Insight into ALKBH8-related intellectual developmental disability based on the first pathogenic missense variant.

ALKBH8 is a methyltransferase that modifies tRNAs by methylating the anticodon wobble uridine residue. The syndrome of ALKBH8-related intellectual developmental disability (MRT71) has thus far been reported solely in the context of homozygous truncating variants that cluster in the last exon. This raises interesting questions about the disease mechanism, because these variants are predicted to escape nonsense mediated decay and yet they appear to be loss of function. Furthermore, the limited class of reported variants complicates the future interpretation of missense variants in ALKBH8. Here, we report a consanguineous family in which two children with MRT71-compatible phenotype are homozygous for a novel missense variant in the methyltransferase domain. We confirm the pathogenicity of this variant by demonstrating complete absence of ALKBH8-dependent modifications in patient cells. Targeted proteomics analysis of ALKBH8 indicates that the variant does not lead to loss of ALKBH8 protein expression. This report adds to the clinical delineation of MRT71, confirms loss of function of ALKBH8 as the disease mechanism and expands the repertoire of its molecular lesions.

APC7 mediates ubiquitin signaling in constitutive heterochromatin in the developing mammalian brain.

Neurodevelopmental cognitive disorders provide insights into mechanisms of human brain development. Here, we report an intellectual disability syndrome caused by the loss of APC7, a core component of the E3 ubiquitin ligase anaphase promoting complex (APC). In mechanistic studies, we uncover a critical role for APC7 during the recruitment and ubiquitination of APC substrates. In proteomics analyses of the brain from mice harboring the patient-specific APC7 mutation, we identify the chromatin-associated protein Ki-67 as an APC7-dependent substrate of the APC in neurons. Conditional knockout of the APC coactivator protein Cdh1, but not Cdc20, leads to the accumulation of Ki-67 protein in neurons in vivo, suggesting that APC7 is required for the function of Cdh1-APC in the brain. Deregulated neuronal Ki-67 upon APC7 loss localizes predominantly to constitutive heterochromatin. Our findings define an essential function for APC7 and Cdh1-APC in neuronal heterochromatin regulation, with implications for understanding human brain development and disease.

Intellectual-disability-associated mutations in the ceramide transport protein gene CERT1 lead to aberrant function and subcellular distribution.

The lipid molecule ceramide is transported from the endoplasmic reticulum to the Golgi apparatus for sphingomyelin production via the ceramide transport protein (CERT), encoded by CERT1. Hyperphosphorylation of CERT's serine-repeat motif (SRM) decreases its functionality. Some forms of inherited intellectual disability (ID) have been associated with a serine-to-leucine substitution in the SRM (S132L mutation) and a glycine-to-arginine substitution outside the SRM (G243R mutation) in CERT; however, it is unclear if mutations outside the SRM disrupt the control of CERT functionality. In the current investigation, we identified a new CERT1 variant (dupAA) in a patient with mild ID that resulted from a frameshift at the C-terminus of CERT1. However, familial analysis revealed that the dupAA variant was not associated with ID, allowing us to utilize it as a disease-matched negative control for CERT1 variants that are associated with ID. Biochemical analysis showed that G243R and S132L, but not dupAA, impair SRM hyperphosphorylation and render the CERT variants excessively active. Additionally, both S132L and G243R mutations but not dupAA caused the proteins to be distributed in a punctate subcellular manner. On the basis of these findings, we infer that the majority of ID-associated CERT variants may impair SRM phosphorylation-dependent repression, resulting in an increase in sphingomyelin production concurrent with CERT subcellular redistribution.

Protein Phosphatase 2A (PP2A) mutations in brain function, development, and neurologic disease.

By removing Ser/Thr-specific phosphorylations in a multitude of protein substrates in diverse tissues, Protein Phosphatase type 2A (PP2A) enzymes play essential regulatory roles in cellular signalling and physiology, including in brain function and development. Here, we review current knowledge on PP2A gene mutations causally involved in neurodevelopmental disorders and intellectual disability, focusing on PPP2CA, PPP2R1A and PPP2R5D. We provide insights into the impact of these mutations on PP2A structure, substrate specificity and potential function in neurobiology and brain development.

Noncanonical protein kinase A activation by oligomerization of regulatory subunits as revealed by inherited Carney complex mutations.

