WO2020221315A1 - 基于甲基化修饰的肿瘤标记物stamp-ep8及其应用 - Google Patents

基于甲基化修饰的肿瘤标记物stamp-ep8及其应用 Download PDF

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WO2020221315A1
WO2020221315A1 PCT/CN2020/087885 CN2020087885W WO2020221315A1 WO 2020221315 A1 WO2020221315 A1 WO 2020221315A1 CN 2020087885 W CN2020087885 W CN 2020087885W WO 2020221315 A1 WO2020221315 A1 WO 2020221315A1
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cancer
seq
methylation
samples
polynucleotide
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PCT/CN2020/087885
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French (fr)
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李振艳
罗怀兵
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上海奕谱生物科技有限公司
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Priority to US17/594,792 priority Critical patent/US20220298577A1/en
Priority to EP20798283.6A priority patent/EP3964578A4/en
Priority to JP2021564600A priority patent/JP7383051B2/ja
Publication of WO2020221315A1 publication Critical patent/WO2020221315A1/zh
Priority to JP2023093508A priority patent/JP2023118716A/ja

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/154—Methylation markers

Definitions

  • the present invention belongs to the field of disease diagnostic markers. More specifically, the present invention relates to a specific Tumor Aligned Methylation of Pan-cancer (Specific Tumor Aligned Methylation of Pan-cancer) based on methylation modification and applications thereof.
  • tumor markers were mainly cell secretions such as hormones, enzymes, and proteins, such as carcinoembryonic antigen (CEA), alpha fetal antigen (AFP), etc., which can be used as markers for various tumors such as gastric cancer and liver cancer.
  • CEA carcinoembryonic antigen
  • AFP alpha fetal antigen
  • CA125 Antigen-like 125
  • PSA prostate-specific antigen
  • Epigenetics is a discipline that studies the heritable changes in gene function without changes in the DNA sequence of genes, which ultimately leads to phenotypic changes.
  • Epigenetics mainly includes biochemical processes such as DNA methylation, histone modification, and microRNA level changes.
  • DNA methylation is one of the epigenetic mechanisms, which refers to the use of S-adenosylmethionine (SAM) as the methyl donor under the catalysis of DNA methyltransferase (DMT) in the organism.
  • SAM S-adenosylmethionine
  • DMT DNA methyltransferase
  • Body the process of transferring methyl groups to specific bases.
  • DNA methylation mainly occurs on the C of 5'-CpG-3' to produce 5-methylcytosine (5mC).
  • Liquid biopsy technology is a technology that uses circulating tumor cells or circulating tumor DNA in the blood as the detection target to diagnose and predict tumors.
  • This technology has many shortcomings: First, the sensitivity and specificity are not high enough, the tumor itself is very heterogeneous, and contains a variety of subtypes of cell populations, and clinical samples, especially blood samples, account for a very small proportion of tumor DNA
  • Existing tumor markers are difficult to meet the sensitivity of clinical requirements, and it is easy to cause misdiagnosis in the clinic;
  • a marker only has a good effect on one or a few tumors, and the source of DNA in the blood is very complicated. Therefore, the existing tumor markers cannot cope with the problems of complex tumor source and metastasis. Due to the existence of these complex conditions, it is difficult for many DNA methylation tumor markers to have a unified standard for use in clinical applications, which seriously affects the sensitivity and accuracy of the markers.
  • the purpose of the present invention is to provide a method for detecting tumors by using DNA methylation modification as a tumor marker and using abnormal hypermethylation at specific sites in tumors.
  • an isolated polynucleotide including: (a) a polynucleotide of the nucleotide sequence shown in SEQ ID NO:1; (b) a fragment of the polynucleotide of (a), and There is at least one modified CpG site (such as 2 to 191, more specifically 3, 5, 10, 15, 20, 25, 30, 40, 50, 60, 70, 90, 110, 130, 150, 170, 180, 190); and/or (c) nucleic acid complementary to the polynucleotide or fragment of (a) or (b) above (such as the polynucleotide of the nucleotide sequence shown in SEQ ID NO: 3) .
  • modified CpG site such as 2 to 191, more specifically 3, 5, 10, 15, 20, 25, 30, 40, 50, 60, 70, 90, 110, 130, 150, 170, 180, 190
  • nucleic acid complementary to the polynucleotide or fragment of (a) or (b) above such as the polynucleotide of
  • the modification includes 5-methylation modification, 5-hydroxymethylation modification, 5-aldehyde methylation modification or 5-carboxymethylation modification.
  • the polynucleotide fragments include: bases 204 to 223 (including methylation sites 021 to 024); SEQ ID NO: 1 or 2 The 1st to 478th bases (including the 001 to 040 methylation sites); SEQ ID NO: 1 or the 513th to 1040th bases (including the 041 to 077 methylation sites); SEQ ID NO: 1 or 2 bases 1082-1602 (including 078-114 methylation sites); SEQ ID NO: 1 or 2 bases 1621-2117 (including 115-153 methylation sites) Or bases 2160-2700 in SEQ ID NO: 1 or 2 (including methylation sites 154-191).
  • an isolated polynucleotide which is converted from the polynucleotide, corresponds to the sequence of the aforementioned first aspect, and the cytosine C of the modified CpG site is unchanged, and is not The modified cytosine is converted to T or U.
  • the polynucleotide in a preferred embodiment, it is converted from the polynucleotide corresponding to the first aspect described above through treatment with bisulfite or bisulfite.
  • the polynucleotide includes: (d) a polynucleotide of the nucleotide sequence shown in SEQ ID NO: 2 or SEQ ID NO: 4; (e) the polynucleotide of (d) above Acid fragments, and at least one modified CpG site (such as 2 to 191, more specifically 3, 5, 10, 15, 20, 25, 30, 40, 50, 60, 70, 90, 110 , 130, 150, 170, 180, 190).
  • the polynucleotide fragments include: bases 204 to 223 (including methylation sites 021 to 024); SEQ ID NO: 1 or 2 The 1st to 478th bases (including the 001 to 040 methylation sites); SEQ ID NO: 1 or the 513th to 1040th bases (including the 041 to 077 methylation sites); SEQ ID NO: 1 or 2 bases 1082-1602 (including 078-114 methylation sites); SEQ ID NO: 1 or 2 bases 1621-2117 (including 115-153 methylation sites) Bases); SEQ ID NO: 1 or 2 bases 2160-2700 (including methylation sites 154-191); positions 2478-2497 (corresponding to methylation sites 021-024 ) Bases; SEQ ID NO: 4 or 3 bases 2223-2700 (corresponding to methylation sites 001-040); SEQ ID NO: 4 or 3 bases 1661-2188 (corresponding At 041-077 methylation sites); SEQ ID NO: 4 or 3 bases 1099-1619
  • the use of the polynucleotide of the aforementioned first or second aspect is provided for preparing tumor detection reagents or kits.
  • the tumor includes (but is not limited to): hematological tumors such as leukemia, lymphoma, multiple myeloma; gynecological and reproductive system tumors such as breast cancer, ovarian cancer, cervical cancer, vulvar cancer, testicular cancer , Prostate cancer, penile cancer; digestive system tumors such as esophageal cancer (esophageal cancer), gastric cancer, colorectal cancer, liver cancer, pancreatic cancer, bile duct and gallbladder cancer; respiratory system tumors such as lung cancer, pleiomas; nervous system tumors such as glioma , Neuroblastoma, meningioma; head and neck tumors such as oral cancer, tongue cancer, laryngeal cancer, nasopharyngeal cancer; urinary system tumors such as kidney cancer, bladder cancer, skin and other systems such as skin cancer, melanoma, osteosarcoma , Liposarcoma, thyroid cancer.
  • the tumor samples include but are not limited to: tissue samples, paraffin-embedded samples, blood samples, pleural effusion samples, alveolar lavage fluid samples, ascites and lavage fluid samples, bile samples, feces Samples, urine samples, saliva samples, sputum samples, cerebrospinal fluid samples, cell smear samples, cervical smear or brush smear samples, tissue and cell biopsy samples.
  • a method for preparing a tumor detection reagent comprising: providing the polynucleotide of the first aspect or the second aspect, using the full-length or fragment of the polynucleotide As the target sequence, design a detection reagent that specifically detects the modification of the CpG site of the target sequence; wherein, there is at least one (such as 2 to 191, more specifically 3, 5, 10, 15) in the target sequence. , 20, 25, 30, 40, 50, 60, 70, 90, 110, 130, 150, 170, 180, 190) modified CpG sites; preferably, the detection reagents include (but are not limited to ): Primer, probe.
