遗传性非息肉病性结直肠癌中错配修复基因甲基化的研究
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摘要
背景遗传性非息肉病性结直肠癌(hereditary nonpolyposis colorectal cancer, HNPCC)是一种常染色体显性遗传性疾病,由错配修复(mismatch repair, MMR)基因种系突变引起,占所有结直肠癌的5%-10%。HNPCC最有效的治疗手段是内镜下或外科手术切除肿瘤,如果能早期发现原发和复发肿瘤,并及时治疗,可以明显改善预后。MMR基因种系突变的检测已经成为HNPCC确诊的金标准,但是较多的研究提示部分家系(包括一些高度微卫星不稳定肿瘤家系)始终找不到已知MMR基因种系突变。有研究表明错配修复基因启动子区CpG岛的过度甲基化也是导致基因失活、肿瘤发生的一种机制。迄今,有关HNPCC患者基因组DNA中MLH1、MSH2过甲基化检测报道较少,检测MMR基因的甲基化可能为HNPCC的筛选和监测提供一种手段,并为HNPCC的诊断、治疗提供一个新的思路。
目的了解国人遗传性非息肉病性结直肠癌中错配修复基因MLH1、MSH2启动子区CpG岛的过甲基化状态,探讨MLH1、MSH2基因DNA甲基化与遗传性非息肉病性结直肠癌发生的关系,从而为HNPCC的筛选和监测提供一种手段,并为HNPCC的诊断、治疗提供一个新的思路。
材料与方法
研究对象我科收集的106个HNPPC家系中先证者,经微小突变检测和大片断缺失检测后,以未发现种系突变的47个HNPCC患者标本为研究对象。后期,又增加符合阿姆斯特丹标准Ⅱ(AC-Ⅱ)的同一家系标本4个,这些患者先期已证实有MSH2基因突变。
方法
1)基因组DNA提取:按试剂盒提供的步骤进行操作,所提取的基因组DNA进行琼脂糖凝胶电泳检测,使用紫外分光光度计进行定量后于-20℃保存。
2)基因组DNA的硫化处理:按试剂盒提供的步骤进行操作,对至少1μg的基因组DNA进行转化,处理后的DNA于-20℃保存。
3)用MSP法检测MLH1、MSH2基因启动子区的甲基化状态:同一标本以甲基化引物和非甲基化引物分别进行PCR扩增。PCR产物在2%的琼脂糖凝胶上电泳。紫外灯下观察目的条带。判定标准:若扩增出M(methylation)的特异性条带,则说明有甲基化:仅有M条带为完全甲基化,同时有M条带和U(unmethylation)条带为部分甲基化;若仅扩增出U的特异性条带,则说明无甲基化。
结果(1)在47个标本中,发现6个存在MLH1基因启动子区过甲基化,发生率为12.8%。分析MSP电泳图可见,存在MLH1基因启动子区甲基化的6个标本包括:4个完全甲基化,2个部分甲基化;(2)在36个标本中,发现27个存在MSH2基因启动子甲基化,发生率75.0%,其余9个(25.0%)为非甲基化。由电泳图可见,存在MSH2基因启动子甲基化的27个标本只扩增出特异性条带M,表现为完全甲基化,其余标本仅扩增出特异性条带U,表示这9个标本除未检出微小突变和大片段缺失外,也未发现有甲基化现象;(3)后加入4个标本(同属一家系,且已证实有MSH2基因突变),由电泳图可见,存在MSH2基因启动子完全甲基化。
结论(1)在遗传性非息肉病性结直肠癌中,MSH2基因启动子区的CpG岛有高频率的甲基化,是肿瘤发生过程中的一个重要因素;而MLH1基因启动子区甲基化在遗传性非息肉病性结直肠癌中相对少见;(2)在实验中发现,DNA甲基化主要发生在AC-II家系中,提示基因过甲基化可能有遗传的特性,即表遗传学机制可能具备遗传性;(3)在其中一个家系中,MSH2种系突变合并MSH2基因启动子CpG岛过度甲基化现象,除证实基因甲基化状态和种系突变一样具备遗传特性外,也提示二者对肿瘤的发生可能有协同作用。
Background Hereditary nonpolyposis colorectal cancer is an autosomal dominant genetic disease, caused by mismatch repair gene mutations, and accounts for about 5% -10% of all colorectal cancers. The most effective treatment of HNPCC is endoscopic and surgical removal of the tumor. If the primary and recurrent tumor can be detected earlier and radical surgery can be carried out, which can significantly improve the prognosis. MMR gene mutation of the detection has become the gold standard HNPCC confirmed, but the some researchers found that known species of MMR gene mutation can’t be found in some families(including some microsatellite instability-high tumor family), and further study showed that mismatch repair gene promoter CpG island methylation is the the cause of gene inactivation and tumor. So far, genomic DNA in patients with HNPCC, reports about the methylation of MLH1, MSH2 are less. Detection of genomic DNA methylation may be HNPCC screening and monitoring to provide a means for HNPCC and the diagnosis, treatment, to provide a new way of thinking.
