A genome assembly and transcriptome atlas of the inbred Babraham pig to illuminate porcine immunogenetic variation.

The inbred Babraham pig serves as a valuable biomedical model for research due to its high level of homozygosity, including in the major histocompatibility complex (MHC) loci and likely other important immune-related gene complexes, which are generally highly diverse in outbred populations. As the ability to control for this diversity using inbred organisms is of great utility, we sought to improve this resource by generating a long-read whole genome assembly and transcriptome atlas of a Babraham pig. The genome was de novo assembled using PacBio long reads and error-corrected using Illumina short reads. Assembled contigs were then mapped to the porcine reference assembly, Sscrofa11.1, to generate chromosome-level scaffolds. The resulting TPI_Babraham_pig_v1 assembly is nearly as contiguous as Sscrofa11.1 with a contig N50 of 34.95 Mb and contig L50 of 23. The remaining sequence gaps are generally the result of poor assembly across large and highly repetitive regions such as the centromeres and tandemly duplicated gene families, including immune-related gene complexes, that often vary in gene content between haplotypes. We also further confirm homozygosity across the Babraham MHC and characterize the allele content and tissue expression of several other immune-related gene complexes, including the antibody and T cell receptor loci, the natural killer complex, and the leukocyte receptor complex. The Babraham pig genome assembly provides an alternate highly contiguous porcine genome assembly as a resource for the livestock genomics community. The assembly will also aid biomedical and veterinary research that utilizes this animal model such as when controlling for genetic variation is critical.

Prevalence of Individuals With Multiple Diagnosed Genetic Diseases in the Undiagnosed Diseases Network.

Report the prevalence of multiple genetic diseases in the Undiagnosed Diseases Network (UDN) cohort in the post-exome-sequencing era. UDN subjects underwent genome sequencing before inclusion in the cohort. Records of all UDN subjects until January 2024 were analyzed. The number of diagnoses, proportion of molecular versus nonmolecular (i.e., not attributable to a discretely identifiable genetic change) diagnoses, and the inheritance patterns of the genetic diagnoses were determined. Of 2799 subjects, 766 (27.4%) had diagnoses. Of these 766, 95.4% had one diagnosis, 4.0% had two diagnoses, and 0.5% had three diagnoses. Of the diagnosed subjects, 93.4% had a genetic disease, and 6.5% had a nonmolecular disease. Of subjects with two diagnoses, both diagnoses were molecular in 90.3%, while 9.7% had one molecular and one nonmolecular diagnosis. All four subjects with three diagnoses had three molecular diagnoses. 4.2% of diagnosed subjects in the UDN had more than one molecular diagnosis, with four individuals having three concurrent Mendelian diagnoses. Additionally, three subjects had concurrent molecular and nonmolecular diagnoses. Given that numerous UDN subjects had a negative genome sequence prior to UDN enrollment, multiple molecular diagnoses may contribute to diagnostic uncertainty even with genome sequencing, as may concurrent nonmolecular disease.

Changing Nepali children's attitudes toward conservation through a participatory education program.

People's attitude toward wildlife is known to affect the success of conservation programs. Public education can increase support for conservation measures by providing the knowledge necessary to make informed decisions, improve pro-environmental behaviour and potentially enlist participation. To that end, we developed a participatory conservation education program in Nepal with a target audience of secondary school students. Our lessons emphasized ecological balance by highlighting the roles of individual species, including the rhesus macaque (Macaca mulatta). This species presents a challenge for educating the public about maintaining balance in nature, because although an important seed disperser and species of religious significance, macaques are frequently involved in human-wildlife conflict and considered a pest species. We evaluated the impact of our program by surveying students from six socioeconomically similar schools, four that participated in our program and two that had not. Our questionnaire was comprised of knowledge questions about local wildlife and conservation issues, an open-ended listing exercise to determine species preferences, and statements that elicited students' opinions regarding empathy toward animals. Knowledge about and attitude toward wildlife, including M. mulatta, were greater in students that participated in the program. More importantly, these students were more likely to understand the role individual species play in a balanced ecosystem, how human behaviour can negatively impact wildlife, and how they could participate in conservation efforts. Gender was not a factor in any response. We discuss the merits of using a participatory learning platform in a standalone education program that has long-term purpose, funding, and buy-in.

