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Yun Ren,* Xiaona Luo,* Haiyan Tong, Simei Wang, Jinbin Yan, Longlong Lin, Yucai Chen Department of Neurology, Shanghai Children’s Hospital, School of Medicine, Shanghai JiaoTong University, Shanghai, People’s Republic of China*These authors contributed equally to this workCorrespondence: Yucai Chen, Email chenyc@shchildren.com.cnBackground: The IQ motif and Sec7 domain ArfGEF 2 (IQSEC2), an X-linked gene that encodes the BRAG1 protein, is a guanine nucleotide exchange factor for the ADP ribosylation factor (ARF) protein family in the small guanosine triphosphate (GTP) binding protein. Mutations in this gene result in disorders such as intellectual disability (ID) and epilepsy. In this study, we analyze the clinical features of two patients with IQSEC2-mutation-related disease and discuss their possible pathogenesis.Methods: The two patients were diagnosed with ID and epilepsy. Genetic testing was performed using whole-exome sequencing, and the three-dimensional protein structure was analyzed. UCSC Genome Browser was used to analyze the conservation of IQSEC2 in different species. We compared IQSEC2 expression in the proband families with that in a control group, as well as the expression of the postsynaptic identity protein 95 (PSD-95), synapse-associated protein 97 (SAP97), ADP ribosylation factor 6 (ARF-6), and insulin receptor substrate 53kDa (IRSP53) genes interacting with IQSEC2.Results: We identified two semi-zygote mutations located in conserved positions in different species: an unreported de novo mutation, C.3576C>A (p. Tyr1192*), and a known mutation, c.2983C>T (p. Arg995Trp). IQSEC2 mutations resulted in significant changes in the predicted three-dimensional protein structure, while its expression in the two probands was significantly lower than that in the age-matched control group, and IQSEC2 expression in proband 1 was lower than that in his family members. The expression levels of PSD-95, ARF-6, and SAP97, IRSP 53, which interact with IQSEC2, were also significantly different from those in the family members and age-matched healthy children.Conclusion: The clinical phenotype resulting from IQSEC2 mutations can be explained by the significant decrease in its expression, loss of function of the mutant protein, and change in the expression of related genes. Our results provide novel insights into the molecular phenotype conferred by the IQSEC2 variants.Keywords: IQSEC2, expression level, intellectual disability, infantile spasm, related genes

Recent insights into IQSEC2 disease are summarized in this review as follows: (1) Exome sequencing of IQSEC2 patient DNA has led to the identification of numerous missense mutations that delineate at least six and possibly seven essential functional domains present in the IQSEC2 gene. (2) Experiments using IQSEC2 transgenic and knockout (KO) mouse models have recapitulated the presence of autistic-like behavior and epileptic seizures in affected animals; however, seizure severity and etiology appear to vary considerably between models. (3) Studies in IQSEC2 KO mice reveal that IQSEC2 is involved in inhibitory as well as stimulatory neurotransmission. The overall picture appears to be that mutated or absent IQSEC2 arrests neuronal development, resulting in immature neuronal networks. Subsequent maturation is aberrant, leading to increased inhibition and reduced neuronal transmission. (4) The levels of Arf6-GTP remain constitutively high in IQSEC2 knockout mice despite the absence of IQSEC2 protein, indicating impaired regulation of the Arf6 guanine nucleotide exchange cycle. (5) A new therapy that has been shown to reduce the seizure burden for the IQSEC2 A350V mutation is heat treatment. Induction of the heat shock response may be responsible for this therapeutic effect. [ABSTRACT FROM AUTHOR]

Purposeful induction of fever for healing, including the treatment of epilepsy, was used over 2000 years ago by Hippocrates. More recently, fever has been demonstrated to rescue behavioral abnormalities in children with autism. However, the mechanism of fever benefit has remained elusive due in large part to the lack of appropriate human disease models recapitulating the fever effect. Pathological mutations in the IQSEC2 gene are frequently seen in children presenting with intellectual disability, autism and epilepsy. We recently described a murine A350V IQSEC2 disease model, which recapitulates important aspects of the human A350V IQSEC2 disease phenotype and the favorable response to a prolonged and sustained rise in body core temperature in a child with the mutation. Our goal has been to use this system to understand the mechanism of fever benefit and then develop drugs that can mimic this effect and reduce IQSEC2-associated morbidity. In this study, we first demonstrate a reduction in seizures in the mouse model following brief periods of heat therapy, similar to what was observed in a child with the mutation. We then show that brief heat therapy is associated with the correction of synaptic dysfunction in neuronal cultures of A350V mice, likely mediated by Arf6-GTP. [ABSTRACT FROM AUTHOR]

