Biology subjects

Weckhuysen, S.

Publications and source records attributed to Weckhuysen, S..

9 recordsLinked to original sources

bioRxiv2025

KCNQ2 Loss-of-Function variants disrupt neuronal maturation via early hyperexcitability followed by maladaptive network remodeling

Loss-of-function (LOF) variants in the potassium channel subunit KCNQ2 cause a spectrum of neonatal epilepsies from self-limiting familial neonatal epilepsy (SeLFNE) to severe developmental and epileptic encephalopathy (DEE). To dissect the developmental consequences of KCNQ2 LOF, we conducted a longitudinal and multimodal comparative analysis in a human neuronal model generated from patients with KCNQ2-DEE and KCNQ2-SeLFNE. KCNQ2-LOF induced a biphasic network dysfunction, with early Kv7-driven hyperexcitability rescued by acute Retigabine (RTG) treatment, followed by maladaptive remodeling in the opposite direction. Transcriptomic analysis mirrored this biphasic dynamic trajectory, revealing an initial upregulation followed by a subsequent downregulation of synaptic genes. Structural analysis showed a steeper decline in presynaptic density alongside a distal shift in the axon initial segment (AIS) throughout maturation, and impaired AIS plasticity at later stages. Overall, KCNQ2-LOF disrupts human neuronal maturation through dynamic, biphasic changes in function, gene expression and structure, offering insights into disease mechanisms and therapeutic options.

neuroscience↗

bioRxiv2024

CONSTITUTIVE OPENING OF THE Kv7.2 PORE ACTIVATION GATE CAUSES KCNQ2-DEVELOPMENTAL ENCEPHALOPATHY

Pathogenic variants in KCNQ2 encoding for Kv7.2 voltage-gated potassium channel subunits cause developmental encephalopathies (KCNQ2-encephalopathies), both with and without epilepsy. We herein describe the clinical, in vitro and in silico features of two encephalopathy-causing variants (A317T, L318V) in Kv7.2 affecting two consecutive residues in the S6 activation gate undergoing large structural rearrangements during pore opening. Currents through these mutant channels displayed increased density, hyperpolarizing shifts in activation gating, and insensitivity to phosphatidylinositol 4,5-bisphosphate (PIP2), a critical regulator of Kv7 channel function; all these features are consistent with a strong gain-of-function effect. An increase in single-channel open probability, with no change in membrane abundance or single-channel conductance, was responsible for the observed gain-of-function effects. All-atoms Molecular Dynamics simulations revealed that the mutations widened the inner pore gate and stabilized a constitutively open channel configuration in the closed state, with minimal effects on the open conformation. Thus, a PIP2-independent stabilization of the inner pore gate open configuration is a novel molecular pathogenetic mechanism for KCNQ2-developmental encephalopathies.

biophysics↗

bioRxiv2024

Electrophysiological signatures of a developmental delay in a stem cell model of KCNQ2 developmental and epileptic encephalopathy

BackgroundKCNQ2, encoding KV7.2 ion channels, has emerged as one of the prominent genes causing early onset seizures with developmental delay (KCNQ2 developmental and epileptic encephalopathy; KCNQ2-DEE). KCNQ2 de novo loss-of-function (LOF) and associated neuronal hyperexcitability have been accepted as mechanisms contributing to seizures. To investigate the developmental impact of KCNQ2 LOF, we generated patient iPSC-derived models for two previously reported de novo variants, p.(Arg325Gly) and p.(Gly315Arg), linked to severe congenital DEE. MethodsFunctional investigation of the two variants was initially performed in Xenopus laevis oocyte system. Patient-derived iPSC lines were differentiated using NGN2- and embryoid body-based protocols yielding neurons roughly corresponding to mid- and mid-late gestational stages, respectively. KV7- mediated M-current, passive neuronal properties, action potential generation and spontaneous oscillatory network activities were analysed with whole-cell patch clamping. FindingsStudied KCNQ2 variants showed LOF with a dominant-negative effect in the heterologous system. Reduced M-currents in patient iPSC-derived neurons corroborated a LOF as the main pathomechanism. Interestingly, this led to the reduced neuronal firing of the early neurons and to a disruption of complex oscillatory activity, with significantly reduced duration and amplitude of these events in patient iPSC-derived neurons. InterpretationWe provide experimental evidence for changing roles of the M-current throughout development and place disease variant-mediated M-current reduction in the context of the neuronal maturation in the prenatal brain. Based on the reduced neuronal firing and disrupted oscillatory activity seen in patient iPSC-derived neurons, we propose that a delayed/impaired maturation of neuronal and network properties underlies KCNQ-DEE caused by LOF variants.

