Pharmacological inhibition of STING reduces neuroinflammation-mediated damage post-traumatic brain injury.

BACKGROUND AND PURPOSE: Traumatic brain injury (TBI) remains a major public health concern worldwide with unmet effective treatment. Stimulator of interferon genes (STING) and its downstream type-I interferon (IFN) signalling are now appreciated to be involved in TBI pathogenesis. Compelling evidence have shown that STING and type-I IFNs are key in mediating the detrimental neuroinflammatory response after TBI. Therefore, pharmacological inhibition of STING presents a viable therapeutic opportunity in combating the detrimental neuroinflammatory response after TBI. EXPERIMENTAL APPROACH: This study investigated the neuroprotective effects of the small-molecule STING inhibitor n-(4-iodophenyl)-5-nitrofuran-2-carboxamide (C-176) in the controlled cortical impact mouse model of TBI in 10- to 12-week-old male mice. Thirty minutes post-controlled cortical impact surgery, a single 750-nmol dose of C-176 or saline (vehicle) was administered intravenously. Analysis was conducted 2 h and 24 h post-TBI. KEY RESULTS: Mice administered C-176 had significantly smaller cortical lesion area when compared to vehicle-treated mice 24 h post-TBI. Quantitative temporal gait analysis conducted using DigiGait™ showed C-176 administration attenuated TBI-induced impairments in gait symmetry, stride frequency and forelimb stance width. C-176-treated mice displayed a significant reduction in striatal gene expression of pro-inflammatory cytokines Tnf-α, Il-1ß and Cxcl10 compared to their vehicle-treated counterparts 2 h post-TBI. CONCLUSION AND IMPLICATIONS: This study demonstrates the neuroprotective activity of C-176 in ameliorating acute neuroinflammation and preventing white matter neurodegeneration post-TBI. This study highlights the therapeutic potential of small-molecule inhibitors targeting STING for the treatment of trauma-induced inflammation and neuroprotective potential.

A novel 11 base pair deletion in KMT2C resulting in Kleefstra syndrome 2.

BACKGROUND: Haploinsufficiency of the Lysine Methyltransferase 2C (KMT2C) gene results in the autosomal dominant disorder, Kleefstra syndrome 2. It is an extremely rare neurodevelopmental condition, with 14 previous reports describing varied clinical manifestations including dysmorphic features, delayed psychomotor development and delayed growth. METHODS: Here, we describe a female with global developmental delay, attention deficit disorder, dyspraxia, short stature and subtle non-specific dysmorphic features. To identify causative mutations, whole exome sequencing was performed on the proband and her younger brother with discrete clinical presentation. RESULTS: Whole exome sequencing identified a novel de novo heterozygous 11 bp deletion in KMT2C (c.1759_1769del), resulting in a frameshift mutation and early termination of the protein (p.Gln587SerfsTer7). This variant is the second-most N-terminal reported mutation, located 4171 amino acids upstream of the critical enzymatically active SET domain (required for chromatin modification and histone methylation). CONCLUSION: The majority of the other reported mutations are frameshift mutations upstream of the SET domain and are predicted to result in protein truncation. It is thought that truncation of the SET domain, results functionally in an inability to modify chromatin through histone methylation. This report expands the clinical and genetic characterisation of Kleefstra syndrome 2.

Severe neonatal onset neuroregression with paroxysmal dystonia and apnoea: Expanding the phenotypic and genotypic spectrum of CARS2-related mitochondrial disease.

