Search

BackgroundIn up to 20% of patients with systemic sclerosis (SSc) none of the established SSc-specific autoantibodies are present [1]. Notwithstanding, in many of these patients high-titer autoantibodies can be detected on the HEp-2 indirect immunofluorescence assay (HEp-2 IIFA) which suggests the presence of an autoantibody to an intracellular protein expressed by the HEp-2 cell line. Immunoprecipitation of unlabeled cell extract followed by gel-free liquid chromatography tandem mass-spectrometry analysis has the potential to identify new autoantigens in an unbiased manner.ObjectivesTo identify new autoantigens through immunoprecipitation combined with liquid chromatography-tandem mass spectrometry (IP + LC-MS/MS) in HEp-2 IIFA-positive patients with SSc in whom none of the established SSc-specific autoantibody specificities are present.MethodsForty-nine patients from the University Hospitals Leuven that fulfilled the EULAR-ACR 2013 classification criteria for systemic sclerosis or LeRoy and Medsger’s criteria for early systemic sclerosis and who were negative on the EliA CTD Screen (Thermo Fisher Scientific, United States), which includes centromere protein B, topoisomerase I, RNA polymerase III, fibrillarin, PM-Scl and U1 ribonucleoprotein, were identified. Immunoprecipitation was performed by incubation of sera of these patients (1/30 in 300 µl Tris-buffered saline) with Pierce A/G magnetic beads, subsequent cross-linking with bissulfosuccinimidyl suberate (BS3) followed by incubation with nuclear extract of HeLa cells (100-150 µg) overnight at 4°C. The eluted protein was analyzed through liquid chromatography with tandem mass spectrometry. Mass spectrometry data were matched against the Uniprot Homo Sapiens database with the Mascot search engine. Candidate autoantigens were confirmed through immunoprecipitation followed by western blot of the eluate with target-specific polyclonal rabbit antibodies or western blot of recombinant protein incubated with sera of the index patients.ResultsWe identified multiple new autoantigens, including the THO complex subunit 1 (THOC1) and other subunits of the THO complex in 3 patients, nuclear valosin-containing protein like-2 (NVL) in 2 patients, nucleolar and coiled-body phosphoprotein 1 (NOLC1) and multiple interacting proteins in 1 patient, probable 28S rRNA (cytosine(4447)-C(5))-methyltransferase (NOP2) in 1 patient, telomeric repeat-binding factor 2 (TERF2) and TERF2-interacting protein (TERF2IP) in 1 patient and regulator of chromosome condensation 1 (RCC1) in 1 patient. The new targets were confirmed through immunoprecipitation-western blot or western blot of recombinant protein incubated with sera (Figure 1). Furthermore, in 10 patients known SSc-associated autoantigens were strongly immunoprecipitated including multiple Th/To subunits in 5 patients, RuvBL1/2 in 2 patients, multiple PM-Scl subunits in 2 patients (who both were negative on the EliA CTD Screen), and fibrillarin in 1 patient (who was also negative on the EliA CTD .).Figure 1.Immunoprecipitation-western blot with target-specific rabbit polyclonal antibody (1/500-2000 dilution), numbers corresponding to order of description of patients, HC healthy control. NE nuclear extract. RP recombinant protein WB-RP western blot of recombinant protein incubated with patient’s sera.ConclusionMultiple new autoantigens were identified and confirmed in patients with SSc without previously identified autoantibody specificity. Further evaluation of reactivity against the newly identified autoantigens in patients with SSc with known autoantibody specificities and other cohorts is required. IP + LC-MS/MS can identify new and established autoantigens in patients with SSc.References[1]Meier FMP, Frommer KW, Dinser R, et al. Update on the profile of the EUSTAR cohort: an analysis of the EULAR Scleroderma Trials and Research group database. Ann Rheum Dis 2012;71:1355–60. doi:10.1136/annrheumdis-2011-200742Disclosure of InterestsJean-Baptiste Vulsteke: None declared, Daniel Blockmans: None declared, Petra De Haes: None declared, Steven Vanderschueren: None declared, Patrick Verschueren: None declared, Kristl G Claeys: None declared, Wim Wuyts Grant/research support from: Boehringer-Ingelheim, Galapagos, Roche, Jan Leo Lenaerts: None declared, Ellen De Langhe: None declared, Xavier Bossuyt Consultant of: Inova Diagnostics, Thermo Fisher Scientific.

International audience; 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 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.

Charcot-Marie-Tooth disease (CMT) is the most common neuromuscular disorder. It represents a group of clinically and genetically heterogeneous inherited neuropathies. Here, we review the results of molecular genetic investigations and the clinical and neurophysiological features of the different CMT subtypes. The products of genes associated with CMT phenotypes are important for the neuronal structure maintenance, axonal transport, nerve signal transduction and functions related to the cellular integrity. Identifying the molecular basis of CMT and studying the relevant genes and their functions is important to understand the pathophysiological mechanisms of these neurodegenerative disorders, and the processes involved in the normal development and function of the peripheral nervous system. The results of molecular genetic investigations have impact on the appropriate diagnosis, genetic counselling and possible new therapeutic options for CMT patients.