Skip to main content

Thank you for visiting You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

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



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.


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.


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.”


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.


  • 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.

This is a preview of subscription content, access via your institution

Access options

Buy article

Get time limited or full article access on ReadCube.


All prices are NET prices.

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.


  1. Quaderi, N. A. et al. Opitz G/BBB syndrome, a defect of midline development, is due to mutations in a new RING finger gene on Xp22. Nat. Genet. 17, 285–291 (1997).

    CAS  Article  Google Scholar 

  2. Kruszka, P. et al. Mutations in SPECC1L, encoding sperm antigen with calponin homology and coiled-coil domains 1-like, are found in some cases of autosomal dominant Opitz G/BBB syndrome. J. Med. Genet. 52, 104–110 (2015).

    CAS  Article  Google Scholar 

  3. Robin, N. H. et al. Opitz syndrome is genetically heterogeneous, with one locus on Xp22, and a second locus on 22q11.2. Nat. Genet. 11, 459–461 (1995).

    CAS  Article  Google Scholar 

  4. Gaudenz, K. et al. Opitz G/BBB syndrome in Xp22: mutations in the MID1 gene cluster in the carboxy-terminal domain. Am. J. Hum. Genet. 63, 703–710 (1998).

    CAS  Article  Google Scholar 

  5. Cox, T. C. et al. New mutations in MID1 provide support for loss of function as the cause of X-linked Opitz syndrome. Hum. Mol. Genet. 9, 2553–2562 (2000).

    CAS  Article  Google Scholar 

  6. Pinson, L. et al. Embryonic expression of the human MID1 gene and its mutations in Opitz syndrome. J. Med. Genet. 41, 381–386 (2004).

    CAS  Article  Google Scholar 

  7. So, J. et al. Mild phenotypes in a series of patients with Opitz GBBB syndrome with MID1 mutations. Am. J. Med. Genet. A 132A, 1–7 (2005).

    Article  Google Scholar 

  8. Mnayer, L., Khuri, S., Merheby, H. A., Meroni, G. & Elsas, L. J. A structure-function study of MID1 mutations associated with a mild Opitz phenotype. Mol. Genet. Metab. 87, 198–203 (2006).

    CAS  Article  Google Scholar 

  9. Ferrentino, R., Bassi, M. T., Chitayat, D., Tabolacci, E. & Meroni, G. MID1 mutation screening in a large cohort of Opitz G/BBB syndrome patients: twenty-nine novel mutations identified. Hum. Mutat. 28, 206–207 (2007).

    Article  Google Scholar 

  10. Reymond, A. et al. The tripartite motif family identifies cell compartments. EMBO J. 20, 2140–2151 (2001).

    CAS  Article  Google Scholar 

  11. Trockenbacher, A. et al. MID1, mutated in Opitz syndrome, encodes an ubiquitin ligase that targets phosphatase 2A for degradation. Nat. Genet. 29, 287–294 (2001).

    CAS  Article  Google Scholar 

  12. Liu, E., Knutzen, C. A., Krauss, S., Schweiger, S. & Chiang, G. G. Control of mTORC1 signaling by the Opitz syndrome protein MID1. Proc. Natl Acad. Sci. USA 108, 8680–8685 (2011).

    CAS  Article  Google Scholar 

  13. Carracedo, A. & Pandolfi, P. P. The PTEN-PI3K pathway: of feedbacks and cross-talks. Oncogene 27, 5527–5541 (2008).

    CAS  Article  Google Scholar 

  14. Short, K. M. & Cox, T. C. Subclassification of the RBCC/TRIM superfamily reveals a novel motif necessary for microtubule binding. J. Biol. Chem. 281, 8970–8980 (2006).

    CAS  Article  Google Scholar 

  15. Gholkar, A. A. et al. The X-linked-intellectual-disability-associated ubiquitin ligase Mid2 interacts with astrin and regulates astrin levels to promote cell division. Cell Rep. 14, 180–188 (2016).

