- Email: [email protected]
Genetic and biochemical findings in Chinese children with Leigh syndrome
Journal of Clinical Neuroscience 20 (2013) 1591–1594
Contents lists available at ScienceDirect
Journal of Clinical Neuroscience journal homepage: www.elsevier.com/locate/jocn
Laboratory studies
Genetic and biochemical findings in Chinese children with Leigh syndrome Yan-Yan Ma a, Tong-Fei Wu b, Yu-Peng Liu b, Qiao Wang b, Jin-Qing Song b, Xi-Yuan Li b, Xiu-Yu Shi a, Wei-Na Zhang a, Meng Zhao a, Lin-Yan Hu a, Yan-Ling Yang b, , Li-Ping Zou a,⇑, a b
Department of Pediatrics, Chinese Liberation Army General Hospital, No. 28 Fu-xing Road, Haidian District, Beijing 100853, People’s Republic of China Department of Pediatrics, Peking University First Hospital, Beijing, People’s Republic of China
a r t i c l e
i n f o
Article history: Received 27 October 2012 Accepted 24 March 2013
Keywords: Leigh syndrome Mitochondrial disorders Mitochondrial gene Mitochondrial respiratory complex deficiency
a b s t r a c t This study investigated the genetic and enzymological features of Leigh syndrome due to respiratory chain complex deficiency in Chinese patients. The clinical features of 75 patients were recorded. Mitochondrial respiratory chain enzyme activities were determined via spectrophotometry. Mitochondrial gene sequence analysis was performed in 23 patients. Five core pedigrees were investigated via restriction fragment length polymorphism and gene sequencing. Psychomotor retardation (55%), motor regression (20%), weakness (29%), and epilepsy (25%) were the most frequent manifestations. Sixty-four patients (85.3%) had isolated respiratory complex deficiencies: complex I was seen in 28 patients (37.3%); complex II, seven (9.3%); complex III, six (8%); complex IV, ten (13.3%); and complex V, 13 patients (17.3%). Eleven patients (14.7%) had combined complex deficiencies. Mitochondrial DNA mutations were detected in 10 patients. Eight point mutations were found in mitochondrial structural genes: m.4833A > G in ND2, m.10191T > C in ND3, m.12338T > C and m.13513G > A in ND5, m.14502T > C and m.14487T > C in ND6, m.8108A > G in COXII, and m.8993T > G in ATPase6. Three mutations were found in tRNA genes: m.4395A > G in tRNA-Gln, m.10454T > C in tRNA-Arg, and m.5587T > C in tRNA-Ala. One patient and their mother both had the m.12338T > C and m.8993T > C mutations. In conclusion, mitochondrial respiratory chain complex I deficiency and structural gene mutations frequently occur in Chinese Leigh syndrome patients. Ó 2013 Elsevier Ltd. All rights reserved.
1. Introduction Leigh syndrome was first reported by Denis Leigh in 1951.1 Thus far, this disorder has been proven to be clinically and genetically heterogeneous. Leigh syndrome most commonly presents as a progressive dysfunction of the central nervous system. Mitochondrial respiratory chain enzyme assay and gene analysis are needed for a precise etiological diagnosis of the disease. Deficiencies can occur in isolated or combined respiratory chain complexes, and a wide spectrum of biochemical data and clinical presentations has been documented. However, respiratory chain complex defects in Chinese patients with Leigh syndrome have not been studied in the past few decades because of the lack of clinically acceptable methods for conducting enzyme assays of respiratory chain complexes in China. In principle, the ideal tissue for clinical diagnosis is one that clinically expresses the disease, such as the brain, kidney, myocardium, liver, or endocrine glands. Because patients
⇑ Corresponding author. Tel.: +86 10 5549 9316; fax: +86 10 6613 4261.
E-mail address: [email protected] (L.-P. Zou). Li-ping Zou and Yan-Ling Yang have contributed equally to the manuscript.
0967-5868/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.jocn.2013.03.034
and their parents are less likely to accept a traumatic biopsy examination in comparison to a blood test, it is difficult to apply such a measure of respiratory chain complex I–V activity using biopsied tissue in China. A few pathogenic mutations have been found in Chinese patients with Leigh syndrome via genetic screening alone.2 The biochemical and genetic features of Leigh syndrome in the Chinese population, however, remain unknown. The present study investigated the etiology of Leigh syndrome in 75 Chinese patients with a view to determining the biochemical and genetic characteristics of this syndrome in the Chinese population.