Disorders of mitochondrial function are a collectively common group of genetic diseases in which deficits in core mitochondrial translation machinery, including aminoacyl tRNA synthetases, are key players. Biallelic variants in the CARS2 gene (NM_024537.4), which encodes the mitochondrial aminoacyl-tRNA synthetase for cysteine (CARS2, mt-aaRScys; MIM*612800), result in childhood onset epileptic encephalopathy and complex movement disorder with combined oxidative phosphorylation deficiency (MIM#616672). Prior to this report, eight unique pathogenic variants in the CARS2 gene had been reported in seven individuals. Here, we describe a male who presented in the third week of life with apnoea. He rapidly deteriorated with paroxysmal dystonic crises and apnoea resulting in death at 16 weeks. He had no evidence of seizure activity or multisystem disease and had normal brain imaging. Skeletal muscle biopsy revealed a combined disorder of oxidative phosphorylation. Whole-exome sequencing identified biallelic variants in the CARS2 gene: one novel (c.1478T>C, p.Phe493Ser), and one previously reported (c.655G>A, p.Ala219Thr; rs727505361). Northern blot analysis of RNA isolated from the patient's fibroblasts confirmed a clear defect in aminoacylation of the mitochondrial tRNA for cysteine (mt-tRNACys). To our knowledge, this is the earliest reported case of CARS2 deficiency with severe, early onset dystonia and apnoea, without epilepsy.

Cochlear nerve deficiency in SOX11-related Coffin-Siris syndrome.

The phenotypic spectrum of SOX11-related Coffin-Siris syndrome (CSS) is expanding with reports of new associations. SOX11 is implicated in neurogenesis and inner ear development. Cochlear nerve deficiency, absence or hypoplasia, is commonly associated with cochlear canal stenosis or with CHARGE syndrome, a monogenic condition that affects inner ear development. SOX11 is a transcription factor essential for neuronal identity, highly correlated with the expression of CHD7, which regulates SOX11. We present two unrelated probands, each with novel de novo SOX11 likely pathogenic variants and phenotypic manifestations of CSS including global developmental delay, growth deficiency, and hypoplastic nails. They have unilateral sensorineural hearing loss due to cochlear nerve deficiency confirmed on MRI. SOX11 is implicated in sensory neuron survival and maturation. It is highly expressed in the developing inner ear. Homozygous ablation of SOX11 in a mouse model resulted in a reduction in sensory neuron survival and decreased axonal growth. A heterozygous knockout mice model had hearing impairment with grossly normal inner ear structures like the two probands reported. We propose cochlear nerve deficiency as a new phenotypic feature of SOX11-related CSS. Magnetic resonance imaging is useful in delineating the cochlear nerve deficiency and other CSS-related brain malformations.

Novel PRMT7 mutation in a rare case of dysmorphism and intellectual disability.

Protein arginine N-methyltransferase 7 (PRMT7) encodes an arginine methyltransferase central to a number of fundamental biological processes, mutations in which result in an autosomal recessive developmental disorder characterized by short stature, brachydactyly, intellectual developmental disability and seizures (SBIDDS). To date, fewer than 15 patients with biallelic mutations in PRMT7 have been documented. Here we report brothers from a consanguineous Iraqi family presenting with a developmental disorder characterized by global developmental delay, shortened stature, facial dysmorphisms, brachydactyly, and kidney dysfunction. In both affected brothers, whole genome sequencing (WGS) identified a novel homozygous substitution in PRMT7 (ENST00000339507.5), c.1097 G > A (p.Cys366Tyr), considered to account for the majority of the phenotypic presentation. Rare compound heterozygous mutations in the dysplasia-associated perlecan-encoding HSPG2 gene (ENST00000374695.3) were also found (c.10721-2dupA, p.Ser71Asn and c.212 G > A), potentially accounting for the kidney dysfunction. In addition to expanding the known mutational spectrum of variably expressive PRMT7 mutations alongside potential digenic inheritance with HSPG2, this report underlines the diagnostic utility of a WGS-guided analysis in the detection of rare genetic disorders.

Dominant monoallelic variant in the PAK2 gene causes Knobloch syndrome type 2.

