Regional and bilateral MRI and gene signatures in facioscapulohumeral dystrophy: implications for clinical trial design and mechanisms of disease progression

Abstract

Identifying the aberrant expression of DUX4 in skeletal muscle as the cause of facioscapulohumeral dystrophy (FSHD) has led to rational therapeutic development and clinical trials. Several studies support the use of MRI characteristics and the expression of DUX4-regulated genes in muscle biopsies as biomarkers of FSHD disease activity and progression. We performed lower-extremity MRI and muscle biopsies in the mid-portion of the tibialis anterior (TA) muscles bilaterally in FSHD subjects and validated our prior reports of the strong association between MRI characteristics and expression of genes regulated by DUX4 and other gene categories associated with FSHD disease activity. We further show that measurements of normalized fat content in the entire TA muscle strongly predict molecular signatures in the mid-portion of the TA, indicating that regional biopsies can accurately measure progression in the whole muscle and providing a strong basis for inclusion of MRI and molecular biomarkers in clinical trial design. An unanticipated finding was the strong correlations of molecular signatures in the bilateral comparisons, including markers of B-cells and other immune cell populations, suggesting that a systemic immune cell infiltration of skeletal muscle might have a role in disease progression.

Keywords: DUX4; MRI; complement; facioscapulohumeral dystrophy.

Figure 1

Comparison of regional fat fraction and total muscle fat percentage. (a) At the lower levels of fat infiltration, whole muscle fat percent shows increased values relative to regional fat fraction, possibly due to earlier fat infiltration in the distal and proximal ends of the muscle compared to the region of the biopsy that was generally near the middle of the TA muscle. (b) Fat infiltration percent over the length of each TA muscle from distal (0) to proximal (100) for STIR− and STIR+ muscles. STIR− muscles show fat infiltration in the distal and proximal regions with the exception of muscles with very high fat infiltration that have likely lost prior STIR+ signal, whereas STIR+ muscles show fat infiltration progressing into the central portion of the muscle. (c) Correlation between RNA sequencing inference of fat content with regional or whole muscle MRI measurements of fat content. (d) STIR+ muscles show higher whole muscle fat content with the exception of muscles that have progressed to very high fat percentages.

Figure 2

Muscles with whole muscle fat percent greater than 20% and MRI+ muscles have high DUX4 scores that correlate with functional and histopathological measures. (a) Scatter plot showing that MRI+ muscles mostly have elevated DUX4 scores, and that MRI+ muscles with greater than whole muscle fat infiltration of greater than 20% have uniformly high DUX4 scores. DUX4 scores plotted on a log scale emphasizes the difference between the levels in the historical control muscle biopsies (shaded area indicates the 95% confidence interval (CI) for the distribution of the DUX4 scores in the control biopsies). (b) Same as in (a) but plotted on a linear scale. (c) Logistic regression predicting the occurrence of a DUX4 score > 0.5. The predictors include the whole muscle fat percent and STIR status of the muscle biopsies and outcome is the occurrence of DUX4 score > 0.5. STIR− muscles indicated in blue, STIR+ in red. Dashed gray line indicates all muscles regardless of STIR status. (c) Scatter plot showing the correlation between (d) TA muscle strength in kg and the DUX4 score; (e) the clinical severity score (CSS) and the DUX4 score; (f) the histopathology score and the DUX4 score. STIR− muscles indicated in blue, STIR+ in red.

Figure 3

Baskets of genes in different categories distinguish between STIR−, and STIR+ muscles. (a) Distribution of the basket scores in FSHD STIR+ compared to STIR− muscle, and also compared to historic controls (quadriceps biopsies from non-FSHD, unaffected subjects (see Methods)). Top panel plotted on a linear scale and bottom panel on a log scale to show separation from control samples. (b) Correlation between the basket scores in each biopsy. (c) Heatmap illustrating row-wise z-score of expression levels for the indicated basket gene. STIR+ muscle exhibit elevated expression compared to STIR−. The muscle-low samples are included here to demonstrate their distinct attributes characterized by low-level DUX4 signatures but elevated in ECM, inflammatory, and complement activation.

Figure 4

Bilateral comparisons of fat infiltration, TA strength, and gene baskets. Scatter plots show correlation between R and L TA for whole muscle fat infiltration (a) and TA strength (b). (c) Whisker plots showing the correlation between the R and L TA for each basket of genes indicated. Each dot represents the Pearson correlation for each gene in the basket and was calculated based on the gene expression level in TPM. The diamond represents the average of the basket.

Figure 5

Methylation levels show intra-subject correlation bilaterally not with other parameters. (a) Methylation levels in pathologic 4qA-short and 4qA-long alleles. (b) Bilateral correlation of methylation levels. (c) Methylation levels are not associated with MRI STIR signal. (d) Correlations between methylation levels and the indicated parameter do not show any moderate or strong correlations.

Figure 6

Dot plots illustrating the relationship between basket scores of bilateral muscle biopsies and complement scoring graded on the scale of 1, 2, or 3.