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SH3 and cysteine-rich domain-containing protein 3 (STAC3) is an essential component of the skeletal muscle excitation-contraction coupling (ECC) machinery, though its role and function are not yet completely understood. Here, we report 18 patients carrying a homozygous p.(Trp284Ser) STAC3 variant in addition to a patient compound heterozygous for the p.(Trp284Ser) and a novel splice site change (c.997-1G > T). Clinical severity ranged from prenatal onset with severe features at birth, to a milder and slowly progressive congenital myopathy phenotype. A malignant hyperthermia (MH)-like reaction had occurred in several patients. The functional analysis demonstrated impaired ECC. In particular, KCl-induced membrane depolarization resulted in significantly reduced sarcoplasmic reticulum Ca2+ release. Co-immunoprecipitation of STAC3 with CaV 1.1 in patients and control muscle samples showed that the protein interaction between STAC3 and CaV 1.1 was not significantly affected by the STAC3 variants. This study demonstrates that STAC3 gene analysis should be included in the diagnostic work up of patients of any ethnicity presenting with congenital myopathy, in particular if a history of MH-like episodes is reported. While the precise pathomechanism remains to be elucidated, our functional characterization of STAC3 variants revealed that defective ECC is not a result of CaV 1.1 sarcolemma mislocalization or impaired STAC3-CaV 1.1 interaction.

Controversy still exists about the pro- or antithrombotic side effects of contrast media used in daily medical practice. Recent reports have shown that various contrast media, including ionic compounds, have deleterious prothrombotic actions. A new evaluation of these adverse side effects is reported here, with the study of the dose–effect relationship. Two ionic (ioxaglate and diatrizoate) and two non-ionic contrast media (iopamidol and iohexol) were studied. Experiments were done on 22 groups of 5 Wistar male rats each, using a Laser Argon-induced thrombosis model in mesenteric microvessels. Three parameters were studied: the number of laser beams needed to induce platelet thrombus formation, the number of emboli, and the duration of embolization. Platelet count and platelet aggregation also were determined. Iopamidol and iohexol induced a significant rise in both the number of emboli and the duration of embolization in mesenteric microvessels at doses up to 1 mL/kg. Ioxaglate and diatrizoate also significantly increased these parameters at doses up to 2 mL/kg. All the products tested decreased platelet count, inducing a −17 to −30% variation from control values. Diatrizoate and ioxaglate inhibited platelet aggregation, while iopamidol and Iohexol behaved as activators. All non-ionic, and to a lesser extent, all ionic contrast media demonstrated prothrombotic properties.

Nemaline myopathy (NM) is a disorder of the skeletal muscle thin filament characterised by muscle dysfunction and electron-dense protein accumulations (nemaline bodies). Pathogenic mutations have been described in nine genes to date, but the genetic basis remains unknown in many cases. We used whole exome sequencing (WES) in two families with NM and subsequent gene sequencing in over 540 additional genetically unresolved NM patients to identify and characterise a new genetic cause of NM. We developed a knock-down zebrafish model of this condition and used immunohistochemistry, western blotting, single-fibre contractility studies and recombinant protein studies to characterise the expression, localisation and biochemical functions of the new disease-related protein. We identified homozygous or compound heterozygous variants in LMOD3, which encodes leiomodin-3 (Lmod3) in 21 patients from 14 families. Affected individuals had severe generalised weakness and hypotonia, and most affected individuals died in the neonatal period. We demonstrated that Lmod3 is expressed from early muscle differentiation, localises to thin filaments with enrichment at the pointed ends, and has strong actin nucleating activity. Loss of Lmod3 in patient muscle results in shortening and disorganisation of thin filaments. Knockdown of lmod3 in the zebrafish replicates this phenotype. These findings define a new genetic subtype of congenital myopathy and demonstrate an essential, previously unrecognised role for Lmod3 in the regulation of sarcomeric thin filaments in skeletal muscle.

