Silvia Torelli

Exon skipping and dystrophin restoration in patients with Duchenne muscular dystrophy after systemic phosphorodiamidate morpholino oligomer treatment: an open-label, phase 2, dose-escalation study

Summary Background We report clinical safety and biochemical efficacy from a dose-ranging study of intravenously administered AVI-4658 phosphorodiamidate morpholino oligomer (PMO) in patients with Duchenne muscular dystrophy. Method We undertook an open-label, phase 2, dose-escalation study (0·5, 1·0, 2·0, 4·0, 10·0, and 20·0 mg/kg bodyweight) in ambulant patients with Duchenne muscular dystrophy aged 5–15 years with amenable deletions in DMD. Participants had a muscle biopsy before starting treatment and after 12 weekly intravenous infusions of AVI-4658. The primary study objective was to assess safety and tolerability of AVI-4658. The secondary objectives were pharmacokinetic properties and the ability of AVI-4658 to induce exon 51 skipping and dystrophin restoration by RT-PCR, immunohistochemistry, and immunoblotting. The study is registered, number NCT00844597. Findings 19 patients took part in the study. AVI-4658 was well tolerated with no drug-related serious adverse events. AVI-4658 induced exon 51 skipping in all cohorts and new dystrophin protein expression in a significant dose-dependent (p=0·0203), but variable, manner in boys from cohort 3 (dose 2 mg/kg) onwards. Seven patients responded to treatment, in whom mean dystrophin fluorescence intensity increased from 8·9% (95% CI 7·1–10·6) to 16·4% (10·8–22·0) of normal control after treatment (p=0·0287). The three patients with the greatest responses to treatment had 21%, 15%, and 55% dystrophin-positive fibres after treatment and these findings were confirmed with western blot, which showed an increase after treatment of protein levels from 2% to 18%, from 0·9% to 17%, and from 0% to 7·7% of normal muscle, respectively. The dystrophin-associated proteins α-sarcoglycan and neuronal nitric oxide synthase were also restored at the sarcolemma. Analysis of the inflammatory infiltrate indicated a reduction of cytotoxic T cells in the post-treatment muscle biopsies in the two high-dose cohorts. Interpretation The safety and biochemical efficacy that we present show the potential of AVI-4658 to become a disease-modifying drug for Duchenne muscular dystrophy. Funding UK Medical Research Council; AVI BioPharma.

Dystrophin and mutations: one gene, several proteins, multiple phenotypes

A large and complex gene on the X chromosome encodes dystrophin. Many mutations have been described in this gene, most of which affect the expression of the muscle isoform, the best-known protein product of this locus. These mutations result in the Duchenne and Becker muscular dystrophies (DMD and BMD). However, there are several other tissue specific isoforms of dystrophin, some exclusively or predominantly expressed in the brain or the retina. Mutations affecting the correct expression of these tissue-specific isoforms have been associated with the CNS involvement common in DMD. Rare mutations also account for the allelic disorder X-linked dilated cardiomyopathy, in which dystrophin expression or function is affected mostly or exclusively in the heart. Genotype definition of the dystrophin gene in patients with dystrophinopathies has taught us much about functionally important domains of the protein itself and has provided insights into several regulatory mechanisms governing the gene expression profile. Here, we focus on current understanding of the genotype-phenotype relation for mutations in the dystrophin gene and their implications for gene functions.

Mutations in the fukutin-related protein gene (FKRP) cause a form of congenital muscular dystrophy with secondary laminin alpha2 deficiency and abnormal glycosylation of alpha-dystroglycan.

The congenital muscular dystrophies (CMD) are a heterogeneous group of autosomal recessive disorders presenting in infancy with muscle weakness, contractures, and dystrophic changes on skeletal-muscle biopsy. Structural brain defects, with or without mental retardation, are additional features of several CMD syndromes. Approximately 40% of patients with CMD have a primary deficiency (MDC1A) of the laminin alpha2 chain of merosin (laminin-2) due to mutations in the LAMA2 gene. In addition, a secondary deficiency of laminin alpha2 is apparent in some CMD syndromes, including MDC1B, which is mapped to chromosome 1q42, and both muscle-eye-brain disease (MEB) and Fukuyama CMD (FCMD), two forms with severe brain involvement. The FCMD gene encodes a protein of unknown function, fukutin, though sequence analysis predicts it to be a phosphoryl-ligand transferase. Here we identify the gene for a new member of the fukutin protein family (fukutin related protein [FKRP]), mapping to human chromosome 19q13.3. We report the genomic organization of the FKRP gene and its pattern of tissue expression. Mutations in the FKRP gene have been identified in seven families with CMD characterized by disease onset in the first weeks of life and a severe phenotype with inability to walk, muscle hypertrophy, marked elevation of serum creatine kinase, and normal brain structure and function. Affected individuals had a secondary deficiency of laminin alpha2 expression. In addition, they had both a marked decrease in immunostaining of muscle alpha-dystroglycan and a reduction in its molecular weight on western blot analysis. We suggest these abnormalities of alpha-dystroglycan are caused by its defective glycosylation and are integral to the pathology seen in MDC1C.