2. Patients and methods 2.1. Patients We recruited 75 Chinese patients with Leigh syndrome with a mean age of 4.5 years (range, 3–18 years), who were admitted in the past 32 months. These patients presented with neuromuscular symptoms and signs that progressively worsened. Leigh syndrome was diagnosed on the basis of the clinical manifestations, biochemical features and characteristic brain MRI scans.3 Typical
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aminoacidopathies, organic acidurias, and mitochondrial b-oxidation defects were excluded via analyses of blood amino acids and acylcarnitines, and urinary organic acids. The parents of all patients gave informed consent. This study was approved by the hospital Ethics Committee following the tenets of the Declaration of Helsinki. 2.2. Methods 2.2.1. Assays of mitochondrial complex I to V enzyme activity The activities of mitochondrial respiratory chain enzymes I to V were detected via spectrophotometry. This method has been previously described in detail.4 2.2.2. Mitochondrial gene sequence analysis and restriction fragment length polymorphism analysis Mitochondrial gene sequence analysis was performed in 23 patients via direct sequencing. The sequence results were then compared with the revised Cambridge reference sequence5 GenBank ID: NC_012920.1. Patients with point mutations and their parents underwent polymerase chain reaction–restriction fragment length polymorphism analysis. This method has been previously described in detail.5 3. Results 3.1. Clinical features Among the 75 patients with Leigh syndrome due to respiratory chain complex deficiency, 49 were boys and 26 were girls. The male:female ratio was 1.9:1. The parents of the patients were healthy and nonconsanguineous. Ten patients (13%) had a positive family history of neuromuscular diseases. Their affected siblings died of encephalopathies or multiple organ failure. A mitochondrial defect was suspected, but a definitive diagnosis had not been reached. Nine (12%) patients had abnormal presentations in the fetal stage, which included intrauterine growth retardation, threatened abortion or fetal distress. The onset of symptoms dated back to the first year of life in 47 patients (63%), and the first 3 years of life in 62 patients (83%). Of the 47 patients who presented in the first year of life, four died of respiratory failure during the follow-up period, one each at the age of 10 months, 11 months, 1 year, and 2 years. Diverse clinical manifestations were present in the study patients. The predominant presentations were abnormalities in the central nervous system. Motor regression occurred after a period of normal development that lasted from 8 months to 14 years. Fourteen patients (19%) developed an abnormal motor presentation after contracting an infection, particularly a respiratory or intestinal infection. Two patients had motor regression after undergoing circumcision surgery. One patient presented with psychomotor retardation after vaccination for epidemic encephalitis at the age of 2 years. The detailed clinical manifestations are listed in Table 1. 3.2. Respiratory chain complex activity Isolated respiratory chain complex defects were identified in 64 patients (85.3%), and combined complex defects in 11 (14.7%). The activities of these enzyme complexes ranged from 23% to 48% of the lowest control value in isolated complex defects. Four types of varied combined complex deficiencies were found in 11 patients. The detailed information is shown in Fig. 1 and Table 2. We also identified 10 patients with mitochondrial DNA mutations. Of these patients, six had a complex I deficiency, two had a
complex V deficiency, and two had a combined deficiency (I and V, IV and V), as shown in Table 3. 3.3. Mitochondrial gene sequence analysis Mitochondrial DNA mutations were detected in 10 of 23 patients who underwent mitochondrial gene sequence analysis. Eight point mutations were found in mitochondrial structural genes (ND2, ND3, ND5, ND6, COXII, and ATPase6), and three point mutations were found in tRNA genes (Table 3). 3.4. Core pedigree analysis Gene analysis was performed in five families via restriction fragment length polymorphism and direct sequence analyses. A patient with a complex I deficiency and his mother carried the same m.12338T > C mutation. The mutation loads were 62% and 51% in the patient and his mother, respectively. The m.8993T > C mutation was found in a patient with respiratory chain complex V deficiency and her mother. The mutation loads were 90% and 62% in this patient and her mother, respectively. 