Poster Presentation 47th Lorne Genome Conference 2026

Transdifferentiation of Human Dermal Fibroblasts to iNeurons and iAstrocytes for Functional Variant Studies (133274)

Olivia Robinson 1 2 , Alison Gardner 1 3 , Emmylou Nicolas-Martinez 1 2 , Jamie V Zhang 1 2 , Tarin C John 1 2 , Tracy Dudding 4 , Jean-Vianney Barnier 5 , Jozef Gecz 1 3 , Lachlan Jolly 1 2
  1. The Robinson Research Institute, The University of Adelaide, Adelaide, SA, Australia
  2. School of Biomedicine, The University of Adelaide, Adelaide, SA, Australia
  3. School of Medicine, The University of Adelaide, Adelaide, SA, Australia
  4. NSW Genetics of Learning Disability (GOLD) Service, Hunter Genetics, Newcastle, NSW, Australia
  5. Institut des Neurosciences Paris-Saclay, Université Paris-Saclay, Saclay, France

We defined a list of 516 silent neurological disease genes (SNGs) which display limited or no expression in clinically accessible tissues of blood and skin forgoing functional RNA and protein based investigations of disease associated genetic variants. We therefore investigated whether transdifferentiation of patient derived human dermal fibroblasts (HDFs) directly into induced Neurons (iNeurons) or iAstrocytes could induce the endogenous expression of SNGs providing an approach to study the impact of variants on the mRNA of SNGs.

Overexpression of cell fate directing transcription factors ASCL1 & NEUROG2 and SOX9 & NFIB, in conjunction with small molecules successfully generated iNeurons and iAstrocytes over 3-6 weeks as confirmed by immunofluorescent based marker gene analysis and short- and long-read RNAseq profiling. Transcriptome profiles correlated well with respective cell types derived from human pluripotent stem cells. Importantly, iNeurons expressed 193/516 (37.4%) SNGs, and iAstrocytes expressed 153/516 (29.7%). 264/516 (51%) SNGs were expressed in either transdifferentiated cell type. Expressed SNGs recapitulated expression levels and isoform diversity consistent with adult human cortex.

Patient derived iNeurons were subsequently used to investigate disease associated variants in SNGs. Three nonsense PAK3 variants were proven to be regulated by nonsense mediated mRNA decay (NMD) confirming loss-of-function (LOF) as the disease mechanism1. A variant of unknown significance in DLG3 predicted to impact splicing (DLG3 c.840+4A>G) was shown to result in a 3.8-fold reduction in expression. Nanopore sequencing revealed the variant caused a 31bp insertion of intron 5 leading to a premature stop codon and NMD, thus confirming variant pathogenicity via a LOF mechanism. The impacts of both PAK3 and DLG3 variants were subsequently confirmed using an orthologous CRISPRa gene transactivation approach.

These data support use of transdifferentiation as a robust and highly reproducible method for functionally investigating the pathogenicity and mechanism of disease associated with variants impacting SNGs.

  1. 1.Nicolas-Martinez EC, Robinson O, Pflueger C, Gardner A, Corbett MA, Ritchie T, et al. RNA variant assessment using transactivation and transdifferentiation. American journal of human genetics. 2024;111(8):1673–99.