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). Using these techniques in the past year we have elucidated the etiology of PIGA-related CNS dysplasia (Johnston et al, 2012) and PIK3CA-related fibroadipose overgrowth (Lindhurst et al, 2012). We have performed functional studies to correlate mutations with in vitro functioning of actin and we have performed extensive cell culture and mutation analysis studies to correlate the distribution of AKT1 mutations with the type of skin manifestations (Lindhurst et al, in Press) Genotype-Phenotype studies in mosaic disorders In the coming year we propose to extend our work correlating the manifestations of mosaic fibroadipose overgrowth with the type and distribution of mutations in PIK3CA, which we discovered this past year. We are developing 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. 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 then propose to move on to next generation sequencing analysis using the intrapatient exome mosaic comparison approach that we have pioneered. We have also come into possession of a large number of autopsy samples from a patient with classical clinical features of Proteus syndrome and will perform a histologic-mutational correlation with these samples to understand the range of distribution in a patient and the potential correlation with mutation load. Exome analysis of novel germline phenotypes We have had success over a number of years in delineating the spectrum of mutations associated with GLI3-related disorders. Yet, in spite of these successes, a substantial number of patients have been negative for known mutations. We propose to analyze a cohort of 15 such patients using exome sequencing to elucidate the etiology of these 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. As well, while this activating mutation provides a very tempting therapeutic target, again we are limited in our ability to do preclinical 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 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. We are also 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 can measure the potential effect of these agents for future clinical development.

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Doucet, Meggie E; Bloomhardt, Hadley M; Moroz, Krzysztof et al. (2016) Lack of mutation-histopathology correlation in a patient with Proteus syndrome. Am J Med Genet A 170:1422-32
Keppler-Noreuil, Kim M; Baker, Eva H; Sapp, Julie C et al. (2016) Somatic AKT1 mutations cause meningiomas colocalizing with a characteristic pattern of cranial hyperostosis. Am J Med Genet A 170:2605-10
Bennett, James T; Tan, Tiong Yang; Alcantara, Diana et al. (2016) Mosaic Activating Mutations in FGFR1 Cause Encephalocraniocutaneous Lipomatosis. Am J Hum Genet 98:579-87
Wentzensen, Ingrid M; Johnston, Jennifer J; Patton, John H et al. (2016) Exome sequencing identifies a mutation in OFD1 in a male with Joubert syndrome, orofaciodigital spectrum anomalies and complex polydactyly. Hum Genome Var 3:15069
Keppler-Noreuil, Kim M; Rios, Jonathan J; Parker, Victoria E R et al. (2015) PIK3CA-related overgrowth spectrum (PROS): diagnostic and testing eligibility criteria, differential diagnosis, and evaluation. Am J Med Genet A 167A:287-95
Johnston, Jennifer J; Sanchez-Contreras, Monica Y; Keppler-Noreuil, Kim M et al. (2015) A Point Mutation in PDGFRB Causes Autosomal-Dominant Penttinen Syndrome. Am J Hum Genet 97:465-74
Hannoush, H; Sachdev, V; Brofferio, A et al. (2015) Myocardial fat overgrowth in Proteus syndrome. Am J Med Genet A 167A:103-10
Russell, Bianca; Johnston, Jennifer J; Biesecker, Leslie G et al. (2015) Clinical management of patients with ASXL1 mutations and Bohring-Opitz syndrome, emphasizing the need for Wilms tumor surveillance. Am J Med Genet A 167A:2122-31
Wentzensen, Ingrid M; Johnston, Jennifer J; Keppler-Noreuil, Kim et al. (2015) Exome sequencing identifies novel mutations in C5orf42 in patients with Joubert syndrome with oral-facial-digital anomalies. Hum Genome Var 2:15045
Lindhurst, Marjorie J; Yourick, Miranda R; Yu, Yi et al. (2015) Repression of AKT signaling by ARQ 092 in cells and tissues from patients with Proteus syndrome. Sci Rep 5:17162

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