4. Discussion Leigh syndrome is a common phenotype of mitochondrial disorders in children. The estimated incidence is about 1 per 40 000 live births.3 Leigh syndrome usually manifests in infancy or early childhood, and clinical onset is commonly seen in the first 2 years of life.6 In rare cases, however, Leigh syndrome can present in adolescents or young adults, with the reported age at clinical onset varying from 14 to 43 years.7 The clinical onset of Leigh syndrome has been reported to occur in the first year of life in 91% of patients.2 In the present study, however, clinical onset in the first year of life was seen in only 63% of patients, whereas onset in the first 3 years of life was seen in 83% of patients. The majority of patients with Leigh syndrome present with regressive dysfunction of the central nervous system. In our study, the main clinical manifestations included motor retardation (55% of patients), weakness (29%), epilepsy (25%), growth retardation (20%), and vomiting (9%). These results are consistent with those of other studies. Thus, Leigh syndrome was confirmed to be a common clinical phenotype of mitochondrial disorders in China. The biochemical features of Leigh syndrome are heterogeneous. A deficiency in the activity of one or more respiratory chain complexes results in Leigh syndrome. Deficiency in either complex I or IV is reported to be the most common cause of Leigh syndrome.8 Deficiency in complex II, III, or V is a minor cause of Leigh syndrome. Skladal et al. reported that the most frequent biochemical defect was a respiratory chain complex IV deficiency, followed by complex I deficiency, and combined deficiencies.9 In the present study, the activities of respiratory chain complexes in the leukocytes were assessed in 75 Chinese patients with Leigh syndrome for the first time. The results revealed that isolated complex I deficiency was the most frequent defect in Chinese patients with Leigh syndrome, followed by combined deficiency. Mitochondrial DNA mutations were detected in 10 patients. Structural gene mutations were frequent among the patients, followed by tRNA gene mutations. Taylor et al. first reported the m.10191T > G mutation in the ND3 gene, which transforms serine into hydrophobic proline (S45P) in highly conserved regions. McFarland et al. revealed that the ND3 subunit may play an important catalytic role in electron transport and ubiquinone binding.10 The m.10191T > G mutation was also reported in several children with Leigh syndrome.6,10 In the present study, two patients had the m.10191T > G mutation, and these patients biochemically
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Table 1 Clinical features of 75 patients with Leigh syndrome and a subset of 10 patients with mtDNA mutations (from 23 patients who underwent mitochondrial gene sequence analysis) Clinical features
Leigh syndrome
Sex (M/F) Age at onset Neonatal period (0–1 month) Infancy (1 month–1 year) Infancy (1–3 years) Childhood (3–18 years) Abnormal family history Abnormal pregnancy Low birth weight Poor feeding Swallowing difficulty Preceding infection or operation history Initial symptom Psychomotor retardation Motor developmental retardation Motor regression Weakness, fatigue Epilepsy Nystagmus Ptosis Ophthalmoplegia Vomiting Hypotonia Ataxia Hyperlacticacidemia High pyruvate level
Leigh syndrome with mitochondrial DNA mutation
Patients (n = 75)
Percentage (%)
Patients (n = 10)
Percentage (%)
49/26
65/35
3/7
13/30
3 44 15 13 10 9 7 12 5 14
4 59 20 17 13 12 9 16 7 19
0 6 1 3 2 1 2 2 1 1
0 26 4 13 9 4 9 9 4 4
15 26 15 22 19 4 3 2 7 29 8 63 47
20 35 20 29 25 5 4 3 9 39 11 84 63
2 3 1 1 6 0 1 0 1 4 0 7 4
9 13 4 4 26 0 4 0 4 17 0 30 17
Fig. 1. Graph showing the activities of respiratory chain complexes (A) and the activity as a ratio of the rate of citrate synthase (B) in 75 patients with Leigh syndrome.
Table 2 Type of respiratory chain complex deficiency in 75 Chinese patients with Leigh syndrome Type of deficiency
Complex I Complex II Complex III Complex IV Complex V Combined complex I and IV I and V III and V IV and V
Leigh syndrome
Leigh syndrome with mitochondrial DNA mutation
Patients (n = 75)
Percentage (%)
Patients (n = 23)
Percentage (%)
28 7 6 10 13
37.3 9.3 8.0 13.3 17.3
9 0 1 3 5
39.1 0.0 4.3 13.0 21.7
5 3 2 1
6.7 4.0 2.7 1.3
3 1 0 1
13.0 4.3 0.0 4.3
presented with respiratory chain complex I deficiency. We propose that the m.10191T > G mutation is one of the most common genotypes of Leigh syndrome in the Chinese population. The m.13513G > A and m.12338T > C mutations in the ND5 gene were detected in one and two patients, respectively. The former
affects an evolutionarily conserved amino acid (D393N) of the ND5 subunit. This mutation has been reported in not only patients with mitochondrial encephalomyopathy, lactic acidosis, and stroke-like episodes (MELAS), but also patients with a combined syndrome of MELAS and Leber hereditary optic neuropathy
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Table 3 Clinical features of 10 Chinese patients with Leigh syndrome caused by mtDNA mutation Patient
Sex
Age
Age at onset
Gene
Mutation
Mitochondrial RNA or protein
Complex deficiency
1 2 3 4 5 6 7 8 9 10
Male Female Male Male Female Male Male Male Male Female
22 mo 17 mo 18 yr 14 yr 11 yr 22 mo 24 mo 15 mo 2 yr 2 yr
6 mo 11 mo 12 yr 6 yr 8 yr 7 mo 3 mo 11 mo 17 mo 2 mo
ND3; ND5 ND6 ND5 ND3 ND2 tRNA-GlnCOXII; ND6 ND5 tRNA-Arg tRNA-Ala ATPase6
10191T > C; 12338T>C 14487T > C 13513G > A 10191T > C 4833A > G 4395A > G; 8108A > G; 14502T > C 12338T > C 10454 T > C 5587T > C 8993T > C
S-P, M-T M-V D-N S-P T-A tRNA-Gln; I-V, I-V M-T tRNA-Arg tRNA-Ala L-R
I I I I I I I and V IV and V V V
mo = months, yr = years.
