Conceived and designed the experiments: GT MP ER. Performed the experiments: GT MP TC. Analyzed the data: GT MP M. Monforte M. Mirabella EI RF FL PO. Contributed reagents/materials/analysis tools: GT ER. Wrote the paper: GT MP ER.
The authors have declared that no competing interests exist.
Facioscapulohumeral muscular dystrophy (FSHD) is one of the most common muscular dystrophies and is characterized by a non-conventional genetic mechanism activated by pathogenic D4Z4 repeat contractions. By muscle Magnetic Resonance Imaging (MRI) we observed that T2-short tau inversion recovery (T2-STIR) sequences identify two different conditions in which each muscle can be found before the irreversible dystrophic alteration, marked as T1-weighted sequence hyperintensity, takes place. We studied these conditions in order to obtain further information on the molecular mechanisms involved in the selective wasting of single muscles or muscle groups in this disease.
Histopathology, gene expression profiling and real time PCR were performed on biopsies from FSHD muscles with different MRI pattern (T1-weighted normal/T2-STIR normal and T1-weighted normal/T2-STIR hyperintense). Data were compared with those from inflammatory myopathies, dysferlinopathies and normal controls. In order to validate obtained results, two additional FSHD samples with different MRI pattern were analyzed.
Myopathic and inflammatory changes characterized T2-STIR hyperintense FSHD muscles, at variance with T2-STIR normal muscles. These two states could be easily distinguished from each other by their transcriptional profile. The comparison between T2-STIR hyperintense FSHD muscles and inflammatory myopathy muscles showed peculiar changes, although many alterations were shared among these conditions.
At the single muscle level, different stages of the disease correspond to the two MRI patterns. T2-STIR hyperintense FSHD muscles are more similar to inflammatory myopathies than to T2-STIR normal FSHD muscles or other muscular dystrophies, and share with them upregulation of genes involved in innate and adaptive immunity. Our data suggest that selective inflammation, together with perturbation in biological processes such as neoangiogenesis, lipid metabolism and adipokine production, may contribute to the sequential bursts of muscle degeneration that involve individual muscles in an asynchronous manner in this disease.
Facioscapulohumeral muscular dystrophy (FSHD) is an inherited myopathy often characterized by asymmetric muscle involvement
FSHD is associated with a contraction in a macrosatellite repeat array in the 4q35 chromosomal region D4Z4
In muscle disease, microarray gene expression analyses have been widely used to characterize molecular aspects of muscle pathology
Muscle magnetic resonance imaging (MRI) has been lately introduced into clinical and research practice as a non-invasive and sensitive technique to evaluate the involvement of individual muscles in different myopathies
To characterize the pathological changes associated with T2-STIR hyperintensity in FSHD muscle tissue, we performed histopathology and microarray analysis of a set of FSHD biopsies and compared T1-W normal/T2-STIR hyperintense muscle (T2-STIR +) with muscle showing absence of pathological signs on both T1-W and T2-STIR images (T2-STIR -) (
The Ethics Committee of the Catholic University School of Medicine approved this study. Written informed consent was obtained from all patients.
