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Observation |

Leigh Syndrome Associated With Mitochondrial Complex I Deficiency Due to a Novel Mutation in the NDUFS1 Gene FREE

Miguel A. Martín, PhD; Alberto Blázquez, BSc; Luis G. Gutierrez-Solana, MD; Daniel Fernández-Moreira, PharmB; Paz Briones, PhD; Antoni L. Andreu, MD, PhD; Rafael Garesse, PhD; Yolanda Campos, PhD; Joaquín Arenas, PhD
[+] Author Affiliations

Author Affiliations: Centro de Investigación, Hospital Universitario 12 de Octubre (Drs Martín, Campos, and Arenas and Messrs Blázquez and Fernández-Moreira), Servicio de Neurología, Hospital Infantil Universitario Niño Jesús (Dr Gutierrez-Solana), Instituto Investigaciones Biomédicas “Alberto Sols” Universidad Autónoma de Madrid–Consejo Superior de Investigaciones Cientificas, Departamento de Bioquímica, Facultad de Medicina, Universidad Autónoma de Madrid (Dr Garesse), Madrid; and Institut de Bioquímica Clínica, Corporació Sanitaria Clínic y Consejo Superior de Investigaciones Cientificas (Dr Briones) and Centre d’Investigacions en Bioquímica i Biología Molecular, Hospital Universitari Vall d’Hebron (Dr Andreu), Barcelona, Spain. Dr Martín and Mr Blázquez contributed equally to this work.


Arch Neurol. 2005;62(4):659-661. doi:10.1001/archneur.62.4.659.
Text Size: A A A
Published online

ABSTRACT

Background  Mutations in the nuclear-encoded subunits of complex I of the mitochondrial respiratory chain are a recognized cause of Leigh syndrome (LS). Recently, 6 mutations in the NDUFS1 gene were identified in 3 families.

Objective  To describe a Spanish family with LS, complex I deficiency in muscle, and a novel mutation in the NDUFS1 gene.

Design  Using molecular genetic approaches, we identified the underlying molecular defect in a patient with LS with a complex I defect.

Patient  The proband was a child who displayed the clinical features of LS.

Results  Muscle biochemistry results showed a complex I defect of the mitochondrial respiratory chain. Sequencing analysis of the mitochondrial DNA–encoded ND genes, the nuclear DNA–encoded NDUFV1, NDUFS1, NDUFS2, NDUFS4, NDUFS6, NDUFS7, NDUFS8, and NDUFAB1 genes, and the complex I assembly factor CIA30 gene revealed a novel homozygous L231V mutation (c.691C→G) in the NDUFS1 gene. The parents were heterozygous carriers of the L231V mutation.

Conclusions  Identifying nuclear mutations as a cause of respiratory chain disorders will enhance the possibility of prenatal diagnosis and help us understand how molecular defects can lead to complex I deficiency.

Figures in this Article

Leigh syndrome (LS) (Online Mendelian Inheritance in Man 256000) is a devastating neurodegenerative disorder characterized neuropathologically by focal bilaterally symmetrical lesions, especially in the thalamus and brainstem regions, and clinically by psychomotor retardation, respiratory difficulties, nystagmus, ophthalmoparesis, optic atrophy, ataxia, and dystonia.1 In most patients, mitochondrial respiratory chain defects and pyruvate dehydrogenase complex deficiency are the underlying causes of the disease.2 Mitochondrial respiratory chain complex I (nicotinamide adenine dinucleotide:ubiquinone oxidoreductase) contains at least 46 subunits, 7 of which are encoded by mitochondrial DNA (mtDNA).2 Various mutations in a few subunits of complex I encoded by nuclear DNA (nDNA) (NDUFV1, NDUFV2, NDUFS1, NDUFS2, NDUFS3, NDUFS4, NDUFS7, and NDUFS8)310 are associated with LS or Leigh-like disease in patients with complex I deficiency. The NDUFS1 gene encodes the largest (75-kDa subunit) protein of complex I.11 Recently, 6 mutations in the NDUFS1 gene were identified in 3 families with LS or Leigh-like disease and complex I deficiency (Online Mendelian Inheritance in Man 157655).5 Herein, we describe a Spanish patient with LS, complex I deficiency in muscle, and a novel mutation in the NDUFS1 gene.

