Downloaded from http://jmg.bmj.com/ on September 8, 2016 - Published by group.bmj.com New loci ORIGINAL ARTICLE Defective NDUFA9 as a novel cause of neonatally fatal complex I disease B J C van den Bosch,1,2 M Gerards,1,2 W Sluiter,3 A P A Stegmann,1 E L C Jongen,1 D M E I Hellebrekers,1 R Oegema,4 E H Lambrichs,1 H Prokisch,5,6 K Danhauser,5,6 K Schoonderwoerd,4 I F M de Coo,7 H J M Smeets1,2 < An additional table is ABSTRACT patterns. Mutations in nuclear mitochondrial genes published online only. To view Background Mitochondrial disorders are associated with include genes encoding assembly factors, complex this file please visit the journal abnormalities of the oxidative phosphorylation (OXPHOS) subunits of the OXPHOS system, and genes online (http://jmg.bmj.com/ content/49/1.toc). system and cause significant morbidity and mortality in involved in mitochondrial maintenance or metab- the population. The extensive clinical and genetic olism in general.4 Mutations in different genes can 1Department of Clinical Genetics, Unit Clinical heterogeneity of these disorders due to a broad variety lead to similar phenotypes, while mutations in the Genomics, Maastricht University of mutations in several hundreds of candidate genes, same gene can give rise to different phenotypes, Medical Centre, Maastricht, The encoded by either the mitochondrial DNA (mtDNA) or illustrating the clinical and genetic heterogeneity of Netherlands 2 nuclear DNA (nDNA), impedes a straightforward genetic these diseases. Nuclear genes are likely the major School for Oncology and diagnosis. A new disease gene is presented here, Developmental Biology, cause of mitochondrial disease, especially in Maastricht University Medical identified in a single Kurdish patient born from affected children, and, based on the mitochondrial Centre, Maastricht, The consanguineous parents with neonatally fatal Leigh proteome, potentially more than 1500 genes could Netherlands syndrome and complex I deficiency. be involved.5 Pathogenic mutations have been 3 Centre for Lysosomal and Methods and results Using homozygosity mapping and identified in w100 genes during the past years. Metabolic Diseases, Erasmus MC, Rotterdam, The subsequent positional candidate gene analysis, a total However, mutations are often limited to only one Netherlands region of 255.8 Mb containing 136 possible or a few patients and families. The genetic cause 4Department of Clinical mitochondrial genes was identified. A pathogenic still remains unknown in a large proportion of Genetics, Erasmus MC, mutation was found in the complex I subunit encoding patients with mitochondrial disease.6 Rotterdam, The Netherlands the NDUFA9 gene, changing a highly conserved arginine 5Institute of Human Genetics, Several approaches exist to identify gene defects at position 321 to proline. This is the first disease- in patients with mitochondrial disease, such as Technische Universita¨t e Mu¨nchen, Munich, Germany causing mutation ever reported for NDUFA9. Complex I conventional sequencing of candidate genes,5 7 6 e Institute of Human Genetics, activity was restored in fibroblasts of the patient by family based approaches,68 10 and next generation e Helmholtz Zentrum Mu¨nchen, lentiviral transduction with wild type but not mutant sequencing.11 13 Consanguinity or suspected relat- German Research Centre for Environmental Health, NDUFA9, confirming that the mutation causes the edness of the parents of OXPHOS patients makes Neuherberg, Germany complex I deficiency and related disease. homozygosity mapping followed by candidate gene 7Department of Neurology, Conclusions The data show that homozygosity mapping analysis a rapid and effective approach. Here we Erasmus MC, Rotterdam, The and candidate gene analysis remain an efficient way to report the first pathogenic mutation in NDUFA9 in Netherlands detect mutations even in small consanguineous a patient from consanguineous parents with fatal fi Correspondence to pedigrees with OXPHOS deficiency, especially when the Leigh syndrome and complex I de ciency using this Dr B J C van den Bosch, enzyme deficiency in fibroblasts allows appropriate approach. Subsequently, the pathogenicity of the Department of Genetics and Cell candidate gene selection and functional mutation was confirmed by complementation Biology, Maastricht University, complementation. studies in complex I deficient fibroblasts of the P.O. Box 616, 6200 MD Maastricht, The Netherlands; patient. The procedure from the initial single [email protected] nucleotide polymorphism (SNP) array analysis until the identification of the mutation took about BJCB and MG contributed 3 weeks, indicating that this type of approach is equally to this work. INTRODUCTION efficient in terms of success rate, time, and costs. Mitochondrial