Stefania Assereto

Clinical and genetic heterogeneity of branching enzyme deficiency (glycogenosis type IV)

Background: Glycogen storage disease type IV (GSD-IV) is a clinically heterogeneous autosomal recessive disorder due to glycogen branching enzyme (GBE) deficiency and resulting in the accumulation of an amylopectin-like polysaccharide. The typical presentation is liver disease of childhood, progressing to lethal cirrhosis. The neuromuscular form of GSD-IV varies in onset (perinatal, congenital, juvenile, or adult) and severity. Objective: To identify the molecular bases of different neuromuscular forms of GSD-IV and to establish possible genotype/phenotype correlations. Methods: Eight patients with GBE deficiency had different neuromuscular presentations: three had fetal akinesia deformation sequence (FADS), three had congenital myopathy, one had juvenile myopathy, and one had combined myopathic and hepatic features. In all patients, the promoter and the entire coding region of the GBE gene at the RNA and genomic level were sequenced. Results: Nine novel mutations were identified, including nonsense, missense, deletion, insertion, and splice-junction mutations. The three cases with FADS were homozygous, whereas all other cases were compound heterozygotes. Conclusions: This study expands the spectrum of mutations in the GBE gene and confirms that the neuromuscular presentation of GSD-IV is clinically and genetically heterogeneous.

Novel Compound Heterozygous Mutations in TBC1D24 Cause Familial Malignant Migrating Partial Seizures of Infancy

Early‐onset epileptic encephalopathies (EOEEs) are a group of rare devastating epileptic syndromes of infancy characterized by severe drug‐resistant seizures and electroencephalographic abnormalities. The current study aims to determine the genetic etiology of a familial form of EOEE fulfilling the diagnosis criteria for malignant migrating partial seizures of infancy (MMPSI). We identified two inherited novel mutations in TBC1D24 in two affected siblings. Mutations severely impaired TBC1D24 expression and function, which is critical for maturation of neuronal circuits. The screening of TBC1D24 in an additional set of eight MMPSI patients was negative. TBC1D24 loss of function has been associated to idiopathic infantile myoclonic epilepsy, as well as to drug‐resistant early‐onset epilepsy with intellectual disability. Here, we describe a familial form of MMPSI due to mutation in TBC1D24, revealing a devastating epileptic phenotype associated with TBC1D24 dysfunction.

Therapeutic potential of proteasome inhibition in Duchenne and Becker muscular dystrophies.

Duchenne muscular dystrophy (DMD) and its milder allelic variant, Becker muscular dystrophy (BMD), result from mutations of the dystrophin gene and lead to progressive muscle deterioration. Enhanced activation of proteasomal degradation underlies critical steps in the pathogenesis of the DMD/BMD dystrophic process. Previously, we demonstrated that treatment with the proteasome inhibitor MG-132 rescues the cell membrane localization of dystrophin and the dystrophin glycoprotein complex in mdx mice, a natural genetic mouse model of DMD. The current work aims to thoroughly define the therapeutic potential in dystrophinopathies of Velcade, a drug that selectively blocks the ubiquitin-proteasome pathway. Velcade is particularly intriguing since it has been approved for the treatment of multiple myeloma. Therefore, its side effects in humans have been explored. Velcade effects were analyzed through two independent methodological approaches. First, we administered the drug systemically in mdx mice over a 2-week period. In this system, Velcade restores the membrane expression of dystrophin and dystrophin glycoprotein complex members and improves the dystrophic phenotype. In a second approach, we treated with the compound explants from muscle biopsies of DMD or BMD patients. We show that the inhibition of the proteasome pathway up-regulates dystrophin, alpha-sarcoglycan, and beta-dystroglycan protein levels in explants from BMD patients, whereas it increases the proteins of the dystrophin glycoprotein complex in DMD cases.

