Heterotopia are organized structures consisting of mixed elements of neurons, glia and neuronal progenitors arranged in an architectural pattern inappropriate to the considered tissue. Heterotopia are observed in pathological preparations of the cerebral cortex and the cerebellum as a feature of many neurological disorders yet the mechanisms driving their development remain unclear. We have analyzed 28 Dandy Walker Malformation (DWM) human fetal cerebella and found that more than 40% of cases contain heterotopia. DWM is the most common structural birth defect of the human cerebellum and is characterized by hypoplasia and foliation defects. A subset of cases are caused by loss of FOXC1, a transcription factor expressed in the mesenchyme adjacent to the developing cerebellum. We previously reported that in mice, loss of FOXC1 causes loss of the mesenchyme-secreted factor SDF1-alpha. Further, loss of SDF1-alpha is sufficient to cause cerebellar heterotopia, highlighting the importance of mesenchymal signaling in the maintenance of the highly stereotypical laminar architecture of the mature cerebellum. We hypothesized that granule neuronal progenitors (GCPs) adjacent to the mesenchyme are the primary cellular target of SDF1-alpha mesenchymal signaling and thus, the primary cell type driving heterotopia formation. To test our hypothesis, we specifically deleted the SDF1-alpha receptor from just GCPs using a Cre-loxP genetic scheme in mice. We determined that loss of SDF1-alpha in just this one cell type caused their premature migration into the developing cerebellar anlage and secondarily caused other cerebellar cell types to form structured heterotopia. We also observed defects in cerebellar foliation in the treatment group. Our data further highlights the importance of SDF1-alpha dependent mesenchymal signaling in cerebellar development and identifies heterotopia as a new phenotype in DWM.

Infants born prematurely often suffer from hypoxia-ischemia and inflammatory responses that can lead to cognitive delay and behavioral problems. A novel rodent preterm brain injury model is being developed to simulate histological and behavioral changes seen in preterm brain injury. It was hypothesized that injured pups {in-utero hypoxia-ischemia followed by post-natal lipopolysaccharide (LPS)} will have a significant early behavioral deficit compared to controls. The rodent model of preterm brain injury includes: intrauterine hypoxia at embryological day 18, with postnatal LPS administration from Postnatal (P) day 2 to P4. In order to assess behavioral changes I conducted early reflex and motor skill tests on rat pups from ages P8 to P13. Early reflex testing included: righting reflex, negative geotaxis, and cliff aversion. Early motor testing included: ambulation, front limb suspension, hindlimb suspension, and grip strength. There was no significant difference seen in early reflex testing between control vs injured. Significant difference (P<0.05) was observed between control (n=19) vs injured (n = 11) in early motor testing: grip strength impulse, ambulation, hindlimb suspension and front limb suspension. Based on these findings, the novel preterm brain injury model is producing a motor deficit in the injured groups compared with the control group. Going forward, we will continue to repeat this experiment to verify the results and further elucidate what aspects of behavioral development are impacted most by this injury model.

Joubert syndrome (JS) is a neurodevelopmental disorder characterized by developmental delay and a specific hindbrain malformation resulting in the “molar tooth sign” (MTS) as seen on MRI. Individuals with JS have variable intellectual disabilities, polydactyly, retinal dystrophy and cystic kidney disease. JS is classified as a ciliopathy—disorders with overlapping phenotypes affecting 1/1000 live births combined. Currently, 36 genes when mutated cause JS, and all their encoded products localize in and around the primary cilium—a cellular organelle important for signaling and human development. We recently identified the newest JS-associated gene ARMC9 and its function, like the molecular mechanism(s) underlying JS, is unknown. Our preliminary data suggest that ARMC9 is required for cilium stability by promoting post-translational modifications (PTM) of ciliary microtubules. In stably ciliated, fixed (non-living) cells, ARMC9 localizes to the ciliary base, but to understand ARMC9’s role in cilium stability, we must determine ARMC9 localization during ciliogenesis (ciliary growth). We hypothesize that ARMC9 redistributes to the ciliary tip to promote tubulin PTMs during ciliogenesis. To test this hypothesis, I am using GFP and BFP (green and blue fluorescent protein) tags to visualize ARMC9 and primary cilia respectively. I created 5HT₆-BFP, a DNA vector encoding a BFP-tagged serotonin receptor fragment called 5HT₆ which naturally localizes to cilia. Currently, I am optimizing the transfection of 5HT₆-BFP and ARMC9-GFP into control cells for live imaging. As suggested by our data, I expect to see ARMC9 at the ciliary tip during growth, then redistribution to the base once the organelle is built. Determining where ARMC9 localizes during ciliogenesis will give us additional insight into its mechanism of action and role in JS. Ultimately, this work will allow us to identify therapeutic targets to mitigate the progressive features of JS and improve the quality of life of affected individuals.

