Biallelic variants in ZNF526 cause a severe neurodevelopmental disorder with microcephaly, bilateral cataract, epilepsy and simplified gyration.

BACKGROUND: Next-generation sequencing, combined with international pooling of cases, has impressively enhanced the discovery of genes responsible for Mendelian neurodevelopmental disorders, particularly in individuals affected by clinically undiagnosed diseases. To date, biallelic missense variants in ZNF526 gene, encoding a Krüppel-type zinc-finger protein, have been reported in three families with non-syndromic intellectual disability. METHODS: Here, we describe five individuals from four unrelated families with an undiagnosed neurodevelopmental disorder in which we performed exome sequencing, on a combination of trio-based (4 subjects) or single probands (1 subject). RESULTS: We identified five patients from four unrelated families with homozygous ZNF526 variants by whole exome sequencing. Four had variants resulting in truncation of ZNF526; they were affected by severe prenatal and postnatal microcephaly (ranging from -4 SD to -8 SD), profound psychomotor delay, hypertonic-dystonic movements, epilepsy and simplified gyral pattern on MRI. All of them also displayed bilateral progressive cataracts. A fifth patient had a homozygous missense variant and a slightly less severe disorder, with postnatal microcephaly (-2 SD), progressive bilateral cataracts, severe intellectual disability and unremarkable brain MRI.Mutant znf526 zebrafish larvae had notable malformations of the eye and central nervous system, resembling findings seen in the human holoprosencephaly spectrum. CONCLUSION: Our findings support the role of ZNF526 biallelic variants in a complex neurodevelopmental disorder, primarily affecting brain and eyes, resulting in severe microcephaly, simplified gyral pattern, epileptic encephalopathy and bilateral cataracts.

Ste20-like Kinase Is Critical for Inhibitory Synapse Maintenance and Its Deficiency Confers a Developmental Dendritopathy.

The size and structure of the dendritic arbor play important roles in determining how synaptic inputs of neurons are converted to action potential output. The regulatory mechanisms governing the development of dendrites, however, are insufficiently understood. The evolutionary conserved Ste20/Hippo kinase pathway has been proposed to play an important role in regulating the formation and maintenance of dendritic architecture. A key element of this pathway, Ste20-like kinase (SLK), regulates cytoskeletal dynamics in non-neuronal cells and is strongly expressed throughout neuronal development. However, its function in neurons is unknown. We show that, during development of mouse cortical neurons, SLK has a surprisingly specific role for proper elaboration of higher, ≥ third-order dendrites both in male and in female mice. Moreover, we demonstrate that SLK is required to maintain excitation-inhibition balance. Specifically, SLK knockdown caused a selective loss of inhibitory synapses and functional inhibition after postnatal day 15, whereas excitatory neurotransmission was unaffected. Finally, we show that this mechanism may be relevant for human disease, as dysmorphic neurons within human cortical malformations revealed significant loss of SLK expression. Overall, the present data identify SLK as a key regulator of both dendritic complexity during development and inhibitory synapse maintenance.SIGNIFICANCE STATEMENT We show that dysmorphic neurons of human epileptogenic brain lesions have decreased levels of the Ste20-like kinase (SLK). Decreasing SLK expression in mouse neurons revealed that SLK has essential functions in forming the neuronal dendritic tree and in maintaining inhibitory connections with neighboring neurons.

Mosaic and non-mosaic protocadherin 19 mutation leads to neuronal hyperexcitability in zebrafish.

Epilepsy is one of the most common neurological disorders. The X-linked gene PCDH19 is associated with sporadic and familial epilepsy in humans, typically with early-onset clustering seizures and intellectual disability in females but not in so-called 'carrier' males, suggesting that mosaic PCDH19 expression is required to produce epilepsy. To characterize the role of loss of PCDH19 function in epilepsy, we generated zebrafish with truncating pcdh19 variants. Evaluating zebrafish larvae for electrophysiological abnormalities, we observed hyperexcitability phenotypes in both mosaic and non-mosaic pcdh19+/- and pcdh19-/- mutant larvae. Thus, we demonstrate that the key feature of epilepsy-network hyperexcitability-can be modeled effectively in zebrafish, even though overt spontaneous seizure-like swim patterns were not observed. Further, zebrafish with non-mosaic pcdh19 mutation displayed reduced numbers of inhibitory interneurons suggesting a potential cellular basis for the observed hyperexcitability. Our findings in both mosaic and non-mosaic pcdh19 mutant zebrafish challenge the prevailing theory that mosaicism governs all PCDH19-related phenotypes and point to interneuron-mediated mechanisms underlying these phenotypes.

