title: PMD patient mutations reveal a long-distance intronic interaction that regulates PLP1/DM20 alternative splicing.
abstract: Alternative splicing of the proteolipid protein 1 gene (PLP1) produces two forms, PLP1 and DM20, due to alternative use of 5' splice sites with the same acceptor site in intron 3. The PLP1 form predominates in central nervous system RNA. Mutations that reduce the ratio of PLP1 to DM20, whether mutant or normal protein is formed, result in the X-linked leukodystrophy Pelizaeus-Merzbacher disease (PMD). We investigated the ability of sequences throughout PLP1 intron 3 to regulate alternative splicing using a splicing minigene construct transfected into the oligodendrocyte cell line, Oli-neu. Our data reveal that the alternative splice of PLP1 is regulated by a long-distance interaction between two highly conserved elements that are separated by 581 bases within the 1071-base intron 3. Further, our data suggest that a base-pairing secondary structure forms between these two elements, and we demonstrate that mutations of either element designed to destabilize the secondary structure decreased the PLP1/DM20 ratio, while swap mutations designed to restore the structure brought the PLP1/DM20 ratio to near normal levels. Sequence analysis of intron 3 in families with clinical symptoms of PMD who did not have coding-region mutations revealed mutations that segregated with disease in three families. We showed that these patient mutations, which potentially destabilize the secondary structure, also reduced the PLP1/DM20 ratio. This is the first report of patient mutations causing disease by disruption of a long-distance intronic interaction controlling alternative splicing. This finding has important implications for molecular diagnostics of PMD.
authors.affiliation: Nemours Biomedical Research, Alfred I. duPont Hospital for Children, Wilmington, DE 19803, USA.
authors.forename: Jennifer R
authors.initials: JR
authors.lastname: Taube
authors.affiliation: Nemours Biomedical Research, Alfred I. duPont Hospital for Children, Wilmington, DE 19803, USA.
authors.forename: Karen
authors.initials: K
authors.lastname: Sperle
authors.affiliation: Nemours Biomedical Research, Alfred I. duPont Hospital for Children, Wilmington, DE 19803, USA.
authors.forename: Linda
authors.initials: L
authors.lastname: Banser
authors.affiliation: Department of Child Neurology, DNA Laboratory, 2nd School of Medicine, Charles University and University Hospital Motol, 150 06 Prague 5, Czech Republic.
authors.forename: Pavel
authors.initials: P
authors.lastname: Seeman
authors.affiliation: Department of Pediatrics, Cebu Institute of Medicine, 6000 Cebu City, Philippines.
authors.forename: Barbra Charina V
authors.initials: BC
authors.lastname: Cavan
authors.affiliation: Department of Neurology, University of Rochester Medical Center, Rochester, NY 14642, USA.
authors.forename: James Y
authors.initials: JY
authors.lastname: Garbern
authors.affiliation: Nemours Biomedical Research, Alfred I. duPont Hospital for Children, Wilmington, DE 19803, USA, Department of Biological Sciences, University of Delaware, Newark, DE 19716, USA and Department of Pediatrics, Jefferson Medical College, Thomas Jefferson University, Philadelphia, PA 19107, USA ghobson@nemours.org.
authors.forename: Grace M
authors.initials: GM
authors.lastname: Hobson
mesh_terms: D017398:Alternative Splicing; D020029:Base Pairing; D002460:Cell Line; D005260:Female; D006801:Humans; D007438:Introns; D008297:Male; D008958:Models, Molecular; D009154:Mutation; D018991:Myelin Proteolipid Protein / Q000235:genetics* / Q000378:metabolism; D009690:Nucleic Acid Conformation; D009836:Oligodendroglia / Q000378:metabolism; D010375:Pedigree; D020371:Pelizaeus-Merzbacher Disease / Q000235:genetics*; D012333:RNA, Messenger / Q000737:chemistry* / Q000378:metabolism; D017422:Sequence Analysis, DNA
chemical_list: D018991:Myelin Proteolipid Protein; C472930:PLP1 protein, human; D012333:RNA, Messenger