A unifying genetic model for facioscapulohumeral muscular dystrophy

Abstract

Facioscapulohumeral muscular dystrophy (FSHD) is a common form of muscular dystrophy in adults that is foremost characterized by progressive wasting of muscles in the upper body. FSHD is associated with contraction of D4Z4 macrosatellite repeats on chromosome 4q35, but this contraction is pathogenic only in certain "permissive" chromosomal backgrounds. Here, we show that FSHD patients carry specific single-nucleotide polymorphisms in the chromosomal region distal to the last D4Z4 repeat. This FSHD-predisposing configuration creates a canonical polyadenylation signal for transcripts derived from DUX4, a double homeobox gene of unknown function that straddles the last repeat unit and the adjacent sequence. Transfection studies revealed that DUX4 transcripts are efficiently polyadenylated and are more stable when expressed from permissive chromosomes. These findings suggest that FSHD arises through a toxic gain of function attributable to the stabilized distal DUX4 transcript.

Fig. 1

Schematic overview showing the minimal genetic requirement for FSHD1. (A) The D4Z4 repeat array on chromosome 4q35 (open triangles) and its homologue on chromosome 10q26 (closed triangles), indicating the location of the simple sequence length polymorphism (SSLP) and 4qA/4qB polymorphisms that define the genetic background of the repeat. Patients with FSHD1 have a D4Z4 repeat array size of 1-10 units on 4qA but not on 4qB or 10q chromosomes. (B) Schematic of the D4Z4 repeat and flanking sequences on A, A-L (with an extended distal D4Z4 repeat unit) and B chromosomes. Each D4Z4 unit is defined by the KpnI restriction site (K). The proximal and distal regions that were sequenced are indicated. The exons of DUX4 are indicated as grey boxes numbered 1-3.

Fig. 2

DUX4 expression analysis after transfection of the distal D4Z4 unit and flanking pLAM sequence into C2C12 mouse myoblast cells. (A) Northern blot analysis of C2C12 mouse myoblast cells transfected with genomic constructs (see fig 2B) derived from permissive (lanes 1-4) or non-permissive chromosomes (lanes 6, 7 and 9) or constructs in which the poly (A) signals from non-permissive chromosomes are replaced by those from permissive chromosomes (4PAS; lanes 8 and 10) and vice versa (10mPAS; lane 5). Only constructs with canonical poly (A) signals show a DUX4 transcript. Co-transfected EGFP gene was used as a control. (B) Schematic of the transfected sequence including the distal D4Z4 unit and flanking pLAM sequence, The DUX4 gene and the poly(A) signal are part of the transfected sequence. The main spliced DUX4 transcript and its open reading frame (ORF) are indicated. The primers used to quantify polyadenylation efficiency are indicated. The boxed area shows the design of the assay to infer the polyadenylation efficiency of the DUX4 poly(A) signal. The forward primer straddles exons 2 and 3 and is therefore specific for the spliced DUX4 transcript. The primer set Proximal to the poly(A) Signal (PAS) and a primer set using the same forward primer but a reverse primer Distal to the poly(A) Signal (DAS) are indicated as well as the expected PAS/DAS ratios. C) Bar diagram with the observed PAS/DAS ratios of permissive chromosomes (lanes 3 and 4), non-permissive chromosomes (lanes 6, 7 and 9), and permissive chromosomes in which the poly(A) signal is replaced for a sequence derived from a non-permissive chromosome (10mPAS; lane 5) or vice versa (4PAS; lanes 8 and 10), or of pathogenic chromosomes derived from families F1 and F3 (lanes 1 and 2). Lane 11 is negative control. ND = not detected Bars represent values of quadruple experiments with standard errors of the mean.

Fig. 3

Pedigrees of FSHD1 families with complex pathogenic chromosomes. Families F1-F4 all carry a hybrid D4Z4 repeat that commences with chromosome 10-type repeat units (closed triangles), but end with 4-type repeat units (open triangles). In family F3 a meiotic rearrangement between chromosomes 4 and 10 generated a short hybrid repeat structure on 4A161. In family F4, this pathogenic repeat is located on chromosome 10 and originates from a mitotic D4Z4 contraction in the mildly affected father which is transmitted to his affected son. Family F5 represents a disease chromosome in which in addition to partial deletion of the D4Z4 repeat, the region proximal to the D4Z4 repeat is also deleted.