neuroscience↗

bioRxiv2023

Long-read sequencing and profiling of RNA-binding proteins reveals the pathogenic mechanism of aberrant splicing of an SCN1A poison exon in epilepsy

Pathogenic loss-of-function SCN1A variants cause a spectrum of seizure disorders. We previously identified variants in individuals with SCN1A-related epilepsy that fall in or near a poison exon (PE) in SCN1A intron 20 (20N). We hypothesized these variants lead to increased PE inclusion, which introduces a premature stop codon, and, therefore, reduced abundance of the full-length SCN1A transcript and Nav1.1 protein. We used a splicing reporter assay to interrogate PE inclusion in HEK293T cells. In addition, we used patient-specific induced pluripotent stem cells (iPSCs) differentiated into neurons to quantify 20N inclusion by long and short-read sequencing and Nav1.1 abundance by western blot. We performed RNA-antisense purification with mass spectrometry to identify RNA-binding proteins (RBPs) that could account for the aberrant PE splicing. We demonstrate that variants in/near 20N lead to increased 20N inclusion by long-read sequencing or splicing reporter assay and decreased Nav1.1 abundance. We also identified 28 RBPs that differentially interact with variant constructs compared to wild-type, including SRSF1 and HNRNPL. We propose a model whereby 20N variants disrupt RBP binding to splicing enhancers (SRSF1) and suppressors (HNRNPL), to favor PE inclusion. Overall, we demonstrate that SCN1A 20N variants cause haploinsufficiency and SCN1A-related epilepsies. This work provides insights into the complex control of RBP-mediated PE alternative splicing, with broader implications for PE discovery and identification of pathogenic PE variants in other genetic conditions.

genetics↗

bioRxiv2022

Efficient and accurate prime editing strategy to correct genetic alterations in hiPSC using single EF-1alpha driven all-in-one plasmids

Prime editing (PE) is currently the most effective and versatile technology to make targeted alterations in the genome. Several improvements to the PE machinery have recently been made, and have been tested in a range of model systems, including immortalized cell lines, stem-cells and animal models. While nick RNA (ncRNA)-dependent PE systems like PE3 and PE5 are currently considered to be the most effective, they come with undesired indels or SNVs at the edit locus. Here, we aimed to improve ncRNA-independent systems PE2 and PE4max by generating novel all-in-one (pAIO) plasmids, driven by a tissue-broad EF-1alpha promoter, that is especially suitable for human iPSC models, and linked to a GFP tag for fluorescent based sorting. These novel pAIO systems effectively corrected mutations observed in patients suffering from epileptic encephalopathy, including a truncating SCN1A R612* variant in HEK293T cells and a gain-of-function KCNQ2 R201C variant in patient-derived hiPSC, with edit efficiency up to 50%. We also show that introducing additional silent PAM-removing mutations can negatively influence edit efficiency. Finally, we observed an absence of genome-wide PE off-target effects at pegRNA homology sites. Taken together, our study shows an improved efficacy and accuracy of EF-1alpha driven ncRNA-independent pAIO PE plasmids in hiPSC.

molecular biology↗

bioRxiv2019

Systematic re-annotation of 191 genes associated with early-onset epilepsy unmasks de novo variants linked to Dravet syndrome in novel SCN1A exons

The early infantile epileptic encephalopathies (EIEE) are a group of rare, severe neurodevelopmental disorders, where even the most thorough sequencing studies leave 60-65% of patients without a molecular diagnosis. Here, we explore the incompleteness of transcript models used for exome and genome analysis as one potential explanation for lack of current diagnoses. Therefore, we have updated the GENCODE gene annotation for 191 epilepsy-associated genes, using human brain-derived transcriptomic libraries and other data to build 3,550 novel putative transcript models. The extended transcriptional footprint of these genes allowed for 294 intronic or intergenic variants, found in human mutation databases, to be reclassified as exonic, while a further 70 intronic variants were reclassified as splice-site proximal. Using SCN1A as a case study due to its close phenotype/genotype correlation with Dravet syndrome, we screened 122 people with Dravet syndrome, or a similar phenotype, with a panel of novel exon sequences representing eight established genes and identified two de novo SCN1A variants that now, through improved gene annotation can be ascribed to residing among novel exons. These two (from 122 screened patients, 1.6%) new molecular diagnoses carry significant clinical implications. Furthermore, we identified a previously-classified SCN1A intronic Dravet-associated variant that now lies within a deeply conserved novel exon. Our findings illustrate the potential gains of thorough gene annotation in improving diagnostic yields for genetic disorders. We would expect to find new molecular diagnoses in our 191 genes that were originally suspected by clinicians for patients, with a negative diagnosis.

