Identification of mutations in the MYO9A gene in patients with congenital myasthenic syndrome.

Congenital myasthenic syndromes are a group of rare and genetically heterogenous disorders resulting from defects in the structure and function of the neuromuscular junction. Patients with congenital myasthenic syndrome exhibit fatigable muscle weakness with a variety of accompanying phenotypes depending on the protein affected. A cohort of patients with a clinical diagnosis of congenital myasthenic syndrome that lacked a genetic diagnosis underwent whole exome sequencing in order to identify genetic causation. Missense biallelic mutations in the MYO9A gene, encoding an unconventional myosin, were identified in two unrelated families. Depletion of MYO9A in NSC-34 cells revealed a direct effect of MYO9A on neuronal branching and axon guidance. Morpholino-mediated knockdown of the two MYO9A orthologues in zebrafish, myo9aa/ab, demonstrated a requirement for MYO9A in the formation of the neuromuscular junction during development. The morphants displayed shortened and abnormally branched motor axons, lack of movement within the chorion and abnormal swimming in response to tactile stimulation. We therefore conclude that MYO9A deficiency may affect the presynaptic motor axon, manifesting in congenital myasthenic syndrome. These results highlight the involvement of unconventional myosins in motor axon functionality, as well as the need to look outside traditional neuromuscular junction-specific proteins for further congenital myasthenic syndrome candidate genes.

Mutations in the collagen XII gene define a new form of extracellular matrix-related myopathy.

Bethlem myopathy (BM) [MIM 158810] is a slowly progressive muscle disease characterized by contractures and proximal weakness, which can be caused by mutations in one of the collagen VI genes (COL6A1, COL6A2 and COL6A3). However, there may be additional causal genes to identify as in ∼50% of BM cases no mutations in the COL6 genes are identified. In a cohort of -24 patients with a BM-like phenotype, we first sequenced 12 candidate genes based on their function, including genes for known binding partners of collagen VI, and those enzymes involved in its correct post-translational modification, assembly and secretion. Proceeding to whole-exome sequencing (WES), we identified mutations in the COL12A1 gene, a member of the FACIT collagens (fibril-associated collagens with interrupted triple helices) in five individuals from two families. Both families showed dominant inheritance with a clinical phenotype resembling classical BM. Family 1 had a single-base substitution that led to the replacement of one glycine residue in the triple-helical domain, breaking the Gly-X-Y repeating pattern, and Family 2 had a missense mutation, which created a mutant protein with an unpaired cysteine residue. Abnormality at the protein level was confirmed in both families by the intracellular retention of collagen XII in patient dermal fibroblasts. The mutation in Family 2 leads to the up-regulation of genes associated with the unfolded protein response (UPR) pathway and swollen, dysmorphic rough-ER. We conclude that the spectrum of causative genes in extracellular matrix (ECM)-related myopathies be extended to include COL12A1.

Agrin mutations lead to a congenital myasthenic syndrome with distal muscle weakness and atrophy.

Congenital myasthenic syndromes are a clinically and genetically heterogeneous group of rare diseases resulting from impaired neuromuscular transmission. Their clinical hallmark is fatigable muscle weakness associated with a decremental muscle response to repetitive nerve stimulation and frequently related to postsynaptic defects. Distal myopathies form another clinically and genetically heterogeneous group of primary muscle disorders where weakness and atrophy are restricted to distal muscles, at least initially. In both congenital myasthenic syndromes and distal myopathies, a significant number of patients remain genetically undiagnosed. Here, we report five patients from three unrelated families with a strikingly homogenous clinical entity combining congenital myasthenia with distal muscle weakness and atrophy reminiscent of a distal myopathy. MRI and neurophysiological studies were compatible with mild myopathy restricted to distal limb muscles, but decrement (up to 72%) in response to 3 Hz repetitive nerve stimulation pointed towards a neuromuscular transmission defect. Post-exercise increment (up to 285%) was observed in the distal limb muscles in all cases suggesting presynaptic congenital myasthenic syndrome. Immunofluorescence and ultrastructural analyses of muscle end-plate regions showed synaptic remodelling with denervation-reinnervation events. We performed whole-exome sequencing in two kinships and Sanger sequencing in one isolated case and identified five new recessive mutations in the gene encoding agrin. This synaptic proteoglycan with critical function at the neuromuscular junction was previously found mutated in more typical forms of congenital myasthenic syndrome. In our patients, we found two missense mutations residing in the N-terminal agrin domain, which reduced acetylcholine receptors clustering activity of agrin in vitro. Our findings expand the spectrum of congenital myasthenic syndromes due to agrin mutations and show an unexpected correlation between the mutated gene and the associated phenotype. This provides a good rationale for examining patients with apparent distal myopathy for a neuromuscular transmission disorder and agrin mutations.

