Facioscapulohumeral muscular dystrophy: genetics, gene activation and downstream signalling with regard to recent therapeutic approaches: an update

Figures

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  Fig. 1. Overview of affected muscles in FSHD. The symptoms tend to start at the upper half of the body and then spread to lower body parts involving the pelvic girdle and the leg muscles. There is a high degree of clinical variability in disease severity and affected muscles are generally involved asymmetrically regarding the left–right body axis. In some cases, patients with foot drop can be supported by ankle–foot orthotics (AFOs) and knee-ankle–foot orthotics (KAFOs) [236]. Surgery to attach the scapula to the ribcage can enhance arm motion or alleviate pain [237–239]

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  Fig. 2. Chromosome 4q35.2 in healthy- and in FSHD1-individuals. 11–150 D4Z4 repeats (dark blue) leading to a high methylation level within the D4Z4 macrosatellite (light blue), which represses the transcription of DUX4. There are two possible haplotypes A and B, which are equally common and a Simple Sequence Length Polymorphism (SSLP) proximal to the D4Z4 repeat further classifying haplotype A or B [36]. FSHD only occurs in individuals, which carry the 4qA allele. People with contraction and thus minor methylation at chromosome 4q35 are either also carrying 4qA and have FSHD or 4qB and are healthy. The 4qA haplotype is further classified based on SSLPs proximal to the D4Z4 repeat. Only the common SSLP variant 4A161 and the rare variants 4A159 and 4A168 are reported to correlate with D4Z4 reduced alleles in FSHD patients. This example: 1–10 repeats/ FSHD1; the same conditions of a permissive Haplotype A apply for FSHD2 [36]. Haplotype A further carries the pLAM1 sequence (light green) and a beta-satellite repeat region immediately distal to the D4Z4 repeat (yellow). Detection is depicted via Southern Blot probe p13E-11 (dark green). Chromosome 10q26 is also illustrated as it shows nearly…

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  Fig. 3. DUX4-FL protein. This protein contains 424 amino acids and is expected to have well-defined tertiary structures in each of the two DNA-binding homeodomains (amino acids 19–79 and 94–154) and in the most C-terminal region (amino acids ∼ 365–424). The C-terminal region includes the transcription-activating domain (TAD) and a p300-binding domain. The region between the second homeodomain and the C-terminal domain (amino acids ∼ 155–364) is predicted to be disordered. The protein contains a potential nine amino acid TAD (9aaTAD) at amino acids 371–379 (classified as a 92% match) [74]

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  Fig. 4. Requirements for DUX4 gene expression. DUX4 expression is only possible at the most distal D4Z4 repeat of the 4qA alleles. Exon 3, which is located outside the D4Z4 repeats in the pLAM1 region is carrying the variant ATTAAA that provides a PAS enabling the expression of the most distal copy of the DUX4 gene into DUX4 mRNA and then DUX4 protein [39]. Whilst healthy individuals especially generate the non-toxic splice form of DUX4-S (encodes the amino-terminal part and both homeodomains; does not contain the C-terminal domain) [73], FSHD muscle produces toxic DUX4-FL mRNA [32]

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  Fig. 5. Schematic representation of the methods used for FSHD1 diagnosis. The D4Z4 repeat array is indicated with triangles (in dark blue). D4Z4 repeat units on chromosomes 4 and 10 can be separated because all repeats on 10q contain BlnI restriction sites (whereas all D4Z4 repeats on 4q contain XapI restriction sites) [238]

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  Fig. 6. Overview of DUX4 signalling. Antagonists of DUX4 are depicted on the left; Signalling pathways for DUX4 gene activation have been associated with MAPK, especially p38α/β, bottom); DUX4 is located within the D4Z4 macrosatellite, which interacts with subtelomeric regions and the nuclear lamina via long distance-interactions, influencing (1) methylation levels and (2) gene expression. Interactions found in control cells and in FSHD are depicted at the top of the graphic. (For reasons of clarity, interactions of FSHD2 cells are not illustrated here, but are mentioned in the text.) FAT1 gene is shown to aggravate FSHD, when expressed at low levels. Muscles are more sensitive to gene deregulations and become affected earlier [83]; DUX4 generally requires help of p300/CBP and H3.X/H3.Y for target gene expression; Depicted on the right: DUX4 expression affects multiple genes, which are either upregulated or downregulated by DUX4 protein. ROS seem to have a prominent role in disease mechanism and are probably activated directly by DUX4 itself and indirectly through further targets of DUX4

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  Fig. 7. Model of DUX4 regulation by β-2 adrenergic signalling. β-2AR binding of agonists induces G protein-mediated activation of adenylyl cyclase, which subsequently catalyzes the formation of cAMP. Downstream effectors of cAMP are PKA-dependent and PKA-independent pathways. β-2 agonist-mediated inhibition effects on DUX4 expression are likely mediated through PKA-independent pathways acting through signalling molecules such as phosphatases (PPtases) and MAPKs to effect chromatin modifiers, e.g. lysine methyltransferases (KMTases), to influence transcription of the DUX4 gene [140]

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  Fig. 8. Current approaches for targeted treatment: modulating DUX4 repressive pathways, targeting DUX4 mRNA, DUX4 protein, or cellular downstream effects of DUX4 expression. Regarding DUX4 mRNA LNA gapmer AOs are further illustrated on the right; These are single-stranded, short oligonucleotides containing a DNA portion flanked by LNA. The LNA parts increase the affinity for the target and provide nuclease resistance, DNA parts activate RNase H [207, 240, 241]; The extra methylene group of LNAs is attached between the 2′-O-and the 4′-positions “locks” the ribofuranosyl-ring in its 3′-endo conformation (shown under the magnifying glass) [240]