Splicing defect and functional characterization of the ETFDH c.1049G > A VUS underlying transient MADD: an iPSC and...

Interpreting variants of uncertain significance (VUS) is challenging in patients with atypical or transient metabolic presentations.

We combined induced pluripotent stem cell (iPSC) models and minigene assays to characterize an ETFDH VUS (c.1049G > A) in a neonate who presented with acute metabolic decompensation characterized by fatty acid oxidation defects but showed normalized metabolic profiles by 14 months of age.

Patient-derived iPSCs and minigene assays confirmed that c.1049G > A induces predominant exon 9 skipping, resulting in an in-frame 48-amino acid deletion within the FAD-binding domain. Structural modeling based on the human ETFDH crystal structure revealed that this deletion disrupts the core architecture of the FAD-binding domain, notably by eliminating the critical stabilizing interaction between Arg364 and Glu246, predicting compromised FAD binding affinity. Consistent with this structural defect, Western blot analysis showed markedly reduced ETFDH protein levels (< 10%) in patient cells versus heterozygous parents (~ 40%). Collectively, these data indicate that while the variant causes substantial molecular impairment explaining the neonatal crisis, the presence of residual protein likely supports metabolic homeostasis under non-stressful conditions.

The c.1049G > A variant causes significant splicing disruption and partial protein loss, explaining the transient nature of the neonatal decompensation rather than a classic, persistent MADD phenotype. Furthermore, this study underscores the utility of iPSC models in resolving VUS cases where clinical biochemistry normalizes over time, bridging the gap between ambiguous genetic data and precise clinical management.