B. De Paepe

Inborn oxidative phosphorylation defect as risk factor for propofol infusion syndrome

Propofol is an anesthetic agent widely used for induction and maintenance of anesthesia, and sedation in children. Although generally considered as reliable and safe, administration of propofol can occasionally induce a potentially fatal complication known as propofol infusion syndrome (PRIS). Mitochondrial dysfunction has been implicated in the pathogenesis of PRIS. We report on an adult patient with Leber hereditary optic neuropathy (LHON) who developed PRIS. He was a carrier of the m.3460G>A mutation, one of the major three pathogenic point mutations associated with LHON. The propositus was blind and underwent propofol sedation after severe head injury. Five days after start of propofol infusion, the patient died. The activity of complex I of the oxidative phosphorylation (OXPHOS) system was severely deficient in skeletal muscle. Our observation indicates that fulminate PRIS can occur in an adult patient with an inborn OXPHOS defect and corroborates the hypothesis that PRIS is caused by inhibition of the OXPHOS system.

Immunohistochemical analysis of the oxidative phosphorylation complexes in skeletal muscle from patients with mitochondrial DNA encoded tRNA gene defects.

Background: Mitochondrial diseases display a heterogeneous spectrum of clinical phenotypes and therefore the identification of the underlying gene defect is often a difficult task. Aims: To develop an immunohistochemical approach to stain skeletal muscle for the five multi-protein complexes that organise the oxidative phosphorylation (OXPHOS) in order to improve the diagnostic workup of mitochondrial defects. Methods: OXPHOS complexes were visualised in skeletal muscle tissue using antibodies directed against different subunits. The staining patterns of patients with heteroplasmic defects in mtDNA tRNA genes were compared with those of normal and disease controls. Results: Normal skeletal muscle displayed a checkerboard staining pattern for complexes I to V due to the higher mitochondrial content of slow muscle fibres versus fast fibres. In patients with tRNA defects, a much more heterogeneous staining pattern was observed for complex I (all six patients) and complex IV (4 of 6 patients): a mosaic staining pattern in which individual fibres displayed staining intensities that ranged from strong to negative. Ragged red fibres (RRFs) in patients with MERRF (myoclonic epilepsy and ragged red fibres) were all complex I and IV negative, while in patient with MELAS (mitochondrial myopathy, encephalopathy, lactic acidosis and stroke-like episodes) the majority of RRFs were complex I negative and complex IV positive. Conclusion: Immunohistochemical detection of OXPHOS complexes could represent a valuable additional diagnostic tool for the evaluation of mitochondrial cytopathy. The technique helps to detect heteroplasmic mtDNA defects. Staining for complex I in particular was able to identify two tRNA patients that stayed undetected with routine histochemical evaluation.

Infantile presentation of the mitochondrial A8344G mutation.

E. Scalais, C. Nuttin, S. Seneca, J. Smet, B. De Paepe, J.-J. Martin, R. Stevens, F. Pierart, O. Battisti, W. Lissens, L. De Meirleir and R. Van Coster Division of Paediatric Neurology, Department of Paediatrics, Centre Hospitalier de Luxembourg, Luxembourg; Center for Medical Genetics and Division of Paediatric Neurology, Academic Hospital of the Vrije Universiteit Brussel, Brussels; Division of Paediatric Neurology and Metabolism, Department of Paediatrics, Ghent University Hospital; Institute Born-Bunge, University of Antwerp, Antwerp; and CHC, Clinique Saint-Vincent, Rocourt, Belgium

G.P.21 Proteomic analysis of cultured skin fibroblasts from UCMD patients reveals the involvement of two new cellular pathways

Abstract Ullrich congenital muscular dystrophy (UCMD) is a severe congenital dystrophy caused by collagen VI mutations and resulting in absent or aberrant collagen VI secretion in fibroblasts, the main collagen VI secreting cells. ColVI is a ubiquitous extracellular matrix (ECM) protein that forms a microfibrillar network in close association with the basement membrane in many tissues. ColVI was shown to interact with several ECM components. Although an increased apoptotic rate, a mitochondrial defect and an impairment of the autophagocytic flux have been described in myoblasts and muscle cells from ColVI-deficient mice, the molecular pathways that link ColVI to the basal membrane protein network (fibronectin, etc.) and the downstream intracellular mechanisms are not fully elucidated. We present the results of a proteomic comparison between cultured skin fibroblasts from four unrelated UCMD patients, all carrying the heterozygous de novo p.G284R mutation in the COL6A1 gene, and fibroblasts from controls. Some of the dysregulated proteins observed in the proteomic profiles were confirmed by western blot, qPCR analysis, and fibroblast immunohistochemical staining. A detailed description of the new protein networks will be presented. Our results point to the involvement of two major cellular pathways that have up to now not been described in UCMD fibroblasts, which besides their role in the muscle disease, display typical abnormalities in other tissues like skin and tendons. We propose new insights in the pathophysiology of this matrix disease.