  • a reagent or a combination of reagents which specifically detects the modification of the CpG site of a target sequence, the target sequence being the polynucleotide of any one of the first or second aspects above
  • the full length or fragments of, at least one such as 2 ⁇ 191, more specifically 3,5,10,15,20,25,30,40,50,60,70,90,110,130,150 , 170, 180, 190) modified CpG sites.
  • the reagent or a combination of reagents is directed to a gene sequence (designed based on the gene sequence) containing the target sequence, and the gene sequence includes a gene panel or a gene group.
  • the detection reagent includes: primers and probes.
  • the primer is: the primer shown in SEQ ID NO: 3 and 4; the primer shown in SEQ ID NO: 7 and 8; the primer shown in SEQ ID NO: 9 and 10; SEQ ID NO: primers shown in 11 and 12; primers shown in SEQ ID NOs: 13 and 14; or primers shown in SEQ ID NOs: 15 and 16.
  • the use of the reagent or the combined reagent of the fifth aspect of the present invention is provided for preparing a kit for detecting tumors; preferably, the tumors include (but not limited to): Hematological tumors such as leukemia, lymphoma, multiple myeloma; gynecological and reproductive system tumors such as breast, ovarian, cervical, vulvar, testicular, prostate, penile cancer; digestive system tumors such as esophageal cancer (esophageal cancer) ), gastric cancer, colorectal cancer, liver cancer, pancreatic cancer, bile duct and gallbladder cancer; respiratory system tumors such as lung cancer, pleuromas; nervous system tumors such as glioma, neuroblastoma, meningioma; head and neck tumors such as oral cancer , Tongue cancer, laryngeal cancer, nasopharyngeal cancer; urinary system tumors such as kidney cancer, bladder cancer
  • Hematological tumors such
  • a detection kit which includes a container and the reagents or reagent combinations described above in the container; preferably, each reagent is in a separate container.
  • the kit also includes: bisulfite or bisulfite, DNA purification reagents, DNA extraction reagents, PCR amplification reagents and/or instructions for use (indicating detection operation steps and results judgement standard).
  • an in vitro method for detecting the methylation profile of a sample including: (i) providing a sample and extracting nucleic acid; (ii) detecting the CpG site of the target sequence in the nucleic acid of (i)
  • the target sequence is the polynucleotide described in the first aspect or the polynucleotide in the second aspect transformed therefrom.
  • the analysis methods include: pyrosequencing, bisulfite conversion sequencing, methylation chip method, qPCR, digital PCR, second-generation sequencing, and third-generation sequencing Method, whole genome methylation sequencing method, DNA enrichment detection method, simplified bisulfite sequencing technology, HPLC method, MassArray, methylation-specific PCR (MSP), or a combination thereof and SEQ ID NO:1
  • pyrosequencing bisulfite conversion sequencing
  • methylation chip method qPCR
  • digital PCR digital PCR
  • second-generation sequencing second-generation sequencing
  • third-generation sequencing Method whole genome methylation sequencing method
  • DNA enrichment detection method DNA enrichment detection method
  • simplified bisulfite sequencing technology HPLC method
  • MassArray methylation-specific PCR
  • SEQ ID NO:1 SEQ ID NO:1
  • step (ii) includes: (1) processing the product of (i) to convert unmodified cytosine into uracil; preferably, the modification includes 5-methyl Chemical modification, 5-hydroxymethylation modification, 5-aldehyde methylation modification or 5-carboxymethylation modification; preferably, treatment with bisulfite or bisulfite as described in step (i) Nucleic acid; (2) Analyze the modification of the target sequence described in the nucleic acid processed by (1).
  • the abnormal methylation profile means that C in the polynucleotide CpG is highly methylated.
  • the methylation profile method does not aim to directly obtain the diagnosis result of the disease, or is not a diagnostic method.
  • a tumor diagnosis kit which includes a primer pair designed using the sequence shown in the first or second aspect of the present invention, and a gene panel or gene group containing the sequence, which is passed through DNA
  • the basic state detection obtains the characteristics of normal cells and tumor cells.
  • Figure 1 BSP verification of methylation levels between lung cancer cells and normal lung cells at 001-040 methylation sites in the SEQ ID NO:1 region.
  • the dark box in the figure indicates methylation.
  • Figure 2 BSP verification of the methylation levels of lung cancer cells and normal lung cells at the 041-077 methylation site in the SEQ ID NO:1 region.
  • the dark box in the figure indicates methylation.
  • Figure 3 BSP verification of methylation levels of lung cancer cells and normal lung cells at 078-114 methylation sites in the SEQ ID NO:1 region.
  • the dark box in the figure indicates methylation.
  • Figure 4 BSP verification of methylation levels between lung cancer cells and normal lung cells at 115-153 methylation sites in the SEQ ID NO:1 region.
  • the dark box in the figure indicates methylation.
  • Figure 5 BSP verification of methylation levels between lung cancer cells and normal lung cells at 154-191 methylation sites in the SEQ ID NO:1 region.
  • the dark box in the figure indicates methylation.
  • Figure 8 Comparison of the methylation values of STAMP-EP8 in the adjacent samples and cancer tissue samples for clinical samples of colorectal cancer.
  • Figure 9 For clinical samples of esophageal cancer, comparison of methylation values of STAMP-EP8 in adjacent samples and cancer tissue samples.
  • the inventor is committed to the research of tumor markers. After extensive research and screening, we provide a universal DNA methylation tumor marker STAMP (Specific Tumor Aligned Methylation of Pan-cancer). STAMP is low in normal tissues. Methylation status, STAMP presents a hypermethylation status in tumor tissues, which can be used for clinical tumor detection and as a basis for designing tumor diagnostic reagents.
  • STAMP Specific Tumor Aligned Methylation of Pan-cancer
  • isolated refers to the separation of a substance from its original environment (if it is a natural substance, the original environment is the natural environment).
  • the polynucleotides and polypeptides in the natural state in living cells are not separated and purified, but the same polynucleotides or polypeptides are separated and purified if they are separated from other substances co-existing in the natural state.
  • sample or “sample” includes a substance suitable for detection of DNA methylation status obtained from any individual or isolated tissue, cell, or body fluid (such as plasma).
  • the samples shown may include, but are not limited to: tissue samples, paraffin-embedded samples, blood samples, pleural effusion samples and alveolar lavage fluid samples, ascites and lavage fluid samples, bile samples, stool samples, urine samples , Saliva samples, cerebrospinal fluid samples, cell smear samples, cervical smear or brush smear samples, tissue and cell biopsy samples.
  • high (degree) methylation refers to the presence of high methylation, hydroxymethylation, aldehyde methylation or carboxymethylation modification of CpG in a gene sequence.
  • MSP methylation-specific PCR
  • a PCR reaction with methylation-specific primers can obtain a positive PCR result, which means that the DNA (gene) region under test is in a hypermethylated state .
  • the determination of the hypermethylation state can be analyzed for statistical differences based on the relative value of the methylation state of the control sample.
  • the inventors have conducted extensive and in-depth research and determined the target of STAMP-EP8.
  • the methylation status of the STAMP-EP8 gene sequence region is significantly different between tumor tissues and non-tumor tissues. As long as the abnormal hypermethylation status of the STAMP-EP8 gene sequence region is detected, it can be determined that the subject is at high risk of tumor crowd.
  • the significant difference between tumor tissue and non-tumor tissue presented by STAMP-EP8 exists in a broad spectrum of different types of tumors, including solid tumors and non-solid tumors.
  • the present invention provides an isolated polynucleotide having the nucleotide shown in SEQ ID NO: 1 or SEQ ID NO: 3 (which is the reverse complement of SEQ ID NO: 1) Sequence: In tumor cells, 5-methylcytosine (5mC) is generated at multiple positions of 5'-CpG-3' base C in the polynucleotide sequence.
  • the present invention also includes a polynucleotide fragment of the nucleotide sequence shown in SEQ ID NO: 1 or SEQ ID NO: 3, and there is at least one (such as 2 to 191, more specifically 3, 5, 10, 15, 20, 25, 30, 40, 50, 60, 70, 90, 110, 130, 150, 170, 180, 190) methylated CpG sites.
