Cellular Modeling of CLN6 with IPSC-derived Neurons and Glia.

Neuronal ceroid lipofuscinosis (NCL), type 6 (CLN6) is a neurodegenerative disorder associated with progressive neurodegeneration leading to dementia, seizures, and retinopathy. CLN6 encodes a resident-ER protein involved in trafficking lysosomal proteins to the Golgi. CLN6p deficiency results in lysosomal dysfunction and deposition of storage material comprised of Nile Red + lipids/proteolipids that include subunit C of the mitochondrial ATP synthase (SUBC). White matter involvement has been recently noted in several CLN6 animal models and several CLN6 subjects had neuroimaging was consistent with leukodystrophy. CLN6 patient-derived induced pluripotent stem cells (IPSCs) were generated from several of these subjects. IPSCs were differentiated into oligodendroglia or neurons using well-established small-molecule protocols. A doxycycline-inducible transgenic system expressing neurogenin-2 (the I3N-system) was also used to generate clonal IPSC-lines (I3N-IPSCs) that could be rapidly differentiated into neurons (I3N-neurons). All CLN6 IPSC-derived neural cell lines developed significant storage material, CLN6-I3N-neuron lines revealed significant Nile Red + and SUBC + storage within three and seven days of neuronal induction, respectively. CLN6-I3N-neurons had decreased tripeptidyl peptidase-1 activity, increased Golgi area, along with increased LAMP1 + in cell bodies and neurites. SUBC + signal co-localized with LAMP1 + signal. Bulk-transcriptomic evaluation of control- and CLN6-I3N-neurons identified >1300 differentially-expressed genes (DEGs) with Gene Ontogeny (GO) Enrichment and Canonical Pathway Analyses having significant changes in lysosomal, axonal, synaptic, and neuronal-apoptotic gene pathways. These findings indicate that CLN6-IPSCs and CLN6-I3N-IPSCs are appropriate cellular models for this disorder. These I3N-neuron models may be particularly valuable for developing therapeutic interventions with high-throughput drug screening assays and/or gene therapy.

Clinical efficacy and satisfaction of a digital wheeze detector in a multicentre randomised controlled trial: the WheezeScan study.

Introduction: Wheezing is common in preschool children and its clinical assessment often challenging for caretakers. This study aims to evaluate the impact of a novel digital wheeze detector (WheezeScan™) on disease control in a home care setting. Methods: A multicentre randomised open-label controlled trial was conducted in Berlin, Istanbul and London. Participants aged 4-84 months with a doctor's diagnosis of recurrent wheezing in the past 12 months were included. While the control group followed usual care, the intervention group received the WheezeScan™ for at-home use for 120 days. Parents completed questionnaires regarding their child's respiratory symptoms, disease-related and parental quality of life, and caretaker self-efficacy at baseline (T0), 90 days (T1) and 4 months (T2). Results: A total of 167 children, with a mean±sd age of 3.2±1.6 years, were enrolled in the study (intervention group n=87; control group n=80). There was no statistically significant difference in wheeze control assessed by TRACK (mean difference 3.8, 95% CI -2.3-9.9; p=0.2) at T1 between treatment groups (primary outcome). Children's and parental quality of life and parental self-efficacy were comparable between both groups at T1. The evaluation of device usability and perception showed that parents found it useful. Conclusion: In the current study population, the wheeze detector did not show significant impact on the home management of preschool wheezing. Hence, further research is needed to better understand how the perception and usage behaviour may influence the clinical impact of a digital support.

LUSTR: a new customizable tool for calling genome-wide germline and somatic short tandem repeat variants.

BACKGROUND: Short tandem repeats (STRs) are widely distributed across the human genome and are associated with numerous neurological disorders. However, the extent that STRs contribute to disease is likely under-estimated because of the challenges calling these variants in short read next generation sequencing data. Several computational tools have been developed for STR variant calling, but none fully address all of the complexities associated with this variant class. RESULTS: Here we introduce LUSTR which is designed to address some of the challenges associated with STR variant calling by enabling more flexibility in defining STR loci, allowing for customizable modules to tailor analyses, and expanding the capability to call somatic and multiallelic STR variants. LUSTR is a user-friendly and easily customizable tool for targeted or unbiased genome-wide STR variant screening that can use either predefined or novel genome builds. Using both simulated and real data sets, we demonstrated that LUSTR accurately infers germline and somatic STR expansions in individuals with and without diseases. CONCLUSIONS: LUSTR offers a powerful and user-friendly approach that allows for the identification of STR variants and can facilitate more comprehensive studies evaluating the role of pathogenic STR variants across human diseases.

