Francesco Muntoni

Mutations in the gene encoding lamin A/C cause autosomal dominant Emery-Dreifuss muscular dystrophy

Emery-Dreifuss muscular dystrophy (EDMD) is characterized by early contractures of elbows and Achilles tendons, slowly progressive muscle wasting and weakness, and a cardiomyopathy with conduction blocks which is life-threatening. Two modes of inheritance exist, X-linked (OMIM 310300) and autosomal dominant (EDMD-AD; OMIM 181350). EDMD-AD is clinically identical to the X-linked forms of the disease. Mutations in EMD, the gene encoding emerin, are responsible for the X-linked form. We have mapped the locus for EDMD-AD to an 8-cM interval on chromosome 1q11-q23 in a large French pedigree, and found that the EMD phenotype in four other small families was potentially linked to this locus. This region contains the lamin A/C gene (LMNA), a candidate gene encoding two proteins of the nuclear lamina, lamins A and C, produced by alternative splicing. We identified four mutations in LMNA that co-segregate with the disease phenotype in the five families: one nonsense mutation and three missense mutations. These results are the first identification of mutations in a component of the nuclear lamina as a cause of inherited muscle disorder. Together with mutations in EMD (Refs 5,6), they underscore the potential importance of the nuclear envelope components in the pathogenesis of neuromuscular disorders.

Missense mutations in the rod domain of the lamin A/C gene as causes of dilated cardiomyopathy and conduction-system disease

BACKGROUND Inherited mutations cause approximately 35 percent of cases of dilated cardiomyopathy; however, few genes associated with this disease have been identified. Previously, we located a gene defect that was responsible for autosomal dominant dilated cardiomyopathy and conduction-system disease on chromosome 1p1-q21, where nuclear-envelope proteins lamin A and lamin C are encoded by the LMNA (lamin A/C) gene. Mutations in the head or tail domain of this gene cause Emery-Dreifuss muscular dystrophy, a childhood-onset disease characterized by joint contractures and in some cases by abnormalities of cardiac conduction during adulthood. METHODS We evaluated 11 families with autosomal dominant dilated cardiomyopathy and conduction-system disease. Sequences of the lamin A/C exons were determined in probands from each family, and variants were confirmed by restriction-enzyme digestion. The genotypes of the family members were ascertained. RESULTS Five novel missense mutations were identified: four in the alpha-helical-rod domain of the lamin A/C gene, and one in the lamin C tail domain. Each mutation caused heritable, progressive conduction-system disease (sinus bradycardia, atrioventricular conduction block, or atrial arrhythmias) and dilated cardiomyopathy. Heart failure and sudden death occurred frequently within these families. No family members with mutations had either joint contractures or skeletal myopathy. Serum creatine kinase levels were normal in family members with mutations of the lamin rod but mildly elevated in some family members with a defect in the tail domain of lamin C. CONCLUSIONS Genetic defects in distinct domains of the nuclear-envelope proteins lamin A and lamin C selectively cause dilated cardiomyopathy with conduction-system disease or autosomal dominant Emery-Dreifuss muscular dystrophy. Missense mutations in the rod domain of the lamin A/C gene provide a genetic cause for dilated cardiomyopathy and indicate that this intermediate filament protein has an important role in cardiac conduction and contractility.

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.

Local restoration of dystrophin expression with the morpholino oligomer AVI-4658 in Duchenne muscular dystrophy: a single-blind, placebo-controlled, dose-escalation, proof-of-concept study

Summary Background Mutations that disrupt the open reading frame and prevent full translation of DMD, the gene that encodes dystrophin, underlie the fatal X-linked disease Duchenne muscular dystrophy. Oligonucleotides targeted to splicing elements (splice switching oligonucleotides) in DMD pre-mRNA can lead to exon skipping, restoration of the open reading frame, and the production of functional dystrophin in vitro and in vivo, which could benefit patients with this disorder. Methods We did a single-blind, placebo-controlled, dose-escalation study in patients with DMD recruited nationally, to assess the safety and biochemical efficacy of an intramuscular morpholino splice-switching oligonucleotide (AVI-4658) that skips exon 51 in dystrophin mRNA. Seven patients with Duchenne muscular dystrophy with deletions in the open reading frame of DMD that are responsive to exon 51 skipping were selected on the basis of the preservation of their extensor digitorum brevis (EDB) muscle seen on MRI and the response of cultured fibroblasts from a skin biopsy to AVI-4658. AVI-4658 was injected into the EDB muscle; the contralateral muscle received saline. Muscles were biopsied between 3 and 4 weeks after injection. The primary endpoint was the safety of AVI-4658 and the secondary endpoint was its biochemical efficacy. This trial is registered, number NCT00159250. Findings Two patients received 0·09 mg AVI-4658 in 900 μL (0·9%) saline and five patients received 0·9 mg AVI-4658 in 900 μL saline. No adverse events related to AVI-4658 administration were reported. Intramuscular injection of the higher-dose of AVI-4658 resulted in increased dystrophin expression in all treated EDB muscles, although the results of the immunostaining of EDB-treated muscle for dystrophin were not uniform. In the areas of the immunostained sections that were adjacent to the needle track through which AVI-4658 was given, 44–79% of myofibres had increased expression of dystrophin. In randomly chosen sections of treated EDB muscles, the mean intensity of dystrophin staining ranged from 22% to 32% of the mean intensity of dystrophin in healthy control muscles (mean 26·4%), and the mean intensity was 17% (range 11–21%) greater than the intensity in the contralateral saline-treated muscle (one-sample paired t test p=0·002). In the dystrophin-positive fibres, the intensity of dystrophin staining was up to 42% of that in healthy muscle. We showed expression of dystrophin at the expected molecular weight in the AVI-4658-treated muscle by immunoblot. Interpretation Intramuscular AVI-4658 was safe and induced the expression of dystrophin locally within treated muscles. This proof-of-concept study has led to an ongoing systemic clinical trial of AVI-4658 in patients with DMD. Funding UK Department of Health.