Rise of long-distance urology transfer during the COVID-19 pandemic: Identifying factors to enhance transfers of care efficiency and clinical outcomes.

OBJECTIVE: The objective of this study is to identify variables associated with clinical outcomes after urologic transfers before and during the COVID pandemic. METHODS: After IRB approval, a retrospective chart review was performed on adult patients transferred to our institution from 01/01/2018 to 12/31/2019 ("pre-COVID") and from 01/02/2020 to 12/31/2022 ("COVID"). We identified demographics, origin hospitals, ICD-10 pre- and post-transfer diagnoses, distance of transfer, and post-transfer CPT codes. RESULTS: During the study period, our adult urology service accepted 160 transfers with a mean patient age of 71 years. A total of 49/160 (30%) of subjects made up the "pre-COVID" cohort and 111/160 (70%) made up the "COVID" cohort. There were 11/111 (10%) transfers of >100 miles in the COVID period but 0/49 in the pre-COVID period (p = 0.02). Patients from the COVID period waited on average 1.2 days longer for a procedure after transfer compared to pre-COVID period (p = 0.03). The time until a patient's surgical procedure after transfer was a significant predictor of length of stay > 5 days (OR 1.91, CI 1.43 - 2.58, p < 0.01). Different diagnosis upon re-evaluation after transfer was associated with a decreased rate of subsequent readmission (OR 0.30, CI 0.09-0.97, p = 0.05). CONCLUSIONS: Long-distance transfer, even >100 miles (which we termed "mega-transfers"), was a new pandemic-related phenomenon at our institution. Delays in definitive care and changes in diagnoses after transfer were associated with readmission and length of stay. Our findings illustrate the importance of inter-institutional communication, diagnostic accuracy, and post discharge planning when managing transfer patients.

Alterations of cerebrovascular reactivity following pediatric mild traumatic brain injury are independent of neurodevelopmental changes.

Cerebrovascular dysfunction following mild traumatic brain injury (mTBI) is understudied relative to other microstructural injuries, especially during neurodevelopment. The blood-oxygen level dependent response was used to investigate cerebrovascular reactivity (CVR) in response to hypercapnia following pediatric mTBI (pmTBI; ages 8-18 years), as well as pseudocontinuous arterial spin labeling to measure cerebral blood flow (CBF). Data were collected ∼1-week (N = 107) and 4 months (N = 73) post-injury. Sex- and age-matched healthy controls (HC) underwent identical examinations at comparable time points (N = 110 and N = 91). Subtle clinical and cognitive deficits existed at ∼1 week that resolved for some, but not all domains at 4 months post-injury. At both visits, pmTBI showed an increased maximal fit between end-tidal CO2 regressor and the cerebrovascular response across multiple regions (primarily fronto-temporal), as well as increased latency to maximal fit in independent regions (primarily posterior). Hypoperfusion was also noted within the bilateral cerebellum. A biphasic relationship existed between CVR amplitude and age (i.e., positive until 14.5 years, negative thereafter) in both gray and white matter, but these neurodevelopment effects did not moderate injury effects. CVR metrics were not associated with post-concussive symptoms or cognitive deficits. In conclusion, cerebrovascular dysfunction may persist for up to four months following pmTBI.

Development of a National Ménière's Disease Registry: A Feasibility Study.

