WO2019024578A1 - 用于检测肝肿瘤良恶性的基因标志物、试剂盒及检测方法 - Google Patents

用于检测肝肿瘤良恶性的基因标志物、试剂盒及检测方法 Download PDF

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WO2019024578A1
WO2019024578A1 PCT/CN2018/088132 CN2018088132W WO2019024578A1 WO 2019024578 A1 WO2019024578 A1 WO 2019024578A1 CN 2018088132 W CN2018088132 W CN 2018088132W WO 2019024578 A1 WO2019024578 A1 WO 2019024578A1
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hydroxymethylcytosine
benign
content
liver
gene marker
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French (fr)
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陆星宇
宋艳群
彭莱
张子谋
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上海易毕恩生物技术有限公司
上海易毕恩基因科技有限公司
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    • 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
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    • 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
    • C12Q2600/112—Disease subtyping, staging or classification
    • 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

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  • the invention relates to the technical field of clinical molecular diagnosis of benign and malignant liver tumors. Specifically, the present invention relates to a genetic marker, a kit for detecting benign and malignant liver tumors, and a method for detecting benign and malignant liver tumors.
  • Tumors are divided into two major categories of benign tumors and malignant tumors. Benign tumors grow slowly, and generally have no harm to health and life except for the influence of the place where the vital parts occupy. Malignant tumors grow rapidly, compete with the human body for nutrition, produce harmful metabolites, destroy the normal organ structure of the human body, and are extremely harmful to human health. If not treated in time, it will be life-threatening.
  • Common benign liver tumors include hepatocellular adenomas, hepatic duct cell adenomas, adrenal residual tumors, hemangiomas, hamartomas, and the like. Hepatic malignancy refers to hepatocellular carcinoma, which is the liver cancer we usually talk about. Liver cancer is one of the most common global malignancies. According to the statistics of the World Health Organization in 2008, there are 748,300 new cases each year and 695,900 deaths, of which more than 50% occur in China.
  • Both benign and malignant tumors are fundamentally different in terms of cell morphology, tissue structure, growth pattern, growth rate and impact on the human body, so the treatment methods are different. Treating a malignant tumor as a benign tumor will delay the patient. Conversely, treating a benign tumor as a malignant tumor not only causes a mental burden on the patient, but also causes the patient to suffer and suffer as a result of taking many unnecessary treatments. However, some benign and malignant tumors have similar growth patterns, which cause great difficulties in diagnosis. If there is a benign tumor, the tumor grows faster and looks like a malignant tumor; while some malignant tumors may grow slowly like a benign tumor. Even some tumors have two characteristics of benign and malignant. Although the tumor cells are benign and have a complete capsule, it is multi-central growth. After treatment, the recurrence rate is higher than that of general benign tumors. These characteristics look like malignant tumors, which are called critical tumors. In addition, some benign tumors may become malignant during their development, and also cause clinical attention.
  • the invention successfully analyzes the content of 5-hydroxymethylcytosine (5-hmC) in each gene by high-throughput sequencing of benign liver tumor samples and liver cancer samples, and unexpectedly finds a plurality of extremely high Information can be used to detect genetic markers of benign and malignant liver tumors.
  • a first aspect of the invention relates to a genetic marker for detecting benign and malignant liver tumors, comprising one or more of the following genes: FAT atypical cadherin 1 (FAT1), estrogen-related receptor gamma (ESRRG) ), sex determining gene y chromosome region cassette family member 9 (SOX9), cilia complex subunit 1 (EVC), chromosome 1 open reading frame 125 antibody (AXDND1), tyrosin (RELN), transferrin (TF), TNF receptor superfamily member (TNFRSF11B), pancreas and duodenum with round frame 1 (PDX1), alpha-acid glycoprotein 2 (ORM2).
  • the genetic markers include FAT1, ESRRG, SOX9, EVC, AXDND1, RELN, TF, TNFRSF11B, PDX1 and ORM2.
  • the invention also relates to the use of the above gene marker for detecting benign and malignant liver tumors, and detecting the content of 5-hydroxymethylcytosine in the lung cancer gene marker by high-throughput sequencing, thereby determining the benignness of the liver tumor.
  • a second aspect of the invention relates to a method for detecting benign and malignant liver tumors, comprising the steps of:
  • the sample is a free DNA fragment in the body fluid of the subject, or is derived from intact genomic DNA in organelles, cells, and tissues.
  • body fluids are blood, urine, sweat, sputum, feces, cerebrospinal fluid, ascites, pleural effusion, bile, pancreatic juice, and the like.
  • the 5-hmC content of the genetic markers of the invention can be determined by any method known to those skilled in the art, including, for example, but not limited to, glucosylation, restriction endonucleases Method, chemical labeling method, precipitation method combined with high-throughput sequencing method, single molecule real-time sequencing method (SMRT), oxidized bisulfite sequencing method (OxBS-Seq), and the like.
  • the principle of the glucosylation method is to transfer glucose to the hydroxyl group in the presence of glucose donor substrate uridine nucleoside diphosphate glucose (UDP-Glu) using T4 phage ⁇ -glucose transferase ( ⁇ -GT).
  • ⁇ -Glucosyl-5-hydroxymethylcytosine (5-ghmC) was produced. Isotopically labeled substrates can also be used for quantification. Restriction endonuclease and chemical labeling methods were further developed based on the glucosylation method. The principle of the restriction endonuclease method is that the glucosylation reaction changes the enzymatic cleavage properties of some restriction enzymes.
