Glucocorticoid-Treated Mice Are an Inappropriate Positive Control for Long-Term Preclinical Studies in the mdx Mouse

Dmd(mdx) (mdx) mice are used as a genetic and biochemical model of dystrophin deficiency. The long-term consequences of glucocorticoid (GC) treatment on dystrophin-deficient skeletal and heart muscle are not yet known. Here we used systematic phenotyping to assess the long-term consequences of GC treatment in mdx mice. Our investigation addressed not only the effects of GC on the disease phenotype but also the question of whether GCs can be used as a positive control for preclinical drug evaluations.We performed nine pre-clinical efficacy trials (treated N = 129, untreated N = 106) of different durations in 9-to-50-week-old dystrophic mdx mice over a 3-year time period using standardized methods. In all these trials, we used either 1 mg/kg body weight of prednisone or 5 mg/kg body weight of prednisolone as positive controls to compare the efficacy of various test drugs. Data from untreated controls and GC-treated mice in the various trials have been pooled and analyzed to assess the effects of GCs on dystrophin-deficient skeletal and cardiac muscles of mdx mice. Our results indicate that continuous GC treatment results in early (e.g., at 50 days) improvements in normalized parameters such as grip strength, motor coordination and maximal in vitro force contractions on isolated EDL muscle, but these initial benefits are followed by a progressive loss of muscle strength after 100 days. We also found a significant increase in heart fibrosis that is reflected in a significant deterioration in cardiac systolic function after 100 days of treatment.Continuous administration of prednisone to mdx mice initially improves skeletal muscle strength, but further therapy result in deterioration of muscle strength and cardiac function associated with enhanced cardiac fibrosis. These results suggest that GCs may not serve as an appropriate positive control for long-term mdx mouse preclinical trials

Deflazacort for the treatment of Duchenne Dystrophy: A systematic review

BACKGROUND: To complete a systematic review and meta-analysis based on the clinical question: Is Deflazacort (DFZ), a prednisolone derivative, an effective therapy for improving strength, with acceptable side effects, in children with Duchenne Dystrophy (DD)? METHODS: MEDLINE, EMBASE, Current Contents, Dissertation Abstracts, Health Star, PsychINFO and Cochrane, were searched using the following inclusion criteria: 1) A randomized controlled trial comparing DFZ with placebo or another therapy; 2) Male participants age 2–18 years with DD; 3) Outcomes of (a) any form of strength or functional testing, or (b) any form of side effect. RESULTS: Fifteen studies of potential relevance were identified, with five meeting the inclusion criteria. These five studies included 291 children and were published in English language journals between 1994 and 2000. Two studies compared DFZ versus placebo, two studies compared DFZ with prednisone and one study had both placebo and prednisone comparisions. Two large trials were identified that have not been published in article format. Due to the heterogeneity in outcome measures and the inconsistent reporting of summary statistics a meta-analytic approach could not be taken. CONCLUSIONS: Examining individual studies it appears that DFZ improves strength and functional outcomes compared to placebo, but it remains unclear if it has a benefit over prednisone on similar outcomes. Two trials found that DFZ causes less weight gain than prednisone

An Alpha-Catulin Homologue Controls Neuromuscular Function through Localization of the Dystrophin Complex and BK Channels in Caenorhabditis elegans

The large conductance, voltage- and calcium-dependent potassium (BK) channel serves as a major negative feedback regulator of calcium-mediated physiological processes and has been implicated in muscle dysfunction and neurological disorders. In addition to membrane depolarization, activation of the BK channel requires a rise in cytosolic calcium. Localization of the BK channel near calcium channels is therefore critical for its function. In a genetic screen designed to isolate novel regulators of the Caenorhabditis elegans BK channel, SLO-1, we identified ctn-1, which encodes an α-catulin homologue with homology to the cytoskeletal proteins α-catenin and vinculin. ctn-1 mutants resemble slo-1 loss-of-function mutants, as well as mutants with a compromised dystrophin complex. We determined that CTN-1 uses two distinct mechanisms to localize SLO-1 in muscles and neurons. In muscles, CTN-1 utilizes the dystrophin complex to localize SLO-1 channels near L-type calcium channels. In neurons, CTN-1 is involved in localizing SLO-1 to a specific domain independent of the dystrophin complex. Our results demonstrate that CTN-1 ensures the localization of SLO-1 within calcium nanodomains, thereby playing a crucial role in muscles and neurons

β1-Syntrophin Modulation by miR-222 in mdx Mice

Background: In mdx mice, the absence of dystrophin leads to the deficiency of other components of the dystrophin-glycoprotein complex (DAPC), making skeletal muscle fibers more susceptible to necrosis. The mechanisms involved in the disappearance of the DAPC are not completely understood. The muscles of mdx mice express normal amounts of mRNA for the DAPC components, thus suggesting post-transcriptional regulation. Methodology/Principal Findings: We investigated the hypothesis that DAPC reduction could be associated with the microRNA system. Among the possible microRNAs (miRs) found to be upregulated in the skeletal muscle tissue of mdx compared to wt mice, we demonstrated that miR-222 specifically binds to the 3′-UTR of β1-syntrophin and participates in the downregulation of β1-syntrophin. In addition, we documented an altered regulation of the 3′-UTR of β1-syntrophin in muscle tissue from dystrophic mice. Conclusion/Significance: These results show the importance of the microRNA system in the regulation of DAPC components in dystrophic muscle, and suggest a potential role of miRs in the pathophysiology of dystrophy. © 2010 De Arcangelis et al

