Single-cell spatial transcriptomics reveals a dystrophic trajectory following a developmental bifurcation of myoblast cell fates in facioscapulohumeral muscular dystrophy.

Facioscapulohumeral muscular dystrophy (FSHD) is linked to abnormal derepression of the transcription activator DUX4. This effect is localized to a low percentage of cells, requiring single-cell analysis. However, single-cell/nucleus RNA-seq cannot fully capture the transcriptome of multinucleated large myotubes. To circumvent these issues, we use multiplexed error-robust fluorescent in situ hybridization (MERFISH) spatial transcriptomics that allows profiling of RNA transcripts at a subcellular resolution. We simultaneously examined spatial distributions of 140 genes, including 24 direct DUX4 targets, in in vitro differentiated myotubes and unfused mononuclear cells (MNCs) of control, isogenic D4Z4 contraction mutant and FSHD patient samples, as well as the individual nuclei within them. We find myocyte nuclei segregate into two clusters defined by the expression of DUX4 target genes, which is exclusively found in patient/mutant nuclei, whereas MNCs cluster based on developmental states. Patient/mutant myotubes are found in "FSHD-hi" and "FSHD-lo" states with the former signified by high DUX4 target expression and decreased muscle gene expression. Pseudotime analyses reveal a clear bifurcation of myoblast differentiation into control and FSHD-hi myotube branches, with variable numbers of DUX4 target-expressing nuclei found in multinucleated FSHD-hi myotubes. Gene coexpression modules related to extracellular matrix and stress gene ontologies are significantly altered in patient/mutant myotubes compared with the control. We also identify distinct subpathways within the DUX4 gene network that may differentially contribute to the disease transcriptomic phenotype. Taken together, our MERFISH-based study provides effective gene network profiling of multinucleated cells and identifies FSHD-induced transcriptomic alterations during myoblast differentiation.

A long ncRNA links copy number variation to a polycomb/trithorax epigenetic switch in FSHD muscular dystrophy.

Repetitive sequences account for more than 50% of the human genome. Facioscapulohumeral muscular dystrophy (FSHD) is an autosomal-dominant disease associated with reduction in the copy number of the D4Z4 repeat mapping to 4q35. By an unknown mechanism, D4Z4 deletion causes an epigenetic switch leading to de-repression of 4q35 genes. Here we show that the Polycomb group of epigenetic repressors targets D4Z4 in healthy subjects and that D4Z4 deletion is associated with reduced Polycomb silencing in FSHD patients. We identify DBE-T, a chromatin-associated noncoding RNA produced selectively in FSHD patients that coordinates de-repression of 4q35 genes. DBE-T recruits the Trithorax group protein Ash1L to the FSHD locus, driving histone H3 lysine 36 dimethylation, chromatin remodeling, and 4q35 gene transcription. This study provides insights into the biological function of repetitive sequences in regulating gene expression and shows how mutations of such elements can influence the progression of a human genetic disease.

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.

FSHD muscle shows perturbation in fibroadipogenic progenitor cells, mitochondrial function and alternative splicing independently of inflammation.

Facioscapulohumeral muscular dystrophy (FSHD) is a prevalent, incurable myopathy. FSHD is highly heterogeneous, with patients following a variety of clinical trajectories, complicating clinical trials. Skeletal muscle in FSHD undergoes fibrosis and fatty replacement that can be accelerated by inflammation, adding to heterogeneity. Well controlled molecular studies are thus essential to both categorize FSHD patients into distinct subtypes and understand pathomechanisms. Here, we further analyzed RNA-sequencing data from 24 FSHD patients, each of whom donated a biopsy from both a non-inflamed (TIRM-) and inflamed (TIRM+) muscle, and 15 FSHD patients who donated peripheral blood mononucleated cells (PBMCs), alongside non-affected control individuals. Differential gene expression analysis identified suppression of mitochondrial biogenesis and up-regulation of fibroadipogenic progenitor (FAP) gene expression in FSHD muscle, which was particularly marked on inflamed samples. PBMCs demonstrated suppression of antigen presentation in FSHD. Gene expression deconvolution revealed FAP expansion as a consistent feature of FSHD muscle, via meta-analysis of 7 independent transcriptomic datasets. Clustering of muscle biopsies separated patients in an unbiased manner into clinically mild and severe subtypes, independently of known disease modifiers (age, sex, D4Z4 repeat length). Lastly, the first genome-wide analysis of alternative splicing in FSHD muscle revealed perturbation of autophagy, BMP2 and HMGB1 signalling. Overall, our findings reveal molecular subtypes of FSHD with clinical relevance and identify novel pathomechanisms for this highly heterogeneous condition.