  • MspI and HpaII recognize the same sequence (CCGG), but their sensitivity to methylation status is different: MspI recognizes and cleaves 5-methylcytosine (5-mC) And 5-hmC, but not 5-ghmC; HpaII only cleaves completely unmodified sites, and any modification on cytosine (5-mC, 5-hmC, 5-ghmC) blocks cleavage. If the CpG site contains 5-hmC, the band can be detected after glycosylation and enzymatic hydrolysis, and there is no band in the unglycosylated control reaction; qPCR can also be used for quantitative analysis.
  • restriction enzymes also have a hindrance to 5-ghmC digestion, and can be applied to 5-hmC detection (eg, GmrSD, MspJI, PvuRts1I, TaqI, etc.).
  • the principle of the chemical labeling method is to chemically modify the glucose on the substrate of the enzyme reaction into UDP-6-N3-glucose, and transfer 6-N3-glucose to the position of the hydroxymethyl group to form N3-5ghmC. Subsequently, a single molecule of biotin was added to each 5-hmC by click chemistry, combined with next-generation high-throughput DNA sequencing technology or single-molecule sequencing technology to analyze the distribution of 5-hmC in genomic DNA.
  • the precipitation method is to modify 5-hmC in a special way and then specifically capture it from genomic DNA and perform sequencing analysis.
  • Oxidized bisulfite sequencing is the first method to quantify 5-hmC with single base resolution. Firstly, 5-hmC is subjected to KRuO4 oxidation treatment to produce 5-formylcytosine (5fC), and then heavy Sulfite sequencing. In this process, 5-hmC is first oxidized to 5fC, and then deaminated to form U. Usually, quantitative detection of 5-hmC is performed simultaneously using a variety of detection methods.
  • the 5-hmC content of the genetic markers of the invention is determined using chemical labeling in conjunction with high throughput sequencing.
  • the method of determining the 5-hmC content of a genetic marker of the present invention comprises the steps of: fragmenting DNA from a liver cancer patient and a benign liver tumor patient; repairing the fragmented DNA end and The ends are filled; the end-filled DNA is ligated to the sequencing linker to obtain a ligation product; the 5-hydroxymethylcytosine in the ligation product is labeled by a labeling reaction; the DNA containing the 5-hydroxymethylcytosine tag is enriched The fragment is obtained, and the enriched product is obtained; the enriched product is subjected to PCR amplification to obtain a sequencing library; the sequencing library is subjected to high-throughput sequencing to obtain a sequencing result; and the content of 5-hydroxymethylcytosine is determined according to the sequencing result.
  • the labeling reaction comprises: i) covalent attachment of a sugar having a modifying group to a methylol group of 5-hydroxymethylcytosine using a glycosyltransferase, and ii) direct or indirect attachment of biotin Click on the chemical substrate to react with 5-hydroxymethylcytosine with a modifying group.
  • step i) and step ii) may be carried out sequentially or simultaneously in one reaction. This labeling method reduces the amount of sample required for sequencing, and the biotin tag on 5-hydroxymethylcytosine allows it to display higher kinetic signals in sequencing, improving the accuracy of nucleotide recognition.
  • the glycosyltransferase includes, but is not limited to, T4 phage ⁇ -glucosyltransferase ( ⁇ -GT), T4 bacteriophage ⁇ -glucosyltransferase ( ⁇ -GT), and the same Or a similarly active derivative, analog, or recombinase;
  • the saccharide with a modifying group includes, but is not limited to, a saccharide with an azide modification (eg, 6-N3-glucose) or with other chemical modifications a saccharide (e.g., a carbonyl group, a thiol group, a hydroxyl group, a carboxyl group, a carbon-carbon double bond, a carbon-carbon triple bond, a disulfide bond, an amine group, an amide group, a diene, etc.), among which a saccharide modified with azide is preferred.
  • the chemical group for indirectly linking the biotin and the click chemical substrate includes, but is not limited to, a carbonyl group, a thiol group, a hydroxyl group, a carboxyl group, a carbon-carbon double bond, a carbon-carbon triple bond, a disulfide bond, an amine group, Amido group, diene.
  • the DNA fragment containing the 5-hmC label is preferably enriched by a solid phase material.
  • a DNA fragment containing a 5-hydroxymethylcytosine label can be bound to a solid phase material by a solid phase affinity reaction or other specific binding reaction, and then the unbound DNA fragment can be removed by multiple washings.
  • Solid phase materials include, but are not limited to, silicon wafers or other chips with surface modification, such as artificial polymer beads (preferably 1 nm to 100 um in diameter), magnetic beads (preferably 1 nm to 100 um in diameter), agarose beads, etc. (Preferably from 1 nm to 100 um in diameter).
  • PCR amplification is preferably performed directly on the solid phase to prepare a sequencing library.
  • the amplified product can be recovered and subjected to a second round of PCR amplification to prepare a sequencing library.
  • the second round of PCR amplification can be performed using conventional methods known to those skilled in the art.
  • one or more purification steps may be further included in the process of preparing the sequencing library. Any purification kit known or commercially available to those skilled in the art can be used in the present invention. Purification methods include, but are not limited to, gel electrophoresis gel recovery, silica gel membrane spin column method, magnetic bead method, ethanol or isopropanol precipitation method, or a combination thereof.
  • the sequencing library is quality checked prior to high throughput sequencing.
  • the library is subjected to fragment size analysis and the concentration of the library is absolutely quantified using the qPCR method. Sequencing libraries that pass quality checks can be used for high throughput sequencing. Then, a certain number (1-96) of libraries containing different barcodes were mixed at the same concentration and sequenced according to the standard on-line method of the second generation sequencer to obtain sequencing results.
  • Various second generation sequencing platforms and related reagents known in the art can be used in the present invention.
  • the sequencing results are preferably aligned with a standard human genome reference sequence, and the sequences in which the gene markers of the invention are aligned are selected, ie, the alignment sites and gene features (eg, groups) are selected.
