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Opitz syndrome: improving clinical interpretation of intronic variants in MID1 gene

Abstract

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 MID1 are rarely investigated in Opitz syndrome.

  • MID1 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 MID1 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.

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Fig. 1: Clinical and molecular findings of individual 1.
Fig. 2: Molecular findings of individual 2 and 3.

Data availability

The datasets generated and analyzed during the current study are available from the corresponding author on reasonable request.

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Acknowledgements

The authors thank the families for their kind availability in sharing the findings within the scientific community. We acknowledge S.W. Tompson (Department of Ophthalmology and Visual Sciences, University of Wisconsin-Madison) for providing pSPL3 vector.

Funding

This work was supported by the Ricerca Corrente 2018–2020 Program from the Italian Ministry of Health and Regione Puglia.

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Contributions

L.M., G.M. and M.C. conceived and designed the work that led to the submission, acquired data, and played an important role in interpreting the results. F.R., M.M., G.N. and C.F. conducted functional studies. L.B. and S.M. analyzed the exome-sequencing data. L.M., G.M. and M.C. wrote the manuscript. All authors revised the manuscript. All of the authors read and approved the final manuscript.

Corresponding author

Correspondence to Lucia Micale.

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Micale, L., Russo, F., Mascaro, M. et al. Opitz syndrome: improving clinical interpretation of intronic variants in MID1 gene. Pediatr Res (2022). https://doi.org/10.1038/s41390-022-02237-y

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