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Phenotypic Differences Between T → C and T → G Mutations at nt 8993 of Mitochondrial DNA in Leigh Syndrome
Phenotypic Differences Between T 3 C and T 3 G Mutations at nt 8993 of Mitochondrial DNA in Leigh Syndrome Tatsuya Fujii, MD, PhD*, Haruo Hattori, MD, PhD†, Yoshihisa Higuchi, MD†, Masahiro Tsuji, MD†, and Izuru Mitsuyoshi, MD, PhD‡ This study reports on a patient with Leigh syndrome with a T-to-C mutation at nucleotide 8993 of mitochondrial deoxyribonucleic acid (T8993C). The authors reviewed 10 Leigh syndrome patients, including ours, with T8993C. Compared with 18 reported patients with Leigh syndrome caused by a T-to-G mutation at nucleotide 8993 (T8993G), Leigh syndrome with T8993C was characterized by a significantly higher frequency of ataxia (P < 0.01). None of the reviewed T8993C-associated Leigh syndrome patients had retinitis pigmentosa, which is one of the characteristic findings in Leigh syndrome with T8993G. The milder symptoms of T8993C-Leigh syndrome can be explained by the milder complex V dysfunction; however, the higher frequency of ataxia in T8993C-Leigh syndrome requires more study. © 1998 by Elsevier Science Inc. All rights reserved. Fujii T, Hattori H, Higuchi Y, Tsuji M, Mitsuyoshi I. Phenotypic differences between T 3 C and T 3 G mutations at nt 8993 of mitochondrial DNA in Leigh syndrome. Pediatr Neurol 1998;18:275-277.
From the *Department of Pediatrics; Shiga Medical Center for Children; Shiga, Japan; †Department of Pediatrics; Kyoto University Faculty of Medicine; Kyoto, Japan; and ‡Department of Pediatrics; Utano National Hospital; Kyoto, Japan.
© 1998 by Elsevier Science Inc. All rights reserved. PII S0887-8994(97)00187-2 ● 0887-8994/98/$19.00
Introduction Leigh syndrome (LS) (subacute necrotizing encephalomyelopathy) is a progressive neurodegenerative disorder of infancy or childhood characterized by various clinical presentations, including psychomotor regression, hypotonia, ataxia, and brainstem dysfunction. A definite diagnosis depends on the characteristic postmortem neuropathology in the basal ganglia and brainstem, but a premortem diagnosis is possible based on magnetic resonance imaging findings and clinical features. Defects of mitochondrial enzymes, which include nicotinamide adenine dinucleotide-coenzyme Q (CoQ) reductase (complex I) [1], cytochrome c oxidase (complex IV) [2], pyruvate dehydrogenase complex (PDHC) [3], and succinate-CoQ reductase (complex II) [4], have been associated with LS. Mutations of mitochondrial deoxyribonucleic acid (mtDNA) have also been described, including a T-to-G mutation at nucleotide (nt) 8993, an A-to-G mutation at nt 8344, single deletions, and depletion [5]. Nucleotide 8993 is located in the adenosinetriphosphatase 6 gene of mtDNA. A T-to-G mutation at this nucleotide (T8993G) was initially described in a family with neurogenic weakness, ataxia, and retinitis pigmentosa (NARP) [6]. Then, Tatuch et al. [7] reported that T8993G can cause LS when the percentage of abnormal mtDNA is high, and T8993G has been described as a common cause of LS [8]. In 1993 a T-to-C mutation at nt 8993 was described in a family with LS [9]. T8993C is much more rare than T8993G, and thus the phenotype of T8993C-associated LS has not been fully characterized. This study reports on a patient with LS caused by T8993C and compares the phenotypic characteristics of T8993C-LS with those of T8993G-LS based on the clinical features of previously reported patients and ours. Case Report A 2-year-old girl was the product a normal pregnancy and delivery. The family history was unremarkable. Her development was normal until 8 months of age when she stopped smiling. At age 11 months, she could speak a few words and walk with assistance. Thereafter, she gradually lost her previously acquired skills. She had mild hypotonia with normal deep tendon reflexes. Ataxia was not noted. She was indifferent to pain, such as on blood sampling. The optic fundi were normal, and nystagmus was not noted. Nerve conduction velocities were normal. The serum levels of lactate and pyruvate were normal, but the cerebrospinal fluid level of lactate was slightly elevated (2.2 mmol/L). Urinary organic acids
Communications should be addressed to: Dr. Fujii; Department of Pediatrics; Shiga Medical Center for Children; 5-7-30 Moriyama; Moriyama-City; Shiga 524, Japan. Received April 14, 1997; accepted August 22, 1997.
