Episodic Ataxia Research Articles

Postural control in episodic ataxia type 2: no evidence for increased vestibular excitability.

Patients with episodic ataxia type 2 (EA2) suffer from recurrent paroxysmal episodes of vertigo and oscillopsia. Pathophysiologically, altered neuronal excitability has been suspected. Vestibular excitability in 22 EA2 patients and 22 age-matched healthy participants was compared. Galvanic vestibular stimulation (GVS) was used to assess vestibular excitability by vestibular motion perception thresholds and mean postural sway velocity during various visual and proprioceptive conditions in the two groups. Control stimuli using sham and no GVS were established to identify the specificity of GVS-induced postural sway. In the baseline condition, EA2 patients showed larger postural instability. However, motion perception thresholds and the increase in mean postural sway velocity during vestibular stimulation (stimulation ratio) did not differ between groups. Postural sway during suprathreshold GVS increased with the vestibular motion perception threshold in EA2 patients, in contrast to healthy participants. The larger postural unsteadiness of EA2 patients probably reflects their progressive cerebellar degeneration. It is not related to abnormal visual (Romberg's ratio) or proprioceptive control of stance. Postural unsteadiness during vestibular stimulation does not indicate altered vestibular excitability in EA2 patients. However, vestibular stimulation increasingly destabilized postural control of EA2 patients with higher motion perception thresholds when proprioceptive information was diminished. This conclusion, however, is restricted to the postural control of EA2 patients in the interval between the vestibulo-cerebellar episodes.

CACNA1A haploinsufficiency leads to reduced synaptic function and increased intrinsic excitability.

Haploinsufficiency of the CACNA1A gene, encoding the pore-forming α1 subunit of P/Q-type voltage-gated calcium channels, is associated with a clinically variable phenotype ranging from cerebellar ataxia, to neurodevelopmental syndromes with epilepsy and intellectual disability. To understand the pathological mechanisms of CACNA1A loss-of-function variants, we characterized a human neuronal model for CACNA1A haploinsufficiency, by differentiating isogenic induced pluripotent stem cell lines into glutamatergic neurons, and investigated the effect of CACNA1A haploinsufficiency on mature neuronal networks through a combination of electrophysiology, gene expression analysis, and in silico modeling. We observed an altered network synchronization in CACNA1A+/- networks alongside synaptic deficits, notably marked by an augmented contribution of GluA2-lacking AMPA receptors. Intriguingly, these synaptic perturbations coexisted with increased non-synaptically driven activity, as characterized by inhibition of NMDA and AMPA receptors on micro-electrode arrays. Single-cell electrophysiology and gene expression analysis corroborated this increased intrinsic excitability through reduced potassium channel function and expression. Moreover, we observed partial mitigation of the CACNA1A+/- network phenotype by 4-aminopyridine, a therapeutic intervention for episodic ataxia type 2. Positive modulation of KCa2 channels could reverse the CACNA1A+/- network electrophysiological phenotype. In summary, our study pioneers the characterization of a human induced pluripotent stem cell-derived neuronal model for CACNA1A haploinsufficiency, and has unveiled novel mechanistic insights. Beyond showcasing synaptic deficits, this neuronal model exhibited increased intrinsic excitability mediated by diminished potassium channel function, underscoring its potential as a therapeutic discovery platform with predictive validity.

Intrafamilial neurological phenotypic variability due to either biallelic or monoallelic pathogenic variants in CACNA1A.

