技术领域technical field
本发明涉及生物技术领域，具体而言，涉及一种特异甲基化位点作为乳腺癌分子分型诊断标志物的应用。The invention relates to the field of biotechnology, in particular to the application of a specific methylation site as a diagnostic marker for molecular typing of breast cancer.
背景技术Background technique
液体活检技术被MIT technology评为2015十大科技进展，为肿瘤耐药生物标志物研究提供了新的思路。液体活检相较于组织活检优势众多，如非介入性、可重复性地获得肿瘤样本，副作用小、操作简便、成本较低、检测速度快。Liquid biopsy technology was rated as one of the top ten scientific and technological advances in 2015 by MIT technology, providing a new idea for the study of tumor drug resistance biomarkers. Compared with tissue biopsy, liquid biopsy has many advantages, such as non-invasive and reproducible tumor samples, less side effects, simple operation, lower cost, and faster detection speed.
循环肿瘤DNA(cfDNA)携带了肿瘤DNA的突变和甲基化信息，利用cfDNA作为肿瘤标志物，检测结果的假阳性率低，灵敏度高，准确度高。大多数蛋白质肿瘤标志物能在血液中存在数周，cfDNA的半衰期只有不到两个小时，因此可以利用cfDNA实时监控肿瘤的发展。cfDNA逐渐成为肿瘤液体活检的经典检测对象之一。但由于cfDNA含量极低(纳克级别)、降解严重，液体活检发展遭遇瓶颈，正面临巨大的挑战。Circulating tumor DNA (cfDNA) carries the mutation and methylation information of tumor DNA. Using cfDNA as a tumor marker has low false positive rate, high sensitivity and high accuracy. Most protein tumor markers can exist in the blood for several weeks, and the half-life of cfDNA is less than two hours, so cfDNA can be used to monitor the development of tumors in real time. cfDNA has gradually become one of the classic detection objects of tumor liquid biopsy. However, due to the extremely low content of cfDNA (nanogram level) and serious degradation, the development of liquid biopsy has encountered a bottleneck and is facing huge challenges.
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，损伤，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，从而导致的癌症的发生。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癌基因的激活、抑癌基因的失活以及后续肿瘤的发生发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，肿瘤的发生时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异常调节并积累的结果。Cancer, also known as malignant tumor, is a disease caused by the abnormality of the mechanism of controlling cell growth and proliferation. Under normal circumstances, the growth and proliferation of cells are tightly regulated by some genes that regulate growth and development, including oncogenes and tumor suppressor genes, and maintain a normal physiological state; Under the influence of these factors, it leads to the activation of oncogenes and the inactivation of tumor suppressor genes in the body, which leads to the occurrence of cancer. In addition to gene mutation, abnormal regulation of epigenetics such as histone modification and DNA methylation also plays an important role in the activation of oncogenes, the inactivation of tumor suppressor genes, and the subsequent occurrence and development of tumors. Abnormalities in epigenetic modification have been found in a variety of cancers. In short, tumorigenesis is an extremely complex process, which is the result of abnormal regulation and accumulation of oncogenes, tumor suppressor genes, and epigenetic modifications in the body.
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。DNA methylation is a common epigenetic modification. Under the catalysis of DNA methyltransferase, the methyl group provided by S-adenosylmethionine is used to form the fifth carbon atom of cytosine. Cytosine converts cytosine into 5-methylcytosine, which plays a vital role in the regulation of gene expression.
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，它是癌症发生中的复发事件。近年来许多研究证明， DNA的甲基化可以作为一种用药指导的生物标志物。DNA methylation is closely related to the occurrence of cancer, and abnormal DNA methylation has been found in many cancers. DNA methylation has a certain stability, which is a recurrent event in carcinogenesis. In recent years, many studies have proved that DNA methylation can be used as a biomarker for drug guidance.
目前乳腺癌的分子分型的黄金诊断依据是组织免疫切片，由于检测需获取乳腺组织，给病人带来较大的痛苦。同时，乳腺癌的分子分型是临床指导用药的指南，目前，还没有微创或无创的方式检测乳腺癌的分子分型，所以寻找一种方便准确的分子分型标志物对于乳腺癌的用药指导具十分迫切。At present, the golden diagnosis basis of molecular typing of breast cancer is tissue immunology section, which brings great pain to patients because breast tissue needs to be obtained for detection. At the same time, the molecular typing of breast cancer is a guideline for clinical guidance of drug use. At present, there is no minimally invasive or non-invasive way to detect the molecular typing of breast cancer. Therefore, looking for a convenient and accurate molecular typing marker for breast cancer drug use Guidance is urgent.
发明内容Contents of the invention
本发明旨在提供一种特异甲基化位点作为乳腺癌分子分型诊断标志物的应用，以解决现有技术中没有微创或无创的方式检测乳腺癌的分子分型的方法的技术问题。The present invention aims to provide the application of a specific methylation site as a diagnostic marker for molecular typing of breast cancer to solve the technical problems of methods for detecting molecular typing of breast cancer in a minimally invasive or non-invasive way in the prior art .
为了实现上述目的，根据本发明的一个方面，提供了一种检测受试者待测样品中cfDNA 甲基化位点的甲基化水平的试剂在制备试剂盒中的用途。其中，试剂盒用于选自以下用途中的一项或多项：乳腺癌分子分型的诊断、乳腺癌患病风险的评估、乳腺癌治疗效果的评估和乳腺癌治疗药物的筛选；甲基化位点选自chr10：39097581，chr10：88386316，chr11：105772483， chr11：113528661，chr1：163455492，chr1：164069493，chr1：173149222，chr1：201310696， chr12：29221374，chr1：233270329，chr1：242355017，chr14：60666657，chr1：59426963， chr16：46420219，chr18：61618871，chr21：20247091，chr21：23757716，chr2：36975610， chr2：87314115，chr2：89834041，chr2：89837315，chr2：89858946，chr3：115818550，chr3： 144975142，chr4：164139191，chr4：189484234，chr4：3579745，chr4：47297495，chr4：71802931， chr5：42079715，chr6：100477543，chr6：153594933，chr6：40877403，chr6：75723365，chr7： 103135406，chr7：148667863，chr7：43817571，chr8：27578331，chr8：46896342和chr8： 75989278中的一个或多个。In order to achieve the above purpose, according to one aspect of the present invention, a reagent for detecting the methylation level of a cfDNA methylation site in a sample to be tested is provided in the preparation of a kit. Wherein, the kit is used for one or more of the following purposes: diagnosis of breast cancer molecular typing, assessment of breast cancer risk, assessment of breast cancer treatment effect and screening of breast cancer treatment drugs;化位点选自chr10：39097581，chr10：88386316，chr11：105772483， chr11：113528661，chr1：163455492，chr1：164069493，chr1：173149222，chr1：201310696， chr12：29221374，chr1：233270329，chr1：242355017，chr14 ：60666657，chr1：59426963， chr16：46420219，chr18：61618871，chr21：20247091，chr21：23757716，chr2：36975610， chr2：87314115，chr2：89834041，chr2：89837315，chr2：89858946，chr3：115818550，chr3： 144975142 ，chr4：164139191，chr4：189484234，chr4：3579745，chr4：47297495，chr4：71802931， chr5：42079715，chr6：100477543，chr6：153594933，chr6：40877403，chr6：75723365，chr7： 103135406，chr7：148667863，chr7 One or more of chr8: 43817571, chr8: 27578331, chr8: 46896342 and chr8: 75989278.
进一步地，待测样品为乳腺癌患者的血液或血浆样本。Further, the samples to be tested are blood or plasma samples of breast cancer patients.
进一步地，乳腺癌患者包括不同分子分型的乳腺癌患者。Further, breast cancer patients include breast cancer patients with different molecular types.
进一步地，甲基化位点为chr10：39097581，chr10：88386316，chr11：105772483，chr11： 113528661，chr1：163455492，chr1：164069493，chr1：173149222，chr1：201310696，chr12： 29221374，chr1：233270329，chr1：242355017，chr14：60666657，chr1：59426963，chr16： 46420219，chr18：61618871，chr21：20247091，chr21：23757716，chr2：36975610，chr2： 87314115，chr2：89834041，chr2：89837315，chr2：89858946，chr3：115818550，chr3：144975142， chr4：164139191，chr4：189484234，chr4：3579745，chr4：47297495，chr4：71802931，chr5： 42079715，chr6：100477543，chr6：153594933，chr6：40877403，chr6：75723365，chr7：103135406， chr7：148667863，chr7：43817571，chr8：27578331，chr8：46896342和chr8：75989278。进一步地，甲基化位点为chr10：39097581，chr10：88386316，chr11：105772483，chr11： 113528661，chr1：163455492，chr1：164069493，chr1：173149222，chr1：201310696，chr12： 29221374，chr1：233270329，chr1 ：242355017，chr14：60666657，chr1：59426963，chr16： 46420219，chr18：61618871，chr21：20247091，chr21：23757716，chr2：36975610，chr2： 87314115，chr2：89834041，chr2：89837315，chr2：89858946，chr3：115818550 ，chr3：144975142， chr4：164139191，chr4：189484234，chr4：3579745，chr4：47297495，chr4：71802931，chr5： 42079715，chr6：100477543，chr6：153594933，chr6：40877403，chr6：75723365，chr7：103135406， chr7 : 148667863, chr7: 43817571, chr8: 27578331, chr8: 46896342 and chr8: 75989278.
进一步地，检测受试者待测样品中甲基化位点的甲基化水平所采用的方法为焦磷酸测序、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、定量和/或定性的亚硫酸氢盐特异性聚合酶链式反应、数字聚合酶链式反应、联合亚硫酸氢盐的靶向测序、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或质谱。Further, the method used to detect the methylation level of the methylation site in the sample to be tested is pyrosequencing, bisulfite sequencing, quantitative and/or qualitative methylation-specific polymerase chain quantitative and/or qualitative bisulfite-specific polymerase chain reaction, digital polymerase chain reaction, targeted sequencing combined with bisulfite, Southern blotting, genomic scanning with restriction landmarks, single-nucleus Nucleotide primer extension, CpG island microarray, single nucleotide primer extension SNUPE, combined sodium bisulfite restriction endonuclease assay or mass spectrometry.
根据本发明的另一方面，提供了一种用于检测受试者待测样品中cfDNA甲基化位点的甲基化水平的试剂盒。该试剂盒中包含检测受试者待测样品中甲基化位点的甲基化水平的试剂，试剂盒用于选自以下用途中的一项或多项：乳腺癌分子分型的诊断、乳腺癌患病风险的评估、乳腺癌癌治疗效果的评估和乳腺癌治疗药物的筛选；甲基化位点选自chr10：39097581，chr10： 88386316，chr11：105772483，chr11：113528661，chr1：163455492，chr1：164069493，chr1： 173149222，chr1：201310696，chr12：29221374，chr1：233270329，chr1：242355017，chr14： 60666657，chr1：59426963，chr16：46420219，chr18：61618871，chr21：20247091，chr21： 23757716，chr2：36975610，chr2：87314115，chr2：89834041，chr2：89837315，chr2：89858946，chr3：115818550，chr3：144975142，chr4：164139191，chr4：189484234，chr4：3579745， chr4：47297495，chr4：71802931，chr5：42079715，chr6：100477543，chr6：153594933，chr6： 40877403，chr6：75723365，chr7：103135406，chr7：148667863，chr7：43817571，chr8：27578331， chr8：46896342和chr8：75989278中的一个或多个。According to another aspect of the present invention, a kit for detecting the methylation level of a cfDNA methylation site in a test sample of a subject is provided. The kit contains reagents for detecting the methylation level of the methylation site in the sample to be tested, and the kit is used for one or more of the following purposes: diagnosis of breast cancer molecular typing, Assessment of breast cancer risk, assessment of breast cancer treatment effect and screening of breast cancer therapeutic drugs; methylation sites are selected from chr10: 39097581, chr10: 88386316, chr11: 105772483, chr11: 113528661, chr1: 163455492, chr1：164069493，chr1： 173149222，chr1：201310696，chr12：29221374，chr1：233270329，chr1：242355017，chr14： 60666657，chr1：59426963，chr16：46420219，chr18：61618871，chr21：20247091，chr21： 23757716，chr2： 36975610，chr2：87314115，chr2：89834041，chr2：89837315，chr2：89858946，chr3：115818550，chr3：144975142，chr4：164139191，chr4：189484234，chr4：3579745， chr4：47297495，chr4：71802931，chr5：42079715， chr6: 100477543, chr6: 153594933, chr6: 40877403, chr6: 75723365, chr7: 103135406, chr7: 148667863, chr7: 43817571, chr8: 27578331, one or more of chr8: 46896342 and 989
进一步地，待测样品为乳腺癌患者的血液或血浆样本。Further, the samples to be tested are blood or plasma samples of breast cancer patients.
进一步地，患者为不同分子分型的乳腺癌患者。Further, the patients are breast cancer patients with different molecular types.