Fujii et al: Leigh Syndrome With T8993C 275
Table 1. Frequencies of symptoms (%) in Leigh syndrome with T8993C and T8993G
T8993C (n 5 10) Age of onset Hypotonia Developmental delay Ataxia Peripheral neuropathy Optic atrophy Retinitis pigmentosa Nystagmus Seizures
6 months-56 years (median 5 4 years) 70 50 90† 30 10 0 20 30
T8993G (n 5 18)* 4-5 months 89 78 28 28 33 39 11 67
* Santorelli et al. [8]. P , 0.01; for all other values, P . 0.05.
†
and serum amino acids were normal. T2-weighted magnetic resonance imaging revealed symmetric high-signal areas in the putamen, periaqueduct, substantia nigra, and inferior colliculus, bilaterally. A muscle biopsy revealed a myogenic change without ragged red fibers. At age 2 years, she could still stand only with support, but she stayed sitting or lying most of the time; occasionally she choked while drinking fluids.
Methods Total DNA was extracted from peripheral leukocytes. Polymerase chain reaction (PCR) and restriction fragment length polymorphism (RFLP) analyses were performed to detect either T8993G or T8993C as described [8]. Statistical significance was determined by means of x2 analysis with Yates’ correction.
Results PCR-RFLP demonstrated that the patient had the T8993C mutation. Although we did not determine the amount of mutant mtDNA, normal mtDNA was almost undetectable in the gel, indicating that most of the mtDNA from the patient’s leukocytes was mutant. The same analysis was performed for her parents and older sister, who were apparently normal. The mother and sister showed heteroplasmic mutant mtDNA, indicating that the patient’s T8993C was maternally inherited (data not shown). Ten patients, including ours, with T8993C-associated LS have been described [5,9-11]. We excluded another eight T8993C-LS patients summarized by Santorelli et al. [11] because the clinical features of each individual were not described; therefore, we could not confirm the diagnosis of LS. Table 1 compares the clinical features of these 10 T8993C-LS patients with those of the T8993G-LS ones summarized by Santorelli et al. (Table 1) [8]. The frequency of ataxia was significantly higher in T8993C-LS than in T8993G-LS (P , 0.01). The age of onset was later in T8993C. Even if we exclude an unusual patient with an onset age of 56 years, the onset age range in T8993C was 6 months to 9 years (i.e., later than in T8993G). None of the T8993C-LS patients had retinitis pigmentosa, which
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Santorelli et al. reported was one of the characteristics of T8993G-LS [8]; however, the difference was not significant. Discussion Because the underlying biochemical or molecular defects of LS are variable, the phenotypes of LS are naturally diverse. A variable X-inactivation pattern for a deficiency of the E1a subunit of PDHC and a heteroplasmic load of mtDNA mutations are other causes of the variability, which make it difficult to clarify the genotype-phenotype correlation. Comparison between the T8993C and T8993G phenotypes is intriguing because the mutations are of the same nucleotide; if the different mutations cause merely a difference in the degree of impairment of complex V activity, the severity of the symptoms should be the only difference. The ATP production rate, which reflects the complex V activity in nt 8993 mutations, was reported to be less impaired in T8993C [11], and the later age of onset of T8993C-LS indicates that the difference may be in the severity. On the other hand, the most characteristic feature of T8993C-LS is a significantly higher frequency of ataxia. If the clinical difference between T8993C and T8993G is due to the difference in the severity of the enzymatic impairment, with T8993C causing a less severe deficit, the higher frequency of ataxia in T8993C-LS needs another explanation. Because mutant mitochondria commonly show different distributions in different tissues, one can assume that the cerebellar neuronal cells in T8993C-LS may harbor