**5. Direct role of D4Z4: The double homeobox gene 4 (DUX4)**

The D4Z4 unit has been completely sequenced (Hewitt et al., 1994; Winokur et al., 1994 Lee et al., 1995). Each D4Z4 repeat unit contains an ORF with a double homeobox putatively encoding the DUX4 protein. *DUX4* belongs to a family of highly homologous genes scattered throughout the genome. One almost identical copy, named *DUX4c*, is located 42 kb proximal to the repeat array. Based on the presence of the molecular signature 4A-PAS, which should allow the stabilization of the mRNA from the distal copy of the *DUX4* gene; it has been proposed that FSHD arises through a toxic gain-of-function mechanism attributable to the pathological expression of *DUX4* mRNA (Lemmers et al., 2010a). More detailed analysis of *DUX4* expression shows that the *DUX4* pre-mRNA can be alternatively spliced and apparently, the FSHD muscle expresses a different splice form of *DUX4* mRNA compared to control muscle (Snider et al., 2010). It is important to note that *DUX4* is a rare transcript and the amount of *DUX4* has been estimated in one copy per cell. To explain how such low expression level of *DUX4* can cause FSHD, it has been proposed that in FSHD muscle the DUX4 protein may exert its toxic effect in a small subset of nuclei, which express a relatively abundant amount of *DUX4* transcript. The possible toxic effect of DUX4 has been inferred on the basis of in vitro and in vivo studies (Kowaljow et al., 2007; Bosnakovski et al., 2008; Wallace et al., 2011) in which *DUX4* was expressed at very high levels. Thus in order to explain FSHD pathogenesis, it is difficult to reconcile those experimental observations with the very limited amount of *DUX4* detected in human muscle cells.
