Objective Mutations in the genes encoding the extracellular matrix protein collagen VI (ColVI) cause a spectrum of disorders with variable inheritance including Ullrich congenital muscular dystrophy, Bethlem myopathy, and intermediate phenotypes. We extensively characterized, at the clinical, cellular, and molecular levels, 49 patients with onset in the first 2 years of life to investigate genotype‐phenotype correlations. Methods Patients were classified into 3 groups: early‐severe (18%), moderate‐progressive (53%), and mild (29%). ColVI secretion was analyzed in patient‐derived skin fibroblasts. Chain‐specific transcript levels were quantified by quantitative reverse transcriptase polymerase chain reaction (qRT‐PCR), and mutation identification was performed by sequencing of complementary DNA. Results ColVI secretion was altered in all fibroblast cultures studied. We identified 56 mutations, mostly novel and private. Dominant de novo mutations were detected in 61% of the cases. Importantly, mutations causing premature termination codons (PTCs) or in‐frame insertions strikingly destabilized the corresponding transcripts. Homozygous PTC‐causing mutations in the triple helix domains led to the most severe phenotypes (ambulation never achieved), whereas dominant de novo in‐frame exon skipping and glycine missense mutations were identified in patients of the moderate‐progressive group (loss of ambulation). Interpretation This work emphasizes that the diagnosis of early onset ColVI myopathies is arduous and time‐consuming, and demonstrates that quantitative RT‐PCR is a helpful tool for the identification of some mutation‐bearing genes. Moreover, the clinical classification proposed allowed genotype‐phenotype relationships to be explored, and may be useful in the design of future clinical trials. ANN NEUROL 2010;68:511–520

Retinoblastoma is the most frequent intraocular malignancy in children, originating from a maturing cone precursor in the developing retina. Little is known on the molecular basis underlying the biological and clinical behavior of this cancer. Here, using multi-omics data, we demonstrate the existence of two retinoblastoma subtypes. Subtype 1, of earlier onset, includes most of the heritable forms. It harbors few genetic alterations other than the initiating RB1 inactivation and corresponds to differentiated tumors expressing mature cone markers. By contrast, subtype 2 tumors harbor frequent recurrent genetic alterations including MYCN-amplification. They express markers of less differentiated cone together with neuronal/ganglion cell markers with marked inter- and intra-tumor heterogeneity. The cone dedifferentiation in subtype 2 is associated with stemness features including low immune and interferon response, E2F and MYC/MYCN activation and a higher propensity for metastasis. The recognition of these two subtypes, one maintaining a cone-differentiated state, and the other, more aggressive, associated with cone dedifferentiation and expression of neuronal markers, opens up important biological and clinical perspectives for retinoblastomas.

We report four heterozygous dynamin 2 (DNM2) mutations in five centronuclear myopathy patients aged 1 to 15 years. They all presented with neonatal hypotonia with weak suckling. Thereafter, their phenotype progressively improved. All patients demonstrated muscle weakness prominent in the lower limbs, and most of them also presented with facial weakness, open mouth, arched palate, ptosis, and ophthalmoparesis. Electrophysiology showed only myopathic changes, and muscle biopsies showed central nuclei and type 1 fiber hypotrophy and predominance. Our results expand the phenotypic spectrum of dynamin 2-related centronuclear myopathy from the classic mild form to the more severe neonatal phenotype.

(2011) Neuropathology and Applied Neurobiology 37, 271-284 Recessive RYR1 mutations cause unusual congenital myopathy with prominent nuclear internalization and large areas of myofibrillar disorganization Aims: To report the clinical, pathological and genetic findings in a group of patients with a previously not described phenotype of congenital myopathy due to recessive mutations in the gene encoding the type 1 muscle ryanodine receptor channel (RYR1). Methods: Seven unrelated patients shared a predominant axial and proximal weakness of varying severity, with onset during the neonatal period, associated with bilateral ptosis and ophthalmoparesis, and unusual muscle biopsy features at light and electron microscopic levels. Results: Muscle biopsy histochemistry revealed a peculiar morphological pattern characterized by numerous internalized myonuclei in up to 51% of fibres and large areas of myofibrillar disorganization with undefined borders. Ultrastructurally, such areas frequently occupied the whole myofibre cross section and extended to a moderate number of sarcomeres in length. Molecular genetic investigations identified recessive mutations in the ryanodine receptor (RYR1) gene in six compound heterozygous patients and one homozygous patient. Nine mutations are novel and four have already been reported either as pathogenic recessive mutations or as changes affecting a residue associated 271Neuropathology and Applied Neurobiology (2011Neurobiology ( ), 37, 271-284 doi: 10.1111Neurobiology ( /j.1365Neurobiology ( -2990Neurobiology ( .2010 with dominant malignant hyperthermia susceptibility. Only two mutations were located in the C-terminal transmembrane domain whereas the others were distributed throughout the cytoplasmic region of RyR1. Conclusion: Our data enlarge the spectrum of RYR1 mutations and highlight their clinical and morphological heterogeneity.A congenital myopathy featuring ptosis and external ophthalmoplegia, concomitant with the novel histopathological phenotype showing fibres with large, poorly delimited areas of myofibrillar disorganization and internal nuclei, is highly suggestive of an RYR1-related congenital myopathy.

