Bruce Hamilton - US grants

This core unit provides crucial molecular resources for each of the research projects, including: preparation and cataloging of DNA sample preparation and storage, genotyping, physical mapping and genetic marker development, informatic support, DNA sequencing, and identification of polymorphisms at targeted loci as candidate functional changes. 1. Preparation and storage of genomic DNA (gDNA). Consistent and reliable DNA samples for genotyping (in analyses of linkage, association, and candidate functional sequence changes) are essential to Projects 1 and 2. We collect 50 ml of EDTA-anticoagulated whole blood from each individual in the clinic. We prepare gDNA from the white cells in small amounts and the remaining blood is stored frozen for future use. 2. DNA Sequencing. Strategy. Each of Projects 1-5 has suggested biological candidate genes as potential contributors to genetic variation in autonomic control of blood pressure. Testing these candidate genes requires linkage, association, and haplotype analysis of known markers and more particularly of single point and haplotype alleles enriched in hypertensive subjects and their first-degree relatives. 3. Genotyping. Strategy and Methods. As described under Sequencing above, we use a two-tiered strategy to maximize efficiency. (1) The first tier involves direct PCR-based resequencing of candidate loci to improve the density and appropriateness of polymorphism selection for larger-scale genotyping. (2) In the second tier of the strategy, derived marker combinations are selected and used to allow efficient discrimination of haplotypes. Obtained genotype data is subsequently analyzed for statistical significance and population parameters in the informatics core, Core B (Informatics and statistical genetics), including additional population-based controls for likely accuracy. 4. Allele-specific expression analysis. Strategy. To detect the effects of allelic variants define in this program on expression level, this core will provide expression analysis based on allelic detection from genotyped samples and heterologous promoter fusions.

[unreadable] DESCRIPTION (provided by applicant): Disruptions in the normal development of the cerebellum result in clinically significant malformations whose mechanistic bases are poorly understood. This proposal uses a combination of established and emerging genetic and genomic methods to dissect molecular mechanisms of cerebellar vermis malformation in a new mouse model resembling Dandy-Walker malformations. Homozygotes for the nur12 mutation show show nearly complete agenesis of the vermis and choroid plexus, cystic dilation of the fourth ventricle, and anterior malrotation of cerebellar structures within the posterior fossa. As preliminary data for defining molecular mechanisms relevant to features shared between this mutant and human patients, the applicant has identified a null mutation of a zinc finger transcription in nur12 mutants. The applicant has begun to identify developmental sequelae to this mutation, including profound effects on proliferation of neural progenitor cells. The three specific aims will (1) establish an allelic series at the locus and investigate sources of interindividual variation in the severity (hemispheric involvement) of null allele; (2) define cellular and developmental mechanisms of the nur12 malformation, including tests for the roles of BMP/SMAD and EBF signaling pathway, through a combination of in situ labeling, marker gene analyses and signaling response measurements in culture; and (3) identify molecular targets of ZFP423 activity in cerebellum development by transcriptional profiling at sequential stages of development and by identifying overlaps in promoter occupancy among ZFP423, BMP-activated SMADs and EBF factors using a highly parallel platform for genome-wide location analysis for transcription factor binding. Relevance: This proposal will identify mechanisms relevant to the Dandy-Walker malformation - a severe defect found in 1/25,000 -1/30,000 births. Preliminary results suggest that the mechanisms acting here affect the ability of precursor cells to continue dividing, which may have therapeutic application for both prenatally diagnosed malformations and pediatric brain cancers. [unreadable] [unreadable] [unreadable]