进一步地，甲基化位点为chr10：39097581，chr10：88386316，chr11：105772483，chr11： 113528661，chr1：163455492，chr1：164069493，chr1：173149222，chr1：201310696，chr12： 29221374，chr1：233270329，chr1：242355017，chr14：60666657，chr1：59426963，chr16： 46420219，chr18：61618871，chr21：20247091，chr21：23757716，chr2：36975610，chr2： 87314115，chr2：89834041，chr2：89837315，chr2：89858946，chr3：115818550，chr3：144975142， chr4：164139191，chr4：189484234，chr4：3579745，chr4：47297495，chr4：71802931，chr5： 42079715，chr6：100477543，chr6：153594933，chr6：40877403，chr6：75723365，chr7：103135406， chr7：148667863，chr7：43817571，chr8：27578331，chr8：46896342和chr8：75989278。进一步地，甲基化位点为chr10：39097581，chr10：88386316，chr11：105772483，chr11： 113528661，chr1：163455492，chr1：164069493，chr1：173149222，chr1：201310696，chr12： 29221374，chr1：233270329，chr1 ：242355017，chr14：60666657，chr1：59426963，chr16： 46420219，chr18：61618871，chr21：20247091，chr21：23757716，chr2：36975610，chr2： 87314115，chr2：89834041，chr2：89837315，chr2：89858946，chr3：115818550 ，chr3：144975142， chr4：164139191，chr4：189484234，chr4：3579745，chr4：47297495，chr4：71802931，chr5： 42079715，chr6：100477543，chr6：153594933，chr6：40877403，chr6：75723365，chr7：103135406， chr7 : 148667863, chr7: 43817571, chr8: 27578331, chr8: 46896342 and chr8: 75989278.
进一步地，检测受试者待测样品中甲基化位点的甲基化水平的试剂为用于焦磷酸测序、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、定量和/或定性的亚硫酸氢盐特异性聚合酶链式反应、数字聚合酶链式反应、联合亚硫酸氢盐的靶向测序、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或质谱检测的试剂。Further, the reagent for detecting the methylation level of the methylation site in the subject's test sample is a methylation-specific polymerase chain for pyrosequencing, bisulfite sequencing, quantitative and/or qualitative quantitative and/or qualitative bisulfite-specific polymerase chain reaction, digital polymerase chain reaction, targeted sequencing combined with bisulfite, Southern blotting, genomic scanning with restriction landmarks, single-nucleus Reagents for nucleotide primer extension, CpG island microarray, single nucleotide primer extension SNUPE, combined sodium bisulfite restriction endonuclease assay or mass spectrometry detection.
根据本发明的再一方面，提供了一种用于治疗乳腺癌的药物筛选的方法。该方法包括检测受试者待测样品中cfDNA甲基化位点的甲基化水平的步骤；甲基化位点选自chr10： 39097581，chr10：88386316，chr11：105772483，chr11：113528661，chr1：163455492，chr1： 164069493，chr1：173149222，chr1：201310696，chr12：29221374，chr1：233270329，chr1： 242355017，chr14：60666657，chr1：59426963，chr16：46420219，chr18：61618871，chr21： 20247091，chr21：23757716，chr2：36975610，chr2：87314115，chr2：89834041，chr2：89837315， chr2：89858946，chr3：115818550，chr3：144975142，chr4：164139191，chr4：189484234， chr4：3579745，chr4：47297495，chr4：71802931，chr5：42079715，chr6：100477543，chr6： 153594933，chr6：40877403，chr6：75723365，chr7：103135406，chr7：148667863，chr7： 43817571，chr8：27578331，chr8：46896342和chr8：75989278中的一个或多个。According to yet another aspect of the present invention, a method for screening drugs for treating breast cancer is provided. The method includes the step of detecting the methylation level of the cfDNA methylation site in the sample to be tested; the methylation site is selected from chr10: 39097581, chr10: 88386316, chr11: 105772483, chr11: 113528661, chr1: 163455492，chr1： 164069493，chr1：173149222，chr1：201310696，chr12：29221374，chr1：233270329，chr1： 242355017，chr14：60666657，chr1：59426963，chr16：46420219，chr18：61618871，chr21： 20247091，chr21：23757716， chr2：36975610，chr2：87314115，chr2：89834041，chr2：89837315， chr2：89858946，chr3：115818550，chr3：144975142，chr4：164139191，chr4：189484234， chr4：3579745，chr4：47297495，chr4：71802931，chr5： 42079715，chr6：100477543，chr6： 153594933，chr6：40877403，chr6：75723365，chr7：103135406，chr7：148667863，chr7： 43817571，chr8：27578331，chr8：46896342和chr8：75989278中的一个或多个。
进一步地，当与治疗前或者使用筛选药物前相比，选自chr10：39097581，chr10：88386316， chr11：105772483，chr11：113528661，chr1：163455492，chr1：164069493，chr1：173149222， chr1：201310696，chr12：29221374，chr1：233270329，chr1：242355017，chr14：60666657， chr1：59426963，chr16：46420219，chr18：61618871，chr21：20247091，chr21：23757716， chr2：36975610，chr2：87314115，chr2：89834041，chr2：89837315，chr2：89858946，chr3： 115818550，chr3：144975142，chr4：164139191，chr4：189484234，chr4：3579745，chr4： 47297495，chr4：71802931，chr5：42079715，chr6：100477543，chr6：153594933，chr6：40877403， chr6：75723365，chr7：103135406，chr7：148667863，chr7：43817571，chr8：27578331，chr8： 46896342和chr8：75989278中的一个或多个甲基化位点的甲基化水平升高或降低时，表明用于筛选的药物有效。Further, when compared with before treatment or before using screening drugs, chr10: 39097581, chr10: 88386316, chr11: 105772483, chr11: 113528661, chr1: 163455492, chr1: 164069493, chr1: 173149222, chr1: 2962, chr1: 2013 ：29221374，chr1：233270329，chr1：242355017，chr14：60666657， chr1：59426963，chr16：46420219，chr18：61618871，chr21：20247091，chr21：23757716， chr2：36975610，chr2：87314115，chr2：89834041，chr2：89837315 ，chr2：89858946，chr3： 115818550，chr3：144975142，chr4：164139191，chr4：189484234，chr4：3579745，chr4： 47297495，chr4：71802931，chr5：42079715，chr6：100477543，chr6：153594933，chr6：40877403， chr6 chr7: 103135406, chr7: 148667863, chr7: 43817571, chr8: 27578331, chr8: 46896342 and chr8: 75989278 increased or decreased methylation levels at one or more of the methylation sites indicated by Drugs that are screened are effective.
进一步地，待测样品为乳腺癌患者的血液或血浆样本。Further, the samples to be tested are blood or plasma samples of breast cancer patients.
进一步地，乳腺癌患者包括不同分子分型的乳腺癌患者。Further, breast cancer patients include breast cancer patients with different molecular types.
进一步地，甲基化位点为chr10：39097581，chr10：88386316，chr11：105772483，chr11： 113528661，chr1：163455492，chr1：164069493，chr1：173149222，chr1：201310696，chr12： 29221374，chr1：233270329，chr1：242355017，chr14：60666657，chr1：59426963，chr16： 46420219，chr18：61618871，chr21：20247091，chr21：23757716，chr2：36975610，chr2： 87314115，chr2：89834041，chr2：89837315，chr2：89858946，chr3：115818550，chr3：144975142， chr4：164139191，chr4：189484234，chr4：3579745，chr4：47297495，chr4：71802931，chr5： 42079715，chr6：100477543，chr6：153594933，chr6：40877403，chr6：75723365，chr7：103135406， chr7：148667863，chr7：43817571，chr8：27578331，chr8：46896342和chr8：75989278。进一步地，甲基化位点为chr10：39097581，chr10：88386316，chr11：105772483，chr11： 113528661，chr1：163455492，chr1：164069493，chr1：173149222，chr1：201310696，chr12： 29221374，chr1：233270329，chr1 ：242355017，chr14：60666657，chr1：59426963，chr16： 46420219，chr18：61618871，chr21：20247091，chr21：23757716，chr2：36975610，chr2： 87314115，chr2：89834041，chr2：89837315，chr2：89858946，chr3：115818550 ，chr3：144975142， chr4：164139191，chr4：189484234，chr4：3579745，chr4：47297495，chr4：71802931，chr5： 42079715，chr6：100477543，chr6：153594933，chr6：40877403，chr6：75723365，chr7：103135406， chr7 : 148667863, chr7: 43817571, chr8: 27578331, chr8: 46896342 and chr8: 75989278.
进一步地，检测受试者待测样品中甲基化位点的甲基化水平所采用的方法为焦磷酸测序、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、定量和/或定性的亚硫酸氢盐特异性聚合酶链式反应、数字聚合酶链式反应、联合亚硫酸氢盐的靶向测序、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或质谱。Further, the method used to detect the methylation level of the methylation site in the sample to be tested is pyrosequencing, bisulfite sequencing, quantitative and/or qualitative methylation-specific polymerase chain quantitative and/or qualitative bisulfite-specific polymerase chain reaction, digital polymerase chain reaction, targeted sequencing combined with bisulfite, Southern blotting, genomic scanning with restriction landmarks, single-nucleus Nucleotide primer extension, CpG island microarray, single nucleotide primer extension SNUPE, combined sodium bisulfite restriction endonuclease assay or mass spectrometry.
根据本发明的又一方面，提供了一种用于乳腺癌分子分型的诊断、患病风险评估、治疗效果评估或药物筛选的装置。该装置包括检测受试者待测样品中cfDNA甲基化位点的甲基化水平的模块；甲基化位点选自chr10：39097581，chr10：88386316，chr11：105772483，chr11：113528661，chr1：163455492，chr1：164069493，chr1：173149222，chr1：201310696，chr12： 29221374，chr1：233270329，chr1：242355017，chr14：60666657，chr1：59426963，chr16： 46420219，chr18：61618871，chr21：20247091，chr21：23757716，chr2：36975610，chr2： 87314115，chr2：89834041，chr2：89837315，chr2：89858946，chr3：115818550，chr3：144975142， chr4：164139191，chr4：189484234，chr4：3579745，chr4：47297495，chr4：71802931，chr5： 42079715，chr6：100477543，chr6：153594933，chr6：40877403，chr6：75723365，chr7：103135406， chr7：148667863，chr7：43817571，chr8：27578331，chr8：46896342和chr8：75989278中的一个或多个。According to yet another aspect of the present invention, a device for diagnosis of breast cancer molecular typing, disease risk assessment, treatment effect assessment or drug screening is provided. The device includes a module for detecting the methylation level of the cfDNA methylation site in the sample to be tested; the methylation site is selected from chr10: 39097581, chr10: 88386316, chr11: 105772483, chr11: 113528661, chr1: 163455492，chr1：164069493，chr1：173149222，chr1：201310696，chr12： 29221374，chr1：233270329，chr1：242355017，chr14：60666657，chr1：59426963，chr16： 46420219，chr18：61618871，chr21：20247091，chr21：23757716， chr2：36975610，chr2： 87314115，chr2：89834041，chr2：89837315，chr2：89858946，chr3：115818550，chr3：144975142， chr4：164139191，chr4：189484234，chr4：3579745，chr4：47297495，chr4：71802931，chr5： 42079715，chr6：100477543，chr6：153594933，chr6：40877403，chr6：75723365，chr7：103135406， chr7：148667863，chr7：43817571，chr8：27578331，chr8：46896342和chr8：75989278中的一个或多个。
进一步地，装置还包括评估模块，评估模块用于将受试者检测值与不同分子分型的乳腺癌患者检测值相比，当选自chr10：39097581，chr10：88386316，chr11：105772483，chr11： 113528661，chr1：163455492，chr1：164069493，chr1：173149222，chr1：201310696，chr12： 29221374，chr1：233270329，chr1：242355017，chr14：60666657，chr1：59426963，chr16： 46420219，chr18：61618871，chr21：20247091，chr21：23757716，chr2：36975610，chr2： 87314115，chr2：89834041，chr2：89837315，chr2：89858946，chr3：115818550，chr3：144975142， chr4：164139191，chr4：189484234，chr4：3579745，chr4：47297495，chr4：71802931，chr5： 42079715，chr6：100477543，chr6：153594933，chr6：40877403，chr6：75723365，chr7：103135406， chr7：148667863，chr7：43817571，chr8：27578331，chr8：46896342和chr8：75989278中的一个或多个位点的甲基化变化与已知分型的患者的检测值相比是同趋势变化，可判断与该已知分型的患者分子型相同。Further, the device also includes an evaluation module, which is used to compare the detection value of the subject with the detection value of breast cancer patients with different molecular types, when selected from chr10: 39097581, chr10: 88386316, chr11: 105772483, chr11: 113528661 ，chr1：163455492，chr1：164069493，chr1：173149222，chr1：201310696，chr12： 29221374，chr1：233270329，chr1：242355017，chr14：60666657，chr1：59426963，chr16： 46420219，chr18：61618871，chr21：20247091，chr21 ：23757716，chr2：36975610，chr2： 87314115，chr2：89834041，chr2：89837315，chr2：89858946，chr3：115818550，chr3：144975142， chr4：164139191，chr4：189484234，chr4：3579745，chr4：47297495，chr4：71802931 ，chr5： 42079715，chr6：100477543，chr6：153594933，chr6：40877403，chr6：75723365，chr7：103135406， chr7：148667863，chr7：43817571，chr8：27578331，chr8：46896342和chr8：75989278中的一个或多个The methylation change of the site is in the same trend as the detection value of the patient with known typing, and it can be judged that the molecular type of the patient with known typing is the same.