  • the number of reads of the coincident region of the protein modification site, transcription factor binding site, gene exon intron region, and gene promoter, etc., to represent the level of modification of 5-hmC on the gene, thereby determining 5-hmC The amount on the genetic marker.
  • the sequencing results are first cleared of low-quality sequencing sites prior to the alignment, wherein factors that measure the quality of the sequencing sites include, but are not limited to, base quality, reads mass, GC content, repeat sequences, and number of Overrepresented sequences.
  • factors that measure the quality of the sequencing sites include, but are not limited to, base quality, reads mass, GC content, repeat sequences, and number of Overrepresented sequences.
  • determining the 5-hmC content of the gene marker means determining the 5-hmC content of the full length of the gene marker or determining the 5-hmC content of a fragment of the gene marker or combination.
  • the 5-hmC content of the gene marker in the benign liver tumor sample is used as a reference, and the corresponding gene marker in the liver cancer subject sample is 5 -hmC content is standardized.
  • the 5-hmC content of the same gene marker in the benign liver tumor sample and the liver cancer subject sample is X and Y, respectively, and the standardized 5-hmC content of the gene marker in the liver cancer subject sample is Y/ X.
  • the standardized 5-hmC content of each gene marker is mathematically correlated to obtain a score, thereby obtaining a detection result based on the score.
  • mathematical association refers to any computational or machine learning method that correlates the 5-hmC content of a genetic marker from a biological sample with a liver tumor diagnosis.
  • computing methods or tools can be selected to provide the mathematical associations of the present invention, such as elastic network regularization, decision trees, generalized linear models, logistic regression, highest score pairs, neural networks, linear and Quadratic Discriminant Analysis (LQA and QDA), Naive Bayes, Random Forest, and Support Vector Machines.
  • the specific steps for mathematically correlating the standardized 5-hmC content of each gene marker and obtaining a score are as follows: multiplying the normalized 5-hmC content of each gene marker by a weighting coefficient to obtain the gene The predictor of the marker t; the predictor t of each gene marker is added to obtain a total predictor T; the total predictor T is subjected to Logistic conversion to obtain a score P; if P>0.5, the subject sample suffers Liver cancer; if P ⁇ 0.5, the subject has a benign liver tumor.
  • the weighting factor described herein refers to the art by the art in consideration of factors that may affect the 5-hmC content (eg, subject area, age, sex, below, smoking history, drinking history, family history, etc.)
  • factors that may affect the 5-hmC content eg, subject area, age, sex, below, smoking history, drinking history, family history, etc.
  • a third aspect of the present invention also relates to a kit for detecting benign and malignant liver tumors using the above gene marker, which comprises reagents and instructions for determining the 5-hmC content of the above gene marker.
  • Agents for determining the 5-hmC content of a genetic marker are known to those skilled in the art, such as T4 bacteriophage beta-glucose transferase and isotopic labeling (for glucosylation), restriction enzymes (for restriction) Endonuclease method), glycosyltransferase and biotin (for chemical labeling), reagents for PCR and sequencing, and the like.
  • the method for detecting benign and malignant liver tumors of the present invention is based on the 5-hmC content of the gene marker, and thus a wider range of DNA sample sources can be used. Therefore, the present invention has the following advantages: (1) safe and non-invasive, even if the asymptomatic population has high acceptance of the test; (2) a wide range of DNA sources, no detection blind spots in imaging; (3) high accuracy It has higher sensitivity and specificity for liver cancer, and is more suitable for distinguishing between benign and malignant liver tumors; (4) easy to operate, good user experience, and easy to monitor the dynamic development of liver tumors.
  • the gene marker of the invention can be combined with other clinical indicators to provide a more accurate judgment for subsequent screening, diagnosis and treatment of liver tumors.
  • Figure 1 is a graph showing a control of a malignant liver tumor sample and a benign liver tumor sample of the present invention.
  • 10 ng of plasma DNA was extracted from samples from 20 liver cancer patients and 20 benign liver tumor patients, respectively. This step can be carried out using any method and reagent suitable for extracting plasma DNA well known to those skilled in the art.
  • the purified labeled product obtained in the above procedure was added to the magnetic bead mixture, and mixed for 15 minutes in a rotary mixer to sufficiently bind.
  • the amplified product was purified using Ampure XP beads to give a final sequencing library.
  • the obtained sequencing library was subjected to concentration determination by qPCR, and the DNA fragment size content in the library was determined using Agilent 2100.
  • the sequencing libraries passed the QC were mixed at the same concentration and sequenced using an Illumina Hiseq 4000.
  • the obtained sequencing results were subjected to preliminary quality control evaluation, and after the low-quality sequencing sites were cleared, the readings that met the sequencing quality standards were compared with the human standard genomic reference sequences using the Bowtie 2 tool.
  • the feature counts and HtSeq-Count tools were then used to count the number of reads to determine the 5-hmC content of each gene marker.
  • the factors that may affect the 5-hmC content were used as covariates, and the weighting coefficients of each gene marker were obtained by logistic regression and elastic network regularization. The results are shown in Table 1.
  • Table 1 Average normalized 5-hmC content and weighting factor of liver tumor gene markers of the present invention
  • the average normalized 5-hmC content refers to the ratio of the average 5-hmC content of the gene marker in the liver cancer sample to the average 5-hmC content of the same gene marker in the benign liver tumor sample.
  • the 5-hmC content of the liver tumor gene marker of the present invention is significantly different between the benign liver tumor sample and the liver cancer sample.
  • liver tumor gene markers of the present invention for distinguishing between benign and malignant liver tumors.
  • the 5-hmC content of the 10 liver cancer gene markers of the present invention in the first batch of 164 samples was determined according to the method of Example 1.
