PROJECT SUMMARY/ABSTRACT Epigenetic dysregulation is a shared mechanism of many developmental disorders. One such disorder, Tatton-Brown-Rahman Syndrome (TBRS), results from heterozygous mutations in the de novo DNA methyltransferase, DNMT3A. TBRS is characterized by intellectual disability, seizures, overgrowth, multiple developmental abnormalities, and a predisposition to various malignancies. Since its 2014 discovery, more than 200 patients with TBRS have been identified. In the blood, patients with TBRS exhibit decreased methylation at developmental gene clusters, and abnormal tissue development in patients with TBRS results in numerous lifelong complications. However, a major gap in knowledge is that the mechanism by which DNMT3A regulates development of non-hematopoietic tissues remains unknown. Our lab has identified two individuals who exhibit a mixture of wild-type and DNMT3A-mutant cells throughout their bodies, such that they are constitutive mosaics. The distribution of mutant cells is highly variable across their tissues, and concomitant analysis of wild-type and mutant cells reveals methylation differences that suggest a role of DNMT3A in tissue development. We hypothesize that DNMT3A wild-type and mutant cells have distinct propensities to form different tissues when directly competed with one another. Understanding which tissues are favored by mutant cells in somatic mosaics will lend insight into the pathogenesis of TBRS. To test our central hypothesis, I have established a novel mouse model of somatic mosaicism. I will use this mouse and an in vitro cell culture system to address two aims: 1) I will investigate the effect of Dnmt3a loss on the differentiation capacity of specific stem cell populations, and 2) I will investigate the downstream contribution of Dnmt3a-mutant versus wild-type cells to post-natal mouse tissues. Our long-term goal is to identify tissues in which DNMT3A loss confers a selective advantage or disadvantage and to explore the mechanism of this differential tissue composition. The project is designed to prepare me for a career as an academic pediatric oncologist specializing in inherited developmental and cancer predisposition syndromes. The Goodell lab has made seminal discoveries in DNMT3A biology and has a long track-record of successful MSTP trainees. The collaborative training environment at Baylor College of Medicine, with its state-of-the-art technology cores and prime location in the Texas Medical Center will facilitate the success of this project. In the future, the results of these studies may inform the clinical care of patients with TBRS by identifying tissues in which a DNMT3A mutation is poorly tolerated. The studies may also help to inform genetic counselling of parents of patients with TBRS about their risk of recurrence by characterizing expansion or depletion of DNMT3A-mutant cells in gonadal tissues. Finally, these results will expand our understanding of how mutant cells that appear in early embryogenesis compete with wild-type cells to shape normal and disordered development.

PROJECT NARRATIVE The human body is a mixture of cells that carry different gene mutations, which may confer tissue-context- dependent advantages or disadvantages. This phenomenon of somatic mosaicism has public health relevance because the expansion of cell populations with pathological mutations, such as those in the leukemia-driver gene DNMT3A, can contribute to cancer and developmental disorders. We plan to model somatic mosaicism using both cell lines and mice that we have developed with the long-term goal of identifying tissues in which DNMT3A loss confers a selective advantage or disadvantage, as well as the mechanism of this differential tissue composition.