Facioscapulohumeral Dystrophy: Incomplete Suppression of a Retrotransposed Gene

Abstract

Each unit of the D4Z4 macrosatellite repeat contains a retrotransposed gene encoding the DUX4 double-homeobox transcription factor. Facioscapulohumeral dystrophy (FSHD) is caused by deletion of a subset of the D4Z4 units in the subtelomeric region of chromosome 4. Although it has been reported that the deletion of D4Z4 units induces the pathological expression of DUX4 mRNA, the association of DUX4 mRNA expression with FSHD has not been rigorously investigated, nor has any human tissue been identified that normally expresses DUX4 mRNA or protein. We show that FSHD muscle expresses a different splice form of DUX4 mRNA compared to control muscle. Control muscle produces low amounts of a splice form of DUX4 encoding only the amino-terminal portion of DUX4. FSHD muscle produces low amounts of a DUX4 mRNA that encodes the full-length DUX4 protein. The low abundance of full-length DUX4 mRNA in FSHD muscle cells represents a small subset of nuclei producing a relatively high abundance of DUX4 mRNA and protein. In contrast to control skeletal muscle and most other somatic tissues, full-length DUX4 transcript and protein is expressed at relatively abundant levels in human testis, most likely in the germ-line cells. Induced pluripotent (iPS) cells also express full-length DUX4 and differentiation of control iPS cells to embryoid bodies suppresses expression of full-length DUX4, whereas expression of full-length DUX4 persists in differentiated FSHD iPS cells. Together, these findings indicate that full-length DUX4 is normally expressed at specific developmental stages and is suppressed in most somatic tissues. The contraction of the D4Z4 repeat in FSHD results in a less efficient suppression of the full-length DUX4 mRNA in skeletal muscle cells. Therefore, FSHD represents the first human disease to be associated with the incomplete developmental silencing of a retrogene array normally expressed early in development.

A unique library of myogenic cells from facioscapulohumeral muscular dystrophy subjects and unaffected relatives: family, disease and cell function

“…3,5,6 Furthermore, expression of this long form of DUX4 is detected in only a small percentage (B0.1%) of nuclei in cultures of FSHD subject-derived myogenic cells. 6 Data acquisition for our study was completed before publication of this disease model, so we did not include analyses of the long form of DUX4 here. In our study, we found that expression of a panel of genes, myotube formation, and stressor responses were, on average, indistinguishable between FSHD and unaffected cell cultures.…”

“…6,[8][9][10][11][12][13][14][15] Some of these studies have suggested possible FSHD-specific phenotypes, including increased sensitivity to oxidative stress, 14,16 altered myotube morphology, 14 and altered expression of FRG1 18 or FRG2. 12 We found that proliferating and differentiating cultures of both FSHD and unaffected cells expressed similar patterns of FRG2 mRNA, and a recent study also found FRG2 mRNA in unaffected myoblasts and myotubes.…”

“…[8][9][10][11][12][13][14][15] Such studies have suggested several possible FSHD-specific phenotypes, including increased sensitivity to oxidative stress 14,16 and altered gene expression, [17][18][19][20] as well as expression of the long form of DUX4. 5,6 These findings have, however, sometimes not been consistent among laboratories. Potential confounding variables include (i) muscles used for biopsy, (ii) genetic backgrounds of unaffected and diseased donors, (iii) extent of disease progression in the donor muscles, (iv) methods used to derive and purify myogenic cells, (v) culture conditions, (vi) number of different individual donors available, or (vii) replicative age of the cells.…”

“…4 Recent work has suggested that both types of FSHD are characterized by expression and stabilization of mRNA produced from an open reading frame in the most telomeric D4Z4 repeat that encodes a potentially toxic protein: a long isoform of DUX4. 3,5,6 Weakness in FSHD appears to be of myogenic, rather than neurogenic, origin. 7 Many studies have, therefore, used primary myogenic cell cultures derived from FSHD subject biopsies to examine potential pathogenic mechanisms and therapeutic strategies.…”

Double SMCHD1 variants in FSHD2: the synergistic effect of two SMCHD1 variants on D4Z4 hypomethylation and disease penetrance in FSHD2

“…4 Both forms are associated with partial chromatin relaxation of the D4Z4 macrosatellite repeat array on the subtelomere of the long arm of chromosome 4 and transcriptional derepression of the D4Z4 unit-encoded DUX4 retrogene in skeletal muscle. [5][6][7][8][9] DUX4 is a germ line transcription factor that is normally repressed in somatic cells. 7 Its expression in skeletal muscle activates genes involved in germ line and early stem cell development, as well as specific classes of repeat elements, and overexpression of DUX4 in somatic cells causes cell death.…”