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

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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.