进一步地，评估模块还用于将治疗前或者使用筛选药物前的受试者检测值与治疗后或者使用筛选药物后的受试者检测值相比，当选自chr10：39097581，chr10：88386316，chr11： 105772483，chr11：113528661，chr1：163455492，chr1：164069493，chr1：173149222，chr1： 201310696，chr12：29221374，chr1：233270329，chr1：242355017，chr14：60666657，chr1： 59426963，chr16：46420219，chr18：61618871，chr21：20247091，chr21：23757716，chr2： 36975610，chr2：87314115，chr2：89834041，chr2：89837315，chr2：89858946，chr3：115818550， chr3：144975142，chr4：164139191，chr4：189484234，chr4：3579745，chr4：47297495，chr4： 71802931，chr5：42079715，chr6：100477543，chr6：153594933，chr6：40877403，chr6：75723365， chr7：103135406，chr7：148667863，chr7：43817571，chr8：27578331，chr8：46896342和 chr8：75989278中的一个或多个甲基化位点的甲基化水平升高或降低时，表明受试者的治疗有效或用于筛选的药物有效。Further, the evaluation module is also used to compare the test value of the subject before treatment or before using the screening drug with the test value of the subject after treatment or using the screening drug, when selected from chr10: 39097581, chr10: 88386316, chr11 ： 105772483，chr11：113528661，chr1：163455492，chr1：164069493，chr1：173149222，chr1： 201310696，chr12：29221374，chr1：233270329，chr1：242355017，chr14：60666657，chr1： 59426963，chr16：46420219，chr18：61618871 ，chr21：20247091，chr21：23757716，chr2： 36975610，chr2：87314115，chr2：89834041，chr2：89837315，chr2：89858946，chr3：115818550， chr3：144975142，chr4：164139191，chr4：189484234，chr4：3579745，chr4 ：47297495，chr4： 71802931，chr5：42079715，chr6：100477543，chr6：153594933，chr6：40877403，chr6：75723365， chr7：103135406，chr7：148667863，chr7：43817571，chr8：27578331，chr8：46896342和chr8：75989278 When the methylation level of one or more methylation sites in the test is increased or decreased, it indicates that the subject's treatment is effective or the drug used for screening is effective.
进一步地，待测样品为乳腺癌患者的血液或血浆样本。Further, the samples to be tested are blood or plasma samples of breast cancer patients.
进一步地，乳腺癌患者包括不同分子分型的乳腺癌患者。Further, breast cancer patients include breast cancer patients with different molecular types.
进一步地，甲基化位点为chr10：39097581，chr10：88386316，chr11：105772483，chr11： 113528661，chr1：163455492，chr1：164069493，chr1：173149222，chr1：201310696，chr12： 29221374，chr1：233270329，chr1：242355017，chr14：60666657，chr1：59426963，chr16： 46420219，chr18：61618871，chr21：20247091，chr21：23757716，chr2：36975610，chr2： 87314115，chr2：89834041，chr2：89837315，chr2：89858946，chr3：115818550，chr3：144975142， chr4：164139191，chr4：189484234，chr4：3579745，chr4：47297495，chr4：71802931，chr5： 42079715，chr6：100477543，chr6：153594933，chr6：40877403，chr6：75723365，chr7：103135406， chr7：148667863，chr7：43817571，chr8：27578331，chr8：46896342和chr8：75989278。进一步地，甲基化位点为chr10：39097581，chr10：88386316，chr11：105772483，chr11： 113528661，chr1：163455492，chr1：164069493，chr1：173149222，chr1：201310696，chr12： 29221374，chr1：233270329，chr1 ：242355017，chr14：60666657，chr1：59426963，chr16： 46420219，chr18：61618871，chr21：20247091，chr21：23757716，chr2：36975610，chr2： 87314115，chr2：89834041，chr2：89837315，chr2：89858946，chr3：115818550 ，chr3：144975142， chr4：164139191，chr4：189484234，chr4：3579745，chr4：47297495，chr4：71802931，chr5： 42079715，chr6：100477543，chr6：153594933，chr6：40877403，chr6：75723365，chr7：103135406， chr7 : 148667863, chr7: 43817571, chr8: 27578331, chr8: 46896342 and chr8: 75989278.
进一步地，检测受试者待测样品中甲基化位点的甲基化水平所采用的方法为焦磷酸测序、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、定量和/或定性的亚硫酸氢盐特异性聚合酶链式反应、数字聚合酶链式反应、联合亚硫酸氢盐的靶向测序、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或质谱。Further, the method used to detect the methylation level of the methylation site in the sample to be tested is pyrosequencing, bisulfite sequencing, quantitative and/or qualitative methylation-specific polymerase chain quantitative and/or qualitative bisulfite-specific polymerase chain reaction, digital polymerase chain reaction, targeted sequencing combined with bisulfite, Southern blotting, genomic scanning with restriction landmarks, single-nucleus Nucleotide primer extension, CpG island microarray, single nucleotide primer extension SNUPE, combined sodium bisulfite restriction endonuclease assay or mass spectrometry.
根据本发明的再一方面，提供了一种用于乳腺癌分子分型的诊断、患病风险评估、治疗效果评估和药物筛选的生物标志物。该生物标志物为cfDNA甲基化位点，甲基化位点选自 chr10：39097581，chr10：88386316，chr11：105772483，chr11：113528661，chr1：163455492， chr1：164069493，chr1：173149222，chr1：201310696，chr12：29221374，chr1：233270329， chr1：242355017，chr14：60666657，chr1：59426963，chr16：46420219，chr18：61618871， chr21：20247091，chr21：23757716，chr2：36975610，chr2：87314115，chr2：89834041，chr2： 89837315，chr2：89858946，chr3：115818550，chr3：144975142，chr4：164139191，chr4： 189484234，chr4：3579745，chr4：47297495，chr4：71802931，chr5：42079715，chr6：100477543， chr6：153594933，chr6：40877403，chr6：75723365，chr7：103135406，chr7：148667863， chr7：43817571，chr8：27578331，chr8：46896342和chr8：75989278中的一个或多个。According to yet another aspect of the present invention, a biomarker for diagnosis of breast cancer molecular typing, disease risk assessment, treatment effect assessment and drug screening is provided. The biomarker is a cfDNA methylation site, and the methylation site is selected from chr10: 39097581, chr1: 88386316, chr11: 105772483, chr11: 113528661, chr1: 163455492, chr1: 164069493, chr1: 173149222, chr1: 2013 ，chr12：29221374，chr1：233270329， chr1：242355017，chr14：60666657，chr1：59426963，chr16：46420219，chr18：61618871， chr21：20247091，chr21：23757716，chr2：36975610，chr2：87314115，chr2：89834041，chr2 ： 89837315，chr2：89858946，chr3：115818550，chr3：144975142，chr4：164139191，chr4： 189484234，chr4：3579745，chr4：47297495，chr4：71802931，chr5：42079715，chr6：100477543， chr6：153594933，chr6：40877403 , one or more of chr6: 75723365, chr7: 103135406, chr7: 148667863, chr7: 43817571, chr8: 27578331, chr8: 46896342 and chr8: 75989278.
根据本发明的又一方面，提供了一种筛选与疾病相关的特异染色体甲基化位点的方法。该方法包括以下步骤：a)获取不同分子分型的乳腺癌患者血液样本；b)从a)步骤获得的样本中分离得到血浆；c)从b)步骤的血浆中提取cfDNA；d)对c)步提取的cfDNA进行甲基化建库；e)确定样本的全基因组甲基化水平；f)分析cfDNA甲基化信息；以及g)筛选不同分子分型的乳腺癌患者之间具有明显差异的甲基化保守位点，即为与疾病相关的特异染色体甲基化位点。According to yet another aspect of the present invention, a method for screening specific chromosomal methylation sites associated with diseases is provided. The method comprises the following steps: a) obtaining blood samples of breast cancer patients with different molecular types; b) separating plasma from the sample obtained in step a); c) extracting cfDNA from the plasma in step b); ) The cfDNA extracted in the first step is used to build a methylation library; e) determine the genome-wide methylation level of the sample; f) analyze the cfDNA methylation information; and g) screen for significant differences between breast cancer patients with different molecular types The conserved methylation site of the gene is the specific chromosomal methylation site associated with the disease.
进一步地，具有明显差异是指不同分子分型的乳腺癌患者样本比较，甲基化水平变化在 0.1以上，且p值小于等于0.05的位点；所述变化为升高或降低。Further, a significant difference refers to the site where the methylation level changes by more than 0.1 and the p value is less than or equal to 0.05 compared with breast cancer patient samples of different molecular types; the change is increased or decreased.
进一步地，患者为不同分子分型的乳腺癌患者。Further, the patients are breast cancer patients with different molecular types.
根据本发明的再一方面，提供了一种乳腺癌分子分型的诊断、患病风险评估或治疗效果评估的方法。该方法包括检测受试者待测样品中cfDNA甲基化位点的甲基化水平的步骤；甲基化位点选自chr10：39097581，chr10：88386316，chr11：105772483，chr11：113528661， chr1：163455492，chr1：164069493，chr1：173149222，chr1：201310696，chr12：29221374， chr1：233270329，chr1：242355017，chr14：60666657，chr1：59426963，chr16：46420219， chr18：61618871，chr21：20247091，chr21：23757716，chr2：36975610，chr2：87314115， chr2：89834041，chr2：89837315，chr2：89858946，chr3：115818550，chr3：144975142，chr4： 164139191，chr4：189484234，chr4：3579745，chr4：47297495，chr4：71802931，chr5：42079715， chr6：100477543，chr6：153594933，chr6：40877403，chr6：75723365，chr7：103135406， chr7：148667863，chr7：43817571，chr8：27578331，chr8：46896342和chr8：75989278中的一个或多个。According to yet another aspect of the present invention, a method for diagnosing molecular typing of breast cancer, assessing disease risk or evaluating therapeutic effect is provided. The method includes the step of detecting the methylation level of the cfDNA methylation site in the sample to be tested; the methylation site is selected from chr10: 39097581, chr10: 88386316, chr11: 105772483, chr11: 113528661, chr1: 163455492，chr1：164069493，chr1：173149222，chr1：201310696，chr12：29221374， chr1：233270329，chr1：242355017，chr14：60666657，chr1：59426963，chr16：46420219， chr18：61618871，chr21：20247091，chr21：23757716， chr2：36975610，chr2：87314115， chr2：89834041，chr2：89837315，chr2：89858946，chr3：115818550，chr3：144975142，chr4： 164139191，chr4：189484234，chr4：3579745，chr4：47297495，chr4：71802931，chr5： 42079715， chr6：100477543，chr6：153594933，chr6：40877403，chr6：75723365，chr7：103135406， chr7：148667863，chr7：43817571，chr8：27578331，chr8：46896342和chr8：75989278中的一个或多个。
进一步地，将受试者检测值与不同分子分型的乳腺癌患者检测值相比，当选自chr10： 39097581，chr10：88386316，chr11：105772483，chr11：113528661，chr1：163455492，chr1： 164069493，chr1：173149222，chr1：201310696，chr12：29221374，chr1：233270329，chr1： 242355017，chr14：60666657，chr1：59426963，chr16：46420219，chr18：61618871，chr21： 20247091，chr21：23757716，chr2：36975610，chr2：87314115，chr2：89834041，chr2：89837315， chr2：89858946，chr3：115818550，chr3：144975142，chr4：164139191，chr4：189484234， chr4：3579745，chr4：47297495，chr4：71802931，chr5：42079715，chr6：100477543，chr6： 153594933，chr6：40877403，chr6：75723365，chr7：103135406，chr7：148667863，chr7： 43817571，chr8：27578331，chr8：46896342和chr8：75989278中的一个或多个位点的甲基化变化与已知分型的患者的检测值相比是同趋势变化，可判断与该已知分型的患者分子型相同。Further, the test value of the subject is compared with the test value of breast cancer patients with different molecular types, when selected from chr10: 39097581, chr10: 88386316, chr11: 105772483, chr11: 113528661, chr1: 163455492, chr1: 164069493, chr1 ：173149222，chr1：201310696，chr12：29221374，chr1：233270329，chr1： 242355017，chr14：60666657，chr1：59426963，chr16：46420219，chr18：61618871，chr21： 20247091，chr21：23757716，chr2：36975610，chr2：87314115 ，chr2：89834041，chr2：89837315， chr2：89858946，chr3：115818550，chr3：144975142，chr4：164139191，chr4：189484234， chr4：3579745，chr4：47297495，chr4：71802931，chr5：42079715，chr6：100477543，chr6 One or more methylation changes in chr: 153594933, chr6: 40877403, chr6: 75723365, chr7: 103135406, chr7: 148667863, chr7: 43817571, chr8: 27578331, chr8: 46896342 and chr8: 75989278 are associated with known sites Compared with the detected value of the typed patient, the trend changes, and it can be judged that the molecular type is the same as that of the known typed patient.
