Martin Arlt: University of Michigan, Ann Arbor — Faculty Assistant Research Scientist of Human Genetics in Department of Human Genetics

Abstract: Copy number variants (CNVs) are chromosomal deletions and duplications that play a major role in human variation, genetic disease, and cancer. Non-recurrent CNVs, which account for most de novo, disease-associated CNVs are thought to arise through aberrant DNA replication and nonhomologous repair of DNA damage. Very little is known about the genetic factors and environmental stressors that influence this important class of mutation. Using a model mammalian cell culture system, we have demonstrated that replication stress is an effective inducer of de novo CNVs. These CNV mutations occur at sites across the genome and at hotspot loci with no difference in size or breakpoint structures. These hotspots provide ideal targets for studies of risk factors involved in CNV formation. The key determining factor of a hotspot locus is the presence of a very large, actively transcribed gene, with deletion CNVs arising preferentially near the center of these regions. This pattern mimics many non-recurrent CNVs in genomic disease and cancer, indicating that CNVs arising in late-replicating, large transcription units are important to human genomic instability, and model a broader class of disease-associated, non-recurrent CNVs.

While our experimental approach is powerful, it is also very time- and resource-intensive, not lending itself easily to high-throughput analyses. I am taking advantage of our predictive model to develop a rapid assay for CNV formation. Using CRISPR-Cas9, I will insert a reporter cassette containing the HSV-TK selectable marker into CNV hotspots in multiple cell lines. HSV-TK confers ganciclovir sensitivity on cells. Following experimental exposure, cells that acquire a CNV that disrupts HSV-TK are ganciclovir-resistant, allowing selection of cells with a de novo CNV. Surviving cells are quantified to determine the effects of experimental manipulation on CNV mutation rate. Thus, hotspot CNV formation can be monitored as a proxy for genome-wide non-recurrent CNVs without the need for time-consuming clonal isolation and whole genome analyses. This rapid screen will inform my future research, providing a valuable tool to rapidly assess the effects of large numbers of genes and environmental agents on CNV formation. 

Host: Don Sakaguchi

Refreshments will be served outside Room 1414 in the Molecular Biology Building before the seminar at 3:45 p.m.