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Background: Biallelic mutations in TBC1-domain containing kinase (TBCK) lead to hypotonia, global developmental delay with severe cognitive and motor deficits, and variable presentation of dysmorphic facial features and brain malformations. It remains unclear whether hypotonia in these individuals is purely neurogenic, or also caused by progressive muscle disease.Methods: Whole exome sequencing was performed on a family diagnosed with nonspecific myopathic changes by means of histological analysis and immunohistochemistry of muscle biopsy samples.Results: A novel homozygous truncation in TBCK was found in two sisters diagnosed with muscle disease and severe psychomotor delay. TBCK was completely absent in these patients.Conclusions: Our findings identify a novel early truncating variant in TBCK associated with a severe presentation and add muscle disease to the variability of phenotypes associated with TBCK mutations. Inconsistent genotype/phenotype correlation could be ascribed to the multiple roles of TBCK in intracellular signaling and endolysosomal function in different tissues. [ABSTRACT FROM AUTHOR]

• We detected by whole exome sequencing 2 truncating LAMA 2 mutations in two siblings. • The proband had very subtle at first undetected reduction in laminin-α2 expression. • Laminin alpha2 reduction was more apparent by immunoblotting. • Brain MRI was normal and clinical presentation unusual, with stable progression. LAMA 2 mutations cause the most frequent congenital muscular dystrophy subtype MDC1A and a variety of milder phenotypes, characterized by total or partial laminin-α2 deficiency. In both severe and milder cases brain MRI invariably shows abnormal white matter signal intensity. We report clinical, histopathological, imaging and genetic data on two siblings with very subtle, and at first undetected, reduction in laminin-α2 expression, and brain MRI showing minor non-specific abnormalities. Clinical features in the female proband were characterized by muscle weakness involving neck and axial muscles, and pelvic girdle and distal lower limb muscles, reduced tendon reflexes and pes cavus. Clinical features in a younger brother were similar, and remained stable in both siblings during the follow up. Whole exome sequencing (WES) detected two heterozygous truncating LAMA 2 mutations. Brain MRI in combination with laminin-α2 immunohistochemistry might not be sufficient and WES might be the only means to reach a diagnosis. [ABSTRACT FROM AUTHOR]

The Biobank was established in 1986 as part of the routine diagnostic activity of the Division of Neuromuscular Diseases and Neuroimmunology, of the Carlo Besta Neurological Institute. It stores muscle tissue, cells and DNA from patients with neuromuscular diseases. The biobank provides samples as a service to the scientific community conducting research on neuromuscular disorders. Samples are from patients affected by different forms of muscular dystrophy, including the severe congenital and Duchenne muscular dystrophies, as well as limb girdle muscular dystrophies, congenital myopathies, distal and myofibrillar myopathies, inflammatory myopathies, and metabolic myopathies. Different types of biomaterials are frequently available from a single patient. The Biobank is founding partner of the EuroBioBank network, the first operating network of biobanks for rare diseases in Europe, and of the Italian Telethon Network of Genetic Biobanks. The involvement of the biobank into both networks has been instrumental for standardization of procedures and activities, implementation of sample access policies, and compliance with ELSI requirements. The biobank, with about 13000 biospecimens stored in total at the time of writing, constitutes a key source of biological samples for researchers worldwide. [ABSTRACT FROM AUTHOR]

An autosomal dominant protein aggregate myopathy, characterized by high plasma creatine kinase and calsequestrin-1 (CASQ1) accumulation in skeletal muscle, has been recently associated with a missense mutation in CASQ1 gene. The mutation replaces an evolutionarily-conserved aspartic acid with glycine at position 244 (p.D244G) of CASQ1, the main sarcoplasmic reticulum (SR) Ca2+ binding and storage protein localized at the terminal cisternae of skeletal muscle cells. Here, immunocytochemical analysis of myotubes, differentiated from muscle-derived primary myoblasts, shows that sarcoplasmic vacuolar aggregations positive for CASQ1 are significantly larger in CASQ1-mutated cells than control cells. A strong co-immuno staining of both RyR1 and CASQ1 was also noted in the vacuoles of myotubes and muscle biopsies derived from patients. Electrophysiological recordings and sarcoplasmic Ca2+ measurements provide evidence for less Ca2+ release from the SR of mutated myotubes when compared to that of controls. These findings further clarify the pathogenic nature of the p.D244G variant and point out defects in sarcoplasmic Ca2+ homeostasis as a mechanism underlying this human disease, which could be distinctly classified as “CASQ1-couplonopathy”. [ABSTRACT FROM AUTHOR]

