Patrizia Ciscato

Facioscapulohumeral muscular dystrophy in mice overexpressing FRG1

Facioscapulohumeral muscular dystrophy (FSHD) is an autosomal dominant neuromuscular disorder that is not due to a classical mutation within a protein-coding gene. Instead, almost all FSHD patients carry deletions of an integral number of tandem 3.3-kilobase repeat units, termed D4Z4, located on chromosome 4q35 (ref. 3). D4Z4 contains a transcriptional silencer whose deletion leads to inappropriate overexpression in FSHD skeletal muscle of 4q35 genes located upstream of D4Z4 (ref. 4). To identify the gene responsible for FSHD pathogenesis, we generated transgenic mice selectively overexpressing in skeletal muscle the 4q35 genes FRG1, FRG2 or ANT1. We find that FRG1 transgenic mice develop a muscular dystrophy with features characteristic of the human disease; by contrast, FRG2 and ANT1 transgenic mice seem normal. FRG1 is a nuclear protein and several lines of evidence suggest it is involved in pre-messenger RNA splicing. We find that in muscle of FRG1 transgenic mice and FSHD patients, specific pre-mRNAs undergo aberrant alternative splicing. Collectively, our results suggest that FSHD results from inappropriate overexpression of FRG1 in skeletal muscle, which leads to abnormal alternative splicing of specific pre-mRNAs.

β-enolase deficiency, a new metabolic myopathy of distal glycolysis

A severe muscle enolase deficiency, with 5% of residual activity, was detected in a 47‐year‐old man affected with exercise intolerance and myalgias. No rise of serum lactate was observed with the ischemic forearm exercise. Ultrastructural analysis showed focal sarcoplasmic accumulation of glycogen β particles. The enzyme enolase catalyzes the interconversion of 2‐phosphoglycerate and phosphoenolpyruvate. In adult human muscle, over 90% of enolase activity is accounted for by the β‐enolase subunit, the protein product of the ENO3 gene. The β‐enolase protein was dramatically reduced in the muscle of our patient, by both immunohistochemistry and immunoblotting, while α‐enolase was normally represented. The ENO3 gene of our patient carries two heterozygous missense mutations affecting highly conserved amino acid residues: a G467A transition changing a glycine residue at position 156 to aspartate, in close proximity to the catalytic site, and a G1121A transition changing a glycine to glutamate at position 374. These mutations were probably inherited as autosomal recessive traits since the mother was heterozygous for the G467A and a sister was heterozygous for the G1121A transition. Our data suggest that ENO3 mutations result in decreased stability of mutant β‐enolase. Muscle β‐enolase deficiency should be considered in the differential diagnosis of metabolic myopathies due to inherited defects of distal glycolysis.

Retrospective study of a large population of patients with asymptomatic or minimally symptomatic raised serum creatine kinase levels

Abstract A retrospective evaluation of asymptomatic subjects with persistent elevation of serum creatine kinase (CK) levels (hyperCKemia) was made in order to verify the presence of subclinical myopathy or idiopathic hyperCKemia and to define the most appropriate diagnostic pathway. Persistently increased serum CK levels are occasionally encountered in healthy individuals. In 1980 Rowland coined for them the term idiopathic hyperCKemia. Despite the increase of scientific knowledge, several healthy subjects with hyperCKemia still represent a problem for the clinician.We made a retrospective evaluation of 114 asymptomatic or minimally symptomatic individuals with incidentally detected persistent hyperCKemia. They underwent neurological examination and laboratory/instrumental evaluation. Skeletal muscle biopsy was performed and thoroughly investigated. Biochemical and genetic investigations were added in selected cases. Logistic regression analysis was applied.We diagnosed a neuromuscular disorder in 21 patients (18.4 %), and found, by muscle biopsy and/or EMG, pathological but not conclusive findings in 57 subjects (50 %). The statistic correlation between elevated serum CK levels and the probability of making a diagnosis changed according to the age of the patient. Conclusions Muscle biopsy is the basic tool for screening asymptomatic subjects with hyperCkemia. It allowed us to make a diagnosis of disease in 18.4 % of patients, and to detect skeletal muscle abnormalities in 38.6 % of the subjects. Interestingly, 31.6 % of individuals had completely normal muscle findings. These best fit the “diagnosis” of idiopathic hyperCKemia.

