Investigating the emerging role of spliceosomal variants in craniofacial developmental disorders

  • Katherine Wood

Student thesis: Phd


The developmental craniofacial disorders Mandibulofacial Dysostosis Guion-Almeida type (MFDGA) and Burn-McKeown syndrome (BMKS) are caused by pathogenic variants in the core spliceosomal U5 small nuclear ribonucleoprotein factors EFTUD2 and TXNL4A, respectively. Patients with MFDGA have heterozygous loss-of-function EFTUD2 variants resulting in EFTUD2 haploinsufficiency. BMKS is caused by biallelic variants in TXNL4A leading to reduced TXNL4A expression. While previous yeast and zebrafish models have been developed to investigate these disorders, there have been no human cell models of MFDGA or BMKS and it is unclear how a reduction in expression of these spliceosomal factors results in defective craniofacial development. Here, human cell line models were developed to investigate MFDGA and BMKS. An EFTUD2-knockdown HEK293 cell line modelling MFDGA was generated using a CRISPR-Cas9 nickase strategy, and the functional and transcriptomic properties of the knockdown cells were characterised using functional assays and RNA sequencing (RNA-Seq) analysis. EFTUD2-knockdown cells showed diminished proliferation, cell cycle defects, increased sensitivity to endoplasmic stress, and widespread changes in gene expression and splicing of genes with functions relevant to craniofacial development and shared pre-mRNA sequence properties. To model BMKS, induced pluripotent stem cells (iPSCs) were generated from an individual with BMKS and her unaffected mother. iPSCs were differentiated into induced neural crest cells (iNCCs), the most disease-relevant cell type, and properties of the patient cells were compared to cells from her mother and unrelated control lines using functional assays and RNA-Seq analysis. Patient cells showed defective/delayed iNCC differentiation, including defects in the epithelial-to-mesenchymal transition and a defective response to WNT signalling, with the corresponding mis-splicing of TCF7L2, a critical gene in the canonical WNT pathway. In addition, two new diagnoses of BMKS were made by analysis of whole-genome sequencing data available from the Genomics England 100,000 Genomes Project and reverse phenotyping. Finally, the 34 base pair deletions in the promoter region of TXNL4A in patients with BMKS were investigated, identifying a critical promoter element required for gene expression and proposing alternative potential genotypes which could be causative in BMKS. Taken together, this work has provided detailed insights into the mechanisms underlying MFDGA and BMKS and has developed pipelines for investigating other rare genetic disorders in the future.
Date of Award31 Aug 2021
Original languageEnglish
Awarding Institution
  • The University of Manchester
SupervisorRaymond O'Keefe (Supervisor), William Newman (Supervisor) & Kathryn Hentges (Supervisor)


  • Developmental disorder
  • Craniofacial disorder
  • U5 snRNP
  • Spliceosome
  • Burn-McKeown syndrome
  • Pre-mRNA splicing
  • Mandibulofacial dysostosis Guion-Almeida type

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