Severe inflammatory myopathy in a pulmonary carcinoma patient treated with Pembrolizumab: An alert for myologists.

Immune-related adverse events (irAE) during the administration of immune-checkpoint inhibitors (ICIs) become more evident due to the increased use of these therapies. To remind the importance of early recognition of this phenomenon, we report a paradigmatic case characterized by severe systemic inflammatory myopathy and severe cardiac involvement that abruptly precipitated in an untoward ending after one single dose of Pembrolizumab.

A CAV3 microdeletion differentially affects skeletal muscle and myocardium.

BACKGROUND: Caveolin-3 is the muscle-specific protein product of the caveolin gene family and an integral membrane component of caveolae. Mutations in the gene encoding caveolin-3 (CAV3) underlie four distinct disorders of skeletal muscle: the autosomal dominant form of limb-girdle muscular dystrophy type 1C (LGMD-1C), rippling muscle disease (RMD), sporadic and familial forms of hyperCKemia, and distal myopathy. OBJECTIVE: To characterize a multigenerational Italian family affected by an autosomal dominant myopathic disorder and to assess the expression of caveolin-3, dystrophin, dystrophin-associated glycoproteins, and neuronal nitric oxide synthase in the myocardium of an affected patient. METHODS: Clinical analysis involved 15 family members. Skeletal muscle expression of sarcolemmal proteins was evaluated by immunohistochemistry and western blot analysis in three affected individuals. Caveolar structures were analyzed through electron microscopy in muscle biopsies and in one heart biopsy. RESULTS: CAV3 genetic analysis showed a heterozygous 3-bp microdeletion (328-330del) in affected individuals, resulting in the loss of a phenylalanine (Phe97del) in the transmembrane domain. In the skeletal muscle, the mutation was associated with severe caveolin-3 deficiency and caveolar disorganization, whereas the expression of the other analyzed muscle proteins was unaltered. Remarkably, caveolin-3 was expressed in myocardium at a level corresponding to about 60% of that of control individuals and was correctly localized at the myocardial cell membranes, with preservation of cardiac myofiber caveolar structures. Clinical analysis revealed the concomitant presence in this family of the following phenotypes: RMD, LGMD, and hyperCKemia. CONCLUSIONS: Intrafamilial phenotypic heterogeneity is associated with caveolin-3 Phe97 microdeletion. The molecular network interacting with caveolin-3 in skeletal muscle and heart may differ.

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.

A subpopulation of murine bone marrow cells fully differentiates along the myogenic pathway and participates in muscle repair in the mdx dystrophic mouse.

Bone marrow (BM) transplantation in mice suggests the existence of pluripotent cells able to differentiate into skeletal muscle tissue, although sustained myofiber reconstitution has not yet been achieved. We investigated the myogenic potential of mouse BM cells and evaluated whether a BM fraction enriched for cells expressing skeletal muscle markers would ameliorate muscle repair, when compared to whole BM, into the dystrophic mdx mouse. We demonstrate that cells expressing striated-muscle-specific proteins are already present in the BM independently from experimentally forced myogenic conversion. We observed the presence of both markers of early myogenic program such as Pax3, Myf5, MyoD, desmin, and late myogenesis such as myosin heavy chain and alpha-sarcomeric actin. These myogenic cells are more represented in the early nonadherent BM fraction, which generates clones able to fully differentiate into myotubes. Transplantation in mdx mice by intravenous injection of whole BM and a tenfold BM myogenic enriched fraction resulted in BM reconstitution and limited dystrophin restoration. Taken together, these data show that a fraction of BM cells have a definite potential for differentiation along the skeletal muscle pathway and can be recruited by muscle repair mechanisms. They also indicate that factors limiting the degree of muscle recruitment and the host stem cell competition should be assessed in order to evaluate the usefulness of BM-derived myogenic cells into the context of cell-mediated gene therapy of inherited muscle diseases.

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

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 defects. Abnormalities 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.

Beta-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 beta 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 beta-enolase subunit, the protein product of the ENO3 gene. The beta-enolase protein was dramatically reduced in the muscle of our patient, by both immunohistochemistry and immunoblotting, while alpha-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 beta-enolase. Muscle beta-enolase deficiency should be considered in the differential diagnosis of metabolic myopathies due to inherited defects of distal glycolysis.

A sporadic, atypical case of desminopathy: morphological and immunological characterization.

Recently, abnormal expression of cyclin-dependent kinases was proposed as a possible cause of desminopathy. We describe an atypical case clinically characterized by severe respiratory distress. Muscle biopsy showed subsarcolemmal and intracytoplasmic accumulation areas, which intensively stained with anti-desmin antibodies and contained electrondense filamentous material at ultrastructural level. WB analysis showed 30% increased desmin signal compared to controls. Positive immunostain for CDC2 kinase, CDK2 and emerin and nuclear matrix-associated protein were, found in desmin-positive fibres.