Pathogenic heterozygous variants in CACNA1A are associated with familial hemiplegic migraine, episodic ataxia type 2 and spinocerebellar ataxia type 6, and more recently, neurodevelopmental disorders. We describe a severe, early-onset phenotype including severe muscular hypotonia, early-onset epileptic seizures, apnoea, optic atrophy and dysphagia in three siblings carrying compound heterozygous frameshift variants in CACNA1A. Two male patients died at the age of 5 or 14 months of suspected SIDS or severe developmental epileptic encephalopathy (DEE) with refractory seizures and apnoea. A male child (index patient) developed severe early-onset DEE including seizures and ictal apnoea at the age of 4 weeks. Another male child developed generalized epilepsy and mild intellectual impairment in late infancy, and his mother and his maternal uncle were identified as carriers of a known CACNA1A pathogenic variant [c.2602delG heterozygous, p. (Ala868Profs*24)] with a diagnosis of episodic ataxia type 2. This maternal pathogenic variant c.2602delG was detected in the index patient and child 2. Trio-Exome sequencing identified an additional heterozygous pathogenic variant in the CACNA1A gene, c.5476delC, p.(His1826Thrfs*30) in the index patient and child 2, which was inherited from the asymptomatic father. In conclusion, the novel compound heterozygosity for two frameshift pathogenic variants, maternally [c.2602delG, p.(Ala868Profs*24)] and paternally [c.5476delC, p.(His1826Thrfs*3)] is associated with a severe phenotype of early-onset DEE. This observation highlights the necessity of additional analyses to clarify unusual phenotypes even if a pathogenic variant has already been identified, and expands the clinical spectrum of CACNA1A-related disorders.

Identification and characterisation of pathogenic and non-pathogenic FGF14 repeat expansions

Repeat expansions in FGF14 cause autosomal dominant late-onset cerebellar ataxia (SCA27B) with estimated pathogenic thresholds of 250 (incomplete penetrance) and 300 AAG repeats (full penetrance), but the sequence of pathogenic and non-pathogenic expansions remains unexplored. Here, we demonstrate that STRling and ExpansionHunter accurately detect FGF14 expansions from short-read genome data using outlier approaches. By combining long-range PCR and nanopore sequencing in 169 patients with cerebellar ataxia and 802 controls, we compare FGF14 expansion alleles, including interruptions and flanking regions. Uninterrupted AAG expansions are significantly enriched in patients with ataxia from a lower threshold (180–200 repeats) than previously reported based on expansion size alone. Conversely, AAGGAG hexameric expansions are equally frequent in patients and controls. Distinct 5’ flanking regions, interruptions and pre-repeat sequences correlate with repeat size. Furthermore, pure AAG (pathogenic) and AAGGAG (non-pathogenic) repeats form different secondary structures. Regardless of expansion size, SCA27B is a recognizable clinical entity characterized by frequent episodic ataxia and downbeat nystagmus, similar to the presentation observed in a family with a previously unreported nonsense variant (SCA27A). Overall, this study suggests that SCA27B is a major overlooked cause of adult-onset ataxia, accounting for 23–31% of unsolved patients. We strongly recommend re-evaluating pathogenic thresholds and integrating expansion sequencing into the molecular diagnostic process.

A positively charged residue at the Kv1.1 T1 interface is critical for voltage-dependent activation and gating kinetics.

Within the tetramerization domain (T1) of most voltage-gated potassium channels (Kv) are highly conserved charged residues that line the T1-T1 interface. We investigated the Kv1.1 residue R86 located at the narrowest region of the T1 interface. A Kv1.1 R86Q mutation was reported in a child diagnosed with lower limb dyskinesia (Set KK, Ghosh D, Huq AHM, Luat AF. Mov Disord Clin Pract 4: 784-786, 2017). The child did not present with episodic ataxia 1 (EA1) symptoms typically associated with Kv1.1 loss-of-function mutations. We characterized the electrophysiological outcome of the R86Q substitution by expressing Kv1.1 in Xenopus laevis oocytes. Mutated α-subunits were able to form functional channels that pass delayed rectifier currents. Oocytes that expressed only mutated α-subunits produced a significant reduction in Kv1.1 current and showed a positive shift in voltage dependence of activation. In addition, there was substantially slower activation and faster deactivation implying a reduction in the time the channel is in its open state. Oocytes co-injected with both mutated and wild-type cRNA in equal amounts, to mimic the heterozygous condition of the disease, showed a decrease in current amplitude at -10 mV, a positive shift in activation voltage-dependence and faster deactivation kinetics when compared with the wild-type channel. These findings indicate that T1 plays a role in Kv1.1's voltage-dependent activation and in its kinetics of activation and deactivation.NEW & NOTEWORTHY This is the first Kv1.1 study to characterize the electrophysiological and structural phenotype of a tetramerization (T1) domain mutation. Surprisingly, the mutated α-subunits were able to tetramerize, albeit with different gating kinetics and voltage dependence. This novel finding points to a clear role of T1 in the channel's voltage dependence and gating. Mimicking the heterozygous condition resulted in milder alterations in channel function when compared with previously reported mutations. This is in agreement with the child's milder symptoms.

