CN101153336B - 检测dna甲基化程度的方法和试剂盒 - Google Patents
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Abstract
本发明涉及检测或监测包含至少两个甲基化敏感限制酶识别位点的靶DNA序列的甲基化程度的方法，该方法包括以识别所述甲基化敏感限制酶识别位点的一种或多种甲基化敏感限制酶消化含有所述靶DNA序列的样品；以及定量消化后样品中残留的靶DNA序列，其中得到的靶DNA序列的水平表明甲基化程度。相应地，本发明还提供了检测、预后或监测个体中癌症的试剂盒。
Description
技术领域
本发明涉及检测DNA序列甲基化程度的方法，以及利用DNA序列的甲基化程度来检测、预后或监测个体中癌症的试剂盒。
发明背景
已熟知肿瘤抑制基因在肿瘤细胞中被甲基化。因而，甲基化标志物已被建议用于检测或监测患者癌症。已有多种方法被提议用于检测这些甲基化的序列。
甲基化特异PCR(MSP)是检测甲基化或未甲基化的DNA最常用的方法。MSP涉及亚硫酸氢盐转化的步骤。用亚硫酸氢钠使胞嘧啶脱氨基成为尿嘧啶，而5-甲基胞嘧啶保持完好。甲基化特异PCR使用靶向亚硫酸氢盐诱导的序列改变的PCR引物，以特异性扩增甲基化的或未甲基化的等位基因。亚硫酸氢盐转化破坏约95％的DNA。由于血清或血浆中DNA的浓度通常很低，DNA减少95％导致检出率小于50％。
其它方法使用特异消化甲基化或未甲基化DNA的限制性内切酶。特异消化甲基化DNA的酶很少。但是，特异消化未甲基化DNA的酶很容易得到。检测方法然后确立消化是否发生，并且因此允许检测所关注DNA是否甲基化或未甲基化(这取决于所用酶的特异性)，其与癌症相关或不相关。
以前已提出过甲基化敏感的酶消化。例如，Silva et al，BritishJournal of Cancer，80：1262-1264，1999进行了甲基化敏感的酶消化及之后的PCR。但是，如作者Yegnasubramanian et al，Nucleic AcidsResearch，Vol.34，No.3，2006e19所指出的，这些方法被产生的假阳性数所困扰。
本发明寻求提供甲基化敏感的检测的更好方法，其排除或减少假阳性和/或假阴性。
发明内容
根据本发明的第一方面，提供了检测或监测包含至少两个甲基化敏感限制酶识别位点的靶DNA序列的甲基化程度的方法，所述方法包括：
(a)以识别所述甲基化敏感限制酶识别位点的一种或多种甲基化敏感限制酶消化含有所述靶DNA序列的样品；以及
(b)定量消化后所述样品中残留的所述靶DNA序列，
其中步骤(b)中得到的所述靶DNA序列的水平表明所述甲基化程度。
本发明的方法利用甲基化敏感限制酶在多个位点切割靶DNA序列，大大提高了检测的准确性。
相应地，本发明还提供了使用选自来自个体的血液、血浆、血清、唾液、尿的生物样品检测或监测癌症的方法，所述方法包括：
(a)从所述生物样品获得DNA；
(b)使用一种或多种甲基化敏感限制酶消化该DNA样品；
(c)在步骤(b)之后量化或检测靶DNA序列，其中靶DNA序列含有至少两个甲基化敏感限制酶的识别位点；以及
(d)将来自该个体的该DNA序列的水平与正常标准比较，以检测、预测或监测癌症。
在本发明的优选实施方案中，将聚合酶链式反应(PCR)或实时定量聚合酶链式反应(Q-PCR)用于定量靶DNA序列。优选地，该甲基化敏感限制酶识别未甲基化的DNA序列。该靶序列为癌症患者的易被甲基化的序列，因此正常患者的未甲基化的靶序列被消化，不被聚合酶链式反应扩增，而在癌症患者中，靶序列被甲基化并且不被酶消化，随后可被例如使用聚合酶链式反应量化或检测。
本发明的方法可用于预测个体的癌症易感性、评估个体中癌症的阶段、预测个体整体存活的可能性、预测个体的复发的可能性或评估个体中治疗的有效性。
根据本发明的第二方面，提供了检测、预后或监测个体中癌症的试剂盒，包括
(i)至少一种甲基化敏感限制酶；以及
(ii)定量来自所述个体的样品中靶DNA序列的单元，
其中所述靶DNA序列在癌症中表现出异常甲基化模式，并且含有所述甲基化敏感限制酶能识别的至少两个甲基化敏感限制酶识别位点。
