Dystrophin myonuclear domain restoration governs treatment efficacy in dystrophic muscle.

Dystrophin is essential for muscle health: its sarcolemmal absence causes the fatal, X-linked condition, Duchenne muscular dystrophy (DMD). However, its normal, spatial organization remains poorly understood, which hinders the interpretation of efficacy of its therapeutic restoration. Using female reporter mice heterozygous for fluorescently tagged dystrophin (DmdEGFP), we here reveal that dystrophin distribution is unexpectedly compartmentalized, being restricted to myonuclear-defined sarcolemmal territories extending ~80 µm, which we called "basal sarcolemmal dystrophin units (BSDUs)." These territories were further specialized at myotendinous junctions, where both Dmd transcripts and dystrophin protein were enriched. Genome-level correction in X-linked muscular dystrophy mice via CRISPR/Cas9 gene editing restored a mosaic of separated dystrophin domains, whereas transcript-level Dmd correction, following treatment with tricyclo-DNA antisense oligonucleotides, restored dystrophin initially at junctions before extending along the entire fiber-with levels ~2% sufficient to moderate the dystrophic process. We conclude that widespread restoration of fiber dystrophin is likely critical for therapeutic success in DMD, perhaps most importantly, at muscle-tendon junctions.

POMK mutation in a family with congenital muscular dystrophy with merosin deficiency, hypomyelination, mild hearing deficit and intellectual disability.

BACKGROUND: Congenital muscular dystrophies (CMD) with hypoglycosylation of α-dystroglycan are clinically and genetically heterogeneous disorders that are often associated with brain malformations and eye defects. Presently, 16 proteins are known whose dysfunction impedes glycosylation of α-dystroglycan and leads to secondary dystroglycanopathy. OBJECTIVE: To identify the cause of CMD with secondary merosin deficiency, hypomyelination and intellectual disability in two siblings from a consanguineous family. METHODS: Autozygosity mapping followed by whole exome sequencing and immunochemistry were used to discover and verify a new genetic defect in two siblings with CMD. RESULTS: We identified a homozygous missense mutation (c.325C>T, p.Q109*) in protein O-mannosyl kinase (POMK) that encodes a glycosylation-specific kinase (SGK196) required for function of the dystroglycan complex. The protein was absent from skeletal muscle and skin fibroblasts of the patients. In patient muscle, ß-dystroglycan was normally expressed at the sarcolemma, while α-dystroglycan failed to do so. Further, we detected co-localisation of POMK with desmin at the costameres in healthy muscle, and a substantial loss of desmin from the patient muscle. CONCLUSIONS: Homozygous truncating mutations in POMK lead to CMD with secondary merosin deficiency, hypomyelination and intellectual disability. Loss of desmin suggests that failure of proper α-dystroglycan glycosylation impedes the binding to extracellular matrix proteins and also affects the cytoskeleton.

Mature T-cell lymphomagenesis induced by retroviral insertional activation of Janus kinase 1.

Retroviral vectors (RVs) are powerful tools in clinical gene therapy. However, stable genomic integration of RVs can be oncogenic, as reported in several animal models and in clinical trials. Previously, we observed that T-cell receptor (TCR) polyclonal mature T cells are resistant to transformation after gammaretroviral transfer of (proto-)oncogenes, whereas TCR-oligoclonal T cells were transformable in the same setting. Here, we describe the induction of a mature T-cell lymphoma (MTCL) in TCR-oligoclonal OT-I transgenic T cells, transduced with an enhanced green fluorescent protein (EGFP)-encoding gammaretroviral vector. The tumor cells were of a mature T-cell phenotype and serially transplantable. Integration site analysis revealed a proviral hit in Janus kinase 1 (Jak1), which resulted in Jak1 overexpression and Jak/STAT-pathway activation, particularly via signal transducer and activator of transcription 3 (STAT3). Specific inhibition of Jak1 markedly delayed tumor growth. A systematic meta-analysis of available gene expression data on human mature T-cell lymphomas/leukemias confirmed the relevance of Jak/STAT overexpression in sporadic human T-cell tumorigenesis. To our knowledge, this is the first study to describe RV-associated insertional mutagenesis in mature T cells.

MR imaging helps predict time from symptom onset in patients with acute stroke: implications for patients with unknown onset time.

PURPOSE: To assess the value of magnetic resonance (MR) imaging parameters as surrogate markers of stroke duration. MATERIALS AND METHODS: The study was approved by the Ethics Committee of Ile de France III and was found to conform to generally accepted scientific principles and ethical standards. The authors studied 130 patients with acute stroke of known onset time who underwent 1.5-T MR imaging within 12 hours of the onset of stroke symptoms. Fluid-attenuated inversion recovery (FLAIR), diffusion-weighted (DW) imaging, and apparent diffusion coefficient (ADC) ratios were computed by using three-dimensional regions of interest to outline signal intensity changes on DW images and then projecting them onto the contralateral hemisphere. Imaging ratios in 63 patients who underwent imaging 0-3 hours after symptom onset were compared with those in 67 patients who underwent imaging more than 3 hours after onset by using the Student t test and receiver operating characteristic curves. The accuracy (sensitivity, specificity, and 95% confidence intervals [CIs]) of lesion visibility on FLAIR images in the prediction of a stroke onset time of less than 3 hours was assessed by two independent observers. RESULTS: Differences in imaging ratios between patients imaged 0-3 hours after symptom onset and those imaged more than 3 hours after onset were statistically significant (P < .001). The FLAIR ratio showed a positive correlation with the time from symptom onset (Pearson correlation coefficient, 0.63). Receiver operating characteristic curves indicated that the FLAIR ratio could reliably identify patients imaged 0-3 hours after symptom onset, reaching 90% sensitivity (95% CI: 83%, 98%) and 93% specificity (95% CI: 86%, 99%) when using a 7% cutoff. Stroke imaged within 3 hours could also be identified by means of visual inspection of FLAIR and DW MR images, with 94% sensitivity (95% CI: 88%, 100%) and 97% specificity (95% CI: 93%, 101%). CONCLUSION: Signal intensity changes on 1.5-T FLAIR MR images can be used as a surrogate marker of stroke age, either qualitatively or quantitatively. This suggests that MR imaging might be used as a "clock" for determining stroke age in patients with an unknown onset time, potentially increasing the number of patients who are eligible for thrombolysis.