Adult-onset neurodegeneration in XMEN disease.

BACKGROUND: XMEN (X-linked immunodeficiency with magnesium defect, Epstein-Barr virus (EBV), and N-linked glycosylation defect) disease results from loss-of-function mutations in MAGT1, a protein that serves as a magnesium transporter and a subunit of the oligosaccharyltransferase (OST) complex. MAGT1 deficiency disrupts N-linked glycosylation, a critical regulator of immune function. XMEN results in recurrent EBV infections and a propensity for EBV-driven malignancies. Although XMEN is recognized as a systemic congenital disorder of glycosylation (CDG), its neurological involvement is rare and poorly characterized. CASES: Two young men, ages 32 and 33, are described here with truncating mutations in MAGT1, progressive behavioral changes, and neurodegenerative symptoms. These features manifested well into adulthood. Both patients still presented with many of the molecular and clinical hallmarks of the typical XMEN patient, including chronic EBV viremia and decreased expression of NKG2D. CONCLUSION: While previously unrecognized, XMEN may include prominent and disabling CNS manifestations. How MAGT1 deficiency directly or indirectly contributes to neurodegeneration remains unclear. Elucidating this mechanism may contribute to the understanding of neurodegeneration more broadly.

Spectrum of LYST mutations in Chediak-Higashi syndrome: a report of novel variants and a comprehensive review of the literature.

INTRODUCTION: Chediak-Higashi syndrome (CHS) is a rare autosomal recessive disorder characterised by partial oculocutaneous albinism, a bleeding diathesis, immunological dysfunction and neurological impairment. Bi-allelic loss-of-function variants in LYST cause CHS. LYST encodes the lysosomal trafficking regulator, a highly conserved 429 kDa cytoplasmic protein with an unknown function. METHODS: To further our understanding of the pathogenesis of CHS, we conducted clinical evaluations on individuals with CHS enrolled in our natural history study. Using genomic DNA Sanger sequencing, we identified novel pathogenic LYST variants. Additionally, we performed an extensive literature review to curate reported LYST variants and classified these novel and reported variants according to the American College of Medical Genetics/Association for Molecular Pathology variant interpretation guidelines. RESULTS: Our investigation unveiled 11 novel pathogenic LYST variants in eight patients with a clinical diagnosis of CHS, substantiated by the presence of pathognomonic giant intracellular granules. From these novel variants, together with a comprehensive review of the literature, we compiled a total of 147 variants in LYST, including 61 frameshift variants (41%), 44 nonsense variants (30%), 23 missense variants (16%), 13 splice site variants or small genomic deletions for which the coding effect is unknown (9%), 5 in-frame variants (3%) and 1 start-loss variant (1%). Notably, a genotype-phenotype correlation emerged, whereby individuals harbouring at least one missense or in-frame variant generally resulted in milder disease, while those with two nonsense or frameshift variants generally had more severe disease. CONCLUSION: The identification of novel pathogenic LYST variants and improvements in variant classification will provide earlier diagnoses and improved care to individuals with CHS.

Sustained OMA1-mediated integrated stress response is beneficial for spastic ataxia type 5.