在所述癌症为肝细胞癌的本发明优选实施方案中，所述试剂盒优选还包括在样品中定量甲胎蛋白的单元。在所述癌症为鼻咽癌的本发明优选实施方案中，所述试剂盒优选还包括在样品中定量EBV DNA的单元。
附图简要说明
图1显示患者血浆中甲基化的RASSF1A的浓度。
图2显示手术切除肝细胞癌(HCC)后血浆甲基化RASSF1A水平的改变。
图3显示HCC患者中AFP和甲基化的RASSF1A序列的循环浓度之间没有相关性。
图4显示HBV携带者中AFP和甲基化的RASSF1A序列的循环浓度之间没有相关性。
图5显示血浆甲基化RASSF1A的定量分析可以补充用于HCC检测的AFP分析。
图6显示手术切除前血浆中甲基化RASSF1A的浓度预测手术切除后患者的存活。
图7显示手术切除后血浆中甲基化RASSF1A的浓度的减小。
图8显示鼻咽癌(NPC)患者血浆中检测到的甲基化RASSF1A的浓度与Epstein-Barr病毒(EBV)的DNA浓度相关。
图9显示RASSF1A基因的启动子和第一个外显子的基因组序列。甲基化敏感限制酶BstUI的识别序列用下划线标出，并且PCR检测的靶序列用粗体突出。靶序列中有5个BstUI酶限制位点。
具体实施方式
本发明提供评估、诊断、预测或监测个体中肿瘤的存在或进展的方法。该方法涉及使用甲基化敏感限制酶消化DNA序列。选择含有至少两个限制位点的靶DNA序列，这两个限制位点可以是甲基化的或未甲基化的。优选使用特定的甲基化敏感限制酶实施本方法，与甲基化序列相比，该特定的甲基化敏感限制酶优先切割未甲基化的序列。甲基化序列保持未被消化并被检测到。在未甲基化序列的至少一个甲基化敏感限制酶位点的消化导致该靶序列不能被检测到或不可扩增。因此甲基化序列可与未甲基化序列区分开。本发明的一个实施方案中，在诸如癌症患者的血浆或血清的生物样品中检测到的未被切割的靶序列的数量高于来自于健康的或没有癌症的个体的同类型生物样品中证实的数量，这是因为与健康个体相比，癌症患者中该靶序列高度甲基化。
或者，可以使用切割甲基化DNA的限制酶。未甲基化的DNA序列未被消化并且可被检测到。本发明的另一实施方案中，与来自于没有癌症的个体的同类型生物样品中证实的数量相比，在诸如癌症患者的血浆或血清的生物样品中检测到较低数量的未被切割的靶序列。
本发明的优选实施方案中，通过PCR扩增检测靶序列。可以使用实时定量PCR。选择特定的引物序列，使得使用该引物扩增的序列中存在至少两个甲基化敏感限制酶位点。本发明的方法不使用亚硫酸氢钠。
使用合适方法(如PCR)的扩增被用于检测未被切割的靶序列，由此确定未被限制酶切割的甲基化DNA的存在。
根据本发明，可以使用任何适合的甲基化敏感限制酶。下表I列出了切割未甲基化的DNA的甲基化敏感限制酶的例子。
表I：甲基化敏感限制酶的例子
| 酶 | 识别序列 | CpG甲基化对酶限制性的影响* |
| AatII | GACGTC | 阻止 |
| AjiI | CACGTC | 阻止 |
| BstUI，Bsh1236I | CGCG | 阻止 |
| Bsh1285I | CGRYCG | 阻止 |
| BshTI | ACCGGT | 阻止 |
| Bsp68I | TCGCGA | 阻止 |
| Bsp119I | TTCGAA | 阻止 |
| Bsp143II | RGCGCY | 阻止 |
| Bsu15I | ATCGAT | 阻止 |
| CseI | GACGC | 阻止 |
| Cfr10I | RCCGGY | 阻止 |
| Cfr42I | CCGCGG | 阻止 |
| CpoI | CGGWCCG | 阻止 |
| Eco47III | AGCGCT | 阻止 |
| Eco52I | CGGCCG | 阻止 |
| Eco72I | CACGTG | 阻止 |
| Eco105I | TACGTA | 阻止 |
| EheI | GGCGCC | 阻止 |
| Esp3I | CGTCTC | 阻止 |
| FspAI | RTGCGCAY | 阻止 |
| HhaI；Hin6I | GCGC | 阻止 |
| Hin1I | GRCGYC | 阻止 |