Objective: The aim is to search for a pattern of muscle involvement in patients with nemaline myopathy due to mutations in the NEB gene and to correlate MRI findings with clinical severity. Methods: The series consisted of seven patients followed in two different French centers and for whom whole body MRI (WBMRI) was done. T1-TSE sequences in axial and frontal views were performed. Results: the clinical phenotype was determined by the severity of motor and respiratory involvement. Two patients had severe phenotype: wheelchair bound and total dependence on mechanical ventilation. Two cases had moderate phenotype: able to walk but limitation of activity outside due to fatigue, severe restrictive lung disease. Three patients have mild phenotype: complete autonomy, proximal weakness after prolonged activity, and no need for ventilator support. WBMRI showed in all patients marked involvement of tongue and tibialis anterior muscles. Other muscles also altered were lateral pterygoid, soleus and extensor digitorum (severe cases); temporal, abdominal belt and soleus muscles (moderate); extensor digitorum (mild). Discussion: NEB-mutated patients present a wide spectrum of severity. A positive correlation between the clinical severity and the degree of muscle abnormalities is observed. Although there is variability in the distribution of the abnormalities in trunk and proximal limbs, a cranial and distal limb involvement is identified. Conclusion: Selective involvement of the tongue and tibialis anterior muscles in NEB mutated patients makes WBMRI a useful tool for diagnosis of nemaline myopathy.

CAP-disease is a rare congenital myopathy characterized by the presence of demarcated areas of subsarcolemmal myofibrillar disorganization that are devoid of myosin ATPase, stained with NADH-TR and strongly immunolabeled for desmin. Mutations in ACTA1, TPM2 or TPM3 have been reported so far. We report on 5 cases: 1 sporadic severe case and four patients from 2 families, respectively. In family 1 mother and daughter were affected; in family 2, two siblings manifested the disease. Mutations in the three documented genes were excluded. Age at onset ranged from birth to childhood. The main clinical phenotype consisted of clear dysmorphic features with elongated face and high-arched palate associated with axial, lower and upper limbs muscle weakness. The most severe cases presented early respiratory distress, and feeding difficulties. One patient had arthrogryposis. Muscle biopsies performed at different ages (from newborn to adult age) showed severe changes with marked variation of fiber size, increased internal nuclei, and type I fiber predominance. Several fibers contained a variable number of CAP in subsarcolemal areas. By electron microscopy, well-delineated CAP structures were constantly encountered. These showed disorganization of myofibrils, loss of thick filaments, and thickened fragments of Z bands without nemaline rod bodies. CAP contained segments of sarcomeres constituted by fragments of Z-lines from where arising thin filaments (”butterfly-like” aspect). Furthermore dense thin filamentous material was found in the subsarcolemmal region and less frequently between the myofibrils. In longitudinal sections, the Z lines appeared occasionally jagged and partially misaligned in some myofibrils. No basal membrane abnormalities were observed. To identify the causative mutation whole exome sequencing is ongoing. In conclusion, we attempt to characterize the clinical and morphological particularities of five cases presenting CAP disease, not-related to known genes.

The congenital myopathies with protein aggregates and inclusions are a subgroup of structural congenital myopathies characterized by aggregation of proteins in muscle. They include Nemaline Myopathy, Core Rod Myopathy, Cap Disease, Reducing body Myopathy, Cylindrical spirals Myopathy, and other entities presenting less distinct protein inclusions. This subgroup represents around 40 % of all biopsies with a structural congenital myopathy. We analysed 154 patients whose muscle was referred to our lab. Pathological diagnosis was achieved combining histochemical and ultrastructural analysis. Nemaline Myopathy was encountered in 103 (67%) patients, 14 (9%) patients had Core Rod myopathy, 12 (8%) Cap Disease, 16 (10%) Reducing Body Myopathy, 3 (2%) Cylindrical spirals Myopathy and 6 (4%) patients presented less distinct protein aggregates. Standard histoenzymology oriented the genetic analysis in around two third of our patients. In one third of cases extended immunohistochemical and ultrastructural studies were pivotal for the constitution of homogenous cohorts, and the orientation of molecular analysis. Genetic diagnosis was achieved in 76 (49%) patients through Sanger sequencing or next generation sequencing. NEB was mutated in 26 patients (34%), ACTA1 in 25 (33%), FHL1 in 14 (18%), TPM2 in 5 (7%) and TPM3 in 3 (4%), RYR1 in 2 (3%), and KLHL40 in 1 (1%). Our study demonstrates that detailed muscle biopsies analysis represents an essential tool in the diagnostic workup of congenital myopathies with protein aggregates and inclusions. Ultrastructural analysis turned to be fundamental in establishing the diagnosis in muscle biopsies from newborn and child patients. A combined clinic-histopathological and genetic approach will certainly help in the identification of the underlying molecular defect in patients without a genetic diagnosis.

