- Email: [email protected]
Novel mutations in the TK2 gene associated with fatal mitochondrial DNA depletion myopathy
Neuromuscular Disorders 18 (2008) 557–560 www.elsevier.com/locate/nmd
Case report
Novel mutations in the TK2 gene associated with fatal mitochondrial DNA depletion myopathy Emma Blakely a, Langping He a, Julie L. Gardner a, Gavin Hudson a, John Walter b, Imelda Hughes c, Douglass M. Turnbull a,d, Robert W. Taylor a,d,* a
Mitochondrial Research Group, The Medical School, Newcastle University, Newcastle Upon Tyne NE2 4HH, UK b Willink Biochemical Genetics Unit, Royal Manchester Children’s Hospital, Manchester, UK c Department of Neurology, Royal Manchester Children’s Hospital, Manchester, UK d Institutes of Human Genetics and Neuroscience, Newcastle University, Newcastle Upon Tyne, UK Received 21 January 2008; received in revised form 31 March 2008; accepted 14 April 2008
Abstract Mitochondrial DNA depletion syndromes are a heterogeneous group of childhood neurological disorders characterised by a quantitative abnormality of mitochondrial DNA. We describe two siblings who presented at 8 months and 14 months with myopathy, which rapidly progressed and resulted in death by respiratory failure at age 14 and 18 months, respectively. Muscle biopsy revealed marked respiratory chain defects, with real-time PCR confirming a dramatic depletion of mitochondrial DNA. Sequencing of the thymidine kinase 2 (TK2) gene revealed two, novel heterozygous mutations (p.Q87X and p.N100S) with parental DNA analysis confirming the transmission of mutated alleles. Ó 2008 Elsevier B.V. All rights reserved. Keywords: Mitochondrial DNA; Myopathy; Depletion syndrome; TK2 mutations
1. Introduction Mitochondrial DNA (mtDNA) depletion syndromes are autosomal recessive disorders of infancy or childhood, characterised by a quantitative decrease in mtDNA copy number in affected organs [1]. Mutations in several nuclear genes, the protein products of which play key roles in maintaining mitochondrial DNA integrity or the intramitochondrial nucleotide pool, have been associated with a spectrum of clinical presentations. Mutations in DGUOK, POLG1, SUCLA2, MPV17 – and more recently PEO1 – have all been shown to cause a hepatocerebral form of mtDNA depletion syndrome leading to encephalopathy and fatal, infantile liver disease including Alpers syndrome *
Corresponding author. Address: Mitochondrial Research Group, The Medical School, Newcastle University, Newcastle Upon Tyne NE2 4HH, UK. Tel.: +44 1912223685; fax: +44 1912228553. E-mail address: [email protected] (R.W. Taylor). 0960-8966/$ - see front matter Ó 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.nmd.2008.04.014
([2] and references within). Mutations in the thymidine kinase 2 (TK2) gene cause a progressive, myopathic form of mtDNA depletion syndrome [3], with some patients demonstrating clinical features including variable brain involvement, ptosis, ophthalmoplegia, nephropathy and optic neuropathy [3–7]. More recently, mutations in RRM2B, encoding the small subunit of p53-inducible ribonucleotide reductase, have also been identified in patients with myopathic mtDNA depletion disorder [8]. Here we report the clinical, biochemical and molecular genetic investigations of a Caucasian family from the UK in which two siblings had died in early infancy with mitochondrial DNA depletion myopathy due to novel heterozygous TK2 gene mutations. 2. Case report and methods The male index case (II-4) was the 4th child born to healthy, non-consanguineous Caucasian parents (Fig. 1).