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.

POMT2 mutations cause alpha-dystroglycan hypoglycosylation and Walker-Warburg syndrome

Background: Walker-Warburg syndrome (WWS) is an autosomal recessive condition characterised by congenital muscular dystrophy, structural brain defects, and eye malformations. Typical brain abnormalities are hydrocephalus, lissencephaly, agenesis of the corpus callosum, fusion of the hemispheres, cerebellar hypoplasia, and neuronal overmigration, which causes a cobblestone cortex. Ocular abnormalities include cataract, microphthalmia, buphthalmos, and Peters anomaly. WWS patients show defective O-glycosylation of α-dystroglycan (α-DG), which plays a key role in bridging the cytoskeleton of muscle and CNS cells with extracellular matrix proteins, important for muscle integrity and neuronal migration. In 20% of the WWS patients, hypoglycosylation results from mutations in either the protein O-mannosyltransferase 1 (POMT1), fukutin, or fukutin related protein (FKRP) genes. The other genes for this highly heterogeneous disorder remain to be identified. Objective: To look for mutations in POMT2 as a cause of WWS, as both POMT1 and POMT2 are required to achieve protein O-mannosyltransferase activity. Methods: A candidate gene approach combined with homozygosity mapping. Results: Homozygosity was found for the POMT2 locus at 14q24.3 in four of 11 consanguineous WWS families. Homozygous POMT2 mutations were present in two of these families as well as in one patient from another cohort of six WWS families. Immunohistochemistry in muscle showed severely reduced levels of glycosylated α-DG, which is consistent with the postulated role for POMT2 in the O-mannosylation pathway. Conclusions: A fourth causative gene for WWS was uncovered. These genes account for approximately one third of the WWS cases. Several more genes are anticipated, which are likely to play a role in glycosylation of α-DG.

Mutations in the gene encoding immunoglobulin μ-binding protein 2 cause spinal muscular atrophy with respiratory distress type 1

Classic spinal muscular atrophy (SMA) is caused by mutations in the telomeric copy of SMN1. Its product is involved in various cellular processes, including cytoplasmic assembly of spliceosomal small nuclear ribonucleoproteins, pre-mRNA processing and activation of transcription. Spinal muscular atrophy with respiratory distress (SMARD) is clinically and genetically distinct from SMA. Here we demonstrate that SMARD type 1 (SMARD1) results from mutations in the gene encoding immunoglobulin μ-binding protein 2 (IGHMBP2; on chromosome 11q13.2–q13.4). In six SMARD1 families, we detected three recessive missense mutations (exons 5, 11 and 12), two nonsense mutations (exons 2 and 5), one frameshift deletion (exon 5) and one splice donor-site mutation (intron 13). Mutations in mouse Ighmbp2 (ref. 14) have been shown to be responsible for spinal muscular atrophy in the neuromuscular degeneration (nmd) mouse, whose phenotype resembles the SMARD1 phenotype. Like the SMN1 product, IGHMBP2 colocalizes with the RNA-processing machinery in both the cytoplasm and the nucleus. Our results show that IGHMBP2 is the second gene found to be defective in spinal muscular atrophy, and indicate that IGHMBP2 and SMN share common functions important for motor neuron maintenance and integrity in mammals.

