Background Facioscapulohumeral dystrophy (FSHD) is associated with partial chromatin relaxation of the DUX4 retrogene containing D4Z4 macrosatellite repeats on chromosome 4, and transcriptional de-repression of DUX4 in skeletal muscle. The common form of FSHD, FSHD1, is caused by a D4Z4 repeat array contraction. The less common form, FSHD2, is generally caused by heterozygous variants in SMCHD1.
Methods We employed whole exome sequencing combined with Sanger sequencing to screen uncharacterised FSHD2 patients for extra-exonic SMCHD1 mutations. We also used CRISPR-Cas9 genome editing to repair a pathogenic intronic SMCHD1 variant from patient myoblasts.
Results We identified intronic SMCHD1 variants in two FSHD families. In the first family, an intronic variant resulted in partial intron retention and inclusion of the distal 14 nucleotides of intron 13 into the transcript. In the second family, a deep intronic variant in intron 34 resulted in exonisation of 53 nucleotides of intron 34. In both families, the aberrant transcripts are predicted to be non-functional. Deleting the pseudo-exon by CRISPR-Cas9 mediated genome editing in primary and immortalised myoblasts from the index case of the second family restored wild-type SMCHD1 expression to a level that resulted in efficient suppression of DUX4.
Conclusions The estimated intronic mutation frequency of almost 2% in FSHD2, as exemplified by the two novel intronic SMCHD1 variants identified here, emphasises the importance of screening for intronic variants in SMCHD1. Furthermore, the efficient suppression of DUX4 after restoring SMCHD1 levels by genome editing of the mutant allele provides further guidance for therapeutic strategies.
- Muscular Dystrophy
- CRISPR-Associated Protein 9
- intronic variant
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RG and MLvdB contributed equally.
Correction notice This article has been corrected since it was published Online First. Figures 1, 3 and 4 have been replaced with better-quality versions. The content of the figures has not been changed.
Contributors RG, MLvdB, JB, RJLFL, RCH, MAFVG and SMvdM contributed to the concept and study design. RG, MLvdB, RJLFL, JB, PJvdV, IMW, NvdS, SJT, IM, RCH and MAFVG provided materials, collected the data and analysed or interpreted the data. JS, NG, SS and RT provided materials, recruited patients and performed the clinical evaluation of patients. RG, MLvdB and SMvdM wrote the manuscript. All authors revised and approved the final version. RG submitted the manuscript. RG and MLvdB contributed equally.
Funding This study was supported by grants from the US National Institutes of Health (NIH), National Institute of Neurological Disorders and Stroke (NINDS) P01NS069539, and National Institute of Arthritis and Musculoskeletal and Skin Diseases (NIAMS) R01AR045203 and R01AR066248, the Prinses Beatrix Spierfonds (W.OP14-01; W.OR11-18; W.OR14-04), the European Union Framework Programme 7 (agreement 2012-305121, NEUROMICS) and Spieren voor Spieren.
Competing interests NG is co-founder of NTrans Technologies, a company developing gene editing therapies to treat monogenetic disease. The authors are members of the European Reference Network for Rare Neuromuscular Diseases (ERN EURO-NMD).
Patient consent for publication Not required.
Provenance and peer review Not commissioned; externally peer reviewed.
Data availability statement All data relevant to the study are included in the article or uploaded as online supplementary information.