| HpaII | CCGG | 阻止 |
| Kpn2I | TCCGGA | 阻止 |
| MluI | ACGCGT | 阻止 |
| NotI | GCGGCCGC | 阻止 |
| NsbI | TGCGCA | 阻止 |
| PauI | GCGCGC | 阻止 |
| PdiI | GCCGGC | 阻止 |
| Pfl23II | CGTACG | 阻止 |
| Ppu21I | YACGTR | 阻止 |
| Psp1406I | AACGTT | 阻止 |
| PvuI | CGATCG | 阻止 |
| SalI | GTCGAC | 阻止 |
| SgsI | GGCGCGCC | 阻止 |
| SmaI | CCCGGG | 阻止 |
| SmuI | CCCGC | 阻止 |
| SsiI | CCGC | 阻止 |
| TaiI | ACGT | 阻止 |
| TauI | GCSGC | 阻止 |
识别序列中的字母代码代表不同的核苷酸组合，总结如下：R＝G或A；Y＝C或T；W＝A或T；M＝A或C；K＝G或T；S＝C或G；H＝A、C或T；V＝A、C或G；B＝C、G或T；D＝A、G或T；N＝G、A、T或C。
识别序列中的CpG二核苷酸用下划线标出。这些CpG二核苷酸的胞嘧啶残基易被甲基化。*识别序列中的CpG二核苷酸的胞嘧啶的甲基化防止该靶序列的酶切割。
所述靶序列包含两个或多个甲基化敏感限制酶位点。这些位点可以被相同或不同的酶识别。但是，如此选择这些位点，以使得当使用与甲基化序列相比优先切割未甲基化的序列的酶时，每个序列中至少两个位点当其未甲基化时被消化。
在不太优选的实施方案中，该靶序列含有至少两个被优先切割甲基化序列的限制酶切割的位点。该两个或多个位点可以被相同或不同的酶切割。
根据本发明，可以使用任何适合的DNA甲基化标志物。这些DNA甲基化标志物是所选定序列与正常个体相比在癌症患者中显示不同甲基化模式的标志物。选择适当的标志物使得待扩增序列含有至少两个甲基化敏感限制酶位点。通常这些甲基化标志物是这样的基因，即其启动子和/或编码序列在癌症患者中被甲基化。优选地，该选定序列在非癌的(non-cancer)或没有癌症的(cancer-free)个体中未甲基化或以较低程度甲基化。
适合的标志物包括例如RASSF1A。实际上，已证明RASSF1A用于检测或监测个体癌症时特别有效。因此，根据本发明的另一方面，提供了使用选自来自个体的血液、血浆、血清、唾液、尿的生物样品检测或监测癌症的方法，所述方法包括：
(a)从所述生物样品获得DNA；
(b)使用一种或多种甲基化敏感限制酶消化该DNA样品；
(c)在步骤(b)之后，量化或检测靶DNA序列，其中该靶DNA序列是包含部分或全部RASSF1A的序列；以及
(d)将来自该个体的该DNA序列的水平与正常标准比较，以检测、预测或监测癌症。
下表II列出了与RASSF1A启动子超甲基化相关的肿瘤类型。
表II：不同癌症类型中RASSF1A启动子超甲基化的频率
(按频率降序排列)
| 癌症类型 | RASSF1A启动子超甲基化的频率 |
| 肝 | 93％-100％ |
| 乳腺 | 49％-95％ |
| 肺(小细胞) | 79％-88％ |
| *** | 71％-83％ |
| 黑素瘤， | 41％-75％ |
| 胰腺 | 64％ |
| 肾 | 36％-64％ |
| 膀胱 | 47％-60％ |
| 结肠 | 12％-45％ |
| 卵巢 | 30％-40％ |
| 肺(非小细胞) | 28％-40％ |
表III列出了其它优选的在肿瘤中表现超甲基化的肿瘤抑制基因。
表III：癌症中其启动子区域经常被甲基化失活的肿瘤抑制基因的例子
| 肿瘤抑制基因 | 其中基因可能被异常甲基化的癌症类型 |
| APC | 结肠直肠、乳腺、头和颈、食道、膀胱、***、胃、肺、肾 |
| DAP-kinase | 胰腺、胃、肺、结肠直肠、乳腺、子宫颈、鼻咽 |
| E-cadherin | 乳腺、肺、胃、结肠直肠、***、膀胱、子宫颈、肾 |
| GSTPI | 肺、胃、膀胱、***、乳腺、子宫颈 |
