Abstract
An exonic missense mutation, c.436C>G, in the PLP1 gene of a patient affected by the hypomyelinating leukodystrophy, Pelizaeus–Merzbacher disease, has previously been found to be responsible for the alteration of the canonical alternative splicing profile of the PLP1 gene leading to the loss of the longer PLP isoform. Here we show that the presence of the c.436C>G mutation served to introduce regulatory motifs that appear to be responsible for the perturbed splicing pattern that led to loss of the major PLP transcript. With the aim of disrupting the interaction between the PLP1 splicing regulatory motifs and their cognate splicing factors, we designed an antisense oligonucleotide-based in vitro correction protocol that successfully restored PLP transcript production in oligodendrocyte precursor cells.
Highlights
Pelizaeus–Merzbacher disease (PMD, MIM #312080) and Xlinked paraplegia type 2 (SPG2, MIM #312920) are allelic hypomyelinating leukodystrophies caused by mutations in the PLP1 gene (MIM #300401)
Since no RNA sample was available from the patient, the functional effect of the c.436C>G mutation was investigated using a recombinant LacZ-PLP1-LacZ in-frame minigene containing a PLP1 gene exon 2 – exon 4 fragment cloned into the pcDNA3.1/V5-His-TOPO/LacZ vector
Comparison of the mRNAs generated through the transfection of the wild-type (c.436C) and mutated (c.436G) versions of the construct into Oli-neu cells, demonstrated that the c.436C>G, expected to result in a missense p.L146V mutation, led instead to the loss of the PLP transcript encoding the major isoform of the PLP1 gene, whereas the shorter DM20 version of the PLP1 transcript was produced normally [8]
Summary
Pelizaeus–Merzbacher disease (PMD, MIM #312080) and Xlinked paraplegia type 2 (SPG2, MIM #312920) are allelic hypomyelinating leukodystrophies caused by mutations in the PLP1 gene (MIM #300401). We previously reported a mildly affected PMD patient with a c.436C>G mutation located in exon 3B, and demonstrated that this mutation, which would be expected to substitute leucine at residue 146 with a valine (p.L146V), resulted instead in the loss of the PLP isoform [8].
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