Differential Tractography: A Biomarker for Neuronal Function in Neurodegenerative Disease.

GM1 gangliosidosis is an ultra-rare inherited neurodegenerative lysosomal storage disorder caused by biallelic mutations in the GLB1 gene. GM1 is uniformly fatal and has no approved therapies, although clinical trials investigating gene therapy as a potential treatment for this condition are underway. Novel outcome measures or biomarkers demonstrating the longitudinal effects of GM1 and potential recovery due to therapeutic intervention are urgently needed to establish efficacy of potential therapeutics. One promising tool is differential tractography, a novel imaging modality utilizing serial diffusion weighted imaging (DWI) to quantify longitudinal changes in white matter microstructure. In this study, we present the novel use of differential tractography in quantifying the progression of GM1 alongside age-matched neurotypical controls. We analyzed 113 DWI scans from 16 GM1 patients and 32 age-matched neurotypical controls to investigate longitudinal changes in white matter pathology. GM1 patients showed white matter degradation evident by both the number and size of fiber tract loss. In contrast, neurotypical controls showed longitudinal white matter improvements as evident by both the number and size of fiber tract growth. We also corroborated these findings by documenting significant correlations between cognitive global impression (CGI) scores of clinical presentations and our differential tractography derived metrics in our GM1 cohort. Specifically, GM1 patients who lost more neuronal fiber tracts also had a worse clinical presentation. This result demonstrates the importance of differential tractography as an important biomarker for disease progression in GM1 patients with potential extension to other neurodegenerative diseases and therapeutic intervention.

Quantitative reliability assessment of brain MRI volumetric measurements in type II GM1 gangliosidosis patients.

Purpose: GM1-gangliosidosis (GM1) leads to extensive neurodegenerative changes and atrophy that precludes the use of automated MRI segmentation techniques for generating brain volumetrics. We developed a standardized segmentation protocol for brain MRIs of patients with type II GM1 and then assessed the inter- and intra-rater reliability of this methodology. The volumetric data may be used as a biomarker of disease burden and progression, and standardized methodology may support research into the natural history of the disease which is currently lacking in the literature. Approach: Twenty-five brain MRIs were included in this study from 22 type II GM1 patients of which 8 were late-infantile subtype and 14 were juvenile subtype. The following structures were segmented by two rating teams on a slice-by-slice basis: whole brain, ventricles, cerebellum, lentiform nucleus, thalamus, corpus callosum, and caudate nucleus. The inter- and intra-rater reliability of the segmentation method was assessed with an intraclass correlation coefficient as well as Sorensen-Dice and Jaccard coefficients. Results: Based on the Sorensen-Dice and Jaccard coefficients, the inter- and intra-rater reliability of the segmentation method was significantly better for the juvenile patients compared to late-infantile (p < 0.01). In addition, the agreement between the two rater teams and within themselves can be considered good with all p-values < 0.05. Conclusions: The standardized segmentation approach described here has good inter- and intra-rater reliability and may provide greater accuracy and reproducibility for neuromorphological studies in this group of patients and help to further expand our understanding of the natural history of this disease.

Biallelic PI4KA Mutations Disrupt B-Cell Metabolism and Cause B-Cell Lymphopenia and Hypogammaglobulinemia.

PURPOSE: PI4KA-related disorder is a highly clinically variable condition characterized by neurological (limb spasticity, developmental delay, intellectual disability, seizures, ataxia, nystagmus) and gastrointestinal (inflammatory bowel disease and multiple intestinal atresia) manifestations. Although features consistent with immunodeficiency (autoimmunity/autoinflammation and recurrent infections) have been reported in a subset of patients, the burden of B-cell deficiency and hypogammaglobulinemia has not been extensively investigated. We sought to describe the clinical presentation and manifestations of patients with PI4KA-related disorder and to investigate the metabolic consequences of biallelic PI4KA variants in B cells. METHODS: Clinical data from patients with PI4KA variants were obtained. Multi-omics analyses combining transcriptome, proteome, lipidome and metabolome analyses in conjunction with functional assays were performed in EBV-transformed B cells. RESULTS: Clinical and laboratory data of 13 patients were collected. Recurrent infections (7/13), autoimmune/autoinflammatory manifestations (5/13), B-cell deficiency (8/13) and hypogammaglobulinemia (8/13) were frequently observed. Patients' B cells frequently showed increased transitional and decreased switched memory B-cell subsets. Pathway analyses based on differentially expressed transcripts and proteins confirmed the central role of PI4KA in B cell differentiation with altered B-cell receptor (BCR) complex and signalling. By altering lipids production and tricarboxylic acid cycle regulation, and causing increased endoplasmic reticulum stress, biallelic PI4KA mutations disrupt B cell metabolism inducing mitochondrial dysfunction. As a result, B cells show hyperactive PI3K/mTOR pathway, increased autophagy and deranged cytoskeleton organization. CONCLUSION: By altering lipid metabolism and TCA cycle, impairing mitochondrial activity, hyperactivating mTOR pathway and increasing autophagy, PI4KA-related disorder causes a syndromic inborn error of immunity presenting with B-cell deficiency and hypogammaglobulinemia.