Ménière's disease is a disabling condition causing vertigo and hearing loss yet remains incompletely understood. Registry studies have the potential to answer important questions about phenotypes and natural history of clinical conditions. The aim of this study was to explore the feasibility of a patient-centered national Ménière's disease registry. This was an observational study carried out at 4 state-funded hospitals and 4 independent clinics, within 3 distinct urban and rural regions within the UK. Adults with Ménière's disease were eligible to participate. A range of patient reported data, questionnaire data and clinical data (audiometric, radiological, and specialist balance testing data) was inputted into a bespoke database. The study recruited 411 participants. The majority of participants chose online recruitment (73%) and 27% chose via paper-based methods for participation. A small majority (57%) of participants were female. 96% of participants were of white ethnicity. Data completeness from online or postal data collection was similar. Around 20% of participants had audiological evidence of bilateral Ménière's disease. This feasibility study has successfully piloted methods for recruitment of hundreds of participants diagnosed with Ménière's disease. Participants actively contributed their data to a robust and extensive data collection platform. The positive outcomes from this initial feasibility study are anticipated to serve as a foundation for the future expansion of the registry. This expansion holds the potential to address a broad spectrum of request, encompassing all aspects of the nature of Ménière's disease.

Novel UROD mutation for porphyria cutanea tarda, type 2: a case report.

Background: Porphyria cutanea tarda (PCT) is usually caused by acquired defects in uroporphyrinogen decarboxylase (UROD) activity in the liver. This more common form of PCT is called type 1 PCT. Major known risk factors for PCT include iron overload, such as occurs due to mutations in HFE, associated with classical hereditary hemochromatosis, chronic hepatitis C infection, heavy alcohol use, tobacco use, and estrogen therapy. In addition, in about 25% of patients with PCT, namely, those with PCT type 2, an inherited partial defect in UROD activity is found. In such persons, this partial defect, which is found in all cells, including hepatocytes, red blood cells, and others, contributes to the development of biochemically and clinically active disease. Case Description: Herein we describe salient features of a man in his eighth decade of life with onset of clinical PCT. Among risk factors were heavy alcohol and tobacco use. Genetic testing revealed a novel mutation in one of his alleles of the UROD gene, namely, c.224 G>C; p. Arg 75 Pro, and enzymatic testing revealed that red blood cell UROD activity was decreased by 50%. This mutation in the UROD gene is predicted to have a major effect on protein structure and function, confirmed by the 50% decrease in activity of the enzyme. Conclusions: The previously undescribed mutation in UROD, found in this man, namely, c.224 G>C; p. Arg 75 Pro is pathogenic.

Assessing Chronic Ear Symptoms in Bone-Conduction Hearing Implant (BCHI) Patients Using the Chronic Otitis Media Benefit Inventory (COMBI) Score.

OBJECTIVE: This study aimed to determine improvement in health-related quality of life (HRQoL) using a validated disease-specific patient-reported outcome measure (PROM) questionnaire in patients undergoing bone-conduction hearing implant (BCHI) insertion. STUDY DESIGN: A mixed retrospective and prospective correlational study. SETTING: Single tertiary referral center in the United Kingdom. PATIENTS: All adult patients undergoing their first BCHI over 6 years (April 1, 2017, to March 3, 2023). MAIN OUTCOME MEASURES: The Chronic Otitis Media Benefit Inventory (COMBI) score (postintervention) and the Glasgow Health Status Inventory (GHSI) (pre-and post-BCHI questionnaire). RESULTS: Improvements were seen across all COMBI domains. The mean total COMBI score was 46.3 (standard deviation = 5.3). Although expected significant improvements were seen in hearing and social domains, there were also notable gains in ear symptoms and reduced medical intervention post-BCHI. There was a statistically significant improvement in all GHSI scores post-BCHI (median total difference 67.1, p < 0.0001). CONCLUSIONS: This study reports very favorable outcomes for BCHI patients using two different PROMs: COMBI and GHSI. Although these PROMs complement each other, they also offer different perspectives on the same cohort of patients, with COMBI providing a unique insight into specific ear symptoms. This is the first reported study using this complement of PROMS in BCHI patients and offers further evidence for the wide-reaching improvements BCHI can have for patients.

A directional total variation minimization algorithm for isotropic resolution in digital breast tomosynthesis.