(LHON).11 The same mutation was later identified in Leigh syndrome.12 Sudo et al. investigated 85 patients with Leigh syndrome, and revealed that six patients (7%) aged 9 months to 5 years had the m.13513G > A mutation. Thus, this mutation is probably a common cause of Leigh syndrome.13 The m.12338T > C mutation has only been reported in Chinese families with LHON.14 The m.12338T > C mutation causes the replacement of methionine in the first residue with threonine, which shortens two residues of the ND5 subunit.14 In the current study, the m.12338T > C mutation was detected in one patient with isolated respiratory chain complex I deficiency and in another patient with combined complex I and V deficiency. The patient with the isolated complex I deficiency also had the m.10191T > C mutation. The other patient with the m.12338T > C mutation presented with growth retardation, hypotonia, and epilepsy, and died of respiratory failure after 10 months. His mother carried the same mutation, but presented normally. The m.14487T > C mutation in the ND6 gene results in a broad spectrum of phenotypes, including Leigh syndrome, MELAS, and LHON. This missense mutation (M63V) is located in the most conserved transmembrane region of the ND6 subunit. Dermaut et al. concluded that the homoplasmic m.14487T > C mutation leads to severe Leigh syndrome phenotypes with onset during infancy. Lower heteroplasmy levels cause progressive myoclonic epilepsy with onset during adulthood.15 The m.14502T > C mutation in the ND6 gene has only been reported in Chinese families with LHON.16 This mutation leads to the replacement of this isoleucine with valine. A study revealed that the m.14502T > C mutation may have an important function in determining the phenotype of LHON.17 In the present study, coexisting m.14502T > C and m.4395A > G mutations were found in a patient with respiratory chain complex I deficiency. The m.14502T > C mutation may be a common genotype of Leigh syndrome in the Chinese population and may have a role in modifying the onset of this disorder. We conclude that Leigh syndrome is a common phenotype of mitochondrial disorders in China. Sixty-three percent of Chinese patients with Leigh syndrome due to respiratory chain complex deficiency had clinical onset within the first year of life. Isolated respiratory complex I deficiency was the most frequent defect in Chinese patients, followed by combined deficiencies of respiratory complexes. Mutations in mitochondrial structural genes form the main genetic basis of Leigh syndrome in Chinese patients. Conflicts of interest/disclosures The authors declare that they have no financial or other conflicts of interest in relation to this research and its publication.
Acknowledgments We are grateful for the technical support and collaboration of Dr. Weiyue Gu from Bio Road Biotech Co., Ltd, Beijing, China. This work was supported by the National Nature Science Foundation of China (No. 30872794), the 12th Five-year Plan National Key Technology R & D Program from the Ministry of Science and Technology (2012BAI09B04) and Beijing Nature Science Foundation (No. 7081002). References 1. Leigh D. Subacute necrotizing encephalomyelopathy in an infant. J Neurol Neurosurg Psychiatry 1951;14:216–21. 2. Yang YL, Sun F, Zhang Y, et al. Clinical and laboratory survey of 65 Chinese patients with Leigh syndrome. Chin Med J (Engl) 2006;119:373–7. 3. Rahman S, Blok RB, Dahl HH, et al. Leigh syndrome: clinical features and biochemical and DNA abnormalities. Ann Neurol 1996;39:343–51. 4. Ma YY, Zhang XL, Wu TF, et al. Analysis of the mitochondrial complex I-V enzyme activities of peripheral leukocytes in oxidative phosphorylation disorders. J Child Neurol 2011;26:974–9. 5. Ma YY, Wu TF, Liu YP, et al. Heterogeneity of six children and their mothers with mitochondrial DNA 3243 A > G mutation. Mitochondrial DNA 2013; 24:297–302. 6. Lebon S, Chol M, Benit P, et al. Recurrent de novo mitochondrial DNA mutations in respiratory chain deficiency. J Med Genet 2003;40:896–9. 7. McKelvie P, Infeld B, Marotta R, et al. Late-adult onset Leigh syndrome. J Clin Neurosci 2012;19:195–202. 8. Distelmaier F, Koopman WJ, van Den Heuvel LP, et al. Mitochondrial complex I deficiency: from organelle dysfunction to clinical disease. Brain 2009;132: 833–42. 9. Skladal D, Sudmeier C, Konstantopoulou V, et al. The clinical spectrum of mitochondrial disease in 75 pediatric patients. Clin Pediatr (Phila) 2003;42: 703–10. 10. McFarland R, Kirby DM, Fowler KJ, et al. De novo mutations in the mitochondrial ND3 gene as a cause of infantile mitochondrial encephalopathy and complex I deficiency. Ann Neurol 2004;55:58–64. 11. Pulkes T, Eunson L, Patterson V, et al. The mitochondrial DNA G13513A transition in ND5 is associated with a LHON/MELAS overlap syndrome and may be a frequent cause of MELAS. Ann Neurol 1999;46:916–9. 12. Chol M, Lebon S, Benit P, et al. The mitochondrial DNA G13513A MELAS mutation in the NADH dehydrogenase 5 gene is a frequent cause of Leigh-like syndrome with isolated complex I deficiency. J Med Genet 2003;40:188–91. 13. Sudo A, Honzawa S, Nonaka I, et al. Leigh syndrome caused by mitochondrial DNA G13513A mutation: frequency and clinical features in Japan. J Hum Genet 2004;49:92–6. 14. Liu XL, Zhou X, Zhou J, et al. Leber’s hereditary optic neuropathy is associated with the T12338C mutation in mitochondrial ND5 gene in six Han Chinese families. Ophthalmology 2011;118:978–85. 15. Dermaut B, Seneca S, Dom L, et al. Progressive myoclonic epilepsy as an adultonset manifestation of Leigh syndrome due to m.14487T>C. J Neurol Neurosurg Psychiatry 2010;81:90–3. 16. Zhao F, Guan M, Zhou X, et al. Leber’s hereditary optic neuropathy is associated with mitochondrial ND6 T14502C mutation. Biochem Biophys Res Commun 2009;389:466–72. 17. Zhang J, Zhou X, Zhou J, et al. Mitochondrial ND6 T14502C variant may modulate the phenotypic expression of LHON-associated G11778A mutation in four Chinese families. Biochem Biophys Res Commun 2010;399:647–53.
