Cis D4Z4 repeat duplications associated with facioscapulohumeral muscular dystrophy type 2

Facioscapulohumeral muscular dystrophy, known in genetic forms FSHD1 and FSHD2, is associated with D4Z4 repeat array chromatin relaxation and somatic derepression of DUX4 located in D4Z4. A complete copy of DUX4 is present on 4qA chromosomes, but not on the D4Z4-like repeats of chromosomes 4qB or 10. Normally, the D4Z4 repeat varies between 8 and 100 units, while in FSHD1 it is only 1-10 units. In the rare genetic form FSHD2, a combination of a 4qA allele with a D4Z4 repeat size of 8-20 units and heterozygous pathogenic variants in the chromatin modifier SMCHD1 causes DUX4 derepression and disease. In this study, we identified 11/79 (14%) FSHD2 patients with unusually large 4qA alleles of 21-70 D4Z4 units. By a combination of Southern blotting and molecular combing, we show that 8/11 (73%) of these unusually large 4qA alleles represent duplication alleles in which the long D4Z4 repeat arrays are followed by a small FSHD-sized D4Z4 repeat array duplication. We also show that these duplication alleles are associated with DUX4 expression. This duplication allele frequency is significantly higher than in controls (2.9%), FSHD1 patients (1.4%) and in FSHD2 patients with typical 4qA alleles of 8-20 D4Z4 units (1.5%). Segregation analysis shows that, similar to typical 8-20 units FSHD2 alleles, duplication alleles only cause FSHD in combination with a pathogenic variant in SMCHD1. We conclude that cis duplications of D4Z4 repeats explain DUX4 expression and disease presentation in FSHD2 families with unusual long D4Z4 repeats on 4qA chromosomes.