Regional and bilateral MRI and gene signatures in facioscapulohumeral dystrophy: 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.

Validation of the association between MRI and gene signatures in facioscapulohumeral dystrophy muscle: implications for clinical trial design.

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, but reproducibility across studies needs further validation. 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. Together with moderate-to-strong correlations of gene signatures and MRI characteristics between the TA muscles bilaterally, these results suggest a whole muscle model of disease progression and provide a strong basis for inclusion of MRI and molecular biomarkers in clinical trial design.

Breast Cancer Screening in High-risk Women During Pregnancy and Lactation.

Women who are at high risk of developing breast cancer warrant screening that is often initiated at younger ages than in average-risk women; this is usually with a combination of annual mammography and breast MRI. Compared to average-risk women, those at high risk are more frequently recommended to undergo screening during childbearing age and thus potentially during pregnancy and lactation. Understanding the appropriate use of screening breast imaging during pregnancy and lactation can be challenging due to limited data defining the evidence-based roles of the different imaging modalities, including mammography, US, and MRI. There have also been assumptions about the diagnostic accuracy of these modalities secondary to physiological changes. This scientific review discusses the current state of evidence- and expert-based guidelines and data for breast imaging screening of high-risk pregnant and/or lactating women, and the clinical and imaging presentations of breast cancer for these women.

Computed Tomography Assessment of Gastric Band Slippage.

Background: The purpose of this study was to develop and validate reliable computed tomography (CT) imaging criteria for the diagnosis of gastric band slippage. Material and Methods: We retrospectively evaluated 67 patients for gastric band slippage using CT. Of these, 14 had surgically proven gastric band slippage (study group), 22 had their gastric bands removed for reasons other than slippage (control group 1), and 31 did not require removal (control group 2). All of the studies were read independently by two radiologists in a blinded fashion. The "O" sign, phi angle, amount of inferior displacement from the esophageal hiatus, and gastric pouch size were used to create CT diagnostic criteria. Standard statistical methods were used. Results: There was good overall interobserver agreement for diagnosis of gastric band slippage using CT diagnostic criteria (kappa = 0.83). Agreement was excellent for the "O" sign (kappa = 0.93) and phi angle (intraclass correlation coefficient = 0.976). The "O" sign, inferior displacement from the hiatus >3.5 cm, and gastric pouch volume >55 cm3 each had 100% positive predictive value. A phi angle <20° or >60° had the highest negative predictive value (NPV) (98%). Of all CT diagnostic criteria, enlarged gastric pouch size was most correlated with band slippage with an AUC of 0.991. Conclusion: All four imaging parameters were useful in evaluating for gastric band slippage on CT, with good interobserver agreement. Of these parameters, enlarged gastric pouch size was most correlated with slippage and abnormal phi angle had the highest NPV.

MRI-informed muscle biopsies correlate MRI with pathology and DUX4 target gene expression in FSHD.

Facioscapulohumeral muscular dystrophy (FSHD) is a common, dominantly inherited disease caused by the epigenetic de-repression of the DUX4 gene, a transcription factor normally repressed in skeletal muscle. As targeted therapies are now possible in FSHD, a better understanding of the relationship between DUX4 activity, muscle pathology and muscle magnetic resonance imaging (MRI) changes is crucial both to understand disease mechanisms and for the design of future clinical trials. Here, we performed MRIs of the lower extremities in 36 individuals with FSHD, followed by needle muscle biopsies in safely accessible muscles. We examined the correlation between MRI characteristics, muscle pathology and expression of DUX4 target genes. Results show that the presence of elevated MRI short tau inversion recovery signal has substantial predictive value in identifying muscles with active disease as determined by histopathology and DUX4 target gene expression. In addition, DUX4 target gene expression was detected only in FSHD-affected muscles and not in control muscles. These results support the use of MRI to identify FSHD muscles most likely to have active disease and higher levels of DUX4 target gene expression and might be useful in early phase therapeutic trials to demonstrate target engagement in therapies aiming to suppress DUX4 expression.

Alfalfa-derived HSP70 administered intranasally improves insulin sensitivity in mice.

