| dc.description.abstracteng | Bloom syndrome (BS) is an autosomal recessive rare disorder clinically characterized by primary microcephaly, growth deficiency, short stature, photosensitivity, immunodeficiency, and cancer predisposition. BS is caused by biallelic, compound heterozygous, or homozygous loss-of-function (LoF) mutations in BLM, which encodes the BLM RecQ-like helicase. BLM has important roles during DNA replication and repair processes. Recently, additional genes associated with BS-like phenotypes were identified by whole exome sequencing strategies, namely autosomal recessively inherited LoF mutations in TOP3A, RMI1, and RMI2. TOP3A encodes the DNA topoisomerase III al- pha, which is able to decatenate single-stranded DNA molecules, while RMI1 and RMI2 code for the DNA interacting proteins RecQ-mediated genome instability protein-1 and -2. In general, BLM, TOP3A, RMI1, and RMI2 form together the BTRR multiprotein complex. The BTRR complex participates in fundamental cellular processes for DNA replication and repair. The most well-studied role of the BTRR complex is to dissolve DNA intermediates during the homologous recombination DNA repair, namely the double Holliday junctions. Failure in proper dissolution of such structures can lead to crossovers between the sister chromatids, an event called the sister chromatid exchange (SCE). Increased SCE rates are prominent cellular characteristics of BS cells. In sum, the BTRR complex is important for the protection of genome stability based on its roles in DNA replication and repair processes. To broaden the clinical and mutational signatures of BS, I aimed to collect and characterize patients with BS-like phenotypes. For this reason, a BS flyer informing about phenotypic characteristics and mechanisms of BS, as well as the details of this project was prepared in German and Turkish languages. This flyer was printed and distributed to clinicians, human genetics institutes, and genetics centers in both countries. Prior to this doctoral thesis, eight patients with BS phenotype were diagnosed by molecular genetic approaches. During my project, I assessed the clinical and mutational findings of patients and compared the data between patients. Homozygous LoF mutations were found in BLM in six patients and one homozygous LoF variant was detected in RMI1 in two patients from a consanguineous family. One of the pathogenic BLM variants was a novel mutation. Further, the phenotypic characteristics were milder in RMI1-associated patients when compared to BLM-associated patients in terms of skin findings and immunodeficiency in BS. Next, I investigated the transcriptional changes in BLM-deficient cells. Three BS patient-derived fibroblast cell lines were subjected to single-cell transcriptome sequencing (scRNAseq) using ICELL8 Single-Cell System and a high data quality was achieved. Differentially expressed genes and pathway analyses in BS samples in comparison to wild-type (WT) fibroblasts revealed highly significant terms in relation to the BS pathogenesis, such as chromosome segregation or mitosis-related terms. Many known interaction partners of BLM from the Fanconi anemia pathway were significantly upregulated. Analysis of cell cycle stages, which was possible to perform with the single-cell data, revealed that there was no difference in cell cycle stages between samples. Furthermore, several genes associated with primary microcephaly were deregulated in BS single cells, among which were NCAPG2, NCAPH, and NCAPD2. These transcripts encode members of the condensin I/II complexes, hence gene expression levels of condensin I/II complex were analyzed in detail and found highly upregulated. The overexpression of condensin I/II complex genes was a novel link in BLM deficiency. In addition, replication stress revealed a mild sensitivity of BS cells in comparison to WT according to the transcriptional changes. I also generated isogenic BTRR complex-deficient induced pluripotent stem cell (iPSC) lines via CRISPR/Cas9 genome editing technology. Cell lines having biallelic truncating mutations in genes encoding members of the BTRR complex, namely BLM, TOP3A, and RMI1, maintained the pluripotency after genome editing experiments. The lack of full-length proteins for the corresponding gene was confirmed via Western blot for every knockout (KO) cell line. The generated cell lines were characterized in terms of cellular phenotypes of the BTRR complex deficiency. SCE rates of BLM-KO, TOP3A-KO, and RMI1-KO iPSCs, were significantly higher than the wild-type parental control. Interestingly, the SCE frequency was lower in the RMI1-KO clone than BLM- and TOP3A-KO clones, although it was still significantly higher than the wild type. Mitotic errors such as chromatin bridges and lagging chromatin were observed in the KO iPSC samples, only RMI1-KO did not show a significant difference. Ultrafine anaphase bridges (UFB) were quantified in the generated KO samples and all three BTRR complex-deficient cell lines showed significantly increased rates of UFBs, implying that the BTRR complex deficiency resulted in unresolved UFBs independent of which member of the BTRR complex was impaired. Next, transcriptional changes of BTRR complex-deficient iPSCs were determined by scRNAseq. The overall transcriptional profiles were similar in KO-iPSCs and the parental wild type, yet the TOP3A-KO clone showed a possible sensitivity to replication stress. In summary, by the use of several different cell models for BS, the data generated in this project is of interest for further characterization of the BTRR complex. Transcriptional changes in BS can further shed light on the pathogenesis of BS. The isogenic iPSC lines provide a source to study the roles and effects of each member of the complex while providing therapeutic screening opportunities. Future differentiation approaches will provide additional insights into disease-associated mechanisms of BS phenotypes such as microcephaly or cancer predisposition in somatic cells. | de |