Objective To study the methylationg status of CpG island on promoter region mismatch repair gene MLH1 and MSH2 in Chinese in hereditary nonpolyposis colorectal cancer, and to explore the relations between MLH1, MSH2 promoter region of the CpG island methylation status of mismatch repair gene inactivation and hereditary non-polyposis colorectal cancer occurrence and development of the relations, So as to HNPCC screening and monitoring to provide a means for HNPCC and the diagnosis, treatment, to provide a new way of thinking.
Materials and Methods
Specimens
* Surpported by the Nature Science Foundation of Beijing(NO.7062064)
47 samples without micromutation and large fragment deletion are from 106 samples which have been confirmed HNPCC. Later, four cases from the same family( Amsterdam Criteria II) were added, and MSH2 gene mutation has been confirmed.
Methods
1)DNA extraction: follow step description of the kit, and the extraction of genomic DNA was detected by agarose gel electrophoresis, the concentration of the DNA was assayed using spectrophotometry, then stored at -20℃.
2)Genomic DNA sulfurize treatment: follow step description of the kit, at least 1μg genomic DNA was transformed, then stored at -20℃.
3)MSP: PCR amplifications were carried out in the same sample, using methylation primers and non-methylation primers respectively. PCR products were detected on the 2% agarose gel electrophoresis, observe the strap under UV lamps. Criteria: the specific fragment M illustrates methylation: complete methylation (fragment M), partial methylation(fragment M and U); non-methylation (fragment U).
Results
1) In 47 examples, hypermethylation of MLH1 promoter was detected in 6 examples(12.8%), by analyzing the electropherogram of methylation, 4 examples were completely methylated, and 2 examples were partial methylated;
2)In 36 examples, hypermethylation of MSH2 promoter was detected in 27 examples(75.0%), and all of them were complete methylation; the remain (25.0%) were non-methylation.
3) Complete methylation of MSH2 promoter was detected in the four augmented examples.
Conclusions
(1)In HNPCC, there is a high frequency of methylation in MSH2 gene promoter region, and it’s an important factor in the course of development associated with HNPCC. MLH1 gene promoter methylation is relative rare;
(2)In the experiment, DNA methylation occurred mainly in the AC-II family, and DNA methylation of HNPCC may shows genetic characteristics;
(3)Methylation was detected in a family which has confirmed MSH2 gene mutation, it is confirmed that gene methylation has inheritance as genetic mutation. Both may play synergies to the incidence of HNPCC.
目的了解国人遗传性非息肉病性结直肠癌中错配修复基因MLH1、MSH2启动子区CpG岛的过甲基化状态,探讨MLH1、MSH2基因DNA甲基化与遗传性非息肉病性结直肠癌发生的关系,从而为HNPCC的筛选和监测提供一种手段,并为HNPCC的诊断、治疗提供一个新的思路。
材料与方法
研究对象我科收集的106个HNPPC家系中先证者,经微小突变检测和大片断缺失检测后,以未发现种系突变的47个HNPCC患者标本为研究对象。后期,又增加符合阿姆斯特丹标准Ⅱ(AC-Ⅱ)的同一家系标本4个,这些患者先期已证实有MSH2基因突变。
方法
1)基因组DNA提取:按试剂盒提供的步骤进行操作,所提取的基因组DNA进行琼脂糖凝胶电泳检测,使用紫外分光光度计进行定量后于-20℃保存。
2)基因组DNA的硫化处理:按试剂盒提供的步骤进行操作,对至少1μg的基因组DNA进行转化,处理后的DNA于-20℃保存。
3)用MSP法检测MLH1、MSH2基因启动子区的甲基化状态:同一标本以甲基化引物和非甲基化引物分别进行PCR扩增。PCR产物在2%的琼脂糖凝胶上电泳。紫外灯下观察目的条带。判定标准:若扩增出M(methylation)的特异性条带,则说明有甲基化:仅有M条带为完全甲基化,同时有M条带和U(unmethylation)条带为部分甲基化;若仅扩增出U的特异性条带,则说明无甲基化。
结果(1)在47个标本中,发现6个存在MLH1基因启动子区过甲基化,发生率为12.8%。分析MSP电泳图可见,存在MLH1基因启动子区甲基化的6个标本包括:4个完全甲基化,2个部分甲基化;(2)在36个标本中,发现27个存在MSH2基因启动子甲基化,发生率75.0%,其余9个(25.0%)为非甲基化。由电泳图可见,存在MSH2基因启动子甲基化的27个标本只扩增出特异性条带M,表现为完全甲基化,其余标本仅扩增出特异性条带U,表示这9个标本除未检出微小突变和大片段缺失外,也未发现有甲基化现象;(3)后加入4个标本(同属一家系,且已证实有MSH2基因突变),由电泳图可见,存在MSH2基因启动子完全甲基化。
结论(1)在遗传性非息肉病性结直肠癌中,MSH2基因启动子区的CpG岛有高频率的甲基化,是肿瘤发生过程中的一个重要因素;而MLH1基因启动子区甲基化在遗传性非息肉病性结直肠癌中相对少见;(2)在实验中发现,DNA甲基化主要发生在AC-II家系中,提示基因过甲基化可能有遗传的特性,即表遗传学机制可能具备遗传性;(3)在其中一个家系中,MSH2种系突变合并MSH2基因启动子CpG岛过度甲基化现象,除证实基因甲基化状态和种系突变一样具备遗传特性外,也提示二者对肿瘤的发生可能有协同作用。
