Memory formation, or plasticity, is the brain’s ability to change and adapt in response to new information through altering the strength and effectiveness of communication at the synapse. Synaptic plasticity plays a major role in the brain’s capacity to incorporate past experiences into stored memories and it has become one of the most intensively researched subjects in neuroscience. Neuroscientists typically evaluate synapse function using enriched, homogenized preparations of synapses, called synaptosomes. However, synaptosome preparations contain a mix of neuronal and non-neuronal contamination along with active and inactive synapses that are impossible to separate, making it difficult to draw specific conclusions about synapse activity. To address these challenges and understand the molecular mechanisms behind synaptic plasticity, my lab has developed a synaptic TAG- an engineered protein that localizes to recently active synapses. The TAG construct, containing an extracellular CD4 protein whose expression is driven by activity-dependent regulatory elements, will be electroporated into the developing motor cortex of mice in utero. The mice will later be trained to run on a rotarod, a test commonly used to assess motor learning. After the mice have learned this task, the differences in their synapses will be evaluated by sectioning and staining the motor cortex with fluorescent antibodies. The number of distinct fluorescent TAGs on the synapses on a given number of neurons will be counted through confocal imaging. My hypothesis is that I will see an increase in the number of TAGs on the synapses of a trained mice, which will demonstrate that the TAG is synaptic in vivo and activity dependent. The completion of this experiment will allow my lab to identify learning-associated proteins using magnetic cell sorting, mass spectrometry, and RNA sequencing. Understanding learning-associated protein interactions will shed light on how their malfunctions contribute to Autism Spectrum Disorders or neuropsychiatric disorders.

Hypoxic-Ischemic Encephalopathy (HIE) is a brain injury that commonly causes mortality in neonates. Current treatment consists of therapeutic hypothermia, but close to 50% of affected infants still have a poor outcome (death or severe disability). In order to discover new effective therapies, it is important to compare how different treatments affect the brain in animal studies. The research laboratory has developed a ferret model of HIE because the ferret brain has more complex gyrification compared to rodents. Animals underwent unilateral carotid ligation at postnatal age 17 days (P17), in which one side of the carotid artery was restricted temporarily and the other was restricted permanently. The animals then received periods of hypoxia and hyperoxia. To better quantify the extent of injury, a system involving measurements of the gyri, sulci, and cerebellar exposure was developed. Ex vivo brain measurements were collected from a population of 63 ferret kits at age P42, and adjusted by the weight and sex of the animal. These measurements included the lengths of: the longitudinal fissure (anterior and posterior), lateral sulci, suprasylvian sulci, coronal sulci, pseudosylvian sulci, ansinate sulci, cruciate sulci, presylvian sulci, lateral gyri, suprasylvian gyri, sigmoid gyri (anterior and posterior), coronal gyri, ectosylvian gyri (anterior and posterior), orbital gyri, and the exposure of the cerebellum. In injured animals, significant changes in the longitudinal fissure, ansinate sulci, left coronal sulci, cruciate sulci, presylvian sulci, posterior sigmoid sulci, and exposure of the cerebellum were seen compared to littermate controls. The implications of this measurement system include the ability to accurately characterize the degree of injury in animals with an hypoxic-ischemic brain injury, which will help to show whether potential treatments are neuroprotective.

Autism spectrum disorder (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 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 could be observed with ube3a transgenic autism model mice. Rapamycin is administered to ube3a transgenic mice to normalize deficits in social interaction and repetitive behavior, two main behavioral hallmarks of autism. Mice are assessed using a three-chamber social interaction and repetitive self-grooming test. Successful behavioral rescue by rapamycin would be seen as increased social interaction and decreased repetitive grooming, similar to behaviors seen in wild type control mice. This would suggest that therapeutic treatment of the mTOR/AKT signaling pathway could be a viable target for patients with 15q11-q13 duplication.

 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.

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 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.

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.