Hexosamine biosynthetic pathway mutations cause neuromuscular transmission defect.

Neuromuscular junctions (NMJs) are synapses that transmit impulses from motor neurons to skeletal muscle fibers leading to muscle contraction. Study of hereditary disorders of neuromuscular transmission, termed congenital myasthenic syndromes (CMS), has helped elucidate fundamental processes influencing development and function of the nerve-muscle synapse. Using genetic linkage, we find 18 different biallelic mutations in the gene encoding glutamine-fructose-6-phosphate transaminase 1 (GFPT1) in 13 unrelated families with an autosomal recessive CMS. Consistent with these data, downregulation of the GFPT1 ortholog gfpt1 in zebrafish embryos altered muscle fiber morphology and impaired neuromuscular junction development. GFPT1 is the key enzyme of the hexosamine pathway yielding the amino sugar UDP-N-acetylglucosamine, an essential substrate for protein glycosylation. Our findings provide further impetus to study the glycobiology of NMJ and synapses in general.

Congenital myasthenic syndromes due to mutations in ALG2 and ALG14.

Congenital myasthenic syndromes are a heterogeneous group of inherited disorders that arise from impaired signal transmission at the neuromuscular synapse. They are characterized by fatigable muscle weakness. We performed linkage analysis, whole-exome and whole-genome sequencing to determine the underlying defect in patients with an inherited limb-girdle pattern of myasthenic weakness. We identify ALG14 and ALG2 as novel genes in which mutations cause a congenital myasthenic syndrome. Through analogy with yeast, ALG14 is thought to form a multiglycosyltransferase complex with ALG13 and DPAGT1 that catalyses the first two committed steps of asparagine-linked protein glycosylation. We show that ALG14 is concentrated at the muscle motor endplates and small interfering RNA silencing of ALG14 results in reduced cell-surface expression of muscle acetylcholine receptor expressed in human embryonic kidney 293 cells. ALG2 is an alpha-1,3-mannosyltransferase that also catalyses early steps in the asparagine-linked glycosylation pathway. Mutations were identified in two kinships, with mutation ALG2p.Val68Gly found to severely reduce ALG2 expression both in patient muscle, and in cell cultures. Identification of DPAGT1, ALG14 and ALG2 mutations as a cause of congenital myasthenic syndrome underscores the importance of asparagine-linked protein glycosylation for proper functioning of the neuromuscular junction. These syndromes form part of the wider spectrum of congenital disorders of glycosylation caused by impaired asparagine-linked glycosylation. It is likely that further genes encoding components of this pathway will be associated with congenital myasthenic syndromes or impaired neuromuscular transmission as part of a more severe multisystem disorder. Our findings suggest that treatment with cholinesterase inhibitors may improve muscle function in many of the congenital disorders of glycosylation.

Dok-7 promotes slow muscle integrity as well as neuromuscular junction formation in a zebrafish model of congenital myasthenic syndromes.

