Raymond L. White - US grants

We propose to develop informative, multi-allelic genetic marker loci in the human for the purpose of constructing a linkage map and, ultimately, for the stusy and diagnosis of human genetic disease. Our marker system is based on DNA sequence polymorphisms which are detected by recombinant DNA probes which define specific loci and restriction enzymes which sample DNA sequence. We will construct an archival resource comprised of DNA and cell lines from a large number of individuals from within a single large MOrmon pedigree. Genotypes will be determined by pedigree analysis.

Several thousand clinical disorders with documented Mendelian inheritance remain of unknown genetic etiology. Recently, the approximate chromosomal location of the unknown mutant gene at the origin of several clinical diseases has been determined by linkage to newly available DNa markers. Furthermore, knowledge of map location has led to the eventual identification of several mutant genes. Precise mapping information, therefore, can make many important human genes that are defined only by their morbid phenotype accessible to gene cloning. Primary genetic maps of markers adequate for the initial localization of mutant genes are now available for most human chromosomes and should be available soon for the remainder. These widely spaced markers usually provide an average resolution of 10 to 20 cM. Gene identification and cloning, however, require mapping to Within 1 cM in order to confine the search for a mutant gene to a region of only a few metabases, containing only a few candidate genes. Precise mapping of mutant genes by linkage analysis in families require a genetic map of a much greater density. We propose to isolate 2,500 highly polymorphic DNA markers for a specific subset of human chromosomes. Genotypes will be defined for these markers in a set of 59 families with large sibship. We will construct genetic maps of these markers which should afford a resolution of the order of a Centimorgan. We expect these markers will lead to the identification of the genetic mutation involved in numerous classical Mendelian conditions. They may make significant contributions to many ongoing programs aim at mapping, identifying and cloning genes responsible for common disorders such as cancer, cardiovascular disease, or mental illness. Lastly, these markers should serve as primary reference points in the orientation and ordering of extensive cosmid maps prior to large scale genomic sequencing.

The faculty and administration of the University of Utah propose to continue development of a Cancer Research Center, firmly based on our strengths in cancer research (molecular, cellular, genetic, epidemiological, pre-therapy, and therapy) and certain unusual demographic characteristics of our region and its population. The scientific programs involve over 80 funded Investigator-members In 3 schools and colleges of the University and 4 Salt Lake city hospitals. The overall goal of the Cancer Center is to contribute to the national effort to decrease the morbidity and mortality of cancer. The means by which we propose to approach this goal can be stated as our objectives. 1. Facilitate and stimulate interdisciplinary research by providing an academic and administrative link among investigators. 2. Strengthen existing research programs by providing support for core equipment and services used by multiple investigators. 3. Strengthen our programs by assisting in recruitment of faculty to add our strengths and to fill current gaps in expertise. 4. Facilitate transfer from the laboratory to the clinic new findings which may reduce the incidence of cancer, facilitate its early detection, or improve its treatment. 5. Provide a scientific environment attractive for graduate research education and develop financial support for graduate students, the future leaders in cancer research. 6. Develop improved and augmented facilities for cancer research. The interdepartmental center will derive financial support from a variety of institutional, state, private and federal sources, including this Cancel Support Grant.

health science research analysis /evaluation; biomedical facility;

The Huntsman Cancer Institute (HCI) is committed to the development of new approaches to reducing the morbidity and mortality due to cancer. The main focus is on the major adult epithelial tumors, breast, prostate, colon, and aerodigestive. A major research theme of the HCI is the development of new approaches to very early diagnosis and treatment based on the emerging knowledge of the earliest molecular changes in tumorigenesis. This builds on our traditional strength in molecular genetics, which has opened a window into the earliest changes in carcinogenesis. Traditional, discipline-oriented Programs will continue to underlie our primary organizational structure. However, our primary approach will be implemented through a series of new, interdisciplinary, translational Programs focused on the molecular genetics, biology, diagnosis, and therapy of the individual tumor types.

This study will perform genetic linkage analyses of breast cancer families to identify kindreds in which breast cancer is linked to the BRCA1 losus at chromosome 17q. Also, female carriers of the disease-associated BRCA1 allele will be identified, the breast epithelial cells will be cultured and characterization of the in vitro phenotype of BRCA1 cell strains will be done.