snRNA-seq analysis in multinucleated myogenic FSHD cells identifies heterogeneous FSHD transcriptome signatures associated with embryonic-like program activation and oxidative stress-induced apoptosis.

The sporadic nature of DUX4 expression in FSHD muscle challenges comparative transcriptome analyses between FSHD and control samples. A variety of DUX4 and FSHD-associated transcriptional changes have been identified, but bulk RNA-seq strategies prohibit comprehensive analysis of their spatiotemporal relation, interdependence and role in the disease process. In this study, we used single-nucleus RNA-sequencing of nuclei isolated from patient- and control-derived multinucleated primary myotubes to investigate the cellular heterogeneity in FSHD. Taking advantage of the increased resolution in snRNA-sequencing of fully differentiated myotubes, two distinct populations of DUX4-affected nuclei could be defined by their transcriptional profiles. Our data provides insights into the differences between these two populations and suggests heterogeneity in two well-known FSHD-associated transcriptional aberrations: increased oxidative stress and inhibition of myogenic differentiation. Additionally, we provide evidence that DUX4-affected nuclei share transcriptome features with early embryonic cells beyond the well-described cleavage stage, progressing into the 8-cell and blastocyst stages. Altogether, our data suggests that the FSHD transcriptional profile is defined by a mixture of individual and sometimes mutually exclusive DUX4-induced responses and cellular state-dependent downstream effects.

Muscle eosinophilia is a hallmark of chronic disease in facioscapulohumeral muscular dystrophy.

Facioscapulohumeral muscular dystrophy (FSHD) is a progressive myopathy caused by the aberrant increased expression of the DUX4 retrogene in skeletal muscle cells. The DUX4 gene encodes a transcription factor that functions in zygotic genome activation and then is silenced in most adult somatic tissues. DUX4 expression in FSHD disrupts normal muscle cell function; however, the downstream pathogenic mechanisms are still unclear. Histologically, FSHD affected muscles show a characteristic dystrophic phenotype that is often accompanied by a pronounced immune cell infiltration, but the role of the immune system in FSHD is not understood. Previously, we used ACTA1;FLExDUX4 FSHD-like mouse models varying in severity as discovery tools to identify increased Interleukin 6 and microRNA-206 levels as serum biomarkers for FSHD disease severity. In this study, we use the ACTA1;FLExDUX4 chronic FSHD-like mouse model to provide insight into the immune response to DUX4 expression in skeletal muscles. We demonstrate that these FSHD-like muscles are enriched with the chemoattractant eotaxin and the cytotoxic eosinophil peroxidase, and exhibit muscle eosinophilia. We further identified muscle fibers with positive staining for eosinophil peroxidase in human FSHD muscle. Our data supports that skeletal muscle eosinophilia is a hallmark of FSHD pathology.

Deciphering D4Z4 CpG methylation gradients in fascioscapulohumeral muscular dystrophy using nanopore sequencing.