Variable Phenotypes in the Same Patient with PRRT2-Associated Disorders

AbstractMutations in the PRRT2 gene lead to a spectrum of diseases with a common pathophysiology including self-limited (familial) infantile epilepsy and paroxysmal kinesigenic dyskinesia as well as other paroxysmal diseases involving movement and headache disorders. Atypical phenotypes, associated with episodic ataxia, epilepsy, hemiplegic migraine, developmental delay, and intellectual disability, have been reported in approximately 5% of the patients, which is probably an underestimation. Here, we present three patients with variable PRRT2 phenotypes in each patient. In the first two patients, the manifestations were characterized by episodes of nonepileptic paroxysms and focal seizures starting in the first years of life with good response to carbamazepine. One of them had no family history either of epilepsy or nonepileptic motor manifestations. The other patient simultaneously developed epileptic spasms. Neurodevelopment was normal in both. The third patient presented with early-onset focal epilepsy that was resistant to antiseizure medications and evolved to spike-wave activation in sleep associated with cognitive impairment and ataxia. In this patient, in addition to the mutation in the PRRT2 gene, a novel pathogenic SCN1A variant was identified. The distinct clinical presentations in the same patient observed in our cases confirm the broad spectrum of PRRT2-associated diseases.

Anti-CASPR2 Antibody-Associated Syndrome Presenting With Episodic Ataxia

Anti-CASPR2 Antibody-Associated Syndrome Presenting With Episodic Ataxia

Anti-CASPR2 antibody-associated syndrome presenting with episodic ataxia

Anti-CASPR2 antibody-associated syndrome presenting with episodic ataxia

СЛУЧАЙ ИЗ ПРАКТИКИ: ЭПИЗОДИЧЕСКАЯ ПАРОКСИЗМАЛЬНАЯ МОЗЖЕЧКОВАЯ АТАКСИЯ 2 ТИПА

Instability walking is a fairly common complaint made by patients who come to be examined by a neurologist in polyclinics and hospitals. From a medical point of view, this symptom is included in the concept of "ataxia". Neurologists distinguish four types of ataxia depending on the localization of the pathological process: cortical, cerebellar, vestibular, and sensitive. There are often combinations of different types of ataxia in the same patient, for example, cortical-vestibular, sensitive-cerebellar. In addition to establishing the variant of the atactic syndrome, there is an urgent question of verifying the etiological factor that led to this pathology. Pathological heredity becomes the least obvious. In this article, we draw attention to a similar case from practice in order to form the alertness of doctors regarding hereditary coordination disorders. Episodic ataxia is a group of rare hereditary diseases of the nervous system, the main clinical manifestation of which are paroxysms of cerebellar ataxia of varying duration and frequency. The most studied from a genetic and clinical point of view are episodic cerebellar ataxia of the first and second types. The authors present a clinical case of episodic paroxysmal cerebellar ataxia type 2 in a 22-year-old patient. The diagnosis of the disease was based on a typical clinical picture and confirmed by a molecular genetic study that revealed a mutation in the CACNA1A gene. The article discusses aspects of the pathogenesis of the main forms of episodic paroxysmal ataxia, age of onset, frequency and duration of seizures, features of the clinical picture, neurological status. This clinical observation draws the attention of practitioners to the possibilities of modern methods of molecular genetic diagnosis and treatment of these conditions.

