ABSTRACT: Background: A genetic variant MED12 c.3423C>T,p.Arg1138Trp, classified as a variant of uncertain significance, was identified in a female patient with multiple congenital anomalies and developmental delay. MED12, located on the X chromosome, is a subunit of the Mediator complex which is involved in regulation of transcription and RNA polymerase II activity. The functional consequence of these MED12 genetic variants in disease is undetermined, and insight into disease mechanism is required. Method: We compare disease in seven female MED12 p.Arg1138Trp patients with a disease phenotype including Blepharophimosis – intellectual disability syndrome, Maat-Kievet-Brunner MKB type, Choleostasis-pigmentary retinopathy-cleft palate syndrome, and non-syndromic MED12-related condition. Inducible pluripotent stem cells were derived from patient PBMCs, followed by neural disease modelling for transcriptomics and functional protein analysis. Results: The phenotype for female patients with the MED12 c.3423C>T, p.Arg1138Trp genetic variant, indicated overlapping and divergent clinical features. In comparison of MED12 genetic variant neural cells, derived from patient induced pluripotent stem cells (iPSCs), identified altered expression levels for genes that regulate RNA polymerase II activity (ZNF558, ZIM2, ZFP3, TBR1, PEG3, EMX1, DMRTA2), transcription and pre-mRNA processing (ZIM2, and SNRPN) and neural development (BMP3, CNP, EBF2, EMX1, EOMES, LXH5, NHLH1, UNCX). Transcriptomics analysis further revealed a reduction in pathways related to axon development, forebrain differentiation, and cell specification (neural cell specification); and significant upregulation of pathway gene sets related to pre-ribosome complex (nucleolus) in the MED12 genetic variant cells. Comparison of MED12 wild-type and MED12 p.Arg1138Trp neural progenitor cells indicated similar MED12 transcript and protein expression. However, there was a decrease in MED12L expression, at the transcript and protein level, and significantly reduced MED12L localisation to the nucleolus in the MED12 p.Arg1138Trp cells. Conclusion: Patient iPSC derived stimulated for neural cell differentiation indicate delayed neural development in MED112 p.Arg1138Trp expressing cells providing functional evidence of disease aetiology. Disease mechanism in genetic variant neural cells was linked to decreased localisation of MED12L to the nucleolus and dysregulation of the pre-ribosome complex.
