title: U1 snRNA mis-binding: a new cause of CMT1B.
abstract: We report the molecular characterization of two splice mutations in two different French families affected with a late onset form of Charcot-Marie-Tooth disease type 1B (CMT1B), an autosomal dominant inherited disorder caused by mutations in the myelin protein zero gene. The first substitution, c.306G>A, located in exon 3, does not change the codon p.Val102Val but is co-transmitted with the disease in the first family. The second substitution, c.675+3dup, is an insertion of a T at position +3 of intron 5. To identify the functional impact of these nucleotide changes on splicing and because no RNA sample was available, we used in silico prediction and in vitro splicing assay. Mutation c.306G>A increases the strength of a preexisting cryptic donor site at position c.304 which becomes stronger than the normal donor site of intron 3. This variation creates a sequence that better matches the U1 small nuclear RNA (snRNA) binding consensus, and HeLa cells, transfected with the mutant minigene, produce a truncated exon 3 messenger RNA (mRNA). Mutation c.675+3dup was predicted to abolish the donor site of intron 5, and, indeed, HeLa cells transfected with the mutant minigene completely skip exon 5 from the transcript. The mutated sequence abolishes U1 snRNA binding and co-transfection of a mutated complementary U1 snRNA restored exon 5 inclusion in the mRNA. This work provides valuable information regarding the molecular basis of two forms of late onset of CMT1B, U1 snRNA mis-binding, and provides more evidence that a "silent" polymorphism may be a disease causing mutation.
authors.affiliation: Laboratoire de Cardiogénétique Moléculaire, Centre de Biologie et Pathologie Est, Hospices Civils de Lyon, Bron, France.
authors.forename: Hervé
authors.initials: H
authors.lastname: Crehalet
authors.forename: Philippe
authors.initials: P
authors.lastname: Latour
authors.forename: Véronique
authors.initials: V
authors.lastname: Bonnet
authors.forename: Shahram
authors.initials: S
authors.lastname: Attarian
authors.forename: Pierre
authors.initials: P
authors.lastname: Labauge
authors.forename: Nathalie
authors.initials: N
authors.lastname: Bonello
authors.forename: Rafaelle
authors.initials: R
authors.lastname: Bernard
authors.forename: Gilles
authors.initials: G
authors.lastname: Millat
authors.forename: Robert
authors.initials: R
authors.lastname: Rousson
authors.forename: Dominique
authors.initials: D
authors.lastname: Bozon
mesh_terms: D000328:Adult; D002607:Charcot-Marie-Tooth Disease / Q000175:diagnosis* / Q000235:genetics*; D005091:Exons; D005260:Female; D006367:HeLa Cells; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins / Q000235:genetics*; D007438:Introns; D008297:Male; D008875:Middle Aged; D009154:Mutation; D010375:Pedigree; D010750:Phosphoproteins / Q000235:genetics*; D011110:Polymorphism, Genetic; D012326:RNA Splicing; D012342:RNA, Small Nuclear / Q000378:metabolism*
chemical_list: D047908:Intracellular Signaling Peptides and Proteins; C115628:MPZL1 protein, human; D010750:Phosphoproteins; D012342:RNA, Small Nuclear; C405483:U1 small nuclear RNA