进一步地，待测样品为乳腺癌患者的血液或血浆样本。Further, the samples to be tested are blood or plasma samples of breast cancer patients.
进一步地，乳腺癌患者包括不同分子分型的乳腺癌患者。Further, breast cancer patients include breast cancer patients with different molecular types.
进一步地，甲基化位点为chr10：39097581，chr10：88386316，chr11：105772483，chr11： 113528661，chr1：163455492，chr1：164069493，chr1：173149222，chr1：201310696，chr12： 29221374，chr1：233270329，chr1：242355017，chr14：60666657，chr1：59426963，chr16： 46420219，chr18：61618871，chr21：20247091，chr21：23757716，chr2：36975610，chr2： 87314115，chr2：89834041，chr2：89837315，chr2：89858946，chr3：115818550，chr3：144975142， chr4：164139191，chr4：189484234，chr4：3579745，chr4：47297495，chr4：71802931，chr5： 42079715，chr6：100477543，chr6：153594933，chr6：40877403，chr6：75723365，chr7：103135406，chr7：148667863，chr7：43817571，chr8：27578331，chr8：46896342和chr8：75989278。进一步地，甲基化位点为chr10：39097581，chr10：88386316，chr11：105772483，chr11： 113528661，chr1：163455492，chr1：164069493，chr1：173149222，chr1：201310696，chr12： 29221374，chr1：233270329，chr1 ：242355017，chr14：60666657，chr1：59426963，chr16： 46420219，chr18：61618871，chr21：20247091，chr21：23757716，chr2：36975610，chr2： 87314115，chr2：89834041，chr2：89837315，chr2：89858946，chr3：115818550 ，chr3：144975142， chr4：164139191，chr4：189484234，chr4：3579745，chr4：47297495，chr4：71802931，chr5： 42079715，chr6：100477543，chr6：153594933，chr6：40877403，chr6：75723365，chr7：103135406，chr7 : 148667863, chr7: 43817571, chr8: 27578331, chr8: 46896342 and chr8: 75989278.
进一步地，检测受试者待测样品中甲基化位点的甲基化水平所采用的方法为焦磷酸测序、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、定量和/或定性的亚硫酸氢盐特异性聚合酶链式反应、数字聚合酶链式反应、联合亚硫酸氢盐的靶向测序、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或质谱。Further, the method used to detect the methylation level of the methylation site in the sample to be tested is pyrosequencing, bisulfite sequencing, quantitative and/or qualitative methylation-specific polymerase chain quantitative and/or qualitative bisulfite-specific polymerase chain reaction, digital polymerase chain reaction, targeted sequencing combined with bisulfite, Southern blotting, genomic scanning with restriction landmarks, single-nucleus Nucleotide primer extension, CpG island microarray, single nucleotide primer extension SNUPE, combined sodium bisulfite restriction endonuclease assay or mass spectrometry.
根据本发明的又一方面，提供了一种cfDNA特异甲基化位点作为生物标志物用于乳腺癌分子分型的诊断、患病风险评估、治疗效果评估和药物筛选的用途。其中，甲基化位点选自 chr10：39097581，chr10：88386316，chr11：105772483，chr11：113528661，chr1：163455492， chr1：164069493，chr1：173149222，chr1：201310696，chr12：29221374，chr1：233270329， chr1：242355017，chr14：60666657，chr1：59426963，chr16：46420219，chr18：61618871， chr21：20247091，chr21：23757716，chr2：36975610，chr2：87314115，chr2：89834041，chr2： 89837315，chr2：89858946，chr3：115818550，chr3：144975142，chr4：164139191，chr4： 189484234，chr4：3579745，chr4：47297495，chr4：71802931，chr5：42079715，chr6：100477543， chr6：153594933，chr6：40877403，chr6：75723365，chr7：103135406，chr7：148667863， chr7：43817571，chr8：27578331，chr8：46896342和chr8：75989278中的一个或多个。According to yet another aspect of the present invention, a specific methylation site of cfDNA is provided as a biomarker for the diagnosis of breast cancer molecular typing, disease risk assessment, treatment effect assessment and drug screening.其中，甲基化位点选自chr10：39097581，chr10：88386316，chr11：105772483，chr11：113528661，chr1：163455492， chr1：164069493，chr1：173149222，chr1：201310696，chr12：29221374，chr1：233270329， chr1 ：242355017，chr14：60666657，chr1：59426963，chr16：46420219，chr18：61618871， chr21：20247091，chr21：23757716，chr2：36975610，chr2：87314115，chr2：89834041，chr2： 89837315，chr2：89858946，chr3：115818550 ，chr3：144975142，chr4：164139191，chr4： 189484234，chr4：3579745，chr4：47297495，chr4：71802931，chr5：42079715，chr6：100477543， chr6：153594933，chr6：40877403，chr6：75723365，chr7：103135406，chr7 One or more of chr7: 148667863, chr7: 43817571, chr8: 27578331, chr8: 46896342 and chr8: 75989278.
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，并以乳腺癌为例筛选到与乳腺癌的诊断相关的40个甲基化位点。所筛选到的特异甲基化位点作为乳腺癌的分子分型诊断标志物，可用于无创的方式检测乳腺癌的分子分型等疾病诊断。The present invention establishes a method and standard for screening disease-related chromosomal methylation sites, and takes breast cancer as an example to screen 40 methylation sites related to the diagnosis of breast cancer. The screened specific methylation sites are used as diagnostic markers for molecular typing of breast cancer, and can be used for non-invasive detection of molecular typing of breast cancer and other disease diagnoses.
附图说明Description of drawings
构成本申请的一部分的说明书附图用来提供对本发明的进一步理解，本发明的示意性实施例及其说明用于解释本发明，并不构成对本发明的不当限定。在附图中：The accompanying drawings constituting a part of the present application are used to provide a further understanding of the present invention, and the schematic embodiments and descriptions of the present invention are used to explain the present invention, and do not constitute an improper limitation of the present invention. In the attached picture:
图1A、1B和1C分别示出了实施例3中不同分子分型LuminalA、LuminalB和三阴性的样本中分别筛选出的均值差异甲基化位点为5641、1691、1739个位点的甲基化水平热图；Figures 1A, 1B, and 1C show the methyl groups of 5641, 1691, and 1739 sites with mean differential methylation sites screened out from samples of different molecular types LuminalA, LuminalB, and triple-negative in Example 3, respectively. heat map of the level of transformation;
图2A、2B和2C分别示出了实施例4中不同分子分型LuminalA、LuminalB和三阴性的样本差异的差异显著性程度p<0.05的甲基化位点的甲基化水平热图；Figures 2A, 2B and 2C respectively show the heat map of the methylation level of the methylation site with a significance degree of difference p<0.05 between samples of different molecular types LuminalA, LuminalB and triple negative in Example 4;
图3A、3B和3C分别示出了实施例5中在不同分子分型LuminalA、LuminalB和三阴性具有显著差异的甲基化位点分别为1472、956、1025的甲基化水平热图；Figures 3A, 3B and 3C respectively show the methylation level heat maps of 1472, 956 and 1025 methylation sites with significant differences in different molecular types LuminalA, LuminalB and triple negative in Example 5;
图4A、4B和4C分别示出了实施例6中筛选出的不同分子分型LuminalA、LuminalB和三阴性最终的位点，分别为9、17、15个，以及根据这位点的甲基化水平进行样本聚类的结果；以及Figures 4A, 4B and 4C show the final sites of different molecular typing LuminalA, LuminalB and triple negative screened in Example 6, respectively 9, 17, and 15, and the methylation according to this site The result of clustering samples horizontally; and
图5示出了实施例6中筛选出的40个位点，根据这些位点的甲基化水平进行样本聚类的结果。Fig. 5 shows the results of sample clustering based on the methylation levels of the 40 sites screened in Example 6.
具体实施方式Detailed ways
需要说明的是，在不冲突的情况下，本申请中的实施例及实施例中的特征可以相互组合。下面将参考附图并结合实施例来详细说明本发明。It should be noted that, in the case of no conflict, the embodiments in the present application and the features in the embodiments can be combined with each other. The present invention will be described in detail below with reference to the accompanying drawings and examples.
本发明的发明人建立了统一、标准的与疾病检测特定位点的甲基化水平的变化情况来辅助诊断乳腺癌分子分型的方法，并筛选到与乳腺癌分子分型诊断相关的甲基化位点，由此完成了本发明。The inventors of the present invention have established a unified and standard method for assisting the diagnosis of breast cancer molecular typing by the change of methylation level at specific sites for disease detection, and screened the methylation levels related to breast cancer molecular typing diagnosis. site, thus completing the present invention.
在本发明中，“分子分型诊断”、“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，“分子分型诊断”是对不同分型进行判断，“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。In the present invention, "molecular typing diagnosis", "disease risk assessment" and "treatment effect assessment" have well-known meanings in the art, for example, "molecular typing diagnosis" is to judge different types, "disease risk assessment" "Risk assessment" is to assess the risk of disease and the risk of recurrence after treatment, and "treatment effect assessment" is to assess whether there is a therapeutic effect, for example, if the symptoms are relieved or disappeared, the lesions are reduced or disappeared, or the disease If there is no progress, the treatment is effective.
在本发明中，“LuminalA”、“LuminalB”、“三阴性”具有不同的含义，LuminalA是指ER 或PR是阳性，Her2为阴性的样本；LuminalB是ER或PR是阳性，Her2为阴性或阳性的样本；三阴性是指ER、PR、Her2均为阴性的样本。In the present invention, "LuminalA", "LuminalB", and "triple negative" have different meanings. LuminalA refers to samples that are positive for ER or PR and negative for Her2; LuminalB refers to samples that are positive for ER or PR and negative or positive for Her2 samples; triple negative refers to samples that are negative for ER, PR, and Her2.
在本发明中，“甲基化”是指CpG位点的甲基化。In the present invention, "methylation" refers to the methylation of CpG sites.
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。In the present invention, the methylation level can be represented by methods known in the art, for example, it can be represented by the ratio of the methylated cytosine C detected during sequencing to the total detected C, specifically the beta value (beta value ), the value range is 0~1 or 0~100%. In some embodiments, the expression method of methylation level is β value (beta value), and the value range is 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值的差值大于等于0.1。In the present invention, the method for judging the increase or decrease of methylation level is that the difference of β value is greater than or equal to 0.1.
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。In the present invention, "p value" and "q value" have meanings and calculation methods known in the art, for example, refer to "Biostatistics with Experimental Design", for example, p value = hypothesis is correct but rejected probability = negative individual The number/total number is a test probability for the sample data.
根据本发明一种典型的实施方式，提供一种检测受试者待测样品中cfDNA甲基化位点的甲基化水平的试剂在制备试剂盒中的用途。其中，试剂盒用于选自以下用途中的一项或多项：乳腺癌分子分型的诊断、乳腺癌患病风险的评估、乳腺癌治疗效果的评估和乳腺癌治疗药物的筛选；甲基化位点选自chr10：39097581，chr10：88386316，chr11：105772483，chr11： 113528661，chr1：163455492，chr1：164069493，chr1：173149222，chr1：201310696，chr12： 29221374，chr1：233270329，chr1：242355017，chr14：60666657，chr1：59426963，chr16： 46420219，chr18：61618871，chr21：20247091，chr21：23757716，chr2：36975610，chr2： 87314115，chr2：89834041，chr2：89837315，chr2：89858946，chr3：115818550，chr3：144975142， chr4：164139191，chr4：189484234，chr4：3579745，chr4：47297495，chr4：71802931，chr5： 42079715，chr6：100477543，chr6：153594933，chr6：40877403，chr6：75723365，chr7：103135406， chr7：148667863，chr7：43817571，chr8：27578331，chr8：46896342和chr8：75989278中的一个或多个。According to a typical embodiment of the present invention, there is provided a use of a reagent for detecting the methylation level of cfDNA methylation sites in a test sample of a subject in the preparation of a kit. Wherein, the kit is used for one or more of the following purposes: diagnosis of breast cancer molecular typing, assessment of breast cancer risk, assessment of breast cancer treatment effect and screening of breast cancer treatment drugs;化位点选自chr10：39097581，chr10：88386316，chr11：105772483，chr11： 113528661，chr1：163455492，chr1：164069493，chr1：173149222，chr1：201310696，chr12： 29221374，chr1：233270329，chr1：242355017，chr14 ：60666657，chr1：59426963，chr16： 46420219，chr18：61618871，chr21：20247091，chr21：23757716，chr2：36975610，chr2： 87314115，chr2：89834041，chr2：89837315，chr2：89858946，chr3：115818550，chr3：144975142 ， chr4：164139191，chr4：189484234，chr4：3579745，chr4：47297495，chr4：71802931，chr5： 42079715，chr6：100477543，chr6：153594933，chr6：40877403，chr6：75723365，chr7：103135406， chr7：148667863，chr7 One or more of chr8:43817571, chr8:27578331, chr8:46896342 and chr8:75989278.