Familial dilated cardiomyopathy ☆: Evidence for genetic and phenotypic heterogeneity

OBJECTIVES This study was performed to evaluate the characteristics, mode of inheritance and etiology of familial dilated cardiomyopathy (FDC). BACKGROUND A genetic form of disease transmission has been identified in a relevant proportion of patients with dilated cardiomyopathy (DCM). Variable clinical characteristics and patterns of inheritance, and an increased frequency of cardiac antibodies have been reported. An analysis of FDC may improve the understanding of the disease and the management of patients. METHODS Of 350 consecutive patients with idiopathic DCM, 281 relatives from 60 families were examined. Family studies included clinical examination, electrocardiography, echocardiography and blood sampling. Of the 60 DCM index patients examined, 39 were attributable to FDC and 21 were due to sporadic DCM. Clinical features, histology, mode of inheritance and autoimmune serology were examined, molecular genetic studies were undertaken and the difference between familial and sporadic forms was analyzed. RESULTS Only a younger age (p = 0.0005) and a higher ejection fraction (p = 0.03) could clinically distinguish FDC patients from those with sporadic DCM. However, a number of distinct subtypes of FDC were identified: 1) autosomal dominant, the most frequent form (56%); 2) autosomal recessive (16%), characterized by worse prognosis; 3) X-linked FDC (10%), with different mutations of the dystrophin gene; 4) a novel form of autosomal dominant DCM with subclinical skeletal muscle disease (7.7%); 5) FDC with conduction defects (2.6%), and 6) rare unclassifiable forms (7.7%). The forms with skeletal muscle involvement were characterized by a restrictive filling pattern; the forms with isolated cardiomyopathy had an increased frequency of organ-specific cardiac autoantibodies. Histologic signs of myocarditis were frequent and nonspecific. CONCLUSIONS Familial dilated cardiomyopathy is frequent, cannot be predicted on a clinical or morphologic basis and requires family screening for identification. The phenotypic heterogeneity, different patterns of transmission, different frequencies of cardiac autoantibodies and the initial molecular genetic data indicate that multiple genes and pathogenetic mechanisms can lead to FDC.OBJECTIVES This study was performed to evaluate the characteristics, mode of inheritance and etiology of familial dilated cardiomyopathy (FDC). BACKGROUND A genetic form of disease transmission has been identified in a relevant proportion of patients with dilated cardiomyopathy (DCM). Variable clinical characteristics and patterns of inheritance, and an increased frequency of cardiac antibodies have been reported. An analysis of FDC may improve the understanding of the disease and the management of patients. METHODS Of 350 consecutive patients with idiopathic DCM, 281 relatives from 60 families were examined. Family studies included clinical examination, electrocardiography, echocardiography and blood sampling. Of the 60 DCM index patients examined, 39 were attributable to FDC and 21 were due to sporadic DCM. Clinical features, histology, mode of inheritance and autoimmune serology were examined, molecular genetic studies were undertaken and the difference between familial and sporadic forms was analyzed. RESULTS Only a younger age (p = 0.0005) and a higher ejection fraction (p = 0.03) could clinically distinguish FDC patients from those with sporadic DCM. However, a number of distinct subtypes of FDC were identified: 1) autosomal dominant, the most frequent form (56%); 2) autosomal recessive (16%), characterized by worse prognosis; 3) X-linked FDC (10%), with different mutations of the dystrophin gene; 4) a novel form of autosomal dominant DCM with subclinical skeletal muscle disease (7.7%); 5) FDC with conduction defects (2.6%), and 6) rare unclassifiable forms (7.7%). The forms with skeletal muscle involvement were characterized by a restrictive filling pattern; the forms with isolated cardiomyopathy had an increased frequency of organ-specific cardiac autoantibodies. Histologic signs of myocarditis were frequent and nonspecific. CONCLUSIONS Familial dilated cardiomyopathy is frequent, cannot be predicted on a clinical or morphologic basis and requires family screening for identification. The phenotypic heterogeneity, different patterns of transmission, different frequencies of cardiac autoantibodies and the initial molecular genetic data indicate that multiple genes and pathogenetic mechanisms can lead to FDC.

Lack of myostatin results in excessive muscle growth but impaired force generation

The lack of myostatin promotes growth of skeletal muscle, and blockade of its activity has been proposed as a treatment for various muscle-wasting disorders. Here, we have examined two independent mouse lines that harbor mutations in the myostatin gene, constitutive null (Mstn−/−) and compact (Berlin High Line, BEHc/c). We report that, despite a larger muscle mass relative to age-matched wild types, there was no increase in maximum tetanic force generation, but that when expressed as a function of muscle size (specific force), muscles of myostatin-deficient mice were weaker than wild-type muscles. In addition, Mstn−/− muscle contracted and relaxed faster during a single twitch and had a marked increase in the number of type IIb fibers relative to wild-type controls. This change was also accompanied by a significant increase in type IIB fibers containing tubular aggregates. Moreover, the ratio of mitochondrial DNA to nuclear DNA and mitochondria number were decreased in myostatin-deficient muscle, suggesting a mitochondrial depletion. Overall, our results suggest that lack of myostatin compromises force production in association with loss of oxidative characteristics of skeletal muscle.