Single-nucleus RNA-seq identifies divergent populations of FSHD2 myotube nuclei.

FSHD is characterized by the misexpression of DUX4 in skeletal muscle. Although DUX4 upregulation is thought to be the pathogenic cause of FSHD, DUX4 is lowly expressed in patient samples, and analysis of the consequences of DUX4 expression has largely relied on artificial overexpression. To better understand the native expression profile of DUX4 and its targets, we performed bulk RNA-seq on a 6-day differentiation time-course in primary FSHD2 patient myoblasts. We identify a set of 54 genes upregulated in FSHD2 cells, termed FSHD-induced genes. Using single-cell and single-nucleus RNA-seq on myoblasts and differentiated myotubes, respectively, we captured, for the first time, DUX4 expressed at the single-nucleus level in a native state. We identified two populations of FSHD myotube nuclei based on low or high enrichment of DUX4 and FSHD-induced genes ("FSHD-Lo" and "FSHD Hi", respectively). FSHD-Hi myotube nuclei coexpress multiple DUX4 target genes including DUXA, LEUTX and ZSCAN4, and also upregulate cell cycle-related genes with significant enrichment of E2F target genes and p53 signaling activation. We found more FSHD-Hi nuclei than DUX4-positive nuclei, and confirmed with in situ RNA/protein detection that DUX4 transcribed in only one or two nuclei is sufficient for DUX4 protein to activate target genes across multiple nuclei within the same myotube. DUXA (the DUX4 paralog) is more widely expressed than DUX4, and depletion of DUXA suppressed the expression of LEUTX and ZSCAN4 in late, but not early, differentiation. The results suggest that the DUXA can take over the role of DUX4 to maintain target gene expression. These results provide a possible explanation as to why it is easier to detect DUX4 target genes than DUX4 itself in patient cells and raise the possibility of a self-sustaining network of gene dysregulation triggered by the limited DUX4 expression

A checklist for clinical trials in rare disease: Obstacles and anticipatory actions-lessons learned from the FOR-DMD trial

Background: Trials in rare diseases have many challenges, among which are the need to set up multiple sites in different countries to achieve recruitment targets and the divergent landscape of clinical trial regulations in those countries. Over the past years, there have been initiatives to facilitate the process of international study set-up, but the fruits of these deliberations require time to be operationally in place. FOR-DMD (Finding the Optimum Steroid Regimen for Duchenne Muscular Dystrophy) is an academic-led clinical trial which aims to find the optimum steroid regimen for Duchenne muscular dystrophy, funded by the National Institutes of Health (NIH) for 5 years (July 2010 to June 2015), anticipating that all sites (40 across the USA, Canada, the UK, Germany and Italy) would be open to recruitment from July 2011. However, study start-up was significantly delayed and recruitment did not start until January 2013. Method: The FOR-DMD study is used as an example to identify systematic problems in the set-up of international, multi-centre clinical trials. The full timeline of the FOR-DMD study, from funding approval to site activation, was collated and reviewed. Systematic issues were identified and grouped into (1) study set-up, e.g. drug procurement; (2) country set-up, e.g. competent authority applications; and (3) site set-up, e.g. contracts, to identify the main causes of delay and suggest areas where anticipatory action could overcome these obstacles in future studies. Results: Time from the first contact to site activation across countries ranged from 6 to 24 months. Reasons of delay were universal (sponsor agreement, drug procurement, budgetary constraints), country specific (complexity and diversity of regulatory processes, indemnity requirements) and site specific (contracting and approvals). The main identified obstacles included (1) issues related to drug supply, (2) NIH requirements regarding contracting with non-US sites, (3) differing regulatory requirements in the five participating countries, (4) lack of national harmonisation with contracting and the requirement to negotiate terms and contract individually with each site and (5) diversity of languages needed for study materials. Additionally, as with many academic-led studies, the FOR-DMD study did not have access to the infrastructure and expertise that a contracted research organisation could provide, organisations often employed in pharmaceutical-sponsored studies. This delay impacted recruitment, challenged the clinical relevance of the study outcomes and potentially delayed the delivery of the best treatment to patients. Conclusion: Based on the FOR-DMD experience, and as an interim solution, we have devised a checklist of steps to not only anticipate and minimise delays in academic international trial initiation but also identify obstacles that will require a concerted effort on the part of many stakeholders to mitigate