Heat shock protein (HSP) 70 is an abundant cytosolic chaperone protein that is deficient in insulin-sensitive tissues in diabetes and unhealthy aging, and is considered a longevity target. It is also protective in neurological disease models. Using HSP70 purified from alfalfa and administered as an intranasal solution, we tested in whether the administration of Hsp70 to diet-induced diabetic mice would improve insulin sensitivity. Both the 10 and 40 µg given three times per week for 26 days significantly improved the response to insulin. The HSP70 was found to pass into the olfactory bulbs within 4-6 hours of a single dose. These results suggest that a relatively inexpensive, plentiful source of HSP70 administered in a simple, non-invasive manner, has therapeutic potential in diabetes.

Deep characterization of a common D4Z4 variant identifies biallelic DUX4 expression as a modifier for disease penetrance in FSHD2.

Facioscapulohumeral muscular dystrophy is caused by incomplete repression of the transcription factor DUX4 in skeletal muscle as a consequence of D4Z4 macrosatellite repeat contraction in chromosome 4q35 (FSHD1) or variants in genes encoding D4Z4 chromatin repressors (FSHD2). A clinical hallmark of FSHD is variability in onset and progression suggesting the presence of disease modifiers. A well-known cis modifier is the polymorphic DUX4 polyadenylation signal (PAS) that defines FSHD permissive alleles: D4Z4 chromatin relaxation on non-permissive alleles which lack the DUX4-PAS cannot cause disease in the absence of stable DUX4 mRNA. We have explored the nature and relevance of a common variant of the major FSHD haplotype 4A161, which is defined by 1.6 kb size difference of the most distal D4Z4 repeat unit. While the short variant (4A161S) has been extensively studied, we demonstrate that the long variant (4A161L) is relatively common in the European population, is capable of expressing DUX4, but that DUX4 mRNA processing differs from 4A161S. While we do not find evidence for a difference in disease severity between FSHD carriers of an 4A161S or 4A161L allele, our study does uncover biallelic DUX4 expression in FSHD2 patients. Compared to control individuals, we observed an increased frequency of FSHD2 patients homozygous for disease permissive alleles, and who are thus capable of biallelic DUX4 expression, while SMCHD1 variant carriers with only one permissive allele were significantly more often asymptomatic. This suggests that biallelic DUX4 expression lowers the threshold for disease presentation and is a modifier for disease severity in FSHD2.

Smchd1 haploinsufficiency exacerbates the phenotype of a transgenic FSHD1 mouse model.

In humans, a copy of the DUX4 retrogene is located in each unit of the D4Z4 macrosatellite repeat that normally comprises 8-100 units. The D4Z4 repeat has heterochromatic features and does not express DUX4 in somatic cells. Individuals with facioscapulohumeral muscular dystrophy (FSHD) have a partial failure of somatic DUX4 repression resulting in the presence of DUX4 protein in sporadic muscle nuclei. Somatic DUX4 derepression is caused by contraction of the D4Z4 repeat to 1-10 units (FSHD1) or by heterozygous mutations in genes responsible for maintaining the D4Z4 chromatin structure in a repressive state (FSHD2). One of the FSHD2 genes is the structural maintenance of chromosomes hinge domain 1 (SMCHD1) gene. SMCHD1 mutations have also been identified in FSHD1; patients carrying a contracted D4Z4 repeat and a SMCHD1 mutation are more severely affected than relatives with only a contracted repeat or a SMCHD1 mutation. To evaluate the modifier role of SMCHD1, we crossbred mice carrying a contracted D4Z4 repeat (D4Z4-2.5 mice) with mice that are haploinsufficient for Smchd1 (Smchd1MommeD1 mice). D4Z4-2.5/Smchd1MommeD1 mice presented with a significantly reduced body weight and developed skin lesions. The same skin lesions, albeit in a milder form, were also observed in D4Z4-2.5 mice, suggesting that reduced Smchd1 levels aggravate disease in the D4Z4-2.5 mouse model. Our study emphasizes the evolutionary conservation of the SMCHD1-dependent epigenetic regulation of the D4Z4 repeat array and further suggests that the D4Z4-2.5/Smchd1MommeD1 mouse model may be used to unravel the function of DUX4 in non-muscle tissues like the skin.

BET bromodomain inhibitors and agonists of the beta-2 adrenergic receptor identified in screens for compounds that inhibit DUX4 expression in FSHD muscle cells.

