MON-699 RNA-Seq Meta-Analysis of the EBF2 p.E165X Variant in Lipodystrophy Implicates the Role of EBF2 in Mesoderm Induction and Adipogenesis

Abstract Disclosure: M.C. Foss de Freitas: None. A.M. da Rocha: None. D. Gilio: None. E.A. Oral: Regeneron Pharmaceuticals, Chiesi Rare Disease Group, Amryt Pharmaceuticals, Marea Therapeutics, Fractyl Laboratories, Rejuvenate Bio, Ionis Pharmaceuticals Inc., Phythm Pharmaceuticals, Novo Nordisk, Morphic Medical. T. Chun: None. Background: We have recently reported the discovery of a stop gain variant in the early B-cell factor 2 (EBF2 8:26033143C>A, c.493G>T, p.E165X) in a patient with atypical partial lipodystrophy. To gain deeper insights into the potential dysregulation of target genes caused by this variant, we conducted a comprehensive meta-analysis of bulk RNA-seq data. Our goal was to investigate the transcriptional effects of the gene variant compared to the wild-type sequence in human preadipocytes and to assess the impact of mesoderm induction on iPSC-derived cells. Methods: Peripheral blood mononuclear cells (PBMCs) from a patient with the EBF2 variant and a non-affected family member were reprogrammed into induced pluripotent stem cells (iPSCs). These iPSCs were treated with CHIR99021 (CHIR) to induce mesodermal differentiation through WNT signaling. Simultaneously, we used lentivirus constructs to express control-GFP, full-length EBF2 (EBF2-full), and the EBF2 p.E165X variant (EBF2-mut) in human preadipocytes, followed by adipogenic stimulation. Bulk RNA sequencing was performed on both untreated and CHIR-treated iPSCs, and a meta-analysis was executed using software tools such as DEseq2, metaRNAseq, and clusterProfiler. The Fisher method and GO enrichment analysis (using org.Hs.eg.db) were used to identify transcriptional changes, applying thresholds of an adjusted p-value of 0.05 and a log2 fold change of 1. Results: Our comprehensive analysis identified 1,414 genes significantly altered in the presence of the EBF2 variant. Specifically, preadipocytes containing the EBF2 variant showed significant changes in the expression of 502 genes, whereas CHIR-treated iPSCs revealed regulation of 77 genes. We looked at genes that were differentially expressed in both datasets, human pre-adipocytes with EBF2 variant and CHIR-treated patient-derived iPSC, with consistent up or down-regulation, and found 56 genes. Among them, the most up-regulated (adjusted p-value<0.0001) were HLA-C, HSPA1A, STON2, and MMP15, while COL4A1, FLT1, VLDLR, CLU, and AFAP1L2 were downregulated. Pathway enrichment analyses underscore significant regulation in pathways such as ECM organization, cell-substrate adhesion, and wound healing. These pathways are essential for structural integrity, cellular signaling, and tissue repair processes, indicating that both the EBF2 variant effect and CHIR-induced gene regulation are involved in mesodermal lineage commitment and adipocyte differentiation. Conclusions: Our findings provide valuable insights into the transcriptional networks dysregulated by the EBF2 variant, offering potential targets for therapeutic intervention. This study lays the groundwork for understanding the molecular landscapes influenced by the EBF2 variant and mesoderm induction, paving the way for future exploration of targeted strategies and metabolic regulation therapies. Presentation: Monday, July 14, 2025