Intronic SMCHD1 variants in FSHD: testing the potential for CRISPR-Cas9 genome editing.

Autor:	Goossens R; Human Genetics, Leiden University Medical Center, Leiden, The Netherlands., van den Boogaard ML; Human Genetics, Leiden University Medical Center, Leiden, The Netherlands., Lemmers RJLF; Human Genetics, Leiden University Medical Center, Leiden, The Netherlands., Balog J; Human Genetics, Leiden University Medical Center, Leiden, The Netherlands., van der Vliet PJ; Human Genetics, Leiden University Medical Center, Leiden, The Netherlands., Willemsen IM; Human Genetics, Leiden University Medical Center, Leiden, The Netherlands., Schouten J; Hubrecht Institute-KNAW and University Medical Center, Utrecht, The Netherlands.; Clinical Sciences of Companion Animals, Faculty of Veterinary Medicine, Utrecht, The Netherlands., Maggio I; Department of Cell and Chemical Biology, Leiden University Medical Center, Leiden, The Netherlands.; Department of Pediatrics, Leiden University Medical Center, Leiden, The Netherlands., van der Stoep N; Center for Human and Clinical Genetics, Leiden University Medical Center, Leiden, The Netherlands., Hoeben RC; Department of Cell and Chemical Biology, Leiden University Medical Center, Leiden, The Netherlands., Tapscott SJ; Division of Biology, Fred Hutchinson Cancer Research Center, Seattle, Washington, USA., Geijsen N; Hubrecht Institute-KNAW and University Medical Center, Utrecht, The Netherlands.; Clinical Sciences of Companion Animals, Faculty of Veterinary Medicine, Utrecht, The Netherlands., Gonçalves MAFV; Department of Cell and Chemical Biology, Leiden University Medical Center, Leiden, The Netherlands., Sacconi S; Peripheral Nervous System, Muscle and ALS Department, Université Côte d'Azur, Nice, France.; Institute for Research on Cancer and Aging of Nice, Faculty of Medicine, Université Côte d'Azur, Nice, France., Tawil R; Department of Neurology, University of Rochester Medical Center, Rochester, New York, USA., van der Maarel SM; Human Genetics, Leiden University Medical Center, Leiden, The Netherlands S.M.van_der_Maarel@lumc.nl.
Jazyk:	angličtina
Zdroj:	Journal of medical genetics [J Med Genet] 2019 Dec; Vol. 56 (12), pp. 828-837. Date of Electronic Publication: 2019 Nov 01.
DOI:	10.1136/jmedgenet-2019-106402
Abstrakt:	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. Competing Interests: 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). (© Author(s) (or their employer(s)) 2019. No commercial re-use. See rights and permissions. Published by BMJ.)
Databáze:	MEDLINE
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