Desmin and Vimentin as markers of regeneration in muscle diseases

SummaryLocalization and distribution of desmin and vimentin have been studied in different neuromuscular disorders using monoclonal antibodies. We have demonstrated that vimentin, although virtually absent in normal human muscle fibers, is expressed in regenerating fibers in different neuromuscular disorders. Moreover, these fibers showed a strong positivity with desmin antibodies. In normal muscle fibers desmin is only localized at Z-line level. These results suggest that desmin and vimentin may be over-expressed during muscle regeneration processes, probably because of their importance in the structural organization of the sarcomere.

Retrospective study of a large population of patients affected with mitochondrial disorders: clinical, morphological and molecular genetic evaluation.

Abstract Mitochondrial disorders are human genetic diseases with extremely variable clinical and genetic features. To better define them, we made a genotype-phenotype correlation in a series of 207 affected patients, and we examined most of them with six laboratory examinations (serum CK and basal lactate levels, EMG, cardiac and EEG studies, neuroradiology). We found that, depending on the genetic abnormality, hyperckemia occurs most often with either chronic progressive external ophthalmoplegia (CPEO) and ptosis or with limb weakness. Myopathic EMGs are more common than limb weakness, except in patients with A8344G mutations. Peripheral neuropathy, when present, is always axonal. About 80 % of patients with A3243G and A8344G mutations have high basal lactate levels, whereas pure CPEO is never associated with increased lactate levels. Cardiac abnormalities mostly consist of conduction defectsAbnormalities on CT or MRI of the brain are relatively common in A3243G mutations independently of the clinical phenotype. Patients with multiple mtDNA deletions are somehow “protected” against the development of abnormalities with any of the tests. We conclude that, despite the phenotypic heterogeneity of mitochondrial disorders, correlation of clinical features and laboratory findings may give the clinician important clues to the genetic defect, allowing earlier diagnosis and counselling.

Congenital myopathy associated with abnormal accumulation of desmin and dystrophin

We studied a 5-yr-old boy clinically presenting congenital myopathy. Muscle biopsy showed sarcoplasmic accumulation of desmin filaments leading to diagnosis of desmin storage myopathy. An immunohistochemical study of other cytoskeletal proteins (actin, alpha-actinin, vimentin and dystrophin) was performed. Desmin positive areas reacted strongly with anti-mid-rod and C-terminus dystrophin antibodies. Probed with the same antibodies by Western blot, desmin and dystrophin showed normal molecular size but densitometric analysis demonstrated a parallel increase of both proteins. Our results indicate that intrasarcoplasmic desmin storage is associated with an abnormal accumulation of dystrophin. Since no other cytoskeletal proteins are accumulated this finding seems to be specific and suggests a possible structural and functional association between these two proteins in striated muscle.

A CAV3 microdeletion differentially affects skeletal muscle and myocardium.

To the Editor: In their article, Cagliani et al. observed a 40% reduction of caveolin-3 in the myocardium of a single patient and concluded that this mutation affects skeletal muscle and myocardium differentially. The findings raise concerns. Was cardiac involvement absent in the patients because there were no comprehensive cardiac investigations? At least history, clinical cardiologic examination, ECG, echocardiography, and 24hour ECG are necessary to assess cardiac involvement. Only rudimentary data of these investigations were provided for Patients III-1, III-4, and IV-1. Since cardiac involvement may also develop during the disease course, it is important to regularly follow up. Reduced myocardial caveolin-3 in Patient III-1 may be independent of the underlying mutation and could be also influenced by coronary heart disease or extracorporal circulation during surgery. In rabbits, chronic myocardial hypoxia increased nitric oxide synthase and simultaneously reduced caveolin-3. Was myocardial biopsy taken from a region supplied by a stenosed or normal coronary artery? Was the microdeletion also detected in the myocardium? Possibly hyper-CK-emia in Patient III-2 erroneously led to the diagnosis of myocardial infarction, although the patient died from cardiac involvement in caveolinopathy. Did Patient III-1 have myocardial thickening from arterial hypertension? Since caveolin-3 reduction manifests in the skeletal muscle with various different phenotypes, CI may be also heterogeneous, even within a single family. How do the authors explain that caveolin-3 knockout mice develop severe cardiomyopathy with hypertrophic cardiomyocytes, while humans with caveolin-3 mutations seem to show few cardiac abnormalities? How is it explained that increased nitric oxide synthase results in decrease of caveolin-3 and the development of hypertrophic cardiomyopathy in mice but not in humans? That reduction of caveolin-3 leads to cardiomyopathy is supported by inhibition of hypertrophy of rat cardiomyocytes by adenovirus-mediated overexpression of caveolin-3. On the contrary, overexpression of caveolin-3 induces severe cardiomyopathy in mice. Which are the modifying factors that led to muscle but not to cardiac disease? Figure 4 shows the muscle of a control subject. Did all control subjects undergo muscle biopsy? Atypical absences in Patient IV-1 may be associated with the expression of caveolin-3 also in endothelial cells and astrocytes of the brain. Besides skeletal muscle, myocardium, and brain, caveolin-3 also occurs in the smooth muscle. Did the authors find involvement also of smooth musclecontaining organs? To demonstrate cardiac involvement in caveolinopathies, thorough cardiologic examination, regular follow-ups, and investigations not only of a single patient but all mutation carriers are required.