  • the subject sample has liver cancer; if P ⁇ 0.5, the subject has a benign liver tumor.
  • Figure 1 shows the results of distinguishing the batch of samples in accordance with the method of the present invention. As shown in Figure 1, the method of the invention is capable of achieving 95% sensitivity and 96% specificity.

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Abstract

本发明提供了用于检测肝肿瘤良恶性的基因标志物、试剂盒及检测方法。通过高通量测序检测FAT非典型钙粘蛋白1、雌激素相关受体γ、性别决定基因y染色体区域盒家族成员9、纤毛状络合物子单元1、1号染色体开放阅读框125抗体、络丝蛋白、转铁蛋白、TNF受体超家族成员、胰腺和十二指肠同圆框1、α-酸性糖蛋白2中一个或两个以上标志基因中5-羟甲基胞嘧啶的含量，从而判定肝肿瘤良恶性

Description

用于检测肝肿瘤良恶性的基因标志物、试剂盒及检测方法 技术领域
本发明涉及肝肿瘤良恶性的临床分子诊断技术领域。具体的，本发明涉及用于检测肝肿瘤良恶性的基因标志物、试剂盒及肝肿瘤良恶性的检测方法。
背景技术
肿瘤分良性肿瘤和恶性肿瘤两大类。良性肿瘤生长缓慢，除在要害部位占位有影响外，一般对健康和生命没有危害。恶性肿瘤生长迅速，与人体争夺营养，产生有害代谢产物，破坏人体正常器官组织结构，对人体健康极为有害，如不及时进行有效治疗将会夺人生命。常见的肝良性肿瘤有肝细胞腺瘤、肝管细胞腺瘤、肾上腺残余瘤、血管瘤、错构瘤等。肝恶性肿瘤就是指肝细胞肝癌，即我们通常讲的肝癌。肝癌是最常见的全球恶性肿瘤之一。据世界卫生组织2008年统计，全球每年新发病748300例，死亡695900例，其中50％以上发生在中国。
良性肿瘤与恶性肿瘤二者在细胞形态、组织结构、生长方式、增长速度和对人体影响等方面均有本质的不同，所以治疗方式上也不一样。把恶性肿瘤当作良性肿瘤治疗就会贻误病人。反之，若把良性肿瘤当作恶性肿瘤治疗，不仅造成病人精神上负担，而且由于采取许多不必要的治疗手段，致使病人遭受痛苦和损失。但某些良性肿瘤和恶性肿瘤有着相近似的生长形式，给诊断造成很大的困难。如有的良性 肿瘤生长较快，状似恶性肿瘤；而有的恶性肿瘤有可能生长缓慢很象良性肿瘤。甚至有的肿瘤本身就具有良性恶性的两种特点，虽然肿瘤细胞表现为良性，而且有完整包膜，但它是多中心性生长，治疗后较一般良性肿瘤复发率高，就其临床的某些特点来看很似恶性肿瘤，固称之为临界性肿瘤。此外，某些良性肿瘤在其发展过程中有变为恶性的可能，也要引起临床的重视。
因此，针对肝良性肿瘤和恶性肿瘤寻找诊断标志物，提高对肝良性肿瘤与恶性肿瘤区分鉴别能力，对临床诊断具有非常重要的意义。
发明内容
本发明通过对良性肝肿瘤样品与肝癌样品进行高通量测序，并对其中各基因上的5-羟甲基胞嘧啶(5-hmC)含量进行分析，出乎意料地发现了多个极具信息的可用于检测肝肿瘤良恶性的基因标志物。
因此，本发明的第一个方面涉及用于检测肝肿瘤良恶性的基因标志物，包括一个或两个以上以下基因：FAT非典型钙粘蛋白1(FAT1)、雌激素相关受体γ(ESRRG)、性别决定基因y染色体区域盒家族成员9(SOX9)、纤毛状络合物子单元1(EVC)、1号染色体开放阅读框125抗体(AXDND1)、络丝蛋白(RELN)、转铁蛋白(TF)、TNF受体超家族成员(TNFRSF11B)、胰腺和十二指肠同圆框1(PDX1)、α-酸性糖蛋白2(ORM2)。优选的，所述基因标志物包括FAT1、ESRRG、SOX9、EVC、AXDND1、RELN、TF、TNFRSF11B、PDX1和ORM2。
本发明还涉及上述基因标志物在检测肝肿瘤良恶性中的用途，通过高通量测序检测肺癌基因标志物中5-羟甲基胞嘧啶的含量，从而 判定肝肿瘤的良性。
本发明的第二个方面涉及用于检测肝肿瘤良恶性的方法，包括以下步骤：
a)测定良性肝肿瘤样品和肝癌受试者样品中本发明所述的基因标志物的5-hmC的含量；
b)用良性肝肿瘤样品中所述基因标志物的5-hmC含量作为参照，将肝癌受试者样品中对应的基因标志物的5-hmC含量标准化；
c)对经标准化的所述基因标志物的5-hmC含量进行数学关联，并获得评分；
d)根据所述评分获得区分结果。
在一个实施方案中，所述样品是受试者体液中游离的DNA片段，或来源于细胞器、细胞以及组织中的完整基因组DNA。其中，体液是血液、尿液、汗液、痰液、粪便、脑脊液、腹水、胸水、胆汁、胰腺液等。