Heterotopia are organized structures consisting of mixed cellular and neuronal elements arranged in a clear architectural pattern inappropriate to the considered tissue. Heterotopia are observed in the cerebral cortex and the cerebellum as a feature of many neurological disorders yet the we know little about the mechanisms driving their formation. We have analyzed a substantial number of Dandy Walker malformation (DWM) human fetal cerebella and found that a significant number of cases contain heterotopia. DWM is the most common structural birth defect of the human cerebellum and is characterized by an enlarged posterior fossa, enlarged fourth ventricle, and cerebellar vermis hypoplasia. A subset of cases are caused by loss of FOXC1, a transcription factor expressed in the mesenchyme during development. Our group has previously shown that loss of FOXC1 in mice causes loss of the mesenchyme-secreted factor SDF1alpha. Further, loss of SDF1alpha is sufficient to cause cerebellar heterotopia. This emphasizes the importance of mesenchymal signaling in the maintenance and development of the clear laminar architecture of the mature cerebellum. We hypothesized that granule neuronal progenitors (GCPs) are the primary cellular target of SDF1alpha mesenchymal signaling and therefore the main cell type causing heterotopia formation. To test our hypothesis, we excised the receptor for SDF1alpha from just GCPs in mice. Our findings show that loss of SDF1alpha in GCPs causes them to prematurely migrate into the developing cerebellar anlage and also caused other cerebellar cell types to form structured heterotopia. We observed defects in cerebellar foliation in the treatment group. Our data emphasizes the importance of SDF1-alpha dependent mesenchymal signaling in cerebellar development and identifies heterotopia as a new phenotype in DWM.

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 sequencing approach (ATAC-Seq) to quantify chromatin accessibility - a feature of non-coding regulatory elements. I then used bioinformatics techniques such as genome track assembly, peak calling, and gene ontology to compare my organoid dataset to adult human retina data we previously generated and to a published dataset on 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 have yielded 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 will drive progress towards regenerating damaged photoreceptor cells in the future.

The most recent National Vital Statistics Report reports that approximately 9.85% of babies in the United States are born preterm, with 72% of those born late-preterm (at 34-36 weeks of gestation). Using neonatal ferrets at age 17 days old, the Juul lab in the Division of Neonatology at the University of Washington Medical Center has developed a preliminary model of brain injury to mimic late-preterm neonatal injuries. In this species-specific adaptation of the Vannucci Model, the left carotid artery is permanently ligated, along with a temporary (4h) occlusion of the right carotid artery. Ferrets are then exposed to periods of hypoxia and hyperoxia. By looking at data and outcomes from our surgeries, I aim to examine the effects of certain surgical parameters on ferret mortality. These parameters include: time the animal is exposed to isoflurane, the length of surgery, and the amount of time the animal is given to recover between surgery and hypoxia. Aside from mortality, I will also analyze the effects of these parameters on respiratory rate after surgery as well as gross brain injury and data from behavioral testing in an attempt to discern the level of injury in living animals and the most common predictors of death in those that died prior to their determined endpoint.

Chronic non-bacterial osteomyelitis (CNO), or chronic recurrent multifocal osteomyelitis (CRMO), is an auto-inflammatory bone condition that causes persistent bone pain, poor growth, and other complications. Physical exams, laboratory tests and radiographs are not sensitive in detecting the active disease. MRI is the gold standard assessment for diagnosis. Patient reported outcome (PRO) measurements have not been commonly used to determine the disease impact on the physical health of affected children. Pain score is not accurate in assessing disease activity due to the confounding effects from other associated conditions such as amplified pain syndrome. Consensus treatment plans (CTPs) were developed by the Childhood Arthritis and Rheumatology Research Alliance (CARRA). In this study, a prospective patient registry based on CTPs is used to compare the effects of different treatment plans. This study was approved by the Seattle Children’s Institutional Review Board (#1232). The center is the coordinating site of this multicenter observational study, Chronic nonbacterial Osteomyelitis International Registry (CHOIR). Inclusion criteria are: ≤21 years old at enrollment and CNO diagnosis. Consent and assents are obtained. Standard of care is provided to the subjects and no investigational intervention is performed. Detailed clinical information, laboratory, and imaging data along with patient/parent reported outcome measurements (PROMIS questionnaires) and childhood health assessment questionnaire (CHAQ) are collected at baseline and follow-up visits. We aim to enroll 2,000 subjects worldwide and follow up with subjects for at least 5 years. Our center has enrolled 74 subjects between June 2018-present and collected PROs at baseline visits. Data is being entered into REDCap database for further analysis. PROs collected throughout the course of the study will be correlated with imaging results and expectedly determine the effectiveness of commonly used medications for patients. The data will establish measurements for physicians to estimate disease burden and treatment responses in children with CNO.

