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Mitochondrial Complex I Deficiency Caused by a Deleterious NDUFA11 Mutation Itai Berger, MD,1 Eli Hershkovitz, MD,2 Avraham Shaag, PhD,3 Simon Edvardson, MD,1 Ann Saada, PhD,3 and Orly Elpeleg, MD3 Complex I deficiency is the most common respiratory chain defect, clinically manifesting by severe neonatal lactic acidosis, Leigh’s disease, or various combinations of cardiac, hepatic, and renal disorders. Using homozygosity mapping, we identified a splice-site mutation in the NDUFA11 gene in six patients from three unrelated families. The patients presented with encephalocardiomyopathy or fatal infantile lactic acidemia. The mutation is predicted to abolish the first transmembrane domain of the gene product, thereby destabilizing the enzymatic complex. Mutation analysis of the NDUFA11 is warranted in isolated complex I deficiency presenting with infantile lactic acidemia or encephalocardiomyopathy. Ann Neurol 2008;63:405– 408 Isolated NADH:ubiquinone oxidoreductase (complex I) deficiency is the most common respiratory chain de- From the 1Pediatric Neurology Unit, Hadassah-Hebrew University Medical Center, Jerusalem; 2Department of Pediatrics, Soroka Medical Center, Faculty of Health Sciences, Ben-Gurion University of the Negev, Beer-Sheva; and 3The Metabolic Disease Unit, Hadassah-Hebrew University Medical Center, Jerusalem, Israel. Received Aug 27, 2007, and in revised form Nov 13. Accepted for publication Nov 29, 2007. Published online February 27, 2008, in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/ana.21332 Address correspondence to Prof Elpeleg, Metabolic Disease Unit, Hadassah-Hebrew University Medical Center, Jerusalem, 91120, Israel. E-mail: email@example.com fect, diagnosed in one third of the patients with OXPHOS dysfunction.1 Five clinical phenotypes are frequently seen in these patients: severe neonatal lactic acidosis, Leigh’s disease, cardiomyopathy-encephalopathy, hepatopathy-tubulopathy, and leukodystrophy with macrocephaly.2,3 Complex I is the largest and most intricate of the five mitochondrial respiratory chain complexes. It consists of 45 proteins, 7 encoded by the mitochondrial DNA and the remaining by nuclear DNA. Mutations in mitochondrial DNA–encoded genes are detected in only 20% of the children with isolated complex I deficiency,4,5 suggesting that most patients harbor mutations in nuclear genes that encode structural subunits or assembly factors. Heretofore extensive sequence determination of candidate genes demonstrated mutations in 10 structural subunits genes (reviewed in Janssen and colleagues6) and in 2 assembly factors of complex I.7,8 Still, molecular diagnosis is lacking for more than 50% of the patients.9 This report is the result of our effort to identify the diseased genes in patients with isolated complex I deficiency originating from 20 consanguineous families using homozygosity mapping. Case Reports Patient A-II-1, B-II-2, and B-II-4 (Fig 1) were the products of first-cousin marriages. They were born at term, birth weight 1,710 to 2,400gm (⫺2.2 to ⫺0.745 standard deviation score for 37 weeks of gestation). The physical examinations were unremarkable, but at 10 to 24 hours of age the patients became apneic and hypotensive with severe metabolic acidosis and hyperlactatemia (pH 6.80 – 6.90; blood lactate 10 –15mM; N ⬍ 2.1mM). Echocardiogram, normal at birth, disclosed biventricular hyperplasia on the second week of life and the patients died at 6 to 40 days of age because of intractable acidosis. Muscle biopsy was performed in Patients A-II-1 and B-II-2. The parents and the two living sons in Family B were healthy. Patients C-IV-1, C-IV-2, and C-IV-7 were born to first-cousins parents of Israeli-Bedouin origin. At term, their birth weights were 1,900 to 2,900gm (⫺1.8 to ⫹0.3 standard deviation score). The physical examination at birth was unremarkable, but their early psychomotor development was slow. Repeated examinations demonstrated acquired microcephaly, marked generalized hypotonia, muscle weakness, and paucity of voluntary movements. None of the patients was ever able to roll or lift the head in the prone position. The deep tendon reflexes were normally elicited without clonus. No extrapyramidal movements were observed. Hearing was normal, but visual fixation was impaired with nystagmus in one patient and bilateral optic atrophy in another. Convulsive disorder appeared at 4 months in one patient. Echocardiogram at birth was © 2008 American Neurological Association Published by Wiley-Liss, Inc., through