The goal of this project is to understand the cellular roles of the Adenomatous Polyposis Coli (APC) protein, with special attention to its potential functions in the cell nucleus. These studies have a central theme: the interaction between APC and the cell adhesion/signaling molecule beta-catenin. Beta-catenin constitutes the final step in the Wnt signaling pathway, complexing with Tcf/LEF-1 transcription factors, translocating into the nucleus and activating expression of target genes. In addition to its role in Wnt signaling, beta-catenin is a functional component of cell-cell adhesion junction, placing it in a prime position to link intercellular communication with intracellular signal transduction. Because APC can promote proteasome-dependent degradation of Beta-catenin, it is the ultimate regulator of beta-catenin activity. Given its ability to regulate beta-catenin protein, it is likely that control of beta-catenin mediated transcription is a major function of APC. Aim 1 proposes to employ powerful genomic screening technologies in a comprehensive scan of beta-catenin/Tcf-mediated transcription in the presence of absence of functional APC protein. The rationale is two-fold: the events downstream of APC/beta-catenin are key mechanisms in tumorigenesis, and the beta-catenin-induced genes are potential targets for anti-polyp drug development. APC protein and beta-catenin can interact in the cell's nucleus as well in the cytoplasm, raising the possibility that APC targets nuclear beta- catenin for proteasome-dependent degradation. Aim 2 uses a combination of protein half-life assays, cell fractionation procedures, and proteasome- inhibiting agents to explore the possibility. The presence of APC in both the cytoplasm and the nucleus suggests that APC relays Wnt signaling information between these two compartments. This hypothesis, combined with the identification of a nuclear export signal in the APC protein, provides the basis for Aim 3, an exploration of whether APC is engaged as a nuclear shuttle protein. Microinjection of nuclei with purified peptides and antibodies, followed by immunofluorescence analysis will be used to track the movement of APC and beta-catenin between the nuclear and cytoplasmic compartments under a variety of conditions. Together, the experiments proposed here focus on understanding the molecular mechanisms underlying the regulatory functions of APC protein and determining how mutations in APC alter these functions, leading to colon polyps and cancer.

DESCRIPTION: (Applicant's Description) Inherited predispositions to specific cancers due to high penetrance alleles of genes such as APC and BRCA1 account for only a small percentage of our populations cancer burden. Genetic epidemiological evidence suggests, however, that a much higher percentage of individuals may be at risk due to the inheritance of alleles, or combinations of alleles, that individually show only a relatively low cancer penetrance. This makes it difficult to analyze the associated patterns of inheritance, localize the loci and identify the specific genes involved. Large sample populations are now required to map and identify these genes. We propose establishing a consortium consisting of the Universities of Colorado, New Mexico and Utah whose primary purpose will be to enroll a study population and create an infrastructure to support this next generation of exploration of the genetics of cancer. A population-based approach is needed to understand the population significance of future findings. State cancer registries provide an appropriate avenue of ascertainment. It is also important to develop a study population enriched for predisposed individuals. Our approach, therefore, is to ascertain individuals with one or more relatives who have been diagnosed with the specific cancer. Specifically, we propose initially to contact each individual diagnosed with breast, prostate, or colorectal cancer in calendar 1998 and 1999 and determine: 1) whether they are willing to participate and 2) have first-degree relatives affected with their same cancer. If so, they will be recontacted and asked to provide more detailed information regarding family history, as well as demographic and epidemiologic information. Selected index cases will be requested to complete questionnaires, which will include the names of their first-degree relatives and their permission to contact these relatives. The first-degree relatives will then be contacted and invited to enroll as members of the Cancer Genetics Network. This approach will create a study population of family members who have had cancer and who are at risk for cancer. We will also offer enrollment to appropriate individuals who have already contacted our high-risk clinics or are enrolled in ongoing clinical protocols of familial cancer; for example, Colorado will extend their current research interests by collecting lung cases. In addition, we will establish or enhance clinical and laboratory core facilities, as well as the necessary capabilities in genetic counseling, informatics and database management. As study groups are enrolled, DNA samples may be collected and pilot projects initiated; these include sib-pair studies and candidate-gene analysis funded by the Huntsman Cancer Institute and the University of Colorado Cancer Center.

