DYZ1阵列鉴别男性单卵双生个体的法医学基础与应用研究

How to discriminate monozygotic twins (MZT) using genetic markers for forensic application remains unresolved. Although there are quantity of DNA methylation differences within MZT pairs, the lower stability and tissue differences of DNA methylation might limit the use of it for forensic use. Next generation sequencing (NGS) has been used to find single nucleotide polymorphism (SNP) or copy number variations (CNV) within MZT pairs. But it was difficult to perform it like finding a needle in the hay, and the cost was extremely high. A recent study reported that DYZ1 array showed sequence variation between MZT males, including SNP, CNV and In/del. DYZ1 represents heterochromatic region of the human Y chromosomes, any changes taking place between the two males of MZT after twining could be recorded faithfully in this region. DYZ1 was identified as a 3.4kb band and a normal male human Y chromosome contains approximately 3000~4300 copies of the DYZ1 array. These characters make DYZ1 array relatively easy to measure and analyze. Therefore, this study will focus on analysing the DYZ1 array differences within large number of male MZT pairs and develop DYZ1 sequencing method, PCR-RFLP method and CNV analysis techniques to discriminate male MZT. Through comprehensive evaluating the forensic values of these techniques from basic theory to practical application, ultimately resolve the problem of forensic discrimination of male MZT. In addition, we will study the tissue differences and the genetic stability of DYZ1 array to discover the mutation types and mechanisms, which is helpful for understanding the function of DYZ1.

目前法医学鉴别单卵双生子（MZT）的问题依然未得到有效解决。DNA甲基化虽在MZT间存在大量差异，但较低的稳定性及组织差异性可能限制其法医学应用。新一代测序技术寻找MZT间的单核苷酸多态性（SNP）或拷贝数变异（CNV）则如同大海捞针，且成本高昂。最近发现DYZ1阵列在男性MZT间存在许多差异，包括碱基插入/缺失、SNP及CNV。DYZ1是人类Y染色体异染色质的主要区域，能忠实记录MZT胚胎分离之后发生的变异。其长度为3.4kb，正常男性个体有3000~4300个拷贝，易于检测。综上，本项目拟研究大样本男性MZT间DYZ1阵列差异，建立DYZ1阵列靶向测序技术、PCR-RFLP技术和CNV分析方法，从基础理论到实际应用全面评估其法医学价值，解决法医学鉴别男性MZT个体的难题。研究DYZ1阵列的组织差异性及遗传稳定性，阐明DYZ1阵列的变异类型及机制，为深入理解DYZ1的功能奠定理论基础。

同卵双生子因具有几乎完全相同的遗传信息，二者的鉴识一直是法医遗传学领域未解的疑难问题。本项目聚焦人类基因组中突变率较高的区域，探索同卵双生个体全基因组中存在的罕见微小变异，并建立新的检验技术。.DYZ1阵列是位于Y染色体异染色质区域的高度串联重复序列，单拷贝长度3564bp，其序列由“TTCCA”及其衍生序列组成，且多拷贝分布。DYZ1阵列无编码功能，不参与染色体重组，能够忠实的记录个体出生后的变异情况。本研究建立了两种方法检测DYZ1阵列：Sanger测序技术（第一代测序技术）和单分子测序技术（SMRT）技术（第三代测序技术），后者通量高，读长长，能够一次性测通3.5kb DYZ1阵列，测序结果无须拼接，并且准确性佳，覆盖度高，重复性，步骤简便。应用SMRT技术检测3对男性同卵双生子DYZ1阵列，未发现序列差异。.快速突变Y-STR是Y染色体中另一种突变率较高的遗传标记，突变率高于1 ×10-2。本研究建立首个全快速突变Y-STR下一代测序复合检测体系，成功分型15个快速突变Y-STR基因座，具有准确性、一致性、特异性和可重复性。检测38对男性同卵双生子，发现5对样本两个体间存在同等位基因差异，即等位基因长度相同但序列不同，表明同卵双生个体之间存在快速突变Y-STR序列差异，该体系为解决同卵双生子鉴定难题提供了有效的技术方案。.人类线粒体基因组（mtGenome）是核外遗传物质，容易受到细胞内各种氧化反应影响且无修复系统，其突变率是核基因组突变率的10倍。本研究建立了单分子测序mtGenome技术，方便快捷，特异高效，准确性和可重复性均较好，且能发现mtGenome的低水平变异。应用该技术检测16对同卵双生子，每对个体之间均存在不同数量低水平变异差异，表明mtGenome低水平变异可以作为区分同卵双生个体的法医遗传标记，该体系为解决同卵双生子鉴定难题提供了新的技术方案。