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E. Blakely et al. / Neuromuscular Disorders 18 (2008) 557–560
a
b I:1
II:1
II:2 II:3
c
d
I:2
II:4 II:5
e
f 82
c.259C>T, (Q87X)
*
c.299A>G, (N100S)
*
H. sapiens p.Q87X p.N100S P. troglodytes M. musculus R. norvegicus
104
PPDKEQE--KEKKSVICVEGNIASG PPDKE* PPDKEQE--KEKKSVICVEGSIASG PPDKEQE--KEKKSVICVEGNIASG PPDKDRENDKEKKAVVCIEGNIASG PPDKDREKDKEKKAVVCIEGNIASG
Fig. 1. Analysis of novel TK2 mutations. (a) Pedigree of the family highlighting the index case (arrow). Transverse-orientated muscle sections from the index case were stained or reacted for (b) H&E, (c) succinate dehydrogenase (SDH) activity and (d) cytochrome c oxidase (COX) activity, revealing marked evidence of muscle regeneration and inflammatory cell infiltrate, with an increased staining of some hypertrophied fibres for succinate dehydrogenase activity. Approximately 50% of all fibres were deficient in histocytochemical COX activity. (e) Sequencing electropherograms highlighting the presence of recessive p.Q87X and p.N100S mutations in patient II-4, the index case. (f) Multiple sequence alignment of human TK2 protein with that from other mammalian species, highlighting the amino acid conservation in this region of the protein. The mutated amino acids (p.Q87X and p.N100S) are highlighted in bold.
He presented at the age of 14 months with a 1 month history of poor head control, reduced feeding and increasing difficulty with crawling and sitting. His past history was otherwise unremarkable and his early development had been normal. On examination, growth was normal with a height, weight and head circumference on the 50th centile. He was alert with normal eye movements and no ptosis. He had no facial weakness but had marked head lag, generalised muscle weakness and absent reflexes. His serum creatine kinase was raised at 802 IU/l (normal < 300 IU/l), but lactate and pyruvate levels were both normal. He was commenced on oral ubiquinone (30 mg tds), but there was progressive worsening of his myopathy over the next 4 months and he died at home at the age of 18 months. The parents have three healthy daughters who are alive and well (ages 12 years, 9 years and 2 years), but their first child (II-1) had presented in a similar manner at 8 months of age and died from respiratory failure aged 14 months. Prior to her death, she was found to have normal cardiac structure and function but a raised serum creatine kinase and abnormal liver function tests; blood and CSF lactate were normal but a muscle biopsy had suggested abnormal mitochondria and a generalised decrease in the activities of respiratory chain complexes I, III and IV. A screen at that time for mtDNA rearrangements and common mtDNA point mutations revealed no obvious abnormalities but no further tissue was available for analysis. Standard histological and histochemical analyses were performed on frozen sections (10 lm) of quadriceps muscle. Muscle biopsy of the index case demonstrated a variation in fibre size (6–45 lm), with evidence of degenerate fibres associating with inflammatory cell infiltrate. Some hypertrophied fibres had increased staining for succinate dehydrogenase activity, and approximately 50% of all
fibres were cytochrome c oxidase-deficient (Fig. 1). Ultrastructural studies showed fibres with abnormally shaped mitochondria and focal increase in lipid. The activities of the respiratory chain complexes and the matrix marker citrate synthase were determined in a muscle homogenate, revealing a severe decrease in the activities of respiratory chain complexes I and IV and a normal activity of complex II, suggestive of mtDNA involvement (Table 1). Total DNA was extracted from muscle and used to screen for mtDNA rearrangements by long-range PCR, whilst a screen for a maternally inherited mtDNA point mutation was undertaken by sequencing the entire mitochondrial genome. No abnormalities were detected. Assessment of mtDNA copy number was by a quantitative, real-time PCR assay using an ABI PRISM 7000 sequence detection system (Foster City, CA), and primers and fluorogenic probes to amplify regions of the mtDNA-encoded MTND1 gene and the nuclear-encoded 18S rRNA gene; the efficiency of PCR amplifications and validation of this assay was performed as previously described [9]. Real-time
Table 1 Respiratory chain complex activities in skeletal muscle homogenate from the index case (patient II-4) Complex
Controls (n = 20; mean ± SD)
Patient
Complex I/CS Complex II/CS Complex IV/CS
0.166 ± 0.04 0.208 ± 0.070 1.805 ± 0.550
0.006 0.159 0.121
Enzyme activities are expressed as nmol NADH oxidised min 1 unit citrate synthase 1 for complex I, nmol DCPIP reduced min 1 unit citrate synthase 1 for complex II (succinate:ubiquinone-1 reductase) and the apparent first-order rate constant s 1 unit citrate synthase 1 for complex IV (103). DCPIP = 2,6-dichlorophenol-indophenol; SD = standard deviation.
