表观遗传学在法医学应用探讨

表观遗传学指没有DNA序列变化的、可遗传的基因表达改变。主要包括DNA甲基化、X染色体剂量补偿、组蛋白修饰、基因组印记、表观基因组学和人类表观基因组计划等方面的内容。在法医实际检案中常遇到一些特殊问题,如妊娠期胎儿父权的认定,单亲鉴定中亲代必需等位基因的确定,同卵双生子的区分,微量组织来源的鉴定等。本文对表观遗传学的基本内容和在法医学中的应用进行综述,以期为相关研究及实践提供参考。     

DNA甲基化在法医学中的应用前景及其检测方法新进展

DNA甲基化是一种重要的表观遗传标记。新近的一些研究表明,DNA甲基化在二联体亲权鉴定、同卵双生子法医学个体甄别等案例中可能具有一定的应用前景,可作为STR或SNP等经典遗传标记的有益补充。目前基于甲基化敏感的限制性核酸内切酶、重亚硫酸盐转化以及甲基化CpG结合蛋白等原理已建立了一系列的DNA甲基化检测方法。甲基化敏感的单核苷酸引物延伸、实时荧光PCR、甲基化特异性PCR、甲基化特异性多重连接反应依赖性探针扩增、光纤微珠芯片等位点特异性DNA甲基化检测方法都可用于已知CpG位点甲基化状态的检测并可能在法医学实验室具有一定的应用前景;AIMS、HELP、COMPARE-MS等通过对基因组范围内的DNA甲基化扫描分析,可发现具有潜在法医学应用价值的DNA甲基化位点。     

同卵双生子外周血DNA甲基化谱的差异

目的通过比较不同个体外周血DNA甲基化谱的差异,评估DNA甲基化在同卵双生子个体甄别中的应用价值。方法在知情同意基础上获得22对同卵双生子外周血样。抽提基因组DNA后进行重亚硫酸盐转化．采用Illuraina公司的人27k甲基化微珠芯片检测基因组27578个CpG位点的甲基化程度（启值）。依据常染色体CpG位点的序值,采用欧氏距离计算方法计算同卵双生子间以及同性男ll的无关个体间的表观遗传距离。比较同卵双生子对与无关个体对两组不同人群间的表观遗传距离差异。结果同卵双生子对人群以及无关个体对人群中的男性个体对与女性个体对的表观遗传距离差异均无统计学意义（P值分别为0．0695和0．4825）。同卵双生子对的表观遗传距离显著低于无关个体对人群（中位数：6．02νs7．20,P=0．0002）．但两组人群的表观遗传距离均显著大于4．00（P〈0．0001）。结论同卵双生子间的外周血DNA甲基化谱差异显著．DNA甲基化是进行同卵双生子个体甄别的有效生物学标记。     

DNA甲基化与年龄推断

年龄推断是法医学研究的重要内容。近年来表观遗传学研究发现,基因组DNA甲基化总体水平随年龄增加而降低,同时部分位点的甲基化水平却随年龄增加而升高。通过检测DNA甲基化水平,构建与之相关的年龄变化模型,可用于推断个体年龄。本文对DNA甲基化与个体年龄的相关性、适用于法医学领域的检测方法及其在法医学年龄推断方面的应用前景进行综述,希望能为相关研究和应用提供参考。     

DNA甲基化检测方法及其法医学应用研究进展

DNA甲基化是表观遗传标记的重要组成部分,参与基因表达的调控,在生物发育、衰老以及肿瘤学等领域受到广泛关注。由于具有相对稳定性、可遗传、含量丰富、随龄变化等特点,在法医学领域,DNA甲基化可以作为DNA序列相关经典遗传标记的有效补充,用于年龄推断、组织体液来源检测、同卵双生子的鉴定等。DNA甲基化的检测方法主要有基于甲基化敏感限制性内切酶、重亚硫酸盐转化以及甲基化CpG结合蛋白等原理而建立的一系列方法,近年研究表明,第三代测序技术也可用于DNA甲基化检测。本文就DNA甲基化检测方法及在法医学领域的应用研究进行回顾与综述,以期为法医学实践提供参考。     

DNA甲基化在法医DNA分析中的应用

表观遗传学在生命的发生、发展过程中起着十分重要的作用。DNA甲基化作为表观遗传的一个重要方面,不仅参与多种基因的表达调控,与机体的发育、肿瘤发生等密切相关,而且具有可遗传性、相对稳定性、亲缘特异性、基因组中含量丰富等特点,已证实适用于法医DNA分析。本文对近年来DNA甲基化在印迹基因、同卵双生子鉴定、年龄、性别推断方面的研究与应用进行回顾与综述,以期为在法医学及相关领域中应用提供参考。     

同卵双生子甄别策略探讨

同卵双生子(MZ)之间存在抗体库差异、甲基化修饰差异和基因突变体差异,甲基化修饰差异的比较可望成为甄别MZ的手段之一,抗体库差异和基因突变体差异比较的方法在甄别MZ方面亦有潜在价值.当前,对MZ的表现遗传以及基因突变等领域的研究还很有限,而抗体库技术的应用方法也有待探讨,因此MZ识别技术的应用前景和发展空间需要进一步摸索与完善.     