  • the aforementioned polynucleotides or fragments can also be used to design detection reagents or detection kits.
  • the polynucleotide fragment is, for example, a fragment containing bases 281 to 309 in SEQ ID NO:1 (containing CpG sites 017 to 020). Antisense strands of the above fragments are also available. These fragments are examples of preferred embodiments of the present invention; according to the information provided by the present invention, other fragments can also be selected.
  • a gene panel or gene group containing the nucleotide sequence or sequence fragment shown in SEQ ID NO: 1 or SEQ ID NO: 2 is also included in the present invention.
  • the characteristics of normal cells and tumor cells can also be obtained through DNA methylation status detection.
  • polynucleotides can be used as key regions in the genome for people to analyze their methylation status, and their methylation status can be analyzed through various techniques known in the art. Any technique that can be used to analyze the methylation status can be applied to the present invention.
  • the present invention also provides a polynucleotide obtained after the above-mentioned polynucleotide (including its complementary strand (antisense strand)) is treated with bisulfite or bisulfite, including: SEQ ID NO: 2 or SEQ ID NO: A polynucleotide of the nucleotide sequence shown in 4. These polynucleotides can also be used to design detection reagents or detection kits.
  • the present invention also includes a fragment of the polynucleotide obtained by treating the above-mentioned polynucleotide or its antisense strand with bisulfite or bisulfite, and in which there is at least one methylated CpG site (such as 2 ⁇ 191, more specifically 3, 5, 10, 15, 20, 25, 30, 40, 50, 60, 70, 90, 110, 130, 150, 170, 180, 190).
  • the number of each CpG site in the antisense strand corresponding to the sense strand is easily obtained according to the content provided by the present invention.
  • a detection reagent designed based on the polynucleotide sequence is also provided for in vitro detection of the methylation profile of the polynucleotide in the sample.
  • the detection methods and reagents known in the art for determining the sequence of the genome, its variation and methylation status can be applied to the present invention.
  • the present invention provides a method for preparing a tumor detection reagent, including: providing the polynucleotide, using the full length or fragment of the polynucleotide as a target sequence, and designing a detection reagent that specifically detects the target sequence ; Wherein, there is at least one methylated CpG site in the target sequence.
  • the detection reagents of the present invention include, but are not limited to: primers, probes, etc.
  • the reagent is, for example, a pair of primers.
  • the design of primers is known to those skilled in the art.
  • the two primers are on both sides of the specific sequence of the target gene to be amplified (including CpG Within the sequence, the complement to CpG is for the gene region that was originally methylated, and the complement to TpG is for the gene region that was originally demethylated).
  • CpG Within the sequence, the complement to CpG is for the gene region that was originally methylated, and the complement to TpG is for the gene region that was originally demethylated.
  • the primers are: the primers shown in SEQ ID NOs: 5 and 6, which can obtain the amplification products containing the CpG sites No. 001 to 040 in SEQ ID NO:1; SEQ The primers shown in ID NO: 7 and 8 can obtain the amplified products containing the CpG sites of No. 041 to 077 in SEQ ID NO:1; the primers shown in SEQ ID NOs: 9 and 10 can be obtained containing SEQ ID NO: 1 amplified product of CpG sites No. 078 to 114; primers shown in SEQ ID NOs: 11 and 12, which can obtain amplified products containing CpG sites No.
  • the reagent can also be a reagent combination (primer combination), including more than one set of primers, so that the multiple polynucleotides can be amplified separately.
  • the present invention also provides a kit for detecting the methylation profile of polynucleotides in a sample in vitro.
  • the kit includes a container and the aforementioned primer pair in the container.
  • the kit may also include various reagents required for DNA extraction, DNA purification, PCR amplification and the like.
  • kit can also include instructions for use, which indicate the detection operation steps and result judgment criteria, so as to facilitate the application by those skilled in the art.
  • the methylation profile of polynucleotides can be determined by existing techniques (such as methylation-specific PCR (MSP) or real-time quantitative methylation-specific PCR, Methylight), or other techniques that are still under development and will The technology was developed to proceed.
  • MSP methylation-specific PCR
  • Methylight real-time quantitative methylation-specific PCR
  • Quantitative methylation-specific PCR can also be used to detect methylation levels. This method is based on a continuous optical monitoring of fluorescent PCR, which is more sensitive than the MSP method. Its high throughput and avoid using electrophoresis method to analyze its results.
  • an in vitro method for detecting the methylation profile of polynucleotides in a sample is also provided.
  • the method is based on the principle that: bisulfite or bisulfite can convert unmethylated cytosine into uracil, which is converted into thymine in the subsequent PCR amplification process, and methylated
  • the cytosine of cytosine remains unchanged; therefore, after the polynucleotide is treated with bisulfite or bisulfite, the methylated site produces a polynucleotide polymorphism (SNP) similar to a C/T.
  • SNP polynucleotide polymorphism
  • the method of the present invention includes: (a) providing a sample and extracting genomic DNA; (b) treating the genomic DNA described in step (a) with bisulfite or bisulfite, so that there is no The methylated cytosine is converted to uracil; (c) the genomic DNA processed in step (b) is analyzed for abnormal methylation patterns.
  • the method of the present invention can be used to: (i) detect samples of subjects to analyze whether the subjects have tumors; (ii) distinguish groups at high risk of tumors.
  • the method described may also be a situation where the purpose is not to obtain direct disease diagnosis results.
  • DNA methylation is detected by PCR amplification and pyrosequencing.
  • PCR amplification and pyrosequencing Those skilled in the art should understand that this method is not limited to this method in practical applications, and other DNA methylation detection methods are also possible.
  • the primers used are not limited to those provided in the examples.
  • genomic DNA is treated with bisulfite, unmethylated cytosine is converted to uracil, and then converted to thymine in the subsequent PCR process, which reduces the sequence complexity of the genome and makes PCR amplify specific targets The difficulty of the fragment increases. Therefore, it is preferable to adopt nested PCR amplification, design two pairs of primers on the outer periphery and inner periphery, carry out two rounds of PCR amplification reaction, and use the amplified product of the first round as the template for the second round of amplification, which can effectively improve the amplification. Efficiency and specificity.
  • the detection method available in the present invention is not limited to this.
  • the method of the present invention has very high accuracy when used in the diagnosis of clinical tumors.
  • the invention can be applied to the fields of tumor auxiliary diagnosis, curative effect judgment, prognosis monitoring, etc., and has high clinical application value.
  • the inventors After repeated research and screening, the inventors obtained a tumor marker STAMP-EP8 whose sequence is shown in SEQ ID NO:1 (chr8:23562476-23565175, Human/hg19), in which the underlined base is methylated CpG Site, the number below the underline indicates the site number.
  • SEQ ID NO: 2 (where Y stands for C or U):
  • SEQ ID NO: 3 The reverse complementary sequence of the nucleotide sequence shown in SEQ ID NO: 1 is as follows SEQ ID NO: 3:
  • SEQ ID NO: 4 (where Y represents C or U):
  • the sequencing method after the bisulfite treatment is as follows:
  • 2% agarose gel electrophoresis detects the specificity of PCR fragments, cuts the gel to recover the target fragments, connects and inserts the T vector, transforms competent E. coli, spreads the plate, and picks the clones for sequencing the next day. Fragment picking more than 10 clones for Sanger sequencing.
  • DNA extraction extract the experimental group and control group DNA separately; this experiment uses phenol-chloroform extraction method, but not limited to this method;
  • Primer design According to the characteristics of STAMP-EP8 SEQ ID NO:1, design PCR amplification primers and pyrosequencing primers, and detect the methylation value of CpG site No.021-024 as the STAMP-EP8 methylation value
  • the representative of PCR primer amplification sequence, pyrosequencing primer sequence, pyrosequencing computer detection sequence and detection site are shown in SEQ ID NO: 15-18, as shown in Table 2;
  • PCR amplification and agarose gel electrophoresis the bisulfite-treated sample is used as a PCR template for PCR amplification, and the amplified product is identified by agarose gel electrophoresis to identify the specificity of PCR amplification;
  • STAMP-EP8 methylation value calculation Pyrosequencing can independently detect the methylation of a single CpG site in the target area, and calculate the average methylation of all CpG sites as the STAMP-EP8 in the sample Methylation value
  • Results Figure 9 shows that in clinical samples of esophageal cancer, the methylation value of STAMP-EP8 in the experimental group was significantly higher than that of adjacent tissues.
