WO2020155982A1 - Shh型髓母细胞瘤甲基化位点的应用 - Google Patents

Shh型髓母细胞瘤甲基化位点的应用 Download PDF

Info

Publication number
WO2020155982A1
WO2020155982A1 PCT/CN2019/129127 CN2019129127W WO2020155982A1 WO 2020155982 A1 WO2020155982 A1 WO 2020155982A1 CN 2019129127 W CN2019129127 W CN 2019129127W WO 2020155982 A1 WO2020155982 A1 WO 2020155982A1
Authority
WO
WIPO (PCT)
Prior art keywords
value
shh
medulloblastoma
methylation
type
Prior art date
Application number
PCT/CN2019/129127
Other languages
English (en)
French (fr)
Inventor
刘晶星
赵薇薇
于世辉
孙明明
陈白雪
胡昌明
毕欣
王怡摄
Original Assignee
广州金域医学检验中心有限公司
广州金域医学检验集团股份有限公司
广州市金域转化医学研究院有限公司
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 广州金域医学检验中心有限公司, 广州金域医学检验集团股份有限公司, 广州市金域转化医学研究院有限公司 filed Critical 广州金域医学检验中心有限公司
Priority to US17/289,803 priority Critical patent/US20210395830A1/en
Publication of WO2020155982A1 publication Critical patent/WO2020155982A1/zh

Links

Images

Classifications

    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12QMEASURING 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
    • C12Q1/00Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C12Q1/6886Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12NMICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09Recombinant DNA-technology
    • C12N15/11DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12QMEASURING 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
    • C12Q2600/00Oligonucleotides characterized by their use
    • C12Q2600/154Methylation markers