DNA甲基化在组织/体液来源鉴定中的研究进展

对可疑生物样本的组织/体液进行来源鉴别是重建犯罪现场、推断犯罪性质等侦查活动中极为重要的一环。对表观遗传学理论的研究证明运用基因组中存在的组织特异性DNA甲基化差异位点(t DMRs)可以对组织/体液进行来源鉴别。本文旨在通过对近年来DNA甲基化在法医学领域用于鉴定人体组织/体液来源方面的研究成果进行阐述,试图用所得到的信息来分析DNA甲基化作为一种组织/体液鉴定遗传学标记的可能性、优劣点及其应用价值和发展前景,以期能为法医工作者的相关研究及实践提供参考。     

DNA甲基化标记检测方法及其法医学应用

DNA甲基化是重要的表观遗传修饰,主要在转录水平上调控基因的表达。DNA甲基化在人类基因组中含量丰富、分布广泛;甲基化谱有时空特异性、细胞特异性和亲源特异性。新近研究表明DNA甲基化在组织体液鉴定、同卵双生子鉴定、年龄推断、性别推断和亲子鉴定等方面具有一定的应用价值,有望成为一种新的法医学遗传标记。本文就DNA甲基化检测方法的研究进展及法医学应用进行综述,以期为法医学实践提供参考。     

DNA甲基化与个体年龄的相关性研究进展

年龄推断在法医实践中占有重要的地位。分子生物学的巨大进展使得人们借助于生物学标记推断个体年龄成为可能。作为表观遗传学重要组成部分的DNA甲基化,在机体生长、发育、衰老的过程中存在着动态变化过程,通过检测DNA甲基化改变,有望构建与之相关的年龄变化模式,用以推断个体年龄。本文着重介绍DNA甲基化水平的改变与个体年龄的相关性及其在判断个体年龄方面的前景。     

A novel multiplex assay system based on 10 methylation markers for forensic identification of body fluids

Identifying the source of body fluids found at a crime scene is an essential forensic step. Some methods based on DNA methylation played significant role in body fluids identification. Since DNA methylation is related to multiple factors, such as race, age, and diseases, it is necessary to know the methylation profile of a given population. In this study, we tested 19 body fluid-specific methylation markers in a Chinese Han population. A novel multiplex assay system based on the selected markers with smaller variation in methylation and stronger tissue-specific methylation were developed for the identification of body fluids. The multiplex assay were tested in 265 body fluid samples. A random forest model was established to predict the tissue source based on the methylation data of the 10 markers. The multiplex assay was evaluated by testing the sensitivity, the mixtures, and old samples. For the result, the novel multiplex assay based on 10 selected methylation markers presented good methylation profiles in all tested samples. The random forest model worked extremely well in predicting the source of body fluids, with an accuracy of 100% and 97.5% in training data and test data, respectively. The multiplex assay could accurately predict the tissue source from 0.5 ng genomic DNA, six-months-old samples and distinguish the minor component from a mixture of two components. Our results indicated that the methylation multiplex assay and the random forest model could provide a convenient tool for forensic practitioners in body fluid identification.     

印记基因H19上游高甲基化区SNPs多态性研究

目的建立简单、高效的DNA甲基化标记和SNPs联合检测技术，并用于H19基因上游高甲基化区两组SNPs群体遗传学检测。方法用PCR—DGGE技术对232例武汉汉族无关个体H19基因上游启动子区H19FR1和H19FR2单倍型进行检测；同时选用两种甲基化敏感的限制酶（msRE）HpaⅡ和HhaⅠ，检测H19FR等位基因亲代来源。结果H19FR1区检出5种单倍型、9种表型组合，其个体识别能力（DP）、多态性信息含量（PIC）和非父排除率（PE）分别为0．803、0．58和0．322；H19FR2区检出2种单倍型、3种表型组合，其DP、PIC和PE值分别为0．626、0．37和0．162。测序结果显示，片段H19FR1含有a7342g、a7357g和g7547a3个SNPs与1个g7351c点突变；H19FR2仅含aS097g1个SNP。msREHpaⅡ或HhaⅠ可消化个体母源等位基因，PDP-DGGE分析仅能检测到父源等位基因。结论PDP-DGGE是一种简单、灵敏、高效的DNA甲基化标记和SNPs联合分析技术，其在进行多态性分型同时还可以确定等位基因的亲代来源，具有较高的法医学应用价值。     

Application of fragment analysis based on methylation status mobility difference to identify vaginal secretions

