Bone health in Duchenne muscular dystrophy: clinical and biochemical correlates.

PURPOSE: An increased fracture risk is commonly reported in Duchenne muscular dystrophy (DMD). Our aim was to investigate bone mineral density (BMD) and bone turnover, including sclerostin, and their association with markers of cardiac and respiratory performance in a cohort of DMD subjects. METHODS: In this single center, cross sectional observational study, lumbar spine (LS) BMD Z-scores, C-terminal telopeptide of procollagen type I (CTX) and osteocalcin (BGP), as bone resorption and formation markers, respectively, and sclerostin were assessed. Left ventricular ejection fraction (LVEF) and forced vital capacity (FVC) were evaluated. Clinical prevalent fractures were also recorded. RESULTS: Thirty-one patients [median age = 14 (12-21.5) years] were studied. Ambulant subjects had higher LS BMD Z-scores compared with non-ambulant ones and subjects with prevalent clinical fractures [n = 9 (29%)] showed lower LS BMD Z-scores compared with subjects without fractures. LS BMD Z-scores were positively correlated with FVC (r = 0.50; p = 0.01), but not with glucocorticoid use, and FVC was positively associated with BGP (r = 0.55; p = 0.02). In non-ambulant subjects, LS BMD Z-scores were associated with BMI (r = 0.54; p = 0.02) and sclerostin was associated with age (r = 0.44; p = 0.05). Age, BMI, FVC and sclerostin were independently associated with LS BMD Z-score in a stepwise multiple regression analysis. Older age, lower BMI, FVC and sclerostin were associated with lower LS BMD Z-scores. CONCLUSION: In a cohort of DMD patients, our data confirm low LS BMD Z-scores, mainly in non-ambulant subjects and irrespective of the glucocorticoid use, and suggest that FVC and sclerostin are independently associated with LS BMD Z-scores.

A Phase 1/2 Study of Flavocoxid, an Oral NF-κB Inhibitor, in Duchenne Muscular Dystrophy.

Flavocoxid is a blended extract containing baicalin and catechin with potent antioxidant and anti-inflammatory properties due to the inhibition of the cyclooxygenase (COX) and 5-lipoxygenase (5-LOX) enzymes, nuclear factor-κB (NF-κB), tumor necrosis factor (TNF)-alpha, and the mitogen-activated protein kinases (MAPKs) pathways. This phase 1/2 study was designed to assess the safety and tolerability of flavocoxid in patients with Duchenne muscular dystrophy (DMD). Thirty-four patients were recruited: 17 were treated with flavocoxid at an oral dose of 250 or 500 mg, according to body weight, for one year; 17 did not receive flavocoxid and served as controls. The treatment was well tolerated and nobody dropped out. Flavocoxid induced a significant reduction in serum interleukin (IL)-1 beta and TNF-alpha only in the group of DMD boys on add-on therapy (flavocoxid added to steroids for at least six months). The decrease in IL-1 beta was higher in younger boys. The serum H2O2 concentrations significantly decreased in patients treated with flavocoxid alone with a secondary reduction of serum glutathione peroxidase (GPx) levels, especially in younger boys. The exploratory outcome measures failed to show significant effects but there was a trend showing that the younger boys who received treatment were faster at performing the Gowers' maneuver, while the older boys who received treatment were faster at doing the 10-m walk test (10MWT). Therefore, a double-blind, placebo-controlled study for at least two/three years is warranted to verify flavocoxid as a steroid substitute or as add-on therapy to steroids.

Mobility shift of beta-dystroglycan as a marker of GMPPB gene-related muscular dystrophy.

BACKGROUND: Defects in glycosylation of alpha-dystroglycan (α-DG) cause autosomal-recessive disorders with wide clinical and genetic heterogeneity, with phenotypes ranging from congenital muscular dystrophies to milder limb girdle muscular dystrophies. Patients show variable reduction of immunoreactivity to antibodies specific for glycoepitopes of α-DG on a muscle biopsy. Recessive mutations in 18 genes, including guanosine diphosphate mannose pyrophosphorylase B (GMPPB), have been reported to date. With no specific clinical and pathological handles, diagnosis requires parallel or sequential analysis of all known genes. METHODS: We describe clinical, genetic and biochemical findings of 21 patients with GMPPB-associated dystroglycanopathy. RESULTS: We report eight novel mutations and further expand current knowledge on clinical and muscle MRI features of this condition. In addition, we report a consistent shift in the mobility of beta-dystroglycan (ß-DG) on Western blot analysis of all patients analysed by this mean. This was only observed in patients with GMPPB in our large dystroglycanopathy cohort. We further demonstrate that this mobility shift in patients with GMPPB was due to abnormal N-linked glycosylation of ß-DG. CONCLUSIONS: Our data demonstrate that a change in ß-DG electrophoretic mobility in patients with dystroglycanopathy is a distinctive marker of the molecular defect in GMPPB.