  • pancreatic cancer adjacent to cancer were obtained clinically as the control group, and 4 samples of pancreatic cancer were obtained as the experimental group. According to the pyrophosphate test procedure in Example 3, the STAMP-EP8 methylation level was analyzed.

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Abstract

提供了一种分离的多核苷酸及其用途，其是SEQ ID NO:1所示多核苷酸或其存在修饰的CpG位点的片段或互补核酸。还提供了多核苷酸用于制备肿瘤检测试剂或试剂盒的用途，制备方法，试剂及其用途，试剂盒，以及体外检测样品的甲基化谱式的方法。

Description

基于甲基化修饰的肿瘤标记物STAMP-EP8及其应用 技术领域
本发明属于疾病诊断标记物领域，更具体地，本发明涉及基于甲基化修饰的肿瘤标记物STAMP(Specific Tumor Aligned Methylation of Pan-cancer)及其应用。
背景技术
肿瘤被认为是一种遗传性疾病的观念在本领域持续几十年。人类几次系统的大规模测序证实了癌组织中体细胞的突变数量明显少于预期，这些结果暗示了癌症并非简单的遗传性疾病。
为了实现肿瘤的诊断，近年来许多新型的肿瘤标记物被发现并用于临床诊断。1980年之前，肿瘤标记物主要是一些激素、酶类、蛋白质等细胞分泌物，例如癌胚抗原(CEA)、甲胎抗原(AFP)等可以作为胃癌和肝癌等多种肿瘤的标记物，糖类抗原125(CA125)可以作为宫颈癌的标记物，前列腺特异性抗原(PSA)可作为前列腺癌标记物，目前这一类肿瘤标记物虽然临床仍在用，但其敏感性与准确性已难以满足临床需求。
越来越多的证据表明，表观遗传调控的细小变化在肿瘤中的重要作用。表观遗传学是研究基因在不发生DNA序列改变的情况下，基因功能发生的可遗传的变化，并最终导致了表型的变化的一门学科。表观遗传学主要包括DNA甲基化(DNA methylation)，组蛋白修饰(histone modification)，microRNA水平变化等生化过程。DNA甲基化是表观遗传学机制之一，其是指生物体内在DNA甲基转移酶(DNA methyltransferase，DMT)的催化下，以S-腺苷甲硫氨酸(SAM)为甲基供体，将甲基转移到特定的碱基上的过程。在哺乳动物中DNA甲基化主要发生在5’-CpG-3’的C上，生成5-甲基胞嘧啶(5mC)。
液体活检技术是以血中循环肿瘤细胞或循环肿瘤DNA为检测靶标对肿瘤进行诊断和预测的技术。该技术存在诸多不足：首先，敏感性与特异性不够高，肿瘤本身有很大的异质性，包含多种亚型的细胞群，而临床样本尤其是血液样本，肿瘤DNA所占比例非常小，现有的肿瘤标记物难以满足临床要求的敏感性，在临床容易造成误诊；其次，一种标记物只针对一种或少数几种肿瘤有较好效果，而血液中的DNA来源非常复杂，因此现有的肿瘤标记物无法应对复杂的肿瘤来源、转移等问题。由于这些复杂情况的存在，使得很多DNA甲基化肿瘤标记物在应用于临床时难以有统一的使用标准，严重影响标记物的敏感度以及准确性。
在本发明人的前期研究中，找到了一部分基于甲基化修饰的肿瘤标记物，但是，仍然有必要找到更多的新型肿瘤标志物，从而为肿瘤的诊断提供更多的途径。
发明内容
本发明的目的在于提供以DNA甲基化修饰作为肿瘤标记物，利用肿瘤中特异性位点异常高甲基化现象来检测肿瘤的方法。
在本发明的第一方面，提供分离的多核苷酸，包括：(a)SEQ ID NO:1所示核苷酸序列的多核苷酸；(b)(a)的多核苷酸的片段，且其中存在至少1个修饰的CpG位点(如2～191个，更具体如3，5，10，15，20，25，30，40，50，60，70，90，110，130，150，170，180，190个)；和/或(c)与上述(a)或(b)的多核苷酸或片段互补的核酸(如SEQ ID NO:3所示核苷酸序列的多核苷酸)。
在一个优选例中，所述修饰包括5-甲基化修饰、5-羟甲基化修饰、5-醛甲基化修饰或5-羧甲基化修饰。
在另一优选例中，(b)中，所述的多核苷酸的片段中存在：第204～223位(包含021～024号甲基化位点)碱基；SEQ ID NO:1或2中第1～478位碱基(包含001～040号甲基化位点)；SEQ ID NO:1或2中第513～1040位碱基(包含041～077号甲基化位点)；SEQ ID NO:1或2中第1082～1602位碱基(包含078～114号甲基化位点)；SEQ ID NO:1或2中第1621～2117位碱基(包含115～153号甲基化位点)；或SEQ ID NO:1或2中第2160～2700位碱基(包含154～191号甲基化位点)。
在本发明的第二方面，提供分离的多核苷酸，其由所述的多核苷酸转变而来，对应于前述第一方面的序列，其修饰的CpG位点的胞嘧啶C不变，非修饰的胞嘧啶转为T或U。
在一个优选例中，其由对应于前述第一方面的多核苷酸经过亚硫酸氢盐或重亚硫酸氢盐处理转变而来。在另一优选例中，所述的多核苷酸包括：(d)SEQ ID NO:2或SEQ ID NO:4所示核苷酸序列的多核苷酸；(e)上述(d)的多核苷酸的片段，且其中存在至少1个修饰的CpG位点(如2～191个，更具体如3，5，10，15，20，25，30，40，50，60，70，90，110，130，150，170，180，190个)。
在另一优选例中，(e)中，所述的多核苷酸的片段中存在：第204～223位(包含021～024号甲基化位点)碱基；SEQ ID NO:1或2中第1～478位碱基(包含001～040号甲基化位点)；SEQ ID NO:1或2中第513～1040位碱基(包含041～077号甲基化位点)；SEQ ID NO:1或2中第1082～1602位碱基(包含078～114号甲基化位点)；SEQ ID NO:1或2中第1621～2117位碱基(包含115～153号甲基化位点)；SEQ ID NO:1或2中第2160～2700位碱基(包含154～191号甲基化位点)；第2478～2497位(对应于021～024号甲基化位点)碱基；SEQ ID NO:4或3中第2223～2700位碱基(对应于001～040号甲基化位点)；SEQ ID NO:4或3中第1661～2188位碱基(对应于041～077号甲基化位点)；SEQ ID NO:4或3中第1099～1619位碱基(对应于078～114号 甲基化位点)；SEQ ID NO:4或3中第584～1080位碱基(对应于115～153号甲基化位点)；或SEQ ID NO:4或3中第1～541位碱基(对应于154～191号甲基化位点)。
在本发明的第三方面，提供前述第一方面或第二方面所述的多核苷酸的用途，用于制备肿瘤的检测试剂或试剂盒。
在一个优选例中，所述肿瘤包括(但不限于)：血液系统肿瘤如白血病，淋巴瘤，多发性骨髓瘤；妇科及生殖系统肿瘤如乳腺癌，卵巢癌，宫颈癌，外阴癌，睾丸癌，前列腺癌，阴茎癌；消化系统肿瘤如食道癌(食管癌)，胃癌，结直肠癌，肝癌，胰腺癌，胆管及胆囊癌；呼吸系统肿瘤如肺癌，胸膜瘤；神经系统肿瘤如胶质瘤，神经母细胞瘤，脑膜瘤；头颈部肿瘤如口腔癌，舌癌，喉癌，鼻咽癌；泌尿系统肿瘤如肾癌，膀胱癌，皮肤及其他系统如皮肤癌、黑色素瘤、骨肉瘤，脂肪肉瘤，甲状腺癌。