De novo variants in the RNU4-2 snRNA cause a frequent neurodevelopmental syndrome.

Around 60% of individuals with neurodevelopmental disorders (NDD) remain undiagnosed after comprehensive genetic testing, primarily of protein-coding genes1. Large genome-sequenced cohorts are improving our ability to discover new diagnoses in the non-coding genome. Here we identify the non-coding RNA RNU4-2 as a syndromic NDD gene. RNU4-2 encodes the U4 small nuclear RNA (snRNA), which is a critical component of the U4/U6.U5 tri-snRNP complex of the major spliceosome2. We identify an 18 base pair region of RNU4-2 mapping to two structural elements in the U4/U6 snRNA duplex (the T-loop and stem III) that is severely depleted of variation in the general population, but in which we identify heterozygous variants in 115 individuals with NDD. Most individuals (77.4%) have the same highly recurrent single base insertion (n.64_65insT). In 54 individuals in whom it could be determined, the de novo variants were all on the maternal allele. We demonstrate that RNU4-2 is highly expressed in the developing human brain, in contrast to RNU4-1 and other U4 homologues. Using RNA sequencing, we show how 5' splice-site use is systematically disrupted in individuals with RNU4-2 variants, consistent with the known role of this region during spliceosome activation. Finally, we estimate that variants in this 18 base pair region explain 0.4% of individuals with NDD. This work underscores the importance of non-coding genes in rare disorders and will provide a diagnosis to thousands of individuals with NDD worldwide.

GM1 gangliosidosis type II: Results of a 10-year prospective study.

PURPOSE: GM1 gangliosidosis (GM1) a lysosomal disorder caused by pathogenic variants in GLB1, is characterized by relentless neurodegeneration. There are no approved treatments. METHODS: Forty-one individuals with type II (late-infantile and juvenile) GM1 participated in a single-site prospective observational study. RESULTS: Classification of 37 distinct variants using American College of Medical Genetics and Genomics criteria resulted in the upgrade of 6 and the submission of 4 new variants. In contrast to type I infantile disease, children with type II had normal or near normal hearing and did not have cherry-red maculae or hepatosplenomegaly. Some older children with juvenile onset disease developed thickened aortic and/or mitral valves. Serial magnetic resonance images demonstrated progressive brain atrophy, more pronounced in late infantile patients. Magnetic resonance spectroscopy showed worsening elevation of myo-inositol and deficit of N-acetyl aspartate that were strongly correlated with scores on the Vineland Adaptive Behavior Scale, progressing more rapidly in late infantile compared with juvenile onset disease. CONCLUSION: Serial phenotyping of type II GM1 patients expands the understanding of disease progression and clarifies common misconceptions about type II patients; these are pivotal steps toward more timely diagnosis and better supportive care. The data amassed through this 10-year effort will serve as a robust comparator for ongoing and future therapeutic trials.

De novo variants in the non-coding spliceosomal snRNA gene RNU4-2 are a frequent cause of syndromic neurodevelopmental disorders.

