Unveiling the crucial neuronal role of the proteasomal ATPase subunit gene PSMC5 in neurodevelopmental proteasomopathies.

Neurodevelopmental proteasomopathies represent a distinctive category of neurodevelopmental disorders (NDD) characterized by genetic variations within the 26S proteasome, a protein complex governing eukaryotic cellular protein homeostasis. In our comprehensive study, we identified 23 unique variants in PSMC5 , which encodes the AAA-ATPase proteasome subunit PSMC5/Rpt6, causing syndromic NDD in 38 unrelated individuals. Overexpression of PSMC5 variants altered human hippocampal neuron morphology, while PSMC5 knockdown led to impaired reversal learning in flies and loss of excitatory synapses in rat hippocampal neurons. PSMC5 loss-of-function resulted in abnormal protein aggregation, profoundly impacting innate immune signaling, mitophagy rates, and lipid metabolism in affected individuals. Importantly, targeting key components of the integrated stress response, such as PKR and GCN2 kinases, ameliorated immune dysregulations in cells from affected individuals. These findings significantly advance our understanding of the molecular mechanisms underlying neurodevelopmental proteasomopathies, provide links to research in neurodegenerative diseases, and open up potential therapeutic avenues.

The clinical and genetic spectrum of autosomal-recessive TOR1A-related disorders.

In the field of rare diseases, progress in molecular diagnostics led to the recognition that variants linked to autosomal-dominant neurodegenerative diseases of later onset can, in the context of biallelic inheritance, cause devastating neurodevelopmental disorders and infantile or childhood-onset neurodegeneration. TOR1A-associated arthrogryposis multiplex congenita 5 (AMC5) is a rare neurodevelopmental disorder arising from biallelic variants in TOR1A, a gene that in the heterozygous state is associated with torsion dystonia-1 (DYT1 or DYT-TOR1A), an early-onset dystonia with reduced penetrance. While 15 individuals with AMC5-TOR1A have been reported (less than 10 in detail), a systematic investigation of the full disease-associated spectrum has not been conducted. Here, we assess the clinical, radiological and molecular characteristics of 57 individuals from 40 families with biallelic variants in TOR1A. Median age at last follow-up was 3 years (0-24 years). Most individuals presented with severe congenital flexion contractures (95%) and variable developmental delay (79%). Motor symptoms were reported in 79% and included lower limb spasticity and pyramidal signs, as well as gait disturbances. Facial dysmorphism was an integral part of the phenotype, with key features being a broad/full nasal tip, narrowing of the forehead and full cheeks. Analysis of disease-associated manifestations delineated a phenotypic spectrum ranging from normal cognition and mild gait disturbance to congenital arthrogryposis, global developmental delay, intellectual disability, absent speech and inability to walk. In a subset, the presentation was consistent with foetal akinesia deformation sequence with severe intrauterine abnormalities. Survival was 71%, with higher mortality in males. Death occurred at a median age of 1.2 months (1 week-9 years), due to respiratory failure, cardiac arrest or sepsis. Analysis of brain MRI studies identified non-specific neuroimaging features, including a hypoplastic corpus callosum (72%), foci of signal abnormality in the subcortical and periventricular white matter (55%), diffuse white matter volume loss (45%), mega cisterna magna (36%) and arachnoid cysts (27%). The molecular spectrum included 22 distinct variants, defining a mutational hotspot in the C-terminal domain of the Torsin-1A protein. Genotype-phenotype analysis revealed an association of missense variants in the 3-helix bundle domain to an attenuated phenotype, while missense variants near the Walker A/B motif as well as biallelic truncating variants were linked to early death. In summary, this systematic cross-sectional analysis of a large cohort of individuals with biallelic TOR1A variants across a wide age-range delineates the clinical and genetic spectrum of TOR1A-related autosomal-recessive disease and highlights potential predictors for disease severity and survival.

Phenotypic and genetic spectrum of ATP6V1A encephalopathy: a disorder of lysosomal homeostasis.