在另一优选例中，所述肿瘤的样本包括但不限于：组织样本、石蜡包埋样本、血液样本、胸腔积液样本以及肺泡灌洗液样本、腹水及灌洗液样本、胆汁样本、粪便样本、尿液样本、唾液样本、痰液样本、脑脊液样本、细胞涂片样本、宫颈刮片或刷片样本、组织及细胞活检样本。
在本发明的第四方面，提供一种制备肿瘤检测试剂的方法，所述方法包括：提供前述第一方面或第二方面所述的多核苷酸，以所述多核苷酸的全长或片段作为靶序列，设计特异性检测该靶序列的CpG位点修饰情况的检测试剂；其中，所述的靶序列中存在至少1个(如2～191个，更具体如3，5，10，15，20，25，30，40，50，60，70，90，110，130，150，170，180，190个)修饰的CpG位点；较佳地，所述的检测试剂包括(但不限于)：引物，探针。
在本发明的第五方面，提供试剂或组合的试剂，其特异性检测靶序列的CpG位点修饰情况，所述的靶序列是前述第一方面或第二方面任一所述的多核苷酸的全长或片段，其中存在至少1个(如2～191个，更具体如3，5，10，15，20，25，30，40，50，60，70，90，110，130，150，170，180，190个)修饰的CpG位点。
在一个优选例中，所述的试剂或组合的试剂针对包含所述靶序列的基因序列(基于所述基因序列而设计)，所述的基因序列包括基因Panel或基因群组。
在另一优选例中，所述的检测试剂包括：引物，探针。
在另一优选例中，所述的引物为：SEQ ID NO:3和4所示的引物；SEQ ID NO:7和8所示的引物；SEQ ID NO:9和10所示的引物；SEQ ID NO:11和12所示的引物；SEQ ID NO:13和14所示的引物；或SEQ ID NO:15和16所示的引物。
在本发明的第六方面，提供本发明的第五方面所述的试剂或组合的试剂的用途，用于制备检测肿瘤的试剂盒；较佳地，所述的肿瘤包括(但不限于)：血液系统肿瘤如白血病，淋巴瘤，多发性骨髓瘤；妇科及生殖系统肿瘤如乳腺癌，卵巢癌，宫颈癌，外阴癌，睾丸癌，前列腺癌，阴茎癌；消化系统肿瘤如食道癌(食管癌)，胃癌，结直 肠癌，肝癌，胰腺癌，胆管及胆囊癌；呼吸系统肿瘤如肺癌，胸膜瘤；神经系统肿瘤如胶质瘤，神经母细胞瘤，脑膜瘤；头颈部肿瘤如口腔癌，舌癌，喉癌，鼻咽癌；泌尿系统肿瘤如肾癌，膀胱癌，皮肤及其他系统如皮肤癌、黑色素瘤、骨肉瘤，脂肪肉瘤，甲状腺癌。
在本发明的第七方面，提供一种检测试剂盒，其包括：容器，以及位于容器中的前面所述的试剂或试剂组合；较佳地，每一种试剂位于一个独立的容器中。
在一个优选例中，所述的试剂盒中还包括：亚硫酸氢盐或重亚硫酸氢盐，DNA纯化试剂，DNA提取试剂，PCR扩增试剂和/或使用说明书(标明检测操作步骤和结果判定标准)。
在本发明的第八方面，提供一种体外检测样品的甲基化谱式的方法，包括：(i)提供样品，提取核酸；(ii)检测(i)的核酸中靶序列的CpG位点修饰情况，所述的靶序列是前述第一方面所述的多核苷酸或由其转变而来的前述第二方面所述的多核苷酸。
在一个优选例中，步骤(3)中，分析的方法包括：焦磷酸测序法、重亚硫酸盐转化测序法、甲基化芯片法、qPCR法、数字PCR法、二代测序法、三代测序法、全基因组甲基化测序法、DNA富集检测法、简化亚硫酸氢盐测序技术、HPLC法、MassArray、甲基化特异PCR(MSP)、或它们的组合以及SEQ ID NO:1所示序列中部分或全部甲基化位点的组合基因群组体外检测方法及体内示踪检测方法。并且，其它其他甲基化检测方法及未来新开发的甲基化检测方法也可被应用于本发明中。
在另一优选例中，步骤(ii)包括：(1)对(i)的产物进行处理，使其中未发生修饰的胞嘧啶转化为尿嘧啶；较佳地，所述修饰包括5-甲基化修饰、5-羟甲基化修饰、5-醛甲基化修饰或5-羧甲基化修饰；较佳地，利用亚硫酸氢盐或重亚硫酸氢盐处理步骤(i)所述的核酸；(2)分析经(1)处理的核酸中所述的靶序列的修饰情况。
在另一优选例中，所述的甲基化谱式异常是指该多核苷酸CpG中的C发生高度甲基化。
在另一优选例中，所述的甲基化谱式的方法不以直接获得疾病的诊断结果为目的，或不是诊断性的方法。
在本发明的第九方面，提供一种肿瘤诊断试剂盒，包括利用本发明的第一方面或第二方面所示序列设计的引物对以及包含该序列的基因Panel或基因群组，通过DNA甲基化状态检测获取正常细胞和肿瘤细胞的特征。
本发明的其它方面由于本文的公开内容，对本领域的技术人员而言是显而易见的。
附图说明
图1、SEQ ID NO:1区域中001-040甲基化位点肺癌细胞与正常肺细胞甲基化水 平BSP验证。图中深色方框表示呈现甲基化。
图2、SEQ ID NO:1区域中041-077甲基化位点肺癌细胞与正常肺细胞甲基化水平BSP验证。图中深色方框表示呈现甲基化。
图3、SEQ ID NO:1区域中078-114甲基化位点肺癌细胞与正常肺细胞甲基化水平BSP验证。图中深色方框表示呈现甲基化。
图4、SEQ ID NO:1区域中115-153甲基化位点肺癌细胞与正常肺细胞甲基化水平BSP验证。图中深色方框表示呈现甲基化。
图5、SEQ ID NO:1区域中154-191甲基化位点肺癌细胞与正常肺细胞甲基化水平BSP验证。图中深色方框表示呈现甲基化。
图6、针对白血病临床样本，STAMP-EP8在非癌组织样本以及癌组织样本中的甲基化值比较。
图7、针对乳腺癌临床样本，STAMP-EP8在癌旁样本以及癌组织样本中的甲基化值比较。
图8、针对结直肠癌临床样本，STAMP-EP8在癌旁样本以及癌组织样本中的甲基化值比较。
图9、针对食管癌临床样本，STAMP-EP8在癌旁样本以及癌组织样本中的甲基化值比较。
图10、针对肝癌临床样本，STAMP-EP8在癌旁样本以及癌组织样本中的甲基化值比较。
图11、针对肺癌临床样本，STAMP-EP8在癌旁样本以及癌组织样本中的甲基化值比较。
图12、针对胰腺癌临床样本，STAMP-EP8在癌旁样本以及癌组织样本中的甲基化值比较。
具体实施方式
本发明人致力于肿瘤标志物的研究，经过广泛的研究筛选，提供一种通用型的DNA甲基化肿瘤标志物STAMP(Specific Tumor Aligned Methylation of Pan-cancer)，在正常的组织中STAMP处于低甲基化状态，在肿瘤组织中STAMP呈高甲基化状态，可用于临床肿瘤的检测，以及用于作为设计肿瘤诊断试剂的基础。
术语
如本文所用，“分离的”是指物质从其原始环境中分离出来(如果是天然的物质，原始环境即是天然环境)。如活体细胞内的天然状态下的多核苷酸和多肽是没有分离纯化的，但同样的多核苷酸或多肽如从天然状态中同存在的其他物质中分开，则为分 离纯化的。
如本文所用，“样本”或“样品”包括从任何个体或分离的组织、细胞或体液(如血浆)中获得的、适合于DNA甲基化状态检测的物质。例如，所示的样本可以包括但不限于：组织样本、石蜡包埋样本、血液样本、胸腔积液样本以及肺泡灌洗液样本、腹水及灌洗液样本、胆汁样本、粪便样本、尿液样本、唾液样本、脑脊液样本、细胞涂片样本、宫颈刮片或刷片样本、组织及细胞活检样本。
如本文所用，“高(度)甲基化”是指在一个基因序列中CpG存在高度甲基化、羟甲基化、醛甲基化或羧甲基化修饰。例如，以甲基化特异PCR(MSP)分析手段而言，以甲基化特异性引物所进行的PCR反应可获得阳性的PCR结果即可认为该受试的DNA(基因)区处于高甲基化状态。例如，以实时定量甲基化特异性PCR而言，高甲基化状态的判定可根据其对照样品的甲基化状态的相对值分析统计学差异。