An optimization-based image reconstruction algorithm is developed for contrast enhanced digital breast tomosynthesis (DBT) using dual-energy scanning. The algorithm minimizes directional total variation (TV) with a data discrepancy and non-negativity constraints. Iodinated contrast agent (ICA) imaging is performed by reconstructing images from dual-energy DBT data followed by weighted subtraction. Physical DBT data is acquired with a Siemens Mammomat scanner of a structured breast phantom with ICA inserts. Results are shown for both directional TV minimization and filtered back-projection for reference. It is seen that directional TV is able to substantially reduce depth blur for the ICA objects.

Ultra-Performance Liquid Chromatography (UPLC) Analysis of Heme Biosynthesis Intermediates.

The utilization of ultra-performance liquid chromatography (UPLC) to analyze the various intermediates in the heme biosynthetic pathway is presented. The first product, ALA, was derivatized to a highly fluorescent pyrrolizine; PBG, the second intermediate, was enzymatically converted to uroporphyrinogen, and all the porphyrinogen intermediates were oxidized in acid to form fluorescent porphyrins. Heme was measured as hemin. The stable porphyrin forms of the intermediates, are then resolved and quantified by UPLC. Further details about the various methods are discussed to promote successful UPLC analyses. Method variations that may be preferable in certain situations are also presented.

Response of the human choroid to short-term changes in eyelid and periocular temperature.

CLINICAL RELEVANCE: Choroidal thickness measurement is gaining popularity in clinical practice and research as an early indicator of myopia progression. Understanding the influence of temperature on choroidal thickness changes will improve the reliability of the measures. BACKGROUND: It has been suggested that environmental temperature may affect choroidal thickness and blood flow, with potential implications for ocular disease and refractive development. This study investigates the effect of changes in eyelid/ocular adnexa temperature on choroidal thickness. METHODS: In a paired-eye study, 20 young, healthy subjects received a warm stimulus (heat pack) over one closed eye and simultaneously a cold stimulus (ice pack) over the other for 10 min. Eyelid temperatures were monitored with thermal probes, and optical coherence tomography scans of the retina and choroid were taken before and after heating and cooling, and then every 5 min during a 15-min recovery period. Retinal and choroidal thicknesses were measured across the macular region (6 mm), including the subfoveal (1 mm), parafoveal (1-3 mm), and perifoveal (3-5 mm) regions, and compared between the cooled and warmed eyes. RESULTS: When the thermal stimuli were applied, eyelid surface temperatures changed predictably and remained significantly different (by approximately 10-15°C) between the eyes after 2 min (p < .001). Relative to the warmed eye, macular choroidal thickness in the cooled eye increased significantly after 10 min of treatment (p = .004). This choroidal thickening response occurred in the subfoveal, parafoveal, and perifoveal regions (all p < .05). Upon removal of the thermal stimuli, choroidal thickness rapidly returned to the baseline and was no longer different between the cooled and warmed eye (p = .641). CONCLUSION: Cooling the anterior eye by application of a cold stimulus directly onto the closed eyelid caused a small but significant increase in choroidal thickness relative to warming the anterior eye, demonstrating that the choroid can modulate its thickness rapidly and transiently in response to local temperature changes.

Implementation of an Enhanced Recovery After Surgery Protocol for Cranial Vault Remodeling Procedures.

BACKGROUND: Enhanced recovery after surgery (ERAS) protocols have been implemented across surgical disciplines, including cranial vault remodeling for craniosynostosis. The authors aim to describe the implementation of an ERAS protocol for cranial vault remodeling procedures performed for patients with craniosynostosis at a tertiary care hospital. DESCRIPTION: Institutional review board approval was received. All patients undergoing a cranial remodeling procedure for craniosynostosis at the authors' institution over a 10-year period were collected (n = 168). Patient and craniosynostosis demographics were collected as well as operative details. Primary outcome measures were intensive care unit length of stay (ICU LOS) and narcotic usage. Chi squared and independent t-tests were employed to determine significance. A significance value of 0.05 was utilized. RESULTS: During the time examined, there were 168 primary cranial vault remodeling procedures performed at the authors' institution - all of which were included in the analysis. Use of the ERAS protocol was associated with decreased initial 24-hour morphine equivalent usage (p < 0.01) and decreased total morphine equivalent usage (p < 0.01). Patients using the ERAS protocol experienced a shorter ICU LOS (p < 0.01), but the total hospital length of stay was unchanged. CONCLUSION: This study reiterates the benefit of developing and implementing an ERAS protocol for patients undergoing cranial vault remodeling procedures. The protocol resulted in an overall decreased ICU LOS and a decrease in narcotic use. This has implications for ways to maximize hospital reimbursement for these procedures, as well as potentially improve outcomes.

