21三体综合征21qCpG岛DNA甲基化水平研究
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摘要
实验目的:21三体综合征是由于21号染色体不分离所导致的一种染色体病,表现为严重的智力发育障碍和多发性先天畸形,是临床最常见的严重出生缺陷之一,由于该病缺乏有效的治疗手段,给社会、家庭带来异常沉重的负担,因而进行孕早期筛查和产前染色体诊断是降低其发生率的有效途径。但目前仍未见21三体患者CpG岛DNA甲基化状态的相关报道,因此,研究21三体患者CpG岛甲基化状态,这对探索更有效、更特异的用于21三体产前风险评估的表观遗传学分子靶点具有重要的科学意义和临床实践价值。
实验方法:运用HpaII–McrBC PCR(HM-PCR)方法筛检分析21qCpG岛甲基化状态。首先从UCSC数据库获得21qCpG岛DNA序列,并与华大基因炎黄数据库比对,验证序列和引物。然后建立HM-PCR检测方法,并利用HM—PCR技术筛查和分析了150例21三体患者21q上2690个CpG岛,并将患者CpG岛甲基化状态与正常个体进行比较。
实验结果:本研究首先验证了149个CpG岛的DNA序列和148对CpG岛引物的准确性,明确序列和引物在中国汉族人群运用的可行性。建立了简化的HM—PCR方法。HM—PCR筛查发现21三体患者21号染色体长臂的CpG岛中, 98个无甲基化,30个完全甲基化,12个杂合甲基化,8个不完全甲基化,在健康个体21号染色体长臂的148个CpG岛中也得到完全相同结果。有4个杂合甲基化CpG岛(编号:11,15,27,71)与日本人存在差异,日本人群呈现出无甲基化状态;有1个杂合甲基化CpG岛(编号:93)与日本人不一致,日本人群呈现出完全甲基化状态。这种差异是否是种族之间的差异,尚需对这5个CpG岛进行重亚硫酸盐测序确认。第103号CpG岛没有HpaII和HhaI酶切位点,因此,需要利用重亚硫酸盐测序检测。
实验结论:21三体患者与健康个体之间在21qCpG岛上不存在DNA甲基化状态的差异;21qCpG岛DNA甲基化状态在同一种族内高度一致,那么21qCpG岛DNA甲基化状态可否为种族间识别的表观遗传学标记,尚需重亚硫酸盐测序法予以证实。
Background: Trisomy 21 is a chromosomal condition caused by the presence of all or part of an extra 21st chromosome. Often Trisomy 21 is associated with some impairment of cognitive ability and physical growth, and is one of the high incidence birth defect. Individuals with Trisomy 21 are incurable, so prenatal screens for Trisomy 21 are likely lead to reductions in overall social welfare burden. Although some scientists reported a few epigenetic markers for the prenatal screen of Trisomy 21, there is still short of the research about the DNA methylation status of CpG islands in 21q. Therefore exploring the DNA methylation status of CpG islands in 21q is essential for developing a series of specific and effective epigenetic marker for the prenatal screen of Trisomy 21.
Method: First DNA sequences of CpG islands in 21q from USCS database were achieved, and 149 sequences and 148 pairs of primers in BGI YH database were aligned. Then the HM-PCR assay was improved, to determine the DNA methylation status of CpG islands in patients with Trisomy 21. Finally there were totally 2690 CpG island analyzed by HM-PCR assay, and a comparison of the DNA methylation status of CpG islands were made between the control and patients with Trisomy 21
Results: According to the results of alignment in BGI YH database, 149 DNA sequences of CpG islands in 21q and 148 pairs of primers for 148 CpG islands are feasible to be used in HM-PCR. HM-PCR assay developed by Yamada were further improved to a simple assay by optimizing experimental condition. Using the improved HM-PCR assay, the results showed that there was no difference in the DNA methylation status of 21q CpG islands between patients with Trisomy 21 and the control. Totally 148 CpG islands in 21q were screened, including 98 null methylation, 30 complete methylation, 12 composite methylation and 8 incomplete methylation. However, 4 null methylation CpG islands (No. 11, 15, 27, 71) and 1complete methylation (No.93) in Japanese were composite methylation in Chinese. The 5 CpG islands were different in DNA methylation level between Japanese and Chinese. Whether the difference is due to the race difference or not, the bisulfite sequencing will be used to detect the DNA methylation status of the 5 CpG islands. Another one CpG island (No.103) will be detected by bisulfite sequencing, because the DNA sequence is no HpaII and HhaI recognition site.