| hMLH1 | 胃、结肠直肠、子宫颈、肝、食道、肺、卵巢、*** |
| MGMT | 肺、结肠直肠、膀胱、子宫颈、乳腺、食道、***、鼻咽、肾 |
| NORE1A | 肾、肺、乳腺、结肠 |
| p14 | 结肠直肠、膀胱、鼻咽、肾、胃、乳腺 |
| p15 | 膀胱、鼻咽、肾、多发性骨髓瘤、结肠直肠、肺、卵巢、胃胃 |
| P16INK4a | 肺、胃、膀胱、子宫颈、鼻咽、乳腺、***、肾、肝、结肠直肠、胰腺、白血病、多发性骨髓瘤、甲状腺 |
| RARbeta | 肺、乳腺、鼻咽、***、肾、胃 |
| SOCS1 | 结肠直肠、白血病、胃、卵巢、肝、胰腺 |
| Rb | 眼癌、肺、食道、胃 |
| VHL | 肾 |
依照本发明的方法，从患者取得或获得样品。适合的样品包括血液、血浆、血清、唾液和尿。依照本发明使用的样品包括全血、血浆或血清。从全血中制备血清或血浆的方法为本领域技术人员所熟知。例如，血液可被置于含有EDTA或诸如Vacutainer SST(BectonDickenson，Franklin Lake，NJ，USA)的专门市售产品的试管中以防止血液凝结，随后可以通过离心从全血中获得血浆。血液凝结后可通过离心或不通过离心获得血清。如果使用离心，通常但不专有地以适当的速度进行，如1500-3000x g。再被转移到新试管进行DNA提取之前，可对血浆或血清进行更多的离心步骤。
优选地，使用合适的DNA提取技术从样品中提取DNA。对于本领域技术人员来说，DNA提取是常规内容。已知从生物样品(包括血液)中提取DNA的许多方法。可遵循DNA制备的一般方法，例如Sambrook and Russell，Molecular Cloning a Laboratory Manual(分子克隆：实验室手册)，3rdEdition(2001)所描述的。还可以使用各种可购得的试剂或试剂盒从血液样品中获得DNA。
依照本发明，使含有DNA的样品与一种或多种在特定条件下优先切割未甲基化的DNA的限制酶共同孵育，该特定条件为当靶序列中存在两个或更多处于未甲基化状态的限制酶位点时，所述限制酶可以在至少一个这样的位点处切割靶序列。依照本发明的另一方面，含有DNA的样品与一种或多种在特定条件下只切割甲基化DNA的限制酶共同孵育，该特定条件为当靶序列中存在两个或更多处于甲基化状态的限制酶位点时，该限制酶可以在至少一个这样的位点处切割靶序列。
优选地，样品在允许完全消化的条件下孵育。这可以通过例如增加孵育时间和/或增加酶的用量来达到。通常，样品与100活性单位的甲基化敏感限制酶一起孵育多至16小时。对于本领域技术人员来说，根据所用酶的量建立合适的条件是常规内容。
孵育后，检测未切的靶序列。优选地，通过例如聚合酶链式反应(PCR)扩增来检测这些序列。
设计DNA引物以扩增含有至少两个甲基化敏感限制酶位点的序列。可以通过考察DNA甲基化标志物并且确定这些标志物中被甲基化敏感限制酶识别的限制酶位点来确定这样的序列。例如使用表I中确定的甲基化敏感限制酶的识别序列，可以在表III所列的甲基化标志物中确定合适靶序列。
使用RASSF1A作为例子，靶序列可以包含RASSF1A基因的部分或全部启动子序列和/或外显子1。图9显示该启动子和外显子1的序列(SEQ ID NO.1)。优选实施方案中，该用于检测的靶序列在图9中用粗体突出。
当使用甲基化敏感限制酶时，根据具体个体中该靶序列的甲基化状态，会检测到不同量的该靶序列。在本发明的优选实施方案中，使用优先切割未甲基化DNA的甲基化敏感限制酶，则该靶序列不会在未甲基化状态时(如在健康个体中)被检测到。但是，如果该靶序列被甲基化(如在来自癌症患者的选定样品中)，该靶序列不被限制酶切割，因此该靶序列可以通过PCR检测到。
因此，该方法可用于确定靶序列的甲基化状态，并且提供该个体的癌症状态的指示。
