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The PIK3R1 gene encodes three proteins - p85$\alpha$, p50$\alpha$ and p55$\alpha$ - that are regulatory subunits of Class IA phosphoinositide 3-kinases (PI3Ks). These regulatory subunits heterodimerise with one of three catalytic subunit isoforms, namely p110$\alpha$, p110$\beta$, or p110$\delta$. Class IA PI3Ks are critical enzymes involved in fundamental metabolic and mitogenic signalling pathways. This thesis describes the delineation of biochemical and molecular mechanisms whereby PIK3R1 mutations cause diverse disease phenotypes observed in SHORT syndrome (defined by Short stature, Hyperextensibility, Ocular depression, Rieger anomaly and Teething delay), the primary immunodeficiency Activated PI3K-$\delta$ Syndrome 2 (APDS2), and cancer. Initial studies of purified wildtype or mutant PI3K complexes, utilising a modified PI3K fluorescence polarisation lipid kinase assay, established that SHORT syndrome-associated p85$\alpha$ mutations impaired phosphotyrosine peptide-stimulated PI3K activity when heterodimerised with either of the Class IA catalytic subunit isoforms. Two cancer-associated mutations assessed using the same assay demonstrated differential effects on PI3K function, causing either basal activation or impaired phosphotyrosine peptide-stimulated PI3K activity. To examine the effect of SHORT syndrome-associated p85$\alpha$ mutations in insulin-responsive cell types, 3T3-L1 preadipocyte models with conditional overexpression of p85$\alpha$ Y657X or p85$\alpha$ R649W were generated. Doxycycline-induced overexpression of mutant p85$\alpha$ attenuated insulin-stimulated Akt phosphorylation due to reduced insulin-stimulated association of p85$\alpha$/p110$\alpha$ heterodimers with either IRS1 or IRS2. This in turn resulted in impaired downstream signalling as indicated by low adipogenic efficiency. Cells and tissues isolated from Pik3r1$^{WT/Y657X}$ knock-in mice also demonstrated decreased insulin-stimulated Akt phosphorylation. Observations from a system with endogenous expression of mutant p85$\alpha$ Y657X supported the results obtained in the 3T3-L1 p85$\alpha$ overexpression models. The final part of this thesis focussed on a PIK3R1 exon skipping mutant (p85$\alpha$ $\Delta$Ex11) that confers PI3K activation in lymphocytes and causes APDS2. APDS2 patients have an immune-restricted phenotype, even though the mutation occurs within the ubiquitously expressed PIK3R1. To investigate this phenomenon, the doxycycline-inducible system was used to model overexpression of p85$\alpha$ $\Delta$Ex11, as well as an activating p110$\alpha$ H1047R mutation associated with cancer, in 3T3-L1 preadipocytes. Surprisingly, given that APDS2 is not normally associated with metabolic or growth problems, high overexpression of p85$\alpha$ $\Delta$Ex11 severely attenuated insulin-stimulated Akt phosphorylation and adipocyte differentiation. There was also reduced insulin-stimulated recruitment of p110$\alpha$ to either IRS1 or IRS2, and impaired heterodimerisation of p85$\alpha$ $\Delta$Ex11 with p110$\alpha$. Collectively, the data presented in this thesis contributes to the developing knowledge of PIK3R1-related diseases. In particular, these studies provided novel insights into the biochemical and molecular mechanisms of SHORT syndrome-associated p85$\alpha$ mutations. Additionally, these data delivered further understanding of potential mechanisms underlying the immune-specific phenotype of APDS2 caused by PIK3R1 mutations.

ObjectivesSignificant gaps exist in the understanding of the presentation of genetic conditions across the lifespan, particularly during the prenatal period. This study aimed to describe the limitations of prenatal phenotyping by detailing the differences between prenatal and postnatal evaluations of neonates with genetic conditions.Methods We conducted a retrospective chart review of neonates with genetic diagnoses who previously received a detailed prenatal phenotype evaluation by fetal ultrasound, MRI, and echocardiogram at the Cincinnati Children’s Fetal Care Center (CCFCC) between July 2018 and October 2022. Details of the prenatal and postnatal phenotypes were collected using Human Phenotype Ontology (HPO) terms to compare findings between the time points. ResultsBetween July 2018 and October 2022, there were 85 neonates with genetic diagnoses who were prenatally evaluated in the CCFCC; these patients either received diagnoses prenatally (n=38), postnatally (n=45), or differing diagnoses before and after birth (n=2). The number of HPO terms significantly increased after postnatal evaluation (mean: 8.45) compared to what was identified prenatally at time of referral (mean: 3.45) (p

The field of genetics is constantly evolving. New advances in bioinformatics and computational approaches are leading to exciting new developments in our ability to treat and prevent diseases. Computational genetics provides valuable insights into the complex mechanisms and layers of biological communication that shape an organism's phenotype. Understanding these mechanisms is critical to advancing human health.The study of diseases in genetics requires a comprehensive understanding of the interactions between various biological processes, including gene expression, protein synthesis, RNA, metabolism, and cell-cell communication. To effectively address the root causes of such diseases, multi-disciplinary approaches that integrate information from different levels of biological organization are increasingly needed. Network analysis, also known as graph theory analysis, provides a solution by allowing the visualization and quantification of these relationships.This paper focuses on the features and functions of sPyderMIM, a program I designed to automate the construction and analysis of genotype, phenotype, and protein interaction networks constructed from clinical and genetic disease data retrieved from the OMIM (Online Mendelian Inheritance in Man) database and protein interaction data retrieved from the IntAct database. I aim to demonstrate the potential utility of sPyderMIM in facilitating the exploration of genetic and protein relationships underlying disease progression.

