Medical & Molecular Genetics Department Theses & Dissertations
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Item Modulation of Splicing Factor Function and Alternative Splicing Outcomes(2022-06) Chen, Steven Xiwei; Liu, Yunlong; Lu, Xiongbin; Schneider, Bryan P.; Wek, Ronald C.Alternative RNA splicing is an important means of genetic control and transcriptome diversity. Alternative splicing events are frequently studied independently, and coordinated splicing controlled by common factors is often overlooked: The molecular mechanisms by which splicing regulators promote or repress specific pre-mRNA processing are still not yet well understood. It is well known that splicing factors can regulate splicing in a context-dependent manner, and the search for modulation of splicing factor activity via direct or indirect mechanisms is a worthwhile pursuit towards explaining context-dependent activity. We hypothesized that the combined analysis of hundreds of consortium RNA-seq datasets could identify trans-acting “modulators” whose expression is correlated with differential effects of a splicing factor on its target splice events in mRNAs. We first tested a genome-wide approach to identify relationships between RNA-binding proteins and their inferred modulators in kidney cancer. We then applied a more targeted approach to identify novel modulators of splicing factor SRSF1 function over dozens of its intron retention splicing targets in a neurological context using hundreds of dorsolateral prefrontal cortex samples. Our hypothesized model was further strengthened with the incorporation of genetic variants to impute gene expression in a Mendelian randomization-based approach. The modulators of intron retention splicing we identified may be associated with risk variants linked to Alzheimer’s Disease, among other neurological disorders, to explain disease-causing splicing mechanisms. Our strategy can be widely used to identify modulators of RNA-binding proteins involved in tissue-specific alternative splicing.Item The Genetic Architecture of Alzheimer's Disease Endophenotypes(2022-05) Jacobson, Tanner Young; Saykin, Andrew J.; Nho, Kwangsik; Foroud, Tatiana; Zhang, Chi; Cao, ShaAlzheimer’s Disease (AD) is one of the most common forms of dementia and is known to have a strong genetic component, but known genetic loci do not fully account for the observed genetic heritability of late onset AD. This genetic complexity is further complicated by disease heterogeneity, with non-uniform presentation and progression of AD neuropathology. Endophenotypes lie upstream of observed AD clinical outcomes and downstream of genetic contributors, allowing for a biological understanding of genetic effects. Understanding the genetic architecture of AD endophenotypes can aid in breaking down AD genetic complexity and heterogeneity. In this study we utilized a variety of models to evaluate the genetic contributors to pathological change and heterogeneity in the top markers of AD pathology: amyloid, tau, neurodegeneration, and cerebrovascular (A/T/N/V framework). Additional composite quantitative measures of cognitive performance were used to relate to downstream AD presentation. These biomarkers allow the investigation of genetic effects contributing to the disease over the stages of disease progression from amyloid deposition to neurofibrillary tangle formation, disruption of metabolism, brain atrophy, and finally to clinical outcomes. First, we performed genome-wide association studies (GWAS) for AD endophenotypes at baseline using a cross-sectional regression model. This method identified sixteen novel or replicated loci, with six (SRSF10, MAPT, XKR3, KIAA1671, ZNF826P, and LOC100507506) associated across multiple A/T/N biomarkers. Cross-sectional data was further utilized to identify three genetic loci (BACH2, EP300, PACRG-AS1) that showed disease stage specific interaction effects. We built upon those results by performing a longitudinal association analysis with linear-mixed effects modeling. Gene enrichment analysis of these results identified 19 significant genetic regions associated with linear longitudinal change in AD endophenotypes. To further break down longitudinal heterogeneity, a latent class mixed model approach was utilized to identify subgroups of longitudinal progression within cognitive and MRI measures, with 16 genetic loci associated with membership in different classes. The genetic patterns of these subgroups show biological relevance in AD. The methods and results from this study provide insight into the complex genetic architecture of AD endophenotypes and a foundation to build upon for future studies into AD genetic architecture.Item Linking Osteocyte Oxygen Sensing and Biomineralization via