCR products reaches saturation and no longer increases exponentially. The PCR amplification curve in this stage forms a plateau, and the increase of the Ct value becomes slow.

[0049] In some embodiments of the present invention, the preset threshold is determined based on the representative value of the Ct values obtained by the same method for population non-colorectal cancer samples and / or population colorectal cancer samples. The representative value is selected from the mean, mode, median, first quartile or third quartile.

[0050] The third aspect of the present invention provides a kit for diagnosing colorectal cancer or predicting the risk of colorectal cancer based on detecting the methylation level of a specific region of the ALX4 gene by DNA immunoprecipitation. The methylation region is chr11:44311570-44311644. The kit includes a 5-methylcytosine antibody, a primer pair targeting the methylation region, and a probe. The primer pair is shown as SEQ ID No.1 and SEQ ID No.2, and the probe is shown as SEQ ID No.3.

[0051] In some embodiments of the present invention, the diagnosis is an early diagnosis. Specifically, the early stage is stage 0-I or II of CRC.

[0052] Advantages of the present invention

[0053] Compared with the prior art, the present invention has the following technical effects:

[0054] The present invention provides the methylation level of a specific region of the ALX4 gene as a marker for the early diagnosis or prediction of colorectal cancer, enriching the choices of those skilled in the art.

[0055] The present invention uses the methylation DNA immunoprecipitation technique to detect the methylation level of a specific region of the ALX4 gene, which has high sensitivity and strong specificity and has very important clinical application value.

[0056] Based on the methylation level of the ALX4 gene, the present invention can perform early detection of colorectal cancer and provide more marker choices for early screening of colorectal cancer. BRIEF DESCRIPTION OF THE DRAWINGS

[0057] Figure 1 Shows the significantly different regions of the methylation level of the ALX4 gene (IGV visualization) in 8 randomly selected colorectal cancer positive samples and 8 normal healthy individuals.

[0058] Figure 2 Shows the significant difference in the ALX4 methylation level (RPM index) between 548 colorectal cancer positive samples and 389 normal healthy samples.

[0059] Figure 3 Shows the flow chart of the ALX4 methylation gene detection in Example 2 of the present invention.

[0060] Figure 4 Shows an amplification curve (A) based on qPCR detection by the methylation DNA immunoprecipitation enrichment method and an amplification curve (B) based on qPCR detection after bisulfite conversion treatment.

[0061] Figure 5 Shows the ROC curves (A) based on qPCR detection by the methylation DNA immunoprecipitation enrichment method and the ROC curves (B) based on qPCR detection after bisulfite conversion treatment for 85 samples.

[0062] Figure 6 Shows the methylation level of chr11-44309888:44310051 of the ALX4 gene (IGV visualization) in 4 colorectal cancer positive samples and 4 normal healthy individuals. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0063] Unless otherwise specified, implied from the context, or conventional in the art, all parts and percentages in this application are based on weight, and the testing and characterization methods used are contemporaneous with the filing date of this application. Where applicable, any patents, patent applications, or publications referred to in this application are hereby incorporated by reference in their entirety, as well as their equivalent family patents, particularly the definitions of relevant terms in the art disclosed in these documents. If the definition of a specific term disclosed in the prior art is inconsistent with any definition provided in this application, the definition of the term provided in this application shall prevail.

[0064] The numerical ranges in this application are approximate values, and thus, unless otherwise specified, they may include values outside the range. The numerical range includes all values from the lower limit value to the upper limit value in increments of one unit, provided that there is an interval of at least two units between any lower value and any higher value. For ranges that include values less than 1 or include fractions greater than 1 (such as 1.1, 1.5, etc.), one unit is appropriately regarded as 0.0001, 0.001, 0.01, or 0.1. For ranges that include single-digit numbers less than 10 (such as 1 to 5), one unit is typically regarded as 0.1. These are merely specific examples of what is intended to be expressed, and all possible combinations of the values between the lowest and highest values listed are considered to be clearly recited in this application.

[0065] The terms "comprising", "including", "having", and their derivatives do not exclude the presence of any other components, steps, or processes, regardless of whether or not these other components, steps, or processes are disclosed in this application. To eliminate any doubt, unless explicitly stated otherwise, all compositions in this application that use the terms "comprising", "including", or "having" may contain any additional additives, excipients, or compounds. In contrast, the term "consisting essentially of" excludes any other components, steps, or processes from the scope of anything recited below the term, except for those necessary for the operating properties. The term "consisting of" does not include any components, steps, or processes not specifically described or listed. Unless explicitly stated otherwise, the term "or" refers to the individual members listed or any combination thereof.

