PAX7 target gene repression is a superior FSHD biomarker than DUX4 target gene activation, associating with pathological severity and identifying FSHD at the single-cell level

Abstract

Facioscapulohumeral muscular dystrophy (FSHD) is a prevalent, incurable skeletal myopathy. The condition is linked to hypomethylation of the D4Z4 macrosatellite repeat at chromosome 4q35, leading to epigenetic derepression of the transcription factor DUX4; coupled with a permissive 4qA haplotype supplying a poly(A) signal. DUX4 may drive FSHD pathology via both induction of target genes and inhibition of the function of the myogenic master regulator PAX7. Biomarkers for FSHD have focused on DUX4 target gene expression. We have, however, reported that PAX7 target gene repression is a hallmark of FSHD skeletal muscle. Here we demonstrate that PAX7 target gene repression is an equivalent biomarker to DUX4 target gene expression when considering RNA-Sequencing data from magnetic resonance imaging-guided muscle biopsies. Moreover, PAX7 target gene repression correlates with active disease, independent to DUX4 target gene expression. PAX7 target genes are also repressed in RNA-Sequencing data from single cells, representing a significantly better biomarker of FSHD cells than DUX4 target gene expression. Importantly, PAX7 target gene repression is a significant biomarker in the majority of FSHD cells that are DUX4 target gene negative, and on which the DUX4 biomarker is indiscriminate. To facilitate the evaluation of validated biomarkers we provide a simple tool that outputs biomarker values from a normalized expression data matrix. In summary, PAX7 target gene repression in FSHD correlates with disease severity, independently of DUX4 target gene expression. At the single-cell level, PAX7 target gene repression can efficiently discriminate FSHD cells, even when no DUX4 target genes are detectable.

Lymphocytes contribute to DUX4 target genes in FSHD muscle biopsies

“…For DUX4, and each of the 3 DUX4 target gene expression signatures, we computed the mean expression of the genes in each of the LCL, myoblast and myotube samples, to generate a single sample score, as previously described 23,24 . Scores were then z -normalised within patient-matched control groups and their performances as biomarkers of FSHD status evaluated using ROC curves, separately for LCLs, myoblasts and myotubes (Fig.…”

“…PAX7 target gene repression and DUX4 target gene activation are however, independently associated with the degree of histological inflammation and active disease in a recent MRI guided FSHD muscle biopsy study 26 , implying that both mechanisms contribute to pathology 24 . Given that DUX4 is expressed at such low levels in patient muscle cells, the question remains as to which cells are expressing DUX4 and its target genes in these highly inflamed biopsies?…”

“…DUX4 shows homology with the homeodomains of the myogenic master regulator PAX7 and a competitive interaction has been proposed between the two proteins 19,25 , with PAX7 homeodomains able to substitute those of DUX4 without affecting certain DUX4 functions 25 . We demonstrated that a biomarker based on suppression of PAX7 target genes hallmarks FSHD muscle biopsies, as well as isolated myoblasts, significantly outperforming DUX4 target gene expression 23,24 .…”

“…However, DUX4 detection in FSHD patient derived myogenic cultures is notoriously difficult, with expression reported to be as low as 1/1000-1/5000 cells in myoblasts and 1/200 myonuclei in differentiated myotubes 20,22 . DUX4 target gene expression has been proposed as a biomarker of FSHD muscle biopsies 21 , but we have demonstrated via meta-analysis that its discriminatory power is generally underwhelming: appreciable levels of DUX4 target genes are detectable only in muscle biopsies which have been preselected for active disease/inflammation, via MRI metrics of T1 and STIR positivity 23,24 . Given this disappointing result, we investigated other biomarkers for FSHD muscle biopsies.…”

DNA aptamers against the DUX4 protein reveal novel therapeutic implications for FSHD

“…18 In FSHD cells PAX7 target genes are repressed. 79,80 Replacing the HDs of DUX4 with the corresponding domains of PAX7 lead to a fully toxic DUX4 mutant. 18 The binding motif of PAX7 is only 10 bases long and contains a major difference at position 9 in comparison to the analogous position in the DUX4 motif.…”