Both gain- and loss-of-function variants of KCNA1 are associated with paroxysmal kinesigenic dyskinesia

KCNA1 is the coding gene for Kv1.1 voltage-gated potassium-channel α subunit. Three variants of KCNA1 have been reported to manifest as paroxysmal kinesigenic dyskinesia (PKD), but the correlation between them remains unclear due to the phenotypic complexity of KCNA1 variants as well as the rarity of PKD cases. Using the whole exome sequencing followed by Sanger sequencing, we screen for potential pathogenic KCNA1 variants in patients clinically diagnosed with paroxysmal movement disorders and identify three previously unreported missense variants of KCNA1 in three unrelated Chinese families. The proband of one family (c.496G>A, p.A166T) manifests as episodic ataxia type 1, and the other two (c.877G>A, p.V293I and c.1112C>A, p.T371A) manifest as PKD. The pathogenicity of these variants is confirmed by functional studies, suggesting that p.A166T and p.T371A cause a loss-of-function of the channel, while p.V293I leads to a gain-of-function with the property of voltage-dependent gating and activation kinetic affected. By reviewing the locations of PKD-manifested KCNA1 variants in Kv1.1 protein, we find that these variants tend to cluster around the pore domain, which is similar to epilepsy. Thus, our study strengthens the correlation between KCNA1 variants and PKD and provides more information on genotype–phenotype correlations of KCNA1 channelopathy.

Delineation of ADPRHL2 Variants: Report of Two New Patients with Review of the Literature.

ADPRHL2 is involved in posttranslational modification and is known to have a role in physiological functions such as cell signaling, DNA repair, gene control, cell death, and response to stress. Recently, a group of neurological disorders due to ADPRHL2 variants is described, characterized by childhood-onset, stress-induced variable movement disorders, neuropathy, seizures, and neurodegenerative course. We present the diagnostic pathway of two pediatric patients with episodic dystonia and ataxia, who later had a neurodegenerative course complicated by central hypoventilation syndrome due to the same homozygous ADPRHL2 variant. We conducted a systematic literature search and data extraction procedure following the Preferred Reporting Items for Systematic Review and Meta-Analysis 2020 statement in terms of patients with ADPRHL2 variants, from 2018 up to 3 February, 2023. In total, 12 articles describing 47 patients were included in the final analysis. Median age at symptom onset was 2 (0.7-25) years, with the most common presenting symptoms being gait problems (n = 19, 40.4%), seizures (n = 16, 34%), ataxia (n = 13, 27.6%), and weakness (n = 10, 21.2%). Triggering factors (28/47; 59.5%) and regression (28/43; 60.4%), axonal polyneuropathy (9/23; 39.1%), and cerebral and cerebellar atrophy with white matter changes (28/36; 77.7%) were the other clues. The fatality rate and median age of death were 44.6% (n = 21) and 7 (2-34) years, respectively. ADPRHL2 variants should be considered in the context of episodic, stress-induced pediatric and adult-onset movement disorders and seizures.

Episodic ataxia type 2 with a novel missense variant (Leu602Arg) in CACNA1A

Autosomal dominant episodic ataxia type 2 (EA2) is caused by variants in CACNA1A. We examined a 20-year-old male with EA symptoms from a Japanese family with hereditary EA. Cerebellar atrophy was not evident, but single photon emission computed tomography showed cerebellar hypoperfusion. We identified a novel nonsynonymous variant in CACNA1A, NM_001127222.2:c.1805T>G (p.Leu602Arg), which is predicted to be functionally deleterious; therefore, this variant is likely responsible for EA2 in this pedigree.

Integration of multi-omics technologies for molecular diagnosis in ataxia patients.