more mutant mitochondria than the cells in other parts of the brain. However, such a different distribution of mutant mitochondria within the brain, depending on different mutations, is unlikely. One possibility is that ataxia appears later in the course of LS and that patients with T8993G, who exhibit faster progression, may die or be severely disabled before the ataxia appears. This may explain why our patient, who is 2 years old currently, did not have ataxia, unlike all other reported patients with T8993C, who were older than 3 years of age except for one whose age is unknown but whose onset age was 20 months. Retinitis pigmentosa is found in about one third of T8993G-LS patients [8]. In our series of T8993C-LS patients, none had retinitis pigmentosa; however, the difference was not statistically significant. Santorelli et al. [11] reported that 25% of their series of T8993C-LS patients had retinitis pigmentosa, suggesting that it may be a finding as common as in T8993G-LS. T8993G causes either NARP or LS, and which phenotype a patient develops depends on the percentage of mutant mitochondria, and LS patients have a higher percentage [7]. This phenomenon indicates that NARP may be a milder form of the T8993G syndrome, with LS being a more severe form. If this is the case, T8993C, which affects the same enzyme, with milder clinical features and enzymatic deficit, is ex-
pected to cause NARP or a related symptomatology more frequently than does T8993G. However, only one NARP patient with T8993C has been reported so far [11]. Whether or not there are differences in the frequencies of the various nt 8993-associated syndromes between T8993C and T8993G is unknown. Further study is necessary to characterize T8993C-LS and other T8993C-associated syndromes and to explain the phenotypical difference between T8993C-LS and T8993G-LS. This study was partly supported by The Shiga Medical Science Association for International Cooperation. Part of the original manuscript was presented at the 39th annual meeting of the Japanese Society of Inherited Metabolic Diseases held in Tokyo, November 14-16, 1996.
References [1] Fujii T, Ito M, Okuno T, Mutoh K, Nishikomori R, Mikawa H. Complex I (reduced nicotinamide-adenine dinucleotide-coenzyme Q reductase) deficiency in two patients with probable Leigh syndrome. J Pediatr 1990;116:84-7. [2] Willems JL, Monnens LAH, Trijbels JMF, et al. Leigh encephalomyelopathy in a patient with cytochrome c oxidase deficiency in muscle tissue. Pediatrics 1977;60:850-7.
[3] Blass JP, Cedarbaum SD, Dunn HG. Biochemical abnormalities in Leigh disease. Lancet 1976;1:1237-8. [4] Bourgeron T, Rustin P, Chretien D, et al. Mutation of a nuclear succinate dehydrogenase gene results in mitochondrial respiratory chain deficiency. Nat Genet 1995;11:144-9. [5] Rahman S, Blok RB, Dahl HH, et al. Leigh syndrome: Clinical features and biochemical and DNA abnormalities. Ann Neurol 1996;39: 343-51. [6] Holt IJ, Harding AE, Petty RK, Morgan-Hughes JA. A new mitochondrial disease associated with mitochondrial DNA heteroplasmy. Am J Hum Genet 1990;46:428-33. [7] Tatuch Y, Christodoulou J, Feigenbaum A, et al. Heteroplasmic mtDNA mutation (T¡ G) at 8993 can cause Leigh disease when the percentage of abnormal mtDNA is high. Am J Hum Genet 1992;50: 852-8. [8] Santorelli FM, Shanske S, Macaya A, DeVivo DC, DiMauro S. The mutation at nt 8993 of mitochondrial DNA is a common cause of Leigh’s syndrome. Ann Neurol 1993;34:827-34. [9] de Vries DD, van Engelen BG, Gabreels FJ, Ruitenbeek W, van Oost BA. A second missense mutation in the mitochondrial ATPase 6 gene in Leigh’s syndrome. Ann Neurol 1993;34:410-2. [10] Santorelli FM, Shanske S, Jain KD, Tick D, Schon EA, DiMauro S. A T 3 C mutation at nt 8993 of mitochondrial DNA in a child with Leigh syndrome. Neurology 1994;44:972-4. [11] Santorelli FM, Mak SC, Vazquez Memije E, et al. Clinical heterogeneity associated with the mitochondrial DNA T8993C point mutation. Pediatr Res 1996;39:914-7.
