Downloaded from http://jmg.bmj.com/ on December 5, 2017 - Published by group.bmj.com Genotype-phenotype correlations ORIGINAL ARTICLE Mutations in SDHD lead to autosomal recessive encephalomyopathy and isolated mitochondrial complex II deficiency Christopher Benjamin Jackson,1,2 Jean-Marc Nuoffer,3 Dagmar Hahn,3 Holger Prokisch,4,5 Birgit Haberberger,4 Matthias Gautschi,3,6 Annemarie Häberli,3 Sabina Gallati,1 André Schaller1 ▸ Additional material is ABSTRACT succinate to fumarate, is formed by the two larger published online only. To view Background Defects of the mitochondrial respiratory hydrophilic subunits, SDHA and SDHB, which also please visit the journal online (10.1136/jmedgenet-2013- chain complex II (succinate dehydrogenase (SDH) harbour the redox cofactors that participate in elec- 101932) complex) are extremely rare. Of the four nuclear encoded tron transfer to ubiquinone. The cofactor FAD is 1 proteins composing complex II, only mutations in the covalently bound to SDHA which provides the suc- Division of Human Genetics, fl fi Departments of Paediatrics and 70 kDa avoprotein (SDHA) and the recently identi ed cinate binding site, and SDHB possesses three Fe-S Clinical Research, University of complex II assembly factor (SDHAF1) have been found to centres which mediate the electron transfer to ubi- Bern, Bern, Switzerland be causative for mitochondrial respiratory chain diseases. quinone. The smaller hydrophobic SDHC and 2 Graduate School for Cellular Mutations in the other three subunits (SDHB, SDHC, SDHD subunits constitute the membrane anchor and Biomedical Sciences, SDHD) and the second assembly factor (SDHAF2) have and ubiquinone binding sites of CII and localise University of Bern, Bern, 2–4 Switzerland so far only been associated with hereditary CII to the inner mitochondrial membrane. 3Institute of Clinical Chemistry, paragangliomas and phaeochromocytomas. Recessive To date, two dedicated CII assembly factors, University Hospital Bern, Bern, germline mutations in SDHB have recently been SDHAF15 and SDHAF2,6 are known and required Switzerland 4 associated with complex II deficiency and leukodystrophy for stable assembly and full activity of CII. Complex Institute of Human Genetics, fi Helmholtz Zentrum München, in one patient. II de ciency is a rare condition in humans and 7 München, Germany Methods and results We present the clinical and accounts for only 2–4% of OXPHOS defects. Its 5Institute of Human Genetics, molecular investigations of the first patient with clinical presentation is highly variable, ranging from Technische Universität biochemical evidence of a severe isolated complex II early onset encephalomyopathies to tumour suscepti- München, München, Germany fi SDHA 6Division of Paediatric de ciency due to compound heterozygous SDHD gene bility in adults. Recessive mutations in are pre- Endocrinology, Diabetology mutations. The patient presented with early progressive dominantly associated with Leigh’s syndrome in and Metabolism, Department encephalomyopathy due to compound heterozygous p. children or in a single case with late-onset neurode- of Paediatrics, University of E69 K and p.*164Lext*3 SDHD mutations. Native generative disease with progressive optic atrophy, Bern, Bern, Switzerland polyacrylamide gel electrophoresis and western blotting ataxia, and myopathy.8 Recently, mutations in SDHA 9 Correspondence to demonstrated an impaired complex II assembly. have been linked with dilated cardiomyopathy. In Dr André Schaller, Division of Complementation of a patient cell line additionally contrast, heterozygous mutations in the other three Human Genetics, Departments supported the pathogenicity of the novel identified structural subunits, SDHB, SDHC,andSDHD,are of Paediatrics and Clinical mutations in SDHD. responsible for dominantly inherited paragangliomas Research, Inselspital Bern, fi 10–12 Bern CH-3010, Switzerland; Conclusions This report describes the rst case of (PGL) and phaeochromocytomas. However, [email protected] isolated complex II deficiency due to recessive SDHD recessive germline mutations in SDHB are also asso- germline mutations. We therefore recommend screening ciated with a primary mitochondrial disease as shown CBJ and J-MN contributed for all SDH genes in isolated complex II deficiencies. It recently in a patient with leukodystrophy.13 equally. further emphasises the importance of appropriate genetic Mutations in SDHAF1 have been associated with a Received 18 July 2013 counselling to the family with regard to SDHD mutations highly progressive infantile leukoencephalopathy Revised 21 November 2013 and their role in tumorigenesis. accompanied by an isolated complex II deficiency,5 Accepted 27 November 2013 whereas mutations in SDHAF2 have been associated Published Online