Optimization of Internally Deleted Dystrophin Constructs.

Duchenne muscular dystrophy (DMD) is a severe, genetic muscle disease caused by the absence of the sarcolemmal protein dystrophin. Gene replacement therapy is considered a potential strategy for the treatment of DMD, aiming to restore the missing protein. Although the elements of the dystrophin molecule have been identified and studies in transgenic mdx mice have explored the importance of a number of these structural domains, the resulting modified dystrophin protein products that have been developed so far are only partially characterized in relation to their structure and function in vivo. To optimize a dystrophin cDNA construct for therapeutic application we designed and produced four human minidystrophins within the packaging capacity of lentiviral vectors. Two novel minidystrophins retained the centrally located neuronal nitric oxide synthase (nNOS)-anchoring domain in order to achieve sarcolemmal nNOS restoration, which is lost in most internally deleted dystrophin constructs. Functionality of the resulting truncated dystrophin proteins was investigated in muscle of adult dystrophin-deficient mdx mice followed by a battery of detailed immunohistochemical and morphometric tests. This initial assessment aimed to determine the overall suitability of various constructs for cloning into lentiviral vectors for ex vivo gene delivery to stem cells for future preclinical studies.

Global N-linked Glycosylation is Not Significantly Impaired in Myoblasts in Congenital Myasthenic Syndromes Caused by Defective Glutamine-Fructose-6-Phosphate Transaminase 1 (GFPT1).

Glutamine-fructose-6-phosphate transaminase 1 (GFPT1) is the first enzyme of the hexosamine biosynthetic pathway. It transfers an amino group from glutamine to fructose-6-phosphate to yield glucosamine-6-phosphate, thus providing the precursor for uridine diphosphate N-acetylglucosamine (UDP-GlcNAc) synthesis. UDP-GlcNAc is an essential substrate for all mammalian glycosylation biosynthetic pathways and N-glycan branching is especially sensitive to alterations in the concentration of this sugar nucleotide. It has been reported that GFPT1 mutations lead to a distinct sub-class of congenital myasthenic syndromes (CMS) termed "limb-girdle CMS with tubular aggregates". CMS are hereditary neuromuscular transmission disorders in which neuromuscular junctions are impaired. To investigate whether alterations in protein glycosylation at the neuromuscular junction might be involved in this impairment, we have employed mass spectrometric strategies to study the N-glycomes of myoblasts and myotubes derived from two healthy controls, three GFPT1 patients, and four patients with other muscular diseases, namely CMS caused by mutations in DOK7, myopathy caused by mutations in MTND5, limb girdle muscular dystrophy type 2A (LGMD2A), and Pompe disease. A comparison of the relative abundances of bi-, tri-, and tetra-antennary N-glycans in each of the cell preparations revealed that all samples exhibited broadly similar levels of branching. Moreover, although some differences were observed in the relative abundances of some of the N-glycan constituents, these variations were modest and were not confined to the GFPT1 samples. Therefore, GFPT1 mutations in CMS patients do not appear to compromise global N-glycosylation in muscle cells.

ANO5 gene analysis in a large cohort of patients with anoctaminopathy: confirmation of male prevalence and high occurrence of the common exon 5 gene mutation.

Limb girdle muscular dystrophy type 2L or anoctaminopathy is a condition mainly characterized by adult onset proximal lower limb muscular weakness and raised CK values, due to recessive ANO5 gene mutations. An exon 5 founder mutation (c.191dupA) has been identified in most of the British and German LGMD2L patients so far reported. We aimed to further investigate the prevalence and spectrum of ANO5 gene mutations and related clinical phenotypes, by screening 205 undiagnosed patients referred to our molecular service with a clinical suspicion of anoctaminopathy. A total of 42 unrelated patients had two ANO5 mutations (21%), whereas 14 carried a single change. We identified 34 pathogenic changes, 15 of which are novel. The c.191dupA mutation represents 61% of mutated alleles and appears to be less prevalent in non-Northern European populations. Retrospective clinical analysis corroborates the prevalently proximal lower limb phenotype, the male predominance and absence of major cardiac or respiratory involvement. Identification of cases with isolated hyperCKaemia and very late symptomatic male and female subjects confirms the extension of the phenotypic spectrum of the disease. Anoctaminopathy appears to be one of the most common adult muscular dystrophies in Northern Europe, with a prevalence of about 20%-25% in unselected undiagnosed cases.

Abnormal vascular development in zebrafish models for fukutin and FKRP deficiency.

Fukutin and fukutin-related protein (FKRP) are involved in the glycosylation of α-dystroglycan, a key receptor for basement membrane proteins. Aberrant α-dystroglycan glycosylation leads to a broad spectrum of disorders, ranging from limb girdle muscular dystrophy to Walker-Warburg syndrome. This is the first study investigating a role of fukutin and FKRP-mediated glycosylation in angiogenesis. Transgenic zebrafish expressing enhanced green fluorescent protein in blood vessels were treated with morpholino antisense oligonucleotides that blocked the expression of fukutin, FKRP and dystroglycan. All morphant fish showed muscle damage and vascular abnormalities at day 1 post-fertilization. Intersegmental vessels of somites failed to reach the dorsal longitudinal anastomosis and in more severe phenotypes retracted further or were in some cases even completely missing. In contrast, the eye vasculature was distorted in both fukutin and FKRP morphants, but not in dystroglycan morphants or control fish. The eye size was also smaller in the fukutin and FKRP morphants when compared with dystroglycan knockdown fish and controls. In general, the fukutin morphant fish had the most severe skeletal muscle and eye phenotype. Our findings suggest that fukutin and FKRP have functions that affect ocular development in zebrafish independently of dystroglycan. Despite anecdotal reports about vascular abnormalities in patients affected by dystroglycanopathies, the clinical relevance of such lesions remains unclear and should be subject to further review and investigations.

Long-term blocking of calcium channels in mdx mice results in differential effects on heart and skeletal muscle.

The disease mechanisms underlying dystrophin-deficient muscular dystrophy are complex, involving not only muscle membrane fragility, but also dysregulated calcium homeostasis. Specifically, it has been proposed that calcium channels directly initiate a cascade of pathological events by allowing calcium ions to enter the cell. The objective of this study was to investigate the effect of chronically blocking calcium channels with the aminoglycoside antibiotic streptomycin from onset of disease in the mdx mouse model of Duchenne muscular dystrophy (DMD). Treatment in utero onwards delayed onset of dystrophic symptoms in the limb muscle of young mdx mice, but did not prevent degeneration and regeneration events occurring later in the disease course. Long-term treatment had a positive effect on limb muscle pathology, reduced fibrosis, increased sarcolemmal stability, and promoted muscle regeneration in older mice. However, streptomycin treatment did not show positive effects in diaphragm or heart muscle, and heart pathology was worsened. Thus, blocking calcium channels even before disease onset does not prevent dystrophy, making this an unlikely treatment for DMD. These findings highlight the importance of analyzing several time points throughout the life of the treated mice, as well as analyzing many tissues, to get a complete picture of treatment efficacy.