Sodium channelopathies in neurodevelopmental disorders.

The voltage-gated sodium channel α-subunit genes comprise a highly conserved gene family. Mutations of three of these genes, SCN1A, SCN2A and SCN8A, are responsible for a significant burden of neurological disease. Recent progress in identification and functional characterization of patient variants is generating new insights and novel approaches to therapy for these devastating disorders. Here we review the basic elements of sodium channel function that are used to characterize patient variants. We summarize a large body of work using global and conditional mouse mutants to characterize the in vivo roles of these channels. We provide an overview of the neurological disorders associated with mutations of the human genes and examples of the effects of patient mutations on channel function. Finally, we highlight therapeutic interventions that are emerging from new insights into mechanisms of sodium channelopathies.

The chloride antiporter CLCN7 is a modifier of lysosome dysfunction in FIG4 and VAC14 mutants.

The phosphatase FIG4 and the scaffold protein VAC14 function in the biosynthesis of PI(3,5)P2, a signaling lipid that inhibits the lysosomal chloride transporter ClC-7. Loss-of-function mutations of FIG4 and VAC14 reduce PI(3,5)P2 and result in lysosomal disorders characterized by accumulation of enlarged lysosomes and neurodegeneration. Similarly, a gain of function mutation of CLCN7 encoding ClC-7 also results in enlarged lysosomes. We therefore tested the ability of reduced CLCN7 expression to compensate for loss of FIG4 or VAC14. Knock-out of CLCN7 corrected lysosomal swelling and partially corrected lysosomal hyperacidification in FIG4 null cell cultures. Knockout of the related transporter CLCN6 (ClC-6) in FIG4 null cells did not affect the lysosome phenotype. In the Fig4 null mouse, reduction of ClC-7 by expression of the dominant negative CLCN7 variant p.Gly215Arg improved growth and neurological function and increased lifespan by 20%. These observations demonstrate a role for the CLCN7 chloride transporter in pathogenesis of FIG4 and VAC14 disorders. Reduction of CLCN7 provides a new target for treatment of FIG4 and VAC14 deficiencies that lack specific therapies, such as Charcot-Marie-Tooth Type 4J and Yunis-Varón syndrome.

Long-Term Downregulation of the Sodium Channel Gene Scn8a Is Therapeutic in Mouse Models of SCN8A Epilepsy.

OBJECTIVE: De novo mutations of the voltage-gated sodium channel gene SCN8A cause developmental and epileptic encephalopathy (DEE). Most pathogenic variants result in gain-of-function changes in activity of the sodium channel Nav1.6, poorly controlled seizures, and significant comorbidities. In previous work, an antisense oligonucleotide (ASO) reduced Scn8a transcripts and increased lifespan after neonatal administration to a mouse model. Here, we tested long-term ASO treatment initiated after seizure onset, as required for clinical application. METHODS: ASO treatment was initiated after observation of a convulsive seizure and repeated at 4 to 6 week intervals for 1 year. We also tested the long-term efficacy of an AAV10-short hairpin RNA (shRNA) virus administered on P1. RESULTS: Repeated treatment with the Scn8a ASO initiated after seizure onset provided long-term survival and reduced seizure frequency during a 12 month observation period. A single treatment with viral shRNA was also protective during 12 months of observation. INTERPRETATION: Downregulation of Scn8a expression that is initiated after the onset of seizures is effective for long-term treatment in a model of SCN8A-DEE. Repeated ASO administration or a single dose of viral shRNA prevented seizures and extended survival through 12 months of observation. ANN NEUROL 2024;95:754-759.

Dentate gyrus granule cells are a locus of pathology in Scn8a developmental encephalopathy.