本发明的方法可能另外包括量化或检测对照序列。选择在癌症中不显示异常甲基化模式的序列作为对照序列。依照本发明的优选方面，选择含有至少两个甲基化敏感限制酶识别位点的对照序列。优选地，选择含有与靶DNA序列相同数目的甲基化敏感限制酶识别位点的对照序列。通常，通过扩增检测该对照序列的存在或缺失，该扩增通过甲基化敏感限制酶消化后的聚合酶链式反应完成。这类对照序列可用于评估一种或多种甲基化敏感限制酶的消化程度。例如，如果甲基化敏感限制酶消化后可检测到对照序列，其表明消化不完全并且可以重复该方法以确保发生完全消化。优选地，选择含有与靶序列中相同的甲基化敏感限制酶识别位点的对照序列。优选实施方案中，该对照序列是β肌动蛋白基因序列。
本方法可用于评估个体的肿瘤状态。该方法可用于例如癌症的诊断和/或预后。该方法还可用于例如治疗过程中癌症进程的监测。该方法还可用于监测随时间发生的甲基化水平的变化，例如评估个体对肿瘤的易感性和疾病的进程。该方法还可用于预测疾病的后果或预测治疗成功的可能性。
在本发明的优选方面，该靶序列是RASSF1A并且被用于癌症的诊断。例如，RASSF1A甲基化可用于检测和监测肝细胞癌或鼻咽癌。该方法在监测乙型肝炎携带者或丙型肝炎携带者向肝细胞癌转化的易感性中特别有用。
在本发明的另一方面，还提供了检测、预后或监测个体中癌症的试剂盒。该试剂盒包括至少一种甲基化敏感限制酶；以及用于定量来自该个体的样品中靶DNA序列的单元，其中该靶DNA序列在癌症中表现出异常甲基化模式，并且含有所述甲基化敏感限制酶能识别的至少两个甲基化敏感限制酶识别位点。
在优选的实施方案中，靶DNA序列至少是RASSF1A的一部分，所采用的甲基化敏感限制性酶为BstUI。本发明的试剂盒还优选包含用于检测样品中对照DNA序列(如β肌动蛋白基因序列)的单元。所述的定量靶DNA序列或对照DNA序列的单元一般包括用于PCR或Q-PCR的试剂，例如引物、DNA聚合酶、核苷酸、缓冲液等，用于Q-PCR的试剂还包括探针，优选荧光团标记的探针。
在本发明的优选实施方案中用于定量RASSF1A序列的PCR或Q-PCR所用引物为SEQ ID NO.2和SEQ ID NO.3，Q-PCR所用的探针包含SEQ ID NO.4的序列；用于定量β肌动蛋白基因序列的PCR或Q-PCR所用引物为SEQ ID NO.5和SEQ ID NO.6，Q-PCR所用的探针包含SEQ ID NO.7的序列。
对于用于肝细胞癌(HCC)的试剂盒，本发明优选还包括检测甲胎蛋白的单元。甲胎蛋白是已知的肝细胞癌(HCC)的标志物，其检测方法和所用试剂为本领域技术人员所熟知。对于用于鼻咽癌(APC)的试剂盒，本发明优选还包括检测EBV DNA的单元。EBV DNA是已知的鼻咽癌标志物，其检测方法和所用试剂也为本领域技术人员所熟知。将靶DNA序列的甲基化程度检测和特定的标志物的检测进行结合，可大大提高检测的准确性。
以下将利用下列实施例更详细地描述本发明。
实施例
征募了63个肝细胞癌(HCC)患者以及两组对照。第一对照组由15个没有慢性乙型肝炎感染的健康对照个体组成。第二对照组由慢性乙型肝炎携带者组成。对于每一个HCC患者，征募一个性别和年龄匹配的慢性乙型肝炎携带者作为对照。慢性乙型肝炎携带者具有发展HCC的更高危险，并且因此成为HCC筛选的靶群体。从每一个研究个体收集4毫升静脉血到含有EDTA的试管中。1,600g离心血液样品10分钟，并且再次以16,000g离心上清液10分钟。使用QIAamp mini kit(Qiagen，Hilden，Germany)从800μL血浆中提取DNA并且用50μLH2O洗脱。用100U的BstUI酶消化35微升血浆DNA，消化在1X消化缓冲液中在60℃下进行16小时。
通过实时PCR确定血浆RASSF1A序列的浓度，该实时PCR使用引物5’AGCCTGAGCTCATTGAGCTG3’(SEQIDNO.2)和5’ACCAGCTGCCGTGTGG3’(SEQIDNO.3)，和探针5’(FAM)-CCAACGCGCTGCGCAT-(MGB)3’(SEQ ID NO.4)。每一反应含有1X TaqMan Universal PCR Master Mix(Applied Biosystems，FosterCity’USA)、引物各300nM和85nM探针。7.15微升酶消化的血浆DNA混合物(相当于5μL未消化的血浆DNA)用作每一个PCR反应的模板。温度模式设定为50℃2分钟、95℃10分钟、50个循环的95℃15秒和60℃1分钟。所有的反应以两个重复进行并取平均值。RASSF1A扩增子包含5个BstUI识别位点。