Many genes have been found to be associated with microcephaly, a condition characterized by a smaller head size. Through exome-sequencing, we identified two de novo missense mutations in an actin polymerization gene, ARPC4, in four patients with microcephaly, mild developmental delay, and mild intellectual disability. To understand the molecular mechanisms that lead to the disease phenotype observed in the patients, I used a variety of genetic and biological assays. I began by using CADD, a measurement used to predict the deleteriousness of single nucleotide variants. The mutations identified in the patients were shown to be in highly conserved loci, indicating they might be pathogenic. We proceeded to study the functional effect of the pathogenic variants in actin polymerization. Actin is an essential component of a cell’s cytoskeleton which gives the cell its structure and aids in cell movement and cell division. I was able to establish fibroblast (skin) cell lines from two patients and perform an immunofluorescence assay staining for actin in patients and control fibroblasts. Additionally, I used western-blot, a technique used to detect a specific protein in a sample, to further quantify the amount of actin present in the patients’ cells. We were able to observe a greater abundance of actin filaments in the control sample compared to the patients’ cell. The results from this study elucidate the impact of ARPC4 in actin polymerization, establishing actin deficiency as a clinically recognizable cause of microcephaly.

The American Academy of Pediatrics recommends 10 preventive care visits, known as “well child care” for all children during the first three years of life. Multiple innovations in the delivery of these visits have been proposed, including group visits, team-based care, and e-visits. While some of these interventions have evidence of effectiveness, it is critical to understand how changes to delivery of care will impact utilization, and thus costs of health-related services for children. The objective of this study is to develop a parent-reported utilization survey to allow a full assessment of potential costs for making improvements to the structure of well child care. After a review of the literature, we developed the utilization survey, which included health and health-related services applicable to infants and new mothers. We included utilization for multiple services, including emergency department visits, inpatient hospitalizations, urgent care visits, early intervention services, maternal mental health services, support groups for new mothers, dental visits, and case management/social services. Each component collects data on how many times the child and/or parent utilized each service and the reason for service use. Each service is also matched to an estimated average cost, specific to California and Washington state. Once completed, the survey will be used in a large, cluster randomized controlled trial of a redesigned system for the delivery of well-child care at ten community clinics, serving a primarily Medicaid-insured population of children. This utilization survey may also be applicable to other efforts to implement well-child care delivery design in other settings.