在一个实施方案中，本发明所述的基因标志物的5-hmC含量可通过本领域技术人员已知的任何方法进行测定，例如包括但不限于，葡糖基化法、限制性内切酶法、化学标记法、与高通量测序方法联用的沉淀法、单分子实时测序法(SMRT)、氧化重亚硫酸盐测序法(OxBS-Seq)等。葡糖基化法的原理是采用T4噬菌体β-葡萄糖转移酶(β-GT),在葡萄糖供体底物尿核苷二磷酸葡萄糖(UDP-Glu)存在下,将葡萄糖转移至羟基位置,从而生成β-葡萄糖基-5-羟甲基胞嘧啶(5-ghmC)。同时可采用同位素标记底物进行定量。在葡糖基化法基础上 进一步发展出限制性内切酶法和化学标记法。限制性内切酶法的原理是：葡糖基化反应改变了一些限制性内切酶的酶切特性。甲基化依赖的限制性内切酶MspI和HpaII可识别同样的序列(CCGG),但它们对甲基化状态的敏感性是不同:MspI识别并切割5-甲基胞嘧啶(5-mC)和5-hmC,但不能切割5-ghmC；HpaII只切割完全未修饰的位点,胞嘧啶上的任何修饰(5-mC、5-hmC、5-ghmC)均阻碍切割。若CpG位点含有5-hmC,那么糖基化、酶解之后能检测到条带,未糖基化对照反应中没有条带；同时可采用qPCR进行定量分析。另外,其他限制性内切酶也同样存在阻碍5-ghmC酶切的情况,可应用于5-hmC检测(如:GmrSD,MspJI,PvuRts1I,TaqI等)。化学标记法的原理是：将酶反应底物上的葡萄糖进行化学修饰转变成UDP-6-N3-glucose,将6-N3-glucose转移到羟甲基位置,生成N3-5ghmC。随后,通过点击化学方法在每个5-hmC上添加一分子生物素,结合下一代高通量DNA测序技术或单分子测序技术,可分析5-hmC在基因组DNA中的分布情况。沉淀法是将5-hmC用特殊方式修饰后再将其特异性地从基因组DNA中捕获下来，并进行测序分析。氧化重亚硫酸盐测序法是首个以单碱基分辨率对5-hmC进行定量测序的方法.首先将5-hmC进行KRuO4氧化处理,生成5-甲酰胞嘧啶(5fC),然后采用重亚硫酸盐测序。在此过程中,5-hmC先氧化为5fC,而后脱氨形成U。通常，同时采用多种检测方法对5-hmC进行定量检测。
在本发明的一个实施方案中，利用化学标记法结合高通量测序来测定本发明的基因标志物的5-hmC含量。在该具体的实施方案中，测 定本发明的基因标志物的5-hmC含量的方法包括以下步骤：将来自肝癌患者和良性肝肿瘤患者的DNA片段化；将所述片段化的DNA末端修复并末端补齐；将末端补齐的DNA与测序接头连接，获得连接产物；通过标记反应对连接产物中的5-羟甲基胞嘧啶进行标记；富集含有5-羟甲基胞嘧啶标记的DNA片段，获得富集产物；对富集产物进行PCR扩增，获得测序文库；对测序文库进行高通量测序，获得测序结果；根据测序结果确定5-羟甲基胞嘧啶在基因上的含量。其中，标记反应包括：i)利用糖基转移酶将带有修饰基团的糖共价连接到5-羟甲基胞嘧啶的羟甲基上，和ii)将直接或间接连有生物素的点击化学底物与带有修饰基团的5-羟甲基胞嘧啶反应。其中，步骤i)和步骤ii)可以按顺序进行，也可以在一个反应中同时进行。这种标记方法减少了测序所需的样本量，且5-羟甲基胞嘧啶上的生物素标签使其在测序中显示出更高的动力学信号，提高了核苷酸识别的准确性。在该实施方案中，所述糖基转移酶包括但不限于：T4噬菌体β-葡糖基转移酶(β-GT)、T4噬菌体α-葡糖基转移酶(α-GT)及其具有相同或相似活性的衍生物、类似物、或重组酶；所述带有修饰基团的糖包括但不限于：带有叠氮修饰的糖类(例如6-N3-葡萄糖)或带有其他化学修饰(例如羰基、巯基、羟基、羧基、碳-碳双键、碳-碳三键、二硫键、胺基、酰胺基、双烯等)的糖类，其中优选带有叠氮修饰的糖类；所述用于间接连接生物素和点击化学底物的化学基团包括但不限于：羰基、巯基、羟基、羧基、碳-碳双键、碳-碳三键、二硫键、胺基、酰胺基、双烯。在该实施方案中，优选通过固相材料来 富集含有5-hmC标记的DNA片段。具体地，可以通过固相亲和反应或其他特异性结合反应将含有5-羟甲基胞嘧啶标记的DNA片段结合在固相材料上，然后通过多次洗涤去除未结合的DNA片段。固相材料包括但不限于带有表面修饰的硅片或其他芯片，例如人工高分子小球(优选直径为1nm-100um)、磁性小球(优选直径为1nm-100um)、琼脂糖小球等(优选直径为1nm-100um)。固相富集中所用的洗涤液是本领域技术人员熟知的缓冲液，包括但不限于：含有Tris-HCl、MOPS、HEPES(pH＝6.0-10.0，浓度在1mM到1M之间)、NaCl(0-2M)或表面活性剂如Tween20(0.01％-5％)的缓冲液。在该实施方案中，优选直接在固相上进行PCR扩增从而制备测序文库。如有需要，在固相上进行PCR扩增后，可以回收扩增产物后进行第二轮PCR扩增来制备测序文库。所述第二轮PCR扩增可用本领域技术人员已知的常规方法进行。任选地，在制备测序文库的过程中可进一步包括一个或多个纯化步骤。本领域技术人员知晓的或可商购的任何纯化试剂盒均可用于本发明。纯化方法包括但不限于：凝胶电泳切胶回收、硅胶膜离心柱法、磁珠法、乙醇或异丙醇沉淀法或其组合。任选地，在高通量测序之前，对测序文库进行质量检查。例如，对文库进行片段大小分析并使用qPCR方法对文库的浓度进行绝对定量。通过质量检查的测序文库可用于高通量测序。然后将一定数量(1-96个)含有不同barcode的文库按相同浓度混匀并根据二代测序仪的标准上机方法上机测序，获得测序结果。本领域已知的各种二代测序平台及其相关的试剂可用于本发明。
在本发明的一个实施方案中，优选将测序结果与标准人类基因组参考序列进行比对，挑选出其中比对到本发明基因标志物上的序列，即选择比对位点与基因特征(如组蛋白修饰位点、转录因子结合位点、基因外显子内含子区域以及基因启动子等)重合区域的读段数量，以代表5-hmC在该基因上的修饰水平，从而测定5-hmC在该基因标志物上的含量。优选在进行比对前，首先将测序结果清除低质量测序位点，其中衡量测序位点质量的因素包括但不限于：碱基质量、reads质量、GC含量、重复序列和Overrepresented序列数量等。该步骤中涉及的各种比对软件和分析方法是本领域已知的。
