Overall Approach of the laboratory The laboratory uses a translational research approach to study human malformations. In the clinical arena (study HG200388-01), we operate several clinical research protocols to assess the range of severity, spectrum of malformations, natural history of pleiotropic developmental and overgrowth disorders and therapeutic studies. We use the tools of modern molecular biology to determine the molecular pathogenesis of these disorders. These include high throughput sequencing, positional cloning, microarray expression and microarray CGH analysis, cell and tissue culture studies to assess cell biologic functions and abnormalities of gene products, and the creation and analysis of animal models of human genetic disease (mouse and zebrafish), and now we are moving into therapeutic approaches to the diseases. Overgrowth syndromes Building on our prior successes with PIK3CA-related fibroadipose overgrowth (Lindhurst et al, 2012) and Proteus syndrome (Lindhurst et al, 2011), we have now identified targets for therapeutic intervention. These discoveries have shown that the mutations that cause mosaic overgrowth also are major contributors to the pathogenesis of cancer. Because of the intense work in the pharmaceutical industry on cancer drugs, we can exploit those resources and repurpose them for therapeutics of overgrowth. We are using single-cell cloned fibroblast lines from patients with Proteus syndrome to test therapeutic agents. By titrating dosage of agents against assays of cell death and proliferation we have measured the potential effect of these agents for future clinical development. These experiments are being completed and have been submitted for presentation at the Am Soc Hum Genet meeting in 2014. We have also successfully correlated the distribution of AKT1 mutations with the type of skin manifestations (Lindhurst et al, 2014) and we are currently working on a detailed mutational analysis of an autopsy case of Proteus syndrome where we have correlated mutational load with histology in more than 50 tissue samples, by far the most detailed study of its kind in humans. We have also convened an international group to set clinical standards of diagnosis and workup for patients with PIK3CA-related overgrowth and have founded an international consortium to develop cooperative trials for pharmacologic treatment of this family of disorders. Modeling Proteus syndrome Following on our discovery of the cause of this disorder in 2011 we have been pursuing a strategy to model this disorder. While the Happle hypothesis (mosaicism for a mutation lethal in the non-mosaic state) is completely consistent with all recognized features of the disorder, it is impossible to prove this in human studies. To that end, we have undertaken efforts to create a mouse model of Proteus syndrome by creating a conditional knock-in allele for the p.Glu17Lys mutation (the mutation that affects all known patients with this disorder). We have made excellent progress on this project with extensive genetic engineering accomplished to make and validate the construct. The construct has been injected into animals and is currently being screened. Genotype-Phenotype studies in mosaic disorders We have developed tissue sampling and culture methods coupled with custom-engineered mutation assays to detect the 5 known mutations in these genes in patients thought to be affected with this disorder and now routinely perform this as a patient screening method. This will allow us to support the activities in our clinical research project (HG200388-01) to reclassify these phenotypes. For any patients in whom this analysis is negative, we feed these samples into our next generation sequencing analysis pipeline using the intrapatient exome mosaic comparison approach that we have pioneered. Exome analysis of novel germline phenotypes In this past year we have used exome analysis to evaluate about 20 distinctive phenotypes, with the elucidation of probable genetic etiologies for 5 of these (which is quite similar to the success rates for other groups. We are currently pursuing two of these with functional studies to assess the pathogenicity of the mutations we have identified.
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