Pathogenic variants in IQ motif and SEC7 domain containing protein 2 (IQSEC2) gene cause a variety of neurodevelopmental disorders, with intellectual disability as a uniform feature. We report five cases, each with a novel missense variant in the pleckstrin homology (PH) domain of the IQSEC2 protein. Male patients all present with moderate to profound intellectual disability, significant delays or absent language and speech and variable seizures. We describe the phenotypic spectrum associated with missense variants in PH domain of IQSEC2, further delineating the genotype–phenotype correlation for this X‐linked gene. [ABSTRACT FROM AUTHOR]

Recent insights into IQSEC2 disease are summarized in this review as follows: (1) Exome sequencing of IQSEC2 patient DNA has led to the identification of numerous missense mutations that delineate at least six and possibly seven essential functional domains present in the IQSEC2 gene. (2) Experiments using IQSEC2 transgenic and knockout (KO) mouse models have recapitulated the presence of autistic-like behavior and epileptic seizures in affected animals; however, seizure severity and etiology appear to vary considerably between models. (3) Studies in IQSEC2 KO mice reveal that IQSEC2 is involved in inhibitory as well as stimulatory neurotransmission. The overall picture appears to be that mutated or absent IQSEC2 arrests neuronal development, resulting in immature neuronal networks. Subsequent maturation is aberrant, leading to increased inhibition and reduced neuronal transmission. (4) The levels of Arf6-GTP remain constitutively high in IQSEC2 knockout mice despite the absence of IQSEC2 protein, indicating impaired regulation of the Arf6 guanine nucleotide exchange cycle. (5) A new therapy that has been shown to reduce the seizure burden for the IQSEC2 A350V mutation is heat treatment. Induction of the heat shock response may be responsible for this therapeutic effect.

Purposeful induction of fever for healing, including the treatment of epilepsy, was used over 2000 years ago by Hippocrates. More recently, fever has been demonstrated to rescue behavioral abnormalities in children with autism. However, the mechanism of fever benefit has remained elusive due in large part to the lack of appropriate human disease models recapitulating the fever effect. Pathological mutations in the IQSEC2 gene are frequently seen in children presenting with intellectual disability, autism and epilepsy. We recently described a murine A350V IQSEC2 disease model, which recapitulates important aspects of the human A350V IQSEC2 disease phenotype and the favorable response to a prolonged and sustained rise in body core temperature in a child with the mutation. Our goal has been to use this system to understand the mechanism of fever benefit and then develop drugs that can mimic this effect and reduce IQSEC2-associated morbidity. In this study, we first demonstrate a reduction in seizures in the mouse model following brief periods of heat therapy, similar to what was observed in a child with the mutation. We then show that brief heat therapy is associated with the correction of synaptic dysfunction in neuronal cultures of A350V mice, likely mediated by Arf6-GTP.

Objectives Mutations in the human IQSEC2 gene are associated with drug-resistant epilepsy and severe behavioral dysfunction. We have focused on understanding one human IQSEC2 missense mutation (A350V) for which we have created a corresponding A350V IQSEC2 mouse model by CRISPR which demonstrates seizures when the mice are 15–20 days old and impaired social vocalizations in adulthood. We observed that a child with the A350V mutation stops having seizures when experiencing a fever of greater than 38 °C. In this study, we first sought to determine if we could recapitulate this phenomenon in A350V 15–20 day old mice using a previously established protocol to raise body temperature to 39 °C achieved by housing the mice at 37 °C. We then sought to determine if mice in whom seizure activity had been prevented as pups would develop social vocalization activity in adulthood. Methods 15–20 day old A350V male mice were housed either at 37 °C or 22 °C. Ultrasonic vocalizations of these mice were assessed at 8–10 weeks in response to a female stimulus. Results Housing of 15–20 day old A350V mice at 37 °C resulted in a reduction in lethal seizures to 2% (1/41) compared to 45% (48/108) in mice housed at 22 °C, p = 0.0001. Adult A350V mice who had been housed at 37 °C as pups displayed a significant improvement in the production of social vocalizations. Conclusion Raising the body temperature by raising the ambient temperature might provide a means to reduce seizures associated with the A350V IQSEC2 mutation and thereby allow for an improved neurodevelopmental trajectory.

A child with a A350V IQSEC2 missense mutation resulting in drug‐resistant epilepsy stops having seizures when he has a fever. We demonstrate that raising the body temperature of the child using a commercial Jacuzzi dramatically reduces his seizures and appears to improve his social behavioral interactions. [ABSTRACT FROM AUTHOR]

Abstract A child with a A350V IQSEC2 missense mutation resulting in drug‐resistant epilepsy stops having seizures when he has a fever. We demonstrate that raising the body temperature of the child using a commercial Jacuzzi dramatically reduces his seizures and appears to improve his social behavioral interactions.