Background Hereditary nonpolyposis colorectal cancer is an autosomal dominant genetic disease, caused by mismatch repair gene mutations, and accounts for about 5% -10% of all colorectal cancers. The most effective treatment of HNPCC is endoscopic and surgical removal of the tumor. If the primary and recurrent tumor can be detected earlier and radical surgery can be carried out, which can significantly improve the prognosis. MMR gene mutation of the detection has become the gold standard HNPCC confirmed, but the some researchers found that known species of MMR gene mutation can’t be found in some families(including some microsatellite instability-high tumor family), and further study showed that mismatch repair gene promoter CpG island methylation is the the cause of gene inactivation and tumor. So far, genomic DNA in patients with HNPCC, reports about the methylation of MLH1, MSH2 are less. Detection of genomic DNA methylation may be HNPCC screening and monitoring to provide a means for HNPCC and the diagnosis, treatment, to provide a new way of thinking.
Objective To study the methylationg status of CpG island on promoter region mismatch repair gene MLH1 and MSH2 in Chinese in hereditary nonpolyposis colorectal cancer, and to explore the relations between MLH1, MSH2 promoter region of the CpG island methylation status of mismatch repair gene inactivation and hereditary non-polyposis colorectal cancer occurrence and development of the relations, So as to HNPCC screening and monitoring to provide a means for HNPCC and the diagnosis, treatment, to provide a new way of thinking.
Materials and Methods
Specimens
* Surpported by the Nature Science Foundation of Beijing(NO.7062064)
47 samples without micromutation and large fragment deletion are from 106 samples which have been confirmed HNPCC. Later, four cases from the same family( Amsterdam Criteria II) were added, and MSH2 gene mutation has been confirmed.
Methods
1)DNA extraction: follow step description of the kit, and the extraction of genomic DNA was detected by agarose gel electrophoresis, the concentration of the DNA was assayed using spectrophotometry, then stored at -20℃.
2)Genomic DNA sulfurize treatment: follow step description of the kit, at least 1μg genomic DNA was transformed, then stored at -20℃.
3)MSP: PCR amplifications were carried out in the same sample, using methylation primers and non-methylation primers respectively. PCR products were detected on the 2% agarose gel electrophoresis, observe the strap under UV lamps. Criteria: the specific fragment M illustrates methylation: complete methylation (fragment M), partial methylation(fragment M and U); non-methylation (fragment U).
Results
1) In 47 examples, hypermethylation of MLH1 promoter was detected in 6 examples(12.8%), by analyzing the electropherogram of methylation, 4 examples were completely methylated, and 2 examples were partial methylated;
2)In 36 examples, hypermethylation of MSH2 promoter was detected in 27 examples(75.0%), and all of them were complete methylation; the remain (25.0%) were non-methylation.
3) Complete methylation of MSH2 promoter was detected in the four augmented examples.
Conclusions
(1)In HNPCC, there is a high frequency of methylation in MSH2 gene promoter region, and it’s an important factor in the course of development associated with HNPCC. MLH1 gene promoter methylation is relative rare;
(2)In the experiment, DNA methylation occurred mainly in the AC-II family, and DNA methylation of HNPCC may shows genetic characteristics;
(3)Methylation was detected in a family which has confirmed MSH2 gene mutation, it is confirmed that gene methylation has inheritance as genetic mutation. Both may play synergies to the incidence of HNPCC.
引文
1. Lynch HT, Smyr k T. Hereditary nonpolyposis colorectal cancer (Lynch syndrome).An update dreview. Cancer, 1996, 78:1149-1167
2. Wagner A, Tops C, Wijnen J T. Genetic testing in hereditary non-polyposis colorectal cancer families with a MSH2, MLH1,or MSH6 mutation. J Med Genet, 2002, 39(11):833
3. Nicolaides NC, Papadopoulos N, Liu B, et al. Mutations of two PMS homologues in hereditary nonpolyposis colon cancer[J]. Nature, 1994, 371(6492):75-80.
