National registry of patients with Fukuyama congenital muscular dystrophy in Japan.

Fukuyama congenital muscular dystrophy (FCMD) is the second most common form of muscular dystrophy in the Japanese population and is caused by mutations in the fukutin (FKTN) gene. In 2011, the Japan Muscular Dystrophy Association (JMDA) developed a nationwide registry of genetically confirmed patients with FCMD. We retrospectively reviewed the registry dataset of patients with FCMD to obtain data, including age, sex, developmental milestones, intellectual level, complications, and primary treatments. In total, 207 patients with FCMD (104 boys and 103 girls) were registered by the end of September 2013. Mean patient age at first registration was 8.1 ±â€¯7.8 years (median, 6 years; range, 0-42 years). A homozygous 3-kb founder insertion mutation in the FKTN gene was present in 80% of registrants, whereas 20% had a compound heterozygous mutation. Sixty-nine patients (33%) had febrile seizures and/or epilepsy. Myopia was the most frequently detected abnormality (8.7%), followed by strabismus (5.9%). Overall, 16% of patients required respiratory support and this percentage increased with age. Cardiac dysfunction was detected in 16%, and dysphagia was observed in 22% of patients with FCMD. The FCMD patient registry is useful for clarifying the natural history of FCMD and recruiting patients for clinical trials.

B3GALNT2-Related Dystroglycanopathy: Expansion of the Phenotype with Novel Mutation Associated with Muscle-Eye-Brain Disease, Walker-Warburg Syndrome, Epileptic Encephalopathy-West Syndrome, and Sensorineural Hearing Loss.

Mutations in B3GALNT2, encoding a glycosyltransferase enzyme involved in α-dystroglycan glycosylation, have been recently associated with dystroglycanopathy, a well-recognized subtype of congenital muscular dystrophy (CMD). Only a few cases have been reported with B3GALNT2-related dystroglycanopathy with variable severity ranging from mild CMD to severe muscle-eye-brain disease. Here, we describe a child with a novel homozygous nonsense mutation in B3GALNT2. The affected child has severe neurological disease since birth, including muscle disease manifested as hypotonia, muscle weakness, and wasting with elevated creatine kinase, eye disease including microphthalmia and blindness, brain disease with extensive brain malformations including massive hydrocephalus, diffuse cobblestone-lissencephaly, deformed craniocervical junction, and pontocerebellar hypoplasia. The clinical and radiologic findings are compatible with a diagnosis of severe muscle-eye-brain disease and more specifically Walker-Warburg syndrome. A more distinct aspect of the clinical phenotype in this child is the presence of refractory epilepsy in the form of epileptic spasms, epileptic encephalopathy, and West syndrome, as well as sensorineural hearing loss. These findings could expand the phenotype of B3GALNT2-related dystroglycanopathy. In this report, we also provide a detailed review of previously reported cases with B3GALNT2-related dystroglycanopathy and compare them to our reported child. In addition, we study the genotype-phenotype correlation in these cases.

Mechanistic aspects of the formation of α-dystroglycan and therapeutic research for the treatment of α-dystroglycanopathy: A review.

α-Dystroglycanopathy, an autosomal recessive disease, is associated with the development of a variety of diseases, including muscular dystrophy. In humans, α-dystroglycanopathy includes various types of congenital muscular dystrophy such as Fukuyama type congenital muscular dystrophy (FCMD), muscle eye brain disease (MEB), and the Walker Warburg syndrome (WWS), and types of limb girdle muscular dystrophy 2I (LGMD2I). α-Dystroglycanopathy share a common etiology, since it is invariably caused by gene mutations that are associated with the O-mannose glycosylation pathway of α-dystroglycan (α-DG). α-DG is a central member of the dystrophin glycoprotein complex (DGC) family in peripheral membranes, and the proper glycosylation of α-DG is essential for it to bind to extracellular matrix proteins, such as laminin, to cell components. The disruption of this ligand-binding is thought to result in damage to cell membrane integration, leading to the development of muscular dystrophy. Clinical manifestations of α-dystroglycanopathy frequently include mild to severe alterations in the central nervous system and optical manifestations in addition to muscular dystrophy. Eighteen causative genes for α-dystroglycanopathy have been identified to date, and it is likely that more will be reported in the near future. These findings have stimulated extensive and energetic investigations in this research field, and novel glycosylation pathways have been implicated in the process. At the same time, the use of gene therapy, antisense therapy, and enzymatic supplementation have been evaluated as therapeutic possibilities for some types of α-dystroglycanopathy. Here we review the molecular and clinical findings associated with α-dystroglycanopathy and the development of therapeutic approaches, by comparing the approaches with the development of Duchenne muscular dystrophy.