BACKGROUND: Facioscapulohumeral dystrophy (FSHD) is a progressive muscle disease caused by mutations that lead to epigenetic derepression and inappropriate transcription of the double homeobox 4 (DUX4) gene in skeletal muscle. Drugs that enhance the repression of DUX4 and prevent its expression in skeletal muscle cells therefore represent candidate therapies for FSHD. METHODS: We screened an aggregated chemical library enriched for compounds with epigenetic activities and the Pharmakon 1600 library composed of compounds that have reached clinical testing to identify molecules that decrease DUX4 expression as monitored by the levels of DUX4 target genes in FSHD patient-derived skeletal muscle cell cultures. RESULTS: Our screens identified several classes of molecules that include inhibitors of the bromodomain and extra-terminal (BET) family of proteins and agonists of the beta-2 adrenergic receptor. Further studies showed that compounds from these two classes suppress the expression of DUX4 messenger RNA (mRNA) by blocking the activity of bromodomain-containing protein 4 (BRD4) or by increasing cyclic adenosine monophosphate (cAMP) levels, respectively. CONCLUSIONS: These data uncover pathways involved in the regulation of DUX4 expression in somatic cells, provide potential candidate classes of compounds for FSHD therapeutic development, and create an important opportunity for mechanistic studies that may uncover additional therapeutic targets.

Virtual unenhanced imaging of the liver with third-generation dual-source dual-energy CT and advanced modeled iterative reconstruction.

OBJECTIVES: To compare image quality and diagnostic accuracy for the detection of liver lesions of virtual unenhanced (VU) images based on third-generation dual-source dual- energy computed tomography (DECT) compared to conventional unenhanced (CU) images. METHODS: Thirty patients underwent triphasic abdominal CT consisting of single-energy CU (120kV, 147 ref.mAs) and dual-energy CT arterial and portal-venous phase acquisitions (100/Sn150kV, 180/90 ref.mAs). VU images were generated from arterial (AVU) and portal venous (PVU) phases. CU, AVU and PVU datasets were reconstructed. Quantitative image quality analysis was performed and two abdominal radiologists independently analyzed all datasets to evaluate image quality and identify liver lesions. Radiation dose was recorded and potential radiation dose reduction was estimated. RESULTS: Image quality was rated diagnostic in 100% of the VU datasets. The mean subjective image quality of the CU datasets was higher than that of VU images (p<0.0001). No significant difference was observed in the mean attenuation values of the liver parenchyma (p>0.99) and hypoattenuating liver lesions (p≥0.21) between CU, AVU and PVU. However, a significant reduction in the attenuation values of calcified lesions (p<0.0001), metallic clips (p<0.0001) and gallstones (p≤0.047) was observed in the AVU and PVU images compared with CU images. A total of 122 liver lesions were found in 25 patients. VU images were more sensitive than CU images for detection of small hypoattenuating liver lesions (≤1cm). However, CU images were more sensitive than VU for calcified liver lesions. The mean radiation dose reduction achievable by avoiding the unenhanced acquisition was 32.9%±1.1% (p<0.01). CONCLUSIONS: Third-generation DSCT VU images of the liver provide diagnostic image quality and improve small (≤1cm) liver lesion detection; however calcified liver lesions can be missed due to complete subtraction.

Model systems of DUX4 expression recapitulate the transcriptional profile of FSHD cells.

Facioscapulohumeral dystrophy (FSHD) is caused by the mis-expression of the double-homeodomain transcription factor DUX4 in skeletal muscle cells. Many different cell culture models have been developed to study the pathophysiology of FSHD, frequently based on endogenous expression of DUX4 in FSHD cells or by mis-expression of DUX4 in control human muscle cells. Although results generated using each model are generally consistent, differences have also been reported, making it unclear which model(s) faithfully recapitulate DUX4 and FSHD biology. In this study, we systematically compared RNA-seq data generated from three different models of FSHD­lentiviral-based DUX4 expression in myoblasts, doxycycline-inducible DUX4 in myoblasts, and differentiated human FSHD myocytes expressing endogenous DUX4­and show that the DUX4-associated gene expression signatures of each dataset are highly correlated (Pearson's correlation coefficient, r ∼ 0.75-0.85). The few robust differences were attributable to different states of cell differentiation and other differences in experimental design. Our study describes a model system for inducible DUX4 expression that enables reproducible and synchronized experiments and validates the fidelity and FSHD relevance of multiple distinct models of DUX4 expression.