FGG23: Implications for Chronic Kidney Disease(2022-05) Noonan, Megan L.; White, Kenneth E.; Kota, Janaiah; Graham, Brett H.; Thompson, William R.FGF23 is an osteocyte produced hormone necessary for maintaining systemic phosphate handling, and thus bone structure and function in both rare and common disorders such as chronic kidney disease (CKD). FGF23 is a critical factor in CKD, with elevated levels causing alterations in mineral metabolism and increased odds for mortality. However, the mechanisms directing the production of key modulators of skeletal homeostasis and biomineralization within osteocytes, and how this is altered in chronic kidney disease, remain unclear. The experimental focus of this dissertation was to dissect the molecular systems and role of oxygen sensing in the regulated production of FGF23. In CKD, up to 75% of patients have anemia and concomitant marked elevations in FGF23, increasing mortality odds. Anemia is a potent driver of FGF23 secretion, therefore, current and emerging therapies, including recombinant EPO and the hypoxia inducible factorprolyl hydroxylase inhibitors (HIF-PHI) FG-4592 and BAY 85-3934, were used to improve anemia in the adenine diet-induced mouse model of CKD. In the mice with CKD, iFGF23 was markedly elevated in control mice but was attenuated by 65-85% after delivery of EPO or HIF-PHI, with no changes in serum phosphate. This was associated with improved systemic iron utilization and reductions in mRNA markers of renal fibrosis. In osteocyte-like cell cultures treated with HIF-PHI, integrative RNAseq and ATACseq analysis identified candidate genes upregulated in response to mimicked hypoxia, concomitant with elevated Fgf23 expression. These genes were found to be downregulated in CKD bone, therefore, knock-out cells were generated using CRISPR/Cas9 technology. These cells were found to be functionally similar to in vivo conditional knockout models that have enhanced bone mass and elevated FGF23. Taken together, these results further define novel factors involved in the regulation of FGF23 and identify new therapeutic targets.Item Identifying Racial/Ethnic Differences in Clinical Trial Enrollment, Drug Response, and Genetic Biomarkers of Taxane Induced Peripheral Neuropathy in African American Breast Cancer Patients(2021-08) Shah, Ebony; Skaar, Todd C.; Radovich, Milan; Quinney, Sara; Liu, YunlongMy first aim identified enrollment patterns and variables that predict enrollment in a diverse underserved population and evaluated barriers to enrollment. We analyzed data from the INGENIOUS, (Indiana GENomics Implementation and Opportunity for the UnderServed), pharmacogenomics implementation clinical trial conducted at a community hospital for underserved subjects and a statewide healthcare system. Our main finding revealed, African-Americans were less likely to refuse the study than non-Hispanic Whites (Safety net, OR =0.68, p<0.002; Academic hospital, OR=0.64, p<0.001), using a logistic regression model. The most frequent barriers to enrollment included not being interested, being too busy, transportation, and illness in African-American and non-Hispanic White subjects. In conclusion, improving research awareness, widening the inclusion criteria, and hiring recruiters who represent potential enrollees, should improve enrollment in African-Americans and other diverse populations. My 2nd research aim evaluated racial/ethnic differences in pharmacokinetics, safety, efficacy, and pharmacogenetics in 213 new molecular entities (NMEs). The current approved drug label for NMEs between 2014 to 2018 was updated in the FDA database. A qualitative analysis revealed ~ 9% (n=20/213) of NMEs reported racial/ethnic differences in the approved product label for PK, safety, efficacy, and/or pharmacogenetics. In conclusion, evaluating racial/ethnic differences in drug exposure and response early in the drug development program is essential to providing recommendations for different racial/ethnic subpopulations. My final aim 3, identified genetic biomarkers of Taxane Induced Peripheral Neuropathy (TIPN) in African-American breast cancer patients. We used an innovative computational tool, ALDY, to identify genetic variants in CYP2C8, CYP3A4, and CYP3A5 in 207 breast cancer subjects. TaqMan SNP genotyping for SNP, rs776746 (T>C) was performed in 160 subjects. Subjects were collapsed into three metabolizer groups; normal, intermediate, and poor metabolizer to test the association of peripheral neuropathy, dose reductions and CYP2C8/CYP3A5 metabolizer status. A logistic regression revealed CYP2C8 metabolizer status is associated with grades 3-4 peripheral neuropathy (p=0.04, OR= 2.21). CYP2C8*2 was modestly associated with dose reductions. In conclusion, evaluating