IQSEC2 mutations are associated with IQSEC2‐related intellectual disability (ID). Phenotypic spectrum has been better defined in the last few years by the increasing number of reported cases although the genotype–phenotype relationship for IQSEC2 remains overall complex. As for IQSEC2‐related ID a wide phenotypic diversity has been described in Rett syndrome (RTT). Several patients harboring IQSEC2 mutations present with clinical symptoms similar to RTT and some cases meet most of the criteria for classic RTT. With the aim of establishing a genotype–phenotype correlation, we collected data of 16 patients harboring IQSEC2 point mutations (15 of them previously unreported) and of five novel patients carrying CNVs encompassing IQSEC2. Most of our patients surprisingly shared a moderate‐to‐mild phenotype. The similarities in the clinical course between our mild cases and patients with milder forms of atypical RTT reinforce the hypothesis that also IQSEC2 mutated patients may lay under the wide clinical spectrum of RTT and thus IQSEC2 should be considered in the differential diagnosis. Our data confirm that position, type of variant and gender are crucial for IQSEC2‐associated phenotype delineation. [ABSTRACT FROM AUTHOR]

Objectives Mutations in the human IQSEC2 gene are associated with drug-resistant epilepsy and severe behavioral dysfunction. We have focused on understanding one human IQSEC2 missense mutation (A350V) for which we have created a corresponding A350V IQSEC2 mouse model by CRISPR which demonstrates seizures when the mice are 15–20 days old and impaired social vocalizations in adulthood. We observed that a child with the A350V mutation stops having seizures when experiencing a fever of greater than 38 °C. In this study, we first sought to determine if we could recapitulate this phenomenon in A350V 15–20 day old mice using a previously established protocol to raise body temperature to 39 °C achieved by housing the mice at 37 °C. We then sought to determine if mice in whom seizure activity had been prevented as pups would develop social vocalization activity in adulthood. Methods 15–20 day old A350V male mice were housed either at 37 °C or 22 °C. Ultrasonic vocalizations of these mice were assessed at 8–10 weeks in response to a female stimulus. Results Housing of 15–20 day old A350V mice at 37 °C resulted in a reduction in lethal seizures to 2% (1/41) compared to 45% (48/108) in mice housed at 22 °C, p = 0.0001. Adult A350V mice who had been housed at 37 °C as pups displayed a significant improvement in the production of social vocalizations. Conclusion Raising the body temperature by raising the ambient temperature might provide a means to reduce seizures associated with the A350V IQSEC2 mutation and thereby allow for an improved neurodevelopmental trajectory. [ABSTRACT FROM AUTHOR]

IQSEC2 gene mutations are associated with epilepsy, autism, and intellectual disability. The primary function IQSEC2, mediated via its Sec 7 domain, is to act as a guanine nucleotide exchange factor for ARF6. We sought to develop a molecular model, which may explain the aberrant Sec 7 activity on ARF6 of different human IQSEC2 mutations. We integrated experimental data of IQSEC2 mutants with protein structure prediction by the RaptorX server combined with molecular modeling and molecular dynamics simulations. Normally, apocalmodulin (apoCM) binds to IQSEC2 resulting in its N-terminal fragment inhibiting access of its Sec 7 domain to ARF6. An increase in Ca2+ concentration destabilizes the interaction of IQSEC2 with apoCM and removes steric hindrance of Sec 7 binding with ARF6. Mutations at amino acid residue 350 of IQSEC2 result in loss of steric hindrance of Sec 7 binding with ARF6 leading to constitutive activation of ARF6 by Sec 7. On the other hand, a mutation at amino acid residue 359 of IQSEC2 results in constitutive hindrance of Sec 7 binding to ARF6 leading to the loss of the ability of IQSEC2 to activate ARF6. These studies provide a model for dysregulation of IQSEC2 Sec 7 activity by mutant IQSEC2 proteins.Communicated by Ramaswamy H. Sarma.