4.张宏盛剑秋耿洪刚等多重连接依赖的探针扩增技术检测中国人遗传非息肉病性大肠癌错配修复基因大片断缺失.中国医学科学院学报,2006,28(6),837-838.
5. Vasen HF, Watson P, Mecklin JP, et al. New clinical criteria for hereditary nonpolyposis colorectal cancer (HNPCC, Lynch syndrome) proposed by the International Collaborative group on HNPCC[J]. Gastroenterology, 1999, 116(6):1453-1456.
6. Baba S. Clinical applications of genetic studies in hereditary colorectal cancer.In: Baba S, ed. New strategies for treatment of hereditary colorectal cancer[M].Tokyo: Churchill Livingston, 1996,17(2):137-152 .
7. Rodriguez-Bigas MA, Boland CR, Hamilton SR, et al. A National Cancer Institute Workshop on Hereditary Nonpolyposis Colorectal Cancer Syndrome: meeting highlights and Bethesda guidelines[J]. J Natl Cancer Inst, 1997, 89(23):1758-1762.
8. Yu Z, Chen J, Ford BN, et al. Human DNA repair systems: An review. Environ Mole Mutagen, 1999, 33:3-20.
9. Fink D, Aebi S, Howell SB: The role of DNA mismatch repair in drug resistance. Clin Cancer Res, 1998, 4: 1-6.
10.金黑鹰崔龙等遗传性非息肉病性大肠癌的诊治进展[J].国外医学(肿瘤学分册),2002,29:387-389.
11. Eshleman JR, Lang EZ, Bowerfind GK, et al. Increased mutation rate at the hprt locus accompanies microsatellite instability in colon cancer. Oncogene, 1995, 10 (1): 33-37.
12. Jones PA, Takai D. The role of DNA methylation in mammalian epigenetics.Science, 2001, 293(5532):1068-1070.
13. Miyakura Y, Sugano K, Konishi F, et al. Methylation profile of the MLH1 promoterregion and their relationship to colorectal carcinogenesis. Genes Chromosomes Cancer, 2003, 36:17-25.
14. Murata H, Khattar NH, Kang Y, et al. Genetic and epigenetic modification of mismatch repair genes hMSH2 and hMLH1 in sporadic breast cancer with microsatellite instability. Oncogene, 2002, 21: 5696-5703.
15. Kane MF, Loda M, Gaida GM, et al. Methylation of the hMLH1 promoter correlates with lack of expression of hMLH1 in sporadic colon tumors and mismatch repair-defective human tumor cell lines. Cancer Res, 1997(57): 808-811.
16. Menigatti M, Di Gregorio C , Borghi F, et al .Methylation pattern of different regions of the MLH1 promoter and silencing of gene expression in hereditary and sporadic colorectal cancer[ J] .Genes Chormosomes Cancer,2001,31(4):357-361.
17. Wheeler JM ,Loukola A , Aaltonen LA, et al .The role of hypermethylation of the hMLH1 pormoter region in HNPCC versus MSI+ sporadic colorectal cancers[J ] . J Med Genet, 2000, 37( 8):588-592.
18. Fleisher AS, Esteller M, Tamura G, et al. Hypermethylation of the hMLH1 gene promoter is associated with microsatellite instability in early human gastric neoplasia[J]. Oncogene,2001,20(3):329-335.
19. Grunau C, Clark S J, Rosenthal A. Bisulfite genomic sequencing: systematic investigation of critical experimental parameters[J]. Nucleic Acids Res, 2001, 29(13):E651-657.
20. Cunningham JM, Christensen ER, Tester DJ, et al. Hypermethylation of the hMLH1 promoter in colon cancer with microsatellite instability[J].Cancer Res,1998,58(15): 3455-3460 .
21. Boland CR, Thibodeau SN, Hamilton SR, et al. A National Cancer Institute Workshop on Microsatellite Instability for cancer detection and familial predisposition: development of international criteria for the determination of microsatellite instability in colorectal cancer[J].Cancer Res,1998;58(22):5248-5257.
22.张翠娟,李晓明,丘礼武,等. DNA错配修复基因甲基化在肝细胞癌发生发展中的作用.中华病理学杂志,2004,33(5):433-436.
23.盛剑秋,陈香宇,孙自勤,等.北方人群HNPCC微卫星不稳定性和错配修复基因突变规律研究.胃肠病学和肝病学杂志,2008,17(4):287-290.
24. Deng G,Chen A,Hong J,et al.Methylation of CpG in a small region of the hMLH1 promoter invariably correlates with the absence of gene expression[J].Cancer Res,1999,59(9):2029-2033 .