Germline alterations of the gene encoding PTEN, a canonical tumor suppressor, contributes to risk of both benign and malignant neoplasia and neurodevelopmental phenotypes, meaning germline PTEN mutations associate with specific rare cancer syndromes (e.g., Cowden syndrome) and autism spectrum disorder (ASD). To describe the PTEN-associated phenotypic heterogeneity, the molecular diagnosis PTEN Hamartoma Tumor Syndrome (PHTS) was established encompassing several clinical entities. The cancer and autism association is a striking, perplexing juxtaposition, inspiring study. Given the mature connection between cancer processes and canonical aspects of PTEN function (i.e. suppression of PI3K/AKT/mTOR signaling), we have focused on PTEN function in neurodevelopmental pathologies like ASD. To investigate this somewhat intractable question (it is challenging to study molecular features of brains in living individuals), we developed two complementary murine models of Pten disruption. The Ptenm3m4 model exhibits cytoplasmic-predominant expression, while the PtenY68H model exhibits nuclear-predominant expression. Prior study of both models found behavioral, cellular, and molecular abnormalities reminiscent of autism. These models are suited for insight into PTEN-ASD pathophysiology and understanding the neurobiological effects of localization-associated, separation-of-function mutants. Herein, we demonstrate that the Ptenm3m4 model shows aberrant alternative splicing with associated changes in splicing factors, U2af2 and Srrm4. Additionally, a multi-omic survey of the Ptenm3m4 brain found dysregulation of molecules and pathways implicated in autism biology, identifying Pten and Psd-95 as regulatory hubs important to initiating and perpetuating observed phenotypes. Intriguingly, these molecular phenotypes undergird numerous changes in neurobiology, including changes in neural stem cell (NSC) maturation, oligodendrocyte (OL) differentiation, and microglia activation. In the PtenY68H model, we found deficits in sociability and increased perseverative behavior, while finding upregulation of genes involved in neuroinflammation, microglia activation, and behavior (e.g., oxytocin). Following the modeling, we sought a comprehensive, clinical perspective on the functional effects of PTEN mutation. Thus, we leveraged variant functionalizing data from deep mutational scanning studies (DMS) compared to PHTS outcomes. We established that variant effects on lipid phosphatase activity and protein stability predict pathogenicity, head circumference, disease burden, and cancer risk (including early onset). This work provides insight on PTEN-ASD pathogenesis and PTEN genotype-phenotype relationships, working toward improved clinical management of PHTS.

New proteins primarily evolve through recombining modular protein domains with discrete structure and function. Often domain recombination combines a catalytic functioning domain with a sensing domain, so protein function can be regulated by different stimuli. This form of domain recombination-based evolution underlies the intricate signaling networks that allow our cells and by extension our bodies to sense and respond to stimuli. Protein engineers mimic nature by combining domains with desirable properties into new useful combinations never seen in nature. This approach for generating synthetic multi-domain proteins has yielded groundbreaking therapeutics and tools for biology, most notably Car-T cancer therapies and GCaMP calcium sensors. However, domain recombination is challenging and requiring years of iterative optimization but unlike evolution we don’t have millions of years to spare. Both, our basic understanding of the biophysical principles of how proteins evolve and how to better engineer proteins are limited by a lack of domain compatibility rules. In the work presented in this thesis, we sought out to apply massively parallel domain insertion experiments and learn rules for domain compatibility. As our target protein, we used ion channels as they are an attractive engineering target and ion channels evolved through extensive domain recombination. Initially we started with a small set of 3 inserted domains inserted into all amino acid positions of a potassium channel kir2.1. We successfully engineered a light-switchable potassium channel that could be used by neuroscientists, however we found a tremendous amount of variability that necessitated expanding out to a broader sample of domain recombination space. Before we could achieve this goal, we needed to improve experimental pipelines because the methods the domain insertion field used at the time were not scalable nor generalizable. We developed a new domain insertion library generation method, SPINE, that yielded near perfect libraries. SPINE allowed us to expand out to over 700 different inserted domains with which we exhaustively sampled insertional space and developed a mechanistic model of domain recombination. We then expanded outward to several additional recipient channels to benchmark our work. Overall, we made major strides towards the goal of a mechanistic model for assembling protein domains. We expect this body of work will provide a foundation that will make domain-based engineering more effective and improve our understanding of the fundamentals of how proteins evolve.

Phelan-McDermid syndrome is a developmental disability syndrome associated with deletions of the terminal end of one copy of chromosome 22q13. The observed chromosomal aberrations include simple terminal deletions, interstitial deletions, deletions and duplications, and duplications without deletions. All patients have some degree of developmental disability and many also have hypotonia, autism, minor dysmorphic features, and seizures. I performed an epidemiological and cytogenetic investigation to better understand the etiology of Phelan-McDermid syndrome and to provide information to patients and their families, clinicians, and researchers investigating developmental disabilities. Deletions vary widely in size, from 60 kb to more than 9 Mb, but almost all cases are missing one copy of the subtelomeric gene SHANK3, a candidate gene for neurological features. The results of this study established that larger deletions are associated with more severe disability establishing the rationale to investigate the role of additional genes or genomic regions for clinical features. Statistical association analyses identified specific genomic regions as associated with 22 clinical features. In particular, speech is highly correlated with deletion size indicating that speech-related genes or genomic elements located in genomic bands 22q13.2q13.31 may be critical in determining a patient's ability to communicate verbally. The use of protein interaction networks identified candidate genes within these narrowed genomic regions. Also, a longitudinal assessment of phenotypes observed among individuals aged 0.4 to 64 years established significant variation of phenotypes by age, such that future investigations need to take age into account when conducting genotype-phenotype studies. In particular, we find that behavioral difficulties subside and low muscle tone becomes less prominent as children age, however seizures, autism, and some chronic diseases become more apparent in teens and adults.