Implementation of an Enhanced Recovery After Surgery Protocol for Cleft Palate Repair.

OBJECTIVE: This study examines an Enhanced Recovery After Surgery (ERAS) protocol for patients with cleft palate and hypothesizes that patients who followed the protocol would have decreased hospital length of stay and decreased narcotic usage than those who did not. DESIGN: Retrospective cohort study. SETTING: The study takes place at a single tertiary children's hospital. PATIENTS: All patients who underwent cleft palate repair during a 10-year period (n = 242). INTERVENTIONS: All patients underwent cleft palate repair with the most recent cohort following a new ERAS protocol. MAIN OUTCOME MEASURES: Primary outcomes included hospital length of stay and narcotic usage in the first 24 hours after surgery. RESULTS: Use of local bupivacaine during surgery was associated with decreased initial 24-hour morphine equivalent usage: 2.25 vs 3.38 mg morphine equivalent (MME) (P < 0.01), and a decreased hospital length of stay: 1.71 days vs 2.27 days (P < 0.01). The highest 24-hour morphine equivalent a patient consumed prior to the ERAS protocol implementation was 24.53 MME, compared with 6.3 MME after implementation. Utilization of the ERAS protocol was found to be associated with a decreased hospital length of stay: 1.67 vs 2.18 days (P < 0.01). CONCLUSIONS: Use of the proposed ERAS protocol may lead to lower narcotic usage and decreased length of stay.

Longitudinal Speech and Fistula Outcomes Following Primary Cleft Palate Repair at a Single Institution.

INTRODUCTION: Fistula formation and velopharyngeal insufficiency (VPI) are complications of cleft palate repair that often require surgical correction. The goal of the present study was to examine a single institution's experience with cleft palate repair with respect to fistula formation and need for surgery to correct velopharyngeal dysfunction. METHODS: Institutional review board approval was obtained. Patient demographics and operative details over a 10-year period were collected. Primary outcomes measured were development of fistula and need for surgery to correct VPI. Chi-square tests and independent t tests were utilized to determine significance (0.05). RESULTS: Following exclusion of patients without enough information for analysis, 242 patients were included in the study. Fistulas were reported in 21.5% of patients, and surgery to correct velopharyngeal dysfunction was needed in 10.7% of patients. Two-stage palate repair was associated with need for surgery to correct VPI (P = 0.014). Furlow palatoplasty was associated with decreased rate of fistula formation (P = 0.002) and decreased need for surgery to correct VPI (P = 0.014). CONCLUSION: This study reiterates much of the literature regarding differing cleft palate repair techniques. A 2-stage palate repair is often touted as having less growth restriction, but the present study suggests this may yield an increased need for surgery to correct VPI. Prior studies of Furlow palatoplasty have demonstrated an association with higher rates of fistula formation. The present study demonstrated a decreased rate of fistula formation with the Furlow technique, which may be due to the use of the Children's Hospital of Philadelphia modification. This study suggests clinically superior outcomes of the Furlow palatoplasty over other techniques.

Mandibular Distraction in Patients With Pierre Robin Sequence: A Multisurgeon Experience.