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，并以乳腺癌为例筛选到与乳腺癌的诊断相关的40个甲基化位点。所筛选到的特异甲基化位点作为乳腺癌的分子分型诊断标志物，可用于无创的方式检测乳腺癌的分子分型等疾病诊断。The present invention establishes a method and standard for screening disease-related chromosomal methylation sites, and takes breast cancer as an example to screen 40 methylation sites related to the diagnosis of breast cancer. The screened specific methylation sites are used as diagnostic markers for molecular typing of breast cancer, and can be used for non-invasive detection of molecular typing of breast cancer and other disease diagnoses.
优选的，待测样品为乳腺癌患者的血液或血浆样本，该样本方便采集，不会给患者带来过多的痛苦。优选的，乳腺癌患者包括不同分子分型的乳腺癌患者。Preferably, the sample to be tested is a blood or plasma sample of a breast cancer patient, which is convenient to collect and will not cause too much pain to the patient. Preferably, breast cancer patients include breast cancer patients with different molecular types.
优选的，甲基化位点为chr10：39097581，chr10：88386316，chr11：105772483，chr11： 113528661，chr1：163455492，chr1：164069493，chr1：173149222，chr1：201310696，chr12： 29221374，chr1：233270329，chr1：242355017，chr14：60666657，chr1：59426963，chr16： 46420219，chr18：61618871，chr21：20247091，chr21：23757716，chr2：36975610，chr2： 87314115，chr2：89834041，chr2：89837315，chr2：89858946，chr3：115818550，chr3：144975142， chr4：164139191，chr4：189484234，chr4：3579745，chr4：47297495，chr4：71802931，chr5： 42079715，chr6：100477543，chr6：153594933，chr6：40877403，chr6：75723365，chr7：103135406， chr7：148667863，chr7：43817571，chr8：27578331，chr8：46896342和chr8：75989278，同时对40个甲基化位点进行检测，准确率更高。优选的，甲基化位点为chr10：39097581，chr10：88386316，chr11：105772483，chr11： 113528661，chr1：163455492，chr1：164069493，chr1：173149222，chr1：201310696，chr12： 29221374，chr1：233270329，chr1 ：242355017，chr14：60666657，chr1：59426963，chr16： 46420219，chr18：61618871，chr21：20247091，chr21：23757716，chr2：36975610，chr2： 87314115，chr2：89834041，chr2：89837315，chr2：89858946，chr3：115818550 ，chr3：144975142， chr4：164139191，chr4：189484234，chr4：3579745，chr4：47297495，chr4：71802931，chr5： 42079715，chr6：100477543，chr6：153594933，chr6：40877403，chr6：75723365，chr7：103135406， chr7 : 148667863, chr7: 43817571, chr8: 27578331, chr8: 46896342 and chr8: 75989278, while detecting 40 methylation sites, the accuracy rate is higher.
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)。In the present invention, the kit may also contain one or more reagents selected from the following: dNTP, buffer, DNA polymerase, restriction endonuclease (including methyl-specific endonuclease enzymes) and markers (including fluorescent markers, chemiluminescent markers and radioactive markers, etc.).
根据本发明一种典型的实施方式，检测受试者待测样品中甲基化位点的甲基化水平所采用的方法为焦磷酸测序、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、定量和/或定性的亚硫酸氢盐特异性聚合酶链式反应、数字聚合酶链式反应、联合亚硫酸氢盐的靶向测序、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或质谱。According to a typical embodiment of the present invention, the method used to detect the methylation level of the methylation site in the subject's test sample is pyrosequencing, bisulfite sequencing, quantitative and/or qualitative formazan methylation-specific polymerase chain reaction, quantitative and/or qualitative bisulfite-specific polymerase chain reaction, digital polymerase chain reaction, targeted sequencing combined with bisulfite, Southern blotting, restriction Genomic scanning of sex landmarks, single nucleotide primer extension, CpG island microarray, single nucleotide primer extension SNUPE, combined sodium bisulfite restriction endonuclease analysis or mass spectrometry.
根据本发明一种典型的实施方式，提供一种用于检测受试者待测样品中cfDNA甲基化位点的甲基化水平的试剂盒。该试剂盒中包含检测受试者待测样品中甲基化位点的甲基化水平的试剂，试剂盒用于选自以下用途中的一项或多项：乳腺癌分子分型的诊断、乳腺癌患病风险的评估、乳腺癌癌治疗效果的评估和乳腺癌治疗药物的筛选；甲基化位点选自chr10： 39097581，chr10：88386316，chr11：105772483，chr11：113528661，chr1：163455492，chr1： 164069493，chr1：173149222，chr1：201310696，chr12：29221374，chr1：233270329，chr1： 242355017，chr14：60666657，chr1：59426963，chr16：46420219，chr18：61618871，chr21： 20247091，chr21：23757716，chr2：36975610，chr2：87314115，chr2：89834041，chr2：89837315， chr2：89858946，chr3：115818550，chr3：144975142，chr4：164139191，chr4：189484234， chr4：3579745，chr4：47297495，chr4：71802931，chr5：42079715，chr6：100477543，chr6： 153594933，chr6：40877403，chr6：75723365，chr7：103135406，chr7：148667863，chr7： 43817571，chr8：27578331，chr8：46896342和chr8：75989278中的一个或多个。According to a typical embodiment of the present invention, a kit for detecting the methylation level of a cfDNA methylation site in a test sample of a subject is provided. The kit contains reagents for detecting the methylation level of the methylation site in the sample to be tested, and the kit is used for one or more of the following purposes: diagnosis of breast cancer molecular typing, Assessment of breast cancer risk, assessment of breast cancer treatment effect and screening of breast cancer therapeutic drugs; methylation sites are selected from chr10: 39097581, chr10: 88386316, chr11: 105772483, chr11: 113528661, chr1: 163455492, chr1： 164069493，chr1：173149222，chr1：201310696，chr12：29221374，chr1：233270329，chr1： 242355017，chr14：60666657，chr1：59426963，chr16：46420219，chr18：61618871，chr21： 20247091，chr21：23757716，chr2： 36975610，chr2：87314115，chr2：89834041，chr2：89837315， chr2：89858946，chr3：115818550，chr3：144975142，chr4：164139191，chr4：189484234， chr4：3579745，chr4：47297495，chr4：71802931，chr5：42079715， chr6: 100477543, chr6: 153594933, chr6: 40877403, chr6: 75723365, chr7: 103135406, chr7: 148667863, chr7: 43817571, chr8: 27578331, one or more of chr8: 46896342 and 982
优选的，待测样品为乳腺癌患者的血液或血浆样本。Preferably, the sample to be tested is a blood or plasma sample of a breast cancer patient.
优选的，患者为不同分子分型的乳腺癌患者。Preferably, the patients are breast cancer patients with different molecular types.
优选的，甲基化位点为chr10：39097581，chr10：88386316，chr11：105772483，chr11： 113528661，chr1：163455492，chr1：164069493，chr1：173149222，chr1：201310696，chr12： 29221374，chr1：233270329，chr1：242355017，chr14：60666657，chr1：59426963，chr16： 46420219，chr18：61618871，chr21：20247091，chr21：23757716，chr2：36975610，chr2： 87314115，chr2：89834041，chr2：89837315，chr2：89858946，chr3：115818550，chr3：144975142， chr4：164139191，chr4：189484234，chr4：3579745，chr4：47297495，chr4：71802931，chr5：42079715，chr6：100477543，chr6：153594933，chr6：40877403，chr6：75723365，chr7：103135406， chr7：148667863，chr7：43817571，chr8：27578331，chr8：46896342和chr8：75989278。优选的，甲基化位点为chr10：39097581，chr10：88386316，chr11：105772483，chr11： 113528661，chr1：163455492，chr1：164069493，chr1：173149222，chr1：201310696，chr12： 29221374，chr1：233270329，chr1 ：242355017，chr14：60666657，chr1：59426963，chr16： 46420219，chr18：61618871，chr21：20247091，chr21：23757716，chr2：36975610，chr2： 87314115，chr2：89834041，chr2：89837315，chr2：89858946，chr3：115818550 ，chr3：144975142， chr4：164139191，chr4：189484234，chr4：3579745，chr4：47297495，chr4：71802931，chr5：42079715，chr6：100477543，chr6：153594933，chr6：40877403，chr6：75723365，chr7：103135406， chr7 : 148667863, chr7: 43817571, chr8: 27578331, chr8: 46896342 and chr8: 75989278.
根据本发明一种典型的实施方式，检测受试者待测样品中甲基化位点的甲基化水平的试剂为用于焦磷酸测序、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、定量和/或定性的亚硫酸氢盐特异性聚合酶链式反应、数字聚合酶链式反应、联合亚硫酸氢盐的靶向测序、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或质谱检测的试剂。According to a typical embodiment of the present invention, the reagent for detecting the methylation level of the methylation site in the subject's test sample is a methylation reagent used for pyrosequencing, bisulfite sequencing, quantitative and/or qualitative methylation-specific polymerase chain reaction, quantitative and/or qualitative bisulfite-specific polymerase chain reaction, digital polymerase chain reaction, targeted sequencing combined with bisulfite, Southern blotting, restriction Reagents for landmark genomic scanning, single nucleotide primer extension, CpG island microarray, single nucleotide primer extension SNUPE, combined sodium bisulfite restriction endonuclease assay or mass spectrometry detection.
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)。In the present invention, the kit may also contain one or more reagents selected from the following: dNTP, buffer, DNA polymerase, restriction endonuclease (including methyl-specific endonuclease enzymes) and markers (including fluorescent markers, chemiluminescent markers and radioactive markers, etc.).
根据本发明一种典型的实施方式，提供一种用于治疗乳腺癌的药物筛选的方法。该方法包括检测受试者待测样品中cfDNA甲基化位点的甲基化水平的步骤；甲基化位点选自chr10： 39097581，chr10：88386316，chr11：105772483，chr11：113528661，chr1：163455492，chr1： 164069493，chr1：173149222，chr1：201310696，chr12：29221374，chr1：233270329，chr1： 242355017，chr14：60666657，chr1：59426963，chr16：46420219，chr18：61618871，chr21： 20247091，chr21：23757716，chr2：36975610，chr2：87314115，chr2：89834041，chr2：89837315， chr2：89858946，chr3：115818550，chr3：144975142，chr4：164139191，chr4：189484234， chr4：3579745，chr4：47297495，chr4：71802931，chr5：42079715，chr6：100477543，chr6： 153594933，chr6：40877403，chr6：75723365，chr7：103135406，chr7：148667863，chr7： 43817571，chr8：27578331，chr8：46896342和chr8：75989278中的一个或多个。According to a typical embodiment of the present invention, a drug screening method for treating breast cancer is provided. The method includes the step of detecting the methylation level of the cfDNA methylation site in the sample to be tested; the methylation site is selected from chr10: 39097581, chr10: 88386316, chr11: 105772483, chr11: 113528661, chr1: 163455492，chr1： 164069493，chr1：173149222，chr1：201310696，chr12：29221374，chr1：233270329，chr1： 242355017，chr14：60666657，chr1：59426963，chr16：46420219，chr18：61618871，chr21： 20247091，chr21：23757716， chr2：36975610，chr2：87314115，chr2：89834041，chr2：89837315， chr2：89858946，chr3：115818550，chr3：144975142，chr4：164139191，chr4：189484234， chr4：3579745，chr4：47297495，chr4：71802931，chr5： 42079715，chr6：100477543，chr6： 153594933，chr6：40877403，chr6：75723365，chr7：103135406，chr7：148667863，chr7： 43817571，chr8：27578331，chr8：46896342和chr8：75989278中的一个或多个。
优选的，当与治疗前或者使用筛选药物前相比，选自chr10：39097581，chr10：88386316， chr11：105772483，chr11：113528661，chr1：163455492，chr1：164069493，chr1：173149222， chr1：201310696，chr12：29221374，chr1：233270329，chr1：242355017，chr14：60666657， chr1：59426963，chr16：46420219，chr18：61618871，chr21：20247091，chr21：23757716， chr2：36975610，chr2：87314115，chr2：89834041，chr2：89837315，chr2：89858946，chr3： 115818550，chr3：144975142，chr4：164139191，chr4：189484234，chr4：3579745，chr4： 47297495，chr4：71802931，chr5：42079715，chr6：100477543，chr6：153594933，chr6：40877403， chr6：75723365，chr7：103135406，chr7：148667863，chr7：43817571，chr8：27578331，chr8： 46896342和chr8：75989278中的一个或多个甲基化位点的甲基化水平升高或降低时，表明用于筛选的药物有效。Preferably, when compared with before treatment or before using screening drugs, chr10: 39097581, chr10: 88386316, chr11: 105772483, chr11: 113528661, chr1: 163455492, chr1: 164069493, chr1: 173149222, chr1: 2962, chr1: 2013 ：29221374，chr1：233270329，chr1：242355017，chr14：60666657， chr1：59426963，chr16：46420219，chr18：61618871，chr21：20247091，chr21：23757716， chr2：36975610，chr2：87314115，chr2：89834041，chr2：89837315 ，chr2：89858946，chr3： 115818550，chr3：144975142，chr4：164139191，chr4：189484234，chr4：3579745，chr4： 47297495，chr4：71802931，chr5：42079715，chr6：100477543，chr6：153594933，chr6：40877403， chr6 chr7: 103135406, chr7: 148667863, chr7: 43817571, chr8: 27578331, chr8: 46896342 and chr8: 75989278 increased or decreased methylation levels at one or more of the methylation sites indicated by Drugs that are screened are effective.
