Reply to Letter to the Editor regarding the article “ <scp>SCN1A</scp> gain of function effects in Dravet syndrome: Insights into clinical phenotypes and therapeutic implications”

We sincerely appreciate the opportunity to reply to the letter from Dr. Müller-Wöhrstein et al. regarding our paper.1 We are grateful for their insightful reevaluation, raising important points in the discussion. In their letter, the authors highlighted the challenges and the need for cautious interpretation of functional analyses, especially for ion channels, by re-evaluating the five SCN1A variants we previously classified as gain-of-function (GOF) or “mixed,” using their newly developed in silico prediction tool, SCION.2 We concur that the functional analysis of SCN1A variants is complex and that the currently available approaches, including those used in our study, have significant limitations. Although patch-clamp electrophysiology remains the gold standard for direct in vitro biophysical characterization, its technical demands and multiparametric nature, including current density, activation, fast inactivation, recovery from inactivation, and persistent current, impose limitations.3, 4 As more parameters are integrated, classification into a simple binary framework of GOF or loss-of-function (LOF) becomes increasingly difficult.3, 5 Moreover, neuronal dysfunction from a given variant may not be solely attributable to altered channel activity but may also result from impaired protein trafficking or expression, implying in vivo properties could differ from electrophysiological data.3 Recent emerging in silico tools, such as paralogue-based patch-clamp studies,6 and machine learning-based predictors, such as funNCion7 and SCION,2 offer alternative approaches for evaluating SCN1A variant function. Although these tools show relatively high concordance with patch-clamp data2, 6 and expand our interpretive capabilities, their clinical utility remains limited. We acknowledge these constraints and highlight the need for more accurate and clinically translatable prediction tools. Despite these methodological limitations, our study, as well as a few previous studies8-10 proposed the previously unrecognized possibility that non-LOF forms of Dravet syndrome (DS) may exist, and no clear evidence has disproved this hypothesis thus far. Our study also suggests that there may be subtle differences in the clinical course and antiseizure medication responsiveness between the LOF and non-LOF groups, based on a combination of experimental and computational approaches,1, 11 even among individuals who satisfy the standard clinical criteria for DS. DS is a clinically heterogeneous disorder with variability in seizure severity, response to antiseizure medications, and developmental outcomes, despite shared diagnostic criteria.12 Where such variability originated is a scientifically and clinically important challenge, and we deem that functional insights from genetic variants could be a potential approach to clarify its mechanisms. In conclusion, while challenges remain in variant functional assessment, its interpretation is becoming increasingly important for therapeutic decision-making, including the potential application of molecular therapies. Our findings open the discussion that, although likely rare, not all DS cases arise exclusively from LOF variants. The complexity of SCN1A-related disorders highlights the need for multidimensional approaches that integrate the clinical, genetic, and functional evidence. We hope that our article raises awareness of the importance of genetic variant identification, as well as the added value of functional characterization. Further research is warranted to validate our observations and to advance personalized therapeutic strategies for SCN1A-related disorders. This work was supported in part by grants from an intramural fund from the National Center of Neurology and Psychiatry (Y. Kobayashi, K. Inoue), the Japan Agency for Medical Research and Development (AMED) under grant numbers: JP24ek0109674, JP24ek0109760, JP24ek0109617, JP24ek0109648, and JP24ek0109677 (N. Matsumoto); Japan Society for the Promotion of Science (JSPS) KAKENHI under grant number JP24K02230 (N. Matsumoto); and the Takeda Science Foundation (N. Matsumoto). We confirm that we have read the Journal's position on issues involved in ethical publication and affirm that this report is consistent with those guidelines. Ethics approval was obtained from the ethics board of the National Center of Neurology and Psychiatry (number: A2024-037). The data that support the findings of this study are available from the corresponding author upon reasonable request.