Around 60% of individuals with neurodevelopmental disorders (NDD) remain undiagnosed after comprehensive genetic testing, primarily of protein-coding genes1. Increasingly, large genome-sequenced cohorts are improving our ability to discover new diagnoses in the non-coding genome. Here, we identify the non-coding RNA RNU4-2 as a novel syndromic NDD gene. RNU4-2 encodes the U4 small nuclear RNA (snRNA), which is a critical component of the U4/U6.U5 tri-snRNP complex of the major spliceosome2. We identify an 18 bp region of RNU4-2 mapping to two structural elements in the U4/U6 snRNA duplex (the T-loop and Stem III) that is severely depleted of variation in the general population, but in which we identify heterozygous variants in 119 individuals with NDD. The vast majority of individuals (77.3%) have the same highly recurrent single base-pair insertion (n.64_65insT). We estimate that variants in this region explain 0.41% of individuals with NDD. We demonstrate that RNU4-2 is highly expressed in the developing human brain, in contrast to its contiguous counterpart RNU4-1 and other U4 homologs, supporting RNU4-2's role as the primary U4 transcript in the brain. Overall, this work underscores the importance of non-coding genes in rare disorders. It will provide a diagnosis to thousands of individuals with NDD worldwide and pave the way for the development of effective treatments for these individuals.

GM1 Gangliosidosis Type II: Results of a 10-Year Prospective Study.

Purpose: GM1 gangliosidosis (GM1) is an ultra-rare lysosomal storage disease caused by pathogenic variants in galactosidase beta 1 (GLB1; NM_000404), primarily characterized by neurodegeneration, often in children. There are no approved treatments for GM1, but clinical trials using gene therapy (NCT03952637, NCT04713475) and small molecule substrate inhibitors (NCT04221451) are ongoing. Understanding the natural history of GM1 is essential for timely diagnosis, facilitating better supportive care, and contextualizing the results of therapeutic trials. Methods: Forty-one individuals with type II GM1 (n=17 late infantile and n=24 juvenile onset) participated in a single-site prospective observational study. Here, we describe the results of extensive multisystem assessment batteries, including clinical labs, neuroimaging, physiological exams, and behavioral assessments. Results: Classification of 37 distinct variants in this cohort was performed according to ACMG criteria and resulted in the upgrade of six and the submission of four new variants to pathogenic or likely pathogenic. In contrast to type I infantile, children with type II disease exhibited normal or near normal hearing and did not have cherry red maculae or significant hepatosplenomegaly. Some older children with juvenile onset developed thickened aortic and/or mitral valves with regurgitation. Serial MRIs demonstrated progressive brain atrophy that were more pronounced in those with late infantile onset. MR spectroscopy showed worsening elevation of myo-inositol and deficit of N-acetyl aspartate that were strongly correlated with scores on the Vineland Adaptive Behavior Scale and progress more rapidly in late infantile than juvenile onset disease. Conclusion: The comprehensive serial phenotyping of type II GM1 patients expands the understanding of disease progression and clarifies some common misconceptions about type II patients. Findings from this 10-year endeavor are a pivotal step toward more timely diagnosis and better supportive care for patients. The wealth of data amassed through this effort will serve as a robust comparator for ongoing and future therapeutic trials.

The spectrum of neurological presentation in individuals affected by TBL1XR1 gene defects.

BACKGROUND: TBL1XR1 encodes a F-box-like/WD40 repeat-containing protein that plays a role in transcription mediated by nuclear receptors and is a known genetic cause of neurodevelopmental disease of childhood (OMIM# 608628). Yet the developmental trajectory and progression of neurologic symptoms over time remains poorly understood. METHODS: We developed and distributed a survey to two closed Facebook groups devoted to families of patients with TBL1XR1-related disorder. The survey consisted of 14 subsections focused upon the developmental trajectories of cognitive, behavioral, motor, and other neurological abnormalities. Data were collected and managed using REDCap electronic data capture tools. RESULTS: Caregivers of 41 patients with a TBL1XR1-related disorder completed the cross-sectional survey. All reported variants affecting a single amino acid, including missense mutations and in-frame deletions, were found in the WD40 repeat regions of Tbl1xr1. These are domains considered important for protein-protein interactions that may plausibly underlie disease pathology. The majority of patients were diagnosed with a neurologic condition before they received their genetic diagnosis. Language appeared most significantly affected with only a minority of the cohort achieving more advanced milestones in this domain. CONCLUSION: TBL1XR1-related disorder encompasses a spectrum of clinical presentations, marked by early developmental delay ranging in severity, with a subset of patients experiencing developmental regression in later childhood.

Thrombotic microangiopathy following systemic AAV administration is dependent on anti-capsid antibodies.

