- Email: [email protected]
A novel mutation in the mitochondrial tRNAPhe gene associated with mitochondrial myopathy
Neuromuscular Disorders 14 (2004) 46–50 www.elsevier.com/locate/nmd
A novel mutation in the mitochondrial tRNAPhe gene associated with mitochondrial myopathy A.-R. Moslemia,*, C. Lindbergb, J. Toftc, E. Holmed, G. Kollberga, A. Oldforsa a
Department of Pathology, Sahlgrenska University Hospital, 41345 Go¨teborg, Sweden Department of Neurology, Sahlgrenska University Hospital, 41345 Go¨teborg, Sweden c Department of Neurology, South Elfsborg Hospital, 41345 Bora˚s, Sweden d Department of Clinical Chemistry, Sahlgrenska University Hospital, 41345 Go¨teborg, Sweden b
Received 20 March 2003; received in revised form 1 July 2003; accepted 10 July 2003
Abstract We report a novel heteroplasmic T ! C mutation at nt position 582 within the mitochondrial tRNAPhe gene of a 70-year-old woman with mitochondrial myopathy. No other family members were affected, suggesting that our patient was a sporadic case. The muscle showed frequent ragged red fibers and 43% cytochrome c oxidase deficient fibers. The mutation alters a conserved base pairing in the aminoacyl acceptor stem. The mutation load was 70% in muscle homogenate and varied from 0 to 95% in individual muscle fiber segments. Cytochrome c oxidase-negative fibers showed significantly higher levels of mutated mtDNA (.75%) than Cytochrome c oxidase-positive fibers ð, 55%Þ: This mutation adds to the previously described four pathogenic mutations in the tRNAPhe gene. q 2003 Elsevier B.V. All rights reserved. Keywords: Myopathy; Cytochrome c oxidase deficiency; tRNA; Mutation; mtDNA
1. Introduction
2. Material and methods
The human mitochondrial DNA (mtDNA) is a circular double-stranded molecule of 16.6 kb, which encodes 13 subunits of the respiratory chain enzymes, 22 tRNA and 2 rRNA genes [1]. Mutations in mtDNA have been reported to be a major cause of mitochondrial myopathy [2]. To date more than 100 pathogenic mtDNA tRNA gene mutations have been reported in association with a wide spectrum of clinical manifestations [3]. Pathogenic point mutations in tRNA genes are usually associated with cytochrome c oxidase (COX) deficiency and abnormal proliferation of mitochondria in muscle tissue. The majority of pathogenic tRNA mutations are transmitted maternally and sporadic cases are rare. In this study we report a novel heteroplasmic mutation in the aminoacyl acceptor stem region of tRNAPhe gene in muscle tissue of a 70-year-old woman with mitochondrial myopathy and encephalopathy.
2.1. Case report
* Corresponding author. Tel: þ 46-31-3423335; fax: þ 46-31-417283. E-mail address: [email protected] (A.-R. Moslemi). 0960-8966/$ - see front matter q 2003 Elsevier B.V. All rights reserved. doi:10.1016/S0960-8966(03)00168-8
The patient was a 70-year-old woman, who had experienced slowly progressive bilateral ptosis from age 50. She had had slightly impaired balance during her entire life. Her mother, her only sister and two of her three children were free of neuromuscular symptoms. Her youngest daughter, aged 39 had generalized muscle pain compatible with the fibromyalgia syndrome and no signs of muscle disease. At age 67 the patient was operated upon because of atrio-ventricular re-entry tachycardia. One year later she experienced weakness in her legs, muscle fatigue and soreness after moderate physical activity. Neurological examination at age 70 showed bilateral ptosis, and impaired vertical and horizontal eye movements. There was slight to moderate weakness in neck flexion, hip flexion and knee extension. She was slower than expected in repetitive hand and feet movements, but had no change in muscle tone. There was no limb ataxia. Tendon reflexes were normal, and the Babinski sign was absent. Laboratory investigations revealed increased plasma lactate both at rest 2.8 (normal , 1.7 mmol/l) and after
A.-R. Moslemi et al. / Neuromuscular Disorders 14 (2004) 46–50
47
Fig. 1. Serial sections of skeletal muscle. (a) Enzyme-histochemical staining of COX and succinate dehydrogenase. Cytochrome c oxidase deficient fibers are blue, while normal fibers are brown. The arrow indicates one of the COX-deficient ragged red fibers. (b) Immuno-histochemical demonstration of COX subunit II. The arrow indicates one of the COX-deficient ragged red fibers, which shows reduced expression of COX II. (c) Immuno-histochemical demonstration of COX subunit IV. The arrow indicates one of the COX-deficient ragged red fibers, which shows increased expression of COX IV.
