Phenotypic Behavior of Caveolin-3 Mutations That Cause Autosomal Dominant Limb Girdle Muscular Dystrophy (LGMD-1C) RETENTION OF LGMD-1C CAVEOLIN-3 MUTANTS WITHIN THE GOLGI COMPLEX

Caveolin-3, a muscle-specific caveolin-related protein, is the principal structural protein of caveolae membrane domains in striated muscle cell types (cardiac and skeletal). Autosomal dominant limb girdle muscular dystrophy (LGMD-1C) in humans is due to mutations within the caveolin-3 gene: (i) a 9-base pair microdeletion that removes three amino acids within the caveolin scaffolding domain (DeltaTFT) or (ii) a missense mutation within the membrane spanning domain (P --> L). The molecular mechanisms by which these two mutations cause muscular dystrophy remain unknown. Here, we investigate the phenotypic behavior of these caveolin-3 mutations using heterologous expression. Wild type caveolin-3 or caveolin-3 mutants were transiently expressed in NIH 3T3 cells. LGMD-1C mutants of caveolin-3 (DeltaTFT or P --> L) were primarily retained at the level of a perinuclear compartment that we identified as the Golgi complex in double-labeling experiments, while wild type caveolin-3 was efficiently targeted to the plasma membrane. In accordance with these observations, caveolin-3 mutants formed oligomers of a much larger size than wild type caveolin-3 and were excluded from caveolae-enriched membrane fractions as seen by sucrose density gradient centrifugation. In addition, these caveolin-3 mutants were expressed at significantly lower levels and had a dramatically shortened half-life of approximately 45-60 min. However, caveolin-3 mutants were palmitoylated to the same extent as wild type caveolin-3, indicating that targeting to the plasma membrane is not required for palmitoylation of caveolin-3. In conclusion, we show that LGMD-1C mutations lead to formation of unstable high molecular mass aggregates of caveolin-3 that are retained within the Golgi complex and are not targeted to the plasma membrane. Consistent with its autosomal dominant form of genetic transmission, we demonstrate that LGMD-1C mutants of caveolin-3 behave in a dominant-negative fashion, causing the retention of wild type caveolin-3 at the level of the Golgi. These data provide a molecular explanation for why caveolin-3 levels are down-regulated in patients with this form of limb girdle muscular dystrophy (LGMD-1C)

dysferlin in a hyperckaemic patient with caveolin 3 mutation and in c2c12 cells after p38 map kinase inhibition

Dysferlin is a plasma membrane protein of skeletal muscle whose deficiency causes Miyoshi myopathy, limb girdle muscular dystrophy 2B and distal anterior compartment myopathy. Recent studies have reported that dysferlin is implicated in membrane repair mechanism and coimmunoprecipitates with caveolin 3 in human skeletal muscle. Caveolin 3 is a principal structural protein of caveolae membrane domains in striated muscle cells and cardiac myocytes. Mutations of caveolin 3 gene (CAV3) cause different diseases and where caveolin 3 expression is defective, dysferlin localization is abnormal. We describe the alteration of dysferlin expression and localization in skeletal muscle from a patient with raised serum creatine kinase (hyperCKaemia), whose reduction of caveolin 3 is caused by a CAV3 P28L mutation. Moreover, we performed a study on dysferlin interaction with caveolin 3 in C2C12 cells. We show the association of dysferlin to cellular membrane of C2C12 myotubes and the low affinity link between dysferlin and caveolin 3 by immunoprecipitation techniques. We also reproduced caveolinopathy conditions in C2C12 cells by a selective p38 MAP kinase inhibition with SB203580, which blocks the expression of caveolin 3. In this model, myoblasts do not fuse into myotubes and we found that dysferlin expression is reduced. These results underline the importance of dysferlin-caveolin 3 relationship for skeletal muscle integrity and propose a cellular model to clarify the dysferlin alteration mechanisms in caveolinopathies

Clinical and translational implications of the caveolin gene family: lessons from mouse models and human genetic disorders.