Non Hodgkin Lymphoma (NHL) affects over 70,000 people in the United States each year. Inappropriate activation of B-cell receptor signaling is associated with NHL, and therapies that target this pathway are showing promising results in the clinic. One example is ibrutinib, a small molecule inhibitor of Bruton’s Tyrosine Kinase (BTK). While ibrutinib has improved the prognosis for the majority of NHL patients, many patients eventually develop resistance to ibrutinib due to mutations in BTK and its downstream target Phospholipase C gamma 2 (PLCG2). Thus, it is important to understand how these mutations affect cell signaling, with the ultimate goal of designing new strategies to mitigate ibrutinib resistance. Previous studies in the James Lab suggest that long-term treatment of cultured cell lines with ibrutinib leads to increased cellular reactive oxygen species (ROS). However, it is unclear if ROS contribute to ibrutinib resistance or if they are a byproduct of other mechanisms that drive resistance. I hypothesize that cells with mutations commonly seen in ibrutinib-resistant patients may also exhibit increased ROS, and these cells may be sensitive to small molecules that regulate ROS. In order to test this hypothesis, I used CRISPR/cas9 gene editing with homology-directed repair to generate lymphoma cell lines that express a leucine-to-phenylalanine mutation in PLCG2. This is a well-described mutation in ibrutinib-resistant patients. Preliminary analysis shows that these mutant cells are resistant to ibrutinib. I measured ROS in these cells by ROS-Glo luminescence. Then, I cultured the cells with ROS-regulating compounds including SOD-1 and NADPH oxidase inhibitors to assess how ROS affect their viability. My goal is to clarify the role of ROS in the acquisition of ibrutinib resistance. These studies may provide justification for further pre-clinical evaluation of small molecule regulators of ROS in lymphoma, especially among subjects with acquired ibrutinib resistance.

Familial caregivers often serve as the primary coordinator of care for children with medical complexity (CMC). However, they currently lack a central tool to organize their child’s health information from multiple fragmented information sources and support their unique information needs. The primary objective of this study was to develop a user-centered, care organization tool by incorporating the information needs and preferences of CMC caregivers. Following a user-centered design methodology, we first conducted interviews with CMC caregivers between 3/16-11/16 to understand their information needs and elicit their preferences for a care organization tool. Next, we conducted design sessions with caregivers between 8/17-12/17, which involved content categorization activities, drawing exercises, and scenario-based prototype testing. Between design sessions, we applied content analysis to interview and design session transcripts, and incorporated findings into the ongoing development of a functional prototype. We conducted interviews with 17 caregivers and design sessions with 6 caregivers. We grouped findings into two categories: design and implementation. An overarching theme of both categories was the need for customizability, as the primary purpose of the tool differed based on the caregiver’s main information needs. In terms of design, caregivers voiced a desire for both mobile and web interfaces, with a customizable home screen, containing templates to track medications, symptoms and major events, and outline communication preferences based on their child’s developmental status. In terms of implementation, caregivers were agreeable to the idea of a cloud-based tool which would provide easy access to emergency information. However, they wanted control over customizable permission settings for others to view and/or edit other content within the tool. We developed a novel, user-centered, cloud-based care organization tool for CMC families by engaging caregivers in the design process. Next steps include examining prototype usability, effectiveness, and integration with provider health records.

Inferior olive (IO) is part of the brain stem that projects their climbing fibers in the cerebellum's Purkinje cells and receives input from the deep cerebellar nuclei DCN to regulate the strength of electrical coupling in the IO. The structure of the IO contains a high density of gap junctions that are responsible for weak spontaneous subthreshold oscillation (STO). The weak electric potential STO can depolarize the neuron and open the N-methyl-D-aspartate receptor (NMDA) channel that is located close to the gap junction by removing its Mg ion. The activation of the NMDA receptor can play a role in strengthening the STO, increasing its synchrony, and inducing a new STO. Increase the synchrony in IO may contribute to the precision learning of the motor skills. Therefore, our study was done on mice in vivo, and extracellular recording technique was used to record from IO before and after the addition of NMDA. It was found that adding NMDA changed the firing of the neuron relative to itself. Second, NMDA decreased the power spectra density's raw power in both groups 2 Hz and 3-6 Hz, however, there was some increasing in the PSD at 5 Hz in the group of neurons that fire with 2 Hz. Third, the coherence value between the neuron and the local field potential (LFP) decreased after the addition of NMDA. There was an exaption in which the coherence value increased at specific frequences. Finally, we found that a neuron showed phase locking its firing activity with the LFP, which was in the negative firing phase. In conclusion, NMDA receptor plays a role in increasing the synchrony of neuronal electrical synapses in IO, involves in the IO rhythmogenesis, and may add temporal precision to coordinate the muscle activity.

