Abstract
Mutations in the choline acetyltransferase (CHAT) gene cause congenital myasthenic syndrome (CMS). Episodic apnea is frequently observed in patients with CMS due to CHAT mutations (CMS-CHAT), and muscle hypotonia at birth or in early infancy is also common. We report two siblings with compound heterozygous mutations in the CHAT gene: c.1231G > A (missense) and c.752 + 2 T > C (splice site). To confirm the splice site mutation induces a splicing variant, we performed a minigene assay and demonstrated that the splice site mutation, c.752 + 2 T > C, results in complete exon skipping. AlphaFold2 analysis predicted that the skipped exon constitutes an α helix, a highly conserved core structural element of ChAT. These structural alterations in ChAT may underlie the clinical phenotype associated with these mutations.
Data availability
All data generated or analysed during this study are included in this published article and its Supplementary Information Files. The original data from this study are available from the corresponding author upon reasonable request. The accession numbers of c.752 + 2 T > C and c.1231G > A are SCV006087544 and SCV006087545 on ClinVar (https://www.ncbi.nlm.nih.gov/clinvar/), respectively.
References
Engel, A. G., Shen, X. M., Selcen, D. & Sine, S. M. Congenital myasthenic syndromes: pathogenesis, diagnosis, and treatment. Lancet Neurol. 14, 420–434. https://doi.org/10.1016/S1474-4422(14)70201-7 (2015).
Ohno, K., Ohkawara, B., Shen, X. M., Selcen, D. & Engel, A. G. Clinical and pathologic features of congenital myasthenic syndromes caused by 35 genes—a comprehensive review. Int. J. Mol. Sci. https://doi.org/10.3390/ijms24043730 (2023).
Engel, A. G., Ohno, K., Shen, X. M. & Sine, S. M. Congenital myasthenic syndromes: multiple molecular targets at the neuromuscular junction. Ann. N. Y. Acad. Sci. 998, 138–160. https://doi.org/10.1196/annals.1254.016 (2003).
Ohno, K. et al. Choline acetyltransferase mutations cause myasthenic syndrome associated with episodic apnea in humans. Proc. Natl. Acad. Sci. USA 98, 2017–2022. https://doi.org/10.1073/pnas.98.4.2017 (2001).
Steinhaus, R. et al. MutationTaster2021. Nucleic Acids Res. 2021(49), W446–W451. https://doi.org/10.1093/nar/gkab266 (2021).
Ng, P. C. & Henikoff, S. Predicting deleterious amino acid substitutions. Genome Res. 11, 863–874. https://doi.org/10.1101/gr.176601 (2021).
Adzhubei, I. A. et al. A method and server for predicting damaging missense mutations. Nat. Methods 7, 248–249. https://doi.org/10.1038/nmeth0410-248 (2010).
Adzhubei, A. A., Sternberg, M. J. & Makarov, A. A. Polyproline-II helix in proteins: structure and function. J. Mol. Biol. 425, 2100–2132. https://doi.org/10.1016/j.jmb.2013.03.018 (2013).
Jaganathan, K. et al. Predicting splicing from primary sequence with deep learning. Cell 176, 535–548. https://doi.org/10.1016/j.cell.2018.12.015 (2019).
Zhang, Y. et al. Congenital myasthenic syndrome caused by a novel hemizygous. Front. Pediatr. 8, 185. https://doi.org/10.3389/fped.2020.00185 (2020).
Liu, Z. et al. Compound heterozygous. Front. Pharmacol. 10, 259. https://doi.org/10.3389/fphar.2019.00259 (2019).
Murtazina, A. et al. Mild phenotype of. Front. Pediatr. 12, 1280394. https://doi.org/10.3389/fped.2024.1280394 (2024).
Arredondo, J. et al. Choline acetyltransferase mutations causing congenital myasthenic syndrome: molecular findings and genotype-phenotype correlations. Hum. Mutat. 36, 881–893. https://doi.org/10.1002/humu.22823 (2015).
Schara, U. et al. Long-term follow-up in patients with congenital myasthenic syndrome due to CHAT mutations. Eur. J. Paediatr. Neurol. 14, 326–333. https://doi.org/10.1016/j.ejpn.2009.09.009 (2010).
Natera-de Benito, D. et al. Advancing the understanding of vesicle-associated membrane protein 1-related congenital myasthenic syndrome: phenotypic insights, favorable response to 3,4-diaminopyridine, and clinical characterization of five new cases. Pediatr. Neurol. 157, 5–13. https://doi.org/10.1016/j.pediatrneurol.2024.04.027 (2024).
Fujii, T. et al. Basic and clinical aspects of non-neuronal acetylcholine: expression of an independent, non-neuronal cholinergic system in lymphocytes and its clinical significance in immunotherapy. J. Pharmacol. Sci. 106, 186–192. https://doi.org/10.1254/jphs.fm0070109 (2008).
Cai, Y. et al. Choline acetyltransferase structure reveals distribution of mutations that cause motor disorders. EMBO J. 23, 2047–2058. https://doi.org/10.1038/sj.emboj.7600221 (2004).
Shen, X. M. et al. Functional consequences and structural interpretation of mutations of human choline acetyltransferase. Hum. Mutat. 32, 1259–1267. https://doi.org/10.1002/humu.21560 (2011).
Jumper, J. et al. Highly accurate protein structure prediction with AlphaFold. Nature 596, 583–589. https://doi.org/10.1038/s41586-021-03819-2 (2021).
Mirdita, M. et al. ColabFold: making protein folding accessible to all. Nat. Methods 19, 679–682. https://doi.org/10.1038/s41592-022-01488-1 (2022).
Acknowledgements
We would like to thank Prof. Kinji Ohno (Nagoya University of Arts and Sciences) for valuable discussions. We also thank Dr. R. Ichikawa and Dr. H. Hino for his advice and K. Iwamura and Y. Moriya for their support. We would like to thank Editage (www.editage.jp) for English language editing.
Funding
This work was supported by JSPS KAKENHI Grant Numbers 22K11315 and 25K14457 (to S.K.), 25K21078 (to N.W.), and 24K02208 (to Y.O.), and by AMED under Grant Number JP24ek0109760.
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S.K. and N.T. designed the study and wrote the manuscript with support from M.K. and Y.O., H.K., S.F., Y.N., and T.O. performed the genetic analysis and evaluation. T.M. and M.K. conducted the three-dimensional structure building and analysis. S.K. and N.W. carried out the minigene assay. N.T. and M.K. collected the clinical data. All authors approved the final manuscript.
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Kikuchi, S., Wada, N., Mariya, T. et al. Compound heterozygous CHAT gene mutations, a missense and a splice site variant, in two siblings with congenital myasthenic syndrome. Sci Rep (2026). https://doi.org/10.1038/s41598-026-39759-y
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DOI: https://doi.org/10.1038/s41598-026-39759-y