E. Blakely et al. / Neuromuscular Disorders 18 (2008) 557–560
PCR analysis demonstrated a marked depletion of mtDNA copy number in skeletal muscle from patient II-4, with mtDNA levels at <5% of aged-match controls. We amplified the 10 exons of the TK2 gene using intronic primers [4] and directly compared these to the recently updated reference sequence of human TK2 (GenBank Accession No.: NM_004614). Sequencing analysis of the entire TK2 gene coding region in patient II-4 revealed two novel, heterozygous changes – a c.259C>T transition in exon 2 and a c.299A>G transition in exon 3 predicting nonsense (p.Q87X) and missense (p.N100S) mutations, respectively (Fig. 1). Subsequent analysis of blood DNA from the patient’s mother and father confirmed transmission of both mutated alleles, c.259C>T from the father and c.299A>G from the mother. During our investigation of patient II-4, the mother conceived a fifth pregnancy, giving birth to another daughter (II-5). Presymptomatic genetic testing was requested by the parents at 5 months of age, which happily failed to identify either of the causative TK2 mutations. 3. Discussion Mitochondrial depletion syndromes are autosomal recessive disorders characterised by a reduction in mitochondrial DNA copy number in clinically affected tissues. A balanced supply of deoxyribonucleoside triphosphates (dNTPs) is required for DNA polymerase c to faithfully replicate the mitochondrial genome, the mitochondrial dNTP pool being maintained both through the import of cytosolic dNTPs and by salvaging deoxyribonucleosides within the organelle itself. A number of nuclear-encoded enzymes are involved in these processes, including thymidine kinase 1 (TK1) within the cytosol, and thymidine kinase 2 (TK2) and deoxyguanosine kinase (dGK) within mitochondria, both key enzymes of the mitochondrial salvage pathway. TK2 is a deoxyribonucleoside kinase which is responsible for the phosphorylation of deoxythymidine, deoxycytidine and deoxyuridine [3]. Since the first description of TK2 mutations leading to the myopathic form of mtDNA depletion syndrome in 2001, approximately 20 different pathogenic mutations have now been reported, distributed throughout the TK2 coding sequence (recently reviewed in [5]). The salient clinical and laboratory findings in 20 patients with TK2 mutations have recently been described in detail [5]. Similar to our patients, all affected children presented with proximal weakness and hypotonia with a mean age at onset of 11.4 months. Death in nearly 90% of cases was due to respiratory failure, at a mean age of 42.1 months – somewhat later than the two affected siblings reported here. As noted by these authors, the clinical spectrum also includes spinal muscular atrophy, rigid spine syndrome and severe myopathy with motor regression. Screening of the SMN1 gene was not considered in the index case given the investigation of an affected sibling (II-1) had strongly suggested a mitochondrial aetiology.