Religious rhetoric has been a common tool in politics for the majority of American history, from Thomas Jefferson being “unfit to lead a Christian Nation” to Ronald Reagan’s “City Upon a Hill”. Although the Constitution declares the government to be secular, members of Congress, to varying degrees, regularly invoke religious imagery and rhetoric in the public communications. This study seeks to understand why. I examine congressional religious rhetoric using a unique data source of congressional webpages from the massive .GOV collection of the Internet Archive Project. I hypothesize that members' personal beliefs and electoral strategies help to explain differences in website content. I measure the personal religiosity of members using a carefully constructed scale of involvement in their religious communities. I measure district religiosity using information about district-level church attendance and prayer frequency from the Cooperative Congressional Election Survey. Finally, I draw from established religion lexicons to measure religious emphasis in congressional website content. I find positive, statistically significant support for both hypotheses. This work sheds light on politicians in particular members of congress are actively seeking to brand themselves through the internet for political purposes.

Previous studies have shown that adolescents with type 1 diabetes (T1D) benefit from being involved in support groups by significantly decreasing their perceived diabetes burden and effectively increasing their knowledge of the disease. The purpose of this study was to assess the feasibility of Instagram as a platform to build peer-support among adolescents with T1D. Participants (13-18 years old with T1D for >6 months) were asked to post photos each week for three months based on predetermined topics related to diabetes management. At the end of each month, participants attended a focus group to discuss their photos using the Photovoice method. Feasibility was assessed through enrollment and retention, number of Instagram posts, post-study questionnaire, and qualitative analysis of the focus groups. Of 24 eligible participants, 16 enrolled (67%) with 3 dropping out prior to the end of the study. The number of photos posted over 3 months ranged from 13-31, and the number of likes given ranged from 5-34. Among the 11 participants who completed a follow-up questionnaire, 55% reported that they very much enjoyed participating in the program, and 95% of participants reported “relating to the photos posted.” Additionally, several themes emerged from qualitative analysis of focus group transcripts regarding their experience using the Instagram intervention, including: establishment of a diabetes community, change in participants’ perspective of the disease, and efficacy of using Instagram, such as direct messaging and having multiple accounts. The results of this study show that Instagram is both a feasible and acceptable form of social media in which to build peer-support. Future research should investigate the use of social media support groups to examine improvement of self-esteem, benefit-finding, and social support using validated tools in adolescents with diabetes.

Non-coding regions account for approximately 98% of the genome and play a significant role in regulating and maintaining levels of gene expression via transcriptional and translational control mechanisms. Our lab focuses on the functional characterization of non-coding regulatory regions in the human retina and identifying mutations within these elements that contribute to inherited retinal diseases. In pursuit of this long-term goal, my aim is to establish an in vitro model to dissect non-coding regulatory elements in the human retina. Retinal organoid cultures are a promising new system for studying the development of the human eye, however it is currently unknown if organoids recapitulate all of the features of the adult human retina. Based on similarities of cell types and gene expression, we hypothesized that retinal organoids recapitulate some non-coding regulatory features of the normal human retina. To identify both shared and specific non-coding regulatory elements, I profiled the epigenetic landscape of developing human retinal organoids using a genome-wide method for assessing chromatin accessibility (ATAC-Seq). Chromatin accessibility is a feature of non-coding regulatory elements which ATAC-Seq revealed in human retinal organoids. I then used bioinformatic techniques to compare my organoid dataset to previous data we generated from the adult human retina and published data from the developing human retina. We found distinct sets of non-coding regulatory elements that were shared between retinal organoids and the human retina, as well as elements that were unique to each system. These comparative data will equip us with additional insight on which non-coding elements may influence retinal disease phenotypes. Furthermore, these data will potentially give us a clearer understanding of the epigenetic landscape of the developing and adult retina in a context that may drive progress towards regenerating damaged photoreceptor cells in the future.