25. Arnold C N,Goel A,Boland C R.Role of hMLH1 promoter hypermethylation in drug resistance to 5&fluorouracil in colorectal cancer cell lines[J].Int J Cancer,2003, 106(1):66-73.
26. Tsun L C, Siu T Y. et al, Heritable germline epimutation of MSH2 in a family with hereditary nonpolyposis colorectal cancer[J].Nature Genetics[J], 2000,38(10): 1178-1183.
27. Herman JG, Graff JR, Myohanen S, et al. Methylation-specific PCR: a novel PCR assay for methylation status of CpG islands [J].Proc Natl Acad Sci USA, 1996, 93:9821-9826.
28.张云,刘泽军.DNA甲基化测定法引物的网上设计.中华检验医学杂志[J],2004,27(7);426.
29. Boland CR, Thibodeau SN, Hamilton SR, et al. A National Cancer Institute Workshop on Microsatellite Instability for cancer detection and familial predisposition: development of international criteria for the determination of microsatellite instability in colorectal cancer[J].Cancer Res, 1998;58(22): 5248-5257.
30.白桦,邓大君. CpG岛甲基化检测技术比较[J].国外医学分子生物学分册, 2003, 25(2):121-125 .
31. Schouten JP, McElgunn CJ, Waaijer R, et al. Relative quantification of 40 nucleic acid sequences by multiplex ligation-dependent probe amplification [J]. Nucleic Acids Res, 2002, 30(12):e57.
1. Lynch HT, Smyrk T. Hereditary nonpolyposis colorectal cancer (Lynch syndrome).An updated review[J]. Cancer, 1996, 78(6):1149-1167 .
2. Wagner A, Tops C, Wijnen JT , et al. Genetic testing in hereditary non-polyposis colorectal cancer families with a MSH2, MLH1,or MSH6 mutation[J]. J Med Genet. 2002, 39(11):833-837 .
3. Nicolaides NC, Papadopoulos N, Liu B, et al. Mutations of two PMS homologues in hereditary nonpolyposis colon cancer[J]. Nature,1994, 371(6492):75-80.
4.金黑鹰崔龙等遗传性非息肉病性大肠癌的诊治进展[J].国外医学(肿瘤学分册),2002,29:387-389.
5. Bird A P. The essentials of DNA methylation[J].Cell,1992,70(1):5-8.
6. Asad Umar C, Richard Boland, et al. Revised Bethesda Guidelines for Hereditary Nonpolyposis Colorectal Cancer(Lynch Syndrome) and Microsatellite Instability[J]. Journal of the National Cancer Institute, 2004,96(4):261-268.
7. Lengauer C, Kinzler KW, Vogelstein B. DNA methylation and genetic instability in colorectal cancer cells[J].Proc Natl Acad Sci USA,1997,94:2545-2550.
8.韩英.遗传性大肠癌的临床研究现状及进展[J].临床内科杂志.2007,24(8):519-520.
9. Aaltonen LA, Salovaara R, Kristo P, et al. Incidence of hereditary nonpolyposis colorectal cancer and the feasibility of molecular screening for the disease[J]. N Engl J Med 1998,338(21):1481-1487.
10. Lamberti C, Kruse R, Ruelfs C, et al. Microsatellite instability—A useful diagnostic tool to select patients at high risk for hereditary non-polyposis colorectal cancer:A study in different groups of patients with colorectal cancer[J]. Gut.1999,44(6):839-843.
11. Samowitz WS, Curtin K, Lin HH, et al: The colon cancer burden of genetically defined hereditary nonpolyposis colon cancer[J]. Gastroenterology.2001,121(4):830-838.
12. Salovaara R, Loukola A, Kristo P, et al: Population-based molecular detection of hereditary nonpolyposis colorectal cancer[J]. J Clin Oncol.2000,18(11):2193-2200.
13. Herman J G,Merlo A, Mao L, et al. Inactivation of the CDKN2/p16/MISI gene is frequently associated with aberrant DNA metheylation in all common human cancer[J].Cancer Res,1995,55(20):4525-4530.
14. Gazzoli, I., Loda, M., Garber, J., Syngal, S. & Kolodner, R.D. A hereditary nonpolyposis colorectal carcinoma case associated with hypermethylation of the MLH1 gene in normal tissue and loss of heterozygosity of the unmethylated allele in the resulting microsatellite instability-high tumor[J]. Cancer Res.2002,62(14):3925–3928.
15. Young J, Simms LA, Biden KG.et al. Features of colorectal cancers with high-level microsatellite instability occurring in familial and sporadic settings: parallel pathways of tumorigenesis[J]. Am J Pathol 2001; 159(6):2107-2116.
