386 A novel truncating variant of EBF2 disrupts human adipocyte differentiation in lipodystrophy syndromes: an example of a discovery from a clinical translational pipeline

OBJECTIVES/GOALS: Aiming to better understand the molecular pathogenesis of familial partial lipodystrophy (PL), we initiated whole-exome sequencing for our patients with PL syndromes. A novel variant of early B cell factor 2 (EBF2) was identified. Here we report the biological impact of a novel tru...

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Main Authors: Maria C. Foss-Freitas, Noel Wys, Miriam Udler, Lynne Pais, Andre Monteiro da Rocha, Ormond A. MacDougald, Elif A. Oral, Tae-Hwa Chun
Format: Article
Language:English
Published: Cambridge University Press 2023-04-01
Series:Journal of Clinical and Translational Science
Online Access:https://www.cambridge.org/core/product/identifier/S2059866123004223/type/journal_article
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author Maria C. Foss-Freitas
Noel Wys
Miriam Udler
Lynne Pais
Andre Monteiro da Rocha
Ormond A. MacDougald
Elif A. Oral
Tae-Hwa Chun
author_facet Maria C. Foss-Freitas
Noel Wys
Miriam Udler
Lynne Pais
Andre Monteiro da Rocha
Ormond A. MacDougald
Elif A. Oral
Tae-Hwa Chun
author_sort Maria C. Foss-Freitas
collection DOAJ
description OBJECTIVES/GOALS: Aiming to better understand the molecular pathogenesis of familial partial lipodystrophy (PL), we initiated whole-exome sequencing for our patients with PL 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/STUDY POPULATION: Using 3T3-L1 and human primary subcutaneous preadipocytes, we performed loss-of-function and gene rescue experiments. All cells were cultured in DMEM with 10% bovine calf serum (Invitrogen) at 5% CO2. After lentivirus transfection, cells were grown to confluence and then exposed to adipogenesis induction media containing dexamethasone (0.25µM), insulin (1µg/ml) and isobutyl methylxanthine (0.5 mM). 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/ANTICIPATED RESULTS: Patient was found to carry a heterozygous nonsense mutation in exon 6 of EBF2, causing the premature termination of the protein at amino acid position 165. Adipogenesis was significantly suppressed in 3T3L1 cells when endogenous Ebf2 was suppressed with siRNA and lentiviral shRNA. Adipocytes with suppressed Ebf2 expression showed 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 did not. Of note, 3T3-L1 cells transfected with the EBF2 variant displayed impaired adipogenesis, suggesting a dominant-negative effect of the EBF2 variant on adipogenesis. We confirmed the dominant effect of the EBF2 variant in human adipocyte differentiation. DISCUSSION/SIGNIFICANCE: Our data suggest that EBF2 is indispensable for adipogenesis. The loss of function and dominant-negative effect of the truncating variant of EBF2 likely plays a pathogenic role in PL. Whole exome sequencing of PL patients and ex-vivo functional analysis help identify novel gene variants and better understand the molecular pathogenesis of PL.
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spelling doaj.art-116ab8def40f405ebc8bd2e8ef5872e92023-04-24T05:55:57ZengCambridge University PressJournal of Clinical and Translational Science2059-86612023-04-01711411510.1017/cts.2023.422386 A novel truncating variant of EBF2 disrupts human adipocyte differentiation in lipodystrophy syndromes: an example of a discovery from a clinical translational pipelineMaria C. Foss-Freitas0Noel Wys1Miriam Udler2Lynne Pais3Andre Monteiro da Rocha4Ormond A. MacDougald5Elif A. Oral6Tae-Hwa Chun7University of MichiganUniversity of MichiganHarvard Medical SchoolHarvard Medical SchoolUniversity of MichiganUniversity of MichiganUniversity of MichiganUniversity of MichiganOBJECTIVES/GOALS: Aiming to better understand the molecular pathogenesis of familial partial lipodystrophy (PL), we initiated whole-exome sequencing for our patients with PL 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/STUDY POPULATION: Using 3T3-L1 and human primary subcutaneous preadipocytes, we performed loss-of-function and gene rescue experiments. All cells were cultured in DMEM with 10% bovine calf serum (Invitrogen) at 5% CO2. After lentivirus transfection, cells were grown to confluence and then exposed to adipogenesis induction media containing dexamethasone (0.25µM), insulin (1µg/ml) and isobutyl methylxanthine (0.5 mM). 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/ANTICIPATED RESULTS: Patient was found to carry a heterozygous nonsense mutation in exon 6 of EBF2, causing the premature termination of the protein at amino acid position 165. Adipogenesis was significantly suppressed in 3T3L1 cells when endogenous Ebf2 was suppressed with siRNA and lentiviral shRNA. Adipocytes with suppressed Ebf2 expression showed 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 did not. Of note, 3T3-L1 cells transfected with the EBF2 variant displayed impaired adipogenesis, suggesting a dominant-negative effect of the EBF2 variant on adipogenesis. We confirmed the dominant effect of the EBF2 variant in human adipocyte differentiation. DISCUSSION/SIGNIFICANCE: Our data suggest that EBF2 is indispensable for adipogenesis. The loss of function and dominant-negative effect of the truncating variant of EBF2 likely plays a pathogenic role in PL. Whole exome sequencing of PL patients and ex-vivo functional analysis help identify novel gene variants and better understand the molecular pathogenesis of PL.https://www.cambridge.org/core/product/identifier/S2059866123004223/type/journal_article
spellingShingle Maria C. Foss-Freitas
Noel Wys
Miriam Udler
Lynne Pais
Andre Monteiro da Rocha
Ormond A. MacDougald
Elif A. Oral
Tae-Hwa Chun
386 A novel truncating variant of EBF2 disrupts human adipocyte differentiation in lipodystrophy syndromes: an example of a discovery from a clinical translational pipeline
Journal of Clinical and Translational Science
title 386 A novel truncating variant of EBF2 disrupts human adipocyte differentiation in lipodystrophy syndromes: an example of a discovery from a clinical translational pipeline
title_full 386 A novel truncating variant of EBF2 disrupts human adipocyte differentiation in lipodystrophy syndromes: an example of a discovery from a clinical translational pipeline
title_fullStr 386 A novel truncating variant of EBF2 disrupts human adipocyte differentiation in lipodystrophy syndromes: an example of a discovery from a clinical translational pipeline
title_full_unstemmed 386 A novel truncating variant of EBF2 disrupts human adipocyte differentiation in lipodystrophy syndromes: an example of a discovery from a clinical translational pipeline
title_short 386 A novel truncating variant of EBF2 disrupts human adipocyte differentiation in lipodystrophy syndromes: an example of a discovery from a clinical translational pipeline
title_sort 386 a novel truncating variant of ebf2 disrupts human adipocyte differentiation in lipodystrophy syndromes an example of a discovery from a clinical translational pipeline
url https://www.cambridge.org/core/product/identifier/S2059866123004223/type/journal_article
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