Vacuolar-type H+-ATPase (V-ATPase) is a multimeric complex present in a variety of cellular membranes that acts as an ATP-dependent proton pump and plays a key role in pH homeostasis and intracellular signalling pathways. In humans, 22 autosomal genes encode for a redundant set of subunits allowing the composition of diverse V-ATPase complexes with specific properties and expression. Sixteen subunits have been linked to human disease. Here we describe 26 patients harbouring 20 distinct pathogenic de novo missense ATP6V1A variants, mainly clustering within the ATP synthase α/ß family-nucleotide-binding domain. At a mean age of 7 years (extremes: 6 weeks, youngest deceased patient to 22 years, oldest patient) clinical pictures included early lethal encephalopathies with rapidly progressive massive brain atrophy, severe developmental epileptic encephalopathies and static intellectual disability with epilepsy. The first clinical manifestation was early hypotonia, in 70%; 81% developed epilepsy, manifested as developmental epileptic encephalopathies in 58% of the cohort and with infantile spasms in 62%; 63% of developmental epileptic encephalopathies failed to achieve any developmental, communicative or motor skills. Less severe outcomes were observed in 23% of patients who, at a mean age of 10 years and 6 months, exhibited moderate intellectual disability, with independent walking and variable epilepsy. None of the patients developed communicative language. Microcephaly (38%) and amelogenesis imperfecta/enamel dysplasia (42%) were additional clinical features. Brain MRI demonstrated hypomyelination and generalized atrophy in 68%. Atrophy was progressive in all eight individuals undergoing repeated MRIs. Fibroblasts of two patients with developmental epileptic encephalopathies showed decreased LAMP1 expression, Lysotracker staining and increased organelle pH, consistent with lysosomal impairment and loss of V-ATPase function. Fibroblasts of two patients with milder disease, exhibited a different phenotype with increased Lysotracker staining, decreased organelle pH and no significant modification in LAMP1 expression. Quantification of substrates for lysosomal enzymes in cellular extracts from four patients revealed discrete accumulation. Transmission electron microscopy of fibroblasts of four patients with variable severity and of induced pluripotent stem cell-derived neurons from two patients with developmental epileptic encephalopathies showed electron-dense inclusions, lipid droplets, osmiophilic material and lamellated membrane structures resembling phospholipids. Quantitative assessment in induced pluripotent stem cell-derived neurons identified significantly smaller lysosomes. ATP6V1A-related encephalopathy represents a new paradigm among lysosomal disorders. It results from a dysfunctional endo-lysosomal membrane protein causing altered pH homeostasis. Its pathophysiology implies intracellular accumulation of substrates whose composition remains unclear, and a combination of developmental brain abnormalities and neurodegenerative changes established during prenatal and early postanal development, whose severity is variably determined by specific pathogenic variants.

Consolidation of the clinical and genetic definition of a SOX4-related neurodevelopmental syndrome.

BACKGROUND: A neurodevelopmental syndrome was recently reported in four patients with SOX4 heterozygous missense variants in the high-mobility-group (HMG) DNA-binding domain. The present study aimed to consolidate clinical and genetic knowledge of this syndrome. METHODS: We newly identified 17 patients with SOX4 variants, predicted variant pathogenicity using in silico tests and in vitro functional assays and analysed the patients' phenotypes. RESULTS: All variants were novel, distinct and heterozygous. Seven HMG-domain missense and five stop-gain variants were classified as pathogenic or likely pathogenic variant (L/PV) as they precluded SOX4 transcriptional activity in vitro. Five HMG-domain and non-HMG-domain missense variants were classified as of uncertain significance (VUS) due to negative results from functional tests. When known, inheritance was de novo or from a mosaic unaffected or non-mosaic affected parent for patients with L/PV, and from a non-mosaic asymptomatic or affected parent for patients with VUS. All patients had neurodevelopmental, neurological and dysmorphic features, and at least one cardiovascular, ophthalmological, musculoskeletal or other somatic anomaly. Patients with L/PV were overall more affected than patients with VUS. They resembled patients with other neurodevelopmental diseases, including the SOX11-related and Coffin-Siris (CSS) syndromes, but lacked the most specific features of CSS. CONCLUSION: These findings consolidate evidence of a fairly non-specific neurodevelopmental syndrome due to SOX4 haploinsufficiency in neurogenesis and multiple other developmental processes.

SUFU haploinsufficiency causes a recognisable neurodevelopmental phenotype at the mild end of the Joubert syndrome spectrum.