DICER/AGO-dependent epigenetic silencing of D4Z4 repeats enhanced by exogenous siRNA suggests mechanisms and therapies for FSHD.

Facioscapulohumeral muscular dystrophy (FSHD) is caused by the aberrant expression of the DUX4 transcription factor in skeletal muscle. The DUX4 retrogene is encoded in the D4Z4 macrosatellite repeat array, and smaller array size or a mutation in the SMCHD1 gene results in inefficient epigenetic repression of DUX4 in skeletal muscle, causing FSHD1 and FSHD2, respectively. Previously we showed that the entire D4Z4 repeat is bi-directionally transcribed with the generation of small si- or miRNA-like fragments and suggested that these might suppress DUX4 expression through the endogenous RNAi pathway. Here we show that exogenous siRNA targeting the region upstream of the DUX4 transcription start site suppressed DUX4 mRNA expression and increased both H3K9 methylation and AGO2 recruitment. In contrast, similarly targeted MOE-gapmer antisense oligonucleotides that degrade RNA but do not engage the RNAi pathway did not repress DUX4 expression. In addition, knockdown of DICER or AGO2 using either siRNA or MOE-gapmer chemistries resulted in the induction of DUX4 expression in control muscle cells that normally do not express DUX4, indicating that the endogenous RNAi pathway is necessary to maintain repression of DUX4 in control muscle cells. Together these data demonstrate a role of the endogenous RNAi pathway in repeat-mediated epigenetic repression of the D4Z4 macrosatellite repeat, and show that enhancing the activity of this pathway by supplying exogenous siRNA oligonucleotides represents a potential therapeutic approach to silencing DUX4 in FSHD.

A feedback loop between nonsense-mediated decay and the retrogene DUX4 in facioscapulohumeral muscular dystrophy.

Facioscapulohumeral muscular dystrophy (FSHD) is a muscular dystrophy caused by inefficient epigenetic repression of the D4Z4 macrosatellite array and somatic expression of the DUX4 retrogene. DUX4 is a double homeobox transcription factor that is normally expressed in the testis and causes apoptosis and FSHD when misexpressed in skeletal muscle. The mechanism(s) of DUX4 toxicity in muscle is incompletely understood. We report that DUX4-triggered proteolytic degradation of UPF1, a central component of the nonsense-mediated decay (NMD) machinery, is associated with profound NMD inhibition, resulting in global accumulation of RNAs normally degraded as NMD substrates. DUX4 mRNA is itself degraded by NMD, such that inhibition of NMD by DUX4 protein stabilizes DUX4 mRNA through a double-negative feedback loop in FSHD muscle cells. This feedback loop illustrates an unexpected mode of autoregulatory behavior of a transcription factor, is consistent with 'bursts' of DUX4 expression in FSHD muscle, and has implications for FSHD pathogenesis.

DUX4-induced gene expression is the major molecular signature in FSHD skeletal muscle.

Facioscapulohumeral dystrophy (FSHD) is caused by decreased epigenetic repression of the D4Z4 macrosatellite array and recent studies have shown that this results in the expression of low levels of the DUX4 mRNA in skeletal muscle. Several other mechanisms have been suggested for FSHD pathophysiology and it remains unknown whether DUX4 expression can account for most of the molecular changes seen in FSHD. Since DUX4 is a transcription factor, we used RNA-seq to measure gene expression in muscle cells transduced with DUX4, and in muscle cells and biopsies from control and FSHD individuals. We show that DUX4 target gene expression is the major molecular signature in FSHD muscle together with a gene expression signature consistent with an immune cell infiltration. In addition, one unaffected individual without a known FSHD-causing mutation showed the expression of DUX4 target genes. This individual has a sibling with FSHD and also without a known FSHD-causing mutation, suggesting the presence of an unidentified modifier locus for DUX4 expression and FSHD. These findings demonstrate that the expression of DUX4 accounts for the majority of the gene expression changes in FSHD skeletal muscle together with an immune cell infiltration.