基因标志物
为了寻找对于诊断肿瘤有用的靶标，本发明人经过了广泛而深入的研究，确定了STAMP-EP8这一靶标。STAMP-EP8基因序列区域的甲基化状态在肿瘤组织和非肿瘤组织之间存在显著的差异，只要检测到STAMP-EP8基因序列区域存在异常高甲基化状态，即可判定该受检者属于肿瘤高危人群。并且，STAMP-EP8呈现的这种在在肿瘤组织和非肿瘤组织之间存的显著差异广谱地存在于不同种类的肿瘤中，包括实体瘤以及非实体瘤。
因此，本发明提供了分离的多核苷酸，所述的多核苷酸，具有SEQ ID NO:1或SEQ ID NO:3(为SEQ ID NO:1的反向互补序列)所示的核苷酸序列，在肿瘤细胞内，该多核苷酸序列中多处5’-CpG-3’的碱基C位置上，生成5-甲基胞嘧啶(5mC)。本发明也包含SEQ ID NO:1或SEQ ID NO:3所示核苷酸序列的多核苷酸的片段，且其中存在至少1个(如2～191个，更具体如3，5，10，15，20，25，30，40，50，60，70，90，110，130，150，170，180，190个)甲基化CpG位点。上述的多核苷酸或片段也可以应用于设计检测试剂或检测试剂盒。
在本发明的一些具体实施例中，所述的多核苷酸的片段例如是：包含SEQ ID NO:1中第281～309位碱基的片段(含有第017～020号CpG位点)。上述片段的反义链也是可用的。这些片段是本发明的较佳实施方式的举例；根据本发明提供的信息，也可以选择其它的片段。
此外，包含SEQ ID NO:1或SEQ ID NO:2所示核苷酸序列或序列片段的基因Panel或基因群组也被包含在本发明中。针对所述的基因Panel或基因群组，也可以通过DNA甲基化状态检测获取正常细胞和肿瘤细胞的特征。
上述的多核苷酸可以作为基因组中人们分析甲基化状态的关键区域，通过各种本 领域已知的技术来分析它们的甲基化状态。任何可用于分析甲基化状态的技术均可被应用于本发明中。
上述的多核苷酸在经过亚硫酸氢盐或重亚硫酸氢盐处理后，其中未发生甲基化的胞嘧啶转化为尿嘧啶，而发生甲基化的胞嘧啶保持不变。
因此，本发明还提供了上述多核苷酸(包括其互补链(反义链))经过亚硫酸氢盐或重亚硫酸氢盐处理后获得的多核苷酸，包括：SEQ ID NO:2或SEQ ID NO:4所示核苷酸序列的多核苷酸。这些多核苷酸也可以应用于设计检测试剂或检测试剂盒。
本发明也包含上述多核苷酸或其反义链经过亚硫酸氢盐或重亚硫酸氢盐处理后获得的多核苷酸的片段，且其中存在至少1个甲基化CpG位点(如2～191个，更具体如3，5，10，15，20，25，30，40，50，60，70，90，110，130，150，170，180，190个)。反义链中各个CpG位点相应于正义链的编号是根据本发明所提供的内容易于获得的。
检测试剂及试剂盒
基于本发明的新发现，还提供了基于所述的多核苷酸序列设计的检测试剂，用于体外检测样品中多核苷酸的甲基化谱式。本领域已知的确定基因组的序列、其变异及甲基化状态的检测方法和试剂均可被应用于本发明中。
因此，本发明提供了一种制备肿瘤检测试剂的方法，包括：提供所述的多核苷酸，以所述多核苷酸的全长或片段作为靶序列，设计特异性检测该靶序列的检测试剂；其中，所述的靶序列中存在至少1个甲基化CpG位点。
本发明所述的检测试剂包括但不限于：引物，探针，等等。
所述的试剂例如是引物对，在得知了多核苷酸的序列后，设计引物是本领域技术人员已知的，两个引物在将被扩增的目标基因特定序列的两侧(包含CpG序列在内，与其中CpG互补为针对原为甲基化的基因区，而与其中TpG互补为针对原为去甲基化的基因区)。应理解，根据本发明的新发现，针对所述靶序列上的不同位置的CpG位点或其组合，本领域技术人员可以设计出多种引物或探针或其它类型的检测试剂，这些均应被包含在本发明的技术方案中。在本发明的优选实施例中，所述的引物为：SEQ ID NO:5和6所示的引物，其可以获得包含SEQ ID NO:1中001～040号CpG位点的扩增产物；SEQ ID NO:7和8所示的引物，其可以获得包含SEQ ID NO:1中041～077号CpG位点的扩增产物；SEQ ID NO:9和10所示的引物，其可以获得包含SEQ ID NO:1中078～114号CpG位点的扩增产物；SEQ ID NO:11和12所示的引物，其可以获得包含SEQ ID NO:1中115～153号CpG位点的扩增产物；SEQ ID NO:13和14所示的引物，其可以获得包含SEQ ID NO:1中154～191号CpG位点的扩增产物；或SEQ ID NO:15和16所示的引物，其可以获得包含SEQ ID NO:1中021-024 号CpG位点的扩增产物。
所述的试剂也可以是试剂组合(引物组合)，包括多于一组的引物，从而可分别扩增上述的多条多核苷酸。
本发明还提供了体外检测样品中多核苷酸的甲基化谱式的试剂盒，该试剂盒包括：容器，以及位于容器中的上述引物对。
此外，所述的试剂盒中还可包括用于提取DNA、DNA纯化、PCR扩增等所需的各种试剂。
此外，所述的试剂盒中还可包括使用说明书，其中标明检测操作步骤和结果判定标准，以便于本领域技术人员应用。
检测方法
测定多核苷酸的甲基化谱式可通过已有的技术(如甲基化特异性PCR(MSP)或实时定量甲基化特异性PCR，Methylight)来进行，或其它仍在发展中和将被开发出来的技术来进行。
检测甲基化水平时也可使用定量甲基化特异性PCR(QMSP)的方法。这种方法是基于一种荧光PCR的持续性的光学监控，其较MSP方法更为敏感。其通量高并避免了用电泳方法对其结果进行分析。
其他可用的技术还有：焦磷酸测序法、重亚硫酸盐转化测序法、qPCR法、二代测序法、全基因组甲基化测序法、DNA富集检测法、简化亚硫酸氢盐测序技术或HPLC法以及组合基因群组检测法等该领域常规方法。应理解，在本发明的新揭示的基础上，本领域公知的这些技术以及即将发展的一些技术，均可被应用于本发明中。
作为本发明的优选方式，还提供了一种体外检测样品中多核苷酸的甲基化谱式的方法。所述的方法基于的原理是：亚硫酸氢盐或重亚硫酸氢盐可以将未甲基化的胞嘧啶转化为尿嘧啶，在后续的PCR扩增过程中转变为胸腺嘧啶，而甲基化的胞嘧啶保持不变；因而，经过亚硫酸氢盐或重亚硫酸氢盐处理多核苷酸后，甲基化的位点产生类似于一个C/T的多核苷酸多态性(SNP)。基于上述原理来鉴定检测样品中多核苷酸的甲基化谱式，可以有效区分出甲基化与非甲基化的胞嘧啶。
本发明所述的方法包括：包括：(a)提供样品，提取基因组DNA；(b)利用亚硫酸氢盐或重亚硫酸氢盐处理步骤(a)所述的基因组DNA，从而基因组DNA中未甲基化的胞嘧啶转化为尿嘧啶；(c)分析经步骤(b)处理的基因组DNA中是否存在甲基化谱式异常。
本发明的方法可用于：(i)对受试者样品进行检测，分析受试者是否患有肿瘤；(ii)区分肿瘤高危人群。所述的方法也可以是不以获得直接的疾病诊断结果为目的的情形。
在本发明的优选实施例中，通过PCR扩增及焦磷酸测序法检测DNA甲基化，本领域人员应理解，实际应用中并不限于该方法，其它DNA甲基化检测方法亦可。在进行PCR扩增中，所应用的引物也不限于是实施例中所提供的。
由于基因组DNA经过重亚硫酸盐处理后，非甲基化的胞嘧啶转变为尿嘧啶，在后续的PCR过程中又转换为胸腺嘧啶，会降低基因组的序列复杂度，使得PCR扩增出特异目标片段的难度增大。因此优选地可采用巢式PCR扩增，设计外围与内围两对引物，进行两轮PCR扩增反应，以第一轮的扩增产物作为第二轮扩增的模板，可以有效提高扩增的效率与特异性。然而应理解，本发明中可用的检测方法并不限于此。