Knobloch syndrome is an autosomal recessive phenotype mainly characterized by retinal detachment and encephalocele caused by biallelic pathogenic variants in the COL18A1 gene. However, there are patients clinically diagnosed as Knobloch syndrome with unknown molecular etiology not linked to COL18A1. We studied an historical pedigree (published in 1998) designated as KNO2 (Knobloch type 2 syndrome with intellectual disability, autistic behavior, retinal degeneration, encephalocele). Whole exome sequencing of the two affected siblings and the normal parents resulted in the identification of a PAK2 non-synonymous substitution p.(Glu435Lys) as a causative variant. The variant was monoallelic and apparently de novo in both siblings indicating a likely germ-line mosaicism in one of the parents; the mosaicism, however, could not be observed after deep sequencing of blood parental DNA. PAK2 encodes a member of a small group of serine/threonine kinases; these P21-activating kinases (PAKs) are essential in signal transduction and cellular regulation (cytoskeletal dynamics, cell motility, death and survival signaling and cell cycle progression). Structural analysis of the PAK2 p.(Glu435Lys) variant that is located in the kinase domain of the protein predicts a possible compromise in the kinase activity. Functional analysis of the p.(Glu435Lys) PAK2 variant in transfected HEK293T cells results in a partial loss of the kinase activity. PAK2 has been previously suggested as an autism-related gene. Our results show that PAK2-induced phenotypic spectrum is broad and not fully understood. We conclude that the KNO2 syndrome in the studied family is dominant and caused by a deleterious variant in the PAK2 gene.

The Complexity of the cGAS-STING Pathway in CNS Pathologies.

Neuroinflammation driven by type-I interferons in the CNS is well established to exacerbate the progression of many CNS pathologies both acute and chronic. The role of adaptor protein Stimulator of Interferon Genes (STING) is increasingly appreciated to instigate type-I IFN-mediated neuroinflammation. As an upstream regulator of type-I IFNs, STING modulation presents a novel therapeutic opportunity to mediate inflammation in the CNS. This review will detail the current knowledge of protective and detrimental STING activity in acute and chronic CNS pathologies and the current therapeutic avenues being explored.

DOORS syndrome and a recurrent truncating ATP6V1B2 variant.

PURPOSE: Biallelic variants in TBC1D24, which encodes a protein that regulates vesicular transport, are frequently identified in patients with DOORS (deafness, onychodystrophy, osteodystrophy, intellectual disability [previously referred to as mental retardation], and seizures) syndrome. The aim of the study was to identify a genetic cause in families with DOORS syndrome and without a TBC1D24 variant. METHODS: Exome or Sanger sequencing was performed in individuals with a clinical diagnosis of DOORS syndrome without TBC1D24 variants. RESULTS: We identified the same truncating variant in ATP6V1B2 (NM_001693.4:c.1516C>T; p.Arg506*) in nine individuals from eight unrelated families with DOORS syndrome. This variant was already reported in individuals with dominant deafness onychodystrophy (DDOD) syndrome. Deafness was present in all individuals, along with onychodystrophy and abnormal fingers and/or toes. All families but one had developmental delay or intellectual disability and five individuals had epilepsy. We also describe two additional families with DDOD syndrome in whom the same variant was found. CONCLUSION: We expand the phenotype associated with ATP6V1B2 and propose another causal gene for DOORS syndrome. This finding suggests that DDOD and DOORS syndromes might lie on a spectrum of clinically and molecularly related conditions.

STING-Mediated Autophagy Is Protective against H2O2-Induced Cell Death.

Stimulator of interferon genes (STING)-mediated type-I interferon signaling is a well characterized instigator of the innate immune response following bacterial or viral infections in the periphery. Emerging evidence has recently linked STING to various neuropathological conditions, however, both protective and deleterious effects of the pathway have been reported. Elevated oxidative stress, such as neuroinflammation, is a feature of a number of neuropathologies, therefore, this study investigated the role of the STING pathway in cell death induced by elevated oxidative stress. Here, we report that the H2O2-induced activation of the STING pathway is protective against cell death in wildtype (WT) MEFSV40 cells as compared to STING-/- MEF SV40 cells. This protective effect of STING can be attributed, in part, to an increase in autophagy flux with an increased LC3II/I ratio identified in H2O2-treated WT cells as compared to STING-/- cells. STING-/- cells also exhibited impaired autophagic flux as indicated by p62, LC3-II and LAMP2 accumulation following H2O2 treatment, suggestive of an impairment at the autophagosome-lysosomal fusion step. This indicates a previously unrecognized role for STING in maintaining efficient autophagy flux and protecting against H2O2-induced cell death. This finding supports a multifaceted role for the STING pathway in the underlying cellular mechanisms contributing to the pathogenesis of neurological disorders.

