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Therefore, we wanted to further investigate possible effects of VPA on the KV3.2 channel.
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Therefore, an analysis on a model system with a respective genetic background (e.g., iPSC-derived neurons) from both the healthy mother and the affected patient would be beneficial to possibly better explain the phenotype.
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However, the phenotypic spectrum and genotype-phenotype correlation of GABRA1 remain undetermined.
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We could demonstrate that also variants of uncertain significance in KCNC2 classified by the ACMG criteria can have a significant effect on the channel function and therefore be associated to different epilepsy phenotypes.
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It is still unknown whether UBE3A overexpression alone is sufficient to cause these phenotypes.
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This suggest an (secondary) effect of ANKB effect on sodium channel function in the AIS, but more research would be necessary to understand how ANKB haploinsufficiency affects ion channels in these patients.Variants in ANK2 were initially associated with non-syndromic ASD (4–10).
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We specifically addressed the question of how loss of ANKB neurons affects the neuronal network
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These findings suggested a potential molecular subregional effect, which warrants a larger case-cohort study to further determine.
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The family history of the other variant with unclear mode of inheritance, N530H, was negative for the mother (gDNA of unaffected father not available; father deceased).
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The challenge in modifying variants with incomplete penetrance is to understand the complex interplay of genetic and environmental factors that determine an individual’s phenotype.
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It has been speculated that the effect on the NSD of gANKB might underlie the phenotypic difference with the missense variants that cause cardiac arrhythmia (16,17)
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These results support a role for other non-imprinted duplicated genes in the development of these deficits, and suggest that one extra copy of the non-imprinted genes in the PatDup neurons may be insufficient to replicate the synaptic and input resistance phenotypes seen in idic Dup15q neurons that have 2 extra copies ...
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Future studies may explore the therapeutic potential of modulating these neuronal network characteristics.
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On the basis of a limited number of individual reports typically from genome-sequencing studies, ANK2 has been proposed as a candidate susceptibility gene for autism spectrum disorder (ASD) (4–10).
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We used these lines to determine the functional differences between Dup15q and corrected neurons and investigate the role of UBE3A in Dup15q cellular phenotypes.
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AbstractPurposeTo characterize a novel neurodevelopmental syndrome due to loss-of-function (LoF) variants in Ankyrin 2 (ANK2), and to explore the effects on neuronal network dynamics and homeostatic plasticity in human-induced pluripotent stem cell-derived neurons.
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The data presented here support that KCNC2 variants with uncertain significance may also be causative for various forms of epilepsy, as they show changes in the current amplitude and activation and deactivation kinetics of the channel, depending on the variant.
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Here, we describe that also variants of uncertain significance in KCNC2 may also be the underlying genetic etiology for different epilepsy syndromes.
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However, if AIS plasticity is important as a homeostatic mechanism for neuronal excitability, how do ANKB-deficient neurons fine-tune their excitability according to ongoing levels of input activity?
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Nevertheless, for many variants, the functional testing in respective in vitro or in vivo systems is not available.
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We did not include axonal analysis in our ANKB-deficient neurons, but it would be useful to investigate whether LoF variants targeting both 220-kDa and 440-kDa isoforms would affect axonal structures as shown in other studies (18,34).
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Disentangling the specific contribution of AIS structure to excitability changes is therefore challenging.
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It is possible that a longer treatment time may be required to reverse these phenotypes in mature neurons, or that optimal connectivity achieved by co-culture with astrocytes or growth as organoids may better support phenotypic rescue.
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One critical question for Dup15q is whether a single gene drives the cellular and patient phenotypes.
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Although these models might be valid for studying the cellular and behavioral effects of Ube3a overexpression, they fail to encompass the full range of severe Dup15q phenotypes and replicate the complex genetics of the disease.
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Unfortunately, we could not explain the beneficial effect of VPA in several described patients by an effect of the drug on the KV3.2 channel.
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It is possible that the increased AIS structure in ANK2+/− iNeurons is a compensation effect to the increased neuronal excitability.
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Moreover, epileptic children with Dup15q have an increased risk for sudden unexpected death in epilepsy (SUDEP) (Friedman et al., 2016), therefore, understanding the cellular and molecular mechanisms that lead to seizure generation is of great importance.
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Future experiments will determine if redistribution of voltage-gated ion channels on the AIS plays a role in fine-tuning neuronal excitability in ANKB-deficient neurons.