METHODS

REPORT OF A CASE

The first child (a girl) of healthy nonconsanguineous parents (aged 30 years) of Spanish origin was born after a term pregnancy (birth weight, 3560 g). She was hospitalized at age 8½ months for recurrent episodes of vomiting, floppiness, and growth retardation. She presented with irritability, horizontal nystagmus, and generalized hypotonia. Tendon reflexes were hyperactive and symmetric. Babinski sign was negative. An electroencephalogram dis played a normal pattern. Brain magnetic resonance imaging showed bilateral lesions affecting the substantia nigra and midbrain. Cardiologic and ophthalmologic examination findings were normal. Laboratory data revealed increased lactate levels in blood (24 mg/dL; normal, <20 mg/dL) and in cerebrospinal fluid (30 mg/dL; normal, <15 mg/dL). In fibroblasts, pyruvate dehydrogenase activity was normal, and pyruvate oxidation was in the lower reference limit. Muscle morphologic structure was normal except for mild atrophy of type 2 fibers. Her status worsened 5 months later, when she developed respiratory insufficiency and horizontal and vertical nystagmus. Her tendon reflexes became highly hyperactive, with a surface area enlargement and presence of Babinski sign. She died at age 14 months. Her younger brother had a similar clinical picture and died at age 8 months, after an acute episode of respiratory failure.

BIOCHEMICAL AND MOLECULAR GENETIC STUDIES

An appropriate institutional review board approved this work, and informed consent was obtained from the child’s parents. Respiratory chain enzymes in muscle homogenate were measured by methods reported elsewhere.12

DNA was isolated from muscle and blood from the patient and from blood from her parents. The mtDNA-encoded ND subunits were amplified using suitable primers.13 The coding region and exon and intron boundaries of the nuclear-encoded complex I subunits of the NDUFV1, NDUFS1, NDUFS2, NDUFS3, NDUFS4, NDUFS7, NDUFS8, and NDUFAB1 genes, as well as the complex I assembly factor CIA30 gene, were amplified as previously described310,14,15 or by using novel intronic primers. Polymerase chain reaction products were purified by electrophoresis in 2% agarose gel and sequenced directly, using the ABI PRISM dRhodamine Terminator Cycle Sequencing Kit in an ABI PRISM 310 genetic analyzer (Applied Biosystems, Foster City, Calif). Nucleotide changes were further confirmed by polymerase chain reaction–restriction fragment length polymorphism methods (Figure). One hundred healthy control subjects (200 alleles) were screened by polymerase chain reaction–restriction fragment length polymorphism methods to rule out the presence of the mutation in the healthy population. Unfortunately, tissue specimens were not available from the proband’s brother.

Place holder to copy figure label and caption
Figure.

Polymerase chain reaction–restriction fragment length polymorphism results (A), sequencing (B), and sequence alignment of the NDUFS1 protein from various species (C). A, Lane 1: control DNA. Lane 2: muscle DNA from the patient. Lane 3: blood DNA from the mother. Lane 4: blood DNA from the father. The L231V mutation was detected by digesting the NDUFS1 exon 8 fragment with the restriction enzyme Sdu I. The 312–base pair (bp) wild-type DNA was digested by Sdu I into 2 fragments of 180 and 132 bp, whereas the 312-bp fragment remained uncut in the patient, who was homozygous for the mutation. B, Electrophoretograms showing the change in the patient (top) and the normal sequence in a control subject (bottom). Asterisk represents the nucleotide substitution. C, Evolutionary conservation of the 231 amino acid residue (arrow) of the NDUFS1 protein.

Graphic Jump Location

RESULTS

The activities of respiratory chain complexes in muscle showed a single defect of nicotinamide adenine dinucleotide:ubiquinone oxidoreductase (complex I), accounting for 25% of the mean of the control subjects (Table>). Given the clinical picture and the biochemical findings, we searched for the underlying molecular alteration of this defect. Sequencing analysis of the genes listed in the “Methods” section showed a novel homozygous missense mutation (L231V) that replaces a leucine by a valine in the 231 amino acid residue of the protein as a result of a c.691C→G transversion in exon 8 of the NDUFS1 gene (Figure). Additional nucleotide changes were not found. The parents were heterozygous carriers of the L231V mutation.