在本发明的一个实施方案中，测定基因标志物的5-hmC含量是指测定该基因标志物全长上的5-hmC含量或测定该基因标志物上某一片段的5-hmC含量或其组合。
根据本发明，在测定各基因标志物上5-hmC含量之后，用良性肝肿瘤样品中所述基因标志物的5-hmC含量作为参照，将肝癌受试者样品中对应的基因标志物的5-hmC含量标准化。举例而言，良性肝肿瘤样品和肝癌受试者样品中同一基因标志物的5-hmC含量分别为X和Y，则肝癌受试者样品中该基因标志物的标准化5-hmC含量为Y/X。根据本发明，在数据标准化后，对各基因标志物的标准化5-hmC含量进行数学关联以获得评分，从而根据所述评分获得检测结果。如本文所用，“数学关联”是指将来自生物样品的基因标志物的5-hmC含量与肝肿瘤诊断结果相关联的任何计算方法或机器学习方法。本领域普通技术人员理解，可选择不同的计算方法或工具用于提供本发明的数 学关联，例如弹性网络正则化、决策树、广义线性模型、逻辑回归、最高分值对、神经网络、线性和二次判别式分析(LQA和QDA)、朴素贝叶斯、随机森林和支持向量机。
在本发明的一个实施方案中，对各基因标志物的标准化5-hmC含量进行数学关联并获得评分的具体步骤如下：将各基因标志物的标准化5-hmC含量乘以加权系数，获得该基因标志物的预测因子t；将各基因标志物的预测因子t相加，获得总预测因子T；将总预测因子T经过Logistic转换获得评分P；若P>0.5，则该受试者样品患有肝癌；若P≤0.5，则该受试者患有良性肝肿瘤。本文所述的加权系数是指在考虑可能影响5-hmC含量的因素(例如受试者地域、年龄、性别、低于、吸烟史、饮酒史、家族史等)的情况下，通过本领域技术人员已知的各种高级统计分析方法获得的系数。
本发明第三个方面还涉及利用上述基因标志物进行肝肿瘤良恶性检测的试剂盒，其包括用于测定上述基因标志物的5-hmC含量的试剂和说明书。用于测定基因标志物的5-hmC含量的试剂是本领域技术人员已知的，例如T4噬菌体β-葡萄糖转移酶和同位素标记(对于葡糖基化法)、限制性内切酶(对于限制性内切酶法)、糖基转移酶和生物素(对于化学标记法)、PCR和测序所用试剂等。
与现有技术相比，本发明检测肝肿瘤良恶性的方法是基于基因标志物上的5-hmC含量，因此可以使用更为广泛的DNA样品来源。因此，本发明具有以下几个优点：(1)安全无创，即使无症状人群也对该检测接受度高；(2)DNA来源广泛，不存在影像学中的检测盲区； (3)准确性高，对肝癌有较高的灵敏度和特异性，更适用于肝肿瘤良恶性的区分；(4)操作方便，用户体验好，容易进行肝肿瘤发展的动态监测。本发明的基因标志物可与其他临床指标相结合，为肝肿瘤后续的筛查、诊断、治疗提供更准确的判断。
附图说明
图1是本发明区恶性肝肿瘤样品和良性肝肿瘤样品对照的曲线图。
具体实施方式
下面结合实施例及附图对本发明进行详细说明，以使本领域技术人员更好的理解本发明，并能予以实施。
实施例1.肝肿瘤基因标志物的筛选
1)抽提血浆DNA：
从来自20位肝癌患者和20位良性肝肿瘤患者的样品中分别抽提10ng血浆DNA。可利用本领域技术人员所熟知的任何适用于抽提血浆DNA的方法、和试剂进行此步骤。
2)将血浆DNA进行末端补齐、悬A并与测序接头连接：
根据Kapa Hyper Perp Kit说明书制备含有50uL血浆DNA、7uL End Repair & A-Tailing Buffer和3uL End Repair & A-Tailing Enzyme mix的反应混合液(总体积为60uL)，在20℃温浴30分钟，然后在65℃温浴30分钟。在1.5mL低吸附EP管中配置以下连接反应混合物：5uL Nuclease free water，30uL Ligation Buffer以及10uL DNA Ligase。向45uL连接反应混合物中加入5uL的测序接 头，混合，于20℃加热20分钟，然后保持于4℃。使用AmpureXP beads对反应产物进行纯化，用20uL含Tris-HCl(10mM，pH＝8.0)及EDTA(0.1mM)的缓冲液进行洗脱获得最终的DNA连接样品。
3)标记5-羟甲基胞嘧啶：
制备总体积为26uL的标记反应混合液：叠氮修饰的二磷酸尿苷葡萄糖(即UDP-N3-Glu，终浓度为50uM)、β-GT(终浓度为1uM)、Mg2+(终浓度为25mM)、HEPES(pH＝8.0，终浓度为50mM)和来自上述步骤的20uL DNA。将混合液在37℃温浴1小时。取出混合液，用AmpureXP beads纯化，获得纯化的20uL DNA。
然后在上述纯化的20uL DNA中加入1uL连接有生物素的二苯基环辛炔(DBCO-Biotin)，于37℃反应2小时，接着用AmpureXP beads纯化，获得纯化的标记产物。
4)固相富集含有标记的5-羟甲基胞嘧啶的DNA片段：
首先，按以下步骤准备磁珠：取出0.5uL C1 streptadvin beads(life technology)并加入100uL缓冲液(5mM Tris，pH＝7.5，1M NaCl，0.02％Tween20)，涡旋混合30秒，然后用100uL洗涤液(5mM Tris，pH＝7.5，1M NaCl，0.02％Tween20)洗涤磁珠3次，最后加入25uL结合缓冲液(10mM Tris，pH＝7.5，2M NaCl，0.04％Tween20或其他表面活性剂)，并混合均匀。