Fascioscapulohumeral muscular dystrophy (FSHD) is caused by a unique genetic mechanism that relies on contraction and hypomethylation of the D4Z4 macrosatellite array on the Chromosome 4q telomere allowing ectopic expression of the DUX4 gene in skeletal muscle. Genetic analysis is difficult because of the large size and repetitive nature of the array, a nearly identical array on the 10q telomere, and the presence of divergent D4Z4 arrays scattered throughout the genome. Here, we combine nanopore long-read sequencing with Cas9-targeted enrichment of 4q and 10q D4Z4 arrays for comprehensive genetic analysis including determination of the length of the 4q and 10q D4Z4 arrays with base-pair resolution. In the same assay, we differentiate 4q from 10q telomeric sequences, determine A/B haplotype, identify paralogous D4Z4 sequences elsewhere in the genome, and estimate methylation for all CpGs in the array. Asymmetric, length-dependent methylation gradients were observed in the 4q and 10q D4Z4 arrays that reach a hypermethylation point at approximately 10 D4Z4 repeat units, consistent with the known threshold of pathogenic D4Z4 contractions. High resolution analysis of individual D4Z4 repeat methylation revealed areas of low methylation near the CTCF/insulator region and areas of high methylation immediately preceding the DUX4 transcriptional start site. Within the DUX4 exons, we observed a waxing/waning methylation pattern with a 180-nucleotide periodicity, consistent with phased nucleosomes. Targeted nanopore sequencing complements recently developed molecular combing and optical mapping approaches to genetic analysis for FSHD by adding precision of the length measurement, base-pair resolution sequencing, and quantitative methylation analysis.

The transcription factor DUX4 orchestrates translational reprogramming by broadly suppressing translation efficiency and promoting expression of DUX4-induced mRNAs.

Translational control is critical for cell fate transitions during development, lineage specification, and tumorigenesis. Here, we show that the transcription factor double homeobox protein 4 (DUX4), and its previously characterized transcriptional program, broadly regulates translation to change the cellular proteome. DUX4 is a key regulator of zygotic genome activation in human embryos, whereas misexpression of DUX4 causes facioscapulohumeral muscular dystrophy (FSHD) and is associated with MHC-I suppression and immune evasion in cancer. We report that translation initiation and elongation factors are disrupted downstream of DUX4 expression in human myoblasts. Genome-wide translation profiling identified mRNAs susceptible to DUX4-induced translation inhibition, including those encoding antigen presentation factors and muscle lineage proteins, while DUX4-induced mRNAs were robustly translated. Endogenous expression of DUX4 in human FSHD myotubes and cancer cell lines also correlated with reduced protein synthesis and MHC-I presentation. Our findings reveal that DUX4 orchestrates cell state conversion by suppressing the cellular proteome while maintaining translation of DUX4-induced mRNAs to promote an early developmental program.

SMCHD1 activates the expression of genes required for the expansion of human myoblasts.

SMCHD1 is an epigenetic regulatory protein known to modulate the targeted repression of large chromatin domains. Diminished SMCHD1 function in muscle fibers causes Facioscapulohumeral Muscular Dystrophy (FSHD2) through derepression of the D4Z4 chromatin domain, an event which permits the aberrant expression of the disease-causing gene DUX4. Given that SMCHD1 plays a broader role in establishing the cellular epigenome, we examined whether loss of SMCHD1 function might affect muscle homeostasis through additional mechanisms. Here we show that acute depletion of SMCHD1 results in a DUX4-independent defect in myoblast proliferation. Genomic and transcriptomic experiments determined that SMCHD1 associates with enhancers of genes controlling cell cycle to activate their expression. Amongst these cell cycle regulatory genes, we identified LAP2 as a key target of SMCHD1 required for the expansion of myoblasts, where the ectopic expression of LAP2 rescues the proliferation defect of SMCHD1-depleted cells. Thus, the epigenetic regulator SMCHD1 can play the role of a transcriptional co-activator for maintaining the expression of genes required for muscle progenitor expansion. This DUX4-independent role for SMCHD1 in myoblasts suggests that the pathology of FSHD2 may be a consequence of defective muscle regeneration in addition to the muscle wasting caused by spurious DUX4 expression.

Human DUX4 and porcine DUXC activate similar early embryonic programs in pig muscle cells: implications for preclinical models of FSHD.

