Lymphatic malformations (LM) are congenital vascular malformations resulting from dysregulated growth of lymphatic vessels. LM can cause difficulty in breathing, swallowing, or eating, and in rare cases, infection or death. LM are associated with postzygotic somatic mutations in PIK3CA, which encodes for the catalytic subunit of PI3K and regulates cell growth and proliferation. Three hotspot mutations cause the majority of LM: p.E545K, p.E542K, and p.H1047R. These mutations occur at very low level in the affected tissue; usually at <10% variant allele fraction (VAF) which makes diagnosis challenging. We hypothesize that variant allele fraction (VAF) differs throughout the vascular malformation and will correlate with location in the lesion. We used droplet digital polymerase chain reaction to detect PIK3CA mutations in 8 LM subjects with 95 samples including lesion, muscle, skin, and fat. We then mapped the VAF to magnetic resonance imaging to assess genetic heterogeneity by sample location. Globally, no sample had a VAF greater than 10% indicating that assaying multiple samples does not increase the observed VAF compared to other studies where one sample was assayed. Within patients, VAF varies within the lesion, ranging from undetectable to 10%. Non-lesion tissue, including skin, and most muscle samples, had no mutation detected. All four fat samples and two muscle samples were positive for mutation with VAF <2%. We did not identify any correlations between VAF level and location within the lesion. Though we didn’t identify a correlation between VAF and location in our data, these findings will be used for further analysis including understanding how cell type composition within the samples correlates with VAF. Our goal is that this dataset will help us explore the development of LM and eventually provide information useful for the advancement of targeted therapies for future pediatric patients.

Autism Spectrum Disorder (ASD) is a neurodevelopmental disorder characterized by the presence of repetitive patterns of behaviors and deficits in social communication and interaction. Attention to social information is a key component of the development of social communication. Previous studies used eye tracking to examine visual scanning patterns associated with social attention in children with ASD and neurotypical children. Eye tracking is used to both identify the atypical patterns of social attention and to predict clinical outcomes in ASD. Although atypical eye gazing patterns are considered as potential biomarkers, researchers commonly consider data loss in eye tracking as error or noise, and rarely investigate it more thoroughly. In this proposal, we hypothesize that loss of data is a potential signature of core social motivation issue when a social video is playing, and, rather than being a nuisance variable, which reflects the broader continuum of social attentional-motivational challenges faced by individuals with ASD. We used eye tracking to confirm previous findings on atypical attention patterns, and further utilize behavior coding to examine the three types of causes of data loss including blinking, non-compliant behaviors, and technical error. We hypothesize that data loss due to blinking is associated with a lack of social motivation and that data loss due to non-compliant behaviors is associated with executive function. Social motivation and executive function were measured by parent reports. Exploring data loss in eye tracking may help reveal comprehensive and fundamental factors of diminished social motivation and neurocognition in ASD.

Autism spectrum disorders (ASD) are a group of neurodevelopmental disorders characterized by impairments in social interaction and repetitive behaviors. One of the most common mutations leading to ASD is 15q11-q13 duplication, a CNV mutation where extra copies of a chromosomal region are expressed. The major gene within this region is ube3a. My lab has generated a mice model with increased copies of this gene and demonstrated a novel deficit in phosphorylated-AKT, a key protein in the mTOR/AKT signaling pathway. Other mice models of autism have exhibited deficits in this pathway and have rescued behavioral deficits with a drug, rapamycin. Rapamycin is an inhibitor of a key protein of the mTOR/AKT pathway suggesting a similar behavioral rescue might be observed with ube3a transgenic autism model mice. Behavioral testing of 4 groups of 20 mice, wild type and ube3a transgenic mice treated with and without rapamycin, found that there were no baseline social deficits in ube3a transgenic mice not treated with rapamycin. This is not similar to previously published findings and could be due to a number of reasons. It can be difficult to get consistent results with mice behavioral testing. In order to assess if rapamycin had its intended effect in restoring deficit levels of phospho-AKT, protein levels will be assessed. Brain slices from the hippocampus of behaviorally tested mice will be analyzed by western blot, a method of identifying and measuring specific proteins. Successful rapamycin treatment would be observed as increased levels of phospho-AKT, similar to wild type mice. This would suggest that repeated mice behavioral testing is needed and therapeutic treatment of the mTOR/AKT signaling pathway could be a viable target for patients with 15q11-q13 duplication.

