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October 22, 2002; 59 (8) Articles

Mitochondrial DNA depletion

Mutations in thymidine kinase gene with myopathy and SMA

M. Mancuso, L. Salviati, S. Sacconi, D. Otaegui, P. Camaño, A. Marina, S. Bacman, C.T. Moraes, J.R. Carlo, M. Garcia, M. Garcia-Alvarez, L. Monzon, A.B. Naini, M. Hirano, E. Bonilla, A.L. Taratuto, S. DiMauro, T.H. Vu
First published October 22, 2002, DOI: https://doi.org/10.1212/01.WNL.0000028689.93049.9A
M. Mancuso
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L. Salviati
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S. Sacconi
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D. Otaegui
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P. Camaño
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A. Marina
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S. Bacman
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C.T. Moraes
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J.R. Carlo
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M. Garcia
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M. Garcia-Alvarez
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M. Hirano
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E. Bonilla
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Citation
Mitochondrial DNA depletion
Mutations in thymidine kinase gene with myopathy and SMA
M. Mancuso, L. Salviati, S. Sacconi, D. Otaegui, P. Camaño, A. Marina, S. Bacman, C.T. Moraes, J.R. Carlo, M. Garcia, M. Garcia-Alvarez, L. Monzon, A.B. Naini, M. Hirano, E. Bonilla, A.L. Taratuto, S. DiMauro, T.H. Vu
Neurology Oct 2002, 59 (8) 1197-1202; DOI: 10.1212/01.WNL.0000028689.93049.9A

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Abstract

Background: The mitochondrial DNA (mtDNA) depletion syndrome (MDS) is an autosomal recessive disorder of early childhood characterized by decreased mtDNA copy number in affected tissues. Recently, MDS has been linked to mutations in two genes involved in deoxyribonucleotide (dNTP) metabolism: thymidine kinase 2 (TK2) and deoxy-guanosine kinase (dGK). Mutations in TK2 have been associated with the myopathic form of MDS, and mutations in dGK with the hepatoencephalopathic form.

Objectives: To further characterize the frequency and clinical spectrum of these mutations, the authors screened 20 patients with myopathic MDS.

Results: No patient had dGK gene mutations, but four patients from two families had TK2 mutations. Two siblings were compound heterozygous for a previously reported H90N mutation and a novel T77M mutation. The other siblings harbored a homozygous I22M mutation, and one of them had evidence of lower motor neuron disease. The pathogenicity of these mutations was confirmed by reduced TK2 activity in muscle (28% to 37% of controls).

Conclusions: These results show that the clinical expression of TK2 mutations is not limited to myopathy and that the myopathic form of MDS is genetically heterogeneous.

Mitochondrial DNA (mtDNA) depletion syndrome (MDS) is a devastating disorder that was first described in 1991.1 Unlike other mitochondrial diseases, MDS is characterized by a quantitative, rather than qualitative, defect of mtDNA. Affected tissues have markedly reduced mtDNA copy number, which impairs the synthesis of respiratory chain components. MDS can be tissue-specific or multisystemic,2 and is inherited as an autosomal recessive trait. In late 2001, mutations in two nuclear genes were identified in patients with MDS: changes in the deoxyguanosine kinase (dGK) gene were identified in patients with the hepatocerebral form,3 and changes in the thymidine kinase 2 (TK2) gene were associated with the myopathic form.4

These findings prompted us to screen a series of 20 patients with mtDNA depletion in muscle for mutations in TK2 and dGK, in order to better define the frequency and phenotypic expression of mutations in these genes.

Patients and methods.

Patients.

Twenty patients with severe mtDNA depletion (70% to 97%) in muscle were included in this study. These patients fulfilled the diagnostic criteria for the myopathic form of MDS.2,5⇓ These criteria include clear evidence of mtDNA depletion by Southern blot, histochemical evidence of cytochrome c oxidase (COX) deficiency, plus at least two of the following features: ragged-red fibers (RRF), lactic acidosis, multiple defects of respiratory chain enzymes, or hyperCKemia. All patients were asymptomatic at birth but soon thereafter developed weakness and hypotonia, often with elevated serum creatine kinase (CK) levels. None had seizures or cognitive delay. Heart, liver, and kidney functions were normal in all cases. Here we describe only the clinical data of the two sets of patients in whom we found TK2 mutations.

Family 1.

Patient 1, a Hispanic boy, was born to nonconsanguineous parents. He was normal until 12 months of age, when he developed frequent falls and progressive gait impairment, leading to inability to walk by age 26 months. Examination at 2 years of age showed shoulder and hip girdle muscle weakness, inability to stand, and hypotonia, but no ophthalmoplegia or facial weakness. He also developed respiratory insufficiency and was dependent on mechanical ventilation by 3 years of age. He died at 40 months of age. Laboratory investigations showed nonspecific organic aciduria and elevated serum CK levels (1,238 U/L, normal <200). A younger sister was asymptomatic, and an older sister (Patient 2) was similarly affected, with a more slowly evolving course. At age 16 months she began to have frequent falls, limb weakness, and gait abnormality, and at age 4 years she was no longer able to walk. She had elevated serum CK levels (950 U/L) and lactic acidosis (12 mmol/L, normal <2.2).