BACKGROUNDSystemic administration of adeno-associated virus (AAV) can trigger life-threatening inflammatory responses, including thrombotic microangiopathy (TMA), acute kidney injury due to atypical hemolytic uremic syndrome-like complement activation, immune-mediated myocardial inflammation, and hepatic toxicity.METHODSWe describe the kinetics of immune activation following systemic AAV serotype 9 (AAV9) administration in 38 individuals following 2 distinct prophylactic immunomodulation regimens. Group 1 received corticosteroids and Group 2 received rituximab plus sirolimus in addition to steroids to prevent anti-AAV antibody formation.RESULTSGroup 1 participants had a rapid increase in immunoglobulin M (IgM) and IgG. Increase in D-dimer, decline in platelet count, and complement activation are indicative of TMA. All Group 1 participants demonstrated activation of both classical and alternative complement pathways, as indicated by depleted C4 and elevated soluble C5b-9, Ba, and Bb antigens. Group 2 patients did not have a significant change in IgM or IgG and had minimal complement activation.CONCLUSIONSThis study demonstrates that TMA in the setting of AAV gene therapy is antibody dependent (classical pathway) and amplified by the alternative complement pathway. Critical time points and interventions are identified to allow for management of immune-mediated events that impact the safety and efficacy of systemic gene therapy.

Risks and benefits of anesthesia for combined pediatric procedures in the NIH undiagnosed diseases program.

PURPOSE: The NIH Undiagnosed Diseases Program (UDP) aims to provide diagnoses to patients who have previously received exhaustive evaluations yet remain undiagnosed. Patients undergo procedural anesthesia for deep phenotyping for analysis with genomic testing. METHODS: A retrospective chart review was performed to determine the safety and benefit of procedural anesthesia in pediatric patients in the UDP. Adverse perioperative events were classified as anesthesia-related complications or peri-procedural complications. The contribution of procedures performed under anesthesia to arriving at a diagnosis was also determined. RESULTS: From 2008 to 2020, 249 pediatric patients in the UDP underwent anesthesia for diagnostic procedures. The majority had a severe systemic disease (American Society for Anesthesiology status III, 79%) and/or a neurologic condition (91%). Perioperative events occurred in 45 patients; six of these were attributed to anesthesia. All patients recovered fully without sequelae. Nearly half of the 249 patients (49%) received a diagnosis, and almost all these diagnoses (88%) took advantage of information gleaned from procedures performed under anesthesia. CONCLUSIONS: The benefits of anesthesia involving multiple diagnostic procedures in a well-coordinated, multidisciplinary, research setting, such as in the pediatric UDP, outweigh the risks.

A pentasaccharide for monitoring pharmacodynamic response to gene therapy in GM1 gangliosidosis.

BACKGROUND: GM1 gangliosidosis is a rare, fatal, neurodegenerative disease caused by mutations in the GLB1 gene and deficiency in ß-galactosidase. Delay of symptom onset and increase in lifespan in a GM1 gangliosidosis cat model after adeno-associated viral (AAV) gene therapy treatment provide the basis for AAV gene therapy trials. The availability of validated biomarkers would greatly improve assessment of therapeutic efficacy. METHODS: The liquid chromatography-tandem mass spectrometry (LC-MS/MS) was used to screen oligosaccharides as potential biomarkers for GM1 gangliosidosis. The structures of pentasaccharide biomarkers were determined with mass spectrometry, as well as chemical and enzymatic degradations. Comparison of LC-MS/MS data of endogenous and synthetic compounds confirmed the identification. The study samples were analyzed with fully validated LC-MS/MS methods. FINDINGS: We identified two pentasaccharide biomarkers, H3N2a and H3N2b, that were elevated more than 18-fold in patient plasma, cerebrospinal fluid (CSF), and urine. Only H3N2b was detectable in the cat model, and it was negatively correlated with ß-galactosidase activity. Following intravenous (IV) AAV9 gene therapy treatment, reduction of H3N2b was observed in central nervous system, urine, plasma, and CSF samples from the cat model and in urine, plasma, and CSF samples from a patient. Reduction of H3N2b accurately reflected normalization of neuropathology in the cat model and improvement of clinical outcomes in the patient. INTERPRETATIONS: These results demonstrate that H3N2b is a useful pharmacodynamic biomarker to evaluate the efficacy of gene therapy for GM1 gangliosidosis. H3N2b will facilitate the translation of gene therapy from animal models to patients. FUNDING: This work was supported by grants U01NS114156, R01HD060576, ZIAHG200409, and P30 DK020579 from the National Institutes of Health (NIH) and a grant from National Tay-Sachs and Allied Diseases Association Inc.