Respiratory management of patients with Fukuyama congenital muscular dystrophy.

BACKGROUND: Fukuyama congenital muscular dystrophy (FCMD), characterized by intellectual impairment associated with cortical migration defects, is an autosomal recessive disorder caused by mutation in the fukutin gene. It is the second most common type of muscular dystrophy in Japan. Respiratory dysfunction, along with cardiomyopathy, can be life-threatening in patients with advanced-stage FCMD. However, few reports have focused on this issue. METHODS: We retrospectively studied respiratory dysfunction and therapeutic management in 48 genetically diagnosed FCMD patients (mean age 11.0 years; range 3.6-31.9 years). RESULTS: Mechanical ventilation was initiated at a median age of 12.1 years in 16 patients, 14 of whom received non-invasive positive pressure ventilation (NPPV) while the other 2 underwent tracheostomy with invasive ventilation (TIV). The two TIV cases had unexpectedly required the initiation of ventilatory support at the ages of 15.7 and 18.0 years, respectively, because of unsuccessful extubation followed by serious respiratory infections, despite rather good respiratory function before these episodes. Patients carrying a compound heterozygous founder mutation or with a severe phenotype tended to need ventilatory support 2-3 years earlier than homozygous patients and those with the typical or mild phenotype. Mechanical insufflation-exsufflation (MI-E) interventions were also employed in six patients with serious dysphagia and were well-tolerated in all cases. CONCLUSION: For respiratory management, it is important to regularly evaluate respiratory function in FCMD patients over 10 years of age, since intellectual impairment and insomnia often mask the signs of respiratory dysfunction. Most patients, despite poor cooperation due to intellectual impairment, can tolerate NPPV and MI-E provided that a carefully worked-out plan is adopted.

[Current status and future prospects of research on Fukuyama muscular dystrophy].

Fukuyama congenital muscular dystrophy(FCMD) is a second common childhood muscular dystrophy in Japan. All FCMD patients have ancestral insertion of the SVA retrotransposal element into fukutin. We show that aberrant mRNA splicing induced by SVA exon-trapping caused FCMD. Introduction of 3 cocktailed antisense oligonucleotides(AONs) targeting around these splice sites prevented pathogenic splicing in FCMD patient cells and model mice, and normalized protein production and functions of Fukutin as well as O-glycosylation of α-dystroglycan. We show the promise of splicing modulation therapy as the first radical clinical treatment for FCMD in the near future. We also show that fukutin is prerequisite to ameliorate muscular dystrophic phenotype by myofiber-selective LARGE expression. Recent advances in FCMD are discussed.

Fukutin is prerequisite to ameliorate muscular dystrophic phenotype by myofiber-selective LARGE expression.

α-Dystroglycanopathy (α-DGP) is a group of muscular dystrophy characterized by abnormal glycosylation of α-dystroglycan (α-DG), including Fukuyama congenital muscular dystrophy (FCMD), muscle-eye-brain disease, Walker-Warburg syndrome, and congenital muscular dystrophy type 1D (MDC1D), etc. LARGE, the causative gene for MDC1D, encodes a glycosyltransferase to form [-3Xyl-α1,3GlcAß1-] polymer in the terminal end of the post-phosphoryl moiety, which is essential for α-DG function. It has been proposed that LARGE possesses the great potential to rescue glycosylation defects in α-DGPs regardless of causative genes. However, the in vivo therapeutic benefit of using LARGE activity is controversial. To explore the conditions needed for successful LARGE gene therapy, here we used Large-deficient and fukutin-deficient mouse models for MDC1D and FCMD, respectively. Myofibre-selective LARGE expression via systemic adeno-associated viral gene transfer ameliorated dystrophic pathology of Large-deficient mice even when intervention occurred after disease manifestation. However, the same strategy failed to ameliorate the dystrophic phenotype of fukutin-conditional knockout mice. Furthermore, forced expression of Large in fukutin-deficient embryonic stem cells also failed to recover α-DG glycosylation, however coexpression with fukutin strongly enhanced α-DG glycosylation. Together, our data demonstrated that fukutin is required for LARGE-dependent rescue of α-DG glycosylation, and thus suggesting new directions for LARGE-utilizing therapy targeted to myofibres.

