New Clinical and Immunofluoresence Data of Collagen VI-Related Myopathy: A Single Center Cohort of 69 Patients.

Pathogenetic mechanism recognition and proof-of-concept clinical trials were performed in our patients affected by collagen VI-related myopathies. This study, which included 69 patients, aimed to identify innovative clinical data to better design future trials. Among the patients, 33 had Bethlem myopathy (BM), 24 had Ullrich congenital muscular dystrophy (UCMD), 7 had an intermediate phenotype (INTM), and five had myosclerosis myopathy (MM). We obtained data on muscle strength, the degree of contracture, immunofluorescence, and genetics. In our BM group, only one third had a knee extension strength greater than 50% of the predicted value, while only one in ten showed similar retention of elbow flexion. These findings should be considered when recruiting BM patients for future trials. All the MM patients had axial and limb contractures that limited both the flexion and extension ranges of motion, and a limitation in mouth opening. The immunofluorescence analysis of collagen VI in 55 biopsies from 37 patients confirmed the correlation between collagen VI defects and the severity of the clinical phenotype. However, biopsies from the same patient or from patients with the same mutation taken at different times showed a progressive increase in protein expression with age. The new finding of the time-dependent modulation of collagen VI expression should be considered in genetic correction trials.

Mutation spectrum in the large GTPase dynamin 2, and genotype-phenotype correlation in autosomal dominant centronuclear myopathy.

Centronuclear myopathy (CNM) is a genetically heterogeneous disorder associated with general skeletal muscle weakness, type I fiber predominance and atrophy, and abnormally centralized nuclei. Autosomal dominant CNM is due to mutations in the large GTPase dynamin 2 (DNM2), a mechanochemical enzyme regulating cytoskeleton and membrane trafficking in cells. To date, 40 families with CNM-related DNM2 mutations have been described, and here we report 60 additional families encompassing a broad genotypic and phenotypic spectrum. In total, 18 different mutations are reported in 100 families and our cohort harbors nine known and four new mutations, including the first splice-site mutation. Genotype-phenotype correlation hypotheses are drawn from the published and new data, and allow an efficient screening strategy for molecular diagnosis. In addition to CNM, dissimilar DNM2 mutations are associated with Charcot-Marie-Tooth (CMT) peripheral neuropathy (CMTD1B and CMT2M), suggesting a tissue-specific impact of the mutations. In this study, we discuss the possible clinical overlap of CNM and CMT, and the biological significance of the respective mutations based on the known functions of dynamin 2 and its protein structure. Defects in membrane trafficking due to DNM2 mutations potentially represent a common pathological mechanism in CNM and CMT.

Cationic PMMA nanoparticles bind and deliver antisense oligoribonucleotides allowing restoration of dystrophin expression in the mdx mouse.

For subsets of Duchenne muscular dystrophy (DMD) mutations, antisense oligoribonucleotide (AON)-mediated exon skipping has proven to be efficacious in restoring the expression of dystrophin protein. In the mdx murine model systemic delivery of AON, recognizing the splice donor of dystrophin exon 23, has shown proof of concept. Here, we show that using cationic polymethylmethacrylate (PMMA) (marked as T1) nanoparticles loaded with a low dose of 2'-O-methyl-phosphorothioate (2'OMePS) AON delivered by weekly intraperitoneal (IP) injection (0.9 mg/kg/week), could restore dystrophin expression in body-wide striated muscles. Delivery of an identical dose of naked AON did not result in detectable dystrophin expression. Transcription, western, and immunohistochemical analysis showed increased levels of dystrophin transcript and protein, and correct localization at the sarcolemma. This study shows that T1 nanoparticles have the capacity to bind and convoy AONs in body-wide muscle tissues and to reduce the dose required for dystrophin rescue. By immunofluorescence and electron microscopy studies, we highlighted the diffusion pathways of this compound. This nonviral approach may valuably improve the therapeutic usage of AONs in DMD as well as the delivery of RNA molecules with many implications in both basic research and medicine.

Identification of novel GDAP1 mutations causing autosomal recessive Charcot-Marie-Tooth disease.

Mutations in the ganglioside-induced differentiation-associated protein 1 gene cause either autosomal recessive demyelinating Charcot-Marie-Tooth disease type 4A or autosomal recessive axonal Charcot-Marie-Tooth disease with vocal cord paresis. We sequenced the ganglioside-induced differentiation-associated protein 1 gene in 138 patients from 119 unrelated families diagnosed with either demyelinating or axonal autosomal recessive Charcot-Marie-Tooth disease. We detected six distinct mutant alleles in four families, four of which are novel. Electrophysiological studies show severely slowed motor nerve conduction velocities with severely reduced compound muscle action potentials. However, one patient had a normal conduction velocity in the ulnar nerve. Based on the electrophysiological tests, patients with ganglioside-induced differentiation-associated protein 1 mutations will therefore be classified as either axonal or demyelinating Charcot-Marie-Tooth disease. The neuropathological aspect shows a divergent pattern; nerve biopsies taken from two siblings at the same age and sharing the same ganglioside-induced differentiation-associated protein 1 gene mutation showed a dissimilar severity stage.

Refined mapping of the HMSNR critical gene region--construction of a high-density integrated genetic and physical map.

Hereditary motor and sensory neuropathy russe, a form of autosomal recessive Charcot-Marie-Tooth disease, is a rare disorder found in several Roma families from Europe. The gene has been mapped to a 1Mb region on 10q22. Detailed analysis led to the exclusion of 22 candidate genes and the assembly of a high-density genetic map comprising 141 polymorphic markers. Extensive genotyping in an extended sample of affected families resulted in a 10-fold reduction of the critical hereditary motor and sensory neuropathy russe gene region, which is now contained within a single completely sequenced BAC clone. The fact that no sequence variant has been detected in the known genes in the critical region indicates that the hereditary motor and sensory neuropathy russe mutation affects a novel gene that remains to be identified.