BACKGROUND: Joubert syndrome (JS) is a recessively inherited ciliopathy characterised by congenital ocular motor apraxia (COMA), developmental delay (DD), intellectual disability, ataxia, multiorgan involvement, and a unique cerebellar and brainstem malformation. Over 40 JS-associated genes are known with a diagnostic yield of 60%-75%.In 2018, we reported homozygous hypomorphic missense variants of the SUFU gene in two families with mild JS. Recently, heterozygous truncating SUFU variants were identified in families with dominantly inherited COMA, occasionally associated with mild DD and subtle cerebellar anomalies. METHODS: We reanalysed next generation sequencing (NGS) data in two cohorts comprising 1097 probands referred for genetic testing of JS genes. RESULTS: Heterozygous truncating and splice-site SUFU variants were detected in 22 patients from 17 families (1.5%) with strong male prevalence (86%), and in 8 asymptomatic parents. Patients presented with COMA, hypotonia, ataxia and mild DD, and only a third manifested intellectual disability of variable severity. Brain MRI showed consistent findings characterised by vermis hypoplasia, superior cerebellar dysplasia and subtle-to-mild abnormalities of the superior cerebellar peduncles. The same pattern was observed in two out of three tested asymptomatic parents. CONCLUSION: Heterozygous truncating or splice-site SUFU variants cause a novel neurodevelopmental syndrome encompassing COMA and mild JS, which likely represent overlapping entities. Variants can arise de novo or be inherited from a healthy parent, representing the first cause of JS with dominant inheritance and reduced penetrance. Awareness of this condition will increase the diagnostic yield of JS genetic testing, and allow appropriate counselling about prognosis, medical monitoring and recurrence risk.

Expanding the phenotype of Wiedemann-Steiner syndrome: Craniovertebral junction anomalies.

Wiedemann-Steiner syndrome (WDSTS) is a rare autosomal dominant condition caused by heterozygous loss of function variants in the KMT2A (MLL) gene, encoding a lysine N-methyltransferase that mediates a histone methylation pattern specific for epigenetic transcriptional activation. WDSTS is characterized by a distinctive facial phenotype, hypertrichosis, short stature, developmental delay, intellectual disability, congenital malformations, and skeletal anomalies. Recently, a few patients have been reported having abnormal skeletal development of the cervical spine. Here we describe 11 such individuals, all with KMT2A de novo loss-of-function variants: 10 showed craniovertebral junction anomalies, while an 11th patient had a cervical abnormality in C7. By evaluating clinical and diagnostic imaging data we characterized these anomalies, which consist primarily of fused cervical vertebrae, C1 and C2 abnormalities, small foramen magnum and Chiari malformation type I. Craniovertebral anomalies in WDSTS patients have been largely disregarded so far, but the increasing number of reports suggests that they may be an intrinsic feature of this syndrome. Specific investigation strategies should be considered for early identification and prevention of craniovertebral junction complications in WDSTS patients.

The variability of SMARCA4-related Coffin-Siris syndrome: Do nonsense candidate variants add to milder phenotypes?

SMARCA4 encodes a central ATPase subunit in the BRG1-/BRM-associated factors (BAF) or polybromo-associated BAF (PBAF) complex in humans, which is responsible in part for chromatin remodeling and transcriptional regulation. Variants in this and other genes encoding BAF/PBAF complexes have been implicated in Coffin-Siris Syndrome, a multiple congenital anomaly syndrome classically characterized by learning and developmental differences, coarse facial features, hypertrichosis, and underdevelopment of the fifth digits/nails of the hands and feet. Individuals with SMARCA4 variants have been previously reported and appear to display a variable phenotype. We describe here a cohort of 15 unrelated individuals with SMARCA4 variants from the Coffin-Siris syndrome/BAF pathway disorders registry who further display variability in severity and degrees of learning impairment and health issues. Within this cohort, we also report two individuals with novel nonsense variants who appear to have a phenotype of milder learning/behavioral differences and no organ-system involvement.

Genotype-phenotype correlation at codon 1740 of SETD2.

