Cis D4Z4 repeat duplications associated with facioscapulohumeral muscular dystrophy type 2

Abstract

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.

FSHD1 and FSHD2 form a disease continuum

“…This is consistent with the recent observation that affected SMCHD1 mutation carriers with unusually long D4Z4 repeats carry small D4Z4 duplications. 40,41 It is also consistent with observations that DUX4 derepression in FSHD2 depends on the functional consequence of the SMCHD1 mutation and the size of the Figure 2 Clinical assessments of the facioscapulohumeral dystrophy (FSHD) subpopulations (A) Significant differences in the age-corrected clinical severity score (CSS) between FSHD type 1 (FSHD1) (8-10) and FSHD1 (4-7) (p = 0.0009***), FSHD1+ FSHD type 2 (FSHD2) (p = 0.0002***), or FSHD2 (p < 0.0001****) subgroups are observed. A significant difference between FSHD1 (4-7) and FSHD2 is also present (p = 0.048*).…”

Deciphering the complexity of the 4q and 10q subtelomeres by molecular combing in healthy individuals and patients with facioscapulohumeral dystrophy

“…The duplication segregates with a short D4Z4 array on the second 4q allele in 3/11 cases or mutation in SMCHD1 in 5/10 families suggesting that it might be associated with FSHD1 or FSHD2, respectively. Yet, the cis-duplication is the only genetic defect that segregates with clinical signs of the disease in 2/10 of our cases and in a proportion of cases later reported by Lemmers et al 37 suggesting that it might also be causal in a small number of patients. 25 Since our initial publication describing this recurrent 4q35 rearrangement, we have identified nine additional patients displaying the same…”

“…Strikingly, the vast majority of them (7 out of 11) carries a very short (1-3 units) 10q allele and a 4q chromosome with more than 20 units escaping the recent definition of FSHD2 proposed as a subclass of patients carrying between 8-20 repeated units, with cis-duplication carriers included in this subgroup based on the size of the most proximal D4Z4 array and regardless of the size of the proximal one. 37 Thus, if the definition of FSHD2 as individuals carrying 8-20 repeats can be applied to many patients, recent examples including those previously described 25 or those reported here (figure 1) indicate that a significant number of patients clinically diagnosed with FSHD cannot be classified in this way or might be discarded if this restrictive classification is applied. Interestingly, in our cohort of patients comprising 426 cases clinically diagnosed with FSHD, the percentage of patients carrying a mutation in SMCHD1 is close to the percentage of patients in whom we found an additional 10q allele and lower than the total percentage of patients with atypical genomic features (table 1).…”

Consequences of epigenetic derepression in facioscapulohumeral muscular dystrophy

“…Although FSHD2 is often referred to as the contraction‐independent form of FSHD, analysis of a large number of unrelated controls and FSHD2 patients reveals a repeat size dependency in these patients as well. While the median number of D4Z4 units in controls is 33.7 units, in FSHD2 this is significantly lower with a median of 16.8 units . Sacconi et al provided further evidence for the hypothesis that FSHD1 and FSHD2 form a disease continuum .…”

“…Additionally, certain unique cases of FSHD2 which were originally thought to have unusually long 4qA alleles (>20 units) can be explained by the presence of D4Z4 duplication events. These cases present as FSHD2 in which a long D4Z4 repeat on a 4qA allele is followed by, or preceded by, a duplication of the D4Z4 repeat, which is of an FSHD2‐compatible size (ie, <20 units) . Therefore, it is tempting to speculate that there is a repeat size threshold for any type of FSHD.…”