Here we review the clinical and translational implications of the caveolin gene family for understanding the pathogenesis of human diseases, including breast and prostate cancers, pulmonary hypertension, cardiomyopathy, diabetes, and muscular dystrophy. Detailed phenotypic analysis of caveolin knockout mice has served to highlight the crucial role of a caveolin deficiency in the pathogenesis of many human disease processes. Mutations in the human caveolin genes are associated with a number of established genetic disorders (such as breast cancer, lipodystrophy, muscular dystrophy, and cardiomyopathy), making the caveolins important and novel targets for drug development. The implementation of new strategies for caveolin replacement therapy-including caveolin mimetic peptides-is ongoing

Caveolin-3 Null Mice Show a Loss of Caveolae, Changes in the Microdomain Distribution of the Dystrophin-Glycoprotein Complex, and T-tubule Abnormalities

Caveolin-3, a muscle-specific caveolin-related protein, is the principal structural protein of caveolae membrane domains in striated muscle cells. Recently, we identified a novel autosomal dominant form of limb-girdle muscular dystrophy (LGMD-1C) in humans that is due to mutations within the coding sequence of the human caveolin-3 gene (3p25). These LGMD-1C mutations lead to an approximately 95% reduction in caveolin-3 protein expression, i.e. a caveolin-3 deficiency. Here, we created a caveolin-3 null (CAV3 -/-) mouse model, using standard homologous recombination techniques, to mimic a caveolin-3 deficiency. We show that these mice lack caveolin-3 protein expression and sarcolemmal caveolae membranes. In addition, analysis of skeletal muscle tissue from these caveolin-3 null mice reveals: (i) mild myopathic changes; (ii) an exclusion of the dystrophin-glycoprotein complex from lipid raft domains; and (iii) abnormalities in the organization of the T-tubule system, with dilated and longitudinally oriented T-tubules. These results have clear mechanistic implications for understanding the pathogenesis of LGMD-1C at a molecular level

Hyperkinetic stereotyped movements in a boy with biallelic CNTNAP2 variants

SCV was supported by a Max Planck Research Group awarded by the Max Planck Gesellschaft, a Human Frontiers Science Program Grant (RGP0058/2016), and a UKRI Future Leaders Fellowship (MR/T021985/1). MA was supported by an International Max Planck Research School (IMPRS) PhD Fellowship from the Max Planck Institute for Psycholinguistics.Background:  Heterozygous variants in CNTNAP2 have been implicated in a wide range of neurological phenotypes, including intellectual disability (ID), epilepsy, autistic spectrum disorder (ASD), and impaired language. However, heterozygous variants can also be found in unaffected individuals. Biallelic CNTNAP2 variants are rarer and cause a well-defined genetic syndrome known as CASPR2 deficiency disorder, a condition characterised by ID, early-onset refractory epilepsy, language impairment, and autistic features. Case-report : A 7-year-old boy presented with hyperkinetic stereotyped movements that started during early infancy and persisted over childhood. Abnormal movements consisted of rhythmic and repetitive shaking of the four limbs, with evident stereotypic features. Additional clinical features included ID, attention deficit-hyperactivity disorder (ADHD), ASD, and speech impairment, consistent with CASPR2 deficiency disorder. Whole-genome array comparative genomic hybridization detected a maternally inherited 0.402 Mb duplication, which involved intron 1, exon 2, and intron 2 of CNTNAP2 (c.97 +?_209-?dup). The affected region in intron 1 contains a binding site for the transcription factor FOXP2, potentially leading to abnormal CNTNAP2 expression regulation. Sanger sequencing of the coding region of CNTNAP2 also identified a paternally-inherited missense variant c.2752C > T, p.(Leu918Phe). Conclusion : This case expands the molecular and phenotypic spectrum of CASPR2 deficiency disorder, suggesting that Hyperkinetic stereotyped movements may be a rare, yet significant, clinical feature of this complex neurological disorder. Furthermore, the identification of an in-frame, largely non-coding duplication in CNTNAP2 points to a sophisticated underlying molecular mechanism, likely involving impaired FOXP2 binding.Publisher PDFPeer reviewe