Frequency and characterisation of anoctamin 5 mutations in a cohort of Italian limb-girdle muscular dystrophy patients

Limb-girdle muscular dystrophy (LGMD) 2L, caused by mutations in the anoctamin 5 (ANO5) gene, is the third most common LGMD in Northern and Central Europe, where the c.191dupA mutation causes the majority of cases. We evaluated data from 228 Italian LGMD patients to determine the prevalence of LGMD2L and the c.191dupA mutation, and to describe the clinical, muscle biopsy, and magnetic resonance imaging findings in these patients. Forty-three patients who lacked molecular diagnosis were studied for ANO5 mutations, and four novel mutations were found in three probands. Only one proband carried the c.191dupA mutation, which was compound heterozygous with c.2516T>G. Two probands were homozygous for the c.1627dupA and c.397A>T mutations, respectively, while a fourth proband had a compound heterozygous status (c.220C>T and c.1609T>C). Therefore occurrence and molecular epidemiology of LGMD2L in this Italian cohort differed from those observed in other European countries. ANO5 mutations accounted for ∼2% of our sample. Affected patients exhibited benign progression with variable onset and an absence of cardiac and respiratory impairment; muscle biopsy generally showed mild signs, except when performed on the quadriceps muscles; MRI showed predominant involvement of the posterior thigh. Overall these common clinical, morphological and imaging findings could be useful in differential diagnosis.

Polyneuropathy due to cobalamin deficiency in the rat

In the present study, we investigated the peripheral nervous system (PNS) (both in terms of its ultrastructure and in terms of its function) of rats made cobalamin (Cbl)-deficient either through total gastrectomy or through prolonged feeding on a Cbl-deficient diet. In both these types of Cbl-deficient neuropathies we found: (a) ultrastructurally, intramyelin and endoneural edema, with no or minimal axonal damage in the PNS, in dorsal root ganglia, and the ventral and dorsal rootlets of the spinal cord; (b) electrophysiologically, a significant reduction in the nerve conduction velocity, consistent with that reported in (a); (c) morphometrically, a significant reduction in the density of myelinated fibers both in the sciatic nerve and in the peroneal nerve. All these pathological changes were reversed by chronic postoperative administration of Cbl into totally gastrectomized (TGX)-rats, hinting at the specificity of the damage itself in relation to the permanent Cbl-deficient status of the TGX-rats. No signs of segmental demyelination or remyelination were found. We also observed a turning of type I fibers into type II fibers in the soleus muscle of all our Cbl-deficient rats, however the Cbl deficiency had been induced. This muscular change was still present in TGX- and Cbl-treated rats, and it cannot be related to a malnutrition status, since it has been observed also in rats fed a Cbl-deficient diet. All these results demonstrate that Cbl deficiency strongly affects rat PNS within different parameters.

Clinical, morphological and immunological evaluation of six patients with dysferlin deficiency

Limb girdle muscular dystrophy (LGMD) type 2B and distal Miyoshi myopathy (MM) are caused by mutations in a recently discovered mammalian gene coding for a skeletal muscle protein called dysferlin. The protein is normally expressed at the skeletal muscle level and absent or reduced in affected patients. We selected a clinically heterogeneous population of Italian myopathic patients with clinical evidence of myopathy and/or hyperCKemia, EMG myopathic pattern, and no alterations of the dystrophin-sarcoglycan complex. Calpain, merosin, emerin and caveolin were also tested and found normal in all patients. Dysferlin immunohistochemical and Western blot analyses allowed us to identify six patients with dysferlin deficiency: one with distal myopathy, four with limb girdle myopathy and one with hyperCKemia. No apoptosis was found in any of the six muscle specimens, although expression of the pro-apoptotic Fas antigen was mildly increased in two cases. Inflammatory reactions were present in two of the six cases, but we found no evidence of immune-mediated processes.