AFG3L2 is a mitochondrial protease exerting protein quality control in the inner mitochondrial membrane. Heterozygous AFG3L2 mutations cause spinocerebellar ataxia type 28 (SCA28) or dominant optic atrophy type 12 (DOA12), while biallelic AFG3L2 mutations result in the rare and severe spastic ataxia type 5 (SPAX5). The clinical spectrum of SPAX5 includes childhood-onset cerebellar ataxia, spasticity, dystonia and myoclonic epilepsy. We previously reported that the absence or mutation of AFG3L2 leads to the accumulation of mitochondria-encoded proteins, causing the overactivation of the stress-sensitive protease OMA1, which over-processes OPA1, leading to mitochondrial fragmentation. Recently, OMA1 has been identified as the pivotal player communicating mitochondrial stress to the cytosol via a pathway involving the inner mitochondrial membrane protein DELE1 and the cytosolic kinase HRI, thus eliciting the integrated stress response. In general, the integrated stress response reduces global protein synthesis and drives the expression of cytoprotective genes that allow cells to endure proteotoxic stress. However, the relevance of the OMA1-DELE1-HRI axis in vivo, and especially in a human CNS disease context, has been poorly documented thus far. In this work, we demonstrated that mitochondrial proteotoxicity in the absence/mutation of AFG3L2 activates the OMA1-DELE1-HRI pathway eliciting the integrated stress response. We found enhanced OMA1-dependent processing of DELE1 upon depletion of AFG3L2. Also, in both skin fibroblasts from SPAX5 patients (including a novel case) and in the cerebellum of Afg3l2-/- mice we detected increased phosphorylation of the α-subunit of the eukaryotic translation initiation factor 2 (eIF2α), increased levels of ATF4 and strong upregulation of its downstream targets (Chop, Chac1, Ppp1r15a and Ffg21). Silencing of DELE1 or HRI in SPAX5 fibroblasts (where OMA1 is overactivated at basal state) reduces eIF2α phosphorylation and affects cell growth. In agreement, pharmacological potentiation of integrated stress response via Sephin-1, a drug that selectively inhibits the stress-induced eIF2alpha phosphatase GADD34 (encoded by Ppp1r15a), improved cell growth of SPAX5 fibroblasts and cell survival and dendritic arborization ex vivo in primary Afg3l2-/- Purkinje neurons. Notably, Sephin-1 treatment in vivo extended the lifespan of Afg3l2-/- mice, improved Purkinje neuron morphology, mitochondrial ultrastructure and respiratory capacity. These data indicate that activation of the OMA1-DELE1-HRI pathway is protective in the context of SPAX5. Pharmacological tuning of the integrated stress response may represent a future therapeutic strategy for SPAX5 and other cerebellar ataxias caused by impaired mitochondrial proteostasis.

An ANXA11 P93S variant dysregulates TDP-43 and causes corticobasal syndrome.

As genetic testing has become more accessible and affordable, variants of uncertain significance (VUS) are increasingly identified, and determining whether these variants play causal roles in disease is a major challenge. The known disease-associated Annexin A11 (ANXA11) mutations result in ANXA11 aggregation, alterations in lysosomal-RNA granule co-trafficking, and TDP-43 mis-localization and present as amyotrophic lateral sclerosis or frontotemporal dementia. We identified a novel VUS in ANXA11 (P93S) in a kindred with corticobasal syndrome and unique radiographic features that segregated with disease. We then queried neurodegenerative disorder clinic databases to identify the phenotypic spread of ANXA11 mutations. Multi-modal computational analysis of this variant was performed and the effect of this VUS on ANXA11 function and TDP-43 biology was characterized in iPSC-derived neurons. Single-cell sequencing and proteomic analysis of iPSC-derived neurons and microglia were used to determine the multiomic signature of this VUS. Mutations in ANXA11 were found in association with clinically diagnosed corticobasal syndrome, thereby establishing corticobasal syndrome as part of ANXA11 clinical spectrum. In iPSC-derived neurons expressing mutant ANXA11, we found decreased colocalization of lysosomes and decreased neuritic RNA as well as decreased nuclear TDP-43 and increased formation of cryptic exons compared to controls. Multiomic assessment of the P93S variant in iPSC-derived neurons and microglia indicates that the pathogenic omic signature in neurons is modest compared to microglia. Additionally, omic studies reveal that immune dysregulation and interferon signaling pathways in microglia are central to disease. Collectively, these findings identify a new pathogenic variant in ANXA11, expand the range of clinical syndromes caused by ANXA11 mutations, and implicate both neuronal and microglia dysfunction in ANXA11 pathophysiology. This work illustrates the potential for iPSC-derived cellular models to revolutionize the variant annotation process and provides a generalizable approach to determining causality of novel variants across genes.

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.

An oligodendrocyte silencer element underlies the pathogenic impact of lamin B1 structural variants.