Abrogation of type-I interferon signalling alters the microglial response to Aß1-42.

Neuroinflammation and accompanying microglial dysfunction are now appreciated to be involved in Alzheimer's disease (AD) pathogenesis. Critical to the process of neuroinflammation are the type-I interferon (IFN) family of cytokines. Efforts to phenotypically characterize microglia within AD identify distinct populations associated with type-I IFN signalling, yet how this affects underlying microglial function is yet to be fully elucidated. Here we demonstrate that Aß1-42 exposure increases bioactive levels of type-I IFN produced by primary microglia alongside increased expression of type-I IFN related genes. Primary microglia isolated from brains of APPswePS1ΔE9 mice with ablated type-I IFN signalling show an increased phagocytic ability to uptake FITC-Aß1-42. Correlative assessment of plaque sizes in aged APPswePS1ΔE9 mice with abrogated type-I IFN signalling show unchanged deposition levels. Microglia from these mice did however show alterations in morphology. This data further highlights the role of type-I IFN signalling within microglia and identifies a role in phagocytosis. As such, targeting both microglial and global type-I IFN signalling presents as a novel therapeutic strategy for AD management.

The use of bioactive matrices in regenerative therapies for traumatic brain injury.

Functional deficits due to neuronal loss are a common theme across multiple neuropathologies, including traumatic brain injury (TBI). Apart from mitigating cell death, another approach to treating brain injuries involves re-establishing the neural circuitry at the lesion site by utilizing exogeneous and/or endogenous stem cells to achieve functional recovery. While there has been limited success, the emergence of new bioactive matrices that promote neural repair introduces new perspectives on the development of regenerative therapies for TBI. This review briefly discusses current development on cell-based therapies and the use of bioactive matrices, hydrogels in particular, when incorporated in regenerative therapies. Desirable characteristics of bioactive matrices that have been shown to augment neural repair in TBI models were identified and further discussed. Understanding the relative outcomes of newly developed biomaterials implanted in vivo can better guide the development of biomaterials as a therapeutic strategy, for biomaterial-based cellular therapies are still in their nascent stages. Nonetheless, the value of bioactive matrices as a treatment for acute brain injuries should be appreciated and further developed. STATEMENT OF SIGNIFICANCE: Cell-based therapies have received attention as an alternative therapeutic strategy to improve clinical outcome post-traumatic brain injury but have achieved limited success. Whilst the incorporation of newly developed biomaterials in regenerative therapies has shown promise in augmenting neural repair, studies have revealed new hurdles which must be overcome to improve their therapeutic efficacy. This review discusses the recent development of cell-based therapies with a specific focus on the use of bioactive matrices in the form of hydrogels, to complement cell transplantation within the injured brain. Moreover, this review consolidates in vivo animal studies that demonstrate relative functional outcome upon the implantation of different biomaterials to highlight their desirable traits to guide their development for regenerative therapies in traumatic brain injury.

9q34.3 microduplications lead to neurodevelopmental disorders through EHMT1 overexpression.