Mutations in the X-linked gene IQSEC2 are associated with multiple cases of epilepsy, epileptic encephalopathy, intellectual disability and autism spectrum disorder, the mechanistic understanding and successful treatment of which remain a significant challenge in IQSEC2 and related neurodevelopmental genetic diseases. To investigate disease etiology, we studied behaviors and synaptic function in IQSEC2 deficient mice. Hemizygous Iqsec2 null males exhibit growth deficits, hyperambulation and hyperanxiety phenotypes. Adult hemizygotes experience lethal spontaneous seizures, but paradoxically have a significantly increased threshold to electrically induced limbic seizures and relative resistance to chemically induced seizures. Although there are no gross defects in brain morphology, hemizygotes exhibit stark hippocampal reactive astrogliosis. Electrophysiological recordings of hippocampal neurons reveal increased excitatory drive specifically onto interneurons, and significant alterations in intrinsic electrical properties specific to the interneuron population. As they age, hemizygotes also develop an increased abundance of parvalbumin-positive interneurons in the hippocampus, neurons in which IQSEC2 is expressed in addition to the excitatory neurons. These findings point to a novel role of IQSEC2 in hippocampal interneuron synaptic function and development with implications for a class of intractable neurodevelopmental diseases.

Aim: Lennox–Gastaut syndrome (LGS) is a severe type of childhood-onset epilepsy with multiple types of seizures, specific discharges on electroencephalography, and intellectual disability. However, LGS-related genes are largely unknown. To identify causative genes related to LGS, we collected and analyzed data from a three-generation Korean family in which one member had LGS and two had intellectual disability. Methods: Genomic DNAs were extracted from blood samples of all participants and used in whole-exome sequencing (WES). Genetic variants were detected by the Genome Analysis Toolkit and confirmed by Sanger sequencing. Variant pathogenicity was evaluated by prediction programs and the American College of Medical Genetics criteria. The LGS patient had generalized slow spike-and-wave discharges, multiple types of seizures, and developmental delay. Results: Analyses of the WES data from the family revealed a novel variant (c.1048G>A, p.Ala350Thr) in the IQ motif and Sec7 domain 2 (IQSEC2). This variant is within a highly evolutionarily conserved IQ-like motif, indicating a decrease in the calmodulin-binding capacity or α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid transmission. The hemizygous variant in the male with LGS was a maternally inherited X-linked variant from the heterozygous maternal grandmother and mother, both of whom had intellectual disability. Conclusion: These findings indicate that the variant of IQSEC2 triggered both LGS and intellectual disability dependent on sex in this family. We report a novel X-linked inherited IQSEC2 variant for LGS and intellectual disability, which enhances the spectrum of variants in the IQ-like motif of IQSEC2. [ABSTRACT FROM AUTHOR]

IQSEC2 is a guanine nucleotide exchange factor (GEF) for ADP-ribosylation factor 6 (Arf6), of which protein is exclusively localized to the postsynaptic density of the excitatory synapse. Human genome studies have revealed that the IQSEC2 gene is associated with X-linked neurodevelopmental disorders, such as intellectual disability (ID), epilepsy, and autism. In this study, we examined the behavior and synapse function in IQSEC2 knockout (KO) mice that we generated using CRIPSR/Cas9-mediated genome editing to solve the relevance between IQSEC2 deficiency and the pathophysiology of neurodevelopmental disorders. IQSEC2 KO mice exhibited autistic behaviors, such as overgrooming and social deficits. We identified that up-regulation of c-Fos expression in the medial prefrontal cortex (mPFC) induced by social stimulation was significantly attenuated in IQSEC2 KO mice. Whole cell electrophysiological recording identified that synaptic transmissions mediated by α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPAR), N-methyl-D-aspartate receptor (NMDAR), and γ-aminobutyric acid receptor (GABAR) were significantly decreased in pyramidal neurons in layer 5 of the mPFC in IQSEC2 KO mice. Reexpression of IQSEC2 isoform 1 in the mPFC of IQSEC2 KO mice using adeno-associated virus (AAV) rescued both synaptic and social deficits, suggesting that impaired synaptic function in the mPFC is responsible for social deficits in IQSEC2 KO mice.

We have recently described an A350V mutation in IQSEC2 associated with intellectual disability, autism and epilepsy. We sought to understand the molecular pathophysiology of this mutation with the goal of developing targets for drug intervention. We demonstrate here that the A350V mutation results in interference with the binding of apocalmodulin to the IQ domain of IQSEC2. We further demonstrate that this mutation results in constitutive activation of the guanine nucleotide exchange factor (GEF) activity of IQSEC2 resulting in increased production of the active form of Arf6. In a CRISPR generated mouse model of the A350V IQSEC2 mutation, we demonstrate that the surface expression of GluA2 AMPA receptors in mouse hippocampal tissue was significantly reduced in A350V IQSEC2 mutant mice compared to wild type IQSEC2 mice and that there is a significant reduction in basal synaptic transmission in the hippocampus of A350V IQSEC2 mice compared to wild type IQSEC2 mice. Finally, the A350V IQSEC2 mice demonstrated increased activity, abnormal social behavior and learning as compared to wild type IQSEC2 mice. These findings suggest a model of how the A350V mutation in IQSEC2 may mediate disease with implications for targets for drug therapy. These studies provide a paradigm for a personalized approach to precision therapy for a disease that heretofore has no therapy.