Background: Episodic ataxias are rare neurological disorders characterized by recurring episodes of imbalance and coordination difficulties. Obtaining definitive molecular diagnoses poses challenges, as clinical presentation is highly heterogeneous, and literature on the underlying genetics is limited. While the advent of high-throughput sequencing technologies has significantly contributed to Mendelian disorders genetics, interpretation of variants of uncertain significance and other limitations inherent to individual methods still leaves many patients undiagnosed. This study aimed to investigate the utility of multi-omics for the identification and validation of molecular candidates in a cohort of complex cases of ataxia with episodic presentation. Methods: Eight patients lacking molecular diagnosis despite extensive clinical examination were recruited following standard genetic testing. Whole genome and RNA sequencing were performed on samples isolated from peripheral blood mononuclear cells. Integration of expression and splicing data facilitated genomic variants prioritization. Subsequently, long-read sequencing played a crucial role in the validation of those candidate variants. Results: Whole genome sequencing uncovered pathogenic variants in four genes (SPG7, ATXN2, ELOVL4, PMPCB). A missense and a nonsense variant, both previously reported as likely pathogenic, configured in trans in individual #1 (SPG7: c.2228T>C/p.I743T, c.1861C>T/p.Q621*). An ATXN2 microsatellite expansion (CAG32) in another late-onset case. In two separate individuals, intronic variants near splice sites (ELOVL4: c.541 + 5G>A; PMPCB: c.1154 + 5G>C) were predicted to induce loss-of-function splicing, but had never been reported as disease-causing. Long-read sequencing confirmed the compound heterozygous variants configuration, repeat expansion length, as well as splicing landscape for those pathogenic variants. A potential genetic modifier of the ATXN2 expansion was discovered in ZFYVE26 (c.3022C>T/p.R1008*). Conclusion: Despite failure to identify pathogenic variants through clinical genetic testing, the multi-omics approach enabled the molecular diagnosis in 50% of patients, also giving valuable insights for variant prioritization in remaining cases. The findings demonstrate the value of long-read sequencing for the validation of candidate variants in various scenarios. Our study demonstrates the effectiveness of leveraging complementary omics technologies to unravel the underlying genetics in patients with unresolved rare diseases such as ataxia. Molecular diagnoses not only hold significant promise in improving patient care management, but also alleviates the burden of diagnostic odysseys, more broadly enhancing quality of life.

Intellectual Disability in Episodic Ataxia Type 2: Beyond Paroxysmal Vertigo and Ataxia.

Episodic ataxia type 2 (EA2) is characterized by recurrent vertigo and ataxia due to mutations in CACNA1A that encodes the α1A-subunit of the P/Q-type voltage-gated calcium channel. This study aimed to determine intellectual function in EA2. During 2019-2023, 13 patients (6 males, age range=10-52 years, median age=29 years) with a genetically confirmed diagnosis of EA2 had their intellectual function evaluated using the Korean versions of the Wechsler Intelligence Scales (version IV) for adults or children in 3 referral-based university hospitals in South Korea. The full-scale intelligence quotients (FSIQs) among the 13 patients were below the average (90-109) in 11, low average (80-89) in 5 (38.5%), borderline (70-79) in 1 (7.7%), and indicated intellectual disability (≤69) in 5 (38.5%). These patterns of cognitive impairments were observed in all four of the following subtests: verbal comprehension, perceptual reasoning, working memory, and processing speed. The FSIQ was not correlated with the ages at onset for vertigo and ataxia (Pearson correlation: p=0.40). Patients with EA2 may have hidden intellectual disabilities even without a history of epilepsy or administration of antiepileptic drugs, and should be considered for genetic counseling and therapeutic interventions. Given the availability of medication to control episodic vertigo and ataxia, early diagnosis and management are important in preventing irreversible brain dysfunction in EA2.

Chapter 8 - The episodic ataxias

The primary episodic ataxias (EAs) are a group of autosomal-dominant disorders characterized by transient recurrent incoordination and truncal instability, often triggered by physical exertion or emotional stress and variably associated with progressive baseline ataxia. There are now nine designated subtypes EA1-9 (OMIM) and late onset cerebellar ataxia with episodic features as newly designated SCA27B, based largely on genetic loci. Mutations have been identified in multiple individuals and families in 4 of the 9 EA subtypes, mostly with the onset before adulthood. This chapter focuses on the clinical assessment and management of EA, genetic diagnosis, and neurophysiologic consequences of the causative mutations in the best characterized EA syndromes: EA1 caused by mutations in KCNA1 encoding a neuronal voltage-gated potassium channel, EA2 caused by mutations in CACNA1A encoding a neuronal voltage-gated calcium channel, EA6 caused by mutations in SLC1A3 encoding a glutamate transporter that is also an anion channel, and SCA27B with late onset episodic ataxia caused by an intronic trinucleotide repeat in FGF14 encoding fibroblast growth factor 14 important in regulating the distribution of voltage-gated sodium channels in the cerebellar Purkinje and granule cells. The study of EA has illuminated previously unrecognized but important roles of ion channels and transporters in brain function with shared mechanisms underlying cerebellar ataxia, migraine, and epilepsy.

