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

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. A study cohort comprising 34 FSHD subjects, 16 female and 18 male, aged 21 to 69 (mean 47.1±14SD) was recruited at three sites of Seattle Paul D. Wellstone Muscular Dystrophy Specialized Research Center, including University of Washington Medical Center, University of Rochester Medical Center, and Kansas University Medical Center. All participants underwent initial functional testing, including lower extremity dorsiflexor strength measurement, lower extremity MRI, and needle muscle biopsy of both left and right tibialis anterior (TA) muscles. Muscle biopsies were processed for histology, RNA extraction, and sequencing. DNA was also extracted for bisulfite sequencing (BSS). Out of 68 attempted muscle biopsies, 64 yielded sufficient RNA for sequencing and met the criteria and an RNA integrity number > 4.