The role of non-coding regulatory elements and how they might contribute to tissue type specificity of disease phenotypes is poorly understood. Autosomal Dominant Leukodystrophy (ADLD) is a fatal, adult-onset, neurological disorder that is characterized by extensive CNS demyelination. Most cases of ADLD are caused by tandem genomic duplications involving the lamin B1 gene ( LMNB1 ) while a small subset are caused by genomic deletions upstream of the gene. Utilizing data from recently identified families that carry LMNB1 gene duplications but do not exhibit demyelination, ADLD patient tissues, CRISPR modified cell lines and mouse models, we have identified a novel silencer element that is lost in ADLD patients and that specifically targets overexpression to oligodendrocytes. This element consists of CTCF binding sites that mediate three-dimensional chromatin looping involving the LMNB1 and the recruitment of the PRC2 repressor complex. Loss of the silencer element in ADLD identifies a previously unknown role for silencer elements in tissue specificity and disease causation.

Genomic Diagnoses for Ectopic Intracerebral Calcifications.

Background and Objectives: Ectopic intracerebral calcifications (EICs) in the basal ganglia, thalamus, cerebellum, or white matter are seen in a variety of disease states or may be found incidentally on brain imaging. The clinical significance and proportion of cases attributable to an underlying genetic cause is unknown. Methods: This retrospective cohort study details the clinical, imaging, and genomic findings of 44 patients with EICs who had no established diagnosis despite extensive medical workup. Results: In total, 15 of 44 patients received a diagnosis through genomic testing explaining their calcifications, and 2 more received a diagnosis that has not been previously associated with EICs. Six of the 15 were found to have one of the 4 genes (PDGFB, PDGFRB, SLC20A2, and XPR1) conventionally associated with the phenotypic term "idiopathic basal ganglia calcifications." Discussion: These findings support the use of genomic testing for symptomatic patients with EICs.

Germline loss-of-function PAM variants are enriched in subjects with pituitary hypersecretion.

Introduction: Pituitary adenomas (PAs) are common, usually benign tumors of the anterior pituitary gland which, for the most part, have no known genetic cause. PAs are associated with major clinical effects due to hormonal dysregulation and tumoral impingement on vital brain structures. PAM encodes a multifunctional protein responsible for the essential C-terminal amidation of secreted peptides. Methods: Following the identification of a loss-of-function variant (p.Arg703Gln) in the peptidylglycine a-amidating monooxygenase (PAM) gene in a family with pituitary gigantism, we investigated 299 individuals with sporadic PAs and 17 familial isolated PA kindreds for PAM variants. Genetic screening was performed by germline and tumor sequencing and germline copy number variation (CNV) analysis. Results: In germline DNA, we detected seven heterozygous, likely pathogenic missense, truncating, and regulatory SNVs. These SNVs were found in sporadic subjects with growth hormone excess (p.Gly552Arg and p.Phe759Ser), pediatric Cushing disease (c.-133T>C and p.His778fs), or different types of PAs (c.-361G>A, p.Ser539Trp, and p.Asp563Gly). The SNVs were functionally tested in vitro for protein expression and trafficking by Western blotting, splicing by minigene assays, and amidation activity in cell lysates and serum samples. These analyses confirmed a deleterious effect on protein expression and/or function. By interrogating 200,000 exomes from the UK Biobank, we confirmed a significant association of the PAM gene and rare PAM SNVs with diagnoses linked to pituitary gland hyperfunction. Conclusion: The identification of PAM as a candidate gene associated with pituitary hypersecretion opens the possibility of developing novel therapeutics based on altering PAM function.

Pathological variants in TOP3A cause distinct disorders of mitochondrial and nuclear genome stability.

Topoisomerase 3α (TOP3A) is an enzyme that removes torsional strain and interlinks between DNA molecules. TOP3A localises to both the nucleus and mitochondria, with the two isoforms playing specialised roles in DNA recombination and replication respectively. Pathogenic variants in TOP3A can cause a disorder similar to Bloom syndrome, which results from bi-allelic pathogenic variants in BLM, encoding a nuclear-binding partner of TOP3A. In this work, we describe 11 individuals from 9 families with an adult-onset mitochondrial disease resulting from bi-allelic TOP3A gene variants. The majority of patients have a consistent clinical phenotype characterised by bilateral ptosis, ophthalmoplegia, myopathy and axonal sensory-motor neuropathy. We present a comprehensive characterisation of the effect of TOP3A variants, from individuals with mitochondrial disease and Bloom-like syndrome, upon mtDNA maintenance and different aspects of enzyme function. Based on these results, we suggest a model whereby the overall severity of the TOP3A catalytic defect determines the clinical outcome, with milder variants causing adult-onset mitochondrial disease and more severe variants causing a Bloom-like syndrome with mitochondrial dysfunction in childhood.