New developments in exon skipping and splice modulation therapies for neuromuscular diseases.

INTRODUCTION: Antisense oligonucleotide (AON) therapy is a form of treatment for genetic or infectious diseases using small, synthetic DNA-like molecules called AONs. Recent advances in the development of AONs that show improved stability and increased sequence specificity have led to clinical trials for several neuromuscular diseases. Impressive preclinical and clinical data are published regarding the usage of AONs in exon-skipping and splice modulation strategies to increase dystrophin production in Duchenne muscular dystrophy (DMD) and survival of motor neuron (SMN) production in spinal muscular atrophy (SMA). AREAS COVERED: In this review, we focus on the current progress and challenges of exon-skipping and splice modulation therapies. In addition, we discuss the recent failure of the Phase III clinical trials of exon 51 skipping (drisapersen) for DMD. EXPERT OPINION: The main approach of AON therapy in DMD and SMA is to rescue ('knock up' or increase) target proteins through exon skipping or exon inclusion; conversely, most conventional antisense drugs are designed to knock down (inhibit) the target. Encouraging preclinical data using this 'knock up' approach are also reported to rescue dysferlinopathies, including limb-girdle muscular dystrophy type 2B, Miyoshi myopathy, distal myopathy with anterior tibial onset and Fukuyama congenital muscular dystrophy.

Mislocalization of fukutin protein by disease-causing missense mutations can be rescued with treatments directed at folding amelioration.

Fukuyama-type congenital muscular dystrophy (FCMD), the second most common childhood muscular dystrophy in Japan, is caused by alterations in the fukutin gene. Mutations in fukutin cause abnormal glycosylation of α-dystroglycan, a cell surface laminin receptor; however, the exact function and pathophysiological role of fukutin are unclear. Although the most prevalent mutation in Japan is a founder retrotransposal insertion, point mutations leading to abnormal glycosylation of α-dystroglycan have been reported, both in Japan and elsewhere. To understand better the molecular pathogenesis of fukutin-deficient muscular dystrophies, we constructed 13 disease-causing missense fukutin mutations and examined their pathological impact on cellular localization and α-dystroglycan glycosylation. When expressed in C2C12 myoblast cells, wild-type fukutin localizes to the Golgi apparatus, whereas the missense mutants A170E, H172R, H186R, and Y371C instead accumulated in the endoplasmic reticulum. Protein O-mannose ß1,2-N-acetylglucosaminyltransferase 1 (POMGnT1) also mislocalizes when co-expressed with these missense mutants. The results of nocodazole and brefeldin A experiments suggested that these mutant proteins were not transported to the Golgi via the anterograde pathway. Furthermore, we found that low temperature culture or curcumin treatment corrected the subcellular location of these missense mutants. Expression studies using fukutin-null mouse embryonic stem cells showed that the activity responsible for generating the laminin-binding glycan of α-dystroglycan was retained in these mutants. Together, our results suggest that some disease-causing missense mutations cause abnormal folding and localization of fukutin protein, and therefore we propose that folding amelioration directed at correcting the cellular localization may provide a therapeutic benefit to glycosylation-deficient muscular dystrophies.

Pathogenic exon-trapping by SVA retrotransposon and rescue in Fukuyama muscular dystrophy.

