Abstract
Pelizaeus–Merzbacher disease (PMD) is a rare Mendelian disorder characterised by central nervous system hypomyelination. PMD typically manifests in infancy or early childhood and is caused by mutations in proteolipid protein-1 (PLP1). However, variants in several other genes including gap junction protein gamma 2 (GJC2) can also cause a similar phenotype and are referred to PMD-like disease (PMLD). Whole-exome sequencing in two siblings presenting with clinical symptoms of PMD revealed a homozygous variant in the arginyl-tRNA synthetase (RARS) gene: NM_002887.3: c.[5A>G] p.(Asp2Gly). Subsequent screening of a PMD cohort without a genetic diagnosis identified an unrelated individual with novel compound heterozygous variants including a missense variant c.[1367C>T] p.(Ser456Leu) and a de novo deletion c.[1846_1847delTA] p.(Tyr616Leufs*6). Protein levels of RARS and the multi-tRNA synthetase complex into which it assembles were found to be significantly reduced by 80 and 90% by western blotting and Blue native-PAGE respectively using patient fibroblast extracts. As RARS is involved in protein synthesis whereby it attaches arginine to its cognate tRNA, patient cells were studied to determine their ability to proliferate with limiting amounts of this essential amino acid. Patient fibroblasts cultured in medium with limited arginine at 30 °C and 40 °C, showed a significant decrease in fibroblast proliferation (P<0.001) compared to control cells, suggestive of inefficiency of protein synthesis in the patient cells. Our functional studies provide further evidence that RARS is a PMD-causing gene.
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Acknowledgements
We are grateful to the NSW Biochemical Genetics Service for establishing initial fibroblast line cultures, and the Molecular Genetics Department for initial DNA extraction, both located at the Western Sydney Genetics Program, Children’s Hospital at Westmead. We are also grateful to Dr Grace Hobson, Alfred I DuPont Hospital for Children, Wilmington, DE, USA, for provision of DNA samples for Sanger sequencing, none of which were shown to have RARS variations. Finally, we are grateful to Dr Shanti Balasubramaniam (Western Sydney Genetics Program, Children’s Hospital at Westmead) and Dr Sebastian Lunke (Translational Genomics Unit, Victorian Clinical Genetics Services) for acquisition and interpretation of some data relating to patient 1. All procedures followed were in accordance with the ethical standards of the responsible committee on human experimentation (institutional and national) and with the Helsinki Declaration of 1975, as revised in 2000. Informed consent was obtained from all patients included in the study as approved by the Human Research Ethics Committee of the Sydney Children’s Hospitals Network and the Institutional Review Board of Johns Hopkins Medicine.
Author contributions
MN: design and implementation of structural and functional studies, data analysis and manuscript preparation. WG and LR: contribution to design of structural and functional studies, data analysis and manuscript preparation. NS and CB: performance of whole-exome sequencing, implementation of bioinformatics, data analysis and manuscript preparation. KP: interpretation of MRI images for the Australian patients and manuscript preparation. BU: Initial molecular genetic screening and provision DNA sample for the German patient and manuscript preparation. CW: provision of clinical information for the German patient and manuscript preparation. RO: clinical diagnosis and management of the Australian patients and manuscript preparation. JC: overall oversight of the research project, clinical interface, data analysis and manuscript preparation.
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Nafisinia, M., Sobreira, N., Riley, L. et al. Mutations in RARS cause a hypomyelination disorder akin to Pelizaeus–Merzbacher disease. Eur J Hum Genet 25, 1134–1141 (2017). https://doi.org/10.1038/ejhg.2017.119
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DOI: https://doi.org/10.1038/ejhg.2017.119
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