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To better understand how ANKB affects neuronal network activity, we analyzed the effect of heterozygous loss of ANKB on somatodendritic morphology and on the structure and location of the AIS.
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To establish whether UBE3A overexpression is required for Dup15q neuronal deficits, we generated an isogenic control line for a Dup15q patient-derived induced pluripotent stem cell line.
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In Patient 6, we cannot exclude an additive effect of the maternally inherited ALG13 variant.
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Therefore, our data suggest a role for 220-kDa ANKB in limiting dendritic networks through a mechanism that still needs to be identified.
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To explore whether UBE3A overexpression was sufficient to cause Dup15q cellular phenotypes, we differentiated neurons from an iPSC line derived from an individual with a paternal interstitial duplication.
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However, this model has poor face validity as there were no behavioral phenotypes with maternal duplication, whereas paternal duplication resulted in subtle autism-like phenotypes (Nakatani et al., 2009).
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Satterstrom et al. have reported six protein truncating variants (PTVs), four missense variants and two synonymous (15), whereas further phenotypic data were not specified.
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Further studies will provide a better understanding whether this compensation mechanism applies during AIS plasticity.
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We further explored the effects of heterozygous LoF variants targeting both 220-kDa and 440-kDa isoforms of the ANKB protein in a human model for heterozygous isogenic ANKB-deficient neurons.
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Future studies to identify these additional gene(s) and investigate how excess UBE3A and/or other genes cause the neuronal pathophysiology may open the door for the discovery of new therapeutic approaches and guide research efforts toward creating a more comprehensive animal model that will better encompass Dup15q beha...
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Further studies are needed to clarify the potential effect of this variant on channel localization and the interaction with Ankyrin G as well as the T1 domain which is present in all KV2, KV 3, and KV 4 channels and is required for tetramerization of KV subunits within the same subfamily (15).
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Because 220-kDa ANKB also localizes to dendrites, it is important to note that the authors found no particular role for 220-kDa ANKB in organelle transport in dendrites, further suggesting a role for 220-kDa ANKB in the development of dendrites.
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Nevertheless, the contribution of other duplicated genes in the region to Dup15q phenotypes is unknown.
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The other two variants tested here were declared as likely pathogenic because the family history of the patient with the I465V variant showed febrile seizures in the father and in the first grade cousin, which might suggest that the variant is inherited, but we could not prove this further due to the unavailability of ...
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Nonetheless, even if duplication of the non-imprinted genes contributes to the cellular phenotypes, our data suggest that normalization of UBE3A alone may ameliorate the majority of neuronal phenotypes in our human in vitro model system.
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Our iPSC model system is not ideal for teasing out these differences.
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In this work, we mainly wanted to test if variants in KCNC2 that were defined as variants of uncertain significance (VUS) according to the ACMG criteria, also show a functional effect and can be associated to the epilepsy phenotype.
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Future research is necessary to distinguish the differences between nonsyndromic ASD and the wider range of NDDs.
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Although the mechanism of action remains poorly understood, VPA has been used for decades in the treatment of epilepsy (17).
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Again, more complex model systems such as iPSC-derived neurons from affected patients with appropriate expression of different ion channels may be needed to elucidate the underlying mechanism.
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The similar, but still not exactly the identical, pathophysiological effects could be due to adjacent spatial proximity of the two variants, as predicted by Alphafold (12, 13), and therefore lead to similar functional changes in the channel upon replacement of the corresponding amino acid.
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This isogenic pair enabled us to investigate the role of UBE3A, a gene thought to play a major role in the disorder, in the development of Dup15q cellular phenotypes.
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The goal of this study was to determine the role of UBE3A overexpression in Dup15q neuronal phenotypes.
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The generation of animal models for Dup15q has been difficult.
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Therefore, we investigated one variant which was inherited from the healthy mother from a patient with DEE (S333T), one de novo variant from a patient with MAE (F382C) and two variants with unknown mode of inheritance from patients with focal epilepsy (I465V) and GGE (N530H).
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Unravelling the pathways linking fine-tuning mechanisms for neuronal excitability to impaired AIS plasticity in ANKB-deficient neurons could uncover potential new targets for epilepsy treatment, where fine-scale control over neuronal excitability represents a major therapeutic challenge for ANK2-related patients with I...
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Nevertheless, the exact role of UBE3A has not been determined.
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This suggests that therapeutics that target UBE3A might influence neuronal excitability and potentially alter the disease course of individuals with Dup15q.