Family 2.

Patient 3, a 2-year-old Hispanic girl, was born at term from nonconsanguineous parents after an uneventful pregnancy. Severe weakness and hypotonia were evident since the first months of life. She had recurrent hospitalizations because of episodic vomiting and failure to thrive with persistent metabolic acidosis. Motor development was delayed. Results of echocardiogram, brain CT, and MRI were normal. EMG was compatible with primary muscle disorder without evidence of neuropathy. At 2 years of age, she had an acute respiratory infection that required mechanical ventilation. She died soon thereafter. Her 3-year-old brother (Patient 4) was normal until the age of 15 months, when he developed increasing lumbar lordosis and waddling gait. Arm and cervical muscles were involved later. At the age of 2 years, he lost his ability to walk. At the age of 3 years, he had severe proximal limb weakness, muscle wasting, areflexia, and scoliosis. Cognitive functions and language were normal. Results of nerve conduction studies were normal, but EMG showed chronic partial denervation, with fibrillations and severe loss of motor unit potentials. These electrophysiologic findings were compatible with spinal muscular atrophy. Serum CK and lactate were mildly increased. He is still alive at 48 months of age.

Muscle morphology and histochemistry.

Routine histologic studies and histochemical staining for COX and succinate dehydrogenase (SDH) were performed as described.6

Biochemical analysis.

Respiratory chain enzyme activities in muscle were measured as described.7

DNA analysis.

Total DNA from patients’ muscle was extracted using standard protocols.8

Southern blot analysis and quantification of mtDNA was performed as described.1,2⇓ Mutations were detected by single strand conformational polymorphism (SSCP) analysis. We used a set of primers that amplified all the exons with flanking intronic sequences of TK2 (see the supplementary table at www.neurology.org). Reactions were performed in 25 μL of 10 mM Tris-HCl (pH 8.9) containing 0.4 μM each of the forward and reverse oligonucleotide; 1.5 mM MgCl2; 0.2 mM each of dATP, dGTP, and dTTP; 0.02 mM dCTP; 1 μCi of α-32[P] dCTP; and 1.25 units of Taq DNA polymerase (Roche, Indianapolis, IN). PCR conditions for exons 1, 6, 8, and 9 were 94 °C for 3 minutes, followed by 35 cycles of 94 °C for 1 minute, 55 °C for 1 minute, 72 °C for 30 seconds, and a final extension step at 72 °C for 7 minutes. PCR conditions other exons were 94 °C for 1 minute, 56 °C for 1 minute, and 72 °C for 30 seconds, for 28 cycles, and a final step at 72 °C for 7 minutes. Samples were denatured and separated on 6% MDE polyacrylamide gel (BME, Rockland, ME) with 5% glycerol, according to the manufacturer’s protocol. Conformations of single-stranded DNA were visualized by autoradiography using BIOMAX film (Kodak, Rochester, NY). Samples with abnormal patterns were directly sequenced using the ABI PRISM Big Dye Terminator Cycle Sequencing Ready Reaction Kit and a 310 Automatic Sequencer (Applied Biosystems, Foster City, CA). The presence of the mutations was confirmed by PCR-RFLP analysis. Mutation screening for dGK was performed as described.3

Determination of TK2 activity.

TK2 activity was measured radiochemically.9

The structure of TK2 was visualized by the programs MOLSCRIPT and RASTER 3D.10 The mutations were checked with the program O11 in a deoxyribonucleosides kinase (dNK)–based model.

Results.

All 20 patients had COX-negative fibers with a mosaic distribution on muscle biopsies. In Patient 4, routine histology of muscle revealed neurogenic abnormalities with large groups of atrophic fibers and separate groups of hypertrophic fibers (figure 1a). Histochemistry showed severe reduction of COX activity in a mosaic pattern (figure 1b).

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Figure 1. Muscle histochemistry in Patient 4. (a) Trichrome stain illustrates hypertrophic fibers intermixed with groups of atrophic fibers (black arrow). (b) Cytochrome c oxidase stain shows reduced enzymatic activity in hypertrophic as well as in groups of atrophic fibers. ×120.

The patients also had markedly decreased activities of respiratory chain complexes containing mtDNA-encoded subunits (complexes I, III, and IV). Biochemical data for patients 1 through 3 are reported in the table. Southern blot analysis showed significant reduction of the mtDNA/nuclear DNA ratio in muscle biopsies from all patients. In patients with TK2 mutations, the degree of mtDNA depletion ranged from 86 to 94% (figure 2).