SAT0374 The euromyositis registry: an international description of myositis

Background The idiopathic inflammatory myopathies (IIM) represent a rare and heterogeneous group of multisystem autoimmune diseases. The rarity of IIM has hampered research efforts, resulting in remarkably limited therapeutic evidence. The EuroMyositis Registry was created to pool resources and expertise across the international IIM research community. Objectives To describe the phenotypic characteristics of different IIM subtypes. Associations with malignancy, interstitial lung disease (ILD), cardiac involvement and dysphagia were assessed. Methods Pooled data from the EuroMyositis Registry (from Belgium, China, Czech Republic, Hungary, Italy, Mexico, Norway, Sweden, Switzerland, UK, Vietnam) were obtained. Associations were assessed using logistic regression and multinomial logistic regression with polymyositis (PM) as the reference group. Results Data regarding 3,196 patients were analysed. The UK was the largest contributor (n=1,307). The most common diagnoses were dermatomyositis (34%), PM (32%) and connective tissue disease (CTD)-overlap myositis (15%). In those with anti-synthetase syndrome (7%), 85% had muscle weakness, 86% ILD and 58% arthritis. Overall, 43% had a myositis specific antibody, most commonly anti-Jo1 autoantibodies (20%). Glucocorticoid usage was noted in 98%. Most commonly used disease modifying agents were methotrexate (71%) and azathioprine (50%). Malignancy occurred in 9% and was associated with a diagnosis of dermatomyositis (Relative Risk Ratio [RRR] 1.68, 95% CI 1.09–2.56, p=0.018). Cardiac involvement occurred in 9%, most commonly in those with CTD-overlap myositis (13%), and was associated with a higher Health Assessment Questionnaire disability index (1 versus 0.75, OR 1.40, 95% CI 1.03–1.91, p=0.031). Dysphagia occurred in 39% and was associated with a diagnosis of CTD-overlap myositis (RRR 2.25, 95% CI 1.61–3.15, p<0.001). Conclusions This large international cohort demonstrates the heterogeneity of IIM and the burden of associated malignancy, ILD, cardiac and gastrointestinal involvement. The EuroMyositis Registry facilitates international collaborative research outputs, underlining the benefits of harmonised data collection methodology between centres. Acknowledgements This work was supported by researchers at the National Institute for Health Research (NIHR) Biomedical Research Unit. The views expressed are those of the authors and not necessarily those of the UK National Health Service, the NIHR or the UK Department of Health Disclosure of Interest J. Lilleker: None declared, J. Vencovsky: None declared, G. Wang: None declared, L. Wedderburn Grant/research support from: The UK JDM Cohort and Biomarker study is supported by grants from the NIHR and Myositis UK, L. Diederichsen: None declared, J. Schmidt: None declared, P. Jordan: None declared, O. Benveniste: None declared, M. G. Danieli: None declared, K. Dankό: None declared, N. T. P. Thuy: None declared, M. Vazquez-Del Mercado: None declared, Ø. Molberg: None declared, B. De Paepe: None declared, J. De Bleecker: None declared, B. Maurer Grant/research support from: AbbVie, Protagen, EMDO, Novartis, Roche, Actelion, N. Pipitone Speakers bureau: GRAPPA Workshop, Alfa-Wassermann, N. McHugh: None declared, Z. Betteridge: None declared, P. New: None declared, R. Cooper: None declared, W. Ollier: None declared, J. Lamb Grant/research support from: MedImmune, N. S. Krogh: None declared, I. Lundberg Grant/research support from: Astra-Zeneca, Bristol-Myers Squibb, Consultant for: Bristol-Myers Squibb, Idera, H. Chinoy Grant/research support from: MedImmune, Novartis

PP03.14 – 2978: Massive early leukoencephalopathy caused by a pathogenic mutation in IBA57: A new clinical presentation for this recently described gene defect