Autism Spectrum Disorder (ASD) is a chronic developmental disorder classified by deficits in social communication and restricted or repetitive patterns of behavior. A highly comorbid disorder with ASD is Attention Deficit Hyperactivity Disorder (ADHD) characterized by inattention, hyperactivity, and impulsivity. Deficits in empathy and facial affect recognition have been observed in children with ASD and ADHD. Through the APEX 5-week intensive Summer Treatment Program (STP), the UW Autism Center integrates naturalistic therapy for children diagnosed with ASD and/or ADHD aged 6 to 12. Within this evidence-based program, children develop social and behavioral skills through structured recreational and learning activities. During the summer of 2019, we collected the Positive and Negative Affect Schedule for Children (PANAS-C) from 120 participants ages 6-12 weekly. The PANAS-C measures youth anxiety and mood by asking participants to rate each emotion, five positive and five negative, on a point scale from 1 (not at all) to 5 (very much). Through multiple regression analyses, we aim to examine child affect through the PANAS-C within the context of variables such as age, gender, and diagnosis. We hypothesize that during participation in the summer treatment program, positive affect ratings will increase as negative affect ratings decrease. We expect that the combination of integrative social skills training, positive reinforcement, and acquisition of behavioral skills, will increase positive affect for children in this setting.

The maternal bond is an intimate attachment between a primary caregiver (PC) and their infant which provides the infant with security, facilitating physical, social, and emotional development. A sensitive and responsive environment, such as the presence of healthy maternal bonds, guides an infant’s neurodevelopment. Changes in mood and emotional state can alter the care a PC provides and cause difficulties in bonding with their infant, impacting the baby’s psychological and physical development. In infants, the mechanisms by which development may be impacted are unknown. Recently, research has shown early atypical attention to visual pop-out in autism spectrum disorders. Attention to visual pop-out describes our cognitive ability to quickly identify differing objects presented among similar looking ones. In this project, we investigated whether PC mental health affects attention to visual pop-out in infants. Participants included 50 infants who were assessed at 6 and 12 months of age. PCs completed the Beck Depression Inventory (BDI) and Beck Anxiety Inventory (BAI) at both timepoints. Infants watched a 4-minute visual pop-out paradigm, which included social (face) and non-social (shape) trials. We assessed responses to visual pop-outs and explored whether BDI and BAI scores correlated with visual pop-out performance at 6 and 12 months. We also investigated whether BDI and BAI scores influenced the development of attention to visual pop-out between 6 and 12 months. We hypothesized infants of primary caregivers who report more (a) depressive and (b) anxious symptoms will demonstrate weaker identification of the pop-out during social trials compared to their peers but be unaffected during nonsocial trials. This study will help deepen our understanding of the impact of maternal depression and anxiety on infant development and help health providers identify and support families.

Malaria, a disease caused by Plasmodium parasites, is an enormous public health burden, especially in resource-poor areas of the world. After the female Anopheles mosquito deposits the Plasmodium sporozoite stage to the host through its saliva during blood feeding, the sporozoite quickly makes its way to the liver where it selectively invades a hepatocyte. The parasite replicates within the host cell, eventually re-entering the blood stream where it causes symptomatic infection. My project focuses on the liver stage of malaria, where complex hepatocyte signaling pathways contribute to parasite development and replication. Previously, our lab demonstrated that host signaling pathways that control lipid peroxidation are crucial to regulating liver stage infection during the first 24 hours. Inhibiting SLC7a11, a protein associated with the regulation of lipid peroxidation, led to increased peroxidated lipids within the infected cell and reduced Plasmodium liver stage parasite infection. Interestingly, lipid peroxides were localized to the infected hepatocyte, leading us to question the kinetic and spatial distribution of peroxidated lipids within the infected hepatocyte. I cultured Hepa 1-6 cells and infected them with Plasmodium yoelii sporozoites. After infection, I treated the cultures with drugs that promote or inhibit SLC7a11. Lipid peroxide levels and localization were observed by fluorescent microscopy at six hours post-infection and mean fluorescent intensity was quantified. At six hours post-infection, I observed no significant difference in lipid peroxidation when comparing infected and uninfected cells, suggesting that lipid peroxidation in infected cells occurs at some point between 6 and 24 hours. This project will allow us to gain a better understanding of how the lipid peroxidation pathway contributes to limiting Plasmodium infection within the liver and how it might be targeted by therapeutics to selectively kill parasites without harming the host.