Developmental and epileptic encephalopathies (DEEs) are epileptic disorders characterized by severe, recurrent seizures and intellectual disability. The Mefford Lab works to discover novel genetic causes of pediatric DEEs. We hypothesize that early-onset DEEs in severely affected patients with two healthy parents are caused by pathogenic de novo variants—disease-causing variants in the genome of the patient that are not inherited from either parent. Comparing sequence data in a trio that consists of an affected patient (proband) and two unaffected biological parents allows for efficient detection of de novo mutations and suggests a genetic cause for the associated phenotype. Trios are submitted for whole exome sequencing (WES) to obtain sequencing data from all the protein-coding regions of their genome. Occasionally, exome sequencing is unsuccessful for one or more members of the trio and alternative methods are needed to sequence and identify potentially pathogenic variants in these trios. I am working on identifying potentially causative variants in patients for whom one or more parent(s) did not successfully complete WES. I analyzed WES data to identify likely pathogenic variants in 10 probands by filtering for protein-changing variants that are not present in >100,000 healthy individuals. I used single molecule molecular inversion probes to perform simultaneous, targeted sequencing of the identified variants in the complete trios to determine which variants were de novo. The existence and inheritance of de novo variants was confirmed using Sanger sequencing. I have identified and confirmed a de novo variant in one patient in HIVEP2, which encodes a transcription-regulating protein expressed in the brain. I am currently completing the process of sequencing and filtering variants on additional trios. The further identification of de novo variants in these patients will be useful in better understanding the complex genetic causes and genotypic-phenotypic associations of epilepsy, and helpful in the clinical setting for improving and individualizing treatment of the disorder.

Developmental Epileptic Encephalopathy (DEE) is a severe form of epilepsy in children and infants characterized by prominent EEG (electroencephalography) abnormalities disrupting brain function. Identifying the genetic causes of DEE will help researchers develop medical treatments for affected patients. Approximately 5% of DEE cases involve a copy number variation (CNV), where a region of DNA has been duplicated or deleted in a gene, causing the disease. Historically, this type of mutation has been difficult to detect using sequence data. To analyze the large amount of sequence data available in the Mefford Lab, I am creating an algorithm that will analyze smMIP (single molecule molecular inversion probe) data to identify CNVs in patient DNA that are likely causal for DEE. The algorithm is a two-step process. First, z-scores are assigned to the number of smMIP reads at each target site of each patient sample to numerically assess how many standard deviations it is from the mean. Next, regions of contiguous significant z-scores will be extracted as areas of top interest to verify as being a CNV. I will run the completed algorithm with smMIP data from the Mefford Lab's unsolved DEE cases. Since CNVs have historically been difficult to detect, it is unclear what percent of these unsolved cases will contain a CNV. It might be that, due to previous lack of detection, CNVs play a more significant role in DEE than previously thought. The opposite might also be true if a large majority of unresolved cases are due to other types of mutations, e.g. point mutations. Regardless, the algorithm will shed light on new causative CNVs and allow the Mefford Lab to solve more patient cases. Additionally, a successful CNV calling algorithm can be shared with other labs to aid them in their research.