Ectopic expression of transcription factor BATF3 induces B-cell lymphomas in a murine B-cell transplantation model.

The mechanisms involved in malignant transformation of mature B and T lymphocytes are still poorly understood. In a previous study, we compared gene expression profiles of the tumor cells of Hodgkin lymphoma (HL) and anaplastic large cell lymphoma (ALCL) to their normal cellular counterparts and found the basic leucine zipper protein ATF-like 3 (BATF3) to be significantly upregulated in the tumor cells of both entities. To assess the oncogenic potential of BATF3 in lymphomagenesis and to dissect the molecular interactions of BATF3 in lymphoma cells, we retrovirally transduced murine mature T and B cells with a BATF3-encoding viral vector and transplanted each population into Rag1-deficient recipients. Intriguingly, BATF3-expressing B lymphocytes readily induced B-cell lymphomas after characteristic latencies, whereas T-cell transplanted animals remained healthy throughout the observation time. Further analyses revealed a germinal center B-cell-like phenotype of most BATF3-initiated lymphomas. In a multiple myeloma cell line, BATF3 inhibited BLIMP1 expression, potentially illuminating an oncogenic action of BATF3 in B-cell lymphomagenesis. In conclusion, BATF3 overexpression induces malignant transformation of mature B cells and might serve as a potential target in B-cell lymphoma treatment.

Characterization of a Dmd (EGFP) reporter mouse as a tool to investigate dystrophin expression.

BACKGROUND: Dystrophin is a rod-shaped cytoplasmic protein that provides sarcolemmal stability as a structural link between the cytoskeleton and the extracellular matrix via the dystrophin-associated protein complex (DAPC). Mutations in the dystrophin-encoding DMD gene cause X-linked dystrophinopathies with variable phenotypes, the most severe being Duchenne muscular dystrophy (DMD) characterized by progressive muscle wasting and fibrosis. However, dystrophin deficiency does not only impair the function of skeletal and heart muscle but may also affect other organ systems such as the brain, eye, and gastrointestinal tract. The generation of a dystrophin reporter mouse would facilitate research into dystrophin muscular and extramuscular pathophysiology without the need for immunostaining. RESULTS: We generated a Dmd (EGFP) reporter mouse through the in-frame insertion of the EGFP coding sequence behind the last Dmd exon 79, which is known to be expressed in all major dystrophin isoforms. We analyzed EGFP and dystrophin expression in various tissues and at the single muscle fiber level. Immunostaining of various members of the DAPC was done to confirm the correct subsarcolemmal location of dystrophin-binding partners. We found strong natural EGFP fluorescence at all expected sites of dystrophin expression in the skeletal and smooth muscle, heart, brain, and retina. EGFP fluorescence exactly colocalized with dystrophin immunostaining. In the skeletal muscle, dystrophin and other proteins of the DAPC were expressed at their correct sarcolemmal/subsarcolemmal localization. Skeletal muscle maintained normal tissue architecture, suggesting the correct function of the dystrophin-EGFP fusion protein. EGFP expression could be easily verified in isolated myofibers as well as in satellite cell-derived myotubes. CONCLUSIONS: The novel dystrophin reporter mouse provides a valuable tool for direct visualization of dystrophin expression and will allow the study of dystrophin expression in vivo and in vitro in various tissues by live cell imaging.

Three-dimensional dynamic time-resolved contrast-enhanced MRA using parallel imaging and a variable rate k-space sampling strategy in intracranial arteriovenous malformations.

PURPOSE: To evaluate the effectiveness of three-dimensional (3D) dynamic time-resolved contrast-enhanced MRA (TR-CE-MRA) using a combination of a parallel imaging technique (ASSET: array spatial sensitivity encoding technique) and a time-resolved method (TRICKS: time-resolved imaging of contrast kinetics) and to compare it with 3D dynamic TR-CE-MRA using ASSET alone in the assessment of intracranial arteriovenous malformations (AVMs). MATERIALS AND METHODS: Twenty consecutive patients with angiographically confirmed AVMs were investigated using both 3D dynamic TR-CE-MRA techniques. Examinations were compared with respect to image quality, spatial resolution, number and type of feeders and drainers, nidus size, presence of early venous filling and temporal resolution. Digital subtraction angiography was used as standard of reference. RESULTS: The higher temporal and spatial resolution of 3D dynamic TR-CE-MRA TRICKS ASSET allowed a better assessment of intracranial vascular malformations, namely better depiction of feeders, drainers and better detection of early venous drainage. There was no significant difference between them in terms of nidus size. CONCLUSION: 3D dynamic TR-CE-MRA combining parallel imaging and a time-resolved method with subsecond and submillimeter resolution could become the first-line investigation technique in both diagnosis and follow-up of intracranial AVMs.