pharmacogenetic and pharmacokinetic studies of paclitaxel and CYP2C8 is important. These studies may lead to clinical actionable prescribing of paclitaxel and improve the tolerance and efficacy in African-American breast cancer patients.Item The Impact of SBF2 on Taxane-Induced Peripheral Neuropathy(2021-05) Cunningham, Geneva Mari; Schneider, Bryan P.; Radovich, Milan; Liu, Yunlong; Skaar, Todd; Meyer, Jason; Ivan, MirceaThe main focus of this study is to determine the impact of Set-Binding Factor 2 (SBF2) on human-derived neurons in the context of taxane-induced peripheral neuropathy. Taxane-induced peripheral neuropathy (TIPN) is a devastating survivorship issue for many cancer patients; SBF2 has been previously identified as a potential germline predictor that has been found to be significantly associated with severe TIPN in African American (AA) patients. The work described here provides ex vivo support for the use of SBF2 as a genotypic biomarker to identify a priori which patients are at a higher risk of manifesting severe TIPN. This study demonstrates that diminished expression of SBF2 exacerbated the effect of paclitaxel on viability and morphology and altered the functional response of a neuronal model exposed to paclitaxel treatment. Furthermore, transcriptomic work showed that reduced expression of SBF2 in a neuronal model treated with paclitaxel impacted the expression of genes that modulate stress-induced cell death and pain threshold. Altogether, these findings suggest that SBF2 plays a role in the development of TIPN. This work sheds light on the pathways potentially involving SBF2 that can be studied to further evaluate the function of this gene in neurons and its contribution to severe TIPN. Further functional approaches investigating these pathways will be pivotal in elucidating the underlying biological mechanism for this toxicity and identifying novel targeted therapeutic strategies to prevent or treat TIPN.Item Lipidomic Dysregulation in Alzheimer's Disease: Relation to Genetics, Neuroimaging and Other Biomarkers(2021-04) Bernath, Megan M.; Saykin, Andrew J.; Nho, Kwangsik; Herbert, Brittney-Shea; Lahiri, Debomoy K.; Lamb, Bruce T.; Risacher, Shannon L.Large-scale genome-wide association studies for Alzheimer’s disease (AD) have identified more than 20 risk loci and several pathways including lipid metabolism. Lipids are fundamental to cellular structure and organization, where they compose biological bilayer membranes surrounding the cell. In their structural role, lipids provide a scaffold for cell signaling, such as neurotransmission. There is a large body of evidence linking lipids and AD, yet the relationship between AD pathogenesis and lipid dyshomeostasis is not well understood. Here, we performed manual PubMed searches to identify the most studied lipid classes and risk genes in AD. We discussed pathological alterations of the key lipids and their potential contribution to the recent NIA-AA “A/T/N” framework. We also summarized what is known between the key lipids and etiological hypotheses of AD. Finally, we characterized relationship of the key lipids with AD genomic risk factors to identify possible downstream mechanisms of lipid dysfunction in AD. There is a large body of evidence linking lipids and AD, yet the relationship between AD pathogenesis and lipid dyshomeostasis is not well understood. In particular, we investigated the association between triglyceride (TG) species and AD. The overall goal was to test the hypothesis that TGs would associate with AD endophenotypes, based on their fatty acid composition. Diagnostic groups (cognitively normal older adults (CN), mild cognitive impairment (MCI), and AD) differed on two principal components extracted from 84 serum TG levels. Fish oil-type and olive oil-type TGs were significantly lower in MCI and AD compared to CN. Next, association analysis of TG principal components with “A/T/N/V” (amyloid-β, tau, neurodegeneration, and cerebrovascular) biomarkers for AD showed that the fish oil-type and olive oil-type TGs were also significantly associated with atrophy on MRI. Finally, a mixed model regression analysis investigated the association between baseline TGs and longitudinal changes of AD endophenotypes to show that olive oil-type TGs predicted changes in AD brain atrophy. Our results indicate that a specific subcategory of TGs is associated with an early prodromal stage of cognitive impairment and early-stage biomarkers for AD, providing the foundation for future therapeutic development related to TG metabolism.Item The Role of MCTP2 in Health and Disease(2021-01) Alkhouli, Mohammed A.; Ware, Stephanie M.; Firulli, Anthony B.; Payne, R. Mark; Wek, Ronald C.MCTP2 (multiple C2 domain transmembrane containing protein 