Human DUX4 and its mouse ortholog Dux are normally expressed in the early embryo-the 4-cell or 2-cell cleavage stage embryo, respectively-and activate a portion of the first wave of zygotic gene expression. DUX4 is epigenetically suppressed in nearly all somatic tissue, whereas facioscapulohumeral dystrophy (FSHD)-causing mutations result in its aberrant expression in skeletal muscle, transcriptional activation of the early embryonic program and subsequent muscle pathology. Although DUX4 and Dux both activate an early totipotent transcriptional program, divergence of their DNA binding domains limits the use of DUX4 expressed in mice as a preclinical model for FSHD. In this study, we identify the porcine DUXC messenger ribonucleic acid expressed in early development and show that both pig DUXC and human DUX4 robustly activate a highly similar early embryonic program in pig muscle cells. These results support further investigation of pig preclinical models for FSHD.

Autosomal dominant in cis D4Z4 repeat array duplication alleles in facioscapulohumeral dystrophy.

Facioscapulohumeral dystrophy (FSHD) has a unique genetic aetiology resulting in partial chromatin relaxation of the D4Z4 macrosatellite repeat array on 4qter. This D4Z4 chromatin relaxation facilitates inappropriate expression of the transcription factor DUX4 in skeletal muscle. DUX4 is encoded by a retrogene that is embedded within the distal region of the D4Z4 repeat array. In the European population, the D4Z4 repeat array is usually organized in a single array that ranges between 8 and 100 units. D4Z4 chromatin relaxation and DUX4 derepression in FSHD is most often caused by repeat array contraction to 1-10 units (FSHD1) or by a digenic mechanism requiring pathogenic variants in a D4Z4 chromatin repressor like SMCHD1, combined with a repeat array between 8 and 20 units (FSHD2). With a prevalence of 1.5% in the European population, in cis duplications of the D4Z4 repeat array, where two adjacent D4Z4 arrays are interrupted by a spacer sequence, are relatively common but their relationship to FSHD is not well understood. In cis duplication alleles were shown to be pathogenic in FSHD2 patients; however, there is inconsistent evidence for the necessity of an SMCHD1 mutation for disease development. To explore the pathogenic nature of these alleles we compared in cis duplication alleles in FSHD patients with or without pathogenic SMCHD1 variant. For both groups we showed duplication-allele-specific DUX4 expression. We studied these alleles in detail using pulsed-field gel electrophoresis-based Southern blotting and molecular combing, emphasizing the challenges in the characterization of these rearrangements. Nanopore sequencing was instrumental to study the composition and methylation of the duplicated D4Z4 repeat arrays and to identify the breakpoints and the spacer sequence between the arrays. By comparing the composition of the D4Z4 repeat array of in cis duplication alleles in both groups, we found that specific combinations of proximal and distal repeat array sizes determine their pathogenicity. Supported by our algorithm to predict pathogenicity, diagnostic laboratories should now be furnished to accurately interpret these in cis D4Z4 repeat array duplications, alleles that can easily be missed in routine settings.

Facioscapulohumeral Muscular Dystrophy is Associated With Altered Myoblast Proteome Dynamics.

Proteomic studies in facioscapulohumeral muscular dystrophy (FSHD) could offer new insight into disease mechanisms underpinned by post-transcriptional processes. We used stable isotope (deuterium oxide; D2O) labeling and peptide mass spectrometry to investigate the abundance and turnover rates of proteins in cultured muscle cells from two individuals affected by FSHD and their unaffected siblings (UASb). We measured the abundance of 4420 proteins and the turnover rate of 2324 proteins in each (n = 4) myoblast sample. FSHD myoblasts exhibited a greater abundance but slower turnover rate of subunits of mitochondrial respiratory complexes and mitochondrial ribosomal proteins, which may indicate an accumulation of "older" less viable mitochondrial proteins in myoblasts from individuals affected by FSHD. Treatment with a 2'-O-methoxyethyl modified antisense oligonucleotide targeting exon 3 of the double homeobox 4 (DUX4) transcript tended to reverse mitochondrial protein dysregulation in FSHD myoblasts, indicating the effect on mitochondrial proteins may be a DUX4-dependent mechanism. Our results highlight the importance of post-transcriptional processes and protein turnover in FSHD pathology and provide a resource for the FSHD research community to explore this burgeoning aspect of FSHD.