Contents lists available at ScienceDirect
Journal of Clinical Neuroscience journal homepage: www.elsevier.com/locate/jocn
Laboratory studies
Genetic and biochemical findings in Chinese children with Leigh syndrome Yan-Yan Ma a, Tong-Fei Wu b, Yu-Peng Liu b, Qiao Wang b, Jin-Qing Song b, Xi-Yuan Li b, Xiu-Yu Shi a, Wei-Na Zhang a, Meng Zhao a, Lin-Yan Hu a, Yan-Ling Yang b, , Li-Ping Zou a,⇑, a b
Department of Pediatrics, Chinese Liberation Army General Hospital, No. 28 Fu-xing Road, Haidian District, Beijing 100853, People’s Republic of China Department of Pediatrics, Peking University First Hospital, Beijing, People’s Republic of China
a r t i c l e
i n f o
Article history: Received 27 October 2012 Accepted 24 March 2013
Keywords: Leigh syndrome Mitochondrial disorders Mitochondrial gene Mitochondrial respiratory complex deficiency
a b s t r a c t This study investigated the genetic and enzymological features of Leigh syndrome due to respiratory chain complex deficiency in Chinese patients. The clinical features of 75 patients were recorded. Mitochondrial respiratory chain enzyme activities were determined via spectrophotometry. Mitochondrial gene sequence analysis was performed in 23 patients. Five core pedigrees were investigated via restriction fragment length polymorphism and gene sequencing. Psychomotor retardation (55%), motor regression (20%), weakness (29%), and epilepsy (25%) were the most frequent manifestations. Sixty-four patients (85.3%) had isolated respiratory complex deficiencies: complex I was seen in 28 patients (37.3%); complex II, seven (9.3%); complex III, six (8%); complex IV, ten (13.3%); and complex V, 13 patients (17.3%). Eleven patients (14.7%) had combined complex deficiencies. Mitochondrial DNA mutations were detected in 10 patients. Eight point mutations were found in mitochondrial structural genes: m.4833A > G in ND2, m.10191T > C in ND3, m.12338T > C and m.13513G > A in ND5, m.14502T > C and m.14487T > C in ND6, m.8108A > G in COXII, and m.8993T > G in ATPase6. Three mutations were found in tRNA genes: m.4395A > G in tRNA-Gln, m.10454T > C in tRNA-Arg, and m.5587T > C in tRNA-Ala. One patient and their mother both had the m.12338T > C and m.8993T > C mutations. In conclusion, mitochondrial respiratory chain complex I deficiency and structural gene mutations frequently occur in Chinese Leigh syndrome patients. Ó 2013 Elsevier Ltd. All rights reserved.
1. Introduction Leigh syndrome was first reported by Denis Leigh in 1951.1 Thus far, this disorder has been proven to be clinically and genetically heterogeneous. Leigh syndrome most commonly presents as a progressive dysfunction of the central nervous system. Mitochondrial respiratory chain enzyme assay and gene analysis are needed for a precise etiological diagnosis of the disease. Deficiencies can occur in isolated or combined respiratory chain complexes, and a wide spectrum of biochemical data and clinical presentations has been documented. However, respiratory chain complex defects in Chinese patients with Leigh syndrome have not been studied in the past few decades because of the lack of clinically acceptable methods for conducting enzyme assays of respiratory chain complexes in China. In principle, the ideal tissue for clinical diagnosis is one that clinically expresses the disease, such as the brain, kidney, myocardium, liver, or endocrine glands. Because patients
⇑ Corresponding author. Tel.: +86 10 5549 9316; fax: +86 10 6613 4261.
E-mail address: [email protected] (L.-P. Zou). Li-ping Zou and Yan-Ling Yang have contributed equally to the manuscript.
0967-5868/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.jocn.2013.03.034
and their parents are less likely to accept a traumatic biopsy examination in comparison to a blood test, it is difficult to apply such a measure of respiratory chain complex I–V activity using biopsied tissue in China. A few pathogenic mutations have been found in Chinese patients with Leigh syndrome via genetic screening alone.2 The biochemical and genetic features of Leigh syndrome in the Chinese population, however, remain unknown. The present study investigated the etiology of Leigh syndrome in 75 Chinese patients with a view to determining the biochemical and genetic characteristics of this syndrome in the Chinese population.