为确保限制酶消化的完全性，对每一个消化的样品进行靶向β-肌动蛋白基因的实时PCR，该实时PCR使用引物5’GCGCCGTTCCGAAAGTT3’(SEQIDNO.5)和5’CGGCGGATCGGCAAA3’(SEQIDNO.6)以及探针5’(FAM)-ACCGCCGAGACCGCGTC-(MGB)3’(SEQ ID NO.7)。通过亚硫酸氢盐测序，显示β-肌动蛋白基因启动子在血细胞和HCC组织中完全未甲基化。β肌动蛋白基因序列扩增子与RASSF1A扩增子有相似大小，并且含有相同数目的BstUI酶限制位点。
为研究未甲基化的RASSF1A和β肌动蛋白基因序列的酶消化效率是否相似，用100U的BstUI酶消化1μg等份的血沉棕黄层(buffycoat)，消化进行不同的时长(15分钟、30分钟、45分钟、60分钟、90分钟、120分钟、3小时、4小时、5小时、6小时、12小时和16小时)。酶消化后测量每一个样品中RASSF1A和β肌动蛋白基因序列的浓度。如图1所示，这些血沉棕黄层DNA中RASSF1A和β肌动蛋白基因序列的浓度显示正相关(r＝0.986，P<0.0001，Pearson相关)。由于未甲基化的RASSF1A和β肌动蛋白基因序列的酶消化效率相似，所以β肌动蛋白基因序列在经消化的血清DNA样品中的缺失应反映未甲基化的RASSF1A序列的完全消化。因此，所有β肌动蛋白基因序列信号为阳性的样品都经进一步的酶消化直到在经消化的血清DNA样品中检测不到β肌动蛋白基因序列。
BstUI酶消化后，在63个HCC患者中的59个(93％)和63个匹配的慢性乙型肝炎携带者中的37个(58％)的血浆中检测到RASSF1A序列。图2显示了这些结果。HCC患者和慢性肝炎B携带者的中位数血浆RASSF1A浓度分别为770拷贝/mL和118拷贝/mL。相反，在15个健康对象中任何一个的血浆中都检测不到RASSF1A。
不被消化的RASSF1A的血浆浓度没有显示与甲胎蛋白(AFP)水平的任何显著线性相关，其中甲胎蛋白为已建立的HCC的肿瘤标志物。图3和图4显示了这些结果。在这点上，我们已经证明血浆甲基化RASSF1A的定量分析可以补充用于HCC检测的AFP分析。图5显示了这些结果。在我们的实验组中，62个患者中的16个有小于10μg/L的低水平AFP。以300拷贝/mL为界，血浆RASSF1A分析鉴定了这16个有假阴性AFP结果的HCC患者中的13个(81％)。
图6证明了外科手术前血浆RASSF1A浓度小于550拷贝/mL的HCC患者的存活可能性好于那些水平高于550拷贝/mL的患者(p＝0.0359，Kaplan-Meier存活分析)。
从手术切除肿瘤后1个月的HCC患者收集血液样品。肿瘤切除前就有可检测到的血浆RASSF1A的59个患者中，45人(76％)显示手术后浓度减小。图7显示了这些结果。这45个患者中，13个在切除肿瘤后检测不到RASSF1A。中位数RASSF1A浓度从770拷贝/mL下降到250拷贝/mL(p<0.0001，Wilcoxon测验)。
为进一步研究甲基化敏感限制酶消化后血浆RASSF1A的定量分析是否是具有RASSF1A异常甲基化的癌症的通用标志物，征募了67个鼻咽癌(NPC)患者研究抗酶消化的RASSF1A的血浆浓度与Epstein-Barr病毒(EBV)DNA的相关性。血浆EBV DNA是NPC的已建立标志物并且已被证实反映肿瘤量(Lo YMD，Chan LY，Lo KW，Leung SF，Zhang J，Chan AT，Lee JC，Hjelm NM，Johnson PJ，Huang DP，Quantitative analysis of cell-free Epstein-Barr virus DNA in plasma ofpatients with nasopharyngeal carcinoma(鼻咽癌患者血浆中无细胞的Epstein-Barr病毒的定量分析).Cancer Res1999；59:1188-91)。图8显示了这些结果。EBV DNA和抗酶消化的RASSF1A序列可分别在65(94％)和39(58％)个患者的血浆中检测到。可检测到抗酶消化的RASSF1A和EBV DNA的患者中，这两个DNA序列的血浆浓度显示正相关(r＝0.38，p＝0.019，Spearman相关)。