Loss-of-function mutations in SCN4A cause severe foetal hypokinesia or 'classical' congenital myopathy.

Congenital myopathies are a clinically and genetically heterogeneous group of muscle disorders characterized by congenital or early-onset hypotonia and muscle weakness, and specific pathological features on muscle biopsy. The phenotype ranges from foetal akinesia resulting in in utero or neonatal mortality, to milder disorders that are not life-limiting. Over the past decade, more than 20 new congenital myopathy genes have been identified. Most encode proteins involved in muscle contraction; however, mutations in ion channel-encoding genes are increasingly being recognized as a cause of this group of disorders. SCN4A encodes the α-subunit of the skeletal muscle voltage-gated sodium channel (Nav1.4). This channel is essential for the generation and propagation of the muscle action potential crucial to muscle contraction. Dominant SCN4A gain-of-function mutations are a well-established cause of myotonia and periodic paralysis. Using whole exome sequencing, we identified homozygous or compound heterozygous SCN4A mutations in a cohort of 11 individuals from six unrelated kindreds with congenital myopathy. Affected members developed in utero- or neonatal-onset muscle weakness of variable severity. In seven cases, severe muscle weakness resulted in death during the third trimester or shortly after birth. The remaining four cases had marked congenital or neonatal-onset hypotonia and weakness associated with mild-to-moderate facial and neck weakness, significant neonatal-onset respiratory and swallowing difficulties and childhood-onset spinal deformities. All four surviving cohort members experienced clinical improvement in the first decade of life. Muscle biopsies showed myopathic features including fibre size variability, presence of fibrofatty tissue of varying severity, without specific structural abnormalities. Electrophysiology suggested a myopathic process, without myotonia. In vitro functional assessment in HEK293 cells of the impact of the identified SCN4A mutations showed loss-of-function of the mutant Nav1.4 channels. All, apart from one, of the mutations either caused fully non-functional channels, or resulted in a reduced channel activity. Each of the affected cases carried at least one full loss-of-function mutation. In five out of six families, a second loss-of-function mutation was present on the trans allele. These functional results provide convincing evidence for the pathogenicity of the identified mutations and suggest that different degrees of loss-of-function in mutant Nav1.4 channels are associated with attenuation of the skeletal muscle action potential amplitude to a level insufficient to support normal muscle function. The results demonstrate that recessive loss-of-function SCN4A mutations should be considered in patients with a congenital myopathy.

Modulation of neuronal nitric oxide synthase and apoptosis by the isoflavone genistein in Mdx mice.

Dystrophin lack in DMD causes neuronal nitric oxide synthase (nNOS) membrane delocalization which in turn promotes functional muscle ischemia, and exacerbates muscle injury. Apoptosis and the exhaustion of muscle regenerative capacity are implicated in Duchenne muscular dystrophy (DMD) pathogenesis and therefore are relevant therapeutic targets. Genistein has been reported to have pro-proliferative effects, promoting G1/S cell phase transition through the induction of cyclin D1, and anti-apoptotic properties. We previously showed that genistein could reduce muscle necrosis and enhance regeneration with an augmented number of myogenin-positive satellite cells and myonuclei, ameliorating muscle function in mdx mice. In this study we evaluated the underlying mechanisms of genistein effect on muscle specimens of mdx and wild type mice treated for five weeks with genistein (2 mg/kg/i.p. daily) or vehicle. Western blot analysis show that genistein increased cyclin D1 and nNOS expression; and showed an antiapoptotic effect, modulating the expression of BAX and Bcl-2. Our results suggest that this isoflavone might enhance the regenerative spurt in mdx mice muscle restoring nNOS, promoting G1/S phase transition in muscle cell, and inhibiting apoptosis. Further studies with longer time treatment or using different experimental approaches are needed to better investigate the underlying mechanisms of such results.