Definitions

  • the present invention relates to the technical field of tumor typing detection, in particular to the application of a methylation site of SHH medulloblastoma.
  • Medulloblastoma is the most common malignant brain tumor in children. Approximately 85% of medulloblastomas occur in children under the age of 18, and 1 in 5 children with brain tumors. . In recent years, the survival rate of patients with medulloblastoma has improved, but the mortality rate is still high. Even patients who are successfully treated often suffer from neurological and endocrine sequelae.
  • medulloblastoma The incidence of medulloblastoma is about 0.71 per 100,000. In the United States, 400 to 500 children are affected each year. The peak incidence of the disease is 3 to 6 years old. This disease is very rare in people after the age of 50. There is no report that the biochemical environment affects the incidence of medulloblastoma. However, certain genetic diseases can lead to the occurrence of medulloblastoma. About 7% of the patients' germ cell genes will be mutated, and a few medulloblastomas are inherited in families.
  • medulloblastoma is not a single disease, but a brain tumor composed of many different molecular subtypes. Each subtype has significant differences in genetics, demographics, and clinical characteristics. The most recent mainstream opinion believes that medulloblastoma has only four core subtypes: WNT, SHH, Group3 and Group4. There are also significant differences in age distribution between different subtypes. SHH subtypes present a typical double-peak distribution in different age groups. SHH type is more common in infants and adults. About 59% of adult medulloblastomas are of the SHH subtype, while the vast majority of WNT subtypes occur in children. Group3 subtypes are common in infants, while Groups subtypes are more common in children. And adults.
  • Identifying different subtypes is not only of great significance for understanding medulloblastoma, but also provides help for clinical diagnosis and treatment. For example, W1NT subtypes often have a better prognosis after receiving standard treatment; and for SHH subtypes of medulloblastoma , The use of some small molecule pathway inhibitors is very helpful for the disease.
  • the current diagnosis of SHH medulloblastoma mainly refers to its gene expression profile, which requires RNA transcriptome sequencing, high cost, long experiment and analysis cycle, because RNA is easy to degrade, and the technical requirements for experimental operations are also high, so the experiment failed The rate and experimental error are relatively high.
  • the kit and system only need to detect a few methylation sites in a targeted manner to type and confirm the SHH type of medulloblastoma.
  • the detection cost is less than 1/10 of that of transcriptome sequencing, it also It has the characteristics of short experiment period, DNA detection object, and high experiment success rate.
  • a reagent for detecting the degree of methylation of at least one gene locus of ROBO4, OTX2OS1, AHRR, DKK4 and PART1 genes in biological samples in preparing SHH medulloblastoma diagnostic reagents or diagnostic equipment.
  • the inventors analyzed and compared data from a large number of SHH-type medulloblastoma samples and other types of medulloblastoma samples (including other subtypes in medulloblastoma: WNT, Group3, and Group4) and found that the above-mentioned genes
  • the degree of methylation at the site can be used as a reference indicator for the diagnosis of SHH medulloblastoma. Only a few methylation sites need to be detected directionally to type and confirm the SHH type of medulloblastoma. It reduces the difficulty of diagnosis of SHH medulloblastoma and reduces the cost.
  • the aforementioned biological sample may be a postoperative tissue or a tumor tissue sample obtained by other methods.
  • the above-mentioned product can be a kit or an integrated detection device.
  • the invention also discloses a detection kit for SHH medulloblastoma, which includes reagents for detecting the degree of methylation of at least one of the following gene sites: ROBO4, OTX2OS1, AHRR, DKK4, PART1.
  • the above detection kit can be used to detect the degree of methylation of at least one gene locus of ROBO4, OTX2OS1, AHRR, DKK4, PART1 in biological samples, thereby assisting the diagnosis of SHH medulloblastoma or providing medication guidance.
  • the above detection methods can use conventional detection methods such as methylation chips, methylation-specific PCR, bisulfite sequencing, or other methods that can detect the degree of methylation at the above sites.
  • the locus in the ROBO4 gene includes: at least one locus of cg20419291, cg09684429 and cg19764370.
  • the locus in the OTX2OS1 gene includes at least one locus of cg22967396, cg23974194, cg04548856, cg14248715, and cg05732979.
  • the locus in the AHRR gene includes at least one locus of cg02385153, cg24064903, cg24980413, and cg16336872.
  • the locus in the DKK4 gene includes at least one of cg02571142 and cg09297903.
  • the locus in the PART1 gene is cg26353176.
  • the invention also discloses a SHH type medulloblastoma detection system, which includes the following modules:
  • the detection module uses the aforementioned detection kit to detect the degree of methylation at the corresponding site;
  • the analysis module obtains the above-mentioned detection results and compares them with the predetermined determination value of the degree of methylation at each site to obtain the detection result of SHH medulloblastoma;
  • the above detection system only needs to detect a few methylation sites in a targeted manner to type and confirm the SHH type of medulloblastoma, thereby reducing the difficulty of diagnosis of SHH medulloblastoma and reducing the cost.
  • the degree of methylation is a methylation value b-value.
  • the b-value refers to the methylation ratio of the site, and the b-value is used as the criterion for determining the degree of methylation, which has a simple and intuitive effect.
  • the b-value of cg20419291 is greater than 0.520
  • the b-value of cg09684429 is greater than 0.371
  • the b-value of cg19764370 is greater than 0.641
  • the b-value of cg22967396 is greater than 0.317
  • the b-value of cg23974194 The value is greater than 0.387
  • the b-value of cg04548856 is greater than 0.389
  • the b-value of cg14248715 is greater than 0.619
  • the b-value of cg05732979 is greater than 0.404
  • the b-value of cg02385153 is greater than 0.594
  • the b-value of cg24064903 is greater than 0.412
  • the b-value of cg24980413 is greater than 0.754
  • the b-value of cg16336872 is greater than 0.733
  • the inventors After screening and comparing a large number of samples, the inventors finally obtained the optimal cut-off value for distinguishing SHH type from other types of medulloblastoma. Using this value as a criterion can provide a basis for the diagnosis of SHH type medulloblastoma. .
  • the present invention has the following beneficial effects:
  • the application of the methylation site of SHH-type medulloblastoma of the present invention is the result of analyzing and comparing the data of a large number of SHH-type medulloblastoma samples and other samples.
  • the ROBO4, OTX2OS1, The degree of methylation in the AHRR, DKK4, PART1 genes can be used as a reference indicator for the diagnosis of SHH medulloblastoma. Only a few methylation sites need to be oriented to detect SHH medulloblastoma. Classification and confirmation, thereby reducing the difficulty of diagnosis of SHH medulloblastoma and reducing costs.
  • Figure 1 is a box diagram of the b-value values of cg26353176, cg16336872, cg23974194 and cg22967396 in Example 2;
  • Figure 2 is a box diagram of b-value values at cg02571142, cg09684429, cg02385153 and cg05732979 in Example 2;
  • Figure 3 is a box diagram of b-value values at cg14248715, cg20419291, cg04548856 and cg24064903 in Example 2;
  • Figure 4 is a box diagram of b-value values at cg19764370, cg09297903 and cg24980413 in Example 2;
  • Figure 5 is a graph showing the ROC curves of cg26353176, cg16336872, cg23974194 and cg22967396 in Example 2;
  • Figure 6 is a graph showing the ROC curves of cg02571142, cg09684429, cg02385153 and cg05732979 in Example 2;
  • Figure 7 is a graph showing the ROC curves of cg14248715, cg20419291, cg04548856 and cg24064903 in Example 2;
  • Figure 8 is a graph showing the ROC curve of cg19764370, cg09297903 and cg24980413 in Example 2;
  • FIG. 9 is a box diagram of the b-value value of ATP7B gene locus methylation in Example 2.
  • the commercial Illumina HumanMethylation450 BeadChip is used to detect methylation, and it is carried out in accordance with conventional experimental procedures.
  • the main experimental procedures include:
  • Example 1 Using the data of Example 1 as a database, a model based on a single methylation site was first established, and then highly discriminating methylation sites were selected to construct a comprehensive model based on a series of methylation sites. Then, according to the b-value value of the selected methylation site (one or more), the eigenvalue vector of a sample is constructed, and each sample corresponds to a tumor type (SHH medulloblastoma or OTHERS). This establishes an SVM classification model, and then obtains significant differences in methylation sites, as shown in the following table.
  • SHH medulloblastoma or OTHERS This establishes an SVM classification model, and then obtains significant differences in methylation sites, as shown in the following table.
  • Figure 1-4 is a boxplot of b-value values at different locations.
  • the b-value values corresponding to the middle horizontal line of the square box in the figure represent the average value.
  • the degree of methylation of the above sites in SHH medulloblastoma is higher than that of other types. Based on this, the degree of hypermethylation of the above sites can be used as an auxiliary for the diagnosis of SHH medulloblastoma index.
  • ROC curve (receiver operating characteristic curve, that is, ROC curve) of the above-mentioned points of the subject, and the result is shown in Figure 5, where the abscissa is the false positive rate and the ordinate is the true positive Rate, AUC represents the area under the curve. The closer the AUC value of the ROC curve is to 1, the higher the diagnostic value of the indicator.
  • the ROC-AUC value of cg26353176 is 1.00
  • the ROC-AUC value of cg16336872 is 1.00
  • the ROC-AUC value of cg23974194 is 1.00
  • the ROC-AUC value of cg22967396 is 0.99
  • the ROC-AUC value of cg02571142 is 0.99.
  • ROC-AUC value 0.99, cg09684429 ROC-AUC value 1.00, cg02385153 ROC-AUC value 1.00, cg05732979 ROC-AUC value 0.99, cg14248715 ROC-AUC value 0.99, cg20419291 ROC-AUC value 1.00, cg04548856 ROC -AUC value is 0.99, the ROC-AUC value of cg24064903 is 1.00, the ROC-AUC value of cg19764370 is 1.00, the ROC-AUC value of cg09297903 is 1.00, the ROC-AUC value of cg24980413 is 0.99, all above 0.99, indicating the above position
  • the degree of spot methylation has higher recognition ability for SHH medulloblastoma.
  • the b-value critical value is calculated according to the established model, as shown in the following table.
  • FPR false positive rate, false positive rate
  • TPR true positive rate, true positive rate
  • the FPR and TPR when the above-mentioned b-value critical value is used as the classification boundary are shown in the above table.
  • the selection of the critical value makes the FPR and TPR reach the optimal balance, that is, the tangent point of the tangent at the convex hull on the ROC curve.
  • Standards can provide a good basis for the diagnosis of SHH medulloblastoma.

