插入型染色体易位的突变机制与致病机理

Chromosomal insertion translocation (IT) is one type of genomic structural rearrangements commonly resulting in a duplication or a deletion of disease-causing genes in offspring of the carriers. Due to two-breakpoint-broken and followed insertion translocation of a chromosome segment into a non-homologous chromosome or a non-adjacent locus on the same chromosome or the other homologue, ITs are commonly classified as complex chromosomal abnormalities (≥3 breakpoints) and usually confirmed by conventional karyotyping or FISH analysis. However, due to the limitation of current techniques of detection and resolution, incidence of ITs is estimated as low as 1 in 500 and little is known about the molecular mechanisms of their formation. In our prelim study of 6 individuals with ITs by our published low-pass whole-genome sequencing (6X) approach with mate-pair library (3-8kb), the percentage of complex insertional rearrangements (≥6 breakpoints) is much higher than the previous study (66.7% vs 21.1). Therefore, we propose to apply this approach to 40 samples with ITs (confirmed by karyotype or FISH) for investigation of the rate of complex insertional rearrangements as well as the repairing mechanism based on the breakpoint sequences with the aid of Sanger sequencing. In addition, with parental DNA samples, we are going to identify the cryptic variants near the breakpoints regions for further discussion of their contribution of the occurrence of ITs. Furthermore, parental origin of the affected chromosomes will be determined by the SNP typing information obtained from mate-pair library sequencing data. In order to study the add-on value of genomic variants combining with ITs, high read-depth (30X) whole-genome sequencing will be performed for each member of three trios, in each of which proband and inherited parent are with distinct phenotypes. Combination of RNA-seq and cell morphological study from nephrogenic intermedia mesoderm differentiated from established iPSCs of proband and inherited parent, we aim to identify the genomic variants contributing to the distinct phenotypes (renal anomalies found in proband but absent in inherited parent). Taking together, we propose this project to investigate the mutagenesis mechanism study of ITs and their pathogenicity combining with other genomic variants, which will provide genetic information for clinical counselling and early prevention of ITs.

插入型染色体易位，是一种较为复杂的染色体结构变异类型，其致病模式常为携带者后代出现致病基因的缺失或重复。由于现有技术手段及分辨率的限制，导致大多数插入型易位难以发现，其突变机制和致病模型未明。本项目团队利用前期工作开发并发表的基于大片段建库（3-8kb）全基因组低深度测序分析方法（6X），对6例样本进行研究发现复杂重组率高达67%，提示可能存在不同的致病机制。本项目拟利用此项技术，分析40例插入片段大小及位置不同的事件，结合断点序列讨论简单型和复杂型插入易位的修复机制，并结合父母样本信息，讨论引发事件的致病机理及是否存在父母源倾向。同时对其中三个遗传型插入型易位患者但亲代和子代表型差异的家系进行全基因组30X测序分析，结合来源于iPSC细胞分化的中胚层肾源性细胞的形态学和RNA分析，全面讨论其致病机理。冀希望通过此研究，能够了解其插入型易位的发生机制，为临床咨询和早期防治提供遗传学基础。

本项目团队利用我们新开发的DNA长片段建库和低深度高通量全基因组测序方法，对插入片段大小不同的插入型易位insertion事件进行测序分析。首先，我们确定了染色体插入的复杂性和断点修复机制：我们收集的40例病例中，总体复杂插入比例达到70%（28/40），比基于基因芯片判定的复杂插入比例（16/76）显著性提高（卡方检验 p < 0.00001）。利用特异性PCR和Sanger测序进行断点研究，79%的断点可进行断点特征分析。其中，断点存在简单插入序列，简单插入型样本比复杂插入型样本显著性提高（卡方检验 p < 0.00001），即简单插入更倾向于由DNA复制作为其修复机制。其次，我们确定插入型易位能够稳定的传代，而自发性插入存在较强的父源性自发突变倾向：26个遗传自父母的插入的断点分析结果显示断点与父母携带者本身一致，提示此类变异可稳定传代。而对14个自发插入进行SNP分析，10例可分辨，其中9例来源于父亲的染色体，另1例为来源于母亲的染色体。证明了存在父源性倾向（90%）。最后，我们利用基因组测序和RNA分析，证明了基因组双诊断是促发差异表型的原因：对3个存在非平衡性插入型易位但存在差异表型的家系进行研究，发现其中两例存在额外的点突变，可解释子代存在严重的表型，而父亲（也带有insertion）为轻微或无表型。对家系1的先证者和其父亲（携带者）的 iPSCs 向目的细胞中胚层肾脏样细胞分化。先证者的中胚层肾脏样细胞的增殖和凋亡与父亲相比（无肾脏异常）都存在显著性差异。而对两者细胞进行RNA测序，发现ANKRD11的RNA特征和先证者的血样RNA分析一致，即部分ANKRD11转录本变短，且此转录本的表达量较样本的正常转录本表达量低，然而蛋白分析并未检测到此基因的表达，考虑与组织特异性相关。本项目研究促进了解插入型易位的发生和致病机制，为临床咨询和早期防治提供遗传学基础。