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Upstream open reading frame-introducing variants in patients with primary familial brain calcification

European Journal of Human Genetics volume 32, pages 779–785 (2024)Cite this article

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Abstract

More than 50% of patients with primary familial brain calcification (PFBC), a rare neurological disorder, remain genetically unexplained. While some causative genes are yet to be identified, variants in non-coding regions of known genes may represent a source of missed diagnoses. We hypothesized that 5ʹ-Untranslated Region (UTR) variants introducing an AUG codon may initiate mRNA translation and result in a loss of function in some of the PFBC genes. After reannotation of exome sequencing data of 113 unrelated PFBC probands, we identified two upstream AUG-introducing variants in the 5’UTR of PDGFB. One, NM_002608.4:c.-373C>G, segregated with PFBC in the family. It was predicted to create an upstream open reading frame (ORF). The other one, NM_002608.4:c.-318C>T, was found in a simplex case. It was predicted to result in an ORF overlapping the natural ORF with a frameshift. In a GFP reporter assay, both variants were associated with a dramatic decrease in GFP levels, and, after restoring the reading frame with the GFP sequence, the c.-318C>T variant was associated with a strong initiation of translation as measured by western blotting. Overall, we found upstream AUG-introducing variants in the 5’UTR of PDGFB in 2/113 (1.7%) undiagnosed PFBC cases. Such variants thus represent a source of putative pathogenic variants.

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Fig. 1: Schematic representation of the identified PDGFB c.-373C>G and c.-318C>T variants and molecular constructs.
Fig. 2: Reduced pedigrees of Family EXT-986 (left panel) and EXT-2263 (right panel).
Fig. 3: Functional impact of the PDGFB c.-373C>G and c.-318C>T variants on GFP expression using an in vitro GFP reporter assay.

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Acknowledgements

We are grateful to the patients and their families.

Funding

Funding

We thank SFN and JNLF for A.B. research fellowship. This study was supported by grants from the French National Research Agency (CALCIPHOS, ANR-17-CE14-0008 to GN) and from Conseil Régional de Haute Normandie—APERC 2014 no. 2014-19 in the context of Appel d’Offres Jeunes Chercheurs (CHU de Rouen to GN). This study was cosupported by European Union and Région Normandie, more specifically in the context of the Recherche Innovation Normandie (RIN 2018 to GN). Europe gets involved in Normandie with the European Regional Development Fund.The CEA-CNRGH sequencing platform was supported by the France Génomique National infrastructure, funded as part of the « Investissements d’Avenir » program managed by the Agence Nationale pour la Recherche (contract ANR-10-INBS-09).

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Author notes

  1. These authors contributed equally: Anne Rovelet-Lecrux, Antoine Bonnevalle, Magalie Lecourtois, Gaël Nicolas.

Authors and Affiliations

  1. Univ Rouen Normandie, Inserm U1245 and CHU Rouen, Department of Genetics, CNRMAJ and Reference Center for Neurogenetics Disorders, F-76000, Rouen, France

    Anne Rovelet-Lecrux, Olivier Quenez, Wandrille Delcroix, Kévin Cassinari, Anne-Claire Richard, Magalie Lecourtois & Gaël Nicolas

  2. Univ Rouen Normandie, Inserm U1245 and CHU Rouen, Department of Neurology, CNRMAJ and Reference Center for Neurogenetics Disorders, F-76000, Rouen, France

    Antoine Bonnevalle

  3. Université Paris‐Saclay, CEA, Centre National de Recherche en Génomique Humaine (CNRGH), 91057, Evry, France

    Anne Boland & Jean-François Deleuze

  4. Department of Medical Genetics, National Reference Center for Rare Diseases ‘Neurogenetic’, Pellegrin Hospital, Bordeaux University Hospital, and University of Bordeaux, CNRS, INCIA, UMR 5287, NRGen Team, Bordeaux, France

    Cyril Goizet

  5. Department of Neurology, University-Hospital of Angers, 49933, Angers, France

    Alice Rucar & Christophe Verny

  6. Unité MitoVasc, UMR CNRS 6015, INSERM U1083, 49933, Angers, France

    Alice Rucar & Christophe Verny

  7. AP-HM, Hôpital Timone, Département de Génétique Médicale, Marseille, France

    Karine Nguyen

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  1. Anne Rovelet-Lecrux
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Contributions

Study design and study supervision: GN, ML. Data acquisition and data analysis: all authors. Drafting the manuscript: GN, ARL, ML.

Corresponding author

Correspondence to Gaël Nicolas.

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The authors declare no competing interests.

Ethical approval

This study was approved by the CERDE ethics committee of the Rouen University Hospital (E2023-40) and has therefore been performed in accordance with the ethical standards laid down in the 1964 Declaration of Helsinki and its later amendments.

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Rovelet-Lecrux, A., Bonnevalle, A., Quenez, O. et al. Upstream open reading frame-introducing variants in patients with primary familial brain calcification. Eur J Hum Genet 32, 779–785 (2024). https://doi.org/10.1038/s41431-024-01580-4

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