Fujii et al: Leigh Syndrome With T8993C 277
From the *Department of Pediatrics; Shiga Medical Center for Children; Shiga, Japan; †Department of Pediatrics; Kyoto University Faculty of Medicine; Kyoto, Japan; and ‡Department of Pediatrics; Utano National Hospital; Kyoto, Japan.
© 1998 by Elsevier Science Inc. All rights reserved. PII S0887-8994(97)00187-2 ● 0887-8994/98/$19.00
Introduction Leigh syndrome (LS) (subacute necrotizing encephalomyelopathy) is a progressive neurodegenerative disorder of infancy or childhood characterized by various clinical presentations, including psychomotor regression, hypotonia, ataxia, and brainstem dysfunction. A definite diagnosis depends on the characteristic postmortem neuropathology in the basal ganglia and brainstem, but a premortem diagnosis is possible based on magnetic resonance imaging findings and clinical features. Defects of mitochondrial enzymes, which include nicotinamide adenine dinucleotide-coenzyme Q (CoQ) reductase (complex I) [1], cytochrome c oxidase (complex IV) [2], pyruvate dehydrogenase complex (PDHC) [3], and succinate-CoQ reductase (complex II) [4], have been associated with LS. Mutations of mitochondrial deoxyribonucleic acid (mtDNA) have also been described, including a T-to-G mutation at nucleotide (nt) 8993, an A-to-G mutation at nt 8344, single deletions, and depletion [5]. Nucleotide 8993 is located in the adenosinetriphosphatase 6 gene of mtDNA. A T-to-G mutation at this nucleotide (T8993G) was initially described in a family with neurogenic weakness, ataxia, and retinitis pigmentosa (NARP) [6]. Then, Tatuch et al. [7] reported that T8993G can cause LS when the percentage of abnormal mtDNA is high, and T8993G has been described as a common cause of LS [8]. In 1993 a T-to-C mutation at nt 8993 was described in a family with LS [9]. T8993C is much more rare than T8993G, and thus the phenotype of T8993C-associated LS has not been fully characterized. This study reports on a patient with LS caused by T8993C and compares the phenotypic characteristics of T8993C-LS with those of T8993G-LS based on the clinical features of previously reported patients and ours. Case Report A 2-year-old girl was the product a normal pregnancy and delivery. The family history was unremarkable. Her development was normal until 8 months of age when she stopped smiling. At age 11 months, she could speak a few words and walk with assistance. Thereafter, she gradually lost her previously acquired skills. She had mild hypotonia with normal deep tendon reflexes. Ataxia was not noted. She was indifferent to pain, such as on blood sampling. The optic fundi were normal, and nystagmus was not noted. Nerve conduction velocities were normal. The serum levels of lactate and pyruvate were normal, but the cerebrospinal fluid level of lactate was slightly elevated (2.2 mmol/L). Urinary organic acids
Communications should be addressed to: Dr. Fujii; Department of Pediatrics; Shiga Medical Center for Children; 5-7-30 Moriyama; Moriyama-City; Shiga 524, Japan. Received April 14, 1997; accepted August 22, 1997.