Phenotypic spectrum associated with mutations in the fukutin-related protein gene

We describe 22 patients with mutations in the fukutin‐related protein (FKPR) gene. Four patients had congenital muscular dystrophy (MDC1C), with presentation at birth, severe weakness and inability to stand unsupported. The other 18 had limb girdle muscular dystrophy (LGMD2I). Eleven showed a Duchenne‐like course with loss of ambulation in the early teens while 7 had a milder phenotype. Muscle biopsy invariably showed abnormal expression of a‐dystroglycan. MDC1C patients either carried 2 missense or 1 missense and 1 nonsense mutations. Patients with LGMD2I shared a common mutation (C826A,Leu276Ileu) and their phenotypic severity was correlated with the second allelic mutation. Ann Neurol 2003;53:537–542

Abnormalities in α-dystroglycan expression in MDC1C and LGMD2I muscular dystrophies

We recently identified mutations in the fukutin related protein (FKRP) gene in patients with congenital muscular dystrophy type 1C (MDC1C) and limb girdle muscular dystrophy type 2I (LGMD2I). The sarcolemma of these patients typically displays an immunocytochemical reduction of α-dystroglycan. In this report we extend these observations and report a clear correlation between the residual expression of α-dystroglycan and the phenotype. Three broad categories were identified. Patients at the severe end of the clinical spectrum (MDC1C) were compound heterozygote between a null allele and a missense mutation or carried two missense mutations and displayed a profound depletion of α-dystroglycan. Patients with LGMD with a Duchenne-like severity typically had a moderate reduction in α-dystroglycan and were compound heterozygotes between a common C826A (Leu276Ileu) FKRP mutation and either a missense or a nonsense mutation. Individuals with the milder form of LGMD2I were almost invariably homozygous for the Leu276Ile FKRP mutation and showed a variable but subtle alteration in α-dystroglycan immunolabeling. Our data therefore suggest a correlation between a reduction in α-dystroglycan, the mutation and the clinical phenotype in MDC1C and LGMD2I which supports the hypothesis that dystroglycan plays a central role in the pathogenesis of these disorders.

Defective glycosylation in muscular dystrophy

CONTEXT Over the past 15 years the causative genes of several inherited muscular dystrophies have been identified. These genes encode sarcolemmal, extracellular matrix, sarcomeric, and nuclear envelope proteins. Although the post-translational processing of muscle proteins has a significant role in their correct assembly and function, these processes have not been shown to be primarily involved in the pathogenesis of muscular dystrophies until recently. In the past 18 months, four different forms of inherited muscular dystrophy in human beings have been associated with mutations in genes encoding for putative glycosyltransferases. Aberrant glycosylation of alpha-dystroglycan, an external membrane protein expressed in muscle, brain, and other tissues, is a common feature in these disorders. alpha-dystroglycan is highly glycosylated, its sugar components varying in different tissues and controlling its interaction with extracellular matrix partners. Disrupted glycosylation of alpha-dystroglycan results in a loss of these interactions, giving rise to both progressive muscle degeneration and abnormal neuronal migration in the brain. STARTING POINT Kevin Campbell and colleagues have recently demonstrated that patients with muscle-eye-brain disease (MEB) and Fukuyama congenital muscular dystrophy (FCMD), as well as the myodystrophy (myd) mouse, have an abnormally glycosyated form of alpha-dystroglycan (Nature 2002; 418: 417-22 and 422-25). The abnormally glycosylated protein did not bind to three of its extracellular matrix ligands, laminin alpha2 chain, agrin, and neurexin. The investigators also showed that a neuronal migration disorder occurs in both the myd mouse and in a brain-restricted alpha-dystroglycan knock-out mouse that is similar to that seen in patients with MEB and FCMD. These results identify alpha-dystroglycan as having an essential role in both muscle and brain development and function. WHERE NEXT Emphasis is moving away from identifying the protein components of the muscle fibre that are involved in muscular dystrophies towards the post-translational processing of proteins and the enzymes involved in these modifications. This opens up new avenues of research. Abnormal glycosylation of alpha-dystroglycan may underlie other as yet uncharacterised forms of muscular dystrophy and neuronal migration disorders.

Loss-of-function mutations in MICU1 cause a brain and muscle disorder linked to primary alterations in mitochondrial calcium signaling

Mitochondrial Ca2+ uptake has key roles in cell life and death. Physiological Ca2+ signaling regulates aerobic metabolism, whereas pathological Ca2+ overload triggers cell death. Mitochondrial Ca2+ uptake is mediated by the Ca2+ uniporter complex in the inner mitochondrial membrane, which comprises MCU, a Ca2+-selective ion channel, and its regulator, MICU1. Here we report mutations of MICU1 in individuals with a disease phenotype characterized by proximal myopathy, learning difficulties and a progressive extrapyramidal movement disorder. In fibroblasts from subjects with MICU1 mutations, agonist-induced mitochondrial Ca2+ uptake at low cytosolic Ca2+ concentrations was increased, and cytosolic Ca2+ signals were reduced. Although resting mitochondrial membrane potential was unchanged in MICU1-deficient cells, the mitochondrial network was severely fragmented. Whereas the pathophysiology of muscular dystrophy and the core myopathies involves abnormal mitochondrial Ca2+ handling, the phenotype associated with MICU1 deficiency is caused by a primary defect in mitochondrial Ca2+ signaling, demonstrating the crucial role of mitochondrial Ca2+ uptake in humans.