Unrelated, genetically confirmed (D4Z4 EcoRI fragment <40Kb) FSHD patients who had undergone lower limb muscle MRI were considered as candidates for the study. MRI was performed as described elsewhere
Sample | MRI signal | Muscle | Age | Biopsy | Histology | Gene expression |
1 | T1-W normal/T2-STIR hyperintense | biceps femoris | 37 | open | marked myopathic changes, endomysial and perimysial connective tissue +, adipocytes ++, endomysial and perivascular inflammatory infiltrates +++ | yes |
2* | T1-W normal/T2-STIR hyperintense | paravertebral | 31 | open | marked myopathic changes, endomysial and perimysial connective tissue ++, adipocytes +, necrotic fibers with invading macrophages ++, endomysial and perivascular inflammatory infiltrates +++ | yes |
3 | T1-W normal/T2-STIR hyperintense | quadriceps | 37 | needle | moderate myopathic changes, endomysial inflammatory infiltrates + | not available |
4 | T1-W normal/T2-STIR hyperintense | quadriceps | 38 | needle | moderate myopathic changes, necrotic and regenerating fibers +, endomysial and perivascular inflammatory infiltrates + | not available |
5 | T1-W normal/T2-STIR hyperintense | quadriceps | 20 | needle | moderate/marked myopathic changes, interstitial oedema and slightly enlarged vessels, adipocytes +, necrotic fibers +, endomysial and perivascular inflammatory infiltrates ++ | yes |
6 | T1-W normal/T2-STIR hyperintense | biceps femoris | 50 | needle | not available | yes |
7 | T1-W and T2-STIR normal | quadriceps | 25 | needle | mild myopathic changes, no inflammation | yes |
8 | T1-W and T2-STIR normal | quadriceps | 33 | needle | mild myopathic changes, no inflammation | not available |
9 | T1-W and T2-STIR normal | quadriceps | 51 | needle | mild myopathic changes, no inflammation | yes |
10 | T1-W and T2-STIR normal | quadriceps | 36 | needle | minimal myopathic changes, no inflammation | not available |
11 | T1-W and T2-STIR normal | quadriceps | 26 | needle | minimal myopathic changes, no inflammation | yes |
12* | T1-W and T2-STIR normal | quadriceps | 31 | needle | mild myopathic changes, no inflammation | yes |
T2-STIR + validation | T1-W normal/T2-STIR hyperintense | quadriceps | 36 | needle | not available | yes |
T2-STIR – validation | T1-W and T2-STIR normal | quadriceps | 36 | needle | not available | yes |
+ = mild increase, ++ = moderate increase, +++ = marked increase. * samples from the same patient.
We also included in the study 7 normal controls (age range 18–58), 7 immunopathologically characterized inflammatory myopathies (IM) (2 dermatomyositis, DM, 2 polymyositis, PM, 1 necrotizing myopathy and 2 IM with nonspecific histopathological features) (age range 23–73) and 4 dysferlinopathies (LGMD2B) (age range 28–35). Non-FSHD samples were obtained for diagnostic purposes.
To provide a biological validation of our findings, two additional biopsies were performed in FSHD muscles showing different MRI patterns (T2-STIR + validation and T2-STIR – validation) and analyzed.
Eleven FSHD samples (5 T2-STIR + and 6 T2-STIR −) were available for histological analysis, which included hematoxylin-eosin and, whenever possible, routine diagnostic stainings (nicotinamide adenine dinucleotide dehydrogenase tetrazolium reductase, succinate dehydrogenase, cytochrome-C oxidase, modified Gomori trichrome, alkaline phosphatase, and periodic acid-Schiff). Immunohistochemistry protocol for inflammatory markers and antibodies used are described elsewhere
Eight FSHD samples (4 T2-STIR + and 4 T2-STIR −) were available for gene expression analysis. Total RNA was extracted by TriZol (TriZol reagent, Invitrogen, Carlsbad, CA, USA) and further purified using the RNAeasy mini kit following the RNA cleanup protocol as indicated by the manufacturer (Qiagen, Valencia, CA, USA). RNA purity and integrity were assessed by spectrophotometric analysis and agarose gel electrophoresis. We made use of the Illumina BeadChips technology to analyze the expression of >31,000 annotated genes with more than 47,000 probes using the HumanHT-12v4 Expression BeadChip (Illumina Inc., San Diego, CA, USA). Gene expression data were extracted and normalized using Illumina Genome Studio and analyzed using BRB-ArrayTools, developed by R. Simon and BRB-ArrayTools Development Team. To discriminate the gene expression variations that characterize T2-STIR + FSHD muscles with respect to T2-STIR – FSHD and IM muscles we computed the probability of genes being differentially expressed between the classes using the unequal variance