Landscapes

  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Organic Chemistry (AREA)
  • Engineering & Computer Science (AREA)
  • Genetics & Genomics (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Immunology (AREA)
  • Pathology (AREA)
  • Analytical Chemistry (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Engineering & Computer Science (AREA)
  • Biotechnology (AREA)
  • Molecular Biology (AREA)
  • General Health & Medical Sciences (AREA)
  • Biophysics (AREA)
  • Physics & Mathematics (AREA)
  • Biochemistry (AREA)
  • Microbiology (AREA)
  • Biomedical Technology (AREA)
  • Oncology (AREA)
  • Hospice & Palliative Care (AREA)
  • Plant Pathology (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

本发明公开了一种SHH型髓母细胞瘤甲基化位点的应用,属于肿瘤分型检测技术领域。具体为一种检测生物样本中ROBO4、OTX2OS1、AHRR、DKK4、PART1基因中至少一个基因位点甲基化程度的试剂在制备SHH型髓母细胞瘤诊断试剂或诊断设备中的应用。本发明人通过对大量SHH型髓母细胞瘤样本和其它型样本的数据进行分析和对比后发现,上述基因中的位点甲基化程度能够作为诊断SHH型髓母细胞瘤的参考指标,只需要定向检测几个甲基化位点,即可对髓母细胞瘤的SHH型进行分型和确认,从而降低了SHH型髓母细胞瘤的诊断难度并降低了成本。

