Developmental epileptic encephalopathies (DEEs) are a group of severe neurological disorders that present with seizures in early infancy and developmental delays. A genetic cause can be identified in up to 50% of affected individuals and is most commonly a de novo genetic change. However, for some patients a genetic change is not evident through DNA sequencing. In this research project, we are using RNA sequencing to search for and identify abnormal splicing events that may be due to splice-altering variants in the genome of previously undiagnosed patients. Splicing occurs naturally in unaffected and affected individuals; it is the process of introns being removed from mRNA transcripts and exons being joined. The process of RNA sequencing allows us to discover abnormal splicing errors by looking at splice junctions, which are sites on the intron and exon border where splicing normally occurs. We search for abnormal splicing defects using RNA sequencing by comparing the splice-junctions from our patients to a reference database of healthy controls (Genotype-Tissue Expression Project), filtering out common splice junctions, allowing us to identify unique and abnormal splice junctions. If we identify abnormal splicing, we will also investigate the genome of the patient to determine whether there is an underlying DNA change. Using RNA sequencing, we hope to provide a genetic diagnosis for a subset of our undiagnosed patients and for more individuals affected by DEE in the future.

Rehabilitation research has demonstrated the benefits of early powered mobility intervention for children with disabilities, from improving developmental skills to empowering children to better participate in family and community life. However, traditional powered mobility devices are often stigmatizing, costly, and require specialized transportation due to their size and weight. One alternative early powered mobility option that has sought to address several of these concerns, The Go Baby Go Mobility and Socialization Project, provides children with disabilities a means of socially welcoming, early independent mobility experiences through custom safety and accessibility modifications to commercially available toy ride-on cars. The Go Baby Go project has resulted in promising pilot research along with community-based outreach and education in collaboration with families, clinicians, and engineers. However, a means to efficiently track car performance in real-world environments without the presence of a researcher has been lacking. Therefore, the purpose of our project was to develop a customized data logger and companion Arduino code with the ability to collect real-time data from the cars as families use them in their homes and communities. Our multidisciplinary team has created and implemented a system which gathers car performance data automatically via integrated sensors, including the number and duration of switch activations, frequency and duration of use, outdoor location, distance traveled, and driving terrain. Housed in a simple, waterproof food storage container, the data logger is integrated into the car’s electronics and powered by the car battery, with data stored on a micro-SD card. Preliminary analysis of results from eight cars in local communities is ongoing and will be shared; initial feasibility of our system for real-world tracking without undue research presence or caregiver reporting burden is promising. Future research goals include full quantitative analysis of car use patterns to improve technology design and implementation in community settings.

Overdoses induced by fentanyl-laced cocaine are skyrocketing today. Meanwhile, in 2017, cocaine was reported as the fourth most prevalent substance used on college campuses. Even more alarming, research shows a modest decline in young people’s disapproval and perceived harmfulness of experimental cocaine use. Favorable depictions of cocaine use in the media contributes to these trends today: the entertainment industry continues to advertise cocaine as a drug of privilege, partying, and success. Conversely, studies investigating Adderall use found college students view cocaine use negatively. Students harshly judged their peers who exposed themselves to cocaine’s hazardous nature and high risks. This contradictory evidence makes it difficult to judge how problematic college students’ views on experimental cocaine use may be. This study aims at sorting this puzzle. The project tests the hypotheses that students think one-time use results in more benefits than risks to the individual. To assess this hypothesis, I use a web-based survey adapted from the so-called “prototype-willingness model” (PWM). The PWM is built on the idea that young people share very clear social images of risky behaviors. The favorability of these images is positively correlated with willingness to engage in risky behavior. Willingness, rather than planned intent, is the primary factor responsible for engaging in risky behaviors if presented with the opportunity. I examine the social image of experimental cocaine use and its favorability in order to predict willingness to use. Predicting how a college student may react when given the opportunity to use is crucial for future prevention planning, as cocaine use is a more pressing issue than ever.

Many genes are associated with microcephaly, a condition characterized by a small head size. Through exome-sequencing, we identified de novo missense mutations in an actin polymerization gene, ARPC4, in four individuals with microcephaly, mild developmental delay, and mild intellectual disability. ARPC4 is involved in actin filament formation. Actin is an essential component of the cell’s cytoskeleton that gives the cell its structure and aids in cell movement and division. The goal of this research project is to understand the molecular mechanisms that lead to the disease phenotype observed in these patients. I began by using CADD, a measurement used to predict the deleteriousness of single nucleotide variants. The mutations identified in the patients were located in highly conserved loci, indicating they might be pathogenic. To provide further evidence of the pathogenicity of the ARPC4 variants, we were interested in determining the functional effect these variants have on actin polymerization – specifically, the role of ARPC4 in this mechanism. I established fibroblast cell lines from two patients with the same ARPC4 mutation. I performed immunofluorescence staining for actin to quantify the amount of actin present in the patients and control cell lines. Preliminary data from this experiment suggests that there is greater abundance of actin filaments in the control sample compared to the patients’ cells. To observe the effect of decreased actin abundance on cell migration, I executed a scratch migration assay. The results from this study elucidate the impact of ARPC4 in actin polymerization, and establish actin deficiency as a clinically recognizable cause of microcephaly.