然后，在磁珠混合液中加入上述步骤获得的纯化的标记产物，并在旋转混合器中混合15min使其充分结合。
最后，用100uL洗涤液(5mM Tris，pH＝7.5，1M NaCl，0.02％ Tween20)洗涤磁珠3次，离心去掉上清液，加入23.75uL不含核酸酶的水。
5)PCR扩增：
向上述步骤的最终体系中加入25uL的2X PCR master mix和1.25uL PCR引物(总体积为50uL)，按照下述PCR反应循环的温度和条件进行扩增：
Figure PCTCN2018088132-appb-000001
将扩增产物用AmpureXP beads纯化，得到最终测序文库。
6)对测序文库进行质检后进行高通量测序：
将获得的测序文库通过qPCR进行浓度测定，并用Agilent2100对文库中DNA片段大小含量进行确定。将通过质检的测序文库以相同浓度混合，用Illumina Hiseq 4000进行测序。
7)确定各基因标志物的5-hmC含量和加权系数：
将获得的测序结果进行初步质控评估，清除低质量测序位点后，将达到测序质量标准的读段利用Bowtie2工具与人类标准基因组参考序列进行比较。然后利用featureCounts和HtSeq-Count工具来统计读段数量以确定各基因标志物的5-hmC含量。同时利用高通量测序结果，将可能影响5-hmC含量的因素作为共变量，通过逻辑回归和弹性网络正则化获得各基因标志物的加权系数。结果如表1所示。
表1：本发明的肝肿瘤基因标志物的平均标准化5-hmC含量和加 权系数
Figure PCTCN2018088132-appb-000002
如上所述，平均标准化5-hmC含量是指肝癌样品中该基因标志物的平均5-hmC含量与良性肝肿瘤样品中同一基因标志物的平均5-hmC含量之比。从表1可以看出，本发明的肝肿瘤基因标志物的5-hmC含量在良性肝肿瘤样品中和肝癌样品中存在显著差异。
实施例2.肝肿瘤基因标志物的有效性
本实施例验证本发明的肝肿瘤基因标志物用于区分肝肿瘤良恶性的有效性。
根据实施例1的方法测定第一批164个样品(82例肝癌和82例良性肝肿瘤)中本发明所述的10个肝癌基因标志物的5-hmC含量。
将各基因标志物的标准化5-hmC含量乘以该标志物在实施例1中对应的加权系数，获得该基因标志物的预测因子t，之后将各基因标志物的预测因子t相加，获得总预测因子T，然后将总预测因子T根据以下公式经过Logistic转换获得评分P：
Figure PCTCN2018088132-appb-000003
若P>0.5，则该受试者样品患有肝癌；若P≤0.5，则该受试者患有良性肝肿瘤。
图1示出了根据本发明的方法区分该批样品的结果。如图1所示，本发明的方法能够达到95％的灵敏度和96％的特异性。
最后应当说明的是，以上内容仅用以说明本发明的技术方案，而非对本发明保护范围的限制，本领域的普通技术人员对本发明的技术方案进行的简单修改或者等同替换，均不脱离本发明技术方案的实质和范围。

Claims (10)

  1. 基因标志物用于检测肝肿瘤良恶性的用途，通过高通量测序检测肝肿瘤基因标志物中5-羟甲基胞嘧啶的含量，从而判定肝肿瘤良恶性，包括一个或两个以上以下基因：FAT非典型钙粘蛋白1(FAT1)、雌激素相关受体γ(ESRRG)、性别决定基因y染色体区域盒家族成员9(SOX9)、纤毛状络合物子单元1(EVC)、1号染色体开放阅读框125抗体(AXDND1)、络丝蛋白(RELN)、转铁蛋白(TF)、TNF受体超家族成员(TNFRSF11B)、胰腺和十二指肠同圆框1(PDX1)、α-酸性糖蛋白2(ORM2)。
  2. 根据权利要求1所述的用途，其特征在于：所述基因标志物包括包括FAT1、ESRRG、SOX9、EVC、AXDND1、RELN、TF、TNFRSF11B、PDX1和ORM2。
  3. 一种用于检测肝肿瘤良恶性的方法，包括以下步骤：
    a)测定良性肝肿瘤样品和肝癌受试者样品中权利要求1和2所述的基因标志物的5-羟甲基胞嘧啶的含量；
    b)用良性肝肿瘤样品中所述基因标志物的5-羟甲基胞嘧啶含量作为参照，将肝癌受试者样品中对应的基因标志物的5-羟甲基胞嘧啶含量标准化，良性肝肿瘤样品和肝癌受试者样品中同一基因标志物的5-羟甲基胞嘧啶含量分别为X和Y，则肝癌受试者样品中该基因标志物的标准化5-羟甲基胞嘧啶含量为Y/X；
    c)对步骤b)中经标准化的所述基因标志物的5-羟甲基胞嘧啶含量进行数学关联，并获得评分P；
    d)根据所述评分P大小得到肝肿瘤良恶性的检测结果。
  4. 根据权利要求3所述的方法，其特征在于步骤a)包括如下步骤：将来自肝癌患者和良性肝肿瘤患者的DNA片段化；将所述片段化的DNA末端修复并末端补齐；将末端补齐的DNA与测序接头连接，获得连接产物；通过标记反应对连接产物中的5-羟甲基胞嘧啶进行标记；富集含有5-羟甲基胞嘧啶标记的DNA片段，获得富集产物；对富集产物进行PCR扩增，获得测序文库；对测序文库进行高通量测序，获得测序结果；根据测序结果确定5-羟甲基胞嘧啶在基因上的含量。
  5. 根据权利要求4所述的方法，其特征在于：所述标记反应包括：i)利用糖基转移酶将带有修饰基团的糖共价连接到5-羟甲基胞嘧啶的羟甲基上，和ii)将直接或间接连有生物素的点击化学底物与带有修饰基团的5-羟甲基胞嘧啶反应。
  6. 根据权利要求3、4或5所述的方法，其特征在于：所述步骤