Gain-of-function mutations in SCN8A cause developmental and epileptic encephalopathy (DEE), a disorder characterized by early-onset refractory seizures, deficits in motor and intellectual functions, and increased risk of sudden unexpected death in epilepsy. Altered activity of neurons in the corticohippocampal circuit has been reported in mouse models of DEE. We examined the effect of chronic seizures on gene expression in the hippocampus by single-nucleus RNA sequencing in mice expressing the patient mutation SCN8A-p.Asn1768Asp (N1768D). One hundred and eighty four differentially expressed genes were identified in dentate gyrus granule cells, many more than in other cell types. Electrophysiological recording from dentate gyrus granule cells demonstrated an elevated firing rate. Targeted reduction of Scn8a expression in the dentate gyrus by viral delivery of an shRNA resulted in doubling of median survival time from 4 months to 8 months, whereas delivery of shRNA to the CA1 and CA3 regions did not result in lengthened survival. These data indicate that granule cells of the dentate gyrus are a specific locus of pathology in SCN8A-DEE.

Biallelic loss-of-function variants of SLC12A9 cause lysosome dysfunction and a syndromic neurodevelopmental disorder.

PURPOSE: Pathogenic variants of FIG4 generate enlarged lysosomes and neurological and developmental disorders. To identify additional genes regulating lysosomal volume, we carried out a genome-wide activation screen to detect suppression of enlarged lysosomes in FIG4-/- cells. METHODS: The CRISPR-a gene activation screen utilized sgRNAs from the promoters of protein-coding genes. Fluorescence-activated cell sorting separated cells with correction of the enlarged lysosomes from uncorrected cells. Patient variants of SLC12A9 were identified by exome or genome sequencing and studied by segregation analysis and clinical characterization. RESULTS: Overexpression of SLC12A9, a solute co-transporter, corrected lysosomal swelling in FIG4-/- cells. SLC12A9 (NP_064631.2) colocalized with LAMP2 at the lysosome membrane. Biallelic variants of SLC12A9 were identified in 3 unrelated probands with neurodevelopmental disorders. Common features included intellectual disability, skeletal and brain structural abnormalities, congenital heart defects, and hypopigmented hair. Patient 1 was homozygous for nonsense variant p.(Arg615∗), patient 2 was compound heterozygous for p.(Ser109Lysfs∗20) and a large deletion, and proband 3 was compound heterozygous for p.(Glu290Glyfs∗36) and p.(Asn552Lys). Fibroblasts from proband 1 contained enlarged lysosomes that were corrected by wild-type SLC12A9 cDNA. Patient variant p.(Asn552Lys) failed to correct the lysosomal defect. CONCLUSION: Impaired function of SLC12A9 results in enlarged lysosomes and a recessive disorder with a recognizable neurodevelopmental phenotype.

Spontaneous seizures and elevated seizure susceptibility in response to somatic mutation of sodium channel Scn8a in the mouse.

De novo mutations of neuronal sodium channels are responsible for ~5% of developmental and epileptic encephalopathies, but the role of somatic mutation of these genes in adult-onset epilepsy is not known. We evaluated the role of post-zygotic somatic mutation by adult activation of a conditional allele of the pathogenic variant Scn8aR1872W in the mouse. After activation of CAG-Cre-ER by tamoxifen, the mutant transcript was expressed throughout the brain at a level proportional to tamoxifen dose. The threshold for generation of spontaneous seizures was reached when the proportion of mutant transcript reached 8% of total Scn8a transcript, equivalent to expression of the epileptogenic variant in 16% of heterozygous neurons. Expression below this level did not result in spontaneous seizures, but did increase susceptibility to seizure induction by kainate or auditory stimulation. The relatively high threshold for spontaneous seizures indicates that somatic mutation of sodium channels is unlikely to contribute to the elevated incidence of epilepsy in the elderly population. However, somatic mutation could increase susceptibility to other seizure stimuli.

Chloroquine corrects enlarged lysosomes in FIG4 null cells and reduces neurodegeneration in Fig4 null mice.