Table Graphic Jump LocationTable. Activities of Mitochondrial Respiratory Chain Complexes in Muscle Homogenate*

COMMENT

Mutations in the nDNA-encoded complex I NDUFV1, NDUFV2, NDUFS1, NDUFS2, NDUFS3, NDUFS4, NDUFS7, and NDUFS8 genes have been documented in patients with LS, although often the underlying molecular defect remains unknown.310 In 3 nonrelated families, Bénit et al5 identified 6 mutations in the NDUFS1 gene, which encodes the largest subunit of complex I. Interestingly, 3 of these mutations (amino acids 222, 241, and 252) lie in a highly evolutionary conserved stretch of the protein encompassing the most C-terminal cysteine residue, potentially involved in the ligation of iron-sulfur clusters.5

We describe a Spanish girl with LS, whose younger brother died of a similar condition. The proband had a complex I defect in muscle and harbored a novel homozygous missense mutation (L231V) in the NDUFS1 gene. The mutation was consistently heterozygous in blood DNA from the healthy parents, suggesting autosomal recessive inheritance. Several lines of evidence support the pathogenicity of the mutation, including the following: (1) the patient had a single complex I defect in muscle; (2) it was the only nucleotide change found in the entire coding region and in the intron and exon boundaries of the gene; (3) no additional pathogenic mutations were found in the other complex I genes analyzed; (4) the mutation was absent in 200 alleles from 100 healthy controls of similar ethnic background; and (5) although the amino acid substitution does not result in a polarity change, the mutation is highly conserved during evolution and is situated in a region of the protein subunit where 3 other mutations were found (Figure).5

Reliable prenatal diagnosis is a difficult task in many cases of mitochondrial respiratory chain disorders. The identification of mutations in nuclear genes in families with a clear-cut pattern of autosomal recessive inheritance makes it possible to predict diagnosis in the fetus.16,17

In addition to this family, we analyzed the nuclear-encoded mitochondrial genes described herein in a series of 13 pediatric patients with LS or Leigh-like disease with a complex I defect and found no additional patients with mutations in these genes. In other reports, frequencies of patients with these mutations ranged between 17% and 25%.2,5 Only 7.7% of our patients with LS or Leigh-like disease and an isolated complex I defect harbor mutations in these genes.

CONCLUSIONS

We describe a family with LS, complex I deficiency, and a novel mutation in the NDUFS1 gene. The rapidly progressive nature of the disease, absence of effective treatment, and commonly fatal course of the disease make prenatal diagnosis a valuable tool in families with this condition. Identifying nuclear mutations as a cause of mitochondrial respiratory chain disorders will enhance the possibility of prenatal diagnosis and help us understand how molecular defects can lead to complex I deficiency.

ARTICLE INFORMATION

Correspondence: Joaquín Arenas, PhD, Centro de Investigación, Hospital Universitario 12 de Octubre, Avenida de Córdoba sin número, 28041 Madrid, Spain (jarenas.hdoc@salud.madrid.org).

Accepted for Publication: April 8, 2004.

Author Contributions:Study concept and design: Martín, Blázquez, and Arenas. Acquisition of data: Martín, Blázquez, Gutierrez-Solana, Fernández-Moreira, Briones, and Campos. Analysis and interpretation of data: Martín, Blázquez, Gutierrez-Solana, Fernández-Moreira, Briones, Andreu, Garesse, Campos, and Arenas. Drafting of the manuscript: Martín, Blázquez, Gutierrez-Solana, Fernández-Moreira, and Arenas. Critical revision of the manuscript for important intellectual content: Martín, Blázquez, Briones, Andreu, Garesse, Campos, and Arenas. Obtained funding: Martín and Arenas. Study supervision: Martín and Arenas.

Funding/Support: This study was supported by grants FIS 01/1426 and FIS PI030224 from the Fondo de Investigación Sanitaria, Ministerio de Sanidad y Consumo, Madrid, and by grant FIS G03/011 from the Spanish Mitochondrial Diseases Network, Coordination Center in Madrid. Mr Blázquez was supported by post-Médico Interno Residente contract FIS CM0300007, Mr Fernández-Moreira by grant FIS PI030224, and Dr Campos by research contract ISC III 98/3166 from the Instituto de Salud Carlos III, Madrid.

Acknowledgment: We are grateful to Pilar del Hoyo and Sara Jiménez, the technicians who worked on the respiratory chain activities.