In skeletal muscle and neural crest cells, SMCHD1 regulates biological pathways relevant for Bosma syndrome and facioscapulohumeral dystrophy phenotype.

Many genetic syndromes are linked to mutations in genes encoding factors that guide chromatin organization. Among them, several distinct rare genetic diseases are linked to mutations in SMCHD1 that encodes the structural maintenance of chromosomes flexible hinge domain containing 1 chromatin-associated factor. In humans, its function as well as the impact of its mutations remains poorly defined. To fill this gap, we determined the episignature associated with heterozygous SMCHD1 variants in primary cells and cell lineages derived from induced pluripotent stem cells for Bosma arhinia and microphthalmia syndrome (BAMS) and type 2 facioscapulohumeral dystrophy (FSHD2). In human tissues, SMCHD1 regulates the distribution of methylated CpGs, H3K27 trimethylation and CTCF at repressed chromatin but also at euchromatin. Based on the exploration of tissues affected either in FSHD or in BAMS, i.e. skeletal muscle fibers and neural crest stem cells, respectively, our results emphasize multiple functions for SMCHD1, in chromatin compaction, chromatin insulation and gene regulation with variable targets or phenotypical outcomes. We concluded that in rare genetic diseases, SMCHD1 variants impact gene expression in two ways: (i) by changing the chromatin context at a number of euchromatin loci or (ii) by directly regulating some loci encoding master transcription factors required for cell fate determination and tissue differentiation.

WDR5 is required for DUX4 expression and its pathological effects in FSHD muscular dystrophy.

Facioscapulohumeral muscular dystrophy (FSHD) is one of the most prevalent neuromuscular disorders. The disease is linked to copy number reduction and/or epigenetic alterations of the D4Z4 macrosatellite on chromosome 4q35 and associated with aberrant gain of expression of the transcription factor DUX4, which triggers a pro-apoptotic transcriptional program leading to muscle wasting. As today, no cure or therapeutic option is available to FSHD patients. Given its centrality in FSHD, blocking DUX4 expression with small molecule drugs is an attractive option. We previously showed that the long non protein-coding RNA DBE-T is required for aberrant DUX4 expression in FSHD. Using affinity purification followed by proteomics, here we identified the chromatin remodeling protein WDR5 as a novel DBE-T interactor and a key player required for the biological activity of the lncRNA. We found that WDR5 is required for the expression of DUX4 and its targets in primary FSHD muscle cells. Moreover, targeting WDR5 rescues both cell viability and myogenic differentiation of FSHD patient cells. Notably, comparable results were obtained by pharmacological inhibition of WDR5. Importantly, WDR5 targeting was safe to healthy donor muscle cells. Our results support a pivotal role of WDR5 in the activation of DUX4 expression identifying a druggable target for an innovative therapeutic approach for FSHD.

Progesterone may be a regulator and B12 could be an indicator of the proximal D4Z4 repeat methylation status on 4q35ter.

Facioscapulohumeral dystrophy (FSHD) has a hypomethylation-related epigenetic background and exhibits a different course in male and female patients. The differences between males and females have been linked to the levels of sex hormones. This study is the first to investigate the possible effect of these hormones on methylation status. We hypothesized that the levels of sex-related hormones, estradiol, testosterone, progesterone, and prolactin might be associated with the methylation status of the proximal part of the D4Z4. We also investigated the effect of fT3, folic acid, and vitamin B12 levels. We collected blood from 28 FSHD patients and 28 controls. DNA was extracted from each individual for bisulfite methylation analysis and serum was separated for biochemical analysis of estradiol, testosterone, progesterone, prolactin, fT3, folic acid, and B12 analysis. Methylation analysis was specified to the DR1, 5P regions and the proximal region covering both DR1 and 5P. Methylation levels were compared between FSHD patients and controls. The correlation of methylation levels with estradiol, testosterone, progesterone, prolactin, fT3, folic acid, and B12 was investigated. We found that the 5P region and the proximal region were significantly hypomethylated in FSHD patients compared to the controls, but not the DR1 region. Male patients exhibited a significant reduction in DNA methylation compared to male controls. Older FSHD patients exhibited a notable decrease in fT3 levels and hypomethylation of the 5P region. Analyses of each CpG revealed seven hypomethylated positions that were significantly different from the control group. Two of the positions demonstrated a correlation with progesterone in the control group. With the exception of one position, the methylation levels were inversely correlated with vitamin B12 in FSHD patients. The results of our study indicate that the methylation of the proximal D4Z4 region, particularly at specific positions, may be associated with progesterone. In addition, vitamin B12 may be an indicator of hypomethylation. We suggest that examining position-specific methylations may be a useful approach for the development of epigenetic treatment modalities.