Introduction: Protein aggregation is a common cause of neuropathology. The protein aggregation myopathy Limb-Girdle Muscular Dystrophy 1D (LGMD1D) is caused by mutations of amino acids Phe89 or Phe93 of DNAJB6, a co-chaperone of the HSP70 anti-aggregation protein. Another DNAJB6 mutation, Pro96Arg, was found to cause a distal-onset myopathy in one family. Results: We detail the mutational, neuropathological, neurophysiological, neurological and radiological features of five new DNAJB6-myopathy families. One has the known Phe93Leu mutation and classic late-onset slowly progressive LGMD1D. Two have different mutations of Phe91 causing a variant childhood-onset severe limb-girdle myopathy. One has a Phe100Val mutation and distal-onset myopathy, unique early bulbar involvement, and a gender-modified wide age-of-onset range. The last has childhood-onset severe distal-onset myopathy and the first non-missense DNAJB6 mutation, c.346 + 5G > A, causing a splicing defect that entirely eliminates DNAJB6's G/F domain (ΔG/F), the domain that harbours all other mutations. Clinical and imaging examinations reveal that muscles considered uninvolved in DNAJB6-myopathy, e.g. lateral gastrocnemii, are affected in our patients with new mutations. Mutational modelling based on the known structure of the bacterial DNAJ2 protein indicates that all past and present mutated residues cluster within 15 Å in the G/F domain and all disturb the interface of this domain with the protein's J domain that confers the interaction with HSP70. Conclusions: Our patients expand the phenotypic spectrum of DNAJB6-myopathy and allow tentative genotype-phenotype specifications. Combining with previous studies, the clinical severity spectrum is as follows: ΔG/F and Phe91 mutations, most severe; Phe100, Pro96, Phe89 mutations, intermediate; and Phe93, least severe. As it stands presently, proximal G/F domain mutations (Phe89, Phe91, Phe93) cause proximal limb-girdle myopathy, while distal G/F mutations (Pro96, Phe100) cause distal-onset myopathy. While all mutations affect the G/F-J interaction, each likely does so in different unknown extents or ways. One mutation, ΔG/F, causes its associated severe distal-onset myopathy phenotype in a clear way, through generation of a G/ F domain-lacking DNAJB6 protein. [ABSTRACT FROM AUTHOR]

Several growth factors (e.g., transforming growth factors beta and alpha, basic fibroblast growth factor), produced by hypothalamic astrocytes, participate in the control of hypothalamic gonadotrophin-releasing hormone (GnRH) neurons. On this basis, we have hypothesized that steroid hormones, like estrogens and progestagens, influence the GnRH neurons by modulating in glial cells the synthesis and the release of these growth factors. Data reported here indicate that the expression of transforming growth factor beta 1 is modulated in hypothalamic astrocytes by a progesterone derivative (i.e., dihydroprogesterone), while estrogens modulate that of basic fibroblast growth factor. Moreover, it is interesting to highlight that the effect of estrogens on basic fibroblast growth factor is mediated by another growth factor (i.e., transforming growth factor alpha). Altogether, the present findings support the concept that steroid hormones and growth factors act in an integrated manner at the level of hypothalamic astrocytes, thus adding a further piece of knowledge in the understanding of the mechanisms controlling GnRH neurons. [ABSTRACT FROM AUTHOR]

Abstract Exosomes, natural carriers of mRNAs, non-coding RNAs and proteins between donor and recipient cells, actively contribute to cell-cell communication. We investigated the potential pro-fibrotic role of exosomes released by muscle-derived fibroblasts of Duchenne muscular dystrophy (DMD) patients, and of miRNAs carried by exosomes. By fibrosis focused array analysis we found that exosomes from DMD fibroblasts, had significantly higher levels of miR-199a-5p, a miRNA up-regulated in fibrotic conditions, compared to control exosomes, while levels in myoblast-derived exosomes were not increased. In control fibroblasts, exposure to DMD fibroblast-derived exosomes induced a myofibroblastic phenotype with increase in α-smooth actin, collagen and fibronectin transcript and protein expression, soluble collagen production and deposition, cell proliferation, and activation of Akt and ERK signaling, while exposure to control exosomes did not. Transfecting control fibroblasts or loading control exosomes with miR-199a-5p mimic or inhibitor induced opposing effects on fibrosis-related mRNAs and proteins, on collagen production and Akt and ERK pathways. Finally, injection of DMD fibroblast-derived exosomes into mouse tibialis anterior muscle after cardiotoxin-induced necrosis, produced greater fibrosis than control exosomes. Our findings indicate that exosomes produced by local fibroblasts in the DMD muscle are able to induce phenotypic conversion of normal fibroblasts to myofibroblasts thereby increasing the fibrotic response. This conversion is related to transfer of high levels of miR-199a-5p and to reduction of its target caveolin-1; both, therefore, are potential therapeutic targets in muscle fibrosis. Highlights • DMD fibroblast-derived exosomes induced a myofibroblastic phenotype in control fibroblasts. • DMD fibroblasts had significantly higher levels of the fibrosis-related miR-199a-5p. • Fibroblast transfection or exosome loading with miR-199a-5p mimic or inhibitor induced opposing effects on fibrosis. • Injection of exosomes into mouse tibialis anterior muscle produced greater fibrosis than control exosomes. [ABSTRACT FROM AUTHOR]