Familial mutations of the protein kinase A (PKA) R1α regulatory subunit lead to a generalized predisposition for a wide range of tumors, from pituitary adenomas to pancreatic and liver cancers, commonly referred to as Carney complex (CNC). CNC mutations are known to cause overactivation of PKA, but the molecular mechanisms underlying such kinase overactivity are not fully understood in the context of the canonical cAMP-dependent activation of PKA. Here, we show that oligomerization-induced sequestration of R1α from the catalytic subunit of PKA (C) is a viable mechanism of PKA activation that can explain the CNC phenotype. Our investigations focus on comparative analyses at the level of structure, unfolding, aggregation, and kinase inhibition profiles of wild-type (wt) PKA R1α, the A211D and G287W CNC mutants, as well as the cognate acrodysostosis type 1 (ACRDYS1) mutations A211T and G287E. The latter exhibit a phenotype opposite to CNC with suboptimal PKA activation compared with wt. Overall, our results show that CNC mutations not only perturb the classical cAMP-dependent allosteric activation pathway of PKA, but also amplify significantly more than the cognate ACRDYS1 mutations nonclassical and previously unappreciated activation pathways, such as oligomerization-induced losses of the PKA R1α inhibitory function.

Two intronic cis-acting variants in both alleles of the POLR3A gene cause progressive spastic ataxia with hypodontia.

POLR3A encodes the largest subunit of the DNA-dependent RNA polymerase III. Pathogenic variants in this gene are associated with dysregulation of tRNA production and other non-coding RNAs. POLR3A-related disorders include variable phenotypes. The genotype-phenotype correlation is still unclear. Phenotypic analysis and exome sequencing were performed in four affected siblings diagnosed clinically with hereditary spastic ataxia, two healthy siblings and their unaffected mother. All four affected siblings (ages 46-55) had similar clinical features of early childhood-onset hypodontia and adolescent-onset progressive spastic ataxia. None had progeria, gonadal dysfunction or dysmorphism. All affected individuals had biallelic POLR3A pathogenic variants composed by two cis-acting intronic splicing-altering variants, c.1909 + 22G > A and c.3337-11 T > C. The two healthy siblings had wild-type alleles. The mother and another unaffected sibling were heterozygous for the allele containing both variants. This is the first report addressing the clinical consequence associated with homozygosity for a unique pathogenic intronic allele in the POLR3A gene. This allele was previously reported in compound heterozygous combinations in patients with Wiedemann-Rautenstrauch syndrome, a severe progeroid POLR3A-associated phenotype. We show that homozygosity for this allele is associated with spastic ataxia with hypodontia, and not with progeroid features. These findings contribute to the characterization of genotype-phenotype correlation in POLR3A-related disorders.

Cardiac expression and location of hexokinase changes in a mouse model of pure creatine deficiency.

Creatine kinase (CK) is considered the main phosphotransfer system in the heart, important for overcoming diffusion restrictions and regulating mitochondrial respiration. It is substrate limited in creatine-deficient mice lacking l-arginine:glycine amidinotransferase (AGAT) or guanidinoacetate N-methyltranferase (GAMT). Our aim was to determine the expression, activity, and mitochondrial coupling of hexokinase (HK) and adenylate kinase (AK), as these represent alternative energy transfer systems. In permeabilized cardiomyocytes, we assessed how much endogenous ADP generated by HK, AK, or CK stimulated mitochondrial respiration and how much was channeled to mitochondria. In whole heart homogenates, and cytosolic and mitochondrial fractions, we measured the activities of AK, CK, and HK. Lastly, we assessed the expression of the major HK, AK, and CK isoforms. Overall, respiration stimulated by HK, AK, and CK was ∼25, 90, and 80%, respectively, of the maximal respiration rate, and ∼20, 0, and 25%, respectively, was channeled to the mitochondria. The activity, distribution, and expression of HK, AK, and CK did not change in GAMT knockout (KO) mice. In AGAT KO mice, we found no changes in AK, but we found a higher HK activity in the mitochondrial fraction, greater expression of HK I, but a lower stimulation of respiration by HK. Our findings suggest that mouse hearts depend less on phosphotransfer systems to facilitate ADP flux across the mitochondrial membrane. In AGAT KO mice, which are a model of pure creatine deficiency, the changes in HK may reflect changes in metabolism as well as influence mitochondrial regulation and reactive oxygen species production.NEW & NOTEWORTHY In creatine-deficient AGAT-/- and GAMT-/- mice, the myocardial creatine kinase system is substrate limited. It is unknown whether subcellular localization and mitochondrial ADP channeling by hexokinase and adenylate kinase may compensate as alternative phosphotransfer systems. Our results show no changes in adenylate kinase, which is the main alternative to creatine kinase in heart. However, we found increased expression and activity of hexokinase I in AGAT-/- cardiomyocytes. This could affect mitochondrial regulation and reactive oxygen species production.

