EFTUD2 missense variants disrupt protein function and splicing in mandibulofacial dysostosis Guion‐Almeida type

Pathogenic variants in the core spliceosome U5 small nuclear ribonucleoprotein gene EFTUD2/SNU114 cause the craniofacial disorder mandibulofacial dysostosis Guion‐Almeida type (MFDGA). MFDGA‐associated variants in EFTUD2 comprise large deletions encompassing EFTUD2, intragenic deletions and single nucleotide truncating or missense variants. These variants are predicted to result in haploinsufficiency by loss‐of‐function of the variant allele. While the contribution of deletions within EFTUD2 to allele loss‐of‐function are self‐evident, the mechanisms by which missense variants are disease‐causing have not been characterized functionally. Combining bioinformatics software prediction, yeast functional growth assays, and a minigene (MG) splicing assay, we have characterized how MFDGA missense variants result in EFTUD2 loss‐of‐function. Only four of 19 assessed missense variants cause EFTUD2 loss‐of‐function through altered protein function when modeled in yeast. Of the remaining 15 missense variants, five altered the normal splicing pattern of EFTUD2 pre‐messenger RNA predominantly through exon skipping or cryptic splice site activation, leading to the introduction of a premature termination codon. Comparison of bioinformatic predictors for each missense variant revealed a disparity amongst different software packages and, in many cases, an inability to correctly predict changes in splicing subsequently determined by MG interrogation. This study highlights the need for laboratory‐based validation of bioinformatic predictions for EFTUD2 missense variants. We have carried out a functional analysis of missense variants in EFTUD2 associated with mandibulofacial dysostosis Guion‐Almeida type. Using a combination of bioinformatics software prediction, yeast functional growth assays and a minigene splicing assay, we have characterised how missense variants result in EFTUD2 loss‐of‐function. We find that EFTUD2 missense variants not only affect protein function but also splicing of the EFTUD2 pre‐messenger RNA that is not predicted well by currently available bioinformatics programs.