Conclusion: There is no difference in the DNA methylation status of 21q CpG islands between patients with Trisomy 21 and the control. The hypothesis that the homogeneity of DNA methylation status of 21q CpG islands in Chinese indicates that DNA methylation is a epigenetic marker to distinguish the different race, is need to be verified by bisulfite sequencing to detect the DNA methylation status.
实验方法:运用HpaII–McrBC PCR(HM-PCR)方法筛检分析21qCpG岛甲基化状态。首先从UCSC数据库获得21qCpG岛DNA序列,并与华大基因炎黄数据库比对,验证序列和引物。然后建立HM-PCR检测方法,并利用HM—PCR技术筛查和分析了150例21三体患者21q上2690个CpG岛,并将患者CpG岛甲基化状态与正常个体进行比较。
实验结果:本研究首先验证了149个CpG岛的DNA序列和148对CpG岛引物的准确性,明确序列和引物在中国汉族人群运用的可行性。建立了简化的HM—PCR方法。HM—PCR筛查发现21三体患者21号染色体长臂的CpG岛中, 98个无甲基化,30个完全甲基化,12个杂合甲基化,8个不完全甲基化,在健康个体21号染色体长臂的148个CpG岛中也得到完全相同结果。有4个杂合甲基化CpG岛(编号:11,15,27,71)与日本人存在差异,日本人群呈现出无甲基化状态;有1个杂合甲基化CpG岛(编号:93)与日本人不一致,日本人群呈现出完全甲基化状态。这种差异是否是种族之间的差异,尚需对这5个CpG岛进行重亚硫酸盐测序确认。第103号CpG岛没有HpaII和HhaI酶切位点,因此,需要利用重亚硫酸盐测序检测。
实验结论:21三体患者与健康个体之间在21qCpG岛上不存在DNA甲基化状态的差异;21qCpG岛DNA甲基化状态在同一种族内高度一致,那么21qCpG岛DNA甲基化状态可否为种族间识别的表观遗传学标记,尚需重亚硫酸盐测序法予以证实。
Background: Trisomy 21 is a chromosomal condition caused by the presence of all or part of an extra 21st chromosome. Often Trisomy 21 is associated with some impairment of cognitive ability and physical growth, and is one of the high incidence birth defect. Individuals with Trisomy 21 are incurable, so prenatal screens for Trisomy 21 are likely lead to reductions in overall social welfare burden. Although some scientists reported a few epigenetic markers for the prenatal screen of Trisomy 21, there is still short of the research about the DNA methylation status of CpG islands in 21q. Therefore exploring the DNA methylation status of CpG islands in 21q is essential for developing a series of specific and effective epigenetic marker for the prenatal screen of Trisomy 21.
Method: First DNA sequences of CpG islands in 21q from USCS database were achieved, and 149 sequences and 148 pairs of primers in BGI YH database were aligned. Then the HM-PCR assay was improved, to determine the DNA methylation status of CpG islands in patients with Trisomy 21. Finally there were totally 2690 CpG island analyzed by HM-PCR assay, and a comparison of the DNA methylation status of CpG islands were made between the control and patients with Trisomy 21
Results: According to the results of alignment in BGI YH database, 149 DNA sequences of CpG islands in 21q and 148 pairs of primers for 148 CpG islands are feasible to be used in HM-PCR. HM-PCR assay developed by Yamada were further improved to a simple assay by optimizing experimental condition. Using the improved HM-PCR assay, the results showed that there was no difference in the DNA methylation status of 21q CpG islands between patients with Trisomy 21 and the control. Totally 148 CpG islands in 21q were screened, including 98 null methylation, 30 complete methylation, 12 composite methylation and 8 incomplete methylation. However, 4 null methylation CpG islands (No. 11, 15, 27, 71) and 1complete methylation (No.93) in Japanese were composite methylation in Chinese. The 5 CpG islands were different in DNA methylation level between Japanese and Chinese. Whether the difference is due to the race difference or not, the bisulfite sequencing will be used to detect the DNA methylation status of the 5 CpG islands. Another one CpG island (No.103) will be detected by bisulfite sequencing, because the DNA sequence is no HpaII and HhaI recognition site.
