WO2016115967A1 - Y染色体甲基化位点作为前列腺癌诊断标志物的应用 - Google Patents

Y染色体甲基化位点作为前列腺癌诊断标志物的应用 Download PDF

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WO2016115967A1
WO2016115967A1 PCT/CN2015/099922 CN2015099922W WO2016115967A1 WO 2016115967 A1 WO2016115967 A1 WO 2016115967A1 CN 2015099922 W CN2015099922 W CN 2015099922W WO 2016115967 A1 WO2016115967 A1 WO 2016115967A1
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methylation
chromosome
sample
prostate cancer
site
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孙英丽
姚鲁帅
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中国科学院北京基因组研究所
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Priority to EP15878635.0A priority patent/EP3249051B1/en
Priority to KR1020177021536A priority patent/KR102067607B1/ko
Priority to US15/544,735 priority patent/US10648039B2/en
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Definitions

  • the invention relates to the application of a methylation site as a diagnostic marker for diseases, in particular to the application of a Y chromosome methylation site as a diagnostic marker for prostate cancer.
  • DNA methylation is a common epigenetic modification that uses the methyl group provided by S-adenosylmethionine to catalyze the fifth carbon atom of cytosine under the catalysis of DNA methyltransferase.
  • the base which converts cytosine to 5-methylcytosine, plays a crucial role in the regulation of gene expression.
  • Cancer also known as a malignant tumor, is a disease caused by an abnormality that controls the growth and proliferation of cells.
  • cell growth and proliferation are closely regulated by some genes regulating growth and development including oncogenes and tumor suppressor genes, maintaining normal physiological state; various factors such as radiation, chemical pollution, viral infection and endocrine imbalance Under the influence of it, the activation of oncogenes in the body and the inactivation of tumor suppressor genes lead to the occurrence of cancer.
  • epigenetic abnormal regulation such as histone modification and DNA methylation plays an important role in the development of tumors, and abnormal expression is found in various cancers.
  • the occurrence of tumors is an extremely complicated process, which is the result of abnormal regulation and accumulation of oncogenes, tumor suppressor genes and apparent modifications in vivo.
  • DNA methylation is closely related to the occurrence of cancer, and DNA methylation abnormalities are found in many cancers. DNA methylation has a certain stability and it is an early event in the development of cancer. In recent years, many studies have shown that DNA methylation abnormalities can be used as a biomarker for cancer diagnosis. Currently, DNA methylation abnormalities in many specific gene promoter regions are used as a potential marker for prostate cancer diagnosis.
  • the main diagnostic methods for prostate cancer are rectal diagnosis, serum PSA detection, and tissue immunosection detection.
  • Tissue immunoslices are the golden diagnostic method for prostate cancer.
  • PSA is currently used as a marker for the early diagnosis of prostate cancer, but its detection is not very accurate, there is a certain gray area. Therefore, finding a convenient and accurate diagnostic marker is very urgent for the early diagnosis of prostate cancer.
  • the inventors of the present invention have established a uniform and standard disease-related methylation site screening method and screened for a Y chromosome methylation site associated with prostate cancer, thereby completing the present invention.
  • a first aspect of the invention relates to the use of an agent for detecting a methylation level of a Y chromosome methylation site in a sample to be tested in a subject, the kit being for use in one of the following uses Item or multiple: diagnosis of prostate cancer, assessment of risk of prostate cancer, evaluation of therapeutic effect of prostate cancer, and screening of therapeutic drugs for prostate cancer; said Y chromosome methylation site is selected from the group consisting of cg03052502, cg04462340, cg05163709, One or several of cg05544622, cg14466580, and cg27539833.
  • the sample to be tested is selected from the group consisting of tissue, urine (eg, urine after prostate massage), and prostatic fluid.
  • the sample to be tested is selected from the group consisting of urine (eg, urine after prostate massage) and prostatic fluid.
  • Y chromosome methylation site is selected from the group consisting of cg05163709 and cg27539833.
  • the method of detecting methylation level of a Y chromosome methylation site in a sample to be tested in a subject is selected from pyrosequencing, bisulfite measurement Sequencing, Quantitative and/or Qualitative Methylation-Specific Polymerase Chain Reaction, Southern Blotting, Restriction Marker Genome Scanning, Single Nucleotide Primer Extension, CpG Island Microarray, Single Nucleotide Primer Extension (SNUPE) , combined with sodium bisulfite restriction endonuclease analysis and mass spectrometry.
  • pyrosequencing bisulfite measurement Sequencing
  • Quantitative and/or Qualitative Methylation-Specific Polymerase Chain Reaction Southern Blotting, Restriction Marker Genome Scanning, Single Nucleotide Primer Extension, CpG Island Microarray, Single Nucleotide Primer Extension (SNUPE) , combined with sodium bisulfite restriction endonuclease analysis and mass
  • the oligonucleotide primer is used to amplify a nucleotide sequence comprising a Y chromosome methylation site.
  • a second aspect of the invention relates to a kit comprising an agent for detecting a methylation level of a Y chromosome methylation site in a sample to be tested in a subject, the kit being for use in one or more of the following uses Item: Diagnosis of prostate cancer, assessment of risk of prostate cancer, evaluation of therapeutic effect of prostate cancer, and screening of therapeutic drugs for prostate cancer; said Y chromosome methylation site is selected from the group consisting of cg03052502, cg04462340, cg05163709, cg05544622, cg14466580 And one or several of cg27539833.
  • the sample to be tested is selected from the group consisting of tissue, urine (eg, urine after prostate massage), and prostatic fluid.
  • the sample to be tested is selected from the group consisting of urine (eg, urine after prostate massage) and prostatic fluid.
  • Y chromosome methylation site is selected from the group consisting of cg05163709 and cg27539833.
  • the method of detecting a methylation level of a Y chromosome methylation site in a sample to be tested in a subject is selected from the group consisting of pyrosequencing, bisulfite sequencing, quantification, and/or characterization Methylation-specific polymerase chain reaction, Southern blotting, restriction landmark genomic scanning, single nucleotide primer extension, CpG island microarray, single nucleotide primer extension (SNUPE), combined with sodium bisulfite limitation Endonuclease analysis and mass spectrometry.