Whole‐exome sequencing has established IQSEC2 as a neurodevelopmental disability gene. The IQSEC2 variant phenotype includes developmental delay, intellectual disability, epilepsy, hypotonia, autism, developmental regression, microcephaly and stereotypies but is yet to be fully described. Presented here are 14 new patients with IQSEC2 variants. In addition to the established features, we observed: gait ataxia in 7 of 9 (77.8%), drooling in 9 of 14 (64.2%), early feeding difficulties in 7 of 14 (50%), structural brain abnormalities in 6 of 13 (46.2%), brachycephaly in 5 of 14 (35.7%), and scoliosis and paroxysms of laughter each in 4 of 14 (28.6%). We suggest that these are features of the IQSEC2‐related disorder. Gastrostomy requirement, plagiocephaly, strabismus and cortical blindness, each seen in 2 of 14 (14.3%), may also be associated. Shared facial features were noted in 8 of 14 patients, and shared hair patterning was identified in 5 of 14 patients. This study further delineates the IQSEC2 phenotypic spectrum and supports the notion of an emerging IQSEC2 syndrome. We draw parallels between the IQSEC2‐related disorder and the Angelman‐/Rett‐/Pitt‐Hopkins syndrome group of conditions and recommend the addition of IQSEC2 to epilepsy and developmental delay gene panels. We observed discordant phenotypes in monozygotic twins and apparent gonadal mosaicism, which has implications for recurrence risk counselling in the IQSEC2‐related disorder. Features found in patients with IQSEC2 variants. [ABSTRACT FROM AUTHOR]

We have recently described an A350V mutation in IQSEC2 associated with intellectual disability, autism and epilepsy. We sought to understand the molecular pathophysiology of this mutation with the goal of developing targets for drug intervention. We demonstrate here that the A350V mutation results in interference with the binding of apocalmodulin to the IQ domain of IQSEC2. We further demonstrate that this mutation results in constitutive activation of the guanine nucleotide exchange factor (GEF) activity of IQSEC2 resulting in increased production of the active form of Arf6. In a CRISPR generated mouse model of the A350V IQSEC2 mutation, we demonstrate that the surface expression of GluA2 AMPA receptors in mouse hippocampal tissue was significantly reduced in A350V IQSEC2 mutant mice compared to wild type IQSEC2 mice and that there is a significant reduction in basal synaptic transmission in the hippocampus of A350V IQSEC2 mice compared to wild type IQSEC2 mice. Finally, the A350V IQSEC2 mice demonstrated increased activity, abnormal social behavior and learning as compared to wild type IQSEC2 mice. These findings suggest a model of how the A350V mutation in IQSEC2 may mediate disease with implications for targets for drug therapy. These studies provide a paradigm for a personalized approach to precision therapy for a disease that heretofore has no therapy. [ABSTRACT FROM AUTHOR]

The IQSEC2‐ related disorders represent a spectrum of X‐chromosome phenotypes with intellectual disability (ID) as the cardinal feature. Here, we review the increasing number of reported families and isolated cases have been reported with a variety of different pathogenic variants. The spectrum of clinical features is expanding with early‐onset seizures as a frequent comorbidity in both affected male and female patients. There is a growing number of female patients with de novo loss‐of‐function variants in IQSEC2 have a more severe phenotype than the heterozygous state would predict, particularly if IQSEC2 is thought to escape X‐inactivation. Interestingly, these findings highlight that the classical understanding of X‐linked inheritance does not readily explain the emergence of these affected females, warranting further investigations into the underlying mechanisms. IQSEC2‐ related disorders represent a spectrum of X‐chromosome phenotypes of intellectual disability, more recently expanding with early‐onset seizures as a frequent comorbidity in both affected male and female patients. There is a growing number of female patients with de novo loss‐of‐function variants in IQSEC2 have a more severe phenotype than the heterozygous state would predict, particularly if IQSEC2 is thought to escape X‐inactivation. The classical understanding of X‐linked inheritance does not readily explain the emergence of these affected females. [ABSTRACT FROM AUTHOR]

Key Clinical Message The neurodevelopmental progression of a school-aged child with a spontaneous IQSEC2 mutation has demonstrated apparent regression of milestones and language. Seizures associated with the disorder have been refractory to medical treatment. Late treatment of autism in this child has led to improved social skills. [ABSTRACT FROM AUTHOR]

