Expression microarray profiling of FSHD vs control muscle biopsy samples has been done, but with little consistency in the results.31,32,69–71
This is probably due partly to the use of very different types of microarrays, none of which were the recently improved, exon-based microarrays. Moreover, studies of muscle are complicated by various extents of contamination with non-muscle cells. In addition, myogenesis-specific changes in muscle tissue would be obscured by the very low percentage of satellite cells in muscle tissue. Recently, four multi-gene expression studies were reported for FSHD and control myoblasts and myotubes that used either exon-based microarrays53,72
or panels of genes for qRT-PCR.60,61
Although different experimental methods or types of samples were used, which probably contribute to considerable differences between the gene expression profiles, these studies present evidence for disease-linked dysregulation of many genes in FSHD myoblasts and myotubes.
First, we summarize results from our expression profiling of immunocytochemically characterized myoblast and myotube preparations53
using myoblast cultures that were ~70% confluent or that had been in differentiation medium for 4–6 days. Of the ~17 000 analyzed genes on the exon-based microarray, 295 and 797 were significantly dysregulated in FSHD vs control myoblasts and myotubes, respectively (fold change >2; adjusted P
<0.01). Many genes that displayed disease-related dysregulation (for example, genes for muscle structure, mitochondrial function and signal transduction) exhibited a dampening, but importantly, not the absence of normal myogenesis-specific expression changes. Some critical myogenesis-associated genes (for example, MYOD1
) displayed normal levels of expression in FSHD myoblasts and myotubes, consistent with the normal growth and differentiation of the FSHD myoblasts and normal appearance of FSHD myotubes. However, other regulatory genes (for example, MEF2A
and all four of the Argonaute genes) that could have widespread effects on gene expression were dysregulated in FSHD myogenic cells.53
That 60 genes showed about 4–16-fold downregulated RNA levels in FSHD vs control myotube preparations indicates a high percentage of the nuclei displaying FSHD-associated dysregulation of transcription.
Our findings suggest that FSHD-related changes in gene expression contribute to abnormalities in muscle function and structure in FSHD. Among the most overrepresented functional terms associated with upregulated genes in FSHD vs control myotubes were inflammation, fatty acid elongation in mitochondria and extracellular matrix.53
These could be relevant to the inflammation, fatty acid infiltration or fibrosis that has been observed in a varying percentages of FSHD muscle biopsies.18,73–76
For example, expression of the pro-inflammatory genes IL6
was upregulated in FSHD myotubes (fold change >2; adjusted P
<0.01). The fibrosis-associated CTGF, which was found to be upregulated at the RNA level in FSHD vs control muscle32
and at the RNA and protein level in muscle fibers from patients with other muscular dystrophies,77
was upregulated threefold in FSHD vs control myotubes.53
Secondly, in an expression profiling study similar to ours, Cheli et al.72
concluded that there was specific dysregulation in myoblasts and myotubes from FSHD patients vs. controls. However, their data are unconvincing because of the lack of assessment of the quality of their myoblast and myotube preparations by immunocytochemistry. Indeed, paradoxically, they reported no muscle-related terms among 177 functional terms associated with genes differentially expressed in control myoblast vs control myotube preparations. In contrast, as expected, in our microarray study all six of the top functional terms for genes displaying differential expression in control myoblasts vs control myotubes had the term ‘muscle’ in them.53
In addition, Cheli et al.
reported <4% overlap between several hundred genes with dysregulation in FSHD vs control cells at the myoblast stage and those dysregulated at the myotube stage, unlike in our study in which there was 48% overlap between myoblasts and myotubes for the genes with FSHD dysregulation (fold-change >2; P
Thirdly, Homma et al.60
analyzed by qRT-PCR the relative expression of 64 test genes and 11 control genes in well-characterized FSHD and control myoblasts at five time points before or after induction of differentiation to myotubes.60
Their subjects were cohorts of affected and unaffected family members, with myoblasts generated from the deltoid and bicep biopsies of each subject. A hierarchical clustering analysis demonstrated that there were strong cohort-related groupings among the expression profiles and that most of the correlations in expression profiles between samples from closely related patients were stronger than the correlations between pairs of FSHD samples or pairs of control samples. The authors then analyzed the average differences in expression between the FSHD and control samples at each time point using standard t
-tests and concluded that there were ‘no consistent, overall differences in mRNA expression patterns or levels’ between FSHD and control myoblasts. However, this analysis was statistically flawed in two critical respects: (1) the measurements at each time point were treated as independent rather than as repeated measures from each subject and (2) there was no adjustment for the variation associated with the sample cohorts. To remedy these deficiencies and thereby greatly increase the power to detect statistically significant FSHD-related differences, we fit mixed-effects models to predict the PCR-derived Ct value as a function of time and/or sample type for their data from the last three differentiation time points (2, 4 and 7 days in differentiation medium). We used nested random intercepts to account for varying baseline levels among and within each cohort, and modeled the effect of FSHD status as an additive term and, where statistically significant, as an adjustment to the time-related slope coefficient. By this re-analysis, we identified 13 genes with significant FSHD-related dysregulation of expression from their data (CXCL11
, upregulated; ACTN3
, downregulated). The SLC25A4
downregulation is contrary to previous findings,24,30,78
and the specificity of FRG2B
probes remains to be demonstrated for this gene, which has very similar sequences throughout the genome. In our expression profiling, six of the 64 genes common to the study of Homma et al.60
displayed significant FSHD-related downregulation of at least twofold (DES
). Three of these were also significantly downregulated in our re-evaluation of the data of Homma et al.