Novel TRIM32 mutation in sarcotubular myopathy

Tripartite motif-containing protein 32 (TRIM32) is a member of the TRIM ubiquitin E3 ligases which ubiquitinates different substrates in muscle including sarcomeric proteins. Mutations in TRIM32 are associated with Limb-Girdle Muscular Dystrophy 2H. In a 66 old woman with disto-proximal myopathy, we identified a novel homozygous mutation of TRIM32 gene c.1781G > A (p. Ser594Asn) localised in the c-terminus NHL domain. Mutations of this domain have been also associated to Sarcotubular Myopathy (STM), a form of distal myopathy with peculiar features in muscle biopsy, now considered in the spectrum of LGMD2H. Muscle biopsy revealed severe abnormalities of the myofibrillar network with core like areas, lobulated fibres, whorled fibres and multiple vacuoles. Desmin and Myotilin stainings also pointed to accumulation as in Myofibrillar Myopathy. This report further confirms that STM and LGMD2H represent the same disorder and suggests to consider TRIM32 mutations in the genetic diagnosis of Sarcotubular Myopathy and Myofibrillar Myopathy

Validation of a score tool for measurement of histological severity in juvenile dermatomyositis and association with clinical severity of disease.

OBJECTIVES: To study muscle biopsy tissue from patients with juvenile dermatomyositis (JDM) in order to test the reliability of a score tool designed to quantify the severity of histological abnormalities when applied to biceps humeri in addition to quadriceps femoris. Additionally, to evaluate whether elements of the tool correlate with clinical measures of disease severity. METHODS: 55 patients with JDM with muscle biopsy tissue and clinical data available were included. Biopsy samples (33 quadriceps, 22 biceps) were prepared and stained using standardised protocols. A Latin square design was used by the International Juvenile Dermatomyositis Biopsy Consensus Group to score cases using our previously published score tool. Reliability was assessed by intraclass correlation coefficient (ICC) and scorer agreement (α) by assessing variation in scorers' ratings. Scores from the most reliable tool items correlated with clinical measures of disease activity at the time of biopsy. RESULTS: Inter- and intraobserver agreement was good or high for many tool items, including overall assessment of severity using a Visual Analogue Scale. The tool functioned equally well on biceps and quadriceps samples. A modified tool using the most reliable score items showed good correlation with measures of disease activity. CONCLUSIONS: The JDM biopsy score tool has high inter- and intraobserver agreement and can be used on both biceps and quadriceps muscle tissue. Importantly, the modified tool correlates well with clinical measures of disease activity. We propose that standardised assessment of muscle biopsy tissue should be considered in diagnostic investigation and clinical trials in JDM

Caveolin-3 Directly Interacts with the C-terminal Tail of β-Dystroglycan IDENTIFICATION OF A CENTRAL WW-LIKE DOMAIN WITHIN CAVEOLIN FAMILY MEMBERS

Caveolin-3, the most recently recognized member of the caveolin gene family, is muscle-specific and is found in both cardiac and skeletal muscle, as well as smooth muscle cells. Several independent lines of evidence indicate that caveolin-3 is localized to the sarcolemma, where it associates with the dystrophin-glycoprotein complex. However, it remains unknown which component of the dystrophin complex interacts with caveolin-3. Here, we demonstrate that caveolin-3 directly interacts with b-dystroglycan, an integral membrane component of the dystrophin complex. Our results indicate that caveolin-3 co-localizes, co-fractionates, and coimmunoprecipitates with a fusion protein containing the cytoplasmic tail of b-dystroglycan. In addition, we show that a novel WW-like domain within caveolin-3 directly recognizes the extreme C terminus of b-dystroglycan that contains a PPXY motif. As the WW domain of dystrophin recognizes the same site within b-dystroglycan, we also demonstrate that caveolin-3 can effectively block the interaction of dystrophin with b-dystroglycan. In this regard, interaction of caveolin-3 with b-dystroglycan may competitively regulate the recruitment of dystrophin to the sarcolemma. We discuss the possible implications of our findings in the context of Duchenne muscular dystrophy

