Array-CGH in the context of chromosomal instability and cancer

Abstract

DNA from clinical specimens is often available only in limited quantities. In pre-implantation diagnostics and analysis of micrometastasis, even merely single cells can be obtained. Therefore whole genome amplification strategies for the subsequent assessment of copy number are required. The first aim of the thesis was to establish the exact resolution limits for the analysis of copy number alterations in single cells and small cell numbers. The latest array technologies and in house developed algorithms for the analysis of single cells were used. Our data suggest that a reliable identification of copy number alterations as small as 500 kb in cell pools (5 or 10 cells), and of 2.6-3.0 Mb in single cells is possible. The characterization of a single gene disrupted by a balanced translocation can easily be linked to a phenotype and is a promising approach to provide insight into gene functions and disease entities. For this purpose several sophisticated techniques have evolved, as e.g. the flow-sorting of the translocation chromosomes and subsequent hybridization on high-resolution arrays, or the analysis with a next-generation sequencing approach. Yet, the flow-sorting of chromosomes is a highly sophisticated method which can only be performed by a few labs due to the special equipment which is required. The second aim of the thesis was to establish the fine-mapping of balanced translocations to the basepair level using chromosomes isolated with laser-capture microdissection, a widely available technique. This approach was exemplified by the finemapping of a breakpoint in a boy with a split hand and foot malformation syndrome. In colorectal cancer copy number alterations and mutations are frequent and can possibly drive tumorigenesis. The same is true for a rare non-melanoma skin cancer called blastic plasmacytoid dendritic cell neoplasm (BPDCN). The third aim was to generate a detailed map of copy number alterations in colorectal cancer and BPDCN to summarize the affected genes in pathways and to correlate these findings with the outcome of the patients. The risk of being diagnosed with colorectal cancer increases dramatically at the age of 50. Several attempts to mimic the human cancerogenesis in mouse models and to investigate very early lesions have been made. The fourth aim was to investigate basal intestinal crypts of telomerase deficient mice with a conditional p53 knockout in the intestine for chromosomal instability. We showed that the deletion of p53 impairs the clearance of chromosomal-instable stem cells in aging telomere-dysfunctional mice.

Date of Award	2011
Original language	English
Awarding Institution	Medical University Graz
Supervisor	Michael Speicher (Supervisor) & Michael Trauner (Supervisor)