A recurrent COL6A1 pseudoexon insertion causes muscular dystrophy and is effectively targeted by splice-correction therapies
Véronique Bolduc, … , Francesco Muntoni, Carsten G. Bönnemann
Véronique Bolduc, … , Francesco Muntoni, Carsten G. Bönnemann
Published March 21, 2019
Citation Information: JCI Insight. 2019;4(6):e124403. https://doi.org/10.1172/jci.insight.124403.
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Research Article Muscle biology Therapeutics

A recurrent COL6A1 pseudoexon insertion causes muscular dystrophy and is effectively targeted by splice-correction therapies

Abstract

The clinical application of advanced next-generation sequencing technologies is increasingly uncovering novel classes of mutations that may serve as potential targets for precision medicine therapeutics. Here, we show that a deep intronic splice defect in the COL6A1 gene, originally discovered by applying muscle RNA sequencing in patients with clinical findings of collagen VI–related dystrophy (COL6-RD), inserts an in-frame pseudoexon into COL6A1 mRNA, encodes a mutant collagen α1(VI) protein that exerts a dominant-negative effect on collagen VI matrix assembly, and provides a unique opportunity for splice-correction approaches aimed at restoring normal gene expression. Using splice-modulating antisense oligomers, we efficiently skipped the pseudoexon in patient-derived fibroblast cultures and restored a wild-type matrix. Similarly, we used CRISPR/Cas9 to precisely delete an intronic sequence containing the pseudoexon and efficiently abolish its inclusion while preserving wild-type splicing. Considering that this splice defect is emerging as one of the single most frequent mutations in COL6-RD, the design of specific and effective splice-correction therapies offers a promising path for clinical translation.

Authors

Véronique Bolduc, A. Reghan Foley, Herimela Solomon-Degefa, Apurva Sarathy, Sandra Donkervoort, Ying Hu, Grace S. Chen, Katherine Sizov, Matthew Nalls, Haiyan Zhou, Sara Aguti, Beryl B. Cummings, Monkol Lek, Taru Tukiainen, Jamie L. Marshall, Oded Regev, Dina Marek-Yagel, Anna Sarkozy, Russell J. Butterfield, Cristina Jou, Cecilia Jimenez-Mallebrera, Yan Li, Corine Gartioux, Kamel Mamchaoui, Valérie Allamand, Francesca Gualandi, Alessandra Ferlini, Eric Hanssen, the COL6A1 Intron 11 Study Group, Steve D. Wilton, Shireen R. Lamandé, Daniel G. MacArthur, Raimund Wagener, Francesco Muntoni, Carsten G. Bönnemann

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Figure 1

A deep intronic COL6A1 mutation creates a donor splice site, prompting the insertion of a pseudoexonic sequence.

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A deep intronic COL6A1 mutation creates a donor splice site, prompting t...
(A) Immunofluorescence images of muscle biopsy sections of patient US1 and one unaffected control costained for collagen VI (red), basement membrane marker laminin (green), and with nuclear stain DAPI (blue). Scale bars: 50 μm. (B) Sashimi plots comparing skeletal muscle biopsy (top) and cultured dermal fibroblast (bottom) RNA sequencing reads from patient UK1, at the COL6A1 intron 11 locus. (C) Genomic DNA chromatograms for the COL6A1 +189C>T mutation testing in family US2. (D) Schematics of the COL6A1 +189C>T mutation locus depicting the pseudoexon sequence boundaries, and splicing outcomes. Splice-modulating antisense oligomers targeting the pseudoexon pre-mRNA sequence are used to promote its skipping as a therapeutic strategy. (E) Splicing reporter (minigene) constructs were prepared by subcloning various segments of COL6A1 genomic DNA (intervening sequence 11 [IVS-11], exons 11 to 13 [Ex-11-13], or exons 10 to 13 [Ex-10-13]) into the pET01 vector (top). Minigenes were transfected into HEK293T cells, and RNA was isolated 24 hours later. Electrophoretic gel image (bottom) represents reverse transcription PCR (RT-PCR) products, amplified using pET01 primers (arrows). Composition of the amplicons is exemplified for Ex-10-13 (right). L = molecular weight ladder. Gel representative of 3 transfections. (F) The pseudoexon percent spliced in (PSI) index was calculated from the RNA sequencing data of 4 muscle specimens and 1 dermis-derived cultured fibroblast specimen, and indicates the percentage of COL6A1 transcript reads that include the pseudoexon. (G and H) Detection of the 72-nucleotide pseudoexon expression in 3 cultured dermal fibroblasts and their corresponding muscle biopsy specimens (G), or in patient F1’s muscle biopsy, cultured dermis- and muscle-derived fibroblasts (H), using RT-PCR. Primers spanning COL6A1 exons 10 to 20 (G), or exons 10 to 15 (H) were used for amplification. RT-PCR gel images were quantified by densitometry (ImageJ) to determine the percentage pseudoexon expression (% p-exon, indicated below each lane and representing the average of 3 to 4 gel quantifications). (I) Location of the single-nucleotide polymorphism rs1980982 relative to the +189C>T mutation site. cDNA from US14 fibroblasts, in whom the “T” allele of rs1980982 is located in cis to the +189T mutation (below), was amplified with primers located in exons 10 and 19 (arrows) for subsequent shotgun cloning.