Description

SHH型髓母细胞瘤甲基化位点的应用
优先权
本申请要求2019年02月01号提交到中国专利局的专利的优先权,具体如下:申请号为CN201910105563.X、申请名称为SHH型髓母细胞瘤甲基化位点的应用。其全部内容通过引用结合在本申请中。
技术领域
本发明涉及肿瘤分型检测技术领域,特别是涉及一种SHH型髓母细胞瘤甲基化位点的应用。
背景技术
髓母细胞瘤是儿童最常见的恶性脑肿瘤,大约有85%的髓母细胞瘤发生于18岁以下的儿童,而每5例患有脑肿瘤的儿童中,就有1例髓母细胞瘤。近年来,髓母细胞瘤患者的生存率有所改善,但其病死率仍然很高,即使是治疗成功的患者也常常发生神经、内分泌方面的后遗症。
髓母细胞瘤的发病率大约为0.71/10万人,在美国每年有400~500例的儿童患病,该病发病高峰在3~6岁,此病在50岁以后的人群中十分罕见。目前尚没有生化环境对髓母细胞瘤发病率有所影响的报道。但是,某些遗传疾病会导致髓母细胞瘤的发生,约有7%的患者生殖细胞基因会发生变异,也有少数髓母细胞瘤呈家族性遗传。
目前,国际上已经认识到髓母细胞瘤不是单一的疾病,而是多种不同分子亚型组成的脑肿瘤。各个亚型在遗传学、人口统计学和临床特点上均有显著差别。最近的主流观点认为,髓母细胞瘤只有4种核心的亚型:即WNT、SHH、Group3和Group4。不同亚型之间年龄分布也有显著的不同,SHH亚型在不同年龄段呈现典型的双高峰分布。SHH型好发于婴儿和成人,大约59%的成人髓母细胞瘤是SHH亚型,而绝大部分WNT亚型出现在儿童患者,Group3亚型常见于婴儿,而Groups亚型却多发于儿童和成人。
识别不同亚型不仅对理解髓母细胞瘤有很大的意义,而且还可为临床诊治提供帮助,比如:W1NT亚型往往接受标准治疗后预后较好;而对于SHH亚型的髓母细胞瘤,使用一些小分子的通路抑制剂对病情很有帮助。
然而,目前诊断SHH型髓母细胞瘤主要参考其基因表达图谱,需要RNA转录组测序,成本高,实验及分析周期长,因RNA容易降解,对实验操作的技术要求也较高,因此实验失败率和实验误差都相对较高。
发明内容
基于此,有必要针对上述问题,提供一种SHH型髓母细胞瘤甲基化位点的应用,将该SHH型髓母细胞瘤甲基化位点应用于检测试剂盒及系统中,采用该试剂盒及系统,只需要定向检测几个甲基化位点,即可对髓母细胞瘤的SHH型进行分型和确认,除检测成本不到转录组测序的1/10的优点外,还具有实验周期短,检测对象为DNA,实验成功率高的特点。
一种检测生物样本中ROBO4、OTX2OS1、AHRR、DKK4、PART1基因中至少一个基因位点甲基化程度的试剂在制备SHH型髓母细胞瘤诊断试剂或诊断设备中的应用。
本发明人通过对大量SHH型髓母细胞瘤样本和其它型髓母细胞瘤样本(包括髓母细胞瘤中的其它亚型:WNT、Group3和Group4)的数据进行分析和对比后发现,上述基因中的位点甲基化程度能够作为诊断SHH型髓母细胞瘤的参考指标,只需要定向检测几个甲基化位点,即可对髓母细胞瘤的SHH型进行分型和确认,从而降低了SHH型髓母细胞瘤的诊断难度并降低了成本。
上述生物样本可以为术后组织或其他方法获取的肿瘤组织样本。
可以理解的,上述产品可以是试剂盒,也可以是一体化检测设备。
本发明还公开了一种SHH型髓母细胞瘤的检测试剂盒,包括检测以下至少一个基因位点甲基化程度的试剂:ROBO4、OTX2OS1、AHRR、DKK4、PART1。
上述检测试剂盒可用于检测生物样本中ROBO4、OTX2OS1、AHRR、DKK4、PART1中至少一个基因位点的甲基化程度,从而辅助SHH型髓母细胞瘤诊断或进行用药指导。
可以理解的,上述检测方法可以采用甲基化芯片、甲基化特异性PCR、亚硫酸氢盐测序法等常规检测方法,或者其它能够检测出上述位点甲基化程度的方法均可。
在其中一个实施例中,所述ROBO4基因中的位点包括:cg20419291、cg09684429和cg19764370中的至少一个位点。
在其中一个实施例中,所述OTX2OS1基因中的位点包括:cg22967396、cg23974194、cg04548856、cg14248715和cg05732979中的至少一个位点。
在其中一个实施例中,所述AHRR基因中的位点包括:cg02385153、cg24064903、cg24980413和cg16336872中的至少一个位点。
在其中一个实施例中,所述DKK4基因中的位点包括:cg02571142和cg09297903中的至少一个位点。
在其中一个实施例中,所述PART1基因中的位点为cg26353176。
本发明还公开了一种SHH型髓母细胞瘤的检测系统,包括以下模块:
检测模块,采用上述的检测试剂盒检测相应的位点甲基化程度;
分析模块,获取上述检测结果,与预定的各位点甲基化程度判定值进行对比,得出SHH型髓母细胞瘤的检测结果;
上述检测系统,只需要定向检测几个甲基化位点,即可对髓母细胞瘤的SHH型进行分型和确认,从而降低了SHH型髓母细胞瘤的诊断难度并降低了成本。
在其中一个实施例中,所述甲基化程度为甲基化值b-value。b-value指位点的甲基化比例,以b-value作为甲基化程度的判断标准,具有简便、直观的效果。