disorders are clinical phenotypes Received 30 August 2011 Accepted 18 October 2011 associated with abnormalities of the oxidative Published Online First phosphorylation (OXPHOS) system. OXPHOS SUBJECTS AND METHODS 23 November 2011 disorders are among the most common inherited Clinical findings metabolic disorders, with a prevalence of 1 in The patient was a boy born as the first and only 5000e8000, and cause significant morbidity and child from consanguineous Kurdish parents (second mortality in the population.1 They can present at cousins, region Kurdistan in Iraq) at 41 weeks of any age and in any tissue. A broad range of clinical gestation with a birth weight of 3.040 kg (À1 SD) features has been associated with defects in the and an occipitofrontal circumference (OFC) of mitochondrial DNA (mtDNA) and in nuclear genes 33.8 cm (À1 SD). No dysmorphic features were encoding essential proteins involved in the noted. A mildly depressed child was seen at birth OXPHOS system.23Because of this dual genetic with intact functioning of the cranial nerves and control, segregation of OXPHOS disorders can be a normal somatic motor and sensory system with complex with maternal, autosomal recessive, symmetric reflexes. After birth, the child deterio- autosomal dominant or X linked inheritance rated and suffered from a combined respiratory and 10 J Med Genet 2012;49:10e15. doi:10.1136/jmedgenet-2011-100466 Downloaded from http://jmg.bmj.com/ on September 8, 2016 - Published by group.bmj.com New loci Figure 1 Brain MRI leading to the diagnosis of Leigh syndrome. (A) Axial T2 weighted image shows high signal intensity in the basal ganglia and thalami bilaterally (open arrows). In the right thalamus, signs of striatal necrosis can be seen (closed arrow). The cerebral white matter is hyperintense. (B) Sagittal T2 weighted image, showing diffuse cerebral and cerebellar atrophy. metabolic acidosis with increased lactate values up to 10 mM Homozygosity mapping 12 h postpartum (hyperlactataemia >2.5 mM). A metabolic Homozygosity mapping was performed with the Affymetrix investigation in blood and urine revealed normal amino acid GeneChip Human Mapping 250 K NspI SNP array using values, a slightly decreased total carnitine value with a slightly peripheral blood DNA of the patient. The DNA was processed elevated alanine value and an increased lactate/pyruvate ratio and labelled according to the instructions of the manufacturer. (>20). In urine, lactic aciduria was present. Morphological Genotypes were generated by the Affymetrix GeneChip Geno- investigations in muscle, including electron microscopy, did not typing Analysis Software (GTYPE). For the identification of reveal any abnormalities. Mitochondrial oxygen consumption homozygous regions the online tool ‘HomozygosityMapper’ was decreased and an isolated complex I deficiency was present was used.15 Candidate regions were selected in which at least in muscle (biopsy taken from right quadriceps muscle) and 400 consecutive SNPs were homozygous in the patient. fibroblasts (respectively, 29% and 11% activity compared to control values). The boy developed profound hearing loss, Sequence analysis apnoeas associated with brainstem abnormalities, and retinitis Exons and flanking intronic regions of 40nt from the NDUFA9 pigmentosa. A brain MRI at day 6 showed diffuse loss of gene were PCR amplified using intronic primers (Biolegio, online supratentorial white matter and brain stem volume with T2 supplementary table 1). The other candidate gene associated with hyperintensities of the basal nuclei (thalamus and putamen) and complex I, NDUFB2, was not sequenced after the pathogenic a right-sided focal thalamic lesion compatible with striatal NDUAF9 mutation was identified. PCR products were directly ischaemia and focal necrosis, leading to a diagnosis of Leigh sequenced with the PRISM Ready Reaction Sequencing Kit syndrome (figure 1). Electroencephalograms (EEGs) taken at (Perkin-Elmer Life Sciences, Boston, MA, USA) on an ABI3730 1 day, 1 week, 2 and 3 weeks of age showed increased aberra- automatic sequencer (Applied Biosystems, Foster City, CA, USA). tions of the normal pattern with multifocal sharp waves and a discontinuous pattern. No overt epileptic seizures were seen. Blue native (BN)-polyacrylamide gel electrophoresis and His legs became increasingly hypertonic with choreadystonic western blotting movements from all limbs. At 3 weeks he needed ventilatory Mitoplasts from patient and control fibroblasts were isolated as support. He died 1 month after birth due to respiratory insuffi- previously described.16 Mitochondrial protein (15 mg) were ciency. Deletions and mutations in the mtDNA were excluded as analysed on polyacrylamide gradient gels (4e16%; Invitrogen, a cause of disease by, respectively,