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Table 1 Mitochondrial respiratory chain enzyme and deoxyribonucleoside kinase activity in patients’ muscles

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Figure 2. Autoradiograph of Southern blot of total DNA extracted from muscle of Patients 1 (P1), 2 (P2), and 3 (P3) and an age-matched control (C). The total DNA was hybridized simultaneously with two 32P-labeled probes—human mtDNA and a cloned fragment of the human 18S ribosomal RNA gene (nuclear DNA).

None of our patients had mutations in the dGK gene, but we found TK2 mutations in two sibships. Patients 1 and 2 of Family 1 were compound heterozygous for the previously described C→A substitution at nt 268 (H90N)4 and for a novel C→T transition at nt 228, which changes a threonine to a methionine at position 77 (T77M) (see parts a and b of the supplementary figure at www.neurology.org). We also found a G→A transition at nt 267, which is probably a neutral polymorphism because it does not change the amino acid sequence of the protein. Patients 3 and 4 harbored a homozygous C→G transversion at nt 66 (see part c of the supplementary figure at www.neurology.org), changing isoleucine at position 22 to methionine (I22M). The presence of the mutations was confirmed by RFLP analysis (data not shown). None of these mutations was present in 120 control alleles.

In agreement with these molecular findings, TK2 activity was decreased in muscle from the two patients so studied (see the table).

Discussion.

MDS encompasses a heterogeneous group of disorders characterized by severe reduction in mtDNA copy number whereas the number of mitochondria is increased, as shown by the RRF in muscle biopsies.1 Depletion of mtDNA can occur as a secondary phenomenon; for example, as a result of nucleoside analogue antiretroviral therapy.12,13⇓ In MDS, however, mtDNA depletion is considered a primary process, and the degree of depletion is generally proportional to the severity of the phenotype. MDS is not uncommon,5,14⇓ onset is in early infancy, and clinical expression can be multisystemic or limited to individual tissues. Patients with the myopathic form of MDS usually present soon after birth with progressive weakness, hypotonia, and areflexia, and die of respiratory failure before 10 years of age.

The discovery of mutations in the thymidine phosphorylase (TP) gene in patients with mitochondrial neurogastrointestinal encephalomyopathy (MNGIE), a disorder associated with multiple deletions and depletion of mtDNA in muscle,15,16⇓ indicated that imbalances in mitochondrial dNTP pool can affect mtDNA integrity. In late 2001, mutations were reported in the TK2 gene in patients with the myopathic form of MDS4 and in the dGK gene in patients with the hepatocerebral form.3

The TK2 gene is located on chromosome 16 and encodes a 234–amino acid polypeptide, which is synthesized in the cytoplasm, then imported into the mitochondrial matrix. It catalyzes the transfer of a phosphate group from ATP to thymidine or deoxycytidine. In contrast, TK1, a cytosolic protein encoded by a gene located on chromosome 17,17 can phosphorylate only thymidine. TK2 belongs to a family of closely related proteins that comprises three other deoxynucleoside kinases of vertebrates—TK1, dGK, and deoxycytidine kinase (dCK)—plus Drosophila melanogaster dNK, a multifunctional enzyme capable of phosphorylating all four deoxynucleosides.18

In eukaryotic cells, the mitochondria and cytosol have distinct dNTP pools. Because the enzymes necessary for de novo dNTP synthesis are not present in mitochondria (figure 3), the mitochondrial pool is maintained either by importing cytosolic dNDP or dNTP through dedicated transporters (mitochondrial deoxynucleotide carrier and mitochondrial dCTP deoxynucleotide transporter19,20⇓) or by salvaging deoxynucleosides within the mitochondria. In nonreplicating cells, cytosolic dNTP synthesis is downregulated, and substrates for mtDNA synthesis derive mostly from the mitochondrial salvage pathway, which depends on two enzymes, dGK and TK2. The combined action of the two proteins allows synthesis of all four dNTP needed for mtDNA replication.21,22⇓ These enzymes have been extensively studied because, in addition to their physiologic substrates, they can also phosphorylate a number of antiviral and antineoplastic agents.18 The crystal structures of human dGK and D melanogaster dNK have been characterized. Owing to the high degree of sequence homology between TK2 and dNK, it is possible to construct a reliable model of TK2 based on the dNK structure,18 and this allows us to infer which structural alteration could be induced by the current mutations (figure 4).