Nemaline myopathy (NM) is a rare congenital myopathy characterised by hypotonia, muscle weakness, and often skeletal muscle deformities with the presence of nemaline bodies (rods) in the muscle biopsy. The nebulin (NEB) gene is the most commonly mutated and is thought to account for approximately 50% of genetically diagnosed cases of NM. We undertook a detailed muscle morphological analysis of 14 NEB-mutated NM patients with different clinical forms to define muscle pathological patterns and correlate them with clinical course and genotype. Three groups were identified according to clinical severity. Group 1 (n = 5) comprises severe/lethal NM and biopsy in the first days of life. Group 2 (n = 4) includes intermediate NM and biopsy in infancy. Group 3 (n = 5) comprises typical/mild NM and biopsy in childhood or early adult life. Biopsies underwent histoenzymological, immunohistochemical and ultrastructural analysis. Fibre type distribution patterns, rod characteristics, distribution and localization were investigated. Contractile performance was studied in muscle fibre preparations isolated from seven muscle biopsies from each of the three groups. G1 showed significant myofibrillar dissociation and smallness with scattered globular rods in one third of fibres; there was no type 1 predominance. G2 presented milder sarcomeric dissociation, dispersed or clustered nemaline bodies, and type 1 predominance/uniformity. In contrast, G3 had well-delimited clusters of subsarcolemmal elongated rods and type 1 uniformity without sarcomeric alterations. In accordance with the clinical and morphological data, functional studies revealed markedly low forces in muscle bundles from G1 and a better contractile performance in muscle bundles from biopsies of patients from G2, and G3.In conclusion NEB-mutated NM patients present a wide spectrum of morphological features. It is difficult to establish firm genotype phenotype correlation. Interestingly, there was a correlation between clinical severity on the one hand and the degree of sarcomeric dissociation and contractility efficiency on the other. By contrast the percentage of fibres occupied by rods, as well as the quantity and the sub sarcolemmal position of rods, appears to inversely correlate with severity. Based on our observations, we propose myofibrillar dissociation and changes in contractility as an important cause of muscle weakness in NEB-mutated NM patients.

Several morphological phenotypes have been associated to RYR1-recessive myopathies. We recharacterized the RYR1-recessive morphological spectrum by a large monocentric study performed on 54 muscle biopsies from a large cohort of 48 genetically confirmed patients, using histoenzymology, immunohistochemistry, and ultrastructural studies. We also analysed the level of RyR1 expression in patients’ muscle biopsies. We defined “dusty cores” the irregular areas of myofibrillar disorganisation characterised by a reddish-purple granular material deposition with uneven oxidative stain and devoid of ATPase activity, which represent the characteristic lesion in muscle biopsy in 54% of patients. We named Dusty Core Disease (DuCD) the corresponding entity of congenital myopathy. Dusty cores had peculiar histological and ultrastructural characteristics compared to the other core diseases. DuCD muscle biopsies also showed nuclear centralization and type1 fibre predominance. Dusty cores were not observed in other core myopathies and centronuclear myopathies. The other morphological groups in our cohort of patients were: Central Core (CCD: 21%), Core-Rod (C&R:15%) and Type1 predominance “plus” (T1P+:10%). DuCD group was associated to an earlier disease onset, a more severe clinical phenotype and a lowest level of RyR1 expression in muscle, compared to the other groups. Variants located in the bridge solenoid and the pore domains were more frequent in DuCD patients. In conclusion, DuCD is the most frequent histopathological presentation of RYR1-recessive myopathies. Dusty cores represent the unifying morphological lesion among the DuCD pathology spectrum and are the morphological hallmark for the recessive form of disease.Electronic supplementary materialThe online version of this article (10.1186/s40478-018-0655-5) contains supplementary material, which is available to authorized users.

The N-glycolylated ganglioside NeuGc-GM3 has been described in solid tumors such as breast carcinoma, nonsmall cell lung cancer, and melanoma, but is usually not detected in normal human cells. Our aim was to evaluate the presence of NeuGc-GM3 in pediatric neuroectodermal tumors by immunohistochemistry. Twenty-seven archival cases of neuroblastoma and Ewing sarcoma family of tumors (ESFT) were analyzed. Formalin-fixed, paraffin-embedded tumor samples were cut into 5 μm sections. The monoclonal antibody 14F7, a mouse IgG1 that specifically recognizes NeuGc-GM3, and a peroxidase-labeled polymer conjugated to secondary antibodies were used. Presence of NeuGc-GM3 was evident in 23 of 27 cases (85%), with an average of about 70% of positive tumors cells. Immunoreactivity was moderate to intense in most tumors, showing a diffuse cytoplasmic and membranous staining, although cases of ESFT demonstrated a fine granular cytoplasmic pattern. No significant differences were observed between neuroblastoma with and without NMYC oncogene amplification, suggesting that expression of NeuGc-GM3 is preserved in more aggressive cancers. Until now, the expression of N-glycolylated gangliosides in pediatric neuroectodermal tumors has not been investigated. The present study evidenced the expression of NeuGc-GM3 in a high proportion of neuroectodermal tumors, suggesting its potential utility as a specific target of immunotherapy.

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