2) encodes a protein with poorly understood roles in lipid metabolism and lipid droplet biogenesis. Genetic studies previously identified variations in MCTP2 in conjunction with left ventricular outflow tract obstructive forms of congenital heart disease (CHD). This dissertation research aimed to delineate the biomedical significance of Mctp2 by investigating its expression and consequences of its genetic deletion in mouse models. Temporal and spatial expression of Mctp2 was investigated by RT-PCR and in-situ hybridization. A novel isoform, designated as isoform 2 in mice, results from alternative pre-mRNA splicing. Similar levels of Mctp2 isoforms 1 and 2 are present in embryonic tissues, whereas isoform 1 is preferentially expressed in adult tissues with high lipid metabolism. During mouse embryonic development, in-situ hybridization suggests expression of Mctp2 at the gut tube, liver bud and near the pharyngeal arches from E8.5 – E10.5. Given association of MCTP2 with CHD, the biological significance of Mctp2 was addressed using gene trap (GT) and conditional mouse models. Survival of Mctp2 GT mice was dependent on the genetic background strain, suggesting a role for strain-specific modifiers. Conditional knockout of Mctp2 in cardiac progenitor cells displayed no effect on survival. The role of Mctp2 in cardiac development remains to be delineated. The role of Mctp2 in cardiac function was addressed in both mouse models. Initial findings suggest Mctp2 allele dosage effects on the development of heart failure. GT mice lacking one, or both, copies of Mctp2 display cardiac systolic dysfunction, with upregulation of heart failure markers at 50 weeks of age in heterozygotes and increases in cardiac fibrosis in homozygotes. Systemic conditional deletion of Mctp2 did not show heart failure phenotypes using the strain protective from lethality. However, cardiac specific deletion of Mctp2 using the Nkx2.5-Cre driver, a line that is sensitized for cardiac dysfunction, led to decreased ejection fraction and fractional shortening in mice with conditional deletion of both copies of Mctp2 as well as Mctp2 dosage dependent penetrance of cardiac dilation. These studies of knockout mice suggest a role for Mctp2 in maintenance of cardiac function and possible genetic interaction with Nkx2.5.Item Multi-Modality Plasma-Based Detection of Minimal Residual Disease in Triple-Negative Breast Cancer(2019-07) Chen, Yu-Hsiang; Radovich, Milan; Medical & Molecular Genetics; Ivan, Mircea; Li, Lang; Liu, Yunlong; Schneider, Bryan P.; Skaar, Todd C.Triple-negative breast cancers (TNBCs) are pathologically defined by the absence of estrogen, progesterone, and HER2 receptors. Compared to other breast cancers, TNBC has a relatively high mortality. In addition, TNBC patients are more likely to relapse in the first few years after treatment, and experiencing a shorter median time from recurrence to death. Detecting the presence of tumor in patients who are technically “disease-free” after neoadjuvant chemotherapy and surgery as early as possible might be able to predict recurrence of patients, and then provide timely intervention for additional therapy. To this end, I applied the analysis of “liquid biopsies” for early detection of minimal residual disease (MRD) on early-stage TNBC patients using next-generation sequencing. For the first part of this study, I focused on detecting circulating tumor DNA (ctDNA) from TNBC patients after neoadjuvant chemotherapy and surgery. First, patient-specific somatic mutations were identified by sequencing primary tumors. From these data, 82% of the patients had at least one TP53 mutation, followed by 16% of the patients having at least one PIK3CA mutation. Next, I sequenced matched plasma samples collected after surgery to identify ctDNA with the same mutations. I observed that by detecting corresponding ctDNA I was able to predict rapid recurrence, but not distant recurrence. To increase the sensitivity of MRD detection, in the second part I developed a strategy to co-detect ctDNA along with circulating tumor RNA (ctRNA). An advantage of ctRNA is its active release into the circulation from living cancer cells. Preliminary data showed that more mutant molecules were identified after incorporating ctRNA with ctDNA detection in a metastatic breast cancer setting. A validation study in early-stage TNBC is in progress. In summary, my study suggests that co-detection of ctDNA and ctRNA could be a potential solution for the early detection of disease recurrence.Item Pathophysiological role of MicroRNA-29 in pancreatic ductal adenocarcinoma(2018-05-23) Kwon, Jason Jae-Hyuk; Kota, Janaiah; Korc, Murray; Liu, Yunlong; Wek, RonaldPancreatic ductal adenocarcinoma (PDAC) is a highly lethal malignancy