IRES-Mediated Translation of Utrophin A Is Enhanced by Glucocorticoid Treatment in Skeletal Muscle Cells

Glucocorticoids are currently the only drug treatment recognized to benefit Duchenne muscular dystrophy (DMD) patients. The nature of the mechanisms underlying the beneficial effects remains incompletely understood but may involve an increase in the expression of utrophin. Here, we show that treatment of myotubes with 6α−methylprednisolone-21 sodium succinate (PDN) results in enhanced expression of utrophin A without concomitant increases in mRNA levels thereby suggesting that translational regulation contributes to the increase. In agreement with this, we show that PDN treatment of cells transfected with monocistronic reporter constructs harbouring the utrophin A 5′UTR, causes an increase in reporter protein expression while leaving levels of reporter mRNAs unchanged. Using bicistronic reporter assays, we further demonstrate that PDN enhances activity of an Internal Ribosome Entry Site (IRES) located within the utrophin A 5′UTR. Analysis of polysomes demonstrate that PDN causes an overall reduction in polysome-associated mRNAs indicating that global translation rates are depressed under these conditions. Importantly, PDN causes an increase in the polysome association of endogenous utrophin A mRNAs and reporter mRNAs harbouring the utrophin A 5′UTR. Additional experiments identified a distinct region within the utrophin A 5′UTR that contains the inducible IRES activity. Together, these studies demonstrate that a translational regulatory mechanism involving increased IRES activation mediates, at least partially, the enhanced expression of utrophin A in muscle cells treated with glucocorticoids. Targeting the utrophin A IRES may thus offer an important and novel therapeutic avenue for developing drugs appropriate for DMD patients

Noncanonical DNA Motifs as Transactivation Targets by Wild Type and Mutant p53

Sequence-specific binding by the human p53 master regulator is critical to its tumor suppressor activity in response to environmental stresses. p53 binds as a tetramer to two decameric half-sites separated by 0–13 nucleotides (nt), originally defined by the consensus RRRCWWGYYY (n = 0–13) RRRCWWGYYY. To better understand the role of sequence, organization, and level of p53 on transactivation at target response elements (REs) by wild type (WT) and mutant p53, we deconstructed the functional p53 canonical consensus sequence using budding yeast and human cell systems. Contrary to early reports on binding in vitro, small increases in distance between decamer half-sites greatly reduces p53 transactivation, as demonstrated for the natural TIGER RE. This was confirmed with human cell extracts using a newly developed, semi–in vitro microsphere binding assay. These results contrast with the synergistic increase in transactivation from a pair of weak, full-site REs in the MDM2 promoter that are separated by an evolutionary conserved 17 bp spacer. Surprisingly, there can be substantial transactivation at noncanonical ½-(a single decamer) and ¾-sites, some of which were originally classified as biologically relevant canonical consensus sequences including PIDD and Apaf-1. p53 family members p63 and p73 yielded similar results. Efficient transactivation from noncanonical elements requires tetrameric p53, and the presence of the carboxy terminal, non-specific DNA binding domain enhanced transactivation from noncanonical sequences. Our findings demonstrate that RE sequence, organization, and level of p53 can strongly impact p53-mediated transactivation, thereby changing the view of what constitutes a functional p53 target. Importantly, inclusion of ½- and ¾-site REs greatly expands the p53 master regulatory network

Pompe disease diagnosis and management guideline

ACMG standards and guidelines are designed primarily as an educational resource for physicians and other health care providers to help them provide quality medical genetic services. Adherence to these standards and guidelines does not necessarily ensure a successful medical outcome. These standards and guidelines should not be considered inclusive of all proper procedures and tests or exclusive of other procedures and tests that are reasonably directed to obtaining the same results. in determining the propriety of any specific procedure or test, the geneticist should apply his or her own professional judgment to the specific clinical circumstances presented by the individual patient or specimen. It may be prudent, however, to document in the patient's record the rationale for any significant deviation from these standards and guidelines.Duke Univ, Med Ctr, Durham, NC 27706 USAOregon Hlth Sci Univ, Portland, OR 97201 USANYU, Sch Med, New York, NY USAUniv Florida, Coll Med, Powell Gene Therapy Ctr, Gainesville, FL 32611 USAIndiana Univ, Bloomington, in 47405 USAUniv Miami, Miller Sch Med, Coral Gables, FL 33124 USAHarvard Univ, Childrens Hosp, Sch Med, Cambridge, MA 02138 USAUniversidade Federal de São Paulo, São Paulo, BrazilColumbia Univ, New York, NY 10027 USANYU, Bellevue Hosp, Sch Med, New York, NY USAColumbia Univ, Med Ctr, New York, NY 10027 USAUniversidade Federal de São Paulo, São Paulo, BrazilWeb of Scienc