Efficient silencing of the multicopy DUX4 gene by ABE-mediated start codon mutation in human embryos

Facioscapulohumeral muscular dystrophy (FSHD) is associated with aberrant epigenetic regulation of the chromosome 4q35 D4Z4 macrosatellite repeat. The resulting DNA hypomethylation and relaxation of epigenetic repression leads to increased expression of the deleterious DUX4-fl mRNA encoded within the distal D4Z4 repeat. With the typical late onset of muscle weakness, prevalence of asymptomatic individuals, and an autosomal dominant mode of inheritance, FSHD is often passed on from one generation to the next and affects multiple individuals within a family. Here we have characterized unique collections of 114 lymphoblastoid cell lines (LCLs) generated from 12 multigenerational FSHD families, including 56 LCLs from large, genetically homogeneous families in Utah. We found robust expression of DUX4-fl in most FSHD LCLs and a good correlation between DNA hypomethylation and repeat length. In addition, DUX4-fl levels can be manipulated using epigenetic drugs as in myocytes, suggesting that some epigenetic pathways regulating DUX4-fl in myocytes are maintained in LCLs. Overall, these FSHD LCLs provide an alternative cellular model in which to study many aspects of D4Z4, DUX4, and FSHD gene regulation in a background of low genetic variation. Significantly, these non-adherent immortal LCLs are amenable for high-throughput screening of potential therapeutics targeting DUX4-fl mRNA or protein expression.