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Indiana University-Purdue University Indianapolis (IUPUI) / Heritable disorders of phosphate handling are the most common cause of hypophosphatemic rickets in developed countries. Isolated renal phosphate wasting and subsequent low serum phosphate concentrations may result from a number of genetic disorders that include: autosomal dominant hypophosphatemic rickets (ADHR), X-linked hypophosphatemic rickets (XLH), and autosomal recessive hypophosphatemic rickets (ARHR). Fibroblast growth factor-23 (FGF23), identified as the causative gene in ADHR, is produced in bone and plays a central role in kidney phosphate regulation. Increased serum concentrations of FGF23 lead to renal phosphate wasting through down regulation of renal sodium-phosphate co-transporters. However, the molecular mechanisms of FGF23 bioactivity in hormonal phosphate regulation are largely unknown. An experimental focus of this dissertation was to investigate the molecular mechanisms of FGF23-mediated phosphate regulation in the bone-kidney hormonal axis. To this end, the role of Dentin Matrix Protein 1 (DMP1), newly identified as the gene responsible for ARHR, was further defined by the identification of a novel large deletion as well as testing the molecular consequences of DMP1 mutations. FGF23 requires a signaling complex composed of Klotho and an FGFR for bioactivity, however, the location and composition of the signaling complex is unknown. Klotho localizes to the renal distal convoluted tubule, whereas the sodium phosphate co-transporters are expressed within the renal proximal tubules. The molecular mechanisms of FGF23 signaling were investigated by isolating a novel marker of FGF23 bioactivity using array technology, determining the location of initial FGF23 signaling in the kidney, and by identifying a novel mutation in a receptor upstream of FGF23 production. Taken together, these results increase the knowledge of the molecular mechanisms of phosphate homeostasis in relation to FGF23 bioactivity, leading to the identification of potentially novel therapeutic targets. / indefinitely

Indiana University-Purdue University Indianapolis (IUPUI) / Aniridia, a congenital ocular disorder caused by haploinsufficiency of transcription factor PAX6, is characterized by complete or partial iris hypoplasia with associated foveal hypoplasia. Brain imaging performed in patients heterozygous for PAX6 mutations often reveal absence of the brain anterior or posterior commissure, absence of the pineal gland, and a present but reduced in size corpus callosum. Renal coloboma syndrome, another autosomal dominant inherited disease, is characterized by hypodysplastic kidneys and optic nerve defects, and is caused by haploinsufficiency of transcription factor PAX2. In the first part of this thesis we investigated the role of these Pax genes in neural development, by generating an allelic series of knock-in models at the Pax6 locus. We showed that Pax6(5a) and Pax2 could not replace Pax6 for its auto-regulation in lens induction or for neural differentiation in retina. In brain development, however, we demonstrated that cell proliferation in the cerebral cortex and dorsoventral patterning of the telencephalon and neural tube was partially rescued in either knock-in mutant. We believe our novel findings not only reveal Pax-protein functional specificity during neural development, but may also be utilized to understand the aberrant molecular mechanism that result in aniridia and/or renal coloboma syndrome. Aphakia (lack of lens) is a rare human congenital disorder with its genetic etiology largely unknown. In the second part of this thesis, we show that homozygous deletion of Nf1, the Ras GTPase gene underlying human neurofibromatosis type 1 syndrome, caused lens dysgenesis in mouse. While early lens specification proceeded normally in Nf1 mutants, lens induction was disrupted due to deficient cell proliferation. Further analysis showed that ERK signaling was initially elevated in invaginating lens placode, but by lens vesicle stage, Ras signaling antagonist Sprouty2 was up regulated, followed by rapid decrease in ERK phosphorylation. Only after intraperitoneal treatment of U0126, an inhibitor of ERK phosphorylation, was lens development restored in Nf1 mutants. Hyperactive RAS-MAPK signaling is known to cause neuro-cardiofacial-cutaneous (NCFC) syndromes in human. As a member of NCFC family genes, Nf1 represents the first example that attenuation of Ras-MAPK kinase signaling pathway is essential for normal lens development.

Despite decades of effort, the mechanistic underpinnings of many cancers remain unsolved It has increasingly become appreciated that cancers can be more readily classified by their transcriptional identities rather than by genomics alone. A fuller understanding of the mechanistic connections between the aberrant genomics leading to the transcriptional dysregulation of tumors is key to both improving our knowledge of cancer biology as well as developing more precise and effective therapeutics. This thesis explores the development and application of a network based multi-omic master regulator framework designed to elucidate these pathways. In Chapter 2 we apply this analysis across 20 tumor types from the Cancer Genome Atlas and in doing so identify 407 key master regulators responsible for canalizing a high percentage of the driver genetics present across these samples. Further evaluation of these key regulators revealed a highly modular structure, indicating that the regulators work in coordinated groups to implement a variety of key cancer hallmarks. Genetic and pharmacological validation assays confirmed the predicted interactions and biological phenotypes. Chapter 3 focuses on the application of this analytical framework specifically on gastroesophageal tumors. Using a more fine-grained approach we find 15 distinct subtypes across a cohort of these heterogenous tumors. These subtypes align well with previously identified features of these cancers but also reveal novel genomic associations and key master regulators that can serve as potential avenues for therapeutic treatment.