  • the oligonucleotide primer is used to amplify a nucleotide sequence comprising a Y chromosome methylation site.
  • a third aspect of the invention relates to a diagnosis, risk assessment and treatment of prostate cancer
  • a method for therapeutic effect evaluation and drug screening comprising the step of detecting a methylation level of a Y chromosome methylation site in a sample to be tested; the Y chromosome methylation site is selected from the group consisting of cg03052502, cg04462340 One or more of cg05163709, cg05544622, cg14466580, and cg27539833.
  • methylation level of one or more methylation sites selected from the group consisting of cg03052502, cg04462340, cg05544622, cg14466580, and cg27539833 is decreased when compared to a normal sample or a normal reference value, indicating Subjects have a high risk of prostate cancer or prostate cancer; when the methylation level of methylation site cg05163709 is elevated compared to normal or normal reference values, the subject has prostate cancer or The risk of prostate cancer is high.
  • the methylation level of one or more methylation sites selected from the group consisting of cg03052502, cg04462340, cg05544622, cg14466580, and cg27539833 is elevated when compared to before or prior to use of the screening drug , indicating that the subject's therapeutically effective or drug for screening is effective; when the methylation level of the methylation site cg05163709 is decreased compared to before or prior to the use of the screening drug, indicating that the subject is therapeutically effective or The drug used for screening is effective.
  • the sample to be tested is selected from the group consisting of tissue, urine (eg, urine after prostate massage), and prostatic fluid.
  • the sample to be tested is selected from the group consisting of urine (eg, urine after prostate massage) and prostatic fluid.
  • Y chromosome methylation site is selected from the group consisting of cg05163709 and cg27539833.
  • the method of detecting a methylation level of a Y chromosome methylation site in a sample to be tested in a subject is selected from the group consisting of pyrosequencing, bisulfite sequencing, quantification, and/or characterization Methylation-specific polymerase chain reaction, Southern blotting, restriction landmark genomic scanning, single nucleotide primer extension, CpG island microarray, single nucleotide primer extension (SNUPE), combined with sodium bisulfite limitation Endonuclease analysis and mass spectrometry.
  • the method of methylation level of a body methylation site includes the step of using an oligonucleotide primer for amplifying a nucleotide sequence containing a Y chromosome methylation site.
  • the fourth aspect of the invention relates to the use of a Y chromosome methylation site as a biomarker for the diagnosis, risk assessment, therapeutic effect evaluation and drug screening of prostate cancer, wherein the Y chromosome methylation site is selected from the group consisting of cg03052502 One or more of cg04462340, cg05163709, cg05544622, cg14466580, and cg27539833.
  • the diagnosis of prostate cancer, the assessment of the risk of disease, the evaluation of therapeutic effects, and the screening of drugs for treating prostate cancer can be performed by detecting the methylation levels of these Y chromosome methylation sites.
  • a fifth aspect of the invention relates to a biomarker for use in diagnosis, risk assessment, therapeutic effect evaluation and drug screening of prostate cancer, said biomarker being a Y chromosome methylation site, said Y chromosome methyl
  • the chemistry site is selected from one or more of cg03052502, cg04462340, cg05163709, cg05544622, cg14466580, and cg27539833.
  • the diagnosis of prostate cancer, the assessment of the risk of disease, the evaluation of therapeutic effects, and the screening of drugs for treating prostate cancer can be performed by detecting the methylation levels of these Y chromosome methylation sites.
  • a sixth aspect of the invention relates to a method for screening a disease-related chromosomal methylation site, the method comprising: 1) obtaining a disease sample and a normal sample of a patient; 2) determining a chromosomal methylation of the disease sample and the normal sample 3) Screening for methylated conserved sites based on chromosomal methylation information of normal samples; 4) Screening for a significant difference in disease samples from normal samples based on chromosomal methylation information of disease samples and normal samples The basement site; 5) combines the results of steps 3) and 4) to obtain a methylated conserved site with significant differences, ie, a chromosome methylation site associated with the disease.
  • cancer such as prostate cancer
  • the chromosome is an autosomal or sex chromosome (eg, a Y chromosome).
  • tissue eg, cancerous tissue
  • blood e.g., urine
  • feces e.g., feces
  • tissue fluid e.g, prostatic fluid
  • methylation conserved site refers to a methylation site having a standard deviation SD value of methylation level of 0.25 or less in a normal sample.
  • the methylation site having a significant difference is a site in which the methylation level changes by more than 0.2 and the p-value and the q-value are both less than or equal to 0.01 compared to the normal sample. The change is increased or decreased.
  • the invention also relates to a method of diagnosing prostate cancer, the method comprising the step of screening for a Y chromosome methylation site associated with prostate cancer.
  • the step of screening for a Y chromosome methylation site associated with prostate cancer comprises: 1) obtaining a disease sample and a normal sample of a prostate cancer patient; 2) determining a Y chromosome of the disease sample and the normal sample basement information; 3) Screening for methylation conserved sites based on Y chromosome methylation information of normal samples; 4) Screening of disease samples based on Y chromosome methylation information of disease samples and normal samples is more significant than normal samples Differential methylation sites; 5) Combining the results of steps 3) and 4) to obtain a methylated conserved site with significant differences, ie, a chromosomal methylation site associated with prostate cancer.
  • tissue eg, cancerous tissue
  • blood e.g, urine after prostate massage
  • feces or tissue fluid e.g, prostatic fluid
  • methylation conserved site is a methylation site having a standard deviation SD value of methylation level of 0.25 or less in a normal sample.
  • the methylation site having a significant difference is a site in which the methylation level changes by more than 0.2 and the p-value and the q-value are both less than or equal to 0.01 compared to the normal sample. The change is increased or decreased.
  • diagnosis is to judge whether or not the disease is present
  • risk risk assessment is to assess the magnitude of the risk of the disease and the risk of recurrence after treatment
  • evaluation of treatment effect It is evaluated whether or not there is a therapeutic effect, for example, if the symptoms are alleviated, disappeared, the lesion is reduced or disappeared, or the disease is no longer progressing, the treatment is effective.
