Michael Wangler, MD - US grants

DESCRIPTION (provided by applicant): The proposal describes a five-year mentored laboratory training experience designed to lead to an independent academic career in clinically-relevant basic science. The applicant holds an M.D. degree, and has completed specialty training and board certification in pediatrics and is currently completing sub-specialty training in Medical Genetics. The career development plan includes a period of mentored research training which will include learning research techniques and concepts supplemented by didactic training, seminars, lab meetings, journal clubs, national and international meetings, an advisory committee and meetings with the mentor. The research environment provides the best intellectual environment and the best technology available and gives the applicant the opportunity to be guided in learning powerful techniques such as electron microscopy and electrophysiology. The research seeks to improve our understanding of peroxisomal biogenesis disorders at the molecular level by focusing on peroxisomal biogenesis in Drosophila. Peroxisomes are ubiquitous organelles in eukaryotes, generated by a set of evolutionarily conserved proteins encoded by the pex genes. Mutations in pex loci in humans lead to Peroxisomal Biogenesis Disorders (PBD), diseases with devastating neurologic consequences. The nervous system complications of PBD have been characterized but their mechanism is not known. Drosophila provides a good model system to add to our knowledge of peroxisomal biogenesis defects. Very little is known about the pex genes in Drosophila. We have selected pex2 and pex16 for analysis. Because of the novelty of this approach very minimal tools are available to study peroxisomes in Drosophila, we will therefore generate additional tools to allow for a precise and thorough analysis of pex2 and pex16 including null alleles, tagged genomic constructs, and assays for the characterization of peroxisome structure and function. We will explore the interaction of peroxisomes with other organelles by testing the hypothesis that peroxisomal loss affects mitochondrial function. Finally, we will build on our preliminary data showing electrophysiologic defects in pex16 P-element insertion mutants by defining the mechanisms by which defective peroxisomal biogenesis lead to exocytic defects in synaptic transmission. This research will create a broader understanding of the effect of peroxisomal biogenesis on the nervous system. This could have clinical implications for patients with peroxisomal disorders. This research will also occur in an environment dedicated to training the applicant to pursue this research further as an independent scientist. PUBLIC HEALTH RELEVANCE: This research studies a group of diseases in which the patient lacks an important cellular organelle named peroxisomes leading to problems in body chemistry. Researchers use fruit flies with similar genetic alterations to study the problems that result from lacking peroxisomes. The research could lead to a better understanding of these diseases for which there are no effective treatments.

PROJECT SUMMARY DROSOPHILA CORE The goal of the Drosophila Core (DC) of the Model Organism Screening Center (MOSC) is to provide in vivo functional information for evolutionarily conserved genes and variants that are likely to underlie specific pathological phenotypes of UDN patients. This will support the molecular diagnosis and facilitate the development of potential therapeutic strategies for rare diseases. To accomplish this goal, the leadership team proposes to use numerous novel strategies, technologies and transgenic strains. Using these tools, our DC will rapidly generate strong loss-of-function (LOF) alleles of potential disease associated genes in Drosophila and characterize their phenotypes. Next, we will determine if the human homolog can rescue these phenotypes to provide a strong molecular link between the Drosophila and human homologs. If rescue is obtained, we will examine the effect of the UDN variants. We will further characterize expression patterns and assess subcellular localization of proteins using the MiMIC and CRIMIC technologies, a novel gene/protein tagging strategy that we recently developed. For genes that have strong LOF phenotypes that can be rescued by the human cDNA, we will perform detailed secondary phenotypic analyses to determine the role of the gene/protein in the organ systems that are affected in the UDN human patients. The proposed pipeline will be highly cost efficient compared to many other options, and will generate high-quality reproducible data in Drosophila that can be directly or indirectly linked to human disease phenotypes.

PROJECT SUMMARY The proposed Phase II continuation of the Model Organism Screening Center (MOSC) for the Undiagnosed Diseases Network (UDN) builds on extensive tools, reagents, and pipelines that we established in Phase I. The leadership team represents international expertise in Drosophila, zebrafish, and human medical genetics. In Phase I, the MOSC developed an interface in the UDN Gateway that supports submission of cases, genes, and variants that the clinical sites propose for model organism studies. The MOSC also developed MARRVEL (www.MARRVEL.org), an online tool that integrates human and model organism data and helps to prioritize variants. The MOSC further established collaborations with independently funded collections of rare disease cohorts at Baylor College of Medicine (BCM) that we use to identify matches to UDN phenotypes and genetic variants. The MOSC holds regular conference calls with clinical sites and provides tools that help them to select variants. The MOSC will continue calls with the sites to review informatic analyses and then assign variants (estimated at 45 per year) for in-depth model organism studies of variants and genes. In the Drosophila Core at BCM, approximately 30 genes and variants per year are studied with innovative technology in flies. For 15 additional genes/variants that affect vertebrate-specific genes or biology, the Zebrafish Core at the University of Oregon will induce new mutations in zebrafish with CRISPR/Cas9 followed by high throughput phenotypic analyses. The MOSC will also develop tools and reagents for future variant studies in Drosophila and zebrafish for an additional 100 genes per year. We will share these additional resources with the broader research community through an innovative ModelMatcher service and will also prioritize genes for generation of mouse knock-outs in collaboration with the Knockout Mouse Phenotyping Project (KOMP). Thus, the proposed center will provide informatics selection, human genetics expertise, broad versatile model organism resources, and in-depth studies of UDN cases to aid in diagnosis. The MOSC employs the most innovative technologies in human genomics and Drosophila and zebrafish genetics, based on extensive previous experience modeling human disease.

