Biology subjects

Scheffer, I. E.

Publications and source records attributed to Scheffer, I. E..

10 recordsLinked to original sources

bioRxiv2026

SYNGAP1 haploinsufficiency disrupts early neurodevelopment and accelerates intrinsic neuronal maturation in human patient-derived models

SYNGAP1 developmental and epileptic encephalopathy (DEE) is a severe neurodevelopmental disorder characterised by intellectual disability, developmental delay, and refractory epilepsy caused by heterozygous variants in SYNGAP1, which encodes Synaptic Ras GTPase-activating protein 1. While SYNGAP1 is best known for its role at the postsynaptic density, increasing evidence indicates that haploinsufficiency also disrupts early neurodevelopment. Here, we used patient-derived induced pluripotent stem cell (iPSC) models to investigate early neurodevelopmental and neuronal phenotypes associated with SYNGAP1 haploinsufficiency. iPSCs derived from a female patient carrying the frameshift variant p.Leu150Valfs*6 were differentiated into two complementary models: micropatterned neural rosettes representing early neuroepithelial organisation and NGN2-induced excitatory neurons representing postmitotic functional development. Patient-derived neural rosettes displayed enlarged, dysmorphic lumens, indicating disrupted neuroepithelial organisation at the earliest stages of brain development. Transcriptomic profiling revealed widespread dysregulation of genes involved in neurodevelopment, cell adhesion and ion channel regulation, including coordinated downregulation of protocadherin family members. Whole-cell patch-clamp electrophysiology demonstrated reduced input resistance, larger action potential amplitudes, and increased inward and outward current densities, consistent with accelerated intrinsic neuronal maturation rather than generalized hyperexcitability. Together, these complementary findings demonstrate that SYNGAP1 haploinsufficiency disrupts early human brain development and accelerates intrinsic neuronal maturation, with pathogenic mechanisms emerging before synaptogenesis and extending beyond SYNGAP1s established synaptic role.

neuroscience↗

bioRxiv2026

Knowledge Inclusive Machine Learning for Disease Gene Prioritisation

The predictive performance of machine learning models depends on the context available to them. In disease gene prioritisation, this context comprises two forms: specific context from sample-level experimental data, such as gene expression and protein-protein interaction networks, and general context from accumulated and curated biological knowledge capturing established relationships among genes, diseases, and pathways. Neither is sufficient alone: experimental data are sensitive to dataset-specific noise and lack broader biological grounding, while curated knowledge lacks the resolution required for gene-level discrimination. Consequently, most machine learning approaches relying solely on experimental data risk learning spurious correlations rather than underlying biology. Here we introduce Knowledge Inclusive Machine Learning (KIML), a paradigm that integrates both context types within a unified analytical pipeline. KIML combines experimental data with two types of general context: literature-derived representations from PubMed and structured biomedical knowledge graphs. We evaluate the approach on Developmental and Epileptic Encephalopathy and benchmark it against recent methods using publicly available datasets. Performance is assessed using temporal-split evaluation and biological evaluations, including ontology enrichment analysis. KIML consistently outperforms existing approaches, providing improved predictive accuracy and biologically meaningful insights. Furthermore, the framework generates interpretable explanations of gene prioritisation and demonstrates strong generalisability across six additional diseases.

genetics↗

bioRxiv2026

Activating Ras-MAPK pathway variants drive hippocampal clonal competition in human epilepsy

