Transcriptional profiling in facioscapulohumeral muscular dystrophy to identify candidate biomarkers

Abstract

Facioscapulohumeral muscular dystrophy (FSHD) is a progressive neuromuscular disorder caused by contractions of repetitive elements within the macrosatellite D4Z4 on chromosome 4q35. The pathophysiology of FSHD is unknown and, as a result, there is currently no effective treatment available for this disease. To better understand the pathophysiology of FSHD and develop mRNA-based biomarkers of affected muscles, we compared global analysis of gene expression in two distinct muscles obtained from a large number of FSHD subjects and their unaffected first-degree relatives. Gene expression in two muscle types was analyzed using GeneChip Gene 1.0 ST arrays: biceps, which typically shows an early and severe disease involvement; and deltoid, which is relatively uninvolved. For both muscle types, the expression differences were mild: using relaxed cutoffs for differential expression (fold change ≥1.2; nominal P value <0.01), we identified 191 and 110 genes differentially expressed between affected and control samples of biceps and deltoid muscle tissues, respectively, with 29 genes in common. Controlling for a false-discovery rate of <0.25 reduced the number of differentially expressed genes in biceps to 188 and in deltoid to 7. Expression levels of 15 genes altered in this study were used as a "molecular signature" in a validation study of an additional 26 subjects and predicted them as FSHD or control with 90% accuracy based on biceps and 80% accuracy based on deltoids.

Fig. 1.

Volcano plots of expression differences between FSHD subjects and controls in biceps (A) and deltoid muscles (B). The horizontal axis shows log₂ (fold change), with vertical lines indicating cutoffs of 1.2-fold change either down (red) or up (green) relative to controls. The vertical axis shows –log₁₀ (P value), with the horizontal gray line indicating the cutoff of nominal P = 0.01. Genes that passed both cutoffs and also satisfied FDR < 0.25 are colored red (down in FSHD) or green (up in FSHD).

Fig. 2.

Correlation between log₂ fold-change values of 15 validation genes from microarrays and qPCR for FSHD subjects vs. controls comparisons in biceps (A) and deltoid muscles (B). The horizontal axis represents the log₂ fold change between FSHD and control samples from qPCR and the vertical axis represents the log₂ fold change from microarrays. Symbols in each scatter plot represent individual genes, with the shape of a symbol indicating the statistical significance of the difference between FSHD and control samples for that gene (square, P < 0.01 in qPCR; triangle, P < 0.01 in array; open circle, P < 0.01 in both; black dot, P < 0.01 in neither). The solid gray line with slope 1 indicates perfect agreement, and the dashed gray line indicates the line of best fit to the data (with slope, intercept, and correlation coefficient as indicated in the plots).