A/S, Danmark), and Texas Rede-conjugated sheep-antimouse antibodies (Amersham Biosciences, Sweden).
a subanaerobic threshold exercise test [4]. Urinary lactate excretion was 29 mmol/mmol creatinine (normal , 20). Cerebrospinal fluid (CSF) lactate was elevated to 2.1 mmol/l (normal , 1.8). CSF albumin was normal but CSF Tau was elevated to 417 ng/l (normal , 400). MRI of the brain showed small bilateral lesions compatible with ischemic areas. Neuropsychological tests revealed impaired memory for verbal material as well as reduced visual attention, confirming the existence of an encephalopathy.
2.3. Biochemical analysis Isolation of mitochondria, oximetric measurements of fresh mitochondria, and spectrophotometric enzyme analyses were performed as previously described [6]. 2.4. Analysis of mtDNA
2.2. Morphological analysis of muscle Total DNA was extracted from frozen skeletal muscle tissue or peripheral blood leucocytes using the DNA Extraction Kit (Qiagen, Hilden, Germany) according to the manufacturer’s instructions. The entire genome of mtDNA was amplified by a long expand polymerase chain reaction (LX-PCR) using two sets of primer pairs as previously described [7]. Point mutations were searched for in the 22 mitochondrial tRNA genes by direct sequencing of the purified PCR products as previously described [8]. Single COX-deficient and normal muscle fibers were dissected as previously described [5]. To analyze the proportion of the A582G mutation in muscle
Open muscle biopsy specimens were obtained from the proband and her 39-year-old daughter with fibromyalgia. Morphological and enzyme-histochemical analysis were performed as previously described [5]. Immuno-histochemical staining of COX subunit II and IV was performed by incubating 8-mm thick cryostat sections with a mixture of polyclonal rabbit antibodies directed towards COX subunit II (a generous gift from A. Lombes) and mouse monoclonal antibodies towards COX subunit IV (a generous gift from DiMauro). As secondary antibodies we used FITC-conjugated swine-anti-rabbit antibodies (DAKO Table 1 Respiratory rates and enzyme activities in isolated skeletal muscle mitochondria
Proband Control range a
Pyruvate þ malate (nmol O/min £ mg protein)
Succinate þ rotenone (nmol O/min £ mg protein)
Ascorbate þ TMPDa (nmol O/min £ mg protein)
COX (rate constant 1/min £ mg protein)
NADH ferricyanide reductase (mmol/min £ mg protein)
Succinate cytochrome-c reductase (mmol/min £ mg protein)
Citrate synthase (mmol/min £ mg protein)
84 95–118
86 98– 151
211 214 –339
3.9 7.7–11.3
4.2 3.1–8.5
0.19 0.21–31
3.0 1.3–2.5
TMPD, N; N; N 0 ; N 0 -tetra methyl-p-phenylenediamine.
48
A.-R. Moslemi et al. / Neuromuscular Disorders 14 (2004) 46–50
3.3. Analysis of mtDNA
Fig. 2. Sequence analysis of the mitochondrial tRNA genes in patient’s muscle homogenate showing a heteroplasmic T582C mutation.
homogenate and single muscle fibers, polymerase chain reaction (PCR) was performed using a forward (F) primer corresponding to nt 511– 530 and a backward (B) primer corresponding to nt 780– 761 (sequence numbering according to the revised Cambridge sequence) [9]. The lysate of one single muscle fiber segment or 50 ng DNA from muscle homogenate was added to the master mixture (ReddyMix PCR Master Mix; ABgene, Epsom, UK). The PCR amplification was performed for 35 cycles including denaturation at 95 8C for 30 s, primer annealing at 57 8C for 30 s and primer extension at 72 8C for 30 s. Before the last cycle,10 pmol fluorescin-labelled (6-FAM) F primer was added. After digestion of the PCR amplified fragment with 5 units SnabI, which cleaved the mutated mtDNA, the relative percentages of mutated and wild-type mtDNA were calculated using GeneScan software (Applied Biosystem, CA) as described previously [10].