Objective Unraveling the underlying molecular defect in a patient with early onset progressive leukoencephalopathy and combined OXPHOS deficiency involving complex I and II. Methods After homozygosity mapping and whole exome sequencing a candidate gene was identified. Pathogenicity was assessed by means of functional studies in HeLa cells and western blotting in patient material. Results In a patient presenting with psychomotor retardation, massive white matter alterations and the presence of high lactate were detected on MRI, and a combined complex I and II deficiency was found in skeletal muscle. This combined complex deficiency is very suggestive of a defective iron-sulfur cluster (ISC) biosynthesis, a suspicion which was further strengthened by absence of lipoic residues in aKGDH and PDH. Homozygosity mapping revealed several candidate genes involved in ISC biogenesis: NFU1, BOLA3, IBA57 and ABCB10. Whole exome sequencing identified a homozygous variant in IBA57 [c.436C> T. (p.Arg146Trp)]. Complementation studies in HeLa cells depleted of wild type IBA57 and transfected with mutant IBA57 showed deficient lipoylation and lowered expression of a complex II subunit. Conclusion By combining a biochemical and molecular approach the underlying genetic cause for this patients phenotype could be unraveled. This is the third report of a pathogenic variant in IBA57 and discloses the clinical and neuroradiological heterogeneity of this gene defect. At the biochemical level a combined complex I and II deficiency and absence of lipoylation seems to be a uniform finding.

G.P.215

Ullrich congenital muscular dystrophy (UCMD) is a severe congenital dystrophy caused by collagen VI (COL VI) mutations and resulting in absent or aberrant collagen VI secretion in fibroblasts. The link between the mutated collagen VI, and Col VI synthesis, secretion into the ECM, interaction with the cell, mitochondrial defect, cell death, ... is not well characterized. The objective of our study was to provide additional proteomics based insight into the cellular impact of the defective ColVI in cultured skin fibroblasts. To reach this objective, a quantitative iTRAQ based LC-MALDI method was used to compare the proteome in cultured skin fibroblasts from UCMD patients with control fibroblasts. We used western blot as a confirmatory method to assess the dysregulation of proteome identified candidates. The most important findings were the differences in protein expression patterns linked to ER collagen-chaperone functioning and collagen I synthesis (alpha 1 chain, alpha 2 chain) together with fibronectin. A number of proteins involved in the formation of adhesions points that form an important link between the ECM and the intracellular space were found dysregulated. Additionally, Immunofluorescence (IF) for vinculin evaluated by several independent observers showed an increased number of focal adhesions on Col VI deficient fibroblasts. Finally IF collagen V evidenced disturbed extracellular network formation with intracellular retention of the protein. Our findings suggest that the dysregulation of the extracellular network in Col VI deficient fibroblasts possibly leads to intracellular stress and compensatory mechanisms. Proteins involved in molecular pathways such us the mitochondrial defect and the impairment of the autophagocytic flux, implicated in UCMD muscle fibers, were not found as differentially regulated in our proteomics experiment on fibroblasts, suggesting different pathophysiological mechanisms at play in these two important cell types in UCMD.

O16 – 1908 A homozygous mutation in IB57 involved in intramitochondrial iron-sulfur cluster synthesis causes severe encephalopathy and mypathy in two neonates

Background: Combined OXPHOS deficiencies involving complexes I and II have recently been detected in patients with deficient iron-sulfur cluster (ISC) biogenesis. So far, patients were reported with pathogenic mutations in NFU1 and BOLA3 presenting with severe encephalomyopathy at young age. Objective: Two siblings with combined deficiency of complex I and II were investigated for possible defect in ISC. Patients and methods: The siblings presented soon after birth with severe encephalomyopathy and died in the neonatal period. Biochemical investigations showed increased lactate in serum and increased glycine in CSF. Considering the consanguineous descent a search for genes in homozygous regions related to ISC metabolism was performed. Results: Isolating IBA57 as a strong candidate gene, sequencing detected a homozygous mutation (c.941A>C) in the two siblings and a heterozygous carrier status in both parents. Western blotting showed a severe decrease of CRM for the IBA57 protein. The protein amount in the complexes I and II was significantly decreased. Transfection experiments in HeLa cells demonstrated that the mutation was pathogenic and that excessive degradation of the IBA57 protein was responsible for the defective ISC biosynthesis. Conclusion: This is the first report of a pathogenic mutation in IBA57 in human.