Maternal microchimerism (MMc) is the transfer of maternal cells to the fetus across the placenta during pregnancy. MMc is found in both normal and abnormal pregnancies. The Harrington lab has previously shown that the amount of MMc increases with Plasmodium falciparum infections during pregnancies. In the same cohort, children with detectable levels of MMc were more likely to become infected with malaria but interestingly less likely to experience symptoms, suggesting that these maternal cells may regulate or educate the infant immune response against malaria. I aim to assess whether maternal cells selectively proliferate in the infant during the first infection with Plasmodium falciparum in order to mount an immune response by comparing the levels of MMc throughout three time points. To investigate the role of maternal cells in the infant immune response, I will test blood samples of infants exposed to placental malaria from three time points (a cord blood sample, a blood sample immediately preceding the infant’s first parasitemia, and a blood sample immediately following the infant’s first parasitemia) for the presence and amount of MMc. I hypothesize that MMc will be found in higher quantities in blood samples following an infant’s first infection with parasitemia, indicating that maternal cells help mount an immune response in the infant. Blood samples stored as dried blood spots (DBS) have been received from Ugandan field sites. First, the DNA was extracted from DBS samples. After comparison of class II HLA markers between a mom and her offspring, quantitative PCR (qPCR) will be used to target a unique, maternal HLA-class II marker in order to quantify MMc levels. The levels of MMc will be compared across the three time points. The results of my research have important implications for the current understanding of how MMc regulates the infant immune system.

In recent years, numerous studies have investigated the transfer of rare cells between a mother and her fetus during pregnancy, a phenomenon known as microchimerism. Maternal microchimerism (MMc) refers to maternal cells transferred to the fetus, whereas fetal microchimerism (FMc) refers to fetal cells transferred to the mother. FMc is found in both normal and abnormal pregnancies, and in particular is increased in settings of placental dysfunction such as pre-eclampsia. Plasmodium falciparum infection during pregnancy may lead to sequestration of infected erythrocytes in the placenta, known as placental malaria, that results in a phenotype similar to pre-eclampsia. The Harrington lab previously found that the fetus acquires more MMc in the setting of placental malaria. My research project investigates the reciprocal transfer of FMc to the mother in this setting. I hypothesize that malaria infection during pregnancy is associated with an increased prevalence of FMc. To complete this project, high-quality genomic DNA was extracted from samples gathered from a cohort of women in Mali. These samples came from women both with and without malaria infection during their pregnancy. After collecting the DNA, HLA-typing between a mother and her offspring were compared to determine a unique marker of fetal DNA. I am currently using quantitative PCR to amplify fetal alleles in the background of the mother in order to quantify FMc. Lastly, I will compare the level of assessed FMc in malaria exposed and non-exposed women. The findings from this research project have the potential to contribute important information about the complex role of microchimerism in immune function.