Posterior superior temporal cortex connectivity is related to social communication in toddlers.

The second year of life is a time when social communication skills typically develop, but this growth may be slower in toddlers with language delay. In the current study, we examined how brain functional connectivity is related to social communication abilities in a sample of 12-24 month-old toddlers including those with typical development (TD) and those with language delays (LD). We used an a-priori, seed-based approach to identify regions forming a functional network with the left posterior superior temporal cortex (LpSTC), a region associated with language and social communication in older children and adults. Social communication and language abilities were assessed using the Communication and Symbolic Behavior Scales (CSBS) and Mullen Scales of Early Learning. We found a significant association between concurrent CSBS scores and functional connectivity between the LpSTC and the right posterior superior temporal cortex (RpSTC), with greater connectivity between these regions associated with better social communication abilities. However, functional connectivity was not related to rate of change or language outcomes at 36 months of age. These data suggest an early marker of low communication abilities may be decreased connectivity between the left and right pSTC. Future longitudinal studies should test whether this neurobiological feature is predictive of later social or communication impairments.

Complex effects on CaV2.1 channel gating caused by a CACNA1A variant associated with a severe neurodevelopmental disorder.

P/Q-type Ca2+ currents mediated by CaV2.1 channels are essential for active neurotransmitter release at neuromuscular junctions and many central synapses. Mutations in CACNA1A, the gene encoding the principal CaV2.1 α1A subunit, cause a broad spectrum of neurological disorders. Typically, gain-of-function (GOF) mutations are associated with migraine and epilepsy while loss-of-function (LOF) mutations are causative for episodic and congenital ataxias. However, a cluster of severe CaV2.1 channelopathies have overlapping presentations which suggests that channel dysfunction in these disorders cannot always be defined bimodally as GOF or LOF. In particular, the R1667P mutation causes focal seizures, generalized hypotonia, dysarthria, congenital ataxia and, in one case, cerebral edema leading ultimately to death. Here, we demonstrate that the R1667P mutation causes both channel GOF (hyperpolarizing voltage-dependence of activation, slowed deactivation) and LOF (slowed activation kinetics) when expressed heterologously in tsA-201 cells. We also observed a substantial reduction in Ca2+ current density in this heterologous system. These changes in channel gating and availability/expression manifested in diminished Ca2+ flux during action potential-like stimuli. However, the integrated Ca2+ fluxes were no different when normalized to tail current amplitude measured upon repolarization from the reversal potential. In summary, our findings indicate a complex functional effect of R1667P and support the idea that pathological missense mutations in CaV2.1 may not represent exclusively GOF or LOF.

Linkage-specific deubiquitylation by OTUD5 defines an embryonic pathway intolerant to genomic variation.

Reversible modification of proteins with linkage-specific ubiquitin chains is critical for intracellular signaling. Information on physiological roles and underlying mechanisms of particular ubiquitin linkages during human development are limited. Here, relying on genomic constraint scores, we identify 10 patients with multiple congenital anomalies caused by hemizygous variants in OTUD5, encoding a K48/K63 linkage-specific deubiquitylase. By studying these mutations, we find that OTUD5 controls neuroectodermal differentiation through cleaving K48-linked ubiquitin chains to counteract degradation of select chromatin regulators (e.g., ARID1A/B, histone deacetylase 2, and HCF1), mutations of which underlie diseases that exhibit phenotypic overlap with OTUD5 patients. Loss of OTUD5 during differentiation leads to less accessible chromatin at neuroectodermal enhancers and aberrant gene expression. Our study describes a previously unidentified disorder we name LINKED (LINKage-specific deubiquitylation deficiency-induced Embryonic Defects) syndrome and reveals linkage-specific ubiquitin cleavage from chromatin remodelers as an essential signaling mode that coordinates chromatin remodeling during embryogenesis.

DYRK1A pathogenic variants in two patients with syndromic intellectual disability and a review of the literature.

