Intronic variants in FSHD: testing the potential for CRISPR-Cas9 genome editing.

BACKGROUND

Facioscapulohumeral dystrophy (FSHD) is associated with partial chromatin relaxation of the retrogene containing D4Z4 macrosatellite repeats on chromosome 4, and transcriptional de-repression of 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 .

METHODS

We employed whole exome sequencing combined with Sanger sequencing to screen uncharacterised FSHD2 patients for extra-exonic mutations. We also used CRISPR-Cas9 genome editing to repair a pathogenic intronic variant from patient myoblasts.

RESULTS

We identified intronic 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 .

CONCLUSIONS

The estimated intronic mutation frequency of almost 2% in FSHD2, as exemplified by the two novel intronic variants identified here, emphasises the importance of screening for intronic variants in . Furthermore, the efficient suppression of after restoring SMCHD1 levels by genome editing of the mutant allele provides further guidance for therapeutic strategies.