TNF-Blockade for Primary Stroke Prevention in Adenosine Deaminase 2 Deficiency: A Case Series.

OBJECTIVES: Adenosine deaminase 2 deficiency (DADA2) is a genetic, neurologic, and systemic vasculitis syndrome, which can lead to recurrent strokes, typically lacunar. In the cohort of now 60 patients followed up at the NIH Clinical Center (NIH CC), no patient has had a stroke since starting tumor necrosis factor (TNF) blockade. We present a family with multiple affected children to highlight the importance of TNF blockade not just as secondary stroke prevention but also as primary stroke prevention in genetically affected but clinically asymptomatic patients. METHODS: A proband with recurrent cryptogenic strokes was referred for evaluation at the NIH CC. The parents and 3 clinically asymptomatic siblings were also evaluated. RESULTS: The proband was diagnosed with DADA2 based on biochemical testing; her antiplatelet therapies were discontinued, and she was started on TNF blockade for secondary stroke prevention. Her 3 asymptomatic siblings were subsequently tested and 2 were found to be biochemically affected. One of them elected to start TNF blockade for primary stroke prevention and the other sibling declined this approach and experienced a stroke. A second genetic sequence variant was subsequently identified in the ADA2 gene. DISCUSSION: This family illustrates the importance of testing for DADA2 in young patients with cryptogenic stroke, given the hemorrhagic risks with antiplatelet drugs in these patients and effectiveness of TNF blockade as secondary stroke prevention. In addition, this family highlights the importance of screening all siblings of affected patients because they may be presymptomatic, and we advocate starting TNF blockade for primary stroke prevention in those who are found to be genetically or biochemically affected.

MYH2-associated myopathy caused by a novel splice-site variant.

MYH2 encodes MyHCIIa, a myosin heavy chain found in fast type 2A fibers. Pathogenic variants in this gene have previously been implicated in dominant and recessive forms of myopathy. Three individuals reported here are part of a family in which four generations of individuals are affected by a slowly progressive, predominantly proximal myopathy in an autosomal dominant inheritance pattern. Affected individuals in this family lacked classic features of an MYH2-associated myopathy such as congenital contractures and ophthalmoplegia. A novel variant, MYH2 c.5673+1G>C, was detected in the proband and subsequently found to segregate with disease in five additional family members. Further studies demonstrated that this variant affects splicing, resulting in novel transcripts. These data and muscle biopsy findings in the proband, indicate that this family's MYH2 variant is causative of their myopathy, adding to our understanding of the clinical and molecular characteristics of the disease.

Gene expression changes in Tay-Sachs disease begin early in fetal brain development.

Treatment of monogenic disorders has historically relied on symptomatic management with limited ability to target primary molecular deficits. However, recent advances in gene therapy and related technologies aim to correct these underlying deficiencies, raising the possibility of disease management or even prevention for diseases that can be treated pre-symptomatically. Tay-Sachs disease (TSD) would be one such candidate, however very little is known about the presymptomatic stage of TSD. To better understand the effects of TSD on brain development, we evaluated the transcriptomes of human fetal brain samples with biallelic pathogenic variants in HEXA. We identified dramatic changes in the transcriptome, suggesting a perturbation of normal development. We also observed a shift in the expression of the sphingolipid metabolic pathway away from production of the HEXA substrate, GM2 ganglioside, presumptively to compensate for dysfunction of the enzyme. However, we do not observe transcriptomic signatures of end-stage disease, suggesting that developmental perturbations precede neurodegeneration. To our knowledge, this is the first report of the relationship between fetal disease pathology in juvenile onset TSD and the analysis of gene expression in fetal TSD tissues. This study highlights the need to better understand the "pre-symptomatic" stage of disease to set realistic expectations for patients receiving early therapeutic intervention.