BACKGROUND: DYRK1A-Related Intellectual Disability Syndrome is a rare autosomal dominant condition characterized by intellectual disability, speech and language delays, microcephaly, facial dysmorphism, and feeding difficulties. Affected individuals represent simplex cases that result from de novo heterozygous pathogenic variants in DYRK1A (OMIM 614104), or chromosomal structural rearrangements involving the DYRK1A locus. Due to the rarity of DYRK1A-Related Intellectual Disability Syndrome, the spectrum of symptoms associated with this disease has not been completely defined. METHODS AND RESULTS: We present two unrelated cases of DYRK1A-Related Intellectual Disability Syndrome resulting from variants in DYRK1A. Both probands presented to the National Institutes of Health (NIH) with multiple dysmorphic facial features, primary microcephaly, absent or minimal speech, feeding difficulties, and cognitive impairment; features that have been previously reported in individuals with DYRK1A. During NIH evaluation, additional features of enlarged cerebral subarachnoid spaces, retinal vascular tortuosity, and bilateral anomalous large optic discs with increased cup-to-disc ratio were identified in the first proband and multiple ophthalmologic abnormalities and sensorineural hearing loss were identified in the second proband. CONCLUSION: We recommend that the workup of future of patients include a comprehensive eye exam. Early establishment of physical, occupational, and speech therapy may help in the management of ataxia, hypertonia, and speech impairments common in these patients.

The peroxisomal disorder spectrum and Heimler syndrome: Deep phenotyping and review of the literature.

The spectrum of peroxisomal disorders is wide and comprises individuals that die in the first year of life, as well as people with sensorineural hearing loss, retinal dystrophy and amelogenesis imperfecta. In this article, we describe three patients; two diagnosed with Heimler syndrome and a third one with a mild-intermediate phenotype. We arrived at these diagnoses by conducting complete ophthalmic (National Eye Institute), auditory (National Institute of Deafness and Other Communication Disorders), and dental (National Institute of Dental and Craniofacial Research) evaluations, as well as laboratory and genetic testing. Retinal degeneration with macular cystic changes, amelogenesis imperfecta, and sensorineural hearing loss were features shared by the three patients. Patients A and C had pathogenic variants in PEX1 and Patient B, in PEX6. Besides analyzing these cases, we review the literature regarding mild peroxisomal disorders, their pathophysiology, genetics, differential diagnosis, diagnostic methods, and management. We suggest that peroxisomal disorders are considered in every child with sensorineural hearing loss and retinal degeneration. These patients should have a dental evaluation to rule out amelogenesis imperfecta as well as audiologic examination and laboratory testing including peroxisomal biomarkers and genetic testing. Appropriate diagnosis can lead to better genetic counseling and management of the associated comorbidities.

The undiagnosed diseases program: Approach to diagnosis.

Undiagnosed and rare conditions are collectively common and affect millions of people worldwide. The NIH Undiagnosed Diseases Program (UDP) strives to achieve both a comprehensive diagnosis and a better understanding of the mechanisms of disease for many of these individuals. Through the careful review of records, a well-orchestrated inpatient evaluation, genomic sequencing and testing, and with the use of emerging strategies such as matchmaking programs, the UDP succeeds nearly 30 percent of the time for these highly selective cases. Although the UDP process is built on a unique set of resources, case examples demonstrate steps genetic professionals can take, in both clinical and research settings, to arrive at a diagnosis for their most challenging cases.

Linked-read genome sequencing identifies biallelic pathogenic variants in DONSON as a novel cause of Meier-Gorlin syndrome.

MATERIAL: Linked-read whole genome sequencing (WGS) presents a new opportunity for cost-efficient singleton sequencing in place of traditional trio-based designs while generating informative-phased variants, effective for recessive disorders when parental DNA is unavailable. METHODS: We have applied linked-read WGS to identify novel causes of Meier-Gorlin syndrome (MGORS), a condition recognised by short stature, microtia and patella hypo/aplasia. There are eight genes associated with MGORS to date, all encoding essential components involved in establishing and initiating DNA replication. RESULTS: Our successful phasing of linked-read data led to the identification of biallelic rare variants in four individuals (24% of our cohort) in DONSON, a recently established DNA replication fork surveillance factor. The variants include five novel missense and one deep intronic variant. All were demonstrated to be deleterious to function; the missense variants all disrupted the nuclear localisation of DONSON, while the intronic variant created a novel splice site that generated an out-of-frame transcript with no residual canonical transcript produced. CONCLUSION: Variants in DONSON have previously been associated with extreme microcephaly, short stature and limb anomalies and perinatal lethal microcephaly-micromelia syndrome. Our novel genetic findings extend the complicated spectrum of phenotypes associated with DONSON variants and promote novel hypotheses for the role of DONSON in DNA replication. While our findings reiterate that MGORS is a disorder of DNA replication, the pathophysiology is obviously complex. This successful identification of a novel disease gene for MGORS highlights the utility of linked-read WGS as a successful technology to be considered in the genetic studies of recessive conditions.

