A functional genomic strategy for the fine mapping of a tumor suppressor gene interval: application to chromosome 3p12
aDepartment of Genetics, Harvard Medical School, Boston, MA, United States, bDepartment of Molecular Genetics, UT-MD Anderson Cancer Center, Houston, TX, United States, cCenter for Genome Information, Department of Environmental Health, University of Cincinnati, Cincinnati, OH, United States
AIM: Our laboratory has previously defined a novel tumor suppressor locus NRC-1 (non-papillary renal carcinoma-1) (OMIM ID 604442) by functional genomic approaches in which a defined fragment of chromosome 3p was transferred into a renal cell carcinoma (RCC) cell line and assayed for tumor suppression in vivo. Functional complementation of the region resulted in the identification of a critical interval within 3p12 containing the NRC-1 locus that mediated RCC tumor suppression independently of tumor microenvironment, histologic type of RCC or the presence of inactivating mutations in the VHL (von Hippel Lindau) gene. VHL is a 3p25 tumor suppressor that underlies a cancer predisposition syndrome in which germline mutation results in a number of histologically diverse tumor types, including RCC. These data implicate the NRC-1 locus either downstream of VHL or in an independent pathway to tumorigenesis in the kidney. The aim of this study was to develop a functional genomic strategy to fine map the NRC-1 interval and identify candidates for this important tumor suppressor gene. METHOD: To address this question, we first examined a VHL kindred with a preponderance of pancreatic islet cell tumors (PICT), because PICT and RCC are two of the main tumor types that progress to malignancy in VHL. Loss of heterozygosity (LOH) was observed for all informative 3p markers proximal to VHL on chromosome 3p in the VHL kindreds understudy as well as high frequency LOH in sporadic PICT coincident with regions of interstitial deletion in sporadic RCC. Thus, 3p LOH (within 3p21 and 3p12) was found at high frequency subsequent to germline VHL mutation and cyst formation, indicating that loss of 3p21 or 3p12 loci may be a prerequisite for malignant conversion in VHL-associated RCC and PICT, and suggesting that the 3p12 interval harbors a tumor suppressor gene that could be involved in the genesis of sporadic RCC and in progression to malignancy of VHL-associated RCC and PICT. To identify candidate genes in this interval, we devised a strategy that combines the utility of microcell hybrids and complementation mapping with a novel technology utilizing STS markers and QPCR to precisely define boundaries of functional tumor suppressor activity by determination of DNA copy number differences between hybrid and parental genomes. The approach is broadly applicable to any functional genomic analysis that relies on genetic complementation with a theoretical physical resolution expected to be as high as only a few base pairs. CONCLUSIONS: Using a functional genomic strategy, the NRC-1 critical interval has been precisely mapped which should allow the identification of candidates for the 3p12 tumor suppressor gene.
Paper presented at the International Symposium on Predictive Oncology and Intervention Strategies; Nice, France; February 7 - 10, 2004; in oral session 896 (Tumor suppressor genes).