MRI-informed muscle biopsies correlate MRI with pathology and DUX4 target gene expression in FSHD

Abstract

Facioscapulohumeral muscular dystrophy (FSHD) is a common, dominantly inherited disease caused by the epigenetic de-repression of the DUX4 gene, a transcription factor normally repressed in skeletal muscle. As targeted therapies are now possible in FSHD, a better understanding of the relationship between DUX4 activity, muscle pathology and muscle magnetic resonance imaging (MRI) changes is crucial both to understand disease mechanisms and for the design of future clinical trials. Here, we performed MRIs of the lower extremities in 36 individuals with FSHD, followed by needle muscle biopsies in safely accessible muscles. We examined the correlation between MRI characteristics, muscle pathology and expression of DUX4 target genes. Results show that the presence of elevated MRI short tau inversion recovery signal has substantial predictive value in identifying muscles with active disease as determined by histopathology and DUX4 target gene expression. In addition, DUX4 target gene expression was detected only in FSHD-affected muscles and not in control muscles. These results support the use of MRI to identify FSHD muscles most likely to have active disease and higher levels of DUX4 target gene expression and might be useful in early phase therapeutic trials to demonstrate target engagement in therapies aiming to suppress DUX4 expression.

Figures

Figure 1

Relative expression of four candidate biomarkers in muscle biopsies from control and FSHD muscles. The sum of relative RNA-Seq expression for the genes LEUTX, KHDC1L, TRIM43 and PRAMEF2 was plotted for each RNA-Seq sample. Biopsies from FSHD individuals are indicated by an asterisk (*), whereas control muscle biopsies do not have an asterisk. A heat map of the relative expression of each of the four genes is shown at the top of the graph, and the graph plots the sum of reads for all four genes. The size of the spot reflects the number of candidate biomarker genes above a threshold level, therefore the smallest spot indicates no biomarkers above threshold, whereas the largest spot indicates all four biomarkers above threshold. The color coding divides the samples into four groups based on the relative expression for all four biomarkers.

Figure 2

Biomarkers’ RNA-Seq expression in biopsy samples from MRI normal versus STIR+ samples: mean (SD) value in muscle biopsies from MRI normal muscle is 1.4 (SD 2.4) and in muscle biopsies from T2 STIR+ muscles is 7.7 (SD 5.7); the difference is statistically significant with a P < 0.001.

Figure 3

Principle component analysis based on total RNA sequencing data. Colors identify groups defined in Figure 1 based on the key at the bottom of this figure. Although FSHD groups 1, 2, 3 and 4 generally show progressively distinction from the group 1 controls, samples 01-0037 and 32-0016 represent outliers in the FSHD group 1 samples.

Figure 4

Boxplots showing the read counts scaled by log10 of RPKM for a subset of the immune/inflammation genes in the GSEA that distinguished the outlier group 1 FSHD samples (01-0037 and 32-0016) from control samples. Note that these same genes tend to be progressively elevated in groups 2, 3 and 4. The boxplots show the line for the median value, the box shows the interquartile range and the whiskers show the lowest and highest data point within the 1.5 interquartile range. The colored points represent expression in the two outlier samples.

Figure 5

Boxplots showing the read counts scaled by log10 RPKM for a subset of the extracellular matrix and other genes in the GSEA that distinguished the outlier group 1 FSHD samples (01-0037 and 32-0016) from control samples. Note that these same genes tend to be progressively elevated in groups 2, 3 and 4. The boxplots show the line for the median value, the box shows the interquartile range and the whiskers show the lowest and highest data point within the 1.5 interquartile range. The colored points represent expression in the two outlier samples.

Figure 6

Immunohistochemical staining of muscle sections using C5b-9 (MAC) antibody (Agilent/Dako Clone aE11). (A) Muscle section from a normal control showing staining in larger perimysial vessels but no staining of endomysial capillaries. (B) A positive control section from an individual with dermatomyositis showing extensive, predominantly perifasicular, deposition of MAC in endomysial capillaries. (C, D, E) Show sections from FSHD subjects with positive MAC capillary staining (arrows); also visible in E is a small necrotic fiber (circle) with sarcolemmal MAC staining. (F) An adjacent section stained with hematoxylin and eosin showing the same atrophic necrotic fiber (circle).