在其中一个实施例中,所述分析模块中,若cg20419291的b-value大于0.520、cg09684429的b-value大于0.371、cg19764370的b-value大于0.641、cg22967396的b-value大于0.317、cg23974194的b-value大于0.387、cg04548856的b-value大于0.389、cg14248715的b-value大于0.619、cg05732979的b-value大于0.404、cg02385153的b-value大于0.594、cg24064903的b-value大于0.412、cg24980413的b-value大于0.754、cg16336872的b-value大于0.733、cg02571142的b-value大于0.430、cg09297903的b-value大于0.440、和/或cg26353176的b-value大于0.564,则提示为SHH型髓母细胞瘤。
本发明人经过大量样本的筛选和比对,最终得出区分SHH型与其他类型髓母细胞瘤的最优临界值,以该值为判断标准,能够为SHH型髓母细胞瘤的诊断提供依据。
与现有技术相比,本发明具有以下有益效果:
本发明的一种SHH型髓母细胞瘤甲基化位点的应用,是发明人通过对大量SHH型髓母细胞瘤样本和其它样本的数据进行分析和对比后发现,采用该ROBO4、OTX2OS1、AHRR、DKK4、PART1基因中的位点甲基化程度能够作为诊断SHH型髓母细胞瘤的参考指标,只需要定向检测几个甲基化位点,即可对髓母细胞瘤的SHH型进行分型和确认,从而降低了SHH型髓母细胞瘤的诊断难度并降低了成本。
附图说明
图1为实施例2中cg26353176、cg16336872、cg23974194和cg22967396位点b-value值盒图;
图2为实施例2中cg02571142、cg09684429、cg02385153和cg05732979位点b-value值盒图;
图3为实施例2中cg14248715、cg20419291、cg04548856和cg24064903位点b-value值盒图;
图4为实施例2中cg19764370、cg09297903和cg24980413位点b-value值盒图;
图5为实施例2中cg26353176、cg16336872、cg23974194和cg22967396位点ROC曲线图;
图6为实施例2中cg02571142、cg09684429、cg02385153和cg05732979位点ROC曲线图;
图7为实施例2中cg14248715、cg20419291、cg04548856和cg24064903位点ROC曲线图;
图8为实施例2中cg19764370、cg09297903和cg24980413位点ROC曲线图;
图9为实施例2中ATP7B基因位点甲基化b-value值盒图。
具体实施方式
为了便于理解本发明,下面将参照相关附图对本发明进行更全面的描述。附图中给出了本发明的较佳实施例。但是,本发明可以以许多不同的形式来实现,并不限于本文所描述的实施例。相反地,提供这些实施例的目的是使对本发明的公开内容的理解更加透彻全面。
除非另有定义,本文所使用的所有的技术和科学术语与属于本发明的技术领域的技术人员通常理解的含义相同。本文中在本发明的说明书中所使用的术语只是为了描述具体的实施例的目的,不是旨在于限制本发明。本文所使用的术语“和/或”包括一个或多个相关的所列项目的任意的和所有的组合。
实施例1
样本收集及检测。
一、样本收集。
收集SHH型髓母细胞瘤患者术后组织样本223例,以及除SHH外其他型髓母细胞瘤患者术后组织样本540例,即OTHERS样本(包括髓母细胞瘤中的其它亚型:WNT、Group3和Group4)。
二、样本检测。
以商品化的Illumina HumanMethylation450 BeadChip芯片检测甲基化,按照常规实验步骤进行,主要实验流程包括:
1、提取样本DNA并扩增;
2、使用酶打断DNA使之片段化;
3、分离纯化DNA片段并加上接头;
4、DNA片段与芯片杂交;
5、芯片上机检测。
实施例2
甲基化位点的筛选。
一、甲基化位点初筛。
以实施例1的数据作为数据库,先建立基于单个甲基化位点的模型,然后挑选高区分度的甲基化位点构建基于一系列甲基化位点的综合模型。再根据选定的甲基化位点(一个或多个)的b-value值构成一个样本的特征值向量,同时每个样本对应一个肿瘤分型(SHH型髓母细胞瘤或OTHERS),由此建立一个SVM分类模型,进而得出显著性差异的甲基化位点,具体如下表所示。
表1.SHH型髓母细胞瘤和OTHERS具有显著性差异的甲基化位点
位点 基因 所在染色体 位点染色体坐标 正/负链
cg20419291 ROBO4 chr11 124767974 +
cg09684429 ROBO4 chr11 124768015 -
cg19764370 ROBO4 chr11 124767933 +
cg22967396 OTX2OS1 chr14 57283942 +
cg23974194 OTX2OS1 chr14 57284319 -
cg04548856 OTX2OS1 chr14 57284528 -
cg14248715 OTX2OS1 chr14 57284096 -
cg05732979 OTX2OS1 chr14 57284219 +
cg02385153 AHRR chr5 404766 +