2. Patients and methods 2.1. Patients We recruited 75 Chinese patients with Leigh syndrome with a mean age of 4.5 years (range, 3–18 years), who were admitted in the past 32 months. These patients presented with neuromuscular symptoms and signs that progressively worsened. Leigh syndrome was diagnosed on the basis of the clinical manifestations, biochemical features and characteristic brain MRI scans.3 Typical
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aminoacidopathies, organic acidurias, and mitochondrial b-oxidation defects were excluded via analyses of blood amino acids and acylcarnitines, and urinary organic acids. The parents of all patients gave informed consent. This study was approved by the hospital Ethics Committee following the tenets of the Declaration of Helsinki. 2.2. Methods 2.2.1. Assays of mitochondrial complex I to V enzyme activity The activities of mitochondrial respiratory chain enzymes I to V were detected via spectrophotometry. This method has been previously described in detail.4 2.2.2. Mitochondrial gene sequence analysis and restriction fragment length polymorphism analysis Mitochondrial gene sequence analysis was performed in 23 patients via direct sequencing. The sequence results were then compared with the revised Cambridge reference sequence5 GenBank ID: NC_012920.1. Patients with point mutations and their parents underwent polymerase chain reaction–restriction fragment length polymorphism analysis. This method has been previously described in detail.5 3. Results 3.1. Clinical features Among the 75 patients with Leigh syndrome due to respiratory chain complex deficiency, 49 were boys and 26 were girls. The male:female ratio was 1.9:1. The parents of the patients were healthy and nonconsanguineous. Ten patients (13%) had a positive family history of neuromuscular diseases. Their affected siblings died of encephalopathies or multiple organ failure. A mitochondrial defect was suspected, but a definitive diagnosis had not been reached. Nine (12%) patients had abnormal presentations in the fetal stage, which included intrauterine growth retardation, threatened abortion or fetal distress. The onset of symptoms dated back to the first year of life in 47 patients (63%), and the first 3 years of life in 62 patients (83%). Of the 47 patients who presented in the first year of life, four died of respiratory failure during the follow-up period, one each at the age of 10 months, 11 months, 1 year, and 2 years. Diverse clinical manifestations were present in the study patients. The predominant presentations were abnormalities in the central nervous system. Motor regression occurred after a period of normal development that lasted from 8 months to 14 years. Fourteen patients (19%) developed an abnormal motor presentation after contracting an infection, particularly a respiratory or intestinal infection. Two patients had motor regression after undergoing circumcision surgery. One patient presented with psychomotor retardation after vaccination for epidemic encephalitis at the age of 2 years. The detailed clinical manifestations are listed in Table 1. 3.2. Respiratory chain complex activity Isolated respiratory chain complex defects were identified in 64 patients (85.3%), and combined complex defects in 11 (14.7%). The activities of these enzyme complexes ranged from 23% to 48% of the lowest control value in isolated complex defects. Four types of varied combined complex deficiencies were found in 11 patients. The detailed information is shown in Fig. 1 and Table 2. We also identified 10 patients with mitochondrial DNA mutations. Of these patients, six had a complex I deficiency, two had a
complex V deficiency, and two had a combined deficiency (I and V, IV and V), as shown in Table 3. 3.3. Mitochondrial gene sequence analysis Mitochondrial DNA mutations were detected in 10 of 23 patients who underwent mitochondrial gene sequence analysis. Eight point mutations were found in mitochondrial structural genes (ND2, ND3, ND5, ND6, COXII, and ATPase6), and three point mutations were found in tRNA genes (Table 3). 3.4. Core pedigree analysis Gene analysis was performed in five families via restriction fragment length polymorphism and direct sequence analyses. A patient with a complex I deficiency and his mother carried the same m.12338T > C mutation. The mutation loads were 62% and 51% in the patient and his mother, respectively. The m.8993T > C mutation was found in a patient with respiratory chain complex V deficiency and her mother. The mutation loads were 90% and 62% in this patient and her mother, respectively. 