). Of the 64 genes, 50, including FSHD candidate gene PITX1
displayed no significant differential expression in either study. The P
-value for the relationship between the direction of differential expression between the two studies was 0.09 (Fisher’s exact test), providing some evidence of an association, although not at a statistically significant level.
Given the lack of normalization of the qRT-PCR data from test genes to standard genes by Homma et al.
and their unconventional use of a pre-amplification for 14 cycles before the real-time PCR, the finding of significant downregulation for three genes in both of these studies is noteworthy. Moreover, all three of these genes are strongly upregulated in control myotubes vs 19 different non-muscle cell type,53
and were seen as upregulated in myoblasts vs non-myogenic cells in RNA-seq (http://genome.ucsc.edu/ENCODE/
, Tom Gingeras, Cold Spring Harbor). This is consistent with our finding that one of the most prominent classes of genes to be dysregulated in FSHD myotubes was genes normally upregulated during myogenesis. In summary, the conclusion of Homma et al.
that FSHD and control myogenic precursors have indistinguishable patterns of gene expression is not supported by our re-analysis of their data.
In the last of these recent expression studies, Geng et al.61
transduced human myoblast cultures with DUX4-fl or control constructs and profiled differential gene expression with an expression microarray 24 h after transduction. The short time of incubation was probably intended to minimize the contribution of toxic effects of induced DUX4-fl
expression to myoblasts, although it is likely that such effects still altered expression of many genes. Among the more than 1000 genes that were significantly dysregulated (fold change >2; false discovery rate <0.01) by the DUX4-fl construct was a small group of genes strongly upregulated due to transduction with DUX4-fl and expressed specifically in germ cells or during early development. By qRT-PCR, Geng et al.
showed that six of these genes (ZSCAN4
) were expressed at moderate levels in normal testis and confluent FSHD myoblasts. The steady–state levels of their RNAs were usually much lower in FSHD skeletal muscle samples, and they displayed little or no expression in control muscle or confluent control myoblasts. These results are consistent with roles for these genes in gametogenesis and abnormal regenerative myogenesis in FSHD.
The only one of these six genes with a known function is ZSCAN4/Zscan4
, which is expressed specifically at the late two-cell stage and promotes the normal progression of mouse embryos to the four-cell stage.67
The transient expression of this gene regulates pluripotency, genome stability, and telomere stability and can upregulate expression of several hundred genes during the late stages of induced pluripotent stem cell formation with major changes in phenotypic outcome.79
We noticed that among the genes regulated by Zscan4
are the murine homologs of KHDC1
all of which were found to be upregulated by DUX4-fl transduction.61
The first two of these genes were also analyzed in human samples and shown to be testis- and FSHD-associated.61
The 1.9-kb enhancer and promoter region of ZSCAN4
contains four binding sites for DUX4-fl protein and was responsive to strong upregulation by transduced DUX-fl in a reporter gene assay using a human rhabdosarcoma cell line.61
Given its ability to upregulate many genes during early differentiation, this gene might be one of the earliest to be dysregulated by inappropriate expression of DUX4-fl in the FSHD muscle lineage and could have a major role in establishing the FSHD transcription phenotype.
Four of the above six testis/FSHD muscle-lineage genes, ZSCAN4
, were included in our expression array study.53
We found that ZSCAN4
were upregulated ~4-, 5- and 2-fold (adjusted P
), respectively, in FSHD vs control myotubes with only about 1.5-fold upregulation in FSHD cells at the myoblast stage. Some of the hundreds of changes in gene expression from myoblasts to myotubes53
may be responsible for our observing a stronger FSHD-associated upregulation of levels of these transcripts at the myotube stage. We used myoblasts from 70% confluent cultures, which are not committed to myotube formation, unlike Geng et al.61
who used confluent myoblast cultures, and this may account for their higher FSHD-specific upregulation of these genes at the myoblast stage. The testis association of these genes probably reflects the finding that the only normal postnatal tissue shown to express DUX4-fl RNA and protein is testis.6
Another testis-associated gene, CCNA1
, which encodes a meiosis-associated cyclin, was strongly upregulated in control myoblasts transduced with a DUX4-fl expression construct in the study of Geng et al.61
Analogously, we found strong upregulation of this gene, 3.6- and 24-fold, in FSHD vs control myoblasts and myotubes, respectively (adjusted P
<0.01 and 10−6
In summary, several studies indicate that multiple genes are dysregulated in normal-appearing FSHD myoblasts and myotubes.