cg24064903 AHRR chr5 404910 +
cg24980413 AHRR chr5 346987 -
cg16336872 AHRR chr5 435267 -
cg02571142 DKK4 chr8 42234803 -
cg09297903 DKK4 chr8 42234798 -
cg26353176 PART1 chr5 59783906 -
注:+表示正链,-表示负链。
二、甲基化位点验证。
1、b-value值对比。
统计上述位点在SHH型髓母细胞瘤和其它(OTHERS)患者中的b-value值,结果如下表和图1-4所示。
表2.SHH型髓母细胞瘤和OTHERS的b-value值对比
Figure PCTCN2019129127-appb-000001
Figure PCTCN2019129127-appb-000002
图1-4为不同位点b-value值盒图(boxplot),图中方形盒子的中间横线所对应的b-value值表示平均值。
通过上述结果可以看出,以上位点在SHH型髓母细胞瘤中的甲基化程度高于其它类型,据此,可将上述位点的高甲基化程度作为SHH型髓母细胞瘤诊断的辅助指标。
2、roc-auc验证。
绘制受试者上述位点的ROC曲线(受试者工作特征曲线,receiver operating characteristic curve,即ROC曲线),结果如图5-所示,图中横坐标为假阳性率,纵坐标为真阳性率,AUC表示曲线下面积,该ROC曲线的AUC值越接近1,表示该指标有更高的诊断价值。
通过上述结果可以看出,cg26353176的ROC-AUC值为1.00、cg16336872的ROC-AUC值为1.00、cg23974194的ROC-AUC值为1.00、cg22967396的ROC-AUC值为0.99、cg02571142的ROC-AUC值为0.99、cg09684429的ROC-AUC值为1.00、cg02385153的ROC-AUC值为1.00、cg05732979的ROC-AUC值为0.99、cg14248715的ROC-AUC值为0.99、cg20419291的ROC-AUC值为1.00、cg04548856的ROC-AUC值为0.99、cg24064903的ROC-AUC值为1.00、cg19764370的ROC-AUC值为1.00、cg09297903的ROC-AUC值为1.00、cg24980413的ROC-AUC值为0.99,均在0.99以上,表示上述位点甲基化程度对SHH型髓母细胞瘤具有越高的识别能力。
3、SHH型髓母细胞瘤各位点b-value值分析。
3.1各位点b-value值分析。
从上对所得SHH型髓母细胞瘤各高区分度甲基化位点b-value值进行分析,从结果中可以看出,SHH型髓母细胞瘤中b-value值集中度较高,SHH型和其他类型的区分较开,可以此作为一个辅助判断标准。
3.2各位点b-value临界值分析
根据所建立的模型计算出b-value临界值,如下表所示。
表3.b-value临界值
位点 b-value临界值 FPR TPR
cg26353176 0.564 0.017 0.996
cg16336872 0.733 0.030 0.991
cg23974194 0.387 0.004 0.964
cg22967396 0.317 0.009 0.955
cg02571142 0.430 0.020 0.991
cg09684429 0.371 0.020 0.996
cg02385153 0.594 0.011 0.987
cg05732979 0.404 0.006 0.955
cg14248715 0.619 0.004 0.955
cg20419291 0.520 0.013 0.996
cg04548856 0.389 0.009 0.969
cg24064903 0.412 0.007 0.987
cg19764370 0.641 0.006 0.996
cg09297903 0.440 0.028 0.991
cg24980413 0.754 0.035 0.991
FPR:false positive rate,假阳性率;TPR:true positive rate,真阳性率。
上述b-value临界值作为分类边界时的FPR和TPR如上表所示,该临界值的选取使FPR和TPR达到最优平衡,即ROC曲线上凸包处切线的切点,以该值为判断标准,能够为SHH型髓母细胞瘤的诊断提供较好的依据。
4、对照验证。
随机选取ATP7B基因,检测并分析其中部分位点的甲基化水平,结果如图9所示,图9为ATP7B基因在SHH型髓母细胞瘤和OTHERS组中的各位点甲基化b-value值的盒图,结果表示两组样本间甲基化几乎无差异。
以上所述实施例的各技术特征可以进行任意的组合,为使描述简洁,未对上述实施例中的各个技术特征所有可能的组合都进行描述,然而,只要这些技术特征的组合不存在矛盾,都应当认为是本说明书记载的范围。
以上所述实施例仅表达了本发明的几种实施方式,其描述较为具体和详细,但并不能因此而理解为对发明专利范围的限制。应当指出的是,对于本领域的普通技术人员来说,在不脱离本发明构思的前提下,还可以做出若干变形和改进,这些都属于本发明的保护范围。因此,本发明专利的保护范围应以所附权利要求为准。