The small signalling adaptor protein Dok-7 has recently been reported as an essential protein of the neuromuscular junction (NMJ). Mutations resulting in partial loss of Dok-7 activity cause a distinct limb-girdle subtype of the inherited NMJ disorder congenital myasthenic syndromes (CMSs), whereas complete loss of Dok-7 results in a lethal phenotype in both mice and humans. Here we describe the zebrafish orthologue of Dok-7 and study its in vivo function. Dok-7 deficiency leads to motility defects in zebrafish embryos and larvae. The relative importance of Dok-7 at different stages of NMJ development varies; it is crucial for the earliest step, the formation of acetylcholine receptor (AChR) clusters in the middle of the muscle fibre prior to motor neuron contact. At later stages, presence of Dok-7 is not absolutely essential, as focal and non-focal synapses do form when Dok-7 expression is downregulated. These contacts however are smaller than in the wild-type zebrafish, reminiscent of the neuromuscular endplate pathology seen in patients with DOK7 mutations. Intriguingly, we also observed changes in slow muscle fibre arrangement; previously, Dok-7 has not been linked to functions other than postsynaptic AChR clustering. Our results suggest an additional role of Dok-7 in muscle. This role seems to be independent of the muscle-specific tyrosine kinase MuSK, the known binding partner of Dok-7 at the NMJ. Our findings in the zebrafish model contribute to a better understanding of the signalling pathways at the NMJ and the pathomechanisms of DOK7 CMSs.

A founder mutation in Anoctamin 5 is a major cause of limb-girdle muscular dystrophy.

The limb-girdle muscular dystrophies are a group of disorders with wide genetic and clinical heterogeneity. Recently, mutations in the ANO5 gene, which encodes a putative calcium-activated chloride channel belonging to the Anoctamin family of proteins, were identified in five families with one of two previously identified disorders, limb-girdle muscular dystrophy 2L and non-dysferlin Miyoshi muscular dystrophy. We screened a candidate group of 64 patients from 59 British and German kindreds and found the truncating mutation, c.191dupA in exon 5 of ANO5 in 20 patients, homozygously in 15 and in compound heterozygosity with other ANO5 variants in the rest. An intragenic single nucleotide polymorphism and an extragenic microsatellite marker are in linkage disequilibrium with the mutation, suggesting a founder effect in the Northern European population. We have further defined the clinical phenotype of ANO5-associated muscular dystrophy. Patients show adult onset proximal lower limb weakness with highly raised serum creatine kinase values (average 4500 IU/l) and frequent muscle atrophy and asymmetry of muscle involvement. Onset varies from the early 20 s to 50 s and the weakness is generally slowly progressive, with most patients remaining ambulant for several decades. Distal presentation is much less common but a milder degree of distal lower limb weakness is often observed. Upper limb strength is only mildly affected and cardiac and respiratory function is normal. Females appear less frequently affected. In the North of England population we have identified eight patients with ANO5 mutations, suggesting a minimum prevalence of 0.27/100,000, twice as common as dysferlinopathy. We suggest that mutations in ANO5 represent a relatively common cause of adult onset muscular dystrophy with high serum creatine kinase and that mutation screening, particularly of the common mutation c.191dupA, should be an early step in the diagnostic algorithm of adult limb-girdle muscular dystrophy patients.

Attenuated muscle regeneration is a key factor in dysferlin-deficient muscular dystrophy.

Skeletal muscle requires an efficient and active membrane repair system to overcome the rigours of frequent contraction. Dysferlin is a component of that system and absence of dysferlin causes muscular dystrophy (dysferlinopathy) characterized by adult onset muscle weakness, high serum creatine kinase levels and a prominent inflammatory infiltrate. We have observed that dysferlinopathy patient biopsies show an excess of immature fibres and therefore investigated the role of dysferlin in muscle regeneration. Using notexin-induced muscle damage, we have shown that regeneration is attenuated in a mouse model of dysferlinopathy, with delayed removal of necrotic fibres, an extended inflammatory phase and delayed functional recovery. Satellite cell activation and myoblast fusion appear normal, but there is a reduction in early neutrophil recruitment in regenerating and also needle wounded muscle in dysferlin-deficient mice. Primary mouse dysferlinopathy myoblast cultures show reduced cytokine release upon stimulation, indicating that the secretion of chemotactic molecules is impaired. We suggest an extension to the muscle membrane repair model, where in addition to fusing patch repair vesicles with the sarcolemma dysferlin is also involved in the release of chemotactic agents. Reduced neutrophil recruitment results in incomplete cycles of regeneration in dysferlinopathy which combines with the membrane repair deficit to ultimately trigger dystrophic pathology. This study reveals a novel pathomechanism affecting muscle regeneration and maintenance in dysferlinopathy and highlights enhancement of the neutrophil response as a potential therapeutic avenue in these disorders.