Minute amounts of hamartin wildtype rescue the emergence of tuber-like lesions in conditional Tsc1 ablated mice.

Tuberous sclerosis (TSC) is a phacomatosis associated with highly differentiated malformations including tubers in the brain. Those are composed of large dysplastic neurons and 'giant cells'. Cortical tubers are frequent causes of chronic seizures and resemble neuropathologically focal cortical dysplasias (FCD) type IIb. Patients with FCDIIb, however, lack additional stigmata of TSC. Mutations and allelic variants of the TSC1 gene have been observed in patients with tubers as well as FCDIIb. Those include hamartin(R692X) and hamartin(R786X), stop mutants frequent in TSC patients and hamartin(H732Y) frequent in FCDIIb. Expression of these variants in cell culture led to aberrant distribution of corresponding proteins. We here scrutinized morphological and structural effects of these TSC1 variants by intraventricular in utero electroporation (IUE), genetically mimicking the discrete focal character and a somatic postzygotic mosaicism of the lesion, focusing on the gene dosage required for tuber-like lesions to emerge in Tsc1(flox/flox) mice. Expression of only hamartin(R692X) as well as hamartin(R786X) led to a 2-fold enlargement of neurons with high pS6 immunoreactivity, stressing their in vivo pathogenic potential. Co-electroporation of the different aberrant alleles and varying amounts of wildtype TSC1 surprisingly revealed already minimal amounts of functional hamartin to be sufficient for phenotype rescue. This result strongly calls for further studies to unravel new mechanisms for substantial silencing of the second allele in cortical tubers, as proposed by Knudson's '2-hit hypothesis'. The rescuing effects may provide a promising basis for gene therapies aiming at reconstituting hamartin expression in tubers.

LRP12 silencing during brain development results in cortical dyslamination and seizure sensitization.

Correct positioning and differentiation of neurons during brain development is a key precondition for proper function. Focal cortical dysplasias (FCDs) are increasingly recognized as causes of therapy refractory epilepsies. Neuropathological analyses of respective surgical specimens from neurosurgery for seizure control often reveal aberrant cortical architecture and/or aberrantly shaped neurons in FCDs. However, the molecular pathogenesis particularly of FCDs with aberrant lamination (so-called FCD type I) is largely unresolved. Lipoproteins and particularly low-density lipoprotein receptor-related protein 12 (LRP12) are involved in brain development. Here, we have examined a potential role of LRP12 in the pathogenesis of FCDs. In vitro knockdown of LRP12 in primary neurons results in impaired neuronal arborization. In vivo ablation of LRP12 by intraventricularly in utero electroporated shRNAs elicits cortical maldevelopment, i.e. aberrant lamination by malpositioning of upper cortical layer neurons. Subsequent epilepsy phenotyping revealed pentylenetetrazol (PTZ)-induced seizures to be aggravated in cortical LRP12-silenced mice. Our data demonstrates IUE mediated cortical gene silencing as an excellent approach to study the role of distinct molecules for epilepsy associated focal brain lesions and suggests LRP12 and lipoprotein homeostasis as potential molecular target structures for the emergence of epilepsy-associated FCDs.

Non-paraneoplastic limbic encephalitis and central nervous HHV-6B reactivation: Causality or coincidence?

Autoantibody-related encephalopathies represent an important differential diagnosis in adult onset epilepsy. Here, we report the case of a 25-year-old patient with new-onset epilepsy and psychotic syndrome, who underwent biopsy resection for etiological classification. MRI analysis and neuropathological examination showed a T-lymphocytic dominated encephalitis with involvement of the limbic system. An indirect immunohistochemistry approach identified autoantibodies against glutamic acid decarboxylase (GAD) in cerebral spinal fluid and serum, which were confirmed by affinity purification / mass spectrometry analysis. Further examinations revealed evidence of chromosomally integrated human herpes virus type 6B (HHV-6B). However, astrocytic expression of HHV-6 lytic protein was detected by double immunofluorescence analysis. The cerebral expression of HHV-6 antigen, a clinical improvement under antiviral therapy as well as an initial finding of HHV-6 IgM antibodies strongly argue for an additional active HHV-6B infection. Review of the literature reveals singular reports of patients with GAD antibody-positive limbic encephalitis and central nervous system infections with HHV-6B. Since herpes simplex virus encephalitis has been recently reported as a trigger of N-methyl-D-aspartate receptor antibody encephalitis, it is tempting to speculate that HHV-6B infections may trigger a non-paraneoplastic form of limbic encephalitis in a parallel cascade.