Developmental epileptic encephalopathies (DEE) are severe brain disorders characterized by early onset seizures, developmental delays, neurological deficits, and regression. The Mefford lab works to find the genetic causes of DEE, which can usually be linked to a single de novo variant in each patient (mutation present in the patient that was not inherited from either parent). De novo variants in candidate genes were discovered through whole exome sequencing (WES). We validated these variants using Sanger sequencing. Of the 291 de novo variants identified by WES, 105 validated. In order to find support for the causative nature of variants in these genes, a cohort of 898 DEE patients is currently being screened for variants in 53 candidate genes identified through WES (T7). The targeted sequencing is done using smMIPs (single-molecule Molecular Inversion Probes) to simultaneously capture all exons of T7 genes in our cohort, requiring over 2,000 MIPs. However, uniform capture of target regions with smMIPS can be problematic. Some easily sequenced areas are over-captured while tougher regions are under-captured, leading to a need for rebalancing. We tested T7 smMIPs at concentrations of 1X and 10X to provide data on capture efficiency and then compiled to provide the best concentrations per smMIP in the final run. The result was 91% of reads falling on-target with 88% of smMIPs producing at least 8 reads. We hope to find multiple variants in the same gene across multiple patients in our cohort to confidently identify new DEE genes. Confirmation of new genes will help to advance our understanding of these severe disorders and pave the way for more effective treatments in the future.

Brain tumors are the most common form of pediatric cancers after leukemia. Deregulation of the family of MYC (avian myelocytomatosis viral oncogene homolog) transcription factors in some forms of pediatric brain cancer correlates with very poor prognosis. It is believed that overexpression of MYC may play a role in sustaining aggressive brain tumor cell populations in diffuse intrinsic pontine glioma (DIPG), medulloblastoma, and atypical teratoid/rhabdoid (AT/RT) pediatric brain tumor types. A targeted therapy capable of inhibiting MYC transcription factors may be a promising option for children with MYC-upregulated brain tumors. We addressed the role of MYC genes in the tumorigenesis of several patient-derived pediatric cell lines with upregulation of MYC transcription factors. Via a lentiviral delivery agent, we attempted to knock down MYC transcription factions through the expression of doxycycline induced omomyc—a 90 amino acid, MYC-derived, polypeptide that interferes with normal MYC activity by outcompeting for the enhancer binding sites that MYC normally binds to. We found that MYC inhibition, using omomyc, halted the growth of tumor cells in over half of the patient-derived primary cell cultures with MYC overexpression. 6 out of 11 lines showed significant inhibition of tumorigenesis through the induction of omomyc. This suggests that the mechanism of action of omomyc, as a MYC inhibitor, may serve as a model for drug development against MYC for pediatric brain cancer.

Early recognition and reporting of concussions in youth sports is crucial in ensuring proper treatment and minimizing possible consequences. All US states have passed legislation requiring that youth sport stakeholders be provided with annual education about concussion, ostensibly because this knowledge will help them engage in key safety promoting behaviors, including reporting symptoms of a suspected concussion. By conducting a systematic review of peer reviewed published studies that measure concussion knowledge, we ask how youth athlete concussion knowledge influences concussion reporting. The hypothesis is that better concussion knowledge is positively correlated with increased reporting of concussions. A systematic review of peer reviewed research published between 2009 and 2018 was conducted in November 2017. Inclusion criteria were 1) measuring concussion knowledge using a quantitative survey measure, 2) the athletes were collegiate age and younger. This resulted in 127 studies. Subsequently, studies were reviewed by two independent coders to determine whether they measured concussion reporting behavior or behavioral intentions. Data was collected in order to assess the strength of association between knowledge and behavior. While data synthesis is ongoing, preliminary results suggest that the association between knowledge and concussion reporting behavior or intention is minimal. Interventions are needed to increase reporting of suspected concussions, however, current results suggest that education focusing solely on increasing an athlete's knowledge about concussions may not be sufficient. Future research should focus on motivators of concussion reporting behavior in youth athletes and explore intervention methods that address evidence-based determinants of this important safety behavior.