[0066] In order to make the technical problems, technical solutions, and beneficial effects solved by the present invention more clearly understood, the present invention will be further described in detail below in conjunction with embodiments.

[0067] The following examples are used herein to illustrate the preferred embodiments of the present invention. Those skilled in the art will understand that the techniques disclosed in the following examples represent techniques that the inventors have found can be used to implement the present invention, and thus can be regarded as preferred embodiments for implementing the present invention. However, those skilled in the art should understand from this specification that many modifications can be made to the specific embodiments disclosed herein, and still obtain the same or similar results, without departing from the spirit or scope of the present invention.

[0068] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which this invention belongs. All publications, patent applications, patents, and other references mentioned herein and their incorporated materials will be incorporated by reference.

[0069] Those skilled in the art will recognize or be able to ascertain, using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the claims.

[0070] Unless otherwise specified, the experimental methods in the following examples are all conventional methods. Unless otherwise specified, the instruments and equipment used in the following examples are all conventional laboratory instruments and equipment; unless otherwise specified, the test materials used in the following examples are all obtained from regular biochemical reagent stores.

[0071] Example 1 Discovery of Colorectal Cancer-Specific Methylated Genes

[0072] In order to screen for biomarkers specifically methylated in colorectal cancer, the present invention collected blood samples, corresponding homologous cancer tissues and paired adjacent cancer tissues (if any) from a total of 548 clinical colorectal cancer patients from the First Affiliated Hospital of Zhejiang University School of Medicine and several other central hospitals, as well as blood samples and corresponding tissue samples (if any) from 389 clinical colorectal cancer-negative patients. It is required that the differentially methylated genes or regions screened in the blood samples be verified in the tissue samples at the same time to be more reliable methylation markers with clinical significance.

[0073] 1. Preparation of Methylated DNA Sample Library

[0074] (1) DNA Extraction

[0075] The extraction of cfDNA was performed using a commercial company's extraction kit according to the instructions in the manual.

[0076] Use Qubit 4.0 and Qsep 100 to perform quality control on the concentration and fragment distribution of nucleic acids respectively. The yield of cfDNA extracted from 2 mL of human plasma should be greater than 5 ng, and there should be an enrichment peak at or near 167 bp for cfDNA. When the yield is greater than 50 ng, use Qsep 100 capillary electrophoresis for fragmentation quality control. When there is large fragment contamination, use magnetic beads for fragment screening to remove large fragments.

[0077] Extract genomic DNA from samples such as cancer tissues, adjacent tissues, and normal tissues using a conventional commercial kit or self-prepared reagent according to the method described in the instruction manual. Genomic DNA can be fragmented by sonication or enzymatic digestion to obtain DNA with a length of about 200 bp, which is convenient for library construction.

[0078] (2) Library construction

[0079] A commercial company's library construction kit can be used according to the method described in the instruction manual. For example, use the Rapid Plus DNA LibPrep Kit for illumina (Cat. No. RK20208, ABclonal) or the VAHTS Universal ProDNA Library Prep Kit for illumina Vazyme (Cat. No. ND608-02, Novoprotein) and other similar kits for end repair, adding an "A" tail, and connecting with Adapters. In this example, the VAHTS Universal Pro DNA Library Prep Kit for illumina Vazyme (Cat. No. ND608-02, Novoprotein) is used for library construction.

[0080] (3) Methylated DNA immunoprecipitation

[0081] The methylation enrichment of cfDNA and tissue genomic DNA is carried out in different reactions. Each methylation enrichment reaction can simultaneously mix 12 - 100 cfDNA libraries (the input amount of each cfDNA sample library is about 10 ng), and can simultaneously mix 10 - 24 genomic DNA libraries (the input amount of each genomic DNA library is about 100 ng). The methylation enrichment based on the principle of 5mC antibody is operated according to the method described in the instruction manual of a commercial company's methylation enrichment kit or self-prepared reagent. In this example, the zymoMeDIP kit (product number D5101-A) is used. Then, purify the reaction after methylation enrichment according to the instruction manual, and perform 10 - 12 rounds of conventional PCR amplification with a universal sequencing primer pair to obtain a methylated DNA fragment library with a yield of more than 500 ng for each reaction.