Neonatal factors related to survival and intellectual and developmental outcome of patients with early-onset urea cycle disorders.

PURPOSE: We aimed to identify prognostic factors for survival and long-term intellectual and developmental outcome in neonatal patients with early-onset urea cycle disorders (UCD) experiencing hyperammonaemic coma. METHODS: We retrospectively analysed ammonia (NH3) and glutamine levels, electroencephalogram and brain images obtained during neonatal coma of UCD patients born between 1995 and 2011 and managed at a single centre and correlated them to survival and intellectual and developmental outcome. RESULTS: We included 38 neonates suffering from deficiencies of argininosuccinate synthetase (ASSD, N = 12), ornithine transcarbamylase (OTCD, N = 10), carbamoylphosphate synthetase 1 (CPSD, N = 7), argininosuccinate lyase (ASLD, N = 7), N-acetylglutamate synthase (NAGS, N = 1) or arginase (ARGD, N = 1). Symptoms occurred earlier in mitochondrial than in cytosolic UCD. Sixty-eight percent of patients survived, with a mean (standard deviation-SD) follow-up of 10.4 (5.3) years. Mortality was mostly observed in OTCD (N = 7/10) and CPSD (N = 4/7) patients. Plasma NH3 level during the neonatal period, expressed as area under the curve, but not glutamine level was associated with mortality (p = .044 and p = .610). 62.1% of the patients had normal intellectual and developmental outcome. Intellectual and developmental outcome tended to correlate with UCD subtype (p = .052). No difference in plasma NH3 or glutamine level during the neonatal period among developmental outcomes was identified. EEG severity was linked to UCD subtypes (p = .004), ammonia levels (p = .037), duration of coma (p = .043), and mortality during the neonatal period (p = .020). Status epilepticus was recorded in 6 patients, 3 of whom died neonatally, 1 developed a severe intellectual disability while the 2 last patients had a normal development. CONCLUSION: UCD subtypes differed by survival rate, intellectual and developmental outcome and EEG features in the neonatal period. Hyperammonaemia expressed as area under the curve was associated with survival but not with intellectual and developmental outcome whereas glutamine was not associated with one of these outcomes. Prognostic value of video-EEG monitoring and the association between status epilepticus and mortality should be assessed in neonatal hyperammonaemic coma in further studies.

A missense mutation in the catalytic domain of O-GlcNAc transferase links perturbations in protein O-GlcNAcylation to X-linked intellectual disability.

X-linked intellectual disabilities (XLID) are common developmental disorders. The enzyme O-GlcNAc transferase encoded by OGT, a recently discovered XLID gene, attaches O-GlcNAc to nuclear and cytoplasmic proteins. As few missense mutations have been described, it is unclear what the aetiology of the patient phenotypes is. Here, we report the discovery of a missense mutation in the catalytic domain of OGT in an XLID patient. X-ray crystallography reveals that this variant leads to structural rearrangements in the catalytic domain. The mutation reduces in vitro OGT activity on substrate peptides/protein. Mouse embryonic stem cells carrying the mutation reveal reduced O-GlcNAcase (OGA) and global O-GlcNAc levels. These data suggest a direct link between changes in the O-GlcNAcome and intellectual disability observed in patients carrying OGT mutations.

Lysine acetyltransferase 8 is involved in cerebral development and syndromic intellectual disability.

Epigenetic integrity is critical for many eukaryotic cellular processes. An important question is how different epigenetic regulators control development and influence disease. Lysine acetyltransferase 8 (KAT8) is critical for acetylation of histone H4 at lysine 16 (H4K16), an evolutionarily conserved epigenetic mark. It is unclear what roles KAT8 plays in cerebral development and human disease. Here, we report that cerebrum-specific knockout mice displayed cerebral hypoplasia in the neocortex and hippocampus, along with improper neural stem and progenitor cell (NSPC) development. Mutant cerebrocortical neuroepithelia exhibited faulty proliferation, aberrant neurogenesis, massive apoptosis, and scant H4K16 propionylation. Mutant NSPCs formed poor neurospheres, and pharmacological KAT8 inhibition abolished neurosphere formation. Moreover, we describe KAT8 variants in 9 patients with intellectual disability, seizures, autism, dysmorphisms, and other anomalies. The variants altered chromobarrel and catalytic domains of KAT8, thereby impairing nucleosomal H4K16 acetylation. Valproate was effective for treating epilepsy in at least 2 of the individuals. This study uncovers a critical role of KAT8 in cerebral and NSPC development, identifies 9 individuals with KAT8 variants, and links deficient H4K16 acylation directly to intellectual disability, epilepsy, and other developmental anomalies.