Inter-society consensus document on treatment and prevention of bronchiolitis in newborns and infants

Acute bronchiolitis is the leading cause of lower respiratory tract infection and hospitalization in children less than 1 year of age worldwide. It is usually a mild disease, but some children may develop severe symptoms, requiring hospital admission and ventilatory support in the ICU. Infants with pre-existing risk factors (prematurity, bronchopulmonary dysplasia, congenital heart diseases and immunodeficiency) may be predisposed to a severe form of the disease. Clinical diagnosis of bronchiolitis is manly based on medical history and physical examination (rhinorrhea, cough, crackles, wheezing and signs of respiratory distress). Etiological diagnosis, with antigen or genome detection to identify viruses involved, may have a role in reducing hospital transmission of the infection. Criteria for hospitalization include low oxygen saturation (<90-92%), moderate-to-severe respiratory distress, dehydration and presence of apnea. Children with pre-existing risk factors should be carefully assessed. To date, there is no specific treatment for viral bronchiolitis, and the mainstay of therapy is supportive care. This consists of nasal suctioning and nebulized 3% hypertonic saline, assisted feeding and hydration, humidified O2 delivery. The possible role of any pharmacological approach is still debated, and till now there is no evidence to support the use of bronchodilators, corticosteroids, chest physiotherapy, antibiotics or antivirals. Nebulized adrenaline may be sometimes useful in the emergency room. Nebulized adrenaline can be useful in the hospital setting for treatment as needed. Lacking a specific etiological treatment, prophylaxis and prevention, especially in children at high risk of severe infection, have a fundamental role. Environmental preventive measures minimize viral transmission in hospital, in the outpatient setting and at home. Pharmacological prophylaxis with palivizumab for RSV bronchiolitis is indicated in specific categories of children at risk during the epidemic period. Viral bronchiolitis, especially in the case of severe form, may correlate with an increased incidence of recurrent wheezing in pre-schooled children and with asthma at school age. The aim of this document is to provide a multidisciplinary update on the current recommendations for the management and prevention of bronchiolitis, in order to share useful indications, identify gaps in knowledge and drive future research

Serum levels of VCAM-1 are associated with survival in patients treated with nivolumab for NSCLC

Background High circulating levels of cellular adhesion molecules (CAMs) in non-small cell lung cancer (NSCLC) have been supposed to act as a negative prognostic factor. Here, we explored the predictive role of pre-treatment levels of CAMs in previously treated patients receiving nivolumab for NSCLC. Materials and methods Seventy one patients with advanced NSCLC, treated with nivolumab at the dose of 3 mg/kg every 14 days, were enrolled. Maximum follow-up time was 3 years. Serum levels of Vascular Cell Adhesion Molecule-1 (VCAM-1) and Intracellular Adhesion Molecule-1 (ICAM-1) were measured at baseline and before each nivolumab administration. Endpoints of the study were a composite outcome of survival &gt;= 2 years or absence of disease progression at the end of the follow-up, and the overall survival. Results Composite outcome and overall survival were positively associated with VCAM-1 baseline levels and with the reduction of VCAM-1 during the treatment. After adjustment for potential confounders, the change in VCAM-1 serum levels during the treatment was an independent predictor of overall survival. Conclusions High baseline serum levels of VCAM-1 are associated with a longer survival in patients treated with nivolumab as second line treatment for NSCLC. Surviving patients experience also a significant reduction in CAMs expression during the treatment. Hence, CAMs might be promising prognostic factors in patients with NSCLC underoing immunotherapy