The SET domain containing 2, histone lysine methyltransferase encoded by SETD2 is a dual-function methyltransferase for histones and microtubules and plays an important role for transcriptional regulation, genomic stability, and cytoskeletal functions. Specifically, SETD2 is associated with trimethylation of histone H3 at lysine 36 (H3K36me3) and methylation of α-tubulin at lysine 40. Heterozygous loss of function and missense variants have previously been described with Luscan-Lumish syndrome (LLS), which is characterized by overgrowth, neurodevelopmental features, and absence of overt congenital anomalies. We have identified 15 individuals with de novo variants in codon 1740 of SETD2 whose features differ from those with LLS. Group 1 consists of 12 individuals with heterozygous variant c.5218C>T p.(Arg1740Trp) and Group 2 consists of 3 individuals with heterozygous variant c.5219G>A p.(Arg1740Gln). The phenotype of Group 1 includes microcephaly, profound intellectual disability, congenital anomalies affecting several organ systems, and similar facial features. Individuals in Group 2 had moderate to severe intellectual disability, low normal head circumference, and absence of additional major congenital anomalies. While LLS is likely due to loss of function of SETD2, the clinical features seen in individuals with variants affecting codon 1740 are more severe suggesting an alternative mechanism, such as gain of function, effects on epigenetic regulation, or posttranslational modification of the cytoskeleton. Our report is a prime example of different mutations in the same gene causing diverging phenotypes and the features observed in Group 1 suggest a new clinically recognizable syndrome uniquely associated with the heterozygous variant c.5218C>T p.(Arg1740Trp) in SETD2.

Body Temperature Responses During Phases of Work in Human Remains Detection Dogs Undergoing a Simulated Deployment.

Body temperature responses were recorded during phases of work (waiting-to-work in close proximity to search site, active work in a search site, and post-work recovery crated in vehicle) in human remains detection dogs during search training. State or federally certified human remains detection dogs (n = 8) completed eight iterations of searching across multiple novel search environments to detect numerous scent sources including partial and complete, buried, hidden, or fully visible human remains. Internal temperature (Tgi) of the body was measured continuously using an ingestible thermistor in the gastrointestinal tract. Mean total phase times were: waiting-to-work: 9.17 min (±2.27); active work: 8:58 min (±2:49); and post-work recovery: 24:04 min (±10.59). Tgi was impacted by phase of work (p < 0.001) with a small increase during active work, with mean peak temperature 39.4 °C (±0.34 °C) during that period. Tgi continued to increase for a mean of 6:37 (±6:04) min into the post-work recovery phase in the handler's vehicle with a mean peak Tgi of 39.66 °C (±0.41 °C). No significant increase in temperature was measured during the waiting-to-work phase, suggesting behaviors typical of anticipation of work did not appear to contribute to overall body temperature increase during the waiting-to-work recovery cycle. Continued increase of gastrointestinal body temperature several minutes after cessation of exercise indicates that risk of heat injury does not immediately stop when the dog stops exercising, although none of the dogs in this study reached clinically concerning body temperatures or displayed any behavioral signs suggestive of pending heat injury. More work is needed to better understand the impact of vehicle crating on post-work recovery temperatures in dogs.

Trauma Management of Military Working Dogs.

There are about 2,500 war and military service dogs in service, with about 700 serving at any given time overseas. Military Working Dogs (MWDs) are critical assets for military police, special operations units, and others operating in today's combat environment. The expectation, given the significant combat multiplier impact of these dogs and the intense bond between the handler and dog, is that injured working dogs will receive the same level of care as any injured U.S. military personnel. Veterinary care is available at multiple locations throughout theater, and the veterinary healthcare team is the MWD's primary provider. Yet, human healthcare providers (HCPs) may be the only medical personnel available to MWDs that are gravely ill or injured. As most HCPs are unfamiliar with medical care of dogs, the Joint Trauma System published a Clinical Practice Guideline (CPG), a set of detailed clinical guidelines for managing life-threatening problems of MWDs encountered in combat operations. The CPG is available at the JTS website. This article is covers the most common urgent MWD care challenges HCPs may face.

Refining the phenotype associated with GNB1 mutations: Clinical data on 18 newly identified patients and review of the literature.