经过针对临床样本的研究验证，本发明的方法用于诊断临床肿瘤时，准确性非常高。本发明可应用于肿瘤辅助诊断、疗效判定、预后监测等等领域，具有很高的临床应用价值。
下面结合具体实施例，进一步阐述本发明。应理解，这些实施例仅用于说明本发明而不用于限制本发明的范围。下列实施例中未注明具体条件的实验方法，通常按照常规条件如J.萨姆布鲁克等编著，分子克隆实验指南，第三版，科学出版社，2002中所述的条件，或按照制造厂商所建议的条件。
实施例1、针对STAMP-EP8检测的核酸序列
经过反复研究筛选，本发明人获得一种肿瘤标记物STAMP-EP8，其序列如下SEQ ID NO:1(chr8:23562476-23565175，Human/hg19)所示，其中下划线标示碱基为甲基化CpG位点，下划线下面的数字表该位点的编号。
Figure PCTCN2020087885-appb-000001
Figure PCTCN2020087885-appb-000002
上述SEQ ID NO:1序列经重亚硫酸盐处理后序列如下SEQ ID NO:2(其中Y代表C或U)：
Figure PCTCN2020087885-appb-000003
Figure PCTCN2020087885-appb-000004
上述SEQ ID NO:1所示核苷酸序列的反向互补序列如下SEQ ID NO:3：
Figure PCTCN2020087885-appb-000005
Figure PCTCN2020087885-appb-000006
上述SEQ ID NO:3序列经重亚硫酸盐处理后序列如下SEQ ID NO:4(其中Y代表C或U)：
Figure PCTCN2020087885-appb-000007
Figure PCTCN2020087885-appb-000008
实施例2、STAMP-EP8CpG位点在肿瘤细胞与非肿瘤细胞的甲基化差异—亚硫酸氢盐处理后测序法(BSP-Bisulfite Sequencing PCR)
该亚硫酸氢盐处理后测序法步骤如下：
1.提取肺癌细胞系A549与正常肺细胞系MRC5基因组DNA；
2.分别用重亚硫酸盐处理提取的肺癌细胞系A549与正常肺细胞系MRC5基因组DNA，作为后续PCR扩增的模板；
3.根据SEQ ID NO:1的序列设计扩增引物(SEQ ID NO:5～14)，如表1，进行扩增。
4.PCR扩增之后，2％琼脂糖凝胶电泳检测PCR片段特异性，切胶回收目的片段，连接插入T载体，转化感受态大肠杆菌，涂菌板，第二天挑克隆测序，每个片段挑取大于10个克隆进行Sanger测序。
表1
Figure PCTCN2020087885-appb-000009
Figure PCTCN2020087885-appb-000010
SEQ ID NO:1区域中001-040甲基化位点肺癌细胞与正常肺细胞甲基化水平BSP验证结果如图1，显示肺癌细胞STAMP-EP8甲基化水平显著高于正常肺细胞。
SEQ ID NO:1区域中041-077甲基化位点肺癌细胞与正常肺细胞甲基化水平BSP验证结果如图2，显示肺癌细胞STAMP-EP8甲基化水平显著高于正常肺细胞。
SEQ ID NO:1区域中078-114甲基化位点肺癌细胞与正常肺细胞甲基化水平BSP验证结果如图3，显示肺癌细胞STAMP-EP8甲基化水平显著高于正常肺细胞。
SEQ ID NO:1区域中115-153甲基化位点肺癌细胞与正常肺细胞甲基化水平BSP验证结果如图4，显示肺癌细胞STAMP-EP8甲基化水平显著高于正常肺细胞。
SEQ ID NO:1区域中154-191甲基化位点肺癌细胞与正常肺细胞甲基化水平BSP验证结果如图5，显示肺癌细胞STAMP-EP8甲基化水平显著高于正常肺细胞。
实施例3、STAMP-EP8CpG位点在肿瘤细胞与非肿瘤细胞的甲基化差异—焦磷酸测序法(pyrosequencing)
该焦磷酸测序法步骤如下：
1.获取临床样本：从临床获取癌旁/非癌-癌组织样本，癌旁/非癌样本作为对照组，癌组织样本作为肿瘤检测实验组；
2.DNA提取：分别提取实验组和对照组DNA；本实验用酚氯仿抽提法，但不限于该方法；
3.重亚硫酸盐处理：以重亚硫酸盐处理提取的DNA样本，严格按照步骤操作；本实验中用ZYMO Research公司的EZ DNA Methylation-Gold Kit，货号D5006，但不限于该试剂盒；
4.引物设计：根据STAMP-EP8序列SEQ ID NO：1特点，设计PCR扩增引物与焦磷酸测序引物，检测021-024号CpG位点的甲基化值，作为STAMP-EP8甲基化值的代表，PCR引物扩增序列、焦磷酸测序引物序列、焦磷酸测序上机检测序列以及检测位点如SEQ ID NO:15～18所示，如表2；
5.PCR扩增及琼脂糖凝胶电泳：以重亚硫酸盐处理后的样本作为PCR的模板，进行PCR扩增，扩增后的产物通过琼脂糖凝胶电泳鉴定PCR扩增的特异性；
6.焦磷酸测序：通过QIAGEN公司的Pyro Mark Q96 ID焦磷酸测序仪进行检测， 严格按照说明书步骤进行操作；
7.STAMP-EP8甲基化值计算：焦磷酸测序可以独立检测出目标区域内单个CpG位点的甲基化情况，计算所有CpG位点甲基化平均值作为STAMP-EP8在该样本中的甲基化值；
8.结果分析：比较癌旁/非癌对照组与肿瘤实验组STAMP-EP8甲基化值。
表2
Figure PCTCN2020087885-appb-000011
实施例4、白血病临床样本验证-焦磷酸测序法
从临床获取8例非白血病骨髓涂片样本作为对照组，获取8例白血病骨髓涂片样本作为实验组，按照以上实施例3的焦磷酸检验步骤，比较对照组与实验组STAMP-EP8甲基化水平。
结果如图6，显示在白血病临床样本中，STAMP-EP8在实验组中甲基化值显著高于非癌组织。
实施例5、乳腺癌临床样本验证-焦磷酸测序法
临床获取5例乳腺癌癌旁样本作为对照组，获取5例乳腺癌组织样本作为实验组，按照以上实施例3的焦磷酸检验步骤，比较对照组与实验组STAMP-EP8甲基化水平。
结果如图7，显示在乳腺癌临床样本中，STAMP-EP8在实验组中甲基化值显著高于癌旁组织。
实施例6、结直肠癌临床样本验证-焦磷酸测序法
临床获取8例结直肠癌癌旁样本作为对照组，获取8例结直肠癌样本作为实验组，按照实施例3的焦磷酸检验步骤，分析STAMP-EP8甲基化水平。
结果如图8，显示在结直肠癌临床样本中，STAMP-EP8在实验组中甲基化值显著高于癌旁组织。
实施例7、食管癌临床样本验证-焦磷酸测序法
临床获取10例食管癌癌旁样本作为对照组，获取10例食管癌样本作为实验组，按照实施例3的焦磷酸检验步骤，分析STAMP-EP8甲基化水平。
结果图9，显示在食管癌临床样本中，STAMP-EP8在实验组中甲基化值显著高于癌旁组织。
实施例8、肝癌临床样本验证-焦磷酸测序法
临床获取8例肝癌癌旁样本作为对照组，获取8例肝癌样本作为实验组，按照实施例3的焦磷酸检验步骤，分析STAMP-EP8甲基化水平。
结果如图10，显示在肝癌临床样本中，STAMP-EP8在实验组中甲基化值显著高于癌旁组织。
实施例9、肺癌临床样本验证-焦磷酸测序法
临床获取4例肺癌癌旁样本作为对照组，获取4例肺癌样本作为实验组，按照实施例3的焦磷酸检验步骤，分析STAMP-EP8甲基化水平。