The adenomatous polyp is an almost essential intermediate in the progression of normal intestinal epithelium to gastrointestinal cancer. Strong support for this view has been provided by clinical and molecular genetic studies of the inherited syndrome, adenomatous polyposis coli (APC), and of sporadic colorectal polyps, thus providing one of the most important windows into the pathogenesis of colon cancer. In pursuing the molecular basis of APC, we identified a tumor suppressor gene, APC, that is now recognized as a primary step in polyp development and progression to cancer. The theme of this Program focuses on the adenomatous polyp: laboratory analysis of its genetic origins, and the molecular and cellular biology of the polyp as a precursor to cancer. Studies of the cellular and molecular changes resulting from mutation of APC form the basis for Project one; our investigations will address functional aspects of the APC protein with regard to its subcellular localization, post-translational regulation and novel binding partners. The second will employ cellular, molecular, biological and genetic approaches to examine the role of cyclooxygenase, a key target of effective chemopreventative agents, in the genesis and progression of adenomatous polyps. Project 3 examines the molecular mechanisms connecting mutations in genes encoding DNA mismatch repair enzymes, which are implicated in a separate form of inherited colon cancer, with observed changes in colon epithelial cells. The fourth seeks to determine the genetic basis for heterogeneity in an attenuated form of the APC syndrome. The fifth project is designed to identify the basis for familial forms of colon cancer that cannot be attributed to mutations in APC or mismatch repair genes. Our preliminary results suggests that several unidentified genes likely account for additional forms of familial colon cancer; in the course of this project, we will identify affected families and carry out initial linkage studies. The Program will support administrative, clinical, tissue procurement and cell culture cores. Use of these core facilities is widespread, as there is extensive interaction among the various projects. In summary, this Program provides an integrated approach to the major precursor to colon cancer, the adenomatous polyp. Our approach is multi-disciplinary with interactive Projects based on molecular and cellular biology, genetics, and clinical characterization. These studies of the adenomatous polyp of the colon offer an unusual and important opportunity for the discovery of new approaches to the prevention of colon cancer through detection and elimination of its precursor, the adenomatous polyp.

DESCRIPTION (provided by applicant): Tobacco use is the leading cause of preventable disease, disability, and death. Excessive alcohol consumption is the number-three cause of preventable death in the United States. Despite the fact that addiction represents more than 40% of brain-related illnesses, there is a dearth of innovative treatments. Alcohol and nicotine addiction are often treated as separate disorders even though most people with alcohol use disorders also smoke, and continued tobacco use during abstinence from alcohol leads to significantly higher relapse rates. These findings suggest that alcohol and nicotine addictions may develop, and depend on, common pathways. Recently, a large number of human genetic association studies have implicated the neuronal nicotinic receptors (nAChRs), such as the a5 nAChR subunit as playing a critical role in developing alcohol and nicotine dependence processes and recent molecular studies indicate that the a5 nAChR subunit changes the activity of a4b2* nAChRs. Our main objective is to apply a multidisciplinary approach that integrates basic and clinical research to define the molecular basis of the role of nAChRs in ethanol and nicotine consumption and substance use disorders with the goal of generating medications and treatment strategies. In the first part of the project, we will combine behavior and electrophysiology studies and there are two objectives: 1) to characterize the role of the a5* nAChRs in the behavioral effects of ethanol and nicotine and varenicline and 2) to measure the expression and synaptic properties of a4b2* nAChRs responses in dopamine neurons in the ventral tegmental area, a brain region that plays a key role in the reinforcing properties of nicotine and ethanol. These studies have been greatly facilitated by an innovative drinking model we developed that enables ethanol and nicotine to be consumed together without the need for saccharin. In the second part of the project, we will combine clinical studies and genetics to determine whether genetic variants in nAChRs correlate with response to varenicline in a cohort clinically characterized for nicotine dependence and hazardous alcohol use. There are two major objectives: 1) to measure the effectiveness of varenicline, as a treatment for hazardous alcohol use and 2) to genotype the subjects and assess whether polymorphisms in the genes encoding for nAChRs moderate the effect of varenicline to reduce heavy drinking. The linking of genetic analyses with human responses to varenicline will provide a means by which we can measure both the efficacy of varenicline for diminishing alcohol use disorders and to determine whether there are underlying genetic differences in responses to varenicline. Our overall goal is to accelerate the development of more effective medications, and to improve and personalize treatment strategies for substance use disorders. PUBLIC HEALTH RELEVANCE: We have developed a multidisciplinary collaborative research program focused on defining the molecular basis of the role of neuronal nicotinic receptors (nAChRs) in ethanol and nicotine consumption and substance use disorders with the goal of generating medications and treatment strategies. We propose to combine behavior and electrophysiology to define the molecular basis of the role of a5* nAChRs in ethanol and nicotine consumption and determine whether genetic variants in nAChRs correlate with response to varenicline in a cohort clinically characterized for nicotine dependence and hazardous alcohol use. The research garnered from this proposal will facilitate the development of medications that target nAChRs for the treatment of alcohol and substance use disorders.