Angela Amati

Clinical features and new molecular findings in muscle phosphofructokinase deficiency (GSD type VII)

Muscle phosphofructokinase (PFKM) deficiency, a rare disorder of glycogen metabolism also known as glycogen storage disease type VII (GSDVII), is characterized by exercise intolerance, myalgias, cramps and episodic myoglobinuria associated with compensated hemolytic anaemia and hyperuricemia. We studied five patients with PFKM deficiency coming from different Italian regions. All probands showed exercise intolerance, hyperCKemia, cramps and myoglobinuria. One patient had a mild hypertrophic cardiomyopathy. Biochemical studies revealed residual PFK activity ranging from 1 to 5%. Molecular genetic analysis identified four novel mutations in the PFKM gene. In our series of patients, clinical and laboratory features were similar in all but one patient, who had an unusual phenotype characterized by 25 ears disease history, high CK levels, hypertrophic cardiomyopathy with paroxysmal atrial fibrillation without fixed muscle weakness.

Overexpression of autophagic proteins in the skeletal muscle of sporadic inclusion body myositis

Sporadic inclusion body myositis (s‐IBM) is characterized by rimmed vacuole formation and misfolded protein accumulation. Intracellular protein aggregates are cleared by autophagy. When autophagy is blocked aggregates accumulate, resulting in abnormal rimmed vacuole formation. This study investigated the autophagy–lysosome pathway contribution to rimmed vacuole accumulation.

A new locus on 3p23-p25 for an autosomal-dominant limb-girdle muscular dystrophy, LGMD1H.

Limb-girdle muscular dystrophies (LGMDs) are a genetically heterogeneous group of neuromuscular disorders with a selective or predominant involvement of shoulder and pelvic girdles. We clinically examined 19 members in a four-generation Italian family with autosomal-dominant LGMD. A total of 11 subjects were affected. Clinical findings showed variable expressivity in terms of age at onset and disease severity. Five subjects presented with a slowly progressive proximal muscle weakness, in both upper and lower limbs, with onset during the fourth–fifth decade of life, which fulfilled the consensus diagnostic criteria for LGMD. Earlier onset of the disease was observed in a group of patients presenting with muscle weakness and/or calf hypertrophy, and/or occasionally high CK and lactate serum levels. Two muscle biopsies showed morphological findings compatible with MD associated with subsarcolemmal accumulation of mitochondria and the presence of multiple mitochondrial DNA deletions. A genome-wide scan performed using microsatellite markers mapped the disease on chromosome 3p23–p25.1 locus in a 25-cM region between markers D3S1263 and D3S3685. The highest two-point LOD score was 3.26 (θ=0) at marker D3S1286 and D3S3613, whereas non-parametric analysis reached a P-value=0.0004. Four candidate genes within the refined region were analysed but did not reveal any mutations. Our findings further expand the clinical and genetic heterogeneity of LGMDs.

Mitochondrial genome aberrations in skeletal muscle of patients with motor neuron disease.

Abstract Our objective was to assess the role of defects of mitochondrial function as contributing factors in the pathogenesis and/or progression of amyotrophic lateral sclerosis (ALS); mitochondrial genome structural alterations were investigated. DNA lesions, point alterations and gross rearrangements were screened by specific applications of real-time PCR including an optimized rapid gene-specific method for the accurate quantification of mitochondrial DNA (mtDNA) lesions as well as sequencing on skeletal muscle biopsies of three patients presenting with motor neuron disease. We found a higher frequency of mtDNA lesions, including multiple deletions, particularly in the only SOD1 mutated patient as well as in a patient negative for mutations in SOD1 but presenting a severe form of the disease. The occurrence and the extent of mtDNA lesions of the cases here presented were consistent in all the examined clinical phenotypes of ALS (SOD1 related ALS, bulbar onset, spinal onset) and correlated with the severity of clinical course of the illness and with the presence of SOD1 mutation as well. In conclusion, the strong association with mtDNA damages supports the hypothesis that mitochondrial dysfunction in skeletal muscle may contribute to the pathogenesis and progression of ALS.