PROJECT SUMMARY The UCSD Genetics Training Program (GTP) is designed to provide advanced training in Genetics and Genomics to predoctoral students beginning in their second graduate year. While several degree-granting umbrella programs at UCSD include genetics or genomics research, GTP is uniquely the point of integration across programs and builds both lateral and vertical cohorts of PhD students interested in the history, practice and future applications of Genetics and Genomics in life and health sciences. Mentor laboratories encompass a broad range of basic science and clinical/translation research and span a range of organisms?including microbial, plant, experimental animal, and human subjects?but share a focus on genetic, epigenetic and genomic mechanisms and approaches. Students who have committed to thesis research in one of these laboratories enter the GTP and may be selected for support by this training grant. The GTP curriculum cuts across traditional graduate programs in participating schools, divisions and departments. GTP students take graduate core courses in Genetics and Quantitative Methods and participate in a weekly journal club during graduate years 2-4. The journal club includes rotating topics in contemporary genetics or classic, landmark papers relevant to the intellectual development of the field. GTP students select quarterly topics in consultation with participating faculty. Journal club papers and discussion are used to assess methods and logic, experimental design, data analysis, and responsible conduct of research in additional to the quarterly research topics around which they are selected. A program-wide annual retreat, organized by year 5 students and a standing faculty committee, includes invited outside keynote speakers, research presentations by program faculty and students. GTP has more than 30-40 students training in any given year, for whom we are requesting 16 training grant slots.

DESCRIPTION (provided by applicant): The ciliopathies comprise a spectrum of disorders unified by defects in primary cilia. Clinical presentations range from primary involvement of a single organ (most often hindbrain, kidney, liver or eye) to more severe presentations, such as Meckel syndrome, with severe and pleiotropic developmental phenotypes in several organs. Several genes have been and continue to be identified for ciliopathy disorders, with the overwhelming majority encoding structural components of primary cilia. Regulatory genes that control cilium-dependent signaling and modifier genes that control the outcome of ciliary defects are only beginning to be tied to pathogenic mechanisms. This project focuses on the synthetic lethal interaction between a transcriptional regulator that control ciliary phenotypes, Zfp423, and an unknown modifier gene. Zfp423 encodes a 30-zinc finger transcription factor required in several signal transduction pathways and in multiple organ systems. Animals that lack Zfp423 have prominent brain malformations with a high frequency of hydrocephalus as well as defects in several peripheral tissues. The distribution of phenotypes in surviving animals is dependent on both modifier genes and apparently stochastic processes. However, on the most commonly used strain background, no mutant animals survive. Genetic mapping identifies a single major locus linked to embryonic and perinatal lethality. The aims of this proposal will identify this synthetic lethal modifier locus, elucidate its mechanism and place it in the context of other genes in the ciliopathy network.

Project Summary: This project focuses on cellular, genetic, and molecular mechanisms that underlie developmental abnormalities in ZNF423-realted ciliopathy. The ciliopathies comprise a spectrum of disorders unified by defects in primary cilia. Clinical presentations range from primary involvement of a single organ (most often hindbrain, kidney, liver or eye) to more severe presentations, such as Meckel syndrome, with severe and pleiotropic developmental phenotypes in several organs. Several genes have been identified for ciliopathy disorders, with the overwhelming majority encoding physical components of primary cilia. Regulatory genes that control cilium-dependent signaling and genetic modifiers that control the outcome of ciliary defects are only beginning to be tied to pathogenic mechanisms. This project focuses on the role and mechanisms of ZNF423, a constitutively nuclear transcriptional regulatory protein mutated in Joubert syndrome (JBTS19) and nephronophthisis (NPHP14) patients. ZNF423 is thought to comprise an integrative node among several transcriptional complexes that respond to classical developmental signals. As ZNF423 expression is also developmentally dynamic, the extent to which phenotypes are cell autonomous, rather than defects in reciprocal intercellular signaling, remains unclear. Aim 1 will use recently developed genetic tools (MADM) to assess cell autonomy by creating a simple platform for inducing and marking mitotic clones in situ. Because patient mutations are individually rare, often found on only one allele, and found in subjects with a range of presentations, it remains unclear what fraction of patients is attributable to ZNF423 and which ZNF423 mutations are truly pathogenic. Aim 2 will use genome editing in a sensitive and well-validated mouse model to test phenotypic effect of patient-derived mutations. It remains unclear whether specific targets of ZNF423 activity might be able to modulate phenotype. Aim 3 will determine whether decreasing activity of newly identified ZNF423-repressed genes, whose expression is increased in both knockdown cell and mutant animals, can improve cilium-dependent functions and emergent phenotypes in the Zfp423 mouse model.