The Rac3 GTPase in Neuronal Development, Neurodevelopmental Disorders, and Cancer.

Rho family small guanosine triphosphatases (GTPases) are important regulators of the cytoskeleton, and are critical in many aspects of cellular and developmental biology, as well as in pathological processes such as intellectual disability and cancer. Of the three members of the family, Rac3 has a more restricted expression in normal tissues compared to the ubiquitous member of the family, Rac1. The Rac3 polypeptide is highly similar to Rac1, and orthologues of the gene for Rac3 have been found only in vertebrates, indicating the late appearance of this gene during evolution. Increasing evidence over the past few years indicates that Rac3 plays an important role in neuronal development and in tumor progression, with specificities that distinguish the functions of Rac3 from the established functions of Rac1 in these processes. Here, results highlighting the importance of Rac3 in distinct aspects of neuronal development and tumor cell biology are presented, in support of the non-redundant role of different members of the two Rac GTPases in physiological and pathological processes.

Glucose Phosphate Isomerase Deficiency: High Prevalence of p.Arg347His Mutation in Indian Population Associated with Severe Hereditary Non-Spherocytic Hemolytic Anemia Coupled with Neurological Dysfunction.

OBJECTIVES: Glucose-6-phosphate isomerase (GPI) deficiency is an autosomal recessive genetic disorder causing hereditary non-spherocytic hemolytic anemia (HNSHA) coupled with a neurological disorder. The aim of this study was to identify GPI genetic defects in a cohort of Indian patients with HNSHA coupled with neurological dysfunction. METHODS: Thirty-five patients were screened for GPI deficiency in the HNSHA patient group; some were having neurological dysfunction. Enzyme activity was measured by spectrophotometric method. The genetic study was done by single-stranded conformation polymorphism (SSCP) analysis, restriction fragment length polymorphism (RFLP) analysis by the restriction enzyme AciI for p.Arg347His (p.R347H) and confirmation by Sanger's sequencing. RESULTS: Out of 35 patients, 15 showed 35% to 70% loss of GPI activity, leading to neurological problems with HNSHA. Genetic analysis of PCR products of exon 12 of the GPI gene showed altered mobility on SSCP gel. Sanger's sequencing revealed a homozygous c1040G > A mutation predicting a p.Arg347His replacement which abolishes AciI restriction site. The molecular modeling analysis suggests p.Arg347 is involved in dimerization of the enzyme. Also, this mutation generates a more labile enzyme which alters its three-dimensional structure and function. CONCLUSIONS: This report describes the high prevalence of p.Arg347His pathogenic variant identified in Indian GPI deficient patients with hemolytic anemia and neuromuscular impairment. It suggests that neuromuscular impairment with hemolytic anemia cases could be investigated for p.Arg347His pathogenic variant causing GPI deficiency because of neuroleukin activity present in the GPI monomer which has neuroleukin action at the same active site and generates neuromuscular problems as well as hemolytic anemia.

ITPase deficiency causes a Martsolf-like syndrome with a lethal infantile dilated cardiomyopathy.

Typical Martsolf syndrome is characterized by congenital cataracts, postnatal microcephaly, developmental delay, hypotonia, short stature and biallelic hypomorphic mutations in either RAB3GAP1 or RAB3GAP2. Genetic analysis of 85 unrelated "mutation negative" probands with Martsolf or Martsolf-like syndromes identified two individuals with different homozygous null mutations in ITPA, the gene encoding inosine triphosphate pyrophosphatase (ITPase). Both probands were from multiplex families with a consistent, lethal and highly distinctive disorder; a Martsolf-like syndrome with infantile-onset dilated cardiomyopathy. Severe ITPase-deficiency has been previously reported with infantile epileptic encephalopathy (MIM 616647). ITPase acts to prevent incorporation of inosine bases (rI/dI) into RNA and DNA. In Itpa-null cells dI was undetectable in genomic DNA. dI could be identified at a low level in mtDNA without detectable mitochondrial genome instability, mtDNA depletion or biochemical dysfunction of the mitochondria. rI accumulation was detectable in proband-derived lymphoblastoid RNA. In Itpa-null mouse embryos rI was detectable in the brain and kidney with the highest level seen in the embryonic heart (rI at 1 in 385 bases). Transcriptome and proteome analysis in mutant cells revealed no major differences with controls. The rate of transcription and the total amount of cellular RNA also appeared normal. rI accumulation in RNA-and by implication rI production-correlates with the severity of organ dysfunction in ITPase deficiency but the basis of the cellulopathy remains cryptic. While we cannot exclude cumulative minor effects, there are no major anomalies in the production, processing, stability and/or translation of mRNA.