Kilquist syndrome: A novel syndromic hearing loss disorder caused by homozygous deletion of SLC12A2.

Syndromic sensorineural hearing loss is multigenic and associated with malformations of the ear and other organ systems. Herein we describe a child admitted to the NIH Undiagnosed Diseases Program with global developmental delay, sensorineural hearing loss, gastrointestinal abnormalities, and absent salivation. Next-generation sequencing revealed a uniparental isodisomy in chromosome 5, and a 22 kb homozygous deletion in SLC12A2, which encodes for sodium, potassium, and chloride transporter in the basolateral membrane of secretory epithelia. Functional studies using patient-derived fibroblasts showed truncated SLC12A2 transcripts and markedly reduced protein abundance when compared with control. Loss of Slc12a2 in mice has been shown to lead to deafness, abnormal neuronal growth and migration, severe gastrointestinal abnormalities, and absent salivation. Together with the described phenotype of the Slc12a2-knockout mouse model, our results suggest that the absence of functional SLC12A2 causes a new genetic syndrome and is crucial for the development of auditory, neurologic, and gastrointestinal tissues.

Prospective longitudinal overnight video-EEG evaluation in Phelan-McDermid Syndrome.

OBJECTIVE: Phelan-McDermid Syndrome (PMS) is a rare genetic condition associated with loss of function mutations, including deletions, in the chromosome 22q13 region. This PMS phenotype includes intellectual disability, often minimal to absent verbal skills, and other neurologic features including autism spectrum disorder and seizures. Reports indicate seizures and abnormal electroencephalograms (EEGs) in this population, but previous studies do not describe EEG findings during sleep or prognostic value of abnormal EEG over any time period. METHODS: During a natural history study, 16 consecutively enrolled participants (mean age 10years) with PMS underwent both routine (approximately 25min) and overnight (average 9.65h) video-EEG, in addition to genetic testing, neurodevelopmental assessment, neurological examination, and epilepsy phenotyping. Over 240h of EEG, data was recorded. Comparison of findings from the routine EEG was made with prolonged EEG acquired during awake and sleep the same night. In a subset of nine participants, the overnight EEG was repeated one or more years later to observe the natural evolution and prognostic value of any abnormalities noted at baseline. RESULTS: A history of epilepsy, with multiple seizure types, was confirmed in seven of the 16 participants, giving a prevalence of 43.8% in this cohort. All but one EEG was abnormal (15 of 16), and 75% (12 of 16) showed epileptiform activity. Of these, only 25% of participants (3 of 12) showed definitive epileptiform discharges during the routine study. Overnight EEGs (sleep included) did not show any clinical events consistent with seizures or electrophic seizures, however, overnight EEG showed either more frequent and/or more definitive epileptiform activity in 68.75% (11 of 16) participants. All seven of the 16 participants who had previously been diagnosed with epilepsy showed epileptiform abnormalities. In addition to a wide range of epileptiform activity observed, generalized slowing with poor background organization was frequently noted. Follow-up EEG confirmed persistence of abnormal discharges, but none of the abnormal EEGs showed evolution to electrographic seizures. Clinically, there was no emergence of epilepsy or significant developmental regression noted in the time frame observed. CONCLUSIONS: This is the first and most abundant prolonged awake and sleep video-EEG data recorded in a PMS cohort to date. The importance of overnight prolonged EEGs is highlighted by findings from this study, as they can be used to document the varied topographies of EEG abnormalities in conditions such as PMS, which are often missed during routine EEG studies. While the long-term significance of the EEG abnormalities found (beyond 1year) remains uncertain despite their persistence over time, these findings do underscore the current clinical recommendation that overnight prolonged EEG studies (with sleep) should be conducted in individuals with PMS.