3. Results
LX-PCR analysis excluded large rearrangements in the mtDNA of our patient. Sequence analysis of the mitochondrial tRNA genes revealed a heteroplasmic T582C substitution mutation in the aminoacyl acceptor stem of tRNAPhe (Fig. 2). The proportion of mutated mtDNA was 70% in muscle homogenate. The distribution of mtDNA with the tRNAPhe T582C mutation was studied by single fiber PCR analysis of COX-negative and COX-positive muscle fibers. The levels of mutated mtDNA in single muscle fibers are presented in Fig. 3. The average level of the T582C mutation was 86% (ranging from 75 to 95%) in ten COXnegative muscle fibers and 15% (ranging from 0 to 55%) in ten COX-positive fibers. PCR-RFLP analysis using SnabI showed that the mutation was not detected in blood leucocytes or hair shafts of the patient. Neither was it detected in mtDNA extracted from muscle tissue of the patient’s daughter, nor in the blood leucocytes of 100 healthy controls.
4. Discussion Numerous mutations in mitochondrial tRNA genes have been described in association with diverse clinical phenotypes. Some tRNA genes appear to be frequently affected, such as the tRNAleu(UUR) gene. Four different mutations in the tRNAPhe have been described. They were associated with tubulointerstitial nephritis and stroke [11], mitochondrial myopathy [12], acute rhabdomyolysis [13] and mitochondrial encephalomyopathy with lactic acidosis and stroke-like episodes (MELAS) [14], respectively. The latter was a heteroplasmic G583A point mutation within the aminoacyl acceptor stem of the tRNAPhe. Here we report a fifth point mutation in tRNAPhe. The mutation fulfils accepted criteria for pathogenicity. First, the mutation disrupts a conserved Watson – Crick T-A base pairing
3.1. Muscle morphology Enzyme-histochemical examination of the patient’s muscle biopsy specimens showed reduced COX activity in the majority of the muscle fibers and numerous ragged red fibers (Fig. 1a). Immuno-histochemical analysis of COX subunit II and IV showed reduced expression of COX II and increased expression of COX IV in COX deficient ragged red fibers (Fig. 1b and c). The muscle biopsy of the patient’s daughter was normal. 3.2. Biochemical analysis The results from biochemical analysis of muscle mitochondria are shown in Table 1. There was a slight general reduction of the respiratory rate and marked reduction of the COX activity.
Fig. 3. Result of single fiber analysis of the T582C mutation in COXnegative and COX-positive muscle fibers. The proportion of the mutation in COX-negative muscle fibers is significantly higher than in COX-positive muscle fibers indicating that a high level of the mutation is associated with respiratory chain deficiency in single fibers.
A.-R. Moslemi et al. / Neuromuscular Disorders 14 (2004) 46–50
49
for a causal association with the T582C mutation. Lack of family history of neuromuscular disease suggests that our patient was a sporadic case. The marked difference in mutant load between muscle and blood has been demonstrated in some other sporadic cases of mtDNA mutation disorders [17 –20]. This different pattern of segregation of pathogenic mtDNA mutations in post-mitotic tissues and tissues composed of rapidly dividing cells could be explained by the appearance of such mutations during oogenesis or early embryogenesis and random genetic drift [20]. The novel tRNAPhe mutation described here further underlines the role of mtDNA tRNA mutations as a cause of mitochondrial myopathy and provides new insights in the pathogenesis of mitochondrial diseases.
Acknowledgements This study was supported by grants from the Swedish Medical Research Council (Project no. 7122 and 10823).
References
Fig. 4. Schematic illustration of the mitochondrial tRNAPhe. (a) Cloverleaf structure, showing the T582C mutation in the aminoacyl acceptor stem. (b) Sequence comparison of 50 and 30 aminoacyl acceptor stem of tRNA among different species, demonstrating that the base pairing at position 582 is strictly conserved.