To define the minimum fold-change threshold we could confidently interpret as a true change given the size of our study sample, we made use of the SampleSize R package implemented in BRB-ArrayTools (Sample size determination in microarray experiments for class comparison and prognostic classification
Routine histopathology displayed moderate to marked myopathic features in T2-STIR + FSHD muscles (
To visualize the correlation relationships among our FSHD samples we made use of multidimensional scaling (MDS) analysis, by which samples are positioned in a 3D space on the basis of first three principal components of variability. Based on the expression level of all the 29045 probesets showing detection p-value greater than 0.05, FSHD samples formed two separate clusters corresponding to T2-STIR + and T2-STIR – muscles. A similar clustering pattern of the samples could be observed by Hierarchical clustering analysis (
(A) Unsupervised hierarchical clustering showing correlation relationships among samples. T2-STIR + FSHD samples are evidently separated from T2-STIR – FSHDs and group with IM (framed in red). T2-STIR – FSHDs show higher correlation with normal controls and one of the dysferlinopathies (framed in green). (B) Multidimensional scaling. Each sample is represented by a sphere in a 3D space. Samples with similar expression profile are shown close together. Note the peculiar and distinct spatial collocation of T2-STIR + FSHD samples clustering together with IM. (C) Scatter plot representation of average transcript expression levels in T2-STIR + FSHD muscles (
To describe at the molecular level the on-going process responsible for the observed differences between MRI patterns of FSHD muscle, we compared the expression profiles of T2-STIR + and T2-STIR – FSHD muscles. We first assessed the power of statistical analysis and estimated that our sample size of 4 samples per class was sufficient to confidently identify differences larger than 1log2, if taking into account the 75th percentile of the variance distribution.
By using a random variance
We also tested the hypothesis that individual gene sets were statistically different between the classes by using the gene set class comparison tool implemented in BRB-ArrayTools to query the BioCarta, Gene Ontology, and KEGG databases (
We used MDS to plot our T2-STIR+ and T2-STIR- samples in a 3D gene expression space together with muscles from normal individuals and patients affected by different types of muscle diseases. As shown in
Functional category | Official Full Name | Official Symbol | Fold change | p-value |
|
Angiogenin | ANG | 2,3725 | 0,0041 |
Angiopoietin-like 2 | ANGPTL2 | 2,5002 | 0,0014 | |
CD44 molecule | CD44 | 2,4375 | 0,0035 | |
CD34 molecule | CD34 | 2,2935 | 0,0047 | |
CD47 molecule | CD47 | 1,6579 | 0,0013 | |
Filamin B beta | FLNB | 2,2672 | 0,0056 | |
Von Willebrand factor | VWF | 1,9606 | 0,0331 | |
Slit homolog 3 | SLIT3 | 2,6961 | 0,0047 | |
Dedicator of cytokinesis 1 | DOCK1 | 1,8760 | 0,0024 | |
Secreted frizzled-related protein 1 | SFRP1 | 4,8032 | 0,0026 | |
Frizzled homolog 4 | FZD4 | 2,1590 | 0,0051 | |
Frizzled homolog 7 | FZD7 | 1,3833 | 0,0401 | |
Cell death-inducing DFFA-like effector a | CIDEA | 9,7264 | 0,0009 | |
Cell death-inducing DFFA-like effector c | CIDEC | 8,5623 | 0,0038 | |
Perilipin 1 | PLIN | 5,6435 | 0,0017 | |
Glycerol-3-phosphate acyltransferase | GPAM | 4,7128 | 0,0023 | |
Alcohol dehydrogenase 1A | ADH1A | 4,1328 | 0,0016 | |
Adiponectin, C1Q and collagen domain containing | ADIPOQ | 2,6172 | 0,0019 | |
C1q and tumor necrosis factor related protein 1 | C1QTNF1 | 2,6181 | 0,0013 | |
Leptin | LEP | 3,9142 | 0,0544 |
All of the cited genes were expressed to a higher extent in T2-STIR + FSHD muscles.