559
Similar to other reported patients, our case also showed an elevated serum creatine kinase level and both COX-deficient fibres and SDH-hypereactive fibres on muscle biopsy. Whilst a decrease in muscle mtDNA copy number should indicate screening of the TK2 gene, it is worth noting the recent description of a case with a milder clinical course and no significant evidence of mtDNA depletion in muscle with a recognised, homozygous TK2 mutation [7]. The two affected siblings we describe died in early infancy with a mitochondrial DNA depletion myopathy due to two novel heterozygous TK2 gene mutations, a c.259C>T transition in exon 2 and a c.299A>G transition in exon 3. To our knowledge, this is the first British family to be described with TK2 mutations. The c.259C>T transition results in a nonsense p.Q87X mutation, whilst the c.299A>G transition predicts a missense p.N100S mutation. Both mutations show high evolutionary conservation (Fig. 1), with Q87X being the first pathogenic mutation to be described in exon 2. The p.N100S mutation lies within a domain of the TK2 protein that is invariant across other cellular deoxyribonucleoside kinases, including dGK, dCK and the Drosophila deoxyribonucleoside kinase, dNK [10], inferring functional significance of this amino acid change, whereas the p.Q87X mutation predicts the premature truncation of the translated TK2 protein. Further, direct evidence of a functional defect can easily be obtained by determining residual TK2 activity in patient cell lines, but unfortunately this was not possible in this case. In regard to the mutation nomenclature, it is worth noting that the GenBank accession number for the human TK2 sequence has recently been updated (NM_004614 (gi: 92859640) replacing the previous version, XM_007855 (gi: 12739950)). The new version includes a novel exon 1, and as such the numbering of previously identified TK2 mutations now begins from the first A (nucleotide 1) of the new ATG initiating methionine codon (first amino acid). The mutations described here are numbered accordingly. Sequencing of parental blood DNA samples confirmed recessive inheritance of the mutated alleles, with the p.Q87X mutation being inherited from the father and p.N100S mutation inherited from the mother. Soon after the identification of the causative mutations in the index case, the parents requested presymptomatic testing for their fifth child at the age of 5 months old, in whom neither mutation was detected. Presymptomatic genetic testing in this additional sibling further reinforces the use of molecular testing in the antenatal detection of mutations in key genes which when mutated, lead to mitochondrial respiratory chain deficiency.
Acknowledgements We thank Gavin Falkous for excellent technical assistance, together with the Wellcome Trust, the Muscular Dystrophy Campaign, the Newcastle upon Tyne Hospitals Foundation NHS Trust and the Department of Health
560
E. Blakely et al. / Neuromuscular Disorders 18 (2008) 557–560
(NCG Rare Mitochondrial Disorders of Adults and Children Service) for their continuing financial support. References [1] Elpeleg O. Inherited mitochondrial DNA depletion. Pediatr Res 2003;54:153–9. [2] Hakonen AH, Isohanni P, Paetau A, Herva R, Suomalainen A, Lo¨nnqvist T. Recessive twinkle mutations in early onset encephalopathy with mtDNA depletion. Brain 2007;130:3032–40. [3] Saada A, Shaag A, Mandel H, Nevo Y, Eriksson S, Elpeleg O. Mutant mitochondrial thymidine kinase in mitochondrial DNA depletion myopathy. Nat Genet 2001;29:342–4. [4] Mancuso M, Salviati L, Sacconi S, et al. Mitochondrial DNA depletion: mutation in thymidine kinase gene with myopathy and SMA. Neurology 2002;59:1197–202.
[5] Oskoui M, Davidzon G, Pascual J, et al. Clinical spectrum of mitochondrial DNA depletion due to mutations in the thymidine kinase 2 gene. Arch Neurol 2006;63:1122–6. [6] Vila` MR, Segovia-Silvestre T, Gamez J, et al. Reversion of mtDNA depletion in a patient with TK2 deficiency. Neurology 2003;60:1203–5. [7] Leshinsky-Silver E, Michelson M, Cohen S, et al. A defect in the thymidine kinase 2 gene causing isolated mitochondrial myopathy without mtDNA depletion. Eur J Paediatr Neurol 2007 [Epub ahead of print]. [8] Bourdon A, Minai L, Serre V, et al. Mutation of RRM2B, encoding p53-controlled ribonucleotide reductase (p53R2), causes severe mitochondrial DNA depletion. Nat Genet 2007;39:776–80. [9] He L, Chinnery PF, Durham SE, et al. Detection and quantification of mitochondrial DNA deletions in individual cells by real-time PCR. Nucleic Acids Res 2002;30:e68. [10] Johansson K, Ramaswamy S, Ljungcrantz C, et al. Structural basis for substrate specificities of cellular deoxyribonucleoside kinases. Nat Struct Biol 2001;8:616–20.