Loss-of-function mutations of FIG4 impair the biosynthesis of PI(3,5)P2 and are responsible for rare genetic disorders including Yunis-Varón Syndrome and Charcot-Marie-Tooth Disease Type 4 J. Cultured cells deficient in FIG4 accumulate enlarged lysosomes with hyperacidic pH, due in part to impaired regulation of lysosomal ion channels and elevated intra-lysosomal osmotic pressure. We evaluated the effects of the FDA approved drug chloroquine, which is known to reduce lysosome acidity, on FIG4 deficient cell culture and on a mouse model. Chloroquine corrected the enlarged lysosomes in FIG4 null cells. In null mice, addition of chloroquine to the drinking water slowed progression of the disorder. Growth and mobility were dramatically improved during the first month of life, and spongiform degeneration of the nervous system was reduced. The median survival of Fig4 null mice was increased from 4 weeks for untreated mutants to 8 weeks with chloroquine treatment (p < 0.009). Chloroquine thus corrects the lysosomal swelling in cultured cells and ameliorates Fig4 deficiency in vivo. The improved phenotype of mice with complete loss of Fig4 suggests that chloroquine could be beneficial FIG2 in partial loss-of-function disorders such as Charcot-Marie-Tooth Type 4 J.

Postictal Death Is Associated with Tonic Phase Apnea in a Mouse Model of Sudden Unexpected Death in Epilepsy.

OBJECTIVE: Sudden unexpected death in epilepsy (SUDEP) is an unpredictable and devastating comorbidity of epilepsy that is believed to be due to cardiorespiratory failure immediately after generalized convulsive seizures. METHODS: We performed cardiorespiratory monitoring of seizure-induced death in mice carrying either a p.Arg1872Trp or p.Asn1768Asp mutation in a single Scn8a allele-mutations identified from patients who died from SUDEP-and of seizure-induced death in pentylenetetrazole-treated wild-type mice. RESULTS: The primary cause of seizure-induced death for all mice was apnea, as (1) apnea began during a seizure and continued for tens of minutes until terminal asystole, and (2) death was prevented by mechanical ventilation. Fatal seizures always included a tonic phase that was coincident with apnea. This tonic phase apnea was not sufficient to produce death, as it also occurred during many nonfatal seizures; however, all seizures that were fatal had tonic phase apnea. We also made the novel observation that continuous tonic diaphragm contraction occurred during tonic phase apnea, which likely contributes to apnea by preventing exhalation, and this was only fatal when breathing did not resume after the tonic phase ended. Finally, recorded seizures from a patient with developmental epileptic encephalopathy with a previously undocumented SCN8A likely pathogenic variant (p.Leu257Val) revealed similarities to those of the mice, namely, an extended tonic phase that was accompanied by apnea. INTERPRETATION: We conclude that apnea coincident with the tonic phase of a seizure, and subsequent failure to resume breathing, are the determining events that cause seizure-induced death in Scn8a mutant mice. ANN NEUROL 2021;89:1023-1035.

Genetic interaction between Scn8a and potassium channel genes Kcna1 and Kcnq2.

Voltage-gated sodium and potassium channels regulate the initiation and termination of neuronal action potentials. Gain-of-function mutations of sodium channel Scn8a and loss-of-function mutations of potassium channels Kcna1 and Kcnq2 increase neuronal activity and lead to seizure disorders. We tested the hypothesis that reducing the expression of Scn8a would compensate for loss-of-function mutations of Kcna1 or Kcnq2. Scn8a expression was reduced by the administration of an antisense oligonucleotide (ASO). This treatment lengthened the survival of the Kcn1a and Kcnq2 mutants, and reduced the seizure frequency in the Kcnq2 mutant mice. These observations suggest that reduction of SCN8A may be therapeutic for genetic epilepsies resulting from mutations in these potassium channel genes.

The PIKfyve complex regulates the early melanosome homeostasis required for physiological amyloid formation.