Sarcoglycan deficiency in a large Italian population of myopathic patients.

Autosomal recessive limb-girdle muscular dystrophies are a heterogeneous group of genetic diseases with a wide spectrum of clinical severity and age of onset; mutations in the gene encoding the dystrophin-associated sarcoglycan proteins (alpha, beta, gamma and delta) have recently been shown to cause some cases of these myopathies (primary sarcoglycanopathies, types 2D, 2E, 2C and 2F, respectively). In this study we have examined a large population of Italian myopathic patients to determine the frequency of (alpha-, beta- and gamma-sarcoglycan deficiency and to correlate molecular defects with clinical phenotypes; to exclude the presence of primary dystrophinopathies both genetic and immunological analysis of dystrophin was performed. We report 12 patients (10 male and 2 female) with deficiency of either one or more sarcoglycan proteins. They were aged 8-56 years with onset between 4 and 30 years of age; they all presented with either mild, moderate or severe limb-girdle involvement associated with elevated blood creatine kinase levels and myopathic pattern at EMG; one was also affected with a mild dilation cardiomyopathy. All patients, except one, showed pathological muscle histological changes. Absence of all three proteins always correlates with severe forms, whereas mild protein deficiencies or isolated partial alpha-sarcoglycan deficiency correlate with either severe, moderate or mild forms.

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.

Asymptomatic familial hyperCKemia associated with desmin accumulation in skeletal muscle.

We describe a family, two brothers and their mother, who came to our observation because of slight to moderate hyperCKemia. The younger brother, who had the highest CK values, was only suffering from episodic myalgia, the other two members of the family were asymptomatic. Neurological examination was normal. Both brothers underwent muscle biopsy which was significant for the presence of abnormal sarcoplasmic areas of desmin accumulation. So far, desmin abnormalities have never been reported in patients with such a mild neuromuscular pattern. We discuss possible correlations between severity of clinical phenotype and degree of desmin accumulation.

Utrophin expression during human fetal development.

Utrophin, a protein encoded by chromosome 6 is highly homologous to the cysteine-rich domain and most of the C-terminal domain of dystrophin. In order to clarify its functional role we analyzed its expression during human fetal development. We carried out immunohistochemical analysis on muscle from normal human fetuses at different ages of gestation using an antibody directed against a specific COOH-terminal sequence of the protein. In addition, we stained serial sections with antibodies against dystrophin and alpha-bungarotoxin FITC-BTX. Our findings show that, at week 9 of gestation, utrophin is diffusely expressed in the cytoplasm. From week 12 to 22 the immunostaining is still cytoplasmic, though the reaction intensity progressively decreases. Moreover we observed a strong reaction in fetal nerve at week 18 and 22. There was no correlation between utrophin expression and progressive dystrophin membrane localization.

High serum creatine kinase levels associated with cylindrical spirals at muscle biopsy.

We studied the muscle biopsy from an asymptomatic patient with high serum creatine kinase values. Subsarcolemmal and intermyofibrillar granular inclusions were seen at the light microscopy level. Ultrastructural observation showed clusters of cylindrical spirals (CS). CS are nonspecific, morphological finding, so far reported only in a few cases, presenting with a wide variety of clinical phenotypes. The case we describe is peculiar because of the complete lack of clinical symptoms. The nature of the CS is unknown; we studied a possible alteration of cytoskeletal proteins using a set of different antibodies against these structures, but none of them reacted with CS. Also, since CS have been described in association with mitochondrial abnormalities, and since in our case CS were strongly positive when stained for succinate dehydrogenase, we performed specific immunohistochemical and genetic studies which ruled out any major mitochondrial alterations.

Anionic phospholipids calcium binding sites in Duchenne and murine X-linked muscular dystrophy.

Duchenne muscular dystrophy (DMD) and murine X-linked muscular dystrophy (mdx) are genetically homologous and both characterized by absence of dystrophin. The function of this protein is not defined nor is the pathogenesis of the severe muscle necrosis and progressive weakness found in DMD but not in mdx. Recently we found that anionic phospholipid (AP) calcium binding sites are lacking at the muscle cell surface in DMD and we correlated these data with dystrophin deficiency and muscle necrosis. In order to verify the role of AP lack in the pathogenesis of muscle necrosis in DMD we studied the ultrastructural localization of these Ca++ receptors in mdx muscle membrane showing that they are normally represented as they are in control mouse and normal human muscle. The absence of AP in DMD compared with a normal distribution in mdx suggests that these calcium binding site alterations play an important and specific role in muscle fiber necrosis.

Desmin and vimentin as markers of regeneration in muscle diseases.

Localization 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.

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.