High-resolution breakpoint junction mapping of proximally extended D4Z4 deletions in FSHD1 reveals evidence for a founder effect.

Facioscapulohumeral muscular dystrophy (FSHD) is an inherited myopathy clinically characterized by weakness in the facial, shoulder girdle and upper a muscles. FSHD is caused by chromatin relaxation of the D4Z4 macrosatellite repeat, mostly by a repeat contraction, facilitating ectopic expression of DUX4 in skeletal muscle. Genetic diagnosis for FSHD is generally based on the sizing and haplotyping of the D4Z4 repeat on chromosome 4 by Southern blotting (SB), molecular combing or single-molecule optical mapping, which is usually straight forward but can be complicated by atypical rearrangements of the D4Z4 repeat. One of these rearrangements is a D4Z4 proximally extended deletion (DPED) allele, where not only the D4Z4 repeat is partially deleted, but also sequences immediately proximal to the repeat are lost, which can impede accurate diagnosis in all genetic methods. Previously, we identified several DPED alleles in FSHD and estimated the size of the proximal deletions by a complex pulsed-field gel electrophoresis and SB strategy. Here, using the next-generation sequencing, we have defined the breakpoint junctions of these DPED alleles at the base pair resolution in 12 FSHD families and 4 control individuals facilitating a PCR-based diagnosis of these DPED alleles. Our resultsshow that half of the DPED alleles are derivates of an ancient founder allele. For some DPED alleles, we found that genetic elements are deleted such as DUX4c, FRG2, DBE-T and myogenic enhancers necessitating re-evaluation of their role in FSHD pathogenesis.

Elevated plasma complement components in facioscapulohumeral dystrophy.

Advances in understanding the pathophysiology of facioscapulohumeral dystrophy (FSHD) have led to several therapeutic approaches entering clinical trials and an increased need to develop biomarkers of disease activity and progression. Multiple prior studies have shown early elevation of RNAs encoding components of the complement pathways and relatively widespread activated complement complexes by immunodetection in FSHD muscle. The current study tested plasma from two independent cohorts of FSHD and control subjects and found elevated complement components in both FSHD cohorts. Combining subjects from both cohorts identified complement factors that best distinguished FSHD and controls. Within the FSHD group, a subset of subjects showed elevation in multiple complement components. Together these findings suggest the need for future studies to determine whether measurements of complement activation can be used as a non-invasive measurement of FSHD disease activity, progression and/or response to therapies. In addition, with the ongoing expansion of complement therapeutic approaches, consideration for precision-based targeting of this pathway is appropriate.

Canine DUXC: implications for DUX4 retrotransposition and preclinical models of FSHD.

