母血浆胎儿DNA甲基化标记的法医学应用基础研究

Given the procedure-related risks of conventional prenatal paternity diagnosis, it would be ideal if genetic analysis of the fetus could be performed non-invasively. Recent researches suggest that the fetal epigenetics marker in maternal plasma was one of the possible solutions to this problem. Supported by the National Natural Science Foundation of China (grant no. 81001352), we employed the high-throughput Illumina HumanMethylation450 BeadChip array to screen hypermethylated CpG sites in fetal placental chronic villus samples (CVS) versus maternal blood cells (MBC) in a genome-wide basis. 144 candidate fetalepigenetics sequences, which harbor differential methylated CpG sites and one polymorphic SNP were identified. As a preliminarily study, 3 candidate regions of PSMB8, SKI and CHST11 gene from this array data set were selected for further validation in maternal plasma. The validation results showed that the hypermathylated versions of these candidate sequences were detectable specifically in pregnant women and possessed the identical SNP genotypes with the fetal tissue, demonstrating their potentials for non-invasive pre-birth paternity testing. The present study is aimed to perform the comprehensive and systematic validation of the 450K Array databy comparing the mathylation patterns in MBC and CVS, using bisulfite conversion of genomic DNA and subsequent high-throughput pyrosequencing. After validation, to confirm the fetal specificity and the sensitivity of the candidate markers, we will perform real-time PCR after methylation-sensitive restriction enzyme (MSRE) digestion to quantitatively detect methylated sequences in the plasma of 1st-, 2nd-, and 3rd- trimester pregnant women, as well as the collected age-matched non-pregnant healthy women. Furthermore, to evaluate the diagnostic accuracy of the candidate markers in revealingthe fetal exact SNP genotype, the genotypes of the SNPs located within the resultant amplified regions for the MSRE-digested maternal plasma, maternal blood cells and the fetal tissue DNA, will be determined by a MALDI-TOF mass spectrometer respectively. Based on the genotyping results and statistical analysis, the forensic significance of the candidate fetal epigenetic markers in maternal plasma will be also evaluated, thus providing a possible solution to the establishment of non-invasive fetal paternity test.

如何建立无创性的方法进行胎儿亲子鉴定是法医学难题之一。表观遗传学研究提示DNA甲基化标记可能是解决这一难题的线索。课题组在青年基金项目资助下，利用芯片技术在全基因组范围内筛选出144个含SNP的胎儿特异高甲基化DNA序列；预试验选取其中3个候选序列（PSMB8、SKI及CHST11）进行验证，证实其特异存在于孕妇血浆中，并与相应胎儿的SNP分型一致，提示在无创性胎儿亲子鉴定研究中的良好潜力。本项目将采用重亚硫酸盐处理结合高通量焦磷酸测序技术，对芯片筛选结果进行全面验证，确定所有候选序列的甲基化模式；定量检测通过验证的候选序列在孕妇血浆中的特异性以及不同孕期血浆中的浓度；选取高特异性和高浓度的候选序列，使用质谱技术进行SNP分型，并与相应胎儿组织作一致性比较，观察其揭示胎儿遗传信息的准确性；基于以上实验结果进行法医学统计分析，评价其法医学意义，为建立无创性胎儿亲子鉴定方法提供可能的解决方案。

如何建立无创性的方法进行胎儿亲子鉴定是法医学难题之一。孕妇血浆游离胎儿DNA片段携带着整个胎儿染色体组的遗传信息，被认为是实现无创产前亲子鉴定的最理想胎儿检材。采用目前常规的PCR-STR分型策略，由于受检测灵敏度限制以及STR常见的影子峰等因素的影响，获得的有效位点数目非常有限，且难以保证检测结果的可重复性和准确性。新一代测序技术的发展，为游离胎儿DNA的法医遗传学研究提供了广阔的前景。本项目基于第三代遗传标记单核苷酸多态性（SNP）位点，分别使用Ion Torrent和Illumina两大主流高通量测序平台，选择短扩增子扩增和探针捕获等两种建库方法对不同孕期的母血浆游离DNA开展了法医遗传学的研究，取得了以下重要研究结果： 1）通过数据库筛选和实验室验证，在全基因组范围内获得一套足够数量的、适于母血浆游离胎儿DNA法医遗传学分析的SNP新型遗传标记。2）基于高通量测序平台，建立适于胎儿游离DNA遗传学分析的高信息量、高通量的检测体系，其中包括微量母血浆游离胎儿DNA提取、文库制备和高通量测序等技术环节。3）根据结果制定相应的数据解析策略，构建无创胎儿亲权鉴定的父权指数（PI）值算法；相关算法分别在由已知生物学父亲或无关男性个体构成的真、假家系中进行验证，并与常规STR分型方法作比较，系统评估其准确性和法庭科学有效性。本项目为建立精确、有效、适用于各孕期（特别是早孕期）的无创产前亲子鉴定技术方法提供理论和应用基础。具有重要的科学意义和应用价值。