16. Fleisher AS, Esteller M, Tamura G, et al. Hypermethylation of the hMLH1 gene promoter is associated with microsatellite instability in early human gastric neoplasia[J]. Oncogene,2001,20(3):329-335.
17. Tsun L C, Siu T Y. et al,Heritable germline epimutation of MSH2 in a family with hereditary nonpolyposis colorectal cancer[J]. Nature Genetics[J],2000,38(10): 1178-1183.
18. Toyota M, Itoh F, and Imai K. DNA methylation and gastrointestinal malignances: functional consequences and clinical implications[J].J Gastroenterol, 2000, 35(10):727-734.
19.白桦,邓大君. CpG岛甲基化检测技术比较[J].国外医学分子生物学分册,2003, 25(2):121.
20. Schouten JP, Mcelgunn CJ, Waaijer R, et al. Relative quantification of 40 nucleic acid sequences by multiplex ligation-dependent probe amplification[J]. Nucleic Acids Res,2002,30(12):e57.
21. Wheeler JM. Epigenetics, mismatch repair genes and colorectal cancer[J]. Ann R Coll Surg Engl. 2005;87(1):15-20.
22. Cheung P, Lau P. Epigenetic regulation by histone methylation and histone variants[J]. Mol Endocrinol. 2005;19(3):563-573.
23. Bassal S, El-Osta A. DNA damage detection and repair, and the involvement of epigenetic states[J]. Hum Mutat. 2005;25(2):101-109.
24. Verma M, Maruvada P, Srivastava S. Epigenetics and cancer[J]. Crit Rev Clin Lab Sci. 2004;41(5-6):585-607.
25. Golubovsky M, Manton KG. Three-generation approach in biodemography is based on the developmental profiles and the epigenetics of female gametes[J]. Front Biosci.2005,10(5):187-191.
26. Laird PW. The power and promise of DNA methylation markers[J]. Nat Rev Cancer,2003,3(4):253-266.
27. Whitman SP, Liu S, Vukosavljevic T. et al. The MLL partial tandem duplication: evidence for recessive gain-of-function in acute myeloid leukemia identifies a novel patient subgroup for molecular-targeted therapy.Blood.2005,106(1):345~352.
28. Costello JF, Plass C. Methylation matters[J].J Med Genet,2001,38(5):285-303.
2. Wagner A, Tops C, Wijnen J T. Genetic testing in hereditary non-polyposis colorectal cancer families with a MSH2, MLH1,or MSH6 mutation. J Med Genet, 2002, 39(11):833
3. Nicolaides NC, Papadopoulos N, Liu B, et al. Mutations of two PMS homologues in hereditary nonpolyposis colon cancer[J]. Nature, 1994, 371(6492):75-80.
4.张宏盛剑秋耿洪刚等多重连接依赖的探针扩增技术检测中国人遗传非息肉病性大肠癌错配修复基因大片断缺失.中国医学科学院学报,2006,28(6),837-838.
5. Vasen HF, Watson P, Mecklin JP, et al. New clinical criteria for hereditary nonpolyposis colorectal cancer (HNPCC, Lynch syndrome) proposed by the International Collaborative group on HNPCC[J]. Gastroenterology, 1999, 116(6):1453-1456.
6. Baba S. Clinical applications of genetic studies in hereditary colorectal cancer.In: Baba S, ed. New strategies for treatment of hereditary colorectal cancer[M].Tokyo: Churchill Livingston, 1996,17(2):137-152 .
7. Rodriguez-Bigas MA, Boland CR, Hamilton SR, et al. A National Cancer Institute Workshop on Hereditary Nonpolyposis Colorectal Cancer Syndrome: meeting highlights and Bethesda guidelines[J]. J Natl Cancer Inst, 1997, 89(23):1758-1762.
8. Yu Z, Chen J, Ford BN, et al. Human DNA repair systems: An review. Environ Mole Mutagen, 1999, 33:3-20.
9. Fink D, Aebi S, Howell SB: The role of DNA mismatch repair in drug resistance. Clin Cancer Res, 1998, 4: 1-6.
10.金黑鹰崔龙等遗传性非息肉病性大肠癌的诊治进展[J].国外医学(肿瘤学分册),2002,29:387-389.
11. Eshleman JR, Lang EZ, Bowerfind GK, et al. Increased mutation rate at the hprt locus accompanies microsatellite instability in colon cancer. Oncogene, 1995, 10 (1): 33-37.
12. Jones PA, Takai D. The role of DNA methylation in mammalian epigenetics.Science, 2001, 293(5532):1068-1070.
13. Miyakura Y, Sugano K, Konishi F, et al. Methylation profile of the MLH1 promoterregion and their relationship to colorectal carcinogenesis. Genes Chromosomes Cancer, 2003, 36:17-25.