genomics↗

bioRxiv2019

Ultra-rare genetic variation in the epilepsies: a whole-exome sequencing study of 17,606 individuals

Sequencing-based studies have identified novel risk genes for rare, severe epilepsies and revealed a role of rare deleterious variation in common epilepsies. To identify the shared and distinct ultra-rare genetic risk factors for rare and common epilepsies, we performed a whole-exome sequencing (WES) analysis of 9,170 epilepsy-affected individuals and 8,364 controls of European ancestry. We focused on three phenotypic groups; the rare but severe developmental and epileptic encephalopathies (DEE), and the commoner phenotypes of genetic generalized epilepsy (GGE) and non-acquired focal epilepsy (NAFE). We observed that compared to controls, individuals with any type of epilepsy carried an excess of ultra-rare, deleterious variants in constrained genes and in genes previously associated with epilepsy, with the strongest enrichment seen in DEE and the least in NAFE. Moreover, we found that inhibitory GABAA receptor genes were enriched for missense variants across all three classes of epilepsy, while no enrichment was seen in excitatory receptor genes. The larger gene groups for the GABAergic pathway or cation channels also showed a significant mutational burden in DEE and GGE. Although no single gene surpassed exome-wide significance among individuals with GGE or NAFE, highly constrained genes and genes encoding ion channels were among the top associations, including CACNA1G, EEF1A2, and GABRG2 for GGE and LGI1, TRIM3, and GABRG2 for NAFE. Our study confirms a convergence in the genetics of common and rare epilepsies associated with ultra-rare coding variation and highlights a ubiquitous role for GABAergic inhibition in epilepsy etiology in the largest epilepsy WES study to date.

genetics↗

bioRxiv2017

Gene family information facilitates variant interpretation and identification of disease-associated genes

Differentiating risk-conferring from benign missense variants, and therefore optimal calculation of gene-variant burden, represent a major challenge in particular for rare and genetic heterogeneous disorders. While orthologous gene conservation is commonly employed in variant annotation, approximately 80% of known disease-associated genes are paralogs and belong to gene families. It has not been thoroughly investigated how gene family information can be utilized for disease gene discovery and variant interpretation. We developed a paralog conservation score to empirically evaluate whether paralog conserved or nonconserved sites of in-human paralogs are important for protein function. Using this score, we demonstrate that disease-associated missense variants are significantly enriched at paralog conserved sites across all disease groups and disease inheritance models tested. Next, we assessed whether gene family information could assist in discovering novel disease-associated genes. We subsequently developed a gene family de novo enrichment framework that identified 43 exome-wide enriched gene families including 98 de novo variant carrying genes in more than 10k neurodevelopmental disorder patients. 33 gene family enriched genes represent novel candidate genes which are brain expressed and variant constrained in neurodevelopmental disorders.

genetics↗

bioRxiv2017

The Spectrum Of De Novo Variants In Neurodevelopmental Disorders With Epilepsy

Epilepsy is a frequent feature of neurodevelopmental disorders (NDD) but little is known about genetic differences between NDD with and without epilepsy. We analyzed de novo variants (DNV) in 6753 parent-offspring trios ascertained for different NDD. In the subset of 1942 individuals with NDD with epilepsy, we identified 33 genes with a significant excess of DNV, of which SNAP25 and GABRB2 had previously only limited evidence for disease association. Joint analysis of all individuals with NDD also implicated CACNA1E as a novel disease gene. Comparing NDD with and without epilepsy, we found missense DNV, DNV in specific genes, age of recruitment and severity of intellectual disability to be associated with epilepsy. We further demonstrate to what extent our results impact current genetic testing as well as treatment, emphasizing the benefit of accurate genetic diagnosis in NDD with epilepsy.

genetics↗