Activation of PKC-β isoforms mediates HNE-induced MCP-1 release by macrophages

Abstract 4-Hydroxynonenal (HNE) in the concentration range detectable in many pathophysiologic conditions is able to modulate signal transduction cascades and gene expression. Here, we report the stimulating effect of 1 μM HNE on the release of the monocyte chemotactic protein-1 (MCP-1) by murine macrophages. MCP-1-increased export following 1-h cell treatment with HNE proved to be comparable to that exerted by standard amounts of bacterial lipopolysaccharide (LPS). However, the key molecular event in HNE-induced secretion of MCP-1 appeared to be the increased activity of β-PKC isoforms, which are recognized as playing a role in the regulation of cell protein transport and secretion. On the other hand, in LPS-stimulated cells, the δ isoform was seen to be involved and was probably related to LPS-mediated effects on MCP-1 expression and synthesis. In conclusion, HNE might interact with other pro-inflammatory stimuli, like LPS, in a concerted amplification of MCP-1 production and secretion.

Caveolin-3 T78M and T78K missense mutations lead to different phenotypes in vivo and in vitro.

Caveolins are the principal protein components of caveolae, invaginations of the plasma membrane involved in cell signaling and trafficking. Caveolin-3 (Cav-3) is the muscle-specific isoform of the caveolin family and mutations in the CAV3 gene lead to a large group of neuromuscular disorders. In unrelated patients, we identified two distinct CAV3 mutations involving the same codon 78. Patient 1, affected by dilated cardiomyopathy and limb girdle muscular dystrophy (LGMD)-1C, shows an autosomal recessive mutation converting threonine to methionine (T78M). Patient 2, affected by isolated familiar hyperCKemia, shows an autosomal dominant mutation converting threonine to lysine (T78K). Cav-3 wild type (WT) and Cav-3 mutations were transiently transfected into Cos-7 cells. Cav-3 WT and Cav-3 T78M mutant localized at the plasma membrane, whereas Cav-3 T78K was retained in a perinuclear compartment. Cav-3 T78K expression was decreased by 87% when compared with Cav-3 WT, whereas Cav-3 T78M protein levels were unchanged. To evaluate whether Cav-3 T78K and Cav-3 T78M mutants behaved with a dominant negative pattern, Cos-7 cells were cotransfected with green fluorescent protein (GFP)-Cav-3 WT in combination with either mutant or WT Cav-3. When cotransfected with Cav-3 WT or Cav-3 T78M, GFP-Cav-3 WT was localized at the plasma membrane, as expected. However, when cotransfected with Cav-3 T78K, GFP-Cav-3 WT was retained in a perinuclear compartment, and its protein levels were reduced by 60%, suggesting a dominant negative action. Accordingly, Cav-3 protein levels in muscles from a biopsy of patient 2 (T78K mutation) were reduced by 80%. In conclusion, CAV3 T78M and T78K mutations lead to distinct disorders showing different clinical features and inheritance, and displaying distinct phenotypes in vitro.

Progressive exercise intolerance associated with a new muscle-restricted nonsense mutation (G142X) in the mitochondrial cytochrome b gene

We report a novel nonsense mitochondrial cytochrome b mutation (G15170A) in a 40‐year‐old woman with progressive exercise intolerance and lactic acidosis. Muscle biopsy showed several cytochrome c oxidase–positive ragged‐red fibers, and reduced activities of respiratory chain complexes I and III. This mutation, resulting in the loss of 228 amino acids of the protein, was very abundant in the patients muscle, but undetectable in lymphocytes and fibroblasts. Clinical and laboratory data indicate that this defect is the primary cause of the disease, thus adding a new mutation in the cytochrome b gene among the growing number of patients with exercise intolerance and lactic acidosis. Muscle Nerve 28: 508–511, 2003

Mitochondrial Myopathy and Respiratory Failure Associated With a New Mutation in the Mitochondrial Transfer Ribonucleic Acid Glutamic Acid Gene

We report a novel T14687C mutation in the mitochondrial transfer ribonucleic acid glutamic acid gene in a 16-year-old boy with myopathy and lactic acidosis, retinopathy, and progressive respiratory failure leading to death. A muscle biopsy showed cytochrome c oxidase—negative ragged-red fibers, and biochemical analysis of the respiratory chain enzymes in muscle homogenate revealed complex I and complex IV deficiencies. The mutation, which affects the trinucleotide (TΨC) loop, was nearly homoplasmic in the muscle DNA of the proband, but it was absent in his blood and in the blood from the asymptomatic mother, suggesting that it may have been a spontaneous somatic mutation in muscle. (J Child Neurol 2003;18:300—303).