  • a "disease sample” refers to a tissue sample in which a lesion is located or a tissue sample associated with the disease, and a disease sample such as prostate cancer includes both a cancer tissue sample of prostate cancer and a prostatic fluid sample associated with prostate cancer. And urine samples, especially urine samples that have been massaged by the prostate.
  • normal sample and "normal sample” have the same meaning and refer to a sample of normal tissue corresponding to a "disease sample”, for example, when the disease sample is a cancer tissue sample, the normal sample may be adjacent to the cancer. Tissue sample; when the disease sample is a blood or urine sample after the disease, the normal sample may be a blood sample or a urine sample before the disease.
  • the "normal reference value" has a meaning well-known in the art, and refers to a range of normal values of an index obtained on the basis of a certain number of normal samples. For example, based on a certain number of normal samples, Y can be obtained.
  • the normal reference value has a reference meaning when it is determined whether an index of a certain sample is within a normal range.
  • the kit may further contain one or more reagents selected from the group consisting of dNTPs, buffers, DNA polymerases, restriction endonucleases (including methyl-specific endonucleases). Enzymes, and labels (including fluorescent labels, chemiluminescent labels, and radioactive labels, etc.).
  • a method for designing an oligonucleotide primer for detecting the methylation level of the methylation site is known in the art based on a methylation site on the genome, and the oligonucleotide primer is known in the art.
  • the nucleic acid sequence in which the methylation site is located is capable of complementing or hybridizing.
  • the number of the oligonucleotide primers is at least one or a group of primers, for example, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8 At least 9, at least 10, at least 11 or at least 12 , which complements or hybridizes to the nucleic acid sequence in which the methylation site is located.
  • the primer that amplifies the cg05163709 site is:
  • R 5'-BIOTIN-CAACCTAATAAAAAACTATACAAACACAT (SEQ ID NO: 2);
  • S-primer ATAAGTATGTTTAATTATTGTTTAAG (SEQ ID NO: 3).
  • the primer that amplifies the cg27539833 site is:
  • R 5'-BIOTIN-AATTTACCACAATACACAAAAAACTAACTA CTTA (SEQ ID NO: 5);
  • S-primer AGATTTTAGTAGTTTTTTGTCGTTA (SEQ ID NO: 6).
  • the expression "cg" of the methylation site on the chromosome is a nomenclature for each methylation site according to the 450K chip of Illumina Co., the meaning of which is known in the art, which is represented by The methylation site is in one-to-one correspondence with a specific methylation site on the chromosome, and a person skilled in the art can accurately and uniquely correspond to a human chromosome according to the site represented by the method.
  • methylation refers to methylation of a CpG site.
  • the level of methylation can be expressed by a method known in the art, for example, it can be expressed by the ratio of methylated cytosine C detected in sequencing to the total C detected, specifically ⁇ value (beta_value) ), the value range is 0-1 or 0-100%.
  • the level of methylation is expressed as a beta value (beta_value) with a range of 0-1.
  • the method of judging the increase or decrease of the methylation level is such that the difference in the ⁇ value (for example, the difference between the samples) is 0.2 or more.
  • the "SD value” that is, the standard deviation value
  • the method of meaning calculation can refer to "Biostatistical Experimental Design” (Ming Daoxu, China Agriculture Press, Fourth Edition, August 2010).
  • the present invention establishes methods and criteria for screening disease-related chromosomal methylation sites, and screens for six Y-chromosome methylation sites associated with prostate cancer diagnosis using prostate cancer as an example.
  • the screened Y chromosome methylation site can be used as a diagnostic marker for prostate cancer for early and rapid diagnosis of the disease.
  • Figure 1 shows the methylation level heat map of 75 conserved sites screened in adjacent tissues.
  • Figure 2 shows 37 methylation sites with significant changes in cancer tissues and adjacent tissues.
  • Figures 3 and 4 show 75 methylation sites conserved in adjacent tissues (Fig. 3), of which 6 showed significant changes in cancer tissues (Fig. 4).
  • Figure 5 shows the methylation levels of the final 6 sites screened (top panel) and the proportion of significant changes in each of the 66 cancer tissues (lower panel, black marker represents significant change).
  • Figure 6 shows the DNA methylation levels in the prostate biopsy positive and negative points cg05163709, cg27539833 detected by urine samples.
  • Figure 7 is a comparison of the methylation of points cg05163709 and cg27539833 as diagnostic markers versus PSA diagnostic efficiency.
  • Tissues were collected from prostate cancer patients undergoing cancer cutting surgery, and cancerous tissues and adjacent tissues were differentiated by tissue immunoassay by experienced physicians and collected accordingly (samples were provided by Shanghai Changhai Hospital).
  • the paracancerous tissues of 66 patients with prostate cancer were collected, and DNA was extracted and amplified (QIAamp DNA Mini Kit (Cat. No. 51306)).
  • the whole genome methylation level of 66 samples was determined by DNA methylation chip Illumina 450K (Infinium HumanMethylation450 BeadChip array).
  • the raw data obtained by scanning was generated by GenomeStudio software according to Illumina official Methylation Analysis Algorithms.
  • the methylation level of one locus is Raw data.
  • the site is filtered to give the methylation level of the filtered site, ie Norm data.
  • the methylation level of each site is represented by a beta value (0-1).
  • the methylation information of the Y chromosome was divided and analyzed and compared. According to the SD value of the methylation level ( ⁇ value) between the samples, 75 methylated conserved sites (SD value ⁇ 0.25) were screened. See Figure 1 for the results.
  • IMA Illumina Methylation Analyzer
  • Example 2 According to the results in Example 2 and Example 3, the intersection of the two (ie, methylation sites conserved in the paracancerous tissues and significantly changed in the cancer tissues), a total of 6 sites cg03052502, cg04462340 , cg05163709, cg05544622, cg14466580 and cg27539833 (see Figures 3 and 4), the specific information of these six sites are shown in Table 1.
  • a urine sample of a prostate cancer patient and a normal person is obtained by prostate massage, and DNA is extracted therefrom.
  • the methylation levels of points cg05163709 and cg27539833 were obtained by pyrosequencing, and the changes in methylation levels in the urine samples of the two sites between the negative and positive samples of the prostate biopsy were compared and analyzed, as shown in Fig. 7.