within the aminoacyl acceptor stem of the tRNA (Fig. 4). This disruption may affect the stability of the acceptor stem hairpin structure [15,16] leading to an overall secondary structure alteration of the tRNAPhe molecule. Second, the mutation was heteroplasmic and the proportion of the mutated mtDNA in COX-negative muscle fibers was significantly higher than in COX-positive muscle fibers. The threshold level of the mutation for COX-deficiency appears to be between 55 and 75% because the lowest percentage of the mutation in COX-deficient muscle fibers was 75% and the highest proportion in COX-positive fibers was 55%. Third, the mutation was not described previously as a neutral polymorphism and was absent in 100 controls. Fourth the mutation was not identified in the patient’s daughter who had normal muscle biopsy. Results from neuropsychological tests and MRI showed that our patient suffered from encephalopathy, which may be due to mitochondrial dysfunction, but there is no definite evidence
[1] Anderson S, Bankier AT, Barrell BG, de Bruijn MH, Coulson AR, Drouin J, et al. Sequence and organization of the human mitochondrial genome. Nature 1981;290:457 –65. [2] Larsson NG, Oldfors A. Mitochondrial myopathies. Acta Physiol Scand 2001;17:385–93. [3] Mitomap: a human mitochondrial genom database. Atlanta, GA, USA: Center for Molecular Medicine, Emory University; 2002, http:// www.mitomap.org. [4] Nashef L, Lane RJ. Screening for mitochondrial cytopathies: the subanaerobic threshold exercise test (SATET). J Neurol Neurosurg Psychiatry 1989;52:1090 –4. [5] Oldfors A, Moslemi AR, Fyhr IM, Holme E, Larsson NG, Lindberg C. Mitochondrial DNA deletions in muscle fibers in inclusion body myositis. J Neuropathol Exp Neurol 1995;54:581–7. [6] Tulinius MH, Holme E, Kristiansson B, Larsson NG, Oldfors A. Mitochondrial encephalomyopathies in childhood. I. Biochemical and morphologic investigations. J Pediatr 1991;119:242–50. [7] Moslemi AR, Selimovic N, Bergh CH, Oldfors A. Fatal dilated cardiomyopathy associated with a mitochondrial DNA deletion. Cardiology 2000;94:68–71. [8] Houshmand M, Larsson NG, Holme E, Oldfors A, Tulinius MH, Andersen O. Automatic sequencing of mitochondrial tRNA genes in patients with mitochondrial encephalomyopathy. Biochim Biophys Acta 1994;1226:49 –55. [9] Andrews RM, Kubacka I, Chinnery PF, Lightowlers RN, Turnbull DM, Howell N. Reanalysis and revision of the Cambridge reference sequence for human mitochondrial DNA. Nat Genet 1999;23:147. [10] Moslemi AR, Tulinius M, Holme E, Oldfors A. Threshold expression of the tRNA(Lys) A8344G mutation in single muscle fibres. Neuromuscul Disord 1998;8:345– 9. [11] Tzen CY, Tsai JD, Wu TY, Chen BF, Chen ML, Lin SP, et al. Tubulointerstitial nephritis associated with a novel mitochondrial point mutation. Kidney Int 2001;59:846–54. [12] Kleinle S, Schneider V, Moosmann P, Brandner S, Krahenbuhl S, Liechti-Gallati S. A novel mitochondrial tRNA(Phe) mutation
50
[13]
[14]
[15]
[16]
A.-R. Moslemi et al. / Neuromuscular Disorders 14 (2004) 46–50 inhibiting anticodon stem formation associated with a muscle disease. Biochem Biophys Res Commun 1998;247:112–5. Chinnery PF, Johnson MA, Taylor RW, Lightowlers RN, Turnbull DM. A novel mitochondrial tRNA phenylalanine mutation presenting with acute rhabdomyolysis. Ann Neurol 1997;41:408 –10. Hanna MG, Nelson IP, Morgan-Hughes JA, Wood NW. MELAS: a new disease associated mitochondrial DNA mutation and evidence for further genetic heterogeneity. J Neurol Neurosurg Psychiatry 1998; 65:512–7. Tiranti V, D’Agruma L, Pareyson D, Mora M, Carrara F, Zelante L, et al. A novel mutation in the mitochondrial tRNA(Val) gene associated with a complex neurological presentation. Ann Neurol 1998;43:98 –101. Yasukawa T, Hino N, Suzuki T, Watanabe K, Ueda T, Ohta S. A pathogenic point mutation reduces stability of mitochondrial mutant tRNA(Ile). Nucleic Acids Res 2000;28:3779 –84.
[17] Moraes CT, Ciacci F, Bonilla E, Ionasescu V, Schon EA, DiMauro S. A mitochondrial tRNA anticodon swap associated with a muscle disease. Nat Genet 1993;4:284–8. [18] Moraes CT, Ciacci F, Bonilla E, Jansen C, Hirano M, Rao N, et al. Two novel pathogenic mitochondrial DNA mutations affecting organelle number and protein synthesis. Is the tRNA(Leu(UUR)) gene an etiologic hot spot? J Clin Invest 1993;92:2906–15. [19] Nishigaki Y, Bonilla E, Shanske S, Gaskin DA, DiMauro S, Hirano M. Exercise-induced muscle ‘burning,’ fatigue, and hyper-CKemia: mtDNA T10010C mutation in tRNA(Gly). Neurology 2002;58: 1282–5. [20] Fu K, Hartlen R, Johns T, Genge A, Karpati G, Shoubridge EA. A novel heteroplasmic tRNAleu(CUN) mtDNA point mutation in a sporadic patient with mitochondrial encephalomyopathy segregates rapidly in skeletal muscle and suggests an approach to therapy. Hum Mol Genet 1996;5:1835–40.