Case report
Novel mutations in the TK2 gene associated with fatal mitochondrial DNA depletion myopathy Emma Blakely a, Langping He a, Julie L. Gardner a, Gavin Hudson a, John Walter b, Imelda Hughes c, Douglass M. Turnbull a,d, Robert W. Taylor a,d,* a
Mitochondrial Research Group, The Medical School, Newcastle University, Newcastle Upon Tyne NE2 4HH, UK b Willink Biochemical Genetics Unit, Royal Manchester Children’s Hospital, Manchester, UK c Department of Neurology, Royal Manchester Children’s Hospital, Manchester, UK d Institutes of Human Genetics and Neuroscience, Newcastle University, Newcastle Upon Tyne, UK Received 21 January 2008; received in revised form 31 March 2008; accepted 14 April 2008
Abstract Mitochondrial DNA depletion syndromes are a heterogeneous group of childhood neurological disorders characterised by a quantitative abnormality of mitochondrial DNA. We describe two siblings who presented at 8 months and 14 months with myopathy, which rapidly progressed and resulted in death by respiratory failure at age 14 and 18 months, respectively. Muscle biopsy revealed marked respiratory chain defects, with real-time PCR confirming a dramatic depletion of mitochondrial DNA. Sequencing of the thymidine kinase 2 (TK2) gene revealed two, novel heterozygous mutations (p.Q87X and p.N100S) with parental DNA analysis confirming the transmission of mutated alleles. Ó 2008 Elsevier B.V. All rights reserved. Keywords: Mitochondrial DNA; Myopathy; Depletion syndrome; TK2 mutations
1. Introduction Mitochondrial DNA (mtDNA) depletion syndromes are autosomal recessive disorders of infancy or childhood, characterised by a quantitative decrease in mtDNA copy number in affected organs [1]. Mutations in several nuclear genes, the protein products of which play key roles in maintaining mitochondrial DNA integrity or the intramitochondrial nucleotide pool, have been associated with a spectrum of clinical presentations. Mutations in DGUOK, POLG1, SUCLA2, MPV17 – and more recently PEO1 – have all been shown to cause a hepatocerebral form of mtDNA depletion syndrome leading to encephalopathy and fatal, infantile liver disease including Alpers syndrome *
Corresponding author. Address: Mitochondrial Research Group, The Medical School, Newcastle University, Newcastle Upon Tyne NE2 4HH, UK. Tel.: +44 1912223685; fax: +44 1912228553. E-mail address: [email protected] (R.W. Taylor). 0960-8966/$ - see front matter Ó 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.nmd.2008.04.014
([2] and references within). Mutations in the thymidine kinase 2 (TK2) gene cause a progressive, myopathic form of mtDNA depletion syndrome [3], with some patients demonstrating clinical features including variable brain involvement, ptosis, ophthalmoplegia, nephropathy and optic neuropathy [3–7]. More recently, mutations in RRM2B, encoding the small subunit of p53-inducible ribonucleotide reductase, have also been identified in patients with myopathic mtDNA depletion disorder [8]. Here we report the clinical, biochemical and molecular genetic investigations of a Caucasian family from the UK in which two siblings had died in early infancy with mitochondrial DNA depletion myopathy due to novel heterozygous TK2 gene mutations. 2. Case report and methods The male index case (II-4) was the 4th child born to healthy, non-consanguineous Caucasian parents (Fig. 1).