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Figure 3. Schematic representation of mitochondrial nucleotide metabolism. In patients, thymidine and cytidine are decreased because of low TK2 and dGK activity, resulting in the nucleotide pool imbalance and the suppression of mtDNA replication. dGK = deoxyguanosine kinase; TK = thymidine kinase; TP = thymidine phosphorylase; nDNA = nuclear DNA; mtDNA = mitochondrial DNA; dTMP = deoxythymidine monophosphate; dTTP = deoxythymidine triphosphate; dNTP = deoxinucleotide triphosphate; dNDP = deoxynucleotide diphosphate; DNC = deoxynucleotide transporter; dNT2 = 5(3)-deoxyribonucleotidase; dAMP = deoxyadenine monophosphate; dATP = deoxyadenine triphosphate; dGMP = deoxyguanosine monophosphate; dGTP = deoxyguanosine triphosphate; dCMP = deoxycytidine monophosphate; dCTP = deoxycytidine triphosphate.

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Figure 4. Thymidine kinase 2 (TK2) mutations mapped onto the deoxyribonucleosides kinase (dNK) structure. (a) Ribbon representation of the dNK dimer, with each subunit represented by a different color. The nucleotide molecule is represented in space filling with oxygen (red), nitrogen (blue), carbon (green,) and phosphate (yellow) atoms. The side chains of the mutated residues are shown in ball-and-stick with the same atom colors. (b) Detail of the nucleoside binding site (yellow ball-and-stick). The conserved residues among deoxyribonucleoside kinases are shown in violet in the ribbon diagram. Mutant residues are represent by the same colors as (a). Important residues related to the nucleoside are represented in salmon. (c) The blue arrow indicates the P-loop region. The red ring reveals the hydrophobic face of the β-sheet including Ile22.

All three mutations are near the active site (figure 4b). Thr77 and His90 are in the α4 helix (figure 4a and b), which is important for enzyme dimerization and nucleoside recognition. The change from threonine to a larger residue, such as methionine, at position 77 may disrupt the packing or force a displacement of the α helix. The conserved Gln79, which interacts with the nucleoside base, is only a half helix turn away from this mutation, and a helix displacement at position 77 is likely to have an adverse effect on the function of the critical Gln79 residue (see figure 4b). Similarly, His90 is in the C-terminal region of the α4 helix, and forms hydrogen bonds with the main chain of three conserved residues (amino acids 104 through 106, including Arg105) that have catalytic function. The substitution of His90 with asparagine, a residue with a shorter side chain, may impair these interactions and thereby alter the orientation of these critical residues. In agreement with this interpretation, this position is occupied by a histidine (in TK2 and dNK) or by a glutamine (in dGK and dCK), which is similar to histidine in size (see figure 4a). Ile22 occupies a central position in the β-sheet that forms the base of the phosphate-binding P-loop, and is surrounded by aliphatic residues (leucines 102 and 149, and valine 100 and 151) (see figure 4c). These residues form a hydrophobic patch where the α1 and α9 helices pack. The substitution of isoleucine with methionine, a residue with lower propensity to form β-sheets,23 could destabilize the protein fold or the β-strand where the residue sits, thus displacing the P-loop and impairing catalytic activity. These mutations, however, do not inactivate the enzyme completely, as residual TK2 activity was still detectable in our patients’ muscle.

The clinical presentation of TK2-related MDS is more heterogeneous than initially reported,4 as illustrated by Patient 4, who had evidence suggestive of lower motor neuron involvement. The association between MDS and lower motor neuron phenotype has already been reported.24 Surprisingly, the sister of Patient 4, who harbored the same mutation, had pure myopathy. The reasons for this phenotypic heterogeneity are unknown; genetic or environmental factors may contribute to modulate the expressivity of the mutation.

In Patient 1, the reason for organic aciduria is unclear, but association between MDS and organic aciduria has been described25 and is considered an epiphenomenon associated with respiratory chain defects.

Finally, even assuming that we may have overlooked some mutations, our data indicate that TK2 mutations account for only a small percentage of patients (11% in our series, considering each set of siblings as one patient) with the myopathic form of MDS. This suggests that defects in other genes must be involved in the etiology of myopathic MDS. As dGK is not mutated in patients with myopathy, we are screening negative patients for mutations in other candidate genes involved in dNTP metabolism.

Acknowledgments

Supported by NIH grants PO1HD32062 and NS11766 and by a grant from the Muscular Dystrophy Association. T.H.V. is supported by NIH K02 NS02235 and an Irving Clinical Research Career award. L.S. is supported by grant 439b from Telethon Italia and by a scholarship from the University of Padova. P.C. and D.O. are supported by Fondo Investigación Sanitaria grant 01/0108–02 and by grants from Fundación Ilundain and Gobierno Vasco.

Footnotes

  • Additional material related to this article can be found on the Neurology Web site. Go to www.neurology.org and scroll down the Table of Contents for the October 22 issue to find the title link for this article.

  • Received March 19, 2002.
  • Accepted June 20, 2002.

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  • Anterior nerve cell disease
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