While X-linked intellectual disability (XLID) syndromes pose a diagnostic challenge for clinicians, an increasing number of recognized disorders and their genetic etiologies are providing answers for patients and their families. The availability of clinical exome sequencing is broadening the ability to identify mutations in genes previously unrecognized as causing XLID. In recent years, the IQSEC2 gene, located at Xp11.22, has emerged as the cause of multiple cases of both nonsyndromic and syndromic XLID. Herein we present a case series of six individuals (five males, one female) with intellectual disability and seizures found to have alterations in IQSEC2. In all cases, the diagnostic odyssey was extensive and expensive, often including invasive testing such as muscle biopsies, before ultimately reaching the diagnosis. We report these cases to demonstrate the exhaustive work-up prior to finding the changes in IQSEC2 gene, recommend that this gene be considered earlier in the diagnostic evaluation of individuals with global developmental delay, microcephaly, and severe, intractable epilepsy, and support the use of intellectual disability panels including IQSEC2 in the first-line evaluation of these patients. [ABSTRACT FROM AUTHOR]

Several genes have been implicated in Rett syndrome (RTT) in its typical and variant forms. We applied next-generation sequencing (NGS) to evaluate for mutations in known or new candidate genes in patients with variant forms of Rett or Rett-like phenotypes of unknown molecular aetiology. In the first step, we used NGS with a custom panel including MECP2, CDKL5, FOXG1, MEF2C and IQSEC2. In addition to a FOXG1 mutation in a patient with all core features of the congenital variant of RTT, we identified a missense (p. Ser240Thr) in CDKL5 in a patient who appeared to be seizure free. This missense was maternally inherited with opposite allele expression ratios in the proband and her mother. In the asymptomatic mother, the mutated copy of the CDKL5 gene was inactivated in 90% of blood cells. We also identified a premature stop codon (p.Arg926*) in IQSEC2 in a patient with a Rett-like phenotype. Finally, exome sequencing enabled us to characterize a heterozygous de novo missense (p. Val408Ala) in KCNA2 encoding the potassium channel Kv 1.2 in a girl with infantile-onset seizures variant of RTT. Our study expands the genetic heterogeneity of RTT and RTT-like phenotypes. Moreover, we report the first familial case of CDKL5-related disease. [ABSTRACT FROM AUTHOR]

Disease gene discovery in neurodevelopmental disorders, including X-linked intellectual disability (XLID) has recently been accelerated by next-generation DNA sequencing approaches. To date, more than 100 human X chromosome genes involved in neuronal signaling pathways and networks implicated in cognitive function have been identified. Despite these advances, the mutations underlying disease in a large number of XLID families remained unresolved. We report the resolution of MRX78, a large family with six affected males and seven affected females, showing X-linked inheritance. Although a previous linkage study had mapped the locus to the short arm of chromosome X (Xp11.4-p11.23), this region contained too many candidate genes to be analyzed using conventional approaches. However, our X-chromosome exome resequencing, bioinformatics analysis and inheritance testing revealed a missense mutation (c.C2366T, p.A789V) in IQSEC2, encoding a neuronal GDP-GTP exchange factor for Arf family GTPases (ArfGEF) previously implicated in XLID. Molecular modeling of IQSEC2 revealed that the A789V substitution results in the insertion of a larger side-chain into a hydrophobic pocket in the catalytic Sec7 domain of IQSEC2. The A789V change is predicted to result in numerous clashes with adjacent amino acids and disruption of local folding of the Sec7 domain. Consistent with this finding, functional assays revealed that recombinant IQSEC2A789V was not able to catalyze GDP-GTP exchange on Arf6 as efficiently as wild-type IQSEC2. Taken together, these results strongly suggest that the A789V mutation in IQSEC2 is the underlying cause of XLID in the MRX78 family.

The article presents a study on the novel missense mutation A789V in IQSEC2 that underlies X-linked intellectual disability in the MRX78 gene family.

Intellectual disability (ID) is a very heterogeneous disorder with over 100 ID genes located on the X chromosome alone. Of these, KDM5C and IQSEC2 are located adjacent to each other at the Xp11.22 locus. While mutations in either of these genes are associated with severe ID in males, female carriers are mostly unaffected. Here, we report on a female patient with severe ID and autistic features carrying a de novo 0.4 Mb deletion containing six coding genes including KDM5C and IQSEC2 . X-inactivation analysis revealed skewing in a lymphocyte-derived cell line from this patient with preferential inactivation of the mutant X chromosome. As the brain-expressed KDM5C and IQSEC2 genes escape X-inactivation, deletion of these alleles could still be detrimental despite skewing of X-inactivation. Indeed, mutations in either of both genes have been reported in a few female ID patients. Expression analysis in the patients' cell line revealed decreased KDM5C mRNA levels compared to female controls. IQSEC2 levels could not be compared due to very low expression in blood. Overall, our data suggest that heterozygous loss-of-function of the escape genes KDM5C and/or IQSEC2 can contribute to severe ID in female patients and should be taken into account in diagnostics. [ABSTRACT FROM AUTHOR]