优选的，待测样品为乳腺癌患者的血液或血浆样本。Preferably, the sample to be tested is a blood or plasma sample of a breast cancer patient.
优选的，乳腺癌患者包括不同分子分型的乳腺癌患者。Preferably, breast cancer patients include breast cancer patients with different molecular types.
优选的，甲基化位点为chr10：39097581，chr10：88386316，chr11：105772483，chr11： 113528661，chr1：163455492，chr1：164069493，chr1：173149222，chr1：201310696，chr12： 29221374，chr1：233270329，chr1：242355017，chr14：60666657，chr1：59426963，chr16： 46420219，chr18：61618871，chr21：20247091，chr21：23757716，chr2：36975610，chr2： 87314115，chr2：89834041，chr2：89837315，chr2：89858946，chr3：115818550，chr3：144975142， chr4：164139191，chr4：189484234，chr4：3579745，chr4：47297495，chr4：71802931，chr5：42079715，chr6：100477543，chr6：153594933，chr6：40877403，chr6：75723365，chr7：103135406， chr7：148667863，chr7：43817571，chr8：27578331，chr8：46896342和chr8：75989278。优选的，甲基化位点为chr10：39097581，chr10：88386316，chr11：105772483，chr11： 113528661，chr1：163455492，chr1：164069493，chr1：173149222，chr1：201310696，chr12： 29221374，chr1：233270329，chr1 ：242355017，chr14：60666657，chr1：59426963，chr16： 46420219，chr18：61618871，chr21：20247091，chr21：23757716，chr2：36975610，chr2： 87314115，chr2：89834041，chr2：89837315，chr2：89858946，chr3：115818550 ，chr3：144975142， chr4：164139191，chr4：189484234，chr4：3579745，chr4：47297495，chr4：71802931，chr5：42079715，chr6：100477543，chr6：153594933，chr6：40877403，chr6：75723365，chr7：103135406， chr7 : 148667863, chr7: 43817571, chr8: 27578331, chr8: 46896342 and chr8: 75989278.
根据本发明一种典型的实施方式，检测受试者待测样品中甲基化位点的甲基化水平所采用的方法为焦磷酸测序、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、定量和/或定性的亚硫酸氢盐特异性聚合酶链式反应、数字聚合酶链式反应、联合亚硫酸氢盐的靶向测序、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或质谱。According to a typical embodiment of the present invention, the method used to detect the methylation level of the methylation site in the subject's test sample is pyrosequencing, bisulfite sequencing, quantitative and/or qualitative formazan methylation-specific polymerase chain reaction, quantitative and/or qualitative bisulfite-specific polymerase chain reaction, digital polymerase chain reaction, targeted sequencing combined with bisulfite, Southern blotting, restriction Genomic scanning of sex landmarks, single nucleotide primer extension, CpG island microarray, single nucleotide primer extension SNUPE, combined sodium bisulfite restriction endonuclease analysis or mass spectrometry.
根据本发明一种典型的实施方式，检测受试者待测样品中甲基化位点的甲基化水平的方法包括使用寡核苷酸引物扩增含有甲基化位点的核苷酸序列的步骤。According to a typical embodiment of the present invention, the method for detecting the methylation level of the methylation site in the subject's test sample comprises using oligonucleotide primers to amplify the nucleotide sequence containing the methylation site A step of.
根据本发明一种典型的实施方式，提供一种用于乳腺癌分子分型的诊断、患病风险评估、治疗效果评估或药物筛选的装置。该装置包括检测受试者待测样品中cfDNA甲基化位点的甲基化水平的模块；甲基化位点选自chr10：39097581，chr10：88386316，chr11：105772483， chr11：113528661，chr1：163455492，chr1：164069493，chr1：173149222，chr1：201310696， chr12：29221374，chr1：233270329，chr1：242355017，chr14：60666657，chr1：59426963， chr16：46420219，chr18：61618871，chr21：20247091，chr21：23757716，chr2：36975610， chr2：87314115，chr2：89834041，chr2：89837315，chr2：89858946，chr3：115818550，chr3： 144975142，chr4：164139191，chr4：189484234，chr4：3579745，chr4：47297495，chr4：71802931， chr5：42079715，chr6：100477543，chr6：153594933，chr6：40877403，chr6：75723365，chr7： 103135406，chr7：148667863，chr7：43817571，chr8：27578331，chr8：46896342和chr8： 75989278中的一个或多个。According to a typical embodiment of the present invention, a device for diagnosing molecular typing of breast cancer, assessing disease risk, evaluating therapeutic effect or screening drugs is provided. The device includes a module for detecting the methylation level of the cfDNA methylation site in the sample to be tested; the methylation site is selected from chr10: 39097581, chr10: 88386316, chr11: 105772483, chr11: 113528661, chr1: 163455492，chr1：164069493，chr1：173149222，chr1：201310696， chr12：29221374，chr1：233270329，chr1：242355017，chr14：60666657，chr1：59426963， chr16：46420219，chr18：61618871，chr21：20247091，chr21：23757716， chr2：36975610， chr2：87314115，chr2：89834041，chr2：89837315，chr2：89858946，chr3：115818550，chr3： 144975142，chr4：164139191，chr4：189484234，chr4：3579745，chr4：47297495，chr4：71802931， chr5： 42079715，chr6：100477543，chr6：153594933，chr6：40877403，chr6：75723365，chr7： 103135406，chr7：148667863，chr7：43817571，chr8：27578331，chr8：46896342和chr8： 75989278中的一个或多个。
根据本发明一种典型的实施方式，装置还包括评估模块，评估模块用于将受试者检测值与不同分子分型的乳腺癌患者检测值相比，当选自chr10：39097581，chr10：88386316，chr11： 105772483，chr11：113528661，chr1：163455492，chr1：164069493，chr1：173149222，chr1： 201310696，chr12：29221374，chr1：233270329，chr1：242355017，chr14：60666657，chr1： 59426963，chr16：46420219，chr18：61618871，chr21：20247091，chr21：23757716，chr2： 36975610，chr2：87314115，chr2：89834041，chr2：89837315，chr2：89858946，chr3：115818550， chr3：144975142，chr4：164139191，chr4：189484234，chr4：3579745，chr4：47297495，chr4： 71802931，chr5：42079715，chr6：100477543，chr6：153594933，chr6：40877403，chr6：75723365， chr7：103135406，chr7：148667863，chr7：43817571，chr8：27578331，chr8：46896342和 chr8：75989278中的一个或多个位点的甲基化变化与已知分型的患者的检测值相比是同趋势变化，可判断与该已知分型的患者分子型相同。According to a typical embodiment of the present invention, the device further includes an evaluation module, which is used to compare the detection value of the subject with the detection value of breast cancer patients with different molecular types, when selected from chr10: 39097581, chr10: 88386316, chr11： 105772483，chr11：113528661，chr1：163455492，chr1：164069493，chr1：173149222，chr1： 201310696，chr12：29221374，chr1：233270329，chr1：242355017，chr14：60666657，chr1： 59426963，chr16：46420219，chr18： 61618871，chr21：20247091，chr21：23757716，chr2： 36975610，chr2：87314115，chr2：89834041，chr2：89837315，chr2：89858946，chr3：115818550， chr3：144975142，chr4：164139191，chr4：189484234，chr4：3579745， chr4：47297495，chr4： 71802931，chr5：42079715，chr6：100477543，chr6：153594933，chr6：40877403，chr6：75723365， chr7：103135406，chr7：148667863，chr7：43817571，chr8：27578331，chr8：46896342和chr8： The methylation changes of one or more sites in 75989278 are in the same trend compared with the detected values of patients with known typing, and it can be judged that the molecular type is the same as that of patients with known typing.
根据本发明一种典型的实施方式，评估模块还用于将治疗前或者使用筛选药物前的受试者检测值与治疗后或者使用筛选药物后的受试者检测值相比，当选自chr10：39097581，chr10： 88386316，chr11：105772483，chr11：113528661，chr1：163455492，chr1：164069493，chr1： 173149222，chr1：201310696，chr12：29221374，chr1：233270329，chr1：242355017，chr14： 60666657，chr1：59426963，chr16：46420219，chr18：61618871，chr21：20247091，chr21： 23757716，chr2：36975610，chr2：87314115，chr2：89834041，chr2：89837315，chr2：89858946， chr3：115818550，chr3：144975142，chr4：164139191，chr4：189484234，chr4：3579745， chr4：47297495，chr4：71802931，chr5：42079715，chr6：100477543，chr6：153594933，chr6：40877403，chr6：75723365，chr7：103135406，chr7：148667863，chr7：43817571，chr8：27578331， chr8：46896342和chr8：75989278中的一个或多个甲基化位点的甲基化水平升高或降低时，表明受试者的治疗有效或用于筛选的药物有效。According to a typical embodiment of the present invention, the evaluation module is also used to compare the test value of the subject before treatment or using the screening drug with the test value of the subject after treatment or using the screening drug, when selected from chr10: 39097581，chr10： 88386316，chr11：105772483，chr11：113528661，chr1：163455492，chr1：164069493，chr1： 173149222，chr1：201310696，chr12：29221374，chr1：233270329，chr1：242355017，chr14： 60666657，chr1：59426963， chr16：46420219，chr18：61618871，chr21：20247091，chr21： 23757716，chr2：36975610，chr2：87314115，chr2：89834041，chr2：89837315，chr2：89858946， chr3：115818550，chr3：144975142，chr4：164139191，chr4： 189484234，chr4：3579745， chr4：47297495，chr4：71802931，chr5：42079715，chr6：100477543，chr6：153594933，chr6：40877403，chr6：75723365，chr7：103135406，chr7：148667863，chr7：43817571，chr8：27578331， When the methylation level of one or more methylation sites in chr8: 46896342 and chr8: 75989278 increases or decreases, it indicates that the subject's treatment is effective or the drug used for screening is effective.
优选的，待测样品为乳腺癌患者的血液或血浆样本。Preferably, the sample to be tested is a blood or plasma sample of a breast cancer patient.
优选的，乳腺癌患者包括不同分子分型的乳腺癌患者。Preferably, breast cancer patients include breast cancer patients with different molecular types.
优选的，甲基化位点为chr10：39097581，chr10：88386316，chr11：105772483，chr11： 113528661，chr1：163455492，chr1：164069493，chr1：173149222，chr1：201310696，chr12： 29221374，chr1：233270329，chr1：242355017，chr14：60666657，chr1：59426963，chr16： 46420219，chr18：61618871，chr21：20247091，chr21：23757716，chr2：36975610，chr2： 87314115，chr2：89834041，chr2：89837315，chr2：89858946，chr3：115818550，chr3：144975142， chr4：164139191，chr4：189484234，chr4：3579745，chr4：47297495，chr4：71802931，chr5： 42079715，chr6：100477543，chr6：153594933，chr6：40877403，chr6：75723365，chr7：103135406， chr7：148667863，chr7：43817571，chr8：27578331，chr8：46896342和chr8：75989278。优选的，甲基化位点为chr10：39097581，chr10：88386316，chr11：105772483，chr11： 113528661，chr1：163455492，chr1：164069493，chr1：173149222，chr1：201310696，chr12： 29221374，chr1：233270329，chr1 ：242355017，chr14：60666657，chr1：59426963，chr16： 46420219，chr18：61618871，chr21：20247091，chr21：23757716，chr2：36975610，chr2： 87314115，chr2：89834041，chr2：89837315，chr2：89858946，chr3：115818550 ，chr3：144975142， chr4：164139191，chr4：189484234，chr4：3579745，chr4：47297495，chr4：71802931，chr5： 42079715，chr6：100477543，chr6：153594933，chr6：40877403，chr6：75723365，chr7：103135406， chr7 : 148667863, chr7: 43817571, chr8: 27578331, chr8: 46896342 and chr8: 75989278.