A novel mutation in the mitochondrial tRNAPhe gene associated with mitochondrial myopathy A.-R. Moslemia,*, C. Lindbergb, J. Toftc, E. Holmed, G. Kollberga, A. Oldforsa a
Department of Pathology, Sahlgrenska University Hospital, 41345 Go¨teborg, Sweden Department of Neurology, Sahlgrenska University Hospital, 41345 Go¨teborg, Sweden c Department of Neurology, South Elfsborg Hospital, 41345 Bora˚s, Sweden d Department of Clinical Chemistry, Sahlgrenska University Hospital, 41345 Go¨teborg, Sweden b
Received 20 March 2003; received in revised form 1 July 2003; accepted 10 July 2003
Abstract We report a novel heteroplasmic T ! C mutation at nt position 582 within the mitochondrial tRNAPhe gene of a 70-year-old woman with mitochondrial myopathy. No other family members were affected, suggesting that our patient was a sporadic case. The muscle showed frequent ragged red fibers and 43% cytochrome c oxidase deficient fibers. The mutation alters a conserved base pairing in the aminoacyl acceptor stem. The mutation load was 70% in muscle homogenate and varied from 0 to 95% in individual muscle fiber segments. Cytochrome c oxidase-negative fibers showed significantly higher levels of mutated mtDNA (.75%) than Cytochrome c oxidase-positive fibers ð, 55%Þ: This mutation adds to the previously described four pathogenic mutations in the tRNAPhe gene. q 2003 Elsevier B.V. All rights reserved. Keywords: Myopathy; Cytochrome c oxidase deficiency; tRNA; Mutation; mtDNA
1. Introduction
2. Material and methods
The human mitochondrial DNA (mtDNA) is a circular double-stranded molecule of 16.6 kb, which encodes 13 subunits of the respiratory chain enzymes, 22 tRNA and 2 rRNA genes [1]. Mutations in mtDNA have been reported to be a major cause of mitochondrial myopathy [2]. To date more than 100 pathogenic mtDNA tRNA gene mutations have been reported in association with a wide spectrum of clinical manifestations [3]. Pathogenic point mutations in tRNA genes are usually associated with cytochrome c oxidase (COX) deficiency and abnormal proliferation of mitochondria in muscle tissue. The majority of pathogenic tRNA mutations are transmitted maternally and sporadic cases are rare. In this study we report a novel heteroplasmic mutation in the aminoacyl acceptor stem region of tRNAPhe gene in muscle tissue of a 70-year-old woman with mitochondrial myopathy and encephalopathy.
2.1. Case report
* Corresponding author. Tel: þ 46-31-3423335; fax: þ 46-31-417283. E-mail address: [email protected] (A.-R. Moslemi). 0960-8966/$ - see front matter q 2003 Elsevier B.V. All rights reserved. doi:10.1016/S0960-8966(03)00168-8
The patient was a 70-year-old woman, who had experienced slowly progressive bilateral ptosis from age 50. She had had slightly impaired balance during her entire life. Her mother, her only sister and two of her three children were free of neuromuscular symptoms. Her youngest daughter, aged 39 had generalized muscle pain compatible with the fibromyalgia syndrome and no signs of muscle disease. At age 67 the patient was operated upon because of atrio-ventricular re-entry tachycardia. One year later she experienced weakness in her legs, muscle fatigue and soreness after moderate physical activity. Neurological examination at age 70 showed bilateral ptosis, and impaired vertical and horizontal eye movements. There was slight to moderate weakness in neck flexion, hip flexion and knee extension. She was slower than expected in repetitive hand and feet movements, but had no change in muscle tone. There was no limb ataxia. Tendon reflexes were normal, and the Babinski sign was absent. Laboratory investigations revealed increased plasma lactate both at rest 2.8 (normal , 1.7 mmol/l) and after
A.-R. Moslemi et al. / Neuromuscular Disorders 14 (2004) 46–50
47
Fig. 1. Serial sections of skeletal muscle. (a) Enzyme-histochemical staining of COX and succinate dehydrogenase. Cytochrome c oxidase deficient fibers are blue, while normal fibers are brown. The arrow indicates one of the COX-deficient ragged red fibers. (b) Immuno-histochemical demonstration of COX subunit II. The arrow indicates one of the COX-deficient ragged red fibers, which shows reduced expression of COX II. (c) Immuno-histochemical demonstration of COX subunit IV. The arrow indicates one of the COX-deficient ragged red fibers, which shows increased expression of COX IV.