Developing a pathway to clinical trials for CACNA1A-related epilepsies: A patient organization perspective.

CACNA1A-related disorders are rare neurodevelopmental disorders linked to variants in the CACNA1A gene. This gene encodes the α1 subunit of the P/Q-type calcium channel Cav2.1, which is globally expressed in the brain and crucial for fast synaptic neurotransmission. The broad spectrum of CACNA1A-related neurological disorders includes developmental and epileptic encephalopathies, familial hemiplegic migraine type 1, episodic ataxia type 2, spinocerebellar ataxia type 6, together with unclassified presentations with developmental delay, ataxia, intellectual disability, autism spectrum disorder, and language impairment. The severity of each disorder is also highly variable. The spectrum of CACNA1A-related seizures is broad across both loss-of-function and gain-of-function variants and includes absence seizures, focal seizures with altered consciousness, generalized tonic-clonic seizures, tonic seizures, status epilepticus, and infantile spasms. Furthermore, over half of CACNA1A-related epilepsies are refractory to current therapies. To date, almost 1700 CACNA1A variants have been reported in ClinVar, with over 400 listed as Pathogenic or Likely Pathogenic, but with limited-to-no clinical or functional data. Robust genotype-phenotype studies and impacts of variants on protein structure and function have also yet to be established. As a result, there are few definitive treatment options for CACNA1A-related epilepsies. The CACNA1A Foundation has set out to change the landscape of available and effective treatments and improve the quality of life for those living with CACNA1A-related disorders, including epilepsy. Established in March 2020, the Foundation has built a robust preclinical toolbox that includes patient-derived induced pluripotent stem cells and novel disease models, launched clinical trial readiness initiatives, and organized a global CACNA1A Research Network. This Research Network is currently composed of over 60 scientists and clinicians committed to collaborating to accelerate the path to CACNA1A-specific treatments and one day, a cure.

Selective block of human Kv1.1 channels and an epilepsy-associated gain-of-function mutation by AETX-K peptide.

Dysfunction of the human voltage-gated K+ channel Kv1.1 has been associated with epilepsy, multiple sclerosis, episodic ataxia, myokymia, and cardiorespiratory dysregulation. We report here that AETX-K, a sea anemone type I (SAK1) peptide toxin we isolated from a phage display library, blocks Kv1.1 with high affinity (Ki ~ 1.6 pM) and notable specificity, inhibiting other Kv channels we tested a million-fold less well. Nuclear magnetic resonance (NMR) was employed both to determine the three-dimensional structure of AETX-K, showing it to employ a classic SAK1 scaffold while exhibiting a unique electrostatic potential surface, and to visualize AETX-K bound to the Kv1.1 pore domain embedded in lipoprotein nanodiscs. Study of Kv1.1 in Xenopus oocytes with AETX-K and point variants using electrophysiology demonstrated the blocking mechanism to employ a toxin-channel configuration we have described before whereby AETX-K Lys23 , two positions away on the toxin interaction surface from the classical blocking residue, enters the pore deeply enough to interact with K+ ions traversing the pathway from the opposite side of the membrane. The mutant channel Kv1.1-L296 F is associated with pharmaco-resistant multifocal epilepsy in infants because it significantly increases K+ currents by facilitating opening and slowing closure of the channels. Consistent with the therapeutic potential of AETX-K for Kv1.1 gain-of-function-associated diseases, AETX-K at 4 pM decreased Kv1.1-L296 F currents to wild-type levels; further, populations of heteromeric channels formed by co-expression Kv1.1 and Kv1.2, as found in many neurons, showed a Ki of ~10 nM even though homomeric Kv1.2 channels were insensitive to the toxin (Ki > 2000 nM).