Nemaline myopathy (NM) is a congenital disorder associating muscle weakness with rods in muscle biopsy. Clinical presentation is variable spanning from severe to mild forms. Seven genes are associated with NM. The nebulin (NEB) gene is the most commonly mutated accounting for 50% of cases. We performed a muscle morphological analysis of thirteen NEB-mutated patients with different clinical forms to define pathological patterns and clinical-morphological correlations. Four groups were identified according to clinical severity and age at biopsy. Group 1 ( n = 4) comprises severe/lethal NM and biopsy in first days. Group 2 ( n = 3) comprehends severe NM and biopsy after one month. Group 3 ( n = 3) comprises typical NM and biopsy in childhood, and group 4 ( n = 2) patients with mild NM and biopsy in adulthood. Molecular diagnosis was done using dHPLC/Sanger-sequencing in six patients and next-generation sequencing in seven. Biopsies underwent histoenzymological, immunohistochemical and ultrastructural analysis. Fiber type distribution, rod characteristics, distribution and localization were investigated. All patients presented NEB mutations consistent with AR inheritance. G1 showed type 2 predominance and scattered squared rods in 1/3 of fibers. Ultrastructural analysis revealed high percentage of fibers with sarcomeric disarray. G2 showed a variable pattern of fiber type distribution spanning from slight type 2 predominance to type 1 uniformity. Rods presented variable distribution and shape. Ultrastructural analysis revealed rare fibers with sarcomeric disarray. In contrast, G3 and G4 presented a homogeneous type 1 uniformity associated with well-delimited subsarcolemmal and/or cytoplasmic elongated rods without sarcomeric alterations. In conclusions we (1) identified an unreported association of type 2 predominance in muscle biopsy of patients presenting severe NEB-related NM; (2) suggest a direct correlation between the association of sarcomeric disarray and clinical severity.

Megaconial myopathies are known as rare and severe autosomal recessive, early-onset pediatric diseases caused by mutations in the CHKB gene, characterized on muscle biopsy by the presence of giant mitochondria. We present here a 50-year old male, stemming from a Jewish consanguineous Lebanese family, who developed progressive muscle wasting and weakness form age 5 years on. The course was slowly progressive; the patient remained ambulant until age 37 years. He displayed episodes of regressive paroxysmal supraventricular tachyarrhythmia at age 46 and 47 years, and a severe restrictive respiratory insufficiency from his early thirties. The patient underwent three muscle biopsies, at age 28, 39 and 49 years. Diagnosis was suspected on the last biopsy, showing an uneven distribution of the intermyofibrillary network associating peripheral giant mitochondria with central areas devoid of organelles. Electron microscopy confirmed the presence of numerous giant mitochondria spanning up to two or more sarcomeres and reaching 1.2–1.8 μm in length and 0.6–0.8 μm in width. Retrospective analysis of the two first biopsies found some similar abnormalities. The patient harbours a homozygous c.499A > T (p.Ile167Phe) substitution in exon 4 of the mutation of the CHKB gene. A whole-body MRI was performed at the age of 42. A diffuse and severe fatty replacement (Mercuri 4) of muscles was detectable. Masticatory muscles and tongue as well as forearms, rectus femoris at thighs and anterior compartment of the legs were totally or partially preserved. This case, resembling a congenital myopathy, differs from the usual pattern of megaconial myopathies and expands the clinical spectrum of this disorder.