AHNAK, a novel component of the dysferlin protein complex, redistributes to the cytoplasm with dysferlin during skeletal muscle regeneration.

Mutations in dysferlin cause limb girdle muscular dystrophy 2B, Miyoshi myopathy and distal anterior compartment myopathy. Dysferlin is proposed to play a role in muscle membrane repair. To gain functional insight into the molecular mechanisms of dysferlin, we have searched for dysferlin-interacting proteins in skeletal muscle. By coimmunoprecipitation coupled with mass spectrometry, we demonstrate that AHNAK interacts with dysferlin. We defined the binding sites in dysferlin and AHNAK as the C2A domain in dysferlin and the carboxyterminal domain of AHNAK by glutathione S-transferase (GST)-pull down assays. As expected, the N-terminal domain of myoferlin also interacts with the carboxyterminal domain of AHNAK. In normal skeletal muscle, dysferlin and AHNAK colocalize at the sarcolemmal membrane and T-tubules. In dysferlinopathies, reduction or absence of dysferlin correlates with a secondary muscle-specific loss of AHNAK. Moreover, in regenerating rat muscle, dysferlin and AHNAK showed a marked increase and cytoplasmic localization, consistent with the direct interaction between them. Our data suggest that dysferlin participates in the recruitment and stabilization of AHNAK to the sarcolemma and that AHNAK plays a role in dysferlin membrane repair process. It may also have significant implications for understanding the biology of AHNAK-containing exocytotic vesicles, "enlargosomes," in plasma membrane remodeling and repair.

Autologous skeletal muscle derived cells expressing a novel functional dystrophin provide a potential therapy for Duchenne Muscular Dystrophy.

Autologous stem cells that have been genetically modified to express dystrophin are a possible means of treating Duchenne Muscular Dystrophy (DMD). To maximize the therapeutic effect, dystrophin construct needs to contain as many functional motifs as possible, within the packaging capacity of the viral vector. Existing dystrophin constructs used for transduction of muscle stem cells do not contain the nNOS binding site, an important functional motif within the dystrophin gene. In this proof-of-concept study, using stem cells derived from skeletal muscle of a DMD patient (mdcs) transplanted into an immunodeficient mouse model of DMD, we report that two novel dystrophin constructs, C1 (ΔR3-R13) and C2 (ΔH2-R23), can be lentivirally transduced into mdcs and produce dystrophin. These dystrophin proteins were functional in vivo, as members of the dystrophin glycoprotein complex were restored in muscle fibres containing donor-derived dystrophin. In muscle fibres derived from cells that had been transduced with construct C1, the largest dystrophin construct packaged into a lentiviral system, nNOS was restored. The combination of autologous stem cells and a lentivirus expressing a novel dystrophin construct which optimally restores proteins of the dystrophin glycoprotein complex may have therapeutic application for all DMD patients, regardless of their dystrophin mutation.

The differential gene expression profiles of proximal and distal muscle groups are altered in pre-pathological dysferlin-deficient mice.

The selective pattern of muscle involvement is a key feature of muscular dystrophies. Dysferlinopathy is a good model for studying this process since it shows variable muscle involvement that can be highly selective even in individual patients. The transcriptomes of proximal and distal muscles from wildtype C57BL/10 and dysferlin deficient C57BL/10.SJL-Dysf mice at a prepathological stage were assessed using the Affymetrix oligonucleotide-microarray system. We detected significant variation in gene expression between proximal and distal muscle in wildtype mice. Dysferlin defiency, even in the absence of pathological changes, altered this proximal distal difference but with little specific overlap with previous microarray analyses of dysferlinopathy. In conclusion, proximal and distal muscle groups show distinct patterns of gene expression and respond differently to dysferlin deficiency. This has implications for the selection of muscles for future microarray analyses, and also offers new routes for investigating the selectivity of muscle involvement in muscular dystrophies.