A/S, Danmark), and Texas Rede-conjugated sheep-antimouse antibodies (Amersham Biosciences, Sweden).
a subanaerobic threshold exercise test [4]. Urinary lactate excretion was 29 mmol/mmol creatinine (normal , 20). Cerebrospinal fluid (CSF) lactate was elevated to 2.1 mmol/l (normal , 1.8). CSF albumin was normal but CSF Tau was elevated to 417 ng/l (normal , 400). MRI of the brain showed small bilateral lesions compatible with ischemic areas. Neuropsychological tests revealed impaired memory for verbal material as well as reduced visual attention, confirming the existence of an encephalopathy.
2.3. Biochemical analysis Isolation of mitochondria, oximetric measurements of fresh mitochondria, and spectrophotometric enzyme analyses were performed as previously described [6]. 2.4. Analysis of mtDNA
2.2. Morphological analysis of muscle Total DNA was extracted from frozen skeletal muscle tissue or peripheral blood leucocytes using the DNA Extraction Kit (Qiagen, Hilden, Germany) according to the manufacturer’s instructions. The entire genome of mtDNA was amplified by a long expand polymerase chain reaction (LX-PCR) using two sets of primer pairs as previously described [7]. Point mutations were searched for in the 22 mitochondrial tRNA genes by direct sequencing of the purified PCR products as previously described [8]. Single COX-deficient and normal muscle fibers were dissected as previously described [5]. To analyze the proportion of the A582G mutation in muscle
Open muscle biopsy specimens were obtained from the proband and her 39-year-old daughter with fibromyalgia. Morphological and enzyme-histochemical analysis were performed as previously described [5]. Immuno-histochemical staining of COX subunit II and IV was performed by incubating 8-mm thick cryostat sections with a mixture of polyclonal rabbit antibodies directed towards COX subunit II (a generous gift from A. Lombes) and mouse monoclonal antibodies towards COX subunit IV (a generous gift from DiMauro). As secondary antibodies we used FITC-conjugated swine-anti-rabbit antibodies (DAKO Table 1 Respiratory rates and enzyme activities in isolated skeletal muscle mitochondria
Proband Control range a
Pyruvate þ malate (nmol O/min £ mg protein)
Succinate þ rotenone (nmol O/min £ mg protein)
Ascorbate þ TMPDa (nmol O/min £ mg protein)
COX (rate constant 1/min £ mg protein)
NADH ferricyanide reductase (mmol/min £ mg protein)
Succinate cytochrome-c reductase (mmol/min £ mg protein)
Citrate synthase (mmol/min £ mg protein)
84 95–118
86 98– 151
211 214 –339
3.9 7.7–11.3
4.2 3.1–8.5
0.19 0.21–31
3.0 1.3–2.5
TMPD, N; N; N 0 ; N 0 -tetra methyl-p-phenylenediamine.
48
A.-R. Moslemi et al. / Neuromuscular Disorders 14 (2004) 46–50
3.3. Analysis of mtDNA
Fig. 2. Sequence analysis of the mitochondrial tRNA genes in patient’s muscle homogenate showing a heteroplasmic T582C mutation.
homogenate and single muscle fibers, polymerase chain reaction (PCR) was performed using a forward (F) primer corresponding to nt 511– 530 and a backward (B) primer corresponding to nt 780– 761 (sequence numbering according to the revised Cambridge sequence) [9]. The lysate of one single muscle fiber segment or 50 ng DNA from muscle homogenate was added to the master mixture (ReddyMix PCR Master Mix; ABgene, Epsom, UK). The PCR amplification was performed for 35 cycles including denaturation at 95 8C for 30 s, primer annealing at 57 8C for 30 s and primer extension at 72 8C for 30 s. Before the last cycle,10 pmol fluorescin-labelled (6-FAM) F primer was added. After digestion of the PCR amplified fragment with 5 units SnabI, which cleaved the mutated mtDNA, the relative percentages of mutated and wild-type mtDNA were calculated using GeneScan software (Applied Biosystem, CA) as described previously [10].
3. Results
LX-PCR analysis excluded large rearrangements in the mtDNA of our patient. Sequence analysis of the mitochondrial tRNA genes revealed a heteroplasmic T582C substitution mutation in the aminoacyl acceptor stem of tRNAPhe (Fig. 2). The proportion of mutated mtDNA was 70% in muscle homogenate. The distribution of mtDNA with the tRNAPhe T582C mutation was studied by single fiber PCR analysis of COX-negative and COX-positive muscle fibers. The levels of mutated mtDNA in single muscle fibers are presented in Fig. 3. The average level of the T582C mutation was 86% (ranging from 75 to 95%) in ten COXnegative muscle fibers and 15% (ranging from 0 to 55%) in ten COX-positive fibers. PCR-RFLP analysis using SnabI showed that the mutation was not detected in blood leucocytes or hair shafts of the patient. Neither was it detected in mtDNA extracted from muscle tissue of the patient’s daughter, nor in the blood leucocytes of 100 healthy controls.