[0082] 2. Prepare the DNA probe library

[0083] (1) Probe design

[0084] The inventors designed probes in accordance with the principle of full coverage without intersection for the sequences within the range including the ALX4 gene, its promoter region, the first exon (exon1), 1 kb upstream of the start codon, as well as the gene intron and the intergenic region between adjacent genes.

[0085] The inventors designed a set of hybridization capture DNA probe libraries consisting of 55 DNA probes, spanning a range of nearly 53 kbp, covering all possible CpG sites.

[0086] (2) Probe synthesis

[0087] The probe coverage information is shown in Table 1.

[0088] Table 1 Probe coverage information

[0089]

[0090]

[0091] Customize the probe panel at Nuoanda (Nanjing) Biotechnology Co., Ltd.

[0092] 3. DNA capture probe hybridization

[0093] Use NadPrep hybridization capture reagent (Cat.No.REF1005101, Nuoanda) for liquid-phase hybridization capture. The hybridization capture reaction can be single hybridization or multiple hybridizations. The total input amount of the MeDIP amplification library for each hybridization capture reaction should be in the range of 300 ng to 8 μg. For the purified library of 500 ng (if less than 500 ng, all will be input), add Human Cot DNA and Nad Nano Blockers, place them in a vacuum concentrator preheated to 42 °C and dry at a rotation speed of 1000 rpm; after drying, add the prepared hybridization reaction solution (containing the above probe panel), oscillate and centrifuge instantaneously, and hybridize and capture for 4 - 16 hours under the hybridization program: 95 °C / 30 sec; 65 °C / Hold (100 °C hot lid); then add the washed streptavidin magnetic beads to the hybridization system and incubate for 40 minutes, vortex and mix evenly every 10 minutes during this period to ensure that the magnetic beads are completely resuspended; it should be noted that the reaction temperature for hybridization capture is the conventional 65 °C, rather than 63 °C for methylation probes designed based on bisulfite conversion.

[0094] After the hybridization capture reaction is completed, wash the bound magnetic beads with the four washing solutions provided in the kit. Discard the residual liquid in each step. Finally, add 20 μL of nuclease-free water and gently vortex to mix well.

[0095] 4. PCR Amplification and Purification after Hybridization Capture

[0096] Perform PCR amplification on the products after hybridization capture. Use the amplification reagents in VAHTS Universal Pro DNA Library Prep Kit for illumina (Cat.No.ND608-02, Vazyme) for the experiment, and the number of cycles is 12 - 13. After the amplification is completed, purify the products with an equal volume of VAHTS DNA Clean Beads (Cat.No.N411-03, Vazyme) to obtain a relatively pure hybridization capture library. Use Qubit 4.0 to quantitatively measure the library concentration, and use Qsep100 fully automatic nucleic acid and protein analyzer to detect the fragment size of the library.

[0097] 5. Library Loading for Sequencing and Bioinformatics Analysis

[0098] Dilute the concentration of the library to be loaded to 4 nM, mix it according to the proportion of the required data volume, and the total data volume should not exceed 120 G. After mixing, take out 5 μL of the library, add 5 μL of 0.2 N NaOH, pipette and mix well, and denature for 5 minutes. Immediately after that, add 990 μL of HT1 Buffer (REF: 15058251, illumina), vortex to mix well, then take out 105 μL and add 1295 μL of HT1 Buffer, vortex to mix well to obtain the library for loading, and the concentration is 1.5 pM.

[0099] The sequencer is NextSeq 550Dx produced by illumina. Use High Output Reagent Cartridge v2 (REF: 15057929, illumina) (300 cycles), High Output Flow Cell Cartridge v2.5 (REF: 20022408, illumina) and Buffer Cartridge v2 (REF: 15057941, illumina). Add 1300 μL of the library for loading to the sample position of High Output Reagent Cartridge v2, and put each reagent in turn to start sequencing. This example uses paired-end sequencing, and the total duration is about 30 hours.

[0100] 6. Quality Control of Sequencing Data

[0101] The off-machine data was quality controlled using Fastp (version 0.22.0) to remove low-quality bases. The overall Q20 of the clean data was above 90%, and Q30 was above 85%. The average sequencing depth was about 300×. The average on-target rate of the probes in the above probe combination was above 80%, indicating that the detection of cancer-related methylation regions based on the combination of methylation immunoprecipitation and liquid hybridization capture probes in this example is feasible and effective.