DUX4 binding to retroelements creates promoters that are active in FSHD muscle and testis.

The human double-homeodomain retrogene DUX4 is expressed in the testis and epigenetically repressed in somatic tissues. Facioscapulohumeral muscular dystrophy (FSHD) is caused by mutations that decrease the epigenetic repression of DUX4 in somatic tissues and result in mis-expression of this transcription factor in skeletal muscle. DUX4 binds sites in the human genome that contain a double-homeobox sequence motif, including sites in unique regions of the genome as well as many sites in repetitive elements. Using ChIP-seq and RNA-seq on myoblasts transduced with DUX4 we show that DUX4 binds and activates transcription of mammalian apparent LTR-retrotransposons (MaLRs), endogenous retrovirus (ERVL and ERVK) elements, and pericentromeric satellite HSATII sequences. Some DUX4-activated MaLR and ERV elements create novel promoters for genes, long non-coding RNAs, and antisense transcripts. Many of these novel transcripts are expressed in FSHD muscle cells but not control cells, and thus might contribute to FSHD pathology. For example, HEY1, a repressor of myogenesis, is activated by DUX4 through a MaLR promoter. DUX4-bound motifs, including those in repetitive elements, show evolutionary conservation and some repeat-initiated transcripts are expressed in healthy testis, the normal expression site of DUX4, but more rarely in other somatic tissues. Testis expression patterns are known to have evolved rapidly in mammals, but the mechanisms behind this rapid change have not yet been identified: our results suggest that mobilization of MaLR and ERV elements during mammalian evolution altered germline gene expression patterns through transcriptional activation by DUX4. Our findings demonstrate a role for DUX4 and repetitive elements in mammalian germline evolution and in FSHD muscular dystrophy.

Intrinsic epigenetic regulation of the D4Z4 macrosatellite repeat in a transgenic mouse model for FSHD.

Facioscapulohumeral dystrophy (FSHD) is a progressive muscular dystrophy caused by decreased epigenetic repression of the D4Z4 macrosatellite repeats and ectopic expression of DUX4, a retrogene encoding a germline transcription factor encoded in each repeat. Unaffected individuals generally have more than 10 repeats arrayed in the subtelomeric region of chromosome 4, whereas the most common form of FSHD (FSHD1) is caused by a contraction of the array to fewer than 10 repeats, associated with decreased epigenetic repression and variegated expression of DUX4 in skeletal muscle. We have generated transgenic mice carrying D4Z4 arrays from an FSHD1 allele and from a control allele. These mice recapitulate important epigenetic and DUX4 expression attributes seen in patients and controls, respectively, including high DUX4 expression levels in the germline, (incomplete) epigenetic repression in somatic tissue, and FSHD-specific variegated DUX4 expression in sporadic muscle nuclei associated with D4Z4 chromatin relaxation. In addition we show that DUX4 is able to activate similar functional gene groups in mouse muscle cells as it does in human muscle cells. These transgenic mice therefore represent a valuable animal model for FSHD and will be a useful resource to study the molecular mechanisms underlying FSHD and to test new therapeutic intervention strategies.

Inhibition of CD26/DPP-IV enhances donor muscle cell engraftment and stimulates sustained donor cell proliferation.

BACKGROUND: Transplantation of myogenic stem cells possesses great potential for long-term repair of dystrophic muscle. In murine-to-murine transplantation experiments, CXCR4 expression marks a population of adult murine satellite cells with robust engraftment potential in mdx mice, and CXCR4-positive murine muscle-derived SP cells home more effectively to dystrophic muscle after intra-arterial delivery in mdx5cv mice. Together, these data suggest that CXCR4 plays an important role in donor cell engraftment. Therefore, we sought to translate these results to a clinically relevant canine-to-canine allogeneic transplant model for Duchenne muscular dystrophy (DMD) and determine if CXCR4 is important for donor cell engraftment. METHODS: In this study, we used a canine-to-murine xenotransplantation model to quantitatively compare canine muscle cell engraftment, and test the most effective cell population and modulating factor in a canine model of DMD using allogeneic transplantation experiments. RESULTS: We show that CXCR4 expressing cells are important for donor muscle cell engraftment, yet FACS sorted CXCR4-positive cells display decreased engraftment efficiency. However, diprotin A, a positive modulator of CXCR4-SDF-1 binding, significantly enhanced engraftment and stimulated sustained proliferation of donor cells in vivo. Furthermore, the canine-to-murine xenotransplantation model accurately predicted results in canine-to-canine muscle cell transplantation. CONCLUSIONS: Therefore, these results establish the efficacy of diprotin A in stimulating muscle cell engraftment, and highlight the pre-clinical utility of a xenotransplantation model in assessing the relative efficacy of muscle stem cell populations.

