SAT-408 Characterization of a Novel Patient-derived EEFSEC Affecting Selenoprotein Synthesis and Thyroid Hormone Conversion

Abstract Disclosure: M. Noh: None. S. Erdenebileg: None. C. Park: None. Y. Han: None. J. Cha: None. T. Aoki: None. J. Kim: None. M. Kang: None. J. Yoo: None. M. Murakami: None. H. Lee: None. S. Lee: None. Thyroid hormone is a key regulator of metabolic processes. Its secretion begins with the hypothalamus releasing thyrotropin-releasing hormone (TRH), which stimulates the anterior pituitary to release thyroid-stimulating hormone (TSH). TSH induces the thyroid gland to produce and secrete thyroxine (T4) and triiodothyronine (T3) at a ratio of approximately 14:1. In peripheral tissues, T4 is converted into T3 by deiodinases (DIOs), with T3 subsequently regulating gene expression and metabolic functions. Hypothyroidism, characterized by insufficient thyroid hormone production, is typically treated with T4. While this treatment is effective for most patients, some do not respond, indicating possible defects in the conversion of T4 to T3. Previous studies have linked such defects to mutations in the DIO2 or SECISBP2 genes, which impact DIO synthesis, a selenoprotein. In this study, we identified a novel mutation in the EEFSEC gene in a post-surgical hypothyroidism patient unresponsive to T4 therapy and aimed to investigate its role in thyroid hormone metabolism. Whole exome sequencing (WES) was performed to identify potential genetic variations contributing to the patient’s condition. Among several genetic mutations, the EEFSEC gene mutation was identified as the most significantly pathologic. DIO2 activity assays and quantitative RT-PCR (qRT-PCR) were conducted on primary cells derived from the patient’s fibroblasts to assess DIO2 activity and transcript levels. Despite normal or elevated baseline DIO2 activity in the patient’s cells, cAMP stimulation failed to enhance DIO2 activity, suggesting defective transcriptional regulation. In contrast, control cells showed increased DIO2 activity following cAMP treatment. To further investigate the role of the EEFSEC mutation, we knocked out the EEFSEC gene in a cell line and introduced either the wild-type or mutant EEFSEC gene. This allowed us to assess whether the patient's phenotype could be replicated and linked to the mutation’s effect on DIO2 activity. In these cell models, EEFSEC gene knock-out significantly reduced glutathione peroxidase (GPX) activity, suggesting compromised selenoprotein synthesis. Rescue assays showed that the mutant EEFSEC gene restored GPX activity more effectively than the wild-type gene, consistent with the behavior observed in patient-derived cells. AlphaFold structural predictions indicated that the EEFSEC mutation caused structural alterations likely affecting its function. These findings suggest that the EEFSEC mutation impacts selenoprotein synthesis through a novel mechanism, highlighting a complex and previously unrecognized role for EEFSEC in thyroid hormone metabolism. This mechanism warrants further investigation and could pave the way for new therapeutic strategies for patients with thyroid hormone metabolism defects who are resistant to conventional T4 therapy. Presentation: Saturday, July 12, 2025