Conclusion: There is no difference in the DNA methylation status of 21q CpG islands between patients with Trisomy 21 and the control. The hypothesis that the homogeneity of DNA methylation status of 21q CpG islands in Chinese indicates that DNA methylation is a epigenetic marker to distinguish the different race, is need to be verified by bisulfite sequencing to detect the DNA methylation status.
引文
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[1] Wolffe AP, Matzke MA.Epigenetics:regulation through repression [J].Science,1999,286(5439):481～486.
[2]吴南,桂建芳。组蛋白变体及组蛋白替换[J].遗传. 2006, 28(4): 493～500.Bird A.Perceptions of epigenetics[J]. Nature, 2007, 447(7143): 396～398.
[3] JonesPA,Baylin SB.The fundamental role of epigenetic events in cancer[J].Nat Rev Genet, 2002,3(6):415～442.
[4]董玉玮,侯进慧,朱必才,李培青,庞永红。表观遗传学的相关概念和研究进展[J]。生物学杂志,2005, 22(1): 1～3
[5] Devinoy E,Rijnkels M. Epigenetics in mammary gland biology and cancer [J].J Mammary Gland Biol Nenplasia. 2010, 15(1): 1～4.
[6] Sharrard RM,Royds JA,Rogers S,et a1.Patterns of methylation of the c-myc gene in human coloreetal cancer progression [J]. Br J Cancer,1992, 65(5): 667～672.
[7] Old RW, Crea F, Puszyk W, Hultén MA. Candidate epigenetic biomarkers for non-invasive prenatal diagnosis of Down syndrome [J]. Reprod Biomed Online. 2007, 15(2): 227~235.
[8] Bird A P. CpG-rich islands and the function of DNA methylation [J]. Nature, 1986, 321: 209~213.
[9] Riggs A D, Jones P D. 5-methylcytosine, gene regulation and cancer [J]. Adv Cancer Res, 1984, 40: 1~30.
[10] Gardiner-Garden M, Frommer M.CpG islands in vertebrate genomes [J]. J Mol Biol. 1987,196(2):261~282.
[11] Cottrell SE. Molecular diagnostic applications of DNA methylation technology [J]. Clin Biochem. 2004, 37(7): 595~604.
[12] Jones PA, Baylin SB. The fundamental role of epigenetic events in cancer [J]. Nat Rev Genet. 2002, 3(6): 415~428.
[13] Daura-Oller E, Cabre M, Montero MA, Paternain JL, Romeu A.Specific gene hypomethylation and cancer: New insights into coding region feature trends [J]. Bioinformation. 2009. 3 (8): 340~343.
[14] Adkins RM, Krushkal J, Tylavsky FA, Thomas F. Racial differences in gene-specific DNA methylation levels are present at birth [J]. Birth Defects Res A Clin Mol Teratol. 2011,doi: 10.1002/bdra.20770.
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[18] Kwabi-Addo B, Wang S, Chung W, Jelinek J, Patierno SR, Wang BD, Andrawis R, Lee NH, Apprey V, Issa JP, Ittmann M.Identification of differentially methylated genes in normal prostate tissues from African American and Caucasian men [J]. Clin Cancer Res. 2010, 16(14): 3539~3547.
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[2]吴南,桂建芳。组蛋白变体及组蛋白替换[J].遗传. 2006, 28(4): 493～500.Bird A.Perceptions of epigenetics[J]. Nature, 2007, 447(7143): 396～398.
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[7] Old RW, Crea F, Puszyk W, Hultén MA. Candidate epigenetic biomarkers for non-invasive prenatal diagnosis of Down syndrome [J]. Reprod Biomed Online. 2007, 15(2): 227~235.
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[22] Powell MA, Mutch DG, Rader JS, Herzog TJ, Huang TH, Goodfellow PJ. Ribosomal DNA methylation in patients with endometrial carcinoma: an independent prognostic marker [J]. Cancer. 2002; 94(11):2941~2952.
[23] Christensen BC, Kelsey KT, Zheng S, Houseman EA, Marsit CJ, Wrensch MR, Wiemels JL, Nelson HH, Karagas MR, Kushi LH, Kwan ML, Wiencke JK.Breast cancer DNA methylation profiles are associated with tumor size and alcohol and folate intake [J]. PLoS Genet. 2010, 6(7):e1001043.
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