Adolescents with newly diagnosed T1D are at risk for poor physical and psychosocial outcomes. We explored associations between glycemic control (A1C) with diabetes-distress, resilience, and psychosocial comorbidities (e.g., depression) over the first five years of diagnosis. Adolescents, aged 10-17, with newly diagnosed T1D completed validated diabetes-distress and resilience scales one-year post-diagnosis. Psychosocial comorbidities and A1C were extracted from patient charts for 5-years from diagnosis, and A1C values were averaged per year. Regression analyses were used to investigate associations between resilience, diabetes-distress and psychosocial comorbidities with A1C. A1C was assessed annually up to five years post-diagnosis. At one-year post-diagnosis, N=60 adolescents (M=13.22±2.09 years) completed distress (M=27.97±7.01) and resilience scales (M=40.35±17.10). Average A1C at 1-year was 7.73± 1.57 and at 5-years was 8.78 ±1.92. 14% of the sample had at least one psychosocial comorbidity at diagnosis. Between years 1-5 post diagnosis, 28.6% of the sample had at least one comorbidity. The most common comorbidities were depression and anxiety. Diabetes-distress was associated with average A1C in the second year (F(1,29)=4.397, p=.045, R2=.132), third year, (F(1,27)=6.596, p=.016, R2=.196), fourth year, (F(1,24)=10.196, p=.004, R2=.298), and fifth year post-diagnosis (F(1,19)=10.665, p=.004, R2=.360). Resilience was associated with average A1C in the second year (F(1,29)=6.848, p=.014, R2=.191) and fifth years (F(1,19)=4.790, p=.041, R2=.201) post-diagnosis. Total psychosocial comorbidities at diagnosis was associated with average A1C in the second year (F(1,49)=2.209, p<.01), third year (F(1,45)=7.925, p<.01), and fifth year (F(1,28)=7.919, p<.01) post diagnosis. The first year of diagnosis for adolescents with T1D is crucial for detecting patients who are at a higher risk for developing poorer health outcomes. Adolescents who present with psychosocial comorbidities at diagnosis and report poor resilience and high distress one year later are at risk for subsequent poor glycemic control. 

Chronic nonbacterial osteomyelitis (CNO) is a chronic autoinflammatory bone disease that typically affects children. It can cause complications such as bone deformities and vertebral fractures. Previous studies were retrospective in nature. The Childhood Arthritis and Rheumatology Research Alliance (CARRA) CNO workgroup has created a consensus treatment plan for children with CNO refractory to NSAIDs and/or having spinal lesions. In order to determine the relative effectiveness of commonly used second-line medications, a longitudinal prospective study is needed. This study was approved by the Seattle Children’s Institutional Review Board (#1232). The CHronic nonbacterial Osteomyelitis International Registry (CHOIR) enrolls subjects under 21 years of age with a diagnosis of CNO, refractory to NSAID and/or having spinal lesions. The goal is to enroll 2000 patients and follow for at least 5 years. Demographic, clinical, laboratory, imaging data as well as patient/parent-reported outcomes were collected at baseline and follow up visits. 136 subjects were enrolled across 8 centers between June 2018 and January 2020, with 40 more centers anticipated to join. Data entry and data analysis are ongoing. We are presenting an inception cohort of over one hundred subjects. We expect that the longitudinal data will allow us to distinguish the responses from patients to different second-line medications. The patient/parent-reported outcomes are also expected to be an important component of the overall assessment. This is the first ever prospective longitudinal multicenter study on children with CNO. Results are expected to be highly applicable to improve clinical care.

Executive function (EF), a broad term for an individual’s higher-order cognitive abilities, has been shown to be an important factor in proper development of play in childhood. Children with autism spectrum disorder (ASD) have been noted to score significantly lower on tasks requiring EF and are often noted to engage in more simplistic levels of play compared to typically developing peers. We investigated within-group associations of average play level for children with ASD, as observed during parent-child play sessions, in relation to parent-reported EF scores, as measured by the Behavior Rating Inventory of Executive Function (BRIEF). Participants with ASD (n = 28, age = 3-11 years) and participants with typical development (n = 27, age = 2-7 years) engaged in a video-recorded, 15-minute unscripted parent-child play session. Blind coders determined the child’s level of play, ranging from object manipulations to pretend play, on a numeric scale of 1-13. The highest level of play was coded at each one-minute epoch of engagement using Behavior Observation Research Interactive Software. Participant’s play scores were averaged and analyzed with their BRIEF scores using Pearson’s correlations. Results indicated no significant correlation between average play level and the BRIEF working memory, planning, and inhibition subscales, with Pearson’s correlations ranging from less than .01 to 0.03 (p > 0.8). Likewise, for participants with typical development, there was no correlation between average play level and BRIEF global composite scores, with Pearson’s correlations less than 0.01 (p > 0.9). Our current analysis did not account for parental support of the child’s play, which may contribute to why parent-reported EF scores did not relate to child play level in these unscripted parent-child play sessions. Future directions include examining the relationship between EF and play in children with other developmental disabilities.