Mitochondrial genome large rearrangements in the skeletal muscle of a patient with PMA

Sir, Progressive muscular atrophy is a motor neuron disease (MND) characterized by selective lower motor neuron involvement [1]. MND pathogenesis is unknown and different mechanisms, including oxidative stress, may be involved [2]. Mitochondrial (mt) abnormalities have been observed in motor neurons of patients with MND [2], and MND patients carrying pathogenic mtDNA alteration have been reported [3]. A 63-year-old woman presented with a 4-year history of proximal upper limb muscle weakness and hypotrophy. Informed consent was obtained. Neurological examination revealed muscle weakness, hypotrophy and fasciculations with the absence of deep tendon reflexes in upper limbs. Cranial nerves and lower limbs function were normal. Laboratory examinations, SOD1, and SMA analysis were normal. Electromyography of the four limb muscles showed diffuse chronic denervation with fibrillations. Motor, sensory evoked potentials were normal. Magnetic Resonance Imaging (MRI) of brain was normal, whilst MRI of the spinal cord revealed cervical and lumbar disks protrusion. The muscular weakness slowly

Molecular analysis in a family presenting with a mild form of late-onset autosomal dominant chronic progressive external ophthalmoplegia.

Nuclear genes affecting mitochondrial genome stability were screened in an Italian family presenting with autosomal dominant progressive external ophthalmoplegia (adPEO) associated with multiple mitochondrial DNA (mtDNA) deletions. We report on a heterozygous c.907C>T (p.R303W) mutation found in the N-terminal domain of the human mitochondrial DNA helicase, Twinkle protein, in six members of a family, in which two individuals manifested late-onset PEO and morphological and molecular signs of mitochondrial dysfunction along with two carriers who are presently free of disease manifestation. We also investigated if the p.R303W mutation in PEO1 gene affected the relative copy number of mitochondrial DNA genomes.

Mitochondrial Disease Mimicking Polymyositis: A Case Report

Abstract: The authors report on a 34-year-old woman who had developed severe weakness and reduction in grip strength in both upper and lower limbs. Laboratory blood tests revealed increased levels of muscle enzyme. The presence of progressive bilateral ptosis and external ophthalmoplegia raised the suspicion of a mitochondrial disease, subsequently confirmed by deltoid biopsy and genetic analysis of mitochondrial DNA that showed a deletion indicative of Kearns–Sayre syndrome. In this report we emphasise the need for a differential diagnosis between myositis and other myopathies, particularly the mitochondrial ones.