甲基化敏感限制酶消化后，分析来自这67个NPC患者的29个肿瘤样品的RASSF1A。酶消化后，RASSF1A序列可在这29个病例中的25个中检测到(86％)。对于这25个RASSF1A基因在肿瘤中超甲基化的病例，酶消化后，RASSF1A序列可在20个病例(80％)的血浆中检测到。该检测率显著好于之前的研究所报告的情况，该研究使用甲基化特异PCR检测血浆中超甲基化的RASSF1A序列(Wong TS，Kwong DL，ShamJS，Wei WI，Kwong YL，Yuen AP，Quantita tiveplasmahypermethylated DN Amarkers of undifferentiated nasopharyngealcarcinoma(无差异的鼻咽癌的定量血浆超甲基化DNA标志物)，ClinCancer Res.2004；10:2401-6)。在该报告中，甲基化的RASSF1A只在5％的NPC患者的血浆中检测到。
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序列表
<110>香港中文大学
<120>检测DNA甲基化程度的方法和试剂盒
<130>06F1150-WDZ
<140>US60/847，499
<141>2006-09-27
<160>7
<170>Patent In version3.3
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<211>1381
<212>DNA
<213>Homo sapiens
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<210>2
<211>20
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<213>用于RASSF1A的PCR引物
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<211>16
<212>DNA
<213>用于RASSF1A的PCR引物
<400>3
<210>4
<211>16
<212>DNA
<213>用于RASSF1A的Q-PCR探针
<400>4
<210>5
<211>17
<212>DNA
<213>用于β-肌动蛋白基因的PCR引物
<400>5
<210>6
<211>15
<212>DNA
<213>用于β-肌动蛋白基因的PCR引物
<400>6
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<213>用于β-肌动蛋白基因的Q-PCR探针
<400>7
Claims (9)
1.检测、预后或监测个体癌症的试剂盒，包括
(i)BstUI酶；以及
(ii)定量来自所述个体的样品中RASSF1A外显子1或其片段的单元，包括用于聚合酶链式反应或实时定量聚合酶链式反应的试剂，所述试剂包括引物SEQ ID NO.2和SEQ ID NO.3，其中所述RASSF1A外显子1或其片段序列在癌症中表现出异常甲基化模式，并且含有所述BstUI酶能识别的至少两个甲基化敏感限制酶识别位点，
(iii)定量作为对照DNA序列的β肌动蛋白基因序列至少一部分的单元，包括用于聚合酶链式反应或实时定量聚合酶链式反应的引物SEQ ID NO.5和SEQ ID NO.6，所述对照DNA序列不在所述癌症中表现出异常甲基化模式。
2.如权利要求1所述的试剂盒，其中用于定量RASSF1A外显子1或其片段的实时定量聚合酶链式反应的试剂包括探针SEQ ID NO.4，定量β肌动蛋白基因序列至少一部分的单元包括用于实时定量聚合酶链式反应的探针SEQ ID NO.7。
3.如权利要求1所述的试剂盒，其中所述试剂盒还包括用于定量所述样品中甲胎蛋白的单元。
