European Journal of Human Genetics (2011) 19, 138–144 & 2011 Macmillan Publishers Limited All rights reserved 1018-4813/11 www.nature.com/ejhg ARTICLE Mitochondrial dysfunction and organic aciduria in five patients carrying mutations in the Ras-MAPK pathway Tjitske Kleefstra1,8, Saskia B Wortmann2,8, Richard JT Rodenburg2,3, Ernie MHF Bongers1, Kinga Hadzsiev4, Cees Noordam5, Lambert P van den Heuvel2,3, Willy M Nillesen1, Katalin Hollody4, Gabrielle Gillessen-Kaesbach6, Martin Lammens7, Jan AM Smeitink2, Ineke van der Burgt1 and Eva Morava*,2 Various syndromes of the Ras-mitogen-activated protein kinase (MAPK) pathway, including the Noonan, Cardio-Facio-Cutaneous, LEOPARD and Costello syndromes, share the common features of craniofacial dysmorphisms, heart defect and short stature. In a subgroup of patients, severe muscle hypotonia, central nervous system involvement and failure to thrive occur as well. In this study we report on five children diagnosed initially with classic metabolic and clinical symptoms of an oxidative phosphorylation disorder. Later in the course of the disease, the children presented with characteristic features of Ras-MAPK pathway-related syndromes, leading to the reevaluation of the initial diagnosis. In the five patients, in addition to the oxidative phosphorylation disorder, disease-causing mutations were detected in the Ras-MAPK pathway. Three of the patients also carried a second, mitochondrial genetic alteration, which was asymptomatically present in their healthy relatives. Did we miss the correct diagnosis in the first place or is mitochondrial dysfunction directly related to Ras-MAPK pathway defects? The Ras- MAPK pathway is known to have various targets, including proteins in the mitochondrial membrane influencing mitochondrial morphology and dynamics. Prospective screening of 18 patients with various Ras-MAPK pathway defects detected biochemical signs of disturbed oxidative phosphorylation in three additional children. We concluded that only a specific, metabolically vulnerable sub-population of patients with Ras-MAPK pathway mutations presents with mitochondrial dysfunction and a more severe, early-onset disease. We postulate that patients with Ras-MAPK mutations have an increased susceptibility, but a second metabolic hit is needed to cause the clinical manifestation of mitochondrial dysfunction. European Journal of Human Genetics (2011) 19, 138–144; doi:10.1038/ejhg.2010.171; published online 10 November 2010 Keywords: Cardio-Facio-Cutaneous syndrome; HRAS; PTPN11; mitochondrial encephalomyopathy; 3-methylglutaconic aciduria; ethylmalonic aciduria INTRODUCTION Mitochondrial dysfunction is the most common inborn error of It is well established that heterozygous germline mutations in metabolism in children, the diagnosis of which is based on charac- genes encoding participants of the Ras-mitogen-activated protein teristic clinical symptoms of multisystem involvement and on the kinase (MAPK) pathway cause Noonan syndrome (NS) and the presence of metabolic markers. Clinical diagnosis is possible using a related disorders, Cardio-Facio-Cutaneous (CFC), LEOPARD and validated scoring system (mitochondrial disease criteria, MDC score) Costello (CS) syndromes.1 These are clinically characterized by considering clinical signs and symptoms, as well as biochemical distinctive facial appearance, heart defects, ectodermal abnormalities, abnormalities (eg, lactic acidemia, elevated serum alanine and urinary variable cognitive defects, motor delay and susceptibility to certain excretion of certain organic acids and Krebs’ cycle intermediates).3 malignancies. Mutations in PTPN11, RAF1 and NF1 have been The ultimate diagnosis is based on the measurement of abnormal ATP identified in patients with NS/LEOPARD syndrome and neurofibro- production from substrate oxidation and the presence of oxidative matosis type 1. HRAS mutations have been identified in 80–90% of phosphorylation (OXPHOS) enzyme complex deficiency in muscle patients with CS, and BRAF and MEK1/2 mutations were found in specimen or cultured fibroblasts.4 CFC patients. KRAS mutations have been identified in NS and CFC. Recently, it was shown that the clinical presentation of congenital SOS, SHOC2, NRAS and RAF1 mutations were found in 10–30% myopathy with excess of muscle spindles and hypertrophic of NS patients without PTPN11 and KRAS mutations, respectively. cardiomyopathy is caused by HRAS germline mutations.5 Moreover, Loss-of-function mutations in SPRED1 were identified in neuro- mitochondrial dysfunction with multiple enzyme deficiencies of fibromatosis type I-like syndrome. All molecules regulate the the OXPHOS system has been described in one patient with Ras-MAPK cascade.2 CFC syndrome carrying a BRAF mutation and showing muscular 