Robert F. Clark, Ph.D. - US grants

Genetic linkage studies in early onset-familial Alzheimer's disease (AD) families have determined that mutations in at least three loci will cause the disease: the amyloid beta-protein precursor gene, the presenilin-1 gene (PS-1), and the presenilin-2 gene (PS-2). While it has been reported that beta-amyloid production is abnormal in fibroblasts from families carrying PS-1 and PS-2 mutations, and a homologues protein in C. elegans affects Notch signaling, the function of the presenilin gene is presently unknown. Therefore, the long-term objective of this application is to determine the function of the presenilin protein and other presenilin- interacting proteins. Drosophila melanogaster is an ideal model organism for determining the function of uncharacterized genes, which in this case is made even more ideal as most studies of Notch signaling have been carried out in Drosophila. Using the high conservation among conservation among presenilin genes, our lab has successfully coned the gene in Drosophila. The first specific aim of this project will be to characterize this gene in Drosophila, studying its gene structure, its developmental transcription pattern, and its localization in embryos, larvae, pupae, and adult flies, DNA sequencing. Southern and Northern blotting techniques, and light microscopy will be used to carry out this part of the project. The second specific aim will be to produce null mutations of this gene and study their effects in Drosophila. P-element insertion mutagenesis will be used to produce the mutant flies, and the flies will be characterized genetically. The third specific aim will be to determine which genes interact with the presenilin gene. Fly genetics and yeast two-hybrid approaches will be used to find these presinilin- interacting genes. This project will lead to a greater understanding about the biology and function of the presenilin genes, the major cause of early- onset familial Alzheimer's disease. By determining the functions of the presenilins, and their interactions with other genes, a greater understanding of the pathogenesis of AD will be gained, which may lead eventually to improved treatment of the disease. Additionally, a long- term study of these presenilin-interacting proteins will help us to identify other human loci which may cause or influence AD in both early-onset and late-onset cases.

DESCRIPTION (provided by applicant): The overall objective of the Molecular Atlas of Lung Development Program (LungMAP) is to develop a molecular atlas of the developing lung from human and mouse to serve as a unique reference resource for the research community. The objective of our application is to serve as the Data Coordinating Center (DCC) for the Molecular Atlas of Lung Development Program (LungMAP-DCC). We have taken several innovative and cost-effective approaches to build the LungMAP-DCC that will greatly benefit the research community and promote a greater understanding of molecular lung development. We have created a multi-disciplinary team of investigators from Duke Clinical Research Institute, RTI International, and Cincinnati Children's Hospital Medical Center to lead and operationalize the LungMAP-DCC. We are leveraging the strengths of two highly experienced investigators as multiple PIs (Drs. Palmer and Clark) and we will apply novel data management and bioinformatics approaches to create the Bioinformatics REsource ATlas for the Healthy lung (BREATH) database. The DCC will perform data collection, integration, and analysis; develop and maintain the LungMAP database and website; and coordinate research activities of the Human Tissue Core (HTC) and the Research Centers (RCs). We will complement these goals with the following Specific Aims that support the success of consortium: Specific Aim 1) Administrative Coordinating Infrastructure: The DCC will serve as the administrative infrastructure that facilitates collaboration among our multidisciplinary team, RC, and the HTC by 1) providing expertise in molecular lung development, clinical design, statistics, and bioinformatics; and 2) the creation of consortium-wide priorities and policies, communication plan, and resource catalog and Specific Aim 2) LungMAP Portal and BREATH (Bioinformatics REsource ATlas for the Healthy lung): The DCC will build the centralized data repository and public interface for LungMAP by creating and managing 1) BREATH; 2) LungMAP Website; 3) standard operating procedures for data management; 4) existing lung development results; 5) ontologies for lung development, structures, and cross species comparisons; 6) experimental data from RCs and biologic sample data from the HTC; and 7) novel tools to analyze the experimental data. This application leverages the complementary strengths of multi-disciplinary research teams with clinical pulmonary expertise, basic understanding of lung molecular development, successful leadership of multicenter research networks, and in-depth computer programing, database development, and bioinformatics skills that will create a highly functional, integrated, publically accessible platform that will facilitate analysis of data generated by the RCs and HTC to advance our understanding of molecular lung development. (End of Abstract)

? DESCRIPTION (provided by applicant): Preterm birth (PTB)-related disorders are the leading cause of perinatal morbidity and mortality. Inflammation exhibits a strong association with these disorders; cytokines are markers for inflammation. Several studies have suggested that fetal genotypes may contribute to the inflammatory response at the feto-maternal interface, which may have implications for membrane rupture, spontaneous PTB, and fetal injury. Although associations have been found between maternal genotypes and PTB-related disorders, few studies have examined and identified fetal genotypes that are strongly associated with the morbidities and mortality associated with PTB, and those that have demonstrated modest association require validation/replication. Thus, the primary aims of the proposed investigation are to identify fetal genetic variation associated with higher risk of PTB-related outcomes, and to identify cytokines that explain this association. By identification of high-risk neonates, the proposed analyses will enhance understanding of the pathophysiology and biological mechanisms leading to PTB-associated outcomes and suggest intervention strategies. Using existing data from the Neonatal Research Network (NRN), the proposed plan will involve analyses on 1,030 samples with genome-wide genotype data and protein expression data at five time points for 23 cytokines. The specific aims of this investigation are to (1) delineate relationships between ¿inflammatory cascade¿ genetic markers and their associated cytokine protein expression levels that may be relevant to PTB-related outcomes, and (2) investigate whether any of the ¿genetic markers and¿cytokines studied suggest differential risk of PTB-related outcomes. The results of this study should allow a complete narrative of the associations among cytokine genetic markers, cytokine protein expression and outcomes, including death, neurodevelopmental impairment (NDI), bronchopulmonary dysplasia (BPD), intraventricular hemorrhage (IVH), retinopathy of prematurity (ROP), cerebral palsy (CP), Candida positive culture, hearing impairment, and bilateral blindness. These goals will be accomplished by (1) testing ¿genetic¿ associations to identify variants in one gene that affect ¿cytokine¿ protein expression levels ¿or are associated with PTB-related outcomes¿; (2) using quantitative trait loci (QTL) analysis to look for QTLs that are associated with cytokine protein expression levels ¿or with PTB-related outcomes¿; (3) elucidating associations between protein levels of 23 cytokines and PTB-related outcomes through Cox Proportional Hazards (PH) survival models with time-dependent covariates (TDC); (4) testing for interaction effects between cytokines and other neonatal variables to identify effect modifiers; and (5) relating these cytokine-outcome results to genotype-cytokine results. If successful, a link from gene to cytokine to outcome will result. We plan scientific publications and presentations from this work, and, guided by the results of the analysis with additional samples to conduct detailed explorations of the gene to cytokine to outcome narrative, we will submit an NICHD R01 application.