  • Cg05163709 F: GGAAAGGGGTGATTAAATATTTAGTTA (SEQ ID NO: 1);
  • R 5'-BIOTIN-CAACCTAATAAAAAACTATACAAACACAT (SEQ ID NO: 2);
  • S-primer ATAAGTATGTTTAATTATTGTTTAAG (SEQ ID NO: 3).
  • R 5'-BIOTIN-AATTTACCACAATACACAAAAAACTAACTA CTTA (SEQ ID NO: 5);
  • S-primer AGATTTTAGTAGTTTTTTGTCGTTA (SEQ ID NO: 6).
  • Example 6 point cg05163709 methylation as a diagnostic marker is more efficient than PSA
  • the diagnostic efficiency of the methylation of points cg05163709 and cg27539833 was analyzed by the receiver operating curve (ROC) as a diagnostic marker, and it was found that although the area under the ROC curve of point cg27539833 was AUC (0.729) and PSA (0.753) There is no obvious advantage compared to the point, but the AUC (0.944) of point cg05163709 is significantly better than PSA (0.753), which has higher sensitivity (93.9%) and specificity (83.3%) as a diagnostic marker for prostate cancer.
  • ROC receiver operating curve

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Abstract

本发明公开了甲基化位点作为疾病诊断标志物的应用，具体涉及Y染色体甲基化位点作为前列腺癌诊断标志物的应用。更具体地，本发明确立了筛选疾病相关的染色体甲基化位点的方法和标准，并以前列腺癌为例筛选到与前列腺癌的诊断相关的六个Y染色体甲基化位点。所筛选到的Y染色体甲基化位点作为前列腺癌的诊断标志物可以用于疾病的早期、快速诊断。

Description

Y染色体甲基化位点作为前列腺癌诊断标志物的应用 技术领域
本发明涉及甲基化位点作为疾病诊断标志物的应用，具体涉及Y染色体甲基化位点作为前列腺癌诊断标志物的应用。
背景技术
DNA甲基化是一种常见的表观遗传(epigenetic)修饰，在DNA甲基转移酶催化下，利用S－腺苷甲硫氨酸提供的甲基，将胞嘧啶的第5位碳原子甲基化，从而使胞嘧啶转化为5-甲基胞嘧啶，其对基因的表达调控有着至关重要的作用。
癌症，又被称为恶性肿瘤，是由控制细胞生长增殖机制的失常而引起的疾病。正常情况下，细胞的生长增殖由包括癌基因和抑癌基因在内的一些调控生长发育的基因严密调控，保持正常的生理状态；在受到辐射，化学污染，病毒感染以及内分泌失衡等多种因素的影响下，导致体内癌基因的激活以及抑癌基因的失活，从而导致癌症的发生。除了基因突变之外，表观遗传如组蛋白修饰、DNA甲基化等异常调节也在肿瘤的发生发展过程中起着重要的作用，在多种癌症中均有发现表观修饰的异常。总之，肿瘤的发生是一个极其复杂的过程，是由于体内癌基因、抑癌基因以及表观修饰等出现异常调节并积累的结果。
DNA甲基化与癌症的发生有着密切的关系，在许多癌症中都发现存在DNA甲基化异常的现象。DNA甲基化具有一定的稳定性，它是癌症发生中的早期事件。近年来许多研究证明，DNA的甲基化异常可以作为一种癌症诊断的生物标志物。目前，许多特定基因启动子区的DNA甲基化异常被用来作为前列腺癌诊断的一种潜在标志物。
目前前列腺癌的主要诊断方法是直肠诊断、血清PSA检测、组织免疫切片检测等，其中组织免疫切片是前列腺癌的黄金诊断方法。 但由于其首先要获得病人的前列腺组织，给病人带来较大的痛苦，不太适合前期诊断。PSA是目前用于前列腺癌前期诊断的一种标志物，但是其检测并不十分精确，存在一定的灰区。所以寻找一种方便准确的诊断标志物对于前列腺癌的早期诊断十分迫切。
同时，现有技术中存在各种针对DNA甲基化监测的方法，比如CN104062334A所公开的一种针对DNA甲基化监测的定量分析方法，再比如DNA甲基化芯片技术。但是，现阶段在判断甲基化是否异常时缺乏一个统一的、较为准确的标准，无法通过特定位点甲基化水平的变化尽早预测患有癌症的风险。
发明内容
本发明的发明人建立了统一、标准的与疾病相关的甲基化位点筛选方法，并筛选到与前列腺癌相关的Y染色体甲基化位点，由此完成了本发明。
本发明的第一方面涉及检测受试者待测样品中Y染色体甲基化位点的甲基化水平的试剂在制备试剂盒中的用途，所述试剂盒用于选自以下用途中的一项或多项：前列腺癌的诊断、前列腺癌患病风险的评估、前列腺癌治疗效果的评估和前列腺癌治疗药物的筛选；所述的Y染色体甲基化位点选自cg03052502、cg04462340、cg05163709、cg05544622、cg14466580和cg27539833中的一个或几个。
在一些实施方案中，其中所述的待测样品选自组织、尿液(例如为前列腺按摩后的尿液)和前列腺液。
在一些实施方案中，其中所述的待测样品选自尿液(例如为前列腺按摩后的尿液)和前列腺液。
在一些实施方案中，其中所述的Y染色体甲基化位点选自cg05163709和cg27539833。