Claims (10)

  1. 检测生物样本中ROBO4、OTX2OS1、AHRR、DKK4、PART1基因中至少一个基因位点甲基化程度的试剂在制备SHH型髓母细胞瘤诊断试剂或诊断设备中的应用。
  2. 一种SHH型髓母细胞瘤的检测试剂盒,其特征在于,包括检测以下至少一个基因位点甲基化程度的试剂:ROBO4、OTX2OS1、AHRR、DKK4、PART1。
  3. 根据权利要求2所述的SHH型髓母细胞瘤的检测试剂盒,其特征在于,所述ROBO4基因中的位点包括:cg20419291、cg09684429和cg19764370中的至少一个位点。
  4. 根据权利要求2所述的SHH型髓母细胞瘤的检测试剂盒,其特征在于,所述OTX2OS1基因中的位点包括:cg22967396、cg23974194、cg04548856、cg14248715和cg05732979中的至少一个位点。
  5. 根据权利要求2所述的SHH型髓母细胞瘤的检测试剂盒,其特征在于,所述AHRR基因中的位点包括:cg02385153、cg24064903、cg24980413和cg16336872中的至少一个位点。
  6. 根据权利要求2所述的SHH型髓母细胞瘤的检测试剂盒,其特征在于,所述DKK4基因中的位点包括:cg02571142和cg09297903中的至少一个位点。
  7. 根据权利要求2所述的SHH型髓母细胞瘤的检测试剂盒,其特征在于,所述PART1基因中的位点为cg26353176。
  8. 一种SHH型髓母细胞瘤的检测系统,其特征在于,包括以下模块:
    检测模块,采用权利要求2-7任一项所述的检测试剂盒检测相应的位点甲基化程度;
    分析模块,获取上述检测结果,与预定的各位点甲基化程度判定值进行对比,得出SHH型髓母细胞瘤的检测结果;
  9. 根据权利要求8所述的SHH型髓母细胞瘤的检测系统,其特征在于,所述甲基化程度为甲基化值b-value。
  10. 根据权利要求9所述的SHH型髓母细胞瘤的检测系统,其特征在于,所述分析模块中,若cg20419291的b-value大于0.520、cg09684429的b-value大于0.371、cg19764370的 b-value大于0.641、cg22967396的b-value大于0.317、cg23974194的b-value大于0.387、cg04548856的b-value大于0.389、cg14248715的b-value大于0.619、cg05732979的b-value大于0.404、cg02385153的b-value大于0.594、cg24064903的b-value大于0.412、cg24980413的b-value大于0.754、cg16336872的b-value大于0.733、cg02571142的b-value大于0.430、cg09297903的b-value大于0.440、和/或cg26353176的b-value大于0.564,则提示为SHH型髓母细胞瘤。
PCT/CN2019/129127 2019-02-01 2019-12-27 Shh型髓母细胞瘤甲基化位点的应用 WO2020155982A1 (zh)