DUX4 activates germline genes, retroelements, and immune mediators: implications for facioscapulohumeral dystrophy.

Facioscapulohumeral dystrophy (FSHD) is one of the most common inherited muscular dystrophies. The causative gene remains controversial and the mechanism of pathophysiology unknown. Here we identify genes associated with germline and early stem cell development as targets of the DUX4 transcription factor, a leading candidate gene for FSHD. The genes regulated by DUX4 are reliably detected in FSHD muscle but not in controls, providing direct support for the model that misexpression of DUX4 is a causal factor for FSHD. Additionally, we show that DUX4 binds and activates LTR elements from a class of MaLR endogenous primate retrotransposons and suppresses the innate immune response to viral infection, at least in part through the activation of DEFB103, a human defensin that can inhibit muscle differentiation. These findings suggest specific mechanisms of FSHD pathology and identify candidate biomarkers for disease diagnosis and progression.

Facioscapulohumeral dystrophy: incomplete suppression of a retrotransposed gene.

Each unit of the D4Z4 macrosatellite repeat contains a retrotransposed gene encoding the DUX4 double-homeobox transcription factor. Facioscapulohumeral dystrophy (FSHD) is caused by deletion of a subset of the D4Z4 units in the subtelomeric region of chromosome 4. Although it has been reported that the deletion of D4Z4 units induces the pathological expression of DUX4 mRNA, the association of DUX4 mRNA expression with FSHD has not been rigorously investigated, nor has any human tissue been identified that normally expresses DUX4 mRNA or protein. We show that FSHD muscle expresses a different splice form of DUX4 mRNA compared to control muscle. Control muscle produces low amounts of a splice form of DUX4 encoding only the amino-terminal portion of DUX4. FSHD muscle produces low amounts of a DUX4 mRNA that encodes the full-length DUX4 protein. The low abundance of full-length DUX4 mRNA in FSHD muscle cells represents a small subset of nuclei producing a relatively high abundance of DUX4 mRNA and protein. In contrast to control skeletal muscle and most other somatic tissues, full-length DUX4 transcript and protein is expressed at relatively abundant levels in human testis, most likely in the germ-line cells. Induced pluripotent (iPS) cells also express full-length DUX4 and differentiation of control iPS cells to embryoid bodies suppresses expression of full-length DUX4, whereas expression of full-length DUX4 persists in differentiated FSHD iPS cells. Together, these findings indicate that full-length DUX4 is normally expressed at specific developmental stages and is suppressed in most somatic tissues. The contraction of the D4Z4 repeat in FSHD results in a less efficient suppression of the full-length DUX4 mRNA in skeletal muscle cells. Therefore, FSHD represents the first human disease to be associated with the incomplete developmental silencing of a retrogene array normally expressed early in development.

A unifying genetic model for facioscapulohumeral muscular dystrophy.

Facioscapulohumeral muscular dystrophy (FSHD) is a common form of muscular dystrophy in adults that is foremost characterized by progressive wasting of muscles in the upper body. FSHD is associated with contraction of D4Z4 macrosatellite repeats on chromosome 4q35, but this contraction is pathogenic only in certain "permissive" chromosomal backgrounds. Here, we show that FSHD patients carry specific single-nucleotide polymorphisms in the chromosomal region distal to the last D4Z4 repeat. This FSHD-predisposing configuration creates a canonical polyadenylation signal for transcripts derived from DUX4, a double homeobox gene of unknown function that straddles the last repeat unit and the adjacent sequence. Transfection studies revealed that DUX4 transcripts are efficiently polyadenylated and are more stable when expressed from permissive chromosomes. These findings suggest that FSHD arises through a toxic gain of function attributable to the stabilized distal DUX4 transcript.