UNIVERSITY OF CINCINNATI _____________ , 20 _____ I,______________________________________________, hereby submit this as part of the requirements for the degree of: ________________________________________________ in: ________________________________________________ It is entitled: ________________________________________________ ________________________________________________ ________________________________________________ ________________________________________________ Approved by: ________________________ ________________________ ________________________ ________________________ ________________________ THE BALANCED, RECIPROCAL TRANSLOCATION OF CHROMOSOMAL SUBBANDS 12q15 AND 14q24 AND ALTERED GENE EXPRESSION IN UTERINE LEIOMYOMA A dissertation submitted to the Division of Research and Advanced Studies of the University of Cincinnati in partial fulfillment of the requirements for the degree of DOCTORATE OF PHILOSOPHY (Ph.D.) in the Department of Molecular Genetics, Biochemistry and Microbiology of the College of Medicine 2002 by Susan E. Ingraham B.S., University of Wisconsin, 1990 Committee Chair: Anil G. Menon Abstract A balanced, reciprocal translocation between chromosomes 12 and 14 is frequently observed in uterine leiomyoma (UL), a common benign smooth muscle tumor. Cytogenetic evidence suggests that t(12;14)(q15;q24) is an early event and therefore may represent one of the important steps in UL pathogenesis. The breakpoints of t(12;14)(q15;q24) have been localized to unusually large genomic regions (approximately 0.5 Mb) on each of the involved chromosomes. In this study, we investigated the molecular events associated with this translocation, specifically the transcription of genes within the breakpoint regions on 14q24 and 12q15. t(12;14)(q15;q24) had previously been associated with activation of HMGA2 on chromosome 12, a gene implicated in promoting cell proliferation, particularly of lipoid tissues. We analyzed this gene and discovered several novel transcripts from 12q15 that are embedded within HMGA2. On chromosome 14, the breakpoint in one UL was found to lie within RAD51L1, a putative DNA repair and recombination gene. Subsequent analysis of two additional t(12;14) UL breakpoints refined the chromosome 14 breakpoint region to between 700 and 1200 kb. The large size of the breakpoint regions and the mapping of breakpoints well outside both HMGA2 and RAD51L1 suggested that the translocation may alter the structure and long-range regulatory controls of genes including but perhaps not limited to HMGA2 and RAD51L1. To test this hypothesis, an expression map was developed which consisted of ESTs and genes within and flanking both breakpoint regions. Expression of these markers was tested in matched normal and t(12;14) UL tissue samples to identify a domain of altered expression on chromosomes 12 and 14. HMGA2 and the three novel ESTs embedded within HMGA2, A15, B6, and D12, were overexpressed six- to more than twenty-fold, while RAD51L1 and other ESTs on chromosome 14 were not consistently or significantly altered in UL. Positional cloning of the UL breakpoint region and mapping of the domain of altered expression in tumors sets the stage for understanding the molecular mechanism for the pathogenesis of UL. Dedication This dissertation is dedicated to several people who taught me by their example that life goes on in spite of everything. John Bosanec, Judy Ingraham, Roy Lynch, and Alissa Winnard, this work is for you, for without your inspiration it might not have been accomplished at all. Acknowledgements I thank James Liu, Frank Dill, Sheila Blanck and Urvashi Surti for collecting the leiomyoma samples which were critical to this work; and Bruce Aronow, Kathy Saalfeld, David Witte, Tim Hubbell, and Betty Davis for their technical expertise. Judy Harmony, Les Myatt, Bob Colbert, and Terri Berning, administrators of the Physician Scientist Training Program, deserve recognition of their tremendous efforts which have made the MD/PhD program at the University of Cincinnati one of the best and most student-focused in the country. My appreciation also to Sohaib Khan and Susan Eder, administrators of NIH Training Grant T32 CA59268 which has supported me financially and intellectually for the past three years. I thank those who have served on my thesis committee, Anil Menon, Joanna Groden, Jim Stringer, Sue Heffelfinger, Bob Arceci, and Shelley Barton, for their insight and guidance; and my colleagues in the Menon lab (past and present), Vasily Ivanenkov, Martha Jiang, Chuck Klanke, Carissa Krane, Roy Lynch, Shodimu Olufemi, Keya Sen, Zheng Yuan Shan, Katie Smith, Teresa Smolarek, Yan Ru Su, Jennifer Towne, Jessica Woo, Fan Yang, and Shiping Zhang for their friendship and support. My deepest gratitude to Alissa and Phil Winnard and Katie and Doug Smith for seeing me through unexpected hardships and getting me back on my feet with extraordinary kindness and efficiency. And finally, I thank my family, Janet, Andy and Benjamin Ingraham Dwyer and Sam and Judy Ingraham, for always having more faith than I did that what I was doing was important and valuable, even though they rarely understood any of it. Table of Contents List of Figures..................................................................................................................5 List of Tables....................................................................................................................8 List of Symbols and Abbreviations................................................................................9 Cytogenetic Notation........................................................................................................................ 12 Genes and Their Products................................................................................................................ 12 Units of Measure.............................................................................................................................. 12 Chapter 1: Introduction ...............................................................................................13 EPIDEMIOLOGY AND PATHOLOGY OF LEIOMYOMA ......................................................13 HORMONAL RESPONSIVENESS AND REGULATION OF UL .............................................14 ANIMAL MODELS OF UL..............................................................................................18 LEIOMYOMA CYTOGENETICS .......................................................................................19 CHROMOSOMAL TRANSLOCATIONS IN HUMAN DISEASE..............................................25 POSITIONAL CLONING OF THE UL TRANSLOCATION ....................................................27 GENE REGULATION BY ACTION AT A DISTANCE ..........................................................29 CANDIDATE GENES ON CHROMOSOME 12: HMGA2 ....................................................33 CANDIDATE GENES ON CHROMOSOME 14: RAD51L1..................................................39 DNA REPAIR IN HUMAN DISEASE ...............................................................................42 ANALYSIS OF GENE EXPRESSION IN T(12;14)(Q15;Q24) UTERINE LEIOMYOMATA ......43 Mapping of Chromosome 14 Breakpoints Relative to RAD51L1..................................................... 43 Identification of EST Markers Significantly Altered in Expression by t(12;14)............................... 44 - 1 - Initial Characterization of Novel 12q15 Transcripts A15, B6, and D12 in Normal Tissue and in UL .......................................................................................................................................................... 45 STATEMENT OF PREMISE AND DEVELOPMENT OF HYPOTHESIS ....................................46 FORMAL STATEMENT OF HYPOTHESIS..........................................................................46 Chapter 2: Materials & Methods................................................................................47 RAD51L1 DISRUPTION BY T(12;14)(Q15;Q24) IN A UTERINE LEIOMYOMA .................47 cDNA Cloning .................................................................................................................................. 47 Polymerase Chain Reaction (PCR).................................................................................................. 49 MAPPING OF ADDITIONAL UL CHROMOSOME 14 BREAKPOINTS .................................49 IDENTIFICATION OF NOVEL TRANSCRIPTS OVEREXPRESSED IN UL..............................50 Exon Trapping.................................................................................................................................. 50 EST Markers .................................................................................................................................... 50 Northern Hybridizations .................................................................................................................. 51 Rapid Amplification of cDNA ends (RACE)..................................................................................... 52 t(12;14) Tumor And Normal Myometrial Samples........................................................................... 54 Tumor and Normal Myometrial Samples Not Analyzed Cytogenetically......................................... 54 cDNA Synthesis from Frozen Tissue Samples.................................................................................. 57 Relative Reverse Transcriptase-Coupled Polymerase Chain Reaction (RT-PCR)........................... 57