Mutations in the DNAJB6 gene have been associated with the autosomal dominant limb girdle muscular dystrophy type 1D (LGMD1D), a disorder characterized by abnormal protein aggregates and rimmed vacuoles in muscle fibers. DNAJB6 is a ubiquitously expressed Hsp40 co-chaperone characterized by a J domain that specifies Hsp70 functions in the cellular environment. DNAJB6 is also a potent inhibitor of expanded polyglutamine (polyQ) aggregation preventing aggregate toxicity in cells. In DNAJB6 -mutated patients this anti-aggregation property is significantly reduced, albeit not completely lost. To elucidate the pathogenetic mechanisms underlying the DNAJB6-related myopathy, animal models have been created showing that, indeed, conditional muscular expression of a DNAJB6 mutant in the mouse causes a LGMD1D myofibrillary muscle tissue phenotype. Both mutations and phenotypes reported until recently were rather homogeneous, being exclusively missense mutations of a few amino acids of the protein G/F domain, and with a phenotype characterized by adult-onset slowly progressive muscular dystrophy predominantly affecting proximal muscles. Lately, several novel mutations and new phenotypes of DNAJB6 have been described. These mutations once more affect the G/F domain of DNAJB6 with missense changes and a splice site mutation; and the phenotypes include childhood onset and distal involvement of muscles, or childhood-onset LGMD1D with loss of ambulation in early adulthood and respiratory involvement. Thus, the spectrum of DNAJB6 -related phenotypes is widening. Although our knowledge about the role of DNAJB6 in the pathogenesis of muscle diseases has made great progression, several questions remain unsolved, including why a ubiquitous protein affects only, or predominantly, skeletal muscle; why only the G/F domain is involved; and what is the possible role of the DNAJB6a isoform. Clarification of these issues will provide clues to implement possible therapeutic strategies for DNAJB6-related myopathies.

An autosomal dominant protein aggregate myopathy, characterized by high plasma creatine kinase and calsequestrin-1 (CASQ1) accumulation in skeletal muscle, has been recently associated with a missense mutation in CASQ1 gene. The mutation replaces an evolutionarily-conserved aspartic acid with glycine at position 244 (p.D244G) of CASQ1, the main sarcoplasmic reticulum (SR) Ca2+ binding and storage protein localized at the terminal cisternae of skeletal muscle cells. Here, immunocytochemical analysis of myotubes, differentiated from muscle-derived primary myoblasts, shows that sarcoplasmic vacuolar aggregations positive for CASQ1 are significantly larger in CASQ1-mutated cells than control cells. A strong co-immuno staining of both RyR1 and CASQ1 was also noted in the vacuoles of myotubes and muscle biopsies derived from patients. Electrophysiological recordings and sarcoplasmic Ca2+ measurements provide evidence for less Ca2+ release from the SR of mutated myotubes when compared to that of controls. These findings further clarify the pathogenic nature of the p.D244G variant and point out defects in sarcoplasmic Ca2+ homeostasis as a mechanism underlying this human disease, which could be distinctly classified as "CASQ1-couplonopathy".

The adult-onset form of Pompe disease had a wide clinical spectrum, ranging from asymptomatic patients with increased CK to muscle cramps and pain syndrome or rigid-spine syndrome. In addition clinical severity and disease progression are greatly variable. We report on a family with 3 siblings characterized by an unusual adult-onset Pompe disease including dysphagia and weakness of tongue, axial and limb-girdle muscles, in association with atypical globular inclusions in muscle fibres. Our study confirms the great clinical and histological variability of adult-onset Pompe disease and further supports the need of careful evaluation of bulbar function in patients affected by this pathology.