Level of residual enzyme activity modulates the phenotype in phosphoglycerate kinase deficiency.

OBJECTIVE: To study the variable clinical picture and exercise tolerance of patients with phosphoglycerate kinase (PGK) 1 deficiency and how it relates to residual PGK enzyme activity. METHODS: In this case series study, we evaluated 7 boys and men from 5 families with PGK1 deficiency. Five had pure muscle symptoms, while 2 also had mild intellectual disability with or without anemia. Muscle glycolytic and oxidative capacities were evaluated by an ischemic forearm exercise test and by cycle ergometry. RESULTS: Enzyme levels of PGK were 4% to 9% of normal in red cells and 5% to10% in muscle in pure myopathy patients and 2.6% in both muscle and red cells in the 2 patients with multisystem involvement. Patients with pure myopathy had greater increases in lactate with ischemic exercise (2-3 mmol/L) vs the 2 multisystem-affected patients (<1 mmol/L). Myopathy patients had higher oxidative capacity in cycle exercise vs multisystem affected patients (≈30 vs ≈15 mL/kg per minute). One multisystem-affected patient developed frank myoglobinuria after the short exercise test. CONCLUSIONS: This case series study of PGK1 deficiency suggests that the level of impaired glycolysis in PGK deficiency is a major determinant of phenotype. Lower glycolytic capacity in PGK1 deficiency seems to result in multisystem involvement and increased susceptibility to exertional rhabdomyolysis.

Pharmacological Inhibition of ERK Signaling Rescues Pathophysiology and Behavioral Phenotype Associated with 16p11.2 Chromosomal Deletion in Mice.

The human 16p11.2 microdeletion is one of the most common gene copy number variations linked to autism, but the pathophysiology associated with this chromosomal abnormality is largely unknown. The 593 kb deletion contains the ERK1 gene and other genes that converge onto the ERK/MAP kinase pathway. Perturbations in ERK signaling are linked to a group of related neurodevelopmental disorders hallmarked by intellectual disability, including autism. We report that mice harboring the 16p11.2 deletion exhibit a paradoxical elevation of ERK activity, cortical cytoarchitecture abnormalities and behavioral deficits. Importantly, we show that treatment with a novel ERK pathway inhibitor during a critical period of brain development rescues the molecular, anatomical and behavioral deficits in the 16p11.2 deletion mice. The ERK inhibitor treatment administered to adult mice ameliorates a subset of these behavioral deficits. Our findings provide evidence for potential targeted therapeutic intervention in 16p11.2 deletion carriers.SIGNIFICANCE STATEMENT The ERK/MAPK pathway is genetically linked to autism spectrum disorders and other syndromes typified by intellectual disability. We provide direct evidence connecting the ERK/MAP kinases to the developmental abnormalities in neurogenesis and cortical cytoarchitecture associated with the 16p11.2 chromosomal deletion. Most importantly, we demonstrate that treatment with a novel ERK-specific inhibitor during development rescues aberrant cortical cytoarchitecture and restores normal levels of cell-cycle regulators during cortical neurogenesis. These treatments partially reverse the behavioral deficits observed in the 16p11.2del mouse model, including hyperactivity, memory as well as olfaction, and maternal behavior. We also report a rescue of a subset of these deficits upon treatment of adult 16p11.2del mice. These data provide a strong rationale for therapeutic approaches to this disorder.

Molecular basis of Tousled-Like Kinase 2 activation.

Tousled-like kinases (TLKs) are required for genome stability and normal development in numerous organisms and have been implicated in breast cancer and intellectual disability. In humans, the similar TLK1 and TLK2 interact with each other and TLK activity enhances ASF1 histone binding and is inhibited by the DNA damage response, although the molecular mechanisms of TLK regulation remain unclear. Here we describe the crystal structure of the TLK2 kinase domain. We show that the coiled-coil domains mediate dimerization and are essential for activation through ordered autophosphorylation that promotes higher order oligomers that locally increase TLK2 activity. We show that TLK2 mutations involved in intellectual disability impair kinase activity, and the docking of several small-molecule inhibitors of TLK activity suggest that the crystal structure will be useful for guiding the rationale design of new inhibition strategies. Together our results provide insights into the structure and molecular regulation of the TLKs.

