Abstract
Disclosure: M.C. Foss de Freitas: Advisory Board Member; Self; PTC Therapeutics. N. Wys: None. M. Udler: None. L. Pais: None. A. Monteiro da Rocha: None. O.A. MacDougald: None. E.A. Oral: Consulting Fee; Self; Regeneron Pharmaceuticals, Aegerion Pharmaceuticals, Ionis Pharmaceuticals Inc., Third Rock Ventures, Rejuvenate Inc. Grant Recipient; Self; Regeneron Pharmaceuticals, Aegerion Pharmaceuticals, Ionis Pharmaceuticals Inc., Novo Nordisk, Fractyl Laboratories, Rhythm pharmaceuticals, GID Dynamics. Other; Self; Aegerion Pharmaceuticals. T. Chun: None. Objective/Goals: Partial Lipodystrophy is characterized by the gradual loss of adipose tissues, which progresses after birth. The genetic and molecular underpinning of many cases of partial lipodystrophy have not been fully understood. Aiming to better understand the molecular pathogenesis of familial partial lipodystrophy, we initiated deep sequencing for our patients with partial lipodystrophy syndromes. A novel variant of early B cell factor 2 (EBF2) was identified. Here we report the biological impact of a novel truncating EBF2 variant. Methods: We performed loss-of-function and gene rescue experiments using 3T3-L1 and human primary subcutaneous preadipocytes. All cells were cultured in DMEM with 10% bovine calf serum (Invitrogen). After lentivirus transfection (Full-length EBF2 or truncated variant EBF2), cells were grown to confluence and then exposed to adipogenesis induction media containing dexamethasone, insulin and isobutyl methylxanthine. Total RNA was extracted using RNeasy Mini Kit (Qiagen), and cDNA was synthesized using IScript (Bio-Rad). Real-time qPCR was performed using TaqMan probes for Pparg and Fabp4, two key adipogenesis markers. Results: The patient was found to carry a heterozygous nonsense mutation in exon 6 of 16 (NM_022659.4) of EBF2, causing the premature termination of the protein at amino acid position 165 (EBF2 8:26033143 C>A (hg38), c.493G>T, p.Glu165Ter). Adipogenesis was significantly suppressed in 3T3-L1 cells when endogenous Ebf2 was suppressed with siRNA and lentiviral shRNA. Adipocytes with suppressed Ebf2 expression showed a marked reduction of intracellular lipid content and Pparg and Fabp4 expression (>80% reduction). With lentiviral gene transfer, EBF2 fully rescued adipogenic potential, whereas the truncated variant EBF2 p.Glu165Ter did not. Of note, 3T3-L1 cells transfected with the EBF2 variant impaired adipogenesis, suggesting an inhibitory effect of the EBF2 variant on adipogenesis. We next confirmed the inhibition in adipogenesis with the EBF2 variant in primary human preadipocytes from subcutaneous adipose tissues. Using lentiviral gene transfer in human preadipocytes, we observed that the full-length EBF2 increased the gene expression of PPARg and FABP4 while the truncated EBF2 variant did not. Conclusion: Our data suggest that EBF2 is indispensable for adipogenesis of mouse and human preadipocytes. The truncating variant of EBF2 p.Glu165Ter likely plays a pathogenic role in the partial lipodystrophy presentation of this patient. Whole genome (or exome) sequencing undertaken in patients with partial lipodystrophy and subsequent biological functional analysis may lead to identification of novel mechanisms driving novel discoveries. Presentation: Friday, June 16, 2023