Mis-expression of DUX4 in skeletal muscle causes facioscapulohumeral muscular dystrophy (FSHD). Human DUX4 and mouse Dux are retrogenes derived from retrotransposition of the mRNA from the parental DUXC gene. Primates and rodents have lost the parental DUXC gene, and it is unknown whether DUXC had a similar role in driving an early pluripotent transcriptional program. Dogs and other Laurasiatherians have retained DUXC, providing an opportunity to determine the functional similarity to the retrotransposed DUX4 and Dux. Here, we identify the expression of two isoforms of DUXC mRNA in canine testis tissues: one encoding the canonical double homeodomain protein (DUXC), similar to DUX4/Dux, and a second that includes an in-frame alternative exon that disrupts the conserved amino acid sequence of the first homeodomain (DUXC-ALT). The expression of DUXC in canine cells induces a pluripotent program similar to DUX4 and Dux and induces the expression of a similar set of retrotransposons of the ERV/MaLR and LINE-1 families, as well as pericentromeric satellite repeats; whereas DUXC-ALT did not robustly activate gene expression in these assays. Important for preclinical models of FSHD, human DUX4 and canine DUXC show higher conservation of their homeodomains and corresponding binding motifs compared with the conservation between human DUX4 and mouse Dux, and human DUX4 activates a highly similar transcriptional program in canine cells. Together, these findings show that retrotransposition resulted in the loss of an alternatively spliced isoform and that DUXC containing mammals might be good candidates for certain preclinical models ofFSHD.

Late-onset myopathies.

PURPOSE OF REVIEW: Late-onset myopathies are defined as muscle diseases that begin after the age of 50 years. Some myopathies present classically in the elderly, whereas others may have a variable age of onset, including late-onset presentation. The purpose of this review is to summarize and comment on the most recent evidence regarding the main diagnosis of late-onset myopathies focusing on genetic causes. RECENT FINDINGS: Although late-onset myopathies (LOM) are expected to be predominantly acquired myopathies, some common genetic myopathies, such as facioscapulohumeral muscular dystrophy (FSHD), can present late in life, usually with an atypical presentation. In addition, metabolic myopathies, which are classically early-onset diseases, are also diagnoses to be considered, particularly as they may be treatable. Late-onset multiple acyl-CoA dehydrogenase deficiency (MADD) has recently been identified as a cause of subacute LOM with a dramatic response to riboflavin supplementation. SUMMARY: Inclusion body myositis is the most frequent of all LOM. Myotonic dystrophy type 2, FSHD and oculopharyngeal muscular dystrophy are the most frequent causes of genetic LOM. We summarize the major differential diagnoses and the clinical features on clinical examination that are suggestive of a genetic diagnosis to provide a diagnostic approach.

Comprehensive genetic analysis of facioscapulohumeral muscular dystrophy by Nanopore long-read whole-genome sequencing.

BACKGROUND: Facioscapulohumeral muscular dystrophy (FSHD) is a high-prevalence autosomal dominant neuromuscular disease characterized by significant clinical and genetic heterogeneity. Genetic diagnosis of FSHD remains a challenge because it cannot be detected by standard sequencing methods and requires a complex diagnosis workflow. METHODS: We developed a comprehensive genetic FSHD detection method based on Oxford Nanopore Technologies (ONT) whole-genome sequencing. Using a case-control design, we applied this procedure to 29 samples and compared the results with those from optical genome mapping (OGM), bisulfite sequencing (BSS), and whole-exome sequencing (WES). RESULTS: Using our ONT-based method, we identified 59 haplotypes (35 4qA and 24 4qB) among the 29 samples (including a mosaic sample), as well as the number of D4Z4 repeat units (RUs). The pathogenetic D4Z4 RU contraction identified by our ONT-based method showed 100% concordance with OGM results. The methylation levels of the most distal D4Z4 RU and the double homeobox 4 gene (DUX4) detected by ONT sequencing are highly consistent with the BSS results and showed excellent diagnostic efficiency. Additionally, our ONT-based method provided an independent methylation profile analysis of two permissive 4qA alleles, reflecting a more accurate scenario than traditional BSS. The ONT-based method detected 17 variations in three FSHD2-related genes from nine samples, showing 100% concordance with WES. CONCLUSIONS: Our ONT-based FSHD detection method is a comprehensive method for identifying pathogenetic D4Z4 RU contractions, methylation level alterations, allele-specific methylation of two 4qA haplotypes, and variations in FSHD2-related genes, which will all greatly improve genetic testing for FSHD.