Mutations in GDP-Mannose Pyrophosphorylase B Cause Congenital and Limb-Girdle Muscular Dystrophies Associated with Hypoglycosylation of α-Dystroglycan

Congenital muscular dystrophies with hypoglycosylation of α-dystroglycan (α-DG) are a heterogeneous group of disorders often associated with brain and eye defects in addition to muscular dystrophy. Causative variants in 14 genes thought to be involved in the glycosylation of α-DG have been identified thus far. Allelic mutations in these genes might also cause milder limb-girdle muscular dystrophy phenotypes. Using a combination of exome and Sanger sequencing in eight unrelated individuals, we present evidence that mutations in guanosine diphosphate mannose (GDP-mannose) pyrophosphorylase B (GMPPB) can result in muscular dystrophy variants with hypoglycosylated α-DG. GMPPB catalyzes the formation of GDP-mannose from GTP and mannose-1-phosphate. GDP-mannose is required for O-mannosylation of proteins, including α-DG, and it is the substrate of cytosolic mannosyltransferases. We found reduced α-DG glycosylation in the muscle biopsies of affected individuals and in available fibroblasts. Overexpression of wild-type GMPPB in fibroblasts from an affected individual partially restored glycosylation of α-DG. Whereas wild-type GMPPB localized to the cytoplasm, five of the identified missense mutations caused formation of aggregates in the cytoplasm or near membrane protrusions. Additionally, knockdown of the GMPPB ortholog in zebrafish caused structural muscle defects with decreased motility, eye abnormalities, and reduced glycosylation of α-DG. Together, these data indicate that GMPPB mutations are responsible for congenital and limb-girdle muscular dystrophies with hypoglycosylation of α-DG.

Defective glycosylation in congenital muscular dystrophies

Purpose of reviewThe recent identification of mutations in five genes coding for proteins with putative or demonstrated glycosyltransferase activity has shed light on a novel mechanism responsible for muscular dystrophy. Abnormal glycosylation of α-dystroglycan appears to be a common finding in all these conditions. Surprisingly, the disease severity due to mutations in several of these genes is extremely variable. This article provides an overview of the clinical, biochemical and genetic advances that have been made over the last year in this field. Recent findingsMutations in the human LARGE gene, a putative glycosyltransferase mutated in the myodystrophy mouse, have now been identified in a form of human muscular dystrophy. In addition, the clinical variability of patients with mutations in the genes encoding fukutin, protein O-linked mannose β1,2-N-acetylglucosaminyltransferase 1 and the fukutin-related protein has been significantly expanded. Disease severity in patients with mutations in the gene encoding the fukutin-related protein varies from a severe prenatal form of congenital muscular dystrophy with cobblestone lissencephaly and structural eye defects to a mild form of limb-girdle muscular dystrophy with onset in adult life and neither brain nor eye involvement. SummaryGlycosylation disorders represent a rapidly growing and common group of muscular dystrophies. Accurate genetic diagnosis can now be made for five forms, and it is anticipated that several other variants will eventually fall into these categories.

Mutations in B3GALNT2 Cause Congenital Muscular Dystrophy and Hypoglycosylation of α-Dystroglycan

Mutations in several known or putative glycosyltransferases cause glycosylation defects in α-dystroglycan (α-DG), an integral component of the dystrophin glycoprotein complex. The hypoglycosylation reduces the ability of α-DG to bind laminin and other extracellular matrix ligands and is responsible for the pathogenesis of an inherited subset of muscular dystrophies known as the dystroglycanopathies. By exome and Sanger sequencing we identified two individuals affected by a dystroglycanopathy with mutations in β-1,3-N-acetylgalactosaminyltransferase 2 (B3GALNT2). B3GALNT2 transfers N-acetyl galactosamine (GalNAc) in a β-1,3 linkage to N-acetyl glucosamine (GlcNAc). A subsequent study of a separate cohort of individuals identified recessive mutations in four additional cases that were all affected by dystroglycanopathy with structural brain involvement. We show that functional dystroglycan glycosylation was reduced in the fibroblasts and muscle (when available) of these individuals via flow cytometry, immunoblotting, and immunocytochemistry. B3GALNT2 localized to the endoplasmic reticulum, and this localization was perturbed by some of the missense mutations identified. Moreover, knockdown of b3galnt2 in zebrafish recapitulated the human congenital muscular dystrophy phenotype with reduced motility, brain abnormalities, and disordered muscle fibers with evidence of damage to both the myosepta and the sarcolemma. Functional dystroglycan glycosylation was also reduced in the b3galnt2 knockdown zebrafish embryos. Together these results demonstrate a role for B3GALNT2 in the glycosylation of α-DG and show that B3GALNT2 mutations can cause dystroglycanopathy with muscle and brain involvement.