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Hence, the use of induced pluripotent stem cell (iPSC)-derived neurons from Dup15q patients represents an excellent first step to study the cellular mechanisms of the disease and identify the relevant genes that contribute to the disease phenotypes.
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Experimental in vitro data of ANK2+/−-derived cultures on MEAs showed an increased neuronal network excitability, suggesting that the ALG13 variant would not contribute to the increased network activity.
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We considered that another possible mechanism by which variants in ANKB affect ASD risk could be through its role in the determination of length and location of the axon initial segment (AIS) (18).
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In the preprint of Fu et al., variant counts and phenotypic data are not accessible.
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Therefore, effects on other proteins are likely to play a more important role in achieving seizure freedom in patients.
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These results indicate that UBE3A overexpression is necessary for most Dup15q cellular phenotypes but also suggest a role for other genes in the duplicated region.
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UBE3A, which encodes an E3 ubiquitin ligase, is likely a major driver of Dup15q because UBE3A is the only imprinted gene expressed solely from the maternal allele.
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The presumed LoF in the context of the heart could be different in the brain, yet the effect of one variant has not been studied in both the heart and the brain.
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However, the observed hyperactive neuronal network could also be explained by several other factors that co-occur with changes in excitability.
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Therefore, it is not possible to evaluate the phenotype–genotype relationship.
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It is unknown whether modifier genes or polygenic interactions also partially contribute to phenotypic heterogeneity.
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Normalizing UBE3A expression at 16 weeks failed to fully reverse many phenotypes in our experimental paradigm.
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Additionally, we were interested in the effect of valproic acid, which was demonstrated to be particularly effective in some of the KCNC2 cases described previously (9).
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Overexpression of UBE3A is likely a major driver of Dup15q phenotypes.
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This account highlights the challenges in diagnosing and managing primary Coenzyme Q10 deficiency, especially when it presents later in life with atypical features such as stroke-like episodes.
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These data may suggest that ANKB primarily localizes sodium channels to the soma to compensate for loss of dendritic sodium channels.
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Animal models of UBE3A overexpression have been used to study the syndrome, but with limited success in recapitulating the full range of Dup15q phenotypes (Smith et al., 2011; Copping et al., 2017).
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The extra genetic material in Dup15q includes approximately 20 genes (Germain et al., 2014), and active research is devoted toward understanding and identifying the genes that contribute to phenotypes associated with the disorder.
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Animal models of UBE3A overexpression have been used to study the syndrome, but with limited success in recapitulating the full range of Dup15q phenotypes (Smith et al., 2011; Copping et al., 2017). Maternal duplication of the syntenic mouse region also failed to replicate behavioral and physiological phenotypes (Nakat...
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It is worth noting that, in Angelman syndrome model mice, the loss of UBE3A in GABAergic neurons has been shown to be a principal cause of circuit hyperexcitability (Judson et al., 2016), thus it will be important in future studies to explore the specific contribution of UBE3A overexpression in inhibitory neurons to ch...
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Previously, we described 18 patients harboring pathogenic variants or variants of uncertain significance in the KCNC2 gene but the functional proof of the latter has not been performed yet (9).
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We sought to determine how LoF variants in ANK2 affect neuronal network activity in human neurons derived from human-induced pluripotent stem cells (hiPSCs).
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Using these lines, we normalized UBE3A levels with antisense oligonucleotides (ASOs) in Dup15q neurons to determine whether UBE3A overexpression is necessary for establishing cellular phenotypes.
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However, in our electrophysiological investigations, no change on the behavior of KV3.2 channels could be observed, suggesting that the therapeutic effect of VPA may be explained by other mechanisms.
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We have functionally investigated four more of these variants of unclear significance to show their effect in different phenotypes (MAE) and to analyze their potential pathogenic effect (DEE, GGE and FE).
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Nevertheless, the construct validity of this model came into question, as the C terminal of Ube3a was tagged with a FLAG tag that affected the catalytic function of the protein.
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However, there may also be specific therapeutic windows for particular phenotypes.
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Here, we report the functional characteristics of three additional KCNC2 variants of uncertain significance and one variant classified as pathogenic.
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We also tested one pathogenic variant that has not functionally characterized before.
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In daily work, one major problem in the interpretation of exome sequencing is that many variants of unknown relevance for the phenotype are detected.
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However, some phenotypes, most notably synaptic phenotypes and input resistance, were not completely dependent on UBE3A overexpression, suggesting an important role for other genes in the duplicated region.
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