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Comprehensive Analysis Of Potential Downstream Target Genes Of The Interaction Of Twist1 Mutation And Environmental Factors In Craniosynostosis
Eloise Stanton, BA1, Janet Sanchez, BS2, Mark Urata, MD, DDS3, Yang Chai, DDS, PhD2.
1Keck School of Medicine, Los Angeles, CA, USA, 2Ostrow School of Dentistry, University of Southern California, Los Angeles, CA, USA, 3Children's Hospital Los Angeles, Los Angeles, CA, USA.

PURPOSE: Craniosynostosis is a congenital defect characterized by the premature fusion of calvarial sutures. While the genetic basis of craniosynostosis can be identified in about a quarter of patients, the pathophysiology underlying development of this disease is mostly unknown. Mutation in TWIST1 leads to Saethre-Chotzen syndrome with coronal synostosis. Our recent study using a Twist1+/- mouse model has shown evidence that environmental factors, including in utero exposure to the selective serotonin reuptake inhibitor citalopram, can exacerbate disease incidence and severity. In order to understand the molecular regulatory mechanism of the synergistic effect of Twist1+/- and exposure to citalopram on the development of craniosynostosis, we have performed RNA sequencing analysis at P7 to uncover the downstream target genes affected in the coronal suture prior to its fusion.
METHODS: Sixteen Twist1+/- mutant mice and wild-type (WT) mice with or without in utero citalopram exposure (20 mg/kg per day) were generated: WT (n=5), Twist1+/- (n=4), WT + citalopram (n=3), and Twist1+/- + citalopram (n=4). At P7, the mice were sacrificed, and the coronal sutures were dissected under a microscope and subjected to RNA extraction. The samples were analyzed for quality, and then Hitech sequencing was conducted at the core facility at University of California, Los Angeles. Post-sequencing data analysis was performed using Partek Flow and Ingenuity Pathway Analysis.
RESULTS: Twist1+/- mice exposed to citalopram demonstrated significant upregulation of genes involved in the pathophysiology of osteoarthritis [S100a8: 4.616; S100a9: 3.815] in comparison to Twist1+/- mice without citalopram exposure. WT mice exposed to citalopram demonstrated a significant downregulation of genes in the Igf1 signaling pathway [Igfbp4: -1.476; Igfbp5: -1.447] compared to WT mice without citalopram exposure. Twist1+/- mice without citalopram treatment had significant downregulation of genes involved in Ephrin signaling [Epha3: -1.562; Cxcl12: -1.279; Grin2b: -2.357] in comparison to WT mice without citalopram treatment. Finally, Twist1+/- mice with citalopram exposure had significant downregulation of growth factor receptors in the STAT3 pathway [Fgfr3: -1.262; Igf1r: -1.246; Igf2r: -1.306] in comparison to WT mice with citalopram exposure.
CONCLUSION: RNA sequencing data provide valuable insights into the potential signaling pathways and regulatory mechanisms involved immediately preceding suture fusion. Future in vivo studies focusing on implicated pathways will guide further mechanistic investigation into the pathophysiology that leads to craniosynostosis. By analyzing the interplay of the environment and genetics involved, we can better understand how to prevent and treat this devastating disease. Supported by R01 DE030901, NIDCR, NIH


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