同卵双生子发育过程中的序列变异及其法医学应用研究

Monozygotic (MZ) twins have been assumed to be genetically identical, which makes it impossible to distinguish one from the other by using the forensic DNA typing systems. How to discriminate the MZ twins has become one of the big challenges in forensic society. Previous studies suggested that the changes in epigenetic modification, expression level of microRNAs, as well as composition of antibodies have potential impact on phenotypic discordance observed in MZ twins. The changes at these molecular levels were found to be highly susceptible to environmental factors, which immensely reduced their potential application for forensic discrimination of MZ twins. In fact, recent studies have suggested MZ twins could accumulate somatic mutations after the zygote splits, suggesting they will be genetically unidentical over the course of ontogenesis. In support of it, variations in copy number and mitochondrial genome have been observed between pairs of MZ twins in our pilot studies. In this proposed study, by using high-throughput sequencing technology, we will measure the whole genome in blood samples collected from age-series MZ twins. We will obtain a comprehensive picture of sequence variation taking place during the development of MZ twins. By testing the robust of sequence variation in different tissues, ages, as well as a large set of population samples, we will construct the strategy for forensic discrimination of the MZ pairs. These findings will reveal the age-related sequence variation during the development of MZ twins, which will provide the scientific basis and technical support for forensic discrimination of MZ twins.

同卵双生子理论上具有完全相同的遗传信息，因而基于传统遗传标记的DNA分型方法均不能对其进行区分，其个体甄别成为目前法医学界的科学难题。前期研究表明表观遗传修饰、microRNAs表达水平以及抗体库组成的改变对同卵双生子表型变化有潜在影响，但这些分子水平变化极易受环境因素影响，因而不能有效应用于司法实践。近期研究表明同卵双生子在受精卵分裂后可以积累体细胞突变，提示着它们在个体发育过程中基因组水平上将产生差异。我们最新研究表明同卵双生子在拷贝数以及线粒体序列上存在差异。在此基础之上，本项目将利用高通量测序技术，测量不同年龄阶段同卵双生子血液中的基因组序列。通过比较分析，探讨同卵双生子序列变异随着个体发育的变化规律及其在不同年龄、不同组织以及大规模同卵双生子人群中的稳定性，建立适用于检材与同卵双生子个体识别的技术方案，为同卵双生子个体甄别提供科学依据和技术手段。

同卵双生子是由一个受精卵一分为二形成的两个相同胚胎发育而来，理论上具有完全相同的遗传信息，这使得基于传统遗传标记的DNA分型方法均不能对其进行区分。前期研究表明表观遗传修饰、microRNAs表达量以及抗体库组成的改变对同卵双胞胎表型变化有潜在影响。然而，这些分子水平的变化被认为极易受环境因素的影响，从而极大降低了它们用于同卵双生子甄别的可能性。事实上，近期研究表明同卵双胞胎在受精卵分裂后可以积累体细胞突变，提示着它们在个体发育过程中基因组水平上将产生差异。本项目围绕同卵双生子发育过程中的序列变异展开研究，探讨了同卵双生子之间的序列变异用于同卵双生子个体识别的法医学应用价值。项目首先通过DNA-seq测序技术测定了不同年龄阶段同卵双生子外周血中的全基因组序列，经过系统分析后发现同卵双生子序列变异在不同双生子中的重叠性均低于0.0073，结合项目组前期在同卵双生子之间开展的CNV研究，提示了同卵双生子序列变异的个体随机性。同时，通过对不同年龄阶段同卵双生子序列变异的比较分析推测出同卵双生子序列变异很有可能发生在个体发育早期。据此提出了同卵双生子甄别的核心问题是识别出同卵双生子之间真实存在的序列变异。随后，项目组设计了一套结合了WGS、靶向高通量测序和Sanger测序三个层层递进检测技术的同卵双生子序列变异甄别技术流程用来识别和验证同卵双胞胎之间真实存在的SNVs。这一技术流程通过对同卵双生子和回访样本的分析以及双盲测实验被证实可以有效用于同卵双生子的个体识别。本项目的实施为解决同卵双生子个体识别这一法医学难题提供了坚实的科学依据和有效的技术方案。