在一些实施方案中，其中所述的检测受试者待测样品中Y染色体甲基化位点的甲基化水平的方法选自焦磷酸测序、亚硫酸氢盐测 序、定量和/或定性的甲基化特异性聚合酶链式反应、DNA印迹法、限制性界标基因组扫描、单核苷酸引物延伸、CpG岛微阵列、单核苷酸引物延伸(SNUPE)、联合亚硫酸氢钠限制性内切酶分析法和质谱。
在一些实施方案中，其中所述的试剂为寡核苷酸引物，所述寡核苷酸引物用于扩增含有Y染色体甲基化位点的核苷酸序列。
本发明第二方面涉及试剂盒，其含有检测受试者待测样品中Y染色体甲基化位点的甲基化水平的试剂，所述试剂盒用于选自以下用途中的一项或多项：前列腺癌的诊断、前列腺癌患病风险的评估、前列腺癌治疗效果的评估和前列腺癌治疗药物的筛选；所述的Y染色体甲基化位点选自cg03052502、cg04462340、cg05163709、cg05544622、cg14466580和cg27539833中的一个或几个。
在一些实施方案中，其中所述的待测样品选自组织、尿液(例如为前列腺按摩后的尿液)和前列腺液。
在一些实施方案中，其中所述的待测样品选自尿液(例如为前列腺按摩后的尿液)和前列腺液。
在一些实施方案中，其中所述的Y染色体甲基化位点选自cg05163709和cg27539833。
在一些实施方案中，其中所述的检测受试者待测样品中Y染色体甲基化位点的甲基化水平的方法选自焦磷酸测序、亚硫酸氢盐测序、定量和/或定性的甲基化特异性聚合酶链式反应、DNA印迹法、限制性界标基因组扫描、单核苷酸引物延伸、CpG岛微阵列、单核苷酸引物延伸(SNUPE)、联合亚硫酸氢钠限制性内切酶分析法和质谱。
在一些实施方案中，其中所述的试剂为寡核苷酸引物，所述寡核苷酸引物用于扩增含有Y染色体甲基化位点的核苷酸序列。
本发明第三方面涉及一种前列腺癌的诊断、患病风险评估、治 疗效果评估和药物筛选的方法，所述方法包括检测受试者待测样品中Y染色体甲基化位点的甲基化水平的步骤；所述Y染色体甲基化位点选自cg03052502、cg04462340、cg05163709、cg05544622、cg14466580和cg27539833中的一个或几个。
在一些实施方案中，其中，当与正常样品或正常参考值相比，选自cg03052502、cg04462340、cg05544622、cg14466580和cg27539833中的一个或几个甲基化位点的甲基化水平降低时，表明受试者患有前列腺癌或罹患前列腺癌的风险高；当与正常样品或正常参考值相比，甲基化位点cg05163709的甲基化水平升高时，表明受试者患有前列腺癌或罹患前列腺癌的风险高。
在一些实施方案中，其中，当与治疗前或者使用筛选药物前相比，选自cg03052502、cg04462340、cg05544622、cg14466580和cg27539833中的一个或几个甲基化位点的甲基化水平升高时，表明受试者的治疗有效或用于筛选的药物有效；当与治疗前或者使用筛选药物前相比，甲基化位点cg05163709的甲基化水平降低时，表明受试者的治疗有效或用于筛选的药物有效。
在一些实施方案中，其中所述的待测样品选自组织、尿液(例如为前列腺按摩后的尿液)和前列腺液。
在一些实施方案中，其中所述的待测样品选自尿液(例如为前列腺按摩后的尿液)和前列腺液。
在一些实施方案中，其中所述的Y染色体甲基化位点选自cg05163709和cg27539833。
在一些实施方案中，其中所述的检测受试者待测样品中Y染色体甲基化位点的甲基化水平的方法选自焦磷酸测序、亚硫酸氢盐测序、定量和/或定性的甲基化特异性聚合酶链式反应、DNA印迹法、限制性界标基因组扫描、单核苷酸引物延伸、CpG岛微阵列、单核苷酸引物延伸(SNUPE)、联合亚硫酸氢钠限制性内切酶分析法和质谱。
在一些实施方案中，其中所述的检测受试者待测样品中Y染色 体甲基化位点的甲基化水平的方法包括使用寡核苷酸引物的步骤，所述寡核苷酸引物用于扩增含有Y染色体甲基化位点的核苷酸序列。
本发明第四方面涉及Y染色体甲基化位点作为生物标志物用于前列腺癌的诊断、患病风险评估、治疗效果评估和药物筛选的用途，所述Y染色体甲基化位点选自cg03052502、cg04462340、cg05163709、cg05544622、cg14466580和cg27539833中的一个或几个。
在一些实施方案中，可以通过检测这些Y染色体甲基化位点的甲基化水平以进行前列腺癌的诊断、患病风险的评估、治疗效果的评估和治疗前列腺癌药物的筛选。
本发明第五方面涉及用于前列腺癌的诊断、患病风险评估、治疗效果评估和药物筛选的生物标志物，所述生物标志物为Y染色体甲基化位点，所述的Y染色体甲基化位点选自cg03052502、cg04462340、cg05163709、cg05544622、cg14466580和cg27539833中的一个或几个。
在一些实施方案中，可以通过检测这些Y染色体甲基化位点的甲基化水平以进行前列腺癌的诊断、患病风险的评估、治疗效果的评估和治疗前列腺癌药物的筛选。
本发明第六方面涉及一种筛选与疾病相关的染色体甲基化位点的方法，所述方法包括：1)获取患者的疾病样本和正常样本；2)确定疾病样本和正常样本的染色体甲基化信息；3)根据正常样本的染色体甲基化信息筛选甲基化保守位点；4)根据疾病样本和正常样本的染色体甲基化信息筛选疾病样本中与正常样本相比具有明显差异的甲基化位点；5)将步骤3)和4)的结果结合，获得具有明显差异的甲基化保守位点，即为与疾病相关的染色体甲基化位点。
在一些实施方案中，其中所述的疾病为癌症，例如为前列腺癌。
在一些实施方案中，其中所述的染色体为常染色体或性染色体(例如Y染色体)。
在一些实施方案中，其中所述的样本来源于组织(例如癌组织)、血液、尿液、粪便或组织液(例如前列腺液)。
在一些实施方案中，其中所述的甲基化保守位点是指在正常样本中甲基化水平的标准偏差SD值小于等于0.25的甲基化位点。
在一些实施方案中，其中所述的具有明显差异的甲基化位点是指疾病样本和正常样本比较，甲基化水平变化在0.2以上，且p值和q值均小于等于0.01的位点；所述的变化为升高或降低。