PROJECT SUMMARY Here we propose to advance the goal of NHGRI to implement genomic medicine and focus on individuals who have not been able to afford DNA testing. The research takes place in the Department of Molecular and Human Genetics at Baylor College of Medicine (BCM) and Texas Childrens Hospital (TCH). Our team of clinicians, geneticists, computer scientists, genomicists and model organism researchers has had a five-year term of success with the Undiagnosed Diseases Network (UDN) Model Organisms Screening Center (MOSC). This has included successfully identifying a number of new disease genes such as EBF3, IRF2BPL, NACC1, TBX2, TOMM70, CDK19, ACOX1, WDR37, and ATP5F1D. We propose to recruit 100 individuals from an underserved population in Houston, Texas with suspected rare disease and without the means to pay for DNA sequencing through insurance. We will provide whole-exome sequencing which will generate a CAP/CLIA report that we anticipate could diagnose 35-40 individuals per year. The remaining individuals will then be converted to a family-based trio exome design. All the sequencing costs of this project will be covered by philanthrophic donation to our hospital and are not budgeted to the grant. We will make every effort to diagnose the remaining 60 individuals per year through machine learning and informatics using the MARRVEL platform, Drosophila functional studies of candidate genes and through ongoing 6 month, 12 month and 2 year follow-up with the patients where we will use matchmaking efforts such as GeneMatcher and Matchmaker exchange as well as our own genomic databases from the UDN and other studies to come to a genetic diagnosis. All subjects will receive genetic counseling from a trained team and will provide us with valuable medical, psychological and social data to guide how genomic implementation in an underserved population is perceived, impacts care and impacts the family. This work will not only produce novel insights into rare disease, diagnosis for undiagnosed families and an expanded role for genomics, it will guide us in the future to provide genomics and functional research to serve all individuals regardless of their ability to pay.

PROJECT SUMMARY Peroxisomes are fundamental sub-cellular organelles present in all eukaryotic cells. Peroxisomes participate in a number of biochemical pathways including catabolism of very-long-chain fatty acids, branched chain fatty acids, and bile acids, the biosynthesis of plasmalogen lipids, and mediate a number of crucial biological processes. Human diseases due to lack of peroxisomes are severe multisystem diseases These conditions, called peroxisome biogenesis disorders, Zellweger-spectrum disorders (PBD-ZSD) illustrate how peroxisomes are required for human health. Insights from studies in PBD-ZSD have been applied to common disease such as Alzheimer?s disease. In order to probe the molecular mechanisms that underlie PBD-ZSD I use genomics, untargeted metabolomics and genetic technology in Drosophila. I am a dedicated physician-scientist devoting my career to the study of PBD-ZSD, having made several contributions. First, I have used metabolomics to define a pattern of biochemical abnormalities or a ?PBD-ZSD Metabolome? a characteristic signature of these diseases that interestingly includes reduced sphingomyelins, a previously unrecognized biomarker of PBD-ZSD. Second, my lab has used innovative genetic technology in Drosophila to further probe consequences of peroxisomal biology for neurons. For example, in a large forward genetic screen on the Drosophila X- chromosome we identified novel genes that alter peroxisomes in vivo and we have shown these are candidate neurological disease genes. Finally, using genomics I have developed a track record of diagnosing undiagnosed individuals who have novel or unique mutations in genes such as ACOX1, DNM1L, PEX1 and PEX16, and these studies point to novel genetic mechanisms for peroxisomal disease. Based on my studies of sphingomyelin I hypothesize that peroxisomal dysfunction leads to altered composition of the side-chains of sphingomyelins resulting in impaired neurological function in PBD-ZSD. I also propose, based on animal model studies that peroxisomes are required both during development and during aging for nervous system function. Finally, my preliminary data suggests that de novo mutations can impact peroxisomal genes, which are traditionally considered ?autosomal recessive? and can be an important mechanism for peroxisomal disease. In this proposal we use clinical studies, unique model organism technology and genomic and metabolomic technology to test these hypotheses and advance studies of PBD- ZSD towards better diagnosis, treatment and improved quality of life for patients.