Mesial (a.k.a., medial) temporal lobe epilepsy (MTLE) is the most common focal epilepsy1,2 and, in drug-resistant cases, is treated by surgical removal of the anterior temporal lobe, which often shows neuronal loss and gliosis consistent with hippocampal sclerosis (HS)2. MTLE with HS has minimal contribution from germline genetic variation3, and is associated with prior precipitating insults such as prolonged childhood seizures and head trauma4-6. Somatic variants in Ras-MAPK pathway genes were recently reported in a few MTLE surgical specimens7,8, but their prevalence, clinical relevance, and underlying biological mechanisms remain unknown. Targeted duplex sequencing of hippocampal DNA from 462 surgical resections revealed significant enrichment of deleterious somatic variants in MTLE versus controls, with >40% of MTLE specimens harboring activating Ras-MAPK variants in PTPN11, NF1, BRAF, KRAS, and twelve genes not previously associated with focal epilepsy. Eight Ras-MAPK genes showed positive clonal selection in MTLE. Increased somatic variant burden predicted worse surgical outcome. Somatic Ras-MAPK variants at ultra-low (<0.5%) variant allele fractions were associated with older seizure onset and HS pathology, supporting a late prenatal or postnatal origin. Ras-MAPK variants in MTLE were enriched in cells derived from hippocampal progenitors--neurons, astrocytes, oligodendrocytes--in line with the known neuronal hyperexcitability and seizures induced by Ras-MAPK overactivation9,10; in contrast, Alzheimer disease hippocampi exhibited microglial enrichment of Ras-MAPK variants, consistent with prior reports11. Single-nucleus RNA sequencing showed increased expression of Ras-MAPK genes in neurons and upregulation of pathways mediating neurogenesis and neural development in MTLE. Functional validation of novel, recurrent PTPN11 variants confirmed gain-of-function, while cellular modeling in induced pluripotent stem cells demonstrated proliferative/survival advantages for mutant cells in mosaic culture. Overall, our data suggest that somatic Ras-MAPK variants and acquired risk factors may converge on clonal competition in the hippocampus to modulate epilepsy risk.

genetics↗

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

Optical genome mapping enables accurate repeat expansion testing

Short tandem repeats (STRs) are amongst the most abundant class of variations in human genomes and are meiotically and mitotically unstable which leads to expansions and contractions. STR expansions are frequently associated with genetic disorders, with the size of expansions often correlating with the severity and age of onset. Therefore, being able to accurately detect the total repeat expansion length and to identify potential somatic repeat instability is important. Current standard of care (SOC) diagnostic assays include laborious repeat-primed PCR-based tests as well as Southern blotting, which are unable to precisely determine long repeat expansions and/or require a separate set-up for each locus. Sequencing-based assays have proven their potential for the genome-wide detection of repeat expansions but have not yet replaced these diagnostic assays due to their inaccuracy to detect long repeat expansions (short-read sequencing) and their costs (long-read sequencing). Here, we tested whether optical genome mapping (OGM) can efficiently and accurately identify the STR length and assess the stability of known repeat expansions. We performed OGM for 85 samples with known clinically relevant repeat expansions in DMPK, CNBP and RFC1, causing myotonic dystrophy type 1 and 2 and cerebellar ataxia, neuropathy and vestibular areflexia syndrome (CANVAS), respectively. After performing OGM, we applied three different repeat expansion detection workflows, i.e. manual de novo assembly, local guided assembly (local-GA) and molecule distance script of which the latter two were developed as part of this study. The first two workflows estimated the repeat size for each of the two alleles, while the third workflow was used to detect potential somatic instability. The estimated repeat sizes were compared to the repeat sizes reported after the SOC and concordance between the results was determined. All except one known repeat expansions above the pathogenic repeat size threshold were detected by OGM, and allelic differences were distinguishable, either between wildtype and expanded alleles, or two expanded alleles for recessive cases. An apparent strength of OGM over current SOC methods was the more accurate length measurement, especially for very long repeat expansion alleles, with no upper size limit. In addition, OGM enabled the detection of somatic repeat instability, which was detected in 9/30 DMPK, 23/25 CNBP and 4/30 RFC1 samples, leveraging the analysis of intact, native DNA molecules. In conclusion, for tandem repeat expansions larger than [~]300 bp, OGM provides an efficient method to identify exact repeat lengths and somatic repeat instability with high confidence across multiple loci simultaneously, enabling the potential to provide a significantly improved and generic genome-wide assay for repeat expansion disorders.

genomics↗

bioRxiv2023

Atenolol reduces cardiac-mediated mortality in a genetic mouse model of sudden unexpected death in epilepsy