4. Discussion Numerous mutations in mitochondrial tRNA genes have been described in association with diverse clinical phenotypes. Some tRNA genes appear to be frequently affected, such as the tRNAleu(UUR) gene. Four different mutations in the tRNAPhe have been described. They were associated with tubulointerstitial nephritis and stroke [11], mitochondrial myopathy [12], acute rhabdomyolysis [13] and mitochondrial encephalomyopathy with lactic acidosis and stroke-like episodes (MELAS) [14], respectively. The latter was a heteroplasmic G583A point mutation within the aminoacyl acceptor stem of the tRNAPhe. Here we report a fifth point mutation in tRNAPhe. The mutation fulfils accepted criteria for pathogenicity. First, the mutation disrupts a conserved Watson – Crick T-A base pairing
3.1. Muscle morphology Enzyme-histochemical examination of the patient’s muscle biopsy specimens showed reduced COX activity in the majority of the muscle fibers and numerous ragged red fibers (Fig. 1a). Immuno-histochemical analysis of COX subunit II and IV showed reduced expression of COX II and increased expression of COX IV in COX deficient ragged red fibers (Fig. 1b and c). The muscle biopsy of the patient’s daughter was normal. 3.2. Biochemical analysis The results from biochemical analysis of muscle mitochondria are shown in Table 1. There was a slight general reduction of the respiratory rate and marked reduction of the COX activity.
Fig. 3. Result of single fiber analysis of the T582C mutation in COXnegative and COX-positive muscle fibers. The proportion of the mutation in COX-negative muscle fibers is significantly higher than in COX-positive muscle fibers indicating that a high level of the mutation is associated with respiratory chain deficiency in single fibers.
A.-R. Moslemi et al. / Neuromuscular Disorders 14 (2004) 46–50
49
for a causal association with the T582C mutation. Lack of family history of neuromuscular disease suggests that our patient was a sporadic case. The marked difference in mutant load between muscle and blood has been demonstrated in some other sporadic cases of mtDNA mutation disorders [17 –20]. This different pattern of segregation of pathogenic mtDNA mutations in post-mitotic tissues and tissues composed of rapidly dividing cells could be explained by the appearance of such mutations during oogenesis or early embryogenesis and random genetic drift [20]. The novel tRNAPhe mutation described here further underlines the role of mtDNA tRNA mutations as a cause of mitochondrial myopathy and provides new insights in the pathogenesis of mitochondrial diseases.
Acknowledgements This study was supported by grants from the Swedish Medical Research Council (Project no. 7122 and 10823).
References
Fig. 4. Schematic illustration of the mitochondrial tRNAPhe. (a) Cloverleaf structure, showing the T582C mutation in the aminoacyl acceptor stem. (b) Sequence comparison of 50 and 30 aminoacyl acceptor stem of tRNA among different species, demonstrating that the base pairing at position 582 is strictly conserved.