Ovarian cancer is the fifth most common cause of cancer death in females and the most lethal gynecologic cancer. Globally, an estimated 240,000 people are diagnosed with ovarian cancer each year, with 22,530 new cases in the United States in 2019. Parity, oral contraceptive use, and lactation are protective, while early menarche, late menopause, and nulliparity have opposite effects. The “incessant ovulation” theory has thus emerged, in which a higher number of ovulations may be a cause of epithelial ovarian cancer (EOC). However, the mechanisms of this theory are unknown; one possibility is that the chance of acquiring a cancer-initiating pathogenic variant increases with each ovulatory cycle because of a microenvironment that promotes DNA damage. In this dissertation, we aimed to leverage genetic epidemiologic data to test this potential mechanism by evaluating the presence of gene-environment interaction between DNA repair capacity (measured through the presence of pathogenic variants in DNA repair genes) and lifetime ovulatory years (LOY). In the first aim of this dissertation, we conducted a systematic review and meta-analysis, following Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines, to formally evaluate the strength of evidence and to generate summary point estimates for the association between LOY and EOC. We then executed two analytic aims to evaluate if the presence of pathogenic variants in DNA repair genes exacerbated the increase in ovarian cancer risk associated with LOY. In Aim 2, we evaluated interaction on the additive scale in the United Kingdom (UK) Biobank through use of a novel DNA repair capacity score developed in this dissertation, measured by quantifying the number of pathogenic variants present per individual from a list of 163 DNA repair genes, using whole exome sequencing (WES) data. In Aim 3, we evaluated the presence of interaction between pathogenic BRCA1/2 status and LOY in the Breast Cancer Family Registry (BCFR), a cohort enriched for familial risk. In both empirical aims, we assessed the presence of interaction on the additive scale using the relative excess risk due to interaction (RERI) formula. We compared results across the two empirical aims. We found the relationship between lifetime ovulations and ovarian cancer risk to be consistent and replicable in the published literature. In pooled estimates from 22 published studies, a one-year increase in LOYs was associated with a 4% (3-6%) increased risk of ovarian cancer and those with a high number of ovulations (compared to low LOYs) had a 2.15-fold (95% CI 1.82, 2.54) increased risk of ovarian cancer. We also confirmed the positive association between increasing LOYs and ovarian cancer risk in the UK Biobank and the BCFR cohorts. Although interaction on the additive scale was not detected, there were strong positive associations between pathogenic variants in DNA repair genes and ovarian cancer risk. In the UK Biobank, the presence of at least one pathogenic variant in a DNA repair gene was associated with a significant 27% increased risk of epithelial ovarian cancer (EOC) (95% CI 5-55%). Among women at high risk of ovarian cancer due to family history of breast and/or ovarian cancer, there was a strong relationship between BRCA1/2 pathogenic variants and ovarian cancer, regardless of the number of ovulations experienced. The association between LOY and ovarian cancer was found to be consistent and replicable, despite differences in study design, covariates, and measurement. We also detected robust evidence that increasing lifetime ovulations and pathogenic DNA repair variants were associated with ovarian cancer risk. Such variants were exceedingly rare in both cohorts, which limited power to detect interaction in an already rare cancer. Despite such associations, there was no evidence of synergy between LOY and impaired DNA repair capacity, but rather, high LOY and impaired DNA repair capacity may be independent risk factors of ovarian cancer. Each exposure may describe a separate class of women at increased risk of ovarian cancer that should be targeted for future prevention and screening strategies.

Developmental and epileptic encephalopathies (DEE) represent a set of rare but devastating and largely intractable childhood epilepsies. While mouse models have made innumerable contributions to understanding the genetic basis of neurological diseases, only a small fraction of missense, gain-of-function DEE variants has been modeled in mice. In this dissertation, we focus on one DEE gene, ARHGEF9. With fewer than fifty ARHGEF9 patients reported to date, ARHGEF9 can be considered one of the rare players in DEE. ARHGEF9 encodes a brain-specific protein also known as collybistin (CB), a guanine nucleotide exchange factor and an essential regulator of inhibitory postsynaptic density. In Chapter 3 and Chapter 4, we present results and ongoing studies from our efforts to unravel the pathological mechanism of ARHGEF9 DEE. We studied the G55A variant on the SH3 domain, which was discovered in one severe case of DEE. Using a novel Arhgef9G55A mouse model, we examined behavioral, cellular, and electrophysiological consequences of Arhgef9G55A. Results demonstrate that the Arhgef9G55A mouse model is an adequate ARHGEF9 DEE model, because it phenocopies key aspects of human ARHGEF9 DEE. We showed the interesting protein aggregation phenotype caused by the G55A variant. Specifically, in Arhgef9G55A/Y neurons, CB forms protein aggregates at the proximal AIS, leading to dramatic disruptions in inhibitory postsynaptic components at the AIS. Furthermore, electrophysiological studies revealed significant changes in intrinsic neuronal excitability and synaptic transmission in Arhgef9G55A/Y brains. The work within this dissertation shows that the G55A variant disrupts axon initial segment structure and functions. In Chapter 2, we review and summarize current understandings on AIS structure and functions. We highlight the central role of the AIS in initiating action potential and integrating synaptic inputs through axo-axonic synapses. Based on our experimental results, we propose that disruptions in AIS function are closely tied to the pathophysiology of ARHGEF9 DEE. Aside from the clinical significance of our study, we demonstrate the important role of CB at the AIS. We propose that CB is a specific stabilizer of axo-axonic synapses. The difference in the requirement of CB in inhibitory synapse formation in different neuronal compartments could be a core molecular machinery underlying the functional diversity of inhibitory inputs.