REFERENCES

DiMauro  SSchon  EA Mitochondrial respiratory-chain diseases. N Engl J Med 2003;3482656- 2668
PubMed Link to Article
Loeffen  JLSmeitink  JATrijbels  JM  et al.  Isolated complex I deficiency in children: clinical, biochemical and genetic aspects. Hum Mutat 2000;15123- 134
PubMed Link to Article
Schuelke  MSmeitink  JMariman  E  et al.  Mutant NDUFV1 subunit of mitochondrial complex I causes leukodystrophy and myoclonic epilepsy. Nat Genet 1999;21260- 261
PubMed Link to Article
Bénit  PBeugnot  RChretien  D  et al.  Mutant NDUFV2 subunit of mitochondrial complex I causes early onset hypertrophic cardiomyopathy and encephalopathy. Hum Mutat 2003;21582- 586
PubMed Link to Article
Bénit  PChretien  DKadhom  N  et al.  Large-scale deletion and point mutations of the nuclear NDUFV1 and NDUFS1 genes in mitochondrial complex I deficiency. Am J Hum Genet 2001;681344- 1352
PubMed Link to Article
Loeffen  JElpeleg  OSmeitink  J  et al.  Mutations in the complex I NDUFS2 gene of patients with cardiomyopathy and encephalomyopathy. Ann Neurol 2001;49195- 201
PubMed Link to Article
Bénit  PSlama  ACartault  F  et al.  Mutant NDUFS3 subunit of mitochondrial complex I causes Leigh syndrome. J Med Genet 2004;4114- 17
PubMed Link to Article
van den Heuvel  LRuitenbeek  WSmeets  R  et al.  Demonstration of a new pathogenic mutation in human complex I deficiency: a 5-bp duplication in the nuclear gene encoding the 18-kD (AQDQ) subunit. Am J Hum Genet 1998;62262- 268
PubMed Link to Article
Triepels  RHvan den Heuvel  LPLoeffen  JL  et al.  Leigh syndrome associated with a mutation in the NDUFS7 (PSST) nuclear encoded subunit of complex I. Ann Neurol 1999;45787- 790
PubMed Link to Article
Loeffen  JSmeitink  JTriepels  R  et al.  The first nuclear-encoded complex I mutation in a patient with Leigh syndrome. Am J Hum Genet 1998;631598- 1608
PubMed Link to Article
Smeitink  JALoeffen  JLTriepels  RHSmeets  RJTrijbels  JMvan den Heuvel  LP Nuclear genes of human complex I of the mitochondrial electron transport chain: state of the art. Hum Mol Genet 1998;71573- 1579
PubMed Link to Article
Martinez  Bdel Hoyo  PMartin  MAArenas  JPerez-Castillo  ASantos  A Thyroid hormone regulates oxidative phosphorylation in the cerebral cortex and striatum of neonatal rats. J Neurochem 2001;781054- 1063
PubMed Link to Article
Andrews  RMKubacka  IChinnery  PFLightowlers  RNTurnbull  DMHowell  N Reanalysis and revision of the Cambridge reference sequence for human mitochondrial DNA [letter]. Nat Genet 1999;23147
PubMed Link to Article
Triepels  RSmeitink  JLoeffen  J  et al.  The human nuclear-encoded acyl carrier subunit (NDUFAB1) of the mitochondrial complex I in human pathology. J Inherit Metab Dis 1999;22163- 173
PubMed Link to Article
Janssen  RSmeitink  JSmeets  Rvan den Heuvel  L CIA30 complex I assembly factor: a candidate for human complex I deficiency? Hum Genet 2002;110264- 270
PubMed Link to Article
Niers  LESmeitink  JATrijbels  JMSengers  RCJanssen  AJvan den Heuvel  LP Prenatal diagnosis of NADH:ubiquinone oxidoreductase deficiency. Prenat Diagn 2001;21871- 880
PubMed Link to Article
Schuelke  MDetjen  Avan den Heuvel  L  et al.  New nuclear encoded mitochondrial mutation illustrates pitfalls in prenatal diagnosis by biochemical methods. Clin Chem 2002;48772- 775
PubMed

Figures

Place holder to copy figure label and caption
Figure.

Polymerase chain reaction–restriction fragment length polymorphism results (A), sequencing (B), and sequence alignment of the NDUFS1 protein from various species (C). A, Lane 1: control DNA. Lane 2: muscle DNA from the patient. Lane 3: blood DNA from the mother. Lane 4: blood DNA from the father. The L231V mutation was detected by digesting the NDUFS1 exon 8 fragment with the restriction enzyme Sdu I. The 312–base pair (bp) wild-type DNA was digested by Sdu I into 2 fragments of 180 and 132 bp, whereas the 312-bp fragment remained uncut in the patient, who was homozygous for the mutation. B, Electrophoretograms showing the change in the patient (top) and the normal sequence in a control subject (bottom). Asterisk represents the nucleotide substitution. C, Evolutionary conservation of the 231 amino acid residue (arrow) of the NDUFS1 protein.

