A number of candidate genes have been proposed in FSHD based on selective upregulation in FSHD-derived tissue or cell lines.22, 27, 28, 29, 30
Yet many of these findings have been difficult to confirm resulting in divergent hypotheses regarding the underlying pathophysiology of FSHD. In this study, utilizing a uniform and systematic approach, the expression levels of the FSHD candidate genes FRG1
were determined in primary myoblasts and myotubes as well as in biopsy material of affected quadriceps muscle. We did not observe consistent and significant deregulation of the FSHD candidate genes we examined in all samples tested, either at the RNA, or at the protein level. Only FRG2
showed increased expression levels in FSHD samples and then only in myotubes at the RNA level. Although the FRG2
expression level was very low in both myoblasts and muscle biopsy samples, expression was consistently observed in myotubes from FSHD patients and not in controls. This observation corroborates an earlier report showing the transcriptional upregulation of chromosome 4 and 10 copies of FRG2
in FSHD myotubes only.28
is involved in FSHD pathogenesis remains doubtful, as muscle-specific overexpression of FRG2
in mice does not result in a muscular dystrophy phenotype.27
Moreover, individuals with proximal deletion encompassing FRG2
have phenotypically typical FSHD.17
For all other genes tested, we did not observe expression level differences, which is in line with23, 25, 26
but also in contrast to22, 27, 29, 30
data described before. Most of these studies were performed with limited sample size and sometimes included samples isolated from different muscle groups. Perhaps the gene of greatest relevance given the conflicting data is FRG1
as it is still considered one of the leading candidate genes in FSHD as muscle-specific overexpression, at high levels, in mice causes a muscular dystrophy phenotype.27
In our studies, no difference in FRG1
expression was observed between FSHD and controls either at the RNA or protein level. Moreover, for RNA expression studies in muscle we confirmed our results by testing expression using two separate experiments with different reference methods, utilizing the same samples. In addition, there was no relationship between expression levels and deletion size or between expression levels and the pathological grading of the muscle in FSHD-derived samples as for all other genes tested. As FRG1 is a nuclear protein, it is important to emphasize that for the protein studies the FRG1 expression levels were compared with both total protein levels using tubulin or ponceau staining as well as nuclear protein levels using histone 2B. In addition, we found no clear upregulation of PITX1
, a gene that has recently been reported to undergo DUX4
Only in the myotubes a trend toward upregulation was observed. It should be noted, however, that the reported deregulation of PITX1
was found in muscle samples of the biceps and deltoid muscle, whereas quadriceps muscle was used in our study.
The quadriceps muscle was chosen in this study because it is easily accessible for needle biopsy and because it is relatively spared early in FSHD. Nevertheless, quantitative muscle strength testing35
shows more involvement of this muscle group than is suspected by bedside examination. Indeed, all the FSHD biopsy samples in our study showed mild-to-moderate pathological changes (). Therefore, possible observed changes in this muscle are more likely to be early changes in FSHD pathophysiology and not secondary changes caused by muscle wasting or damage, which could explain some of the conflicting data regarding myoblast phenotypes from affected and unaffected muscle.25, 36, 37
The importance of sample size in verifying selective deregulation of FSHD candidate genes is illustrated by the high inter sample variability in RNA and protein expression for several target genes. Expression level differences determined with a limited number of samples can result in unreliable results. In biopsy material this can be caused by variability in the presence of non-muscle cells (eg, blood cells, endothelial cells and so on). Of note is that CRYM
showed high RNA and protein expression level differences in the muscle biopsy samples, which did not seem to be disease related. Recently, Reed et al30
reported that CRYM
protein levels are upregulated in FSHD deltoid muscle. In this study, three FSHD samples were compared with two controls. In our study, we cannot confirm a FSHD-specific upregulation of CRYM
in FSHD quadriceps muscle biopsies (n
=10). In contrast, some control samples showed high CRYM
RNA and protein expression levels, while most had barely detectable expression levels. These results clearly show the heterogeneity of biological samples in studying RNA and protein expression levels and emphasize the need to examine large sample sizes and especially the need for adequate normal control samples to understand the intrinsic variability in expression of a particular gene in normal tissue.
This study clearly shows that none of the FSHD candidate genes, except for FRG2
, are upregulated at the levels described earlier in some studies.22, 27, 28, 29, 30
We cannot exclude low levels of deregulation that is beyond the sensitivity of our methods or exclude the possibility that one or more of the candidate genes show a transient spatiotemporal deregulation under conditions we have not tested. Our study emphasizes the need for large collections of identically processed samples to investigate FSHD-specific transcriptional changes to overcome sample heterogeneity. As humans are genetically and phenotypically heterogeneous in nature, a more promising approach to investigate the role of the established candidate genes for FSHD might be to elucidate their cellular function and relate that function to FSHD pathology.
A paper by Masny and et al
utilizing a different approach to study a potential cis
effect in FSHD reached a similar conclusion. The authors studied native transcripts emanating from chromosome 4 by RNA–DNA FISH to quantitatively establish the expression level of the nascent transcript from each allele. Masny and co-workers found no difference in expression level between the normal and contracted allele, providing further evidence against a cis
effect of transcriptional deregulation of 4qter genes in FSHD.
Recent evidence offers an alternative hypothesis to pathophysiology of FSHD. As all patients with FSHD share a change in the chromatin structure of D4Z4 on chromosome 4qA161,16, 19, 20
it might well be that a shift in balance of the complex transcriptional activity recently discovered to emanate from the repeat29, 31
is causally related to the pathology.