Facioscapulohumeral muscular dystrophy family studies of DUX4 expression: evidence for disease modifiers and a quantitative model of pathogenesis

Abstract

Facioscapulohumeral muscular dystrophy (FSHD), the most prevalent myopathy afflicting both children and adults, is predominantly associated with contractions in the 4q35-localized macrosatellite D4Z4 repeat array. Recent studies have proposed that FSHD pathology is caused by the misexpression of the DUX4 (double homeobox 4) gene resulting in production of a pathogenic protein, DUX4-FL, which has been detected in FSHD, but not in unaffected control myogenic cells and muscle tissue. Here, we report the analysis of DUX4 mRNA and protein expression in a much larger collection of myogenic cells and muscle biopsies derived from biceps and deltoid muscles of FSHD affected subjects and their unaffected first-degree relatives. We confirmed that stable DUX4-fl mRNA and protein were expressed in myogenic cells and muscle tissues derived from FSHD affected subjects, including several genetically diagnosed adult FSHD subjects yet to show clinical manifestations of the disease in the assayed muscles. In addition, we report DUX4-fl mRNA and protein expression in muscle biopsies and myogenic cells from genetically unaffected relatives of the FSHD subjects, although at a significantly lower frequency. These results establish that DUX4-fl expression per se is not sufficient for FSHD muscle pathology and indicate that quantitative modifiers of DUX4-fl expression and/or function and family genetic background are determinants of FSHD muscle disease progression.

Figure 1.

Polyadenylated DUX4-fl mRNA was expressed in cultures of myogenic cells derived from FSHD and control subjects. Schematic for DUX4 mRNA splicing and detection by RT-PCR based on the model (15) for (A) FSHD and (B) control cells. Locations of oligonucleotide primers are indicated with arrows. Nested RT-PCR analysis of polyadenylated DUX4 mRNA from CD56+ myogenic cells derived from biceps (bic) and deltoid (del) muscle biopsies for (C) cohort 09, (D) cohort 15, (E) cohort 16 and (F) cohort 20 following differentiation for 0 or 4 days, as indicated. All RT-PCRs were repeated at least three times, all products were sequenced and the presence of spliced DUX4-fl mRNA (arrow) was confirmed in cultures from FSHD subjects (09Adel, 9Abic, 15Abic, 15Bbic, 16Adel, 16Abic, 20Adel, 20Abic) and from control individuals not containing an FSHD D4Z4 contraction (16Udel, 16Ubic, 20Udel, 20Ubic). Gray boxes indicate DUX4-fl expression in unaffected cell cultures. RT–PCR for GAPDH mRNA expression controlled for integrity of the mRNA and first strand cDNA synthesis.

Figure 2.

DUX4-FL protein was detected in FSHD and control myogenic cells. Differentiated CD56+ myogenic cells derived from FSHD subjects (A–H, I and M), or control subjects (J–L and N–P) were immunostained for DUX4-FL (brown). DUX4-FL positive nuclei (white arrows) and DUX4-FL negative nuclei (white arrowheads) were observed. (Q) Summary of DUX4-FL immunostaining; gray shading indicates unaffected controls expressing DUX4-FL; asterisk indicates negative control cells with two non-permissive B haplotype subtelomeres.

Figure 3.

The total numbers of DUX4-FL expressing nuclei are significantly higher in myogenic cultures from affected (A) versus unaffected (U) subjects. Counts of DUX4-FL positive nuclei, determined by ICC, per 10 000 nuclei are shown. From left to right, columns show FSHD biceps (A.bic, n= 18, including 5 asymptomatic in green), unaffected biceps (U.bic, n= 9, including 2 4qB/4qB in blue), FSHD deltoid (A.del, n= 16, including 4 asymptomatic in green), and unaffected deltoid samples (U.del, n= 9 including 2 4qB/4qB in blue). Gray boxes show range from 25th percentile to 75th percentile, with the median shown as a bold grey line. Within each column, samples are ordered by increasing length of shortest 4qA EcoRI/BlnI fragment, with 4qB/4qB haplotypes at far right (ties broken arbitrarily). The difference between affected and unaffected samples is highly significant (p = 0.001; likelihood-ratio test), the difference between muscle types is non-significant (p = 0.4), and the interaction between muscle type and disease status is mildly significant (p = 0.03). (All P-values exclude the 4qB/4qB samples, which lack DUX4-FL permissive alleles.)

Figure 4.

Polyadenylated DUX4-fl mRNA was expressed in muscle biopsies from control subjects and FSHD subjects. Polyadenylated and spliced DUX4-fl mRNA (arrow) was identified by nested RT-PCR and confirmed by sequencing in muscle biopsies. (A) Cohort 09 shows DUX4-fl exclusively in FSHD subjects. (B) Cohorts 17 and 20 show DUX4-fl mRNA expression in control biopsies from unaffected subjects without the FSHD-linked D4Z4 deletion. (C) Cohorts 15, 28 and 29 show DUX4-fl mRNA expression in biopsies from FSHD subjects exhibiting no apparent weakness in the biopsied muscle (15Bbic, 28Bbic, 29Bbic). RT-PCRs for GAPDH mRNA controlled for mRNA integrity and first strand cDNA synthesis.

Figure 5.

Digital RT-PCR shows variation in DUX4-fl mRNA detection due to low levels of transcript in muscle biopsy. Blind RT-PCRs were performed in six replicates using mRNA derived from muscle biopsies from the indicated subjects. GAPDH RT-PCRs controlled for mRNA integrity and cDNA synthesis.