De novo germline mutations in GNB1 have been associated with a neurodevelopmental phenotype. To date, 28 patients with variants classified as pathogenic have been reported. We add 18 patients with de novo mutations to this cohort, including a patient with mosaicism for a GNB1 mutation who presented with a milder phenotype. Consistent with previous reports, developmental delay in these patients was moderate to severe, and more than half of the patients were non-ambulatory and nonverbal. The most observed substitution affects the p.Ile80 residue encoded in exon 6, with 28% of patients carrying a variant at this residue. Dystonia and growth delay were observed more frequently in patients carrying variants in this residue, suggesting a potential genotype-phenotype correlation. In the new cohort of 18 patients, 50% of males had genitourinary anomalies and 61% of patients had gastrointestinal anomalies, suggesting a possible association of these findings with variants in GNB1. In addition, cutaneous mastocytosis, reported once before in a patient with a GNB1 variant, was observed in three additional patients, providing further evidence for an association to GNB1. We will review clinical and molecular data of these new cases and all previously reported cases to further define the phenotype and establish possible genotype-phenotype correlations.

Causes of Death in Military Working Dogs During Operation Iraqi Freedom and Operation Enduring Freedom, 2001-2013.

BACKGROUND: Military working dogs (MWDs) are a major asset in the theater of operations. Their unique abilities make them ideal for tasks such as tracking, patrol, and scent detection. MWDs deployed to a war zone are exposed to harsh environments and battlefield dangers that increase their risk of disease, injuries, and death. Although canines have been used extensively in Operation Iraqi Freedom (OIF) and Operation Enduring Freedom (OEF), no published studies have reported detailed causes of death among MWDs deployed to these conflicts. MATERIALS AND METHODS: Potential cases were defined as U.S. military-owned MWDs that died while deployed in Iraq (OIF) or Afghanistan (OEF) from January 1, 2001 through December 31, 2013 and identified from both official sources and unofficial sources, that is, online searches. Cases included in this study were limited to MWDs with data on cause of death obtained by abstraction from official veterinary treatment records (VTRs) from the Department of Defense Military Working Dog Veterinary Service, Joint Base San Antonio-Lackland Air Force Base, San Antonio, Texas, and Special Operations Forces units. RESULTS: We identified 92 MWDs that died while deployed to OEF/OIF from 2001 through 2013 and had cause of death information from official VTRs. For both OEF and OIF, the most common training program was Multi-Purpose Canine (36.5% and 51.7%, respectively), followed by Improvised Explosive Detector Dog for OEF (34.9%) and Patrol Explosive Detector Dog for OIF (34.5%). Injuries were the primary cause of death for 77.2% of the MWDs for which we had cause of death data. The most frequent external injuries were gunshot wounds (GSW) (31.5%), explosion or blast (26.1%), and heat stress (9.8%). The proportion of deaths due to GSW was similar for OEF and OIF (30.2% vs. and 34.5%, respectively). However, a greater proportion of MWDs died from explosions during OEF than during OIF (30.2% vs. 17.2%, respectively). Diseases were the cause of death in 23.0% of the MWDs. The most common diseases were gastric dilation and volvulus (GDV, n = 3), pleuritis (n = 2), and sepsis (n = 3). Two deaths were associated with anesthesia-related medical procedures. A total of 8.7% of cases were missing cause of death, 8.7% were missing age, 32.6% of cases were missing data on necropsy, and 14.1% were missing data on final disposition of the body. Other variables of interest including number of deployments and duration of training had a very high proportion of missing values and thus could not be analyzed. CONCLUSIONS: Our study is the most comprehensive to date that reports causes of death of MWDs deployed to OIF and OEF. However, limitations in the available data lessen the potential of our results to inform improvements in training and point of injury medical care. Better documentation in VTRs and systematic data collection into an official MWD trauma registry could lead to improved training and facilitate further development and evaluation of guidelines to improve care of wounded MWDs in future conflicts.

Best practice recommendations for prehospital veterinary care of dogs and cats.

OBJECTIVE: To examine available evidence on prehospital care in human and veterinary trauma and emergency medicine and develop best practice guidelines for use by both paramedical and nonparamedical personnel in the approach to the prehospital care of dogs and cats. DESIGN: Systematic evaluation of the literature gathered via medical databases searches of Medline, CAB abstracts, and Google Scholar. SYNTHESIS: From a review and systematic evaluation of the available evidence, consensus guidelines on the approach to prehospital care of dogs and cats in 18 scenarios were developed. CONCLUSIONS: Due to the lack of current evidence in the veterinary prehospital arena, best practice guidelines were developed as an initial platform. Recommendations were based on a review of pertinent human and available veterinary literature as well as a consensus of the authors' professional opinions. It is anticipated that evidence-based additions will be made in the future.