Epilepsy is a wide classification of neurological disorders that vary in symptoms, severity, age of onset, and demographics. Developmental and epileptic encephalopathies (DEE) are severe forms of epilepsy that involve both early-onset seizures and cognitive and behavioral impairments. One major cause of DEE is genetic variation. However, only 24-40% of DEE cases receive a molecular diagnosis. Our goal is to identify novel genetic variants that cause DEE via sequencing. In DEE, many of the variants associated are de novo, meaning they are novel mutations not inherited from either parent. Because of this, we are able to utilize segregation testing to identify the inheritance of a variant and determine if a variant is potentially pathogenic. From whole exome sequencing data on 200 patient and parent trios, we identified de novo variants in genes not previously associated with DEE. We then narrowed down our list of potential DEE genes to 53 candidate genes via Sanger sequencing. Using a sequencing technique called single molecule Molecular Inversion Probes (smMIPs), we simultaneously and accurately sequenced these 53 potential DEE genes in our cohort of 898 DEE patients, looking for additional de novo variants. These variants include missense mutations, frameshift insertions and deletions, and premature stop codons. Once potentially pathogenic variants are identified in the patient genome, we screened the parents using specific smMIPs selected for the patient-determined target regions to determine inheritance. If the gene validated as de novo, we can investigate the potential role of the affected protein in the phenotype. Our hope is that by identifying additional de novo variants in some of these 53 candidate genes, we can identify novel genes associated with DEE and provide answers to families about the cause of their child's condition. In the future, we hope the genetic understanding of DEE could lead to better, genotype-driven treatments.

 Developmental and epileptic encephalopathies (DEEs) are an early-onset form of epilepsy that result in aggressive and recurring seizures, possibly leading to 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 RNA, generating a protein that is non-functional, partially functional, or aberrantly expressed. If aberrant splicing occurs, it could have pathogenic consequences, but predicting which variants near splice sites will have an effect on splicing is difficult. This raises the need to investigate splice-site variants. My work is concentrated on potential splice site variants in three genes associated with DEE: SYNGAP1, SCN1B, and WWOX. SCN1B codes for voltage-gated channels within the brain, SYNGAP1 is associated with postsynaptic signaling, and WWOX acts as a tumor suppressor and is vital in brain development. DEE genes are primarily expressed in the brain, a tissue that is unavailable from most patients, making splicing studies in these genes difficult. I was able to successfully amplify targeted regions of SCN1B, SYNGAP1, and WWOX in RNA isolated from blood and fibroblast, demonstrating the efficacy of using these more easily accessible tissues in my studies. The lab has now collected fibroblast samples from DEE patients with potential splice site variants in WWOX and SCN1B. I will use RNA extracted from these fibroblasts to confirm whether the splice site variants in WWOX and SCN1B cause aberrant splicing, and what the specific consequences of this aberrant splicing is on the protein-coding sequence of these genes. In the future, we also plan on collecting fibroblasts samples with variants from SYNGAP1 as well.

Joubert syndrome (JS) is a rare recessive genetic disorder characterized by hindbrain malformations appearing as the “molar tooth sign” on axial brain imaging. Causal genetic variants for JS need to fulfill three criteria: They need to be 1) rare, 2) deleterious, and 3) present in both alleles in the patient. Patients in our cohort are sequenced for rare predicted-deleterious variants (RDVs) to identify variants that satisfy the first two criteria. To determine whether the segregation pattern of the variants satisfies the third criterion, we sequence both parents, each of whom should carry only one of the variants. My research uses Next-Generation Sequencing to determine the segregation pattern for families in whom RDVs have already been identified. Specifically, I performed targeted-sequencing of putative causal variants in the parents using molecular inversion probes, and then I used the sequencing data to determine the segregation pattern. In most families, one RDV will be present in each parent, indicating that they affect both alleles in the child, because these variants are robust candidates that have passed the filters for being rare and deleterious. If both RDVs are present in one parent, then they have to be on the same allele and cannot be the cause of JS in the family. In these families, we will have to consider other variants or perform additional sequencing to identify the causal variants. Ultimately, the goal of my research is to elucidate the genetic basis of JS to improve prognostic and risk recurrence information, provide diagnostic, carrier and prenatal testing, and delineate the biological mechanisms of JS that will be the targets of future precision therapies.