First with hereditary paranganglioma.6 23 December 2013 INTRODUCTION The mitochondrial oxidative phosphorylation SUBJECT AND METHODS (OXPHOS) system is the final biochemical pathway Informed consent was obtained from all participat- in the production of adenosine triphosphate (ATP). ing individuals and the study was approved by the Defects in the OXPHOS system result in devastat- local ethical committee. ing, mainly multisystem, diseases.1 The system con- sists of five multiprotein complexes. The individual Patient subunits are encoded by either the mitochondrial The patient was the second female child of non- or the nuclear genome. Only the succinate consanguineous Swiss parents, with normal preg- To cite: Jackson CB, dehydrogenase (SDH) complex (complex II, CII) is nancy and birth. Clinical examination was normal. Nuoffer J-M, Hahn D, et al. entirely encoded by the nuclear genome. It is com- There was no relevant family history. J Med Genet 2014;51: posed of the four subunits SDHA, SDHB, SDHC, After respiratory syncytial virus (RSV) bronchio- – 170 175. and SDHD. The catalytic core, which dehydrates litis at 3 months of age, the parents observed a 170 Jackson CB, et al. J Med Genet 2014;51:170–175. doi:10.1136/jmedgenet-2013-101932 Downloaded from http://jmg.bmj.com/ on December 5, 2017 - Published by group.bmj.com Genotype-phenotype correlations developmental regression with reduced motor activity, followed SDHAF1, and SDHAF2 were amplified from genomic DNA by by a slower pace of psychomotor development. At 10 months means of PCR using primers listed in the online supplementary the patient had pendular nystagmus in all directions, muscle data. Mutation analysis of the amplified exons was performed hypotonia, and severely retarded development (Griffith by single strand conformation polymorphism (SSCP) as Developmental Quotient (DQ) 53). Biochemical analyses were described previously.17 PCR products were sequenced using unremarkable, including normal lactate, glucose, electrolytes, BigDye Terminator Chemistry (Applied Biosystems) and sepa- liver enzymes, blood gases, amino acids and congenital disorders rated on an ABI 3100 DNA Sequencer. Data were analysed with of glycosylation (CDG) screening and organic acids. Brain MRI, SeqScape V.2.1.1software (Applied Biosystems). EEG, and screening for hearing were normal. The ocular fundi were normal apart from bilateral pale papillae. SDS and native-PAGE and western blotting By 2 years, the patient had developed secondary microceph- SDS-PAGE aly, ataxia and dystonia, and her vision had deteriorated. For western blotting 7 μg of isolated mitochondria of fibroblasts Routine laboratory results were always normal outside severe and 10 μg of muscle homogenate were separated by 15% catabolic episodes. A further extended metabolic work-up was SDS-PAGE (sodium dodecyl sulfate- polyacrylamide gel electro- normal. A second brain MRI and spectroscopy were equally phoresis). The membrane was cut into two pieces between 37 kDa normal apart from a relatively low NAA peak. No abnormal and 50 kDa. Proteins were visualised using antibodies against peak could be detected, including for lactate or succinate. The complex II 70 kDa (SDHA, upper part) and 30 kDa (SDHB, lower parents later refused further follow-up imaging, because they part) subunit. The membrane hybridised with the antibody against had observed developmental regression after each MRI in the SDHB was stripped and probed again with a porin antibody. All context of prolonged fasting. primary antibodies were purchased from Mitoscience/Abcam. For Subsequently, several episodes of developmental regression detection, blots were treated with appropriate horseradish perox- were observed. They occurred after periods of prolonged fasting ide (HRP)-conjugated immunoglobulins, stained with ECL (GE or one of her frequent respiratory or gastrointestinal infections. Healthcare) and exposed to film. Her maximum developmental age was 9–11 months at 3–4 years of age. Native-PAGE After 4.5 years of age, she developed polymorphic epileptic sei- For western blotting 15 μg of isolated fibroblast mitochondria zures, and later also continuous intractable myoclonic movements. and 25 μg of 600×g supernatants of muscle homogenates were At 7 years of age, in the context of aspiration pneumonia, she had centrifuged (30 min, 4°C, 13 000 rpm) and the oxidative phos- lactic acidosis (lactate 10.2 mmol/L), with an increased anion gap; phorylation complexes were solubilised by 5 mg digitonin per creatine kinase (CK) was 2496 U/L (reference range (NR) <154 U/ mg protein before separation by native 4.5–13% PAGE as L), aspartate aminotransferase (ASAT) 177 U/L (NR 11–47 U/L), recommended.18 After blotting, the membrane was cut below and alanine aminotransferase (ALAT)