Fukuyama muscular dystrophy (FCMD; MIM253800), one of the most common autosomal recessive disorders in Japan, was the first human disease found to result from ancestral insertion of a SINE-VNTR-Alu (SVA) retrotransposon into a causative gene. In FCMD, the SVA insertion occurs in the 3' untranslated region (UTR) of the fukutin gene. The pathogenic mechanism for FCMD is unknown, and no effective clinical treatments exist. Here we show that aberrant messenger RNA (mRNA) splicing, induced by SVA exon-trapping, underlies the molecular pathogenesis of FCMD. Quantitative mRNA analysis pinpointed a region that was missing from transcripts in patients with FCMD. This region spans part of the 3' end of the fukutin coding region, a proximal part of the 3' UTR and the SVA insertion. Correspondingly, fukutin mRNA transcripts in patients with FCMD and SVA knock-in model mice were shorter than the expected length. Sequence analysis revealed an abnormal splicing event, provoked by a strong acceptor site in SVA and a rare alternative donor site in fukutin exon 10. The resulting product truncates the fukutin carboxy (C) terminus and adds 129 amino acids encoded by the SVA. Introduction of antisense oligonucleotides (AONs) targeting the splice acceptor, the predicted exonic splicing enhancer and the intronic splicing enhancer prevented pathogenic exon-trapping by SVA in cells of patients with FCMD and model mice, rescuing normal fukutin mRNA expression and protein production. AON treatment also restored fukutin functions, including O-glycosylation of α-dystroglycan (α-DG) and laminin binding by α-DG. Moreover, we observe exon-trapping in other SVA insertions associated with disease (hypercholesterolemia, neutral lipid storage disease) and human-specific SVA insertion in a novel gene. Thus, although splicing into SVA is known, we have discovered in human disease a role for SVA-mediated exon-trapping and demonstrated the promise of splicing modulation therapy as the first radical clinical treatment for FCMD and other SVA-mediated diseases.

Muscular dystrophies due to glycosylation defects: diagnosis and therapeutic strategies.

PURPOSE OF REVIEW: Dystroglycanopathies are a common group of diseases characterized by a reduction in α-dystroglycan glycosylation. This review discusses the recent novel discovery of additional dystroglycanopathy variants and progress in dystroglycanopathy animal models. RECENT FINDINGS: Several novel glycosyltransferase genes have been found to be responsible for a dystroglycanopathy phenotype, and in addition recessive mutations in DAG1 have been identified for the first time in a primary dystroglycanopathy. Studies in dystroglycanopathy mouse models have clarified some aspects of the structural defects observed in the central nervous system and in the eye, whereas a study in zebrafish implicates unfolded protein response in the pathogenesis of two of the secondary dystroglycanopathies. SUMMARY: Improved understanding of the molecular bases of dystroglycanopathies will lead to more precise diagnosis and genetic counseling; therapeutic strategies are being developed and tested in the preclinical models and it is hoped that these observations will pave the way to therapeutic interventions in humans.

[Emerging novel therapeutic strategy for α-dystroglycanopathy by Large].

The past decade of researches have revealed mutations of known or putative glycosyltransferases in several types of muscular dystrophy, including Fukuyama-type congenital muscular dystrophy. In these disorders, the function of α-dystroglycan is severely decreased, therefore they are called α-dystroglycanopathy. Recently, putative glycosyltransferase Large was shown to restore the defective function of α-dystroglycan, thus, it is an intriguing idea to apply this effect to the therapy of α-dystroglycanopathy. In the present study, we sought to test this possibility. Using several cultured cell lines, we confirmed that the overexpression of Large results in hyperglycosylation and marked enhancement of the function of α-dystroglycan. For this effect, the whole luminal domain of Large was shown to be necessary using several deletion constructs. We further generated transgenic mice overexpressing Large ubiquitously. In each tissue of the mice, the glycosylation of α-dystroglycan and its laminin binding activity was remarkably increased. Moreover, the morphological analyses on each tissue stained by H-E revealed no significant abnormality in the transgenic mice, suggesting no serious side effects by the overexpression of Large. Taken together, these results indicate that the restoration of the function of α-dystroglycan by Large should be an important molecular target to develop therapeutic strategies for α-dystroglycanopathy.