Chronic Non-bacterial Osteomyelitis (CNO), also known as Chronic Recurrent Multifocal Osteomyelitis (CRMO), is an auto inflammatory bone condition that causes persistent bone pain, poor growth, and complications such as vertebral compression fracture. Understanding the possible epidemiological risk factors impacting the disease will provide all physicians including pediatric rheumatologists a better comprehension of the disease when diagnosing and treating CNO. The objectives of this study are to: 1) determine the annual incidence rate of CNO from King County; 2) compare the time to diagnosis from 2000-2019.Subjects were identified by the patient list maintained within Division of Rheumatology, Seattle Children’s Hospital. Diagnosis will be ascertained by chart review upon IRB approval based on published diagnostic criteria. Demographic and basic clinical information including the date of disease onset and physician diagnosis, first visit to a rheumatologist, and geographic location will be extracted. Public census data from King County will be used to calculate the annual incidence rate of CNO. Descriptive statistics will be performed and a nonparametric test will be used to determine the difference of time to diagnosis over time. A geographic mapping across WWAMI region for all cases will be generated to identify any patterns of case clustering. 250 subjects were identified with diagnosis of CNO between 2000 and 2019. Chart extraction will be completed after IRB approval. I expect the annual incidence rate calculated based on recent years (2015-2019) will be similar or greater than previously reported. I expect that the time to diagnosis is shortened during recent years likely due to the rise of awareness of CNO. From a large single center in the United States, I will be able to estimate the annual incidence rate of CNO in children. Continuous efforts of raising the awareness of CNO are needed to identify all cases for optimal care.

Previous studies show that when a parent moves their infant to a second sleep location at night, it is often less safe than the original location. The objectives of this study were to assess the safety of infant second sleep locations consistent with the American Academy of Pediatrics Safe Sleep recommendations and analyze demographic factors associated with the occurrence of second sleep locations. We conducted a self-report survey of 1500 randomly selected parents with children <12 months using Qualtrics, an online survey platform. Questions focused on participant’s recent infant sleep behaviors, including sleep position, co-sleeping, and sleep location at 2 timepoints: sleep onset and after nighttime waking. We examined summary statistics of demographics for all participants and used T-tests and bivariate logistic regression to compare demographic characteristics of parents who reported a second sleep location with those who did not. Safe sleep was defined as: supine position, not co-sleeping, and in a crib, bassinet, or playard. A composite score examined if all safe sleep behaviors were used at both timepoints. We found that 39% of participants reported a second sleep location for their infant the previous night. Of parents who reported a second sleep location: 28% met all 3 safe sleep criteria at sleep onset, only 9% met all 3 criteria at both timepoints, and 66% of those parents did not meet the 3 criteria at either timepoint. Demographics associated with higher odds of reporting a second sleep location were parental age <29 years, Hispanic parents, preterm babies, first-time parents, babies <6 lbs, and parents identifying as the baby’s primary caretaker 6 days a week. Our findings show the prevalence of unsafe infant sleep behaviors used by parents throughout nighttime, highlighting a gap in our current approach to educating parents on safe sleep behaviors to minimize infant sleep mortality rates.

Chronic Nonbacterial Osteomyelitis (CNO) is a severe and occult autoinflammatory bone disease of unknown cause. CNO predominantly affects children and young adults, with older literature reporting estimated annual incidence rates of 0.4 per 100,000 children. A timely diagnosis is challenging, but critical, because CNO may result in permanent bone damage, long-term disability, and disfigurement when left untreated. Diagnostic criteria were proposed by three groups. These sets of criteria were derived from single-center cohorts and none has been validated. In the proposed initiative, we developed a consensus among internationally recognized experts based on the analysis of a large international cohort of pediatric CNO patients and pediatric disease controls in order to develop and validate classification criteria for pediatric CNO. IRB approval was obtained at each site. CNO cases and mimicker cases were identified. Data were collected retrospectively using uniform case report form. The demographic, symptoms, physical examination, lab result and imaging feature were obtained. Descriptive statistics were performed. Our international group have collected a total of 360 cases including 226 CNO and 134 mimicker cases from 18 centers in 4 continents. 53% of subjects were females and 69% were whites. The typical site of bone pain is the lower extremity (231, 65%) followed by the lower torso (83, 24%). The most frequent imaging technique performed is the MRI (326, 91%). The top three mimicker diseases were infectious osteomyelitis (35, 10%), primary malignant bone disease (27, 8%) and juvenile idiopathic arthritis (24, 7%). The diagnosis of CNO was most commonly based on imaging features (116, 55%) and bone biopsies (61, 29%). A large international case repository was successfully created by using predetermined data collection form. These datasets can aid the development of classification criteria and future validation.