Cardiomyocyte Regeneration in the mdx Mouse Model of Nonischemic Cardiomyopathy.

Endogenous regeneration has been demonstrated in the mammalian heart after ischemic injury. However, approximately one-third of cases of heart failure are secondary to nonischemic heart disease and cardiac regeneration in these cases remains relatively unexplored. We, therefore, aimed at quantifying the rate of new cardiomyocyte formation at different stages of nonischemic cardiomyopathy. Six-, 12-, 29-, and 44-week-old mdx mice received a 7 day pulse of BrdU. Quantification of isolated cardiomyocyte nuclei was undertaken using cytometric analysis to exclude nondiploid nuclei. Between 6-7 and 12-13 weeks, there was a statistically significant increase in the number of BrdU-labeled nuclei in the mdx hearts compared with wild-type controls. This difference was lost by the 29-30 week time point, and a significant decrease in cardiomyocyte generation was observed in both the control and mdx hearts by 44-45 weeks. Immunohistochemical analysis demonstrated BrdU-labeled nuclei exclusively in mononucleated cardiomyocytes. This study demonstrates cardiomyocyte regeneration in a nonischemic model of mammalian cardiomyopathy, controlling for changes in nuclear ploidy, which is lost with age, and confirms a decrease in baseline rates of cardiomyocyte regeneration with aging. While not attempting to address the cellular source of regeneration, it confirms the potential utility of innate regeneration as a therapeutic target.

Absence of Cardiac Benefit with Early Combination ACE Inhibitor and Beta Blocker Treatment in mdx Mice.

Most patients with Duchenne muscular dystrophy (DMD) will develop cardiomyopathy; however, the evidence for prophylactic treatment of children with cardiac medications is limited. We have used the mdx mouse model of DMD to assess if early combination treatment with beta blocker (BB) and ACE inhibitor (AI) is superior to single treatment with either one of these drugs. Mice were assessed with cardiac MRI (ventricular structure and function, in vivo calcium influx (manganese-enhanced MRI)), pressure-volume loops, and histopathology. Combination treatment did not show benefits over treatment with AI or BB alone. Indeed, some beneficial aspects of BB and AI were lost when used in combination. None of the treatments impacted RV function. Combination treatment had no significant effect on sarcolemmal damage or histopathology. The study suggests that combined BB and AI may not confer an advantage at an early stage in DMD cardiomyopathy. However, limitations of the mdx model should be considered.

Assessment of ventricular function in mouse models of muscular dystrophy: a comparison of MRI with conductance catheter.

Outcomes of clinical trials depend on the quality of preceding preclinical research, yet functional assays and outcome measures for mouse models of disease are often poorly standardized or inappropriate. Muscular dystrophies are associated with cardiomyopathy so preclinical research requires reliable measures of cardiac function in animal models of the disease. MRI and conductance catheter were compared as preclinical tools to detect cardiomyopathy in two mouse models of muscular dystrophy. Sgcd-/-, mdx and C57Bl10 mice (n = 7/group) were assessed by catheter following MRI at an early stage of cardiomyopathy. Volumetric measurements were higher from MRI compared to catheter. In particular, by catheter, the measurement of end-systolic volume (and its related measures) was disproportionately lower in dystrophic mice compared to controls. This was related to greater calculated parallel conductance in dystrophic mice. Catheter highlighted differences in pressure generation between the two models while MRI detected differences in left ventricular hypertrophy and right ventricular function. Although MRI and conductance catheter provide unique but complimentary information regarding the nature of cardiomyopathy in dystrophic mice, we present the possibility that pathology itself may affect the accuracy of the catheter technique and that particular caution must be taken when interpreting catheter volume data in dystrophic mice.

