Svetlana Gorokhova

Improving molecular diagnosis of distal myopathies by targeted next-generation sequencing

Distal myopathies are a heterogeneous group of muscle diseases sharing the clinical pattern of predominant weakness in the feet and/or hands. The classical approach for molecular diagnosis is based on targeted gene-by-gene analysis guided by currently existing combinatorial algorithms.1 Many patients remain undiagnosed. Within the last 5 years, next-generation sequencing (NGS) has emerged as a successful and rapid approach to simultaneously analyse multiple genes in neuromuscular disorders.2 Our objective was to evaluate the efficiency of a targeted NGS approach using a panel of neuromuscular genes on patients with distal myopathies. We first tested its validity in a control group of six index cases (IC) with known molecular diagnosis. Then, we prospectively evaluated this approach by testing a group of 17 IC without molecular diagnosis. ### Patients We prospectively included 54 patients (37 IC and 17 relatives) with a diagnosis of distal myopathy, followed at the Neuromuscular Diseases and ALS Reference Centre of La Timone Hospital, Marseille, France, between 1989 and 2014. Among these 37 IC previously explored by Sanger sequencing, 20 IC had an identified molecular diagnosis: six IC constituted the control group. The remaining 17 undiagnosed IC constituted the test group. A targeted-NGS approach was used to search mutations in 298 neuromuscular genes in both groups. Samples analysed in this study have been prepared and stored by the Center of Biological Resources, Department of Medical Genetics, La Timone Hospital, Marseille, and used following the ethical recommendations of our institution and according to the Declaration of Helsinki. All included patients gave their written consent prior to …

Uncoupling of Molecular Maturation from Peripheral Target Innervation in Nociceptors Expressing a Chimeric TrkA/TrkC Receptor

Neurotrophins and their receptors control a number of cellular processes, such as survival, gene expression and axonal growth, by activating multiple signalling pathways in peripheral neurons. Whether each of these pathways controls a distinct developmental process remains unknown. Here we describe a novel knock-in mouse model expressing a chimeric TrkA/TrkC (TrkAC) receptor from TrkA locus. In these mice, prospective nociceptors survived, segregated into appropriate peptidergic and nonpeptidergic subsets, projected normally to distinct laminae of the dorsal spinal cord, but displayed aberrant peripheral target innervation. This study provides the first in vivo evidence that intracellular parts of different Trk receptors are interchangeable to promote survival and maturation of nociceptors and shows that these developmental processes can be uncoupled from peripheral target innervation. Moreover, adult homozygous TrkAC knock-in mice displayed severe deficits in acute and tissue injury-induced pain, representing the first viable adult Trk mouse mutant with a pain phenotype.

Direct evidence for the interaction of stathmin along the length and the plus-end of microtubules in cells

Stathmin is a prominent destabilizer of microtubules (MTs). Extensive in vitro studies have strongly suggested that stathmin could act by sequestering tubulin and/or by binding to MT tips. In cells, the molecular mechanisms of stathmin binding to tubulin and/or MTs and its implications for the MT dynamics remain unexplored. By using immunofluorescence resonance energy transfer and fluorescence recovery after photobleaching, we analyzed the ability of stathmin and its phosphorylated forms (on Ser16, −25, −38, and −63) to interact with tubulin and MTs in A549 cells. Consistent with in vitro studies, we detected stathmin–tubulin interactions at the MT plus ends and in the cytosol. Of interest, we also observed a novel pool of stathmin bound along the MT. Expression of truncated stathmin and use of MT‐stabilizing taxol further showed that the C‐terminal domain of stathmin is the main contributor to this binding and that the phosphorylation state of stathmin plays a role in its binding along the MT wall. Our findings demonstrate that stathmin binds directly along the MT wall. This pool of stathmin would be readily available to participate in protofilament dissociation when the moving plus end of a depolymerizing MT reaches stathmin molecules.—Nouar, R., Breuzard, G., Bastonero, S., Gorokhova, S., Barbier, P., Devred, F., Kovacic, H., Peyrot, V. Direct evidence for the interaction of stathmin along the length and the plus end of microtubules in cells. FASEB J. 30, 3202–3215 (2016). www.fasebj.org

Genetic Characterization of a French Cohort of GNE‐mutation negative inclusion body myopathy patients with exome sequencing

Hereditary inclusion body myopathy (hIBM) refers to a group of clinically and genetically heterogeneous diseases. The overlapping histochemical features of hIBM with other genetic disorders lead to low diagnostic rates with targeted single‐gene sequencing. This is true for the most prevalent form of hIBM, GNEpathy. Therefore, we used whole‐exome sequencing (WES) to determine whether a cohort of clinically suspected GNEpathy patients undiagnosed by targeted GNE analysis could be genetically characterized.

Comparing targeted exome and whole exome approaches for genetic diagnosis of neuromuscular disorders

Massively parallel sequencing is rapidly becoming a widely used method in genetic diagnostics. However, there is still no clear consensus as to which approach can most efficiently identify the pathogenic mutations carried by a given patient, while avoiding false negative and false positive results. We developed a targeted exome approach (MyoPanel2) in order to optimize genetic diagnosis of neuromuscular disorders. Using this approach, we were able to analyse 306 genes known to be mutated in myopathies as well as in related disorders, obtaining 98.8% target sequence coverage at 20 ×. Moreover, MyoPanel2 was able to detect 99.7% of 11,467 known mutations responsible for neuromuscular disorders. We have then used several quality control parameters to compare performance of the targeted exome approach with that of whole exome sequencing. The results of this pilot study of 140 DNA samples suggest that targeted exome sequencing approach is an efficient genetic diagnostic test for most neuromuscular diseases.

Spindle-Derived NT3 in Sensorimotor Connections: Principal Role at Later Stages

The circuit between muscle spindles, a subtype of proprioceptors (Ia afferents), and motor neurons is established during late embryonic development and is essential for proper coordination and movement. Muscle spindles, the specialized sensory structures that respond to muscle stretch, are induced

Novel Pathogenic Variants in a French Cohort Widen the Mutational Spectrum of GNE Myopathy

Background: GNE myopathy is a rare autosomal recessively inherited muscle disease resulting from mutations in the gene encoding GNE (UDP-N-acetylglucosamine-2-epimerase/N-acetylmannosamine kinase), a key enzyme in sialic acid biosynthesis. 154 different pathogenic variants have been previously associated with GNE myopathy. Objective: Describe novel pathogenic variants associated with GNE myopathy in a large French cohort. Methods: We analyzed mutational data from 32 GNE myopathy index patients. Novel, as well as previously published pathogenic variants, were examined for possible deleterious effects on splicing. Results: We describe 13 novel pathogenic variants in GNE, identified in the first large French cohort reported to date. We also find that 6 published pathogenic variants might have a previously unrecognized deleterious effect on splicing. Conclusions: Novel pathogenic GNE variants described here raise the total number of different pathogenic variants reported to 167, complementing the recently published GNE mutation update. Our novel findings on possible splice-disrupting effects by several variants suggest that the pathogenicity mechanism of these variants could be reinterpreted, expanding our knowledge about the GNE mutational spectrum.