Geração de iPSCs derivadas de pacientes com TEA e desenvolvimento de organoides cerebrais para a investigação das alterações funcionais associadas às variantes no gene SCN2A

Abstract

INTRODUCTION: Autism spectrum disorders (ASD) are a group of conditions that impact social interaction, communication, and behavior. In recent years, numerous mutations in various genes have been associated with ASD. Among these genes, pathogenic variants in SCN2A have shown a strong statistical correlation with ASD. The SCN2A gene encodes the alpha subunit of the voltage-gated sodium channel Nav1.2, which is highly expressed in excitatory pyramidal neurons during brain development. These neurons are crucial for cortical organization, excitability, and synaptogenesis, and are common targets of various genetic variants that may contribute to the ASD phenotype. There is a significant scientific demand for the development of studies and models that help establish genotype-phenotype correlations. In recent years, induced pluripotent stem cells (iPSCs) and their derivatives, including brain organoids, have been proposed as essential tools for this purpose. This work hypothesizes that brain organoid models, using iPSCs from patients with ASD, can significantly contribute to the analysis of genotype-phenotype correlations in different contexts and mutations, including SCN2A variants. METHODS AND RESULTS: In this study, four iPSC clones were generated and characterized from peripheral blood mononuclear cells (PBMCs) of two patients with ASD carrying pathogenic variants in the SCN2A gene. The iPSCs were rigorously characterized through the following assays: (i) RT-PCR, flow cytometry, and immunofluorescence analyses confirmed the expression of pluripotency markers; (ii) the embryoid body formation assay demonstrated the capacity for differentiation into the three germ layers; (iii) sequencing analysis confirmed the presence of SCN2A variants; (iv) STR analysis authenticated the cell lines; and (v) karyotype analysis demonstrated the chromosomal integrity of the cell lines. Finally, brain organoids were generated from these cells, providing a robust platform for future investigations into the pathophysiological mechanisms of ASD, especially those involving SCN2A. A literature review on the role of brain organoid models derived from iPSCs was conducted, highlighting their use in recapitulating critical aspects of human brain development and allowing the analysis of specific genetic variations and their influences on molecular and cellular pathways. Collectively, the analysis demonstrates that mutations in ASD risk genes, such as SCN2A, converge on a common phenotype of asynchronous neuronal development, affecting both excitatory and inhibitory neurons. Furthermore, brain organoids, due to their cellular composition, organization, and maturation state, allow the investigation of environmental factors and their interactions with genetic variants in ASD, underscoring the relevance of this model for understanding the complex neurobiology of ASD. CONCLUSION: The results of the presented studies indicate that brain organoids derived from iPSCs are valuable tools for investigating the genetic and environmental mechanisms underlying ASD, particularly regarding the SCN2A gene. These models offer a unique opportunity to establish genotype-phenotype correlations and develop more effective therapeutic approaches. The use of brain organoids can help elucidate the mechanisms of ASD development and contribute to the advancement of personalized medicine, providing new perspectives for the treatment and understanding of this complex condition