The metabolism of PI(3,5)P2 is regulated by the PIKfyve, VAC14 and FIG4 complex, mutations in which are associated with hypopigmentation in mice. These pigmentation defects indicate a key, but as yet unexplored, physiological relevance of this complex in the biogenesis of melanosomes. Here, we show that PIKfyve activity regulates formation of amyloid matrix composed of PMEL protein within the early endosomes in melanocytes, called stage I melanosomes. PIKfyve activity controls the membrane remodeling of stage I melanosomes, which regulates PMEL abundance, sorting and processing. PIKfyve activity also affects stage I melanosome kiss-and-run interactions with lysosomes, which are required for PMEL amyloidogenesis and the establishment of melanosome identity. Mechanistically, PIKfyve activity promotes both the formation of membrane tubules from stage I melanosomes and their release by modulating endosomal actin branching. Taken together, our data indicate that PIKfyve activity is a key regulator of the melanosomal import-export machinery that fine tunes the formation of functional amyloid fibrils in melanosomes and the maintenance of melanosome identity.This article has an associated First Person interview with the first author of the paper.

Antisense Oligonucleotide Therapy for Neurodevelopmental Disorders.

Antisense oligonucleotides (ASOs) are short oligonucleotides that can modify gene expression and mRNA splicing in the nervous system. The FDA has approved ASOs for treatment of ten genetic disorders, with many applications currently in the pipeline. We describe the molecular mechanisms of ASO treatment for four neurodevelopmental and neuromuscular disorders. The ASO nusinersen is a general treatment for mutations of SMN1 in spinal muscular atrophy that corrects the splicing defect in the SMN2 gene. Milasen is a patient-specific ASO that rescues splicing of CNL7 in Batten's disease. STK-001 is an ASO that increases expression of the sodium channel gene SCN1A by exclusion of a poison exon. An ASO that reduces the abundance of the SCN8A mRNA is therapeutic in mouse models of developmental and epileptic encephalopathy. These examples demonstrate the variety of mechanisms and range of applications of ASOs for treatment of neurodevelopmental disorders.

Scn8a Antisense Oligonucleotide Is Protective in Mouse Models of SCN8A Encephalopathy and Dravet Syndrome.

OBJECTIVE: SCN8A encephalopathy is a developmental and epileptic encephalopathy (DEE) caused by de novo gain-of-function mutations of sodium channel Nav 1.6 that result in neuronal hyperactivity. Affected individuals exhibit early onset drug-resistant seizures, developmental delay, and cognitive impairment. This study was carried out to determine whether reducing the abundance of the Scn8a transcript with an antisense oligonucleotide (ASO) would delay seizure onset and prolong survival in a mouse model of SCN8A encephalopathy. METHODS: ASO treatment was tested in a conditional mouse model with Cre-dependent expression of the pathogenic patient SCN8A mutation p.Arg1872Trp (R1872W). This model exhibits early onset of seizures, rapid progression, and 100% penetrance. An Scn1a +/- haploinsufficient mouse model of Dravet syndrome was also treated. ASO was administered by intracerebroventricular injection at postnatal day 2, followed in some cases by stereotactic injection at postnatal day 30. RESULTS: We observed a dose-dependent increase in length of survival from 15 to 65 days in the Scn8a-R1872W/+ mice treated with ASO. Electroencephalographic recordings were normal prior to seizure onset. Weight gain and activity in an open field were unaffected, but treated mice were less active in a wheel running assay. A single treatment with Scn8a ASO extended survival of Dravet syndrome mice from 3 weeks to >5 months. INTERPRETATION: Reduction of Scn8a transcript by 25 to 50% delayed seizure onset and lethality in mouse models of SCN8A encephalopathy and Dravet syndrome. Reduction of SCN8A transcript is a promising approach to treatment of intractable childhood epilepsies. Ann Neurol 2020;87:339-346.

The MAP1B Binding Domain of Nav1.6 Is Required for Stable Expression at the Axon Initial Segment.