Both copy number losses and gains occur within subtelomeric 9q34 region without common breakpoints. The microdeletions cause Kleefstra syndrome (KS), whose responsible gene is EHMT1. A 9q34 duplication syndrome (9q34 DS) had been reported in literature, but it has never been characterized by a detailed molecular analysis of the gene content and endpoints. To the best of our knowledge, we report on the first patient carrying the smallest 9q34.3 duplication containing EHMT1 as the only relevant gene. We compared him with 21 reported patients described here as carrying 9q34.3 duplications encompassing the entire gene and extending within ~ 3 Mb. By surveying the available clinical and molecular cytogenetic data, we were able to discover that similar neurodevelopmental disorders (NDDs) were shared by patient carriers of even very differently sized duplications. Moreover, some facial features of the 9q34 DS were more represented than those of KS. However, an accurate in silico analysis of the genes mapped in all the duplications allowed us to support EHMT1 as being sufficient to cause a NDD phenotype. Wider patient cohorts are needed to ascertain whether the rearrangements have full causative role or simply confer the susceptibility to NDDs and possibly to identify the cognitive and behavioral profile associated with the increased dosage of EHMT1.

Inflammation in Traumatic Brain Injury: Roles for Toxic A1 Astrocytes and Microglial-Astrocytic Crosstalk.

Traumatic brain injury triggers neuroinflammation that may contribute to progressive neurodegeneration. We investigated patterns of recruitment of astrocytes and microglia to inflammation after brain trauma by firstly characterising expression profiles over time of marker genes following TBI, and secondly by monitoring glial morphologies reflecting inflammatory responses in a rat model of traumatic brain injury (i.e. the lateral fluid percussion injury). Gene expression profiles revealed early elevation of expression of astrocytic marker glial fibrillary acidic protein relative to microglial marker allograft inflammatory factor 1 (also known as ionized calcium-binding adapter molecule 1). Adult rat brains collected at day 7 after injury were processed for immunohistochemistry with allograft inflammatory factor 1, glial fibrillary acidic protein and complement C3 (marker of bad/disruptive astrocytic A1 phenotype). Astrocytes positive for glial fibrillary acidic protein and complement C3 were significant increased in the injured cortex and displayed more complex patterns of arbourisation with significantly increased bifurcations. Our observations suggested that traumatic brain injury changed the phenotype of microglia from a ramified appearance with long, thin, highly branched processes to a swollen amoeboid shape in the injured cortex. These findings suggest differential glial activation with astrocytes likely undergoing strategic changes in morphology and function. Whilst a detailed analysis is needed of temporal patterns of glial activation, ours is the first evidence of a role for the bad/disruptive astrocytic A1 phenotype in an open head model of traumatic brain injury.

The involvement of microglia in Alzheimer's disease: a new dog in the fight.

First described clinically in 1906, Alzheimer's disease (AD) is the most common neurodegenerative disease and form of dementia worldwide. Despite its prevalence, only five therapies are currently approved for AD, all dealing with the symptoms rather than the underlying causes of the disease. A multitude of experimental evidence has suggested that the once thought inconsequential process of neuroinflammation does, in fact, contribute to the AD pathogenesis. One such CNS cell type critical to this process are microglia. Plastic in nature with varied roles, microglia are emerging as key contributors to AD pathology. This review will focus on the role of microglia in the neuroinflammatory response in AD, highlighting recent studies implicating aberrant changes in microglial function in disease progression. Of critical note is that with these advances, a reconceptualization of the framework in which we view microglia is required. LINKED ARTICLES: This article is part of a themed section on Therapeutics for Dementia and Alzheimer's Disease: New Directions for Precision Medicine. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v176.18/issuetoc.

STING-mediated type-I interferons contribute to the neuroinflammatory process and detrimental effects following traumatic brain injury.