OBJECTIVE: Mandibular distraction osteogenesis (MDO) is rapidly becoming a standard of care for management of patients with severe Pierre Robin sequence. The tongue is brought forward to alleviate airway obstruction. This study will look at an institutional, multisurgeon experience with MDO over 10 years. DESIGN: A retrospective chart review was conducted. SETTING: All patients who underwent MDO at the authors' institution from 2012 to 2022 were included. Three craniofacial surgeons performed all interventions. PATIENTS: Demographics, preoperative and postoperative respiratory and feeding status, and distraction data were collected for 27 patients meeting inclusion criteria. MAIN OUTCOME MEASURES: Primary outcomes were avoidance of a gastrostomy tube, avoidance of a tracheostomy, discharge from hospital on room air, and complications. A significance value of 0.05 was utilized. RESULTS: The average age at MDO was 135 days, mean activation phase was 13.6 days, mean distraction length was 14.9 mm, and mean consolidation phase was 64.2 days. A longer activation phase was associated with discharge with a gastrostomy tube and a shorter activation phase was associated with discharge on full oral feeds. The ability to discharge on room air was associated with a shorter latency phase, shorter activation phase, and decreased distance of distraction. CONCLUSIONS: The goal of MDO is to achieve full oral feeds with no respiratory support. Several different latency periods were used in this study, and a short latency period was demonstrated to be safe.

Patients Requiring Multiple Surgeries for Velopharyngeal Insufficiency: Findings From a 10-Year Retrospective Review.

This study analyzed patients undergoing multiple surgeries for velopharyngeal insufficiency (VPI) and reviewed their preoperative evaluations and techniques selected for subsequent surgeries. A retrospective chart review was performed including patients having undergone multiple surgeries for VPI at the authors' institution between 2012 and 2022. All patients were evaluated and managed at the author's institution under the direction of 4 senior surgeons. The objective of this study was to provide insight into preoperative evaluation, surgical technique selection, and other factors that may contribute to patients who require multiple VPI surgeries. Of 71 patients having undergone surgery for VPI, 8 required at least 1 additional operation for persistent VPI following the initial intervention. Six patients who initially underwent a superiorly based posterior pharyngeal flap (PPF) required additional surgery, and for those patients' subsequent operations, 5 different techniques were used throughout their treatment. Of the remaining 2 patients who had multiple VPI surgeries, 1 initially received autologous fat transfer and 1 initially underwent a furlow palatoplasty. Finally, 0 patients that initially underwent buccal flaps or collagen injection required further unplanned surgical intervention. This study demonstrates the importance of selecting an appropriate surgical approach when operating on patients following a failed VPI surgery. The algorithm developed from these findings emphasizes the importance of properly identifying the anatomical deficiency resulting from the failed intervention. A review of the advantages of nasopharyngoscopy as a preoperative evaluative technique of velopharyngeal form and function when compared to lateral barium video fluoroscopy was also included.

Parkinson's disease cerebrovascular reactivity pattern: A feasibility study.

A mounting body of research points to cerebrovascular dysfunction as a fundamental element in the pathophysiology of Parkinson's disease (PD). In the current feasibility study, blood-oxygen-level-dependent (BOLD) MRI was used to measure cerebrovascular reactivity (CVR) in response to hypercapnia in 26 PD patients and 16 healthy controls (HC), and aimed to find a multivariate pattern specific to PD. Whole-brain maps of CVR amplitude (i.e., magnitude of response to CO2) and latency (i.e., time to reach maximum amplitude) were computed, which were further analyzed using scaled sub-profile model principal component analysis (SSM-PCA) with leave-one-out cross-validation. A meaningful pattern based on CVR latency was identified, which was named the PD CVR pattern (PD-CVRP). This pattern was characterized by relatively increased latency in basal ganglia, sensorimotor cortex, supplementary motor area, thalamus and visual cortex, as well as decreased latency in the cerebral white matter, relative to HC. There were no significant associations with clinical measures, though sample size may have limited our ability to detect significant associations. In summary, the PD-CVRP highlights the importance of cerebrovascular dysfunction in PD, and may be a potential biomarker for future clinical research and practice.

Heterozygous ZNHIT3 variants within the 17q12 recurrent deletion region are associated with Mayer-Rokitansky-Kuster Hauser (MRKH) syndrome.