本发明还涉及一种诊断前列腺癌的方法，所述方法包括筛选与前列腺癌相关的Y染色体甲基化位点的步骤。
在一些实施方案中，所述筛选与前列腺癌相关的Y染色体甲基化位点的步骤包括：1)获取前列腺癌患者的疾病样本和正常样本；2)确定疾病样本和正常样本的Y染色体甲基化信息；3)根据正常样本的Y染色体甲基化信息筛选甲基化保守位点；4)根据疾病样本和正常样本的Y染色体甲基化信息筛选疾病样本中与正常样本相比具有明显差异的甲基化位点；5)将步骤3)和4)的结果结合，获得具有明显差异的甲基化保守位点，即为与前列腺癌相关的染色体甲基化位点。
在一些实施方案中，其中所述的样本来源于组织(例如癌组织)、血液、尿液(例如为前列腺按摩后的尿液)、粪便或组织液(例如前列腺液)。
在一些实施方案中，其中所述的甲基化保守位点是指在正常样本中甲基化水平的标准方差SD值小于等于0.25的甲基化位点。
在一些实施方案中，其中所述的具有明显差异的甲基化位点是指疾病样本和正常样本比较，甲基化水平变化在0.2以上，且p值和q值均小于等于0.01的位点；所述的变化为升高或降低。
在本发明中，“诊断”、“患病风险评估”和“治疗效果评估” 具有本领域公知的含义，例如，“诊断”是对是否患有该疾病进行判断，“患病风险评估”是对患病风险的大小以及治疗后复发风险的大小进行评估，“治疗效果评估”是对是否具有治疗效果进行评估，例如，如果症状减轻、消失，病灶减小或消失，或者疾病不再进展，则治疗有效。
在本发明中，“疾病样本”是指病灶所在的组织样本或者和该疾病相关的组织样本，例如前列腺癌的疾病样本既包括前列腺癌的癌组织样本，也包括和前列腺癌相关的前列腺液样本和尿液样本，特别是经过前列腺按摩的尿液样本。
在本发明中，“正常样本”和“正常样品”具有相同的含义，是指与“疾病样本”相对应的正常组织的样本，例如当疾病样本为癌组织样本时，正常样本可以为癌旁组织样本；当疾病样本为患病后的血液或尿液样本时，正常样本可以为患病前的血液样本或尿液样本。
在本发明中，“正常参考值”具有本领域公知的含义，是指在一定数量的正常样本基础上得到的某指标的正常值范围，例如，在一定数量的正常样本基础上，可以得到Y染色体某甲基化位点的甲基化水平的正常参考值范围。当判定某一样本的某指标是否在正常范围内时，该正常参考值具有参考意义。
在本发明中，所述的试剂盒中还可以含有选自以下的一种或多种的试剂：dNTP，缓冲液，DNA聚合酶，限制性内切核酸酶(包括甲基特异性内切核酸酶)，和标记物(包括荧光标记物、化学发光标记物和放射标记物等)。
在本发明中，根据基因组上某甲基化位点，设计用于检测该甲基化位点的甲基化水平的寡核苷酸引物的方法为本领域所公知，该寡核苷酸引物与该甲基化位点所处的核酸序列能够互补或杂交。
在本发明中，所述寡核苷酸引物的数量为至少一个或一组引物，例如至少2个、至少3个、至少4个、至少5个、至少6个、至少7个、至少8个、至少9个、至少10个、至少11个或至少12 个，其互补或杂交于所述甲基化位点所处的核酸序列。
在本发明的具体实施方案中，扩增cg05163709位点的引物为：
F:GGAAAGGGGTGATTAAATATTTAGTTA(SEQ ID NO：1)；
R:5’-BIOTIN-CAACCTAATAAAAAACTATACAAACACAT(SEQ ID NO：2)；
S-primer:ATAAGTATGTTTAATTATTGTTTAAG(SEQ ID NO：3)。
在本发明的具体实施方案中，扩增cg27539833位点的引物为：
F:GGAATAGTTTAGTTAAAGAAAAAGGTTAAGAT(SEQ ID NO：4)；
R:5’-BIOTIN-AATTTACCACAATACACAAAAAACTAACTA CTTA(SEQ ID NO：5)；
S-primer:AGATTTTAGTAGTTTTTTGTCGTTA(SEQ ID NO：6)。
在本发明中，染色体上甲基化位点的表示方法“cg”是根据Illumina公司的450K芯片对每个甲基化位点的命名，该表示方法的含义为本领域所公知，其表示的甲基化位点与染色体上的特定甲基化位点一一对应，本领域技术人员可以根据该方法表示的位点准确、唯一地对应到人染色体上。
在本发明中，“甲基化”是指CpG位点的甲基化。
在本发明中，甲基化水平可以采用本领域公知的表示方法，例如可以用测序时检测到的甲基化的胞嘧啶C占检测到的总C的比例来表示，具体为β值(beta_value)，数值范围为0-1或者0-100％。在一些实施方案中，甲基化水平的表示方法为β值(beta_value)，数值范围为0-1。
在本发明中，甲基化水平的升高或降低的判断方法为β值的差值(例如样本间的差值)大于等于0.2。
在本发明中，“SD值”即标准偏差值，具有本领域公知的含 义和计算方法，例如可参考《生物统计附实验设计》(明道绪，中国农业出版社，2010年8月第四版)。
在本发明中，“p值”和“q值”具有本领域公知的含义和计算方法，例如可参考《生物统计附实验设计》，例如p值＝假设是正确但是被拒绝的概率＝阴性个数/总个数,是对与样本数据的一个检验概率；q值＝被拒绝但却是正确的概率＝假阳性/推测为阳性的个数,是对你得到的推论的一种检验概率,是基于P值计算出来的.可以说q值是对p值的再统计。
发明的有益效果
本发明确立了筛选疾病相关的染色体甲基化位点的方法和标准，并以前列腺癌为例筛选到与前列腺癌的诊断相关的六个Y染色体甲基化位点。所筛选到的Y染色体甲基化位点作为前列腺癌的诊断标志物可以用于疾病的早期、快速诊断。
附图说明
图1为癌旁组织中筛选出的75个保守位点的甲基化水平热图。
图2为癌组织和癌旁组织对比筛选出的37个具有明显变化的甲基化位点。
图3、图4为75个在癌旁组织中保守的甲基化位点(图3)，其中6个在癌组织中发生了明显的变化(图4)。
图5为筛选出的最终6个位点的甲基化水平(上图)，以及每个位点在66例癌组织中发生明显变化的比例(下图，黑色标记代表明显变化)。
图6为通过尿液样本检测到的点cg05163709、cg27539833在前列腺穿刺活检阳性和阴性中的DNA甲基化水平。