[0102] 7. Analysis of methylation difference regions of the ALX4 gene related to colorectal cancer

[0103] The DiffBind tool (version 3.8.4) was used to screen for differential peaks between tumors and non-tumors. Two algorithms, DESeq and EdgeR, were used, and the regions within the intersection and within the panel were preferentially screened.

[0104] In this example, from the regions of the ALX4 gene covered by 55 probes, 3 probe-targeted characteristic methylation regions with the most significant differences between the colorectal cancer group and the non-colorectal cancer group were screened out: chr11:44311452-44311645 (Intergenic). The visualization of the IGV map of this characteristic methylation region (i.e., the specific region) is shown in Figure 1 .

[0105] The methylated CpG sites of this characteristic methylation region are shown in Table 2:

[0106] Table 2 CpG sites of the characteristic methylation region

[0107] Chromosomal location Start site End site chr11 44311462 44311463 chr11 44311480 44311481 chr11 44311517 44311518 chr11 44311520 44311521 chr11 44311522 44311523 chr11 44311557 44311558 chr11 44311570 44311571 chr11 44311574 44311575 chr11 44311581 44311582 chr11 44311608 44311609 chr11 44311642 44311643

[0108] The RPM index (Reads per million mapped reads) of this characteristic methylation difference region was analyzed in 548 colorectal cancer positive samples and 389 normal healthy population samples. The P value was 0.000019, indicating a significant difference, as shown in Figure 2 .

[0109] Example 2 Detection of ALX4 methylated gene in clinical samples and comparison of different treatment regimens

[0110] To further verify the clinical performance of the ALX4 gene methylation differential region related to colorectal cancer in CRC plasma samples, the inventors used the qPCR method to detect 35 plasma samples clinically diagnosed with CRC and 50 plasma control samples with negative colonoscopy results. Among the 35 plasma samples clinically diagnosed with CRC, there were 5 samples in CRC stage I, 7 samples in CRC stage II, 12 samples in CRC stage III, and 11 samples in CRC stage IV.

[0111] The flowchart of ALX4 methylation gene detection is as Figure 3 shown, specifically:

[0112] (1) DNA extraction

[0113] The extraction of cfDNA was performed using a commercial company's extraction kit according to the instructions in the manual.

[0114] The concentration and fragment distribution of nucleic acids were quality controlled using Qubit4.0 and Qsep100 respectively. The yield of cfDNA extracted from 4 mL of human plasma should be greater than 10 ng, and there should be an enrichment peak at 167 bp or nearby for cfDNA. When the yield is greater than 50 ng, Qsep100 capillary electrophoresis is required for fragmentation quality control. When there is contamination with large fragments, magnetic beads are used for fragment screening to remove large fragments.

[0115] (2) Methylated DNA treatment

[0116] ① Methylated DNA immunoprecipitation

[0117] Take 1 / 2 of the total amount of the above-extracted nucleic acids for cfDNA methylation enrichment, which is carried out in different reactions. For methylation enrichment based on the 5mC antibody principle, the zymoMeDIP kit (product number D5101-A) is used, and after the methylation enrichment reaction, purification is carried out according to the manual, and the elution volume is 50 μL.

[0118] ② Bisulfite conversion of methylated DNA

[0119] Take 1 / 2 of the total amount of the above-extracted nucleic acids for bisulfite treatment of cfDNA methylation, which is carried out in different reactions. For the treatment of methylated DNA based on the bisulfite conversion principle, the DNA conversion kit (EZ DNA Methylation Kit, D5002) of ZYMO RESEARCH Biotechnology Company is used for bisulfite treatment of DNA. The elution volume is 50 μL.

[0120] (3) qPCR detection

[0121] The primers and probes were synthesized by Shanghai BioGeno Technology Co., Ltd., and the specific sequence information is as follows:

[0122] The sequences of Taqman MGB probe primers enriched by methylated DNA immunoprecipitation are shown in Table 3.

[0123] Table 3 Taqman MGB probe primers enriched by methylated DNA immunoprecipitation

[0124]

[0125] The 3'-end of the probe is labeled with MGB.