Fujii et al: Leigh Syndrome With T8993C 275
Table 1. Frequencies of symptoms (%) in Leigh syndrome with T8993C and T8993G
T8993C (n 5 10) Age of onset Hypotonia Developmental delay Ataxia Peripheral neuropathy Optic atrophy Retinitis pigmentosa Nystagmus Seizures
6 months-56 years (median 5 4 years) 70 50 90† 30 10 0 20 30
T8993G (n 5 18)* 4-5 months 89 78 28 28 33 39 11 67
* Santorelli et al. [8]. P , 0.01; for all other values, P . 0.05.
†
and serum amino acids were normal. T2-weighted magnetic resonance imaging revealed symmetric high-signal areas in the putamen, periaqueduct, substantia nigra, and inferior colliculus, bilaterally. A muscle biopsy revealed a myogenic change without ragged red fibers. At age 2 years, she could still stand only with support, but she stayed sitting or lying most of the time; occasionally she choked while drinking fluids.
Methods Total DNA was extracted from peripheral leukocytes. Polymerase chain reaction (PCR) and restriction fragment length polymorphism (RFLP) analyses were performed to detect either T8993G or T8993C as described [8]. Statistical significance was determined by means of x2 analysis with Yates’ correction.
Results PCR-RFLP demonstrated that the patient had the T8993C mutation. Although we did not determine the amount of mutant mtDNA, normal mtDNA was almost undetectable in the gel, indicating that most of the mtDNA from the patient’s leukocytes was mutant. The same analysis was performed for her parents and older sister, who were apparently normal. The mother and sister showed heteroplasmic mutant mtDNA, indicating that the patient’s T8993C was maternally inherited (data not shown). Ten patients, including ours, with T8993C-associated LS have been described [5,9-11]. We excluded another eight T8993C-LS patients summarized by Santorelli et al. [11] because the clinical features of each individual were not described; therefore, we could not confirm the diagnosis of LS. Table 1 compares the clinical features of these 10 T8993C-LS patients with those of the T8993G-LS ones summarized by Santorelli et al. (Table 1) [8]. The frequency of ataxia was significantly higher in T8993C-LS than in T8993G-LS (P , 0.01). The age of onset was later in T8993C. Even if we exclude an unusual patient with an onset age of 56 years, the onset age range in T8993C was 6 months to 9 years (i.e., later than in T8993G). None of the T8993C-LS patients had retinitis pigmentosa, which
276
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Santorelli et al. reported was one of the characteristics of T8993G-LS [8]; however, the difference was not significant. Discussion Because the underlying biochemical or molecular defects of LS are variable, the phenotypes of LS are naturally diverse. A variable X-inactivation pattern for a deficiency of the E1a subunit of PDHC and a heteroplasmic load of mtDNA mutations are other causes of the variability, which make it difficult to clarify the genotype-phenotype correlation. Comparison between the T8993C and T8993G phenotypes is intriguing because the mutations are of the same nucleotide; if the different mutations cause merely a difference in the degree of impairment of complex V activity, the severity of the symptoms should be the only difference. The ATP production rate, which reflects the complex V activity in nt 8993 mutations, was reported to be less impaired in T8993C [11], and the later age of onset of T8993C-LS indicates that the difference may be in the severity. On the other hand, the most characteristic feature of T8993C-LS is a significantly higher frequency of ataxia. If the clinical difference between T8993C and T8993G is due to the difference in the severity of the enzymatic impairment, with T8993C causing a less severe deficit, the higher frequency of ataxia in T8993C-LS needs another explanation. Because mutant mitochondria commonly show different distributions in different tissues, one can assume that the cerebellar neuronal cells in T8993C-LS may harbor more mutant mitochondria