Objective: The objective of this study was to investigate the genetic etiology of the X-linked disorder "Hypomyelination of Early Myelinating Structures" (HEMS). Methods: We included 16 patients from 10 families diagnosed with HEMS by brain MRI criteria. Exome sequencing was used to search for causal mutations. In silico analysis of effects of the mutations on splicing and RNA folding was performed. In vitro gene splicing was examined in RNA from patients' fibroblasts and an immortalized immature oligodendrocyte cell line after transfection with mutant minigene splicing constructs. Results: All patients had unusual hemizygous mutations of PLP1 located in exon 3B (one deletion, one missense and two silent), which is spliced out in isoform DM20, or in intron 3 (five mutations). The deletion led to truncation of PLP1, but not DM20. Four mutations were predicted to affect PLP1/DM20 alternative splicing by creating exonic splicing silencer motifs or new splice donor sites or by affecting the local RNA structure of the PLP1 splice donor site. Four deep intronic mutations were predicted to destabilize a long-distance interaction structure in the secondary PLP1 RNA fragment involved in regulating PLP1/DM20 alternative splicing. Splicing studies in fibroblasts and transfected cells confirmed a decreased PLP1/DM20 ratio. Interpretation: Brain structures that normally myelinate early are poorly myelinated in HEMS, while they are the best myelinated structures in Pelizaeus-Merzbacher disease, also caused by PLP1 alterations. Our data extend the phenotypic spectrum of PLP1-related disorders indicating that normal PLP1/DM20 alternative splicing is essential for early myelination and support the need to include intron 3 in diagnostic sequencing.

Altered PLP1 splicing causes hypomyelination of early myelinating structures / S. Kevelam, J. Taube, R. van Spaendonk, E. Bertini, K. Sperle, M. Tarnopolsky, D. Tonduti, E. Valente, L. Travaglini, E. Sistermans, G. Bernard, C. Catsman-Berrevoets, C. van Karnebeek, J. Ostergaard, R. Friederich, M. Elsaid, J. Schieving, M. Tarailo-Graovac, S. Orcesi, M. Steenweg, C. van Berkel, Q. Waisfisz, T. Abbink, M. van der Knaap, G. Hobson, N. Wolf. - In: ANNALS OF CLINICAL AND TRANSLATIONAL NEUROLOGY. - ISSN 2328-9503. - 2:6(2015 Jun), pp. 648-661. [10.1002/acn3.203]

Altered PLP1 splicing causes hypomyelination of early myelinating structures

D. Tonduti;
2015

Abstract

Objective: The objective of this study was to investigate the genetic etiology of the X-linked disorder "Hypomyelination of Early Myelinating Structures" (HEMS). Methods: We included 16 patients from 10 families diagnosed with HEMS by brain MRI criteria. Exome sequencing was used to search for causal mutations. In silico analysis of effects of the mutations on splicing and RNA folding was performed. In vitro gene splicing was examined in RNA from patients' fibroblasts and an immortalized immature oligodendrocyte cell line after transfection with mutant minigene splicing constructs. Results: All patients had unusual hemizygous mutations of PLP1 located in exon 3B (one deletion, one missense and two silent), which is spliced out in isoform DM20, or in intron 3 (five mutations). The deletion led to truncation of PLP1, but not DM20. Four mutations were predicted to affect PLP1/DM20 alternative splicing by creating exonic splicing silencer motifs or new splice donor sites or by affecting the local RNA structure of the PLP1 splice donor site. Four deep intronic mutations were predicted to destabilize a long-distance interaction structure in the secondary PLP1 RNA fragment involved in regulating PLP1/DM20 alternative splicing. Splicing studies in fibroblasts and transfected cells confirmed a decreased PLP1/DM20 ratio. Interpretation: Brain structures that normally myelinate early are poorly myelinated in HEMS, while they are the best myelinated structures in Pelizaeus-Merzbacher disease, also caused by PLP1 alterations. Our data extend the phenotypic spectrum of PLP1-related disorders indicating that normal PLP1/DM20 alternative splicing is essential for early myelination and support the need to include intron 3 in diagnostic sequencing.
Settore MED/39 - Neuropsichiatria Infantile
   Understanding the basis of cerebellar and brainstem congenital defects: from clinical and molecular characterisation to the development of a novel neuroembryonic in vitro model
   CBCD
   European Commission
   260888
giu-2015
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/2434/938712
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