[0126] Using the enriched product after methylated DNA immunoprecipitation as the template, PCR amplification was carried out. The final concentration of each primer was 10 μM. The PCR reaction system was 5 μL of enriched template DNA, 2.5 μL of the premixed solution containing the above primers; 17.5 μL of PCR reaction solution reagent (2×Rapid Taq Master Mix), and the total volume was made up to 35 μL with water. The PCR reaction conditions were as follows: 95°C for 5 minutes, 95°C for 15 seconds, 60°C for 40 seconds, and amplified for 48 cycles.

[0127] The sequences of Taqman MGB probe primers after bisulfite conversion are shown in Table 4.

[0128] Table 4 Taqman MGB probe primers after bisulfite conversion

[0129]

[0130] The 3'-end of the probe is labeled with MGB.

[0131] Using the DNA after bisulfite conversion as the template, PCR amplification was carried out. The final concentration of each primer was 10 μM. The PCR reaction system was 5 μL of enriched template DNA, 2.5 μL of the premixed solution containing the above primers; 17.5 μL of PCR reaction solution reagent (2×Rapid Taq Master Mix), and the total volume was made up to 35 μL with water. The PCR reaction conditions were as follows: 95°C for 5 minutes, 95°C for 15 seconds, 60°C for 40 seconds, and amplified for 48 cycles.

[0132] (4) Analysis of clinical sample test results

[0133] The off-machine data was analyzed. The detection results of 85 samples by using the method of enriching methylated DNA immunoprecipitation and the method of treating DNA with bisulfite and then using qPCR are shown in Table 5.

[0134] Table 5 Summary table of qPCR detection results of 85 samples

[0135]

[0136]

[0137]

[0138]

[0139] ALX4 Figure 4 Examples of qPCR amplification curves of samples from the same CRC patient (sample number: 101672) using the methylated DNA immunoprecipitation enrichment method and the bisulfite-treated DNA method are shown. Among them, the Ct value of qPCR amplification after methylated DNA immunoprecipitation enrichment in Figure A is 29.84, and the Ct value of qPCR amplification of bisulfite-treated DNA in Figure B is 39.14. There is an obvious advantage in qPCR detection after methylated DNA immunoprecipitation enrichment.

[0140] Set the Ct value of the sample with the detection result of Undetermined to 48, and draw the ROC curves respectively, as Figure 5 shown. The areas under the ROC curves (AUC) obtained based on the two different methods are 0.840 and 0.683 respectively. According to the ROC curves, set the cut-off values for different methods: for qPCR detection based on the methylated DNA immunoprecipitation enrichment method, the cut-off value is set to Ct = 32.11; for qPCR detection based on bisulfite conversion treatment, the cut-off value is set to Ct = 35.82. If the Ct value of the ALX4 gene amplification of the sample to be monitored is equal to or lower than the set cut-off value, the sample is determined to be a positive sample, otherwise it is determined to be a negative sample. Thus, the detection results of 85 samples are statistically analyzed.

[0141] Table 6 shows the comparison between qPCR detection based on the methylated DNA immunoprecipitation enrichment method and the colonoscopy examination results (gold standard). Table 7 shows the comparison between the qPCR detection results based on bisulfite conversion treatment and the colonoscopy examination results. Table 8 shows the comparison between the qPCR detection results based on the methylated DNA immunoprecipitation enrichment method and the qPCR detection results based on bisulfite conversion treatment.

[0142] Table 6 Comparison of qPCR detection results based on the methylated DNA immunoprecipitation enrichment method with colonoscopy examination results

[0143]

[0144]

[0145] Table 7 Comparison of qPCR detection results based on bisulfite conversion treatment with colonoscopy examination results

[0146]

[0147] Table 8 Comparison of enrichment methods based on methylated DNA immunoprecipitation and bisulfite conversion treatment methods

[0148]

[0149] As can be seen from Tables 6 - 8, when the methylation differential region of the ALX4 gene was verified on the qPCR detection platform based on the enrichment method of methylated DNA immunoprecipitation, it had higher sensitivity (82.86%) for CRC and also maintained high specificity (82.00%) for non - colorectal cancer samples, with an accuracy of 82.35%. Its overall performance was superior to that of the qPCR detection with bisulfite conversion treatment.