Wiley Subscription Services 405 Fig 1. The families’ pedigrees. Gray symbols designate patients. Numbered symbols represent individuals whose DNA samples were available for analysis. normal, but hypertrophy of the myocardial walls and septum (wall thickness, 6mm; normal for age, 2.5–3mm) with mild dilatation of the left ventricle was present at 3 to 4 months of age. Plasma lactate level was increased (3.2–10mM), and the patients suffered from exacerbation of the acidosis during intercurrent infections. Complete blood count, liver transaminases, and creatine kinase concentration were normal. The electroencephalogram was normal, and brain computed tomography scan performed in two patients demonstrated generalized brain atrophy. Two patients died at 18 months and 4 years of age during an intercurrent infection. One patient was alive at 6 months at the time of writing this report. Muscle biopsy was obtained only from Patient C-IV-1, but DNA was available from all the members of Family C. The parents in Family C and their four living offspring were healthy. Results Enzymatic activities of the five respiratory chain complexes were determined in isolated muscle mitochondria from Patients A-II-1, B-II-2, and C-IV-1, and in isolated fibroblast mitochondria from Patient B-II-2 by standard spectrophotometric methods and normalized to citrate synthase.10 The activity of complex I in muscle mitochondria, determined as rotenone-sensitive NADH: cytochrome c oxidoreductase, NADH:ubiquinone reductase, and NADH:ferricyanide reductase, was reduced to 4 to 10%, 13 to 27%, and 19 to 39% of the control mean, respectively. In mitochondria isolated from fibroblasts, NADH:ubiquinone reductase activity was reduced to 45% of the control mean. The activities of the other complexes were within the reference range. To localize the mutated gene, we performed homozygosity mapping using GeneChip Human Mapping 406 Annals of Neurology Vol 63 No 3 March 2008 250K Nsp Array of Affymetrix11 in the DNA samples of Patients A-II-1, B-II-2, and C-IV-1. DNA analysis was approved by the Hadassah Ethical Review Committee. Patient A-II-1 was homozygous for 7 regions including 12.5Mb on chromosome 19 at 2.50 to 14.65Mb (rs7260635 to rs10407394). Patients B-II-2 and C-IV-1 were also homozygous for multiple regions, including a large region on chromosome 19 (0 –21.6 and 0 –13.7Mb, respectively). The three patients, each originating from a different family, shared an identical haplotype over 342 single nucleotide polymorphism markers (2.50 –13.7Mb). Within the genomic region detected at Patient B-II-2’s DNA, there were three known complex I subunit genes: NDUFA11, NDUFA7, and NDUFB7. Sequence analysis of the exons and flanking intronic sequences of the genes showed a single mutation, a g3a at intervening sequence (IVS) 1 ⫹5 of the NDUFA11 gene (Fig 2). The six patients whose DNA was available for analysis were homozygous for the mutation, and we did not find homozygosity for the mutation among the healthy family members. The parents in Families B and C, and five of the six healthy sibs in these families were heterozygous for the mutation and one healthy sib was homozygous for the normal allele. No carriers were detected among 52 ethnicity-matched control subjects. Although the identical haplotype on chromosome 19 was indicative of a founder mutation, the three couples could not recall any relationship among the families. According to the splice-site prediction software, NatGene2 (http://www.cbs.dtu.dk/services/NetGene2/), the mutation abolished the donor splice site of IVS1. In agreement, two NDUFA11 complementary DNA transcripts were amplified from the patients’ fibroblasts, using primers 5⬘-TATTTCTGGACGCATTCTGC -3⬘ and 5-TTCTGCAGGCTGGAGGTC -3⬘. The normal transcript was 484bp long, and the shorter (406bp) lacked the 3⬘-end 78bp of exon 1 (see Fig 2). Real-time polymerase chain reaction of the two transcripts in complementary DNA produced from the patient fibroblasts detected a 2:1 ratio of the wild-type to the mutant transcript. These findings suggest that IVS1⫹5g3a mutation in the NDUFA11 gene is a “leaky” mutation, reducing the splice score at the normal site with the consequent activation of a cryptic splice site at 19 to 20bp of exon 1. Finally, none of the complex I–deficient patients originating from the remaining 17 consanguineous families had a homozygous region that encompassed the NDUFA11 gene. Hence the gene sequence was not determined in other complex I–deficient patients. Discussion The six patients reported hereby presented with encephalocardiomyopathy