558
E. Blakely et al. / Neuromuscular Disorders 18 (2008) 557–560
a
b I:1
II:1
II:2 II:3
c
d
I:2
II:4 II:5
e
f 82
c.259C>T, (Q87X)
*
c.299A>G, (N100S)
*
H. sapiens p.Q87X p.N100S P. troglodytes M. musculus R. norvegicus
104
PPDKEQE--KEKKSVICVEGNIASG PPDKE* PPDKEQE--KEKKSVICVEGSIASG PPDKEQE--KEKKSVICVEGNIASG PPDKDRENDKEKKAVVCIEGNIASG PPDKDREKDKEKKAVVCIEGNIASG
Fig. 1. Analysis of novel TK2 mutations. (a) Pedigree of the family highlighting the index case (arrow). Transverse-orientated muscle sections from the index case were stained or reacted for (b) H&E, (c) succinate dehydrogenase (SDH) activity and (d) cytochrome c oxidase (COX) activity, revealing marked evidence of muscle regeneration and inflammatory cell infiltrate, with an increased staining of some hypertrophied fibres for succinate dehydrogenase activity. Approximately 50% of all fibres were deficient in histocytochemical COX activity. (e) Sequencing electropherograms highlighting the presence of recessive p.Q87X and p.N100S mutations in patient II-4, the index case. (f) Multiple sequence alignment of human TK2 protein with that from other mammalian species, highlighting the amino acid conservation in this region of the protein. The mutated amino acids (p.Q87X and p.N100S) are highlighted in bold.
He presented at the age of 14 months with a 1 month history of poor head control, reduced feeding and increasing difficulty with crawling and sitting. His past history was otherwise unremarkable and his early development had been normal. On examination, growth was normal with a height, weight and head circumference on the 50th centile. He was alert with normal eye movements and no ptosis. He had no facial weakness but had marked head lag, generalised muscle weakness and absent reflexes. His serum creatine kinase was raised at 802 IU/l (normal < 300 IU/l), but lactate and pyruvate levels were both normal. He was commenced on oral ubiquinone (30 mg tds), but there was progressive worsening of his myopathy over the next 4 months and he died at home at the age of 18 months. The parents have three healthy daughters who are alive and well (ages 12 years, 9 years and 2 years), but their first child (II-1) had presented in a similar manner at 8 months of age and died from respiratory failure aged 14 months. Prior to her death, she was found to have normal cardiac structure and function but a raised serum creatine kinase and abnormal liver function tests; blood and CSF lactate were normal but a muscle biopsy had suggested abnormal mitochondria and a generalised decrease in the activities of respiratory chain complexes I, III and IV. A screen at that time for mtDNA rearrangements and common mtDNA point mutations revealed no obvious abnormalities but no further tissue was available for analysis. Standard histological and histochemical analyses were performed on frozen sections (10 lm) of quadriceps muscle. Muscle biopsy of the index case demonstrated a variation in fibre size (6–45 lm), with evidence of degenerate fibres associating with inflammatory cell infiltrate. Some hypertrophied fibres had increased staining for succinate dehydrogenase activity, and approximately 50% of all
fibres were cytochrome c oxidase-deficient (Fig. 1). Ultrastructural studies showed fibres with abnormally shaped mitochondria and focal increase in lipid. The activities of the respiratory chain complexes and the matrix marker citrate synthase were determined in a muscle homogenate, revealing a severe decrease in the activities of respiratory chain complexes I and IV and a normal activity of complex II, suggestive of mtDNA involvement (Table 1). Total DNA was extracted from muscle and used to screen for mtDNA rearrangements by long-range PCR, whilst a screen for a maternally inherited mtDNA point mutation was undertaken by sequencing the entire mitochondrial genome. No abnormalities were detected. Assessment of mtDNA copy number was by a quantitative, real-time PCR assay using an ABI PRISM 7000 sequence detection system (Foster City, CA), and primers and fluorogenic probes to amplify regions of the mtDNA-encoded MTND1 gene and the nuclear-encoded 18S rRNA gene; the efficiency of PCR amplifications and validation of this assay was performed as previously described [9]. Real-time
Table 1 Respiratory chain complex activities in skeletal muscle homogenate from the index case (patient II-4) Complex
Controls (n = 20; mean ± SD)
Patient
Complex I/CS Complex II/CS Complex IV/CS
0.166 ± 0.04 0.208 ± 0.070 1.805 ± 0.550
0.006 0.159 0.121
Enzyme activities are expressed as nmol NADH oxidised min 1 unit citrate synthase 1 for complex I, nmol DCPIP reduced min 1 unit citrate synthase 1 for complex II (succinate:ubiquinone-1 reductase) and the apparent first-order rate constant s 1 unit citrate synthase 1 for complex IV (103). DCPIP = 2,6-dichlorophenol-indophenol; SD = standard deviation.
