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Ann Clin Transl Neurol. 2015 Jun;2(6):648-61. doi: 10.1002/acn3.203. Epub 2015 May 1.

Altered PLP1 splicing causes hypomyelination of early myelinating structures.

Author information

1
Department of Child Neurology, VU University Medical Center Amsterdam, The Netherlands ; Neuroscience Campus Amsterdam, VU University Amsterdam, The Netherlands.
2
Nemours Biomedical Research, Alfred I. duPont Hospital for Children Wilmington, Delaware.
3
Department of Clinical Genetics, VU University Medical Center Amsterdam, The Netherlands.
4
Unit for Neuromuscular and Neurodegenerative Diseases, Laboratory of Molecular Medicine, Bambino Gesu' Children's Research Hospital, IRCCS Rome, Italy.
5
Department of Pediatrics, McMaster Children's Hospital Hamilton, Ontario, Canada.
6
Child Neuropsychiatry Unit, Department of Brain and Behavioral Sciences, University of Pavia Pavia, Italy.
7
Department of Medicine and Surgery, University of Salerno Salerno, Italy ; CSS-Mendel Institute, IRCCS Casa Sollievo della Sofferenza San Giovanni Rotondo, Italy.
8
Division of Pediatric Neurology, Departments of Pediatrics, Neurology and Neurosurgery, Montreal Children's Hospital, McGill University Health Center Montreal, Quebec, Canada.
9
Department of Pediatric Neurology, Erasmus University Hospital - Sophia Children's Hospital Rotterdam, The Netherlands.
10
Division of Biochemical Diseases, Department of Pediatrics, BC Children's Hospital, Centre for Molecular Medicine and Therapeutics, University of British Columbia Vancouver, Canada.
11
Centre for Rare diseases, Department of Paediatrics, Aarhus University Hospital Aarhus, Denmark.
12
Department of Child Neurology, Kaiser Permanente Pediatric Specialties Roseville, California.
13
Department of Pediatric Neurology, Hamad Medical Corp Doha, Qatar.
14
Department of Child Neurology, Radboud University Medical Center Nijmegen, The Netherlands.
15
Department of Medical Genetics, University of British Colombia Vancouver, Canada.
16
Child Neurology and Psychiatry Unit, C. Mondino National Neurological Institute Pavia, Italy.
17
Department of Child Neurology, VU University Medical Center Amsterdam, The Netherlands.
18
Department of Child Neurology, VU University Medical Center Amsterdam, The Netherlands ; Neuroscience Campus Amsterdam, VU University Amsterdam, The Netherlands ; Department of Functional Genomics, Center for Neurogenomics and Cognitive Research, VU University Amsterdam, The Netherlands.
19
Nemours Biomedical Research, Alfred I. duPont Hospital for Children Wilmington, Delaware ; Department of Biological Sciences, University of Delaware Newark, Delaware ; Department of Pediatrics, Jefferson Medical College, Thomas Jefferson University Philadelphia, Pennsylvania.

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.

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