title: Altered PLP1 splicing causes hypomyelination of early myelinating structures.
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.
  
  
  CONCLUSIONS
  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.
authors.affiliation: Department of Child Neurology, VU University Medical Center Amsterdam, The Netherlands ; Neuroscience Campus Amsterdam, VU University Amsterdam, The Netherlands.
authors.forename: Sietske H
authors.initials: SH
authors.lastname: Kevelam
authors.affiliation: Nemours Biomedical Research, Alfred I. duPont Hospital for Children Wilmington, Delaware.
authors.forename: Jennifer R
authors.initials: JR
authors.lastname: Taube
authors.affiliation: Department of Clinical Genetics, VU University Medical Center Amsterdam, The Netherlands.
authors.forename: Rosalina M L
authors.initials: RM
authors.lastname: van Spaendonk
authors.affiliation: Unit for Neuromuscular and Neurodegenerative Diseases, Laboratory of Molecular Medicine, Bambino Gesu' Children's Research Hospital, IRCCS Rome, Italy.
authors.forename: Enrico
authors.initials: E
authors.lastname: Bertini
authors.affiliation: Nemours Biomedical Research, Alfred I. duPont Hospital for Children Wilmington, Delaware.
authors.forename: Karen
authors.initials: K
authors.lastname: Sperle
authors.affiliation: Department of Pediatrics, McMaster Children's Hospital Hamilton, Ontario, Canada.
authors.forename: Mark
authors.initials: M
authors.lastname: Tarnopolsky
authors.affiliation: Child Neuropsychiatry Unit, Department of Brain and Behavioral Sciences, University of Pavia Pavia, Italy.
authors.forename: Davide
authors.initials: D
authors.lastname: Tonduti
authors.affiliation: Department of Medicine and Surgery, University of Salerno Salerno, Italy ; CSS-Mendel Institute, IRCCS Casa Sollievo della Sofferenza San Giovanni Rotondo, Italy.
authors.forename: Enza Maria
authors.initials: EM
authors.lastname: Valente
authors.affiliation: Unit for Neuromuscular and Neurodegenerative Diseases, Laboratory of Molecular Medicine, Bambino Gesu' Children's Research Hospital, IRCCS Rome, Italy.
authors.forename: Lorena
authors.initials: L
authors.lastname: Travaglini
authors.affiliation: Department of Clinical Genetics, VU University Medical Center Amsterdam, The Netherlands.
authors.forename: Erik A
authors.initials: EA
authors.lastname: Sistermans
authors.affiliation: Division of Pediatric Neurology, Departments of Pediatrics, Neurology and Neurosurgery, Montreal Children's Hospital, McGill University Health Center Montreal, Quebec, Canada.
authors.forename: Geneviève
authors.initials: G
authors.lastname: Bernard
authors.affiliation: Department of Pediatric Neurology, Erasmus University Hospital - Sophia Children's Hospital Rotterdam, The Netherlands.
authors.forename: Coriene E
authors.initials: CE
authors.lastname: Catsman-Berrevoets
authors.affiliation: Division of Biochemical Diseases, Department of Pediatrics, BC Children's Hospital, Centre for Molecular Medicine and Therapeutics, University of British Columbia Vancouver, Canada.
authors.forename: Clara D M
authors.initials: CD
authors.lastname: van Karnebeek
authors.affiliation: Centre for Rare diseases, Department of Paediatrics, Aarhus University Hospital Aarhus, Denmark.
authors.forename: John R
authors.initials: JR
authors.lastname: Østergaard
authors.affiliation: Department of Child Neurology, Kaiser Permanente Pediatric Specialties Roseville, California.
authors.forename: Richard L
authors.initials: RL
authors.lastname: Friederich
authors.affiliation: Department of Pediatric Neurology, Hamad Medical Corp Doha, Qatar.
authors.forename: Mahmoud
authors.initials: M
authors.lastname: Fawzi Elsaid
authors.affiliation: Department of Child Neurology, Radboud University Medical Center Nijmegen, The Netherlands.
authors.forename: Jolanda H
authors.initials: JH
authors.lastname: Schieving
authors.affiliation: Department of Medical Genetics, University of British Colombia Vancouver, Canada.
authors.forename: Maja
authors.initials: M
authors.lastname: Tarailo-Graovac
authors.affiliation: Child Neurology and Psychiatry Unit, C. Mondino National Neurological Institute Pavia, Italy.
authors.forename: Simona
authors.initials: S
authors.lastname: Orcesi
authors.affiliation: Department of Child Neurology, VU University Medical Center Amsterdam, The Netherlands ; Neuroscience Campus Amsterdam, VU University Amsterdam, The Netherlands.
authors.forename: Marjan E
authors.initials: ME
authors.lastname: Steenweg
authors.affiliation: Department of Child Neurology, VU University Medical Center Amsterdam, The Netherlands.
authors.forename: Carola G M
authors.initials: CG
authors.lastname: van Berkel
authors.affiliation: Department of Clinical Genetics, VU University Medical Center Amsterdam, The Netherlands.
authors.forename: Quinten
authors.initials: Q
authors.lastname: Waisfisz
authors.affiliation: Department of Child Neurology, VU University Medical Center Amsterdam, The Netherlands ; Neuroscience Campus Amsterdam, VU University Amsterdam, The Netherlands.
authors.forename: Truus E M
authors.initials: TE
authors.lastname: Abbink
authors.affiliation: 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.
authors.forename: Marjo S
authors.initials: MS
authors.lastname: van der Knaap
authors.affiliation: 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.
authors.forename: Grace M
authors.initials: GM
authors.lastname: Hobson
authors.affiliation: Department of Child Neurology, VU University Medical Center Amsterdam, The Netherlands ; Neuroscience Campus Amsterdam, VU University Amsterdam, The Netherlands.
authors.forename: Nicole I
authors.initials: NI
authors.lastname: Wolf