within the aminoacyl acceptor stem of the tRNA (Fig. 4). This disruption may affect the stability of the acceptor stem hairpin structure [15,16] leading to an overall secondary structure alteration of the tRNAPhe molecule. Second, the mutation was heteroplasmic and the proportion of the mutated mtDNA in COX-negative muscle fibers was significantly higher than in COX-positive muscle fibers. The threshold level of the mutation for COX-deficiency appears to be between 55 and 75% because the lowest percentage of the mutation in COX-deficient muscle fibers was 75% and the highest proportion in COX-positive fibers was 55%. Third, the mutation was not described previously as a neutral polymorphism and was absent in 100 controls. Fourth the mutation was not identified in the patient’s daughter who had normal muscle biopsy. Results from neuropsychological tests and MRI showed that our patient suffered from encephalopathy, which may be due to mitochondrial dysfunction, but there is no definite evidence
[1] Anderson S, Bankier AT, Barrell BG, de Bruijn MH, Coulson AR, Drouin J, et al. Sequence and organization of the human mitochondrial genome. Nature 1981;290:457 –65. [2] Larsson NG, Oldfors A. Mitochondrial myopathies. Acta Physiol Scand 2001;17:385–93. [3] Mitomap: a human mitochondrial genom database. Atlanta, GA, USA: Center for Molecular Medicine, Emory University; 2002, http:// www.mitomap.org. [4] Nashef L, Lane RJ. Screening for mitochondrial cytopathies: the subanaerobic threshold exercise test (SATET). J Neurol Neurosurg Psychiatry 1989;52:1090 –4. [5] Oldfors A, Moslemi AR, Fyhr IM, Holme E, Larsson NG, Lindberg C. Mitochondrial DNA deletions in muscle fibers in inclusion body myositis. J Neuropathol Exp Neurol 1995;54:581–7. [6] Tulinius MH, Holme E, Kristiansson B, Larsson NG, Oldfors A. Mitochondrial encephalomyopathies in childhood. I. Biochemical and morphologic investigations. J Pediatr 1991;119:242–50. [7] Moslemi AR, Selimovic N, Bergh CH, Oldfors A. Fatal dilated cardiomyopathy associated with a mitochondrial DNA deletion. Cardiology 2000;94:68–71. [8] Houshmand M, Larsson NG, Holme E, Oldfors A, Tulinius MH, Andersen O. Automatic sequencing of mitochondrial tRNA genes in patients with mitochondrial encephalomyopathy. Biochim Biophys Acta 1994;1226:49 –55. [9] Andrews RM, Kubacka I, Chinnery PF, Lightowlers RN, Turnbull DM, Howell N. Reanalysis and revision of the Cambridge reference sequence for human mitochondrial DNA. Nat Genet 1999;23:147. [10] Moslemi AR, Tulinius M, Holme E, Oldfors A. Threshold expression of the tRNA(Lys) A8344G mutation in single muscle fibres. Neuromuscul Disord 1998;8:345– 9. [11] Tzen CY, Tsai JD, Wu TY, Chen BF, Chen ML, Lin SP, et al. Tubulointerstitial nephritis associated with a novel mitochondrial point mutation. Kidney Int 2001;59:846–54. [12] Kleinle S, Schneider V, Moosmann P, Brandner S, Krahenbuhl S, Liechti-Gallati S. A novel mitochondrial tRNA(Phe) mutation
50
[13]
[14]
[15]
[16]
A.-R. Moslemi et al. / Neuromuscular Disorders 14 (2004) 46–50 inhibiting anticodon stem formation associated with a muscle disease. Biochem Biophys Res Commun 1998;247:112–5. Chinnery PF, Johnson MA, Taylor RW, Lightowlers RN, Turnbull DM. A novel mitochondrial tRNA phenylalanine mutation presenting with acute rhabdomyolysis. Ann Neurol 1997;41:408 –10. Hanna MG, Nelson IP, Morgan-Hughes JA, Wood NW. MELAS: a new disease associated mitochondrial DNA mutation and evidence for further genetic heterogeneity. J Neurol Neurosurg Psychiatry 1998; 65:512–7. Tiranti V, D’Agruma L, Pareyson D, Mora M, Carrara F, Zelante L, et al. A novel mutation in the mitochondrial tRNA(Val) gene associated with a complex neurological presentation. Ann Neurol 1998;43:98 –101. Yasukawa T, Hino N, Suzuki T, Watanabe K, Ueda T, Ohta S. A pathogenic point mutation reduces stability of mitochondrial mutant tRNA(Ile). Nucleic Acids Res 2000;28:3779 –84.
[17] Moraes CT, Ciacci F, Bonilla E, Ionasescu V, Schon EA, DiMauro S. A mitochondrial tRNA anticodon swap associated with a muscle disease. Nat Genet 1993;4:284–8. [18] Moraes CT, Ciacci F, Bonilla E, Jansen C, Hirano M, Rao N, et al. Two novel pathogenic mitochondrial DNA mutations affecting organelle number and protein synthesis. Is the tRNA(Leu(UUR)) gene an etiologic hot spot? J Clin Invest 1993;92:2906–15. [19] Nishigaki Y, Bonilla E, Shanske S, Gaskin DA, DiMauro S, Hirano M. Exercise-induced muscle ‘burning,’ fatigue, and hyper-CKemia: mtDNA T10010C mutation in tRNA(Gly). Neurology 2002;58: 1282–5. [20] Fu K, Hartlen R, Johns T, Genge A, Karpati G, Shoubridge EA. A novel heteroplasmic tRNAleu(CUN) mtDNA point mutation in a sporadic patient with mitochondrial encephalomyopathy segregates rapidly in skeletal muscle and suggests an approach to therapy. Hum Mol Genet 1996;5:1835–40.