Identifying vaginal secretions attaching or adhering to a suspect’s belongings would be beneficial for reconstructing the events that have taken place during a sexual assault. The present study describes a novel approach to identify vaginal secretions by fragment analysis using capillary electrophoresis, based on the mobility differences of PCR amplicons from bisulfite-treated DNA depending on methylation status. We targeted three genome regions including each of three vaginal secretion-specific methylated CpG sites reported previously: cg25416153, cg09765089, and cg14991487. In all three genome regions, the amplicon peaks for methylated genomic DNA (gDNA) sequences were only detected in vaginal samples, whereas samples of other body fluids (blood, saliva, semen, and deposit on skin surface) only showed amplicon peaks for unmethylated gDNA sequences. In vaginal secretions, the methylation ratio of each of the three targeted regions between samples was variable, while the ratios at the three regions in each sample were similar. Furthermore, commercial vaginal epithelial cells were completely methylated at the three regions. Therefore, vaginal secretion-specific methylation may derive from vaginal epithelial cells present in the sample.In forensic cases with a limited amount of DNA, the reproducibility of a detected peak using the present method is not high due to degradation of DNA by bisulfite treatment and subsequent stochastic PCR bias. However, it was possible to detect peaks from methylated DNA sequences by performing PCR and capillary electrophoresis in triplicate after bisulfite treatment, even when bisulfite treatment was performed using 0.5 ng of gDNA from vaginal secretions. In addition, the level of methylation at each targeted region was found to be stable in vaginal secretions stored for 1 year at room temperature. Therefore, we conclude that detection of the visual peak from vaginal secretion-specific methylated DNA sequence is useful to prove the presence of vaginal secretions. This approach has the potential to analyze multiple marker regions simultaneously, and may provide a new multiplex assay to identify various body fluids.     

DNA甲基化标记法医学应用探讨

CpG的胞嘧啶在DNA复制后多数被甲基化,5-甲基胞嘧啶的分布是贮存表遗传信息的主要形式。近年来研究表明,DNA甲基化标记具有信息含量丰富、相对稳定、检测和结果处理方便、可与SNP联合分析等优点,是一种新的强有力的遗传分析工具。基因组的甲基化差异可用甲基化敏感性限制酶、重亚硫酸盐转化、Maxam-Gilbert裂解等技术来分析。人类基因组甲基化谱有时空特异性、亲源特异性、病理特异性等特征,在法医亲子鉴定、个人识别等方面有潜在应用价值。     

外周血DNA甲基化谱与吸烟的关系

目的通过比较吸烟与不吸烟个体外周血DNA甲基化谱的差异,评估DNA甲基化在区分吸烟与不吸烟人群中的应用价值。方法根据下载于NIH的GEO公共数据库的人类全基因组DNA甲基化数据,运用Student’s T检验和聚类分析的方法评估外周血特定CpG位点DNA甲基化程度在吸烟与不吸烟人群中的差异。结果特定Cp G位点上,非吸烟人群与吸烟人群的DNA甲基化有显著区别。结论检测人外周血的甲基化情况可以区分吸烟和非吸烟人群。     

印记基因5个SNP分型及亲代来源的检测

目的 采用复合PCR-Snapshot联合甲基化敏感酶切技术,检测印记基因中5个SNP的甲基化状态、印记亲代来源及分型.方法 选择15例亲子鉴定已证实为亲生关系的家系样本,采用单碱基延伸复合检测技术,检测家系样本IGF2AS rs1003483、SNURF rs220028、SNURF rs4906939、DLGAP2 rs6558478、SIM2 rs737380等5个SNP分型,同时选用核酸内切酶(McrBC)和甲基化敏感的限制酶(msRE) HhaⅠ、HpaⅡ消化子代DNA,验证印记基因的亲代来源.结果 经用本文方法检测,证实rs1003483为父源印记;rs220028、rs4906939为母源印记;rs6558478及rs737380未在差异甲基化区,不能确定其印记亲代来源.结论 复合PCR-Snapshot联合甲基化敏感酶切技术简单、高效,在检测多个SNP分型的同时可确定亲代来源,可在相关研究和实践中选用.     

Use of the genomic matching technique to complement multiplex STR profiling reduces DNA profiling costs in high volume crimes and intelligence led screens

The genomic matching technique (GMT) targets duplicated polymorphic sequences within genomic blocks in the human major histocompatibility complex (MHC), differentiating between individuals at the DNA level using a single primer pair per block. The GMT is currently used to supplement human leukocyte antigen (HLA) typing to match donor and recipient pairs for bone marrow transplantation and has the potential to be employed as a powerful exclusion tool in forensic biology. The GMT is highly reproducible, produces DNA profiles from less than 1 ng of DNA and was successfully employed to profile a range of forensic samples including buccal swabs, handled objects and fingerprints. Furthermore, GMT profiles from a single genomic block in the MHC are likely to be more discriminatory than known highly polymorphic short tandem repeat (STR) loci such as ACTBP2. As such, the GMT can reduce the cost of investigations that require profiling of multiple suspects or samples from one or more crime scenes and could be extended to profile genomic blocks in other polymorphic genetic systems in the human genome.     

DNA甲基化及其与个体年龄相关性研究

近年来表观遗传学研究发现,基因组DNA甲基化总体水平随年龄增加而降低,同时部分位点的甲基化水平却随年龄增加而升高。本文重点介绍了DNA甲基化与年龄的相关性及其在年龄推断方面的研究进展,旨在为法医学个体年龄推断的研究提供一种新的思路。