Hereditary nonpolyposis colorectal cancer (HNPCC; OMIM 120435-6) is an autosomal dominant inherited syndrome caused by germline mutations in DNA mismatch repair (MMR) genes such as hMSH2 and hMLH1. With the loss of both alleles, DNA mismatch repair activity is completely lost and mutation rates increase significantly in specific regions of the genome called microsatellites. This phenomenon, referred to as microsatellite instability (MSI), leads to accelerated accumulation of frameshift mutations in specific tumor suppressors and other genes. Inactivation of these tumor suppressor genes contributes to stepwise neoplastic progression. This proposal is focused on two hypotheses regarding MSI and its relationship to DNA repair genes and HNPCC. The first hypothesis is that different DNA replication and repair genes influence microsatellite instability and mutation rates in human tumor suppressors and other cancer-related genes. While the effects of the MMR genes, MSH2 and MLH1, on MSI and mutation rates have been studied, there are other genes which contribute to MSI but are not as well characterized. The second hypothesis is that there are other human genes, as yet unidentified, which are susceptible to MSI mutations, and thus may be important contributors to colon epithelial neoplastic development in HNPCC. Although a number of genes vulnerable to MSI have been discovered, undoubtedly there are others that contribute to tumorigenesis. In this proposal, the principal investigator describes a strategy using S. cerevisiae as a model organism system to test these hypotheses and expand the genetic studies of MSI-related mutations in human tumor genes. Using isogenic S. cerevisiae strains with deletion mutations for a variety of DNA repair and replication (such as polymerases) genes I will test the effects these mutants have on human gene microsatellite instability. This quantitative data will be used to discern different DNA repair and replication genes' contribution to MSI in human genes. In addition, I will measure the mutation rates of selected tumor suppressor microsatellites in MMR-deficient mammalian cell lines to study the conservation of MSI between yeast and higher eukaryotes. I will identify hypermutable microsatellites in the coding region of new human genes that participate in neoplastic development in HNPCC-related malignancies. To accomplish this goal, I will screen a cDNA library derived from normal colon tissue for new genes which are susceptible to microsatellite instability mutations.

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Clinical Investigator Award (CIA) (K08)
Project #
1K08CA096879-01
Application #
6521912
Study Section
Subcommittee G - Education (NCI)
Program Officer
Myrick, Dorkina C
Project Start
2002-07-26
Project End
2007-06-30
Budget Start
2002-07-26
Budget End
2002-12-31
Support Year
1
Fiscal Year
2002
Total Cost
$61,398
Indirect Cost
Name
Stanford University
Department
Internal Medicine/Medicine
Type
Schools of Medicine
DUNS #
800771545
City
Stanford
State
CA
Country
United States
Zip Code
94305
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Ji, Hanlee P; Morales, Shannon; Welch, Katrina et al. (2012) Identification of a novel deletion mutant strain in Saccharomyces cerevisiae that results in a microsatellite instability phenotype. Biodiscovery :
Flaherty, Patrick; Natsoulis, Georges; Muralidharan, Omkar et al. (2012) Ultrasensitive detection of rare mutations using next-generation targeted resequencing. Nucleic Acids Res 40:e2
Newburger, Daniel E; Natsoulis, Georges; Grimes, Sue et al. (2012) The Human OligoGenome Resource: a database of oligonucleotide capture probes for resequencing target regions across the human genome. Nucleic Acids Res 40:D1137-43
Myllykangas, Samuel; Buenrostro, Jason D; Natsoulis, Georges et al. (2011) Efficient targeted resequencing of human germline and cancer genomes by oligonucleotide-selective sequencing. Nat Biotechnol 29:1024-7
Kim, Redecca Y; Xu, Hua; Myllykangas, Samuel et al. (2011) Genetic-based biomarkers and next-generation sequencing: the future of personalized care in colorectal cancer. Per Med 8:331-345
Natsoulis, Georges; Bell, John M; Xu, Hua et al. (2011) A flexible approach for highly multiplexed candidate gene targeted resequencing. PLoS One 6:e21088
Myllykangas, Samuel; Natsoulis, Georges; Bell, John M et al. (2011) Targeted sequencing library preparation by genomic DNA circularization. BMC Biotechnol 11:122
Myllykangas, Samuel; Ji, Hanlee P (2010) Targeted deep resequencing of the human cancer genome using next-generation technologies. Biotechnol Genet Eng Rev 27:135-58
Ji, Hanlee; Welch, Katrina (2009) Molecular inversion probe assay for allelic quantitation. Methods Mol Biol 556:67-87

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