Further validating the consistency of the expression data within the identified functional categories, we observed co-regulation of a number of functionally related genes (
Co-regulation of functionally related genes: (A–F) members of complement system, (G–H) co-regulated and selective induction in T2-STIR + FSHD muscles can be observed for SFRP1 and its receptor FZD4 and (I–L) for adipose tissue genes and inflammatory adipokines.
We performed a technical validation of our microarray experiment and verified by real time PCR some of the gene expression changes identified by microarray analysis (
Real time PCR analysis of selected gene expression. All the pathological samples display alterations consistent with the gene chip expression level. Changes in transcript abundance are expressed as log2 ratio to control mean.
To further validate at the biological level the consistency of obtained results we prospectively performed two additional biopsies, one in a FSHD T2-STIR + and one in a FSHD T2-STIR – muscle and generated expression profiles for these two muscle specimens. We normalized the two new arrays together with the overall set of FSHD arrays previously generated and performed a correlation analysis. As can be observed in
Based on the signal level of the two probesets interrogating
Other genes previously suggested to play a role in FSHD pathogenesis like
Histopathological differences that emerge from the comparison of FSHD T2-STIR + with T2-STIR – muscles include myopathic alterations, increased endomysial spaces, presence of adipocytes in perimysial areas and scattered necrotic phenomena. All our T2-STIR + FSHD samples displayed inflammatory changes on immunohistochemistry
Together with innate immunity, adaptive immunity processes and in particular T-cell mediated immune responses were induced. Accordingly, we have described increased presence of activated circulating CD8+ T-cells in FSHD patients displaying T2-STIR hyperintense muscles
T2-STIR + FSHD samples appear more similar to IM, and in particular PM, while T2-STIR – muscles group with normal controls. Although many transcriptional alterations were shared between T2-STIR + FSHD and IM muscles, differences were present that mainly regarded the overexpression in T2-STIR + FSHD muscles of a number of proangiogenic factors and surface proteins involved in cell-cell adhesion and cell-ECM interactions (
Several T2-STIR + FSHD upregulated genes can be ascribed to adipocyte metabolism. SFRP1 itself has proadipogenic activity
In FSHD, different muscles of each patient can, at the same time, display different stages of the dystrophic process, characterized by peculiar and distinct histological and molecular alterations. Thus, even though the aim of our study was not to identify what distinguishes FSHD from normal muscle, it emerges that taking one “snapshot” of the pathologic process could be not enough to identify the process itself. This strict dependency on the timing and the muscle in which the “snapshot” is taken could have contributed to the diverse results obtained in different FSHD gene expression studies
The early stages of involvement of each FSHD muscle, defined by the absence of T1-W hyperintensity, are identifiable through muscle MRI based on their T2-STIR normal or hyperintense signal. MRI with T2-STIR sequences, marking different phases of the disease at the single muscle level, is a useful and reliable tool in monitoring the progression of involvement of FSHD muscles and with this aim it should be taken into consideration in the future clinical trials in this disease.
The pronounced similarity with PM and DM of the T2-STIR + FSHD muscle transcriptional profile, characterized by a marked upregulation of genes involved in innate and adaptive immune response, suggests that a selective and multifocal inflammatory process may play an active role in the development of dystrophic changes and consequently in disease advancement at single muscle level. However, some aspects of our analysis may be limited in power due to the moderate sample size. Further studies are needed to clarify the exact significance of the specific biological processes active in T2-STIR + muscles in determining muscle damage, in a phase in which tissue modifications are still theoretically reversible.
(TIF)
(TIF)
(TIF)
(TIF)
(DOC)
(DOC)
(DOC)
(DOC)
(DOC)
We greatly acknowledge the patients for their participation in the study.