E. Blakely et al. / Neuromuscular Disorders 18 (2008) 557–560
PCR analysis demonstrated a marked depletion of mtDNA copy number in skeletal muscle from patient II-4, with mtDNA levels at <5% of aged-match controls. We amplified the 10 exons of the TK2 gene using intronic primers [4] and directly compared these to the recently updated reference sequence of human TK2 (GenBank Accession No.: NM_004614). Sequencing analysis of the entire TK2 gene coding region in patient II-4 revealed two novel, heterozygous changes – a c.259C>T transition in exon 2 and a c.299A>G transition in exon 3 predicting nonsense (p.Q87X) and missense (p.N100S) mutations, respectively (Fig. 1). Subsequent analysis of blood DNA from the patient’s mother and father confirmed transmission of both mutated alleles, c.259C>T from the father and c.299A>G from the mother. During our investigation of patient II-4, the mother conceived a fifth pregnancy, giving birth to another daughter (II-5). Presymptomatic genetic testing was requested by the parents at 5 months of age, which happily failed to identify either of the causative TK2 mutations. 3. Discussion Mitochondrial depletion syndromes are autosomal recessive disorders characterised by a reduction in mitochondrial DNA copy number in clinically affected tissues. A balanced supply of deoxyribonucleoside triphosphates (dNTPs) is required for DNA polymerase c to faithfully replicate the mitochondrial genome, the mitochondrial dNTP pool being maintained both through the import of cytosolic dNTPs and by salvaging deoxyribonucleosides within the organelle itself. A number of nuclear-encoded enzymes are involved in these processes, including thymidine kinase 1 (TK1) within the cytosol, and thymidine kinase 2 (TK2) and deoxyguanosine kinase (dGK) within mitochondria, both key enzymes of the mitochondrial salvage pathway. TK2 is a deoxyribonucleoside kinase which is responsible for the phosphorylation of deoxythymidine, deoxycytidine and deoxyuridine [3]. Since the first description of TK2 mutations leading to the myopathic form of mtDNA depletion syndrome in 2001, approximately 20 different pathogenic mutations have now been reported, distributed throughout the TK2 coding sequence (recently reviewed in [5]). The salient clinical and laboratory findings in 20 patients with TK2 mutations have recently been described in detail [5]. Similar to our patients, all affected children presented with proximal weakness and hypotonia with a mean age at onset of 11.4 months. Death in nearly 90% of cases was due to respiratory failure, at a mean age of 42.1 months – somewhat later than the two affected siblings reported here. As noted by these authors, the clinical spectrum also includes spinal muscular atrophy, rigid spine syndrome and severe myopathy with motor regression. Screening of the SMN1 gene was not considered in the index case given the investigation of an affected sibling (II-1) had strongly suggested a mitochondrial aetiology.