BACKGROUND: Traumatic brain injury (TBI) represents a major cause of disability and death worldwide with sustained neuroinflammation and autophagy dysfunction contributing to the cellular damage. Stimulator of interferon genes (STING)-induced type-I interferon (IFN) signalling is known to be essential in mounting the innate immune response against infections and cell injury in the periphery, but its role in the CNS remains unclear. We previously identified the type-I IFN pathway as a key mediator of neuroinflammation and neuronal cell death in TBI. However, the modulation of the type-I IFN and neuroinflammatory responses by STING and its contribution to autophagy and neuronal cell death after TBI has not been explored. METHODS: C57BL/6J wild-type (WT) and STING-/- mice (8-10-week-old males) were subjected to controlled cortical impact (CCI) surgery and brains analysed by QPCR, Western blot and immunohistochemical analyses at 2 h or 24 h. STING expression was also analysed by QPCR in post-mortem human brain samples. RESULTS: A significant upregulation in STING expression was identified in late trauma human brain samples that was confirmed in wild-type mice at 2 h and 24 h after CCI. This correlated with an elevated pro-inflammatory cytokine profile with increased TNF-α, IL-6, IL-1ß and type-I IFN (IFN-α and IFN-ß) levels. This expression was suppressed in the STING-/- mice with a smaller lesion volume in the knockout animals at 24 h post CCI. Wild-type mice also displayed increased levels of autophagy markers, LC3-II, p62 and LAMP2 after TBI; however, STING-/- mice showed reduced LAMP2 expression suggesting a role for STING in driving dysfunctional autophagy after TBI. CONCLUSION: Our data implicates a detrimental role for STING in mediating the TBI-induced neuroinflammatory response and autophagy dysfunction, potentially identifying a new therapeutic target for reducing cellular damage in TBI.

STAC3 variants cause a congenital myopathy with distinctive dysmorphic features and malignant hyperthermia susceptibility.

SH3 and cysteine-rich domain-containing protein 3 (STAC3) is an essential component of the skeletal muscle excitation-contraction coupling (ECC) machinery, though its role and function are not yet completely understood. Here, we report 18 patients carrying a homozygous p.(Trp284Ser) STAC3 variant in addition to a patient compound heterozygous for the p.(Trp284Ser) and a novel splice site change (c.997-1G > T). Clinical severity ranged from prenatal onset with severe features at birth, to a milder and slowly progressive congenital myopathy phenotype. A malignant hyperthermia (MH)-like reaction had occurred in several patients. The functional analysis demonstrated impaired ECC. In particular, KCl-induced membrane depolarization resulted in significantly reduced sarcoplasmic reticulum Ca2+ release. Co-immunoprecipitation of STAC3 with CaV 1.1 in patients and control muscle samples showed that the protein interaction between STAC3 and CaV 1.1 was not significantly affected by the STAC3 variants. This study demonstrates that STAC3 gene analysis should be included in the diagnostic work up of patients of any ethnicity presenting with congenital myopathy, in particular if a history of MH-like episodes is reported. While the precise pathomechanism remains to be elucidated, our functional characterization of STAC3 variants revealed that defective ECC is not a result of CaV 1.1 sarcolemma mislocalization or impaired STAC3-CaV 1.1 interaction.

Genetic variation affecting DNA methylation and the human imprinting disorder, Beckwith-Wiedemann syndrome.