IQSEC2 is an X-linked gene localized to the post synaptic density encoding a GTP exchange factor that regulates NMDA mediated changes in synaptic function. Mutations in the IQSEC2 gene are associated with drug resistant epilepsy, intellectual disability and autism. Precision medicine based therapeutics to treat IQSEC2 associated epilepsy requires the development and characterization of mutation specific animal models. To date no EEG recordings have been presented for any mouse model of any IQSEC2 mutation showing seizures. In this study we characterize the seizures and EEG brain wave abnormalities present in mice with a A350V IQSEC2 missense mutation that is associated with drug resistant epilepsy in man. We show that seizures are associated with a greater than 40% mortality rate in male mice and occur exclusively from post-natal day 16–20. EEG recordings of mouse pups during this window demonstrate seizures and the presence of spikes with a marked increase in delta waves. EEG recordings in adult male mice have persistent excessive slow frequency activity and spikes, but seizures were not recorded. RNAseq analysis of the hippocampi of mice prior to the development of seizures demonstrated marked abnormalities in canonical pathways involved in synaptogenesis and dendritic maturation with the most prominently dysregulated gene being that for TRH suggesting a potential target for therapy given the previous demonstration of TRH to decrease seizures in several forms of drug resistant epilepsy. • First EEG based documentation of seizures in mice with a IQSEC2 mutation. • Marked abnormalities in delta waves consistent with cognitive dysfunction. • RNAseq data identifies dysfunction in pathways as targets for precision therapy. [ABSTRACT FROM AUTHOR]

Intellectual disability is a heterogeneous disorder with a wide phenotypic spectrum. Over 1,700 OMIM genes have been associated with this condition, many of which reside on the X-chromosome. The IQSEC2 gene is located on chromosome Xp11.22 and is known to play a significant role in the maintenance and homeostasis of the brain. Mutations in IQSEC2 have been historically associated with nonsyndromic X-linked intellectual disability. Case reports of affected probands show phenotypic overlap with conditions associated with pathogenic MECP2, FOXG1, CDKL5, and MEF2C gene mutations. Affected individuals, however, have also been identified as presenting with additional clinical features including seizures, autistic-behavior, psychiatric problems, and delayed language skills. To our knowledge, only 5 deleterious mutations and 2 intragenic duplications have been previously reported in IQSEC2. Here we report two novel IQSEC2 de novo truncating mutations identified through diagnostic exome sequencing in two severely affected unrelated male probands manifesting developmental delay, seizures, hypotonia, plagiocephaly, and abnormal MRI findings. Overall, diagnostic exome sequencing established a molecular diagnosis for two patients in whom traditional testing methods were uninformative while expanding on the mutational and phenotypic spectrum. In addition, our data suggests that IQSEC2 may be more common than previously appreciated, accounting for approximately 9 % (2/22) of positive findings among patients with seizures referred for diagnostic exome sequencing. Further, these data supports recently published data suggesting that IQSEC2 plays a more significant role in the development of X-linked intellectual disability with seizures than previously anticipated. [ABSTRACT FROM AUTHOR]

IQSEC2 is a guanine nucleotide exchange factor (GEF) for ADP-ribosylation factor 6 (Arf6), of which protein is exclusively localized to the postsynaptic density of the excitatory synapse. Human genome studies have revealed that the IQSEC2 gene is associated with X-linked neurodevelopmental disorders, such as intellectual disability (ID), epilepsy, and autism. In this study, we examined the behavior and synapse function in IQSEC2 knockout (KO) mice that we generated using CRIPSR/Cas9-mediated genome editing to solve the relevance between IQSEC2 deficiency and the pathophysiology of neurodevelopmental disorders. IQSEC2 KO mice exhibited autistic behaviors, such as overgrooming and social deficits. We identified that up-regulation of c-Fos expression in the medial prefrontal cortex (mPFC) induced by social stimulation was significantly attenuated in IQSEC2 KO mice. Whole cell electrophysiological recording identified that synaptic transmissions mediated by α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPAR), N-methyl-D-aspartate receptor (NMDAR), and γ-aminobutyric acid receptor (GABAR) were significantly decreased in pyramidal neurons in layer 5 of the mPFC in IQSEC2 KO mice. Reexpression of IQSEC2 isoform 1 in the mPFC of IQSEC2 KO mice using adeno-associated virus (AAV) rescued both synaptic and social deficits, suggesting that impaired synaptic function in the mPFC is responsible for social deficits in IQSEC2 KO mice. [ABSTRACT FROM AUTHOR]