Germline loss-of-function PAM variants are enriched in subjects with pituitary hypersecretion.

Pituitary adenomas (PAs) are common, usually benign tumors of the anterior pituitary gland which, for the most part, have no known genetic cause. PAs are associated with major clinical effects due to hormonal dysregulation and tumoral impingement on vital brain structures. Following the identification of a loss-of-function variant (p.Arg703Gln) in the PAM gene in a family with pituitary gigantism, we investigated 299 individuals with sporadic PAs and 17 familial isolated pituitary adenomas kindreds for PAM variants. PAM encodes a multifunctional protein responsible for the essential C-terminal amidation of secreted peptides. Genetic screening was performed by germline and tumor sequencing and germline copy number variation (CNV) analysis. No germline CNVs or somatic single nucleotide variants (SNVs) were identified. We detected seven likely pathogenic heterozygous missense, truncating, and regulatory SNVs. These SNVs were found in sporadic subjects with GH excess (p.Gly552Arg and p.Phe759Ser), pediatric Cushing disease (c.-133T>C and p.His778fs), or with different types of PAs (c.-361G>A, p.Ser539Trp, and p.Asp563Gly). The SNVs were functionally tested in vitro for protein expression and trafficking by Western blotting, for splicing by minigene assays, and for amidation activity in cell lysates and serum samples. These analyses confirmed a deleterious effect on protein expression and/or function. By interrogating 200,000 exomes from the UK Biobank, we confirmed a significant association of the PAM gene and rare PAM SNVs to diagnoses linked to pituitary gland hyperfunction. Identification of PAM as a candidate gene associated with pituitary hypersecretion opens the possibility of developing novel therapeutics based on altering PAM function.

Association of Brain Injury Biomarkers and Circulatory Shock Following Moderate-Severe Traumatic Brain Injury: A TRACK-TBI Study.

INTRODUCTION: Early circulatory shock following traumatic brain injury (TBI) is a multifactorial process; however, the impact of brain injury biomarkers on the risk of shock has not been evaluated. We examined the association between neuronal injury biomarker levels and the development of circulatory shock following moderate-severe TBI. METHODS: In this retrospective cohort study, we examined adults with moderate-severe TBI (Glasgow Coma Scale score <13) enrolled in the TRACK-TBI study, an 18-center prospective TBI cohort study. The exposures were day-1 levels of neuronal injury biomarkers (glial fibrillary acidic protein, ubiquitin C-terminal hydrolase-L1 [UCH-L1], S100 calcium-binding protein B [S100B], neuron-specific enolase), and of an inflammatory biomarker (high-sensitivity C-reactive protein). The primary outcome was the development of circulatory shock, defined as cardiovascular Sequential Organ Failure Assessment Score ≥2 within 72 hours of admission. Association between day-1 biomarker levels and the development of circulatory shock was assessed with regression analysis. RESULTS: The study included 392 subjects, with a mean age of 40 years; 314 (80%) were male and 165 (42%) developed circulatory shock. Median (interquartile range) day-1 levels of UCH-L1 (994.8 [518.7 to 1988.2] pg/mL vs. 548.1 [280.2 to 1151.9] pg/mL; P <0.0001) and S100B (0.47 µg/mL [0.25 to 0.88] vs. 0.27 [0.16 to 0.46] µg/mL; P <0.0001) were elevated in those who developed early circulatory shock compared with those who did not. In multivariable regression, there were associations between levels of both UCH-L1 (odds ratio, 1.63 [95% confidence interval, 1.25-2.12]; P <0.0005) and S100B (odds ratio, 1.73 [95% confidence interval 1.27-2.36]; P <0.0005) with the development of circulatory shock. CONCLUSION: Neuronal injury biomarkers may provide the improved mechanistic understanding and possibly early identification of patients at risk for early circulatory shock following moderate-severe TBI.

Repeat expansions nested within tandem CNVs: a unique structural change in GLS exemplifies the diagnostic challenges of non-coding pathogenic variation.