Aquaporin-4 expression is severely reduced in human sarcoglycanopathies and dysferlinopathies

Aquaporin-4 (AQP4) is the major water channel expressed in fast-twitch skeletal muscle fibers. AQP4 is reduced in Duchenne and Becker Muscular Dystrophies, but not in caveolinopathies, thus suggesting an interaction with dystrophin or with members of the dystrophin-glycoprotein complex (DGC) rather than a nonspecific effect due to muscle membrane damage. To establish the role of sarcoglycans in AQP4 decrease occurring in muscular dystrophy, AQP4 expression was analyzed in muscle biopsies from patients affected by Limb Girdle Muscular Dystrophies (LGMDs) 2C-F genetically confirmed. In all the LGMD 2C-F (2α-, 1β-, 2γ-, 1δ-deficiency), AQP4 was severely decreased. This effect was associated to a marked reduction in α1-syntrophin levels. In control muscle AQP4 did not show a direct interaction with any of the four sarcoglycans but, it co-immunoprecipitated with α1-syntrophin, indicating that this modular protein may link AQP4 levels with the DGC complex. To determine whether AQP4 expression could be affected in other LGMDs due to the defect of a membrane protein not associated to the dystrophin complex, we examined AQP4 expression in 6 patients affected by dysferlin deficiency genetically confirmed. All the patients displayed a reduction of the water channel, and AQP4 expression appeared to correlate with the severity of the muscle histopathological lesions. However, differently from what observed in the sarcoglycans, α1-syntrophin expression was normal or just slightly reduced. These results seem to indicate an additional mechanism of regulation of AQP4 levels in muscle cells.In accordance with a specific effect of membrane muscle disorders, AQP4 protein levels were not changed in 3 mitochondrial and 3 metabolic myopathies. In conclusion, AQP4 expression and membrane localization are markedly reduced in LGMD 2B-2F. The role of AQP4 in the degenerative mechanism occurring in these diseases will be the object of our future research.

Electroclinical presentation and genotype-phenotype relationships in patients with Unverricht-Lundborg disease carrying compound heterozygous CSTB point and indel mutations.

Purpose:  Unverricht‐Lundborg disease (EPM1A) is frequently due to an unstable expansion of a dodecamer repeat in the CSTB gene, whereas other types of mutations are rare. EPM1A due to homozygous expansion has a rather stereotyped presentation with prominent action myoclonus. We describe eight patients with five different compound heterozygous CSTB point or indel mutations in order to highlight their particular phenotypical presentations and evaluate their genotype–phenotype relationships.

Mitochondrial DNA Deletion in a Child With Mitochondrial Encephalomyopathy, Growth Hormone Deficiency, and Hypoparathyroidism

We report an 11-year-old boy with short stature, bilateral ptosis, sensorineural hearing loss, muscle weakness, and endocrine abnormalities. Brain magnetic resonance imaging (MRI) showed a bilateral abnormal signal in the globus pallidus and in the midbrain tegment. Muscle biopsy specimens showed ragged red and cytochrome c oxidase negative fibers, and biochemical analysis of muscle homogenate showed a partial defect of complex I and IV activities of the respiratory chain enzymes. Analysis of mitochondrial DNA by a polymerase chain reaction screening procedure and Southern blot revealed a novel heteroplasmic single mitochondrial DNA deletion of 7.8 kb in different tissues. This deletion was absent in the blood DNA of his mother and brother. This case further expands and confirms the wide clinical spectrum of mitochondrial disorders associated with single large-scale mitochondrial DNA deletions. (J Child Neurol 2006;21:983—985; DOI 10.2310/7010.2006.00218).