Youth sports participation can have many benefits, including positive psychosocial development and lifelong healthy activity levels. However, such benefits are often not realized due to attrition from sport. Parents play a key role in shaping youth sport experiences. High pressure sports parenting and disruptive sideline behavior contribute to attrition. Interventions aimed at reducing such parent behaviors exist; however, little is known about their effectiveness. The purpose of this narrative review was to synthesize the extant research on sports parenting interventions. A narrative literature review was conducted between December 2018 and February 2019. Studies were selected if they contained a critically evaluated sports parenting intervention (inclusive, but not limited to education and policies) for parents of children 18 or younger participating in organized sports. Additional articles were obtained via pearling of reference lists. Study quality was appraised using the Downs & Black checklist. Our preliminary search found 13 articles, which included 8 educational interventions and 6 policies. The majority of educational interventions were related to sportsmanship; few included any elements of sports psychology. Many educational interventions were implemented using online videos and non-interactive online modules. Evaluations were of poor methodological quality, consisting primarily of limited-scope qualitative evaluations. No randomized controlled trials have as-yet been published on this topic. High quality research into the effectiveness of sports parenting education and sideline behavior policies is needed to better understand whether existing approaches are effectively changing this important parenting behavior. Future research should seek to use randomized designs and measure behavioral outcomes. Further, studies should move beyond efficacy measures and collect implementation data to understand how policies and educational programs are being used in a range of youth sport settings.

Distal Arthrogryposis (DA) syndromes are a group of disorders with a wide breadth of phenotypic severity. A previous study found that individuals with recessive Spondylocarpotarsal Synostosis Syndrome were compound heterozygous, that is, there was the presence of two different variant alleles at a specific gene locus, for a coding variant found in the gene Myosin Heavy Chain 3 (MYH3) and a non-coding variant found at the location c.-9+1G>A (rs557849165), also in the same gene. We are interested in discovering if individuals with DA and MYH3 variants are carriers for this variant, c.-9+1G>A, to explain more severe phenotypes seen. We hypothesize that rs557849165 has a role in phenotype severity of individuals with DA. We screened our cohort of individuals with DA and MYH3 variants in the previously unscreened non-coding region of exon 2. We did not detect the variant in any individuals screened in our first cohort. Individuals that did not previously produce results for analysis are being re-screened for the same variant. Further investigation is being conducted on additional DA candidates to better identify the genotype-phenotype relationship of various forms within DA1, DA2A, and DA2B. Some cases of DA are more severe than others due to specific variants in MYH3 and we are trying to better understand the effects these variants have on these individuals with DA.

 The Diabetes Community Care Ambassador (DCCA) Program represents a promising intervention for improving glycemic control in youth with type 1 diabetes (T1D) struggling with diabetes management. In this novel program, lay health workers called DCCAs supported youth with T1D with poor glycemic control (HbA1 >8.5%) and their caregivers through home visits, school visits, and phone support, and connected participants with legal support. Given the findings that youth with T1D in the DCCA Program demonstrated improved glycemic control, we carried out a follow up qualitative study to understand the salient components of the DCCA program that promoted improved diabetes self-management from the youth and caregiver participant perspectives. We conducted semi-structured exit interviews with a subset of DCCA Program participants (18 caregivers, 14 youth). Participants reported on how the DCCA was helpful in supporting the youth’s diabetes management and how DCCA support compared to care provided by the multidisciplinary diabetes care team. A qualitative content analysis was carried out and emergent themes were identified. We found that within the home environment, participants felt the DCCAs helped youth address underlying weaknesses in their diabetes management routine and provided accountability for self-care goals between visits with their diabetes provider. DCCAs also provided psychosocial support that youth and caregivers believed they did not sufficiently receive from their usual multidisciplinary diabetes care team. At school, caregivers, in particular, felt that DCCAs successfully advocated on behalf of the student’s interests to facilitate diabetes management at school and also provided diabetes education to school staff. Finally in the community setting, caregivers shared that DCCAs helped to connect them to legal and support resources and better navigate the healthcare system. Our study results highlight the value of using lay health workers in providing individualized assistance for youth with T1D in supporting diabetes management outside of the ambulatory care setting.