Mutations in the X-linked gene IQSEC2 are associated with multiple cases of epilepsy, epileptic encephalopathy, intellectual disability and autism spectrum disorder, the mechanistic understanding and successful treatment of which remain a significant challenge in IQSEC2 and related neurodevelopmental genetic diseases. To investigate disease etiology, we studied behaviors and synaptic function in IQSEC2 deficient mice. Hemizygous Iqsec2 null males exhibit growth deficits, hyperambulation and hyperanxiety phenotypes. Adult hemizygotes experience lethal spontaneous seizures, but paradoxically have a significantly increased threshold to electrically induced limbic seizures and relative resistance to chemically induced seizures. Although there are no gross defects in brain morphology, hemizygotes exhibit stark hippocampal reactive astrogliosis. Electrophysiological recordings of hippocampal neurons reveal increased excitatory drive specifically onto interneurons, and significant alterations in intrinsic electrical properties specific to the interneuron population. As they age, hemizygotes also develop an increased abundance of parvalbumin-positive interneurons in the hippocampus, neurons in which IQSEC2 is expressed in addition to the excitatory neurons. These findings point to a novel role of IQSEC2 in hippocampal interneuron synaptic function and development with implications for a class of intractable neurodevelopmental diseases. • Iqsec2 knockout mice display growth and adult behavior deficits. • Iqsec2 knockout mice exhibit increased adulthood mortality due to lethal seizure but are relatively resistant to induced seizures. • IQSEC2 expression is strongest in the hippocampus and loss results in hippocampus specific gliosis. • IQSEC2 loss results in aberrant excitatory input onto interneurons and in altered intrinsic properties of interneurons. • IQSEC2 is expressed in parvalbumin interneurons, and IQSEC2 loss results in an increase in parvalbumin interneurons in adult hippocampus. [ABSTRACT FROM AUTHOR]

Pathogenic variants in the IQSEC2 gene including nonsense, frameshift, splice-alterations, deletions, and missense changes have been identified in individuals with X-linked mental retardation. Although highly variable, clinical features may include hypotonia, moderate to severe delayed psychomotor development, intellectual disability, speech deficits, refractory seizures, autistic features, and stereotypical movements. Females with de novo variants have been described with classical features. In contrast, the phenotype in carrier females identified through an affected male may range from asymptomatic to mild intellectual disability. We present male (N = 2) and female (N = 3) probands ascertained via diagnostic exome sequencing with distinct variant types in the IQSEC2 gene encompassing a spectrum of phenotypic severity with patient sex, variant type and inheritance hypothesized to drive disease penetrance and expressivity. All of these patients demonstrated epilepsy, global developmental delays, intellectual disability, and constipation. Our data support that de novo , truncating variants correlate with severe disease in both female and male patients harboring an IQSEC2 alteration. Missense variants in male and female patients may account for a milder disease overall, with more severe symptoms in males than females. We also present the first confirmed case of parental mosaicism, which has implications regarding counseling for recurrence risk. These data further delineate a genotype-phenotype correlation of IQSEC2 variation. [ABSTRACT FROM AUTHOR]

X-linked intellectual disability (XLID) is known to contribute up to 10% of intellectual disability (ID) in males and could explain the increased ratio of affected males observed in patients with ID. Over the past decade, next-generation sequencing has clearly stimulated the gene discovery process and has become part of the diagnostic procedure. We have performed targeted next-generation sequencing of 82 XLID genes on 61 non-related male patients with suggestive non-syndromic XLID. These patients were initially referred to the molecular genetics laboratory to exclude Fragile X Syndrome. The cohort includes 47 male patients with suggestive X-linked family history of ID meaning that they had half-brothers or maternal cousins or uncles affected; and 14 male patients with ID and affected brothers whose mothers show skewed X-inactivation. Sequencing data analysis identified 17 candidate variants in 16 patients. Seven families could be re-contacted and variant segregation analysis of the respective eight candidate variants was performed: HUWE1 , IQSEC2 , MAOA , MED12 , PHF8 , SLC6A8 , SLC9A6 , and SYN1 . Our results show the utility of targeted next-generation sequencing in unravelling the genetic origin of XLID, especially in retrospective cases. Variant segregation and additional studies like RNA sequencing and biochemical assays also helped in re-evaluating and further classifying the genetic variants found., Competing Interests: The authors declare no conflict of interest.