优选的，检测受试者待测样品中甲基化位点的甲基化水平所采用的方法为焦磷酸测序、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、定量和/或定性的亚硫酸氢盐特异性聚合酶链式反应、数字聚合酶链式反应、联合亚硫酸氢盐的靶向测序、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或质谱。Preferably, the method used to detect the methylation level of the methylation site in the sample to be tested is pyrosequencing, bisulfite sequencing, quantitative and/or qualitative methylation-specific polymerase chain quantitative and/or qualitative bisulfite-specific polymerase chain reaction, digital polymerase chain reaction, targeted sequencing combined with bisulfite, Southern blotting, genomic scanning with restriction landmarks, single-nucleus Nucleotide primer extension, CpG island microarray, single nucleotide primer extension SNUPE, combined sodium bisulfite restriction endonuclease assay or mass spectrometry.
根据本发明一种典型的实施方式，提供一种用于乳腺癌分子分型的诊断、患病风险评估、治疗效果评估和药物筛选的生物标志物。该生物标志物为cfDNA甲基化位点，甲基化位点选自chr10：39097581，chr10：88386316，chr11：105772483，chr11：113528661，chr1：163455492， chr1：164069493，chr1：173149222，chr1：201310696，chr12：29221374，chr1：233270329， chr1：242355017，chr14：60666657，chr1：59426963，chr16：46420219，chr18：61618871， chr21：20247091，chr21：23757716，chr2：36975610，chr2：87314115，chr2：89834041，chr2： 89837315，chr2：89858946，chr3：115818550，chr3：144975142，chr4：164139191，chr4： 189484234，chr4：3579745，chr4：47297495，chr4：71802931，chr5：42079715，chr6：100477543， chr6：153594933，chr6：40877403，chr6：75723365，chr7：103135406，chr7：148667863， chr7：43817571，chr8：27578331，chr8：46896342和chr8：75989278中的一个或多个。According to a typical embodiment of the present invention, a biomarker for the diagnosis of breast cancer molecular typing, disease risk assessment, treatment effect assessment and drug screening is provided. The biomarker is a cfDNA methylation site, and the methylation site is selected from chr10: 39097581, chr1: 88386316, chr11: 105772483, chr11: 113528661, chr1: 163455492, chr1: 164069493, chr1: 173149222, chr1: 2013 ，chr12：29221374，chr1：233270329， chr1：242355017，chr14：60666657，chr1：59426963，chr16：46420219，chr18：61618871， chr21：20247091，chr21：23757716，chr2：36975610，chr2：87314115，chr2：89834041，chr2 ： 89837315，chr2：89858946，chr3：115818550，chr3：144975142，chr4：164139191，chr4： 189484234，chr4：3579745，chr4：47297495，chr4：71802931，chr5：42079715，chr6：100477543， chr6：153594933，chr6：40877403 , one or more of chr6: 75723365, chr7: 103135406, chr7: 148667863, chr7: 43817571, chr8: 27578331, chr8: 46896342 and chr8: 75989278.
根据本发明一种典型的实施方式，提供一种筛选与疾病相关的特异染色体甲基化位点的方法。该方法包括以下步骤：a)获取不同分子分型的乳腺癌患者血液样本；b)从a)步骤获得的样本中分离得到血浆；c)从b)步骤的血浆中提取cfDNA；d)对c)步提取的cfDNA 进行甲基化建库；e)确定样本的全基因组甲基化水平；f)分析cfDNA甲基化信息；以及g) 筛选不同分子分型的乳腺癌患者之间具有明显差异的甲基化保守位点，即为与疾病相关的特异染色体甲基化位点。根据本发明一种典型的实施方式，具有明显差异是指不同分子分型的乳腺癌患者样本比较，甲基化水平变化在0.1以上，且p值小于等于0.05的位点；变化为升高或降低。详细筛选过程如下：经过0.1、0.15、0.2、0.25、0.3等不同差异标准的尝试之后，最终确定了明显差异为不同分子分型癌症样本之间甲基化水平均值的差的绝对值大于等于0.1，即|mean(betas of non relapse samples)–mean(betas of relapse samples)|≥0.1。这个标准的确立，保证了既能有明显的分类效果，又能保留尽可能多的有效位点以供下一步筛选。利用limma软件包，根据乳腺癌不同分子分型患者的cfDNA甲基化水平比对，经过0.1、0.5、 0.01、0.001等不同差异标准的尝试之后，最终确定了明显差异为p值小于等于0.05，即 t.test(betas of non relapse samples,betas of relapse samples)≤0.05。这个标准的确立，保证了既能有明显的分类效果，又能保留尽可能多的有效位点以供下一步筛选。According to a typical embodiment of the present invention, a method for screening specific chromosomal methylation sites associated with diseases is provided. The method comprises the following steps: a) obtaining blood samples of breast cancer patients with different molecular types; b) separating plasma from the sample obtained in step a); c) extracting cfDNA from the plasma in step b); ) The cfDNA extracted in the first step is used to build a methylation library; e) determine the genome-wide methylation level of the sample; f) analyze the cfDNA methylation information; and g) screen for significant differences between breast cancer patients with different molecular types The conserved methylation site of the gene is the specific chromosomal methylation site associated with the disease. According to a typical embodiment of the present invention, having a significant difference refers to the site where the methylation level changes by more than 0.1 and the p value is less than or equal to 0.05 compared with breast cancer patient samples of different molecular types; the change is increased or reduce. The detailed screening process is as follows: After trying different difference standards such as 0.1, 0.15, 0.2, 0.25, 0.3, etc., it is finally determined that the obvious difference is that the absolute value of the difference in the mean value of methylation levels between cancer samples of different molecular types is greater than or equal to 0.1 , ie |mean(betas of non relapse samples)–mean(betas of relapse samples)|≥0.1. The establishment of this standard ensures not only the obvious classification effect, but also retaining as many effective sites as possible for the next step of screening. Using the limma software package, according to the comparison of the cfDNA methylation levels of patients with different molecular types of breast cancer, after trying different difference standards such as 0.1, 0.5, 0.01, and 0.001, it was finally determined that the significant difference was that the p value was less than or equal to 0.05. That is, t.test(betas of non relapse samples, betas of relapse samples)≤0.05. The establishment of this standard ensures not only the obvious classification effect, but also retaining as many effective sites as possible for the next step of screening.
在一些实施方案中，详细筛选过程如下：筛选出两者的交集即在某种分子分型患者中保守而在其他分子分型患者中发生明显变化的甲基化位点，即|mean(betas of nonrelapse samples) –mean(betas of relapse samples)|≥0.1&&t.test(betas of nonrelapse samples,betas of relapse samples)≤0.05。In some embodiments, the detailed screening process is as follows: the intersection of the two is selected, that is, the methylation sites that are conserved in patients with a certain molecular type and significantly changed in patients with other molecular types, that is |mean(betas of nonrelapse samples) –mean(betas of relapse samples)|≥0.1&&t.test(betas of nonrelapse samples,betas of relapse samples)≤0.05.
在一些实施方案中，其中所述的作为乳腺癌分子分型诊断标志物的甲基化位点是指在某个分子分型样本中保守而在其他分子分型样本中发生异常甲基化，且对分类贡献程度最大的位点。详细筛选过程如下：利用Lasso算法筛选出贡献程度大的位点。经过不同位点数量的尝试之后，最终选定定了LuminalA、LuminalB、三阴性中贡献程度最大的位点，分别是9，17， 15个位点。这样既保证了有明显的分类效果，又能保留尽可能少的有效位点以方便临床快速检验。之后我们将各自差异的位点合并，发现有40个位点，可以仅根据这40个位点的甲基化水平对样本进行了聚类,聚类结果符合临床预期。这40个甲基化位点可以作为乳腺癌分子分型诊断的标志物。In some embodiments, the methylation site used as a diagnostic marker for molecular typing of breast cancer refers to a methylation site that is conserved in a certain molecular typing sample but abnormally methylated in other molecular typing samples, and the locus with the greatest contribution to the classification. The detailed screening process is as follows: Use the Lasso algorithm to screen out the sites with a large contribution. After trying different numbers of sites, the sites with the greatest contribution in LuminalA, LuminalB, and triple negatives were finally selected, which were 9, 17, and 15 sites, respectively. This not only ensures a significant classification effect, but also retains as few effective sites as possible to facilitate rapid clinical testing. Afterwards, we merged the different sites and found that there are 40 sites, and the samples can be clustered only according to the methylation level of these 40 sites, and the clustering results are in line with clinical expectations. These 40 methylation sites can be used as markers for molecular typing and diagnosis of breast cancer.
根据本发明一种典型的实施方式，患者为不同分子分型的乳腺癌患者。According to a typical embodiment of the present invention, the patients are breast cancer patients with different molecular types.
根据本发明一种典型的实施方式，提供一种乳腺癌分子分型的诊断、患病风险评估或治疗效果评估的方法。该方法包括检测受试者待测样品中cfDNA甲基化位点的甲基化水平的步骤；甲基化位点选自chr10：39097581，chr10：88386316，chr11：105772483，chr11：113528661， chr1：163455492，chr1：164069493，chr1：173149222，chr1：201310696，chr12：29221374， chr1：233270329，chr1：242355017，chr14：60666657，chr1：59426963，chr16：46420219， chr18：61618871，chr21：20247091，chr21：23757716，chr2：36975610，chr2：87314115， chr2：89834041，chr2：89837315，chr2：89858946，chr3：115818550，chr3：144975142，chr4： 164139191，chr4：189484234，chr4：3579745，chr4：47297495，chr4：71802931，chr5：42079715， chr6：100477543，chr6：153594933，chr6：40877403，chr6：75723365，chr7：103135406， chr7：148667863，chr7：43817571，chr8：27578331，chr8：46896342和chr8：75989278中的一个或多个。According to a typical embodiment of the present invention, a method for diagnosing molecular typing of breast cancer, evaluating disease risk or evaluating treatment effect is provided. The method includes the step of detecting the methylation level of the cfDNA methylation site in the sample to be tested; the methylation site is selected from chr10: 39097581, chr10: 88386316, chr11: 105772483, chr11: 113528661, chr1: 163455492，chr1：164069493，chr1：173149222，chr1：201310696，chr12：29221374， chr1：233270329，chr1：242355017，chr14：60666657，chr1：59426963，chr16：46420219， chr18：61618871，chr21：20247091，chr21：23757716， chr2：36975610，chr2：87314115， chr2：89834041，chr2：89837315，chr2：89858946，chr3：115818550，chr3：144975142，chr4： 164139191，chr4：189484234，chr4：3579745，chr4：47297495，chr4：71802931，chr5： 42079715， chr6：100477543，chr6：153594933，chr6：40877403，chr6：75723365，chr7：103135406， chr7：148667863，chr7：43817571，chr8：27578331，chr8：46896342和chr8：75989278中的一个或多个。
优选的，将受试者检测值与不同分子分型的乳腺癌患者检测值相比，当选自chr10： 39097581，chr10：88386316，chr11：105772483，chr11：113528661，chr1：163455492，chr1： 164069493，chr1：173149222，chr1：201310696，chr12：29221374，chr1：233270329，chr1： 242355017，chr14：60666657，chr1：59426963，chr16：46420219，chr18：61618871，chr21： 20247091，chr21：23757716，chr2：36975610，chr2：87314115，chr2：89834041，chr2：89837315， chr2：89858946，chr3：115818550，chr3：144975142，chr4：164139191，chr4：189484234， chr4：3579745，chr4：47297495，chr4：71802931，chr5：42079715，chr6：100477543，chr6： 153594933，chr6：40877403，chr6：75723365，chr7：103135406，chr7：148667863，chr7： 43817571，chr8：27578331，chr8：46896342和chr8：75989278中的一个或多个位点的甲基化变化与已知分型的患者的检测值相比是同趋势变化，可判断与该已知分型的患者分子型相同。Preferably, the detection value of the subject is compared with the detection value of breast cancer patients with different molecular types, when selected from chr10: 39097581, chr10: 88386316, chr11: 105772483, chr11: 113528661, chr1: 163455492, chr1: 164069493, chr1 ：173149222，chr1：201310696，chr12：29221374，chr1：233270329，chr1： 242355017，chr14：60666657，chr1：59426963，chr16：46420219，chr18：61618871，chr21： 20247091，chr21：23757716，chr2：36975610，chr2：87314115 ，chr2：89834041，chr2：89837315， chr2：89858946，chr3：115818550，chr3：144975142，chr4：164139191，chr4：189484234， chr4：3579745，chr4：47297495，chr4：71802931，chr5：42079715，chr6：100477543，chr6 One or more methylation changes in chr: 153594933, chr6: 40877403, chr6: 75723365, chr7: 103135406, chr7: 148667863, chr7: 43817571, chr8: 27578331, chr8: 46896342 and chr8: 75989278 are associated with known sites Compared with the detected value of the typed patient, the trend changes, and it can be judged that the molecular type is the same as that of the known typed patient.
优选的，待测样品为乳腺癌患者的血液或血浆样本。Preferably, the sample to be tested is a blood or plasma sample of a breast cancer patient.
优选的，乳腺癌患者包括不同分子分型的乳腺癌患者。Preferably, breast cancer patients include breast cancer patients with different molecular types.
