Date of Award
Doctor of Philosophy (PhD)
Neurofibromatosis type 1 (NF1) is a common neurodevelopmental disorder (NDD) characterized by remarkable phenotypic variability, where affected children manifest a spectrum of central nervous system (CNS) abnormalities, including brain tumors, impairments in attention, behavior, learning disabilities, and an increased incidence of autism spectrum disorder (ASD). A significant barrier to the implementation of precision medicine strategies for children with NF1 is a lack of prognostic risk factors to guide clinical management. However, emerging population-based genotype-phenotype association studies have suggested that the germline NF1 gene mutation may represent one clinically actionable risk factor for NF1-associated neurodevelopmental abnormalities. As a critical step in interpreting the significance of these reported genotype-phenotype correlations, we sought to determine whether germline NF1 gene mutations differentially affected human brain development, while controlling for other important confounding factors (e.g., sex, genomic differences). For these studies, we generated an isogenic series of CRISPR/Cas9-engineered human induced pluripotent stem cells (hiPSCs) harboring seven different intragenic NF1 patient germline NF1 gene mutations. Using this experimental platform, we established 2D neural progenitor cell (NPC) and astrocyte cultures, as well as three-dimensional (3D) human cerebral organoids (hCOs). While all mutations similarly increased proliferation and RAS activity in 2D NPCs and astrocytes, we observed striking differences between NF1 mutations on 2D NPC dopamine levels and 3D NPC proliferation, apoptosis, and neuronal differentiation in developing hCOs. Importantly, identical abnormalities were observed using patient-derived hiPSC 2D and 3D cultures bearing the same NF1 gene mutations, thus establishing differential effects of the germline NF1 gene mutation on human brain development. Based on these findings, we next sought to determine the cellular and molecular etiologies that underlie the severe developmental delays and intellectual disability (IQ < 70) seen in children with a specific type of NF1 mutation involving heterozygous deletion of the NF1 gene and several contiguous genes (NF1-total gene deletion [NF1-TGD]). Using hCOs from three NF1 patients harboring NF1-TGDs, we identified both NPC proliferation and neuronal maturation abnormalities in NF1-TGD hCOs. While increased NPC proliferation resulted from decreased NF1/RAS regulation, the neuronal differentiation, survival, and maturation defects were caused by reduced cytokine receptor-like factor 3 (CRLF3) expression and impaired RhoA signaling. Furthermore, we demonstrated a higher autistic trait burden in NF1 patients harboring a deleterious germline mutation in the CRLF3 gene (c.1166T>C, p.Leu389Pro). Taken together, this body of work conceptually advances the field by (1) demonstrating differential effects of NF1 mutations at the cellular and tissue levels in humans, establishing that the germline NF1 gene mutation is one important factor that underlies clinical variability in this monogenic syndrome, and (2) revealing a new causative gene within the NF1-TGD locus responsible for hCO neuronal abnormalities and autism in children with NF1.
Chair and Committee
David H. Gutmann
Wegscheid, Michelle L., "Genetic risk factors for neurodevelopmental disorders: insights from hiPSC-cerebral organoids" (2021). Arts & Sciences Electronic Theses and Dissertations. 2628.