DNA甲基化作为法医学组织标记的可行性研究
摘要
背景鉴别在犯罪现场发现的斑痕等人体检材的组织来源,对判断案件类型和证明犯罪事实有重要的作用。当代法庭科学对微量、陈旧生物证据的应用,使组织鉴别变得更为必要,同时在技术是也提出了更高的要求。探索新型的分子组织标记,研究开发与当代DNA分析技术相匹配的组织鉴别技术,是目前法医物证学的研究热点之一。
目的本研究调查人体主要组织胶质纤维酸性蛋白(Glial fibrillary acidic protein,GFAP)基因和DEAD盒多肽4(DEAD (Asp-Glu-Ala-Asp) box polypeptide 4,DDX4)基因的启动子甲基化水平,评估GFAP启动子低甲基化作为脑组织、DDX4启动子低甲基化作为精液斑组织标记的可行性,初步探讨DNA甲基化作为法医组织标记的意义。
方法应用联合亚硫酸氢盐的限制酶法(Combined Bisulfite Restriction Analysis,COBRA),一种半定量的DNA甲基化检测技术,调查6例尸体主要组织(大脑、心脏、肺、肝、胰、脾、肾、皮肤)和17例精液的GFAP和DDX4启动子甲基化水平,对数据进行单因素方差分析并确定判别值。
结果人类大脑组织GFAP启动子甲基化水平均低于45.82%((X|￣)_(brain)=44.70%,S_(brain)=8.38%),非脑组织均高于58.60%((X|￣)_(non-brain)=68.01%, S_(non-brain)=1.42%),以52.87%为判别值,可以有效的区别脑组织与非脑组织。人精液DDX4启动子甲基化水平均低于50.41 % ((X|￣)sperm=11.52%_(EcoRI), 11.88%_(TaqI) ; S_(sperm)=8.98%_(EcoRI), 10.25%_(TaqI)) ,体细胞组织均高于75.41%((X|￣)_(somatic)=82.73%_(EcoRI), 82.94%_(TaqI);S_(somatic)=3.54%_(EcoRI), 3.91%_(TaqI)),以49.85%(EcoRI位点)或50.58%(TaqI位点)为判别值,可以有效的区别精液斑组织与非精液斑组织。
结论大脑组织的GFAP启动子甲基化水平显著低与非脑组织,精液DDX4启动子甲基化水平显著低与非精液的体细胞组织,GFAP启动子低甲基化可作为脑组织特异标记,DDX4启动子低甲基化可作为精液特异标记。考虑到法医学检材的特殊性和DNA标记的优势,组织特异性甲基化变异位点(methylation-variable positions,MVPs)可能是一种理想的分子组织标记。筛选一组合适的组织特异性MVPs位点,开发通用的基于DNA甲基化的组织ID系统,可能是解决目前法医组织鉴定问题的有效途径。
Background and Objective The identification of tissue origins can be of crucial importance in forensic casework. Method for tiny and aged tissues discrimination remains a challenge due to many cross reaction existing in immunodetection and unstable mRNA. DNA methylation is an epigenetic mark on the mammalian genome. There are numerous tissue-dependent and differentially methylated regions (T-DMRs) in the unique sequences distributed throughout the genome. Recently study had shown that the detection tissue specific DNA methylation is a suitable technique to identify neuronal tissue in raw meat products which is prohibited by BSE (Bovine Spongiform Encephalopathy) legislation. Cell/tissue-specific expressions of various genes had been explained by the binding of cell/tissue specific transcription factors to their promoters. Taken together, in order to support a model in which the promoter regions special methylate pattern of gene are accessible for discriminate one tissue from the other. To study the feasibility of DNA methylation as forensic tissue markers, the promoter of human GFAP and DDX4 gene were selected as samples.
Method Methylation levels of GFAP and DDX4 promoter of human tissue samples including brain, heart, lungs, liver, pancreas, spleen, kidney, skin, and semen(n=6) were detected by the combined bisulfite restriction enzyme analysis (COBRA), a semiquantitative DNA methylation analytical method. One-way variance of data was carried out and discrimate values were defined.
Result The results showed that the methylation level of GFAP promotor in all huamn brain were lower than 45.82%((X|￣)_(brain)=44.70%, S_(brain)=8.38%), while that in all non-brain human tissues were larger than 58.60%((X|￣)_(non-brain)=68.01%, S_(non-brain)=1.42%),; When the discriminant value was set at 52.87%, brain and non-brain huamn tissues can be discriminated effectively. The methylation level of DDX4 promotor in all semen were lower than 50.41 % ((X|￣)_(sperm)=11.52%_(EcoRI), 11.88%_(TaqI) ; S_(sperm)=8.98%_(EcoRI), 10.25%_(TaqI)), while that in all somatic tissues were larger than 75.41 % ((X|￣)_(somatic)=82.73%_(EcoRI), 82.94%_(TaqI) ; S_(somatic)=3.54%_(EcoRI), 3.91%_(TaqI)). When the discriminant value was set at 49.85%(EcoRIsite)or 50.58%(TaqI site), semen and non- semen huamn tissues can be discriminated effectively.
Conclusion Methylation level of GFAP promoter in brain tissue(including gray and white matter) was significantly lower than that in non-brain tissues, DNA methylation level of GFAP promoter can be used as a brain tissue-specific markers.
Methylation level of DDX4 promoter in semen was significantly lower than that in somatic tissue, DNA methylation level of DDX4 promoter can be used as a semen-specific markers.