    CAS  Article  Google Scholar 

  16. Zanchetta, M. E., Napolitano, L. M. R., Maddalo, D. & Meroni, G. The E3 ubiquitin ligase MID1/TRIM18 promotes atypical ubiquitination of the BRCA2-associated factor 35, BRAF35. Biochim. Biophys. Acta Mol. Cell Res. 1864, 1844–1854 (2017).

    CAS  Article  Google Scholar 

  17. Zanchetta, M. E. & Meroni, G. Emerging roles of the TRIM E3 ubiquitin ligases MID1 and MID2 in cytokinesis. Front. Physiol. 10, 274 (2019).

    Article  Google Scholar 

  18. Brnich, S. E. et al. Recommendations for application of the functional evidence PS3/BS3 criterion using the ACMG/AMP sequence variant interpretation framework. Genome Med. 12, 3 (2019).

    Article  Google Scholar 

  19. Richards, S. et al. Standards and guidelines for the interpretation of sequence variants: a Joint Consensus Recommendation of the American College of Medical Genetics and Genomics and the Association for Molecular Pathology. Genet. Med. 17, 405–424 (2015).

    Article  Google Scholar 

  20. Tompson, S. W. & Young, T. L. Assaying the effects of splice site variants by exon trapping in a mammalian cell line. Bio Protoc. 7, e2281 (2017).

  21. Abou, Tayoun et al. Recommendations for interpreting the loss of function PVS1 ACMG/AMP variant criterion. Hum. Mutat. 39, 1517–1524 (2018).

    Article  Google Scholar 

  22. Steffensen, A. Y. et al. Functional characterization of BRCA1 gene variants by mini-gene splicing assay. Eur. J. Hum. Genet. 22, 1362–1368 (2014).

    CAS  Article  Google Scholar 

  23. Fraile-Bethencourt, E. et al. Functional classification of DNA variants by hybrid minigenes: Identification of 30 spliceogenic variants of BRCA2 exons 17 and 18. PLoS Genet. 13, e1006691 (2017).

    Article  Google Scholar 

  24. Giorgi, G. et al. Validation of CFTR intronic variants identified during cystic fibrosis population screening by a minigene splicing assay. Clin. Chem. Lab. Med. 53, 1719–1723 (2015).

    CAS  Article  Google Scholar 

  25. Villate, O. et al. Functional analyses of a novel splice variant in the CHD7 gene, found by next generation sequencing, confirm its pathogenicity in a Spanish patient and diagnose him with CHARGE Syndrome. Front. Genet. 9, 7 (2018).

    Article  Google Scholar 

  26. Morbidoni, V. et al. Hybrid minigene assay: an efficient tool to characterize mRNA splicing profiles of NF1 variants. Cancers 13, 999 (2021).

  27. Hentze, M. W. & Kulozik, A. E. A perfect message: RNA surveillance and nonsense-mediated decay. Cell 96, 307–310 (1999).

    CAS  Article  Google Scholar 

  28. Linde, L. et al. The efficiency of nonsense-mediated mRNA decay is an inherent character and varies among different cells. Eur. J. Hum. Genet. 15, 1156–1162 (2007).

    CAS  Article  Google Scholar 

  29. Lejeune, F. Nonsense-mediated mRNA decay, a finely regulated mechanism. Biomedicines 10, 141 (2022).

  30. Sarkar, H. et al. Nonsense-mediated mRNA decay efficiency varies in choroideremia providing a target to boost small molecule therapeutics. Hum. Mol. Genet. 28, 1865–1871 (2019).

    CAS  Article  Google Scholar 

Download references


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.


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

Author information

Authors and Affiliations



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.

Ethics declarations

COMPETING interests

The authors declare no competing interests.

Ethics approval and consent to participate

Written informed consent was obtained from all individuals for publication.

Additional information

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Supplementary information

Rights and permissions

Springer Nature or its licensor holds exclusive rights to this article under a publishing agreement with the author(s) or other rightsholder(s); author self-archiving of the accepted manuscript version of this article is solely governed by the terms of such publishing agreement and applicable law.

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Micale, L., Russo, F., Mascaro, M. et al. Opitz syndrome: improving clinical interpretation of intronic variants in MID1 gene. Pediatr Res (2022).

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • DOI:


Quick links