Hemophilia A is an X-linked bleeding disorder caused by the absence or defective function of plasma coagulation factor VIII (FVIII). A significant complication with hemophilia A treatment by FVIII transfusions is the development of inhibitory antibodies to the FVIII protein. This project uses a combined treatment of weekly intramuscular injections of an engineered FVIII DNA plasmid vaccine with liver-directed gene therapy using a DNA plasmid encoding the FVIII protein to treat hemophilia A in mice. This strategy was designed to suppress the immune response in order to achieve persistent expression of FVIII without inducing the formation of anti-FVIII antibodies. To test the success of this experiment, blood samples were collected at various time points throughout the project and the plasma was used in FVIII antigen and FVIII inhibitor assays. Clotting assays were performed to test the activities of FVIII and FVIII inhibitors. This project consisted of three different experimental groups: a group that started FVIII vaccines before liver-directed gene therapy, a group that started FVIII vaccines after liver-directed gene therapy, and a control group that received PBS only combined with gene therapy. We hypothesized that the groups receiving the engineered FVIII plasmid vaccine should have decreased levels of inhibitor antibodies and increased expression of the FVIII protein compared to the group receiving PBS only. If this project succeeds using hemophilia A as a model, this strategy could potentially be used in other disorders with undesired immune responses.

Cystic fibrosis (CF) is a genetic disorder affecting the lungs, and chronic polymicrobial lung infections are responsible for decreased life expectancy and poor quality of life of CF patients. Staphylococcus aureus (SA) is a microbe commonly found in the respiratory tract of CF patients, and this organism adapts within the lung environment to establish chronic infections. Among the most common bacterial adaptations is the emergence of mutants known as small colony variants (SCVs). There are multiple subtypes of SCVs that arise from mutations in different metabolic pathways. Recent studies have demonstrated that SCVs are prevalent in the CF respiratory tract, are more difficult to treat with antibiotics, and are associated with worse lung health. SCVs are very difficult to detect in clinical laboratories, thus complicating the selection of appropriate treatment by physicians to improve the health of CF patients. The goal of this study is to determine if SCVs can be more readily detected than with standard culture by using mass spectrometry to identify proteins that distinguish these variants from normal colony S. aureus. Matrix Assisted Laser Desorption/Ionization-Time of Flight (MALDI-TOF) will be used to identify proteins unique to specific SCV subtypes by separating those proteins using ionization and Tandem Mass Spectrometry. This analysis will generate isolate-specific spectra of peaks which will subsequently be compared to each other using Principal Coordinate Analysis (PCoA). We hypothesize this technique will identify differences between proteins produced by each SCV type, which can then be distinguished from normal colony S. aureus, allowing the rapid identification of these variants. As a result, we anticipate the detection of SCV’s will improve, which will help inform physicians to select appropriate treatments to target SCVs.

Sports-related concussion data in high school, collegiate, and professional athletes is well documented. However, limited data exists in the middle-school-aged population. The purpose of this study was to better understand the mean number of diagnosed concussions among 9 to 14-year-old football and soccer players. Prior to the Fall 2019 season, 262 athletes from two youth sports leagues in the Greater Seattle Area self-reported all prior diagnosed concussions. Overall, a small percentage (20.61%) of athletes reported at least one previously diagnosed concussion (1 concussion, n = 45; 2 concussions, n = 7; 3+ concussions, n = 2). There was no significant difference in concussion history among athletes of the three sports. Football (n = 120), boys’ soccer (n = 71), and girls’ soccer (n = 71) reported a mean (SD) of 0.28 (0.57), 0.32 (0.77), and 0.14 (0.35) concussions, respectively. The mean number of diagnosed concussions was highest in boys’ soccer, then football, and lowest in girls’ soccer. Our results were different when compared to another study which shows that the concussion rates per 1,000 athlete exposures were highest in football, followed by girls’ soccer, and lowest in boys’ soccer. These results show that the number of youth concussions may be different among football, boys’ soccer, and girls’ soccer. Further research could provide a better understanding of concussion diagnoses in youth sports.

