Background Loss-of-function variants in MID1 are the most common cause of Opitz G/BBB syndrome (OS). The interpretation of intronic variants affecting the splicing is a rising issue in OS. Methods Exon sequencing of a 2-year-old boy with OS showed that he was a carrier of the de novo c.1286-10G>T variant in MID1. In silico predictions and minigene assays explored the effect of the variant on splicing. The minigene approach was also applied to two previously identified MID1 c.864+1G>T and c.1285+1G>T variants. Results Minigene assay demonstrated that the c.1286-10G>T variant generated the inclusion of eight nucleotides that predicted generation of a frameshift. The c.864+1G>T and c.1285+1G>T variants resulted in an in-frame deletion predicted to generate a shorter MID1 protein. In hemizygous males, this allowed reclassification of all the identified variants from "of unknown significance" to "likely pathogenic." Conclusions Minigene assay supports functional effects from MID1 intronic variants. This paves the way to the introduction of similar second-tier investigations in the molecular diagnostics workflow of OS. Impact Causative intronic variants in are rarely investigated in Opitz syndrome. is not expressed in blood and mRNA studies are hardly accessible in routine diagnostics. Minigene assay is an alternative for assessing the effect of intronic variants on splicing. This is the first study characterizing the molecular consequences of three variants for diagnostic purposes and demonstrating the efficacy of minigene assays in supporting their clinical interpretation. Review of the criteria according to the American College of Medical Genetics reassessed all variants as likely pathogenic.