图7为点cg05163709、cg27539833甲基化作为诊断标志物与PSA诊断效率的对比。
具体实施方式
下面将结合实施例对本发明的实施方案进行详细描述，但是本领域技术人员将会理解，下列实施例仅用于说明本发明，而不应视为限定本发明的范围。实施例中未注明具体条件者，按照常规条件或制造商建议的条件进行。所用试剂或仪器未注明生产厂商者，均为可以通过市购获得的常规产品。
实施例1前列腺癌癌组织及癌旁组织的收集鉴定
从前列腺癌病人癌症切割手术中搜集组织，并经具有丰富经验的医师通过组织免疫切片将癌组织和癌旁组织区分，并对应收集(样本由上海长海医院提供)。
实施例2保守甲基化位点的筛选
搜集获取66例前列腺癌病人的癌旁组织，从中提取DNA并进行扩增(QIAamp DNA Mini Kit(Cat.No.51306))。利用DNA甲基化芯片Illumina 450K(Infinium HumanMethylation450 BeadChip array)测得66例样本的全基因组甲基化水平，将扫描得到的原始数据，通过GenomeStudio软件，根据illumina官方Methylation Analysis Algorithms生成含有每个样品每个位点的甲基化水平即Raw data，然后，经过对不同荧光、探针类型引起的偏差校正及归一化、位点过滤，给出过滤后位点的甲基化水平，即Norm data，每个位点的甲基化水平用β值(0-1)表示。分取其中Y染色体甲基化信息，进行分析对比，根据样本间的甲基化水平(β值)的SD值筛选出75个甲基化保守位点(SD值≤0.25)。结果参见图1。
实施例3明显差异的甲基化位点的筛选
利用Illumina Methylation Analyzer(IMA)软件包，根据66对癌组织和癌旁组织间的Y染色体上DNA甲基化水平(β值)进行比对，筛选出37个在癌组织中具有明显变化的DNA甲基化位点，即Δβ≥0.2 (即癌组织和癌旁组织之间β值的差值≥0.2)，且p值≤0.01的位点，见图2。
实施例4筛选出发生明显变化的保守性位点
根据实施例2和实施例3中的结果，筛选出两者的交集(即在癌旁组织中保守并且在癌组织中发生明显变化的甲基化位点)，共6个位点cg03052502、cg04462340、cg05163709、cg05544622、cg14466580和cg27539833(见图3和图4)，这6个位点的具体信息见表1。
表1 在癌组织中发生明显变化的甲基化位点的具体信息
Figure PCTCN2015099922-appb-000001
将这6个位点在每一对癌组织-癌旁组织中对比检测，计算出有多少比例的癌组织中确实发生了明显的甲基化变化(见图5)。这6个交集位点即为在癌组织中发生明显甲基化水平变化的保守位点，可以作为前列腺癌诊断的标志物。
实施例5检测尿液样本中点cg05163709和cg27539833的甲基化水平
通过前列腺按摩获得前列腺癌病人和正常人的尿液样本，并从中提取DNA。利用焦磷酸测序获得点cg05163709和cg27539833的甲基化水平，对比分析前列腺活体穿刺阴性和阳性样本间这两个位点在尿液样本中甲基化水平的变化，如图7。
各引物的序列如下：
cg05163709:F:GGAAAGGGGTGATTAAATATTTAGTTA(SEQ ID NO：1)；
R:5’-BIOTIN-CAACCTAATAAAAAACTATACAAACACAT(SEQ ID NO：2)；
S-primer:ATAAGTATGTTTAATTATTGTTTAAG(SEQ ID NO：3)。
Cg27539833
F:GGAATAGTTTAGTTAAAGAAAAAGGTTAAGAT(SEQ ID NO：4)；
R:5’-BIOTIN-AATTTACCACAATACACAAAAAACTAACTA CTTA(SEQ ID NO：5)；
S-primer:AGATTTTAGTAGTTTTTTGTCGTTA(SEQ ID NO：6)。
实施例6点cg05163709甲基化作为诊断标志物的诊断效率优于PSA
根据实施例5的实验结果，通过接受者操作曲线(ROC)，分析点cg05163709和cg27539833甲基化作为诊断标志物的诊断效率，发现虽然点cg27539833的ROC曲线下面积AUC(0.729)与PSA(0.753)相比并没有明显优势，但是点cg05163709的AUC(0.944)明显优于PSA(0.753)，其作为前列腺癌诊断标志物具有较高的灵敏性(93.9％)和特异性(83.3％)。
尽管本发明的具体实施方式已经得到详细的描述，本领域技术人员将会理解。根据已经公开的所有教导，可以对那些细节进行各种修改和替换，这些改变均在本发明的保护范围之内。本发明的全部范围由所附权利要求及其任何等同物给出。

Claims (30)

  1. 检测受试者待测样品中Y染色体甲基化位点的甲基化水平的试剂在制备试剂盒中的用途，所述试剂盒用于选自以下用途中的一项或多项：前列腺癌的诊断、前列腺癌患病风险的评估、前列腺癌治疗效果的评估和前列腺癌治疗药物的筛选；所述的Y染色体甲基化位点选自cg03052502、cg04462340、cg05163709、cg05544622、cg14466580和cg27539833中的一个或几个。
  2. 权利要求1的用途，其中所述的待测样品选自组织、尿液(例如为前列腺按摩后的尿液)和前列腺液。
  3. 权利要求2的用途，其中所述的待测样品选自尿液(例如为前列腺按摩后的尿液)和前列腺液。
  4. 权利要求3的用途，其中所述的Y染色体甲基化位点选自cg05163709和cg27539833。
  5. 权利要求1的用途，其中所述的检测受试者待测样品中Y染色体甲基化位点的甲基化水平的方法选自焦磷酸测序、亚硫酸氢盐测序、定量和/或定性的甲基化特异性聚合酶链式反应、DNA印迹法、限制性界标基因组扫描、单核苷酸引物延伸、CpG岛微阵列、单核苷酸引物延伸(SNUPE)、联合亚硫酸氢钠限制性内切酶分析法和质谱。