Prevalence of youth with Type-2 Diabetes (T2D) is increasing. Youth with T2D are at risk for cardiovascular disease and are advised to take on lifestyle change behaviors to improve outcomes, such as exercise, improving diet, and managing stress. The process of goal setting includes determining what needs to be accomplished and creating a plan to achieve this result. Goal setting has been found to help create healthy habits that can aid in the management of disease. While previous work outlines the importance of goal setting in T2D, little is known about the goals set by youth with T2D and whether they influence one’s health. For this study, we sought to examine the content of goals amongst youth with T2D and to examine whether the type of goal is associated with glycemic control. One hundred and seven youth with Type-2 Diabetes completed a clinical intake survey during their clinic visit at Seattle Children’s Hospital in 2017. The goals were categorized into altering blood sugar and blood glucose intake (N=27, 25.32%), changing eating habits (N=20, 18.69%), insulin management (N=15, 14.02%), improving or decreasing exercise (N=12, 11.21%) and decreasing highs or lows through exercise (N=5, 4.67%). Our next step (currently in progress), is to examine the association of these goal categories with A1C to better understand whether the type of goal relates to concurrent glycemic control. We anticipate that those with higher A1C may choose goals related to exercise and eating habits more than those who have lower A1C and are in good control. These results will inform future interventions to use goals within this population to provide tailored education to improve outcomes in youth with T2D.

Palatal shelf elevation is an essential morphogenetic process during closure of the secondary palate. It has been proposed that palatal shelf elevation results from an intrinsic elevating force while also relying on extrinsic factors associated with development of other orofacial structures, such as the lower jaw and tongue. While in vitro palate culture is commonly utilized to study palate shelf elevation, it requires removal of the tongue or mandible prior to culture, which prevents any determination of the role of extrinsic factors in regulating palatal shelf elevation. Therefore, the aim of our project was to improve upon palate culture methods to better mimic in vivo conditions of palate development for studying the mechanism of palatal shelf elevation. We modified culture conditions to maintain the integrity of the oral cavity by leaving the tongue and mandible in place and cultured these mouse palate explants in suspension in dynamic conditions. Using this technique, the palatal shelves successfully elevated after 24-48 hour culture and displayed morphology like that of in vivo elevation, suggesting that our method improves upon previous culture techniques for investigation of the roles of both intrinsic and extrinsic factors during secondary palate development.

Vaccine hesitancy, or negative beliefs and attitudes about vaccines, is a key driver to under-immunization in the United States, which can lead to an increase in vaccine preventable diseases (VPDs). Although adolescents are among the populations at risk for VPDs, little research focuses on vaccine beliefs and vaccine hesitancy in this age group, especially college students. College students have both greater independence and increased autonomy in healthcare decisions including the decision to receive HPV, Tdap, Flu, and meningococcal vaccinations. However, only a small percentage of college students receive all the recommended vaccines. The purpose of this study is to explore and identify factors that influence college student's perceptions about vaccine safety and reliability using cluster mapping. We enrolled 65 current undergraduate college students from the University of Washington, who are between 18 and 23 years old. Participants generated statements about factors that influence college student's perceptions about vaccine safety and reliability. These statements were linked and ranked using Concept System's software (Concept Systems, Inc., Ithaca, NY), and the resulting data was used to generate a cluster map, which was interpreted in focus groups. 25 students participated in generating statements and were 56% female, 44% Caucasian, and have an average age of 19.8 years. After eliminating redundancy, the open ended question yielded 102 unique responses. Examples include: "affordability of vaccinations" and "social media". For the next stage, 25 participants who were 48% female, 52% Caucasian, and have an average age of 19.75 years grouped these statements into clusters. Participants on averaged created 7.5 clusters, which included cluster titles such as: "personal beliefs", "vaccine accessibility", and "media about vaccines". The generated cluster map will provide researchers with invaluable insights that may aid in the development of interventions and may help achieve our long-term goal of increasing vaccine uptake among college students.