The molecular basis of mullerian aplasia, also known as Mayer-Rokitansky-Kuster Hauser (MRKH) or congenital absence of the uterus and vagina, is largely unknown. We applied a multifaceted genetic approach to studying the pathogenesis of MRKH including exome sequencing of trios and duos, genome sequencing of families, qPCR, RT-PCR, and Sanger sequencing to detect intragenic deletions, insertions, splice variants, single nucleotide variants, and rearrangements in 132 persons with MRKH. We identified two heterozygous variants in ZNHIT3 localized to a commonly involved CNV region at chromosome 17q12 in two different families with MRKH. One is a frameshift, truncating variant that is predicted to interfere with steroid hormone binding of the LxxLL sequence of the C-terminal region. The second variant is a double missense/stopgain variant. Both variants impair protein expression in vitro. In addition, four more probands with MRKH harbored the stopgain variant without the nearby missense variant. In total, 6/132 (4.5%) of patients studied, including five with associated anomalies (type 2 MRKH), had ZNHIT3 variants that impair function in vitro. Our findings implicate ZNHIT3 as an important gene associated with MRKH within the 17q12 CNV region.

Clustered de novo start-loss variants in GLUL result in a developmental and epileptic encephalopathy via stabilization of glutamine synthetase.

Glutamine synthetase (GS), encoded by GLUL, catalyzes the conversion of glutamate to glutamine. GS is pivotal for the generation of the neurotransmitters glutamate and gamma-aminobutyric acid and is the primary mechanism of ammonia detoxification in the brain. GS levels are regulated post-translationally by an N-terminal degron that enables the ubiquitin-mediated degradation of GS in a glutamine-induced manner. GS deficiency in humans is known to lead to neurological defects and death in infancy, yet how dysregulation of the degron-mediated control of GS levels might affect neurodevelopment is unknown. We ascertained nine individuals with severe developmental delay, seizures, and white matter abnormalities but normal plasma and cerebrospinal fluid biochemistry with de novo variants in GLUL. Seven out of nine were start-loss variants and two out of nine disrupted 5' UTR splicing resulting in splice exclusion of the initiation codon. Using transfection-based expression systems and mass spectrometry, these variants were shown to lead to translation initiation of GS from methionine 18, downstream of the N-terminal degron motif, resulting in a protein that is stable and enzymatically competent but insensitive to negative feedback by glutamine. Analysis of human single-cell transcriptomes demonstrated that GLUL is widely expressed in neuro- and glial-progenitor cells and mature astrocytes but not in post-mitotic neurons. One individual with a start-loss GLUL variant demonstrated periventricular nodular heterotopia, a neuronal migration disorder, yet overexpression of stabilized GS in mice using in utero electroporation demonstrated no migratory deficits. These findings underline the importance of tight regulation of glutamine metabolism during neurodevelopment in humans.

Undiagnosed Disease Network collaborative approach in diagnosing rare disease in a patient with a mosaic CACNA1D variant.

The Undiagnosed Disease Network (UDN) is comprised of clinical and research experts collaborating to diagnose rare disease. The UDN is funded by the National Institutes of Health and includes 12 different clinical sites (About Us, 2022). Here we highlight the success of collaborative efforts within the UDN Clinical Site at Vanderbilt University Medical Center (VUMC) in utilizing a cohort of experts in bioinformatics, structural biology, and genetics specialists in diagnosing rare disease. Our UDN team identified a de novo mosaic CACNA1D variant c.2299T>C in a 5-year-old female with a history of global developmental delay, dystonia, dyskinesis, and seizures. Using a collaborative multidisciplinary approach, our VUMC UDN team diagnosed the participant with Primary Aldosteronism, Seizures, and Neurologic abnormalities (PASNA) OMIM: 615474 due to a rare mosaic CACNA1D variant (O'Neill, 2013). Interestingly, this patient was mosaic, a phenotypic trait previously unreported in PASNA cases. This report highlights the importance of a multidisciplinary approach in diagnosing rare disease.