and responds poorly to current therapies. Thus, it is imperative to develop novel treatments for PDAC. Dense fibrotic stroma associated with PDAC abrogates drug perfusion into the tumor, and pancreatic stellate cells (PSCs) are the major stromal cells responsible for fibrosis. Activated PSCs produce pro-inflammatory factors and secrete an excessive amount of extracellular matrix (ECM) proteins, the major stromal proteins in PDAC. MicroRNAs (miRNAs) are conserved small non-coding RNAs that regulate gene expression by binding to the 3′UTR of target mRNA transcripts, causing translational repression or degradation. A single miRNA regulates several targets within intracellular networks and can have a profound impact on normal physiology. miR-29 has been previously reported to have anti-fibrotic and tumor suppressive roles in various cancers. We found miR-29 expression was significantly decreased in activated PSCs and pancreatic cancer cells in vitro, in vivo models, as well as in PDAC patient biopsies. Through in vitro studies in activated PSC, we found that miR-29 inhibited the expression of ECM proteins and reduced cancer growth when co-cultured with pancreatic cancer cells. miR-29 overexpression in pancreatic cancer cells decreased their invasive potential and sensitized chemoresistant cancer cells to gemcitabine treatment by inhibiting autophagy through the direct targeting of two essential, autophagy related genes, TFEB and ATG9A. In developing therapies and for in vivo functional studies, viral-based gene delivery is a powerful tool to target the pancreas. We tested various self-complementary recombinant adeno-associated virus (scAAV) serotypes in normal mice (C57BL/6) and in a KrasG12D-driven pancreatic cancer mouse model via systemic and intraductal delivery methods. We found that retrograde intraductal delivery of scAAV6 safely targeted the pancreas/neoplasm with the greatest efficiency. Our findings provide a better understanding of miR-29 in pancreatic cancer and demonstrates its potential therapeutic use to target PDAC.Item The role of Bruton's tyrosine kinase and PI3K p110δ in mutant SHP2-induced juvenile myelomonocytic leukemia(2018) Deng, Lisa; Chan, Rebecca, J.; Kapur, Reuben; Herbert, Brittney-Shea; Ware, Stephanie M.; Yoder, MervinJuvenile myelomonocytic leukemia (JMML) is an aggressive myeloproliferative neoplasm that lacks effective chemotherapies. Most commonly, patients have gain-of-function (GOF) oncogenic mutations in SHP2, leading to hyperactivation of ERK and AKT and hyperproliferation of cells in response to granulocyte macrophage-colony stimulating factor (GM-CSF). Our lab previously showed that p110δ, the hematopoietic-specific catalytic subunit of phosphoinositide 3-kinase, is a crucial mediator of mutant Shp2-induced GM-CSF hypersensitivity in vitro. We treated oncogenic Shp2-expressing mice with a p110δ inhibitor and showed that the strong effect our lab observed in vitro translated into reduced splenomegaly and prolonged survival in vivo. We investigated molecules potentially cooperating with p110δ signaling and discovered that Bruton’s tyrosine kinase (BTK) is hyperphosphorylated in GOF Shp2 myeloid cells. We used specific BTK and p110δ inhibitors to demonstrate that BTK cooperates with p110δ to hyperactivate Akt/Erk and to promote hyperproliferation. GOF Shp2-expressing mice treated in vivo with the drug combination targeting p110δ and BTK have significantly decreased splenomegaly and WBC counts. We also explored the mechanism of BTK signaling and hypothesized that B cell adaptor for PI3K (BCAP) mediated BTK upregulation of PI3K activity. In mutant Shp2 macrophages, we observed BCAP phosphorylation specifically in the larger isoforms needed for PI3K activation, and BTK inhibition led to a dose-dependent reduction in this phosphorylation. We also demonstrated reduced interaction between BCAP and the PI3K regulatory p85α subunit bearing mutated SH2 domains. Finally, we investigated the effects of mutated DNA methyltransferase 3A (Dnmt3a) in conjunction with GOF Shp2. Double mutant mice quickly became moribund with pronounced splenomegaly and leukocytosis. There was an expansion of mature myeloid cells in the periphery and myeloid progenitors in the bone marrow, plus anemia with evidence of compensatory erythropoiesis in the spleen. Our findings show that the myeloproliferative neoplasm caused by GOF Shp2 is due to hyperactive p110δ, and this is further promoted by BTK, which forms a positive feedback loop with PI3K and BCAP, thus leading to more Akt/Erk hyperphosphorylation and more hyperproliferation in response to GM-CSF. The dual inhibition of p110δ and BTK represents a novel effective treatment strategy for JMML and other diseases induced by oncogenic Shp2.