[0150] (5) Analysis of the detection results of clinical colorectal cancer staging samples

[0151] Among the above 35 plasma samples clinically diagnosed as CRC, there were 5 samples in CRC stage I, 7 samples in CRC stage II, 12 samples in CRC stage III, and 11 samples in CRC stage IV. The detection and statistical analysis of samples with different CRC pathological stages by qPCR detection based on the enrichment method of methylated DNA immunoprecipitation and qPCR detection with bisulfite conversion treatment are shown in Tables 9 - 10:

[0152] Table 9 Detection of samples with different CRC pathological stages

[0153]

[0154] Table 10 Statistical analysis of sensitivity for different CRC pathological stages

[0155]

[0156]

[0157] As can be seen from Tables 9 - 10, when the methylation differential region of the ALX4 gene was verified on the qPCR detection platform based on the enrichment method of methylated DNA immunoprecipitation, it also maintained high sensitivity (80.00%) for CRC stage I samples. Its performance for early CRC detection was superior to that of the qPCR detection with bisulfite conversion treatment, providing a new potential biomarker for the early detection of colorectal cancer.

[0158] Example 3 Detection results of different methylation regions

[0159] To further illustrate the performance of the ALX4 gene methylation differential region related to colorectal cancer screened by the present invention, the inventor selected different methylation regions (different from the screened methylation region), specifically the methylation region disclosed in the prior art (CN112195243A): chr11:44309888:44310051. IGV map analysis was performed on 4 colorectal cancer positive samples and 4 normal intestinal negative samples, as Figure 6 shown. It can be clearly seen from the figure that the methylation level captured in the region of chr11:44309888:44310051 is relatively low and cannot distinguish between 4 colorectal cancer positive samples and 4 normal intestinal negative samples, further proving that the ALX4 gene methylation differential region related to colorectal cancer screened by the present invention has obvious advantages.

[0160] All documents mentioned in the present invention are cited in this application for reference as if each document was cited separately for reference. In addition, it should be understood that after reading the above teachings of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the appended claims of this application.

Claims

1. Use of a reagent for detecting the methylation level of a specific region of the ALX4 gene in the preparation of a kit for diagnosing colorectal cancer or predicting the risk of colorectal cancer, characterized in that, The specific region includes at least a part of chr11:44311452-44311645.

2. The application according to claim 1, wherein The detection reagent includes a methylation region enrichment or methylation treatment reagent, and also includes a qPCR detection reagent.

3. The application according to claim 2, wherein The methylation region enrichment reagent includes a methylated DNA antibody.

4. The application according to claim 3, characterized in that The qPCR detection reagent includes a primer pair and a probe targeting the specific region.

5. The application according to claim 4, wherein The specific region is chr11:44311570-44311644, the primer pair is as shown in SEQ ID No.1 and SEQ ID No.2, and the probe is as shown in SEQ ID No.

3.

6. The application according to claim 2, characterized in that The methylation treatment reagent includes bisulfite.

7. The application according to claim 6, wherein The qPCR detection reagent includes a primer pair and a probe targeting the treated specific region.

8. The application according to claim 7, characterized in that The specific region is chr11:44311570-44311644, the primer pair is as shown in SEQ ID No.4 and SEQ ID No.5, and the probe is as shown in SEQ ID No.

6.

9. Use of a detection reagent for the methylation level of a specific region of the ALX4 gene in the preparation of a kit for diagnosing colorectal cancer or predicting the risk of colorectal cancer based on the following method: S1, obtaining a cfDNA sample in a biological sample of a subject; S2, enriching the methylation region of the cfDNA sample or performing methylation treatment; S3, using the product enriched or treated in step S2 as a template, and performing qPCR amplification with a primer pair and a probe targeting the untreated or treated specific region; If there is a typical amplification curve and the Ct value is not greater than a preset threshold, it is diagnosed that the subject has colorectal cancer or has a risk of developing colorectal cancer. The specific region includes at least a part of chr11:44311452-44311645.

10. A kit for diagnosing colorectal cancer or predicting the risk of colorectal cancer by detecting specific regions of the ALX4 gene based on DNA immunoprecipitation, characterized in that, The specific region is chr11:44311570-44311644, the kit includes a 5-methylcytosine antibody, a primer pair and a probe targeting the specific region, the primer pair is as shown in SEQ IDNo.1 and SEQ IDNo.2, and the probe is as shown in SEQ ID No.3.

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