Sudden Unexpected Death in Epilepsy (SUDEP) is the leading cause of premature mortality in epilepsy. Genetic cardiac risk factors, including loss-of-function KCNH2 variants, have been linked to SUDEP. We hypothesised that seizures and LQTS interact to increase SUDEP risk. To investigate this, we crossed Kcnh2+/- and Gabrg2R43Q/+ mice that model LQTS and genetic epilepsy, respectively. Electrocorticography and electrocardiogram confirmed that Kcnh2+/- mice had a LQTS phenotype, while Gabrg2R43Q/+ mice displayed spontaneous seizures. Double mutant mice (Gabrg2R43Q/+/Kcnh2+/-) had both seizure and LQTS phenotypes that were indistinguishable from the respective single mutant mice. Survival analysis revealed that Gabrg2R43Q/+/Kcnh2+/- mice experienced a disproportionate higher rate of seizure-related death. Long-term oral administration of atenolol, a cardiac-selective {beta}-blocker, significantly improved survival in the Gabrg2R43Q/+/Kcnh2+/- mice. An additional mouse model, Hcn1M294L/+/Kcnh2+/-, based on a HCN1 developmental epileptic encephalopathy variant, also experienced a disproportionately higher rate of premature death that was rescued by atenolol. Kcnh2+/- mice also spent more time in ventricular arrhythmia during proconvulsant-induced seizures. Overall, the data implicates cardiac and loss-of-function KCNH2 variants as an important risk factor, and the potential repurposing of {beta}- blockers as a prevention strategy, for SUDEP in a subset of epilepsy patients.

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↗

bioRxiv2021

Loss-of-function variants in the cardiac Kv11.1 channel as a genetic biomarker for SUDEP

ObjectiveTo compare the frequency and impact on channel function of KCNH2 variants in SUDEP patients with epilepsy controls comprising patients older than 50 years, a group with low SUDEP risk, and establish loss-of-function KCNH2 variants as predictive biomarkers of SUDEP risk. MethodsWe searched for KCNH2 variants with a minor allele frequency of < 5%. Functional analysis in Xenopus laevis oocytes was performed for all KCNH2 variants identified. ResultsKCNH2 variants were found in 11.1% (10/90) of SUDEP individuals compared to 6.0% (20/332) of epilepsy controls (p = 0.11). Loss-of-function KCNH2 variants, defined as causing > 20% reduction in maximal amplitude, were observed in 8.9% (8/90) SUDEP patients compared to 3.3% (11/332) epilepsy controls suggesting about three-fold enrichment (nominal p = 0.04). KCNH2 variants that did not change channel function occurred at a similar frequency in SUDEP (2.2%; 2/90) and epilepsy control (2.7%; 9/332) cohorts (p > 0.99). Rare KCNH2 variants (< 1% allele frequency) associated with greater loss of function and an [~]11-fold enrichment in the SUDEP cohort (nominal p = 0.03). In silico tools were unable to predict the impact of a variant on function highlighting the need for electrophysiological analysis. ConclusionsThese data show that loss-of-function KCNH2 variants are enriched in SUDEP patients and suggest that cardiac mechanisms contribute to SUDEP risk. We propose that genetic screening in combination with functional analysis can identify loss-of-function KCNH2 variants that could act as biomarkers of an individuals SUDEP risk.

neuroscience↗

bioRxiv2019

Tracing Autism Traits in Large Multiplex Families to Identify Endophenotypes of the Broader Autism Phenotype

Families comprising many individuals with Autism Spectrum Disorder (ASD) may carry a dominant predisposing mutation. Our aim was to use rigorous phenotyping of the Broader Autism Phenotype (BAP) in large multiplex ASD families to identify endophenotypes of the BAP for future genetic studies. We evaluated ASD/BAP features using standardised tests and a semi-structured interview to assess social, intellectual, executive and adaptive functioning in 109 individuals, including two large multiplex families (Family A: 30; Family B: 34) and an independent sample of small families (n=45). Our protocol identified four psychological endophenotypes of the BAP that were evident in both samples, and showed high sensitivity (97%) and specificity (82%) for individuals classified with the BAP. The patterns of inheritance of these endophenotypes varied in the two large families, supporting their utility for identifying genes in autism.

neuroscience↗

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↗