1Department of Human Genetics, Radboud University Nijmegen Medical Centre, Nijmegen, The Netherlands; 2Nijmegen Center for Mitochondrial Disorders at the Department of Pediatrics and the Institute for Genetic and Metabolic Disease (IGMD), Radboud University Nijmegen Medical Centre, Nijmegen, The Netherlands; 3Department of Laboratory Medicine, Radboud University Nijmegen Medical Centre, Nijmegen, The Netherlands; 4Department of Human Genetics and Pediatrics, Radboud University Nijmegen Medical Centre, University of Pecs, Pecs, Hungary; 5Department of Pediatric Endocrinology, Radboud University Nijmegen Medical Centre, Nijmegen, The Netherlands; 6Institute for Human Genetics, University Luebeck, Luebeck, Germany; 7Department of Pathology, Radboud University Nijmegen Medical Centre, Nijmegen, The Netherlands *Correspondence: Dr E Morava, Radboud University Nijmegen Medical Centre, Nijmegen Center for Mitochondrial Disorders at the Department of Pediatrics and the Institute for Genetic and Metabolic Disease (IGMD), PO Box 9101, 6500 HB Nijmegen, The Netherlands. Tel:+31 24 361 9470; Fax:+31 24 361 6428; E-mail: [email protected] 8These authors contributed equally to this work. Received 18 January 2010; revised 31 August 2010; accepted 22 September 2010; published online 10 November 2010 Mutations in MAPK and mitochondrial dysfunction TKleefstraet al 139 coenzyme Q deficiency. This suggests a functional connection between under general anesthesia and by obtaining the adjacent skin sample during the Ras-MAPK pathway and mitochondrial function.6 biopsy.3 The samples were used for histological, histochemical (hematoxin Interestingly, the role of mitochondrial DNA (mtDNA) mutations has eosin, succinate dehydrogenase (SDH), COX, NADH immune staining and been implicated before in a patient with NS, carrying not PTPN11 electron microscopy) and detailed biochemical investigations. mutations but a heterozygous 3-bp deletion in the beta myosin heavy After biopsy, muscle tissue was immediately placed in ice-cold SETH buffer (0.25 mol/l sucrose, 2 mmol/l EDTA, 10 mmol/l Tris, 5Â104 IU/l heparin, chain gene associated with seven mtDNA alterations. Unfortunately, pH 7.4) and transported to the laboratory within 15 min. Fat and connective familiality and functional assays in that patient were not available. The tissue samples were removed. Muscle tissue was minced with a Sorvall TC2 authors raised the possibility, however, that the mtDNA mutations tissue chopper, homogenized in SETH buffer and centrifuged at 600 g. A portion 7 might have a role in phenotypic presentation. Another recent study of the 600 g supernatant was used for measuring oxidation rates and ATP underlines these findings by showing evidence that the mtDNA haplo- production rates. The remaining 600 g supernatant was frozen in 100-ml aliquots group ‘R’ is associated with NS in South India. In seven patients with the in liquid nitrogen and kept at –80 1C for enzymatic measurements.4 typical clinical picture of NS, mutation analysis of PTPN11 failed to OXPHOS was evaluated by determining the ATP production rate from show alterations. In contrast, a total of 146 mtDNA mutations, five of pyruvate oxidation in fresh muscle and the activity of the enzyme complexes which were novel and exclusively observed in NS patients, were found.8 I–V of the respiratory chain by spectrophotometric assays in frozen muscle 3,4,10 Furthermore, another study could show lower mitochondrial membrane and under standard condition-cultured fibroblasts. Incubations were performed at 37 1C in 20-ml glass incubation vials potential and lower ATP content as well as higher levels of reactive for 20 min. The vials for measuring 14C-labeled substrate oxidations contained oxygen species (ROS) in mouse fibroblast cell lines with constitutively a small plastic tube with 0.2 ml of hyamine. Incubation volume was 9 active SHP2 (as found in NS patients) compared with wild type. 0.5 ml, containing 30 mmol/l potassium phosphate, 75 mmol/l potassium We encountered several patients presenting with clinical and meta- chloride, 8 mmol/l Tris, 1.6 mmol/l EDTA, 5 mmol/l MgCl2 and 0.2 mmol/l bolic features of OXPHOS dysfunction, encephalomyopathy and lactic p1,p5-di(adenosine-5¢) pentaphosphate (myo-adenylate kinase inhibitor). To acidemia in infancy, who later developed clinical features suggesting regenerate ADP by creatine kinase in the 600 g supernatant, 20 mmol/l creatine defects in genes involved in the Ras-MAPK pathway. To collect was added to all ADP-containing incubations.4 information about the association of mitochondrial dysfunction and ATP and phosphocreatine were measured in the supernatant. Samples were Ras-MAPK pathway defects, we systematically studied clinical, bio- thawed, placed on ice for 5 min and centrifuged