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Mitochondrial complex I deficiency caused by a deleterious NDUFA11 mutation.

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Mitochondrial Complex I
Deficiency Caused by a
Deleterious NDUFA11
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
( DOI: 10.1002/ana.21332
Address correspondence to Prof Elpeleg, Metabolic Disease Unit,
Hadassah-Hebrew University Medical Center, Jerusalem, 91120, Israel. E-mail:
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
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
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.
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
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 (, 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.
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
(, 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.
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Annals of Neurology
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