559
Similar to other reported patients, our case also showed an elevated serum creatine kinase level and both COX-deficient fibres and SDH-hypereactive fibres on muscle biopsy. Whilst a decrease in muscle mtDNA copy number should indicate screening of the TK2 gene, it is worth noting the recent description of a case with a milder clinical course and no significant evidence of mtDNA depletion in muscle with a recognised, homozygous TK2 mutation [7]. The two affected siblings we describe died in early infancy with a mitochondrial DNA depletion myopathy due to two novel heterozygous TK2 gene mutations, a c.259C>T transition in exon 2 and a c.299A>G transition in exon 3. To our knowledge, this is the first British family to be described with TK2 mutations. The c.259C>T transition results in a nonsense p.Q87X mutation, whilst the c.299A>G transition predicts a missense p.N100S mutation. Both mutations show high evolutionary conservation (Fig. 1), with Q87X being the first pathogenic mutation to be described in exon 2. The p.N100S mutation lies within a domain of the TK2 protein that is invariant across other cellular deoxyribonucleoside kinases, including dGK, dCK and the Drosophila deoxyribonucleoside kinase, dNK [10], inferring functional significance of this amino acid change, whereas the p.Q87X mutation predicts the premature truncation of the translated TK2 protein. Further, direct evidence of a functional defect can easily be obtained by determining residual TK2 activity in patient cell lines, but unfortunately this was not possible in this case. In regard to the mutation nomenclature, it is worth noting that the GenBank accession number for the human TK2 sequence has recently been updated (NM_004614 (gi: 92859640) replacing the previous version, XM_007855 (gi: 12739950)). The new version includes a novel exon 1, and as such the numbering of previously identified TK2 mutations now begins from the first A (nucleotide 1) of the new ATG initiating methionine codon (first amino acid). The mutations described here are numbered accordingly. Sequencing of parental blood DNA samples confirmed recessive inheritance of the mutated alleles, with the p.Q87X mutation being inherited from the father and p.N100S mutation inherited from the mother. Soon after the identification of the causative mutations in the index case, the parents requested presymptomatic testing for their fifth child at the age of 5 months old, in whom neither mutation was detected. Presymptomatic genetic testing in this additional sibling further reinforces the use of molecular testing in the antenatal detection of mutations in key genes which when mutated, lead to mitochondrial respiratory chain deficiency.
Acknowledgements We thank Gavin Falkous for excellent technical assistance, together with the Wellcome Trust, the Muscular Dystrophy Campaign, the Newcastle upon Tyne Hospitals Foundation NHS Trust and the Department of Health
560
E. Blakely et al. / Neuromuscular Disorders 18 (2008) 557–560
(NCG Rare Mitochondrial Disorders of Adults and Children Service) for their continuing financial support. References [1] Elpeleg O. Inherited mitochondrial DNA depletion. Pediatr Res 2003;54:153–9. [2] Hakonen AH, Isohanni P, Paetau A, Herva R, Suomalainen A, Lo¨nnqvist T. Recessive twinkle mutations in early onset encephalopathy with mtDNA depletion. Brain 2007;130:3032–40. [3] Saada A, Shaag A, Mandel H, Nevo Y, Eriksson S, Elpeleg O. Mutant mitochondrial thymidine kinase in mitochondrial DNA depletion myopathy. Nat Genet 2001;29:342–4. [4] Mancuso M, Salviati L, Sacconi S, et al. Mitochondrial DNA depletion: mutation in thymidine kinase gene with myopathy and SMA. Neurology 2002;59:1197–202.
[5] Oskoui M, Davidzon G, Pascual J, et al. Clinical spectrum of mitochondrial DNA depletion due to mutations in the thymidine kinase 2 gene. Arch Neurol 2006;63:1122–6. [6] Vila` MR, Segovia-Silvestre T, Gamez J, et al. Reversion of mtDNA depletion in a patient with TK2 deficiency. Neurology 2003;60:1203–5. [7] Leshinsky-Silver E, Michelson M, Cohen S, et al. A defect in the thymidine kinase 2 gene causing isolated mitochondrial myopathy without mtDNA depletion. Eur J Paediatr Neurol 2007 [Epub ahead of print]. [8] Bourdon A, Minai L, Serre V, et al. Mutation of RRM2B, encoding p53-controlled ribonucleotide reductase (p53R2), causes severe mitochondrial DNA depletion. Nat Genet 2007;39:776–80. [9] He L, Chinnery PF, Durham SE, et al. Detection and quantification of mitochondrial DNA deletions in individual cells by real-time PCR. Nucleic Acids Res 2002;30:e68. [10] Johansson K, Ramaswamy S, Ljungcrantz C, et al. Structural basis for substrate specificities of cellular deoxyribonucleoside kinases. Nat Struct Biol 2001;8:616–20.