The neuromuscular disorder facioscapulohumeral muscular dystrophy (FSHD) results from integral deletions of the subtelomeric repeat D4Z4 on chromosome 4q. A disruption of chromatin structure affecting gene expression is thought to underlie the pathophysiology. The global gene expression profiling of mature muscle tissue presented here provides the first insight into an FSHD-specific defect in myogenic differentiation. FSHD expression profiles generated by oligonucleotide microarrays were compared with those from normal muscle as well as other types of muscular dystrophies (DMD, aSGD) in order to determine FSHD-specific changes. In addition, matched biopsies (affected and unaffected muscle) from individuals with FSHD served to monitor expression changes during the progression of the disease as well as to diminish non-specific changes resulting from individual variability. Among genes altered in an FSHD-specific and highly significant manner, many are involved in myogenic differentiation and suggest a partial block in the normal differentiation program. Indeed, many of the transcripts affected in FSHD represent direct targets of the transcription factor MyoD. Additional mis-expressed genes confirm a diminished capacity to buffer oxidative stress, as demonstrated in FSHD myoblasts. This enhanced vulnerability of proliferative stage myoblasts to reactive oxygen species is also disease-specific, further implicating a defect in FSHD muscle satellite cells. Importantly, none of the genes localizing to the FSHD region at 4q35 were found to exhibit a significantly altered pattern of expression in FSHD muscle. This finding was corroborated by expression analysis of FSHD muscle using a custom cDNA microarray containing 51 genes and ESTs from the 4q35 region. Disruptions in FSHD myogenesis and oxidative capacity may therefore not arise from a position effect mechanism as has been previously suggested, but rather from a global effect on gene regulation. Improper nuclear localization of 4qter is discussed as an alternative model for FSHD gene regulation and pathogenesis.

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Facioscapulohumeral muscular dystrophy (FSHD) may be a new member of the class of neuromuscular diseases (NMD) due to defects in the nuclear envelope. Unlike other NMDs with primary defects in nuclear envelope proteins, however, FSHD may result from inappropriate chromatin interactions at the envelope. 3D Immuno-FISH and a novel method of 3D by 2D analysis using NucProfile were developed to examine nuclear organization of the FSHD genomic region. In contrast to most other telomeres, the FSHD region at 4q35.2 localizes to the nuclear periphery. This localization is consistent in normal myoblasts, myotubes, fibroblasts and lymphoblasts, does not vary significantly throughout the cell cycle, and is independent of chromosome territory effects. The nuclear lamina protein lamin A/C is required for FSHD region chromatin localization to the nuclear envelope, as the association is lost in lamin A/C null fibroblasts. As both normal and affected alleles (deleted for the subtelomeric repeat D4Z4) localize to the nuclear periphery, FSHD likely arises instead from improper interactions with transcription factors or chromatin modifiers at the nuclear envelope. Interestingly, it is not D4Z4 itself that mediates interaction with the envelope, as sequences proximal to D4Z4 (i.e. D4S139) localize closer to the nuclear periphery, perhaps accounting for the chromosome 4 specificity of the disease.

Autosomal dominant facioscapulohumeral muscular dystrophy (FSHD) is likely caused by epigenetic alterations in chromatin involving contraction of the D4Z4 repeat array near the telomere of chromosome 4q. The precise mechanism by which deletions of D4Z4 influence gene expression in FSHD is not yet resolved. Regulatory models include a cis effect on proximal gene transcription (position effect), DNA looping, non-coding RNA, nuclear localization and trans-effects. To directly test whether deletions of D4Z4 affect gene expression in cis, nascent RNA was examined in single myonuclei so that transcription from each allele could be measured independently. FSHD and control myotubes (differentiated myoblasts) were subjected to sequential RNA-DNA FISH. A total of 16 genes in the FSHD region (FRG2, TUBB4Q, FRG1, FAT1, F11, KLKB1, CYP4V2, TLR3, SORBS2, PDLIM3 (ALP), LRP2BP, ING2, SNX25, SLC25A4 (ANT1), HELT and IRF2) were examined for interallelic variation in RNA expression within individual myonuclei. Sequential DNA hybridization with a unique 4q35 chromosome probe was then applied to confirm the localization of nascent RNA to 4q. A D4Z4 probe, labeled with a third fluorochrome, distinguished between the deleted and normal allele in FSHD nuclei. Our data do not support an FSHD model in which contracted D4Z4 arrays induce altered transcription in cis from 4q35 genes, even for those genes (FRG1, FRG2 and SLC25A4 (ANT1)) for which such an effect has been proposed.