Discipline of Genetics, School of Life Sciences, University of KwaZulu-Natal, Westville Campus, Durban, South Africa.
Discipline of Genetics, School of Life Sciences, University of KwaZulu-Natal, Westville Campus, Durban, South Africa; South African Sugarcane Research Institute, Durban, South Africa.
Forensic Sci Int Genet. 2020 Nov;49:102392. doi: 10.1016/j.fsigen.2020.102392. Epub 2020 Sep 13.
Differential DNA methylation in human tissues has been widely used to develop markers for body fluid identification in forensics. In the present study, identification of potential tissue specific differentially methylated regions (tDMRs) was based on mining differentially expressed genes in surrogate tissues for blood, saliva, semen and vaginal fluid. Genes specifically over expressed in one of the surrogate tissues viz: blood, salivary glands, testis, prostrate, cervix, uterus and ovary were identified from genome wide expression datasets. We hypothesized that over expression in surrogate tissues for body fluids could be correlated with differential methylation. Methylation information from two methylation datasets, NGSmethDB and ENCODE were integrated and heavily methylated gene body CpG islands (CGI) representing the body fluids were extracted. From a total of 53 potential genes the present study reports, two genes, ZNF282 and HPCAL1 which were preferentially expressed in cervix with comparatively reduced expression in other surrogate tissues. Methylated CGIs were targeted to design primers for methylation specific PCR (MSP) and bisulphite sequencing (BS). The ZNF282 CpG sites displayed semen-specific hypomethylation while HPCAL1 CpGs showed saliva-specific hypomethylation. Clone-based bisulphite sequencing also revealed significant hypomethylation in the target body fluids. To evaluate the stability of methylation profiles, the ZNF282 tDMR was tested and each body fluid was subjected to five different forensic simulated conditions (dry at room temperature, wet in an exicator, outside on the ground, sprayed with alcohol and sprayed with bleach) for 50 days. Under the condition "outside on the ground", saliva showed a significant decrease in methylation level by bisulphite sequencing analysis over time. Complete methylation profiles were obtained only for vaginal fluid under all conditions and no differences in methylation levels were observed for this fluid after 50 days. Thus, ZNF282 and HPCAL1 tDMRs can be used as reliable semen and saliva identification markers respectively.
人体组织中的差异 DNA 甲基化已被广泛用于开发法医体液鉴定的标志物。在本研究中，基于对血液、唾液、精液和阴道液替代组织中差异表达基因的挖掘，鉴定了潜在的组织特异性差异甲基化区域（tDMR）。从全基因组表达数据集鉴定了在一种替代组织中特异性过表达的基因，即血液、唾液腺、睾丸、前列腺、宫颈、子宫和卵巢。我们假设体液替代组织中的过表达可能与差异甲基化相关。从两个甲基化数据集 NGSmethDB 和 ENCODE 中整合甲基化信息，并提取代表体液的重度甲基化基因体 CpG 岛（CGI）。本研究共报告了 53 个潜在基因，其中 2 个基因 ZNF282 和 HPCAL1 在宫颈中优先表达，而在其他替代组织中的表达相对较少。靶向甲基化 CGI 设计用于甲基化特异性 PCR（MSP）和亚硫酸氢盐测序（BS）的引物。ZNF282 的 CpG 位点显示精液特异性低甲基化，而 HPCAL1 的 CpG 显示唾液特异性低甲基化。克隆亚硫酸氢盐测序也揭示了目标体液中的显著低甲基化。为了评估甲基化谱的稳定性，测试了 ZNF282 tDMR，每个体液都经过 50 天的五种不同法医模拟条件（室温干燥、干燥器中湿润、室外地面、喷洒酒精和喷洒漂白剂）处理。在“室外地面”条件下，唾液的甲基化水平随时间推移通过亚硫酸氢盐测序分析显示出显著下降。仅在所有条件下获得阴道液的完整甲基化谱，并且在 50 天后未观察到该液体的甲基化水平差异。因此，ZNF282 和 HPCAL1 tDMR 可分别用作可靠的精液和唾液鉴定标志物。