Priority Applications (1)

Application Number Priority Date Filing Date Title
US17/289,803 US20210395830A1 (en) 2019-02-01 2019-12-27 Uses of methylated loci in sonic hedgehog medulloblastoma

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN201910105563.X 2019-02-01
CN201910105563.XA CN109777875B (zh) 2019-02-01 2019-02-01 Shh型髓母细胞瘤甲基化位点的应用

Publications (1)

Publication Number Publication Date
WO2020155982A1 true WO2020155982A1 (zh) 2020-08-06

Family

ID=66504059

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2019/129127 WO2020155982A1 (zh) 2019-02-01 2019-12-27 Shh型髓母细胞瘤甲基化位点的应用

Country Status (3)

Country Link
US (1) US20210395830A1 (zh)
CN (1) CN109777875B (zh)
WO (1) WO2020155982A1 (zh)

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN109777875B (zh) * 2019-02-01 2020-01-17 广州金域医学检验中心有限公司 Shh型髓母细胞瘤甲基化位点的应用

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN108728533A (zh) * 2017-04-20 2018-11-02 常青 用于髓母细胞瘤分子分型的基因群以及snca基因作为4型髓母细胞瘤的生物标志物的用途
CN109182517A (zh) * 2018-09-07 2019-01-11 杭州可帮基因科技有限公司 一组用于髓母细胞瘤分子分型的基因及其应用
CN109777875A (zh) * 2019-02-01 2019-05-21 广州金域医学检验中心有限公司 Shh型髓母细胞瘤甲基化位点的应用

Family Cites Families (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6783933B1 (en) * 1999-09-15 2004-08-31 The Johns Hopkins University School Of Medicine CACNA1G polynucleotide, polypeptide and methods of use therefor
CN102311953B (zh) * 2011-09-23 2013-12-18 上海市肿瘤研究所 尿液诊断膀胱癌的方法和试剂盒
CN104056277B (zh) * 2013-03-21 2019-07-05 中国人民解放军第二军医大学 Mlh1基因或其表达产物在dkk4高表达的结直肠癌中的应用
US20170356051A1 (en) * 2014-10-17 2017-12-14 Tohoku University Method for estimating sensitivity to drug therapy for colorectal cancer

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN108728533A (zh) * 2017-04-20 2018-11-02 常青 用于髓母细胞瘤分子分型的基因群以及snca基因作为4型髓母细胞瘤的生物标志物的用途
CN109182517A (zh) * 2018-09-07 2019-01-11 杭州可帮基因科技有限公司 一组用于髓母细胞瘤分子分型的基因及其应用
CN109777875A (zh) * 2019-02-01 2019-05-21 广州金域医学检验中心有限公司 Shh型髓母细胞瘤甲基化位点的应用

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
XU, BAOYAN: "Scanning of Differentially methylated genes associated with phenotype in CpG islands DNA of whole genome between HBV infected monozygotic twin pairs", DOCTORAL DISSERTATION, no. 11, 15 November 2006 (2006-11-15), pages 1 - 114, XP009522389 *

Also Published As

Publication number Publication date
CN109777875B (zh) 2020-01-17
CN109777875A (zh) 2019-05-21
US20210395830A1 (en) 2021-12-23

Similar Documents

Publication Publication Date Title
JP6695392B2 (ja) ゲノム配列決定を使用する胎児染色体異数性の診断
CN106462670B (zh) 超深度测序中的罕见变体召集
EP3704264B1 (en) Using nucleic acid size range for noninvasive prenatal testing and cancer detection
US20230170048A1 (en) Systems and methods for classifying patients with respect to multiple cancer classes
CN113462781A (zh) 使用血浆dna的尺寸和数目畸变检测癌症
CN113151474A (zh) 用于癌症检测的血浆dna突变分析
US20200325546A1 (en) Enhancement of cancer screening using cell-free viral nucleic acids
EP3973080A1 (en) Systems and methods for determining whether a subject has a cancer condition using transfer learning
CA3167633A1 (en) Systems and methods for calling variants using methylation sequencing data
WO2020155982A1 (zh) Shh型髓母细胞瘤甲基化位点的应用
EP3596229A1 (en) Method and system for nucleic acid sequencing
Granda 5 Complete screening of CFTR gene mutations in cystic fibrosis patients from Eastern Hungary

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 19912562

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 19912562

Country of ref document: EP

Kind code of ref document: A1

122 Ep: pct application non-entry in european phase

Ref document number: 19912562

Country of ref document: EP

Kind code of ref document: A1