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.

OTUD7A Regulates Neurodevelopmental Phenotypes in the 15q13.3 Microdeletion Syndrome.

Copy-number variations (CNVs) are strong risk factors for neurodevelopmental and psychiatric disorders. The 15q13.3 microdeletion syndrome region contains up to ten genes and is associated with numerous conditions, including autism spectrum disorder (ASD), epilepsy, schizophrenia, and intellectual disability; however, the mechanisms underlying the pathogenesis of 15q13.3 microdeletion syndrome remain unknown. We combined whole-genome sequencing, human brain gene expression (proteome and transcriptome), and a mouse model with a syntenic heterozygous deletion (Df(h15q13)/+ mice) and determined that the microdeletion results in abnormal development of cortical dendritic spines and dendrite outgrowth. Analysis of large-scale genomic, transcriptomic, and proteomic data identified OTUD7A as a critical gene for brain function. OTUD7A was found to localize to dendritic and spine compartments in cortical neurons, and its reduced levels in Df(h15q13)/+ cortical neurons contributed to the dendritic spine and dendrite outgrowth deficits. Our results reveal OTUD7A as a major regulatory gene for 15q13.3 microdeletion syndrome phenotypes that contribute to the disease mechanism through abnormal cortical neuron morphological development.

Otud7a Knockout Mice Recapitulate Many Neurological Features of 15q13.3 Microdeletion Syndrome.

15q13.3 microdeletion syndrome is characterized by a wide spectrum of neurodevelopmental disorders, including developmental delay, intellectual disability, epilepsy, language impairment, abnormal behaviors, neuropsychiatric disorders, and hypotonia. This syndrome is caused by a deletion on chromosome 15q, which typically encompasses six genes. Here, through studies on OTU deubiquitinase 7A (Otud7a) knockout mice, we identify OTUD7A as a critical gene responsible for many of the cardinal phenotypes associated with 15q13.3 microdeletion syndrome. Otud7a-null mice show reduced body weight, developmental delay, abnormal electroencephalography patterns and seizures, reduced ultrasonic vocalizations, decreased grip strength, impaired motor learning/motor coordination, and reduced acoustic startle. We show that OTUD7A localizes to dendritic spines and that Otud7a-null mice have decreased dendritic spine density compared to their wild-type littermates. Furthermore, frequency of miniature excitatory postsynaptic currents (mEPSCs) is reduced in the frontal cortex of Otud7a-null mice, suggesting a role of Otud7a in regulation of dendritic spine density and glutamatergic synaptic transmission. Taken together, our results suggest decreased OTUD7A dosage as a major contributor to the neurodevelopmental phenotypes associated with 15q13.3 microdeletion syndrome, through the misregulation of dendritic spine density and activity.

Mutations causing acrodysostosis-2 facilitate activation of phosphodiesterase 4D3.

Type 2 acrodysostosis (ACRDYS2), a rare developmental skeletal dysplasia characterized by short stature, severe brachydactyly and facial dysostosis, is caused by mutations in the phosphodiesterase (PDE) 4D (PDE4D) gene. Several arguments suggest that the mutations should result in inappropriately increased PDE4D activity, however, no direct evidence supporting this hypothesis has been presented, and the functional consequences of the mutations remain unclear. We evaluated the impact of four different PDE4D mutations causing ACRDYS2 located in different functional domains on the activity of PDE4D3 expressed in Chinese hamster ovary cells. Three independent approaches were used: the direct measurement of PDE activity in cell lysates, the evaluation of intracellular cAMP levels using an EPAC-based (exchange factor directly activated by cAMP) bioluminescence resonance energy transfer sensor , and the assessment of PDE4D3 activation based on electrophoretic mobility. Our findings indicate that PDE4D3s carrying the ACRDYS2 mutations are more easily activated by protein kinase A-induced phosphorylation than WT PDE4D3. This occurs over a wide range of intracellular cAMP concentrations, including basal conditions, and result in increased hydrolytic activity. Our results provide new information concerning the mechanism whereby the mutations identified in the ACRDYS2 dysregulate PDE4D activity, and give insights into rare diseases involving the cAMP signaling pathway. These findings may offer new perspectives into the selection of specific PDE inhibitors and possible therapeutic intervention for these patients.