As a proof of concept study, the sample size, methylation marker selected and techniques used here may not be suitable for the forensic practice. However, considering the characteristics of forensic samples and the advantages of DNA markers, tissue specific methylation-variable positions (MVPs) may be ideal tissue makers. Screening for different methylation regions (DMR) among tissues, developing a universally adapted DNA methylation-based tissue ID system may be a solution to the present problems in forensic tissue identification.
目的本研究调查人体主要组织胶质纤维酸性蛋白(Glial fibrillary acidic protein,GFAP)基因和DEAD盒多肽4(DEAD (Asp-Glu-Ala-Asp) box polypeptide 4,DDX4)基因的启动子甲基化水平,评估GFAP启动子低甲基化作为脑组织、DDX4启动子低甲基化作为精液斑组织标记的可行性,初步探讨DNA甲基化作为法医组织标记的意义。
方法应用联合亚硫酸氢盐的限制酶法(Combined Bisulfite Restriction Analysis,COBRA),一种半定量的DNA甲基化检测技术,调查6例尸体主要组织(大脑、心脏、肺、肝、胰、脾、肾、皮肤)和17例精液的GFAP和DDX4启动子甲基化水平,对数据进行单因素方差分析并确定判别值。
结果人类大脑组织GFAP启动子甲基化水平均低于45.82%((X|￣)_(brain)=44.70%,S_(brain)=8.38%),非脑组织均高于58.60%((X|￣)_(non-brain)=68.01%, S_(non-brain)=1.42%),以52.87%为判别值,可以有效的区别脑组织与非脑组织。人精液DDX4启动子甲基化水平均低于50.41 % ((X|￣)sperm=11.52%_(EcoRI), 11.88%_(TaqI) ; S_(sperm)=8.98%_(EcoRI), 10.25%_(TaqI)) ,体细胞组织均高于75.41%((X|￣)_(somatic)=82.73%_(EcoRI), 82.94%_(TaqI);S_(somatic)=3.54%_(EcoRI), 3.91%_(TaqI)),以49.85%(EcoRI位点)或50.58%(TaqI位点)为判别值,可以有效的区别精液斑组织与非精液斑组织。
结论大脑组织的GFAP启动子甲基化水平显著低与非脑组织,精液DDX4启动子甲基化水平显著低与非精液的体细胞组织,GFAP启动子低甲基化可作为脑组织特异标记,DDX4启动子低甲基化可作为精液特异标记。考虑到法医学检材的特殊性和DNA标记的优势,组织特异性甲基化变异位点(methylation-variable positions,MVPs)可能是一种理想的分子组织标记。筛选一组合适的组织特异性MVPs位点,开发通用的基于DNA甲基化的组织ID系统,可能是解决目前法医组织鉴定问题的有效途径。
Background and Objective The identification of tissue origins can be of crucial importance in forensic casework. Method for tiny and aged tissues discrimination remains a challenge due to many cross reaction existing in immunodetection and unstable mRNA. DNA methylation is an epigenetic mark on the mammalian genome. There are numerous tissue-dependent and differentially methylated regions (T-DMRs) in the unique sequences distributed throughout the genome. Recently study had shown that the detection tissue specific DNA methylation is a suitable technique to identify neuronal tissue in raw meat products which is prohibited by BSE (Bovine Spongiform Encephalopathy) legislation. Cell/tissue-specific expressions of various genes had been explained by the binding of cell/tissue specific transcription factors to their promoters. Taken together, in order to support a model in which the promoter regions special methylate pattern of gene are accessible for discriminate one tissue from the other. To study the feasibility of DNA methylation as forensic tissue markers, the promoter of human GFAP and DDX4 gene were selected as samples.
Method Methylation levels of GFAP and DDX4 promoter of human tissue samples including brain, heart, lungs, liver, pancreas, spleen, kidney, skin, and semen(n=6) were detected by the combined bisulfite restriction enzyme analysis (COBRA), a semiquantitative DNA methylation analytical method. One-way variance of data was carried out and discrimate values were defined.
Result The results showed that the methylation level of GFAP promotor in all huamn brain were lower than 45.82%((X|￣)_(brain)=44.70%, S_(brain)=8.38%), while that in all non-brain human tissues were larger than 58.60%((X|￣)_(non-brain)=68.01%, S_(non-brain)=1.42%),; When the discriminant value was set at 52.87%, brain and non-brain huamn tissues can be discriminated effectively. The methylation level of DDX4 promotor in all semen were lower than 50.41 % ((X|￣)_(sperm)=11.52%_(EcoRI), 11.88%_(TaqI) ; S_(sperm)=8.98%_(EcoRI), 10.25%_(TaqI)), while that in all somatic tissues were larger than 75.41 % ((X|￣)_(somatic)=82.73%_(EcoRI), 82.94%_(TaqI) ; S_(somatic)=3.54%_(EcoRI), 3.91%_(TaqI)). When the discriminant value was set at 49.85%(EcoRIsite)or 50.58%(TaqI site), semen and non- semen huamn tissues can be discriminated effectively.
Conclusion Methylation level of GFAP promoter in brain tissue(including gray and white matter) was significantly lower than that in non-brain tissues, DNA methylation level of GFAP promoter can be used as a brain tissue-specific markers.
Methylation level of DDX4 promoter in semen was significantly lower than that in somatic tissue, DNA methylation level of DDX4 promoter can be used as a semen-specific markers.
As a proof of concept study, the sample size, methylation marker selected and techniques used here may not be suitable for the forensic practice. However, considering the characteristics of forensic samples and the advantages of DNA markers, tissue specific methylation-variable positions (MVPs) may be ideal tissue makers. Screening for different methylation regions (DMR) among tissues, developing a universally adapted DNA methylation-based tissue ID system may be a solution to the present problems in forensic tissue identification.
引文
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