Nav1.6 (SCN8A) is a major voltage-gated sodium channel in the mammalian CNS, and is highly concentrated at the axon initial segment (AIS). As previously demonstrated, the microtubule associated protein MAP1B binds the cytoplasmic N terminus of Nav1.6, and this interaction is disrupted by the mutation p.VAVP(77-80)AAAA. We now demonstrate that this mutation results in WT expression levels on the somatic surface but reduced surface expression at the AIS of cultured rat embryonic hippocampal neurons from both sexes. The mutation of the MAP1B binding domain did not impair vesicular trafficking and preferential delivery of Nav1.6 to the AIS; nor was the diffusion of AIS inserted channels altered relative to WT. However, the reduced AIS surface expression of the MAP1B mutant was restored to WT levels by inhibiting endocytosis with Dynasore, indicating that compartment-specific endocytosis was responsible for the lack of AIS accumulation. Interestingly, the lack of AIS targeting resulted in an elevated percentage of persistent current, suggesting that this late current originates predominantly in the soma. No differences in the voltage dependence of activation or inactivation were detected in the MAP1B binding mutant relative to WT channel. We hypothesize that MAP1B binding to the WT Nav1.6 masks an endocytic motif, thus allowing long-term stability on the AIS surface. This work identifies a critical and important new role for MAP1B in the regulation of neuronal excitability and adds to our understanding of AIS maintenance and plasticity, in addition to identifying new target residues for pathogenic mutations of SCN8ASIGNIFICANCE STATEMENT Nav1.6 is a major voltage-gated sodium channel in human brain, where it regulates neuronal activity due to its localization at the axon initial segment (AIS). Nav1.6 mutations cause epilepsy, intellectual disability, and movement disorders. In the present work, we show that loss of interaction with MAP1B within the Nav1.6 N terminus reduces the steady-state abundance of Nav1.6 at the AIS. The effect is due to increased Nav1.6 endocytosis at this neuronal compartment rather than a failure of forward trafficking to the AIS. This work confirms a new biological role of MAP1B in the regulation of sodium channel localization and will contribute to future analysis of patient mutations in the cytoplasmic N terminus of Nav1.6.

Protective role of the lipid phosphatase Fig4 in the adult nervous system.

The signaling lipid phosphatidylinositol 3,5-bisphosphate, PI(3,5)P2, functions in vesicular trafficking through the endo-lysosomal compartment. Cellular levels of PI(3,5)P2 are regulated by an enzyme complex comprised of the kinase PIKFYVE, the phosphatase FIG4, and the scaffold protein VAC14. Mutations of human FIG4 cause inherited disorders including Charcot-Marie-Tooth disease type 4J, polymicrogyria with epilepsy, and Yunis-Varón syndrome. Constitutive Fig4-/- mice exhibit intention tremor, spongiform degeneration of neural tissue, hypomyelination, and juvenile lethality. To determine whether PI(3,5)P2 is required in the adult, we generated Fig4flox/-; CAG-creER mice and carried out tamoxifen-induced gene ablation. Global ablation in adulthood leads to wasting, tremor, and motor impairment. Death follows within 2 months of tamoxifen treatment, demonstrating a life-long requirement for Fig4. Histological examinations of the sciatic nerve revealed profound Wallerian degeneration of myelinated fibers, but not C-fiber axons in Remak bundles. In optic nerve sections, myelinated fibers appear morphologically intact and carry compound action potentials at normal velocity and amplitude. However, when iKO mice are challenged with a chemical white matter lesion, repair of damaged CNS myelin is significantly delayed, demonstrating a novel role for Fig4 in remyelination. Thus, in the adult PNS Fig4 is required to protect myelinated axons from Wallerian degeneration. In the adult CNS, Fig4 is dispensable for fiber stability and nerve conduction, but is required for the timely repair of damaged white matter. The greater vulnerability of the PNS to Fig4 deficiency in the mouse is consistent with clinical observations in patients with Charcot-Marie-Tooth disease.