or with fatal infantile lactic acidemia attributed to isolated complex I deficiency. In view of their young age and the consanguineous pedi- Fig 2. The NDUFA11 mutation at the genomic and complementary DNA (cDNA) levels. NDUFA11 exon 1-IVS1 splice junction sequence of (A) a patient, (B) a carrier, and (C) a healthy control subject. The mutation site is indicated by an arrow. (D) Polymerase chain reaction (PCR) amplification of NDUFA11 cDNA of a patient (lane 1), a carrier (lane 2), and a control subject (lane 3). A 100bp ladder is shown in lane 4. The mutant 406bp product is indicated by a white arrow on the left. Note a faint band of the normal size (484 nt) product in the patient’s lane. (E) NDUFA11 cDNA produced from patient’s fibroblasts showing the normal transcript (wild type [WT]), with the 3⬘-end of exon 1 spliced to exon 2, superimposed on the mutant transcript (MUT) with the activated cryptic splice site within exon 1 (at base pair 19) spliced to exon 2. grees, a mutation in one of the nuclear genes was sought. The large number of known candidate genes, including the 38 structural subunit genes and an unknown number of assembly factors, led us to perform homozygosity mapping in the consanguineous families rather than direct sequencing. This approach facilitated the identification of a splice-site mutation in NDUFA11 gene. Leigh’s and Leigh-like syndromes are the commonest presentations of complex I deficiency in young age, and mutations in six genes have already been identified in these patients (NDUFS1, NDUFS3, NDUFS4, NDUFS7, NDUFS8, and NDUFV1).6 Early-onset encephalocardiomyopathy has been reported previously in patients harboring mutations in complex I genes NDUFS2 and NDUFV2.12,13 Heretofore no complex I gene was associated with fatal infantile lactic acidemia. Our data provide a molecular cause for this widely reported group of patients. The identification of a single mutation in the NDUFA11 gene in patients with two different clinical presentations is suggestive of a modifier gene effect. The mutation perturbs the splicing efficiency at exon 1-IVS1 junction, as predicted in silico and as indicated by the presence of both wild-type and mutant transcripts in our patients’ fibroblasts. Hence we speculate that it is the mutant/wild-type transcript ratio in the respective tissues that dictates the phenotype. The production of a wildtype transcript in the presence of an exon-intron junction mutation is generally dependent on the splice-site score of the wild-type junction, the presence and conservation of exonic splicing enhancer domains, and the tissue concentration of the splicing factors. Slight variability in these factors may determine the clinical phenotype of our patients. For this reason, the transcripts ratio in a patient’s fibroblasts may not reflect the situation in the heart or brain of the same patient. Complex I has an L-shape structure with a hydrophobic arm embedded in the lipid membrane and a hydrophilic, peripheral arm protruding into the matrix.14 The NDUFA11 subunit is a unique component of complex I: It has an acetylated N terminus, no presequence, and contains four potential transmembrane helices. It is homologous to subunit 21.3b from complex I in Neurospora crassa and is related to Tim17, Tim22, and Tim23, which are involved in protein translocation across the inner membrane.15 It has been assumed that the NDUFA11 protein is located at the small connection between the peripheral arm and the membrane arm of complex I, anchoring the peripheral arm to the inner membrane. Disruption of the NDUFA11 homolog in Neurospora crassa resulted in an incomplete assembly of the complex.16 A software program that identifies transmembrane domains, ProtScale (http://us.expasy.org/tools/protscale.html), predicts that the mutant transcript will lack its first transmembrane domain, suggesting that the mutation in our patients could destabilize the enzymatic complex. This is the first report of a mutation in the human NDUFA11 gene. Mutation analysis of this gene is warranted in patients with complex I deficiency presenting in infancy with encephalocardiomyopathy or with fatal lactic acidemia. This work was supported by the United Mitochondrial Disease Foundation (07-204B, O.E.) and by the Joint Research Fund of the Hebrew University and Hadassah Medical Organization (S.E.). We gratefully acknowledge the collaboration of the patients’ families and C. Belaiche for OXPHOS analysis. References 1. Kirby DM, Crawford M, Cleary MA, et al. 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