FSHD presents with high and unpredictable inter- and intrafamilial clinical variability in disease onset and progression.1
To provide patients with a better prognosis it would be beneficial to identify genetic or epigenetic markers that correlate with disease severity. Earlier studies failed to identify correlations between genetic markers (D4Z4 repeat size) or epigenetic markers (D4Z4 methylation) that can be used in the clinic.8,10
The focus of the present study was to investigate possible relationships between the epigenetic changes occurring in primary fibroblasts and myoblasts of FSHD1 and FSHD2 patients at the promoter region of DUX4 and the clinical severity defined by the CSS or MPS. As a measure of chromatin changes first we introduced the chromatin compaction score, ChCS, in which the relative enrichment of H3K9me3 at D4Z4 is divided by the relative enrichment of H3K4me2. The advantage of such a score is that it circumvents the correlation calculation problem of ChIP experiments studying repetitive sequences. Different samples contain different numbers of D4Z4 units and our experimental procedure detects all repeat units on chromosomes 4 and 10. A comparison between different samples is only reliable if the measured signal can be correlated with D4Z4 unit numbers. Since both H3K9me3 and H3K4me2 are present in every unit, their ratio, as calculated in the ChCS, gives a value per unit.
Statistical analysis revealed a significant difference between the ChCS of control and FSHD1 and between control and FSHD2 in fibroblast and myoblast cultures. These results, showing a significant decrease in ChCS in FSHD cell lines, confirm the previously reported epigenetic alterations at D4Z4 suggesting that the D4Z4 array in FSHD patients is less compact than in controls.4
Comparison of ChCS values between the disease subtypes separately in fibroblast and myoblast samples showed that independent of cell type FSHD1 ChCS values show high variation while FSHD2 ChCS values are lower and less variable. This might be explained by a much more widespread chromatin relaxation over all four repeats in FSHD2, while in FSHD1 the changes may be more restricted to the pathogenic allele. This is also observed for the loss of DNA methylation at D4Z4.5
We also detected tissue-specific differences in the chromatin landscape at D4Z4 arrays of fibroblast and myoblast cultures with control myoblast ChCS values being higher than control fibroblast ChCS values. Genome-wide ChIP-seq studies have shown that the chromatin landscape in a particular cell type reflects its transcriptional activity and is therefore cell type-specific.11
Our results show that ChCS values are also cell-type specific with a more compact chromatin organization of D4Z4 in myoblasts to prevent expression of flDUX4
. This may explain the specific tissue involvement in FSHD.
Correlation analysis of the ChCS and age-corrected CSS revealed a significant relationship in male fibroblast patient samples (p = 0.028). Correlation analysis of all fibroblast samples gave borderline significance (p = 0.062). Gender differences for disease penetrance and severity have been reported and showed that males are typically more severely affected than females.12
The weak correlation may be caused by the small sample size or because of technical reasons. In our ChIP assays we measure collectively the chromatin status of each DUX4
promoter in the entire array, rather than specifically interrogating the most telomeric unit from which flDUX4
originates. Moreover, in FSHD1 we are not only measuring the DUX4
promoters of the pathogenic allele, but instead all D4Z4 and D4Z4-like arrays on chromosomes 4 and 10. Considering that in FSHD2 the chromatin status of all four chromosomes seems to be affected, this limitation should not apply to FSHD2. Nevertheless, also in FSHD2 cases we could not detect relationship between ChCS and age corrected CSS possibly due to small sample size.
Statistical analysis of myoblast data could not detect significant relationship between ChCS and age corrected CSS in any combination of samples (). Myoblasts are more sensitive to culturing conditions, often less homogeneous and can quickly transform to a pre-differentiation state, factors that may all affect their epigenetic profile. An alternative explanation might be that myoblast cultures carry a ‘history of disease’ that may affect the chromatin status of D4Z4 while fibroblasts, being derived from a non-affected tissue are not expected to have such history.
Irrespective of the explanation, if a negative correlation between the ChCS and CSS in fibroblast samples can be confirmed in larger group of patients, it would provide the first prognostic marker that can be measured in a relatively accessible tissue. In addition, since we could observe significant ChCS changes in myoblasts and fibroblasts, it is tempting to speculate that this score can also be used for prenatal diagnosis of FSHD2, a condition for which currently no validated diagnostic test exists.
An interesting observation from our study is that there is strong correlation between the CSS and the MPS of the vastus lateralis muscle. These results indicate that the pathology observed in the vastus lateralis muscle, typically not clinically affected in the early stages of FSHD, provides a good reflection of overall disease severity. This result shows that the vastus lateralis muscle, easily and safely accessible by needle biopsy techniques, is an ideal muscle to study for surrogate markers of overall disease severity. A non-invasive modality such as MRI to score the involvement of the vastus lateralis would be even more preferable. A study of lower extremity MRI in FSHD using a simple scoring system based on T1-W sequences was recently completed.13
This study showed that the combined MRI score of all lower extremity muscles was highly correlated with CSS. Whether this correlation holds true when the MRI score is based on the score of only the vastus lateralis remains to be determined.
In summary, our study of a large cohort of patient and control fibroblast and myoblast cultures confirms earlier reports of D4Z4 chromatin relaxation in FSHD and introduces a practical chromatin compaction score (ChCS). We found a near-significant correlation between the ChCS and severity in patient fibroblast samples and identified an unexpected correlation between the CSS and the MPS. We propose that factors in addition to locus intrinsic properties, influence disease severity in FSHD.