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

基因组不稳定性是由编码具有重要基因组维持功能的蛋白质的基因突变引起的选择性遗传性单基因疾病的共同致病机制。新一代测序（NGS）技术的最新进展使鉴定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中基因组维持的改变和疾病相关的染色体不稳定机制的理解仍然有限。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了编码BTR复合体成员（BLM、TOP3A、RMI1、RMI2）及其相互作用伙伴BRCA1的基因的致病突变。BTR复合体及其组分在DNA复制和有丝分裂中发挥重要作用，这对维持基因组稳定性至关重要。该项目旨在剖析BTR复合物本身或相关结合伙伴的不同组分突变的相关性，并将其分子效应与复制应激和结构/数字染色体不稳定性联系起来。我们将定义突变特征和BTR复合物功能障碍的临床谱，并使用来自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，我们将系统地确定与基因组不稳定性和有丝分裂改变以及不同BTR复合物基因型的复制应激和DNA损伤反应有关的特定细胞表型。特别是与Holger Bastians （SP2）和Markus Räschle （SP6）四轴染色体形成合作，将探讨姐妹染色单体交换，超细后期桥的形成，滞后染色体和其他有丝分裂错误的差异。详细分析复制压力和DNA损伤将与Matthias Dobbelstein （SP4）合作完成。此外，由于与Bloom综合征相关的原发性小头畸形的分子发病机制尚不清楚，该项目旨在深入了解神经发生过程中BTR复合物相关的DNA复制和有丝分裂改变对细胞的影响。为了系统地分析染色体不稳定性，我们还将与中央服务项目SP-Z合作，利用其生物信息学管道和先进的新方法，例如，通过低覆盖率全基因组测序检测拷贝数变化，或通过模板链特异性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BTR复杂疾病的分子发病机制中有丝分裂改变和DNA复制胁迫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.