Development and Application of an Ultrasensitive Hybridization-Based ELISA Method for the Determination of Peptide-Conjugated Phosphorodiamidate Morpholino Oligonucleotides.

Antisense oligonucleotide (AON)-induced exon skipping is one of the most promising strategies for treating Duchenne muscular dystrophy (DMD) and other rare monogenic conditions. Phosphorodiamidate morpholino oligonucleotides (PMOs) and 2'-O-methyl phosphorothioate (2'OMe) are two of the most advanced AONs in development. The next generation of peptide-conjugated PMO (P-PMO) is also showing great promise, but to advance these therapies it is essential to determine the pharmacokinetic and biodistribution (PK/BD) profile using a suitable method to detect AON levels in blood and tissue samples. An enzyme-linked immunosorbent assay (ELISA)-based method, which shows greater sensitivity than the liquid chromatography-mass spectrometry method, is the method of choice for 2'OMe detection in preclinical and clinical studies. However, no such assay has been developed for PMO/P-PMO detection, and we have, therefore, developed an ultrasensitive hybridization-based ELISA for this purpose. The assay has a linear detection range of 5-250 pM (R(2)>0.99) in mouse serum and tissue lysates. The sensitivity was sufficient for determining the 24-h PK/BD profile of PMO and P-PMO injected at standard doses (12.5 mg/kg) in mdx mice, the dystrophin-deficient mouse model for DMD. The assay demonstrated an accuracy approaching 100% with precision values under 12%. This provides a powerful cost-effective assay for the purpose of accelerating the development of these emerging therapeutic agents.

Dystrophin quantification: Biological and translational research implications.

OBJECTIVE: We formed a multi-institution collaboration in order to compare dystrophin quantification methods, reach a consensus on the most reliable method, and report its biological significance in the context of clinical trials. METHODS: Five laboratories with expertise in dystrophin quantification performed a data-driven comparative analysis of a single reference set of normal and dystrophinopathy muscle biopsies using quantitative immunohistochemistry and Western blotting. We developed standardized protocols and assessed inter- and intralaboratory variability over a wide range of dystrophin expression levels. RESULTS: Results from the different laboratories were highly concordant with minimal inter- and intralaboratory variability, particularly with quantitative immunohistochemistry. There was a good level of agreement between data generated by immunohistochemistry and Western blotting, although immunohistochemistry was more sensitive. Furthermore, mean dystrophin levels determined by alternative quantitative immunohistochemistry methods were highly comparable. CONCLUSIONS: Considering the biological function of dystrophin at the sarcolemma, our data indicate that the combined use of quantitative immunohistochemistry and Western blotting are reliable biochemical outcome measures for Duchenne muscular dystrophy clinical trials, and that standardized protocols can be comparable between competent laboratories. The methodology validated in our study will facilitate the development of experimental therapies focused on dystrophin production and their regulatory approval.

Mutations in the Mitochondrial Citrate Carrier SLC25A1 are Associated with Impaired Neuromuscular Transmission.

BACKGROUND AND OBJECTIVE: Congenital myasthenic syndromes are rare inherited disorders characterized by fatigable weakness caused by malfunction of the neuromuscular junction. We performed whole exome sequencing to unravel the genetic aetiology in an English sib pair with clinical features suggestive of congenital myasthenia. METHODS: We used homozygosity mapping and whole exome sequencing to identify the candidate gene variants. Mutant protein expression and function were assessed in vitro and a knockdown zebrafish model was generated to assess neuromuscular junction development. RESULTS: We identified a novel homozygous missense mutation in the SLC25A1 gene, encoding the mitochondrial citrate carrier. Mutant SLC25A1 showed abnormal carrier function. SLC25A1 has recently been linked to a severe, often lethal clinical phenotype. Our patients had a milder phenotype presenting primarily as a neuromuscular (NMJ) junction defect. Of note, a previously reported patient with different compound heterozygous missense mutations of SLC25A1 has since been shown to suffer from a neuromuscular transmission defect. Using knockdown of SLC25A1 expression in zebrafish, we were able to mirror the human disease in terms of variable brain, eye and cardiac involvement. Importantly, we show clear abnormalities in the neuromuscular junction, regardless of the severity of the phenotype. CONCLUSIONS: Based on the axonal outgrowth defects seen in SLC25A1 knockdown zebrafish, we hypothesize that the neuromuscular junction impairment may be related to pre-synaptic nerve terminal abnormalities. Our findings highlight the complex machinery required to ensure efficient neuromuscular function, beyond the proteomes exclusive to the neuromuscular synapse.