Graphic Jump Location

Tables

Table Graphic Jump LocationTable. Activities of Mitochondrial Respiratory Chain Complexes in Muscle Homogenate*

References

DiMauro  SSchon  EA Mitochondrial respiratory-chain diseases. N Engl J Med 2003;3482656- 2668
PubMed Link to Article
Loeffen  JLSmeitink  JATrijbels  JM  et al.  Isolated complex I deficiency in children: clinical, biochemical and genetic aspects. Hum Mutat 2000;15123- 134
PubMed Link to Article
Schuelke  MSmeitink  JMariman  E  et al.  Mutant NDUFV1 subunit of mitochondrial complex I causes leukodystrophy and myoclonic epilepsy. Nat Genet 1999;21260- 261
PubMed Link to Article
Bénit  PBeugnot  RChretien  D  et al.  Mutant NDUFV2 subunit of mitochondrial complex I causes early onset hypertrophic cardiomyopathy and encephalopathy. Hum Mutat 2003;21582- 586
PubMed Link to Article
Bénit  PChretien  DKadhom  N  et al.  Large-scale deletion and point mutations of the nuclear NDUFV1 and NDUFS1 genes in mitochondrial complex I deficiency. Am J Hum Genet 2001;681344- 1352
PubMed Link to Article
Loeffen  JElpeleg  OSmeitink  J  et al.  Mutations in the complex I NDUFS2 gene of patients with cardiomyopathy and encephalomyopathy. Ann Neurol 2001;49195- 201
PubMed Link to Article
Bénit  PSlama  ACartault  F  et al.  Mutant NDUFS3 subunit of mitochondrial complex I causes Leigh syndrome. J Med Genet 2004;4114- 17
PubMed Link to Article
van den Heuvel  LRuitenbeek  WSmeets  R  et al.  Demonstration of a new pathogenic mutation in human complex I deficiency: a 5-bp duplication in the nuclear gene encoding the 18-kD (AQDQ) subunit. Am J Hum Genet 1998;62262- 268
PubMed Link to Article
Triepels  RHvan den Heuvel  LPLoeffen  JL  et al.  Leigh syndrome associated with a mutation in the NDUFS7 (PSST) nuclear encoded subunit of complex I. Ann Neurol 1999;45787- 790
PubMed Link to Article
Loeffen  JSmeitink  JTriepels  R  et al.  The first nuclear-encoded complex I mutation in a patient with Leigh syndrome. Am J Hum Genet 1998;631598- 1608
PubMed Link to Article
Smeitink  JALoeffen  JLTriepels  RHSmeets  RJTrijbels  JMvan den Heuvel  LP Nuclear genes of human complex I of the mitochondrial electron transport chain: state of the art. Hum Mol Genet 1998;71573- 1579
PubMed Link to Article
Martinez  Bdel Hoyo  PMartin  MAArenas  JPerez-Castillo  ASantos  A Thyroid hormone regulates oxidative phosphorylation in the cerebral cortex and striatum of neonatal rats. J Neurochem 2001;781054- 1063
PubMed Link to Article
Andrews  RMKubacka  IChinnery  PFLightowlers  RNTurnbull  DMHowell  N Reanalysis and revision of the Cambridge reference sequence for human mitochondrial DNA [letter]. Nat Genet 1999;23147
PubMed Link to Article
Triepels  RSmeitink  JLoeffen  J  et al.  The human nuclear-encoded acyl carrier subunit (NDUFAB1) of the mitochondrial complex I in human pathology. J Inherit Metab Dis 1999;22163- 173
PubMed Link to Article
Janssen  RSmeitink  JSmeets  Rvan den Heuvel  L CIA30 complex I assembly factor: a candidate for human complex I deficiency? Hum Genet 2002;110264- 270
PubMed Link to Article
Niers  LESmeitink  JATrijbels  JMSengers  RCJanssen  AJvan den Heuvel  LP Prenatal diagnosis of NADH:ubiquinone oxidoreductase deficiency. Prenat Diagn 2001;21871- 880
PubMed Link to Article
Schuelke  MDetjen  Avan den Heuvel  L  et al.  New nuclear encoded mitochondrial mutation illustrates pitfalls in prenatal diagnosis by biochemical methods. Clin Chem 2002;48772- 775
PubMed

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