New cause of CMT 1B: U1 snRNA mis-binding

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Neurogenetics. 2009 May 28.

U1 snRNA mis-binding: a new cause of CMT1B.

Crehalet H, Latour P, Bonnet V, Attarian S, Labauge P, Bonello N, Bernard R,

Millat G, Rousson R, Bozon D.

Laboratoire de Cardiogénétique Moléculaire, Centre de Biologie et Pathologie

Est, Hospices Civils de Lyon, Bron, France.

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.