Mutations at a split codon in the GTPase-encoding domain of OPA1 cause dominant optic atrophy through different molecular mechanisms.

Exonic (i.e. coding) variants in genes associated with disease can exert pathogenic effects both at the protein and mRNA level, either by altering the amino acid sequence or by affecting pre-mRNA splicing. The latter is often neglected due to the lack of RNA analyses in genetic diagnostic testing. In this study we considered both pathomechanisms and performed a comprehensive analysis of nine exonic nucleotide changes in OPA1, which is the major gene underlying autosomal dominant optic atrophy (DOA) and is characterized by pronounced allelic heterogeneity. We focused on the GTPase-encoding domain of OPA1, which harbors most of the missense variants associated with DOA. Given that the consensus splice sites extend into the exons, we chose a split codon, namely codon 438, for our analyses. Variants at this codon are the second most common cause of disease in our large cohort of DOA patients harboring disease-causing variants in OPA1. In silico splice predictions, heterologous splice assays, analysis of patient's RNA when available, and protein modeling revealed different molecular outcomes for variants at codon 438. The wildtype aspartate residue at amino acid position 438 is directly involved in the dimerization of OPA1 monomers. We found that six amino acid substitutions at codon 438 (i.e. all substitutions of the first and second nucleotide of the codon) destabilized dimerization while only substitutions of the first nucleotide of the codon caused exon skipping. Our study highlights the value of combining RNA analysis and protein modeling approaches to accurately assign patients to future precision therapies.