Girl with intermittent ataxia, myoclonus and poor scholastic perfomance – A case report

A numerous number of disorders are being linked with ataxia and myoclonus. Majority of causes are genetic and a growing number of genes are being connected with myoclonus-ataxia syndromes (MAS), due to recent advances in genetic techniques like Next generation Sequencing (NGS). We report a rare case of progressive myoclonus and ataxia, in a young girl who presented with action induced myoclonus, episodic ataxia and cognitive regression.

Molecular insights into disease-associated glutamate transporter (EAAT1 / SLC1A3) variants using in silico and in vitro approaches

Glutamate is an essential excitatory neurotransmitter and an intermediate for energy metabolism. Depending on the tumor site, cancer cells have increased or decreased expression of excitatory amino acid transporter 1 or 2 (EAAT1/2, SLC1A3/2) to regulate glutamate uptake for the benefit of tumor growth. Thus, EAAT1/2 may be an attractive target for therapeutic intervention in oncology. Genetic variation of EAAT1 has been associated with rare cases of episodic ataxia, but the occurrence and functional contribution of EAAT1 mutants in other diseases, such as cancer, is poorly understood. Here, 105 unique somatic EAAT1 mutations were identified in cancer patients from the Genomic Data Commons dataset. Using EAAT1 crystal structures and in silico studies, eight mutations were selected based on their close proximity to the orthosteric or allosteric ligand binding sites and the predicted change in ligand binding affinity. In vitro functional assessment in a live-cell, impedance-based phenotypic assay demonstrated that these mutants differentially affect L-glutamate and L-aspartate transport, as well as the inhibitory potency of an orthosteric (TFB-TBOA) and allosteric (UCPH-101) inhibitor. Moreover, two episodic ataxia-related mutants displayed functional responses that were in line with literature, which confirmed the validity of our assay. Of note, ataxia-related mutant M128R displayed inhibitor-induced functional responses never described before. Finally, molecular dynamics (MD) simulations were performed to gain mechanistic insights into the observed functional effects. Taken together, the results in this work demonstrate 1) the suitability of the label-free phenotypic method to assess functional variation of EAAT1 mutants and 2) the opportunity and challenges of using in silico techniques to rationalize the in vitro phenotype of disease-relevant mutants.

Clinical and electrophysiological features of SCN8A variants causing episodic or chronic ataxia

Variants in SCN8A are associated with a spectrum of epilepsies and neurodevelopmental disorders. Ataxia as a predominant symptom of SCN8A variation has not been well studied. We set out to investigate disease mechanisms and genotype-phenotype correlations of SCN8A-related ataxia. We collected genetic and electro-clinical data of ten individuals from nine unrelated families carrying novel SCN8A variants associated with chronic progressive or episodic ataxia. Electrophysiological characterizations of these variants were performed in ND7/23cells and cultured neurons. Variants associated with chronic progressive ataxia either decreased Na+ current densities and shifted activation curves towards more depolarized potentials (p.Asn995Asp, p.Lys1498Glu and p.Trp1266Cys) or resulted in a premature stop codon (p.Trp937Ter). Three variants (p.Arg847Gln and biallelic p.Arg191Trp/p.Asp1525Tyr) were associated with episodic ataxia causing loss-of-function by decreasing Na+ current densities or a hyperpolarizing shift of the inactivation curve. Two additional episodic ataxia-associated variants caused mixed gain- and loss-of function effects in ND7/23cells and were further examined in primary murine hippocampal neuronal cultures. Neuronal firing in excitatory neurons was increased by p.Arg1629His, but decreased by p.Glu1201Lys. Neuronal firing in inhibitory neurons was decreased for both variants. No functional effect was observed for p.Arg1913Trp. In four individuals, treatment with sodium channel blockers exacerbated symptoms. We identified episodic or chronic ataxia as predominant phenotypes caused by variants in SCN8A. Genotype-phenotype correlations revealed a more pronounced loss-of-function effect for variants causing chronic ataxia. Sodium channel blockers should be avoided under these conditions. BMBF, DFG, the Italian Ministry of Health, University of Tuebingen.