Muscular dystrophy in dysferlin-deficient mouse models.

Mutations in the dysferlin gene result in the development of a range of early adult-onset, progressive muscular dystrophies, collectively known as the dysferlinopathies. There is currently no effective treatment for these disorders. Several spontaneous and engineered alleles at the mouse dysferlin locus have been isolated and these dysferlin-deficient mouse strains are providing valuable insights into the role dysferlin plays in skeletal muscle physiology, heart function, and the regulation of the innate immune system. In addition, mouse models of dysferlinopathy are now widely used to test novel therapeutic strategies. Each dysferlin-deficient mouse strain has been characterised to varying degrees using a variety of histological and functional assays, occasionally producing results inconsistent with other strains. Here, we review each mouse model and physiological changes in various systems which accompany their muscle disease with emphasis on the how the disease process develops in different mouse models of dysferlinopathy. This review highlights the urgent requirement for standardised assays and outcome measures that will unify and coordinate research efforts throughout the field, procedures that are necessary if potential therapies are to be tested efficiently and effectively.

Exon skipping and gene transfer restore dystrophin expression in human induced pluripotent stem cells-cardiomyocytes harboring DMD mutations.

With an incidence of ∼1:3,500 to 5,000 in male children, Duchenne muscular dystrophy (DMD) is an X-linked disorder in which progressive muscle degeneration occurs and affected boys usually die in their twenties or thirties. Cardiac involvement occurs in 90% of patients and heart failure accounts for up to 40% of deaths. To enable new therapeutics such as gene therapy and exon skipping to be tested in human cardiomyocytes, we produced human induced pluripotent stem cells (hiPSC) from seven patients harboring mutations across the DMD gene. Mutations were retained during differentiation and analysis indicated the cardiomyocytes showed a dystrophic gene expression profile. Antisense oligonucleotide-mediated skipping of exon 51 restored dystrophin expression to ∼30% of normal levels in hiPSC-cardiomyocytes carrying exon 47-50 or 48-50 deletions. Alternatively, delivery of a dystrophin minigene to cardiomyocytes with a deletion in exon 35 or a point mutation in exon 70 allowed expression levels similar to those seen in healthy cells. This demonstrates that DMD hiPSC-cardiomyocytes provide a novel tool to evaluate whether new therapeutics can restore dystrophin expression in the heart.

Efficient and fast functional screening of microdystrophin constructs in vivo and in vitro for therapy of duchenne muscular dystrophy.

Duchenne muscular dystrophy (DMD) is an X-linked, lethal genetic disorder affecting the skeletal muscle compartment, and is caused by mutation(s) in the dystrophin gene. Gene delivery of microdystrophin constructs using adeno-associated virus (AAV) and antisense-mediated exon skipping restoring the genetic reading frame are two of the most promising therapeutic strategies for DMD. Both approaches use microdystrophin proteins either directly as a desired construct for gene delivery, using the capacity-limited AAV vectors, or as the therapeutic outcome of gene splicing. Although functionality of the resulting artificial dystrophin proteins can be predicted in silico, experimental evidence usually obtained in transgenic mice is required before human trials. However, the enormous number of potential constructs makes screening assays for dystrophin protein function in vitro and in vivo highly desirable. Here we present data showing that functionality of microdystrophins can be assessed using relatively simple and fast techniques.