The nucleus is the information center of the cell, acting as a hub for DNA storage, regulation, and replication. Therefore, transport of molecules into and out of the nucleus is vital for proper biological functioning. This transport is tightly regulated via the nuclear membrane, and a collection of proteins called nuclear pore proteins that interact with molecular signals. These signals allow molecules that cannot passively diffuse through the nuclear membrane (typically greater than 60 kD) to be shuttled into or out of the nucleus via a variety of pathways. Like all biological pathways, this process can be disrupted and lead to phenotypic abnormalities. Our lab identified two siblings with biallelic variants in NUP188, a known nuclear pore protein; we identified four additional cases in three families through collaborators. Clinical features include brain abnormalities with thin corpus callosum, progressive microcephaly, severely delayed myelination; congenital cataracts; mild dysmorphic features; and hypoventilation leading to death in infancy. In order to better understand our patients’ phenotype, we are investigating nuclear import pathways as a potential mechanism of disease. Using green fluorescent protein (GFP), we can directly visualize protein localization in living cells, without the use of additional stains such as immunohistochemistry. Based on size restriction of the nuclear pore, we used a vector construct with three repeats of GFP and our nuclear localization signal (NLS). Specifically, we are investigating four main NLS pathways: Importin α+β (SV40 NLS), Kap β2 (hnRNP NLS), Importin β (CREB NLS), and no NLS. We have been able to successfully create a viral vector for each NLS. Next steps include optimizing expression in patient cell lines. This approach will allow us to quantify and visualize discrepancies between patient and control localization patterns, leading to better understanding of causes of patient phenotype and potential novel therapies.

Developmental and Epileptic Encephalopathies (DEEs) are a group of severe epilepsy disorders in children and infants characterized by prominent EEG (electroencephalography) abnormalities that disrupt brain function leading to cognitive decline. Identifying genetic causes of DEE is a key step to help researchers develop and personalize medical treatments for affected patients. Approximately 5% of DEE cases are caused by a copy number variation (CNV), where a region of DNA involving a disease gene has been duplicated or deleted. Historically, this type of mutation has been difficult to detect using sequence data. To address this, I have written a multi-step algorithm that analyzes smMIP (single molecule molecular inversion probe) targeted DNA resequencing data for known DEE genes to identify CNVs in patients’ DNA that are potentially disease causing. I have run this algorithm on the large collection of smMIP data for 1158 DEE patients available in the Mefford Lab and identified several potential CNVs. Among these, three CNVs ranging in size from 250,000-2,790,000 base pairs, each involving a DEE gene - GNB1, GRIA2, and UHRF1BP1L - were validated by a second method, array CGH, the current gold standard for CNV validation. To date, the validation rate of high-confidence CNV candidates is 50% or higher. Currently I am expanding the algorithm’s functionality to include the ability to selectively search for single-exon CNVs, which are as small as 500 base pairs, are more challenging to detect, have largely been missed by all CNV detection methods, but could still be pathogenic. To do this, I am leveraging the power of intersecting duplicate smMIP datasets to improve the sensitivity of single-exon CNV detection. As any disruption of a pathogenic DEE gene could be disease causing, inclusion of these smaller CNVs will increase our ability to solve DEE cases and improve patient care.

 Developmental and epileptic encephalopathies (DEEs) are an early-onset form of epilepsy characterized by intractable seizures and severe cognitive and developmental impairment. While most genetic variants that cause DEE reside in the coding regions of genes, splice-site variants can also be pathogenic. Splice-site variants are changes in the DNA close to, or on, the exon-intron boundary, which can cause aberrant splicing, resulting in exon exclusion or intron inclusion within spliced mRNA, generating a protein that is non-functional, partially functional, or aberrantly expressed. Aberrant splicing can have pathogenic consequences, but predicting which variants near splice-sites will have an effect on splicing is difficult. I am studying three potential splice-site variants in three genes associated with DEE: SYNGAP1, SCN1B, and WWOX. I used RNA extracted from fibroblasts from three DEE patients, each with one of these variants, to confirm whether the splice-site variants cause aberrant splicing and what the predicted consequences of this aberrant splicing is on the protein. I have been able to discover the effects of the variants on splicing in WWOX and SYNGAP1. In SYNGAP1, a synonymous variant at the exon-intron junction caused an exon 4 deletion, resulting in a severely truncated protein (SYNGAP1:p.Glu120Alafs*20). In WWOX, a duplication which included exon 5 resulted in a transcript with the inclusion of two copies of exon 5, leading to a frameshift mutation and predicted truncated protein (WWOX:p.His173Glyfs*13). We are still investigating the effects of the intronic variant in SCN1B, which appears to decrease expression of the gene. We are using nonsense-mediated decay inhibitors in order to better understand the mechanism through which this decreased expression occurs. This research could potentially improve the care of these patients by providing genetic evidence of the causes of DEE, which can be the basis for advancing better treatments.

 In higher education, diversity has become a widespread initiative; taking the form of diversity councils, required diversity courses and campus-wide diversity statements. The aim is to be more than a physical grouping of all people, but have an inclusion of all perspectives. However, who bears the burden of these diversity initiatives? Instructors and professional staff provide resources and serve as outlets to students who are in need of space to share personal experience and themselves go through additional emotional labor. Emotional labor is the process of managing feelings/emotions to fulfill the emotional requirements of a job and often is not talked about. Research has shown that instructors teaching diversity courses experience a large amount of emotional labor, and a high demand for support from students. This project complements the existing research by understanding the emotional labor of non-teaching professionals who work directly in diversity offices, serving underrepresented groups of students. The primary research question is: how do non-teaching professionals working with underrepresented populations experience and understand emotional labor in higher education? Through interviews with non-teaching professionals working with underrepresented groups of students, this research sheds light on the behind-the-scenes work they do to support students and the personal costs and benefits of the emotional labor they provide. These findings help us more accurately assess the necessary investment institutions of higher education need to make in order to foster a truly diverse environment. This study will open up the conversation around diversity initiatives by examining how exclusionary institutions of higher education achieve inclusivity and show who is bearing the additional work to create an equitable, inclusive community for all.