14. Murata H, Khattar NH, Kang Y, et al. Genetic and epigenetic modification of mismatch repair genes hMSH2 and hMLH1 in sporadic breast cancer with microsatellite instability. Oncogene, 2002, 21: 5696-5703.
15. Kane MF, Loda M, Gaida GM, et al. Methylation of the hMLH1 promoter correlates with lack of expression of hMLH1 in sporadic colon tumors and mismatch repair-defective human tumor cell lines. Cancer Res, 1997(57): 808-811.
16. Menigatti M, Di Gregorio C , Borghi F, et al .Methylation pattern of different regions of the MLH1 promoter and silencing of gene expression in hereditary and sporadic colorectal cancer[ J] .Genes Chormosomes Cancer,2001,31(4):357-361.
17. Wheeler JM ,Loukola A , Aaltonen LA, et al .The role of hypermethylation of the hMLH1 pormoter region in HNPCC versus MSI+ sporadic colorectal cancers[J ] . J Med Genet, 2000, 37( 8):588-592.
18. Fleisher AS, Esteller M, Tamura G, et al. Hypermethylation of the hMLH1 gene promoter is associated with microsatellite instability in early human gastric neoplasia[J]. Oncogene,2001,20(3):329-335.
19. Grunau C, Clark S J, Rosenthal A. Bisulfite genomic sequencing: systematic investigation of critical experimental parameters[J]. Nucleic Acids Res, 2001, 29(13):E651-657.
20. Cunningham JM, Christensen ER, Tester DJ, et al. Hypermethylation of the hMLH1 promoter in colon cancer with microsatellite instability[J].Cancer Res,1998,58(15): 3455-3460 .
21. Boland CR, Thibodeau SN, Hamilton SR, et al. A National Cancer Institute Workshop on Microsatellite Instability for cancer detection and familial predisposition: development of international criteria for the determination of microsatellite instability in colorectal cancer[J].Cancer Res,1998;58(22):5248-5257.
22.张翠娟,李晓明,丘礼武,等. DNA错配修复基因甲基化在肝细胞癌发生发展中的作用.中华病理学杂志,2004,33(5):433-436.
23.盛剑秋,陈香宇,孙自勤,等.北方人群HNPCC微卫星不稳定性和错配修复基因突变规律研究.胃肠病学和肝病学杂志,2008,17(4):287-290.
24. Deng G,Chen A,Hong J,et al.Methylation of CpG in a small region of the hMLH1 promoter invariably correlates with the absence of gene expression[J].Cancer Res,1999,59(9):2029-2033 .
25. Arnold C N,Goel A,Boland C R.Role of hMLH1 promoter hypermethylation in drug resistance to 5&fluorouracil in colorectal cancer cell lines[J].Int J Cancer,2003, 106(1):66-73.
26. Tsun L C, Siu T Y. et al, Heritable germline epimutation of MSH2 in a family with hereditary nonpolyposis colorectal cancer[J].Nature Genetics[J], 2000,38(10): 1178-1183.
27. Herman JG, Graff JR, Myohanen S, et al. Methylation-specific PCR: a novel PCR assay for methylation status of CpG islands [J].Proc Natl Acad Sci USA, 1996, 93:9821-9826.
28.张云,刘泽军.DNA甲基化测定法引物的网上设计.中华检验医学杂志[J],2004,27(7);426.
29. Boland CR, Thibodeau SN, Hamilton SR, et al. A National Cancer Institute Workshop on Microsatellite Instability for cancer detection and familial predisposition: development of international criteria for the determination of microsatellite instability in colorectal cancer[J].Cancer Res, 1998;58(22): 5248-5257.
30.白桦,邓大君. CpG岛甲基化检测技术比较[J].国外医学分子生物学分册, 2003, 25(2):121-125 .
31. Schouten JP, McElgunn CJ, Waaijer R, et al. Relative quantification of 40 nucleic acid sequences by multiplex ligation-dependent probe amplification [J]. Nucleic Acids Res, 2002, 30(12):e57.
1. Lynch HT, Smyrk T. Hereditary nonpolyposis colorectal cancer (Lynch syndrome).An updated review[J]. Cancer, 1996, 78(6):1149-1167 .
2. Wagner A, Tops C, Wijnen JT , et al. Genetic testing in hereditary non-polyposis colorectal cancer families with a MSH2, MLH1,or MSH6 mutation[J]. J Med Genet. 2002, 39(11):833-837 .
3. Nicolaides NC, Papadopoulos N, Liu B, et al. Mutations of two PMS homologues in hereditary nonpolyposis colon cancer[J]. Nature,1994, 371(6492):75-80.
4.金黑鹰崔龙等遗传性非息肉病性大肠癌的诊治进展[J].国外医学(肿瘤学分册),2002,29:387-389.