    (a)是测定所述基因标志物全长或其片段上的5-hmC的含量。
  7. 根据权利要求3、4或5所述的方法，其特征在于步骤c)包括如下步骤：将各基因标志物的标准化5-hmC含量乘以加权系数，获得该基因标志物的预测因子t；将各基因标志物的预测因子t相加，获得总预测因子T；将总预测因子T经过Logistic转换获得评分P；若P>0.5，则该受试者样品患有肝癌；若P≤0.5，则该受试者患有良性肝肿瘤。
  8. 根据权利要求3所述的方法，其特征在于：所述样品是来自受试者体液中游离的DNA片段，或来源于细胞器、细胞以及组织中的 完整基因组DNA。
  9. 根据权利要求8所述的方法，其特征在于：所述体液是血液、尿液、汗液、痰液、粪便、脑脊液、腹水、胸水、胆汁或胰腺液。
  10. 一种用于检测肺癌的试剂盒，其特征在于包括：
    a)用于测定权利要求1或2所述的基因标志物的5-hmC含量的试剂；
    和b)说明书；
    所述5-羟甲基胞嘧啶含量是指所述基因标志物全长或其片段上的5-羟甲基胞嘧啶的含量。
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CN108103196A (zh) * 2018-02-05 2018-06-01 上海易毕恩生物技术有限公司 一种用于检测食管癌的基因标志物及其用途和检测方法
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Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN103091492A (zh) * 2011-11-04 2013-05-08 中国科学院上海生命科学研究院 诊断癌症的试剂及试剂盒
CN106755464A (zh) * 2017-01-11 2017-05-31 上海易毕恩基因科技有限公司 用于筛选肠癌和/或胃癌的基因标志物的方法、用该方法筛选的基因标志物及其用途
CN107142320A (zh) * 2017-06-16 2017-09-08 上海易毕恩基因科技有限公司 用于检测肝癌的基因标志物及其用途
CN107164508A (zh) * 2017-06-16 2017-09-15 上海易毕恩基因科技有限公司 用于检测肝癌的基因标志物及其用途
CN107365845A (zh) * 2017-08-04 2017-11-21 上海易毕恩生物技术有限公司 用于检测肺癌的基因标志物、试剂盒及肺癌检测方法
CN107385050A (zh) * 2017-08-04 2017-11-24 上海易毕恩生物技术有限公司 用于检测胰腺癌的基因标志物、试剂盒及胰腺癌检测方法

Family Cites Families (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP2549399A1 (en) * 2011-07-19 2013-01-23 Koninklijke Philips Electronics N.V. Assessment of Wnt pathway activity using probabilistic modeling of target gene expression
GB201115095D0 (en) * 2011-09-01 2011-10-19 Singapore Volition Pte Ltd Method for detecting nucleosomes containing nucleotides
US20140030727A1 (en) * 2012-01-20 2014-01-30 Gerd PFEIFER Loss of 5-hydroxymethylcytosine as a biomarker for cancer
CN105648537B (zh) * 2016-03-02 2018-06-29 上海易毕恩基因科技有限公司 Dna5-甲基胞嘧啶与5-羟甲基胞嘧啶基因图谱测序方法

Patent Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN103091492A (zh) * 2011-11-04 2013-05-08 中国科学院上海生命科学研究院 诊断癌症的试剂及试剂盒
CN106755464A (zh) * 2017-01-11 2017-05-31 上海易毕恩基因科技有限公司 用于筛选肠癌和/或胃癌的基因标志物的方法、用该方法筛选的基因标志物及其用途
CN107142320A (zh) * 2017-06-16 2017-09-08 上海易毕恩基因科技有限公司 用于检测肝癌的基因标志物及其用途
CN107164508A (zh) * 2017-06-16 2017-09-15 上海易毕恩基因科技有限公司 用于检测肝癌的基因标志物及其用途
CN107365845A (zh) * 2017-08-04 2017-11-21 上海易毕恩生物技术有限公司 用于检测肺癌的基因标志物、试剂盒及肺癌检测方法
CN107385050A (zh) * 2017-08-04 2017-11-24 上海易毕恩生物技术有限公司 用于检测胰腺癌的基因标志物、试剂盒及胰腺癌检测方法

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