4.如权利要求1所述的试剂盒，其中所述试剂盒还包括用于定量所述样品中EBV DNA的单元。
5.如权利要求1所述的试剂盒，其中在所述对照DNA序列中存在所述至少两个甲基化敏感限制酶识别位点。
6.如权利要求5所述的试剂盒，其中相同数量的甲基化限制酶识别位点存在于所述对照DNA序列和所述RASSF1A外显子1或其片段中。
7.如权利要求1所述的试剂盒，其中所述癌症为肝细胞癌或鼻咽癌。
8.如权利要求1所述的试剂盒，其中所述个体为乙型肝炎或丙型肝炎携带者。
9.如权利要求1所述的试剂盒，其中所述样品为血液、血浆、血清、唾液或尿。
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2007
- 2007-04-23 CN CN2007100969763A patent/CN101153336B/zh active Active
- 2007-04-23 TW TW096114234A patent/TWI335354B/zh active
- 2007-09-26 WO PCT/GB2007/003674 patent/WO2008038000A1/en active Application Filing
- 2007-09-26 US US11/861,809 patent/US9371566B2/en active Active
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2008
- 2008-05-05 HK HK08104936.1A patent/HK1115167A1/xx unknown
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2014
- 2014-05-22 US US14/284,724 patent/US20140272975A1/en not_active Abandoned
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2016
- 2016-05-23 US US15/162,258 patent/US10435754B2/en active Active - Reinstated
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2019
- 2019-08-29 US US16/555,609 patent/US11898208B2/en active Active
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Also Published As
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|---|---|
| US10435754B2 (en) | 2019-10-08 |
| WO2008038000A8 (en) | 2009-07-16 |
| TWI335354B (en) | 2011-01-01 |
| US20080081338A1 (en) | 2008-04-03 |
| US20200071771A1 (en) | 2020-03-05 |
| US20170101685A1 (en) | 2017-04-13 |
| US9371566B2 (en) | 2016-06-21 |
| US11898208B2 (en) | 2024-02-13 |
| CN101153336A (zh) | 2008-04-02 |
| US20140272975A1 (en) | 2014-09-18 |
| TW200815755A (en) | 2008-04-01 |
| WO2008038000A1 (en) | 2008-04-03 |
| HK1115167A1 (en) | 2008-11-21 |
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