Different molecular signatures in magnetic resonance imaging-staged facioscapulohumeral muscular dystrophy muscles

Abstract

Background: 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.

Methods: 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.

Results: 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.

Conclusions: 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.

Figure 1. Class discovery graphical displays and scatter plot.

(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 (n = 4) compared to T2-STIR – FSHD muscles (n = 4) or to inflammatory myopathies (n = 7).

Figure 2. Schematic representation of the molecular pathways modulated in T2-STIR + FSHD samples.

Figure 3. Expression levels of single genes across samples.

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.

Figure 4. Real time PCR.

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.

Figure 5. Unsupervised hierarchical clustering of the validation experiment.