5. Bird A P. The essentials of DNA methylation[J].Cell,1992,70(1):5-8.
6. Asad Umar C, Richard Boland, et al. Revised Bethesda Guidelines for Hereditary Nonpolyposis Colorectal Cancer(Lynch Syndrome) and Microsatellite Instability[J]. Journal of the National Cancer Institute, 2004,96(4):261-268.
7. Lengauer C, Kinzler KW, Vogelstein B. DNA methylation and genetic instability in colorectal cancer cells[J].Proc Natl Acad Sci USA,1997,94:2545-2550.
8.韩英.遗传性大肠癌的临床研究现状及进展[J].临床内科杂志.2007,24(8):519-520.
9. Aaltonen LA, Salovaara R, Kristo P, et al. Incidence of hereditary nonpolyposis colorectal cancer and the feasibility of molecular screening for the disease[J]. N Engl J Med 1998,338(21):1481-1487.
10. Lamberti C, Kruse R, Ruelfs C, et al. Microsatellite instability—A useful diagnostic tool to select patients at high risk for hereditary non-polyposis colorectal cancer:A study in different groups of patients with colorectal cancer[J]. Gut.1999,44(6):839-843.
11. Samowitz WS, Curtin K, Lin HH, et al: The colon cancer burden of genetically defined hereditary nonpolyposis colon cancer[J]. Gastroenterology.2001,121(4):830-838.
12. Salovaara R, Loukola A, Kristo P, et al: Population-based molecular detection of hereditary nonpolyposis colorectal cancer[J]. J Clin Oncol.2000,18(11):2193-2200.
13. Herman J G,Merlo A, Mao L, et al. Inactivation of the CDKN2/p16/MISI gene is frequently associated with aberrant DNA metheylation in all common human cancer[J].Cancer Res,1995,55(20):4525-4530.
14. Gazzoli, I., Loda, M., Garber, J., Syngal, S. & Kolodner, R.D. A hereditary nonpolyposis colorectal carcinoma case associated with hypermethylation of the MLH1 gene in normal tissue and loss of heterozygosity of the unmethylated allele in the resulting microsatellite instability-high tumor[J]. Cancer Res.2002,62(14):3925–3928.
15. Young J, Simms LA, Biden KG.et al. Features of colorectal cancers with high-level microsatellite instability occurring in familial and sporadic settings: parallel pathways of tumorigenesis[J]. Am J Pathol 2001; 159(6):2107-2116.
16. Fleisher AS, Esteller M, Tamura G, et al. Hypermethylation of the hMLH1 gene promoter is associated with microsatellite instability in early human gastric neoplasia[J]. Oncogene,2001,20(3):329-335.
17. Tsun L C, Siu T Y. et al,Heritable germline epimutation of MSH2 in a family with hereditary nonpolyposis colorectal cancer[J]. Nature Genetics[J],2000,38(10): 1178-1183.
18. Toyota M, Itoh F, and Imai K. DNA methylation and gastrointestinal malignances: functional consequences and clinical implications[J].J Gastroenterol, 2000, 35(10):727-734.
19.白桦,邓大君. CpG岛甲基化检测技术比较[J].国外医学分子生物学分册,2003, 25(2):121.
20. Schouten JP, Mcelgunn CJ, Waaijer R, et al. Relative quantification of 40 nucleic acid sequences by multiplex ligation-dependent probe amplification[J]. Nucleic Acids Res,2002,30(12):e57.
21. Wheeler JM. Epigenetics, mismatch repair genes and colorectal cancer[J]. Ann R Coll Surg Engl. 2005;87(1):15-20.
22. Cheung P, Lau P. Epigenetic regulation by histone methylation and histone variants[J]. Mol Endocrinol. 2005;19(3):563-573.
23. Bassal S, El-Osta A. DNA damage detection and repair, and the involvement of epigenetic states[J]. Hum Mutat. 2005;25(2):101-109.
24. Verma M, Maruvada P, Srivastava S. Epigenetics and cancer[J]. Crit Rev Clin Lab Sci. 2004;41(5-6):585-607.
25. Golubovsky M, Manton KG. Three-generation approach in biodemography is based on the developmental profiles and the epigenetics of female gametes[J]. Front Biosci.2005,10(5):187-191.
26. Laird PW. The power and promise of DNA methylation markers[J]. Nat Rev Cancer,2003,3(4):253-266.
27. Whitman SP, Liu S, Vukosavljevic T. et al. The MLL partial tandem duplication: evidence for recessive gain-of-function in acute myeloid leukemia identifies a novel patient subgroup for molecular-targeted therapy.Blood.2005,106(1):345~352.
28. Costello JF, Plass C. Methylation matters[J].J Med Genet,2001,38(5):285-303.