Dandy Walker malformation (DWM) is the most common human cerebellar malformation, affecting 1 in every 3000 live births. DWM is an imaging diagnosis that is characterized by three features: cerebellar vermis hypoplasia, an enlarged posterior fossa, and an enlarged fourth ventricle. Although recent advances in neuroimaging have improved diagnosis of DWM, virtually nothing is known about the cellular and histological defects that lead to DWM during brain development. One major reason is that little human specific data is available describing the histology of normal and abnormal human fetal cerebellar development. Currently, there is limited published fetal pathology of DWM. Few comparative analyses are available and most studies are confounded by lack of molecular confirmations of diagnoses. We have carried out the first comprehensive prenatal histo-pathological analysis of human DWM. Our results indicate a significant reduction in foliar complexity the developing human cerebellum. We also observe aberrations in the developmental trajectories of specific cell types like Purkinje cells, and progenitor zones like the rhombic lip. Significantly, proliferation and self-renewal of rhombic lip progenitors is reduced leading to hypoplasia, particularly of the posterior lobe. Through our analysis of the human fetal DW cerebellum, we begin to directly address the developmental pathology of human DWM beyond that of the mouse models that share similar pathology. Our studies will fundamentally improve our view and understanding of the biology of the human cerebellar development and give us insights on the developmental pathogenesis of DWM.

Joubert syndrome (JS) is a genetic neurodevelopmental disorder that affects ~1 in 100,000 live births. JS is diagnosed by a distinctive hindbrain malformation that manifests as the “molar tooth sign” on axial brain imaging. Remarkably, >40 genes have been associated with JS, making it one of the most genetically heterogeneous Mendelian conditions. The clinical and brain imaging features of people with JS display a broad range of severity. In fact, we have identified a substantial number of individuals without the molar tooth sign but that have imaging features suggestive of JS. It is not known whether these “JS-like” patients represent the mild end of the phenotypic spectrum associated with variants in JS genes or a different set of genetic disorders. It is also not known whether these JS-like patients are at risk for the progressive retinal, kidney and liver disease seen in some JS patients. To answer these questions, I performed targeted DNA sequencing of the JS genes in JS-like patients, and I used an in-house bioinformatics pipeline to identify predicted-pathogenic variants. We hypothesize that a large subset of JS-like patients will have genetic causes in JS genes. If this hypothesis is supported, we will expand the phenotypic spectrum associated with variants in JS genes and improve the medical care of JS-like patients by supporting monitoring of JS-associated progressive features and sequencing of JS genes in these patients. This will also be proof of concept for evaluating mild clinical presentations of other conditions to determine if they share the same genetic causes.

Congenital heart defects have been linked to numerous genes, but many of the genes responsible are not yet identified. The purpose of this research is to identify the unknown genetic causes of human congenital heart defects, utilizing zebrafish as a model organism. Using CRISPR-Cas9 to edit the genome of zebrafish, we are creating mutations in genes we predict are involved in human congenital heart defects. Our lab has used a CRISPR-based screen in zebrafish to identify new genes that, when knocked out, lead to defective heart development in zebrafish embryos. For this research project, our questions are: Can we associate specific, CRISPR-induced genetic mutations in these genes with our heart-defective zebrafish embryos? And, can we genetically engineer heritable mutations in these respective genes in zebrafish? The methods used in this project involve using several sets of DNA oligonucleotide primers to assess where and how the CRISPR reagents have altered the screened candidate genes. We have analyzed three genes—grpel1, pomp, and psmd6—each with four CRISPR target sites. The primer testing and animal genotyping have been done using PCR, gel electrophoresis, and gel imaging, with genotyping also requiring restriction digests. Our results have been promising. First, we determined which CRISPR target sites are effective for each gene. Second, we successfully identified CRISPR-induced mutations in F0-generation animals for each of these three genes. Third, for the pomp gene, we identified germline-transmission of a specific CRISPR mutation corresponding with heart-defective embryos. This result identifies pomp as a new candidate gene for heart defects. A key implication of these findings is that we can successfully create lineages of zebrafish carrying mutations in these new heart defect genes. Our work will allow for further testing and a better understanding of the genetics behind heart development.