Development of patient-specific iPSC-based epilepsy models and identification of differentially expressed genes for disease mechanisms

Abstract

The successful establishment of CLCNKB-mutant patient-specific iPSC lines and their transcriptomic characterization provide a valuable tool for studying the molecular basis of epilepsy. These findings underscore the potential of iPSC-based models to advance our understanding of disease mechanisms and support the development of novel diagnostic and therapeutic strategies. Moreover, this study demonstrates the applicability of iPSCs for epilepsy research and their promise in the field of personalized medicine.

Introduction

Epilepsy remains a highly heterogeneous neurological disorder, often presenting challenges for traditional animal models that fail to replicate the specific genetic nuances of human patients. While many cases are linked to ion channel mutations, the precise molecular pathways connecting genotype to seizure phenotype are often unclear.

This research addresses a specific clinical case involving a proband with recurrent seizures and motor impairment. The patient carries a novel mutation in CLCNKB (a chloride channel gene). Intriguingly, the patient's father carries the same mutation but remains asymptomatic. By creating a "disease-in-a-dish" model, the authors aimed to explore why this mutation manifests as epilepsy in the son and identify dysregulated genes contributing to the disease state.

Methodology

The study employed a comprehensive cellular reprogramming and transcriptomic pipeline to model the disease. Peripheral blood mononuclear cells (PBMCs) were collected from the affected son, the carrier father, and the healthy mother. These cells were reprogrammed into induced pluripotent stem cells (iPSCs) using the non-integrating Sendai virus method.

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The resulting iPSC lines underwent rigorous validation, including G-band karyotyping, alkaline phosphatase staining, and immunofluorescence for pluripotency markers such as OCT4, SOX2, and NANOG. Teratoma assays in mice confirmed the cells' ability to differentiate into all three germ layers. Following validation, RNA sequencing (RNA-seq) was performed to compare gene expression profiles across the family members, with differential expression analysis conducted via the DESeq2 package.

Results and Transcriptomic Analysis

The primary dataset generated was the transcriptomic profile of the patient-specific iPSC lines (NCBI BioProject: SAMN48836013). The analysis compared the "Experimental Group" (affected son and carrier father) against the "Control Group" (healthy mother).

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Key findings from the transcriptomic analysis include:

• Differential Expression: The team identified a total of 1,819 differentially expressed genes (DEGs). Applying stricter filters (Fold Change > 5 and p < 0.05) narrowed this to 301 high-confidence DEGs.
• Upregulation of CLCNKB: Interestingly, CLCNKB expression was upregulated in the patient compared to the control, likely as a compensatory mechanism for the non-functional truncated proteins produced by the mutation.
• Top Candidates: Significant downregulation was observed in genes such as XIST, RAB17, PDGFB, MYOCD, and LARGE1, while NLRP2 and DDX3Y were among the top upregulated genes.

Discussion: The Genotype-Phenotype Gap

The most striking observation is the disparity between the father and son, who share the same CLCNKB mutation (c.228C > A, p.Cys76Ter). Structural modeling suggests this mutation leads to a truncated protein lacking essential functional domains, disrupting chloride homeostasis. However, since only the son is symptomatic, the epilepsy in this family is likely polygenic or influenced by epigenetic factors.

The downregulation of LARGE1 is particularly noteworthy. While typically associated with muscular dystrophy, LARGE1 is involved in glycosylation and plays a role in neural development and synaptic plasticity. This provides a novel link between glycosylation pathways and epilepsy. Furthermore, the downregulation of MYOCD (associated with muscle atrophy) may explain the patient's specific clinical symptoms of muscle weakness and motor impairment alongside seizures.

Limitations and Future Work

While this study provides a robust foundation, several limitations remain. The transcriptomic analysis was performed on undifferentiated iPSCs, which do not fully replicate the electrical environment of mature neurons. Future work will involve differentiating these lines into mature neurons or brain organoids for electrophysiological studies (e.g., patch-clamping) to functionally validate the roles of LARGE1 and MYOCD. Additionally, while the authors hypothesize that the nonsense mutation triggers nonsense-mediated mRNA decay (NMD), this requires further experimental verification. As a case study of a single family, broader cohort validation is necessary to generalize these findings to other CLCNKB-related pathologies.

@inproceedings{wu2025development,
  author = {Jianfeng Wu and Siqi Huang and Lihao Chen and Yinghong Yang and Shunhan Zhang and Jiajia Xian and Xiaoyan Ma and Furong Ma and Shaoying Li and Shuicheng Yan and Yingjun Xie and Xiaofang Sun},
  title = {{Development of patient-specific iPSC-based epilepsy models and identification of differentially expressed genes for disease mechanisms}},
  booktitle = {Frontiers in Neuroscience},
  year = {2025},
  doi = {10.3389/fnins.2025.1582255},
}