Gabra2 is a genetic modifier of Scn8a encephalopathy in the mouse.

OBJECTIVE: SCN8A encephalopathy is a developmental epileptic encephalopathy typically caused by de novo gain-of-function mutations in Nav 1.6. Severely affected individuals exhibit refractory seizures, developmental delay, cognitive disabilities, movement disorders, and elevated risk of sudden death. Patients with the identical SCN8A variant can differ in clinical course, suggesting a role for modifier genes in determining disease severity. The identification of genetic modifiers contributes to understanding disease pathogenesis and suggesting therapeutic interventions. METHODS: We generated F1 and F2 crosses between inbred mouse strains and mice carrying the human pathogenic variants SCN8A-R1872W and SCN8A-N1768D. Quantitative trait locus (QTL) analysis of seizure-related phenotypes was used for chromosomal mapping of modifier loci. RESULTS: In an F2 cross between strain SJL/J and C57BL/6J mice carrying the patient mutation R1872W, we identified a major QTL on chromosome 5 containing the Gabra2 gene. Strain C57BL/6J carries a splice site mutation that reduces expression of Gabra2, encoding the α2 subunit of the aminobutyric acid type A receptor. The protective wild-type allele of Gabra2 from strain SJL/J delays the age at seizure onset and extends life span of the Scn8a mutant mice. Additional Scn8a modifiers were observed in the F2 cross and in an F1 cross with strain C3HeB/FeJ. SIGNIFICANCE: These studies demonstrate that the SJL/J strain carries multiple modifiers with protective effects against seizures induced by gain-of-function mutations in Scn8a. Homozygosity for the hypomorphic variant of Gabra2 in strain C57BL/6J is associated with early seizure onset and short life span. GABRA2 is a potential therapeutic target for SCN8A encephalopathy.

Prominent role of forebrain excitatory neurons in SCN8A encephalopathy.

De novo mutations of the sodium channel gene SCN8A result in an epileptic encephalopathy with refractory seizures, developmental delay, and elevated risk of sudden death. p.Arg1872Trp is a recurrent de novo SCN8A mutation reported in 14 unrelated individuals with epileptic encephalopathy that included seizure onset in the prenatal or infantile period and severe verbal and ambulatory comorbidities. The major biophysical effect of the mutation was previously shown to be impaired channel inactivation accompanied by increased current density. We have generated a conditional mouse mutation in which expression of this severe gain-of-function mutation is dependent upon Cre recombinase. Global activation of p.Arg1872Trp by EIIa-Cre resulted in convulsive seizures and lethality at 2 weeks of age. Neural activation of the p.Arg1872Trp mutation by Nestin-Cre also resulted in early onset seizures and death. Restriction of p.Arg1872Trp expression to excitatory neurons using Emx1-Cre recapitulated seizures and juvenile lethality between 1 and 2 months of age. In contrast, activation of p.Arg1872Trp in inhibitory neurons by Gad2-Cre or Dlx5/6-Cre did not induce seizures or overt neurological dysfunction. The sodium channel modulator GS967/Prax330 prolonged survival of mice with global expression of R1872W and also modulated the activity of the mutant channel in transfected cells. Activation of the p.Arg1872Trp mutation in adult mice was sufficient to generate seizures and death, indicating that successful therapy will require lifelong treatment. These findings provide insight into the pathogenic mechanism of this gain-of-function mutation of SCN8A and identify excitatory neurons as critical targets for therapeutic intervention.

Neuronal hyperexcitability in a mouse model of SCN8A epileptic encephalopathy.