BACKGROUND: Beckwith-Wiedemann syndrome (BWS) is an imprinting disorder with a population frequency of approximately 1 in 10,000. The most common epigenetic defect in BWS is a loss of methylation (LOM) at the 11p15.5 imprinting centre, KCNQ1OT1 TSS-DMR, and affects 50% of cases. We hypothesised that genetic factors linked to folate metabolism may play a role in BWS predisposition via effects on methylation maintenance at KCNQ1OT1 TSS-DMR. RESULTS: Single nucleotide variants (SNVs) in the folate pathway affecting methylenetetrahydrofolate reductase (MTHFR), methionine synthase reductase (MTRR), 5-methyltetrahydrofolate-homocysteine S-methyltransferase (MTR), cystathionine beta-synthase (CBS) and methionine adenosyltransferase (MAT1A) were examined in 55 BWS patients with KCNQ1OT1 TSS-DMR LOM and in 100 unaffected cases. MTHFR rs1801133: C>T was more prevalent in BWS with KCNQ1OT1 TSS-DMR LOM (p < 0.017); however, the relationship was not significant when the Bonferroni correction for multiple testing was applied (significance, p = 0.0036). None of the remaining 13 SNVs were significantly different in the two populations tested. The DNMT1 locus was screened in 53 BWS cases, and three rare missense variants were identified in each of three patients: rs138841970: C>T, rs150331990: A>G and rs757460628: G>A encoding NP_001124295 p.Arg136Cys, p.His1118Arg and p.Arg1223His, respectively. These variants have population frequencies of less than 1 in 1000 and were absent from 100 control cases. Functional characterization using a hemimethylated DNA trapping assay revealed a reduced methyltransferase activity relative to wild-type DNMT1 for each variant ranging from 40 to 70% reduction in activity. CONCLUSIONS: This study is the first to examine folate pathway genetics in BWS and to identify rare DNMT1 missense variants in affected individuals. Our data suggests that reduced DNMT1 activity could affect maintenance of methylation at KCNQ1OT1 TSS-DMR in some cases of BWS, possibly via a maternal effect in the early embryo. Larger cohort studies are warranted to further interrogate the relationship between impaired MTHFR enzymatic activity attributable to MTHFR rs1801133: C>T, dietary folate intake and BWS.

Generation and characterisation of a parkin-Pacrg knockout mouse line and a Pacrg knockout mouse line.

Mutations in PARK2 (parkin) can result in Parkinson's disease (PD). Parkin shares a bidirectional promoter with parkin coregulated gene (PACRG) and the transcriptional start sites are separated by only ~200 bp. Bidirectionally regulated genes have been shown to function in common biological pathways. Mice lacking parkin have largely failed to recapitulate the dopaminergic neuronal loss and movement impairments seen in individuals with parkin-mediated PD. We aimed to investigate the function of PACRG and test the hypothesis that parkin and PACRG function in a common pathway by generating and characterizing two novel knockout mouse lines harbouring loss of both parkin and Pacrg or Pacrg alone. Successful modification of the targeted allele was confirmed at the genomic, transcriptional and steady state protein levels for both genes. At 18-20 months of age, there were no significant differences in the behaviour of parental and mutant lines when assessed by openfield, rotarod and balance beam. Subsequent neuropathological examination suggested there was no gross abnormality of the dopaminergic system in the substantia nigra and no significant difference in the number of dopaminergic neurons in either knockout model compared to wildtype mice.

O-GlcNAc transferase missense mutations linked to X-linked intellectual disability deregulate genes involved in cell fate determination and signaling.

It is estimated that ∼1% of the world's population has intellectual disability, with males affected more often than females. OGT is an X-linked gene encoding for the enzyme O-GlcNAc transferase (OGT), which carries out the reversible addition of N-acetylglucosamine (GlcNAc) to Ser/Thr residues of its intracellular substrates. Three missense mutations in the tetratricopeptide (TPR) repeats of OGT have recently been reported to cause X-linked intellectual disability (XLID). Here, we report the discovery of two additional novel missense mutations (c.775 G>A, p.A259T, and c.1016 A>G, p.E339G) in the TPR domain of OGT that segregate with XLID in affected families. Characterization of all five of these XLID missense variants of OGT demonstrates modest declines in thermodynamic stability and/or activities of the variants. We engineered each of the mutations into a male human embryonic stem cell line using CRISPR/Cas9. Investigation of the global O-GlcNAc profile as well as OGT and O-GlcNAc hydrolase levels by Western blotting showed no gross changes in steady-state levels in the engineered lines. However, analyses of the differential transcriptomes of the OGT variant-expressing stem cells revealed shared deregulation of genes involved in cell fate determination and liver X receptor/retinoid X receptor signaling, which has been implicated in neuronal development. Thus, here we reveal two additional mutations encoding residues in the TPR regions of OGT that appear causal for XLID and provide evidence that the relatively stable and active TPR variants may share a common, unelucidated mechanism of altering gene expression profiles in human embryonic stem cells.