Prioritizing MCDC test cases by spectral analysis of Boolean functions

Test case prioritization aims at scheduling test cases in an order that improves some performance goal. One performance goal is a measure of how quickly faults are detected. Such prioritization can be performed by exploiting the Fault Exposing Potential (FEP) parameters associated to the test cases. FEP is usually approximated by mutation analysis under certain fault assumptions. Although this technique is effective, it could be relatively expensive compared to the other prioritization techniques. This study proposes a cost-effective FEP approximation for prioritizing Modified Condition Decision Coverage (MCDC) test cases. A strict negative correlation between the FEP of a MCDC test case and the influence value of the associated input condition allows to order the test cases easily without the need of an extensive mutation analysis. The method is entirely based on mathematics and it provides useful insight into how spectral analysis of Boolean functions can benefit software testing

Epigenetics of a tandem DNA repeat: chromatin DNaseI sensitivity and opposite methylation changes in cancers

DNA methylation and chromatin DNaseI sensitivity were analyzed in and adjacent to D4Z4 repeat arrays, which consist of 1 to ∼100 tandem 3.3-kb units at subtelomeric 4q and 10q. D4Z4 displayed hypomethylation in some cancers and hypermethylation in others relative to normal tissues. Surprisingly, in cancers with extensive D4Z4 methylation there was a barrier to hypermethylation spreading to the beginning of this disease-associated array (facioscapulohumeral muscular dystrophy, FSHD) despite sequence conservation in repeat units throughout the array. We infer a different chromatin structure at the proximal end of the array than at interior repeats, consistent with results from chromatin DNaseI sensitivity assays indicating a boundary element near the beginning of the array. The relative chromatin DNaseI sensitivity in FSHD and control myoblasts and lymphoblasts was as follows: a non-genic D4Z4-adjacent sequence (p13E-11, array-proximal)> untranscribed gene standards > D4Z4 arrays> constitutive heterochromatin (satellite 2; P < 10−4 for all comparisons). Cancers displaying D4Z4 hypermethylation also exhibited a hypermethylation-resistant subregion within the 3.3-kb D4Z4 repeat units. This subregion contains runs of G that form G-quadruplexes in vitro. Unusual DNA structures might contribute to topological constraints that link short 4q D4Z4 arrays to FSHD and make long ones phenotypically neutral

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

Asymmetric Bidirectional Transcription from the FSHD-Causing D4Z4 Array Modulates DUX4 Production

Facioscapulohumeral Disease (FSHD) is a dominantly inherited progressive myopathy associated with aberrant production of the transcription factor, Double Homeobox Protein 4 (DUX4). The expression of DUX4 depends on an open chromatin conformation of the D4Z4 macrosatellite array and a specific haplotype on chromosome 4. Even when these requirements are met, DUX4 transcripts and protein are only detectable in a subset of cells indicating that additional constraints govern DUX4 production. Since the direction of transcription, along with the production of non-coding antisense transcripts is an important regulatory feature of other macrosatellite repeats, we developed constructs that contain the non-coding region of a single D4Z4 unit flanked by genes that report transcriptional activity in the sense and antisense directions. We found that D4Z4 contains two promoters that initiate sense and antisense transcription within the array, and that antisense transcription predominates. Transcriptional start sites for the antisense transcripts, as well as D4Z4 regions that regulate the balance of sense and antisense transcripts were identified. We show that the choice of transcriptional direction is reversible but not mutually exclusive, since sense and antisense reporter activity was often present in the same cell and simultaneously upregulated during myotube formation. Similarly, levels of endogenous sense and antisense D4Z4 transcripts were upregulated in FSHD myotubes. These studies offer insight into the autonomous distribution of muscle weakness that is characteristic of FSHD

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 arm 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, 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 Southern blot strategy. Here, using 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 results show 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

DNaseI hypersensitivity at gene-poor, FSH dystrophy-linked 4q35.2

A subtelomeric region, 4q35.2, is implicated in facioscapulohumeral muscular dystrophy (FSHD), a dominant disease thought to involve local pathogenic changes in chromatin. FSHD patients have too few copies of a tandem 3.3-kb repeat (D4Z4) at 4q35.2. No phenotype is associated with having few copies of an almost identical repeat at 10q26.3. Standard expression analyses have not given definitive answers as to the genes involved. To investigate the pathogenic effects of short D4Z4 arrays on gene expression in the very gene-poor 4q35.2 and to find chromatin landmarks there for transcription control, unannotated genes and chromatin structure, we mapped DNaseI-hypersensitive (DH) sites in FSHD and control myoblasts. Using custom tiling arrays (DNase-chip), we found unexpectedly many DH sites in the two large gene deserts in this 4-Mb region. One site was seen preferentially in FSHD myoblasts. Several others were mapped >0.7 Mb from genes known to be active in the muscle lineage and were also observed in cultured fibroblasts, but not in lymphoid, myeloid or hepatic cells. Their selective occurrence in cells derived from mesoderm suggests functionality. Our findings indicate that the gene desert regions of 4q35.2 may have functional significance, possibly also to FSHD, despite their paucity of known genes