4. Discussion Leigh syndrome is a common phenotype of mitochondrial disorders in children. The estimated incidence is about 1 per 40 000 live births.3 Leigh syndrome usually manifests in infancy or early childhood, and clinical onset is commonly seen in the first 2 years of life.6 In rare cases, however, Leigh syndrome can present in adolescents or young adults, with the reported age at clinical onset varying from 14 to 43 years.7 The clinical onset of Leigh syndrome has been reported to occur in the first year of life in 91% of patients.2 In the present study, however, clinical onset in the first year of life was seen in only 63% of patients, whereas onset in the first 3 years of life was seen in 83% of patients. The majority of patients with Leigh syndrome present with regressive dysfunction of the central nervous system. In our study, the main clinical manifestations included motor retardation (55% of patients), weakness (29%), epilepsy (25%), growth retardation (20%), and vomiting (9%). These results are consistent with those of other studies. Thus, Leigh syndrome was confirmed to be a common clinical phenotype of mitochondrial disorders in China. The biochemical features of Leigh syndrome are heterogeneous. A deficiency in the activity of one or more respiratory chain complexes results in Leigh syndrome. Deficiency in either complex I or IV is reported to be the most common cause of Leigh syndrome.8 Deficiency in complex II, III, or V is a minor cause of Leigh syndrome. Skladal et al. reported that the most frequent biochemical defect was a respiratory chain complex IV deficiency, followed by complex I deficiency, and combined deficiencies.9 In the present study, the activities of respiratory chain complexes in the leukocytes were assessed in 75 Chinese patients with Leigh syndrome for the first time. The results revealed that isolated complex I deficiency was the most frequent defect in Chinese patients with Leigh syndrome, followed by combined deficiency. Mitochondrial DNA mutations were detected in 10 patients. Structural gene mutations were frequent among the patients, followed by tRNA gene mutations. Taylor et al. first reported the m.10191T > G mutation in the ND3 gene, which transforms serine into hydrophobic proline (S45P) in highly conserved regions. McFarland et al. revealed that the ND3 subunit may play an important catalytic role in electron transport and ubiquinone binding.10 The m.10191T > G mutation was also reported in several children with Leigh syndrome.6,10 In the present study, two patients had the m.10191T > G mutation, and these patients biochemically
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Table 1 Clinical features of 75 patients with Leigh syndrome and a subset of 10 patients with mtDNA mutations (from 23 patients who underwent mitochondrial gene sequence analysis) Clinical features
Leigh syndrome
Sex (M/F) Age at onset Neonatal period (0–1 month) Infancy (1 month–1 year) Infancy (1–3 years) Childhood (3–18 years) Abnormal family history Abnormal pregnancy Low birth weight Poor feeding Swallowing difficulty Preceding infection or operation history Initial symptom Psychomotor retardation Motor developmental retardation Motor regression Weakness, fatigue Epilepsy Nystagmus Ptosis Ophthalmoplegia Vomiting Hypotonia Ataxia Hyperlacticacidemia High pyruvate level
Leigh syndrome with mitochondrial DNA mutation
Patients (n = 75)
Percentage (%)
Patients (n = 10)
Percentage (%)
49/26
65/35
3/7
13/30
3 44 15 13 10 9 7 12 5 14
4 59 20 17 13 12 9 16 7 19
0 6 1 3 2 1 2 2 1 1
0 26 4 13 9 4 9 9 4 4
15 26 15 22 19 4 3 2 7 29 8 63 47
20 35 20 29 25 5 4 3 9 39 11 84 63
2 3 1 1 6 0 1 0 1 4 0 7 4
9 13 4 4 26 0 4 0 4 17 0 30 17
Fig. 1. Graph showing the activities of respiratory chain complexes (A) and the activity as a ratio of the rate of citrate synthase (B) in 75 patients with Leigh syndrome.
Table 2 Type of respiratory chain complex deficiency in 75 Chinese patients with Leigh syndrome Type of deficiency
Complex I Complex II Complex III Complex IV Complex V Combined complex I and IV I and V III and V IV and V
Leigh syndrome
Leigh syndrome with mitochondrial DNA mutation
Patients (n = 75)
Percentage (%)
Patients (n = 23)
Percentage (%)
28 7 6 10 13
37.3 9.3 8.0 13.3 17.3
9 0 1 3 5
39.1 0.0 4.3 13.0 21.7
5 3 2 1
6.7 4.0 2.7 1.3
3 1 0 1
13.0 4.3 0.0 4.3
presented with respiratory chain complex I deficiency. We propose that the m.10191T > G mutation is one of the most common genotypes of Leigh syndrome in the Chinese population. The m.13513G > A and m.12338T > C mutations in the ND5 gene were detected in one and two patients, respectively. The former
affects an evolutionarily conserved amino acid (D393N) of the ND5 subunit. This mutation has been reported in not only patients with mitochondrial encephalomyopathy, lactic acidosis, and stroke-like episodes (MELAS), but also patients with a combined syndrome of MELAS and Leber hereditary optic neuropathy
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Table 3 Clinical features of 10 Chinese patients with Leigh syndrome caused by mtDNA mutation Patient
Sex
Age
Age at onset
Gene
Mutation
Mitochondrial RNA or protein
Complex deficiency
1 2 3 4 5 6 7 8 9 10
Male Female Male Male Female Male Male Male Male Female
22 mo 17 mo 18 yr 14 yr 11 yr 22 mo 24 mo 15 mo 2 yr 2 yr
6 mo 11 mo 12 yr 6 yr 8 yr 7 mo 3 mo 11 mo 17 mo 2 mo
ND3; ND5 ND6 ND5 ND3 ND2 tRNA-GlnCOXII; ND6 ND5 tRNA-Arg tRNA-Ala ATPase6
10191T > C; 12338T>C 14487T > C 13513G > A 10191T > C 4833A > G 4395A > G; 8108A > G; 14502T > C 12338T > C 10454 T > C 5587T > C 8993T > C
S-P, M-T M-V D-N S-P T-A tRNA-Gln; I-V, I-V M-T tRNA-Arg tRNA-Ala L-R
I I I I I I I and V IV and V V V
mo = months, yr = years.