  6. 权利要求1的用途，其中所述的试剂为寡核苷酸引物，所述寡核苷酸引物用于扩增含有Y染色体甲基化位点的核苷酸序列。
  7. 试剂盒，其含有检测受试者待测样品中Y染色体甲基化位点 的甲基化水平的试剂，所述试剂盒用于选自以下用途中的一项或多项：前列腺癌的诊断、前列腺癌患病风险的评估、前列腺癌治疗效果的评估和前列腺癌治疗药物的筛选；所述的Y染色体甲基化位点选自cg03052502、cg04462340、cg05163709、cg05544622、cg14466580和cg27539833中的一个或几个。
  8. 权利要求7的试剂盒，其中所述的待测样品选自组织、尿液(例如为前列腺按摩后的尿液)和前列腺液。
  9. 权利要求8的试剂盒，其中所述的待测样品选自尿液(例如为前列腺按摩后的尿液)和前列腺液。
  10. 权利要求9的用途，其中所述的Y染色体甲基化位点选自cg05163709和cg27539833。
  11. 权利要求7的试剂盒，其中所述的检测受试者待测样品中Y染色体甲基化位点的甲基化水平的方法选自焦磷酸测序、亚硫酸氢盐测序、定量和/或定性的甲基化特异性聚合酶链式反应、DNA印迹法、限制性界标基因组扫描、单核苷酸引物延伸、CpG岛微阵列、单核苷酸引物延伸(SNUPE)、联合亚硫酸氢钠限制性内切酶分析法和质谱。
  12. 权利要求7的试剂盒，其中所述的试剂为寡核苷酸引物，所述寡核苷酸引物用于扩增含有Y染色体甲基化位点的核苷酸序列。
  13. 一种前列腺癌的诊断、患病风险评估、治疗效果评估和药物筛选的方法，所述方法包括检测受试者待测样品中Y染色体甲基化位点的甲基化水平的步骤；所述Y染色体甲基化位点选自cg03052502、cg04462340、cg05163709、cg05544622、cg14466580和 cg27539833中的一个或几个。
  14. 权利要求13的方法，其中，当与正常样品或正常参考值相比，选自cg03052502、cg04462340、cg05544622、cg14466580和cg27539833中的一个或几个甲基化位点的甲基化水平降低时，表明受试者患有前列腺癌或罹患前列腺癌的风险高；当与正常样品或正常参考值相比，甲基化位点cg05163709的甲基化水平升高时，表明受试者患有前列腺癌或罹患前列腺癌的风险高。
  15. 权利要求13的方法，其中，当与治疗前或者使用筛选药物前相比，选自cg03052502、cg04462340、cg05544622、cg14466580和cg27539833中的一个或几个甲基化位点的甲基化水平升高时，表明受试者的治疗有效或用于筛选的药物有效；当与治疗前或者使用筛选药物前相比，甲基化位点cg05163709的甲基化水平降低时，表明受试者的治疗有效或用于筛选的药物有效。
  16. 权利要求13的方法，其中所述的待测样品选自组织、尿液(例如为前列腺按摩后的尿液)和前列腺液。
  17. 权利要求13的方法，其中所述的待测样品选自尿液(例如为前列腺按摩后的尿液)和前列腺液。
  18. 权利要求13的方法，其中所述的Y染色体甲基化位点选自cg05163709和cg27539833。
  19. 权利要求13的方法，其中所述的检测受试者待测样品中Y染色体甲基化位点的甲基化水平的方法选自焦磷酸测序、亚硫酸氢盐测序、定量和/或定性的甲基化特异性聚合酶链式反应、DNA印迹法、限制性界标基因组扫描、单核苷酸引物延伸、CpG岛微阵列、 单核苷酸引物延伸(SNUPE)、联合亚硫酸氢钠限制性内切酶分析法和质谱。
  20. 权利要求13的方法，其中所述的检测受试者待测样品中Y染色体甲基化位点的甲基化水平的方法包括使用寡核苷酸引物的步骤，所述寡核苷酸引物用于扩增含有Y染色体甲基化位点的核苷酸序列。
  21. Y染色体甲基化位点作为生物标志物用于前列腺癌的诊断、患病风险评估、治疗效果评估和药物筛选的用途，所述Y染色体甲基化位点选自cg03052502、cg04462340、cg05163709、cg05544622、cg14466580和cg27539833中的一个或几个。
  22. 用于前列腺癌的诊断、患病风险评估、治疗效果评估和药物筛选的生物标志物，所述生物标志物为Y染色体甲基化位点，所述的Y染色体甲基化位点选自cg03052502、cg04462340、cg05163709、cg05544622、cg14466580和cg27539833中的一个或几个。
  23. 一种筛选与疾病相关的染色体甲基化位点的方法，所述方法包括：1)获取患者的疾病样本和正常样本；2)确定疾病样本和正常样本的染色体甲基化信息；3)根据正常样本的染色体甲基化信息筛选甲基化保守位点；4)根据疾病样本和正常样本的染色体甲基化信息筛选疾病样本中与正常样本相比具有明显差异的甲基化位点；5)将步骤3)和4)的结果结合，获得具有明显差异的甲基化保守位点，即为与疾病相关的染色体甲基化位点。
  24. 权利要求23的方法，其中所述的疾病为癌症，例如为前列腺癌。
  25. 权利要求23的方法，其中所述的染色体为常染色体或性染色体(例如Y染色体)。
  26. 权利要求23的方法，其中所述的样本来源于组织(例如癌组织)、血液、尿液、粪便或组织液(例如前列腺液)。
  27. 权利要求23的方法，其中所述的甲基化保守位点是指在正常样本中甲基化水平的标准方差SD值小于等于0.25的甲基化位点。
  28. 权利要求23的方法，其中所述的具有明显差异的甲基化位点是指疾病样本和正常样本比较，甲基化水平变化在0.2以上，且p值和q值均小于等于0.01的位点；所述的变化为升高或降低。
  29. 一种诊断前列腺癌的方法，所述方法包括筛选与前列腺癌相关的Y染色体甲基化位点的步骤。
  30. 权利要求29的方法，所述筛选与前列腺癌相关的Y染色体甲基化位点的步骤包括权利要求23－29任一项所述的方法。
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