结果如图11，显示在肺癌临床样本中，STAMP-EP8在实验组中甲基化值显著高于癌旁组织。
实施例10、胰腺癌临床样本验证-焦磷酸测序法
临床获取4例胰腺癌癌旁样本作为对照组，获取4例胰腺癌样本作为实验组，按照实施例3的焦磷酸检验步骤，分析STAMP-EP8甲基化水平。
结果如图12，显示在胰腺癌临床样本中，STAMP-EP8在实验组中甲基化值显著高于癌旁组织。
在本发明提及的所有文献都在本申请中引用作为参考，就如同每一篇文献被单独引用作为参考那样。此外应理解，在阅读了本发明的上述讲授内容之后，本领域技术人员可以对本发明作各种改动或修改，这些等价形式同样落于本申请所附权利要求书所限定的范围。

Claims (18)

  1. 分离的多核苷酸，其特征在于，包括：
    (a)SEQ ID NO:1所示核苷酸序列的多核苷酸；
    (b)(a)的多核苷酸的片段，且其中存在至少1个修饰的CpG位点；和/或
    (c)与上述(a)或(b)的多核苷酸或片段互补的核酸。
  2. 如权利要求1所述的分离的多核苷酸，其特征在于，所述修饰包括5-甲基化修饰、5-羟甲基化修饰、5-醛甲基化修饰或5-羧甲基化修饰。
  3. 如权利要求1所述的分离的多核苷酸，其特征在于，(b)中，所述的多核苷酸的片段中存在：
    SEQ ID NO:1或2中第204～223位碱基；
    SEQ ID NO:1或2中第1～478位碱基；
    SEQ ID NO:1或2中第513～1040位碱基；
    SEQ ID NO:1或2中第1082～1602位碱基；
    SEQ ID NO:1或2中第1621～2117位碱基；或
    SEQ ID NO:1或2中第2160～2700位碱基。
  4. 分离的多核苷酸，其特征在于，其由权利要求1、2或3所述的多核苷酸转变而来，对应于权利要求1或3的序列，其修饰的CpG位点的胞嘧啶C不变，非修饰的胞嘧啶转为T或U。
  5. 如权利要求4所述的多核苷酸，其特征在于，包括：
    (d)SEQ ID NO:2或SEQ ID NO:4所示核苷酸序列的多核苷酸；
    (e)上述(d)的多核苷酸的片段，且其中存在至少1个修饰的CpG位点。
  6. 如权利要求5所述的多核苷酸，其特征在于，(e)中，所述的多核苷酸的片段中存在：
    SEQ ID NO:1或2中第204～223位碱基；
    SEQ ID NO:1或2中第1～478位碱基；
    SEQ ID NO:1或2中第513～1040位碱基；
    SEQ ID NO:1或2中第1082～1602位碱基；
    SEQ ID NO:1或2中第1621～2117位碱基；
    SEQ ID NO:1或2中第2160～2700位碱基；
    SEQ ID NO:4或3中第2478～2497位碱基；
    SEQ ID NO:4或3中第2223～2700位碱基；
    SEQ ID NO:4或3中第1661～2188位碱基；
    SEQ ID NO:4或3中第1099～1619位碱基；
    SEQ ID NO:4或3中第584～1080位碱基；或
    SEQ ID NO:4或3中第1～541位碱基。
  7. 权利要求1～6任一所述的多核苷酸的用途，其特征在于，用于制备肿瘤的检测试剂或试剂盒。
  8. 如权利要求7所述的用途，其特征在于，所述肿瘤包括：血液系统肿瘤如白血病，淋巴瘤，多发性骨髓瘤；妇科及生殖系统肿瘤如乳腺癌，卵巢癌，宫颈癌，外阴癌，睾丸癌，前列腺癌，阴茎癌；消化系统肿瘤如食道癌，胃癌，结直肠癌，肝癌，胰腺癌，胆管及胆囊癌；呼吸系统肿瘤如肺癌，胸膜瘤；神经系统肿瘤如胶质瘤，神经母细胞瘤，脑膜瘤；头颈部肿瘤如口腔癌，舌癌，喉癌，鼻咽癌；泌尿系统肿瘤如肾癌，膀胱癌，皮肤及其他系统如皮肤癌、黑色素瘤、骨肉瘤，脂肪肉瘤，甲状腺癌。
  9. 如权利要求7或8所述的用途，其特征在于，所述肿瘤的样本包括：组织样本、石蜡包埋样本、血液样本、胸腔积液样本以及肺泡灌洗液样本、腹水及灌洗液样本、胆汁样本、粪便样本、尿液样本、唾液样本、痰液样本、脑脊液样本、细胞涂片样本、宫颈刮片或刷片样本、组织及细胞活检样本。
  10. 一种制备肿瘤检测试剂的方法，其特征在于，所述方法包括：提供权利要求1～6任一所述的多核苷酸，以所述多核苷酸全长或片段作为靶序列，设计特异性检测该靶序列的CpG位点修饰情况的检测试剂；其中，所述的靶序列中存在至少1个修饰的CpG位点；较佳地，所述的检测试剂包括：引物，探针。
  11. 试剂或组合的试剂，其特征在于，其特异性检测靶序列的CpG位点修饰情况，所述的靶序列是权利要求1～6任一所述的多核苷酸的全长或片段，其中存在至少1个修饰的CpG位点。
  12. 如权利要求11所述的试剂或组合的试剂，其特征在于，所述的试剂或组合的试剂针对包含所述靶序列的基因序列，所述的基因序列包括基因Panel或基因群组。
  13. 如权利要求11所述的试剂或组合的试剂，其特征在于，所述的试剂或组合的试剂包括：引物或探针；较佳地，所述的引物为：
    SEQ ID NO:5和6所示的引物；
    SEQ ID NO:7和8所示的引物；
    SEQ ID NO:9和10所示的引物；
    SEQ ID NO:11和12所示的引物；
    SEQ ID NO:13和14所示的引物；或
    SEQ ID NO:15和16所示的引物。
  14. 权利要求11～13任一所述的试剂或组合的试剂的用途，用于制备检测肿瘤的试剂盒；较佳地，所述的肿瘤包括：血液系统肿瘤如白血病，淋巴瘤，多发性骨髓瘤；妇科及生殖系统肿瘤如乳腺癌，卵巢癌，宫颈癌，外阴癌，睾丸癌，前列腺癌， 阴茎癌；消化系统肿瘤如食道癌，胃癌，结直肠癌，肝癌，胰腺癌，胆管及胆囊癌；呼吸系统肿瘤如肺癌，胸膜瘤；神经系统肿瘤如胶质瘤，神经母细胞瘤，脑膜瘤；头颈部肿瘤如口腔癌，舌癌，喉癌，鼻咽癌；泌尿系统肿瘤如肾癌，膀胱癌，皮肤及其他系统如皮肤癌、黑色素瘤、骨肉瘤，脂肪肉瘤，甲状腺癌。
  15. 一种检测试剂盒，其特征在于，其包括：
    容器，以及位于容器中的权利要求11～13任一所述的试剂或试剂组合。
  16. 一种体外检测样品的甲基化谱式的方法，其特征在于，包括：
    (i)提供样品，提取核酸；
    (ii)检测(i)的核酸中靶序列的CpG位点修饰情况，所述的靶序列是权利要求1～3任一所述的多核苷酸或由其转变而来的权利要求3～6任一所述的多核苷酸。
  17. 如权利要求16所述的方法，其特征在于，步骤(3)中，分析的方法包括：焦磷酸测序法、重亚硫酸盐转化测序法、甲基化芯片法、qPCR法、数字PCR法、二代测序法、三代测序法、全基因组甲基化测序法、DNA富集检测法、简化亚硫酸氢盐测序技术、HPLC法、MassArray、甲基化特异PCR、或它们的组合以及SEQ ID NO:1所示序列中部分或全部甲基化位点的组合基因群组体外检测方法及体内示踪检测方法。
  18. 如权利要求16所述的方法，其特征在于，步骤(ii)包括：
    (1)对(i)的产物进行处理，使其中未发生修饰的胞嘧啶转化为尿嘧啶；较佳地，所述修饰包括5-甲基化修饰、5-羟甲基化修饰、5-醛甲基化修饰或5-羧甲基化修饰；较佳地，利用亚硫酸氢盐或重亚硫酸氢盐处理步骤(i)所述的核酸；
    (2)分析经(1)处理的核酸中所述的靶序列的修饰情况。
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