Glutaminase deficiency has recently been associated with ataxia and developmental delay due to repeat expansions in the 5'UTR of the glutaminase (GLS) gene. Patients with the described GLS repeat expansion may indeed remain undiagnosed due to the rarity of this variant, the challenge of its detection and the recency of its discovery. In this study, we combined advanced bioinformatics screening of ~3000 genomes and ~1500 exomes with optical genome mapping and long-read sequencing for confirmation studies. We identified two GLS families, previously intensely and unsuccessfully analyzed. One family carries an unusual and complex structural change involving a homozygous repeat expansion nested within a quadruplication event in the 5'UTR of GLS. Glutaminase deficiency and its metabolic consequences were validated by in-depth biochemical analysis. The identified GLS patients showed progressive early-onset ataxia, cognitive deficits, pyramidal tract damage and optic atrophy, thus demonstrating susceptibility of several specific neuron populations to glutaminase deficiency. This large-scale screening study demonstrates the ability of bioinformatics analysis-validated by latest state-of-the-art technologies (optical genome mapping and long-read sequencing)-to effectively flag complex repeat expansions using short-read datasets and thus facilitate diagnosis of ultra-rare disorders.

A de novo hexokinase 1 (HK1) variant presenting as Boucher-Neuhäuser syndrome.

Boucher-Neuhäuser syndrome (BNHS) is characterized by chorioretinal dystrophy, hypogonadotropic hypogonadism, and cerebellar dysfunction and atrophy. The disorder has been associated with biallelic pathogenic variants in the patatin-like phospholipase domain-containing protein 6 (PNPLA6) gene. We present an individual with a clinical diagnosis consistent with BNHS who lacked any PNPLA6 variants but on quartet family exome sequencing had a de novo variant in the hexokinase 1 (HK1) gene (NM_000188.2 [GRCh37/hg19]: g.71139826G>A, c.1240G>A, p.Gly414Arg), suggesting genetic heterogeneity for BNHS. Longitudinal follow-up indicated neurological deterioration, neuropsychiatric symptoms, and progressive cerebellar atrophy. The BNHS phenotype overlaps and expands the known HK1 genotypic and phenotypic spectrum. Individuals with variants in HK1 should undergo evaluation for hypogonadotropic hypogonadism, potentially amenable to treatment.

Genomic analysis, immunomodulation and deep phenotyping of patients with nodding syndrome.

The aetiology of nodding syndrome remains unclear, and comprehensive genotyping and phenotyping data from patients remain sparse. Our objectives were to characterize the phenotype of patients with nodding syndrome, investigate potential contributors to disease aetiology, and evaluate response to immunotherapy. This cohort study investigated members of a single-family unit from Lamwo District, Uganda. The participants for this study were selected by the Ugandan Ministry of Health as representative for nodding syndrome and with a conducive family structure for genomic analyses. Of the eight family members who participated in the study at the National Institutes of Health (NIH) Clinical Center, three had nodding syndrome. The three affected patients were extensively evaluated with metagenomic sequencing for infectious pathogens, exome sequencing, spinal fluid immune analyses, neurometabolic and toxicology testing, continuous electroencephalography and neuroimaging. Five unaffected family members underwent a subset of testing for comparison. A distinctive interictal pattern of sleep-activated bursts of generalized and multifocal epileptiform discharges and slowing was observed in two patients. Brain imaging showed two patients had mild generalized cerebral atrophy, and both patients and unaffected family members had excessive metal deposition in the basal ganglia. Trace metal biochemical evaluation was normal. CSF was non-inflammatory and one patient had CSF-restricted oligoclonal bands. Onchocerca volvulus-specific antibodies were present in all patients and skin snips were negative for active onchocerciasis. Metagenomic sequencing of serum and CSF revealed hepatitis B virus in the serum of one patient. Vitamin B6 metabolites were borderline low in all family members and CSF pyridoxine metabolites were normal. Mitochondrial DNA testing was normal. Exome sequencing did not identify potentially causal candidate gene variants. Nodding syndrome is characterized by a distinctive pattern of sleep-activated epileptiform activity. The associated growth stunting may be due to hypothalamic dysfunction. Extensive testing years after disease onset did not clarify a causal aetiology. A trial of immunomodulation (plasmapheresis in two patients and intravenous immunoglobulin in one patient) was given without short-term effect, but longer-term follow-up was not possible to fully assess any benefit of this intervention.