Impaired chromosome integrity caused by mutations in members of the BTR complex

基因组不稳定性是选择性遗传性单基因疾病的共同致病机制，这些疾病是由编码对基因组维持具有重要功能的蛋白质的基因突变引起的。下一代测序(NGS)技术的最新进展使识别与基因组不稳定综合征相关的新基因成为可能。然而，我们对有丝分裂细胞基因组维持的变化和与疾病相关的染色体不稳定的机制的了解仍然有限。我们之前在Bloom综合征患者中进行的基于NGS的基因鉴定研究发现，BTR复合体成员(BLM、TOP3A、RMI1、RMI2)及其相互作用伙伴BRCA1的编码基因发生了致病突变。BTR复合体及其组分在DNA复制和有丝分裂中发挥着重要作用，而DNA复制和有丝分裂是维持基因组稳定所必需的。该项目旨在剖析BTR复合体本身或相关结合伙伴不同成分中突变的相关性，并将它们对复制压力和结构/数字染色体不稳定性的分子效应联系起来。我们将定义BTR复合体功能障碍的突变特征和临床谱系，并使用患者的原代成纤维细胞、由这些成纤维细胞产生的iPS细胞以及CRISPR/Cas9衍生的突变HCT116细胞，系统地确定关于基因组不稳定性和有丝分裂变化的特定细胞表型，以及不同BTR复合体基因的复制应激和DNA损伤反应。具体地说，将与Holger bastians(SP2)和Markus Räschle(SP6)合作，探索姐妹染色单体交换、超细后期桥的形成、落后染色体和其他有丝分裂错误方面的差异。将与Matthias Dobbelstein(SP4)合作进行复制压力和DNA损伤的详细分析。此外，由于与Bloom综合征相关的原发性小头畸形症的分子发病机制尚不清楚，该项目旨在为BTR复合体相关的DNA复制和有丝分裂在神经发生过程中的细胞后果提供见解。为了对染色体不稳定性进行系统分析，我们还将与中央服务项目SP-Z合作，利用其生物信息学流水线和复杂的新方法，例如在低覆盖率全基因组测序中检测拷贝数变异，或通过模板链特定测序在单细胞中确定拷贝数中性染色体重排。预期的结果将为有丝分裂改变和DNA复制应激在BTR复杂疾病的分子发病机制中的串扰提供新的疾病相关见解。

Genomic instability is a shared pathogenic mechanism of selected inherited, monogenic disorders caused by mutations in genes encoding proteins with important functions for genomic maintenance. Recent advances in next-generation sequencing (NGS) technologies have allowed the identification of novel genes associated with genomic instability syndromes. However, our understanding of alterations of genomic maintenance in mitotic cells and mechanisms of disease-associated chromosome instability is still limited. Previous NGS-based gene identification studies that we performed in patients with Bloom syndrome identified causative mutations in genes encoding members of the BTR complex (BLM, TOP3A, RMI1, RMI2) and its interacting partner BRCA1. The BTR complex and its components play important roles in DNA replication and mitosis that are essential for maintaining genomic stability. This project aims at dissecting the relevance of mutations in different components of the BTR complex itself or associated binding partners and at linking their molecular effects on replication stress and structural/numerical chromosome instability. We will define mutational signatures and the clinical spectrum of BTR complex dysfunction and, using primary fibroblasts from patients, iPS cells generated from these fibroblasts as well as CRISPR/Cas9-derived mutant HCT116 cells, we will systematically determine specific cellular phenotypes regarding genomic instability and mitotic alterations as well as replication stress and DNA damage response for different BTR complex genotypes. Specifically and in collaboration with Holger Bastians (SP2) and Markus Räschle (SP6) quadriradial chromosome formation, differences in sister chromatid exchange, formation of ultrafine anaphase bridges, lagging chromosomes, and other mitotic errors will be explored. Detailed analyses of replication stress and DNA damage will be done in collaboration with Matthias Dobbelstein (SP4). Furthermore, as the molecular pathogenesis of primary microcephaly associated with Bloom syndrome is not yet understood, the project aims at providing insights into the cellular consequences of BTR complex-related alterations of DNA replication and mitosis during neurogenesis. For our systematic analysis of chromosome instability, we will also collaborate with the central service project SP-Z and make use of its bioinformatics pipeline and sophisticated novel methods, e.g. to detect copy number variations in low-coverage whole-genome sequencing or to determine copy-number neutral chromosome rearrangements by template-strand-specific sequencing in single cells. The expected results will give new disease-related insights into the crosstalk of mitotic alterations and DNA replications stress in the molecular pathogenesis of BTR complex disorders.