Factors Modifying Neurodevelopmental Disorder-Related Phenotypes in Scn2a K1422E Mice

Echevarria-Cooper, Dennis Michael

doi:https://doi.org/10.21985/n2-exg7-bf35

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Factors Modifying Neurodevelopmental Disorder-Related Phenotypes in Scn2a K1422E Mice

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SCN2A encodes the NaV1.2 voltage-gated sodium channel, which is thought to contribute to the development of the central nervous system. Pathogenic variants in SCN2A have been associated with neurodevelopmental disorders (NDD), including developmental and epileptic encephalopathies (DEE), intellectual disability (ID), and autism spectrum disorder (ASD). These disorders represent a significant public health burden, affecting roughly 1 in 6 children in the United States. However, the pathogenic mechanisms of SCN2A variants are complex and require further elucidation. For example, sex differences have been reported in a number of NDD, including epilepsy and ASD. Furthermore, recurrent and inherited variants in SCN2A show wide phenotypic heterogeneity among individuals. This suggests that genetic and environmental factors may contribute to variable expressivity of the phenotype. Some SCN2A variants fit into a framework wherein gain-of-function missense variants that increase neuronal excitability lead to DEE, while loss-of-function variants that reduce neuronal excitability lead to ID/ASD. One unique case less easily classified using this framework is the de novo missense variant SCN2A-p.K1422E, associated with infant-onset developmental delay, infantile spasms, and features of ASD. Prior structure-function studies demonstrated that K1422E substitution alters NaV1.2 ion selectivity, conferring calcium permeability, lowering overall conductance, and conferring resistance to tetrodotoxin (TTX). As part of this work and based on heterologous expression data, a compartmental neuron model incorporating K1422E channels was developed that predicted reductions in peak action potential (AP) speed. To further investigate the effects of the p.K1422E variant on cellular excitability and neurological/neurobehavioral phenotypes, a novel mouse model (Scn2aK1422E) was generated using CRISPR/Cas9 genome editing. Excitatory neurons in neocortex exhibited features indicative of functional K1422E-containing NaV1.2 channels, including lower current density with a TTX-insensitive component, reduced AP speed, and aberrant calcium influx occurring during the NaV-mediated rising phase of the AP. Analysis of behaving animals revealed a mix of phenotypes, including infrequent spontaneous seizures, altered susceptibility to chemically induced seizures, reduced anxiety-like behavior and alterations in olfactory-guided social behavior. Scn2aK1422E females showed a reproducible, non-unimodal distribution of flurothyl-induced seizure thresholds. Women with epilepsy often show a cyclical pattern of altered seizure susceptibility during specific phases of the menstrual cycle which can be attributed to fluctuations in hormones and corresponding changes in neurosteroid levels. To determine whether the estrous cycle affects susceptibility to flurothyl-induced seizures, estrous cycle monitoring was performed in female mice that had undergone ovariectomy (OVX), sham surgery, or no treatment prior to seizure induction. Removing the influence of circulating sex hormones via OVX did not affect the non-unimodal distribution of flurothyl seizure thresholds observed in Scn2aK1422E females. Additionally, flurothyl seizure thresholds were not associated with estrous cycle stage in mice that underwent sham surgery or were untreated. Interestingly, untreated Scn2aK1422E females showed evidence of disrupted estrous cyclicity, an effect not previously described in a genetic epilepsy model. Genetic modifiers can contribute to variability in disease phenotypes associated with rare driver mutations. Accordingly, different genetic backgrounds across inbred rodent strains have been shown to influence disease-related phenotypes, including those associated with Scn2a-related NDD. The Scn2aK1422E mouse model was developed as an isogenic line maintained on the C57BL/6J (B6) strain. To determine if background strain affects phenotype severity in the Scn2aK1422E mouse model, phenotypes of Scn2aK1422E mice on B6 and [DBA/2J×B6] F1 hybrid strains were compared. Convergent evidence from neurobehavioral assays demonstrated lower anxiety-like behavior in Scn2aK1422E mice compared to WT and further suggested that this effect is more pronounced on the B6 background compared to the F1D2 background. Although there were no strain-dependent differences in occurrence of rare spontaneous seizures, response to the chemoconvulsant kainic acid (KA) revealed differences in seizure generalization and lethality risk, with variation based on strain and sex. The work described in this dissertation establishes the Scn2aK1422E mouse model as a sharable resource for future studies on the consequences of altered NaV1.2 ion selectivity and serves as a case study highlighting the importance of considering modifying factors of disease phenotypes that present as complex traits. Continued examination of phenotypic heterogeneity in the Scn2aK1422E mouse model using diverse genetic reference panels may enable the identification of highly penetrant phenotypes and modifier genes that could provide clues about the primary pathogenic mechanism of the K1422E variant. In addition, examining strain level effects could reveal genetic backgrounds with unique susceptibility profiles that would be relevant for future studies on specific traits such as seizure susceptibility. Properly validated animal models can help refine genotype-phenotype correlations for SCN2A-related disorders, enhance our understanding of disease mechanisms, and support the development of targeted therapeutic strategies.

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