(LHON).11 The same mutation was later identified in Leigh syndrome.12 Sudo et al. investigated 85 patients with Leigh syndrome, and revealed that six patients (7%) aged 9 months to 5 years had the m.13513G > A mutation. Thus, this mutation is probably a common cause of Leigh syndrome.13 The m.12338T > C mutation has only been reported in Chinese families with LHON.14 The m.12338T > C mutation causes the replacement of methionine in the first residue with threonine, which shortens two residues of the ND5 subunit.14 In the current study, the m.12338T > C mutation was detected in one patient with isolated respiratory chain complex I deficiency and in another patient with combined complex I and V deficiency. The patient with the isolated complex I deficiency also had the m.10191T > C mutation. The other patient with the m.12338T > C mutation presented with growth retardation, hypotonia, and epilepsy, and died of respiratory failure after 10 months. His mother carried the same mutation, but presented normally. The m.14487T > C mutation in the ND6 gene results in a broad spectrum of phenotypes, including Leigh syndrome, MELAS, and LHON. This missense mutation (M63V) is located in the most conserved transmembrane region of the ND6 subunit. Dermaut et al. concluded that the homoplasmic m.14487T > C mutation leads to severe Leigh syndrome phenotypes with onset during infancy. Lower heteroplasmy levels cause progressive myoclonic epilepsy with onset during adulthood.15 The m.14502T > C mutation in the ND6 gene has only been reported in Chinese families with LHON.16 This mutation leads to the replacement of this isoleucine with valine. A study revealed that the m.14502T > C mutation may have an important function in determining the phenotype of LHON.17 In the present study, coexisting m.14502T > C and m.4395A > G mutations were found in a patient with respiratory chain complex I deficiency. The m.14502T > C mutation may be a common genotype of Leigh syndrome in the Chinese population and may have a role in modifying the onset of this disorder. We conclude that Leigh syndrome is a common phenotype of mitochondrial disorders in China. Sixty-three percent of Chinese patients with Leigh syndrome due to respiratory chain complex deficiency had clinical onset within the first year of life. Isolated respiratory complex I deficiency was the most frequent defect in Chinese patients, followed by combined deficiencies of respiratory complexes. Mutations in mitochondrial structural genes form the main genetic basis of Leigh syndrome in Chinese patients. Conflicts of interest/disclosures The authors declare that they have no financial or other conflicts of interest in relation to this research and its publication.
Acknowledgments We are grateful for the technical support and collaboration of Dr. Weiyue Gu from Bio Road Biotech Co., Ltd, Beijing, China. This work was supported by the National Nature Science Foundation of China (No. 30872794), the 12th Five-year Plan National Key Technology R & D Program from the Ministry of Science and Technology (2012BAI09B04) and Beijing Nature Science Foundation (No. 7081002). References 1. Leigh D. Subacute necrotizing encephalomyelopathy in an infant. J Neurol Neurosurg Psychiatry 1951;14:216–21. 2. Yang YL, Sun F, Zhang Y, et al. Clinical and laboratory survey of 65 Chinese patients with Leigh syndrome. Chin Med J (Engl) 2006;119:373–7. 3. Rahman S, Blok RB, Dahl HH, et al. Leigh syndrome: clinical features and biochemical and DNA abnormalities. Ann Neurol 1996;39:343–51. 4. Ma YY, Zhang XL, Wu TF, et al. Analysis of the mitochondrial complex I-V enzyme activities of peripheral leukocytes in oxidative phosphorylation disorders. J Child Neurol 2011;26:974–9. 5. Ma YY, Wu TF, Liu YP, et al. Heterogeneity of six children and their mothers with mitochondrial DNA 3243 A > G mutation. Mitochondrial DNA 2013; 24:297–302. 6. Lebon S, Chol M, Benit P, et al. Recurrent de novo mitochondrial DNA mutations in respiratory chain deficiency. J Med Genet 2003;40:896–9. 7. McKelvie P, Infeld B, Marotta R, et al. Late-adult onset Leigh syndrome. J Clin Neurosci 2012;19:195–202. 8. Distelmaier F, Koopman WJ, van Den Heuvel LP, et al. Mitochondrial complex I deficiency: from organelle dysfunction to clinical disease. Brain 2009;132: 833–42. 9. Skladal D, Sudmeier C, Konstantopoulou V, et al. The clinical spectrum of mitochondrial disease in 75 pediatric patients. Clin Pediatr (Phila) 2003;42: 703–10. 10. McFarland R, Kirby DM, Fowler KJ, et al. De novo mutations in the mitochondrial ND3 gene as a cause of infantile mitochondrial encephalopathy and complex I deficiency. Ann Neurol 2004;55:58–64. 11. Pulkes T, Eunson L, Patterson V, et al. The mitochondrial DNA G13513A transition in ND5 is associated with a LHON/MELAS overlap syndrome and may be a frequent cause of MELAS. Ann Neurol 1999;46:916–9. 12. Chol M, Lebon S, Benit P, et al. The mitochondrial DNA G13513A MELAS mutation in the NADH dehydrogenase 5 gene is a frequent cause of Leigh-like syndrome with isolated complex I deficiency. J Med Genet 2003;40:188–91. 13. Sudo A, Honzawa S, Nonaka I, et al. Leigh syndrome caused by mitochondrial DNA G13513A mutation: frequency and clinical features in Japan. J Hum Genet 2004;49:92–6. 14. Liu XL, Zhou X, Zhou J, et al. Leber’s hereditary optic neuropathy is associated with the T12338C mutation in mitochondrial ND5 gene in six Han Chinese families. Ophthalmology 2011;118:978–85. 15. Dermaut B, Seneca S, Dom L, et al. Progressive myoclonic epilepsy as an adultonset manifestation of Leigh syndrome due to m.14487T>C. J Neurol Neurosurg Psychiatry 2010;81:90–3. 16. Zhao F, Guan M, Zhou X, et al. Leber’s hereditary optic neuropathy is associated with mitochondrial ND6 T14502C mutation. Biochem Biophys Res Commun 2009;389:466–72. 17. Zhang J, Zhou X, Zhou J, et al. Mitochondrial ND6 T14502C variant may modulate the phenotypic expression of LHON-associated G11778A mutation in four Chinese families. Biochem Biophys Res Commun 2010;399:647–53.