Patients with early infantile epileptic encephalopathy (EIEE) experience severe seizures and cognitive impairment and are at increased risk for sudden unexpected death in epilepsy (SUDEP). EIEE13 [Online Mendelian Inheritance in Man (OMIM) # 614558] is caused by de novo missense mutations in the voltage-gated sodium channel gene SCN8A Here, we investigated the neuronal phenotype of a mouse model expressing the gain-of-function SCN8A patient mutation, p.Asn1768Asp (Nav1.6-N1768D). Our results revealed regional and neuronal subtype specificity in the effects of the N1768D mutation. Acutely dissociated hippocampal neurons from Scn8aN1768D/+ mice showed increases in persistent sodium current (INa) density in CA1 pyramidal but not bipolar neurons. In CA3, INa,P was increased in both bipolar and pyramidal neurons. Measurement of action potential (AP) firing in Scn8aN1768D/+ pyramidal neurons in brain slices revealed early afterdepolarization (EAD)-like AP waveforms in CA1 but not in CA3 hippocampal or layer II/III neocortical neurons. The maximum spike frequency evoked by depolarizing current injections in Scn8aN1768D/+ CA1, but not CA3 or neocortical, pyramidal cells was significantly reduced compared with WT. Spontaneous firing was observed in subsets of neurons in CA1 and CA3, but not in the neocortex. The EAD-like waveforms of Scn8aN1768D/+ CA1 hippocampal neurons were blocked by tetrodotoxin, riluzole, and SN-6, implicating elevated persistent INa and reverse mode Na/Ca exchange in the mechanism of hyperexcitability. Our results demonstrate that Scn8a plays a vital role in neuronal excitability and provide insight into the mechanism and future treatment of epileptogenesis in EIEE13.

The splicing regulator Rbfox2 is required for both cerebellar development and mature motor function.

The Rbfox proteins (Rbfox1, Rbfox2, and Rbfox3) regulate the alternative splicing of many important neuronal transcripts and have been implicated in a variety of neurological disorders. However, their roles in brain development and function are not well understood, in part due to redundancy in their activities. Here we show that, unlike Rbfox1 deletion, the CNS-specific deletion of Rbfox2 disrupts cerebellar development. Genome-wide analysis of Rbfox2(-/-) brain RNA identifies numerous splicing changes altering proteins important both for brain development and mature neuronal function. To separate developmental defects from alterations in the physiology of mature cells, Rbfox1 and Rbfox2 were deleted from mature Purkinje cells, resulting in highly irregular firing. Notably, the Scn8a mRNA encoding the Na(v)1.6 sodium channel, a key mediator of Purkinje cell pacemaking, is improperly spliced in RbFox2 and Rbfox1 mutant brains, leading to highly reduced protein expression. Thus, Rbfox2 protein controls a post-transcriptional program required for proper brain development. Rbfox2 is subsequently required with Rbfox1 to maintain mature neuronal physiology, specifically Purkinje cell pacemaking, through their shared control of sodium channel transcript splicing.

Cerebral hypomyelination associated with biallelic variants of FIG4.

The lipid phosphatase gene FIG4 is responsible for Yunis-Varón syndrome and Charcot-Marie-Tooth disease Type 4J, a peripheral neuropathy. We now describe four families with FIG4 variants and prominent abnormalities of central nervous system (CNS) white matter (leukoencephalopathy), with onset in early childhood, ranging from severe hypomyelination to mild undermyelination, in addition to peripheral neuropathy. Affected individuals inherited biallelic FIG4 variants from heterozygous parents. Cultured fibroblasts exhibit enlarged vacuoles characteristic of FIG4 dysfunction. Two unrelated families segregate the same G > A variant in the +1 position of intron 21 in the homozygous state in one family and compound heterozygous in the other. This mutation in the splice donor site of exon 21 results in read-through from exon 20 into intron 20 and truncation of the final 115 C-terminal amino acids of FIG4, with retention of partial function. The observed CNS white matter disorder in these families is consistent with the myelination defects in the FIG4 null mouse and the known role of FIG4 in oligodendrocyte maturation. The families described here the expanded clinical spectrum of FIG4 deficiency to include leukoencephalopathy.