优选的，甲基化位点为chr10：39097581，chr10：88386316，chr11：105772483，chr11： 113528661，chr1：163455492，chr1：164069493，chr1：173149222，chr1：201310696，chr12： 29221374，chr1：233270329，chr1：242355017，chr14：60666657，chr1：59426963，chr16： 46420219，chr18：61618871，chr21：20247091，chr21：23757716，chr2：36975610，chr2： 87314115，chr2：89834041，chr2：89837315，chr2：89858946，chr3：115818550，chr3：144975142， chr4：164139191，chr4：189484234，chr4：3579745，chr4：47297495，chr4：71802931，chr5： 42079715，chr6：100477543，chr6：153594933，chr6：40877403，chr6：75723365，chr7：103135406， chr7：148667863，chr7：43817571，chr8：27578331，chr8：46896342和chr8：75989278。优选的，甲基化位点为chr10：39097581，chr10：88386316，chr11：105772483，chr11： 113528661，chr1：163455492，chr1：164069493，chr1：173149222，chr1：201310696，chr12： 29221374，chr1：233270329，chr1 ：242355017，chr14：60666657，chr1：59426963，chr16： 46420219，chr18：61618871，chr21：20247091，chr21：23757716，chr2：36975610，chr2： 87314115，chr2：89834041，chr2：89837315，chr2：89858946，chr3：115818550 ，chr3：144975142， chr4：164139191，chr4：189484234，chr4：3579745，chr4：47297495，chr4：71802931，chr5： 42079715，chr6：100477543，chr6：153594933，chr6：40877403，chr6：75723365，chr7：103135406， chr7 : 148667863, chr7: 43817571, chr8: 27578331, chr8: 46896342 and chr8: 75989278.
根据本发明一种典型的实施方式，检测受试者待测样品中甲基化位点的甲基化水平所采用的方法为焦磷酸测序、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、定量和/或定性的亚硫酸氢盐特异性聚合酶链式反应、数字聚合酶链式反应、联合亚硫酸氢盐的靶向测序、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或质谱。According to a typical embodiment of the present invention, the method used to detect the methylation level of the methylation site in the subject's test sample is pyrosequencing, bisulfite sequencing, quantitative and/or qualitative formazan methylation-specific polymerase chain reaction, quantitative and/or qualitative bisulfite-specific polymerase chain reaction, digital polymerase chain reaction, targeted sequencing combined with bisulfite, Southern blotting, restriction Genomic scanning of sex landmarks, single nucleotide primer extension, CpG island microarray, single nucleotide primer extension SNUPE, combined sodium bisulfite restriction endonuclease analysis or mass spectrometry.
根据本发明一种典型的实施方式，提供一种cfDNA特异甲基化位点作为生物标志物用于乳腺癌分子分型的诊断、患病风险评估、治疗效果评估和药物筛选的用途。其中，甲基化位点选自chr10：39097581，chr10：88386316，chr11：105772483，chr11：113528661，chr1： 163455492，chr1：164069493，chr1：173149222，chr1：201310696，chr12：29221374，chr1： 233270329，chr1：242355017，chr14：60666657，chr1：59426963，chr16：46420219，chr18： 61618871，chr21：20247091，chr21：23757716，chr2：36975610，chr2：87314115，chr2：89834041， chr2：89837315，chr2：89858946，chr3：115818550，chr3：144975142，chr4：164139191， chr4：189484234，chr4：3579745，chr4：47297495，chr4：71802931，chr5：42079715，chr6： 100477543，chr6：153594933，chr6：40877403，chr6：75723365，chr7：103135406，chr7： 148667863，chr7：43817571，chr8：27578331，chr8：46896342和chr8：75989278中的一个或多个。According to a typical embodiment of the present invention, a specific methylation site of cfDNA is provided as a biomarker for the diagnosis of breast cancer molecular typing, disease risk assessment, treatment effect assessment and drug screening.其中，甲基化位点选自chr10：39097581，chr10：88386316，chr11：105772483，chr11：113528661，chr1： 163455492，chr1：164069493，chr1：173149222，chr1：201310696，chr12：29221374，chr1： 233270329，chr1 ：242355017，chr14：60666657，chr1：59426963，chr16：46420219，chr18： 61618871，chr21：20247091，chr21：23757716，chr2：36975610，chr2：87314115，chr2：89834041， chr2：89837315，chr2：89858946，chr3：115818550 ，chr3：144975142，chr4：164139191， chr4：189484234，chr4：3579745，chr4：47297495，chr4：71802931，chr5：42079715，chr6： 100477543，chr6：153594933，chr6：40877403，chr6：75723365，chr7：103135406，chr7 One or more of: 148667863, chr7: 43817571, chr8: 27578331, chr8: 46896342, and chr8: 75989278.
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。Embodiments of the present invention will be described in detail below in conjunction with examples, but those skilled in the art will understand that the following examples are only used to illustrate the present invention, and should not be considered as limiting the scope of the present invention. Those who do not indicate the specific conditions in the examples are carried out according to the conventional conditions or the conditions suggested by the manufacturer. The reagents or instruments used were not indicated by the manufacturer, and they were all commercially available conventional products.
实施例1Example 1
乳腺癌症患者血液样本的收集鉴定Collection and identification of blood samples from breast cancer patients
搜集不同分子分型的患者血液样本，并经具有丰富经验的医师通过验证收集(样本由天津肿瘤医院提供)。Blood samples from patients with different molecular types were collected and collected through verification by experienced physicians (the samples were provided by Tianjin Cancer Hospital).
实施例2Example 2
乳腺癌患者cfDNA提取及建库测序Extraction and library sequencing of cfDNA in breast cancer patients
搜集获取各种乳腺癌分子分型患者的血液样本，并从血浆中提取cfDNA。利用QIAamp Circulating Nucleic Acid Kit试剂盒。Blood samples from patients with various breast cancer molecular types were collected, and cfDNA was extracted from plasma. Utilize the QIAamp Circulating Nucleic Acid Kit.
具体操作步骤如下：1)利用密度梯度离心法，以1900g的转速离心，将红细胞、白细胞和血浆分离，在最上层清澈的为血浆，将血浆取出，再利用14000rpm的转速，进行再次高速离心，上层为血浆；2)将1)提取的血浆加入已经加了蛋白酶k的50ml离心管中，再加入裂解液ACL(已含有CarrierRNA)，得到第一混合液；3)将2)中最后所得第一混合液混匀，放于60℃水浴30分钟；4)将完成3)后第一混合液从水浴中取出，加入ACB，按1：1.8的比列，即1ml血浆，1.8mlACB，得第二混合液；5)将4)中得到的第二混合液利用真空泵抽滤，抽滤完后，利用ACW1、ACW2和无水乙醇进行洗涤；6)14000rpm空离3分钟，56℃蒸干去除乙醇；7)利用30ul水洗脱，得到cfDNA；8)将7)中所得的cfDNA进行甲基化建库。The specific operation steps are as follows: 1) Use the density gradient centrifugation method to centrifuge at a speed of 1900g to separate red blood cells, white blood cells and plasma. The clear top layer is plasma, take out the plasma, and then use a speed of 14000rpm to perform high-speed centrifugation again. The upper layer is plasma; 2) Add the extracted plasma from 1) to a 50ml centrifuge tube that has been added with proteinase K, and then add the lysate ACL (which already contains CarrierRNA) to obtain the first mixed solution; 3) Add the last obtained second mixture in 2) 1. Mix the mixed solution and put it in a water bath at 60°C for 30 minutes; 4) Take out the first mixed solution from the water bath after 3) is completed, add ACB, and set it according to the ratio of 1:1.8, that is, 1ml plasma, 1.8ml ACB, to obtain the first 2 mixed solution; 5) Use the vacuum pump to filter the second mixed solution obtained in 4), and wash it with ACW1, ACW2 and absolute ethanol after the suction filtration; 6) Evacuate at 14000rpm for 3 minutes, evaporate to dryness at 56°C and remove ethanol; 7) eluted with 30 ul of water to obtain cfDNA; 8) methylated the cfDNA obtained in 7) to build a library.
利用全基因组DNA甲基化二代测序仪X-ten，测得样本的全基因组甲基化水平，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，将全部甲基化信息，进行分析对比。Using the whole genome DNA methylation next-generation sequencer X-ten, the whole genome methylation level of the sample is measured, and the methylation level of each site of each sample is generated according to illumina official Methylation Analysis Algorithms, that is, Raw data, Analyze and compare all the methylation information.
实施例3Example 3
明显差异的甲基化位点的筛选Screening of significantly different methylation sites
经过0.1、0.15、0.2、0.25、0.3等不同差异标准的尝试之后，最终确定了明显差异为乳腺癌某个分子分型的样本和其他分子分型样本之间甲基化水平均值的差的绝对值大于等于0.1。这个标准的确立，保证了既能有明显的分类效果，又能保留尽可能多的有效位点以供下一步筛选，参见图1A、1B和1C。After trying different difference standards such as 0.1, 0.15, 0.2, 0.25, 0.3, etc., the absolute difference between the mean difference of methylation level between samples of a certain molecular type of breast cancer and samples of other molecular types was finally determined. The value is greater than or equal to 0.1. The establishment of this standard ensures not only a clear classification effect, but also retaining as many effective sites as possible for the next step of screening, see Figures 1A, 1B and 1C.
实施例4Example 4
利用limma软件包，根据乳腺癌某种分子分型和其他分子分型患者的cfDNA甲基化水平比对，经过0.1、0.5、0.01、0.001等不同差异标准的尝试之后，最终确定了明显差异为p值小于等于0.05。这个标准的确立，保证了既能有明显的分类效果，又能保留尽可能多的有效位点以供下一步筛选。根据筛选后确立的标准绘制了图，参见图2A、2B和2C。Using the limma software package, according to the comparison of the cfDNA methylation level of patients with a certain molecular type of breast cancer and other molecular types, after trying different difference standards such as 0.1, 0.5, 0.01, and 0.001, the obvious difference was finally determined as p-value less than or equal to 0.05. The establishment of this standard ensures not only the obvious classification effect, but also retaining as many effective sites as possible for the next step of screening. Plots were drawn according to criteria established after screening, see Figures 2A, 2B and 2C.
实施例5Example 5
筛选出发生明显变化的位点Screen out sites with significant changes
根据实施例3和实施例4中的结果，筛选出两者的交集即在不同分子分型中发生明显变化的甲基化位点，结果参见图3A、3B和3C。According to the results in Example 3 and Example 4, the intersection of the two, that is, the methylation sites that have changed significantly in different molecular typing, was screened out, and the results are shown in Figures 3A, 3B and 3C.
实施例6Example 6
进一步筛选出作为乳腺癌分子分型诊断标志物的甲基化位点根据实施例5结果，利用 Lasso算法筛选出贡献程度大的位点。经过不同位点数量的尝试之后，最终选定定了LuminalA、 LuminalB、三阴性中贡献程度最大的位点，分别是9，17，15个位点。这样既保证了有明显的分类效果，又能保留尽可能少的有效位点以方便临床快速检验，结果参见图4A、4B和4C。之后我们将各自差异的位点合并，发现有40个位点，可以仅根据这40个位点的甲基化水平对样本进行了聚类,聚类结果符合临床预期。这40个甲基化位点可以作为乳腺癌分子分型诊断的标志物。结果参见图5。Further screening out methylation sites as diagnostic markers for molecular typing of breast cancer According to the results of Example 5, sites with a large contribution were screened out using the Lasso algorithm. After trying different numbers of loci, the loci with the greatest contribution in LuminalA, LuminalB, and triple negative were finally selected, which were 9, 17, and 15 loci, respectively. This not only ensures a clear classification effect, but also retains as few effective sites as possible to facilitate rapid clinical testing. The results are shown in Figures 4A, 4B and 4C. Afterwards, we merged the different sites and found that there were 40 sites, and the samples could be clustered only according to the methylation level of these 40 sites, and the clustering results were in line with clinical expectations. These 40 methylation sites can be used as markers for molecular typing and diagnosis of breast cancer. See Figure 5 for the results.
以上所述仅为本发明的优选实施例而已，并不用于限制本发明，对于本领域的技术人员来说，本发明可以有各种更改和变化。凡在本发明的精神和原则之内，所作的任何修改、等同替换、改进等，均应包含在本发明的保护范围之内。The above descriptions are only preferred embodiments of the present invention, and are not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and changes. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principles of the present invention shall be included within the protection scope of the present invention.
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| CN201810507023.XACN108676879A (en) | 2018-05-24 | 2018-05-24 | Application of specific methylation sites as diagnostic markers for molecular typing of breast cancer |
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| CN201810507023.XACN108676879A (en) | 2018-05-24 | 2018-05-24 | Application of specific methylation sites as diagnostic markers for molecular typing of breast cancer |
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| CN108676879Atrue CN108676879A (en) | 2018-10-19 |
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| CN201810507023.XAPendingCN108676879A (en) | 2018-05-24 | 2018-05-24 | Application of specific methylation sites as diagnostic markers for molecular typing of breast cancer |
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