A rare case of multiple sclerosis and McArdle disease

Multiple sclerosis (MS) might share clinical features with muscular diseases (MD); however, only sporadic association between MS and MD has been reported [1–3]. A 44-year-old woman presented with a family history of MS and MD. Her mother and her uncle were wheelchair bound due to a progressive muscle weakness and wasting in the four limbs; two sisters were affected by MS. Patient clinical history revealed severe obesity and autoimmune thyroiditis. Neurological symptoms started at 39 years of age with progressive muscle weakness and dysesthesias in the left limbs. She developed ataxia and urinary urgency at 41 years of age. Brain, spinal MRI showed several T2-hyperintense lesions involving frontal, parietal, temporoparietal white matter, corpus callosum, semioval centers and cervical spinal cord (Fig. 1). Visualand sensorial-evoked potential showed, respectively, a bilateral delay of P100 and a delay in right N13 wave; brainstem-evoked potentials were normal. Cerebrospinal fluid analysis showed intrathecal oligoclonal IgG synthesis. MS was diagnosed and interferon beta-1a was initiated (but early suspended due to liver toxicity). Then azathioprine was begun but the development of herpetic stomatitis required interruption. On our observation, the patient showed ataxic/spastic gate, possible only for 300 m, with nystagmus. The clinical assessment of the upper limbs showed diffuse muscle weakness, wasting of the scapular girdle, and brisk tendon reflexes. In lower limbs muscle weakness, wasting of the pelvic girdle, calf pseudohypertrophy and normal deep tendon reflexes were observed. Left hemisomatic tactile, pain hypoesthesia, moderate hypopallesthesia, and urinary urgency were also observed. EDSS score was 4.5. Laboratory assessment showed a slight CK increase (350 U/L), a decrease in D-vitamin content (10 ng/mL). A careful neuromuscular evaluation was performed to better elucidate the possible cause of the predominant pelvic and scapular girdles muscle weakness and wasting, which is not usual in patients with relapsing remitting MS. Electromyography was myopathic. Muscular biopsy of the right deltoid showed caliber variability of the muscle fibers, a subsarcolemmal increased mitochondrial proliferation was present in some fibers on SDH. Histoenzymatic analysis revealed the absence of myophosphorylase enzymatic activity confirmed in scheletric muscle homogenate (0.02 nmol/min/mg protein; normal: 0.4 ± 0.12). Molecular analysis of the 20 exons and flanking intronic boundaries of myophosphorylase (PGYM) gene, conducted by polymerase chain reaction and direct sequencing, revealed a transition G[A at codon 220 with no aminoacid change (Q220Q) in compound heterozygosity with IVS14?1G[A. None of the family members gave the informed consent to the genetic counseling. Treatment with glatiramer acetate was started. In the following 3 years the patient developed an optic neuritis, recovering after methylprednisolone treatment, and a worsening of gait imbalance and urinary urgency, only partially improving after steroids. She presented also partial complex seizures fully controlled with levetiracetam. & Stefano Zoccolella stefzoc@hotmail.it

activation of autophagy and suspended apoptosis in skeletal muscle of inclusion body myositis

Inclusion Body Myositis (IBM) is characterized by rimmed vacuole formation and misfolded protein accumulation, both depending on lysosome dysfunction. In skeletal muscle, selective protein degradation is allowed by macroautophagy. A proper balance in degradation and accumulation of proteins and organelles is critical for cell survival. Extracellular signal-regulated protein kinase (ERK1/2) is essential in cell survival, but recent evidence suggests that it is also necessary for autophagy. Alteration in subcellular localization of ERK promotes cell death either via autophagic death or via apoptosis upstream caspase-3. Moreover, in IBM myocytes there is no convincing evidence for apoptosis. Here, we correlated the expression level of autophagic and apoptotic molecules with that of ERK2 by analysing, with immunohistochemistry (IHC) and western blot (WB) methods, immunolocalization and expression of a panel of molecules directly involved and/or associated with the disease histopathogenesis: coated vesicles protein clathrin, mannose-6-phosphate receptor (M6PR), autophagy related proteins Beclin1 and ATG5, microtubule associated protein light chain LC3a and LC3b, Apoptotic Protease Activating Factor 1 (APAF1), Caspase-3, ERK2, and the specific IBM marker SMI31. Muscle biopsy specimens were obtained from 10 patients with sporadic IBM, 1 familial IBM patient, 1 amyotrophic lateral sclerosis patient, 1 patient with polymyositis with prominent mitochondrial pathology and 9 non myophatic patients as control specimens. IHC studies of expression and colocalization revealed an increase of clathrin, Beclin1, ATG5, and LC3 immunoreactivity, mainly observed in the sarcoplasm of small, atrophic fibres in all diseased specimens compared to controls. By WB analysis, expression level of both APAF1 and Caspase-3 did not significantly change between patients and controls, whereas the level of expression of ERK2 and autophagy markers seemed to inversely correlate. The results demonstrated that transport of newly synthesized lysosome enzymes and formation of autophagic vacuoles are both activated in IBM muscle. ERK2 phosphorylating activity is probably involved in rescue attempt to overcome the cell injury rather than directly stimulating the cell death. During IBM, the apoptotic cascade seems to be suspended, however,under the effect of cytotoxic stimuli, protective autophagy may switch to autophagic programmed cell death.