POGZ truncating alleles cause syndromic intellectual disability.

BACKGROUND: Large-scale cohort-based whole exome sequencing of individuals with neurodevelopmental disorders (NDDs) has identified numerous novel candidate disease genes; however, detailed phenotypic information is often lacking in such studies. De novo mutations in pogo transposable element with zinc finger domain (POGZ) have been identified in six independent and diverse cohorts of individuals with NDDs ranging from autism spectrum disorder to developmental delay. METHODS: Whole exome sequencing was performed on five unrelated individuals. Sanger sequencing was used to validate variants and segregate mutations with the phenotype in available family members. RESULTS: We identified heterozygous truncating mutations in POGZ in five unrelated individuals, which were confirmed to be de novo or not present in available parental samples. Careful review of the phenotypes revealed shared features that included developmental delay, intellectual disability, hypotonia, behavioral abnormalities, and similar facial characteristics. Variable features included short stature, microcephaly, strabismus and hearing loss. CONCLUSIONS: While POGZ has been associated with neurodevelopmental disorders in large cohort studies, our data suggest that loss of function variants in POGZ lead to an identifiable syndrome of NDD with specific phenotypic traits. This study exemplifies the era of human reverse clinical genomics ushered in by large disease-directed cohort studies; first defining a new syndrome molecularly and, only subsequently, phenotypically.

Review of Canine Deaths While in Service in US Civilian Law Enforcement (2002-2012).

BACKGROUND: Working dogs have been proven effective in multiple military and law enforcement applications. Similar to their human counterparts, understanding mortality while still in service can help improve treatment of injuries, and improve equipment and training, to potentially reduce deaths. This is a retrospective study to characterize mortality of working dogs used in civilian law enforcement. METHODS: Reported causes of death were gathered from two working dog and law enforcement officer memorial websites. RESULTS: Of the 867 civilian law enforcement dogs reported to these memorial websites from 2002 to 2012 with reported causes of death while in service, the deaths of 318 were categorized as traumatic. The leading reported causes of traumatic death or euthanasia include trauma as a result of a vehicle strike, 25.8% (n=82); heatstroke, 24.8% (n=79); and penetrating ballistic trauma, 23.0% (n=73). CONCLUSION: Although the information gathered was from online sources, this study casts some light on the risks that civilian law enforcement dogs undergo as part of the tasks to which they are assigned. These data underscore the need for a comprehensive database for this specialized population of working dogs to provide the robust, reliable data needed to develop prevention and treatment strategies for this valuable resource.

Gunshot wounds in military working dogs in Operation Enduring Freedom and Operation Iraqi Freedom: 29 cases (2003-2009).

OBJECTIVE: To describe the patient population, injuries, and treatment received on the battlefield, and ultimate outcome of U.S. military working dogs that incurred gunshot wound (GSW) injury in Operation Enduring Freedom (Afghanistan) or Operation Iraqi Freedom (Iraq). DESIGN: Retrospective study between January 2003 and December 2009. ANIMALS: Twenty-nine military working dogs from the U.S. military with confirmed GSW injuries incurred in combat in Operation Enduring Freedom or Operation Iraqi Freedom. INTERVENTIONS: None. MEASUREMENTS AND MAIN RESULTS: Clinical data from battlefield treatment, which includes care from the point of injury through arrival to, but not including, a designated veterinary treatment facility. Twenty-nine dogs were injured between 2003 and 2009. All but one of the injuries were from high caliber, high velocity weapons. Of the 29 injured dogs, 11 survived the injuries and 18 died (38% survival rate). Of the dogs that died, all but 1 died from catastrophic nonsurvivable injuries before treatment or evacuation could be instituted. The thorax was the most common site of injury (50%) followed by extremity wounds (46%). The leading cause of death from GSWs was from thoracic wounds, followed by head wounds. Dogs with extremity wounds as their only injury were most likely to survive, and dogs with multiple injuries were least likely to survive. All surviving dogs received treatment at the point of injury by military medics and dog handlers consistent with Tactical Combat Casualty Care guidelines for combat injuries in human service members. Of the 11 that survived, all dogs returned to full duty with subsequent deployment to combat zones. Location of wounds and injury severity at the time of presentation to veterinary care was not correlated with length of time until return to duty.