Zebrafish models of candidate human epilepsy-associated genes provide evidence of hyperexcitability.

Hundreds of novel candidate human epilepsy-associated genes have been identified thanks to advancements in next-generation sequencing and large genome-wide association studies, but establishing genetic etiology requires functional validation. We generated a list of >2200 candidate epilepsy-associated genes, of which 81 were determined suitable for the generation of loss-of-function zebrafish models via CRISPR/Cas9 gene editing. Of those 81 crispants, 48 were successfully established as stable mutant lines and assessed for seizure-like swim patterns in a primary F2 screen. Evidence of seizure-like behavior was present in 5 (arfgef1, kcnd2, kcnv1, ubr5, wnt8b) of the 48 mutant lines assessed. Further characterization of those 5 lines provided evidence for epileptiform activity via electrophysiology in kcnd2 and wnt8b mutants. Additionally, arfgef1 and wnt8b mutants showed a decrease in the number of inhibitory interneurons in the optic tectum of larval animals. Furthermore, RNAseq revealed convergent transcriptional abnormalities between mutant lines, consistent with their developmental defects and hyperexcitable phenotypes. These zebrafish models provide strongest experimental evidence supporting the role of ARFGEF1, KCND2, and WNT8B in human epilepsy and further demonstrate the utility of this model system for evaluating candidate human epilepsy genes.

Zebrafish models of candidate human epilepsy-associated genes provide evidence of hyperexcitability.

Hundreds of novel candidate human epilepsy-associated genes have been identified thanks to advancements in next-generation sequencing and large genome-wide association studies, but establishing genetic etiology requires functional validation. We generated a list of >2,200 candidate epilepsy-associated genes, of which 48 were developed into stable loss-of-function (LOF) zebrafish models. Of those 48, evidence of seizure-like behavior was present in 5 (arfgef1, kcnd2, kcnv1, ubr5, and wnt8b). Further characterization provided evidence for epileptiform activity via electrophysiology in kcnd2 and wnt8b mutants. Additionally, arfgef1 and wnt8b mutants showed a decrease in the number of inhibitory interneurons in the optic tectum of larval animals. Further, RNA sequencing (RNA-seq) revealed convergent transcriptional abnormalities between mutant lines, consistent with their developmental defects and hyperexcitable phenotypes. These zebrafish models provide strongest experimental evidence supporting the role of ARFGEF1, KCND2, and WNT8B in human epilepsy and further demonstrate the utility of this model system for evaluating candidate human epilepsy genes.

Abundance of LRP12 C-rs9694676 allelic promoter variant in epilepsy-associated gangliogliomas.

AIMS: Chronic epilepsy associated gangliogliomas (GGs) represent tumors composed of irregularly distributed, often dysmorphic, neurons and neoplastic astroglia. The pathogenesis of GGs is largely unknown. Low-density lipoprotein receptor-related protein 12 (LRP12) is critical for brain development and involved in tumorigenesis of non-cerebral neoplasms. MAIN METHODS: Here, we have examined a potential role of LRP12 in the pathogenesis of GGs by a combination of mRNA quantification and molecular-biological in vitro assays. KEY FINDINGS: We observed a significant increase of the single nucleotide polymorphism (SNP) rs9694676 C-allele, located in the LRP12 promoter, in GGs compared to normal control individuals. C-allele expression is correlated with abundant seizure frequency. Expression of LRP12 was lower in GGs than in control brain. In luciferase assays, the C-allele of rs9694676 decreases both, the basal LRP12 core promoter activity and the stimulatory effect of the transcription factor (TF) STAT5a. SIGNIFICANCE: Accumulation of functional promoter-associated allelic variants with impact on the transcriptional regulation of LRP12 provides a new pathomechanism for GGs, i.e. highly differentiated epileptogenic brain tumors.