Growing concern regarding brain injury in football has encouraged research on head impact exposure (HIE), incidence of concussion, and strategies to reduce injury risk. Reducing HIE in games has presented challenges due to the physical nature of the sport. However, limiting the amount of full-contact in practice may present a means for both decreasing HIE and minimizing concussion risk. In the past few years, guidelines limiting contact have been proposed, including decreasing the intensity of contact (limiting "two-a-days"), decreasing the number of full-contact practices, and eliminating practice drills associated with greater contact (such as the "Oklahama drill"). A few states have even modified their existing concussion legislation to include provisions limiting contact. The goal of our study is to review the literature regarding limitations on contact as a means for improving safety in football and to evaluate whether evidence supports this approach as a means for mitigating risk. We have identified studies assessing the impact of limitations on contact for either reducing HIE or concussive risk in football. We have then categorized the resulting studies by level of evidence, and summarized our findings. We concluded that reduced contact should result in a decrease in HIE and thereby a decrease in injury rate. 

Hypoxic-ischemic encephalopathy (HIE) and inflammatory responses are commonly seen in premature infants which 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 inflammation with lipopolysaccharide (LPS) + hypoxia + hyperoxia] will have a significant late behavioral deficit compared to controls. The rodent model of preterm brain injury includes: intrauterine hypoxia at embryological day 18, with LPS administration on Postnatal (P) day 2 followed by hypoxia (8% oxygen) and hyperoxia (80% oxygen). In order to assess late behavioral effect of early neonatal injury, I conducted motor tests on rats. The motor testing included: gait analysis via CatWalk XT and Rotarod analysis. For the Rotarod analysis, I performed testing on both, the injured rats and controls, on P28 to test their locomotor ability. Gait analysis was performed on P35. The results will be tested for significant differences between the groups. Future research will be conducted by repeating this experiment to verify these results and clarify what aspects of late behavior are impacted most by this injury model.

Autism Spectrum Disorder (ASD) is a neurodevelopmental condition associated with deficits in social interaction and the presence of restricted patterns of behavior. ASD is clinically and phenotypically heterogeneous. The most recent update to the diagnostic definitions for mental disorders (DSM-5) provides additional flexibility for capturing the diverse array of phenotypes shown by diagnosed individuals. Despite categorized behavioral symptoms across core and comorbid dimensions of ASD, the neural mechanisms are unknown. Studies have shown atypical neuroanatomy and functional connectivity of the brains of individuals with ASD, suggesting a neural etiology. Here, we focus our attention on the superior temporal sulcus (STS), a region of the brain demonstrated to play a role in processing communication, social information, and theory of mind. Our research question evaluates whether neural activity in STS is affected by behavioral phenotype in children with ASD. We speculate that neural activity between individuals with ASD varies significantly more than typically developing (TD) children, who show more clustered, similar outcomes. Functional near infrared spectroscopy (fNIRS) distinguishes concentrations of oxygenated (HbO) and deoxygenated (HbR) hemoglobin in regions of cortical vasculature, signifying neurovascular coupling. This study uses HbO and HbR measured as subjects watch the events of a social scene on a monitor and analyzes ASD individuals’ deviance from average TD activity. Eye-tracking, clinical assessments, and parent questionnaires are considered to extrapolate possible correlations between ASD individuals’ variation in neural outcome and the behavioral phenotype expressed. We expect that individuals showing higher degree of neural variance from TD activity will show more severe autism behavioral phenotype. It is possible that in the search for neural mechanisms of ASD, considering behavioral factors signifying atypical variance could reveal significant differences. This exploratory analysis aims to examine the wide range of possible influential factors on neural heterogeneity within the social brain of individuals with ASD.