than the cells in other parts of the brain. However, such a different distribution of mutant mitochondria within the brain, depending on different mutations, is unlikely. One possibility is that ataxia appears later in the course of LS and that patients with T8993G, who exhibit faster progression, may die or be severely disabled before the ataxia appears. This may explain why our patient, who is 2 years old currently, did not have ataxia, unlike all other reported patients with T8993C, who were older than 3 years of age except for one whose age is unknown but whose onset age was 20 months. Retinitis pigmentosa is found in about one third of T8993G-LS patients [8]. In our series of T8993C-LS patients, none had retinitis pigmentosa; however, the difference was not statistically significant. Santorelli et al. [11] reported that 25% of their series of T8993C-LS patients had retinitis pigmentosa, suggesting that it may be a finding as common as in T8993G-LS. T8993G causes either NARP or LS, and which phenotype a patient develops depends on the percentage of mutant mitochondria, and LS patients have a higher percentage [7]. This phenomenon indicates that NARP may be a milder form of the T8993G syndrome, with LS being a more severe form. If this is the case, T8993C, which affects the same enzyme, with milder clinical features and enzymatic deficit, is ex-
pected to cause NARP or a related symptomatology more frequently than does T8993G. However, only one NARP patient with T8993C has been reported so far [11]. Whether or not there are differences in the frequencies of the various nt 8993-associated syndromes between T8993C and T8993G is unknown. Further study is necessary to characterize T8993C-LS and other T8993C-associated syndromes and to explain the phenotypical difference between T8993C-LS and T8993G-LS. This study was partly supported by The Shiga Medical Science Association for International Cooperation. Part of the original manuscript was presented at the 39th annual meeting of the Japanese Society of Inherited Metabolic Diseases held in Tokyo, November 14-16, 1996.
References [1] Fujii T, Ito M, Okuno T, Mutoh K, Nishikomori R, Mikawa H. Complex I (reduced nicotinamide-adenine dinucleotide-coenzyme Q reductase) deficiency in two patients with probable Leigh syndrome. J Pediatr 1990;116:84-7. [2] Willems JL, Monnens LAH, Trijbels JMF, et al. Leigh encephalomyelopathy in a patient with cytochrome c oxidase deficiency in muscle tissue. Pediatrics 1977;60:850-7.
[3] Blass JP, Cedarbaum SD, Dunn HG. Biochemical abnormalities in Leigh disease. Lancet 1976;1:1237-8. [4] Bourgeron T, Rustin P, Chretien D, et al. Mutation of a nuclear succinate dehydrogenase gene results in mitochondrial respiratory chain deficiency. Nat Genet 1995;11:144-9. [5] Rahman S, Blok RB, Dahl HH, et al. Leigh syndrome: Clinical features and biochemical and DNA abnormalities. Ann Neurol 1996;39: 343-51. [6] Holt IJ, Harding AE, Petty RK, Morgan-Hughes JA. A new mitochondrial disease associated with mitochondrial DNA heteroplasmy. Am J Hum Genet 1990;46:428-33. [7] Tatuch Y, Christodoulou J, Feigenbaum A, et al. Heteroplasmic mtDNA mutation (T¡ G) at 8993 can cause Leigh disease when the percentage of abnormal mtDNA is high. Am J Hum Genet 1992;50: 852-8. [8] Santorelli FM, Shanske S, Macaya A, DeVivo DC, DiMauro S. The mutation at nt 8993 of mitochondrial DNA is a common cause of Leigh’s syndrome. Ann Neurol 1993;34:827-34. [9] de Vries DD, van Engelen BG, Gabreels FJ, Ruitenbeek W, van Oost BA. A second missense mutation in the mitochondrial ATPase 6 gene in Leigh’s syndrome. Ann Neurol 1993;34:410-2. [10] Santorelli FM, Shanske S, Jain KD, Tick D, Schon EA, DiMauro S. A T 3 C mutation at nt 8993 of mitochondrial DNA in a child with Leigh syndrome. Neurology 1994;44:972-4. [11] Santorelli FM, Mak SC, Vazquez Memije E, et al. Clinical heterogeneity associated with the mitochondrial DNA T8993C point mutation. Pediatr Res 1996;39:914-7.
Fujii et al: Leigh Syndrome With T8993C 277